Synthesis and Secretion of Hepatic Lipase by Rat Hepatocytes
Hepatic lipase (HL) is a glycoprotein postulated to play a central role in HDL metabolism, converting HDL2 to HDL3 by hydrolyzing lipoprotein phospholipids. In order to study factors regulating HL levels, we characterized an isolated hepatocyte preparation in which synthesis and secretion of the enzyme could be studied using metabolic labeling. Furthermore, the ability of total RNA extracted from liver to direct the synthesis of immunoisolatable HL in a cell-free in vitro translation (IVT) system was determined. Hepatocytes were isolated by collagenase perfusion of rat livers, suspended in DMEM to a final concentration of 10 mg dry weight/ml, and were kept at 37° with continuous gassing (95% O2/5% CO2) in a shaking water bath. After 1 hr of incubation in methionine-free medium, the cells were suspended in DMEM containing 50 U/ml heparin and 300 μCi/ml [35S]methionine. Newly synthesized HL was detected by immunoisolation of the enzyme from cell extracts and incubation medium using the IgG fraction of a specific rabbit antirat HL antibody. This antibody was raised against rat HL purified to homogeneity from heparin-perfused rat livers. The antibody inhibited postheparin HL activity but did not neutralize lipoprotein lipase activity. Immunoisolation of HL was verified by (1) its specific competition with purified HL enzyme, (2) its inability to be isolated by nonimmune rabbit IgG, and (3) its comigration with purified HL on SDS-PAGE. After SDS-PAGE and fluorography, newly synthesized HL was quantitated by densitometry. Hepatic lipase synthesized by hepatocytes was clearly demonstrated in cytosol 1 hr after addition of label (densitometry units × 104:0, 0.1, 7.1, 14.2, and 66.6 at time 0, 72, 1, 2, and 3 hr, respectively). The majority of labeled HL, however, was secreted and labeled HL progressively accumulated in the medium throughout the 3-hr incubation. The [35S]methionine incorporated into HL was 0.01% of that incorporated into newly synthesized and secreted albumin. Immunoisolatable HL from both incubation medium and cellular cytosol migrated with a molecular weight of 56,900 on SDS-PAGE. The progressive accumulation of HL in the incubation medium represented specific secretion and not release following cell death since (1) hepatocytes exhibited >75% viability by trypan blue exclusion after 6 hr of incubation, and (2) LDH activity in the medium, a measure of hepatocellular integrity, remained relatively constant throughout the incubation period. Total RNA was extracted from rat liver using the guanidium isothiocyanate method and isolated by CsCl gradient centrifugation. Total RNA (20 μg) added to a cell-free IVT system directed the incorporation of [35S]methionine into an immunoisolatable band of 53,000 molecular weight on SDS-PAGE. This difference in molecular weight can be explained by the absence of posttranslational processing of HL in the IVT system. Importantly, a band of similar molecular weight was noted in the immunosiolate of HL from cellular cytosol, suggesting the existence of a precursor form of the enzyme. Thus, we have shown that isolated rat hepatocytes can be used to examine the regulation of synthesis and secretion of HL. Moreover, translatable HL mRNA can be quantitated using cell-free IVT systems.
KeywordsHigh Density Lipoprotein2 Hepatic Lipase Metabolic Label Collagenase Perfusion Hepatic Lipase Activity
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