Interesting study on test and lipids, c-reactive protein, insulin and more

Sorry it is so lengthy, but I wanted everyone to see all of the study. Of course some of the figures couldn't format over on the change (if you want, you can pm me and I can figure out a way to let you see the original. It is on MDConsult, so if anone has a registration or membership, you can view it there)
I just wanted to point a few things out. First off, this a pretty valid study. It is a controlled, double blinded, randomized trial which is pretty much gold standard. There was appropriate time on AAS (20weeks) and a good followup (16 weeks). Pretty good sample size (of the 61, only 53 completed entire study). They also used a nice dosage range in the 5 groups: 25,50,125,300 and 600 mg of TestE/week. All of the participants were relatively young and healthy. Diets and exercise were kept pretty much constant and consistent between groups. And all men were chemically castrated before Test administration with a GnRH agonist, so endogenous Test can't factor into the equation and they are able to get a clean equal start among men.
Anyways, the good part is what the study set out to accomplish. The main outcome was to measure the baseline change in plasma lipids, apolipoproteins, insulin sensitivity, and C-reactive protein. All of which are indicated to play a role in coronary heart disease, and cardiovascular related morbidities and mortalities. If you can get through the study, I think you will find it pretty interesting. Only the 600mg/wk group showed any difference in significant clinical outcomes.
So try to read through it, and decide for yourself the usefulness of it. Remember though, this is just ONE study of 53 relatively normal, healthy men. But it is interesting nevertheless. If you have any questions, post em and lets discuss them.


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Endocrine Care Of Special Interest to the Practice of Endocrinology
The Effects of Varying Doses of T on Insulin Sensitivity, Plasma Lipids, Apolipoproteins, and C-Reactive Protein in Healthy Young Men

ATAM B. SINGH 1
STANLEY HSIA 1
PETAR ALAUPOVIC 2
INDRANI SINHA-HIKIM 1
LINDA WOODHOUSE 1
THOMAS A. BUCHANAN 3
RUOQUING SHEN 1
RACHELLE BROSS 3
NANCY BERMAN 4
SHALENDER BHASIN 1



Abbreviations:

AIRG
Acute insulin response to glucoseCRP
Creactive proteinDI
glucose disposition indexHDL-C
high density lipoprotein cholesterolLDL-C
low density lipoprotein cholesterolSG
glucose effectiveness indexSI
insulin sensitivity indexVLDL-C
very low density lipoprotein cholesterol

The effects of T supplementation on insulin sensitivity, inflammation-sensitive markers, and apolipoproteins remain poorly understood. We do not know whether T's effects on plasma lipids, apolipoproteins, and insulin sensitivity are dose dependent, or whether significant anabolic effects can be achieved at T doses that do not adversely affect these cardiovascular risk factors. To determine the effects of different doses of T, 61 eugonadal men, 18–35 yr of age, were randomly assigned to 1 of 5 groups to receive monthly injections of long-acting GnRH agonist to suppress endogenous T secretion and weekly injections of 25, 50, 125, 300, or 600 mg T enanthate for 20 wk. Dietary energy and protein intakes were standardized. Combined administration of GnRH agonist and graded doses of T enanthate resulted in nadir T concentrations of 253, 306, 542, 1345, and 2370 ng/dl at the 25-, 50-, 125-, 300-, and 600-mg doses, respectively. Plasma high density lipoprotein cholesterol and apolipoprotein A–I concentrations were inversely correlated with total and free T concentrations and were significantly decreased only in the 600 mg/wk group (change in high density lipoprotein cholesterol: −8 ± 2 mg/dl; P = 0.0005; change in apolipoprotein A–I: −16 ± 2 mg/dl; P = 0.0001). Serum total cholesterol, low density lipoprotein cholesterol, very low density lipoprotein cholesterol, triglycerides, apolipoprotein B, and apolipoprotein C-III were not significantly correlated with T dose or concentration. There was no significant change in total cholesterol, low density lipoprotein cholesterol, very low density lipoprotein cholesterol, triglycerides, apolipoprotein B, or apolipoprotein C-III levels at any dose. The insulin sensitivity index, glucose effectiveness, and acute insulin response to glucose, derived from the insulin-modified, frequently sampled, iv glucose tolerance test using the Bergman minimal model, did not change significantly at any dose. Circulating levels of C-reactive protein were not correlated with T concentrations and did not change with treatment in any group. Significant increments in fat-free mass, muscle size, and strength were observed at doses that did not affect cardiovascular risk factors.

