胰岛β细胞胰岛素分泌的反馈调节

胰岛β细胞分泌的胰岛素可反馈调节其自身的分泌。胰岛素通过存在于β细胞的胰岛素受体信号转导系统直接作用于β细胞,抑制其内质网上的Ca2 -ATP酶活性,使胞浆内钙离子浓度升高,激活蛋白激酶C,诱导胰岛素分泌,对β细胞分泌起正反馈调节;还可通过旁分泌作用于β细胞周围的δ细胞及α细胞,对其自身分泌进行负反馈调节。同时,胰岛素反馈调节途径还受白细胞介素 -1β、肿瘤坏死因子-α、儿茶酚胺以及瘦素等多种因素影响。     

胰岛素受体底物及其基因多态性与胰岛素抵抗

胰岛素受体底物(IRS)是胰岛素信息传递的重要介质。IRS-1主要介导胰岛素在外周组织的效应，调节β细胞分泌；IRS-2主要介导胰岛素在肝脏的作用，对β细胞发育有重要影响；IRS-3、IRS-4的作用尚存争论。IRS-1、IRS-2基因多态性可致肝脏、外周组织及β细胞胰岛素抵抗；IRS-3、IRS-4基因多态性尚缺乏相关的报道。     

新型胰岛素分泌模式调节剂——那格列奈

控制2型糖尿病（T2DM）的最终目的是保存胰岛β细胞。因为早在血糖升高前15～20年，胰岛β细胞分泌胰岛素的能力巳经丧失50％～70％，为此，大多数抗糖尿病（DM）药物需长期服用，随着时间的推移，β细胞功能会逐渐丧失，使血糖控制达标越来越困难，故临床只关注血糖控制是不够的，治疗的目标应是改变糖尿病的自然病程，长期保护β细胞。正如国际胰岛素分泌研究组主席Cerasi教授一言以蔽之：胰岛素生成，胰岛素分泌及2型糖尿病：问题的核心在于β细胞。     

痛风患者胰岛素抵抗与胰岛β细胞早期分泌相改变的研究

近年来 ,痛风的发生率明显增加 ,其发生除与遗传因素有关外 ,还与环境因素 (动物蛋白摄入过多 )有关 ,高尿酸血症为其特点。临床发现 ,高尿酸血症患者常伴有肥胖、高甘油三酯、糖耐量异常、高血压与糖尿病〔1〕。因血清尿酸浓度与胰岛素抵抗程度以及服糖后胰岛素反应性呈正相关 ,故高尿酸血症患者可具有胰岛素抵抗综合征的临床特征 ;其不仅容易发生痛风 ,还可引起胰岛素抵抗 ,从而加速血管病变和糖耐量异常的发生和发展〔2 ,3〕。我们对 2 6例痛风患者的胰岛素抵抗以及左旋精氨酸 ( L- ARG)刺激后胰岛 β细胞早期分泌相进行了观察 ,现报告…     

Ca2+调控的胰岛素分泌

胰岛β细胞内胰岛素分泌颗粒胞裂外排,从而分泌胰岛素。该过程是由胰岛素分泌颗粒局部Ca^2＋浓度的迅速升高而触发的。胰岛β细胞内Ca^2＋浓度的迅速升高主要源于经质膜上钙通道的Ca^2＋内流和胞内钙库Ca^2＋释放。细胞外钙内流主要通过电压依赖型钙通道,而胞内Ca^2＋释放可能来自三磷酸肌醇敏感钙库、尼克酰胺腺嘌呤二核苷酸磷酸（NAADP）敏感钙库、ryanodine敏感钙库及线粒体钙库等。不同情况下的胰岛素分泌或胰岛素分泌的不同阶段可能由不同来源的Ca^2＋触发或增强,两者相互协调,在胰岛素分泌中起重要作用。因此,对β细胞内钙信号的产生及调控途径的深入研究有利于进一步阐明糖尿病的发病机制,并为糖尿病的治疗提供新思路。     

激素敏感性脂肪酶与胰岛素分泌

激素敏感性脂肪酶 (HSL)是脂肪酸代谢的关键酶 ,除了脂肪组织外 ,在多种组织也有表达。最近研究发现胰岛 β细胞中有HSL的表达 ,其表达和脂解活性也受激素、葡萄糖、脂质水平的影响 ,并且HSL可能在葡萄糖刺激的胰岛素分泌中发挥着重要的作用。     

