替米沙坦上调内脏脂肪组织的脂联素表达并改善胰岛素抵抗

目的 探讨替米沙坦对体外原代培养的人前脂肪细胞和OLETF大鼠脂肪组织中脂联素表达以及胰岛素敏感性的影响.方法 提取人腹部皮下和内脏的前脂肪细胞,原代培养并传代后接种于无血清培养基,分为对照组（H-N）、吡格列酮组（H-P）和替米沙坦（H-T）组.葡萄糖消耗试验测定细胞的胰岛素敏感性,实时荧光定量PCR（RT-PCR）技术测定细胞脂联素mRNA的表达,放射免疫法测定培养基上清脂联素的分泌量.4周龄雄性OLETF大鼠28只,初始体重150～180 g.高脂喂养,分为对照组（O-HFD组,10只）、吡格列酮组（O-P组,8只）和替米沙坦组（O-T组,10只）.口服葡萄糖耐量实验和高胰岛素-正糖钳夹实验检测胰岛素抵抗指数和60 ～ 120 min葡萄糖输注速度以测定大鼠胰岛素抵抗水平,RT-PCR和Western blotting技术检测大鼠皮下和内脏脂肪组织脂联素的mRNA和蛋白表达.计量资料均数比较应用单因素方差分析.结果 H-T组和H-P组内脏前脂肪细胞的葡萄糖消耗量较之H-N组明显升高[分别为（5.6±1.6）、（4.4±1.6）、（2.0±0.8）mmol/L,F=20.240,P＜0.05].O-T和O-P组大鼠高胰岛素-正糖钳夹实验得到的60 ～ 120 min葡萄糖输注速度明显高于O-HFD组大鼠[分别为（18±5）、（20±4）、（10±3） mg·kg-1·min-1,F=8.136,P＜0.05].O-T和O-P组大鼠的HOMA胰岛素抵抗指数也显著低于O-HFD组大鼠（分别为10.0±8.6、5.5±2.0、17.8±10.1,F=5.784,P＜0.05）.替米沙坦可降低高脂饲养OLETF大鼠升高的腹围、内脏脂肪系数和血清游离脂肪酸水平,并改善大鼠的空腹高血糖,差别有统计学意义[分别为（24.0±2.0）比（26.5±2.7） cm、5.8％±2.4％比8.6％±2.4％、（2.8±0.7）比（5.3±1.8）μg/L、（10±6）比（15±7） mmoL/L,均P＜0.05].H-T较之H-N组原代培养人前脂肪细胞和O-T较之O-HFD组大鼠脂肪组织脂联素的mRNA表达和蛋白分泌量均明显上调（均P＜0.05）.H-T较之H-P组人内脏脂肪细胞和O-T较之O-P组大鼠内脏脂肪组织脂联素mRNA和蛋白表达水平均升高或有上升的趋势（分别为59.9±2.0比6.0±1.6、1.807±0.297比1.332±0.112、4.43±2.57比1.71±0.57、2.6±0.9比1.9±0.5,均P＜0.05）.结论 替米沙坦具有提高OLETF大鼠与人前脂肪细胞胰岛素敏感性、缓解大鼠的内脏性肥胖和改善大鼠糖脂代谢功能紊乱的作用,并很可能通过上调脂肪尤其是内脏脂肪的脂联素表达来完成.     

