Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting enzyme of gluconeogenesis. This metabolically important enzyme is unique in that it has no known allosteric modifiers, and all of the regulation of its activity is exerted at the level of gene expression. The expression of the PEPCK gene in liver is elevated in most forms of diabetes, and plays a major contributory role in the hyperglycemia characteristic of this disease. In this study, we initiated studies to determine the molecular basis for the increased PEPCK gene expression in diabetes. RNase protection assays of RNA isolated from control, streptozotocin-induced diabetic, and insulin-treated diabetic rat liver indicated that PEPCK mRNA levels are elevated two- to threefold in diabetic rat liver compared to controls. Nuclear run-on assays indicated that the increased PEPCK mRNA levels can be fully accounted for by changes in the transcription rate of the gene. We next initiated characterization of the cAMP response element binding protein (CREB) in diabetic rat liver, since it is known to play a major role in mediating the it is known to play a major role in mediating the basal transcriptional activity of the PEPCK gene as well as the cAMP-dependent stimulation of PEPCK gene transcription, the latter through the phosphorylation of serine 133 of CREB. Western blot analysis of nuclear lysates prepared from rat livers indicated that CREB protein levels in diabetic rat liver nuclei were similar to those of controls. However, using an antibody which specifically recognizes the serine 133-phosphorylated form of CREB, we found that the levels of phospho-CREB were significantly decreased in diabetic rat liver, an effect which insulin treatment reversed. This observation suggests that overexpression of the PEPCK gene in diabetes is not linked to the cAMP signaling system in liver.
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PMID:The phosphorylation state of the cAMP response element binding protein is decreased in diabetic rat liver. 748 14

Insulin-dependent diabetes mellitus (IDDM) is associated with elevated plasma triglyceride levels that normalize after insulin administration. The observation that overexpression of the apoC-III gene in transgenic mice can cause hypertriglyceridemia and other evidence implicating apoC-III in the regulation of triglyceride levels prompted us to examine whether apoC-III might be involved in the hypertriglyceridemia associated with IDDM. To this end, the regulation of apoC-III gene expression was studied in the streptozotocin-treated mouse model of IDDM. In the insulin-deficient diabetic state, these mice have elevated glucose and triglyceride levels and a 1.4- to 1.5-fold increase in hepatic apoC-III mRNA levels, by Northern analysis as well as quantitative solution hybridization RNase protection assay. Insulin treatment normalized the glucose and triglyceride levels and diminished hepatic apoC-III mRNA levels by 59%. Analysis of transcription rates using the nuclear run-on technique demonstrated that the changes in hepatic apoC-III mRNA levels were the results of changes in the transcriptional activity of the gene. To determine the role of insulin in the regulation of apoC-III transcription, HepG2 cells were transfected with an apoC-III reporter construct, and treated with different insulin concentrations. The results demonstrated that insulin treatment induced a dose-dependent down-regulation of apoC-III transcriptional activity. These data suggest that the apoC-III transcriptional changes seen in animals are caused by differences in insulin concentrations. Assuming that apoC-III mRNA levels reflect the synthesis and secretion of the protein, these results present the possibility that overexpression of the apoC-III gene could contribute to the hypertriglyceridemia observed in IDDM.
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PMID:Transcriptional regulation of the apoC-III gene by insulin in diabetic mice: correlation with changes in plasma triglyceride levels. 786 70

