柠檬皮多酚成分分析及其对胰岛素抵抗HepG2细胞糖代谢的影响

目的:探究柠檬皮多酚（Limon Peel Polyphenols,LPP）的组成成分,并研究其对胰岛素抵抗（Insulin Resistance,IR）的HepG2细胞糖代谢的影响。方法:采用高效液相色谱串联四级杆飞行时间质谱法（HPLC-QTOF-MS）分析LPP组成,利用HepG2细胞建立胰岛素抵抗模型,用LPP作用于IR-HepG2细胞,通过测定细胞葡萄糖消耗量初步探究LPP对糖代谢的影响,再通过测定糖原含量和己糖激酶（Hexokinase,HK）、丙酮酸激酶（Pyruvate Kinase,PK）、磷酸烯醇式丙酮酸羧基酶（Phosphoenol Pyruvate Carboxykinase,PEPCK）、葡萄糖六磷酸酶（Glucose-6-Phosphatase,G6Pase）的活性,探究LPP调节细胞糖代谢的作用途径。结果:经过HPLC-QTOF-MS分析出12种物质,主要为黄酮及其苷类成分;在糖代谢研究方面,与模型组相比浓度为0.1~2 mg/mL柠檬皮多酚可显著提高细胞葡萄糖消耗量（P<0.05）,且当浓度为0.5 mg/mL时其提高IR-HepG2细胞糖原含量和HK、PK活性,降低PEPCK和G6Pase活性的能力最接近于二甲双胍阳性对照。结论:柠檬皮多酚可以缓解IR-HepG2细胞的胰岛素抵抗状态,并能通过促进糖原合成、提高糖酵解关键酶活性、降低糖异生酶活力的方式调节糖代谢水平,为后续的体内研究提供了数据支持,并为未来开发功能性产品提供了理论依据。     

糖苷结构对苦荞黄酮促进葡萄糖在C2C12小鼠骨骼肌细胞中摄取作用的影响

苦荞是芦丁含量最高的粮食作物,但不同的加工过程会导致芦丁降解为单糖苷结构的异槲皮素及其前体槲皮素,进而影响生物活性。本研究比较芦丁及其降解产物（槲皮素、异槲皮素）这三类含有不同糖苷结构的黄酮在C2C12小鼠骨骼肌细胞中促进葡萄糖摄取的功效差异,并探究其作用机制。研究结果表明,苦荞与水接触会导致苦荞中的核心黄酮组分-芦丁降解为槲皮素。芦丁及其降解产物（槲皮素、异槲皮素）均能有效促进C2C12细胞对葡萄糖的摄取,作用顺序为:槲皮素>异槲皮素>芦丁。糖苷键的增加会降低槲皮素促进骨骼肌细胞糖摄取的效果。芦丁和异槲皮素不能激活胰岛素依赖型信号通路中的IRS-1表达及AKT的磷酸化,但高浓度下（400 μmol/L）,芦丁和异槲皮素可以通过非胰岛素依赖型的信号通路中的p-AMPK/p-ACC促进葡萄糖摄取作用,且异槲皮素的作用大于芦丁。槲皮素虽然抑制了IRS-1的表达及AKT的磷酸化,但槲皮素通过AMPK/ACC的磷酸化显著了促进C2C12细胞对葡萄糖的摄取。本研究揭示芦丁及其降解产物在细胞水平上的降血糖作用及机理的差异,对于充分理解苦荞黄酮降血糖机制提供了科学依据。     

