ER and Nutrient Stress Promote Assembly of Respiratory Chain Supercomplexes through the PERK-eIF2α Axis.

Endoplasmic reticulum (ER) stress and unfolded protein response are energetically challenging under nutrient stress conditions. However, the regulatory mechanisms that control the energetic demand under nutrient and ER stress are largely unknown. Here we show that ER stress and glucose deprivation stimulate mitochondrial bioenergetics and formation of respiratory supercomplexes (SCs) through protein kinase R-like ER kinase (PERK). Genetic ablation or pharmacological inhibition of PERK suppresses nutrient and ER stress-mediated increases in SC levels and reduces oxidative phosphorylation-dependent ATP production. Conversely, PERK activation augments respiratory SCs. The PERK-eIF2α-ATF4 axis increases supercomplex assembly factor 1 (SCAF1 or COX7A2L), promoting SCs and enhanced mitochondrial respiration. PERK activation is sufficient to rescue bioenergetic defects caused by complex I missense mutations derived from mitochondrial disease patients. These studies have identified an energetic communication between ER and mitochondria, with implications in cell survival and diseases associated with mitochondrial failures.

Insight into the Molecular Mechanism of Diabetic Kidney Disease and the Role of Metformin in Its Pathogenesis.

Diabetic kidney disease (DKD) is one of the leading causes of death among patients diagnosed with diabetes mellitus. Despite the growing knowledge about the pathogenesis of DKD, we still do not have effective direct pharmacotherapy. Accurate blood sugar control is essential in slowing down DKD. It seems that metformin has a positive impact on kidneys and this effect is not only mediated by its hypoglycemic action, but also by direct molecular regulation of pathways involved in DKD. The molecular mechanism of DKD is complex and we can distinguish polyol, hexosamine, PKC, and AGE pathways which play key roles in the development and progression of this disease. Each of these pathways is overactivated in a hyperglycemic environment and it seems that most of them may be regulated by metformin. In this article, we summarize the knowledge about DKD pathogenesis and the potential mechanism of the nephroprotective effect of metformin. Additionally, we describe the impact of metformin on glomerular endothelial cells and podocytes, which are harmed in DKD.

Insulin Resistance Does Not Impair Mechanical Overload-Stimulated Glucose Uptake, but Does Alter the Metabolic Fate of Glucose in Mouse Muscle.

Skeletal muscle glucose uptake and glucose metabolism are impaired in insulin resistance. Mechanical overload stimulates glucose uptake into insulin-resistant muscle; yet the mechanisms underlying this beneficial effect remain poorly understood. This study examined whether a differential partitioning of glucose metabolism is part of the mechanosensitive mechanism underlying overload-stimulated glucose uptake in insulin-resistant muscle. Mice were fed a high-fat diet to induce insulin resistance. Plantaris muscle overload was induced by unilateral synergist ablation. After 5 days, muscles were excised for the following measurements: (1) [3H]-2-deoxyglucose uptake; (2) glycogen; 3) [5-3H]-glucose flux through glycolysis; (4) lactate secretion; (5) metabolites; and (6) immunoblots. Overload increased glucose uptake ~80% in both insulin-sensitive and insulin-resistant muscles. Overload increased glycogen content ~20% and this was enhanced to ~40% in the insulin-resistant muscle. Overload did not alter glycolytic flux, but did increase muscle lactate secretion 40-50%. In both insulin-sensitive and insulin-resistant muscles, overload increased 6-phosphogluconate levels ~150% and decreased NADP:NADPH ~60%, indicating pentose phosphate pathway activation. Overload increased protein O-GlcNAcylation ~45% and this was enhanced to ~55% in the insulin-resistant muscle, indicating hexosamine pathway activation. In conclusion, insulin resistance does not impair mechanical overload-stimulated glucose uptake but does alter the metabolic fate of glucose in muscle.

Sialic acid glycoengineering using N-acetylmannosamine and sialic acid analogs.

