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1.  Testosterone Dose-Response Relationships in Hysterectomized Women with and without Oophorectomy: Effects on Sexual Function, Body Composition, Muscle Performance and Physical Function in a Randomized Trial 
Menopause (New York, N.Y.)  2014;21(6):612-623.
To determine dose-dependent effects of testosterone on sexual function, body composition, muscle performance, and physical function in hysterectomized women with and without oophorectomy.
71 menopausal women who previously underwent hysterectomy with or without oophorectomy with total testosterone<31ng/dl or free testosterone<3.5 pg/ml received a standardized transdermal estradiol regimen during the 12-week run-in period, and were then randomized to receive weekly IM injections of placebo, or 3, 6.25, 12.5 or 25 mg testosterone enanthate for 24 weeks. Total and free testosterone levels were measured by LC-MS/MS and equilibrium dialysis, respectively. The primary outcome was change in sexual function measured using Brief Index of Sexual Function (BISF-W); Secondary outcomes included changes in sexual activity, sexual distress, DeRogatis Inventory of Sexual Function, lean (LBM) and fat mass, muscle strength and power, and physical function.
71 women were randomized; five groups were similar at baseline. 62 women with analyzable data for the primary outcome were included in the final analysis. Mean on-treatment total testosterone concentrations were 19, 78, 102, 128 and 210ng/dl in the placebo, 3, 6.25, 12.5 and 25-mg groups, respectively. Changes in composite BISF-W scores, thoughts-desire, arousal, frequency of sexual activity, LBM, chest-press power and loaded stair-climb power were significantly related to increases in free testosterone concentrations; changes were significantly greater in women assigned to the 25-mg group when compared to placebo but not at the lower dose groups. Sexual activity increased by 2.7 encounters per week in 25-mg group. Frequency of androgenic adverse events was low.
Testosterone administration in hysterectomized women with and without oophorectomy for 24-weeks was associated with dose and concentration-dependent gains in several domains of sexual function, LBM, chest-press power and loaded stair-climb power. Long-term trials are needed to weigh improvements in these outcomes against potential long-term adverse effects.
PMCID: PMC4701202  PMID: 24281237
Testosterone; menopause; oophorectomy; hysterectomy; sexual function; body composition; muscle performance; physical function
2.  Nutritional Recommendations for the Management of Sarcopenia 
The Society for Sarcopenia, Cachexia, and Wasting Disease convened an expert panel to develop nutritional recommendations for prevention and management of sarcopenia. Exercise (both resistance and aerobic) in combination with adequate protein and energy intake is the key component of the prevention and management of sarcopenia. Adequate protein supplementation alone only slows loss of muscle mass. Adequate protein intake (leucine-enriched balanced amino acids and possibly creatine) may enhance muscle strength. Low 25(0H) vitamin D levels require vitamin D replacement.
PMCID: PMC4623318  PMID: 20627179
Clinical trials (London, England)  2014;11(3):362-375.
The prevalence of low testosterone levels in men increases with age, as does the prevalence of decreased mobility, sexual function, self-perceived vitality, cognitive abilities, bone mineral density, and glucose tolerance, and of increased anemia and coronary artery disease. Similar changes occur in men who have low serum testosterone concentrations due to known pituitary or testicular disease, and testosterone treatment improves the abnormalities. Prior studies of the effect of testosterone treatment in elderly men, however, have produced equivocal results.
To describe a coordinated set of clinical trials designed to avoid the pitfalls of prior studies and determine definitively if testosterone treatment of elderly men with low testosterone is efficacious in improving symptoms and objective measures of age-associated conditions.
We present the scientific and clinical rationale for the decisions made in the design of this trial.
