The liver plays a central role in physiological and pathological conditions by switching from a carbohydrate to fatty acid-based metabolism. Carnitine palmitoyltransferase-l (CPT-I) is the key regulated step in the hormone-induced changes in mitochondrial fatty acid oxidation mediated via the cAMP signaling system. Malonyl-CoA inhibits CPT-I activity and represents the control point for fatty acid oxidation. The mechanism for the dramatically decreased malonyl-CoA sensitivity of CPT-I in fasting and diabetes has not been uncovered. We have shown 1) regulation of CPT-I involves the covalent modification of the CPT-I protein by phosphorylation and dephosphorylation, 2) 50 percent of mitochondrial CPT-I localizes with contact sites, 3) the localization of CPT-I in contact sites is enhanced in diabetic ketoacidosis, 4) inhibition kinetics of CPT-I in liver mitochondrial contact sites from diabetic ketoacidotic rats differs markedly from insulin-treated diabetic rats. Our hypothesis is that in the hormonal milieu resulting in the activation of hepatic protein kinases, CPT-I is phosphorylated leading to resistance to malonyl-CoA inhibition; thus more malonyl-CoA is required to effect the same degree of inhibition, but without a change in the velocity of CPT-I. We hypothesize that contact sites serve as docking domains for protein kinases and protein phosphatases involved in CPT-I regulation. The phosphorylation / dephosphorylation-based regulation of CPT-I occurs in contact sites where the phosphorylated CPT-I predominantly exists.
Aim 1 is to determine the amino acid sequence of the phosphorylated peptide following digestion of the immunoprecipitated CPT-I.
Aim 2 is to identify the kinase(s) for phosphorylation and the phosphatase(s) for dephosphorylation of CPT-I. The studies will address the functional consequences of CPT-I phosphorylation in isolated hepatocytes.
Aim 3 will examine the relationship between CPT-I kinetics and phosphorylation in liver of diabetic ketoacidotic rats.
Aim 4 approaches whether contact sites provide docking domains for kinase(s) and phosphatase(s), as well as, for liver isoform of CPT-I.
In Aim 5 malonyl-CoA content of the liver will be determined under the various metabolic states. The proposed studies will establish at the molecular and biochemical level the phosphorylation cycle of CPT-I and how it relates to the kinetics of CPT-I in liver.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK066107-03
Application #
7225597
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Blondel, Olivier
Project Start
2005-07-11
Project End
2009-04-30
Budget Start
2007-05-01
Budget End
2008-04-30
Support Year
3
Fiscal Year
2007
Total Cost
$263,690
Indirect Cost
Name
Case Western Reserve University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
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Kerner, Janos; Lee, Kwangwon; Hoppel, Charles L (2011) Post-translational modifications of mitochondrial outer membrane proteins. Free Radic Res 45:16-28
Lee, Kwangwon; Kerner, Janos; Hoppel, Charles L (2011) Mitochondrial carnitine palmitoyltransferase 1a (CPT1a) is part of an outer membrane fatty acid transfer complex. J Biol Chem 286:25655-62
Minkler, Paul E; Hoppel, Charles L (2010) Separation and characterization of cardiolipin molecular species by reverse-phase ion pair high-performance liquid chromatography-mass spectrometry. J Lipid Res 51:856-65
Distler, Anne M; Kerner, Janos; Hoppel, Charles L (2009) Mass spectrometric demonstration of the presence of liver carnitine palmitoyltransferase-I (CPT-I) in heart mitochondria of adult rats. Biochim Biophys Acta 1794:431-7
Chegary, Malika; Brinke, Heleen te; Ruiter, Jos P N et al. (2009) Mitochondrial long chain fatty acid beta-oxidation in man and mouse. Biochim Biophys Acta 1791:806-15
Distler, Anne M; Kerner, Janos; Lee, Kwangwon et al. (2009) Post-translational modifications of mitochondrial outer membrane proteins. Methods Enzymol 457:97-115
Minkler, Paul E; Kerner, Janos; Ingalls, Stephen T et al. (2008) Novel isolation procedure for short-, medium-, and long-chain acyl-coenzyme A esters from tissue. Anal Biochem 376:275-6
Kerner, Janos; Parland, William K; Minkler, Paul E et al. (2008) Rat liver mitochondrial carnitine palmitoyltransferase-I, hepatic carnitine, and malonyl-CoA: effect of starvation. Arch Physiol Biochem 114:161-70

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