磷酸二酯酶抑制剂诱导的大鼠胰岛素抵抗对糖脂代谢的影响

目的:探讨选择性磷酸二酯酶III(PDE₃)抑制剂米力农(milrinone)对大鼠胰岛素分泌、血糖、血浆游离脂肪酸(FFA)的影响和剂量依赖关系,及其在胰岛素钳夹状态下对大鼠糖代谢和胰岛素敏感性的影响。方法:由植入导管给予大鼠不同剂量的米力农(1、5、25μmol/kg)在不同时相测定血糖、血浆FFA和胰岛素水平并与对照组比较。在清醒状态下建立大鼠高胰岛素-正常血糖钳夹技术,并在钳夹120min时分别经导管给予米力农(25μmol/kg)和25%二甲基亚砜(DMSO,对照组)。采用气相色谱-质谱仪(GC-MS)测定糖代谢率。结果:3个不同剂量米力农组血浆FFA浓度明显高于对照组和给药前,在注射后2min,各组FFA升高的百分数为:50%、52%、55%(1、5、25μmol/kg)。在5、25μmol/kg组血浆胰岛素水平也明显高于对照组和给药前,仅25μmol/kg组血糖浓度高于对照组和给药前。在胰岛素钳夹研究中,米力农处理组大鼠血浆FFA明显高于给药前,肝糖输出(HGP)也明显高于给药前。葡萄糖输注率(GIR)明显低于对照组和给药前。结论:米力农损害了胰岛素抑制脂解和肝糖输出的能力及胰岛素介导的外周组织糖的利用。因此,米力农处理可能在体内诱导了一个急性胰岛素抵抗。     

脂质灌注对大鼠血浆抵抗素和ghrelin的影响*

目的：探讨脂质灌注对大鼠血浆抵抗素和ghrelin的影响。 方法： 采用正糖钳夹技术，在钳夹前后分别测定生理盐水对照组和脂质灌注组血浆抵抗素和ghrelin浓度，并用［3H］-葡萄糖作为示踪剂测定外周组织和肝糖的代谢。 结果： 脂质灌注组大鼠血浆游离脂肪酸（FFA）明显增加（P<0.01），葡萄糖输注率（GIR）明显降低（P<0.01）。对照组肝糖产率（HGP）明显被抑制（88%）。脂质输注组胰岛素对HGP的抑制作用明显减弱。在钳夹期间，脂质组与对照组比葡萄糖清除率（GRd）轻度降低。在正糖钳夹术结束时，对照组血浆ghrelin水平与钳夹前相比明显降低(P<0.05)。4 h的脂质灌注也引起了血浆ghrelin浓度的明显下降(P<0.05)，但是在钳夹结束时和对照组比没有明显差异。相关性分析表明空腹血浆ghrelin水平与空腹胰岛素和血糖呈明显负相关（r=-0.52和r=-0.61, P<0.05）。脂质灌注后大鼠血浆抵抗素水平较灌注前和对照组明显升高（P<0.01），空腹血浆抵抗素浓度与空腹FFA（r=0.68, P<0.01）、血糖（r=0.66, P<0.01）呈明显正相关。 结论： 脂质灌注诱导了肝脏和外周的胰岛素抵抗，抵抗素在胰岛素抵抗的形成中可能具有重要作用。高胰岛素血症，而不是游离脂肪酸，降低了大鼠循环ghrelin水平。     

