Regulation of lipoprotein lipase synthesis by recombinant tumor necrosis factor--the primary regulatory role of the hormone in 3T3-L1 adipocytes

Tumor necrosis factor (TNF), a protein homologous to cachectin, has been implicated in mediating cachexia. This effect at least in part has been suggested to occur through the influence of the hormone on adipose tissue metabolism. Using fully differentiated 3T3-L1 adipocytes as a model system, we have been investigating the effects of recombinant TNF (rTNF) on key features of adipocyte metabolism. Exposure of fully differentiated 3T3-L1 adipocytes to recombinant tumor necrosis factor resulted in a dose and time-dependent suppression of the activity of lipoprotein lipase. The loss in activity results from an effect on the synthesis of the enzyme, as determined by a decreased incorporation of [35S]methionine into immunoprecipitable lipoprotein lipase. No effect of rTNF on the half-life of the enzyme was observed. General protein synthesis, as judged by [35S]methionine incorporation into acid-insoluble protein, was minimally affected by exposure of the cells to rTNF; this was further confirmed by sodium dodecyl sulfate-polyacrylamide gel analysis of total cellular protein. As opposed to our previously reported results with crude preparations of TNF, no effect on either the ability of the adipocytes to synthesize and store or mobilize triacylglycerol was observed. Our results are consistent with the hypothesis that other hormones present in crude preparations of TNF acting either alone or synergistically with TNF play a major role in the further metabolic derangements associated with adipose tissue during cachexia.     

Dibutyryl cyclic AMP decreases the rate of lipoprotein lipase synthesis in cultured adipocytes

The regulation of avian lipoprotein lipase by dibutyryl cyclic AMP in cultured adipocytes was studied with quantitative and specific methods for the measurements of enzyme catalytic activity, enzyme protein mass, and immunoadsorption of labeled enzyme. Incubation of adipocytes in 0.5 mM dibutyryl cyclic AMP plus 0.5 mM theophylline results in a time-dependent decrease in cell lipoprotein lipase catalytic activity. The activity is decreased by 70% in 4 h and over 90% by 12 h. The decrease in cellular catalytic activity is due to a decrease in both enzyme content and enzyme catalytic efficiency. 4 h after exposure of adipocytes to cAMP, enzyme protein was decreased from 3.58 +/- 0.5 to 1.92 +/- 0.1 ng/dish and specific activity from 15.1 +/- 2.1 to 8.4 +/- 1.1 nmol/ng. In the presence of 0.5 mM theophylline, the dibutyryl cyclic AMP-mediated decrease in lipoprotein lipase activity was half-maximal at less than 25 microM dibutyryl cyclic AMP. The rate of lipoprotein lipase synthesis was estimated by measuring the incorporation of L-[35S]methionine into enzyme protein during 30 min. A method for the quantitative immunoadsorption of lipoprotein lipase from cell lysates was developed. Utilizing this immunoadsorption technique, the rate of incorporation of L-[35S]methionine into lipoprotein lipase was 0.0026 +/- 0.002%, when expressed as a percentage of that incorporated into total trichloroacetic acid-precipitable counts. By 2 h after exposure of adipocytes to 0.5 mM dibutyryl cAMP, the relative synthesis rate had already decreased to 64 +/- 4% of the control rate. After 16 h the synthesis rate was 43.2 +/- 13.8% of the control rate. The observed decreased synthesis rate could account for most of the decreased cellular enzyme content and diminished enzyme secretion rate.     

Enhanced release and synthesis of lipoprotein lipase in rat heart cell cultures exposed to high concentrations of Hepes

While attempting to optimize conditions for synthesis of lipoprotein lipase by cultured heart cells, we encountered an unexpected rise in enzyme activity when media were supplemented inadvertently with 100 mM Hepes buffer (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid). This finding was further investigated and optimal results were obtained at pH 7.0-7.2. The increase in lipoprotein lipase activity was time dependent; after 3-6 h there was a rise in medium activity but cellular activity increased only after 24 h. The increased enzyme activity was defined as lipoprotein lipase by inhibition with antiserum to rat adipose tissue lipoprotein lipase. A 72-h exposure to Hepes resulted in a 30% increase in the incorporation of [35S]methionine into cellular proteins and a 2-fold increase into heparin-releasable proteins. Using heparin Sepharose chromatography and stepwise elution, a lipoprotein lipase enriched fraction was recovered with 2 M NaCl. The amount of [35S]methionine and [3H]galactose incorporated into protein of this fraction derived from Hepes-treated cells was 2-6-fold that of controls. A 4-fold increase in cellular lipoprotein lipase mass in Hepes-treated cells was shown by immunoblotting. Results obtained with Hepes-conditioned medium suggest the presence of cell-derived compounds that enhance release and subsequent synthesis of lipoprotein lipase. The effect of Hepes-conditioned medium on lipoprotein lipase resembled to some extent that of the addition of heparin. Therefore, it appears that when Hepes is first added to the culture medium, it might promote a release of heparan sulfate or related compounds, possibly by virtue of its negatively charged sulfonic acid residue. The accumulated heparan sulfate could then promote a sustained release of lipoprotein lipase into the culture medium which in turn leads to increased enzyme synthesis.     

