葡萄糖、维生素C对普安银鲫卵黄囊仔鱼发育中LPL和HL活性的影响

通过设置不同浓度的葡萄糖溶液和维生素C溶液分别添加到普安银鲫的孵化水体中,直至卵黄囊消失完全,探究葡萄糖和维生素C溶液分别浸泡对普安银鲫(Carassius auratus gibelio)卵黄囊仔鱼发育中脂蛋白脂酶(LPL)和肝脂酶(HL)活性的影响。葡萄糖浓度为0、5、10、15和20 g/L;维生素C浓度为0、20、25、30和35 mg/L,记录孵化时间、孵化率及仔鱼成活率,并测定了最适葡萄糖浓度组、维生素C浓度组与对照组中普安银鲫卵黄囊仔鱼发育中LPL和HL活性。结果显示:普安银鲫卵黄囊仔鱼发育中对照组与维生素C组LPL比活力与全活力呈"下降-上升"的变化趋势,HL比活力与全活力均呈上升趋势。葡萄糖组LPL和HL比活力与全活力呈上升趋势,在混合营养期与外源营养期,LPL和HL比活力与全活力显著高于对照组(P0.05),而维生素C组LPL和HL比活力与全活力仅稍高于对照组,但HL全活力在内源营养期显著高于对照组(P0.05)。研究表明:适宜水平的葡萄糖溶液可通过调节脂类代谢酶的活性来维持机体内脂质代谢的动态平衡,同时适宜水平的维生素C溶液能促进脂质代谢。     

维生素C对普安银鲫早期发育中氧化损伤及总抗氧化能力的影响

本实验旨在探究普安银鲫(Carassius auratus gibelio)早期发育过程中氧化损伤程度和总抗氧化能力的变化,及添加维生素C(Vc)后对它们的影响。采用生化方法测定了普安银鲫早期发育过程中丙二醛(MDA)含量、蛋白质羰基(PCA)含量、乳酸脱氢酶(LDH)的活性和总抗氧化能力(T-AOC)。(1)30 mg/L的Vc溶液中仔鱼孵化率和成活率最高,分别高出对照组(Vc浓度为0 mg/L)16.2%和17.8%;(2)普安银鲫早期发育过程中丙二醛、蛋白质羰基含量及乳酸脱氢酶活性均呈"升高-降低"的趋势,均在原肠中期达到最高,总抗氧化能力逐渐增加;(3)30 mg/L的Vc组中丙二醛、蛋白质羰基含量及乳酸脱氢酶的活性显著低于对照组(P0.05),总抗氧化能力显著高于对照组(P0.05)。结果表明,(1)普安银鲫胚胎期受到的氧化损伤比胚后严重;(2)适宜浓度的维生素C能提高胚胎及仔鱼的抗氧化能力,有效降低胚胎及仔鱼受到的氧化损伤,提高其孵化率及存活率。     

维生素C多聚磷酸酯对小鼠肝脏脂质过氧化物和抗氧化物酶的影响

为研究维生素C多聚磷酸酯对小鼠肝脏脂质过氧化物和抗氧化物酶的影响 ,我们设置了 4个实验组 ,采用 2 4只小鼠 ,饵料中 35 %维生素C多聚磷酸酯的添加量依次为 0、 5 0 0、 2 5 0 0和 5 0 0 0mg/kg ,喂食 4周后取其肝脏 ,用硫代巴比妥酸分光光度测脂质过氧化物的含量 ,用亚硝酸盐形成法测定超氧化物歧化酶的活性 ,用分光光度法测过氧化氢酶和谷胱甘肽过氧化物酶的活性。结果表明 ,维生素C多聚磷酸酯对小鼠肝脏脂质过氧化物没有明显影响 ,但随着维生素C多聚磷酸酯添加量的增加 ,脂质过氧化物有减少的趋势。维生素C多聚磷酸酯添加量为 2 5 0 0和 5 0 0 0mg/kg的两组 ,其超氧化物歧化酶的活性明显高于对照组和维生素C多聚磷酸酯添加量为 5 0 0mg/kg组 ;过氧化氢酶的活性明显高于对照组。维生素C多聚磷酸酯添加量为5 0 0 0mg/kg组 ,其谷胱甘肽过氧化物酶的活性明显高于其它三组。表明高剂量的维生素C多聚磷酸酯能促进小鼠抗氧化物酶的活性 ,但促进不同抗氧化物酶活性所需的维生素C多聚磷酸酯的量不同     

