Abstract 3878: Glucagon-Like Peptide-1 Metabolite Protects the Myocardium Against Ischemia-Reperfusion Injury in Diabetes Mellitus

BackgroundThe dipeptidyl peptidase-4 (DPP-4) inhibitors Sitagliptin and Vildagliptin lower blood glucose by augmenting endogenous levels of glucagon-like peptide-1 (GLP-1), an incretin which also confers cardioprotection. As such, we hypothesized that treatment with DPP-4 inhibitors are also cardioprotective.MethodsIn ex vivo experiments: Male Sprague–Dawley rats were randomized to receive by oral gavage either Vildagliptin (20 mg/kg/day), Sitagliptin (100 mg/kg/day), or water for 2 weeks. Excised hearts were Langendorff-perfused with buffer containing either 5 mmol/L or 11 mmol/L glucose and subjected to 35 minutes ischaemia/120 minutes reperfusion. In in vivo experiments: Male young Wistar and Sprague–Dawley rats, middle aged Wistar and Goto-Kakizaki diabetic rats were randomized to receive by oral gavage either Sitagliptin (100 mg/kg/day), or water for 2 weeks. Rats were then subjected to 30 minutes ischaemia/120 minutes reperfusion and infarct size ascertained.ResultsTwo weeks pre-treatment with either Vildagliptin or Sitagliptin reduced ex vivo myocardial infarction (MI) size in hearts perfused with buffer containing 11 mmol/L glucose but not 5 mmol/L glucose. This effect was abolished by Exendin 9–39 (GLP-1 receptor antagonist) and H-89 (PKA antagonist). Treatment of perfused hearts with native GLP-1 was also glucose-sensitive, reducing MI size, at glucose concentrations 7, 9, and 11 mmol/L but not at 5 mmol/L. Finally, Sitagliptin reduced in vivo MI size in middle aged Wistar (7-8 mmol/L glucose) and Goto-Kakizaki (9-10 mmol/L glucose) rats where blood glucose was elevated, but not in young Wistar (5 mmol/L glucose) or Sprague–Dawley (5 mmol/L glucose) rats, where blood glucose was normal.ConclusionsWe find that chronic treatment with DPP-4 inhibitors reduced MI size, via the GLP-1 receptor-PKA pathway, in a glucose-dependent manner. Glucose-sensitive cardioprotection of endogenous GLP-1 in diabetic patients may in part explain why intensive control of serum glucose levels has been associated with increased cardiovascular risk.

Insulin action on glucose uptake by soleus muscles of nonobese diabetic mice and streptozotocin diabetic mice

The changes in the pharmacological responses to cesium were examined in streptozotocin(STZ)-induced diabetic mice. An acute administration of cesium chloride (10 mEqCs+/kg IP) to non-diabetic control mice elicited increased salivation and inhibition of respiration followed by death in about half of the animals examined. These effects of cesium were diminished in STZ-diabetic mice. LD50 for acute cesium was higher in STZ-diabetic mice (14.3 mEq/kg) than non-diabetic buffer controls (11.7 mEq/kg): however, subchronic administration of cesium did not decrease the LD50 in STZ-diabetic mice. The sleeping time induced by pentobarbital was reduced in STZ-diabetic mice and the reduction of the pentobarbital-induced hypnosis was reversed by subchronic cesium pretreatment but not by acute cesium administration. Methamphetamine-induced mortality was increased in STZ-diabetic mice and acute administration of cesium decreased the toxicity in both control and diabetic mice. Inhibition of locomotor activity elicited by acute single injection of cesium chloride was observed in both STZ-diabetic and non-diabetic mice. These results indicate that responses to cesium as well as centrally-acting drugs are affected differentially in STZ-diabetic mice.

