Purification and characterization of hepatic triacylglycerol lipase isolated from rainbow trout,Oncorhynchus mykiss

Trialcylglycerol (TG) lipase was isolated and partially purified from rainbow trout liver. Triacylglycerol lipase activity was assayed by measuring¹⁴C-oleic acid release from¹⁴C-triolein.¹⁴C-oleic acid release was linear for up to two hours. Optimal activity occurred at pH 7.0 and 15°C. Most of the lipase activity was recovered in the cytosolic fraction. A 27,000-fold purification was achieved after Sepharose (Bio-gel A 0.5 M, 200–400 mesh) chromatography of a resuspended 20% ammonium sulfate fraction. The molecular weight of the trout hepatic lipase as determined by size-exclusion chromatography and by SDS-polyacrylamide gel electrophoresis was 40–43 kD. Lipase-mediated hydrolysis of TG resulted in the production of diacylglycerols, monoacylglycerols, and fatty acids. Kinetic analysis indicated that V_max=0.016 nmol/h/mg protein and that K_m=0.28 mM triolein. Lipolytic activity was enhanced in the presence of cAMP/ATP-Mg²⁺. These results suggest that the liver of trout possesses a neutral TG lipase that is responsible for mobilizing stored TG and is catalytically activated by phosphorylation.A part of this work was presented at the Annual Meeting of the American Society of Zoologists, December 26–30, 1990, San Antonio, TX.     

siRNA干扰ATGL基因表达对猪脂肪细胞脂肪分解的抑制作用

【目的】探讨猪脂肪甘油三酯水解酶(adipose triglyceride lipase,ATGL)基因在脂肪细胞脂解中的作用。【方法】构建针对猪ATGL基因CDS区113和1 358位点的慢病毒干扰载体,包装后用其感染猪成熟脂肪细胞,检测ATGL和激素敏感脂酶(hormone-sensitive lipase,HSL)基因mRNA及其蛋白的表达情况;用甘油试剂盒检测细胞甘油释放量。【结果】成功构建了ATGL基因的慢病毒干扰载体,用其感染猪成熟脂肪细胞后,RT-PCR分析和Western印迹检测结果表明,试验组脂肪细胞ATGL的表达显著下调,HSL的表达没有明显变化;甘油释放量显著降低,其中针对ATGL基因CDS区113位点的siRNA1对ATGL mRNA的干扰效率达55%,甘油释放量降低了49%。【结论】成功构建了猪ATGL基因慢病毒干扰载体,其能显著降低ATGL基因mRNA及其蛋白在猪成熟脂肪细胞中的表达,降低脂肪细胞的甘油释放量,表明ATGL在猪脂肪细胞脂解中有着重要作用,且113位点是ATGL基因CDS区的最佳干扰靶位点。     

Adipose tissue gene expression in obese dogs after weight loss

Body weight (BW) mainly depends on a balance between fat storage (lipogenesis) and fat mobilization (lipolysis) in adipocytes. BW changes play a role in insulin resistance (IR), the inability of insulin target tissue to respond to physiological levels of insulin. This results in inhibition of lipogenesis and stimulation of lipolysis. Weight gain leads to IR whereas, weight loss improves insulin sensitivity (IS). The aim of this study was to evaluate the effect of weight loss and recovery of IS on the expression of genes involved in lipogenesis and lipolysis in weight losing dogs. Gene expression was studied in both subcutaneous and visceral adipose tissue. Obese dogs received a hypoenergetic low fat high protein diet (0.6 x NRC recommendation). Before and after weight loss, IS was assessed using the euglycaemic hyperinsulinaemic clamp. Gene expression of IRS-2, SREBP, intracellular insulin effectors, ACC, FAS, FABP, ADRP, PEPCK, lipogenesis key proteins, perilipin and HSL, lipolysis key proteins were quantified using real-time RT-PCR in subcutaneous and visceral fat. BW decreased from 15.2 +/- 0.5 to 11.4 +/- 0.4 kg (p < 0.05) over 78 +/- 8 days. When obese, dogs were insulin resistant. After weight loss, IS was improved. In the subcutaneous adipose tissue, the expression of only the IRS-2 was increased. In the visceral adipose tissue, the expression of the genes involved in the lipogenesis was decreased whereas one of the genes implied in the lipolysis did not change. The expression profile of genes involved in lipid metabolism, as measured after weight loss, is indicative for a lower lipogenesis after weight loss than in obese dogs. Our results also confirm dramatic differences in the lipid metabolism of visceral and subcutaneous fat. They should be completed by comparing gene expression during weight losing and normal weight steady state.     

