Common Bodybuilding myths , by Nandi

[Merc.]

This a great article by Karl Hoffman( was know as Nandi on the boards ).. he has some great articles I wll be posting some more also ...

Karl was a true guru ... He was a Great person ( RIP Karl )...

EDIT : I have added a bunch of some of Karls best work ... So I am posting his articles all in this thread as a Tribute to Karl .. and so the newer peeps could check out some of his BEST reads.. There have been peeps that posted his articles on here ( over the many years), but i think it is cool to have one thread dedicated to him.. He was a very valued member in the bodybuilding and scientific worlds.. I think he at least deserves a thread on here to Honor him..... It is a lot of reading , but well worth it .. Lots to be learned in this thread..


Common Bodybuilding myths,
A Few Common Bodybuilding Myths
by Karl Hoffman



Based on my experience of having been either a moderator or administrator on three Anabolic Fitness boards, I’ve put together a collection of what I feel are, for lack of a better term, a few of the most prevalent “bodybuilding myths.” These are topics that are discussed often and at great length, usually accompanied by much misinformation. In some cases “myth” might be an inappropriate term. A better term might be “bodybuilding dogma based on little or no evidence.” Some might even argue that is unfair in some cases, and that these are simply “controversial topics.” These are not myths held by the general public about bodybuilders and drugs, such as “anabolic steroids invariably cause ‘roid rage,’ or anabolic steroids lead to permanent impotence. They are rather what I believe are widespread misconceptions within the bodybuilding community itself about problems encountered and practices employed by the participants themselves.



ESTROGENIC FAT


Estrogen makes a person fat, doesn’t it? Well, women do have a higher body fat content in general than do men, especially in the gluteofemoral (hips and buttocks) region. Is estrogen really the cause of this gender dimorphism in adiposity? Probably not. In fact, there are a wealth of data that implicate estrogen as both an anorectic and antiadipogenic hormone. It is much more likely that progesterone is the culprit in supporting higher levels of gluteofemoral fat in women (1). The model described in (1) has progesterone as the lipogenic hormone. Before menopause, both estrodiol and progesterone are secreted by the ovaries. After menopause, estrone becomes the primary circulating estrogen produced from aromatization of adrenal androgens (primarily the aromatization of androstenedione to estrone by adipose tissue), while progesterone levels drop dramatically since adrenal production of progesterone is minimal.

In premenopausal women, progesterone increases lipoprotein lipase activity, which is greater in the gluteofemoral region, while estrogen suppresses it. Lipoprotein lipase is the body’s primary fat storage enzyme; it is responsible for allowing fats to leave the circulation and enter adipocytes. The progesterone wins out however and before menopause, women tend to have more gluteofemoral fat and less abdominal fat.

Why do women have more gluteofemoral fat while men have more central (abdominal) fat? One popular theory is that women hold fat in the gluteofemoral region where it is far removed from the liver and has fewer fat mobilizing enzymes/more fat retaining enzymes than in men. Men hold fat in the visceral and abdominal subcutaneous region where it is closer to the liver and richer in fat mobilizing enzymes. Proximity to the liver is a factor because the portal circulation connects abdominal fat deposits directly to the liver. Free fatty acids released from abdominal deposits can act directly on the liver to promote gluconeogenesis, providing the body with a ready supply of glucose for “fight or flight” situations.

From an adaptational viewpoint, women's fat is designed to be stored until needed for lactation and child rearing. Men's fat on the other hand is designed to be readily mobilized for fight or flight situations during defense and hunting. This theory may be a bit simplistic as well as sexist; but it does make sense to some degree.

Most likely the notion of estrogenic fat originated from the belief that estrogen upregulates alpha 2 receptors in fat cells, retarding lipolysis. This may be just one facet of estrogen’s actions. If one looks at the net result of estrogen’s effects, to quote a leading expert in the field


“Testosterone and GH inhibit LPL and stimulate lipolysis markedly. Oestrogens seem to exert net effects similar to those of testosterone.” (2)

For example, animal studies have shown that testosterone promotes alpha 2 adrenoreceptor mediated antilipolytic activity, just as it promotes beta adrenoreceptor mediated lipolysis.

Interestingly, recent research has even attributed at least part of testosterone's fat burning properties to its local aromatization to estradiol (3). For instance when testosterone is administered along with an aromatase inhibitor, LPL activity increases, showing that the testosterone itself is devoid of any ability to lower LPL. (4)

There are a number of animal studies where estradiol administration led to significant weight and fat loss. Citing just one, for example:


"The administration of 17 beta-estradiol (500 micrograms/kg, 2 or 4 weeks) to male rats significantly reduced the body weight...Basal lipolysis and adrenaline-induced lipolysis [due to increase in HSL action] were also significantly enhanced in the epididymal adipose tissue from the male rat treated either with 7 mg/kg estradiol 12 h ahead or with 500 micrograms/kg estradiol for 2 weeks. These results indicate that estradiol exerts strong effects on metabolism of the adipose and these effects seems to be mediated through cyclic-AMP." (5)

This research indicates that in addition to the abovementioned inhibition of LPL, estrogen also stimulates the lipolytic enzyme hormone sensitive lipase.

Some of the most compelling evidence for the antiadipogenic effect of estrogen in both males and females comes from studies of estrogen receptor knockout mice and humans with aromatase deficiency. Both the afflicted humans and the knockout mice exhibit obesity. A detailed look at this topic can be found here:




I also mentioned that estrogen is a potent hunger-suppressing hormone. Research is a bit sketchier here, but the effect is thought to be due to an estrogen-induced inhibition in melanin-concentrating hormone (MCH) signaling (6). MCH is a neuropeptide found in the hypothalamus that is also thought to be involved in leptin’s regulation of appetite. Leptin, an anorectic hormone secreted from the adipose tissue, acts on the specific receptor present on its target neurons in the brain, and suppresses the expression of both MCH and its receptor. So we see that the actions of both estrogen and leptin are at least partly mediated through interactions with MCH.



LONG TERM USE OF T3 WILL ULTIMATELY DAMAGE YOUR THYROID


This notion was dispensed with in Mind & Muscle #10. Here is a brief recap of the relevant findings.

The first study that looked at thyroid function and recovery under the influence of exogenous thyroid hormone was undertaken by Greer (7). He looked at patients who were misdiagnosed as being hypothyroid and put on thyroid hormone replacement for as long as 30 years. When the medication was withdrawn, their thyroids quickly returned to normal.

Here is a remark about Greer's classic paper from a later author:


"In 1951, Greer reported the pattern of recovery of thyroid function after stopping suppressive treatment with thyroid hormone in euthyroid [normal] subjects based on sequential measurements of their thyroidal uptake of radioiodine. He observed that after withdrawal of exogenous thyroid therapy, thyroid function, in terms of radioiodine uptake, returned to normal in most subjects within two weeks. He further observed that thyroid function returned as rapidly in those subjects whose glands had been depressed by several years of thyroid medication as it did in those whose gland had been depressed for only a few days" (8)

These results have been subsequently verified in several studies (8,9) and a large number of trials where T3 was used to treat obesity. So, contrary to what has been stated in the bodybuilding literature, there is no evidence that long-term thyroid supplementation will somehow damage your thyroid gland.



ANDROGENS SUPPRESS THE IMMUNE SYSTEM


This is a grossly oversimplified description of the effects of androgens on immunity. The immune system is comprised of two “arms,” so called humor and cellular immunity. Humoral immunity involves the production of antibodies, and is primarily responsible for targeting extracellular pathogens. Cellular, or cell-mediated immunity involves the action of white blood cells including macrophages, neutrophils, and NK (natural killer) cells. These cells mount an attack on invading pathogens and are responsible for the clearance of intracellular pathogens, virus infected cells, and tumor cells. The cell-mediated response is also responsible for the development of inflammation.

Immune cells known as helper T cells, or Th cells, determine which response—humoral or cellular—the body will mount. There are three subsets of Th cells, Th0, Th1 and Th2 cells. The Th1 cells drive the cellular response, whereas Th2 cells control the humoral response. The two Th subsets are mutually inhibitory. Chemicals called cytokines secreted by Th1 cells suppress Th2 cells, and vice versa. The Th0 cells are precursor cells that can give rise to both Th1 and Th2 cells. This process is illustrated schematically here:




Numerous studies have shown that androgens as well as high levels of estrogens such as occur during pregnancy stimulate Th2 cells and hence promote humoral immunity. So in this sense, androgens are immune stimulating. However, as mentioned, the two arms of the immune system inhibit each other, so by virtue of stimulating Th2 cells and humoral immunity, cellular immunity is suppressed. A nice schematic illustration of this process can be found here:




How exactly do androgens stimulate humoral immunity? One idea is that androgens directly stimulate the production of the cytokine interleukin 10, IL-10, by T cells (10). As illustrated in the first link above, when Th2 cells secrete IL-10, this cytokine has a direct suppressive effect on Th1 cells and hence on the cellular immune response.

Probably the most important clinical effect of the suppression of cellular immunity by androgens is the resulting suppression of the inflammatory response. Androgens have been used with varying degrees of success to ameliorate the symptoms of some autoimmune inflammatory diseases like rheumatoid arthritis. Bodybuilders and other athletes commonly remark how certain anabolic steroids, like testosterone and nandrolone, help to alleviate the inflammation associated with injury or overuse.




ORAL ANABOLIC STEROIDS STIMULATE HEPATIC IGF-1 PRODUCTION


It’s never been completely clear to me exactly how this notion originated. As far as I can tell it was part of the dubious Class I/Class II theory of steroid action that was spawned on anabolic boards and now generally considered meritless. But many people still seem to believe that oral anabolic steroids such as methandrostenelone (Dianabol) and stanozolol (Winstrol) act directly on the liver to stimulate the production of insulin like growth factor (IGF-1) independently of any increase in growth hormone production. As most readers are aware, normally the pituitary gland secretes growth hormone (GH), and the GH then acts on the liver to stimulate the production of IGF-1. In fact, some “experts” have claimed that it is essential to include an oral steroid in any cycle for this reason.

Some oral androgens have been shown to increase IGF-1 levels, but these same drugs also elevate GH levels. So any increase in circulating liver-derived IGF-1 is almost certainly due to an increase in GH. There is not much research in this area to fall back on, but oxandrolone (10) and methandrostenolone (12) have both been shown to elevate GH in humans. Interestingly, when methandrostenolone was administered to rats whose pituitary glands had been removed, it demonstrated no anabolic effects, suggesting that GH secretion is important to the growth promoting effects of Dianabol (13).

Also, as was demonstrated in (14) and a number of other studies, plain old testosterone increases both GH and IGF-1 production. Perhaps most importantly, testosterone has been shown to stimulate the production of IGF-1 directly in muscle tissue, where it acts in an autocrine manner to stimulate growth (15). Locally produced IGF-1 is believed to play a more important role in muscle growth than does liver-derived IGF-1. So this renders moot the argument that it is necessary to incorporate an oral steroid in a cycle in order to elevate hepatic IGF-1 levels.



ANABOLIC STEROIDS DOWNREGULATE THE ANDROGEN RECEPTOR


Or is it upregulate? It seems there are two schools of thought on this, with the answer probably lying somewhere in between. Short-term in vitro and in vivo studies generally show that androgens upregulate the androgen receptor (AR) in skeletal muscle. For example, in humans given 15 mg of oxandrolone daily for 5 days, the skeletal muscle AR density nearly doubled (15). When exposed to testosterone in vitro, skeletal muscle AR expression increased significantly (16).

In longer-term studies the picture is somewhat different. One study looked at AR expression in androgen treated sedentary rats vs nontreated exercised rats over 8 weeks. To quote from the abstract:


Results show that contractile muscular activity always increased the quantity of receptors whereas the steroid treatment decreased it. Thus for EDL (extensorum digitorum longus) and SOL (soleus) of control trained rats the quantity of receptors was 0.78 and 0.82 fmol/mg protein, respectively, compared to 0.23 and 0.43 fmol/mg protein for sedentary testosterone-treated rats. (17)

In long term studies in humans we get yet a different picture. In work conducted by Sheffield-Moore et. al., (18) older men were supplemented with testosterone so as to bring their testosterone levels into the mid-to-high physiological range. Androgen receptor expression had more than doubled after one month of treatment, yet by 6 months had returned to baseline. This pattern suggested to the authors that cycling androgen replacement much as bodybuilders cycle AAS might be a viable strategy:


This pattern of AR expression raises the possibility that cycling of testosterone administration could produce effects on skeletal muscle analogous to continuous administration. Such a paradigm would be beneficial by administering significantly less testosterone for similar anabolic outcomes, thus minimizing the possibility of side effects.

So despite the passion with which advocates of either AR upregulation or downregulation defend their positions, the research is equivocal. Would exercise combined with AAS maintain increased AR expression, or would the addition of exercise serve to offset the AAS induced AR downregulation observed in the study by Bricout et al? These are just a couple of questions that require further research, and could lead to answers on why exercise combined with AAS use is so much more productive than simply using steroids alone when it comes to building muscle mass.



PROGESTERONE AND PROLACTIN INDUCED GYNECOMASTIA


Before delving into this subject, I’d like to say first and foremost, that in users of anabolic/androgenic steroids (AAS) the first step in combating the development of gynecomastia, or male breast enlargement, is to eliminate the causative agent: the anabolic steroid. Drug-induced gynecomastia almost invariably resolves on its own when a person quits taking the drugs responsible for it, if caught before permanent fibrosis develops. Unfortunately, most AAS users don’t want to employ this simple approach, for obvious reasons, so the foregoing will all be under the assumption that a person wants to prevent or treat gyno and still continue steroid use.

In the belief that certain anabolic steroids increase prolactin levels as well as act as agonists at the progesterone receptor, some have advocated the use of antiprolactin agents, like bromocriptine, or progesterone receptor blockers like RU-486 to treat AAS related gynecomastia, in lieu of more traditional drugs like tamoxifen.

In truth, the etiology of gynecomastia is unknown and a number of agents including estrogens, progestins, GH, IGF-1, and prolactin may be involved. However, most authorities believe that a decreased (T+DHT)/E ratio is central to the development of gyno, and that blocking the effects of estrogen, or increasing T + DHT levels, is central to ameliorating the problem.

Regarding prolactin, androgens decrease prolactin levels whereas estrogens increase prolactin. Non-aromatizing androgens have never been shown to elevate prolactin levels in humans, but testosterone has, due to its aromatization to estradiol (19). Prolactin secreting tumors, or prolactinomas, are often associated with gyno. But in these cases the prolactin is believed to induce gyno by suppressing testosterone production: “Prolactinomas that are sufficiently large to cause gynecomastia do so as a result of impairment of gonadotropin secretion and secondary hypogonadism”. (20). However, this is a moot issue in AAS users whose gonadotropin secretion is already blunted.

According to research cited in (20), prolactin may have a direct stimulatory effect on mammary tissue development, but only in the presence of high estrogen levels:


The presence of mild hyperprolactinaemia is therefore not uncommon in patients with estrogen excess. Significant primary hyperprolactinaemia, on the other hand, may directly stimulate epithelial cell proliferation in an estrogen-primed breast, causing epithelial cell proliferation and gynaecomastia.

So rather than focusing solely on lowering prolactin levels which may be elevated in users of aromatizing androgens, attacking estrogen should be the first line of action.

GH and IGF-1 are considered critical to the proliferation of mammary tissue. An excellent review of the role played by these hormones, as well as a general overview of gynecomastia can be found here:




Since elevated GH and IGF-1 are considered important to the anabolic effect of AAS, it would be impractical and counterproductive to attempt to prevent gynecomastia by blocking GH/IGF.

Progesterone acts in concert with estrogen to promote breast development, and at least part of any role played by synthetic progestins may be to stimulate IGF-1 production in the breast. But again, blocking the action of progesterone or synthetic progestins is not practical. Specific progesterone receptor antagonists like RU-486 block not only the progesterone receptor, but the androgen receptor as well, and have actually been associated with the development of gynecomastia (21). In any case, progesterone is thought to act on the breast to enhance the effects of estrogen (22) so once again, attacking estrogen is the easiest and most logical approach.

DHT gel (Andractim) or a generic knockoff might help as well. DHT is thought to act as an aromatase inhibitor (23) and perhaps compete directly with estrogen for binding at the estrogen receptor (24). DHT has been used in several case reports and controlled trials to successfully treat gynecomastia. So perhaps a viable strategy would be to combine DHT gel with tamoxifen. I would recommend tamoxifen rather than an aromatase inhibitor due to the simple fact that tamoxifen has been widely used in numerous controlled studies to succesfully treat gynecomastia, whereas the evidence to support the efficacy of aromatase inhibitors is scanty at best.

Undoubtedly, due to space limitations, I have left out a number of what are surely many readers’ pet myths. Perhaps in a future issue we can address more of these myths and questionable notions. Feedback is always welcome, and if readers wish to submit their ideas for myths that need to be examined in the future, please feel free to contact Mind & Muscle with your ideas.



References:

(1) Price TM, O'Brien SN, Welter BH, George R, Anandjiwala J, Kilgore M. Am J Obstet Gynecol 1998 Jan;178(1 Pt 1):101-7

(2) Bjorntorp P. Hum Reprod 1997 Oct;12 Suppl 1:21-5

(3) Ramirez ME, McMurry MP, Wiebke GA, Felten KJ, Ren K, Meikle AW, Iverius PH Metabolism 1997 Feb;46(2):179-85

(4) Zmuda JM, Fahrenbach MC, Younkin BT, Bausserman LL, Terry RB, Catlin DH, Thompson PD. Metabolism 1993 Apr;42(4):446-50

(5) Tomita T, Yonekura I, Okada T, Hayashi E
Horm Metab Res 1984 Oct;16(10):525-8

(6) Mystkowski P, Seeley RJ, Hahn TM, Baskin DG, Havel PJ, Matsumoto AM, Wilkinson CW, Peacock-Kinzig K, Blake KA, Schwartz MW. J Neurosci 2000 Nov 15;20(22):8637-42

(7) Greer,M. N Engl J Med 244:385, 1951

(8) Vagenakis AG, Braverman LE, Azizi F, Portinay GI, Ingbar SH. N Engl J Med 1975 Oct 2;293(14):681-4

(9) Krugman LG, Hershman JM, Chopra IJ, Levine GA, Pekary E, Geffner DL, Chua Teco GN J Clin Endocrinol Metab 1975 Jul;41(1):70-80

(10) Liva SM, Voskuhl RR J Immunol 2001 Aug 15;167(4):2060-7

(11) Ulloa-Aguirre A, Blizzard RM, Garcia-Rubi E, Rogol AD, Link K, Christie CM, Johnson ML, Veldhuis J Clin Endocrinol Metab 1990 Oct;71(4):846-54

(12) Hochman IH, Laron Z Horm Metab Res 1970 Sep;2(5):260-4
.
(13) Steinetz BG, Giannina T, Butler M, Popick F
Endocrinology 1972 May;90(5):1396-8

(14) Ferrando AA, Sheffield-Moore M, Yeckel CW, Gilkison C, Jiang J, Achacosa A, Lieberman SA, Tipton K, Wolfe RR, Urban RJ.
Am J Physiol Endocrinol Metab 2002 Mar;282(3):E601-7

(15) Sheffield-Moore M, Urban RJ, Wolf SE, Jiang J, Catlin DH, Herndon DN, Wolfe RR,
Ferrando AA
J Clin Endocrinol Metab 1999 Aug;84(8):2705-11

(16) Doumit ME, Cook DR, Merkel RA..Endocrinology 1996 Apr;137(4):1385-94

(17) Bricout VA, Germain PS, Serrurier BD, Guezennec CY.Cell Mol Biol (Noisy-le-grand) 1994 May;40(3):291-4

(18) Ferrando AA, Sheffield-Moore M, Yeckel CW, Gilkison C, Jiang J, Achacosa A, Lieberman SA, Tipton K, Wolfe RR, Urban RJ.
Am J Physiol Endocrinol Metab 2002 Mar;282(3):E601-7

(19) Nicoletti I, Filipponi P, Fedeli L, Ambrosi F, Gregorini G, Santeusanio F
Acta Endocrinol (Copenh) 1984 Feb;105(2):167-72

(20) Ismail AA, Barth JH.Ann Clin Biochem 2001 Nov;38(Pt 6):596-607

(21) Grunberg SM, Weiss MH, Spitz IM, Ahmadi J, Sadun A, Russell CA, Lucci L, Stevenson LL J Neurosurg 1991 Jun;74(6):861-6

(22) Nomura K, Suzuki H, Saji M, Horiba N, Ujihara M, Tsushima T, Demura H, Shizume K
J Clin Endocrinol Metab 1988 Jan;66(1):230-2

(23) Perel E, Stolee KH, Kharlip L, Blackstein ME, Killinger DW
J Clin Endocrinol Metab 1984 Mar;58(3):467-72

(24) Casey RW, Wilson JD.
J Clin Invest 1984 Dec;74(6):2272-8

[Hazard]

Awesome post merc..... Lots of good info in there.....

~Haz~

[Hazard]

The T3 one I have heard about a few times and for some reason..... I still get nervous taking it LOL

~Haz~

[Merc.]

The T3 one I have heard about a few times and for some reason..... I still get nervous taking it LOL

~Haz~

Sup Haz...


I know quite a few peeps that are really afraid to take any thyroid hormones..



Merc.

[sigman roid]

Good read Merc

keep em coming

[Swifto]

I learnt alot of what I know by reading Nandi's posts and BigCat's (who learnt from Nandi). Karl was a true "guru" and way ahead of his time.

RIP Karl.

[Merc.]

Good read Merc

keep em coming

[CHUCKYthentic]

Merc strikes again! great info, good to have you frequenting the board again

[Merc.]

I learnt alot of what I know by reading Nandi's posts and BigCat's (who learnt from Nandi). Karl was a true "guru" and way ahead of his time.

RIP Karl.

Yea he was so ahead of his time is it insane ....




Merc.

[Critical Mass]

Great info here.

[Merc.]

Merc strikes again! great info, good to have you frequenting the board again

Thank You Chucky .. I have been able to have a bit more free time .. I have been so busy ( with work and all kinds of stuff ) it is crazy..






Merc.

[Merc.]

Great info here.

Thank you Omna ... I have quite a few of his articles that I have saved over the years ( peeps have posted his articles on here through out the years, but there is a lot of new peeps that havent seen his work ).. So I am going to post up some of his best articles here ( so we have one place with some of his best work .. If others have any good reads by Nandi post them up also ..




Merc.

[Merc.]

Heres another great one ...


Thyroid Hormone for Weight loss, by Karl Hoffman
Physiologic and Metabolic Effects
by Karl Hoffman


Introduction


It has been over 100 years since the discovery by Magnus-Levy that thyroid hormones play a central role in energy homeostasis, and 75 years since the hormones were first used for weight loss. Despite this great length of time, the precise mechanisms by which thyroid hormones exert their calorigenic effect are not completely characterized, and still actively debated. Despite numerous clinical studies having shown that the administration of thyroid hormone induces weight loss, it is not currently indicated as a weight loss agent. This is probably due to the number of side effects observed during thyroid hormone use at the relatively high doses used in the majority of obesity treatment studies. These deleterious effects include cardiac problems such as tachycardia and atrial arrhythmias, loss of muscle mass as well as fat, increased bone resorption and muscle weakness. Nevertheless, thyroid hormones, particularly triiodothyronine (T3) are a mainstay in the arsenal of drugs used by bodybuilders for fat loss. The widespread underground use of T3 warrants an understanding of its mechanism of action, as well as a knowledge of how it is most effectively and safely used, with an eye to minimizing side effects.



Thyroid Function and Physiology


Before jumping right into a discussion of the use of thyroid hormone for fat loss, a little review of thyroid function and physiology might be in order. The thyroid gland secretes two hormones of interest to us, thyroxine (T4) and triiodothyronine (T3). T3 is considered the physiologically active hormone, and T4 is converted peripherally into T3 by the action of the enzyme deiodinase. The bulk of the body's T3 (about 80%) comes from this conversion. The secretion of T4 is under the control of Thyroid Stimulating Hormone (TSH) which is produced by the pituitary gland. TSH secretion is in turn controlled through release of Thyrotropin Releasing Hormone which is produced in the hypothalamus. This is analogous to testosterone production, where GnRH from the hypothalamus causes the pituitary to release LH, which in turn stimulates the testes to produce testosterone.

In addition to T3, it has recently been recognized that there exist two additional active metabolites of T3: 3,5 and 3,3' diiodothyronines, which we will collectively call T2. Studies have shown that 3,3'-T2 may be more effective in raising resting metabolic rate when hypothyroid subjects are treated with T3, than when normal (euthyroid) subjects are given T3. Therefore in normal subjects 3,5-T2 may be the principal active metabolite of T3 (1)

Like the hypothalamic-pituitary-gonadal axis, the thyroid gland is under negative feedback control. When T3 levels go up, TSH secretion is suppressed. This is the mechanism whereby exogenous thyroid hormone suppresses natural thyroid hormone production. There is a difference though between the way anabolic steroids suppress natural testosterone production and the way T3 suppresses the thyroid. With steroids, the longer and heavier the cycle is, the longer your natural testosterone is suppressed. This is not the case with exogenous thyroid hormone.

An early study that looked at thyroid function and recovery under the influence of exogenous thyroid hormone was undertaken by Greer (2). He looked at patients who were misdiagnosed as being hypothyroid and put on thyroid hormone replacement for as long as 30 years. When the medication was withdrawn, their thyroids quickly returned to normal.

Here is a remark about Greer's classic paper from a later author:


"In 1951, Greer reported the pattern of recovery of thyroid function after stopping suppressive treatment with thyroid hormone in euthyroid [normal] subjects based on sequential measurements of their thyroidal uptake of radioiodine. He observed that after withdrawal of exogenous thyroid therapy, thyroid function, in terms of radioiodine uptake, returned to normal in most subjects within two weeks. He further observed that thyroid function returned as rapidly in those subjects whose glands had been depressed by several years of thyroid medication as it did in those whose gland had been depressed for only a few days" (3)

These results have been subsequently verified in several studies.(3)(4) So contrary to what has been stated in the bodybuilding literature, there is no evidence that long term thyroid supplementation will somehow damage your thyroid gland. Nevertheless, most bodybuilders will choose to cycle their T3 (or T4 which in most cases works just as well) as part of a cutting strategy, since T3 is catabolic with respect to muscle just as it is with fat. As previously mentioned, long term T3 induced hyperthyroidism is also catabolic to bone as well as muscle.

The proviso about T4 vs T3 for weight loss alluded to above needs some elaboration. There have been a number of studies that have shown that during starvation, or when carbohydrate intake is reduced to approximately 25 to 50 grams per day, levels of deiodinase decline, hindering the conversion of T4 to the physiologically active T3.(5) From an evolutionary standpoint this makes sense: during periods of starvation the body, teleologically speaking, would like to reduce its basal metabolic rate to preserve fat and especially muscle stores. However, a recent study demonstrating the effectiveness and safety of the ketogenic diet for weight loss recorded no change in circulating T3 levels.(6) So this issue not completely settled. Nevertheless, persons contemplating thyroid supplementation during ketogenic dieting might prefer T3 over T4 since the bulk of the research does suggest a decline in the peripheral conversion of T4 to T3 during low carb dieting.

Now that we have reviewed a little about thyroid function, let's consider just how it is that thyroid hormone exerts its fat burning effects.



Increased Oxidative Energy Metabolism


Thyroid hormone has long been recognized as a major regulator of the oxidative metabolism of energy producing substrates (food or stored substrates like fat, muscle, and glycogen) by the mitochondria. The mitochondria are often called the "cell's powerhouses" because this is where foodstuffs are turned into useful energy in the form of ATP. T3 and T2 increase the flux of nutrients into the mitochondria as well as the rate at which they are oxidized, by increasing the activities of the enzymes involved in the oxidative metabolic pathway. The increased rate of oxidation is reflected by an increase in oxygen consumption by the body.

T3 and T2 appear to act by different mechanisms to produce different results. T2 is believed to act on the mitochondria directly, increasing the rate of mitochondrial respiration, with a consequent increase in ATP production. T3 on the other hand acts at the nuclear level, inducing the transcription of genes controlling energy metabolism, primarily the genes for so-called uncoupling proteins, or UCP (see below). The time course of these two actions is quite different. T2 begins to increase mitochondrial respiration and metabolic rate immediately. T3 on the other hand requires a day or longer to increase RMR since the synthesis of new proteins, the UCP, is required (1).

There are a number of putative mechanisms whereby T2 is believed to increase mitochondrial energy production rates, resulting in increased ATP levels. These include an increased influx of Ca++ into the mitochondria, with a resulting increase in mitochondrial dehydrogenases. This in turn would lead to an increase in reduced substrates available for oxidation. An increase in cytochrome oxidase activity has also been observed. This would hasten the reduction of O2, speeding up respiration. These and a number of other proposed mechanisms for the action of T2 are reviewed by Lannie et al.(7)

What is the fate of the extra ATP produced during hyperthyroidism? There are a number of ways by which the increased ATP promotes an increase in metabolic activity, including the following:

Increased Na+/K+ATPase. This is the enzyme responsible for controlling the Na/K pump, which regulates the relative intracellular and extracellular concentrations of these ions, maintaining the normal transmembrane ion gradient. Sestoft(7) has estimated this effect may account for up to to 10% of the increased ATP usage.


Increased Ca++-dependent ATPase. The intracellular concentration of calcium must be kept lower than the extracellular concentration to maintain normal cellular function. ATP is required to pump out excess calcium. It has been estimated that 10% of a cell's energy expenditure is used just to maintain Ca++ homeostasis. (1)


Substrate cycling. Hyperthyroidism induces a futile cycle of lipogenesis/lipolysis in fat cells. The stored triglycerides are broken down into free fatty acids and glycerol, then reformed back into triglycerides again. This is an energy dependent process that utilizes some of the excess ATP produced in the hyperthyroid state (8). Futile cycling has been estimated to use approximately 15% of the excess ATP created during hyperthyroidism (8)


Increased Heart Work. This puts perhaps the greatest single demand on ATP usage, with increased heart rate and force of contraction accounting for up to 30% to 40% of ATP usage in hyperthyroidism (9)



Mitochondrial Uncoupling


As mentioned, the mitochondria are often characterized as the cell's powerhouse. They convert foodstuffs into ATP, which is used to fuel all the body's metabolic processes. Much research suggests that T3, like another much more potent agent DNP, has the ability to uncouple oxidation of substrates from ATP production. T3 is believed to increase the production of so called uncoupling proteins. Uncoupling protein (UCP) is a transporter family that is present in the mitochondrial inner membrane, and as its name suggests, it uncouples respiration from ATP synthesis by dissipating the transmembrane proton gradient as heat. Instead of useful ATP being produced from energy substrates, heat is generated instead. There are conflicting studies about the importance of T3 induced uncoupling. Animal studies have demonstrated an actual increase in ATP production commensurate with increased oxygen consumption as we discussed above. Other studies in humans have shown that in fact uncoupling in skeletal muscle does occur. This would contribute to T3 induced thermogenesis, with a resulting increase in basal metabolic rate.(10)

To make up for the deficit in ATP production (as well as provide fuel for the extra ATP production discussed above) more substrates must be burned for fuel, resulting in fat loss. Unfortunately, along with the fat that is burned, some protein from muscle is also catabolized for energy. This is the downside of T3 use, and the reason many people choose to use an anabolic steroid or prohormone during a T3 cycle to help preserve muscle mass. Studies have shown this to be an effective strategy (11). (Muscle glycogen is also more rapidly depleted, and less efficiently stored during hyperthyroidism. This may account for some of the muscle weakness generally associated with T3 use.)

Countering T3 induced muscle loss with AAS or prohormones makes sense from a physiological viewpoint as well. Thyroid hormone muscle protein breakdown is mainly mediated via the so-called ubiquitin-proteasome pathway. (12). (There are several independent metabolic pathways of protein breakdown in the body. For instance, another pathway, the lysosomal pathway, is responsible for the accelerated rate of muscle protein breakdown during and after exercise.) Testosterone administration has been shown to decrease ubiquitin-proteasome activity. (13) So AAS specifically target the muscle protein breakdown process stimulated by T3.

What may not be an effective strategy to maintain muscle mass during a T3 cycle is the use of exogenous growth hormone (GH). Studies have shown that when GH and T3 are administered concurrently, the increased nitrogen retention normally associated with GH use is abolished. This has been attributed to the observation that T3 increases levels of insulin like growth factor binding protein, reducing the bioavailability of igf-1 (14). Nevertheless, GH has fat burning properties independent of igf-1, so using GH with T3 would act additively to speed fat burning, but with little if any preservation of lean body mass. So again, if GH is used in conjunction with T3, anabolic steroid/prohormone use would be indicated.



