高脂饮食大鼠的胰岛素抵抗及肝脂肪酸的代谢
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摘要
糖尿病主要分为1型和2型糖尿病,2型糖尿病患者占糖尿病患病人数90%以上。胰岛素抵抗(insulin resistance, IR)是2型糖尿病的基本特征。虽然造成胰岛素抵抗的机制还没有搞清楚,但游离脂肪酸(free fatty acid, FFA),如软脂酸(C16:0)、硬脂酸(C18:0)、二十二烷酸(C22:0)、二十四烷酸(C24:0)在血浆中的升高,以及脂肪在肝组织的沉积,引起人们高度关注。静脉注射英脱利匹特(Intralipid)能提高循环中的FFA浓度,可导致胰岛素抵抗的发生,证实FFA可能是胰岛素抵抗的独立危险因素。那么在高脂食物诱导的胰岛素抵抗中, FFA是否是引起胰岛素抵抗的关键因素之一呢?
肝脏是胰岛素抵抗和脂肪酸代谢的重要器官。肝脏脂肪酸的代谢变化对血中脂肪酸水平和其它组织脂肪酸的代谢有重大的影响。FFA是过氧化物酶体增殖物激活受体α(PPARα)的天然配体。正常生理条件下,当血中游离脂肪酸增多时,FFA激活PPARα,使参与脂肪酸摄入,脂肪酸氧化的某些蛋白表达增多,从而加速肝脏的脂肪酸摄取以及线粒体和过氧化物酶体的脂肪酸β-氧化。与此同时,肝脏通过二酰基甘油酰基转移酶(DGAT)的催化,将摄入的脂肪酸合成脂肪,通过极低密度脂蛋白(VLDL)转运到肝外。胰岛素抵抗和糖尿病状态下,脂肪在肝组织沉积是否与脂肪合成的限速酶DGAT的表达增强有关?肝中FFA水平以及某些极长链脂肪酸的增加,是否与肝脂肪酸分解代谢紊乱,脂肪酸的氧化不足所致有关?未见报道。
短链、中链以及长链的脂肪酸在线粒体经β-氧化、三羧酸循环、氧化磷酸化生成水和二氧化碳以及ATP的过程中,在呼吸链水平可产生氧自由基;而极长链脂肪酸在过氧化物酶体经β-氧化的过程中,脂酰CoA氧化酶以O2为直接受氢体,产生过氧化氢。因此,脂肪酸,特别是极长链脂肪酸在细胞内的蓄积,不仅可引起生物膜中磷脂所含脂肪酸种类的变化,而且还会产生大量的活性氧,损伤细胞的结构和功能。高脂食物引起胰岛素抵抗和糖尿病,对肝脏的脂毒性,是否是因脂肪酸,特别是极长链脂肪酸在肝脏中堆积,通过直接的细胞毒和间接产生活性氧所致呢?
为了研究这些问题,我们用高脂饮食长期喂养SD大鼠诱导胰岛素抵抗并进一步形成糖尿病,并给原代培养的肝细胞造成高脂肪酸环境。从整体和细胞水平,观察血、肝组织、肝细胞的脂肪酸的量和种类的变化,以及脂肪酸的去路(合成脂肪和脂肪酸β-氧化)和不同途径活性(线粒体和过氧化物酶体)的变化,探讨过氧化物酶体脂肪酸β-氧化与高脂饮食诱导的胰岛素抵抗和2型糖尿病肝游离脂肪酸量和种类变化的关系,以及肝脂肪变性形成的机制。
第一部分高脂饮食与胰岛素抵抗的发生
目的:通过动态观察血液生化指标的变化,探讨高脂饲料诱导大鼠产生胰岛素抵抗的过程中游离脂肪酸与胰岛素抵抗的关系。
方法:雄性Sprague-Dawley(SD)大鼠,随机分为高脂组(HF)和对照组(Con),Con组喂养标准饲料,HF组喂养高脂饲料,脂类以猪油为主,热量比占59.8%。HF组分别于2、4、6和8周,用口服糖耐量试验(OGTT),胰岛素敏感指数(ISI)和正常葡萄糖-高胰岛素血糖钳夹(euglycaemic hyperinsulinaemic clamp)等方法检测胰岛素抵抗;用快速血糖测定仪检测血糖,用氧化酶法测定血中甘油三酯、胆固醇,用放免法检测血胰岛素浓度,用铜试剂显色法检测血清中总脂肪酸浓度。8周末,部分HF组大鼠诱导糖尿病,组成糖尿病组(DM)。各组继续喂养6周后处死,用气相色谱法检测血中脂肪酸谱的变化(详细操作见第二部分)。
结果:
1体重及血生化指标
喂养4周时HF组大鼠体重明显超过Con组(HF组为286.0±16.0g,Con组为250.2±15.1g,P<0.01),血中甘油三酯浓度增高(HF组为1.14±0.14 mmol/L,Con组为0.92±0.17 mmol/L,P<0.01),FFA总量增多(HF组为323.52±35.83μmol/L,Con组为272.65±50.21μmol/L,P<0.05),但此时空腹血糖、血中胆固醇和胰岛素浓度差异无统计学意义(P>0.05);6周时HF组胆固醇(2.05±0.29 mmol/L)和胰岛素(28.43±14.5μIU/ml)浓度高于Con组胆固醇(1.69±0.14 mmol/L)和胰岛素(15.28±3.97μIU/ml),差异有统计学意义(P<0.01和P<0.01),两组的空腹血糖差异仍无统计学意义;8周时HF组大鼠空腹血糖(5.85±0.10 mmol/L)明显高于Con组(5.32±0.39 mmol/L),差异有统计学意义(P<0.05),HF组血中总FFA浓度为406.69±70.47μmol/L,高于Con组299.52±59.17μmol/L(P<0.01)。随着喂养时间的增加,两组大鼠血中葡萄糖、甘油三酯、胆固醇、脂肪酸和胰岛素浓度的差异均增大。
2胰岛素敏感指数(ISI)
高脂饲料喂养2周,HF组和Con组的ISI分别为-4.53±0.19和-4.41±0.28,两组差异无统计学意义(P>0.05)。喂养4周、6周和8周,HF组和Con组的ISI分别为-4.71±0.21和-4.47±0.25、-4.95±0.52和-4.45±0.30、-5.31±0.26和-4.60±0.14,两组相比差异均有统计学意义(P<0.05, P<0.01和P<0.01)。高脂饲料喂养4周时,HF组大鼠开始对胰岛素的敏感度下降,并且,随喂养时间的延长下降幅度逐渐增大。
3糖耐量结果
大鼠空腹12h,用50%葡萄糖空腹灌胃(2g/kg)。2周时,OGTT各时间点两组血糖均无差别;4周时,HF组30和60min血糖浓度增高(P<0.05, P<0.01),0和120min差异无统计学意义;6周时,HF组30,60和120min血糖浓度明显高于Con组(P<0.05, P<0.01, P<0.01),而两组0min血糖差异仍无统计学意义;8周时,两组大鼠各时间点血糖均出现差异(P<0.05, P<0.05, P<0.01, P<0.01)。从高脂喂养4周开始,HF组糖耐量出现异常。
4血糖钳夹结果
大鼠高脂喂养6周,葡萄糖钳夹实验结果表明HF组输注率20.44±1.99mg/kg.min较Con组27.97±1.72 mg/kg.min减低(P <0.01),表明HF组大鼠出现明显的胰岛素抵抗。
5 IR与血脂的相关性
4周时,甘油三酯与ISI的相关系数是-0.368(P>0.05);胆固醇与ISI的相关系数是-0.288(P>0.05);总FFA与ISI的相关系数是-0.511(P<0.05)。8周时,甘油三酯与ISI的相关系数是-0.771 (P<0.01);胆固醇与ISI的相关系数是-0.820 (P<0.01);总FFA与ISI的相关系数是-0.856(P<0.01)。4周时,总FFA与ISI具有相关性,甘油三酯和胆固醇与ISI无相关性;8周时三者与ISI均具有相关性。
6血中游离脂肪酸谱的变化(14周)
在本试验条件下C18:1、C18:2和C18:3未能完全分离(文中统称为C18烯),但其它游离脂肪酸分离较好。与Con组相比,DM组血清FFA除C20:0(18.91±2.49 mg/L)、C24:0(4.18±1.05 mg/L)浓度不变(P>0.05), C20:5(19.33±4.47 mg/L)浓度降低外(P<0.05),其他C16:0(112.21±13.40 mg/L)、C18:0(2.84±13.73 mg/L)、C18:0烯(218.32±33.95 mg/L)、C20:4(63.50±12.02 mg/L)、C22:0(5.75±1.40 mg/L)、C22:6(6.13±0.79 mg/L)、C26:0(8.61±1.47 mg/L)浓度均升高(P<0.05); HF组C16:0(73.37±13.03 mg/L )、C18:0(51.40±7.87 mg/L)、C18烯( 176.07±22.27 mg/L )、C22:6(5.33±1.48 mg/L)浓度浓度升高,C20:5(17.71±2.95 mg/L)浓度降低(P<0.05),其它脂肪酸C20:0(17.06±2.32 mg/L)、C20:4(35.43±6.90 mg/L)、C22:0(4.42±1.36 mg/L)、C24:0(3.79±0.84 mg/L)、C26:0(5.60±1.20 mg/L)浓度不变(P>0.05)。
各游离脂肪酸占总脂肪酸相对百分比也发生变化。DM组大鼠血清中C18烯(32.14±3.50) %、C20:4(1.85±0.26) %、C22:6(9.49±1.35) %和C26:0(1.18±0.18) %上升,其它C16:0、C18:0、C20:0、C20:5、C22:0和C24:0不变。HF组在血清中C18烯比值(34.96±3.21)%上升,C20:5(0.64±0.09)%下降(P<0.05),C16:0、C18:0、C20:0、C20:4、C22:0、C22:6、C24:0和C26:0不变(P>0.05)。DM组的C20:4、C22:6和C26:0相对百分比高于HF组(P<0.05)。
小结:
1胰岛素抵抗的发生和抵抗程度与高脂饮食诱导的时间有关,胰岛素抵抗加大到一定程度,血糖浓度明显增加。
2高脂诱导的胰岛素抵抗与血游离脂肪酸浓度的升高有密切关系。
第二部分高脂诱导的胰岛素抵抗大鼠肝中脂质的变化
目的:观察高脂诱导胰岛素抵抗状态下,肝脏脂质(脂肪和游离脂肪酸)的量以及肝脂肪酸β-氧化活性的变化,探讨高脂诱导胰岛素抵抗与肝脏脂肪酸代谢的关系。
方法:为了快速获得胰岛素抵抗并发糖尿病的模型,给高脂诱导8周产生胰岛素抵抗的大鼠,禁食12h后,一次性腹腔注射2% STZ (27mg/kg)。注射后72h,尾静脉采血,测定禁食12h后的空腹血糖,血糖浓度大于7.8 mmol/L的大鼠继续高脂喂养6周,为胰岛素抵抗并发糖尿病组(DM)。同时HF组大鼠和Con组大鼠分别用高脂饲料和标准饲料继续喂养6周。各组随机选择8只大鼠,进行下一步试验。动物禁食12h,3%戊巴比妥60mg/kg腹腔麻醉后动脉插管取血,血清用于血总脂肪酸(铜试剂显色法)和游离脂肪酸谱(气相色谱法)的测定;取肝脏,用于测定肝组织的脂肪沉积(油红O染色法)和脂肪的量(酶法),脂肪酸β-氧化的活性(紫外吸收法)以及肝游离脂肪酸谱(气相色谱法)。
结果:
1肝组织脂肪的含量
Con组肝组织脂肪的含量为11.30±1.89 mg/g,HF组为34.94±4.93 mg/g, DM组为51.47±7.77mg/g。DM组脂肪含量是Con组4.55倍,是HF组的1.47倍。各组之间差异均有统计学意义(P<0.01)。
2油红染色检测肝组织脂肪沉积油红染色显示肝组织细胞轮廓清晰,Con组仅发现极少量脂肪滴的存在;HF组和DM组出现大量弥漫性脂肪滴,表明高脂喂养的胰岛素抵抗大鼠肝组织脂肪沉积。
3肝组织脂肪酸β-氧化活性
3.1脂肪酸β-氧化总活性DM组和HF组脂肪酸β-氧化总活性(10.76±0.81mU/mg pro,9.10±1.31mU/mg pro)明显高于Con组(6.46±1.07 mU/mg pro,P<0.01)。
3.2过氧化物酶体脂肪酸β-氧化活性DM组和HF组过氧化物酶体脂肪酸β-氧化活性(4.41±1.10 mU/mg pro, 4.13±0.82 mU/mg pro)明显高于Con组(3.21±0.55 mU/mg pro,P<0.05)。
