低脂饮食和有氧运动干预对高脂大鼠脂肪组织蛋白激酶B表达的影响

目的 :观察低脂饮食和有氧运动对高脂大鼠脂肪组织中蛋白激酶B(PKB)表达的影响。方法 :选取雄性Wistar大鼠 4 0只 ,随机分为正常对照组 ( 10只 )和模型组 ( 30只 )。模型组大鼠给予高脂喂养 ,4周后 ,再随机分为 3组 :胰岛素抵抗组 ,继续高脂饮食 ;低脂饮食组 ,给予低脂饮食 ;有氧运动组 ,继续高脂饮食 +有氧运动。干预 6周后 ,蛋白印迹法检测大鼠脂肪组织中胰岛素刺激PKB表达的含量变化。结果 :低脂饮食组和有氧运动组大鼠空腹血糖 (FBG)、甘油三酯 (TG)、胆固醇 (TC)、内脏脂肪及胰岛素抵抗指数 (HOMA IR)下降 ,胰岛素敏感指数 (ISI)显著升高。而PKB蛋白表达分别增加了 16 .1%和 19.2 %(P <0 .0 1)。结论 :低脂饮食和有氧运动能改善胰岛素抵抗 ,可能与增加脂肪组织中PKB蛋白表达有关。     

多囊卵巢综合征骨骼肌胰岛素抵抗大鼠模型的建立

背景：研究表明胰岛素抵抗在多囊卵巢综合征的发生与发展过程中起重要作用,建立理想的多囊卵巢综合征骨骼肌胰岛素抵抗动物模型是研究该疾病的基础。 目的：探讨建立较为理想的多囊卵巢综合征骨骼肌胰岛素抵抗大鼠模型的方法。 方法：将八九周龄SD雌性大鼠随机分为模型组和对照组。模型组给予胰岛素联合人绒毛膜促性腺激素皮下注射,并以高脂饲料和50 g/L葡萄糖水喂养,对照组皮下注射生理盐水,常规饮食喂养。 结果与结论：造模6周后,模型组大鼠卵巢体积明显增大,且呈多囊性改变;血清睾酮、黄体生成素、空腹血糖和胰岛素水平高于对照组;骨骼肌组织中葡萄糖转运蛋白4表达明显低于对照组,且其葡萄糖转运蛋白4阳性颗粒靠近骨骼肌细胞膜边缘者较少。可见胰岛素联合人绒毛膜促性腺激素皮下注射,并饲以高脂饲料和50 g/L葡萄糖水是建立多囊卵巢综合征骨骼肌胰岛素抵抗大鼠模型较为理想的方法。     

宫内发育迟缓大鼠胰腺肝脏骨骼肌中胰岛素受体底物的表达

目的分析宫内发育迟缓（IUGR）大鼠胰腺、肝脏、骨骼肌中胰岛素受体底物1（IRS-1）和2（IRS-2）mRNA和蛋白水平的表达,探讨IUGR个体发生胰岛素抵抗的机制。方法采用母孕期低蛋白饮食法建立IUGR大鼠模型,以模型鼠产下的仔鼠为IUGR组;以孕期予正常饮食母鼠产下的仔鼠为对照组。应用RT-PCR法检测各组仔鼠在出生后0、3和8周时点胰腺、肝脏和骨骼肌中IRS-1、IRS-2 mRNA表达水平,采用Western blot检测IRS-1和IRS-2蛋白表达水平。检测3和8周时点仔鼠空腹血糖和血清胰岛素水平,计算胰岛素抵抗指数。结果①IUGR组出生后0和3周体重显著低于对照组;8周时点IUGR组体重显著高于对照组。②IUGR组出生后0、3和8周时点胰腺、肝脏IRS-2 mRNA和蛋白表达水平低于对照组;IRS-1 mRNA和蛋白表达水平与对照组差异无统计学意义;骨骼肌IRS-1 mRNA和蛋白表达水平均显著低于对照组;IRS-2蛋白表达水平与对照组差异无统计学意义。③至8周时点,骨骼肌IRS-1 mRNA和蛋白表达水平的下降幅度较胰腺和肝脏组织更为明显;肝脏IRS-2 mRNA和蛋白表达水平的下降幅度较胰腺和骨骼肌组织更为明显。④至8周时点,IUGR组空腹血糖水平与对照组差异无统计学意义,胰岛素水平和空腹胰岛素抵抗指数较对照组显著升高。结论 IUGR大鼠胰腺、肝脏和骨骼肌组织均存在IRS-1或IRS-2 mRNA和蛋白表达水平的下降,可能是发生胰岛素抵抗的机制之一。     

