Malonyl-CoA-independent Acute Control of Hepatic Carnitine Palmitoyltransferase I Activity

Abstract

The mechanism of malonyl-CoA-independent acute control of hepatic carnitine palmitoyltransferase I (CPT-I) activity was investigated. In a first series of experiments, the possible involvement of the cytoskeleton in the control of CPT-I activity was studied. The results of these investigations can be summarized as follows. (i) Very mild treatment of permeabilized hepatocytes with trypsin produced around 50% stimulation of CPT-I activity. This effect was absent in cells that had been pretreated with okadaic acid (OA) and seemed to be due to the action of trypsin on cell component(s) distinct from CPT-I. (ii) Incubation of intact hepatocytes with 3,3-iminodipropionitrile, a disruptor of intermediate filaments, increased CPT-I activity in a non-additive manner with respect to OA. Taxol, a stabilizer of the cytoskeleton, prevented the OA-and 3,3-iminodipropionitrile-induced stimulation of CPT-I. (iii) CPT-I activity in isolated mitochondria was depressed in a dose-dependent fashion by the addition of a total cytoskeleton fraction and a cytokeratin-enriched cytoskeletal fraction, the latter being 3 times more potent than the former. In a second series of experiments, the possible link between Ca 2؉ /calmodulin-dependent protein kinase II (Ca 2؉ /CM-PKII) and the cytoskeleton was studied in the context of CPT-I regulation. The data of these experiments indicate that (i) purified Ca 2؉ /CM-PKII activated CPT-I in permeabilized hepatocytes but not in isolated mitochondria, (ii) purified Ca 2؉ /CM-PKII abrogated the inhibition of CPT-I of isolated mitochondria induced by a cytokeratin-enriched fraction, and (iii) the Ca 2؉ /CM-PKII inhibitor KN-62 prevented the OA-induced phosphorylation of cytokeratins in intact hepatocytes. Results thus support a novel mechanism of short-term control of hepatic CPT-I activity which may rely on the cascade Ca 2؉ /CM-PKII activation 3 cytokeratin phosphorylation 3 CPT-I de-inhibition.Mitochondrial fatty acid oxidation in liver provides a major source of energy to this organ and supplies extrahepatic tissues with ketone bodies as a glucose-replacing fuel (1, 2). Carnitine palmitoyltransferase I (CPT-I), 1 the carnitine palmitoyltransferase of the mitochondrial outer membrane, catalyzes the pace-setting step of long-chain fatty acid translocation into the mitochondrial matrix (1-5). Moreover, recent determination of flux control coefficients of the enzymes involved in hepatic long-chain fatty acid oxidation shows that CPT-I plays a pivotal role in controlling the flux through this pathway under different substrate concentrations and pathophysiological states (6, 7). CPT-I is subject to long-term regulation in response to alterations in the nutritional and hormonal status of the animal (1, 2, 5). Short-term control of CPT-I activity involves inhibition by malonyl-CoA, the product of the reaction catalyzed by acetyl-CoA carboxylase (8). Since the latter enzyme is a key regulatory site of fatty acid synthesis de novo (cf. Refs. 1-5), malonyl-CoA inhibition of CPT-I allows an elegant explanati...

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“…Like the AMP-activated protein kinase, PKA may stimulate CPT-1, a key enzyme of fatty acid oxidation, by two mechanisms: one is by decreasing malonyl-CoA levels through phosphorylation and inactivation of ACC, and the other is by modulating the interactions between CPT-1 and the cytoskeleton through phosphorylation of intermediate filaments in a malonyl-CoA-independent manner (23,24). Our results suggest that PKA activation is a key step for the leptin-induced ROS generation in BAEC.…”

Liver denervation affects hepatocyte mitochondrial fatty acid transport capacity

“…contact sites or outer membrane) 39 -and of regulation mediated by the cytoskeleton. 40 Therefore, in our system short-term regulation of enzyme activity was not addressed, nor was its interaction with malonyl CoA. Long-term regulation of enzyme activity, regarding its catalytic capacity, but not its concentration, since results are expressed in terms of mg of protein, was the focus of the present study.…”

Identification of a novel malonyl-CoA IC50 for CPT-I: implications for predicting in vivo fatty acid oxidation rates

“…P/O ratios), several physiologically relevant mitochondrial characteristics are altered during the isolation procedure [6-8]. Additionally, the removal of the cytoskeleton during the isolation procedure may alter the inherent regulatory mechanisms associated with LCFA oxidation as the cytoskeleton has previously been shown to influence CPT-I activity [9,10] and other mitochondrial regulatory processes [11,12]. In contrast to isolated mitochondria, permeabilized skeletal muscle fibres (PMF) maintain the inherent cytoskeletal architecture and mitochondrial morphology while the sarcolemma is selectively permeabilzed [13,14].…”