日本鬼鲉胚胎发育及仔、稚鱼形态学观察

对日本鬼鲉的胚胎及仔稚鱼形态发育进行观察,描述了各发育时期的发育时序和形态特征。日本鬼鲉的受精卵呈正圆球形,无色透明,无油球,平均卵径为(1.42±0.04)mm,呈浮性。在水温(22±0.5)℃,盐度31‰的条件下,历时约52h10min完成孵化。初孵仔鱼全长(3.04±0.13)mm,卵黄囊长(2.23±0.08)mm,卵黄囊高为(1.65±0.06)mm,在(21±0.5)℃水温下,仔鱼孵化后3d内营内源性营养(卵黄囊仔鱼),第4天卵黄囊完全消失,开口摄食轮虫。13d全长(7.12±0.35)mm,背鳍鳍条出现进入稚鱼早期,15d全长(7.92±0.61)mm鱼体及各鳍开始出现金黄色斑纹,25d全长(13.66±0.55)mm,整个鱼体呈黄褐色,完成变态进入幼鱼期,开始营底栖生活。同时观察了自胚胎发育至幼鱼期鱼苗体表色素分布的变化及其器官发育的形态变化。     

美洲鲥胚胎及仔稚鱼的发育

对美洲鲥(Alosa sapidissima)早期生活史阶段的生长发育特征进行了观察和测量,描述了胚胎和仔、稚鱼的生长发育特征。美洲鲥受精卵球形、无油球,为沉性卵,卵径2.85—3.28 mm。在水温20.3℃—21.9℃孵化条件下,经过82h孵化出膜,根据其胚胎发育过程的形态特征,胚胎发育分为受精卵、卵裂期、囊胚期、原肠胚期、神经胚期、器官形成期和出膜期7个发育阶段。美洲鲥初孵仔鱼全长为(8.56±0.36)mm,其卵黄囊体积为(4.57±0.77)mm3。1日龄仔鱼脑部发育明显,口张开,肛门开通,胸鳍形成。2日龄仔鱼卵黄囊体积(0.71±0.23)mm3,只有刚孵化的15.54%。3日龄仔鱼经过1d的混合营养期,卵黄被完全吸收,4日龄仔鱼完全营外源性营养,卵黄囊的体积(V)随孵化时间(h)的变化方程为V=4.1583e-0.0356h(R2=0.9901)。此后,背鳍鳍条、尾鳍鳍条、臀鳍鳍条和腹鳍鳍条相继在晚期仔鱼出现,9日龄仔鱼尾椎开始弯曲,21日龄仔鱼尾椎弯曲完成。27日龄鱼鳞开始形成,到33日龄稚鱼全身披鳞,个体发育进入幼鱼期,仔稚鱼期间的生长模型方程为:TL=0.0049D2+0.5091D+9.2578(R2=0.9885,TL为全长,D为日龄)。     

鳜消化系统器官发生的组织学

利用形态学观察和连续组织切片技术,对出膜后0-35d的鳜仔稚鱼消化系统胚后发育的组织学特征进行了系统研究.结果表明,试验水温为18.0-20.0℃时,鳜初孵仔鱼消化道仅为一段位于脊索下方、卵黄囊上方的实心细胞索,卵黄囊呈椭圆形,含有油球.孵化后第2天,口和肛门形成,消化道贯通.孵化后第4天,消化道上皮细胞出现分化,肝脏和胰脏出现,仔鱼开始由内源性营养向外源性营养转变.孵化后第5天,仔鱼开口摄食,消化道分化成口咽腔、食道、胃、前肠和后肠.孵化后第9天,卵黄囊完全被吸收.此后随着鱼体的生长,消化系统从结构和功能上逐步发育完善和成熟.孵化后第7天前肠中出现空泡,孵化后第8天仔鱼后肠中发现有嗜曙红颗粒,表明肠上皮细胞吸收了脂肪和蛋白质.在孵化后第13天,出现胃腺,标志着稚鱼期的开始.     

