UMLS:C0011849 - FACTA Search

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Query: UMLS:C0011849 (diabetes)
277,896 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Engineered insulinoma cell lines may represent an alternative to isolated islets for transplantation therapy of type 1 diabetes. Success of this approach may require development of cell lines that can withstand cytokine-mediated damage. To this end, we have cultured INS-1 insulinoma cells in increasing concentrations of interleukin-1beta (IL-1beta) + gamma-interferon (IFN-gamma), with approximate weekly iterations over an 8-week period. Based on the C,N diphenyl-N'-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium+ ++ bromide (MTT) viability assay, the selected cells, termed INS-1res, were 100% viable after 5 days of treatment with 10 ng/ml of IL-1beta. These cells were also 78 +/- 1.2% viable after 5 days of exposure to the combination of 10 ng/ml IL-1beta and 100 U/ml IFN-gamma, whereas parental INS-1 cells treated in the same manner were only 0.3 +/- 0.03% viable. INS-1res cells were also resistant to treatment with supernatants from activated rat peripheral blood mononuclear cells, whereas only 20% of parental INS-1 cells survived such treatment. The resistance to IL-1beta conferred by this procedure was stable, whereas the partial resistance to IFN-gamma was transient but reinducible by culture in the presence of cytokines. Stable transfection of INS-1res cells with a plasmid containing the human insulin cDNA and expansion of the transfected colonies in the absence of cytokines produced cell lines that were on average more resistant to IL-1beta + IFN-gamma (53 +/- 11%) than similarly transfected clones derived from parental INS-1 cells (15 +/- 7%). Importantly, several INS-1res-derived clones retained the capacity to secrete insulin in response to glucose concentrations over the normal physiological range. With regard to the mechanism by which selection was conferred, we found normal levels of IFN-gamma receptor mRNA, but a 60% reduction in expression of the IL-1 receptor type I (IL-1RI) in INS-1res cells compared with parental INS-1 cells. IL-1beta signaling through p38 MAP kinase was found to be normal in INS-1res cells, suggesting that their expression of IL-1RI is sufficient to maintain cytokine action. However, normal IL-1beta-mediated translocation of NF-kappaB and induction of inducible nitric oxide synthase expression and nitric oxide production was severely impaired in the INS-1res cell lines, suggesting a mechanism for the IL-1beta resistance. In sum, this study defines a strategy for isolation of cytokine-resistant beta-cell lines and provides a new system for studying the mechanisms by which such resistance can be achieved.
Diabetes 2000 Apr
PMID:Selection of insulinoma cell lines with resistance to interleukin-1beta- and gamma-interferon-induced cytotoxicity. 1087 Nov 93

Hepatocyte nuclear factors 3 (HNF-3 alpha, -3 beta and -3 gamma) belong to an evolutionarily conserved family of transcription factors that are critical for diverse biological processes such as development, differentiation and metabolism. Gene expression studies have shown that HNF3 proteins are critical regulators of the early-onset type 2 diabetes genes HNF-1 alpha, HNF-4 alpha and IPF-1/PDX-1 (MODY3, 1 and 4, respectively) and of glucagon transcription and pancreatic alpha-cell function. In this study, we investigated whether genetic variation in the genes encoding HNF-3 alpha, HNF-3 beta and HNF-3 gamma predisposes humans to hyperglycemic or hypoglycemic syndromes. In addition, we report the cloning and partial nucleotide sequence of the human HNF-3 alpha, -3 beta and -3 gamma genes. Mutation screening included 96 subjects with type 2 diabetes mellitus, as well as one family with persistent neonatal hypoglycemia. No functional mutations were detected in the coding sequences of the three HNF-3 genes. Our results suggest that mutations in HNF-3 genes are not a common cause of type 2 diabetes mellitus. The data provided will facilitate genetic studies in other populations and will advance our understanding of the role HNF-3 plays in the development of diabetes mellitus and other metabolic disorders of glucose homeostasis.
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PMID:The human HNF-3 genes: cloning, partial sequence and mutation screening in patients with impaired glucose homeostasis. 1089 56

