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 ,     

Utilisation of triacylglycerol and non-esterified fatty acid by the working rat heart: myocardial lipid substrate preference

Utilisation and subsequent metabolic fate (oxidation; tissue lipid deposition) of non-esterified fatty acid (NEFA), very-low-density lipoprotein-triacylglycerol (VLDL-TAG), and chylomicron-triacylglycerol (CM-TAG) alone or in combination by isolated working rat heart were examined. Cardiac mechanical function was maintained regardless of lipid substrate used. NEFA and CM-TAG were assimilated to a greater extent than VLDL-TAG; CM-TAG utilisation (76+/-10 nmol fatty acid/min per g wet wt.; n=8), but not VLDL-TAG utilisation (16+/-2 nmol fatty acid/min per g wet wt.; n=8), was suppressed in the presence of NEFA, but TAG (CM or VLDL) did not alter NEFA utilisation (57+/-9 nmol fatty acid/min per g wet wt.; n=8). Most (about 75%) of the lipid utilised was oxidised. In the presence of NEFA, CM-TAG deposition as tissue lipid was preserved, despite decreased CM-TAG oxidation; metabolic fate of VLDL-TAG was unaffected by NEFA. TAG (CM or VLDL) in the perfusate tended to decrease lipoprotein lipase (LPL) activity; this may be a reflection of increased LPL turnover in the presence of lipoproteins.     

Metabolism of very-low-density lipoprotein and chylomicrons by streptozotocin-induced diabetic rat heart: effects of diabetes and lipoprotein preference

Very-low-density lipoprotein (VLDL) and chylomicrons (CM) are major sources of fatty acid supply to the heart, but little is known about their metabolism in diabetic myocardium. To investigate this, working hearts isolated from control rats and diabetic rats 2 wk following streptozotocin (STZ) injection were perfused with control and diabetic lipoproteins. Analysis of the diabetic lipoproteins showed that both VLDL and CM were altered compared with control lipoproteins; both were smaller and had different apolipoprotein composition. Heparin-releasable lipoprotein lipase (HR-LPL) activity was increased in STZ-induced diabetic hearts, but tissue residual LPL activity was decreased; moreover, diabetic lipoproteins stimulated HR-LPL activity in both diabetic and control hearts. Diabetic hearts oxidized lipoprotein-triacylglycerol (TAG) to a significantly greater extent than controls (>80% compared with deposition as tissue lipid), and the oxidation rate of exogenous lipoprotein-TAG was increased significantly in diabetic hearts regardless of TAG source. Significantly increased intracardiomyocyte TAG accumulation was found in diabetic hearts, although cardiac mechanical function was not inhibited, suggesting that lipotoxicity precedes impaired cardiac performance. Glucose oxidation was significantly decreased in diabetic hearts; additionally, however, diabetic lipoproteins decreased glucose oxidation in diabetic and control hearts. These results demonstrate increased TAG-rich lipoprotein metabolism concomitant with decreased glucose oxidation in type 1 diabetic hearts, and the alterations in cardiac lipoprotein metabolism may be due to the properties of diabetic TAG-rich lipoproteins as well as the diabetic state of the myocardium. These changes were not related to cardiomyopathy at this early stage of diabetes.     

Postabsorptive VLDL‐TG Fatty Acid Storage in Adipose Tissue in Lean and Obese Women

Adipose tissue lipoprotein lipase (LPL) is a necessary enzyme for storage of very‐low‐density lipoprotein–triglyceride (VLDL‐TG), but whether it is a rate‐determining step is unknown. To test this hypothesis we included 10 upper‐body obese (UBO), 11 lower‐body obese (LBO), and 8 lean women. We infused ex vivo‐labeled VLDL‐¹⁴C‐TG and then performed adipose tissue biopsies to understand the relationship between VLDL‐TG storage and LPL activity in femoral and upper‐body subcutaneous fat. Both fractional tracer storage and rate of storage of the VLDL‐TG tracer were evaluated. VLDL‐TG storage was also examined as a function of regional adipose tissue blood flow (ATBF), insulin, VLDL‐TG turnover, regional fat mass, fat‐free mass (FFM), and fat cell size. LPL activity per adipocyte was significantly greater in obese than lean women but not significantly different per gram lipid. Both VLDL‐TG fractional tracer storage per kg lipid and VLDL‐TG storage rate per kg lipid were similar in abdominal and femoral fat in all three groups and were not significantly different between groups. Multiple regression analysis identified FFM and femoral fat mass as significant independent predictors of VLDL‐TG fractional tracer storage and insulin as a significant predictor of VLDL‐TG fatty acid storage rate. LPL activity, ATBF, and VLDL‐TG turnover did not predict VLDL‐TG storage. We conclude that lower FFM and greater plasma insulin are associated with greater VLDL‐TG deposition in abdominal subcutaneous and femoral fat. Greater femoral fat mass signals greater femoral VLDL‐TG storage. We suggest that the differences in VLDL‐TG storage in abdominal and femoral fat that occur with progressive obesity are regulated through mechanisms other than LPL activity.     