Andregenic Receptor Modulation


Administration of T3 has been shown to upregulate the so-called beta 2 adrenergic receptor in fat tissue. What is the significance of this effect for fat loss? Before fat can be used as fuel, it must be mobilized from the fat cells where it is stored. An enzyme called Hormone Sensitive Lipase (HSL) is the rate-controlling enzyme in lipolysis, or fat mobilization. The body produces two catecholamines, epinephrine and norepinephrine, which bind to the beta 2 receptor and activate HSL. The upregulation of the beta 2 receptor due to T3 results in an increased ability of catecholamines to activate HSL, leading to increased lipolysis.

Bodybuilders often use drugs like clenbuterol, which bind to the beta 2 receptors and activate them in the same way as the body's endogenous catecholamines. The use of clenbuterol along with T3 can produce an additive lipolytic effect: T3 increases the number of receptors, while clenbuterol binds to the receptors activating HSL and increasing lipolysis. Since clenbuterol itself downregulates the beta 2 receptor, most bodybuilders use clenbuterol in a two week on/ two week off cycle, the rationale being that this minimizes downregulation and allows receptor recovery. Another option is to use the antihistamine ketotifen concurrently with the clenbuterol. Studies have shown that ketotifen attenuates the beta 2 receptor downregulation caused by clenbuterol (15). Moreover, research in AIDS patients has shown that ketotifen blocks the production of the proinflammatory and catabolic cytokine TNF-alpha (16). This may be of relevance to bodybuilders since there is evidence showing TNF lowers both testosterone and IGF-1 levels quite significantly (17) (18), while strenuous exercise elevates TNF levels. (19)

Besides increasing beta 2 receptor density in adipose tissue, T3 upregulates this receptor in human skeletal muscle (12). This has some very intriguing if somewhat speculative implications for the combined use of clenbuterol and T3. Animal studies have shown that catecholamines, particularly clenbuterol, inhibit Ca++ dependent skeletal muscle proteolysis (20). Like the lysosomal and ubiquitin-proteasome pathways discussed above, Ca++ regulated proteolysis is yet another way for the body to degrade muscle protein. Again the implications are enticing: Increased beta 2 receptor density from T3 use, coupled with the beta 2 agonist clenbuterol, could slow this pathway of muscle catabolism.

Another adrenergic receptor important to lipolysis is the alpha 2 receptor, which impedes fat mobilization by counteracting the effects of the beta 2 receptor. There are some conflicting studies about the effects of T3 on the alpha 2 receptor, with studies showing either a downregulation (21) or no effect (22). If T3 does in fact downregulate alpha 2 receptors, this would further aid lipolysis.

Studies in rats have shown that inducing hyperthyroidism increases the lipolytic beta 3 receptor density in white adipose tissue by 70% (23). Beta 3 receptors are abundant in human white adipose tissue as well, and if T3 administration has the same effect in humans, this could could contribute significantly to T3 induced fat loss. This might also argue for taking a currently available beta 3 agonist such as octopamine along with T3 and perhaps clenbuterol.



Decreased Phosphodiesterase Expression


In hyperthyroid patients as well as in normal subjects given T3, levels of the enzyme phosphodiesterase are lowered in fat cells (20). When lipolytic hormones like epinephrine (adrenaline) bind to the beta 2 receptor described above, they initiate a signaling cascade mediated by the so called “second messenger” cyclic AMP (cAMP). cAMP in turn acts on other cellular enzymes to initiate and maintain lipolysis. The original signal is terminated when cAMP is degraded by the enzyme phosphodiesterase. Clearly, maintaining elevated cAMP levels, by lowering phosphodiesterase concentrations with T3, will prolong lipolysis.

As an aside, caffeine is thought to exert at least a portion of its lipolytic action by lowering phosphodiesterase in fat cells. Interestingly, Viagra and Cialis are also phosphodiesterase inhibitors but their action seems to be limited to relaxing vascular smooth muscles.



Increased Growth Hormone Secretion


In vitro, animal, and human studies have all demonstrated that T3 administration increases growth hormone production. (24)(25) Since GH is calorigenic aside from any increase in igf-1, elevated GH may contribute to some of the fat burning associated with T3 administration. This effect may obviate the need for the use of expensive recombinant HGH, as mentioned above.



Decreased Insulin Secretion


Insulin is well known as a lipogenic hormone. It promotes fat storage by facilitating the uptake of fatty acids by adipocytes, and reducing lipid oxidation in muscle tissue. Several studies have shown that thyroid hormone is associated with glucose intolerance resulting from decreased glucose stimulated insulin secretion (26).

This defect in insulin secretion is believed to result from an increase in the rate of apoptosis (programmed cell death) of pancreatic beta cells as a direct effect of thyroid hormone excess.(27) This process is reversible, since when thyroid hormone is withdrawn the rate of beta cell replication increases until homeostasis returns. However, there are conflicting studies regarding the effects of T3 on insulin. For example, Dimitriadis et al (28) showed a decrease in glucose stimulated insulin secretion, consistent with (25), but an increase in basal insulin. They also observed increased insulin clearance, with a compensatory increase in basal insulin secretion.

So if in fact the hyperthyroid state is associated with lower insulin levels, this could explain a portion of hyperthyroid stimulated lipolysis. The obvious downside here is that insulin is also an anabolic hormone. Basal insulin concentration is thought to limit the action of the ubiquitin-proteasome degradative pathway of muscle protein breakdown (29). Of course supplementing with insulin during T3 use would be counterproductive. However, as mentioned above, anabolic steroids inhibit ubiquitin-proteasome activity, so their use could counter any loss in muscle anabolism resulting from a drop insulin levels.



The Future


As mentioned at the beginning of this article, a major roadblock in the adoption of T3 by the medical community as an antiobesity agent is its deleterious effect on the heart. Recent research has identified two isoforms of the thyroid hormone receptor, TRalpha and TRbeta. The TRalpha-form may preferentially regulate the heart rate, and an experimental agent, GC-1, has been developed that selectively binds the TRbeta receptor, with minimal effects on the heart (30). The distribution and actions of TRalpha and TRbeta throughout the body are not yet well characterized. However should it turn out that TRalpha is specific to the heart, then drugs like GC-1 may turn out to be effective fat burning agents with a much safer profile that T3 or T4.

One alleged “futuristic” agent that is here now is T2, or 3,5-Di-iodo-L-thyronine, the T3 metabolite discussed above. Unfortunately, this product does not live up to its hype. It has been claimed to be as or more effective that T3 for fat burning with minimal suppression of endogenous thyroid production. Regarding the relative effectiveness of T2 as a lipolytic agent, and its effect on TSH, this topic was thoroughly covered in a recent article by Bryan Haycock in Muscle Monthly:




All of my research into this subject has led me to the same conclusion reached by Mr. Haycock. That is, T2 is only slightly less suppressive of TSH than is T3, and only packs a portion of the lipolytic punch of T3, with no ability to increase the expression of the UCPs, which is a major determinant of the action of thyroid hormone.



Summary


We have discussed a number of ways by which T3, and its active metabolite T2 act to increase resting energy expenditure. Also discussed were some drawbacks of T3 use, such as cardiac stress, as well as the potential loss of muscle mass. It is ironic that the latter may be of more concern to many bodybuilders that the other more serious potential impacts on health. Nevertheless, used moderately and for short periods (a couple of months or less) in people with no preexisting cardiovascular disease T3 has a relatively safe medical profile, compared to other lipolytic agents like DNP. Perhaps most importantly we have presented substantial evidence that even the long-term use of supraphysiological levels of T3 does not damage the thyroid gland.


References:

(1) Endocrinology 2002 Feb;143(2):504-10 Are the effects of T3 on resting metabolic rate in euthyroid rats entirely caused by T3 itself? Moreno M, Lombardi A, Beneduce L, Silvestri E, Pinna G, Goglia F, Lanni A.

(2)(Greer,M. N Engl J Med 244:385, 1951)

(3)N Engl J Med 1975 Oct 2;293(14):681-4 Recovery of pituitary thyrotropic function after withdrawal of prolonged thyroid-suppression therapy. Vagenakis AG, Braverman LE, Azizi F, Portinay GI, Ingbar SH.

(4) J Clin Endocrinol Metab 1975 Jul;41(1):70-80 Patterns off recovery of the hypothalamic-pituitary-thyroid axis in patients taken of chronic thyroid therapy. Krugman LG, Hershman JM, Chopra IJ, Levine GA, Pekary E, Geffner DL, Chua Teco GN

(5) Int J Obes 1983;7(2):123-31 The effect of a low-calorie diet alone and in combination with triiodothyronine therapy on weight loss and hypophyseal thyroid function in obesity. Koppeschaar HP, Meinders AE, Schwarz F.

(6) Am J Med 2002 Jul;113(1):30-6 Effect of 6-month adherence to a very low carbohydrate diet program. Westman EC, Yancy WS, Edman JS, Tomlin KF, Perkins CE.

(7) J Endocrinol Invest 2001 Dec;24(11):897-913 Control of energy metabolism by iodothyronines.
Lanni A, Moreno M, Lombardi A, de Lange P, Goglia F

(8) Clin Endocrinol (Oxf) 1980 Nov;13(5):489-506 Metabolic aspects of the calorigenic effect of thyroid hormone in mammals. Sestoft L.

(9)Annu Rev Nutr 1995;15:263-91 Thermogenesis and thyroid function. Freake HC, Oppenheimer JH.

(10) J Clin Invest 2001 Sep;108(5):733-7 Effect of triiodothyronine on mitochondrial energy coupling in human skeletal muscle. Lebon V, Dufour S, Petersen KF, Ren J, Jucker BM, Slezak LA, Cline GW, Rothman DL, Shulman GI.

(11)J Clin Endocrinol Metab 1999 Jan;84(1):207-12 Testosterone administration preserves protein balance but not muscle strength during 28 days of bed rest. Zachwieja JJ, Smith SR, Lovejoy JC, Rood JC, Windhauser MM, Bray GA.

(12) Genome Res 2002 Feb;12(2):281-91 In vivo regulation of human skeletal muscle gene expression by thyroid hormone. Clement K, Viguerie N, Diehn M, Alizadeh A, Barbe P, Thalamas C, Storey JD, Brown PO, Barsh GS, Langin D.

(13) J Clin Endocrinol Metab 2003 Jan;88(1):358-62 Related Articles, Links Differential anabolic effects of testosterone and amino Acid feeding in older men. Ferrando AA, Sheffield-Moore M, Paddon-Jones D, Wolfe RR, Urban RJ.

(14) J Hepatol 1996 Mar;24(3):313-9 Effects of long-term growth hormone (GH) and triiodothyronine (T3) administration on functional hepatic nitrogen clearance in normal man. Wolthers T, Grofte T, Moller N, Vilstrup H, Jorgensen JO.

(15) Cardiovasc Res 1998 Oct;40(1):211-22 Terbutaline-induced desensitization of human cardiac beta 2-adrenoceptor-mediated positive inotropic effects: attenuation by ketotifen. Poller U, Fuchs B, Gorf A, Jakubetz J, Radke J, Ponicke K, Brodde OE.

(16) Eur J Clin Pharmacol 1996;50(3):167-70 Ketotifen in HIV-infected patients: effects on body weight and release of TNF-alpha. Ockenga J, Rohde F, Suttmann U, Herbarth L, Ballmaier M, Schedel I.

(17)Endocrinology 1998 Jun;139(6):2863-8 Tumor necrosis factor-alpha inhibits leydig cell steroidogenesis through a decrease in steroidogenic acute regulatory protein expression. Mauduit C, Gasnier F, Rey C, Chauvin MA, Stocco DM, Louisot P, Benahmed M.

(18) Growth Horm IGF Res 2001 Aug;11(4):250-60 Tissue-specific regulation of IGF-I and IGF-binding proteins in response to TNFalpha. Lang CH, Nystrom GJ, Frost RA.

(19) Exerc Immunol Rev 2001;7:18-31 Exercise and cytokines with particular focus on muscle-derived IL-6. Pedersen BK, Steensberg A, Fischer C, Keller C, Ostrowski K, Schjerling P.

(20) Am J Physiol Endocrinol Metab 2001 Sep;281(3):E449-54 Catecholamines inhibit Ca(2+)-dependent proteolysis in rat skeletal muscle through beta(2)-adrenoceptors and cAMP. Navegantes LC, Resano NM, Migliorini RH, Kettelhut IC

(21) J Clin Endocrinol Metab 2002 Feb;87(2):630-4 Regulation of human adipocyte gene expression by thyroid hormone Viguerie N, Millet L, Avizou S, Vidal H, Larrouy D, Langin D.

(22) Metabolism 1987 Nov;36(11):1031-9 Alpha 2- and beta-adrenergic receptor binding and action in gluteal adipocytes from patients with hypothyroidism and hyperthyroidism. Richelsen B, Sorensen NS

(23) Br J Pharmacol 2000 Feb;129(3):448-56 Regulation of beta 1- and beta 3-adrenergic agonist-stimulated lipolytic response in hyperthyroid and hypothyroid rat white adipocytes. Germack R, Starzec A, Perret GY

(24) Braz J Med Biol Res 1994 May;27(5):1269-72 Role of thyroid hormone in the control of growth hormone gene expression. Volpato CB, Nunes MT.

(25) Am J Physiol 1999 Aug;277(2 Pt 1):E370-9 Related Articles, Links Low-dose T(3) improves the bed rest model of simulated weightlessness in men and women. Lovejoy JC, Smith SR, Zachwieja JJ, Bray GA, Windhauser MM, Wickersham PJ, Veldhuis JD, Tulley R, de la Bretonne JA.

(26) Life Sci 2002 Jul 19;71(9):1059-70 Evidence for a deficient pancreatic beta-cell response in a rat model of hyperthyroidism. Fukuchi M, Shimabukuro M, Shimajiri Y, Oshiro Y, Higa M, Akamine H, Komiya I, Takasu N.

(27) Diabetologia 2002 Jun;45(6):851-5 Thyroxine induces pancreatic beta cell apoptosis in rats.
Jorns A, Tiedge M, Lenzen S.

(28) Am J Physiol 1985 May;248(5 Pt 1):E593-601 Effect of thyroid hormone excess on action, secretion, and metabolism of insulin in humans.= Dimitriadis G, Baker B, Marsh H, Mandarino L, Rizza R, Bergman R, Haymond M, Gerich J

(29) Curr Opin Clin Nutr Metab Care 2000 Jan;3(1):67-71 Effects of insulin on muscle tissue.
Wolfe RR.

(30) J Steroid Biochem Mol Biol 2001 Jan-Mar;76(1-5):31-42 Selective modulation of thyroid hormone receptor action. Baxter JD, Dillmann WH, West BL, Huber R, Furlow JD, Fletterick RJ, Webb P, Apriletti JW, Scanlan TS.



Merc.

[Skippa]

Great finds merc!

Good read

[Merc.]

Growth Hormone and the athlete,
Recombinant Growth Hormone and the Athlete
by Karl Hoffman


In last month’s issue of Mind and Muscle (M&M #14) we looked at how growth hormone has been used in a number of trials to successfully induce weight loss in obese humans.

In order to better understand how GH affects this weight loss we also discussed in some detail how growth hormone and fat cells interact with one another. In this review of the existing literature, I would like to look at another growing use of recombinant GH: its use to increase athletic performance and increase muscle mass. There are much less data to guide us here than was available in our discussion of GH treatment of obesity. Further, the scientific literature contrasts starkly with the vast number of anecdotal reports of dramatic improvement in athletic performance and muscle mass seen with GH use. The scientific literature paints a rather bleak picture of recombinant GH as an ergogenic aid.

The positive results of some of the obesity trials discussed in Mind & Muscle #14 do suggest that GH might be beneficial to athletes and bodybuilders for weight loss while maintaining lean body mass. In fact, the studies in which recombinant GH has been administered to athletes and healthy young adults have yielded mixed results in terms of changes in strength and body composition, with the data often being difficult to interpret. This will be evident upon looking in detail at the research. For example, Yarasheski et al (1) looked at the effect of 14 days of recombinant GH administration (40 mcg/kg/day) on muscle protein synthesis rates in experienced weight lifters. The authors concluded that short-term GH administration neither increased the fractional rate of skeletal muscle protein synthesis nor did it reduce the rate of whole body protein breakdown despite significantly elevated levels of circulating IGF-1. This is in contrast to research that has shown that GH administration in normal, healthy humans in the postabsorptive state increases net muscle amino acid balance during the period of GH infusion (2). This anabolic effect is evidently short lived, since as mentioned, long-term studies show no increase in muscle mass. Note that in the study by Yarasheski et al protein synthesis/breakdown rates were measured several hours after the last GH injection, not during an infusion as in (2). Nevertheless, IGF-1 levels were still elevated 2 fold above baseline when Yarasheski et al collected their data.

As an aside, in another interesting study (3) that looked at the short-term infusion of a combination of GH and insulin, GH once again appeared to increase protein synthesis, but it also blunted the normal antiproteolytic effects of insulin.

Yarasheski et al (4) conducted another study in which GH was administered to healthy young men in conjunction with a resistance training program. The authors measured a number of parameters: change in body composition; muscle strength improvement; whole body protein turnover; and fractional muscle protein synthesis rate. Compared to placebo, the GH treated group showed a significantly larger increase in fat free mass. However, due to the rapid gain in this mass and the rapid loss after treatment ended, the authors attributed this gain primarily to water retention. There was no difference in strength gains between the GH and placebo treated groups. The GH treated group showed an increase in whole body protein synthesis but no change in fractional skeletal muscle synthesis rate. From this, and the lack of strength gains and muscle circumference, the authors deduce that the net protein accretion was not in the form of skeletal muscle.

Deyssig et al (5) conducted a similar study in trained power athletes. One group was given rhGH at 0.09 U/kgBW day while another was given placebo. Both groups participated in a resistance training program for six weeks. At the end of the study period changes in strength and body composition were measured in both groups. Again there was no difference between the two groups in the parameters measured. The authors concluded that GH treatment had no effect on strength or body composition in highly trained strength athletes.

Crist et al (6) examined the effects of six weeks of rhGH administration (30 – 50 mcg/kg, 3 days per week) in a group of young, highly conditioned (resistance and aerobic trained) men and women. FFM increased more (2.7 kg) and body fat decreased more (1.5 kg) during the GH treatment period than during the six-week placebo treatment period. It is unclear however whether the increase in FFM was due to any accumulation of skeletal muscle (contractile) protein. The study did demonstrate a greater fat loss during the GH period. This is consistent with some of the research presented last issue of M&M showing that GH treatment is capable of promoting fat loss.

In bodybuilders wishing to lower their body fat levels to what is humanly feasible, GH may be a viable option if one is willing to accept the possibility of some unhealthful side effects. In competitive endurance or strength athletes, as opposed to bodybuilders, the detrimental effects of GH use on performance may argue against its use. In a review of the topic (7) Rennie cites recent research conducted at the Danish Institute of Sports Medicine where GH administration to trained athletes actually impaired their performance (8). In these studies healthy endurance trained athletes were unable to complete accustomed cycling tasks after administration of exogenous hGH. The authors suggest that this could be a result of an observed increase in plasma lactate in the GH group compared to placebo. The significantly elevated lactate could result from the inhibition of the enzyme pyruvate dehydrogenase (PDH) by high levels of fatty acids released during GH-stimulated lipolysis. With PDH thus inhibited, pyruvate, produced from the glycolysis of glucose, is unable to enter the mitochondrial citric acid cycle and accumulates instead as lactate. One problem with this theory however, is that despite the increase in plasma free fatty acids observed by the authors, there was no apparent increase in lipid oxidation. The latter would be expected to be required to inhibit PDH. In any case, by whatever mechanism, GH administration clearly adversely affected cycling performance in this experiment.

Although the research described above looked at the acute effects of GH administration on athletic performance, there are chronic effects as well that could be detrimental to the athlete. Insulin resistance is a common side effect of GH use and would be expected to reduce glucose availability to muscle. GH administration also results in the impairment of muscle and liver glycogen storage. These latter effects, limited liver and muscle glycogen storage, could have a serious impact on recovery from strenuous exercise, as well as negatively impact performance itself as a result of decreased glycogen availability. The edema associated with GH administration could also impair athletic performance, as might the arthralgia experienced by many GH users. Rennie even cites the possibility that the fatty acidemia resulting from GH-induced lipolysis could promote cardiac arrhythmia during intense exercise. Although remarks such as this are reminiscent of some of the hyperbole from the medical community regarding anabolic steroids, there is probably some degree of legitimacy to the concerns of Rennie and others who have stressed the potential seriousness of GH related side effects. Athletes should at least be aware that concern exists over such things as potentially fatal as arrhythmia.

In addition to the potentially detrimental derangements in glucose metabolism mentioned above, GH administration in humans has been shown to induce a shift in muscle fiber type from type 2a to 2x (9, 10). The latter has been characterized as the “default” fiber type since the proportion of 2x fibers to type1 and type 2a is relatively high in “couch potatoes” compared to strength and power athletes. Resistance training induces a shift in the opposite direction from type 2x to 2a. During detraining, the muscle fiber type shifts back to 2x. The training induced shift is interpreted as an adaptive mechanism to the increased demands placed upon the muscle. If GH administration induces a shift in muscle fiber type away from the trained state, this could have negative implications for strength and power athletes.

Why, in light of all this negative evidence for any strength or muscle mass increase resulting from exogenous GH, is the bodybuilding literature replete with anecdotal reports of impressive gains in muscle mass and strength? And what motivates athletes to use GH in light of the negative research and side effects? One obvious possibility is that the research results are wrong or incomplete. But assuming they are not for the sake of furthering the discussion, another conceivable explanation for the reported gains in muscle mass are the lipolytic effects of GH discussed above. Bodybuilders could easily be mistaking enhanced definition for an increase in muscle. GH associated water retention could also add to the feeling that mass has increased. Certain anabolic steroids such as Dianabol and Deca Durabolin are notorious for causing water retention. These same drugs also have a reputation for increasing the resistance exercise induced muscle “pump”, contributing to a feeling of increased strength. The water retention from exogenous GH could have the same effect. Additionally, athletes and even researchers have noted that in elite athletes, studies would probably be unable to detect with statistical significance a 1 or 2 percent increase in performance that could result from GH use, and would make all the difference in the world to an elite athlete. Arguing against this is the observation that performances in a number of Olympic events such as shotput, discus, and javelin, particularly among women, have deteriorated since routine testing for anabolic steroids was implemented. It is very likely that these athletes who formerly were heavy users of anabolic steroids are now using rhGH, but it does not seem to be helping their performance. And perhaps the most obvious reason that many athletes and bodybuilders use GH is that the competition is using it.

In summary, despite numerous anecdotal reports to the contrary, to quote from (7),


The results of studies of muscle protein synthesis, body composition, and strength in healthy young to middle aged humans tell a different tale: so far no robust, credible study has been able to show clear effects of either medium to long term rhGH administration, alone or in combination with a variety of training protocols or anabolic steroids, on muscle protein synthesis, mass or strength.

These results, coupled with the possibility that GH use could significantly compromise training and performance, as described in (8), make a fairly strong argument against the use of GH in sport.



References

(1) Yarasheski KE, Zachweija JJ, Angelopoulos TJ, Bier DM Short-term growth hormone treatment does not increase muscle protein synthesis in experienced weight lifters. J Appl Physiol. 1993 Jun;74(6):3073-6.

(2) Fryburg DA, Gelfand RA, Barrett EJ. Growth hormone acutely stimulates forearm muscle protein synthesis in normal humans. Am J Physiol. 1991 Mar;260(3 Pt 1):E499-504

(3) Fryburg DA, Louard RJ, Gerow KE, Gelfand RA, Barrett EJ. Growth hormone stimulates skeletal muscle protein synthesis and antagonizes insulin's antiproteolytic action in humans. Diabetes. 1992 Apr;41(4):424-9

(4) Yarasheski KE, Campbell JA, Smith K, Rennie MJ, Holloszy JO, Bier DM. Effect of growth hormone and resistance exercise on muscle growth in young men. Am J Physiol. 1992 Mar;262(3 Pt 1):E261-7

(5) Deyssig R, Frisch H, Blum WF, Waldhor T. Effect of growth hormone treatment on hormonal parameters, body composition and strength in athletes. Acta Endocrinol (Copenh). 1993 Apr;128(4):313-8.

(6) Crist DM, Peake GT, Egan PA, Waters DL. Body composition response to exogenous GH during training in highly conditioned adults. J Appl Physiol. 1988 Aug;65(2):579-84.

(7) Rennie MJ.Claims for the anabolic effects of growth hormone: a case of the emperor's new clothes? Br J Sports Med. 2003 Apr;37(2):100-5.

(8) Lange KH, Larsson B, Flyvbjerg A, Dall R, Bennekou M, Rasmussen MH, Orskov H, Kjaer M. Acute growth hormone administration causes exaggerated increases in plasma lactate and glycerol during moderate to high intensity bicycling in trained young men. J Clin Endocrinol Metab. 2002 Nov;87(11):4966-75.

(9) Hennessey JV, Chromiak JA, DellaVentura S, Reinert SE, Puhl J, Kiel DP, Rosen CJ, Vandenburgh H, MacLean DB. Growth hormone administration and exercise effects on muscle fiber type and diameter in moderately frail older people. J Am Geriatr Soc. 2001 Jul;49(7):852-8.

(10) Lange KH, Andersen JL, Beyer N, Isaksson F, Larsson B, Rasmussen MH, Juul A, Bulow J, Kjaer M. GH administration changes myosin heavy chain isoforms in skeletal muscle but does not augment muscle strength or hypertrophy, either alone or combined with resistance exercise training in healthy elderly men. J Clin Endocrinol Metab. 2002 Feb;87(2):513-23

[Merc.]

And this complements the other GH article...


Growth Hormone and Obesity,
by Karl Hoffman


INTRODUCTION


The observation that growth hormone (GH) secretion is impaired in obesity, and is reversible upon weight loss, has prompted a great deal of research that has helped us understand how GH acts on adipocytes to regulate lipolysis and lipogenesis. Reciprocally, we are beginning to understand how adipocytes, as secretory organs, contribute to the regulation of GH secretion. The impaired secretion of GH in obesity, as well as the predominantly lipolytic effects of GH has prompted a number of studies where GH has been successfully used to induce significant weight loss in obese patients.

In this brief overview, I’d like to first look at the effects of GH on adipocyte function, then address the converse subject of adipocyte regulation of GH secretion, with particular emphasis on how obesity impairs GH secretion. Finally, we will look at how GH has been used therapeutically to treat obesity.



PHYSIOLOGICAL EFFECTS OF GH ON ADIPOCYTES


Two enzymes active in adipocytes which are of paramount importance in regulating lipogenesis (fat accumulation) and lipolysis (the breakdown of stored triglycerides into free fatty acids [FFA]) are lipoprotein lipase (LPL) and Hormone Sensitive Lipase (HSL); both are affected by GH. The accumulation of triglycerides in adipose tissue is controlled primarily by LPL. Triglycerides are transported to fat cells for storage in the form of very low-density lipoproteins (VLDL) and chylomicrons. LPL is synthesized by adipocytes and then secreted to the intracellular space, after which it attaches to the luminal portion of the vascular endothelium of the vessels supplying the adipocytes. There it hydrolyzes the triglyceride fraction of the VLDL and chylomicron particles, releasing FFA that are taken up by adipocytes. GH has been shown to have an inhibitory effect on adipose LPL (1,2), with a more pronounced reduction of LPL activity in intra-abdominal fat deposits than in subcutaneous fat (3). Exactly how GH inhibits LPL is unclear. GH treatment does not seem to affect LPL gene expression or mRNA levels, so it is assumed that the effect is post-translational, with GH somehow interfering with the activity of the enzyme (1). In any case, the net effect is that GH reduces the uptake of free fatty acids by fat cells, a clear antiadipogenic effect.

It should be noted that a number of other hormones affect LPL activity in significant ways. Insulin is the hormone with the greatest ability to stimulate LPL activity, contributing to the well-known lipogenic effect of this hormone. Conversely catecholamines (e.g. epinephrine) are strong downregulators of LPL, contributing to their ability to block fat accumulation. Testosterone and estrogen both inhibit LPL, contributing to their fat burning properties (4).

The second enzyme that dominates adipocyte metabolism is Hormone Sensitive Lipase (HSL). HSL is responsible for the hydrolysis of stored triglycerides to glycerol and free fatty acids. Thus hydrolyzed, the FFA can leave the adipocyte and travel in the blood to other tissues where they can be used as fuel, primarily in working muscle (As with fats entering adipocytes, the glycerol portion of the triglyceride must be removed in order for free fatty acids to leave the fat cell). GH amplifies the action of HSL in two ways. First, HSL is activated by catecholamines that act as agonists at beta 1, beta 2, and possibly beta 3 receptors in adipocytes. This is how sympathomimetic drugs like ephedrine and clenbuterol stimulate fat burning: they act as beta agonists to stimulate HSL. GH has been shown to be capable of inducing beta 2 receptors in adipocytes; more beta 2 receptors mean more HSL activity (5). As an aside, this is one way androgens promote lipolysis as well, via the upregulation of beta adrenoreceptors. Beta receptors employ the “second messenger” cyclic AMP (cAMP) to relay their signal within the cell that ultimately activates HSL. The signal is terminated by the enzyme phosphodiesterase. GH has been shown to have the ability to block phosphodiesterase, prolonging the activity of HSL (5). So we see GH promotes lipolysis via HSL by two routes: it upregulates the receptors that activate HSL, and it prolongs the signaling that keeps HSL functioning.

Besides affecting the metabolic functioning of adipocytes, GH controls adipocyte differentiation and proliferation. Differentiation refers to the process whereby immature preadipocytes activate the genes that direct them onto the path to becoming fully functioning mature adipocytes capable of carrying out the metabolic and secretory processes described above, as well as storing lipids. Proliferation refers to the increase in cell number via repeated cell division. The actions of GH are mixed here. We know that GH stimulates the hepatic production of Insulin-like Growth Factor 1 (IGF-1), which is responsible for many of the metabolic and perhaps anabolic actions of GH. It has been shown that IGF-1 is capable of stimulating the proliferation of preadipocytes, increasing the pool of potential adult fat cells (6). On the other hand, GH itself inhibits the differentiation of these precursor cells into adult adipocytes. Despite these contradictory effects of GH/IGF-1 on adipocyte proliferation and differentiation, the net effect of GH treatment in obese subjects in a number of studies is one of reduced adiposity.



FREE FATTY ACIDS AND GH SECRETION


GH and FFA function together in a regulatory feedback fashion. We have seen above how GH stimulates lipolysis, resulting in elevated levels of FFA. FFA in turn act back in a negative feedback manner to inhibit GH secretion. Circulating free fatty acids, elevated in obesity, are thought to be partly responsible for the suppression of GH seen in this condition (Plasma levels of FFA are elevated in obesity primarily because a greater than normal amount of FFA is released from the expanded adipose tissue mass even though the rate of lipolysis from individual fat cells appears to be normal).

It is generally accepted that circulating FFA rapidly partition into the plasma membranes of pituitary cells which secrete GH. This is believed to alter the function of proteins embedded in the plasma membrane, perturbing intracellular signaling and inhibiting GH release (9). Animal studies have shown that FFA are also capable of acting directly on the hypothalamus to increase the release of somatostatin, with a resulting inhibitory effect on GH release. It is controversial whether this hypothalamic effect exists in humans (10). No known stimulus for GH release seems to be able to escape the suppressive effects of elevated FFA. As just one example of relevance to athletes, exercise is a well-known stimulus for GH release. Seemingly paradoxically, exercise also elevates FFA acid levels, as lipolysis increases in order to supply FFA to muscle to serve as a fuel source. However, when nicotinic acid, a potent inhibitor of FFA release from adipocytes is administered during exercise, the low FFA levels resulting from nicotinic acid feeding were associated with a 3- to 6-fold increase in concentrations of human growth hormone throughout exercise. Exercise performance was also negatively impacted by the lack of availability of FFA as a fuel substrate (11). This could have practical implications for anyone using nicotinic acid to elevate HDL cholesterol levels, as many anabolic steroid using athletes are known to do (Anabolic steroids in general, and oral 17 alpha alkylated steroids in particular, are known to significantly lower HDL, or “good” cholesterol).

Somewhat surprisingly, in light of the evidence discussed above that FFA inhibit GH release, GH secretion is increased during fasting both in obese and normal subjects after administration of GHRH, despite an increase in fasting related FFA levels. This has been cited as contradictory to the theory that FFA impair GH secretion in obesity (12). However, as mentioned above, ghrelin may be more important than GHRH in stimulating GH release during fasting. While FFA do reduce the ability of ghrelin to stimulate GH release, ghrelin is partially refractory to this inhibitory effect of FFA. So it is possible that the results described in (12) were confounded by the effects of ghrelin on GH during fasting.

