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.

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.

The nuclear receptor peroxisome proliferator-activated receptor ␥ (PPAR-␥) is an important target in diabetes therapy, but its direct role, if any, in the restoration of islet function has remained controversial. To identify potential molecular mechanisms of PPAR-␥ in the islet, we treated diabetic or glucose-intolerant mice with the PPAR-␥ agonist pioglitazone or with a control. Treated mice exhibited significantly improved glycemic control, corresponding to increased serum insulin and enhanced glucose-stimulated insulin release and Ca 2؉ responses from isolated islets in vitro. This improved islet function was at least partially attributed to significant upregulation of the islet genes Irs1, SERCA, Ins1/2, and Glut2 in treated animals. The restoration of the Ins1/2 and Glut2 genes corresponded to a two-to threefold increase in the euchromatin marker histone H3 dimethyl-Lys4 at their respective promoters and was coincident with increased nuclear occupancy of the islet methyltransferase Set7/9. Analysis of diabetic islets in vitro suggested that these effects resulting from the presence of the PPAR-␥ agonist may be secondary to improvements in endoplasmic reticulum stress. Consistent with this possibility, incubation of thapsigargin-treated INS-1 ␤ cells with the PPAR-␥ agonist resulted in the reduction of endoplasmic reticulum stress and restoration of Pdx1 protein levels and Set7/9 nuclear occupancy. We conclude that PPAR-␥ agonists exert a direct effect in diabetic islets to reduce endoplasmic reticulum stress and enhance Pdx1 levels, leading to favorable alterations of the islet gene chromatin architecture.Type 2 diabetes mellitus results from a combination of insulin resistance and progressive islet dysfunction (46). In many individuals, ␤-cell failure may precede the clinical diagnosis of diabetes, and landmark studies such as the United Kingdom Prospective Diabetes Study have shown a continued decrement in ␤-cell function despite treatment intervention with sulfonylureas, metformin, and insulin (52). Thiazolidinediones are orally active agents used in the treatment of type 2 diabetes that act as agonists for the nuclear transcription factor peroxisome proliferator-activated receptor ␥ (PPAR-␥) (60). Although thiazolidinediones are classically thought to act as peripheral insulin sensitizers, there is growing evidence from studies of human and animal models that these agents may also act to preserve and/or enhance ␤-cell function in the setting of progressive type 2 diabetes and insulin resistance (3, 12). PPAR-␥ is known to be expressed in the pancreatic islet (8, 48), and PPAR-responsive elements have been identified in the promoters of genes involved in glucose-stimulated insulin secretion, including Glut2, Gck, and Pdx1 (16,21,26,27,33). Reports from studies of ␤-cell lines, rodent models of progressive type 2 diabetes, and humans at risk for type 2 diabetes suggest that PPAR-␥ agonist administration leads to preservation of islet mass and function (10,13,18,22,25,33,57,58).Whereas the studies noted ab...

Elevated ratios of circulating unmethylated to methylated preproinsulin (INS) DNA have been suggested to reflect β-cell death in type 1 diabetes (T1D). We tested the hypothesis that absolute levels (rather than ratios) of unmethylated and methylated INS DNA differ between subjects with new-onset T1D and control subjects and assessed longitudinal changes in these parameters. We used droplet digital PCR to measure levels of unmethylated and methylated INS DNA in serum from subjects at T1D onset and at 8 weeks and 1 year post-onset. Compared with control subjects, levels of both unmethylated and methylated INS DNA were elevated at T1D onset. At 8 weeks post-onset, methylated INS DNA remained elevated, but unmethylated INS DNA fell. At 1 year postonset, both unmethylated and methylated INS DNA returned to control levels. Subjects with obesity, type 2 diabetes, and autoimmune hepatitis exhibited lower levels of unmethylated and methylated INS compared with subjects with T1D at onset and no differences compared with control subjects. Our study shows that elevations in both unmethylated and methylated INS DNA occurs in new-onset T1D and that levels of these DNA species change during T1D evolution. Our work emphasizes the need to consider absolute levels of differentially methylated DNA species as potential biomarkers of disease.

OBJECTIVE-12/15-lipoxygenase (12/15-LO), one of a family of fatty acid oxidoreductase enzymes, reacts with polyenoic fatty acids to produce proinflammatory lipids. 12/15-LO is expressed in macrophages and pancreatic ␤-cells. It enhances interleukin 12 production by macrophages, and several of its products induce apoptosis of ␤-cells at nanomolar concentrations in vitro. We had previously demonstrated a role for 12/15-LO in ␤-cell damage in the streptozotocin model of diabetes. Since the gene encoding 12/15-LO (gene designation Alox15) lies within the Idd4 diabetes susceptibility interval in NOD mice, we hypothesized that 12/15-LO is also a key regulator of diabetes susceptibility in the NOD mouse. RESEARCH DESIGN AND METHODS-We developed NOD mice carrying an inactivated 12/15-LO locus (NOD-Alox15null ) using a "speed congenic" protocol, and the mice were monitored for development of insulitis and diabetes.RESULTS-NOD mice deficient in 12/15-LO develop diabetes at a markedly reduced rate compared with NOD mice (2.5 vs. Ͼ60% in females by 30 weeks). Nondiabetic female NOD-Alox15 null mice demonstrate improved glucose tolerance, as well as significantly reduced severity of insulitis and improved ␤-cell mass, when compared with age-matched nondiabetic NOD females. Disease resistance is associated with decreased numbers of islet-infiltrating activated macrophages at 4 weeks of age in NOD-Alox15 null mice, preceding the development of insulitis. Subsequently, islet-associated infiltrates are characterized by decreased numbers of CD4 ϩ T cells and increased Foxp3 ϩ cells.CONCLUSIONS-These results suggest an important role for 12/15-LO in conferring susceptibility to autoimmune diabetes in NOD mice through its effects on macrophage recruitment or activation.

