罗格列酮对自发性2型糖尿病大鼠主动脉内皮细胞保护作用的实验研究

目的研究罗格列酮(胰岛素增敏剂)对自发性2型糖尿病(OLETF)大鼠血管内皮细胞的保护作用及其作用机制。方法将OLETF大鼠随机分为2组:治疗组与未治组,LETO选同种系同周龄LETO大鼠作为对照组。治疗组每日给予罗格列酮2mg·kg-1灌胃。分别于16、24周龄,观察主动脉组织形态学改变;测定主动脉胰岛素受体底物1(IRS-1)、磷脂酰肌醇3激酶(PI-3K)中p85α亚单位和内皮型一氧化氮合酶(eNOS)的蛋白表达。结果治疗组主动脉内皮细胞及血管壁病变明显减轻,主动脉IRS-1和eNOS的蛋白表达均显著高于同期未治组。结论罗格列酮可能通过干预胰岛素受体后信号传导途径,而具有直接的血管保护效应。     

罗格列酮对高糖诱导的β细胞胰岛素分泌功能的影响

目的 探讨罗格列酮对高糖诱导的β细胞胰岛素分泌功能及胰腺十二指肠同源盒因子-1(PDX-1)mRNA表达的影响.方法 应用正常浓度葡萄糖、正常浓度葡萄糖 罗格列酮、高浓度葡萄糖、高浓度葡萄糖 罗格列酮培养RIN-m细胞(大鼠胰岛细胞瘤细胞系).采用放射免疫的方法检测胰岛素水平,RT-PCR方法检测PDX-1及胰岛素mRNA表达水平.结果 (1)长期高糖培养引起RIN-m细胞胰岛素分泌水平降低(P<0.01).长期高糖作用使RIN-m细胞PDX-1及胰岛素mRNA的表达水平显著下降(P<0.01).(2)罗格列酮早期干预可以增加高糖环境下RIN-m细胞胰岛索分泌水平(P<0.01).罗格列酮早期干预可以显著上调高糖环境下RIN-m细胞PDX-1及胰岛素mRNA的表达水平(P<0.01).结论 罗格列酮可以通过上调PDX-1和胰岛素基因表达,延缓高糖环境下的β细胞胰岛素合成和分泌功能的下降.     

二甲双胍在慢性高胰岛素处理的Hep G2细胞对胰岛素受体后信号转导的调节作用(英文)

目的:研究慢性高胰岛素对胰岛素受体后信号转导的影响及其与胰岛素抵抗的关系,并探讨二甲双胍治疗胰岛素抵抗是否通过胰岛素信号转导途径的介导。方法:人类肝癌细胞系(Hep G2)在无血清条件下首先与100nmol/L高浓度胰岛素预温育16h以产生胰岛素抵抗细胞模型,并观察不同浓度(0.01-10mmol/L)的二甲双胍对胰岛素受体后磷酯酰肌醇3激酶(PI3K)途径信号转导的影响。结果:高浓度胰岛素100nmol/L慢性处理引起了IRβ、IRS1和IRS2的酪氨酸磷酸化和蛋白表达水平的下调,p85与IRS的相互作用也有显著降低。生理治疗浓度的(0.01-0.1mmol/L)二甲双胍逆转了这种慢性高胰岛素引起的信号下调,细胞用0.1mmol/L二甲双胍预温育后,IRB、IRS1和IRS2的磷酸化反应水平分别增加了2.7倍(P<0.01),6.8倍(P<0.01)和2.3倍(P<0.01),p85与IRS1的相互作用从34％增加至86％(P<0.01),与IRS2的相互作用从30％增加至92％(P<0.01)。相反,药物浓度(1-10mmol/L)的二甲双胍进一步抑制了IRB、IRS的磷酸化及IRS与p85的相互作用。结论:高浓度胰岛素慢性处理可引起胰岛素受体后PI3K途径信号转导的下调,二甲双胍对胰岛素信号转导的作用可能是其治疗胰岛素抵抗的主要分子机制。     

