Binge Ethanol Drinking Produces Sexually Divergent and Distinct Changes in Nucleus Accumbens Signaling Cascades and Pathways in Adult C57BL/6J Mice.

We previously determined that repeated binge ethanol drinking produced sex differences in the regulation of signaling downstream of Group 1 metabotropic glutamate receptors in the nucleus accumbens (NAc) of adult C57BL/6J mice. The purpose of the present study was to characterize RNA expression differences in the NAc of adult male and female C57BL/6J mice following 7 binge ethanol drinking sessions, when compared with controls consuming water. This binge drinking procedure produced high intakes (average >2.2 g/kg/30 min) and blood ethanol concentrations (average >1.3 mg/ml). Mice were euthanized at 24 h after the 7th binge session, and focused qPCR array analysis was employed on NAc tissue to quantify expression levels of 384 genes in a customized Mouse Mood Disorder array, with a focus on glutamatergic signaling (3 arrays/group). We identified significant regulation of 50 genes in male mice and 70 genes in female mice after 7 ethanol binges. Notably, 14 genes were regulated in both males and females, representing common targets to binge ethanol drinking. However, expression of 10 of these 14 genes was strongly dimorphic (e.g., opposite regulation for genes such as Crhr2, Fos, Nos1, and Star), and only 4 of the 14 genes were regulated in the same direction (Drd5, Grm4, Ranbp9, and Reln). Interestingly, the top 30 regulated genes by binge ethanol drinking for each sex differed markedly in the male and female mice, and this divergent neuroadaptive response in the NAc could result in dysregulation of distinct biological pathways between the sexes. Characterization of the expression differences with Ingenuity Pathway Analysis was used to identify Canonical Pathways, Upstream Regulators, and significant Biological Functions. Expression differences suggested that hormone signaling and immune function were altered by binge drinking in female mice, whereas neurotransmitter metabolism was a central target of binge ethanol drinking in male mice. Thus, these results indicate that the transcriptional response to repeated binge ethanol drinking was strongly influenced by sex, and they emphasize the importance of considering sex in the development of potential pharmacotherapeutic targets for the treatment of alcohol use disorder.

The androgen receptor in bone marrow progenitor cells negatively regulates fat mass.

It is well established that testosterone negatively regulates fat mass in humans and mice; however, the mechanism by which testosterone exerts these effects is poorly understood. We and others have shown that deletion of the androgen receptor (AR) in male mice results in a phenotype that mimics the three key clinical aspects of hypogonadism in human males; increased fat mass and decreased bone and muscle mass. We now show that replacement of the Ar gene specifically in mesenchymal progenitor cells (PCs) residing in the bone marrow of Global-ARKO mice, in the absence of the AR in all other tissues (PC-AR Gene Replacements), completely attenuates their increased fat accumulation. Inguinal subcutaneous white adipose tissue and intra-abdominal retroperitoneal visceral adipose tissue depots in PC-AR Gene Replacement mice were 50-80% lower than wild-type (WT) and 75-90% lower than Global-ARKO controls at 12 weeks of age. The marked decrease in subcutaneous and visceral fat mass in PC-AR Gene Replacements was associated with an increase in the number of small adipocytes and a healthier metabolic profile compared to WT controls, characterised by normal serum leptin and elevated serum adiponectin levels. Euglycaemic/hyperinsulinaemic clamp studies reveal that the PC-AR Gene Replacement mice have improved whole-body insulin sensitivity with higher glucose infusion rates compared to WT mice and increased glucose uptake into subcutaneous and intra-abdominal fat. In conclusion, these data provide the first evidence for an action of androgens via the AR in mesenchymal bone marrow PCs to negatively regulate fat mass and improve metabolic function.

Prefrontal cortex expression of chromatin modifier genes in male WSP and WSR mice changes across ethanol dependence, withdrawal, and abstinence.

