Molecular basis for negative regulation of the glucagon receptor.

Members of the class B family of G protein-coupled receptors (GPCRs) bind peptide hormones and have causal roles in many diseases, ranging from diabetes and osteoporosis to anxiety. Although peptide, small-molecule, and antibody inhibitors of these GPCRs have been identified, structure-based descriptions of receptor antagonism are scarce. Here we report the mechanisms of glucagon receptor inhibition by blocking antibodies targeting the receptor's extracellular domain (ECD). These studies uncovered a role for the ECD as an intrinsic negative regulator of receptor activity. The crystal structure of the ECD in complex with the Fab fragment of one antibody, mAb1, reveals that this antibody inhibits glucagon receptor by occluding a surface extending across the entire hormone-binding cleft. A second antibody, mAb23, blocks glucagon binding and inhibits basal receptor activity, indicating that it is an inverse agonist and that the ECD can negatively regulate receptor activity independent of ligand binding. Biochemical analyses of receptor mutants in the context of a high-resolution ECD structure show that this previously unrecognized inhibitory activity of the ECD involves an interaction with the third extracellular loop of the receptor and suggest that glucagon-mediated structural changes in the ECD accompany receptor activation. These studies have implications for the design of drugs to treat class B GPCR-related diseases, including the potential for developing novel allosteric regulators that target the ECDs of these receptors.

Exenatide blocks JAK1-STAT1 in pancreatic beta cells.

Exenatide (Ex-4) is an antidiabetic drug that acts through the glucagon-like peptide 1 receptor and has recently been approved for the treatment of type 2 diabetes mellitus. Ex-4 also has been shown to affect beta cell gene expression and increase beta cell mass in rodent models of type 1 diabetes mellitus, but the mechanisms are not fully understood. We therefore analyzed the pathways affected by Ex-4 in human islets by using oligonucleotide microarrays and the PathwayStudio software (Ariadne Genomics, Rockville, MD). We identified the JAK1-STAT1 pathway as a novel target of Ex-4 and confirmed the Ex-4-mediated down-regulation of JAK1 and STAT1 by quantitative reverse transcription-polymerase chain reaction in human islets and INS-1 cells. JAK1-STAT1 is the major signaling pathway mediating the interferon gamma effects on beta cell apoptosis in type 1 diabetes mellitus. Thus, these findings suggest that Ex-4 treatment may also be beneficial in type 1 diabetes mellitus, where it may help protect beta cells from cytokine-induced cell death by inhibiting JAK1-STAT1.

Increased insulin translation from an insulin splice-variant overexpressed in diabetes, obesity, and insulin resistance.

Type 2 diabetes occurs when pancreatic beta-cells become unable to compensate for the underlying insulin resistance. Insulin secretion requires beta-cell insulin stores to be replenished by insulin biosynthesis, which is mainly regulated at the translational level. Such translational regulation often involves the 5'-untranslated region. Recently, we identified a human insulin splice-variant (SPV) altering only the 5'-untranslated region and conferring increased translation efficiency. We now describe a mouse SPV (mSPV) that is found in the cytoplasm and exhibits increased translation efficiency resulting in more normal (prepro)insulin protein per RNA. The RNA stability of mSPV is not increased, but the predicted secondary RNA structure is altered, which may facilitate translation. To determine the role of mSPV in insulin resistance and diabetes, mSPV expression was measured by quantitative real-time RT-PCR in islets from three diabetic and/or insulin-resistant, obese and nonobese, mouse models (BTBRob/ob, C57BL/6ob/ob, and C57BL/6azip). Interestingly, mSPV expression was significantly higher in all diabetic/insulin-resistant mice compared with wild-type littermates and was dramatically induced in primary mouse islets incubated at high glucose. This raises the possibility that the mSPV may represent a compensatory beta-cell mechanism to enhance insulin biosynthesis when insulin requirements are elevated by hyperglycemia/insulin resistance.

