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Activation of the unfolded protein response in high glucose treated endothelial cells is mediated by methylglyoxal.

ABSTRACT: Metabolic dysfunction of endothelial cells in hyperglycemia contributes to the development of vascular complications of diabetes where increased reactive glycating agent, methylglyoxal (MG), is involved. We assessed if increased MG glycation induced proteotoxic stress, identifying related metabolic drivers and protein targets. Human aortal endothelial cells (HAECs) were incubated in high glucose concentration (20?mM versus 5?mM control) in vitro for 3-6 days. Flux of glucose metabolism, MG formation and glycation and changes in cytosolic protein abundances, MG modification and proteotoxic responses were assessed. Similar studies were performed with human microvascular endothelial HMEC-1 cells where similar outcomes were observed. HAECs exposed to high glucose concentration showed increased cellular concentration of MG (2.27?±?0.21 versus 1.28?±?0.03 pmol/10⁶ cells, P?6 cells/day; P?

SUBMITTER: Irshad Z

PROVIDER: S-EPMC6536510 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Activation of the unfolded protein response in high glucose treated endothelial cells is mediated by methylglyoxal.

Irshad Zehra Z Xue Mingzhan M Ashour Amal A Larkin James R JR Thornalley Paul J PJ Rabbani Naila N

Scientific reports 20190527 1

Metabolic dysfunction of endothelial cells in hyperglycemia contributes to the development of vascular complications of diabetes where increased reactive glycating agent, methylglyoxal (MG), is involved. We assessed if increased MG glycation induced proteotoxic stress, identifying related metabolic drivers and protein targets. Human aortal endothelial cells (HAECs) were incubated in high glucose concentration (20 mM versus 5 mM control) in vitro for 3-6 days. Flux of glucose metabolism, MG forma ...[more]

PMID: 31133647

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Project description:The unfolded protein response (UPR) detects increased misfolded proteins and activates protein refolding, protein degradation and inflammatory responses. UPR sensors in the endoplasmic reticulum, IRE1α and PERK, bind and are activated by proteins with unexpected surface hydrophobicity, whereas sensor ATF6 is activated by proteolytic cleavage when released from complexation with protein disulfide isomerases (PDIs). Metabolic dysfunction leading to the formation of misfolded proteins with surface hydrophobicity and disruption of ATF6-PDI complexes leading to activation of UPR sensors remains unclear. The cellular concentration of reactive dicarbonyl metabolite, methylglyoxal (MG), is increased in impaired metabolic health, producing increased MG-modified cellular proteins. Herein we assessed the effect of high glucose concentration and related increased cellular MG on activation status of IRE1α, PERK and ATF6. Human aortal endothelial cells and HMEC-1 microvascular endothelial cells were incubated in low and high glucose concentration to model blood glucose control, with increase or decrease of MG by silencing or increasing expression of glyoxalase 1 (Glo1), which metabolizes MG. Increased MG induced by high glucose concentration activated IRE1α, PERK and ATF6 and related downstream signalling leading to increased chaperone, apoptotic and inflammatory gene expression. Correction of increased MG by increasing Glo1 expression prevented UPR activation. MG modification of proteins produces surface hydrophobicity through arginine-derived hydroimidazolone MG-H1 formation, with related protein unfolding and preferentially targets PDIs and chaperone pathways for modification. It thereby poses a major challenge to proteostasis and activates UPR sensors. Pharmacological decrease of MG with Glo1 inducer, trans-resveratrol and hesperetin in combination, offers a novel treatment strategy to counter UPR-related cell dysfunction, particularly in hyperglycemia associated with diabetes.

