When mammals fast, glucose homeostasis is achieved by triggering expression of gluconeogenic genes in response to glucagon and glucocorticoids. The pathways act synergistically to induce gluconeogenesis (glucose synthesis), although the underlying mechanism has not been determined. Here we show that mice carrying a targeted disruption of the cyclic AMP (cAMP) response element binding (CREB) protein gene, or overexpressing a dominant-negative CREB inhibitor, exhibit fasting hypoglycaemia [corrected] and reduced expression of gluconeogenic enzymes. CREB was found to induce expression of the gluconeogenic programme through the nuclear receptor coactivator PGC-1, which is shown here to be a direct target for CREB regulation in vivo. Overexpression of PGC-1 in CREB-deficient mice restored glucose homeostasis and rescued expression of gluconeogenic genes. In transient assays, PGC-1 potentiated glucocorticoid induction of the gene for phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in gluconeogenesis. PGC-1 promotes cooperativity between cyclic AMP and glucocorticoid signalling pathways during hepatic gluconeogenesis. Fasting hyperglycaemia is strongly correlated with type II diabetes, so our results suggest that the activation of PGC-1 by CREB in liver contributes importantly to the pathogenesis of this disease.

Sir2 and insulin/IGF-1 are the major pathways that impinge upon aging in lower organisms. In Caenorhabditis elegans a possible genetic link between Sir2 and the insulin/IGF-1 pathway has been reported. Here we investigate such a link in mammals. We show that Sirt1 positively regulates insulin secretion in pancreatic β cells. Sirt1 represses the uncoupling protein (UCP) gene UCP2 by binding directly to the UCP2 promoter. In β cell lines in which Sirt1 is reduced by SiRNA, UCP2 levels are elevated and insulin secretion is blunted. The up-regulation of UCP2 is associated with a failure of cells to increase ATP levels after glucose stimulation. Knockdown of UCP2 restores the ability to secrete insulin in cells with reduced Sirt1, showing that UCP2 causes the defect in glucose-stimulated insulin secretion. Food deprivation induces UCP2 in mouse pancreas, which may occur via a reduction in NAD (a derivative of niacin) levels in the pancreas and down-regulation of Sirt1. Sirt1 knockout mice display constitutively high UCP2 expression. Our findings show that Sirt1 regulates UCP2 in β cells to affect insulin secretion.

The incretin hormone GLP1 promotes islet-cell survival via the second messenger cAMP. Here we show that mice deficient in the activity of CREB, caused by expression of a dominant-negative A-CREB transgene in pan-creatic-cells, develop diabetes secondary to-cell apoptosis. Remarkably, A-CREB severely disrupted expression of IRS2, an insulin signaling pathway component that is shown here to be a direct target for CREB action in vivo. As induction of IRS2 by cAMP enhanced activation of the survival kinase Akt in response to insulin and IGF-1, our results demonstrate a novel mechanism by which opposing pathways cooperate in promoting cell survival. Supplemental material is available at http://www.genesdev.org.

The helix-loop-helix (HLH) protein NEUROD1 (also known as BETA2) functions as a regulatory switch for endocrine pancreatic development. In mice homozygous for a targeted disruption of Neurod, pancreatic islet morphogenesis is abnormal and overt diabetes develops due in part to inadequate expression of the insulin gene 1 (Ins2). NEUROD1, following its heterodimerization with the ubiquitous HLH protein E47, regulates insulin gene (INS) expression by binding to a critical E-box motif on the INS promoter 2 . Here we describe two mutations in NEUROD1, which are associated with the development of type 2 diabetes in the heterozygous state. The first, a missense mutation at Arg 111 in the DNA-binding domain, abolishes E-box binding activity of NEUROD1. The second mutation gives rise to a truncated polypeptide lacking the carboxy-terminal trans-activation domain, a region that associates with the co-activators CBP and p300 (refs 3,4). The clinical profile of patients with the truncated NEUROD1 polypeptide is more severe than that of patients with the Arg 111 mutation. Our findings suggest that deficient binding of NEUROD1 or binding of a transcriptionally inactive NEUROD1 polypeptide to target promoters in pancreatic islets leads to the development of type 2 diabetes in humans.NEUROD1 contains two exons and has been mapped to chromosome 2q (refs 5,6). We did not examine exon 1 because it is not translated 7 . Exon 2 encodes a protein with several distinct domains (Fig. 1a). We screened exon 2 and the flanking intron sequences for DNA sequence differences by direct sequencing of DNA samples from 94 individuals with type 2 diabetes. Each was the index case through which we ascertained 94 large families for the presence of diabetes segregating as an autosomal dominant disorder 8,9 .We examined exon 2 in all index cases and found four variants of the published sequence. The first was a single base-pair substitution, G→A, in codon 45 that results in an Ala→Thr substitution in the amino terminus of NEUROD1. The frequency of the threonine variant was similar in 94 index cases and in 96 unrelated non-diabetic individuals (32.9% and 35.9%, respectively). Similarly, this polymorphism was not associated with Fig. 1 Sequence differences found in NEUROD1. a, Schematic organization of NEUROD1 and its domains. Numbers refer to the amino acids bordering the domains. The details of the HLH domain are shown at the top. Filled arrows indicate mutations and the dotted arrows indicate the amino acid variants identified in NEUROD1.The borders were determined based on mammalian homology using published data 2 . 'tx/p300' indicates the transactivation domain as well as the p300-interacting region of NEUROD1 (refs 3,4). b, Alignment of the first 30 aa of the bHLH domain of NEUROD1 with other members of the basic HLH (bHLH) family. Residues responsible for DNA contact are underlined. The Arg 111 residue of NEUROD1 and the corresponding residues of MYOD and E47 are shown [16][17][18] (italics). c, A fragment of NEUROD1 sequence is shown, with the 2...

