279-OR: Hydroxylation Regulation of Hepatic Gluconeogenesis via Prolyl-Hydroxylase 3 in Mice

Hepatic gluconeogenesis is critical for metabolic adaption in response to fasting, and its abnormal elevation contributes to hyperglycemia and type 2 diabetes. However, whether and how hydroxylation regulates hepatic gluconeogenesis remains largely unknown. Here we identified prolyl hydroxylase 3 (PHD3) of the hydroxylase family as a key regulator for hepatic gluconeogenesis. Liver-specific knockout of PHD3 (PHD3 LKO) mice show attenuated fasting gluconeogenic genes, glycaemia and hepatic capacity to produce glucose as evidenced by a pyruvate tolerance test. The efficacies of glucagon or adenylyl cyclase activator forskolin on stimulating the expression of gluconeogenic enzymes, such as PEPCK and G6Pase, and on glucose output are abolished in PHD3-/- mouse primary hepatocytes. To determine whether the prolyl hydroxylase activity is required for PHD3’s effects on promoting gluconeogenesis, liver-specific prolyl hydroxylase-deficient knockin mice with alanine mutation in PHD3 at Pro196 (PHD3 KI) were generated. Similar to PHD3 LKO mice, PHD3 KI mice exhibit attenuated fasting gluconeogenic genes, glycaemia and hepatic capacity to produce glucose, suggesting that PHD3 regulates hepatic gluconeogenesis in a proline hydroxylase-dependent manner. Moreover, several cellular proteins that associate with PHD3 were identified by comprehensive protein analysis using LC-MS/MS followed by bioinformatic analysis including GSK3β, IRS2 and OGT, which may act as PHD3 substrates and play roles in mediating PHD3’s effects on regulating glucose metabolism. Intriguingly, expression levels of PHD3 are increased in the liver diet-induced insulin resistant mice. Together, these results demonstrate that hydroxylation plays a critical role in the regulation of hepatic gluconeogenesis under physiological and insulin resistant conditions. Hyperactivation of PHD3 may underscore the potential role of PHD3 in the development of sustained gluconeogenesis, hyperglycemia and type 2 diabetes.