AMPK-dependent Repression of Hepatic Gluconeogenesis via Disruption of CREB·CRTC2 Complex by Orphan Nuclear Receptor Small Heterodimer Partner

Abstract

Orphan nuclear receptor small heterodimer partner (SHP) plays a key role in transcriptional repression of gluconeogenic enzyme gene expression. Here, we show that SHP inhibited protein kinase A-mediated transcriptional activity of cAMP-response element-binding protein (CREB), a major regulator of glucose metabolism, to modulate hepatic gluconeogenic gene expression. Deletion analysis of phosphoenolpyruvate carboxykinase (PEPCK) promoter demonstrated that SHP inhibited forskolin-mediated induction of PEPCK gene transcription via inhibition of CREB transcriptional activity. In vivo imaging demonstrated that SHP inhibited CREB-regulated transcription coactivator 2 (CRTC2)-mediated cAMP-response elementdriven promoter activity. Furthermore, overexpression of SHP using adenovirus SHP decreased CRTC2-dependent elevations in blood glucose levels and PEPCK or glucose-6-phosphatase (G6Pase) expression in mice. SHP and CREB physically interacted and were co-localized in vivo. Importantly, SHP inhibited both wild type CRTC2 and S171A (constitutively active form of CRTC2) coactivator activity and disrupted CRTC2 recruitment on the PEPCK gene promoter. In addition, metformin or overexpression of a constitutively active form of AMPK (Ad-CA-AMPK) inhibited S171A-mediated PEPCK and G6Pase gene expression, and hepatic glucose production and knockdown of SHP partially relieved the metformin-and Ad-CA-AMPK-mediated repression of hepatic gluconeogenic enzyme gene expression in primary rat hepatocytes. In conclusion, our results suggest that a delayed effect of metformin-mediated induction of SHP gene expression inhibits CREB-dependent hepatic gluconeogenesis.Glucose homeostasis is regulated by the opposing actions of insulin and glucagon (1-3), and glucose production in the liver is controlled primarily by gluconeogenesis (4). The regulation of hepatic gluconeogenesis involves the transcriptional regulation of key metabolic enzymes, including PEPCK 6 and G6Pase. The gluconeogenic program is largely regulated at the level of transcription and the process is coordinated by CREB via its direct binding to the cAMP-response element (CRE) site on the promoter of PEPCK, G6Pase, or PGC-1␣ (PPAR␥ coactivator-1␣) (5).Metformin has been shown to activate AMP-activated protein kinase (AMPK) via an LKB1-dependent mechanism (6). AMPK is a serine/threonine kinase that functions as an intracellular energy sensor and has been implicated in the modulation of glucose and fatty acid metabolism (7). AMPK is activated by physiological stimuli, including exercise, muscle contraction, and hormones, such as adiponectin and leptin, as well as by physiological stresses, glucose deprivation, hypoxia, oxidative stress, and osmotic shock conditions (8, 9). In the liver, activation of AMPK suppresses hepatic gluconeogenesis acutely by direct phosphorylation of its substrates, including CREB-binding protein (CBP) (10), CRTC2 (11), and GSK3␤ (glycogen synthase kinase 3␤) (12). Recent studies also suggest that AMPK induces SHP gene expression and inhibits hepatic g...

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