Detrimental effects of homocysteine on insulin release and apoptosis of pancreatic beta cells

OBJECTIVE: To study the effects of homocysteine (Hcy) on the insulin secretion and apoptosis of pancreatic beta cells. METHODS: Clonal mouse pancreatic beta cells of the line NIT-1 were cultured and exposed to Hcy of 50, 100, 250, 500 and 1000 micromol/L for 6, 12, 24, and 48 hours respectively, then glucose of the concentrations of 5.6 mmol/L or 16.7 mmol/L was added for 1 h, and then the insulin concentration in the culture medium was determined by radioimmunoassay, and MTT method was used to detect the survival rate of the cells. NIT-1 cells were exposed to Hcy of the concentrations of 0, 50, 100, 250, 500 and 1000 micromol/L respectively for 24 h, another NIT-1 cells were exposed to Hcy of the final concentration of 250 micromol/L for 0, 6, 12, and 48 h respectively, then flow cytometry (FC) was used to detect the apoptosis of the cells. After exposure to Hcy of the concentrations of 50, 100, 250, 500 and 1000 micromol/L for 72 h DNA agarose gel electrophoresis was performed. RESULTS: Hcy inhibited the basal and glucose-induced insulin secretion in a time- and dose-dependent manner. The insulin secretion amounts of the NIT-1 cells after exposure to 50 micromol/L Hcy for 24 hours and to 100 micromol/L Hcy for 12 hours were significantly lower by 17.1% and 10.8% compared with the control group (all P < 0.01). Incubated with 100 micromol/L Hcy for 12 hours and 24 hours respectively, the survival rates of the NIT-1 cells were 94.56% and 87.93% respectively (P < 0.05, P < 0.01). Incubated with 100 micromol/L Hcy for 24 hours, the apoptosis rate of the NIT-1 cells was 7.21% (P < 0.01); and incubated with 250 micromol/L Hcy for 12 hours, the apoptosis rate of the NIT-1 cells was 12.93% (P < 0.01). Both FC and agarose gel electrophoresis indicated that Hcy induced cell apoptosis time- and concentration-dependently. CONCLUSION: Hcy impairs insulin secretion and induces cell apoptosis of pancreatic beta cells time- and concentration-dependently.     

胰淀素对大鼠胰岛素和胰高血糖素分泌的影响

目的研究胰淀素对大鼠胰岛素和胰高血糖素分泌的影响。方法应用胶原酶消化和不连续密度梯度离心技术建立离体大鼠胰岛模型,研究不同浓度胰淀素对胰岛素和胰高血糖素分泌的影响。结果不同浓度的葡萄糖增加胰岛素的分泌,抑制胰高血糖素的分泌,并都呈线性相关。葡萄糖浓度为0、5.6、11.2mmol/L时,孵育1h,胰淀素(10-10~10-5mol/L)显著抑制胰高血糖素的分泌(P<0.05),且胰高血糖素的分泌与胰淀素浓度呈负相关(P<0.01)。葡萄糖浓度为5.6mmol/L时,不同浓度的胰淀素增加胰岛素分泌,而且两者呈正相关(P<0.05);但葡萄糖浓度为11.2和16.7mmol/L时,10-5mol/L胰淀素对胰岛素的分泌有明显抑制作用(P<0.05)。结论胰淀素对胰高血糖素的分泌有直接抑制作用;在葡萄糖浓度为5.6mmol/L时,胰淀素能增加胰岛素分泌,抑制胰高血糖素分泌;高糖时,高浓度的胰淀素抑制胰岛素分泌。     

