Characterization of lipoprotein

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 oligisaccharide 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 heparinreleasable 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. O n 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 3%-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 pulselabeled 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. I 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. In addition, the maturation of N-linked oligosaccharides on LPL was associated with an increased intracellular turnover.-Simsolo, R. B., J. M. Ong, and P. A. Kern. Characterization of lipoprotein lipase activity, secretion, and degradation at different steps of post-translational processing in primary cultures of rat adipocytes. J Lipid Res. 1992. 33: 1777-1784. Supplementary key words camycin deoxymannojirimycin glucosidase mannosidase glycosylation castanospermine tuniLipoprotein lipase (LPL) is a central enzyme in lipid metabolism. The enzyme is synthesized and secreted by adipocytes, and then transported to the capillary endothelium, where hydrolysis of the triglyceride core of circulating VLDL and chylomicrons takes place (1, 2). Recent studies of LPL gene expression have indicated that there are multiple sites of transcriptional and posttranscriptional regulation. Rat adipocytes demonstrate an increase in LPL mRNA in response to insulin (3, 4), and a decrease in response to dexamethasone (5). On the other hand, both rats and humans demonstrate increases in LPL activity after feeding due to post-transcriptional changes, characterized by an increase in LPL specific activity (6, 7). The importance of LPL glycosylation in the development of LPL activity has been demonstrated in several studies. When adipocytes were treated with tunicamycin, or cultured in glucose-free medium, a deglycosylated form of LPL was synthesized that had low or absent LPL activity and was not secreted from the cell (8-10). Several studies have further examined LPL post-translational processing using various inhibitors of oligosaccharide processing. One study suggested that cells produced active and secretable LPL after inhibiting cellular mannosidases I and 11, as well as after inhibition of glucosidases I and I1 with methyldeoxynojirimycin (MDN) (11). These data suggested that only core glycosylation was necessary for fully developed LPL activity and secretion. On the other hand, studies with hepatic lipase, which is structurally similar to LPL and part of the same gene family, demonstrated that hepatic lipase was active and secreted Abbreviations: LPL, lipoprotein lipase; DMJ, deoxymannojirimycin; MDN, methyldeoxynojirimycin; HL, hepatic lipase; VLDL, very low density lipoprotein; FFA, free fatty acids; E A , trichloroacetic acid; HR, LPL activity in adipocyte fraction released by heparin; EXT, LPL activity in fraction remaining after heparin release in cell extracts. 'To whom correspondence should be addressed at: Division of Endocrinology, Becker 131, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048. Journal of Lipid Research Volume 33, 1992 1777 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 1, 2013 w w w .j.org D ow nladed fom after the action of glucosidase I and I1 on the core oligosaccharide (12). A recent study examined LPL processing in Chinese hamster ovary (CHO) cells (13), which secrete large amounts of LPL (14), and also found that the trimming of terminal glucose residues from the oligosaccharide in the rough endoplasmic reticulum (RER) was essential for further processing and secretion. This study used primary cultures of rat adipocytes, and was designed to determine what steps in LPL glycosylation are necessary for LPL activity and secretion, and also to determine whether different forms of LPL differ in their rate of degradation. As with hepatic lipase (12), cleavage of the terminal glucoses from the core N-linked oligosaccharide was a critical step in the development of LPL activity and in LPL secretion from the adipocyte. MATERIALS AND METHODS Preparation of adipocytes Adipocytes were prepared from epididymal fat pads of male Sprague-Dawley rats, as described previously (3). Cells were cultured for 24 h in Medium 199 (Irvine Scientific, Santa Ana, CA) containing 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), and were then washed in the same medium, followed by the addition of the indicated concentrations of either castanospermine, 1-deoxymannojirimycin (DMJ), N-methyldeoxynojirimycin (MDN), swainsonine, or tunicamycin (Sigma, St. Louis, MO). Measurement of LPL activity LPL catalytic activity was measured in the adipocyte fraction released by heparin (HR), as well as in the fraction remaining after heparin release in cell extracts, which is referred to as EXT, as described previously (8). In brief, adipocytes were incubated in phosphate-buffered saline (PBS) containing 13 pglml heparin (Fisher Scientific Co.) for 30 min at 37OC. An aliquot of this buffer was then assayed as described below. The cells were then washed and the EXT fraction was prepared by homogenizing the cells in buffer containing deoxycholate and heparin, as described previously (6). LPL activity was then measured in the supernatant after centrifugation. LPL activity was determined as described previously (15) using a [3H]triolein-containing substrate emulsified with lecithin, and containing normal human serum as a source of apoC-11. Activity was expressed as nEq FFA released/min per 106 cells. LPL immunoreactive mass Previous studies have described the measurement of LPL immunoreactive mass by enzyme-linked immunosorbent assay (ELISA) (16, 17). In brief, samples for LPL immunoreactive mass were prepared as described above for LPL activity, except protease inhibitors were present in all the buffers. Affinity-purified chicken antibovine LPL antibodies are used as a capture antibody, and biotinylated affinity-purified anti-LPL antibody, followed by streptavidin-peroxidase, is used as the indicator antibody. The concentration of LPL was then calculated using the standard curve for bovine LPL, and expressed as ng/106 cells. [35S]methionine labeling, immunoprecipitation, and