Sialic acids cap the glycans of cell surface glycoproteins and glycolipids. They are involved in a multitude of biological processes and aberrant sialic acid expression is associated with several pathologies. Sialic acids modulate the characteristics and functions of glycoproteins and regulate cell-cell as well as cell-extracellular matrix interactions. Pathogens such as influenza virus use sialic acids to infect host cells and cancer cells exploit sialic acids to escape from the host's immune system. The introduction of unnatural sialic acids with different functionalities into surface glycans enables the study of the broad biological functions of these sugars and presents a therapeutic option to intervene with pathological processes involving sialic acids. Multiple chemically modified sialic acid analogs can be directly utilized by cells for sialoglycan synthesis. Alternatively, analogs of the natural sialic acid precursor sugar N-Acetylmannosamine (ManNAc) can be introduced into the sialic acid biosynthesis pathway resulting in the intracellular conversion into the corresponding sialic acid analog. Both, ManNAc and sialic acid analogs, have been employed successfully for a large variety of glycoengineering applications such as glycan imaging, targeting toxins to tumor cells, inhibiting pathogen binding, or altering immune cell activity. However, there are significant differences between ManNAc and sialic acid analogs with respect to their chemical modification potential and cellular metabolism that should be considered in sialic acid glycoengineering experiments.

Hexosamine pathway regulates StarD7 expression in JEG-3 cells.

StarD7 is a lipid binding protein involved in the delivery of phosphatidylcholine to the mitochondria whose promoter is activated by Wnt/ß-catenin signaling. Although the majority of glucose enters glycolysis, ~ 2-5% of it can be metabolized via the hexosamine biosynthetic pathway (HBP). Considering that HBP has been implicated in the regulation of ß-catenin we explored if changes in glucose levels modulate StarD7 expression by the HBP in trophoblast cells. We found an increase in StarD7 as well as in ß-catenin expression following high-glucose (25 mM) treatment in JEG-3 cells; these effects were abolished in the presence of HBP inhibitors. Moreover, since HBP is able to promote unfolded protein response (UPR) the protein levels of GRP78, Ire1α, calnexin, p-eIF2α and total eIF2α as well as XBP1 mRNA was measured. Our results indicate that a diminution in glucose concentration leads to a decrease in StarD7 expression and an increase in the UPR markers: GRP78 and Ire1α. Conversely, an increase in glucose is associated to high StarD7 levels and low GRP78 expression, phospho-eIF2α and XBP1 splicing, although Ire1α remains high when cells are restored to high glucose. Taken together these findings indicate that glucose modulates StarD7 and ß-catenin expression through the HBP associated to UPR, suggesting the existence of a link between UPR and HBP in trophoblast cells. This is the first study reporting the effects of glucose on StarD7 in trophoblast cells. These data highlight the importance to explore the role of StarD7 in placenta disorders related to nutrient availability.

Impaired O-linked N-acetylglucosaminylation in the endoplasmic reticulum by mutated epidermal growth factor (EGF) domain-specific O-linked N-acetylglucosamine transferase found in Adams-Oliver syndrome.

Epidermal growth factor (EGF) domain-specific O-linked N-acetylglucosamine (EOGT) is an endoplasmic reticulum (ER)-resident O-linked N-acetylglucosamine (O-GlcNAc) transferase that acts on EGF domain-containing proteins such as Notch receptors. Recently, mutations in EOGT have been reported in patients with Adams-Oliver syndrome (AOS). Here, we have characterized enzymatic properties of mouse EOGT and EOGT mutants associated with AOS. Simultaneous expression of EOGT with Notch1 EGF repeats in human embryonic kidney 293T (HEK293T) cells led to immunoreactivity with the CTD110.6 antibody in the ER. Consistent with the GlcNAc modification in the ER, the enzymatic properties of EOGT are distinct from those of Golgi-resident GlcNAc transferases; the pH optimum of EOGT ranges from 7.0 to 7.5, and the Km value for UDP N-acetylglucosamine (UDP-GlcNAc) is 25 µm. Despite the relatively low Km value for UDP-GlcNAc, EOGT-catalyzed GlcNAcylation depends on the hexosamine pathway, as revealed by the increased O-GlcNAcylation of Notch1 EGF repeats upon supplementation with hexosamine, suggesting differential regulation of the luminal UDP-GlcNAc concentration in the ER and Golgi. As compared with wild-type EOGT, O-GlcNAcylation in the ER is nearly abolished in HEK293T cells exogenously expressing EOGT variants associated with AOS. Introduction of the W207S mutation resulted in degradation of the protein via the ubiquitin-proteasome pathway, although the stability and ER localization of EOGT(R377Q) were not affected. Importantly, the interaction between UDP-GlcNAc and EOGT(R377Q) was impaired without adversely affecting the acceptor substrate interaction. These results suggest that impaired glycosyltransferase activity in mutant EOGT proteins and the consequent defective O-GlcNAcylation in the ER constitute the molecular basis for AOS.