Clinical evidence shows that circulating levels of adipocyte fatty-acid-binding protein (A-FABP) are elevated in patients with diabetes and closely associated with ischaemic heart disease. Patients with diabetes are more susceptible to myocardial ischaemia/reperfusion (MI/R) injury. The experiments in the present study investigated the role of A-FABP in MI/R injury with or without diabetes. Non-diabetic and diabetic (streptozotocin-induced) A-FABP knockout and wild-type mice were subjected to MI/R or sham intervention. After MI/R, A-FABP knockout mice exhibited reductions in myocardial infarct size, apoptotic index, oxidative and nitrative stress, and inflammation. These reductions were accompanied by an improved left ventricular function compared with the relative controls under non-diabetic or diabetic conditions. After diabetes induction, A-FABP knockout mice exhibited a preserved cardiac function compared with that in wild-type mice. Endothelial cells, but not cardiomyocytes, were identified as the most likely source of cardiac A-FABP. Cardiac and circulating A-FABP levels were significantly increased in mice with diabetes or MI/R. Diabetes-induced superoxide anion production was significantly elevated in wild-type mice, but diminished in A-FABP knockout mice, and this elevation contributed to the exaggeration of MI/R-induced cardiac injury. Phosphorylation of endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO) were enhanced in both diabetic and non-diabetic A-FABP knockout mice after MI/R injury, but diminished in wild-type mice. The beneficial effects of A-FABP deficiency on MI/R injury were abolished by the NOS inhibitor N(G)-nitro-L-arginine methyl ester. Thus, A-FABP deficiency protects mice against MI/R-induced and/or diabetes-induced cardiac injury at least partially through activation of the eNOS/NO pathway and reduction in superoxide anion production.

Sildenafil, a potent inhibitor of phosphodiesterase type 5, has recently been investigated in animal models of myocardial ischemia-reperfusion (MI/R) injury. Previous studies have suggested that the protective effects of sildenafil are mediated via activation of endothelial nitric oxide (NO) synthesis (eNOS) and inducible NOS (iNOS). To further investigate the protective mechanism of sildenafil, we subjected wild-type, eNOS, and iNOS null animals to 30 min of myocardial ischemia and 24 h of reperfusion. Treatment with 0.06 mg/kg sildenafil 5 min before reperfusion significantly reduced myocardial infarct size in wild-type, eNOS null mice (eNOS(-/-)), and iNOS(-/-) animals. Additionally, the low dose utilized in this study did not alter myocardial cGMP. These results suggest that acute low-dose sildenafil-mediated cardioprotection is independent of eNOS, iNOS, and cGMP. In a second series of experiments, we investigated sildenafil in db/db diabetic mice subjected to MI/R. We found that sildenafil failed to protect diabetic mice against MI/R. However, NO(.) donor therapy was found to significantly protect against MI/R injury in both nondiabetic and diabetic mice, suggesting that protection could be conferred in diabetic mice and that the upstream modulator of soluble guanylyl cyclase, NO(.), may mediate protection independent of cGMP signaling. The present study suggests that further research is needed to delineate the precise mechanisms by which sildenafil exerts cardioprotection.

glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted from gut enteroendocrine L cells in response to nutrient ingestion that potentiates glucose-stimulated insulin secretion via direct actions on the islet β-cell GLP-1 receptor (GLP-1R) ([4][1]). Because of these properties, manipulation