半滑舌鳎仔鱼的摄食与生长

在水温23.4-24.0℃、盐度33.00、pH7.78-8.02的培养条件下,半滑舌鳎初孵仔鱼全长为3.55±0.161mm(n=60),依照公式4/3π.R/2.(r/2)2计算其卵黄囊体积为0.606±0.106mm3(n=60)。1日龄仔鱼,全长为4.99±0.211mm(n=10),卵黄囊体积较初孵仔鱼缩小近10倍,约为0.066±0.008mm3(n=10);2日龄,仔鱼的巡游模式建立,仔鱼全长为5.61±0.069mm,卵黄囊体积为0.030±0.002mm3(n=10),口初开;2.5日龄,口完全裂开,口裂0.24±0.024mm(n=10),仔鱼进入摄食期;3日龄,仔鱼的外源性摄食关系初步建立;4日龄,仔鱼的摄食率达100%,完成了内源性营养向外源性营养的转换;5日龄,仔鱼的卵黄完全被吸收,仅剩聚成一团的小油球,仔鱼的混合营养期持续2.5天时间;21日龄,稚鱼全长为12.96±0.611mm(n=11),仍有40%的个体残余的油球还没有完全被吸收,其体积仅为0.0000005±0.000003mm3(n=11)。仔鱼发育过程中,其长度的生长存在内源性营养阶段的快速生长、混合营养阶段的慢速生长以及饥饿期间的负生长三个生长期相,平均增长率为0.45mm/d,依照TL=aD3 bD2 cD d方程式对仔鱼的全长与日龄进行回归,其生长模型为TL(mm)=0.0026D3-0.0704D2 0.7993D 3.55(R2=0.9811,n=324)。仔鱼耐受饥饿的时间临界点发生在孵化后第10天(即9日龄)。仔鱼具有摄食能力的时间约6天,不可逆转饥饿期的时间约3天。残余的油球较长时间的存在,相对地延长了仔鱼混合营养期的时间,对仔鱼的发育、生长和存活有着至关重要的作用。5-20日龄的个体都具有胸角这个明显的形态学特征,只是饥饿个体和不可逆转饥饿期个体的胸角比摄食个体更为明显和尖锐,胸角不能作为区分健康仔鱼和饥饿仔鱼的形态学依据之一[动物学报51(6)1023-1033,2005]。     

微流水培养条件下斑鳜仔鱼的摄食与生长

在孵化环道连续微流水培养、水温(24±2)℃条件下,斑鳜(Siniperca scherzeri Steindachner)初孵仔鱼全长为(4.87±0.10)mm(n=50),卵黄囊体积为(1.461±0.172)mm3(n=50),油球直径为(0.47±0.04)mm(n=50).仔鱼孵出12h,胸鳍增大,具有一定阵发性水平游动能力,1日龄巡游模式建立;2日龄口膜消失,开始主动摄食,进入混合营养期,3 日龄外源性摄食关系完全建立.5日龄仔鱼的卵黄和油球全部消失.进入外源营养期;15日龄全长达到(13.72±0.76)mm(n=12).仔鱼发育过程中,其全长生长存在内源性营养阶段的较快速生长,混合营养阶段的慢速生长以及外源性营养阶段的快速生长三个生长期相,平均增长率为0.59 mm/d,对仔鱼全长TL(mm)与日龄D(d)进行同归,其生长模型为:TL=-0.0004D3+0.0283D2+0.2159D + 4.9335(R2=0.985,n=261).2-15 日龄,口宽与全长呈正比关系.仔鱼从初孵到PNR仅为5-6d,具有摄食能力的时间4d,仔鱼依赖外源性营养开始时间较早,对饥饿的耐受力较差.     