Hyperglycaemic conditions perturb mouse oocyte in vitro developmental competence via beta-O-linked glycosylation of heat shock protein 90.

STUDY QUESTION: What is the effect of beta-O-linked glycosylation (O-GlcNAcylation) on specific proteins in the cumulus-oocyte complex (COC) under hyperglycaemic conditions? SUMMARY ANSWER: Heat shock protein 90 (HSP90) was identified and confirmed as being O-GlcNAcylated in mouse COCs under hyperglycaemic conditions (modelled using glucosamine), causing detrimental outcomes for embryo development. WHAT IS KNOWN ALREADY: O-GlcNAcylation of proteins occurs as a result of increased activity of the hexosamine biosynthesis pathway, which provides substrates for cumulus matrix production during COC maturation, and also for O-GlcNAcylation. COCs matured under hyperglycaemic conditions have decreased developmental competence, mediated at least in part through the mechanism of increased O-GlcNAcylation. STUDY DESIGN, SIZE, DURATION: This study was designed to examine the effect of hyperglycaemic conditions (using the hyperglycaemic mimetic, glucosamine) on O-GlcNAc levels in the mouse COC, and furthermore to identify potential candidate proteins which are targets of this modification, and their roles in oocyte maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS: COCs from 21-day-old superovulated CBA × C57BL6 F1 hybrid female mice were matured in vitro (IVM). Levels of O-GlcNAcylated proteins, HSP90 and O-GlcNAc transferase (OGT, the enzyme responsible for O-GlcNAcylation) in COCs were measured using western blot, and localization observed using immunocytochemistry. For glycosylated HSP90 levels, and to test OGT-HSP90 interaction, immunoprecipitation was performed prior to western blotting. Embryo development was assessed using in vitro fertilization and embryo culture post-maturation. MAIN RESULTS AND THE ROLE OF CHANCE: Addition of the hyperglycaemic mimetic glucosamine to IVM medium for mouse COCs increased detectable O-GlcNAcylated protein levels (by western blot and immunocytochemistry), and this effect was reversed using an OGT inhibitor (P < 0.05). HSP90 was identified as a target of O-GlcNAcylation in the COC, and inhibition of HSP90 during IVM reversed glucosamine-induced decreases in oocyte developmental competence (P < 0.05). We also demonstrated the novel finding of an association between HSP90 and OGT in COCs, suggesting a possible client-chaperone relationship. LIMITATIONS, REASONS FOR CAUTION: In vitro maturation of COCs was used so that treatment time could be limited to the 17 h of maturation prior to ovulation. Additionally, glucosamine, a hyperglycaemic mimetic, was used because it specifically activates the hexosamine pathway which provides the O-GlcNAc moieties. The results in this study should be confirmed using in vivo models of hyperglycaemia and different HSP90 inhibitors. WIDER IMPLICATIONS OF THE FINDINGS: This study leads to a new understanding of how diabetes influences oocyte competence and provides insight into possible therapeutic interventions based on inhibiting HSP90 to improve oocyte quality. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a programme grant from the National Health and Medical Research Council, Australia, ID 453556. J.G.T. is a recipient of funding from and a consultant to Cook Medical Pty Ltd. The other authors have no conflicts of interest to declare.

Metabolites that feed upper glycolytic branches support glucose independent human cytomegalovirus replication.

The broad tissue distribution and cell tropism of human cytomegalovirus indicates that the virus successfully replicates in tissues with various nutrient environments. HCMV requires and reprograms central carbon metabolism for viral replication. However, many studies focus on reprogramming of metabolism in high nutrient conditions that do not recapitulate physiological nutrient environments in the body. In this study, we investigate how HCMV successfully replicates when nutrients are suboptimal. We limited glucose following HCMV infection to determine how glucose supports virus replication and how nutrients potentially present in the physiological environment contribute to successful glucose independent HCMV replication. Glucose is required for HCMV viral genome synthesis, viral protein production and glycosylation, and virus production. However, supplement of glucose-free cultures with uridine, ribose, or UDP-GlcNAc-metabolites that support upper glycolytic branches-resulted in partially restored viral genome synthesis and subsequent partial restoration of viral protein levels. Low levels of virus production were also restored. Supplementing lower glycolysis in glucose-free cultures using pyruvate had no effect on virus replication. These results indicate nutrients that support upper glycolytic branches like the pentose phosphate pathway and hexosamine pathway can compensate for glucose during HCMV replication to support low levels of virus production. More broadly, our findings suggest that HCMV could successfully replicate in diverse metabolic niches, including those in the body with low levels of glucose, through alternative nutrient usage.