Alcohol-use disorder (AUD) is a relapsing disorder associated with excessive ethanol consumption. Recent studies support the involvement of epigenetic mechanisms in the development of AUD. Studies carried out so far have focused on a few specific epigenetic modifications. The goal of this project was to investigate gene expression changes of epigenetic regulators that mediate a broad array of chromatin modifications after chronic alcohol exposure, chronic alcohol exposure followed by 8 h withdrawal, and chronic alcohol exposure followed by 21 days of abstinence in Withdrawal-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) selected mouse lines. We found that chronic vapor exposure to highly intoxicating levels of ethanol alters the expression of several chromatin remodeling genes measured by quantitative PCR array analyses. The identified effects were independent of selected lines, which, however, displayed baseline differences in epigenetic gene expression. We reported dysregulation in the expression of genes involved in histone acetylation, deacetylation, lysine and arginine methylation and ubiquitinationhylation during chronic ethanol exposure and withdrawal, but not after 21 days of abstinence. Ethanol-induced changes are consistent with decreased histone acetylation and with decreased deposition of the permissive ubiquitination mark H2BK120ub, associated with reduced transcription. On the other hand, ethanol-induced changes in the expression of genes involved in histone lysine methylation are consistent with increased transcription. The net result of these modifications on gene expression is likely to depend on the combination of the specific histone tail modifications present at a given time on a given promoter. Since alcohol does not modulate gene expression unidirectionally, it is not surprising that alcohol does not unidirectionally alter chromatin structure toward a closed or open state, as suggested by the results of this study.

A neurotoxic alcohol exposure paradigm does not induce hepatic encephalopathy.

Alcohol abuse is associated with neurological dysfunction, brain morphological deficits and frank neurotoxicity. Although these disruptions may be a secondary effect due to hepatic encephalopathy, no clear evidence of causality is available. This study examined whether a 72h period of alcohol intoxication known to induce physical dependence, followed by a single withdrawal, was sufficient to induce signs of hepatic encephalopathy in male and female mice. Animals were continuously intoxicated via alcohol vapor inhalation, a procedure previously shown to induce significant neurotoxicity in female mice. At peak synchronized withdrawal (8h following the end of alcohol exposure), blood samples were taken and levels of several liver-regulated markers and brain swelling were characterized. Glutathione levels were also determined in the medial frontal cortex (mFC) and hippocampus. Results revealed elevated levels of cholesterol, albumin, alkaline phosphatase (ALP), alanine aminotransferase (ALT) and decreased levels of blood urea nitrogen and total bilirubin in alcohol-exposed male and female groups compared to controls. Brain water weight was not affected by alcohol exposure, though males tended to have slightly more water weight overall. Alcohol exposure led to reductions in tissue levels of glutathione in both the hippocampus and mFC which may indicate increased oxidative stress. Combined, these results suggest that hepatic encephalopathy does not appear to play a significant role in the neurotoxicity observed following alcohol exposure in this model.

Functional regulation of PI3K-associated signaling in the accumbens by binge alcohol drinking in male but not female mice.

It is well established that binge alcohol consumption produces alterations in Group 1 metabotropic glutamate receptors (mGlus) and related signaling cascades in the nucleus accumbens (NAC) of adult male mice, but female and adolescent mice have not been examined. Thus, the first set of studies determined whether repeated binge alcohol consumption produced similar alterations in protein and mRNA levels of Group 1 mGlu-associated signaling molecules in the NAC of male and female adult and adolescent mice. The adult (9 weeks) and adolescent (4 weeks) C57BL/6J mice were exposed to 7 binge alcohol sessions every 3rd day while controls drank water. Repeated binge alcohol consumption produced sexually divergent changes in protein levels and mRNA expression for Group 1 mGlus and downstream signaling molecules in the NAC, but there was no effect of age. Binge alcohol intake decreased mGlu5 levels in females, whereas it decreased indices of phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), 4E-binding protein 1, and p70 ribosomal protein S6 kinase in males. Expression of genes encoding mGlu1, mGlu5, the NR2A subunit of the NMDA receptor, and Homer2 were all decreased by binge alcohol consumption in males, while females were relatively resistant (only phosphoinositide-dependent protein kinase 1 was decreased). The functional implication of these differences was investigated in a separate study by inhibiting mTOR in the NAC (via infusions of rapamycin) before binge drinking sessions. Rapamycin (50 and 100 ng/side) significantly decreased binge alcohol consumption in males, while consumption in females was unaffected. Altogether these results highlight that mTOR signaling in the NAC was necessary to maintain binge alcohol consumption only in male mice and that binge drinking recruits sexually divergent signaling cascades downstream of PI3K and presumably, Group 1 mGlus. Importantly, these findings emphasize that sex should be considered in the development of potential pharmacotherapeutic targets.

Astrocyte Dysfunction Induced by Alcohol in Females but Not Males.