In any case, GH is generally low in obesity, and as a consequence there is a loss of the usual lipolytic effect of GH seen in normal individuals. This has prompted the experimental use of GH to attempt to reverse obesity in a number of studies.



INCREASED GH CLEARANCE RATE IN OBESITY


Studies have shown besides decreased production of GH in obesity, GH clearance rates are increased as well. While not necessarily being an effect directly attributable to the action of adipocytes on GH, it does contribute to lower overall GH plasma levels (13). Not well understood, this phenomenon has been attributed to either increased glomerular filtration of GH, changes in liver metabolism, or accelerated processing by excessive body fat stores.



EFFECT OF ADIPOSE TISSUE ON IGF-1


Despite the fact that GH levels are typically depressed in obesity, total serum IGF-1 levels are normal or high, and free IGF-1 levels are consistently elevated (5). This may seem surprising since—as discussed above—IGF-1 is normally produced in the liver under the stimulus of GH. One might expect the opposite to be observed: low GH in obesity leading to low circulating IGF-1. However, the observation that IGF-1 mRNA levels in fat cells are nearly as high as those found in the liver has led to the suggestion that adipocytes could contribute significantly to circulating levels of IGF-1 (5). If this is the case, then the normal negative feedback of IGF-1 on GH secretion could contribute in part to the depressed levels of GH seen in obesity. Adipocytes seem to secrete IGF-1 in response to GH, and in obesity, individual fat cells may secrete less IGF-1 than in normal subjects. The net overall effect of the increased number of fat cells in obese subjects would offset this, leading to the observed elevation in IGF-1. The depressed GH due to elevated IGF-1 in obesity provides another rationale for the use of GH to treat obesity.



INHIBITION OF GH SECRETION AND SIGNALING BY INSULIN


Insulin resistance and hyperinsulinemia are often associated with obesity. Research has shown that both normal physiological levels of insulin (14) as well as obesity-associated hyperinsulinemia blunt the GH response to GHRH and may contribute to the GH deficiency seen in obesity (15). Although the exact mechanism by which insulin regulates GH secretion is not known, a number of possibilities exist. Specific insulin binding sites have been found in both rat and human anterior pituitary adenoma cells. Inhibition of GH synthesis and release, and suppression of GH mRNA content, has also been observed when pituitary cells are exposed to insulin. So insulin could have a direct inhibitory effect on the pituitary. Insulin receptors are also present in the hypothalamus, so it is possible insulin is acting there. It has also been suggested that insulin could inhibit GH release by lowering plasma amino acid levels, since amino acids stimulate GH release. It has also been observed that insulin lowers circulating levels of the potent GH secretagogue ghrelin (16).

In vitro, insulin has also been shown in nonhepatic tissue to block the translocation of the GH receptor from the cytosol to the cell surface, with the effect of inhibiting binding of GH to its receptor. This may be another way hyperinsulinemia associated with obesity disrupts GH signaling (17)



GROWTH HORMONE THERAPY TO TREAT OBESITY


We have discussed a number of reasons why GH might potentially be of therapeutic use in the treatment of obesity due to its lipolytic action. Nevertheless, the results of trials have been inconsistent. This inconsistency, coupled with side effects of treatment which include insulin resistance, edema, arthralgia, and carpel tunnel syndrome to name a few, has prompted some critics to take a strong stand against the use of GH to treat obesity:



OBJECTIVE: To summarize the reports in the literature regarding the effect of growth hormone (GH) treatment of obesity. RESEARCH METHODS AND PROCEDURES: Clinical trials of GH treatment of obese adults were reviewed and summarized. Specifically, information regarding the effects of GH on body fat and body fat distribution, glucose tolerance/insulin resistance, and adverse consequences of treatment were recorded. RESULTS: GH administered together with hypocaloric diets did not enhance fat loss or preserve lean tissue mass. No studies provided strong evidence for an independent beneficial effect of GH on visceral adiposity. In all but one study, glucose tolerance during GH treatment suffered relative to placebo. CONCLUSION: The bulk of studies indicate little or no beneficial effects of GH treatment of obesity despite the low serum GH concentrations associated with obesity (18).


Despite the harsh tone of these investigators, a number of studies have shown a positive effect of GH on fat loss, with the abovementioned side effects being reversible upon termination of treatment. Additionally, countless anecdotal reports by bodybuilders and athletes contribute to the evidence that GH can be efficacious for fat loss.

In stark contrast to the assessment of the GH trials in (18) are reports by Lucidi et al (19) and Nam et al (20) that cite a number of studies where “GH is effective in reducing fat mass, especially visceral fat” (20). Nam et al discuss why some studies may have shown negative results. In their paper, the authors reported significantly enhanced fat loss (1.6 fold) compared to placebo, with a greater loss in visceral fat and an increase in lean body mass (20). Kim et al used low dosages of GH (0.18 U/kg Ideal Body Weight/week) and a hypocaloric diet, and believed this accounted for at least part of the success of their trial. They point out that one of the well known and dose dependent side effects of GH administration is insulin resistance and hyperinsulinemia. Insulin is well known to be an adipogenic hormone, and the hyperinsulinemia that often accompanies GH treatment could offset the lipolytic effect of the administered GH. To quote from the authors,



In addition, as the product of GH-induced lipolysis, FFA has been considered to be the principle factor in peripheral insulin resistance. These findings suggest that GH-induced hyperinsulinemia may antagonize the lipolytic effect of GH. In our study, GH treatment did not induce a further increase in insulin levels. [This] suggest[s] that although GH might induce insulin insensitivity and hyperinsulinemia, low-dose GH therapy with diet restriction in obesity could overwhelm the antilipolytic action of insulin.

The frequency of side effects depends on the dose of GH. We observed only minor side effects which spontaneously subsided, indicating that the dose of GH in this study was lower in comparison with other studies (20).


So it may very well be that many of the studies that failed to demonstrate weight loss after GH administration employed excessively high doses, which either aggravated pre-existing hyperinsulinemia or subsequently induced hyperinsulinemia, which offset any lipolytic effects of GH.

We have discussed a number of ways by which GH promotes lipolysis, the main effect being to stimulate Hormone Sensitive Lipase in adipocytes. But lipolysis, the term used to describe the mobilization of fatty acids so that they can potentially be used as fuel, is not the same thing as the actual oxidation of those fatty acids for energy in muscle tissue. Perhaps the failure of some trials to show fat loss during GH treatment is a result of a failure to oxidize the lipids that GH makes available as a potential fuel source. This seems not to be the case however, as research has shown that GH actually increases lipid oxidation at the expense of glucose oxidation by activating the so called glucose-fatty acid cycle where the preferential use of fat as a fuel substrate inhibits the use of glucose as fuel (21) (This process actually provides a mechanistic explanation of how GH administration induces insulin resistance: when more fatty acids are used as fuel, cells take up less glucose for use as a fuel substrate, leading to glucose intolerance). In addition to promoting the preferential use of fat as a fuel substrate by increasing its availability through enhanced lipolysis, GH also appears to directly stimulate the oxidation of lipids, perhaps by upregulating key mitochondrial enzymes involved in lipid oxidation (22).

Moreover, another well-known effect of growth hormone is to slow skeletal muscle breakdown during fasting (23). Teleologically speaking, the body secretes GH during periods of caloric restriction in an attempt to preserve skeletal muscle at the expense of increased fat oxidation for fuel. So during periods of caloric restriction, GH is responsible for less reliance on glucose and protein for energy, with fat being preferentially oxidized.



References

(1) J Endocrinol Invest. 1999;22(5 Suppl):10-5 Effect of growth hormone on adipose tissue and skeletal muscle lipoprotein lipase activity in humans. Richelsen B

(2) J Endocrinol Invest. 1999;22(5 Suppl):2-9 Effects of growth hormone on lipoprotein lipase and hepatic lipase. Oscarsson J, Ottosson M, Eden S.

(3) J Endocrinol. 1993 May;137(2):203-11. Influence of growth hormone deficiency on growth and body composition in rats: site-specific effects upon adipose tissue development. Flint DJ, Gardner MJ.

(4) Hum Reprod. 1997 Oct;12 Suppl 1:21-5. Hormonal control of regional fat distribution.Bjorntorp P.

(5) Horm Res. 2000;53 Suppl 1:87-97. Growth hormone and adipocyte function in obesity Nam SY, Marcus C.

(6) Pediatr Res 1996 Sep;40(3):450-6 Mitogenic and antiadipogenic properties of human growth hormone in differentiating human adipocyte precursor cells in primary culture. Wabitsch M, Braun S, Hauner H, Heinze E, Ilondo MM, Shymko R, De Meyts P, Teller WM

(7) Endocrinology. 2003 Mar;144(3):967-74. Interrelationship between the novel peptide ghrelin and somatostatin/growth hormone-releasing hormone in regulation of pulsatile growth hormone secretion. Tannenbaum GS, Epelbaum J, Bowers CY.

(8) Am J Physiol Endocrinol Metab. 2003 Jul;285(1):E163-E170. Epub 2003 Apr 01. Pulsatile and nocturnal growth hormone secretions in men do not require periodic declines of somatostatin. Dimaraki EV, Jaffe CA, Bowers CY, Marbach P, Barkan AL.

(9) Pombo M, Pombo CM, Astorga R, Cordido F, Popovic V, Garcia-Mayor RV, Dieguez C, Casanueva FF. Regulation of growth hormone secretion by signals produced by the adipose tissue. J Endocrinol Invest 22(5 Suppl):22-6 1999

(10) Endocrinology. 1998 Dec;139(12):4811-9. Hypothalamic mediated action of free fatty acid on growth hormone secretion in sheep. Briard N, Rico-Gomez M, Guillaume V, Sauze N, Vuaroqueaux V, Dadoun F, Le Bouc Y, Oliver C, Dutour A.

(11) Med Sci Sports Exerc. 1995 Jul;27(7):1057-62. Physiological and performance responses to nicotinic-acid ingestion during exercise. Murray R, Bartoli WP, Eddy DE, Horn MK

(12) J Clin Endocrinol Metab. 1988 Mar;66(3):489-94. Enhanced growth hormone (GH) responsiveness to GH-releasing hormone after dietary manipulation in obese and nonobese subjects. Kelijman M, Frohman LA.

(13) Am J Physiol. 1999 Nov;277(5 Pt 1):E824-9. Influence of obesity and body fat distribution on growth hormone kinetics in humans. Langendonk JG, Meinders AE, Burggraaf J, Frolich M, Roelen CA, Schoemaker RC, Cohen AF, Pijl H.

(14) J Clin Endocrinol Metab. 1997 Jul;82(7):2239-43. Evidence for an inhibitory effect of physiological levels of insulin on the growth hormone (GH) response to GH-releasing hormone in healthy subjects. Lanzi R, Manzoni MF, Andreotti AC, Malighetti ME, Bianchi E, Sereni LP, Caumo A, Luzi L, Pontiroli AE.

(15) Metabolism 1999 Sep;48(9):1152-6 Elevated insulin levels contribute to the reduced growth hormone (GH) response to GH-releasing hormone in obese subjects. Lanzi R, Luzi L, Caumo A, Andreotti AC, Manzoni MF, Malighetti ME, Sereni LP, Pontiroli AE.

(16) Am J Physiol Endocrinol Metab. 2003 Feb;284(2):E313-6. The influence of insulin on circulating ghrelin. Flanagan DE, Evans ML, Monsod TP, Rife F, Heptulla RA, Tamborlane WV, Sherwin RS.

(17) Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11381-6. Insulin and insulin-like growth factor-I acutely inhibit surface translocation of growth hormone receptors in osteoblasts: a novel mechanism of growth hormone receptor regulation. Leung KC, Waters MJ, Markus I, Baumbach WR, Ho KK.

(18) Obes Res. 2003 Feb;11(2):170-5. Effects of growth hormone administration in human obesity. Shadid S, Jensen MD.

(19) J Clin Endocrinol Metab. 2002 Jul;87(7):3105-9. Short-term treatment with low doses of recombinant human GH stimulates lipolysis in visceral obese men. Lucidi P, Parlanti N, Piccioni F, Santeusanio F, De Feo P

(20) Int J Obes Relat Metab Disord. 2001 Aug;25(8):1101-7. Low-dose growth hormone treatment combined with diet restriction decreases insulin resistance by reducing visceral fat and increasing muscle mass in obese type 2 diabetic patients. Nam SY, Kim KR, Cha BS, Song YD, Lim SK, Lee HC, Huh KB.

(21) J Clin Endocrinol Metab. 2003 Apr;88(4):1455-63. Growth hormone replacement therapy induces insulin resistance by activating the glucose-fatty acid cycle. Bramnert M, Segerlantz M, Laurila E, Daugaard JR, Manhem P, Groop L.

(22) J Clin Endocrinol Metab. 1997 Dec;82(12):4208-13. Stimulation of mitochondrial fatty acid oxidation by growth hormone in human fibroblasts. Leung KC, Ho KK.

(23) Diabetes. 2001 Jan;50(1):96-104. The protein-retaining effects of growth hormone during fasting involve inhibition of muscle-protein breakdown. Norrelund H, Nair KS, Jorgensen JO, Christiansen JS, Moller N


Merc.

[Merc.]

Bromocriptine for Weight Loss
by Karl Hoffman



Introduction


Editor's note: Given how this piece more or less assails Lyle McDonald's e-book dealing with Bromocriptine, in will by this token be controversial. Lyle will be given the opportunity to retort either in the form of an article, or on our debate board, perhaps both, whatever medium of response as how may he choose.

Bromocriptine, a drug that mimics the action of the naturally occurring neurotransmitter dopamine, has a long history of use by body builders and life extension enthusiasts. The drug originally gained popularity due to its reputation for acting as a mild growth hormone secretagogue. This is paradoxical, since in people suffering from acr*****ly, or growth hormone excess, bromocriptine has the opposite effect: it actually lowers GH levels. Bromocriptine has a number of other legitimate medical uses, including treatment of Parkinson's Disease and the lowering of prolactin levels in people suffering from prolactin secreting tumors. It has also been used successfully to treat hyperprolactinemia (elevated prolactin) that often occurs as a side effect of the administration of antipsychotic medications.



Bromocriptine and Weight Loss


Lately however, bromocriptine has gained prominence particularly on the World Wide Web as a weight loss agent. Perhaps no single person is more responsible for this resurgence of interest in the drug than Lyle McDonald, who popularized its anti-obesity properties in his recently published e-book (1). There are studies both in animals (2) and humans (3),(4) that support the ability of bromocriptine to reduce weight and body fat. The exact mechanism whereby dopaminergic agonists induce weight loss has not been elucidated.

Studies in animals have shown mixed effects of dopaminergic agonists on lipid metabolism. When dopamine and SKF 38393, a D2 receptor agonist were administered to genetically obese mice, antilipogenic effects were observed in the liver, but a combination of both lipolytic and antilipolytic effects were demonstrated in adipose tissue. In adipose tissue lipoprotein lipase activity was decreased (an antilipogenic effect) where as beta-agonist stimulated lipolysis was decreased (5). Additionally, obese mice treated with dopaminergic agonists also exhibited reduced de novo lipogenesis (2). This is the process whereby dietary carbohydrates are converted to fat. Interestingly, while de novo lipogenesis is important in animals, its contribution to fat deposition in humans is relatively unimportant (6). When humans ingest excess carbohydrates, rather than being stored as fat, the carbohydrates are preferentially used as fuel, preserving fat stores that would have otherwise been oxidized. Here is a case where the results of animal studies do not necessarily carry the same implications for humans.

Dopamine has also been implicated in appetite control. It has been postulated that dopamine modulates appetite by providing a reward stimulus, and that obese individuals have lower levels of dopamine receptors in certain portions of the brain (7). Hence to achieve the same "reward" from eating as normal individuals, obese individuals must eat more. This would provide some rationale for the treatment of obese individuals with dopaminergic agonists, but it is unclear whether normal body weight individuals possessing a normal density of dopamine receptors and/or normal dopaminergic activity in the brain would benefit from such treatment. The authors of the previously cited paper also acknowledged that it was unclear whether the relative paucity of dopamine receptors in the obese subjects was a cause or a result of their overeating. Since eating elevates dopamine levels, the brain could be compensating for elevated dopamine in chronic overeaters by downregulating the dopamine receptors. This latter possibility could call into question the use of dopaminergic agonists for appetite control. The result of increasing dopamine levels with agonists could lead to a further downregulation of the D2 receptors, leading to an increased desire to eat in order to further elevate dopamine levels. In a review of the above-cited study, Dr. Joseph Frascella of NIDA’s [National Institute of Drug Abuse] Division of Treatment Research commented on this positve feedback effect on D2 receptor downregulation:

“This deficiency could be a double-edged sword that cuts both ways. First, the reduced reward experienced by people with this deficiency may make them more likely to engage in addictive behaviors. Then, the addictive behavior itself could make the deficit worse as the brain further lowers D2 levels in response to constant overstimulation of the reward pathway. “In the end, they could be much worse off biologically than when they started,”




Another mechanism by which dopamine could suppress appetite is by antagonizing neuropeptide Y (NPY). As fat stores decrease during dieting, leptin levels fall. This signals an increase in NPY, which is a potent hunger inducing neuropeptide. Treatment of genetically obese mice with bromocriptine led to a decrease in the elevated levels of hypothalamic NPY in these animals (2). Again, the implications of these observations to normal humans are unclear.

Both obesity and cocaine addiction have been linked to the dopaminergic reward pathway. As we have been discussing, food consumption elevates dopamine level, leading to a reward stimulus. In the case of cocaine, the traditional view has been that cocaine blocks the cellular dopamine transporter, blocking dopamine reuptake and increasing extracellular dopamine levels, again leading to reinforcing reward. However, this view has been called into question by the 1998 publication of a study showing that mice lacking the dopamine transporter develop cocaine addiction (8). According to the dopamine reward model of cocaine addiction, the lack of the dopamine transporter should have maintained chronically elevated dopamine levels, obviating any reward derived from cocaine use. Nevertheless the mice became addicted. The authors suggested that other neurotransmitter pathways, such as those mediated by serotonin may play a more important role in addiction. These ideas are supported by the fact that dopaminergic agonists, including bromocriptine, have not been useful in treating cocaine addiction (9).

Is it possible that the food driven reward mechanism is also dopamine independent, or at least only partially dependent on dopamine? The successful use of fenfluramine as an anorectic agent suggests this could be the case. Fenfluramine stimulates the release of serotonin and is a potent reuptake inhibitor of serotonin into nerve endings. This increases levels of serotonin in the nerve synapse, increasing levels of serotonergic nerve transmission. In both animals and humans, fenfluramine induces lack of appetite leading to weight loss. Fenfluramine was withdrawn from the market in 1997 due to findings that its use was associated with valvular heart disease.



Side Effects of Bromocriptine Treatment


As with the majority of drugs bromocriptine has a number of well characterized side effects that seem more unpleasant than dangerous, and often abate during treatment. These include nausea, orthostatic hypotension, headaches, abdominal discomfort, nasal congestion, fatigue and constipation. Besides these there are two other potential side effects that are not as well characterized, that are controversial, and that are of particular interest to bodybuilders and other athletes. The first I would like to address is the possibility that bromocriptine may lower testosterone levels in normal men, as well as increase the ovarian aromatization of testosterone to estrogen in women. The second is the potential bromocriptine may have to suppress the immune system in normal humans.



Bromocriptine and Steroidgenesis


It has been appreciated for decades that elevated levels of prolactin in males (hyperprolactinemia) can suppress testosterone production. Hyperprolactinemia disrupts the hypothalamic-pituitary-gonadal axis in women as well, leading to amenorrhea and infertility. Since bromocriptine lowers prolactin levels, when bromocriptine is administered to these patients, normal sexual function is usually restored. What is less well known is that studies done both in vitro and in humans suggest that hypoprolactinemia (low prolactin levels) also leads to suppressed testosterone production. So prolactin appears to exert a biphasic effect: too much or too little can disrupt testicular function. Normal physiological levels of prolactin appear to be necessary for normal gonadal function. (10) (11). To quote from Marin-Lopez et al, (10), where sulpiride and bromocriptine were used respectively to induce hyper and hypoprolactinemia in normal males


"the hyperprolactinemia induced a low basal level of testosterone with a higher response of this steroid to hCG...while the loss of the trophic effect of prolactin on gonadal steroidogenesis, as seen in hypoprolactinemia produces a decrease of basal testosterone levels without any alteration of the response of this steroid to hCG. We conclude that prolactin plays an important role in the steroidogenesis of Leydig cells in normal men.'' (11)
Confusing the issue is the fact that several other studies both in vitro and in vivo have shown either no effect or an increase in testosterone production due to both prolactin and bromocriptine administration (12) (13).

A number of experimental observations have led to several theories that could possibly explain how bromocriptine induced hypoprolactinemia suppresses testosterone production. Kovacevic and Sarac (14) proposed that bromocriptine competitively inhibits androgen production at the level of the testicular enzymes 17 alpha-hydroxylase and/or 17,20-lyase. These enzymes act at intermediate steps in the testicular production of testosterone. Aisaka et al. observed a decrease in luteinizing hormone (LH) levels that was mirrored by a decrease in testosterone after bromocriptine administration, suggesting that bromocriptine directly inhibits LH secretion from the pituitary (15). As we know, luteinizing hormone, or LH, secreted from the pituitary gland acts directly on testicular Leydig cells to stimulate testosterone secretion.

On the other hand Suescun et al. observed a decrease in circulating testosterone after bromocriptine administration in men with no decrease in LH levels (16), consistent with a direct testicular action of bromocriptine, as proposed by Kovacevic.

Other studies have shown that lowering prolactin decreases the binding of LH to the Leydig cell LH receptor, with a concomittent reduction in androgen production (17). These researchers concluded that


These results suggest that under normal conditions, endogenous prolactin plays a key role in maintaining the functional integrity of rat Leydig cells." (16)
So perhaps by either lowering the affinity of the LH receptor to LH, or by directly decreasing LH receptor number, bromocriptine could lower testosterone production.

As is obvious from the conflicting studies, and the variety of proposed mechanisms for bromocriptine induced testosterone suppression, there is much to be learned about the role of prolactin in maintaining normal testicular steroidogenesis.

All of the studies thus far cited have been carried out in men. What about the effects of bromocriptine in normal women? As mentioned earlier in the article, hyperprolactinemia inhibits ovulation in both animals and humans. One interesting study showed that prolactin administered to rats decreased levels of ovarian aromatase. Conversely, when bromocriptine was administered, ovarian aromatase was increased (18). Is this of any relevance to human females? Perhaps, since the same phenomenon is observed during the follicular phase of the menstrual cycle: bromocriptine increases the estradiol/testosterone ratio as a result of increased aromatization of testosterone to estrogen (19).



Bromocriptine and the Immune System


A number of studies have shown that prolactin stimulates certain aspects of immunity, and that by lowering prolactin with bromocriptine many of the symptoms of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus can be ameliorated. A recent review by McMurray (20) provides an excellent overview of this topic. While this is certainly hopeful news for people suffering from these diseases, what are the implications for the immune lowering effects of bromocriptine in healthy people?

One possibility is that by suppressing the prolactin mediated immune response, an individual could become more susceptible to tumor formation. (21). As Matera et al pointed out (22), prolactin specifically heightens the response of the cellular arm of the immune system. Recalling some basic immunology, the immune system has two components, the cellular arm and the humoral arm. The former is responsible for the direct attack on pathogens, while the latter involves the production of antibodies that mark pathogens for destruction, as well as creating a class of "memory" B cells that once primed by exposure to a pathogen, will respond vigorously the next time the body encounters the same invader. The cellular response is orchestrated by so called Th1 helper cells. The humoral response on the other hand is governed by another T cell subclass, the Th2 helper cells. These two T cell subclasses keep each other in check by controlling the production of cytokines that are mutually suppressive. So any immune stimulus that activates Th1 cells will suppress the Th2 response, and vice versa. So by suppressing prolactin (which heightens the cellular immune respons), cellular immunity is weakened.

One point of possible concern is that many persons who are using or contemplating the use of bromocriptine also use anabolic-androgenic steroids (AAS). Like bromocriptine, AAS suppress the cellular arm of the immune system. They do so by stimulating Th2 cells, which as we discussed above, suppress the Th1 driven cellular response. So users of both AAS and bromocriptine could receive a double dose of immune suppression, leaving the body open to attack by pathogens, or conceivably more susceptible to the development of tumors. This latter possibility is potentially heightend by the fact that many AAS elevate IGF-1 levels, which have been implicated in tumorogenesis.

It has also been demonstrated that prolactin opposes the immunosuppressive effects of glucocorticoids (21). Bromocriptine, by virtue of prolactin suppression, therefore may leave an overtrained or stressed individual more prone to the deleterious effects of elevated cortisol levels, including increased muscle catabolism, and an impaired immune response to exercise induced muscle damage, which is essential to growth.(Post exercise muscle damage is a powerful catalyst for growth. The muscles repair and rebuild so that they are bigger and stronger that before training. Suppressing this post exercise inflammation, whether with cortisol or NSAIDS, hinders growth.)



Bromocriptine and Cognitive Impairment


Dopaminergic agonists including bromocriptine exhibit mixed effects on cognitive ability when used to treat Parkinson’s patients, as well when administered to normal subjects. Swainson et al (23) showed that when administered to Parkinson’s patients, bromocriptine improved working spatial memory, while at the same time impairing so called “reversal learning” skills. This effect was subsequently reproduced in normal volunteers (24). In a reversal task, subjects have to learn that a previously rewarded stimulus now lacks reward, while the formerly neutral or negative stimulus now receives reward. This involves two stages: ceasing to respond to the former stimulus, while simultaneously learning to respond to a new stimulus.

A real world example of working spatial memory is learning to adapt and respond to changing situations, as one would face during a chess game. An example of a reversal learning skill is the ability of a child who is repeatedly reprimanded for chatting to classmates to realize that it is expected of her to participate in classroom discussions.

In the experiment carried out by Swainson et al (24) mentioned above, normal volunteers were administered 1.25 mg of bromocriptine. One aspect of the study that is interesting is that only the subjects who showed relatively poor working spatial memory before bromocriptine administration showed an improvement after being given the drug, whereas no such correlation existed for reversal learning. Bromocriptine impaired reversal learning independently of a subject’s pre-administration reversal learning skills. The bromocriptine treated subjects performed better on the second round of reversal learning testing, which suggested to the researchers that bromocriptine impairs this aspect of cognition in novel situations more strongly than in familiar ones.

Although there is no concrete explanation for the bromocriptine induced cognitive impairment, the authors theorize it is due to dopamine overload of the neural circuitry involved in reverse learning. Jentsch et al (25) showed that monkeys exhibited a similar impairment in reversal learning after cocaine administration. In this experiment food was placed under one of three novel objects and the monkeys learned to lift that object to obtain the food. When the food was subsequently placed under a different one of the three objects, placebo treated monkeys quickly learned to lift the new object to get the food, while the cocaine treated monkeys continued to lift the original object in search of food.

The parallels between this experiment and the human reversal learning experiment are not surprising since cocaine elevates dopamine levels in certain portions of the brain, just as bromocriptine acts as a dopaminergic agonist in the brain. Dopaminegic overload is likely the cause of the impaired reverse learning in both experiments.




Summary


Just as users of AAS owe it to themselves to become familiar with the impact these drugs can have on a person's health, so should users, or potential users, of bromocriptine be aware of the possible health implications of its use. I've tried to point out some of the potential benefits of bromocriptine use (e.g. weight loss, treatment of autoimmune diseases) as well as some possible drawbacks (impaired gonadal function; immunosuppression). Like just about every other agent used to improve performance or lose weight, bromocriptine has its potential dark side as well.

Feel free to debate and critique this piece. You may also of course contact the author. Critique and discuss here.


References:

(1) Bromocriptine; McDonald, Lyle

(2) Int J Obes Relat Metab Disord 1999 Apr;23(4):425-31
Biochemical mechanisms responsible for the attenuation of diabetic and obese conditions in ob/ob mice treated with dopaminergic agonists.
Scislowski PW, Tozzo E, Zhang Y, Phaneuf S, Prevelige R, Cincotta AH.

(3) Diabetes Care 1996 Jun;19(6):667-70
Bromocriptine (Ergoset) reduces body weight and improves glucose tolerance in obese subjects.
Cincotta AH, Meier AH.
.
(4) Eur J Endocrinol 2002 Jul;147(1):77-84
Dopaminergic tone and obesity: an insight from prolactinomas treated with bromocriptine.
Doknic M, Pekic S, Zarkovic M, Medic-Stojanoska M, Dieguez C, Casanueva F, Popovic V.

(5) Metabolism 1999 Aug;48(8):1033-40
Bromocriptine/SKF38393 treatment ameliorates dyslipidemia in ob/ob mice.
Zhang Y, Scislowski PW, Prevelige R, Phaneuf S, Cincotta AH.

(6) Am J Clin Nutr 2001 Dec;74(6):707-8
Am J Clin Nutr. 2001 Dec;74(6):737-46.
No common energy currency: de novo lipogenesis as the road less traveled.
Hellerstein MK.

(7) Lancet 2001 Feb 3;357(9253):354-7
Brain dopamine and obesity.
Wang GJ, Volkow ND, Logan J, Pappas NR, Wong CT, Zhu W, Netusil N, Fowler JS.

(8) Nat Neurosci 1998 Jun;1(2):132-7
Cocaine self-administration in dopamine-transporter knockout mice.
Rocha BA, Fumagalli F, Gainetdinov RR, Jones SR, Ator R, Giros B, Miller GW, Caron MG.

(9) Expert Opin Investig Drugs 2002 Apr;11(4):491-9
The potential of dopamine agonists in drug addiction.
Kosten TR, George TP, Kosten TA

(10) Endocrinology 2001 Jan;142(1):308-18
Biphasic action of prolactin in the regulation of murine Leydig tumor cell functions.
Manna PR, El-Hefnawy T, Kero J, Huhtaniemi IT.

(11) Invest Clin 1996 Sep;37(3):153-66
Leydig cell function in hyper- or hypoprolactinemic states in healthy men
Marin-Lopez G, Vilchez-Martinez J, Hernandez-Yanez L, Torres-Morales A, Bishop W.

(12) Biol Reprod 1978 Feb;18(1):44-54
Hormonal interactions in regulation of androgen secretion.
Bartke A, Hafiez AA, Bex FJ, Dalterio S.

(13) Clin Endocrinol (Oxf) 1982 Oct;17(4):345-52
Relationship of changes in serum concentrations of prolactin and testosterone during dopaminergic modulation in males.
Nakagawa K, Obara T, Matsubara M, Kubo M.

(14) J Steroid Biochem Mol Biol 1993 Dec;46(6):841-5
Bromocriptine-induced inhibition of hydroxylase/lyase activity of adult rat Leydig cells.
Kovacevic R, Sarac M.

(15) Nippon Naibunpi Gakkai Zasshi 1985 Jun 20;61(6):701-9
The effects of bromocriptine on the pulsatile pattern and the circadian profile of gonadotropins and testosterone secretion in normal adult men
Aisaka K, Ogawa T, Mori H, Kigawa T.

(16) J Androl 1985 Jan-Feb;6(1):10-4
Induced hypoprolactinemia and testicular steroidogenesis in man.
Suescun MO, Scorticati C, Chiauzzi VA, Chemes HE, Rivarola MA, Calandra RS

(17) Arch Androl 1979 Nov;3(3):219-30
Prolactin and Leydig cell responsiveness to LH/hCG in the rat.
Purvis K, Clausen OP, Olsen A, Haug E, Hansson V.

(18) Biol Reprod 1983 Sep;29(2):342-6
Inhibition of ovarian aromatase by prolactin in vivo.
Tsai-Morris CH, Ghosh M, Hirshfield AN, Wise PM, Brodie AM.

(19) Fertil Steril 1989 Jul;52(1):51-4
Prolactin suppression by bromocriptine stimulates aromatization of testosterone to estradiol in women.
Martikainen H, Ronnberg L, Puistola U, Tapanainen J, Orava M, Kauppila A.

(20) Semin Arthritis Rheum 2001 Aug;31(1):21-32
Bromocriptine in rheumatic and autoimmune diseases.
McMurray RW.

(21)J Neuroimmunol 2000 Sep 1;109(1):47-55
Prolactin in autoimmunity and antitumor defence.
Matera L, Mori M, Geuna M, Buttiglieri S, Palestro G.

(22) Brain Behav Immun 1992 Dec;6(4):394-408
Prolactin and prolactin secretagogues reverse immunosuppression in mice treated with cysteamine, glucocorticoids, or cyclosporin-A.
Bernton E, Bryant H, Holaday J, Dave J.