Maternal loading with very low-density lipoproteins stimulates fetal surfactant synthesis

We examined whether administration of very low-density lipoproteins (VLDL) to pregnant rats increases surfactant phosphatidylcholine (PtdCho) content in fetal pre-type II alveolar epithelial cells. VLDL-triglycerides are hydrolyzed to fatty acids by lipoprotein lipase (LPL), an enzyme activated by heparin. Fatty acids released by LPL can incorporate into the PtdCho molecule or activate the key biosynthetic enzyme cytidylyltransferase (CCT). Dams were given BSA, heparin, VLDL, or VLDL with heparin intravenously. Radiolabeled VLDL given to the pregnant rat crossed the placenta and was distributed systemically in the fetus and incorporated into disaturated PtdCho (DSPtdCho) in pre-type II cells. Maternal administration of VLDL with heparin increased DSPtdCho content in cells by 45% compared with control (P < 0.05). VLDL produced a dose-dependent, saturable, and selective increase in CCT activity. VLDL did not significantly alter immunoreactive CCT content but increased palmitic, stearic, and oleic acids in pre-type II cells. Furthermore, hypertriglyceridemic apolipoprotein E knockout mice contained significantly greater levels of DSPtdCho content in alveolar lavage and CCT activity compared with either LDL receptor knockout mice or wild-type controls that have normal serum triglycerides. Thus the nutritional or genetic modulation of serum VLDL-triglycerides provides specific fatty acids that stimulate PtdCho synthesis and CCT activity thereby increasing surfactant content.     

Mechanism of the cholesteryl ester transfer protein-mediated uptake of high density lipoprotein cholesteryl esters by Hep G2 cells

Plasma cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters (CE) between lipoproteins and was reported to also directly mediate the uptake of high density lipoprotein (HDL) CE by human Hep G2 cells and fibroblasts. The present study investigates that uptake and its relationship to a pathway for "selective uptake" of HDL CE that does not require CETP. HDL3 labeled in both the CE and apoprotein moieties was incubated with Hep G2 cells. During 4-h incubations, CE tracer was selectively taken up from doubly labeled HDL3 in excess of apoA-I tracer, and added CETP did not modify that uptake. However, during 18-20-h incubations, CETP stimulated the uptake of CE tracer more than 4-fold without modifying the uptake of apoA-I tracer. This suggested that secreted products, perhaps lipoproteins, might be required for the CETP effect. Four inhibitors of lipoprotein uptake via low density lipoprotein (LDL) receptors (heparin, monensin, an antibody against the LDL receptor, and antibodies against the receptor binding domains of apoB and apoE) effectively blocked the CETP stimulation of CE tracer uptake. Heparin caused an increase in CE tracer in a d less than 1.063 g/ml fraction of the medium that more than accounted for the heparin blockade of CETP-stimulated CE uptake. CETP did not affect the uptake of doubly labeled HDL3 by human fibroblasts, even at twice plasma levels of activity, and heparin did not modify uptake of HDL3 tracers. Thus the CETP effect on Hep G2 cells can be accounted for by transfer of HDL CE to secreted lipoproteins which are then retaken up, and there is no evidence for a direct effect of CETP on cellular uptake of HDL CE.     

The isolation and partial characterization of the serum lipoproteins and apolipoproteins of the rainbow trout.