New Horizons in Diabetic Neuropathies: An Updated Review on their Pathology, Diagnosis, Mechanism, Screening Techniques, Pharmacological, and Future Approaches.

BACKGROUND: One of the largest problems for global public health is diabetes mellitus (DM) and its micro and macrovascular consequences. Although prevention, diagnosis, and treatment have generally improved, its incidence is predicted to keep rising over the coming years. Due to the intricacy of the molecular mechanisms, which include inflammation, oxidative stress, and angiogenesis, among others, discovering treatments to stop or slow the course of diabetic complications is still a current unmet need. METHODS: The pathogenesis and development of diabetic neuropathies may be explained by a wide variety of molecular pathways, hexosamine pathways, such as MAPK pathway, PARP pathway, oxidative stress pathway polyol (sorbitol) pathway, cyclooxygenase pathway, and lipoxygenase pathway. Although diabetic neuropathies can be treated symptomatically, there are limited options for treating the underlying cause. RESULT: Various pathways and screening models involved in diabetic neuropathies are discussed, along with their possible outcomes. Moreover, both medicinal and non-medical approaches to therapy are also explored. CONCLUSION: This study highlights the probable involvement of several processes and pathways in the establishment of diabetic neuropathies and presents in-depth knowledge of new therapeutic approaches intended to stop, delay, or reverse different types of diabetic complications.

GFPT2/GFAT2 and AMDHD2 act in tandem to control the hexosamine pathway.

The hexosamine biosynthetic pathway (HBP) produces the essential metabolite UDP-GlcNAc and plays a key role in metabolism, health, and aging. The HBP is controlled by its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFPT/GFAT) that is directly inhibited by UDP-GlcNAc in a feedback loop. HBP regulation by GFPT is well studied but other HBP regulators have remained obscure. Elevated UDP-GlcNAc levels counteract the glycosylation toxin tunicamycin (TM), and thus we screened for TM resistance in haploid mouse embryonic stem cells (mESCs) using random chemical mutagenesis to determine alternative HBP regulation. We identified the N-acetylglucosamine deacetylase AMDHD2 that catalyzes a reverse reaction in the HBP and its loss strongly elevated UDP-GlcNAc. To better understand AMDHD2, we solved the crystal structure and found that loss-of-function (LOF) is caused by protein destabilization or interference with its catalytic activity. Finally, we show that mESCs express AMDHD2 together with GFPT2 instead of the more common paralog GFPT1. Compared with GFPT1, GFPT2 had a much lower sensitivity to UDP-GlcNAc inhibition, explaining how AMDHD2 LOF resulted in HBP activation. This HBP configuration in which AMDHD2 serves to balance GFPT2 activity was also observed in other mESCs and, consistently, the GFPT2:GFPT1 ratio decreased with differentiation of human embryonic stem cells. Taken together, our data reveal a critical function of AMDHD2 in limiting UDP-GlcNAc production in cells that use GFPT2 for metabolite entry into the HBP.

The Effect of Vitamin D on Cellular Pathways of Diabetic Nephropathy.

BACKGROUND: Diabetic nephropathy is one of the most important microvascular complications and a major cause of morbidity and mortality in diabetic patients. This study was designed to investigate the effect of vitamin D on the expression of three key genes involved in the development of diabetic nephropathy. METHODS: Twenty-four male Sprague-Dawley rats were randomly divided into three groups. The first group served as control and the other two groups received intraperitoneal injections of 45 mg/kg STZ to develop diabetes. The groups were treated for four weeks either with placebo or two vitamin D injections of 20,000 IU/kg. Serum glucose, insulin, and HbA1c levels, and AGE cellular receptor (RAGE), aldose reductase (AR) and glutamine: fructose-6-phosphate aminotransferase (GFAT) gene expression were assessed in kidney tissue at the end of the experiment. RESULTS: Vitamin D treatment resulted in a significant increase in insulin concentration, which could improve hyperglycaemia in diabetic rats. Serum HbA1c decreased slightly but insignificantly following the vitamin D injections. In addition, expression of GFAT, a key regulatory enzyme in the hexosamine pathway, was significantly reduced following vitamin D administration. CONCLUSION: Vitamin D may reduce diabetic nephropathy not only by improving blood glucose and insulin levels, but also by modulating hexosamine pathways in kidney.

