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1.  Age Trends in Estradiol and Estrone Levels Measured Using Liquid Chromatography Tandem Mass Spectrometry in Community-Dwelling Men of the Framingham Heart Study 
Background.
Age trends in estradiol and estrone levels in men and how lifestyle factors, comorbid conditions, testosterone, and sex hormone–binding globulin affect these age trends remain poorly understood, and were examined in men of the Framingham Heart Study.
Methods.
Estrone and estradiol concentrations were measured in morning fasting samples using liquid chromatography tandem mass spectrometry in men of Framingham Offspring Generation. Free estradiol was calculated using a law of mass action equation.
Results.
There were 1,461 eligible men (mean age [±SD] 61.1±9.5 years and body mass index [BMI] 28.8±4.5kg/m2). Total estradiol and estrone were positively associated with age, but free estradiol was negatively associated with age. Age-related increase in total estrone was greater than that in total estradiol. Estrone was positively associated with smoking, BMI, and testosterone, and total and free estradiol with diabetes, BMI, testosterone, and comorbid conditions; additionally, free estradiol was associated negatively with smoking. Collectively, age, BMI, testosterone, and other health and behavioral factors explained only 18% of variance in estradiol, and 9% of variance in estrone levels. Men in the highest quintile of estrone levels had significantly higher age and BMI, and a higher prevalence of smoking, diabetes, and cardiovascular disease than others, whereas those in the highest quintile of estradiol had higher BMI than others.
Conclusions.
Total estrone and estradiol levels in men, measured using liquid chromatography tandem mass spectrometry, revealed significant age-related increases that were only partially accounted for by cross-sectional differences in BMI, diabetes status, and other comorbidities and health behaviors. Longitudinal studies are needed to confirm these findings.
doi:10.1093/gerona/gls216
PMCID: PMC3660115  PMID: 23105044
Age trends; Estrogen levels in men; LC-MS/MS; Age-related changes in estrone and estradiol; Determinants of estrogen levels in men.
2.  The Effect of Testosterone on Mood and Well-being in Men with Erectile Dysfunction in a Randomized, Placebo-Controlled Trial 
Andrology  2013;1(3):475-482.
The relationship between testosterone, well-being and mood is poorly understood. We investigated the effect of testosterone supplementation on mood, well-being, and self-reported health in men with erectile dysfunction (ED) and low serum testosterone levels. This was a randomized, double-blind, placebo-controlled trial (ClinicalTrials.gov registration number NCT00512707) in which 140 men, 40 to 70-years, with ED and low serum testosterone levels were first optimized on sildenafil alone for 3 to 7-weeks and then randomized to receive either sildenafil plus testosterone gel (n = 70) or sildenafil plus placebo (n = 70) gel for 14-weeks. Using multiple imputations and generalized linear regression, we compared psychological changes in well-being, evaluated by the Psychological General Well-Being Index, and mood, evaluated by Derogatis Affects Balance Scale. Mood and well-being scores were similar between the two groups at baseline and did not substantially change during the administration of sildenafil or after randomization to testosterone. Our findings show that the addition of testosterone to sildenafil in men with ED and low serum testosterone levels was not associated with improvement in either well-being or mood.
doi:10.1111/j.2047-2927.2013.00075.x
PMCID: PMC3630276  PMID: 23494931
Erectile dysfunction; testosterone replacement; mood; affectivity balance; well-being; androgen deficiency
3.  Testosterone Administration Inhibits Hepcidin Transcription and is Associated with Increased Iron Incorporation into Red Blood Cells 
Aging cell  2013;12(2):280-291.
Testosterone administration increases hemoglobin levels and has been used to treat anemia of chronic disease. Erythrocytosis is the most frequent adverse event associated with testosterone therapy of hypogonadal men, especially older men. However, the mechanisms by which testosterone increases hemoglobin remain unknown.
Testosterone administration in male and female mice was associated with a greater increase in hemoglobin and hematocrit, reticulocyte count, reticulocyte hemoglobin concentration, and serum iron and transferring saturation than placebo. Testosterone downregulated hepatic hepcidin mRNA expression, upregulated renal erythropoietin mRNA expression, and increased erythropoietin levels. Testosterone-induced suppression of hepcidin expression was independent of its effects on erythropoietin or hypoxia-sensing mechanisms. Transgenic mice with liver-specific constitutive hepcidin over-expression failed to exhibit the expected increase in hemoglobin in response to testosterone administration. Testosterone upregulated splenic ferroportin expression and reduced iron retention in spleen. After intravenous administration of transferrin-bound 58Fe, the amount of 58Fe incorporated into red blood cells was significantly greater in testosterone-treated mice than in placebo-treated mice. Serum from testosterone-treated mice stimulated hemoglobin synthesis in K562 erythroleukemia cells more than that from vehicle-treated mice. Testosterone administration promoted the association of androgen receptor (AR) with Smad1 and Smad4 to reduce their binding to BMP-response elements in hepcidin promoter in the liver. Ectopic expression of AR in hepatocytes suppressed hepcidin transcription; this effect was blocked dose-dependently by AR antagonist flutamide. Testosterone did not affect hepcidin mRNA stability. Conclusion: Testosterone inhibits hepcidin transcription through its interaction with BMP-Smad signaling. Testosterone administration is associated with increased iron incorporation into red blood cells.
doi:10.1111/acel.12052
PMCID: PMC3602280  PMID: 23399021
4.  Risk Factors Associated With Cardiovascular Events During Testosterone Administration in Older Men With Mobility Limitation 
Background.
Testosterone in Older Men with Mobility Limitations Trial found an increased incidence of cardiovascular events in men randomized to testosterone, resulting in enrollment cessation by trial's Data and Safety Monitoring Board. We evaluated changes in gonadal hormones and markers of inflammation and coagulation to elucidate risk factors associated with cardiovascular events.
Methods.