1. VLD (very-low-density), LD (low-density) and HD (high-density) lipoproteins were isolated from the serum of trout (Salmo gairdneri Richardson). 2. Each lipoprotein class resembled that of the human in immunological reactivity, electrophoretic behaviour and appearance in the electron microscope. Trout LD lipoprotein, however, was of greater density than human LD lipoprotein. 3. The trout lipoproteins have lipid compositions which are similar to those of the corresponding human components, except for their high contents of long-chain unsaturated fatty acids. 4. HD and LD lipoproteins were immunologically non-identical, whereas LD lipoproteins possessed antigenic determinants in common with VLD lipoproteins. 5. VLD and HD lipoproteins each contained at least seven different apoproteins, whereas LD liprotein was composed largely of a single apoprotein which resembled human apolipoprotein B. 6. At least one, and possibly three, apoprotein of trout HD lipoprotein showed features which resemble human apoprotein A-1.7. The broad similarity between the trout and human lipoprotein systems suggests that both arose from common ancestral genes early in evolutionary history.     

Effects of a prudent diet containing either lean beef and mutton or fish and skinless chicken on the plasma lipoproteins and fatty acid composition of triacylglycerol and cholesteryl ester of hypercholesterolemic subjects

In this two-phase crossover study, 39 hypercholesterolemic subjects followed a prudent diet with either lean red meat or fish and skinless chicken (treatment groups), and 13 subjects (reference group) followed their habitual diet. Fasting blood samples were analyzed for plasma total cholesterol, triacylglycerol (TAG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein one- and two-cholesterol, apolipoprotein-B, very low density lipoprotein cholesterol, and very low density lipoprotein TAG, and fatty acid composition of plasma TAG and cholesteryl ester (CE). Body mass and blood pressure were determined. Seven-day dietary records were kept once at baseline and twice during the treatment periods. Significant differences were observed in dietary intake between the baseline and treatment diets and between the two treatment diets. HDL-C (P < 0.05) and diastolic blood pressure (P < 0.01) were higher in patients on the red meat diet than in those on the chicken-fish diet. No other significant differences in lipoproteins were observed between the effects of the two treatment diets. The linoleic acid (%), eicosapentaenoic acid (%), and the eicosapentaenoic acid/arachidonic acid ratios in TAG and CE were higher (P < 0.01) in subjects on the chicken-fish diet than in those on the red meat diet. In conclusion, this study showed that the effect of two lipid-lowering diets containing either lean red meat or skinless chicken and fish on the atherogenic lipoproteins did not differ significantly. A prudent diet with skinless chicken and fish, however, had a more favorable effect on the fatty acid composition of the plasma TAG and the CE than did the lean red meat diet.     

Brown adipose tissue activity controls triglyceride clearance

Brown adipose tissue (BAT) burns fatty acids for heat production to defend the body against cold and has recently been shown to be present in humans. Triglyceride-rich lipoproteins (TRLs) transport lipids in the bloodstream, where the fatty acid moieties are liberated by the action of lipoprotein lipase (LPL). Peripheral organs such as muscle and adipose tissue take up the fatty acids, whereas the remaining cholesterol-rich remnant particles are cleared by the liver. Elevated plasma triglyceride concentrations and prolonged circulation of cholesterol-rich remnants, especially in diabetic dyslipidemia, are risk factors for cardiovascular disease. However, the precise biological role of BAT for TRL clearance remains unclear. Here we show that increased BAT activity induced by short-term cold exposure controls TRL metabolism in mice. Cold exposure drastically accelerated plasma clearance of triglycerides as a result of increased uptake into BAT, a process crucially dependent on local LPL activity and transmembrane receptor CD36. In pathophysiological settings, cold exposure corrected hyperlipidemia and improved deleterious effects of insulin resistance. In conclusion, BAT activity controls vascular lipoprotein homeostasis by inducing a metabolic program that boosts TRL turnover and channels lipids into BAT. Activation of BAT might be a therapeutic approach to reduce elevated triglyceride concentrations and combat obesity in humans.     

Potentially conflicting metabolic demands of diving and exercise in seals

Metabolic replacement rates (Ra) for glucose and free fatty acids (FFA) were determined during rest, exercise, and diving conditions in the gray seal using bolus injections of radiotracers. In the exercise experiments the seal swam at a metabolic rate elevated twofold over resting Ra for glucose and FFA while resting were similar to values found in terrestrial mammals and other marine mammal species. During exercise periods glucose turnover increased slightly while FFA turnover changes were variable. However, the energetic demands of exercise could not be met by the increase in the replacement rates of glucose or FFA even if both were completely oxidized. Under diving conditions the tracer pool displayed radically different specific activity curves indicative of the changes in perfusion and metabolic rate associated with a strong dive response. Since the radiotracer curves during exercise and diving differed qualitatively and quantitatively, it is possible that similar studies on freely diving animals can be used to assess the role of the diving response during underwater swimming in nature.     

