Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytokines, such as IL-1beta and TNF-alpha, contribute to pancreatic beta-cell death in type 1 diabetes mellitus. The transcription factor nuclear factor-kappaB (NF-kappaB) mediates cytokine-induced beta-cell apoptosis. Paradoxically, NF-kappaB has mostly antiapoptotic effects in other cell types. The cellular actions of NF-kappaB depend on the cell type, the nature and duration of the stimulus, the periodicity, and the degree of activity of the particular dimers involved. To clarify the reasons behind the proapoptotic effects of NF-kappaB in pancreatic beta-cells, we compared the pattern of cytokine-induced NF-kappaB activation between rat insulin-producing cells (INS-1E cells) and fibroblasts (208F cells). NF-kappaB activation was induced in INS-1E cells and in 208F cells after exposure to cytokines, but apoptosis was induced only in INS-1E cells, with a more pronounced proapoptotic effect of IL-1beta than of TNF-alpha. NF-kappaB activation in IL-1beta-exposed INS-1E cells was earlier and more marked as compared with TNF-alpha-exposed INS-1E cells or IL-1beta-exposed 208F cells. Both cytokines induced a prolonged (up to 48 h) and stable NF-kappaB activation in INS-1E cells, whereas IL-1beta induced an oscillatory NF-kappaB activation in 208F cells. p65/p65 and p65/p50 were the predominant NF-kappaB dimers in IL-1beta-exposed INS-1E cells and 208F cells, respectively. IL-1beta induced a differential usage of cis-elements in the inducible nitric oxide synthase promoter region in the two cell-lines and an increase in ERK1/2 activity in INS-1E cells but not in 208F cells. Cytokine-induced expression of IkappaB isoforms and other NF-kappaB target genes (Fas, MCP-1, and inducible nitric oxide synthase) was severalfold higher in INS-1E cells than in 208F cells. These results suggest that cytokine-induced NF-kappaB activation in insulin-producing cells is more rapid, marked, and sustained than in fibroblasts, which correlates with a more pronounced activation of downstream genes and a proapoptotic outcome.
Mol Endocrinol 2006 Aug
PMID:Cytokine-induced proapoptotic gene expression in insulin-producing cells is related to rapid, sustained, and nonoscillatory nuclear factor-kappaB activation. 1655 31

Tight regulation of fatty acid metabolism in pancreatic beta-cells is important for beta-cell viability and function. Chronic exposure to elevated concentrations of fatty acid is associated with beta-cell lipotoxicity. Glucose is known to repress fatty acid oxidation and hence to augment the toxicity of fatty acids. The peroxisome proliferator activated receptor alpha (PPARalpha) is a key activator of genes involved in beta-cell fatty acid oxidation, and transcription of the PPARalpha gene has been shown to be repressed by increasing concentrations of glucose in beta-cells. However, the mechanism underlying this transcriptional repression by glucose remains unclear. Here we report that glucose-induced repression of PPARalpha gene expression in INS-1E cells is independent of beta-cell excitation and insulin secretion but requires activation of protein phosphatase 2A in a process involving inactivation of the AMP-activated protein kinase (AMPK). Pharmacological activation of AMPK at high glucose concentrations interferes with glucose repression of PPARalpha and PPARalpha target genes in INS-1E cells as well as in rat islets. Specific knock-down of the catalytic AMPK-subunit AMPKalpha2 but not AMPKalpha1 using RNAi suppressed PPARalpha expression, thereby mimicking the effect of glucose. These results indicate that activation of protein phosphatase 2A and subsequent inactivation of AMPK is necessary for glucose repression of PPARalpha expression in pancreatic beta-cells.
J Mol Endocrinol 2006 Apr
PMID:Glucose-induced repression of PPARalpha gene expression in pancreatic beta-cells involves PP2A activation and AMPK inactivation. 1659

