Development of lipoprotein lipase in cultured 3T3-L1 cells

The 3T3-L1 mouse fibroblast resembles an adipocyte after reaching a confluent stage of growth. Lipoprotein lipase activity was released with heparin and was present in acetone-ether extracts of these cells. During the early post-confluent period both activities increased rapidly. A wide variation in enzyme activities was noted in subclones suggesting that spontaneous heritable change continues to take place in these cells. Since lipoprotein lipase activity was measurable before triglyceride accumulation, it may be the earliest marker of adipocyte expression in this line. This system appears to offer a unique opportunity to study the processes of cellular differentiation and fat metabolism $\text{in vitro}$.     

Ionic currents in avian granulosa cells

Transmembrane ionic currents have been recorded in single granulosa cells from the laying hen using the whole-cell patch-clamp technique. Under voltage-clamp conditions, depolarizing voltage steps evoked currents composed of a fast inactivating inward component and a delayed outward component. The former was activated at voltages more positive than -50 mV and was fully inactivated within 500 ms. It was blocked by D600 (methoxyverapamil) and by cobalt, suggesting that it is a calcium current. The latter displayed inward rectification and did not inactivate during long duration pulses. It was blocked by tetraethylammonium indicating that it is a potassium current. This is the first evidence of the existence of potassium and calcium transmembrane currents in granulosa cells.     

Insulin regulation of lipoprotein lipase in cultured 3T3-L1 cells

Lipoprotein lipase activity is produced by the 3T3-L1 cell an established mouse fibroblast line which resembles an adipocyte after reaching a confluent stage of growth. Since insulin has been shown to be an important regulator of lipoprotein lipase in other mammalian systems, a two hour incubation period was utilized to determine if insulin could enhance an acute response of enzyme activity. Over the range of concentrations tested (0.4, 4.0 and 40 ng/ml), insulin increased lipoprotein lipase activity in acetone ether powders of cells (intracellular enzyme) and the activity secreted into the culture medium. A simultaneous decrease in lipoprotein lipase activity releasable with heparin in a subsequent incubation (membrane bound activity) indicates two distinct effects of insulin on the enzyme in this system.     

Cell-free translation of avian adipose tissue lipoprotein lipase messenger RNA

Poly(A)+ mRNA isolated from chicken adipose tissue directed cell-free translation in a rabbit reticulocyte lysate system. Immunoadsorption with polyclonal antibodies against lipoprotein lipase detected a protein of 56 +/- 2 kDa. Immunodetection of this protein was prevented by inclusion of purified lipoprotein lipase in the assay mixture. Identification of the 56 kDa protein as lipoprotein lipase was confirmed by immunoadsorption to the monoclonal antibody CAL 1-11. Inclusion of dog pancreatic microsomal membranes in the translation system resulted in isolation of an additional protein of 62 kDa. Treatment of the 62 kDa protein with endo-beta-N-acetylglycosaminidase H or endo-beta-N-acetylglucosaminidase F decreased the observed molecular mass to that of the primary translation product, indicating that the increase in molecular mass resulted from the addition of N-linked oligosaccharides. Starving and refeeding chickens prior to poly(A)+ mRNA isolation resulted in a 3-fold increase in the amount of immunodetectable lipoprotein lipase synthesized.     

Metabolism of progesterone by avian granulosa cells in culture

Previous studies have demonstrated that progesterone is the primary product of steroidogenesis in avian granulosa cells during short-term incubation. However, during more prolonged culture, lasting several days, the progesterone content in the medium was found to decrease progressively, indicating in vitro metabolic conversion. In the present study we have isolated and identified a number of progesterone metabolites. Granulosa cells, isolated from mature ovarian follicles of laying hens, were cultured in medium 199 supplemented with fetal calf serum and containing [14C]progesterone. After 4 days in culture, cells + media were extracted and the radioactive metabolites separated and identified by TLC, HPLC and GC-MS. Several of the metabolites were further characterized by derivatization and crystallization to constant specific activity. A total of 24 radioactive substances was detected. Of these, 15 have been positively identified, 5 tentatively and the remaining 4 are unidentified. The principal metabolite, representing more than 45% of the total radioactivity, was identified as 3 alpha-hydroxy-5 beta-pregnan-20-one. In addition, significant amounts of 3 alpha-hydroxy-5 alpha-pregnan-20-one (5.76%), 5 beta-pregnane-3,20-dione (3.05%), and 5 alpha-pregnane-3,20-dione (2.95%) were detected and identified. The results indicate that avian granulosa cells possess 3 alpha-hydroxy-steroid dehydrogenase (3 alpha-HSD), 17 beta-HSD, 20 alpha-HSD, 20 beta-HSD, 17 alpha-hydroxylase, C17-20-lyase and 5 alpha- and 5 beta-reductase activities. These enzyme activities may convert progesterone to biologically inactive or less active metabolites. However, a functional role for some of these metabolites cannot be ruled out.     