We designed The Testosterone Trials as a coordinated set of seven trials to determine if testosterone treatment of elderly men with low serum testosterone concentrations and also symptoms and objective evidence of impaired mobility and/or diminished libido and/or reduced vitality would be efficacious in improving mobility (Physical Function Trial), sexual function (Sexual Function Trial), fatigue (Vitality Trial), cognitive function (Cognitive Function Trial), hemoglobin (Anemia Trial), bone density (Bone Trial), and coronary artery plaque volume (Cardiovascular Trial). The scientific advantages of this coordination were common eligibility criteria, treatment and monitoring and the ability to pool safety data. The logistical advantages were a single steering committee, data coordinating center and data safety monitoring board (DSMB), the same clinical trial sites, and the possibility of men participating in multiple trials. The major consideration in subject selection was setting the eligibility criterion for serum testosterone low enough to ensure that the men were unequivocally testosterone deficient, but not so low as to preclude sufficient enrollment or eventual generalizability of the results. The major considerations in choosing primary end points for each trial were identifying those of the highest clinical importance and identifying the minimum clinically important differences between treatment arms for sample size estimation.
Potential Limitations
Setting the serum testosterone concentration sufficiently low to ensure that most men would be unequivocally testosterone deficient, as well as many other entry criteria, resulted in screening approximately 30 men in person to randomize one subject.
The Testosterone Trials were designed to determine definitively if testosterone treatment of elderly men with low testosterone would have any clinical benefit. Designing The Testosterone Trials as a coordinated set of seven trials afforded many important scientific and logistical advantages but required an intensive recruitment and screening effort.
PMCID: PMC4182174  PMID: 24686158
clinical trial; testosterone; hypogonadism; aging; mobility; sexual function; vitality; cognitive function; anemia; CT angiography; volumetric BMD; bone strength
4.  Dehydroepiandrosterone Secretion in Healthy Older Men and Women: Effects of Testosterone and Growth Hormone Administration in Older Men 
Aging is associated with diminished gonadal steroid and GH/IGF-I axis activity; whether these changes contribute to the parallel declines of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) production is unknown, as are the effects of sex steroid and/or GH administration on DHEA and DHEAS production.
Our objective was to evaluate morning DHEAS concentrations and nocturnal DHEA secretory dynamics in healthy older men and women, before and after chronic administration of sex steroid(s) alone, GH alone, sex steroid(s) combined with GH, or placebo alone.
We compared nocturnal DHEA secretory dynamics (2000 h to 0800 h, sampling every 20 min, analyzed by multiparameter deconvolution and approximate entropy algorithms) in healthy older (65–88 yr) men (n = 68) and women (n = 36), both before and after 26 wk of administration of sex steroid(s) alone [testosterone (T) in men or estrogen/progesterone in women], GH alone, sex steroid(s) combined with GH, or placebo alone.
Morning concentrations of DHEAS were lower; nocturnal DHEA pulsatile production rate, burst frequency, and amplitude were higher; and half-life was shorter in women (P < 0.05). Nocturnal integrated DHEA concentrations, total production rate, and approximate entropy did not differ significantly by sex. Because of small treatment group sizes in women, only hormone intervention results in men are presented. In men, T and T plus GH administration significantly decreased nocturnal integrated DHEA but not morning DHEAS concentrations. GH alone exerted no significant effects on nocturnal DHEA secretion or morning DHEAS.
Spontaneous nocturnal DHEA secretion is sexually dimorphic in healthy older individuals, and T administration decreases nocturnal DHEA secretion in older men. The clinical significance of sex steroid modulation of DHEA secretion in older persons remains to be elucidated.
PMCID: PMC4575787  PMID: 16926252
5.  Lopinavir/Ritonavir Impairs Physical Strength in Association with Reduced Igf1 Expression in Skeletal Muscle of Older Mice 
Late-middle age HIV patients are prone to fatigue despite effective viral control by antiretroviral therapies. Rodent models to recapitulate this phenotype are still not available.
Drug treatment may compromise muscle strength and physical performance more in older individuals with pre-existing metabolic disorders than normal young ones.