Over a wide range of doses, including those associated with significant gains in fat-free mass and muscle size, T had no adverse effect on insulin sensitivity, plasma lipids, apolipoproteins, or C-reactive protein. Only the highest dose of T (600 mg/wk) was associated with a reduction in plasma high density lipoprotein cholesterol and apolipoprotein A–I. Long-term studies are needed to determine whether T supplementation of older men with low T levels affects atherosclerosis progression. ( J Clin Endocrinol Metab 87: 136–143, 2002)


THERE IS CONSIDERABLE interest in exploring the use of T supplementation to prevent or reverse the loss of muscle mass and function, preserve bone mass, and improve health-related quality of life in older men with low T levels [1] [2] [3] [4] [5] [6] and in those with chronic diseases [7] . However, the risks and benefits of T supplementation remain poorly understood. Although T supplementation increases fat-free mass in healthy hypogonadal men [8] [9] [10] [11] [12] , older men with low T levels [2] [3] [4] [5] and human immunodeficiency virusinfected men with weight loss [7] , we do not know whether these beneficial effects can be achieved without adverse effects on the risk of cardiovascular disease.

Previous studies suggest that different androgen-dependent processes have different T dose requirements [13] [14] [15] [16] . Thus, sexual function can be maintained at T concentrations at the lower end of the normal male range [13] [14] [15] [16] ; in contrast, the effects of T on muscle mass and strength vary depending on T dose and circulating T concentrations [16] . It is not known whether clinically meaningful gains in muscle mass and strength can be achieved at T concentrations that do not adversely affect cardiovascular risk. Therefore, the objective of this study was to determine the dose-dependent effects of T on several risk factors of atherosclerotic heart disease in healthy young men. We wanted to determine the range of T doses and concentrations through which T could be safely administered without adversely affecting important cardiovascular risk factors, including plasma lipids, apolipoproteins, insulin sensitivity, and C-reactive protein (CRP).

The effects of T on insulin sensitivity remain controversial; some studies have suggested that physiological T replacement improves insulin sensitivity in middle-aged men with low T levels [17] [18] [19] . In contrast, administration of supra-physiological doses of T to castrated male rats [20] and of anabolic steroids to women [21] and power lifters [22] has
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been shown to induce insulin resistance, suggesting that the relationship between circulating T concentrations and insulin sensitivity is complex. We considered the hypothesis that the relationship between serum T concentrations and insulin sensitivity is curvilinear, such that both subphysiological and supraphysiological T concentrations might adversely affect insulin sensitivity.

The inflammation-sensitive marker, CRP, has emerged as an important marker of cardiovascular risk that predicts the risk of heart disease independent of plasma lipids [23] . The effects of T on CRP have not been previously studied and were also the subject of this investigation.

In this study we investigated the relationship of T dose and concentrations to plasma levels of lipids, apolipoprotein, and CRP and measures of insulin sensitivity in healthy young men. Testicular T production was suppressed in healthy men by the administration of a long-acting GnRH agonist. We then created different circulating T concentrations by administering graded doses of T enanthate. In men in whom endogenous T production has been suppressed by GnRH agonist administration, circulating T concentrations are proportional to the administered T dose [16] . This approach minimizes the heterogeneity in serum T levels that results from varying degrees of suppression of endogenous T production by administration of exogenous androgen alone.


Subjects and Methods
Study design

This was a double blind, randomized study that consisted of a 4-wk control period, a 20-wk treatment period, and a 16-wk recovery period. The details of the study design have been previously described [16] .
Participants

Participants were healthy young men, 18–35 yr of age, with normal serum T levels. These men were within 90–120% of their ideal body weight. We excluded men with any known illness or those who had used androgenic steroids in the preceding year or were planning to participate in competitive sports events in the subsequent year. Each participant provided written, informed consent. The institutional review boards of Charles R. Drew University of Medicine and Science (Los Angeles, CA) and Harbor-University of California-Los Angeles Research and Education Institute (Torrance, CA) approved the study protocol.
Randomization

