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
Ca2+/calmodulin-dependent protein kinase
II (Ca2+/CM-PKII) and the cytoskeleton was studied in the
context of CPT-I regulation. The data of these experiments indicate
that (i) purified Ca2+/CM-PKII activated CPT-I in
permeabilized hepatocytes but not in isolated mitochondria, (ii)
purified Ca2+/CM-PKII abrogated the inhibition of CPT-I of
isolated mitochondria induced by a cytokeratin-enriched fraction, and
(iii) the Ca2+/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 Ca2+/CM-PKII
activation
cytokeratin phosphorylation
CPT-I de-inhibition.
 |
INTRODUCTION |
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 explanation for the coordinate control of the
partition of hepatic fatty acids into esterification and oxidation. As
a matter of fact, evidence has accumulated during the last two decades highlighting the physiological importance of malonyl-CoA inhibition of
CPT-I not only in liver but also in extrahepatic tissues (1, 5).
During the last years, however, a novel mechanism of control of hepatic
CPT-I activity has been put forward. Studies using permeabilized
hepatocytes have shown that various agents exert short-term changes in
CPT-I activity in parallel with changes in the rate of long-chain fatty
acid oxidation (3, 9). These short-term changes in hepatic CPT-I
activity are assumed to be mediated by a malonyl-CoA-independent
mechanism, since they survive cell permabilization, extensive washing
of the permeabilized cells (to allow complete removal of malonyl-CoA),
and subsequent preincubation of the cell ghosts at 37 °C before
determination of CPT-I activity (to allow recovery of the original
conformational state of CPT-I) (10). Evidence has also been presented
showing that the stimulation of hepatic CPT-I by the phosphatase
inhibitor okadaic acid (OA), used as a model compound to study the
short-term regulation of CPT-I, does not involve the direct
phosphorylation of CPT-I (10). It has been recently shown that the
OA-induced stimulation of CPT-I is prevented by KN-62, an inhibitor of
Ca2+/calmodulin-dependent protein kinase II
(Ca2+/CM-PKII) (11), and by taxol, a stabilizer of
the cytoskeleton (12). These observations suggest that both activation
of Ca2+/CM-PKII and disruption of the cytoskeleton may be
necessary for the OA-induced stimulation of CPT-I to be demonstrated.
It is conceivable that these two processes may be related, since
Ca2+/CM-PKII is one of the protein kinases more actively
involved in the control of the integrity of the cytoskeleton by
phosphorylating cytoskeletal proteins (13). However, the events
underlying this novel mechanism of control of CPT-I activity are as yet
unknown. The present work was thus undertaken to study the molecular
basis of the malonyl-CoA-independent short-term control of hepatic
CPT-I activity.
 |
EXPERIMENTAL PROCEDURES |
Materials--
L-[methyl-3H]Carnitine,
carrier-free [32P]Pi,
[1-14C] acetyl-CoA, the ECL detection kit, and the
protein kinase C assay kit were from Amersham International (Amersham,
Bucks, United Kingdom). Tetradecylglycidate was kindly donated by Dr.
J. M. Lowenstein (Brandeis University, Waltham, MA). The
anti-CPT-I polyclonal antibody (raised against peptide residues
428-441 of rat liver CPT-I) was kindly given by Dr. V. A. Zammit
(Hannah Research Institute, Ayr, United Kingdom). The anti-cytokeratin
8, anti-cytokeratin 18, and anti-actin monoclonal antibodies were
kindly given by Dr. P. M. P. Van Bergen en Henegouwen
(Utrecht University, The Netherlands). Colchicine and cytochalasin B
were kindly gifted by Dr. J. M. Andreu (CIB, Madrid, Spain).
3,3'-Iminodipropionitrile (IDPN) was from Acros Chimica (Geel,
Belgium). OA, KN-62, A23187, and classical protein kinase C were from
Calbiochem (San Diego, CA). Ca2+/CM-PKII was from Biomol
(Plymouth Meeting, PA). Purified cytokeratins 8 and 18 were from ICN
Pharmaceuticals (Costa Mesa, CA). cAMP-dependent protein
kinase, trypsin, and the anti-actin and anti-pan cytokeratin monoclonal
antibodies were from Sigma.
Isolation and Incubation of Hepatocytes--
Male Wistar rats
(200-250 g) which had free access to food and water were used in all
experiments. Hepatocytes were isolated by the collagenase perfusion
method and routinely incubated in Krebs-Henseleit bicarbonate buffer
(pH 7.4) supplemented with 10 mM glucose and 1% (w/v)
defatted and dialyzed bovine serum albumin as described before (9).
Assay of CPT-I Activity in Isolated
Mitochondria--
Mitochondria were isolated either from hepatocytes
or from intact liver and CPT-I activity was measured as the
malonyl-CoA-sensitive incorporation of radiolabeled
L-carnitine into palmitoylcarnitine exactly as described
before (10). When CPT-I activity was determined in suspensions of
mitochondria containing cytoskeletal fractions (Figs. 3 and 4), CPT
activity in the cytoskeletal fractions was subtracted from the CPT-I
activity experimentally determined. In any event, CPT activity
determined in those cytoskeletal fractions was always marginal and on
the basis of protein content never accounted for more than 5% of the
CPT-I activity measured in mitochondrial suspensions. Preparations of
mitochondria were practically devoid of peroxisomes, as judged from the
low recovery of catalase activity (<5%) in those preparations.
