硒对大鼠急性缺血心肌溶酶体膜的影响

结扎大鼠冠状动脉，造成急性心肌梗塞模型，观察硒对缺血心肌溶酶体酶的影响．结果表明，心肌缺血１小时后，缺血区心肌溶酶体酸性磷酸酶（ＡＣＰ）、组织蛋白酶Ｄ（ＣＤ）的游离酶活性（Ｆ）及游离酶活性／总酶活性比值（Ｆ／Ｔ）均明显升高，ＧＳｌｌ－Ｐｘ活性显著降低，脂质过氧化物含量（ＭＤＡ）明显增加。硒预处理大鼠，心肌急性缺血后．Ｆ及Ｆ／Ｔ显著下降，ＧＳＨ－Ｐｘ明显升高，ＭＤＡ含量显著降低。硒可能通过提高缺血心肌ＧＳＨ－Ｐｘ活性，阻止自由基介导的脂质过氧化，增加溶酶体膜的稳定性，从而减轻心肌缺血性损伤。     

DAG—PKC信号传导通路与糖尿病血管并发症

高血糖可引起多种组织细胞内二脂酰甘油（ＤＡＧ）水平升高，ＤＡＧ可激活蛋白激酶Ｃ（ＰＫＣ）。最近研究表明，持续高血糖引起的ＤＡＧ－ＰＫＣ系统激活与糖尿病多种血管异常关系密切。ＰＫＣ是由一个多基因家族编码的多肽类物质，至少有１１种亚型。在多外ＰＫＣ亚型中，β、γ亚型在糖尿病动物的血管系统中优先被激活。选择性ＰＫＣ－β抑制剂（ＬＹ３３３５３１）能逆转或减轻啮类糖尿病动物的血管异常，其对糖尿病血管并发症预     

高血糖对血管细胞内信息传导的影响——糖尿病血管并发症发…

作者观察了高血糖对大，小血管细胞内信息传导的影响。结果表明血管细胞暴露在高糖环境下，细胞内二酰基甘油（ＤＡＧ）合成明显增加，ＤＡＧ含量升高，进而激活了蛋白激酶Ｃ（ＰＫＣ）。因此，高血糖可以使细胞内ＤＡＧ－ＰＫＣ传导通络处于活化状态，从而影响血管内一系列生理功能，这可能是糖尿病血管并发症发生的一个重要机制。     

维生素E与糖尿病

简要介绍糖尿病血管并发症发病机制中的二酰甘油（ＤＡＧ）－蛋白激酶Ｃ（ＰＫＣ）通路学说，以揭示高血糖状态下ＤＡＧ合成增加并激活ＰＫＣ，造成血管通透性、收缩性、血液流变学改变的机理。同时介绍作为ＰＫＣ抑制剂维生素Ｅ（ＶＥ）在糖尿病治疗中的应用     

糖尿病大鼠肾小球二酰基甘油-蛋白激酶C通路的变化

本研究旨在利用二酰基甘油 (ＤＡＧ)激酶法和ＡＴＰ底物磷酸化的方法 ,检测糖尿病大鼠肾小球ＤＡＧ水平和蛋白激酶Ｃ(ＰＫＣ)活性变化 ,以探讨肾小球ＤＡＧ ＰＫＣ通路的变化在肾小球血流动力学和肾脏形态学改变中所起的作用。一、材料与方法1.动物模型的制备 :体重 2 2 0～ 2 4 0ｇ雄性Ｗｉｓｔａｒ大鼠 5 0只 (山东大学实验动物中心提供 ) ,分为正常对照组和糖尿病组。将链脲佐菌素 (Ｓｉｇｍａ公司 )溶于 0 .1ｍｏｌ/Ｌ枸橼酸钠缓冲液 (ｐＨ 4 .2 )中 ,腹腔内注射 (6 5ｍｇ/ｋｇ) ,2 4ｈ大鼠血糖开始升高 ,以 4 8ｈ后血糖≥ 16 .5ｍｍｏ…     

维生素E与糖尿病

简要介绍糖尿病血管并发症发病机制中的二酰甘油（ＤＡＧ）－蛋白激酶Ｃ（ＰＫＣ）通路学说，以揭示高血糖状态下ＤＡＧ合成增加并激活ＰＫＣ，造成血管通透性，收缩性，血液流变学改变的机理，同时介绍作为ＰＫＣ抑制维生素Ｅ（ＶＥ）在糖尿病治疗中的应用。     