Men aged 65 years or more, with mobility limitation, total testosterone 100–350 ng/dL, or free testosterone less than 50 pg/mL, were randomized to placebo or 10 g testosterone gel daily for 6 months. Changes in total and free testosterone, estradiol and estrone, C-reactive protein, interleukin 6, fibrinogen, plasminogen activator inhibitor-1, and pro-brain naturetic peptide were compared between groups and within the testosterone group between subjects who experienced cardiovascular events and those who did not.
Results.
Of 209 men randomized (mean age 74 years), gonadal hormones and biomarkers were available in 179 men. Baseline body mass index, gonadal hormones, lipids, Framingham risk scores, and other biomarkers were similar in the two treatment groups. Within the testosterone group, the 6-month increase in free testosterone was significantly greater in men who experienced cardiovascular events than in those who did not [mean (95% confidence interval), 10.6 (4.6–16.7) vs 5.2 (3.0–7.5) ng/dL, p = .05]. In multivariable logistic regression analysis, the change in the serum levels of free testosterone was associated with cardiovascular events.
Conclusion.
Mobility-limited older men who experienced cardiovascular events had greater increases in serum free testosterone levels than those who did not.
doi:10.1093/gerona/gls138
PMCID: PMC3598355  PMID: 22562960
Testosterone; Older men; Mobility limitation; Cardiovascular disease
5.  Inhibition of in vitro and in vivo brown fat differentiation program by myostatin 
Obesity (Silver Spring, Md.)  2013;21(6):1180-1188.
Obesity arises mainly due to the imbalance between energy storage and its expenditure. Metabolically active brown adipose tissue (BAT) has recently been detected in humans and has been proposed as a new target for anti-obesity therapy because of its unique capacity to regulate energy expenditure. Myostatin (Mst), a negative regulator of muscle mass, has been identified as a potential target to regulate overall body composition. While the beneficial effects of Mst inhibition on muscle mass are well known, its role in the regulation of lipid metabolism, and energy expenditure is not very clear. We tested the effects of Mst inhibition on the gene regulatory networks that control BAT differentiation using both in vivo and in vitro model systems. PRDM16 and UCP1, two key regulators of brown fat differentiation were significantly up regulated in levator-ani (LA) and gastrocnemius (Gastroc) muscles as well as in epididymal (Epi) and subcutaneous (SC) fat pads isolated from Mst knock out (Mst KO) male mice compared to wild type (WT) mice. Using mouse embryonic fibroblast (MEFs) primary cultures obtained from Mst KO group compared to the WT group undergoing adipogenic differentiation, we also demonstrate a significant increase in select genes and proteins that improve lipid metabolism and energy expenditure. Furthermore, treatment of Mst KO MEFs with recombinant Mst protein significantly inhibited the gene expression levels of UCP1, PRDM16, PGC1-α/β as well as BMP7. Future studies to extend these findings and explore the therapeutic potential of Mst inhibition on metabolic disorders are warranted.
doi:10.1002/oby.20117
PMCID: PMC3735638  PMID: 23868854
6.  Testosterone Improves the Regeneration of Old and Young Mouse Skeletal Muscle 
Aging is associated with loss of muscle mass and strength, reduced satellite cell number, and lower regenerative potential. Testosterone increases muscle mass, strength, and satellite cell number in humans; however, the effects of testosterone on the regenerative potential of skeletal muscle are unclear. Here, we investigated the effect of testosterone on the skeletal muscle regeneration of young (2-month-old) and aged (24-month-old) male mice. We show that testosterone increases the number of proliferating satellite cells in regenerating “tibialis anterior” muscle of young and aged castrated mice 2 and 4 days postinjury. Testosterone supplementation increases the number and the cross-sectional area of regenerating fibers in both classes of age 4 days postinjury. Testosterone increases satellite cell activation and proliferation and the regeneration of both young and aged mouse muscle. These data suggest prospective application of androgens to improve the regenerating potential of the aged human skeletal muscle.
doi:10.1093/gerona/gls083
PMCID: PMC3598367  PMID: 22499765
Muscle regeneration; Testosterone
7.  Clinical correlates of sex steroids and gonadotropins in men over the late adulthood : the Framingham Heart Study 
Background
Low serum concentrations of sex steroids and gonadotropins in men have been associated with increased cardiometabolic risk and mortality, but the clinical correlates of these hormones in men over the late adulthood are less clearly understood.
Methods
We analyzed up to five serial measurements of total testosterone (TT), dehydroepiandrosterone sulfate (DHEAS), follicle stimulating hormone (FSH), luteinizing hormone (LH), and total estradiol (EST) in older men in the original cohort of the Framingham Heart Study to determine the short- (2-years; 1,165 person-observations in 528 individuals) and long-term (up to 10-years follow-up; 2,520 person-observations in 835 individuals with mean baseline age: 71.2 years) clinical correlates of these sex steroids and gonadotropins using multilevel modelling and Generalized Estimating Equations.
Results
Age, body mass index, and pre-existing type 2 diabetes were inversely related to long-term TT concentrations, whereas higher systolic blood pressure showed a positive association. Furthermore, age and pre-existing cardiovascular disease (CVD) were inversely and HDL cholesterol concentrations positively associated with long-term DHEAS concentrations. Analyses of short-term changes revealed age was inversely related to DHEAS, but positively related to FSH and LH concentrations.
Conclusion
Our community-based study identified modifiable correlates of decreasing TT and DHEAS concentrations in elderly men, suggesting that maintenance of a low CVD risk factor burden may mitigate the age-related decline of these hormones over the late adulthood.
doi:10.1111/j.1365-2605.2012.01285.x
PMCID: PMC3434249  PMID: 22640232
sex steroids; gonadotropins; testosterone; aging male; Framingham Heart Study
8.  AAV-Mediated Administration of Myostatin Pro-Peptide Mutant in Adult Ldlr Null Mice Reduces Diet-Induced Hepatosteatosis and Arteriosclerosis 
PLoS ONE  2013;8(8):e71017.
