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PubMed1991

Coordinated regulation of glutamine:fructose-6-phosphate amidotransferase activity by insulin, glucose, and glutamine. Role of hexosamine biosynthesis in enzyme regulation.

We reported previously that glutamine:F-6-P amidotransferase (GFAT) plays an integral role in the development of insulin resistance by directing the flow of incoming glucose into the hexosamine biosynthesis pathway. To determine whether the enzymatic activity of GFAT is altered during desensitization of the glucose transport system, we treated isolated rat adipocytes with various combinations of insulin, glucose, and glutamine. Treatment with insulin or glucose alone (or in combination) failed to reduce cytosolic GFAT activity after 4 h, whereas combined treatment with all three components elicited a progressive loss of GFAT activity that was rapid (t1/2 of 2 h) and extensive (70% loss). A pronounced loss of GFAT activity was also seen in cells exposed to glucosamine, an agent known to directly enter the hexosamine pathway (55% loss at 4 h, ED50 of 360 microM). Moreover, a close correlation was observed between the induction of desensitization and the loss of GFAT activity as a function of glucose, insulin, glutamine, and glucosamine concentrations. When total intracellular hexosamine products were measured, we found that hexosamine formation was unaltered by insulin or glucose (or a combination) but was elevated by greater than 4-fold in the presence of insulin, glucose, and glutamine (t1/2 of 22 min), a condition known to cause both desensitization and loss of GFAT activity. Additional studies indicated that the loss of GFAT activity under desensitizing conditions is not due to allosteric regulation since removal of potential allosteric factors from the cytosol of desensitized cells by G-25 column chromatography failed to restore enzyme activity. Overall, these studies indicate that 1) GFAT is an insulin-regulated enzyme; however, control of enzyme activity is not due to a direct action of insulin, but rather is mediated by insulin-induced enhancement of glucose uptake; 2) the routing of incoming glucose through the hexosamine pathway and the formation of hexosamine products appears to regulate GFAT activity; and 3) the progressive loss of GFAT activity over several hours is probably not due to allosteric regulation.

Adipose Tissue↗

PubMed1980

Studies of the intercellular matrix of growth plates from dwarf and homozygous nonaffected Alaskan Malamutes: collagen and hexosamine.

This study was performed to compare the extractability of dwarf growth plate collagen and hexosamine and that of homozygous nonaffected Malamutes and to measure the activity of three of the enzymes involved in the post-translational modifications of the collagen molecule. No significant differences were found in the activity of prolyl hydroxylase or lysyl oxidase in the dwarf growth plates. Lysyl hydroxylase activity in the dwarf was decreased to 22% and 33% that of the activity present in the homozygous nonaffected growth plates. Amino acid analysis of the collagen isolated from dwarf growth plates failed to reveal any decrease in hydroxylysine content. Growth plates were extracted with either 1 M sodium chloride or 4 M guanidine hydrochloride. The extracts were applied to a DEAE-cellulose column. Amino acid analyses of the material which did not bind to DEAE revealed a slight decrease in the amount of guanidine-extractable hydroxyproline in the dwarf but a 60-fold increase in the amount of salt-extractable hydroxyproline in the dwarf growth plates. Material which eluted with 1 M sodium choloride was analyzed for hexosamine. There was a 10-fold increase in the amount of salt-extractable hexosamine present in the dwarf growth plates, whereas no significant differences were observed in the guanidine-extracted material. Hexosamine analysis of the growth plates revealed a significant increase in the total amount of hexosamine present in the dwarf growth plates. SDS-polyacrylamide gels of the material which did not bind to DEAE as well as the pepsin digested, 0.9M sodium chloride precipitated collagen demonstrated the presence of only type II collagen.

Animals↗

PubMed2005

Acid hydrolysis and quantitative determination of total hexosamines of an exopolysaccharide produced by Citrobacter sp.

