Fasting-induced KLF9Regulates Hepatic Gluconeogenesis Through PGC-1α

Chronic glucocorticoid therapy has serious side effects, including diabetes, hypertention and fatty liver. However, the molecular mechanisms responsible for steroid-induced diabetes are not well understood. KLF9is highly expressed in the liver; however, the physiological function of hepatic KLF9remains unexplored. Peroxisome proliferators-activated receptor y coactivator-1α (PGC-1α) is capable of coactivating a number of nuclear receptors and other transcription factors involved in the regulation of multiple metabolic processes. In the present study, to exploring the role of KLF9in hepatic gluconeogenesis, we mainly use western blot, real time PCR, dual-luciferase reporter assay, chromatin immunoprecipitation assay, glucose output assay with mouse primary hepatocytes and diabetic mouse models through overexression and knockdown KLF9respectively. We also examined the blood glucose levels and serum insulin levels in dexamethasone treated mice, ob/ob, db/db and high fat diet-induced obese mice through overexpression and knockdown KLF9in livers.Here, we show that hepatic KLF9gene expression is induced by dexamethasone and fasting. Dexamethasone induces the expression of KLF9through activating glucocorticoid receptor (GR) in mouse primary hepatocytes. Treatment with the GR antagonist RU486almost completely abolishes the DEX-mediated induction of KLF9and its target genes expression in primary hepatocytes. The transfection of the GR expression plasmid into HepG2cells causes a significant activation of the KLF9promoter-reporter gene in dual luciferase reporter assay, and chromatin immunoprecipitation (ChIP) assays indicate that endogenous GR proteins were recruited to the KLF9gene promoter region containing GR response element, suggesting that KLF9is a direct target gene of GR in vivo. KLF9induced hepatic gluconeogenesis through PGC-1α. The modest induction of KLF9expression in primary hepatocytes strongly stimulates PGC-1α gene expression. KLF9activates the promoter of PGC-1α gene, and chromatin immunoprecipitation assay indicates GR can directly bind to PGC-la gene promoter, thereby activating the gluconeogenic program and stimulating hepatocytes glucose output. Knockdown KLF9in primary hepatocytes leads to the decrease of glucose output and the expression of PEPCK and G6Pase, whereas PGC-1α rescue experiment can restore the effects caused by deficience in KLF9. We found that the knockdown of PGC-1α significantly reduced KLF9activity on the expression of the PEPCK and G6Pase genes and cellular glucose production. The adenovirus-mediated overexpression of KLF9in mouse livers markedly increases the blood glucose level, the serum insulin level and impairs glucose tolerance and insulin sensitivity. Conversely, KLF9-deficient C57BL/6J mice display lowered blood glucose levels and serum insulin levels. Overexpression of PGC-1α in KLF9-deficient mice also rescues the blood glucose levels. All the results demonstrate that KLF9is a significant regulator in hepatic glucose metabolism. Moreover, the knockdown of KLF9in the livers of different diabetic mouse models, including dexamethasone-treated, ob/ob, db/db and diet-induced obese mice, significantly alleviates insulin resistance and hyperglycemia. These results suggest a critical role for KLF9, as a central target of glucocorticoids, in the regulation of hepatic glucose metabolism. This study provides a novel target for the treatment of type2diabetes, specifically glucocorticoid therapy-induced diabetes.