Synthesis and secretion of lipoprotein lipase in 3T3-L1 adipocytes. Demonstration of inactive forms of lipase in cells

3T3-L1 adipocytes in culture incorporated [35S]methionine into a protein which could be immunoprecipitated with chicken antiserum to bovine lipoprotein lipase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed this protein had an Mr of 55,000, similar to that of bovine lipoprotein lipase, and accounted for 0.1-0.5% of total protein synthesis in the adipocytes. Lipoprotein lipase protein was present in small amounts in confluent 3T3-L1 fibroblasts, and the amount increased many-fold as the cells differentiated into adipocytes. This increase was accompanied by parallel increases in cellular lipase activity and secretion. When cells were grown with [35S]methionine, the amount of label incorporated into lipoprotein lipase increased for 2 h and then leveled off. Pulse-chase experiments showed that half-life of newly synthesized lipase was about 1 h. Turnover of lipoprotein lipase in control cells involved both release to the medium and intracellular degradation. When N-linked glycosylation was blocked by tunicamycin, the cells synthesized a form of lipase that had a smaller Mr (48,000), was catalytically inactive, and was not released to the medium. Radioimmunoassay demonstrated that 3T3-L1 adipocytes contained an unexpectedly large amount of lipoprotein lipase protein. 55% of the enzyme protein in acetone/ether powder of the cells was insoluble in 50 mM NH3/NH4Cl at pH 8.1, a solution commonly used to extract lipoprotein lipase; 27% of the lipase protein was soluble but did not bind to heparin-Sepharose and had very low lipase activity; and the remaining 13% was soluble, bound to heparin-Sepharose, and had high lipolytic activity. About one-half of the lipase released spontaneously to the medium was inactive, and lipase inactivation proceeded in the medium with little loss of enzyme protein. Lipoprotein lipase released heparin, in contrast, was fully active and more stable. When protein synthesis was blocked by cycloheximide, the level of lipoprotein lipase activity in adipocytes decreased more rapidly than the amount of lipase protein in the cells. Most of the inactive lipoprotein lipase in adipocytes probably results from dissociation of active dimeric lipase, but some could be a precursor of active enzyme.     

Regulation of lipoprotein lipase synthesis in 3T3-L1 adipocytes by cachectin. Further proof for identity with tumour necrosis factor.

We investigated the mechanism by which the endotoxin-induced macrophage secretory protein cachectin is able to suppress the activity of lipoprotein lipase in 3T3-L1 adipocytes. The loss in activity results from an effect on the synthesis of the enzyme, as determined by a decreased incorporation of [35S]methionine into immunoprecipitable lipoprotein lipase. The results were nearly identical whether crude conditioned medium or a highly purified preparation was utilized as a source of cachectin. [35S]Methionine incorporation into acid-precipitable protein was minimally affected by purified cachectin, suggesting that the suppression of the lipoprotein lipase was not due to a general suppression of protein synthesis. These results, taken together with our previous work, provide additional evidence that cachectin and tumour necrosis factor are functionally identical.     

