The Regulation Mechanism Of Androgen On Sex Hormone-Binding Globulin Expression

Backgroud and ObjectiveHuman testosterone in plasma about 66%is bound to sex hormone-binding globulin(SHBG),31%to albumin,and 1.5%to transcortin,only 2%is unbound and only the unbound hormone is biologically active and available to tissues.Thus,the circulating SHBG level is a major determinant of the metabolic clearance of testosterone,modulating the access of testosterone to their target tissues.SHBG mainly expressed in the liver and secreted into the blood,regulating blood androgen levels;in addition to the liver,the ovaries are also a small amount of expression,regulating ovarian local androgen levels,affecting female reproductive function.About 70%-80%of patient with polycystic ovary syndrome(PCOS)are hyperandrogenism,and PCOS patients have low levels of serum SHBG than non-PCOS patients.There are assumption that androgen suppress the expression of SHBG,but little known about the mechanisms.Adenosine monophosphate activated protein kinase(AMPK)modulate cellular metabolism and to control cell growth.Peroxisome proliferator-activated receptor y(PPARy)and hepatocyte transcription factor(HNF-4?)are downstream regulatory factors after AMPK activation,and studies have shown that PPAR? and HNF-4? are competitive with the SHBG promoter footprint structure;the former inhibits the expression of SHBG while the latter promotes SHBG expression.This study investigated the relationship among androgen/AR?AMPK activation,the expression of PPARy and HNE-4?,and the expression of SHBG.Methods and Results1.In this study,in order to verify the effect of androgen/AR on the expression of SHBG and verify whether AMPK phosphorylation?PPAR? and HNF-4? expression were changed,human hepatocellular carcinoma cell line HepG2 and normal liver cell line L02 and ovarian granule cancer cell line KGN were treated with DHT(androgen receptor agonists)and flutamide(androgen receptor antagonist).The rusults show that the expression of the expression of SHBG in L02 and KGN was increased,but decreased in HepG2 compared with the blank control group and DF group.The expression of p-AMPK protein expression was higher than that in blank control group and DF group in LO2?HepG2 and KGN.The expression ofHNF-4? protein expression were not obvious changed in L02,HepG2 and KGN compared with the blank control group and DF group.The expression of PPARy decreased in L02 and KGN,but increased in HepG2 compared with the blank control group and DF group.2.In order to verify whether the expression changes of SHBG,PPARy and HNF-4a were consistent with DHT treatment after AMPK activation,the above cell lines were treated with AICAR(AMPK agonist))and Compound-C(AMPK antagonist).The results showed that the expression of SHBG in L02 increased after AICAR treatment,but decreased in HepG2 compared with blank control group and Compound-C group.The expression of PPARy decreased in L02,but increased in HepG2 compared with blank control group and Compound-C group.The expression of HNF-4a were not obvious changed in HepG2 and KGN.The experimental results were consistent with DHT treatment.But the expression of HNF-4a decreased in L02 after Compound-C treatment compared with control group and AICAR group.3.In order to verify whether the effect of androgen on expression of SHBG in liver and ovarian granulosa cells in the body is consistent with the results in vitro cell levels.We selected 21-day-old female SD rats,daily subcutaneous injection of dehydroepiandrosterone(DHEA)to construct PCOS model,the control group were injected with equal volume of oil.After 35 days of continuous injection,WB method was used to detect the expression of SHBG,p-AMPK,PPARy and HNF-4a in the liver.The expression of SHBG and PPARy in ovarian granulosa cells detected by immunohistochemistry.ELISA used to detect serum SHBG levels.The result show that the expression of SHBG was decreased and the expression of p-AMPK and PPAR? was increased and the expression of HNF-4a was not changed in the HA group compared with the control group in liver.The expression of SHBG and PPARy in the ovarian granulosa cells increased.The serum SHBG levels decreased compared with the control group.Conclusion1.The effect of androgen on the expression of SHBG was different between in L02 and HepG2.The expression of SHBG promoted in L02,but the expression of SHBG inhibited in HepG2 after DHT treated.2.The expression of PPARy protein inhibited by activating AMPK by activating AMPK,which weakened the inhibitory effect of PPARy on SHBG expression and increased the expression of SHBG in L02 and KGN.The expression of PPARy promoted enhances the inhibitory effect of PPAR? on SHBG expression,which reduces the expression of SHBG in HepG2.3.Hepatic SHBG expression and secretion is difference between in in vivo and in vitro.The expression of SHBG decreased,the phosphorylation level of AMPK was increased and the expression of PPARy increased in liver,which was consistent with that of HepG2 cells in vitro.The expression of SHBG in ovarian granulosa cells increased consistent with the results of in vitro KGN and GC cells.4.The expression of SHBG in ovarian granulosa cells in vivo was consistent with that in vitro,and the expression of SHBG promoted by androgen treatment.