软脂酸诱导HepG2细胞胰岛素抵抗及花生四烯酸防治作用的机制研究

目的 探讨高浓度软脂酸(PA)诱导HepG2细胞胰岛素抵抗(IR)的机制及花生四烯酸(AA)对IR的防治作用。方法 (1)用高浓度软脂酸(PA)或10^-7mol／L高胰岛素(HI)培养HepG2细胞建立具有IR的细胞模型，测定培养液中葡萄糖含量及细胞内糖原含量作为鉴定指标；(2)用Western blot检测胞内糖原合酶(GS)和蛋白激酶B(PKB)蛋白水平；(3)用磷脂酰肌醇3激酶(P13K)抑制剂Wortmannin(WT)探讨其对胰岛素信号通路的影响；(4)观察AA是否对PA引起的IR有防治作用。结果 (1)0．20mmol／L PA或川培养HepG2细胞36h后，培养液中葡萄糖含量极显著增高，细胞内糖原含量极显著减少；(2)高浓度PA使磷酸化的PKB(P-Ser473)蛋白水平显著减少，磷酸化的糖原合酶(P-Ser641 GS)蛋白水平极显著增加；(3)WT使对照组GS活性及胞内糖原含量极显著减少，HI组和PA组胞内糖原含量均无统计学差异，但各实验组PKB活性都极显著减少；(4)PA AA组培养液中葡萄糖含量显著低于PA组，GS和PKB活性及胞内糖原含量显著增加。结论 高浓度PA或HI培养HepG2细胞能够诱导IR，其机制可能是其引起胰岛素信号传递途径中自PKB下游到GS之间的信号通路受阻所致。AA能改善PA引起的IR。     

PKC在软脂酸诱导的肝细胞胰岛素抵抗信号转导中的作用

目的探讨蛋白激酶PKC在软脂酸（PA）诱导的肝细胞胰岛素抵抗中的作用。方法将DMEM培养基中含0．25mmol／L的软脂酸（PA组）与HepG2细胞共同培养24h，并设立正常对照组（Control组）。加入PKC抑制剂chelerythrine chloride（CC），胰岛素刺激后分光光度酶偶联速率法测定胞内糖异生限速酶磷酸烯醇式丙酮酸羧激酶（PEPCK）活性，Western blot测定胞内胰岛素受体底物2（IRS-2）蛋白水平。结果PA组与Control组相比，基础及胰岛素刺激的PEPCK活性升高（P〈0．05）；加入CC与否，Control组IRS-2蛋白水平存在明显差异（P〈0．05），PA组中却无明显变化（P〉0．05），而PEPCK活性在Control组、PA组均无明显改变（P〉0．05）。结论细胞胰岛素抵抗时，糖异生的关键酶活性以及胰岛素信号通路的信号蛋白异常改变，胰岛素信号转导PKC通路可能存在传导障碍。     

腺苷酸活化蛋白激酶与胰岛素抵抗

哺乳动物组织中，腺苷酸活化蛋白激酶（AMPK）是细胞内重要的能量代谢感受器，属于代谢敏感性蛋白激酶家族。它是调节多种代谢过程的重要信号分子，对于糖脂代谢尤有积极的调节作用。有证据表明，在抗糖尿病药物中，二甲双胍和噻唑烷二酮类药物均是通过激活AMPK而发挥胰岛素增敏作用的。     

腺苷酸活化蛋白激酶与胰岛素抵抗

腺苷酸活化蛋白激酶（AMP-activated protein kinase, AMPK）是一类重要的蛋白激酶,通过改变细胞代谢和调节基因转录恢复细胞ATP水平。AMPK参与了肌肉收缩介导的葡萄糖转运和脂肪酸氧化,抑制肝脏葡萄糖、胆固醇和甘油三酯产生,并具有调节食物摄取和体重的作用。AMPK信号通路是目前具有吸引力的治疗肥胖、胰岛素抵抗、2型糖尿病和其它代谢病的药理靶点。     

