Although resveratrol has widely been studied for its potential health benefits, little is known about its metabolic effects in humans. Our aims were to determine whether the polyphenol resveratrol improves insulin sensitivity in type 2 diabetic patients and to gain some insight into the mechanism of its action. After an initial general examination (including blood chemistry), nineteen patients enrolled in the 4-weeklong double-blind study were randomly assigned into two groups: a resveratrol group receiving oral 2 £ 5 mg resveratrol and a control group receiving placebo. Before and after the second and fourth weeks of the trial, insulin resistance/sensitivity, creatinine-normalised ortho-tyrosine level in urine samples (as a measure of oxidative stress), incretin levels and phosphorylated protein kinase B (pAkt):protein kinase B (Akt) ratio in platelets were assessed and statistically analysed. After the fourth week, resveratrol significantly decreased insulin resistance (homeostasis model of assessment for insulin resistance) and urinary ortho-tyrosine excretion, while it increased the pAkt:Akt ratio in platelets. On the other hand, it had no effect on parameters that relate to b-cell function (i.e. homeostasis model of assessment of b-cell function). The present study shows for the first time that resveratrol improves insulin sensitivity in humans, which might be due to a resveratrol-induced decrease in oxidative stress that leads to a more efficient insulin signalling via the Akt pathway.Key words: Resveratrol: Type 2 diabetes: Insulin sensitivity: Oxidative stress: Akt Despite the rather high average daily fat intake in France, compared with other European countries, epidemiological surveys document a relatively low rate of cardiovascular mortality. This phenomenon, which is often called the French paradox, is thought to be explained by a fairly high red wine consumption by the French (1,2) . Red wine is known to be rich in various polyphenolic compounds that might have a variety of health benefits. Among these polyphenols, the stilbene derivative resveratrol seems to be the most vigorously studied, which is probably due to the fact that it apparently affects a wide array of physiological and biochemical processes as shown in animal and cell culture studies (3) . On the other hand, human studies with conclusive results on resveratrol are regrettably lacking.Resveratrol is considered to have beneficial effects on the cardiovascular system, as it has been found to improve vasodilatation (4) , ischaemic preconditioning (5,6) , both of which seem to be the result of the activation of the endothelial NO synthase enzyme (7) , and to inhibit both platelet aggregation (3) and vascular smooth muscle cell proliferation (8) . In addition, resveratrol has also been demonstrated to show anti-inflammatory (9) and anti-tumour activities (3) , and it might even have considerable anti-ageing properties as it provokes changes in cell signalling that mimics those found upon energy restriction (3) .Oxidative stress, whi...

The cardiomyocyte circadian clock directly regulates multiple myocardial functions in a time-of-day-dependent manner, including gene expression, metabolism, contractility, and ischemic tolerance. These same biological processes are also directly influenced by modification of proteins by monosaccharides of O-linked ␤-N-acetylglucosamine (O-GlcNAc). Because the circadian clock and protein O-GlcNAcylation have common regulatory roles in the heart, we hypothesized that a relationship exists between the two. We report that total cardiac protein O-GlcNAc levels exhibit a diurnal variation in mouse hearts, peaking during the active/awake phase. Genetic ablation of the circadian clock specifically in cardiomyocytes in vivo abolishes diurnal variations in cardiac O-GlcNAc levels. These time-ofday-dependent variations appear to be mediated by clock-dependent regulation of O-GlcNAc transferase and O-GlcNAcase protein levels, glucose metabolism/uptake, and glutamine synthesis in an NAD-independent manner. We also identify the clock component Bmal1 as an O-GlcNAc-modified protein.Increasing protein O-GlcNAcylation (through pharmacological inhibition of O-GlcNAcase) results in diminished Per2 protein levels, time-of-day-dependent induction of bmal1 gene expression, and phase advances in the suprachiasmatic nucleus clock. Collectively, these data suggest that the cardiomyocyte circadian clock increases protein O-GlcNAcylation in the heart during the active/ awake phase through coordinated regulation of the hexosamine biosynthetic pathway and that protein O-GlcNAcylation in turn influences the timing of the circadian clock.Circadian clocks have emerged as critical regulators of energy metabolism (1, 2). Animal models wherein components of these cell autonomous mechanisms are genetically manipulated invariably exhibit altered energy balance, resulting in overt metabolic phenotypes (e.g. obesity or leanness). This concept is exemplified when either Clock or Bmal1 (two transcription factors at the core of the mammalian clock) are disrupted; Clock⌬19 mutant mice are obesity-prone, whereas Bmal1 null mice are lean (3, 4). Appreciation for links between circadian clocks and metabolism has grown further through demonstration that perturbations in metabolism (e.g. changes in nutrient availability, models of obesity, and diabetes mellitus, etc.) in turn influence the clock mechanism (5-7). Collectively, these observations have fueled identification of a number of posttranslational mediators that facilitate the interdependence of circadian clocks with metabolism. These include phosphorylation, ubiquitination, acetylation, and ribosylation of critical clock and/or metabolic components in time-of-day-dependent manners (1, 8 -14).Defining the role of a specific cell autonomous circadian clock in metabolic regulation through the use of animal models wherein clock components are genetically altered in a ubiquitous fashion is often hampered by the fact that time-of-day-dependent rhythms are altered at multiple levels (e.g. behavioral, neurohum...

