Chronic exposure to leucine in vitro induces β-cell dysfunction in INS-1E cells and mouse islets

Chronic hyperglycemia and hyperlipidemia cause deleterious effects on β-cell function. Interestingly, increased circulating amino acid (AA) levels are also a characteristic of the prediabetic and diabetic state. The chronic effects of AAs on β-cell function remain to be determined. Isolated mouse islets and INS-1E cells were incubated with or without excess leucine. After 72?h, leucine increased basal insulin secretion and impaired glucose-stimulated insulin secretion in both mouse islets and INS-1E cells, corroborating the existence of aminoacidotoxicity-induced β-cell dysfunction. This took place concomitantly with alterations in proteins and genes involved in insulin granule transport, trafficking (e.g. collapsin response mediator protein 2 and GTP-binding nuclear protein Ran), insulin signal transduction (proteasome subunit α type 6), and the oxidative phosphorylation pathway (cytochrome c oxidase). Leucine downregulated insulin 1 gene expression but upregulated pancreas duodenum homeobox 1 and insulin 2 mRNA expressions. Importantly, cholesterol (CH) accumulated in INS-1E cells concomitantly with upregulation of enzymes involved in CH biosynthesis (e.g. 3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate (diphospho) decarboxylase, and squalene epoxidase) and LDL receptor, whereas triglyceride content was decreased. Our findings indicate that chronic exposure to elevated levels of leucine may have detrimental effects on both β-cell function and insulin sensitivity. Aminoacidotoxicity may play a pathogenic role in the development of type 2 diabetes.     

脂联素对胰岛β细胞的保护作用

脂联素是新发现的由脂肪细胞分泌的一种糖蛋白,参与调节糖脂代谢、炎性反应等病理生理过程,并可改善胰岛素抵抗.血浆脂联素水平与胰岛β细胞功能独立相关且与第一时相胰岛素分泌呈正相关;可减少基础胰岛素释放而增加葡萄糖刺激的胰岛素分泌(GSIS),阻止胰岛β细胞功能的进一步恶化.并且还可拮抗炎性反应因子及脂肪酸诱导的胰岛β细胞凋亡和功能缺陷,从而发挥对胰岛β细胞的保护作用.本文拟对脂联素的生物学作用及新发现的其对胰岛β细胞的保护作用进行综述.     

G6PD up-regulation promotes pancreatic beta-cell dysfunction

Increased reactive oxygen species (ROS) induce pancreatic β-cell dysfunction during progressive type 2 diabetes. Glucose-6-phosphate dehydrogenase (G6PD) is a reduced nicotinamide adenine dinucleotide phosphate-producing enzyme that plays a key role in cellular reduction/oxidation regulation. We have investigated whether variations in G6PD contribute to β-cell dysfunction through regulation of ROS accumulation and β-cell gene expression. When the level of G6PD expression in pancreatic islets was examined in several diabetic animal models, such as db/db mice and OLEFT rats, G6PD expression was evidently up-regulated in pancreatic islets in diabetic animals. To investigate the effect of G6PD on β-cell dysfunction, we assessed the levels of cellular ROS, glucose-stimulated insulin secretion and β-cell apoptosis in G6PD-overexpressing pancreatic β-cells. In INS-1 cells, G6PD overexpression augmented ROS accumulation associated with increased expression of prooxidative enzymes, such as inducible nitric oxide synthase and reduced nicotinamide adenine dinucleotide phosphate oxidase. G6PD up-regulation also caused decrease in glucose-stimulated insulin secretion in INS-1 cells and primary pancreatic islets. Moreover, elevated G6PD expression led to β-cell apoptosis, concomitant with the increase in proapoptotic gene expression. On the contrary, suppression of G6PD with small interference RNA attenuated palmitate-induced β-cell apoptosis. Together, these data suggest that up-regulation of G6PD in pancreatic β-cells would induce β-cell dysregulation through ROS accumulation in the development of type 2 diabetes.     

