野生型人LPL及联合突变体原核表达载体构建及抗体制备

目的构建野生型人脂蛋白脂酶(LPL)和联合突变体的pET28a( )原核表达载体,蛋白纯化后制备突变型多克隆抗体。方法采用RT-PCR技术从人肾周脂肪组织中获得野生型LPLcDNA,酶切和测序鉴定后插入原核表达载体pET28a( ),通过两次定点诱变得到pET28a( )-Asn291Ser和Lys312inC联合突变LPL,酶切和测序鉴定后分别转染大肠杆菌BL21。异丙基-β-D-硫代半乳糖苷(IPTG)诱导,SDS-PAGE电泳分析;镍柱亲和层析纯化,Western blotting鉴定;将纯化后的突变型LPL蛋白免疫新西兰大白兔,Western blotting检测血清突变型多克隆抗体滴度。结果经酶切和测序鉴定,所构建的野生型LPL及联合突变LPL的基因片段正确;两种重组质粒转染大肠杆菌BL21后,均呈高效表达;SDS-PAGE电泳分析及Western blotting鉴定表明,诱导纯化后的蛋白为目的蛋白;兔抗突变型LPL的多克隆抗体滴度达1∶100000。结论成功构建pET28a( )-野生型LPL及pET28a( )-Asn291Ser和Lys312inC联合突变LPL,同时利用获得的高纯度蛋白制备了高效价突变型LPL多克隆抗体,为进一步进行野生型LPL和突变型LPL蛋白的体内试验奠定了基础。     

兔实验性动脉粥样硬化血浆脂蛋白脂酶和肝脂酶活性的变化

初步建立了兔肝素化血浆脂蛋白脂酶(LPL)和肝脂酶(HL)活性的测定方法,两种脂酶活性以水解三油酸甘油酯(TG)生成游离脂肪酸(FFA)的含量表示。测得雄性日本大耳白兔肝素化后15min血浆LPL、HL活性最大,批内差异分别为4.96%、3.26%;正常兔LPL、HL分别为12.15±4.07μmol·L~(-1)FFA/h·ml、17.11±6.80μmol·L~(-1)FFA/h·ml;实验兔喂饲胆固醇12wk形成动脉粥样硬化(AS)时LPL、HL活性分别为7.94±3.36μmol·L~(-1)FFA/h·ml、4.54±1.52μmol·L~(-1)FFA/h·ml,两者均较正常兔明显降低(P<0.005)。提示:在高胆固醇负荷时,LPL、NL活性降低可能促进了AS的形成。     

Role of the liver in the degradation of very low density lipoproteins: a study of lipolysis by heparin releasable liver lipase and uptake during isolated rat liver perfusion

Abstract . The role of the liver and of a heparin-releas-able liver lipase in the metabolism of very low density lipoprotein (VLDL) was investigated in vitro and during recycling rat liver perfusion. Rat plasma VLDL and nascent hepatic VLDL were labelled biosyntheti-cally in their lipid moieties. Incubation in vitro of VLDL with the lipase caused hydrolysis of VLDL-tri-glycerides (>80%) and VLDL-phosphatidylcholine (> 30%). Nascent VLDL was a better substrate for the enzyme. The hydrolytic activities were inhibited by 70–90% when rat plasma (10–30 vol%) was added to the incubation mixture.
VLDL-triglycerides and cholesterol esters were taken up by the liver during 180 min recycling perfusion. The rate of disappearance of nascent VLDL was faster than that of plasma VLDL (half-life times of 56.2 ±13.9 and 125.0±24.8 min respectively). Injection of heparin into the perfusion medium caused accelerated uptake of the hydrolysed VLDL-triglycer-ide by the liver. Addition of plasma ( d > 1.006 g/ml) to the perfusion at a concentration of 10 vol% delayed the rate of disappearance of VLDL from the perfusate by about 50–75%.
These studies have established the capacity of the hepatic lipase to hydrolyse VLDL-lipids and the ability of the liver to degrade nascent and plasma VLDL particles. These two activities, however, are depressed by plasma and therefore previous studies of VLDL metabolism may have to be re-examined when based on incubations or perfusions in the absence of plasma.     

LIPASES AND LIPID ABSORPTION IN THE NEWBORN

Intragastric lipolysis resulting from the action of lingual lipase occurs in preterm infants and may be important quantitatively in the digestion of fat.     

Studies of lipoprotein metabolism in a patient with fish-eye disease

In the rare familial disorder fish-eye disease, hypertriglyceridaemia is associated with elevated levels of very-low-density lipoprotein (VLDL) and enrichment of low-density lipoprotein (LDL) with triglyceride. The kinetic basis of the dyslipoproteinaemia was investigated by studying the metabolism of the apolipoprotein-B moeity of VLDL, intermediate-density lipoprotein (IDL) and LDL in a 68-year-old woman with this condition. The major kinetic abnormality was a pronounced reduction in the rate of fractional conversion of VLDL-B to IDL-B and of IDL-B to LDL-B, suggesting that the dyslipoproteinaemia represents accumulation in plasma of partly degraded products of VLDL metabolism. This kinetic disorder has features in common with type-III hyperlipoproteinaemia. In studies in vitro no defect in the enzyme, activator or substrate components of the lipoprotein lipase or hepatic lipase systems was observed.     

