Inositol-1,4,5-trisphosphate receptor regulates hepatic gluconeogenesis in fasting and diabetes

Abstract

In the fasted state, increases in circulating glucagon promote hepatic glucose production through induction of the gluconeogenic program. Triggering of the cyclic AMP pathway increases gluconeogenic gene expression via the de-phosphorylation of the CREB co-activator CRTC2 (ref. 1). Glucagon promotes CRTC2 dephosphorylation in part through the protein kinase A (PKA)-mediated inhibition of the CRTC2 kinase SIK2. A number of Ser/Thr phosphatases seem to be capable of dephosphorylating CRTC2 (refs 2, 3), but the mechanisms by which hormonal cues regulate these enzymes remain unclear. Here we show in mice that glucagon stimulates CRTC2 dephosphorylation in hepatocytes by mobilizing intracellular calcium stores and activating the calcium/calmodulin-dependent Ser/Thr-phosphatase calcineurin (also known as PP3CA). Glucagon increased cytosolic calcium concentration through the PKA-mediated phosphorylation of inositol-1,4,5-trisphosphate receptors (InsP(3)Rs), which associate with CRTC2. After their activation, InsP(3)Rs enhanced gluconeogenic gene expression by promoting the calcineurin-mediated dephosphorylation of CRTC2. During feeding, increases in insulin signalling reduced CRTC2 activity via the AKT-mediated inactivation of InsP(3)Rs. InsP(3)R activity was increased in diabetes, leading to upregulation of the gluconeogenic program. As hepatic downregulation of InsP(3)Rs and calcineurin improved circulating glucose levels in insulin resistance, these results demonstrate how interactions between cAMP and calcium pathways at the level of the InsP(3)R modulate hepatic glucose production under fasting conditions and in diabetes.

Figure 1

Calcineurin promotes CRTC2 activation during fasting. A. Effect of Ser/Thr phosphatase inhibitors (okadaic acid (OA), cyclosporin A (CsA) on CRTC2 dephosphorylation and CRE-luciferase (luc) reporter activation (*P < 0.001; n=3). B. Effect of glucagon (Gcg) on dephosphorylation (left) and activity (right) of wild-type (WT) and calcineurin-defective (ΔCalna) CRTC2 in hepatocytes (*P < 0.001; n=3). C. Effect of calcineurin A over-expression (left) or knockdown (right) on CRTC2 dephosphorylation (top), CRE-luc reporter activity (middle, *P < 0.001; n=3), and glucose output (bottom, *P < 0.001; n=3) from hepatocytes. D. Effect of hepatic calcineurin over-expression (left) or knockdown (right) on CRE-luc activity, gluconeogenic gene (Pck1, G6pc) expression, and blood glucose concentrations in 6–8 hour fasted mice (*P < 0.01; n=5). For this and other figures, data are shown as mean ± s.e.m.

Figure 2

Glucagon stimulates CRTC2 dephosphorylation via activation of InsP3 receptors. A. Effect of glucagon (Gcg) on calcium mobilization in hepatocytes by fluorescence imaging. Calcium mobilization and calcineurin activation following knockdown of all three InsP3R family members shown (*P < 0.001; n=3). B. Effect of calcium chelator (BAPTA) on CRTC2 dephosphorylation and CRE-luc activation (*P < 0.001; n=3). C. Effect of InsP3R depletion on CRTC2 dephosphorylation, CRE-luc activity, and glucose output from hepatocytes (*P < 0.001; n=3). D. Effect of hepatic InsP3R knockdown on CRE-luc activity, blood glucose, and gluconeogenic gene expression (*P < 0.01; n=5).

Figure 3

Glucagon stimulates CRTC2 activity via PKA-dependent phosphorylation of InsP3Rs. A. and B. Immunoblots of InsP3R1 immunoprecipitates using phospho-PKA substrate antiserum to show effect of H89 (A) and Ala mutations (B) at one or both (DM) PKA consensus sites (Ser1589, Ser1756) on InsP3R1 phosphorylation in hepatocytes exposed to glucagon (Gcg). Effect of wild-type and PKA-mutant InsP3R1 on calcium mobilization in response to Gcg (B) shown (*P < 0.001; n=3). C. and D. Effect of wild-type or PKA-defective InsP3R1 (DM) on calcineurin (Calna) activation (C) and CRTC2 dephosphorylation (C), as well as CRE-luc activation (D) and glucose output (D) from hepatocytes (*P < 0.001; n=3). E. Effect of wild-type and PKA-defective InsP3R1 on hepatic CRE-luc activity, fasting blood glucose, and gluconeogenic gene expression (G6pc, Pck1) (*P < 0.01 versus wild-type; n=5). F. Co-immunoprecipitation of CRTC2 with InsP3R1 in primary hepatocytes. Exposure to glucagon (100 nM; 15 minutes) indicated. Input levels of CRTC2 and InsP3R1 in nuclear (Nu) and post-nuclear (p/Nu) supernatant fractions shown.

Figure 4

InsP3R activity is upregulated in diabetes. A. Hepatic CRE-luc and calcineurin activity in lean and db/db mice (*P < 0.001; n=5). B. Immunoblots showing relative amounts and phosphorylation of InsP3R family members in livers of ad libitum fed lean, db/db, or ob/ob mice. InsP3R phosphorylation at PKA or AKT sites indicated. C. Effect of RNAi-mediated depletion of InsP3Rs or calcineurin A on CRE-luc activity, gluconeogenic gene expression, and hepatic glucose production in db/db mice, determined by pyruvate tolerance testing (*P < 0.01; n=5).