E-cigarettes and Western Diet: Important Metabolic Risk Factors for Hepatic Diseases.

The use of electronic nicotine delivery systems (ENDS), also known as e-cigarettes, with a variety of e-liquids/e-juices, is increasing at an alarming rate among adolescents who do not realize the potential harmful health effects. This study examines the harmful effects of ENDS on the liver. Apolipoprotein E null (ApoE-/-) mice on a western diet (WD) were exposed to saline or ENDS with 2.4% nicotine aerosol for 12 weeks using our mouse ENDS exposure model system, which delivers nicotine to mice and leads to equivalent serum cotinine levels found in human cigarette users. ApoE-/- mice on a WD exposed to ENDS exhibited a marked increase in hepatic lipid accumulation compared with ApoE-/- on a similar diet exposed to saline aerosol. The detrimental effects of ENDS on hepatic steatosis were associated with significantly greater oxidative stress, increased hepatic triglyceride levels, and increased hepatocyte apoptosis, independent of adenosine monophosphate-activated protein kinase signaling. In addition, hepatic RNA sequencing analysis revealed that 433 genes were differentially expressed in ENDS-exposed mice on WD compared with saline-exposed mice. Functional analysis indicates that genes associated with lipid metabolism, cholesterol biosynthesis, and circadian rhythm were most significantly altered in the liver in response to ENDS. Conclusion: These results demonstrate profound adverse effects of ENDS on the liver. This is important information for regulatory agencies as they regulate ENDS.

Nicotine plus a high-fat diet triggers cardiomyocyte apoptosis.

Cigarette smoking is an important risk factor for diabetes, cardiovascular disease and non-alcoholic fatty liver disease. The health risk associated with smoking can be aggravated by obesity. Smoking might also trigger cardiomyocyte (CM) apoptosis. Given that CM apoptosis has been implicated as a potential mechanism in the development of cardiomyopathy and heart failure, we characterize the key signaling pathways in nicotine plus high-fat diet (HFD)-induced CM apoptosis. Adult C57BL6 male mice were fed a normal diet (ND) or HFD and received twice-daily intraperitoneal (IP) injections of nicotine (0.75 mg/kg body weight [BW]) or saline for 16 weeks. An additional group of nicotine-treated mice on HFD received twice-daily IP injections of mecamylamine (1 mg/kg BW), a non-selective nicotinic acetylcholine receptor antagonist, for 16 weeks. Nicotine when combined with HFD led to a massive increase in CM apoptosis that was fully prevented by mecamylamine treatment. Induction of CM apoptosis was associated with increased oxidative stress and activation of caspase-2-mediated intrinsic pathway signaling coupled with inactivation of AMP-activated protein kinase (AMPK). Furthermore, nicotine treatment significantly (P < 0.05) attenuated the HFD-induced decrease in fibroblast growth factor 21 (FGF21) and silent information regulator 1 (SIRT1). We conclude that nicotine, when combined with HFD, triggers CM apoptosis through the generation of oxidative stress and inactivation of AMPK together with the activation of caspase-2-mediated intrinsic apoptotic signaling independently of FGF21 and SIRT1.

Testosterone is essential for skeletal muscle growth in aged mice in a heterochronic parabiosis model.

As humans age, they lose both muscle mass and strength (sarcopenia). Testosterone, a circulating hormone, progressively declines in aging and is associated with loss of muscle mass and strength. The surgical joining of a young and old mouse (heterochronic parabiosis) activates Notch signaling and restores muscle regenerative potential in aged mice. We hypothesize that testosterone is at least one of the factors required for the improvement seen in muscles in old mice in heterochronic parabiosis with young mice. To test this hypothesis, we established the following heterochronic parabioses between young (Y; 5 months old) and old (O; 22-23 months old) C57BL6 male mice: (1) Y:O; (2) castrated Y:O (ØY:O); (3) castrated + testosterone-treated Y:O (ØY + T:O). A group of normal young mice received empty implants, and old mice were used as controls. Parabiotic pairings were maintained for 4 weeks prior to analysis. Serum testosterone levels were three-fold higher in young than in old mice. The ØY + T:O pairing demonstrated significantly elevated levels of serum testosterone and an improvement in gastrocnemius muscle weight, muscle ultrastructure, muscle fiber cross-sectional area, and Notch-1 expression in old mice. These changes were not present in aged mice in the ØY:O pairing. These data indicate that testosterone has a critical role in mediating the improved muscle mass and ultrastructure seen in an experimental model of heterochronic parabiosis.