铬对实验性心肌损伤保护机制探讨

为探讨铬对实验性心肌损伤的保护作用机制，选用Ｗｉｓｔａｒ大鼠腹腔内注射铬，尾静脉注射阿霉素，复制动物模型，用荧光偏振法测定心肌线粒体膜流动性，低温电子自旋共振技术测定心肌及肝脏自由基含量，同时观察抗氧化酶及心肌酶活性变化。结果表明单纯注射阿霉素大鼠心肌线粒体膜流动性降低，血清ＬＤＨ及ＣＰＫ活性增强，心肌及肝脏自由基含量升高，全血、心肌及肝脏ＧＳＨ－ｐｘ活性降低，血清ＳＯＤ活性增强，心肌及肝脏ＳＯＤ     

牛磺酸对心功能不全心肌细胞保护作用的初步研究

作者在３１例心功能不全病人及离体大鼠心脏缺血－再灌注模型上给予牛磺酸治疗后，观察铜、锌超氧化物歧化酶（ＣｕＺｎＳＯＤ）、脂质过氧化终产物丙二醛（ＭＤＡ）水平的改变及其与心功能变化的关系。结果发现心功能不全患者的超氧化物歧化酶（ＳＯＤ）、ＭＤＡ随心衰严重程度而增加，应用牛磺酸治疗２周后，血中ＳＯＤ、ＭＤＡ水平显著降低，且随疗程的延长效果更显著。牛磺酸与血管扩张剂、利尿剂及强心剂联合应用，较单用牛磺酸治疗效果更佳。大鼠心脏缺血、再灌注损伤后，心功能降低，心肌组织脂质过氧化终产物ＭＤＡ的含量明显增加，补充牛磺酸后，心功能明显改善，ＭＤＡ显著降低，证实牛磺酸是正常机体的内源性抗氧化剂，具有清除自由基的作用，因此有可能作为临床防治心衰的辅助药物。     

硒和维生素E对大鼠心肌腺苷酸及氧化损伤的影响

目的：研究低硒（Ｓｅ）低维生素Ｅ（ＶＥ）饮食大鼠心肌腺苷酸含量和氧化损伤的关系。方法：使用高效液相测定心肌腺苷酸含量和使用生化方法测定心肌ＭＤＡ含量和ＳＯＤ、ＧＳＨ－Ｐｘ活性。结果：与补充Ｓｅ和／或ＶＥ饮食大鼠相比，低Ｓｅ低ＶＥ饮食大鼠心肌ＡＭＰ，ＡＤＰ含量无明显差异，而ＡＴＰ含量明显降低，ＭＤＡ含量增加，ＳＯＤ，ＧＳＨ－Ｐｘ活性降低，表明Ｓｅ和ＶＥ影响ＡＴＰ含量与其抗氧化作用有关，而其中以联合补充Ｓｅ和ＶＥ效果最佳     

糖尿病大鼠心肌GluT_4mRNA的表达水平

目的　探讨ＧｌｕＴ４ ｍＲＮＡ的表达水平与糖尿病心肌病的相关性。方法采用逆转录聚合酶链式反应的方法对链脲佐菌素 （ＳＴＺ）诱导的糖尿病大鼠心肌 ＧｌｕＴ４ ｍＲＮＡ的表达水平进行检测并与正常对照组大鼠对比。结果糖尿病大鼠心肌 ＧｌｕＴ４ ｍＲＮＡ的表 达水平明显低于正常对照组（Ｐ＜０．０１）。结论在发生糖尿病的同时,大鼠心肌细胞ＧｌｕＴ４ ｍＲＮＡ的表达受抑制,含量降低,导致心肌的能量代谢紊乱,引起糖尿病心肌病。     

高血糖对血管细胞内信息传导的影响——糖尿病血管并发症发病机制的探讨

作者观察了高血糖对大、小血管细胞内信息传导约影响。结果表明血管细胞暴露在高糖环境下,细胞内二酰基甘油(DAG)合成明显增加,DAG含量升高,进而激活了蛋白激酶C(PKC)。因此,高血糖可以使细胞内DAG-PKC传导通路处于活化状态,从而影响血管的一系列生理功能。这可能是糖尿病血管并发症发生的一个重要机制。     

Preferential elevation of protein kinase C isoform beta II and diacylglycerol levels in the aorta and heart of diabetic rats: differential reversibility to glycemic control by islet cell transplantation.

