Effect of dietary fatty acids on lipoprotein lipase gene expression in the liver and visceral adipose tissue of fed and starved red sea bream Pagrus major

Juvenile red sea bream Pagrus major were fed either a commercial diet (diet 1) or diets supplemented with 10% oleate (diet 2), 5% oleate+5% linoleate (diet 3) or 5% oleate+5% n-3 polyunsaturated fatty acid mixture (diet 4) for 4 weeks. Following the conditioning period, the effects of dietary fatty acids on lipoprotein lipase (LPL) gene expression in the liver and visceral adipose tissue of fed (5 h post-feeding) and starved (48 h post-feeding) fish were investigated by competitive polymerase chain reaction. Fish liver showed substantial LPL mRNA expression that is not found in adult rat liver. When compared with diet 1, diets 2-4 tended to increase the LPL mRNA level in the liver, but tended to decrease it in the visceral adipose tissue under the fed condition. The reciprocal regulation of the liver and visceral adipose LPL mRNA abundance by dietary fatty acids was comparable to that of rat brown and white adipose tissue, respectively. The change in the LPL mRNA level by fatty acids was not completely consistent with the degree of fatty acid unsaturation. Our results indicate that the regulatory effect of dietary fatty acids on LPL gene expression was tissue-specific and related to feeding conditions, but was not solely dependent on the degree of unsaturation of fatty acids.     

海水鱼真鲷脂蛋白脂肪酶基因cDNA序列与组织表达

为研究脊椎动物真鲷脂蛋白脂肪酶 (LPL)结构与功能关系以及探讨动脉粥样硬化形成机理 ,通过构建cDNA文库 ,克隆对动脉粥样硬化表现抗性的海水鱼真鲷LPL基因cDNA全序列 .再通过PCR方法扩增基因组DNA ,获取内含子 9及其两侧序列以确定外显子 10的大小 ,最后通过RT PCR ,以 β肌动蛋白为外参照 ,比较真鲷在食用两种脂肪含量不同饲料和摄食状态不同的处理条件下 ,肝脏和腹腔肠系膜脂肪组织LPLmRNA的相对水平 .从腹腔肠系膜脂肪组织cDNA文库中克隆出LPLcDNA序列 ,其完整的开放阅读框架由 15 36bp组成 ,编码 5 11个氨基酸残基 .与哺乳类不同 ,真鲷LPL基因外显子 10的开始部分是翻译的 .LPL的催化位点、二硫键位点、N 糖基化位点、肝素结合区、脂质结合位点、介导脂蛋白与低密度脂蛋白受体结合位点、二聚体形成位点等主要功能域在真骨鱼类真鲷与其它脊椎动物间基本保守 ,但肝素结合区的碱性氨基酸残基含量较人类减少 ,并在结合脂质底物的疏水环套中出现插入片段 .与哺乳类不同 ,真鲷LPL基因在成体肝脏存在诱导性表达 ,而在其腹腔肠系膜脂肪组织则存在与哺乳类相似的组成性表达 .当真鲷喂食高脂饲料时 ,其饱食状态下肝脏LPLmRNA水平升高 ,但对其腹腔肠系膜脂肪组织LPL表达没有影响 .当真鲷喂食标准商业饲料时 ,     

Insulin regulation of lipoprotein lipase (LPL) activity and expression in gilthead sea bream (Sparus aurata)

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein metabolism by virtue of its capacity to hydrolyze triglycerides circulating in the form of lipoprotein particles. Here we analyzed the fasting effects of LPL in gilthead sea bream (Sparus aurata) and also present the first study in fish of the role of insulin as a potential modulator of both LPL activity and expression. Fasting for 2 weeks provoked a clear decrease in adipose tissue LPL activity, concomitant with lower levels of plasma insulin, while no effects were observed in red muscle. To elucidate the specific role of insulin, increases of plasma insulin were experimentally induced by arginine and insulin injections. However, arginine predominantly stimulated glucagon over insulin secretion in this fish species while LPL activity did not change significantly in adipose tissue. Instead, insulin administration induced an increase in adipose tissue LPL activity 3 h after the injection, whereas LPL activity in red muscle was not affected. Changes in LPL activity were accompanied by an increase in LPL mRNA levels in the adipose tissue of insulin-injected gilthead sea bream, although changes in LPL expression were delayed in time with respect to variations in LPL activity. Finally, LPL mRNA levels in red muscle were similar between control and insulin-injected gilthead sea bream, suggesting that insulin does not play a direct role in the regulation of LPL in this tissue. The current study shows that LPL activity is regulated by nutritional condition and underscores the importance of insulin as a modulator of LPL activity and expression in the adipose tissue of gilthead sea bream.     