Intracellular localization of lipoprotein lipase in adipose cells

Subcellular localization of lipoprotein lipase has been examined in differentiated Ob17 adipose cells. No patent activity is detectable in carefully homogenized cells. All latent activity can be unmasked by disrupting membrane structures with neutral detergents. The sequestration of lipoprotein lipase in closed membrane structures is supported by experiments of immunotitration with anti-lipoprotein lipase antibodies and by experiments showing a full protection of the masked activity against proteolytic attack by trypsin. The intracellular distribution of lipoprotein lipase investigated by immunofluorescence staining and by isopycnic centrifugation indicates that a large proportion of the enzyme is located in the Golgi apparatus, in which the activation of the enzyme is likely to take place (C. Vannier et al. (1985) J. Biol. Chem. 260, 4424-4431). Altogether, the results are in favor of a localization of lipoprotein lipase in adipose cells as being typical of that of a secretory protein and underline the absence of lipoprotein lipase in the cell cytoplasm.     

Synthesis and regulation of lipoprotein lipase in the hippocampus

Lipoprotein lipase (LPL) expression was determined in adult rat hippocampus and compared to enzyme expression in other brain regions. Hippocampus LPL mRNA levels were at least 2.5-fold higher than those detected in the cerebral cortex, cerebellum, and remaining brain regions. Enzyme mass and activity levels in the hippocampus were also increased to a similar degree. De novo synthesis of LPL in the hippocampus was confirmed by [35S]methionine-labeling of the tissue and identification of a 57 kDa protein obtained by immunoprecipitation. Addition of an excess amount of bovine LPL completely prevented the immunoprecipitation of this protein. The effect of nutritional modulations on brain LPL activity was determined after a 12-h fast. While no significant changes were observed in other regions of the brain, hippocampus LPL activity in fasted rats increased by 60% compared to the fed control group. Simultaneously, fasting reduced adipose LPL activity by 60%. Intraperitoneal injection of ACTH over a 5-day period had no effect on hippocampus LPL activity, while adipose LPL levels increased 2.3-fold and heart LPL levels decreased 1.4-fold. We conclude that LPL is synthesized, active and regulated in a tissue-specific manner in the adult rat hippocampus.     

Transcytosis of lipoprotein lipase across cultured endothelial cells requires both heparan sulfate proteoglycans and the very low density lipoprotein receptor

Lipoprotein lipase (LPL), the major enzyme responsible for the hydrolysis of circulating lipoprotein triglyceride molecules, is synthesized in myocytes and adipocytes but functions while bound to heparan sulfate proteoglycans (HSPGs) on the luminal surface of vascular endothelial cells. This requires transfer of LPL from the abluminal side to the luminal side of endothelial cells. Studies were performed to investigate the mechanisms of LPL transcytosis using cultured monolayers of bovine aortic endothelial cells. We tested whether HSPGs and members of the low density lipoprotein (LDL) receptor superfamily were involved in transfer of LPL from the basolateral to the apical side of cultured endothelial cells. Heparinase/heparinitase treatment of the basolateral cell surface or addition of heparin to the basolateral medium decreased the movement of LPL. This suggested a requirement for HSPGs. To assess the role of receptors, we used either receptor-associated protein, the 39-kDa inhibitor of ligand binding to the LDL receptor-related protein and the very low density lipoprotein (VLDL) receptor, or specific receptor antibodies. Receptor-associated protein reduced (125)I-LPL and LPL activity transfer across the monolayers. When the basolateral surface of the cells was treated with antibodies, only anti-VLDL receptor antibodies inhibited transcytosis. Moreover, overexpression of the VLDL receptor using adenoviral-mediated gene transfer increased LPL transcytosis. Thus, movement of active LPL across endothelial cells involves both HSPGs and VLDL receptor.     