During the hydrolysis of an exopolysaccharide (EPS) produced by Citrobacter sp., the maximum liberation of hexosamine was obtained with 6 M HCl at 115 degrees C in an autoclave for 1 h. The glycosidic bond energy and degree of acetylation of the hexosamine in EPS were approximately 77 kJ mol(-1) and 61%, respectively. Thermal destruction of the hexosamines and the effect of salt on the hexosamine determination could be minimized under the optimized hydrolytic conditions. Using a modified Elson-Morgan method, maximum total hexosamine concentration was determined to be 3.2 g l(-1) (29% of crude EPS) after 96 h of fed-batch culture.

Acids↗

PubMed1987

Quantitative gas chromatographic measurement of glycosaminoglycan hexosamines in urine and plasma.

A method is described for the quantitative determination of urine and plasma glycosaminoglycans (GAGs) by gas chromatography of the acetylated amino sugars. GAGs were first recovered by precipitation from urine with alkyltrimethylammonium bromide and from plasma by mini-column chromatography after papain digestion. Urine samples (24) analysed for total hexosamines by gas chromatography and for uronic acid by colorimetry had a correlation coefficient of 0.85. The within-run coefficient of variation (C.V.) for nineteen samples from a pooled urine was 5.2% for total hexosamines and that for the ratio of galactosamine to total hexosamines was 3.7%. The corresponding C.V. values for twelve plasma samples from a common pool were 6.5 and 3.7%. The mean ratio of galactosamine to total hexosamine in ten pre-breakfast spot urines was 51.5%. The corresponding ratio in the plasma from twenty adolescent blood donors was 76.3% and the mean total hexosamine content of the GAGs was 47.36 mumol/l.

Adult↗

PubMedDate not supplied

Glucose-induced insulin resistance of phosphatidylinositol 3'-OH kinase and AKT/PKB is mediated by the hexosamine biosynthesis pathway.

Hyperglycemia is responsible for many of the vascular complications and metabolic derangements seen in diabetes. One potential regulator of the effects of glucose is the hexosamine biosynthesis pathway (HBP). Glutamine: fructose-6-phosphate amidotransferase (GFA), the first and rate-limiting enzyme in this pathway, catalyzes the transfer of an amino group from glutamine to fructose-6-phosphate to form glucosamine-6-phosphate. Overexpression of GFA in rat-1 fibroblasts results in insulin resistance for glycogen synthase (GS) activity, and renders these cells more sensitive to the effects of glucose. Using rat-1 cells, we examine further the mechanisms whereby hexosamines lead to insulin resistance. Insulin stimulated GS activity was found to occur via a PI-3 kinase (PI-3K)-dependent pathway as wortmannin, an inhibitor of PI-3K, blocked insulin's ability to stimulate GS activity. Subsequently, we examined the effects of hexosamines on PI-3K and Akt/PKB activity. Cells were cultured in 1 mM glucose (low glucose, LG), 20 mM glucose (high glucose, HG), or 1 mM glucose plus 3 mM glucosamine (GlcN) for 16--20 h. After treatment with insulin (100 nM) for 5 min, cell extracts were assayed for IRS-1 associated and total PI-3K activity. At LG, insulin increased PI-3K activity by 43%. There was no insulin stimulation of PI-3K activity in cells cultured in HG or GlcN. There was a trend for IRS-1 protein levels to decrease in HG but not GlcN. PI-3K protein levels were not altered by HG or GlcN. Finally PKB activity was assayed. At LG, insulin stimulated PKB activity. Again, both HG and GlcN significantly reduced insulin's ability to stimulate PKB activity. We conclude that the hexosamine-mediated insulin resistance of GS activity seen in rat-1 cells is mediated by hexosamine regulation of PI-3K and PKB.

Animals↗

PubMedDate not supplied

Hexosamines as mediators of nutrient sensing and regulation in diabetes.

