Expression of lipoprotein lipase in different human subcutaneous adipose tissue regions

Steady state expression of lipoprotein lipase was compared in abdominal and gluteal subcutaneous adipose tissue of nonobese men and women. In both regions enzyme activity and lipoprotein lipase mRNA levels were significantly higher in women than in men. In men the enzyme activity was higher in abdominal than in gluteal adipose tissue (P less than 0.01) whereas the opposite was observed in women (P less than 0.05). In both sexes, however, lipoprotein lipase mRNA levels were threefold higher in the abdominal as compared to the gluteal site, whether they were determined in isolated fat cells or in fat segments (P less than 0.01). This regional difference persisted when the mRNA values were expressed as a function of the mRNA concentration for beta-actin. There was a correlation between the two adipose tissue regions as regards the values for enzyme activity and mRNA level (r = 0.6-0.8). Northern blot analysis revealed two mRNA species of 3.5 and 3.7 kilobases, respectively. It is concluded that there are regional variations in the steady state expression of lipoprotein lipase in human subcutaneous adipose tissue. This involves site variations in gene expression as well as posttranslational modification of lipoprotein lipase enzyme activity and may contribute to the characteristic variations in adipose tissue mass and distribution between men and women.     

Hormone-sensitive lipase from bovine adipose tissue

Hormone-sensitive lipase has been purified to near homogeneity from bovine perirenal adipose tissue. The purification method involves isoelectric precipitation at pH 5.0, followed by partial solubilisation in Triton N-101 and ion-exchange chromatography on DE-52. After additional solubilisation, the enzyme is further purified by chromatography on phenyl-Sepharose and heparin-Sepharose. This procedure can be completed within three working days and yields approx. 30 units of enzyme with a specific activity of 30 U/mg. The enzyme has been identified as a polypeptide of Mr 84 000 by affinity labelling with [3H]diisopropyl fluorophosphate. This polypeptide comprises approx. 60-80% of the protein in the final preparation, as judged by scanning densitometry of SDS-polyacrylamide gels stained with silver or with Coomassie blue R. The polypeptide of Mr 84 000 serves as a substrate for cyclic AMP-dependent protein kinase, phosphorylation correlating with activation of the lipase. Polyclonal antibody to the lipase has been raised in a rabbit and shown to specifically cross-react with the Mr 84 000 subunit.     

p-nitrophenyl butyrate hydrolyzing activity of hormone-sensitive lipase from bovine adipose tissue

The "esterase" activity of hormone-sensitive lipase (HSL) was studied using water-soluble p-nitrophenyl butyrate (PNPB) as a substrate. Bovine adipose tissue HSL was purified to near homogeneity by precipitation at pH 5.0, followed by chromatography on DEAE-cellulose, phenyl-Sepharose, and high performance ion-exchange columns on Mono Q and Mono S. The purified preparation hydrolyzed emulsified triolein and cholesteryl oleate (CO), and water-soluble PNPB. In the two last steps of purification, the elution profile of the CO-hydrolyzing activity coincided with that of PNPB-hydrolyzing activity. The HSL was adsorbed to heparin-Sepharose and the CO- and PNPB-hydrolyzing activities were eluted together in the same peak. Diisopropylfluorophosphate (DFP) strongly inhibited the HSL activities and the inhibition profiles of the triolein-; CO-, and PNPB-hydrolyzing activities were essentially identical. Only one polypeptide of Mr 84,000 in partial purified HSL fraction was labeled by affinity labeling with [3H]DFP. On digestion of the enzyme with trypsin, the triolein- and CO-hydrolyzing activities were lost more rapidly than the PNPB-hydrolyzing activity. Phosphorylation increased the triolein-hydrolyzing activity to 40% more than that of the control, but did not affect the CO- and PNPB-hydrolyzing activities.     

Sites of lipoprotein lipase activity in adipose tissue perfused with chylomicrons. Electron microscope cytochemical study

Lipoprotein lipase activity was studied in rat parametrial adipose tissue perfused with chylomicrons and in gelatin blocks containing postheparin plasma and chylomicrons. The tissues and blocks were fixed in glutaraldehyde and incubated in 0.035 M CaCl₂-0.1 M Tris medium (pH 8.3) at 38°C. The doubly labeled chylomicron triglycerides (glycerol-³H and palmitate-¹⁴C) in the tissues and blocks were hydrolyzed during incubation to free fatty acids (FFA) and the FFA remained in the specimens; hydrolysis was inhibited by 0.004 M diethyl paranitrophenyl phosphate (E-600). Incubated blocks and tissue were treated with 0.05 M Pb(NO₃)₂, postfixed in OsO₄, dehydrated with acetone, embedded in Epon, and examined by electron microscopy. The incubated blocks contained electronlucent areas and granular and laminar precipitates at sites of hydrolysis. Similar precipitates were found in incubated tissue, within vacuoles and microvesicles of capillary endothelium, and in the subendothelial space (between the endothelium and pericytes), but not in the capillary lumen or in or near fat cells. The cytochemical reaction was greatly reduced, in blocks and tissues incubated with E-600. It is concluded that plasma glycerides are hydrolyzed by lipoprotein lipase in capillary endothelial cells and in the subendothelial space of adipose tissue and that glycerides across the endothelial cells within a membrane-bounded system.     