2型糖尿病小鼠骨骼肌细胞受体后胰岛素信号转导蛋白的下降调节

目的研究2型糖尿病小鼠骨骼肌细胞受体后胰岛素信号转导蛋白:胰岛素受体底物-1(IRS-1)、磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(PKB,Akt)的蛋白表达和磷酸化情况。方法雌性C57BL/6J小鼠予高脂、高糖膳食,制成2型糖尿病动物模型,提取完整的骨骼肌用胰岛素刺激2、15和30min。Westernblot技术检测IRS-1、PI3-Kp85α、PKB的蛋白水平,免疫沉淀技术检测IRS-1酪氨酸磷酸化水平。结果对照组、2型糖尿病模型组骨骼肌细胞的信号转导蛋白IRS-1、PKB未发现数量上的不同,2型糖尿病模型组的PI3Kp85的蛋白比对照组明显减少(P<0·05),在基础状态下,对照组和2型糖尿病模型组的IRS-1、PKB磷酸化水平相似,但2型糖尿病模型组胰岛素刺激后的这些蛋白磷酸化反应曲线上升幅度较之对照组明显降低。结论2型糖尿病小鼠骨骼肌细胞存在受体后胰岛素信号传导蛋白的下降调节。     

厄贝沙坦和罗格列酮对胰岛素抵抗SD大鼠脂蛋白脂酶的影响

目的比较厄贝沙坦与罗格列酮对胰岛素抵抗(IR)SD大鼠血压、胰岛素敏感性及脂蛋白脂酶(LPL)含量的变化。方法 6周龄SD大鼠30只用果糖喂养4周,造成IR之后随机分为3组,其中2组分别给予厄贝沙坦和罗格列酮治疗4周。8周后行收缩压、空腹血糖、空腹血清胰岛素(FINS)、三酰甘油(TG)、胆固醇(TC)、极低密度脂蛋白(VLDL)、游离脂肪酸(FFA)及LPL含量测定。结果果糖喂养组的血压、FINS、TG、及FFA均高于药物治疗组(P<0.01),而胰岛素敏感指数(ISI)及LPL含量均低于药物治疗组(P<0.01)。厄贝沙坦组的血压降低较罗格列酮组明显(P<0.01),而血脂高于罗格列酮组(P<0.01),低于果糖喂养组(P<0.01)。结论厄贝沙坦较罗格列酮降压作用明显,罗格列酮改善IR和降脂作用优于厄贝沙坦。     

山药多糖对2型糖尿病大鼠肾病的预防作用研究

目的:研究山药多糖对2型糖尿病大鼠肾病的预防作用。方法:采用链脲佐菌素(STZ)腹腔注射的方法复制2型糖尿病大鼠模型,用山药多糖干预治疗4周,检测血清胰岛素及胰高血糖素、肾组织胰岛素受体(InsR)、胰岛素受体底物-(lIRS-l)、磷酯酰肌醇3激酶(PI-3K)的表达水平。结果:山药多糖可显著升高模型大鼠胰岛素水平(P<0.01),显著降低胰高血糖素水平(P<0.01),显著升高模型大鼠肾组织InsR、IRS-1、PI-3K表达水平(P<0.05)。结论:山药多糖对2型糖尿病大鼠的治疗作用机制可能与提高糖尿病大鼠肾组织中InsR、IRS-1、PI-3K水平,增加组织对胰岛素的敏感性,改善胰岛素信号传导有关。     

罗格列酮钠治疗糖耐量减低合并非酒精性脂肪肝患者的疗效观察

目的:观察罗格列酮钠治疗糖耐量减低(IGT)合并非酒精性脂肪肝(NAFL)患者的疗效。方法:将IGT合并NAFL患者80例随机分为2组,分别给予罗格列酮钠或二甲双胍治疗,观察24wk后2组血糖、血清胰岛素、血脂、肝功能及脂肪肝的变化情况。结果:治疗后2组服糖后2h血糖、空腹胰岛素(FINS)和胰岛素抵抗指数均较治疗前明显降低(P<0.05或P<0.01),罗格列酮组FINS水平较二甲双胍组降低更明显(P<0.05);罗格列酮组服糖后2h胰岛素、丙氨酸氨基转移酶较治疗前明显降低(P<0.05或P<0.01);罗格列酮组脂肪肝好转率为65.0%,明显高于二甲双胍组的37.5%(P<0.01)。结论:罗格列酮钠治疗IGT合并NAFL安全、有效,其在改善胰岛素抵抗的同时对脂肪肝有明显的改善作用。     