In the present study, we have measured protein kinase C (PKC) specific activities and total diacylglycerol (DAG) level in the aorta and heart of rats, which showed that after 2 weeks of streptozotocin (STZ)-induced diabetes, membranous PKC specific activity and total DAG content were increased significantly by 88% and 40% in the aorta and by 21% and 72% in the heart, respectively. Hyperglycemia was identified as being a causal factor since elevated glucose levels increased DAG levels in cultured aortic endothelial and smooth muscle cells. Analysis by immunoblotting revealed that only alpha and beta II PKC isoenzymes are detected in these two tissues and vascular cells among those studied. In STZ-induced diabetic rats, beta II isoenzyme is preferentially increased in both aorta and heart, whereas PKC alpha did not change significantly. The increases in membranous PKC specific activity and DAG level are observed in both spontaneous diabetes-prone diabetic BB rats as well as in STZ-induced diabetic BB and Sprague-Dawley rats, which persisted for up to 5 weeks. After 2 weeks of diabetes without treatment, the normalization of blood glucose levels for up to 3 weeks with islet cell transplants in STZ-induced diabetic BB rats reversed the biochemical changes only in the heart, but not in the aorta. These results suggest that PKC activity and DAG level may be persistently activated in the macrovascular tissues from diabetic animals and indicate a possible role for these biochemical parameters in the development of diabetic chronic vascular complications.     

Protein kinase C and the development of diabetic vascular complications.

Hyperglycemic control in diabetes is key to preventing the development and progression of vascular complications such as retinopathy, nephropathy and neuropathy. Increased activation of the diacylglycerol (DAG)-protein kinase C (PKC) signal transduction pathway has been identified in vascular tissues from diabetic animals, and in vascular cells exposed to elevated glucose. Vascular abnormalities associated with glucose-induced PKC activation leading to increased synthesis of DAG include altered vascular blood flow, extracellular matrix deposition, basement membrane thickening, increased permeability and neovascularization. Preferential activation of the PKCbeta isoform by elevated glucose is reported to occur in a variety of vascular tissues. This has lead to the development of LY333531, a PKCbeta isoform specific inhibitor, which has shown potential in animal models to be an orally effective and nontoxic therapy able to produce significant improvements in diabetic retinopathy, nephropathy, neuropathy and cardiac dysfunction. Additionally, the antioxidant vitamin E has been identified as an inhibitor of the DAG-PKC pathway, and shows promise in reducing vascular complications in animal models of diabetes. Given the overwhelming evidence indicating a role for PKC activation in contributing to the development of diabetic vascular complications, pharmacological therapies that can modulate this pathway, particularly with PKC isoform selectivity, show great promise for treatment of vascular complications, even in the presence of hyperglycemia.     

Interrelated effects of insulin and glucose on diacylglycerol-protein kinase-C signalling in rat adipocytes and solei muscle in vitro and in vivo in diabetic rats

The effects of insulin and glucose, alone and combined, on diacylglycerol (DAG), protein kinase-C (PKC), and glucose transport were compared in rat adipocytes and solei incubated in medium containing 0-20 mM glucose. In both tissues insulin rapidly stimulated [3H]DAG production from [3H]glycerol; extracellular glucose masked this effect in adipocytes, but not in solei. [3H]Glucose was avidly converted to DAG in adipocytes, and this conversion was enhanced by insulin. In contrast, [3H]glucose was poorly converted to DAG in solei. Glucose alone (5-20 mM) stimulated PKC translocation in adipocytes, but not in solei. Insulin stimulated PKC translocation in both tissues at all glucose concentrations. However, glucose modulated this effect of insulin in adipocytes by 1) decreasing cytosolic PKC and the absolute amount of PKC translocated, and 2) promoting apparent turnover of membrane PKC. In contrast, in solei, glucose did not affect PKC levels or translocation responses to insulin. In keeping with DAG-PKC signalling, the relative glucose transport effects of insulin were influenced by extracellular glucose in adipocytes, but not in solei. These results suggest that 1) glucose-induced PKC translocation requires metabolism of glucose to DAG; 2) glucose activates DAG-PKC signalling in adipocytes, but not in solei; 3) insulin activates DAG-PKC signalling in both tissues at all glucose levels; and 4) glucose may modulate the effects of insulin on DAG-PKC signalling in adipocytes, but not in solei. Consistent with in vitro results, in solei taken directly from diabetic rats, membrane PKC was decreased, and cytosolic PKC was increased, presumably reflecting diminished PKC translocation due to hypoinsulinemia. In contrast, in adipose tissue, cytosolic PKC was decreased, presumably reflecting hyperglycemia-induced PKC translocation. Accordingly, DAG levels were increased in adipose tissue, but not in solei, in diabetic rats, and insulin increased DAG in both tissues.     