GLP1-derived nonapeptide GLP1(28-36)amide protects pancreatic β-cells from glucolipotoxicity

Type 2 diabetes, often associated with obesity, results from a deficiency of insulin production and action manifested in increased blood levels of glucose and lipids that further promote insulin resistance and impair insulin secretion. Glucolipotoxicity caused by elevated plasma glucose and lipid levels is a major cause of impaired glucose-stimulated insulin secretion from pancreatic β-cells, due to increased oxidative stress, and insulin resistance. Glucagon-like peptide-1 (GLP1), an insulinotropic glucoincretin hormone, is known to promote β-cell survival via its actions on its G-protein-coupled receptor on β-cells. Here, we report that a nonapeptide, GLP1(28-36)amide, derived from the C-terminal domain of the insulinotropic GLP1, exerts cytoprotective actions on INS-1 β-cells and on dispersed human islet cells in vitro in conditions of glucolipotoxicity and increased oxidative stress independently of the GLP1 receptor. The nonapeptide appears to enter preferably stressed, glucolipotoxic cells compared with normal unstressed cells. It targets mitochondria and improves impaired mitochondrial membrane potential, increases cellular ATP levels, inhibits cytochrome c release, caspase activation, and apoptosis, and enhances the viability and survival of INS-1 β-cells. We propose that GLP1(28-36)amide might be useful in alleviating β-cell stress and might improve β-cell functions and survival.     

高糖对小鼠胰岛和胰岛b细胞株INS-1E的长期作用

目的探讨高糖对胰岛B细胞INS-1E和小鼠胰岛的慢性作用。方法雌性NMRI小风6-10周龄,苯巴比妥腹腔注射麻醉,应用胶原酶技术消化胰腺分离胰岛。传代培养的INS-1E细胞和分离的小鼠胰岛分别于含11．1,25．0mmol/L葡萄糖的RPMll640培养液中培养72h,然后于含3．3,16．7mmol／L葡萄糖的Krebs-Ringer缓冲液中培养60min,留取上清液行胰岛素测定。INS-1E细胞在含不同浓度的葡萄糖的RPMI1640培养液中培养72h,提取其总RNA,合成相应的cDNA,再行RT-PCR检测胰十二指肠同源异形盒-1（Pdxl）,胰岛素1（Insl）,胰岛素2（Ins2）和葡萄糖转运子2（Glut2）的基因表达。结果高糖培养后INS-1E细胞和小鼠胰岛的基础INS分泌增加[INS-1E细胞：（10．47±0．78）vs（7．71±0．59）ng／10000细胞,P〈0．01;小鼠胰岛：（3．85±0．26）vs（2．18±0．21）μg／L,P〈0．001],糖刺激的INS分泌减少[INS-1E细胞：（17．11±1．98）vs（30．76±2．20）ng／10000细胞,P〈0．001;小鼠胰岛：（14．78±1．03）VS（20．46±1．49）μg／L,P〈0．01];高糖处理后INS-1E细胞的Pdxl,Insl,Ins2和Glut2的mRNA水平下降。结论高糖对胰岛β细胞具有慢性毒性作用。     

β-Amyloid-induced cognitive dysfunction impairs glucose homeostasis by increasing insulin resistance and decreasing β-cell mass in non-diabetic and diabetic rats

