The Role Of Adipose H6PDH In The Pathogenesis Of Obesity And Insulin Resistance

Aim:Obesity is a severe metabolic disease for public health threat that dramatically increases the risk of type 2 diabetes(T2DM)and metabolic syndrome(MS).Glucocorticoids(GCs),one of the steroids secreted by adrenal cortex regulate the metabolism of glucose and fat.Excessive GCs generation and intake will induce obesity and insulin resistance.11β-hydroxysteroid dehydrogenase type 1(11β-HSD1)activates adipose GCs production depending on endoplasmic reticulum NADPH,which has been known as a major etiologic factor of obesity and metabolic syndrome.Hexose-6-phosphate dehydrogenase(H6PDH)is the key enzyme for NADPH generation at endoplasmic reticulum and regulate the activity of 11β-HSD1.However,the role and molecular mechanism of H6PDH in the regulation of adipose GCs metabolism and inducing obesity and insulin resistance are still unclear.We aimed to observe the changes of metabolic phenotypes in adipose tissue-specific H6PDH overexpression and knockout mouse models,and to explore the role and molecular mechanism of adipose H6PDH in regulating fat GCs metabolism and inducing obesity and insulin resistance.These data will provide new target and theoretical evidence for prevention and treatment of obesity and metabolic syndrome in clinic.Methods:1.The role and molecular mechanism of adipose H6PDH overexpression in obesity and metabolic syndromeThe litters from fat-specific overexpression H6PDH(aP2-H6PDH-Tg)mice,which has been established by our research group,were genotyped.aP2-H6PDH and wild-type(WT)male mice were clarified.Adult male transgenic mice and their age-matched were maintained on either control diet or high fat diet(HFD)and were divided into 4 groups:aP2-H6PDH mice with control diet;aP2-H6PDH mice with HFD diet;WT mice with control diet;WT mice with HFD.GTT and ITT were performed.The mice were sacrificed at 24weeks.Blood samples and adipose fat depots and non-adipose tissues were collected.1)Body weight,fat depots,GTT and ITT were monitored and recorded.2)Plasma corticosterone and free fatty acid were detected.3)Lipid droplet accumulation was monitored using Oil Red O staining.4)Adipose corticosterone and the activities of H6PDH and llβ-HSD1 were measured.5)The pathways involving obesity were investigated by real time RT-PCR and Western blot,including adipose and non-adipose tissue H6PDH,adipose GCs pathway(11β-HSD1,GR);the key enzymes of lipogenesis(ACC,ACL,FAS,PEPCK);obese genes(C/EBPα,PPARγ,SREBP,Leptin);Endoplasmic reticulum stress(IRElα/XBP1);Akt/GSK3β pathway and browning marker genes(CD137,TBX GLUT4).2.The role of adipose-specific knockout H6PDH in improved metabolic phenotype(1)Adipose-specific knockout mice of H6PDH(H6PDHAcKO)were achieved by breeding a floxed H6PDH allele mice with an adipocyte-specific expression Cre line.(2)The litters from adipose-specific knockout H6PDH(H6PDHAcKO)mice were genotyped and H6PDHAcKO and Fl/Fl male mice were screened.Male knockout mice and their age-matched mice were maintained on either control diet.GTT and ITT were performed.The mice were sacrificed at 18weeks at fating for 12h or non-fasting conditions and divided into 4 groups:H6PDHAcKO at fasting status;H6PDHAcKO at non-fasting status;Flox at fasting status;Flox at non-fasting status.Blood samples and adipose fat depots and non-adipose tissue were collected.1)Body weight,fat depots,GTT and ITT were monitored and recorded.2)Plasma corticosterone,insulin and free fatty acid at fasting and non-fasting status were detected3)Adipocyte size was measured by HE staining4)Adipose corticosterone and the activities of H6PDH and 11 β-HSD1 were measured5)Adipose and non-adipose H6PDH expression level were detected by real time RT-PCR and Western blot;The lipogenesis and lipolysis of adipose tissue were also investigated including the key enzyme of lipogenesis(ACC,ACL);the limiting enzyme of lipolysis(HSL,ATGL)Result:1.The role and molecular mechanism of adipose H6PDH overexpression in obesity and metabolic syndrome(1)aP2-H6PDH mice showed increased abdominal fat accumulation,which is paralleled by elevated lipid synthesis associated with induction of lipogenic transcriptor C/EBPa and PPARy mRNA levels within adipose tissue(2)aP2-H6PDH mice fed a high-fat diet gained more abdominal visceral fat mass coupled with activation of GSK3β and induction of XBP1/IREla,but reduced pThr308 Akt/PKB content and browning gene CD137 and GLUT4 mRNA levels within the visceral adipose tissue than WT controls(3)aP2-H6PDH mice had impaired glucose tolerance and insulin sensitivity and worsen insulin resistance induce by high-fat diet(4)aP2-H6PDH mice fed a high-fat diet exhibited elevated levels of intra-adipose GCs with induction of adipose 11β-HSD1,but did not change the circulation GCs levels 2.The role of adipose specific knockout H6PDH in improved metabolic phenotype(1)H6PDHAcKOmice exhibited almost complete absence of H6PDH expression and in adipose tissue,did not affect H6PDH expression in non-adipose tissues(2)H6PDHAcKOmice decreased abdominal fat mass,especially for mesenteric fat,which was paralleled by decreased adipose lipogenic ACC and ACL gene expression and reduction in their transcription factor C/EBPa mRNA levels(3)H6PDHA,KOmice reduced fasting blood glucose levels and improved glucose tolerance and insulin sensitivity(4)H6PDHAcKOmice decreased key enzymes of lipolysis(ATGL and HSL)expression in adipose tissue and plasma free fatty acid(FFA)levels(5)H6PDHA,KOmice exhibited decreased intra-adipose 11β-HSD1 expression,activity and intra-adipose GCs levels,but did not change the circulation GCs levelsConclusion:(1)Adipose H6PDH overexpression and knockout mice may be used as ideal animal models to explore human metabolic syndrome(2)Adipose H6PDH causes the changes of fatty metabolism related phenotypes through regulating the expression of 11β-HSD1 and activating local GCs production(3)Stimulating transcription factor CEBPα、PPARγ,inducing Endoplasmic reticulum stress,activating Akt/GSK3β pathway and reducing white fat browning are the potential pathogenesis of abdominal fat accumulation induced by adipose H6PDH overexpression.(4)Adipose H6PDH overexpression impairs glucose tolerance and insulin sensitivity,and worsens abdominal obesity and insulin resistance induced by high-fat diet;on the contrary,adipose H6PDH knockout inhibits lipogenesis and improves fat distribution,and also decreases lipolysis and elevates insulin sensitivity(5)Adipose H6PDH overexpression and knockout mice changed intra-adipose glucocorticoid,but did not find the effect on plasma GCs levels.