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3-hydroxy-3-methylglutaryl CoA reductase in cultured hepatocytes

Regulation by Heterologous Lipoproteins and Hormones
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      Abstract

      Regulation of the key enzyme of cholesterol synthesis, 3-hydroxy-3-methylglutaryl CoA reductase (EC: 1.1.1.34), by heterologous human lipoproteins and hormones was studied in a maintenance culture of rat hepatocytes. The liver cells were cultured under hormone and serum free conditions and maintained differentiated morphology and specific function.
      Under control conditions total HMG-CoA reductase increased by 50% after 24 h culture compared to 0 h values immediately after isolation. Thereafter a plateau of enzyme activity was reached lasting until 48 h, with a slight decline at 72 h. Concomitantly the “expressed” enzyme activity increased steadily, probably through dephosphorylation of latent reductase, the activation was largely complete at 48 h.
      During the steady state period of total reductase VLDL added to the medium at concentrations up to 50 μg/ml protein had no effect on HMG-CoA reductase activity. In contrast, LDL suppressed the enzyme in a dose-dependent fashion to 40% of controls at 100 μg/ml. On the other hand, HDL had the opposite effect with a significant induction up to 252% of controls at 50 μg/ml. Insulin also caused a comparable dose-dependent stimulation of enzyme activity at 10−8 and 10−7 M, whereas glucagon inhibited reductase activity. Compared to the insulin action, triiodothyronine and triamcinolone prompted a minor, but still significant increase of reductase activity. Insulin and triamcinolone acted synergistically, but the combination of triamcinolone and triiodothyronine was only additive. All hormonal inductions of reductase could be blocked by cycloheximide.
      The present data establish that HMG-CoA reductase of maintenance cultured hepatocytes is subject to a complex regulation by heterologous lipoproteins as well as pancreatic, adrenal and thyroid hormones.

      Keywords

      Abbreviations:

      HMG-CoA reductase: 3-hydroxy-3-methylglutaryl CoA reductase (), VLDL: Very low density lipoproteins (d: < 1.006 g/ml) (), LDL: Low density lipoproteins (d: 1.019-1.063 g/ml) (), HDL: High density lipoproteins (d: 1.063–1.210 g/ml) ()
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      References

