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Donald A. Mcclain - One of the best experts on this subject based on the ideXlab platform.

  • increased Hexosamine pathway flux and high fat feeding are not additive in inducing insulin resistance evidence for a shared pathway
    Amino Acids, 2011
    Co-Authors: Robert C Cooksey, Donald A. Mcclain
    Abstract:

    Excess fatty acids and carbohydrates have both been implicated in the pathogenesis of type 2 diabetes, and both can reproduce essential features of the disease including insulin resistance and beta cell failure. It has been proposed that both nutrients may regulate metabolism through a common fuel sensing mechanism, namely Hexosamine synthesis. We have previously shown that transgenic overexpression of the rate-limiting enzyme for Hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), targeted to muscle and fat, leads to insulin resistance mediated by increased O-linked glycosylation of nuclear and cytosolic proteins. We report here that Hexosamine-induced insulin resistance is not additive with that induced by high fat feeding. In control mice fed a high fat diet, glucose disposal rates during euglycemic hyperinsulinemia were decreased by 37% (p < 0.02) compared to mice on a low fat diet. Transgenic mice overexpressing GFA and fed a low fat diet exhibited a 51% decrease in glucose disposal compared to controls on a low fat diet (p < 0.001), but no further decrease was evident in the transgenic mice fed a high fat diet. Decreased glucose disposal rates were mirrored by increases in skeletal muscle levels of the principal end product of the Hexosamine pathway, UDP-N-acetyl glucosamine. Serum leptin levels, which are modulated both by feeding and Hexosamine flux, also show no additivity in their stimulation by GFA overexpression and high fat feeding. These data are consistent with a shared nutrient sensing pathway for high fat and carbohydrate fluxes and a common pathway by which glucose and lipids induce insulin resistance.

  • chronic Hexosamine flux stimulates fatty acid oxidation by activating amp activated protein kinase in adipocytes
    Journal of Biological Chemistry, 2007
    Co-Authors: Bai Luo, Robert C Cooksey, Glendon J Parker, Yudi Soesanto, Mark Evans, Deborah Jones, Donald A. Mcclain
    Abstract:

    The Hexosamine biosynthesis pathway (HBP) serves as a nutrient sensor and has been implicated in the development of type 2 diabetes. We previously demonstrated that fatty acid oxidation was enhanced in transgenic mouse adipocytes, wherein the rate-limiting enzyme of the HBP, glutamine:fructose-6-phosphate amidotransferase (GFA), was overexpressed. To explore the molecular mechanism of the HBP-induced fatty acid oxidation in adipocytes, we studied AMP-activated protein kinase (AMPK), an energy sensor that stimulates fatty acid oxidation by regulating acetyl-CoA carboxylase (ACC) activity. Phosphorylation and activity of AMPK were increased in transgenic fat pads and in 3T3L1 adipocytes treated with glucosamine to stimulate Hexosamine flux. Glucosamine also stimulated phosphorylation of ACC and fatty acid oxidation in 3T3L1 adipocytes, and these stimulatory effects were diminished by adenovirus-mediated expression of a dominant negative AMPK in 3T3L1 adipocytes. Conversely, blocking the HBP with a GFA inhibitor reduced AMPK activity, ACC phosphorylation, and fatty acid oxidation. These changes are not explained by alterations in the cellular AMP/ATP ratio. Further demonstrating that AMPK is regulated by the HBP, we found that AMPK was recognized by succinylated wheat germ agglutinin, which specifically binds O-GlcNAc. The levels of AMPK in succinylated wheat germ agglutinin precipitates correlated with Hexosamine flux in mouse fat pads and 3T3L1 adipocytes. Moreover, removal of O-GlcNAc by hexosaminidase reduced AMPK activity. We conclude that chronically high Hexosamine flux stimulates fatty acid oxidation by activating AMPK in adipocytes, in part through O-linked glycosylation.

  • adipocytes with increased Hexosamine flux exhibit insulin resistance increased glucose uptake and increased synthesis and storage of lipid
    American Journal of Physiology-endocrinology and Metabolism, 2005
    Co-Authors: Donald A. Mcclain, Mark Hazel, Glendon J Parker, Robert C Cooksey
    Abstract:

    The Hexosamine signaling pathway has been shown to serve a nutrient-sensing function. We have previously shown that overexpression of the rate-limiting enzyme for Hexosamine synthesis (glutamine-fr...

  • overexpression of glutamine fructose 6 phosphate amidotransferase in the liver of transgenic mice results in enhanced glycogen storage hyperlipidemia obesity and impaired glucose tolerance
    Diabetes, 2000
    Co-Authors: Geddati Veerababu, Robert C Cooksey, Errol D Crook, Leon F. Hebert, Marc C. Daniels, Jiping Tang, Rosemary T Hoffman, Donald A. Mcclain
    Abstract:

