The Role Of Protein Phosphatase4in IL-6-induced Hepatic Insulin Resistance

Objectives:Diabetes mellitus is caused by absolute or relative insufficient insulin and inflammation is one of the main characteristics of diabetes mellitus. Type2diabetes may account for more than90%of all diagnosed cases of diabetes. Low-grade chronic inflammation is a feature of type2diabetes and appears to play a pathogenetic role in insulin resistance. And the proinflammatory cytokines have also been shown to be elevated in type2diabetes. Of these proinflammatory cytokines, IL-6has been shown to have the strongest correlation with insulin resistance and type2diabetes. The elevation of circulating IL-6is highly related to increased blood glucose, decreased glucose tolerance, and decreased insulin sensitivity. Protein phosphatase4(PP4) is a member of PP2A-family and plays important role in many cellular processes. It has been reported that PP4is involved in the down-regulation of IRS4and hepatic glucose metabolism. In the present study, we established the IL-6-induced insulin resistance models in vivo and in vitro and analyzed the expression and phosphatase activity of PP4in hepatocytes and liver. Moreover, we explored the role of PP4in IL-6-induced hepatic insulin resistance.Methods:We established cellular insulin resistance model by treatment of human HepG2cells and C57BL/6mouse primary hepatocytes with10ng/ml IL-6for18h or15h. Implanting the IL-6osmotic pumps to the back subcuticle of mice was used to establish IL-6-induced insulin resistance animal model. The HepG2cell clones expressing HA-PP4and FLAG-PP4RL were obtained by G418selection. The expression of PP4in HepG2cells and primary hepatocytes was knock-downed by transfection of PP4-siRNA and PP4-shRNA. Moreover, Ad-PP4-shRNA was delivered to the livers in C57BL/6mice via tail veil injection before implanting the IL-6osmotic pumps to the back subcuticle of mice. Similarly, Ad-PP4-shRNA was also delivered to the livers in db/db mice via tail veil injection. Serum insulin and IL-6levels were detected by ELISA, and glycogen content was measured by Anthrone method. The expressions of the G6Pase, PEPCK and PGC1-α were analyzed by Real-time PCR, and the levels of JNK, P-JNK, IRS1, P-IRS1, AKT, P-AKT, GSK, P-GSKand PP4were detected by Western blot. Phosphatase activity of PP4was also analyzed with immunoprecipitation and phosphatase activity assay. In addition, the interaction between PP4and IRS1was measured by co-immunoprecipitation and Western blot.Results:The db/db mice displayed increased levels of serum GLU, insulin and IL-6, and decreased ISI. We also found decreased glycogen levels in the liver of db/db mice, suggesting a state of insulin resistance. Importantly, levels of phosphorylated JNK and IRS1, and PEPCK expression were elevated in liver of db/db mice, accompanied by decreased IRS1expression and phosphorylated levels of GSK and AKT. It is noteworthy that elevated PP4activity and expression were detected in liver of db/db mice, suggesting that PP4may be involved in IL-6-induced insulin resistance. To extend these observations from db/db mice to the cellular models of insulin resistance, human HepG2cells and C57BL/6mouse primary hepatocytes were treated with IL-6to induce insulin resistance. These two cellular models displayed a state of insulin resistance, as assessed by declined glycogen content, elevated expression of gluconeogenesis-related genes and impaired insulin signaling. The expression and activity of PP4were also increased. Moreover, C57BL/6mice implanted the IL-6osmotic pumps to the back subcuticle displayed declined glycogen content, elevated expression of gluconeogenesis-related genes and impaired insulin signaling, accompanied by reduction of PP4expression and activity. Taken together, these data suggest that PP4may play a key role in IL-6-induced insulin resistance.In order to further assess whether PP4is involved in IL-6-induced insulin resistance and explore the possible mechanism, HA-PP4, PP4-siRNA, PP4-shRNA and PP4RL were used to investigate the effect of PP4expression and activity on IL-6-induced hepatic insulin resistance. The results suggested that up-regulation of PP4expression resulted in decreased glycogen content in HepG2cells and impaired glycogen synthesis and gluconeogenesis signaling. In contrast, down-regulation of PP4expression led to increase of glycogen content in HepG2cells and C57BL/6mouse primary hepatocytes and liver of C57BL/6mice, accompanied by restoration of insulin signaling. Similarly, inhibition of PP4activity in HepG2cells also led to elevated glycogen content. Moreover, the interaction between PP4and IRS1was assessed by co-immunoprecipitation and Western blot.In order to further clarify the role of PP4in db/db mice, we suppressed the expression of PP4in the liver of db/db mice. Ad-PP4-shRNA was delivered to the liver of db/db mice by tail vein injection. The knockdown of PP4in mouse liver significantly reduces blood glucose levels in db/db mice. Moreover, PP4knockdown greatly improves glucose tolerance and pyruvate intolerance.Conclusions:1. In cellular or animal insulin resistance models, the expression and activity of PP4were increased in hepatocytes and livers.2. PP4functions as a positive regulator of IL-6-signaling and a linker between inflammation and hepatic insulin resistance.3. PP4plays a key role in IL-6-induced hepatic insulin resistance via regulating IRS1by activation of JNK or interaction with IRS1directly.4. Reduced expression of PP4in the livers of db/db mice could effectively reverse insulin resistance.