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However, no effect of AlGAR, ZMP, or 5’-AMP ora CPT-
1 activity ira either of the twa systems coníd be detected
(results raot showra>.
VELASCO ET AL.
Malonyl-CoA-dependent and .independent mechan¿sms
are invol ved in tite AJCAR-induced stimulation
of CPT-1? It has been showra that CPT-I is reversibly
serasitized to malonyl-CoA inhibition by malonyl-CoA
itself[reviewed ira Ref. (11)]. Thus, whera mitoehondíla
are incubated ira the abserace or preserace of malorayl-
CoA, the enzyme becomes less or more serasitive to irabibitiora,
respectively (11). This effect is also observed ira
whole-cell systems: incubatiora of isolated hepatocytes
witb compounds thaI decrease intracellular malorayl-
CoA leveis makes the enzyme acquire a relaxed coraformation
which exhibits high activity arad low serasitivity
to inhibitiora by malonyl-CoA ira a subsequerat assay
(11). The opposite ensues whera hepatocytes are exposed
to compounds that iracrease malonyl-CoA conceratration,
i.e, Ihe enzyme acquires a tight coraformatiora
with low activity arad enhaneed sensitivity to inhibitiora
by malorayl-CoA (11). Because permeabilization of Ihe
plasma membrane of Ihe hepatocyte leads lo a large
dilutiora of cytosolic comporaents iracludirag malonyl-CoA
(22), arad the transitiora between Ihe two different sensi-
tivity states is very fasí at 37
0C (11), tbe coraformational
state of CPT-I within the intaet hepatocyte is
only retairaed for very short periods of time ira permeabilized
celís [cf. Ref. (15)]. The permeabilized-hepatocyte
system tbus allows monitorirag the conformational
state of CPT-I when tbe assay of erazyme activity is
performed at different times after permeabilization.
We used Ihis experimental approach lo leal whether
the stimulation of CPT-I observed ira AICAR-treated
hepatocytes aetuaily iravolves a deerease of intracellular
malonyl-CoA levela. As atated aboye, the AlGARiradueed
atimulation of [‘4G]palmitate oxidatiora correlated
well with the AICAR-iraduced stimulation of CPT-
1 whera erazyme activity was determiraed 20 s after permeabilizatiora
of Ihe hepalocyte plasma membrane
(Pig. 1). Ira control incubatioras, a lag phase ira Ihe CPT-
1 assay was observed at very short reaction times (Fig.
2). This lag phase seems tobe dueto the coraformational
corastrainís of the CPT-I protein iraduced by intracellular
malonyl-CoA [cf. Ref. (15)], arad it rapidly disappears
after complete leakage of malorayl-CoA from the
permeabilized ceils, allowing relaxation of the enzyme
(Fig. 2). Ira contrast, by depletirag intracellular malorayl-
CoA, ALGAR was indeed able to eliminate Ihe lag pbase
inhererat to the permeabilized-ceil assay of CPT-I activity
(Fig. 2). The magnitude of the AICAR-induced stimulation
of CPT-I was reduced lo 45 t 14% at 40 s afler
permeabilizatiora arad lo 30 t 12% at 60 s after permeabilization
(Fig. 2), indicating thaI differences in the
conformational state of the CPT-1 protein belweera con-
o
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2’—
oa
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o 60
Reaction time (s)
¡ ¡ 1 ¡
FIO. 2. Effact of AICAR on CP’I’-l activity at dilfororat reaction
timos. Hepatocytes were proincubated for 15 mira ira tSe abseraco (O)
or ira the preserace (e) of0.5 mM AICAR. Subsoqueratly, aliquots wOre
removed to dotermirae CPT-I activity at difToront reaction times by
method A. Noto that enzyme volocity (arad not product accurnulatiora)
‘a represerated ora tSey-axis. Results corresporad to six different Sopatocyte
preparratioras.
trol arad AICAR-treated celís are disappearing. However,
a constaral 20—25% stimulatiora of CPT-I activity
was observed ira MCAR-treated hepatocytes up to 140
a after hepatocyte permeabilizatiora (Fig. 2). Ira aur ayatem,
this time is believed to ‘be more than eraough to
allow reversal of coraformational corastrairata of CPT-I
induced by charages ira intraceilular malarayl-CoA conceníratiora
[cf. Ref. (11)].
To obtain more direcí evidence for the involvemeral of
a malonyl-CoA-independent componení ira the AlGARiraduced
atimulatiora of CPT-L erazyme aetivity was determiraed
by a more complex procedure whicb eliminates
any posaible interfereraceofmalorayl-CoA. Ira such
ara assay (method B), hepalocytes are penneabilized
with digitonira, followed by rapid arad extensive washíng
of permeabilized celís to allaw complete remaval of
malorayl-CoA. Thera, permeabilized celís are iracubated
al 370C for up la 15 mira prior lo determinatiora of erazyme
activity, so thaI any conformational carastrairal of
the CPT-I enzyme will disappear (15). The use of tbis
pracedure corroborated thaI a 20—25% atimulation of
CPT-I by AlGAR is exerted by a malorayl-CaA-iradeperadení
mecharaism (Fig. 3). Mareover, Ihe magrailude of
tbe stimulaliora of CPT-I as delermiraed by method B
was ideratical with reactiora times of 20, 60, arad 120 s
(resulta nol shown). Note thaI for determinaban of
CPT-I activity by method B cytosolie proteira that
leaked from the permeabilized cella is removed prior
to determinatiora of erazyme aetivity, arad so erazyme
specific activity (referred lo as milligrama of prolein)
waa always higher ira melbod B Ihan ira method A (note
to Table 1).
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