    To examine the effect of increased Hexosamine flux in liver, the rate-limiting enzyme in Hexosamine biosynthesis (glutamine:fructose-6-phosphate amidotransferase [GFA]) was overexpressed in transgenic mice using the PEPCK promoter. Liver from random-fed transgenic mice had 1.6-fold higher GFA activity compared with nontransgenic control littermates (276 +/- 24 pmol x mg(-1) x min(-1) in transgenic mice vs. 176 +/- 18 pmol x mg(-1) x min(-1) in controls, P < 0.05) and higher levels of the Hexosamine end product UDP-N-acetyl glucosamine (288 +/- 11 pmol/g in transgenic mice vs. 233 +/- 10 pmol/g in controls, P < 0.001). Younger transgenic mice compared with control mice had lower fasting serum glucose (4.8 +/- 0.5 mmol/l in transgenic mice vs. 6.5 +/- 0.8 mmol/l in controls, P < 0.05) without higher insulin levels (48.0 +/- 7.8 pmol/l in transgenic mice vs. 56.4 +/- 5.4 pmol/l in controls, P = NS); insulin levels were significantly lower in transgenic males (P < 0.05). At 6 months of age, transgenic animals had normal insulin sensitivity by the hyperinsulinemic clamp technique. Hepatic glycogen content was higher in the transgenic mice (108.6 +/- 5.2 pmol/g in transgenic mice vs. 32.8 +/- 1.3 micromol/g in controls, P < 0.01), associated with an inappropriate activation of glycogen synthase. Serum levels of free fatty acids (FFAs) and triglycerides were also elevated (FFAs, 0.67 +/- 0.03 mmol/l in transgenic mice vs. 0.14 +/- 0.01 in controls; triglycerides, 1.34 +/- 0.15 mmol/l in transgenic mice vs. 0.38 +/- 0.01 in controls, P < 0.01). Older transgenic mice became heavier than control mice and exhibited relative glucose intolerance and insulin resistance. The glucose disposal rate at 8 months of age was 154 +/- 5 mg x kg(-1) x min(-1) in transgenic mice vs. 191 +/- 6 mg x kg(-1) x min(-1) in controls (P < 0.05). We conclude that Hexosamines are mediators of glucose sensing for the regulation of hepatic glycogen and lipid metabolism. Increased Hexosamine flux in the liver signals a shift toward fuel storage, resulting ultimately in obesity and insulin resistance.

  • transgenic mice with increased Hexosamine flux specifically targeted to beta cells exhibit hyperinsulinemia and peripheral insulin resistance
    Diabetes, 2000
    Co-Authors: Jiping Tang, Robert C Cooksey, John L. Neidigh, Donald A. Mcclain
    Abstract:

    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.

Robert C Cooksey - One of the best experts on this subject based on the ideXlab platform.

  • increased Hexosamine pathway flux and high fat feeding are not additive in inducing insulin resistance evidence for a shared pathway
    Amino Acids, 2011
    Co-Authors: Robert C Cooksey, Donald A. Mcclain
    Abstract:

    Excess fatty acids and carbohydrates have both been implicated in the pathogenesis of type 2 diabetes, and both can reproduce essential features of the disease including insulin resistance and beta cell failure. It has been proposed that both nutrients may regulate metabolism through a common fuel sensing mechanism, namely Hexosamine synthesis. We have previously shown that transgenic overexpression of the rate-limiting enzyme for Hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), targeted to muscle and fat, leads to insulin resistance mediated by increased O-linked glycosylation of nuclear and cytosolic proteins. We report here that Hexosamine-induced insulin resistance is not additive with that induced by high fat feeding. In control mice fed a high fat diet, glucose disposal rates during euglycemic hyperinsulinemia were decreased by 37% (p < 0.02) compared to mice on a low fat diet. Transgenic mice overexpressing GFA and fed a low fat diet exhibited a 51% decrease in glucose disposal compared to controls on a low fat diet (p < 0.001), but no further decrease was evident in the transgenic mice fed a high fat diet. Decreased glucose disposal rates were mirrored by increases in skeletal muscle levels of the principal end product of the Hexosamine pathway, UDP-N-acetyl glucosamine. Serum leptin levels, which are modulated both by feeding and Hexosamine flux, also show no additivity in their stimulation by GFA overexpression and high fat feeding. These data are consistent with a shared nutrient sensing pathway for high fat and carbohydrate fluxes and a common pathway by which glucose and lipids induce insulin resistance.

  • chronic Hexosamine flux stimulates fatty acid oxidation by activating amp activated protein kinase in adipocytes
    Journal of Biological Chemistry, 2007
    Co-Authors: Bai Luo, Robert C Cooksey, Glendon J Parker, Yudi Soesanto, Mark Evans, Deborah Jones, Donald A. Mcclain
    Abstract:

    The Hexosamine biosynthesis pathway (HBP) serves as a nutrient sensor and has been implicated in the development of type 2 diabetes. We previously demonstrated that fatty acid oxidation was enhanced in transgenic mouse adipocytes, wherein the rate-limiting enzyme of the HBP, glutamine:fructose-6-phosphate amidotransferase (GFA), was overexpressed. To explore the molecular mechanism of the HBP-induced fatty acid oxidation in adipocytes, we studied AMP-activated protein kinase (AMPK), an energy sensor that stimulates fatty acid oxidation by regulating acetyl-CoA carboxylase (ACC) activity. Phosphorylation and activity of AMPK were increased in transgenic fat pads and in 3T3L1 adipocytes treated with glucosamine to stimulate Hexosamine flux. Glucosamine also stimulated phosphorylation of ACC and fatty acid oxidation in 3T3L1 adipocytes, and these stimulatory effects were diminished by adenovirus-mediated expression of a dominant negative AMPK in 3T3L1 adipocytes. Conversely, blocking the HBP with a GFA inhibitor reduced AMPK activity, ACC phosphorylation, and fatty acid oxidation. These changes are not explained by alterations in the cellular AMP/ATP ratio. Further demonstrating that AMPK is regulated by the HBP, we found that AMPK was recognized by succinylated wheat germ agglutinin, which specifically binds O-GlcNAc. The levels of AMPK in succinylated wheat germ agglutinin precipitates correlated with Hexosamine flux in mouse fat pads and 3T3L1 adipocytes. Moreover, removal of O-GlcNAc by hexosaminidase reduced AMPK activity. We conclude that chronically high Hexosamine flux stimulates fatty acid oxidation by activating AMPK in adipocytes, in part through O-linked glycosylation.

  • Hexosamines regulate sensitivity of glucose stimulated insulin secretion in β cells
    American Journal of Physiology-endocrinology and Metabolism, 2006
    Co-Authors: Robert C Cooksey, Sumitha Pusuluri, Mark Hazel, Deborah A. Mcclain
    Abstract:

    Hexosamines serve a nutrient-sensing function through enzymatic O-glycosylation of proteins. We previously characterized transgenic (Tg) mice with overexpression of the rate-limiting enzyme in Hexosamine production, glutamine:fructose-6-phosphate amidotransferase, in β-cells. Animals were hyperinsulinemic, resulting in peripheral insulin resistance. Glucose tolerance deteriorated with age, and males developed diabetes. We therefore examined islet function in these mice by perifusion in vitro. Young (2-mo-old) Tg animals had enhanced sensitivity to glucose of insulin secretion. Insulin secretion was maximal at 20 mM and half maximal at 9.9 ± 0.5 mM glucose in Tg islets compared with maximal at 30 mM and half maximal at 13.5 ± 0.7 mM glucose in wild type (WT; P < 0.005). Young Tg animals secreted more insulin in response to 20 mM glucose (Tg, 1,254 ± 311; WT, 425 ± 231 pg·islet−1·35 min−1; P < 0.01). Islets from older (8-mo-old) Tg mice became desensitized to glucose, with half-maximal secretion at 16.1 ± 0...

  • adipocytes with increased Hexosamine flux exhibit insulin resistance increased glucose uptake and increased synthesis and storage of lipid
    American Journal of Physiology-endocrinology and Metabolism, 2005
    Co-Authors: Donald A. Mcclain, Mark Hazel, Glendon J Parker, Robert C Cooksey
    Abstract:

    The Hexosamine signaling pathway has been shown to serve a nutrient-sensing function. We have previously shown that overexpression of the rate-limiting enzyme for Hexosamine synthesis (glutamine-fr...

  • overexpression of glutamine fructose 6 phosphate amidotransferase in the liver of transgenic mice results in enhanced glycogen storage hyperlipidemia obesity and impaired glucose tolerance
    Diabetes, 2000
    Co-Authors: Geddati Veerababu, Robert C Cooksey, Errol D Crook, Leon F. Hebert, Marc C. Daniels, Jiping Tang, Rosemary T Hoffman, Donald A. Mcclain
    Abstract:

    To examine the effect of increased Hexosamine flux in liver, the rate-limiting enzyme in Hexosamine biosynthesis (glutamine:fructose-6-phosphate amidotransferase [GFA]) was overexpressed in transgenic mice using the PEPCK promoter. Liver from random-fed transgenic mice had 1.6-fold higher GFA activity compared with nontransgenic control littermates (276 +/- 24 pmol x mg(-1) x min(-1) in transgenic mice vs. 176 +/- 18 pmol x mg(-1) x min(-1) in controls, P < 0.05) and higher levels of the Hexosamine end product UDP-N-acetyl glucosamine (288 +/- 11 pmol/g in transgenic mice vs. 233 +/- 10 pmol/g in controls, P < 0.001). Younger transgenic mice compared with control mice had lower fasting serum glucose (4.8 +/- 0.5 mmol/l in transgenic mice vs. 6.5 +/- 0.8 mmol/l in controls, P < 0.05) without higher insulin levels (48.0 +/- 7.8 pmol/l in transgenic mice vs. 56.4 +/- 5.4 pmol/l in controls, P = NS); insulin levels were significantly lower in transgenic males (P < 0.05). At 6 months of age, transgenic animals had normal insulin sensitivity by the hyperinsulinemic clamp technique. Hepatic glycogen content was higher in the transgenic mice (108.6 +/- 5.2 pmol/g in transgenic mice vs. 32.8 +/- 1.3 micromol/g in controls, P < 0.01), associated with an inappropriate activation of glycogen synthase. Serum levels of free fatty acids (FFAs) and triglycerides were also elevated (FFAs, 0.67 +/- 0.03 mmol/l in transgenic mice vs. 0.14 +/- 0.01 in controls; triglycerides, 1.34 +/- 0.15 mmol/l in transgenic mice vs. 0.38 +/- 0.01 in controls, P < 0.01). Older transgenic mice became heavier than control mice and exhibited relative glucose intolerance and insulin resistance. The glucose disposal rate at 8 months of age was 154 +/- 5 mg x kg(-1) x min(-1) in transgenic mice vs. 191 +/- 6 mg x kg(-1) x min(-1) in controls (P < 0.05). We conclude that Hexosamines are mediators of glucose sensing for the regulation of hepatic glycogen and lipid metabolism. Increased Hexosamine flux in the liver signals a shift toward fuel storage, resulting ultimately in obesity and insulin resistance.