Both unacylated ghrelin (UAG) and acylated ghrelin (AG) exert metabolic effects. To investigate the interactions between AG and UAG on ghrelin receptors we evaluated the effects of AG and UAG on INS-1E rat insulinoma cells, using insulin secretion after 30min static incubation as a read-out. A possible involvement of the growth hormone secretagogue receptor type 1a (GHS-R1a) or the corticotropin-releasing factor 2 (CRF2) receptor (CRF2R), as a putative receptor for UAG, was also studied determining their mRNA expression and the functional effects of receptor antagonists on insulin release. Both UAG and AG stimulated insulin release dose-dependently in the nanomolar range. The AG-induced insulin output was antagonized by two GHS-R1a antagonists ([d-Lys(3)]GHRP-6 and BIM28163), which did not block UAG actions. These effects occurred in the presence of low levels of GHS-R1a mRNA. Neither CRF2R expression nor effects of the CRF2R antagonist (astressin(2)B) on insulin output were observed. In conclusion, we provide a sensitive and reproducible assay for specific effects of UAG, which in this study is responsible for insulin release by INS-1E cells. Our data support the existence of a specific receptor for UAG, other than the CRF2R and GHS-R1a. The stimulatory effect on insulin secretion by AG in this cell line is mediated by the GHS-R1a.
Mol Cell Endocrinol 2006 Jun 07
PMID:Unacylated ghrelin is active on the INS-1E rat insulinoma cell line independently of the growth hormone secretagogue receptor type 1a and the corticotropin releasing factor 2 receptor. 1664 96

In cells lacking expression of Ca(2+)-mobilizing G proteins, coexpression of human GPR40 and Galpha(q) allowed medium- and long-chain fatty acids to elevate intracellular [Ca(2+)]. This was also observed when human embryonic kidney (HEK) 293 cells were transfected with a GPR40-Galpha(q) fusion protein. The kinetic of elevation of intracellular [Ca(2+)] slowed with increasing fatty acid chain length, suggesting different ligand on-rates, whereas the addition of fatty acid-free bovine serum albumin reduced signals, presumably by binding the fatty acids. To allow effective ligand equilibration, GPR40-Galpha(q) was used in guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assays. After expression of GPR40-Galpha(q) in HEK293 cells and membrane preparation basal binding of [(35)S]GTPgammaSinGalpha(q) immunoprecipitates was high and not elevated substantially by fatty acids. However, treatment of membranes with fatty acid-free bovine serum albumin reduced the basal [(35)S]GTPgammaS binding in a concentration-dependent manner and allowed the responsiveness and pharmacology at GPR40 of each of the fatty acids thiazolidinediones and a novel small-molecule agonist to be uncovered. Membranes of rat INS-1E cells that express GPR40 endogenously provided similar observations. The high apparent constitutive activity of GPR40-Galpha(q) was also reversed by a small-molecule GPR40 antagonist, and basal [(35)S]GTPgammaS binding was prevented by the selective Galpha(q)/Galpha(11) inhibitor YM-254890. The current studies provide novel insights into the pharmacology of GPR40 and indicate that G protein-coupled receptors which respond to fatty acids, and potentially to other lipid ligands, can be occupied by endogenous agonists before assay and that this may mask the pharmacology of the receptor and may be mistaken for high levels of constitutive activity.
Mol Pharmacol 2007 Apr
PMID:Uncovering the pharmacology of the G protein-coupled receptor GPR40: high apparent constitutive activity in guanosine 5'-O-(3-[35S]thio)triphosphate binding studies reflects binding of an endogenous agonist. 1720 Apr 19

Type 1 diabetes is a metabolic disorder caused by loss of insulin-producing pancreatic beta-cells. Expression of insulin in non-beta-cells to create beta-cell surrogates has been tried to treat type 1 diabetes. Enteroendocrine K cells have characteristics similar to pancreatic beta-cells, such as a glucose-sensing system and insulin-processing proteases. In this study, we genetically engineered an enteroendocrine cell line (STC-1) to express insulin under the control of the glucose-dependent insulinotropic polypeptide promoter. We screened clones and chose one, Gi-INS-7, based on its high production of insulin. Gi-INS-7 cells expressed glucose transporter 2 (GLUT2) and glucokinase (GK) and secreted insulin in response to elevated glucose levels in vitro. To determine whether Gi-INS-7 cells can control blood glucose levels in diabetic mice, we transplanted these cells under the kidney capsule of streptozotocin (STZ)-induced diabetic mice and found that blood glucose levels became normal within 2 weeks of transplantation. In addition, glucose tolerance tests in mice that became normoglycemic after transplantation with Gi-INS-7 cells showed that exogenous glucose was cleared appropriately. These results suggest that engineered K cells may be promising surrogate beta-cells for possible therapeutic use for the treatment of type 1 diabetes.
Mol Ther 2007 Jun
PMID:Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice. 1729 98

