吡格列酮对胰岛素抵抗HepG2细胞胰岛素受体底物表达的影响

目的探讨毗格列酮对胰岛索抵抗（IR）HepG2细胞胰岛素受体底物（IRS）蛋白表达的影响。方法胰岛素抵抗HepG2细胞模型建立后,培养液中加入吡格列酮共同孵育,观察吡格列酮对模型细胞葡萄糖掺入率的影响;应用免疫细胞化学染色法观察吡格列酮对IR HepG2细胞IRS-1、IRS-2表达的影响。结果与模型细胞组比较,1×10^-5mol／L吡格列酮显著提高了HepG2细胞的葡萄糖掺入率（P〈0．01）,使IRHepG2细胞IRS-1、IRS-2蛋白的表达显著增加（P〈0．05）。结论吡格列酮的胰岛素增敏作用可能与胰岛素信号转导分子IRS-1、IRS-2蛋白的表达增强有关。     

蜕皮甾酮对2型糖尿病大鼠肝细胞IR S-2蛋白表达的影响

目的：探讨蜕皮甾酮对2型糖尿病大鼠肝细胞胰岛素受体底物2（ IRS-2）蛋白表达的影响。方法选择42只大鼠,14只作为正常组,常规饲料喂养,余下28只先以高脂喂养加小剂量STZ注射诱导2型糖尿病大鼠模型,造模成功后,随机分为模型组和治疗组,分别给予高脂饲料喂养、高脂饲料喂养＋蜕皮甾酮灌胃治疗。5周后取大鼠肝组织标本,应用免疫组化和RT-PCR技术检测IRS-2蛋白和mRNA含量。结果正常组和治疗组肝细胞IRS-2蛋白及mRNA含量均明显高于模型组（ P＜0．05或＜0．01）。结论蜕皮甾酮可提高2型糖尿病大鼠肝细胞IRS-2蛋白和mRNA含量,这可能是蜕皮甾酮改善2型糖尿病大鼠胰岛素抵抗的机制之一。     

胰岛素抵抗HepG2细胞模型的建立及鉴定

目的 建立呈胰岛素抵抗的ＨｅｐＧ２细胞模型。方法 将ＨｅｐＧ２细胞置于１０＾－７ｍｏｌ／Ｌ胰岛素培养液中２４小时,使ＨｅｐＧ２细胞胰岛素受体充分下调,检测燕比较下调和未下调的ＨｅｐＧ２细胞,糖,脂类代谢水平,发现：下调ＨｅｐＧ２细胞胰岛素受体减少了５６％,将ＤＲ－ＨｅｐＧ２细胞于不同浓度胰岛素培养液中,胰岛素受体数 明显减少,当培养液中胰岛素的浓度为１０＾－７ｍｏｌ／Ｌ时,ＤＲ－ＨｅｐＧ２细胞胰岛     

胰岛素抵抗细胞模型的胰岛素降解酶表达

目的 了解胰岛素降解酶（ＩＤＥ）基因、酶蛋白表达水平与胰岛素敏感性的关系,并初步探讨胰岛素降解抑制剂氯喹改善胰岛素敏感性的分子机制。方法 以的代大鼠肝细胞胰岛抵抗（ＩＲ）模型为研究对象,分别采用免疫印迹技术和逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术检测ＩＲ细胞模型的ＩＤＥ酶蛋白表达（ＥＩＰ）及基因表达水平（ＥＩＧ）,同时检测ＩＤＥ活性和反映细胞胰岛素敏感性的＾１４Ｃ－２－脱氧葡萄糖掺入率及＾１４Ｃ     

