Insulin resistance of glycogen synthase mediated by o-linked N-acetylglucosamine

We have investigated the mechanism by which high concentrations of glucose inhibit insulin stimulation of glycogen synthase. In NIH-3T3-L1 adipocytes cultured in low glucose (LG; 2.5 mm), the half-maximal activation concentration (A(0.5)) of glucose 6-phosphate was 162 +/- 15 microm. Exposure to either high glucose (HG; 20 mm) or glucosamine (GlcN; 10 mm) increased the A(0.5) to 558 +/- 61 or 612 +/- 34 microm. Insulin treatment with LG reduced the A(0.5) to 96 +/- 10 microm, but cells cultured with HG or GlcN were insulin-resistant (A(0.5) = 287 +/- 27 or 561 +/- 77 microm). Insulin resistance was not explained by increased phosphorylation of synthase. In fact, culture with GlcN decreased phosphorylation to 61% of the levels seen in cells cultured in LG. Hexosamine flux and subsequent enzymatic protein O-glycosylation have been postulated to mediate nutrient sensing and insulin resistance. Glycogen synthase is modified by O-linked N-acetylglucosamine, and the level of glycosylation increased in cells treated with HG or GlcN. Treatment of synthase in vitro with protein phosphatase 1 increased basal synthase activity from cells cultured in LG to 54% of total activity but was less effective with synthase from cells cultured in HG or GlcN, increasing basal activity to only 13 or 16%. After enzymatic removal of O-GlcNAc, however, subsequent digestion with phosphatase increased basal activity to over 73% for LG, HG, and GlcN. We conclude that O-GlcNAc modification of glycogen synthase results in the retention of the enzyme in a glucose 6-phosphate-dependent state and contributes to the reduced activation of the enzyme in insulin resistance.     

Hyperglycemia reduces nitric oxide synthase and glycogen synthase activity in endothelial cells.

Hyperglycemia is considered a primary cause of diabetic vascular complications. A hallmark of vascular disease is endothelial cell dysfunction characterized by diminished nitric-oxide (NO)-dependent phenomena such as vasodilation, angiogenesis, and vascular maintenance. This study was designed to investigate the effects of a high level of D-glucose on endothelial NO response, oxidative stress, and glucose metabolism. Bovine aortic endothelial cells (BAECs) were pretreated with a high concentration of glucose (HG) (22 mmol/L) for at least 2 weeks and compared with control cells exposed to 5 mmol/L glucose (NG). The effect of chronic hyperglycemia on endothelial NO-synthase (eNOS) activity and expression, glycogen synthase (GS) activity, extracellular-signal-regulated kinase (ERK 1,2), p38, Akt expression, and Cu/Zn superoxide-dismutse (SOD-1) activity and expression were determined. Western blot analysis showed that eNOS protein expression decreased in HG cells and was accompanied by diminished eNOS activity. The activity of GS was also significantly lower in the HG cells than in NG cells, 25.0+/-17.4 and 89+/-22.5 nmol UDP-glucose.mg protein(-1)x min(-1), respectively. Western blot analysis revealed a 40-60% decrease in ERK 1,2 and p38 protein levels, small modification of phosphorylated Akt expression, and a 30% increase in SOD-1 protein expression in HG cells. Although SOD expression was increased, no change was observed in SOD activity. These results support the findings that vascular dysfunction due to exposure to pathologically high D-glucose concentrations may be caused by impairment of the NO pathway and increased oxidative stress accompanied by altered glucose metabolism.     

Evidence of O-linked N-acetylglucosamine in diabetic nephropathy

AimsThere is increasing evidence that O-linked N-acetylglucosamine (O-GlcNAc) plays an important role in cell signaling pathways. It has also been reported that increases in O-GlcNAc contribute to the development of diabetes and diabetic complications; however, little is known about O-GlcNAc levels in diabetic nephropathy (DNP). Therefore the goal of this study was to determine whether O-GlcNAc could be detected in human kidney biopsy specimens, and if so to examine whether O-GlcNAc levels were increased in the kidneys of patients with DNP compared to the non-diabetic individuals.Main methodsKidney biopsy specimens were obtained from type-2 diabetic patients (n = 6) and patients diagnosed with thin basement membrane nephropathy (n = 7) were used as non-diabetic controls. O-GlcNAc levels were assessed by immunohistochemistry using the anti-O-GlcNAc antibody CTD110.6.Key findingsWe show that O-GlcNAc modification of proteins can be detected in the human kidney biopsy specimens. Furthermore, in diabetic patients, we found significantly increased numbers of O-GlcNAc positive cells in the glomeruli and significantly elevated staining in the tubuli (both in the nucleus and in the cytosol). In addition we also observed an intense, granular O-GlcNAc staining specifically in diabetic tubuli.SignificanceIn light of the increase in O-GlcNAc staining in the diabetic patients, we propose that increased O-GlcNAc levels might contribute to the development of diabetic nephropathy.     