Molecular characterization of lipoprotein lipase, hepatic lipase and pancreatic lipase genes: effects of fasting and refeeding on their gene expression in red sea bream Pagrus major

To investigate the nutritional regulation of lipid metabolism in fish, molecular characterization of lipases was conducted in red sea bream Pagrus major, and the effects of fasting and refeeding on their gene expression was examined. Together with data from a previous study, a total of four lipase genes were identified and characterized as lipoprotein lipase (LPL), hepatic lipase (HL) and pancreatic lipase (PL). These four lipase genes, termed LPL1, LPL2, HL and PL, share a high degree of similarity. LPL1 and LPL2 genes were expressed in various tissues including adipose tissue, gill, heart and hepatopancreas. HL gene was exclusively expressed in hepatopancreas. PL gene expression was detected in hepatopancreas and adipose tissue. Red sea bream LPL1 and LPL2 gene expression levels in hepatopancreas were increased during 48 h of fasting and decreased after refeeding, whereas no significant change in the expression levels of LPL1 and LPL2 was observed in adipose tissue, indicating that LPL1 and LPL2 gene expression is regulated in a tissue-specific manner in response to the nutritional state of fish. HL and PL gene expression was not affected by fasting and refeeding. The results of this study suggested that LPL, HL and PL gene expression is under different regulatory mechanisms in red sea bream with respect to the tissue-specificities and their nutritional regulation.     

Abundant and constant expression of uncoupling protein 2 in the liver of red sea bream Pagrus major

Four overlapping cDNA fragments encoding a partial sequence for uncoupling protein 2 (UCP2) were amplified by PCR using degenerate primers from the liver of a marine teleost fish, red sea bream (Pagrus major). The partial sequence was 674 bp long, encoding 224 amino acids. The deduced amino acid sequence from the cDNA partial sequence contained the signature motifs for mitochondrial transporter protein and revealed positional identity higher than 72.8% with UCP2 from mammals. The fish UCP2 gene was highly expressed in the liver but almost undetectable in the visceral mesenteric adipose tissue. Using beta-actin as control, the UCP2 mRNA level was determined to be at least 20-fold higher in the liver than in the visceral mesenteric adipose tissues. Neither 48 h starvation nor high lipid diet had any significant effect on liver UCP2 gene expression, indicating that the abundant UCP2 gene expression was stable and might have some basic function in a fish liver that always contains high lipid content. The striking contrast of UCP2 gene expression in the two fish fat-depot organs is consistent with their large differences in oxidative capacity. We suggest that the fish liver may adapt to a constantly high fat deposit by maintaining high UCP2 expression to constrain reactive oxygen species (ROS) production and protect hepatocytes from apoptosis.     

饲料脂肪水平对瓦氏黄颡鱼生长及脂蛋白脂酶基因表达的影响

研究采用脂肪水平分别为4.7%、7.9%、10.9%、15.4%、18.9%的五种等氮配合饲料饲喂瓦氏黄颡鱼早期幼鱼,进行了为期30d的生长实验,探讨了瓦氏黄颡鱼早期幼鱼的脂肪需求。并克隆了瓦氏黄颡鱼脂蛋白脂酶(LPL)cDNA序列片段,采用实时荧光定量PCR研究了饲料脂肪水平对肝脏LPL基因表达水平的影响。结果表明,饲料脂肪水平从4.7%增加到10.9%显著促进了瓦氏黄颡鱼早期幼鱼的生长(P<0.05)。饲料脂肪水平显著影响了实验鱼的鱼体体成分,随着饲料脂肪水平的升高,鱼体干物质和脂肪含量显著增加而蛋白含量显著下降(P<0.05)。高脂诱导了瓦氏黄颡鱼肝脏LPL基因表达,摄食15.4%、18.9%这两组较高脂肪水平的实验鱼肝脏LPLmRNA表达水平显著升高(P<0.05)。根据特定生长率通过折线回归分析得出瓦氏黄颡鱼早期幼鱼最适脂肪水平为11.2%。       