Deborah A. Mcclain - One of the best experts on this subject based on the ideXlab platform.

  • Hexosamines regulate sensitivity of glucose stimulated insulin secretion in β cells
    American Journal of Physiology-endocrinology and Metabolism, 2006
    Co-Authors: Robert C Cooksey, Sumitha Pusuluri, Mark Hazel, Deborah A. Mcclain
    Abstract:

    Hexosamines serve a nutrient-sensing function through enzymatic O-glycosylation of proteins. We previously characterized transgenic (Tg) mice with overexpression of the rate-limiting enzyme in Hexosamine production, glutamine:fructose-6-phosphate amidotransferase, in β-cells. Animals were hyperinsulinemic, resulting in peripheral insulin resistance. Glucose tolerance deteriorated with age, and males developed diabetes. We therefore examined islet function in these mice by perifusion in vitro. Young (2-mo-old) Tg animals had enhanced sensitivity to glucose of insulin secretion. Insulin secretion was maximal at 20 mM and half maximal at 9.9 ± 0.5 mM glucose in Tg islets compared with maximal at 30 mM and half maximal at 13.5 ± 0.7 mM glucose in wild type (WT; P < 0.005). Young Tg animals secreted more insulin in response to 20 mM glucose (Tg, 1,254 ± 311; WT, 425 ± 231 pg·islet−1·35 min−1; P < 0.01). Islets from older (8-mo-old) Tg mice became desensitized to glucose, with half-maximal secretion at 16.1 ± 0...

  • Hexosamines as mediators of nutrient sensing and regulation in diabetes
    Journal of Diabetes and Its Complications, 2002
    Co-Authors: Deborah A. Mcclain
    Abstract:

    Abstract 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.

  • Hexosamines as mediators of nutrient sensing relevance to obesity insulin resistance and diabetes
    Current Opinion in Endocrinology & Diabetes, 2001
    Co-Authors: Deborah A. Mcclain
    Abstract:

    High concentrations of glucose induce insulin resistance, impair insulin secretion, and affect hepatic glucose production in a manner that mirrors type 2 diabetes. High concentrations of 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 in several model systems. For example, with overexpression of the rate-limiting enzyme for Hexosamine synthesis in transgenic mice, skeletal muscle becomes insulin resistant, the liver synthesizes excess fatty acid, and the β cells increase insulin secretion. Thus, excess Hexosamine flux leads to a coordinated response whereby fuel is shunted toward long-term storage, mirroring the thrifty phenotype. However when these same adaptive changes occur chronically, they ultimately result in obesity, hyperlipidemia, β-cell failure, and type 2 diabetes. Recent work indicates that these effects may be the result of enzymatic O-linked glycosylation of proteins and that this glycosylation is regulated by the levels of the end-product of the Hexosamine pathway, UDP-N-acetyl glucosamine. The results suggest a mechanism by which chronic overnutrition leads to the phenotype of type 2 diabetes.

  • Hexosamines stimulate leptin production in transgenic mice
    Endocrinology, 2000
    Co-Authors: Deborah A. Mcclain, Robert C Cooksey, Thomas Alexander, Robert V Considine
    Abstract:

    ABSTRACT Hexosamine flux has been shown to mediate aspects of nutrient sensing in insulin sensitive tissues and has been hypothesized to represent a satiety signal that results in shunting of fuel toward storage as fat. It has been recently reported that in vitro treatment of fat and muscle cells with Hexosamines and acute glucosamine infusion in intact rats stimulate leptin secretin. In order to investigate the effects of chronic, physiologic increases in Hexosamine flux on leptin we have examined leptin mRNA and serum leptin in mice overexpressing the rate-limiting enzyme for Hexosamine synthesis, GFA, in muscle and fat. Increased levels of UDP-N-acetylglucosamine, the principal end-product of the Hexosamine pathway were seen in transgenic fat, consistent with the overexpression of GFA. After overnight fasting, the transgenic mice were hyperleptinemic compared to littermate controls (4.5 ± 0.5 ng/ml in transgenic, 2.8 ± 0.2 in control, p = 0.005) despite equal body weights. In the random-fed state, the ...

  • Hexosamines and insulin resistance
    Diabetes, 1996
    Co-Authors: Deborah A. Mcclain, Errol D Crook
    Abstract:

    Glucose is an important regulator of cell growth and metabolism. Thus, it is likely that some of the adverse effects of hyperglycemia are reflections of normal regulation by abnormal concentrations of glucose. How the cell senses glucose, however, is still incompletely understood. Evidence has been presented that the Hexosamine biosynthesis pathway serves this function for regulation of aspects of glucose uptake, glycogen synthesis, glycolysis, and synthesis of growth factors. Excess Hexosamine flux causes insulin resistance in cultured cells, tissues, and intact animals. Further evidence for the possible role of this pathway in normal glucose homeostasis and disease is that the level of activity of the rate-limiting enzyme in Hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase, is correlated with glucose disposal rates (GDRs) in normal humans and transgenic mice.

