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Type 2 diabetes has emerged as an important risk factor for poor prognosis in hospitalized patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hyperglycemia can independently predispose the patients with SARS-CoV-2 to adverse outcomes, irrespective of the diagnosis of diabetes.1 Despite the accentuated risk, there is limited evidence about the complex interplay of undiagnosed diabetes and SARS-CoV-2 pneumonia on the clinical outcomes. We propose to investigate the cardiovascular events and clinical outcomes in patients with undiagnosed and type 2 diabetes hospitalized with SARS-CoV-2 pneumonia. Methods This retrospective study included patients hospitalized with SARS-CoV-2 pneumonia between March 7, 2020, through March 30, 2021. The diagnosis of SARS-CoV-2 pneumonia required a positive reverse transcriptase polymerase chain reaction (RT-PCR) for SARS-CoV-2, presence of new or worsening pulmonary infiltrates on computed tomography scan or chest X-ray, and at least one of the following: new or increased cough, a temperature of >37.8 °C or dyspnea. The diagnosis of undiagnosed diabetes was determined in patients with no previous history of diabetes and glycated hemoglobin (HbA1c) ≥ 6.5% (48 mmol/mol) or an admission serum glucose ≥ 200 mg/dL (≥ 11.1 mmol/mol). The outcomes included in-hospital cardiovascular events, intensive care unit (ICU) admissions and in-hospital mortality. Logistic regression was used to estimate adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for association of undiagnosed diabetes and type 2 diabetes for individual outcomes. Results Among the 1,645 adults hospitalized with SARS-CoV-2 pneumonia, 107 (10.8%) patients were identified with undiagnosed diabetes and 634 (39%) had established diagnosis of type 2 diabetes. Patients with undiagnosed diabetes had increased odds of developing cardiovascular events (OR: 1.73, 95% CI 1. 07, 2.80), ICU admissions (OR: 1.61, 95% CI 1.10, 2.34), and mortality (OR: 1.77, 95% CI 1. 02, 3. 07), compared to patients type 2 diabetes and no diabetes. A prior history of type 2 diabetes was not significantly associated with primary study outcomes. Conclusions Patients with undiagnosed diabetes hospitalized with SARS-CoV-2 pneumonia have an increased risk of developing cardiovascular complications and overall poor clinical outcomes compared to patients with type 2 diabetes and no diabetes. Implementation of healthcare-wide strategies for screening and early intervention of prediabetes and diabetes are needed to improve the disease outcomes in the current pandemic and future public health threats. References Khunti, K., et al., COVID-19, Hyperglycemia, and New-Onset Diabetes. Diabetes Care, 2021. 44(12): p. 2645-2655. Presentation: Saturday, June 11, 2022 1:00 p.m. - 2:00 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Electronic nicotine delivery systems or electronic cigarettes (e-cigarettes) are becoming exceptionally popular in the world as an alternative to conventional nicotine cigarettes, both among smokers and people who have never smoked. Nicotine can induce lipolysis in adipose tissue, leading to increased serum free fatty acids (FFAs). Increased levels of FFAs are one of the key elements in generating a pro-inflammatory response and lead to ectopic lipid accumulation, lipotoxicity, mitochondrial dysfunction, and DNA damage. Our laboratory has shown that e-cigarettes induce cardiac dysfunction associated with oxidative stress and inflammatory phenotype. We investigated the effects of acipimox, an antihyperlipidemic drug that blocks lipolysis, on e-cigarettes-induced cardiac dysfunction and its associated inflammatory signals and oxidative stress. C57BL/6J wild type mice on high fat diet were exposed to saline, e-cigarette with nicotine (2.4%), e-cigarette (2.4%) plus acipimox for 12 weeks. Fractional shortening and ejection fraction was decreased in mice exposed to e-cigarette (2.4%) compared with saline and acipimox. Therefore, acipimox normalized the e-cigarette-induced cardiac dysfunction. Transcriptomic evaluation with Gene Set Enrichment Analysis revealed that e-cigarette treated mice had genes enriched in the G2/M DNA damage checkpoint pathways. These transcriptomic changes were normalized by acipimox. Mice exposed to e-cigarettes had increased circulating levels of M-CSF, IL-6, and FFAs, which were decreased by acipimox. Mice exposed to e-cigarette (2.4%) had increased cardiac Heme oxygenase 1, a marker of oxidative stress. Additionally, treatment with e-cigarette (2.4%) increased the apurinic/apyrimidinic sites. This marker of oxidative DNA damage was normalized by acipimox. Understanding the consequences of e-cigarette use on the cardiovascular system is directly relevant to developing policies related to e-cigarette use. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Background Sedentary lifestyles and changes in diet are fueling the worldwide epidemic of obesity and the prevalence of NAFLD and insulin resistance (IR). It has been predicted that NAFLD and its more advanced form, nonalcoholic steatohepatitis (NASH) will be the next epidemic in chronic liver diseases, and NAFLD/NASH will soon replace viral hepatitis as the primary cause of end-stage liver failure and transplantation. NAFLD is a significant risk factor for IR and cardiovascular diseases, and it highlights the importance of finding a way to control the epidemic. CARF is a multifunctional stress-responsive gene and was found to be reduced in response to metabolic stress in the fatty liver of diet-induced obesity mice (DIO). We also showed that CARF expression was down-regulated in palmitate (PA)-treated HepG2 cells. In this study, we aim to understand the consequences of the reduced expression of CARF on the development of NAFLD. Methods We performed RNA seq, RT-PCR and western blotting (WB) to evaluate the effect of CARF knockdown on lipid metabolism in HepG2 cells. Differentially expressed genes were analyzed by gene sort enrichment analysis (GSEA) and Ingenuity Pathway Analysis (IPA) to identify the metabolic pathways affected by CARF depletion. Apoptosis was assessed by TUNEL assay. To induce ER stress, HepG2 cells were treated with thapsigargin and evaluated its effect on CARF. Results By performing Sh-RNA mediated CARF knockdown, RNA-seq analysis, RT-PCR, western blotting (WB), we were able to show that ER-stress and de-novo lipogenesis pathways were significantly affected in HepG2 cells. We showed that GRP78, CHOP, PERK, ERN1a, genes associated with ER-stress were upregulated in CARF-depleted HepG2 cells. Additionally, thapsigargin-induced ER stress was found to reduce the expression of CARF along with the increased expression of ER stress marker genes. We also showed that overexpression of CARF mitigated the thapsigargin-induced ER stress suggesting that CARF protects against ER stress in HepG2 cells. In addition, the genes associated with triglyceride biosynthesis GPAT3, GPAM were significantly upregulated, indicating that lipogenesis was triggered by silencing of CARF in HepG2 cells. BODIPY staining and TG assay confirmed that silencing of CARF enhanced triglyceride biosynthesis in HepG2 cells. Furthermore, we showed that the expression of antioxidant genes GPX2, GPX3, and TXRND3 decreased, resulting in enhanced oxidative stress and higher apoptosis in CARF depleted cells. Conclusion We identified novel roles of CARF regulating cellular ER-Stress, lipid metabolism, and oxidative stress, and its impairment in response to metabolic stress could lead to the development of NAFLD in obese patients with metabolic abnormalities. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 12:42 p.m. - 12:47 p.m.

