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...

Rationale B cells are abundant in the adventitia of normal and diseased vessels. Yet, the molecular and cellular mechanisms mediating homing of B cells to the vessel wall and B cell effects on atherosclerosis are poorly understood. Inhibitor of Differentiation-3 (Id3), is important for atheroprotection in mice and polymorphism in the human ID3 gene has been implicated as a potential risk marker of atherosclerosis in humans. Yet the role of Id3 in B cell regulation of atherosclerosis is unknown. Objective To determine if Id3 regulates B cell homing to the aorta and atheroprotection, and identify molecular and cellular mechanisms mediating this effect. Methods and Results Loss of Id3 in Apoe−/− mice resulted in early and increased atherosclerosis. Flow cytometry revealed a defect in Id3−/− Apoe−/− mice in the number of B cells in the aorta, but not the spleen, lymph nodes and circulation. Similarly, B cells transferred from Id3−/− Apoe−/− mice into B cell deficient micereconstituted spleen, lymph node and blood similarly to B cells from Id3+/+ Apoe−/− mice, but aortic reconstitution and B cell-mediated inhibition of diet-induced atherosclerosis was significantly impaired. In addition to retarding initiation of atherosclerosis, B cells homed to regions of existing atherosclerosis, reduced macrophage content in plaque and attenuated progression of disease. The chemokine receptor, CCR6, was identified as an important Id3 target mediating aortic homing and atheroprotection. Conclusions Together, these results are the first to identify the Id3-CCR6 pathway in B cells and demonstrate its role in aortic B cell homing and B cell mediated protection from early atherosclerosis.

Inflammation is a key pathological process in the progression of atherosclerosis and type 2 diabetes. 12/15-lipoxygenase (12-LO), an enzyme involved in fatty acid metabolism, may contribute to inflammatory damage triggered by stressors such as obesity and insulin resistance. We hypothesized that mice lacking 12-LO are protected against inflammatory-mediated damage associated with a "western" diet. To test this hypothesis, age-matched male 12-LO knockout (12-LOKO) and wild-type C57BL/6 (B6) mice were fed either a standard chow or western diet and assessed for several inflammatory markers. Western-fed B6 mice showed expected reductions in glucose and insulin tolerance compared with chow-fed mice. In contrast, western-fed 12-LOKO mice maintained glucose and insulin tolerance similar to chow-fed mice. Circulating proinflammatory cytokines, tumor necrosis factor-alpha and interleukin-6, were increased in western B6 mice but not 12-LOKO mice, whereas the reported protective adipokine, adiponectin, was decreased only in western B6 mice. 12-LO activity was significantly elevated by western diet in islets from B6 mice. Islets from 12-LOKO mice did not show western-diet-induced islet hyperplasia or increases in caspase-3 apoptotic staining observed in western-fed B6 mice. Islets from 12-LOKO mice were also protected from reduced glucose-stimulated insulin secretion observed in islets from western-fed B6 mice. In visceral fat, macrophage numbers and monocyte chemoattractant protein-1 expression were elevated in western B6 mice but not 12-LOKO mice. These data suggest that 12-LO activation plays a role in western-diet-induced damage in visceral fat and islets. Inhibiting 12-LO may provide a new therapeutic approach to prevent inflammation-mediated metabolic consequences of excess fat intake.

