Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0011849 (diabetes)
 277,896 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutamine:fructose-6-phosphate amidotransferase (GFAT) has recently been shown to be an insulin-regulated enzyme that plays a key role in the induction of insulin resistance in cultured cells. As a first step in understanding the molecular regulation of this enzyme the human form of this enzyme has been cloned and the functional protein has been expressed in Escherichia coli. A 3.1-kilobase cDNA was isolated which contains the complete coding region of 681 amino acids. Expression of the cDNA in E. coli produced a protein of approximately 77 kDa and increased GFAT activity 4.5-fold over endogenous bacterial levels. Recombinant GFAT activity was inhibited 51% by UDP-GlcNAc whereas bacterial GFAT activity was insensitive to inhibition by UDP-GlcNAc. On the basis of these results we conclude that: 1) functional human GFAT protein was expressed, and 2) the cloned human cDNA encodes both the catalytic and regulatory domains of GFAT since the recombinant GFAT was sensitive to UDP-GlcNAc. Overall, the development of cloned GFAT molecular probes should provide new insights into the development of insulin resistance by allowing quantitation of GFAT mRNA levels in pathophysiological states such as non-insulin-dependent diabetes mellitus and obesity.
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PMID:Molecular cloning, cDNA sequence, and bacterial expression of human glutamine:fructose-6-phosphate amidotransferase. 146 20

Confluent human retinal pigmented epithelial cells were cultured on microcarrier beads in the presence of 5.6 or 26 mmol/l glucose with or without the aldose-reductase inhibitor Sorbinil (200 microM) for 2 wk. At the end of the incubation period, perchloric acid extracts were prepared and analyzed by 31P nuclear magnetic resonance spectroscopy. As assessed by this method, the phosphorylated metabolites of cells incubated with 5.6 or 26 mmol/l glucose differed significantly in the concentrations of a number of uridine diphosphate (UDP)-conjugated monosaccharides, which were elevated two- to threefold in cells incubated in 26 mmol/l glucose over control samples. The affected metabolites were identified (through a series of spiking experiments) to be UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, and UDP-glucuronic acid. Coincubation of the cells with Sorbinil 200 microM in the presence of 26 mmol/l glucose had no effect on this accumulation. Under normal circumstances, these molecules selectively and sequentially are incorporated into the polysaccharide chains of glycosaminoglycans (GAGs), whose presence and distribution in the basement membranes is affected adversely by diabetes mellitus. These data suggest that the availability of the monosaccharide precursor is not the rate-limiting step for GAG synthesis in the presence of pathologic glucose concentrations. Thus, the lost GAG content in the basement membranes of diabetic patients may be caused by changes elsewhere in the biosynthesis and/or catabolism of the polysaccharide-linked protein molecules.
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PMID:Human retinal pigment epithelial cells cultured in hyperglycemic media accumulate increased amounts of glycosaminoglycan precursors. 163 9

Optimal assay conditions have been determined in human liver preparations for the catalytic transfer of mannose and N-acetylglucosamine from GDP-mannose and UDP-N-acetylglucosamine, respectively, to dolichyl phosphate. Both enzymatic reactions have an absolute requirement for divalent cation (5 mmol/l Mn2+ optimal), detergent (Triton X-100 or Nonidet P-40) and dolichyl phosphate (as acceptor substrate) and both reactions have optimal activity at a pH value of 7.8. Preliminary characterization of the glycolipid products for both enzymatic reactions indicates that phosphorylated dolichol is the major acceptor substrate for radiolabeled mannose and N-acetylglucosamine. The activity levels and specific activities of dolichyl phosphate-mannosyltransferase are comparable in liver homogenates from normal controls and patients with cystic fibrosis and diabetes mellitus. The activity levels and specific activities of dolichyl phosphate-N-acetylglucosaminyltransferase are comparable in liver homogenates from normal controls and patients with cystic fibrosis and diabetes mellitus but considerably lower than the activity levels of dolichyl phosphate-mannosyltransferase. It appears that two of the initial steps of the lipid-mediated glycosylation pathway are normal in livers from patients with cystic fibrosis and diabetes mellitus.
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PMID:Dolichyl phosphate-mannosyltransferase and dolichyl phosphate-N-acetylglucosaminyltransferase activities in liver preparations from normal controls and patients with cystic fibrosis and diabetes mellitus. 622 43