Kaletra was given to overweight male mice at late-middle age and normal young adults; both on a rodent diet containing 30% fat calorie. Body composition and grip strength were measured at baseline and after drug treatment. Rota-rod running, insulin and glucose tolerance were measured at the end of the experiment. Drug effect on metabolic activity and spontaneous movements were assessed using the metabolic cage system. Representative muscle and fat tissue were analyzed for protein and mRNA expression. Selected findings were tested using murine C2C12 myotubes.
Kaletra reduced grip strength in both young and older mice but impaired rotarod performance only in the old. Spontaneous movements were also reduced in Kaletra-treated old mice. Kaletra reduced IGF-1 expression in all muscle groups tested for the old and in cultured myotubes but to a less extent in the muscle of young animals. Reduced IGF-1 expression correlated with increased expression of muscle-specific atrogene MAFbx and MuRF1. Kaletra also increased abdominal fat mass markedly in the old animals and to a less extend in the young.
Long-term Kaletra intake aggravated abdominal obesity and impaired muscle strength. This effect was worse in older animals than in normal young adults.
PMCID: PMC4524660  PMID: 26251758
HIV protease inhibitor; Age and overweight; Insulin resistance; Insulin-like growth factor 1; Muscle; Adipose tissue; Physical strength and mobility
6.  Testosterone Induces Erythrocytosis via Increased Erythropoietin and Suppressed Hepcidin: Evidence for a New Erythropoietin/Hemoglobin Set Point 
The mechanisms by which testosterone increases hemoglobin and hematocrit remain unclear.
We assessed the hormonal and hematologic responses to testosterone administration in a clinical trial in which older men with mobility limitation were randomized to either placebo or testosterone gel daily for 6 months.
The 7%–10% increase in hemoglobin and hematocrit, respectively, with testosterone administration was associated with significantly increased erythropoietin (EPO) levels and decreased ferritin and hepcidin levels at 1 and 3 months. At 6 months, EPO and hepcidin levels returned toward baseline in spite of continued testosterone administration, but EPO levels remained nonsuppressed even though elevated hemoglobin and hematocrit higher than at baseline, suggesting a new set point. Consistent with increased iron utilization, soluble transferrin receptor (sTR) levels and ratio of sTR/log ferritin increased significantly in testosterone-treated men. Hormonal and hematologic responses were similar in anemic participants. The majority of testosterone-treated anemic participants increased their hemoglobin into normal range.
Testosterone-induced increase in hemoglobin and hematocrit is associated with stimulation of EPO and reduced ferritin and hepcidin concentrations. We propose that testosterone stimulates erythropoiesis by stimulating EPO and recalibrating the set point of EPO in relation to hemoglobin and by increasing iron utilization for erythropoiesis.
PMCID: PMC4022090  PMID: 24158761
Testosterone; Erythropoietin; Hepcidin; Ferritin.
7.  Testosterone Inhibits Adipogenic Differentiation in 3T3-L1 Cells: Nuclear Translocation of Androgen Receptor Complex with β-Catenin and T-Cell Factor 4 May Bypass Canonical Wnt Signaling to Down-Regulate Adipogenic Transcription Factors 
Endocrinology  2005;147(1):141-154.
Testosterone supplementation in men decreases fat mass; however, the mechanisms by which it inhibits fat mass are unknown. We hypothesized that testosterone inhibits adipogenic differentiation of preadipocytes by activation of androgen receptor (AR)/β-catenin interaction and subsequent translocation of this complex to the nucleus thereby bypassing canonical Wnt signaling. We tested this hypothesis in 3T3-L1 cells that differentiate to form fat cells in adipogenic medium. We found that these cells express AR and that testosterone and dihydrotestosterone dose-dependently inhibited adipogenic differentiation as analyzed by Oil Red O staining and down-regulation of CCAAT/enhancer binding protein-α and -δ and peroxisome proliferator-activated receptor-γ2 protein and mRNA. These inhibitory effects of androgens were partially blocked by flutamide or bicalutamide. Androgen treatment was associated with nuclear translocation of β-catenin and AR. Immunoprecipitation studies dem onstrated association of β-catenin with AR and T-cell factor 4 (TCF4) in the presence of androgens. Transfection of TCF4 cDNA inhibited adipogenic differentiation, whereas a dominant negative TCF4 cDNA construct induced adipogenesis and blocked testosterone's inhibitory effects. Our gene array analysis indicates that testosterone treatment led to activation of some Wnt target genes. Expression of constitutively activated AR fused with VP-16 did not inhibit the expression of CCAAT/enhancer binding protein-α in the absence of androgens. Testosterone and dihydrotestosterone inhibit adipocyte differentiation in vitro through an AR-mediated nuclear translocation of β-catenin and activation of downstream Wnt signaling. These data provide evidence for a regulatory role for androgens in inhibiting adipogenic differentiation and a mechanistic explanation consistent with the observed reduction in fat mass in men treated with androgens.
