3T3-L1脂肪细胞对MIN6胰岛素细胞中Kir6.2表达的抑制作用

为明确脂肪细胞对胰岛素细胞中KATP通道表达的直接影响,MIN6胰岛素细胞被分为两组：一组为对照组,一组与分化的3T3-L1脂肪细胞共培养1周。运用半定量RT-PCR方法测定MIN6细胞中KATP通道蛋白Kir6．2的表达变化,Fura-2荧光方法测定MIN6细胞内钙浓度的变化,放射免疫测定方法明确MIN6细胞的胰岛素分泌功能。结果显示,与3T3-L1脂肪细胞共培养1周后,MIN6细胞中Kir6．2的表达明显减少,其表达水平降低为对照组的65．3％。对照组MIN6细胞在0．1mmoi／L甲苯磺丁脲(KATP通道关闭剂)的刺激下,表现为细胞内钙水平显著性升高和胰岛素分泌显著性增加,而共培养组MIN6细胞则失去了甲苯磺丁脲刺激所引起的细胞内钙升高及胰岛素分泌反应。以上实验结果表明,3T3-L1脂肪细胞可以通过分泌一些活性因子直接降低MIN6细胞中KATP通道蛋白的表达和合成,损害MIN6细胞的胰岛素分泌功能。实验结果提示脂肪细胞直接参与2型糖尿病中胰岛β细胞功能障碍的发生。     

脂肪酸诱导胰岛β细胞凋亡过程中FoxO1对MTP的转录调控研究

目的研究微粒体甘油三酯转移蛋白MTP在脂肪酸诱导的胰岛B细胞凋亡过程中,转录水平受FoxO1调控的情况。方法脂肪酸处理胰岛8细胞系MIN6细胞,MTF和Hoechst染色检测细胞活力和凋亡情况;ReahimePCR检测MTP相对表达量;染色质免疫共沉淀技术检验FoxO1与肘即启动子区的结合情况;荧光素酶报告基因系统检测Fox01对MTP的转录调控情况。结果脂肪酸处理引起MIN6细胞活力下降、凋亡增加,使MIN6细胞中MTP mRNA水平上升;糖尿病模型小鼠胰岛中MTPmRNA水平上升;转录因子FoxO1的过表达可上调MTP的转录活性;ChIP—PCR结果显示FoxO1能与MZP的启动子区相结合。结论MTP在脂肪酸诱导胰岛B细胞凋亡的过程中,作为转录因子FoxO1的下游靶基因,转录水平受到FoxO1的调控。     

沉默长链非编码RNA Meg3损伤小鼠胰岛细胞的胰岛素分泌功能

MEG3是一种长链非编码RNA。已有研究证明,鼠源Meg3参与小鼠诱导多能干细胞、神经元和视网膜的分化过程。最新报道,MEG3在人胰岛β细胞中高表达,但其对维持成年胰岛β细胞的功能尚不清楚。本研究旨在探讨Meg3在小鼠胰岛细胞胰岛素分泌功能中的作用。实时定量PCR揭示,与Balb/c小鼠心、肝、脾、肺、肌、肾等组织/器官比较,Meg3在胰腺组织中高表达。在非糖尿病小鼠发生自发性糖尿病的第8、12周,Meg3在胰岛中的表达水平分别下调24%±8%和29%±9% (P<0.01);而当血糖升高20 mmol/L,小鼠胰岛中Meg3表达下调72%±16%(P<0.01)。在MIN6细胞中采用RNA干扰敲减Meg3的表达,在高糖浓度（20 mmol/L）刺激条件下,胰岛素分泌显著减少。小鼠静脉注射siRNA,结合血糖测定或葡萄糖耐受试验（IPGTT）显示,si-Meg3小鼠血清胰岛素水平显著下降。注射葡萄糖前血糖升高,注射葡萄糖后耐受能力降低;免疫组化分析显示,si-Meg3小鼠胰岛素阳性细胞的面积减少。实验结果提示,Meg3通过参与胰岛素的合成和分泌维持成年小鼠胰岛功能。Meg3表达失调可能参与I型糖尿病（T1DM）发病过程。     