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)     

Heparin decreases the degradation rate of lipoprotein lipase in adipocytes

The mechanism responsible for the stimulation of secretion of lipoprotein lipase by heparin in cultured cells was studied with avian adipocytes in culture. Immunoprecipitation followed by electrophoresis and fluorography were used to isolate and quantitate the radiolabeled enzyme, whereas total lipoprotein lipase was quantitated by radioimmunoassay. Rates of synthesis of lipoprotein lipase were not different for control or heparin treatments as judged by incorporation of L-[35S]methionine counts into lipoprotein lipase during a 20-min pulse. This observation was corroborated in pulse-chase experiments where the calculation of total lipoprotein lipase synthesis, based on the rate of change in enzyme-specific activity during the chase, showed no difference between control (8.13 +/- 3.1) and heparin treatments (9.1 +/- 5.3 ng/h/60-mm dish). Secretion rates of enzyme were calculated from measurements of the radioactivity of the secreted enzyme and the cellular enzyme-specific activity. Degradation rates were calculated by difference between synthesis and secretion rates of enzyme. In control cells 76% of the synthesized enzyme was degraded. Addition of heparin to the culture medium reduced the degradation rate to 21% of the synthetic rate. The presence of heparin in cell media resulted in a decrease in apparent intracellular retention half-time for secreted enzyme from 160 +/- 44 min to 25 +/- 1 min. The above data demonstrate that the increase in lipoprotein lipase protein secretion, observed upon addition of heparin to cultured adipocytes, is due to a decreased degradation rate with no change in synthetic rate. Finally, newly synthesized lipoprotein lipase in cultured adipocytes is secreted constitutively and there is no evidence that it is stored in an intracellular pool.     

Synthesis of lipoprotein lipase in the liver of newborn rats and localization of the enzyme by immunofluorescence.

In newborn rats, lipoprotein lipase (LPL) activity was higher in the liver than in several other tissues, such as heart, diaphragm or lungs, and accounted for about 3% of total LPL activity in the body. There was no significant correlation between LPL activity in liver and in plasma. Thus transport of the enzyme from extrahepatic tissues was probably not the major source of LPL in liver. To study LPL biosynthesis directly, newborn rats were injected intraperitoneally with [35S]methionine, and LPL was isolated by immunoprecipitation and separation by SDS/polyacrylamide-gel electrophoresis. Radioactivity in LPL increased with a similar time course in all tissues studied, including the liver. Substantial synthesis of LPL was also demonstrated in isolated perfused livers from newborn rats, whereas synthesis was low in livers from adult rats. There was strong LPL immunofluorescence in livers from newborn rats, mainly within sinusoids and along the walls of larger vessels. This labelling disappeared after perfusion with heparin, which indicates that much of the enzyme is in contact with blood and can take part in lipoprotein metabolism.     

Nutritional regulation of lipoprotein lipase in guinea pig tissues

Glucose transport in guinea pig adipocytes has been shown to be markedly resistant to stimulation by insulin. Lipoprotein lipase is another transport catalyst in adipose tissue which is believed to be regulated by insulin. We have therefore studied how feeding-fasting affects lipoprotein lipase activity in guinea pig tissues. There was an even more marked decrease in adipose tissue lipoprotein lipase activity on fasting in guinea pigs (10-20 fold) than in rats or mice (4-5 fold). In adipocytes, the activity decreased only 2.5-4.5 fold; most of the change was in extracellular lipoprotein lipase. On glucose refeeding, the activity was rapidly restored. In the first 4 hours after glucose administration extracellular lipoprotein lipase activity increased to more than 10 times the amount present in adipocytes. After cycloheximide, lipoprotein lipase activity decreased with a half-life of 22 min. It is concluded that lipoprotein lipase is rapidly produced and turned over in guinea pig adipose tissue, and that the system is quite sensitive to feeding-fasting. In contrast to adipose tissue, there was no significant change in lipoprotein lipase activity in any other tissue on fasting. There was a strong correlation between the activities in heart and diaphragm muscle, but this correlation was independent of feeding-fasting.     

Hyperlipoproteinemia type I in a patient with active lipoprotein lipase in adipose tissue and indications of defective transport of the enzyme

This paper presents a case of typical hyperlipoproteinemia type I in a young woman. Her serum triglycerides varied between 2 and 90 mmol/l and she had substantial amounts of apolipoprotein B-48 in fasting plasma. She had no detectable lipoprotein lipase (LPL) activity in post-heparin plasma (less than 0.2 percent of normal). Southern blot analysis suggested no major defect in her LPL gene and Northern blot analysis of adipose tissue RNA showed normal-sized LPL-mRNA. A 2-h [35S]methionine incorporation experiment with adipose tissue pieces in vitro showed that she produced normal-sized LPL and had LPL catalytic activity in the tissue. The amounts were, however, only 5-10% of control. No detectable LPL radioactivity or catalytic activity was released from patient tissue even in the presence of heparin in the incubations. Immunofluorescent staining of adipose tissue biopsies from the patient showed LPL immunoreactivity only in adipocytes and little or none within the capillaries. Treatment of immunoprecipitated labeled LPL with endoglycosidase H showed that the oligosaccharide chains on her enzyme were of the high-mannose type and not processed as in controls. Taken together the data suggest that the patient synthesizes a relatively normal LPL protein which is core-glycosylated and folded into active enzyme as in normal subjects, but is not effectively transported via the Golgi to the cell surface.     