Pathological Perturbations in Diabetic Retinopathy: Hyperglycemia, AGEs, Oxidative Stress and Inflammatory Pathways.

Diabetic retinopathy (DR) remains the leading cause of blindness in working-aged adults around the world. The proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME) are the severe vision threatening stages of the disorder. Although, a huge body of research exists in elaborating the pathological mechanisms that lead to the development of DR, the certainty and the correlation amongst these pathways remain ambiguous. The complexity of DR lies in the multifactorial pathological perturbations that are instrumental in both the disease development and its progression. Therefore, a holistic perspective with an understanding of these pathways and their correlation may explain the pathogenesis of DR as a unifying mechanism. Hyperglycemia, oxidative stress and inflammatory pathways are the crucial components that are implicated in the pathogenesis of DR. Of these, hyperglycemia appears to be the initiating central component around which other pathological processes operate. Thus, this review discusses the role of hyperglycemia, oxidative stress and inflammation in the pathogenesis of DR, and highlights the cross-talk amongst these pathways in an attempt to understand the complex interplay of these mechanisms. Further, an effort has been made to identify the knowledge gap and the key players in each pathway that may serve as potential therapeutic drug targets.

Diabetes-related cardiomyopathy: The sweet story of glucose overload from epidemiology to cellular pathways.

Type 2 diabetes (T2D) is a major risk factor for heart failure (HF). Although the number of cases of myocardial infarction in the T2D population has been reduced by 25% over the last 10 years, the incidence of HF is continuously increasing, making it the most worrying diabetes complication. This strongly reinforces the urgent need for innovative therapeutic interventions to prevent cardiac dysfunction in T2D patients. To this end, epidemiological, imaging and animal studies have aimed to highlight the mechanisms involved in the development of diabetic cardiomyopathy. Epidemiological observations clearly show that hyperglycaemia correlates with severity of cardiac dysfunction and mortality in T2D patients. Both animal and cellular studies have demonstrated that, in the context of diabetes, the heart loses its ability to utilize glucose, therefore leading to glucose overload in cardiomyocytes that, in turn, promotes oxidative stress, accumulation of advanced glycation end-products (AGEs) and chronic activation of the hexosamine pathway. These have all been found to activate apoptosis and to alter heart contractility, calcium signalling and mitochondrial function. Although, in the past, tight glycaemic control has failed to improve cardiac function in T2D patients, recent clinical trials have reported cardiovascular benefit with hypoglycaemic antidiabetic drugs of the SGLT2-inhibitor family. This review, based on clinical evidence from mechanistic studies as well as several large clinical trials, covers 15 years of research, and strongly supports the idea that hyperglycaemia and glucose overload play a central role in the pathophysiology of diabetic cardiomyopathy.

Vitamin D suppresses cellular pathways of diabetes complication in liver.

OBJECTIVES: The aim of this study was to investigate the effect of vitamin D on glucose metabolism, as well as the expression of five key genes involved in the development of diabetes complications in liver tissue of diabetic rats. MATERIALS AND METHODS: Twenty-four male Sprague-Dawley rats were randomly divided into three groups (8 rats in each group). The first group served as control and the other two groups received an intraperitoneal injection of 45 mg/kg streptozotocin to develop diabetes. Groups were treated for four weeks either with placebo or vitamin D (two injections of 20000 IU/kg). Thereafter, serum levels of glucose, insulin and HbA1c were assessed. Liver tissue was examined for the level of advanced glycation end products (AGEs) and the gene expression of AGE cellular receptor (AGER), glyoxalase-1 (GLO-1), aldose reductase (AR), O-linked N-acetylglucosamine transferase (OGT) and glutamine/ fructose-6-phosphate aminotransferase (GFAT). RESULTS: Vitamin D injection resulted in a significant increase in plasma level of 25-hydroxycholecalciferol, which could improve hyperglycemia about 11% compared to placebo-receiving diabetic rats (P=0.005). Insulin level increased as a result of vitamin D treatment compared to control (3.31±0.65 vs. 2.15±0.79; P= 0.01). Serum HbA1c and liver AGE concentrations had a slight but insignificant reduction following vitamin D intake. Moreover, a significant decline was observed in gene expression of AGER and OGT in liver tissue (P=0.04 and P<0.001 respectively). CONCLUSION: Vitamin D might contribute in ameliorating diabetes complications not only by improving blood glucose and insulin levels, but also by suppressing AGER and OGT gene expression in the liver.

Liver glucose metabolism in humans.

