【作者】 卢雁；

【导师】 韩萍；

【作者基本信息】 中国医科大学 ， 内科学， 2007， 博士

【摘要】 目的糖尿病是一种多病因的代谢性疾病,随着20世纪社会的进步和生活方式的改变,糖尿病已日益成为威胁人类健康的卫生保健问题。胰岛素抵抗(IR)及胰岛β细胞功能受损是2型糖尿病发病的两大病理生理基础。2型糖尿病与肥胖紧密相关已经成为一种共识。肥胖者由于体内脂肪组织的过度积聚,导致释放过多的游离脂肪酸(Free Fatty Acid,FFA),进而引发一系列病理生理反应,称为“脂毒性”,该学说包括了FFA引起的IR以及β细胞结构和功能的改变,这与FFA直接作用于酶类,离子通道,信号分子/转录因子有关,亦与FFA诱导活性氧簇(ROS)有关。IR与胰岛素信号转导的各个环节、调控糖脂代谢的多种基因的表达和多态性相关。胰岛素受体信号转导通路异常包括受体前通路异常;受体水平的异常;受体后异常(包括IRS-1基因突变,GluT4异常等)。目前对胰岛素受体后途经研究较多,在外周靶组织,胰岛素与其受体结合后,激活β亚单位上的酪氨酸,使其磷酸化,导致胞质内酪氨酸激酶活性升高,胰岛素受体底物(IRS-1)的酪氨酸磷酸化,SH2的特定氨基酸的序列与IRS-1上磷酸化酪氨酸结合,磷脂酰激醇3激酶(PI-3K)被激活,使GLUT4向细胞膜转位,细胞摄取葡萄糖,出现胰岛素效应称为胰岛素信号转导的PI-3K途径。PKC是细胞内信号传导通路上的一个重要成员,催化胰岛素受体(IR)和胰岛素受体底物-1(IRS-1)蛋白的丝氨酸/苏氨酸的磷酸化反应,使磷酸肌醇-3激酶(PI-3K)的活性降低,减弱细胞内信号传导。同时,PKC是氧化应激的介导物,能活化细胞NAD(P)H氧化酶,诱导ROS的合成以及随后的脂质过氧化,同时使还原型谷胱甘肽酶(GSH)转化为氧化型谷胱甘肽酶(GSSG),多种抗氧化酶活性降低:ROS又作为类似于第二信使的信号分子,激活许多氧化还原敏感性信号通路,这些通路包括核因子κB(NFκB)、p38丝裂原活化蛋白激酶(p38MAPK)、JNK、己糖胺等。这些通路激活后,使氧化还原敏感性丝氨酸/苏氨酸激酶信号级联活化,抑制了胰岛素刺激的酪氨酸磷酸化。结果,胰岛素信号传导通路下游信号分子如PI-3K、AKt等的相关性和(或)活性降低,GluT4的表达和转运下降,减少了胰岛素的生物效应,此为FFA通过PKC间接诱导氧化应激导致的胰岛素抵抗。另外,FFA水平的升高可直接使ROS增多,通过氧化应激降低β细胞中特异表达的转录因子—胰腺和十二指肠的同源形基因因子-1(PDX-1)的表达,从而抑制胰岛β细胞胰岛素的基因转录。虽然许多资料发现输注脂肪乳后的3～4小时,行正血糖高胰岛素钳夹试验证实肝糖生成增加,但目前FFA升高,尤其是FFA慢性升高引起IR的机制尚未完全阐明。我们给Wistar大鼠延长静脉输注脂肪乳至48小时,模拟糖尿病患者脂代谢紊乱内环境,观察慢性FFA升高造成的脂毒性对胰岛素抵抗的影响,探明PKC-δ在其中的作用,以及其对胰岛素信号通路上的GluT-4和胰岛β细胞合成胰岛素的影响。实验方法一、动物模型的制备1、正常雄性Wistar大鼠重250～300克,给予常规饲料喂养并自由饮水,日夜周期为12小时,经过3～5天的环境适应。2、大鼠颈部动、静脉置管术:术前禁食12小时,10%水合氯醛(0.35ml/kg)腹腔麻醉后,进行颈部动静脉插管:导管分别插入右侧颈静脉以输注液体,左侧颈动脉以抽取动脉血留取样本。静脉导管置于右心房水平,动脉导管置于主动脉水平。两侧的导管均经皮下延续至背部外置,导管内充以60%PVP和肝素(1000~U/ml)的混合液保持导管通畅,防止血液返流,末端用金属钉封闭。所有大鼠术后均恢复3天,每天观察大鼠状况及测体重。二、实验分组:各亚组8只Wistar鼠(一)、盐水组(SAL)1、盐水基础亚组:静脉输注生理盐水0.3ml/h48小时。2、盐水钳夹亚组:静脉输注生理盐水0.3 ml/h48小时,输注的最后2小时行高胰岛素正血糖钳夹试验(胰岛素输注率为5 mU.kg-1.min-1)。(二)、脂肪乳组(IH)1、脂肪乳基础亚组:48小时静脉输注脂肪乳(20%脂肪乳+20 U/ml肝素),0.3 ml/h。2、脂肪乳钳夹亚组:48小时静脉输注脂肪乳,0.3 ml/h,最后2小时行高胰岛素正血糖钳夹试验,胰岛素输注率为5 mU.kg-1.min-1。每亚组实验结束,大鼠行腹腔麻醉,45秒内取肝脏,胰腺,骨骼肌,脂肪等组织,部分4%多聚甲醛固定、包埋,部分液氮冷冻后-70℃冰箱保存。三、检测指标及实验方法1、大鼠行48小时持续静脉输液(国内目前尚没有输液48小时的报道,最长7小时),钳夹亚组行高胰岛素-正血糖钳夹试验(评价胰岛素抵抗金标准)。2、葡萄糖氧化酶法检测血糖;放射免疫方法检测血浆胰岛素;比色法检测血浆游离脂肪酸水平。3、2,4-二硝基苯肼法检测肝脏羰基蛋白含量。4、Western Blot法检测肝脏PKC-δ以及GluT-4在细胞浆和细胞膜的蛋白表达。5、免疫组化方法比较两组胰腺组织胰岛素的含量,RT-PCR方法观察胰腺PDX-1mRNA表达的情况。四、统计分析应用SPSS10.0软件进行统计学分析。各组数据用(?)±s表示。两组数据间用t-检验,P＜0.05认为有统计学意义。实验结果1、48小时输注脂肪乳与盐水组比较FFA升高2.1倍,空腹血糖升高了1.26mmol/L,葡萄糖输注率降低了58.6%(P＜0.001)。2、脂肪乳组肝脏氧化损伤的指示物羰基蛋白水平较盐水组高3.2倍(P＜0.001)。3、盐水组,脂肪乳组肝脏细胞膜PKC-δ密度值分别为55.12±2.37,99.48±4.52;细胞浆PKC-δ密度值分别为49.37±1.96,21.64±0.83;盐水组与脂肪乳组肝脏胞膜PKC-δ/胞浆PKC-δ比值分别为1.12,4.60(P＜0.001)。4、盐水组、脂肪乳组肝脏细胞膜GluT-4密度值分别为37.69±1.20,16.28±0.55;细胞浆GluT-4密度值分别为20.34±1.03,41.92±1.74;盐水组与脂肪乳组肝脏胞膜GluT-4/胞浆GluT-4比值分别为1.85,0.39(P＜0.001)。脂肪乳组肝细胞膜GluT-4/肝细胞浆GluT-4的比值是对照组的21%(P＜0.001)。5、脂肪乳组胰腺组织中胰岛素的表达明显减少,为盐水组的55.60%;脂肪乳组、盐水组胰腺PDX-1mRNA的相对表达量分别为0.78±0.04,1.10±0.07,脂肪乳组为盐水组的71%(P＜0.05)。结论1、持续静脉48小时输注脂肪乳造成了空腹FFA及静脉血糖明显升高,高胰岛素正血糖钳夹实验后三十分钟两组平均血糖无统计学差异,但脂肪乳组葡萄糖输注率明显下降,证明高胰岛素正血糖钳夹试验成功;48小时输注脂肪乳造成了大鼠胰岛素抵抗。2、慢性脂毒性引起肝脏羰基蛋白含量升高,证实氧化应激在脂毒性造成的IR中起到了重要作用。3、脂肪乳组肝细胞膜PKC-δ的表达是盐水组的1.8倍,而肝脏细胞浆PKC-δ的表达为盐水组的24%,说明氧化应激可能通过诱导肝脏PKC-δ从细胞浆到细胞膜的转位引起IR,48小时输注脂肪乳引起的IR与PKC-δ的激活转位有关。4、脂肪乳组肝细胞膜GluT-4表达减少,因此减少了胰岛素的生物效应,导致葡萄糖利用下降。5、持续静脉48小时输注脂肪乳可以造成胰腺PDX-1mRNA及胰岛素表达下降,从而影响了胰岛β细胞胰岛素基因合成及表达。更多还原