Minocycline suppresses oxidative stress and attenuates fetal cardiac myocyte apoptosis triggered by in utero cocaine exposure.

This study investigates the molecular mechanisms by which minocycline, a second generation tetracycline, prevents cardiac myocyte death induced by in utero cocaine exposure. Timed mated pregnant Sprague-Dawley (SD) rats received one of the following treatments twice daily from embryonic (E) day 15-21 (E15-E21): (i) intraperitoneal (IP) injections of saline (control); (ii) IP injections of cocaine (15 mg/kg BW); and (iii) IP injections of cocaine + oral administration of 25 mg/kg BW of minocycline. Pups were killed on postnatal day 15 (P15). Additional pregnant dams received twice daily IP injections of cocaine (from E15-E21) + oral administration of a relatively higher (37.5 mg/kg BW) dose of minocycline. Minocycline treatment continued from E15 until the pups were sacrificed on P15. In utero cocaine exposure resulted in an increase in oxidative stress and fetal cardiac myocyte apoptosis through activation of c-Jun-NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK)-mediated mitochondria-dependent apoptotic pathway. Continued minocycline treatment from E15 through P15 significantly prevented oxidative stress, kinase activation, perturbation of BAX/BCL-2 ratio, cytochrome c release, caspase activation, and attenuated fetal cardiac myocyte apoptosis after prenatal cocaine exposure. These results demonstrate in vivo cardioprotective effects of minocycline in preventing fetal cardiac myocyte death after prenatal cocaine exposure. Given its proven clinical safety and ability to cross the placental barrier and enter into the fetal circulation, minocycline may be an effective therapy for preventing cardiac consequences of in utero cocaine exposure.

A novel cystine based antioxidant attenuates oxidative stress and hepatic steatosis in diet-induced obese mice.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver pathologies and is associated with obesity and the metabolic syndrome. Here, we investigated the molecular mechanisms by which a novel cystine based glutathione precursor with added selenomethionine (F1) prevents hepatic steatosis in a moderate high fat dietary model of NAFLD. Adult (8 weeks old), male apolipoprotein E (ApoE)-/- mice were fed with a normal diet (ND) or high fat diet (HFD), consisting of 21% fat and 0.21% cholesterol, with or without dietary supplementation of F1 (3 g/kg food) for 16 weeks. Compared with ApoE-/- mice fed with ND with or without F1, ApoE-/- mice fed with HFD exhibited significant weight gain, hepatomegaly, and increased serum cholesterol and triglycerides levels with no change in serum albumin levels. High resolution light and electron microscopy revealed micro-and macro-vesicular steatosis in ApoE-/- mice fed on a HFD. HFD-induced obesity also led to increased lipogenesis, oxidative stress, activation of c-Jun-NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), perturbation of the BAX/BCL-2 rheostat, hepatocyte apoptosis, and activation of caspases 9 and 3. F1 fully prevented the adverse effects of HFD on serum triglyceride levels, body and liver weights, and hepatic steatosis and substantially attenuated HFD-induced increase in lipogenesis, oxidative stress, kinase activation, apoptotic signaling, and hepatocyte ultrastructural abnormalities. These results demonstrate that administration of F1, a glutathione precursor, ameliorates HFD-induced hepatic steatosis in ApoE-/- mice and emphasizes the role of oxidative stress in diet-induced obesity and hepatic steatosis.

Effects of a novel cystine-based glutathione precursor on oxidative stress in vascular smooth muscle cells.

