The Mechanism Of The Effect Of Hepatic HRD1 On The Reproductive Function Of Female Mice By Regulating Energy Metabolism Through FGF21

ObjectivesInfertility has become one of the most serious problems.Exploring the molecular mechanism of infertility is imperative.Liver plays an important role in energy metabolism which affects female reproductive functions.As a ubiquitin ligase mainly expressed in liver,HRD1 regulates energy expenditure.But the relationship between HRD1 and fertility is still not clear.And it is necessary to establish a molecular link between energy and fertility.Energy metabolism and reproductive function of female mice were explored to clarify how liver works in a molecular way,based on the liver specific HRD1 knockout mouse models in our laboratory.Our study enriches the biological function of HRD1 and reveals the molecular mechanism of infertility and provides a reliable theoretical for the prevention and treatment of infertility in the future.MethodsFirst,HRD1 KO mice were employed to observe the reproductive function and energy metabolism and to detect the level of FGF21.Secondly,to verify the PERK-ATF4-CHOP and CREBH/PPARα pathway and to detect the protein interaction between HRD1 and CREBH.Finally,to observe the influence on female reproduction after High Fat Diet.Results1.The effect of HRD1 on fertility of female mice.HRD1 KO mice display the reduction of body weight and body length.We also observed that female KO mice were infertile and displayed a prolonged diestrus.The increased energy expenditure could suggest the reason of reduced body weight in KO mice.To study the role of HRD1 in energy metabolism,HRD1KO mice and their littermate controls were placed on a HFD at the age of 6 weeks.As expected,the control mice gradually gained body weight on the HFD,but the body weight gain of KO mice was significantly reduced.However,liver-specific HRD1 deletion protects mice from HFD-induced obesity and hepatic steatosis.Further,the accumulation of subcutaneous white adipose tissue and lipid levels were dramatically lower in HRD1 KO mice.Histological analysis detected a significant increase in browning of the white adipose tissue in HRD1 KO mice.And mice with liver HRD1 deletion display other key beneficial effects of FGF21 overexpression.2.The molecular mechanism of HRD1 regulates FGF21 in liver.It was reported that FGF21 expression is regulated by the ER stress-associated transcription factors including ATF4 and CREBH.Our results excluding the possibility that HRD1 regulates FGF21 transcription through targeting ATF4.Interestingly,our study found CREBH but not PPARa was increased in the HRD1 KO liver.Further,Co-IP and Western blotting analysis detected that HRD1 interacted with CREBH and its overexpression dramatically enhanced CREBH ubiquitination.These results indicate that HRD1 regulates FGF21 through HRD1-CREBH-FGF21 pathway.3.The effect of energy metabolism on fertility of female mice.We discovered that female infertility of liver-specific HRD1 knockout mice could be fully rescued by additional energy supplementation upon HFD feeding.These results suggested energy expenditure consequently leads to female fertility.Conclusion1.Liver specific HRD1 deletion leads to infertility,weight loss and body fat loss in female mice,and including energy expenditure increased.2.The deletion of liver HRD1 significantly increased the expression of FGF21 on both protein levels and mRNA levels.3.HRD1 did not regulate FGF21 through PERK-ATF4-CHOP pathway and PPARα.4.HRD1 interacts with CREBH and promotes its ubiquitination mediated degradation.HRD1 regulated FGF21 through HRD1-CREBH-FGF21 pathway.5.Energy supplementary rescued infertility caused by liver specific HRD1 deletion.