High concentrations of glucose induce insulin resistance, impair insulin secretion, and affect hepatic glucose production in a manner that mirrors Type 2 diabetes, and hexosamines mimic many of these effects. This has led to the hypothesis that cells use hexosamine flux as a glucose- and satiety-sensing pathway. The hexosamine hypothesis for glucose sensing has been validated by overexpressing the rate-limiting enzyme for hexosamine synthesis, glutamine: fructose-6-phosphate amidotransferase (GFA) in several tissues including muscle, liver, fat, and beta cells. With overexpression of GFA in transgenic animals, skeletal muscle becomes insulin resistant, the liver synthesizes excess fatty acid, and the beta cell secretes excess insulin leading to hyperinsulinemia. Thus, excess hexosamine flux leads to a coordinated response whereby fuel is shunted toward long-term storage, mirroring the "thrifty phenotype." Chronically, however, these same adaptive changes result ultimately in obesity, hyperlipidemia, beta cell failure, and Type 2 diabetes. These results suggest a mechanism by which chronic overnutrition leads to the phenotype of Type 2 diabetes.

Animals↗

PubMed2000

Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation.

The hexosamine pathway has been implicated in the pathogenesis of diabetic complications. We determined first that hyperglycemia induced a decrease in glyceraldehyde-3-phosphate dehydrogenase activity in bovine aortic endothelial cells via increased production of mitochondrial superoxide and a concomitant 2.4-fold increase in hexosamine pathway activity. Both decreased glyceraldehyde-3-phosphate dehydrogenase activity and increased hexosamine pathway activity were prevented completely by an inhibitor of electron transport complex II (thenoyltrifluoroacetone), an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenylhydrazone), a superoxide dismutase mimetic [manganese (III) tetrakis(4-benzoic acid) porphyrin], overexpression of either uncoupling protein 1 or manganese superoxide dismutase, and azaserine, an inhibitor of the rate-limiting enzyme in the hexosamine pathway (glutamine:fructose-6-phosphate amidotransferase). Immunoprecipitation of Sp1 followed by Western blotting with antibodies to O-linked GlcNAc, phosphoserine, and phosphothreonine showed that hyperglycemia increased GlcNAc by 1.7-fold, decreased phosphoserine by 80%, and decreased phosphothreonine by 70%. The same inhibitors prevented all these changes. Hyperglycemia increased expression from a transforming growth factor-beta(1) promoter luciferase reporter by 2-fold and increased expression from a (-740 to +44) plasminogen activator inhibitor-1 promoter luciferase reporter gene by nearly 3-fold. Inhibition of mitochondrial superoxide production or the glucosamine pathway prevented all these changes. Hyperglycemia increased expression from an 85-bp truncated plasminogen activator inhibitor-1 (PAI-1) promoter luciferase reporter containing two Sp1 sites in a similar fashion (3.8-fold). In contrast, hyperglycemia had no effect when the two Sp1 sites were mutated. Thus, hyperglycemia-induced mitochondrial superoxide overproduction increases hexosamine synthesis and O-glycosylation of Sp1, which activates expression of genes that contribute to the pathogenesis of diabetic complications.

Acetylglucosamine↗

PubMed2001

Activation of the hexosamine pathway leads to deterioration of pancreatic beta-cell function through the induction of oxidative stress.

It is known well that activation of the hexosamine pathway causes insulin resistance, but how this activation influences pancreatic beta-cell function remains unclear. In this study, we found that in isolated rat islets adenovirus-mediated overexpression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the first and rate-limiting enzyme of the hexosamine pathway, leads to deterioration of beta-cell function, which is similar to that found in diabetes. Overexpression of GFAT or treatment with glucosamine results in impaired glucose-stimulated insulin secretion and reduction in the expression levels of several beta-cell specific genes (insulin, GLUT2, and glucokinase). Additionally, the DNA binding activity of PDX-1, an important transcription factor for these three genes, was markedly reduced. These phenomena were not mimicked by the induction of O-linked glycosylation with an inhibitor of O-GlcNAcase, PUGNAc. It was also found that glucosamine increases hydrogen peroxide levels and that several hexosamine pathway-mediated changes were suppressed by treatment with the antioxidant N-acetyl-l-cysteine. In conclusion, activation of the hexosamine pathway leads to deterioration of beta-cell function through the induction of oxidative stress rather than O-linked glycosylation. Thus, the hexosamine pathway may contribute to the deterioration of beta-cell function found in diabetes.