Lipoprotein lipase from rainbow trout differs in several respects from the enzyme in mammals

Previously we found lipase activity with characteristics similar to lipoprotein lipase (LPL) in tissues from rainbow trout [Biochim. Biophys. Acta 1255 (1995) 205], whereas no equivalent to the related hepatic lipase could be found. An equivalent to apolipoprotein CII was also identified and characterized [Gene 254 (2000) 189]. We present here the full nucleotide sequence for LPL from rainbow trout (Oncorhynchus mykiss) and have investigated some properties of the enzyme. In contrast to what has been found in mammals, LPL mRNA was expressed in livers of adult trout. This indicates that trout LPL carries out functions that hepatic lipase has evolved to take over in mammals. Trout LPL was unstable at 37 degrees C compared with bovine and human LPL. Two sequence differences that may relate to the instability are that trout LPL lacks the disulfide bridge in the C-terminal domain and lacks Pro(258). This residue is conserved in LPL from all mammals and has been shown to be critical for enzyme stability at 37 degrees C. On chromatography on heparin-Sepharose trout and chicken LPL eluted at higher salt concentration than bovine (or other mammalian) LPL. The C-terminal end of LPL has been implied in heparin binding and the higher heparin affinity of the trout and chicken enzymes may be because they have 17 and 15 extra amino acid residues at the C-terminal end, of which three residues are positively charged.     

Activation of trout adipose tissue lipoprotein lipase by trout apoproteins

In rainbow trout (Salmo gairdnerii) lipoprotein profiles change during the annual sexual cycle. Among other factors, lipoprotein lipase (LPL) activity might play a role. This enzyme is activated by trout serum suggesting the existence of a cofactor corresponding to apoprotein CII in this species. In the present study, we determined more accurately some characteristics of the enzyme activity inhibited by 0.3 M NaCl. Trout serum and high density lipoproteins (HDL) activated both rat and trout adipose tissue LPLs. A fraction of apo HDL obtained by gel filtration also activated the enzyme. The mean Mr was 10,000. Isoelectric focusing of the same fraction gave several bands of proteins with apparent pI in the range of 4.2-4.9. These results show that in trout, LPL is activated by a cofactor similar to that in mammals, the apo CII. In addition, a fraction mainly containing apo AI (+ traces of apo C) activated trout LPL and reinforced the activation by apo CII. These findings suggest that trout apo AI may promote the activating effect of apo CII on trout LPL.     

Exercise training and the stress response in rainbow trout, Salmo gairdneri Richardson

Plasma levels of catecholamines, cortisol, and glucose were monitored in rainbow trout during a 6-week forced swimming exercise programme. Compared to resting non-exercised controls, resting trained fish had lower levels of epinephrine, norephinephrine, cortisol, and glucose during the last 3 weeks of training. Initially, trained fish that were swimming had higher levels of epinephrine than resting trained fish. After 2 weeks of exercise, swimming did not significantly elevate epinephrine levels in trained fish. Glucose levels were consistently greater in swimming fish than in resting fish. At the end of the training period, exercised trout had lower (15–20%) oxygen consumption rates while resting or swimming than unexercised fish.
After a 5-month forced swimming exercise programme plasma levels of catecholamines and glucose were monitored in trained and untrained cannulated rainbow trout after 2 min of mild agitation. Trained fish showed an immediate (within 1 min) increase in the levels of epinephrine, but not norepinephrine and a delayed (within 15 min) increase in the levels of plasma glucose. Epinephrine levels returned to pre-stress levels within 15 min. Untrained fish had no significant increase in the plasma levels of norepinephrine, epinephrine, or glucose.     

Modifications of plasma lipoproteins after lipase activation in patients with chylomicronemia

