Adipocyte Lipoprotein Lipase Research Articles

Antibodies against amino acids 1-15 of tumor necrosis factor block its binding to cell-surface receptor.

Human tumor necrosis factor (hTNF) mediates a variety of biologic activities, which are dependent on the attachment of hTNF to cell-surface receptors. To identify regions of the hTNF protein involved in binding hTNF to its receptor, we prepared five synthetic peptides [hTNF-(1-15), hTNF-(1-31), hTNF-(65-79), hTNF-(98-111), and hTNF-(124-141)] and two hydroxylamine cleavage fragments [hTNF-(1-39) and hTNF-(40-157)] of hTNF. The hTNF-synthetic peptides and hTNF fragments were tested in hTNF receptor binding assays and in two biologic assays: cytolysis of tumor cells and suppression of lipoprotein lipase in adipocytes. Neither the synthetic peptides nor hTNF fragments were active agonists or antagonists in these assays. The synthetic peptides were also conjugated to thyroglobulin, and peptide-specific antisera were raised. All five peptide-thyroglobulin conjugates induced antibody responses to the immunizing peptide and to hTNF. Each antiserum was tested for antagonist activity in hTNF binding assays. Only antisera raised against hTNF-(1-15) or hTNF-(1-31) and antisera against whole hTNF blocked binding. IgGs purified from these three antisera also block hTNF-induced cytolysis and lipoprotein lipase suppression. We conclude that antibodies that recognize the N-terminus of hTNF block the attachment of hTNF to its cellular receptor and inhibit the biologic effects of hTNF.

Mechanisms for turnover of lipoprotein lipase in guinea pig adipocytes

Guinea-pig adipocytes released lipoprotein lipase activity to the medium without depletion of cellassociated lipoprotein lipase activity. Heparin caused immediate release of 20–25% of the lipase activity to the medium, and also enhanced the continued release. After addition of cycloheximide, cell-associated lipoprotein lipase activity decreased rapidly. Release of lipase activity to the medium continued unabated for about 30 min, but there was little release thereafter. The release accounted for only about 25% of the initial lipoprotein lipase activity in the absence and about 50% in the presence of heparin. In pulse-chase experiments with [ 35S]methionine, labeled lipoprotein lipase appeared in the medium within 40 min, and most of the release occurred during the first h of chase. In a 4-h chase the total (cells + medium) amount of labeled lipase decreased to 34%. Thus, degradation was a main fate of the lipase. Heparin markedly increased the amount of labeled lipase that was released to the medium and decreased the amount that was degraded. Heparin did not change the time-course for the release, and the amount of labeled lipase degraded was proportional to the amount not released to the medium, indicating that the effect of heparin was primarily on release, not on degradation as such. This study demonstrates that adipocytes synthesize lipoprotein lipase in excess of what is being released, and that the excess is rapidly degraded.

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.

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.

Environmental control of energy metabolism in rats

Food availability was arranged so that episodes of feeding were separated by long periods of deprivation. Distinctly different contexts were associated with each condition. For other animals, relatively short periods of deprivation stood in contrast to relatively long opportunities to free-feed, and were also embedded in widely differing physical contexts. These temporal relationships among the conditions were adjusted to sharpen the saliency of each metabolic condition and thereby enhance its associability with the distinctive environment in which each occurred. To avoid the possibility of circadian entrainment, the feeding or deprivation episodes occurred at unpredictable times according to a variable-time schedule. Following several training cycles and an extended period of free-feeding in a neutral environment, the animals were reexposed to the various contexts and sacrificed for metabolite assays. The environment predictive of feeding elevated adipocyte lipoprotein lipase activity and lowered the levels of serum free fatty acids. The deprivation context boosted serum triglycerides and blood urea nitrogen. The conditioned responses were all of a compensatory nature: Feeding cues resulted in accelerated caloric deposition, deprivation cues elicited conditioned mobilization of stored energy. While protein catabolism and several indices of fat metabolism appear to be conditionable, no evidence of environmental control of glycemic responses was observed.

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.

