乙肝病毒X蛋白结合蛋白通过下调PEPCK的表达抑制肝脏糖异生（英文）

乙肝病毒X蛋白结合蛋白(hepatitis B X-interacting protein,HBXIP)可以调节乳腺癌中糖代谢重编程.为了研究HBXIP在生理条件下对糖代谢的调节作用及机制,本研究利用Cre/lox P重组酶系统成功构建了肝脏组织中HBXIP特异敲除小鼠.当小鼠接受刺激后,与正常组小鼠相比,肝脏HBXIP敲除小鼠表现基础糖代谢功能异常,如葡萄糖、丙酮酸;相对于对照小鼠,肝脏HBXIP敲除小鼠对糖异生和胰岛素耐受性减弱. RT-PCR、Western blot实验和免疫组化实验结果表明,HBXIP敲除小鼠肝脏组织中糖异生关键酶磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxykinase,PEPCK)表达显著增加. QRT-PCR分析30例临床肝组织中HBXIP m RNA和PEPCK m RNA表达水平发现,HBXIP与PEPCK表达水平呈负相关.荧光素酶报告基因实验和Ch IP实验结果表明HBXIP可以在基因转录水平调节PEPCK表达.以上结果表明,HBXIP通过调节糖异生关键酶PEPCK的表达参与调控小鼠肝脏糖异生.     

分泌性卷曲相关蛋白5表达上调对小鼠肝脏糖代谢的影响

分泌性卷曲相关蛋白5(secreted frizzled-related protein 5,SFRP5)是一种抗炎脂肪因子,它与糖尿病密切相关,具有抗糖尿病作用。将8周龄的C57BL/6J小鼠随机分为4组:普食-空载病毒组(SD+Ad-GFP组,n=10)、普食-SFRP5过表达病毒组(SD+Ad-SFRP5组,n=10)、高脂-空载病毒组(HFD+Ad-GFP组,n=10)、高脂-SFRP5过表达病毒组(HFD+Ad-SFRP5组,n=10),各组喂养13周后,利用定量PCR技术及Western-blot技术,分别检测小鼠肝脏糖异生关键酶磷酸烯醇式丙酮酸羧激酶(phosphoenolpyruvate carboxykinase,PEPCK)和葡萄糖-6-磷酸酶(glucose-6-phosphatase,G6Pase)mRNA及蛋白质水平的变化情况,以及肝脏经典胰岛素信号通路的改变。结果显示:高脂喂养组中,SFRP5表达上调可使肝脏糖异生指标PEPCK和G6Pase的mRNA及蛋白质相对表达水平均降低(P0.05),而且SFRP5过表达可使肝脏经典胰岛素信号通路指标胰岛素受体(InsR)及蛋白激酶B(AKT)的磷酸化水平增高(P0.05)。以上结果说明,C57BL/6J小鼠体内过表达SFRP5可能通过降低肝脏糖异生,增强胰岛素信号通路,从而改善机体胰岛素抵抗状态。     

低盐度可诱导鲈鱼胞浆型PEPCK基因表达

磷酸烯醇式丙酮酸羧激酶(PEPCK)催化草酰乙酸生成磷酸烯醇式丙酮酸,是糖异生途径的第1个限速酶.本研究用SMARTRACE技术从鲈鱼肝脏中分离克隆了PEPCK基因的全长cDNA序列.该基因全长2215bp,包含1个123bp的5′非翻译区和217bp的3′非翻译区,开放阅读框为1875bp,编码1个由624个氨基酸组成的蛋白质,该蛋白理论分子量为69.1kD,等电点为5.87.氨基酸序列分析表明,与其它动物的胞浆型PEPCK相似性很高,与黑鲷为94.2%,与大西洋鲑为86.4%,与人为75.9%,而与该鱼线粒体型PEPCK氨基酸同源性只有70.6%.系统发育分析显示,该蛋白首先与其它动物的cPEPCK聚成一支,然后再与鱼类的mPEPCK成簇,认为该PEPCK属于胞浆型.同时用RT-PCR分析了PEPCK基因在10个组织中的表达,结果表明只有在肝脏、消化道和肾脏有较高的表达.将鲈鱼从盐度为25的海水转入盐度为12的海水48h后,肝脏和肾脏的PEPCK基因表达有增加.实验结果表明,本实验克隆的为鲈鱼胞浆型PEPCK,低盐度可诱导其表达.     