PTEN在心力衰竭心肌MAPK/PKC信号通路的作用

目的 观察充血性心力衰竭病人心肌重构病理过程中肿瘤抑制因子PTEN（phosphatase and tensin homolog tumor suppressor,PTEN）及丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）与蛋白激酶C（protein kinase C,PKC）的作用.方法 通过手术取材,选择因瓣膜性心脏病接受二尖瓣置换术的心力衰竭病人39例,正常对照38例（其中8例来自意外伤亡的器官捐献者）.竞争蛋白结合法检测心肌组织PKC及MAPK活性,免疫沉淀法检测PTEN蛋白表达.结果 心力衰竭病人心肌组织呈典型的重构心肌的病理改变.心肌组织PTEN表达蛋白光吸收（absorbance,A）与β肌动蛋白光吸收比值（PTEN/β-actin）随心功能恶化而降低,各心力衰竭组与正常组相比,差异有统计学意义（P＜0.01）;相反,心力衰竭病人心肌组织PKC和MAPK活性明显高于对照组（P＜0.01）,并随心功能恶化其表达逐渐增加,各心力衰竭组与正常组比较,差异有统计学意义（P＜0.01）.结论 PTEN及MAPK与PKC信号通路共同参与调节CHF病人心肌重构的病理过程,PTEN在心肌重构病理过程中起负性调节作用.     

Tissue and isoform-selective activation of protein kinase C in insulin-resistant obese Zucker rats - effects of feeding.

The mechanisms of insulin resistance in the obese Zucker rat have not been clearly established but increased diacylglycerol-protein kinase C (DAG-PKC) signalling has been associated with decreased glucose utilisation in states of insulin resistance and non-insulin-dependent diabetes mellitus. The purpose of this study was to characterise tissue- and isoform-selective differences in DAG-PKC signalling in insulin-sensitive tissues from obese Zucker rats, and to assess the effects of feeding on DAG-PKC pathways. Groups of male obese (fa/fa, n=24) and lean (fa/-, n=24) Zucker rats were studied after baseline measurements of fasting serum glucose, triglycerides, insulin and oral glucose tolerance tests. Liver, epididymal fat and soleus muscle samples were obtained from fed and overnight-fasted rats for measurements of DAG, PKC activity and individual PKC isoforms in cytosol and membrane fractions. Obese rats were heavier (488+/-7 vs 315+/-9 g) with fasting hyperglycaemia (10.5+/-0.8 vs 7.7+/-0.1 mM) and hyperinsulinaemia (7167+/-363 vs 251+/-62 pM) relative to lean controls. In fasted rats, PKC activity in the membrane fraction of liver was significantly higher in the obese group (174+/-16 vs 108+/-12 pmol/min/mg protein, P<0.05) but there were no differences in muscle and fat. The fed state was associated with increased DAG levels and threefold higher PKC activity in muscle tissue of obese rats, and increased expression of the major muscle isoforms, PKC-theta and PKC-epsilon: e.g. PKC activity in the membrane fraction of muscle from obese animals was 283+/-42 (fed) vs 107+/-20 pmol/min/mg protein (fasting) compared with 197+/-27 (fed) and 154+/-21 pmol/min/mg protein (fasting) in lean rats. In conclusion, hepatic PKC activity is higher in obese rats under basal fasting conditions and feeding-induced activation of DAG-PKC signalling occurs selectively in muscle of obese (fa/fa) rats due to increased DAG-mediated activation and/or synthesis of PKC-theta and PKC-epsilon. These changes in PKC are likely to exacerbate the hyperglycaemia and hypertriglyceridaemia associated with obesity-induced diabetes.     