吡格列酮对脂质灌注大鼠糖代谢和PPAR-γ表达的影响

目的：探讨吡格列酮 (Pio) 对游离脂肪酸诱导的胰岛素抵抗大鼠糖代谢和PPAR-γ表达的影响。方法:采用扩展正糖钳夹实验和［3-3H］标记葡萄糖示踪技术，观察了4 h脂质灌注导致大鼠血浆游离脂肪酸（FFA）升高引起糖代谢和脂肪组织PPAR-γ表达变化及Pio处理后的影响。 结果:在钳夹稳态期，对照组（N组）血浆FFA水平明显降低，而脂质灌注组（L组）和吡格列酮+脂质组（P/L组）FFA水平明显升高。 P/L组葡萄糖输注率(GIR)较N组明显降低（P<0.01）, 而L组又明显低于P/L组（P<0.01）；N组和P/L组肝糖输出 (HGP) 与基础值相比被明显抑制达85%（均P<0.01），在L组，胰岛素对HGP的抑制作用受到明显障碍（仅抑制8.7%）。L组和P/L组葡萄糖清除率（GRd）明显低于N组（P<0.01）。P/L组脂肪组织PPAR-γ表达明显增加。 结论:脂质灌注诱导了大鼠胰岛素抵抗。吡格列酮干预使大鼠脂肪组织PPAR-γ表达明显增加，并抑制了内源性肝糖产生，从而部分逆转了脂质诱导的胰岛素抵抗。     

N-乙酰半胱氨酸改善高游离脂肪酸所致大鼠外周胰岛素抵抗

目的 探讨N-乙酰半胱氨酸(NAC)对高游离脂肪酸(FFA)导致的外周胰岛素抵抗的影响及机制.方法 SD大鼠随机分为对照组(NS组,12只)、脂肪乳输注组(FFA组,13只)和脂肪乳 NAC组(NAC组,12只).输注48 h,(1)测血浆硝基酪氨酸,丙二醛(MDA)和还原型谷胱甘肽(GSH)水平;(2)高胰岛素正糖钳夹试验,评价外周胰岛素抵抗程度;(3)实时荧光定量PCR方法测定肌肉组织胰岛素受体底物-1(IRS-1)、胰岛素受体底物-2(IRS-2)mRNA表达.结果 (1)FFA组硝基酪氨酸,MDA高于NS组,GSH低于NS组,NAC分别改善28.6%,33.1%,22.9%(P＜0.05);(2)FFA组葡萄糖输注率(GIR)比NS组降低(P＜0.05),用NAC后GIR升高36.6%(P＜0.05);(3)FFA组肌肉组织IRS-1、IRS-2 mRNA表达比NS组降低87.7%、50.7%(P＜0.05);NAC组肌肉组织IRS-1、IRS-2表达比FFA组增加370.1%、46.2%,(P＜0.05).结论 NAC干预能改善高FFA所致的外周胰岛素信号传导障碍,逆转外周胰岛素抵抗,可能与NAC纠正机体氧化及抗氧化失衡有关.     

高饱和脂肪酸、高不饱和脂肪酸、高糖不同饮食诱导高血压伴胰岛素抵抗大鼠的研究

目的:对高饱和脂肪酸、高不饱和脂肪酸和高糖不同饮食诱导高血压伴胰岛素抵抗大鼠及相关因素的研究。方法:用高饱和脂肪酸、高不饱和脂肪酸和高糖饮食喂养成年Wistar大鼠24周,连续监测体重（BW）、血糖（FBG）、胰岛素（FBI）、总胆固醇（TC）、甘油三酯（TG）和血清游离脂肪酸（FFA）及血清一氧化氮代谢产物NO-2/NO-3 的水平 ；用光电鼠尾血压测定仪测定鼠尾平均收缩压（SBP）；用正常葡萄糖高胰岛素钳夹技术的葡萄糖输注率（GIR）评价胰岛素抵抗（IR）；用Myography微血管测定技术观察肾动脉对乙酰胆碱（Ach）舒张反应（EDV）。结果:各实验组经喂养24周TG、TC、FFA、FBG、INS升高，NO-2/NO-3、 GIR减低；各实验组SBP明显高于对照组；各实验组大鼠离体肾动脉环对Ach内皮依赖性舒张反应（EDV）显著弱于对照组；采用相关和多元线性回归方法（逐步回归法）分析BW、TG、TC、FFA、FBG、FBI、NO-2/NO-3、GIR、对SBP的影响，结果SBP与TG、TC、FFA、FBG、FBI呈明显正相关，与NO-2/NO-3、 GIR、EDV呈明显负相关，经多元回归分析剔除TC、TG、FBI、NO-2/NO-3，SBP只与FBG、FFA呈明显正相关、与GIR、EDV呈明显负相关。结论:高饱和脂肪酸饮食、高不饱和脂肪酸饮食和高糖饮食均可诱导伴胰岛素抵抗的高血压大鼠模型。     