The posttranslational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide ␤-N-acetylglucosamine (O-GlcNAc) is a highly dynamic and ubiquitous protein modification. Protein O-GlcNAcylation is rapidly emerging as a key regulator of critical biological processes including nuclear transport, translation and transcription, signal transduction, cytoskeletal reorganization, proteasomal degradation, and apoptosis. Increased levels of O-GlcNAc have been implicated as a pathogenic contributor to glucose toxicity and insulin resistance, which are both major hallmarks of diabetes mellitus and diabetes-related cardiovascular complications. Conversely, there is a growing body of data demonstrating that the acute activation of O-GlcNAc levels is an endogenous stress response designed to enhance cell survival. Reports on the effect of altered O-GlcNAc levels on the heart and cardiovascular system have been growing rapidly over the past few years and have implicated a role for O-GlcNAc in contributing to the adverse effects of diabetes on cardiovascular function as well as mediating the response to ischemic injury. Here, we summarize our present understanding of protein O-GlcNAcylation and its effect on the regulation of cardiovascular function. We examine the pathways regulating protein O-GlcNAcylation and discuss, in more detail, our understanding of the role of O-GlcNAc in both mediating the adverse effects of diabetes as well as its role in mediating cellular protective mechanisms in the cardiovascular system. In addition, we also explore the parallels between O-GlcNAc signaling and redox signaling, as an alternative paradigm for understanding the role of O-GlcNAcylation in regulating cell function. hexosamine biosynthesis; protein O-glycosylation; ␤-N-acetylglucosamine transferase; diabetes mellitus POSTTRANSLATIONAL MODIFICATION (PTM) of proteins is a common mechanism for the modulation of protein function, location, and turnover. Although protein phosphorylation is probably the most widely studied form of PTM, there are many other PTMs, including acylation, ubiquitylation, methylation, acetylation, thiolation, nitration, and glycosylation (107). The focus of this review is protein glycosylation, specifically, O-glycosylation of nuclear and cytoplasmic proteins. Classical protein glycosylation occurs in the endoplasmic reticulum and Golgi, leading to the formation of stable and complex elongated oligosaccharide structures via both N-linkage on asparagine and O-linkage on the hydroxy amino acids serine and threonine in addition to hydroxyproline, hydroxylysine, and tyrosine residues of proteins that become secreted or membrane component glycoproteins (189). In contrast, glycosylation of nuclear and cytoplasmic proteins is a rapid and dynamic modification of serine or threonine residues by the O-linked attachment of the monosaccharide ␤-N-acetylglucosamine (OGlcNAc) (97,195); this process is referred to as protein O-GlcNAcylation to contrast it wi...

ObjectiveThe hexosamine biosynthesis pathway (HBP) flux and protein O-linked N-acetyl-glucosamine (O-GlcNAc) levels have been implicated in mediating the adverse effects of diabetes in the cardiovascular system. Activation of these pathways with glucosamine has been shown to mimic some of the diabetes-induced functional and structural changes in the heart; however, the effect on cardiac metabolism is not known. Therefore, the primary goal of this study was to determine the effects of glucosamine on cardiac substrate utilization.MethodsIsolated rat hearts were perfused with glucosamine (0–10 mM) to increase HBP flux under normoxic conditions. Metabolic fluxes were determined by 13C-NMR isotopomer analysis; UDP-GlcNAc a precursor of O-GlcNAc synthesis was assessed by HPLC and immunoblot analysis was used to determine O-GlcNAc levels, phospho- and total levels of AMPK and ACC, and membrane levels of FAT/CD36.ResultsGlucosamine caused a dose dependent increase in both UDP-GlcNAc and O-GlcNAc levels, which was associated with a significant increase in palmitate oxidation with a concomitant decrease in lactate and pyruvate oxidation. There was no effect of glucosamine on AMPK or ACC phosphorylation; however, membrane levels of the fatty acid transport protein FAT/CD36 were increased and preliminary studies suggest that FAT/CD36 is a potential target for O-GlcNAcylation.Conclusion/InterpretationThese data demonstrate that acute modulation of HBP and protein O-GlcNAcylation in the heart stimulates fatty acid oxidation, possibly by increasing plasma membrane levels of FAT/CD36, raising the intriguing possibility that the HBP and O-GlcNAc turnover represent a novel, glucose dependent mechanism for regulating cardiac metabolism.