The distribution of actin in cultured ovarian granulosa cells

Ovarian granulosa cells grown on glass coverslips were split by a "sandwich" technique. Using this technique we describe a complex filamentous network in the cytoplasm of cultured granulosa cells that was composed of a branching and anastomosing lattice of filaments 20-40 nm in diameter. Since filament identification is impossible on the basis of size, split cells were decorated with S-1 fragments of rabbit skeletal muscle myosin. It was readily apparent that the major constituent of the filamentous lattice was actin. Actin was organized in large bundles in which individual filaments were longitudinally aligned. Actin was also observed organized in a loose network throughout the remainder of the cytoplasm. Actin appeared to be intimately associated with organelle and plasma membranes. Coated pits were also a site of actin-filament interaction. Filament polarity was generally away from the membrane with which filaments were associated.     

Synthesis and secretion of active lipoprotein lipase in Chinese-hamster ovary (CHO) cells.

Cultured Chinese-hamster ovary cells (CHO cells) were found to produce and secrete a lipase, which was identified as a lipoprotein lipase by the following criteria. Its activity was stimulated by serum and apolipoprotein CII, and was inhibited by high salt concentration. The lipase bound to heparin-agarose and co-eluted with 125I-labelled bovine lipoprotein lipase in a salt gradient. A chicken antiserum to bovine lipoprotein lipase inhibited the activity and precipitated a labelled protein of the same apparent size as bovine lipoprotein lipase from media of CHO cells labelled with [35S]methionine. The lipase activity and secretion were similar in growing cells and in cells that had reached confluency. Hence, lipoprotein lipase appears to be expressed constitutively in CHO cells and is not linked to certain growth conditions, as in pre-adipocyte and macrophage cell lines. At 37 degrees C, but not at 4 degrees C, heparin increased the release of lipase to the medium 2-4-fold. This increased release occurred without depletion of cell-associated lipase activity, suggesting that heparin enhanced release of newly synthesized lipase.     

Maturation and secretion of lipoprotein lipase in cultured adipose cells. I. Intracellular activation of the enzyme

The intracellular pathway and the activation of lipoprotein lipase have been examined in differentiated Ob17 cells. These adipose cells were previously shown to secrete lipoprotein lipase during exposure to heparin. Treatment of the cells with cycloheximide and heparin leads to enzyme depletion, as shown by activity measurement and immunofluorescence microscopy. The repletion phase has been studied in the presence of monensin or carbonyl cyanide m-chlorophenylhydrazone, ionophores known to affect the intracellular transport of membrane and secretory proteins. Monensin-treated cells synthesize fully active lipoprotein lipase. Under these conditions the antigen accumulates in the Golgi apparatus and the heparin-stimulated enzyme release is extensively reduced. Carbonyl cyanide m-chlorophenylhydrazone-treated cells do not contain any enzyme activity but show detectable antigen which accumulates in the endoplasmic reticulum. Competition for binding to immobilized anti-lipoprotein lipase antibodies of mature and endoplasmic reticulum-sequestered antigens is observed. Carbonyl cyanide m-chlorophenylhydrazone removal is rapidly followed by a transient burst of enzyme activity and a redistribution of the antigen in the different subcellular compartments. Therefore, the results show that the activation of lipoprotein lipase is an intracellular event taking place after the enzyme exits from the endoplasmic reticulum and before it reaches the trans-Golgi cisternae.     