复方石斛合剂调节胰岛素受体表达促进HepG2细胞糖代谢

目的 探讨复方石斛合剂增加HepG2细胞胰岛素信号促进葡萄糖代谢的机制.方法 以高胰岛素培养HepG2细胞24 h,诱导胰岛素抵抗(IR)状态,继以10％复方石斛合剂的含药血清干预48 h,测定细胞6-磷酸果糖激酶、异柠檬酸脱氢酶的活性,RT-PCR与免疫印迹胰检测岛素受体mRNA与蛋白水平的表达.结果 高浓度胰岛素能降低6-磷酸果糖激酶、异柠檬酸脱氢酶的活性,降低胰岛素受体的表达;复方石斛合剂的治疗能增加6-磷酸果糖激酶、异柠檬酸脱氢酶的活性(P＜0.05),促进胰岛素受体的转录与翻译水平表达(P＜0.05),逆转高胰岛素的下调影响.结论 高浓度胰岛素可诱发IR,复方石斛合剂能增加HepG2细胞胰岛素受体的表达,提高葡萄糖分解代谢关键酶活性,缓解IR.     

胰岛素受体基因变异与胰岛素抵抗

胰岛素受体基因突变常常引起胰岛素抵抗。它主要通过抑制受体的生物合成，使受体向膜的转运发生障碍，降低受体与胰岛素的亲和力，抑制受体酪氨酸激素酶活性，加速受体的降解，而减少细胞膜表面的胰岛素受体的数目和（或）削弱胰岛素受体的正常功能而最终导致胰岛素抵抗。由于胰岛素受体基因与胰岛素抵抗的密切关系，胰岛素受体基因已成为研究一些与胰岛素抵抗相关疾病的重要的候选基因。有研究推测大约１％￣１０％的非胰岛素依赖型     

长效胰岛素类似物对胰岛素抵抗和β细胞功能的影响

91名2型糖尿病患者分为来得时及诺和灵30R治疗组，测定治疗前后空腹血糖（FPG）。空腹血清免疫反应胰岛素（IRI）、SI、PI水平，计算PI占总胰岛素比例，并比较治疗前后HOMA模型中胰岛素抵抗指数（HOMA-IR）及β细胞功能指数（HOMA-IS）的变化，分析血糖下降对胰岛素抵抗的影响。结果：（1）FPG两治疗组问差异无显著性。（2）HOMA-IS来得时组上升72％；诺和灵30R组上升62％，差异无显著性。HOMA-IR来得时组下降16％。诺和灵30R组下降17％，差异无显著性。下降与空腹及餐后血糖的下降相关。（3）治疗后，来得时组空腹PI及SI水平均略有下降；诺和灵组均略有上升．但均无统计学差异：结论：注射来得时及诺和灵30R均可改善胰岛素抵抗和β细胞功能。     

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。     

蜕皮甾酮对2型糖尿病大鼠肾组织氧化应激的影响

目的探讨蜕皮甾酮对2型糖尿病(T2DM)大鼠肾组织氧化应激的影响。方法选择70只雄性SD大鼠,高脂喂养8周后,对60只用链脲佐菌素腹腔注射建立T2DM模型,然后分T2DM组、蜕皮甾酮治疗组(A组)、阿托伐他汀治疗组(B组)、吡格列酮治疗组(C组);另10只为正常对照组(对照组)。干预治疗6周后杀检,检测各组空腹血糖(FPG)、尿素氮(BUN)、血肌酐(SCr)、血脂及尿肌酐清除率(Ccr),用ELISA法检测肾组织超氧化物岐化酶(SOD)、丙二醛(MDA)、一氧化氮合酶(NOS)。结果与对照组比较,各组FPG、BUN、血脂、Ccr、SOD、MDA、NOS升高,SCr降低,均以T2DM组明显(P<0.05或<0.01);与B、C组比较,A组Ccr、LDL-C、NOS、MDA降低,SOD升高(P<0.05或<0.01)。结论皮甾酮能降低T2DM大鼠的Ccr,其可能通过减少肾组织MDA、NOS,升高SOD发挥抗氧化效应,因而具有防治DKD的作用。     

