Zhan Chen


December 2008

Document Type


Degree Name



Dept. of Science & Engineering


Oregon Health & Science University


Carnitine palmitoyltransferase I (CPTI) catalyzes the conversion of long-chain fatty acyl-CoAs to acylcarnitines in the presence of L-carnitine, the first step in the transport of long chain fatty acids from the cytosol to the mitochondria matrix, a rate-limiting step in ?-oxidation. To determine the role of the N-and C-terminal domains of L-CPTI on malonyl-CoA sensitivity, a series of double site-specific N-and C-terminal mutants were constructed. The mutants that decrease malonyl-CoA sensitivity (?18 and M593A) were counteracted by those that increase malonyl-CoA sensitivity (?19-30 and E590A) resulting in an increase in inhibitor sensitivity higher than either the ?18 or M593A mutant. Furthermore, combining the mutants that decrease malonyl-CoA sensitivity (?18 + M593A) further decreases the sensitivity of the enzyme to the inhibitor, while combining the mutants that increase malonyl-CoA sensitivity (?19-30 + E590A) increased the sensitivity of the enzyme to the inhibitor to a level 100-fold higher than that observed with the wild-type enzyme. Mutant ?19-30+M593A and E590A+M593A exhibited malonyl-CoA sensitivity similar to the wild-type. This data supports the hypothesis that the N-terminal end interacts with a region of the C-terminal end far from the catalytic site. Since the data shows that the mutations are compensatory or additive, we can hypothesize that malonyl-CoA binding could change the interaction of the N-terminal domain with the C-terminal domain resulting in a conformation with less catalytic activity. To address the pathophysiological effect of diminished M-CPTI expression on fatty acid metabolism in vivo, we developed a mouse M-CPTI (muscle isoform) gene knockout heterozygous model. We used a gene targeting strategy in ES cells that resulted in the deletion of exon 6, 7 and part of 5 of M-CPTI, a null allele. Homozygous deficient mice (M-CPTI -/-) were not viable. We conclude that targeted deletion of M-CPTI results in embryonic lethality. However heterozygous (M-CPTI +/-) pups survived. M-CPTI +/- mice became more weak, immobile and inactive than wild-type mice after fasting 48h. The heterozygous mice heart had half of CPTI protein expression and CPT activity of the wild-type mice heart. There was no sex difference on the CPTI activity. In heart and skeletal muscle the heterozygous (M-CPTI +/-) mice showed half CPT mRNA level of the wild-type mice, but the heterozygous (M-CPTI +/-) mice showed more increase in CPT mRNA level than the wild-type mice after fasting for 48h. To gain a further understanding of the role of CPTI in regulating fatty acid metabolism in vivo, we generated a transgenic mouse model that over-expresses human heart malonyl-CoA insensitive mutant M-CPTI in heart. The purpose of this study was to characterize the physiological and metabolic phenotype of localized M-CPTI over-expression in heart, especially the effect of M-CPTI over-expression on obesity and diabetes in vivo. In this study, mice with cardiac-specific expression of human heart wild-type M-CPTI was not found, while mice over-expressing human heart malonyl-CoA insensitive M-CPTI in heart were obtained. An increase in M-CPTI content can upregulate fatty acid metabolism in vivo under basal conditions and stress burden. M-CPTI plays an important role in body weight control, especially for the high fat diet fed mice. Our data set the basis for M-CPTI activation to treat obesity. The over-expression of CPTI leads to increased CPTI activity in isolated mitochondria and may play an important role in control of lipid storage in vivo. We also found that transgenic mice that over-express the mutant cardiac-specific M-CPTI were more susceptible to diabetes.




Div. of Environmental & Biomolecular Systems


School of Medicine



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