3.3线粒体脂肪酸β-氧化的活性
DM组和HF组肝线粒体脂肪酸β-氧化活性(6.35±1.85mU/mg pro,4.97±1.86 mU/mg pro)明显高于Con组(3.25±1.20 mU/mg pro,P<0.01, P<0.05)。
4肝脏总游离脂肪酸含量
DM组和HF组肝总游离脂肪酸含量(112.79±22.24μmol/g,97.01±16.02μmol/g)明显高于Con组(66.03±15.00μmol/g,P<0.01)。表明肝脏脂肪酸增多。
5肝脏游离脂肪酸谱的变化
在本试验条件下C20:5未能检测到,C18:1、C18:2、C18:3未能完全分离(本文通称C18烯)。与Con组相比,DM组C18烯(24.31±3.50 mg/g)、C20:4(5.06±0.82 mg/g)、C22:6(1.41±0.23 mg/g)和C26:0(2.49±0.46 mg/g)含量增多(P<0.05);C18:0(5.56±1.09 mg/g)、C20:0(0.50±0.08 mg/g)、C22:0(0.13±0.02 mg/g)和C24:0(0.12±0.02 mg/g)含量减少(P<0.05);C16:0含量没有变化(P>0.05)。与Con组相比,HF组肝脏中C18烯(21.89±3.92 mg/g)含量增多(P<0.05);C20:0(0.82±0.19 mg/g)、C22:0(0.19±0.08 mg/g)和C24:0(0.14±0.08 mg/g)含量减少(P<0.05);其它(C16:0、C18:0、C20:4、C22:6和C26:0)含量没有变化(P>0.05)。比较DM组和HF组脂肪酸含量发现,C20:0、C22:6和C26:0三种脂肪酸在两组之间存在统计学差异,DM组C20:0浓度低于HF(P<0.05),C22:6和C26:0高于HF组(P<0.05)。
各游离脂肪酸占总脂肪酸相对百分比的变化也发生变化。与Con组相比,DM组游离脂肪酸百分比C18烯(36.67±3.31)%、C22:6(2.59±0.49)%和C26:0 (3.16±0.64)%增多(P<0.05);C20:0(0.16±0.04)%、C22:0(0.16±0.01) %、C24:0(0.44±0.09) %下降(P<0.05);其他(C16:0、C18:0和C20:4)不变。与Con组比较,HF组大鼠肝脏中C18烯(37.24±3.06) %增多(P<0.05);C20:0(0.12±0.03)%、C22:0(0.19±0.04)%、C24:0(0.51±0.11)%比值下降(P<0.05);其它不变(C16:0、C18:0、C20:4、C22:6和C26:0)(P>0.05)。DM组的C22:6和C26:0相对百分比高于HF组(P<0.05)。
小结:
1长期食用高脂食物的大鼠,血游离脂肪酸的增加,促使肝脏线粒体、过氧化物酶体以及总的脂肪酸β-氧化活性增加,但不足以分解肝脏大量摄入的脂肪酸,致使肝组织中脂肪、总的游离脂肪酸以及有的极长链脂肪酸的增多。
2高脂诱导的胰岛素抵抗(单纯抵抗和伴糖尿病的)与肝脏脂肪、游离脂肪酸总量和某些极长链脂肪酸的增多有关。
第三部分高脂诱导的胰岛素抵抗大鼠肝中脂质代谢有关基因的表达变化
目的:通过检测胰岛素抵抗大鼠和胰岛素抵抗伴糖尿病大鼠肝脏与脂肪酸代谢有关酶蛋白基因的表达,探讨肝脏脂质代谢变化的原因。
方法:用PT-PCR法检测参与脂肪酸代谢的有关基因的mRNA水平,免疫印迹检法测D-双功能蛋白(DBP)的蛋白水平。
结果:
1参与甘油三酯合成的二酰基甘油酰基转移酶(DGAT1)mRNA的水平
DM组和HF组的DGAT1 mRNA水平(0.38±0.06,0.26±0.06)均较Con组(0.12±0.02)高(P<0.01,P<0.05)。表明DM组和HF组肝脂肪合成能力增强与DGAT1转录增加有关。
2线粒体脂肪酸β-氧化的限速酶肉毒碱脂酰转移酶Ⅰ(CPTⅠ)mRNA的水平
DM组和HF组(0.58±0.10,0.37±0.04)的CPTⅠmRNA水平均较Con组(0.26±0.02)高(P<0.01,P<0.05),且DM组高于HF组(P<0.01)。表明DM组和HF组肝线粒体分解脂肪酸能力增强与CPTⅠ转录增加有关。
3过氧化物酶体脂肪酸β-氧化途径中的软脂酰辅酶A氧化酶(ACOX1)、降植烷酰辅酶A氧化酶(ACOX3)、L-双功能蛋白(LBP)和D-双功能蛋白(DBP)的mRNA水平
DM组和HF组ACOX1 mRNA的水平(1.51±0.10,1.74±0.25)较Con组(1.12±0.22)髙(P<0.01, P<0.05);DM组和HF组的LBP mRNA水平(0.66±0.12,0.59±0.04)较Con组(0.47±0.09)髙(P<0.01, P<0.05)。提示过氧化物酶体LBP参与的β-氧化途径活性增强。
DM组和HF组(0.88±0.15,1.08±0.19)与Con组(1.01±0.10)的AOX3 mRNA水平差异无统计学意义;而DM组的DBP mRNA水平(0.13±0.01)较Con组和HF组(0.21±0.05,0.26±0.06)低(P<0.05)。提示DM组过氧化物酶体DBP参与的β-氧化途径活性减弱。
4肝DBP蛋白的表达量
DM组肝DBP的蛋白表达量(0.43±0.05)明显低于Con组(0.75±0.12,P<0.01)和HF组(0.81±0.11,P<0.01)。DM组DBP蛋白表达减弱与mRNA转录水平一致,进一步说明DBP表达减少。
5过氧化物酶体增殖物受体α(PPARα)mRNA的变化。
DM组和HF组的PPARαmRNA水平(0.96±0.07,0.90±0.08)均较Con组(0.78±0.07)高(P<0.01,P<0.05)。结合其下游基因CPTⅠ、ACOX1和LBP的转录增强,说明PPARα被激活。
小结:
1高脂诱导的胰岛素抵抗大鼠(单纯抵抗和伴糖尿病的)肝中脂肪的沉积与甘油三酯合成的限速酶DGAT1基因表达增加有关。
2高脂诱导的胰岛素抵抗大鼠(单纯抵抗和伴糖尿病的)肝中线粒体、过氧化物酶体脂肪酸β-氧化活性的增强,与PPARα的转录水平增加并被激活,进而上调CPTⅠ、ACOX1、LBP等基因的表达有关。
3高脂诱导胰岛素抵抗伴糖尿病大鼠肝中某些极长链脂肪酸如C26:0的增加可能与DBP表达减少,DBP参与的脂肪酸β-氧化活性减弱有关。
第四部分游离脂肪酸对原代培养大鼠肝细胞脂肪酸代谢的影响
目的:以原代培养的肝细胞为对象,观察细胞外游离脂肪酸引起肝细胞胰岛素抵抗以及对脂肪酸代谢有关基因表达的影响。证明血中高浓度的游离脂肪酸是高脂饮食诱导胰岛素抵抗的重要因素之一。
方法:采用200-250g正常SD大鼠。麻醉后,用胶原酶Ⅳ灌注肝脏,分离肝细胞。将原代培养的肝细胞随机分为对照组(Con组)和软脂酸组(PA组)。PA组用200μmol/L的软脂酸作用24 h,RT-PCR测定PEPCK1、DGAT1、CPTⅠ、AOX1、LBP和DBP的mRNA水平,Western blotting检测DBP蛋白水平,气相色谱检测肝细胞内游离脂肪酸谱。
结果:
1糖代谢有关基因PEPCK1的mRNA表达PEPCK1在PA组的mRNA的表达量(1.12±0.13)比Con组(0.73±0.15)明显增加( P < 0. 01)。说明胰岛素抑制糖异生作用减弱,胰岛素抵抗出现。
2脂肪酸代谢有关基因的表达
DGAT1在PA组的mRNA量(0.39±0.09)的表达量比Con组(0.25±0.07)明显增加(P<0.05)。CPTⅠ的mRNA量表达量(1.07±0.23)比Con组(0.72±0.16)增加(P<0.05),ACOX1在PA组的mRNA量表达量(2.00±0.22)比Con组(1.61±0.15)增加(P<0.05),LBP在PA组的mRNA量表达量(0.76±0.13)比Con组(0.50±0.09)增加(P<0.01)。DBP在Con组(0.90±0.22)和PA组(0.78±0.18)mRNA量差异无统计学意义(P>0.05)。表明高浓度脂肪酸存在条件下,原代培养的肝细胞脂肪合成和脂肪酸β-氧化增强。
3 DBP蛋白表达
PA组DBP蛋白相对表达量(0.66±0.08),与Con组(0.70±0.09)相比差异无统计学意义(P>0.05)。这一结果与整体试验HF组DBP蛋白的表达相似,而与DM组不同。
小结:
1游离脂肪酸可引起原代培养肝细胞参与脂肪酸代谢的某些基因在转录水平发生改变。这些变化类似于高脂喂养的胰岛素抵抗大鼠肝同种基因变化。
2软脂酸(200μmol/L)未引起培养肝细胞DBP基因表达的变化,这一点与整体研究中糖尿病大鼠不同。
结论:
1血中游离脂肪酸的增加及由此造成的肝组织脂肪和游离脂肪酸的沉积是高脂饮食诱导胰岛素抵抗的重要因素。
2高脂饮食诱导的胰岛素抵抗大鼠(单纯抵抗和伴糖尿病的)肝脏中,脂肪的沉积与甘油三酯合成的限速酶DGAT1的基因表达增加有关。
3高脂饮食诱导的胰岛素抵抗大鼠(单纯抵抗和伴糖尿病的)肝脏中,虽然PPARα的转录水平增加并被激活,进而上调CPTⅠ、ACOX1、LBP等基因的表达,使脂肪酸β-氧化活性增强,但不足以分解肝脏大量摄入的脂肪酸。
4高脂诱导的胰岛素抵抗伴糖尿病大鼠肝中,某些极长链脂肪酸(如C26:0)的增加可能与DBP表达减少,DBP参与的脂肪酸β-氧化活性减弱有关。
Western-like-diet and lifestyle, especially excessive lipid intake, is accused for epidemic of obesity and insulin resistance, as well as the rapid increase of type 2 diabetes. Diabetes can be classified into two types, type 1 and 2. The latter accounts for more than 90% of diabetes. Insulin resistance, the decreased response to insulin, is a key feature of type 2 diabetes. Although how insulin resistance occurs remains unclear,the focus has been concentrated on the elevation of free fatty acid(FFA), such as palmitic acid (C16:0), stearic acid (C18:0), docosanoic acid (C22:0) and isoselachoceric acid (C24:0), and the accumulation of triglyceride in liver. Intralipid infused in vein, which can elevate the level of circulation FFA, results in insulin resistance, proving that FFA may be an independent risk factor in the developement of insulin resistance. Then, whether FFA is one of the key factors in the insulin resistance induced by high-fat diet.