Objectiveβ-Amyloid accumulation in the brain may impair glucose homeostasis in both the brain and peripheral tissues. The present study investigated whether β-amyloid deposition in the hippocampus impairs glucose homeostasis by altering insulin sensitivity, glucose-stimulated insulin secretion or β-cell mass.MethodsMale rats were divided into two groups: a non-diabetic sham group and a diabetic partial pancreatectomized (Px) group. Each group was then subdivided into three treatment groups that received intra-CA1 infusions of β-amyloid (25–35; AMY), β-amyloid (35–25; RAMY; non-plaque forming), or saline at a rate of 3.6 nmol/day for 14 days.ResultsAfter 4 weeks, cognitive function measured by passive avoidance and water maze tests was impaired in non-diabetic rats that received AMY compared with rats that received saline or RAMY. Furthermore, diabetes exacerbated cognitive dysfunction in AMY-infused rats. This was associated with the hyperphosphorylation of tau as a result of attenuated insulin signaling (pAkt→pGSK) through decreased phosphorylation of cAMP responding element binding protein in the hippocampus of non-diabetic and diabetic rats. AMY exacerbated whole-body and hepatic insulin resistance in non-diabetic and diabetic rats. However, AMY potentiated glucose-stimulated insulin secretion in non-diabetic and diabetic rats, but caused decreased β-cell mass via increased β-cell apoptosis and decreased β-cell proliferation. As a result, glucose homeostasis was maintained by potentiating insulin secretion in diabetic rats, but may not be sustainable with further decreases in β-cell mass.ConclusionCognitive dysfunction attributable to β-amyloid accumulation in the hippocampus might be related to disturbed glucose homeostasis due to increased insulin resistance and decreased β-cell mass.     

β-Cell-protective effect of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid as a glutamate dehydrogenase activator in db/db mice

2-Aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) is an activator of glutamate dehydrogenase (GDH), which is a mitochondrial enzyme with an important role in insulin secretion. We investigated the effect of BCH on the high-glucose (HG)-induced reduction in glucose-stimulated insulin secretion (GSIS), the HG/palmitate (PA)-induced reduction in insulin gene expression, and HG/PA-induced β-cell death. We also studied whether long-term treatment with BCH lowers blood glucose and improves β-cell integrity in db/db mice. We evaluated GSIS, insulin gene expression, and DNA fragmentation in INS-1 cells exposed to HG or HG/PA in the presence or absence of BCH. An in vivo study was performed in which 7-week-old diabetic db/db mice were treated with BCH (0.7 g/kg, n = 10) and placebo (n = 10) every other day for 6 weeks. After treatment, an intraperitoneal glucose tolerance test and immunohistological examinations were performed. Treatment with BCH blocked HG-induced GSIS inhibition and the HG/PA-induced reduction in insulin gene expression in INS-1 cells. In addition, BCH significantly reduced HG/PA-induced INS-1 cell death and phospho-JNK level. BCH treatment improved glucose tolerance and insulin secretion in db/db mice. BCH treatment also increased the ratio of insulin-positive β-cells to total islet area (P < 0.05) and reduced the percentage of β-cells expressing cleaved caspase 3 (P < 0.05). In conclusion, the GDH activator BCH improved glycemic control in db/db mice. This anti-diabetic effect may be associated with improved insulin secretion, preserved islet architecture, and reduced β-cell apoptosis.     

Linoleic acid induces Ca2+-induced inactivation of voltage-dependent Ca2+ currents in rat pancreatic beta-cells

Free fatty acids (FFAs) regulate insulin secretion in a complex pattern and induce pancreatic beta-cell dysfunction in type 2 diabetes. Voltage-dependent Ca2+ channels (VDCC) in beta-cells play a major role in regulating insulin secretion. The aim of present study is to clarify the action of the FFA, linoleic acid, on VDCC in beta-cells. The VDCC current in primary cultured rat beta-cells were recorded under nystatin-perforated whole-cell recording configuration. The VDCC was identified as high-voltage-gated Ca2+ channels due to there being no difference in current amplitude under holding potential between -70 and -40 mV. Linoleic acid (10 microM) significantly inhibited VDCC currents in beta-cells, an effect which was fully reversible upon washout. Methyl-linoleic acid, which does not activate G protein coupled receptor (GPR)40, neither did alter VDCC current in rat beta-cells nor did influence linoleic acid-induced inhibition of VDCC currents. Linoleic acid-induced inhibition of VDCC current was not blocked by preincubation of beta-cells with either the specific protein kinase A (PKA) inhibitor, H89, or the PKC inhibitor, chelerythrine. However, pretreatment of beta-cells with thapsigargin, which depletes intracellular Ca2+ stores, completely abolished linoleic acid-induced decrease in VDCC current. Measurement of intracellular Ca2+ concentration ([Ca2+](i)) illustrated that linoleic acid induced an increase in [Ca2+](i) and that thapsigargin pretreatment inhibited this increase. Methyl-linoleic acid neither did induce increase in [Ca2+](i) nor did it block linoleic acid-induced increase in [Ca2+](i). These results suggest that linoleic acid stimulates Ca2+ release from intracellular Ca2+ stores and inhibits VDCC currents in rat pancreatic beta-cells via Ca2+-induced inactivation of VDCC.     