Genetic disruption of myostatin or its related signaling is known to cause strong protection against diet-induced metabolic disorders. The translational value of these prior findings, however, is dependent on whether such metabolically favorable phenotype can be reproduced when myostatin blockade begins at an adult age. Here, we reported that AAV-mediated delivery of a myostatin pro-peptide D76A mutant in adult mice attenuates the development of hepatic steatosis and arteriosclerosis, two common diet-induced metabolic diseases. A single dose of AAV-D76A in adult Ldlr null mice resulted in sustained expression of myostatin pro-peptide in the liver. Compared to vehicle-treated mice, D76A-treated mice gained similar amount of lean and fat mass when fed a high fat diet. However, D76A-treated mice displayed significantly reduced aortic lesions and liver fat, in association with a reduction in hepatic expression of lipogenic genes and improvement in liver insulin sensitivity. This suggests that muscle and fat may not be the primary targets of treatment under our experimental condition. In support to this argument, we show that myostatin directly up-regulated lipogenic genes and increased fat accumulation in cultured liver cells. We also show that both myostatin and its receptor were abundantly expressed in mouse aorta. Cultured aortic endothelial cells responded to myostatin with a reduction in eNOS phosphorylation and an increase in ICAM-1 and VCAM-1 expression. Conclusions: AAV-mediated expression of myostatin pro-peptide D76A mutant in adult Ldlr null mice sustained metabolic protection without remarkable impacts on body lean and fat mass. Further investigations are needed to determine whether direct impact of myostatin on liver and aortic endothelium may contribute to the related metabolic phenotypes.
doi:10.1371/journal.pone.0071017
PMCID: PMC3731267  PMID: 23936482
9.  Testosterone inhibits transforming growth factor-β signaling during myogenic differentiation and proliferation of mouse satellite cells: Potential role of follistatin in mediating testosterone action 
Testosterone (T) administration is associated with increased satellite cell number and skeletal muscle hypertrophy, although there is considerable heterogeneity in the response of different skeletal muscle groups to T in vivo. We investigated the effects of T on the growth and differentiation of satellite cells isolated from levator ani (LA) and gastrocnemius (gastroc) muscles. T up regulated follistatin (Fst) expression, but down regulated the mRNA and protein expression of a number of genes in the transforming growth factor-beta (TGF-β)-signaling pathway. Inhibition of Fst expression by small interfering RNA (siRNA) inhibited myogenic differentiation and blocked the pro-myogenic effects of T. Treatment of satellite cells with T or Fst up regulated the expression of Pax7 and PCNA, and increased their proliferation. T and Fst blocked TGF-β induced inhibition of growth and myogenic differentiation and down regulated TGF-β-dependent transcriptome in both LA and gastroc cells. We conclude that T stimulation of satellite cell proliferation and myogenic differentiation are associated with up regulation of Fst and inhibition of TGF-β-signaling.
doi:10.1016/j.mce.2011.11.019
PMCID: PMC3264813  PMID: 22138414
Transforming growth factor-β; follistatin; myostatin; myosin heavy chain II
10.  Durability of the Effects of Testosterone and Growth Hormone Supplementation in Older Community Dwelling Men: The HORMA Trial 
Clinical endocrinology  2011;75(1):103-111.
Objectives
Determine the durability of anabolic effects and adverse events (AEs) after stopping testosterone and growth hormone supplementation in older men.
Design
Secondary analysis of a double-masked, randomized controlled trial of testosterone gel (5g or 10g/daily) plus rhGH (0, 3, or 5ug/kg/day) with follow-up of outcomes 3-months later.
Participants
108 community-dwelling 65-90 year-old-men.
Measurements
Testosterone and IGF-1 levels, body composition (DEXA), 1-repetition maximum (1-RM) strength, stair-climbing power, quality-of-life (QOL) and activity questionnaires, AEs.
Results
Despite improvements in body composition during treatment, residual benefits 3-months later (week-28) were variable. For participants with improvements exceeding their week-17 median changes, benefits were sustained at week 28 for lean body mass (LBM, 1.45±1.63kg, 45% of week-17 values, p<0.0001-vs-baseline), appendicular skeletal muscle mass (ASMM, 0.71±1.01kg, 42%, p<0.0001), total fat (-1.06±2.18kg, 40%, p<0.0001,), and trunk fat (-0.89±1.42kg, 50%, p<0.0001,); retention of ASMM was associated with greater week-16 protein intake (p=0.01). For 1-RM strength, 39%-43% of week-17 improvements (p≤0.05) were retained and associated with better week-17 strength (p<0.0001), change in testosterone from week-17-to-28 (p=0.004) and baseline PASE (p=0.04). Framingham 10-year cardiovascular risks were low (~14%), didn’t worsen, and improved by week-28 (p=0.0002). The hypothalamic-pituitary-gonadal axis recovered completely.
Conclusions
Durable improvements in muscle mass, strength, and fat mass were retained 3-months after discontinuing hormone supplementation in participants with greater than median body composition changes during treatment, but not in others with smaller gains. AEs largely resolved after intervention discontinuation. Additional strategies may be needed to sustain or augment muscle mass and strength gains achieved during short-term hormone therapy.
doi:10.1111/j.1365-2265.2011.04014.x
PMCID: PMC3529980  PMID: 21521283
Lean body mass; fat mass; muscle performance; quality of life; cardiovascular risks
11.  Testosterone Plus Low-Intensity Physical Training in Late Life Improves Functional Performance, Skeletal Muscle Mitochondrial Biogenesis, and Mitochondrial Quality Control in Male Mice 
PLoS ONE  2012;7(12):e51180.