【Abstract】 Obesity is associated with insulin resistance mainly due to the release of free fatty acids(FFA) from the expanded adipose tissue mass and cytokines.Numerous studies have shown that FFA,which are often elevated in obese individuals have been implicated as an important causative link in the association between obesity,insulin resistance and type 2 diabetes mellitus.An elevation of plasma FFA has been shown to impair insulin action,and to be a risk factor for the development of type 2 diabetes. Contrary to muscle insulin action and glucose metabolism,the effects of FFA on hepatic insulin action and glucose metabolism have not been extensively investigated.Glucose is produced through the pathway of glycogenolysis and gluconeogenesis. The main substrates for gluconeogenesis are free fatty acid(FFAs),glycerol,alanine and lactate,and liver glycogen is the substrate for glycogenolysis.Endogenous plasma FFAs are elevated and are correlated with the rates of basal hepatic glucose production(HGP) in type 2 diabetes.A large number of studies have demonstrated that infusion of intralipid + heparin(IH) increases endogenous glucose production (EGP) during euglycemic clamps.The increase of HGP could be partly attributed to a breakdown of hepatic autoregulation.In addition,FFA may decrease the ability of insulin to suppress HGP(i.e.hepatic insulin resistance) by impairing hepatic insulin action(signaling).This speculation is supported by recent studies in which high fat feeding decreased hepatic IRS-2 associated PI3-Kinase activity.The mechanism of elevation of HGP in type 2 diabetes is still unclear.Previous studies have demonstrated that insulin acts directly at the liver to suppress HGP by acutely inhibiting glycogenolysis(i.e.inhibition breakdown of glycogen.Insulin also indirectly suppresses HGP via its peripheral actions,by reducing the level of gluconeogenic precursors and FFA.Insulin binds to its receptor at the surface of the hepatocytes to initiate its action through signaling molecules. Upon insulin binding to its receptor,insulin receptor tyrosine kinase activity is activated,which results in receptor autophosphorylation.The activated insulin receptor tyrosine kinase also phosphorylates insulin receptor substrates,which include insulin receptor substrate(IRS) 1-4.IRS-2 has been shown to play an important role in hepatic insulin action.IRS-2 knockout mice have impaired hepatic insulin signaling (i.e.impaired PI3-Kinase activity,impaired insulin suppression of HGP),Enhanced insulin-stimulated PI3-kinase activity associated with IRS-1 and IRS-2 was observed in liver from rats fed a high-saturated-fat diet(58%lard,wt/wt) for 2 wk.Oxidative stress is present in type 2 diabetes patients,and can directly induce insulin resistance in vivo.Purpose1.TO determine whether the prolonged elevation of FFA-induced IR is associated with evidence of increased FFA oxidation and/or PKC activation and or decreased GluT-42.Determine whether the prolonged elevation of FFA affect insulin gene expression.Materials and Methods1.Animal modelsNormal Male Wistar rats,weighing 250-300g,were used for experiments.2.Surgical proceduresAfter 3-5 days of adaptation to the facility,rats were anesthetized and indwelling catheters were inserted into the right internal jugular vein for infusions and the left carotid artery for sampling.The venous catheter was extended to the level of the right atrium,and the arterial catheter was advanced to the level of the aortic arch.Both catheters were tunneled subcutaneously and exteriorized.The catheters were filled with a mixture of 60%polyvinylpyrrolidone