Assay of CPT-I Activity in Permeabilized Hepatocytes--
After
incubation of the hepatocytes with the additions indicated in each
case, CPT-I activity was determined in digitonin-permeabilized hepatocytes as the tetradecylglycidate-sensitive incorporation of
radiolabeled L-carnitine into palmitoylcarnitine (9, 10). In the "one-step assay" (Table II), cell permeabilization and assay
of enzyme activity are simultaneously performed (9). In other
experiments, however, CPT-I activity was determined by a more complex
procedure (Fig. 1). In this "two-step" assay, hepatocytes are
permeabilized with digitonin, and then extensively washed with a medium
containing 10 mM Tris-HCl (pH 7.4), 50 mM
potassium fluoride, 100 mM KCl, 2.5 mM EDTA,
and 2.5 mM EGTA. The permeabilized cell pellets were taken
up in that medium with the additions indicated and CPT-I activity was
subsequently determined after preincubation at 37 °C for 5 min
(10).
Western Blot Analysis of CPT-I--
Mitochondrial fractions were
subjected to SDS-PAGE using 10% polyacrylamide gels and proteins were
further transferred onto nitrocellulose membranes. The blots were then
blocked with 5% fat-free dried milk in phosphate-buffered saline
supplemented with 0.1% Tween 20. They were subsequently incubated with
the anti-CPT-I antibody (1:10,000) in phosphate-buffered saline/Tween 20 for 2 h at 4 °C, and washed thoroughly. The blots were then incubated with anti-sheep peroxidase-conjugated secondary antibody (1:10,000) for 1 h at room temperature, and finally subjected to
luminography with an ECL detection kit.
Isolation of Cytoskeletal Fractions--
Isolated hepatocytes
were sedimented (2 min at 100 × g) and resuspended in
a cytoskeleton stabilizing buffer consisting of 10 mM Pipes
(pH 6.8), 0.25 M sucrose, 3 mM
MgCl2, 150 mM KCl, and 1 mM EGTA,
supplemented with a proteinase/inhibitor mixture (11, 15). The fraction
corresponding to total cytoskeleton (Fraction I) as well as the
fraction enriched in intermediate filaments (Fraction II) were
prepared according to van Bergen en Henegouwen et al.
(14).
Western Blot Analysis of Cytoskeletal Proteins--
Cytoskeletal
fractions were subjected to SDS-PAGE using 10% polyacrylamide gels and
proteins were further transferred onto polyvinylidene fluoride
membranes. The blots were then blocked with 2% Protifar (Nutricia,
Zoetermeer, The Netherlands) in 50 mM Tris-HCl (pH 7.8),
100 mM NaCl, and 0.1% Tween 20 (TBST). They were
subsequently incubated with the anti-actin, anti-tubulin, anti-cytokeratin 8, and anti-cytokeratin 18 monoclonal antibodies (1:10,000) in TBST with 0.2% Protifar for 1 h at 4 °C, and
washed thoroughly. The blots were incubated with peroxidase-conjugated secondary antibodies (1:10,000) for 1 h at room temperature, and finally subjected to luminography with the ECL detection kit.
Immunoprecipitation of 32P-Labeled
Cytokeratins--
After isolation, hepatocytes were washed twice in
phosphate-free Dulbecco's modified Eagle's medium supplemented with
1% (w/v) defatted and dialyzed bovine serum albumin. Hepatocytes (6-8
mg of cellular protein in 1.5 ml of the aforementioned medium) were subsequently incubated in that medium at 37 °C for 1 h with 0.2 mCi of [32P]Pi and subsequently exposed to
the additions indicated. One ml of cells was rapidly sedimented (5 s at
12,000 × g) and resuspended in 0.5 ml of 50 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 1% (v/v)
Igepal, 0.5% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS,
supplemented with proteinase inhibitors (11, 15). Further treatment of the samples and immunoprecipitation with the monoclonal anti-pan cytokeratin antibody bound to protein A-Sepharose were performed as
described (11, 15).
Determination of the Stoichiometry of Cytokeratin
Phosphorylation--
The stoichiometry of cytokeratin phosphorylation
was calculated by simultaneously determining (i) the specific
radioactivity of the
-phosphate of intracellular ATP, (ii) the
amount of 32P incorporated into the cytokeratin bands, and
(iii) the mass of protein in those cytokeratin bands.
(i) To determine the specific radioactivity of the
-phosphate of
intracellular ATP, hepatocytes were labeled with 32P for 60 min to achieve steady-state labeling of proteins before addition of the
agonists (16). After the indicated times, 1.0 ml of cells was
precipitated with 0.15 ml of 2 M HClO4. After neutralization with K2CO3, samples were
centrifuged (20,000 × g, 15 min). Supernatants were
filtered through a filter of 0.22-µm pore diameter and subsequently
used for nucleotide separation exactly as described by Gualix et
al. (17). The specific radioactivity of nucleotides was determined
by measuring in parallel the nucleotide concentration after
transformation of the A259 peak areas to masses by correlation with standards and the 32P incorporation
into those peaks (18). The specific activity of the
-phosphate of
ATP was taken to be the difference between the specific activities of
ATP and ADP (18, 19).
(ii) To determine the amount of 32P incorporated into
cytokeratins, immunoprecipitates were obtained and treated exactly as described above, the two labeled bands in the gels were cut out and
their radioactivity was determined.