高脂饮食诱导肥胖模型大鼠骨骼肌组织蛋白酪氨酸磷酸酯酶1B和胰岛素受体底物2的表达

背景：外周组织的胰岛素抵抗是2型糖尿病的主要病因。 目的：观察高脂饮食诱导的肥胖大鼠骨骼肌中蛋白酪氨酸磷酸酯酶1B和胰岛素受体底物2的表达。 方法：将20只SD大鼠随机等分为对照组和高脂组,分别给予常规饲料和高脂饲料喂养12周。 结果和结论：与对照组相比,高脂组大鼠胰岛素敏感指数显著降低(P < 0.01),大鼠葡萄糖耐量受损,胰岛素释放试验提示葡萄糖刺激的胰岛素第一时相分泌受损,骨骼肌组织中蛋白酪氨酸磷酸酯酶1B蛋白表达水平明显增加(P < 0.01),骨骼肌中胰岛素诱导的胰岛素受体底物2磷酸化程度降低(P < 0.01)。提示高脂饮食诱导的肥胖大鼠骨骼肌中蛋白酪氨酸磷酸酯酶1B蛋白表达量升高,使胰岛素诱导的胰岛素受体底物2磷酸化程度降低,可能是肥胖导致胰岛素抵抗的机制之一。   关键词：肥胖;蛋白酪氨酸磷酸酶1B;胰岛素受体底物2;骨骼肌;胰岛素抵抗 doi:10.3969/j.issn.1673-8225.2012.20.020     

地塞米松诱导大鼠胰岛素抵抗机制的研究

观察了大鼠肌注地塞米松１周和３周后的空腹血糖、血清胰岛素及血浆胰高血糖素ｃＡＭＰ、ｃＧＭＰ水平。结果是经地主松处理后、１周组和３周组的大鼠血清胰岛素明显高于对照组；１周组血糖浓度无显著变化，３周组血糖浓度明显升高；血浆胰高血糖素水平无明显变化，未被高血糖，高胰岛素所抑制；１周组血浆ｃＧＭＰ明显下降；３组间的ｃＡＭＰ浓度差异无统计学意义。     

用高脂肪膳食复制胰岛素抵抗大鼠模型

用富含不饱和脂肪酸的高脂肪饮食（脂肪提供59％的热量）喂养28天后．用正常血糖-高血浆胰岛素钳尖技术检测大鼠的胰岛素敏感性。结果发现经高脂肪饮食喂养大鼠的葡萄糖输注率显著低于普通饮食喂养的对照大鼠,而且高脂肪饮食喂养后,大鼠的胰岛素敏感性指数显著下降,空腹血浆胰岛素则显著升高。该结果表明,用高脂肪饮食喂养发可复制出大鼠的胰岛素抵抗模型,而且该方法简便、易行、成功率高,便于广泛应用。     