Chronic alcohol abuse is associated with brain damage in a sex-specific fashion, but the mechanisms involved are poorly described and remain controversial. Previous results have suggested that astrocyte gene expression is influenced by ethanol intoxication and during abstinence in vivo. Here, bioinformatic analysis of astrocyte-enriched ethanol-regulated genes in vivo revealed ubiquitin pathways as an ethanol target, but with sexually dimorphic cytokine signaling and changes associated with brain aging in females and not males. Consistent with this result, astrocyte activation was observed after exposure in female but not male animals, with reduced S100ß levels in the anterior cingulate cortex and increased GFAP(+) cells in the hippocampus. In primary culture, the direct effects of chronic ethanol exposure followed by recovery on sex-specific astrocyte function were examined. Male astrocyte responses were consistent with astrocyte deactivation with reduced GFAP expression during ethanol exposure. In contrast, female astrocytes exhibited increased expression of Tnf, reduced expression of the neuroprotective cytokine Tgfb1, disrupted bioenergetics and reduced excitatory amino acid uptake following exposure or recovery. These results indicate widespread astrocyte dysfunction in ethanol-exposed females and suggest a mechanism that may underlie increased vulnerability to ethanol-induced neurotoxicity in females.

Females uniquely vulnerable to alcohol-induced neurotoxicity show altered glucocorticoid signaling.

Women are more sensitive to the harmful effects of alcohol (EtOH) abuse than men, yet the underlying mechanisms remain poorly understood. Previous gene expression analysis of the medial prefrontal cortex (mPFC) following a chronic intoxication paradigm using continuous 72 h vapor inhalation found that females, but not males, exhibit an inflammatory response at peak withdrawal that is associated with cell damage. Given that glucocorticoids can function as anti-inflammatories, are known to increase with EtOH exposure, and influence neurotoxicity, we hypothesized that males and females may exhibit an altered corticosterone (CORT) response following chronic intoxication. Analysis of serum CORT levels revealed the expected increase during withdrawal with no difference between males and females, while control males but not females exhibited higher CORT concentrations than naive animals. Glucocorticoid signaling characterized using focused qPCR arrays identified a sexually dimorphic response in the mPFC during withdrawal, particularly among astrocyte-enriched genes. These genes include aquaporin-1 (Aqp1), sphingosine kinase 1 (Sphk1) and connective tissue growth factor (Ctgf); genes associated with inflammatory signaling, and tissue damage and repair. Bioinformatic analysis also revealed activation of inflammatory signaling and cell death pathways in females. Confirmation studies showed that female mice exhibited significant neuronal degeneration within the anterior cingulate cortex (ACC). By contrast, EtOH exposure lead to a significant reduction in cell death in males. Thus, distinct glucocorticoid signaling pathways are associated with sexually dimorphic neurotoxicity, suggesting one mechanism by which EtOH-exposed females are particularly vulnerable to the damaging effects of alcohol in the CNS.

Androgen receptor action in osteoblasts in male mice is dependent on their stage of maturation.

Androgen action via the androgen receptor (AR) is essential for normal skeletal growth and bone maintenance post-puberty in males; however, the molecular and cellular mechanisms by which androgens exert their actions in osteoblasts remains relatively unexplored in vivo. To identify autonomous AR actions in osteoblasts independent of AR signaling in other tissues, we compared the extent to which the bone phenotype of the Global-ARKO mouse was restored by replacing the AR in osteoblasts commencing at either the (1) proliferative or (2) mineralization stage of their maturation. In trabecular bone, androgens stimulated trabecular bone accrual during growth via the AR in proliferating osteoblasts and maintained trabecular bone post-puberty via the AR in mineralizing osteoblasts, with its predominant action being to inhibit bone resorption by decreasing the ratio of receptor activator of NF-κB ligand (RANKL) to osteoprotegerin (OPG) gene expression. During growth, replacement of the AR in proliferating but not mineralizing osteoblasts of Global-ARKOs was able to partially restore periosteal circumference, supporting the concept that androgen action in cortical bone to increase bone size during growth is mediated via the AR in proliferating osteoblasts. This study provides further significant insight into the mechanism of androgen action via the AR in osteoblasts, demonstrating that it is dependent on the stage of osteoblast maturation.

The Effect of mGluR5 Antagonism During Binge Drinkingon Subsequent Ethanol Intake in C57BL/6J Mice: Sex- and Age-Induced Differences.

