Malic enzyme and fatty acid synthase are two enzymes in the pathway for de novo biosynthesis of long-chain fatty acids. The relative synthesis rates of these enzymes are regulated by nutritional and hormonal stimuli. In vivo, feeding stimulates and starvation inhibits the synthesis of these enzymes. In chick- embryo hepatocytes in culture, triiodothyronine (T3) and insulin stimulate, and glucagon (via cyclic AMP) inhibits. We have isolated cloned cDNA sequences for malic enzyme and fatty acid synthase and established that enzyme synthesis rates are regulated by controlling the amounts of malic enzyme and fatty acid synthase mRNAs. One exception is stimulation of translation of fatty acid synthase mRNA by insulin. In vivo and in culture, regulation of the level of malic enzyme mRNA involves a combination of transcription and post-transcriptional controls, with the latter predominant. Regulation of fatty acid synthase mRNA level in vivo is primarily transcriptional. The point of regulation in hepatocytes in culture has not been ascertained. Kinetic and inhibitor experiments suggest that there is an intermediate in the T3-induced stimulation of the accumulation of mRNAs for both enzymes. We speculate that cyclic AMP may regulate the activity of that intermediate. Our objective in the next grant period is to understand the molecular mechanisms by which T3 and glucagon regulate expression of the gene for malic enzyme. We plan to determine which step subsequent to transcription initiation in the pathway for gene expression is regulated by T3, and to determine if a step other than degradation of mature mRNA is regulated by glucagon. Infective retroviral vectors will be used to develop a new method for gene transfer into chick-embryo hepatocytes. Putative regulatory sequences in the malic enzyme gene or transcript (hormone regulatory elements) will be tested functionally by attaching them to reporter genes containing hormone-neutral promoters and expressing the hybrid DNAs in hormone-sensitive hepatocytes. To achieve these objectives also will require completion of the cloning and characterization of the malic enzyme gene, a gene of more than 63 kb. Finally, we plan to initiate the identification, purification, and characterization of regulatory factors which bind to hormone regulatory elements in the malic enzyme gene or transcripts, with the ultimate aim of understanding how such factors interact with specific DNA or RNA, and with other regulatory factors, to achieve multifactorial regulation of gene expression.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK021594-15
Application #
3227039
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1987-09-01
Project End
1992-08-31
Budget Start
1991-09-01
Budget End
1992-08-31
Support Year
15
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Iowa
Department
Type
Schools of Medicine
DUNS #
041294109
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Chung, S S; Goodridge, A G (1999) Cis-acting elements in the 5'-flanking DNA of the malic enzyme gene regulate tissue-specific T3-responsiveness in chick embryo fibroblasts. Arch Biochem Biophys 364:1-12
Chung, S S; MacPhee, K G; Goodridge, A G (1999) Effect of the CCAAT/enhancer binding protein on expression of the gene for chicken malic enzyme. Arch Biochem Biophys 364:30-41
Xu, G; Goodridge, A G (1999) Function of a C-rich sequence in the polypyrimidine/polypurine tract of the promoter of the chicken malic enzyme gene depends on promoter context. Arch Biochem Biophys 363:202-12
Thurmond, D C; Baillie, R A; Goodridge, A G (1998) Regulation of the action of steroid/thyroid hormone receptors by medium-chain fatty acids. J Biol Chem 273:15373-81
Thurmond, D C; Goodridge, A G (1998) Characterization of thyroid hormone response elements in the gene for chicken malic enzyme. Factors that influence triiodothyronine responsiveness. J Biol Chem 273:1613-22
Xu, G; Goodridge, A G (1998) A CT repeat in the promoter of the chicken malic enzyme gene is essential for function at an alternative transcription start site. Arch Biochem Biophys 358:83-91
Mounier, C; Chen, W; Klautky, S A et al. (1997) Cyclic AMP-mediated inhibition of transcription of the malic enzyme gene in chick embryo hepatocytes in culture. Characterization of a cis-acting element far upstream of the promoter. J Biol Chem 272:23606-15
Carlisle, T L; Roncero, C; el Khadir-Mounier, C et al. (1996) Malic enzyme gene in chick embryo hepatocytes in culture: clofibrate regulates responsiveness to triiodothyronine. J Lipid Res 37:2088-97
Hodnett, D W; Fantozzi, D A; Thurmond, D C et al. (1996) The chicken malic enzyme gene: structural organization and identification of triiodothyronine response elements in the 5'-flanking DNA. Arch Biochem Biophys 334:309-24
Goodridge, A G; Klautky, S A; Fantozzi, D A et al. (1996) Nutritional and hormonal regulation of expression of the gene for malic enzyme. Prog Nucleic Acid Res Mol Biol 52:89-122

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