小檗碱活化AMPK-eNOS减轻油酸所致人主动脉内皮细胞损伤

目的：考察小檗碱对油酸所致内皮细胞损伤的保护作用,并探究其作用机制。方法：以体外培养人主动 脉内皮细胞系（human aorta endothelial cells,HAEC）为受试对象;分别以油酸、油酸联合小檗碱、油酸联合单 磷酸腺苷活化蛋白激酶（adenosine monophosphate-activated protein kinase,AMPK）激活剂（AICAR）或抑制剂 （Compound C）处理HAEC;采用油红O染色法观察细胞内脂滴合成;采用比色法检测细胞内甘油三酯、总胆 固醇含量,硝酸还原酶法检测NO的水平,2’,7’-二氯荧光黄双乙酸盐探针检测细胞内总活性氧（reactive oxygen species,ROS）水平,Western Blotting检测细胞总AMPK、内皮型一氧化氮合酶（endothelial nitric oxide synthase, eNOS）及其磷酸化（p-AMPK、p-eNOS）水平。结果：油酸可浓度依赖性地抑制细胞增殖,小檗碱组各项指标 较正常对照组无显著性差异,油酸联合小檗碱组的细胞活性较油酸组明显好转（P＜0.01）。与正常对照组比较, 油酸组的脂质浸润程度明显,油酸在不同浓度下使内皮细胞的脂质含量呈剂量依赖性增加,加入小檗碱可明显减 少这种脂质堆积。油酸组的NO水平较正常对照组明显减少,小檗碱能显著抑制油酸所致的内皮NO水平的减少, 并且减少由于油酸所致的ROS水平的升高;油酸抑制了AMPK的活化,使p-AMPK和p-eNOS的蛋白水平明显降低 （P＜0.01）;小檗碱可明显逆转油酸所致AMPK和eNOS磷酸化水平下降,Compound C可拮抗其作用。结论：小檗 碱可减轻油酸所致血管内皮细胞损伤,或与其活化AMPK/eNOS信号相关。     

D-松醇复配Mn2+对HepG2细胞胰岛素抵抗的调节及其作用机制

采用胰岛素诱导HepG2细胞建立胰岛素抵抗模型(IR),研究D-松醇复配Mn2+对细胞葡萄糖消耗量和糖原合成量的影响。实时荧光全定量分析(RT-PCR)实验检测AMPK信号通路相关基因mRNA表达水平。结果表明当胰岛素浓度为1×10-3 mmol/L,作用时间为36 h时,细胞葡萄糖消耗量达到最低,产生最大的胰岛素抵抗效应。与模型对照组相比,当D-松醇浓度为100 mg/L,复配MnSO₄为10 mg/L时,葡萄糖消耗量和糖原含量均极显著升高(p<0.01)。此外,复配组AMPKα-1、AMPKα-2和GLUT4基因表达水平均显著上调(p<0.05),G6Pase基因mRNA表达水平显著下调(p<0.05)。D松醇复配Mn2+可显著促进胰岛素抵抗细胞葡萄糖的利用,增加糖原合成,其作用机制可能涉及AMPK参与的抑制糖异生等生化调控过程起到降血糖作用。     

饲养方式对苏尼特羊肌内脂肪沉积途径AMPK-ACC-CPT1通路和肉品品质的影响

以放牧和舍饲两种饲养条件下12 月龄苏尼特羊股二头肌为实验材料,利用酶联免疫吸附测定法和实时荧光定量聚合酶链式反应法对肌内脂肪沉积动态平衡中脂肪酸β氧化一磷酸腺苷激活蛋白激酶（AMP-activated protein kinase,AMPK）-乙酰辅酶A羧化酶（acetyl-CoA carboxylase,ACC）-肉毒碱脂酰转移酶1（carnitine palmitoyltransferase,CPT1）通路所涉及的相关酶活力和质量浓度、相关基因mRNA表达量及肉品品质等指标进行测定,研究不同饲养方式对AMPK-ACC-CPT1信号通路相关指标、肌内脂肪含量及肉品品质产生的影响。结果表明：放牧饲养组羊股二头肌AMPK质量浓度、AMPK活力、CPT1质量浓度、AMPK mRNA表达量、亮度、黄度和剪切力显著大于舍饲饲养组（P＜0.05）;放牧饲养组羊股二头肌ACC活力、ACC mRNA表达量、肌内脂肪含量显著低于舍饲饲养组（P＜0.05）。由此可见,放牧饲养可以一定程度上激活AMPK-ACC-CPT1通路,从而减少肌内脂肪沉积,增加羊肉亮度和黄度,降低嫩度,进而影响肉品品质。     