Diabetes-aggravated myocardial ischemia-reperfusion (MI/R) injury remains an urgent medical issue, and the molecular mechanisms involved with diabetes and MI/R injury remain largely unknown. Previous studies have shown that inflammation and P2X7 signaling participate in the pathogenesis of the heart under individual conditions. It remains to be explored if P2X7 signaling is exacerbated or alleviated under double insults. We established a high-fat diet and streptozotocin-induced diabetic mouse model, and we compared the differences in immune cell infiltration and P2X7 expression between diabetic and nondiabetic mice after 24 h of reperfusion. The antagonist and agonist of P2X7 were administered before and after MI/R. Our study showed that the MI/R injury of diabetic mice was characterized by increased infarct area, impaired ventricular contractility, more apoptosis, aggravated immune cell infiltration, and overactive P2X7 signaling compared with nondiabetic mice. The major trigger of increased P2X7 was the MI/R-induced recruitment of monocytes and macrophages, and diabetes can be a synergistic factor in this process. Administration of P2X7 agonist eliminated the differences in MI/R injury between nondiabetic mice and diabetic mice. Both 2 wk of brilliant blue G injection before MI/R and acutely administered A438079 at the time of MI/R injury attenuated the role of diabetes in exacerbating MI/R injury, as evidenced by decreased infarct size, improved cardiac function, and inhibition of apoptosis. Additionally, brilliant blue G blockade decreased the heart rate after MI/R, which was accompanied by downregulation of tyrosine hydroxylase expression and nerve growth factor transcription. In conclusion, targeting P2X7 may be a promising strategy for reducing the risk of MI/R injury in diabetes.

Introduction & Objective: Epigenetic processes and inflammation have each emerged as key players in the development of diabetic kidney disease. Here, we compared the consequences of knockout of the epigenetic enzyme and histone demethylase, KDM6B from myeloid cells in diabetic mice and mice with obstructive uropathy. Methods: Myeloid cell KDM6B knockout (KDM6BMyeloidKO) mice were generated by breeding Kdm6bfl/fl mice with LysMCre+mice and compared to LysMCre+ controls (KDM6BCtrl). M-CSF levels were measured by cytokine array of media conditioned by bone marrow-derived macrophages (BMDMs). Mice were studied either 12 weeks after diabetes induction with streptozotocin (STZ) or 1 week after unilateral ureteral obstruction (UUO) surgery. Kidney injury and inflammation were assessed by qRT-PCR. Results: Basal M-CSF secretion was reduced in KDM6BMyeloidKO BMDMs in comparison to KDM6BCtrl BMDMs (KDM6BCtrl 62pg/mL, KDM6BMyeloidKO 4pg/mL, p<0.01), suggestive of a shift towards M1-like polarization. Kidney injury (as assessed by Havcr1 mRNA) was increased ~90-fold in STZ-diabetic mice and ~615-fold in UUO mice. Similarly, kidney inflammation (as assessed by Ccl2 mRNA) was increased ~9-fold in STZ-diabetic mice and ~72-fold in UUO mice. Blood glucose levels were equally elevated in STZ-diabetic KDM6BCtrl and KDM6BMyeloidKO mice. Knockout of KDM6B from myeloid cells attenuated Havcr1 and Ccl2 upregulation in UUO mice but not in STZ-diabetic mice. Conclusion: Kidney inflammation in STZ-diabetic mice is typically mild, and myeloid cell involvement is primarily through M1-like responses that are independent of the histone demethylase KDM6B. In contrast, myeloid KDM6B contributes to the more advanced inflammation and injury associated with obstructive uropathy. These findings underscore the importance of the physiological context in determining the actions of ostensibly ‘epigenetic enzymes’ in kidney disease, including that caused by diabetes. Disclosure L.Y.Q. Hong: None. H. Kaur: None. D. Tran: None. Y. Liu: None. S. Batchu: None. A. Advani: None.

Tissue ischemia promotes vasculogenesis through chemokine-induced recruitment of bone marrow-derived endothelial progenitor cells (EPCs). Diabetes significantly impairs this process. Because hyperglycemia increases reactive oxygen species in a number of cell types, and because many of the defects responsible for impaired vasculogenesis involve HIF1-regulated genes, we hypothesized that HIF1 function is impaired in diabetes because of reactive oxygen species-induced modification of HIF1alpha by the glyoxalase 1 (GLO1) substrate methylglyoxal. Decreasing superoxide in diabetic mice by either transgenic expression of manganese superoxide dismutase or by administration of an superoxide dismutase mimetic corrected post-ischemic defects in neovascularization, oxygen delivery, and chemokine expression, and normalized tissue survival. In hypoxic fibroblasts cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the EPC mobilizing chemokine stromal cell-derived factor-1 (SDF-1) and of vascular epidermal growth factor, which modulates growth and differentiation of recruited EPCs. In hypoxic EPCs cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the SDF-1 receptor CXCR4, and endothelial nitric-oxide synthase, an enzyme essential for EPC mobilization. HIF1alpha modification by methylglyoxal reduced heterodimer formation and HIF1alpha binding to all relevant promoters. These results provide a basis for the rational design of new therapeutics to normalize impaired ischemia-induced vasculogenesis in patients with diabetes.