脂联素、核因子-κB在胰岛素抵抗大鼠表达

目的观察炎症相关因子脂联素、核因子-κB(NF-κB)在胰岛素抵抗大鼠的表达。方法20只Wistar大鼠随机分为空白对照组与胰岛素抵抗组,分别给予基础饮食和高糖高脂饮食8周,应用高血浆胰岛素-正常血糖钳夹技术证明胰岛素抵抗的存在。用全自动生化分析仪测定各组血脂、脂联素等,并应用免疫组化技术测定NF-κB在血管内皮细胞的表达。结果①应用钳夹技术证明,胰岛素抵抗组存在胰岛素抵抗,而空白对照组未出现胰岛素抵抗;②胰岛素抵抗组甘油三酯、胆固醇、低密度脂蛋白、高密度脂蛋白较空白对照组明显升高(P<0.05),而保护性因子脂联素浓度则明显降低(P<0.05);③免疫组化显示,胰岛素抵抗组大鼠血管内皮细胞NF-κB阳性细胞百分率明显多于空白对照组(P<0.05);④脂联素浓度与NF-κB的表达呈明显负相关(r=-0.854,P<0.05);结论胰岛素抵抗时,脂联素浓度降低,NF-κB过表达,二者呈明显负相关。     

多囊卵巢综合征大鼠骨骼肌细胞胰岛素受体底物1及其丝氨酸307位点磷酸化的表达

背景：胰岛素受体底物1的丝氨酸307位点磷酸化程度的增加参与了骨骼肌胰岛素抵抗的发生。 目的：观察多囊卵巢综合征大鼠骨骼肌细胞胰岛素受体底物1的含量及其丝氨酸307位点磷酸化程度的变化。 方法：将大鼠随机分为模型组和对照组,模型组给予人绒毛膜促性腺激素联合胰岛素皮下注射,并配以高脂饮食,构建大鼠多囊卵巢综合征模型;对照组皮下注射生理盐水,正常饲料喂养。 结果与结论：干预6周后,Western blot检测结果显示,与对照组相比,模型组大鼠骨骼肌细胞胰岛素受体底物1表达量显著下降(P < 0.05),而其丝氨酸307位点磷酸化蛋白表达明显升高(P < 0.05)。结果提示,多囊卵巢综合征大鼠骨骼肌细胞中胰岛素受体底物1蛋白表达下调及其丝氨酸307位点磷酸化蛋白表达上调与多囊卵巢综合征大鼠骨骼肌胰岛素抵抗的发生密切相关。     

8-羟基脱氧鸟苷在胰岛素抵抗大鼠骨骼肌的表达及运动、吡格列酮的干预作用

目的:观察氧化应激标志物8-羟基脱氧鸟苷(8-OHdG)在胰岛素抵抗(IR)大鼠骨骼肌的表达及运动、吡格列酮对其的干预作用。方法:40只雄性Wistar大鼠随机分为对照组和模型组,分别给予普通饲料和高糖高脂饲料饲养;8周后再将模型组大鼠随机分为IR组、运动干预组和吡格列酮干预组,后两组分别进行游泳训练及吡格列酮(20mg/kg/天)灌胃,持续8周。16周末测量各组大鼠体重(BW)、空腹血糖(FPG)、空腹胰岛素(FINS),计算胰岛素抵抗指数(HOMA-IR);采用黄嘌呤氧化酶法检测骨骼肌超氧化物歧化酶(SOD)活性、免疫组化法检测骨骼肌8-OHdG表达。结果:(1)IR组大鼠FBG、FINS、HOMA-IR水平升高,骨骼肌8-OHdG表达增强、SOD活性降低;运动、吡格列酮干预后SOD活性增加,其它指标均下降。(2)相关性分析发现,实验大鼠HOMA-IR与8-OHdG表达水平呈正相关,与SOD活性呈负相关。结论:运动、吡格列酮可通过抑制IR大鼠骨骼肌的氧化应激反应,改善IR。     

