Cell study: more arachidonic acid, more muscle growth

[Turkish Juicer]

Cell study: more arachidonic acid, more muscle growth

William Llewellyn was right. About ten years ago Llewellyn came up with the theory that the fatty acid arachidonic acid might just be a key factor in muscle growth. Llewellyn launched a supplement containing arachidonic acid on the market, and financed a study which, although it didn't discover any anabolic effects, did show that the substance enhances performance levels. But arachidonic acid probably does have an anabolic effect too, according to researchers at the University of Auckland in New Zealand.

The body converts the n-6 fatty acid linoleic acid into gamma-linolenic acid, or GLA, which is then converted into arachidonic acid. Arachidonic acid in turn is a precursor of hormone-like signal substances such as PGE2 and PGF2a. The diagram below shows how this all works.



Arachidonic acid has a bad reputation. The fatty acid, or rather, its metabolites are believed to whip up inflammation, and are therefore regarded as being responsible to the negative effects of a diet containing too much n-6 fatty acids. Llewellyn has always maintained that the story isn't as simple as that. Some metabolites of arachidonic acid actually inhibit inflammation. [vpxsports.com January 4, 2013]

Seven years ago Llewellyn's company Molecular Nutrition sponsored a study in which young strength athletes were given arachidonic acid. Their anaerobic capacity increased dramatically, although they didn't become more muscular. In January 2013 a New Zealand PhD researcher James Markworth published the results of an in-vitro study which showed that Llewellyn's theory does seem to hold water.

Markworth exposed C2C12 muscle cells to various concentrations of arachidonic acid. The higher the concentration, the thicker the muscle fibres were that the cells forms and the more muscle protein they manufactured.





Exposure to arachidonic acid inhibited the increase in the total number of muscle fibres, the figure above shows. Click on it for a larger version. Dark bars = muscle cells that were exposed to arachidonic acid.

On the right above you can see that arachidonic acid caused an increase in the number of larger muscle fibres – ones with more than five cell nuclei. That means that arachidonic acid causes muscle fibres to become bigger.

The researchers repeated their experiments with compounds that inhibit the COX-2 enzyme. COX converts arachidonic acid into PGE2. By blocking this almost all of the anabolic effect of arachidonic acid was taken away. Experiments with a non-metabolisable version of arachidonic acid produced no results either.

"The findings of the present study show that an increased availability of free arachidonic acid and subsequent metabolism by the COX-2 pathway have a net stimulatory effect on in vitro skeletal muscle cell growth", the researchers conclude.

Source: Am J Physiol Cell Physiol. 2013 Jan 1;304(1):C56-67.

[Doom44]

Awesome post

[kelkel]

Any more info on it Turk? Lasting effects?

[Turkish Juicer]

Any more info on it Turk? Lasting effects?

I will try to look into it and see if I find any more information on it.

[tigerspawn]

Thanks Turkish

[Turkish Juicer]

Arachidonic Acid and Site-Specific Muscle Growth

Although building muscle without exercise may seem like the ideal endpoint of all anabolic research, this is a feat that just hasn’t been realized yet. There are a number of effective drugs and supplements for augmenting muscle growth, but resistance training is in inseparable part to the success of them all. This fact may be a “given”, but have you ever wondered why that is? Why potent anabolic hormones simply do not build large amounts of muscle by themselves? Although the complete picture is not yet fully understood to science, the answer lies somewhere in the way the muscles respond to exercise; or more specifically, the changes in local chemistry that prime them for the actions of anabolic hormones. It is all about tissue specificity, and the body indeed is adept at directing anabolism only toward those muscles that require it. In this article we will take an up-close look at this tissue-specific response phenomenon, as well as examine the nutrient arachidonic acid, which seems to sit at the very center of it.

