应用细胞工程和转基因技术治疗糖尿病：“胰岛代理细胞”的构建研究

背景：由于胰岛细胞分离技术难度较大，目前糖尿病细胞移植治疗尚缺乏理想的细胞来源。目的：构建具有生理性胰岛素分泌能力胰岛代理细胞系，为糖尿病基因治疗提供有效手段。设计：随机对照的实验研究。地点、材料和干预：本研究在第三军医大学附属新桥医院中心实验室进行。以携葡萄糖激酶(glucokinase，GK)基因的逆转录病毒及携人胰岛素原基因突变体(mutant proinsulin gene，MPIN)的逆转录病毒共同感染人肝癌细胞系HepG2细胞，G418筛选，放免法、Western—blotting及RT-PCR鉴定；挑选联合表达GK及成熟胰岛素的HepG2细胞进行葡萄糖反应性胰岛素分泌反应的检测，以单独表达成熟胰岛素的HepG2细胞作对照。主要观察指标：不同葡萄糖浓度条件下，联合表达GK及成熟胰岛素的HepG2细胞培养上清中的胰岛素浓度。结果：G418筛选3周获阳性细胞克隆，放免法、Western—blotting挑选出两个目的基因表达均较强的HepG2细胞1株，命名为细胞克隆“β”；RT—PCR证实细胞“β”中已有两个目的基因的转录；在细胞“β”中获得葡萄糖反应性胰岛素分泌，葡萄糖浓度约1．75-2．00mmol／L时出现胰岛素分泌高峰；而在单独表达成熟胰岛素的HepG2细胞中，各种葡萄糖浓度引起的胰岛素分泌差异无显著性意义(P&;gt;0．05)。结论：成功构建了具有生理性胰岛素分泌能力的“胰岛代理细胞”系。     

内源性胰岛素替代疗法中葡萄糖激酶基因逆转录病毒表达载体的构建

背景由于胰岛细胞分离技术难度较大,目前糖尿病细胞移植治疗尚缺乏理想的细胞来源。目的构建葡萄糖激酶(Glucokinase,GK)基因逆转录病毒表达载体及稳定的产毒细胞系,为构建具有葡萄糖反应性胰岛素分泌能力的胰岛代理细胞打下基础,可望为糖尿病提供一种更符合生理要求的内源性胰岛素替代疗法。设计非随机非对照的实验研究。地点、材料和干预本研究在解放军第三军医大学附属新桥医院中心实验室进行。将GK质粒(pCMV4-GKZ1)经EcoR1/BamH1双酶切后亚克隆至逆转录病毒载体PLXSN,构建逆转录病毒表达载体PLX-GK,用酶切法和测序法对重组体进行鉴定。然后脂质体介导逆转录病毒表达载体PLX-GK转入包装细胞PA317,筛选病毒滴度较高的稳定产毒细胞系,PCR鉴定。主要观察指标①重组逆转录病毒载体构建与鉴定结果。②病毒滴度鉴定及PCR结果。结果成功构建GK基因逆转录病毒表达载体,经酶切及测序证明目的基因插入位点和读码框架正确、无突变;产毒细胞系的平均病毒滴度为6.8×108CFU/L,筛选出一株病毒滴度为1.8×109CFU/L稳定产毒细胞系PA317/GK,PCR证实GK基因整合入细胞基因组。结论成功构建了携GK基因的逆转录病毒表达载体及高滴度的产毒细胞系。     

