liiiMIIIfl~UDliiiMIII~U - Biblioteca de la Universidad Complutense ...

THE JOURNAL OP WOLOGICAL CI4EM¡SIRY (en revisión)
Malonyl-CoA-independent Acute Control of
Hepat¡c Carnitine Palmitoyltransferase 1 Activity
Role of Ca2~/ca1moduIin-dependent Protein K¡nase II aud Cytoskeletal Component?
Guillermo Velascot, Math J.H. Geelen, and Manuel Guzmánt
From the ‘~Department of Biochemistry and Molecular Biolo~r 1, School of Bio)o~’, Complutense University, 28040-Madrid,
Spain, and the ‘ Laboratory of Veíerina¡y Biochemistry, Schooi of Veterinaay Medicine, Utrecht University, 3508-TD Utrecht,
The Netheriands
This work was supported by granrs froan the Spanish Comisión Interministerial de Ciencia y Tecnología (SAF9Ó/O113) arad
Fondo de Investigación Sanitaria (FIS 97/0039), as well as fror» the Ncthcrlands Foundation for Chemical Research (SON)
and the Netheriands Organization for Sciantific Research (NWO)
-The mechanism of malonyl-CoA-independent acate
control of hepatie carn¡tine palmitoyltransferase 1 (CP’T-I)
acrivity was investigated. In a first series of experiments,
the possible involvement of the cytoskeleton in the control
of CI’T-1 activity was studied (1) Treatment of
pe¡-meabllized hepatocytes with tryps¡n ¡u very mild
conditions produced a ca. 50% stimulation of CPT-1. This
effect was not observed in celis that bad been pretreated
with okadaic acid (QA) and seemed to be due to tbe action
of tryps¡n 00 cdl component(s) distinct from CPT-I. (u)
Incubation of ¡ntact bepatocytes with 3,3’-
iminodipropionitrile (IDPN), a disruptor of intermediare
filaments, ¡nci-eased CPT-1 activity in a non-addit¡ve
manner with respect to OX Taxol, a stabiiizer of tbe
cytoskeleton, prevented the QA- and IDPN-induced
stimulatlon of CPT-l. (iii) CPT-I activity iii isolated
mitochondria ns depressed ¡o a dose-dependent fashion
by the addition of a total-cytoskeleton fraction and a
cytokeratin-enriched cytoskeletal fraction, tbe latrer be¡ng
3 times more potent than the former. lo a second series of
experiments, tbe poss¡ble link between Ca2~/calmodulin-
dependent protein kinase 11 (Ca2/CM-PKII) and the
cytoskeleton ii-as stud¡ed in the contefl of CFI’-1
regulation. Cytokeratin phosphorylation -> CPT-1 de-inhibition.
Mitochondrial fatty acid oxidation in livor provides a
major sourco of energy Lo this organ and supplios
extrahepatic tissuos with ketone bodios as a glucose-
replacing fuel (1,2). Carnitino palmitoyltransforase 1 (CFI’-
1), the outer-mitochondrial-menibrano carnitino
pa]mitoyltransforaso, catalyzos the pace-sotting stop of long-
chain fatty acid translocation hito dio mitochondrial matrix
(1-5). Moreovor, receut determination of flux control
1
coefficients of the enzymes involved in hepatic iong-chain
fatty acid oxidation shows that CPT-I plays a pivotal role in
controlling tho flux through tbk patbway undor different
substrato concontrations and patho-physiological ataros
(6,7). CPT-I is subjectod Lo long-torm rogulation lii
responk to altorations in tho nutrirional aud hormonal
status of tSe animal (1,2,5). Short-term control of CPT-I
acdvity involves inhibition by malonyl-CoA, the product of
the roaction catalyzed by acetyl-CoA carboxylase (8). Since
tho lattor onzyme ja a koy regulatory site of fatty acid
synthosis de novo (cf. 1-5), malonyl-CoA inhibition of CFI’-
1 allows an elegant expianation for tho coordinato control
of tho partition of hepatic fatty acids betweon ostorification
and oxidation. As a matror of fact, ovidonce has
accumulated during tSe lasÉ two docados highlighting tSe
pbysiological importanco of malonyi-CoA inhibition of
CPT-I not only in livor bur also iii extra-hopatic tissues
(1,5).
During tho lasÉ years, howovor, a novel mochanism of
control of hepatic CPT-I activity has beon pur forward.
Srudios using permeabilizod hopatocytos hayo shown that
varinus agonts oxort short-torm changos ¡u CP’I’-I activity iii
parallol with changos ¡u tho rato of long-chain fatty acid
oxidation (3,9). Thoso shorr-rerm changos ira bopatio CPT-l
activity are asaumed to be mediatod by a malonyl-CoAindopendent
mochanism, since they survive cd
pormabilization, extensivo washing of tSe permoabilizod
celis «o aliow complote reoñoval of malonyl-CoA) and
subsequont proincubation of tho coil ghosts at 3TC before
detormination of CPT-I activity (to allow equalization of
tho conformational atate of CPT-I) (10). Evidonco has also
boen prosontod showing thaÉ the stimu¡ation of hepatic
CPT-I by tSe phosphataso inhibitor okadaic acid (OA),
used as a modol compound to study tho shorr-torm
rogulation of CPT-I, doca nor involvo tho direct
phosphoryiation of CPT-I (10). It has been rocontly shown
that tSe OA-inducod stimulation of CPT-I ¡a prevented by
KN-62, an inhibitor of Ca2~/cahnodulin-dependont protoin
kinase II (Ca2~/CM-PKII) (11), aud by taxol, a atabilizer of
the cyroskeleton (12). These obsorvations suggost that both
activation of Ca2~/CM-PKII aud disruption of tho
cytoskoloton may be nocossary for the QA-induced
stimulation of CPT-I Lo be demonstrated. It os conceivable