Abstract
Hepatic triglyceride lipase (H-TGL) is a key lipolytic enzyme in the metabolism of human plasma high density lipoproteins. The enzyme is bound to glycosaminoglycans on endothelial cells in the liver and is immediately released into the circulation after heparin administration. In addition to releasing H-TGL, heparin-like glycosaminoglycans have also been shown to suppress hepatocyte proliferation and to alter tissue-specific gene expression. In the present study, the effects of heparin exposure on the secretion of H-TGL were examined in a human parenchymal hepatoma (HepG2) cell line. The addition of heparin to serum-supplemented medium induced the secretion of H-TGL in a time- and concentration-dependent manner. At 5.4 micrograms/ml heparin, H-TGL levels, as determined by triacyglycerol hydrolase activity, increased 7-fold after a 44-h incubation. Heparin exposure decreased intracellular H-TGL activity from 21.3 to 4.8 nmol of oleic acids released/h/10(8) cells and increased enzyme activity in the medium from 16.2 to 165.3 nmol of oleic acids released/h/10(8) cells. The heparin-induced secretion of H-TGL was associated with increased levels of H-TGL-specific mRNA. The addition of actinomycin D or cycloheximide reversed the heparin-induced increase in H-TGL activity and mRNA. Heparin treatment did not increase the level of actin mRNA suggesting that elevated H-TGL mRNA is due to enhanced tissue-specific expression of H-TGL. Expression of apolipoprotein E, another protein involved in lipoprotein metabolism, also showed induced levels of mRNA by heparin but to a lesser extent than that for H-TGL. We conclude that heparin stimulates the de novo synthesis of H-TGL in liver parenchymal cells in vivo by influencing both transcriptional and post-transcriptional events.
Highlights
Mediately released into the circulation after heparin In the presentstudy, we have investigated the effect of administration
A central problem in eukaryotic biologyinvolves under- pretreated with heparin that assists in the process of hepatic-triglyceride lipase (H-TGL)
Standing the mechanisms by which genes are regulated in a induction upon the second heparin exposure
Summary
Cells were plated and cultured in Falcon gesting that elevateHd-TGL mRNA is due to enhanced T150 flasks withEagle’s minimal essential medium (MEM, GIBCO). Determination of H-TGL Actiuity-To quantitate H-TGL activity, the enzymewas firstisolated by chromatography of conditioned medium or cell lysate on heparin-Sepharose. Heparin Inductionof Hepatic Lipase at 500 X g for 5 min, the medium or lysate discarded, and the pellet wasresuspended in 2 ml of 10 mM potassium phosphate, 150 mM NaCI, pH 7.4 (phosphate-buffered saline). Cells were seeded in Falcon T150 culture flasks and allowed t o attachovernightinMEMcontaining 10% fetal bovine serumas described under "Experimental Procedures." Cells were refed every 24 h by replacing the medium and supplementing it with 1p5g/ml of heparin. At48h after cells were initially exposed to heparin and medium from each flask (from the final 24-h conditioned medium) was harvested andassaved for H-TGL activitv in tridicate. X lo cpm of cDNA/ml of prehybridizationsolution.Blots were washed 2 X 30 min in X SSPE, 0.2% sodium dodecyl sulfate and 10
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