Metabolism of mammary, abdominal, and femoral adipocytes in women before and after menopause

In 23 women before and 11 after menopause, adipocyte size, lipoprotein lipase activity, and lipolytic responsiveness to norepinephrine were compared in different regions of adipose tissue. In premenopausal women femoral adipocytes were characterized by a higher lipoprotein lipase activity than abdominal or mammary adipocytes. On the other hand, lipolytic responsiveness and sensitivity in the latter two was higher than in femoral tissue. In postmenopausal women no differences in lipoprotein lipase or lipolysis were found among the three regions. Consequently, menopause in women seemed to be associated with a diminution of not only the increased lipoprotein lipase activity of femoral adipocytes, but also of the high lipolytic response in abdominal and mammary adipose tissue. It is therefore suggested that female sex steroid hormones exert regionally specific effects, ie, increasing lipoprotein lipase in femoral adipocytes, which therefore become enlarged. It also seems possible that stimulation of lipolysis in abdominal and mammary adipocytes is an effect of sex steroid hormones. From the results obtained it is hypothesized that the secondary sex characteristics of adipose tissue distribution in women might be caused by regionally specific effects of sex steroid hormones on adipocyte metabolism.

Inhibition of adipocyte and whole-tissue lipoprotein lipase by rat high-density lipoprotein

Inhibition of adipocyte and whole-tissue lipoprotein lipase by rat high-density lipoprotein

Regulation of lipoprotein lipase in primary cultures of isolated human adipocytes.

To study the regulation of adipose tissue lipoprotein lipase (LPL) in human adipocytes, omental adipose tissue was obtained from healthy subjects and digested in collagenase. The isolated adipocytes thus obtained were suspended in Medium 199 and cultured at 37 degrees C. Cell viability was demonstrated in adipocytes cultured for up to 72 h by constancy of cell number, cell size, trypan-blue exclusion, and specific 125I-insulin binding. In addition, chloroquine induced an increase in cell-associated 125I-insulin at 24, 48, and 72 h after preparation. Thus, isolated adipocytes retained their ability to bind, internalize, and degrade insulin. LPL was measured as activity secreted into the culture medium (CM), released from cells by heparin (HR), and extracted from cell digests. A broad range of heparin concentrations produced a prompt release of LPL from a rapidly replenishable pool of cellular activity. When cells were cultured in medium containing 10% fetal bovine serum, there was a marked stimulation of CM and HR. The secretory response to serum (CM) correlated strongly with HR 24 h after preparation (rs = 0.731, P less than 0.001). In addition, HR was found to correlate logarithmically and inversely with body mass index (r = -0.731, P less than 0.001). Insulin, at 400 ng/ml only, increased HR by 36 +/- 10%, an effect simulated by lower concentrations of insulin-like growth factor-1 (IGF1). Thus, LPL is produced and regulated in isolated human adipocytes. The degree of adiposity and serum are important regulators of HR activity, whereas insulin is stimulatory only at a pharmacologic concentration. This effect of insulin may be mediated through the IGF1 receptor. Isolated human adipocytes represent a novel and useful system for the study of LPL and lipid metabolism as well as for other aspects of adipocyte biology.

Calcium messenger system: an integrated view.

Calcium messenger system: an integrated view.

Insulin regulation of lipoprotein lipase in cultured isolated rat adipocytes.

The cellular regulation of adipose tissue lipoprotein lipase by insulin was investigated using cultured isolated rat adipocytes. Evidence for sustained cell viability over 3 days included stability of specific [125I]insulin binding and adipocyte number. Lipoprotein lipase was measured in three functional compartments: 1) enzyme activity secreted into the culture medium, 2) activity releasable from cell suspensions by heparin, and 3) activity extractable from cells (after maximal heparin release) in deoxycholate and detergent. One day after preparation, these activities stabilized and were 1.3 +/- 0.2, 1.4 +/- 0.2, and 7.7 +/- 0.9 neq/10(6) cells X min, respectively (n = 24, mean +/- SEM). Insulin, added the day after preparation, produced a dose-dependent (1-400 ng/ml) increase in lipoprotein lipase releasable from cells by heparin at 2, 4, and 24 h. Insulin also increased intracellular enzyme measured as deoxycholate-detergent-solubilized activity extracted from previously heparin-released cells. However, insulin-mediated increases in culture medium enzyme only occurred subsequent to cellular effects. All insulin-mediated effects were prevented by cycloheximide (1 microgram/ml). Thus, insulin increased two cellular pools of adipocyte lipoprotein lipase in a dose-dependent manner, but had no direct effect on enzyme secretion. Overall, cultured isolated rat adipocytes appear to be a valuable system for the study of lipoprotein lipase regulation at the level of the adipocyte.

