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Coordinate Control of 3-Hydroxy-3-Methylglutaryl

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Coordinate Control of 3-Hydroxy-3-Methylglutaryl Proc. Nati. Acad. Sci. USA Vol. 74, No. 4, pp. 1421-1425, April 1977 Biochemistry Regulation of cholesterol synthesis in rat adrenal gland through coordinate control of 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase activities [4-aminopyrazolopyrimidine/acetyl CoA acetyltransferase (acetoacetyl CoA thiolase)/mevalonate kinase/low density lipoprotein/ diurnal variation] S. BALASUBRAMANIAM, JOSEPH L. GOLDSTEIN, AND MICHAEL S. BROWN Division of Medical Genetics, Department of Internal Medicine, University of Texas Health Science Center at Dallas, Dallas, Texas 75235 Communicated by E. R. Stadtman, January 13, 1977 ABSTRACT The activities of cytosolic 3-hydroxy-3-meth- second site of regulation of cholesterol synthesis exists. This site ylglutaryl coenzyme A synthase [3-hydroxy-3-methylglutaryl- involves the reaction catalyzed by HMG CoA synthase [3- CoA acetoacetyl-CoA-lyase (CoA-acetylating), EC 4.1.3.5] and hydroxy-3-methylglutaryl-CoA acetoacetyl-CoA-lyase (CoA- microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase acetylating), EC 4.1.3.5], the enzyme that immediately pre- Imevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.341, two sequential enzymes in the cholesterol biosynthetic cedes HMG CoA reductase in the cholesterol biosynthetic pathway, were shown to be regulated coordinately in the pathway (11, 12) (Fig. 1). The situation in liver is complex, adrenal gland of the rat. When the plasma cholesterol level was however, because HMG CoA is synthesized at two sites within lowered by administration of 4-aminopyrazolopyrimidine, a this tissue (13-15). The vast bulk of the HMG CoA is synthesized treatment known to enhance cholesterol synthesis in the adre- within the mitochondria by a specific intramitochondrial HMG nal, synthase activity in the gland rose by 14- to 29-fold and re- CoA synthase (13) and is cleaved within the mitochondria by ductase activity rose by 50- to 100-fold. The subsequent intra- venous infusion of low density lipoprotein restored the plasma HMG CoA Iyase (EC 4.1.3.4) to produce acetoacetate (16). A cholesterol level and suppressed synthase and reductase ac- much smaller amount of hepatic HMG CoA is synthesized by tivities in parallel. The activities of adrenal 3-hydroxy-3-meth- a distinct HMG CoA synthase within the cytosol (13). This cy- ylglutaryl coenzyme A synthase and reductase were also shown tosolic HMG CoA is reduced by microsomal HMG CoA re- to exhibit a coordinate pattern of diurnal variation with peaks ductase to form mevalonate, which is subsequently converted in both enzymes achieved at the mid-point of the dark cycle. The to cholesterol (Fig. 1). The experiments of Lane and co-workers activity of adrenal acetoacetyl coenzyme A thiolase (acetyl CoA acetyltransferase; acetyl-CoA:acetyl-CoA C-acetyltransferase, have shown that in liver the cytosolic but not the mitochondrial EC 2.3.1.9), the enzyme preceding the synthase in the cholesterol HMG CoA synthase is suppressed by cholesterol feeding (11, biosynthetic pathway, and the activity-of adrenal mevalonate 12). kinase (ATP:mevalonate 5-phosphotransferase, EC 2.7.1.36), the The magnitude of the rise in the activity of HMG CoA re- enzyme following the reductase, were not enhanced by cho- ductase in the adrenal gland of the 4-APP-treated rat suggested lesterol deprivation, and neither exhibited a pattern of diurnal the use of this tissue as a model system to determine whether variation. The coordinate control of 3-hydroxy-3-methylglutaryl a similar degree of regulation at CoA synthase and reductase in rat adrenal gland provides a exists the level of HMG CoA model system to study the biochemical mechanism for the reg- synthase. This model is particularly useful because the adrenal ulation of cholesterol synthesis in a tissue that uses cholesterol gland is not known to produce large amounts of ketone bodies for the synthesis of steroid hormones. and hence should not have large amounts of intramitochondrial HMG CoA synthase. The current results indicate that cytosolic Recent studies have shown that cholesterol synthesis is subject HMG CoA synthase activity in rat adrenal gland is regulated to an extraordinary degree of regulation in the adrenal gland coordinately with microsomal HMG CoA reductase activity and of the rat (1, 2). When plasma cholesterol levels are normal, the that these two enzymes function in concert to supply substrate adrenal gland satisfies its large requirement for cholesterol as for cholesterol biosynthesis during conditions of cholesterol a precursor for steroid hormone synthesis by utilizing the cho- deprivation. lesterol contained in plasma lipoproteins (1-5). However, when the availability of plasma cholesterol is diminished, such as when hepatic lipoprotein secretion is blocked by administration MATERIALS AND METHODS of the drug 4-aminopyrazolopyrimidine (4-APP) (6-9), cho- lesterol