Glucose-stimulated insulin secretion correlates with β-cell lipolysis

Background and aimsLipids are needed for optimal glucose-stimulated insulin secretion (GSIS), and long-chain acyl-CoA (LC-CoA) has been suggested as one candidate molecule active as a lipidic coupling factor. LC-CoAs may be available to the β-cell via uptake of circulating free fatty acids or from hydrolysis of intracellularly stored triglycerides. Inhibition of lipolysis in rat islets using a non-specific lipase inhibitor (orlistat) resulted in blunted GSIS. The aim of this study was to investigate the relationship between GSIS and lipolysis in clonal β-cells and in mouse islets.Methods and resultsINS-1 cells, cultured overnight at 3.3 mM or 11.1 mM glucose, or freshly isolated islets were incubated with 3.3 mM or 16.7 mM glucose for 1 h. Medium samples were collected and analyzed for insulin and glycerol. Triglycerides were measured in both INS-1 cells and islets. There was a dose-dependent glucose-stimulated lipolysis in INS-1 cells, which strongly correlated with insulin secretion (r = 0.85, P < 0.0001). The same phenomenon was observed in mouse islets (r = 0.9, P = 0.013). Low levels of triglycerides, which were observed in INS-1 cells pre-cultured at 3.3 mM glucose, were associated with reduced GSIS.ConclusionsThis study suggests that lipids obtained from lipolysis of intracellular triglycerides are involved in GSIS.     

β-cell metabolic alterations under chronic nutrient overload in rat and human islets

The aim of this study was to assess multifactorial β-cell responses to metabolic perturbations in primary rat and human islets. Treatment of dispersed rat islet cells with elevated glucose and free fatty acids (FFAs, oleate:palmitate = 1:1 v/v) resulted in increases in the size and the number of lipid droplets in β-cells in a time- and concentration-dependent manner. Glucose and FFAs synergistically stimulated the nutrient sensor mammalian target of rapamycin complex 1 (mTORC1). A potent mTORC1 inhibitor, rapamycin (25 nM), significantly reduced triglyceride accumulation in rat islets. Importantly, lipid droplets accumulated only in β-cells but not in α-cells in an mTORC1-dependent manner. Nutrient activation of mTORC1 upregulated the expression of adipose differentiation related protein (ADRP), known to stabilize lipid droplets. Rat islet size and new DNA synthesis also increased under nutrient overload. Insulin secretion into the culture medium increased steadily over a 4-day period without any significant difference between glucose (10 mM) alone and the combination of glucose (10 mM) and FFAs (240 μM). Insulin content and insulin biosynthesis, however, were significantly reduced under the combination of nutrients compared with glucose alone. Elevated nutrients also stimulated lipid droplet formation in human islets in an mTORC1-dependent manner. Unlike rat islets, however, human islets did not increase in size under nutrient overload despite a normal response to nutrients in releasing insulin. The different responses of islet cell growth under nutrient overload appear to impact insulin biosynthesis and storage differently in rat and human islets.     

Processing of proglucagon to GLP-1 in pancreatic α-cells: is this a paracrine mechanism enabling GLP-1 to act on β-cells?