Molecular characterization of gilthead sea bream (Sparus aurata) lipoprotein lipase. Transcriptional regulation by season and nutritional condition in skeletal muscle and fat storage tissues

Lipoprotein lipase (LPL) of gilthead sea bream (Sparus aurata) was cloned and sequenced using a RT-PCR approach completed by 3' and 5'RACE assays. The nucleotide sequence covered 1669 bp with an open reading frame of 525 amino acids, including a putative signal peptide of 23 amino acids long. Sequence alignment and phylogenetic analysis revealed a high degree of conservation among most fish and higher vertebrates, retaining the consensus sequence the polypeptide "lid", the catalytic triad and eight cysteine residues at the N-terminal region. A tissue-specific regulation of LPL was also found on the basis of changes in season and nutritional condition as a result of different dietary protein sources. First, the expression of LPL in mesenteric adipose tissue was several times higher than in liver and skeletal muscle. Secondly, the spring up-regulation of LPL expression in the mesenteric adipose tissue was coincident with a pronounced increase of whole body fat content. Thirdly, the highest expression of LPL in the skeletal muscle was found in summer, which may serve to cover the increased energy demands for muscle growth and protein accretion. Further, in fish fed plant-protein-based diets, hepatic LPL expression was up-regulated whereas an opposite trend was found in the mesenteric adipose tissue, which may contribute to drive dietary lipids towards liver fat storage. Finally, it is of interest that changes in circulating triglyceride (TG) levels support the key role of LPL in the clearance of TG-rich lipoproteins. This study is the first report in fish of a co-regulated expression of LPL in oxidative and fat storage tissues under different physiological conditions.     

Effects of dietary mulberry, Korean red ginseng, and banaba on glucose homeostasis in relation to PPAR-alpha, PPAR-gamma, and LPL mRNA expressions

Despite lack of scientific evidences to support its therapeutic efficacy, the use of herbal supplements has significantly increased. The purpose of this study was to evaluate the effects of traditional anti-diabetic herbs on the progress of diabetes in db/db mice, a typical non-insulin-dependent model. Five different experimental diets were as follows: control diet, 0.5% mulberry leaf water extract diet, 0.5% Korean red ginseng diet, 0.5% banaba leaf water extract diet, and 0.5% combination diet (mulberry leaf water extract/Korean red ginseng/banaba leaf water extract, 1:1:1). Blood levels of glucose, insulin, HbA1c, and triglyceride were measured every 2 weeks. At 12 weeks of age, animals were sacrificed, and tissue mRNA levels of PPAR-alpha, PPAR-gamma, and LPL were determined. Results indicated that mulberry leaf water extract, Korean red ginseng, banaba leaf water extract, and the combination of above herbs effectively reduced blood glucose, insulin, TG, and percent HbA1c in study animals (p<0.05). We also observed that the increased expressions of liver PPAR-alpha mRNA and adipose tissue PPAR-gamma mRNA in animals fed diets supplemented with test herbs. The expression of liver LPL mRNA was also increased with experimental diets containing herbs. The efficacy was highest in animals fed the combination diet for all of the markers used. These results suggest that mulberry leaf water extract, Korean red ginseng, banaba leaf water extract, and the combination of these herbs fed at the level of 0.5% of the diet significantly increase insulin sensitivity, and improve hyperglycemia possibly through regulating PPAR-mediated lipid metabolism.     

Copper transporter 1, metallothionein and glutathione reductase genes are differentially expressed in tissues of sea bream (Sparus aurata) after exposure to dietary or waterborne copper