内源性胰岛素替代疗法中葡萄糖激酶基因逆转录病毒表达载体的构建

背景：由于胰岛细胞分离技术难度较大，目前糖尿病细胞移植治疗尚缺乏理想的细胞来源。目的：构建葡萄糖激酶(Glucokinase，GK)基因逆转录病毒表达载体及稳定的产毒细胞系，为构建具有葡萄糖反应性胰岛素分泌能力的胰岛代理细胞打下基础，可望为糖尿病提供一种更符合生理要求的内源性胰岛素替代疗法。设计：非随机非对照的实验研究。地点、材料和干预：本研究在解放军第三军医大学附属新桥医院中心实验室进行。将GK质粒(pCMV4-GKZl)经EcoR1／BamH1双酶切后亚克隆至逆转录病毒载体PLXSN，构建逆转录病毒表达载体PLX-GK，用酶切法和测序法对重组体进行鉴定。然后脂质体介导逆转录病毒表达载体PLX-GK转入包装细胞PA317，筛选病毒滴度较高的稳定产毒细胞系，PCR鉴定。主要观察指标：①重组逆转录病毒载体构建与鉴定结果。②病毒滴度鉴定及PCR结果。结果：成功构建GK基因逆转录病毒表达载体，经酶切及测序证明目的基因插入位点和读码框架正确、无突变；产毒细胞系的平均病毒滴度为6．8&;#215;10^8CFU／L，筛选出一株病毒滴度为I．8&;#215;10^9CFU／L稳定产毒细胞系PA317／GK，PCR证实GK基因整合入细胞基因组。结论：成功构建了携GK基因的逆转录病毒表达载体及高滴度的产毒细胞系。     

葡萄糖对INS-1细胞活性氧及PTEN表达的调控

目的：研究葡萄糖刺激胰岛口细胞后,是否通过活性氧（ROS）调控第10号染色体缺失的磷酸酶与张力蛋白同源物基因（PTEN）表达而参与胰岛素分泌信号通路的调控。方法：用不同浓度葡萄糖和H2O2短时间干预胰岛细胞系（INS-1细胞）,分别检测细胞分泌胰岛素、细胞内ROS浓度及PTENmRNA表达。结果：（1）随着刺激的葡萄糖、H2O2浓度升高,INS-1细胞内ROS浓度和分泌的胰岛素逐渐升高。（2）随着葡萄糖刺激加大或H2O2浓度逐渐升高,INS-1细胞内PTENmRNA表达先受到抑制,但当刺激浓度超过一定范围后,抑制作用消失。结论：葡萄糖可能通过刺激胰岛细胞内ROS生成,以低浓度ROS作为信号分子抑制PTEN表达,成为调控胰岛素分泌的机制之一。     

二甲双胍对糖脂中毒的大鼠胰岛β细胞胰岛素分泌功能的保护作用

目的：观察二甲双胍对慢性暴露于高糖和高游离脂肪酸的大鼠离体胰岛细胞胰岛素分泌功能的影响。方法：将大鼠离体胰岛细胞培养于5.5-16.7 mmol/L葡萄糖或5.5 mmol/L软脂酸中48 h,再加入不同浓度二甲双胍（0-5 mg/L）培养24 h。收集各组胰岛细胞,加入5.5-16.7 mmol/L葡萄糖刺激培养1 h。收集培养液,用放免法测定其基础及葡萄糖刺激的胰岛素分泌（GSIS）水平。结果：低糖组（5.5 mmol/L葡萄糖）用不同浓度二甲双胍（0-5 mg/L）处理后的β细胞基础及葡萄糖刺激的胰岛素分泌无显著性差异（P〉0.05）;高糖（16.7 mmol/L葡萄糖）及高脂（0.5 mmol/L棕榈酸）组β细胞基础胰岛素分泌较对照组明显增高（P〈0.01）,GSIS较对照组明显降低（P〈0.01）;用2.5-5 mg/L二甲双胍干预后,高糖及高脂组β细胞基础胰岛素分泌较未治疗组明显降低（P〈0.01）,GSIS较未治疗组明显增高（P〈0.01）。结论：低糖环境下,二甲双胍对β细胞胰岛素分泌功能无明显影响;慢性高糖及高脂可致β细胞胰岛素分泌功能受损;而2.5-5 mg/L二甲双胍能改善糖脂毒性所致β细胞胰岛素分泌功能异常。提示二甲双胍降糖效果除了其外周作用外,可能对糖脂中毒的β细胞具有直接保护作用。     