Thioredoxin-interacting protein is stimulated by glucose through a carbohydrate response element and induces beta-cell apoptosis.

Recently, we identified thioredoxin-interacting protein (TXNIP) as the most dramatically glucose-induced gene in our human islet microarray study. TXNIP is a regulator of the cellular redox state, but its role in pancreatic beta-cells and the mechanism of its regulation by glucose remain unknown. We therefore generated a stable transfected beta-cell line (INS-1) overexpressing human TXNIP and found that TXNIP overexpression induced apoptosis as assessed by Bax, Bcl2, caspase-3, and cleaved caspase-9 as well as Hoechst staining. Interestingly, islets of insulin-resistant/diabetic mice (AZIP-F1, BTBRob/ob) demonstrated elevated TXNIP expression, suggesting that TXNIP may play a role in glucotoxicity and the beta-cell loss observed under these conditions. Furthermore, we found that glucose-induced TXNIP transcription is not dependent on glucose metabolism and is mediated by a distinct carbohydrate response element (ChoRE) in the human TXNIP promoter consisting of a perfect nonpalindromic repeat of two E-boxes. Transfection studies demonstrated that this ChoRE was necessary and sufficient to confer glucose responsiveness. Thus, TXNIP is a novel proapoptotic beta-cell gene elevated in insulin resistance/diabetes and up-regulated by glucose through a unique ChoRE and may link glucotoxicity and beta-cell apoptosis.

Insulinomas and expression of an insulin splice variant.

BACKGROUND: Insulinomas are beta-cell tumours characterised by uncontrolled insulin secretion even in the presence of hypoglycaemia. However, the mechanisms allowing such excessive insulin secretion are not known. Insulin secretion can occur only when the beta-cell insulin stores have been replenished by insulin biosynthesis, which is mainly controlled by translation. Such specific translational regulation often involves the 5' untranslated region. We have identified an insulin splice variant in isolated human pancreatic islets of non-diabetic donors that retains 26 bp of intron 1 and thereby changes the 5' untranslated region, but leaves the coding region unchanged. This splice variant has increased translation efficiency in vitro and in vivo compared with native insulin mRNA. However, splice variant expression is less than 1% of native insulin mRNA in normal islets. METHODS: To test whether this splice variant is involved in insulin production by human insulinomas, we extracted RNA from nine laser-captured surgical insulinoma samples and from isolated islets of nine donors who did not have diabetes. We then determined the ratio of splice variant to native insulin mRNA by quantitative real-time RT-PCR. FINDINGS: The mean ratio of the splice variant to native insulin mRNA was increased more than 50-fold in insulinomas compared with normal islets, and this difference was present in all nine human insulinomas. Overexpression of the splice variant therefore seems to be a general characteristic of insulinomas and is estimated to contribute about 90% to insulin synthesis by these tumours. INTERPRETATION: Overexpression of the insulin splice variant with increased translation efficiency in insulinomas might explain how these tumours maintain high levels of insulin synthesis and secretion leading to hyperinsulinaemia-the hallmark of this disease.

Metabolism-independent sugar effects on gene transcription: the role of 3-O-methylglucose.