The liver is a vital organ in fatty acid metabolism and insulin resistance. The variations of fatty acids metabolism in liver have a profound effect on the levels of serum fatty acids and affect the fatty acid metabolism in other organizations. FFA is a natural ligand of peroxisome proliferator-activated receptorα(PPARα). In normal physiological conditions, the increased FFA in the blood activates PPARα, which enhances the expression of proteins involved in the intake and oxidation of fatty acids, and therefore accelerate the intake andβ- oxidation of fatty acids in mitochondria and peroxisome in liver. At the same time, the excessive FFA is esterified to fat in liver, which is catalyzed by diacylglycerol acyltransferases (DGAT), and then diverted to other organs by very low density lipoprotein (VLDL). In insulin resistance and diabetes, whether the elevated levels of total FFA and some fatty acids in liver are caused by the abnormal lipid metabolism and abatedβ-oxidation and whether the fat deposition in liver is related to enhanced expression of DGAT that is a limit enzyme in fat synthesis, have not been reported.
Short, medial and long chain fatty acids can be catabolized in mitochondria byβ-oxidation, tricarboxylic cycle and oxidative phosphorylation to produce ATP and CO2. During this process, oxygen free radicals are produced in respiratory chain level. Very long chain fatty acids must be handled in peroxisomal byβ-oxidation. In this condition, Acyl-CoA oxidases act as a rate-limiting enzyme, which donates electrons directly to molecular oxygen and generates H2O2. Therefore, accumulation of very long chain fatty acids in cells can not only cause changes of fatty acids types contained in phospholipid of biomembrane, but also generate a lot of reactive oxygen species that damage the cell structure and function. Is the lipotoxicity to liver in insulin resistance and diabetes, which were induced by high-fat diet, caused by accumulation of fatty acids especially the very long chain fatty acids?
In order to study these issues, we developed insulin resistance in Sprague-Dawley rat by high-fat diet, and further formation of diabetes, and primary culture hepatocytes in high level fatty acids environment. To explore the relation between peroximalβ-oxidation and the variance of quantity and kinds of FFA in liver of insulin resistance and type 2 diabetes states induced by high-fat diet, the changes in quantity and kinds of fatty acids of blood, liver and primary culture hepatocytes were detected, and the changes of fatty acids outlet(triglycerides synthesis and fatty acid oxidation) and the activities of differentβ-oxidation ways were also detected from integeral and cell level.
PartⅠHigh fat diet and occurrence of insulin resistance
Objective: By observing the dynamic changes in blood biochemical parameters, the relationship between FFA and insulin resistance developement was explored in high-fat diet rat.
Methods: Male SD rats were divided randomly into control group (Con group) and high fat group (HF group). The rats in Con group were fed with standard diet and HF group, high-fat diet. lipid in the high-fat diet consisted mainly of lard-based, accounting for 59.8 percent of calory. After feeding for 2, 4, 6 and 8 weeks in high-fat diet, the rats were detected for insulin resistance by oral glucose tolerance (OGTT), insulin sensitivity index (ISI) and euglycaemic-hyperinsulinaemic clamp. Blood sugar was test with rapid blood glucose detector. Blood triglycerides and cholesterol were measured by oxidase method and blood insulin concentration was detected by RIA, and serum total FFA concentration was detected with detection kit. After 8 weeks, some HF rats developed to dibetes by streptozotocin(DM group). All rats were fed continuely for 6 weeks, and sacrificed, and the blood fatty acids profiles were evaluated by gas chromatography.
Results:
1 Biochemical parameters in the blood
At 4th week, the body weights of HF group exceeded that of Con group(HF. 286.0±16.0g,Con.250.2±15.1g,P<0.01), and so did the blood triglycerides levels (HF. 1.14±0.14 mmol/L,Con. 0.92±0.17 mmol/L,P<0.01) and the FFA levels (HF. 323.52±35.83μmol/L,Con. 272.65±50.21μmol/L,P<0.05). However, fasting blood sugar, cholesterol and blood insulin concentration showed no significant difference (P>0.05) between the two groups at this time point. At 6th week, blood cholesterol (2.05±0.29 mmol/L) and insulin (28.43±14.5μIU/ml) concentrations of HF group were are higher than those of Con group (cholesterol, 1.69±0.14 mmol/L and insulin, 15.28±3.97μIU/ml), there were statistical differences between two groups(P<0.01 and P<0.01). But there was no statistical difference in blood sugar levels between two groups. At 8th week, fasting blood sugar(5.85±0.10 mmol/L) of HF group was obviously higher than that of Ctrl group(5.32±0.39 mmol/L), and the difference was statistically significan(tP<0.05). Total blood FFA concentration in HF group (406.69±70.47μmol/L) was higher than that of Con group(299.52±59.17μmol/L) (P<0.01). Along with the duration of high-fat feeding increases, the differences of blood glucose, triglycerides, cholesterol, FFA and insulin concentration between the two groups were increasing.
2 insulin sensitivity index (ISI)
2 weeks after high fat diet, ISIs in HF group and Con group were -4.53±0.19 and -4.41±0.28, respectively. there was no statistical difference(P>0.05)between the two groups. At the time points of 4,6,8 weeks, ISIs of HF and Ctrl group were -4.71±0.21 and -4.47±0.25, -4.95±0.52 and -4.45±0.30,-5.31±0.26 and -4.60±0.14, respectively. There were statistical differences(P<0.05, P<0.01 and P<0.01)between the two groups. 4 weeks after the rats were fed high fat diet, the insulin sensitivity in HF group rats began to decease, which progressed time dependently.
3 Oral glucose tolerance test (OGTT) After fasting for 12 h, rats were gavaged with 50% glucose (2 g / kg), and then the blood glucose levels were tested. At the time point of 2 weeks, there was no difference at each time point in OGTT. At 4 weeks, 30 min and 60 min blood sugars were higher in HF group than that of Ctrl. group, there were statistical differences. In the rats fed in high-fat for 6 weeks, the blood sugar at 30 min, 60 min and 120 min time points were higer than that of Ctrl group, but at 0 min, the difference has no statistical significance.
4 Result of euglycaemic hyperinsulinaemic clamp
At the time point of 6 weeks, glucose infusion rate(GIR) of HF group was 20.44±1.99mg/kg.min ,which was lower than that of Ctrl. group(27.97±1.72 mg/kg.min) (P <0.01). The results showed that HF group rats developed obvious insulin resistance.
5 Correlation between IR and blood lipid.
At the time point of 4 weeks, correlation coefficient of triglycerides and ISI was -0.368 (P>0.05), that of cholesterol and ISI was -0.288 (P >0.05) and that of FFA and ISI was -0.511(P<0.05). At the time point of 8 weeks, correlation coefficient of triglycerides and ISI was -0.771 (P<0.01), that of cholesterol and ISI was -0.82 (P<0.01) and that of FFA and ISI was -0.856(P<0.01). Blood triglycerides, cholesterol and FFA were correlated to ISI.
6 Changes of blood FFAs
In our research conditions, C18:1,C18:2 and C18:3 could not be separated completely, while other fatty acids were separated well. When compared with Con, the concentrations of C20:0(18.91±2.49 mg/L), C24:0(4.18±1.05 mg/L)of DM group did not change(P>0.05), C20:5(19.33±4.47 mg/L)decreased, and the other fatty acids, such as C16:0, C18:0, C20:4, C22:0, C22:6 and C26:0, increased(P<0.05) In HF group, the concentrations of C16:0 (73.37±13.03 mg/L), C18:0(51.40±7.87 mg/L), C18:1+C18:2+C18:3 (176.07±22.27 mg/L), C22:6(5.33±1.48 mg/L) increased, C20:5 (17.71±2.95 mg/L) decreased ( P<0.05 ), and the concentrations of other fatty acid, such as C20:0, C20:4, C22:0, C24:0 and C26:0, were unchanged (P>0.05), when compared with control group. Percentages of different fatty acids in the total fatty acid were also changed. C18:1+C18:2+C18:3 (32.14±3.50) %, C20:4(1.85±0.26) %, C22:6 (9.49±1.35) % and C26:0(1.18±0.18) % in the serum of DM group increased, and the other fatty acids (C16:0, C18:0, C20:0, C20:5, C22:0 and C24:0) showed no change. C18:1+C18:2+C18:3 (4.96±3.21)% in the serum of HF group increased, C20:5(0.64±0.09)% of HF group decreased(P<0.05), and the other fatty acids (C16:0, C18:0, C20:0, C20:4, C22:0, C22:6, C24:0 and C26:0) showed no change (P<0.05).
Conclusion:
1 Occurrence and degree of insulin resistance were related to the inducing time, blood sugar concentration began to increase obviously when insulin resistance reached to certain degree.
2 Increase of FFA had a close relation with occurrence of insulin resistance induce by high-fat diet.
PartⅡChanges of hepatic lipids in rats with insulin resistance induced by high-fat diet
Objective: To observe changes of hepatic lipids(fat and FFA) and fatty acidβ-oxidation, and investigate the relation between insulin resistance inducing by high fat diet and hepatic fatty acid metabolism.
Methods: In order to establish quickly the animal model with insulin resistance and diabetes, HF group rats that had developed insulin resistance, were administered with 2% STZ (27 mg/kg) by peritoneal injection. The blood sugar levels of tail vains were evaluated 72 hours later. The rats whose fasting blood sugar was higher than 7.8 mmol/L were fed continuely with high-fat diet for 6 weeks, and grouped as insulin resistance complicated with diabetes group (DM). Con and HF groups were also fed for 6 weeks with standard diet and high-fat diet respectively. Eight rats were selected randomly from each group for next test. The rats were intraperitoneally anesthetized with pentobarbital sodium (60 mg/kg) in the fasting state. Blood was collected by carotid artery bloodletting, and the serum was used for the detection of blood total FFA and FFA profiles, and the liver was used for the detection of liver fatty deposition(oil red o staining), amount of fat (oxidase way), fatty acidβ-oxidation activities (ultraviolet absorption) and liver FFA profiles.
Results:
1 Contents of liver fat
Compared with Con group(11.30±1.89 mg/g), content of liver fat of HF group(34.94±4.93 mg/g) and DM group(51.47±7.77mg/g) increased remarkly (P<0.01).
2 Detected fatty deposition in liver by oil red o staining
Little fat drops could be found in Ctrl. group, but more fat drops were found in DM and HF groups. Results of oil red o staining showed that rats with insulin resistance reduced by high-fat diet had severe fat deposition.
3 Fatty acidβ-oxidation of liver tissue
3.1 Total activities of fatty acidβ-oxidation
Compared with Con group (6.46±1.07 mU/mg pro), total activities ofβ-oxidation of DM group (10.76±0.81mU/mg pro) and HF group (9.10±1.31mU/mg pro) increased remarkablely (P<0.01).
3.2 Activities ofβ-oxidation of peroxisome Compared with Con group (3.21±0.55 mU/mg pro), the activities of peroximalβ-oxidation of DM group (4.41±1.10 mU/mg pro) and HF group (4.13±0.82mU/mg pro) increased (P<0.05).
3.3 Activities ofβ-oxidation of mitochondria
Compared with Con group (3.25±1.20 mU/mg pro), the activities of mitochondriaβ-oxidation of DM group (6.35±1.85mU/mg pro) and HF group (4.97±1.86mU/mg pro) increased (P<0.01, P<0.05).
4 Total FFA contents in livers
Compared with Con group (66.03±15.00μmol/g), the total FFA contents of DM group (112.79±22.24μmol/g) and HF group (97.01±16.02μmol/g) increased (P<0.01, P<0.05).
5 Changes of liver FFA profiles measured by gas chromatography
C20:5 could not be detected and C18:1, 18:2, 18:3 could not be separated completely by the gas chromatography equipment we used. In comparison with Con, C18:1+C18:2+C18:3 (24.31±3.50 mg/g), C20:4 (5.06±0.82 mg/g), C22:6 (1.41±0.23 mg/g) and C26:0 (2.49±0.46 mg/g) contained in liver increased (P<0.05), C18:0(5.56±1.09 mg/g), C20:0 (0.50±0.08 mg/g), C22:0 (0.13±0.02 mg/g) and C24:0 (0.12±0.02 mg/g) decreased (P<0.05), and C16:0 was unchanged(P>0.05)in DM group。In HF group the fatty acids C18:1+C18:2+C18:3 (21.89±3.92 mg/g) contained in liver increased (P<0.05), C20:0 (0.82±0.19 mg/g), C22:0 (0.19±0.08 mg/g) and C24:0 (0.14±0.08 mg/g) decreased (P<0.05), and the other fatty acids (C16:0、C18:0、C20:4、C22:6和C26:0) were unchanged (P>0.05) when compared with control group. There were statistic differences of contents of C20:0、C22:6 and C26:0 between DM group and HF group.