Sixty-one eligible men were randomly assigned to 1 of 5 treatment groups. All participants received monthly injections of a long-acting GnRH agonist (Decapeptyl, DebioPharm, R.P., Martigny, Switzerland) to suppress endogenous T production, starting on treatment d 1. In addition, group 1 received 25 mg T enanthate by im injection weekly, group 2 received 50 mg T enanthate weekly, group 3 received 125 mg T enanthate weekly, group 4 received 300 mg T enanthate weekly, and group 5 received 600 mg T enanthate weekly. T administration started on treatment d 1. These doses were selected to create and maintain T concentrations in a range that extended from low normal to supraphysiological levels for healthy young men. Twelve men were assigned each to groups 1, 2, and 3; 11 men were assigned to group 4; and 14 men were assigned to group 5. To assure compliance, the General Clinical Research Center staff administered the T and GnRH agonist injections.
Nutritional intake

For all participants, energy and protein intakes were standardized at 35 kcal/kg•d and 1.2 g/kg•d, respectively. The standardized diet was initiated 2 wk before the start of the treatment. Dietary instructions were reinforced every 4 wk by a nutritionist throughout the study. Adherence to dietary prescription was verified by analysis of 3-d food records and 24-h food recalls every 4 wk.
Exercise stimulus

The subjects were instructed to maintain their usual activity levels and not to undertake strength training or moderate to heavy endurance exercise during the study.
Outcome measures

The main outcome measures were change from baseline in plasma lipids, apolipoproteins, insulin sensitivity, and CRP.

Plasma levels of total cholesterol and triglycerides were determined enzymatically on an Abbott VP-Super System Analyzer, using commercially available reagents [CHOP/PAP, Roche (Mannheim, Germany), for total cholesterol, and Abbott Laboratories, Diagnostics Division (Irwing, TX), for triglycerides]. High density lipoprotein cholesterol (HDL-C) levels were measured by the modified heparin-manganese precipitation procedure of Warnick and Albers [24] .

Very low density lipoprotein cholesterol (VLDL-C), and low density lipoprotein cholesterol (LDL-C) levels were determined by the method of Friedewald et al. [25] . The quantification of apolipoproteins was performed by electroimmunoassay according to the previously described procedures for apolipoprotein A-I [26] , apolipoprotein B [27] , and apolipoprotein C-III [28] . All apolipoprotein measurements were carried out in triplicate. The inter- and intraassay coefficients of variation were 1.2% and 1% for apolipoprotein A-I, 1.3% and 1.2% for apolipoprotein B, and 1.7% and 1.2% for apolipoprotein C-III, respectively.

CRP was measured by a sensitive ELISA (DSL-10-42100 Active ELISA kit, Diagnostics Systems Laboratories, Inc., Webster, TX). Serum total and free T were measured periodically throughout the study. Serum total T levels were measured with an RIA that uses iodinated T as tracer [7] [29] [30] . This assay has a sensitivity of 0.44 ng/dl, and intra- and interassay coefficients of variation of 13.2% and 8.2%, respectively [30] . Free T was separated by an equilibrium dialysis procedure and then measured in the dialysate by an immunoassay [30] . The sensitivity of the free T assay is 0.6 pg/ml, and intra- and interassay coefficients of variation are 4.2% and 12.3%, respectively. Serum E2 levels were measured by an RIA with sensitivity of 2.5 pg/ml. Insulin concentrations were measured by RIA (ICN Biomedical, Inc., Costa Mesa, CA), with inter- and intraassay coefficients of variation of 6.8% and 11.5%, respectively. Plasma glucose levels were measured by the hexokinase-spectrophotometric method using reagents from Raichem, Inc. (San Diego, CA) and run on a Cobas Mira autoanalyzer (Global Medical Instrumentation, Clearwater, MN), giving inter- and intraassay of coefficients of variation of 3.8% and 6.8%, respectively. The lower limits of detection in plasma glucose and insulin assays were 10 mg/dl and 2.5 ìU/ml, respectively.