滇结香花对他克莫司诱导的糖尿病大鼠胰腺的保护作用及机制研究

目的 研究滇结香花乙酸乙酯提取物（ethyl acetate extraction from Edgeworthia gardneri Flos,EG）对他克莫司诱导的糖尿病模型大鼠胰腺的保护作用及其机制。方法 将40只SD大鼠随机分为正常组、模型组、滇结香花组和罗格列酮组,每组10只。采用他克莫司单因素干预制作SD大鼠糖尿病模型,通过检测大鼠空腹血糖、胰岛素、糖化血红蛋白水平,显微镜下观察胰腺组织病理学变化,评价EG降糖和保护胰腺组织的作用。并采用RT-PCR和Western blot分别检测胰腺组织中胰岛素受体底物蛋白2（insulin receptor substrate 2,IRS-2）mRNA和蛋白表达水平,探究EG保护糖尿病胰腺组织的作用机制。结果 与正常组比较,他克莫司诱导的糖尿病模型大鼠血糖、胰岛素抵抗指数、糖化血红蛋白升高,胰岛素、胰岛素分泌指数降低（P<0.05）。与模型组比较,滇结香花组血糖、糖化血红蛋白降低,胰岛素、胰岛素分泌指数升高（P<0.05）,大鼠胰腺组织IRS-2 mRNA表达量和IRS-2蛋白表达量增加（P<0.05）。HE染色观察到滇结香花组大鼠胰腺组织切片中胰岛细胞形态结构明显改善,细胞数目增多,排列较整齐,胞浆分布均匀。结论 EG能降低他克莫司诱导的糖尿病模型大鼠的血糖、糖化血红蛋白水平,其机制可能是通过胰腺胰岛素信号转导通路中胰岛素受体底物,保护胰腺,促进胰岛素分泌,增强糖代谢。     

罗格列酮联合阿司匹林对糖尿病伴代谢综合征的疗效

目的:探讨罗格列酮联合阿司匹林对糖尿病伴代谢综合征的治疗效果。方法:纳入观察和统计的糖尿病伴代谢综合征患者80例,随机分为对照组与观察组,每组40例。对照组治疗药物为阿司匹林肠溶片,观察组治疗药物为罗格列酮分散片加阿司匹林肠溶片,比较两组治疗前后胰岛素、血脂、尿微量白蛋白(M-Alb)和不良反应发生率。结果:两组治疗后与治疗前比较,空腹血清胰岛素(F-INS)和胰岛素抵抗指数(HOMA-IR)水平均明显降低、胰岛素分泌指数(HOMA-IS)水平明显升高(P<0.05或P<0.01),总胆固醇(TC)、甘油三酯(TG)和尿M-Alb水平均明显降低(P<0.05或P<0.01)。观察组治疗后与对照组比较,F-INS和HOMA-IR水平降低更显著(P<0.05)、HOMA-IS水平升高更显著(P<0.01),TC、TG和尿M-Alb水平降低更显著(P<0.05或P<0.01)。两组不良反应发生率差异无统计学意义(P>0.05)。结论:罗格列酮联合阿司匹林治疗糖尿病伴代谢综合征的疗效显著,可使机体血脂与胰岛素抵抗得到有效调节。     

P2Y receptor activation enhances insulin release from pancreatic beta-cells by triggering the cyclic AMP/protein kinase A pathway