Errol D Crook - One of the best experts on this subject based on the ideXlab platform.

  • Hexosamine induced fibronectin protein synthesis in mesangial cells is associated with increases in camp responsive element binding creb phosphorylation and nuclear creb the involvement of protein kinases a and c
    Diabetes, 2001
    Co-Authors: Lalit P Singh, Jack Andy, Vivian Anyamale, Kennieth Greene, M Y Alexander, Errol D Crook
    Abstract:

    Hyperglycemia-induced alterations in mesangial (MES) cell function and extracellular matrix protein accumulation are seen in diabetic glomerulopathy. Recent studies have demonstrated that some of the effects of high glucose (HG) on cellular metabolism are mediated by the Hexosamine biosynthesis pathway (HBP), in which fructose-6-phosphate is converted to glucosamine 6-phosphate by the rate-liming enzyme glutamine:fructose-6-phosphate amidotransferase (GFA). In this study, we investigated the role of HBP on HG-stimulated fibronectin protein synthesis, a matrix component, in SV-40–transformed rat kidney MES cells. Treatment of MES cells with 25 mmol/l glucose (HG) for 48 h increases cellular fibronectin levels by two- to threefold on Western blots when compared with low glucose (5 mmol/l). Glucosamine (GlcN; 1.5 mmol/l), which enters the Hexosamine pathway distal to GFA action, also increases fibronectin synthesis. Azaserine (AZA; 0.5 μmol/l), an inhibitor of GFA, blocks the HG- but not the GlcN-induced fibronectin synthesis. Fibronectin contains cAMP responsive element (CRE) consensus sequences in its promoter and the phosphorylation of CRE-binding protein (CREB) may regulate its expression. On Western blots, HG and GlcN stimulate two- to threefold the phosphorylation of CREB at Ser 133, whereas CREB protein content was unaltered by either HG or GlcN. In addition, nuclear CREB activity was increased by HG and GlcN on gel-shift assays using 32P-CRE oligonucleotides. AZA impeded the HG-enhanced CREB phosphorylation and CRE binding but had no effect on GlcN-mediated CREB phosphorylation and CRE binding. Pharmacologic inhibition of protein kinase C (PKC) and protein kinase A (PKA), which are involved in Hexosamine-mediated matrix production, blocked the CREB phosphorylation and fibronectin synthesis seen in HG and GlcN conditions. We conclude that the effects of HG on fibronectin synthesis in the mesangium are mediated by the HBP possibly via Hexosamine regulation of CREB and PKC/PKA signaling pathways. These results support the hypothesis that the HBP is a sensor and regulator of the actions of glucose in the kidney.

  • overexpression of glutamine fructose 6 phosphate amidotransferase in the liver of transgenic mice results in enhanced glycogen storage hyperlipidemia obesity and impaired glucose tolerance
    Diabetes, 2000
    Co-Authors: Geddati Veerababu, Robert C Cooksey, Errol D Crook, Leon F. Hebert, Marc C. Daniels, Jiping Tang, Rosemary T Hoffman, Donald A. Mcclain
    Abstract:

    To examine the effect of increased Hexosamine flux in liver, the rate-limiting enzyme in Hexosamine biosynthesis (glutamine:fructose-6-phosphate amidotransferase [GFA]) was overexpressed in transgenic mice using the PEPCK promoter. Liver from random-fed transgenic mice had 1.6-fold higher GFA activity compared with nontransgenic control littermates (276 +/- 24 pmol x mg(-1) x min(-1) in transgenic mice vs. 176 +/- 18 pmol x mg(-1) x min(-1) in controls, P < 0.05) and higher levels of the Hexosamine end product UDP-N-acetyl glucosamine (288 +/- 11 pmol/g in transgenic mice vs. 233 +/- 10 pmol/g in controls, P < 0.001). Younger transgenic mice compared with control mice had lower fasting serum glucose (4.8 +/- 0.5 mmol/l in transgenic mice vs. 6.5 +/- 0.8 mmol/l in controls, P < 0.05) without higher insulin levels (48.0 +/- 7.8 pmol/l in transgenic mice vs. 56.4 +/- 5.4 pmol/l in controls, P = NS); insulin levels were significantly lower in transgenic males (P < 0.05). At 6 months of age, transgenic animals had normal insulin sensitivity by the hyperinsulinemic clamp technique. Hepatic glycogen content was higher in the transgenic mice (108.6 +/- 5.2 pmol/g in transgenic mice vs. 32.8 +/- 1.3 micromol/g in controls, P < 0.01), associated with an inappropriate activation of glycogen synthase. Serum levels of free fatty acids (FFAs) and triglycerides were also elevated (FFAs, 0.67 +/- 0.03 mmol/l in transgenic mice vs. 0.14 +/- 0.01 in controls; triglycerides, 1.34 +/- 0.15 mmol/l in transgenic mice vs. 0.38 +/- 0.01 in controls, P < 0.01). Older transgenic mice became heavier than control mice and exhibited relative glucose intolerance and insulin resistance. The glucose disposal rate at 8 months of age was 154 +/- 5 mg x kg(-1) x min(-1) in transgenic mice vs. 191 +/- 6 mg x kg(-1) x min(-1) in controls (P < 0.05). We conclude that Hexosamines are mediators of glucose sensing for the regulation of hepatic glycogen and lipid metabolism. Increased Hexosamine flux in the liver signals a shift toward fuel storage, resulting ultimately in obesity and insulin resistance.