Chronic kidney disease (CKD) is associated with accelerated atherosclerosis and cardiovascular disease, which is largely mediated by oxidative stress. We investigated the effect of three glutathione (GSH) precursors: N-acetyl-cysteine (NAC), cystine as the physiological carrier of cysteine in GSH with added selenomethionine (F1), and NAC fortified with selenomethionine (F2) on oxidative stress induced by spermine (a uremic toxin) in cultured human aortic vascular smooth muscle cells (VSMC). VSMC were exposed to spermine (15 microM) with or without the given antioxidants (dose 50, 100, 200, and 500 microg/ml) or vehicle (control) and assessed for intracellular GSH levels, 4-hydroxy-trans-2-nonenal (4-HNE), and incorporation of 13C from glucose into alanine and protein. Spermine exposure reduced intracellular GSH levels, increased 4-HNE, and impaired glucose metabolism through reduction in pyruvate generation and/or transamination. Treatment with NAC had no effect on intracellular glutathione level. In contrast, F1 maintained intracellular GSH at control levels at all four doses. Subsequent studies performed with 200 microg/ml of F1, F2, or NAC (optimal dose) revealed normalization of 4-HNE, whereas restoration of 13C from glucose to alanine or protein to control values was only noted in the F1 group. Spermine-induced alterations in VSMC ultrastructure were prevented in approximately 90% of cells treated with F1 but only approximately 50% of cells treated with either NAC or F2. In conclusion, F1 was more effective than NAC or F2 in ameliorating spermine-induced reduction in intracellular GSH levels and cellular alterations in VSMC. The cystine-based GSH precursor (F1) is a promising antioxidant, and further studies are needed to examine the effect of this compound in preventing CKD-associated vascular disease.

Inhibition of apoptotic signalling in spermine-treated vascular smooth muscle cells by a novel glutathione precursor.

CKD (chronic kidney disease) is a public health problem, mediated by haemodynamic and non-haemodynamic events including oxidative stress. We investigated the effect of two GSH (glutathione) precursors, NAC (N-acetylcysteine) and cystine as the physiological carrier of cysteine in GSH with added selenomethionine (F1) in preventing spermine (uraemic toxin)-induced apoptosis in cultured human aortic VSMC (vascular smooth muscle cells). VSMCs exposed to spermine (15 microM) with or without antioxidants (doses 50, 100, 200 and 500 microg/ml) were assessed for apoptosis, JNK (c-Jun-NH2-terminal kinase) activation and iNOS (inducible nitric oxide synthase) induction and activation of intrinsic pathway signalling. Spermine exposure resulted in activation of JNK and iNOS induction and apoptosis. NAC and F1 (dose range 50-500 microg/ml) attenuated spermine-induced acceleration of VSMC apoptosis but only F1 (at 200 and 500 microg/ml) maintained spermine-induced apoptosis at control levels. Spermine-induced JNK activation was prevented by 200 microg/ml of both NAC and F1, while iNOS induction was blocked only by F1. Notably, the adverse effects of spermine on BAX/BCL-2 ratio, cytochrome c release and caspase activation was fully attenuated by F1. In conclusion, F1 was more effective than NAC in preventing spermine-induced apoptosis and downstream changes in related signal transduction pathways in VSMCs. Further studies are needed to examine the effect of these compounds in preventing CKD-associated vascular disease.

Involvement of oxidative stress and caspase 2-mediated intrinsic pathway signaling in age-related increase in muscle cell apoptosis in mice.

Apoptosis has been implicated as a mechanism of loss of muscle cells in normal aging and plays an important role in age-related sarcopenia. To test the hypothesis that caspase 2 and c-Jun NH(2)-terminal kinase (JNK)-mediated intrinsic pathway signaling contribute to skeletal muscle cell apoptosis in aging, we compared activation of caspase 2 and JNK and the in vivo expression of 4-hydroxynonenal protein adducts (4-HNE), inducible nitric oxide synthase (iNOS), glucose-6-phosphate dehydrogenase (G6PDH), B-cell lymphoma-2 (BCL-2), BAX, and phospho-BCL-2 in gastrocnemius muscles of young (5 months old) and old (25 months old) mice. A distinct age-related increase in 4-HNE and iNOS expression was readily detected in mice. Increased oxidative stress and iNOS induction were further accompanied by a decrease in G6PDH expression, activation of caspase 2 and JNK, and inactivation of BCL-2 through phosphorylation at serine 70, and caspase 9 activation. Regression analysis further revealed that increased muscle cell death in aging was significantly correlated with changes in the levels of these molecules. Taken together, our data indicate that caspase 2 and JNK-mediated intrinsic pathway signaling is one of the mechanisms involved in age-related increase in muscle cell apoptosis.

α7-Nicotinic Acetylcholine Receptor Agonist Ameliorates Nicotine Plus High-Fat Diet-Induced Hepatic Steatosis in Male Mice by Inhibiting Oxidative Stress and Stimulating AMPK Signaling.