Effect of Experimental Hypertriglyceridaemia on Tissue and Serum Lipoprotein Lipase Activity

Abstract Hypertriglyceridaemia was produced in rats by the intravenous infusion of Intralipid emulsion or of very low density (d < 1.006) rabbit or human lipoproteins (VLDL). Lipoprotein lipase activity was assayed, in tissues removed at the end of infusion, on serum-activated mono- and triolein emulsions at pH 8.6. Hypertriglyceridaemia resulted in a marked decrease in epididymal adipose tissue lipoprotein lipase activity and in an increase in heart enzyme activity. These changes were evident with both mono- and triolein substrates. The effects on adipose tissue enzyme activity seemed roughly dependent on the triglyceride (TG) level and, relative to TG elevation, were most pronounced in the case of VLDL infusion. Serum lipoprotein lipase activity, measured in the absence of heparin, was considerably increased suggesting that the TG-rich material "leached" the adipose tissue enzyme into the circulation. Leaching of lipoprotein lipase from adipose tissue by Intralipid emulsion or VLDL was also demonstrated in an in vitro system devoid of heparin. Contact with the TG-poor, 1.006 < d < 1.063, lipoprotein induced only a small loss in adipose tissue lipoprotein lipase activity, either in vitro or in vivo.
Intracellular lipolytic activity toward mono- and triolein, measured in adipose tissue and heart homogenates at pH 7.2 in the absence of serum, was not significantly affected by TG elevation. Thus, the observed changes in lipoprotein lipase activity seem unrelated to the intracellular lipolytic activity.
It is suggested that the low adipose tissue lipoprotein lipase activity and the retarded TG removal observed in certain hypertriglyceridaemic conditions may be secondary to the increased supply of TG-rich lipoproteins.     

Anti-diabetic action of Punica granatum flower extract: activation of PPAR-gamma and identification of an active component

Peroxisome proliferator-activated receptor (PPAR)-gamma activators are widely used in the treatment of type 2 diabetes because they improve the sensitivity of insulin receptors. Punica granatum flower (PGF) has been used as an anti-diabetic medicine in Unani medicinal literature. The mechanism of actions is, however, unknown. In the current study, we demonstrated that 6-week oral administration of methanol extract from PGF (500 mg/kg, daily) inhibited glucose loading-induced increase of plasma glucose levels in Zucker diabetic fatty rats (ZDF), a genetic animal model for type 2 diabetes, whereas it did not inhibit the increase in Zucker lean rats (ZL). The treatment did not lower the plasma glucose levels in fasted ZDF and ZL rats. Furthermore, RT-PCR results demonstrated that the PGF extract treatment in ZDF rats enhanced cardiac PPAR-gamma mRNA expression and restored the down-regulated cardiac glucose transporter (GLUT)-4 (the insulin-dependent isoform of GLUTs) mRNA. These results suggest that the anti-diabetic activity of PGF extract may result from improved sensitivity of the insulin receptor. From the in vitro studies, we demonstrated that the PGF extract enhanced PPAR-gamma mRNA and protein expression and increased PPAR-gamma-dependent mRNA expression and activity of lipoprotein lipase in human THP-1-differentiated macrophage cells. Phytochemical investigation demonstrated that gallic acid in PGF extract is mostly responsible for this activity. Thus, our findings indicate that PPAR-gamma is a molecular target for PGF extract and its prominent component gallic acid, and provide a better understanding of the potential mechanism of the anti-diabetic action of PGF.     

Lack of Effect of Exogenous or Endogenous Gastric Inhibitory Polypeptide on the Elimination Rate of Intralipid® in Man

Abstract Six healthy subjects were studied by means of an intravenous fat tolerance test on three occasions, in the fasting state, in the postprandial state, and during an intravenous infusion of gastric inhibitory polypeptide (GIP). No effects on the elimination rate of Intralipid® were seen either by endogenous or exogenous GIP.     

Effects of heparin and a semi-synthetic heparin analogue on platelet aggregation, lipoprotein lipase and other laboratory tests in surgical patients

Platelet aggregation, lipoprotein lipase activity, coagulation parameters and routine blood chemistry were measured in a randomised study of 21 surgical patients before, immediately after and 3 months after operation. Sodium heparin 5000 IU was given subcutaneously to 11 patients every 12 hours for 7 days, the first injection 2 hours preoperatively; 10 patients received a semi-synthetic heparin analogue (SSHA 75 mg) in the same manner. The groups were sex and age matched. No conclusive changes were found in platelet aggregation. The increase in lipoprotein lipase activity in SSHA patients 2 hours after injection was significantly greater than in heparin patients. Neither of the two drugs induced significant changes in coagulation parameters or routine blood chemistry. The results indicate a difference in the effect on lipoprotein lipase release between heparin and SSHA at the used dosage schedules.