  • Overexpression of glutamine:fructose-6-phosphate amidotransferase in transgenic mice leads to insulin resistance.
    The Journal of clinical investigation, 1996
    Co-Authors: Leon F. Hebert, Errol D Crook, Alain D. Baron, Marc C. Daniels, Jianxin Zhou, Ramona L. Turner, Shakisha T. Simmons, John L. Neidigh, Jin Su Zhu, Donald A. Mcclain
    Abstract:

    The Hexosamine biosynthetic pathway has been hypothesized to be involved in mediating some of the toxic effects of hyperglycemia. Glutamine:fructose-6-phosphate amidotransferase (GFA), the first and rate limiting enzyme of the Hexosamine biosynthetic pathway, was overexpressed in skeletal muscle and adipose tissue of transgenic mice. A 2.4-fold increase of GFA activity in muscle of the transgenic mice led to weight-dependent hyperinsulinemia in random-fed mice. The hyperinsulinemic-euglycemic clamp technique confirmed that transgenic mice develop insulin resistance, with a glucose disposal rate of 68.5 +/- 3.5 compared with 129.4 +/- 9.4 mg/kg per min (P < 0.001) for littermate controls. The decrease in the glucose disposal rate of the transgenic mice is accompanied by decreased protein but not mRNA levels of the insulin-stimulated glucose transporter (GLUT4). These data support the hypothesis that excessive flux through the Hexosamine biosynthesis pathway mediates adverse regulatory and metabolic effects of hyperglycemia, specifically insulin resistance of glucose disposal. These mice can serve as a model system to study the mechanism for the regulation of glucose homeostasis by Hexosamines.

  • Hexosamines and insulin resistance
    Diabetes, 1996
    Co-Authors: Deborah A. Mcclain, Errol D Crook
    Abstract:

    Glucose is an important regulator of cell growth and metabolism. Thus, it is likely that some of the adverse effects of hyperglycemia are reflections of normal regulation by abnormal concentrations of glucose. How the cell senses glucose, however, is still incompletely understood. Evidence has been presented that the Hexosamine biosynthesis pathway serves this function for regulation of aspects of glucose uptake, glycogen synthesis, glycolysis, and synthesis of growth factors. Excess Hexosamine flux causes insulin resistance in cultured cells, tissues, and intact animals. Further evidence for the possible role of this pathway in normal glucose homeostasis and disease is that the level of activity of the rate-limiting enzyme in Hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase, is correlated with glucose disposal rates (GDRs) in normal humans and transgenic mice.

  • regulation of glycogen synthase and protein phosphatase 1 by Hexosamines
    Diabetes, 1996
    Co-Authors: Errol D Crook, Deborah A. Mcclain
    Abstract:

    The Hexosamine biosynthesis pathway has been hypothesized to be involved in mediating some of the adverse effects of high glucose. We have previously shown that glucose downregulates basal glycogen synthase (GS) activity in Rat-1 cells and that overexpressing the rate-limiting enzyme in the Hexosamine biosynthesis pathway (glutamine:fructose-6-phosphate amidotransferase [GFA]) makes the cells more sensitive to these effects of glucose. GFA overexpression also leads to a reduction in insulin sensitivity of GS. Here we examine the effects of glucose and glucosamine on insulin-stimulated GS activity and on protein phosphatase-1 (PP1) activity. These activities were assayed in cytoplasmic extracts from Rat-1 fibroblasts overexpressing human GFA and cultured in varying glucose concentrations. Both maximal insulin-stimulated GS activity and insulin sensitivity decreased with increasing glucose. Overexpression of GFA leads to a further reduction in insulin sensitivity but not in maximal insulin-stimulated GS activity. Because there were no differences in total (glucose-6-phosphate-dependent) GS activity between cell lines or as a function of glucose concentration, these results most likely reflect a change in the phosphorylation state of the synthase. Activity of PP1, a potential mediator of these effects, was responsive to glucose and Hexosamines. Control cells showed a 9.3 ± 4.3% decrease in PP1 activity with increasing glucose. GFA cells showed a greater response to glucose, with PP1 activity decreasing 34.2 ± 5.5% with increasing glucose. Glucosamine was more potent than glucose in decreasing PP1 activity in control cells. Cells overexpressing the normal human insulin receptor (HIRc-B) were used to facilitate analysis of insulin-stimulated PP1 activity. Stimulation with 1.7 mmol/l insulin led to a 37.6 ± 9.9% increase in PP1 activity in HIRc-B cells cultured in 1 mmol/l glucose, while cells cultured in 5 mmol/l glucosamine or 20 mmol/l glucose demonstrated only 3.79 ± 0.60 or 1.6 ± 0.75% increases, respectively. We conclude that both basal and insulin-stimulable GS and PP1 activity are downregulated by high glucose in fibroblasts and this regulation is mediated by products of the Hexosamine biosynthesis pathway.