养心通脉片对胰岛素抵抗综合征大鼠红细胞胰岛素受体的影响

目的研究养心通脉片对胰岛素抵抗综合征(IRS)大鼠模型胰岛素受体的影响.方法采取\"高脂高糖高盐膳食\"饲养法复制实验性IRS大鼠模型.设养心通脉组、二甲双胍组、病理模型组、健康对照组,治疗30d,观察比较4组大鼠模型\"高亲和力红细胞受体的结合胰岛素位点数(EIR-H)\"和\"低亲和力红细胞受体的结合胰岛素位点数(EIR-L)\".结果IRS病理模型组大鼠的EIR-H和EIR-L的检测值均显著低于健康对照组及二治疗组(P均＜0.01),4组大鼠比较,EIR-H和EIR-L二项指标都呈现健康对照组→养心通脉组→二甲双胍组→病理模型组递减的趋势(P均＜0.01);二治疗组比较,养心通脉组的EIR-H和EIR-L较之二甲双胍组均显著升高(P＜0.01或P＜0.05).结论IRS时,胰岛素受体浓度及亲和力均有明显下调,养心通脉片对EIR的上调作用明显优于二甲双胍片.     

Increased expression of low-affinity insulin receptor isoform and insulin/insulin-like growth factor-I hybrid receptors in term placenta from insulin-resistant women with gestational hypertension

Summary Gestational hypertension is associated with insulin-resistance; insulin and insulin-like growth factor-1 (IGF-1), acting through their receptors, play a role in the growth of the feto-placental unit. Since both receptors are exposed to the maternal circulation, it has been suggested that maternal metabolic abnormalities might affect placental insulin (HIR) and IGF-1 (IGF-1R) receptors. To clarify this issue, we characterized HIR and IGF-1R in placenta at term from normal women, normoinsulinaemic women with gestational hypertension (NGH), and hyperinsulinaemic women with gestational hypertension (HGH). Insulin binding was decreased in HGH women (B/T 0.12±0.03) compared to control and NGH women (B/T 0.18±0.07, p<0.036; and 0.22±0.5, p<0.009 respectively). Receptor affinity was lower in HGH women (ED₅₀ 0.95±0.32 nmol/l) than control and NGH women (ED₅₀ 0.42±0.19 nmol/l, p<0.01; and 0.40±0.1 nmol/l, p<0.007, respectively), whereas low-affinity Ex11⁺ isoform was higher in HGH women (Ex11⁺ 50±7,%) than in control and NGH women (Ex11⁺ 34±9%, p<0.001; and 39±4%, p<0.01, respectively). Increased expression of Ex11⁺ isoform was correlated with ED₅₀ (r=0.71; p<0.002) and insulinaemia (r=0.70, p<0.002). IGF-I binding was increased in HGH women (B/T 0.17±0.03) compared to control and NGH women (B/T 0.09±0.05, p<0.002; and 0.11±0.03, p<0.002, respectively). IGF-IR affinity was similar in the three groups. The percentage of insulin/IGF-I hybrid receptors was increased in HGH women (85±3%) compared to control and NGH women (68±7%, p<0.001; and 63±9%, p<0.001, respectively), and was positively correlated with insulinaemia (r=0.62, p<0.018), ED₅₀ of insulin binding (r=0.62, p<0.05), and maximal IGF-I binding (r=0.69, p<0.004); whereas it was inversely correlated with maximal insulin binding (r=–0.69, p<0.004). Results provide the first evidence for altered expression of insulin/IGF-I hybrids found in insulin-resistance states.Abbreviations IGF-1 Insulin-like growth factor-1 - IGF-1R IGF-1 receptor - HIR human insulin receptor - OGTT oral glucose tolerance test - HGH hyperinsulinaemia with gestational hypertension - NGH normoinsulinaemia with gestational hypertension - PA-C polyclonal antibody C Drs. H. Valensise, Y. Y. Liu, M. Federici have contributed equally to this work     

Decrease in the insulin receptor protein level by anti-insulin receptor antibodies: roles of tyrosine kinase activity and receptor internalization