肿瘤坏死因子α（TNF—α）对SD大鼠胰岛β细胞凋亡的分子机制

目的 探讨TNF—α对体外SD大鼠胰岛β细胞调亡的分子机制。方法 成年雄性SD大鼠胰岛细胞经胶原酶P消化，Ficoll400纯化后，在37℃，5％CO2，RPMI1640培养剂培养7d，铺成单层，用不同浓度TNF-α（0、10、30、50ng／mL）孵育24h。应用放免法测定不同浓度上清夜及葡萄糖刺激时上清液中胰岛素水平；应用RT—PCR检测胰岛细胞Bcl-2和Bax mRNA表达水平。结果 随TNF-α浓度的增加：（1）胰岛β细胞葡萄糖刺激的胰岛素分泌反应逐渐降低；（2）Bax mRNA表达水平表达呈稳步上升趋势，明显高于对照组（P〈0．05）；而Bcl-2mRNA表达水平在10ng／mL时轻度上升．随着剂量的加大（30-50ng/mL）表达开始明显下降，明显低于对照组（P〈0．05）。结论 （1）TNF-α可诱导大鼠胰岛细胞功能缺陷。（2）大鼠胰岛细胞的Bcl-2和Bax mRNA表达水平变化在TNF—α所致的胰岛细胞凋亡中起重要作用。     

正常小鼠离体胰岛功能与垂体腺苷酸环化酶激活肽-38的关系

目的:探讨垂体腺苷酸环化酶激活肽-38(pituitaryadenylatecyclaseac-tivatingpolypeptide-38,PACAP-38)对离体正常小鼠胰岛功能的影响。方法:用胶原酶和DNA酶联合消化法分离NMRI小鼠胰岛,RPMI1640组织培养液过夜培养,采用MilliporeMultiscreen系统观察不同浓度PACAP-38对胰岛功能的影响。放免法测定孵育液中胰岛素和胰高血糖素浓度。结果:①离体胰岛胰岛素的分泌依赖于孵育液中葡萄糖的浓度,PACAP-38显著刺激胰岛素的释放,且刺激强度依赖于葡萄糖浓度和自身浓度。当孵育液中葡萄糖浓度为5mmol/L时,PACAP-38在1×10-9mmol/L以上才能显著刺激胰岛素分泌犤(6.12±0.26)fmol/(min·胰岛)犦;而当孵育液中葡萄糖浓度为10mmol/L时,PACAP-38在1×10-11mmol/L就可显著刺激胰岛素分泌犤(22.87±1.35)fmol/(min·胰岛)犦(t=4.2271～7.7944,P<0.01)。②离体胰岛胰高血糖素的分泌明显受到孵育液中葡萄糖的浓度的抑制。在低糖环境(葡萄糖浓度在0和2.5mmol/L时)中,1×10-9mmol/LPACAP-38可显著抑制胰高血糖素的分泌,且其抑制作用呈明显的浓度依赖型(在葡萄糖浓度为0mmol/L时,IC50=1×10-10mmol/L)。而当孵育液中葡萄糖浓度为5和10mmol/L时,1×10-9mmol/LPACAP-38对胰高血糖素分泌的抑制作用消失。结论:PACAP-38可刺激离     

胰岛β细胞功能评估方法及临床应用

胰岛β细胞功能是指胰岛素脉冲样分泌以及对各种刺激物引起的胰岛素释放或分泌反应以及分泌其他多肽的能力.检测胰岛β细胞功能有很多方法,包括胰岛素脉冲样分泌模式的检测和胰岛β细胞分泌刺激试验(葡萄糖刺激试验和非葡萄糖刺激试验).葡萄糖刺激试验主要有高葡萄糖钳夹技术、静脉葡萄糖耐量试验(IVGTT)结合数学模型分析技术以及临床常用的口服葡萄糖耐量试验(OGTT)等;非糖刺激试验主要有精氨酸试验及胰升糖素试验.另外,还可通过分析胰岛β细胞分泌的其他肽类来判断其功能,如C肽、胰岛素原(PI)、胰淀素等[1].在糖尿病的不同阶段胰岛β细胞功能有其不同特点,选择不同的测定方法对于评估不同阶段糖尿病胰岛β细胞功能有着重要意义.……     

人脐血间充质干细胞向胰岛素分泌细胞分化的研究

目的 探讨脐血间充质干细胞(MSC)向胰岛素分泌细胞分化的潜能及其分化诱导条件.方法 分离脐血有核细胞,将其置于MesencultTM培养基中进行培养,并利用贴壁法进行纯化、扩增.扩增后的脐血MSC用表皮生长因子(EGF)、β-巯基乙醇和高糖进行诱导.采用RT-PCR、胰岛素免疫细胞化学染色方法对诱导后的细胞进行鉴定,定量检测胰岛素分泌水平及其对葡萄糖刺激的反应性;移植到链脲佐菌素诱导的糖尿病小鼠体内,观测其在体内的降糖作用.结果 诱导后细胞形态发生明显变化,细胞变圆且聚集成团;RT-PCR分析显示,细胞表达部分胰岛相关基因;细胞的胰岛素免疫细胞化学染色为阳性;而且细胞能分泌少量[(0.37±0.06)mU/L]胰岛素,并对糖刺激具有反应性,刺激指数为1.76;诱导后的细胞移植能降低糖尿病小鼠的血糖.结论 脐血MSC具有向胰岛素分泌细胞分化的潜能.     