The percentage of each fatty acid in the composition of total fatty acid was also changed. Compared with Con, the percent of C18:1+C18:2+C18:3 (36.67±3.31)%, C22:6 (2.59±0.49)% and C26:0 (3.16±0.64) % increased (P<0.05), that of C20:0 (0.16±0.04) %, C22:0 (0.16±0.01) %, C24:0 (0.44±0.09) % decreased(P<0.05), and that of other fatty acids (C16:0、C18:0 and C20:4) were unchanged in DM group. Compared with Con group, percent fatty acid composition of C18:1+C18:2+C18:3 (37.24±3.06)% of HF group increased (P<0.05), that of C20:0 (0.12±0.03) %, C22:0 (0.19±0.04) % and C24:0 (0.51±0.11) % decreased (P<0.05), and that of other fatty acids (C16:0、C18:0、C20:4、C22:6 and C26:0) were unchanged (P>0.05). Percent fatty acid compositions of C22:6 and C26:0 of DM group were higher than HF group (P<0.05).
Conclusion:
1 Increase of blood FFA of the rats fed with high-fat diet for a long time promoted the activities of mitochondria, peroximal and total oxidation. Although having increased activities ofβ-oxidation, the liver failed to disposal the large amount of fatty acid and it resulted in increase of fat, total fatty acid and some very long chain fatty acid.
2 Insulin resistance induced by high-fat diet correlated with increased hepatic triglycerides, total fatty acids and some very long chain fatty acids.
PartⅢChanges in expression of genes involved in lipid metabolism in liver of high-fat diet induced insulin resistance rat
Objective: To investigate the changes in lipid metabolism of liver by detecting the expression of the genes involved in fatty acid metabolism in liver of insulin resistance rats and diabetes rats.
Methods: The expression of genes involved in fatty acid metabolism was detected with RT-PCR. The level of the D-bifunctional protein(DBP) was detected with western blotting.
Results:
1 The mRNA expression of diacylglycerol acyltransferase 1 (DGAT1) involved in triglyceride synthesis
The DGAT1 mRNA of DM and HF groups (0.38±0.06,0.26±0.06) were higher than Con group (0.12±0.02, P<0.01, P<0.05), which indicated that triglyceride synthesis in DM and HF group increased.
2 The mRNA expression of carnitine palmitoyl transferaseⅠ(CPTⅠ), the rate-limiting enzyme in mitochondria fatty acidβ-oxidation.
The CPT I mRNA of DM and HF groups (0.58±0.10,0.37±0.04) were higher than Con group (0.26±0.02, P<0.01,P<0.05) and that of DM group was higher than HF group (P<0.01), which indicated that mitochondria fatty acidβ-oxidation in DM and HF groups increased.
3 The mRNA expression of fatty acyl-CoA oxidase 1(ACOX1), fatty acyl-CoA oxidase 3 (ACOX3), D-bifunctional protein(DBP), L-bifunctional protein (LBP)
The ACOX1 mRNA expression of DM and HF groups (1.51±0.10,1.74±0.25) was higher than Con group (1.12±0.22, P<0.01,P<0.05) and the LBP mRNA expression of DM and HF groups (0.66±0.12,0.59±0.04) was higher than Con group (0.47±0.09, P<0.01,P<0.05), which indicated that peroxisomal fatty acid oxidation involved of LBP of HF and DM groups was increased.
DM and HF groups compared to Con group(0.88±0.15,1.08±0.19 vs 1.01±0.10, p>0.05), the changes of expression of ACOX3 mRNA have no statistical significance. The DBP mRNA of DM group(0.13±0.01)was lower than Con and HF groups (0.21±0.05,0.26±0.06, P<0.05), which indicated that the activity of fatty acid oxidation in peroxisome involved of DBP was decreased.
4 The changes of peroxisome proliferator-activated receptorα(PPARα) mRNA expression
The PPARαmRNA of DM and HF groups (0.96±0.07,0.90±0.08) were higher than Con group (0.78±0.07, P<0.01,P<0.05) and the transcription of downstream genes (CPTⅠ、ACOX1、LBP) increased , which indicated that mitochondria fatty acid oxidation in DM and HF group increased
5 The expression of DBP protein in liver
The expression of DBP protein in DM group (0.43±0.05, n=4)was lower than Con group(0.75±0.12,P<0.01)and HF group(0.81±0.11,P<0.01). It was coincident with the expression of DBP mRNA, which indicated that the expression of DBP decreased.
Conclusion:
1 In high-fat diet induced insulin resistance rats and diabetes rats, triglyceride accumulated in liver. It associated with increased expression of DGAT1, the rate-limiting enzyme in triglyceride synthesis.
2 In the liver of high-fat diet induced insulin resistance rats and diabetes rats, mitochondria and peroxisome fatty acid oxidation increased. It associated with increased transcription and activation of PPARαand up-regulated genes of CPTⅠ、ACOX1、LBP.
3 In the liver of high-fat-induced insulin resistance and diabetes rats, some very long chain fatty acids ,such as C26:0, were increased. It associated with decreased expression of DBP and decreased activity of fatty acid oxidation involved of DBP.
PartⅣEffects of free fatty acids on metabolism of fatty acids in primary cultured rat hepatocytes
Objective: To observe the effects of FFA which outside of hepatocytes on IR and on expression of the genes which involve in the metabolism of fatty acids based on the primary cultured hepotocytes.
Methods: SD rats weighing between 200 and 250 g were anesthetized with thiopental and hepatocytes were isolated after collagenaseⅣperfusion of the livers. Primary cultured hepatocytes were divided randomly into: control group (Con group), palmitic acid group(PA group). PA group was administered with 200μmol/L PA for 24 h. The expression of PEPCK1, CPTⅠ, DGAT1, AOX1, LBP and DBP mRNA were observed by RT-PCR and the expression of DBP protein were observed by Western blotting.
Results:
1 Expression of PEPCK1 mRNA
The PEPCK1 mRNA level in PA group (1.12±0.13) was significantly higher than that of Con group (0.73±0.15) ( P < 0. 01). The results showed that the effect of insulin’s inhibition to glyconeogenesis was reduced, and IR was proved.
2 Expression of mRNA of genes involved in the metabolism of fatty acids
The DGAT1 mRNA level was higher in PA group (0.39±0.09) than that of Con group (0.25±0.07) (P<0.05). The CPTⅠmRNA level was higher in PA group (1.07±0.23) than that of Con group (0.72±0.16) (P<0.05), the ACOX1 mRNA level was higher in PA group (2.00±0.22) than that of Con group (1.61±0.15) (P<0.05) and the LBP mRNA level was higher in PA group (0.76±0.13) than that of Con group (0.50±0.09) (P<0.01). However, difference of the DBP mRNA level between PA(0.78±0.18) and Con (0.90±0.22) have no statistic significance (P>0.05). The results showed that the metabolism of fatty acids in primary cultured liver cells altered.
3 The protein level of DBP
The DBP protein levels between PA group (0.87±0.11) and Con group (0.80±0.13) have no statistic significance (P>0.05). The result was similar to DBP expression of HF group, but was different from DM group in integral experiment.
Conclusion:
1 The expressions of some genes involved in fatty acids metabolism were altered in transcriptive level by FFA. These changes were similar to the changes of rat liver of insulin resistance induced by high-fat diet.
2 Expression of DBP gene of liver cell cultured in palmitic acid (200μmol/L) was not altered, which is different from that of DM group in integrated research.
Summary:
1 Increase of free fatty acids in blood and accumulations of fat and free fatty acids resulted from the increase of free fatty acids in blood were vital factors that contributed to insulin resistance induced by a long term of high-fat diet.
2 In high-fat-induced insulin resistance rats and diabetes rats, triglyceride accumulates in liver. It associated with increased expression of DGAT1, the rate-limiting enzyme in triglyceride synthesis.
3 In the liver of high-fat-induced insulin resistance rats and diabetes rats, fatty acidβ-oxidation increased. It associated with increased transcription and activation of PPARαand up-regulated genes of CPTⅠ, ACOX1 and LBP. But it failed to consume the large mount of fatty acid uptaken by liver.
4 In the liver of high-fat-induced insulin resistance and diabetes rats, some very long chain fatty acid (C26:0) was increased. It associated with decreased expression of DBP and decreased activity of fatty acid oxidation involved of DBP.
肝脏是胰岛素抵抗和脂肪酸代谢的重要器官。肝脏脂肪酸的代谢变化对血中脂肪酸水平和其它组织脂肪酸的代谢有重大的影响。FFA是过氧化物酶体增殖物激活受体α(PPARα)的天然配体。正常生理条件下,当血中游离脂肪酸增多时,FFA激活PPARα,使参与脂肪酸摄入,脂肪酸氧化的某些蛋白表达增多,从而加速肝脏的脂肪酸摄取以及线粒体和过氧化物酶体的脂肪酸β-氧化。与此同时,肝脏通过二酰基甘油酰基转移酶(DGAT)的催化,将摄入的脂肪酸合成脂肪,通过极低密度脂蛋白(VLDL)转运到肝外。胰岛素抵抗和糖尿病状态下,脂肪在肝组织沉积是否与脂肪合成的限速酶DGAT的表达增强有关?肝中FFA水平以及某些极长链脂肪酸的增加,是否与肝脂肪酸分解代谢紊乱,脂肪酸的氧化不足所致有关?未见报道。
短链、中链以及长链的脂肪酸在线粒体经β-氧化、三羧酸循环、氧化磷酸化生成水和二氧化碳以及ATP的过程中,在呼吸链水平可产生氧自由基;而极长链脂肪酸在过氧化物酶体经β-氧化的过程中,脂酰CoA氧化酶以O2为直接受氢体,产生过氧化氢。因此,脂肪酸,特别是极长链脂肪酸在细胞内的蓄积,不仅可引起生物膜中磷脂所含脂肪酸种类的变化,而且还会产生大量的活性氧,损伤细胞的结构和功能。高脂食物引起胰岛素抵抗和糖尿病,对肝脏的脂毒性,是否是因脂肪酸,特别是极长链脂肪酸在肝脏中堆积,通过直接的细胞毒和间接产生活性氧所致呢?
