白细胞介素1β和干扰素γ对小鼠胰岛素瘤βTC-6细胞胰岛素分泌功能的影响

目的探讨炎性细胞因子白细胞介素1β（IL-1β）、干扰素γ（IFN-γ）对小鼠胰岛β细胞株βTC-6胰岛素分泌功能的影响。方法将βTC-6细胞培养于高糖DMEM培养基,48h后换用无糖KRBH缓冲液培养30min,分别于含0、1．38、5．50和11．10mmol／L葡萄糖的KRBH缓冲液中孵育60min,放射免疫法检测细胞上清液中胰岛素水平（GSIS）。在BTC-6细胞中分别加入不同浓度的IL-1β（0．15、1．50μg／L）和（或）IFN-γ（10、100U／m1）,24h后换用无糖KRBH缓冲液培养30min,在含1．38mmol／L葡萄糖的KRBH缓冲液中孵育60min,放射免疫法检测细胞上清液中胰岛素水平。多组间数据比较采用单因素方差分析,两组间数据比较采用t检验。结果βTC-6细胞在1．38mmol／L葡萄糖刺激时胰岛素分泌达高峰,与无葡萄糖刺激时相比差异有统计学意义[（151±14）对（120±4）mU／L,t=-4．215,P=0．006];5．50、11．10mmol／L葡萄糖刺激时胰岛素分泌较1．38mmol／L葡萄糖刺激时明显下降（均P〈0．05）。与单纯1．38mmol／L葡萄糖刺激组相比,IL-1β（0．15μg／L）组[（85．53±5．06）mU／L对（103．11±0．27）mU／L,t=4．897,P=0．039]、IL-1β（1．50μg／L）组[（62．62±0．64）mU／L对（103．11±0．27）mU／L,t=212．66,P〈0．001]葡萄糖刺激下胰岛素分泌水平显著下降,下降程度与IL-1β的剂量呈正相关;与单纯葡萄糖刺激组相比,IFN-γ（100U／m1）组葡萄糖刺激下胰岛素分泌显著下降[（73．2±1．6）对（105．2±1．8）mU／L,t=19．52,P：0．003];与IL-1β（0．15μg／L）组及IFN-γ（100U／m1）组相比,IL-1β联合IFN-γ组葡萄糖刺激下胰岛素分泌显著下降（F=77．38,P〈0．001）。结论IL-1β和IFN-γ对13TC-6细胞的胰岛素分泌功能有抑制作用,且两者间具有协同效应。     

细胞外信号调节激酶1/2在白介素-1β抑制胰岛β细胞葡萄糖刺激下胰岛素分泌中的作用

目的 探讨细胞外信号调节激酶1/2 (ERK1/2)信号转导通路在人IL-1β抑制小鼠胰岛素瘤胰岛β细胞(βTC-6)葡萄糖刺激下胰岛素分泌反应(GSIS)中的作用. 方法 (1)βTC-6细胞分别于含不同浓度(0、1.38、5.5 mmol/L)葡萄糖的KRBH缓冲液中孵育60 min,放射免疫法检测细胞上清液中胰岛素浓度;(2)βTC-6细胞分别于含不同浓度(0、1.38、5.5 mmol/L)葡萄糖的KRBH缓冲液中孵育5min,蛋白质免疫印迹法检测细胞蛋白中磷酸化ERK1/2及β-actin的水平;(3)βTC-6细胞加入IL-1β(0.15、1.5、15 ng/ml)培养24 h后于含1.38 mmol/L葡萄糖的KRBH缓冲液中孵育60 min,检测上清液中胰岛素浓度;(4)βTG-6细胞加入IL-1β(0.15、1.5、15 ng/ml)培养24 h后于含1.38 mmol/L葡萄糖的KRBH缓冲液中孵育5 min,检测细胞蛋白中磷酸化ERK1/2及β-actin的水平. 结果 (1)βTC-6细胞在1.38 mmol/L葡萄糖刺激时胰岛素分泌及ERK1/2磷酸化水平均达到高峰;(2)IL-1β可抑制βTC-6细胞葡萄糖刺激下的ERK1/2磷酸化及胰岛素分泌,作用与剂量呈正相关. 结论 ERK1/2信号转导通路可能在IL-1β抑制βTC-6细胞葡萄糖刺激下的胰岛素分泌反应中发挥重要作用.     