Background Prostate cancer (PCa) is a leading cause of cancer-associated death among men worldwide. Although localized prostate cancer can be cured by surgery and radiation therapy, metastatic PCa remains a challenge. Androgen therapy (ADT) and androgen signaling inhibitors are the first line of therapy against PCa. However, resistance against these treatments develops, leading to the emergence of castration resistance prostate cancer (CRPC). Studies are required to identify the pathways that contribute to the emergence of CRPC and identify pharmaceutical targets. CARF is a putative transcription regulator reported to play a pivotal role in different cancers, including breast cancer and hepatocarcinoma. We observed that CARF is highly expressed in prostate tumors, but the biological function of CARF in PCa is unknown yet. Methods We performed RNA seq, RT-PCR, and western blot (WB) analyses to show how knockdown of CARF affects the global gene expression in PC3 cells. Gene sort enrichment analysis (GSEA) and Ingenuity Pathway Analysis (IPA) of differentially expressed genes were done to identify the cellular pathways affected upon silencing of CARF. Scratch assay and trans-well migration assay were conducted to show how CARF overexpression influences the motility of the cells. The effect of CARF on PC3 cells growth and proliferation was evaluated by colony-forming and MTT assays. In addition, we analyzed patient datasets to show the relevance of CARF expression, androgen signaling, and epithelial-mesenchymal transformation (EMT) pathways in PCa. Results The silencing of CARF inhibited the growth and proliferation of PC3 cells, suggesting the necessity of CARF for PC3 growth and survival. In agreement, overexpression of CARF enhanced the PC3 cells proliferation. We found that silencing of CARF reduced PC3 cells motility in trans-well assay. Mechanistically, RNA seq analysis after CARF knockdown uncovered that gene of AR signaling and EMT pathways were significantly altered in PC3 cells. RT-PCR and WB data confirmed that silencing of CARF enhanced E-cadherin expression and reduced the expression of N-cadherin in PC3 cells, suggesting that CARF regulates EMT in PCa. Silencing of CARF altered the AR-regulated metastatic genes expression in PC3 cells. Our data revealed that the expression of PMEPA1, a negative regulator of metastasis, was increased, but the expression of SGK1, an inducer of EMT, was decreased in CARF cells. Furthermore, CARF could regulate the prostaglandins metabolism by suppressing the expression of HGPD1 and triggering inflammation and angiogenesis in prostate cancer. Conclusion We conclude that by controlling the AR and EMT signaling pathways, CARF may play a crucial role in the development of metastatic CRPC. Future studies by integrating the RNA seq and Chip-Seq will uncover how CARF regulates the AR and EMT signaling pathways contributing to the development and progression of CRPC and paving a way to find a target for intervention. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m., Sunday, June 12, 2022 1:18 p.m. - 1:23 p.m.

Electronic nicotine delivery systems (ENDS) or electronic cigarettes (e-cigs) are becoming exceptionally popular in the world as an alternative to conventional nicotine cigarettes, both in smokers and people who have never smoked. Use of tobacco products is a major risk factor for diabetes and contribute to non-alcoholic fatty liver disease (NAFLD). Nicotinamide adenine dinucleotide (NAD+) plays a critical role in regulating metabolism and aging. Our laboratory has shown that ENDS induces lipolysis and NAFLD in Apolipoprotein E Knockout (ApoE-/-). We developed an ENDS exposure model that delivers nicotine in a manner similar to that of human ENDS users. ApoE-/- mice were exposed to saline, ENDS without nicotine [ENDS (0%)] and ENDS with 2.4% nicotine [(ENDS (2.4%)] aerosol for 12 weeks. Mice exposed to ENDS (2.4%) had increased apurinic/apyrimidinic (AP) sites (a manifestation of DNA damage) associated with a decreased NAD+/NADH ratio in the liver, in comparison with saline and ENDS (0%). Western blot analysis shows that mice treated with ENDS (2.4%) had increased poly(ADP-ribose) polymerases 1 (PARP-1) activity associated with reduced levels of Sirtuin 1 (SIRT1). Furthermore, hepatic oxidative stress and mitochondrial DNA mutations were increased in mice treated with ENDS (2.4%). These results demonstrate adverse effects of ENDS leading to NAD+ deficiency which is a common central pathological factor of a number of metabolic- and aging-associated diseases.