遮光对刺梨果实和叶片中维生素C与糖含量以及相关酶活性的影响

为探究光照强度减弱对不同发育期的刺梨(Rosa roxburghii Tratt.)果实和叶片中维生素C与糖含量及相关酶活性的影响,以‘贵农5号’刺梨扦插苗为材料,采用遮阳网设置光强减弱20%、40%、60%的3个遮光处理,以自然光照强度为对照(CK),分析不同光照强度条件下刺梨果实和叶片中维生素C含量及相关酶活性以及糖含量的变化。结果表明：(1)光照强度减弱后,不同发育期的刺梨果实和叶片中维生素C含量均显著降低,对照及3个处理间的果实维生素C含量的差异在快速膨大期均达到显著水平。(2)随着光照强度的减弱,刺梨果实和叶片中L 半乳糖 1,4 内酯脱氢酶(GalLDH)、单脱氢抗坏血酸还原酶(MDHAR)和脱氢抗坏血酸还原酶(DHAR)活性均降低,而抗坏血酸氧化酶(AAO)和抗坏血酸过氧化物酶(APX)活性均增强,尤其是果实中MDHAR活性在快速膨大期迅速大幅增强。(3)刺梨果实和叶片中可溶性糖、蔗糖、葡萄糖、果糖含量随光照强度减弱而降低,但果实中葡萄糖含量在快速膨大期增加尤为迅速。(4)果实中维生素C含量与叶片中蔗糖、葡萄糖含量在不同发育期均呈极显著正相关关系,快速膨大期果实中GalLDH、MDHAR活性与维生素C含量呈极显著正相关关系且相关系数均达到最大值,而其APX活性与维生素C含量呈极显著负相关关系且相关系数达到最大值。研究认为,光照强度通过调控刺梨果实中维生素C合成及相关代谢关键酶的活性与叶片中光合产物的含量最终影响果实中维生素C的积累量;弱光环境不利于刺梨果实和叶片中维生素C及糖的积累,其影响在果实快速膨大期尤为明显;良好的光照是保证刺梨果实优良品质的基本条件。     

甘蓝型油菜子油分的积累与某些生理变化关系的研究

油菜种子发育过程中,其内部的生理代谢过程发生了规律性的变化。伴随着种子的发育进程,６－磷酸葡萄糖脱氢酶、异柠檬酸裂解酶、异柠檬酸脱氢酶和琥珀酸脱氢酶的活性均有不同程度的增强。在油分旺盛合成期,６－磷酸葡萄糖脱氢酶和异柠檬酸裂解酶的活性均达到了最大值,而此时,异柠檬酸脱氢酶和琥珀酸脱氢酶的活属于匀增加较慢;在种子的不同发育时期,高含油量品系的６－磷酸葡萄糖脱氢酶和异柠檬酸裂解酶的活性均高于低含油量的     

Thioredoxin-interacting protein regulates lipid metabolism via Akt/mTOR pathway in diabetic kidney disease

Abnormal lipid metabolism contributes to the renal lipid accumulation, which is associated with diabetic kidney disease, but its precise mechanism remains unclear. The growing evidence demonstrates that thioredoxin-interacting protein is involved in regulating cellular glucose and lipid metabolism. Here, we investigated the effects of thioredoxin-interacting protein on lipid accumulation in diabetic kidney disease. In contrast to the diabetic wild-type mice, the physical and biochemical parameters were improved in the diabetic thioredoxin-interacting protein knockout mice. The increased renal lipid accumulation, expression of acetyl-CoA carboxylase, fatty acid synthase and sterol regulatory element binding protein-1, and phosphorylated Akt and mTOR associated with diabetes in wild-type mice was attenuated in diabetic thioredoxin-interacting protein knockout mice. Furthermore, thioredoxin-interacting protein knockout significantly increased the expression of peroxisome proliferator-activated receptor-α, acyl-coenzyme A oxidase 1 and carnitine palmitoyltransferaser 1 in diabetic kidneys. In vitro experiments, using HK-2 cells, revealed that knockdown of thioredoxin-interacting protein inhibited high glucose-mediated lipid accumulation, expression of acetyl-CoA carboxylase, fatty acid synthase and sterol regulatory element binding protein-1, as well as activation of Akt and mTOR. Moreover, knockdown of thioredoxin-interacting protein reversed high glucose-induced reduction of peroxisome proliferator-activated receptor-α, acyl-coenzyme A oxidase 1 and carnitine palmitoyltransferaser 1 expression in HK-2 cells. Importantly, blockade of Akt/mTOR signaling pathway with LY294002, a specific PI3K inhibitor, replicated these effects of thioredoxin-interacting protein silencing. Taken together, these data suggest that thioredoxin-interacting protein deficiency alleviates diabetic renal lipid accumulation through regulation of Akt/mTOR pathway, thioredoxin-interacting protein may be a potential therapeutic target for diabetic kidney disease.     