(23) Neuropsychologia 2000;38(5):596-612
Probabilistic learning and reversal deficits in patients with Parkinson's disease or frontal or temporal lobe lesions: possible adverse effects of dopaminergic medication.
Swainson R, Rogers RD, Sahakian BJ, Summers BA, Polkey CE, Robbins TW.

(24) Psychopharmacology (Berl) 2001 Dec;159(1):10-20
Improved short-term spatial memory but impaired reversal learning following the dopamine D(2) agonist bromocriptine in human volunteers.
Mehta MA, Swainson R, Ogilvie AD, Sahakian J, Robbins TW.

(25) Neuropsychopharmacology 2002 Feb;26(2):183-90
Impairments of reversal learning and response perseveration after repeated, intermittent cocaine administrations to monkeys.
Jentsch JD, Olausson P, De La Garza R 2nd, Taylor JR.


Merc.

[Merc.]

Maybe we need to rename this thread the Karl Hoffman Nandi tribute thread .....




Merc.

[InsaneInTheMembrane]

...man, the more I learn, the less I know

thanx merc

[ranging1]

GREAT POST, think alot fo people will find this interesting

well done mate

[marcus300]

Very wise man, Ive learnt alot from his threads.

[Swifto]

Karl (Nandi) didnt just write articles from abstracts, like a certain someone now excluded from this board (our old friend Marcus). He wrote extensive articles with water tight references and on full papers/texts!!!!!

[Merc.]

The Effects of Anabolic Steroids on Thyroid Function
by Karl Hoffman


One of the more commonly encountered assertions in the bodybuilding literature is that anabolic steroids (AAS) suppress thyroid function. What is the origin of this claim? Is it supported by evidence published in the scientific literature? And perhaps most important is it of any clinical significance, meaning if it does occur is it serious enough to worry about? Before reviewing the evidence for and against AAS induced thyroid impairment a short review of thyroid physiology is probably in order.

The thyroid gland secretes principally the hormone thyroxine (T4) along with small amounts of triiodothyronine (T3). The majority of the T3 in the circulation (about 80%) is formed from the conversion of T4 to T3 by the enzyme deiodinase. Most of this transformation takes place in the liver and kidney. T3 is considered the physiologically active hormone; in this sense T4 can be thought of as a prohormone.

The production within and secretion from the thyroid gland of T4 is under the control of Thyroid Stimulating Hormone (TSH), which is secreted from the pituitary gland. Higher levels of TSH lead to higher rates of hormone production and secretion from the thyroid. TSH in turn is regulated by another hormone secreted from the hypothalamus, thyrotropin-releasing hormone (TRH). TSH levels are also regulated in a negative feedback manner by the levels of circulating thyroid hormone. If extra hormone is administered, transcription of the TSH gene is decreased and less TSH is produced by the pituitary, leading to suppression of natural thyroid hormone production. Similarly, a decrease in the rate of thyroid hormone secretion leads to enhanced TSH production in an attempt to return to homeostasis.

Just as the bulk of circulating androgens and estrogens are bound to sex hormone binding globulin (SHBG), most of the thyroid hormone in circulation is bound to thyroid binding globulin (TBG). And as with SHBG and sex hormones, the levels of TBG influence the levels of total thyroid hormone in circulation. If TBG is depressed, total T4 and T3 levels will go down. An increase in TBG leads to higher values of total thyroid hormone. Note however that the small percentage of T3 and T4 that remain unbound to TBG (0.05% of T4 and 0.5% of T3), the so-called free fraction, is the portion considered physiologically active. So it is quite possible to have lowered total T3 if TBG is low, but still have normal levels of free T3. This condition is not indicative of thyroidal impairment since the bioactive free T3 is normal. Similarly when TBG is elevated total T4 and T3 are high, again with the possibility that physiologically active free hormone levels remain normal.

A number of drugs and medical conditions are capable of elevating TBG and hence total T4 and T3 levels. These include estrogen, oral contraceptives (OC), pregnancy, acute infectious hepatitis, and cirrhosis. Likewise there are drugs and medical conditions that lower TBG. These include cortisol, growth hormone, and very important to this discussion, anabolic steroids. So to recap, if a person were using AAS and had their total T4 and T3 measured, because TBG is low, those total values would register as low, but that would not necessarily mean that the bioactive (free) levels of T3 and T4 are low. This observation will be critical to our discussion of the effects of AAS on the thyroid gland. In a similar vein, a woman using OC might have elevated T4 and T3 because oral contraceptives raise TBG. This would not necessarily warrant a diagnosis of hyperthyroidism, as her free thyroid levels could be perfectly normal.

So we see that in order to assess thyroid function, measuring only the total T4 and/or T3 is inadequate because these values are strongly influenced by TBG levels. Other laboratory tests are required to determine whether low T4, say, is being caused by actual hypothyroidism, or reflects the use of a drug that is simply lowering TBG. One such test is the thyroid hormone binding ratio (THBR; T3 resin uptake). This test is essentially a measure of the number of TBG sites that are occupied by thyroid hormone. In a person who is hyperthyroid (high T4) there are fewer unbound TBG sites/more occupied sites (since obviously there is more thyroid hormone available to bind to them). In this case T3 resin uptake is high. Conversely, in hypothyroidism there are fewer occupied TBG binding sites, and T3 resin uptake is low. In the case where thyroid function is normal but TBG is elevated (oral contraceptives) it turns out T3 resin uptake registers LOW; conversely when TBG is lowered (AAS use), a lab report would show T3 resin uptake reading HIGH.

We can tabulate these various possible outcomes to give a clearer picture of how these two tests can be used to distinguish thyroid dysfunction from mere altered levels of TBG:


SERUM T4 SERUM T3 T3 RESIN UPTAKE
Hyperthyroidism High High High
Hypothyroidism Low Low Low
Normal, on OC High High Low
Normal, on AAS Low Low High


TABLE 1

Perhaps the test most commonly performed test to determine thyroid status measures Thyroid Stimulating Hormone (TSH) levels. Typically in hypothyroidism, the thyroid is not secreting adequate levels of T4, and in an attempt to stimulate the thyroid, the pituitary secretes excess TSH. So in hypothyroidism, TSH is HIGH. The opposite is observed in hyperthyroidism: the excess thyroid hormone in circulation acts back on the pituitary to suppress TSH production. TSH is LOW in hyperthyroidism.

The above applies to so-called primary hypothyroidism/hyperthyroidism where the thyroid gland itself is malfunctioning. In secondary hyper/hypothyroidism the problem lies at the levels of the pituitary or hypothalamus. In this case the pituitary secretes insufficient TSH to stimulate the thyroid, resulting in hypothyroidism with low TSH. In secondary hyperthyroidism the pituitary secretes excess TSH, resulting in hyperthyroidism associated with elevated TSH.

Before the advent of highly sensitive TSH assays, it was common to perform a TRH challenge test. Recall the hypothalamus secretes TRH, in turn stimulating the pituitary to secrete TSH. In the TRH test, a bolus injection of synthetic TRH is administered. The body’s normal response is to secrete increased levels of TSH up to a peak at 20 minutes and then to decrease TSH secretion. In hyperthyroidism, TSH is being suppressed by circulating thyroid hormones so there is a suppressed response to TRH. In primary hypothyroidism, which is due to thyroid dysfunction with normal pituitary function, levels of thyroid hormones are very low and TSH levels are ordinarily raised; however, TSH increases greatly on TRH stimulation yielding an exaggerated response - it reaches a higher peak and does not decline for over an hour. In secondary hypothyroidism (where the pituitary is malfunctioning, not the thyroid) it doesn't matter how much TRH there is, the pituitary cannot make TSH so there is an absent response to TRH stimulation.

Now that we have reviewed the elements of thyroid physiology and gone over the basic tests to determine thyroid function, we are ready to review the literature regarding the effects of anabolic steroids on the thyroid. At the beginning of this review, we asked the question “what is the origin of the claim that AAS impair the thyroid?” The answer perhaps lies in a 1993 paper by Deyssig & Weissel (1). The authors looked at the effects of self-administered AAS use in an admittedly small group of five bodybuilders. Eight additional subjects served as controls. In the AAS using group, total T4, Total T3, and TBG were depressed relative to the control group. Recall this is consistent with the widespread observation that by lowering TBG, AAS lower total T4 and total T3 with no effect on the free hormone levels and hence no effect on TSH. Indeed in this study there was no difference in free T4 and TSH between the AAS group and the controls. Basal free T3 was not measured. So far everything is consistent with normal thyroid function accompanied by AAS induced depression of TBG.

The authors next performed a TRH test. Upon administration of THR, TSH values climbed significantly higher in the AAS group, and the T3 response was significantly lower in the AAS group. Recall that in hypothyroidism there is an exaggerated TSH response to TRH. Quoting from the study, “These T3 and TSH reactions to TRH point to a mild impairment of thyroid function as a consequence of the use of anabolic steroids.” However, stressing the fact that all unstimulated parameters were consistent with the simple suppression of TBG by AAS, the authors conclude that “the results of our cross-sectional study show that high doses of androgenic-anabolic steroids, as are used by some athletes, may impair thyroid function to an extent that is not clinically detectable and probably not relevant.” (Italics added.)

Moreover, when one scrutinizes the data, one sees that out of the five AAS-using subjects, only two had stimulated TSH values higher than the controls, one was only marginally but not significantly higher than in controls, and two had stimulated TSH values in the control range. In addition, no pre-study baseline stimulated TSH values were measured in any subjects. One could argue these facts call into question the authors’ conclusions of “mild impairment of thyroid function”.

How high were the “high doses” used by the participants? The subjects were using a number of different AAS including testosterone, nandrolone, stanozolol, and Dianabol, stacking them as bodybuilders typically do. The average total dose for all drugs combined was 1.26 grams/week, with a range of 740 – 1950 mg/week.

Alen et. al. (2) conducted a study along similar lines. Seven power athletes stacking testosterone, Dianabol, stanozolol, and nandrolone were monitored for (among other things) thyroid function during a 12 week period. In this study, total T3, total T4 and TBG were all depressed during the study period, while T3 resin uptake was elevated. All of these changes are again consistent with AAS induced suppression of TBG, with no direct effect on thyroid function. Interestingly, TSH dropped during the first 8 weeks of the study, and then began to climb. Free T4 dropped marginally but stayed within the normal range. The authors interpreted the data thusly:


It is tempting to suggest that decreases in serum TBG led to decreased protein binding of the thyroid hormones, T4 and T3, which is reflected in the elevated T3U-values [T3 resin uptake]. Increased availability of T4 and T3 would then lead to a compensatory decrease in serum TSH, and this, via decreased thyroid stimulation, would further decrease total concentrations of circulating T4 and T3. The measurements of thyroid function parameters performed support this reasoning. In general our findings suggest that thyroid hormones at the cellular level were not disturbed in our athletes.

While the approximate 20% drop in free T4 observed by Alen et al is suggestive of some degree of thyroid impairment, the consequences of this need to be interpreted carefully. First, free T4 stayed well within the normal range. Second, since free T3 was not measured, we do not know if there was any change in free T3, the metabolically active hormone. Lum et al. have shown that when serum T4 levels drop, the body upregulates the peripheral conversion of T4 to T3, maintaining metabolic homeostasis (3). So it is possible that any drop in free T3 could have been significantly smaller than the observed 20% drop in free T4.

We see here contradictory findings between the two studies discussed so far as regards TSH levels: Deyssig & Weissel observed no change in unstimulated TSH levels, while Alen et al observed a decline in TSH, although the values remained well inside the normal range. The finding consistent between the two studies is the AAS suppressed TBG and the consequent decline in total T4 and T3, and an increase in T3 resin uptake observed by Alen but not measured by Deyssig. Again, quoting from (2): “In relation to the changes in thyroid function parameters measured, we suggest that the primary target of androgen action was TBG biosynthesis.”

In a third study, this time performed by Malarkey et al in AAS-using females, the authors looked at Total T4, FreeT4, TSH, and TBG (4). The authors observed that


Thyroxine-binding proteins also were decreased in the steroid users, as reflected by the low thyroxine binding index and the decrease in total serum thyroxine levels. These latter changes had no significant influence on the biological activity of thyroid hormone, however, because the free thyroxine concentration and the thyroid stimulating hormone level were within normal limits. These findings are similar to those of a previous report of decreased thyroxine-binding globulin in men who were using anabolic steroids [2].

Note here though that while TSH was “within normal limits” it was nevertheless elevated significantly compared to controls (2.5 mU/L vs 0.8 mU/L).

The difference between this study and the previous one by Alen is that in (4) free T4 was unchanged, while in (2) there was a drop in free T4. Also here TSH was elevated in the AAS users while in Alen et al it was depressed. In the current study the combination of normal free T4 but elevated TSH is suggestive of subclinical hypothyroidism. However, to truly meet the criteria required for that diagnosis TSH would have to be elevated above 5.0 mU/L (although some physicians have argued that that threshold should be lowered). Technically, these subjects would be considered euthyroid (normal).

One criticism of the studies examined thus far is that in each case the subjects used a cocktail of anabolic steroids, including ones that aromatize and others that do not. Might there be a difference in the thyroidal effects of the two classes of drugs? A study by Lovejoy et al (5) addressed that question as part of research looking at the broader differences between the metabolic effects of oral (oxandrolone) and parenteral (testosterone) steroids. Lovejoy’s group administered each drug separately to groups of subjects. Testosterone aromatizes to estrogen, and estrogen has an opposite effect on TBG from pure androgens: the former elevates TBG while the latter lowers it. If the primary effects of AAS on measured thyroid parameters result from changes in TBG, then testosterone would be expected to have only a minor effect, the increased androgen and estrogen levels tending to cancel each other’s effects. Indeed this was the case in (5). Testosterone had no significant effect on any parameter measured (T4; TSH; T3 resin uptake; or free thyroxine index, a calculated measure of free T4).

Oxandrolone on the other hand does not aromatize. The oxandrolone group showed a significant decrease in T4 and T3 resin uptake, with no change in TSH. Referring to Table 1 above, we see that the combination of low T4 and low T3 resin uptake is characteristic of hypothyroidism. This is the conclusion the authors arrived at as well, that the oxandrolone group experienced mild hypothyroidism. Again, as in the study by Deyssig & Weissel, even though T4 and T3 resin uptake were low relative to placebo the values fell within the normal range, making the diagnosis of “hypothyroidism” a relative one rather than a clinical one. Clinically all subjects would be classified as euthyroid. And as in the other studies the authors here concluded “these changes were most likely due to the effects of sex steroids on thyroid binding globulin (TBG).” The authors also observed that the Free T4 Index was higher in the oxandrolone group than either the placebo or testosterone groups.

A recent study looked at the effects of short-term methyltestosterone administration to normal subjects (6). The researchers found that total T4, total T3, and TBG were lowered, as we have come to expect. However, TSH and free T4 were elevated as well compared to the subjects’ baseline values. Again all hormone values remained within the normal range. The authors speculate that the elevated TSH and free T4 could be due to increased sensitivity of the thyroid to TRH or decreased sensitivity to hormonal feedback, suggesting some form of mild impaired thyroid function. As with the study by Deyssig, if this functional impairment were real, it would be subclinical and of dubious relevance. Note also that the elevated TSH measured by Daly et al differs from the depressed TSH observed by Alen et al. Daly et al speculate that this may be due to the fact that they sampled blood after six days vs 4 weeks and longer in the study by Alen et al.

A 1984 study by Small et al examined the effects of 10 mg daily of stanozolol, another nonaromatizing steroid, for 14 days in nine healthy subjects (7). This dosage was enough to lower testosterone by 50% and LH by 30%. TBG, T4 and T3 were lowered significantly, with no change in free T4 or TSH. This is the “standard model” of action of androgens on thyroid parameters stressed in endocrinology texts: no change in thyroid function, merely a lowering of TBG with the expected lowering of total thyroid hormone levels, but no effect on the physiologically relevant free hormone levels. To quote from the study,

“The changes found in thyroid hormones are in accord with the well known effects of anabolic steroids on thyroid function tests. Both T3 and T4 fell as a result of the reductions in TBG levels. The lack of change in TSH or Free T4 indicates that important physiological changes of thyroid function do not occur during treatment with stanozolol.”


We can summarize the results of the studies for comparative purposes by tabulating the data in Fig 2.


T4 T3 TSH T3 resin uptake Free T4 TBG
Deyssig low low 0 - 0 low
Alen low low low high low low
Lovejoy (test) 0 - 0 0 0 -
Lovejoy (ox) low - 0 low high -
Malarkey low - high - 0 low
Daly low low high - high low
Small low low 0 - 0 low


Table 2. Summary of measured thyroid parameters ( 0, no change; - unmeasured; )

Fluoxymesterone at 10 mg per day caused the by now familiar drop in TBG and total thyroid hormone levels with no effect on free parameters (8). Quoting from this study,

“Fluoxymesterone administration was accompanied by a reduction in thyroid binding globulin (with associated decreases in T3 and increases in T3 resin uptake). The free T4 index was unaltered, which implies that thyroid function was unchanged.”

Thus far we have looked at studies involving humans. One study that is often cited in the bodybuilding literature as evidence that trenbolone in particular suppresses thyroid function was done in sheep (9). However, in this study only total T4 was measured, not free T4, so we cannot conclude from this research that bioavailable T3 was affected in any way.

Can we make any sense out of the seeming hodgepodge of conflicting data? The only parameters that are consistent from study to study, where they were measured, are depressed total T3 and T4, and TBG. As we have discussed, androgens typically lower TBG, along with total T4 and T3 since the latter are a function of TBG levels. This however does not necessarily reflect thyroid dysfunction since the physiologically significant free fractions of these hormones typically remain in the normal range. If TBG levels change rapidly, however, a period of disequilibrium will exist during which thyroid function will be perturbed. This could explain the low free T4 and TSH observed by Alen as follows: The abrupt drop in TBG leads to a drop in bound T4, but free T4 remains elevated. This causes a shift in hormone from the blood to tissues because of a steeper free T4 concentration gradient. This increases the degradation rate of hormone in peripheral tissues. The increased tissue concentration of T4 signals the pituitary to lower TSH production, which will be reflected by temporarily lowered free T4 until the appropriate thyroid hormone/TBG ratio, and plasma/tissue ratio is reestablished. Alen et al discuss this possibility, and the process is illustrated graphically here:


http://www.thyroidmanager.org/Chapter5/5a-frame.htm

In conclusion then AAS seem to have little if any effect on thyroid function per se. The reports by Deyssig & Weissel, and Daly et al suggest the possibility of a direct action of AAS on the thyroid or pituitary, but their results are inconsistent: The former researchers detected elevated stimulated TSH while the latter saw an increase in basal TSH. Free T4 was unchanged in former group, while it was elevated in the latter. The only consistently reported effect is a depression in total T4, total T3 and TBG. If there is a direct effect of AAS on the thyroid, pituitary, or hypothalamus the studies conducted so far shed little light on the mechanism due to their inconsistent results. And as stressed by Deyssig & Weissel any direct effect of anabolic steroids on the thyroid would likely be of no clinical significance due to its small magnitude.

From a practical standpoint for those concerned that anabolic steroids might suppress the thyroid it is a simple matter to incorporate low dose (25 to 50 mcg/day) T3 into a cycle to enhance fat loss while at the same time only minimally if it all compromising gains in muscle mass (10). In (10) one group of subjects was given T3 alone while the other was given a combination of T3 and testosterone enanthate, 200 mg/week. After 28 days of bed rest, the men in the T3 group lost an average of 3.9 kg of body weight (i.e. from 82.0 ± 7.1 to 78.1 ± 7.1 kg). Body weight in the T3 plus testosterone-treated subjects declined by only 1.0 kg (78.9 ± 4.9 to 77.9 ± 4.9 kg). Lean body mass declined by 1.5 kg in the T3 group, whereas the T3 plus testosterone-treated subjects experienced nearly a 2-kg increase in lean mass (i.e. 1.7 ± 0.9 kg). Of course we don’t know how much mass the test plus T3 group would have gained had they foregone the T3. Nevertheless these are still impressive gains considering the subjects were forced to lie in bed for 28 days with no exercise, and considering that no special dietary measures were imposed to preserve or increase muscle mass.



References:

(1) Deyssig R, Weissel M. Ingestion of androgenic-anabolic steroids induces mild thyroidal impairment in male body builders. J Clin Endocrinol Metab. 1993 Apr;76(4):1069-71

(2) Alen M, Rahkila P, Reinila M, Vihko R. Androgenic-anabolic steroid effects on serum thyroid, pituitary and steroid hormones in athletes. Am J Sports Med. 1987 Jul-Aug;15(4):357-61

(3) Lum SM, Nicoloff JT, Spencer CA, Kaptein EM Peripheral tissue mechanism for maintenance of serum triiodothyronine values in a thyroxine-deficient state in man. J Clin Invest. 1984 Feb;73(2):570-5.

(4) Malarkey WB, Strauss RH, Leizman DJ, Liggett M, Demers LM. Endocrine effects in female weight lifters who self-administer testosterone and anabolic steroids. Am J Obstet Gynecol. 1991 Nov;165(5 Pt 1):1385-90.

(5) Lovejoy JC, Bray GA, Greeson CS, Klemperer M, Morris J, Partington C, Tulley R. Oral anabolic steroid treatment, but not parenteral androgen treatment, decreases abdominal fat in obese, older men Int J Obes Relat Metab Disord. 1995 Sep;19(9):614-24.

(6) Daly RC, Su TP, Schmidt PJ, Pagliaro M, Pickar D, Rubinow DR. Neuroendocrine and behavioral effects of high-dose anabolic steroid administration in male normal volunteers. Psychoneuroendocrinology. 2003 Apr;28(3):317-31

(7) Small M, Beastall GH, Semple CG, Cowan RA, Forbes CD. Alteration of hormone levels in normal males given the anabolic steroid stanozolol. Clin Endocrinol (Oxf). 1984 Jul;21(1):49-55

(8) Spitz IM, Margalioth EJ, Yeger Y, Livshin Y, Zylber-Haran E, Shilo S. Effect of non aromatizable androgens on LHRH and TRH responses in primary testicular failure. Horm Metab Res. 1984 Sep;16(9):492-7.

(9) Donaldson IA, Hart IC, Heitzman RJ. Growth hormone, insulin, prolactin and total thyroxine in the plasma of sheep implanted with the anabolic steroid trenbolone acetate alone or with oestradiol. Res Vet Sci. 1981 Jan;30(1):7-13.

(10) Zachwieja JJ, Smith SR, Lovejoy JC, Rood JC, Windhauser MM, Bray GA. Testosterone administration preserves protein balance but not muscle strength during 28 days of bed rest. J Clin Endocrinol Metab. 1999 Jan;84(1):207-12.


Merc.

[Merc.]

T.T.T




Merc.

[Merc.]

Resistance Exercise and Androgen Levels
by Karl Hoffman




Ratamess Study


Ratamess and coworkers recently published the results of a study that looked at the hormonal profile and androgen receptor content in the vastus lateralis muscle (a portion of the muscles comprising the quadriceps) of men following two exercise protocols1.

The results were a bit surprising in light of some previous studies, and suggest a possible supplement regimen to offset some of the negative effects that were observed.

The Salient Results Of Their Research Were:


An increase in both cortisol and testosterone levels after multiple sets of squats.

A significant downregulation of the androgen receptor in biopsied muscle tissue.
Several previous studies have examined hormonal changes in cortisol, testosterone, and growth hormone (GH) during and following resistance exercise 2-4. In2 Kraemer et.al. observed an increase in both testosterone and GH after heavy resistance exercise.

Hakkinen and Pakarinen observed increases in free and total testosterone, cortisol, and GH after an acute bout of heavy squatting3. Kraemer et.al. examined plasma hormone changes after an intense bout of cycling and noted a significant increase in cortisol4. The current study and the earlier ones cited show a trend of increased cortisol and testosterone immediately after strenuous exercise.



Click Here To Enlarge.
Squats.

EXERCISE DEMONSTRATION


The current study by Ratamass et.al. is the first to look at androgen receptor content in worked muscle immediately post-exercise. While the elevated testosterone that occurs after exercise sounds beneficial, if receptor levels are low, then the increased testosterone would be of less anabolic value than if receptor levels were unchanged or increased.

In fact, a depressed level of AR is exactly what Ratamass and coworkers found. The downregulation of AR coupled with high cortisol levels post-exercise would be expected to make for a metabolic state characterized by net catabolism.

To quote from the current study under investigation,


"...acute hormonal elevations are without context unless subsequent interaction with a specific membrane bound or nuclear receptor occurs and the appropriate signal is transduced."
In other words, what good is the extra testosterone produced during lifting if the receptors aren't there to accept it?

In the current study, 9 young resistance trained men performed two exercise protocols. One consisted of a single set (SS) of 10 reps of heavy squats. The second exercise involved 6 sets of 10 reps of squats (MS). Weights were determined for each individual by measuring their 1 Rep Max (RM) and then having them squat at 80 - 85% of the (RM). The average RM was 330.4 lbs.

1 RM CALCULATOR


Enter the amount of weight you can squat (in pounds) and the number of reps you can sustain it.
Weight You Squat: lbs.
Number Of Reps:

Choose...12345678910




Your 1 RM:


lbs.



Plasma testosterone and cortisol were measured every 15 minutes for 1 hour after both sessions. The vastus lateralis was biopsied to determine AR content 1 hour after training. The results, taken from1 are shown below (Fig 2,3).




Similarly, testosterone did not change in the SS group but showed a transient increase of 20% in the MS group.




FIG 2: Changes In Serum Total Testosterone During
Single Set & Multiple Set Trials.
FIG 3: Changes In Plasma Cortisol During
Single Set & Multiple Set Trials.
The bar graph below (Fig 1) from1 shows relative vastus lateralis AR content at baseline and 1 hour after completion of exercise. The drop in AR content in the worked muscle is clear


FIG 1: Western Blot Analysis Of Androgen Receptor Conentent.
The authors of the present study attribute the decline in androgen receptors to an overall loss of protein due to the demands of strenuous exercise. Cortisol is highly catabolic to proteins and does not discriminate between contractile proteins and noncontractile proteins, such as the androgen receptor, which itself is a protein.

A number of studies have shown that the AR is upregulated after a longer post exercise time period. For example, Bamman & Shipp reported that in humans AR messenger RNA in the vastus lateralis increased 63% and 102% respectively 48 hours following 8 sets of 8 reps of either eccentric (110% of 1 RM) or concentric ( 85% of 1RM) squats5.


RNA:
One of a group of molecules similar in structure to a single strand of DNA. The function of RNA is to carry the information from DNA in the cell's nucleus into the body of the cell, to use the genetic code to assemble proteins, and to comprise part of the ribosomes that serve as the platform on which protein synthesis takes place.



Thus resistance exercise may ultimately upregulate the AR, but the initial response appears to be a catabolic one, based on the current study.

One might be tempted to speculate that the increased testosterone and decreased AR may cancel each other out. This may not be the case. Another interesting finding of this study was the individual baseline 1 RM was independent of plasma testosterone levels, but correlated highly with androgen receptor content. So an individual's AR levels may be more indicative of their strength than their testosterone levels.

Certain anabolic steroids such as Anavar (oxandrolone) that are considered to have a very high anabolic to androgenic ratio are noted for their ability to upregulate the AR6.


Since it is generally believed that protein synthesis peaks in the few hours after a training session, it makes sense to attempt to limit the downregulation of the AR that seems to occur after exercise. One strategy might be to supplement with amino acids, especially Branched Chain Amino Acids rich in leucine.

Besides being anabolic in and of itself, leucine taken as a supplement will be preferentially oxidized for fuel, sparing body proteins, which would likely include the AR.

Another strategy would be to combine a cortisol blocker such as 7-oxo DHEA and/or phosphatidyl serine to the BCAA mix to help limit protein catabolism.

While I don't advocate the use of anabolic steroids, clearly agents such as Anavar which upregulate the AR would likely prove helpful as well.


While elevated cortisol is a likely contributor to protein catabolism, other proteolytic mechanisms may be at work as well. The body has three independent systems for degrading and disposing of proteins.

These are the so-called lysosomal and calcium mediated proteases, and the ATP-ubiquitin dependent proteolytic pathway.

However, cortisol has been implicated in activating the ATP-ubiquitin proteolytic pathway7, which may ultimately be the mechanism by which cortisol exerts its catabolic action; so here again cortisol blockers might help.



We mentioned Anavar above. Besides upregulating the AR, Anavar also antagonizes the catabolic actions of cortisol8. Calcium mediated proteolysis is suppressed by cyclic adenosine monophosphate (cAMP), and forskolin is well know to elevate cAMP. Thus forskolin may be a worthwhile supplement to defend against this pathway of protein breakdown.



Beta adrenergic agonists, either synthetic such as clenbuterol or albuterol, or naturally occurring epinephrine and norepinephrine also elevate cAMP and suppress calcium mediated protein breakdown9.

Ephedrine elevates cAMP directly by binding to beta receptors, and indirectly by increasing levels of the body's naturally occurring hormone/neurotransmitter norepinephrine.

Newly published research also shows that clenbuterol, besides inhibiting calcium dependent proteolysis, also acts to block ATP-ubiquitin mediated protein breakdown10.

Finally, both the lysosomal breakdown of protein and the ATP-ubiquitin proteolytic system are suppressed by insulin11,12, so adequate carbohydrate intake prior to, during and after strenuous exercise should help blunt these pathways of protein breakdown.


Conclusion


Thus we have several strategies for reducing the breakdown of androgen receptor proteins after exercise, some as simple as eating to elevate insulin, as well as perhaps even increasing those receptor numbers with the use of certain anabolic steroids such as oxandrolone.


MR, French DN, Vescovi JD, Silvestre R, Hatfield DL, Fleck SJ, Deschenes MR. Androgen receptor content following heavy resistance exercise in men. J Steroid Biochem Mol Biol. 2005 Jan;93(1):35-42.
Kraemer WJ, Gordon SE, Fleck SJ, Marchitelli LJ, Mello R, Dziados JE, Friedl K, Harman E, Maresh C, Fry AC. Endogenous anabolic hormonal and growth factor responses to heavy resistance exercise in males and females. Int J Sports Med. 1991 Apr;12(2):228-35.
Hakkinen K, Pakarinen A. Acute hormonal responses to two different fatiguing heavy-resistance protocols in male athletes. J Appl Physiol. 1993 Feb;74(2):882-7.
Kraemer WJ, Patton JF, Knuttgen HG, Marchitelli LJ, Cruthirds C, Damokosh A, Harman E, Frykman P, Dziados JE. Hypothalamic-pituitary-adrenal responses to short-duration high-intensity cycle exercise. J Appl Physiol. 1989 Jan;66(1):161-6.
Bamman MM, Shipp JR, Jiang J, Gower BA, Hunter GR, Goodman A, McLafferty CL Jr, Urban RJ. Mechanical load increases muscle IGF-I and androgen receptor mRNA concentrations in humans. Am J Physiol Endocrinol Metab. 2001 Mar;280(3):E383-90.
Sheffield-Moore M, Urban RJ, Wolf SE, Jiang J, Catlin DH, Herndon DN, Wolfe RR, Ferrando AA Short-term oxandrolone administration stimulates net muscle protein synthesis in young men. J Clin Endocrinol Metab. 1999 Aug;84(8):2705-11.
Tiao G, Fagan J, Roegner V, Lieberman M, Wang JJ, Fischer JE, Hasselgren PO. Energy-ubiquitin-dependent muscle proteolysis during sepsis in rats is regulated by glucocorticoids. J Clin Invest. 1996 Jan 15;97(2):339-48.
Zhao J, Bauman WA, Huang R, Caplan AJ, Cardozo C. Oxandrolone blocks glucocorticoid signaling in an androgen receptor-dependent manner. Steroids. 2004 May;69(5):357-66.
Navegantes LC, Resano NM, Migliorini RH, Kettelhut IC. Catecholamines inhibit Ca(2+)-dependent proteolysis in rat skeletal muscle through beta(2)-adrenoceptors and cAMP. Am J Physiol Endocrinol Metab. 2001 Sep;281(3):E449-54.
Yimlamai T, Dodd SL, Borst SE, Park S. Clenbuterol induces muscle-specific attenuation of atrophy through effects on the ubiquitin-proteasome pathway. J Appl Physiol. 2005 Mar 17;
Wolfe RR. Effects of insulin on muscle tissue. Curr Opin Clin Nutr Metab Care. 2000 Jan;3(1):67-71
Bennett RG, Hamel FG, Duckworth WC. Insulin inhibits the ubiquitin-dependent degrading activity of the 26S proteasome. Endocrinology. 2000 Jul;141(7):2508-17.