The indexes for insulin sensitivity (SI ), glucose effectiveness (SG ), and acute insulin response to glucose (AIRG ) were derived from the frequently sampled, iv glucose tolerance test, using the Bergman minimal model [31] [32] . In this procedure insulin and plasma glucose measurements were performed on arterialized plasma obtained at baseline (−30 and −15 min), followed by an iv bolus of glucose (300 mg/kg BW) given within 2 min. Arterialized blood samples were collected at 2, 3, 4, 5, 8, 10, 18, and 20 min after iv glucose administration. An iv injection of regular recombinant human insulin (0.03 U/kg BW) was administered at 20 min, and additional arterialized blood samples were drawn at 28, 32, 40, 60, 70, 120, and 180 min for measurement of plasma insulin and glucose. SI , SG , and AIR G were derived from the minimal model program (version 3.0) using default parameters. The glucose disposition index was calculated as the product of SI and AIRG .
Statistical analyses

All variables were examined for their distribution characteristics. Variables that did not meet the assumption of a normal distribution or heterogeneity of variance were log-transformed and retested.

ANOVA was used to compare the change from baseline (change = posttreatment value minus pretreatment value) in the outcome measures among the five groups. If ANOVA revealed an overall significant
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difference among groups, then those outcome measures were analyzed using paired t test to detect a nonzero change from baseline within each group. P < 0.05 was considered statistically significant; however, significance levels were adjusted for multiple comparisons using Bonferroni's correction.

The relationships between serum total and free T levels and each outcome measure were investigated using linear regression and correlation analyses. We also investigated the correlation between E2 and lipids using correlation analysis. To account for the effect of T on lipids, a multiple regression model, including both E2 and T as independent variables, was also run. The data are presented as the mean ± SEM.

Now the Results:



Results

Participant characteristics
The details of the subject population have been published [16] . Of the 61 men who were randomized, 53 completed the study: 11 in group 1, 7 in group 2, 12 in group 3, 10 in group 4, and 13 in group 5. One subject discontinued treatment because of acne, and the other 7 withdrawals were due to the subject's inability to meet the requirements of the protocol. The 5 groups did not differ significantly with respect to age, height, weight, baseline T levels, fat mass, or fat-free mass [16] . All eligible subjects received 100% of their GnRH agonist injections, and only 1 man in the 50-mg dose group missed 1 T injection.

Nutritional intake
Daily energy and protein intakes were not significantly different among the five groups at baseline (data not shown). The percentages of calories derived from protein, carbohydrate, and fat were not significantly different among the five groups at baseline or during treatment. There was no significant change in caloric or protein intake in any group during the treatment period. The change from baseline in caloric, protein, carbohydrate, and fat intake did not differ significantly among the five treatment groups.

Total and free T, and E2 concentrations
Serum total and free T levels have been previously reported [16] . Mean (± SEM) serum total T concentrations in the five groups, 7 d after previous T injection, were 253 ± 66, 306 ± 58, 570 ± 75, 1345 ± 139, and 2370 ± 150 ng/dl, respectively; the corresponding free T concentrations were 29 ± 5, 32 ± 3, 52 ± 8, 138 ± 21, and 275 ± 30 pg/ml, respectively. Serum E2 concentrations were not significantly different in the five treatment groups at baseline (21.6 ± 2.3, 21.3 ± 3.5, 20.3 ± 3.1, 27.1 ± 1.8, and 19.5 ± 1.6 pg/ml; P = NS), but increased significantly during treatment only in men receiving 300 and 600 mg T enanthate weekly (wk 20 values, 15.2 ± 1.8, 14.2 ± 1.1, 22.8 ± 3.0, 43.0 ± 5.3, and 55.7 ± 5.3; P = 0.0012). Serum E2 concentrations were highly correlated with serum total T concentrations (r = 0.76; P = 0.0001). Serum total T, free T, and E2 levels measured during the last treatment week after the previous injection were linearly dependent on the T dose ( P = 0.0001). In men receiving the 25- and 50-mg doses, nadir total and free T concentrations decreased from baseline and were at or below the lower limit of the normal range for healthy young men. In contrast, serum total and free T concentrations increased significantly from baseline and were in the supraphysiological range in men receiving the 300- and 600-mg doses.

Plasma lipid levels ( Table 1 )
Plasma concentrations of total cholesterol, triglycerides, LDL-C, and VLDL-C did not differ significantly among groups at baseline. There were no significant differences in change from baseline scores for any of these measures among the five treatment groups ( Table 1 ). There was no significant correlation between T dose or serum total and free T concentrations during treatment and change in total cholesterol, triglycerides, LDL-C, and VLDL-C concentrations.