Smoking is a major risk factor for diabetes, cardiovascular disease, and nonalcoholic fatty liver disease. The health risk associated with smoking can be exaggerated by obesity. We hypothesize that nicotine when combined with a high-fat diet (HFD) can also cause ectopic lipid accumulation in skeletal muscle, similar to recently observed hepatic steatosis. Adult C57BL6 male mice were fed a normal chow diet or HFD and received twice-daily ip injections of nicotine (0.75 mg/kg body weight) or saline for 10 weeks. Transmission electron microscopy of the gastrocnemius muscle revealed substantial intramyocellular lipid accumulation in close association with intramyofibrillar mitochondria along with intramyofibrillar mitochondrial swelling and vacuolization in nicotine-treated mice on an HFD compared with mice on an HFD treated with saline. These abnormalities were reversed by acipimox, an inhibitor of lipolysis. Mechanistically, the detrimental effect of nicotine plus HFD on skeletal muscle was associated with significantly increased oxidative stress, plasma free fatty acid, and muscle triglyceride levels coupled with inactivation of AMP-activated protein kinase and activation of its downstream target, acetyl-coenzyme A-carboxylase. We conclude that 1) greater oxidative stress together with inactivation of AMP-activated protein kinase mediates the effect of nicotine on skeletal muscle abnormalities in diet-induced obesity and 2) adipose tissue lipolysis is an important contributor of muscle steatosis and mitochondrial abnormalities.

Smoking is a major risk factor for diabetes and cardiovascular disease and may contribute to nonalcoholic fatty liver disease. We hypothesize that in the presence of nicotine, high-fat diet (HFD) causes more severe hepatic steatosis in obese mice. Adult C57BL6 male mice were fed a normal chow diet or HFD and received twice daily injections of nicotine (0.75 mg/kg body weight, ip) or saline for 10 wk. Light microscopic image analysis revealed significantly higher lipid accumulation in livers from mice on HFD plus nicotine (190 ± 19 μm(2)), compared with mice on HFD alone (28 ± 1.2 μm(2)). A significant reduction in the percent volume of endoplasmic reticulum (67.8%) and glycogen (49.2%) was also noted in hepatocytes from mice on HFD plus nicotine, compared with mice on HFD alone. The additive effects of nicotine on the severity of HFD-induced hepatic steatosis was associated with significantly greater oxidative stress, increased hepatic triglyceride levels, higher incidence of hepatocellular apoptosis, inactivation (dephosphorylation) of AMP-activated protein kinase, and activation of its downstream target acetyl-coenzyme A-carboxylase. Treatment with acipimox, an inhibitor of lipolysis, significantly reduced nicotine plus HFD-induced hepatic lipid accumulation. We conclude that: 1) greater oxidative stress coupled with inactivation of AMP-activated protein kinase mediate the additive effects of nicotine and HFD on hepatic steatosis in obese mice and 2) increased lipolysis is an important contributor to hepatic steatosis. We surmise that nicotine exposure is likely to exacerbate the metabolic abnormalities induced by high-fat intake in obese patients.

Sertoli cells play a key role in triggering and regulating the process of spermatogenesis. Failure of a Sertoli cell to mature functionally will presumably render it incapable of supporting germ cell survival and development that appeared after puberty. Expression of cytokeratin 18 (ck-18) intermediate filaments indicates a state of undifferentiation usually observed in Sertoli cells of prepubertal testis. In this study we demonstrated that local testicular heat treatment of adult monkey with water at 43 C for 30 min once daily for 2 consecutive days was capable of activating reexpression of ck-18 in Sertoli cells, which was coincident with activation of ERK1/2 and Akt kinases. Using primary Sertoli cell culture isolated from adult monkey testis, we further confirmed that the heat treatment of the cells at 43 C could also induce ck-18 reexpression, which was similar to the in vivo treatment. ERK MAPK was also induced by the heat treatment in a time- and protein kinase A (PKA)-dependent manner. After blocking the ERK MAPK signaling pathway, an inhibition of ck-18 expression in the cultured Sertoli cells was observed, and this inhibitory effect was also detected by blocking the PKA activation. However, ck-18 activation in Sertoli cells remained unaltered when the phosphatidylinositol 3-kinase/Akt pathway was blocked. In conclusion, the heat treatment of adult monkey Sertoli cells are capable of inducing a reversible change in the Sertoli cells from an adult differentiated state to an immature-like dedifferentiated state through PKA-ERK MAPK-dependent pathways but not via the phosphatidylinositol 3-kinase/Akt pathway.