软脂酸对HIT—T15细胞线粒体形态功能和胰岛素分泌的影响

目的观察软脂酸（PA）对HIT-T15细胞凋亡、线粒体结构和功能及胰岛素分泌的影响并探讨可能的机制。方法试验分对照组、0．5 mmol／LPA和1．0 mmol／LPA组,透射电镜观察细胞及线粒体形态,流式细胞仪（FC）和原位末端标记法（TUNEL）检测凋亡率,高效液相色谱法（HPLC）检测细胞ATP／ADP,RT—PCR检测过氧化物酶体增殖物激活受体γ共激活因子1（PGC-1）和核呼吸因子1（NRF-1）mRNA,放免法测基础和葡萄糖刺激后胰岛素分泌（GSIS）。结果PA能使线粒体肿胀、嵴破坏;细胞的凋亡率增加,且FC检测高浓度PA组凋亡率增加更显著;ATP／ADP比率下降;PGC-1mRNA和NRF-1mRNA表达增加,高浓度PA组增加更显著;GSIS下降（P均〈0．05）。结论PA导致HIT-T15细胞的线粒体结构和功能的损害及GSIS下降,可能与PGC-1和NRF-1的调节作用有关。     

应用动态血糖监测系统评价格列喹酮对新发2型糖尿病血糖波动的控制及其促泌作用

目的应用动态血糖监测技术评价不同磺脲类药物控制新发2型糖尿病餐后血糖波动的作用特征,对比其胰岛素促泌作用模式的差异,并分析二者的相关性。方法选取2008年3月至12月于解放军总医院门诊就诊病程小于1年的2型糖尿病患者40例,采用随机数字表法将所有研究对象分为3组,分别给予不同磺脲类药物干预1个月：格列喹酮组16例（60mg／早,30mg／晚）,格列齐特组13例（80mg／早,80mg／晚）,格列苯脲组11例（2．5mg／早,2．5mg／晚）。各组研究对象在干预前后均行动态血糖监测（CGMS）和葡萄糖耐量一胰岛素释放试验（OGTF-IRT,分别在服糖前30、0min、糖负荷后15、30、45、60、120和180rain留取静脉血标本）,同时采集空腹血送检各项代谢指标;对比干预前后血糖波动和胰岛素分泌特征的变化,组内治疗前后比较用配对t检验,组间比较采用方差分析。结果格列喹酮和格列奇特组干预后果糖胺较干预前分别下降了17．5％和14．8％[分别为（257±49）、（212±40）μmol／L;（2374-52）、（202±31）Ixmol／L;t=2．098、2．052,均P〈0．05];干预后3组糖化血红蛋白水平均有下降,但差异均无统计学意义（均P〉0．05）;干预后3组的平均血糖波动幅度（MAGE）、平均血糖（MBG）、平均血糖标准差（SDBG）、最大血糖波动幅度（LAGE）、空腹血糖变异系数（CV—FPG）均较干预前有不同程度的改善（均P〈0．05）,其中格列喹酮组和格列奇特组干预后MAGE降幅（分别为42．2％、36．6％）明显优于格列苯脲组的27．0％（分别为6．6±2．3,3．8±2．0;7．0±2．3,4．4±1．6;6．44-1．2,4．74-1．8;t=3．977、2．349、2．977,均P〈0．05）;干预后格列喹酮组和格列奇特组的血糖谱曲线相对平稳,血糖波动特征类似;格列苯脲组清晨空腹血糖偏低,而早餐后、晚餐后血糖升高明显强于其他2组,血糖曲线显著分离;格列喹酮干预后胰岛素分泌速率分别在30和60min达峰,120min降至基线水平;格列奇特与之非常类似;格列苯脲组胰岛素分泌速率达峰时间延迟,峰值更高,高胰岛素血症持续时间相对较长;MAGE与糖负荷后45、60min胰岛素分泌速率显著相关（r=-0．342、-0．386,均P〈0．05）。结论格列喹酮和格列奇特显著降低餐后血糖波动,改善平均血糖控制水平,与其刺激胰岛β细胞快速双峰分泌胰岛素的拟生理促泌模式有关;格列苯脲低血糖发生风险相对较大,与其强而持久的促泌作用有关。     