余甘子提取物对糖尿病大鼠肌肉和脂肪组织胰岛素信号通路的影响

背景：余甘子具有明显的降血糖作用,但其作用机制尚不清楚。 目的：观察余甘子提取物对大鼠骨骼与脂肪组织中胰岛素信号通路相关蛋白的影响。 方法：将30只SD大鼠随机等分为对照组、模型组和余甘子组,后2组以腹腔注射链脲佐菌素建立糖尿病大鼠模型。余甘子组用余甘子提取液灌胃6周,模型组和对照组灌胃同体积的蒸馏水。 结果与结论：与对照组相比,糖尿病大鼠的体质量、空腹血糖、空腹胰岛素和胰岛素抵抗指数均显著升高(P < 0.05),脂肪和肌肉组织磷脂酰肌醇-3-激酶、蛋白激酶B mRNA及葡萄糖转运蛋白4 mRNA和蛋白水平显著下降(P < 0.05,P < 0.01),灌胃余甘子提取物6周后,余甘子组大鼠体质量、空腹血糖、空腹胰岛素和胰岛素抵抗指数均比模型组下降(P < 0.05),脂肪和肌肉组织磷脂酰肌醇-3-激酶、蛋白激酶B和葡萄糖转运蛋白4 mRNA水平明显增加(P < 0.01,P < 0.05),且脂肪组织葡萄糖转运蛋白4 蛋白表达增加(P < 0.01),但肌肉组织葡萄糖转运蛋白4 蛋白差异没有显著性意义。提示余甘子提取物可调控胰岛素介导的磷脂酰肌醇-3-激酶/蛋白激酶B/葡萄糖转运蛋白4信号转导通路,发挥降血糖作用。     

Endurance-training induced changes in skeletal muscle phosphoglycerate kinase of old Wistar rats

Sufficiently intense, long-term, endurance training has been shown in several studies to induce a variety of adaptations in skeletal muscle, including a substantial restoration of the activities of several muscle enzymes which are known to be modified during biological aging. This activity-restoration may reflect either an increase in the amounts of enzyme proteins or an enhancement of the specific activities of these molecules. The present study examined the effect of long-term endurance training on the status of phosphoglycerate kinase in skeletal muscle of old rats, as compared with the enzyme isolated either from non-trained old or young animals. The kinetics of heat inactivation, which differ markedly between young and old forms of phosphoglycerate kinase, were used as a sensitive probe for the status of the enzyme. The results reveal a remarkable similarity between the heat inactivation patterns of phosphoglycerate kinase from the muscle of old, exercise-trained rats and enzyme purified from young animals, while enzyme samples isolated from sedentary old animals are significantly more heat-stable. Adaptation to endurance-training is thus evident at the molecular level, and maintains phosphoglycerate kinase in its young form. The aging of this enzyme has been previously shown to involve only conformational changes, which develop following a reversible partial oxidation of reactive cysteine residues. Whether the adaptation of the enzyme to endurance-training results from enhancement in its turnover rate (i.e., dwell time in the cell becoming too short for modifications to develop) or is due to increased protection against oxidation (being the first step in the enzyme's aging) remains to be studied.     

骨骼肌小热休克蛋白在离心运动后的表达

背景：研究证明离心方式的训练可使骨骼肌产生保护作用,避免离心运动引起的损伤,但是机械负荷引起的小热休克蛋白的保护作用至今却少有报道。 目的：观察骨骼肌小热休克蛋白家族中αB-晶体蛋白在离心运动后的表达,以此探讨机械负荷诱导的小热休克蛋白对骨骼肌细胞的保护作用机制。 方法：将Wistar大鼠随机分为安静对照组和运动训练组。运动训练组使用动物跑台进行6周间歇性离心运动训练,每周训练5 d,安静对照组正常喂养。训练6周休息48 h后,安静对照组与运动训练组随机选出6只大鼠做1次性大负荷离心运动。观察两组血清肌酸激酶变化;蛋白免疫印迹法检测两组腓肠肌αB-晶体蛋白含量变化,用免疫荧光组织化学法分析两组腓肠肌αB-晶体蛋白亚细胞表达特征。 结果与结论：大负荷离心运动后,安静对照组血清肌酸激酶与运动前相比显著增高(P< 0.05),说明骨骼肌细胞出现严重的损伤,而运动训练组这种损伤不明显。蛋白免疫印迹结果表明,运动训练组做一次性大负荷离心运动后αB-晶体蛋白表达水平比安静对照组增加(P < 0.05)。从免疫荧光组化切片可见,αB-晶体蛋白在细胞内发生了移位变化,从胞浆移位于Z盘和细胞膜。提示αB-晶体蛋白在离心运动训练后表达增多,并通过移位于肌细胞Z-盘和细胞膜发挥对骨骼肌细胞的保护作用。     