Influence of nutritional state on lipoprotein lipase activities in the hypothyroid rat

We have investigated the effects of nutritional state on the lipoprotein lipase activities of the experimentally hypothyroid rat. Both short-term effects (i.e., those of a 24 h fast with and without re-feeding) and long-term effects (due to decreased food intake in hypothyroidism) have been studied. The hypothyroid rats had significantly higher lipoprotein lipase activities of adipose tissue and heart muscle. The effect of hypothyroidism on adipose tissue lipoprotein lipase activities was modified by the nutritional state. In rats studied after 24 h fasting, the hypothyroid group had significantly higher lipoprotein lipase activities than weight-matched, age-matched and pair-fed (i.e., semi-starved) control groups. In rats studied in the re-fed state, the effects of hypothyroidism as such were less evident, since the pair-fed group also demonstrated significantly higher enzyme activities than did the other control groups. We have also studied the lipoprotein lipase activities of different enzyme preparations from adipose tissue. The effects of hypothyroidism were most clearly reflected in an increase of heparin-elutable enzyme activity from adipose tissue, whereas adipocyte lipoprotein lipase activity and the lipoprotein lipase secretion rate from adipocytes were affected to a lesser extent. We conclude that alterations in food intake strongly influence the lipoprotein lipase activities in the hypothyroidism. Our data also imply that the increased lipoprotein lipase activity in the hypothyroid state is due to a decreased degradation of the enzyme, both intra- and extracellularly.

Rat adipocyte lipoprotein lipase activity is inhibited by theophylline and stimulated by inosine through adenosine-independent mechanisms

Incubation of rat adipose tissue or isolated rat adipocytes with high (50 mM) but not with low concentrations (0.5 mM) of theophylline results in a decrease of lipoprotein lipase (LPL) activity. This effect is not altered by the addition of adenosine deaminase, indicating that the decrease of adipose LPL activity by theophylline is not due to the competition of theophylline with adenosine. On the contrary, incubation of isolated fat cells with adenosine (0.1 – 100 μM) results in an increase of the intracellular form of LPL activity. As this effect is also observed in cells incubated with adenosine deaminase (40 mU/ml) or with inosine (0.1 – 100 μM) but not in cells incubated with the adenosine analog N 6-phenylisopropyladenosine, it is concluded that the increase in the intracellular form of LPL found after incubation with adenosine is not due to adenosine per se but to inosine generated from the breakdown of endogenous adenosine by adenosine deaminase.

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.

Interrelationship of cyclic AMP, lipolysis, and respiration in brown fat cells.

