Paradloxical effects of clofibrate on liver and muscle metabolismii in rats: Induction of myotonia and alteration of fatty acidl ancd glucose oxidation

Abstract

A B S T R A C T Chronic clofibrate intake, on occasion, results in a muscular syndrome in man. We have investigated the effects of chronic clofibrate administration in rats on the electrical activity of a skeletal muiscle (gastrocnemius), its composition, and its oxidation of palmitate and glucose. These effects have been compared with those in the liver. Clofibrate administration altered electromyographic pattern of gastrocnemius muscle (characteristic of myotonia), decreased its protein content, and impaired its oxidation of palmitate and glucose. These INTRODUCTION Clofibrate has been widely used as a therapeutic agent in the treatment ofpatients with hyperlipidemias. Because clofibrate also lowers triglyceride and cholesterol concentrations in rat plasma, the mechanism of its action and some of its metabolic effects have been extensively studied in this animal model (1). These studies have shown that clofibrate has profound effects on the histology, chemical composition, and biochemical reactions of the liver (2-5). In contrast to liver, the effects of clofibrate on other tissues, particularly skeletal muscle, have not been adequately studied. The importance of such a study is underscored by the clinical observations that clofibrate therapy, on occasion, results in a muscular syndrome which is characterized by muscle weakness, pain, and tenderness, together with a rise in serum creatine phosphokinase and transaminase activity (6-10). In this series of experiments, we have investigated the effect of clofibrate on the electrical activity, composition, and glucose and palmitate oxidation of rat skeletal muscle and have compared these biochemical effects with those in the liver. After finding that fatty acid oxidation is paradoxically affected in these tissues, (increased in the liver and decreased in the muscle) we studied the effect of clofibrate on carnitine concentration and the activity of carnitine palmitoyltransferase in the liver and skeletal muscle. These studies were undertaken in view of the requirement of carnitine and carnitine acyltransferase for the oxidation of fatty acids (11). Furthermore, it has been postulated that these factors play a regulatory role in fatty acid oxidation and ketogenesis Electromyography. Electromyography was performed with a TECA model TE-4 Electromyograph (TECA Corp., White Plains, N. Y.) and a triaxial needle electrode. Control and clofibrate-treated rats were anesthetized with urethane (125 mg/100 g body wt, i.p.), and the gastrocnemius muscle was exposed by incising and reflecting the skin of the hind limb. The triaxial needle electrode was inserted in the gastrocnemius muscle to record the electrical activity. The electrical discharges were visualized on the electromyograph oscilloscope and recorded on a fiber optic printout. After the base line was established, the muscle was stimulated by a gentle movement of the electrode, and the electrical activity was monitored continuously. This process was repeated several times to confirm the reproducibility of the electrical activity. The recording of electrical activity from control and clofibrate-treated rats was done under identical conditions. The observer did not know whether the animals were controls or had been treated with clofibrate. Tissue preparation. Rats were sacrificed by stunning and decapitation 4 h after the last dose of clofibrate. Blood was collected in chilled, heparinized tubes, and a small portion of liver and gastrocnemius muscle was quickly removed and pressed between metal clamps which had been previously cooled in liquid nitrogen. Preparation of plasma and tissue homogenates were followed by the methods previously described (16, 17). Isolation of mitochondria. Mitochondria from the liver and gastrocnemius muscle were isolated as outlined by Hogeboom (18) and Ernster and Nordenbrand (19), respectively. Palmitate and glucose oxidation in vitro. The rate of palmitate oxidation was determined by measuring the rate of "4CO2 production when [1-_4C]palmitate (55.26 mCi/mmol) was incubated with whole homogenates of the tissues as reported previously The rate ofglucose oxidation was determined by measuring the rate of "4CO2 production when [U-14C]glucose (313 mCi/ mmol) was incubated with tissue homogenates. Preliminary experiments were carried out to determine the optimum conditions for glucose oxidation by the muscle and liver homogenates. Several cofactors at varying concentrations were tested for their ability to increase the rate ofglucose oxidation. Maximum rate ofglucose oxidation by the muscle homogenate was achieved when 3 mM ATP was added to the incubation medium. For maximum rate of glucose oxidation by the liver homogenate, the addition of 3 mM ATP and 2 mM NADP was required. These cofactors were added for this study of glucose oxidation. Incubation studies, in duplicate, were carried out in 25-ml Erlenmeyer flasks which contained center wells fitted with tubes. The reaction mixture contained in a final volume of 5.0 ml, 619 ,umol NaCl, 10.6 ,umol MgSO4, 2.7 ,umol KH2PO4, 109.4 ,umol Na2HPO4, 8.7 ,umol NaH2PO4, 15 ,umol ATP, 2.0 ,uCi of D_[U-_4C]glucose, 50 mg of homogenized tissue (6-8 mg protein), and a physiological concentration of glucose (8 mM). For glucose oxidation by the liver, the above incubation mixture also contained 10 ,umol of NADP. Incubation of the reactions and determination of radioactivity were carried out by previously described methods To determine background radioactivity, tissue homogenates were boiled for 1 min and then studied as above. This background activity was subtracted from the amount of 14CO2 produced when glucose was incubated with nonboiled tissue homogenates. The amount (nanomoles) of glucose oxidized was calculated from the amount of radioactivity determined in each vial and corrected for the specific activity of glucos