Effects Of ACE2-ang(1-7)-Mas Mediated Pancreatic Endothelial Function On Beta Cell Function

Objective The aim of this study was to evaluate the effects of Angiotensin-converting enzyme2(ACE2) on glucose homeostasis and islet function in mice.Methods Male wildtype (WT) and ACE2knockout (ACE2KO) mice were divided into chow diet group and long-term high-fat diet (HFD) group for16weeks.(1) Islet function of the animals was evaluated by intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin releasing test (IPIRT). The insulin sensitivity was presented by intraperitoneal insulin tolerance test (IPITT).(2) The pancreas was immunohistochemically stained to analyze the relative content of insulin (IRC), insulin-positive cell density (ICD), microvessel density (MVD) in islets. The apoptosis of islet was evaluated by TUNEL.(3) Pancreatic islets isolated by handpicking following collagenase digestion. NO production was detected by nitrate reductase assay. Insulin levels were measured with the mouse insulin ELIS A kit.(4) The eNOS, ICAM-1, VCAM-1mRNA in islet were observed by RT-PCR.Results(1) There was no significant difference of AUCG between WT mice and KO mice. The AUCG in WH mice was significantly elevated than that in WT mice (p<0.05). The AUCG in KH group was obviously increased than that in KO (p<0.05). Compared with WH mice, increased AUCG in KH mice was observed (p<0.05).(2) The AUCI0-30and AUCI0-5were not different between WT and KO group. Decrease AUCIo-5and increased AUCIo-30were observed in WH group compared with those in WT group (both p<0.05). The AUCIo-30in KH group was not increased when compared with that in KO group.(3)In IPITT, similar glucose disappearance rate was observed in WT and KO mice as well as between KH and WH mice.(4) The IRC, ICD, MVD and NO concentration of islet were slightly decreased in KO mice than those in WT mice, but the difference was not significant. Decreased IRC,NO concentration and increased apoptosis in islet were noted in WH group in comparison with WT group (p<0.05). In comparison with WH mice, KH mice exhibited a decreased IRC,MVD,NO concentration and increased apoptosis in islet (p<0.05).(5) The expression of eNOS, ICAM-1and VCAM-1between WT and KO mice was not significant. WH mice exhibited increased ICAM-1, VCAM-1expression and decreased eNOS expression (all P＜0.05). In comparison with WH mice, reduced eNOS and increased ICAM-1and VCAM-1were observed in KH mice (all P<0.05).Conclusions Loss of ACE2deteriorates impairment of islet β cell function and pancreatic islet endothelial function in mice with long-term high-fat diet. Objective The aim of this study was to evaluate the apoptosis effects of palmitate on MS-1cells. Examine the role of Mas on lipoapoptosis of MS-1cells.Methods (1) We down-regulated Mas expression by siRNA in mice islet microvessel cells MS-1. After transfected with Mas-siRNA, Mas mRNA expression was detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR).(2) Culturing the MS-1cells which were transfected by Mas-siRNA at125uM concerntration of PA for24h. MS-1cells apoptosis was analyzed by Annexin V-FITC/PI flow cytometry. MS-1cells which were induced by siRNA and/or PA was co-cultured with MIN6for24h. Glucose-stimulated insulin secretion (GSIS) in MIN6was examined after co-culturing.(3) Akt-eNOS, JNK, p38-MAPK signaling pathways activation and bcl-2, bax, caspase-3expression in MS-1cells were observed by Western-blotting.(4) NO production from MS-1cells was detected by nitrate reductase assay.(5) The FoxO1, p22phox, TNF-a mRNA in MS-1cells were detected by RT-PCR.Results (1) Mas mRNA expression in Mas-siRNA transfected MS-1cells showed a marked reduction, compared with the negative control.(p<0.05).(2) No significant difference of the percentage of apoptosis cell were indicated between normal MS-1cells and Mas-siRNA transfected MS-1cells. PA induced the MS-1cells apoptosis (p<0.05). The cell apoptosis rates induced by PA increased after being transfected by Mas-siRNA (p<0.05).(3) PA promoted the expression of p-JNK, p-p38, caspase-3,Bax and inhibited the expression of p-Akt, p-eNOS, bcl-2in MS-1cells (all p<0.05). The up-regulation of caspase-3,Bax and down-regulation of p-Akt,p-eNOS, bcl-2were greater after Mas-siRNA transfection (all p<0.05).(4) There was no difference of NO concentration between MS-1cells with and without Mas-siRNA transfection. PA reduced the NO concentrations in MS-1cells (both p<0.05). After transfected by Mas-siRNA, the reduction of NO concentration induced by PA was greater (p<0.05).(5) The expression of FoxO1mRNA was not significant among different MS-1cells. The p22phox, TNF-a mRNA expression were not significant between MS-1cells with and without Mas-siRNA transfection. PA stimulated p22phox, TNF-a mRNA expression in MS-lcells (all p<0.05). The p22phox mRNA expression between MS-1cells with and without Mas-siRNA transfection induced by PA was not significant. Transfection Mas-siRNA markedly increased the TNF-a mRNA expression in MS-1cells induced by PA (P＜0.05).(6) After co-cultured, the basic insulin among four group was not significant. The GSIS in MIN6cells co-cultured with MS-1cells with and without Mas-siRNA was not different. In comparison with MIN6cells co-cultured with MS-1which was not stimulated by PA, the MIN6cells co-cultured with MS-1cells induced by PA exhibited a reduction of GSIS (both p<0.05). With the intervention by PA, the MIN6cells co-cultured with MS-1cells transfected by Mas-siRNA showed a slightly reduced GSIS compared with the MIN6cells co-cultured with normal MS-1cells,but the difference was not significant (P>0.05).Conclusions Mas-siRNA transfection promoted MS-1cells lipoapoptosis induced by PA through down-regulation of Akt-eNOS signaling pathways, bcl-2expression and up-regulation of caspase-3, bax expression.