(iii) To determine the amount of protein in the labeled bands
corresponding to cytokeratins 8 and 18, immunoprecipitates were obtained exactly as described above except that
[32P]Pi was omitted from the hepatocyte
incubation medium. Immunoprecipitates were subjected to SDS-PAGE
together with varying concentrations of purified cytokeratin 8 and
cytokeratin 18. Bands were visualized by the silver staining technique
(20), and the amount of protein in the cytokeratin bands was calculated
by interpolating the values of optical density of the
immunoprecipitated protein bands in the standard curve of protein mass
versus optical density constructed with the commercial,
purified cytokeratins 8 and 18. The standard curve was corrected
for the contaminating proteins present in the commercial preparations
of purified cytokeratins 8 and 18. In order to improve the
visualization of the stained protein bands of the immunoprecipitate
(e.g. Fig. 6), the amount of immunoprecipitate applied for
determination of cytokeratin mass was routinely 3 times the amount used
for other purposes (e.g. cytokeratin phosphorylation, Western blotting). Values of cytokeratin mass inferred were corrected for this factor.
Incubation with Protein Kinases--
Permeabilized hepatocytes
or isolated mitochondria (1.5-2.0 mg of protein/ml), supplemented or
not with cytoskeletal fractions (as indicated in every case), were
incubated at 30 °C for 10 min in either of the following
phosphorylation media, and aliquots of the incubations were
subsequently taken to determine CPT-I activity as described above. (i)
Ca2+/CM-PKII phosphorylation medium contained 50 mM Hepes/KOH (pH 7.4), 100 mM KCl, 1 mM CaCl2, 10 mM MgCl2,
0.1 mM ATP, 30 ng/µl calmodulin, 50 nM OA,
and 0.6 ng/µl purified Ca2+/CM-PKII, essentially as
recommended by the supplier. (ii) cAMP-dependent protein
kinase phosphorylation medium was exactly as described before (10).
(iii) Protein kinase C phosphorylation medium contained 3 milliunits/µl purified protein kinase C and the assay components according to the supplier.
Determination of Malonyl-CoA Concentration--
Intracellular
levels of malonyl-CoA were determined in neutralized perchloric acid
extracts by a radioenzymatic method (15).
Statistical Analysis--
Unless otherwise indicated, results
shown represent the mean ± S.D. of the number of hepatocyte
preparations indicated in each case. Incubations of hepatocytes or
mitochondria as well as enzyme assays were always carried out in
triplicate. Statistical analysis was performed by Student's
t test.
 |
RESULTS |
Effect of Mild Trypsin Digestion on CPT-I Activity--
In a first
set of experiments aimed at determining whether extramitochondrial
components may be involved in the control of CPT-I activity (12),
permeabilized hepatocytes were treated with trypsin in very mild
conditions (low doses, 4 °C, 2 min) and CPT-I activity was
subsequently determined. As shown in Fig. 1, when hepatocytes were incubated
without further additions and then permeabilized with digitonin,
trypsin was able to stimulate CPT-I by approximately 50% in these cell
ghosts. Preincubation of hepatocytes with OA led to a similar
activation of CPT-I in the permeabilized cell system (Fig. 1). However,
trypsin was unable to produce any further stimulation of CPT-I in
ghosts prepared from OA-pretreated hepatocytes (Fig. 1). The
cytoskeletal stabilizer taxol has been shown to prevent the changes in
hepatic CPT-I activity induced by a number of cellular effectors
including OA (12). Likewise, when hepatocytes were pretreated with OA
in combination with taxol, the stimulatory effect of trypsin was
evident (Table I).

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Fig. 1.
Effect of mild trypsin digestion on CPT-I
activity in permeabilized hepatocytes. Hepatocytes were
preincubated for 15 min in the absence ( ) or presence ( ) of 0.5 µM OA. Cells were then permeabilized with digitonin and
thoroughly washed with 40 volumes of digitonin-free medium as described
under "Experimental Procedures." Permeabilized hepatocytes were
subsequently resuspended at 1.5-2.0 mg of protein/ml and treated with
varying concentrations of trypsin at 4 °C. Trypsin action was
stopped after 2 min by addition of 10 mg/ml bovine serum albumin and
immediate washing with 40 volumes of trypsin-free medium. CPT-I
activity was subsequently determined in those permeabilized
hepatocytes. One-hundred percent CPT-I activity was 1.87 ± 0.20 nmol/min × mg of protein. Results correspond to four different
cell preparations. Inset, mitochondria were isolated from
permeabilized hepatocytes that had been treated without (lane
a) or with (lane b) 17.5 µg/ml trypsin, and CPT-I was
detected by Western blotting. The arrow points to the 88-kDa
band.
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Table I
CPT-I activity in permeabilized hepatocytes and isolated mitochondria
after mild trypsin treatment
Hepatocytes were preincubated for 45 min in the absence or presence of
10 µM taxol. Incubations were continued for 15 additional
min with or without 0.5 µM OA. Cells were permeabilized
with digitonin, and ghosts (1.5-2.0 mg of protein/ml) were
subsequently treated with or without 12.5 µg of trypsin/ml for 2 min
at 4°C. After trypsin removal, CPT-I activity was determined in
permeabilized cells or in mitochondria isolated from those
permeabilized hepatocytes. Results correspond to four different
experiments.
