Shalender Bhasin

Context: Free hormone hypothesis (FHH) posits that free testosterone crosses plasma membrane to activate intracellular signaling and is biologically active fraction of circulating testosterone. Widely accepted in other fields, FHH has been debated in andrology. Objective: To disentangle the roles of free and total testosterone, we studied men with greatly elevated sex hormone-binding globulin (SHBG) levels and primary hypogonadism, who had marked divergence in total and free testosterone concentrations, in whom elevated gonadotropins provided independent evidence of primary testicular insufficiency. Design: Prospective hormonal evaluation of men with greatly elevated SHBG. Outcomes: Total and free testosterone, LH, FSH, symptom ascertainment Results: Among 20 men with SHBG>100 nmol/L, 15 met Endocrine Society's criteria for primary hypogonadism: low free testosterone, one or more symptoms/signs, elevated LH and/or FSH. Among 15 men with elevated LH and/or FSH and unequivocally low free testosterone who met the criteria for primary hypogonadism, 4 had total testosterone that exceeded the reference range (>916 ng/dL) and 8 had total testosterone >450 ng/dL, substantially above the lower normal limit. Conclusion: In men with markedly elevated SHBG, in whom total and free testosterone diverged substantially, low free testosterone was associated with elevated gonadotropins, providing independent evidence of primary testicular insufficiency even when total testosterone levels were above the reference range or substantially above the lower normal limit. Although sample size was small, these illustrative “n-of-1” observations in uncommon patients are consistent with the primacy of free testosterone over total testosterone in the hypothalamic–pituitary–testicular feedback loop to regulate LH and FSH, and support the FHH.

Context: Spinal cord injury (SCI) leads to profound muscle atrophy, aerobic deconditioning, and metabolic dysfunction. Exercise-based interventions alone produce modest benefits. Whether testosterone can augment physiologic responses to exercise in this population remains untested. Objective: To evaluate efficacy and safety of home-based intervention combining functional electrical stimulation–assisted leg cycling (FES-LC), arm ergometry (AE) and testosterone compared with FES-LC, AE plus placebo in adults with SCI. Methods: This randomized, placebo-controlled, double-blind trial enrolled 84 adults (76 males and 8 females) aged 19-70 years with SCI (neurologic levels C4-T12; AIS grades A-D). Participants were randomized to multimodality intervention (home-based FES-LC, AE and intramuscular testosterone undecanoate) (n=38) or control intervention (FES-LC, AE plus placebo) (n=46) for 16 weeks. The primary outcome was change in aerobic capacity (peak VO2) during AE cardiopulmonary exercise testing. Secondary outcomes included lean mass, hemoglobin, cardiometabolic markers and safety. Results: Mean (SD) age was 44 (13) yrs and time since injury was 13.9 (13) years). Between-group changes in peak VO2 were not statistically significant. Within-group improvements were larger in multimodality (∼19% increase; 0.10 L/min; 95% CI, 0.02–0.18 L/min) compared to controls (∼6% increase; 0.06 L/min; 95% CI, –0.01-0.13). The multimodality group gained significantly more lean mass (whole-body:1.84 kg, 95% CI: 0.52-3.16, P =.007; lower extremity 0.92 kg, 95% CI: 0.38-1.45, P =.001), and anemia was corrected in a greater proportion of participants. Adverse event rates were similar between groups. Conclusions: A home-based multimodality intervention combining FES-LC, AE, and testosterone was safe and associated with greater improvements in lean mass and hemoglobin. Although between-group differences in aerobic capacity were not statistically significant, greater within-group increases were observed in the multimodality group. These findings may inform future studies of testosterone-augmented exercise interventions for individuals living with SCI.