Glucose effects on cellular functions such as gene expression require, in general, glucose metabolism at least to glucose-6-phosphate (G-6-P). However, the example of thioredoxin-interacting protein (TXNIP), a glucose-regulated gene involved in the cellular redox state and pancreatic beta cell apoptosis, demonstrates that this rule may not always apply. We found that aside form glucose, the nonmetabolizable sugars 2-deoxyglucose, which is still converted to G-6-P as well as 3-O-methylglucose (3-MG), which cannot be phosphorylated by glucokinase, stimulate TXNIP expression. In contrast, incubation of INS-1 beta cells with equimolar amounts (25 mM) of l-glucose or mannitol had no effect on TXNIP expression as measured by real-time RT-PCR, eliminating the possibility of an osmotic effect. Also, glucose uptake into the cell is critical because phloretin, an inhibitor of glucose transporter 2, blunted the glucose effects. Moreover, the 3-MG effect was not restricted to a cell line and was observed in 293 cells and primary human islets. Incubation of INS-1 cells with 30mM mannoheptulose, an inhibitor of glucose metabolism, blunted all glucose-induced gene expression but left the 3-MG effects unaltered. Using transient transfection studies and deletion constructs of the human TXNIP promoter, we found that the effects of glucose and 3-MG were dependent on the same region of the TXNIP promoter containing an E-box repeat carbohydrate response element (ChoRE). Thus, these findings provide the first evidence for regulation of gene expression by 3-MG, which is independent of glucose metabolism and suggest that glucose and 3-MG regulate transcription by two distinct pathways converging at a common ChoRE.

Exenatide inhibits beta-cell apoptosis by decreasing thioredoxin-interacting protein.

Exenatide (Ex-4) is a novel anti-diabetic drug that stimulates insulin secretion and enhances beta-cell mass, but the mechanisms involved are not fully understood. We found that Ex-4 protects INS-1 beta-cells against oxidative stress-induced apoptosis (TUNEL) and also reduces expression (mRNA and protein) of thioredoxin-interacting protein (TXNIP), a pro-apoptotic factor involved in beta-cell glucose toxicity and oxidative stress. This reduction was observed in INS-1 cells, mouse, and human islets as well as in wild-type mice receiving Ex-4 and was accompanied by decreased expression of the apoptotic factors caspase-3 and Bax. To determine whether Ex-4-mediated TXNIP reduction is critical for this inhibition of apoptosis, we stably overexpressed TXNIP in INS-1 cells, which completely blunted the anti-apoptotic Ex-4 effects. Thus, Ex-4 inhibits apoptosis by reducing TXNIP expression and early initiation of Ex-4 treatment may help preserve endogenous beta-cell mass, protect against oxidative stress, and delay type 2 diabetes progression.

Gene expression profiling in INS-1 cells overexpressing thioredoxin-interacting protein.

Thioredoxin-interacting protein (TXNIP) is overexpressed in diabetes and has deleterious effects on pancreatic beta-cells and the cardiovascular system. TXNIP is a regulator of the cellular redox state, but has also been suggested to act as a transcriptional repressor. However, the genes and pathways regulated by TXNIP remain unknown. We therefore compared gene expression in INS-1 insulinoma beta-cells overexpressing TXNIP and control LacZ-overexpressing cells using the Affymetrix 230A rat chip. Analysis with the Bayes methodology revealed 98 differentially expressed genes, 90 of which were down-regulated, consistent with the predicted role of TXNIP as a repressor. Using the PathwayAssist software, we found that affected genes were involved in cell death/survival and insulin secretion, and confirmed these findings by real-time RT-PCR and by functional studies. Thus, aside from regulating the cellular redox, TXNIP does modulate overall gene transcription and thereby may further enhance beta-cell death and impair insulin secretion.

Resistin is expressed in pancreatic islets.

Resistin, a recently described adipocyte factor, is regulated by peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. While resistin has been proposed to mediate insulin resistance in rodents, little is known about human resistin and its expression in pancreatic islets has not been tested. The goal of the present study was therefore to analyze whether resistin, like PPARgamma, is expressed in islets. Human islets from seven donors were analyzed by quantitative RT-PCR revealing resistin expression in all samples. Immunohistochemistry using a resistin-specific antibody on human pancreatic sections localized resistin protein to the islets. Mouse resistin was also detected in the Min6 beta cell line. Interestingly, we found a 4-fold increase in islet resistin expression in insulin resistant A-ZIP transgenic compared to wild-type mice. Our results demonstrate that resistin is expressed in islets and up-regulated in insulin resistance and thereby shed new light on the role of resistin in mice and humans.