Sterol regulatory element-binding protein 1 mediates liver X receptor-beta-induced increases in insulin secretion and insulin messenger ribonucleic acid levels

Liver X receptors (LXRalpha and LXRbeta) regulate glucose and lipid metabolism. Pancreatic beta-cells and INS-1E insulinoma cells express only the LXRbeta isoform. Activation of LXRbeta with the synthetic agonist T0901317 increased glucose-induced insulin secretion and insulin content, whereas deletion of the receptor in LXRbeta knockout mice severely blunted insulin secretion. Analysis of gene expression in LXR agonist-treated INS-1E cells and islets from LXRbeta-deficient mice revealed that LXRbeta positively regulated expression of ATP-binding cassette transporter A1 (ABCA1), sterol regulatory element-binding protein 1 (SREBP-1), insulin, PDX-1, glucokinase, and glucose transporter 2 (Glut2). Down-regulation of SREBP-1 expression with the specific small interfering RNA blocked basal and LXRbeta-induced expression of pancreatic duodenal homeobox 1 (PDX-1), insulin, and Glut2 genes. SREBP-1 small interfering RNA also prevented an increase in insulin secretion and insulin content induced by T0901317. Moreover, 5-(tetradecyloxy)-2-furoic acid, an inhibitor of the SREBP-1 target gene acetyl-coenzyme A carboxylase, blocked T0901317-induced stimulation of insulin secretion. In conclusion, activation of LXRbeta in pancreatic beta-cells increases insulin secretion and insulin mRNA expression via SREBP-1-regulated pathway. These data support the role of LXRbeta, SREBP-1, and cataplerosis/anaplerosis pathways in the control of insulin secretion in pancreatic beta-cells.     

Glucose-induced lipogenesis in pancreatic beta-cells is dependent on SREBP-1

High concentrations of glucose induce de novo fatty acid synthesis in pancreatic β-cells and chronic exposure of elevated glucose and fatty acids synergize to induce accumulation of triglycerides, a phenomenon termed glucolipotoxicity.

Here we investigate the role of sterol-regulatory element binding proteins in glucose-induced lipogenesis in the pancreatic β-cell line INS-1E. We show that glucose induces SREBP-1c expression and SREBP-1 activity independent of insulin secretion and signaling. Using adenoviral expression of SREBP-1c and a SREBP-mutant we show that lipogenic gene expression, de novo fatty acid synthesis and lipid accumulation are induced primarily through sterol-regulatory elements (SREs) and not E-Boxes. Adenoviral expression of a dominant negative SREBP compromises glucose induction of some lipogenic genes and significantly reduces glucose-induction of de novo fatty acid synthesis. Thus, we demonstrate for the first time that SREBP activity is necessary for full glucose induction of de novo fatty acid synthesis in pancreatic β-cells.     

脂毒性导致胰岛β细胞衰竭的机制

脂毒性可通过抑制β细胞胰-十二指肠同源盒因子-1(PDX-1)和肌腱膜纤维肉瘤癌基因同源核A(MafA)的表达,使胰岛素基因表达减少,并通过上调同醇调节元件结合蛋白-1c(SREBP-1c)激活其下游靶分子,导致β细胞胰岛素分泌功能受损.脂毒性还可通过激活氧化应激、导致各种凋亡因子失衡、内质网应激、microRNA途径引起β细胞凋亡.     

