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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Cellular Aspects of Diabetes and Obesity Study Section (CADO)
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Blondel, Olivier
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Case Western Reserve University
Internal Medicine/Medicine
Schools of Medicine
United States
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Rosca, Mariana G; Vazquez, Edwin J; Chen, Qun et al. (2012) Oxidation of fatty acids is the source of increased mitochondrial reactive oxygen species production in kidney cortical tubules in early diabetes. Diabetes 61:2074-83
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