Background: Circulating estradiol is predominantly protein-bound, with human serum albumin (HSA) serving as its major carrier. While traditionally considered a carrier with low affinity and readily reversible binding at a single site, the molecular details and kinetics of estradiol-HSA interactions remain incompletely understood. Methods: We employed equilibrium dialysis, steady-state and time-resolved fluorescence spectroscopy to characterize estradiol-HSA interactions. Surface plasmon resonance (SPR) was used to elucidate the kinetics of estradiol's association and dissociation with HSA. Structural and energetic features of binding were investigated using molecular docking and structure network analyses. Results: Binding isotherms generated using equilibrium dialysis, steady-state and time-resolved fluorescence spectroscopy revealed nonlinear asymmetric binding with apparent Kd that varied as a function of estradiol and HSA concentrations, inconsistent with canonical model of low-affinity, single-site interaction characterized by a fixed Kd. Kinetic analyses by SPR revealed initial rapid association dynamics followed by a slower second phase. Molecular modeling identified a high-affinity estradiol-binding pocket in Sudlow's Site I and two additional low-affinity sites within a highly interconnected hub of structural blocks. Spatially coordinated conformational rearrangements accompanying estradiol partitioning into the high-affinity pocket of Sudlow's Site I and two additional moderate-affinity sites suggest an allosterically-coupled binding architecture that enables albumin to actively regulate estradiol bioavailability across a broad, physiologically relevant concentration range. Conclusion: Estradiol's binding to HSA is a dynamic, multi-equilibrium process driven by ligand-induced conformational rearrangements within HSA; the binding data are inconsistent with canonical model of estradiol-HSA interaction with 1:1 stoichiometry and a fixed Kd.

Context: Based on cross-sectional studies and short longitudinal studies, levels of sex hormones (SHs) change with age in men. However, there are limited data about SHs in men older than age 80 years and few longitudinal studies >10 years. Objective: The aim of this study was to characterize SH concentrations in older men and assess long-term changes. Methods: We studied 477 community-dwelling men, ages 78 to 97 years, who participated in the Osteoporotic Fractures in Men study. Medical history was obtained via clinical visits and questionnaires. Serum sex steroid levels (testosterone, estradiol, estrogen, DHT) were assayed using liquid chromatography/mass spectrometry, free testosterone by equilibrium dialysis, and sex hormone binding globulin (SHBG) by radioimmunoassay. In 215 men, SH levels were compared to levels obtained 14.0 ± 0.65 years earlier. Results: In cross-sectional analyses, mean total testosterone levels were 413.9 ± 180.0, with considerable variation among participants. In 21.4%, total testosterone levels were <275 ng/dL. Similarly, directly measured free testosterone levels were low ( 275 ng/dL at baseline, testosterone levels decreased to <275 ng/dL in 20.5%. Conclusion: In old men, there was a broad range of total testosterone levels, and a significant fraction were in a range considered hypogonadal. In longitudinal analyses, although there was considerable interindividual variation, gradual declines in mean total testosterone continued in the 8th through 10th decades of life.

Testosterone treatment increases muscle mass, maximal voluntary muscle strength, aerobic capacity, and some measures of physical function. Activational and epigenetic mechanisms by which androgens improve muscle mass and physical performance and how to apply these anabolic effects to treat functional limitations associated with aging and disease remain incompletely understood. Testosterone treatment induces hypertrophy of type 1 and 2 muscle fibers and increases muscle progenitor cell numbers by promoting differentiation of mesenchymal progenitor cells into myogenic lineage by an androgen receptor (AR)-mediated pathway. Liganded AR binds to β-catenin, translocates into nucleus where it binds T cell factor-4, and upregulates follistatin that blocks signaling through the TGF-β pathway to promote myogenesis and inhibit adipogenesis. Testosterone increases myoblast proliferation by stimulating polyamine biosynthesis. Stimulation of GH and IGF-1 secretion, intramuscular IGF-1 receptor, and muscle protein synthesis and inhibition of muscle atrophy genes further contribute to testosterone's anabolic effects. Testosterone improves muscle bioenergetics by increasing erythrocytes, oxygen availability, tissue blood flow, and mitochondrial mass and quality. Testosterone increases blood flow by nongenomic mechanisms involving nitric oxide production and calcium and potassium channels in vascular smooth muscle. The conversion of testosterone to 5α-DHT is not required for mediating its anabolic effects. Mechanisms of testosterone's sexually dimorphic epigenetic and tissue-specific activational effects and roles of α-keto reductase and steroid 5α-reductase and 1-carbon and polyamine metabolism in testosterone's actions remain poorly understood. Strategies to translate testosterone-induced muscle mass and strength gains into patient-important improvements in functional performance and health outcomes are needed to enable its clinical applications to treat functional limitations associated with aging and disease.