BACKGROUND: Binge ethanol (EtOH) intake during adolescence leads to an array of behavioral and cognitive consequences including elevated intake of EtOH during adulthood, with female mice showing greater susceptibility than males. Administration of the metabotropic glutamate receptor 5 (mGluR5) antagonist 3-((2-Methyl-1,3-thiazol-4-yl)ethynyl)pyridine (MTEP) has been shown to reduce EtOH self-administration in adult male mice, but little is known about its effect on female and adolescent mice. METHODS: MTEP (0, 10, 20 mg/kg, i.p.) was repeatedly administered to female and male, adult and adolescent C57BL/6J mice during binge sessions using the scheduled high alcohol consumption paradigm. Next, we assessed whether MTEP administration during binge altered the subsequent 24-hour EtOH intake following a period of abstinence. Finally, we investigated whether MTEP administration during binge followed by an abstinence period altered mRNA of glutamatergic genes within the nucleus accumbens of female mice. RESULTS: MTEP significantly decreased binge EtOH intake in all mice, but only female mice exhibited altered subsequent 24-hour EtOH intake. Interestingly, the alteration in subsequent EtOH intake in female animals was age dependent, with adolescent exposure to MTEP during binge decreasing 24-hour intake and adult exposure to MTEP during binge increasing 24-hour intake. Finally, while there were no effects of MTEP pretreatment on the genes examined, there was a robust age effect found during analysis of mGluR1 (Grm1), mGluR5 (Grm5), the NR2A subunit of the NMDA receptor (Grin2a), phosphatidylinositol 3-kinase (Pik3r1), mammalian target of rapamycin (Mtor), and extracellular signal-regulated kinase (Mapk1) mRNA, with adolescent female animals having lower expression than their adult counterparts. CONCLUSIONS: Collectively, the present findings add to existing evidence implicating the contribution of long-term effects of adolescent binge drinking to enhance alcohol abuse in adulthood, while suggesting that mGluR5 antagonism may not be the best pharmacotherapy to treat binge alcohol consumption in female and adolescent animals.

Androgen prevents hypogonadal bone loss via inhibition of resorption mediated by mature osteoblasts/osteocytes.

Androgen receptor (AR) is expressed throughout the osteoblast lineage. Two different AR transgenic families (AR2.3-tg and AR3.6-tg mice) demonstrating overlapping and distinct expression profiles were employed to assess the effects of enhanced androgen sensitivity to ameliorate hypogonadal loss. Two different paradigms of steroid replacement following orchidectomy (ORX) were used as either preventative or therapeutic therapy. ORX was performed in male wild-type (WT), AR2.3-tg and AR3.6-tg mice at 5 months with immediate DHT replacement (prevention, higher turnover) or at 3 months with DHT treatment delayed for 2 months (therapeutic, lower turnover), both with treatment for the last 6 weeks. Dual energy X-ray absorptiometry (DXA), micro-computed tomography (µCT), and histomorphometry were performed. In the prevention model, ORX significantly reduced BMD and BMC in all genotypes compared to sham and DHT was effective at prevention of osteopenia. In the therapeutic model, all genotypes became osteopenic compared to sham, but after a prolonged hypogonadal period, delayed DHT treatment provided little benefit. µCT analysis of mid-shaft total bone in all genotypes generally showed reductions after ORX. Delayed DHT was ineffective at restoring bone volume in any genotype whereas immediate treatment prevented loss only in AR transgenic mice. Cortical thickness also decreased with ORX but immediate DHT treatment was effective to increase thickness only in WT mice, likely due to expansion of marrow volume in both AR-tg lines. In metabolically highly active cancellous bone, ORX resulted in lower bone volume/tissue volume (BV/TV) in all genotypes, consistent among 3 sites measured. Again with delayed treatment, there was little effect of DHT to restore BV/TV, but when administered at the time of ORX, DHT completely prevented the decrease in cancellous bone in all genotypes. Improvement in cancellous bone architecture was seen with immediate DHT replacement that was enhanced in AR transgenic lines compared to WT. In contrast, there were only modest changes in all genotypes using the delayed treatment paradigm. With ORX in both paradigms, trabecular number was decreased while spacing increased. Thus, androgen therapy is effective for the prevention of endosteal and cancellous osteopenia primarily through its anti-resorptive properties, but shows little anabolic action as a therapeutic strategy to restore bone. Given the similarity in response to androgen treatment in both AR transgenic lines, overlapping expression profiles suggest that the target cells mediating androgen action in vivo are mature osteoblast/osteocytes. Combined, these results demonstrate that in the adult mouse, androgen treatment can reduce bone resorption but has little overall anabolic activity.