PPARgamma agonists ameliorate endothelial cell activation via inhibition of diacylglycerol-protein kinase C signaling pathway: role of diacylglycerol kinase

Subject- Peroxisome proliferator-activated receptor (PPAR)-gamma agonists are emerging as potential protectors against inflammatory cardiovascular diseases including atherosclerosis and diabetic complications. However, their molecular mechanism of action within vasculature remains unclear. We report here that PPARgamma agonists, thiazolidinedione class drugs (TZDs), or 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) were capable of activating diacylglycerol (DAG) kinase (DGK), resulting in attenuation of DAG levels and inhibition of protein kinase C (PKC) activation. The PPARgamma agonist-induced DGK was completely blocked by a dominant-negative mutant of PPARgamma, indicating an essential receptor-dependent action. Importantly, the suppression of DAG-PKC signaling pathway was functional linkage to the anti-inflammatory properties of PPARgamma agonists in endothelial cells (EC), characterized by the inhibition of proinflammatory adhesion molecule expression and adherence of monocytes to the activated EC induced by high glucose. These findings thus demonstrate a novel molecular action of PPARgamma agonists to suppress the DAG-PKC signaling pathway via upregulation of an endogenous attenuator, DGK.     

Protein kinase C activation and its pharmacological inhibition in vascular disease

Vascular complications in diabetes mellitus are known to be associated with the activation of the protein kinase C (PKC) pathway through the de novo synthesis of diacylglycerol (DAG) from glycolytic intermediates. Specific PKC isoforms, mainly the beta- and delta-isoforms, have been shown to be persistently activated in diabetic mellitus. Multiple studies have reported that the activation of PKC leads to increased production of extracellular matrix and cytokines, enhances contractility, permeability and vascular cell proliferation, induces the activation of cytosolic phospholipase A2 and inhibits the activity of Na+-K+-ATPase. These events are not only frequently observed in diabetes mellitus but are also involved in the actions of vasoactive agents or oxidative stress. Inhibition of PKC by two different kinds of PKC inhibitors - LY333531, a selective PKC-beta-isoform inhibitor, and vitamin E, d-alpha-tocopheron - were able to prevent or reverse the various vascular dysfunctions in vitro and in vivo. Clinical studies using these compounds are now ongoing to evaluate the significance of DAG-PKC pathway activation in the development of vascular complications in diabetic patients.     

Diacylglycerol kinase zeta rescues G alpha q-induced heart failure in transgenic mice

BACKGROUND: The G alpha q protein-coupled receptor (GPCR) signaling pathway, which includes diacylglycerol (DAG) and protein kinase C (PKC), plays a critical role in the development of cardiac hypertrophy and heart failure (HF). It has been reported that the expression of a constitutively active mutant of the G protein alpha q subunit in the hearts of transgenic mice (G alpha q-TG) induces cardiac hypertrophy and lethal HF. DAG kinase (DGK) catalyzes DAG and controls its cellular levels, thus acting as a regulator of GPCR signaling. It has been found that transgenic mice with cardiac-specific overexpression of DGK zeta (DGK zeta-TG) inhibit GPCR agonist-induced activation of the DAG-PKC signaling and subsequent cardiac hypertrophy, so this study tested the hypothesis that DGK zeta could rescue G alpha q-TG mice from developing HF. METHODS AND RESULTS: Double transgenic mice (G alpha q/DGK zeta-TG) with cardiac-specific overexpression of both DGK zeta and G alpha q were generated by crossing G alpha q-TG with DGK zeta-TG mice, and the pathophysiological consequences were analyzed. DGK zeta prevented cardiac dysfunction, determined by dilatation of left ventricular (LV) dimensions, reduction of LV fractional shortening, and marked increases in LV end-diastolic pressure in G alpha q-TG mice. Translocation of PKC isoforms, phosphorylation activity of c-jun N-terminal kinase and p38 mitogen-activated protein kinase in G alpha q-TG mice were attenuated by DGK zeta. DGK zeta improved the survival rate of G alpha q-TG mice. CONCLUSIONS: These results demonstrate the first evidence that DGK zeta blocks cardiac dysfunction and progression to lethal HF by activated G alpha q protein without detectable adverse effects in the in-vivo heart and suggest that DGK zeta is a novel therapeutic target for HF.