Studies on acetyl-CoA carboxylase and fatty acid synthase from rat mammary gland and mammary tumours

The activities of two lipogenic enzymes, acetyl-CoA carboxylase and fatty acid synthase, were determined in two transplantable mammary adenocarcinomas (13762 and R3230AC) carried by non-pregnant, pregnant and lactating rats, and in mammary tissue of control animals (non-tumour-carrying) of comparable physiological states. During mammary-gland differentiation of control or tumour-carrying animals, the activities of acetyl-CoA carboxylase and fatty acid synthase in the lactating gland increased by about 40--50-fold over the values found in non-pregnant animals. On the other hand, in tumours carried by lactating dams there were only modest increases (1.5--2-fold) in acetyl-CoA carboxylase and fatty acid synthase compared with the neoplasms carried by non-pregnant animals. On the basis of the Km values for different substrates and immunodiffusion and immunotitration data, the fatty acid synthase of neoplastic tissues appeared to be indistinguishable from the control mammary-gland enzyme. However, a comparison of the immunotitration and immunodiffusion experiments indicated that the mammary-gland acetyl-CoA carboxylase might differ from the enzyme present in mammary neoplasms.     

西伯利亚鲟和鲫鱼脑型一氧化氮合酶cDNA片段克隆及序列分析

实验根据脑型一氧化氮合酶氨基酸高度保守序列设计一对简并引物,采用RT-PCR方法扩增并克隆了西伯利亚鲟(Acipenser baeri)和鲫鱼(Carassius auratus)的脑型一氧化氮合酶cDNA片段。西伯利亚鲟cD-NA片段为356 bp,鲫鱼cDNA片段为377 bp。氨基酸序列同源分析发现,西伯利亚鲟与斑马鱼(Brachydaniorerio)的脑型一氧化氮合酶同源性为70%,鲫鱼与斑马鱼的同源性为97.6%。     

Cold stress regulates lipid metabolism via AMPK signalling in Cherax quadricarinatus

Lipids play an important role in protecting poikilotherms from cold stress, but relatively little is known about the regulation of lipid metabolism under cold stress, especially in crustaceans. In the present study, red-clawed crayfish Cherax quadricarinatus was employed as a model organism. Animals were divided into four temperature groups (25, 20, 15 and 9 °C) and treated for 4 weeks, with the 25 °C group serving as a control. The total lipid content in the hepatopancreas as well as the triglyceride, cholesterol and free fatty acid levels in the hemolymph were determined. Lipids stored in the hepatopancreas and hemolymph decreased with decreasing temperature, with changes in the 9 °C group most pronounced, indicating that lipids are the main energy source for crayfish at low temperatures. Furthermore, enzyme activity of lipase, fatty acid synthase, acetyl-CoA carboxylase, and lipoprotein esterase, and gene expression analysis of fatty acid synthase gene, acetyl-CoA carboxylase gene and carnitine palmitoyltransferase gene showed that the digestion, synthesis and oxidation of lipids in the hepatopancreas were inhibited under low temperature stress, but expression of sphingolipid delta-4 desaturase (DEGS) was increased, indicating an increase in the demand for highly unsaturated fatty acids at low temperatures. Analysis of the expression of genes related to the AMP-activated protein kinase (AMPK) signalling pathway revealed that the adiponectin receptor gene was rapidly upregulated at low temperatures, which may in turn activate the expression of the downstream AMPKα gene, thereby inhibiting lipid anabolism.     