高糖高棕榈酸培养对β细胞脂质含量及脂肪酸转位酶表达的影响

目的研究高糖高棕榈酸（PA）培养对β细胞脂质含量及脂肪酸转位酶（FAT/CD36）表达的影响。方法NIT-1细胞分别以5mmol/L葡萄糖（NC组）、25mmol/L葡萄糖（HG组）、0.25mmol/LPA＋5mmol/L葡萄糖（HP组）及0.25mmol/LPA＋25mmol/L葡萄糖（GP组）培养24h后,测定细胞内甘油三酯（TG）含量、胰岛素分泌以及FAT/CD36 mRNA与蛋白的表达。结果（1）与NC组比较,各处理组细胞内TG含量增加,而葡萄糖刺激的胰岛素分泌下降,以GP组变化最明显。（2）在高糖孵育下,HG组和GP组FAT/CD36 mRNA与蛋白表达均较NC组显著增加（P〈0.01）,但HP和GP组与相应的葡萄糖组比较,FAT/CD36表达无明显变化。结论在高棕榈酸的环境下,高糖可进一步促进β细胞脂质沉积,抑制葡萄糖刺激的胰岛素分泌;其中高糖上调脂肪酸转位酶表达可能是其重要机制之一。     

那格列奈改善初发2型糖尿病患者早时相胰岛素分泌临床观察

初发T2DM患者100例,随机分为对照组和治疗组各50例。治疗组予那格列奈（唐力）联合甘精胰岛素（来得时）治疗,对照组予阿卡波糖（拜唐苹）联合甘精胰岛素治疗,疗程16周。治疗前后均做空腹、30、60、120、180分钟胰岛素测定。通过早相分泌指数（△I30／△G30,糖负荷后30分钟胰岛素浓度与血浆葡萄糖浓度的比值）评估胰岛B细胞早时相胰岛素分泌能力。结论：那格列奈能改善初发T2DM患者早时相胰岛素分泌。     

格列奈类药物应用进展

本文就格列奈类药物的作用机制及临床应用作一综述。 1 格列奈类药物作用机制 1．1 恢复胰岛素分泌的第一时相，改善胰岛素抵抗 Tankova等通过研究发现，瑞格列奈治疗2型糖尿病患者静脉葡萄糖耐量试验下胰岛索分泌的第一时相的幅度和胰岛素浓度时间曲线下面积明显增加，Jyoti等通过那格列奈与瑞格列奈进行比较发现，那格列奈组不仅能促使胰岛索第一时相的分泌，而且餐后30分钟后胰岛素的浓度明显高于瑞格列奈，提示那格列奈较瑞格列奈刺激胰岛素早期分泌的作用更强。     