Animals↗

PubMed2002

Hexosamines regulate leptin production in 3T3-L1 adipocytes through transcriptional mechanisms.

This study was undertaken to examine the regulation of leptin gene (LEP) transcription and leptin release by hexosamines in 3T3-L1 adipocytes. Glucosamine (1 mM), an intermediate in hexosamine biosynthesis, increased leptin release to 117.0 +/- 7.3% (P = 0.0430; n = 9) and 134.6 +/- 6.5% of the control value (P = 0.0367; n = 4) by 48 and 96 h, respectively. With 0.01 mM glucosamine, leptin release was increased to 120.0 +/- 3.0% of the control value (P = 0.0069; n = 4) by 96 h of treatment. Glucose at 5 and 20 mM stimulated leptin release to 759 +/- 227% and 1104 +/- 316% of the control value over the 96-h culture period. Inhibition of hexosamine biosynthesis with 6-diazo-5-oxonorleucine (20 microM) reduced glucose-stimulated leptin release 13 +/- 2.3% and 29.9 +/- 6.6% at 24 and 96 h, respectively (n = 4; P < 0.05). A 24-h incubation in 5 mM glucose significantly increased (163.0 +/- 19.3%; n = 7) the activity of a human LEP promoter electroporated into differentiated 3T3-L1 cells. Glucosamine (1 mM; 48 h) also increased LEP promoter activity 170.0 +/- 13.0% (n = 5). Mutation of the three Sp1 binding sites in the LEP construct significantly reduced promoter activity. However, glucose (5 mM; 24 h) and glucosamine (1 mM; 48 h) increased the activity of the mutated promoter to 165 +/- 40% (n = 8) and 143 +/- 13% of the control value (n = 8). Glucosamine significantly increased O-glycosylation of Sp1 by 16.1 +/- 4.5% (P = 0.0305; n = 3). These data demonstrate that glucose and hexosamines regulate leptin production through transcriptional mechanisms localized to the proximal portion of the LEP promoter. Hexosamine-mediated regulation of LEP gene expression does not depend on Sp1 binding to traditional sites on the promoter.

3T3 Cells↗

PubMed2000

Transgenic mice with increased hexosamine flux specifically targeted to beta-cells exhibit hyperinsulinemia and peripheral insulin resistance.

Hexosamines have been shown to mediate effects of hyperglycemia and so-called "glucose toxicity" in insulin-sensitive tissues. To determine the effects of hexosamines on insulin synthesis and secretion, transgenic mice were created to overexpress the rate-limiting enzyme for hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), specifically in beta-cells. GFA activity in islets of heterozygous transgenic mice was elevated 76% compared with littermate controls. The increased GFA activity led to 1.4- and 2.1-fold increased pancreatic insulin content in 2- and 10-month-old transgenic mice, respectively (P < 0.005). Fasting insulin levels were 1.6-fold higher than in littermate controls (P < 0.05). Hyperinsulinemia was evident despite a 28% reduction in insulin mRNA levels. The fasting glucose levels in the transgenic mice equaled that of controls aged 2-4 months but exceeded that of the controls aged 6-10 months (means +/- SE 6.9 +/- 0.2 vs. 5.9 +/- 0.2 mmol/l, P < 0.001). By 8 months, the males were overweight and mildly diabetic (fasting glucose 8.8 +/- 0.5 mmol/l) despite persistent hyperinsulinemia. Insulin resistance was confirmed in both males and females using the euglycemic-hyperinsulinemic clamp technique; glucose disposal rates decreased by 48% in transgenic mice (P < 0.01). Triglyceride levels did not differ, and free fatty acid levels were lower in the transgenic animals. ATP levels were unchanged in the transgenic islets. We conclude that hexosamine biosynthesis is involved in the regulation of insulin content in beta-cells by glucose. Increased hexosamine flux in the beta-cell results in hyperinsulinemia, insulin resistance, and (in males) mild type 2 diabetes.