蛋白激酶C/NF-κB在X线辐射诱导HepG2细胞凋亡中的作用

目的 探讨蛋白激酶C/核因子κB(NF-κB)在X线电离辐射诱导HepG2细胞凋亡中的作用.方法 分对照组、辐射组、PMA(蛋白激酶C激活剂)组、SP(蛋白激酶C抑制剂)组.流式细胞仪检测细胞凋亡率;EMSA 检测NF-κB的激活.免疫印迹法检测IκB的表达.结果 对照组、辐射组、PMA组、SP组细胞凋亡率分别为1.73%、20.90%、3.20%、40.57%.对照组的NF-κB微弱激活,辐射组NF-kB明显激活,并诱导了胞质内IκB的降解;PMA可增强NF-κB的激活,加强胞质内IκB的降解;SP减弱NF-κB的激活,抑制质内IκB降解.结论 蛋白激酶C可能参与了6 Gy X线辐射诱导的HepG2细胞凋亡的保护机制, 发挥抗凋亡作用,且可能是通过激活NF-κB来实现的.     

高浓度胰岛素、葡萄糖对HepG2细胞蛋白激酶C活性的影响

研究高浓度胰岛素,葡萄糖对ＨｅｐＧ２细胞蛋白激酶Ｃ（ＰＫＣ）活性的影响。方法建立具胰岛素抵抗特性受体下调的ＨｅｐＧ２（ＤＲ－ＨｅｐＧ２）细胞模型,测定１０＾－７ｍｏｌ／Ｌ胰岛素、２５ｍｍｏｌ／Ｌ葡萄糖对未处理ＨｅｐＧ２（ＮＤＲ－ＨｅｐＧ２）细胞和ＤＲ－Ｈ细胞膜ＰＫＣ、胞浆ＰＫＣ活性的影响。结果ＤＲ－ＨｅｐＧ２细胞未受高浓度胰岛素、葡萄糖刺激时膜和胞浆ＰＫＣ活性均高于ＮＤＲ－ＨｅｐＧ２细胞（Ｐ〈０．     

高脂喂养的胰岛素抵抗大鼠骨骼肌蛋白激酶B mRNA表达的改变

目的研究高脂饮食喂养的胰岛素抵抗(IR)大鼠胰岛素敏感性及骨骼肌中蛋白激酶B(PKB)mRNA表达改变。方法 70只大鼠随机分为正常对照组和高脂饮食组,高脂饮食组采用高脂喂养的方法建立IR大鼠模型,用高胰岛素-正常葡萄糖钳夹实验测定葡萄糖输注率(GIR),同时测定各组大鼠空腹血糖(FPG)、空腹胰岛素(FINS)等指标的水平,实时荧光定量RT-PCR测定大鼠骨骼肌PKB mRNA表达。结果①高脂喂养4 w后,高脂组FPG、FINS、胰岛素抵抗指数(HOMA-IR)均较正常对照组显著升高,而胰岛素敏感指数(ISI)下降(P<0.05),GIR显著低于正常对照组(P<0.01),说明高脂组存在IR,IR模型制造成功。②与正常对照组相比高脂组PKB mRNA表达明显减弱(P<0.05)。结论高脂饮食喂养的IR大鼠IR的产生与骨骼肌胰岛素刺激PKB表达明显降低有关。     

褪黑素对软脂酸诱导L02细胞胰岛素抵抗的防护作用及机制

目的探讨褪黑素(MT)对软脂酸(PA)诱导L02细胞胰岛素抵抗的防护作用及机制。方法培养L02细胞,分别设立对照组(BSA组)、PA组、PA+MT组、PA+维生素C(Vit C)组。各组药物浓度和孵育时间根据不同的实验需要来确定。用荧光比色法测定各组细胞胞内氧自由基(ROS)水平,Western印迹法检测胰岛素刺激后胞内胰岛素信号蛋白磷酸化水平,包括Y-p-胰岛素受体(IR)、Y-p-胰岛素受体底物(IRS)1/2以及应激敏感激酶p-JNK水平。结果用不同浓度的PA处理L02细胞24 h,细胞内ROS生成量呈现明显的浓度依赖关系,差异有统计学意义(P<0.05);用0.3 mmol/L的PA处理L02细胞不同时间,细胞内ROS生成量存在明显的时间依赖关系,在24 h达到最高,并在36 h后仍维持较高水平(P<0.05);与BSA组相比,PA组ROS含量明显升高(P<0.05)PA+MT组ROS含量无明显变化(P>0.05);PA+MT组ROS生成量明显低于PA组(P<0.05)。PA+Vit C组与PA组相比,ROS的生成量减少,但仍高于PA+MT组(P<0.05)。与BSA组相比,PA组Y-p-IR、Y-p-IRS1/2磷酸化水平减少,而p-JNK磷酸化水平升高;与PA组相比,PA+MT组Y-p-IR、Y-p-IRS1/2磷酸化水平显著升高,p-JNK磷酸化水平明显降低(P<0.05)。结论 PA导致细胞内ROS增加,MT能够清除PA产生的过量ROS。MT通过减少JNK的活化,改善因ROS引起的胰岛素信号传递损伤,对PA诱导的胰岛素抵抗具有防护作用。     