Primary cardiac abnormalities have been frequently reported in patients with diabetes probably due to metabolic consequences of the disease. Approximately 2,000 mRNA species from the heart of streptozotocin-induced diabetic and control rats were compared by the mRNA differential display method, two of eight candidate clones thus isolated (DH1 and 13) were confirmed by Northern blot analysis. The expression of clone 13 was increased in the heart by 3.5-fold (P < 0.05) and decreased in the aorta by twofold (P < 0.05) in diabetes as compared to control. Sequence analysis showed that clone 13 is a rat mitochondrial gene. DH1 was predominantly expressed in the heart with an expression level 6.8-fold higher in the diabetic rats than in control (P < 0.001). Insulin treatment significantly (P < 0.001) normalized the expression of DH1 in the hearts of diabetic rats. DH1 expression was observed in cultured rat cardiomyocytes, but not in aortic smooth muscle cells or in cardiac derived fibroblasts. The expression in cardiomyocytes was regulated by insulin and glucose concentration of culture media. The full length cDNA of DH1 had a single open-reading frame with 85 and 92% amino acid identity to human and mouse UDP-GlcNAc:Gal beta 1-3GalNAc alpha R beta 1-6 N-acetylglucosaminyltransferase (core 2 GlcNAc-T), respectively, a key enzyme determining the structure of O-linked glycosylation. Transient transfection of DH1 cDNA into Cos7 cells conferred core 2 GlcNAc-T enzyme activity. In vivo, core 2 GlcNAc-T activity was increased by 82% (P < 0.05) in diabetic hearts vs controls, while the enzymes GlcNAc-TI and GlcNAc-TV responsible for N-linked glycosylation were unchanged. These results suggest that core 2 GlcNAc-T is specifically induced in the heart by diabetes or hyperglycemia. The induction of this enzyme may be responsible for the increase in the deposition of glycoconjugates and the abnormal functions found in the hearts of diabetic rats.
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PMID:Identification and characterization of a gene regulating enzymatic glycosylation which is induced by diabetes and hyperglycemia specifically in rat cardiac tissue. 756 67

To test the hypothesis that increased flux through the hexosamine biosynthetic pathway can induce insulin resistance in skeletal muscle in vivo, we monitored glucose uptake, glycolysis, and glycogen synthesis during insulin clamp studies in 6-h fasted conscious rats in the presence of a sustained (7-h) increase in glucosamine (GlcN) availability. Euglycemic (approximately 7 mM) insulin (approximately 2,500 pM) clamps with saline or GlcN infusions were performed in control (CON; plasma glucose [PG] = 7.4 +/- 0.2 mM), diabetic (D; PG = 19.7 +/- 1.1), and phlorizin-treated (3-wk) diabetic rats (D + PHL; PG = 7.6 +/- 0.9). 7-h euglycemic hyperinsulinemia with saline did not significantly decrease Rd (360-420 min = 39.2 +/- 3.6 vs. 60-120 min = 42.2 +/- 3.7 mg/kg.min; P = NS). GlcN infusion raised plasma GlcN concentrations to approximately 1.2 mM and increased muscle and liver UDP-GlcNAc levels by 4-5-fold in all groups. GlcN markedly decreased Rd in CON (360-420 min = 30.4 +/- 1.3 vs. 60-120 min = 44.1 +/- 3.5 mg/kg.min; P < 0.01) and D + PHL (360-420 min = 29.4 +/- 2.5 vs. 60-120 min = 43.8 +/- 2.9 mg/kg.min; P < 0.01), but not in D (5-7 h = 21.5 +/- 0.8 vs. 0-2 h = 24.3 +/- 1.1 mg/kg.min; P = NS). Thus, increased GlcN availability induces severe skeletal muscle insulin resistance in normoglycemic but not in chronically hyperglycemic rats. The lack of additive effects of GlcN and chronic hyperglycemia (experimental diabetes) provides support for the hypothesis that increased flux through the GlcN pathway in skeletal muscle may play an important role in glucose-induced insulin resistance in vivo.
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PMID:In vivo glucosamine infusion induces insulin resistance in normoglycemic but not in hyperglycemic conscious rats. 761 83