Effects of High Zinc Levels on the Lipid Synthesis in Rat Hepatocytes

Zinc deficiency impairs the hepatic lipid metabolism. Previous studies were focused on the negative effects of zinc deficiency on the hepatic lipid metabolism. A few studies investigated the effects of high zinc levels on the lipid metabolism in hepatocytes. In this study, rat hepatocytes were cultured and treated with different and high concentrations of zinc to investigate the effects of high zinc levels on the lipid synthesis in hepatocytes in vitro. The levels of hepatocytes functional markers, including alkaline phosphatase, lactate dehydrogenase, and albumin, were significantly higher in the zinc treatment groups than in the control group (p?<?0.05, p?<?0.01). The mRNA and protein levels of sterol regulatory element-binding protein 1c (SREBP-1c) were significantly higher in the zinc treatment groups than in the control group (p?<?0.05, p?<?0.01). Furthermore, the mRNA expression levels of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS) were significantly higher in the medium- and high-dose zinc treatment groups than in the control group (p?<?0.01). The mRNA levels of stearoyl-CoA desaturase-1 (SCD-1) were significantly higher in the high-dose group (p?<?0.01). These results indicate that high levels of zinc increase hepatocytes activity and SREBP-1c expression, which upregulate the expression of ACC1, FAS, and SCD-1, thereby improving the lipid metabolism in the hepatocytes.     

Kinetic Flux Profiling Elucidates Two Independent Acetyl-CoA Biosynthetic Pathways in Plasmodium falciparum

The malaria parasite Plasmodium falciparum depends on glucose to meet its energy requirements during blood-stage development. Although glycolysis is one of the best understood pathways in the parasite, it is unclear if glucose metabolism appreciably contributes to the acetyl-CoA pools required for tricarboxylic acid metabolism (TCA) cycle and fatty acid biosynthesis. P. falciparum possesses a pyruvate dehydrogenase (PDH) complex that is localized to the apicoplast, a specialized quadruple membrane organelle, suggesting that separate acetyl-CoA pools are likely. Herein, we analyze PDH-deficient parasites using rapid stable-isotope labeling and show that PDH does not appreciably contribute to acetyl-CoA synthesis, tricarboxylic acid metabolism, or fatty acid synthesis in blood stage parasites. Rather, we find that acetyl-CoA demands are supplied through a “PDH-like” enzyme and provide evidence that the branched-chain keto acid dehydrogenase (BCKDH) complex is performing this function. We also show that acetyl-CoA synthetase can be a significant contributor to acetyl-CoA biosynthesis. Interestingly, the PDH-like pathway contributes glucose-derived acetyl-CoA to the TCA cycle in a stage-independent process, whereas anapleurotic carbon enters the TCA cycle via a stage-dependent phosphoenolpyruvate carboxylase/phosphoenolpyruvate carboxykinase process that decreases as the parasite matures. Although PDH-deficient parasites have no blood-stage growth defect, they are unable to progress beyond the oocyst phase of the parasite mosquito stage.     

Hepatocyte heterogeneity in the metabolism of fatty acids: discrepancies on zonation of acetyl-CoA carboxylase.

Lipid metabolism appears to be less zonated than carbohydrate and protein metabolism. Studies on the zonation of lipid metabolism have been centered in particular on fatty acid synthesis which, according to the concept of metabolic zonation, should be a predominantly perivenous process while fatty acid oxidation should be periportal. There are, however, conflicting data on the activity gradients of lipogenic enzymes as well as measurements of actual synthesis of fatty acid and very low density lipoprotein. Data obtained by microdissection show a 1.5- to 2-fold higher activity of acetyl-CoA carboxylase and citrate lyase in the perivenous zone in agreement with measurements of the actual rate of fatty acid synthesis in preparations of hepatocyte, enriched in periportal or perivenous cells. On the other hand, results obtained with the dual-digitonin-pulse perfusion technique demonstrate the opposite gradient in the form of a 2- to 3-fold higher specific activity of acetyl-CoA carboxylase in the periportal zone based on measurements of the acetyl-CoA carboxylase protein proper. This specific activity gradient, which applies to male and not female rats, disappears almost completely in the fasted-refed animal, were lipogenesis is strongly induced. In this review we attempt to rationalize these discrepancies in the results as methodological differences which in particular apply to the following parameters: (1) expression of results (reference substance); (2) selectivity of zonal sampling, and (3) differences in methodology of acetyl-CoA carboxylase measurements. It is concluded that these factors could account for the discrepancies, but further studies, in particular on the zonation acetyl-CoA carboxylase mRNA, are required in order to further understand the zonation of lipid metabolism and its possible role in the metabolic regulation of the liver.     

