Request for Funding

Medical Student Research Fellowship for Summer 2002

The mitochondrial transport of long-chain fatty acids is effected by the sequential action of carnitine palmitoyltransferase I (CPT I) (outer membrane) and CPT II (inner membrane), together with a carnitine-acylcarnitine translocase (inner membrane). The system plays a pivotal role in the regulation of fatty acid metabolism by virtue of the unique inhibitability of CPT I by malonyl-CoA, the product of the acetyl-CoA carboxylase reaction. CPT I, unlike CPT II, is also sensitive to pharmacological inhibition by agents such as etomoxir (Et) and 2-bromopalmitate (2-BP). Et, after conversion into its CoA ester, interacts covalently with CPT I in a carnitine-independent fashion, causing irreversible loss of enzyme activity. 2-BP, again upon activation to its CoA form, also becomes an inhibitor of CPT I, but only in the presence of carnitine.
Both Et and 2-BP have proved to be extremely useful agents in studies with isolated mitochondria designed to elucidate structure/function relationships surrounding the CPT proteins. They are also potential inhibitors of CPT I in intact cells both in vitro and in vivo provided that three conditions are met: first, that the inhibitor (as a carboxylic acid) readily enters the cell type under study; second, that the free acid is efficiently activated to its CoA ester by a fatty acyl-CoA synthetase (FACS) within the cell; and third, that the CPT I isoform expressed in that particular tissue (the liver (L) or muscle (M) variant) is sensitive to inhibition by the intracellular concentration of Et-CoA or 2-BP-CoA achieved.
In a number of studies using Et it has been assumed that all three of the above requirements were fulfilled. Also, radiolabeled 2-BP has been proposed as a useful tool for tracking the uptake of natural fatty acids into various tissues in the whole animal. The premise here is that the tracer is efficiently converted into its CoA ester intracellularly and that the latter becomes metabolically trapped because of its inability to enter the b-oxidation or esterification pathways. In the vast majority of these studies it has been presumed that the inhibitors have efficiently inhibited CPT I. Because we have reservations about some of the assumptions made, we feel that a detailed series of studies is in order to test their validity.
We want to administere 2-BP or Et to rats using a variety of dosing regimens and for different periods of time, after which selected organs will be removed for measurement of mitochondrial CPT I activity. Similar studies will be conducted with the perfused rat pancreas and also with isolated islets and an insulinoma (INS-1) cell line. In addition, the ability of Et (in the presence of ATP and CoASH) and 2-BP (in the presence of ATP, CoASH and carnitine), as well as 2-BP-CoA, to inhibit CPT I in isolated mitochondria will be examined.

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