Overactivity of the hexosamine pathway mediates glucose-induced insulin resistance in rat adipocytes. Glutamine:fructose-6-phosphate amidotransferase (GFA) is the rate-limiting enzyme of this pathway. We determined GFA activity in human skeletal muscle biopsies and rates of insulin-stimulated whole-body, oxidative, and nonoxidative glucose disposal using the euglycemic insulin clamp technique combined with indirect calorimetry (insulin infusion rate (1.5 mU x kg-1 x min-1)) in 12 male patients with NIDDM (age 54 +/- 2 years, BMI 27.5 +/- 0.9 kg/m2, fasting plasma glucose 8.5 +/- 0.6 mmol/l) and 9 matched normal men. GFA activity was detectable in human skeletal muscles and completely inhibited by uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) in all subjects. GFA activity was 46% increased in the NIDDM patients compared with the normal subjects (9.5 +/- 1.3 vs. 6.5 +/- 1.2 pmol, P < 0.05). Whole-body glucose uptake was 58% decreased in patients with NIDDM (20 +/- 3 micromol x kg body wt-1 x min-1) compared with normal subjects (47 +/- 4 micromol x kg body wt-1 x min-1, P < 0.001). This decrease was attributable to decreases in both glucose oxidation (9 +/- 1 vs. 15 +/- 1 micromol x kg-1 x min-1, NIDDM patients vs. control subjects, P < 0.002) and nonoxidative glucose disposal (11 +/- 2 vs. 31 +/- 4 micromol x kg-1 x min-1, P < 0.001). In patients with NIDDM, both HbA1c (r= 0.51, P < 0.05) and BMI (r= -0.57, P < 0.05) correlated with whole-body glucose uptake. HbA1c but not BMI or insulin sensitivity was correlated with basal GFA activity (r = -0.57,P < 0.01) in NIDDM patients and control subjects. We conclude that GFA is found in human skeletal muscle and that all this activity is sensitive to feedback inhibition by UDP-GlcNAc. Chronic hyperglycemia is associated with an increase in skeletal muscle GFA activity, suggesting that increased activity of the hexosamine pathway may contribute to glucose toxicity and insulin resistance in humans.
Diabetes 1996 Mar
PMID:Increased glutamine:fructose-6-phosphate amidotransferase activity in skeletal muscle of patients with NIDDM. 859 34