替米沙坦对糖尿病大鼠心肌脂联素及其受体1表达的影响

目的 观察替米沙坦对糖尿病大鼠心肌脂联素及其受体1表达的影响,探讨替米沙坦对糖尿病大鼠心肌病变的保护作用及机制.方法30只雄性Wistar大鼠随机分为对照组,糖尿病组和糖尿病替米沙坦治疗组(替米沙坦组).用高糖高脂饲料加小剂量链脲佐菌素建立糖尿病大鼠模型.成模后,替米沙坦组予以替米沙坦(5 mg·kg~(-1)·d~(-1))灌胃12周.实验结束时,测定心功能,用酶联免疫法检测血浆和心肌脂联素水平,用RT-PCR方法检测心肌脂联素受体1(AdipoR1)mRNA表达,用Western印迹法检测心肌脂联素受体1、磷酸化的磷酸腺苻激活的蛋白激酶-α(AMPK-α)、葡萄糖转运子4(GluT_4)蛋白表达.结果与正常对照组比较,糖尿病组大鼠12周时出现心重/体重增加[(4.38±0.07对2.99±0.67)g/kg,P<0.05],心功能降低,血浆和心肌脂联素水平降低[(1.09±0.03对1.87±0.05)μg/ml,(0.12±0.02对0.21±0.02)μg/mg蛋白,均P<0.05],心肌脂联索受体1 mRNA和蛋白表达降低(0.35±0.08对0.78±0.11,0.34±0.07对0.77±0.08,均P<0.05),心肌磷酸化AMPK-α和GluT_4蛋白表达降低(0.47±0.12对0.72±0.10,0.41±0.09对0.79±0.08,均P<0.05).与糖尿病组比较,替米沙坦治疗12周后心室重/体重降低[(3.49±0.39对4.38±0.07)g/kg,P<0.05],心功能改善.血浆和心肌脂联素水平升高[(1.41±0.02对1.09±0.03)μg/ml,(0.16±0.01对0.12±0.02)μg/mg蛋白,均P<0.05],心肌脂联素受体1 mRNA和蛋白表达增加(0.48±0.06对0.35±0.08,0.47±0.09对0.34±0.07,均P<0.05),心肌磷酸化AMPK-α和GluT_4表达水平均升高(0.57±0.10对0.47±0.12,0.52±0.10对0.41±0.09,均P<0.05).结论糖尿病大鼠心肌脂联素及其受体1的表达水平降低.替米沙坦上调糖尿病大鼠心肌脂联素及其受体1的表达水平,促进心肌葡萄糖氧化,从而改善心功能.     

替米沙坦对非酒精性脂肪性肝炎大鼠血清脂联素、肝脏AdipoR2 mRNA表达及胰岛素抵抗的影响

目的探讨替米沙坦对非酒精性脂肪性肝炎大鼠胰岛素抵抗、血清脂联素及其肝组织脂联素受体R2mRNA表达的影响。方法 35只雄性SD大鼠随机分为正常对照组(NC,n=10)、模型组(FC,n=15)和替米沙坦干预组(FT,n=10)。FC和FT组给予高脂饲料喂养16周诱发脂肪性肝炎,其中FT组于高脂喂养12周后,给予替米沙坦(5mg·kg-1·d-1)灌胃治疗4周。检测血清ALT、AST、空腹血糖、空腹胰岛素和胰岛素抵抗指数(HOMA-IR);应用RT-PCR方法测定大鼠肝组织脂联素受体R2mRNA的表达;ELISA法测定血浆脂联素水平。结果与NC组相比,FC组脂联素及其受体R2mRNA水平均显著降低(P0.01);FT组的脂联素及其受体R2mRNA水平较FC组升高(P0.05)。脂联素及其受体R2mRNA水平均与HOMA-IR呈负相关(r分别为-0.891,-0.686;P0.01,0.05)。结论替米沙坦能上调脂联素及其受体R2的表达,改善非酒精性脂肪性肝炎大鼠的胰岛素抵抗。     

罗格列酮通过上调脂肪脂联素及其受体的表达改善去卵巢大鼠胰岛素抵抗

目的 研究罗格列酮对去卵巢大鼠胰岛素抵抗和脂肪中脂联素及其受体表达的影响,为噻唑烷二酮类药物用于防治绝经后2型糖尿病提供实验依据.方法 36只SD雌性大鼠.随机分为假手术组、去卵巢组和去卵巢+罗格列酮组;检测各组大鼠体重、血压和胰岛素敏感指数的变化.测量内脏脂肪重量,计算体脂含量;逆转录聚合酶链反应检测内脏脂肪中脂联素、脂联素受体的表达.结果 与假手术组相比,去卵巢组大鼠内脏脂肪含量和血压显著增加,胰岛素敏感指数显著降低,内脏脂肪中脂联素、脂联素受体表达显著降低;罗格列酮逆转了上述变化.结论 去卵巢大鼠胰岛素抵抗可能与脂肪脂联素及其受体的表达下调有关;罗格列酮能够上调脂肪脂联素及其受体的表达而改善去卵巢大鼠胰岛素抵抗.     