Proglucagon is cleaved to glucagon by prohormone convertase 2 (PC2) in pancreatic α-cells, but is cleaved to glucagon-like peptide-1 (GLP-1) by PC1 in intestinal L-cells. The aim of this study was to identify mechanisms which switch processing of proglucagon to generate GLP-1 in the pancreas, given that GLP-1 can increase insulin secretion and β-cell mass. The α-cell line, αTC1-6, expressed PC1 at low levels and GLP-1 was detected in cells and in culture media. GLP-1 was also found in isolated human islets and in rat islets cultured for 7 days. High glucose concentrations increased Pc1 gene expression and PC1 protein in rat islets. High glucose (25 mM) also increased GLP-1 but decreased glucagon secretion from αTC1-6 cells suggesting a switch in processing to favour GLP-1. Three G protein-coupled receptors, GPR120, TGR5 and GPR119, implicated in the release of GLP-1 from L-cells are expressed in αTC1-6 cells. Incubation of these cells with an agonist of TGR5 increased PC1 promoter activity and GLP-1 secretion suggesting that this is a mechanism for switching processing to GLP-1 in the pancreas. Treatment of isolated rat islets with streptozotocin caused β-cell toxicity as evidenced by decreased glucose-stimulated insulin secretion. This increased GLP-1 but not glucagon in the islets. In summary, proglucagon can be processed to GLP-1 in pancreatic cells. This process is upregulated by elevated glucose, activation of TGR5 and β-cell destruction. Understanding this phenomenon may lead to advances in therapies to protect β-cell mass, and thereby slow progression from insulin resistance to type 2 diabetes.     

沙奎那韦致大鼠INS-1细胞胰岛素抵抗

目的探讨HIV-1蛋白酶抑制剂沙奎那韦对大鼠INS-1细胞内胰岛素信号转导通路及β细胞功能的影响。方法INS-1细胞经10μmol／L沙奎那韦处理48h后,台盼蓝染色计数细胞,MTT试验评估沙奎那韦对细胞活力的影响,Western印迹法测定100nmol／L胰岛素刺激的细胞裂解产物中的胰岛素信号转导蛋白的含量及其磷酸化,免疫酶标法测定20mmol／L葡萄糖刺激的胰岛素释放量,并用细胞内DNA含量标准化。结果沙奎那韦处理后,INS-1细胞内胰岛素刺激的胰岛素受体底物1（IRS-1）及IRS-2酪氨酸磷酸化和Akt—Thr^308磷酸化分别降低了60％、66％和55％,基础胰岛素分泌速率和葡萄糖刺激的胰岛素释放速率分别下降了39％和49％。结论沙奎那韦可损害胰岛β细胞内胰岛素信号的转导,导致β细胞自身胰岛素抵抗,这一作用可能影响胰岛β细胞功能。     

Expression Analysis of cPLA2 Alpha Interacting TIP60 in Diabetic KKAy and Non-Diabetic C57BL Wild-Type Mice: No Impact of Transient and Stable TIP60 Overexpression on Glucose-Stimulated Insulin Secretion in Pancreatic Beta-Cells

In the present study we investigate the expression levels of cytosolic phospholipase A2 α (cPLA2α) interacting histone acetyl transferase proteins TIP60α and TIP60β in non-diabetic C57BL wild-type mice and obese type 2 diabetic KKAy model mice. The aim was to test our hypothesis that TIP60 plays a regulatory role in glucose-stimulated insulin secretion from pancreatic β-cells. MATERIAL AND METHODS: Ten obese diabetic KKAy mice and ten non-diabetic C57BL mice were fed a standard chow diet. After nine weeks, islet RNA was purified and used to measure TIP60 expression. We investigated the effect of TIP60α and TIP60β on glucose-stimulated insulin secretion by transient and stable overexpression in the pancreatic mouse β-cell line MIN6 and the rat β-cell line INS-1E. RESULTS: We found that non-diabetic C57BL mice and diabetic KKAy mice have the same level of both the α and β splice forms of TIP60. Furthermore, we demonstrated that transient and stable expression of TIP60 in INS-1E cells affects neither glucose-stimulated insulin secretion, insulin output nor cell insulin content. Also susceptibility to developing gluco-toxicity was unaffected. CONCLUSION: TIP60 over-expression does not affect glucose stimulated insulin secretion, insulin content or abnormal β-cell function during glucotoxicity.     