To investigate the mechanism of severe impairment of insulin action in type B insulin resistance, we extracted IgG from the serum of a patient with type B insulin resistance (B-IgG) and analyzed the inhibiting effect of B-IgG not only on insulin signaling but also on IGF-I signaling in Chinese hamster ovary (CHO) cells expressing human insulin receptor or human IGF-I receptor. Preincubation with 1 mg/ml B-IgG prevented insulin-induced phosphorylation of insulin receptor and insulin receptor substrate-1 (IRS-1) but did not alter the IGF-I-induced phosphorylation of the IGF-I receptor and IRS-1. ¹²⁵I-insulin binding was inhibited by 93% after preincubation with BigG at 37° C and was recovered up to 50% of the control value by acid washing. However, when cells were preincubated with B-IgG at 4° C, the insulin binding completely recovered the control value by acid washing. ¹²⁵I-IGF-I binding was not altered by B-IgG preincubation. Immunoblot study revealed that the protein level of the insulin receptor was strongly decreased by preincubation with 1 mg/ml B-IgG at 37° C, but never at 4° C. The IRS-1 protein level did not change by B-IgG preincubation. In order to know the role of the insulin receptor internalization in the inhibiting effect of B-IgG, we employed CHO cells expressing mutant insulin receptors which do not undergo internalization (CHO-K1018R). B-IgG incubation of CHOK1018R at 37° C failed to decrease the protein level of the insulin receptor. The present data indicate that IgG from the diabetic patient with type B insulin resistance decreased insulin receptor protein level, probably due to the enhanced degradation rate of the insulin receptor, in which insulin receptor tyrosine kinase activity and internalization are required for this process. This effect of B-IgG was specific for the insulin receptor with no effect on either IGF-I receptor or IRS-1, as reflected by the IGF-I effectiveness on glycemic control in this patient. Received: 26 September 2001 / Accepted in revised form: 12 July 2002 Correspondence to K. Eguchi     

Delayed intracellular dissociation of the insulin-receptor complex impairs receptor recycling and insulin processing in cultured Epstein-Barr virus-transformed lymphocytes from insulin-resistant subjects

Summary Insulin-receptor internalization and processing are defective in insulin-resistant subjects. To assess the reversibility of these defects, we cultured Epstein-Barr virus-transformed-lymphoblasts from six normal, six obese, and six non-insulin-dependent diabetic (NIDDM) subjects in media containing low (5 mmol/l) or high (25 mmol/l) glucose concentrations, and studied the insulin-receptor internalization and processing in vitro. In cells from normal, obese, and NIDDM subjects cultured in low glucose concentrations, exposure to 100 nmol/l insulin for 30 min at 37C reduced cell-surface ¹²⁵I-insulin binding to a similar extent (82±2, 77±5, and 82±5 % of initial values, respectively). The same results were obtained with cells cultured in high glucose concentrations. In cells cultured under both glucose conditions, and exposed to 100 nmol/l insulin for 30 min at 37C, a complete recovery of the initial ¹²⁵I-insulin binding was observed in normal but not in obese and NIDDM subjects. Release of intracellular insulin and its degradation in vitro was determined by incubating cells with 600 pmol/l of ¹²⁵I-insulin for 60 min at 37C, acid washing cells, and re-incubating in insulin-free buffer at 37C. The radioactivity released by cells was characterized by trichloroacetic acid precipitability, Sephadex G-50 column Chromatograph, and rebinding to fresh cells. Rates of release of internalized radioactivity were reduced in obese and NIDDM subjects (t_1/2=61±9 min, p<0.02; 58±10 min, p<0.05; and 38±4 min in obese, NIDDM, and normal subjects, respectively). The percentage of intact insulin released from cells was significantly higher in obese and NIDDM subjects than in the normal subjects. The t_1/2 of intracellular dissociation of insulin-receptor complexes measured by a polyethylene glycol assay was lower in normal (6±1 min) than in obese (12±2 min, p<0.03) and NIDDM subjects (14±3 min, p<0.02). The results suggest that in insulin-resistant subjects a primary defect in intracellular dissociation of insulin is responsible for alterations of receptor recycling and insulin processing.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - EBV Epstein-Barr virus - RPMI - FCS fetal calf serum - PEG polyethylene glycol - ANOVA analysis of variance     