肝细胞生长因子及葡萄糖对人胰腺干细胞向胰岛素分泌细胞分化的影响

目的 探讨人胰腺干细胞体外扩增及其向胰岛素分泌细胞分化的影响因素.方法 应用剪碎消化法从孕4～6月龄引产的人胎儿胰腺中获得胰岛组织,选用差速贴壁法从胰岛中分离胰腺干细胞,经扩增后定向诱导成具有胰岛素分泌功能和胰岛样结构的胰岛样细胞团,观察肝细胞生长因子和葡萄糖对胰腺干细胞向胰岛素分泌细胞诱导分化以及胰岛样细胞团胰岛素分泌功能的影响.采用流式细胞仪和免疫组织化学法检测扩增前后nestin阳性细胞和ABCG2阳性细胞.行葡萄糖刺激的胰岛素释放试验,以酶联免疫吸附法（ELISA）检测每1×105个扩增细胞经定向诱导后形成的胰岛样细胞团释放的胰岛素量,比较肝细胞生长因子和葡萄糖对定向分化的影响.采用单因素方差分析或多样本均数两两比较进行统计学分析.结果 经过15代连续扩增,nestin阳性细胞和ABCG2阳性细胞分别扩增了27.9倍和37.8倍.扩增后的细胞经15 d定向诱导后形成具有胰岛素分泌功能和胰岛样结构的细胞团.葡萄糖刺激的胰岛素分泌试验显示,当诱导培养基中肝细胞生长因子浓度为0、5、10、15μg/L时,5.6 mmol/L葡萄糖刺激后,胰岛素分泌量分别为5.00、12.93、14.91和11.23μU;25.0 mmol/L葡萄糖刺激后,胰岛素分泌量分别为19.91、35.53、47.43和23.33 μU.诱导培养基中的葡萄糖浓度为0、2.8、5.6和11.2 mmol/L时,5.6 mmol/L葡萄糖刺激后,胰岛素分泌量分别为9.07、14.21、6.91和3.53 μU;25.0 mmol/L葡萄糖刺激后,胰岛素分泌量分别为26.64、48.97、37.63和34.20 μU.结论 人胰腺中nestin和ABCG2阳性胰腺干细胞具有很强的体外扩增能力.扩增后的胰腺干细胞仍然具有形成胰岛素分泌细胞的功能,经体外诱导后能形成胰岛样细胞团.在胰腺干细胞向胰岛素分泌细胞分化诱导过程中,适当浓度的肝细胞生长因子和葡萄糖有利于胰岛样细胞团的形成和胰岛素分泌.     

曲古霉素A和5-氮杂胞苷对高糖诱导损伤的胰岛β细胞的保护作用

目的 探讨高糖诱导下曲古霉素A（TSA）和5-氮杂胞苷（5-AzaC）对胰岛β细胞增殖、凋亡及功能的影响. 方法 体外培养大鼠胰岛素瘤（RIN-m5f）细胞至指数增长期,根据干预方案分为空白对照（NC）组、高糖诱导（HG）组、0.05、0.10 μmol/L TSA干预组、0.63、1.25 μmol/L 5-AzaC干预组、0.10 μmol/L TSA＋1.25 μmol/L 5-AzaC联合干预组.检测RIN-m5f细胞增殖活性、细胞凋亡率、葡萄糖刺激胰岛素分泌能力. 结果 0.05、0.10 μmol/L TSA干预组、0.63、1.25 μmol/L 5-AzaC干预组及联合干预组的细胞增殖活性均高于HG组,而细胞凋亡率均低于HG组（P＜0.05）.在3.3 mmol/L及25 mmol/L葡萄糖刺激时,各组葡萄糖刺激胰岛素分泌量均高于HG组（P＜0.05）. 结论 TSA和5-AzaC可抑制高糖诱导的胰岛β细胞凋亡和恢复β细胞的胰岛素分泌功能.     

细胞外调节蛋白激酶1/2在小鼠胰岛瘤βTC-6细胞胰岛素分泌功能中的作用探讨

目的探讨细胞外调节蛋白激酶(ERK)1/2信号转导通路在βTC-6细胞葡萄糖刺激胰岛素分泌(GSIS)反应中的作用。方法采用不同浓度葡萄糖刺激βTC-6细胞,放射免疫法检测细胞上清液中胰岛素浓度,Western blot检测细胞裂解物ERK1/2磷酸化水平。采用MEK抑制剂PD98059处理βTC-6细胞,放射免疫法检测葡萄糖刺激后上清液中胰岛素浓度,Western blot检测葡萄糖刺激后ERK1/2磷酸化水平。结果βTC-6细胞在1.38mmol/L葡萄糖刺激时,胰岛素分泌和ERK1/2磷酸化水平达高峰。PD98059可抑制葡萄糖刺激下ERK1/2磷酸化及胰岛素分泌,作用与剂量呈正相关。结论ERK1/2信号转导通路可能在βTC-6细胞GSIS中发挥作用。     

Therapeutical concentrations of tolbutamide, glibenclamide, gliclazide and gliquidone at different glucose levels: in vitro effects on pancreatic A- and B-cell function.