PP2Cm overexpression alleviates MI/R injury mediated by a BCAA catabolism defect and oxidative stress in diabetic mice

Streptozotocin (STZ)-induced diabetic mice increased levels of serum glucose, triglyceride and cholesterol, and decreased level of serum insulin. Effects of Bofutsushosan (BOF: Pulvis ledebouriellae compositae: 防風通聖散) and its composed crude drug, gardeniae fructus (GF: 山梔子) were investigated on levels of these diabetic parameters (serum glucose, insulin, triglyceride and cholesterol) in STZ-diabetic mice. BOF and GF were extracted in 10 volumes of distilled water with an automatic extractor “Torobi”. STZ-induced diabetic mice with serum glucose level of over 600 mg/dl at 3 - 4 weeks after intravenous injection of 150 mg/kg STZ were used for experiments. BOF extract, GF extract, geniposide (a main constituent of GF), and glibenclamide were administered intraperitoneally into 3-hour-fasted STZ-diabetic mice. At 6 hours after administration, BOF extract (100 - 300 mg/kg) decreased high levels of serum glucose, triglyceride and cholesterol, and also increased low level of serum insulin in STZ-diabetic mice in a dose-dependent manner, respectively. Anti-diabetic drug glibenclamide (0.3 - 1 mg/kg) as positive control significantly decreased serum glucose and cholesterol levels, and increased serum insulin level in the diabetic mice. GF extract (30 - 300 mg/kg) decreased serum glucose, triglyceride and cholesterol levels but did not affect serum insulin level in the diabetic mice. Geniposide (10 - 100 mg/kg), decreased serum glucose level but did not affect serum insulin and triglyceride levels in the diabetic mice. These results demonstrated that intraperitoneally administrated BOF extract improved abnormal levels of serum glucose, insulin, triglyceride and cholesterol in the STZ-diabetic mice as being similar to glibenclamide. GF extract has an important role in a part of improving actions of BOF in the diabetic mice. The action of GF extract on serum glucose was parallel with the action of geniposide in the diabetic mice, supporting roles of geniposide in anti-hyperglycemic action of GF.

Brief pressure overload of the left ventricle reduces myocardial infarct size via activation of protein kinase C

To develop an oral delivery system of glucagon-like peptide 1 (GLP-1) (28-36) for treating type-2 diabetes, B.S-GLP-1(28-36), a recombinant Bacillus subtilis spores transformed with a plasmid vector encoding five consecutive GLP-1 (28-36) nonapeptides with an enterokinase site was constructed. GLP-1(28-36) nonapeptide was successfully expressed on the surface of B. subtilis spores and validated by Western blot and immunofluorescence. The therapeutic effect of oral administration of B.S-GLP-1(28-36) spores was evaluated in type 2 diabetic model mice. The efficacy of recombinant spores was examined for a period of 13weeks after oral administration in diabetic mice. At the end of the sixth week, diabetic mice with oral administration of BS-GLP-1(28-36) spores showed decreased blood glucose levels from 2·4×10- 2 moll-1 to 1·7×10- 2 moll-1 . By the ninth week, the mean fasting blood glucose level in the experimental group was significantly lower than that in the control group 30min after injection of pyruvate. At the end of the 10th week of oral administration, the blood glucose of the experimental group was significantly lower than that of the control group after intraperitoneal injection of glucose. By the 12th week, fasting blood glucose level and fasting insulin level were measured in all mice, the results showed that the recombinant spores increased the insulin sensitivity of mice. The results of pathological observation showed that the recombinant spores also had a certain protective effect on the liver and islets of mice, and the content of GLP-1(28-36) in the pancreas of the experimental group was increased. The results of this study revealed that GLP-1(28-36) nonapeptides can reduce blood glucose and play an important role in the treatment of type 2 diabetes.