NF-κB抑制剂对胰岛素抵抗大鼠抵抗素、脂联素和脂联素受体表达的影响

高脂喂养大鼠脂肪组织抵抗素、脂联素的表达均明显下降，加入N-乙酰半胱氨酸后脂联素表达增高，脂联素受体1表达无改变。抵抗素和脂联素水平的降低是高脂饲养大鼠体重增加、血糖升高、脂质代谢紊乱和胰岛素抵抗的重要原因。NF-κB通路介导了脂联素对胰岛素敏感性的调节作用，但不影响脂联素受体1的表达。     

胰岛素对大鼠骨骼肌细胞脂联素受体基因表达的影响

目的了解体外胰岛素对原代培养大鼠骨骼肌细胞脂联素受体1表达的影响。方法体外原代培养骨骼肌细胞,应用SYBRGreenⅠ染料建立一种快速、可靠的实时定量PCR,对其主要要素进行优化。观察不同胰岛素浓度不同作用时间下,大鼠骨骼肌细胞脂联素受体基因表达水平的动态变化。结果建立敏感、特异、快速检测脂联素受体1mRNA的实时定量PCR方法,随着胰岛素浓度的增加,脂联素受体1表达逐渐降低。在较低浓度（胰岛素浓度〈1nmol/L）时,脂联素受体1表达的降低无统计学意义,当胰岛素浓度增加到10nmol/L及以上时,骨骼肌细胞脂联素受体1表达的降低有统计学意义（P〈0.05）,这种抑制作用1h后出现,24h后达到高峰。结论成功地建立SYBRGreenⅠ实时定量PCR检测脂联素受体基因的表达方法,体外高胰岛素对骨骼肌细胞脂联素受体1mRNA表达有抑制作用,并呈时间和剂量依赖性。     

脂联素与胰岛素抵抗

脂联素是最近发现的一种脂肪特异性蛋白质、胰岛素、噻唑烷二酮类、β肾上腺能拮抗剂等 ,也参与调节脂联素的分泌与表达 ,动物实验及临床研究均表明 ,脂联素与空腹胰岛素浓度负相关 ,与胰岛素敏感指数正相关 ,罗格列酮治疗可使血浆脂联素明显增加 ,胰岛素敏感性增加。脂联素通过抑制单核细胞黏附 ,减低其细胞周期活性 ,降低血管壁脂蛋白的沉积 ,而抑制动脉硬化过程。     

NF-kB抑制剂对胰岛素抵抗大鼠抵抗素、脂联素和脂联素受体表达的影响

高脂喂养大鼠脂肪组织抵抗素、脂联素的表达均明显下降,加入N-乙酰半胱氨酸后脂联素表达增高,脂联素受体1表达无改变。抵抗素和脂联素水平的降低是高脂饲养大鼠体重增加、血糖升高、脂质代谢紊乱和胰岛素抵抗的重要原因。NF-kB通路介导了脂联素对胰岛素敏感性的调节作用,但不影响脂联素受体1的表达。     

脂联素与胰岛素抵抗

脂肪组织是重要的内分泌器官,能以旁分泌、自分泌和内分泌方式产生生物活性因子或因子样分子,称为脂肪细胞因子,包括肿瘤坏死因子α(tumor necrosis factorα,TNF-α)、     

脂联素受体在胰岛细胞表达,脂联素促进胰岛素的分泌

目的　检测脂联素受体(AR)在大鼠胰岛细胞的表达和脂联素对体外胰岛细胞分泌胰岛素的影响。方法　RT PCR和免疫细胞化学方法检测AR1、AR2的mRNA和蛋白表达;并在体外用脂联素(100μg/L)和不同浓度葡萄糖(3. 3, 5. 6, 16. 7mmol/L)处理胰岛细胞,放免法测定上清液的胰岛素浓度。结果　RT PCR扩增出胰岛AR1和AR2基因,并经直接和亚克隆测序证实;胰岛免疫细胞化学荧光染色AR1和AR2呈阳性;经脂联素处理后的胰岛细胞,在高糖(16. 7mmol/)培养 6~24h,其胰岛素分泌持续增加(均P<0. 05)。结论　胰岛细胞上存在AR1和AR2,以前者为主。在高糖情况下,一定浓度的脂联素可在体外促进胰岛细胞的胰岛素分泌和释放。     

