Hepatic p38α MAPK controls gluconeogenesis via FOXO1 phosphorylation at S273 during glucagon signalling in mice

Aims/hypothesis Hyperglucagonaemia-stimulated hepatic glucose production (HGP) contributes to hyperglycaemia during type 2 diabetes. A better understanding of glucagon action is important to enable efficient therapies to be developed for the treatment of diabetes. Here, we aimed to investigate the role of p38 MAPK family members in glucagon-induced HGP and determine the underlying mechanisms by which p38 MAPK regulates glucagon action. Methods p38 alpha, ss,gamma and delta MAPK siRNAs were transfected into primary hepatocytes, followed by measurement of glucagoninduced HGP. Adeno-associated virus serotype 8 carrying p38a MAPK short hairpin RNA (shRNA) was injected into liverspecific Foxo1 knockout, liver-specific Irs1/Irs2 double knockout and Foxo1(S273D) knockin mice. Foxo1(S273A) knockin mice were fed a high-fat diet for 10 weeks. Pyruvate tolerance tests, glucose tolerance tests, glucagon tolerance tests and insulin tolerance tests were carried out in mice, liver gene expression profiles were analysed and serum triglyceride, insulin and cholesterol levels were measured. Phosphorylation of forkhead box protein O1 (FOXO1) by p38 alpha MAPK in vitro was analysed by LC-MS. Results We found that p38 alpha MAPK, but not the other p38 isoforms, stimulates FOXO1-S273 phosphorylation and increases FOXO1 protein stability, promoting HGP in response to glucagon stimulation. In hepatocytes and mouse models, inhibition of p38 alpha MAPK blocked FOXO1-S273 phosphorylation, decreased FOXO1 levels and significantly impaired glucagon- and fasting-induced HGP. However, the effect of p38a MAPK inhibition on HGP was abolished by FOXO1 deficiency or a Foxo1 point mutation at position 273 from serine to aspartic acid (Foxo1(S273D)) in both hepatocytes and mice. Moreover, an alanine mutation at position 273 (Foxo1(S273A)) decreased glucose production, improved glucose tolerance and increased insulin sensitivity in diet- induced obese mice. Finally, we found that glucagon activates p38 alpha through exchange protein activated by cAMP 2 (EPAC2) signalling in hepatocytes. Conclusions/interpretation This study found that p38 alpha MAPK stimulates FOXO1-S273 phosphorylation to mediate the action of glucagon on glucose homeostasis in both health and disease. The glucagon-induced EPAC2-p38 alpha MAPK-pFOXO1S273 signalling pathway is a potential therapeutic target for the treatment of type 2 diabetes.

Automated summary

This study found that p38 alpha MAPK stimulates FOXO1-S273 phosphorylation to mediate the action of glucagon on glucose homeostasis. The glucagon-induced EPAC2-p38 alpha MAPK-pFOXO1S273 signaling pathway is a potential therapeutic target for the treatment of type 2 diabetes.