Type 1 diabetes is preceded by islet β-cell dysfunction, but the mechanisms leading to β-cell dysfunction have not been rigorously studied. Because immune cell infiltration occurs prior to overt diabetes, we hypothesized that activation of inflammatory cascades and appearance of endoplasmic reticulum (ER) stress in β-cells contributes to insulin secretory defects. Prediabetic nonobese diabetic (NOD) mice and control diabetes-resistant NOD-SCID and CD1 strains were studied for metabolic control and islet function and gene regulation. Prediabetic NOD mice were relatively glucose intolerant and had defective insulin secretion with elevated proinsulin:insulin ratios compared with control strains. Isolated islets from NOD mice displayed age-dependent increases in parameters of ER stress, morphologic alterations in ER structure by electron microscopy, and activation of nuclear factor-κB (NF-κB) target genes. Upon exposure to a mixture of proinflammatory cytokines that mimics the microenvironment of type 1 diabetes, MIN6 β-cells displayed evidence for polyribosomal runoff, a finding consistent with the translational initiation blockade characteristic of ER stress. We conclude that β-cells of prediabetic NOD mice display dysfunction and overt ER stress that may be driven by NF-κB signaling, and strategies that attenuate pathways leading to ER stress may preserve β-cell function in type 1 diabetes.

Intermittent fasting (IF) protects against the development of metabolic diseases and cancer, but whether it can prevent diabetic microvascular complications is not known. In mice, we examined the impact of long-term IF on diabetic retinopathy (DR). Despite no change in glycated hemoglobin, mice on the IF regimen displayed significantly longer survival and a reduction in DR end points, including acellular capillaries and leukocyte infiltration. We hypothesized that IF-mediated changes in the gut microbiota would produce beneficial metabolites and prevent the development of DR. Microbiome analysis revealed increased levels of Firmicutes and decreased Bacteroidetes and Verrucomicrobia. Compared with mice on ad libitum feeding, changes in the microbiome of the mice on IF were associated with increases in gut mucin, goblet cell number, villi length, and reductions in plasma peptidoglycan. Consistent with the known modulatory effects of Firmicutes on bile acid (BA) metabolism, measurement of BAs demonstrated a significant increase of tauroursodeoxycholate (TUDCA), a neuroprotective BA, in on IF but not in on AL feeding. TGR5, the TUDCA receptor, was found in the retinal primary ganglion cells. Expression of TGR5 did not change with IF or diabetes. However, IF reduced retinal TNF-α mRNA, which is a downstream target of TGR5 activation. Pharmacological activation of TGR5 using INT-767 prevented DR in a second diabetic mouse model. These findings support the concept that IF prevents DR by restructuring the microbiota toward species producing TUDCA and subsequent retinal protection by TGR5 activation.

The transcription factor Pdx1 is expressed in the pancreatic ␤-cell, where it is believed to regulate several ␤-cell-specific genes. Whereas binding by Pdx1 to elements of ␤-cell genes has been demonstrated in vitro, almost none of these genes has been demonstrated to be a direct binding target for Pdx1 within cells (where complex chromatin structure exists). To determine which ␤-cell promoters are bound by Pdx1 in vivo, we performed chromatin immunoprecipitation assays using Pdx1 antiserum and chromatin from ␤-TC3 cells and Pdx1-transfected NIH3T3 cells and subsequently quantitated co-immunoprecipitated promoters using realtime PCR. We compared these in vivo findings to parallel immunoprecipitations in which Pdx1 was allowed to bind to promoter fragments in in vitro reactions. Our results show that in all cells Pdx1 binds strongly to the insulin, islet amyloid polypeptide, glucagon, Pdx1, and Pax4 promoters, whereas it does not bind to either the glucose transporter type 2 or albumin promoters. In addition, no binding by Pdx1 to the glucokinase promoter was observed in ␤-cells. In contrast, in in vitro immunoprecipitations, Pdx1 bound all promoters to an extent approximately proportional to the number of Pdx1 binding sites. Our findings suggest a critical role for chromatin structure in directing the promoter binding selectivity of Pdx1 in ␤-cells and non-␤-cells.

