Pleiotropic and age-dependent effects of decreased protein modification by O-linked N-acetylglucosamine on pancreatic ß-cell function and vascularization.

The hexosamine biosynthesis pathway (HBP) regulates the post-translational modification of nuclear and cytoplasmic protein by O-linked N-acetylglucosamine (O-GlcNAc). Numerous studies have demonstrated increased flux through this pathway contributes to the development of ß-cell dysfunction. The effect of decreased O-GlcNAc on the maintenance of normal ß-cell function, however, is not well understood. We studied transgenic mice that over express ß-N-acetylglucosaminidase (O-GlcNAcase), an enzyme that catalyzes the removal of O-GlcNAc from proteins, in the pancreatic ß-cell under control of the rat insulin promoter. 3-4-Month-old O-GlcNAcase transgenic mice have higher glucose excursions with a concomitant decrease in circulating insulin levels, insulin mRNA levels, and total islet insulin content. In older (8-9-month-old) O-GlcNAcase transgenic mice glucose tolerance is no longer impaired. This is associated with increased serum insulin, islet insulin content, and insulin mRNA in the O-GlcNAcase transgenic mice. These improvements in ß-cell function with aging are associated with increased angiogenesis and increased VEGF expression, with parallel increases in activation of Akt and expression of PGC1α. The biphasic effects as a function of age are consistent with published observations of mice with increased O-GlcNAc in islets and demonstrate that O-GlcNAc signaling exerts multiple effects on both insulin secretion and islet survival.

Dietary iron restriction or iron chelation protects from diabetes and loss of beta-cell function in the obese (ob/ob lep-/-) mouse.

Iron overload can cause insulin deficiency, but in some cases this may be insufficient to result in diabetes. We hypothesized that the protective effects of decreased iron would be more significant with increased beta-cell demand and stress. Therefore, we treated the ob/ob mouse model of type 2 diabetes with an iron-restricted diet (35 mg/kg iron) or with an oral iron chelator. Control mice were fed normal chow containing 500 mg/kg iron. Neither treatment resulted in iron deficiency or anemia. The low-iron diet significantly ameliorated diabetes in the mice. The effect was long lasting and reversible. Ob/ob mice on the low-iron diet exhibited significant increases in insulin sensitivity and beta-cell function, consistent with the phenotype in mouse models of hereditary iron overload. The effects were not accounted for by changes in weight or feeding behavior. Treatment with iron chelation had a more dramatic effect, allowing the ob/ob mice to maintain normal glucose tolerance for at least 10.5 wk despite no effect on weight. Although dietary iron restriction preserved beta-cell function in ob/ob mice fed a high-fat diet, the effects on overall glucose levels were less apparent due to a loss of the beneficial effects of iron on insulin sensitivity. Beneficial effects of iron restriction were minimal in wild-type mice on normal chow but were apparent in mice on high-fat diets. We conclude that, even at "normal" levels, iron exerts detrimental effects on beta-cell function that are reversible with dietary restriction or pharmacotherapy.

Regulation of Akt signaling by O-GlcNAc in euglycemia.

The hexosamine biosynthesis pathway (HBP) regulates the posttranslational modification of nuclear and cytoplasmic protein by O-linked N-acetylglucosamine (O-GlcNAc). Numerous studies have demonstrated that, in hyperglycemic conditions, excessive glucose flux through this pathway contributes to the development of insulin resistance. The role of the HBP in euglycemia, however, remains largely unknown. Here we investigated the effect of O-GlcNAc on hepatic Akt signaling at physiological concentrations of glucose. In HepG2 cells cultured in 5 mM glucose, removal of O-GlcNAc by adenoviral-mediated overexpression of O-GlcNAcase increased Akt activity and phosphorylation. We also observed that Akt was recognized by succinylated wheat germ agglutinin (sWGA), which specifically binds O-GlcNAc. Overexpression of O-GlcNAcase in HepG2 cells reduced the levels of Akt in sWGA precipitates. The increased Akt activity was accompanied by increased phosphorylation of Akt substrates and reduced mRNA for glucose-6-phosphatase and phosphoenolpyruvate carboxykinase (PEPCK). The increased Akt activity was not a result of activation of its upstream activator phosphoinositide 3-kinase (PI 3-kinase). Further demonstrating Akt regulation by O-GlcNAc, we found that overexpression of O-GlcNAcase in the livers of euglycemic mice also significantly increased Akt activity, resulting in increased phosphorylation of downstream targets and decreased mRNA for glucose-6-phosphatase. Together, these data suggest that O-GlcNAc regulates Akt signaling in hepatic models under euglycemic conditions.

Glucose deprivation stimulates O-GlcNAc modification of proteins through up-regulation of O-linked N-acetylglucosaminyltransferase.