Histone modifying enzymes contribute to the activation or inactivation of transcription by ultimately catalyzing the unfolding or further compaction, respectively, of chromatin structure. Actively transcribed genes are typically hyperacetylated at Lys residues of histones H3 and H4 and hypermethylated at Lys-4 of histone H3 (H3-K4). To determine whether covalent histone modifications play a role in the ␤ cell-specific expression of the insulin gene, we performed chromatin immunoprecipitation assays using anti-histone antibodies and extracts from ␤ cell lines, non-␤ cell lines, and ES cells, and quantitated specific histone modifications at the insulin promoter by real-time PCR. Our studies reveal that the proximal insulin promoter is hyperacetylated at histone H3 only in ␤ cells. This hyperacetylation is highly correlated to recruitment of the histone acetyltransferase p300 to the proximal promoter in ␤ cells, and is consistent with the role of hyperacetylation in promoting euchromatin formation. We also observed that the proximal insulin promoter of ␤ cells is hypermethylated at H3-K4, and that this modification is correlated to the recruitment of the histone methyltransferase SET7/9 to the promoter. ES cells demonstrate a histone modification pattern intermediate between that of ␤ cells and non-␤ cells, and is consistent with their potential to express the insulin gene. We therefore propose a model in which insulin transcription in the ␤ cell is facilitated by a unique combination of transcription factors that acts in the setting of an open, euchromatic structure of the insulin gene.The pancreatic ␤ cell is exclusively responsible for the synthesis and secretion of insulin. The production of insulin appears to be governed by constraints imposed at the level of transcription of the gene encoding insulin (Ins), 1 and involves an intricate interplay between transcription factors that are known to function as transactivators of the gene. In recent years, specific DNA elements within the proximal ϳ400 base pairs (bp) of the Ins promoter have been mapped precisely and shown to be bound by several major classes of transactivating transcription factors, including homeodomain factors (Pdx1, Lmx1.1), basic helix loop helix factors (NeuroD1, E47), and bZip factors (mMafA) (see Ref. 1 for review). In addition, coactivators such as p300 (by virtue of its interaction with Pdx1, NeuroD1, and E47) have also been suggested to contribute to Ins gene regulation (2, 3). Thus, it is hypothesized that the unique combination of ubiquitous and cell type-specific factors within the ␤ cell results in the transcription of the Ins gene (3-5). However, this hypothesis alone cannot explain why heterologous expression of ␤ cell factors results in activation of the endogenous Ins gene in only limited, "responsive" cell types (e.g. liver, pancreatic ductal cells, and embryonic and intestinal stem cells) (6 -10). Although this finding suggests that "unresponsive" cell types are still missing some critical genetic component (transcription facto...

Expression of the insulin gene is nearly exclusive to the ␤ cells of the pancreatic islets. Although the sequence-specific transcription factors that regulate insulin expression have been well studied, the interrelationship between these factors, chromatin structure, and transcriptional elongation by RNA polymerase II (pol II) has remained undefined. In this regard, recent studies have begun to establish a role for the methylation of histone H3 in the initiation or elongation of transcription by pol II. To determine a role for the transcriptional activator Pdx-1 in the maintenance of chromatin structure and pol II recruitment at the insulin gene, we performed small interfering RNA-mediated knockdown of Pdx-1 in ␤TC3 cells and subsequently studied histone modifications and pol II recruitment by chromatin immunoprecipitation. We demonstrated here that the 50% fall in insulin transcription following knockdown of Pdx-1 is accompanied by a 60% fall in dimethylated histone H3-Lys-4 at the insulin promoter. H3-Lys-4 methylation at the insulin promoter may be mediated, at least partially, by the methyltransferase Set9. Immunohistochemical analysis revealed that Set9 is expressed in an islet-enriched pattern in the pancreas, similar to the pattern of Pdx-1 expression. The recruitment of Set9 to the insulin gene appears to be a consequence of its direct interaction with Pdx-1, and small interfering RNA-mediated knockdown of Set9 attenuates insulin transcription. Pdx-1 knockdown was also associated with an overall shift in the recruitment of pol II isoforms to the insulin gene, from an elongation isoform (Ser(P)-2) to an initiation isoform (Ser(P)-5). Our findings therefore suggest a model whereby Pdx-1 plays a novel role in linking H3-Lys-4 dimethylation and pol II elongation to insulin transcription.During pancreatic development, endocrine and exocrine cell types differentiate from a common endodermal precursor cell via a tightly coordinated sequence of transcriptional events (1, 2). The Hox-like transcription factor Pdx-1 is thought to initiate this cascade by regulating specific genes within the endodermal precursor cell (3, 4). The disruption of pdx-1 expression during development, either by targeted knockout in mice (5, 6) or homozygous mutations in humans (7), can be catastrophic, resulting in the failure of pancreas formation and consequent development of diabetes. Within the mature pancreas, Pdx-1 is necessary for the maintenance and function of endocrine cell types within the islets of Langerhans. In the islet ␤ cell, Pdx-1 is believed to activate a number of critical genes involved in glucose sensing and insulin production, including glucokinase, glut2, and insulin (3,8).Expression of the insulin gene is almost exclusive to the islet ␤ cells. By using small interfering RNA (siRNA) 2 and chromatin immunoprecipitation (ChIP) in isolated mouse islets, we recently demonstrated that Pdx-1 is a direct activator of insulin transcription upon binding to A box DNA elements in the 5Ј-regulatory region of the gene (the ins...