饲料中糖水平对不同食性海水鱼类PEPCK基因表达和酶活性的影响

磷酸烯醇式丙酮酸羧激酶(Phosphoenolpyruvate carboxykinase, PEPCK, E.C.4.1.1.32)是水生生物糖异生代谢的关键限速酶. 实验以杂食性罗非鱼(Oreochromis niloticus)、温和肉食性卵形鲳鲹(Trachinotus ovatus)、凶猛肉食性军曹鱼(Rachycentron canadum)三种不同食性海水养殖鱼类为研究对象, 以糊精为饲料糖源, 分别设置不同饲料糖添加水平(低糖组LD、中糖组MD、高糖组HD)等氮等能饲料, 每种鱼分别随机选取60尾体格均匀的幼鱼进行为期8周的饲养实验, 同时克隆卵形鲳鲹胞质型PEPCK基因cDNA全长序列, 以期探讨不同饲料糖水平对不同食性鱼类PEPCK活性及其mRNA表达的影响. 结果显示: 卵形鲳鲹PEPCK基因cDNA共2652 bp, 含1个编码624个氨基酸的开放阅读框, 三种不同食性海水鱼类PEPCK的生物信息学比较分析显示相似度达90%以上, 在结构和功能上具有较高的保守和同源性. 养殖实验结果显示: 随着饲料糖水平的增加, 三种鱼肝脏中PEPCK酶活性均降低, 其中卵形鲳鲹、军曹鱼HD组PEPCK活性比LD组分别显著降低28.05%和26.03% (P0.05). 而其肝脏中PEPCK mRNA表达水平同样均随饲料碳水化合物水平增加而受到抑制, 其中罗非鱼、卵形鲳鲹、军曹鱼中LD组PEPCK的mRNA分别是HD组的100倍、4.3倍和4.77倍. 结果表明鱼类的糖异生能力可能与其食性有关, 三种鱼PEPCK酶活性与基因表达量随着饲料糖水平的增加而受到显著抑制, 且mRNA表达抑制程度随食性不同而具有较大差异, 以杂食性罗非鱼受抑制程度最高, 凶猛肉食性军曹鱼受抑制程度最低.       

父代糖尿病对子代糖代谢的长期影响

探讨父代糖尿病对子代老年期糖代谢的潜在影响。健康雄性SD大鼠经腹腔注射链脲佐菌素(35 mg/kg)建立雄性大鼠糖尿病模型,并与健康SD雌鼠交配所得子代为处理组(STZ-O组);健康雄鼠与健康雌鼠交配所得子代为正常对照组(CON-O组)。两组动物出生后记录出生体重,标准饲料喂养至18月龄,测定随机血糖含量、血清胰岛素含量;进行葡萄糖耐量试验(glucose tolerance test,GTT)、胰岛素耐量试验(insulin tolerance test,ITT)检测胰岛素抵抗情况;丙酮酸耐量试验(pyruvic acid tolerance test,PTT)检测肝脏糖异生情况;经糖原染色观察肝脏糖原沉积情况;以Real-time PCR检测肝脏组织糖异生途径关键酶磷酸烯醇式丙酮酸羧激酶(PEPCK)、葡萄糖-6-磷酸酶(G-6-P)、果糖-1,6-磷酸酶(FBP)mRNA表达水平。结果表明:父代糖尿病可导致子代在老年期出现胰岛素抵抗,同时伴有肝脏糖异生作用增强。其具体作用机制仍有待进一步研究。     