蜕皮甾酮对胰岛素抵抗HepG2细胞胰岛素受体表达的影响

目的 探讨蜕皮甾酮对胰岛素抵抗(IR)HepG2细胞胰岛素受体(InsR)蛋白表达的影响.方法 建立胰岛素抵抗HepG2细胞模型,培养液中加入蜕皮甾酮孵育,观察蜕皮甾酮及吡格列酮对细胞葡萄糖摄取率的影响;应用免疫组化染色法及Western blot方法观察蜕皮甾酮对IR HepG2细胞InsR蛋白表达的影响. 结果 与模型细胞比较,1×10-5 mol/L蜕皮甾酮可使IR HepG2细胞InsR蛋白的表达显著增加. 结论 蜕皮甾酮的胰岛素增敏作用可能与胰岛素信号转导分子InsR蛋白的表达增强有关.     

贝那普利、替米沙坦对人前脂肪细胞脂联素表达的影响

目的探讨贝那普利、替米沙坦对体外原代培养的人网膜和皮下来源的前脂肪细胞脂联素(APN)表达的影响。方法自12例行电切开腹部手术的健康成年女性腹部皮下和网膜分离前脂肪细胞,分为3组,即无干预正常对照组(NC组)、血管紧张素转换酶抑制剂类药物贝那普利组(ACEI组)和血管紧张素Ⅱ受体拮抗剂类药物替米沙坦组(ARB组),诱导分化共14 d。放射免疫法测定各组APN mRNA表达水平。结果与NC组比较,ACEI组、ARB组体外原代培养的人网膜和皮下前脂肪细胞中APN mRNA表达明显增强。结论贝那普利、替米沙坦可促进网膜来源的前脂肪细胞的分化,在以腹型肥胖为特征的代谢综合征干预方面可能具有广泛的应用前景。     

Associations between glucose tolerance,insulin sensitivity and insulin secretion phenotypes and polymorphisms in adiponectin and adiponectin receptor genes in the Quebec Family Study

Aims Studies suggest that adiponectin (APM1) and its receptors 1 and 2 (AdipoR1 and AdipoR2) play an important role in the development of insulin resistance (IR). Our objective was to examine associations between APM1 (+45T>G, +276G>T and –3971A>G), AdipoR1 (−100G>T and −3882T>C) and AdipoR2 (−35361A>G and –1352G>A) genes single-nucleotide polymorphisms (SNPs) and adiponectin plasma levels, indicators of glucose tolerance, insulin sensitivity (IS) and insulin secretion. Methods Six hundred and twenty-two non-diabetic subjects from the Quebec Family Study (QFS) underwent a 75-g oral glucose tolerance test (OGTT), with measurement of fasting adiponectin, glucose, insulin and C-peptide levels. Indices of glucose tolerance, IS and insulin secretion were derived from fasting and OGTT measurements. Results Significant evidence of association was found between indices of IS and APM1 and AdipoR1 SNPs. The APM1 –3971G/G homozygotes exhibited a reduced area under the curve of insulin during the OGTT (P = 0.007) and higher Cederholm index (P = 0.01) compared to the A/A homozygotes. The APM1+45T>G variant was also associated with fasting (P = 0.002) and 2-h (P = 0.007) glucose values as well as with higher Cederholm index (P = 0.04) and disposition index (P = 0.02). Finally, the AdipoR1−3882T>C SNP was associated with fasting glucose (P = 0.03), the homeostasis model assessment for insulin resistance (P = 0.04) and an index of insulin secretion (P30/G30, P = 0.02). No evidence of association was found with plasma adiponectin levels. Conclusions These results provide evidence for an influence of common SNPs in the APM1 and AdipoR1 genes on different phenotypes of glucose and insulin metabolism associated with increased risk of type 2 diabetes.     