高糖、高脂饮食诱导大鼠胰岛素抵抗和血管舒张功能减弱

目的观察高糖、高饱和及高不饱和脂肪酸饮食对大鼠胰岛素抵抗(IR)和血管内皮依赖性舒张功能的影响。方法成年Wistar大鼠随机分为对照组(NC)、高糖组(HS)、高饱和脂肪酸组(HSF)和高不饱和脂肪酸组(HUF),喂养24周后,用正葡萄糖高胰岛素钳夹技术的葡萄糖输注率(GIR)评价IR;用微血管测定技术观察肾动脉累积舒张反应。结果各实验组GIR减低,以HSF组最低,TG、FFA与GIR明显负相关;各实验组大鼠离体肾动脉环对乙酰胆碱(Ach)舒张反应减弱,HSF、HUF和HS组最大舒张反应(Rmax)分别较对照组下降37.4%、32.7%和27.7%;各实验组离体肾动脉环经左旋精氨酸(L-Arg)孵育后对Ach舒张反应增强,经L-NNA、MB孵育后舒张反应减弱;大鼠离体肾动脉环对Ach内皮依赖性舒张反应与TGI、NS明显负相关,与NO、GIR明显正相关,FFA与NO明显负相关。结论高糖、高饱和脂肪酸和高不饱和脂肪酸饮食均可诱导大鼠IR并伴有血管内皮舒张功能减弱。     

GSK-3及P70S6K在胰岛素抵抗大鼠肌肉中的表达及意义

目的:观察饮食诱导的胰岛素抵抗(IR)大鼠骨骼肌中糖原合成酶-3(GSK-3)及核糖体S6蛋白激酶(P70S6K)的表达及变化情况,并探讨GSK-3和P70S6K在IR发生中的作用及意义.方法:将40只4周龄Wistar大鼠随机分为正常对照组、高糖组、高脂组、高脂高糖饲养组,喂养8周后用高胰岛素-正葡萄糖钳夹技术(钳夹试验)对大鼠进行胰岛素敏感性的评估,采用Western blot法检测各组大鼠骨骼肌中GSK-3及P70S6K的含量,同时测定各组大鼠的附睾脂肪垫重量、血糖(BG)、胰岛素(INS)、甘油三酯(TG)、胆固醇(TC)及游离脂肪酸(FFA)水平、超敏C反应蛋白(hsCRP).结果:高脂高糖组、高脂组大鼠产生明显IR,体重增加(P＜0.01),以附睾脂肪垫的重量增加更为显著(P＜0.01),TG、TC及FFA水平、hsCRP增加(P＜0.01),GSK-3、P70S6K在IR大鼠肌肉中的表达明显升高.结论:高脂高糖饮食可诱导大鼠产生IR,IR大鼠的hsCRP、TG、TC及FFA水平明显增高,大鼠产生IR与炎症反应有关,GSK-3、P70S6K在IR大鼠中的表达明显升高,在胰岛素信号传导中起负相调节作用.     