瘦素对胰岛β细胞胰岛素分泌及葡萄糖信号传递的影响

瘦素是近年发现的一种与肥胖症有关的激素。它通过与下丘脑特异受体结合 ,发挥维持机体正常体重的功能。同时瘦素对胰岛素分泌及胰岛素的外周作用亦存在影响 ,但结果不一 ,对其作用机制尚少有报道。我们研究瘦素对胰岛素分泌的急性与慢性影响 ,并通过观察细胞内游离钙、葡萄糖激酶 (ＧＫ)ｍＲＮＡ表达的变化 ,初步探讨瘦素影响胰岛素分泌的机制。一、研究方法1.胰岛 β细胞培养 :β ＴＣ3 细胞 (由美国爱因斯坦大学Ｅｆｒａｔ教授惠赠 )以含 15 %马血清、2 5 %胎牛血清 (ＰＢＳ)、10 0Ｕ/ｍｌ青霉素、0 1ｍｇ/ｍｌ链霉素的ＤＭＥＭ培养液 …     

瞬时受体电位通道与心律失常的关系

瞬时受体电位通道(TRP)超家族组成超过50种阳离子通道,分布在几乎所有心血管组织中,可以整合多种物理和化学的刺激诱导Ca2+进入胞内。TRP通道家族对心脏节律的维持起着关键作用:TRPC可能通过钙池操纵Ca2+通道参与起搏调控;肥厚心肌中TRPM4表达上调,通过瞬时性内向电流(Iti)参与延迟后除极的发生;TR-PC1和TRPC6参与牵张诱发性心律失常的发生;在缺血缺氧的病理条件下,ATP/UTP的释放激活TRPC3/7引发心电异常。将来有望通过干预TRP活性来开发新一代的抗心律失常药物。     

激素敏感性脂肪酶与胰岛素分泌

激素敏感性脂肪酶(HSL)是脂肪酸代谢的关键酶，除了脂肪组织外，在多种组织也有表达。最近研究发现胰岛β细胞中有HSL的表达，其表达和脂解活性也受激素、葡萄糖、脂质水平的影响，并且HSL可能在葡萄糖刺激的胰岛素分泌中发挥着重要的作用。     

Fat-induced changes in mouse pancreatic islet insulin secretion,insulin biosynthesis and glucose metabolism

Insulin secretion, insulin biosynthesis and islet glucose oxidation were studied in pancreatic islets isolated from fat-fed diabetic mice of both sexes. Insulin secretion from isolated islets was studied after consecutive stimulation with -ketoisocaproic acid + glutamine, glucose, forskolin, and 12-O-tetradecanoylphorbol 13-acetate. Glucose-induced insulin secretion was impaired in islets from fat-fed mice. This was associated with a reduction of approximately 50% in islet glucose oxidation. Islet insulin secretion stimulated by the non-carbohydrate secretagogues tended to be higher in the fat-fed mice, but a statistically significant effect was not observed. Pancreatic insulin content was reduced by 50%, whereas the islet insulin and DNA content was unchanged after fat feeding. Proinsulin mRNA was reduced by 35% in islets from fat-fed mice, and was associated with a reduction of approximately 50% in glucose-stimulated (pro)insulin biosynthesis. It is concluded that the insulin secretory response of islets isolated from fat-fed mice is similar to the secretory pattern known from human type 2, non-insulin-dependent diabetics, and that a defect in islet glucose recognition, resulting in decreased glucose oxidation, may be responsible for the observed insulin secretory and biosynthetic defects seen after glucose stimulation.     