Insulin secretory granules (ISGs) are cytoplasmic organelles of pancreatic beta-cells. They are responsible for the storage and secretion of insulin. To date, only about 30 different proteins have been clearly described to be associated with these organelles. However, data from two-dimensional gel electrophoresis analyses suggested that almost 150 different polypeptides might be present within ISGs. The elucidation of the identity and function of the ISG proteins by proteomics strategies would be of considerable help to further understand some of the underlying mechanisms implicated in ISG biogenesis and trafficking. Furthermore it should give the bases to the comprehension of impaired insulin secretion observed during diabetes. A proteomics analysis of an enriched insulin granule fraction from the rat insulin-secreting cell line INS-1E was performed. The efficacy of the fractionation procedure was assessed by Western blot and electron microscopy. Proteins of the ISG fraction were separated by SDS-PAGE, excised from consecutive gel slices, and tryptically digested. Peptides were analyzed by nano-LC-ESI-MS/MS. This strategy identified 130 different proteins that were classified into four structural groups including intravesicular proteins, membrane proteins, novel proteins, and other proteins. Confocal microscopy analysis demonstrated the association of Rab37 and VAMP8 with ISGs in INS-1E cells. In conclusion, the present study identified 130 proteins from which 110 are new proteins associated with ISGs. The elucidation of their role will further help in the understanding of the mechanisms governing impaired insulin secretion during diabetes.
Mol Cell Proteomics 2007 Jun
PMID:Proteomics analysis of insulin secretory granules. 1731 58

Multiple sequence alignment (MSA) is a crucial first step in the analysis of genomic and proteomic data. Commonly occurring sequence features, such as deletions and insertions, are known to affect the accuracy of MSA programs, but the extent to which alignment accuracy is affected by the positions of insertions and deletions has not been examined independently of other sources of sequence variation. We assessed the performance of 6 popular MSA programs (ClustalW, DIALIGN-T, MAFFT, MUSCLE, PROBCONS, and T-COFFEE) and one experimental program, PRANK, on amino acid sequences that differed only by short regions of deleted residues. The analysis showed that the absence of residues often led to an incorrect placement of gaps in the alignments, even though the sequences were otherwise identical. In data sets containing sequences with partially overlapping deletions, most MSA programs preferentially aligned the gaps vertically at the expense of incorrectly aligning residues in the flanking regions. Of the programs assessed, only DIALIGN-T was able to place overlapping gaps correctly relative to one another, but this was usually context dependent and was observed only in some of the data sets. In data sets containing sequences with non-overlapping deletions, both DIALIGN-T and MAFFT (G-INS-I) were able to align gaps with near-perfect accuracy, but only MAFFT produced the correct alignment consistently. The same was true for data sets that comprised isoforms of alternatively spliced gene products: both DIALIGN-T and MAFFT produced highly accurate alignments, with MAFFT being the more consistent of the 2 programs. Other programs, notably T-COFFEE and ClustalW, were less accurate. For all data sets, alignments produced by different MSA programs differed markedly, indicating that reliance on a single MSA program may give misleading results. It is therefore advisable to use more than one MSA program when dealing with sequences that may contain deletions or insertions, particularly for high-throughput and pipeline applications where manual refinement of each alignment is not practicable.
Mol Biol Evol 2007 Nov
PMID:Mind the gaps: evidence of bias in estimates of multiple sequence alignments. 1770 32