Adenine nucleotides stimulate insulin secretion by binding to P2 receptors of the pancreatic beta-cells; the stimulus-secretion coupling is not yet clearly established and may depend on the receptor subtype. The aim of the present study was to further investigate the mechanism whereby P2Y receptor agonists enhance glucose-induced insulin secretion. Experiments were performed in rat pancreatic islets and in the INS-1 secreting cell line in the presence of a slightly stimulating glucose concentration (8.3 mmol/l). In isolated islets, the P2Y receptor agonist ADPbetaS (50 micromol/l) induced a significant fivefold increase in the cyclic AMP (cAMP) content, from 43.4+/-3.7 fmol/10 islets in controls to 210.6+/-12.0; it still induced a 4.5-fold increase in cAMP content in the absence of calcium. In another series of experiments, ADPbetaS (50 micromol/l) significantly increased glucose-induced insulin secretion from 7.7+/-0.6 ng/3 islets in controls to 11.2+/-1.0. The adenylyl cyclase inhibitor SQ 22,536 (9-[tetrahydro-2-furanyl]-9 H-purin-6-amine; 100 micromol/l), which was ineffective alone, completely prevented the stimulating effect of ADPbetaS. In a set of experiments in which ADPbetaS increased glucose-induced insulin secretion from 10.0+/-0.7 ng/3 islets to 12.6+/-0.8, the inhibitor of cAMP-dependent protein kinase, TPCK (tos-phe-chloromethylketone; 3 micromol/l), which was ineffective alone, also prevented the stimulating effect of ADPbetaS. In incubated INS-1 cells, the P2Y receptor ligand ATPalphaS increased significantly both the content of cAMP and the release of insulin, in a concentration-dependent manner in the range of 50-150 micromol/l; the insulin release was significantly correlated with the cAMP content. In conclusion, the present results show that P2Y receptor agonists, ADPbetaS and ATPalphaS, amplify glucose-induced insulin secretion by activating beta-cell adenylyl cyclase and the subsequent cAMP/protein kinase A signaling pathway.     

Mechanism of suppression of insulin signalling with lignocaine

Lignocaine suppresses insulin-stimulated glucose transport into the cells and insulin-stimulated glycogenesis at doses equivalent to that used in the treatment of muscle pain disorder. We evaluated the direct effect of lignocaine on insulin receptor (IR) kinase activity. After lignocaine (40 mM, approximately equivalent to 1%) or an equal volume (100 microl) saline had been injected into the tibialis anterior muscle of rat, insulin (50 mM g-1 body weight) was administered into the portal vein in vivo. Immunoprecipitation and immunoblotting were used to detect insulin-mediated tyrosine phosphorylation of both IR-beta and insulin receptor substrate (IRS)-1, and insulin-stimulated binding of IRS-1 to p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-K) in the extracted muscle. In the in vitro study, purified IR from rat liver and/or recombinant IRS-1 protein with adenosine triphosphate were incubated with lignocaine (4 or 40 mM). Lignocaine reduced insulin-stimulated tyrosine phosphorylation of IR-beta to 12.6+/-5.7% (P<0.001), and IRS-1 to 32.1+/-18.8% (P<0.01), and also reduced insulin-stimulated binding of IRS-1 to p85 to 27.4+/-12.7% (P<0.001) relative to control (100%) in muscle in vivo. The in vitro study revealed that lignocaine directly inhibited both basal and insulin-stimulated tyrosine phosphorylation of IR. These results indicate that clinically used doses of lignocaine inhibit insulin signalling in skeletal muscle. The inhibitory effect of lignocaine on tyrosine kinase activity of the IR underlies the suppression of insulin signalling with lignocaine.     

Effects of the beta-carbolines, harmane and pinoline, on insulin secretion from isolated human islets of Langerhans

It is well known that certain imidazoline compounds can stimulate insulin secretion and this has been attributed to the activation of imidazoline I(3) binding sites in the pancreatic beta-cell. Recently, it has been proposed that beta-carbolines may be endogenous ligands having activity at imidazoline sites and we have, therefore, studied the effects of beta-carbolines on insulin secretion. The beta-carbolines harmane, norharmane and pinoline increased insulin secretion two- to threefold from isolated human islets of Langerhans. The effects of harmane and pinoline were dose-dependent (EC(50): 5 and 25 microM, respectively) and these agents also blocked the inhibitory effects of the potassium channel agonist, diazoxide, on glucose-induced insulin release. Stimulation of insulin secretion by harmane was glucose-dependent but, unlike the imidazoline I(3) receptor agonist efaroxan, it increased the rate of insulin release beyond that elicited by 20 mM glucose (20 mM glucose alone: 253+/-34% vs. basal; 20 mM glucose plus 100 microM harmane: 327+/-15%; P<0.01). Stimulation of insulin secretion by harmane was attenuated by the imidazoline I(3) receptor antagonist KU14R (2 (2-ethyl 2,3-dihydro-2-benzofuranyl)-2-imidazole) and was reduced when islets were treated with efaroxan for 18 h, prior to the addition of harmane. The results reveal that beta-carbolines can potentiate the rate of insulin secretion from human islets and suggest that these agents may be useful prototypes for the development of novel insulin secretagogues.     