α7-Nicotinic acetylcholine receptor (α7nAChR) agonists confer protection against a wide variety of cytotoxic insults and suppress oxidative stress and apoptosis in various cell systems, including hepatocytes. We recently demonstrated that nicotine, when combined with a high-fat diet (HFD), triggers oxidative stress, activates hepatocyte apoptosis, and exacerbates HFD-induced hepatic steatosis in male mice. This study evaluates whether PNU-282987 (PNU), a specific α7nAChR agonist, is effective in preventing nicotine plus HFD-induced hepatic steatosis. Adult C57BL6 male mice were fed a normal chow diet or HFD with 60% of calories derived from fat and received twice-daily intraperitoneal injections of 0.75 mg/kg body weight (BW) of nicotine, PNU (0.26 mg/kg BW), PNU plus nicotine, or saline for 10 weeks. PNU treatment was effective in attenuating nicotine plus HFD-induced increase in hepatic triglyceride levels, hepatocyte apoptosis, and hepatic steatosis. The preventive effects of PNU on nicotine plus HFD-induced hepatic steatosis were mediated by suppression of oxidative stress and activation of adenosine 5'-monophosphate-activated protein kinase (AMPK) together with inhibition of its downstream target sterol regulatory element binding protein 1c (SREBP1c), fatty acid synthase (FAS), and acetyl-coenzyme A-carboxylase (ACC). We conclude that the α7nAChR agonist PNU protects against nicotine plus HFD-induced hepatic steatosis in obese mice. PNU appears to work at various steps of signaling pathways involving suppression of oxidative stress, activation of AMPK, and inhibition of SREBP1c, FAS, and ACC. α7nAChR agonists may be an effective therapeutic strategy for ameliorating fatty liver disease, especially in obese smokers.

Connection of Nicotine to Diet-Induced Obesity and Non-Alcoholic Fatty Liver Disease: Cellular and Mechanistic Insights.

Non-alcoholic fatty liver disease (NAFLD) poses a serious health hazard affecting 20-40% of adults in the general population in the USA and over 70% of the obese and extremely obese people. In addition to obesity, nicotine is recognized as a risk factor for NAFLD, and it has been reported that nicotine can exaggerate obesity-induced hepatic steatosis. The development of NAFLD has serious clinical complications because of its potential progression from simple hepatic steatosis to non-alcoholic steatohepatitis (NASH), liver cirrhosis, and hepatocellular carcinoma. Multiple mechanisms can be involved in nicotine plus high-fat diet-induced (HFD) hepatic steatosis. Emerging evidence now suggests that nicotine exacerbates hepatic steatosis triggered by HFD, through increased oxidative stress and hepatocellular apoptosis, decreased phosphorylation (inactivation) of adenosine-5-monophosphate-activated protein kinase and, in turn, up-regulation of sterol response-element binding protein 1-c, fatty acid synthase, and activation of acetyl-coenzyme A-carboxylase, leading to increased hepatic lipogenesis. There is also growing evidence that chronic endoplasmic reticulum stress through regulation of several pathways leading to oxidative stress, inflammation, perturbed hepatic lipid homeostasis, apoptosis, and autophagy can induce hepatic steatosis and its progression to NASH. Evidence also suggests a central role of the gut microbiota in obesity and its related disorders, including NAFLD. This review explores the contribution of nicotine and obesity to the development of NAFLD and its molecular underpinning.

Effects of testosterone supplementation on skeletal muscle fiber hypertrophy and satellite cells in community-dwelling older men.