The high affinity copper transporter 1 (Ctr1), metallothionein (MT) and glutathione reductase (GR) are essential for copper uptake, sequestration and defense respectively. Following rearing on a normal commercial diet (12.6+/-0.2 mg kg(-1) Cu), sea bream were fed an experimental control diet lacking mineral mix (7.7+/-0.3 mg kg(-1) Cu), an experimental diet enhanced with Cu (135+/-4 mg kg(-1) Cu) or an experimental diet (7.7+/-0.3 mg kg(-1) Cu) whilst exposed to Cu in water (0.294+/-0.013 mg L(-1)). Fish were sampled at 0, 15 and 30 days after exposures. Fish fed the Cu-enhanced experimental diet showed lower levels of expression of Ctr1 in the intestine and liver compared to fish fed control experimental diets, whilst Ctr1 expression in the gill and kidney was unaffected by excess dietary Cu exposure. Waterborne-Cu exposure increased Ctr1 mRNA levels in the intestine and the kidney compared to experimental controls. Excess dietary Cu exposure had no effect on levels of metallothionein (MT) mRNA, and the only effect of dietary excess Cu on glutathione reductase (GR) mRNA was a decrease in the intestine. Both MT mRNA and GR were increased in the liver and gill after waterborne-Cu exposure, compared to levels in fish fed experimental control low Cu diets. Thus, Ctr1, MT and GR mRNA expression in response to excess Cu is dependent on the route of exposure. Furthermore, the tissue expression profile of sea bream Ctr1 is consistent with the known physiology of copper exposure in fish and indicates a role both in essential copper uptake and in avoidance of excess dietary and waterborne copper influx.     

Dietary diacylglycerol suppresses high fat and high sucrose diet-induced body fat accumulation in C57BL/6J mice

Diacylglycerol (DG) comprises up to approximately 10% of various edible oils. In the present study, we examined the effects of dietary DG consisting mainly of 1,3-species on body weight, body fat accumulation, and mRNA levels of various genes involved in energy homeostasis in obesity-prone C57BL/6J mice. Five-month feeding with the high triacylglycerol (TG) diet (30% TG + 13% sucrose) resulted in significant increases in body weight, visceral fat accumulation, and circulating insulin and leptin levels compared with mice fed the control diet (5% TG). Compared with mice fed the high TG diet, body weight gain and visceral fat weight were reduced by 70% and 79%, respectively, in those fed the high DG diet (30% DG + 13% sucrose). In addition, circulating leptin and insulin levels were reduced to the respective control levels. Compared with high TG feeding, high DG feeding suppressed the elevation of leptin mRNA expression in adipose tissue, and up-regulated acyl-coenzyme (Co)A oxidase and acyl-CoA synthase mRNA expression in the liver. These results indicate that dietary DG is beneficial for suppression of high fat diet-induced body fat accumulation. Furthermore, it is suggested that structural differences in DG and TG, but not the composition of fatty acid, markedly affect nutritional behavior of lipids. -- Murase, T., T. Mizuno, T. Omachi, K. Onizawa, Y. Komine, H. Kondo, T. Hase, and I. Tokimitsu. Dietary diacylglycerol suppresses high fat and high sucrose diet-induced body fat accumulation in C57BL/6J mice. J. Lipid Res. 2001. 42: 372--378.     

Fat metabolism is regulated by altered gene expression of lipogenic enzymes and regulatory factors in liver and adipose tissue but not in semimembranosus muscle of pigs during the fattening period

It has been shown previously that lipid metabolism is regulated by fatty acids (FA) and that thyroid hormones are important regulators of energy metabolism. The effects of weight, dietary fat level and dietary FA profile on thyroid hormone levels and expression of lipogenic genes and tissue FA composition were studied. Sixty-one crossbred gilts weighing 62 ± 5.2 kg BW average were either slaughtered at the beginning of the trial (n = 5) or fed one of seven diets (n = 8 pigs per diet): a semi-synthetic diet formulated to contain a very low level of fat (NF) and six diets based on barley-soybean meal supplemented with approximately 10% fat of different origin and slaughtered at 100 kg BW. The supplemental fats were tallow, high-oleic sunflower oil, sunflower oil (SFO), linseed oil, fat blend (55% tallow, 35% sunflower oil, 10% linseed oil) and fish oil blend (40% fish oil, 60% linseed oil). In general, the dietary FA profiles altered the FA composition of liver, semimembranosus muscle and adipose tissues. Pigs fed the NF diet had the highest free and total triiodothyronine (T3) values followed by pigs fed SFO. Total T3 levels were higher in pigs at 60 kg than in pigs at 100 kg. Correlations between thyroid hormones and genes encoding enzymes of fat synthesis in adipose tissue (acetyl CoA carboxylase (ACACA), fatty acid synthase and stearoyl CoA desaturase (SCD)) and the large differences in expression of lipogenic genes at different weights (60 and 100 kg BW), suggest a role for thyroid hormones and for T3, in particular, in regulating whole animal fat metabolism, with effects brought about by altered expression of lipogenic genes. Liver sterol receptor element binding protein-1 (SREBP1) mRNA content was affected by dietary treatment (P < 0.001) and was correlated with ACACA and SCD, whereas adipose tissue SREBP1 was not correlated with the mRNA abundance of any lipogenic enzyme. Weight and tissue factors showed greater influence on mRNA abundance of genes related with lipid metabolism than diet and tissue FA composition. In the pig, FA synthesis appear to be of greater magnitude in adipose tissue than in the liver as suggested by the higher expression of lipogenic genes in adipose tissue.     