Merc.

[BJJ]

Thanks for this type of thread.
Very informative one.

[Merc.]

Contrarian Endocrinology Revisited: Estrogen for Men?
by: Karl Hoffman


Estrogen has been reported to confer a number of health benefits in both males and females, such as the well described effects of increasing bone mass density. Here I want to examine some aspects of estrogen that may be of more immediate interest to bodybuilders and athletes. These include the lipolytic (fat mobilizing) and fat oxidizing properties of estrogen, as well as its anorectic (appetite suppressing) effect. Finally we will look at an emerging area of research: the relative contributions of the various metabolites of estrogen to the overall effects of the parent steroid.

Estrogen as a Lipolytic Hormone
Are male (and many female) bodybuilders misguided in their attempts to limit their exposure to estrogen? After all, it makes a person fat doesn’t it? And cause gynecomastia? Well, certainly for men using anabolic steroids that aromatize, too much estrogen can lead to gynecomastia in susceptible individuals. But what about those of us not using steroids? Should we treat estrogen as our fat producing enemy and even go so far as to attempt to prevent its production by using aromatase inhibitors? Or block its action at the estrogen receptor with agents like tamoxifen?

In fact, there are a wealth of data that implicate estrogen as both an anorectic and antiadipogenic hormone. It is much more likely that progesterone is the culprit in supporting higher levels of gluteofemoral fat that is prominent in women (men tend to store more fat in the abdominal area) (1). The model described in (1) has progesterone as the lipogenic hormone. Before menopause, both estrodiol and progesterone are secreted by the ovaries. After menopause, estrone becomes the primary circulating estrogen produced from aromatization of adrenal androgens (primarily the aromatization of androstenedione to estrone by adipose tissue), while progesterone levels drop dramatically since adrenal production of progesterone is minimal.

In premenopausal women progesterone increases lipoprotein lipase activity, which is greater in the gluteofemoral region, while estrogen suppresses it. Lipoprotein lipase is the body’s primary fat storage enzyme; it is responsible for allowing fats to leave the circulation and enter adipocytes. The progesterone wins out though and before menopause, women tend to have more gluteofemoral fat and less abdominal fat.

From an adaptational viewpoint, it has been argued that women's fat is designed to be stored until needed for lactation and child rearing. Men's is designed to be readily mobilized for fight or flight situations during defense and hunting. This theory may be a bit simplistic as well as sexist; but it does make sense to some degree.

Most likely the notion of estrogenic fat originated from the belief that estrogen upregulates alpha 2 receptors in fat cells, retarding lipolysis. This may be just one facet of estrogen’s actions. If one looks at the net result of estrogen’s effects, to quote a leading expert in the field “Testosterone and GH inhibit LPL and stimulate lipolysis markedly. Oestrogens seem to exert net effects similar to those of testosterone.” (2)

For example, animal studies have shown that testosterone and the nonaromatizing DHT promote alpha 2 adrenoreceptor mediated antilipolytic activity, just as they promote beta adrenoreceptor mediated lipolysis (25).

Interestingly, recent research has even attributed at least part of testosterone's fat burning properties to its local aromatization to estradiol (3). For example when testosterone is administered along with an aromatase inhibitor, LPL activity has been shown to increase (4). This suggests that the aromatization of testosterone to estradiol is responsible for the noted ability of testosterone to inhibit LPL.

There are a number of animal studies where estradiol administration led to significant weight and fat loss. Citing just one, for example

The administration of 17 beta-estradiol (500 micrograms/kg, 2 or 4 weeks) to male rats significantly reduced the body weight...Basal lipolysis and adrenaline-induced lipolysis [due to increase in HSL action] were also significantly enhanced in the epididymal adipose tissue from the male rat treated either with 7 mg/kg estradiol 12 h ahead or with 500 micrograms/kg estradiol for 2 weeks. These results indicate that estradiol exerts strong effects on metabolism of the adipose and these effects seems to be mediated through cyclic-AMP. (5)
This research indicates that in addition to the abovementioned inhibition of LPL, estrogen also stimulates the lipolytic enzyme hormone sensitive lipase.

Some of the most compelling evidence for the antiadipogenic effect of estrogen in both males and females comes from studies of estrogen receptor knockout mice and humans with aromatase deficiency. Both the afflicted humans and the knockout mice exhibit obesity. A detailed look at this topic can be found in a study of estrogen receptor knockout mice (6) Quoting from that study,

The one known human male lacking ER had a body weight approximately 2 SD greater than normal. However, this individual also had increased height because of a lack of epiphysial plate fusion. Thus, continued growth may mitigate potential increases in WAT that might normally occur because of a lack of ER. However, men and women lacking aromatase manifest truncal obesity. This and the insulin resistance and impaired glucose tolerance observed in both humans lacking ER or aromatase and their murine counterparts emphasize that similar effects accompany loss of ER in both species and strongly suggest ER may regulate adipose tissue in men.
Estrogen as an Anorectic Hormone
I also mentioned that estrogen is a potent anorectic, hunger-suppressing hormone. This effect is thought to be due to an estrogen-induced inhibition in melanin-concentrating hormone (MCH) signaling (7). MCH is a neuropeptide found in the hypothalamus that is also thought to be involved in leptin’s regulation of appetite. Leptin, an anorectic hormone secreted from adipose tissue, acts on the specific receptor present on its target neurons in the brain, and suppresses the expression of both MCH and its receptor. So we see that the actions of both estrogen and leptin are at least partly mediated through interactions with MCH. In rats and mice, intracerebroventricular administration of MCH induces hyperphagia, whereas MCH deficiency induced by targeted gene deletion leads to a hypophagia syndrome and loss of body fat.

Under normal conditions, restricted food availability leads to a drop in leptin. Falling leptin levels in turn elevate Neuropeptide Y (NPY), a hunger inducing peptide, and decrease expression of pro-opiomelanocortin (POMC), the precursor of the anorexic melanocortin -MSH, a hunger suppressing hormone. Both these changes result in elevated MHC, and food seeking behavior is initiated. When high levels of estrogen are present, the normal food seeking brought on by the changes in NPY, POMC, and MHC described above is blocked, as depicted in the diagram below, adapted from (7)

Figure 1. Proposed model of the effects of estrogen on hypothalamic neuronal pathways involved in the regulation of energy balance. A, In response to energy restriction, circulating leptin and insulin levels decrease, resulting in increased gene expression of orexigenic peptides (e.g., NPY) and decreased gene expression of anorexic peptides (e.g., POMC) in neurons of the ARC. These neuronal responses are proposed to increase expression of the orexigenic neuropeptide MCH in neurons of the LHA, which in turn promote increased food intake. B, Estrogen-mediated weight loss and anorexia also lower plasma leptin and insulin, but the expected activation of MCH neurons fails to occur in the presence of estrogen, despite the preservation of "upstream" ARC NPY and POMC neuronal responses to reduced adiposity signaling. This inhibition of MCH neurons by estrogen is hypothesized to contribute to the sustained anorexia observed with chronic estrogen exposure in male rodents. Adapted from Mystkowski et.al. J Neurosci. 2000 Nov 15;20(22):8637-42.
Estrogen Promotes Fat Burning During Exercise
For ethical reasons, the bulk of the research described was carried out in females and male animals. However, one recent study looked at the effects of estrogen administration on energy expenditure in exercising men (8). In this study male subjects cycled for 90 min at an intensity of 65% VO2max following eight days of either estrogen supplementation (2 mg 17beta-estradiol/day) or placebo. Estrogen supplementation significantly decreased carbohydrate oxidation by 5-16% and leucine oxidation by 16% (indicating a sparing effect on glycogen and muscle) whereas it significantly increased lipid oxidation by 22-44% at rest and during exercise. The authors concluded that estrogen influences fuel source selection at rest and during endurance exercise in men characterized by a reduced dependence on amino acids and carbohydrate and an increased reliance on lipids as a fuel source.

The administered dose of estrogen in (8) resulted in an increase in plasma estradiol from a baseline of 125 pM/L to 876 pM/L.

The authors suggest that the change in substrate use during exercise caused by estrogen may result for estrogen related stimulation of beta and possibly alpha adrenergic receptors. I see a problem with this hypothesis, however, since research has shown that beta receptor stimulation during exercise actually has the opposite effect, sparing fat at the expense of glycogen oxidation (9). Whatever the underlying mechanism is, estrogen clearly promotes fat burning in men.

Studies Involving Male to Female Transsexuals
Studies where hormonal treatment was administered to male to female transsexuals are often cited as evidence that estrogen administration to men leads to accumulation of subcutaneous fat.

These studies are typically confounded by the co-administration of progestational antiandrogens along with estradiol (24). The observed increase in subcutaneous fat in these subjects could very well be due to the progestational antiandrogens and the resulting drop in testosterone, which itself is a lipolytic hormone in subcutaneous adipose tissue.

All Estrogens are Not Created Equal
So far we have dwelt on the effects of estrogen itself. However, considerable work has shown that the major metabolites of estradiol and estrone are those hydroxylated (possess an OH group) at either the C-2 or the C-16alpha positions, although forms hydroxylated at the C-4 and C-15alpha are present, but in relatively lesser amounts. There exists a complete divergence in the biological properties of the 2- and 16alpha-hydroxylated metabolites of estradiol. 2-hydroxyestrone (2-OHE1) has been found to exert a modest anti-estrogenic effect in some tissues (10) and is popularly called the “good estrogen”. Studies on its ability to alter Lutienizing Hormone (LH) have yielded variable results, with some studies showing it increases LH production while others report either no change or a slight drop in LH with large doses of 2-OHE1.16alpha-hydroxyestrone on the other is a potent estrogen (11). In addition, it is associated with various cancers and has been shown to be a mutagen (cancer promoting agent).

Figure 2. Pathways of Estradiol metabolism, showing the so-called “good” metabolite 2-hydroxyestrone and the “bad” metabolite 16alpha-hydroxyestrone.
Figure 3. Schematic illustration of the metabolic pathway depicted in figure 2 showing additional estrogenic metabolites affected by DIM.
Phytochemicals such as indole-3-carbinol (I3C) are components of cruciferous vegetables, which exhibit antitumor activity associated with altered carcinogen metabolism and detoxification. The compound 3,3'-diindolylmethane, (DIM), is a major metabolite of I3C now available in supplement form. DIM directs estrogen metabolism away from “bad” estrogen to the good 2-hydroxyestrone/estradiol metabolites. Moreover, DIM itself exhibits antiestrogenic properties according to some researchers, and estrogenic activity according to others, much as if it were a SERM (12, 24). Among the hypothesized mechanisms of chemoprevention by I3C and DIM is their ability to induce a number of phase I enzymes in liver and colon, including cytochrome P450 (CYP) 1A1, CYP1A2, and CYP 3A. Increased activity of phase I drug-metabolizing enzymes can protect against some carcinogens by increasing their rate of oxidative metabolism to less toxic metabolites.

So by taking supplements containing 3,3'-diindolylmethane we can possibly lessen the likelihood of developing prostate and possibly other cancers, since research has shown that 3,3'-diindolylmethane has direct anticancer effects on the prostate independent of its ability to suppress “bad” estrogen (13). However, at least part of the ability of DIM to help prevent prostate cancer may lie is its antiandrogenic as well as antiestrogenic properties. Studies using prostate cancer (LNCaP) cells show that at physiologically obtainable levels DIM acts as a pure androgen antagonist that blocks expression of androgen-responsive genes and inhibits AR nuclear translocation (14).

One theory is that DIM appears to exert its antiestrogenic/antiandrogenic properties by acting as a weak agonist at the so-called aryl hydrocarbon receptor (AhR) (15). The AhR has been extensively studied due to the fact that a number of environmental toxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exert their effects by acting as strong agonists of the AhR. Dioxins are well known to disrupt reproductive activity in animals by deranging gonadal, pituitary, and CNS function. TCDD, like DIM acts as an antiestrogen in numerous tissues. At least part of TCDD’s antiestrogenic activity results from activation of proteosomes that degrade the estrogen receptor (16).

It’s intriguing to speculate whether DIM might also be capable of ER degradation in the pituitary, which we noted possesses AhR receptors and is sensitive to TCDD. If so, DIM might block estrogen related negative feedback on the HPTA. Yet another theory on DIM action proposes that in some tissues it activates the estrogen receptor independently of the presence of estrogen by activating the so-called PKA- and MAPK signaling systems (22).

As noted above, some studies show 2-hydroxyestrone has no suppressive effect on the HPTA while other research shows either suppression or enhancement. Another metabolite of estradiol, depicted in figure 3, is 2-hydroxyestradiol. Animal experiments have shown that administration of 2-hydroxyestradiol can override the suppressive effect of estradiol on pituitary LH secretion in males (17). The same study showed that the bad estrogen 4-hyroxyestradiol was able to suppress LH production

As can be seen in figures 3 and 4, both 2-hydroxyestradiol and 2-hydroxyestrone are methylated during their metabolism by the body. Interestingly, 2-methoxyestradiol (2-MeOE2) has a significantly higher affinity for Sex Hormone Binding Globulin than do estradiol and even testosterone (18). This is significant in that 2-MeOE2, by virtue of its higher affinity for SHBG than testosterone, can displace testosterone from SHBG, possibly enlarging the fraction of free, or bioactive testosterone.

16alpha-hydroxyestrone may play a role in a number of diseases other than cancers. For example, the estrogen found in the synovial fluid of rheumatoid arthritis patients is primarily the proinflammatory 16alpha-hydroxyestrone and may be responsible for the inflammation associated with that disease (19, 20). DIM may prove useful in treating or ameliorating the symptoms of rheumatoid arthritis and other estrogen related autoimmune diseases that primarily affect women. Also of interest is the observation that while estradiol has well-known neuroprotective actions, 2-hydroxy-estradiol appears to be significantly more neuroprotective than its parent, estradiol (21). Of course, standard medical protocols should be followed and a physician’s advice obtained, before self-medicating with DIM to treat any disease.

Since some evidence suggests DIM might generate estrogen metabolites that suppress LH production, it might not be advisable to use DIM as the sole agent during Post Cycle Therapy. When used post cycle with a SERM such as Clomid that stimulates the HPTA, DIM could offer a number of potential health advantages by shunting estrogen metabolites towards the more healthful 2-OH series while at the same time elevating free testosterone levels. Similarly, during a cycle of aromatizable steroids, when estrogen is high and the HPTA is suppressed, DIM use may offer a number of health benefits due to its actions described above.

For a person not using anabolics and unconcerned about the complexities of Post Cycle Therapy, DIM use may confer enough health benefits, such as possible cancer prevention, to warrant its use as a supplement. And, as mentioned above, we may get a boost in free testosterone as well. Long term safety studies in animals have failed to detect any toxicity due to DIM (23).

In summary, while certainly not advocating estrogen supplementation for men, I also believe it is not the evil hormone it is often made out to be. We should accept it for what it is, a naturally occurring part of or normal hormonal milieu, that can be manipulated in form and quantity to better suit the needs of male athletes and bodybuilders by focusing on altering the byproducts of estrogen metabolism.

References
1) Price TM, O'Brien SN, Welter BH, George R, Anandjiwala J, Kilgore M. Am J Obstet Gynecol 1998 Jan;178(1 Pt 1):101-7

2) Bjorntorp P. Hum Reprod 1997 Oct;12 Suppl 1:21-5

3) Ramirez ME, McMurry MP, Wiebke GA, Felten KJ, Ren K, Meikle AW, Iverius PH Metabolism 1997 Feb;46(2):179-85

4) Zmuda JM, Fahrenbach MC, Younkin BT, Bausserman LL, Terry RB, Catlin DH, Thompson PD. Metabolism 1993 Apr;42(4):446-50

5) Tomita T, Yonekura I, Okada T, Hayashi E Horm Metab Res 1984 Oct;16(10):525-8

6) Heine PA, Taylor JA, Iwamoto GA, Lubahn DB, Cooke PS. Increased adipose tissue in male and female estrogen receptor-alpha knockout mice. Proc Natl Acad Sci U S A. 2000 Nov 7;97(23):12729-34

7) Mystkowski P, Seeley RJ, Hahn TM, Baskin DG, Havel PJ, Matsumoto AM, Wilkinson CW, Peacock-Kinzig K, Blake KA, Schwartz MW. J Neurosci 2000 Nov 15;20(22):8637-42

8) Hamadeh MJ, Devries MC, Tarnopolsky MA. Estrogen supplementation reduces whole body leucine and carbohydrate oxidation and increases lipid oxidation in men during endurance exercise. J Clin Endocrinol Metab. 2005 Mar 8

9) Mora-Rodriguez R, Hodgkinson BJ, Byerley LO, Coyle EF. Effects of beta-adrenergic receptor stimulation and blockade on substrate metabolism during submaximal exercise. Am J Physiol Endocrinol Metab. 2001 May;280(5):E752-60.

10) Schneider J, Huh MM, Bradlow HL, Fishman J. Antiestrogen action of 2-hydroxyestrone on MCF-7 human breast cancer cells. J Biol Chem. 1984 Apr 25;259(8):4840-5

11) Dalessandri KM, Firestone GL, Fitch MD, Bradlow HL, Bjeldanes LF. Pilot study: effect of 3,3'-diindolylmethane supplements on urinary hormone metabolites in postmenopausal women with a history of early-stage breast cancer. Nutr Cancer. 2004;50(2):161-7.

12) Chen I, McDougal A, Wang F, Safe S. Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of diindolylmethane. Carcinogenesis. 1998 Sep;19(9):1631-9.

13) Sarkar FH, Li Y. Indole-3-carbinol and prostate cancer. J Nutr. 2004 Dec;134(12 Suppl):3493S-3498S.

14) Le HT, Schaldach CM, Firestone GL, Bjeldanes LF. Plant-derived 3,3'-Diindolylmethane is a strong androgen antagonist in human prostate cancer cells. J Biol Chem. 2003 Jun 6;278(23):21136-45.

15) Chen I, McDougal A, Wang F, Safe S. Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of diindolylmethane. Carcinogenesis. 1998 Sep;19(9):1631-9.

16) Wormke M, Stoner M, Saville B, Walker K, Abdelrahim M, Burghardt R, Safe S. The aryl hydrocarbon receptor mediates degradation of estrogen receptor alpha through activation of proteasomes. Mol Cell Biol. 2003 Mar;23(6):1843-55.

17) Franks S, MacLusky NJ, Naish SJ, Naftolin F. Actions of catechol oestrogens on concentrations of serum luteinizing hormone in the adult castrated rat: various effects of 4-hydroxyoestradiol and 2-hydroxyoestradiol. J Endocrinol. 1981 May;89(2):289-95.

18) Avvakumov GV, Grishkovskaya I, Muller YA, Hammond GL. Crystal structure of human sex hormone-binding globulin in complex with 2-methoxyestradiol reveals the molecular basis for high affinity interactions with C-2 derivatives of estradiol. J Biol Chem. 2002 Nov 22;277(47):45219-25

19) Cutolo M, Sulli A, Capellino S, Villaggio B, Montagna P, Seriolo B, Straub RH Sex hormones influence on the immune system: basic and clinical aspects in autoimmunity. Lupus. 2004;13(9):635-8.

20) Cutolo M, Villaggio B, Seriolo B, Montagna P, Capellino S, Straub RH, Sulli A. Synovial fluid estrogens in rheumatoid arthritis. Autoimmun Rev. 2004 Mar;3(3):193-8.

21) Teepker M, Anthes N, Krieg JC, Vedder H 2-OH-estradiol, an endogenous hormone with neuroprotective functions. J Psychiatr Res. 2003 Nov-Dec;37(6):517-23.

22) Leong H, Riby JE, Firestone GL, Bjeldanes LF. Potent ligand-independent estrogen receptor activation by 3,3'-diindolylmethane is mediated by cross talk between the protein kinase A and mitogen-activated protein kinase signaling pathways. Mol Endocrinol. 2004 Feb;18(2):291-302.

23) Leibelt DA, Hedstrom OR, Fischer KA, Pereira CB, Williams DE. Evaluation of chronic dietary exposure to indole-3-carbinol and absorption-enhanced 3,3'-diindolylmethane in sprague-dawley rats. Toxicol Sci. 2003 Jul;74(1):10-21

24) Elbers JM, Asscheman H, Seidell JC, Gooren LJ. Effects of sex steroid hormones on regional fat depots as assessed by magnetic resonance imaging in transsexuals. Am J Physiol. 1999 Feb;276(2 Pt 1):E317-25.

25) Bouloumie A, Valet P, Dauzats M, Lafontan M, Saulnier-Blache JS. In vivo upregulation of adipocyte alpha 2-adrenoceptors by androgens is consequence of direct action on fat cells. Am J Physiol. 1994 Oct;267(4 Pt 1):C926-31.



Merc.

[Merc.]

Thanks for this type of thread.
Very informative one.

Merc.

[Merc.]

Androgens and Glucorticoids
by: Karl Hoffman



Despite decades of research, there is as yet no universally agreed upon model that explains the observed actions of androgens and anabolic steroids (AAS) when these agents are administered to animals and humans. The classical model of AAS action posits that after an anabolic/androgenic compound binds to the androgen receptor (AR), the AAS/AR complex enters the nucleus of target cells, recruits additional transcription factors, and initiates the transcription of androgen responsive genes. In a recent issue of Mind & Muscle we described how one model attributes the spectrum of effects caused by AAS to activation of different genes by different steroids

We also briefly discussed in that article how some androgens may exert their effects by inhibiting the action of glucocorticoids, catabolic hormones produced by the adrenal glands. It is this proposed mechanism that I would like to look at in more detail in this article.

DIRECT ANTAGONISM OF THE GLUCOCORTICOID RECEPTOR BY ANDROGENS
Glucocorticoids and androgens are generally accepted as having opposing actions in skeletal muscle. The former breaks down muscle during periods of stress in order to provide a supply of amino acids that can be used as fuel substrates. It does this primarily by increasing the activity of the ubiquitin-proteasome proteolytic system in muscle. Glucocorticoids also inhibit the growth of muscle, possibly by increasing levels of myostatin (1). Androgens on the other hand promote muscle hypertrophy and according to some studies, muscle hyperplasia (growth of new muscle fibers) via a number of proposed mechanisms.

Research has shown that at least two AAS, nandrolone and R 1881 (methyltrienolone) competitively bind to the glucocorticoid receptor (GR) in the cytosol of cells. (2) It was observed that androgens were able to displace dexamethasone, a potent synthetic GR agonist, from the GR in a concentration dependent manner. So here we have an example of how AAS might exert an anabolic effect independently of any binding to the androgen receptor (AR). In some respects this result is not surprising, since the GR and the AR share a high degree of sequence homology, meaning simply that the DNA sequences that code for the two receptors are quite similar.

Competitive inhibition of glucocorticoid binding to the GR by androgens could explain how certain androgens with a very weak binding affinity for the AR (e.g. oxymetholone) nevertheless act as potent anabolic agents: they may have a relatively high affinity for the GR. Of course, we also have an alternate model described in the issue of Mind & Muscle linked to above, where low AR binding-affinity AAS may still be capable of activating genes that strongly promote anabolism. Yet a third model, described below, attributes the inhibition of glucocorticoid action by androgens (and vice versa, the inhibition of androgen action by glucocorticoids) to a form of "crosstalk" between the AR and the GR. This interaction between the two receptors inhibits the binding of the receptors to their respective hormone response elements on target genes.

INHIBITION OF GLUCOCORTICOID DEPENDENT GENE EXPRESSION BY ANDROGENS VIA HETERODIMER FORMATION
Interestingly, some androgens and synthetic AAS such as DHT and oxandrolone are unable to displace such potent endogenous glucocorticoids as cortisol from the GR, yet in culture these androgens inhibit the expression of cortisol dependent genes in the presence of cortisol (3, 4). Clearly this observation is incompatible with the simple model of competitive inhibition of the GR by AAS described above. Evidence for lack of competitive inhibition is bolstered by the observation that the cellular presence of the AR is required for the observed glucocorticoid antagonism, at least in the case of oxandrolone (4). Clearly, had the effect been observed in cells lacking the AR, there would have been no possibility of any GR/AR interaction.

Normally, in order for androgens to activate target genes, they must bind to the androgen receptor, which then binds (dimerizes) to another androgen receptor forming a so-called homodimer. The receptors joined in this way are then able to bind stably to the target gene, recruit various transcription factors, and initiate gene transcription, which eventually leads to protein synthesis. What Chen et. al. (1) discovered was that the androgen receptor and the glucocorticoid receptor are capable of forming so called heterodimers that repress transcription of target genes instead of activating the genes, as in the case of androgen or glucocorticoid homodimers. See fig 1.


Figure 1. Schematic illustration of AR/GR heterodimer bound by ionic bond between Arginine (+) and Aspartate (-) ARDBD = androgen receptor DNA Binding Domain; GRDBD = glucocorticoid receptor DNA binding domain. (Adapted from Chen et.al.)

ALTERNATE MECHANISMS OF RECEPTOR CROSSTALK
While Zhao et. al. in their study of glucocorticoid antagonism by oxandrolone did not look specifically for the presence of AR/GR heterodimers, their data are consistent with this model. Nevertheless, as the authors pointed out, potential alternative mechanisms may exist. In order for the AR and the GR to form homo or heterodimers, they must first enter the nucleus bound to their respective ligands. Zhao et. al. constructed a mutant AR that was incapable of translocating into the nucleus. When oxandrolone was added to cell cultures containing the normal GR and the mutant AR, GR dependent gene transcription was still repressed. To quote the authors: "while the mechanisms underlying this surprising finding remain to be defined, these data indicate that repression can occur even when the two receptors are localized in different subcellular compartments". So in this admittedly artificial system where the engineered AR was unable to enter the nucleus and interfere with GR binding, the mutant AR was nevertheless able to communicate with the normal GR to interfere with its action.

Another possible mechanism for mutual repression is so-called squelching, where the two receptors compete for a limited number of transcriptional co-activators. For example, in the case of excess androgen relative to cortisol, more androgen receptors will translocate to the nucleus, effectively using up the co-activators common to both receptors (4). Squelching has been observed in a number of systems. One in particular may have important physiological and environmental implications. The action of estrogen is antagonized by a number of xenobiotic estrogenic compounds (e.g. DDE, a breakdown product of DDT) that bind only very weakly to the estrogen receptor but bind strongly to the so called xenobiotic orphan receptor CAR. Ligand bound CAR has been shown to inhibit the action of estrogen via squelching (5). The possibility exists that residues from xenobiotic estrogens may interfere with female reproductive physiology. Some researchers have attributed the worldwide decline in amphibians to CAR/ER squelching.

HETERODIMER FORMATION AS A MODULATOR OF LIGAND ACTION
Heterodimer formation may have physiological implications as well. The classic model of receptor dimerization posits that receptor dimers are required for stable binding of the receptor to DNA. Chen et. al. suggest another possibility, namely that the ability to form homodimers is an effect secondary in importance to the ability of different receptors to form heterodimers. They suggest that heterodimer formation is a regulatory mechanism that allows the body to respond to changing environmental conditions.

For example, during periods of stress such as induced by starvation, the body’s cortisol to testosterone ratio increases. This would lead to the formation of GR/AR heterodimers and an excess of GR homodimers. The result would be a shutdown of energy consuming anabolic processes and an increase in catabolic processes to provide energy substrates. Squelching could also represent such a regulatory system.

HETERODIMERIZATION IN DIVERSE RECEPTOR CONTROLED SIGNALING SYSTEMS
Before leaving the subject of heterodimerization, it’s logical to ask whether such a phenomenon occurs with other receptor types, and whether there are physiological or clinical implications for such heterodimerization. It turns out the answer is yes on both counts. For example, bradykinin and angiotensin II are compounds in the body that are involved in the regulation of blood pressure. The former binds to the B (2) receptor and acts as a vasodepressor, lowering blood pressure. The latter binds to the AT(1) receptor as a vasopressor, elevating blood pressure (There may be readers with hypertension who use a so called ACE inhibitor such as lisinopril that inhibits the formation of angiotensin II, or an angiotensin receptor antagonist like valsartan, that blocks the action of angiotensin at its receptor). Heterodimerization between AT (1) and B (2) has been observed, and the combined AT (1)/B (2) receptor is hypersensitive to the effects of angiotensin II, leading to high blood pressure (6). The hypertension that accompanies preeclampsia during pregnancy may be the result of such heterodimerization (7).

About half of all Europeans are either homo or heterozygous for an isoform of the growth hormone (GH) receptor gene that lacks exon 3, so called (d3-GHR). In people who possess both the regular GH receptor and the d3 isoform (heterozygous individuals), the two receptor types are capable of forming heterodimers, increasing the sensitivity of the combined receptor to GH. People who are homozygous for d3 are even more sensitive to GH. For example, in children administered GH for short stature, those children homozygous for the d3 allele experience 1.7 to 2 times the growth acceleration than do children that possess the normal allele (8). So it’s very likely that athletes and bodybuilders who self-administer GH experience a degree of responsiveness dependent on whether they are homo or heterozygous for d3, and dependent as well on the extent of heterodimerization of the two isoforms.

Further, it’s been recognized for some time that the estrogen receptor exists in two forms, ER-alpha and ER-beta. The two are capable of forming heterodimers affecting estrogen signaling (9). In fact, it has been suggested that the primary role of ER-beta may be to modulate the effect of ER-alpha, the primary estrogen receptor. ER-beta seems to exert a bodywide repressing effect on ER-alpha dependent transcription (10). In bone, for example, ER-beta activation inhibits the anabolic effect normally exerted by estrogen (10).

These are just a few examples of receptor heterodimerization that may be of clinical importance. The classical picture of a ligand binding to its receptor and the complex initiating gene transcription is blurred now that we see that receptors are able to crosstalk with one another via heterodimerization and other mechanisms like squelching. Perhaps most intriguing is the idea that the requirement for dimerization of hormonal receptors in order that they be active evolved to allow for heterodimerization as a way for receptors to modulate the activity of one another, rather than as simply a way to stabilize DNA binding, as the classical model of receptor action postulates.

References:
(1) Ma K, Mallidis C, Bhasin S, Mahabadi V, Artaza J, Gonzalez-Cadavid N, Arias J, Salehian B.Am J

Glucocorticoid-induced skeletal muscle atrophy is associated with upregulation of myostatin gene expression. Physiol Endocrinol Metab. 2003 Aug;285(2):E363-71

(2) Mayer M, Rosen F.Am J Physiol. Interaction of anabolic steroids with glucocorticoid receptor sites in rat muscle cytosol. 1975 Nov;229(5):1381-6.

(3) Chen S, Wang J, Yu G, Liu W, Pearce D.J Androgen and glucocorticoid receptor heterodimer formation. A possible mechanism for mutual inhibition of transcriptional activity.

Biol Chem. 1997 May 30;272(22):14087-92.

(4) Zhao J, Bauman WA, Huang R, Caplan AJ, Cardozo C. Oxandrolone blocks glucocorticoid signaling in an androgen receptor-dependent manner Steroids. 2004 May;69(5):357-66.

(5) Min G, Kim H, Bae Y, Petz L, Kemper JK Inhibitory cross-talk between estrogen receptor (ER) and constitutively activated androstane receptor (CAR). CAR inhibits ER-mediated signaling pathway by squelching p160 coactivators.J Biol Chem. 2002 Sep 13;277(37):34626-33.

(6) AbdAlla S, Lother H, Quitterer U AT1-receptor heterodimers show enhanced G-protein activation and altered receptor sequestration. Nature. 2000 Sep 7;407(6800):94-8.

(7) AbdAlla S, Lother H, el Massiery A, Quitterer U. Increased AT(1) receptor heterodimers in preeclampsia mediate enhanced angiotensin II responsiveness. Nat Med. 2001 Sep;7(9):1003-9

(8) Dos Santos C, Essioux L, Teinturier C, Tauber M, Goffin V, Bougneres P. A common polymorphism of the growth hormone receptor is associated with increased responsiveness to growth hormone. Nat Genet. 2004 Jul;36(7):720-4

(9) Pettersson K, Grandien K, Kuiper GG, Gustafsson JA. Mouse estrogen receptor beta forms estrogen response element-binding heterodimers with estrogen receptor alpha. Mol Endocrinol. 1997 Sep;11(10):1486-96.

(10) Lindberg MK, Moverare S, Skrtic S, Gao H, Dahlman-Wright K, Gustafsson JA, Ohlsson C Estrogen receptor (ER)-beta reduces ERalpha-regulated gene transcription, supporting a "ying yang" relationship between ERalpha and ERbeta in mice. Mol Endocrinol. 2003 Feb;17(2):203-8

[Merc.]