Topology of O-linked N-acetylglucosamine in murine lymphocytes

A unique form of protein glycosylation in which N-acetylglucosamine monosaccharides are O-glycosidically linked to serine or threonine residues (O-GlcNAc) was initially reported in studies that used purified bovine milk galactosyltransferase to exogenously probe living populations of murine lymphocytes. However, in this same study, detergent latency experiments surprisingly indicated that unlike other known forms of protein glycosylation, O-GlcNAc-modified proteins occur predominantly intracellularly. Since we now know that as little as 5% lysis could have accounted for the putative cell surface O-GlcNAc seen in these earlier studies, and also in the light of recent data on the subcellular localization of the O-GlcNAc glycosyltransferase(s), we decided to critically reexamine the topology and polypeptide distribution of O-GlcNAc in primary cultures of murine lymphocytes that were prepared using improved cell selection techniques that do not involve complement-mediated lysis for cell enrichment. We have also examined two well-characterized T cell hybridoma lines. Under these highly stringent conditions of cell viability, we were unable to detect O-GlcNAc bearing proteins on the cell surfaces of any of these cell types. Also, O-GlcNAc was found on a similar subset of proteins in all of the various lymphocyte cell types. These data suggest that O-GLcNAc is highly restricted to the cytoplasmic/nucleoplasmic compartment of the cell and is found on a similar subset of nuclear and cytoplasmic proteins in functionally different types of lymphocytes.     

O-连接的N-乙酰葡糖胺糖蛋白的研究进展

O-连接的N-乙酰葡糖胺(O-GlcNAc)修饰是普遍存在的翻译后修饰.已有许多的蛋白被发现是O-GlcNAc蛋白.目前,许多分析方法可以检测O-GlcNAc,将其从内膜系统的多种糖基化中区分出来.O-GlcNAc修饰在细胞事件中发挥着重要的功能,O-GlcNAc的调控异常可能会引起某些人类疾病,如癌症、阿尔茨海默病和II型糖尿病.杆状病毒GP41蛋白也是糖蛋白,它介导芽殖型病毒粒子(budded virus,BV)的核衣壳通过胞核.O-GlcNAc的调控研究为探讨GP41蛋白O-GlcNAc的调控作用提供了参考模式.     

Genetic correlational analysis of glycogen synthase kinase-3 beta and prepulse inhibition in inbred mice

In humans, GSK-3β activity is diminished in schizophrenic patients as is prepulse inhibition of the startle response (PPI). We performed a genetic correlational analysis between published PPI values and frontal cortex GSK-3 activity analyzed in our laboratory in 10 inbred mouse strains. This methodology could indicate relevant parameters for study in an animal model. Indeed, we obtained significant correlations between the enzyme's activity and PPI measured by two different methods. This may indicate that investigation of the genetics of GSK-3β regulation holds promise for understanding some of the biochemical underpinnings of schizophrenia.     

COX-2 inhibition prevents insulin-dependent diabetes in low-dose streptozotocin-treated mice

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease believed to be caused by an inflammatory process in the pancreas leading to selective destruction of the beta cells. Inducible cyclooxygenase (COX-2) is expressed under inflammatory conditions and its product prostaglandin E(2) (PGE(2)) is an important inflammation mediator. We report here that administration of the selective COX-2 inhibitor NS-398 prevents the onset of diabetes in mice brought on by multiple low-doses of streptozotocin (STZ). Histological observations indicated that STZ-mediated destruction of beta cells was prevented by NS-398 treatment. Delayed (day 3) administration of NS-398 was also protective in this model. No protective effect was observed when NS-398 was administered prior to a high, toxic dose of STZ. These results demonstrate the critical importance of COX-2 activity in autoimmune destruction of beta cells, and point to the fact that COX-2 inhibition can potentially develop into a preventive therapy against IDDM.     