Stem cell activation in adults can reverse detrimental changes in body composition to reduce fat and increase lean mass in both sexes.

Detrimental changes in body composition are often associated with declining levels of testosterone. Here, we evaluated the notion that multipotent mesenchymal stem cells, that give rise to both fat and muscle tissue, can play a significant role to alter existing body composition in the adult. Transgenic mice with targeted androgen receptor (AR) overexpression in stem cells were employed. Wild-type littermate and AR-transgenic male and female mice were gonadectomized and left untreated for 2 months. After the hypogonadal period, mice were then treated with 5α-dihydrotestosterone (DHT) for 6 weeks. After orchidectomy (ORX), wild-type males have reduced lean mass and increased fat mass compared to shams. DHT treatment was beneficial to partially restore body composition. In wild-type females, ovariectomy (OVX) produced a similar change but there was no improvement with DHT. In targeted AR transgenic mice, DHT treatment increased lean and reduced fat mass to sham levels. In contrast to wild-type females, DHT treatment in female transgenic mice significantly ameliorated the increased fat and decreased lean mass changes that result after OVX. Our results show that DHT administration reduces fat mass and increases lean mass in wild-type males but not females, indicating that wild-type females are not as sensitive to androgen treatment. Because both male and female transgenic mice are more responsive than wild-type, results suggest that body composition remains linked to stem cell fate in the adult and that targeted androgen signaling in stem cells can play a significant role to reverse detrimental changes in body composition in both sexes.

Bone vs. fat: embryonic origin of progenitors determines response to androgen in adipocytes and osteoblasts.

Although androgen is considered an anabolic hormone, the consequences of androgen receptor (AR) overexpression in skeletally-targeted AR-transgenic lines highlight the detrimental effect of enhanced androgen sensitivity on cortical bone quality. A compartment-specific anabolic response is observed only in male and not in female AR3.6-transgenic (tg) mice, with increased periosteal bone formation and calvarial thickening. To identify anabolic signaling cascades that have the potential to increase bone formation, qPCR array analysis was employed to define expression differences between AR3.6-tg and wild-type (WT) periosteal tissue. Notably, categories that were significantly different between the two genotypes included axonal guidance, CNS development and negative regulation of Wnt signaling with a node centered on stem cell pathways. Further, fine mapping of AR3.6-tg calvaria revealed that anabolic thickening in vivo is not uniform across the calvaria, occurring only in frontal and in not parietal bones. Multipotent fraction 1 progenitor populations from both genotypes were cultured separately as frontal bone neural crest stem-like cells (fNCSC) and parietal bone mesenchymal stem-like cells (pMSC). Both osteoblastic and adipogenic differentiation in these progenitor populations was influenced by embryonic lineage and by genotype. Adipogenesis was enhanced in WT fNCSC compared to pMSC, but transgenic cultures showed strong suppression of lipid accumulation only in fNCSC cells. Osteoblastogenesis was significantly increased in transgenic fNCSC cultures compared to WT, with elevated alkaline phosphatase (ALP) activity and induction of mineralization and nodule formation assessed by alizarin red and von Kossa staining. Osteocalcin (OC) and ALP mRNA levels were also increased in fNCSC cultures from AR3.6-tg vs. WT, but in pMSC cultures ALP mRNA levels, mineralization and nodule formation were decreased in AR3.6-tg cells. Expression differences identified by array in long bone periosteal tissue from AR3.6-tg vs. WT were recapitulated in the fNCSC samples while pMSC profiles reflected cortical expression. These observations reveal the opposing effects of androgen signaling on lineage commitment and osteoblast differentiation that is enhanced in cells derived from a neural crest origin but inhibited in cells derived from a mesodermal origin, consistent with in vivo compartment-specific responses to androgen. Combined, these results highlight the complex action of androgen in the body that is dependent on the embryonic lineage and developmental origin of the cell. Further, these data these data suggest that the periosteum surrounding long bone is derived from neural crest.

Body composition changes and inhibition of fat development in vivo implicates androgen in regulation of stem cell lineage allocation.