Exercise and Receptors

A good way to start off this discussion is to first look at the two hormones universally identified as those most integral to the building of muscle tissue, testosterone (representing the androgen class) and IGF-1 (insulin -like growth factor 1; the anabolic end product of GH). Elevation of either hormone, again, really only works well at promoting growth when training is also involved. Given the basic interaction between hormones and receptors, Bamman et al. set out in 2000 to see if receptor modification may play a key role in their tissue specificity[i]. The group of researchers open up their study noting, “… transient elevations in serum anabolic factors such as growth hormones (GH) and testosterone have been documented after a bout of conventional resistance exercise… However, because the hypertrophic response is specific to the loaded muscle(s), activation by systemic hormone would require load-mediated modulation of the hormone’s efficacy in the exercised muscle. Load-mediated modulation of receptor expression or binding affinity in the muscle might explain localization of the growth response…”

Bamman and company proceeded with the first in-depth examination of how androgen and IGF-1 receptor expression is changed in local tissues following exercise. The investigation involved 10 healthy male (7) and female (3) subjects, who were subject to bouts of eccentric and concentric resistance exercise and examined periodically for 48 hours after. Measures were taken of blood hormone levels, as well as local hormone receptor concentrations. The paper goes on to demonstrate some very interesting results. First, testosterone levels actually tended to decline in this study post-exercise. This was attributed to general as well as diurnal variation, the latter of which refers to the way testosterone levels fluctuate throughout the course of the day. IGF-1 levels did increase 48 hours after training, but only slightly (from 349 ng/dl to 416 ng/dl), and only with concentric training. Receptor concentrations, however, were a much different story. Both eccentric and concentric exercise produced significant increases in androgen and IGF-1 receptor concentrations. IGF-1 binding protein 4 (IGFBP-4) was also suppressed, which additionally would increase free IGF-1 and its biological activity. The results demonstrate that sensitivity to both testosterone and IGF-1 is markedly increased in local muscles following training, and present one way in which the body can “direct” their anabolic actions.

Figure 1. The effect of eccentric resistance exercise on androgen receptor, IGF-1 receptor, and IGF-1 binding protein concentrations.

AA – Root Trigger of Hypertrophy

So we have one proposed mechanism contributing to the localization of the growth response: receptor modulation. But how exactly does this modulation come about, and are other things involved too? Perhaps we should go back to the root. We need to see what is going on in your muscles during exercise, and how physiological changes during this window will shape growth in the hours and days to follow. It all begins with the damage caused to muscle fibers during resistance exercise. The coinciding cell disruption causes the release of phospholipase A2, which is responsible for liberating fatty acids from cell membranes. The main target of phospholipase A2 here is the polyunsaturated fatty acid arachidonic acid (AA), which is the most biologically active fatty acid towards skeletal muscle growth. Via the conversion of AA to prostaglandins (PGE2 and PGF2alpha), AA release will regulate protein synthesis and breakdown rates in local tissues, shifting them in favor of growth[i] [ii] [iii]. It is the first trigger in a very long and complex anabolic cascade.

Muscle growth involves more than just protein synthesis, however. For true hypertrophy to occur, immature satellite (stem) cells called myoblasts must be able to differentiate and fuse with mature muscle fibers. The process involves several steps. First, myoblasts are formed through cell proliferation, and stay stored on the outside of the cell. These mononucleated myoblasts will differentiate and fuse, forming a multinucleated immature cell (with several nuclei). This is usually referred to as a nascent myotube, and is the biological target of most molecules that effect muscle cell fusion. Immature myotubes then fuse with mature muscle cells, increasing myonuclear number and cell size. The myotubes will then continue to increase in size through normal protein accretion (protein synthesis). Through this, the growth and strengthening of the muscle fiber is achieved. Studies with PGF2alpha have shown this prostaglandin to strongly support hypertrophy at the second level of fusion, affecting the already formed nascent myotube by increasing its number of nuclei[iv]. This is a sufficient mechanism to support hypertrophy, and we must remember it is but one, and isolated to one (albeit a primary) metabolite of arachidonic acid. There are yet still other things at play.

