Regulation of hepatic beta-hydroxy-beta-methylglutaryl coenzyme A reductase by the interplay of hormones

We have previously established that insulin causes a marked and rapid stimulation of hepatic β-hydroxy-β-methylglutaryl coenzyme A reductase activity in normal and diabetic rats [Biochem. Biophys. Res. Commun.50, 504 (1973)], whereas l-triiodothyronine stimulates the reductase activity to supranormal levels in hypophysectomized rats two days after administration [Proc. Nat. Acad. Sci. (1974) In press]. In the present investigation it is demonstrated that the stimulation of the reductase activity in hypophysectomized-diabetic rats requires the mediation of both insulin and l-triiodothyronine. Neither hormone alone is effective. The rapid stimulation of reductase activity by insulin and the delayed stimulation elicited by l-triiodothyronine are both inhibited by either glucagon or hydrocortisone. Thus, an interplay of hormones regulates reductase activity and consequently cholesterol biosynthesis.     

Mode of interaction of beta-hydroxy-beta-methylglutaryl coenzyme A reductase with strong binding inhibitors: compactin and related compounds

The sodium salts of compactin (1) and trans-6-[2-(2,4- dichloro-6-hydroxyphenyl)ethyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran- 2-one (3) are inhibitors of yeast beta-hydroxy-beta-methylglutaryl coenzyme A (HMG-CoA) reductase. The dissociation constants are 0.24 X 10(-9) and 0.28 X 10(-9) M, respectively. Similar values have been reported for HMG-CoA reductase from mammalian sources [Endo, A., Kuroda, M., & Tanzawa, K. (1976) FEBS Lett. 72, 323; Alberts, A. W., et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3957]. The structures of these compounds marginally resemble that of any substrates of HMG-CoA reductase. We, therefore, investigated the basis for the strong interaction between HMG-CoA reductase and these inhibitors. HMG-CoA and coenzyme A (CoASH), but not reduced nicotinamide adenine dinucleotide phosphate (NADPH), prevent binding of compactin to the enzyme. HMG-CoA, but not CoASH or NADPH, prevents binding of 3 to the enzyme. We also investigated the inhibitory activity of molecules that resemble structural components of compactin. Compactin consists of a moiety resembling 3,5-dihydroxyvaleric acid that is attached to a decalin structure. The sodium salt of DL-3,5-dihydroxyvaleric acid inhibits HMG-CoA reductase competitively with respect to HMG-CoA and noncompetitively with respect to NADPH. The dissociation constant for DL-3,5-dihydroxyvaleric acid, derived from protection against inactivation of enzyme by iodoacetic acid, is (2.1 +/- 0.9) X 10(-2) M. Two decalin derivatives (structurally identical with or closely related to the decalin moiety of compactin) showed no detectable inhibition. If the lack of inhibition is due to their limited solubility, the dissociation constant of these decalin derivatives may be conservatively estimated to be greater than or equal to 0.5 mM. Simultaneous addition of decalin derivatives and DL-3,5-dihydroxyvaleric acid does not lead to enhanced inhibition. The sodium salt of (E)-6-[2-(2-methoxy-1-naphthalenyl)ethenyl]-3,4,5,6- tetrahydro-4-hydroxy-2H-pyran-2-one (6) inhibits HMG-CoA reductase competitively with respect to HMG-CoA and noncompetitively with respect to NADPH. The inhibition constant (vs. HMG-CoA) is 0.8 microM. CoASH does not prevent binding of 6 to enzyme. Compound 6, therefore, behaves analogously to compound 3. We propose that these inhibitors occupy two sites on the enzyme: one site is the hydroxymethylglutaryl binding domain of the enzyme active site and the other site is a hydrophobic pocket located adjacent to the active site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of rat liver beta-hydroxy-beta-methylglutaryl coenzyme. A reductase activity and cholesterol levels of serum and liver in various dietary and hormonal states.