Testosterone supplementation increases muscle mass in older men but has not been shown to consistently improve physical function and activity. It has been hypothesized that physical exercise is required to induce the adaptations necessary for translation of testosterone-induced muscle mass gain into functional improvements. However, the effects of testosterone plus low intensity physical exercise training (T/PT) on functional performance and bioenergetics are unknown. In this pilot study, we tested the hypothesis that combined administration of T/PT would improve functional performance and bioenergetics in male mice late in life more than low-intensity physical training alone. 28-month old male mice were randomized to receive T/PT or vehicle plus physical training (V/PT) for 2 months. Compare to V/PT control, administration of T/PT was associated with improvements in muscle mass, grip strength, spontaneous physical movements, and respiratory activity. These changes were correlated with increased mitochondrial DNA copy number and expression of markers for mitochondrial biogenesis. Mice receiving T/PT also displayed increased expression of key elements for mitochondrial quality control, including markers for mitochondrial fission-and-fusion and mitophagy. Concurrently, mice receiving T/PT also displayed increased expression of markers for reduced tissue oxidative damage and improved muscle quality. Conclusion: Testosterone administered with low-intensity physical training improves grip strength, spontaneous movements, and respiratory activity. These functional improvements were associated with increased muscle mitochondrial biogenesis and improved mitochondrial quality control.
doi:10.1371/journal.pone.0051180
PMCID: PMC3519841  PMID: 23240002
12.  Sex Hormone–Binding Globulin, but Not Testosterone, Is Associated Prospectively and Independently With Incident Metabolic Syndrome in Men 
Diabetes Care  2011;34(11):2464-2470.
OBJECTIVE
The association between total testosterone and metabolic syndrome has prompted speculation that low testosterone contributes to the pathophysiology of metabolic syndrome in men. We determined whether testosterone or sex hormone–binding globulin (SHBG) is independently associated with the risk of metabolic syndrome.
RESEARCH DESIGN AND METHODS
Cross-sectional relationships of hormone levels with metabolic syndrome were assessed in a sample of men in generation 2 of the Framingham Heart Study (FHS) who did not receive testosterone or androgen-deprivation therapy (n = 1,625) and confirmed in a validation sample of men in FHS generation 3 (n = 1,912). Hormone levels in generation 2 examination 7 were related prospectively to incident metabolic syndrome 6.6 years later at examination 8. Testosterone was measured using liquid chromatography–tandem mass spectrometry, SHBG was measured by immunofluorometric assay, and free testosterone was calculated. Metabolic syndrome was defined using the National Cholesterol Education Program Adult Treatment Panel III criteria.
RESULTS
Cross-sectionally, testosterone and SHBG were more strongly associated with metabolic syndrome than free testosterone in the training sample. SHBG, but not testosterone or free testosterone, was significantly associated with metabolic syndrome after adjusting for age, smoking, BMI, and insulin sensitivity (homeostasis model assessment of insulin resistance [HOMA-IR]). These findings were confirmed in a validation sample. Longitudinally, SHBG at examination 7, but not testosterone or free testosterone, was associated with incident metabolic syndrome at examination 8 after adjusting for age, smoking, BMI, and HOMA-IR. Multivariable analyses suggested that age, BMI, and insulin sensitivity independently affect SHBG and testosterone levels and the risk of metabolic syndrome and its components.
CONCLUSIONS
SHBG, but not testosterone, is independently associated with the risk of metabolic syndrome. These data do not reveal an independent prospective relationship between testosterone and metabolic syndrome in men.
doi:10.2337/dc11-0888
PMCID: PMC3198304  PMID: 21926281
13.  Relation of Visceral Adiposity to Circulating Natriuretic Peptides in Ambulatory Individuals 
The American journal of cardiology  2011;108(7):979-984.
Natriuretic peptides have important roles in the regulation of vasomotor tone, salt homeostasis, and ventricular remodeling. Lower natriuretic peptide levels observed in obese individuals may underlie the greater cardiovascular risk associated with obesity. Thus, the aim of this study was to determine whether lower natriuretic peptide levels in obesity are attributable to differences in regional fat distribution. We investigated the relationship of plasma N-terminal pro-B-type natriuretic peptide (N-BNP) with regional adiposity in 1,873 community-based individuals (46% women; mean age 45 years). Subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) volumes were measured by multi-detector computed tomography. In sex-specific, multivariable analyses adjusting for age and blood pressure, log N-BNP was inversely associated with VAT in both men (β −0.11, P<0.001) and women (β −0.19, P<0.001). Log N-BNP was inversely associated with SAT in women only (β −0.14, P<0.001). In models containing both VAT and SAT, only VAT was significantly associated with log N-BNP (men, β −0.137, P<0.001; women, β −0.184, P<0.001). VAT remained associated with log N-BNP even after adjustment for body mass index and waist circumference (β −0.119, P<0.001), and in analyses restricted to non-obese individuals (β −0.114; P<0.001). Adjustment for insulin resistance attenuated the associations of N-BNP with both VAT and SAT. In conclusion, this study demonstrates that circulating N-BNP is related to variation in regional and particularly visceral adiposity. These findings suggest that excess visceral adiposity and concomitant hyperinsulinemia may contribute to the natriuretic peptide “deficiency” observed in obesity.
doi:10.1016/j.amjcard.2011.05.033
PMCID: PMC3175269  PMID: 21813106
adiposity; natriuretic peptides; cardiovascular risk
14.  Clinical Meaningfulness of the Changes in Muscle Performance and Physical Function Associated With Testosterone Administration in Older Men With Mobility Limitation 
Context.
Testosterone in Older Men with Mobility Limitations Trial determined the effects of testosterone on muscle performance and physical function in older men with mobility limitation. Trial’s Data and Safety Monitoring Board recommended enrollment cessation due to increased frequency of adverse events in testosterone arm. The changes in muscle performance and physical function were evaluated in relation to participant’s perception of change.
Methods.