Anabolic Steroid Action In Vitro and In Vivo
by: Karl Hoffman


The traditional view of anabolic steroids is that their anabolic-androgenic potency is to a large extent dependent on the relative binding affinity of a given steroid to the androgen receptor. In an earlier edition of Mind and Muscle Magazine I reviewed some recent research suggesting that while binding affinity is still a factor controlling the action of anabolic steroids, the particular genes activated by any given steroid may be just as important or even more so.

There I made the comments that:

Surprisingly, despite the number of synthetic AAS that have been developed, their modes of action are poorly understood. This holds for the naturally occurring androgens as well. There is some evidence (which we will discuss below) that androgens are able to exert some of their actions independently of the androgen receptor (AR). Antagonism of the glucocorticoid receptor is one possible way androgens may exert an anabolic effect.
Binding affinity to the androgen receptor has also been invoked to explain the differences in potencies and effects of the natural and synthetic androgens. For example, dihydrotestosterone binds the androgen receptor much more strongly than does testosterone at the same concentration, yielding a higher degree of ligand-receptor stability. When the concentration of testosterone is increased however, the receptor stability increases to a level similar to that seen with dihydrotestosterone (1). This has led to the proposal that the weaker androgenic potency of testosterone compared to that of dihydrotestosterone in target tissues such as the prostate resides in testosterone’s weaker interaction with the androgen receptor. Yet it is well known that some steroids which are very potent anabolic agents, such as stanozolol or oxymetholone, bind the AR only very weakly (2). If we assume that AR binding affinity is the sole determinant of an agent’s ability to act via the AR to promote anabolic or androgenic actions, then we are forced into the conclusion that certain potent AAS that bind the AR with negligible affinity must be exerting their anabolic effects via some other routes that do not involve AR binding. Indeed, this has become to a large degree dogma in the bodybuilding literature.
Some interesting recent research has shed light on this problem by showing that AR binding affinity is only partly responsible for the androgen receptor mediated effects of both physiologic androgens and synthetic AAS. In the study I would like to discuss, the authors present evidence for the existence of distinct steroid specific target gene transcription profiles following AR activation (3). In other words, the structures of androgen responsive genes vary in such a way that some genes are more readily activated by certain androgens than by others. The set of genes readily switched on by a given androgen determines the net physiological effect of that androgen. This theory readily explains how an anabolic steroid like oxandrolone, whose AR binding affinity is quite low, can be so anabolic: it happens to preferentially turn on genes whose products promote skeletal muscle anabolism, while failing to activate genes which promote virilization.
In this paper I would like to discuss a different but not necessarily contradictory explanation of why some steroids that appear to bind only weakly to the AR still manage to exert potent anabolic and androgenic effects. The solution to the apparent paradox is a simple one: virtually all binding affinity studies to date have been carried out in vitro. Here we will look at the recently published research by Feldkoren and Andersson [1] who compared the in vivo and in vitro actions of the anabolic steroids stanozolol (Winstrol) and methanedienone (Dianabol). We will see that the interactions of these anabolic steroids with the AR are much different when measured in vitro when compared to measurements carried out in vivo.

In [1] the authors examined the interaction of the above mentioned steroids with the AR by using three different systems: (1) a recombinant AR ligand binding in vitro assay (the modern standard method of expressing binding affinities); (2) a cell based AR-dependent transactivation assay; and (3) an in vivo assay based on steroid induced cytosolic AR depletion in skeletal muscle. The logic behind system (3) is that normally the unbound AR resides in the cytosol of the cell. Upon ligand (steroid) binding the ligand-receptor complex translocates to the nucleus. So the degree of cytosolic depletion of the AR when exposed to a particular steroid serves as a measure of the degree of binding of the steroid to its receptor. The binding affinities of testosterone and 17-alpha methyltestosterone were examined as well.

System (1) measured the in vitro binding strengths of the given steroids by determining how effectively they displaced radiolabeled methyltrienolone (MT) from the recombinant AR. Methyltrienolone binds extremely strongly to the AR and to what degree it can be displaced from the AR serves as the standard assay for measuring the binding strength of any given androgen receptor ligand

By observing the amount of displaced radiolabeled MT as a function of the concentration of the competing steroid, it’s possible to calculate the binding affinity of the competitor. The affinity is usually quantified as the equilibrium dissociation constant, Ki. The subscript i is used to indicate that the competitor inhibited radioligand binding. The Ki is the concentration of the competing ligand that will bind to half the binding sites at equilibrium, in the absence of radioligand or other competitors. If the Ki is low, the affinity of the receptor for the inhibitor is high, and vice versa.

Not surprisingly MT possessed the highest affinity with a Ki of 0.20 nM, the Ki for testosterone and 17alpha-methyltestosterone were 0.80 and 0.90 nM, respectively. Both stanozolol and methanedienone were the least effective competitors with a Ki for stanozolol of 4.5nM and methanedienone of 5.0 nM. The calculated in vitro binding affinities of the various agents are depicted below in Figure 1, excerpted from [1].


Fig. 1. Binding strength of various steroids to the recombinant rat AR. The data presented in Fig. 1 were analyzed to calculate the equilibrium dissociation constant Ki for each steroid mentioned above. T, testosterone; MT, methyltrienolone; 17alpha-MeT, 17alpha-methyltestosterone; S, stanozolol; MA, methanedienone.

So here we see the expected picture from all we have read about various anabolic steroids; namely, certain oral steroids like Winstrol and Dianabol only bind relatively weakly (compared to testosterone, for example) to the AR yet are well known to be quite potent.

System (2) employed a full length recombinant AR and a section of DNA consisting of the androgen response element (the portion of a gene to which the AR-ligand complex binds) spliced to a luciferase reporter gene. These are then inserted into a cell. Luciferase emits light when activated, and the idea here is that when the cell containing this artificial gene complex is exposed to a given steroid, the steroid will bind to the AR, attach to the artificial gene construct, and activate the luciferase. The amount of light emitted is a measure of the binding of the AR-ligand complex to the reporter gene, which in turn is a function of the strength of binding of the ligand to the AR.

Transcriptional activation is usually expressed in terms of so called EC50 of the ligand. EC50 is defined as the molar concentration of a ligand, which produces 50% of the maximum possible response for that ligand. Methyltrienolone was found to be the most effective transcriptional activator with an EC50 of 5 pM (picomoles), in agreement with its high affinity in vitro. The calculated EC50 values for the other steroids were 44pM for 17alpha-methyltestosterone, 52pM for stanozolol, and 79pM for methanedienone; all four steroids induced the same level of maximum transactivation. The researchers did not measure the EC50 of testosterone because it metabolized to the relatively inactive androgen androstenedione by the enzyme 17beta-hydroxysteroid dehydrogenase present in the type of cells used in the experiment. Hence, in intact cells, both stanozolol and methanedienone are potent activators of the AR, of the same order of magnitude as 17alpha-methyltestosterone.

Finally, to test the in vivo strength of the various steroids, rats were injected with each steroid at a dose 0.3 mg/kg of body weight. On hour after treatment, muscle cells were removed from the animals and the cytosolic depletion analysis of the AR was carried out. Methyltrienolone resulted in a 67% reduction of androgen binding sites in muscle cytosol. Stanozolol possessed almost as much activity (44% depletion) as testosterone (55%), followed by methanedienone, which caused a 33% reduction in binding sites. 17alpha-Methyltestosterone demonstrated the lowest degree of cytosolic AR depletion (11%) of all of the AS.

We see then a clear discrepancy between the typically published in vitro binding affinities of various anabolic steroids, and the ability of these steroids to evoke biological responses via classical androgen receptor mediated transcription in both cell based systems and in vivo.

It is interesting to compare the in vivo binding affinities obtained above with previously published binding data. Saartok et.al.[2] measured the binding affinities of a number of anabolic steroids relative to methyltrienolone (MT) using a system based on the binding of steroids to the AR in the cytosols obtained by grinding rat muscle and prostate tissue. Their relative binding affinities (RBA) were calculated as the ratio between the molar concentrations of unlabeled MT and of the competitor required to displace 50% of the radiolabeled MT from cytosolic binding sites (i.e. androgen receptors). If MT is arbitrarily given an RBA of 1, testosterone exhibited an RBA of 0.7; 17alpha-methyltestosterone’s was 0.10; stanozolol 0.03; and methanedienone 0.02.

We immediately see large differences in the results obtained in vivo in [1] compared to the in vitro data published in [2]. For example in [1] the binding affinities between MT and testosterone differed by only 18%. In [2] the difference was 30%. The disparity between studies for stanozolol and methanedienone is even greater. Looking at stanozolol, in [1] we see an affinity difference from MT of 34%. In [2] the difference is enormous, almost 2 orders of magnitude.

References
1. Feldkoren BI, Andersson S. Anabolic-androgenic steroid interaction with rat androgen receptor in vivo and in vitro: a comparative study. J Steroid Biochem Mol Biol. 2005 Apr;94(5):481-7.

2. Saartok T, Dahlberg E, Gustafsson JA. Relative binding affinity of anabolic-androgenic steroids: comparison of the binding to the androgen receptors in skeletal muscle and in prostate, as well as to sex hormone-binding globulin. Endocrinology. 1984 Jun;114 (6):2100-6.




Merc.

[Merc.]

How Does Myostatin Inhibit Muscle Development & Hypertrophy?
by: Karl Hoffman


Myostatin is a member of the family of growth factors known as the transforming growth factor-beta (TGF-beta) family. It is an important negative regulator of embryonic andpostnatal muscle growth. By now we have all seen photographs of the famous myostatin null mice. These mice lackmyostatin, which normally puts the brakes on muscular development. Myostatin null mice generated by gene targetingshow a dramatic and widespread increase in skeletal muscle mass. Individual muscles in myostatin null mice weigh 2- to3-fold more than those of wild-type mice, primarily due to an increased number of muscle fibers without a correspondingincrease in the amount of fat. As well as an increased number of muscle fibers (hyperplasia), the individual muscle fibersin these mice are larger than normal (hypertrophy).

Mutations in the myostatin gene leading to “overmuscled” humans havebeen reported (1). See figure 1 below


Figure 1. Photographs of the Child at the Ages of SixDays and Seven Months (Panel A), Ultrasonograms (Panel B) and Morphometric Analysis (Panel C) of the Muscles of thePatient and a Control Infant, and the Patient's Pedigree (Panel D).

The arrowheads in Panel A indicate the protruding muscles of the patient's thigh andcalf. In Panel B, an ultrasonographic transverse section (linear transducer, 10 MHz) through the middle portion of the thighreveals differences between the patient and a control infant of the same age, sex, and weight. VL denotes vastus lateralis,VI vastus intermedius, VM vastus medialis, RF rectusfemoris, and F femur. In Panel C, retracings of the muscle outlinesand results of the morphometric analysis of the muscle cross-sectional planes of the two infants



also reveal markeddifferences. Panel D shows the patient's pedigree. Solid symbols denote family members who are exceptionally strong,according to information in their clinical history. Square symbols denote male family members, and circles female family members. (Adapted from Schuelke et.al)

There are several reported ways by which myostatin may regulate muscular development. During growth, muscle precursor cells (known as myoblasts) enter the cell cycle and proliferate until a cascade of signals (initiated by the myogenic regulatory factor MyoD) causes myoblasts to withdraw from the cell cycle, differentiating and fusing into multinucleated myotubes. These mature into fully developed muscle fibers. Myostatin appears to downregulate the activity and expression levels of MyoD, thus preventing the differentiation of myoblasts into
myotubes (2). See figure 2 below.



Fig. 2. A model for the role of myostatin during muscle growth and differentiation. A model from Thomas et al. (24) for the role of myostatin during muscle growth, adapted to include the role of myostatin during myogenic differentiation. During myogenic embryogenesis, Myf5 and MyoD specify cells to adopt the myogenic fate. Myoblast proliferation is regulated by myostatin via the up-regulation of p21 and inactivation of Cdk (cyclin-dependent kinase) activity resulting in retinoblastoma (Rb)hypophosphorylation and myoblast cell cycle arrest. In response to a differentiation cue, MyoD becomes fully functional, activating downstream myogenic gene expression, including myogenin and p21, resulting in committed myoblasts that fuse into multinucleated myotubes. Myostatin regulates this process by inhibiting MyoD expression via Smad 3 (another protein in the TGF-beta family) after the differentiation cue resulting in the loss of downstream myogenic gene expression and myogenicdifferentiation. (Adapted from Langley et al)

Satellite cells are myogenic precursor (stem) cells that are quiescent in mature muscle.Injury to the surrounding muscle activates the nearby satellite cells which then reenter the cell cycle, and express myogenic regulatory factors, proliferate, develop into myoblasts, and fuse to the injured muscle, affecting its repair. Satellite cells are responsible for the postnatal growth of muscle. When we damage muscle by performing resistance exercise, we activate satellite cells which then contribute to the observed hypertrophy. Myostatin has been shown to inhibit reentry of satellite cells into the cell cycle, thus rendering them inactive (3).

McCrosky et.al. (3) performed an interesting experiment designed to demonstrate the effects of myostatin on satellite cells. To show the direct inhibitory effects of myostatin on myoblast proliferation, tissue-dissociated satellite cells were isolated from both wild-type and myostatin-null mice. The same number of wild-type and myostatin-null myoblasts were cultured in media, and myostatin was added in increasing concentrations to only myostatin-null cultures. After 48 h of proliferation, the cells were fixed with 10% formal saline and stained with the methylene blue. When recombinant myostatin was added to the media in increasing amounts, the enhanced proliferation rate seen in the myostatin-null myoblasts was reduced to that of the wild-type myoblasts.



Figure 3. A model for the role of Myostatin in postnatal muscle growth. Quiescent satellite cells on muscle fibers are activated in response to muscle injury to give rise to myoblasts. Proliferating myoblasts can either fuse with the existing fiber or differentiate into a nascent myotube. A portion of proliferating myoblasts, however, can revert to become quiescent satellite cells, thus resulting in self-renewal. As myostatin is a negative regulator of cell cycle progression, high levels of myostatin in satellite cells block the activation to maintain quiescence.(From McCroskey et al.)

MYOSTATIN AND ARA70



Myostatin acts in yet another way to inhibit muscle growth. When androgens such as testosterone bind to the androgen receptor (AR), the AR undergoes a series of conformational (shape) changes that allow it to interact with androgen response elements (binding sites) in androgen target genes. ARA70 (androgen receptor associated protein 70) is a so-called AR coregulator that stabilizes the ligand (androgen)-bound AR enhancing the ability of the AR to induce transcription of target genes. Siriett et. al. were recently able to demonstrate that ARA70 is expressed in myoblasts during myogenesis and that myostatin is a potent ownregulator of ARA70 gene expression (4). Siriett et. al. thus propose that the hypertrophy seen in animals that lack myostatin could not only be due to the mechanisms outlined above but could also be due to increased protein synthesis due to enhanced AR activity resulting from increased expression of ARA70. Although the authors of (4) performed their experiments with mice, they showed that murine ARA70 has about an 80% sequence homology to human ARA70 and propose that the same mechanism at work in mice alsosuppresses ARA70 expression in humans.

In addition to ARA70, Siriett et.al. discovered numerous other key genes controlling muscle development and various metabolic pathways that are upregulated in myostatin-null mice. These include the
genes for actin and tropomyosin, both myofibril proteins; muscle creatine kinase which is involved in energy metabolism; phosphorylase kinase, an enzyme involved in glycogen metabolism; ATP synthase, an enzyme involved in mitochondrial ATP synthesis, to name but a few, as well as a number of novel genes with unknown functions.

GLUCOCORTICOIDS UPREGULATE MYOSTATIN




Glucocorticoids are well known to induce muscle atrophy in animals and humans, although the exact mechanism(s) are not well understood. Ma and colleagues (5) recently cloned and characterized the 5'-upstream regulatory region of the human myostatin gene and found that the promoter contains a number of response elements important for muscle growth, including seven putative glucocorticoid response elements (GREs). GREs are sites where the glucocorticoid/receptor complex bind and control gene transcription. They also demonstrated thatdexamethasone dose-dependently increases endogenous myostatin transcription in myocytes through a glucocorticoid receptor-mediated mechanism.

These findings led them to conclude that an increase in myostatin gene expression by glucocorticoids might contribute to the pathogenesis of glucocorticoid-induced skeletal muscle atrophy. Specifically, the intramuscular myostatin mRNA expression in rats treated with dexamethasone for 5 days was significantly (4.50-fold, P < 0.01) higher than that in their pair-fed controls. The myostatin protein expression in these rats was also significantlyhigher (2.6-fold, P < 0.01) than that in their pair-fed controls. Further confirming dexamethasone’s effect on myostatin, administration of RU-486, a potent glucocorticoid antagonist, was given to the dexamethasone treated animals. The action of dexamethasone treatment on myostatin mRNA expression was effectively nullified by RU-486 administration.

Interestingly, the majority of muscle loss in the dexamethasone treated animals occurred in the first 5 days of treatment, whereas animals treated for 10 days did not experience significantly greater muscle loss than those treated for 5 days. This coincides with the observation that, although the marked upregulation of myostatin mRNA and protein expression induced by dexamethasone could be seen for 5 days after treatment, this overexpression
was not sustained by extending the treatment to 10 days. The counterregulatory mechanism is unknown.

MYOSTATIN AND GROWTHHORMONE/IGF-1



We discussed above how myostatin inhibits the normally hypertrophic activity of satellite cells. Growth hormone, on the other hand, may exert its stimulatory effects on muscle protein synthesis, in part, by activating satellite cells (6). Marcell et al (7) observed a significant inverse relation between myostatin levels and GH receptor levels in healthy but aging (>65). It is also well known that GH levels decline with age. Thus it’s possible that the combination of decreasing GH/GHR and increasing myostatin that accompanies aging may contribute to age related muscle loss.

Collectively the data suggest that GH excess in youth may override myostatin's inhibition of satellite cell activation rather than GH directly inhibiting myostatin promoter activity. For example Brill et al (8) examined the effects of GH and/or testosterone administration on body composition, performance, mood, sexual function, bone turnover, and muscle-gene expression in healthy older men. Of relavence to our current discussion, myostatin gene expression was unaffected by either treatment.

MYOSTATIN AUTOREGULATION



In addition to GH/IGF-1 keeping myostatin in check, a negative feedback autoregulatory system for myostatin gene expression appears to exist (9). As previously mentioned, myostatin is a member of the TGF-beta family of growth factors. Human SMAD7 is a 426 amino acid protein. Smad7 acts as an antagonist of TGF-beta signaling. It functions in several different ways. It interacts with the TGF-beta receptor, interfering with the activation of the downstream signaling factors Smad2/3. In addition, it recruits E3-ubiquitin ligases, Smurf1/2, to the activated TGF-beta receptor, resulting in receptor ubiquitination and degradation. Smad7 expression is induced by TGF-beta itself, resulting in an autoregulatory feedback loop in TGF-beta signaling.

Since myostatin belongs to the TGF-beta family, Forbes et al (9) tested the logical proposition that myostatin expression might be autoregulated via Smad7. Indeed, the researchers discovered that auto-regulation by myostatin does appear to be signaled through Smad7, since the expression of the inhibitory Smad7 is induced by myostatin and the over-expression of Smad7 in turn inhibits the myostatin promoter activity

In summary, we have seen several proposed mechanisms through which myostatin regulates muscle growth. It is not known which if any is the predominant mode through which myostatin works, and it is likely that all contribute to myostatin’s action.

References



1) Schuelke M, Wagner KR, Stolz LE, Hubner C, Riebel T, Komen W, Braun T, Tobin JF, Lee SJ. Myostatin mutation associated with gross muscle hypertrophy in a child. N Engl J Med. 2004 Jun 24;350(26):2682-8



2) Langley B, Thomas M, Bishop A, Sharma M, Gilmour S, Kambadur R. Myostatin inhibits myoblast differentiation by down-regulating MyoD expression. J Biol Chem. 2002 Dec 20;277(51):49831-40


3) McCroskery S, Thomas M, Maxwell L, Sharma M, Kambadur R. Myostatin negatively regulates satellite cell activation and self-renewal. J Cell Biol. 2003 Sep 15;162(6):1135-47.


4) Siriett V, Nicholas G, Berry C, Watson T, Hennebry A, Thomas M, Ling N, Sharma M, Kambadur R. Myostatin negatively regulates the expression of the steroid receptor co-factor ARA70. J Cell Physiol. 2005 Aug 18; [Epub ahead of print


5) Ma K, Mallidis C, Bhasin S, Mahabadi V, Artaza J, Gonzalez-Cadavid N, Arias J, Salehian B.Glucocorticoid-induced skeletal muscle atrophy is associated with upregulation of myostatin gene expression. Am J Physiol Endocrinol Metab. 2003 Aug;285(2):E363-71


6) Le Roith D, Bondy C, Yakar S, Liu JL, Butler A. The somatomedin hypothesis: 2001. Endocr Rev. 2001Feb;22(1):53-74.



Merc.

[Merc.]

Anti-Obesity Effect of AMPK
by: Karl Hoffman


AMPK (adenosine 5'-monophosphate-activated protein kinase) is an enzyme found in numerous tissues throughout the body, and has been characterized as a “metabolic master switch” that regulates the activity of a number of target proteins that control metabolism. Specifically, the proteins that are upregulated or down regulated by AMPK control the flux of compounds through the metabolic pathways of hepatic ketogenesis, cholesterol synthesis, lipogenesis, triglyceride synthesis, adipocyte lipolysis, and skeletal muscle fatty acid oxidation. AMPK also controls the contraction-induced uptake of glucose by skeletal muscle and the secretion of insulin by pancreatic beta cells (1).

AMPK activity is modulated by numerous stimuli, including nutrient levels, muscle contraction, hypoxia, and mitochondrial uncoupling agents, such as DNP and rotenone. A commonly used experimental activator of AMPK is AICAR, 5-aminoimidazole-4carboxamide ribonucleoside (2). AMPK is also found in the brain, where its activity is regulated by substances as diverse as adiponectin (3) and alpha lipoic acid (ALA) (4).

After an introductory discussion of the peripheral effects of APMK signaling as they relate to energy sensing, we will focus on how AMPK activation in the central nervous system by the agents mentioned above adiponectin and ALA acts to regulate body weight. We will place specific interest on ALA since it is such a popular nutritional supplement.

Regulation of AMPK Activity
A kinase is an enzyme that catalyzes the transfer of phosphate groups between substrates. Adenosine 5'-monophosphate-activated protein kinase then is an enzyme that is activated by adenosine 5'-monophosphate, or AMP. AMP is formed when two phosphate groups are removed from the parent molecule adenosine triphosphate, or ATP. ATP is comprised of an adenosine molecule bonded to three phosphates; each phosphate bond contains energy, especially the third bond. By breaking the two phosphate bonds and reducing ATP to AMP cells can get the energy to carry out their various processes: ATP -> AMP + 2P + energy

Hawley et.al. (5) showed that AMPK activity is decreased by ATP, so rather than simply being a function of AMP concentration, AMPK activity is sensitive to the AMP to ATP ratio. High ATP levels correspond to high levels of availability of energy in a cell, whereas from the discussion above we see that a low ATP to AMP ratio corresponds to a state of energy depletion. It is for this reason that AMPK is considered to be an “energy sensor”, active when a cell is depleted in energy and inactive when a cell is rich in energy. The job of AMPK is to shut down energy (ATP) consuming processes such as biosynthesis, and activate energy producing processes such as fatty acid oxidation that generate ATP. This will replenish ATP, and raise the ATP to AMP ratio, eventually deactivating AMPK when the cell is replenished with the energy (ATP) it needs.

AMPK AND SKELETAL MUSCLE
The role of AMPK has been studied extensively in exercising skeletal muscle. Before moving on to AMPK and the brain, we can gain much insight into AMPK functioning in general by examining its role in tissue where its activity is well characterized. In broad terms, AMPK activation in skeletal muscle seems to increase glucose transport (6) and fatty acid oxidation (1, 7). This is in keeping with its role as a provider of ATP to energy depleted tissues, e.g. exercised skeletal muscle.

Remember that AMPK’s job is to catalyze the transfer of phosphate groups between molecules (this is what kinases do). One of the best-characterized targets of AMPK activation in skeletal muscle is acetyl-CoA carboxylase (ACC). When AMPK is activated, it phosphorylates ACC, inhibiting its action (8). ACC in turn catalyzes the synthesis of Malonyl-CoA. Malonyl-CoA is a potent inhibitor of carnitine palmitoyltransferase-1 (CPT-1) which controls the entry of fatty acids into the mitochondria where they can be oxidized or turned into ketone bodies (in the liver) for use as fuel in other organs. So the net result of this chain of events is that an increase in muscle AMPK leads to an increase in CPT-1, and an increase in fatty acid oxidation.

AMPK also seems to enhance glucose uptake by skeletal muscle. It may do this by increasing the transcription of the gene for GLUT4, a glucose transporter that acts to shuttle glucose into cells (9).

In addition AMPK elevation in working muscles may enhance the translocation of GLUT4 to the cell surface where it can pick up glucose and carry it into the cell to be used for fuel to generate ATP (10). If AMPK is responsible for the exercise-induced enhanced flux of glucose to muscle, might it have the same effect in adipocytes? From our basic picture of AMPK shutting down energy consuming processes in favor of energy producing ones, we would expect otherwise, since it takes energy to produce triglycerides from free fatty acids taken up by fat cells. This AMPK induced inhibition of fatty acid uptake by adipocytes under the influence of AMPK has been observed (11), where the authors remarked that:

We propose that in contrast to skeletal muscle, in which AMPK stimulation promotes glucose transport to provide ATP as a fuel, AMPK stimulation inhibits insulin-stimulated glucose transport in adipocytes, inhibiting triacylglycerol synthesis, to conserve ATP under conditions of cellular stress (11).
Observant readers may have noted that AMPK stimulation of both glucose uptake and fatty acid oxidation to generate ATP seems to violate a fundamental principle of metabolic control—that of the so called Randle cycle (and reverse Randle cycle). The Randle cycle proposes that the use of fat for fuel precludes the use of glucose for fuel. This observation has not been overlooked by researchers (12). Glycogen synthase is the enzyme responsible for converting glucose into glycogen. AMPK, in line with its overall role of inhibiting biosynthesis, inhibits glycogen synthase, according to some data anyway, that show AMPK acts as a glycogen synthase kinase (13). However, this inhibition is overridden by high intracellular glucose concentrations. Since AMPK activation shuttles more glucose into cells via GLUT4, this excess glucose dominates the suppressive effects of AMPK on glycogen synthase, so considerably more glucose is stored as glycogen when AMPK levels are high. The remaining glucose is used for fuel.

ALPHA LIPOIC ACID DECREASES AMPK IN THE BRAIN AND ELEVATES AMPK IN MUSCLE
Andersson et.al. showed recently that AMPK levels in the hypothalamus control food intake in animals (16). Leptin, which is known to reduce food intake, decreased hypothalamic AMPK activity when administered to laboratory animals. Ghrelin, a hormone with the opposite effect as leptin, increases food intake. When injected, ghrelin caused an increase in food intake along with an increase in AMPK activity in the hypothalamus. AICAR, or 5-amino-4-imidazole carboxamide riboside, mimics the action of AMP on AMPK, increasing AMPK activity.

When AICAR was administered to the animals, food intake increased significantly, as did AMPK activity. All this evidence suggests that low levels of hypothalamic AMPK activity decrease food intake, whereas high levels of AMPK activity increase food intake. This certainly makes sense from what we have learned thus far about the role of AMPK as a metabolic switch. When energy is depleted and AMP levels are high relative to ATP, AMPK is activated and might be expected to signal the animal to eat.

The naturally occurring short-chain fatty acid alpha-Lipoic Acid (ALA) is well known to increase glucose uptake in skeletal muscle of both lean and insulin resistant obese animals. It’s logical to ask if ALA’s action on glucose transport is AMPK dependent. This question was addressed in a recent paper by Kim et.al. (17). The authors found that indeed ALA administration to rats increased AMPK activity in skeletal muscle. Rats fed a diet containing ALA (0.25, 0.5, and 1% of diet by weight) reduced their food intake and lost body weight in a dose dependent manner. Injecting microgram quantities of ALA into the ventricles of the animals’ brains induced the same anorectic effect, suggesting that at least part of the reduction in food intake was due to effects of ALA on the brain.

The animals promptly gained back the weight they lost while on ALA. Upon examination, the organs of the treated rats appeared normal. These facts suggested to the authors that the weight lost while consuming ALA was not due to any illness or toxicity related to ALA.

Calorimetry experiments, which measure energy expenditure, showed that the ALA fed rats had higher metabolic rates than the controls. This is likely due to an observed increase in UCP-1 in brown fat of the ALA treated rats compared to controls. UCP-1 is a so called uncoupling protein that acts in rodents to increase non-shivering thermogenesis. It uncouples mitochondrial respiration from ATP production. Instead, heat is generated. This is a principal means of thermoregulation in rodents. Moreover, UCP-1 was expressed in white adipose tissue in the ALA group. White adipose tissue does not normally contain UCP-1.

Is the increase in UCP-1 in brown fat, and the ectopic expression of UCP-1 in white fat seen in these rodents of any significance to humans? Human infants have considerable brown fat, but we lose it as we become adults, retaining only a small bit. However, UCP-1 is expressed in human brown fat, and studies have shown that low levels of UCP-1 are associated with obesity (18).

To verify that lowered AMPK activity was responsible for the anorexic effect of ALA, rats that had previously been injected with ALA were given cerebroventricular injections of AICAR, which we’ve seen increases AMPK activity. This completely abolished the suppression of food intake seen in the ALA injected rats.

We discussed in our overview of AMPK action how elevated AMPK increases fatty acid oxidation. In the study under discussion, recall that while ALA reduced AMPK activity in the brain, it increased AMPK in rodent peripheral tissues. This led to a loss of fat mass, particularly visceral fat, or fat around the abdominal organs. It is well known that visceral obesity is more unhealthful than excess subcutaneous fat. The authors looked in particular at a strain of genetically obese mice that were leptin resistant.

We mentioned above how leptin administration reduces food intake; it’s for this reason that leptin is often called a “satiety hormone”. Leptin also increases energy expenditure and at one point was considered a potentially promising candidate for an anti-obesity drug, until it was learned that the majority of obese people are leptin resistant. In fact, leptin resistance has been termed a hallmark of obesity. If the effects of ALA seen in rats also occur in humans, ALA could potentially be an effective treatment for leptin resistant obesity.

To summarize the effects of ALA, we see that it reduces food intake; improves glucose uptake and increases lipid oxidation in muscle tissue; elevates basal metabolic rate by increasing the expression of UCP-1 in brown fat; induces UCP-1 expression in white fat; and reduces bodyfat, particularly in the visceral region.

Questions or comments on this article? CLICK HERE to pose your questions and to receive live feedback from Karl Hoffman, as well as the Mind and Muscle staff and fellow readers!
References
(1) Winder WW, Hardie DG. AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes. Am J Physiol. 1999 Jul;277(1 Pt 1):E1-10.

(2) Sullivan JE, Brocklehurst KJ, Marley AE, Carey F, Carling D, Beri RK Inhibition of lipolysis and lipogenesis in isolated rat adipocytes with AICAR, a cell-permeable activator of AMP-activated protein kinase FEBS Lett. 1994 Oct 10;353(1):33-6.

(3) Qi Y, Takahashi N, Hileman SM, Patel HR, Berg AH, Pajvani UB, Scherer PE, Ahima RS. Adiponectin acts in the brain to decrease body weight.

Nat Med. 2004 May;10(5):524-9

(4) Kim MS, Park JY, Namkoong C, Jang PG, Ryu JW, Song HS, Yun JY, Namgoong IS, Ha J, Park IS, Lee IK, Viollet B, Youn JH, Lee HK, Lee KU Anti-obesity effects of alpha-lipoic acid mediated by suppression of hypothalamic AMP-activated protein kinase. Nat Med. 2004 Jul;10(7):727-33.

(5) Hawley SA, Davison M, Woods A, Davies SP, Beri RK, Carling D, Hardie DG. Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase.

J Biol Chem. 1996 Nov 1;271(44):27879-87.

(6) Hayashi T, Hirshman MF, Fujii N, Habinowski SA, Witters LA, Goodyear LJ Metabolic stress and altered glucose transport: activation of AMP-activated protein kinase as a unifying coupling mechanism. Diabetes. 2000 Apr;49(4):527-31

(7) Ruderman NB, Saha AK, Vavvas D, Witters LA. Malonyl-CoA, fuel sensing, and insulin resistance. Am J Physiol. 1999 Jan;276(1 Pt 1):E1-E18

(8) Saha AK, Ruderman NB.. Malonyl-CoA and AMP-activated protein kinase: an expanding partnership

Mol Cell Biochem 2003 Nov;253(1-2):65-70

(9) Med Sci Sports Exerc. 2004 Jul;36(7):1202-6. Regulation of GLUT4 gene expression during exercise. Holmes B, Dohm GL

(10) Diabetes. 1999 Aug;48(8):1667-71 5' AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle. Kurth-Kraczek EJ, Hirshman MF, Goodyear LJ, Winder WW.