The paucity of pharmacokinetic data on androgen formulations in women has hindered clinical trials of testosterone supplementation in women.The objective of this study was to determine the time course and profile of serum testosterone concentrations during treatment with different doses of testosterone gel in postmenopausal women and assess whether estrogen treatment affects the pharmacokinetics of testosterone gel.Postmenopausal women with total testosterone levels less than 33 ng/dl after baseline 24-h sampling were treated with 4.4, 8.8, or 13.2 mg testosterone gel daily for 7 d each in random order, with a 7-d washout period between doses. We studied 13 women who had not received estrogen therapy (group I) and 13 who had received stable estrogen therapy for 3 months or more (group II). Total and free testosterone concentrations were measured for 48 h on the seventh day of each dose administration.Twenty-six women were randomized; of these, 24 were evaluable, 13 in group I and 11 in group II. The average steady-state concentrations (Cav) of serum total and free testosterone increased with increasing testosterone dose and were highly correlated with the dose (dose effect, P0.00001), but were not affected by estrogen therapy (P = 0.43). In both groups, the 4.4-mg dose increased Cav total and free testosterone into the mid- to high-normal range, whereas 8.8- and 13.2-mg doses raised total (Cav: 22.3, 51.6, 80.3, and 92.0 ng/dl in group I; 22.7, 59.8, 82.0, and 114.3 ng/dl in group II at 0, 4.4, 8.8, and 13. 2 mg, respectively) and free testosterone (5.9, 8.4, 11.5,12.8 pg/ml in group I and 5.0,7.6,11.1,10.8 in group II, respectively, at the various doses) above the physiological range. The area under the curve, maximum and minimum concentrations, and the change in Cav for total and free testosterone were dose related and significantly higher during administration of the 13.2-mg dose than during the 0- or 4.4-mg dose; estrogen therapy had no significant effect on these measures. Serum estradiol, LH, FSH, and SHBG levels did not change significantly at any dose. Testosterone gel was well tolerated.Administration of testosterone gel to postmenopausal women raised total and free testosterone concentrations in proportion to the administered dose without affecting estradiol levels. A 4.4-mg dose raised testosterone levels into the mid- to high-normal range. Previous estrogen therapy had no significant effect on testosterone pharmacokinetics over this short duration.

Klinefelter syndrome (XXY males) is the most common sex chromosome aneuploidy. XXY mice were generated by using a four-generation breeding scheme that involves the use of a structurally rearranged Y chromosome, Y*, yielding approximately 50% of the live-born male offspring in the fourth generation with a XXY karyotype. Adult XXY mice have small testes, decreased plasma T levels, and elevated plasma FSH levels. The testes of adult XXY mice contained small seminiferous tubules with intraepithelial vacuolization and absence of germ cells, whereas Leydig cells appeared to be more abundant than their XY littermates. Androgen receptor immunoexpression was localized in Leydig cells and peritubular myoid cells in both XY and XXY mice. Androgen receptor immunoexpression was abundant in the Sertoli cells of XY mice but nearly absent in those of XXY mice. The testicular phenotype was marked by a 23.1% decrease in testis weights in XXY pups beginning at d 7 after birth. Gonocyte numbers were similar in XY and XXY mice at d 1 of age, followed by a 62.6% decrease in the number of gonocytes in the XXY mice on d 3 and further progressive loss in spermatogonia by d 5 and 7. On d 10, only a few spermatogonia remained in the XXY mice. To determine whether the phenotype of XXY mice extended into the neurobehavioral domain, studies were conducted demonstrating impairment of learning and memory function in XXY mice. We conclude that adult XXY mice have testicular failure and learning deficits, similar to its human counterpart, Klinefelter syndrome.

Context: The relationships between testosterone dose and its effects on sexual function, mood, and visuospatial cognition are poorly understood. Objective: To elucidate testosterone dose-response relationships in older men, we examined the effects of graded testosterone doses on sexual function, mood, and visuospatial cognition in healthy, older men (age, 60–75 yr). Setting: This study was performed at the General Clinical Research Center. Intervention/Methods: Subjects each received a long-acting GnRH agonist to suppress endogenous testosterone production and were randomized to receive one of five doses (25, 50, 125, 300, and 600 mg) oftestosteroneenanthateweeklyfor20wk.Questionnaireswereused to evaluate sexual function. Scores for overall sexual function as well as subcomponents of sexual function (libido, sexual activity, and erectile function) were calculated. Results: Changes in overall sexual function (P 0.003) and waking erections (P 0.024) differed by dose. An interaction between libido and being sexually active was observed, such that libido changed by testosterone dose only among men who reported being sexually active at the beginning of the study (P 0.009). Men’s log-transformed free testosterone levels during treatment were positively correlated with overall sexual function (P 0.001), waking erections (P 0.040), spontaneouserections(P0.047),andlibido(P0.027),butnotwith intercourse frequency (P 0.428) or masturbation frequency (P 0.814). No effects of testosterone dose were observed on two measures of mood: Hamilton’s Depression Inventory (P 0.359) and Young’s Mania Scale (P 0.851). The number of trials completed on a computer-based test of visuospatial cognition differed by dose (P0.042), but the number of squares correctly completed on this task did not differ by dose (P 0.159). Conclusions: Different aspects of male behavior respond differently to testosterone. When considered together with previous data from young men, these data indicate that testosterone dose-response relationships for sexual function and visuospatial cognition differ in older and young men. (J Clin Endocrinol Metab 90: 3838–3846, 2005)