Animals↗

PubMed1991

Discovery of a metabolic pathway mediating glucose-induced desensitization of the glucose transport system. Role of hexosamine biosynthesis in the induction of insulin resistance.

Based on our previous finding that desensitization of the insulin-responsive glucose transport system (GTS) requires three components, glucose, insulin, and glutamine, we postulated that the routing of incoming glucose through the hexosamine biosynthesis pathway plays a key role in the development of insulin resistance in primary cultured adipocytes. Two approaches were used to test this hypothesis. First, we assessed whether glucose-induced desensitization of the GTS could be prevented by glutamine analogs that irreversibly inactivate glutamine-requiring enzymes, such as glutamine:fructose-6-phosphate amidotransferase (GFAT) the first and the rate-limiting enzyme in hexosamine biosynthesis. Both O-diazoacetyl-L-serine (azaserine) and 6-diazo-5-oxonorleucine inhibited desensitization in 18-h treated cells without affecting maximal insulin responsiveness in control cells. Moreover, close agreement was seen between the ability of azaserine to prevent desensitization of the GTS in intact adipocytes (70% inhibition, ED50 = 1.1 microM), its ability to inactivate GFAT in intact adipocytes (64% inhibition, ED50 = 1.0 microM) and its ability to inactivate GFAT activity in a cytosolic adipocyte preparation (ED50 = 1.3 microM). From these results we concluded that a glutamine amidotransferase is involved in the induction of insulin resistance. As a second approach, we determined whether glucosamine, an agent known to preferentially enter the hexosamine pathway at a point distal to enzymatic amidation by GFAT, could induce cellular insulin resistance. When adipocytes were exposed to various concentrations of glucosamine for 5 h, progressive desensitization of the GTS was observed (ED50 = 0.36 mM) that culminated in a 40-50% loss of insulin responsiveness. Moreover, we estimated that glucosamine is at least 40 times more potent than glucose in mediating desensitization, since glucosamine entered adipocytes at only one-quarter of the glucose uptake rate, yet induced desensitization at an extra-cellular dose 10 times lower than glucose. In addition, we found that glucosamine-induced desensitization did not require glutamine and was unaffected by azaserine treatment. Thus, we conclude that glucosamine enters the hexosamine-desensitization pathway at a point distal to GFAT amidation. Overall, these studies indicate that a unique metabolic pathway exists in adipocytes that mediates desensitization of the insulin-responsive GTS, and reveal that an early step in this pathway involves the conversion of fructose 6-phosphate to glucosamine 6-phosphate by the first and rate-limiting enzyme of the hexosamine pathway, glutamine:fructose-6-phosphate amidotransferase.

Animals↗

PubMed1998

The relationship between intracellular UDP-N-acetyl hexosamine nucleotide pool and monoclonal antibody production in a mouse hybridoma.

The effect of intracellular UDP-N-acetyl hexosamine accumulation in a murine hybridoma was investigated using tunicamycin and ammonium chloride. The treatment of cells with tunicamycin resulted in the inhibition of glycosylation of the secreted monoclonal antibody (IgGl) and cell growth arrest. Tunicamycin concentrations of 0.01-0.1 microgram ml-1 resulted in the formation of both glycosylated and non-glycosylated heavy chains of the immunoglobulin, whereas complete inhibition of protein glycosylation was observed at higher concentrations of tunicamycin. Tunicamycin treatment also resulted in a dose dependent accumulation of UDP-N-acetyl hexosamine. It was concluded that the specific monoclonal antibody production rate (qMab) was not dependent on the extent of glycosylation. Treatment of cells with NH4Cl also resulted in dose dependent accumulation of UDP-N-acetyl hexosamine. Supplementation of cultures with 10 mM NH4Cl resulted in a 40% reduction in cell growth rate and a 36% increase in the qMab. The data suggest that the reduction in growth rate and not UDP-N-acetyl hexosamine accumulation was the cause of increased qMab in the ammonium supplemented cultures. Ammonium chloride did not affect the extent of protein glycosylation. We conclude that UDP-N-acetyl hexosamine does not act as a mediator of enhanced rates of monoclonal antibody synthesis in the hybridoma cell culture system.