        • Dietschy J.M.
        • Wilson J.D.
        Regulation of cholesterol metabolism.
        N. Engl. J. Med. 1970; 282: 1128
        • Siperstein D.
        • Fagan V.M.
        Feedback control of mevalonate synthesis by dietary cholesterol.
        J. Biol. Chem. 1966; 241: 602
        • Shapiro D.J.
        • Rodwell V.W.
        Regulation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol synthesis.
        J. Biol. Chem. 1971; 246: 3210
        • Redgrave T.G.
        Formation of cholesteryl ester-rich particulate lipid during metabolism of chylomicrons.
        J. Clin. Invest. 1970; 49: 465
        • Cooper A.D.
        • Yu P.Y.S.
        Rates of removal and degradation of chylomicron remnants by isolated perfused rat liver.
        J. Lipid Res. 1978; 19: 635
        • Carrella M.
        • Cooper A.D.
        High affinity binding of chylomicron remnants to rat liver plasma membranes.
        in: 6th edition. Proc. Nat. Acad. Sci. (USA). 76. 1979: 338
        • Sherill B.C.
        Rapid hepatic clearance of the canine lipoprotein containing only the E apoprotein by a high affinity receptor — Identity with the chylomicron remnant transport process.
        J. Biol. Chem. 1980; 255: 1804
        • Nervi F.L.
        • Weis H.J.
        • Dietschy J.M.
        The kinetic characteristics of inhibition of hepatic cholesterogenesis by lipoproteins of intestinal origin.
        J. Biol. Chem. 1975; 250: 4145
        • Nervi F.L.
        • Dietschy J.M.
        Ability of six different lipoprotein fractions to regulate the rate of hepatic cholesterogenesis in vivo.
        J. Biol. Chem. 1975; 250: 8704
        • Andersen J.M.
        • Turley S.D.
        • Dietschy J.M.
        Low and high density lipoproteins and chylomicrons as regulators of rate of cholesterol synthesis of rat liver in vivo.
        in: 6th edition. Proc. Nat. Acad. Sci. (U.S.A.). 76. 1979: 165
        • Eisenberg S.
        • Rachmilewitz D.
        Metabolism of rat plasma very low density lipoprotein, Part 1 (Fate in circulation of the whole lipoprotein).
        Biochim. Biophys. Acta. 1973; 326: 378
        • Stange E.
        • Alavi M.
        • Papenberg J.
        Changes in metabolic properties of rabbit very low density lipoproteins by dietary cholesterol and saturated and polyunsaturated fats.
        Atherosclerosis. 1977; 28: 1
        • Jakoi L.
        • Quarfordt S.H.
        Alterations of rat hepatic cholesterogenesis by heterologous lipoproteins.
        J. Biol. Chem. 1977; 252: 6856
        • Edwards P.A.
        Effect of plasma lipoproteins and lecithin-cholesterol dispersions on the activity of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase of isolated rat hepatocytes.
        Biochim. Biophys. Acts. 1975; 409: 39
        • Calandra S.
        • Tarugi P.
        • Battistini N.
        • Ferrari R.
        Cholesterol synthesis in isolated rat hepatocytes — Effect of homologous and heterologous serum lipoproteins.
        Metabolism. 1979; 28: 843
        • Breslow J.L.
        • Lothrop D.A.
        • Alexander W.C.
        • Lux S.E.
        • Luxoprotein regulation of 3-hydroxy3-methylglutaryl coenzyme A reductase activity in rat liver cell cultures
        J. Biol. Chem. 1977; 252: 2726
        • Breslow J.L.
        • Spaulding D.R.
        • Lothrop D.A.
        • Clowes A.W.
        Effect of lipoprotein on 3-hydroxy3-rnethylglutaryl coenzyme A (HMG-CoA) reductase activity in rat liver cell culture — special suppressant effect of a lipoprotein isolated from hypercholesterolemic rat plasma.
        Biochem. Biophys. Res. Comm. 1975; 67: 119
        • Lakshmanan M.R.
        • Nepokroeff C.M.
        • Ness G.C.
        • Dugan R.E.
        • Porter J.W.
        Stimulation by insulin of rat liver β-hydroxy-β-methylglutaryl coenzyme A reductase and cholesterol-synthesizing activities.
        Biochem. Biophys. Res. Comm. 1973; 50: 704
        • Nepokroeff C.M.
        • Lakshmanan M.R.
        • Ness G.C.
        • Dugan R.E.
        • Porter J.W.
        Regulation of the diurnal rhythm of rat liver β-hydroxy-β-methylglutaryl coenzyme A reductase activity by insulin, glucagon, cyclic AMP and hydrocortisone.
        Arch. Biochem. Biophys. 1974; 160: 387
        • Dugan R.E.
        • Ness G.C.
        • Lakshmanan M.R.
        • Nepokroeff C.M.
        • Porter J.W.
        Regulation of hepatic β-hydroxy-β-methylglutaryl coenzyme A reductase by the interplay of hormones.
        Arch. Biochem. Biophys. 1974; 161: 499
        • Lakshmanan M.R.
        • Dugan R.E.
        • Nepokroeff C.M.
        • Ness G.C.
        • Porter J.W.
        Regulation of rat liver β-hydroxy-β-methylglutaryl coenzyme A reductase activity and cholesterol levels of serum and liver in various dietary and hormonal states.
        Arch. Biochem. Biophys. 1975; 168: 89
        • Huber J.
        • Hamprecht B.
        • Müller O.A.
        • Guder W.
        Tageszeitlicher Rhythmus der Hydroxymethylglutaryl-CoA-Reduktase in der Rattenleber, Part 2 (Rhythmus bei adrenalektomierten Tieren).
        Hoppe-Seyler's Z. Physiol. Chem. 1972; 353: 313
        • Hickman P.E.