Marc C. Daniels - One of the best experts on this subject based on the ideXlab platform.

  • overexpression of glutamine fructose 6 phosphate amidotransferase in the liver of transgenic mice results in enhanced glycogen storage hyperlipidemia obesity and impaired glucose tolerance
    Diabetes, 2000
    Co-Authors: Geddati Veerababu, Robert C Cooksey, Errol D Crook, Leon F. Hebert, Marc C. Daniels, Jiping Tang, Rosemary T Hoffman, Donald A. Mcclain
    Abstract:

    To examine the effect of increased Hexosamine flux in liver, the rate-limiting enzyme in Hexosamine biosynthesis (glutamine:fructose-6-phosphate amidotransferase [GFA]) was overexpressed in transgenic mice using the PEPCK promoter. Liver from random-fed transgenic mice had 1.6-fold higher GFA activity compared with nontransgenic control littermates (276 +/- 24 pmol x mg(-1) x min(-1) in transgenic mice vs. 176 +/- 18 pmol x mg(-1) x min(-1) in controls, P < 0.05) and higher levels of the Hexosamine end product UDP-N-acetyl glucosamine (288 +/- 11 pmol/g in transgenic mice vs. 233 +/- 10 pmol/g in controls, P < 0.001). Younger transgenic mice compared with control mice had lower fasting serum glucose (4.8 +/- 0.5 mmol/l in transgenic mice vs. 6.5 +/- 0.8 mmol/l in controls, P < 0.05) without higher insulin levels (48.0 +/- 7.8 pmol/l in transgenic mice vs. 56.4 +/- 5.4 pmol/l in controls, P = NS); insulin levels were significantly lower in transgenic males (P < 0.05). At 6 months of age, transgenic animals had normal insulin sensitivity by the hyperinsulinemic clamp technique. Hepatic glycogen content was higher in the transgenic mice (108.6 +/- 5.2 pmol/g in transgenic mice vs. 32.8 +/- 1.3 micromol/g in controls, P < 0.01), associated with an inappropriate activation of glycogen synthase. Serum levels of free fatty acids (FFAs) and triglycerides were also elevated (FFAs, 0.67 +/- 0.03 mmol/l in transgenic mice vs. 0.14 +/- 0.01 in controls; triglycerides, 1.34 +/- 0.15 mmol/l in transgenic mice vs. 0.38 +/- 0.01 in controls, P < 0.01). Older transgenic mice became heavier than control mice and exhibited relative glucose intolerance and insulin resistance. The glucose disposal rate at 8 months of age was 154 +/- 5 mg x kg(-1) x min(-1) in transgenic mice vs. 191 +/- 6 mg x kg(-1) x min(-1) in controls (P < 0.05). We conclude that Hexosamines are mediators of glucose sensing for the regulation of hepatic glycogen and lipid metabolism. Increased Hexosamine flux in the liver signals a shift toward fuel storage, resulting ultimately in obesity and insulin resistance.

  • effects of glucosamine infusion on insulin secretion and insulin action in humans
    Diabetes, 2000
    Co-Authors: T Monauni, Donald A. Mcclain, Marc C. Daniels, M G Zenti, A Cretti, Giovanni Targher, Beatrice Caruso, M Caputo, S Del Prato, Andrea Giaccari
    Abstract:

    Glucose toxicity (i.e., glucose-induced reduction in insulin secretion and action) may be mediated by an increased flux through the Hexosamine-phosphate pathway. Glucosamine (GlcN) is widely used to accelerate the Hexosamine pathway flux, independently of glucose. We tested the hypothesis that GlcN can affect insulin secretion and/or action in humans. In 10 healthy subjects, we sequentially performed an intravenous glucose (plus [2-3H]glucose) tolerance test (IVGTT) and a euglycemic insulin clamp during either a saline infusion or a low (1.6 micromol x min(-1) x kg(-1)) or high (5 micromol x min(-1) x kg(-1) [n = 5]) GlcN infusion. Beta-cell secretion, insulin (SI*-IVGTT), and glucose (SG*) action on glucose utilization during the IVGTT were measured according to minimal models of insulin secretion and action. Infusion of GlcN did not affect readily releasable insulin levels, glucose-stimulated insulin secretion (GSIS), or the time constant of secretion, but it increased both the glucose threshold of GSIS (delta approximately 0.5-0.8 mmol/l, P < 0.03-0.01) and plasma fasting glucose levels (delta approximately 0.3-0.5 mmol/l, P < 0.05-0.02). GlcN did not change glucose utilization or intracellular metabolism (glucose oxidation and glucose storage were measured by indirect calorimetry) during the clamp. However, high levels of GlcN caused a decrease in SI*-IVGTT (delta approximately 30%, P < 0.02) and in SG* (delta approximately 40%, P < 0.05). Thus, in humans, acute GlcN infusion recapitulates some metabolic features of human diabetes. It remains to be determined whether acceleration of the Hexosamine pathway can cause insulin resistance at euglycemia in humans.