两种日粮对乌金猪脂肪组织脂类分解代谢相关基因表达的影响

为探讨不同日粮对乌金猪不同生长阶段脂类分解代谢的差异。选用乌金猪为研究对象,以本实验室前期研究确定的乌金猪适宜能量与蛋白水平的日粮和美国NRC(1998)推荐的猪营养需要量为依据,设计和配制乌金猪WJ和NRC日粮。选取体重约23kg的乌金猪36头,公母各半,随机分为2个处理组,每个处理下设3个重复,每个重复6头。分别于30kg、60kg和100kg体重时屠宰,通过real-time PCR比较了4个脂肪分解代谢相关基因在脂肪组织中表达的差异。结果显示:在各个屠宰体重,WJ日粮组HSL基因相对表达量均高于NRC日粮组(P>0.05);在各个屠宰体重,WJ日粮组LPL基因相对表达量均显著高于NRC日粮组(P<0.05);在30kg和60kg屠宰体重,WJ日粮组CPT-1基因的相对表达量显著高于NRC日粮组(P<0.05),但在100kg屠宰体重水平时,WJ日粮组CPT-1基因的相对表达量低于NRC日粮组(P>0.05);在各个屠宰体重,WJ日粮组PPARγ基因的相对表达量均高于NRC日粮组,在30kg屠宰体重达到显著水平(P<0.05)。结果提示:WJ日粮通过上调脂肪分解代谢相关基因的表达促进了脂肪组织脂肪的分解,减少了脂肪沉积。     

原料乳中耐冷菌群及其产酶特点

耐冷菌及其分泌的耐热酶是原料乳中常见污染物,会导致乳制品腐败。通过16S rRNA基因测序分析了分离自上海地区原料乳的耐冷菌群,以及通过平板法研究不同耐冷菌的蛋白酶和脂肪酶活力。结果表明:所有的分离株属于9个菌纲,含33个菌属,其中数量占主要的菌属是假单胞菌(Pseudomonas)(55％)、不动杆菌(Acinetobacter)(10％)和黄杆菌(Flavobacterium)(5％),其余30个菌属占所有菌株数的30％。耐冷菌产酶能力平板法实验显示,大部分的耐冷菌都有产蛋白酶或脂肪酶的能力,不同菌属之间产酶特点存在差异。假单胞菌和黄杆菌主要表现为产蛋白酶活性,克雷伯氏菌(Klebsiella)主要表现为产脂肪酶活性,而大多数的不动杆菌没有产蛋白酶或脂肪酶活性。     

Fucoidan from marine brown algae inhibits lipid accumulation

In this study, we elucidated the inhibitory effect of fucoidan from marine brown algae on the lipid accumulation in differentiated 3T3-L1 adipocytes and its mechanism. The treatment of fucoidan in a dose-dependent manner was examined on lipid inhibition in 3T3-L1 cells by using Oil Red O staining. Fucoidan showed high lipid inhibition activity at 200 μg/mL concentration (P < 0.001). Lipolytic activity in adipocytes is highly dependent on hormone sensitive lipase (HSL), which is one of the most important targets of lipolytic regulation. Here, we examined the biological response of fucoidan on the protein level of lipolysis pathway. The expressed protein levels of total hormone sensitive lipase (HSL) and its activated form, phosphorylated-HSL were significantly increased at concentration of 200 μg/mL fucoidan. Furthermore, insulin-induced 2-deoxy-D-[3H] glucose uptake was decreased up to 51% in fucoidan-treated cells as compared to control. Since increase of HSL and p-HSL expression and decrease of glucose uptake into adipocytes are known to lead to stimulation of lipolysis, our results suggest that fucoidan reduces lipid accumulation by stimulating lipolysis. Therefore, these results suggest that fucoidan can be useful for the prevention or treatment of obesity due to its stimulatory lipolysis.     