HDL-C ( Fig. 1, A-C )
Plasma HDL-C levels did not differ significantly among groups at baseline. Figure 1A shows the change from baseline in plasma HDL-C levels in the five treatment groups (overall ANOVA, P = 0.014). Plasma HDL-C concentrations decreased significantly in men receiving the 600-mg dose of T enanthate (change, −8.3 ± 1.7 mg/dl; P = 0.0005; Fig. 1A ). Plasma HDL-C levels increased numerically in men receiving 25 mg/wk (change, +4.1 ± 2.3 mg/dl) and decreased in the men receiving 125 mg/wk (change, −4.0+1.74), but these changes did not achieve statistical significance after Bonferroni's correction for multiple comparisons.

The changes in plasma HDL-C levels from baseline were inversely correlated with circulating total (r = −0.40; P = 0.0054; Fig. 1B ) and free T (r = −0.30; P = 0.030; Fig. 1C ) concentrations, but not with serum E2 concentrations (r = −0.30; P = 0.052).

Although plasma HDL-C levels decreased during treatment in men receiving the 600-mg dose, by d 252, 16 wk after discontinuation of treatment, plasma HDL-C concentrations were not significantly different from pretreatment values.



TABLE 1 -- Plasma total cholesterol, LDL-cholesterol, VLDLcholesterol, and triglycerides T dose Baseline Week 20
Total cholesterol (mg/dl), overall ANOVA P = 0.518
BEFORE AFTER
25 mg 167 ± 10 180 ± 12
50 mg 177 ± 13 174 ± 7
125 mg 155 ± 5 157 ± 7
300 mg 154 ± 15 150 ± 16
600 mg 158 ± 10 147 ± 8

LDL cholesterol (mg/dl), overall ANOVA P = 0.057
BEFORE AFTER
25 mg 104 ± 8 114 ± 9
50 mg 101 ± 11 102 ± 8
125 mg 84 ± 5 93 ± 4
300 mg 99 ± 15 101 ± 14
600 mg 102 ± 9 100 ± 8

VLDL cholesterol (mg/dl), overall ANOVA P = 0.578
BEFORE AFTER
25 mg 22 ± 5 23 ± 4
50 mg 22 ± 3 24 ± 4
125 mg 20 ± 2 17 ± 2
300 mg 14 ± 3 17 ± 3
600 mg 13–2 16 ± 2

Triglycerides (mg/dl), overall ANOVA P = 0.727
BEFORE AFTER
25 mg 99 ± 21 97 ± 21
50 mg 133 ± 28 126 ± 31
125 mg 105 ± 17 95 ± 16
300 mg 96 ± 18 94 ± 22
600 mg 65 ± 9 81 ± 6
Data are mean ± SEM. Baseline values are pretreatment values obtained during the control period. None of the comparisons vs. zero change were significant after Bonferroni's correction.

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Figure 1. A, Dose-dependent effects of T on change in plasma HDL-C levels. Data are the mean ± SEM change from baseline in plasma HDL-C levels in the five treatment groups. *, P = 0.0005 vs. zero change. Baseline values were obtained during the pretreatment control period, and treatment values during wk 20. The change from baseline was calculated as the difference between wk 20 and baseline values. B, The correlation of serum total T concentrations and change in plasma HDL cholesterol levels. A linear regression model was used to determine the correlation between total T concentrations and plasma HDL-C levels. Serum T levels were measured at nadir, 1 wk after the previous injection. Pearson correlation coefficient, r = −0.40; P = 0.0006. C, The correlation of serum free T concentrations and change in plasma HDL-C levels. A linear model was used to assess the correlation between free T concentrations and plasma HDL-C levels. Serum free T levels were measured at nadir, 1 wk after the previous injection. Pearson correlation coefficient, r = −0.30; P = 0.030.

TABLE 2 -- Plasma apolipoprotein B and C-III concentrations at baseline and during treatment T dose Baseline Week 20
Apolipoprotein B (mg/dl), overall ANOVA P = 0.855
BEFORE AFTER
25 mg 78 ± 4 91 ± 7
50 mg 79 ± 4 87 ± 3
125 mg 81 ± 4 87 ± 5
300 mg 89 ± 8 95 ± 8
600 mg 79 ± 6 87 ± 6

Apolipoprotein C-III (mg/dl), overall ANOVA P = 0.078
BEFORE AFTER
25 mg 22 ± 5 23 ± 4
50 mg 24 ± 3 24 ± 4
125 mg 20 ± 2 17 ± 2
300 mg 14 ± 3 17 ± 3
600 mg 13–2 16 ± 2
Data are mean ± SEM. The P values shown in the table are unadjusted P values. However, none of the comparisons vs. zero change were significant after Bonferroni's correction.