The objective of this study was to determine whether physiological testosterone replacement increases fat-free mass (FFM) and muscle strength and contributes to weight maintenance in HIV-infected women with relative androgen deficiency and weight loss. Fifty-two HIV-infected, medically stable women, 18-50 yr of age, with more than 5% weight loss over 6 months and testosterone levels below 33 ng/dl were randomized into this double-blind, placebo-controlled trial of 24-wk duration. Subjects in the testosterone group applied testosterone patches twice weekly to achieve a nominal delivery of 300 mug testosterone over 24 h. Data were evaluable for 44 women. Serum average total and peak testosterone levels increased significantly in the testosterone group, but did not change in the placebo group. However, there were no significant changes in FFM (testosterone, 0.7 +/- 0.4 kg; placebo, 0.3 +/- 0.4 kg), fat mass (testosterone, 0.3 +/- 0.7 kg; placebo, 0.6 +/- 0.7 kg), or body weight (testosterone, 1.0 +/- 0.9 kg; placebo, 0.9 +/- 0.8 kg) between the two treatment groups. There were no significant changes in leg press strength, leg power, or muscle fatigability in either group. Changes in quality of life, sexual function, cognitive function, and Karnofsky performance scores did not differ significantly between the two groups. High-density lipoprotein cholesterol levels decreased significantly in the testosterone group. The patches were well tolerated. We conclude that physiological testosterone replacement was safe and effective in raising testosterone levels into the mid to high normal range, but did not significantly increase FFM, body weight, or muscle performance in HIV-infected women with low testosterone levels and mild weight loss. Additional studies are needed to fully explore the role of androgens in the regulation of body composition in women.

Previous studies of Delta 4-androstene-3,17-dione (4-androstenedione) administration in men have not demonstrated sustained increments in testosterone levels, fat-free mass (FFM), and muscle strength, and failure to demonstrate androstenedione's androgenic/anabolic effects has stifled efforts to regulate its sales. To determine whether 4-androstenedione has androgenic/anabolic properties, we evaluated its association with androgen receptor (AR) and its effects on myogenesis in vitro. Additionally, we studied the effects of a high dose of 4-androstenedione on testosterone levels, FFM, and muscle strength in hypogonadal men. We determined the dissociation constant (K(d)) for 4-androstenedione using fluorescence anisotropy measurement of competitive displacement of fluorescent androgen from AR ligand-binding domain. AR nuclear translocation and myogenic activity of androstenedione were evaluated in mesenchymal, pluripotent C3H10T1/2 cells, in which androgens stimulate myogenesis through an AR pathway. We determined effects of a high dose of androstenedione (500 mg thrice daily) given for 12 wk on FFM, muscle strength, and hormone levels in nine healthy, hypogonadal men. 4-Androstenedione competitively displaced fluorescent androgen from AR ligand-binding domain with a lower affinity than dihydrotestosterone (K(d), 648 +/- 21 and 10 +/- 0.4 nm, respectively). In C3H10T1/2 cells, 4-androstenedione caused nuclear translocation of AR and stimulated myogenesis, as indicated by a dose-dependent increase in myosin heavy chain II+ myotube area and up-regulation of MyoD protein. Stimulatory effects of 4-androstenedione on myosin heavy chain II+ myotubes and myogenic determination factor expression were attenuated by bicalutamide, an AR antagonist. Administration of 1500 mg 4-androstenedione daily to hypogonadal men significantly increased serum androstenedione, total and free testosterone, estradiol, and estrone levels and suppressed SHBG and high-density lipoprotein cholesterol levels. 4-androstenedione administration was associated with significant gains in FFM (+1.7 +/- 0.5 kg; P = 0.012) and muscle strength in bench press (+4.3 +/- 3.1 kg; P = 0.006) and leg press exercises (+18.8 +/- 17.3 kg; P = 0.045). 4-androstenedione is an androgen that binds AR, induces AR nuclear translocation, and promotes myogenesis in vitro, with substantially lower potency than dihydrotestosterone. 4-androstenedione administration in high doses to hypogonadal men increases testosterone levels, FFM, and muscle strength, although at the dose tested, the anabolic effects in hypogonadal men are likely because of its conversion to testosterone.

Short-term exposure (43 C for 15 min) of the rat testis to mild heat results within 6 h in stage- and cell-specific activation of germ cell apoptosis. Initiation of apoptosis was preceded by a redistribution of Bax from a cytoplasmic to paranuclear localization in heat-susceptible germ cells. Here we show that the relocation of Bax is accompanied by cytosolic translocation of cytochrome c and is associated with activation of the initiator caspase 9 and the executioner caspases 3, 6, and 7 and cleavage of poly(ADP) ribose polymerase. Furthermore, early in apoptosis, a significant amount of Bax also accumulates in endoplasmic reticulum, as assessed by Western blot analyses of fractionated testicular lysates. In additional studies using the FasL-defective gld mice, we have shown that heat-induced germ cell apoptosis is not blocked, thus providing evidence that the Fas signaling system may be dispensable for heat-induced germ cell apoptosis in the testis. Taken together, these results demonstrate that the mitochondria- and possibly also endoplasmic reticulum-dependent pathways are the key apoptotic pathways for heat-induced germ cell death in the testis.

