Research Article| Volume 33, ISSUE 5, P669-678, May 1992

Download started.


Glycerolipid synthesis in isolated adipocytes: substrate dependence and influence of norepinephrine.

Open AccessPublished:May 01, 1992DOI:
      This paper is only available as a PDF. To read, Please Download here.
      Studies of fatty acid (FA) esterification by adipocytes have led to conflicting views with respect to how the process is regulated by norepinephrine (NE). It remains unclear whether NE directly modulates the pathway or whether its effects are indirect and reflect its well-known action to activate lipolysis. Changes in lipolysis can complicate estimation of esterification rates by altering both medium FA and the hydrolysis of newly formed FA esters. In this report, we describe an experimental approach that determined the effect of NE on FA esterification, amidst the complications introduced by activation of lipolysis. Esterification rates were estimated from the simultaneous incorporations (0.1-60 min) of [14C]glucose and [3H]oleate into diglyceride (DG), phospholipid (PL), and triglyceride (TG). Saturation kinetics of incorporation rates, with respect to FA, and more specifically to unbound or albumin-free FA (ubFA), were determined in both basal and NE-treated cells. To obtain true estimates of ester synthesis, incorporation rates were adjusted for label loss from breakdown of labeled esters. Our findings were: 1) In basal versus NE-treated cells, [3H]oleate, on its pathway to esterification, was diluted, respectively, by 2 and 50% of measured cell FA, and the diluting FA appeared derived from lipolysis. 2) Syntheses of PL, DG, and TG, estimated from incorporation of [14C]glucose, saturated at low ubFA. The Km for TG synthesis (0.06 microM) was within the physiological range of ubFA which meant that changes in plasma FA will modulate TG synthesis. PL synthesis, on the other hand (Km less than 0.01 microM), would be largely saturated under physiological conditions. 3) NE treatment increased the molar ratio of FA to albumin in the medium an average 8-fold and ubFA about 87-fold. In addition, NE accelerated hydrolysis of labeled PL and DG. Adjusting incorporation rates for these changes indicated that NE does not directly regulate glyceride synthesis. The assays described should allow estimation of glycerolipid synthesis under various metabolic or disease states and will distinguish direct effects from those reflecting changes in FA concentration or in hydrolysis of labeled FA esters.