葡萄糖对胰岛β细胞功能影响的研究进展

早在上世纪 30年代就已发现 2型糖尿病存在胰岛素分泌功能减低。胰岛 β细胞是高度分化、具有胰岛素分泌功能的内分泌细胞。β细胞对血糖、氨基酸、激素、神经递质等许多刺激因素都有反应 ,但最重要的是葡萄糖。本文将阐述葡萄糖对胰岛 β细胞影响的研究进展。1　血糖对胰岛 β     

Superoxide-mediated activation of uncoupling protein 2 causes pancreatic beta cell dysfunction

Failure to secrete adequate amounts of insulin in response to increasing concentrations of glucose is an important feature of type 2 diabetes. The mechanism for loss of glucose responsiveness is unknown. Uncoupling protein 2 (UCP2), by virtue of its mitochondrial proton leak activity and consequent negative effect on ATP production, impairs glucose-stimulated insulin secretion. Of interest, it has recently been shown that superoxide, when added to isolated mitochondria, activates UCP2-mediated proton leak. Since obesity and chronic hyperglycemia increase mitochondrial superoxide production, as well as UCP2 expression in pancreatic beta cells, a superoxide-UCP2 pathway could contribute importantly to obesity- and hyperglycemia-induced beta cell dysfunction. This study demonstrates that endogenously produced mitochondrial superoxide activates UCP2-mediated proton leak, thus lowering ATP levels and impairing glucose-stimulated insulin secretion. Furthermore, hyperglycemia- and obesity-induced loss of glucose responsiveness is prevented by reduction of mitochondrial superoxide production or gene knockout of UCP2. Importantly, reduction of superoxide has no beneficial effect in the absence of UCP2, and superoxide levels are increased further in the absence of UCP2, demonstrating that the adverse effects of superoxide on beta cell glucose sensing are caused by activation of UCP2. Therefore, superoxide-mediated activation of UCP2 could play an important role in the pathogenesis of beta cell dysfunction and type 2 diabetes.     

Development of non-insulin-dependent diabetes mellitus in the double knockout mice with disruption of insulin receptor substrate-1 and beta cell glucokinase genes. Genetic reconstitution of diabetes as a polygenic disease.

Non-insulin-dependent diabetes mellitus (NIDDM) is considered a polygenic disorder in which insulin resistance and insulin secretory defect are the major etiologic factors. Homozygous mice with insulin receptor substrate-1 (IRS-1) gene knockout showed normal glucose tolerance associated with insulin resistance and compensatory hyperinsulinemia. Heterozygous mice with beta cell glucokinase (GK) gene knockout showed impaired glucose tolerance due to decreased insulin secretion to glucose. To elucidate the interplay between insulin resistance and insulin secretory defect for the development of NIDDM, we generated double knockout mice with disruption of IRS-1 and beta cell GK genes by crossing the mice with each of the single gene knockout. The double knockout mice developed overt diabetes. Blood glucose levels 120 min after intraperitoneal glucose load (1.5 mg/g body wt) were 108 +/- 24 (wild type), 95 +/- 26 (IRS-1 knockout), 159 +/- 68 (GK knockout), and 210 +/- 38 (double knockout) mg/dl (mean +/- SD) (double versus wild type, IRS-1, or GK; P < 0.01). The double knockout mice showed fasting hyperinsulinemia and selective hyperplasia of the beta cells as the IRS-1 knockout mice (fasting insulin levels: 0.38 +/- 0.30 [double knockout], 0.35 +/- 0.27 [IRS-1 knockout] versus 0.25 +/- 0.12 [wild type] ng/ml) (proportion of areas of insulin-positive cells to the pancreas: 1.18 +/- 0.68%; P < 0.01 [double knockout], 1.20 +/- 0.93%; P < 0.05 [IRS-1 knockout] versus 0.54 +/- 0.26% [wild type]), but impaired insulin secretion to glucose (the ratio of increment of insulin to that of glucose during the first 30 min after load: 31 [double knockout] versus 163 [wild type] or 183 [IRS-1 knockout] ng insulin/mg glucose x 10(3)). In conclusion, the genetic abnormalities, each of which is nondiabetogenic by itself, cause overt diabetes if they coexist. This report provides the first genetic reconstitution of NIDDM as a polygenic disorder in mice.     