OBJECTIVE: In this study, we determined the effects of graded doses of testosterone on muscle fiber cross-sectional area (CSA) and satellite cell number and replication in older men. PARTICIPANTS: Healthy men, 60-75 yr old, received a long-acting GnRH agonist to suppress endogenous testosterone production and 25, 50, 125, 300, or 600 mg testosterone enanthate im weekly for 20 wk. METHODS: Immunohistochemistry, light and confocal microscopy, and electron microscopy were used to perform fiber typing and quantitate myonuclear and satellite cell number in vastus lateralis biopsies, obtained before and after 20 wk of treatment. RESULTS: Testosterone administration in older men was associated with dose-dependent increases in CSA of both types I and II fibers. Satellite cell number increased dose dependently at the three highest doses (3% at baseline vs. 6.2, 9.2, and 13.0% at 125, 300, and 600 mg doses, P < 0.05). Testosterone administration was associated with an increase in the number of proliferating cell nuclear antigen+ satellite cells (1.8% at baseline vs. 3.9, 7.5, and 13% at 125, 300, and 600 mg doses, P < 0.005). The expression of activated Notch, examined only in the 300-mg group (baseline, 2.3 vs. 9.0% after treatment, P < 0.005), increased in satellite cells after testosterone treatment. The expression of myogenin (baseline, 6.2 vs. 20.7% after treatment, P < 0.005), examined only in the 300-mg group, increased significantly in muscle fiber nuclei after testosterone treatment, but Numb expression did not change. CONCLUSIONS: Older men respond to graded doses of testosterone with a dose-dependent increase in muscle fiber CSA and satellite cell number. Testosterone-induced skeletal muscle hypertrophy in older men is associated with increased satellite cell replication and activation.

Pharmacokinetics of a testosterone gel in healthy postmenopausal women.

BACKGROUND: The paucity of pharmacokinetic data on androgen formulations in women has hindered clinical trials of testosterone supplementation in women. OBJECTIVE: 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. METHODS: 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. RESULTS: 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, P < 0.00001), 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. CONCLUSIONS: 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.

Dose-dependent effects of testosterone on sexual function, mood, and visuospatial cognition in older men.

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) of testosterone enanthate weekly for 20 wk. Questionnaires were used 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), spontaneous erections (P = 0.047), and libido (P = 0.027), but not with 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 (P = 0.042), 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.

Older men are as responsive as young men to the anabolic effects of graded doses of testosterone on the skeletal muscle.

Although testosterone levels and muscle mass decline with age, many older men have serum testosterone level in the normal range, leading to speculation about whether older men are less sensitive to testosterone. We determined the responsiveness of androgen-dependent outcomes to graded testosterone doses in older men and compared it to that in young men. The participants in this randomized, double-blind trial were 60 ambulatory, healthy, older men, 60-75 yr of age, who had normal serum testosterone levels. Their responses to graded doses of testosterone were compared with previous data in 61 men, 19-35 yr old. The participants received a long-acting GnRH agonist to suppress endogenous testosterone production and 25, 50, 125, 300, or 600 mg testosterone enanthate weekly for 20 wk. Fat-free mass, fat mass, muscle strength, sexual function, mood, visuospatial cognition, hormone levels, and safety measures were evaluated before, during, and after treatment. Of 60 older men who were randomized, 52 completed the study. After adjusting for testosterone dose, changes in serum total testosterone (change, -6.8, -1.9, +16.1, +49.5, and +101.9 nmol/liter at 25, 50, 125, 300, and 600 mg/wk, respectively) and hemoglobin (change, -3.6, +9.9, +20.9, +12.6, and +29.4 g/liter at 25, 50, 125, 300, and 600 mg/wk, respectively) levels were dose-related in older men and significantly greater in older men than young men (each P < 0.0001). The changes in FFM (-0.3, +1.7, +4.2, +5.6, and +7.3 kg, respectively, in five ascending dose groups) and muscle strength in older men were correlated with testosterone dose and concentrations and were not significantly different in young and older men. Changes in fat mass correlated inversely with testosterone dose (r = -0.54; P < 0.001) and were significantly different in young vs. older men (P < 0.0001); young men receiving 25- and 50-mg doses gained more fat mass than older men (P < 0.0001). Mood and visuospatial cognition did not change significantly in either group. Frequency of hematocrit greater than 54%, leg edema, and prostate events were numerically higher in older men than in young men. Older men are as responsive as young men to testosterone's anabolic effects; however, older men have lower testosterone clearance rates, higher increments in hemoglobin, and a higher frequency of adverse effects. Although substantial gains in muscle mass and strength can be realized in older men with supraphysiological testosterone doses, these high doses are associated with a high frequency of adverse effects. The best trade-off was achieved with a testosterone dose (125 mg) that was associated with high normal testosterone levels, low frequency of adverse events, and significant gains in fat-free mass and muscle strength.

Delta-4-androstene-3,17-dione binds androgen receptor, promotes myogenesis in vitro, and increases serum testosterone levels, fat-free mass, and muscle strength in hypogonadal men.