Changes in adipocyte cell size, gene expression of lipid metabolism markers, and lipolytic responses induced by dietary fish oil replacement in gilthead sea bream (Sparus aurata L.)

The effects of fish oil (FO) substitution by 66% vegetable oils in a diet with already 75% vegetable protein (66VO) on adipose tissue lipid metabolism of gilthead sea bream were analysed after a 14-month feeding trial. In the last 3 months of the experiment, a FO diet was administrated to a 66VO group (group 66VO/FO) as a finishing diet. Hormone-sensitive lipase (HSL) activity was measured in adipose tissue and adipocyte size, and HSL, lipoprotein lipase and liver X receptor gene expression in isolated adipocytes, on which lipolysis and glucose uptake experiments were also performed. Lipolysis was measured after incubation with tumour necrosis factor-α (TNFα), linoleic acid, and two conjugated linoleic acid isomers. Glucose uptake was analysed after TNFα or insulin administration. Our results show that FO replacement increased lipolytic activity and adipocyte cell size. The higher proportion of large cells observed in the 66VO group could be involved in their observed lower response to fatty acid treatments and lower insulin sensitivity. The 66VO/FO group showed a moderate return to the FO conditions. Therefore, FO replacement can affect the morphology and metabolism of gilthead sea bream adipocytes which could potentially affect other organs such as the liver.     

Dietary conjugated linoleic acid reduces body fat mass and affects gene expression of proteins regulating energy metabolism in mice

ICR and C57BL/6J mice were fed experimental diets containing either a 2% fatty acid preparation rich in conjugated linoleic acid (CLA) or a preparation rich in linoleic acid and free of CLA for 21 days. CLA greatly decreased weights of white adipose tissue and interscapular brown adipose tissue in the two strains. CLA reduced mRNA levels of glucose transporter 4 (Glut 4) in white and brown adipose tissue of both strains. A CLA-dependent decrease in mRNA levels of peroxisome proliferator activated receptor (PPAR) gamma was seen in interscapular brown adipose tissue of both strains and in white adipose tissue of C57BL/6J but not ICR mice. Dietary CLA was found to cause a decrease in the mRNA levels of uncoupling protein (UCP) 1 in brown adipose tissue when the value was corrected for the expression of a house-keeping gene (beta-actin) in the two strains. Uncorrected values were, however, indistinguishable between the animals fed the CLA diet and CLA-free diet. UCP 3 expression in brown adipose tissue was much lower in mice fed the CLA diet than in those fed the control diet in both strains. In contrast, CLA greatly up-regulated the gene expression of UCP 2 in brown adipose tissue. Dietary CLA also increased UCP 2 mRNA level in skeletal muscle. It is apparent that dietary CLA decreases white and brown adipose tissue mass, accompanying changes in the gene expression of proteins regulating energy metabolism in white and brown adipose tissues, and skeletal muscle of mice.     

Effects of dietary protein and lipid levels on growth and feed utilization of wild‐caught striped sea bream,Lithognathus mormyrus