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To test whether trypsin may cleave CPT-I itself under these digestion
conditions, mitochondria were isolated from control and trypsin-treated
permeabilized hepatocytes and CPT-I was subsequently detected by
Western blotting (Fig. 1). As expected (5), a major band with a
molecular mass of 88 kDa was detected in the blots. In addition, no
differences were observed between the two preparations of mitochondria
(Fig. 1). Thus, the value of relative optical density of the 88-kDa
band of trypsin-treated samples was 100 ± 4% (n = 4), setting at 100% the value for control mitochondria. Furthermore,
CPT-I activity was determined in mitochondria isolated from
permeabilized hepatocytes that had been treated with or without trypsin. As shown in Table I, no differences in CPT-I activity were
evident among the different conditions.
It is worth noting that treatment of permeabilized hepatocytes with
trypsin had no effect on the recovery of total permeabilized cell or
total mitochondrial protein. Thus, when permeabilized hepatocytes at
1.6 ± 0.2 mg of protein (n = 4) were treated
without or with 17.5 µg of trypsin for 2 min at 4 °C and ghosts
were collected after stopping trypsin action as described in legend to
Fig. 1, 1.5 ± 0.1 and 1.5 ± 0.2 mg of ghost protein were
recovered, respectively. Likewise, when mitochondria were isolated from
those ghosts, 0.18 ± 0.04 and 0.19 ± 0.06 mg of protein
were recovered in mitochondria prepared from trypsin-treated and
trypsin-untreated ghosts, respectively.
Since permeabilized hepatocytes also express CPT activity from
peroxisomes and microsomes (cf. Refs. 1-5), the
contribution of CPT-I to total hepatocellular
tetradecylglycidate-sensitive CPT activity was quantified. Thus,
hepatocytes were incubated with 10 µM tetradecylglycidate
for 30 min; purified mitochondria, peroxisomes, and microsomes were
isolated (9), and CPT activity was measured in these fractions. It
turned out that at least 85% of total tetradecylglycidate-sensitive
CPT activity experimentally determined routinely corresponded to CPT-I,
whereas microsomal CPT and peroxisomal CPT together made a minor
contribution (<15%) to the tetradecylglycidate-sensitive CPT pool
under these conditions. Therefore, we believe that determination of
CPT-I activity by our permeabilized hepatocyte procedure is not prone
to substantial error.
Effect of Disruptors of the Cytoskeleton on CPT-I Activity--
OA
and other phosphatase inhibitors produce hyperphosphorylation and
consequently disruption of the cytoskeletal network in several cell
types, including hepatocytes (e.g. Refs. 21 and 22). To test
whether changes in the organization of the cytoskeleton may be related
to parallel changes in CPT-I activity, hepatocytes were incubated with
colchicine (a microtubule disruptor), cytochalasin B (a microfilament
disruptor), and IDPN (an intermediate filament disruptor) (23, 24). As
shown in Table II, neither colchicine nor
cytochalasin B affected CPT-I activity. As a control to prove the
biological activity of these two compounds in hepatocytes, cellular
lipids were prelabeled with [14C]palmitate, and very
low-density lipoprotein output into the medium was monitored (25). As
described previously (25), disruption of microtubules with colchicine
or disruption of microfilaments with cytochalasin B led to a strong
inhibition (>90%) of the output of very low-density lipoprotein
lipids into the medium and to a parallel accumulation of intracellular
lipids.
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Table II
Effect of disruptors of the cytoskeleton on CPT-I activity and
malonyl-CoA levels
Hepatocytes were preincubated for 45 min in the absence or presence of
the modulators of the integrity of the cytoskeleton as indicated.
Incubations were continued for 15 min additional with or without 0.5 µM OA, and then aliquots were taken to determine the
level of malonyl-CoA as well as the activity of CPT-I by the one-step
assay. One-hundred percent values of CPT-I activity and malonyl-CoA
concentration were 1.29 ± 0.22 nmol/min × mg of protein and
73 ± 12 pmol/mg of protein, respectively. Results correspond to
the number of experiments indicated in parentheses for CPT-I activity
and to three different experiments for malonyl-CoA concentration.
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In contrast to colchicine and cytochalasin B, IDPN produced a
significant increase in CPT-I activity (Table II). Interestingly, the
effects of IDPN and OA were basically non-additive (Table II).
Furthermore, stabilization of the cytoskeleton with taxol prevented the
stimulation of CPT-I induced by IDPN and OA, either alone or in
combination (Table II). It should be pointed out that neither taxol nor
IDPN changed by themselves the malonyl-CoA concentration in hepatocytes
(Table II). In addition, neither of these two compounds affected the
OA-induced decrease of intracellular malonyl-CoA levels (Table II).
Effect of Cytoskeletal Fractions on CPT-I Activity--
To further
support the notion that cytoskeletal components may inhibit CPT-I, two
cytoskeletal fractions were isolated from rat hepatocytes to study
their possible inhibitory effect on CPT-I. The composition of these two
fractions is shown in Fig. 2. Thus, the
total cytoskeleton fraction (Fraction I) contained the major components
of microtubules (tubulin), microfilaments (actin), and intermediate
filaments (cytokeratins 8 and 18). The bands in the blots showed the
mobilities expected for the molecular weights of the respective
proteins (14). In the case of cytokeratin 8, a minor band of 45 kDa
(perhaps cytokeratin 18) was sometimes detected in the blots (Fig.