lncRNA TUG1在胰岛β细胞分泌胰岛素中的功能研究

目的:关于lncRNA TUG1在体内外胰岛β细胞分泌胰岛素中的功能研究。方法:通过qRT-PCR检测lncRNA TUG1在小鼠胰腺,脑,肌肉等不同组织的表达。体外干扰MIN6胰岛素瘤细胞系lncRNA TUG1后,通过MTT法和流式细胞计数检测对β细胞增殖和周期影响;通过GSIS检测β细胞不同糖浓度刺激下的胰岛素分泌水平;采用qRT-PCR检测β细胞Insulin及相关特异转录因子Pdx1,Maf A,Neuro D,Glut2的变化;外源性封闭正常成年小鼠中lncRNA TUG1的表达后,采用ELISA法检测对血清胰岛素的影响,采用免疫组化检测对胰岛形态的影响。结果:lncRNA TUG1在胰腺组织中高度表达。干扰lncRNA TUG1后可致β细胞增殖活力受到抑制,糖刺激下的胰岛素分泌水平下降,Insulin及相关特异转录因子Pdx1,Maf A,Neuro D,Glut2减少;外源性封闭正常成年小鼠中lncRNA TUG1的表达后,血清胰岛素减少,胰岛面积减小。结论:干扰lncRNA TUG1后在体内外均可导致胰腺β细胞分泌胰岛素减少,提示lncRNA TUG1可在体内外影响β细胞的胰岛素分泌,lncRNA TUG1是调节胰岛β细胞功能的因素之一。     

INS-1E细胞葡萄糖毒性模型的建立

目的:建立胰岛细胞系INS-1E细胞的葡萄糖毒性模型。方法:将INS-1E细胞分别在不同葡萄糖浓度(5.5 mmol/L、16.7mmol/L、25 mmol/L、30 mmol/L)的1640完全培养基中培养不同时间(48 h、72 h、96 h、120 h),分别在不同时间点取细胞进行细胞功能检测,实时荧光定量PCR法检测胰岛素m RNA的表达,ELISA检测葡萄糖刺激的胰岛素的分泌。结果:与对照组相比,高糖浓度(5.5 mmol/L、16.7 mmol/L、25 mmol/L、30 mmol/L)培养基中培养48 h后,INS-1E细胞的胰岛素合成和分泌的功能均增加(P均0.05),随着培养基中葡萄糖浓度的升高以及培养时间的延长,INS-1E细胞胰岛素合成及分泌的功能逐渐下降,当在葡萄糖浓度为30 mmol/L的培养基中培养120 h后,胰岛素m RNA合成及葡萄糖刺激的胰岛素分泌均显著降低(P均0.01)。结论:INS-1E细胞在30 m M的葡萄糖中培养120 h形成稳定的葡萄糖毒性模型。     

单克隆人胰腺干细胞分化特性的研究

研究1例来源于4月龄男性流产胎儿胰腺组织的单克隆人胰腺干细胞（monoclonal human pancreatic stem cell,mhPSC）系的体内外分化特性。将mhPSCs接种在铺有0．1％明胶的培养皿内,扩增培养3d后,加高糖DMEM诱导液诱导培养25d。相差显微镜下．观察细胞生长状况。采用双硫腙染色法、RT—PCR及葡萄糖刺激释放胰岛素和C肽实验．对体外定向诱导mhPSCs分化为功能性胰岛进行检测。将mhPSCs悬液注射在成年雄性裸鼠腹股沟皮下．注射30d时,取出移植物,采用SP法进行免疫组织化学反应,以检测mhPSCs的体内自然分化潜能。体外扩增培养,mhPSCs贴壁生长,呈多角形上皮样。生长至单层．呈“铺路石”状。体外定向诱导,细胞逐渐由多角形变成圆形,并聚集成类胰岛。诱导培养15d时．形成的类胰岛中少数细胞分化为B细胞,双硫腙染色阳性。诱导培养25d时,多数细胞分化为8细胞,双硫腙染色阳性,转录表达胰岛素的mRNA。用不同浓度葡萄糖刺激．诱导胰岛不仅释放胰岛素和C肽,而且其释放量随糖刺激浓度升高显著增加（0．01〈P〈0．05）。体内分化实验显示,mhPSCs在裸鼠背部形成类畸胎瘤。类畸胎瘤易与裸鼠分离,色白,血管丰富。显著表达pdx1、胰岛素、胰高血糖素、CK、MBP及NF蛋白。该研究结果证实单克隆人胰腺干细胞系体外定向诱导分化为包含大量β细胞的功能性类胰岛,在体内自然分化为胰岛、上皮及神经组织细胞。     

干细胞定向分化胰岛β细胞新进展

在治疗糖尿病领域里,干细胞定向分化成胰岛β细胞是目前新颖又有前景的一种糖尿病治疗策略,不仅克服了注射胰岛素所带来的并发症,还避免了胰岛移植供体来源的短缺不足。目前供体细胞的材料主要有从胰腺中分离出的胰腺干细胞/胰腺祖细胞、胰腺导管细胞、胰腺泡细胞、肝实质细胞、小肠细胞、神经干细胞、ES细胞以及近来研究热点之一的iPS细胞。以上这些细胞都被证明或多或少具备分化成胰岛素分泌的细胞形态的潜力。在体外分化技术方面,国际上有D’Amour法、Lumelsky法等。现对干细胞定向分化胰岛β细胞的一些研究概况作简单评述。     