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     

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。     

In humans the adiponectin receptor R2 is expressed predominantly in adipose tissue and linked to the adipose tissue expression of MMIF‐1

In this study, the regional adipose tissue‐adiponectin (AT‐ADN) and adiponectin receptor (R1 and R2) expression and their relation with metabolic parameters, circulating and AT‐derived cytokine expressions were compared. Paired subcutaneous adipose tissue (SCAT) and visceral adipose tissue (VAT) were taken from 18 lean and 39 obese humans, AT‐mRNA expression of adipokines analysed by RT‐PCR and corresponding serum levels by enzyme‐linked immunosorbent assay (ELISA). R1 and R2 adipocyte expression was compared with 17 other human tissues. ADN‐gene expression was lower in VAT than SCAT [mean (SD) 1.54 (1.1) vs. 2.84 (0.87); p < 0.001], and lower in obese subjects (VAT : p = 0.01;SCAT : p < 0.001). SCAT‐ADN correlated positively with serum ADN (r = 0.33;p = 0.036) but not VAT‐ADN. AT expressions of ADN and macrophage migration inhibiting factor (MMIF), IL18 and cluster of differentiation factor 14 (CD14) in both depots showed inverse correlations. R1 and R2 were expressed ubiquitously and R2 highest in SCAT, and this is much higher (×100) than R1 (×100). R expression was similar in lean and obese subjects and unrelated to the metabolic syndrome, however, receptors correlated with VAT‐MMIF (R 1: r = 0.4;p = 0.008;R 2: r = 0.35,p = 0.02) and SCAT‐MMIF expression (R 2: r = 0.43;p = 0.004). Unlike ADN, its receptors are expressed in many human tissues. Human R2 expression is not highest in the liver but in AT where it is associated with MMIF expression. The adiponectin‐dependent insulin‐sensitizing action of thiazolidinediones is thus probably to differ amongst species with weaker effects on the human liver.     

Circulating adiponectin and adiponectin receptor expression in skeletal muscle: effects of exercise

Excess visceral fat can regulate insulin sensitivity and energy metabolism by releasing adipokines into the circulation which then bind with their cognate receptors in various tissues and alter glucose and lipid metabolism. Circulating levels of adiponectin, which promotes glucose uptake into skeletal muscle and increases fat oxidation rates, are decreased in obesity. Strategies to enhance the insulin-like and insulin-sensitizing actions of adiponectin have been shown to be effective in improving metabolic abnormalities associated with obesity and diabetes. Interestingly, the insulin-sensitizing effects of exercise have similar metabolic effects as adiponectin in that exercise also promotes glucose uptake into muscle and increases rates of fatty acid oxidation. Recent studies have begun to examine the potential role of adiponectin in mediating the insulin-sensitizing action of exercise by investigating changes in plasma adiponectin levels and tissue-specific adiponectin receptor (AdipoR) expression. In this review, we have summarized the key findings to date which suggest that changes in expression of AdipoR isoforms in skeletal muscle, rather than circulating total adiponectin levels, may be of physiological importance.     

超重和肥胖人群脂联素受体mRNA的水平与脂肪细胞因子的相关性

目的 了解两种脂联素受体（AdpR1和AdpR2）在人脂肪组织中的表达水平及其与血清脂肪细胞因子的关系。方法 选取19例超重或肥胖（OW＋Ob）和29例正常体重者（NC），用RT-PCR技术检测腹部皮下脂肪组织（AAT）与大网膜脂肪组织（0AT）中脂联素受体的表达水平，并测FIns、血脂和血清脂联素、TNF-α、FFA等指标。结果 （1）两种脂联素受体mRNA的表达水平在OW＋Ob组和NC组、AAT和OAT之间均无差异；但OW＋Ob患者的血浆TG、VLDL-C、FIns、TNF-α、FFA均高于NC组（P＜0．05～P＜0．01），而血清脂联素低于NC组（P＜0．05）。（2）总体观察对象中，AAT的脂联素受体mRNA表达水平与血清脂联素、FFA、FIns、HOMA-IR水平呈负相关（P＜0．05～P＜0．01）；与ISI呈正相关（P分别＜0．05和＜0．01）。（3）OW＋Ob组中脂联素与BMI、FFA、FIns、HOMA-IR呈负相关（P＜0．05～P＜0．01），与ISI呈正相关（P＜0．01）；FFA与血浆TG、LDL-C、FIns呈正相关（P均＜0．05）。结论 OW＋Ob人群的血清TNF-α、FFA水平高于NC人群，脂联素水平低于NC人群，而脂联素受体mRNA的表达水平在两组间没有差异。脂联素受体、FIns、脂联素、FFA是一组很好的评估胰岛素敏感性的指标。     

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