and heparin(1,000 U/ml) to maintain patency and were closed at the end with a metal pin.The rats were allowed a minimum 3-4 days period of post-surgery recovery before experiments.3.Experimental designThe rats were fasted overnight and randomized to two groups,each of which received Intralipid plus Heparin(IH) infusion(20%Intralipid + 20U/ml heparin,5μl/min) and saline(SAL) was infused respectively with equivolume.The duration of different solution was 48h,and experimental determinations were made in the basal fasting state and during hyperinsulinemic euglycernic clamp.For the basal protocol,the different solution was infused throughout the 48-hour experiments.The Clamp protocols were similar to the Basal protocol with the additional infusion of insulin(5 mU.kg-1.min-1) during the last two hours of the experiments,resulting in plasma insulin levels in the postprandial range.To maintain euglycemia during insulin infusion,a variable infusion of 20%glucose was given intravenously through the jugular catheter and adjusted according to frequent glycemic determination(every 5-10 min).The glucose infusion was adjusted to sustain the normal glucose level.Blood samples for glucose,insulin,FFA were taken during the last 30 min(every 10 min) of each experiment.At the end of the experiments the rats were anesthetized with intraarterial pentobarbital,and the liver was freeze-clamped with pre-cooled aluminum tongs within 45 s of anesthetic injection while infusions were maintained through the jugular vein.4.Laboratory methods(1) Plasma glucose was measured with a Biosen Glucose Analyzer 5030(2) Insulin level was decided by radio-immune method(3) PKC-δtranslocation:Liver samples(250mg) were homogenized by hand-held glass homogenizer in buffer A,the homogenates were centrifuged at 100,000 x g for 1 h at 4oC,and the supernatants were retained as the cytosolic fraction.The pellet was resuspended in buffer B,homogenized by passing through a 23-gauge needle three times,incubated for 15 min on ice,and centrifuged at 100,000 x g for 1 h at 4oC.The supernatant provided the solubilized membrane fraction.The protein concentration in all samples was determined by the detergent-compatible modified Lowry microassay (BioRad),using serum albumin as the standard.Fiftyμg of protein in all samples were mixed with equal volumes of 3 x sample-loading buffer(6.86 M urea,4.29% sodium dodecyl sulphate(SDS),300 mM dithiothreitol,43 mM Tris-HCl(pH 6.8) and left at room temperature for 30 min.The mixture was then vortexed and subjected to SDS-PAGE(10%polyacrylamide).Following electrophoretic separation,proteins were transferred to Immobilon-P membranes.The membranes were then incubated for lh at 4oC in Tris-buffered saline-Tween(TBST) containing 5%non-fat dried milk, pH 7.4.After the blocking step,membranes were washed in rinsing solution(TBST, pH 7.4) and then incubated overnight at a concentration of 1:1000 with an affinity-purified polyclonal antibody specific for PKC-δ.The translocation of the DAG-sensitive isoform of PKC-δ,from cystosol to membrane reflects its activation, and was assessed by comparing immunoblots of the cytosolic and membrane-associated fractions.