(11) Diabetes. 2000 Oct;49(10):1649-56 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes. Salt IP, Connell JM, Gould GW

(12) Biochem Soc Trans. 2003 Dec;31(Pt 6):1157-60 Bypassing the glucose/fatty acid cycle: AMP-activated protein kinase. Carling D, Fryer LG, Woods A, Daniel T, Jarvie SL, Whitrow H

(13) Biochem Soc Trans. 2003 Dec;31(Pt 6):1290-4. Transgenic models--a scientific tool to understand exercise-induced metabolism: the regulatory role of AMPK (5'-AMP-activated protein kinase) in glucose transport and glycogen synthase activity in skeletal muscle. Wojtaszewski JF, Nielsen JN, Jorgensen SB, Frosig C, Birk JB, Richter EA.

(14) J Biol Chem. 2002 Jul 5;277(27):23977-80 AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling. Bolster DR, Crozier SJ, Kimball SR, Jefferson LS

(15) Diabetes. 2002 Jul;51(7):2074-81 Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes. Musi N, Hirshman MF, Nygren J, Svanfeldt M, Bavenholm P, Rooyackers O, Zhou G, Williamson JM, Ljunqvist O, Efendic S, Moller DE, Thorell A, Goodyear LJ.

(16) J Biol Chem. 2004 Mar 26;279(13):12005-8 AMP-activated protein kinase plays a role in the control of food intake. Andersson U, Filipsson K, Abbott CR, Woods A, Smith K, Bloom SR, Carling D, Small CJ.

(17) Nat Med. 2004 Jul;10(7):727-33. Anti-obesity effects of alpha-lipoic acid mediated by suppression of hypothalamic AMP-activated protein kinase. Kim MS, Park JY, Namkoong C, Jang PG, Ryu JW, Song HS, Yun JY, Namgoong IS, Ha J, Park IS, Lee IK, Viollet B, Youn JH, Lee HK, Lee KU.

(18) J Lipid Res. 1998 Apr;39(4):834-44 Uncoupling protein-1 mRNA expression in obese human subjects: the role of sequence variations at the uncoupling protein-1 gene locus. Esterbauer H, Oberkofler H, Liu YM, Breban D, Hell E, Krempler F, Patsch W.


Merc.

[Merc.]

Understanding Androgen Actions
by Karl Hoffman


Researchers as well as athletes and bodybuilders know that besides the two principal physiological androgens, testosterone and dihydrotestosterone, there exist a number of synthetic anabolic-androgenic steroids (AAS) that exhibit diverse biological actions. For example, dihydrotestosterone (DHT) is considered androgenic relative to testosterone since it is essential for the virilization of the external genital organs. On the other hand, DHT is not considered anabolic because it is not active in skeletal muscle (it is enzymatically deactivated); testosterone is anabolic in this regard, being responsible (along with other hormones and growth factors) for the development and maintenance of skeletal muscle. The array of synthetic AAS were developed to meet differing needs and like the physiological androgens differ in their relative anabolic/androgen potency. Some, like methyltestosterone and fluoxymesterone are relatively androgenic (although not as much so as DHT) and are indicated for androgen replacement while others like oxandrolone and stanozolol are relatively more anabolic. Yet despite the wide range of effects and potencies of both the natural and synthetic androgens, to date only one androgen receptor has been identified. What accounts for the diversity of effects of the different AAS?

Surprisingly, despite the number of synthetic AAS that have been developed, their modes of action are poorly understood. This holds for the naturally occurring androgens as well. There is some evidence (which we will discuss below) that androgens are able to exert some of their actions independently of the androgen receptor (AR). Antagonism of the glucocorticoid receptor is one possible way androgens may exert an anabolic effect.

Binding affinity to the androgen receptor has also been invoked to explain the differences in potencies and effects of the natural and synthetic androgens. For example, dihydrotestosterone binds the androgen receptor much more strongly than does testosterone at the same concentration, yielding a higher degree of ligand-receptor stability. When the concentration of testosterone is increased however, the receptor stability increases to a level similar to that seen with dihydrotestosterone (1). This has led to the proposal that the weaker androgenic potency of testosterone compared to that of dihydrotestosterone in target tissues such as the prostate resides in testosterone’s weaker interaction with the androgen receptor. Yet it is well known that some steroids which are very potent anabolic agents, such as stanozolol or oxymetholone, bind the AR only very weakly (2). If we assume that AR binding affinity is the sole determinant of an agent’s ability to act via the AR to promote anabolic or androgenic actions, then we are forced into the conclusion that certain potent AAS that bind the AR with negligible affinity must be exerting their anabolic effects via some other routes that do not involve AR binding. Indeed, this has become to a large degree dogma in the bodybuilding literature.

Some interesting recent research has shed light on this problem by showing that AR binding affinity is only partly responsible for the androgen receptor mediated effects of both physiologic androgens and synthetic AAS. In the study I would like to discuss, the authors present evidence for the existence of distinct steroid specific target gene transcription profiles following AR activation (3). In other words, the structures of androgen responsive genes vary in such a way that some genes are more readily activated by certain androgens than by others. The set of genes readily switched on by a given androgen determines the net physiological effect of that androgen. This theory readily explains how an anabolic steroid like oxandrolone, whose AR binding affinity is quite low, can be so anabolic: it happens to preferentially turn on genes whose products promote skeletal muscle anabolism, while failing to activate genes which promote virilization.

Before looking at this research in detail, a brief review of how androgens activate genes is in order. The AR is generally thought to reside primarily in the cytoplasm of the target cell, bound to so-called heat shock proteins. The androgen (ligand) diffuses into the cytoplasm and binds to part of the receptor called, appropriately enough, the ligand binding domain. The heat shock proteins dissociate from the ligand-receptor complex, the complex dimerizes (binds to another ligand-receptor complex), and then translocates to the nucleus where the target gene(s) is located. The stretch of chromosomal DNA comprising the target gene acts as a template for the synthesis of RNA in a process called transcription.

Part of the gene (depicted below) consists of a promoter region that contains a subsection called the androgen response element (ARE). When the ligand binds to the AR, it induces a conformation change in the ligand-receptor complex that allows the complex to recognize and bind to the specific nucleotide sequence comprising the ARE. There it proceeds to recruit coactivators, which act as “power boosters” that amplify transcription, as well as other transcription factors, which are proteins that are required to initiate transcription of the target gene via RNA polymerase II. The receptor/ligand and coactivators along with perhaps other transcription factors would form a large complex that serves as a sort of platform for RNA polymerase to dock with, allowing the polymerase to begin transcribing the gene. The messenger RNA (mRNA) created from the DNA template of the gene then leaves the nucleus and enters the cytoplasm, where in the process known as translation, the mRNA in turn serves as a template for the construction of a specific protein.

The exons in the gene depicted below contain the segments of DNA that actually code for the protein that will ultimately be transcribed.



Fig 1. Generic gene structure showing exon (protein coding region), RNA polymerase II bound to gene; TATA box; and promoter with bound transcription factors. The androgen/AR complex would bind to a specific region within the promoter, the Androgen Response Element (ARE). From (4)

Upstream from the exon is the region of the promoter called the TATA box. It contains a sequence of seven bases TATAAAA and is a common feature of promoters found in all genes. The base sequences in the remaining upstream portion of the promoter vary from gene to gene.

The authors of the paper under discussion wanted to see how the promoter base sequence affected steroid hormone binding and action. In order to do this, they first constructed a set of so called artificial reporter genes; these consisted first of an exon coding for the enzyme luciferase. Luciferase is found in fireflies and produces luminescence when it acts on its substrate luciferin. To the luciferase exon they then spliced 3 different well-characterized promoters whose base sequences varied greatly. The three resulting artificial genes were designated GRE-OCT-luc; (ARE)2TATA-luc; and MMTV-luc.

The idea here is that if a given androgen is exposed to one of these genes and is able to bind to the promoter and induce transcription of the luciferase gene, detectable light will be emitted in proportion to the effectiveness of that androgen to activate the gene. What are the possible outcomes and interpretations of the experiment? If for example all androgens induce transcription to the same extent in all three genes, then it could be assumed that the structure of a given gene’s promoter would probably not be a determinant in the biological profiles of differing androgens. If on the other hand stanozolol, say, activated only one of the genes, while testosterone activated another, then the different biologic profiles of the two steroids (e.g. their different anabolic/androgenic ratio) could be due in part to the possibility that the two steroids activate different sets of genes in the body, depending on the promoter structure of the gene. If the latter is the case, a particular AAS that only binds the AR weakly could still be quite potent if it turned out to be a strong activator of anabolism promoting genes in skeletal muscle. This obviates the need to invoke non-AR mediated actions for weak androgen receptor agonists (the dubious class I/class II theory of steroid action). Receptor binding may be only part of the picture; promoter binding and the strength of the transcription signal could be equally if not more important than AR affinity in determining the biological effects of a given agent.

Chinese hamster ovary cells (which do not express the androgen receptor or any androgen responsive genes) were transfected with the three genes described above, as well as with a vector expressing the androgen receptor. The cells were treated with varying concentrations of a number of different androgens, including R1881 (methyltrienolone), testosterone, DHT, nandrolone, oxandrolone, androstenedione, and DHEA.

The main result of the study was that the androgens could be divided into two main subgroups based on reporter gene activation. DHT, nandrolone, R1881, and testosterone grouped together statistically based on their activation profile, while the precursor hormones together with the anabolic steroids oxandrolone and stanozolol fell into a separate subgroup based on the reporters they preferentially activated.

There were some interesting individual results. Testosterone showed twice the ability of DHT to activate the GRE-OCT-luc reporter at all concentrations, suggesting that AR binding affinity is certainly not the determinant of gene transcription with this reporter. DHT on the other hand maximally stimulated the (ARE)2TATA-luc construct at 10nM concentration.

The anabolic steroids oxandrolone, nandrolone, and stanozolol were potent activators of the MMTV-luc construct. Remarkably, at 10nM, stanozolol, which has a very weak AR binding affinity exceeded R-1881 induced activity for this reporter despite the fact that R-1881 has one of the highest AR binding affinities of any androgen. Here, once again, we see binding affinity is not the sole determinant of androgen activity.

Another interesting result was the fact that the androgen precursors DHEA and androstenedione were potent AR ligands leading to differential target gene expression. The authors concluded their data potentially support a relevant contribution of testosterone-precursor hormones to mechanisms of in vivo androgen action.

These findings are in accord with earlier work (5) where two different androgen response elements were discovered that showed different T- vs. DHT-induced AR transactivation. In vivo work supports in vitro findings that different androgens are capable of differentially regulating AR responsive genes. In castrated rats, DHT proved more potent at maintaining prostate epithelial cell function, whereas testosterone and DHT were equipotent at inhibiting prostatic apoptosis (programmed cell death) (6). In another study that looked at the effects of testosterone and DHT on prostatic regrowth in castrated rats, testosterone proved to be more potent than DHT in activating genes governing cellular differentiation than those responsible for proliferation. (Differentiation is the process whereby immature cells activate genes that commit them to the path to becoming fully functioning mature cells, whereas proliferation is the process of multiple cell division that leads to an increase in cell number) (7).

Now that we see that steroid receptor agonists activate transcription in part by recruiting coactivators to aid in transcription it is relatively easy to understand how receptor antagonists might block transcription: by inhibiting coactivator binding. This has been well studied for the interaction between the estrogen receptor (ER) and tamoxifen, which acts as an antiestrogen in some tissues. The ligand binding domain of the estrogen receptor consists of a number of amino acid sequences folded into a series of helixes. Different ER ligands can relatively easily change the conformation of one helix in particular, helix 12. When an agonist like estradiol binds the ER, helix 12 takes on a conformation that forms part of the coactivator binding pocket once the ligand/receptor binds to the gene to be transcribed. In contrast, when an estrogen antagonist binds to the ER, the antagonist changes the shape of the ligand binding domain in such a way that helix 12 now bends so as to occupy part of the coactivator binding pocket, blocking coactivator binding. Without a coactivator present, transcription of the gene cannot proceed. It turns out the estrogen receptor contains two regions that can bind coactivators, so called AF-1 and AF-2. Tamoxifen inactivates AF-2, but AF-1 still retains the ability to bind coactivators. Tamoxifen is a Selective Estrogen Receptor Modulator, or SERM; it has the ability to act as an antiestrogen with regard to certain genes, and an estrogen with respect to others, blocking transcription of the former and initiating transcription of the latter.. It is believed that in the case where tamoxifen acts as an antiestrogen, the promoter of the gene in question depends on AF-2 to hold the coactivator in place, and we have seen that tamoxifen renders AF-2 incapable of doing so. With other genes where tamoxifen acts as an agonist, it is believed AF-1, which is unaffected by tamoxifen, functions as the important coactivator binding site.

Pure antiestrogens, such as faslodex, block transcription of all estrogen responsive genes by blocking both coactivator binding sites, AF-1 and AF-2. In this case it is impossible for any coactivator to bind the target gene once faslodex has attached, so transcription cannot proceed.



INDIRECT MECHANISMS OF ANDROGEN ACTION


While the results described above may obviate the need to invoke non-AR mediated mechanisms to explain some of the biological activity of various AAS, such mechanisms nevertheless do exist. For example, androgens undergo differential metabolism in target tissues. DHT is inactive in skeletal muscle because the enzyme 3 alpha-hydroxysteroid dehydrogenase, present in large quantities in skeletal muscle, rapidly metabolizes it. On the other hand, androgen target tissues such as the prostate, skin, and scalp are relatively rich in the 5 alpha reductase enzymes that convert testosterone to DHT, so DHT is considered the active androgen in those tissues.

We also mentioned above the possibility that androgens may exert anabolic activity by binding to and antagonizing the glucocorticoid receptor. Endogenous glucocorticoids such as cortisol exert a catabolic effect on skeletal muscle by activating the ubiquitin proteasome proteolytic pathway and to a lesser extent calcium-dependent protein breakdown. Testosterone seems to be a particularly potent glucocorticoid antagonist (8,9), more so than the anabolic steroid trenbolone (10). Speculating a bit, and using some “contrarian endocrinology”, this may explain the observation commonly made by bodybuilders that trenbolone is a more effective lipolytic agent than is testosterone, since research indicates that cortisol is a predominantly lipolytic hormone:

Cortisol's effects on lipid metabolism are controversial and may involve stimulation of both lipolysis and lipogenesis...In conclusion, the present study unmistakably shows that cortisol in physiological concentrations is a potent stimulus of lipolysis and that this effect prevails equally in both femoral and abdominal adipose tissue. (11)

So by antagonizing the glucocorticoid receptor and blocking the lipolytic effects of cortisol, testosterone could possibly be losing some of its lipolytic power. It has also been proposed that glucocorticoid activity at the gene level is inhibited via androgen interference with the glucocorticoid response element in genes targeted by cortisol (11).

Androgens are capable of stimulating both the production of hepatic insulin like growth factor (IGF-1), as well as local IGF-1 production within skeletal muscle. One often reads in the bodybuilding literature that the former is an attribute only of oral 17-alpha alkylated steroids and occurs by direct action of these steroids on the liver. In fact, testosterone as well as oxandrolone (12) and methandrostenelone (Dianabol) (13, 14) all elevate hepatically derived IGF-1, but secondary to an increase in growth hormone secretion. So these agents are not acting directly on the liver to elevate IGF-1; rather they stimulate pituitary growth hormone secretion, and this GH in turn is responsible for inducing hepatic IGF-1 secretion.

The second effect mentioned above, the local production of IGF-1 within skeletal muscle, may be more important than hepatic IGF-1 production for growth, while hepatically derived IGF-1 may play a more important role in carbohydrate and lipid metabolism (15). The locally produced IGF-1 acts back on the muscle tissue that produced it in an autocrine manner to stimulate growth. Based on their research on testosterone suppression in normal men, Mauras et al concluded that:

[During androgen suppression] [t]here were, however, significant decreases in [intramuscular] mRNA concentrations for IGF-I and a trend toward increased IGFBP-4 gene expression, the main inhibitory binding protein for IGF-I in muscle. The gene expression for actin and myosin in muscle was not altered by the systemic decrease in testosterone concentrations. These observations are congruent with the observation made in elderly men treated with testosterone and suggest that, within skeletal muscle tissue, androgens are necessary for local IGF-I production, independent of GH production and systemic IGF-I concentrations. IGF-I and its type I receptor are ubiquitously expressed in skeletal muscle and appear to be important in both the proliferation and differentiation of skeletal myocytes. Even though the gene expression of actin and myosin, the main contractile proteins of skeletal muscle, were not altered during severe hypogonadism, testosterone deficiency was associated with a marked decrease in measures of muscle strength, indicating that other mechanisms besides changes in muscle protein expression are affected by this severe degree of androgen deficiency (16).

So one of primary anabolic effects of androgens may be their ability to stimulate IGF-1 production in skeletal muscle.

It may be somewhat misleading to call the production of GH and IGF-1, either local or hepatic, an indirect action of androgens in the same sense that glucocorticoid receptor antagonism is. It is likely that the genes for GH and IGF-1 are direct targets for androgens and are activated by the androgen/AR complex, just as any other androgen responsive genes would be.



References

(1) Grino PB, Griffin JE, Wilson JD Endocrinology 1990 Feb;126(2):1165-72

(2) Saartok T, Dahlberg E, Gustafsson JA Endocrinology 1984 Jun;114(6):2100-6

(3) Holterhus PM, Piefke S, Hiort O J Steroid Biochem Mol Biol 2002 Nov;82(4-5):269-75.

(4) Biology. John W. Kimball 1994 by Wm. C. Brown,

(5) Hsiao PW, Thin TH, Lin DL, Chang C. Mol Cell Biochem 2000 Mar;206(1-2):169-75

(6) Wright AS, Thomas LN, Douglas RC, Lazier CB, Rittmaster RS.
J Clin Invest 1996 Dec 1;98(11):2558-63

(7) Dadras SS, Cai X, Abasolo I, Wang Z.Gene Expr 2001;9(4-5):183-

(8) Danhaive PA, Rousseau GG. J Steroid Biochem 1988 Jun;29(6):575-81

(9) Danhaive PA, Rousseau GG J Steroid Biochem 1986 Feb;24(2):481-7

(10) Djurhuus CB, Gravholt CH, Nielsen S, Mengel A, Christiansen JS, Schmitz OE, Moller N.
Am J Physiol Endocrinol Metab 2002 Jul;283(1):E172-7

(11) Hickson RC, Czerwinski SM, Falduto MT, Young AP. Med Sci Sports Exerc 1990 Jun;22(3):331-40 .

(12) Ulloa-Aguirre A, Blizzard RM, Garcia-Rubi E, Rogol AD, Link K, Christie CM, Johnson ML, Veldhuis JD. J Clin Endocrinol Metab 1990 Oct;71(4):846-54

(13) Hochman IH, Laron Z Horm Metab Res 1970 Sep;2(5):260-4

(14) Steinetz BG, Giannina T, Butler M, Popick F.Endocrinology 1972 May;90(5):1396-8

(15) Isaksson OG, Jansson JO, Sjogren K, Ohlsson C.Horm Res 2001;55 Suppl 2:18-21

(16) Mauras N, Hayes V, Welch S, Rini A, Helgeson K, Dokler M, Veldhuis JD, Urban RJ.
J Clin Endocrinol Metab 1998 Jun;83(6):1886-92



Merc.

[Merc.]

Stem Cells as the Target of Androgen Action
by: Karl Hoffman


Testosterone and other androgens are noted for exerting two well-known effects on body composition: skeletal muscle hypertrophy, and a reduction in fat mass. It may be that androgens work in a variety of ways to achieve these effects, but is it possible to formulate one hypothesis of androgen action that can explain both these phenomena? Here I’d like to summarize one such hypothesis put forth recently by Bhasin et al (1) that attempts to do just that.

EFFECTS OF ANDROGENS ON MUSCLE TISSUE
Classically, muscle hypertrophy has been attributed to an increase in the synthesis rate of skeletal muscle protein, including myosin heavy chain. However, this theory fails to explain two well-characterized features of the action of testosterone on skeletal muscle; namely an increase in the number of nuclei contained in muscle cells, as well as the observed increase in muscle satellite cells. In fact, since mature muscle cell nuclei are incapable of mitosis, an increase in satellite cell number is a necessary requirement for the observed increase in nuclei after androgen administration. This is because satellite cells act as donors of nuclei when they fuse with preexisting muscle cells and donate their nuclei to the hypertrophying muscle fibers.

In addition to the observed increase in muscle mass and decrease in fat mass after testosterone administration, an increase in bone mass has also been observed. It would be elegant if this feature of the action of androgens could also be incorporated into one unifying theory of androgen action, without invoking separate mechanisms for this effect.

EFFECTS OF ANDROGENS ON FAT MASS
In previous studies carried out by Bhasin et.al., administration of graded doses of testosterone was associated with a dose-dependent decrease in fat mass, both in the trunk and appendices and in the subcutaneous and deep compartments. Testosterone exerts several effects that may be responsible for these observations. Testosterone seems to limit lipid uptake into fat cells, possibly by its inhibition of the enzyme lipoprotein lipase. Testosterone also stimulates lipolysis partly by increasing the number of beta-adrenergic receptors, which activate the enzyme hormone sensitive lipase, which is responsible for mobilizing stored fat for use as fuel. Additionally, testosterone has been observed to inhibit the differentiation of preadipocytes into mature fat cells, capable of fat storage. Again, while it may be the case that testosterone exerts a myriad of actions on muscle, fat and bone, it would greatly simplify our understanding of testosterone’s effects if one unifying theory of androgen action could be formulated to explain the reciprocal effects of testosterone on muscle and bone on one hand, and fat cells on the other.

THE PLURIPOTENT STEM CELL HYPOTHESIS
In the current paper, the authors hypothesize that androgens control the fate of so called uncommitted pluripotent stem cells of mesodermal origin. Stem cells have the ability to differentiate into several classes of adult cells, depending on which stem cell genes are activated. For example, the body has only a finite supply of satellite cells able to contribute their nuclei to hypertrophying muscle tissue. Stem cells serve as a reservoir for new satellite cells, so the supply of satellite cells is not rapidly exhausted. The same stem cells also possess the ability to follow different lineages, some becoming for instance adipocytes. The authors propose that androgens skew the differentiation of these stem cells away from an adipogenic lineage, towards a myogenic lineage. In other words, under the influence of androgens, such stem cells evolve into muscle tissue instead of fat cells. This neatly explains why androgens both promote muscle growth, and inhibit fat accumulation. The beauty of the theory lies in its simplicity: instead of invoking a myriad of different actions for androgens in their control of body composition, only one mechanism is required.

The figure below summarizes the key elements of this hypothesis. Plus signs indicate effects promoted by testosterone, while minus signs indicate processes inhibited by testosterone. The diagram indicates how testosterone promotes the differentiation of stem cells into muscle tissue (right branch of diagram), while at the same time inhibiting adipogenesis (left branch of diagram).


Fig (1): The effect of testosterone on stem cell differentiation. *Used without permission from the paper by Bhasin et. al (1)

To test their hypothesis, the authors incubated pluripotent stem cells (specifically of the cultured lineage designated C3H10T1/2) with both testosterone and DHT and observed the effects. Commitment of these cells to the myogenic lineage (i.e. their eventual development into muscle tissue) is associated with increased expression of muscle specific transcription factors including MyoD, myogenenin, and myf5, with the eventual expression of MHC II in terminally differentiated cells. All of these factors were observed to increase in a dose dependent manner upon exposure to the androgens.

On the other hand, exposure of the stem cells to androgens decreased the number of adipocytes, downregulating such markers of adipogenic differentiation such as PPAR-gamma. The concentrations of androgens used in the experiment were well within the range found in the plasma of normal adult men. Supraphysiological concentrations of androgens resulted in even greater stimulation of muscle development, explaining at least in part how AAS (anabolic-androgenic steroid) use promotes muscle hypertrophy and leads to fat loss.

References
(1) Bhasin S, Taylor WE, Singh R, Artaza J, Sinha-Hikim I, Jasuja R, Choi H, Gonzalez-Cadavid NF. The mechanisms of androgen effects on body composition: mesenchymal pluripotent cell as the target of androgen action. J Gerontol A Biol Sci Med Sci. 2003 Dec;58(12):M1103-10



Merc.

[BJJ]

This is not a thread, it is an encyclopedia!!!

[Merc.]

This is not a thread, it is an encyclopedia!!!

Lol BJJ .... yea my friend ... lots of GREAT info in this thread.. There is a Lot of reading , but well worth it ...





Merc.

[Merc.]

Pathophysiology of Insulin Resistance and Noninsulin Dependent Diabetes
by: Karl Hoffman



One of modern society's most prevalent health problems is type 2 diabetes, or non-insulin dependent diabetes mellitus, NIDDM. It is estimated that approximately 15 million individuals in the U.S. have NIDDM, 85% of whom are obese. Unlike type I diabetes, or insulin dependent diabetes, which generally manifests itself early in life, type 2 diabetes normally afflicts people later in life, with an age of onset generally later than 30 years. Also unlike with type 1 diabetes, most NIDDM patients retain at least some capacity to secrete insulin until the disease reaches a late stage. Instead, what characterizes the disease is an impaired insulin secretory response to glucose and decreased insulin effectiveness, or insulin resistance. Clinically, insulin resistance is most simply described as a failure of insulin to stimulate normal glucose uptake into its target tissues of muscle and fat. The disease is complex however, and the majority of NIDDM patients initially suffer from hypersecretion of insulin. This is the body's way of compensating for the defective insulin signaling in skeletal muscle that limits glucose uptake. Current thinking is that over a period of many years eventually the hypersecretion of insulin coupled with hyperglycemia from insulin resistance leads to the exhaustion of the pancreatic islet cells which are responsible for insulin secretion, and the eventual failure of the insulin secretory process.

Athletes and bodybuilders have developed a keen interest in insulin resistance, particularly in ways to make muscle more sensitive to the effects of insulin. There are primarily two reasons for this developing interest, other than the obvious concern about reducing the likelihood of developing NIDDM. One belief is that by increasing the muscle’s ability to take up glucose more readily, glycogen stores can be repleted more quickly. The other area of concern is weight management, or more specifically bodyfat reduction. Insulin is a fat storage-promoting hormone. If drugs or supplements can be used that facilitate glucose uptake into skeletal muscle, the reasoning goes, the body will be required to secrete less insulin, and this will reduce fat deposition.

OBESITY AND TYPE 2 DIABETES
The cause and effect relationship between obesity and NIDDM is debated, but there are a number of observations that shed light on how obesity could lead to NIDDM. One is that the connection between obesity and NIDDM is stronger in people exhibiting abdominal subcutaneous and visceral obesity versus peripheral obesity (1). Visceral obesity refers to fat accumulation in the intraperitoneal region around the organs, whereas peripheral obesity refers to fat accumulation in places where most people find it the least attractive: in the hips, love handles, etc. Ironically, the fat we can't see that is stored intrapreritoneally, as well as abdominal subcutaneous fat (the combination is often termed central fat), may be the least healthful.

Central obesity can promote hyperinsulinemia, hyperglycemia, and insulin resistance in a number of ways. For one thing, central adipocytes are larger than peripheral adipocytes, and they have higher levels of enzymes that can break down fats and allow them to enter the bloodstream as free fatty acids (FFA). The portal circulation also connects central adipose tissue to the liver, so the released free fatty acids can act on the liver in several ways. Normally insulin limits production of glucose by the liver, the process known as gluconeogenesis. Free fatty acids block this inhibitory effect of insulin, so the liver puts out inappropriately high levels of glucose, contributing to hyperglycemia. The elevated FFA also slow the metabolic breakdown of insulin by the liver, contributing to hyperinsulinemia (2).

Besides their action on the liver, free fatty acids are also thought to inhibit insulin signaling in muscle tissue (3). This contributes greatly to hyperglycemia, with the pancreas putting out more insulin in an attempt to compensate. Adipocytes also secrete insulin like growth factor (IGF-1). Lest this sounds like a good thing, the IGF-1 acts in a negative feedback way on growth hormone (GH) secretion (4). GH has lipolytic action, stimulating the enzyme hormone sensitive lipase, which mobilizes fat so it can be used as fuel. GH also lowers activity of lipoprotein lipase, the enzyme responsible for the storage of free fatty acids in fat cells. Additionally, GH elevates metabolic rate, perhaps in part by elevating levels of thyroid hormone (T3). To compound matters, hyperinsulinemia has been shown to reduce GH secretion in response to growth hormone releasing hormone (GHRH) in both normal and obese humans (5).

Circulating free fatty acids, elevated in obesity, are also thought to be responsible for the suppression of GH seen in obesity. It is generally accepted that circulating FFA rapidly partition into the plasma membranes of pituitary somatotrophs, the cells which secrete GH. This is believed to alter the function of proteins embedded in the plasma membrane, perturbing intracellular signaling and inhibiting GH release (6).

Another chemical, specifically a cytokine, called tumor necrosis factor alpha, or TNF alpha, also correlates strongly with insulin resistance and may be an early marker for the onset of type 2 diabetes (7). TNF alpha is secreted in relatively large amounts by the adipocytes of obese persons. TNF alpha has been shown to interfere with insulin signaling in muscle and adipose tissue, and may in this way contribute to insulin resistance (8)

Leptin, which acts as a satiety or hunger-suppressing hormone, is released from fat cells and is thought to serve as a signal to reduce food intake. Leptin is expressed more in subcutaneous fat than in visceral fat, so in visceral obesity less leptin may be available to suppress eating, leading to further obesity. Moreover, the hyperinsulinemia of NIDDM is also believed to lead to leptin resistance, abrogating the normal action of leptin to suppress appetite (9).

Besides the aforementioned leptin and TNF-alpha, adipocytes secrete a number of other compounds that may have a bearing on the development of diabetes. The observation that adipocytes are active secretaory organs is part of an emerging picture of fat cells in which they are much more than just passive storage sites for fat. The compounds they secrete have complex actions that work not only on fat cells themselves, but also on other tissues throughout the body.

Resistin, first described in 2001 (10), originally looked very promising as a compound potentially linking obesity with diabetes. Animal studies showed resistin levels are decreased by the anti-diabetic drug rosiglitazone, and increased in diet-induced and genetic forms of obesity. Administration of anti-resistin antibody improves blood sugar and insulin action in mice with diet-induced obesity. Further, treatment of normal mice with recombinant resistin impairs glucose tolerance and insulin action. Insulin-stimulated glucose uptake by adipocytes is enhanced by neutralization of resistin and is reduced by resistin treatment.

Unfortunately, in humans, the relationship between resistin, obesity, and insulin resistance is much less clear. Some research has shown that in human adipose tissue resistin seems to be present at much lower levels than in rodents, and has a considerably different amino acid sequence. There also seems to be a much weaker correlation between resistin levels and obesity.

Adiponectin is another putative candidate linking obesity with diabetes. Like resistin, adiponectin is secreted from adipocytes. Plasma adiponectin concentration is reduced in obese animals and humans and in patients with type 2 diabetes mellitus. Adiponectin appears to stimulate fatty acid oxidation, decreases plasma triglycerides, and improves glucose metabolism by increasing insulin sensitivity. Like resistin, adiponectin levels respond to treatment with antidiabetic drugs. In humans treatment with the drug troglitazone increased serum adiponectin levels by 300% (11).

During prolonged exercise, interleukin-6 (IL-6) is released from skeletal muscle. Current thinking is that IL-6 is a glucose-sensing agent that registers declining levels of muscle glycogen, promotes glucose production by the liver, and improves glucose uptake in skeletal muscle. IL-6 levels increase dramatically when exercise is performed in a glycogen-depleted state. Basal serum IL-6 is higher in type II diabetics. Muscle-derived IL-6 may also work to inhibit the effects of pro-inflammatory cytokines such as TNF, thereby protecting against insulin resistance (12). Confusing matters somewhat is the observation that IL-6 downregulates adiponectin (13), seemingly at odds with IL-6's role as a glucose sensitizing agent. However, one way in which adiponectin regulates blood glucose levels is by inhibiting hepatic glucose production. This is consistent with low levels of adiponectin in NIDDM, which is characterized by hyperglycemia. Since one putative role of IL-6 is to increase glucose availability, it is possible that the downregulation of adiponectin by IL-6 is a mechanism to increase glucose availability to skeletal muscle by increasing hepatic glucose production.

