Effect Of PGC-1? On Porcine Hepatocytes Urea Cycle Through SIRT3 And SIRT5

Under stress or starvation status,the flow of carbon from hepatic amino acid(AA)into central metabolism is promoted by hepatic gluconeogenesis.Under these conditions there is excess ammonia production,which is converted to urea for ammonia detoxification.The aim of the present study was to investigate the effect of peroxisome proliferator-activated receptor ? coactivator-1?(PGC-1?)on procine hepatocytes urea cycle through SIRT3 and SIRT5.The primary hepatocytes were isolated to explore the effect of glucagon(Glu)on PGC-1? and urea cycle.Secondly,PGC-1? overexpressing cell model was established to explore the effect of PGC-1?on SIRT3,SIRT5 expression and ornithine carbamoyltransferase(OTC),carbamoyl-phosphate synthase I(CPS1)activity.The main findings are as follows:(1)The effect of glucagon on PGC-1? and urea cycle in porcine hepatocytes.The primary hepatocytes of porcine were isolated by two-step collagenase perfusion.Compared with non-glucagon group,the mRNA and protein levels of urea cycle enzyme ASS,ASL and ARG in the glucagon group increased(P<0.01).Likewise,the mitochondrial CPS1 and OTC mRNA expression in glucagon group were significantly higher than those in non-glucagon group(P<0.05).But,the protein expression of CPS1 and OTC did not differ(P>0.05)between the two groups.In addition,it was induced a markedly higher expression(P<0.01)of hepatic phosphoenolpyruvate carboxykinase(PEPCK)and glucose-6-phosphatase(G6Pase)both in mRNA and protein levels in the glucagon group.Moreover,glucagon strongly stimulated(P<0.01)the expression of PGC-1?,SIRT3 and SIRT5 in porcine hepatocytes(P<0.01).Compared with Ala group,the concentration of glucose and urea was significantly increased in Glu+Ala group(P<0.05).Compared with NH4 Cl group,the levels of urea in Glu+NH4Cl group,was significantly increased(P<0.01),however,the levels of glucose did not differ(P>0.05).These results indicated that glucagon not only promoted the alanine carbon frame to gluconeogenesis,but also stimulated the key gene expression of the urea production pathway,and promoted the formation of urea by the alanine nitrogen source into the urea cycle.(2)The effect of PGC-1? on hepatocytes SIRT3?SIRT5 expression and OTC?CPS1 activity.Compared with control group,the SIRT3 and SIRT5 mRNA and protein levels were increased(P<0.01)by PGC-1? overexpression.However,the mRNA and protein expression of CPS1 and OTC was not affected in PGC-1?overexpression hepatocytes(P>0.05).The acetylated CPS1 and OTC levels were significantly lower while their activities were significantly higher with PGC-1?overexpression(P<0.01).Compared with Ala group,the Ala+PGC-1? overexpression group significantly increased(P<0.05)concentrations of glucose and urea.Compared with NH4 Cl group,the NH4Cl+PGC-1? overexpression group did not affect glucose concentration,but significantly increased the urea concentration(P<0.05).The results showed that PGC-1? regulated the flow of carbon from AA into central metabolism was promoted by hepatic gluconeogenesis.Meanwhile,it has been regulated the nitrogen source from amino acid into urea cycle through deacetylating CPS1 and OTC by modulation of mitochondrial deacetylases SIRT3 and SIRT5 in hepatocytes to promote ureagenesis.