O-Linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of proteins that functions as a nutrient sensing mechanism. Here we report on regulation of O-GlcNAcylation over a broad range of glucose concentrations. We have discovered a significant induction of O-GlcNAc modification of a limited number of proteins under conditions of glucose deprivation. Beginning 12 h after treatment, glucose-deprived human hepatocellular carcinoma (HepG2) cells demonstrate a 7.8-fold increase in total O-GlcNAc modification compared with cells cultured in normal glucose (5 mm; p = 0.008). Some of the targets of glucose deprivation-induced O-GlcNAcylation are distinct from those modified in response to high glucose (20 mm) or glucosamine (10 mm) treatment, suggesting differential targeting with glucose deprivation and glucose excess. O-GlcNAcylation of glycogen synthase is significantly increased with glucose deprivation, and this O-GlcNAc increase contributes to a 60% decrease (p = 0.004) in glycogen synthase activity. Increased O-GlcNAc modification is not mediated by increased UDP-GlcNAc, the rate-limiting substrate for O-GlcNAcylation. Rather, the mRNA for nucleocytoplasmic O-linked N-acetylglucosaminyltransferase (OGT) increases 3.4-fold within 6 h of glucose deprivation (p = 0.006). Within 12 h, OGT protein increases 1.7-fold (p = 0.01) compared with normal glucose-treated cells. In addition, 12-h glucose deprivation leads to a 49% decrease in O-GlcNAcase protein levels (p = 0.03). We conclude that increased O-GlcNAc modification stimulated by glucose deprivation results from increased OGT and decreased O-GlcNAcase levels and that these changes affect cell metabolism, thus inactivating glycogen synthase.

Protein modification by O-linked GlcNAc reduces angiogenesis by inhibiting Akt activity in endothelial cells.

OBJECTIVE: Glucose flux through the hexosamine biosynthesis pathway (HBP) has been implicated in the development of diabetic vascular complications. O-linked N-acetylglucosamine (O-GlcNAc) modification on protein is the major mechanism mediating the actions of the HBP. Impaired angiogenesis is well-recognized in diabetes; however, the mechanisms are not completely defined. Here, we investigated the role of protein O-GlcNAc modification in angiogenesis. METHODS AND RESULTS: In a mouse aortic ring assay, elevated O-GlcNAc levels induced by high-fat diet, streptozotocin-induced diabetes, or in vitro glucosamine treatment were associated with impaired angiogenesis. In cultured human umbilical vein endothelial cells and EA.hy926 endothelial cells, glucosamine increased protein O-GlcNAc modification and inhibited cell migration and capillary-like structure formation. Conversely, removal of O-GlcNAc by adenoviral-mediated overexpression of O-GlcNAcase improved these steps of angiogenesis. Also, high concentrations of glucose reduced capillary-like structure formation of human umbilical vein endothelial cells. Akt was recognized by an O-GlcNAc specific lectin, and glucosamine increased the amounts of Akt protein in these lectin precipitates. Increased glycosylation paralleled reduced Akt activity in endothelial cells. CONCLUSIONS: These results suggest that elevated protein O-GlcNAc modification through the HBP impairs angiogenesis in endothelial cells, possibly by inhibiting Akt signaling.

Increased glucose disposal and AMP-dependent kinase signaling in a mouse model of hemochromatosis.

Hereditary hemochromatosis is an inherited disorder of increased iron absorption that can result in cirrhosis, diabetes, and other morbidities. We have investigated the mechanisms underlying supranormal glucose tolerance despite decreased insulin secretion in a mouse model of hemochromatosis with deletion of the hemochromatosis gene (Hfe(-/-)). Hfe(-/-) mice on 129Sv or C57BL/6J backgrounds have decreased glucose excursions after challenge compared with controls. In the C57BL/6J/ Hfe(-/-), for example, incremental area under the glucose curve is reduced 52% (p < 0.001) despite decreased serum insulin, and homeostasis model assessment insulin resistance is decreased 50% (p < 0.05). When studied by the euglycemic clamp technique 129Sv/Hfe(-/-) mice exhibit a 20% increase in glucose disposal (p < 0.05) at submaximal insulin but no increase at maximal insulin compared with wild types. [1,2-(13)C]D-glucose clearance from plasma is significantly increased in Hfe(-/-) mice (19%, p < 0.05), and lactate derived from glycolysis is elevated 5.1-fold in Hfe(-/-) mice (p < 0.0001). Basal but not insulin-stimulated glucose uptake is elevated in isolated soleus muscle from Hfe(-/-) mice (p < 0.03). Compared with controls Hfe(-/-) mice exhibit no differences in serum lipid, insulin, glucagon, or thyroid hormone levels; adiponectin levels are elevated 41% (p < 0.05), and the adiponectin message in adipocytes is increased 83% (p = 0.04). Insulin action measured by phosphorylation of Akt is not enhanced in muscle, but phosphorylation of AMP-dependent kinase is increased. We conclude that supranormal glucose tolerance in iron overload is characterized by increased glucose disposal that does not result from increased insulin action. Instead, the Hfe(-/-) mice demonstrate increased adiponectin levels and activation of AMP-dependent kinase.

Chronic hexosamine flux stimulates fatty acid oxidation by activating AMP-activated protein kinase in adipocytes.

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.