The hypothesis proposing that anaplerosis and cataplerosis play an important role in fuel signaling by providing mitochondrially derived coupling factors for stimulation of insulin secretion was tested. A rise in citrate coincided with the initiation of insulin secretion in response to glucose in INS-1 beta-cells. The dose dependence of glucose-stimulated insulin release correlated closely with those of the cellular contents of citrate, malate, and citrate-derived malonyl-CoA. The glucose-induced elevations in citrate, alpha-ketoglutarate, malonyl-CoA, and the 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium reduction state, an index of beta-cell metabolic activity, were unaffected by the Ca2+ chelator EGTA. Glucose induced a rise in both mitochondrial and cytosolic citrate and promoted efflux of citrate from the cells. The latter amounted to approximately 20% of glucose carbons entering the glycolytic pathway. Phenylacetic acid, a pyruvate carboxylase inhibitor, reduced the glucose-induced rise in citrate in INS-1 cells and insulin secretion in both INS-1 cells and rat islets. The results indicate the feasibility of a pyruvate/citrate shuttle in INS-1 beta-cells, allowing the regeneration of NAD+ in the cytosol and the formation of cytosolic acetyl-CoA, malonyl-CoA, and NADPH. The data suggest that anaplerosis and cataplerosis are early signaling events in beta-cell activation that do not require a rise in Ca2+. It is proposed that citrate is a signal of fuel abundance that contributes to beta-cell activation in both the mitochondrial and cytosolic compartments and that a major fate of anaplerotic glucose carbons is external citrate.
Diabetes 2000 May
PMID:Glucose-regulated anaplerosis and cataplerosis in pancreatic beta-cells: possible implication of a pyruvate/citrate shuttle in insulin secretion. 1090 79

IA-2, a member of the protein tyrosine phosphatase family, represents a major target autoantigen in type 1 diabetes. To study the regulation of IA-2 gene expression, we used INS-1 insulinoma cells to analyze beta-cell signal transduction pathways as well as the effect of metabolic and hormonal factors involved in the regulation of the insulin secretory pathway. Quantitative competitive reverse transcriptase-polymerase chain reaction revealed that an increase of cellular cAMP mediated by forskolin (10 micromol/l, 24 h) or 3-isobutyl-1-methylxanthine (100 micromol/l, 24 h) induced maximal stimulation of IA-2 mRNA levels (451 +/- 85 and 338 +/- 86% compared with basal conditions; P < 0.001). In contrast, activation of protein kinase C (PKC) by short-term treatment with phorbol 12-myristate 13-acetate (PMA) (1 micromol/l, 6 h) did not alter IA-2 expression, whereas depletion of PKC by prolonged culturing (24 h) exerted a significant inhibition (57 +/- 24%; P < 0.05). cAMP-dependent upregulation was confirmed by the findings that glucagon (10 micromol/l, 24-48 h) increased levels of IA-2 mRNA (190 +/- 35%; P < 0.05), whereas short-term incubation with high glucose concentration showed no effect. However, prolonged incubation in high glucose (21 mmol/l) induced a time- and dose-dependent increase of IA-2 mRNA expression, reaching maximal values after 144 h (285 +/- 68%; P < 0.05). These studies demonstrate that stimuli of insulin secretion that operate by activation of adenylate cyclase generating cAMP significantly increase IA-2 gene expression. In contrast, activation of PKC by high glucose concentration or PMA exerted no effect, suggesting that IA-2 gene expression is not simply coupled to insulin secretion, but may be involved in the fine regulation of beta-cell function. These findings may be important to clarify the function of IA-2 in beta-cells and elucidate mechanisms involved in the induction of autoimmunity to IA-2.
Diabetes 2000 Jul
PMID:Regulation of the diabetes-associated autoantigen IA-2 in INS-1 pancreatic beta-cells. 1090 70

Glucagon-like peptide 1 (GLP-1), a hormonal activator of adenyl cyclase, stimulates insulin gene transcription, an effect mediated by the cAMP response element (CRE) of the rat insulin I gene promoter (RIP1). Here we demonstrate that the signaling mechanism underlying stimulatory effects of GLP-1 on insulin gene transcription results from protein kinase A (PKA)-independent activation of the RIP1 CRE. Although GLP-1 stimulates cAMP production in rat INS-1 insulinoma cells, we find accompanying activation of a -410-bp RIP1 luciferase construct (-410RIP1-LUC) to exist independently of this second messenger. GLP-1 produced a dose-dependent stimulation of -410RIP1-LUC (EC50 0.43 nmol/l), an effect reproduced by the GLP-1 receptor agonist exendin-4 and abolished by the antagonist exendin(9-39). Activation of RIP1 by GLP-1 was not affected by cotransfection with dominant-negative Gs alpha, was not blocked by cAMP antagonist Rp-cAMPS, and was insensitive to PKA antagonist H-89. Truncation of -410RIP1-LUC to generate -307-, -206-, and -166-bp constructs revealed 2 segments of RIP1 targeted by GLP-1. The first segment, not regulated by forskolin, was located between -410 and -307 bp of the promoter. The second segment, regulated by both GLP-1 and forskolin, included the CRE and was located between -206 and -166 bp. Consistent with these observations, stimulatory effects of GLP-1 at RIP1 were reduced after introduction of delta-182 and delta-183/180 inactivating deletions at the CRE. The action of GLP-1 at -410RIP1-LUC was also reduced by cotransfection with A-CREB, a genetically engineered isoform of the CRE binding protein CREB, which dimerizes with and prevents binding of basic-region-leucine-zipper (bZIP) transcription factors to the CRE. In contrast, the action of GLP-1 at the CRE was not blocked by cotransfection with M1-CREB, an isoform that lacks a consensus serine residue serving as substrate for PKA-mediated phosphorylation. On the basis of these studies, it is proposed that PKA-independent stimulatory actions of GLP-1 at RIP1 are mediated by bZIP transcription factors related in structure but not identical to CREB.
Diabetes 2000 Jul
PMID:Glucagon-like peptide 1 stimulates insulin gene promoter activity by protein kinase A-independent activation of the rat insulin I gene cAMP response element. 1090 73