Men aged 65 years and older, with mobility limitation, total testosterone 100–350 ng/dL, or free testosterone less than 50 pg/mL, were randomized to placebo or 10 g testosterone gel daily for 6 months. Primary outcome was leg-press strength. Secondary outcomes included chest-press strength, stair-climb, 40-m walk, muscle mass, physical activity, self-reported function, and fatigue. Proportions of participants exceeding minimally important difference in study arms were compared.
Results.
Of 209 randomized participants, 165 had follow-up efficacy measures. Mean (SD) age was 74 (5.4) years and short physical performance battery score 7.7 (1.4). Testosterone arm exhibited greater improvements in leg-press strength, chest-press strength and power, and loaded stair-climb than placebo. Compared with placebo, significantly greater proportion of men receiving testosterone improved their leg-press and chest-press strengths (43% vs 18%, p = .01) and stair-climbing power (28% vs 10%, p = .03) more than minimally important difference. Increases in leg-press strength and stair-climbing power were associated with changes in testosterone levels and muscle mass. Physical activity, walking speed, self-reported function, and fatigue did not change.
Conclusions.
Testosterone administration in older men with mobility limitation was associated with patient-important improvements in muscle strength and stair-climbing power. Improvements in muscle strength and only some physical function measures should be weighed against the risk of adverse events in this population.
doi:10.1093/gerona/glr100
PMCID: PMC3202898  PMID: 21697501
Testosterone; Minimally important difference; Mobility limitation; Older men; Function promoting therapies
15.  Value of measuring muscle performance to assess changes in lean mass with testosterone and growth hormone supplementation 
European journal of applied physiology  2011;112(3):1123-1131.
We hypothesized that treatment with testosterone (T) and recombinant human growth hormone (rhGH) would increase lean mass (LM) and muscle strength proportionally and an in a linear manner over 16 weeks. This was a multicenter, randomized, controlled, double-masked investigation of T and rhGH supplementation in older (71 ± 4 years) community-dwelling men. Participants received transdermal T at either 5 or 10 g/day as well as rhGH at 0, 3.0 or 5.0 µg/kg/day for 16 weeks. Body composition was determined by dual-energy X-ray absorptiometry (DEXA) and muscle performance by composite one-repetition maximum (1-RM) strength and strength per unit of lean mass (muscle quality, MQ) for five major muscle groups (upper and lower body) at baseline, week 8 and 17. The average change in total LM at study week 8 compared with baseline was 1.50 ± 1.54 kg (P < 0.0001) in the T only group and 2.64 ± 1.7 (P < 0.0001) in the T + rhGH group and at week 17 was 1.46 ± 1.48 kg (P < 0.0001) in the T only group and 2.14 ± 1.96 kg (P < 0.0001) in the T + rhGH group. 1-RM strength improved modestly in both groups combined (12.0 ± 23.9%, P < 0.0001) at week 8 but at week 17 these changes were twofold greater (24.7 ± 31.0%, P < 0.0001). MQ did not significantly change from baseline to week 8 but increased for the entire cohort, T only, and T + rhGH groups by week 17 (P < 0.001). Despite sizeable increases in LM measurements at week 8, tests of muscle performance did not show substantive improvements at this time point.
doi:10.1007/s00421-011-2077-y
PMCID: PMC3448487  PMID: 21748366
Anabolic hormones; Androgen supplementation; Muscle quality; HORMA study
16.  Adverse Events Associated with Testosterone Administration 
The New England journal of medicine  2010;363(2):109-122.
Background
Testosterone supplementation has been shown to increase muscle mass and strength in healthy older men. The safety and efficacy of testosterone treatment in older men who have limitations in mobility have not been studied.
Methods
Community-dwelling men, 65 years of age or older, with limitations in mobility and a total serum testosterone level of 100 to 350 ng per deciliter (3.5 to 12.1 nmol per liter) or a free serum testosterone level of less than 50 pg per milliliter (173 pmol per liter) were randomly assigned to receive placebo gel or testosterone gel, to be applied daily for 6 months. Adverse events were categorized with the use of the Medical Dictionary for Regulatory Activities classification. The data and safety monitoring board recommended that the trial be discontinued early because there was a significantly higher rate of adverse cardiovascular events in the testosterone group than in the placebo group.
Results
A total of 209 men (mean age, 74 years) were enrolled at the time the trial was terminated. At baseline, there was a high prevalence of hypertension, diabetes, hyperlipidemia, and obesity among the participants. During the course of the study, the testosterone group had higher rates of cardiac, respiratory, and dermatologic events than did the placebo group. A total of 23 subjects in the testosterone group, as compared with 5 in the placebo group, had cardiovascular-related adverse events. The relative risk of a cardiovascular-related adverse event remained constant throughout the 6-month treatment period. As compared with the placebo group, the testosterone group had significantly greater improvements in leg-press and chest-press strength and in stair climbing while carrying a load.
Conclusions
In this population of older men with limitations in mobility and a high prevalence of chronic disease, the application of a testosterone gel was associated with an increased risk of cardiovascular adverse events. The small size of the trial and the unique population prevent broader inferences from being made about the safety of testosterone therapy.
doi:10.1056/NEJMoa1000485
PMCID: PMC3440621  PMID: 20592293
17.  Influence of Sex and Hormone Status on Circulating Natriuretic Peptides 
Objectives
To assess the relationship between sex hormones and natriuretic peptide levels in community-based adults
Background
Women have higher circulating natriuretic peptide concentrations than men, but the mechanisms for these sex-related differences and the impact of hormone therapy are unclear. Experimental studies suggest that androgens may suppress natriuretic peptide secretion.
Methods
We measured plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP), total testosterone, and sex hormone binding globulin (SHBG) in 4,056 men and women (mean age 40±9 years) from the Framingham Heart Study Third Generation cohort. Sex/hormone status was grouped as: 1) men, 2) postmenopausal women not receiving hormone replacement therapy, 3) premenopausal women not receiving hormonal contraceptives, 4) postmenopausal women receiving hormone replacement therapy and 5) premenopausal women receiving hormonal contraceptives.