The classical mouse fancy Agouti gene is responsible for the wild-type coat color where hairs are banded black and yellow. The Agouti gene encodes a 131-amino-acid secreted protein product that regulates phaeomelanin synthesis by melanocytes in mice. Mice with a dominant mutation at this locus, Ay, develop a yellow coat color, obesity, and diabetes, as the result of a deletion that results in ectopic overexpression of the Agouti gene mRNA in all tissues examined. Obesity and diabetes in Ay mutant mice could be caused by circulation of the protein, or localized action in specific tissues as a paracrine factor acting in cell-cell communication. To test these two possibilities, the Agouti cDNA was overexpressed in the skin of transgenic mice using either the Tyrosinase-Related Protein-1 or the keratin-14 (K14) promoter, the latter with and without an intron. The K14 promoter directed high constitutive levels of expression of Agouti mRNA in the skin, and several lines of transgenic mice exhibited coat colors resembling dominant Agouti allele phenotypes. Two highly expressing K14-Agouti transgenic lines, with light-yellow pelage, were analyzed for obesity and hyperglycemia. The transgenic mice were not significantly different from the controls (P > 0.05), indicating that the Agouti product does not act as an endocrine factor. RNase protection assays revealed a correlation between the levels of dorsal and ventral skin expression with pigmentation/phaeomelanin phenotypes. Co-injection experiments with the Agouti transgenes and other transgenes demonstrated co-integration of the two constructs at the same chromosomal site in approximately 95% of F1 progeny, allowing transgene inheritance to be visibly detected.
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PMID:Overexpression of an Agouti cDNA in the skin of transgenic mice recapitulates dominant coat color phenotypes of spontaneous mutants. 857 18

To determine whether defects of muscle glycogen synthase (GS) activity can be acquired by exposure to elevated glucose or insulin levels, human skeletal muscle cells obtained by needle biopsy from normal control subjects were grown in culture for 4-6 weeks followed by 4 days of fusion and differentiation in media containing either normal (5.5 mmol/l glucose and 22 pmol/l insulin) or increased concentrations of glucose (20 mmol/l), insulin (30 micromol/l), or both. After fusion in normal media, acute stimulation by 33 nmol/l insulin for 1 h increased GS fractional velocity (FV) approximately twofold (from 9.01 +/- 1.26 to 16.31 +/- 2.40, P < 0.05). Increasing the media glucose concentration alone to 20 mmol/l during fusion had no effect on basal FV but caused a marginal impairment of the insulin-stimulated GS response (from 8.51 +/- 1.33 to 12.99 +/- 1.90, P = 0.08). Increasing the media insulin concentration to 30 micromol/l during fusion at 5.5 mmol/l glucose also did not alter basal GS FV (10.61 +/- 1.69%) but completely abolished the normal insulin-stimulated increase in GS activity (to 11.63 +/- 1.55%, NS). The combination of high insulin (30 micromol/l) and high glucose (20 mmol/l) during fusion had no greater effect on the FV of either basal (11.66 +/- 2.16%, NS) or insulin-stimulated (9.20 +/- 1.80%, NS) GS activity than high insulin alone. Fusion in hyperinsulinemic media altered the kinetic parameters of GS with a near doubling of the basal Km0.1 and Vmax0.1 for uridinediphospho-glucose. Hyperinsulinemia also totally prevented the normal insulin-stimulated threefold increase in the Vmax0.1 and the 65% decrease in the A0.5 for glucose-6-phosphate. GS mRNA and protein expression, determined by RNase protection assay and immunoblotting, respectively, were unaffected by changes in media conditions. We conclude that exposure of human skeletal muscle cells primarily to high insulin induces severe insulin resistance through multiple acquired posttranslational defects, which affect both the kinetic characteristics and absolute activity of the GS enzyme.
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PMID:Acquired defects of glycogen synthase activity in cultured human skeletal muscle cells: influence of high glucose and insulin levels. 860 59