During muscle regeneration, the mechanism integrating environmental cues at the chromatin of muscle progenitors is unknown. We show that inflammation-activated MKK6-p38 and insulin growth factor 1 (IGF1)-induced PI3K/AKT pathways converge on the chromatin of muscle genes to target distinct components of the muscle transcriptosome. p38 alpha/beta kinases recruit the SWI/SNF chromatin-remodeling complex; AKT1 and 2 promote the association of MyoD with p300 and PCAF acetyltransferases, via direct phosphorylation of p300. Pharmacological or genetic interference with either pathway led to partial assembly of discrete chromatin-bound complexes, which reflected two reversible and distinct cellular phenotypes. Remarkably, PI3K/AKT blockade was permissive for chromatin recruitment of MEF2-SWI/SNF complex, whose remodeling activity was compromised in the absence of MyoD and acetyltransferases. The functional interdependence between p38 and IGF1/PI3K/AKT pathways was further established by the evidence that blockade of AKT chromatin targets was sufficient to prevent the activation of the myogenic program triggered by deliberate activation of p38 signaling.

Inadequate compensatory β cell hyperplasia in insulin-resistant states triggers the development of overt diabetes. The mechanisms that underlie this crucial adaptive response are not fully defined. Here we show that the compensatory islet-growth response to insulin resistance in 2 models -insulin receptor (IR)/IR substrate-1 (IRS-1) double heterozygous mice and liver-specific IR KO (LIRKO) mice -is severely restricted by PDX-1 heterozygosity. Six-month-old IR/IRS-1 and LIRKO mice both showed up to a 10-fold increase in β cell mass, which involved epithelial-to-mesenchymal transition. In both models, superimposition of PDX-1 haploinsufficiency upon the background of insulin resistance completely abrogated the adaptive islet hyperplastic response, and instead the β cells showed apoptosis resulting in premature death of the mice. This study shows that, in postdevelopmental states of β cell growth, PDX-1 is a critical regulator of β cell replication and is required for the compensatory response to insulin resistance.

Ser-133 phosphorylation of CREB within the kinase-inducible domain (KID) promotes target gene activation via complex formation with the KIX domain of the coactivator CBP. Concurrent phosphorylation of CREB at Ser-142 inhibits transcriptional induction via an unknown mechanism. Unstructured in the free state, KID folds into a helical structure upon binding to KIX. Using site-directed mutagenesis based on the NMR structure of the KID:KIX complex, we have examined the mechanisms by which Ser-133 and Ser-142 phosphorylation regulate CREB activity. Our results indicate that phospho-Ser-133 stablizes whereas phospho-Ser-142 disrupts secondary structure-mediated interactions between CREB and CBP. Thus, differential phosphorylation of CREB may form the basis by which upstream signals regulate the specificity of target gene activation.

Protein palmitoylation, by modulating the dynamic interaction between protein and cellular membrane, is involved in a wide range of biological processes, including protein trafficking, sorting, sub-membrane partitioning, protein-protein interaction and cell signaling. To explore the role of protein palmitoylation in adipocytes, we have performed proteomic analysis of palmitoylated proteins in adipose tissue and 3T3-L1 adipocytes and identified more than 800 putative palmitoylated proteins. These include various transporters, enzymes required for lipid and glucose metabolism, regulators of protein trafficking and signaling molecules. Of note, key proteins involved in membrane translocation of the glucose-transporter Glut4 including IRAP, Munc18c, AS160 and Glut4, and signaling proteins in the JAK-STAT pathway including JAK1 and 2, STAT1, 3 and 5A and SHP2 in JAK-STAT, were palmitoylated in cultured adipocytes and primary adipose tissue. Further characterization showed that palmitoylation of Glut4 and IRAP was altered in obesity, and palmitoylation of JAK1 played a regulatory role in JAK1 intracellular localization. Overall, our studies provide evidence to suggest a novel and potentially regulatory role for protein palmitoylation in adipocyte function.