        • Horton B.J.
        • Sabine J.R.
        Effect of adrenalectomy on the diurnal variation of hepatic cholesterogenesis in the rat.
        J. Lipid Res. 1972; 13: 17
        • Edwards P.A.
        Effect of adrenalectomy and hypophysectomy on the circadian rhythm of β-hydroxβ-A-methylglutaryl coenzyme A reductase activity in rat liver.
        J. Biol. Chem. 1973; 248: 2912
        • Mitropoulos K.A.
        • Balasubramaniam S.
        The role of glucocorticoids in the regulation of the diurnal rhythm of hepatic β-hydroxy-(3-methylglutaryl-coenzyme A reductase and cholesterol 7α-hydroxylase.
        Biochem. J. 1976; 160: 49
        • Edwards P.A.
        • Lemongello D.
        • Fogelman A.M.
        The effect of glucagon, norepinephrine, and dibutyryl cyclic AMP on cholesterol efflux and on the activity of 3-hydroxy-3-methylglutaryl CoA reductase in rat hepatocytes.
        J. Lipid Res. 1979; 20: 2
        • Geelen J.H.
        • Gibson D.M.
        Control of lipogenesis in maintenance cultures of freshly isolated hepatocytes.
        in: Tager J.M. Soling H.D. Williamson J.R. Use of Isolated Liver Cells and Kidney Tubules in Metabolic Studies. North-Holland Publishing Company Amsterdam, 1976: 219-230
        • Geelen J.H.
        • Gibson D.M.
        Lipogenesis in maintenance cultures of rat hepatocytes.
        FEBS Lett. 1975; 58: 334
        • Raskin P.
        • McGarry D.
        • Foster D.W.
        Independence of cholesterol and fatty acid biosynthesis from cyclic adenosine monophosphate concentration in the perfused rat liver.
        J. Biol. Chem. 1974; 19: 6029
        • Lin R.C.
        • Snodgrass P.J.
        A steady state of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured rat liver cells and the increase of its activity by dexamethasone.
        FEBS Lett. 1977; 83: 89
        • Johnston D.
        • Cavenee W.K.
        • Ramachandran C.K.
        • Melnykovych G.
        Cholesterol biosynthesis in a variety of cultured cells — Lack of correlation between synthesis and activity of 3-hydroxy-3methylglutaryl coenzyme A reductase caused by dexamethasone.
        Biochim. Biophys. Acta. 1979; 572: 188
        • Goldfarb S.
        • Barber T.A.
        • Pariza M.W.
        • Pugh T.D.
        Lipid synthesis and ultrastructure of adult rat hepatocytes during their first twenty-four hours in culture.
        Exp. Cell Res. 1978; 117: 39
        • Lin R.C.
        • Snodgrass P.J.
        Conversion of inactive 3-hydroxy-3-methylglutaryl-CoA reductase to its active enzyme during culture of rat hepatocytes.
        FEBS Lett. 1980; 109: 171
        • Ingebritsen T.S.
        • Geelen M.J.H.
        • Parker R.A.
        • Evenson K.J.
        • Gibson D.M.
        Modulation of hydroxymethylglutaryl-CoA reductase activity, reductase kinase activity, and cholesterol synthesis in rat hepatocytes in response to insulin and glucagon.
        J. Biol. Chem. 1979; 254: 9986
        • Berry M.N.
        • Friend D.S.
        High-yield preparation of isolated rat liver parenchymal cells.
        J. Cell Biol. 1969; 43: 506
        • Schudt Ch.
        Influence of insulin, glucocorticoids and glucose on glycogen synthase activity in hepatocyte cultures.
        Biochim, Biophys. Acta. 1980; 629: 499
        • Shapiro D.R.
        • Nordstrom J.L.
        • Mitschelen J.J.
        • Rodwell V.W.
        • Schimke R.T.
        Micro assay for 3-hydroxy-3-methylglutaryl-CoA reductase in rat liver and in L-cell fibroblasts.
        Biochim. Biophys. Acta. 1974; 370: 369
        • Stange E.F.
        • Alavi M.
        • Schneider A.
        • Ditschuneit H.
        • Poley J.R.
        Influence of dietary cholesterol, saturated and unsaturated lipid on hydroxymethylglutaryl CoA reductase activity in rabbit intestine and liver.
        J. Lipid Res. 1981; 22: 47
        • Stange E.F.
        • Schneider A.
        • Alavi M.
        • Preclik G.
        • Ditschuneit H.
        Lipoprotein regulation of 3-hydroxy-3-methylglu;aryl CoA reductase in cultured intestine.
        Biochim. Biophys. Acta. 1980; 620: 520
        • Lowry O.H.
        • Rosebrough N.J.
        • Farr A.L.
        • Randall R.J.
        Protein measurement with the Folin phenol reagent.
        J. Biol. Chem. 1951; 193: 265
        • Burton K.
        A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid.
        Biochem. J. 1956; 62: 315
        • Diamondstone T.I.
        Assay of tyrosine transaminase activity by conversion of p-hydroxyphenylpyruvate to p-hydroxybenzaldehyde.
        Anal. Biochem. 1966; 16: 395
        • Izak B.
        • Epstein E.
        • Baginsky E.S.
        Determination of liver microsomal glucose-6-phosphatase.
        Ann. Clin. Lab. Sci. 1977; 7: 169
        • Stange E.
        • Agostini B.
        • Papenberg J.
        Changes in rabbit lipoprotein properties by dietary cholesterol, and saturated and polyunsaturated fats.
        Atherosclerosis. 1975; 22: 125
      1. leig, W.E., Nöther-Fleig, G., Röben, H. and Ditschuneit, H., Hormonal regulation of key gluconeogenic enzymes in cultured hepatocytes, Biochim. Biophys. Acts, Submitted.