Neurogenin3 (ngn3), a basic helix-loop-helix (bHLH) transcription factor, functions as a pro-endocrine factor in the developing pancreas: by itself, it is sufficient to force undifferentiated pancreatic epithelial cells to become islet cells. Because ngn3 expression determines which precursor cells will differentiate into islet cells, the signals that regulate ngn3 expression control islet cell formation. To investigate the factors that control ngn3 gene expression, we mapped the human and mouse ngn3 promoters and delineated transcriptionally active sequences within the human promoter. Surprisingly, the human ngn3 promoter drives transcription in all cell lines tested, including fibroblast cell lines. In contrast, in transgenic animals the promoter drives expression specifically in regions of ngn3 expression in the developing pancreas and gut; and the addition of distal sequences greatly enhances transgene expression. Within the distal enhancer, binding sites for several pancreatic transcription factors, including hepatocyte nuclear factor (HNF)-1 and HNF-3, form a tight cluster. HES1, an inhibitory bHLH factor activated by Notch signaling, binds to the proximal promoter and specifically blocks promoter activity. Together with previous genetic data, these results suggest a model in which the ngn3 gene is activated by the coordinated activities of several pancreatic transcription factors and inhibited by Notch signaling through HES1. Diabetes 50:928 -936, 2001

G protein-coupled receptors have been well described to contribute to the regulation of glucose-stimulated insulin secretion (GSIS). The short-chain fatty acid-sensing G protein-coupled receptor, free fatty acid receptor 2 (FFAR2), is expressed in pancreatic β-cells, and in rodents, its expression is altered during insulin resistance. Thus, we explored the role of FFAR2 in regulating GSIS. First, assessing the phenotype of wild-type and Ffar2(-/-) mice in vivo, we observed no differences with regard to glucose homeostasis on normal or high-fat diet, with a marginally significant defect in insulin secretion in Ffar2(-/-) mice during hyperglycemic clamps. In ex vivo insulin secretion studies, we observed diminished GSIS from Ffar2(-/-) islets relative to wild-type islets under high-glucose conditions. Further, in the presence of acetate, the primary endogenous ligand for FFAR2, we observed FFAR2-dependent potentiation of GSIS, whereas FFAR2-specific agonists resulted in either potentiation or inhibition of GSIS, which we found to result from selective signaling through either Gαq/11 or Gαi/o, respectively. Lastly, in ex vivo insulin secretion studies of human islets, we observed that acetate and FFAR2 agonists elicited different signaling properties at human FFAR2 than at mouse FFAR2. Taken together, our studies reveal that FFAR2 signaling occurs by divergent G protein pathways that can selectively potentiate or inhibit GSIS in mouse islets. Further, we have identified important differences in the response of mouse and human FFAR2 to selective agonists, and we suggest that these differences warrant consideration in the continued investigation of FFAR2 as a novel type 2 diabetes target.

Type 2 diabetes mellitus (T2DM) results from pancreatic ␤ cell failure in the setting of insulin resistance. Heterozygous mutations in the gene encoding the ␤ cell transcription factor pancreatic duodenal homeobox 1 (Pdx1) are associated with both T2DM and maturity onset diabetes of the young (MODY4), and low levels of Pdx1 accompany ␤ cell dysfunction in experimental models of glucotoxicity and diabetes. Here, we find that Pdx1 is required for compensatory ␤ cell mass expansion in response to diet-induced insulin resistance through its roles in promoting ␤ cell survival and compensatory hypertrophy. Pdx1-deficient ␤ cells show evidence of endoplasmic reticulum (ER) stress both in the complex metabolic milieu of high-fat feeding as well as in the setting of acutely reduced Pdx1 expression in the Min6 mouse insulinoma cell line. Further, Pdx1 deficiency enhances ␤ cell susceptibility to ER stressassociated apoptosis. The results of high throughput expression microarray and chromatin occupancy analyses reveal that Pdx1 regulates a broad array of genes involved in diverse functions of the ER, including proper disulfide bond formation, protein folding, and the unfolded protein response. These findings suggest that Pdx1 deficiency leads to a failure of ␤ cell compensation for insulin resistance at least in part by impairing critical functions of the ER.chromatin occupancy ͉ diabetes ͉ gene regulation ͉ islet compensation