The biomechanical integrity of bone in experimental diabetes

The effects of long-term exposure of a pancreatic beta cell line, INS-1, to major free fatty acids (FFA; palmitic acid, oleic acid and linoleic acid) and leptin on insulin secretion and cell viability by C,N-diphenyl-N'-4,5 dimethylthiazol 2-yl tetrazolium bromide (MTT) assay were examined. The cells were incubated with 1 mmol/l of each FFA and 25 or 100 ng/ml leptin, alone or in combination, for 4, 24 or 48 h before the insulin secretion experiments. Palmitic acid (C 16:0) significantly suppressed cell viability, and suppressed insulin secretion at 24 h. Treatment with oleic acid (C 18:1) or linoleic acid (C 18:2) enhanced basal insulin secretion and diminished glucose-stimulated insulin secretion (GSIS) at 48 h. In these groups, there were no differences in cell viability as compared to cells treated without FFA. Leptin did not affect insulin secretion at 4, 24 and 48 h, and in the cells co-treated with FFA and leptin, leptin did not ameliorate lipotoxicity. These results suggest that, in INS-1 cells, different FFA have different patterns of lipotoxicity with chronic exposure, and leptin has little direct effect on insulin secretion.     

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.     

Stevioside Counteracts Beta-Cell Lipotoxicity without Affecting Acetyl CoA Carboxylase

Chronic exposure to high levels of free fatty acids impairs beta-cell function (lipotoxicity). Then basal insulin secretion (BIS) is increased and glucose-stimulated insulin secretion (GSIS) is inhibited. Acetyl CoA carboxylase (ACC) acts as the sensor for insulin secretion in pancreatic beta-cells in response to glucose and other nutrients. Stevioside (SVS), a diterpene glycoside, has recently been shown to prevent glucotoxic effect by regulating ACC activity. The aim of this study was to investigate whether SVS can alleviate impaired beta-cell function by regulating ACC activity. We exposed isolated rat islets and the clonal beta-cell line, INS-1E, to palmitate concentrations of 1.0 or 0.6 mM, respectively, for a period of 24 h to 120 h. The results showed that lipotoxicity occurred in rat islets after 72 h exposure to 1.0 mM palmitate. The lipotoxicity was counteracted by 10(-6) M SVS (n = 8, p < 0.001). Similar results were obtained in INS-1E cells. Neither SVS nor palmitate had any effect on the gene expression of ACC, insulin 2, and glucose transporter 2 in INS-1E cells. In contrast, palmitate significantly increased the gene expression of carnitine palmitoyl transporter 1 (n = 6, p = 0.003). However, the addition of SVS to palmitate did not counteract this effect (n = 6, p = 1.0). During lipotoxicity, SVS did not alter levels of ACC protein, phosphorylated-ACC, ACC activity or glucose uptake. Our results showed that SVS counteracts the impaired insulin secretion during lipotoxicity in rat islets as well as in INS-1E cells without affecting ACC activity.     

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.     

Decreasing IRS-2 expression in pancreatic beta-cells (INS-1) promotes apoptosis, which can be compensated for by introduction of IRS-4 expression

IRS-2 plays a pivotal role in the control of pancreatic beta-cell growth. Here, the effect of altering IRS-2 expression levels in the pancreatic beta-cell line, INS-1, was examined. Adenoviral-mediated increased in IRS-2 protein levels protected against fatty acid (FFA)-induced apoptosis, associated with increased activation of PKB and decreased levels of activated caspase-9. Conversely, decreasing endogenous IRS-2 in INS-1 cells, using adenoviral-mediated expression of IRS-2 antisense, caused a three-fold increase in baseline apoptosis that was further enhanced in the presence of FFA. This was associated with decreased activation of PKB and increased caspase-9 activation. Although IRS-4 is not normally expressed in beta-cells, it was found that adenoviral-mediated introduction of IRS-4 into INS-1 cells enhanced glucose/IGF-1 induced mitogenesis, and protected against FFA-induced apoptosis, similarly to IRS-2. Moreover, expression of IRS-4 in INS-1 cells depleted of IRS-2 levels by IRS-2 antisense, was able to compensate for the lack of IRS-2 and reduce apoptosis in these cells back to normal. Thus, in beta-cells IRS-4 and -2 have similar biological functions. Also, this study further emphasizes the importance of IRS-2 signaling in control of beta-cell survival.     