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

Androgen deficiency is common in men with end stage renal disease (ESRD) on maintenance hemodialysis. Pharmacokinetics of transdermal testosterone in men receiving maintenance hemodialysis have not been studied. Our objective was to compare the pharmacokinetics of a transdermal testosterone system in healthy hypogonadal men and in men with ESRD on maintenance hemodialysis. We recruited 10 healthy hypogonadal men and 8 medically stable men on maintenance hemodialysis, 18--70 yr old, who had serum testosterone less than 300 ng/dL. After baseline sampling during a 24-h control period, two testosterone patches were applied daily for 28 days, to achieve a nominal delivery of 10-mg testosterone daily. In addition to single, pooled samples on days 7, 14, and 21, blood was drawn at 0, 2, 4, 6, 8, and 24 h on day 28 in healthy hypogonadal men and on an interdialytic day (day 21 or 28) as well as a dialysis day (day 21 or 28) in men on hemodialysis. On the dialysis day (day 21 or 28), serum free and total testosterone levels were measured hourly for 4 h before hemodialysis and for 4 h during hemodialysis. The dialysate was sampled for testosterone measurement. Baseline mean + SD total (92 +/- 82 vs. 222 +/- 50 ng/dL) and free (11 +/- 9 vs. 27 +/- 6 pg/mL) testosterone concentrations were lower in healthy hypogonadal men than in men with ESRD. After application of two testosterone patches, serum total and free testosterone concentrations rose into the midnormal range in both groups of men. Time-average, steady state (total testosterone, 506 +/- 88 vs. 516 +/- 86 ng/dL; free testosterone, 55 +/- 9 vs. 67 +/- 11 pg/mL), minimum, and maximum total and free testosterone concentrations were not significantly different between the two groups of men during treatment. Increments in total and free testosterone concentrations above baseline, baseline-subtracted areas under the total and free testosterone curves, and half-life of testosterone elimination (t(1/2), 2.1 +/- 0.1 vs. 2.1 +/- 0.2 h, P = not significant) were not significantly different between the two groups. In men receiving hemodialysis, time-average, steady state, and maximal total and free testosterone concentrations and baseline-subtracted areas under the total and free testosterone curves were higher on dialysis day than on an interdialytic day. On the day of hemodialysis, time-average total and free testosterone concentrations were not significantly different during the 4 h before or during hemodialysis. The amount of testosterone removed in the dialysate (8.4 +/- 1.6 microg during 4 h of hemodialysis) was small compared with the daily testosterone production rates in healthy young men. Serum dihydrotestosterone and estradiol concentrations increased into the normal male range and were not significantly different between the two groups. Percent suppression of LH was greater in men with ESRD than in healthy hypogonadal men. A regimen of two Testoderm TTS testosterone patches (Alza Corp., Mountain View, CA) daily can maintain serum concentrations of total and free testosterone and its metabolites dihydrotestosterone and estradiol in the midnormal range in healthy hypogonadal men and men on hemodialysis. The amount of testosterone cleared by hemodialysis is small, and hemodialysis does not significantly affect serum total and free testosterone concentrations in men treated with the testosterone patch.

Klinefelter syndrome (47,XXY) is the most common sex chromosome aneuploidy in men. Thus, it is important to establish an experimental animal model to explore its underlying molecular mechanisms. Mice with a 41,XXY karyotype were produced by mating wild-type male mice with chimeric female mice carrying male embryonic stem cells. The objectives of the present study were to characterize the testicular phenotype of adult XXY mice and to examine the ontogeny of loss of germ cells in juvenile XXY mice. In the first experiment the testicular phenotypes of four adult XXY mice and four littermate controls (40,XY) were studied. XXY mice were identified by either Southern hybridization or karyotyping and were further confirmed by fluorescence in situ hybridization. The results showed that the testis weights of adult XXY mice (0.02 +/- 0.01 g) were dramatically decreased compared with those of the controls (0.11 +/- 0.01 g). Although no significant differences were apparent in plasma testosterone levels, the mean plasma LH and FSH levels were elevated in adult XXY mice compared with controls. The testicular histology of adult XXY mice showed small seminiferous tubules with varying degrees of intraepithelial vacuolization and a complete absence of germ cells. Hypertrophy and hyperplasia of Leydig cells were observed in the interstitium. Electron microscopic examination showed Sertoli cells containing scanty amounts of cytoplasm and irregular nuclei with prominent nucleoli. The junctional region between Sertoli cells appeared normal. In some tubules, nests of apparently degenerating Sertoli cells were found. In the second experiment the ontogeny of germ cell loss in juvenile XXY mice and their littermate controls was studied. Spermatogonia were found and appeared to be morphologically normal in juvenile XXY mice. Progressive loss of germ cells occurred within 10 days after birth. This resulted in the absence of germ cells in the adult XXY mice. We conclude that a progressive loss of germ cells occurring in early postnatal life results in the complete absence of germ cells in adult XXY mice. The XXY mouse provides an experimental model for its human XXY counterpart, Klinefelter syndrome.

The clinical consequences of androgen deficiency in human immunodeficiency virus (HIV)-infected women remain underappreciated. The pharmacokinetics of transdermally administered testosterone in premenopausal women and HIV-infected women have not been studied. In this study we compared the pharmacokinetics of a novel testosterone matrix transdermal system (TMTDS) in healthy premenopausal women and women infected with HIV. Eight menstruating HIV-infected women, 18-50 yr of age, who had been receiving stable antiretroviral therapy, including a protease inhibitor, for at least 12 weeks and nine healthy, menstruating women of comparable age were enrolled. After baseline sampling during a 24-h control period in the early follicular phase (days 1-6), two TMTDS patches were applied with an expected delivery rate of 300 microg testosterone daily over an application period of 3-4 days. After 72 h, the patches were removed, a second set of two patches was applied, and blood samples were drawn over 96 h. Baseline serum total and free testosterone levels were lower in HIV-infected women than in healthy women. A diurnal rhythm of testosterone secretion, with higher levels in the morning and lower levels in the late afternoon, was apparent in both groups of women. Free testosterone levels were in the midnormal range at baseline in healthy women and increased above the upper limit of normal during TMTDS application. In HIV-infected women, free testosterone levels were in the low normal range at baseline and rose into the upper normal range during patch application. Serum total testosterone levels increased into the midnormal range in HIV-infected women and into the upper normal range in healthy women during patch application. The mean increments in free and total testosterone levels were significantly lower in HIV-infected women than in healthy women. Testosterone bioavailability, expressed as the mean +/- SEM baseline-subtracted area under the total testosterone curve, was significantly greater in healthy women than in HIV-infected women [3323 +/- 566 ng/dL x h (115 +/- 20 nmol/L x h) vs. 1506 +/- 316 ng/dL x h (52 +/- 11 nmol/ L x h); P = 0.016]. Assuming a daily testosterone delivery rate of 300 microg/day, the apparent plasma clearance was significantly higher in HIV-infected women than in healthy women (2531 +/- 469 vs. 1127 +/- 217 L/day1 P = 0.022), respectively. There was no significant change from baseline in serum LH, sex hormone-binding globulin, and estradiol levels in either group. Serum FSH levels showed a greater decrease from baseline in healthy women. A regimen of two testosterone patches applied twice a week can maintain serum total and free testosterone levels in the mid- to upper normal range, respectively, in HIV-infected women with low testosterone levels. During TMTDS application, the increments in serum total and free testosterone levels are lower in HIV-infected women than in healthy women, presumably due to increased plasma clearance or decreased absorption. Further studies are needed to assess the effects of physiological androgen replacement in HIV-infected women.

