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  • The post translational modification O GlcNAcylation is


    The post-translational modification O-GlcNAcylation is also dependent upon glucose fluctuations. O-GlcNAcylation has been linked to insulin resistance and glucose toxicity in diabetes and its comorbidities [11]. O-GlcNAcylation is a dynamic modification tightly regulated by the nutritional sensor UDP-GlcNAc levels. This glycosylation consists of the addition of N-acetyl-beta-d-glucosamine through an O-linkage to serine or threonine of numerous nuclear, cytosolic and mitochondrial proteins [12], [13]. The reaction is catalyzed by the O-linked N-acetylglucosamine transferase (OGT) and dynamically reversed by the N-acetylglucosaminidase (OGA) [14]. Interestingly, O-GlcNAc is analogous to phosphorylation, with which it can competes for the same or adjacent phosphorylation sites [15]. Identified on over 4000 proteins, O-GlcNAcylation plays a critical role in the regulation of Conessine receptor progression and proliferation [16], [17], stress response [18], [19], protein turn over [20] and transcription [21]. Nevertheless, disturbance of O-GlcNAcylation modification on proteins is implicated in the emergence of several pathologies such as cancer, neurodegenerative diseases and diabetes [22]. In this study, we investigated the nutrient-dependent modification of hepatic glucokinase by O-GlcNAcylation and its role on GCK expression in physiological and pathological conditions (hyperglycemia and insulin resistance). The present study leads to the discovery of a new nutrient-dependent regulation of GCK, improving basic knowledge of this critical glucose homeostasis enzyme, and may be critical to optimize the design of novel molecules for treating type 2 diabetes and other metabolic pathologies.
    Material and methods
    Discussion GCK plays a critical role in glucose homeostasis by mediating efficient hepatic extraction of glucose postprandially. Numerous studies demonstrated that liver GCK exerts dominant control on glycogen synthesis and glycolysis. Overexpression of GCK in hepatocytes increase glycogen synthesis related to an increase in the cellular content of glucose-6-phosphate and allosteric activation of glycogen synthase [25]. GCK overexpression also resulted in stimulation of glycolytic flux [26]. Since type 2 diabetes is associated with an impairment of glycogen synthesis, resulting in a decrease of glucose utilization by the liver, GCK was pointed out in these disorders. Evidence for mutations in GCK gene have been associated with maturity onset diabetes of the young type 2 (MODY2) and overexpression of GCK in diabetic hepatocytes from Zucker diabetic rats improved glucose utilization and storage [27]. In the past years, considerable efforts have been made to understand the molecular mechanisms leading to insulin resistance and its subsequent chronic hyperglycemia and hepatic steatosis. Specifically studies have focused the role of O-GlcNAcylation on several transcription factors implicated in lipogenesis (ChREBP), gluconeogenesis (FOXO1 and TORC2) and insulin signaling (insulin receptor substrate, IRS, glycogen synthase, GS and Akt) (for review [21], [28]). Recent studies from our laboratory have shown that the liver fatty acid synthase (FAS) lipogenic enzyme is O-GlcNAcylated providing a new regulatory mechanism for the biosynthesis of lipids in the liver [29]. The current study was undertaken to determine whether O-GlcNAcylation could process on the first regulating enzymes for glucose metabolism, GCK. Considering obesity predisposition modulation by hepatic glucokinase [30] and the fact that O-GlcNAc is abnormally increased in the cells and tissues of diabetic animals and humans, we undertook the study of O-GlcNAcylation levels and GCK expression in a model of human obesity-associated diabetes, the ob/ob mice. In agreement with previous reports, we found an increase in GCK mRNA and protein expression in refed mice. Indeed liver GCK gene expression depends on the fasting refeeding states through insulin which is considered as the primary up-regulator for GCK transcription [31]. Concomitantly an increase in hepatic glucose flux used up to 2 to 3% by the hexosamine biosynthesis pathway lead to increased levels of overall O-GlcNAcylation. The finding of an increased level of Gck mRNA in livers of ob/ob strengthens the observation of Schimomura et al. [32]. Since GCK protein increase is partly explained by a transcriptional mechanism and GCK protein expression is correlated to high O-GlcNAc level, we focused on the role of this post-translational modification on GCK protein expression. In order to determinate whether O-GlcNAc could impact on GCK expression, we used a potent OGA inhibitor, Thiamet-G, to increase O-GlcNAc content in liver of fasted mice. Increasing O-GlcNAcylation level also increases GCK protein level in fasted condition. This observation suggests that O-GlcNAcylation itself is able to increase GCK protein content independently of glucose input. We also demonstrated that GCK is modified in liver by O-GlcNAcylation directly after glucose input. Therefore, we suggest that O-GlcNAc cycling on GCK is a novel way to regulate GCK expression and increase glucose entry into liver cells. Moreover, in both experiments, increase of GCK protein level is not correlated to gene upregulation suggesting that GCK may be stabilized by O-GlcNAcylation. To finally confirm this statement, we knock-down Ogt and confirmed that increased GCK content is dependent of OGT presence and activity. Taken together, this study proposes a new pathway of short-term regulation of GCK in response to glucose (Fig. 4). Indeed, until now, the well-described GCK regulation involved two mechanisms. The long-term regulation promotes GCK transcription and translation following glucose input and insulin-dependent stimulation. The short-term regulation is faster and regulated by binding of regulatory proteins. Importantly, interaction with the nuclear regulatory protein, GKRP is constitutive in low glucose whereas glucose input dissociates the complex, allowing GCK to translocate into the cytoplasm where its activity is carried. So this study reveals a new short term regulation of GCK expression through O-GlcNAc pathway. All regulatory process aims to metabolize quickly and efficiently glucose input.