No abstract

Laczy B, Marsh SA, Brocks CA, Wittmann I, Chatham JC. Inhibition of O-GlcNAcase in perfused rat hearts by NAG-thiazolines at the time of reperfusion is cardioprotective in an O-GlcNAc-dependent manner. Acute increases in O-linked ␤-N-acetylglucosamine (O-GlcNAc) levels of cardiac proteins exert protective effects against ischemiareperfusion (I/R) injury. One strategy to rapidly increase cellular O-GlcNAc levels is inhibition of O-GlcNAcase (OGA), which catalyzes O-GlcNAc removal.Here we tested the cardioprotective efficacy of two novel and highly selective OGA inhibitors, the NAGthiazoline derivatives NAG-Bt and NAG-Ae. Isolated perfused rat hearts were subjected to 20 min global ischemia followed by 60 min reperfusion. At the time of reperfusion, hearts were assigned to the following four groups: 1) untreated control; 2) 50 M NAG-Bt; 3) 100 M NAG-Bt; or 4) 50 M NAG-Ae. All treatment groups significantly increased total O-GlcNAc levels (P Ͻ 0.05 vs. control), and this was significantly correlated with improved contractile function and reduced cardiac troponin I release (P Ͻ 0.05). Immunohistochemistry of normoxic hearts showed intense nuclear O-GlcNAc staining and higher intensity at Z-lines with colocalization of OGlcNAc and the Z-line proteins desmin and vinculin. After I/R, there was a marked loss of both cytosolic and nuclear O-GlcNAcylation and disruption of normal striated Z-line structures. OGA inhibition largely preserved structural integrity and attenuated the loss of OGlcNAcylation; however, nuclear O-GlcNAc levels remained low. Immunoblot analysis confirmed ϳ50% loss in both nuclear and cytosolic O-GlcNAcylation following I/R, which was significantly attenuated by OGA inhibition (P Ͻ 0.05). These data provide further support for the notion that increasing cardiac O-GlcNAc levels by inhibiting OGA may be a clinically relevant approach for ischemic cardioprotection, in part, by preserving the integrity of O-GlcNAcassociated Z-line protein structures.is increasingly recognized as a critical signaling mechanism regulating a diverse range of biological processes in mammalian cells (3,19,44). This attachment of O-GlcNAc to Ser/Thr residues of proteins is frequently considered to be analogous to protein phosphorylation in that it is a highly dynamic, reversible, and tightly regulated enzyme-catalyzed process. Sustained activation of the hexosamine biosynthesis pathway (HBP) and the resulting increase in O-GlcNAcylation has been implicated in the etiology of glucotoxicity and insulin resistance. However, there is rapidly emerging evidence demonstrating that acute activation of these pathways affords protection against a wide range of injury, including cardioprotection against ischemia-reperfusion (I/R) injury in different biological systems (4, 5, 17, 21).We have previously reported that pretreatment with high glucose or glucosamine attenuated cell death following hypoxia-reoxygenation in isolated cardiomyocytes (4, 5). We also demonstrated that administration of glucosamine and glutamine before ischemia resul...

The difference between para-tyrosine levels of the groups is probably due to renal impairment, while there is indirect evidence for an increased tubular secretion or production of ortho-tyrosine in the kidney in diabetic patients with or without CKD.

Endothelial nitric oxide synthase (eNOS) is regulated by phosphorylation of Ser(1177) and Thr(495), which affects NO bioavailability. Cigarette smoke disturbs the eNOS-cGMP-NO pathway and causes decreased NO production. Here the authors investigated the acute effects of cigarette smoke on eNOS phosphorylation, focusing on protein kinases (PKs). Endothelial cell culture was concentration- and time-dependently treated first with cigarette smoke buffer (CSB), then with reduced glutathione (GSH) or various PK inhibitors (H-89, LY-294002, Ro-318425, and ruboxistaurin). eNOS, phospho-Ser(1177)-eNOS, phospho-Thr(495)-eNOS, Akt(PKB), and phospho-Akt protein levels were determined by Western blot. CSB increased the phosphorylation of eNOS at Ser(1177) and more at Thr(495) in a concentration- and time-dependent manner (p < .01, p < .05 versus control, respectively) and resulted in the dissociation of the active dimeric form of eNOS (p < .05). GSH decreased the phosphorylation of eNOS at both sites (p < .05 versus CSB without GSH) and prevented the decrease of dimer eNOS level. CSB treatment also decreased the level of phospho-Ser(473)-Akt (p < .05 versus control). Inhibition of PKA by H-89 did not affect CSB-induced phosphorylation, whereas the PKB inhibitor LY-294002 enhanced it at Ser(1117). The PKC blockers Ro-318425 and ruboxistaurin augmented the CSB-induced phosphorylation at Ser(1177) but decreased phosphorylation at Thr(495) (p < .05 versus CSB). Cigarette smoke causes a disruption of the enzymatically active eNOS dimers and shifts the eNOS phosphorylation to an inhibitory state. Both effects might lead to reduced NO bioavailability. The shift of the eNOS phosphorylation pattern to an inhibitory state seems to be independent of the PKA and phosphoinositol 3-kinase (PI3-K)/Akt pathways, whereas PKC appears to play a key role.