2C), whereas a minor band of 54 kDa (perhaps cytokeratin 8)
was sometimes detected in the blots of cytokeratin 18 (Fig.
2D).

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Fig. 2.
Characterization of cytoskeletal
fractions. A total cytoskeleton fraction (Fraction I) and a
fraction enriched in intermediate-filament components (Fraction II)
were obtained from rat hepatocytes and characterized for their content
in tubulin (A), actin (B), cytokeratin 8 (C), and cytokeratin 18 (D) as described under
"Experimental Procedures." Equal amounts of total protein were
applied on every lane. Values of molecular mass (in kDa) of the various
protein bands are shown on the left-hand side of every
luminogram.
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In contrast to Fraction I, the fraction that was intended to be more
enriched in intermediate filaments (Fraction II) actually contained
more cytokeratins that Fraction I and was practically devoid of actin
and tubulin (Fig. 2). In particular, the content of cytokeratins
(relative to total protein content) of Fraction II was 4.0 ± 1.3 (cytokeratin 8) and 3.7 ± 0.8 (cytokeratin 18) times that of
Fraction I (n = 3).
Isolated rat liver mitochondria were then incubated with the
cytoskeletal fractions and CPT-I activity was subsequently determined. As shown in Fig. 3, the two cytoskeletal
fractions produced a dose-dependent inhibition of CPT-I
activity. In agreement with the effect of IDPN described above, the
fraction that was more enriched in intermediate filament components
(Fraction II) produced a more potent inhibition of CPT-I (Fig. 3).
Fifty percent inhibition of CPT-I by the two fractions occurred at
total cytoskeletal protein:total mitochondrial protein ratios of
0.104 ± 0.025 and 0.032 ± 0.008 for Fraction I and Fraction
II, respectively.

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Fig. 3.
Effect of cytoskeletal fractions on CPT-I
activity. Rat liver mitochondria (1.5-2.0 mg of protein/ml) were
incubated for 30 min with varying amounts of cytoskeletal fractions
( , Fraction I; , Fraction II) and CPT-I activity was subsequently
determined as indicated under "Experimental Procedures."
One-hundred percent CPT-I activity was 8.65 ± 1.11 nmol/min × mg of protein. Results correspond to four different
experiments.
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Effect of Purified Protein Kinases on CPT-I Activity--
On the
basis of the antagonistic effect exerted by KN-62, an inhibitor of
Ca2+/CM-PKII (26), the OA-dependent stimulation
of CPT-I has been suggested to rely on the phosphorylation and
subsequent activation of Ca2+/CM-PKII (11). Hence, purified
autophosphorylated Ca2+/CM-PKII was directly added to
isolated mitochondria or permeabilized hepatocytes and CPT-I activity
was determined. Ca2+/CM-PKII was able to significantly
(p < 0.01) stimulate CPT-I in permeabilized cells
(140 ± 8% stimulation, n = 4) but not in isolated mitochondria (6 ± 7% stimulation, n = 4). In contrast, addition of purified cAMP-dependent
protein kinase or protein kinase C to permeabilized hepatocytes did not
produce any change in CPT-I activity in either isolated mitochondria or
permeabilized hepatocytes (data not shown).
In order to define the cell components that are sufficient for the
malonyl-CoA-independent control of CPT-I to be demonstrated, we
next attempted to reconstitute the whole-cell experimental system in a
simple manner by incubating isolated mitochondria together with
cytoskeletal Fraction II and purified Ca2+/CM-PKII. As
shown in Fig. 4, the inhibition of CPT-I
produced by exposure of isolated mitochondria to cytoskeletal Fraction II was reverted by addition of exogenous Ca2+/CM-PKII.

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Fig. 4.
Effect of Ca2+/CM-PKII
on CPT-I activity. Rat liver mitochondria (1.5-2.0 mg of
protein/ml) were preincubated for 30 min in the absence ( ) or
presence (+) of cytoskeletal Fraction II (0.05-0.06 mg of protein/ml).
Purified Ca2+/CM-PKII was subsequently added (+) or not
( ) to the incubations, which were run for 10 additional min. Aliquots
were subsequently taken to determine CPT-I activity. Results correspond
to four different experiments. *, significantly different
(p < 0.01) from incubations with no additions.
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Phosphorylation of Cytokeratins in Intact Hepatocytes--
To
obtain further evidence for a possible connection between
Ca2+/CM-PKII and intermediate filaments, hepatic
cytokeratin phosphorylation was investigated. The phosphorylation
pattern of purified cytokeratins in vitro may not reflect
their phosphorylation status in more physiological, intact cell systems
(27-29). Therefore, intact hepatocytes were labeled with
32Pi and cytokeratins were immunoprecipitated.
As shown in Fig. 5, two major cytokeratin
bands were phosphorylated upon hepatocyte challenge to OA. These two
bands were assigned to cytokeratins 8 and 18 on the basis of their
molecular mass (54 and 45 kDa, respectively) and high abundance in rat
liver (e.g. Refs. 27-29). Moreover, the OA-induced
phosphorylation of these two bands was prevented by KN-62, the
Ca2+/CM-PKII inhibitor that antagonizes the OA-induced
stimulation of CPT-I (11). Nevertheless, the Ca2+ ionophore
A23187 had no effect on cytokeratin phosphorylation in intact
hepatocytes (Fig. 5, lanes e and f).

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Fig. 5.