Androgens regulate body composition in youth and declining testosterone that occurs with aging is associated with muscle wasting, increased fat mass and osteopenia. Transgenic mice with targeted androgen receptor (AR) over-expression in mesenchymal stem cells (MSC) were generated to explore the role of androgen signaling in the regulation of body composition. Transgenic males, but not females, were shorter and have reduced body weight and visceral fat accumulation. Dual-energy X-ray absorptiometry (DXA) revealed significant reductions in fat mass with a reciprocal increase in lean mass, yet no difference in food consumption or locomotor activity was observed. Adipose tissue weight was normal in brown fat but reduced in both gonadal and perirenal depots, and reduced hyperplasia was observed with smaller adipocyte size in visceral and subcutaneous white adipose tissue. Although serum leptin, adiponectin, triglyceride, and insulin levels were no different between the genotypes, intraperitoneal glucose tolerance testing (IPGTT) showed improved glucose clearance in transgenic males. High levels of the AR transgene are detected in MSCs but not in mature fat tissue. Reduced fibroblast colony forming units indicate fewer progenitor cells resident in the marrow in vivo. Precocious expression of glucose transporter 4 (GLUT4), peroxisome proliferator-activated receptor γ (PPARγ), and CCAAT enhancer-binding protein α (C/EBPα) was observed in proliferating precursor cultures from transgenic mice compared to controls. In more mature cultures, there was little difference between the genotypes. We propose a mechanism where enhanced androgen sensitivity can alter lineage commitment in vivo to reduce progenitor number and fat development, while increasing the expression of key factors to promote smaller adipocytes with improved glucose clearance.

Elevated testosterone in females reveals a robust sex difference in altered androgen levels during chronic alcohol withdrawal.

The endocrine disruption associated with alcohol (ethanol) abuse in both males and females is widely recognized. Ethanol intoxication and withdrawal in males results in significant reductions in androgen levels. Less is known about female alcoholics, and because the changes in testosterone concentrations remain controversial, we systematically characterized changes in sex steroids after chronic ethanol exposure and withdrawal in both sexes. Testosterone and 17ß-estradiol concentrations were determined during chronic high intoxication, over a withdrawal time course, and following a period of abstinence using a genetic model of withdrawal vulnerability, the Withdrawal Seizure-Resistant (WSR) and -Prone (WSP) selected lines. In males, testosterone concentrations were significantly lower in intoxicated WSP mice after chronic ethanol exposure, and were dramatically and transiently reduced during the withdrawal period in both WSR and WSP lines. In contrast, testosterone levels were increased in intoxicated WSP females and in both WSR and WSP mice during withdrawal. Chronic ethanol exposure disrupted normal estrous cycling in WSP mice, associated with hyperandrogenemia while intoxicated. In abstinence, elevated testosterone was observed in both sexes but only in WSR mice. Estrogen levels were modestly reduced during withdrawal in both WSR and WSP lines, predominantly in males. These findings identify a mechanism based on altered androgen signaling that likely contributes to sex-specific responses during withdrawal. However, only WSR mice showed similar elevations in androgen long after withdrawal in both sexes, suggesting that genotype is an important determinant of steroid responses after abstinence. Increased androgen signaling in females as a consequence of chronic ethanol exposure may play an important and relatively uncharacterized role in sexually dimorphic responses to alcohol abuse.

Individual differences in hyperlipidemia and vitamin E status in response to chronic alcohol self-administration in cynomolgus monkeys.