利拉鲁肽抑制高糖诱导的小鼠胰岛细胞损伤

利拉鲁肽(liraglutide, Lira)是胰高血糖素样肽-1的类似物,在糖尿病治疗中发挥重要作用,但利拉鲁肽通过改善胰岛β细胞的功能实现治疗糖尿病的具体机制尚未完全阐明。本研究采用高糖(33 mmol/L)诱导胰岛MIN6细胞48 h建立高糖损伤模型,CCK-8检测发现,与对照组相比,高糖组MIN6细胞活力下降(P<0.05),利拉鲁肽作用高糖组细胞活力升高(P<0.05);小鼠胰岛素和ATP含量检测发现,与对照组相比,高糖组胰岛素分泌降低(P<0.01),ATP含量减少(P<0.001),利拉鲁肽作用高糖组胰岛素释放量增加(P<0.05)和细胞内ATP含量增加(P<0.001);采用活体细胞线粒体膜通道孔(MPTP)荧光检测发现,与对照组相比,高糖组绿色荧光强度降低(P<0.001),利拉鲁肽作用高糖组绿色荧光强度增加(P<0.001);DCFH-DA探针联合流式细胞仪检测细胞活性氧簇(ROS)含量发现,与对照组相比,高糖组ROS水平升高(P<0.001),利拉鲁肽作用高糖组ROS水平降低(P<0.01);细胞内丙二醛...     

DMSO联合高糖体外诱导兔骨髓间充质干细胞分化为胰岛样细胞的研究

该研究旨在探索二甲基亚砜(dimethyl sulphoxide,DMSO)联合高糖体外诱导日本大耳白兔(Lepus brachyurus)骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)分化为胰岛样细胞的可行性及其调控机制。采用不含血清的DMSO联合高糖诱导P3代兔BMSCs分化为胰岛样细胞。倒置显微镜下观察细胞的形态变化;双硫腙染色和免疫荧光染色检测细胞分化;RTq PCR检测胰岛细胞相关基因[Foxa2(forkhead box A2)、Nestin(neuroepithelial stem cell protein)、Pax6(paired box gene 6)、Pdx-1(pancreatic duodenal homeobox-1)、胰岛素]的表达,并以诱导培养前(0 d)细胞、兔骨髓细胞、P3代BMSCs和胰腺组织中胰岛细胞相关基因表达情况作为参照。结果表明,Foxa2、Nestin和Pax6基因不能作为兔BMSCs向胰岛样细胞诱导分化成功的标志基因。DMSO能够激活Pdx-1基因的表达,促进兔BMSCs分化为可分泌胰岛素的胰岛前体细胞。高糖能够促进兔BMSCs分化为胰岛样细胞,并可显著促进Pdx-1和Foxa2基因的表达。     

High passage MIN6 cells have impaired insulin secretion with impaired glucose and lipid oxidation

Type 2 diabetes is a metabolic disorder characterized by the inability of beta-cells to secrete enough insulin to maintain glucose homeostasis. MIN6 cells secrete insulin in response to glucose and other secretagogues, but high passage (HP) MIN6 cells lose their ability to secrete insulin in response to glucose. We hypothesized that metabolism of glucose and lipids were defective in HP MIN6 cells causing impaired glucose stimulated insulin secretion (GSIS). HP MIN6 cells had no first phase and impaired second phase GSIS indicative of global functional impairment. This was coupled with a markedly reduced ATP content at basal and glucose stimulated states. Glucose uptake and oxidation were higher at basal glucose but ATP content failed to increase with glucose. HP MIN6 cells had decreased basal lipid oxidation. This was accompanied by reduced expressions of Glut1, Gck, Pfk, Srebp1c, Ucp2, Sirt3, Nampt. MIN6 cells represent an important model of beta cells which, as passage numbers increased lost first phase but retained partial second phase GSIS, similar to patients early in type 2 diabetes onset. We believe a number of gene expression changes occurred to produce this defect, with emphasis on Sirt3 and Nampt, two genes that have been implicated in maintenance of glucose homeostasis.     