AA and the AR

Although the link between arachidonic acid and muscle growth is well established, if, and how much, the AA cascade is directly involved with another specific aspect of the anabolic response, androgen receptor proliferation and local testosterone sensitivity in the muscles, has not (yet) been. However, there is support for the suspicion that there is a link, beyond the mere coincidence that both AA release and AR density increase during productive workouts. It lies in the examination of androgen receptor concentrations in other tissues. The first is a study on the drug flufenamic acid, which blocks inflammation by preventing arachidonic acid from converting to prostaglandins[v]. This drug was shown to markedly suppressed AR density and transcription in prostate cancer cells, which points to a directly related mechanism. However, because of speculation concerning other modes of action, a definitive conclusion was not made. Not long after, another study of interest was published, however. This one looked at the effects of essential fatty acids on gene expression in mice liver[vi]. After a diet rich in AA, androgen receptor concentrations were shown to increase more than 2.5 fold over control values. Yet another investigation took place about a year later. It was a follow up to the first investigation on flufenamic acid, which used more specific drugs[vii]. Here, the cyclooxygenase inhibitors celecoxib and nimesulide also suppressed androgen receptor levels in prostate cancer cells. Again, none of these studies may have been in skeletal muscle, but they do illustrate a strong effect in other tissues that may (likely) carry over here.

AA, IGF-1, and PI3K

Those familiar with the field of growth hormone research may recognize a key study published back in 1996, which looked at PI3K, and how it related to the protein synthesizing effects of both insulin and IGF-1[ix]. During this investigation, incubation of epitrochlearis muscle explants from mice with IGF-1 significantly increased both inward glucose transport and protein synthesis rates. This was an expected result given the hormone used, of course. But when the PI3K inhibitor wortmannin was added in with the IGF-1, both of these increases were quickly and effectively blunted. The researchers put it very succinctly when commenting “Our results clearly demonstrated that stimulation of PI3 kinase was indispensable for the stimulatory effect of both insulin and IGF-1 on muscle protein synthesis.”

Exactly how AA affects PI3K and the IGF-1 signaling cascade is not entirely known. This does appear to be one of the most complex actions of arachidonic acid, and may involve the interaction of a number of its end products. It may include not only the cyclooxygenase metabolites of AA (prostaglandins), but lipoxygenase metabolite(s) as well, and perhaps involves both modulation of IGF-1 receptor density and signaling ability, through direct and indirect means[x] [xi] [xii] [xiii] [xiv]. Definitive studies in skeletal muscle tissue have not been undertaken, so no exact conclusions can be drawn. What is clear, however, is that arachidonic acid does affect this system, and likewise, should be intensifying the growth producing signals of not only androgens, but Insulin-Like Growth Factor 1 as well (endogenous or exogenous in origin). An in-depth examination of the IGF-1/PI3 kinase system would entail an article all of its own.

Response Modulation

OK, so we know that arachidonic acid sits at the center of the core anabolic/hypertrophic response. Now, here comes the bad news. Levels of this nutrient can vary depending on a number of factors, and one of them happens to be exercise itself. A key effect of regular training is to diminish the responsiveness of the arachidonic acid cascade! This effect occurs via the gradual utilization of membrane-bound arachidonic acid, and its replacement with other fatty acids. As the arachidonic acid content is diminished, the body simply has less to use as substrate, and becomes less able to produce prostaglandins and other active metabolites. When this happens we seem to find it more difficult to trigger growth. It is also one of the reasons we are so much more receptive to training when we are first introduced to lifting, or when we hit the weights after a long period of time off.

Both ends of this equation have been well documented, but independently of one another. To begin with, studies with 19 sedentary middle-aged men in Sweden in 1998 showed that moderate exercise for as little as 6 weeks would measurably lower muscle AA content (it was largely replaced with oleic acid in this study)[xv]. On the other end of this phenomenon, we find a paper that was published about a decade earlier, which followed a group of athletes for one full year[xvi]. This study looked at this response using PGE2 as a marker, and reported a gradual and steady suppression of the prostaglandin system with regular exercise. The longer the subjects were on a regular training schedule, the harder it was for them to generate the same strong prostaglandin response (and logically other products of AA metabolism). Thankfully, dietary intake of AA can be modified (increased) to reverse (even exploit) this phenomenon. As you may be aware, I have been a strong advocate of AA loading for muscle building phases or training (X-Factor™), which is intended to increase AA levels and offer a strong pro-anabolic effect. It is a practice I am happy to say has been very effective, however I will save further product promotions for another time.