Rapid assay for hormone-sensitive lipase activity of adipose tissue

A highly specific and rapid assay for hormone-sensitive lipase activity of rat adipose tissue is described. The method employs emulsified 2,3-di-O-oleyl-[9,10-(3)H(2)]oleoyl glycerol as a substrate; it is very sensitive and is suitable for serial sampling.     

Partial purification of monoglyceride lipase from adipose tissue

A monoglyceride lipase was partly purified from extracts of rat adipose tissue by ammonium sulfate fractionation, alcohol precipitation, and lyophilization, or by ammonium sulfate fractionation, sodium deoxycholate treatment, and a second ammonium sulfate fractionation. Partial purification and heat denaturation showed the lipase to be different from tributyrinase and from an enzyme(s) which hydrolyzes diglycerides and triglycerides. Although the best preparations hydrolyzed monobutyrin this activity decreased with purification, indicating that the enzyme acts on insoluble substrates and is therefore a lipase and not an esterase. Further-more, classification of the enzyme as a lipase is consistent also with its behavior with inhibitors, since low concentrations of esterase inhibitors, e.g., fluoride, sodium deoxycholate, and physostigmine did not inhibit lipolytic activity. Inhibition studies with EDTA, sodium pyrophosphate, protamine, and fluoride showed that the enzyme differs from clearing factor lipase. The enzyme catalyzed hydrolysis of monostearin in the pH range 6.3-9.0, with a maximum at 7.4-7.6.     

Post-translational regulation of lipoprotein lipase activity in adipose tissue.

Changes in adipose-tissue lipoprotein lipase activity that are independent of protein synthesis were investigated in an incubation system in vitro. Under appropriate conditions at 25 degrees C a progressive increase in the enzyme activity occurs that is energy-dependent. Part of the enzyme is rapidly inactivated when the tissue is incubated with adrenaline or adrenaline plus theophylline. The mechanism of this inactivation appears to be distinct from, and to follow, the activation of the enzyme. A hypothesis is presented to account for the results in terms of an activation of the enzyme during obligatory post-translational processing and a catecholamine-regulated inactivation of the enzyme as an alternative to secretion from the adipocyte.     

Effects of glycerol on human adipose tissue triglyceride lipase activity

Glycerol fully protects the human adipose tissue triglyceride lipase against the denaturing effects of high and low temperatures. Under such protection, storage of crude preparations at -10 degrees C or incubation at 50 degrees C resulted in a 1.5-3-fold increase of the measured lipase activity. This increase was shown to be related to enzyme newly released from tissular microparticles present in the samples. Advantage may be taken of these observations to improve greatly the conditions of extraction and storage of this lipase activity.     

Relationship of lipoprotein lipase activity to triglyceride uptake in adipose tissue

Fasted rats injected with actinomycin or fed glucose show increased lipoprotein lipase activity of epididymal adipose tissue. Data from the actinomycin-treated animals showed a direct correlation between the lipoprotein lipase activity and the uptake of lipoprotein triglyceride by the epididymal fat pad in vitro and in vivo. Data from the animals fed glucose confirmed these findings in vitro. These data strongly suggest that lipoprotein lipase plays a major role in triglyceride deposition in adipose tissue.     

Hormone-sensitive lipase of adipose tissue.

Some physiologic aspects of the mobilization and fate of free fatty acids are reviewed. The molecular mechanism of the activation of hormone-sensitive lipase in adipose tissue is then discussed. Recent evidence established that hormone-sensitive lipase, concerned with fat mobilization, is both functionally and immunochemically distinct from lipoprotein lipase, concerned with uptake of plasma triglycerides. Lipoprotein lipase activity is not altered by cyclic AMP-dependent protein kinase. The latter enzyme enhances not only triglyceride hydrolase but also monoglyceride, diglyceride and cholesterol ester hydrolase activities in chicken adipose tissue. Finally, it is shown that the activation of all four acyl hydrolases is reversible, the deactivation being magnesium-dependent. Protein phosphatase fractions from heart and liver active against phosphorylase a can reversibly deactivate adipose tissue hormone-sensitive lipase, implying a low degree of substrate specificity for lipase phosphatase.