leupeptins and Hyperglycemia

Acute hyperglycemia co-presenting with myocardial infarction (in diabetic and non-diabetic individuals) is often associated with a poor prognosis. Although acute hyperglycemia induces oxidative stress that can lead to dysregulation of the ubiquitin-proteasome system (UPS), it is unclear whether increased/decreased UPS is detrimental with ischemia-reperfusion under such conditions. As our earlier data implicated the UPS in cardiac damage, we hypothesized that its inhibition results in cardioprotection with ischemia-reperfusion performed under conditions that simulate acute hyperglycemia.. Ex vivo rat heart perfusions were performed with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM glucose) for 60 min stabilization, followed by 20 min global ischemia and 60 min reperfusion ± 5 µM lactacystin and 10 µM MG-132, respectively. The UPS inhibitors were added during the first 20 min of the reperfusion phase and various cardiac functional parameters evaluated. In parallel experiments, infarct sizes were assessed following 20 min regional ischemia and 120 min reperfusion ± each of the respective UPS inhibitors (added during reperfusion). Heart tissues were collected and analyzed for markers of oxidative stress, UPS activation, inflammation and autophagy.. The proteasome inhibitor doses and treatment duration here employed resulted in partial UPS inhibition during the reperfusion phase. Both lactacystin and MG-132 administration resulted in cardioprotection in our experimental system, with MG-132 showing a greater effect. The proteasome inhibitors also enhanced cardiac superoxide dismutase protein levels (SOD1, SOD2), attenuated pro-inflammatory effects and caused an upregulation of autophagic markers.. This study established that partial proteasome inhibition elicits cardioprotection in hearts exposed to ischemia-reperfusion with acute simulated hyperglycemia. These data reveal that protease inhibition triggered three major protective effects, i.e. (a) enhancing myocardial anti-oxidant defenses, (b) attenuating inflammation, and (c) increasing the autophagic response. Thus the UPS emerges as a unique therapeutic target for the treatment of ischemic heart disease under such conditions.

Topics: Acetylcysteine; Animals; Anti-Inflammatory Agents; Antioxidants; Autophagy; Diabetic Cardiomyopathies; Hyperglycemia; Inflammation Mediators; Isolated Heart Preparation; Leupeptins; Male; Myocardial Reperfusion Injury; Myocardium; Proteasome Inhibitors; Rats, Wistar; Superoxide Dismutase; Superoxide Dismutase-1; Time Factors

The present study tested whether MG132 increases vascular nuclear factor E2-related factor-2 (Nrf2) expression and transcription to provide a therapeutic effect on diabetes-induced pathogenic changes in the aorta. To this end, three-month-old OVE26 diabetic and age-matched control mice were intraperitoneally injected with MG-132, 10  μ g/kg daily for 3 months. OVE26 transgenic type 1 diabetic mice develop hyperglycemia at 2-3 weeks of age and exhibit albuminuria at 3 months of age with mild increases in TNF- α expression and 3-NT accumulation in the aorta. Diabetes-induced significant increases in the wall thickness and structural derangement of aorta were found in OVE26 mice with significant increases in aortic oxidative and nitrosative damage, inflammation, and remodeling at 6 months of diabetes, but not at 3 months of diabetes. However, these pathological changes seen at the 6 months of diabetes were abolished in OVE26 mice treated with MG-132 for 3 months that were also associated with a significant increase in Nrf2 expression in the aorta as well as transcription of downstream genes. These results suggest that chronic treatment with low-dose MG132 can afford an effective therapy for diabetes-induced pathogenic changes in the aorta, which is associated with the increased Nrf2 expression and transcription.

Topics: Albuminuria; Animals; Aorta; Blood Pressure; Diabetes Mellitus, Experimental; Hyperglycemia; Leupeptins; Mice; Mice, Transgenic; NF-E2-Related Factor 2; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Tyrosine; Up-Regulation

Hyperglycemia plays a pivotal role in the development of diabetic nephropathy (DN) and may be related to epigenetic metabolic memory. One of the most crucial epigenetic mechanisms is histone modification, which is associated with the expression of a fibrosis factor in vascular injury. Aim .In this study, we investigated the ubiquitination of histones H2A and H2B to explore the epigenetic mechanisms of DN.. The GMCs were cultured as follows: normal group, high glucose group, mannitol group, and intervention group. After 12 hr, 24 hr, and 48 hr, histones ubiquitination, transforming growth factor-β (TGF-β), and fibronectin (FN) were measured using WB, RT-PCR, and IF.. High glucose can induce the upregulation of FN. H2A ubiquitination in GMCs increased in high glucose group (P < 0.01), whereas it decreased significantly in intervention group (P < 0.05). In contrast, H2B ubiquitination decreased with an increasing concentration of glucose, but it was recovered in the intervention group (P < 0.05). Expression of TGF-β changed in response to abnormal histone ubiquitination.. The high glucose may induce H2A ubiquitination and reduce H2B ubiquitination in GMCs. The changes of histone ubiquitination may be due in part to DN by activating TGF-β signaling pathway.

