Hepatic lipase. Synthesis, processing, and secretion by isolated rat hepatocytes.

284

Endothelial lipase (EL) is a new member of the triglyceride lipase gene family previously reported to have phospholipase activity. Using radiolabeled lipid substrates, we characterized the lipolytic activity of this enzyme in comparison to lipoprotein lipase (LPL) and hepatic lipase (HL) using conditioned medium from cells infected with recombinant adenoviruses encoding each of the enzymes. In the absence of serum, EL had clearly detectable triglyceride lipase activity. Both the triglyceride lipase and phospholipase activities of EL were inhibited in a dose-dependent fashion by the addition of serum. The ratio of triglyceride lipase to phospholipase activity of EL was 0.65, compared with ratios of 24.1 for HL and 139.9 for LPL, placing EL at the opposite end of the lipolytic spectrum from LPL. Neither lipase activity of EL was influenced by the addition of apolipoprotein C-II (apoC-II), indicating that EL, like HL, does not require apoC-II for activation. Like LPL but not HL, both lipase activities of EL were inhibited by 1 M NaCl. The relative ability of EL, versus HL and LPL, to hydrolyze lipids in isolated lipoprotein fractions was also examined using generation of FFAs as an end point.As expected, based on the relative triglyceride lipase activities of the three enzymes, the triglyceride-rich lipoproteins, chylomicrons, VLDL, and IDL, were efficiently hydrolyzed by LPL and HL. EL hydrolyzed HDL more efficiently than the other lipoprotein fractions, and LDL was a poor substrate for all of the enzymes.

Section: Expression Of Enzymesmentioning

confidence: 99%

Characterization of the lipolytic activity of endothelial lipase

McCoy

Sun

Marchadier

et al. 2002

284

“…HL activity is found in liver, adrenals, ovaries, and plasma (6) (Tables 1 and 2). Rat liver cells are known to synthesize and secrete active HL (19,20). Our immunocytochemical findings in mouse liver cells treated with and without monensin demonstrate that mouse hepatocytes can also synthesize and secrete HL.…”

Section: Discussionmentioning

confidence: 60%

“…LPL is also synthesized by liver, but only in newborn rodents (17,18). HL is synthesized and secreted by liver (19,20) and is found on the surface of liver cells (21,22), in the space of Disse (21) and at the luminal surface of liver sinusoids (21,23,24). HL is also found in adrenals and ovaries (25,26), but it is generally thought that HL found in these tissues is synthesized by liver cells (6,(25)(26)(27).…”

mentioning

confidence: 99%

Adrenal and liver in normal and cld/cld mice synthesize and secrete hepatic lipase, but the lipase is inactive in cld/cld mice

Schultz

Blanchette‐Mackie

Scow

2000

Combined lipase deficiency ( cld ) is a recessive mutation in mice that causes a severe lack of lipoprotein lipase (LPL) and hepatic lipase (HL) activities, hyperlipemia, and death within 3 days after birth. Earlier studies showed that inactive LPL and HL were synthesized by cld / cld tissues and that LPL synthesized by cld / cld brown adipocytes was retained in their ER. We report here a study of HL in liver, adrenal, and plasma of normal newborn and cld / cld mice. Immunofluorescence studies showed HL was present in extracellular space, but not in cells, in liver and adrenal of both normal and cld / cld mice. When protein secretion was blocked with monensin, HL was retained intracellularly in liver cell cultures and in incubated adrenal tissues of both groups of mice. These findings demonstrated that HL was synthesized and secreted by liver and adrenal cells in normal newborn and cld / cld mice. HL activities in liver, adrenal, and plasma in cld / cld mice were very low, Ͻ 8% of that in normal newborn mice, indicating that HL synthesized and secreted by cld / cld cells was inactive. Livers of both normal newborn and cld / cld mice synthesized LPL, but the level of LPL activity in cld / cld liver was very low, Ͻ 9% of that in normal liver. Immunofluorescence studies showed that LPL was present intracellularly in liver of cld/cld mice, indicating that LPL was synthesized but not secreted by cld/ cld liver cells. Immunofluorescent LPL was not found in normal newborn liver cells unless the cells were treated with monensin, thus demonstrating that normal liver cells synthesized and secreted LPL. Livers of both groups of mice contained an unidentified alkaline lipase activity which accounted for 34-54% of alkaline lipase activity in normal and 65% of that in cld / cld livers. Our findings indicate that liver and adrenal cells synthesized and secreted HL in both normal newborn and cld / cld mice, but the lipase was inactive in cld/cld mice. That cld / cld liver cells secreted inactive HL while retaining inactive LPL indicates that these closely related lipases were processed differently. -Schultz, C. J., E. J. Blanchette-Mackie, and R. O. Scow. Adrenal and liver in normal and cld/cld mice synthesize and secrete hepatic lipase, but the lipase is inactive in cld / cld mice.