藻蓝色素对胰岛素抵抗HepG2细胞糖代谢的影响

为探究藻蓝色素(phycocyanin, PC)体外改善胰岛素抵抗的作用机制,采用胰岛素体外诱导方式,分别设置5个胰岛素浓度梯度(10-9、10-8、10-7、10-6 、10-5μmol/L)以及6个时间梯度(0、12、24、36、48、72h)处理HepG2细胞,以葡萄糖消耗量和细胞存活率为指标,确定建立胰岛素抵抗型HepG2细胞模型的较佳作用浓度及时间。检测PC干预后HepG2细胞葡萄糖消耗量、糖原合成和存活率,利用RT-qPCR和Western blot探讨PC可能的作用机制。实验结果表明:胰岛素抵抗型HepG2细胞模型最佳诱导时间和浓度为36h、10-7μmol/L;不同浓度的PC可以提升胰岛素抵抗型HepG2细胞的葡萄糖消耗量;PC能够上调正常HepG2和胰岛素抵抗型HepG2细胞中IRS1、IRS2、GLUT1、GLUT4基因的转录水平,并且增加细胞中IRS1、AMPK、GSK-3β以及AKT蛋白的磷酸化水平。研究结果表明,PC能够显著提升胰岛素抵抗型HepG2细胞的葡萄糖消耗量,通过激活胰岛素调节相关的IRS1/AKT信号通路,增加AMPK的磷酸化和葡萄糖转运蛋白GLUT1、GLUT4基因表达,加速HepG2细胞对葡萄糖的利用和改善细胞胰岛素抵抗。研究结果显示,PC在预防或改善2型糖尿病肝脏胰岛素抵抗方面有潜在作用。     

AMPK活化调控的能量代谢对宰后牦牛肉肉色稳定性影响的研究

为探究牦牛肉在宰后成熟过程中单磷酸腺苷激活蛋白激酶（AMPK）活化调控的能量代谢对肉色稳定性的影响,本实验以牦牛背最长肌为研究对象,使用AMPK激活剂（AICAR）和抑制剂（Compound C）对其进行处理,并置于4 ℃下成熟,在相应的成熟时间点对AMPK活性、肉色及能量代谢相关指标进行测定,并进行肉色和能量代谢指标相关性分析。结果表明:成熟至12 h,与对照组相比,AICAR组AMPK活力提高了13.17%,而抑制组降低了9.03%,表明AICAR对AMPK活性有激活作用,而Compound C对AMPK活性有明显的抑制作用。随着成熟时间的延长,三者的L*值、a*值和己糖激酶的活性均呈先上升后下降趋势,b*值、H*值、乳酸含量均呈逐渐上升的趋势,其中L*值、b*值和H*值始终为AICAR组>对照组>Compound C组,a*值始终为AICAR组<对照组相似文献   