PKClambda regulates glucose-induced insulin secretion through modulation of gene expression in pancreatic beta cells

Altered regulation of insulin secretion by glucose is characteristic of individuals with type 2 diabetes mellitus, although the mechanisms that underlie this change remain unclear. We have now generated mice that lack the lambda isoform of PKC in pancreatic beta cells (betaPKClambda(-/-) mice) and show that these animals manifest impaired glucose tolerance and hypoinsulinemia. Furthermore, insulin secretion in response to high concentrations of glucose was impaired, whereas the basal rate of insulin release was increased, in islets isolated from betaPKClambda(-/-) mice. Neither the beta cell mass nor the islet insulin content of betaPKClambda(-/-) mice differed from that of control mice, however. The abundance of mRNAs for Glut2 and HNF3beta was reduced in islets of betaPKClambda(-/-) mice, and the expression of genes regulated by HNF3beta was also affected (that of Sur1 and Kir6.2 genes was reduced, whereas that of hexokinase 1 and hexokinase 2 genes was increased). Normalization of HNF3beta expression by infection of islets from betaPKClambda(-/-) mice with an adenoviral vector significantly reversed the defect in glucose-stimulated insulin secretion. These results indicate that PKClambda plays a prominent role in regulation of glucose-induced insulin secretion by modulating the expression of genes important for beta cell function.     

A radical explanation for glucose-induced beta cell dysfunction

The development of type 2 diabetes requires impaired beta cell function. Hyperglycemia itself causes further decreases in glucose-stimulated insulin secretion. A new study demonstrates that hyperglycemia-induced mitochondrial superoxide production activates uncoupling protein 2, which decreases the ATP/ADP ratio and thus reduces the insulin-secretory response. These data suggest that pharmacologic inhibition of mitochondrial superoxide overproduction in beta cells exposed to hyperglycemia could prevent a positive feed-forward loop of glucotoxicity that drives impaired glucose tolerance toward frank type 2 diabetes.     

Alterations in basal and glucose-stimulated voltage-dependent Ca2+ channel activities in pancreatic beta cells of non-insulin-dependent diabetes mellitus GK rats.

In genetically occurring non-insulin-dependent diabetes mellitus (NIDDM) model rats (GK rats), the activities of L- and T-type Ca2+ channels in pancreatic beta cells are found to be augmented, by measuring the Ba2+ currents via these channels using whole-cell patch-clamp technique, while the patterns of the current-voltage curves are indistinguishable. The hyper-responsiveness of insulin secretion to nonglucose depolarizing stimuli observed in NIDDM beta cells could be the result, therefore, of increased voltage-dependent Ca2+ channel activity. Perforated patch-clamp recordings reveal that the augmentation of L-type Ca2+ channel activity by glucose is markedly less pronounced in GK beta cells than in control beta cells, while glucose-induced augmentation of T-type Ca2+ channel activity is observed neither in the control nor in the GK beta cells. This lack of glucose-induced augmentation of L-type Ca2+ channel activity in GK beta cells might be causatively related to the selective impairment of glucose-induced insulin secretion in NIDDM beta cells, in conjunction with an insufficient plasma membrane depolarization due to impaired closure of the ATP-sensitive K+ channels caused by the disturbed intracellular glucose metabolism in NIDDM beta cells.     

Frataxin deficiency in pancreatic islets causes diabetes due to loss of beta cell mass

Diabetes is caused by an absolute (type 1) or relative (type 2) deficiency of insulin-producing beta cells. We have disrupted expression of the mitochondrial protein frataxin selectively in pancreatic beta cells. Mice were born healthy but subsequently developed impaired glucose tolerance progressing to overt diabetes mellitus. These observations were explained by impairment of insulin secretion due to a loss of beta cell mass in knockout animals. This phenotype was preceded by elevated levels of reactive oxygen species in knockout islets, an increased frequency of apoptosis, and a decreased number of proliferating beta cells. Hence, disruption of the frataxin gene in pancreatic beta cells causes diabetes following cellular growth arrest and apoptosis, paralleled by an increase in reactive oxygen species in islets. These observations might provide insight into the deterioration of beta cell function observed in different subtypes of diabetes in humans.     