Summing It Up

We’ve merely touched on what is an extremely complex system in this article, and perhaps in doing so have raised more questions that we have answered. There is simply not enough time, space, or information to piece together a comprehensive look at the entire arachidonic acid/anabolic response cascade in one sitting. However, we can walk away with somewhat of a fundamental understanding of what is going on. Some main points of interest are summarized below for your review. As mentioned, this is an extremely large system to investigate, which leaves open room for a number of follow up articles with a different focus each. I’ll try my best to make sure more information is presented here, at Avant. Stay Tuned!

Key Points

Arachidonic acid release/metabolism is the core regulator for muscle growth.
AA metabolism increases protein synthesis rates, and also stimulates muscle hypertrophy.
AA metabolism is tied to androgen receptor proliferation and testosterone sensitivity in certain tissues. This is speculated to include muscle.
AA increases IGF-1 signaling via PI3K, and perhaps other mechanisms as well (speculated to include receptor proliferation in muscle).
AA levels in skeletal muscle are decreased with regular resistance training.
AA can be taken as a supplement to increase its stores in muscle tissue, in order to maintain maximum workout productivity and increase muscle hypertrophy.

References

[1] Mechanical load increases muscle IGF-1 and androgen receptor mRNA concentrations in humans. Bamman MM, Shipp JR, Jiang J. gower BA, Hunter GR, Goodman A, McLafferty CL Jr, Urban RJ. AM J Physiol Endogrinol Metab. 2001 Mar; 280(3):E383-90.

[1] Stretch-induced prostaglandins and protein turnover in cultural skeletal muscle. Vandenburgh HH, Hatfaludy S, Sohar I, Shansky J. Am J Physiol. 1990 Aug;259(2 Pt 1): C232-40.

[1] Effect of ibuprofen and acetaminophen on postexercise muscle protein synthesis. Trappe TA, White F, Lambert CP, Cesar D, Hellerstein M, Evans WJ. Am J Physiol Endocrinol Metab. 2002 Mar;282(3):E551-6.

[1] Skeletal muscle PGF(2)(alpha) and PGE(2) in response to eccentric resistance exercise: influence of ibuprofen acetaminophen. Trappe TA, Fluckey JD, White F, Lambert CP, Evans WJ. J Clin Endocrinol Metab. 2001 Oct;86(10):5067-70.

[1] Prostaglandin F2(alpha)stimulates growth of skeletal muscle cells via an NFATC2-dependent pathway. Horsley V, Pavlath GK. J Cell Biol. 2003 Apr 14; 161(1):111-8.

[1] A nonsteroidal anti-inflammatory drug, flufenamic acid, inhibits the expression of the androgen receptor in LNCaP cells. Zhu W, Smith A, Young CY. Endocrinology. 1999 Nov;140(11):5451-4.

[1] Dietary effects of arachidonic-rich fungal oil and fish oil on murine hepatic and hippocampal gene expression. Berger A, Mutch DM, Bruce German J, Roberts MA. Lipids Health Dis. 2002 Oct 21;1(1):2.

[1] The cyclooxygenase 2-specific nonsteroidal anti-inflammatory drugs celecoxib and nimesulide inhibit androgen receptor activity via induction of c-Jun in prostate cancer cells. Pan Y, Zhang JS, Gazi MH, Young CY. Cancer Epidemiol Biomarkers Prev. 2003 Aug;12(8):769-74.

[1] Arachidonic acid activation of translation initiation signaling in vascular smooth muscle cells. Neeli I, Yellaturu CR, Rao GN. Biochem Biophys Res Commun. 2003 Oct 2;309(4):755-61.

[1] Phosphatidylinositol 3-kinase and p70 s6 kinase participate in the regulation of protein turnover in skeletal muscle by insulin and insulin-like growth factor I. Dardevet d, Sornet C, Vary T, Grizard J. Endocrinology. 1996 Oct;137(10):4087-94

[1] Insulin-like growth factor-I antagonizes the antiproliferative effects of cyclooxygenase-2 inhibitors on BxPC-3 pancreatic cancer cells. Levitt RJ, Pollak M. Cancer Res. 2002 Dec 15;62(24):7372-6

[1] COX-2 Regulates the insulin-like growth factor I-induced potentiation of Zn(2+)-toxicity in primary cortical culture. Im JY, Kim D, Lee KW, Kim JB, Lee JK, Kim DS, Lee Yi, Ha KS, Joe CO, Han PL. Mol Pharmacol. 2004 Sep;66(3):368-76.