ARTICLESInterrelationship of cyclic AMP, lipolysis, and respiration in brown fat cellsJN Fain, MD Jacobs, and YC Clement-CormierJN Fain, MD Jacobs, and YC Clement-CormierPublished Online:01 Feb 1973https://doi.org/10.1152/ajplegacy.1973.224.2.346MoreSectionsPDF (1 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByNeed for Ca ions in the lipolytic action of 5-hydroxytryptamine in rat brown adipose tissue12 April 2011 | Journal of Pharmacy and Pharmacology, Vol. 30, No. 1Brown Adipose Tissue: Function and Physiological SignificanceBARBARA CANNON, and JAN NEDERGAARD1 January 2004 | Physiological Reviews, Vol. 84, No. 1Effects of cold exposure on cyclic AMP concentration in plasma, liver, and brown and white adipose tissues in cold-acclimated ratsInternational Journal of Biometeorology, Vol. 33, No. 2Temperature Regulation5-HT Receptors Coupled to Adenylate CyclaseBiochemical Mechanisms of ThermogenesisBrown adipose tissue: molecular mechanisms controlling activity and thermogenesisQuantitative differentiation of α- and β-adrenergic respiratory responses in isolated hamster brown fat cells: Evidence for the presence of an α1-adrenergic componentEuropean Journal of Pharmacology, Vol. 93, No. 3-4Cellular Mechanisms of Heat DissipationThyroid Hormones and ThermogenesisRat adipocyte lipoprotein lipase activity is inhibited by theophylline and stimulated by inosine through adenosine-independent mechanismsBiochemical and Biophysical Research Communications, Vol. 107, No. 1Regulation of Lipid Metabolism by Cyclic NucleotidesThe Brown Fat CellInfluence of Cardiac Glycosides on Myocardial Energy MetabolismLipid biochemistry in the cold-acclimated ratComparative Biochemistry and Physiology Part B: Comparative Biochemistry, Vol. 70, No. 4Alpha1-adrenergic activation of phosphatidylinositol labeling in isolated brown fat cellsBiochemical Pharmacology, Vol. 29, No. 24Hormonal Regulation of Lipid Mobilization from Adipose TissueAdrenergic- and cyclic nucleotide-induced glycerol release from brown adipocytesEuropean Journal of Pharmacology, Vol. 56, No. 1-2Effects of colchicine and trimethoxybenzene on glucose oxidation in isolated brown fat cells from ratsGeneral Pharmacology: The Vascular System, Vol. 10, No. 1EFFECTS OF CHLORPROMAZINE ON LIPOLYSIS IN BROWN AND WHITE ADIPOSE TISSUES OF COLD-EXPOSED RATSJapanese Journal of Pharmacology, Vol. 29, No. 4beta-Adrenergic Receptors in Brown-Adipose Tissue. Characterization and Alteratios during Acclimation of Rats to ColdEuropean Journal of Biochemistry, Vol. 92, No. 1Comparison of isoproterenol, salbutamol and talzolol as lipolytic agents with isolated rodent adipocytesBiochemical Pharmacology, Vol. 27, No. 14Effects of Lipolytic and Antilipolytic Drugs on Metabolism of Adenosine 3': 5'-Monophosphate in Brown Adipose Tissue of Cold Acclimated RatsEuropean Journal of Biochemistry, Vol. 69, No. 1Brown adipose tissue mitochondriaBiochimica et Biophysica Acta (BBA) - Reviews on Bioenergetics, Vol. 416, No. 16 Methods Related to Cyclic AMP and Adenylate CyclaseControl of cardiac sarcolemmal adenylate cyclase and sodium, potassium-activated adenosinetriphosphatase activities.Circulation Research, Vol. 36, No. 1Lack of correlation between cyclic amp synthesis and free fatty acid release in brown fat of cold-adapted ratsBiochemical Pharmacology, Vol. 23, No. 20Control of sn-glycerql 3-phosphate oxidation in brown adipose tissue mitochondria by calcium and ACYL-CoABiochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, Vol. 348, No. 1Cyclic AMP metabolism in brown adipocytes of hamsters exposed to different temperaturesBiochimica et Biophysica Acta (BBA) - General Subjects, Vol. 338, No. 2 More from this issue > Volume 224Issue 2February 1973Pages 346-351 Copyright & PermissionsCopyright © 1973 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1973.224.2.346PubMed4346846History Published online 1 February 1973 Published in print 1 February 1973 Metrics

The mechanism of heparin stimulation of rat adipocyte lipoprotein lipase

Free fat cells and stromal-vascular cells were prepared from rat adipose tissue by incubation with collagenase. NH(4)OH-NH(4)Cl extracts of acetone-ether powders prepared from fat cells contained lipoprotein lipase activity but extracts of stromal-vascular cells did not. Intact fat cells released lipoprotein lipase activity into incubation medium, but intact stromal-vascular cells did not. The lipoprotein lipase activity of the medium was increased when fat cells were incubated with heparin, and this was accompanied by a corresponding decrease in the activity of subsequently prepared fat cell extracts. Heparin did not release lipoprotein lipase activity from stromal-vascular cells. The lipoprotein lipase activity of NH(4)OH-NH(4)Cl extracts of fat cell acetone powders is increased by the presence of heparin during the assay. This increase is not due to preservation of enzyme activity, but to increased binding of lipoprotein lipase to chylomicrons. Protamine sulfate and sodium chloride have little effect on the binding of lipoprotein lipase to chylomicrons, but they inhibit enzyme activity after binding to substrate has occurred. These inhibitors do, however, inhibit the stimulatory effect of heparin on enzyme-substrate binding.