Nitric oxide is a potent anti-apoptotic and cardioprotective molecule in healthy animals. However, recent study demonstrates that overexpression of eNOS exacerbates the liver injury in diabetic animals. whether diabetes may also alter NO's biologic activity in ischaemic/reperfused heart remains unknown. The present experiment was designed to determine whether the nitric oxide donor, S-nitrosoglutathione, may exert different effects on diabetic and non-diabetic myocardial ischaemia/reperfusion (MI/R) injury. Diabetic state was induced in mice by multiple intraperitoneal injections of low-dose streptozotocin (STZ). The control or diabetic mice were subjected to 30minutes ischaemia and 3 or 24hours reperfusion. At 10minutes before reperfusion, diabetic and non-diabetic mice were received an intraperitoneal injection of S-nitrosoglutathione (GSNO, a nitric oxide donor, 1μmol/kg). GSNO attenuated MI/R injury in non-diabetic mice, as measured by improved cardiac function, reduced infarct size and decreased cardiomyocyte apoptosis. In contrast, GSNO failed to attenuate but, rather, aggravated the MI/R injury in diabetic mice. Mechanically, the diabetic heart exhibited an increased nitrative/oxidative stress level, as measured by peroxynitrite formation, compared with non-diabetic mice. Co-administration of GSNO with EUK134 (a peroxynitrite scavenger) or MnTE-2-PyP5 (a superoxide dismutase mimetic) or Apocynin (a NADPH oxidase inhibitor) 10minutes before reperfusion significantly decreased the MI/R-induced peroxynitrite formation and the MI/R injury. Collectively, the present study for the first time demonstrated that diabetes may cause superoxide overproduction, increase NO inactivation and peroxynitrite formation, and thus convert GSNO from a cardioprotective molecule to a cardiotoxic molecule.

The effect of limb remote ischaemic conditioning (RIC) on myocardial infarct (MI) size and left ventricular ejection fraction (LVEF) was investigated in a pre-planned cardiovascular magnetic resonance (CMR) substudy of the CONDI-2/ERIC-PPCI trial. This single-blind multi-centre trial (7 sites in UK and Denmark) included 169 ST-segment elevation myocardial infarction (STEMI) patients who were already randomised to either control (n = 89) or limb RIC (n = 80) (4 × 5 min cycles of arm cuff inflations/deflations) prior to primary percutaneous coronary intervention. CMR was performed acutely and at 6 months. The primary endpoint was MI size on the 6 month CMR scan, expressed as median and interquartile range. In 110 patients with 6-month CMR data, limb RIC did not reduce MI size [RIC: 13.0 (5.1–17.1)% of LV mass; control: 11.1 (7.0–17.8)% of LV mass, P = 0.39], or LVEF, when compared to control. In 162 patients with acute CMR data, limb RIC had no effect on acute MI size, microvascular obstruction and LVEF when compared to control. In a subgroup of anterior STEMI patients, RIC was associated with lower incidence of microvascular obstruction and higher LVEF on the acute scan when compared with control, but this was not associated with an improvement in LVEF at 6 months. In summary, in this pre-planned CMR substudy of the CONDI-2/ERIC-PPCI trial, there was no evidence that limb RIC reduced MI size or improved LVEF at 6 months by CMR, findings which are consistent with the neutral effects of limb RIC on clinical outcomes reported in the main CONDI-2/ERIC-PPCI trial.