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.

Androgen receptor in human skeletal muscle and cultured muscle satellite cells: up-regulation by androgen treatment.

Androgens stimulate myogenesis, but we do not know what cell types within human skeletal muscle express the androgen receptor (AR) protein and are the target of androgen action. Because testosterone promotes the commitment of pluripotent, mesenchymal cells into myogenic lineage, we hypothesized that AR would be expressed in mesenchymal precursor cells in the skeletal muscle. AR expression was evaluated by immunohistochemical staining, confocal immunofluorescence, and immunoelectron microscopy in sections of vastus lateralis from healthy men before and after treatment with a supraphysiological dose of testosterone enanthate. Satellite cell cultures from human skeletal muscle were also tested for AR expression. AR protein was expressed predominantly in satellite cells, identified by their location outside sarcolemma and inside basal lamina, and by CD34 and C-met staining. Many myonuclei in muscle fibers also demonstrated AR immunostaining. Additionally, CD34+ stem cells in the interstitium, fibroblasts, and mast cells expressed AR immunoreactivity. AR expression was also observed in vascular endothelial and smooth muscle cells. Immunoelectron microscopy revealed aggregation of immunogold particles in nucleoli of satellite cells and myonuclei; testosterone treatment increased nucleolar AR density. In enriched cultures of human satellite cells, more than 95% of cells stained for CD34 and C-met, confirming their identity as satellite cells, and expressed AR protein. AR mRNA and protein expression in satellite cell cultures was confirmed by RT-PCR, reverse transcription and real-time PCR, sequencing of RT-PCR product, and Western blot analysis. Incubation of satellite cell cultures with supraphysiological testosterone and dihydrotestosterone concentrations (100 nm testosterone and 30 nm dihydrotestosterone) modestly increased AR protein levels. We conclude that AR is expressed in several cell types in human skeletal muscle, including satellite cells, fibroblasts, CD34+ precursor cells, vascular endothelial, smooth muscle cells, and mast cells. Satellite cells are the predominant site of AR expression. These observations support the hypothesis that androgens increase muscle mass in part by acting on several cell types to regulate the differentiation of mesenchymal precursor cells in the skeletal muscle.

The effects of varying doses of T on insulin sensitivity, plasma lipids, apolipoproteins, and C-reactive protein in healthy young men.

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.

The mechanisms of androgen effects on body composition: mesenchymal pluripotent cell as the target of androgen action.

Testosterone supplementation increases muscle mass primarily by inducing muscle fiber hypertrophy; however, the mechanisms by which testosterone exerts its anabolic effects on the muscle are poorly understood. The prevalent view is that testosterone improves net muscle protein balance by stimulating muscle protein synthesis, decreasing muscle protein degradation, and improving the reutilization of amino acids. However, the muscle protein synthesis hypothesis does not adequately explain testosterone-induced changes in fat mass, myonuclear number, and satellite cell number. We postulate that testosterone promotes the commitment of pluripotent stem cells into the myogenic lineage and inhibits their differentiation into the adipogenic lineage. The hypothesis that the primary site of androgen action is the pluripotent stem cell provides a unifying explanation for the observed reciprocal effects of testosterone on muscle and fat mass.

Nicotine in combination with a high-fat diet causes intramyocellular mitochondrial abnormalities in male mice.

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.

Long-term supplementation with a cystine-based antioxidant delays loss of muscle mass in aging.

Oxidative stress increases with age and is postulated to be a major causal factor for sarcopenia in aging. Here, we examined whether the administration of a cystine-based antioxidant (F1) can alleviate/delay age-specific changes in skeletal muscles. C57BL6 male mice aged 17 months (middle aged) were fed with normal diet with or without supplementation of F1 (3 mg/kg food) for 6 months. Compared with young (5 months old) mice old mice exhibited increased markers of oxidative stress, inflammation, and muscle cell apoptosis and decreased muscle weight. These age-related changes were further associated with inactivation of adenosine-5'-monophosphate-activated protein kinase (AMPK), increased lipogenesis, activation of c-Jun NH2-terminal kinase, and decreased expression of Delta 1, phospho-Akt, and proliferating cell nuclear antigen in aged skeletal muscle. Such alterations were significantly prevented by F1. These results demonstrate the beneficial effects of F1 to attenuate loss of muscle mass associated with aging.