Epidemiological studies support the hypothesis that genetic factors modulate the risk for diabetic nephropathy (DN). Aldose reductase (ALDR1), the rate-limiting enzyme in the polyol pathway, is a potential candidate gene. The present study explores the hypothesis that polymorphisms of the (A-C)n dinucleotide repeat sequence, located 2.1 kb upstream of the transcription start site, modulate ALDR1 gene expression and the risk for DN. We conducted studies at two different institutions, the University of New Mexico Health Sciences Center (UNMHSC), and the Istituto Scientifico H San Raffaele (HSR). There were four groups of volunteers at UNMHSC: group I, normal subjects; group II, patients with insulin-dependent diabetes mellitus (IDDM) without DN; group III, IDDM with DN; and group IV, nondiabetics with kidney disease. At HSR we studied volunteers in groups I, II, and III. ALDR1 genotype was assessed by PCR and fluorescent sequencing of the (A-C)n repeat locus, and ALDR1 messenger ribonucleic acid (mRNA) was measured by ribonuclease protection assay in peripheral blood mononuclear cells. At UNMHSC we identified 10 alleles ranging from Z-10 to Z+8. The prevalence of the Z-2 allele among IDDM patients was increased in those with DN. Sixty percent of group III and 22% of group II were homozygous for Z-2. Moreover, 90% and 67% of groups III and II, respectively, had 1 or more copy of Z-2. In contrast, among nondiabetics, 19% of group IV and 3% of group I were homozygous for Z-2, and 69% and 32%, respectively, had 1 copy or more of Z-2. Among diabetics, homozygosity for the Z-2 allele was associated with renal disease [odds ratio (OR), 5.25; 95% confidence interval, 1.71-17.98; P = 0.005]. ALDR1 mRNA levels were higher in patients with DN (group III; 0.113 +/- 0.050) than in group I (0.068 +/- 0.025), group II (0.042 +/- 0.020), or group IV (0.015 +/- 0.011; P < 0.01). Among diabetics, ALDR1 mRNA levels were higher in Z-2 homozygotes (0.098 +/- 0.06) and Z-2 heterozygotes (0.080 +/- 0.04) than in patients with no Z-2 allele (0.043 +/- 0.02; P < 0.05). In contrast, among nondiabetics, ALDR1 mRNA levels in Z-2 homozygotes (0.034 +/- 0.04) and Z-2 heterozygotes (0.038 +/- 0.03) were similar to levels in patients without a Z-2 allele (0.047 +/- 0.03; P = NS). At HSR we identified eight alleles ranging from Z- 12 to Z+2. The prevalence of the Z-2 allele was higher in group III than in group II. In group III, 43% of the patients were homozygous for Z-2, and 81% had one copy or more of the Z-2 allele. In contrast, in group II, 4% were homozygous for Z-2, and 36% had one copy or more of the Z-2 allele. IDDM patients homozygous for Z-2 had an increased risk for DN compared with those lacking the Z-2 allele (OR, 18; 95% confidence interval, 2-159). IDDM patients who had one copy or more of Z-2 had increased risk (OR, 7.5; 95% confidence interval, 1.9-29.4) for DN compared with those without the Z-2 allele. These results support our hypothesis that environmental-genetic interactions modulate the risk for DN. Specifically, the Z 2 allele, in the presence of diabetes and/or hyperglycemia, is associated with increased ALDR1 expression. This interaction may explain the observed association between the Z-2 allele and DN.
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PMID:Z-2 microsatellite allele is linked to increased expression of the aldose reductase gene in diabetic nephropathy. 970 64

P:eroxisome proliferator-activated receptor-gamma (PPARgamma) is a novel nuclear receptor, which enhances insulin-mediated glucose uptake. Ligands to PPARgamma are currently used as therapy for type II diabetes. Using Western blot analysis, RNase protection assay, and immunostaining, we identified the presence of PPARgamma message and protein in cultured primary rat mesangial cells. Electrophoretic mobility of a labeled PPARgamma response element (PPRE) was retarded in the presence of mesangial cell nuclear extract, suggesting that PPARgamma is functional in these cells. The addition of unlabeled PPRE efficiently competed away the PPARgamma-PPRE protein complex, confirming specificity of binding of the PPARgamma to the PPRE. PPARgamma ligands rosiglitazone (1 to 10 micromol/L) and troglitazone (1 to 10 micromol/L) inhibited platelet-derived growth factor-induced DNA synthesis, measured as bromodeoxyuridine incorporation (P<0.01). This inhibition was dose dependent. When administered in antidiabetic doses to streptozotocin-induced diabetic rats, troglitazone substantially normalized albumin excretion at 3 months (from 687.1 to 137.6 microgram urinary albumin/mg creatinine, P:<0.05) but did not affect hyperglycemia or blood pressure in this model. This treatment also decreased glomerular plasminogen activator inhibitor-1 (PAI-1) expression. These data suggest that PPARgamma activation may directly attenuate diabetic glomerular disease, possibly by inhibiting mesangial growth, which occurs early in the process of diabetic nephropathy, or by inhibiting PAI-1 expression. PAI-1 inhibits the activation of plasmin and matrix metalloproteinase, which degrade extracellular matrix in the glomerulus. Excess glomerular PAI-1 allows the accumulation of extracellular matrix, leading to glomerulosclerosis. These results have therapeutic implications for diabetic nephropathy as well as for proliferative mesangial diseases of the kidney.
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PMID:Expression and function of peroxisome proliferator-activated receptor-gamma in mesangial cells. 1123 Mar 63

