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216 G. Velasco and others pho;phorylation is triggerad by OA (but not by A23187) are requirad for tha stimuiation of CPT-¡. 13V. mas Ide recipiení cf a FEES sbudentship. Thess ¡nvest¡galions mere supported o pan by Ide Nebherlands Faundation fo; Chemical Researod (SON) ¡¡lId linancial aid treo Ide Nethsrlands Organization ter Scieníific Researod (NWO). as melí as by (he Soanish Comísión lníermioister¡aI de Ciencia y Tennologia (5AF93/0281). R Ef ER E NCES ~AcGarry, 1 0.. WeeIb)e. 1 Olabeles .fe
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
Page 1 and 2:
UNiVERSIDAD COMPLUTENSE DE MADRID F
Page 3 and 4: 1. INTRODUCCIÓN ÍNDICE 1.1. ASPEC
Page 5 and 6: L Introducción
Page 7 and 8: Canítulo 1 Introducción en forma
Page 9 and 10: Can/tu/o ¡ 1988) y sales biliares
Page 11 and 12: Capítulo 1 Introducción sistema d
Page 13 and 14: Capitulo 1 Introducción ble la sí
Page 15 and 16: Can¡tu/o 1 introducción Ca racten
Page 17 and 18: Cadhulo 1 Introducción drial exter
Page 19 and 20: Canítulo 1 Introduccián del malon
Page 21 and 22: Capitulo ¡ Introducción Preincuba
Page 23 and 24: Canítulo ¡ BIBLIOGRAFLA Abunirad,
Page 25 and 26: Capítulo ¡ Rodríguez, J.C., Cil-
Page 27 and 28: 2.1. Efectos deJATP extracelulary d
Page 29 and 30: Effects of extracellular ATP Qn hep
Page 31 and 32: Tabla 1. Effect of extracellular A=
Page 33 and 34: EFFECTS OF EXTRACEI 4LULARATP Di HE
Page 35 and 36: EFFECTS OF EXTRACELLTJLAR AIF Di HE
Page 37 and 38: 240 snspansions ware incubatad at 3
Page 39 and 40: Capitulo 2 Bibliograria. DISCUSIÓN
Page 41 and 42: Capitulo 2 INTRODUCCIÓN Resultados
Page 43 and 44: BIOCHEMICAL AND B!OPHYSTCAL RESEARC
Page 45 and 46: Vol. 224, No. 3, 1996 BIOCHEMICAL M
Page 47 and 48: Vol. 224. No. 3, 1996 . BIOCHEMICAL
Page 49 and 50: ajochen. J. (1997)321. 211—216 (P
Page 51 and 52: Table 1 Effect al protejo kinase in
Page 53: ab cd — — — FIgure 4 Eblect o
Page 57 and 58: Immunop~rcipi¡añon of 32P-¡abeil
Page 59 and 60: permeabilizod hepatocytos and CPT-I
Page 61 and 62: (autbors’ urapublisbad rosullis).
Page 63 and 64: TItE JOURNAL OF BIoLoGICA[. CIIEMIS
Page 65 and 66: 2.3. Posible papel fisiológico del
Page 67 and 68: LOSS OF RESPONSE OF CARNITINE PALMI
Page 69 and 70: Lowenstein, Brandeis Uraiversity, W
Page 71 and 72: TABLE 2. Comparative effect of okad
Page 73 and 74: ARCHIVES OF BIOCHEMJSTRY AND BIOPHY
Page 75 and 76: CONTROL OF FATTY ACID OXIDATION BY
Page 77 and 78: -co 100 n H o ocoo E ci, Method 50
Page 79 and 80: CONTROL OF FAflY ACID OXIDATION BY
Page 81 and 82: Vol. 233, No. 1. 1997 íonxc strera
Page 83 and 84: Vol. 233, No. 1, 1997 BIOCIjEMICAL
Page 85 and 86: Capítulo 2 DISCUSIÓN Relvultados
Page 87 and 88: Cavitulo .3 ¡3kcu~ión General y C
Page 89 and 90: Cacílulo 3 Esquema 2 >1. APOPTOSIS
Page 91: Canítulo 3 Discusión Géneral y C
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