Combined lipase deficiency (cld/cld) in mice. Demonstration that an inactive form of lipoprotein lipase is synthesized

Combined lipase deficiency, cld, is a recessive mutation within the T/t complex of mouse chromosome 17. Mice homozygous for this defect display severe functional deficiencies of lipoprotein lipase and the related hepatic lipase. They develop massive hyperchylomicronemia and die within 3 days when allowed to suckle. Heart, diaphragm muscle, and brown adipose tissue of 1-day-old cld/cld and unaffected mice incorporated in vivo [35S]methionine into a protein that could be immunoprecipitated by antilipoprotein lipase serum. The immunoprecipitated protein in all tissues had the same Mr as bovine lipoprotein lipase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proportion of radioactivity in the lipoprotein lipase band to that in total protein was 0.02% in tissues of cld/cld mice and 0.01% in tissues of unaffected mice. There was 2-6 times more lipoprotein lipase-like protein (determined by immunoassay) in tissues of defective mice than in those of unaffected mice. These findings indicate that the cld mutation did not cause deletion of the structural gene for lipoprotein lipase. Lipoprotein lipase activity in heart, diaphragm muscle, brown adipose tissue, and lung of cld/cld mice was less than 5% of that in tissues of unaffected mice. This low activity could be inhibited more than 85% by antilipoprotein lipase serum, but not by nonimmune serum. It is concluded that tissues in cld/cld mice synthesize a lipoprotein lipase-like protein which has subnormal catalytic activity.     

Regulation of lipoprotein lipase. Induction by insulin.

Lipoprotein lipase activity in intact epididymal adipose tissue of fasted rats increased rapidly after treatment with insulin in vivo. In contrast, lipoprotein lipase activity in adipocytes isolated from the contralateral fat pads remained essentially unchanged. When adipocytes were incubated for 30 min at ambient temperature in vitro, about 2 times more lipoprotein lipase activity was found in the medium of cells from insulin-treated rats than in medium from cells of control animals. Following insulin treatment, extracts of tissue acetone powders separated by gel chromatography showed increases in both enzyme activity fractions obtained (designated lipoprotein lipase a and b). However, no consistent differences were observed between fractions derived from adipocyte acetone powders of insulin-treated and control animals. All the observed effects of insulin on lipoprotein lipase activity were abolished by cycloheximide treatment in vivo. These data indicate that following insulin treatment, increased lipoprotein lipase activity in adipose tissue results from enhanced enzyme secretion by the fat cell and subsequent accumulation in the tissue, thus implicating the adipocyte secretory mechanism as a major site of regulation of lipoprotein lipase activity in adipose tissue.     

Regulation of lipoprotein lipase synthesis and 3T3-L1 adipocyte metabolism by recombinant interleukin 1

When fully differentiated 3T3-L1 adipocytes were exposed to purified, recombinant murine interleukin 1 (rIL-1), a dose-dependent suppression of lipoprotein lipase activity was observed. The loss of activity reached a maximum of 60-70% of control and appeared to be due to an effect on the synthesis of the enzyme as judged by a suppression of the ability to incorporate [35S]methionine into immunoprecipitable lipoprotein lipase. There was no general effect on protein synthesis as determined by radiolabel incorporation into acid precipitable protein; however, after a 17 h exposure of the 3T3-L1 cells to recombinant interleukin 1, the synthesis of two proteins (molecular weights, 19,400 and 165,000 daltons) was enhanced several-fold. When the effect of Il-1 on the major metabolic pathways of the adipocyte was investigated, lipolysis as measured by glycerol release from the cells was markedly enhanced after a 17 h incubation with the hormone, while no effect was observed on de novo fatty acid synthesis. These effects on the metabolism of the adipocytes occur at concentration on a basis of molecules per cell, similar (only a 3-fold difference) to those required for stimulation of [3H]thymidine incorporation into mouse thymocyte DNA, suggesting that IL-1 may be a physiologically significant effector of adipocyte metabolism.     