Glutamate decarboxylase 67 contributes to compensatory insulin secretion in aged pancreatic islets

Pancreatic islets play an essential role in regulating blood glucose levels. Age-dependent development of glucose intolerance and insulin resistance results in hyperglycemia, which in turn stimulates insulin synthesis and secretion from aged islets, to fulfill the increased demand for insulin. However, the mechanism underlying enhanced insulin secretion remains unknown. Glutamic acid decarboxylase 67 (GAD67) catalyzes the conversion of glutamate into γ-aminobutyric acid (GABA) and CO₂. Both glutamate and GABA can affect islet function. Here, we investigated the role of GAD67 in insulin secretion in young (3 month old) and aged (24 month old) C57BL/6J male mice. Unlike young mice, aged mice displayed glucose-intolerance and insulin-resistance. However, aged mice secreted more insulin and showed lower fed blood glucose levels than young mice. GAD67 levels in primary islets increased with aging and in response to high glucose levels. Inhibition of GAD67 activity using a potent inhibitor of GAD, 3-mercaptopropionic acid, abrogated glucose-stimulated insulin secretion from a pancreatic β-cell line and from young and aged islets. Collectively, our results suggest that blood glucose levels regulate GAD67 expression, which contributes to β-cell responses to impaired glucose homeostasis caused by advanced aging.     

甲状旁腺素受体1表达对高糖状态下INS-1细胞功能影响研究

目的 观察甲状旁腺受体1(PTH1R)的表达对胰岛β细胞胰岛素合成与分泌功能影响.方法 构建出PTH1R基因沉默的细胞模型后,分别应用放射免疫法观察25 mmol/L D-葡萄糖处理后对照组、阴性基因克隆组(siPTH1R-NC)、PTHrP组及阳性基因序列组(siPTH1R)细胞内胰岛素含量、葡萄糖刺激胰岛素分泌能力、应用Fluo-3/AM 检测INS-1细胞内钙离子浓度及应用INS-1细胞2-脱氧-[3H]-葡萄糖摄入率检测INS-1细胞葡萄糖转运能力.结果 PTHrP组胰岛素分泌能力高于其他3组,siPTH1R组则低于对照组及siPTH1R-NC组(均P＜0.01);PTHrP组中细胞内胰岛素含量显著高于对照组、siPTH1R-NC及siPTH1R组(均P＜0.01),其他3组间差异无统计学意义;PTHrP组中钙离子浓度水平高于其他3组,siPTH1R组低于对照组及siPTH1R-NC组.PTHrP组中细胞2-脱氧-[3H]-葡萄糖摄入率高于其他3组.结论 高糖状态下PTH1R表达水平与INS-1细胞胰岛素合成与分泌功能有关,可能为INS-1细胞自我保护的一种作用.

Abstract：

Objective To observe insulin synthesis and secretion in INS-1 under high glucose, and to clarify the effect of PTH1R. Methods After successful construction of recombinant PTH1R-siRNA vectors in INS-1 cell, insulin secretion and intracellular insulin content of control group, siPTH1R-Negative control group, PTHrP group, and siPTH1R group under 25 mmol/L glucose were measured by radioimmunoassay in INS-1 cell. Intracellular calcium were detected by Fluo-3/AM and the capability of glucose transport was calculated by assaying the uptake of [3H]-2-deoxy-D-glucose in cells.Results Compared with control group, and siPTH1R-NC group, PTHrP group showed increased capability of insulin secretion; PTHrP group had higher intracellular insulin levels than others; PTHrP group showed increased intracellular calcium; the uptake of [3H]-2-deoxy-D-glucose under high glucose after 48h of PTHrP group was increased(all P＜0.01). Conclusion There is a close relationship between PTH1R activation and insulin secretion and synthesis, PTH1R activation may be one of the protective mechanisms in maintaining function of β-cell under high glucose.     

