Carnitine palmitoyltransferase (CPT) is a regulatory membrane enzyme which controls the entry of long-chain fatty acids into the mitochondria for beta-oxidation. The regulation of fatty acid metabolism is a focus of nutritional and pharmacological investigation since fatty acids represent a significant energy source in American diets. Further, CPT has generated clinical interest because of the potential to modify it's activity and produce clinical improvement in cardiac ischemia, diabetes, and trauma/ sepsis, as well as in CPT deficiency. Previous studies in vivo have shown that CPT activity and synthesis increased in starvation, diabetes, with administration of hypolipidemic drugs, in riboflavin deficiency, and with increased dietary fat. In vivo and in vitro studies have shown that increased CPT synthesis is correlated with increased transcription rate of CPT mRNA from DNA and increased CPT mRNA concentrations. In H4IIE cells and hepatocytes CPT mRNA is induced by peroxisomal proliferators, cAMP, dexamethasone, epinephrine, fatty acids and dicarboxylic acids, and repressed by insulin. The first goal of the present proposal is to identify the 5'regulatory response unit of the CPT genomic DNA and to compare it to response units of other enzymes that respond to cAMP and dexamethasone. Since CPT transcription is also induced by fatty acids, we will define the fatty acid responsive element(s) of the CPT genome. The second goal is to assess the impact of specific CPT regulation on fatty acid metabolism. This will be accomplished by infection of rat and human hepatoma cell lines, as well as CPT-deficient human fibroblast lines, with retroviral vector constructs containing the phosphoenolpyruvate carboxykinase (PEPCK) promoter, the mouse mammary tumor virus (MMTV) promoter or the metallothionein promoter with a CPT structural gene (sense constructs) or CPT antisense constructs. Selection of the cells for incorporation of the gene construct will be by resistance to G418 (neo), since the neo element can be included in all of the vectors. CPT over- or under-expression should have characteristic specific effects on lipid synthesis and lipid oxidation. These experiments should give us further insight into the specific function and regulation of carnitine palmitoyltransferase and provide a framework for the study of the regulation of the peroxisomal carnitine octanoyltransferase, and peroxisomal and mitochondrial carnitine acetyltransferase in the future. The proposed studies should also help to define specific conditions under which it would be metabolically fruitful to insert a CPT gene construct into liver.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK039285-04A3
Application #
3239059
Study Section
Medical Biochemistry Study Section (MEDB)
Project Start
1987-02-01
Project End
1994-06-30
Budget Start
1991-08-01
Budget End
1992-06-30
Support Year
4
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
Other Domestic Higher Education
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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Chapin, R B; Brady, P S; Barke, R A et al. (1994) Hepatic CCAAT/enhancer binding protein (C/EBP-alpha and C/EBP-beta) expression changes with riboflavin deficiency, diet restriction and starvation in rats. J Nutr 124:2365-75
Barke, R A; Brady, P S; Brady, L J (1993) The regulation of mitochondrial fatty acid oxidation and hepatic gene expression by catecholamine. J Surg Res 54:95-101
Brady, P S; Park, E A; Hanson, R W et al. (1992) Multiple regulatory elements characterize the promoter region of the 68 kDa carnitine palmitoyltransferase in the rat and the human. Prog Clin Biol Res 375:175-81
Brady, L J; Ramsay, R R; Brady, P S (1991) Regulation of carnitine acyltransferase synthesis in lean and obese Zucker rats by dehydroepiandrosterone and clofibrate. J Nutr 121:525-31
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Brady, P S; Marine, K A; Brady, L J et al. (1989) Co-ordinate induction of hepatic mitochondrial and peroxisomal carnitine acyltransferase synthesis by diet and drugs. Biochem J 260:93-100
Brady, L J; Brady, P S (1989) Regulation of carnitine palmitoyltransferase synthesis in spontaneously diabetic BB Wistar rats. Diabetes 38:65-9
Wang, L; Brady, P S; Brady, L J (1989) Turnover of carnitine palmitoyltransferase mRNA and protein in H4IIE cells. Effect of cyclic AMP and insulin. Biochem J 263:703-8
Brady, P S; Brady, L J (1989) Regulation of carnitine palmitoyltransferase in vivo by glucagon and insulin. Biochem J 258:677-82

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