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.

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.

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.

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.

Additive effects of nicotine and high-fat diet on hepatic steatosis in male mice.

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.

Effect of nicotine on body composition in mice.

Nicotine induces weight loss in both humans and rodents consuming a regular diet; however, the effect of nicotine on body weight and fat composition in rodents consuming a high-fat diet (HFD) has not been well studied. Thus, this study examined the effect of nicotine vs saline on body weight and fat composition in mice fed with either an HFD (62% of kcal from fat) or a standard normal chow diet (NCD) for 7 weeks. Nicotine dose dependently reduced body weight gain in mice that consumed both diets, but this effect was significantly greater in mice on the HFD. Caloric intake was decreased in nicotine-treated mice. Estimates of energy intake suggested that decreased caloric intake accounted for all the reduced weight gain in mice on an NCD and 66% of the reduced weight gain on an HFD. Computed tomography analysis for fat distribution demonstrated that nicotine was effective in reducing abdominal fat in mice that consumed the HFD, with nicotine treatment leading to lower visceral fat. The effect of nicotine on weight loss in mice on an HFD was completely blocked by mecamylamine, a nonselective nicotinic acetylcholine receptor (nAChR) antagonist, but only partially blocked by the α4ß2 nAChR partial agonist/antagonist, varenicline. We conclude that nicotine is effective in preventing HFD-induced weight gain and abdominal fat accumulation.

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.

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.

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.

Testosterone supplementation reverses sarcopenia in aging through regulation of myostatin, c-Jun NH2-terminal kinase, Notch, and Akt signaling pathways.

Aging in rodents and humans is characterized by loss of muscle mass (sarcopenia). Testosterone supplementation increases muscle mass in healthy older men. Here, using a mouse model, we investigated the molecular mechanisms by which testosterone prevents sarcopenia and promotes muscle growth in aging. Aged mice of 22 months of age received a single sc injection of GnRH antagonist every 2 wk to suppress endogenous testosterone production and were implanted subdermally under anesthesia with 0.5 or 1.0 cm testosterone-filled implants for 2 months (n = 15/group). Young and old mice (n = 15/group), of 2 and 22 months of age, respectively, received empty implants and were used as controls. Compared with young animals, a significant (P < 0.05) increase in muscle cell apoptosis coupled with a decrease in gastrocnemius muscles weight (by 16.7%) and muscle fiber cross-sectional area, of both fast and slow fiber types, was noted in old mice. Importantly, such age-related changes were fully reversed by higher dose (1 cm) of testosterone treatment. Testosterone treatment effectively suppressed age-specific increases in oxidative stress, processed myostatin levels, activation of c-Jun NH(2)-terminal kinase, and cyclin-dependent kinase inhibitor p21 in aged muscles. Furthermore, it restored age-related decreases in glucose-6-phosphate dehydrogenase levels, phospho-Akt, and Notch signaling. These alterations were associated with satellite cell proliferation and differentiation. Collectively these results suggest involvement of multiple signal transduction pathways in sarcopenia. Testosterone reverses sarcopenia through stimulation of cellular metabolism and survival pathway together with inhibition of death pathway.

Mouse model of testosterone-induced muscle fiber hypertrophy: involvement of p38 mitogen-activated protein kinase-mediated Notch signaling.

As a prerequisite for studies using mutant mice, we established a mouse model for investigating the molecular mechanisms by which testosterone (T) promotes muscle growth. Groups of six adult male mice (C57BL/6) received one of the following treatments: 1) vehicle (sterile distilled water; normal control) and 2) GnRH antagonist with empty (sham control) or 2 cm T- filled implant. Mice were killed 2, 6, and 8 weeks after treatment. T treatment for 8 weeks resulted in a significant (P<0.001) increase in fiber area of gastrocnemius muscles. T-induced fiber-hypertrophy was accompanied by up-regulation of the Notch ligand Delta 1 and activation of Notch signaling, as evidenced by increase in activated forms of Notch 1 and Notch 2. Consistent with this, we also observed an increase in the number of proliferating cell nuclear antigen (PCNA)-positive nuclei in muscles of T-treated mice, indicating that activation of Notch signaling enhanced cell proliferation. T supplementation not only triggered p38 mitogen-activated protein kinase (MAPK) activation but also concurrently inhibited c-Jun NH(2)-terminal kinase (JNK) activation within 2 weeks of treatment. Concomitant administration of SB203580, a p38 MAPK inhibitor, effectively blocked T-induced activation of Notch signaling and significantly (P<0.001) suppressed PCNA levels. Together, our results indicate that T induces muscle fiber hypertrophy through activation of Notch signaling and the inactivation of JNK together with the activation of p38 MAPK may be critical for T-induced activation of Notch signaling and, as a consequence, muscle fiber hypertrophy.

Mitogen-activated protein kinase signaling in male germ cell apoptosis in the rat.