驱动蛋白-1在小鼠脂肪组织糖脂代谢中的作用研究

目的:本文旨在探讨动物体内水平驱动蛋白-1在脂肪组织糖、脂代谢中的作用。方法:通过Cre/Loxp重组系统构建脂肪组织特异性敲除驱动蛋白-1的小鼠模型,在生理水平观察驱动蛋白-1表达缺陷对小鼠糖代谢、脂代谢和脂肪因子分泌的影响。结果:与六月龄对照组小鼠相比,同月龄驱动蛋白-1敲除小鼠的体重、脂肪组织重量和空腹血糖水平没有显著差异,但是其血清胰岛素水平显著升高;使用葡萄糖耐量试验(GTT)和胰岛素耐量实验(ITT)对小鼠的糖代谢水平进行评估,结果显示驱动蛋白-1敲除小鼠表现为葡萄糖不耐受、胰岛素不耐受;进一步血清检测显示驱动蛋白-1敲除小鼠表现为高甘油三酯血症和血清脂联素水平降低。结论:驱动蛋白-1在脂肪组织中参与调节糖、脂代谢过程,其表达或功能障碍是2型糖尿病等代谢性疾病的一个重要的发病因素。     

乙肝病毒X 蛋白通过CREB 上调HBx结合蛋白促进肝癌细胞迁移

乙型肝炎病毒X蛋白（hepatitis B virus X protein,HBx）对肝癌的发生发展具有十分重要的作用. HBx 具有促进肝癌迁移的作用,但其作用的分子机制不清. 本研究对 HBx 促进肝癌细胞迁移的分子机制进行了探讨. 伤口愈合和 Boyden’s chamber结果表明,HBx 可明显促进肝癌 HepG2 细胞迁移. 在稳定转染 HBx 的 HepG2（HepG2-X）细胞中转染 HBx 结合蛋白（hepatitis B X-interacting protein,HBXIP）的 RNA 干扰片段,可明显抑制 HBx 的促迁移作用. 免疫组化和实时定量 PCR 结果表明,HBXIP 在肝癌组织中显著高表达,并且与 HBx 表达成正相关. 荧光素酶报告基因和免疫印迹结果表明,HBx 显著增强 HBXIP 的启动子活性和蛋白质表达水平. 应用 HBx 的 RNA 干扰处理 HepG2-X 细胞,HBXIP 的启动子活性和蛋白质表达水平明显下降.将 HBXIP 启动子区的cAMP效应元件结合因子（CREB）结合位点突变后,HBx 上调 HBXIP 的作用消失. 应用 CREB 的 RNA 干扰处理肝癌细胞,在启动子水平和蛋白质水平上, HBx 对 HBXIP 的上调作用被显著抑制. 染色质免疫共沉淀结果表明,HBx 能够通过 CREB 结合到 HBXIP 的启动子上,进而发挥激活 HBXIP 的功能. 本研究结果表明,HBx 促进肝癌细胞迁移的作用是通过 CREB 上调 HBXIP 实现的. 这一发现对进一步揭示 HBx 促进肝癌细胞迁移的分子机制具有重要意义.     

十二指肠-空肠转流手术对2型糖尿病大鼠胰岛素抵抗的改善作用

目的建立十二指肠-空肠转流手术(duodenal-jejunal bypass surgery,DJB)动物模型,观察术后GK大鼠胰岛素抵抗情况的变化,研究DJB手术治疗2型糖尿病的机理。方法雄性Wistar大鼠为空白对照组;雄性GK大鼠分为模型对照组和DJB手术组。分别于手术后3、6和9周每组随机抽取6只动物进行高胰岛素-正葡萄糖钳夹实验;钳夹实验结束后1周,检测肝脏Gc K、G6P以及PEPCK mRNA表达情况以及骨骼肌细胞膜GLUT4含量变化。结果术后3周和6周,DJB手术组动物的葡萄糖输注率(GIR)较模型对照组差异无显著性(P0.05),肝脏Gc K、G6P以及PEPCK mRNA表达量较模型对照组差异无显著性(P0.05);术后9周,DJB手术组动物的葡萄糖输注率(GIR)显著高于模型对照组(P0.05),肝脏Gc K表达量DJB手术组显著高于模型对照组(P0.05),而G6P以及PEPCK mRNA表达量显著低于模型对照组(P0.05);DJB手术后3、6和9周,DJB手术组骨骼肌细胞膜GLUT4的含量较模型对照组差异无显著性(P0.05)。结论 DJB手术改善血糖的水平是通过改善体内肝脏组织的胰岛素抵抗,通过调节糖代谢酶的表达,进而提高肝脏葡萄糖摄取并抑制肝脏糖异生作用。在实验周期内,DJB手术对于骨骼肌组织的胰岛素抵抗未发现有明显改善,提示DJB手术治疗2型糖尿病的效果与时间有一定关系。     