The reverse tetracycline-dependent transactivator system was employed in insulinoma INS-1 cells to achieve controlled inducible expression of hepatocyte nuclear factor-1 alpha (HNF1 alpha)-P291fsinsC, the most common mutation associated with subtype 3 of maturity-onset diabetes of the young (MODY3). Nuclear localized HNF1 alpha-P291fsinsC protein exerts its dominant-negative effects by competing with endogenous HNF1 alpha for the cognate DNA-binding site. HNF1 alpha controls multiple genes implicated in pancreatic beta-cell function and notably in metabolism- secretion coupling. In addition to reduced expression of the genes encoding insulin, glucose transporter-2, L-pyruvate kinase, aldolase B and 3-hydroxy-3-methylglutaryl coenzyme A reductase, induction of HNF1 alpha-P291fsinsC also significantly inhibits expression of mitochondrial 2-oxoglutarate dehydrogenase (OGDH) E1 subunit mRNA and protein. OGDH enzyme activity and [(14)C]pyruvate oxidation were also reduced. In contrast, the mRNA and protein levels of mitochondrial uncoupling protein-2 were dramatically increased by HNF1 alpha-P291fsinsC induction. As predicted from this altered gene expression profile, HNF1 alpha-P291fsinsC also inhibits insulin secretory responses to glucose and leucine, correlated with impaired nutrient-evoked mitochondrial ATP production and mitochondrial membrane hyperpolarization. These unprecedented results suggest the molecular mechanism of HNF1 alpha-P291fsinsC causing beta-cell dysfunction.
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PMID:Molecular targets of a human HNF1 alpha mutation responsible for pancreatic beta-cell dysfunction. 1094 8

Non-insulin dependent type 2 diabetes (NIDDM) is a chronic and degenerative disease characterized by elevated glucose serum and the predisposition to the development of vascular complications. In Mexico the incidence of the disease reaches 8%, where one in every ten patients are diagnosed before age 40 (early-onset diabetes). NIDDM is a clinically and genetic heterogeneous entity. Mutations in the glucokinase gene and the genes for the transcription factors HNF-1 alpha, HNF-4 alpha, IPF-1, HNF-1 beta y HNF-3 beta have been demonstrated to cause MODY, a subtype of NIDDM characterized by autosomal dominate pattern of inheritance and an early-onset. Mutations in any of these genes result in deficient insulin synthesis and/or secretion. Five of these genes encode transcription factors that activate the transcription of various genes in pancreatic beta cell including, the insulin gene. Mutations in any of the genes associated to MODY may contribute to the insulin secretion deficiency frequently observed in early-onset type 2 diabetic patients. The structural and functional analysis of these genes, as well as other transcription factors expressed in pancreatic beta cell has allowed their recognition as putative candidate genes involved in the susceptibility to develop the disease.
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PMID:[Genetics of type 2 diabetes mellitus: genes implicated in early onset diabetes]. 1095 13