Expression and localisation of IGF-binding protein mRNAs in regenerating rat skeletal muscle

The expression of the insulin-like growth factor-binding proteins (IGFBP) -3, -4, -5 and -6 was investigated in neonatal, in normal adult and in regenerating rat skeletal muscle. Semi-quantification was done by densitometric scannings of Northern blots. The expression of all investigated IGFBPs, with the exception of IGFBP-5, was higher in neonatal than in adult muscle. During postischaemic regeneration the expression of all IGFBPs increased, but with different time schedules. IGFBP-3 increased transiently during the early phase of regeneration, while IGFBP-4, -5 and -6 increased during the later phase of regeneration. In situ hybridisation on regenerating muscle showed that the expression of the various IGFBPs was cell specific; thus, IGFBP 3 was mainly expressed in macrophages, IGFBP-4 in connective tissue, IGFBP-5 in regenerating muscle cells, and IGFBP-6 in muscle cells, connective tissue and endothelium. Ligand blotting, using 125I-IGF-I as the ligand, showed a number of bands ranging between 24 and 44 kDa. Samples from neonatal and regenerating muscle contained much higher levels of all IGFBPs than those from normal adult muscle. An ordered and cell-specific expression of IGFBPs, allowing a strict regulation of IGF actions, is probably necessary to ensure an optimal regeneration process.     

Hexokinase and adenylate kinase activities in aorta, heart muscle and skeletal muscle from uraemic rats

The effect of parathyroidectomy and/or vitamin D on the development of arterial and myocardial lesions was studied in rats with moderate uraemia. The activities of hexokinase and adenylate kinase in the aorta, myocardium and skeletal muscle were measured and the incidence of aortic calcification and muscle cell necrosis determined. There was a decreased hexokinase activity in the aorta, myocardium and skeletal muscle from uraemic rats. Adenylate kinase showed an increased activity in the same tissues. Parathyroidectomy as well as I-alpha-hydroxycholecalciferol in a dose of 3 ng/100 g b.w. normalized these activities to a great extent. This effect did not occur when 10 ng/100 g b.w. was given. Parathyroidectomy in combination with a low dose of I-alpha-OH-D3 reduced the incidence of myocardial necrosis. Aortic calcifications were found in uraemic animals given 10 ng/100 g b.w. of I-alpha-hydroxycholecalciferol. In this group increased activity of adenylate kinase was found in calcified aortae but not in non-calcified aortae. The study shows that uraemia causes metabolic changes in the aorta, myocardium, and skeletal muscle which may partly be prevented by parathyroidectomy and by low doses of vitamin D. It also indicates some parallelism between these metabolic changes and the development of histologically demonstrable lesions in the aorta.     

Role of protein kinase C during insulin mediated skeletal muscle cell spreading

Protein kinase C (PKC) is known to play important roles in integrin mediated cell spreading. This study investigated the role of PKC during insulin mediated muscle cell spreading, which was independent of integrin α5. We found that PKC-α becomes active and localise to membrane during insulin mediated cell spreading. We also found that PKC activation is essential for cell spreading stimulated by insulin and this activation enhances the cell spreading. PKC activation increased the tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin as well as tyrosine kinase activity of FAK. We also observed that PKC activation enhanced the FAK associated PI 3-kinase activity and also increased the activation of ERK-1/-2. Moreover, the effect of PKC activation on insulin mediated cell spreading as well as tyrosine phosphorylation of FAK and paxillin depends upon integrity of actin cytoskeleton. Thus, PKC is an important signaling protein during insulin mediated muscle cell spreading. This revised version was published online in July 2006 with corrections to the Cover Date.     