不同糖源及糖水平对大菱鲆糖代谢酶活性的影响

采用34双因素实验设计, 以初始质量为(8.060.08) g的大菱鲆幼鱼(Scophthalmus maximus L.)为对象, 研究在饲料中添加3种糖源(葡萄糖、蔗糖和糊精)及4个水平(0、5%、15%、28%)对大菱鲆肝脏糖酵解关键酶己糖激酶(HK)、葡萄糖激酶(GK)、磷酸果糖激酶(PFK)、丙酮酸激酶(PK)和糖异生关键酶磷酸烯醇式丙酮酸羧激酶(PEPCK)、1, 6-二磷酸果糖酶(FBPase)活性的影响。结果表明: 饲料糖添加量从0升高到15%时, 大菱鲆的糖酵解酶GK和PK活性随饲料葡萄糖或糊精含量的增加而增加; 当饲料中葡萄糖或糊精含量为28%时, GK和PK活性有下降的趋势。3种糖源的4个添加水平对HK和PFK活性均无显著影响(P 0.05)。添加不同水平的葡萄糖对大菱鲆糖异生途径的PEPCK活性无显著影响(P 0.05), 但在饲料中葡萄糖添加量为5%时显著促进了FBPase活性(P 0.05), 当葡萄糖添加量升高为15%或28%时, FBPase活性与对照组无显著差异(P 0.05)。糊精作为饲料糖源时抑制了大菱鲆肝脏FBPase和PEPCK的活性, 而添加不同水平的蔗糖对FBPase和PEPCK活性的影响均不显著(P 0.05)。总的来说, 从大菱鲆幼鱼肝脏糖代谢角度而言, 在饲料中添加15%的葡萄糖或糊精时, 可以有效促进大菱鲆肝脏糖酵解能力; 较添加葡萄糖, 糊精在促进大菱鲆肝脏糖酵解的同时对糖异生存在一定程度的抑制。蔗糖作为饲料糖源时, 仅在添加量为28%时显著促进糖酵解酶GK活性, 糖酵解其他酶活性以及糖异生酶活性均不受蔗糖水平的显著影响。       

乙肝病毒X蛋白结合蛋白最新研究进展

乙肝病毒X蛋白结合蛋白(HBXIP)是肝细胞癌变过程的一个关键因素,它能在动物肌肉组织和恶性肿瘤组织中过表达。近年来研究显示:HBXIP能与人体内多种蛋白结合。本文综述了HBXIP蛋白参与细胞凋亡与增殖、细胞周期进程、中心体复制、肿瘤细胞迁移等过程,以期为以HBXIP蛋白为靶标的新的抗乙肝病毒等药物设计提供基础。     

Cytosolic phosphoenolpyruvate carboxykinase does not solely control the rate of hepatic gluconeogenesis in the intact mouse liver

When dietary carbohydrate is unavailable, glucose required to support metabolism in vital tissues is generated via gluconeogenesis in the liver. Expression of phosphoenolpyruvate carboxykinase (PEPCK), commonly considered the control point for liver gluconeogenesis, is normally regulated by circulating hormones to match systemic glucose demand. However, this regulation fails in diabetes. Because other molecular and metabolic factors can also influence gluconeogenesis, the explicit role of PEPCK protein content in the control of gluconeogenesis was unclear. In this study, metabolic control of liver gluconeogenesis was quantified in groups of mice with varying PEPCK protein content. Surprisingly, livers with a 90% reduction in PEPCK content showed only a approximately 40% reduction in gluconeogenic flux, indicating a lower than expected capacity for PEPCK protein content to control gluconeogenesis. However, PEPCK flux correlated tightly with TCA cycle activity, suggesting that under some conditions in mice, PEPCK expression must coordinate with hepatic energy metabolism to control gluconeogenesis.     