蛋白激酶C激活与胰岛素抵抗及糖尿病血管并发症

蛋白激酶C（PKC）是一个由多基因编码的多功能同工酶家族，目前发现至少有12种亚型。高血糖和游离脂肪酸可引起多种组织内二酯酰甘油（DAG）水平升高，DAG可激活PKC。不同的PKC亚型可介导或抑制胰岛素的作用，促进葡萄糖代谢或引起胰岛素抵抗。研究表明，持续高血糖引起的DAG－PKC系统激活与糖尿病多种血管并发症密切相关。选择性PKC－β抑制到LY333531在临床和动物实验中已取得满意疗效。     

蛋白激酶C激活与胰岛素抵抗及糖尿病血管并发症

蛋白激酶C(PKC)是一个由多基因编码的多功能同工酶家族 ,目前发现至少有 12种亚型。高血糖和游离脂肪酸可引起多种组织内二脂酰甘油 (DAG)水平升高 ,DAG可激活PKC。不同的PKC亚型可介导或抑制胰岛素的作用 ,促进葡萄糖代谢或引起胰岛素抵抗。研究表明 ,持续高血糖引起的DAG PKC系统激活与糖尿病多种血管并发症密切相关。选择性PKC β抑制剂LY3335 31在临床和动物实验中已取得满意疗效。     

Inhibition of protein kinase C in multidrug-resistant cells by modulators of multidrug resistance

We have evaluated the protein kinase C (PKC) activity in two series of cultured cell lines presenting the multidrug-resistance (MDR) phenotype and in the corresponding wild-type cells: the human KB 3.1, KB A1 and KB 8.5 cell lines, and the rat C6, C6 0.5 and C6 1V cell lines. We have observed an increase in PKC activity in the MDR cell lines of the KB cell lineage, proportional to their degree of resistance to doxorubicin. In contrast, the MDR cell lines of the C6 cell lineage presented no change (C6 0.5) or even decrease (C6 1V) in PKC activity; the basal level of PKC activity in C6 cells was, however, 50-fold higher than in KB 3.1 cells. We have tested, in these lines, the effect of four modulators of MDR: verapamil, cyclosporin A, quinine and S-9788, on doxorubicin acytotoxicity and on PKC activity. We observed that cyclosporin A and S-9788, which were the most active on MDR reversal, were able to inhibit PKC activity in the KB resistant lines as well as in all C6 lines, whereas verapamil and quinine had only marginal effects on PKC activity. The distribution of PKC isoenzymes was studied by Western blots. The PKC , and isoforms were increased in the KB resistant lines as compared to wild-type cells, which could account for the increase PKC activity we observed. In contrast, PKC and were decreased in C6 1V cells, as expected from the results obtained for total PKC activity, but we also noticed an important decrease in PKC in the C6 0.5 line. Our results suggest that an increase in PKC activity is not an absolute requirement for expression of MDR, provided that the basal level be high enough; and that some modulators may act on MDR, not only through direct P-glycoprotein interaction, but also through P-glycoprotein phosphorylation or expression. The distribution of PKC isoenzymes revealed that the modifications encountered between sensitive and resistant cells mainly concerned , and isoenzymes of PKC.Abbreviations PKC protein kinase C - MDR multidrug resistance     

Contrasting insulin dose-dependent defects in activation of atypical protein kinase C and protein kinase B/Akt in muscles of obese diabetic humans