PMCID: PMC4417624  PMID: 16210377
8.  Testosterone Supplementation Improves Anemia in Aging Male Mice 
Whether aging alone causes anemia is still controversial. In this study, we show that 28-month-old male C57BL/6 mice, maintained in a pathogen-free environment, had significantly lower hemoglobin, hematocrit, and erythrocyte counts than young mice. The anemic condition aggravated further from 28 to 30 months. Old mice displayed increased erythropoietic activity, evidenced by an increase in reticulocyte counts, serum erythropoietin, and splenic expression of erythropoietic genes. An increase in late-stage erythroid progenitors was detected in spleen but not in bone marrow of the old mice. However, old mice also had lower serum iron and transferrin saturation, as well as lower erythrocyte iron incorporation rate. Testosterone supplementation restored serum iron status in old mice to levels similar to that of young adults, further upregulated splenic expression of erythropoietic genes, increased splenic erythroid progenitors, and significantly improved the red cell index. In conclusion, we found that mice can become anemic at very old age without apparent illness. The endogenous compensatory erythropoietic activity was insufficient to normalize the red cell index in old mice, either due to impaired iron homeostasis, ineffective erythropoiesis, or other unknown factors. Testosterone supplementation normalized the iron status and further stimulated splenic erythropoietic activity; both may contribute to improve the anemic condition in the old mice.
PMCID: PMC3991143  PMID: 23974081
Anemia; Aging; Testosterone; Erythropoiesis; Iron bioavailability.
9.  Hormonal regulators of muscle and metabolism in aging (HORMA): design and conduct of a complex, double masked multicenter trial 
Older persons often lose muscle mass, strength, and physical function. This report describes the challenges of conducting a complex clinical investigation assessing the effects of anabolic hormones on body composition, physical function, and metabolism during aging.
HORMA is a multicenter, randomized double masked study of 65–90-year-old community dwelling men with testosterone levels of 150–550 ng/dL and IGF-1 < 167 ng/dL. Subjects were randomized to transdermal testosterone (5 or 10 g/day) and rhGH (0, 3, or 5 μg/kg/day) for 16 weeks. Outcome measures included body composition by DEXA, MRI, and 2H2O dilution; muscle performance (strength, power, and fatigability), VO2peak, measures of physical function, synthesis/breakdown of myofibrillar proteins, other measures of metabolism, and quality of life.
Major challenges included delay in startup caused by need for 7 institutional contracts, creating a 142-page manual of operations, orientation and training, creating a 121-page CRF; enrollment inefficiencies; scheduling 16 evaluations/subject; overnight admissions for invasive procedures and isotope infusions; large data and image management and transfer; quality control at multiples sites; staff turnover; and replacement of a clinical testing site. Impediments were largely solved by implementation of a web-based data entry and eligibility verification; electronic scheduling for multiple study visits; availability of research team members to educate and reassure subjects; more frequent site visits to validate all source documents and reliability of data entry; and intensifying quality control in testing and imaging. The study exceeded the target goal of 108 (n =112) completely evaluable cases. Two interim DSMB meetings confirmed the lack of excessive adverse events, lack of center effects, comparability of subjects, and that distribution of subjects and enrollment will not jeopardize outcomes or generalizability of results.