In vitro association of the phosphatidylinositol 3-kinase regulatory subunit (p85) with the human insulin receptor

The insulin receptor, as a consequence of ligand binding, undergoes autophosphorylation of critical tyrosyl residues within the cytoplasmic portion of its β-subunit. The 85 kDa regulatory subunit of phosphatidylinositol (PI) 3-kinase (p85), an SH2 domain protein, has been implicated as a regulatory molecule in the insulin signal transduction pathway. For the present study, glutathione S-transferase (GST) fusion proteins of p85 SH2 domains were used to determine if such motifs associate directly with the autophosphorylated human insulin receptor. The p85 N + C (amino plus carboxyl) SH2 domains were demonstrated to associate with the autophosphorylated β-subunit, while neither the GTPase activator protein (GAP) N SH2 domain nor the phospholipase C-γ1 (PLCγ1) N + C SH2 domains exhibited measurable affinity for the activated receptor. The p85 N SH2 domain demonstrated weak association with the insulin receptor, while the p85 C SH2 domain alone formed no detectable complexes with the insulin receptor. The association of p85 N + C SH2 domains with the autophosphorylated receptor was competed efficiently by a 15-residue tyrosine-phos-phorylated peptide corresponding to the carboxyl-terminal region of the insulin receptor, but not by phos-phopeptides of similar length derived from the juxtamembrane or regulatory regions. The insulin receptor C domain phosphopeptide inhibited the p85 N i C SH2 domain-insulin receptor complex with an IC_0.5 of 2.3 ± 0.35 μM, whereas a 10-residue phosphopeptide derived from the insulin receptor substrate 1 (IRS-1) competed with an IC_0.5 of 0.54 ± 0.10 μM. These results demonstrate that, in vitro, there is an association between the p85 regulatory protein and the carboxyl-terminal region of the activated insulin receptor that requires the presence of both the N and C SH2 domains. Furthermore, formation of the p85/insulin receptor complex may lead to signaling pathways independent of IRS-1. © Munksgaard 1995.     

Effects of alpha-adrenoceptor antagonists on clonidine-induced inhibition of insulin secretion by isolated pancreatic islets.

The effects of clonidine, yohimbine, corynanthine and prazosin on glucose-induced insulin secretion by incubated or perifused mouse pancreatic islets were investigated. Clonidine (0.1 microM) inhibited glucose-induced insulin secretion alone and in the presence of yohimbine (0.1 microM), corynanthine (10 microM) or prazosin (1 microM). In higher concentrations, yohimbine (1-10 microM) antagonized the inhibitory effect of clonidine (0.1 microM) upon glucose-induced insulin secretion by incubated islets and by perifused islets. The results support the view that adrenergic inhibition of insulin secretion is mediated by alpha 2-adrenoceptors on pancreatic beta-cells.     

Effect of diltiazem on insulin secretion. I. Experiments in vitro.

Effect of diltiazem on glucose-induced insulin secretion was investigated in the rat islets of Langerhans isolated by a collagenase digestion technique. It was found that B-cells, main constituents of isolated islet preparations, had a well-preserved ultrastructural appearance immediately following isolation or after incubation with glucose or glucose and diltiazem. The islets released a large amount of insulin upon stimulation with glucose and CaCl2. Diltiazem (10(-6)-10(-4) M) produced a dose-related inhibition of glucose-induced insulin secretion and this effect was antagonized by the increase in extracellular concentration of CaCl2. The inhibitory effect of diltiazem on the insulin secretion was also counteracted by dibutyryl-3',5'-cyclic AMP or by theophylline. Among calcium-antagonists tested, nifedipine produced the most powerful inhibitory action on the insulin secretion, while the effect of verapamil was similar to or somewhat stronger than that of diltiazem. It was suggested that diltiazem may reduce the intracellular concentration of free calcium ion, thus causing an inhibitory effect on the glucose-induced insulin secretion by the isolated islets of Langerhans.     