HIF-1α is necessary to support gluconeogenesis during liver regeneration

Coordinated recovery of hepatic glucose metabolism is prerequisite for normal liver regeneration. To examine roles of hypoxia inducible factor-1α (HIF-1α) for hepatic glucose homeostasis during the reparative process, we inactivated the gene in hepatocytes in vivo. Following partial hepatectomy (PH), recovery of residual liver weight was initially retarded in the mutant mice by down-regulation of hepatocyte proliferation, but occurred comparably between the mutant and control mice at 72 h after PH. At this time point, the mutant mice showed lowered blood glucose levels with enhanced accumulation of glycogen in the liver. The mutant mice exhibited impairment of hepatic gluconeogenesis as assessed by alanine tolerance test. This appeared to result from reduced expression of PGK-1 and PEPCK since 3-PG, PEP and malate were accumulated to greater extents in the regenerated liver. In conclusion, these findings provide evidence for roles of HIF-1α in the regulation of gluconeogenesis under liver regeneration.     

Hepatic mechanisms of the Walnut antidiabetic effect in mice

The present study was designed to explore the mechanism of action of walnut (the seed of Juglans regia) leaf and ridge on hepatic glucose metabolism in diabetic mice. Experimental diabetes was induced by intravenous administration of streptozotocin (60 mg/kg)and confirmed with an increase of blood glucose, 90–100% of the control, 72 hours later. Isolated extracts from walnut leaf and ridges were administered in a single effective dose of 400 mg/kg orally. Firstly, blood glucose was determined every 1 hour until 5 hours post administration of extracts. In the second experiment, the liver was surgically removed, 2 hours post treatment of diabetic animals with extracts, homogenized and used for measurement of key enzymes of glycogenolysis (glycogen phosphorylase, GP) and gluconeogenesis (phosphoenolpyruvate carboxykinase, PEPCK). Treatment by both leaf and ridge extracts decreased blood glucose and liver PEPCK activity and increased blood insulin and liver GP activity. It is concluded that walnut is able to lower blood glucose through inhibition of hepatic gluconeogenesis and secretion of pancreatic insulin.     

Orphan Nuclear Receptor Small Heterodimer Partner Negatively Regulates Growth Hormone-mediated Induction of Hepatic Gluconeogenesis through Inhibition of Signal Transducer and Activator of Transcription 5 (STAT5) Transactivation

Growth hormone (GH) is a key metabolic regulator mediating glucose and lipid metabolism. Ataxia telangiectasia mutated (ATM) is a member of the phosphatidylinositol 3-kinase superfamily and regulates cell cycle progression. The orphan nuclear receptor small heterodimer partner (SHP: NR0B2) plays a pivotal role in regulating metabolic processes. Here, we studied the role of ATM on GH-dependent regulation of hepatic gluconeogenesis in the liver. GH induced phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase gene expression in primary hepatocytes. GH treatment and adenovirus-mediated STAT5 overexpression in hepatocytes increased glucose production, which was blocked by a JAK2 inhibitor, AG490, dominant negative STAT5, and STAT5 knockdown. We identified a STAT5 binding site on the PEPCK gene promoter using reporter assays and point mutation analysis. Up-regulation of SHP by metformin-mediated activation of the ATM-AMP-activated protein kinase pathway led to inhibition of GH-mediated induction of hepatic gluconeogenesis, which was abolished by an ATM inhibitor, KU-55933. Immunoprecipitation studies showed that SHP physically interacted with STAT5 and inhibited STAT5 recruitment on the PEPCK gene promoter. GH-induced hepatic gluconeogenesis was decreased by either metformin or Ad-SHP, whereas the inhibition by metformin was abolished by SHP knockdown. Finally, the increase of hepatic gluconeogenesis following GH treatment was significantly higher in the liver of SHP null mice compared with that of wild-type mice. Overall, our results suggest that the ATM-AMP-activated protein kinase-SHP network, as a novel mechanism for regulating hepatic glucose homeostasis via a GH-dependent pathway, may be a potential therapeutic target for insulin resistance.