为了研究这些问题,我们用高脂饮食长期喂养SD大鼠诱导胰岛素抵抗并进一步形成糖尿病,并给原代培养的肝细胞造成高脂肪酸环境。从整体和细胞水平,观察血、肝组织、肝细胞的脂肪酸的量和种类的变化,以及脂肪酸的去路(合成脂肪和脂肪酸β-氧化)和不同途径活性(线粒体和过氧化物酶体)的变化,探讨过氧化物酶体脂肪酸β-氧化与高脂饮食诱导的胰岛素抵抗和2型糖尿病肝游离脂肪酸量和种类变化的关系,以及肝脂肪变性形成的机制。
第一部分高脂饮食与胰岛素抵抗的发生
目的:通过动态观察血液生化指标的变化,探讨高脂饲料诱导大鼠产生胰岛素抵抗的过程中游离脂肪酸与胰岛素抵抗的关系。
方法:雄性Sprague-Dawley(SD)大鼠,随机分为高脂组(HF)和对照组(Con),Con组喂养标准饲料,HF组喂养高脂饲料,脂类以猪油为主,热量比占59.8%。HF组分别于2、4、6和8周,用口服糖耐量试验(OGTT),胰岛素敏感指数(ISI)和正常葡萄糖-高胰岛素血糖钳夹(euglycaemic hyperinsulinaemic clamp)等方法检测胰岛素抵抗;用快速血糖测定仪检测血糖,用氧化酶法测定血中甘油三酯、胆固醇,用放免法检测血胰岛素浓度,用铜试剂显色法检测血清中总脂肪酸浓度。8周末,部分HF组大鼠诱导糖尿病,组成糖尿病组(DM)。各组继续喂养6周后处死,用气相色谱法检测血中脂肪酸谱的变化(详细操作见第二部分)。
结果:
1体重及血生化指标
喂养4周时HF组大鼠体重明显超过Con组(HF组为286.0±16.0g,Con组为250.2±15.1g,P<0.01),血中甘油三酯浓度增高(HF组为1.14±0.14 mmol/L,Con组为0.92±0.17 mmol/L,P<0.01),FFA总量增多(HF组为323.52±35.83μmol/L,Con组为272.65±50.21μmol/L,P<0.05),但此时空腹血糖、血中胆固醇和胰岛素浓度差异无统计学意义(P>0.05);6周时HF组胆固醇(2.05±0.29 mmol/L)和胰岛素(28.43±14.5μIU/ml)浓度高于Con组胆固醇(1.69±0.14 mmol/L)和胰岛素(15.28±3.97μIU/ml),差异有统计学意义(P<0.01和P<0.01),两组的空腹血糖差异仍无统计学意义;8周时HF组大鼠空腹血糖(5.85±0.10 mmol/L)明显高于Con组(5.32±0.39 mmol/L),差异有统计学意义(P<0.05),HF组血中总FFA浓度为406.69±70.47μmol/L,高于Con组299.52±59.17μmol/L(P<0.01)。随着喂养时间的增加,两组大鼠血中葡萄糖、甘油三酯、胆固醇、脂肪酸和胰岛素浓度的差异均增大。
2胰岛素敏感指数(ISI)
高脂饲料喂养2周,HF组和Con组的ISI分别为-4.53±0.19和-4.41±0.28,两组差异无统计学意义(P>0.05)。喂养4周、6周和8周,HF组和Con组的ISI分别为-4.71±0.21和-4.47±0.25、-4.95±0.52和-4.45±0.30、-5.31±0.26和-4.60±0.14,两组相比差异均有统计学意义(P<0.05, P<0.01和P<0.01)。高脂饲料喂养4周时,HF组大鼠开始对胰岛素的敏感度下降,并且,随喂养时间的延长下降幅度逐渐增大。
3糖耐量结果
大鼠空腹12h,用50%葡萄糖空腹灌胃(2g/kg)。2周时,OGTT各时间点两组血糖均无差别;4周时,HF组30和60min血糖浓度增高(P<0.05, P<0.01),0和120min差异无统计学意义;6周时,HF组30,60和120min血糖浓度明显高于Con组(P<0.05, P<0.01, P<0.01),而两组0min血糖差异仍无统计学意义;8周时,两组大鼠各时间点血糖均出现差异(P<0.05, P<0.05, P<0.01, P<0.01)。从高脂喂养4周开始,HF组糖耐量出现异常。
4血糖钳夹结果
大鼠高脂喂养6周,葡萄糖钳夹实验结果表明HF组输注率20.44±1.99mg/kg.min较Con组27.97±1.72 mg/kg.min减低(P <0.01),表明HF组大鼠出现明显的胰岛素抵抗。
5 IR与血脂的相关性
4周时,甘油三酯与ISI的相关系数是-0.368(P>0.05);胆固醇与ISI的相关系数是-0.288(P>0.05);总FFA与ISI的相关系数是-0.511(P<0.05)。8周时,甘油三酯与ISI的相关系数是-0.771 (P<0.01);胆固醇与ISI的相关系数是-0.820 (P<0.01);总FFA与ISI的相关系数是-0.856(P<0.01)。4周时,总FFA与ISI具有相关性,甘油三酯和胆固醇与ISI无相关性;8周时三者与ISI均具有相关性。
6血中游离脂肪酸谱的变化(14周)
在本试验条件下C18:1、C18:2和C18:3未能完全分离(文中统称为C18烯),但其它游离脂肪酸分离较好。与Con组相比,DM组血清FFA除C20:0(18.91±2.49 mg/L)、C24:0(4.18±1.05 mg/L)浓度不变(P>0.05), C20:5(19.33±4.47 mg/L)浓度降低外(P<0.05),其他C16:0(112.21±13.40 mg/L)、C18:0(2.84±13.73 mg/L)、C18:0烯(218.32±33.95 mg/L)、C20:4(63.50±12.02 mg/L)、C22:0(5.75±1.40 mg/L)、C22:6(6.13±0.79 mg/L)、C26:0(8.61±1.47 mg/L)浓度均升高(P<0.05); HF组C16:0(73.37±13.03 mg/L )、C18:0(51.40±7.87 mg/L)、C18烯( 176.07±22.27 mg/L )、C22:6(5.33±1.48 mg/L)浓度浓度升高,C20:5(17.71±2.95 mg/L)浓度降低(P<0.05),其它脂肪酸C20:0(17.06±2.32 mg/L)、C20:4(35.43±6.90 mg/L)、C22:0(4.42±1.36 mg/L)、C24:0(3.79±0.84 mg/L)、C26:0(5.60±1.20 mg/L)浓度不变(P>0.05)。
各游离脂肪酸占总脂肪酸相对百分比也发生变化。DM组大鼠血清中C18烯(32.14±3.50) %、C20:4(1.85±0.26) %、C22:6(9.49±1.35) %和C26:0(1.18±0.18) %上升,其它C16:0、C18:0、C20:0、C20:5、C22:0和C24:0不变。HF组在血清中C18烯比值(34.96±3.21)%上升,C20:5(0.64±0.09)%下降(P<0.05),C16:0、C18:0、C20:0、C20:4、C22:0、C22:6、C24:0和C26:0不变(P>0.05)。DM组的C20:4、C22:6和C26:0相对百分比高于HF组(P<0.05)。
小结:
1胰岛素抵抗的发生和抵抗程度与高脂饮食诱导的时间有关,胰岛素抵抗加大到一定程度,血糖浓度明显增加。
2高脂诱导的胰岛素抵抗与血游离脂肪酸浓度的升高有密切关系。
第二部分高脂诱导的胰岛素抵抗大鼠肝中脂质的变化
目的:观察高脂诱导胰岛素抵抗状态下,肝脏脂质(脂肪和游离脂肪酸)的量以及肝脂肪酸β-氧化活性的变化,探讨高脂诱导胰岛素抵抗与肝脏脂肪酸代谢的关系。
方法:为了快速获得胰岛素抵抗并发糖尿病的模型,给高脂诱导8周产生胰岛素抵抗的大鼠,禁食12h后,一次性腹腔注射2% STZ (27mg/kg)。注射后72h,尾静脉采血,测定禁食12h后的空腹血糖,血糖浓度大于7.8 mmol/L的大鼠继续高脂喂养6周,为胰岛素抵抗并发糖尿病组(DM)。同时HF组大鼠和Con组大鼠分别用高脂饲料和标准饲料继续喂养6周。各组随机选择8只大鼠,进行下一步试验。动物禁食12h,3%戊巴比妥60mg/kg腹腔麻醉后动脉插管取血,血清用于血总脂肪酸(铜试剂显色法)和游离脂肪酸谱(气相色谱法)的测定;取肝脏,用于测定肝组织的脂肪沉积(油红O染色法)和脂肪的量(酶法),脂肪酸β-氧化的活性(紫外吸收法)以及肝游离脂肪酸谱(气相色谱法)。
结果:
1肝组织脂肪的含量
Con组肝组织脂肪的含量为11.30±1.89 mg/g,HF组为34.94±4.93 mg/g, DM组为51.47±7.77mg/g。DM组脂肪含量是Con组4.55倍,是HF组的1.47倍。各组之间差异均有统计学意义(P<0.01)。
2油红染色检测肝组织脂肪沉积油红染色显示肝组织细胞轮廓清晰,Con组仅发现极少量脂肪滴的存在;HF组和DM组出现大量弥漫性脂肪滴,表明高脂喂养的胰岛素抵抗大鼠肝组织脂肪沉积。
3肝组织脂肪酸β-氧化活性
3.1脂肪酸β-氧化总活性DM组和HF组脂肪酸β-氧化总活性(10.76±0.81mU/mg pro,9.10±1.31mU/mg pro)明显高于Con组(6.46±1.07 mU/mg pro,P<0.01)。
3.2过氧化物酶体脂肪酸β-氧化活性DM组和HF组过氧化物酶体脂肪酸β-氧化活性(4.41±1.10 mU/mg pro, 4.13±0.82 mU/mg pro)明显高于Con组(3.21±0.55 mU/mg pro,P<0.05)。
3.3线粒体脂肪酸β-氧化的活性
DM组和HF组肝线粒体脂肪酸β-氧化活性(6.35±1.85mU/mg pro,4.97±1.86 mU/mg pro)明显高于Con组(3.25±1.20 mU/mg pro,P<0.01, P<0.05)。
4肝脏总游离脂肪酸含量
DM组和HF组肝总游离脂肪酸含量(112.79±22.24μmol/g,97.01±16.02μmol/g)明显高于Con组(66.03±15.00μmol/g,P<0.01)。表明肝脏脂肪酸增多。
5肝脏游离脂肪酸谱的变化
在本试验条件下C20:5未能检测到,C18:1、C18:2、C18:3未能完全分离(本文通称C18烯)。与Con组相比,DM组C18烯(24.31±3.50 mg/g)、C20:4(5.06±0.82 mg/g)、C22:6(1.41±0.23 mg/g)和C26:0(2.49±0.46 mg/g)含量增多(P<0.05);C18:0(5.56±1.09 mg/g)、C20:0(0.50±0.08 mg/g)、C22:0(0.13±0.02 mg/g)和C24:0(0.12±0.02 mg/g)含量减少(P<0.05);C16:0含量没有变化(P>0.05)。与Con组相比,HF组肝脏中C18烯(21.89±3.92 mg/g)含量增多(P<0.05);C20:0(0.82±0.19 mg/g)、C22:0(0.19±0.08 mg/g)和C24:0(0.14±0.08 mg/g)含量减少(P<0.05);其它(C16:0、C18:0、C20:4、C22:6和C26:0)含量没有变化(P>0.05)。比较DM组和HF组脂肪酸含量发现,C20:0、C22:6和C26:0三种脂肪酸在两组之间存在统计学差异,DM组C20:0浓度低于HF(P<0.05),C22:6和C26:0高于HF组(P<0.05)。
各游离脂肪酸占总脂肪酸相对百分比的变化也发生变化。与Con组相比,DM组游离脂肪酸百分比C18烯(36.67±3.31)%、C22:6(2.59±0.49)%和C26:0 (3.16±0.64)%增多(P<0.05);C20:0(0.16±0.04)%、C22:0(0.16±0.01) %、C24:0(0.44±0.09) %下降(P<0.05);其他(C16:0、C18:0和C20:4)不变。与Con组比较,HF组大鼠肝脏中C18烯(37.24±3.06) %增多(P<0.05);C20:0(0.12±0.03)%、C22:0(0.19±0.04)%、C24:0(0.51±0.11)%比值下降(P<0.05);其它不变(C16:0、C18:0、C20:4、C22:6和C26:0)(P>0.05)。DM组的C22:6和C26:0相对百分比高于HF组(P<0.05)。
小结:
1长期食用高脂食物的大鼠,血游离脂肪酸的增加,促使肝脏线粒体、过氧化物酶体以及总的脂肪酸β-氧化活性增加,但不足以分解肝脏大量摄入的脂肪酸,致使肝组织中脂肪、总的游离脂肪酸以及有的极长链脂肪酸的增多。
2高脂诱导的胰岛素抵抗(单纯抵抗和伴糖尿病的)与肝脏脂肪、游离脂肪酸总量和某些极长链脂肪酸的增多有关。
第三部分高脂诱导的胰岛素抵抗大鼠肝中脂质代谢有关基因的表达变化
目的:通过检测胰岛素抵抗大鼠和胰岛素抵抗伴糖尿病大鼠肝脏与脂肪酸代谢有关酶蛋白基因的表达,探讨肝脏脂质代谢变化的原因。
方法:用PT-PCR法检测参与脂肪酸代谢的有关基因的mRNA水平,免疫印迹检法测D-双功能蛋白(DBP)的蛋白水平。
结果:
1参与甘油三酯合成的二酰基甘油酰基转移酶(DGAT1)mRNA的水平
DM组和HF组的DGAT1 mRNA水平(0.38±0.06,0.26±0.06)均较Con组(0.12±0.02)高(P<0.01,P<0.05)。表明DM组和HF组肝脂肪合成能力增强与DGAT1转录增加有关。
2线粒体脂肪酸β-氧化的限速酶肉毒碱脂酰转移酶Ⅰ(CPTⅠ)mRNA的水平
DM组和HF组(0.58±0.10,0.37±0.04)的CPTⅠmRNA水平均较Con组(0.26±0.02)高(P<0.01,P<0.05),且DM组高于HF组(P<0.01)。表明DM组和HF组肝线粒体分解脂肪酸能力增强与CPTⅠ转录增加有关。
3过氧化物酶体脂肪酸β-氧化途径中的软脂酰辅酶A氧化酶(ACOX1)、降植烷酰辅酶A氧化酶(ACOX3)、L-双功能蛋白(LBP)和D-双功能蛋白(DBP)的mRNA水平
DM组和HF组ACOX1 mRNA的水平(1.51±0.10,1.74±0.25)较Con组(1.12±0.22)髙(P<0.01, P<0.05);DM组和HF组的LBP mRNA水平(0.66±0.12,0.59±0.04)较Con组(0.47±0.09)髙(P<0.01, P<0.05)。提示过氧化物酶体LBP参与的β-氧化途径活性增强。
DM组和HF组(0.88±0.15,1.08±0.19)与Con组(1.01±0.10)的AOX3 mRNA水平差异无统计学意义;而DM组的DBP mRNA水平(0.13±0.01)较Con组和HF组(0.21±0.05,0.26±0.06)低(P<0.05)。提示DM组过氧化物酶体DBP参与的β-氧化途径活性减弱。
4肝DBP蛋白的表达量
DM组肝DBP的蛋白表达量(0.43±0.05)明显低于Con组(0.75±0.12,P<0.01)和HF组(0.81±0.11,P<0.01)。DM组DBP蛋白表达减弱与mRNA转录水平一致,进一步说明DBP表达减少。
5过氧化物酶体增殖物受体α(PPARα)mRNA的变化。
DM组和HF组的PPARαmRNA水平(0.96±0.07,0.90±0.08)均较Con组(0.78±0.07)高(P<0.01,P<0.05)。结合其下游基因CPTⅠ、ACOX1和LBP的转录增强,说明PPARα被激活。
小结:
1高脂诱导的胰岛素抵抗大鼠(单纯抵抗和伴糖尿病的)肝中脂肪的沉积与甘油三酯合成的限速酶DGAT1基因表达增加有关。
2高脂诱导的胰岛素抵抗大鼠(单纯抵抗和伴糖尿病的)肝中线粒体、过氧化物酶体脂肪酸β-氧化活性的增强,与PPARα的转录水平增加并被激活,进而上调CPTⅠ、ACOX1、LBP等基因的表达有关。
3高脂诱导胰岛素抵抗伴糖尿病大鼠肝中某些极长链脂肪酸如C26:0的增加可能与DBP表达减少,DBP参与的脂肪酸β-氧化活性减弱有关。
第四部分游离脂肪酸对原代培养大鼠肝细胞脂肪酸代谢的影响
目的:以原代培养的肝细胞为对象,观察细胞外游离脂肪酸引起肝细胞胰岛素抵抗以及对脂肪酸代谢有关基因表达的影响。证明血中高浓度的游离脂肪酸是高脂饮食诱导胰岛素抵抗的重要因素之一。
方法:采用200-250g正常SD大鼠。麻醉后,用胶原酶Ⅳ灌注肝脏,分离肝细胞。将原代培养的肝细胞随机分为对照组(Con组)和软脂酸组(PA组)。PA组用200μmol/L的软脂酸作用24 h,RT-PCR测定PEPCK1、DGAT1、CPTⅠ、AOX1、LBP和DBP的mRNA水平,Western blotting检测DBP蛋白水平,气相色谱检测肝细胞内游离脂肪酸谱。
结果:
1糖代谢有关基因PEPCK1的mRNA表达PEPCK1在PA组的mRNA的表达量(1.12±0.13)比Con组(0.73±0.15)明显增加( P < 0. 01)。说明胰岛素抑制糖异生作用减弱,胰岛素抵抗出现。