synthesis in the adrenal gland rises by as much as 30-fold Materials. [1-14C]Acetyl CoA (54 mCi/mmol) and DL-3- (1, 2). This increase is associated with a 50- to 200-fold rise in hydroxy-3-methyl[3-14C]glutaryl CoA (26.2 mCi/mmol) were the activity of the microsomal enzyme 3-hydroxy-3-methyl- obtained from New England Nuclear Corp. DL-[2-14C]Mev- glutaryl coenzyme A reductase [HMG CoA reductase; meva- alonic acid lactone (13 mCi/mmol) was purchased from lonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34] Amersham/Searle. (2). These findings are consistent with the view that in rat Animals. Male Sprague-Dawley rats weighing between 300 adrenal gland as in rat liver (10) HMG CoA reductase consti- and 400 g were obtained from Charles River Laboratories. tutes a major rate-controlling step in the cholesterol biosynthetic Unless otherwise indicated, the rats were exposed to a light-dark pathway. cycle consisting of 12 hr of light (0700-1900 hr) and 12 hr of Lane and coworkers have recently demonstrated by cho- darkness for at least 2 weeks prior to use. Throughout these lesterol feeding experiments that in rat and chicken liver a periods of adaptation, the rats had full access to water and formula chow (Wayne's Laboratory, Chicago). Abbreviations: 4-APP, 4-aminopyrazolopyrimidine; CoA, coenzyme In experiments in which 4-APP-treated rats were used, the A; HMG CoA, 3-hydroxy-3-methylglutaryl coenzyme A; LDL, low animals were given daily injections of 4-APP (40 mg/kg of body density lipoprotein. weight intraperitoneally in 10 mM sodium phosphate, pH 3) 1421 Downloaded by guest on September 29, 2021 1422 Biochemistry: Balasubramaniam et al. Proc. NatI.Acad. Sci. USA 74 (1977) Acetoacetyt CoA HMO CoA HM CoA Mevolonate Thiolae Synthase Reductase Kinose 2 ACETYL CoA .-ACETOACETYL CoA7 HMG CoA MEVALONATE7 5-PHOSPHOMEVALONATEue..s* CHOLESTEROL Acetyl CoA ATP FIG. 1. Early steps in the cholesterol biosynthetic pathway. This sequence of reactions emerged from the experiments of Rudney, Lynen, Gould, Popjak, Siperstein, Lane, and others (reviewed in refs. 12 and 13). for 3 days and were killed on the fourth day (2, 8). For intra- [14C]mevalonate in the presence of NADPH and microsomal venous injection of low density lipoprotein, a polyethylene HMG CoA reductase. For this experiment, a scaled-up HMG cannula was introduced into the tail vein while the animal was CoA synthase reaction was conducted, and the reaction was under light anesthesia. Control animals received appropriate stopped by heating for 15 min at 500 to inactivate the HMG volumes of either 10 mM sodium phosphate, pH 3, intraperi- CoA synthase. One aliquot of this mixture was added to 6 M toneally for the 4-APP experiments or saline intravenously for HCI and heated to determine the amount of nonvolatile 14C the lipoprotein experiments. radioactivity. Another aliquot was added to a solution con- Lipoproteins. Human low density lipoprotein (LDL) (den- taining the reaction components for the HMG CoA reductase sity, 1.019-1.063 g/ml) was obtained from the plasma of assay supplemented with adrenal microsomes from a 4-APP- healthy subjects as described previously (17). treated rat. (The microsomes had been subjected to prior Preparation of Adrenal Cytosolic and Microsomal Frac- heating at 500 for 30 min to inactivate traces of adherent HMG tions. Rats were routinely killed at 0900 hr by decapitation, and CoA synthase.) The mixture was incubated at 370 and aliquots blood was collected by drainage from the neck into a tube were removed at different times to determine the amount of containing EDTA. All subsequent steps were carried out at 4°. [14C]mevalonate formed (2). Under these conditions, the The adrenal glands were removed immediately, placed into maximal amount of [14C]mevalonate formed was equal to ice-cold medium containing 0.3 M sucrose, 25 mM 2-mercap- 80-90% of the value expected from the determination of non- toethanol, and 10 mM EDTA, pH 7, and homogenized as pre- volatile "'C radioactivity. viously described (2). The homogenate was subjected to cen- Assay of Microsomal HMG CoA Reductase. The conver- trifugation at 10,000 X g for 10 min and the resulting super- sion of [14C]HMG CoA to ["4C]mevalonate in adrenal micro- natant was spun at 100,000 X g for 1 hr. The pellet from this somes was measured using thin-layer chromatography as pre- latter centrifugation (microsomal fraction) was washed once viously described (2). in the homogenizing buffer, frozen, and stored at -60°. The Assay of Cytosolic Acetoacetyl CoA Thiolase (acetyl-CoA supernatant from the 100,000 X g centrifugation (cytosolic acetyltransferase; acetyl-CoA:acetyl-CoA C-acetyltransferase, fraction) was dialyzed for 24 hr at 40 against buffer containing EC 2.3.1.9). CoA-dependent cleavage of acetoacetyl CoA was 20 mM potassium phosphate at pH 7.2, 0.1 mM EDTA, and 0.5 determined spectrophotometrically by measuring the rate of mM dithiothreitol, after which it was frozen and stored at decrease in absorbance at 300 nm (18).
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