BACKGROUND: Chronic ethanol self-administration induces oxidative stress and exacerbates lipid peroxidation. α-Tocopherol is a potent lipid antioxidant and vitamin that is dependent upon lipoprotein transport for tissue delivery. METHODS: To evaluate the extent to which vitamin E status is deranged by excessive alcohol consumption, monkeys voluntarily drinking ethanol (1.36 to 3.98 g/kg/d for 19 months, n = 11) were compared with nondrinkers (n = 5, control). RESULTS: Three alcohol-drinking animals developed hyperlipidemia with plasma triglyceride levels (1.8 ± 0.9 mM) double those of normolipidemic (NL) drinkers (0.6 ± 0.2) and controls (0.6 ± 0.3, p < 0.05); elevated plasma cholesterol (3.6 ± 0.5 mM) compared with NL drinkers (2.3 ± 0.2, p < 0.05) and controls (2.9 ± 0.3); and lower plasma α-tocopherol per triglycerides (14 ± 6 mmol/mol) than controls (27 ± 8) and NL drinkers (23 ± 6, p < 0.05). Hyperlipidemic monkey liver α-tocopherol (47 ± 15 nmol/g) was lower than NL drinkers (65 ± 13) and controls (70 ± 15, p = 0.080), as was adipose α-tocopherol (84 ± 37 nmol/g) compared with controls (224 ± 118) and NL drinkers (285 ± 234, p < 0.05). Plasma apolipoprotein (apo) CIII increased compared to baseline at both 12 and 19 months in the normolipidemic (p = 0.0016 and p = 0.0028, respectively) and in the hyperlipidemic drinkers (p < 0.05 and p < 0.05, respectively). Plasma apo H concentrations at 19 months were elevated hyperlipidemics (p < 0.05) relative to concentrations in control animals. C-reactive protein (CRP), a marker of inflammation, was increased compared to baseline at both the 12- and 19-month time points in the normolipidemic (p = 0.005 and p = 0.0153, respectively) and hyperlipidemic drinkers (p = 0.016 and p = 0.0201, respectively). CONCLUSION: A subset of alcohol-drinking monkeys showed a predisposition to alcohol-induced hyperlipidemia. The defect in lipid metabolism resulted in lower plasma α-tocopherol per triglycerides and depleted adipose tissue α-tocopherol, and thus decreased vitamin E status.

Androgen receptor overexpression is neuroprotective in experimental stroke.

Male sex is a known risk factor in human stroke. However, the role of the cognate receptor for androgens-the androgen receptor (AR)-in stroke outcome remains unclear. Here, we found that AR mRNA is downregulated in the peri-infarct tissue of gonadally intact male mice subjected to middle cerebral artery occlusion (MCAO) and 6 h reperfusion. We then used genetically engineered mice overexpressing AR in brain (AR-Tg) to compare outcomes from MCAO in intact or castrated males and to evaluate the neuroprotective role of dihydrotestosterone (DHT) replacement in AR-Tg castrates. A further evaluation of AR overexpression in ischemic paradigms was performed using rat PC12 cells transfected with human AR and treated with oxidative and apoptotic stressors. We then studied the role of DHT in cultures overexpressing AR. Our results show (1) ischemia alters the expression of AR by decreasing AR mRNA levels, (2) AR overexpression is protective in vivo against MCAO in intact and castrated AR-Tg mice and in vitro against oxidative and apoptotic stressors in AR-PC12 cells, and (3) DHT does not enhance the protection triggered by AR overexpression in AR-Tg castrated mice nor in AR-PC12 cells.

Signaling pathways implicated in androgen regulation of endocortical bone.

Periosteal expansion is a recognized response to androgen exposure during bone development and in profoundly hypogonadal adults. However, androgen also suppresses endocortical bone formation, indicating that its effects on bone are dichotomous and envelope-specific. In fact, enhanced androgen signaling has been shown to have dramatic detrimental effects on whole bone biomechanical properties in two different transgenic models with skeletally targeted androgen receptor (AR) overexpression. As the mechanisms underlying this response are uncharacterized, we compared patterns of gene expression in periosteum-free cortical bone samples derived from AR-overexpressing transgenic male mice and their wild-type counterparts. We then assessed direct androgen effects in both wild-type and AR-overexpressing osteoblasts in primary culture. Among major signaling pathways associated with bone formation, focused quantitative RT-PCR (qPCR) array-based analysis of endocortical bone gene expression from wild-type vs. transgenic males identified the transforming growth factor-beta (TGF-beta) superfamily and bone morphogenetic protein (BMP) signaling as significantly altered by androgen in vivo. Bioinformatic analyses indicated proliferation, osteoblast differentiation and mineralization as major biological processes affected. Consistent with the in vivo array data and bioinformatic analyses, inhibition of differentiation observed with androgen exposure was reduced by exogenous BMP2 treatment of AR-overexpressing cultures to stimulate BMP signaling, confirming array pathway analysis. In addition, nonaromatizable dihydrotestosterone (DHT) inhibited osteoblast proliferation, differentiation and several indices of mineralization, including mineral accumulation and mineralized nodule formation in primary cultures from both wild-type and AR-transgenic mice. These findings identify a molecular mechanism based on altered BMP signaling that contributes to androgen inhibition of osteoblast differentiation and mineralization. Such detrimental effects of androgen on osteoblast function may underlie the generally disappointing results of androgen therapy.

