Targeted disruption of the CREB coactivator Crtc2 increases insulin sensitivity

Abstract

Under fasting conditions, increases in circulating concentrations of pancreatic glucagon maintain glucose homeostasis through induction of gluconeogenic genes by the CREB coactivator CRTC2. Hepatic CRTC2 activity is elevated in obesity, although the extent to which this cofactor contributes to attendant increases in insulin resistance is unclear. Here we show that mice with a knockout of the CRTC2 gene have decreased circulating glucose concentrations during fasting, due to attenuation of the gluconeogenic program. CRTC2 was found to stimulate hepatic gene expression in part through an N-terminal CREB binding domain that enhanced CREB occupancy over relevant promoters in response to glucagon. Deletion of sequences encoding the CREB binding domain in CRTC2 −/− mice lowered circulating blood glucose concentrations and improved insulin sensitivity in the context of diet-induced obesity. Our results suggest that small molecules that attenuate the CREB-CRTC2 pathway may provide therapeutic benefit to individuals with type 2 diabetes.D uring fasting, increases in hepatic gluconeogenesis ensure energy balance for glucose-dependent tissues such as brain and the red blood cell compartment. Hepatic glucose production is elevated in type 2 diabetes, reflecting decreases in insulin signaling that otherwise inhibit the gluconeogenic program (1-3).Increases in circulating glucagon are thought to trigger gluconeogenic gene expression in part through the cAMP-dependent phosphorylation of the transcription factor CREB and through the dephosphorylation of its cognate coactivator CRTC2 (4-6). In parallel, decreases in circulating insulin concentrations during fasting also stimulate gluconeogenic genes by the dephosphorylation of the forkhead activator FOXO1 (7).Localized in the cytoplasm under basal conditions through a phosphorylation-dependent association with 14-3-3 proteins, CRTC2 shuttles to the nucleus following its dephosphorylation at Ser171, where it mediates induction of cellular genes by binding to the bZIP domain of CREB over relevant promoters (8, 9). The importance of CRTC2 for gluconeogenic gene expression is supported by RNAi-mediated knockdown studies, where acute depletion of CRTC2 lowered hepatic glucose production in fasted mice and by overexpression studies in which phosphorylation-defective, active CRTC2 increases circulating glucose levels under both fasting and feeding conditions (4, 6, 10).To determine the importance of the CRTC2-CREB association for induction of the gluconeogenic program, we characterized mice lacking the conserved N-terminal CREB binding domain of CRTC2. We found that gluconeogenic gene expression and hepatic glucose production were reduced in CRTC2-deficient mice during fasting and in the setting of insulin resistance. Our results support an important role for CRTC2 in mediating effects of fasting signals on hepatic gluconeogenesis.

cAMP-responsive Element-binding Protein (CREB)-regulated Transcription Coactivator 2 (CRTC2) Promotes Glucagon Clearance and Hepatic Amino Acid Catabolism to Regulate Glucose Homeostasis

“…Thus, the degree of hyperglycemia and hormonal milieu are critical to the physiologic outcome of CRTC2 knockdown. This may explain why other models of CRTC2 knockdown have shown more impressive reductions in plasma glucose and glucose production (9,10). Alternatively, such differences may be due to the degree to which CRTC2 expression was reduced or the species used.…”

PRMT5 modulates the metabolic response to fasting signals

“…Cultured mouse primary hepatocytes were plated in 150-mm plates and exposed to glucagon for 1 h. Adenovirally encoded shRNAs were added to cells for 60 h. ChIP assays were performed as described, with minimal modification (20). Briefly, the treated hepatocytes were cross-linked with 1% formaldehyde for 15 min.…”

“…Cellular RNA was isolated by RNeasy kit (Qiagen) from mouse liver tissue or from mouse primary hepatocytes. Mouse primary hepatocytes were plated in six-well plates and infected with adenovirus encoded with indicated shRNAs for 60 h and exposed to glucagon for 1 and 2 h. mRNA levels were measured as described (20).…”

Mechanism of CREB recognition and coactivation by the CREB-regulated transcriptional coactivator CRTC2

“…Gel mobility shift assays were conducted as previously described using a 32 P-labeled oligonucleotide probe containing a somatostatin CRE (35), except purified phosphorylated CREM-α (a kind gift from Young-In Chi, University of Minnesota, Austin, MN), GST-CREB bZip, and wild-type/mutant GST-CRTC2 (1-65) peptides were used for these studies. Tissue Culture and Luciferase Assays.…”