Intra- and extracellular forms of lipoprotein lipase in adipose tissue.

The location of lipoprotein lipase activity in rat adipose tissue was studied using intact epididymal fat pads, isolated adipocytes, and lipoprotein lipase activity secreted from adipocytes as enzyme sources. The enzyme activities of these preparations were characterized by gel filtration. The method used for isolation of adipocytes had been modified to minimize activation of lipoprotein lipase during the procedures. Extracts of intact adipose tissue separated into two major lipoprotein lipase activity peaks, designated "a" and "b", the "a" fraction representing about 30 (fasted rats) to 50% (fed rats) of the total enzyme activity. An intermediate fraction (designated "i") was frequently observed. Extracts of isolated adipocytes from fed rats contained about 35% and those from fasted rats about 65% of the lipoprotein lipase activity present in intact tissue. The "b" fraction constituted 80--97% of the adipocyte lipoprotein lipase activity. In contrast, the enzyme activity secreted from the adipocytes contained only the "a" and "i" fractions. These data implicate the existance of one intracellular form of lipoprotein lipase (corresponding to the "b" fraction), different from extracellular forms of the enzyme (corresponding to fractions "a" and "i"). A transformation of the intracellular to the extracellular forms appears to occur in conjunction with secretion of enzyme from the fat cell.     

Influence of age and exercise training on lipid metabolism in Fischer-344 rats

The influence of training on fatty acid and glyceride synthesis by liver and adipose tissue homogenates of young and old Fischer-344 rats was examined. Four groups of rats (10 animals/group) were studied: young untrained, young trained, old untrained, and old trained. Training of each group was for 10 wk at 75% maximal O2 uptake. Young rats were killed at 6 mo of age and old rats were killed at 27 mo of age. Fatty acid synthesis was assessed by measuring the activities of acetyl-CoA carboxylase, fatty acid synthase, ATP citrate-lyase, "malic" enzyme, and glucose-6-phosphate dehydrogenase. Glyceride synthesis was evaluated by determining the rate of incorporation of [14C]glycerol 3-phosphate into lipids. In addition, lipoprotein lipase activity was measured in acetone-ether powders of adipose tissue from the four groups of rats. In liver, training had no effect on fatty acid or glyceride synthesis in either group. However, aging caused a significant decrease in the activities of four of the lipogenic enzymes but had no effect on glyceride synthesis. Training caused an increase in fatty acid synthase and glyceride synthesis in adipose tissue, and aging decreased lipoprotein lipase activity. It was concluded that training enhances the synthetic capacity of lipids by adipose tissue but that aging had a more profound effect in that the activities of the enzymes involved in these processes were lower in the old rats. Furthermore, the decreased activity of lipoprotein lipase in the older rats may explain the higher plasma triglyceride levels that were observed in these animals.     

Changes with starvation in the rat of the lipoprotein lipase activity and hydrolysis of triacylglycerols from triacylglycerol-rich lipoproteins in adipose tissue preparations.

Lipoprotein lipase activity was higher in fat-pad pieces than in isolated adipocytes from the same fed rats, whereas hydrolysis of triacylglycerols from triacylglycerol-rich lipoproteins was similar in the two preparations when incubated either in basal conditions or in the presence of heparin. In both preparations there was a similar release of lipoprotein lipase activity into the medium during basal incubation, enhanced by the presence of heparin. In fat-pad pieces, but not in isolated adipocytes, incubation with heparin produced a decrease in the lipoprotein lipase activity measured in the tissue preparation. In fat-pad pieces from 24 h-starved rats, lipoprotein lipase activity was the same as in isolated adipocytes from the same animals and incubation with heparin did not affect the appearance of lipoprotein lipase in the medium or the utilization of triacylglycerols from triacylglycerol-rich lipoproteins. These results support the following conclusions. (1) The effectiveness of lipoprotein lipase in adipose tissue preparations in vitro depends more on its availability to the substrate than on its total activity. (2) Heparin acts on adipose tissue preparations from fed animals both by enhancing the release of pre-existing extracellular enzyme (which is absent in isolated adipocytes) and by enhancing the transfer outside the cells of the intracellular (and mainly undetectable) enzyme that is activated in the secretion process. (3) In adipose tissue from starved animals there is not only a decrease in the active extracellular form of lipoprotein lipase activity but also a reduction in the intracellular (and mainly undetectable) pool of the enzyme.