O-GlcNAcylation of connexin 40: a sweet connection between diabetes and endothelial cell dysfunction? Focus on "O-GlcNAcase overexpression reverses coronary endothelial cell dysfunction in type 1 diabetic mice".

CARDIOVASCULAR COMPLICATIONS are the leading cause of morbidity and mortality in patients with diabetes mellitus. The vascular alterations that develop in diabetic patients relate to the pathogenesis of both macrovascular (increased incidence and severity of stroke and myocardial infarction) and microvascular (neuropathy, nephropathy, retinopathy, and erectile dysfunction) systems. Endothelial dysfunction has been implicated in the development of macroand microvascular diseases. Chronic hyperglycemia is believed to constitute a major contributing factor to the pathogenesis of vascular complications in diabetic patients. Among the various mechanisms that have been involved in the so-called glucotoxicity phenomenon, O-linked N-acetylglucosaminylation (O-GlcNAcylation) of proteins is emerging as an important player (for review see Ref. 5). O-GlcNAcylation is a posttranslational reversible modification that involves the addition of N-acetylglucosamine (GlcNAc) on serine or threonine residues of cytosolic and nuclear proteins. Only two enzymes, O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and -N-acetylglucosaminidase (OGA), regulate O-GlcNAc level on protein according to glucose availability (Fig. 1). O-GlcNAcylation has been shown to control the activity, subcellular localization, and stability of numerous proteins, including transcription factors, metabolic enzymes, and signaling molecules. O-GlcNAcylation can also control the phosphorylation of proteins, either by directly competing with the phosphate group for the same residue (yin-yang mechanism) or by regulating the phosphorylation of nearby residues (5). Nitric oxide (NO), the principal endothelium-dependent relaxing factor, is a key component of vascular homeostasis. In diabetes, endothelial cells fail to produce sufficient amounts of NO and induce vasorelaxation in response to endotheliumdependent vasorelaxants (e.g., acetylcholine, bradykinin, and shear stress) (1). Several studies have implicated O-GlcNAc in the deleterious effects of diabetes on NO production. Endothelial NO synthase (eNOS), the enzyme responsible for NO production in endothelial cells, is activated via its phosphorylation on S1177 by Akt. In bovine and rat aortic endothelial cells, as well as human coronary artery endothelial cells, high glucose resulted in increased O-GlcNAc on eNOS and decreased phosphorylation of the protein on the Akt site, suggesting a reciprocal relationship between eNOS phosphorylation and O-GlcNAcylation (5). In addition, Musicki et al. (10), studying erectile function in diabetic rat penis, also observed increased O-GlcNAcylation of eNOS, associated with decreased phosphorylation on S1177 and reduced erectile response to shear stress or VEGF, providing a potential mechanism for diabetes-associated erectile dysfunction. Moreover, Lima et al. (6), using rat artery segments, showed that OGlcNAcylation induced by O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenylcarbamate (PugNAc), an inhibitor of OGA, blunted vascular reactivity to the vasorelaxing effect of acetylcholine, and this was again associated with decreased S1177 phosphorylation on eNOS. Importantly, these alterations in vascular reactivity were not observed in endothelium-denuded vessels, indicating that the effect of PUGNAc was mediated by endothelial cells (6). Impaired angiogenesis also participates in cardiovascular complications in diabetes, through impaired wound healing, exacerbated peripheral limb ischemia, and cardiac mortality associated with reduced collateral vessel development. Elevated O-GlcNAc levels also inhibited the potency of endothelial cells to migrate and form capillary-like tubes. This effect was mediated, at least in part, by O-GlcNAcylation of Akt, which decreased its phosphorylation and, thereby, its activity (7). Therefore, several lines of evidence argue for a contribution of O-GlcNAcylation to hyperglycemia-associated cardiovascular defects. In this issue of American Journal of PhysiologyCell Physiology, Makino et al. (8) add an additional brick to the wall. To determine whether reducing O-GlcNAcylation levels in mouse coronary endothelial cells (MCECs) may improve endothelial dysfunctions in diabetic rats, Makino et al. generated a tetracycline-inducible endothelium-specific OGA transgenic mouse. In the absence of doxycycline, streptozotocin-induced diabetes resulted in an increase in protein O-GlcNAcylation in MCECs, associated with a decrease in OGA expression level. Doxycycline induction restored OGA expression levels and markedly reduced O-GlcNAcylation in MCECs. Overexpression of OGA also restored to normal levels the reduced capillary density in the ventricle myocardium of diabetic rats, an indicator of oxygen transport efficiency and diffusion ability in the muscular tissue. Moreover, using coronary artery rings from these animals, Makino et al. showed that whereas the effect of acetylcholine on endothelium-dependent relaxation and endothelium-derived hyperpolarization-dependent relaxation was markedly impaired in diabetic mice, OGA overexpression fully restored these endothelial cell functions. Neither diabetes nor OGA overexpression affected endothelium-independent relaxation (evaluated using sodium nitroprusside treatment). In agreement with the studies mentioned previously, Makino et al. (8) found that eNOS O-GlcNAcylation was increased in Address for reprint requests and other correspondence: T. Issad, Cochin Institute, Dept. of Endocrinology, Metabolism and Diabetes, 22 Rue Méchain, 75014 Paris, France (e-mail: [email protected]). Am J Physiol Cell Physiol 309: C590–C592, 2015; doi:10.1152/ajpcell.00260.2015. Editorial Focus