Loss of beta-cell function in type 1 and type 2 diabetes leads to metabolic dysregulation and inability to maintain normoglycemia. Noninvasive imaging of beta-cell function in vivo would therefore provide a valuable diagnostic and research tool for quantifying progression to diabetes and response to therapeutic intervention. Because manganese (Mn(2+)) is a longitudinal relaxation time (T1)-shortening magnetic resonance imaging (MRI) contrast agent that enters cells such as pancreatic beta-cells through voltage-gated calcium channels, we hypothesized that Mn(2+)-enhanced MRI of the pancreas after glucose infusion would allow for noninvasive detection of beta-cell function in vivo. To test this hypothesis, we administered glucose and saline challenges intravenously to normal mice and mice given high or low doses of streptozotocin (STZ) to induce diabetes. Serial inversion recovery MRI was subsequently performed after Mn(2+) injection to probe Mn(2+) accumulation in the pancreas. Time-intensity curves of the pancreas (normalized to the liver) fit to a sigmoid function showed a 51% increase in signal plateau height after glucose stimulation relative to saline (P < 0.01) in normal mice. In diabetic mice given a high dose of STZ, only a 9% increase in plateau signal intensity was observed after glucose challenge (P = not significant); in mice given a low dose of STZ, a 20% increase in plateau signal intensity was seen after glucose challenge (P = 0.02). Consistent with these imaging findings, the pancreatic insulin content of high- and low-dose STZ diabetic mice was reduced about 20-fold and 10-fold, respectively, compared with normal mice. We conclude that Mn(2+)-enhanced MRI demonstrates excellent potential as a means for noninvasively monitoring beta-cell function in vivo and may have the sensitivity to detect progressive decreases in function that occur in the diabetic disease process.

High-fat diets lead to obesity, inflammation, and dysglycemia. 12-Lipoxygenase (12-LO) is activated by high-fat diets and catalyzes the oxygenation of cellular arachidonic acid to form proinflammatory intermediates. We hypothesized that 12-LO in the pancreatic islet is sufficient to cause dysglycemia in the setting of high-fat feeding. To test this, we generated pancreas-specific 12-LO knockout mice and studied their metabolic and molecular adaptations to high-fat diets. Whereas knockout mice and control littermates displayed identical weight gain, body fat distribution, and macrophage infiltration into fat, knockout mice exhibited greater adaptive islet hyperplasia, improved insulin secretion, and complete protection from dysglycemia. At the molecular level, 12-LO deletion resulted in increases in islet antioxidant enzymes Sod1 and Gpx1 in response to high-fat feeding. The absence or inhibition of 12-LO led to increases in nuclear Nrf2, a transcription factor responsible for activation of genes encoding antioxidant enzymes. Our data reveal a novel pathway in which islet 12-LO suppresses antioxidant enzymes and prevents the adaptive islet responses in the setting of high-fat diets.O besity, typically a result of diets high in saturated fat content, is directly correlated to insulin resistance and prediabetes. Macrovascular disease consequences, including stroke, myocardial infarction, and mortality increase even as blood sugars rise in the prediabetic phase (1), thus emphasizing the perils of glycemic dysregulation in the absence of frank diabetes. High-fat diets (HFDs) and their consequent insulin resistance lead to adaptive islet hyperplasia, in which basal secretion of insulin is increased in an attempt to compensate for insulin resistance (2). However, this compensatory mechanism can eventually fail in the face of prolonged insulin resistance. Inflammation and oxidative stress are major factors thought to contribute to dysfunction of ␤ cells in the setting of insulin resistance (3, 4). In this regard, ␤ cells have low expression and activity levels of the antioxidant enzymes compared to other metabolic tissues (5), and the pathway(s) that endogenously suppresses production of these antioxidant enzymes in the ␤ cell remains unclear.The lipoxygenases represent a group of enzymes that catalyzes the oxygenation of polyunsaturated fatty acids to form inflammatory lipid intermediates, which have been shown to contribute to oxidative stress (6). In the mouse, 12-lipoxygenase (12-LO) is expressed in several cell types, including macrophages, white adipocytes, and pancreatic islets (7). 12-LO oxygenates membranederived arachidonic acid primarily at the 12-position carbon atom to form 12-hydroperoxyeicosatetraenoic acid (12-HPETE), which is then converted to the more stable form 12-hydroxyeicosatetraenoic acid (12-HETE) (7). The role of 12-LO in the setting of HFDs and obesity has been studied primarily in the context of whole-body 12-LO knockout mice. Recent studies showed that 12-LO knockout mice fed a HFD exhibit r...

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories [1][2][3][4] , variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations 5,6 , but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh 7,8 , in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient 9 , and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a 6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate 6 . This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay 10 . Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.