Apelin对葡萄糖的毒性作用及对胰岛细胞PDX-1表达的影响

目的 探讨Apelin对葡萄糖毒性作用及对胰岛细胞胰十二指肠同源盒蛋白-1(PDX-1)表达的影响.方法 在不同葡萄糖浓度下(正糖组5.6 mmol/L,高糖组16.7 mmol/L,极高糖组33.3 mmol/L),+/-Apelin-36培养胰岛β细胞株NIT-1细胞3d,然后测定基础和葡萄糖刺激后胰岛细胞胰岛素分泌量,测定细胞内胰岛素含量和PDX-1蛋白及mRNA表达.结果 与正糖组比较,高糖组和极高糖组的基础和葡萄糖刺激后胰岛素分泌、细胞内胰岛素均明显减少,PDX-1蛋白表达下降(P＜0.05);与未加Apelin组比较,加Apelin高糖组和极高糖组的基础和葡萄糖刺激后胰岛素分泌、细胞内胰岛素明显减少,PDX-1蛋白表达下降(P＜0.05);6组胰岛细胞的胰岛素水平与PDX-1蛋白表达呈正相关,与PDX-1 mRNA表达无关.结论 Apelin可能通过降低PDX-1蛋白表达参与葡萄糖毒性作用,引起胰岛素分泌减少,从而在糖尿病的发病机制中发挥作用.     

甘油三酯对体外培养大鼠胰岛细胞功能的影响

OBJECTIVE: To study the effect of triglycerides (TGs) on the function of in vitro cultured rat islet cells. METHODS: SD rat islet cells were isolated for monolayer cell culture and treated with TGs at different concentrations (0, 0.5, 2.5, 5.0, 10.0 mmol/L) for 72 h. Insulin level in the cell culture media was measured after glucose loading test, and the deposition of fat droplets in the islet cells observed by oil red O-staining. RESULTS: No obvious effect was observed after a 72-h incubation of the islet cells with TGs at low glucose level (2.8 mmol/L), while with the stimulation of high-level glucose (27.8 mmol/L), the insulin secretion was significantly inhibited by TGs of higher concentrations (5.0, 10.0 mmol/L). The deposition of fat droplets was found in the islet cells after incubation with TGs, with TGs of higher concentrations. CONCLUSION: TGs induces deposition of fat droplets in islet cells in vitro, and may inhibit the insulin-secreting function of the cells.     

Effects of cyclosporine A on insulin content and insulin release in neonatal rat islet cell monolayer cultures]

We investigated the effects of cyclosporine A (CsA) on insulin content and insulin release in neonatal rat islet cell monolayer cultures. Insulin content in 5.5-day-old monolayers was not affected after 12-hour incubation in the medium containing 16.7 mmol/L glucose plus 0.1 or 100 micrograms/ml CsA and 5.6 mmol/L glucose plus 10 micrograms/ml CsA. Insulin release in 7.5-day-old monolayers was inhibited after 12-hour incubation in the medium containing 5.6 or 16.7 mmol/L glucose plus 0.01-100 micrograms/ml CsA in a dose-responsive manner. At 16.7 mmol/L glucose, CsA less than or equal to 0.1 microgram/ml did not affect insulin release, while CsA greater than or equal to 1.0 microgram/ml significantly inhibited insulin release and the inhibitory effect did not return to normal after a 16-hour incubation in CsA-free medium. At 5.6 mmol/L glucose, CsA 1.0 microgram/ml did not affect insulin release. CsA 100 micrograms/ml inhibited insulin release less than it did at 16.7 mmol/L glucose. These experimental data demonstrated that high doses of CsA have inhibitory effects on insulin release in islet monolayer cultures.     

半自动消化法分离纯化人胰岛细胞的实验研究

目的 探索用半自动胰腺消化系统建立大规模人胰岛细胞纯化的可靠方法.方法 使用自制的半自动人胰腺消化分离装置及胶原酶P进行人胰腺的消化分离,用COBE2991细胞分离机及HCA-Ficoll纯化人胰岛细胞.通过DTZ染色,在倒置显微镜下计数胰岛细胞的数量和纯度,用胰岛素释放试验检测胰岛细胞的分泌功能.结果 消化后平均每条胰腺可平均获得(38 6201±78 219)个胰岛细胞当量(IEQ),纯化后平均每条胰腺可获得231 420±28 054IEQ,平均每克胰腺组织可获得(3148±317)IEQ,纯化后胰岛细胞平均纯度为(62.81±2.68)%.纯化后的胰岛细胞对胰岛素释放刺激反应良好,高糖(16.7 mmol/L)时胰岛素的释放量为低糖(3.3 mmol/L)时的3.53倍(P≤0.001).结论 成功建立了半自动人胰岛细胞分离、纯化的方法,纯化的人胰岛细胞具有良好的生物活性.     