Aims/hypothesis Insulin-stimulated glucose transport in muscle is impaired in obesity and type 2 diabetes, but alterations in levels of relevant signalling factors, i.e. atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt), are still uncertain. Clamp studies using maximal insulin concentrations have revealed defects in activation of aPKC, but not PKB, in both obese non-diabetic and obese diabetic subjects. In contrast, clamp studies using submaximal insulin concentrations revealed defects in PKB activation/phosphorylation in obese non-diabetic and diabetic subjects, but changes in aPKC were not reported. The aim of this study was to test the hypothesis that dose-related effects of insulin may account for the reported differences in insulin signalling to PKB in diabetic muscle.Subjects and methods We compared enzymatic activation of aPKC and PKB, and PKB phosphorylation (threonine-308 and serine-473) during hyperinsulinaemic–euglycaemic clamp studies using both submaximal (400–500 pmol/l) and maximal (1400 pmol/l) insulin levels in non-diabetic control and obese diabetic subjects.Results In lean control subjects, the submaximal insulin concentration increased aPKC activity and glucose disposal to approximately 50% of the maximal level and PKBβ activity to 25% of the maximal level, but PKBα activity was not increased. In these subjects, phosphorylation of PKBα and PKBβ was increased to near-maximal levels at submaximal insulin concentrations. In obese diabetic subjects, whereas aPKC activation was defective at submaximal and maximal insulin concentrations, PKBβ activation and the phosphorylation of PKBβ and PKBα were defective at submaximal, but not maximal, insulin concentrations.Conclusions/interpretations Defective PKBβ activation/phosphorylation, seen on submaximal insulin stimulation in diabetic muscle, may largely reflect impaired activation of insulin signalling factors present in concentrations greater than those needed for full PKB activation/phosphorylation. Defective aPKC activation, seen at all insulin levels, appears to reflect, at least partly, an impaired action of distal factors needed for aPKC activation, or poor aPKC responsiveness.     

Metformin improves atypical protein kinase C activation by insulin and phosphatidylinositol-3,4,5-(PO4)3 in muscle of diabetic subjects

Aims/hypothesis Metformin is widely used for treating type 2 diabetes mellitus, but its actions are poorly understood. In addition to diminishing hepatic glucose output, metformin, in muscle, activates 5′-AMP-activated protein kinase (AMPK), which alone increases glucose uptake and glycolysis, diminishes lipid synthesis, and increases oxidation of fatty acids. Moreover, such lipid effects may improve insulin sensitivity and insulin-stimulated glucose uptake. Nevertheless, the effects of metformin on insulin-sensitive signalling factors in human muscle have only been partly characterised to date. Interestingly, other substances that activate AMPK, e.g., aminoimidazole-4-carboxamide-1-β-d-riboside (AICAR), simultaneously activate atypical protein kinase C (aPKC), which appears to be required for the glucose transport effects of AICAR and insulin. Methods Since aPKC activation is defective in type 2 diabetes, we evaluated effects of metformin therapy on aPKC activity in muscles of diabetic subjects during hyperinsulinaemic–euglycaemic clamp studies. Results After metformin therapy for 1 month, basal aPKC activity increased in muscle, with little or no change in insulin-stimulated aPKC activity. Metformin therapy for 8 to 12 months improved insulin-stimulated, as well as basal aPKC activity in muscle. In contrast, IRS-1-dependent phosphatidylinositol (PI) 3-kinase activity and Ser473 phosphorylation of protein kinase B were not altered by metformin therapy, whereas the responsiveness of muscle aPKC to PI-3,4,5-(PO₄)₃, the lipid product of PI 3-kinase, was improved. Conclusions/interpretation These findings suggest that the activation of AMPK by metformin is accompanied by increases in aPKC activity and responsiveness in skeletal muscle, which may contribute to the therapeutic effects of metformin.     

The role of protein kinase B/Akt in insulin-induced inactivation of phosphorylase in rat hepatocytes