Effects of dietary nutrients on substrate and effector levels of lipogenic enzymes, and lipogenesis from tritiated water in rat liver

When fasted rats were refed for 4 days with a carbohydrate and protein diet, a carbohydrate diet (without protein) or a protein diet (without carbohydrate), the effects of dietary nutrients on the fatty acid synthesis from injected tritiated water, the substrate and effector levels of lipogenic enzymes and the enzyme activities were compared in the livers. In the carbohydrate diet group, although acetyl-CoA carboxylase was much induced and citrate was much increased, the activity of acetyl-CoA carboxylase extracted with phosphatase inhibitor and activated with 0.5 mM citrate was low in comparison to the carbohydrate and protein diet group. The physiological activity of acetyl-CoA carboxylase seems to be low. In the protein diet group, the concentrations of glucose 6-phosphate, acetyl-CoA and malonyl-CoA were markedly higher than in the carbohydrate and protein group, whereas the concentrations of oxaloacetate and citrate were lower. The levels of hepatic cAMP and plasma glucagon were high. The activities of acetyl-CoA carboxylase and also fatty acid synthetase were low in the protein group. By feeding fat, the citrate level was not decreased as much as the lipogenic enzyme inductions. Comparing the substrate and effector levels with the Km and Ka values, the activities of acetyl-CoA carboxylase and fatty acid synthetase could be limited by the levels. The fatty acid synthesis from tritiated water corresponded more closely to the acetyl-CoA carboxylase activity (activated 0.5 mM citrate) than to other lipogenic enzyme activities. On the other hand, neither the activities of glucose-6-phosphate dehydrogenase and malic enzyme (even though markedly lowered by diet) nor the levels of their substrates appeared to limit fatty acid synthesis of any of the dietary groups. Thus, it is suggested that under the dietary nutrient manipulation, acetyl-CoA carboxylase activity would be the first candidate of the rate-limiting factor for fatty acid synthesis with the regulations of the enzyme quantity, the substrate and effector levels and the enzyme modification.     

Effects of ethanol feeding on hepatic lipid synthesis

Rats were fed a high-fat, liquid diet containing either 36% of total calories as ethanol or an isocaloric amount of sucrose, for a period up to 35 days. At different time intervals we measured the effects of ethanol administration on the activities of a number of key enzymes involved in hepatic lipid synthesis. At the start of the experimental period the activities of acetyl-CoA carboxylase and fatty acid synthase, measured in liver homogenates, increased in the control as well as in the ethanol-fed group. After 35 days these enzyme activities were still elevated but there were no significant differences between the two groups. In hepatocytes isolated from controls as well as from ethanol-fed rats, short-term incubations with ethanol induced an increase in the rate of fatty acid synthesis and in the activities of acetyl-CoA carboxylase and fatty acid synthase. However, no alterations in the regulation of these enzymes by short-term modulators of lipogenesis were apparent in hepatocytes isolated from alcohol-treated animals. The results do not indicate a major role for the enzymes of de novo fatty acid synthesis in the development of the alcoholic fatty liver. The amount of liver triacylglycerols increased in ethanol-fed rats during the entire treatment period, whereas the hepatic levels of phosphatidylcholine and phosphatidylethanolamine were not affected by ethanol ingestion. Ethanol administration for less than 2 weeks increased the activities of phosphatidate phosphohydrolase, diacylglycerol acyltransferase, and microsomal phosphocholine cytidylyltransferase, whereas the cytosolic activity of phosphocholine cytidylyltransferase was slightly decreased. Upon prolonged ethanol administration the activities of these enzymes were slowly restored to control values after 35 days, suggesting development of some kind of adaptation. It is interesting that, although the activities of phosphatidate phosphohydrolase and diacylglycerol acyltransferase were restored to the levels found in the control rats, this effect was not accompanied by a stabilization or decrease of the concentration of hepatic triacylglycerols.