腺病毒载体转染人HO-1基因增强成人胰岛细胞抗凋亡和胰岛素释放功能的研究

目的了解腺病毒载体转染人HO-1基因对体外培养的成人胰岛的作用,探索基因治疗在胰岛移植中的潜在应用价值。方法将成人胰岛分离纯化后分为3组:转染人HO-1基因组(Ad-HO-1组)、转染EGFP基因组(Ad-EGFP组)及对照组,采用携带人HO-1基因及EGFP基因的腺病毒作为载体对体外培养的成人胰岛进行转染,通过形态学观察、胰岛素释放试验及肿瘤坏死因子(TNF)α及放线菌酮诱导48 h后流式细胞仪检测凋亡。结果Ad-HO-1组的胰岛在高糖刺激下胰岛素分泌量为270 m IU/L±89 m IU/L,高于对照组(182 m IU/L±59 m IU/L)和Ad-EGFP组(189 m IU/L±88 m IU/L,P<0.05);诱导后Ad-HO-1组凋亡细胞的发生率为63%±11%,对照组为91%±11%,两组比较,P<0.01。结论使用腺病毒作为载体对体外培养的成人胰岛转染HO-1基因能够增加胰岛的抗凋亡能力、促进胰岛素的分泌。     

硫酸脱氢表雄酮刺激MIN6细胞胰岛素分泌的机制研究

目的 初步探讨硫酸脱氢表雄酮(DHEAS)促进MIN6细胞胰岛素分泌的机制.方法 选用小鼠胰岛B细胞株MIN6作为实验对象,在葡萄糖浓度为2.8 mmol/L和16.7 mmol/L的条件下,分别以0.1、1、5、10、50μmol/L的DHEAS干预10 min和24h,采用ELISA法测定细胞培养上清液中胰岛素的含量,应用相关试剂盒检测细胞内三磷酸腺苷(ATP)和二磷酸腺苷(ADP)的生物发光水平及比率(ATP/ADP);采用Real-Time PCR法检测不同浓度DHEAS干预24 h的细胞内葡萄糖激酶(GCK) mRNA的表达.结果 两种葡萄糖浓度条件下,以5、10、50 μmol/L DHEAS干预10 min和24 h的MIN6的细胞培养上清液中胰岛素含量和细胞内ATP/ADP比率显著高于空白对照组(P＜0.05).与空白对照组比较,1、5、10、50 μmol/L DHEAS干预24 h的MIN6细胞内GCK mRNA表达显著上调(p＜0.05).结论 DHEAS可能通过增加ATP/ADP的比率,促进MIN6细胞胰岛素的分泌;干预24 h的效应可能与上调GCK mRNA的表达、促进葡萄糖酵解有关.     

EFFECTS OF ACUTE HYPOGLYCEMIA ON THE OREXIN SYSTEM IN RAT

OREXIN isa kind of new neuropeptideisolated from rathypothalamus, which involvedin many physiologicalregulationprogressesvia orexin1receptor(OX R )and orexin2 receptor(OX R ).The physio- 1 2logicalfunctionoforexinisstillnotcompletelyknown ,butitisidentifiedthatorexinis involvedinmany physiologicalprogressesuchasfeeding,sleep,and stress.1However ,itisstillunknown abouttheregulationoforexinsystemby hypo-thalamus,theregulationtopancreasby orexinsystem,and the regulationbetween orexin,bloodglu…