O-Linked N-acetylglucosamine (O-GlcNAc (1-4). In the cell, the HBP converts imported glucose and glucosamine to UDP-GlcNAc. Glutamine:fructose-6-phosphate amidotransferase is the rate-limiting enzyme in this pathway. OGT catalyzes GlcNAc transfer to serine and threonine residues of target proteins, whereas O-O-GlcNAc is known to affect multiple metabolic pathways and has been implicated specifically as a contributor to insulin resistance and type 2 diabetes (5-7). Chronically elevated HBP flux, a result of hyperglycemia, is known to exacerbate metabolic dysregulation in part by targeting metabolic enzymes. For example, in diabetic mice, glycogen synthase (GS) becomes resistant to insulin stimulation as its level of O-GlcNAc modification increases (8, 9). AMP-activated protein kinase is activated in adipocytes with elevated HBP flux, resulting in O-GlcNAc-mediated elevation of fatty acid oxidation (10). To date, the majority of reports of O-GlcNAc-mediated metabolic changes attribute increased O-GlcNAc modification to increased HBP flux. We report a novel and significant induction of O-GlcNAc modification in glucose-deprived HepG2 cells that is independent of increased HBP flux and appears distinct from previously reported stress-induced O-GlcNAc induction. Rather, increased O-GlcNAc with glucose deprivation is mediated by induction of OGT and down-regulation of O-GlcNAcase. Increased O-GlcNAcylation of GS in these conditions contributes to decreased GS activity.

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.

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, streptozotocininduced diabetes, or in vitro glucosamine treatment were associated with impaired angiogenesis. Key Words: hexosamine Ⅲ angiogenesis Ⅲ O-GlcNAc Ⅲ endothelial cells Ⅲ Akt V ascular complications are the leading cause for morbidity and mortality in diabetic patients, and hyperglycemia is the primary factor in their pathogenesis. 1,2 Angiogenesis, the formation of new blood vessels out of preexisting capillaries, appears to play a pivotal role in the development of diabetic vascular complications. 3,4 Clinical and animal studies have demonstrated abnormally enhanced angiogenesis in the retina, leading to diabetic retinopathy. 3 At the same time, impaired angiogenesis in diabetes often leads to reduced wound healing, exacerbated peripheral limb ischemia, and cardiac mortality through reduced collateral vessel development. 5,6 See accompanying article on page 608The effects of hyperglycemia on angiogenesis are not completely understood. Glucose metabolism through the hexosamine biosynthesis pathway (HBP) has been implicated in many of the adverse effects of hyperglycemia, such as insulin resistance in peripheral tissues and diabetic vascular complications. [7][8][9] In this pathway, a relatively small amount of cellular glucose flux is converted to UDP-Nacetylglucosamine (UDP-GlcNAc) and other amino sugars. The rate-limiting step is catalyzed by glutamine:fructose-6-phosphate amidotransferase (GFA). The levels of the product UDP-GlcNAc are proportional to cellular glucose flux and thus able to serve a nutrient sensing function. UDP-GlcNAc, the chief product of the pathway, is the substrate for O-glycosyltransferase (OGT), which catalyzes the O-linked glycosylation of nuclear and cytosolic proteins with an N-acetylglucosamine (O-GlcNAc) moiety on serine and threonine residues. O-GlcNAc can be removed by ␤-O-linked N-acetylglucosaminidase (O-GlcNAcase). 7 The O-GlcNAc modification has been observed in numerous proteins and is thought as the main mechanism mediating the nutrient sensing function of the HBP. 9,10 This posttranslational modification modulates protein function in a manner analogous to protein phosphorylation. Indeed, phosphorylation and O-GlcNAc are reciprocal on some well-studied proteins, such as transcription factor Sp1, 11 glycogen synthase, 12 and endothelial nitric oxide synthase (eNOS), 13 further supporting the dynamic significance of the O-GlcNAc modification.It has been reported that O-GlcNAc levels are elevated in coronary endothelial ...

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.Although there is a major genetic contribution to type 2 diabetes, the largest predisposing factor remains caloric excess and/or obesity. Underlining the importance of this mechanism, excess glucose and lipids themselves can cause the pathological hallmarks of diabetes, insulin resistance, and ␤-cell failure. One pathway by which excess nutrients can contribute to the diabetic phenotype is the hexosamine biosynthesis pathway (HBP) 2 (1-4). In this pathway, a relatively small amount of cellular glucose flux is converted to UDP-GlcNAc and other amino sugars. The rate-limiting step is catalyzed by enzyme glutamine:fructose-6-phosphate amidotransferase (GFA), and the levels of the product UDP-GlcNAc are proportional to cellular glucose flux and thus able to serve a nutrient sensing function. In the short run, hexosamines function as physiologic glucose sensors that serve an adaptive role in directing excess calories toward storage as fat. When chronically stimulated, however, the HBP can also lead to insulin resistance, hyperinsulinemia, hyperlipidemia, and hyperleptinemia (2, 3, 5-7).UDP-GlcNAc, the chief product of the pathway, is the substrate for O-glycosyltransferase, which catalyzes the O-linked glycosylation of nuclear and cytosolic proteins with a single N-acetylglucosamine (O-GlcNAc) moiety on serine and threonine residues (8 -11). It...

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.