Glucagon and insulin secretion in potential diabetes

Summary Insulin and glucagon have been studied in 20 subjects (both of the subjects’ parents were diabetic or in case of only one diabetic parent, the other showed a first degree familiarity of diabetes): 10 showed normal glucose tolerance (‘true prediabetics’) and 10 impaired glucose tolerance (‘genetic chemical diabetes’). Mean insulin response to oral (100 g) and i.v. glucose load (200 mg/kg followed by 20 mg/kg/min for 60 min) and to arginine infusion (25 g in 30 min) was normal in the prediabetics and delayed and higher in the subjects with chemical diabetes as compared to the control group. Glucagon response to arginine was higher, but not significantly, in prediabetics and in subjects with chemical diabetes. In both of these groups glucagon suppression by glucose was not observed. The insulin/glucagon molar ratio was significantly reduced after glucose infusion in these two groups. No correlation was found between insulin and glucagon secretion after arginine or glucose. A possible alteration in the mechanism controlling glucagon secretion even in the earliest phases of diabetes is suggested. This work was supported in part by C.N.R. (Consiglio Nazionale delle Ricerche), Roma, grant # CT 76.01345.04.     

Malic enzyme is present in mouse islets and modulates insulin secretion

Aims/hypothesis  The pyruvate–malate shuttle is a metabolic cycle in pancreatic beta cells and is important for beta cell function. Cytosolic malic enzyme (ME) carries out an essential step in the shuttle by converting malate to pyruvate and generating NADPH. In rat islets the pyruvate–malate shuttle may regulate insulin secretion and it has been shown to play a critical role in adaptation to obesity and insulin resistance. However, ME has not been demonstrated in mouse islets and three reports indicate that mouse islets contain no ME activity. If mouse islets lack ME, rat and mouse islets must regulate insulin secretion by different mechanisms. Methods  We measured ME activity by a fluorometric enzymatic assay and Me mRNA by real-time PCR. ME activity was also measured in streptozotocin-treated mouse islets. FACS-purified beta cells were obtained from MIP-GFP mouse islets, agouti-L obese mouse islets and mouse beta cell line MIN-6. Insulin secretion and NADPH/NADP⁺ ratios were measured in Me siRNA-treated beta cells. Results  ME activity and Me mRNA were present in C57BL/6 mouse islets. ME activity was reduced in streptozotocin-treated mouse islets. ME activity was also measurable in FACS-purified mouse beta cells. In addition, ME activity was significantly increased in obese agouti-L mouse islets and the mouse MIN-6 cell line. Me siRNA inhibited ME activity and reduced glucose-stimulated insulin secretion and also inhibited NADPH products. Conclusions/interpretation  Mouse islets contain ME, which plays a significant role in regulating insulin secretion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users. J. Xu and J. Han contributed equally to this study.     

Adiponectin induces insulin secretion in vitro and in vivo at a low glucose concentration

Aims/hypothesis A decrease in plasma adiponectin levels has been shown to contribute to the development of diabetes. However, it remains uncertain whether adiponectin plays a role in the regulation of insulin secretion. In this study, we investigated whether adiponectin may be involved in the regulation of insulin secretion in vivo and in vitro. Methods The effect of adiponectin on insulin secretion was measured in vitro and in vivo, along with the effects of adiponectin on ATP generation, membrane potentials, Ca²⁺ currents, cytosolic calcium concentration and state of 5′-AMP-activated protein kinase (AMPK). In addition, insulin granule transport was measured by membrane capacitance and total internal reflection fluorescence (TIRF) analysis. Results Adiponectin significantly stimulated insulin secretion from pancreatic islets to approximately 2.3-fold the baseline value in the presence of a glucose concentration of 5.6 mmol/l. Although adiponectin had no effect on ATP generation, membrane potentials, Ca²⁺ currents, cytosolic calcium concentrations or activation status of AMPK, it caused a significant increase of membrane capacitance to approximately 2.3-fold the baseline value. TIRF analysis revealed that adiponectin induced a significant increase in the number of fusion events in mouse pancreatic beta cells under 5.6 mmol/l glucose loading, without affecting the status of previously docked granules. Moreover, intravenous injection of adiponectin significantly increased insulin secretion to approximately 1.6-fold of baseline in C57BL/6 mice. Conclusions/interpretation The above results indicate that adiponectin induces insulin secretion in vitro and in vivo. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users. M. Okamoto and M. Ohara-Imaizumi contributed equally to this study.     