Phosphorylation of cytokeratins in intact
hepatocytes. Hepatocytes were labeled with
32Pi as indicated under "Experimental
Procedures" and further incubated for 15 min in the absence or
presence of 30 µM KN-62. Incubations were continued for
an additional 15-min period with or without 0.5 µM OA or
10 µM A23187. Cells were subsequently disrupted and
cytokeratins were immunoprecipitated with the anti-pan cytokeratin
monoclonal antibody. Immunoprecipitates were subjected to SDS-PAGE and
autoradiography. Lane a, no additions; lane b,
OA; lane c, KN-62; lane d, KN-62 plus OA;
lane e, A23187; lane f, KN-62 plus A23187.
Molecular mass markers (in kDa) are shown on the left-hand
side of the autoradiogram. The experiment was repeated three times and
similar results were obtained.
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To confirm that the 32P-labeled bands do, indeed,
correspond to cytokeratins 8 and 18, and not to proteins which have
co-precipitated with the cytokeratins, a Western blotting analysis of
the immunoprecipitated proteins was performed with the anti-cytokeratin
8 and anti-cytokeratin 18 monoclonal antibodies. The rationale of this
experiment was that it would be most unlikely that proteins different
to cytokeratins would also cross-react with the anti-cytokeratin
antibodies on a Western blot, especially when, as in the present study,
different sources of antibodies are used in the immunoprecipitation and in the blotting. As shown in Fig. 6, the
54-kDa band actually contained cytokeratin 8, whereas cytokeratin 18 was actually present in the 45-kDa band.

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Fig. 6.
Quantification of the amount of cytokeratins
8 and 18 in the immunoprecipitate obtained with the anti-pan
cytokeratin monoclonal antibody. Panel A, the
immunoprecipitate obtained with the anti-mouse-pan cytokeratin
monoclonal antibody was resolved by SDS-PAGE, blotted, incubated with
anti-rat-cytokeratin 8 monoclonal antibody (lane a), or with
anti-rat-cytokeratin 18 monoclonal antibody (lane b), and
developed with an anti-rat peroxidase-conjugated secondary antibody.
The arrows point to the 54-kDa band (c 8, lane a) or the
45-kDa band (c 18, lane b). Panel B, the
immunoprecipitate obtained with the anti-pan cytokeratin monoclonal
antibody (lane a) and varying amounts of commercial purified
cytokeratin 8 (lane b, 10 ng; lane c, 40 ng;
lane d, 100 ng; lane e, 200 ng) or cytokeratin 18 (lane f, 10 ng; lane g, 20 ng; lane h,
40 ng; lane i, 60 ng) were resolved by SDS-PAGE and stained
by the silver staining method. "ab" denotes the heavy
chains of the anti-pan cytokeratin antibody, which have a molecular
mass of about 50 kDa.
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Stoichiometry of Cytokeratin Phosphorylation--
Since
cytokeratins are rather abundant elements of the hepatocyte
cytoskeleton (27-29), it might be argued that the stoichiometry of
cytokeratin phosphorylation might be very low and therefore non-functional. Therefore, the phosphorylation state of
cytokeratins in okadaic acid-treated hepatocytes was determined.
For this purpose, we determined the specific radioactivity of the
-phosphate of intracellular ATP by a high performance liquid
chromatography method, the amount of 32P incorporated into
the cytokeratins, and the amount of cytokeratins in the
immunoprecipitated bands as described under "Experimental Procedures." This calculation assumes that all of the phosphate in
the phosphorylated proteins reaches isotopic equilibrium with the
-phosphate of intracellular ATP after the 60-min labeling period
with [32P]Pi (16, 18). After this 60-min
labeling period, the ratio of specific radioactivities of ATP:ADP:AMP
in two separate experiments was 1.00:0.62:0.15 and 1.00:0.56:0.11
(Table III). These values are in
agreement with those obtained by Holland et al. (18) and
indicate that the
- and the
-phosphates are at isotopic equilibrium with each other, but not with the
-phosphate. Fig. 6
shows a gel used for the calculation of the amount of protein in the
54- and 45-kDa bands. Values of mole of phosphate/mole of cytokeratin
obtained in two separate experiments (Table III) are in agreement with
the observations that cytokeratins 8 and 18 (and mostly the former)
become significantly phosphorylated upon exposure of intact hepatocytes
to phosphatase inhibitors (22, 28), although in the latter two reports
no quantification of cytokeratin phosphorylation was achieved.
Therefore, cytokeratin phosphorylation may be functionally relevant
in our system.
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Table III
Stoichiometry of cytokeratin phosphorylation in isolated hepatocytes
Hepatocytes were labeled by incubation for 1 h with 32P
and further incubated for 15 min in the presence of 0.5 µM OA. Aliquots of the incubations were taken to
determine the specific radioactivity of adenine nucleotides and the
radioactivity incorporated into cytokeratin 8 (CK8) and cytokeratin 18 (CK18). Parallel incubations were performed in 32P-free medium
to determine the amount of protein in the bands corresponding to CK8
and CK18 (see Fig. 6). Two separate experiments were performed to
calculate the stoichiometry of cytokeratin phosphorylation. Further
details are given under "Experimental Procedures."