苦荞清蛋白酶解物对高糖诱导的HepG2细胞胰岛素抵抗的保护作用

目的：建立胰岛素抵抗HepG2细胞模型,观察苦荞清蛋白酶解物（tartary buckwheat albumin hydrolysate,TBAH）对HepG2细胞胰岛素抵抗的影响。方法：采用碱性蛋白酶水解苦荞清蛋白制备TBAH,超滤法截留分子质量小于3 kDa的TBAH;利用高糖高胰岛素诱导HepG2细胞36 h建立胰岛素抵抗模型,以2.5、5、10 μg/mL的TBAH培养细胞24 h。观察TBAH对HepG2细胞葡萄糖代谢的影响;检测细胞氧化损伤指标（一氧化氮（nitric oxide,NO）、丙二醛（malondialdehyde,MDA）、乳酸脱氢酶（lactic dehydrogenase,LDH）、超氧化物歧化酶（superoxide dismutase,SOD）和活性氧（reactive oxygen species,ROS））水平;Western blot检测胰岛素受体底物1（insulin receptor substrate 1,IRS-1）/磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase,PI3K）/蛋白激酶B（protein kinase B,Akt）信号通路中相关蛋白表达情况。结果：与IR模型组相比,TBAH组葡萄糖消耗量显著增多（P＜0.05）,且呈剂量依赖性,同时MDA、NO和ROS水平显著降低（P＜0.01,P＜0.05）,SOD活力显著升高（P＜0.05）,LDH活力显著下降（P＜0.05）;p-IRS-1蛋白表达水平显著降低（P＜0.05）,磷酸化Akt、PI3K、葡萄糖转运蛋白4表达水平极显著上升（P＜0.01）。结论：TBAH通过抑制氧化应激改善胰岛素抵抗。     

人参多糖对氧化应激损伤肝细胞的保护作用机制研究

目的:以氧化应激损伤模型,通过对人参中物质基础的筛选,研究其对氧化应激造成的肝细胞损伤的保护作用及其可能的作用机制的初步探讨。方法:采用浓度为25 μmol/L的过氧化氢溶液建立肝细胞损伤模型,对人参中总蛋白、总多糖、总皂苷的保护作用进行筛选。在此基础上,采用流式细胞术检测其凋亡程度、线粒体膜电位的改变,活性氧的含量变化。并采用ELISA法测肝糖原,超氧化物歧化酶(SOD)、丙二醛(MDA)、葡萄糖-6-磷酸酶(G6P)、磷酸烯醇式丙酮酸羧激酶(PEPCK)及ATP酶等指标的变化情况。结果:经过氧化氢诱导后的肝细胞,细胞活力极显著降低(P<0.01),通过对比人参中活性成分,发现人参多糖的保护作用较强,且呈现浓度依赖性;与模型组比较,人参多糖高剂量组大鼠肝细胞中MDA含量非常显著降低(P<0.001);中、高剂量组大鼠肝细胞中SOD含量显著升高(P<0.05);低剂量组大鼠肝细胞中G6P含量显著升高(P<0.05),中、高剂量组中G6P含量极显著升高(P<0.01);中剂量组大鼠肝细胞中PEPCK的含量显著升高(P<0.05),高剂量组中PEPCK的含量极显著升高(P<0.01);低、中剂量组大鼠肝细胞中ATP酶含量极显著升高(P<0.01);中、高剂量组大鼠肝细胞中肝糖原含量显著升高(P<0.05)。结论:人参多糖通过提高肝细胞中ATP酶的活力,升高线粒体膜电位来恢复肝细胞线粒体的功能,通过恢复G6P与PEPCK的含量来恢复肝脏糖异生的功能,同时通过升高SOD,降低MDA来减弱氧化应激带来的损伤,为后续研究氧化应激造成肝损伤提供一定的基础。     

滇结香花提取物组分1（Fr.1）对C2C12肌细胞胰岛素抵抗的改善作用

探讨滇结香花正己烷提取物组分1(Fr.1)改善棕榈酸(Palmitic acid,PA)诱导C2C12肌细胞产生胰岛素抵抗(Insulin Resistance,IR)的效果及其作用机制。方法:本文采用PA诱导C2C12肌细胞建立稳定的胰岛素抵抗细胞模型,通过检测Fr.1对胰岛素抵抗细胞(C2C12/IR)葡萄糖消耗量、葡萄糖摄取量的变化,研究其对PA介导的AMP活化蛋白激酶(AMPK)、胞内磷脂酰肌醇激酶/丝氨酸苏氨酸蛋白激酶(PI3K/AKT)信号通路中相关基因及蛋白表达的影响。结果:0.75mmol/L PA作用C2C12肌细胞16 h作为最佳建模条件;Fr.1在无细胞毒性范围内(12.5～100μg/mL)可显著增加C2C12/IR对葡萄糖的消耗量及摄取量;Fr.1可明显上调胰岛素受体(Insulin receptors,IRs)、胰岛素受体底物(Insulin receptor substrate-1,IRS-1)、葡萄糖转运蛋白4(Glut4)、PI3K、AMPKα基因的表达水平,并上调p-IRs、p-IRS-1、Glut4、p-AMPKα、磷酸化乙酰辅酶A羧化酶(p-ACC)、p-AKT蛋白质的表达水平,表明滇结香花提取物Fr.1可通过调控多个信号通路改善IR。     