Project description:Defective macroautophagy/autophagy and a failure to initiate the adaptive unfolded protein response (UPR) in response to the endoplasmic reticulum (ER) stress contributes to obesity-associated metabolic dysfunction. However, whether and how unresolved ER stress leads to defects in the autophagy pathway and to the progression of obesity-associated hepatic pathologies remains unclear. Obesity suppresses the expression of hepatic spliced XBP1 (X-box binding protein 1; sXBP1), the key transcription factor that promotes the adaptive UPR. Our RNA-seq analysis revealed that sXBP1 regulates genes involved in lysosomal function in the liver under fasting conditions. Chromatin immunoprecipitation (ChIP) analyzes of both primary hepatocytes and whole livers further showed that sXBP1 occupies the -743 to -523 site of the promoter of Tfeb (transcription factor EB), a master regulator of autophagy and lysosome biogenesis. Notably, this occupancy was significantly reduced in livers from patients with steatosis. In mice, hepatic deletion of Xbp1 (xbp1 LKO) suppressed the transcription of Tfeb as well as autophagy, whereas hepatic overexpression of sXbp1 enhanced Tfeb transcription and autophagy. Moreover, overexpression of Tfeb in the xbp1 LKO mouse liver ameliorated glucose intolerance and steatosis in mice with diet-induced obesity (DIO). Conversely, loss of TFEB function impaired the protective role of sXBP1 in hepatic steatosis in mice with DIO. These data indicate that sXBP1-Tfeb signaling has direct functional consequences in the context of obesity. Collectively, our data provide novel insight into how two organelle stress responses are integrated to protect against obesity-associated metabolic dysfunction.Abbreviations: AAV8: adeno-associated virus serotype 8; ACTB: actin, beta; ANOVA: analysis of variance; ATF6: activating transcription factor-6; ATG: autophagy related; BECN1: beclin 1; BMI: body mass index; ChIP: chromatin immunoprecipitation; CLEAR: coordinated lysosomal expression and regulation; Cre: cre recombinase; DIO: diet-induced obesity; EBSS: Earle's balanced salt solution; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum (ER) to nucleus signaling 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HFD: high-fat diet; h: hours; HSCs: hepatic stellate cells; INS: insulin; L/A: ammonium chloride and leupeptin; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mRNA: messenger RNA; NAFLD: nonalcoholic fatty liver disease; NASH: nonalcoholic steatohepatitis; RD: regular diet; RFP: red fluorescent protein; SERPINA7/TBG: serpin family A member 7; SQSTM1/p62: sequestome 1; sXbp1 LOE: liver-specific overexpression of spliced Xbp1; TFEB: transcription factor EB; TG: thapsigargin; TN: tunicamycin; UPR: unfolded protein response; wks: weeks; WT: wild type; XBP1: X-box binding protein 1; xbp1 LKO: liver-specific Xbp1 knockout.

Project description:Patients suffering from immune-mediated necrotizing myopathies (IMNM) harbor, the pathognomonic myositis-specific auto-antibodies anti-SRP54 or -HMGCR, while about one third of them do not. Activation of chaperone-assisted autophagy was described as being part of the molecular etiology of IMNM. Endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR)-stress accompanied by activation of the unfolded protein response (UPR) often precedes activation of the protein clearance machinery and represents a cellular defense mechanism toward restoration of proteostasis. Here, we show that ER/SR-stress may be part of the molecular etiology of IMNM. To address this assumption, ER/SR-stress related key players covering the three known branches (PERK-mediated, IRE1-mediated, and ATF6-mediated) were investigated on both, the transcript and the protein levels utilizing 39 muscle biopsy specimens derived from IMNM-patients. Our results demonstrate an activation of all three UPR-branches in IMNM, which most likely precedes the activation of the protein clearance machinery. In detail, we identified increased phosphorylation of PERK and eIF2a along with increased expression and protein abundance of ATF4, all well-documented characteristics for the activation of the UPR. Further, we identified increased general XBP1-level, and elevated XBP1 protein levels. Additionally, our transcript studies revealed an increased ATF6-expression, which was confirmed by immunostaining studies indicating a myonuclear translocation of the cleaved ATF6-form toward the forced transcription of UPR-related chaperones. In accordance with that, our data demonstrate an increase of downstream factors including ER/SR co-chaperones and chaperones (e.g., SIL1) indicating an UPR-activation on a broader level with no significant differences between seropositive and seronegative patients. Taken together, one might assume that UPR-activation within muscle fibers might not only serve to restore protein homeostasis, but also enhance sarcolemmal presentation of proteins crucial for attracting immune cells. Since modulation of ER-stress and UPR via application of chemical chaperones became a promising therapeutic treatment approach, our findings might represent the starting point for new interventional concepts.

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Activation of the unfolded protein response in high glucose treated endothelial cells is mediated by methylglyoxal.

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Activation of the unfolded protein response in high glucose treated endothelial cells is mediated by methylglyoxal.

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