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DISCUSSION |
Involvement of Cytoskeletal Components in the Control of CPT-I
Activity--
A number of reports have recently described the
existence of specific interactions between the mitochondrial outer
membrane and cytoskeletal elements (reviewed in Refs. 30 and 31). Four observations in the present report strengthen the notion that cytoskeletal components (most likely cytokeratin intermediate filaments) are involved in the control of hepatic CPT-I activity. First, experiments of mild trypsin digestion suggest that CPT-I may
become activated by cleavage of extramitochondrial (although not
necessarily cytoskeletal) cell component(s). In line with this
observation, Fontaine et al. (32) have recently reported that porin, the mitochondrial outer-membrane pore-forming protein, also
becomes activated in permeabilized hepatocytes upon mild trypsin
digestion of extramitochondrial cell components. It is worth noting
that the digestion conditions employed in the present paper were much
milder than those previously used by Kashfi and Cook (33) to study the
effect of proteolysis on CPT-I, and, therefore, the two types of
experiments are not comparable. In line with our observations, Fraser
et al. (34) did not observe any effect of trypsin on CPT-I
under digestion conditions more or less comparable to ours.
Interestingly, cell pretreatment with OA prevented further activation
of CPT-I by trypsin, suggesting that both OA and trypsin may share a
common mechanism to relieve CPT-I from inhibition. Second, incubation
of intact hepatocytes with IDPN increased CPT-I activity in a basically
non-additive manner with respect to OA, suggesting a common mechanism
of action. Third, 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. Fourth, taxol prevented the OA-induced desensitization of CPT-I to trypsin
activation, as well as the OA- and IDPN-induced stimulation of CPT-I.
In short, all these data suggest that disruption of interactions
between CPT-I and cytoskeletal component(s) may de-inhibit CPT-I and, therefore, increase enzyme activity.
The possibility that CPT-I interacts with cytoskeletal components as
put forward in this paper is in line with the current notion that the
dynamics of mitochondria in living cells may result from specific
interactions of mitochondria with components of the cytoskeleton (30,
31, 35-38). It has been suggested that a function of the interactions
between mitochondria and intermediate filaments may be to locate
mitochondria in precise sites within the cell (30, 31, 39). This idea
is based on both in vitro (39, 40) and in vivo
(41, 42) experiments. Since the organization of intermediate filaments
changes dramatically in a number of liver pathologies (43), the
observations described in the present paper predict that CPT-I activity
as affected by cytoskeletal components may change under
pathophysiological situations in which the organization of the
cytoskeleton is altered, e.g. in transformed cells. In this
respect, Paumen et al. (44) have recently observed that
inhibition of CPT-I with etomoxir leads to a stimulation of ceramide
synthesis and to palmitate-induced cell death. These authors suggested
that cells that express high CPT-I activity are expected to withstand
palmitate-induced apoptosis (44). Thus, we have recently observed that
CPT-I specific activity is similar in mitochondria isolated from
hepatoma cells and normal hepatocytes, but just about half in
permeabilized hepatocytes than in permeabilized hepatoma cells; in
addition, CPT-I is not activated by OA in hepatoma cells (45). These
observations support the notion that in hepatocytes OA liberates CPT-I
from certain constrictions imposed by extramitochondrial cell
components that do not operate either in isolated mitochondria or in
transformed liver cells. Whether liberation of CPT-I from those
potential constrictions may help hepatoma cells to escape from
apoptosis is currently under study in our laboratories. Anyway, it is
worth noting that treatment of hepatocytes with OA, a well known tumor promoter, renders a "CPT-I regulatory phenotype" similar to that shown by hepatoma cells.
Involvement of Ca2+/CM-PKII in the Control of CPT-I
Activity--
Previous experiments in our laboratories have shown that
KN-62, an inhibitor of Ca2+/CM-PKII (26), antagonizes the
OA-induced stimulation of hepatic CPT-I activity (11). Data in the
present report further point to a link between Ca2+/CM-PKII
and the cytoskeleton in the context of CPT-I regulation. This
conclusion is based mostly on three observations. First, purified
Ca2+/CM-PKII was able to activate CPT-I in permeabilized
cells but not in isolated mitochondria. This is in agreement with
previous evidence against the involvement of direct phosphorylation in the OA-induced stimulation of CPT-I (10) and indicates that extramitochondrial cell components are required for the regulation of
CPT-I activity by Ca2+/CM-PKII. In this respect, it is
worth noting that permeabilization of hepatocytes with digitonin seems
to preserve quite well both the general morphology of the cell and the
structure of the cytoskeleton (46), and, therefore, the potential
interactions between the cytoskeleton and cell organelles may remain
basically unaffected upon this type of manipulation. Second, when
isolated mitochondria were incubated with a cytokeratin-enriched
cytoskeletal fraction, purified Ca2+/CM-PKII was able to
abrogate the inhibition of CPT-I induced by that cytokeratin
fraction. It is clear from these experiments that a simple
reconstituted system composed of isolated mitochondria, a
cytokeratin-enriched fraction, and purified Ca2+/CM-PKII
may reflect the situation occurring in the intact hepatocyte, indicating that these three components are sufficient for the malonyl-CoA-independent acute control of CPT-I to be demonstrated in vitro. Third, the Ca2+/CM-PKII inhibitor
KN-62 prevented the OA-induced phosphorylation of cytokeratins in
intact hepatocytes, pointing to a role of Ca2+/CM-PKII on
cytokeratin phosphorylation in these cells (28, 29).
