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Objectives: Assess the prevalence of hypogonadism in older male Veterans by comparing direct measurements of total testosterone (T) and bioavailable testosterone (BioT) versus indirect BioT values derived from existing and newly developed regression analyses., Design: Cohort study., Setting: Malcom Randall VA Medical Center, Gainesville, FL., Participants: Community-dwelling male Veterans aged 60 and older (n = 203)., Measurements: Total T, BioT, albumin, sex hormone-binding globulin (SHBG), and body mass index were evaluated. Blood values were assessed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) and clinical or commercially available immunoassays to compare accuracy among assessment techniques. Existing and newly developed multiple regression analyses were evaluated to assess accuracy in predicting BioT., Results: Total T was 13.80 ± 6.25 nmol/L (398 ± 180 ng/dL) and was low (≤10.4 nmol/L or ≤300 ng/dL) in 34% of participants. SHBG was 58 ± 35 nmol/L and elevated (≥62 nmol/L) in 36% of participants. BioT was 1.94 ± 0.97 nmol/L (56 ± 28 ng/dL), with 72% of participants below the clinical cutoff (≤2.43 nmol/L or ≤70 ng/dL). Albumin was within the normal clinical range. Total T and BioT measured via immunoassay and LC-MS/MS were moderately to highly correlated, with no differences between assessment methods. Several existing predictive equations overestimated BioT by 74% to 166% within our cohort (P < .001). A newly developed regression model that included total T, SHBG, albumin, and age more accurately predicted BioT, with values correlated (r = 0.508, P < .001) and comparable to LC-MS/MS., Conclusion: In our cohort, the prevalence of low total T was higher and low BioT was markedly higher than reported in the general age-matched population, indicating a greater incidence of hypogonadism in older male Veterans. In addition, existing empiric formulae, derived from other populations produced BioT values that were considerably greater than those directly measured, whereas our newly developed regression analysis provides improved predictive capabilities for older male Veterans., (Published by Elsevier Inc.)

Androgens regulate body composition and skeletal muscle mass in males, but the molecular mechanisms are not fully understood. Recently, we demonstrated that trenbolone (a potent synthetic testosterone analogue that is not a substrate for 5-alpha reductase or for aromatase) induces myotrophic effects in skeletal muscle without causing prostate enlargement, which is in contrast to the known prostate enlarging effects of testosterone. These previous results suggest that the 5α-reduction of testosterone is not required for myotrophic action. We now report differential gene expression in response to testosterone versus trenbolone in the highly androgen-sensitive levator ani/bulbocavernosus (LABC) muscle complex of the adult rat after 6weeks of orchiectomy (ORX), using real time PCR. The ORX-induced expression of atrogenes (Muscle RING-finger protein-1 [MuRF1] and atrogin-1) was suppressed by both androgens, with trenbolone producing a greater suppression of atrogin-1 mRNA compared to testosterone. Both androgens elevated expression of anabolic genes (insulin-like growth factor-1 and mechano-growth factor) after ORX. ORX-induced increases in expression of glucocorticoid receptor (GR) mRNA were suppressed by trenbolone treatment, but not testosterone. In ORX animals, testosterone promoted WNT1-inducible-signaling pathway protein 2 (WISP-2) gene expression while trenbolone did not. Testosterone and trenbolone equally enhanced muscle regeneration as shown by increases in LABC mass and in protein expression of embryonic myosin by western blotting. In addition, testosterone increased WISP-2 protein levels. Together, these findings identify specific mechanisms by which testosterone and trenbolone may regulate skeletal muscle maintenance and growth., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Enzyme immunoassays (EIA) are commonly utilized for the evaluation of androgens in biological fluids; however, careful consideration must be given to cross-reactivity with other endogenous sex-steroid hormones. Our purpose was to determine the validity of a commonly-utilized commercially-available dihydrotestosterone (DHT) EIA. Serum samples obtained from older hypogonadal men who participated in a 12-month randomized controlled trial evaluating the effects of testosterone-enanthate (125 mg/week) or vehicle in combination with finasteride (5mg/day) or placebo were assayed for DHT via EIA and using a validated gold-standard LC-MS/MS approach. Additionally, commercially-available (DHT-free) buffer containing graded testosterone doses was evaluated by DHT immunoassay. DHT concentrations measured via EIA were 79% to >1000% higher than values obtained by LC-MS/MS (p<0.05), with the largest differences (415-1128%) occuring in groups receiving finasteride. Both LC-MS/MS and EIA indicated that testosterone-enanthate increased serum DHT to a similar magnitude. In contrast, finasteride-induced reductions in DHT were detected by LC-MS/MS, but not EIA (p<0.05). No significant associations were present for DHT concentrations between measurement techniques. Cross-reactivity of testosterone with the immunoassay ranged from 18% to 99% and DHT concentrations measured by EIA were highly associated with the spiked testosterone concentrations in DHT-free buffer (r=0.885, p<0.001). In conclusion, we provide evidence invalidating a commonly-utilized commercially-available DHT immunoassay because significant cross-reactivity exists between testosterone and the EIA and because the changes in DHT observed via EIA were not associated with a validated gold-standard measurement technique. The cross-reactivity of testosterone is particularly concerning because testsoterone is present in 100-fold greater concentrations than is DHT within the circulation., (Published by Elsevier Inc.)

