The activities of several enzymes involved in the pathway for the de novo synthesis of long-chain fatty acids are low in starved animals and high in animals fed high-carbohydrate diets. The primary regulatory mechanism is transcriptional. We are using malic enzyme as a model to investigate the molecular basis of this phenomenon. Transitions between the fed and starved states are mimicked quantitatively in chick-embryo hepatocytes incubated in chemically-defined medium. Insulin and triiodothyronine (T3) stimulate transcription of the gene for malic enzyme by 30- to 50-fold; glucagon, cAMP, unesterified fatty acids and isoquinoline sulfonamide protein kinase inhibitors inhibit transcription by 80 to 95% or more. Corticosterone and peroxisome-proliferating agents such as clofibrate have no effects on transcription of malic enzyme by themselves but stimulate the response to T3 in cells incubated in culture for more than 3 days. The cell culture system that we have developed is thus ideally suited for analyzing the molecular mechanisms by which these agents regulate transcription of the malic enzyme gene. In next grant period, we propose to identify and characterize cis-acting response elements for T3, cAMP, hexanoate, isoquinoline sulfonamide protein kinase inhibitors, corticosterone and peroxisome-proliferating agents. We also propose to characterize cis-acting elements that interact with the T3 response element and modify its transcriptional response to T3. In these analyses, we will test promoter function of various parts of the malic enzyme gene with a transient transfection assay. Uptake of DNA into chick-embryo hepatocytes will be facilitated by lipofectin. We have already established that a 5.8 kb fragment of 5'-flanking DNA from the malic enzyme gene confers regulation by T3, cAMP and hexanoate on a linked CAT gene. We also propose to identify and characterize the trans-acting factors that bind to the cis-acting elements and mediate the actions of the hormones and metabolites listed above. DNase I and methylation interference footprinting and gel mobility-shift assays will be used to assess binding of protein factors to cis-acting response elements. Finally, we propose to purify those factors that have not been described previously and to clone and characterize the corresponding cDNAs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK021594-18
Application #
2137589
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1987-09-01
Project End
1996-08-31
Budget Start
1994-09-01
Budget End
1995-08-31
Support Year
18
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Iowa
Department
Biochemistry
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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