Additional evidence for the involvement of cytokeratins in the control
of CPT-I activity is given by the lack of effect of A23187 on
cytokeratin phosphorylation in hepatocytes. In this context, challenge
of hepatocytes to OA leads to CPT-I activation and cytokeratin
phosphorylation, whereas elevation of cytosolic free Ca2+
concentration by A23187 has no effect on either CPT-I activity (47) or
cytokeratin phosphorylation (the present paper). The possibility that
liver Ca2+/CM-PKII has a different pattern of activation by
Ca2+/calmodulin and by autophosphorylation than brain
Ca2+/CM-PKII (cf. Ref. 11) is as yet an open
question.
It is worth noting that neither cAMP-dependent protein
kinase nor protein kinase C affected CPT-I activity in permeabilized hepatocytes. This is in line with the observation that neither cAMP-dependent protein kinase inhibitors nor protein kinase
C inhibitors were able to prevent the OA-induced stimulation of CPT-I
(11). As a matter of fact, several reports indicate that, despite their
ability to phosphorylate cytokeratins in vitro, neither of
these two protein kinases play an important role in the direct control
of intermediate filament integrity in intact hepatocytes (28, 48, 49).
In contrast, and in line with data in the present paper,
Ca2+/CM-PKII has been shown to play a major role in the
phosphorylation and functional integrity of hepatic cytokeratins
in vivo (28) as well as in the OA-induced disruption of
hepatic cytoskeleton (21).
Malonyl-CoA-dependent and Malonyl-CoA-independent
Control of CPT-I Activity--
Together with previous observations
(10-12), data in this paper allow for a model that explains the
OA-induced malonyl-CoA-independent control of hepatic CPT-I. As shown
in Scheme I, OA may activate Ca2+/CM-PKII by increasing its degree of phosphorylation
upon inhibition of protein phosphatases 1 and 2A; this effect would be
overcome by KN-62, an inhibitor of Ca2+/CM-PKII
autophosphorylation. Activated Ca2+/CM-PKII would
phosphorylate cytoskeletal components, perhaps cytokeratins 8 and 18, thereby disrupting putative inhibitory interactions between the
cytoskeleton and CPT-I. Stimulation of CPT-I upon disruption of the
cytoskeleton would be also achieved by challenge of intact hepatocytes
to IDPN or by treatment of permeabilized hepatocytes with trypsin in
mild conditions. Stabilization of the cytoskeleton with taxol may
prevent the malonyl-CoA-independent acute stimulation of CPT-I.

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Scheme I.
Proposed model for the
malonyl-CoA-independent acute control of hepatic CPT-I activity.
See the text for abbreviations and further details. +, activation; ,
inhibition.
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It is obvious that the notion that fatty acid translocation into
mitochondria may be controlled by modulation of the interactions between CPT-I and cytoskeletal components (i.e. by a
malonyl-CoA-independent mechanism) does not diminish the importance of
malonyl-CoA as a physiological modulator of CPT-I activity (5, 8). On
the one hand, since the pioneering work of McGarry and co-workers (8,
50), changes in long-chain fatty acid oxidation under many different
pathophysiological situations have been shown to be linked to changes
in intracellular malonyl-CoA concentration and/or changes in the
sensitivity of CPT-I to malonyl-CoA (1, 2, 5). On the other hand,
several observations suggest that malonyl-CoA-dependent and
malonyl-CoA-independent acute control of hepatic CPT-I activity might
operate in concert. First, we have recently shown that stimulation of
the AMP-activated protein kinase, a major protein kinase involved in
the control of hepatic lipid metabolism, leads to an activation of
hepatic CPT-I by malonyl-CoA-dependent and
malonyl-CoA-independent mechanisms (51). Second, a fraction of hepatic
acetyl-CoA carboxylase, the enzyme responsible for the synthesis of
malonyl-CoA, has been recently suggested to be bound to the
cytoskeleton (52). Third, it has been put forward that the 280-kDa
isoform of acetyl-CoA carboxylase might interact with the outer leaflet
of the mitochondrial outer membrane in order to channel malonyl-CoA for
CPT-I inhibition (53). Fourth, the recent observation that the bulk of
the CPT-I protein seems to face the cytoplasmic side of the
mitochondrial outer membrane (34) makes more likely that interactions
between CPT-I and cytoskeletal components might occur. Although the
physiological role of the malonyl-CoA-independent mechanism of
regulation of hepatic CPT-I activity is as yet unknown, it is worth
noting that hormonal challenge of hepatocytes (e.g.
glucagon, insulin) leads to changes in CPT-I activity that parallel
changes in long-chain fatty acid oxidation and that are retained after
washing of the permeabilized cells (3). In the context of the emerging
role of cytoskeletal filamentous networks in intracellular signaling
(54), current research in our laboratories is focussed on the possible
existence of a coordinate control of CPT-I and acetyl-CoA carboxylase
activities by modulation of interactions between the cytoskeleton and
the mitochondrial outer membrane.
We are indebted to Dr. Ismael Galve for
expert technical assistance; Dr. Alejandra Alonso, Dr. Jean Francois
Leterrier, and Dr. Paul M. P. Van Bergen en Henegouwen for advice
in the isolation of cytoskeletal fractions; Dr. David Carling, Dr.
Javier Gualix, and Dr. Antonio Sillero for advice in the
phosphorylation experiments; and Dr. Lambert M.G. Van Golde for advice
in preparing the manuscript.