Regulation of early cholesterol biosynthesis in rat liver: Effects of sterols, bile acids, lovastatin, and BM 15.766 on 3-hydroxy-3-methylglutaryl coenzyme A synthase and acetoacetyl coenzyme A thiolase activities
18
Citation
31
Reference
10
Related Paper
Citation Trend
Abstract:
Cytosolic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase catalyzes the formation of HMG-CoA, the substrate for the rate-controlling enzyme in the cholesterol biosynthetic pathway. To explore the regulation in liver, we developed a new, accurate, and reliable reversed-phase ion-pair chromatographic assay that uses nonradioactive substrates and n -propionyl coenzyme A as an internal recovery standard. The hepatic activities were measured in rats treated with cholesterol, sitosterol, cholic acid, deoxycholic acid, ursodeoxycholic acid, cholestyramine, bile fistula, lovastatin, and BM 15.766, an inhibitor of 7-dehydrocholesterol Δ 7 -reductase, and were compared with microsomal HMG-CoA reductase and cytosolic acetoacetyl coenzyme A (AcAc-CoA) thiolase activities. HMG-CoA synthase activity was effectively suppressed in synchrony with HMG-CoA reductase activity by treatments with cholesterol (−41%, P < .05), cholic acid (−72%, P < .005), and deoxycholic acid (−62%, P < .05). However, ursodeoxycholic acid increased activity 84% ( P < .05) and intravenous sitosterol did not change activity. AcAc-CoA thiolase activities also paralleled HMG-CoA reductase and HMG-CoA synthase activities, but differences were not statistically significant. In contrast to inhibition, up-regulation of hepatic HMG-CoA synthase activities by cholestyramine, bile fistula, and lovastatin was much less than HMG-CoA reductase activities. In addition, BM 15.766 did not stimulate synthase activity, whereas lovastatin increased activity 2.4-fold. Thus, hepatic HMG-CoA synthase activity was regulated coordinately with HMG-CoA reductase, and responded more forcefully to regulatory stimuli than acetoacetyl-CoA thiolase activity but usually less than HMG-CoA reductase.Keywords:
Thiolase
Lovastatin
Coenzyme A
Mevalonic acid
Hydroxymethylglutaryl-CoA reductase
Cholestyramine
CYP8B1
Cytosolic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase catalyzes the formation of HMG-CoA, the substrate for the rate-controlling enzyme in the cholesterol biosynthetic pathway. To explore the regulation in liver, we developed a new, accurate, and reliable reversed-phase ion-pair chromatographic assay that uses nonradioactive substrates and n -propionyl coenzyme A as an internal recovery standard. The hepatic activities were measured in rats treated with cholesterol, sitosterol, cholic acid, deoxycholic acid, ursodeoxycholic acid, cholestyramine, bile fistula, lovastatin, and BM 15.766, an inhibitor of 7-dehydrocholesterol Δ 7 -reductase, and were compared with microsomal HMG-CoA reductase and cytosolic acetoacetyl coenzyme A (AcAc-CoA) thiolase activities. HMG-CoA synthase activity was effectively suppressed in synchrony with HMG-CoA reductase activity by treatments with cholesterol (−41%, P < .05), cholic acid (−72%, P < .005), and deoxycholic acid (−62%, P < .05). However, ursodeoxycholic acid increased activity 84% ( P < .05) and intravenous sitosterol did not change activity. AcAc-CoA thiolase activities also paralleled HMG-CoA reductase and HMG-CoA synthase activities, but differences were not statistically significant. In contrast to inhibition, up-regulation of hepatic HMG-CoA synthase activities by cholestyramine, bile fistula, and lovastatin was much less than HMG-CoA reductase activities. In addition, BM 15.766 did not stimulate synthase activity, whereas lovastatin increased activity 2.4-fold. Thus, hepatic HMG-CoA synthase activity was regulated coordinately with HMG-CoA reductase, and responded more forcefully to regulatory stimuli than acetoacetyl-CoA thiolase activity but usually less than HMG-CoA reductase.
Thiolase
Lovastatin
Coenzyme A
Mevalonic acid
Hydroxymethylglutaryl-CoA reductase
Cholestyramine
CYP8B1
Cite
Citations (18)
Sitosterolemia is a recessively inherited disorder characterized by abnormally increased plasma and tissue plant sterol concentrations. Patients have markedly reduced whole body cholesterol biosynthesis associated with suppressed hepatic, ileal, and mononuclear leukocyte 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme in cholesterol biosynthetic pathway, coupled with significantly increased low density lipoprotein (LDL) receptor expression. To investigate the mechanism of down-regulated cholesterol biosynthesis, we assayed several other key enzymes in the cholesterol biosynthetic pathway including acetoacetyl-CoA thiolase, HMG-CoA synthase, squalene synthase, and 7-dehydrocholesterol Δ7-reductase activities in liver and freshly isolated mononuclear leukocytes from four sitosterolemic patients and 19 controls. Hepatic acetoacetyl-CoA thiolase, HMG-CoA synthase, reductase, and squalene synthase activities were significantly decreased (P < 0.05) –39%, –54%, –76%, and –57%, respectively, and 7-dehydrocholesterol Δ7-reductase activity tended to be lower (–35%) in the sitosterolemic compared with control subjects. The reduced HMG-CoA synthase, reductase, and squalene synthase activities were also found in mononuclear leukocytes from a sitosterolemic patient. Thus, reduced cholesterol synthesis is caused not only by decreased HMG-CoA reductase but also by the coordinate down-regulation of entire pathway of cholesterol biosynthesis. These results suggest that inadequate cholesterol production in sitosterolemia is due to abnormal down-regulation of early, intermediate, and late enzymes in the cholesterol biosynthetic pathway rather than a single inherited defect in the HMG-CoA reductase gene.—Honda, A., G. Salen, L. B. Nguyen, G. S. Tint, A. K. Batta, and S. Shefer. Down-regulation of cholesterol biosynthesis in sitosterolemia: diminished activities of acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-CoA synthase, reductase, squalene synthase, and 7-dehydrocholesterol Δ7-reductase in liver and mononuclear leukocytes. J. Lipid Res. 1998. 39: 44–50.