Aims/hypothesis An insulin signalling pathway leading from activation of protein kinase B (PKB, also known as Akt) to phosphorylation (inactivation) of glycogen synthase kinase-3 (GSK-3) and activation of glycogen synthase is well characterised. However, in hepatocytes, inactivation of GSK-3 is not the main mechanism by which insulin stimulates glycogen synthesis. We therefore tested whether activation of PKB causes inactivation of glycogen phosphorylase. Materials and methods We used a conditionally active form of PKB, produced using recombinant adenovirus, to test the role of acute PKB activation in the control of glycogen phosphorylase and glycogen synthesis in hepatocytes. Results Conditional activation of PKB mimicked the inactivation of phosphorylase, the activation of glycogen synthase, and the stimulation of glycogen synthesis caused by insulin. In contrast, inhibition of GSK-3 caused activation of glycogen synthase but did not mimic the stimulation of glycogen synthesis by insulin. PKB activation and GSK-3 inhibition had additive effects on the activation of glycogen synthase, indicating convergent mechanisms downstream of PKB involving inactivation of either phosphorylase or GSK-3. Glycogen synthesis correlated inversely with the activity of phosphorylase-a, irrespective of whether this was modulated by insulin, by PKB activation or by a selective phosphorylase ligand, supporting an essential role for phosphorylase inactivation in the glycogenic action of insulin in hepatocytes. Conclusions/interpretation In hepatocytes, the acute activation of PKB, but not the inhibition of GSK-3, mimics the stimulation of glycogen synthesis by insulin. This is explained by a pathway downstream of PKB leading to inactivation of phosphorylase, activation of glycogen synthase, and stimulation of glycogen synthesis, independent of the GSK-3 pathway.     

蛋白激酶B调节骨桥蛋白在肝癌HepG2细胞中的表达

目的 转移相关基因骨桥蛋白(OPN)在肝癌中的表达方式、途径尚不清楚，检测OPN在HepG2细胞中转染蛋白激酶B(Akt)前后的表达，旨在探讨磷脂酰肌醇3激酶信号途径中的关键基因Akt与OPN表达的关系。方法 用脂质体介导的基因转染法将含有Akt基因的质粒转染HepG2细胞，并用Western blot鉴定；OPN的表达用Northern blot和Western blot方法检测。结果 Akt基因成功转染HepG2细胞，Western blot能检测到HepG2细胞中外源表达的Akt基因；Northern blot和Western blot检测发现，Akt在核酸和蛋白水平调节OPN的表达；在无血清培养条件下，OPN在HepG2中结构性表达量很少或无表达,转染活性型Akt后OPN表达升高；在有血清培养条件下,HepG2细胞转染缺陷型Akt基因后OPN表达下降。结论 Akt调节转移相关基因OPN在肝癌细胞中的表达，提示可通过使Akt基因失活来阻断OPN产生，从而抑制肝癌转移。     

蛋白激酶C及抑制剂在肺癌耐药中的研究进展

耐药已成为当今肺癌化疗过程中的一大难题,其作用机制至今还不十分清楚.蛋白激酶C(protein kinase C,PKC)作为多种信号传导过程中的枢纽,不仅参与细胞信息传递、分泌、细胞分化、增殖,更重要的是参与肿瘤细胞的凋亡和分化.国内外研究表明通过抑制PKC的活性,减少其表达量可以增加细胞内药物的积聚导致胞内有效浓度的上升,从而降低肿瘤细胞耐药率.PKC抑制剂对肿瘤细胞具有明显的诱导分化、增强细胞毒性、促进细胞凋亡的作用.目前已有部分PKC抑制剂进入了临床的Ⅰ/Ⅱ期研究中,并取得了一定的疗效,通过对其作用机制的进一步深入探讨,有望在肺癌耐药的研究中取得更多的突破.     

The role of protein kinase C in insulin biosynthesis

Activation of protein kinase C (PKC) by the phorbol ester 4-phorbol myristate acetate (4-PMA) stimulated (pro)insulin biosynthesis in collagenase-isolated rat islets of Langerhans, as assessed by measuring the incorporation of [³⁵S]cysteine into proinsulin and insulin after fractionation by high performance liquid chromatography. The stimulatory effects of 4-PMA were observed at a substimulatory concentration of glucose (2 mM) but were not additive to the stimulatory effects of 20 mM glucose on insulin biosynthesis. Prolonged exposure to 4-PMA caused a marked down-regulation of PKC activity in islets. PKC-depleted islets showed a much reduced biosynthetic response to 20 mM glucose, but this was caused, at least in part, by an enhanced basal rate of (pro)insulin synthesis. These elevations in the basal rate of insulin synthesis were not secondary to an inerease in the amount of preproinsulin mRNA in PKC-depleted islets since Northern blot analysis showed that prolonged exposure to 4-PMA, and the subsequent loss of PKC activity, did not detectably alter basal levels of preproinsulin mRNA. These results suggest that the activation of PKC stimulates (pro)insulin synthesis in rat islets by enhancing translation of existing preproinsulin mRNA, and that this may play some part in the biosynthetic responses of -cells to glucose.