Search for variants of the gene-promoter and the potential phosphotyrosine encoding sequence of the insulin receptor substrate-2 gene: evaluation of their relation with alterations in insulin secretion and insulin sensitivity

Aims/hypothesis. The aim of this study was to screen part of the putative promoter sequence in addition to 14 potential phosphotyrosine residues of human IRS-2 for genetic variability which might cause changes in protein expression or function. Furthermore, the potential impact on insulin secretion and sensitivity of a previously identified IRS-2 variant (Gly1057Asp) was analysed Methods. The screenings were carried out by the SSCP-heteroduplex technique on DNA from Type II (non-insulin-dependent) diabetic patients. The impact of the Gly1057Asp variant was analysed in four glucose-tolerant Scandinavian study groups. Results. The results showed no nucleotide substitutions in the promoter sequence, however, a novel heterozygous amino acid variant was identified (Leu647Val). In an association study, the new variant was found in 3 of 413 diabetic patients and in none of 280 glucose tolerant subjects. The variant did not affect the binding of IRS-2 to the insulin receptor or p85α of phosphatidylinositol 3-kinase when measured in the yeast two-hybrid system. Examination of the common Gly1057Asp variant in 363 young healthy subjects and in 228 glucose tolerant offspring of one diabetic parent showed no differences in insulin secretion or insulin sensivity after an intravenous glucose tolerance test. Glucose tolerant middle-aged subjects homozygous for the polymorphism (n = 31), however, had on average a 25 % decrease in fasting serum insulin concentrations (p = 0.009) and 28 % (p = 0.01) and 34 % (p = 0.003) reductions in serum insulin concentrations at 30 and 60 min, respectively, during an OGTT compared with wildtype carriers (n = 107). In a cohort of 639 elderly Swedish men the amino acid variant did not have any detectable impact on insulin secretion after an OGTT. Conclusion/interpretation. No genetic variability was found in the IRS-2 promoter. A rare IRS-2 variant at codon 647 has been identified in Type II diabetic patients. The prevalent codon 1057 polymorphism had no consistent effect on insulin secretion or insulin sensitivity. [Diabetologia (1999) 42: 1244–1249] Received: 25 January 1999 and in final revised form: 30 April 1999     

Pancreatic islet metabolism and redox state during stimulation of insulin secretion with glucose and fructose

Summary The mechanism of potentiation of insulin secretion by fructose was investigated. Twenty mM fructose + 3 mM glucose stimulated insulin secretion in a biphasic manner similar to what is found during stimulation with 20 mM glucose, whereas 20 mM fructose alone did not affect secretion. Fructose utilization was measured as formation of tritiated water from 5-³H-fructose. At 27.8 mM fructose the utilization rate was 258 pmol/2 h/10 islets, which is less than the utilization rate of 2.8 mM glucose. 20 mM glucose increased the islet NADH/NAD⁺ and NADPH/-NADP⁺ redox ratios as well as islet concentration of ATP and PEP. 20 mM fructose + 3 mM glucose did not affect the concentration of ATP and PEP or the NADH/NAD⁺ redox ratio. The NADPH/NADP⁺ ratio was significantly decreased (60%) after 2.5 min incubation with 20 mM fructose + 3 mM glucose. It is concluded that fructose potentiation of insulin secretion is not primarily dependent on fructose metabolism and that any conceivable effect on plasma membrane ion fluxes as caused by a reduction of plasma membrane disulfides, may be caused by mechanisms other than a mere increase in the pyridine nucleotide substrates for the transhydrogenation process.     

Transient receptor potential vanilloid 4-dependent calcium influx and ATP release in mouse and rat gastric epithelia

AIM: To explore the expression of transient receptor potential vanilloid 4 (TRPV4) and its physiological meaning in mouse and rat gastric epithelia.METHODS: RT-PCR and immunochemistry were used to detect TRPV4 mRNA and protein expression in mouse stomach and a rat normal gastric epithelial cell line (RGE1-01), while Ca²⁺-imaging and electrophysiology were used to evaluate TRPV4 channel activity. ATP release was measured by a luciferin-luciferase assay. Gastric emptying was also compared between WT and TRPV4 knockout mice.RESULTS: TRPV4 mRNA and protein were detected in mouse tissues and RGE1-01 cells. A TRPV4-specific agonist (GSK1016790A) increased intracellular Ca²⁺ concentrations and/or evoked TRPV4-like current activities in WT mouse gastric epithelial cells and RGE1-01 cells, but not TRPV4KO cells. GSK1016790A or mechanical stimuli induced ATP release from RGE1-01 cells while TRPV4 knockout mice displayed delayed gastric emptying in vivo.CONCLUSION: TRPV4 is expressed in mouse and rat gastric epithelium and contributes to ATP release and gastric emptying.     

