MALONYL-COA-INDEPENDENT ACUTE CONTROL OF HEPATIC CARNITINE PALMITOYLTRANSFERASE-I ACTIVITY - ROLE OF CA2-DEPENDENT PROTEIN-KINASE-II AND CYTOSKELETAL COMPONENTS( CALMODULIN)

The mechanism of malonyl-CoA-independent acute control of hepatic carn itine palmitoyltransferase I (CPT-I) activity was investigated. In a f irst series of experiments, the possible involvement of the cytoskelet on in the control of CPT-I activity was studied. The results of these investigations can be summarized as follows. (i) Very mild treatment o f permeabilized hepatocytes with trypsin produced around 50% stimulati on of CPT-I activity. This effect was absent in cells that had been pr etreated 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 i ntact hepatocytes with 3,3'-iminodipropionitrile, a disrupter of inter mediate filaments, increased CPT-I activity in a non-additive manner w ith respect to OA, Taxol, a stabilizer of the cytoskeleton, prevented the OA- and 3,3'-iminodipropionitrile-induced stimulation of CPT-I, (i ii) CPT-I activity in isolated mitochondria was depressed in a dose-de pendent 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 po ssible link between Ca2+/calmodulin-dependent protein kinase II (Ca2+/ CM-PKII) and the cytoskeleton was studied in the context of CPT-I regu lation. The data of these experiments indicate that (i) purified Ca2+/ CM-PKII activated CPT-I in permeabilized hepatocytes but not in isolat ed mitochondria, (ii) purified Ca2+/CM-PKII abrogated the inhibition o f CPT-I of isolated mitochondria induced by a cytokeratin-enriched fra ction, and (iii) the Ca2+/CM-PKII inhibitor KN-62 prevented the OA-ind uced phosphorylation of cytokeratins in intact hepatocytes, Results th us support a novel mechanism of short-term control of hepatic CPT-I ac tivity which may rely on the cascade Ca2+/CM-PKII activation --> cytok eratin phosphorylation --> CPT-I de-inhibition.