Nicotinamide adenine dinucleotide (NAD ⁺ ) plays an important role in the innate immune response and is depleted during SARS‐CoV‐2 infection due to increased turnover. It is unknown whether treatment with NAD ⁺ precursors can safely raise NAD ⁺ levels in patients with COVID‐19. To determine whether MIB‐626 ( β‐ nicotinamide mononucleotide), an NAD ⁺ precursor, can safely increase blood NAD ⁺ levels and attenuate acute kidney injury (AKI) and inflammation in hospitalized patients with COVID‐19, 42 adults, ≥ 18 years, hospitalized with COVID‐19 and AKI, were randomized in a 3:2 ratio to MIB‐626 1.0‐g or placebo tablets twice daily for 14 days. Circulating NAD ⁺ and its metabolites, markers of AKI, inflammation, and disease severity, were assessed. MIB‐626 treatment significantly but gradually raised blood NAD ⁺ levels to a peak between 5 to 14 days (16.0 ± 6.9, 25.5 ± 12.6, and 42.6 ± 25.6 μg/mL at baseline, days 5 and 14) and raised plasma concentrations of NAD ⁺ metabolites 1‐methylnicotinamide, N‐methyl, 2‐pyridone, 4‐carboxamide rapidly to a peak by day 3. Changes in serum creatinine, cystatin‐C, and serum markers of AKI did not differ significantly between groups. Serum CRP, IL‐6, and TNFα and indices of disease severity also did not differ between groups. MIB‐626 treatment of patients with COVID‐19 and AKI safely and substantially raised blood NAD ⁺ and plasma concentrations of NAD ⁺ metabolites. Markers of AKI, inflammation, and disease severity did not differ between groups, likely due to the slow rise in NAD ⁺ levels. Future studies should assess whether a rapid increase in NAD ⁺ by parenteral administration can attenuate disease severity and AKI. Trial Registration: ClinicalTrials.gov Identifier: NCT05038488

Using a modified Delphi process, we engaged 28 experts in clinical trials, geriatrics, and research translation to determine if there were consensus around what clinical endpoints should be used for trials evaluating the efficacy of interventions to prevent or treat multiple age-related conditions. Four focus groups developed themes. Statements related to those themes were circulated back to participants in a survey. There was consensus (more than 66% agreed or disagreed) that outcome measures should include multiple health dimensions including-age-related disease, function and patient reported outcomes that reflect participants goals; and be tailored to population characteristics. Experts felt that blood-based biomarkers would be unlikely to be accepted as primary endpoints of efficacy trials. Plausible components mentioned as part of a composite endpoint included mortality, mobility function and the onset of multiple age-related diseases. Our findings provide guidance on acceptable approaches to endpoint selection guiding the design of future geroscience trials.

Context: Whether circulating testosterone, dihydrotestosterone, and estradiol levels or testosterone replacement therapy (TRT) affects the risk of COVID-19 and whether COVID-19 affects response to TRT remains unknown. Objective: The study evaluated whether baseline testosterone, dihydrotestosterone, and estradiol levels or TRT are associated with risk of developing COVID-19 and whether COVID-19 affects treatment response to TRT. Methods: Among 5204 men, aged 45 to 80 years, with hypogonadism in the TRAVERSE trial, 379 developed COVID-19. We compared baseline and on-treatment hormone levels, and safety and efficacy in participants with and without COVID-19 diagnosis. Results: Neither baseline nor on-treatment testosterone, estradiol, and dihydrotestosterone levels prior to COVID-19 differed significantly between men with and without COVID-19 diagnosis. Incidence of COVID-19 was similar in participants randomized to TRT or placebo groups (3-year Kaplan-Meier incidence 8.0% in TRT and 8.6% in placebo group, P = .823). Incidences of COVID-19-related hospitalizations (38.5% vs 32.8%, P = .222) and deaths (12.8% vs 8.9%, P = .247) were similar in the TRT and placebo groups. Changes in hypogonadal symptoms, libido, energy, and hemoglobin/hematocrit in response to TRT were attenuated in testosterone-treated men who developed COVID-19. Incidences of major adverse cardiovascular events, venous thromboembolism, and acute kidney injury were similar in those with COVID-19 diagnosis and those without. Conclusion: In men with hypogonadism and cardiovascular disease (CVD) or increased risk of CVD, baseline and pre-COVID-19 on-treatment testosterone, dihydrotestosterone, and estradiol levels were similar in those who developed COVID-19 and those who did not. TRT did not affect the risk of COVID-19. COVID-19 attenuated the treatment response to TRT.