In both type 1 and type 2 diabetes, pancreatic islet dysfunction results in part from cytokine-mediated inflammation. The ubiquitous eukaryotic translation initiation factor 5A (eIF5A), which is the only protein to contain the amino acid hypusine, contributes to the production of proinflammatory cytokines. We therefore investigated whether eIF5A participates in the inflammatory cascade leading to islet dysfunction during the development of diabetes. As described herein, we found that eIF5A regulates iNOS levels and that eIF5A depletion as well as the inhibition of hypusination protects against glucose intolerance in inflammatory mouse models of diabetes. We observed that following knockdown of eIF5A expression, mice were resistant to β cell loss and the development of hyperglycemia in the low-dose streptozotocin model of diabetes. The depletion of eIF5A led to impaired translation of iNOS-encoding mRNA within the islet. A role for the hypusine residue of eIF5A in islet inflammatory responses was suggested by the observation that inhibition of hypusine synthesis reduced translation of iNOS-encoding mRNA in rodent β cells and human islets and protected mice against the development of glucose intolerance the low-dose streptozotocin model of diabetes. Further analysis revealed that hypusine is required in part for nuclear export of iNOS-encoding mRNA, a process that involved the export protein exportin1. These observations identify the hypusine modification of eIF5A as a potential therapeutic target for preserving islet function under inflammatory conditions.

Like expression of other cell-type-specific genes, expression of the insulin gene depends on the actions of a unique set of nuclear activators. These activators cooperate synergistically in building a transcriptional activation complex that binds to the regulatory domains of the gene and activates the basal RNA polymerase machinery (reviewed in reference 7). The complexity and specificity of the interactions among these activators limit the cell types capable of building a functional activation complex. Dissection of these interactions provides insight into the mechanism by which insulin expression is limited to the correct cell type.In adult mammals, activation of the insulin gene is tightly restricted to the ␤ cells in the pancreatic islets of Langerhans, where it is expressed at high levels. This specificity is reflected in the restricted function of the insulin promoter, the proximal few hundred base pairs of which can replicate the specificity of the intact gene (19,50). Because of the complexity of the intact promoter (9, 13, 24), a short portion of the rat insulin I promoter between bp Ϫ247 and Ϫ197 upstream from the transcription initiation site has been used as a model of the types of synergistic interactions that combine to give the characteristic activity of the full promoter (15). This 50-bp fragment contains at least three distinct DNA-binding sites named E2, A3, and A4 (13). The E and A elements synergize: neither has significant activity on its own, but in combination E and A elements produce ␤-cell specific transcriptional activation (15,23).The E2 element functions as a recognition site for dimers of basic helix-loop-helix (bHLH) proteins, including a heterodimer of the ubiquitous bHLH protein E47/Pan1 and the neuroendocrine specific bHLH protein BETA2/NeuroD1 (35). The A elements each contain the sequence TAAT and have been shown to bind to several homeodomain proteins found in ␤-cell nuclei (12,16,22, 32,37 (16,38,40).The LIM homeodomain protein Lmx1.1 contains two LIM domains that form zinc-binding structures in the amino end of the molecule. The second of these two LIM domains (LIM2) directly binds to the bHLH domain of E47/Pan1 and mediates the synergy between Lmx1.1 and E47/Pan1. This interaction is specific, since analogous domains from other LIM proteins and bHLH proteins cannot substitute for the LIM2 domain of Lmx1.1 or the bHLH domain of E47/Pan1, respectively (20).PDX-1 plays an important role both in the development of the pancreas and in maintaining ␤-cell function. Mice with a targeted disruption of the pdx1 gene selectively lack a pancreas (2, 21, 36). Similar pancreatic agenesis has been found in a human patient with a single nucleotide deletion in the pdx1 gene (46). If the pancreas is allowed to develop with an intact pdx1 gene, and the pdx1 gene is disrupted only in mature ␤ cells, diabetes ensues due to impaired ␤-cell function (3). This