Results
Circulating NT-proBNP was associated with sex/hormone status (overall P<0.0001). Men had lower NT-proBNP than women of all menopause or hormone groups, and women receiving hormonal contraceptives had higher NT-proBNP than women who were not receiving hormone therapy (all P<0.0001). These relationships remained significant after adjusting for age, body mass index, and cardiovascular risk factors. Across sex/hormone status groups, FT decreased and SHBG increased in tandem with increasing NT-proBNP. In sex-specific analyses, NT-proBNP decreased across increasing quartiles of free testosterone in men (P for trend<0.01) and in women (P for trend<0.0001). Adjustment for FT markedly attenuated the association between sex/hormone status and NT-proBNP concentrations.
Conclusions
These findings suggest that lower circulating androgens and the potentiating effect of exogenous female hormone therapy contribute to the higher circulating NT-proBNP concentrations in women.
doi:10.1016/j.jacc.2011.03.042
PMCID: PMC3170816  PMID: 21798425
natriuretic peptides; sex; hormones
18.  Premature expression of a muscle fibrosis axis in chronic HIV infection 
Skeletal Muscle  2012;2:10.
Background
Despite the success of highly active antiretroviral therapy (HAART), HIV infected individuals remain at increased risk for frailty and declines in physical function that are more often observed in older uninfected individuals. This may reflect premature or accelerated muscle aging.
Methods
Skeletal muscle gene expression profiles were evaluated in three uninfected independent microarray datasets including young (19 to 29 years old), middle aged (40 to 45 years old) and older (65 to 85 years old) subjects, and a muscle dataset from HIV infected subjects (36 to 51 years old). Using Bayesian analysis, a ten gene muscle aging signature was identified that distinguished young from old uninfected muscle and included the senescence and cell cycle arrest gene p21/Cip1 (CDKN1A). This ten gene signature was then evaluated in muscle specimens from a cohort of middle aged (30 to 55 years old) HIV infected individuals. Expression of p21/Cip1 and related pathways were validated and further analyzed in a rodent model for HIV infection.
Results
We identify and replicate the expression of a set of muscle aging genes that were prematurely expressed in HIV infected, but not uninfected, middle aged subjects. We validated select genes in a rodent model of chronic HIV infection. Because the signature included p21/Cip1, a cell cycle arrest gene previously associated with muscle aging and fibrosis, we explored pathways related to senescence and fibrosis. In addition to p21/Cip1, we observed HIV associated upregulation of the senescence factor p16INK4a (CDKN2A) and fibrosis associated TGFβ1, CTGF, COL1A1 and COL1A2. Fibrosis in muscle tissue was quantified based on collagen deposition and confirmed to be elevated in association with infection status. Fiber type composition was also measured and displayed a significant increase in slow twitch fibers associated with infection.
Conclusions
The expression of genes associated with a muscle aging signature is prematurely upregulated in HIV infection, with a prominent role for fibrotic pathways. Based on these data, therapeutic interventions that promote muscle function and attenuate pro-fibrotic gene expression should be considered in future studies.
doi:10.1186/2044-5040-2-10
PMCID: PMC3407733  PMID: 22676806
Skeletal muscle; Aging; Gene expression; HIV infection; Senescence
19.  Testosterone Threshold Levels and Lean Tissue Mass Targets Needed to Enhance Skeletal Muscle Strength and Function: The HORMA Trial 
Background.
In the HORMA (Hormonal Regulators of Muscle and Metabolism in Aging) Trial, supplemental testosterone and recombinant human growth hormone (rhGH) enhanced lean body mass, appendicular skeletal muscle mass, muscle performance, and physical function, but there was substantial interindividual variability in outcomes.
Methods.
One hundred and twelve men aged 65–90 years received testosterone gel (5 g/d vs 10 g/d via Leydig cell clamp) and rhGH (0 vs 3 vs 5 μg/kg/d) in a double-masked 2 × 3 factorial design for 16 weeks. Outcomes included lean tissue mass by dual energy x-ray absorptiometry, one-repetition maximum strength, Margaria stair power, and activity questionnaires. We used pathway analysis to determine the relationship between changes in hormone levels, muscle mass, strength, and function.
Results.
Increases in total testosterone of 1046 ng/dL (95% confidence interval = 1040–1051) and 898 ng/dL (95% confidence interval = 892–904) were necessary to achieve median increases in lean body mass of 1.5 kg and appendicular skeletal muscle mass of 0.8 kg, respectively, which were required to significantly enhance one-repetition maximum strength (≥30%). Co-treatment with rhGH lowered the testosterone levels (quantified using liquid chromatography–tandem mass spectrometry) necessary to reach these lean mass thresholds. Changes in one-repetition maximum strength were associated with increases in stair climbing power (r = .26, p = .01). Pathway analysis supported the model that changes in testosterone and insulin-like growth factor 1 levels are related to changes in lean body mass needed to enhance muscle performance and physical function. Testosterone’s effects on physical activity were mediated through a different pathway because testosterone directly affected Physical Activity Score of the Elderly.
Conclusions.
To enhance muscle strength and physical function, threshold improvements in lean body mass and appendicular skeletal muscle mass are necessary and these can be achieved by targeting changes in testosterone levels. rhGH augments the effects of testosterone. To maximize functional improvements, the doses of anabolic hormones should be titrated to achieve target blood levels.
doi:10.1093/gerona/glq183
PMCID: PMC3032430  PMID: 21059836
Testosterone; Growth hormone; Lean body mass; Muscle performance; Physical function
20.  Testosterone Replacement and Resistance Exercise in HIV-Infected Men With Weight Loss and Low Testosterone Levels 
Context
Previous studies of testosterone supplementation in HIV-infected men failed to demonstrate improvement in muscle strength. The effects of resistance exercise combined with testosterone supplementation in HIV-infected men are unknown.