So, in summary, Type 2 diabetes is characterized by reduced sensitivity of muscle tissue to insulin, leading to hyperglycemia, as well as reduced insulin secretion and eventual failure of insulin secretion altogether. Obesity is thought to be a central player in the development of diabetes, leading to derangement in glucose and insulin metabolism via a number of proposed mechanisms.

INSULIN RESISTANCE AND NIDDM: GENETIC ADAPTATION GONE AWRY?
Perhaps the most intriguing theory about how NIDDM originated, and why it has become prevalent in modern society, was one originally formulated by James Neel in 1962 (14).

Neel suggested "that the diabetic genotype is, to employ a somewhat colloquial but expressive term, a "thrifty" genotype, in the sense of being exceptionally efficient in the intake and utilization of food". Neel regarded early humans as generally having lived under conditions of feast or famine. When faced with feasting, individuals who possessed a "quick insulin trigger" to use Neel's term released large amounts of insulin to facilitate more efficient storage of food and minimize urinary glucose loss. We now live in a period where food is relatively abundant (at least in developed countries), people are generally sedentary, and the diet contains large quantities of fat and high glycemic index carbohydrates. Postprandial hyperinsulinemia, once a genetic benefit, now leads to insulin resistance, beta cell failure, and eventual NIDDM. One thing to note here is that according to Neel's theory, muscle insulin resistance is acquired as a result of chronic hyperinsulinemia.

Hypersinsulinemia is believed to interfere with cellular glucose uptake either by a direct effect on the insulin receptor or a disruption of the intracellular insulin-signaling pathway (15).

Cahill (16) and Reaven (17) have looked at the evolution of NIDDM differently. They regard insulin resistance as the fundamental genetic adaptation, rather than an acquired characteristic. Their logic holds that preservation of skeletal muscle mass was the priority for early man. Muscle insulin resistance conserves glucose for use by the central nervous system, decreasing the amount of muscle protein that must be converted into glucose during periods of food deprivation. Hyperinsulinemia may be the direct mechanism for this. Studies have shown that physiologic hyperinsulinemia promotes skeletal muscle anabolism either by stimulating protein synthesis or inhibiting protein breakdown, and stimulating amino acid uptake by muscle (18). Skeletal muscle proteolysis appears to be reduced in NIDDM, consistent with the hypothesis that insulin resistance was selected in order to preserve muscle mass (41). At the same time, resistance to glucose uptake does not extend to amino acid uptake in NIDDM, so contrary to what might be expected, there is no impairment of amino acid transport in cells (42). This is not entirely surprising since glucose and amino acids use different families of transporters to enter cells.

As farming developed and food became relatively more abundant, the development of diabetes was probably held in check by the fact that people remained active, and early crops produced food of a relatively low glycemic index. It was not until the modern era, the abundance of highly processed carbohydrates, and the onset of obesity that the symptoms of insulin resistance and NIDDM began to manifest themselves through the mechanisms described above.

Miller and Colagiuri (19) offer a third explanation for the current high incidence of NIDDM. According to their “carnivore connection” hypothesis, our primate ancestors subsisted on a diet that was rich in carbohydrates, and as a consequence our brains and reproductive systems evolved a requirement for glucose as a source of fuel. The climate changes that accompanied the ice ages beginning about two million years ago forced a relatively high protein diet (from animal meat) on early humans. As we have discussed, hyperglycemia is a hallmark of insulin resistance; insulin resistance would help maintain high blood glucose levels in the face of a shortage of carbohydrates. To quote the authors:

We propose that the low-carbohydrate carnivorous diet would have disadvantaged reproduction in insulin-sensitive individuals and positively selected for individuals with insulin resistance. Natural selection would therefore result in a high proportion of people with genetically determined insulin resistance (19).

An attractive aspect of this theory is that it explains why societies that have switched from a hunter-gatherer lifestyle to an agricultural one relatively recently have higher incidences of type 2 diabetes than do societies with a long history of agriculture, such as northern Europeans. The latter have had longer to readapt genetically to a diet once again high in carbohydrates.

Not surprisingly, there is much evidence both to support and refute each of these three theories. Neel’s ideas have been criticized on the basis of archeological evidence suggesting that famine was actually less common in prehistoric times than it was after the development of agriculture. Cahill and Reaven have been questioned because their theory depends on the unproven assumption that type 2 diabetics retain sensitivity to the antiproteolytic effects of insulin, while at the same time losing sensitivity to the glucose disposing effects of insulin. Miller and Colagiuri have been criticized based on the observation that primates are omnivores more than strict vegetarians, and the diets of our primate ancestors may not have been as carbohydrate rich as the authors assume.

THE THRIFTY PHENOTYPE HYPOTHESIS
In contrast to the above “thrifty genotype” theories, Hales & Barker have proposed that the fetal environment plays a vital role in determining the likelihood of developing NIDDM. Their so-called “thrifty phenotype” theory posits the following chain of events leading up to diabetes in adulthood: (1) the growth of the fetus (and possibly infant) is altered by its nutritional environment (which may include maternal diet-dependent changes in maternal hormones); (2) The fetus adapts to this environment by being nutritionally ‘thrifty’, resulting in decreased fetal growth, islet function and ?-cell mass (low glucose levels require low insulin levels), and other hormonal and metabolic adaptations. In essence, the developing fetus sacrifices organ development, in particular pancreatic development, to ensure adequate energy supplies for the brain to develop (3)—an individual so constituted suffers adverse consequences in adult life if he/she experiences good or supranormal nutrition; (4) both poor insulin secretion and insulin resistance can result from these adaptive processes; (5) the adverse consequences include loss of glucose tolerance and hypertension.

Needless to say, the proponents of the various “thrifty genotype” theories and the “thrifty phenotype” theory have amassed evidence to support their theories and refute those of their rivals. For example, thrifty phenotype proponents argue that since NIDDM generally develops in the post reproductive years, it would not be controlled by natural selection so could not be genetic in origin. The thrifty phenotype theory also can explain why the most westernized societies where obesity is rampant and diet is conducive to the development of NIDDM have rates of the disease far lower than in countries with developing economies: natives of the former countries are much less likely than the latter to suffer from fetal malnourishment. On the other hand this argument can be turned around by genetic theory advocates who note that obesity is increasing along with NIDDM in the West while there is nothing to suggest fetal nutrition is deteriorating.

One attractive feature of the thrifty phenotype theory is that it can be tested in animals. When pregnant rats are fed an isocaloric, protein poor diet their offspring have low birth weight, reduced islet cell mass and vascularization, and an impaired insulin response to glucose. These first generation rats in turn go on to have diabetic pregnancies, and their offspring, exposed to hyperglycemia in utero go on to develop diabetes as adults. The reduced islet cell mass and consequent diminished response of insulin secretion to glucose posited by this theory is also in accord with research showing that reduced insulin secretion is quantitatively more important than insulin resistance in muscle and fat in the development of reduced tolerance to glucose (40).

NUTRIENT SENSING PATHWAYS
THE HEXOSAMINE BIOSYNTHETIC PATHWAY
Consistent with the hypotheses of Neel and others, the body has developed nutrient sensing pathways that may alter metabolism to regulate the storage of energy substrates when nutrient levels are high. One such recently characterized pathway is the hexosamine biosynthetic pathway (HBP). Glucose entering the HBP is ultimately converted to UDP-N-acetylglucosamine, which is believed to play a role in modifying the activity of a number of transcription factors involved in the expression of genes whose transcribed proteins affect energy substrate utilization. HPB activation acts seemingly in a paradoxical way both to induce satiety via leptin secretion and promote fat burning in brown adipose tissue in animals, while at the same time slowing the body's overall metabolic rate. In animal studies this latter effect seems to win out, with a net slowing of the body’s resting metabolic rate (20). The slowing of metabolic rate by HPB activation is accomplished by inhibiting the expression of mitochondrial proteins involved in oxidative phosphorylation and substrate oxidation in skeletal muscle. In other words, energy utilization is slowed in skeletal muscle, allowing excess nutrients to be stored.

Other than possibly contributing to obesity and thereby indirectly promoting insulin resistance, there are other mechanisms where increased glucose flux through the HBP may directly lead to insulin resistance. One fate for dietary glucose is storage as glycogen. The key enzyme responsible for glycogen formation is glycogen synthase. In a normal (non-diabetic) individual, a carbohydrate meal normally results in an increase in insulin secretion. Insulin in turn activates glycogen synthase, promoting storage of a part of the ingested carbohydrates as glycogen. In type 2 diabetes glycogen synthase is resistant to stimulation by insulin. One proposed mechanism for this is increased glucose flux through the HBP. An end product of the HBP, N-acetylglucosamine, has been shown capable of binding to glycogen synthase and significantly reducing its ability to catalyze the conversion of glucose to glycogen (21). So again, we see an adaptation where nutrient intake activates mechanisms for the efficient storage of excess calories as fat. The calories that are not stored as glycogen or used immediately for fuel are shunted into fat storage.

One interesting approach to gain further insight into HBP modulation of substrate storage is to engineer strains of mice that overexpress the rate-limiting enzyme for hexosamine synthesis, glutamine: fructose-6-phosphate amidotransferase (GFA) in tissues including skeletal muscle, liver, fat, and pancreatic beta cells (which recall are responsible for insulin secretion) (22). In these animals, the overactivation of the HPB leads to muscle insulin resistance, excess insulin secretion by the pancreas and excess synthesis of fatty acids by the liver. All these can be considered mechanisms to promote the storage of excess calories, consistent with the thrifty phenotype of Neel, and/or the preservation of muscle mass via hyperinsulinemia ala Cahill and Reaven.

The abovementioned skeletal muscle insulin resistance in transgenic animals overexpressing GFA was shown to result from an inhibition of GLUT4 translocation from intracellular vesicles to the plasma membrane (23). Normally, insulin acts via binding to its receptor to promote migration of the glucose transporter GLUT4 to the cell surface, where it picks up glucose and shuttles it into the cell. This process is inhibited in the presence of an oveactive hexosamine biosynthetic pathway.

We also mentioned above that overactivation of the HBP in transgenic animals leads directly to hyperinsulinemia with subsequent development of peripheral insulin resistance (24). As previously mentioned, hyperinsulinemia is a consistent hallmark of early type 2 diabetes. Arguing from the viewpoint of the "thrifty genotype", hyperinsulinemia may simply be the body's way of compensating for insulin resistance, the fundamental phenotype of the Cahill/Reaven model, where insulin resistance was selected in order to preserve muscle mass during times of low food availability. On the other hand there are data, such as presented in (22) showing that hyperinsulinemia can lead directly to insulin resistance. This model is consistent with Neel's original idea.

In any case, although hyperinsulinemia is characteristic of the early stages if NIDDM, eventually the beta cells fail. This is thought to be a result of the chronic hyperglycemia associated with the disease. After chronic exposure to hyperglycemia, insulin gene transcription and glucose-stimulated insulin secretion are suppressed. Although the exact mechanism is unknown, hyperglycemia has been shown to induce the production of reactive oxygen species at concentrations high enough to be toxic to beta cells (24). Keeping with our theme of the role of the HBP in the development of diabetes, this pathway has been shown to induce beta cell deterioration via the generation of reactive oxygen species (24).

We discussed above how obesity, particularly visceral obesity, contributes to NIDDM through the action of elevated levels of circulating free fatty acids (FFA). High FFA have been implicated in stimulating the HBP (26). Free fatty acids and glucose exert a mutually inhibitory action on the use of one versus the other for fuel. When FFA are readily available as energy substrates, their metabolism leads to high levels of acetyl-CoA within the cellular mitochondria. This acts as a signal to slow the use of glucose as fuel. Specifically, the high concentrations of acetyl-coA inhibit the activity of the enzyme pyruvate dehydrogenase, a rate-limiting enzyme for the complete oxidation of glucose. Additionally, the elevated acetyl-CoA leads to high levels of citrate in the mitochondria. Citrate in turn deactivates another enzyme involved in glycolysis (the use of glucose for fuel), phosphofructokinase. The end result is that dietary glucose is shunted away from use as fuel when FFA levels are high. This expands the pool of glucose available for entry into the HBP, with all the associated deleterious consequences discussed above.

[Merc.]

AMPK SIGNALING
Adenosine 5'-monophosphate-activated protein kinase (AMPK) has been characterized as one of the body’s master metabolic switches. The primary function of AMPK is thought to be as a sensor of ATP status, shutting off ATP consuming processes like lipogenesis and switching on ATP producing processes (like fatty acid oxidation) when ATP levels are low. A kinase is an agent that phosphorylates specific target proteins. AMP kinase is activated by high AMP and low ATP and acts as an energy sensor, regulating such diverse processes as lipolysis and lipogenesis, skeletal muscle fatty acid oxidation, cholesterol synthesis, glucose uptake by adipocytes and muscle tissue, and regulating preproinsulin (insulin precursor) gene expression and insulin secretion in pancreatic islet beta-cells. High concentrations of ATP antagonize the effects of AMP, so it is probably best to say that AMPK responds to the ratio AMP/ATP rather than to absolute values of either.

Since derangements in many of these processes are present in NIDDM, it has been suggested that defects in AMPK signaling could contribute to the disease. If so, AMPK could be a potential target for antidiabetic drugs. For example, in the liver, AMPK activation causes an increase in fatty acid oxidation and inhibition of glucose production. Unrestrained hepatic glucose production is a hallmark of NIDDM. A defect in AMPK signaling in the liver could be responsible at least in part for this. Perhaps even if AMPK signaling were normal in type 2 diabetes, it might be possible to stimulate the signaling pathway to improve symptoms nevertheless.

One agent in particular, AICAR (5-amino-imidazole carboxamide riboside) has been studied extensively for its ability to activate AMPK. It has shown the ability to increase glucose transport in vivo in animals, as well as in vitro in muscle tissue from both normal and diabetic human subjects. In one study exposure of type 2 diabetic skeletal muscle to a combination of insulin and AICAR increased glucose transport and cell-surface GLUT4 content to levels achieved in control subjects.

It has also been observed that AMPK is important in non-insulin mediated glucose uptake by skeletal muscle during contraction. Many of the current treatments for NIDDM focus on improving insulin mediated glucose uptake. But as mentioned, beta cell failure is the end result of NIDDM. If the AMPK regulated non-insulin mediated glucose uptake seen in exercising muscle could be duplicated in resting muscle with drugs, this could represent another potential therapeutic avenue that would not be thwarted by eventual beta cell failure. Here again AICAR or an analog may be promising.

AICAR is a potential drug for the future, but metformin is an antidiabetic drug that is already widely prescribed to treat type 2 diabetes. Its primary mode of action is to reduce hepatic glucose production, thereby improving hyperglycemia, but it is also thought to enhance glucose transport into skeletal muscle as well. Some recent research has shown that metformin may work to improve glucose uptake and glycogen storage in human skeletal muscle by activating AMPK. The increase in AMPK activity was likely due to a change in muscle energy status because ATP and phosphocreatine concentrations were lower after metformin treatment, and we have seen that the trigger to activate AMPK is low ATP status.

PEROXISOME PROLIFERATOR ACTIVATED RECEPTOR GAMMA (PPARg)
PPARg is a member of a family of ligand activated transcription factors. It is found in both white and brown adipose tissue and is involved in the transcriptional activation of an array of adipose tissue specific genes. But perhaps its most important roles are to promote the development of adipose tissue and to inhibit leptin gene expression. In this sense it may be a “thrifty gene”: during times of food shortage, PPARg would tend to promote fat storage; during times of plenty, or in modern times associated with high fat diets, it seems to promote obesity. As we have seen above, obesity may be the most important risk factor for developing NIDDM. So in this way, PPARg may be important to the development of diabetes.

Perhaps no research better illustrates the potential connection between PPARg and the development of insulin resistance than that carried out by Kubota et al (27). These researchers developed heterozygous mice lacking one PPARg gene, but expressing another. (Wild type mice carry two copies of the gene, one inherited from each parent.) The heterozygous mice are designated PPAR+/-; wild type are PPAR+/+. The PPAR+/- strain exhibited reduced adiposity and reduced adipocyte size when placed on a high fat diet, as well as an increase in leptin. They also showed reduced food intake and increased energy expenditure, likely as a result of increased leptin levels. These mice were protected from the development of insulin resistance as well. In contrast, wild type mice with the normal complement of PPARg exhibited adipocyte hypertrophy and obesity on a high fat diet. The increased adiposity would tend to increase systemic levels of TNF-alpha, FFA, and the other adipokines described above, potentially leading to insulin resistance and diabetes.

It is thought that during development and childhood PPARg is responsible primarily for the differentiation of preadipocytes into relatively small adult adipocytes that do not secrete large amounts of the insulin resistance promoting adipokines we discussed above. Later in adulthood, PPARg activation in conjuction with a high fat diet appears to promote adipocyte hypertrophy, obesity, and insulin resistance. This would help explain the apparent paradoxical action of the class of antidiabetic drugs known as the thiazolidinediones, which act to improve insulin sensitivity by acting as PPAR gamma agonists and actually increase the number of fat cells in the body. A large number of relatively small fat cells seem to improve insulin sensitivity, while hypertrophied fat cells have the opposite effect. Interestingly, PPARg antagonists such as the experimental agent SR-202, which have the opposite effect as the thiazolidinediones in that they inhibit adipocyte differentiation, reduce the ability of mice to accumulate fat. This leads to drop in plasma levels of TNFalpha, which as we discussed above is believed to be a key cytokine in promoting insulin resistance (28).

ERGOGENIC AIDS AND INSULIN RESISTANCE
Do any of the agents used as ergogenic aids by athletes and bodybuilders cause insulin resistance? Over the years there have been a number of studies done on how androgens affect glucose sensitivity, some of them conflicting. Virtually all studies show that low testosterone causes insulin resistance. One of the most recent studies done in humans showed that supraphysiological testosterone had no effect on insulin sensitivity but nandrolone actually improved it (29). The doses were 300 mg/week.

In another recent study 600 mg of testosterone enanthate per week had no effect on glucose sensitivity in normal adult men (30). This is the highest dose that I've seen used in any studies. However, when 500 mg of testosterone was administered to obese men, glucose tolerance decreased, while 250 mg increased glucose tolerance (31). Obesity could be having an effect here, as the previously mentioned negative studies were done in non-obese individuals.

Hyperandrogenism is often associated with insulin resistance in women. Whether this is merely an association or an actual causal relationship is debated. In agreement with the latter hypothesis, when normal women were administered methyltestosterone, insulin sensitivity deteriorated (32). This could have widespread implications for women’s health as androgen administration becomes more common in the treatment of menopausal symptoms and sexual dysfunction

In other studies in animals, there appears to be a "window" of testosterone levels around the normal range that optimize insulin sensitivity (33).

So you can see why there is some confusion. We don't know what happens when androgen doses exceed 600 mg/week (It is hardly uncommon for bodybuilders to use doses of anabolic steroids far in excess of 600 mg/wk.), and studies in animals have given results in conflict with those done in men, but in agreement with studies performed in women. And the particular compound used seems to make a difference.

One anabolic agent that unquestionably is capable of causing (temporary) insulin resistance is recombinant human growth hormone, hGH. This side effect of hGH is discussed in detail in the M & M # 14 article on GH use to treat obesity, so I will refer interested readers to that piece for more information.

Hyperthyroidism is associated with elevated plasma glucose, and when rats are given high doses of thyroid hormone both fasting glucose and plasma glucose in response to a glucose load are elevated (34). This hyperglycemia does not appear to be the result of any thyroid hormone induced defect in insulin signaling, but rather decreased insulin secretion in response to glucose. The deceased insulin secretion is most likely a result of thyroid hormone induced apoptosis (programmed cell death) of pancreatic beta cells (35). A second factor contributing to the hyperglycemia seen in hyperthyroidism is increased gluconeogenesis (36). So technically, the elevated plasma glucose seen when supraphysiological doses of T3 or T4 are administered is not due to insulin resistance, but rather to lowered insulin production and increased glucose production from glucogenic substrates.

Caffeine, another common ergogenic aid, decreases insulin sensitivity in healthy subjects. This is most likely due to increased plasma epinephrine and FFA levels associated with caffeine ingestion (37).

Athletes, and in particular bodybuilders, consume a high proportion of protein in their diets. It turns out a protein enriched diet may impair glucose metabolism. Elevated plasma amino acid levels impair glucose uptake, most likely by direct inhibition of muscle glucose transport and/or phosphorylation with a resulting reduction in rates of glycogen synthesis (38).

Finally, in what may be the greatest irony of all for an athlete, eccentric resistance exercise induces a transient whole body insulin resistance. This may result from elevated levels of TNFalpha associated with the muscle damage arising from strenuous eccentric exercise (39). Recall that TNFalpha interferes directly with insulin signaling, in particular with impaired insulin-stimulated IRS-1-associated PI 3-kinase activity. It should be noted however that over the long term both aerobic and resistance exercise training improve glucose sensitivity.

SUMMARY
We have attempted to address what are thought to be the major pathophysiological features of insulin resistance and type 2 diabetes, as well as possible theories about the genetic origin of the disease, and the potential role of the body’s energy sensing mechanisms in the development of diabetes. The treatment has been far from exhaustive; only the better-characterized aspects of the pathophysiology of the disease have been presented. We have emphasized understanding the development of the disease within the context of the different models of NIDDM, which stress the importance of the inheritance of thrifty genes, such as PPARg, which tend to promote fuel storage and lead to insulin resistance under conditions of a modern lifestyle, but were beneficial to our ancestors.


References
(1) Kissebah AH: Intra-abdominal fat: is it a major factor in developing diabetes and coronary artery disease? Diabetes Res Clin Pract Feb;30 Suppl:25-30 1996

(2) Boden G, Chen X, Capulong E, Mozzoli M: Effects of free fatty acids on gluconeogenesis and autoregulation of glucose production in type 2 diabetes.
Diabetes Apr;50(4):810-6 2001

(3) Boden G, Shulman GI:. Free fatty acids in obesity and type 2 diabetes: defining their role in the development of insulin resistance and beta-cell dysfunction.
Eur J Clin Invest Jun;32 Suppl 3:14-23 2002

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[Merc.]

Of Mosquitoes and Men
by: Karl Hoffman


Each year as the tropical storm season worsens, torrential rainfall collects in every depression into which it falls, creating stagnating pools or water which mosquitoes delight in. Malaria, transmitted by mosquitoes of several species, is among the world’s top health problems. 3 million people die from malaria every year.

Before delving into the causes and potential cures, and looking at the role played by testosterone in suppressing the progression of malaria after a person is infected, a primer in immunology might be helpful.

Here is Immunology 101 in a nutshell. The immune system has two "arms of attack": the cell mediated arm and the humoral arm. The cell mediated arm, or cellular immunity, responds to general assaults on the body by sending out immune cells to do things like attack invading organisms, or degrade necrotic tissue, in a non-specific manner. By non-specific it is meant that the immune cells do not recognize the invader as a specific target with which they are familiar. Inflammation is an example of a cell-mediated response. When you get a sliver or strain a muscle the body sends immune cells there to wall off the site, increase blood flow, remove damaged tissue, etc.

Humoral immunity involves B lymphocytes that secrete antibodies that bind to the target and allow immune cells to recognize the target immediately as an invader and launch an attack. When you are vaccinated for something, like smallpox, you are injected with a small inactive piece of the virus. This primes your body to make large numbers of B cell clones that, if ever challenged with smallpox for real, pump out antibodies that mark the virus for destruction by other cells. The big advantage of this system is that it is fast and efficient. The disadvantage is that it is very specific. The cellular response is not as efficient but it works against any invader, not just one for which primed clonal B cells already exist.

Please note that this is a gross simplification since in many diseases humoral and cellular immunity work together to tag and destroy invading organisms.

An important generalization must be made here. Invading organisms that have already entered cells within the body are targets for the cellular immune system. While circulating in the blood stream outside of cells (including blood cells) the pathogens are prone to attack by the humoral arm of the immune system.

There is an emerging model of how the sex steroids regulate the two arms of the immune system. It is thought that testosterone stimulates the humoral arm and suppresses the cellular arm. This paradigm arose from the study of autoimmune diseases which overwhelmingly plague women more than men. The majority of autoimmune diseases involve a cellular immune system gone wild. Since in men testosterone suppresses cellular immunity, men are much less likely to suffer from these diseases, like rheumatoid arthritis. See Figure 1 below.

So when one hears or reads that androgens are anti-inflammatory, this is kind of what it means technically. Some steroids seem to have stronger effects than others. When people say deca (nandrolone) improves joint health because it makes one hold water and lubricates the joints, this is likely not what is really happening. It is an anti-inflammatory because it suppresses cell-mediated immunity, which controls inflammation. It has nothing to do with water.

Why is deca's reputation as an anti-inflammatory better than testosterone's for example? My guess is the minimal aromatization and its progestogenic activity. If you examine Figure 1 below, you will see a couple of interesting things.

First, progesterone, like testosterone (T), stimulates humoral immunity (the TH2 mediated response in the graphic) and suppresses cellular immunity (TH1 response) and the inflammatory response. So progesterone has anti-inflammatory action. Second, estrogen exerts a biphasic effect. At low doses it is pro-inflammatory, stimulating the TH1 arm of the immune system (cellular immunity) and inflammation. Deca then works both as an androgen and a progestin to quell inflammation. Testosterone, by virtue of its aromatization to estrogen is a less effective anti-inflammatory.

Figure 1. Hormonal influences

T helper 1 (TH1) cells secrete pro-inflammatory cytokines and promote cell-mediated immune responses, whereas TH2 cells trigger antibody production. In multiple sclerosis (MS) and rheumatoid arthritis (RA), there are features characteristic of a TH1 immune response directed against autoantigens in the central nervous system and joints, respectively. Pregnancy and systemic lupus erythematosus (SLE) favor a TH2 environment. Sex hormones (such as progesterone) that promote the development of a TH2 response antagonize the emergence of TH1 cells. This may explain why in multiple sclerosis and rheumatoid arthritis disease symptoms improve during pregnancy, whereas in lupus, they do not. (Science, Vol 283, Issue 5406, 1277-1278 , 26 February 1999)

So in summary an overactive TH2 system (due to elevated testosterone) will suppress cell-mediated immunity which is under the control of TH1 cells. Numerous animal studies have shown that the cytokine IFN-gamma is critical in controlling infection. Note from the graphic above that IFN-gamma is secreted by signals emanating from TH1 cells. Since testosterone suppresses TH1 activity, we would expect to see in animals and in patients that low testosterone levels correspond to an increase in parasite clearance, and vice versa. This is exactly what has been observed in a recently published report (1). Patients in that report (1) invariably presented themselves at clinics well after they had been infected. These patients had testosterone levels at about half the normal range. We will return to this point later.

In animal studies, administration of testosterone to Plasmodium chabaudi malaria infected animals resulted in the death of these animals, which are normally capable of clearing the parasite on their own (2).

Returning to our observation made earlier (Patients in (1) invariably presented at clinics well after they had been infected. These patients had testosterone levels at about half the normal range.), it would be extremely unlikely that all these patients coincidentally had low T levels when they were bitten. Rather, it is much more likely that the body itself lowered T levels as a result of the metabolic stress associated with the disease. This concept is bolstered by the fact that in the same patients’ cortisol levels rose under the stress of the illness.

In general, decreased testosterone synthesis and release may be caused by a variety of factors. Activated immune cells called macrophages can secrete nitric oxide (NO), which at high concentrations can inhibit testicular steroidogenesis. Cortisol could act to inhibit testosterone secretion and/or signaling.

This observed drop in testosterone might represent an adaptive host response to prevent immunosuppression by higher testosterone levels. Recall from our discussion above about humoral versus cell mediated immunity that once the malaria-transmitting parasite (some species of Plasmodium) enters cells in the body, cell mediated immunity comes into play to eliminate the invading organisms. In the case of Plasmodium, this pathogen spends virtually its entire time living inside the body’s cells—primarily red blood cells and the liver. It is then logical to anticipate that cellular immunity would be required to rid the body of Plasmodium.

So we see that activated immune cells, macrophages for example, lower testosterone, since this lowered T would result in a milder case of the disease.

There is an emerging paradigm in epidemiology that suggests that diseases start out highly virulent but end up evolving into milder forms lest their victims die, leaving no hosts to colonize. This may very well be the case with the majority of malaria strains. Teleologically speaking, the malaria parasite Plasmodium sp. may be choosing to moderate the effects of its infection to ensure the survival of the host, who can then go on to infect others. Exactly how this process is effected is not well understood.

The same phenomenon of depressed testosterone is seen in other parasitic infections. One of the best studied is toxoplasmosis, caused by the protozoa Toxoplasma. In the case of this infection, researchers determined that the pro-inflammatory cytokine IL-1 beta, released by immune cells known as macrophages during the early phase of the disease, was capable of suppressing GnRH, LH and testosterone (3). This is strikingly similar to malaria, where the cytokines produced during the early acute phase of the disease lower testosterone, in effect putting the brakes on inflammatory damage to organs such as the liver.

As a third and final example of how sex determines the outcome of certain diseases, we will look briefly at leishmaniasis, an endemic tropical disease caused by Leishmania sp., an obligatory intracellular parasite. Men are more prone to suffer this infection, likely due to the discussion above, where we described how testosterone suppresses the arm of the immune system that attacks intracellular parasites. In an interesting experiment with animals, Travi et al (4) reported the following:

Effect of exogenous administration of hormones The administration of the opposing sex hormones to male and female hamsters for 20 days prior to and throughout the course of L. (V.) panamensis infection altered the course of disease evolution. Female hamsters treated with testosterone developed larger cutaneous lesions than untreated females at all time points and in fact developed larger lesions than male animals. This difference in lesion evolution was observed from the 30th to the 90th day, when the experiment was terminated. Androgens had a more pronounced effect on females than estrogens did on males. Male animals treated with estrogens showed a tendency to develop smaller lesions than their untreated controls, but this was not statistically significant” See Figure 2 below.
FIG. 2. Effects of the administration of opposing sex hormones to male and female hamsters infected with L. (V.) panamensis.
Recently weaned male hamsters (n = 8) received a subcutaneous implant (Compudose 200) that released approximately 240 µg of 17 ß-estradiol/day throughout the study period. Recently weaned female hamsters (n = 8) received intramuscular injections of 1 mg of testosterone enanthate (Testoviron-Depot) per hamster twice per week until 3 months p.i. Twenty days after initiation of the sex hormone treatment, the hamsters were inoculated with 106 L. (V.) panamensis stationary-phase promastigotes as described in Materials and Methods. Lesion evolution was determined as described in the legend to Fig. 1, and the results are expressed as the mean (± standard error of the mean) of the lesion size. Female animals treated with testosterone had significantly larger lesions than untreated females (P < 0.05) at all time points. There was no significant difference in lesion size between the estradiol-treated and untreated male animals.

On a rather sad finishing note, one can see from Figure 1 that the high levels of estrogen during pregnancy favor a TH2 environment, suppressing cellular immunity. Malaria is a serious problem for pregnant women and their fetuses in tropical developing countries. It is estimated that malaria-induced low birth weight (LBW) may kill nearly 400,000 African infants each year (5).

The potential upside is that women become resistant to pregnancy malaria over successive pregnancies as they acquire antibodies that recognize placental parasites, suggesting that the development of a vaccine is feasible. It may be possible to use recombinant technology to develop such a vaccine modeled after the natural antimalarial antibodies.

Appendix A. Life cycle of the malaria parasite Plasmodium sp.
References
(1) Muehlenbein MP, Alger J, Cogswell F, James M, Krogstad D. THE REPRODUCTIVE ENDOCRINE RESPONSE TO PLASMODIUM VIVAX INFECTION IN HONDURANS. Am J Trop Med Hyg. 2005 Jul;73(1):178-187
(2) Mossmann H, Benten WP, Galanos C, Freudenberg M, Kuhn-Velten WN, Reinauer H, Wunderlich F. Dietary testosterone suppresses protective responsiveness to Plasmodium chabaudi malaria Life Sci. 1997;60(11):839-48

(3) Oktenli C, Doganci L, Ozgurtas T, Araz RE, Tanyuksel M, Musabak U, Sanisoglu SY, Yesilova Z, Erbil MK, Inal A. Transient hypogonadotrophic hypogonadism in males with acute toxoplasmosis: suppressive effect of interleukin-1 beta on the secretion of GnRH. Hum Reprod. 2004 Apr;19(4):859-66

(4) Travi BL, Osorio Y, Melby PC, Chandrasekar B, Arteaga L, Saravia NG. Gender is a major determinant of the clinical evolution and immune response in hamsters infected with Leishmania spp. Infect Immun. 2002 May;70(5):2288-96

(5) Duffy PE. Maternal immunization and malaria in pregnancy. Vaccine. 2003 Jul 28;21(24):3358-61

[Merc.]

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