Long-Term exposure of INS-1 cells to cis and trans fatty acids influences insulin release and fatty acid oxidation differentially

The importance of elevated levels of fatty acids in the pathogenesis of the deteriorated beta-cell function present in type 2 diabetes has been established. Long-term exposure of the beta-cell to high levels of fatty acids causes enhanced insulin secretion at low glucose (basal insulin release), while glucose-stimulated insulin secretion (GSIS) is decreased or unchanged. We have previously demonstrated that the spatial configuration of fatty acids (cis and trans isomers) is of importance for the acute impact on the beta-cell function. In this study we aimed to elucidate whether the spatial configuration also influenced beta-cell function after long-term exposure. Thus, we compared the effect of 3 days culture of INS-1 cells with cis (cis C 18:1-11) and trans vaccenic acid (trans C 18:1-11), as well as oleic (cis C 18:1-9) and elaidic acid (trans C 18:1-9), on basal and glucose-stimulated insulin release. All fatty acids tested increased basal insulin release; however, a significantly lower basal insulin release was demonstrated for cells cultured with 0.3 to 0.4 mmol/L trans vaccenic acid compared to equimolar levels of the cis isomer. GSIS was not changed by cis or trans vaccenic acid or by oleic acid, whereas it was stimulated by 0.3 to 0.4 mmol/L elaidic acid. The mechanisms behind the fatty acid-induced changes in the beta cells have been linked to changes in glucose and fatty acid oxidation. We demonstrated an increased fatty acid oxidation in beta cells after long-term exposure to all of the tested fatty acids. Interestingly, both trans isomers (trans vaccenic and elaidic acid) induced higher fatty acid oxidation than the cis isomers (cis vaccenic and oleic acid, respectively). No changes in glucose oxidation were found when INS-1 cells were cultured with either of the fatty acids. The increased fatty acid oxidation was associated with an increased content of carnitine palmitoyltransferase I (CPT-I) mRNA, but no difference in the content of CPT-I mRNA to the different fatty acids was found. Insulin mRNA expression in beta cells was not affected by the fatty acids. In conclusion, we have demonstrated that the pathological changes in insulin secretion from INS-1 cells to long-term culture with elevated levels of fatty acids are more pronounced for the cis (cis vaccenic acid and oleic acid) rather than the trans isomers (trans vaccenic acid and elaidic acid). We suggest that this, at least in part, may be explained by a lower fatty acid oxidation in cells cultured with the cis compared to the trans fatty acid isomers. Apparently, the difference in fatty acid oxidation was not caused by an increased induction of CPT-I mRNA, nor by changes in glucose oxidation or insulin mRNA in beta cells chronically exposed to the fatty acids.     

Pioglitazone attenuates fatty acid-induced oxidative stress and apoptosis in pancreatic beta-cells

