Synthesis of hepatic lipase in liver and extrahepatic tissues1

Immunoprecipitations of hepatic lipase from pulselabeled rat liver have demonstrated that hepatic lipase is synthesized in two distinct molecular weight forms, HL-I (M, = 51,000) and HL-I1 (M, = 53,000). Both forms are immunologically related to purified hepatic lipase, but not to lipoprotein lipase. HL-I and HL-11 are also kinetically related and represent different stages of intracellular processing. Glycosidase experiments suggest that HL-I is the high mannose microsomal form of the mature, sialyated HL-I1 enzyme. Hepatic lipase activity was detected in liver and adrenal gland but was absent in brain, heart, kidney, testes, small intestine, lung, and spleen. The adrenal and liver lipase activities were inhibited in a similar dose-dependent manntr by hepatic lipase antiserum. Immunoblot analysis of partially purified adrenal lipase showed an immunoreactive band co-migrating with HL-I1 at 53,000 daltons which was absent in a control blot treated with preimmune serum. Adrenal lipase and authentic hepatic lipase yielded similar peptide maps, confirming the presence of the lipase in adrenal gland. However, incorporation of L-[ 35S]methionine into immunoprecipitable hepatic lipase was not detected in this tissue. In addition, Northern blot analysis showed the presence of hepatic lipase mRNA in liver but not adrenal gland. The presence of hepatic lipase in adrenal gland in the absence of detectable synthesis or messenger suggests that hepatic lipase originates in liver and is transported to this extrahepatic site. -Doolittle, M. H., H. Wong, R. C. Davis, and M. C. Schotz. Synthesis of hepatic lipase in liver and extrahepatic tissues. J. Lipid Res. 1987. 28: 1326-1334. Supplementary key words glycosylation salt-resistant triacylglycerol lipase in adrenal gland immunoblotting and precipitation peptide mapping hepatic lipase mRNA containing chylomicrons and VLDL. In contrast, hepatic lipase appears to prefer lipoproteins deficient in apoC-I1 such as chylomicron remnants, IDL, and HDL2 (for reviews, see refs. 1, 2). The in vivo functions of hepatic lipase are unclear. Studies in which hepatic lipase activity in vivo is blocked by the injection of specific antibodies have suggested that hepatic lipase is the critical enzyme for conversion of IDL to LDL (3-5), and that it may process apoB-48-containing chylomicron remnants for uptake by the liver ( 6 ) as well as convert HDL2 to HDL3 (2). This latter function may be important in mediating reverse cholesterol transport, a process thought to protect extrahepatic tissues from cholesterol accumulation (7). Hepatic lipase has been purified from human (8), canine (9), and rat liver (10, 11). The enzyme which we have purified from heparin perfusates of rat liver migrates on SDS polyacrylamide gels as one major band with an apparent molecular weight of 53,000 (12). Recently, we have isolated and sequenced the full-length rat liver hepatic lipase cDNA clone (13). Hepatic lipase appears to be a classical secretory protein containing a 22-amino acid hydrophobic leader sequence and two potential N-linked glycosylation sites. Based on the derived amino acid sequence, the molecular weight of the mature unglycosylated protein is 53,222. Direct evidence for the synthesis of hepatic lipase in any tissue has not been reported. Hepatic lipase activity is Two major enzymes involved in triacylglycerol and phospholipid metabolism of circulating lipoproteins are hepatic lipase and lipoprotein lipase. Classically, hepatic lipase activity on the basis of its differing protein cofactor and salt requirement. Although both hepatic and lipoproacylglycerol substrates as well as a number Of phosphoAbbreviations: apo, apolipoprotein; VLDL, very low density lipolipoproteins; HDL, high density lipoproteins; HEPES, N-2-hydroxyethylpiperazine-N-ethane-sulfonic acid; PMSF, phenylmethylsulfonyl fluoride; KRB, Krebs-Ringer bicarbonate; PBS, phosphate-buffered trophoresis: Staph A. crude insoluble Protein A from lyophilized lipase activity has been distinguished from lipoprotein proteins; IDL, intermediate density lipoproteins; LDL, low density tein lipase catalyze the Of mono-, d i 2 and trisaline; SDS PAGE, sodium sulfate polyacrylamide gel elec. _ . . . . . _ lipids in vitro, their natural lipoprotein substrate specispecifically hydrolyzes triacylglycerols from apoC-11(M.D.). ~ W b ~ o c o ~ c ~ OWW cells (cowan strain). 'This work was carried out during the tenure of a Research Fellowship ficities are distinctive. For lipoprotein lipase of the American Heart Association-Greater Los Angeles Affiliate 1326 Journal of Lipid Research Volume 28, 1987 by gest, on O cber 8, 2017 w w w .j.org D ow nladed fom secreted by primary cultures of rat and chicken liver parenchymal cells (14-16) and a human hepatoma cell line (17). This lipase activity binds to liver nonparenchymal cells (18, 19) and has been localized by immunocytochemistry to liver endothelium (20), presumably the functional location of the mature enzyme. Hepatic lipase activity, inhibitable by hepatic lipase-specific antibodies, has also been detected in adrenal gland (21-24) and ovary (23, 25, 26). The present study utilizes immunoprecipitation, immunoblotting, and mRNA hybridization techniques to: I) elucidate the steps in liver intracellular processing of newly synthesized hepatic lipase; 2) establish the presence of hepatic lipase in adrenal gland; and 3) determine the capability of adrenal gland to synthesize this enzyme. EXPERIMENTAL PROCEDURES