The 12/15-lipoxygenase enzymes react with fatty acids producing active lipid metabolites that are involved in a number of significant disease states. The latter include type 1 and type 2 diabetes (and associated complications), cardiovascular disease, hypertension, renal disease, and the neurological conditions Alzheimer’s disease and Parkinson’s disease. A number of elegant studies over the last thirty years have contributed to unraveling the role that lipoxygenases play in chronic inflammation. The development of animal models with targeted gene deletions has led to a better understanding of the role that lipoxygenases play in various conditions. Selective inhibitors of the different lipoxygenase isoforms are an active area of investigation, and will be both an important research tool and a promising therapeutic target for treating a wide spectrum of human diseases.
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.
Atrial fibrillation (AF) is the most common form of sustained clinical arrhythmia. We previously mapped an AF locus to chromosome 5p13 in an AF family with sudden death in early childhood. Here we show that the specific AF gene underlying this linkage is NUP155, which encodes a member of the nucleoporins, the components of the nuclear pore complex (NPC). We have identified a homozygous mutation, R391H, in NUP155 that cosegregates with AF, affects nuclear localization of NUP155, and reduces nuclear envelope permeability. Homozygous NUP155(-/-) knockout mice die before E8.5, but heterozygous NUP155(+/-) mice show the AF phenotype. The R391H mutation and reduction of NUP155 are associated with inhibition of both export of Hsp70 mRNA and nuclear import of Hsp70 protein. These human and mouse studies indicate that loss of NUP155 function causes AF by altering mRNA and protein transport and link the NPC to cardiovascular disease.
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.
Inflammation and insulin resistance associated with visceral obesity are important risk factors for the development of type 2 diabetes, atherosclerosis, and the metabolic syndrome. The 12/15‐lipoxygenase (12/15‐LO) enzyme has been linked to inflammatory changes in blood vessels that precede the development of atherosclerosis. The expression and role of 12/15‐LO in adipocytes have not been evaluated. We found that 12/15‐LO mRNA was dramatically upregulated in white epididymal adipocytes of high‐fat fed mice. 12/15‐LO was poorly expressed in 3T3‐L1 fibroblasts and was upregulated during differentiation into adipocytes. Interestingly, the saturated fatty acid palmitate, a major component of high fat diets, augmented expression of 12/15‐LO in vitro. When 3T3‐L1 adipocytes were treated with the 12/15‐LO products, 12‐hydroxyeicosatetranoic acid (12(S)‐HETE) and 12‐hydroperoxyeicosatetraenoic acid (12(S)‐HPETE), expression of proinflammatory cytokine genes, including tumor necrosis factor‐α (TNF‐α), monocyte chemoattractant protein 1 (MCP‐1), interleukin 6 (IL‐6), and IL‐12p40, was upregulated whereas anti‐inflammatory adiponectin gene expression was downregulated. 12/15‐LO products also augmented c‐Jun N‐terminal kinase 1 (JNK‐1) phosphorylation, a known negative regulator of insulin signaling. Consistent with impaired insulin signaling, we found that insulin‐stimulated 3T3‐L1 adipocytes exhibited decreased IRS‐1(Tyr) phosphorylation, increased IRS‐1(Ser) phosphorylation, and impaired Akt phosphorylation when treated with 12/15‐LO product. Taken together, our data suggest that 12/15‐LO products create a proinflammatory state and impair insulin signaling in 3T3‐L1 adipocytes. Because 12/15‐LO expression is upregulated in visceral adipocytes by high‐fat feeding in vivo and also by addition of palmitic acid in vitro, we propose that 12/15‐LO plays a role in promoting inflammation and insulin resistance associated with obesity.
