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Lowenstein, Brandeis Uraiversity, Waltham, MA, USA), a speciflc irreversible inhibitor of CPT-I (cf. [7]).
Incubatioras were cantinued for an additianal 45-mm period ira the preserace or tha absance of varyirag
coracenlirarioras of akadaic acid. Subsequeralily, carnitine palmitoyltrarasferase activity was manitored ira
hapatocyte monolayers [5] ar susperasioras [7].
Far libe determinatiora of CPT-I activity ira isalated mitochoradria, the culture medium from 10-15
F75 flasks was aspirated, celis wara washed ira NaCl/P 1, scraped from libe flasks, arad homogenized ira a
madium coratainirag 10 mM Tris-HCl, pH 7.4, 0.25 M sucrose arad 1 mM EDTA. The resulting crude
bamogenates were directly used for isolation of mitochondria and determinatiora of CPT-I arad glutamate
dehydrogeraase (GDH) activities exactly as described ira [8].
Ststistical analysis
Rasullis shawn represent the means±S.D. of tha raumber of experimenlis iradicated in each case. Each
axperimeratal coraditiara was always carried out at least ira quadruplicate. Sliatistical analysis was performed
by the Studerat’s t tost.
RESULTS AND DISCUSSION
‘¡‘be properties of CPT-I ware studied ira twa hepatoma cdl liraes, namel>’ human HepG2 celís and rat Fao
calís, which are commoraly usad as a modal ro sliudy liver lipid matabolism (cf. [12-14]).As showra ira Table
1, CPT-I specific activity was not sigraificantly different in mitachondria isolated from hepatoma cells than
ira mitochondria isolatad from hepatacytes ira primary culture. Similar qualitative resullis were olitairaed
whera values of enzyma activity ware referred ta mass of mitachondrial protein arad to activity unilis of
GDH, a mirochoradrial marker (Table 1). ‘¡‘bis indicares that no differeraces ira the mass of mitochoradria
are evident araiorag tite threa types of celís.
CPT-I activity was subsequantly moraitorad in a permeabiized-celí system. The use of this assay
allows measuremerar of heparocellular CP’T-I activity in its physialogical environirnerat [7].As showra ira Fig.
1, CF1-I activity ira primary hepatocytes was about half of that ira hepatoma celis. This clearly indicates
that CPT-I activity iraside tite hepatacyte is subjected to certaira corastricti¿ns that do raot aperate ira
hepatoma celís or ira preparatians of purified bepatic mirochoradria. ‘¡‘bis is ira lina with libe recerar idea
lihat interactioras betweera libe mitocharadrial auter membrane arad extra-mitacharadrial celí componanlis,
mast Iikely localizad in the cytoskeleron, might be involved ira the caratrol of hepatic CPT-I activity [8,9].
‘¡‘bese interactioras could be readily lost ira preparations of purified mitochondria.
Ta test libis hyparhesis, the offact of libe phosphaliasa inhibilior okadaic acid on CPT-I activity was
determinad ira lihe libree celís testad. One of libe most remarkable effects elicited by okadaic acid ira
hepatocylias arad arber calI typas is libe hyperphosphoxylaliiora arad subsequerat disruptiora of libe cytoskeleton
(cg. [15,16]).Fig. 1 sbaws that a remarkable 80% srimulation of CPT-I ansued upan exposure of primary
hapaliocytas to akadaic acid. Fifty percarar activariara of CP’1’-I accurred at ca. 10 raM okadaic acid,