Philip A. Bell

Diverse mechanisms of inhibition of pyruvate dehydrogenase kinase by structurally distinct inhibitors.

The mechanism of action of structurally distinct pyruvate dehydrogenase kinase (PDK) inhibitors was examined in assays with experimental contexts ranging from an intact pyruvate dehydrogenase complex (PDC) with and without supplemental ATP or ADP to a synthetic peptide substrate to PDK autophosphorylation. Some compounds directly inhibited the catalytic activity of PDKs. Some of the inhibitor classes tested inhibited autophosphorylation of recombinant PDK1 and PDK2. During these studies, PDC was shown to be directly inhibited by a novel mechanism; the addition of supplemental recombinant PDKs, an effect that is ADP-dependent and partly alleviated by members of each of the compound classes tested. Overall, these data demonstrate that small molecules acting at diverse sites can inhibit PDK activity.

Pharmacological Strategies for Reduction of Lipid Availability

The chronic elevation of circulating free fatty acid (FFA) levels is associated with insulin resistance.’ In normal individuals, excess energy is converted to FFAs by the liver and stored as triglyceride in adipocytes. The stored energy can be mobilized as needed via an increase in the rate of FFA release from the adipocytes. However, if the balance between storage and release is disturbed such that the FFAs become chronically elevated, insulin resistance is usually manifest. Elevated FFA levels have been implicated in inducing metabolic derangements. For example, over 30 years ago, Randle demonstrated that FFAs have an impact on peripheral glucose utilization in muscle, principally via a FFA-induced suppression of pyruvate dehydrogenase (PDH).* Felber has recently shown that FFAs may be playing a role in glycogen storage3 and Grill has suggested that they have an important role in regulating beta-cell function and thereby altering insulin secretion profile^.^ Furthermore, recent studies by Bergman demonstrate that lowering of circulating FFAs is required for suppression of hepatic glucose production following a meal.5 The causative role of FFAs in the development or maintenance of insulin resistance has been difficult to assess. One reason for this could be that measurement of circulating FFA levels may be an inadequate measure of the role of FFAs since stored triglycerides and local fluxes may be major factors as well. In a normal individual, the stored triglyceride in tissues such as the liver, the muscle, and even the islet is relatively low. However, with chronic elevation of circulating FFA, as occurs in obesity, the triglyceride content of all of these tissues is predicted to be higher than normal, so local storage and release of FFAs may become important. One may ask the following question: “If the circulating FFAs were reduced, would insulin sensitivity improve?” Acute experiments in humans suggest that the answer is no. However, such conclusions may be flawed because the locally stored triglyc-

Kinetic, Circular Dichroism and Fluorescence Studies on Heterologously Expressed Carnitine Palmitoyltransferase II

Km estimates for carnitine and palmitoyl-CoA of heterologously expressed rat liver carnitine palmitoyl-transferase-II (rCPT-II) were 950 +/- 27 microM and 34 +/- 6 microM, respectively. Vmax for the enzyme was 1.8 mumol/min/mg purified protein. Consistent with an ordered reaction mechanism in which palmitoyl-CoA binds first, SDZ CPI 975, a reversible carnitine palmitoyltransferase inhibitor containing both carnitine and alkyl moieties, inhibited rCPT-II competitively with carnitine and uncompetitively with palmitoyl-CoA. Substrate-enzyme interactions were examined by circular dichroism (CD) and fluorescence. Both carnitine and palmitoyl-CoA alone induced conformational changes in the enzyme; dissociation constant estimates by CD for carnitine and palmitoyl-CoA were 41 +/- 5 microM and 7 +/- 2 microM, respectively.

EFFECTS OF SUBSTRATE BINDING AND PH ON THE SECONDARY STRUCTURE OF CARNITINE ACETYLTRANSFERASE

Carnitine acetyltransferase (CAT) exists as a monomer in solution as demonstrated by dynamic light scattering measurements. Under these conditions, interactions between CAT and its substrates, L-carnitine and acetyl-CoA, were studied by circular dichroism (CD) and fluorescence spectroscopy over a wide range of substrate concentrations. CD data indicated that the binding of L-carnitine and acetyl-CoA caused changes in the secondary structure of the protein. Quenching of the intrinsic protein fluorescence upon binding of either substrate corroborated these findings. Analysis of the binding data suggests that binding of both substrates to CAT is specific and saturable, and that there is a single binding site (or multiple identical and independent binding sites) on CAT for each substrate. Estimated L-carnitine/CAT dissociation constants were 506 +/- 58 microM and 236 +/- 27 microM in the absence or presence of acetyl-CoA, respectively. The dissociation constant for acetyl-CoA/CAT was estimated at 19 +/- 7 microM. The effect of pH on the secondary structure of the protein was determined in order to investigate the structural cause for the pH-dependent enzymatic activity of CAT. Loss of alpha-helices and a reduction of thermal stability in CAT was detected at both acidic and basic pH. Thus, the reduced catalytic activity of CAT at acidic or basic pH may be due to pH-induced protein unfolding.