The effects of hormones and dietary factors on rat liver β-hydroxy-β-methylglutaryl coenzyme A reductase activity and serum and liver cholesterol levels were tested. Cholestyramine feeding markedly stimulated reductase activity in the livers of rats depleted of insulin or l-triiodothyronine. Therefore, these hormones are not absolute requirements for the stimulation of reductase activity.In hypophysectomized rats, l-triiodothyronine markedly stimulated reductase activity, even when the animals were cholesterol fed or fasted. However, this stimulation was accompanied by a reduction of serum and liver cholesterol levels. In diabetic rats, insulin failed to either stimulate reductase activity after cholesterol feeding, or to depress the level of liver cholesterol. These results are consistent with a model in which cholesterol functions as a feedback repressor of reductase activity.In contrast, a number of dietary and hormonal states produced little or no change in the level of serum and liver cholesterol while producing widely different reductase activities. These results suggest that the cholesterol level does not regulate reductase activity and cholesterol synthesis and that the factors that affect the formation of cholesterol also have a similar effect on its degradation. However, the possibility of a small subcellular pool of cholesterol regulating reductase activity and thus showing a positive correlation cannot be ruled out.The results reported in this paper suggest that the repressor, in a feedback repression model of regulation, should have similar effects on the rate-limiting enzymes of cholesterol synthesis and degradation. In this way a factor that operates through the repressor affects the rates of synthesis and degradation, but not the level of liver and serum cholesterol.     

Regulation of coenzyme A biosynthesis.

Coenzyme A (CoA) and acyl carrier protein are two cofactors in fatty acid metabolism, and both possess a 4'-phosphopantetheine moiety that is metabolically derived from the vitamin pantothenate. We studied the regulation of the metabolic pathway that gives rise to these two cofactors in an Escherichia coli beta-alanine auxotroph, strain SJ16. Identification and quantitation of the intracellular and extracellular beta-alanine-derived metabolites from cells grown on increasing beta-alanine concentrations were performed. The intracellular content of acyl carrier protein was relatively insensitive to beta-alanine input, whereas the CoA content increased as a function of external beta-alanine concentration, reaching a maximum at 8 microM beta-alanine. Further increase in the beta-alanine concentration led to the excretion of pantothenate into the medium. Comparing the amount of pantothenate found outside the cell to the level of intracellular metabolites demonstrates that E. coli is capable of producing 15-fold more pantoic acid than is required to maintain the intracellular CoA content. Therefore, the supply of pantoic acid is not a limiting factor in CoA biosynthesis. Wild-type cells also excreted pantothenate into the medium, showing that the beta-alanine supply is also not rate limiting in CoA biogenesis. Taken together, the results point to pantothenate kinase as the primary enzymatic step that regulates the CoA content of E. coli.     

Mitochondrial biosynthesis of coenzyme A.

All enzymes required for the biosynthesis of CoA from pantothenic acid are present in the particle-free supernatant fraction from rat liver. We now report that also mitochondria have the capacity for biosynthesis of CoA, with 4′-phosphopantetheine as the initial precursor. Rat liver mitochondria do not contain pantothenate kinase, 4′-phosphopantothenoyl-1-synthetase or 4′-phosphopantothenoyl-1-cysteine decarboxylase. Dephospho-CoA pyrophosphorylase and dephospho-CoA kinase are present in the inner mitochondrial membrane, however, at specific activities as high as in cytosol. K_m of mitochondrial dephospho-CoA kinase for dephospho-CoA is about 0.01 mmol/1, which is one order of magnitude lower than reported for the kinase from cytosol.     

The origin of chloroplastic acetyl coenzyme A

Pyruvic dehydrogenase activity has been examined in a number of highly purified leaf organelles. In spinach leaf cell, the major activity is in the mitochrondrion with low activity in isolated chloroplasts. The major source of CO₂ derived from pyruvic acid metabolism in the isolated chloroplast is via the acetolactic synthase reaction localized in the chloroplast. Evidence is presented that the leaf mitochondrion contains both the pyruvic acid dehydrogenase and an acetyl coenzyme A hydrolase. It is suggested that free acetic acid is generated in the mitochrondrion and then moves to the chloroplast where acetyl coenzyme synthetase converts it from the metabolically inert acid to the very metabolically active acetyl coenzyme A.