A fasting inducible switch modulates gluconeogenesis via activator/coactivator exchange

Abstract

During early fasting, increases in skeletal muscle proteolysis liberate free amino acids for hepatic gluconeogenesis in response to pancreatic glucagon. Hepatic glucose output diminishes during the late protein-sparing phase of fasting, when ketone body production by the liver supplies compensatory fuel for glucose-dependent tissues. Glucagon stimulates the gluconeogenic program by triggering the dephosphorylation and nuclear translocation of the CREB regulated transcription coactivator 2 (CRTC2; also known as TORC2), while parallel decreases in insulin signalling augment gluconeogenic gene expression through the dephosphorylation and nuclear shuttling of forkhead box O1 (FOXO1). Here we show that a fasting-inducible switch, consisting of the histone acetyltransferase p300 and the nutrient-sensing deacetylase sirtuin 1 (SIRT1), maintains energy balance in mice through the sequential induction of CRTC2 and FOXO1. After glucagon induction, CRTC2 stimulated gluconeogenic gene expression by an association with p300, which we show here is also activated by dephosphorylation at Ser 89 during fasting. In turn, p300 increased hepatic CRTC2 activity by acetylating it at Lys 628, a site that also targets CRTC2 for degradation after its ubiquitination by the E3 ligase constitutive photomorphogenic protein (COP1). Glucagon effects were attenuated during late fasting, when CRTC2 was downregulated owing to SIRT1-mediated deacetylation and when FOXO1 supported expression of the gluconeogenic program. Disrupting SIRT1 activity, by liver-specific knockout of the Sirt1 gene or by administration of a SIRT1 antagonist, increased CRTC2 activity and glucose output, whereas exposure to SIRT1 agonists reduced them. In view of the reciprocal activation of FOXO1 and its coactivator peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha, encoded by Ppargc1a) by SIRT1 activators, our results illustrate how the exchange of two gluconeogenic regulators during fasting maintains energy balance.

Figure 1. Sequential activation of CRTC2 and FOXO1 during fasting

a, Ad-CRE-luc activity (top) and CRTC2 protein amounts (bottom) in mice fasted for 6 or 24 hours. Intra-peritoneal glucagon injection indicated. b, and c, Effect of 6 or 18 hour fasting on Ad-G6Pase-luc activity (b), G6Pase mRNA amounts (c), and blood glucose concentrations (c) in mice infected with Ad-CRTC2i, Ad-FOXO1i, or (USi) control virus (n=4, (*) P< .05; (**) P< .02; (***) P< .01). d, Top, activities of wild-type or mutant Ad-G6Pase-luc reporters defective in CREB (CREmut) or FOXO1 (IREmut) binding. Mice were fasted for 6 or 18 hours as indicated (n=4, *;P<.05). Bottom, chromatin immunoprecipitation (ChIP) assay showing binding of myc-tagged FOXO1 or Flag-epitope tagged CRTC2 to the G6Pase promoter in HepG2 hepatocytes exposed to FSK for 6 or 18 hours. For panels b, c, d, data are means ± s.e.m.

Figure 2. CBP/P300 promote CRTC2 acetylation during fasting

a, Top, immunoblot of acetylated or ubiquitinated hepatic CRTC2 proteins in 8 and 24 hour fasted mice. Recovery of CRTC2 from P300 immunoprecipitates shown. Bottom, immunoblot of acetylated CRTC2 in HEK293T cells expressing wild-type or Lys628Arg mutant CRTC2. Exposure to FSK indicated. b, Hepatic Ad-CRE-luc activity (top) and circulating glucose levels (bottom) in 8 hr. and 24 hr. fasted mice expressing wild-type or Lys628Arg mutant CRTC2. For blood glucose, (n=3, P <.05). c, Top, immunoblot of phospho-Ser89 P300 protein amounts in primary hepatocytes exposed to glucagon (2 hrs.) followed by insulin (1hr). Effect of Ad-SIK2 RNAi relative to control (USi) shown. Bottom, Ad-CRE-luc reporter activity (left) and G6Pase mRNA amounts (right) in primary hepatocytes expressing wild-type or Ser89Ala mutant P300. Exposure to FSK or glucagon (6hrs.) indicated (n=3; P < .001). d, Top, effect of Ad-P300 RNAi on amounts of acetylated CRTC2 (top) in hepatocytes exposed to glucagon for 1 hour. Bottom, effect of Ad-P300 RNAi on Ad-CRE-luc reporter activity (bottom) in hepatocytes exposed to FSK for 6 hrs (n=3, P < .001). For panels b, c, d, data are means ± s.e.m.

Figure 3. P300/CBP modulate hepatic CRTC2 activity

a, Top, Ad-CRE-luc activity in 6 hour fasted mice expressing Ad-CBP RNAi, Ad-P300 RNAi, or Ad-USi. Bottom, G6Pase mRNA amounts (left) and blood glucose levels (right) in mice expressing Ad-P300 RNAi relative to control. (n=3; * P <.001; ** P <.01). b, Effect of P300/CBP HAT inhibitor Lys-CoA-TAT or control TAT peptide on CRTC2 acetylation (top) and Ad-CRE-luc activity (bottom) in primary hepatocytes exposed to glucagon. (n=3; P <.001). c, Effect of Lys-CoA-TAT on CRTC2 protein amounts (top) and on glucose output (bottom) from primary hepatocytes expressing wild-type or Lys628Arg mutant CRTC2 (n=3; P < .001). d, Top, Ad-CRE-luc activity in 6 hour fasted mice imaged prior to (Pre) or following (Post) IP injection of Lys-CoA-TAT or TAT peptide. Bottom, blood glucose concentrations in 8 hr. fasted mice injected with Lys-coA-TAT or TAT control. For blood glucose levels (n=6; P < .001). For panels a, b, c, d, data are means ± s.e.m.

Figure 4. SIRT1 attenuates CRTC2 activity during fasting

a, Top left, Immunoblot of hepatic CRTC2 protein recovered from SIRT1 IPs from ad libitum fed and 6 hour or 18 hour fasted mice. Right, effect of Ad-SIRT1 expression on Ad-CRE-luc activity in 8 hr. fasted mice (top) and on CRTC2 acetylation in hepatocytes exposed to glucagon (bottom). (n=3; P <.001). b, Top, Ad-CRE-luc activity in primary hepatocytes exposed to SIRT1 activators resveratrol or SRT1720 (n=3; P <.001). Bottom, immunoblot showing amounts of acetylated CRTC2 in livers of Zucker fa/fa obese rats maintained on chow supplemented with SRT1720 (n=4; P <.05). c. Top, immunoblot of acetylated and total CRTC2 protein in primary hepatocytes exposed to Sirtinol and/or glucagon for 4 hours. Bottom, Ad-CRE-luc activity in 6 or 24 hour fasted mice following sirtinol administration. d, Total and acetylated CRTC2 protein amounts (top) and Ad-CRE-luc activity (bottom) in primary hepatocytes from control or liver-specific Sirt1^−/− mice. Exposure to glucagon (6 hours) indicated (n=4; P <.05). For panels a, b, d, data are means ± s.e.m.).