Objective
To determine the effects of testosterone replacement with and without resistance exercise on muscle strength and body composition in HIV-infected men with low testosterone levels and weight loss.
Design and Setting
Placebo-controlled, double-blind, randomized clinical trial conducted from September 1995 to July 1998 at a general clinical research center.
Participants
Sixty-one HIV-infected men aged 18 to 50 years with serum testosterone levels of less than 12.1 nmol/L (349 ng/dL) and weight loss of 5% or more in the previous 6 months, 49 of whom completed the study.
Interventions
Participants were randomly assigned to 1 of 4 groups: placebo, no exercise (n = 14); testosterone enanthate (100 mg/wk intramuscularly), no exercise (n = 17); placebo and exercise (n = 15); or testosterone and exercise (n = 15). Treatment duration was 16 weeks.
Main Outcome Measures
Changes in muscle strength, body weight, thigh muscle volume, and lean body mass compared among the 4 treatment groups.
Results
Body weight increased significantly by 2.6 kg (P<.001) in men receiving testosterone alone and by 2.2 kg (P = .02) in men who exercised alone but did not change in men receiving placebo alone (−0.5 kg; P = .55) or testosterone and exercise (0.7 kg; P = .08). Men treated with testosterone alone, exercise alone, or both experienced significant increases in maximum voluntary muscle strength in leg press (range, 22%–30%), leg curls (range, 18%–36%), bench press (range, 19%–33%), and latissimus pulls (range, 17%–33%). Gains in strength in all exercise categories were greater in men assigned to the testosterone-exercise group or to the exercise-alone group than in those assigned to the placebo-alone group. There was a greater increase in thigh muscle volume in men receiving testosterone alone (mean change, 40 cm3; P<.001 vs zero change) or exercise alone (62 cm3; P = .003) than in men receiving placebo alone (5 cm3; P = .70). Average lean body mass increased by 2.3 kg (P = .004) and 2.6 kg (P<.001), respectively, in men who received testosterone alone or testosterone and exercise but did not change in men receiving placebo alone (0.9 kg; P = .21). Hemoglobin levels increased in men receiving testosterone but not in those receiving placebo.
Conclusion
Our data suggest that testosterone and resistance exercise promote gains in body weight, muscle mass, muscle strength, and lean body mass in HIV-infected men with weight loss and low testosterone levels. Testosterone and exercise together did not produce greater gains than either intervention alone.
PMCID: PMC3173037  PMID: 10683055
21.  Habitual Physical Activity Levels are Associated with Performance in Measures of Physical Function and Mobility in Older Men 
Objectives
To determine whether objectively measured physical activity levels are associated with physical function and mobility in older men.
Design
Cross-sectional.
Setting
Academic research center.
Participants
Eighty-two community-dwelling men ≥ 65 years of age with self-reported mobility limitations were divided into a low activity and a high activity group based on the median average daily physical activity counts of the whole sample.
Measurements
Physical activity by triaxial accelerometers; physical function and mobility by the Short Physical Performance Battery (SPPB), gait speed, stair climb time, and a lift and lower task; aerobic capacity by maximum oxygen consumption (VO2max); and leg press and chest press maximal strength and peak power.
Results
Older men with higher compared to lower physical activity levels demonstrated a > 1.4 point higher mean SPPB score and a 0.35 m/s faster walking speed. They also climbed a standard flight of stairs 1.85 sec faster and completed 60% more shelves in a lift and lower task (all p < 0.01). Muscle strength and power measures, however, were not significantly different between the low and high activity group. Correlation analyses and multiple linear regression models showed that physical activity is positively associated with all physical function and mobility measures, leg press strength, and VO2max.
Conclusion
Older men with higher physical activity levels demonstrate better physical function and mobility than less active peers. Moreover, in older men physical activity levels are predictive of performance in measures of physical function and mobility. Future work is needed to determine whether modifications in physical activity levels can improve or preserve physical performance in later-life.
doi:10.1111/j.1532-5415.2010.03012.x
PMCID: PMC2945416  PMID: 20738436
aging; sarcopenia; muscle strength; disability; exercise
22.  The Dynamic Structure of the Estrogen Receptor 
Journal of Amino Acids  2011;2011:812540.
The estrogen receptor (ER) mediates most of the biological effects of estrogens at the level of gene regulation by interacting through its site-specific DNA and with other coregulatory proteins. In recent years, new information regarding the dynamic structural nature of ER has emerged. The physiological effects of estrogen are manifested through ER's two isoforms, ERα and ERβ. These two isoforms (ERα and ERβ) display distinct regions of sequence homology. The three-dimensional structures of the DNA-binding domain (DBD) and ligand-binding domain (LBD) have been solved, whereas no three-dimensional natively folded structure for the ER N-terminal domain (NTD) is available to date. However, insights about the structural and functional correlations regarding the ER NTD have recently emerged. In this paper, we discuss the knowledge about the structural characteristics of the ER in general and how the structural features of the two isoforms differ, and its subsequent role in gene regulation.
doi:10.4061/2011/812540
PMCID: PMC3268042  PMID: 22312471
23.  Identification of serum biomarkers for aging and anabolic response 
Objective
With the progressive aging of the human population, there is an inexorable decline in muscle mass, strength and function. Anabolic supplementation with testosterone has been shown to effectively restore muscle mass in both young and elderly men. In this study, we were interested in identifying serum factors that change with age in two distinct age groups of healthy men, and whether these factors were affected by testosterone supplementation.
Methods
We measured the protein levels of a number of serum biomarkers using a combination of banked serum samples from older men (60 to 75 years) and younger men (ages 18 to 35), as well as new serum specimens obtained through collaboration. We compared baseline levels of all biomarkers between young and older men. In addition, we evaluated potential changes in these biomarker levels in association with testosterone dose (low dose defined as 125 mg per week or below compared to high dose defined as 300 mg per week or above) in our banked specimens.
