1.Chronic Ethanol Feeding Impairs The Synthesis And Action Of Testosterone In Rats' Testis: Role Of PPARa, PBR And ABP 2.Chronic Ethanol Feeding Impairs The Expression Of GluT4 In Rats Liver Via An AMPK-Mediated Mechanism

Background:Excessive ethanol consumption may be among the causes leading to male infertility, while the precise mechanism is not fully understood. It has been proposed that the decreased testosterone synthesis and secretion caused by ethanol consumption might the possible mechanisms underlying. Numerous clinical and experimental data have shown the effect of ethanol on impaired testosterone synthesis. Testosterone is mainly produced by leydig cells in testis, and the synthesis and secretion of testosterone is mainly regulated by hypothalamus-pituitary-adrenal axis. Ethanol could reportedly affect the activity of HPA axis, which is believed as the possible mechanism of ethanol.Recently, Wanderley MI and Murono EP has demonstrated that in cultured rats leydig cells, both the LH-stimulated and basal testosterone synthesis were decreased significantly by ethanol exposure, thus indicating a direct effect of ethanol on testis leydig cells. Further, it was proposed that the precise step impaired by ethanol on leydig cells might lie on the common pathway of testosterone synthesis, as named as the mitochondria stage.PBR, Peripheral-type Benzodiazepine Receptor, represents the receptors that could recognize benzodiazepines and locate in tissues besides brain. Tissue distribution of PBR shows their abundance in adrenal gland and testis. Recently, itwas raised that PBR is functionally linked to leydig cells testosterone synthesis. PBR is located on the outer membrane in mitochondria and plays a key role in the transport of cholesterol from outer to inner membrane of mitochondria. As is known, the entry of cholesterol from extramitochondria stores into mitochondria is the first step, also the rate-limiting step, of the mitochondria stage of testosterone synthesis. Also, ethanol could reportedly affect PBR expression, which might participate the pathophysiology of many ethanol-relayed disease, such as alcohol sedation and alcoholic hepatitis.PPARa is a regulator of PBR expression and ethanol is known to downregulate PPARa expression. Whether ethanol could affect PBR expression via a PPARa mediated pathway, thereby affect legdig cells testosterone synthesis is unclear.ABP, androgen binding protein, is produced by testis Sertoli cells under stimulation of FSH. ABP exert its biological effect mainly via binding to testosterone, thereby raising testosterone concentrations around contorted seminiferous tubules and stimulating spermatogenesis. The effect of ethanol on ABP expression is unknown. Objective:(1)The effect of chronic ethanol feeding at different doses on testosterone levels in rats testis.(2)The effect of chronic ethanol feeding on the expression of PPARa and PBR and the possible PPARa and PBR mediated mechanisms of ethanol-impaired testosterone synthesis.(3)The changes of ABP expression after ethanol exposure and the possible role of ABP of ethanol's action. Methods:(4)Animal feeding: Forty male Wistar rats, divided into four groups, received either distilled water (control group) or ethanol, which was administered by gastric tube with a single daily dose: 5 g · kg^-1 (large dose group), 2.5 g · kg^-1 (middle dose group) and 0.5 g · kg^-1 (small dose group).(2) Measurement of testis testosterone levels: ITT (Intra-Testis Testosterone) levels were measured using ELISA.? Effects of ethanol on PPARa and PBR: mRNA levels of PPARa and PBR of rats'testis were measured using RT-PCR.?The alternations of ABP expression after ethanol feeding: mRNA levels of ABP were determined using RT-PCR. Results:?Compared with control, ethanol feeding with daily doses of 5g 'kg*1, 2.5g *kg"' and 0.5 g -kg"1 significantly decreased ITT levels by 31.13%(p<0.05), 26.8%(p<0.05) and 4.2%(p<0.05) separately, indicating ethanol might impair testosterone synthesis.? Compared with control, mRNA levels of PBR decreased in all three ethanol-treated groups(p<0.05). As a known regulator of testis testosterone synthesis, the alternations of PBR might contribute, at least partly, to the impaired testosterone synthesis caused by ethanol.?Accompanied with the changes of PBR, PPARa expression also decreased after ethanol exposure(p<0.05). As PPARa is a known positive