Denervation increases protein tyrosine kinase and protein tyrosine phosphatase activities in fast and slow skeletal muscle

We have investigated the expression and regulation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) in fast extensor digitorum longus (EDL) and slow soleus (SOL) in adult rat skele-tal muscles. Biochemical assays revealed significantly greater PTK and PTP activities in SOL than in EDL; these results were confirmed and extended by in-gel assays demonstrating that the PTKs and PTPs detected had higher activity levels in SOL compared to EDL extracts. Although phosophotyrosine proteins were concentrated at the neuromuscular junction (NMJ), PTK and PTP activities were observed in extra-junctional regions of the muscle fiber. Following denervation, we observed significant increases in PTK and PTP activities in both SOL and EDL, and gel-based assays showed an increase in the activities of several PTKs and PTPs. These results suggest that the same PTK and PTPs have different activity levels in fast and slow skeletal muscles and are regulated by nerve-dependent mechanisms. Accepted: 31 May 2001     

Effect of exercise on protein kinase C activity and localization in human skeletal muscle

To investigate the effect of exercise on protein kinase C (PKC) activity and localization in human skeletal muscle, eight healthy men performed cycle ergometer exercise for 40 min at 76 ± 1% the peak pulmonary O₂ uptake     , with muscle samples obtained at rest and after 5 and 40 min of exercise. PKC expression, phosphorylation and activities were examined by immunoblotting and in vitro kinase assays of fractionated and whole tissue preparations. In response to exercise, total PKC activity was slightly higher at 40 min in an enriched membrane fraction, and using a pSer-PKC-substrate motif antibody it was revealed that exercise increased the serine phosphorylation of a ∼50 kDa protein. There were no changes in conventional PKC (cPKC) or PKCθ activities; however, atypical PKC (aPKC) activity was ∼70% higher at 5 and 40 min, and aPKC expression and Thr^410/403 phosphorylation were unaltered by exercise. There were no effects of exercise on the abundance of PKCα, PKCδ, PKCθ and aPKC within cytosolic or enriched membrane fractions of skeletal muscle. These data indicate that aPKC, but not cPKC or PKCθ, are activated by exercise in contracting muscle suggesting a potential role for aPKC in the regulation of skeletal muscle function and metabolism during exercise in humans.     

Hexokinase and adenylate kinase activities in aorta,heart muscle and skeletal muscle from uraemic rats.

The effect of parathyroidectomy and/or vitamin D on the development of arterial and myocardial lesions was studied in rats with moderate uraemia. The activities of hexokinase and adenylate kinase in the aorta, myocardium and skeletal muscle were measured and the incidence of aortic calcification and muscle cell necrosis determined. There was a decreased hexokinase activity in the aorta, myocardium and skeletal muscle from uraemic rats. Adenylate kinase showed an increased activity in the same tissues. Parathyroidectomy as well as I-alpha-hydroxycholecalciferol in a dose of 3 ng/100 g b.w. normalized these activities to a great extent. This effect did not occur when 10 ng/100 g b.w. was given. Parathyroidectomy in combination with a low dose of I-alpha-OH-D3 reduced the incidence of myocardial necrosis. Aortic calcifications were found in uraemic animals given 10 ng/100 g b.w. of I-alpha-hydroxycholecalciferol. In this group increased activity of adenylate kinase was found in calcified aortae but not in non-calcified aortae. The study shows that uraemia causes metabolic changes in the aorta, myocardium, and skeletal muscle which may partly be prevented by parathyroidectomy and by low doses of vitamin D. It also indicates some parallelism between these metabolic changes and the development of histologically demonstrable lesions in the aorta.