Flexibility and rapidly solving evolving problems is critical when conducting highly complex multicenter metabolic studies.
PMCID: PMC4301418  PMID: 17942471
10.  Association of sex steroids, gonadotropins, and their trajectories with clinical cardiovascular disease and all-cause mortality in elderly men from the Framingham Heart Study 
Clinical endocrinology  2013;78(4):629-634.
Emerging data from longitudinal studies suggests that low sex steroid concentrations in men are associated with increased cardiovascular risk and mortality. The impact of longitudinal trajectory patterns from serial sex steroid and gonadotropin measurements on the observed associations is unknown to date.
We prospectively evaluated 254 elderly men (mean age: 75.5 years) of the Framingham Heart Study with up to four serial measurements of serum total testosterone (TT), dehydroepiandrosterone sulfate (DHEAS), follicle stimulating hormone (FSH), luteinizing hormone (LH), and total estradiol (EST); and constructed age- and multivariable-adjusted Cox proportional hazard regression models relating baseline hormone concentrations and their mean, slope, and variation over time (modelled as continuous and categorized into quartiles) to the incidence of clinical cardiovascular disease (CVD) and all-cause mortality at 5-years and 10-years of follow-up.
We observed no association between baseline concentrations of sex steroids, gonadotropins, and their trajectories with incident clinical CVD over 5-years and 10-years follow-up, respectively. Although higher baseline TT concentrations were associated with lower mortality risk at 5-years (hazard ratio per quartile increment, 0.74; 95% confidence interval, 0.56 – 0.98), correction for multiple statistical testing (p <0.005) rendered this association statistically non-significant. Repeat analyses at the 10-year follow-up time point also demonstrated no significant association between sex steroids, gonadotropins, or their trajectories and mortality.
Investigating longitudinal trajectory patterns of serial sex steroid and gonadotropin measurements, the present study found no consistent associations with incident clinical CVD and all-cause mortality risk in elderly men in the community.
PMCID: PMC4161203  PMID: 22901104
sex steroids; gonadotropins; testosterone; men; cardiovascular disease; trajectories; longitudinal; Framingham Heart Study
11.  Circulating Estrone Levels Are Associated Prospectively With Diabetes Risk in Men of the Framingham Heart Study 
Diabetes Care  2013;36(9):2591-2596.
In postmenopausal women and preclinical murine models, estrogen administration reduces diabetes risk; however, the relationship of estradiol and estrone to diabetes in men is poorly understood. We determined the relationship between circulating estradiol and estrone levels and diabetes risk in community-dwelling men of the Framingham Heart Study (FHS).
Cross-sectional relationships of estradiol and estrone levels with diabetes were assessed at examination 7 (1998–2001) in FHS generation 2 men (n = 1,458); prospective associations between hormone levels at examination 7 and incident diabetes were assessed 6.8 years later at examination 8. Type 2 diabetes mellitus was defined as fasting glucose >125 mg/dL, medication use, or both. Estradiol, estrone, and testosterone levels were measured with liquid chromatography–tandem mass spectrometry, and free estradiol and estrone were calculated.
In cross-sectional models, men with elevated estrone and estradiol had 40% and 62% increased likelihoods of existing diabetes per cross-sectional doubling of estrone and estradiol levels, respectively. Free estrone (cross-sectional odds ratio 1.28 [95% CI 1.02–1.62], P = 0.04) was associated with impaired fasting glucose at examination 7. There was an increase in risk of existing diabetes with increasing quartiles of total and free estrone and estradiol and an increase in risk of incident diabetes with increasing quartiles of estrone levels. In multivariate longitudinal analyses, a twofold increase in total or free estrone levels at examination 7 was associated with 77 and 93% increases, respectively, in odds of incident diabetes at examination 8.