切达干酪成熟期间脂肪分解和氧化的研究

主要研究了切达干酪（Cheddar cheese）成熟过程中脂肪的分解和氧化。结果表明：切达干酪在成熟过程中酸度值（ADV）、羰基价（CBV）和硫代巴比妥酸值（TBA）随着时间的延长逐渐增大,过氧化值（POV）呈先增大后减小的趋势。     

Adenosine deaminase and porcine meat quality. I. Effect of dipyridamole on plasma free fatty acids, glucose, lactate and c-AMP in pigs representing high and low red cell adenosine deaminase activity

The effect of dipyridamole--an adenosine uptake inhibitor--on the plasma concentration of free fatty acids (FFA), glucose, lactate and cyclic adenosine monophosphate (cAMP) has been examined in 2 groups of Landrace pigs representing low (Ada 0) and high (Ada A) red cell adenosine deaminase (Ada) activity. Pigs fitted with a jugular vein catheter were given dipyridamole (0.16 mg/kg/min) over a period of 30 min. The infusions were performed 22 h after the last meal at a time where pigs were found to show steady increase and decline in rates of lipolysis and glycogenolysis, respectively. The results showed that lipid mobilization as identified by the plasma FFA concentration was markedly depressed. During the infusion of dipyridamole similar degree of inhibition was seen in Ada 0 and Ada A pigs, however, in the period following the infusion, a significantly stronger suppression persisted in the Ada 0 pigs. Both the blood glucose and lactate level rose distinctly as a result of the dipyridamole treatment. This stimulation of the glycolysis rate was significantly more expressed in Ada 0 pigs compared to that of the Ada A pigs. When theophylline, an antagonist of adenosine, was given together with dipyridamole, the rise in the lactate level was considerably diminished. Dipyridamole also produced a distinct rise in the plasma cAMP levels.     

灭菌猪肉浆中发酵菌株脂质水解和氧化能力分析

【目的】筛选具有较强脂质水解能力和抗氧化能力的发酵菌株,为研发新型发酵剂提供理论基础。【方法】在无菌猪肉浆体系中分别接种木糖葡萄球菌（Staphylococcus xylosus）YSZ11、YCC3,腐生葡萄球菌（Staphylococcus saprophyticus）YCC2,巨球菌（Macrococcus caseolyticus）YZC2、YZC3,并设立不接种发酵菌株为对照组（CK）,测定发酵4 d内猪肉浆pH、过氧化值（peroxide value,POV）、硫代巴比妥酸（thiobarbituric acid reactive substances,TBARS）值、脂肪酶活力、脂质组成和游离脂肪酸含量的变化。【结果】5株发酵菌株均可以降低猪肉浆体系的pH,POV和TBARS值分别为2.51—2.96 mmol·kg^-1和0.21—0.24 mg/100 g,显著低于CK组（P<0.05）。在5组接种发酵菌株的猪肉浆中检测到了中性脂肪酶、酸性脂肪酶和磷脂酶3种酶活力,且后两者活性较高。发酵4 d后,接种发酵菌株组的磷脂含量显著降低（P<0.05）,游离脂肪酸含量增加了21.1%—73.7%,其中饱和脂肪酸总量显著降低,不饱和脂肪酸含量显著增加,尤其是棕榈油酸（C_16:1）、油酸（C_18:1）和亚油酸（C_18:2）含量较高,并检测到亚麻酸（C_18:3）。【结论】本试验中的5株菌株均可抑制脂质的氧化,同时可以分泌脂肪酶促进脂质的水解,增加游离脂肪酸特别是不饱和脂肪酸的含量。其中,腐生葡萄球菌YCC2和木糖葡萄球菌YCC3脂质水解和抗氧化能力较好,对改善发酵肉制品的品质具有更明显的促进作用。     

Bacterial Contribution to Salted Anchovy (Engraulis anchoita Hubbs & Marinni, 1935) Ripening Process

The bacterial populations of the salted anchovy ripening process and its potential role on the development of the typical sensory characteristics of this product were studied. Salted anchovy samples were taken during the ripening process and submitted to microbiological analyses in order to monitor the evolution of bacterial groups. According to the results obtained, the ripening process was dominated by moderate halophilic bacteria. Moreover, many of the isolated strains showed proteolytic, lipolytic, and trimethylamine oxide (TMAO) reductase activities. These activities contribute to the development of the typical flavor of this product and to the increase of total volatile bases observed during ripening.