Ammonium Chloride↗

PubMed2002

Muscle uridine diphosphate-hexosamines do not decrease despite correction of hyperglycemia-induced insulin resistance in type 2 diabetes.

Animal studies suggest that overactivity of the hexosamine pathway, resulting in increased UDP-hexosamines [UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc)] is an important mechanism by which hyperglycemia causes insulin resistance. This study was performed to test this hypothesis in patients with type 2 diabetes mellitus (DM). Eight obese patients with uncontrolled DM type 2 and severe insulin resistance were treated with iv insulin for 28 +/- 6 d aimed at euglycemia. Before and after iv insulin treatment, insulin sensitivity was measured using a hyperinsulinemic euglycemic clamp, and a muscle biopsy was taken for measurement of UDP-GlcNAc, UDP-GalNAc, UDP-glucose, and UDP-galactose levels. Also, isoelectric focusing patterns of serum transferrin and the urinary excretion of glycosaminoglycans as measures of final products of the hexosamine pathway were examined. After euglycemia, insulin resistance improved, as demonstrated by an increase in the glucose infusion rate during the clamp from 12.7 +/- 5.6 to 22.4 +/- 8.8 micro mol/kg.min (P < 0.0005) and a decrease in insulin requirement from 1.7 +/- 0.9 to 1.1 +/- 0.6 U/kg.d (P < 0.005), whereas metabolic control improved. Surprisingly, both UDP-GlcNAc, from 8.81 +/- 1.21 to 12.31 +/- 2.52 nmol/g tissue (P < 0.005), and UDP-GalNAc concentrations, from 4.49 +/- 0.85 to 5.89 +/- 1.55 nmol/g tissue (P < 0.05) increased. Isoelectric focusing patterns of serum transferrin and excretion of glycosaminoglycans were similar before and after euglycemia. In conclusion, after amelioration of hyperglycemia- induced insulin resistance, UDP-hexosamines increased in skeletal muscle of patients with type 2 DM. These results do not support the hypothesis that accumulation of products of the hexosamine pathway plays a major role in hyperglycemia-induced insulin resistance.

Adult↗

PubMed1980

The hexosamine concentration and output in human pure pancreatic juice in chronic pancreatitis.

Hexosamine concentration in human pure pancreatic juice was determined during wash-out phase and secretin stimulation phase. Specimens were collected by endoscopic retrograde catheterization of the papilla at one minute intervals for 20 minutes after intravenous injection of secretin (Eisai, 1 U/kg) and for 10 minutes after pancreozymin injection (Boots, 1 U/kg). In suspected and established chronic pancreatitis (calcifying or non-calcifying), hexosamine concentration was significantly raised during both wash-out phase and secretin phase. Hexosamine output was significantly raised in suspected chronic pancreatitis and non-calcifying chronic pancreatitis during both wash-out phase and secretin phase; in calcifying chronic pancreatitis, no significant increase in hexosamine output was noted during both phases because of decreased secretory volume. Significance of these findings was discussed in relation to the pathogenesis of chronic pancreatitis.

Alcoholism↗

PubMed1985

Hexose and hexosamine concentrations in human pancreatic juice.

Hexose and hexosamine contents were measured in 117 samples of either duodenal or pancreatic juice from 49 subjects. The specimens were obtained by three methods: firstly, through a Dreiling's double lumen tube and with a pancreozymin secretin test performed simultaneously; secondly, by fiber duodenoscopy after an intravenous injection of secretin; thirdly, through a postoperative cannula without stimulation. The hexose content measured by phenol-sulfuric acid reaction correlated well with the hexosamine content measured by the Elson-Morgan method (r = 0.63, p less than 0.005). Since determination of hexose is not as complicated as that of hexosamine, the measurement of hexose content in the pancreatic juice seems to be more useful than measuring hexosamine for obtaining valuable information on pancreatic abnormalities.

