Characterization of lipoprotein lipase activity, secretion, and degradation at different sites of post-translational processing in primary cultures of rat adipocytes.
The regulation of adipose tissue lipoprotein lipase (LPL) by feeding and fasting occurs through post-translational changes in the LPL protein. In addition, LPL activity and secretion are decreased when N-linked glycosylation is inhibited. To better understand the role of oligosaccharide processing in the development of LPL activity and in LPL secretion, primary cultures of rat adipocytes were treated with inhibitors of oligosaccharide processing. LPL catalytic activity from the heparin-releasable fraction of adipocytes was inhibited by more than 70%, with similar decreases in LPL mass, when cells were cultured for 24 h in the presence of either tunicamycin or castanospermine. On the other hand, deoxymannojirimycin (DMJ) and swainsonine had no effect on LPL activity. LPL secretion was examined after pulse-labeling cells with [35S]methionine. The appearance of 35S-labeled LPL in the medium was blocked by treatment of cells with tunicamycin and castanospermine, whereas secretion was not affected by DMJ or swainsonine. To examine the effect of oligosaccharide processing on LPL intracellular degradation, adipocytes were treated with tunicamycin, castanospermine, and DMJ and then pulse-labeled with [35S]methionine, followed by a chase with unlabeled methionine for 120 min. The unglycosylated [35S]LPL that was synthesized in the presence of tunicamycin demonstrated essentially no intracellular degradation. In the presence of castanospermine and DMJ, the half-life of newly synthesized LPL was increased to 81 and 113 min, as compared to 65 min in control cells. Thus, castanospermine-treated adipocytes demonstrated a decrease in LPL activity and secretion, suggesting that the glucosidase-mediated cleavage of terminal glucose residues from oligosaccharides is a critical step in LPL maturation.(ABSTRACT TRUNCATED AT 250 WORDS)
Simsolo
R B
RB
Department of Medicine/Division of Endocrinology, Cedars-Sinai Medical Center, Los Angeles, CA 90048.
Ong
J M
JM
Kern
P A
PA
D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000273:Adipose Tissue / Q000187:drug effects / Q000201:enzymology*; D000818:Animals; D002478:Cells, Cultured; D006596:Hexosaminidases / Q000494:pharmacology; D007212:Indolizines / Q000494:pharmacology; D008071:Lipoprotein Lipase / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D017026:Swainsonine / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology; D054836:Indolizidines
D017485:1-Deoxynojirimycin; D006596:Hexosaminidases; D007212:Indolizines; D008071:Lipoprotein Lipase; D009844:Oligosaccharides; D017026:Swainsonine; D014415:Tunicamycin; C037806:castanospermine; D054836:Indolizidines