ADRP基因在2型糖尿病大鼠脂肪组织中的表达及意义

目的观察实验性2型糖尿病（2-DM）大鼠脂肪组织中脂肪分化相关蛋白（ADRP）基因mRNA表达的情况,探讨ADRP在2-DM发病机制中的作用。方法20只Wistar大鼠随机选取10只为正常对照组,另外10只大鼠高糖高脂饮食伴尾静脉注射小剂量链脲佐菌素（STZ）25mg/kg复制2-DM大鼠模型,6周后空腹取材,放免法测定血清胰岛素（Ins）并计算胰岛素敏感指数（ISI）,铜显色法测定游离脂肪酸（FFA）,RT-PCR法检测脂肪组织ADRP基因mRNA表达水平。结果2-DM大鼠脂肪组织中ADRP基因mRNA表达明显增高;空腹血糖、Ins、FFA水平升高,ISI降低。结论ADRP基因mRNA的表达在2-DM胰岛素抵抗和β细胞功能减退中起重要作用。     

营养性肥胖大鼠血清Orexin A与游离脂肪酸的相关性分析

探讨营养性肥胖胰岛素抵抗大鼠血清增食欲素A(OrexinA)与脂质代谢的相关性。方法构建营养性肥胖型胰岛素抵抗Wistar大鼠模型,采用放射免疫法检测血清OrexinA、血清胰岛素及C肽含量,生化酶法测定大鼠血清甘油三酯(TG)和总胆固醇(TC),生化比色法测定血清游离脂肪酸(FFA),分析血清OrexinA的水平与血清FFA以及胰岛素水平的相关性。结果喂养8w时,高脂饲料喂养的大鼠体重、Lee's指数、血糖、TG及TC值较普通饲料喂养的对照组明显增加(P<0.05);营养性肥胖组大鼠血清FFA、血清OrexinA、胰岛素和C肽含量分别比对照组升高64%、57%、52%和50%(P<0.05)。血清OrexinA的水平与血清FFA、胰岛素、C肽呈显著正相关,相关系数r分别为+0.756(P<0.01)、+0.587(P<0.01)和+0.685(P<0.01)。结论营养性肥胖大鼠血清OrexinA的异常升高,与FFA呈正相关,可能参与胰岛素抵抗和肥胖的发生。     

原发性高血压患者糖负荷后血糖和胰岛素变化对游离脂肪酸水平的影响

目的:探讨糖负荷后血糖和胰岛素变化对血清游离脂肪酸(FFA)水平的影响。方法: 234例高血压病患者[2型糖尿病(DM)20例,糖耐量低减(IGT)74例,正常糖耐量(NGT)140例;男98例,女136例]做口服葡萄糖耐量试验(OGTT),测定0、30、60、120 min时相的葡萄糖、血清胰岛素和FFA水平。结果: 空腹血清FFA浓度(μmol/L):DM组(1 048.7±481.6)显著高于IGT组(706.1±332.1)(P＜0.05)和NGT组(725.8±353.9)(P＜0.05)。DM组OGTT血糖水平显著升高,胰岛素释放曲线呈反应低平,高峰不明显或呈延迟相。3组FFA释放均呈低下,DM组更为显著。30、60、120 min时相的血清FFA水平,3组均无显著差异。结论: 糖尿病患者空腹血清FFA水平升高,OGTT中糖尿病患者胰岛素分泌的绝对不足未能增大DM组FFA水平与IGT及NGT组的差异,相反缩小了与IGT和NGT组的差距,提示体内葡萄糖利用水平对血清FFA浓度可能有重要的影响。     

In vivo insulin sensitivity in the rat determined by euglycemic clamp

Our aim was to develop the glucose clamp (GC) technique in the conscious rat for assessment of in vivo insulin sensitivity. A 2-h euglycemic GC could be performed in chronically cannulated rats using 625 microliter blood. Overnight-fasted rats were infused with porcine insulin (1.67 mU . kg-1 . h-1). Insulin levels of 41 +/- 2 (SE) mU/liter were produced in rats aged 91 +/- 4 days with a 60- to 120-min glucose infusion rate (GIR60-120) of 10.6 +/- 0.6 mg . kg-1 . min-1 (n = 9) during euglycemia. GIR60-120 was significantly (P less than 0.025) reduced in rats aged greater than 130 days (mean, 169 +/- 16 days) to 7.7 +/- 1.2 mg . kg-1 . min-1 (n = 7). Metabolic clearance rate of porcine insulin (46 +/- 3 ml . kg-1 . min-1) and GIR60-120 compared with plateau plasma insulin levels are higher than values reported in humans. The latter may be due to suppression of a higher basal hepatic glucose production or increased potency of porcine compared with native insulin. We conclude that the GC can be accomplished in the rat. When combined with tracer administration and subsequent killing, it should provide a quantitative in vivo measurement of insulin sensitivity in individual tissues.     