2脂肪酸代谢有关基因的表达
DGAT1在PA组的mRNA量(0.39±0.09)的表达量比Con组(0.25±0.07)明显增加(P<0.05)。CPTⅠ的mRNA量表达量(1.07±0.23)比Con组(0.72±0.16)增加(P<0.05),ACOX1在PA组的mRNA量表达量(2.00±0.22)比Con组(1.61±0.15)增加(P<0.05),LBP在PA组的mRNA量表达量(0.76±0.13)比Con组(0.50±0.09)增加(P<0.01)。DBP在Con组(0.90±0.22)和PA组(0.78±0.18)mRNA量差异无统计学意义(P>0.05)。表明高浓度脂肪酸存在条件下,原代培养的肝细胞脂肪合成和脂肪酸β-氧化增强。
3 DBP蛋白表达
PA组DBP蛋白相对表达量(0.66±0.08),与Con组(0.70±0.09)相比差异无统计学意义(P>0.05)。这一结果与整体试验HF组DBP蛋白的表达相似,而与DM组不同。
小结:
1游离脂肪酸可引起原代培养肝细胞参与脂肪酸代谢的某些基因在转录水平发生改变。这些变化类似于高脂喂养的胰岛素抵抗大鼠肝同种基因变化。
2软脂酸(200μmol/L)未引起培养肝细胞DBP基因表达的变化,这一点与整体研究中糖尿病大鼠不同。
结论:
1血中游离脂肪酸的增加及由此造成的肝组织脂肪和游离脂肪酸的沉积是高脂饮食诱导胰岛素抵抗的重要因素。
2高脂饮食诱导的胰岛素抵抗大鼠(单纯抵抗和伴糖尿病的)肝脏中,脂肪的沉积与甘油三酯合成的限速酶DGAT1的基因表达增加有关。
3高脂饮食诱导的胰岛素抵抗大鼠(单纯抵抗和伴糖尿病的)肝脏中,虽然PPARα的转录水平增加并被激活,进而上调CPTⅠ、ACOX1、LBP等基因的表达,使脂肪酸β-氧化活性增强,但不足以分解肝脏大量摄入的脂肪酸。
4高脂诱导的胰岛素抵抗伴糖尿病大鼠肝中,某些极长链脂肪酸(如C26:0)的增加可能与DBP表达减少,DBP参与的脂肪酸β-氧化活性减弱有关。
Western-like-diet and lifestyle, especially excessive lipid intake, is accused for epidemic of obesity and insulin resistance, as well as the rapid increase of type 2 diabetes. Diabetes can be classified into two types, type 1 and 2. The latter accounts for more than 90% of diabetes. Insulin resistance, the decreased response to insulin, is a key feature of type 2 diabetes. Although how insulin resistance occurs remains unclear,the focus has been concentrated on the elevation of free fatty acid(FFA), such as palmitic acid (C16:0), stearic acid (C18:0), docosanoic acid (C22:0) and isoselachoceric acid (C24:0), and the accumulation of triglyceride in liver. Intralipid infused in vein, which can elevate the level of circulation FFA, results in insulin resistance, proving that FFA may be an independent risk factor in the developement of insulin resistance. Then, whether FFA is one of the key factors in the insulin resistance induced by high-fat diet.
The liver is a vital organ in fatty acid metabolism and insulin resistance. The variations of fatty acids metabolism in liver have a profound effect on the levels of serum fatty acids and affect the fatty acid metabolism in other organizations. FFA is a natural ligand of peroxisome proliferator-activated receptorα(PPARα). In normal physiological conditions, the increased FFA in the blood activates PPARα, which enhances the expression of proteins involved in the intake and oxidation of fatty acids, and therefore accelerate the intake andβ- oxidation of fatty acids in mitochondria and peroxisome in liver. At the same time, the excessive FFA is esterified to fat in liver, which is catalyzed by diacylglycerol acyltransferases (DGAT), and then diverted to other organs by very low density lipoprotein (VLDL). In insulin resistance and diabetes, whether the elevated levels of total FFA and some fatty acids in liver are caused by the abnormal lipid metabolism and abatedβ-oxidation and whether the fat deposition in liver is related to enhanced expression of DGAT that is a limit enzyme in fat synthesis, have not been reported.
Short, medial and long chain fatty acids can be catabolized in mitochondria byβ-oxidation, tricarboxylic cycle and oxidative phosphorylation to produce ATP and CO2. During this process, oxygen free radicals are produced in respiratory chain level. Very long chain fatty acids must be handled in peroxisomal byβ-oxidation. In this condition, Acyl-CoA oxidases act as a rate-limiting enzyme, which donates electrons directly to molecular oxygen and generates H2O2. Therefore, accumulation of very long chain fatty acids in cells can not only cause changes of fatty acids types contained in phospholipid of biomembrane, but also generate a lot of reactive oxygen species that damage the cell structure and function. Is the lipotoxicity to liver in insulin resistance and diabetes, which were induced by high-fat diet, caused by accumulation of fatty acids especially the very long chain fatty acids?
In order to study these issues, we developed insulin resistance in Sprague-Dawley rat by high-fat diet, and further formation of diabetes, and primary culture hepatocytes in high level fatty acids environment. To explore the relation between peroximalβ-oxidation and the variance of quantity and kinds of FFA in liver of insulin resistance and type 2 diabetes states induced by high-fat diet, the changes in quantity and kinds of fatty acids of blood, liver and primary culture hepatocytes were detected, and the changes of fatty acids outlet(triglycerides synthesis and fatty acid oxidation) and the activities of differentβ-oxidation ways were also detected from integeral and cell level.
PartⅠHigh fat diet and occurrence of insulin resistance
Objective: By observing the dynamic changes in blood biochemical parameters, the relationship between FFA and insulin resistance developement was explored in high-fat diet rat.
Methods: Male SD rats were divided randomly into control group (Con group) and high fat group (HF group). The rats in Con group were fed with standard diet and HF group, high-fat diet. lipid in the high-fat diet consisted mainly of lard-based, accounting for 59.8 percent of calory. After feeding for 2, 4, 6 and 8 weeks in high-fat diet, the rats were detected for insulin resistance by oral glucose tolerance (OGTT), insulin sensitivity index (ISI) and euglycaemic-hyperinsulinaemic clamp. Blood sugar was test with rapid blood glucose detector. Blood triglycerides and cholesterol were measured by oxidase method and blood insulin concentration was detected by RIA, and serum total FFA concentration was detected with detection kit. After 8 weeks, some HF rats developed to dibetes by streptozotocin(DM group). All rats were fed continuely for 6 weeks, and sacrificed, and the blood fatty acids profiles were evaluated by gas chromatography.
Results:
1 Biochemical parameters in the blood
At 4th week, the body weights of HF group exceeded that of Con group(HF. 286.0±16.0g,Con.250.2±15.1g,P<0.01), and so did the blood triglycerides levels (HF. 1.14±0.14 mmol/L,Con. 0.92±0.17 mmol/L,P<0.01) and the FFA levels (HF. 323.52±35.83μmol/L,Con. 272.65±50.21μmol/L,P<0.05). However, fasting blood sugar, cholesterol and blood insulin concentration showed no significant difference (P>0.05) between the two groups at this time point. At 6th week, blood cholesterol (2.05±0.29 mmol/L) and insulin (28.43±14.5μIU/ml) concentrations of HF group were are higher than those of Con group (cholesterol, 1.69±0.14 mmol/L and insulin, 15.28±3.97μIU/ml), there were statistical differences between two groups(P<0.01 and P<0.01). But there was no statistical difference in blood sugar levels between two groups. At 8th week, fasting blood sugar(5.85±0.10 mmol/L) of HF group was obviously higher than that of Ctrl group(5.32±0.39 mmol/L), and the difference was statistically significan(tP<0.05). Total blood FFA concentration in HF group (406.69±70.47μmol/L) was higher than that of Con group(299.52±59.17μmol/L) (P<0.01). Along with the duration of high-fat feeding increases, the differences of blood glucose, triglycerides, cholesterol, FFA and insulin concentration between the two groups were increasing.