Results
We identified nine serum biomarkers that differed between the young and older subjects. These age-associated biomarkers included: insulin-like growth factor (IGF1), N-terminal propeptide of type III collagen (PIIINP), monokine induced by gamma interferon (MIG), epithelial-derived neutrophil-activating peptide 78 (ENA78), interleukin 7 (IL-7), p40 subunit of interleukin 12 (IL-12p40), macrophage inflammatory protein 1β (MIP-1β), platelet derived growth factor β (PDGFβ) and interferon-inducible protein 10 (IP-10). We further observed testosterone dose-associated changes in some but not all age related markers: IGF1, PIIINP, leptin, MIG and ENA78. Gains in lean mass were confirmed by dual energy X-ray absorptiometry (DEXA).
Conclusions
Results from this study suggest that there are potential phenotypic biomarkers in serum that can be associated with healthy aging and that some but not all of these biomarkers reflect gains in muscle mass upon testosterone administration.
doi:10.1186/1742-4933-8-5
PMCID: PMC3135554  PMID: 21689392
Testosterone; Age; Biomarker
24.  Sex Hormone–Binding Globulin as an Independent Predictor of Incident Type 2 Diabetes Mellitus in Men 
Background.
Low levels of sex hormone–binding globulin (SHBG) and total testosterone (T) in men have been associated with increased risk of type 2 diabetes mellitus (T2DM). As total T and SHBG levels are highly correlated, we determined whether SHBG influences the risk of T2DM through T or whether SHBG is an independent predictor of T2DM.
Methods.
Longitudinal analyses were conducted on men participating in the Massachusetts Male Aging Study, a population-based study of men aged 40–70 years. Of 1,709 men enrolled in 1987–1989, 1,156 were evaluated 7–10 years later and 853 after 15–17 years. Analyses were restricted to 1,128 men without T2DM at baseline.
Results.
Ninety new cases of T2DM were identified. After adjustment for age, body mass index, hypertension, smoking, alcohol intake, and physical activity, the hazard ratio (HR) for incident T2DM was 2.0 for each 1 SD decrease in SHBG (95% confidence interval [CI], 1.42–2.82, p < .001) and 1.29 for each 1 SD decrease in total T (95% CI, 1.01–1.66, p = .04). Free T was not associated with T2DM (HR = 1.03, 95% CI, 0.81–1.31, p = .79). The strong association of T2DM risk with SHBG persisted even after additional adjustment for free T (HR = 2.04, 95% CI, 1.44–2.87, p < .0001) or total T (HR = 1.95, 95% CI, 1.34–2.82, p = .0004).
Conclusions.
SHBG is an independent predictor of incident T2DM even after adjusting for free T or total T. Free T is not significantly associated with T2DM. SHBG may contribute to the risk of T2DM through nonandrogenic mechanisms, which should be investigated as they may provide novel targets for diabetes prevention.
doi:10.1093/gerona/glq002
PMCID: PMC2854882  PMID: 20106959
SHBG; Type-2 diabetes; Testosterone
25.  Eight Common Genetic Variants Associated with Serum DHEAS Levels Suggest a Key Role in Ageing Mechanisms 
PLoS Genetics  2011;7(4):e1002025.
Dehydroepiandrosterone sulphate (DHEAS) is the most abundant circulating steroid secreted by adrenal glands—yet its function is unknown. Its serum concentration declines significantly with increasing age, which has led to speculation that a relative DHEAS deficiency may contribute to the development of common age-related diseases or diminished longevity. We conducted a meta-analysis of genome-wide association data with 14,846 individuals and identified eight independent common SNPs associated with serum DHEAS concentrations. Genes at or near the identified loci include ZKSCAN5 (rs11761528; p = 3.15×10−36), SULT2A1 (rs2637125; p = 2.61×10−19), ARPC1A (rs740160; p = 1.56×10−16), TRIM4 (rs17277546; p = 4.50×10−11), BMF (rs7181230; p = 5.44×10−11), HHEX (rs2497306; p = 4.64×10−9), BCL2L11 (rs6738028; p = 1.72×10−8), and CYP2C9 (rs2185570; p = 2.29×10−8). These genes are associated with type 2 diabetes, lymphoma, actin filament assembly, drug and xenobiotic metabolism, and zinc finger proteins. Several SNPs were associated with changes in gene expression levels, and the related genes are connected to biological pathways linking DHEAS with ageing. This study provides much needed insight into the function of DHEAS.
Author Summary
Dehydroepiandrosterone sulphate (DHEAS), mainly secreted by the adrenal gland, is the most abundant circulating steroid in humans. It shows a significant physiological decline after the age of 25 and diminishes about 95% by the age of 85 years, which has led to speculation that a relative DHEAS deficiency may contribute to the development of common age-related diseases or diminished longevity. Twin- and family-based studies have shown that there is a substantial genetic effect with heritability estimate of 60%, but no specific genes regulating serum DHEAS concentration have been identified to date. Here we take advantage of recent technical and methodological advances to examine the effects of common genetic variants on serum DHEAS concentrations. By examining 14,846 Caucasian individuals, we show that eight common genetic variants are associated with serum DHEAS concentrations. Genes at or near these genetic variants include BCL2L11, ARPC1A, ZKSCAN5, TRIM4, HHEX, CYP2C9, BMF, and SULT2A1. These genes have various associations with steroid hormone metabolism—co-morbidities of ageing including type 2 diabetes, lymphoma, actin filament assembly, drug and xenobiotic metabolism, and zinc finger proteins—suggesting a wider functional role for DHEAS than previously thought.
doi:10.1371/journal.pgen.1002025
PMCID: PMC3077384  PMID: 21533175

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