吸烟减少大鼠骨骼肌细胞胰岛素受体基因表达

应用RT-PCR及免疫组化技术分别检测实验大鼠骨骼肌细胞胰岛素受体基因mRNA及蛋白表达.结果显示正常吸烟组、高脂饲养吸烟组及糖尿病吸烟组大鼠骨骼肌细胞胰岛索受体基因mRNA的表达显著低于各自对照组(0.50±0.06对0.84±0.09,0.38±0.01对0.59±0.05,0.37±0.05对0.55±0.05,均P<0.01),蛋白含量亦明显低于各自对照组(6.99±0.53对8.89±0.36,5.17±0.29对7.53±0.53,2.16±0.56对5.03±0.79,均P<0.01),这可能是长期吸烟导致胰岛素抵抗的分子机制之一.     

Effect of nelfinavir on insulin metabolism, proteasome activity and protein degradation in HepG2 cells

HIV-1 protease inhibitors have revolutionized the treatment of HIV infection, but their use has been associated with lipodystrophy and insulin resistance. One suggestion for this has been the inhibition of insulin-degrading enzyme (IDE). We have previously demonstrated that insulin, through IDE, can inhibit the proteasome, thus decreasing cytosolic protein degradation. We examined whether the protease inhibitor nelfinavir inhibited IDE and its effect on protein degradation both in vitro and in whole cells. 125I-Insulin degradation was measured by trichloroacetic acid precipitation. Proteasome activities were measured using fluorogenic peptide substrates. Cellular protein degradation was measured by prelabelling cells with 3H-leucine and determining the release of TCA-soluble radioactivity. Nelfinavir inhibited IDE in a concentration-dependent manner with 50% inhibition at the maximal concentration tested, 100 microm. Similarly, the chymotrypsin-like and trypsin-like activities of the proteasome were decreased with an IC50 of approximately 3 microm. The ability of insulin to inhibit the proteasome was abrogated by nelfinavir. Treatment of HepG2 cells with 50 microm nelfinavir decreased 125I-insulin degradation and increased cell-associated radioactivity. Insulin alone maximally decreased protein degradation by 15%. Addition of 50 microm nelfinavir inhibited cellular protein degradation by 14% and blunted the effect of insulin. These data show that nelfinavir inhibits IDE, decreases insulin's ability to inhibit protein degradation via the proteasome and provides another possible mechanism for the insulin resistance seen in protease inhibitor-treated HIV patients.     

Angiotensin II type 2 receptor inhibits expression and function of insulin receptor in rat renal proximal tubule cells

Both renin–angiotensin systems and insulin participate in kidney-involved blood pressure regulation. Activation of angiotensin II type 2 receptor (AT₂R) decreases sodium reabsorption in renal proximal tubule (RPT) cells, whereas insulin produces the opposite effect. We presume that AT₂R has an inhibitory effect on insulin receptor expression in RPT cells, which may affect renal sodium transport and therefore be of physiological or pathological significance. Our present study found that activation of AT₂R inhibited insulin receptor expression in a concentration and time-dependent manner in RPT cells from Wistar-Kyoto (WKY) rats. In the presence of a protein kinase C (PKC) inhibitor (PKC inhibitor peptide 19–31, 10^?6 mol/L) or a phosphatidylinositol 3 kinase inhibitor (wortmannin, 10^?6 mol/L), the inhibitory effect of AT₂R on insulin receptor was blocked, indicating that both PKC and phosphatidylinositol 3 kinase were involved in the signaling pathway. There was a linkage between AT₂R and insulin receptor which was determined by both laser confocal microscopy and coimmunoprecipitation. However, the effect of AT₂R activation on insulin receptor expression was different in RPT cells from spontaneously hypertensive rats (SHRs). Being contrary to the effect in WKY RPT cells, AT₂R stimulation increased insulin receptor in SHR RPT cells. Insulin (10^?7 mol/L, 15 minutes) enhanced Na⁺-K⁺-ATPase activity in both WKY and SHR RPT cells. Pretreatment with CGP42112 decreased the stimulatory effect of insulin on Na⁺-K⁺-ATPase activity in WKY RPT cells, whereas pretreatment with CGP42112 increased it in SHR RPT cells. It is suggested that activation of AT₂R inhibits insulin receptor expression and function in RPT cells. The lost inhibitory effect of AT₂R on insulin receptor expression may contribute to the pathophysiology of hypertension.     