        • Van Berkel T.J.C.
        • Kruijt J.K.
        • Van Gent T.
        • Van Tol A.
        Saturable high affinity binding of low density and high density lipoprotein by parenchymal and non-parenchymal cells from rat liver.
        Biochem. Biophys. Res. Comm. 1980; 92: 1002
        • Kovanen P.T.
        • Brown M.S.
        • Goldstein J.L.
        Increased binding of low density lipoprotein to liver membranes from rats treated with 17α-ethinyl estradiol.
        J. Biol. Chem. 1979; 254: 11367
        • Gaertner U.
        Die Regulation der 3-Hydroxy-3-Methylglutaryl-Coenzyme-A-Reduktase von Hepatozyten in prlmärer Zellkultur durch Low Density Lipoprotein.
        Z. Gastroenterol. 1978; 16: 251
        • Daerr W.H.
        • Gianturco S.H.
        • Patsch J.R.
        • Sith L.C.
        • Gotto Jr., A.M.
        Stimulation and suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase in normal human fibroblasts by high density lipoprotein subclasses.
        Biochim. Biophys. Acts. 1980; 619: 287
        • Olefsky J.M.
        • Johnson J.
        • Liu F.
        • Edwards P.A.
        • Baur S.
        Comparison of 125I-Insulin binding and degradation to isolated rat hepatocytes and liver membranes.
        J. Amer. Diabetes Ass. 1975; 24: 801
        • Blackard W.G.
        • Guzelian P.S.
        • Small M.E.
        Down regulation of insulin receptors in primary cultures of adult rat hepatocytes in monolayer.
        Endocrinology. 1978; 103: 548
        • Goodridge A.G.
        • Garay A.
        • Silpananta P.
        Regulation of lipogenesis and the total activities of lipogenic enzymes in a primary culture of hepatocytes from prenatal and early postnatal chicks.
        J. Biol. Chem. 1974; 249: 1469
        • Alberts A.W.
        • Ferguson K.
        • Hennessy S.
        • Vagelos P.R.
        Regulation of lipid synthesis in cultured animal cells.
        J. Biol. Chem. 1974; 249: 5249
        • Geelen J.H.
        • Beynen A.C.
        • Christiansen R.Z.
        • Lepreau-Jose M.
        • Gibson D.M.
        Short-term effects of insulin and glucagon on lipid synthesis in isolated rat hepatocytes — Covariance of acetyl-CoA carboxylase activity and the rate of 3H2O incorporation into fatty acids.
        FEBS Lett. 1978; 95: 326
        • Bhathena S.J.
        • Avigan J.
        • Schreiner M.E.
        Effect of insulin on sterol and fatty acid synthesis and hydroxymethylglutaryl CoA reductase activity in mammalian cells grown in culture.
        in: 6th edition. Proc. Nat. Acad. Sci. (USA). 71. 1974: 2174
        • Goh E.H.
        • Heimberg M.
        Relationship between activity of hepatic 3-hydroxy-3-methylglutarylcoenzyme A reductase and secretion of very-low-density-lipoprotein cholesterol by the isolated perfused liver and in the intact rat.
        Biochem. J. 1979; 184: 1
        • Ramachandran C.K.
        • Gray S.L.
        • Melnykovych G.
        Coordinate repression of cholesterol biosynthesis and cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase by glucocorticoids in HeLa cells.
        Arch. Biochem. Biophys. 1978; 189: 205
        • Ness G.C.
        • Dugan R.E.
        • Lakshmanan M.R.
        • Nepokroeff C.M.
        • Porter J.W.
        Stimulation of hepatic β-hydroxy-β-methylglutaryl coenzyme A reductase activity in hypophysectomized rats by L-triiodothyronine.
        in: 6th edition. Proc. Nat. Aced. Sci. (U.S.A.). 70. 1973: 3839
        • Strand O.
        Effect of D- and L-triiodothyronine and of propylthiouracil on the production of bile acids in the rat.
        J. Lipid Res. 1963; 4: 305
        • Hellström K.
        • Lindstedt S.
        Cholic acid turnover and biliary bile acid composition in humans with abnormal thyroid function.
        J. Lab. Clin. Med. 1963; 63: 666