  • Overexpression of glutamine:fructose-6-phosphate amidotransferase in transgenic mice leads to insulin resistance.
    The Journal of clinical investigation, 1996
    Co-Authors: Leon F. Hebert, Errol D Crook, Alain D. Baron, Marc C. Daniels, Jianxin Zhou, Ramona L. Turner, Shakisha T. Simmons, John L. Neidigh, Jin Su Zhu, Donald A. Mcclain
    Abstract:

    The Hexosamine biosynthetic pathway has been hypothesized to be involved in mediating some of the toxic effects of hyperglycemia. Glutamine:fructose-6-phosphate amidotransferase (GFA), the first and rate limiting enzyme of the Hexosamine biosynthetic pathway, was overexpressed in skeletal muscle and adipose tissue of transgenic mice. A 2.4-fold increase of GFA activity in muscle of the transgenic mice led to weight-dependent hyperinsulinemia in random-fed mice. The hyperinsulinemic-euglycemic clamp technique confirmed that transgenic mice develop insulin resistance, with a glucose disposal rate of 68.5 +/- 3.5 compared with 129.4 +/- 9.4 mg/kg per min (P < 0.001) for littermate controls. The decrease in the glucose disposal rate of the transgenic mice is accompanied by decreased protein but not mRNA levels of the insulin-stimulated glucose transporter (GLUT4). These data support the hypothesis that excessive flux through the Hexosamine biosynthesis pathway mediates adverse regulatory and metabolic effects of hyperglycemia, specifically insulin resistance of glucose disposal. These mice can serve as a model system to study the mechanism for the regulation of glucose homeostasis by Hexosamines.

  • glutamine fructose 6 phosphate amidotransferase activity in cultured human skeletal muscle cells relationship to glucose disposal rate in control and non insulin dependent diabetes mellitus subjects and regulation by glucose and insulin
    Journal of Clinical Investigation, 1996
    Co-Authors: Marc C. Daniels, Theodore P Ciaraldi, Svetlana E Nikoulina, Robert R Henry, Donald A. Mcclain
    Abstract:

    We examined the activity of the rate-limiting enzyme for Hexosamine biosynthesis, glutamine:fructose-6-phosphate amidotransferase (GFA) in human skeletal muscle cultures (HSMC), from 17 nondiabetic control and 13 subjects with non-insulin-dependent diabetes. GFA activity was assayed from HSMC treated with low (5 mM) or high (20 mM) glucose and low (22 pM) or high (30 microM) concentrations of insulin. In control subjects GFA activity decreased with increasing glucose disposal rate (r = -0.68, P < 0.025). In contrast, a positive correlation existed between GFA and glucose disposal in the diabetics (r = 0.86, P < 0.005). Increased GFA activity was also correlated with body mass index in controls but not diabetics. GFA activity was significantly stimulated by high glucose (22%), high insulin (43%), and their combination (61%). GFA activity and its regulation by glucose and insulin were not significantly different in diabetic HSMC. We conclude that glucose and insulin regulate GFA activity in skeletal muscle. More importantly, our results are consistent with a regulatory role for the Hexosamine pathway in human glucose homeostasis. This relationship between Hexosamine biosynthesis and the regulation of glucose metabolism is altered in non-insulin-dependent diabetes.

  • regulation of glycogen synthase by glucose glucosamine and glutamine fructose 6 phosphate amidotransferase
    Diabetes, 1995
    Co-Authors: Errol D Crook, Marc C. Daniels, Jianxin Zhou, John L. Neidigh, Donald A. Mcclain
    Abstract:

    The Hexosamine biosynthesis pathway has been hypothesized to mediate some of the regulatory as well as the deleterious effects of glucose. We have stably overexpressed the cDNA for human glutamine:fructose-6-phosphate amidotransferase (GFA), the rate-limiting enzyme in the Hexosamine biosynthesis pathway, in rat-1 fibroblasts. Two cell lines expressing the human RNA were selected by Northern analysis, and they exhibited 51-95% increases in GFA activity. Insulin-stimulated glycogen synthase (GS) activity and net glycogen synthesis were assayed, and GFA cells revealed decreased insulin sensitivity for both GS and net glycogen synthesis. The ED50 for insulin stimulation of GS was 2.45 ± 0.4 nmol/l insulin in controls and 5.29 ± 1.01 nmol/l in GFA cells ( P 50 was 3.43 ± 0.88 nmol/l in controls and 5.54 ± 0.98 nmol/l in GFA cells ( P P 50 values for glucose were 1.72 ± 0.08 mmol/1 in GFA cells and 5.60 ± 2.05 mmol/l in control cells (P ? 0.05). Glucosamine mimicked the effects of glucose but at a much higher potency with an ED50 of 0.667 ± 0.15 mmol/1 in control cells cultured in low glucose. The changes in activity seen with glucose are not due to the osmotic effects of glucose or the carryover of intracellular effector molecules into the in vitro GS assay. In sum, the stable overexpression of the cDNA for human GFA leads to decreased insulin sensitivity of GS and glycogen synthesis and is accompanied by an increased sensitivity to inhibition of GS by high glucose. We conclude that glucose regulates glycogen synthesis through the metabolism of glucose to Hexosamines.