regulator of PBR expression, the findings further indicates the possible PPARa and PBR mediated pathway in ethanol-impaired testosterone synthesis.?Compared with control, mRNA levels of ABP also decreased in all three ethanol-treated groups(p<0.05), suggesting ethanol could not only impair testosterone synthesis but also could affect the action of testosterone. Conclusions:?Ethanol feeding could impair testis testosterone synthesis at least partly via decreased PPARa and PBR expression.?The alternations of ABP expression might also play a role in ethanol-induced male infertility.Background:It has been proposed that ethanol consumption is an independent risk factor leading to insulin resistance. To date, several studies have shown the effects of ethanol intake on glucose intolerance in muscle, adipocytes, and liver. But the precise molecular mechanism is not fully elucidated. Data available indicate that the primary step impaired by ethanol may likely lie in the downstream of insulin signal pathway. For example, it has been demonstrated that ethanol-induced insulin resistance is neither accompanied with the impaired expression of insulin receptor, IRS and PI3K, nor the impairment of their activities. Glut4 is the final molecular mediating insulin-stimulated glucose disposal, and ethanol reportedly can inhibit its translocation stimulated by insulin, thus makes it a candidate site of ethanol's effect. Although ethanol can inhibit the translocation of glut4, whether ethanol can affect its expression is unclear and results of related studies are controversy.Several cytokines could regulate GluT4 expression, such as MEF and GEF and so on. Recently, AMPK has been proposed as a positive regulator of GluT4 expression. Besides affecting GluT4 expression, the major biological effect of AMPK is to regulate intracellular energy metabolism. Thus, AMPK plays a key role in the linkage between energy metabolism and GluT4 expression. Recently, it has demonstrated that AMPK is involved in the pathophysiology of many energy-relatedinsulin resistance, such as glutoxicity and lipotoxicity. As is known, ethanol is also an substance of high energy. Whether AMPK also participates in the ethanol-induced insulin resistance remains to be elucidated. Objective:?The effect of chronic ethanol feeding at different doses on GluT4 expression in rats liver.?The effect of chronic ethanol feeding on the expression and activity of AMPK and the possible role of AMPK in ethanol-induced insulin resistance. Methods:?Animal feeding: Forty male Wistar rats, divided into four groups, received either distilled water (control group) or ethanol, which was administered by gastric tube with a single daily dose: 5 g ? kg"1 (large dose group), 2.5 g ? kg"1 (middle dose group) and 0.5 g ? kg"1 (small dose group).?Measurement of GluT4 expression: mRNA levels of GluT4 of rats' liver were measured using Real-Time PCR and RT-PCR;Protein levels were further measured using Western blot.?Effects of ethanol on the expression and activity of AMPKa: mRNA levels of alpha 1 subunit and alpha 2 subunit of AMPK were measured using Real-Time PCR and RT-PCR;Protein levels of total AMPK alpha subunit were detected using Western blot;Activities of AMPK were evaluated by measuring the protein levels of phosphated AMPK alpha subunit using Western blot. Results:?Compared with control, Ethanol feeding with daily doses of 5g ? kg"1 and 2.5g ? kg"1 significantly decreased GluT4 expression (PO.05), whereas no significant changes of GluT4 expression was found in 0.5g ? kg"1 alcohol-fed group.?Compared with control, no differences were detected in mRNA levels of alpha 1 subunit and alpha 2 subunit of AMPK in all ethanol-treated groups. Consistently, protein levels of total AMPK alpha subunit were not altered after ethanol feeding. While, the protein levels of phosphated AMPK alpha subunit significantly decreased after ethanol treatment (P<0.05), indicating ethanol exposure might impair AMPKactivity. Conclusions:?Chronic ethanol feeding decreased the expression of GluT4 in rats liver, indicating the onset of insulin resistance and the possible site impaired by ethanol.?Ethanol treatment could impaire the activity of AMPK, while does not affect its expression. AS a known positive regulator of GluT4 expression, the changes of AMPK activity may participate in, at least in part, the ethanol-induced liver insulin resistance.