A feeding trial was carried out to determine the effects of dietary protein and lipid levels on the growth performance and feed utilization of wild‐caught striped sea bream (Lithognathus mormyrus). The experimental fish were collected from a local lagoon (Çardak Lagoon, Çanakkale, Turkey), transferred to the Marine Net Cage Unit and fed by hand to apparent satiation with a commercial sea bream feed (Biomar; 42% crude protein, 16% crude lipid). Approximately 4 weeks were needed to acclimate the fish to farming conditions. No pathological signs were observed and no fish losses occurred during the adaptation period. For the test trials four test diets with different levels of protein and lipid were formulated [low protein and low lipid (LP:LL), low protein and high lipid (LP:HL), high protein and low lipid (HP:LL), and high protein and high lipid (HP:HL)] and fed to L. mormyrus (mean weight 85.0 ± 4.6 g SEM) in the net cages (Ø 2 m, depth 2.5 m) for 60 days. During the experiment water temperature varied between 21.1 and 26.4°C; dissolved oxygen 8.4–9.6 mg L^?1; pH 7.2–8.6; and salinity 23.3–25.6‰. Growth performances of fish fed high protein diets were higher compared to fish fed low protein diets, irrespective of the dietary lipid level (P < 0.05). Feed conversion ratio (FCR) and protein efficiency ratio (PER) were not influenced by dietary protein or lipid levels (P > 0.05). Preliminary results indicate that striped sea bream can be easily adapted to farming conditions in net cages, and that a diet containing 50% crude protein and 15% crude lipid (HP:LL) levels with 23.0 g protein MJ^?1 gross energy of protein/energy ratio would be suitable for striped sea bream growth.     

Molecular characterization of peroxisome proliferator-activated receptors (PPARs) and their gene expression in the differentiating adipocytes of red sea bream Pagrus major

To investigate the molecular mechanism of fish adipocyte differentiation, the three subtypes of PPAR genes (alpha, beta and gamma) were characterized in a marine teleost red sea bream (Pagrus major). The primary structures of red sea bream PPARs exhibited high degrees of similarities to their mammalian counterparts, and their gene expression was detected in various tissues including adipose tissue, heart and hepatopancreas. During the differentiation of primary cultured red sea bream adipocytes, three PPARs showed distinct expression patterns: The alpha subtype showed a transient increase and the beta gene expression tended to increase during adipocyte differentiation whereas the gene expression level of PPARgamma did not change. These results suggest that they play distinct roles in adipocyte differentiation in red sea bream. In the differentiating red sea bream adipocytes, mammalian PPAR agonists, 15-deoxy-Delta(12,14)-prostaglandin J(2), ciglitazone and fenofibrate did not show clear effects on the adipogenic gene expression. However, 2-bromopalmitate increased the PPARgamma and related adipogenic gene expression levels, suggesting the gamma subtype plays a central role in red sea bream adipocyte differentiation and in addition, fatty acid metabolites can be used as modulators of adipocyte function. Thus our study highlighted the roles of PPARs in fish adipocyte differentiation and provided information on the molecular mechanisms of fish adipocyte development.     

Mammalian adipose tissue and muscle are major sources of lipid transfer protein mRNA

The plasma cholesteryl ester transfer protein (CETP) catalyzes the transfer of cholesteryl esters from high density lipoproteins (HDL) to triglyceride-rich lipoproteins and plays a major role in the catabolism of HDL. Lipoprotein lipase (LPL) is the rate-limiting enzyme for hydrolysis of circulating triglyceride and is involved in HDL formation. We show that tissues containing LPL are major sources of CETP mRNA in several mammalian species, including some with low cholesteryl ester transfer activity in plasma. In hamsters, adipose tissue and heart were found to be the richest sources of both CETP and LPL mRNA; in situ hybridization studies showed that the same cell types (i.e. adipocytes or myocytes) contained CETP and LPL mRNA in these tissues. Isolated adipocytes synthesized active CETP. Dietary studies revealed a complex pattern of response of CETP mRNA levels in different tissues, which showed partial similarity to the changes in LPL mRNA abundance. However, high cholesterol diets resulted in increased CETP mRNA abundance in adipose tissue, heart, and skeletal muscle, without equivalent changes in LPL mRNA. Plasma HDL cholesteryl ester levels showed strong inverse correlations with CETP mRNA abundance in adipose tissue. The results suggest a conserved function of CETP in adipose tissue and heart, such as a co-ordinate action with LPL to enhance HDL turnover. Although there is considerable overlap in the tissue- and cell-specific pattern of CETP and LPL gene expression, dietary studies revealed only limited parallelism in response at the mRNA level. The increase in CETP mRNA in peripheral tissues in response to increased dietary cholesterol suggests that local induction of CETP synthesis may help to recycle cholesterol deposited in these tissues during lipolysis of dietary lipoproteins.