Microarray Analysis of Novel Candidate Genes Responsible for Glucose-Stimulated Insulin Secretion in Mouse Pancreatic β Cell Line MIN6

Elucidating the regulation of glucose-stimulated insulin secretion (GSIS) in pancreatic islet β cells is important for understanding and treating diabetes. MIN6 cells, a transformed β-cell line derived from a mouse insulinoma, retain GSIS and are a popular in vitro model for insulin secretion. However, in long-term culture, MIN6 cells'' GSIS capacity is lost. We previously isolated a subclone, MIN6 clone 4, from the parental MIN6 cells, that shows well-regulated insulin secretion in response to glucose, glybenclamide, and KCl, even after prolonged culture. To investigate the molecular mechanisms responsible for preserving GSIS in this subclone, we compared four groups of MIN6 cells: Pr-LP (parental MIN6, low passage number), Pr-HP (parental MIN6, high passage number), C4-LP (MIN6 clone 4, low passage number), and C4-HP (MIN6 clone 4, high passage number). Based on their capacity for GSIS, we designated the Pr-LP, C4-LP, and C4-HP cells as “responder cells.” In a DNA microarray analysis, we identified a group of genes with high expression in responder cells (“responder genes”), but extremely low expression in the Pr-HP cells. Another group of genes (“non-responder genes”) was expressed at high levels in the Pr-HP cells, but at extremely low levels in the responder cells. Some of the responder genes were involved in secretory machinery or glucose metabolism, including Chrebp, Scgn, and Syt7. Among the non-responder genes were Car2, Maf, and Gcg, which are not normally expressed in islet β cells. Interestingly, we found a disproportionate number of known imprinted genes among the responder genes. Our findings suggest that the global expression profiling of GSIS-competent and GSIS-incompetent MIN6 cells will help delineate the gene regulatory networks for insulin secretion.     

Functional Analysis of Novel Candidate Regulators of Insulin Secretion in the MIN6 Mouse Pancreatic β Cell Line

Elucidating the regulation of glucose-stimulated insulin secretion (GSIS) in pancreatic β cells is important for understanding and treating diabetes. The pancreatic β cell line, MIN6, retains GSIS but gradually loses it in long-term culture. The MIN6 subclone, MIN6c4, exhibits well-regulated GSIS even after prolonged culture. We previously used DNA microarray analysis to compare gene expression in the parental MIN6 cells and MIN6c4 cells and identified several differentially regulated genes that may be involved in maintaining GSIS. Here we investigated the potential roles of six of these genes in GSIS: Tmem59l (Transmembrane protein 59 like), Scgn (Secretagogin), Gucy2c (Guanylate cyclase 2c), Slc29a4 (Solute carrier family 29, member 4), Cdhr1 (Cadherin-related family member 1), and Celsr2 (Cadherin EGF LAG seven-pass G-type receptor 2). These genes were knocked down in MIN6c4 cells using lentivirus vectors expressing gene-specific short hairpin RNAs (shRNAs), and the effects of the knockdown on insulin expression and secretion were analyzed. Suppression of Tmem59l, Scgn, and Gucy2c expression resulted in significantly decreased glucose- and/or KCl-stimulated insulin secretion from MIN6c4 cells, while the suppression of Slc29a4 expression resulted in increased insulin secretion. Tmem59l overexpression rescued the phenotype of the Tmem59l knockdown MIN6c4 cells, and immunostaining analysis indicated that the TMEM59L protein colocalized with insulin and GM130, a Golgi complex marker, in MIN6 cells. Collectively, our findings suggested that the proteins encoded by Tmem59l, Scgn, Gucy2c, and Slc29a4 play important roles in regulating GSIS. Detailed studies of these proteins and their functions are expected to provide new insights into the molecular mechanisms involved in insulin secretion.     

Sphingosine 1-phosphate (S1P) regulates glucose-stimulated insulin secretion in pancreatic beta cells

Recent studies suggest that sphingolipid metabolism is altered during type 2 diabetes. Increased levels of the sphingolipid ceramide are associated with insulin resistance. However, a role for sphingolipids in pancreatic beta cell function, or insulin production, and release remains to be established. Our studies in MIN6 cells and mouse pancreatic islets demonstrate that glucose stimulates an intracellular rise in the sphingolipid, sphingosine 1-phosphate (S1P), whereas the levels of ceramide and sphingomyelin remain unchanged. The increase in S1P levels by glucose is due to activation of sphingosine kinase 2 (SphK2). Interestingly, rises in S1P correlate with increased glucose-stimulated insulin secretion (GSIS). Decreasing S1P levels by treatment of MIN6 cells or primary islets with the sphingosine kinase inhibitor reduces GSIS. Moreover, knockdown of SphK2 alone results in decreased GSIS, whereas knockdown of the S1P phosphatase, Sgpp1, leads to a rise in GSIS. Treatment of mice with the sphingosine kinase inhibitor impairs glucose disposal due to decreased plasma insulin levels. Altogether, our data suggest that glucose activates SphK2 in pancreatic beta cells leading to a rise in S1P levels, which is important for GSIS.     