Lipoprotein lipase (LPL) and hepatic lipase (HL) are enzymatic activities involved in lipoprotein metabolism. The purpose of this study was to analyze the physicochemical modifications of plasma lipoproteins produced by LPL activation in two patients with apoC-II deficiency syndrome and by HL activation in two patients with LPL deficiency. LPL activation was achieved by the infusion of normal plasma containing apoC-II and HL was released by the injection of heparin. Lipoproteins were analyzed by ultracentrifugation in a zonal rotor under rate flotation conditions before and after lipase activation. The LPL activation resulted in: a reduction of plasma triglycerides; a reduction of fast-floating very low density lipoprotein (VLDL) concentration; an increase of intermediate density lipoprotein (IDL), which maintained unaltered flotation properties; an increase of low density lipoproteins (LDL) accompanied by modifications of their flotation rates and composition; no significant variations of high density lipoprotein (HDL) levels; and an increase of the HDL flotation rate. The HL activation resulted in: a slight reduction of plasma triglycerides; a reduction of the relative triglyceride content of slow-floating VLDL, IDL, LDL2, and HDL3 accompanied by an increase of phospholipid in VLDL and by an increase of cholesteryl ester in IDL; and a reduction of the HDL flotation rate. These experiments in chylomicronemic patients provide in vivo evidence that LPL and HL are responsible for plasma triglyceride hydrolysis of different lipoproteins, and that LPL is particularly involved in determining the levels and physicochemical properties of LDL. Moreover, in these patients, the LPL activation does not directly change the HDL levels, and LPL or HL does not produce a step-wise conversion of HDL3 to HDL2 (or vice versa) but rather modifies the flotation rates of all the HDL molecules present in plasma.     

脂蛋白脂肪酶在巨噬细胞代谢正常极低密度脂蛋白中的作用

家兔正常极低密度脂蛋白(N—VLDL)可使体外培养的小鼠腹腔巨噬细胞内甘油三酯(TG)增加6～16倍。加入脂蛋白脂肪酶(LPL)抑制剂苯硼酸(BBA)可使培养液内游离脂酸(FFA)水平降低,N—VLDL—TG含量升高、细胞内TG堆积被明显甚至完全抑制。证明了LPL在巨噬细胞摄取N—VLDL—TG中起主要作用。     

Lipoprotein lipase-mediated lipolysis of very low density lipoproteins increases monocyte adhesion to aortic endothelial cells.

Lipoprotein lipase (LPL) bound to vascular endothelial cells hydrolyses triglycerides in plasma lipoproteins. To explore the role of LPL in atherogenesis, the effect of LPL-mediated lipolysis of very low density lipoproteins (VLDL) on monocyte adhesion to endothelial cells was examined. Adhesion of U937 monocytes to porcine aortic endothelial cells that were incubated with VLDL and purified bovine milk LPL was markedly higher than endothelial cells that were incubated with VLDL alone. The increase in monocyte adhesion obtained with VLDL was dependent on the concentration of the lipoprotein, monocyte dose and time of incubation. The increase in adhesion correlated with generation of free fatty acids from the hydrolysis of triglycerides in VLDL by LPL. Furthermore, direct addition of oleic acid to endothelial cells also increased adhesion of monocytes. We postulate that LPL-derived lipolytic products increase monocyte adhesion to vascular endothelium and thereby promote atherogenesis.     

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.     

Fatty acids of Treponema pallidum and Borrelia burgdorferi lipoproteins.

A fundamental ultrastructural feature shared by the spirochetal pathogens Treponema pallidum subsp. pallidum (T. pallidum) and Borrelia burgdorferi, the etiological agents of venereal syphilis and Lyme disease, respectively, is that their most abundant membrane proteins contain covalently attached fatty acids. In this study, we identified the fatty acids covalently bound to lipoproteins of B. burgdorferi and T. pallidum and examined potential acyl donors to these molecules. Palmitate was the predominant fatty acid of both B. burgdorferi and T. pallidum lipoproteins. T. pallidum lipoproteins also contained substantial amounts of stearate, a fatty acid not typically prevalent in prokaryotic lipoproteins. In both spirochetes, the fatty acids of cellular lipids differed from those of their respective lipoproteins. To characterize phospholipids in these organisms, spirochetes were metabolically labeled with [3H]palmitate or [3H]oleate; B. burgdorferi contained only phosphatidylglycerol and phosphatidylcholine, while T. pallidum contained phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and cardiolipin. Although palmitate predominated in the lipoproteins, there were no apparent differences in the incorporation of these two fatty acids into phospholipids (putative acyl donors). Phospholipase A1 and A2 digestion of phosphatidylcholine from B. burgdorferi and T. pallidum labeled with either [3H]palmitate or [3H]oleate also revealed that neither fatty acid was incorporated preferentially into the 1 and 2 positions (potential acyl donor sites) of the glycerol backbone. The combined findings suggest that fatty acid utilization during lipoprotein synthesis is determined largely by the fatty acid specificities of the lipoprotein acyl transferases. These findings also provide the basis for ongoing efforts to elucidate the relationship between lipoprotein acylation and the physiological functions and inflammatory activities of these molecules.