Effects Of ACE2-Ang(1-7)-Mas Axis Mediated Pancreatic Endothelial Cells On Pancreatic Isletβ Cell Function And Its Mechanisms

Objective In this study, we assessed the role of the Angiotensin-converting enzyme2(ACE2) on pancreatic islet (3cells and pancreatic microvascular endothelial cell in mice.Methods ACE2knockout and wild-type mice were fed a high-fat diet or a control diet for16weeks. We performed immunofluorescence and immunohistochemically stained to analyze the relative content of insulin (IRC), insulin-positive cell density (ICD), the expression of CD31, VEGF, caspase-3and iNOS in islets,and we determined the expression levels of tumor necrosis factor-a(TNF-a) and interleukin-1β (IL-1β) in islets. Pancreatic islets were isolated from the mice of group KH and incubated with Ang(1-7) to determine its influences on insulin secretion.Results When fed the control diet, ACE2deficiency did not significantly affect the concentration of IRC and ICD. When fed the high-fat diet, compared with WT group, the the concentration of IRC and ICD were decreased in WH group (P<0.05).The relative content of induced NO synthase(iNOS), caspase-3,TNF-a and IL-1β were increased in WH group in comparison with WT group(P<0.05). However, in comparison with WH mice, KH mice exhibited a increased iNOS, caspase-3,TNF-α and IL-1β in islet, and decreased IRC,ICD concentration (P<0.05), which suggests that ACE2knockout mice are more susceptible to high-fat diet-induced β cell dysfuntion. After ineubation with Ang(1-7),the GSIS of islets isolated from KH mice was improved (P<0.05).When fed the control diet, the islet CD31positive cells were no significant difference in wild-type mice (WT group) and ACE2knockout mice (KO group). In comparison with WT group, the islet CD31positive cells and the expression of VEGF were increased in WH group (P<0.05).When fed the high-fat diet, in comparison with WH mice, KH mice exhibited decreased islet CD31positive cells and VEGF expression(p<0.05).Conclusions ACE2deficiency aggravated stress response in islets of pre-diabetic mouse, leading to islet endothelial cells decreased and increasing islet dysfunction. Objective In this study, we assessed the role of the ACE2-Ang(1-7)-Mas axis on function of islet endothelial cell as well as the protection of palmitate-induced dysfunction.Methods MS-1cells were incubated with Ang (1-7) and NO production was measured.The Akt-eNOS signaling pathway activity in MS-1cells were detected by Western blotting.MS-1cells were treated with palmitate in the presence or absence of Ang(1-7). Expression of AKT, eNOS was detected by western blotting. The percentage of apoptotic cells was determined by DNA fragmentation. Reactive oxygen species(ROS) production was measured using a Reactive Oxygen Species Assay Kit.Results Under physiological conditions,Ang(1-7) significantly increased eNOS and AKT phosphorylation in a time-dependent manner and enhanced NO production of MS-1cells(both P<0.05),these effects was blocked by A-779(both P<0.05), indicating that reinforcing the effect of ACE2-Ang(1-7)-Mas axis by application of Ang(1-7) improved the function of MS-1cells.The expression of ACE2and Mas in MS-1cells decreased significantly after exposure to palmitate for24h (both P<0.05). By contrast, ACE and AT1protein level was increased significantly after palmitate exposure (both P<0.05).Compared with the control group, palmitate decreased the phosphorylation of AKT and eNOS (both P<0.05). Pretreatment of MS-1cells with Ang(1-7) prevented the palmitate-induced decrease in AKT and eNOS activation (both P<0.05). Palmitate significantly enhanced intracellular ROS production and increased lipoapoptosis of MS-1cells (both P<0.05). By contrast, palmitate-exposed cells co-incubated with Ang(1-7) decreased DNA fragmentation and ROS production compared with cells in palmitate group (both P<0.05). The anti-lipoapoptosis effect of Ang(1-7) in MS-1cells were blocked by wotmannin or L-NAME(both P<0.05). Conclusions Reinforcing the effect of ACE2-Ang(1-7)-Mas axis by application of Ang(1-7) improved the function of MS-1cells. When incubated with palmitate and Ang(1-7), Ang(1-7) attenuated palmitate-induced apoptosis by down-regulation of ROS production and up-regulation of Akt-eNOS-NO signaling pathway. Objective MIN6cells preincubated with palmitate were co-cultured with different states of MS-1cells to research the ACE2-Ang(1-7)-Mas axis mediated endothelial function in regulating effect on pancreatic β-cell.Methods MIN6cells which were incubated with palmitate for48h were co-cultured with MS-1cells which were preincubated with Ang (1-7) and Mas, grouped as follows:MIN6group; MIN6+PA group; Co-culture group; Co-culture+A group; Co-culture+A+7group. Glucose-stimulated insulin secretion (GSIS) in MIN6was examined after co-culturing. Insulin mRNA in MIN6cells were detected by RT-PCR. MIN6cells apoptosis was analyzed by Annexin V-FITC/PI flow cytometry. P65expression and JNK, p38-MAPK signaling pathways activation in MIN6cells were detected by Western-blotting.Results Palmitate increased basal insulin secretion but decreased glucose stimulated insulin release in MIN6cells (both P<0.05). Comparison with PA group,glucose stimulated insulin secretion was restored partly in Co-culture group, but the difference was not significant. In Co-culture+A group, the basal insulin secretion showed no difference, the GSIS increased significantly (P<0.05) when compared with Co-culture group,while detecting the increase in insulin mRNA expression in MIN6cells(P<0.05).After exposed to palmitate for24h,the AKT phosphorylation levels in MIN6cells were significantly lower (P<0.05). Compared with MIN6+PA group, the AKT phosphorylation levels in Co-culture group increased, but the difference was not statistically significant, but the AKT phosphorylation levels in Co-culture+A group increased significantly (P<0.05). Palmitate increased apoptosis in MIN6cells (P<0.05), while promoted the production of intracellular ROS and increased the expression of P65protein and p-JNK, p-P38phosphorylation levels in MIN6cells (both P<0.05). Compared with MIN6+PA group, co-culture with MS-1cells reduced apoptosis, decreased ROS concentration and reduced P65, JNK, P38expression in MIN6cells. Compared with Co-culture group, the down-regulation of ROS production and the expression of P65,p-JNK,p-P38were greater in co-culture+PA group.Conclusions Ang(1-7) plays a permissive role in protecting β cell by improving islet endothelial cell function.