Thiolase
Squalene
Coenzyme A
Hydroxymethylglutaryl-CoA reductase
Cite
Citations (45)
Incubating Hep G2 cells for 18 h with triparanol, buthiobate and low concentrations (less than 0.5 microM) of U18666A, inhibitors of desmosterol delta 24-reductase, of lanosterol 14 alpha-demethylase and of squalene-2,3-epoxide cyclase (EC 5.4.99.7) respectively, resulted in a decrease of the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase activity. However, U18666A at concentrations higher than 3 microM increased the HMG-CoA reductase activity in a concentration-dependent manner. None of these inhibitors influenced directly the reductase activity in Hep G2 cell homogenates. Analysis by t.l.c. of 14C-labelled non-saponifiable lipids formed from either [14C]acetate or [14C]mevalonate during the cell incubations confirmed the sites of action of the drugs used. Beside the 14C-labelled substrates of the blocked enzymes and 14C-labelled cholesterol, another non-saponifiable lipid fraction was observed, which behaves as polar sterols on t.l.c. This was the case with triparanol and at those concentrations of U18666A that decreased the reductase activity, suggesting that polar sterols may play a role in suppressing the reductase activity. In the presence of 30 microM-U18666A (sterol formation blocked) the increase produced by simultaneously added compactin could be prevented by addition of mevalonate. This indicates the existence of a non-sterol mevalonate-derived effector in addition to a sterol-dependent regulation. LDL (low-density lipoprotein), which was shown to be able to decrease the compactin-induced increase in reductase activity, could not prevent the U18666A-induced increase. On the contrary, LDL enhanced the U18666A effect, showing that the LDL regulation is not merely the result of introducing cholesterol to the cells.
Desmosterol
Mevalonic acid
Hydroxymethylglutaryl-CoA reductase
Lanosterol
Coenzyme A
Squalene
Hep G2
Cite
Citations (51)
The regulation of mevalonic acid synthesis requires both nonsterol isopentenoid and sterol regulatory signal molecules. A primary target of this multivalent control process is the enzyme which catalyzes mevalonate synthesis: 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34). In this report Staphylococcus aureus alpha-toxin perforated Chinese hamster ovary cells were used to facilitate the identification of isopentenoidogenic reactions and metabolites required for mevalonate-mediated loss of HMG-CoA reductase activity. alpha-Toxin-perforated cells retained the capacity to decrease, upon demand, HMG-CoA reductase activity and protein in response to mevalonate or isopentenoid pyrophosphate esters. Also, it was deduced with highly specific metabolic inhibitors, that conversion of farnesyl 1-diphosphate to squalene was required for mevalonate-mediated suppression of reductase activity. Since squalene (2 microM) did not downregulate reductase activity, pre-squalene pyrophosphate or a derivative, or polyprenyl-1-pyrophosphate-generated inorganic pyrophosphate, or a combination of these metabolites are proposed as candidate regulatory nonsterol isopentenoid signal molecules.
Farnesyl pyrophosphate
Mevalonic acid
Geranylgeranyl pyrophosphate
Squalene
Coenzyme A
Mevalonate pathway
Hydroxymethylglutaryl-CoA reductase
Cite
Citations (15)
Thiolase
Mevalonic acid
Coenzyme A
Cite
Citations (3)
In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining Km values for the overall reaction of 40.3±5.8 μM for (R,S)-HMG-CoA and 81.4±5.3 μM for NADPH; Vmax was 33.55±1.8 units·mg-1. Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control.
Mevalonic acid
Hydroxymethylglutaryl-CoA reductase
Coenzyme A
Leishmania mexicana
Homogenization
Geranylgeraniol
Cite
Citations (19)
Mevalonic acid
Thiolase
Terpene
Mevalonate pathway
Farnesyl diphosphate synthase
Coenzyme A
Cite
Citations (2)
Thiolase
Mevalonic acid
Ergosterol
Wild type
Cite
Citations (39)
Mevalonic acid
Cholestyramine
Hydroxymethylglutaryl-CoA reductase
Coenzyme A
Cite
Citations (19)
Mevalonate pathway
Mevalonic acid
Hydroxymethylglutaryl-CoA reductase
Lovastatin
Coenzyme A
Cite
Citations (61)