Insulin secretion stimulated by allogeneic lymphocytes in an inbred strain of mice.

Effects of intraperitoneal injection of allogeneic lymphocytes on insulin secretion were studied in incubated pancreas slices from BALB/c mice. Injection of allogeneic lymphocytes from C57BL/6J (H2b) mice increased insulin secretion, both in basal and 11-mM glucose-stimulated conditions. This effect was only present when at least 5 X 10(6) or 1 X 10(6) cells were injected (in basal and stimulated conditions, respectively). Glucose-induced insulin secretion (3.3-27.5 mM) was significantly increased in pancreata from mice injected with allogeneic lymphocytes. No effect was observed when glucose was not included in the incubation medium. Intraperitoneal injection of Dextran 70 produced no change in glucose-elicited insulin secretion. There were no differences in glucagon and somatostatin (SRIF) secretion obtained from pancreas of mice injected with allogeneic or syngeneic lymphocytes. Injection of allogeneic cells increases insulin secretion (basal and both phases of 11 mM glucose-stimulated secretion). Puromycin significantly inhibited the second phase of insulin secretion. These results suggest that: Injection of allogeneic lymphocytes raises both basal and glucose-stimulated insulin secretion. This effect seems to be connected with the major histocompatibility complex, and to be related to the number of allogeneic cells injected. Injection of allogeneic lymphocytes seems to sensitize the beta cell response to glucose stimulus. Neither glucagon nor SRIF secretion are altered by alloantigen injection. The stimulatory effect of allogeneic lymphocytes is related, at least in part, to insulin synthesis.     

Engineering of a glucose-responsive surrogate cell for insulin replacement therapy of experimental insulin-dependent diabetes

Glucose responsiveness in the millimolar concentration range is a crucial requirement of a surrogate pancreatic beta cell for insulin replacement therapy of insulin-dependent diabetes. Novel insulin-secreting GK cell clones with millimolar glucose responsiveness were generated from an early-passage glucose-unresponsive RINm5F cell line. This line expressed constitutively both the K(ATP) channel and the GLUT2 glucose transporter; but it had a relative lack of glucokinase. Through overexpression of glucokinase, however, it was possible to generate glucose-responsive clones with a glucokinase-to-hexokinase ratio comparable to that of a normal pancreatic beta cell. This aim, on the other hand, was not achieved through overexpression of the GLUT2 glucose transporter. Raising the expression level of this glucose transporter into the range of rat liver, without correcting the glucokinase-to-hexokinase enzyme ratio, did not render the cells glucose responsive. These glucokinase-overexpressing RINm5F cells also stably maintained their molecular and insulin secretory characteristics in vivo. After implantation into streptozotocin diabetic immunodeficient rats, glucokinase-overexpressing cells retained their insulin responsiveness to physiological glucose stimulation under in vivo conditions. These cells represent a notable step toward the future bioengineering of a surrogate beta cell for insulin replacement therapy in insulin-dependent diabetes mellitus.     

Junctional communication of pancreatic beta cells contributes to the control of insulin secretion and glucose tolerance

Proper insulin secretion requires the coordinated functioning of the numerous beta cells that form pancreatic islets. This coordination depends on a network of communication mechanisms whereby beta cells interact with extracellular signals and adjacent cells via connexin channels. To assess whether connexin-dependent communication plays a role in vivo, we have developed transgenic mice in which connexin 32 (Cx32), one of the vertebrate connexins found in the pancreas, is expressed in beta cells. We show that the altered beta-cell coupling that results from this expression causes reduced insulin secretion in response to physiologically relevant concentrations of glucose and abnormal tolerance to the sugar. These alterations were observed in spite of normal numbers of islets, increased insulin content, and preserved secretory response to glucose by individual beta cells. Moreover, glucose-stimulated islets showed improved electrical synchronization of these cells and increased cytosolic levels of Ca(2+). The results show that connexins contribute to the control of beta cells in vivo and that their excess is detrimental for insulin secretion.