Glucose clamp technique: a method for quantifying insulin secretion and resistance.

Methods for the quantification of beta-cell sensitivity to glucose (hyperglycemic clamp technique) and of tissue sensitivity to insulin (euglycemic insulin clamp technique) are described. Hyperglycemic clamp technique. The plasma glucose concentration is acutely raised to 125 mg/dl above basal levels by a priming infusion of glucose. The desired hyperglycemic plateau is subsequently maintained by adjustment of a variable glucose infusion, based on the negative feedback principle. Because the plasma glucose concentration is held constant, the glucose infusion rate is an index of glucose metabolism. Under these conditions of constant hyperglycemia, the plasma insulin response is biphasic with an early burst of insulin release during the first 6 min followed by a gradually progressive increase in plasma insulin concentration. Euglycemic insulin clamp technique. The plasma insulin concentration is acutely raised and maintained at approximately 100 muU/ml by a prime-continuous infusion of insulin. The plasma glucose concentration is held constant at basal levels by a variable glucose infusion using the negative feedback principle. Under these steady-state conditions of euglycemia, the glucose infusion rate equals glucose uptake by all the tissues in the body and is therefore a measure of tissue sensitivity to exogenous insulin.     

Effects of phlorizin and acipimox on insulin resistance in STZ-diabetic rats.

To evaluate the roles of hyperglycemia and increased plasma FFA level in the development of insulin resistance, we examined the effects of phlorizin and acipimox treatments on tissue sensitivity to insulin in streptozotocin(STZ)-diabetic rats. Insulin sensitivity was assessed with the glucose-insulin clamp technique. Blood glucose concentration was clamped at basal levels of control and diabetic states, and plasma insulin concentrations were clamped at the levels of basal, approximately 60 and approximately 1500 microU/ml. In diabetic rats, the basal blood glucose and plasma FFA levels in the fasting state were elevated, while the plasma insulin concentration was lower than in normal controls. Moreover, diabetic rats became glucose intolerant after intravenous injection of glucose. The metabolic clearance rate(MCR) of glucose showed a decrease of basal and insulin stimulated response in diabetic rats. As results of the glucose-insulin clamp study and intravenous glucose tolerance test, insulin resistance was developed in STZ-diabetic rats. Phlorizin treatment of diabetic rats recovered insulin sensitivity to nearly normal levels and improved glucose tolerance, but had no effect on insulin action in controls. Insulin sensitivity was also improved by acipimox treatment in diabetic rats, but did not reach normal levels. These results show that hyperglycemia is an obvious causative factor of insulin resistance, and increased FFA level may also act on the development of insulin resistance in STZ-diabetic rats.     

Glucose intolerance following chronic metabolic acidosis in man.

The effect of chronic metabolic acidosis (0.1 g/(kg . day) X 3 days) on carbohydrate metabolism was examined with the glucose-clamp technique in 16 healthy volunteers. Hyperglycemic clamp. Plasma glucose concentration is acutely raised and maintained 125 mg/dl above the basal level. Because the glucose concentration is held constant, the glucose infusion rate is an index of glucose metabolism (M). Following NH4Cl, M decreased from 8.95 +/- 1.12 to 7.35 +/- 0.76 (P less than 0.05) despite an increased plasma insulin concentration (I) 23 +/- 9%, P less than 0.05). Consequently the M/I ratio, an index of tissue sensitivity to insulin, decreased by 32 +/- 5% (P less than 0.005). Euglycemic clamp. Plasma insulin concentration is acutely raised and maintained 101 +/- 3 microU/ml above basal and plasma glucose is held constant at the fasting level by a variable glucose infusion (M). Following NH4Cl both M and M/I decreased by 15 +/- 4% (P = 0.005) and 15 +/- 5% (P = 0.01), respectively. Metabolic acidosis had no effect on basal [3-3H]glucose production or the percent of decline (91 +/- 4%) following hyperinsulinemia. Both hyperglycemic and euglycemic clamp studies indicate that impaired glucose metabolism following metabolic acidosis results from impaired tissue sensitivity to insulin.     

