It has been recognized that a major risk factor for coronary heart disease is increased serum cholesterol levels. This finding highlights the importance of achieving a basic understanding, at the molecular level, of the metabolic regulation of isopentenoids in general and cholesterol specifically. The broad aim of this proposal is to assess the role of oxysterol synthesis and/or other isopentenoids in serum (LDL) cholesterol mediated down- regulation of cellular mevalonate synthesis directly and indirectly. We will use a mutant CHO cell line, unable to demethylate 4 alpha-methyl sterols, as our experimental model. The mutant CHO cells' metabolic lesion eliminates the metabolic complexity generated by greater than 20 intermediates between 4,4-dimethyl sterols and cholesterol. As such our changes to detect, isolate, and identify potential regulatory oxysterol(s) synthesized from (3H)mevalonate or preformed (14C/3H)cholesterol are increased. We will pursue a work plan which incorporates the coordinated, kinetic assessment of serum mediated oxysterol synthesis with the onset of the down-regulation for 3-hydroxy, 3-methyl glutaryl coenzyme A (HMG-CoA) reductase/HMG-CoA synthetase activities, and their respective turnover. In addition, we will assess the role of cytochrome P450 dependent processes on the serum mediated, down-regulation cascade. Potential biosynthesized regulatory sterols will be monitored by radio-HPLC, isolated, purified and identified by GC/MS. Radioimmunochemical techniques will be used to assess specific enzyme turnover. We expect our immediate study to yield new information about the obligatory sterologenic events which occur distal to serum lipoprotein uptake and catabolism. Such information should serve as a framework for a new generation of fundamental questions to be addressed with intact animals challenged by a single cholesterol meal. Ultimately we hope that a fundamental knowledge base will be generated to provide the necessary insight for understanding serum mediated control of mevalonate synthesis.
| Plemenitas, A; Watson, J A (1999) Down-regulation of mammalian 3-hydroxy-3-methylglutaryl coenzyme A reductase activity with highly purified liposomal cholesterol. Eur J Biochem 266:317-26 |
| Plemenitas, A; Havel, C M; Watson, J A (1990) Sterol-mediated regulation of mevalonic acid synthesis. Accumulation of 4-carboxysterols as the predominant sterols synthesized in a Chinese hamster ovary cell cholesterol auxotroph (mutant 215). J Biol Chem 265:17012-7 |