Metformin enhances insulin signalling in insulin-dependent and-independent pathways in insulin resistant muscle cells

1 Metformin lowers blood glucose levels in type 2 diabetic patients. To evaluate the insulin sensitizing action of metformin on skeletal muscle cells, we have used C2C12 skeletal muscle cells differentiated in chronic presence or absence of insulin. 2 Metformin was added during the last 24 h of differentiation of the C2C12 myotubes. Insulin-stimulated tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) was determined. 3 Chronic insulin treatment resulted in 60 and 40% reduction in insulin-stimulated tyrosine phosphorylation of IR and IRS-1, respectively. Treatment with metformin was able to increase the tyrosine phosphorylation of IR and IRS-1 by 100 and 90% respectively. 4 Chronic insulin treatment drastically reduced (45%) insulin-stimulated phosphatidyl inositol 3-kinase (PI 3-kinase) activity. Metformin treatment restored PI 3-kinase activity in insulin-resistant myotubes. 5 Insulin-stimulated glucose uptake was impaired in chronically insulin-treated myotubes. Metformin increased basal glucose uptake to significant levels (P<0.05), but metformin did not increase insulin-stimulated glucose transport. 6 All the three mitogen-activated protein kinases (MAPK) were activated by insulin in sensitive myotubes. The activation of p38 MAPK was impaired in resistant myotubes, while ERK and JNK were unaffected. Treatment with metformin enhanced the basal activation levels of p38 in both sensitive and resistant myotubes, but insulin did not further stimulate p38 activation in metformin treated cells. 7 Treatment of cells with p38 inhibitor, SB203580, blocked insulin- and metformin-stimulated glucose uptake as well as p38 activation. 8 Since the effect of metformin on glucose uptake corresponded to p38 MAPK activation, this suggests the potential role p38 in glucose uptake. 9 These data demonstrate the direct insulin sensitizing action of metformin on skeletal muscle cells.     

Rosiglitazone ameliorates abnormal expression and activity of protein tyrosine phosphatase 1B in the skeletal muscle of fat-fed, streptozotocin-treated diabetic rats

Protein tyrosine phosphatase 1B (PTP1B) acts as a physiological negative regulator of insulin signaling by dephosphorylating the activated insulin receptor (IR). Here we examine the role of PTP1B in the insulin-sensitizing action of rosiglitazone (RSG) in skeletal muscle and liver. Fat-fed, streptozotocin-treated rats (10-week-old), an animal model of type II diabetes, and age-matched, nondiabetic controls were treated with RSG (10 micromol kg(-1) day(-1)) for 2 weeks. After RSG treatment, the diabetic rats showed a significant decrease in blood glucose and improved insulin sensitivity. Diabetic rats showed significantly increased levels and activities of PTP1B in the skeletal muscle (1.6- and 2-fold, respectively) and liver (1.7- and 1.8-fold, respectively), thus diminishing insulin signaling in the target tissues. We found that the decreases in insulin-stimulated glucose uptake (55%), tyrosine phosphorylation of IRbeta-subunits (48%), and IR substrate-1 (IRS-1) (39%) in muscles of diabetic rats were normalized after RSG treatment. These effects were associated with 34 and 30% decreases in increased PTP1B levels and activities, respectively, in skeletal muscles of diabetic rats. In contrast, RSG did not affect the increased PTP1B levels and activities or the already reduced insulin-stimulated glycogen synthesis and tyrosine phosphorylation of IRbeta-subunits and IRS-2 in livers of diabetic rats. RSG treatment in normal rats did not significantly change PTP1B activities and levels or protein levels of IRbeta, IRS-1, and -2 in diabetic rats. These data suggest that RSG enhances insulin activity in skeletal muscle of diabetic rats possibly by ameliorating abnormal levels and activities of PTP1B.