Aims:  Thiazolidinediones (TZDs), ligands for peroxisome proliferator–activated receptor γ, are antidiabetic agents that improve hyperglycemia by decreasing insulin resistance in obese diabetic animal models and patients with type 2 diabetes. We have studied whether pioglitazone, a TZD, can exert a direct effect against pancreatic β-cell lipoapoptosis.
Methods:  MIN6 cells were cultured in medium containing either 5.6 (low glucose) or 25 mM glucose (high glucose) in the presence or absence of 0.5 mM palmitate for 48 h. We examined the effect of 10 μM pioglitazone on MIN6 cells on glucose-stimulated insulin secretion, cellular ATP, uncoupling protein-2 (UCP-2) mRNA expression, intracellular triglyceride content, reactive oxygen species production, the number of apoptotic cells and nuclear factor-κB (NF-κB) activity.
Results:  Pioglitazone recovered partly impaired glucose-stimulated insulin secretion and cellular ATP in MIN6 cell exposed to high glucose with 0.5 mM palmitate. Pioglitazone suppressed intracellular triglyceride accumulation in cells exposed to high glucose with 0.5 mM palmitate. Palmitate-induced upregulation of UCP-2 mRNA levels was suppressed by pioglitazone in a dose-dependent manner. Pioglitazone decreased palmitate-induced reactive oxygen species production in MIN6 cells by 24% and in mouse islet cells by 53%. Pioglitazone also decreased palmitate-induced NF-κB activity by 40% and protected β-cells from palmitate-induced apoptosis by 22% in MIN6 cell.
Conclusions:  Pioglitazone attenuated fatty acid–induced oxidative stress and apoptosis in pancreatic β-cells. TZDs might be used as a mean for maintaining β-cell survival and preserving capacity of insulin secretion in patients with diabetes mellitus.     

Repression of sterol regulatory element-binding protein 1-c is involved in the protective effects of exendin-4 in pancreatic β-cell line

Exendin-4 (Ex-4), a long-acting agonist of glucagon-like peptide-1 receptor, is a novel anti-diabetic drug that prevents β-cells against various toxicities. However, the mechanism and molecules mediating the protection procession of Ex-4 are not fully understood. We investigated the protective effect of Ex-4 against lipotoxicity, mediated by a repression of sterol regulatory element-binding protein (SREBP)-1c, a regulator of genes expression involved in fat and cholesterol synthesis. To observe the effect of Ex-4, we evaluated glucose-stimulated insulin secretion (GSIS) and apoptosis in the MIN6 pancreatic β-cell line, which were cultured in DMEM medium containing 500μM palmitate, with or without 10nM Ex-4. We also examined the roles of SREBP-1c in lipotoxicity model by knockdown with si-RNA. Treatment with Ex-4 improved insulin secretion and survival as well as reduced SREBP-1c expression and activity in palmitate-treated MIN6 cells. This improvement was accompanied with an upregulation of PI3K/Akt signaling pathway, and LY294.002, a specific inhibitor of PI3 kinase, abrogated effects of Ex-4 on insulin secretion. Moreover, SREBP-1c in nuclei was increased by the inhibition of PI3 kinase. Lipotoxic effects of palmitate in the insulin secretion and apoptosis were significantly prevented by SREBP-1 knockdown. In conclusion, Ex-4 protects β-cell against palmitate-induced β-cell dysfunction and apoptosis, by inhibiting SREBP-1c expression and activity through the PI3K/Akt signaling pathway.     

Modulation of β-cell function: a translational journey from the bench to the bedside

Both decreased insulin secretion and action contribute to the pathogenesis of type 2 diabetes (T2D) in humans. The insulin receptor and insulin signalling proteins are present in the rodent and human β-cell and modulate cell growth and function. Insulin receptors and insulin signalling proteins in β-cells are critical for compensatory islet growth in response to insulin resistance. Rodents with tissue-specific knockout of the insulin receptor in the β-cell (βIRKO) show reduced first-phase glucose-stimulated insulin secretion (GSIS) and with aging develop glucose intolerance and diabetes, phenotypically similar to the process seen in human T2D. Expression of multiple insulin signalling proteins is reduced in islets of patients with T2D. Insulin potentiates GSIS in isolated human β-cells. Recent studies in humans in vivo show that pre-exposure to insulin increases GSIS, and this effect is diminished in persons with insulin resistance or T2D. β-Cell function correlates to whole-body insulin sensitivity. Together, these findings suggest that pancreatic β-cell dysfunction could be caused by a defect in insulin signalling within β-cell, and β-cell insulin resistance may lead to a loss of β-cell function and/or mass, contributing to the pathophysiology of T2D.