2 insulin sensitivity index (ISI)
2 weeks after high fat diet, ISIs in HF group and Con group were -4.53±0.19 and -4.41±0.28, respectively. there was no statistical difference(P>0.05)between the two groups. At the time points of 4,6,8 weeks, ISIs of HF and Ctrl group were -4.71±0.21 and -4.47±0.25, -4.95±0.52 and -4.45±0.30,-5.31±0.26 and -4.60±0.14, respectively. There were statistical differences(P<0.05, P<0.01 and P<0.01)between the two groups. 4 weeks after the rats were fed high fat diet, the insulin sensitivity in HF group rats began to decease, which progressed time dependently.
3 Oral glucose tolerance test (OGTT) After fasting for 12 h, rats were gavaged with 50% glucose (2 g / kg), and then the blood glucose levels were tested. At the time point of 2 weeks, there was no difference at each time point in OGTT. At 4 weeks, 30 min and 60 min blood sugars were higher in HF group than that of Ctrl. group, there were statistical differences. In the rats fed in high-fat for 6 weeks, the blood sugar at 30 min, 60 min and 120 min time points were higer than that of Ctrl group, but at 0 min, the difference has no statistical significance.
4 Result of euglycaemic hyperinsulinaemic clamp
At the time point of 6 weeks, glucose infusion rate(GIR) of HF group was 20.44±1.99mg/kg.min ,which was lower than that of Ctrl. group(27.97±1.72 mg/kg.min) (P <0.01). The results showed that HF group rats developed obvious insulin resistance.
5 Correlation between IR and blood lipid.
At the time point of 4 weeks, correlation coefficient of triglycerides and ISI was -0.368 (P>0.05), that of cholesterol and ISI was -0.288 (P >0.05) and that of FFA and ISI was -0.511(P<0.05). At the time point of 8 weeks, correlation coefficient of triglycerides and ISI was -0.771 (P<0.01), that of cholesterol and ISI was -0.82 (P<0.01) and that of FFA and ISI was -0.856(P<0.01). Blood triglycerides, cholesterol and FFA were correlated to ISI.
6 Changes of blood FFAs
In our research conditions, C18:1,C18:2 and C18:3 could not be separated completely, while other fatty acids were separated well. When compared with Con, the concentrations of C20:0(18.91±2.49 mg/L), C24:0(4.18±1.05 mg/L)of DM group did not change(P>0.05), C20:5(19.33±4.47 mg/L)decreased, and the other fatty acids, such as C16:0, C18:0, C20:4, C22:0, C22:6 and C26:0, increased(P<0.05) In HF group, the concentrations of C16:0 (73.37±13.03 mg/L), C18:0(51.40±7.87 mg/L), C18:1+C18:2+C18:3 (176.07±22.27 mg/L), C22:6(5.33±1.48 mg/L) increased, C20:5 (17.71±2.95 mg/L) decreased ( P<0.05 ), and the concentrations of other fatty acid, such as C20:0, C20:4, C22:0, C24:0 and C26:0, were unchanged (P>0.05), when compared with control group. Percentages of different fatty acids in the total fatty acid were also changed. C18:1+C18:2+C18:3 (32.14±3.50) %, C20:4(1.85±0.26) %, C22:6 (9.49±1.35) % and C26:0(1.18±0.18) % in the serum of DM group increased, and the other fatty acids (C16:0, C18:0, C20:0, C20:5, C22:0 and C24:0) showed no change. C18:1+C18:2+C18:3 (4.96±3.21)% in the serum of HF group increased, C20:5(0.64±0.09)% of HF group decreased(P<0.05), and the other fatty acids (C16:0, C18:0, C20:0, C20:4, C22:0, C22:6, C24:0 and C26:0) showed no change (P<0.05).
Conclusion:
1 Occurrence and degree of insulin resistance were related to the inducing time, blood sugar concentration began to increase obviously when insulin resistance reached to certain degree.
2 Increase of FFA had a close relation with occurrence of insulin resistance induce by high-fat diet.
PartⅡChanges of hepatic lipids in rats with insulin resistance induced by high-fat diet
Objective: To observe changes of hepatic lipids(fat and FFA) and fatty acidβ-oxidation, and investigate the relation between insulin resistance inducing by high fat diet and hepatic fatty acid metabolism.
Methods: In order to establish quickly the animal model with insulin resistance and diabetes, HF group rats that had developed insulin resistance, were administered with 2% STZ (27 mg/kg) by peritoneal injection. The blood sugar levels of tail vains were evaluated 72 hours later. The rats whose fasting blood sugar was higher than 7.8 mmol/L were fed continuely with high-fat diet for 6 weeks, and grouped as insulin resistance complicated with diabetes group (DM). Con and HF groups were also fed for 6 weeks with standard diet and high-fat diet respectively. Eight rats were selected randomly from each group for next test. The rats were intraperitoneally anesthetized with pentobarbital sodium (60 mg/kg) in the fasting state. Blood was collected by carotid artery bloodletting, and the serum was used for the detection of blood total FFA and FFA profiles, and the liver was used for the detection of liver fatty deposition(oil red o staining), amount of fat (oxidase way), fatty acidβ-oxidation activities (ultraviolet absorption) and liver FFA profiles.
Results:
1 Contents of liver fat
Compared with Con group(11.30±1.89 mg/g), content of liver fat of HF group(34.94±4.93 mg/g) and DM group(51.47±7.77mg/g) increased remarkly (P<0.01).
2 Detected fatty deposition in liver by oil red o staining
Little fat drops could be found in Ctrl. group, but more fat drops were found in DM and HF groups. Results of oil red o staining showed that rats with insulin resistance reduced by high-fat diet had severe fat deposition.
3 Fatty acidβ-oxidation of liver tissue
3.1 Total activities of fatty acidβ-oxidation
Compared with Con group (6.46±1.07 mU/mg pro), total activities ofβ-oxidation of DM group (10.76±0.81mU/mg pro) and HF group (9.10±1.31mU/mg pro) increased remarkablely (P<0.01).
3.2 Activities ofβ-oxidation of peroxisome Compared with Con group (3.21±0.55 mU/mg pro), the activities of peroximalβ-oxidation of DM group (4.41±1.10 mU/mg pro) and HF group (4.13±0.82mU/mg pro) increased (P<0.05).
3.3 Activities ofβ-oxidation of mitochondria
Compared with Con group (3.25±1.20 mU/mg pro), the activities of mitochondriaβ-oxidation of DM group (6.35±1.85mU/mg pro) and HF group (4.97±1.86mU/mg pro) increased (P<0.01, P<0.05).
4 Total FFA contents in livers
Compared with Con group (66.03±15.00μmol/g), the total FFA contents of DM group (112.79±22.24μmol/g) and HF group (97.01±16.02μmol/g) increased (P<0.01, P<0.05).
5 Changes of liver FFA profiles measured by gas chromatography
C20:5 could not be detected and C18:1, 18:2, 18:3 could not be separated completely by the gas chromatography equipment we used. In comparison with Con, C18:1+C18:2+C18:3 (24.31±3.50 mg/g), C20:4 (5.06±0.82 mg/g), C22:6 (1.41±0.23 mg/g) and C26:0 (2.49±0.46 mg/g) contained in liver increased (P<0.05), C18:0(5.56±1.09 mg/g), C20:0 (0.50±0.08 mg/g), C22:0 (0.13±0.02 mg/g) and C24:0 (0.12±0.02 mg/g) decreased (P<0.05), and C16:0 was unchanged(P>0.05)in DM group。In HF group the fatty acids C18:1+C18:2+C18:3 (21.89±3.92 mg/g) contained in liver increased (P<0.05), C20:0 (0.82±0.19 mg/g), C22:0 (0.19±0.08 mg/g) and C24:0 (0.14±0.08 mg/g) decreased (P<0.05), and the other fatty acids (C16:0、C18:0、C20:4、C22:6和C26:0) were unchanged (P>0.05) when compared with control group. There were statistic differences of contents of C20:0、C22:6 and C26:0 between DM group and HF group.
The percentage of each fatty acid in the composition of total fatty acid was also changed. Compared with Con, the percent of C18:1+C18:2+C18:3 (36.67±3.31)%, C22:6 (2.59±0.49)% and C26:0 (3.16±0.64) % increased (P<0.05), that of C20:0 (0.16±0.04) %, C22:0 (0.16±0.01) %, C24:0 (0.44±0.09) % decreased(P<0.05), and that of other fatty acids (C16:0、C18:0 and C20:4) were unchanged in DM group. Compared with Con group, percent fatty acid composition of C18:1+C18:2+C18:3 (37.24±3.06)% of HF group increased (P<0.05), that of C20:0 (0.12±0.03) %, C22:0 (0.19±0.04) % and C24:0 (0.51±0.11) % decreased (P<0.05), and that of other fatty acids (C16:0、C18:0、C20:4、C22:6 and C26:0) were unchanged (P>0.05). Percent fatty acid compositions of C22:6 and C26:0 of DM group were higher than HF group (P<0.05).
Conclusion:
1 Increase of blood FFA of the rats fed with high-fat diet for a long time promoted the activities of mitochondria, peroximal and total oxidation. Although having increased activities ofβ-oxidation, the liver failed to disposal the large amount of fatty acid and it resulted in increase of fat, total fatty acid and some very long chain fatty acid.
2 Insulin resistance induced by high-fat diet correlated with increased hepatic triglycerides, total fatty acids and some very long chain fatty acids.
PartⅢChanges in expression of genes involved in lipid metabolism in liver of high-fat diet induced insulin resistance rat
Objective: To investigate the changes in lipid metabolism of liver by detecting the expression of the genes involved in fatty acid metabolism in liver of insulin resistance rats and diabetes rats.
Methods: The expression of genes involved in fatty acid metabolism was detected with RT-PCR. The level of the D-bifunctional protein(DBP) was detected with western blotting.
Results:
1 The mRNA expression of diacylglycerol acyltransferase 1 (DGAT1) involved in triglyceride synthesis
The DGAT1 mRNA of DM and HF groups (0.38±0.06,0.26±0.06) were higher than Con group (0.12±0.02, P<0.01, P<0.05), which indicated that triglyceride synthesis in DM and HF group increased.
2 The mRNA expression of carnitine palmitoyl transferaseⅠ(CPTⅠ), the rate-limiting enzyme in mitochondria fatty acidβ-oxidation.
The CPT I mRNA of DM and HF groups (0.58±0.10,0.37±0.04) were higher than Con group (0.26±0.02, P<0.01,P<0.05) and that of DM group was higher than HF group (P<0.01), which indicated that mitochondria fatty acidβ-oxidation in DM and HF groups increased.
3 The mRNA expression of fatty acyl-CoA oxidase 1(ACOX1), fatty acyl-CoA oxidase 3 (ACOX3), D-bifunctional protein(DBP), L-bifunctional protein (LBP)
The ACOX1 mRNA expression of DM and HF groups (1.51±0.10,1.74±0.25) was higher than Con group (1.12±0.22, P<0.01,P<0.05) and the LBP mRNA expression of DM and HF groups (0.66±0.12,0.59±0.04) was higher than Con group (0.47±0.09, P<0.01,P<0.05), which indicated that peroxisomal fatty acid oxidation involved of LBP of HF and DM groups was increased.
DM and HF groups compared to Con group(0.88±0.15,1.08±0.19 vs 1.01±0.10, p>0.05), the changes of expression of ACOX3 mRNA have no statistical significance. The DBP mRNA of DM group(0.13±0.01)was lower than Con and HF groups (0.21±0.05,0.26±0.06, P<0.05), which indicated that the activity of fatty acid oxidation in peroxisome involved of DBP was decreased.
4 The changes of peroxisome proliferator-activated receptorα(PPARα) mRNA expression
The PPARαmRNA of DM and HF groups (0.96±0.07,0.90±0.08) were higher than Con group (0.78±0.07, P<0.01,P<0.05) and the transcription of downstream genes (CPTⅠ、ACOX1、LBP) increased , which indicated that mitochondria fatty acid oxidation in DM and HF group increased
5 The expression of DBP protein in liver
The expression of DBP protein in DM group (0.43±0.05, n=4)was lower than Con group(0.75±0.12,P<0.01)and HF group(0.81±0.11,P<0.01). It was coincident with the expression of DBP mRNA, which indicated that the expression of DBP decreased.
Conclusion:
1 In high-fat diet induced insulin resistance rats and diabetes rats, triglyceride accumulated in liver. It associated with increased expression of DGAT1, the rate-limiting enzyme in triglyceride synthesis.