Effect of silymarin on gluconeogenesis and lactate production in exercising rats

In this study, we investigated the effects of silymarin (SM) on gluconeogenesis during exercise in rats. After 4 weeks of exercise, blood samples, liver, and skeletal muscle tissues were collected, and the levels of triglycerides (TG), lactate, peroxisome proliferator activated receptor gamma (PPARγ), phosphoenol pyruvate carboxykinase (PEPCK), pyruvate dehydrogenase kinase 4 (PDK4), and phosphorylated 5-AMP activated protein kinase (AMPK) were measured. The TG and lactate level of the serum were reduced. In addition, the expression of Akt, PEPCK, and PPARγ in liver was decreased as well as the expression of AMPK in muscle. On the contrary, the level of PDK4 in muscle was increased. These results showed that that administration of SM ameliorated exerciseinduced gluconeogenesis and β-oxidation through the regulation of PPARγ, PEPCK, and PDK4. Thus, intake of SM during exercise may improve endurance by modulating of the metabolism of glucose, lipids, and lactate.     

刺梨果酒改善2型糖尿病大鼠糖脂代谢紊乱的研究

为探究刺梨果酒对2型糖尿病大鼠糖脂代谢紊乱的影响及可能机制。采用高脂高糖膳食联合腹腔注射链佐霉素（Streptozocin,STZ）建立2型糖尿病大鼠模型,将造模成功的大鼠分为刺梨果酒高（8 mL/kg）、中（4 mL/kg）、低（2 mL/kg）剂量组和模型组,并设空白对照组,给药期间每2周测一次空腹血糖,实验时间28 d。实验结束后测量血清和肝脏中高密度脂蛋白胆固醇（High-density lipoprotein cholesterol,HDL-C）、果糖胺（fructosamine,FMN）、甘油三酯（Triglyceride,TG）、低密度脂蛋白胆固醇（Low-density lipoprotein cholesterol,LDL-C）、总胆固醇（Total cholesterol,TC）、肝糖原等含量;采用实时灾光定量PCR（real time polymerase chain reaction,RT-PCR）测定肝脏中腺苷酸活化蛋白激酶（AMP-activated protein kinase α,AMPK）、乙酰辅酶A羧化酶（Acetyl-CoA carboxylases alpha,ACACA）、β-羟-β-甲戊二酸单酰辅酶A还原酶（3-hydroxy-3-methylglutaryl coenzyme A reductase,HMG-CoA）、脂肪酸合成酶（fatty acid synthetase,FASN）、和葡萄糖转运载体2（Glucose Transporter 2,GLUT2）、胆固醇7α-羟化酶（Cholesterol 7α-hydroxylase,CYP7A1）等mRNA相对表达量。结果表明,与模型组相比,刺梨果酒可减缓2型糖尿病大鼠体重减轻和多饮、多食的症状;高、中剂量刺梨果酒降低实验大鼠空腹血糖和果糖胺的效果显著（P<0.05）;各剂量组均有降低实验大鼠血清和肝脏TC、TG、LDL-C的含量和升高HDL-C含量的作用,其中高、中剂量效果显著（P<0.05）;低、中、高刺梨果酒均可显著（P<0.05）上调AMPK、GLUT2和ACACA mRNA表达量,低、中、高刺梨果酒均可上调FASN mRNA表达量,其中高、中剂量组上调FASN mRNA表达量显著（P<0.05）;中、高剂量刺梨果酒可显著（P<0.05）下调G6Pase、PEPCK、HMG-COA和CYP7A1 mRNA表达量。结论:刺梨果酒改善2型糖尿病大鼠糖脂代谢紊乱的机制可能与通过抑制内源性胆固醇、增加脂肪的从头合成及提高葡萄糖跨膜转速率有关。     