The objectives of the study were to determine stage-specific changes in the kinetics of germ cell apoptosis induced by administration of exogenous testosterone (T) alone and to examine whether addition of a single testicular heat exposure would enhance the induction of germ cell apoptosis and the suppression of spermatogenesis by T. Adult male rats were implanted with 3-cm SILASTIC brand capsules (Dow Corning Corp.) containing T for up to 6 weeks. Intratesticular T levels declined to 2.9% of control values by 1 week and remained suppressed at 2, 3, and 6 weeks after T administration. The incidence of germ cell apoptosis (expressed as numbers per 100 Sertoli cells) was low in control rats (0-9.52). After T treatment, the mean incidence of apoptosis at stages VII-VIII increased significantly by 1 week (21.43 +/-3.33) and showed further increases by 6 weeks (56.30 +/- 7.47); apoptotic rates remained low at early (I-VI) and later (XII-XIV) stages. To test whether the combination of T with a single testicular heat exposure resulted in more complete suppression of spermatogenesis than either treatment alone, four groups of adult rats received one of the following treatments: 1) a subdermal empty polydimethylsilozane implant, 2) exposure to a single testicular heating (43 C for 15 min) applied on day 14, 3) 3-cm T implant, or 4) 3-cm T implant and a single testicular heat exposure (applied on day 14). All animals were killed at the end of 6 weeks. In the heat-treated group, testis weight and testicular sperm counts were decreased to 65.4% and 28.9% of control levels, respectively. The corresponding values in the T-treated group were 49.7% and 24.9% of control levels, respectively. Notably, addition of heat to T further reduced testis weight to 31.1% of control levels and testicular sperm counts to near zero. Histomorphometric analysis showed that all treatments reduced seminiferous tubular diameter and epithelial and luminal volume, with the greatest decrease after combined T and heat treatment. Heat exposure in animals bearing T implants markedly reduced the number of pachytene spermatocytes and round spermatids through apoptosis, resulting in tubules devoid of mature spermatids. Spermatogonia and preleptotene spermatocytes remained unaffected. These results clearly demonstrate that 1) exogenous T reduces intratesticular T and induces apoptosis mainly at stages VII-VIII within 1-6 weeks; 2) the combined treatment of T and heat markedly inhibits spermatogenesis, resulting in near azoospermia within 6 weeks; and 3) meiosis and spermiogenesis are the most vulnerable phases of spermatogenesis in response to T plus heat treatment. These findings suggest that a combination of hormonal treatment such as T and a physical agent (heat exposure) is more effective in suppressing spermatogenesis than either treatment alone. We hypothesize that combination of two antispermatogenic agents ("two hit") working at separate stages of the spermatogenic cycle will lead to greater male contraceptive efficacy.

Short term exposure of the testis to heat causes degeneration of germ cells. However, the mechanisms underlying this process are poorly understood. The major objectives of this study were to determine whether the heat-induced loss of germ cells in the adult rat occurs via apoptosis, to document its stage-specific and cell-specific distribution, and to examine whether intratesticular testosterone (T) plays any role in the stage specificity of heat-induced germ cell death. Testes of adult male Sprague-Dawley rats were exposed to 22 C (control) or 43 C for 15 min. Animals were killed on days 1, 2, 9, and 56 after heat exposure. Germ cell apoptosis was characterized by DNA gel electrophoresis and in situ terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling assay. The incidence of germ cell apoptosis [apoptotic index (AI)] was quite low in control rats (AI = 0.04–0.1). Mild hyperthermia within 1 or 2 days resulted in a marked activation (AI = 4.7–5.6) of germ cell apoptosis predominantly ...