Apolipoproteins ( Table 2 and Fig. 2, A-C )
There were no significant differences in serum apolipoprotein concentrations between groups at baseline ( Table 2 ). There were significant between-group differences in the change from baseline in apolipoprotein A-1 concentrations (by ANOVA, P = 0.0015; Fig. 2A ). Plasma apolipoprotein A–I levels increased from baseline in men receiving 25 mg T enanthate weekly (change, +9 ± 3 mg/dl; P = 0.021; Fig. 2A ); this increase was not significant after Bonferroni's correction. In contrast, there was a significant decrease in apolipoprotein AI levels in the 600 mg group (change, −16 ± 2 mg/dl; P = 0.0001 vs. zero change; Fig. 2A ).

There was a significant inverse correlation between serum total and free T concentrations and change in apolipoprotein A–I concentrations (total T: r = −0.47; P = 0.0005; free T: r = −0.40; P = 0.0001; Fig. 2, B and C ). Changes in apolipoprotein A–I concentrations were also correlated with E2 concentrations (r = −0.36; P = 0.016). When T concentrations were factored into the analysis, serum E2 concentrations were no longer significantly correlated with change in apolipoprotein A–I concentrations ( P = 0.06).

There was no significant correlation between serum T dose, serum total and free T concentrations, and changes in apolipoprotein B and C-III levels. There were no significant changes in apolipoprotein B and C-III at any dose, and there were no significant differences between groups in the change from baseline in apolipoprotein B or C-III (overall ANOVA,
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Figure 2. A, The effects of different doses of T enanthate on plasma apolipoprotein A–I levels. Data are the mean ± SEM change from baseline in plasma apolipoprotein A–I levels in the five treatment groups. *, P = 0.0005. B, The correlation of serum total T concentration and change in apolipoprotein A–I concentration. A linear regression model was used to assess the correlation between total T and apolipoprotein A–I concentrations. Serum T levels were measured at nadir, 1 wk after the previous injection. Pearson correlation coefficient, r = −0.40; P = 0.002. C, The correlation of serum free T concentrations and change in apolipoprotein A–I. A linear regression model was used to assess the correlation between free T and apolipoprotein A–I concentrations. Serum free T levels were measured at nadir, 1 wk after the previous injection. Pearson correlation coefficient, r = −0.40; P = 0.0001.
P = 0.855; Table 2 ). Changes in apolipoprotein B and C-III were not significantly correlated with E2 concentrations.

SI-Insulin sensitivity index ( Fig. 3A )
There were no significant differences among the five groups in S I at baseline. Also, there were no significant differences in the change from baseline among five groups (overall ANOVA, P = 0.774; Fig. 3A ).

Serum T concentrations were not significantly correlated with S I (r = −0.07; P = 0.875).

SG Glucose effectiveness index( Fig. 3B )
There were no significant differences in SG among the five treatment groups at baseline. The change in SG was not significantly different among the five treatment groups (by ANOVA, P = 0.070; Fig. 3B ). There was no significant correlation between serum total (r = −0.09; P = 0.780) and free (not shown) T concentrations and change in SG .

AIRG Acute Insulin Response to Glucose( Fig. 3C )
There was no significant change from baseline in the AIRG after T treatment in any group, nor were there any significant differences among the five treatment groups (by ANOVA, P = 0.974; Fig. 3C ). The change in AIRG was not significantly correlated with either serum total (r = −0.052; P = 0.140) or free (not shown) T concentrations.

The glucose disposition index (DI), derived as a product of SI and AIRG , did not significantly change from baseline in any group, nor were there any significant differences in DI among the groups ( Fig. 3D ). The change in DI was not significantly correlated with serum T concentrations (r = −0.003; P = 0.982).