Evidence for two distinct potassium channels in avian granulosa cells

Single-channel potassium currents were recorded in avian granulosa cells using the patch-clamp technique. Two types of channel were observed. The smaller of the two channels, gK1, had a conductance of 15 to 30 picosiemens (pS) and was voltage- and calcium-independent. Its null-current potential was -50 mV in the cell-attached recording mode. The other channel, gK2, was infrequently observed in the cell-attached configuration. Its conductance was between 160 and 195 pS. It could be activated by calcium on the cytoplasmic side of the membrane patch in the inside-out configuration. It was also voltage-dependent. These results suggest that fast transmembrane potassium movements may be involved in the membrane voltage regulation of granulosa cells, which in turn may play an important role in the modulation of steroidogenesis and other metabolic activities.     

High-cholesterol diet-induced lipoproteins stimulate lipoprotein lipase secretion in cultured rat alveolar macrophages

We have previously shown that cultured rat alveolar macrophages synthesize and secrete lipoprotein lipase into the medium. The purpose of the present experiments is to examine whether cholesterol-enriched lipoproteins from cholesterol-fed animals have any effects on the lipoprotein lipase secretion and the lipid accumulation in macrophages. Macrophages incubated with the VLDL obtained from rats fed a normal diet secreted 2-fold higher amounts of lipoprotein lipase than those without lipoproteins. Intermediate-, low- and very-low-density lipoproteins from rats fed a high-cholesterol diet also enhanced the lipoprotein lipase secretion. Normal high- and low-density lipoproteins, and high-density lipoproteins from hypercholesterolemic animals did not cause any increase in the lipoprotein lipase secretion. The lipoproteins which stimulated the lipoprotein lipase secretion caused intracellular accumulation of both triacylglycerol and cholesterol. It is speculated that macrophages residing in the environment rich in lipoproteins, especially hypercholesterolemic lipoproteins, take them up and accumulate lipids intracellularly, and that this process links with the lipoprotein lipase secretion. The secreted lipoprotein lipase could facilitate, by degrading lipoproteins, the uptake of lipoprotein lipase-modified lipoproteins. Probably such a series of events is of importance in the foam cell formation of macrophages.     

Secretion and degradation of lipoprotein lipase in cultured adipocytes. Binding of lipoprotein lipase to membrane heparan sulfate proteoglycans is necessary for degradation

Equilibrium-binding data of highly purified 125I-labeled avian lipoprotein lipase to cultured avian adipocytes demonstrate the presence of a class of high affinity binding sites. Analysis of the binding function yielded an association constant of 0.62 x 10(8)M-1 and a maximum binding capacity of 2.1 micrograms/60-mm dish. From a time course of dissociation of 125I-lipoprotein lipase from adipocytes at 4 degrees C, a dissociation rate constant of 6.1 x 10(-5)s-1 was obtained. Pretreatment of cells with heparinase and heparitinase resulted in a quantitative suppression of the high affinity binding component, establishing that lipoprotein lipase is bound to cell surface heparan sulfate proteoglycans. At 37 degrees C, cell surface-bound 125I-lipoprotein lipase is internalized and either degraded or recycled to the medium. The degradation rate constant for 125I-lipoprotein lipase was estimated to be 0.78 h-1. The degradation rate constant was reduced 6-fold when cells were exposed to 100 microM chloroquine, indicating that most of the degradation occurs within the lysosomal compartment. By using cells that had been pulsed with Trans35S-label for 1 h, it was demonstrated that acute treatment with endoglycosidases for up to 1 h resulted in a new lipoprotein lipase secretion rate which was 6-fold higher than that of control cells. Degradation of newly synthesized lipoprotein lipase was essentially blocked 30 min after the initiation of the chase. In other studies it was observed that there were no additive effects of chloroquine and either endoglycosidase or heparin treatment on total lipoprotein lipase levels (intracellular, cell surface, and medium) in adipocyte cultures. These experiments support the hypothesis that the release of lipoprotein lipase from its receptor prevents its internalization and degradation and enhances enzyme efflux from the adipocyte. A new model of lipoprotein lipase secretion in cultured adipocytes is proposed: Newly synthesized lipoprotein lipase is transported to the cell surface where it binds to specific heparan sulfate proteoglycan receptors. The enzyme is either released to the medium or internalized via the receptor, in which case the enzyme is degraded or recycled to the cell surface. Major determinants of enzyme efflux from the cell surface include the number and integrity of receptors, the association constant of the enzyme-receptor complex, and the presence in the medium of competing molecules with high affinity for lipoprotein lipase. In this model, modulation of lipoprotein lipase degradation rate may be a significant mechanism for acute regulation of enzyme efflux independent of changes in the rate of enzyme synthesis.     