In the classical model of isolated perfused rat pancreas four commonly used sulfonylureas--tolbutamide, glibenclamide, gliquidone and gliclazide--were investigated at therapeutical concentrations at three different glucose levels (with 0, 2.22 and 5 mmol/l glucose surrounding) and in the presence of a metabolic stimulus with glucose at 8.33 mmol/l. All the sulfonylureas stimulated the B-cell function. Tolbutamide, gliquidone and gliclazide produced a prompt biphasic hormone release while glibenclamide induced a delayed monophasic insulin secretion. In all cases the amount of insulin released depended on the metabolic condition. As the environmental glucose levels fell, the sulfonylureas' stimulatory effect on the B-cell function decreased. At the therapeutical concentrations we tested, no sulfonylurea influenced A-cell activity whether directly or indirectly via an insulin-mediated paracrine inhibition of glucagon release.     

Glucose modulates the amount, but not the kinetics, of insulin released by sulfonylureas

This study compares the insulin-secretory profiles induced by therapeutical concentrations of four different sulfonylureas—tolbutamide, gliquidone, gliclazide,^* and glibenclamide—and the amount of hormone released by each under different ambient glucose concentrations, using the isolated perfused rat pancreas model. All four sulfonylureas stimulated B-cell function, but the kinetics varied. Tolbutamide, gliquidone, and gliclazide produced a quick, biphasic release, whereas glibenclamide stimulated a delayed monophasic insulin secretion. Dramatic falls in insulin release were observed when ambient glucose concentrations were lowered. Glucagon release was not influenced by any of the sulfonylureas whatever the metabolic condition, neither directly nor indirectly, via an insulin-mediated paracrine inhibition of A-cell activity.     

格列喹酮对糖基化终产物诱导的人肾系膜细胞RANTES表达的影响

目的探讨格列喹酮对糖基化终产物（AGEs）诱导的人肾系膜细胞（HRMC）正常T细胞表达分泌的调节活化蛋白（RANTES）表达的影响。方法运用糖基化修饰的牛血清白蛋白（AGE-BSA）和格列喹酮干预体外培养的HRMC,用半定量RT-PCR检测细胞中RANTES mRNA水平,用ELISA法测定细胞培养上清中RANTES蛋白水平。结果格列喹酮干预组HRMC RANTES的mRNA表达和蛋白分泌低于AGE-BSA组（P〈0.05）,且降低水平呈浓度和时间依赖性。结论格列喹酮能抑制糖基化终产物诱导的HRMC RANTES的表达和分泌。     

Cholinergic stimulation of insulin release from cloned B-cell lines HIT-T15 and RINm5F

Summary Acetylcholine stimulated the release of insulin from cultured HIT-T15 and RINm5F cells in the absence of glucose confirming that the insulin response to cholinergic stimulation is preserved in these two cloned B-cell lines. In addition acetylcholine potentiated glucose stimulated insulin release from HIT-T15 but not from RINm5F cells. Perifused HIT-T15 cells responded to square wave acetylcholine challenge with a monophasic release of insulin while the release profile for RINm5F cells was irregular and reduced. Acetylcholine stimulated insulin release from both cell lines was reduced by atropine but unaffected by hexamethonium confirming a mechanism of action involving muscarinic receptor activation.     