Cytokines released by islet-infiltrating immune cells play a crucial role in beta-cell dysfunction and apoptotic cell death in the pathogenesis of type 1 diabetes and after islet transplantation. RNA studies revealed complex pathways of genes being activated or suppressed during this beta-cell attack. The aim of the present study was to analyze protein changes in insulin-producing INS-1E cells exposed to inflammatory cytokines in vitro using two-dimensional DIGE. Within two different pH ranges we observed 2214 +/- 164 (pH 4-7) and 1641 +/- 73 (pH 6-9) spots. Analysis at three different time points (1, 4, and 24 h of cytokine exposure) revealed that the major changes were taking place only after 24 h. At this time point 158 proteins were altered in expression (4.1%, n = 4, p < or = 0.01) by a combination of interleukin-1beta and interferon-gamma, whereas only 42 and 23 proteins were altered by either of the cytokines alone, giving rise to 199 distinct differentially expressed spots. Identification of 141 of these by MALDI-TOF/TOF revealed proteins playing a role in insulin secretion, cytoskeleton organization, and protein and RNA metabolism as well as proteins associated with endoplasmic reticulum and oxidative stress/defense. We investigated the interactions of these proteins and discovered a significant interaction network (p < 1.27e-05) containing 42 of the identified proteins. This network analysis suggests that proteins of different pathways act coordinately in a beta-cell dysfunction/apoptotic beta-cell death interactome. In addition the data suggest a central role for chaperones and proteins playing a role in RNA metabolism. As many of these identified proteins are regulated at the protein level or undergo post-translational modifications, a proteomics approach, as performed in this study, is required to provide adequate insight into the mechanisms leading to beta-cell dysfunction and apoptosis. The present findings may open new avenues for the understanding and prevention of beta-cell loss in type 1 diabetes.
Mol Cell Proteomics 2007 Dec
PMID:Proteomics analysis of cytokine-induced dysfunction and death in insulin-producing INS-1E cells: new insights into the pathways involved. 1792 Nov 77

Human islet transplantation has great potential as an effective means of treating insulin-dependent diabetes mellitus. Upregulation of inducible nitric oxide synthase (iNOS) and subsequent product of radical nitric oxide (NO) impair islet beta-cell function. Therefore, we hypothesize that iNOS gene silencing will prevent beta-cell death and improve the survival and function of islets. Small interfering RNA duplex (siRNA) inhibited rat iNOS gene expression and NO production in rat beta-cell lines (INS-1E) in a dose- and sequence-dependent manner. iNOS gene silencing also protected these beta-cells from inflammatory cytokine-induced apoptosis and increased their capacity to secret insulin. Three siRNA sequences against human iNOS were then designed and transfected into human islets. Although there was dose- and sequence-dependent iNOS gene silencing and NO production in human islets, the effect of iNOS gene silencing on apoptosis of islets was only moderate, as evidenced by 25-30% reduction in caspase 3 activity and in the percentage of apoptotic cells. Since an islet is a cluster of 200-1,000 cells, the transfection efficiency of lipid/siRNA complexes into human islets was only 21-28%, compared to effective transfection efficiency (>90%) in beta-cell lines. Nevertheless, these results suggest that siRNA may penetrate beyond the periphery into a larger percentage of an islet mass than previously thought.
Mol Pharm
PMID:iNOS gene silencing prevents inflammatory cytokine-induced beta-cell apoptosis. 1821 11

In Drosophila melanogaster a partial loss of ribosomal genes leads to the bobbed phenotype. Magnification is a heritable increase in rDNA that may occur in males carrying a deleted X chromosome with a strong bobbed phenotype. The restriction patterns of X chromosome total rDNA, insertions and spacers from magnified bobbed strains were compared with those of the original bobbed mutations. It was found that magnification modifies restriction patterns and differentially affects gene types, increasing specific genes lacking insertions (INS-). Increases in copy number of genes with type I insertions are generally lower than the total number of INS- genes while type II insertion genes are not perceptibly increased. The recovery of homogeneous progeny from a single premagnified male indicates that the magnification event might take place and become stable very early in the germ line, arguing against magnification being due to extrachromosomal amplification. Additionally, some gene types increase 3.5-fold while others are eliminated, indicating that they could not result from a single unequal cross-over. These results are in good agreement with the existence of partial clustering of rDNA genes according to type, and suggest that magnification could result from local amplification of genes.
Mol Gen Genet 1987 Jun
PMID:Differential magnification of rDNA gene types in bobbed mutants of Drosophila melanogaster. 1881 82


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