Cholecystokinin↗

PubMed1992

HPLC analysis of hexosamine phosphates in biological samples.

Galactosamine is quickly metabolized to galactosamine 1-phosphate in rats treated with this compound. An HPLC method to quantify hexosamine phosphates in biological samples is described, modified from the o-phthaldialdehyde amino acid analysis procedure. o-Phthaldialdehyde derivatives of hexosamines and hexosamine-phosphates can be eluted from a reverse-phase column at different retention times, with a total analysis time of 30 min and without overlapping with free amino acids at physiological concentrations. The standard curves are linear between 1 and 40 nmol. This simple method is more selective and sensitive than previous enzymatic analyses of hexosamine phosphorylation.

Animals↗

PubMed1994

The hexosamine biosynthetic pathway and glucose-induced down regulation of glucose transport in L6 myotubes.

Based on experiments in cultured adipocytes, it has been proposed that glucose-induced down regulation of glucose transport is mediated by the conversion of fructose-6-phosphate to glucosamine-6-phosphate via the first and rate-determining enzyme of the hexosamine biosynthetic pathway, glutamine: fructose-6-phosphate amidotransferase (glutamine hexosephosphate aminotransferase). Evidence for this assertion was: (a) L-glutamine, the provider group for the aminotransferase was essential; (b) two inhibitors of glutamine hexosephosphate aminotransferase, 6-diazo-5-oxonorleucine (L form) and azaserine, blocked glucose-induced down regulation of glucose transport; (c) azaserine inhibited the activity of the aminotransferase, (d) glucosamine, which enters the hexosamine pathway distal to this enzyme was 40-times more potent than glucose; and (e) azaserine was unable to block the effect of glucosamine. Since muscle is quantitatively much more important than adipose tissue for whole body glucose utilization, we sought to determine if the hexosamine pathway was involved in glucose-induced down regulation of glucose transport in L6 myotubes. Glucose was effective, both in the presence and absence of glutamine in the incubation media. Glucosamine was also effective but was as equipotent as glucose. Small amounts of glutamine hexosephosphate aminotransferase were present in the L6 myotubes and although the leucine derivative (20 microM) inhibited the enzyme, it did not impair glucose-induced down regulation of glucose transport. Total GLUT-1 levels were similar when the cells were incubated in the absence or presence of 5 mM glucose or glucosamine although glucosamine was associated with a marked increase in a lower molecular weight band. These results do not suggest that the hexosamine biosynthetic pathway is involved in glucose-induced down regulation of glucose transport in L6 myotubes. Thus, this phenomenon is regulated differently in muscle and fat.

Animals↗

PubMed2002

The hexosamine biosynthesis pathway regulates insulin secretion via protein glycosylation in mouse islets.

The hexosamine biosynthesis pathway plays a role in the modification of cellular proteins via the provision of substrate for addition of O-linked N-acetylglucosamine (GlcNAc). The relative importance of the GlcNAc modification of proteins to insulin secretion from pancreatic beta-cells has not been investigated and so remains unclear. In the present study, we show that inhibition of the hexosamine biosynthesis pathway decreases insulin secretion from mouse islets in response to a number of secretagogues, including glucose. This impairment in beta-cell function could not be attributed to reduced islet insulin content, altered ATP levels, or cell death and was restored with the addition of N-acetylglucosamine, a substrate that enters the pathway below the point of inhibition. Western blot analysis revealed that decreased islet protein glycosylation paralleled the decrease in insulin secretion following inhibition of the pathway. In conclusion, the data suggest a role for the hexosamine biosynthesis pathway in regulating the secretion of insulin by altering protein glycosylation. This finding may have implications for the development of type 2 diabetes, as chronic increase in flux through the hexosamine biosynthesis pathway may lead to the deterioration of beta-cell function via abnormal protein glycosylation.

Acetylglucosamine↗