Organisms respond to infection in a complex manner involving bidirectional interactions between the neuroendocrine and immune systems. Many of the bioactive endocrine/immune factors are synthesized in a precursor form and are expected to be activated by prohormone convertases (PCs). Since patients with both type 1 and type 2 diabetes have an increased incidence and severity of infections, we hypothesized that in a condition of hyperglycemia, these processing enzymes would be activated in an immune tissue, the spleen. To test this hypothesis, we treated rats with intraperitoneal streptozotocin (STZ) (50 mg/kg/day) daily for 5 days and measured splenic PC1 and PC2 mRNA by ribonuclease protection assay. We found that PC1 mRNA was increased 6.0+/-0.02-fold (P<0.05) and PC2 mRNA was increased 1.80+/-0.01-fold (P<0.005) in the spleen of rats that received STZ compared to rats that received vehicle. Western blot indicated that the 75-kDa form of PC1 was the only form of PC1 present in the spleen and that this form increased with STZ treatment. Immunohistochemistry revealed that PC1 was found in both the white pulp (T-lymphocytes) and red pulp (monocytes and macrophages) and that its increase in immunoreactivity occurred primarily in the white pulp. PC2 and pro-opiomelanocortin (POMC, a possible splenic substrate for PC1/PC2) immunoreactivity was found predominantly in the red pulp. STZ induced an increase in splenic PC1 and POMC, but not PC2 protein levels. We conclude that in the STZ model of diabetes, splenic PCs are induced, which could lead to an increased activation of many immune-derived hormones. We speculate that this up-regulation of prohormone converting enzymes may be related to the increased infections seen in patients with both type 1 and type 2 diabetes.
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PMID:Up-regulation of splenic prohormone convertases PC1 and PC2 in diabetic rats. 1173 Sep 86

Although kinins have been associated with the regulation of cardiovascular function in left ventricular hypertrophy (LVH) as a consequence of hypertension, myocardial infarction (MI), and/or diabetic cardiomyopathy, less is known about their receptor regulation under these conditions. We have therefore investigated the bradykinin B1-receptor (B1R) and B2-receptor (B2R) mRNA expression in rat models of MI, LVH and diabetes mellitus (DM). Sprague-Dawley rats (SD) were submitted to permanent ligation of the left descending coronary artery (LAD) to induce a MI, whereas DM was induced by a single injection of streptozotocin (STZ). LVH was induced after thoracic aortic banding (AB). Three weeks after MI, six weeks after STZ injection or six weeks after AB, left ventricular (LV) function was characterized using a Millar-tip catheter. Cardiac B1R- and B2R-mRNA expression were analyzed by specific RNase-protection assays (RPA). LV contractility (dP/dt max) was impaired by 40-48% in rats after induction of MI or DM compared to their controls. However, despite an enormous increase in LV end-diastolic pressure (LEVDP) to 310% after AB, LV contractility did not differ compared to the controls. These hemodynamic changes were accompanied by an up-regulation of cardiac B1R- (MI, 288%; STZ, 215%; AB, 4180%) and B2R-mRNA expression (MI, 122%; STZ, 288%; AB, 96%). Up-regulation of both BK-receptor (BKR) types in early stages of cardiac wound healing induced by ischemia and in chronic stages of cardiac remodeling induced by pressure-overload or by hyperglycemia indicates that kinins play a major role in the complex processes of cardiac tissue injury and repair.
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PMID:Regulation of cardiac bradykinin B1- and B2-receptor mRNA in experimental ischemic, diabetic, and pressure-overload-induced cardiomyopathy. 1248 96