Modulation of insulin receptor signalling by pancreastatin in HTC hepatoma cells

Summary Pancreastatin, a neuropeptide derived from chromogranin A, has a glycogenolytic and counterregulatory effect to insulin in the rat liver. This effect is mediated by calcium and protein kinase C activity. Our aim was to study the possible cross-talk between pancreastatin and the insulin signalling system, by using the well-studied insulin sensitive rat hepatoma HTC cells. First, we checked the counterregulatory effect of pancreastatin on insulin action. Pancreastatin dose-dependently inhibited insulin stimulated glycogen synthesis. This effect was not due to competition for insulin receptors. Moreover, when protein kinase C activation was blocked with staurosporine, this effect of pancreastatin was not observed. Next, we found a dose-dependent inhibition of insulin receptor autophosphorylation by pancreastatin. In addition, phosphorylation of the major substrates of insulin receptor in HTC, i. e. insulin-receptor substrate (IRS)-1/IRS-2 and p62 was also blunted and so was its association with p85 regulatory subunit of phosphatidylinositol-3-kinase. Moreover, the insulin activation of S6 kinase was also blocked by pancreastatin. Again, all these inhibitory effects of pancreastatin were prevented by staurosporine. Furthermore, pancreastatin produced Ser/Thr phosphorylation of insulin receptor by a staurosporine-sensitive mechanism. Finally, we checked the pancreastatin activation of protein kinase C in HTC cells and found that a “classical” isoform of this protein is translocated to the plasma membrane. These findings suggest that pancreastatin could exert its anti-insulin effect in the hepatocyte by interrupting the stimulation of early insulin receptor signalling as a result of phosphorylation. This interaction might have a role in the mechanisms of insulin resistance. [Diabetologia (1999) 42: 317–325] Received: 9 February 1998 and in final revised form: 1 September 1998     

The HIV-1 protease inhibitor indinavir impairs insulin signalling in HepG2 hepatoma cells

Aims/hypothesis. Patients treated with human immunodeficiency virus-1 protease inhibitors often develop impaired glucose tolerance or diabetes, most likely due to an induction of insulin resistance. We therefore investigated whether the protease inhibitor indinavir alters insulin signalling. Methods. We incubated HepG2 cells for 48 h without or with indinavir (100 μmol/l). Subsequently ¹²⁵I-insulin binding to the cells and the effects of insulin stimulation on insulin-receptor substrate-1-phosphorylation, association of phosphatidylinositol 3-kinase with insulin-receptor substrate-1 and Akt-Thr³⁰⁸-phosphorylation were measured. Results. In cells not exposed to indinavir, insulin (100 nmol/l) led to rapid increases of insulin-receptor substrate-1-phosphorylation, association of phosphatidylinositol 3-kinase with insulin-receptor substrate-1 and Akt-phosphorylation during the first 75 s, followed by subsequent decreases. In indinavir-treated cells, these insulin-stimulated increases during the first 75 s were reduced by 30–60 % and this was not associated with alterations in cell number or viability, insulin binding to the cells or cellular insulin-receptor substrate-1-content. Conclusion/interpretation. Effects of indinavir on initial insulin signalling could cause, or contribute to, the metabolic effects of human immunodeficiency virus-1 protease inhibitors. [Diabetologia (2000) 43: 1145–1148] Received: 8 March 2000 and in revised form: 31 May 2000