格列喹酮可促进成肌细胞葡萄糖摄取

目的 观察格列喹酮对小鼠成肌细胞(C2C12)葡萄糖摄取及葡萄糖转运相关蛋白葡萄糖转运体4表达的影响.方法 采用格列喹酮干预体外培养的C2C12细胞,将细胞分为空白组、胰岛素组(1×10-7mol/L胰岛素)、格列喹酮组(1×10-5mol/L格列喹酮)、格列喹酮+胰岛素组(1×10-5mol/L格列喹酮+1×10-7mol/L胰岛素).应用葡萄糖氧化酶法检测细胞培养基中葡萄糖水平,DMEM组葡萄糖含量平均值减去其余各孔葡萄糖含量算得各孔细胞葡萄糖消耗量;用MTT法检测细胞活力;利用液闪计数法检测C2C12细胞葡萄糖摄取量;采用半定量实时聚合酶链反应(RTPCR)检测细胞中葡萄糖转运体4 mRNA水平.应用单因素方差分析进行数据统计.结果 与空白组相比,格列喹酮组C2C12细胞葡萄糖消耗量明显增加[(11.0±0.5)vs(7.9±0.6)mmol/L,q=0.36,P＜0.01];格列喹酮+胰岛素组C2C12细胞葡萄糖消耗量较胰岛素组增多[(13.8±0.7)vs(12.3±0.6)mmol/L,q=0.72,P＜0.01].胰岛素组、格列喹酮组C2C12细胞对葡萄糖的摄取均增加,分别为空白组的(1.68±0.09)、(1.52±0.23)、(1.23±0.16)倍(q值分别为14.78、11.30、5.00,均P＜0.01);格列喹酮组C2C12细胞葡萄糖摄取增加较格列苯脲组明显(q=6.30,P＜0.01),格列喹酮+胰岛素组C2C12细胞葡萄糖摄取为空白组的(1.93±0.12)倍(q=13.04,P＜0.01),且明显高于胰岛素组(q=5.43,P＜0.01).格列喹酮组C2C12细胞葡萄糖转运体4 mRNA水平与空白组比较无明显差异.结论 格列喹酮可促进C2C12细胞对葡萄糖的摄取,增加胰岛素刺激的葡萄糖摄取,并未增加C2C12细胞葡萄糖转运体4基因表达.     

Comparison of insulinotrophic actions of nateglinide with glibenclamide dissociated from absorption in conscious dogs

Nateglinide is more rapidly absorbed than glibenclamide. Therefore, the different absorption kinetics of both drugs were eliminated by intraportal administration in conscious fasted dogs. The plasma insulin profiles were compared under similar kinetic changes in plasma drug concentrations. After a priming dose of nateglinide (1 mg/kg. 5 min) or glibenclamide (40 microg/kg. 5 min), plasma drug concentrations reached a peak at 4 minutes (nateglinide, 80 +/- 5 micromol/L, n = 6 and glibenclamide, 263 +/- 60 nmol/L, n = 6) followed by a sustained level at approximately 30% of the peak concentration at 30 minutes. Nateglinide led to a rapid and constant reduction in arterial glucose of approximately 30% basal, while glibenclamide promoted a gradual decrease to approximately 50% basal at 120 minutes. An increase in plasma insulin level by nateglinide of 4 times basal (218 +/- 58 pmol/L v 47 +/- 3 pmol/L, P <.05, n = 6) occurred at 6 to 10 minutes followed by sustained release of 1.4 times basal (67 +/- 15 pmol/L, n = 6). The insulin surge was more than doubled (484 +/- 209 pmol/L, n = 6) under a euglycemic clamp. Insulin release by glibenclamide increased gradually reaching 10-fold basal (449 +/- 166 pmol/L, n = 6) at 60 minutes. This was not enhanced during a euglycemic clamp. Lowering the primed doses of nateglinide resulted in a diminished peak plasma insulin concentration. In contrast, glibenclamide caused only a slower increase, but eventually reaching a similar peak. By increasing the continuous infusion of nateglinide, the sustained insulin release was not altered. Glibenclamide, but not nateglinide, evoked prompt and sustained insulin release in the continuing presence of the other. These results are consistent with the concept that nateglinide produces a quick, but very short-lived, interaction with sulfonylurea (SU)-receptors on plasma membrane by free access of the drug from the cell exterior. In contrast, glibenclamide promotes a slow and longer interaction with the receptor by distribution of the drug into the cell inferior. We conclude, therefore, that not only the different kinetics of gastrointestinal (GI) absorption, but also the inherent difference in the interaction with beta cells is attributed to the different insulin release characteristics between nateglinide and glibenclamide in vivo.     