Objective: Resistance exercise (RE) stimulates growth hormone (GH) secretion in a load-dependent manner, with heavier loads producing larger GH responses. However, new research demonstrates that low-load RE performed with blood flow restriction (BFR) produces potent GH responses that are similar to or exceed those produced following high-load RE. We hypothesized that low-load RE with vascular restriction would attenuate the known age-related reduction in GH response to RE., Design: In a randomized crossover design, ten young (28 ± 7.8 years) and ten older (67.4 ± 4.6 years) men performed bilateral knee extension RE with low-load [20% of one-repetition maximum (1RM)] with BFR and high-load (80% 1RM) without BFR. GH and lactate were measured every 10 minutes throughout a 150-minute testing session (30 minutes prior to and 120 minutes following completion of the exercise); IGF-I was measured at baseline and 60 minutes post-exercise., Results: Area under the GH curve indicated that both age groups responded similarly to each exercise condition. However, young men had a significantly greater maximal GH response to low-load RE with BFR than the high-load condition without BFR. Additionally, younger men had greater maximal GH concentrations to low-load RE with BFR than older men (p=0.02). The GH responses were marginally correlated to lactate concentration (r=0.13, p=0.002) and IGF-I levels were unchanged with RE., Conclusions: GH responses to low-load RE with vascular restriction are slightly higher than high-load RE without vascular restriction in young men. However, low-load RE with vascular restriction did not attenuate the known age-related reduction in GH response with exercise. These data suggest that while low-load RE with vascular restriction is as effective for inducing a GH response than traditionally-based high-load RE, there is a more potent response in young men., (Copyright © 2012. Published by Elsevier Ltd.)

The hypogonadal state in men is accompanied by substantial decreases in muscle and bone mass and by an increase in adiposity. Most of the strains of orchiectomized (ORX) rat that have been used to model this state display substantial losses in bone, but only subtle changes in adiposity and muscle mass. In order to identify a rat model displaying a robust catabolic response to ORX, we studied three strains: Fischer 344 (F344), Brown Norway and Wistar. ORX caused a significant and sustained decrease in weight gained by F344, but only a trend toward reduced weight gain in Brown Norway rats and a modest reduction weight gain in Wistar rats that was significant only after 56days. ORX suppressed food intake in F344 rats, and to a lesser degree in Brown Norway and Wistar rats. ORX reduced muscle mass significantly in F344 rats, but not in Brown Norway or Wistar rats. ORX increased adiposity moderately in F344 rats and substantially in Wistar rats. ORX caused a marked reduction in prostate mass and increase in bone resorption in all three strains. Thus, F344 was the only strain in which ORX produced substantial decreases in food intake, body weight and muscle mass with increased adiposity and increased bone resorption. We conclude that the F344 rat displays a broad range of catabolic effects following ORX and is the best rat model for studying the androgenic pathway and strategies for reversing catabolic changes induced by hypogonadism.

In several strains of genetically obese and insulin resistant rodents, adipose tissue over expresses mRNA for tumor necrosis factor alpha (TNF-alpha). Our purpose was to determine whether tissue expression of TNF-alpha protein is elevated in rats that are made obese and insulin resistant by administration of a high-fat diet. Young Wistar rats weighing approximately 50 g were fed for 39 days with either normal rat chow (12.4% fat) or a high-fat diet (50% fat). After 33 days, glucose tolerance was assessed and after 39 days, insulin-stimulated transport of [3H]-2-deoxyglucose was assessed in isolated strips of soleus muscle. Rats on the high-fat diet consumed slightly fewer calories but became obese, displaying significant approximately 2-fold increases in the mass of both visceral and subcutaneous fat depots. High-fat feeding also caused a moderate degree of insulin resistance. Fasting serum insulin was significantly increased, as were insulin and glucose concentrations following glucose loading. In isolated strips of soleus muscle, the high-fat diet produced a trend toward a 33% decrease in the insulin-stimulated component of glucose transport (p=0.064). Western analysis of muscle, liver and fat revealed two forms of TNF-alpha, a soluble 17 Kd form (sTNF-alpha) and a 26 Kd membrane form (mTNF-alpha). Both sTNF-alpha and mTNF-alpha were relatively abundant in fat; whereas sTNF-alpha was the predominant form present in muscle and liver. High-fat feeding caused a significant 2-fold increase in muscle sTNF-alpha, along with a trend toward a 54% increase in visceral fat sTNF-alpha (p=0.055). TNF-alpha was undetectable in serum. We conclude that muscle over expression of TNF-alpha occurs during the development of diet-induced obesity and may, in part cause insulin resistance by an autocrine mechanism.