Mitochondrial glycerol phosphate dehydrogenase (mGPD) is abundant in the normal pancreatic insulin cell, but its level is lowered 50% by diabetes. To evaluate mGPD expression, we cloned and characterized the 5'-flanking region of the human mGPD gene. The gene has two alternative first exons and two promoters. The downstream promoter (B) is 10 times more active than the upstream promoter (A) in insulin-secreting cells (INS-1) and HeLa cells. Promoter B has higher activity in INS-1 than in non-beta cells. Deletion and mutation analysis suggested that a NRF-2 binding site at -94 to -101 and an E2F binding site at -208 to -215 are important regulatory cis elements in promoter B. Gel mobility shift assays indicated that the -94 to -101 region binds the NRF-2 protein. When INS-1 cells were maintained in the presence of high glucose (25 mm) for 7 days, mGPD was the only 1 of 6 enzyme activities lowered (53%). mGPD promoter B activity was reduced by 60% in INS-1 cells by the high glucose, but in HepG2 cells and HeLa cells, promoter B activity was unchanged or slightly increased. Deletion analysis indicated the glucose responsiveness was distributed across the region from -340 to -260 in promoter B. The results indicate that mGPD gene transcription in the beta cell is regulated differently from other cells and that decreased mGPD promoter B transcription is at least in part the cause of the decreased beta cell mGPD levels in diabetes.
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PMID:Functional analysis of two promoters for the human mitochondrial glycerol phosphate dehydrogenase gene. 1095 7

To better understand the action of glucose on fatty acid metabolism in the beta-cell and the link between chronically elevated glucose or fatty acids and beta-cell decompensation in adipogenic diabetes, we investigated whether glucose regulates peroxisomal proliferator-activated receptor (PPAR) gene expression in the beta-cell. Islets or INS(832/13) beta-cells exposed to high glucose show a 60-80% reduction in PPARalpha mRNA expression. Oleate, either in the absence or presence of glucose, has no effect. The action of glucose is dose-dependent in the 6-20 mm range and maximal after 6 h. Glucose also causes quantitatively similar reductions in PPARalpha protein and DNA binding activity of this transcription factor. The effect of glucose is blocked by the glucokinase inhibitor mannoheptulose, is partially mimicked by 2-deoxyglucose, and is not blocked by the 3-O-methyl or the 6-deoxy analogues of the sugar that are not phosphorylated. Chronic elevated glucose reduces the expression levels of the PPAR target genes, uncoupling protein 2 and acyl-CoA oxidase, which are involved in fat oxidation and lipid detoxification. A 3-day exposure of INS-1 cells to elevated glucose results in a permanent rise in malonyl-CoA, the inhibition of fat oxidation, and the promotion of fatty acid esterification processes and causes elevated insulin secretion at low glucose. The results suggest that a reduction in PPARalpha gene expression together with a rise in malonyl-CoA plays a role in the coordinated adaptation of beta-cell glucose and lipid metabolism to hyperglycemia and may be implicated in the mechanism of beta-cell "glucolipotoxicity."
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PMID:Glucose down-regulates the expression of the peroxisome proliferator-activated receptor-alpha gene in the pancreatic beta -cell. 1096 13

Mutations in the HNF4alpha gene are associated with the subtype 1 of maturity-onset diabetes of the young (MODY1), which is characterized by impaired insulin secretory response to glucose in pancreatic beta-cells. Hepatocyte nuclear factor 4alpha (HNF4alpha) is a transcription factor critical for liver development and hepatocyte-specific gene expression. However, the role of HNF4alpha in the regulation of pancreatic beta-cell gene expression and its correlation with metabolism secretion coupling have not been previously investigated. The tetracycline-inducible system was employed to achieve tightly controlled expression of both wild type (WT) and dominant-negative mutant (DN) of HNF4alpha in INS-1 cells. The induction of WT-HNF4alpha resulted in a left shift in glucose-stimulated insulin secretion, whereas DN-HNF4alpha selectively impaired nutrient-stimulated insulin release. Induction of DN-HNF4alpha also caused defective mitochondrial function substantiated by reduced [(14)C]pyruvate oxidation, attenuated substrate-evoked mitochondrial membrane hyperpolarization, and blunted nutrient-generated cellular ATP production. Quantitative evaluation of HNF4alpha-regulated pancreatic beta-cell gene expression revealed altered mRNA levels of insulin, glucose transporter-2, L-pyruvate kinase, aldolase B, 2-oxoglutarate dehydrogenase E1 subunit, and mitochondrial uncoupling protein-2. The patterns of HNF4alpha-regulated gene expression are strikingly similar to that of its downstream transcription factor HNF1alpha. Indeed, HNF4alpha changed the HNF1alpha mRNA levels and HNF1alpha promoter luciferase activity through altered HNF4alpha binding. These results demonstrate the importance of HNF4alpha in beta-cell metabolism-secretion coupling.
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PMID:Hepatocyte nuclear factor 4alpha regulates the expression of pancreatic beta -cell genes implicated in glucose metabolism and nutrient-induced insulin secretion. 1096 20

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