Milk performance and glucose metabolism in dairy cows fed rumen-protected fat during mid lactation

Feeding rumen-protected fat (RPF) can improve energy supply for dairy cows but it affects glucose metabolism. Glucose availability is a precondition for high milk production in dairy cows. Therefore, this study investigated endocrine regulation of glucose homeostasis and hepatic gene expression related to glucose production because of RPF feeding in lactating cows. Eighteen Holstein dairy cows during second lactation were fed either a diet containing RPF (mainly C16:0 and C18:1; FD; n = 9) or a control diet based on corn starch (SD; n = 9) for 4 wk starting at 98 d in milk (DIM). Feed intake and milk yield were measured daily and milk composition once a week. Blood samples were taken weekly for analyses of plasma triglyceride, nonesterified fatty acids (NEFA), β-hydroxybutyrate, bilirubin, urea, lactate, glucose, insulin, and glucagon. At 124 DIM, an intravenous glucose tolerance test (GTT; 1 g/kg of BW^0.75) was performed after a 12-h period without food. Blood samples were taken before and 7, 14, 21, and 28 min after glucose administration, and plasma concentrations of glucose, insulin, and glucagon were measured. Glucose half-life as well as areas under the concentration curve for glucose, insulin, and glucagon were calculated. After slaughter at d 28 of treatment, liver samples were taken to measure mRNA abundance of pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase, glucose 6-phosphatase (G6Pase), and facilitative glucose transporter 2. Dry matter intake, but not energy and protein intake, was lower in FD than in SD. Milk yield during lactation decreased more in SD than in FD, and milk protein was lower in FD than in SD. Plasma concentrations of triglycerides and NEFA were higher in FD than in SD. Plasma insulin concentrations were lower and the glucagon:insulin ratios were higher in FD than in SD. Fasting glucose concentration before GTT was lower, and fasting glucagon concentrations tended to be higher in FD than in SD. In liver, fat content tended to be higher and G6Pase mRNA abundance was lower in FD than in SD. Lower hepatic G6Pase mRNA abundance was associated with reduced fasting plasma glucose concentrations, but the glucose-induced insulin response was not affected by RPF feeding. Hepatic G6Pase gene expression might be affected by DMI and might be involved in the regulation of glucose homeostasis in dairy cows, resulting in a lower hepatic glucose output after RPF feeding.     

Korean red ginseng attenuates hepatic lipid accumulation via AMPK activation in human hepatoma cells

In this study, we examined Korean red ginseng (KRG) extract affects on the lipid metabolism in HepG2 cells. Increase in AMP-activated protein kinase (AMPK) phosphorylation was observed when the cells were treated with KRG. Activation of AMPK was also demonstrated by measuring the phosphorylation of acetyl-CoA caboxylase (ACC), a substrate of AMPK. KRG down-regulated gene expressions of sterol regulatory element binding protein 1c (SREBP1c) and its target proteins, such as fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD1) in time- and dose-dependent fashions. In contrast, gene expressions of peroxisome proliferator-activated receptor α (PPARα) and CD36 were increased. These effects were reversed in the presence of compound C, an AMPK inhibitor. However, there were no differences in gene expressions of SREBP2, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, and low-density-lipoprotein receptor (LDLR). Taken together, KRG induced supression of SREBP1c and activation of PPARα via AMPK and these effects seem to be one of anti-hyperlipidemic mechanism of KRG in HepG2 cells.