2 In the liver of high-fat diet induced insulin resistance rats and diabetes rats, mitochondria and peroxisome fatty acid oxidation increased. It associated with increased transcription and activation of PPARαand up-regulated genes of CPTⅠ、ACOX1、LBP.
3 In the liver of high-fat-induced insulin resistance and diabetes rats, some very long chain fatty acids ,such as C26:0, were increased. It associated with decreased expression of DBP and decreased activity of fatty acid oxidation involved of DBP.
PartⅣEffects of free fatty acids on metabolism of fatty acids in primary cultured rat hepatocytes
Objective: To observe the effects of FFA which outside of hepatocytes on IR and on expression of the genes which involve in the metabolism of fatty acids based on the primary cultured hepotocytes.
Methods: SD rats weighing between 200 and 250 g were anesthetized with thiopental and hepatocytes were isolated after collagenaseⅣperfusion of the livers. Primary cultured hepatocytes were divided randomly into: control group (Con group), palmitic acid group(PA group). PA group was administered with 200μmol/L PA for 24 h. The expression of PEPCK1, CPTⅠ, DGAT1, AOX1, LBP and DBP mRNA were observed by RT-PCR and the expression of DBP protein were observed by Western blotting.
Results:
1 Expression of PEPCK1 mRNA
The PEPCK1 mRNA level in PA group (1.12±0.13) was significantly higher than that of Con group (0.73±0.15) ( P < 0. 01). The results showed that the effect of insulin’s inhibition to glyconeogenesis was reduced, and IR was proved.
2 Expression of mRNA of genes involved in the metabolism of fatty acids
The DGAT1 mRNA level was higher in PA group (0.39±0.09) than that of Con group (0.25±0.07) (P<0.05). The CPTⅠmRNA level was higher in PA group (1.07±0.23) than that of Con group (0.72±0.16) (P<0.05), the ACOX1 mRNA level was higher in PA group (2.00±0.22) than that of Con group (1.61±0.15) (P<0.05) and the LBP mRNA level was higher in PA group (0.76±0.13) than that of Con group (0.50±0.09) (P<0.01). However, difference of the DBP mRNA level between PA(0.78±0.18) and Con (0.90±0.22) have no statistic significance (P>0.05). The results showed that the metabolism of fatty acids in primary cultured liver cells altered.
3 The protein level of DBP
The DBP protein levels between PA group (0.87±0.11) and Con group (0.80±0.13) have no statistic significance (P>0.05). The result was similar to DBP expression of HF group, but was different from DM group in integral experiment.
Conclusion:
1 The expressions of some genes involved in fatty acids metabolism were altered in transcriptive level by FFA. These changes were similar to the changes of rat liver of insulin resistance induced by high-fat diet.
2 Expression of DBP gene of liver cell cultured in palmitic acid (200μmol/L) was not altered, which is different from that of DM group in integrated research.
Summary:
1 Increase of free fatty acids in blood and accumulations of fat and free fatty acids resulted from the increase of free fatty acids in blood were vital factors that contributed to insulin resistance induced by a long term of high-fat diet.
2 In high-fat-induced insulin resistance rats and diabetes rats, triglyceride accumulates in liver. It associated with increased expression of DGAT1, the rate-limiting enzyme in triglyceride synthesis.
3 In the liver of high-fat-induced insulin resistance rats and diabetes rats, fatty acidβ-oxidation increased. It associated with increased transcription and activation of PPARαand up-regulated genes of CPTⅠ, ACOX1 and LBP. But it failed to consume the large mount of fatty acid uptaken by liver.
4 In the liver of high-fat-induced insulin resistance and diabetes rats, some very long chain fatty acid (C26:0) was increased. It associated with decreased expression of DBP and decreased activity of fatty acid oxidation involved of DBP.
引文
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4 Asayama K, Sandhir R, Sheikh FG, et al. Increased peroxisomal fatty acid b-oxidation and enhanced expression of peroxisome proliferatoractivated receptor-a in diabetic rat liver. Molecular and Cellular Biochemistry, 1999, 194(1-2): 227~234
5 McGarry, JD. Banting lecture 2001: dysregulation of fatty acid metabolism in the etiology of type 2 diabetes. Diabetes, 2002, 51(1):7~18
6 Sone H, Takahashi A, Lida K, et al Disease model: hyperinsulinemia and insulin resistance Part B-polygenic and other animal models[J]. TRENDS in Molecular Medicine, 2001, 7(8) :373~376
7 Saini KS,Thompson C,Winterford CM,et al. Streptozotocin at low doses induces apoptosis and at high doses causes necrosis in a murine pancreatic beta cell line,INS-1. Biochem Mol Biol Int, 1996,39(6):1229~1236
8 李光伟,潘孝仁,Lillioja ,等. 检测人群胰岛素敏感性的一项新指标. 中华内科杂志, 1993, 32 (10) :656~659
9 陈珺; 张健; 薛启蓂. 气相色谱法检测极长链脂肪酸的诊断价值. 中华检验医学杂志, 2000, 23(6):350~353
10 Greenberg AS, McDaniel ML. Identifying the links between obesity, insulin resistance and β-cell function: potential role of adipocyte-derived cytokines in the pathogenesis of type 2 diabetes. Eur J Clin Invest, 2002,32 (Suppl. 3):24~34
11 Storlien LH, James DE, Burleigh KM, et al. Fat feeding causes widespread in vivo insulin resistance, decreased energy expenditure, and obesity in rats. Am J Physiol, 1986;251(5 Pt 1):E576~583
12 Randle PJ, Garland PB, Hales CN, et al. The glucose fatty-acid cycle: its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet, 1963, 1(7285):785~789
13 Boden G, Jadali F, White J, et al Chen X et al. Effects of fat on insulin-stimulated carbohydrate metabolism in normal men. J Clin Invest, 1991, 88(3):960~966
14 Griffin ME, Marcucci MJ, Cline GW, et al. Free fatty acid-induced insulin resistance is associated with activation of protein kinase C theta and alterations in the insulin signaling cascade. Diabetes, 1999, 48(6):1270~1274
1 Tiikkainen M, Tamminen M, Hakkinen AM, et al. Liver-fat accumulation and insulin resistance in obese women with previous gestational diabetes. Obes Res, 2002, 10(9):859~867
2 Seppala-Lindroos A, Vehkavaara S, Hakkinen AM, et al. Fat accumulation in the liver is associated with defects in insulin suppression of glucose production and serum free fatty acids independent of obesity in normal men. J Clin Endocrinol Metab, 2002, 87(7):3023~3028
3 Samuel VT, Liu ZX, Qu X, et al. Mechanism of hepatic insulin resistance in nonalcoholic fatty liver disease. J Biol Chem, 2004, 279(31):32345~32353
4 王咏波,陈璐璐,周愍,等. 胰岛素治疗对2型糖尿病大鼠肝脂质含量及胰岛素抵抗的影响。中华肝脏病杂志, 2005, 13(6):451~454
5 Markwell MA, Haas SM, Bieber LL, et al. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem, 1978, 87(1): 206~210
6 Lazarow PB, De Duve C. A fatty acyl-CoA oxidizing system in rat liver peroxisomes; enhancement by clofibrate, a hypolipidemic drug. Proc. NatlAcad Sci USA, 1976, 73(6):2043~2046
7 Watt MJ, Holmes AG, Steinberg GR, et al. Reduced plasma FFA availability increases net triacylglycerol degradation, but not GPAT or HSL activity, in human skeletal muscle. Am J Physiol Endocrinol Metab, 2004, 287(1):E120~127
8 Picard F, Naimi N, Richard D, et al. Response of adipose tissue lipoprotein lipase to the cephalic phase of insulin secretion. Diabetes, 1999,48(3):452~459
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10 Baes M, Huyghe S, Carmeliet P,et al. Inactivation of the peroxisomal multifunctional protein-2 in mice impedes the degradation of not only 2-methyl-branched fatty acids and bile acid intermediates but also of very long chain fatty acids. J Biol Chem, 2000, 275(21): 16329~16336
11 李静,宋光耀,姜玲玲等. 实验性糖尿病大鼠肝过氧化物酶体脂肪酸β-氧化及D-双功能蛋白活性变化的初步研究. 生物化学与生物物理进展, 2004, 31(11):1024~1029
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2 Kim JK, Gavrilova O, Chen Y, et al. Mechanism of insulin resistance in A-ZIP/F-1 fatless mice. J Biol Chem, 2000, 275(12):8456~8460
3 Lam TK, Carpentier A, Lewis GF, et al. Mechanisms of the free fatty acid-induced increase in hepatic glucose production. Am J Physiol Endocrinol Metab, 2003, 284(5):E863~873
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5 McGarry, JD. Banting lecture 2001: dysregulation of fatty acid metabolism in the etiology of type 2 diabetes. Diabetes, 2002, 51(1):7~18
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9 陈珺; 张健; 薛启蓂. 气相色谱法检测极长链脂肪酸的诊断价值. 中华检验医学杂志, 2000, 23(6):350~353
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11 Storlien LH, James DE, Burleigh KM, et al. Fat feeding causes widespread in vivo insulin resistance, decreased energy expenditure, and obesity in rats. Am J Physiol, 1986;251(5 Pt 1):E576~583
12 Randle PJ, Garland PB, Hales CN, et al. The glucose fatty-acid cycle: its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet, 1963, 1(7285):785~789
13 Boden G, Jadali F, White J, et al Chen X et al. Effects of fat on insulin-stimulated carbohydrate metabolism in normal men. J Clin Invest, 1991, 88(3):960~966
14 Griffin ME, Marcucci MJ, Cline GW, et al. Free fatty acid-induced insulin resistance is associated with activation of protein kinase C theta and alterations in the insulin signaling cascade. Diabetes, 1999, 48(6):1270~1274
1 Tiikkainen M, Tamminen M, Hakkinen AM, et al. Liver-fat accumulation and insulin resistance in obese women with previous gestational diabetes. Obes Res, 2002, 10(9):859~867
2 Seppala-Lindroos A, Vehkavaara S, Hakkinen AM, et al. Fat accumulation in the liver is associated with defects in insulin suppression of glucose production and serum free fatty acids independent of obesity in normal men. J Clin Endocrinol Metab, 2002, 87(7):3023~3028
3 Samuel VT, Liu ZX, Qu X, et al. Mechanism of hepatic insulin resistance in nonalcoholic fatty liver disease. J Biol Chem, 2004, 279(31):32345~32353
4 王咏波,陈璐璐,周愍,等. 胰岛素治疗对2型糖尿病大鼠肝脂质含量及胰岛素抵抗的影响。中华肝脏病杂志, 2005, 13(6):451~454
5 Markwell MA, Haas SM, Bieber LL, et al. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem, 1978, 87(1): 206~210
6 Lazarow PB, De Duve C. A fatty acyl-CoA oxidizing system in rat liver peroxisomes; enhancement by clofibrate, a hypolipidemic drug. Proc. NatlAcad Sci USA, 1976, 73(6):2043~2046
7 Watt MJ, Holmes AG, Steinberg GR, et al. Reduced plasma FFA availability increases net triacylglycerol degradation, but not GPAT or HSL activity, in human skeletal muscle. Am J Physiol Endocrinol Metab, 2004, 287(1):E120~127
8 Picard F, Naimi N, Richard D, et al. Response of adipose tissue lipoprotein lipase to the cephalic phase of insulin secretion. Diabetes, 1999,48(3):452~459
9 Jiang LL , Kurosawa T, Sato M, et al. Physiological role of D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein. J Biochem, 1997, 121(3):506~513
10 Baes M, Huyghe S, Carmeliet P,et al. Inactivation of the peroxisomal multifunctional protein-2 in mice impedes the degradation of not only 2-methyl-branched fatty acids and bile acid intermediates but also of very long chain fatty acids. J Biol Chem, 2000, 275(21): 16329~16336
11 李静,宋光耀,姜玲玲等. 实验性糖尿病大鼠肝过氧化物酶体脂肪酸β-氧化及D-双功能蛋白活性变化的初步研究. 生物化学与生物物理进展, 2004, 31(11):1024~1029
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