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。     

高游离脂肪酸血症通过氧化应激导致胰岛β细胞功能受损

目的 研究高游离脂肪酸(FFA)血症对胰岛β细胞功能的影响.方法 正常雄性SD大鼠随机分为3组:对照组输注生理盐水,FFA组输注脂肪乳,氮乙酰半胱氨酸(NAC)干预组在输注脂肪乳的同时加用NAC.持续输液2～4天,行静脉葡萄糖耐最试验(IVGTY)和胰腺组织表面灌注试验评价在体及离体情况下胰岛β细胞胰岛素分泌功能.结果 胰腺组织表面灌注结果显示,FFA组脂肪乳输注2天,基础状态下的胰岛素分泌较对照组增加[(55.5±19.4 vs 27.4±6.7)mIU/L,P<0.01],而葡萄糖刺激的胰岛素分泌(GSIS)与对照组并无差别;延长脂肪乳输注到4天,高FFA刺激基础胰岛素分泌的作用消失,且GSIS明显抑制[(46.8±33.0 vs 214.7±27.4)mIU/L,P<0.05].IVG3T结果亦显示脂肪乳输注4天后胰岛素分泌功能受到抑制.但在脂肪乳输注的同时加用抗氧化剂NAC可使β细胞GSIS峰值部分恢复[(165.4±14.8)mIU/L,P<0.01].结论 循环中FFA水平增高可能通过激活氧化应激导致胰岛β细胞功能受损.     

游离脂肪酸对βTc6细胞PDX-1表达及胰岛素分泌能力的影响

目的 探讨游离脂肪酸对胰岛β细胞胰-十二指肠同源盒因子-1(PDX-1)的表达及相应的β细胞增殖活性和胰岛素分泌功能变化的影响.方法 0.25～1.00 mmol/L游离脂肪酸干预小鼠胰岛素瘤细胞系βTc6细胞24～48 h,应用RT-PCR法检测转录因子PDX-1 mRNA表达,四甲基偶氮唑盐法检测细胞的增殖活性,放射免疫法检测葡萄糖刺激的胰岛素分泌水平,并观察细胞形态变化.结果 经0.25～1.00 mmol/L游离脂肪酸干预24 h,βTc6细胞PDX-1 mRNA的转录逐步增加,但随着干预时间进一步延长至48 h,PDX-1 mRNA的转录逐渐回落,特别是用1.00 mmoL/L游离脂肪酸干预48 h后,βTc6细胞PDX-1 mRNA的表达低于空白对照组.经过0.50～1.00 mmol/L游离脂肪酸24～48 h的干预之后,βTc6细胞形态学上呈现凋亡趋势,细胞增殖活力以及葡萄糖刺激的胰岛素分泌功能降低,而0.25 mmol/L游离脂肪酸24 h的干预未见此类现象.结论 短时间低浓度的游离脂肪酸干预可介导β细胞转录因子PDX-1的表达上调,对β细胞的增殖活性和胰岛素分泌能力无明显影响;而长时间高浓度的游离脂肪酸干预则将导致PDX-1 mRNA的表达下调,并使β细胞的增殖活性和胰岛素分泌能力受损.     

Differential impact of acute and chronic lipotoxicity on gene expression in INS-1 cells

Fatty acids induce abnormal insulin secretion, so-called lipotoxicity, which may develop over a period that can span from a few hours to several years. The relationship between insulin secretion patterns and the pace of lipotoxicity development is, however, sparse. In this study acute lipotoxicity was defined as the functional changes in clonal pancreatic beta cells, INS-1 cells, cultured with 400 and 1,000 micromol/L palmitate for 2 days. Chronic lipotoxicity was demonstrated by exposure of INS-1 cells to 50 and 200 micromol/L palmitate for up to 10 weeks. During acute lipotoxicity, basal insulin secretion (BIS), as well as glucose- and fatty acid-stimulated insulin secretion, were reduced after 1,000 micromol/L palmitate exposure. Concomitantly, total cell protein and (3)H-thymidine incorporation were significantly reduced. In chronic lipotoxicity, BIS increased, whereas a decrease in insulin responsiveness to glucose and fatty acid (defined as fold increase in insulin compared with BIS) was observed after 5 weeks in cells cultured with 200 micromol/L palmitate. Carnitine palmitoyltransferase I gene expression was induced by palmitate upon acute, as well as chronic, exposure. Genes involved in the insulin signal pathway may play an important role in the pathogenesis of lipotoxicity in beta cells. Thus, insulin receptor substrate-1 and 2 gene expressions were downregulated during acute lipotoxicity, while insulin receptor gene expression was suppressed in chronic lipotoxicity. In conclusion, insulin secretion and gene expression in INS-1 cells depends on palminate exposure time and concentration.     