Prolonged glucosamine (GlcN) infusion increases the skeletal muscle hexosamine concentration and induces peripheral insulin resistance in conscious rats. IGF-1 and insulin share common steps in signal transduction, and the action of IGF-1 on carbohydrate metabolism is preserved in certain insulin-resistant states. In our study, we attempted to delineate whether increased GlcN availability also impairs the effects of IGF-1 on glucose uptake (Rd), glycolysis, and glycogen synthesis. We performed euglycemic IGF-1 (5 and 15 microg x kg(-1) x min(-1)) and insulin (3 and 18 mU mg x kg(-1) x min(-1)) clamp studies at 0-2 h and 5-7 h in conscious rats (n = 44) during saline or GlcN infusions. GlcN infusion raised plasma GlcN levels to approximately 2.0 mmol/l and skeletal muscle uridinediphospho-n-acetylglucosamine to 80-150 nmol/g (approximately three- to fivefold over basal). During physiological hyperinsulinemia (3 mU x kg(-1) x min(-1), plasma insulin approximately 50 microU/ml), GlcN infusion caused comparable decreases in Rd (15.7 +/- 1.0 [5-7 h] vs. 21.7 +/- 2.3 [0-2 h] mg x kg(-1) x min(-1); P < 0.01) and glycogen synthesis (5.4 +/- 0.5 [5-7 h] vs. 10.4 +/- 1.9 [0-2 h] mg x kg(-1) x min(-1); P < 0.005). Furthermore, GlcN markedly decreased Rd by 7.8 +/- 1.2 mg x kg(-1) x min(-1) (18.7 +/- 0.7 [5-7 h] vs. 26.5 +/- 1.3 [0-2 h] mg x kg(-1) x min(-1); P < 0.001 vs. control) during IGF-1 (5 microg x kg(-1) x min(-1)) clamp studies. This decline was associated with a 26% decrease in the steady-state concentration of skeletal muscle Glc-6-P (286 +/- 45 vs. 386 +/- 36 nmol/g; P < 0.01) and was primarily caused by impaired glycogen synthesis (6.7 +/- 0.5 [5-7 h] vs. 13.9 +/- 0.9 [0-2 h] mg x kg(-1) x min(-1); P < 0.005). The effects of GlcN infusion on glucose disposal (percentage decrease in Rd) were correlated (r2 = 0.803; P < 0.01) with the skeletal muscle concentration of UDP-GlcNAc. To investigate whether IGF-1 can overcome GlcN-induced insulin resistance, GlcN and insulin (18 mU x kg(-1) x min(-1)) were infused for 7 h during euglycemic clamps, and IGF-1 (15 microg x kg(-1) x min(-1)) was superimposed during the final 2 h. GlcN infusion induced severe impairment of insulin action on Rd (39.4 +/- 3.2 [4-5 h] vs. 49.8 +/- 3.6 [1-2 h] mg x kg(-1) x min(-1); P < 0.05), which the addition of IGF-1 failed to improve (35.9 +/- 2.3 [6-7 h] vs. 39.4 +/- 3.2 [4-5 h] mg x kg(-1) x min(-1); P > 0.1). In summary, GlcN induced severe resistance to the actions of both insulin and IGF-1 on glucose uptake and glycogen synthesis, and IGF-1 was unable to overcome GlcN-induced insulin resistance. Thus, it is likely that GlcN causes peripheral insulin resistance acting at a site common to both IGF-1 and insulin signaling pathways.
Diabetes 1996 Dec
PMID:Increased hexosamine availability similarly impairs the action of insulin and IGF-1 on glucose disposal. 892 59

Glutamine:fructose 6-phosphate amidotransferase (GFA) is rate-limiting for hexosamine biosynthesis, while a UDP-GlcNAc beta-N-acetylglucosaminyltransferase (O-GlcNAc transferase) catalyses final O-linked attachment of GlcNAc to serine and threonine residues on intracellular proteins. Increased activity of the hexosamine pathway is a putative mediator of glucose-induced insulin resistance but the mechanisms are unclear. We determined whether O-GlcNAc transferase is found in insulin-sensitive tissues and compared its activity to that of GFA in rat tissues. We also determined whether non-insulin-dependent diabetes mellitus (NIDDM) or acute hyperinsulinaemia alters O-GlcNAc transferase activity in human skeletal muscle. O-GlcNAc transferase was measured using 3H-UDP-GlcNAc and a synthetic cationic peptide substrate containing serine and threonine residues, and GFA was determined by measuring a fluorescent derivative of GlcN6P by HPLC. O-GlcNAc transferase activities were 2-4 fold higher in skeletal muscles and the heart than in the liver, which had the lowest activity, while GFA activity was 14-36-fold higher in submandibular gland and 5-18 fold higher in the liver than in skeletal muscles or the heart. In patients with NIDDM (n = 11), basal O-GlcNAc transferase in skeletal muscle averaged 3.8 +/- 0.3 nmol/mg.min, which was not different from that in normal subjects (3.3 +/- 0.4 nmol/mg.min). A 180-min intravenous insulin infusion (40 mU/m2.min) did not change muscle O-GlcNAc transferase activity in either group. We conclude that O-GlcNAc transferase is widely distributed in insulin-sensitive tissues in the rat and is also found in human skeletal muscle. These findings suggest the possibility that O-linked glycosylation of intracellular proteins is involved in mediating glucose toxicity. O-GlcNAc transferase does not, however, appear to be regulated by either NIDDM or acute hyperinsulinaemia, suggesting that mass action effects determine the extent of O-linked glycosylation under hyperglycaemic conditions.
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PMID:UDP-N-acetylglucosamine transferase and glutamine: fructose 6-phosphate amidotransferase activities in insulin-sensitive tissues. 902 21