Selected line difference in the effects of ethanol dependence and withdrawal on allopregnanolone levels and 5alpha-reductase enzyme activity and expression.

BACKGROUND: Allopregnanolone (ALLO) is a progesterone derivative that rapidly potentiates gamma-aminobutyric acid(A) (GABA(A)) receptor-mediated inhibition and modulates symptoms of ethanol withdrawal. Because clinical and preclinical data indicate that ALLO levels are inversely related to symptoms of withdrawal, the present studies determined whether ethanol dependence and withdrawal differentially altered plasma and cortical ALLO levels in mice selectively bred for differences in ethanol withdrawal severity and determined whether the alterations in ALLO levels corresponded to a concomitant change in activity and expression of the biosynthetic enzyme 5alpha-reductase. METHODS: Male Withdrawal Seizure-Prone (WSP) and -Resistant (WSR) mice were exposed to 72 hours ethanol vapor or air and euthanized at select times following removal from the inhalation chambers. Blood was collected for analysis of ALLO and corticosterone levels by radioimmunoassay. Dissected amygdala, hippocampus, midbrain, and cortex as well as adrenals were examined for 5alpha-reductase enzyme activity and expression levels. RESULTS: Plasma ALLO was decreased significantly only in WSP mice, and this corresponded to a decrease in adrenal 5alpha-reductase expression. Cortical ALLO was decreased up to 54% in WSP mice and up to 46% in WSR mice, with a similar decrease in cortical 5alpha-reductase activity during withdrawal in the lines. While cortical gene expression was significantly decreased during withdrawal in WSP mice, there was a 4-fold increase in expression in the WSR line during withdrawal. Hippocampal 5alpha-reductase activity and gene expression was decreased only in dependent WSP mice. CONCLUSIONS: These results suggest that there are line and brain regional differences in the regulation of the neurosteroid biosynthetic enzyme 5alpha-reductase during ethanol dependence and withdrawal. In conjunction with the finding that WSP mice exhibit reduced sensitivity to ALLO during withdrawal, the present results are consistent with the hypothesis that genetic differences in ethanol withdrawal severity are due, in part, to modulatory effects of GABAergic neurosteroids such as ALLO.

Targeting of androgen receptor in bone reveals a lack of androgen anabolic action and inhibition of osteogenesis: a model for compartment-specific androgen action in the skeleton.

Androgens are anabolic hormones that affect many tissues, including bone. However, an anabolic effect of androgen treatment on bone in eugonadal subjects has not been observed and clinical trials have been disappointing. The androgen receptor (AR) mediates biological responses to androgens. In bone tissue, both AR and the estrogen receptor (ER) are expressed. Since androgens can be converted into estrogen, the specific role of the AR in maintenance of skeletal homoeostasis remains controversial. The goal of this study was to use skeletally targeted overexpression of AR in differentiated osteoblasts as a means of elucidating the specific role(s) for AR transactivation in the mature bone compartment. Transgenic mice overexpressing AR under the control of the 2.3-kb alpha1(I)-collagen promoter fragment showed no difference in body composition, testosterone, or 17ss-estradiol levels. However, transgenic males have reduced serum osteocalcin, CTx and TRAPC5b levels, and a bone phenotype was observed. In cortical bone, high-resolution micro-computed tomography revealed no difference in periosteal perimeter but a significant reduction in cortical bone area due to an enlarged marrow cavity. Endocortical bone formation rate was also significantly inhibited. Biomechanical analyses showed decreased whole bone strength and quality, with significant reductions in all parameters tested. Trabecular morphology was altered, with increased bone volume comprised of more trabeculae that were closer together but not thicker. Expression of genes involved in bone formation and bone resorption was significantly reduced. The consequences of androgen action are compartment-specific; anabolic effects are exhibited exclusively at periosteal surfaces, but in mature osteoblasts androgens inhibited osteogenesis with detrimental effects on matrix quality, bone fragility and whole bone strength. Thus, the present data demonstrate that enhanced androgen signaling targeted to bone results in low bone turnover and inhibition of bone formation by differentiated osteoblasts. These results indicate that direct androgen action in mature osteoblasts is not anabolic, and raise concerns regarding anabolic steroid abuse in the developing skeleton or high-dose treatment in eugonadal adults.