Group X Secretory Phospholipase A2 Regulates Insulin Secretion through a Cyclooxygenase-2-dependent Mechanism

Group X secretory phospholipase A₂ (GX sPLA₂) potently hydrolyzes membrane phospholipids to release arachidonic acid (AA). While AA is an activator of glucose-stimulated insulin secretion (GSIS), its metabolite prostaglandin E2 (PGE2) is a known inhibitor. In this study, we determined that GX sPLA₂ is expressed in insulin-producing cells of mouse pancreatic islets and investigated its role in beta cell function. GSIS was measured in vivo in wild-type (WT) and GX sPLA₂-deficient (GX KO) mice and ex vivo using pancreatic islets isolated from WT and GX KO mice. GSIS was also assessed in vitro using mouse MIN6 pancreatic beta cells with or without GX sPLA₂ overexpression or exogenous addition. GSIS was significantly higher in islets isolated from GX KO mice compared with islets from WT mice. Conversely, GSIS was lower in MIN6 cells overexpressing GX sPLA₂ (MIN6-GX) compared with control (MIN6-C) cells. PGE2 production was significantly higher in MIN6-GX cells compared with MIN6-C cells and this was associated with significantly reduced cellular cAMP. The effect of GX sPLA₂ on GSIS was abolished when cells were treated with NS398 (a COX-2 inhibitor) or L-798,106 (a PGE2-EP3 receptor antagonist). Consistent with enhanced beta cell function, GX KO mice showed significantly increased plasma insulin levels following glucose challenge and were protected from age-related reductions in GSIS and glucose tolerance compared with WT mice. We conclude that GX sPLA₂ plays a previously unrecognized role in negatively regulating pancreatic insulin secretion by augmenting COX-2-dependent PGE2 production.     

Involvement of RhoA/ROCK in insulin secretion of pancreatic β-cells in 3D culture

Cell-cell contacts and interactions between pancreatic β-cells and/or other cell populations within islets are essential for cell survival, insulin secretion, and functional synchronization. Three-dimensional (3D) culture systems supply the ideal microenvironment for islet-like cluster formation and functional maintenance. However, the underlying mechanisms remain unclear. In this study, mouse insulinoma 6 (MIN6) cells were cultured in a rotating 3D culture system to form islet-like aggregates. Glucose-stimulated insulin secretion (GSIS) and the RhoA/ROCK pathway were investigated. In the 3D-cultured MIN6 cells, more endocrine-specific genes were up-regulated, and GSIS was increased to a greater extent than in cells grown in monolayers. RhoA/ROCK inactivation led to F-actin remodeling in the MIN6 cell aggregates and greater insulin exocytosis. The gap junction protein, connexin 36 (Cx36), was up-regulated in MIN6 cell aggregates and RhoA/ROCK-inactivated monolayer cells. GSIS dramatically decreased when Cx36 was knocked down by short interfering RNA and could not be reversed by RhoA/ROCK inactivation. Thus, the RhoA/ROCK signaling pathway is involved in insulin release through the up-regulation of Cx36 expression in 3D-cultured MIN6 cells.     

The plecomacrolide vacuolar-ATPase inhibitor bafilomycin, alters insulin signaling in MIN6 β-cells

Inhibition of endosomal acidification disturbs insulin signaling in both liver and adipose cells. In this study we used MIN6 β cells to determine whether bafilomycin, a potent inhibitor of the proton-translocating vacuolar ATPase, disrupts insulin signaling in islet β cells. Pretreatment of MIN6 cells with varying concentrations of bafilomycin according to a time course revealed concentration and time-dependent changes in phosphorylation of insulin receptor signaling components. Increased phosphorylation of insulin receptor (IR), IRS2 and Akt was prolonged at low bafilomycin concentrations (10 and 50 nmol/L), whereas at high concentrations (100 and 200 nmol/L) phosphorylation rapidly returned to basal levels or below. Akt activation was demonstrated by transient increases in phosphorylation of BAD, cytoplasmic retention of FoxO1 and increased preproinsulin mRNA. Bcl2 expression was also transiently increased but reduced after 30 min exposure to bafilomycin, and this coincided with reduced cell viability. Thus, in β cells inhibition of endosomal acidification by low concentrations of bafilomycin transiently increases insulin signaling, whereas high concentrations promote cell death. Bafilomycin and other agents that interfere with insulin signaling may contribute to diabetes development through disturbing homeostatic control of β cell growth.