Long-term exposure of INS-1 rat insulinoma cells to linoleic acid and glucose in vitro affects cell viability and function through mitochondrial-mediated pathways

Obesity with excessive levels of circulating free fatty acids (FFAs) is tightly linked to the incidence of type 2 diabetes. Insulin resistance of peripheral tissues and pancreatic β-cell dysfunction are two major pathological changes in diabetes and both are facilitated by excessive levels of FFAs and/or glucose. To gain insight into the mitochondrial-mediated mechanisms by which long-term exposure of INS-1 cells to excess FFAs causes β-cell dysfunction, the effects of the unsaturated FFA linoleic acid (C 18:2, n-6) on rat insulinoma INS-1 β cells was investigated. INS-1 cells were incubated with 0, 50, 250 or 500?μM linoleic acid/0.5% (w/v) BSA for 48?h under culture conditions of normal (11.1?mM) or high (25?mM) glucose in serum-free RPMI-1640 medium. Cell viability, apoptosis, glucose-stimulated insulin secretion, Bcl-2, and Bax gene expression levels, mitochondrial membrane potential and cytochrome c release were examined. Linoleic acid 500?μM significantly suppressed cell viability and induced apoptosis when administered in 11.1 and 25?mM glucose culture medium. Compared with control, linoleic acid 500?μM significantly increased Bax expression in 25?mM glucose culture medium but not in 11.1?mM glucose culture medium. Linoleic acid also dose-dependently reduced mitochondrial membrane potential (ΔΨm) and significantly promoted cytochrome c release from mitochondria in both 11.1?mM glucose and 25?mM glucose culture medium, further reducing glucose-stimulated insulin secretion, which is dependent on normal mitochondrial function. With the increase in glucose levels in culture medium, INS-1 β-cell insulin secretion function was deteriorated further. The results of this study indicate that chronic exposure to linoleic acid-induced β-cell dysfunction and apoptosis, which involved a mitochondrial-mediated signal pathway, and increased glucose levels enhanced linoleic acid-induced β-cell dysfunction.     

过氧化物酶体增生因子激活受体α和γ配体对游离脂肪酸介导的β细胞损害的干预作用

目的：研究过氧化物酶体增生因子激活受体α和γ(PPARα和PPARγ)配体对游离脂肪酸(FFA)介导的胰岛β细胞损害的干预作用。方法：应用PPARα配体氯贝丁酯及PPARγ配体曲格列酮(troglitazone,TGZ)和噻唑烷二酮(thiazolidinedione,TZD)处理大鼠胰岛素瘤细胞系β细胞(INS-1细胞),采用细胞活力和DNA片段梯度分析评价PPARα和PPARγ配体对FFA诱导的INS-1细胞损害的影响。结果：INS-1细胞与0．25～1mmol／L的FFA孵育24h后细胞活力下降,1mmol／L的FFA可诱导INS-1细胞发生凋亡。比较是否使用氯贝丁酯(100μmol／L)、TGZ(10μmol／L)和TZD(100μmol/L)处理β细胞的结果,发现这些配体可保护INS-1细胞免于FFA的细胞毒性(包括脂性凋亡)作用。结论：FFA可介导β细胞发生明显的脂毒性和脂性凋亡,应用PPARα和PPARγ配体可能具有保护β细胞免于FFA的细胞毒性的作用。