Although weight loss associated with human immunodeficiency virus (HIV) infection is multifactorial in its pathogenesis, it has been speculated that hypogonadism, a common occurrence in HIV disease, contributes to depletion of lean tissue and muscle dysfunction. We, therefore, examined the effects of testosterone replacement by means of Androderm, a permeation-enhanced, nongenital transdermal system, on lean body mass, body weight, muscle strength, health-related quality of life, and HIV-disease markers. We randomly assigned 41 HIV-infected, ambulatory men, 18–60 yr of age, with serum testosterone levels below 400 ng/dL, to 1 of 2 treatment groups: group I, two placebo patches (n = 21); or group II, two testosterone patches designed to release 5 mg testosterone over 24 h. Eighteen men in the placebo group and 14 men in the testosterone group completed the 12-week treatment. Serum total and free testosterone and dihydrotestosterone levels increased, and LH and FSH levels decreased in the testosterone-treated, but not in the placebo-treated, men. Lean body mass and fat-free mass, measured by dual energy x-ray absorptiometry, increased significantly in men receiving testosterone patches [change in lean body mass,+ 1.345 ± 0.533 kg (P = 0.02 compared to no change); change in fat-free mass, +1.364 ± 0.525 kg (P = 0.02 compared to no change)], but did not change in the placebo group [change in lean body mass, 0.189 ± 0.470 kg (P = NS compared to no change); change in fat-free mass, 0.186 ± 0.470 kg (P = NS compared to no change)]. However, there was no significant difference between the 2 treatment groups in the change in lean body mass. The change in lean body mass during treatment was moderately correlated with the increment in serum testosterone levels (r = 0.41; P = 0.02). The testosterone-treated men experienced a greater decrease in fat mass than those receiving placebo patches (P = 0.04). There was no significant change in body weight in either treatment group. Changes in overall quality of life scores did not correlate with testosterone treatment; however, in the subcategory of role limitation due to emotional problems, the men in the testosterone group improved an average of 43 points of a 0–100 possible score, whereas those in the placebo group did not change. Red cell count increased in the testosterone group (change in red cell count, +0.1 ± 0.1 1012/L) but decreased in the placebo group (change in red cell count, −0.2 ± 0.1 1012/L). CD4+ and CD8+ T cell counts and plasma HIV copy number did not significantly change during treatment. Serum prostate-specific antigen and plasma lipid levels did not change in either treatment group. Testosterone replacement in HIV-infected men with low testosterone levels is safe and is associated with a 1.35-kg gain in lean body mass, a significantly greater reduction in fat mass than that achieved with placebo treatment, an increased red cell count, and an improvement in role limitation due to emotional problems. Further studies are needed to assess whether testosterone supplementation can produce clinically meaningful changes in muscle function and disease outcome in HIV-infected men.

Measurements of total and free testosterone levels in women have lacked precision and accuracy because of limited assay sensitivity. The paucity of normative data on total and free testosterone levels in healthy women has confounded interpretation of androgen levels in women with human immunodeficiency virus (HIV) infection and other disease states. Therefore, the objectives of this study were to develop sensitive assays for the measurement of the low total and free testosterone levels in women to define the range for these hormones during the normal menstrual cycle and assess the total and free testosterone levels in HIV-infected women. By using a larger volume of serum, increasing the incubation time, and reducing the antibody concentration, the sensitivity of the total testosterone assay was increased to 0.008 nmol/L, and that of the free testosterone assay was increased to 2 pmol/L. The mean percent free testosterone was 1.0 +/- 0.1% of the total testosterone. Serum total and free testosterone levels in the follicular and luteal phases were not significantly different, but both demonstrated a modest preovulatory increase, 3 days before the LH peak. Serum total [0.50 +/- 0.32 (14.60 +/- 9.22) vs. 1.2 +/- 0.7 nmol/L (34.3 +/- 21.0 ng/dL); P < 0.0001] and free testosterone levels (5.56 +/- 2.70 (1.58 +/- 0.80) vs. 12.8 +/- 5.5 pmol/L (3.4 +/- 1.7 pg/mL); P < 0.0001) were significantly lower in HIV-infected women (n = 37) than in healthy women (n = 34). Serum total and free testosterone levels were also significantly lower in HIV-infected women who were menstruating normally. There were no significant differences in serum total and free testosterone levels between those who had lost weight and those who had not. Testosterone levels correlated inversely with plasma HIV ribonucleic acid copy number. Serum FSH, but not LH, levels were significantly higher in HIV-infected women than in controls. Using assays with sufficient sensitivity, we defined the range for total and free testosterone levels during the normal menstrual cycle. Serum total and free testosterone levels are lower in HIV-infected women and correlate inversely with plasma HIV ribonucleic acid levels. The hypothesis that androgen deficiency contributes to wasting in HIV-infected women remains to be tested.

Spontaneous death of certain classes of germ cells has been shown to be a constant feature of normal spermatogenesis in a variety of mammalian species, including the human. Recent studies on various animal models have demonstrated that apoptosis is the underlying mechanism of germ cell death during normal spermatogenesis. Withdrawal of gonadotropins and/or testosterone further accelerates the germ cell apoptosis. We examined the involvement of apoptosis in the spontaneous loss of germ cells in men. Testicular samples obtained at autopsy from 5 Chinese and 9 non-Hispanic Caucasian men were analyzed. To identify individual germ cells undergoing apoptosis, we used a modified terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling technique that detects germ cell apoptosis with high sensitivity and specificity. Testicular sections from all 14 subjects exhibited spontaneous occurrence of germ cell apoptosis involving spermatogonia, spermatocytes, and spermatids (apoptotic indexes, 1.6 +/- 0.4 2.5 +/- 0.6, and 5.5 +/- 1.2, respectively). The incidence of spermatogonial (2.8 +/- 0.8 vs. 1.0 +/- 0.2) as well as spermatid (9.3 +/- 2.1 vs. 8.4 +/- 0.9) apoptosis was higher in Chinese than in Caucasian men. A higher incidence of spermatocyte apoptosis was also noted for Chinese (4.4 +/- 1.4) compared to Caucasian (1.9 +/- 0.4) men, but the difference was not statistically significant. These results suggest that germ cell death during normal spermatogenesis in men occurs via apoptosis and provide evidence for ethnic differences in the inherent susceptibility of germ cells to programmed cell death. Our data may also help to explain the greater efficacy of testosterone-induced spermatogenic suppression to azoospermia observed in Asian compared to non-Asian men.