IGF-1 receptor signalling determines the mitogenic potency of insulin analogues in human smooth muscle cells and fibroblasts

Aims/hypothesis Mitogenic activity of insulin and insulin analogues and the involvement of the IGF-1 receptor (IGF-1R) is still a controversial issue. We compared levels of the proteins IGF-1R and insulin receptor (InsR) in fibroblasts and smooth muscle cells from healthy donors and assessed the downstream signalling and growth-promoting activity of insulin and insulin analogues. Methods DNA synthesis was monitored in human fibroblasts and coronary artery smooth muscle cells. Using small interfering RNAs, the levels of IGF-1 and InsR were reduced by 95 and 75%, respectively. Results Enhanced mitogenic potency of insulin and insulin analogues was observed which correlated with increased levels of IGF-1R and/or IRS-1. A reduction in the IGF-1R level significantly blunted stimulation of Akt phosphorylation by IGF-1, AspB10 and glargine by 72, 58 and 40%, respectively. Akt phosphorylation in response to insulin remained unaffected. Silencing of InsR did not significantly alter Akt phosphorylation in response to IGF-1, AspB10 and glargine. IGF-1R knockdown reduced the stimulation of DNA synthesis in response to IGF-1 and glargine to a level identical to that produced by insulin. Conclusions/interpretation These data show a prominent role of IGF-1R/Akt signalling in mediating the mitogenic effects of insulin analogues. Regular insulin stimulates DNA synthesis by exclusively activating InsR, whereas insulin analogues mainly signal through IGF-1R. It is suggested that inter-individual differences in the levels of proteins of the IGF-1R system may function as a critical determinant of the mitogenic potency of insulin analogues.     

GPR40, a free fatty acid receptor on pancreatic beta cells, regulates insulin secretion.

GPR40 was originally isolated from human genomic DNA by degenerate PCR. We isolated GPR40 cDNAs from various species, and precisely analyzed its mRNA expression in rat tissues, and found that GPR40 was highly expressed in beta cells in the islets of rat pancreas. When compared to the cell-surface receptors (i.e., choresistokinin receptor, glucagon-like peptide-1 receptor, and sulfonylurea receptor) that are known to predominantly express in the pancreatic beta cells, GPR40mRNA was comparable to these receptors in mRNA expression levels. In addition, all of pancreatic beta cell lines, which we examined, expressed GPR40mRNA at significant levels. Its highest expression was detected in a mouse beta cell line MIN6. To reveal the function of GPR40, we searched for the ligands of GPR40 by screening more than 1500 compounds. As a result we found that CHO cells expressing GPR40 specifically responded to free fatty acids (FFAs), that is, elevation of intracellular Ca(2+) was detected in these cells. Among FFAs tested, apparent stimulatory activities were detected in C12- to 16-length saturated FFAs (e.g., lauric acid, myristic acid, and palmitic acid) and in both C18- and C22-length unsaturated FFAs (e.g., oleic acid, elaidic acid, linoleic acid, a-linolenic acid, g-linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid) at EC(50) of micro molar range. We found that FFAs induced Ca(2+) influx and activated MAP kinase in CHO cells expressing GPR40. As it is known that the increase of intracellular Ca(2+) promotes insulin secretion, we expected the stimulation of FFAs through GPR40 would promote insulin secretion from pancreatic beta cells. As we expected, FFAs induced glucose-stimulated insulin secretion (GSIS) in MIN6 cells. Our results indicate that GPR40 is a cell-surface receptor for FFAs and regulates insulin secretion from pancreatic beta cells. FFAs are known not only to provide an important energy source as nutrients for the body but also to act as signaling molecules in various cellular processes including insulin secretion. However, the molecular mechanism behind the relationship between insulin secretion and FFAs is little understood. We believe that the discovery of a cell-surface FFA receptor on pancreatic beta cells will provide a clue to resolve the relation between FFAs and insulin secretion, and thus eventually lead to the development of anti-diabetic drugs.