(4)Carbonyl protein:Liver tissue was homogenized at 4℃in a solution containing HEPES,cell debris was removed by centrifugation.Protein concentrations were determined using a standardized assay kiat with bovine serum albumine solution as the standard.Oxidative protein damage,assessed by the formation of carbonyl groups,was measured as described by Levine et al.1 mg of protein was precipitated by addition supernatant discarded.Protein pellets were incubated with and without 2, 4-dinitrophenylhydrazine(DNPH,10 mM in 2 HC1) and were allowed to stand at room temperature for 1 hour,vortexing every 15 minutes.Following incubation, protein was re-precipitated using 20%TCA and the pellet was obtained by centrifugation(8500g) for 3 minutes.The pellets were washed 3 times with ethanol-ethyl acetate(1:1) to remove free DNPH,allowing the samples to stand for 10 minutes each time before discarding the supernatant.The pellet was then re-dissolved in guanidine solution for 1 hour at 37℃.Insoluble material was removed by centrifugation(8500g) for 3 minutes.Carbonyl content was calculated from the sample absorbance at 365 nm compared to their complementary HCl-treated blanks, using a molar absorption coefficient of 22000M-1cm-1.Insulin expression was tested by immunohistochemistry method:1:500 on rat pancreas with overnight incubation and Cy labeled secondary antibody.Suggested tissue fixative is 10%formalin or 4% paraformaldehyde.It is best to cut unfixed frozen tissue and then fix the sections for 10 minutes in 4%paraformaldehyde in PBS followed by 4 x 5 minute rinses in PBS. Suggested permeabilization is 10 minutes in ice cold MeOH.Blocking buffer should contain serum from the same host as the secondary antibody.Optimal working dilutions must be determined by the end user.Result1.After 48h infusion of Intralipid + Heparin,plasma FFA level increased 2.1 fold of SAL,Plasma glucose levels higher with IH vs.SAL infusion in the basal experiments but were maintained at similar levels during the hyperinsulinemic clamps, and GIR of IH decreased 58.6%vs SAL.(P＜0.001).2.To investigate the mechanisms of FFA-induced impairment in hepatic glucose metabolism,we measured carbonyl protein content(to indirectly assess FFA oxidation),IH increased carbonyl protein by 3.2 fold of SAL(P＜0.001).3.IH induced hepatic PKC-δtranslocation from the cytosolic to the membrane fraction in basal study(Membrane/Cytosolic ratio:1.12+0.12 in SAL,4.60+0.36 in IH, P＜0.001).hepatic insulin resistance was associated with oxidative stress along with activation of PKC-δand subsequently with impairment of insulin signaling pathway.4.IH decreased hepatic GluT-4 translocation from the cytosolic to the membrane fraction in basal study,IH decreased GluT-4 translocation by 4.74 fold of SAL. (P＜0.001).5.48h infusion of Intralipid + Heparin affected insulin gene expression,in IH insulin(+) cell decreased 55.60%of SAL,and the level of PDX-1 are 0.78±0.04, 1.10±0.07 respectively in IH and SAL(P＜0.05).Conclusion1.Glucose and FFA levels were both elevated by IH,and IR was significant in IH.2.Oxidative stress has very important function on IR lead by the elevation of FFA.3.PKC-δtranslocation was observed in IH,it indicates that PKC-δplays a key role in insulin resistance induced by prolonged infusion of IH.4.The expression of GluT-4 on membrane decreased by Western Blot in IH,it leads to decreased effects of insulin.5.The level of PDX-1mRNA and insulin measured by RT-PCR and immune histochemistry respectively decreased in IH,insulin gene expression was affected by prolonged infusion of IH.更多还原