CRP C-Reactive Protein ( Table 3 )
Circulating CRP concentrations were not significantly different among the five treatment groups at baseline. There was no significant change in CRP levels in any of the five treatment groups. There were no significant differences in the change from baseline among the five groups (by ANOVA, P = 0.833; Table 3 ). The change in CRP concentrations was not significantly correlated with total or free T concentrations (data not shown).


Discussion

Our data demonstrate that over a wide span of serum T concentrations within the broad physiological range, T administration had no significant effect on plasma lipids, apolipoproteins, insulin sensitivity, and CRP. Only the highest dose of T enanthate (600-mg/wk) that produced supraphysiological T concentrations was associated with significant lowering of plasma HDL-C and apolipoprotein A–I levels. We recently reported that T dose-dependently increases fat-free mass and muscle size [16] ; significant increases in fat-free mass and muscle volume were observed with 125-, 300-, and 600-mg doses. Taken together with the data reported previously, the current study demonstrates that significant gains in fat-free mass, muscle size, and muscle strength can be achieved at T doses that do not significantly affect insulin sensitivity, CRP, plasma lipids, and apolipoproteins in a population of healthy young men.

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Figure 3. The effects of graded doses of T enanthate on the change in measures of insulin sensitivity. Changes in SI (A, left), SG (B, left), AIRG (C, right), and DI (D, right), in healthy young men, measured by the Bergman minimal model, are shown. Data are the mean ± SEM.

TABLE 3 -- Plasma C-reactive protein concentrations at baseline and during the 20th wk of treatment T dose Baseline Week 20 Change from baseline P vs. zero change
T dose Baseline Wk 20 Change from P vs. zero
Baseline change
25 mg 0.81 + 0.39 1.28 + 0.54 0.26 + 0.25 0.353
50 mg 1.85 + 0.42 1.70 + 0.27 −0.8 + 0.26 0.751
125 mg 0.55 + 0.16 0.69 + 0.15 0.14 + 0.16 0.406
300 mg 1.40 = 0.53 1.28 + 0.41 −0.12 + 0.28 0.524
600 mg 0.82 + 0.23 0.85 + 0.29 −.03 + 0.20 0.917
Data are mean ± SEM. Baseline values are pretreatment values obtained during the control period. Change was calculated by subtracting baseline value from wk 20 value.


**Androgen effects on plasma HDL-C depend on the dose, type of androgen used (aromatizable or not), and route of administration (oral or parenteral). Supraphysiological doses of androgens, especially orally administered, nonaromatizable, androgenic steroids, undoubtedly decrease plasma HDL-C levels [33] [34] [35] [36] . When nonaromatizable androgens such as stanozolol are administered, the response is a marked reduction in HDL-C, particularly the HDL-C2 fraction, and a significant increase in LDL-C levels. [33] . However, in several placebo-controlled studies of older men, physiological T replacement had minimal or no effect on plasma HDL-C levels [3] [4] [5] [6] .

Several epidemiological studies have reported a direct relationship between circulating T concentrations and plasma HDL-C levels [37] [38] [39] [40] [41] [42] . For instance, in the Telecom study [38] , men with normal and low T were compared for cardiovascular risk factors. The men with low T had a higher body mass index, waist/hip ratio, systolic blood pressure, fasting and 2 h blood glucose, total cholesterol, LDL-C, apolipoprotein B, and fasting and 2-h plasma insulin and lower values of HDL-C and apolipoprotein A–I. Importantly, SHBG levels were also lower in the low T group. Because SHBG levels affect total T concentrations, it is possible that the association between total T and plasma HDL-C concentrations found in cross-sectional epidemiological studies is a reflection of the effects of higher insulin levels in men with higher body mass index on SHBG concentrations. Indeed, the bioavailable T levels were not significantly different between the two groups in the Telecom study. A direct relationship between plasma T and HDL-C concentrations was also shown in the Multiple Risk Factor Intervention Trial [37] . It is not clear whether the relationship between T and HDL-C is independent of other correlates, particularly the effects of obesity on SHBG concentrations. Factors that may influence both HDL and T include coexisting disease states, obesity and body fat distribution, and lifestyle factors, such as smoking, alcohol, and exercise. Some of the factors that decrease HDL-C may also be associated with lower SHBG levels and might indirectly affect T levels.
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In our study changes in apolipoprotein A–I levels were inversely correlated with serum T concentrations. Because apolipoprotein A–I, a component of HDL particles, is thought to be cardioprotective, and its levels are inversely related to cardiovascular risk, the decrease in apolipoprotein A–I at the 600-mg dose of T enanthate might be viewed as deleterious.