Effects of cytokines on the production of lipoprotein lipase in cultured human macrophages

Macrophages are important cells in the pathogenesis of atherosclerosis because of their tendency to accumulate lipid and become transformed into foam cells. Cultured human monocyte-derived macrophages spontaneously secrete lipoprotein lipase (LPL), and LPL has been linked to increased lipid uptake by these cells. Because secretion of various macrophage products depends on activation by lymphokines, we studied the effects of immunoregulatory lymphokines on LPL secretion by cultured human macrophages. After culturing cells in RPMI 1640 medium with 20% fetal calf serum, recombinant human gamma-interferon (gamma-INF), interleukin-1 (IL-1), and interleukin-2 (IL-2) were added to the medium and LPL secretion was assessed by measuring LPL activity and/or LPL mass in the medium. Gamma-INF suppressed LPL production both when added to freshly plated cultures of human blood monocytes, as well as when added to monocyte/macrophages from mature cultures (day 6) that were producing large amounts of LPL. IL-1 inhibited medium LPL when added to freshly plated cultures, but not when added to mature cultures. On the other hand, IL-2 did not inhibit LPL in freshly plated cultures, but produced a dose-dependent suppression of LPL from mature cultures. None of the cytokines were cytotoxic to macrophages, and cells that were cultured in gamma-INF demonstrated partial recovery from LPL-suppressive doses of the cytokine. After exposure of cells to 50 U/ml of gamma-INF and 50 U/ml of IL-2 for 3 days, LPL mRNA levels, when expressed as LPL/gamma-actin ratios, were 42% and 53% of controls, respectively. Thus, activation of human macrophages in vitro by gamma-INF resulted in a suppression of LPL production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gossypol inhibits aromatase activity in cultured porcine granulosa cells

The present study investigated whether gossypol inhibited aromatase activity in cultured porcine granulosa cells. Aromatase activity was assayed by measuring (3)H-H(2)O released from [1beta-(3)H]-androstenedione. First, immature porcine granulosa cells were cultured with various doses of follicle stimulating hormone (FSH, 1 to 1000 ng/ml) for 1 to 5 d to determine optimal culture conditions for aromatase activity assay. Second, porcine granulosa cells were cultured with or without FSH in the presence or absence of gossypol. Gossypol, at 4 muM, significantly inhibited FSH-induced aromatase activity while showing no effect on basal aromatase activity. Gossypol did not inhibit cell proliferation during cell culture. These results suggest that gossypol inhibits aromatase activity by interfering with FSH induction of aromatase in cultured porcine granulosa cells.     

Ovarian substance induces steroid production in cultured granulosa cells

Summary The rat ovary produces an apparently low molecular weight substance that mimics the action of follitropin (FSH) on ovarian granulosa cells in culture. Similar to FSH action, the ovarian substance (OS) induces temporal cell rounding and, later on, intensive progestin production. However, unlike FSH, OS does not induce accumulation of cyclic AMP (cAMP) in the granulosa cells. The ovarian factor cannot be cAMP as its action is not abolished by phosphodiesterase (PDE) treatment. Neither is it a possible PDE inhibitor, as it does not augment cAMP accumulation in granulosa cells or Friend erythroleukemic cells induced by FSH or PGE₁, respectively. The factor is still active after heating for 20 min at 90° C but is rapidly inactivated by alkali treatment. In addition, treatment with various proteases did not abolish the steroidogenic activity. These findings suggest a possible novel intraovarian regulator of the granulosa cell function. Presented in the symposium on Plant and Animal Physiology in Vitro at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, California, June 6–10, 1982. This work was supported by the United States-Israel Binational Science Foundation, Grant 2656/81. This symposium was supported in part by the following organizations: Bellco Glass, Inc., California Branch of the Tissue Culture Association, Collaborative Research, Hana Media, Hybridtech, K C Biological, Inc., and Millipore Corporation.