A novel post-translational modification in nerve terminals: O-linked N-acetylglucosamine phosphorylation

Protein phosphorylation and glycosylation are the most common post-translational modifications observed in biology, frequently on the same protein. Assembly protein AP180 is a synapse-specific phosphoprotein and O-linked beta-N-acetylglucosamine (O-GlcNAc) modified glycoprotein. AP180 is involved in the assembly of clathrin coated vesicles in synaptic vesicle endocytosis. Unlike other types of O-glycosylation, O-GlcNAc is nucleocytoplasmic and reversible. It was thought to be a terminal modification, that is, the O-GlcNAc was not found to be additionally modified in any way. We now show that AP180 purified from rat brain contains a phosphorylated O-GlcNAc (O-GlcNAc-P) within a highly conserved sequence. O-GlcNAc or O-GlcNAc-P, but not phosphorylation alone, was found at Thr-310. Analysis of synthetic GlcNAc-6-P produced identical fragmentation products to GlcNAc-P from AP180. Direct O-linkage of GlcNAc-P to a Thr residue was confirmed by electron transfer dissociation MS. A second AP180 tryptic peptide was also glycosyl phosphorylated, but the site of modification was not assigned. Sequence similarities suggest there may be a common motif within AP180 involving glycosyl phosphorylation and dual flanking phosphorylation sites within 4 amino acid residues. This novel type of protein glycosyl phosphorylation adds a new signaling mechanism to the regulation of neurotransmission and more complexity to the study of O-GlcNAc modification.     

Methadone inhibition of glycogen synthase and phosphorylase in rat skeletal muscle

The activities of glycogen synthase (I and total) and phosphorylase ($\text{a}$ and total) in crude extracts of isolated extensor digitorum longus and soleus muscles of the rat incubated $\text{in vitro}$ in the absence or presence of methadone were very low. Addition of glycogen during homogenization increased the activities of both enzymes in control muscles. Even at optimal concentrations of glycogen, however, the activities of both enzymes from methadone-treated muscles were significantly lower than their activities in control muscles. The activity of phosphoglucomutase was not altered by incubation with methadone or by homogenization with glycogen. It is suggested that the addition of optimal amounts of glycogen during extraction of the enzymes enhances the extractability of glycogen synthase and increases the activity of phosphorylase by some other mechanism and that these processes are interfered with when the muscles are pretreated with methadone.     

Alloxan is an inhibitor of the enzyme O-linked N-acetylglucosamine transferase

We have previously shown that diabetogenic antibiotic streptozotocin (STZ), an analog of N-acetylglucosamine (GlcNAc), inhibits the enzyme O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase (O-GlcNAcase) which is responsible for the removal of O-GlcNAc from proteins. Alloxan, another beta-cell toxin is a uracil analog. Since the O-GlcNAc transferase (OGT) uses UDP-GlcNAc as a substrate, we investigated whether alloxan might interfere with the process of protein O-glycosylation by blocking OGT, a very abundant enzyme in beta-cells. In isolated pancreatic islets, alloxan almost completely blocked both glucosamine-induced and STZ-induced protein O-GlcNAcylation, suggesting that alloxan indeed was inhibiting (OGT). In order to show definitively that alloxan was inhibiting OGT activity, recombinant OGT was incubated with 0-10 mM alloxan, and OGT activity was measured directly by quantitating UDP-[(3)H]-GlcNAc incorporation into the recombinant protein substrate, nucleoporin p62. Under these conditions, OGT activity was completely inhibited by 1 mM alloxan with half-maximal inhibition achieved at a concentration of 0.1 mM alloxan. Together, these data demonstrate that alloxan is an inhibitor of OGT, and as such, is the first OGT inhibitor described.     

Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine

beta-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification of resident nuclear and cytoplasmic proteins in eukaryotes. Increasing evidence suggests that O-GlcNAc plays a regulatory role in numerous cellular processes. Here we report on the production and characterization of a highly specific mouse monoclonal antibody, MAb CTD110.6, that specifically reacts with O-GlcNAc. The antibody recognizes O-GlcNAc in beta-O-glycosidic linkage to both serine and threonine. We could detect no cross-reactivity with alpha-linked Ser/Thr-O-GlcNAc, alpha-linked Ser-O-linked N-acetylgalactosamine (O-GalNAc), or N-linked oligosaccharides on ovalbumin and immunoglobulin G. The monosaccharide GlcNAc, but not GalNAc, abolishes immunoreactivity, further demonstrating specificity toward O-GlcNAc. Furthermore, galactose capping of O-GlcNAc sites also inhibits CTD110.6 immunoreactivity. Enrichment of GlcNAc-containing glycoproteins using the lectin wheat germ agglutinin dramatically enriches for CTD110.6-reactive proteins. The antibody reacts with a large number of proteins from cytoplasmic and nuclear extracts and readily detects in vivo changes in O-GlcNAc modification. These studies demonstrate that CTD110.6 is highly specific toward O-GlcNAc, with no cross-reactivity toward similar carbohydrate antigens or toward peptide determinants.     