Although both estradiol and estrone exhibit cross-sectional associations with diabetes in men, in longitudinal analyses estrone is a more sensitive marker of diabetes risk than is estradiol.
PMCID: PMC3747918  PMID: 23690532
12.  Effect of Testosterone Administration on Liver Fat in Older Men With Mobility Limitation: Results From a Randomized Controlled Trial 
Androgen receptor (AR) knockout male mice display hepatic steatosis, suggesting that AR signaling may regulate hepatic fat. However, the effects of testosterone replacement on hepatic fat in men are unknown. The aim of this study was to determine the effects of testosterone administration on hepatic fat in older men with mobility limitation and low testosterone levels who were participating in a randomized trial (the Testosterone in Older Men trial).
Two hundred and nine men with mobility limitation and low total or free testosterone were randomized in the parent trial to either placebo or 10-g testosterone gel daily for 6 months. Hepatic fat was determined by magnetic resonance imaging in 73 men (36 in placebo and 37 in testosterone group) using the volumetric method. Insulin sensitivity (homeostatic model assessment–insulin resistance) was derived from fasting glucose and insulin.
Baseline characteristics were similar between the two groups, including liver volumes (1583±363 in the testosterone group vs 1522±271mL in the placebo group, p = .42). Testosterone concentrations increased from 250±72 to 632±363ng/dL in testosterone group but did not change in placebo group. Changes in liver volume during intervention did not differ significantly between groups (p = .5) and were not related to on-treatment testosterone concentrations. The change in homeostatic model assessment–insulin resistance also did not differ significantly between groups and was not related to either baseline or change in liver fat.
Testosterone administration in older men with mobility limitation and low testosterone levels was not associated with a reduction in hepatic fat. Larger trials are needed to determine whether testosterone replacement improves liver fat in men with nonalcoholic hepatic steatosis.
PMCID: PMC3826861  PMID: 23292288
Testosterone; Older men; Liver fat; Insulin resistance.
13.  Age Trends in Estradiol and Estrone Levels Measured Using Liquid Chromatography Tandem Mass Spectrometry in Community-Dwelling Men of the Framingham Heart Study 
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.
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.
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.
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.
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.
14.  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 ( 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.
PMCID: PMC3630276  PMID: 23494931
Erectile dysfunction; testosterone replacement; mood; affectivity balance; well-being; androgen deficiency
15.  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.
PMCID: PMC3602280  PMID: 23399021
16.  Combined administration of testosterone plus an ornithine decarboxylase inhibitor as a selective prostate-sparing anabolic therapy 
Aging Cell  2013;13(2):303-310.
Because of its anabolic effects on muscle, testosterone is being explored as a function-promoting anabolic therapy for functional limitations associated with aging; however, concerns about testosterone’s adverse effects on prostate have inspired efforts to develop strategies that selectively increase muscle mass while sparing the prostate. Testosterone’s promyogenic effects are mediated through upregulation of follistatin. We show here that the administration of recombinant follistatin (rFst) increased muscle mass in mice, but had no effect on prostate mass. Consistent with the results of rFst administration, follistatin transgenic mice with constitutively elevated follistatin levels displayed greater muscle mass than controls, but had similar prostate weights. To elucidate signaling pathways regulated differentially by testosterone and rFst in prostate and muscle, we performed microarray analysis of mRNAs from prostate and levator ani of castrated male mice treated with vehicle, testosterone, or rFst. Testosterone and rFst shared the regulation of many transcripts in levator ani; however, in prostate, 593 transcripts in several growth-promoting pathways were differentially expressed after testosterone treatment, while rFst showed a negligible effect with only 9 transcripts differentially expressed. Among pathways that were differentially responsive to testosterone in prostate, we identified ornithine decarboxylase (Odc1), an enzyme in polyamine biosynthesis, as a testosterone-responsive gene that is unresponsive to rFst. Accordingly, we administered testosterone with and without α-difluoromethylornithine (DFMO), an Odc1 inhibitor, to castrated mice. DFMO selectively blocked testosterone’s effects on prostate, but did not affect testosterone’s anabolic effects on muscle. Co-administration of testosterone and Odc1 inhibitor presents a novel therapeutic strategy for prostate-sparing anabolic therapy.