        • Hubbard R.E.
        • Garrat C.J.
        The composition and fluidity of adipocyte membranes prepared from young and adult rats.
        Biochim. Biophys. Acta. 1980; 600: 701-704
        • Berridge M.J.
        Inositol triphosphate and diacyl-glycerol as second messengers.
        Biochem. J. 1984; 220: 345-360
        • Gorin E.
        • Shafrir E.
        Turnover of adipose tissue triglycerides measured by the rate of synthesis and release of triglyceride-glycerol.
        Biochim. Biophys. Acta. 1963; 70: 109-117
        • Grahn M.F.
        • Davies J.I.
        Lipolytic agents as regulators of fatty acid esterification in rat adipose tissue.
        Biochem. Soc. Trans. 1980; 8: 362-363
        • Efendic S.
        • Ostman J.
        Catecholamines and metabolism of adipose tissue. V. Studies on the incorporation of glucose 1-14C into lipids and re-esterification of FFA by human omental tissue in vitro.
        Acta Med. Scand. 1970; 187: 493-502
        • Cahill G.F.
        • Leboeuf B.
        • Flinn R.B.
        Studies on rat adipose tissue in vitro. VI. Effects of epinephrine on glucose metabolism.
        J. Biol. Chem. 1960; 235: 1250-1264
        • Zinder O.
        • Eisenberg E.
        • Shapiro B.
        Compart-mentation of glycerides in adipose tissue cells. I. The mechanism of free fatty acid release.
        J. Biol. Chem. 1973; 248: 7673-7676
        • Winand J.
        • Furnelle J.
        • Wodon C.
        • Christophe J.
        Spectrum of fatty acids synthesized in situ and metabolic heterogeneity of free fatty acids and glycerides within isolated rat adipocytes.
        Biochim. Biophys. Ada. 1971; 239: 142-153
        • Edens N.K.
        • Leibel R.L.
        • Hirsh J.
        Lipolytic effects on diacylglycerol accumulation in human adipose tissue in vitro.
        J. Lipid Res. 1990; 31: 1351-1359
        • Spector A.A.
        • Steinberg D.
        • Tanaka A.
        Uptake of free fatty acids by rlich ascites tumor cells.
        J. Biol. Chem. 1965; 240: 1032-1041
        • Abumrad N.A.
        • Perkins R.C.
        • Park J.H.
        • Park C.R.
        Mechanism of long chain fatty acid permeation in the isolated adipocyte.
        J. Biol. Chem. 1981; 256: 9183-9191
        • Sorrentino D.
        • Robinson R.B.
        • Kiang C.L.
        • Berk P.D.
        At physiologic albumin/oleate concentrations, oleate uptake by isolated hepatocytes, cardiac myocytes and adipocytes is a saturable function of the unbound oleate concentration.
        J. Clin. Invest. 1989; 84: 1225-1233
        • Rodbell M.
        Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis.
        J. Biol. Chem. 1964; 239: 375-384
        • Abumrad N.A.
        • Park J.H.
        • Park C.R.
        Permeation of long chain fatty acids into adipocytes: kinetics, specificity, and evidence for involvement of a membrane protein.
        J. Biol. Chem. 1984; 259: 8945-8953
        • Harris W.D.
        • Popat P.
        Determination of the phosphorus content of lipids.
        J. Am. Oil Chem. Soc. 1954; 31: 124-127
        • Preiss J.
        • Loomis C.R.
        • Bishop W.R.
        • Stein R.
        • Niedel J.E.
        • Bell R.M.
        Quantitative measurements of sn-l,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells.
        J. Biol. Chem. 1986; 261: 8597-8600
        • Wright T.M.
        • Rangan L.A.
        • Shin H.S.
        • Rabin D.M.
        Kinetic analysis of 1,2-diacylglycerol mass levels in culture fibroblasts. Comparison of stimulation by α thrombin and epidermal growth factor.
        J. Biol. Chem. 1988; 263: 9374-9380
        • Ho R.J.
        • Meng H.C.
        A simple and ultrasensitive method for determination of free fatty acids by radiochemical assay.
        Anal. Biochem. 1969; 31: 426-436
        • Dole V.P.
        Insulin-like action of ribonucleic acid, adenylic acid and adenosine.
        J. Biol. Chem. 1964; 237: 2758-2762
        • Abumrad N.A.
        • Perry P.K.
        • Whitesell R.R.
        Stimulation by epinephrine of the membrane transport of long chain fatty acids in the adipocyte.
        J. Biol. Chem. 1985; 260: 9969-9971
        • Abumrad N.A.
        • Park C.R.
        • Whitesell R.R.
        Catecholamine activation of the membrane transport of long chain fatty acids in adipocytes is mediated by cyclic AMP and protein kinase.
        J. Biol. Chem. 1986; 261: 13082-13086
        • Leibel R.L.
        • Hirsh J.
        A radioisotopic technique for analysis of free fatty acid reesterification in human adipose tissue.
        Am. J. Physiol. 1985; 248: E140-E147
        • Scheider W.
        Dissociation rate of serum albumin-fatty acid complex from stop-flow dielectric study of ligand exchange.
        Biophys. J. 1978; 24: 260-262
        • Weisiger R.A.
        • Ma W.L.
        Uptake of oleate from albumin solutions by rat liver. Failure to detect catalysis of oleate from albumin by an albumin receptor.
        J. Clin. Invest. 1987; 79: 1070-1077
        • Edens N.K.
        • Leibel R.L.
        • Hirsh J.
        Mechanism of free fatty acid re-esterification in human adipocytes in vitro.
        J. Lipid Res. 1990; 31: 1423-1431
        • Mohell N.
        • Wallace M.
        • Fain J.N.
        Alpha 1-adrenergic stimulation of phosphotidylinositol turnover and respiration of brown fat cells.
        Mol. Cell. Pharmacol. 1984; 25: 64-69
        • Elam M.B.
        • Simkevich C.P.
        • Solomon S.S.
        • Wilcox H.G.
        • Heimberg M.
        Stimulation of in vitro triglyceride synthesis in the rat hepatocyte by growth hormone treatment in vivo.
        Endocrinology. 1988; 122: 1397-1402
        • Rodbell M.
        Modulation of lipolysis in adipose tissue by fatty acid concentration in fat cell.
        Ann. NY Acad. Sci. 1965; 131: 302-314
        • Mukerjee P.
        Dimerization of anions of long-chain fatty acids in aqueous solutions and the hydrophobic properties of the acids.
        J. Phys. Chem. 1965; 69: 2821-2827
        • Simpson R.B.
        • Ashbrook J.D.
        • Santos E.C.
        • Spector A.A.
        Partition of fatty acids.
        J. Lipid Res. 1974; 15: 415-422
        • Parfitt G.D
        • Smith A.L.
        Conductivity of sodium dodecyl sulfate solutions below the critical micelle concentration.
        J. Phys. Chem. 1962; 66: 942-943
        • Augert G.
        • Exton J.H.
        Insulin and oxytocin effects on phosphoinositide metabolism in adipocytes.
        J. Biol. Chem. 1988; 263: 3600-3609
        • Farese R.W.
        • Barnes D.E.
        • Davis J.S.
        • Standaert M.L.
        • Pollet R.J.
        Effects of insulin and protein synthesis inhibitors on phospholipid metabolism, diacylglycerol levels, and pyruvate dehydrogenase activity in BC3H-1 cultured myocytes.
        J. Biol. Chem. 1984; 259: 7095-7100
        • Honeyman T.W
        • Strohsnitter W.
        • Scheid C.R.
        • Schimmel R.J.
        Phosphatidic acid and phosphatidylinositol labelling in adipose tissue. Relationship to the metabolic effects of insulin and insulin-like agents.
        Biochem. J. 1983; 212: 489-498
        • Spector A.A.
        • John K.
        • Fletcher J.E.
        Binding of long-chain fatty acids to bovine serum albumin.
        J. Lipid Res. 1969; 10: 56-67