YAP Suppresses Gluconeogenic Gene Expression Via PGC1?

Background:YAP,a transcription factor,is a key regulator of liver size,development and function.In the liver,YAP activation produces hepatomegaly,whereas Yap deletion leads to the development of smaller,dysfunctional livers.YAP is also required for the regenerative response to hepatic injury and frequently activated in pathological growth,such as hepatocellular carcinoma.The massive growth induced by YAP requires ATP,metabolic building blocks and reducing equivalents.Gluconeogenesis,a metabolic process mainly taking place in liver,converts non-carbohydrate sources(like amino acids)into glucose.In times of fasting,it supplies glucose to the brain and other tissues,preventing the development of severe hypoglycemia and death.However,this altruistic response comes at great cost to the hepatocyte:each molecule of glucose generated by the hepatocyte requires six adenosine triphosphate(ATP)equivalents,two nicotinamide adenine dinucleotide hydride(NADH),and two molecules of pyruvate.We would thus expect gluconeogenesis to be tightly regulated in the proliferating hepatocyte,allowing it to balance the needs of the whole organism for glucose with its own needs for anabolic building blocks.The connection between YAP and cellular metabolism is only now emerging.For example,YAP is recently shown to be inhibited by glucagon,a hormone inducing gluconeogenesis.However,the role of YAP in gluconeogenesis control is still unclear.Material and Method:Alb-Cre::rtTAflox/flox::TetO-YAP and YAPflox/flox mouse models were used in this study.In vitro,primary hepatocytes were isolated from both mouse models using a two-step collagenase perfusion method.Constitutively active YAP(YAP S127A overexpression)models were achieved by treating hepatocytes from Alb-Cre::rtTAflox/flox::TetO-YAP mice with doxycycline or infecting hepatocytes from wild type mice with Ad-YAP S127A,whereas Yap deletion models were obtained by incubating hepatocytes from YAPflox/flox mice with Ad-Cre.After treating these primary hepatocytes with glucagon or dexamethasone,real-time PCR was performed to examine whether overexpression of YAP S127A or deletion of Yap could affect the gluconeogenic gene expression.We also infected those primary hepatocytes with Ad-PGC1? and Ad-shPGC1? to investigate whether PGC1? was required for the effects of YAP.Furthermore,chromatin immunoprecipitation(ChIP)assays with antibodies against PGC1? were performed to determine the effects of YAP on the ability of PGC1? to activate transcription.In vivo,we got liver specific Yap knock out by injecting AAV8-TBG Cre(or AAV-Cre)to YAPflox/flox mice.To obtain constitutively active YAP,we administered water with doxycycline to Alb-Cre::rtTAflox/flox::TetO-YAP mice.We tested the body weight,liver weight,blood glucose,IGTT as well as gluconeogenic gene levels for both mouse models.Finally,the relationship between YAP activity and gluconeogenic gene expression were examined using liver microarray data.Results:Knockdown of Yap was confirmed by reduced YAP protein and mRNA levels,and constitutively active YAP was confirmed by increased YAP protein and YAP target mRNA levels.In vitro,Yap deletion increased gluconeogenic gene(G6pc and Pckl)expression approximately two-fold in response to glucagon and dexamethasone.Conversely,constitutively active YAP suppresses the gluconeogenic gene response more than 99%.Glucagon increased PGC1? mRNA levels ten-to twenty-fold,but this effect was blunted by YAP S127A and potentiated by YAP deletion.The ability of PGC1? to activate expression of the gluconeogenic genes was blunted by YAP S127A.Conversely,the deletion of YAP preferentially potentiated PGC1? activation of its gluconeogenic targets.The association of PGC1? with the G6pc promoter,as well as the Pckl promoter was reduced 75%by YAP S127A.Conversely,similar experiments performed in Yap deficient hepatocytes showed that the association of PGC1? with the G6pc and Pckl promoters was enhanced by knockdown of Yap.In the presence of PGC1?,YAP S127A suppressed both G6pc and Pckl 80 to 90%.However,the effects of YAP S127A on G6pc and Pckl were diminished in the absence of PGCla.Therefore,the effects of YAP are mediated by the transcriptional coactivator PGC1?.In vivo,constitutively active YAP lowers plasma glucose levels in response to a glucose challenge,as it increases liver size,whereas Yap deletion had no significant effect on liver size,gluconeogenic gene expression,glucose levels or glucose tolerance.By using microarray studies analysis,we found a strong inverse correlation between YAP activation and gluconeogenic gene expression(r =-0.57,p = 2.3 × 10-6).Conclusion:In summary,our data identify PGC1? and the gluconeogenic genes as important targets of YAP in the liver.The effects of YAP on the gluconeogenic genes would be expected to divert substrates away from the energy consuming process of gluconeogenesis,thereby favoring the anabolic processes necessary for growth.