Topics: Animals; Cells, Cultured; Cysteine Proteinase Inhibitors; Fibronectins; Glucose; Histones; Hyperglycemia; Leupeptins; Mesangial Cells; Rats; Transforming Growth Factor beta; Ubiquitination; Up-Regulation

The development of type 2 diabetes is accompanied by a progressive decline in β-cell mass and function. Vildagliptin, a dipeptidyl peptidase 4 inhibitor, is representative of a new class of antidiabetic agents that act through increasing the expression of glucagon-like peptide-1. The protective effect of this agent on β cells was studied in diabetic mice. Diabetic pancreatic β cell-specific C/EBPB transgenic (TG) mice exhibit decreased β-cell mass associated with increased apoptosis, decreased proliferation, and aggravated endoplasmic reticulum (ER) stress. Vildagliptin was orally administered to the TG mice for a period of 24 weeks, and the protective effects of this agent on β cells were examined, along with the potential molecular mechanism of protection. Vildagliptin ameliorated hyperglycemia in TG mice by increasing the serum concentration of insulin and decreasing the serum concentration of glucagon. This agent also markedly increased β-cell mass, improved aggravated ER stress, and restored attenuated insulin/IGF1 signaling. A decrease in pancreatic and duodenal homeobox 1 expression was also observed in β cells isolated from our mouse model, but this was also restored by vildagliptin treatment. The expression of C/EBPB protein, but not mRNA, was unexpectedly downregulated in vildagliptin-treated TG mice and in exenatide-treated MIN6 cells. Activation of the GLP1 pathway induced proteasome-dependent C/EBPB degradation in β cells as the proteasome inhibitor MG132 restored the downregulation of C/EBPB protein by exenatide. Vildagliptin elicits protective effects on pancreatic β cells, possibly through C/EBPB degradation, and has potential for preventing the progression of type 2 diabetes.

Topics: Adamantane; Animals; Blood Glucose; CCAAT-Enhancer-Binding Protein-beta; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Endoplasmic Reticulum Stress; Exenatide; Gene Expression Regulation; Glucagon; Glucagon-Like Peptide 1; Hyperglycemia; Insulin; Insulin-Secreting Cells; Leupeptins; Mice; Mice, Transgenic; Nitriles; Peptides; Pyrrolidines; Venoms; Vildagliptin

Intercellular communication through gap junctions (GJIC) is most likely relevant to maintaining the integrity of the blood-retinal barrier. In this study, we investigated the mechanism whereby high glucose enhances degradation of connexin 43 (Cx43), thus contributing to a decrease in GJIC. The levels of Cx43 in bovine retinal endothelial cells exposed to high glucose (25 mm) decreased about 50% as compared with controls (5.5 mm glucose). Consistently, the half-life of the protein decreased from 2.3 to 1.9 h. The proteasome inhibitors MG132 and lactacystin prevented the loss of Cx43 induced by high glucose and extended Cx43 half-life. The amount of phosphorylated Cx43 increased in high glucose and after proteasome inhibition. Scrape-loading dye transfer experiments show that high glucose is associated to a decrease of 40% in GJIC. Significantly, this reduction can be reversed by proteasome inhibitors. The decrease in GJIC in cells exposed to high glucose is associated with a loss of Cx43 from the plasma membrane, as demonstrated by immunofluorescence and biotinylation of cell-surface proteins. Results indicate that increased phosphorylation of Cx43 under high glucose is the mechanism targeting Cx43 for degradation by a proteasome-dependent mechanism. Increased degradation of Cx43 and reduction of GJIC in high glucose may be of physiological importance by contributing to endothelial cell dysfunction associated with the breakdown of the blood-retinal barrier in diabetic retinopathy.

Topics: Animals; Cattle; Cell Communication; Cell Membrane; Connexin 43; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Down-Regulation; Endothelial Cells; Gap Junctions; Glucose; Half-Life; Hyperglycemia; Leupeptins; Multienzyme Complexes; Phosphorylation; Proteasome Endopeptidase Complex; Retina; Staurosporine