“…Nascent N-linked glycan chains (GlcNac 2 Man 9 Glc 2 ; where Nac is N-acetylglucosamine, Man is mannose, and Glc is glucose) are added en bloc to secretory proteins as they are co-translationally translocated across the ER membrane. Inhibition of glycosylation by treatment of cells with tunicamycin (TM) prevents secretion of rat HL (22,(24)(25)(26). To determine the role of N-linked glycans in human HL secretion, pulse-chase experiments were performed in the presence of TM ( Fig.…”

Section: N-linked Glycosylation Is Essential For Hl Secretionmentioning

confidence: 99%

“…After transport from the ER, Golgi-specific maturation of N-linked glycans results in an increase in the molecular mass of the secreted enzyme to 53-59 kDa (3,22,23). Inhibition of N-linked glycosylation with tunicamycin (TM), or inhibition of glucose trimming with ER glucosidase inhibitors, results in intracellular retention of inactive rat HL (22,(24)(25)(26). Inhibition of later steps in N-linked glycan processing affects neither HL secretion nor acquisition of enzyme activity (24).…”

mentioning

confidence: 99%

Role of N-linked carbohydrate processing and calnexin in human hepatic lipase secretion

Boedeker

Doolittle

Santamarina-Fojo

et al. 1999

The addition and endoplasmic reticulum (ER) glucosidase processing of N-linked glycans is essential for the secretion of rat hepatic lipase (HL). Human HL is distinct from rat HL by the presence of four as opposed to two N-linked carbohydrate side chains. We examined the role of N-linked glycosylation and calnexin interaction in human HL secretion from Chinese hamster ovary (CHO) cells stably expressing a human HL cDNA. Steady-state and pulse-chase labeling experiments established that human HL was synthesized as an ER-associated precursor containing high mannose N-linked glycans. Secreted HL had a molecular mass of ϳ 65 kDa and contained mature N-linked sugars. Inhibition of N-linked glycosylation with tunicamycin (TM) prevented secretion of HL enzyme activity and protein mass. In contrast, incubation of cells with the ER glucosidase inhibitor, castanospermine (CST), decreased human HL protein secretion by 60%, but allowed 40% of fully active HL to be secreted. HL protein mass and enzyme activity were also recovered from the media of a CHO-derivative cell line genetically deficient in ER glucosidase I activity (Lec23) that was transiently transfected with a human HL cDNA. Co-immunoprecipitation experiments demonstrated that newly synthesized human HL bound to the lectin-like ER chaperone, calnexin, and that this interaction was inhibited by TM and CST.These results suggest that under normal conditions calnexin may increase the efficiency of HL export from the ER. Whereas a significant proportion of human HL can attain activity and become secreted in the absence of glucose trimming and calnexin association, these interrelated processes are nevertheless essential for the expression of full HL activity. -Boedeker, J. C., M. Doolittle, S. Santamarina-Fojo, and A. L. White. Role of N-linked carbohydrate processing and calnexin in human hepatic lipase secretion.