[1] Cyclooxygenase-2 modulates the insulin-like growth factor axis in non-small-cell lung cancer. Pold M, Krysan K. Pold a, Dohadwala M, Heuze-Vourc’h N, Mao JT, Riedl KL, Sharma S, Dubinett SM. Cancer Res. 2004 Sep 15;64(18):6549-55.

[1] Insulin and insulin-like growth factor-I responsiveness and signaling mechanisms in C2C12 satellite cells: effect of differentiation and fusion. Palmer RM, Thompson MG, Knott RM, Campbell GP, Thom A, Morrison KS. Biochim Biophys Acta. 1997 Feb 4;1355(2):167-76

[1] Five-lipoxygenase inhibitors can mediate apoptosis in human breast cancer cell lines through complex eicosanoid interactions. Avis I, Hong SH, Martinez A, Moody T, Choi YH, Trepel J, Das R, Jett M, Mulshin JL. FASEB J. 2001 Sep;15(11):2007-9. Epub 2001 Jul 9.

[1] Andersson, A, Sjodin A, Olsson R, Vessby B. Effects of physical exercise on phospholipid fatty acid composition in skeletal muscle. Am J Physiol. 1998 Mar;274(3 Pt 1):E432-8.

[1] Effects of exercise on parameters of blood coagulation, platelet function and the prostaglandin system. Sinzinger H, Virgolini I. Sports Med, 1988 Oct;6(4):238-45. Review.

Author: William Llewellyn

[Turkish Juicer]

Am J Physiol Cell Physiol. 2013 Jan 1;304(1):C56-67. doi: 10.1152/ajpcell.00038.2012. Epub 2012 Oct 17.

Arachidonic acid supplementation enhances in vitro skeletal muscle cell growth via a COX-2-dependent pathway.

Markworth JF, Cameron-Smith D.

Source: School of Exercise and Nutrition Science, Deakin University, Melbourne, Australia.

Abstract

Arachidonic acid (AA) is the metabolic precursor to a diverse range of downstream bioactive lipid mediators. A positive or negative influence of individual eicosanoid species [e.g., prostaglandins (PGs), leukotrienes, and hydroxyeicosatetraenoic acids] has been implicated in skeletal muscle cell growth and development. The collective role of AA-derived metabolites in physiological states of skeletal muscle growth/atrophy remains unclear. The present study aimed to determine the direct effect of free AA supplementation and subsequent eicosanoid biosynthesis on skeletal myocyte growth in vitro. C2C12 (mouse) skeletal myocytes induced to differentiate with supplemental AA exhibited dose-dependent increases in the size, myonuclear content, and protein accretion of developing myotubes, independent of changes in cell density or the rate/extent of myogenic differentiation. Nonselective (indomethacin) or cyclooxygenase 2 (COX-2)-selective (NS-398) nonsteroidal anti-inflammatory drugs blunted basal myogenesis, an effect that was amplified in the presence of supplemental free AA substrate. The stimulatory effects of AA persisted in preexisting myotubes via a COX-2-dependent (NS-389-sensitive) pathway, specifically implying dependency on downstream PG biosynthesis. AA-stimulated growth was associated with markedly increased secretion of PGF(2α) and PGE(2); however, incubation of myocytes with PG-rich conditioned medium failed to mimic the effects of direct AA supplementation. In vitro AA supplementation stimulates PG release and skeletal muscle cell hypertrophy via a COX-2-dependent pathway.

PMID:23076795 [PubMed - indexed for MEDLINE]

[austinite]

Damnit TJ. This is like 8 days worth of research for me now.

[Ashop]

I would like to see some real world results from bodybuilders/athletes in the gym.
If someone gives this a fair run,,,keep us all posted.

[Armykid93]

I'm really curious about this to. Hopefully someone knows where to get it in a pure bulk form cuz as of yet I've only found expensive supps that have it in it.

[M302_Imola]

I would like to see some real world results from bodybuilders/athletes in the gym.
If someone gives this a fair run,,,keep us all posted.

same here...to many supps sound ideal on paper but yet lead to minimal/no real world results.

Nice post TJ!