The effects of T administration on glucose metabolism and insulin sensitivity remain poorly understood. Total T levels are inversely related to insulin concentrations in several cross-sectional studies [39] [42] [43] [44] [45] . Fasting insulin levels are higher and T and SHBG values are lower in men with type 2 diabetes mellitus [39] . Haffner et al. [39] reported that lower T levels were correlated with a higher waist/hip ratio and lower nonoxidative, whole body glucose disposal in men. Administration of exogenous androgens has been reported to induce glucose intolerance and hyperinsulinemia in some studies [20] [21] [22] , but other studies found no significant changes in glucose tolerance or insulin concentrations with administration of T or 19-nor-T to men even at pharmacological doses [46] . A marked lowering of T levels in the male rat by surgical castration as well as the administration of supraphysiological doses of T induces insulin resistance [20] . This suggests that the relationship between T levels and insulin sensitivity might be curvilinear. Our study failed to find any significant changes in SI , SG , and AIRG in any treatment group over a wide range of serum T concentrations. Our data differ from those of Marin [17] [18] [19] [47] , who reported improvements in insulin sensitivity, blood glucose, and blood pressure after T supplementation in middle-aged men who had visceral obesity and low T levels. In contrast to the studies by Marin et al. [17] [18] [19] [47] , the subjects in our study were younger and had significantly lower fat mass. Marin et al. used euglycemic-hyperinsulinemic clamp, whereas we used frequently sampled iv glucose tolerance test to assess insulin sensitivity; we do not know whether the differences in methodology could have contributed to the observed differences in outcomes. We cannot exclude the possibility that in middle-aged men with midsegment obesity, T might decrease whole body and intraabdominal fat and thereby improve insulin sensitivity.

Serum T levels are inversely correlated with fat mass, particularly visceral fat area [45] [48] . The induction of androgen deficiency in young men is associated with a decrease in lipid oxidation rates and an increase in total fat mass [10] . T replacement of young [8] [9] [12] and older hypogonadal men [5] decreases fat mass. T inhibits uptake of labeled triglycerides and enhances lipid mobilization from the visceral fat [49] . As fat mass is an important determinant of insulin sensitivity [50] , T supplementation would be expected to improve the latter by decreasing fat mass. It is possible that we did not observe changes in insulin sensitivity in this study because our participants were lean.

CRP, an inflammation-sensitive marker, has emerged as an important independent predictor of coronary events [23] [52] . In mice, both constitutive and IL-6-dependent expressions of CRP transgene require T [53] [54] . Furthermore, treatment with T results in an increase in CRP in male rats [54] . Our data did not show a significant change in CRP levels at any dose of T in healthy young men.

In conclusion, only the 600-mg dose of T enanthate, which was associated with highly supraphysiological T concentrations, decreased HDL-C and apolipoprotein A–I levels in healthy young men. Over a wide range of T concentrations, there was no significant change in plasma lipids, apolipoproteins, insulin sensitivity, and CRP concentrations. Whereas significant gains in fat-free mass and muscle size were observed with the 125- and 300-mg doses of T, these doses were not associated with a concurrent deterioration in the lipoprotein profile, CRP levels, or insulin sensitivity. Several studies are in agreement that significant gains in fat-free mass and muscle strength can be accrued in healthy hypogonadal men [8] [9] [10] [11] [12] , older men with low T levels [3] [5] , and human immunodeficiency virus-infected men with weight loss [7] by increasing serum T concentrations from hypogonadal levels into a range that is mid-normal for healthy young men. Thus, physiological T replacement in hypogonadal states may improve muscle mass without adversely affecting the cardiovascular risk factors. Although long-term studies are needed to determine directly the effects of T supplementation on cardiovascular event rates and atherosclerosis progression, our data do not support the idea that T replacement in doses that raise serum T concentrations into the mid to high normal range will necessarily adversely affect the risk of atherosclerotic heart disease.
Acknowledgments

Bio-Technology General Corp. provided T enanthate, and DebioPharm, Inc. (Geneva, Switzerland), provided long-acting GnRH agonist for these studies.
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