Information about normal hepatic glucose metabolism may help to understand pathogenic mechanisms underlying obesity and diabetes mellitus. In addition, liver glucose metabolism is involved in glycosylation reactions and connected with fatty acid metabolism. The liver receives dietary carbohydrates directly from the intestine via the portal vein. Glucokinase phosphorylates glucose to glucose 6-phosphate inside the hepatocyte, ensuring that an adequate flow of glucose enters the cell to be metabolized. Glucose 6-phosphate may proceed to several metabolic pathways. During the post-prandial period, most glucose 6-phosphate is used to synthesize glycogen via the formation of glucose 1-phosphate and UDP-glucose. Minor amounts of UDP-glucose are used to form UDP-glucuronate and UDP-galactose, which are donors of monosaccharide units used in glycosylation. A second pathway of glucose 6-phosphate metabolism is the formation of fructose 6-phosphate, which may either start the hexosamine pathway to produce UDP-N-acetylglucosamine or follow the glycolytic pathway to generate pyruvate and then acetyl-CoA. Acetyl-CoA may enter the tricarboxylic acid (TCA) cycle to be oxidized or may be exported to the cytosol to synthesize fatty acids, when excess glucose is present within the hepatocyte. Finally, glucose 6-phosphate may produce NADPH and ribose 5-phosphate through the pentose phosphate pathway. Glucose metabolism supplies intermediates for glycosylation, a post-translational modification of proteins and lipids that modulates their activity. Congenital deficiency of phosphoglucomutase (PGM)-1 and PGM-3 is associated with impaired glycosylation. In addition to metabolize carbohydrates, the liver produces glucose to be used by other tissues, from glycogen breakdown or from de novo synthesis using primarily lactate and alanine (gluconeogenesis).

Identification of an N-acetylglucosamine kinase essential for UDP-N-acetylglucosamine salvage synthesis in Arabidopsis.

Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) donates GlcNAc for various glycans and glycoconjugates. We previously found that GlcNAc supplementation increases the UDP-GlcNAc content in Arabidopsis; however, the metabolic pathway was undefined. Here, we show that the homolog of human GlcNAc kinase (GNK) is conserved in land plants. Enzymatic assays of the Arabidopsis homologous protein (AtGNK) revealed kinase activity that was highly specific for GlcNAc. We also demonstrate the role of AtGNK in plants by using its knockout mutant, which presents lower UDP-GlcNAc contents and is insensitive to GlcNAc supplementation. Moreover, our results demonstrate the presence of a GlcNAc salvage pathway in plants.

SRT1720 counteracts glucosamine-induced endoplasmic reticulum stress and endothelial dysfunction.

AIMS: We hypothesized that a disproportionate activation of the glucosamine (GlcN) pathway, caused by a prolonged exposure to hyperglycaemia, could impair endothelial integrity promoting endoplasmic reticulum (ER) stress. We also tested the possibility that SRT1720 may be able to counteract GlcN-induced ER stress. METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVECs), human cardiac microvascular endothelial cells, and human retinal endothelial cells were treated with GlcN in the presence or absence of the chemical chaperone phenyl butyric acid (PBA) or SRT1720. Expression of ER stress markers, activation of apoptosis, and pro-inflammatory/pro-thrombotic pathways were evaluated by western blot, real-time RT-PCR, and ELISA assays. GlcN treatment resulted in a significantly increased expression of the major ER stress mediators. ER stress activation was paralleled by increased levels of apoptotic markers and by pro-inflammatory/pro-coagulant pathway activation. In HUVECs, ER stress inhibition by PBA alleviated a GlcN-induced pro-apoptotic and pro-inflammatory/pro-thrombotic state, suggesting a crucial role of ER stress in endothelial dysfunction caused by GlcN. Furthermore, SRT1720 treatment abolished GlcN-induced ER stress and reversed its effects on apoptosis and pro-inflammatory/pro-coagulant pathways. This SRT1720 action was mediated by its ability to modulate Raptor acetylation, thus inhibiting mammalian target of Rapamycin complex 1 (mTORC1)-dependent protein synthesis and alleviating ER overload. CONCLUSIONS: Our data show that GlcN promotes a pro-apoptotic and pro-inflammatory/pro-thrombotic phenotype in endothelial cells by activating ER stress. The observation that SRT1720, inhibiting ER stress, was able to counteract GlcN effects lays the basis for future studies aimed to exploit this drug and cognate compounds in the treatment of endothelial dysfunction and atherosclerosis.