Kim Ravnskjaer

A Fasting Inducible Switch Modulates Gluconeogenesis Via Activator-Coactivator Exchange

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-γ coactivator-1α (PGC-1α, encoded by Ppargc1a) by SIRT1 activators, our results illustrate how the exchange of two gluconeogenic regulators during fasting maintains energy balance.

Cooperative interactions between CBP and TORC2 confer selectivity to CREB target gene expression

A number of hormones and growth factors stimulate gene expression by promoting the phosphorylation of CREB (P‐CREB), thereby enhancing its association with the histone acetylase paralogs p300 and CBP (CBP/p300). Relative to cAMP, stress signals trigger comparable amounts of CREB phosphorylation, but have minimal effects on CRE‐dependent transcription. Here, we show that the latent cytoplasmic coactivator TORC2 mediates target gene activation in response to cAMP signaling by associating with CBP/p300 and increasing its recruitment to a subset of CREB target genes. TORC2 is not activated in response to stress signals, however; and in its absence, P‐CREB is unable to stimulate CRE‐dependent transcription, due to a block in CBP recruitment. The effect of TORC2 on CBP/p300 promoter occupancy appears pivotal because a gain of function mutant CREB polypeptide with increased affinity for CBP restored CRE‐mediated transcription in cells exposed to stress signals. Taken together, these results indicate that TORC2 is one of the long sought after cofactors that mediates the differential effects of cAMP and stress pathways on CREB target gene expression.

Targeted disruption of the CREB coactivator Crtc2 increases insulin sensitivity

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.

PPARdelta is a fatty acid sensor that enhances mitochondrial oxidation in insulin-secreting cells and protects against fatty acid-induced dysfunction.

The peroxisome proliferator-activated receptor δ (PPARδ) is implicated in regulation of mitochondrial processes in a number of tissues, and PPARδ activation is associated with decreased susceptibility to ectopic lipid deposition and metabolic disease. Here, we show that PPARδ is the PPAR subtype expressed at the highest level in insulinoma cells and rat pancreatic islets. Furthermore, PPARδ displays high transcriptional activity and acts in pronounced synergy with retinoid-X-receptor (RXR). Interestingly, unsaturated fatty acids mimic the effects of synthetic PPARδ agonists. Using short hairpin RNA-mediated knockdown, we demonstrate that the ability of unsaturated fatty acids to stimulate fatty acid metabolism is dependent on PPARδ. Activation of PPARδ increases the fatty acid oxidation capacity in INS-1E β-cells, enhances glucose-stimulated insulin secretion (GSIS) from islets, and protects GSIS against adverse effects of prolonged fatty acid exposure. The presented results indicate that the nuclear receptor PPARδ is a fatty acid sensor that adapts β-cell mitochondrial function to long-term changes in unsaturated fatty acid levels. As maintenance of mitochondrial metabolism is essential to preserve β-cell function, these data indicate that dietary or pharmacological activation of PPARδ and RXR may be beneficial in the prevention of β-cell dysfunction.

Glucagon regulates gluconeogenesis through KAT2B- and WDR5-mediated epigenetic effects

Circulating pancreatic glucagon is increased during fasting and maintains glucose balance by stimulating hepatic gluconeogenesis. Glucagon triggering of the cAMP pathway upregulates the gluconeogenic program through the phosphorylation of cAMP response element-binding protein (CREB) and the dephosphorylation of the CREB coactivator CRTC2. Hormonal and nutrient signals are also thought to modulate gluconeogenic gene expression by promoting epigenetic changes that facilitate assembly of the transcriptional machinery. However, the nature of these modifications is unclear. Using mouse models and in vitro assays, we show that histone H3 acetylation at Lys 9 (H3K9Ac) was elevated over gluconeogenic genes and contributed to increased hepatic glucose production during fasting and in diabetes. Dephosphorylation of CRTC2 promoted increased H3K9Ac through recruitment of the lysine acetyltransferase 2B (KAT2B) and WD repeat-containing protein 5 (WDR5), a core subunit of histone methyltransferase (HMT) complexes. KAT2B and WDR5 stimulated the gluconeogenic program through a self-reinforcing cycle, whereby increases in H3K9Ac further potentiated CRTC2 occupancy at CREB binding sites. Depletion of KAT2B or WDR5 decreased gluconeogenic gene expression, consequently breaking the cycle. Administration of a small-molecule KAT2B antagonist lowered circulating blood glucose concentrations in insulin resistance, suggesting that this enzyme may be a useful target for diabetes treatment.

Hepatic Insulin Resistance Following Chronic Activation of the CREB Coactivator CRTC2

Background: Loss of CRTC2 improves insulin sensitivity, but it is unknown if chronic CRTC2 activity causes hepatic insulin resistance. Results: Liver expression of constitutively active CRTC2 increased hepatic glucose production, despite compensatory increases in FOXO1 phosphorylation. Conclusion: Persistent increases in CRTC2 activation promote hepatic insulin resistance. Significance: Disrupting CREB signaling in liver could provide therapeutic benefit against insulin resistance. Under fasting conditions, increases in circulating concentrations of glucagon maintain glucose homeostasis via the induction of hepatic gluconeogenesis. Triggering of the cAMP pathway in hepatocytes stimulates the gluconeogenic program via the PKA-mediated phosphorylation of CREB and dephosphorylation of the cAMP-regulated CREB coactivators CRTC2 and CRTC3. In parallel, decreases in circulating insulin also increase gluconeogenic gene expression via the de-phosphorylation and activation of the forkhead transcription factor FOXO1. Hepatic gluconeogenesis is increased in insulin resistance where it contributes to the attendant hyperglycemia. Whether selective activation of the hepatic CREB/CRTC pathway is sufficient to trigger metabolic changes in other tissues is unclear, however. Modest hepatic expression of a phosphorylation-defective and therefore constitutively active CRTC2S171,275A protein increased gluconeogenic gene expression under fasting as well as feeding conditions. Circulating glucose concentrations were constitutively elevated in CRTC2S171,275A-expressing mice, leading to compensatory increases in circulating insulin concentrations that enhance FOXO1 phosphorylation. Despite accompanying decreases in FOXO1 activity, hepatic gluconeogenic gene expression remained elevated in CRTC2S171,275A mice, demonstrating that chronic increases in CRTC2 activity in the liver are indeed sufficient to promote hepatic insulin resistance and to disrupt glucose homeostasis.

Keystone Symposia on Epigenomics and Chromatin Dynamics: Keystone resort, CO, January 17-22, 2012.

Keystone Symposia kicked off the start of 2012 with two joint meetings on Epigenomics and Chromatin Dynamics and a star-studded list of speakers. Held in Keystone, CO, January 17–22, and organized by Steven Jacobsen and Steven Henikoff and by Bradley Cairns and Geneviève Almouzni, respectively, there was plenty happening in these sessions that it did not seem to matter that the ski-slope conditions were not ideal.