Marked suppression of ghrelin concentration by insulin in Prader-willi syndrome

The plasma ghrelin has been reported to be elevated in Prader-Willi syndrome (PWS) and modulated by insulin. It was hypothesized that insulin might have a more pronounced effect on reducing plasma ghrelin in PWS patients, which would influence appetite. This study investigated the degree of ghrelin suppression using an euglycemic hyperinsulinemic clamp in children with PWS (n=6) and normal children (n=6). After a 90-min infusion of insulin, the plasma ghrelin level decreased from a basal value of 0.86+/-0.15 to 0.58+/-0.12 ng/mL in the controls, and from 2.38+/-0.76 to 1.12+/-0.29 ng/mL in children with PWS (p=0.011). The area under the curve below the baseline level over the 90 min insulin infusion was larger in children with PWS than in controls (-92.82+/-44.4 vs. -10.41+/-2.87 ng/mL/90 min) (p=0.011). The insulin sensitivity measured as the glucose infusion rate at steady state was similar in the two groups (p=0.088). The decrease in the ghrelin levels in response to insulin was more pronounced in the children with PWS than in the controls. However, the level of ghrelin was always higher in the children with PWS during the clamp study. This suggests that even though insulin sensitivity to ghrelin is well maintained, an increase in the baseline ghrelin levels is characteristic of PWS.     

长期高脂喂养大鼠胰岛功能改变与胰岛炎症反应有关

目的： 观察长期高脂喂养诱导的胰岛素抵抗大鼠胰岛炎症反应水平,并探讨其与胰岛功能改变的关联及机制。方法: 8周龄的Wistar大鼠随机分为正常对照组（NC,n=15）和高脂组（HF,n=15）,分别给予普通饲料及高脂饲料喂养。24周后行高胰岛素正葡萄糖钳夹试验检测胰岛素敏感性,行静脉葡萄糖耐量试验(IVGTT)检测β细胞功能,以免疫组化法检测胰岛β细胞内胰岛素相对含量（IRC）以及胰岛内NF-κB与caspase-3的相对浓度,以TUNEL法检测胰岛内细胞凋亡水平,以RT-PCR法检测胰岛内胰岛素原(INS)及白细胞介素（IL）-1β mRNA的相对表达量。结果: HF组葡萄糖输注率（GIR）较NC组显著降低［(5.32±0.90) mg·kg-1·min-1 vs (7.80±0.51) mg·kg-1·min-1,P<0.01］;NC组葡萄糖负荷后胰岛素分泌高峰出现于第5 min,而HF组延迟至第10 min,且10-60 min胰岛素曲线下面积（AUCI）较NC组增加了67.7％（P<0.01）。免疫组化显示,HF组IRC较NC组减少了25.6%,NF-κB、 caspase-3相对含量及单位面积胰岛凋亡细胞数分别增加20.5％、19.1%及2.43倍(均P<0.01)。HF组胰岛IL-1β mRNA相对表达量较NC组增加了1.95倍(P<0.01),INS mRNA 表达水平增加了12.0％（P<0.05）。结论: 长期高脂喂养诱导的胰岛素抵抗大鼠胰岛存在炎症反应,可能通过NF-κB及其介导的凋亡信号通路,与早期胰岛结构及功能损害有关。