In liver cells from diabetic rats, an increased incorporation of labeled glucosamine into cellular and secretory proteins was found, when related to the incorporation of labeled leucine. This increased N-glycosylation was present in the face of decreased synthesis of hepatic cellular and secretory proteins evident from reduced leucine incorporation and diminished glycosyltransferase activity. To elucidate the mechanisms involved we incubated isolated hepatocytes with two N-glycosylation inhibitors: tunicamycin and 2-deoxyglucose. Tunicamycin exerted a marked inhibitory effect on the incorporation rate of labeled glucosamine into proteins in liver cells from diabetic rats, while 2-deoxyglucose had a negligible effect on this process in these cells. These diverse effects might be explained by the fact that tunicamycin acts through strong association with the enzyme catalyzing the first step in glycoprotein synthesis, namely, the transfer of UDP-GlcNAc to dolichol-P (indicating noncompetitive inhibition). This enzyme is reduced in liver cells from diabetic animals. On the other hand, 2-deoxyglucose exerts its effect by being attached to dolichol-P, preventing further elongation of oligosaccharide chain on the protein backbone. This latter effect might be eliminated by excess dolichol-P (indicating competitive inhibition). The dolichol content in liver extract from diabetic rats was about 2.5-fold higher compared with nondiabetic rats (51.6 micrograms/g versus 20.6 micrograms/g wet liver weight). These two lines of evidence confirm the notion that the enhanced enzymatic glycosylation in liver from diabetic animals is maintained by an increased hepatic dolichol concentration, which is most probably related to the hyperglycemia. Thus, the dolichol-N-glycosylation pathway may represent another detrimental aspect of hyperglycemia and may operate by dolichol mass action rather than through glycosylating enzyme activity.
J Diabetes Complications
PMID:Dolichol-mediated enhanced protein N-glycosylation in experimental diabetes--a possible additional deleterious effect of hyperglycemia. 920 1

High intracellular glucose concentrations increase flux though the hexosamine biosynthetic pathway, resulting in elevated UDP-N-acetylglucosamine (GlcNAc) concentrations. The nucleocytoplasmic enzyme O-linked N-acetylglucosaminyltransferase (OGT) uses UDP-GlcNAc as a donor to modify numerous critical substrates, including nuclear pore proteins and transcription factors. Here, we document (a) the overwhelming enrichment of pancreatic OGT transcripts in the beta-cells of the islets of Langerhans, (b) the physiologically significant increase in the level of O-GlcNAc residues present in beta-cells, and (c) the action of streptozotocin, a close analogue of GlcNAc, to selectively inhibit O-GlcNAcase, an enzyme involved in the removal of O-GlcNAc residues. Taken together, these findings suggest that pancreatic beta cells maintain a highly elevated O-GlcNAc metabolism and that the diabetes inducing drug streptozotocin inhibits O-GlcNAcase.
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PMID:Elevated O-linked N-acetylglucosamine metabolism in pancreatic beta-cells. 991 27


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