Exercise capacity of mice genetically lacking muscle glycogen synthase: in mice, muscle glycogen is not essential for exercise

The glucose storage polymer glycogen is generally considered to be an important source of energy for skeletal muscle contraction and a factor in exercise endurance. A genetically modified mouse model lacking muscle glycogen was used to examine whether the absence of the polysaccharide affects the ability of mice to run on a treadmill. The MGSKO mouse has the GYS1 gene, encoding the muscle isoform of glycogen synthase, disrupted so that skeletal muscle totally lacks glycogen. The morphology of the soleus and quadriceps muscles from MGSKO mice appeared normal. MGSKO-null mice, along with wild type littermates, were exercised to exhaustion. There were no significant differences in the work performed by MGSKO mice as compared with their wild type littermates. The amount of liver glycogen consumed during exercise was similar for MGSKO and wild type animals. Fasting reduced exercise endurance, and after overnight fasting, there was a trend to reduced exercise endurance for the MGSKO mice. These studies provide genetic evidence that in mice muscle glycogen is not essential for strenuous exercise and has relatively little effect on endurance.     

O-linked N-acetylglucosamine is present on the extracellular domain of notch receptors

Rare types of glycosylation often occur in a domain-specific manner and are involved in specific biological processes. In particular, O-fucose glycans are reported to regulate the functions of EGF domain-containing proteins such as Notch receptors. In the course of mass spectrometric analysis of O-glycans displayed on Drosophila Notch receptors expressed in S2 cells, we found an unusual O-linked N-acetylhexosamine (HexNAc) modification which occurs at a site distinct from those of O-fucose and O-glucose glycosylations. Modification site mapping by mass spectrometry and amino acid substitution studies revealed that O-HexNAc modification occurs on a serine or threonine located between the fifth and sixth cysteines within the EGF domain. This modification occurs simultaneously along with other closely positioned O-glycosylations. This modification was determined to be O-beta-GlcNAc by galactosyltransferase labeling and beta-N-acetyl-hexosaminidase digestion experiments and by immunoblotting with a specific antibody. O-GlcNAc modification occurs at multiple sites on Notch epidermal growth factor repeats. O-GlcNAc modification was also found on the extracellular domain of Delta, a ligand for Notch receptors. Although the O-GlcNAc modification is known to regulate a wide range of cellular processes, the list of known modified proteins has previously been limited to intracellular proteins in animals. Thus, the finding of O-GlcNAc modification in extracellular environments predicts a distinct glycosylation process that might be associated with a novel regulatory mechanism for Notch receptor activity.     

Hexokinase 2, glycogen synthase and phosphorylase play a key role in muscle glycogen supercompensation

Background

Glycogen-depleting exercise can lead to supercompensation of muscle glycogen stores, but the biochemical mechanisms of this phenomenon are still not completely understood.

Methods

Using chronic low-frequency stimulation (CLFS) as an exercise model, the tibialis anterior muscle of rabbits was stimulated for either 1 or 24 hours, inducing a reduction in glycogen of 90% and 50% respectively. Glycogen recovery was subsequently monitored during 24 hours of rest.

Results

In muscles stimulated for 1 hour, glycogen recovered basal levels during the rest period. However, in those stimulated for 24 hours, glycogen was supercompensated and its levels remained 50% higher than basal levels after 6 hours of rest, although the newly synthesized glycogen had fewer branches. This increase in glycogen correlated with an increase in hexokinase-2 expression and activity, a reduction in the glycogen phosphorylase activity ratio and an increase in the glycogen synthase activity ratio, due to dephosphorylation of site 3a, even in the presence of elevated glycogen stores. During supercompensation there was also an increase in 5′-AMP-activated protein kinase phosphorylation, correlating with a stable reduction in ATP and total purine nucleotide levels.

Conclusions

Glycogen supercompensation requires a coordinated chain of events at two levels in the context of decreased cell energy balance: First, an increase in the glucose phosphorylation capacity of the muscle and secondly, control of the enzymes directly involved in the synthesis and degradation of the glycogen molecule. However, supercompensated glycogen has fewer branches.