Effect of two sulphonylureas on the dose kinetics of glucose-induced insulin release in normal and diabetic subjects.

The effect of two second generation sulphonylureas, gliquidone and glibenclamide, on insulin secretion has been studied in the basal state and in combination with glucose infusions in normal controls, patients with mild maturity-onset diabetes, and subjects with normal glucose tolerance but low insulin response. When injected intravenously, gliquidone caused rapid elevation of plasma insulin, peaking at 5 min in all groups, while glibenclamide induced a slow rise in insulin. Insulin response was somewhat smaller than normal in diabetics and low insulin responders. In all groups, 25 micrograms/kg glibenclamide and 200 micrograms/kg gliquidone were equipotent in generating an insulin response at the basal state. Equipotent amounts of sulphonylureas were combined with glucose in fusions at three different dose levels. The glucose-insulin dose relationships, established by giving glucose alone, demonstrated curves that were flatter, and shifted to the right of the control in diabetics and low insulin responders, the changes being more marked in the former group. Addition of sulphonylurea induced a left shift in the dose-response relationships in controls and low insulin responders; under these conditions the effect of glibenclamide was more pronounced than that of gliquidone. The dose-response relation for glucose-induced insulin release was completely normalized in low responders when sulphonylureas were added. In the group of mild diabetics, insulin response to glucose was enhanced by sulphonylureas only to a modest extent, the dose-response curves remaining grossly abnormal.     

Comparison of the secretory properties of four insulin-secreting cell lines

The insulin-secretory responsiveness of four popular and widely used insulin-secreting cells lines (RINm5F, HIT-T15, INS-1 and BRIN-BD11 cells) to a range of stimuli including glucose, amino acids, neurotransmitters, peptide hormones and sulphonylureas was studied. Differences were seen in the pattern of responsiveness of the cell lines to the various modulators of insulin release. While these studies revealed that INS-1 cells had the highest insulin content, only BRIN-BD11 cells exhibited a significant step-wise insulin secretory response to increasing glucose concentrations. BRIN-BD11 cells also showed pronounced insulin responses to leucine, KIC, L-arginine, L-alanine and palmitic acid. All the cell lines tested gave significant responses to the neurotransmitters carbachol and glibenclamide with increased insulin release. A comparison was made between the functional characteristics of the various cell lines with those of freshly isolated rat islets. This illustrated the general value of each cell line as a model for studies of insulin secretion. Electrofusion-derived BRIN-BD11 cells appeared to closely mimic the glucose sensitivity and overall secretory performance of normal rat islets.     

Hydrogen sulphide reduces insulin secretion from HIT-T15 cells by a KATP channel-dependent pathway

Hydrogen sulphide (H(2)S), a naturally occurring gas exerts physiological effects by opening K(ATP) channels. Anti-diabetic drugs (e.g. glibenclamide) block K(ATP) channels and abrogate H(2)S-mediated physiological responses which suggest that H(2)S may also regulate insulin secretion by pancreatic beta-cells. To investigate this hypothesis, insulin-secreting (HIT-T15) cells were exposed to NaHS (100 microM) and the K(ATP) channel-driven pathway of insulin secretion was tracked with various fluorescent probes. The concentration of insulin released from HIT-T15 cells decreased significantly after NaHS exposure and this effect was reversed by the addition of glibenclamide (10 microM). Cell viability and intracellular ATP and glutathione (GSH) levels remained unchanged, suggesting that changes in insulin secretion were not ATP linked or redox dependent. Through fluorescence imaging studies, it was found that K(+) efflux occurs in cells exposed to NaHS. The hyperpolarised cell membrane, a result of K(+) leaving the cell, prevents the opening of voltage-gated Ca(2+) channels. This subsequently prevents Ca(2+) influx and the release of insulin from HIT-T15 cells. This data suggest that H(2)S reduces insulin secretion by a K(ATP) channel-dependent pathway in HIT-T15 cells. This study reports the molecular mechanism by which H(2)S reduces insulin secretion and provides further insight into a recent observation of increased pancreatic H(2)S production in streptozotocin-diabetic rats.