The presence of advanced glycation end products (AGEs) formed because of hyperglycemia in diabetic patients has been strongly linked to the development of diabetic complications and disturbances in cellular function. In this report, we describe the isolation and identification of novel AGE-binding proteins from diabetic rat kidneys. The proteins were purified by cation exchange and AGE-modified bovine serum albumin (AGE-BSA) affinity chromatography. NH2-terminal and internal sequencing identified the proteins as the NH2-terminal domains of ezrin, radixin, and moesin (ERM proteins). Using BIAcore biosensor analysis, human N-ezrin-(1-324) bound to immobilized AGE-BSA with a KD of 5.3 +/- 2.1 x 10 -7 m, whereas full-length ezrin-(1-586) and C-ezrin-(323-586) did not bind. Other glycated proteins such as AGE-RNase, N in -carboxymethyllysine (CML)-BSA, and glycated human serum albumin isolated from hyperglycemic diabetic sera competed with the immobilized AGE-BSA for binding to N-ezrin, but non-glycated BSA and RNase did not. Thus N-ezrin binds to AGEs in a glycation- and concentration-dependent manner. Phosphorylated ezrin plays a crucial role in cell shape changes, cell attachment, and cell adhesion. The effect of AGE-BSA on ezrin function was studied in a tubulogenesis model in which LLC-PK1 cell tubule formation is dependent on phosphorylated ezrin. Addition of AGE-BSA completely inhibited the ability of the cells to produce tubules. Furthermore, in vitro tyrosine phosphorylation of N-ezrin and ezrin was also inhibited by AGE-BSA. These proteins represent a novel family of intracellular binding molecules for glycated proteins and provide a potential new target for therapeutic intervention in the prevention or treatment of diabetic complications.
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PMID:The amino-terminal domains of the ezrin, radixin, and moesin (ERM) proteins bind advanced glycation end products, an interaction that may play a role in the development of diabetic complications. 1273 2

Glucose uptake into adipose and liver cells is known to up-regulate mRNA levels for various lipogenic enzymes such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC). To determine whether the hexosamine biosynthesis pathway (HBP) mediates glucose regulation of mRNA expression, we treated primary cultured adipocytes for 18 h with insulin (25 ng/ml) and either glucose (20 mm) or glucosamine (2 mm). A ribonuclease protection assay was used to quantitate mRNA levels for FAS, ACC, and glycerol-3-P dehydrogenase (GPDH). Treatment with insulin and various concentrations of d-glucose increased mRNA levels for FAS (280%), ACC (93%), and GPDH (633%) in a dose-dependent manner (ED50 8-16 mm). Mannose similarly elevated mRNA levels, but galactose and fructose were only partially effective. l-glucose had no effect. Omission of glutamine from the culture medium markedly diminished the stimulatory effect of glucose on mRNA expression. Since glutamine is a crucial amide donor in hexosamine biosynthesis, we interpret these data to mean that glucose flux through the HBP is linked to regulation of lipogenesis through control of gene expression. Further evidence for hexosamine regulation was obtained using glucosamine, which is readily transported into adipocytes where it directly enters the HBP. Glucosamine was 15-30 times more potent than glucose in elevating FAS, ACC, and GPDH mRNA levels (ED50 approximately 0.5 mm). In summary: 1) GPDH, FAS, and ACC mRNA levels are upregulated by glucose; 2) glucose-induced up-regulation requires glutamine; and 3) mRNA levels for lipogenic enzymes are up-regulated by glucosamine. Hyperglycemia is the hallmark of diabetes mellitus and leads to insulin resistance, impaired glucose metabolism, and dyslipidemia. We postulate that disease pathophysiology may have a common underlying factor, excessive glucose flux through the HBP.
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PMID:Role of hexosamine biosynthesis in glucose-mediated up-regulation of lipogenic enzyme mRNA levels: effects of glucose, glutamine, and glucosamine on glycerophosphate dehydrogenase, fatty acid synthase, and acetyl-CoA carboxylase mRNA levels. 1275 50


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