Programmed germ cell death is critical for functional spermatogenesis. Increased germ cell apoptosis can be triggered by various regulatory stimuli, including testicular hyperthermia or deprivation of gonadotropins and intratesticular testosterone. We have previously shown the involvement of the mitogen-activated protein kinase (MAPK) 14 in apoptotic signaling of male germ cells across species after hormone deprivation. This study investigates the role of MAPK14 in germ cell apoptosis in rats triggered by testicular hyperthermia. The contributions of the MAPK1/3 and the MAPK8 to male germ cell death were also examined after this intervention. We show that 1) testicular hyperthermia results in induction of both MAPK1/3 and MAPK14 but not MAPK8; 2) inhibition of MAPK1/3 has no effect on the incidence of heat-induced germ cell apoptosis, suggesting that MAPK1/3 signaling may be dispensable for heat-induced male germ cell apoptosis; and 3) activation of MAPK14 and BCL2 phosphorylation are critical for heat-induced male germ cell apoptosis in rats. Thus, unlike the hormone deprivation model, heat stress through activation of the MAPK14 signaling promotes germ cell apoptosis by provoking BCL2 phosphorylation, leading to its inactivation and the subsequent activation of the mitochondria-dependent death pathway. These novel findings point to a critical role of MAPK14 in stage- and cell-specific activation of male germ cell apoptosis triggered by hormone deprivation or heat stress.

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.

Involvement of c-Jun NH2-terminal kinase and nitric oxide-mediated mitochondria-dependent intrinsic pathway signaling in cardiotoxin-induced muscle cell death: role of testosterone.

To test the hypothesis that c-Jun NH2-terminal kinase (JNK) and nitric oxide (NO)-mediated signaling plays an important role in muscle cell apoptosis, we examined the contribution of these molecules in muscle cell apoptosis during cardiotoxin (ctx)-induced muscle injury in mice. Compared to controls, where no apoptosis was detected, the percent of muscle cell apoptosis rose significantly (P < 0.05) at 4 h (27%) after ctx-treatment and increased further progressively up to 16 h posttreatment (80%), before it fell again at 24 h posttreatment (38%). Initiation of apoptosis was preceded by JNK activation and elevated levels of B-cell lymphoma-2 (BCL-2) in the mitochondrial fractions (BAX levels remained unaffected). Ctx treatment also resulted in the inactivation of BCL-2 through phosphorylation at serine 70, thereby perturbing the BAX/BCL-2 rheostat, and the subsequent activation of the cytochrome c-mediated death pathway. Concomitant administration of SP600125, a selective JNK inhibitor, or aminoguanidine (AG), a selected inducible nitric oxide synthase (iNOS) inhibitor, effectively diminished BCL-2 phosphorylation, suppressed cytochrome c release from mitochondria and caspase activation, and significantly prevented ctx-induced muscle cell apoptosis. In additional studies, we examined the role of testosterone in preventing such ctx-induced muscle cell apoptosis. Collectively, the present study emphasizes the role of a new signal transduction pathway involving JNK and iNOS that promotes ctx-induced myocyte apoptosis by provoking BCL-2 phosphorylation, leading to its inactivation, and subsequent activation of the intrinsic pathway signaling. Testosterone therapy has no protective effect in acute muscle injury associated with increased muscle cell death after ctx-treatment.

Effects of transdermal testosterone administration on insulin sensitivity, fat mass and distribution, and markers of inflammation and thrombolysis in human immunodeficiency virus-infected women with mild to moderate weight loss.

OBJECTIVE: To determine the effects of raising serum T levels into the high normal female range by transdermal T administration on insulin sensitivity, fat volume, and markers of inflammation and thrombolysis in HIV-infected women with recent weight loss. DESIGN: Placebo-controlled, randomized clinical trial. SETTING: Academic clinical research center. PATIENT(S): Fifty-two HIV-infected, menstruating women with >5% weight loss over the prior 6 months and current T<33 ng/dL. INTERVENTION(S): Placebo or T patches twice weekly for 24 weeks to achieve nominal delivery of 300 microg T daily. MAIN OUTCOME MEASURE(S): Testosterone by liquid chromatography-tandem mass spectrometry, insulin sensitivity by the frequently sampled intravenous glucose tolerance test (FSIVGT), abdominal and thigh fat volumes by magnetic resonance imaging (MRI), and C-reactive protein (CRP) as a measure of inflammation and plasminogen-activated inhibitor-1 (PAI-1) levels as a marker of thrombolysis. RESULT(S): Serum and free T levels significantly increased into the high normal female range in T-treated but not placebo-treated women. Insulin sensitivity by FSIVGT, whole body, thigh SC, and intra-abdominal fat volumes, and CRP and PAI-1 levels did not change significantly in either group and were not significantly different between the two groups. Fasting insulin increased in the placebo group and fell slightly in the T group, resulting in significant differences in change between groups. CONCLUSION(S): Twenty-four weeks of elevation of serum T levels into the high normal female range in HIV-infected women with mild to moderate weight loss by transdermal T patches did not adversely affect insulin sensitivity, whole-body fat mass or regional fat distribution, or markers of inflammation and thrombolysis. More prolonged and larger studies are needed to determine the effects of higher doses of T on body composition and insulin sensitivity in women.

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.