Consumption of a high fat diet promotes protein O-GlcNAcylation in mouse retina via NR4A1-dependent GFAT2 expression

The incidence of type 2 diabetes, the most common cause of diabetic retinopathy (DR), is rapidly on the rise in developed countries due to overconsumption of calorie rich diets. Using an animal model of diet-induced obesity/pre-diabetes, we evaluated the impact of a diet high in saturated fat (HFD) on O-GlcNAcylation of retinal proteins, as dysregulated O-GlcNAcylation contributes to diabetic complications and evidence supports a role in DR. Protein O-GlcNAcylation was increased in the retina of mice fed a HFD as compared to littermates receiving control chow. Similarly, O-GlcNAcylation was elevated in retinal Müller cells in culture exposed to the saturated fatty acid palmitate or the ceramide analog Cer6. One potential mechanism responsible for elevated O-GlcNAcylation is increased flux through the hexosamine biosynthetic pathway (HBP). Indeed, inhibition of the pathway's rate-limiting enzyme glutamine-fructose-6-phosphate amidotransferase (GFAT) prevented Cer6-induced O-GlcNAcylation. Importantly, expression of the mRNA encoding GFAT2, but not GFAT1 was elevated in both the retina of mice fed a HFD and in retinal cells in culture exposed to palmitate or Cer6. Notably, expression of nuclear receptor subfamily 4 group A member 1 (NR4A1) was increased in the retina of mice fed a HFD and NR4A1 expression was sufficient to promote GFAT2 mRNA expression and O-GlcNAcylation in retinal cells in culture. Whereas palmitate or Cer6 addition to culture medium enhanced NR4A1 and GFAT2 expression, chemical inhibition of NR4A1 transactivation repressed Cer6-induced GFAT2 mRNA expression. Overall, the results support a model wherein HFD increases retinal protein O-GlcNAcylation by promoting NR4A1-dependent GFAT2 expression.     

Multiple Tissue-specific Roles for the O-GlcNAc Post-translational Modification in the Induction of and Complications Arising from Type II Diabetes

In this minireview, we will highlight work in the last 30 years that has clearly demonstrated that the O-GlcNAc modification is nutrient-responsive and plays multiple roles in metabolic regulation of signaling and gene expression. Further, we will examine recent studies that have investigated the impact of O-GlcNAc in a variety of glucose- and insulin-responsive tissues and the roles attributed to O-GlcNAc in the induction of insulin resistance and glucose toxicity, the hallmarks of type II diabetes mellitus. We will also summarize potential causal roles for the O-GlcNAc modification in complications associated with diabetes.     

蛋白分子O-糖基化的研究进展

蛋白分子的氧连接糖基化（O-糖基化）修饰是生物体内必不可少的转录后化学修饰之一,其作用方式类似磷酸化,并且两者之间相互作用,共同调节生物大分子的活性。O-糖基化修饰在生物体的转录、翻译、核运输、细胞骨架的形成以及调节细胞器的功能中发挥着重要的作用。通过影响细胞信号的传导,在细胞吞噬、炎性细胞的迁移以及细胞内大分子物质的循环中也起着重要作用。该文主要通过介绍蛋白分子O-糖基化修饰的基础理论以及。一糖基化修饰作用的几个方面,来简要阐述O-糖基化修饰在生物体内发挥的作用。     

Characterization of O-GlcNAc cycling and proteomic identification of differentially O-GlcNAcylated proteins during G1/S transition

Background

DNA replication represents a critical step of the cell cycle which requires highly controlled and ordered regulatory mechanisms to ensure the integrity of genome duplication. Among a plethora of elements, post-translational modifications (PTMs) ensure the spatiotemporal regulation of pivotal proteins orchestrating cell division. Despite increasing evidences showing that O-GlcNAcylation regulates mitotic events, the impact of this PTM in the early steps of the cell cycle remains poorly understood.

Methods and results

Quiescent MCF7 cells were stimulated by serum mitogens and cell cycle progression was determined by flow cytometry. The levels of O-GlcNAc modified proteins, O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) were examined by Western blotting and OGA activity was measured during the progression of cells towards S phase. A global decrease in O-GlcNAcylation was observed at S phase entry, concomitantly to an increase in the activity of OGA. A combination of two-dimensional electrophoresis, Western blotting and mass spectrometry was then used to detect and identify cell cycle-dependent putative O-GlcNAcylated proteins. 58 cytoplasmic and nuclear proteins differentially O-GlcNAcylated through G1/S transition were identified and the O-GlcNAc variations of Cytokeratin 8, hnRNP K, Caprin-1, Minichromosome Maintenance proteins MCM3, MCM6 and MCM7 were validated by immunoprecipitation.

Conclusions

The dynamics of O-GlcNAc is regulated during G1/S transition and observed on key proteins involved in the cytoskeleton networks, mRNA processing, translation, protein folding and DNA replication.

General significance

Our results led us to propose that O-GlcNAcylation joins the PTMs that take part in the regulation of DNA replication initiation.     

Enzymatic characterization and inhibition of the nuclear variant of human O-GlcNAcase

Increasing cellular O-GlcNAc levels through pharmacological inhibition of O-GlcNAcase, the enzyme responsible for removal of the O-GlcNAc post-translational modification, is being increasingly used to aid in discerning the roles played by this form of intracellular glycosylation. Interestingly, two forms of O-GlcNAcase have been studied; a full-length isoform that is better characterized, and a shorter nuclear-localized variant, arising from failure to splice out one intron, which has not been as well characterized. Given the increasing use of O-GlcNAcase inhibitors as research tools, we felt that a clear understanding of how these inhibitors affect both isoforms of O-GlcNAcase is important for proper interpretation of studies making use of these inhibitors in cell culture and in vivo. Here we describe an enzymatic characterization of the nuclear variant of human O-GlcNAcase. We find that this short nuclear variant of O-GlcNAcase, which has the identical catalytic domain as the full-length enzyme, has similar trends in a pH-rate profile and Taft linear free energy analysis as the full-length enzyme. These findings strongly suggest that both enzymes use broadly similar transition states. Consistent with this interpretation, the short isoform is potently inhibited by several previously described inhibitors of full-length O-GlcNAcase including PUGNAc, NAG-thiazoline, and the selective O-GlcNAcase inhibitor NButGT. These findings contrast with earlier studies and suggest that studies using O-GlcNAcase inhibitors in cultured cells or in vivo can be interpreted with the knowledge that both these forms of O-GlcNAcase are inhibited when present.