In the developing pancreas, the basic helix-loop-helix (bHLH) protein Neurogenin3 (Ngn3) specifies which precursor cells ultimately will become endocrine cells and initiates the islet differentiation program. NeuroD1, a closely related bHLH protein and a downstream target of Ngn3, maintains the differentiation program initiated by Ngn3. We have developed an in vitro model of Ngn3-dependent differentiation by infecting pancreatic duct cell lines with an Ngn3-expressing adenovirus. We found that both Ngn3 and its downstream target NeuroD1 activated the islet differentiation program in these cells by inducing the expression of genes with early roles in the differentiation cascade, as well as genes characteristic of fully differentiated islet cells. Induction of these genes, as exemplified by the insulin1 gene, involved alteration of the local chromatin structure. Interestingly, the subsets of genes activated by Ngn3 and NeuroD1 were not completely overlapping, indicating that these two bHLH proteins serve specific functions in the development of the endocrine pancreas. In addition, microarray gene expression analysis identified a previously uncharacterized group of Ngn3-induced genes with potentially important roles in islet development and function. These studies demonstrate how Ngn3 initiates islet differentiation and provide us with a model for testing methods for producing islet cells for people with diabetes. During pancreatic development, differentiation of endocrine and exocrine cells from a common endodermal progenitor cell requires the coordinated regulation of specific sets of genes. This process can be envisioned as a hierarchy or cascade of transcription factors that initiate and maintain the distinct gene expression programs that define the various pancreatic cell types (1). Among these factors, the basic helix-loop-helix (bHLH) protein Neurogenin3 (Ngn3) plays a dominant role in the specification of the endocrine͞islet cell lineage.During embryonic development, Ngn3 appears transiently in scattered pancreatic epithelial cells (2, 3). Several lines of evidence indicate that the expression of Ngn3 in these undifferentiated cells directs them to an endocrine cell fate and initiates the program of islet differentiation. First, lineage tracing shows that these transient Ngn3-expressing cells differentiate exclusively into islet cells (4). Second, mice homozygous for a targeted deletion of the ngn3 gene fail to generate any islet cells (5). Third, ectopic expression of Ngn3 drives embryonic endoderm to an endocrine fate (2, 3, 6).Ngn3 may play a similar role in the generation of new islet cells postnatally. It has been suggested that cells along the pancreatic ducts may act as progenitors for new islet cells in the postnatal period, although recent lineage tracing experiments suggest that the bulk of newly generated beta cells in adult mice result from the replication of preexisting beta cells (7).Ngn3 initiates islet cell differentiation, but other factors downstream of ngn3 must complete the task. Genetic...
Aims/hypothesis Chronic inflammation in type 2 diabetes is proposed to affect islets as well as insulin target organs. However, the nature of islet inflammation and its effects on islet function in type 2 diabetes remain unclear. Moreover, the immune cell profiles of human islets in healthy and type 2 diabetic conditions are undefined. We aimed to investigate the correlation between proinflammatory cytokine expression, islet leucocyte composition and insulin secretion in type 2 diabetic human islets. Methods Human islets from organ donors with or without type 2 diabetes were studied. First and second phases of glucose-stimulated insulin secretion were determined by perifusion. The expression of inflammatory markers was obtained by quantitative PCR. Immune cells within human islets were analysed by FACS. Results Type 2 diabetic islets, especially those without first-phase insulin secretion, displayed higher CCL2 and TNFa expression than healthy islets. CD45+ leucocytes were elevated in type 2 diabetic islets, to a greater extent in moderately functional type 2 diabetic islets compared with poorly functional ones, and corresponded with elevated ALOX12 but not with CCL2 or TNFa expression. T and B lymphocytes and CD11c+ cells were detectable within both non-diabetic and type 2 diabetic islet leucocytes. Importantly, the proportion of B cells was significantly elevated within type 2 diabetic islets. Conclusions/interpretation Elevated total islet leucocyte content and proinflammatory mediators correlated with islet dysfunction, suggesting that heterogeneous insulitis occurs during the development of islet dysfunction in type 2 diabetes. In addition, the altered B cell content highlights a potential role for the adaptive immune response in islet dysfunction.
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...
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