PMCID: PMC4331775  PMID: 24305501
aging; anti-aging; sarcopenia; sex hormones; skeletal muscle; steroids
17.  Risk Factors Associated With Cardiovascular Events During Testosterone Administration in Older Men With Mobility Limitation 
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.
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.
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.
Mobility-limited older men who experienced cardiovascular events had greater increases in serum free testosterone levels than those who did not.
PMCID: PMC3598355  PMID: 22562960
Testosterone; Older men; Mobility limitation; Cardiovascular disease
18.  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.
PMCID: PMC3735638  PMID: 23868854
19.  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.
PMCID: PMC3598367  PMID: 22499765
Muscle regeneration; Testosterone
20.  The Safety, Pharmacokinetics, and Effects of LGD-4033, a Novel Nonsteroidal Oral, Selective Androgen Receptor Modulator, in Healthy Young Men 
Concerns about potential adverse effects of testosterone on prostate have motivated the development of selective androgen receptor modulators that display tissue-selective activation of androgenic signaling. LGD-4033, a novel nonsteroidal, oral selective androgen receptor modulator, binds androgen receptor with high affinity and selectivity.
To evaluate the safety, tolerability, pharmacokinetics, and effects of ascending doses of LGD-4033 administered daily for 21 days on lean body mass, muscle strength, stair-climbing power, and sex hormones.
In this placebo-controlled study, 76 healthy men (21–50 years) were randomized to placebo or 0.1, 0.3, or 1.0 mg LGD-4033 daily for 21 days. Blood counts, chemistries, lipids, prostate-specific antigen, electrocardiogram, hormones, lean and fat mass, and muscle strength were measured during and for 5 weeks after intervention.
LGD-4033 was well tolerated. There were no drug-related serious adverse events. Frequency of adverse events was similar between active and placebo groups. Hemoglobin, prostate-specific antigen, aspartate aminotransferase, alanine aminotransferase, or QT intervals did not change significantly at any dose. LGD-4033 had a long elimination half-life and dose-proportional accumulation upon multiple dosing. LGD-4033 administration was associated with dose-dependent suppression of total testosterone, sex hormone–binding globulin, high density lipoprotein cholesterol, and triglyceride levels. follicle-stimulating hormone and free testosterone showed significant suppression at 1.0-mg dose only. Lean body mass increased dose dependently, but fat mass did not change significantly. Hormone levels and lipids returned to baseline after treatment discontinuation.
LGD-4033 was safe, had favorable pharmacokinetic profile, and increased lean body mass even during this short period without change in prostate-specific antigen. Longer randomized trials should evaluate its efficacy in improving physical function and health outcomes in select populations.
PMCID: PMC4111291  PMID: 22459616
Selective androgen receptor modulators; SARMs; Sarcopenia; Function promoting anabolic therapies; Cachexia
21.  Clinical correlates of sex steroids and gonadotropins in men over the late adulthood : the Framingham Heart Study 
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.
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.
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.
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.
PMCID: PMC3434249  PMID: 22640232
sex steroids; gonadotropins; testosterone; aging male; Framingham Heart Study
22.  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.
PMCID: PMC3731267  PMID: 23936482
23.  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.
PMCID: PMC3264813  PMID: 22138414
Transforming growth factor-β; follistatin; myostatin; myosin heavy chain II
24.  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.
Determine the durability of anabolic effects and adverse events (AEs) after stopping testosterone and growth hormone supplementation in older men.
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.
108 community-dwelling 65-90 year-old-men.
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.
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.
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.
PMCID: PMC3529980  PMID: 21521283
Lean body mass; fat mass; muscle performance; quality of life; cardiovascular risks
25.  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.
PMCID: PMC3519841  PMID: 23240002

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