Download Uncoupling effect of fatty acids on heart muscle

FEBS
Volume 795. number 1.1,3, 51-54
0 1991 Fcdcra:ion
or European
Uncoupling
Biochemical
Socictics
10502
December
effect of fatty acids on heart muscle mitochondria
submitochondrial particles
V.I. Dedukhova’,
E.N. Mokhova’,
1991
00145793/9l/S3.50
V.P. Skulachev’, A.A. Starkov’,
and V.A. Bobyleva3
and
E. Arrigoni-Martel!i?
’Dcpartrmwr qf Bioettergerics, A.N. Belozersk_v htitm
of Pll~sico-Ci~er,ricaI Biology. Moscow State Uttiwrsity, Moscow
USSR, ‘Siptu- Tall, Potncsia, Ronru. Italy and ‘ltrstitutc of Gcrrcral Patlrolqy.
Modctta, Ilaly.
Received
IO October
119899.
1991
The effect oiATP/ADP-antiporter
inhibitors on palmi1atc-induced
uncoupling was studied in heart muscle mitochondria
and inside-out submitochondrial particles. In both systems palmitatc
is found to decrease the respiration-generated
membrane
potential. In mitochondria.
this effect is
spccitically abolished by carboxyatractylatc
(CAtr) a non-pcnctrating
inhibitor ofantiporter.
In submitochondrial
particles, CAtr does not abolish
the palmitatc-induced
potential decrease. At the same time, bongkrckic acid, a penetrating inhibitor ofthc antiporter.supprcsscs
the palmitatceffcct
on the potential both in mitochondria
and particles. Palmitoyl-CoA
which is known to inhibit the antiporter
in rnitochondria
as well as in particles
decreases the palmi:ate
uncouplingcfficiency
in both these systems. Thcsc data arc in agreement with the bypothcsis that the ATP/ADP-antiporter
is involved in the action of lice l’atty acids as natural uncouplers of oxidativc phosphorylation.
Uncoupling; Fatty acid; ATPIADP-antiportcr;
1. INTRODUCTION
Fatty acids are known to differ from classical protonophore uncouplers in that they penetrate through a
phospholipid bilayer only in their protonated form.
Their anions do not penetrate the bilayer [1,2]. This is
why they fail to uncouple, i.e. to increase the I-I’ conductance, in cytochrome oxidase protcoliposomes [2].
To explain the mechanism of fatty acid uncoupling in
mitochondria. we suggested that there are protein(s) in
the inner mitochondrial membrane that facilitate transport of the fatty acid anionic species [2-51.
In brown fat, which is specialized for heat production
by means of fatty acid-mediated uncoupling, the role of
fatty acid anion porter was postulated to be performed
by the ‘uncoupling protein’ thermogenin [5].
Recently this suggestion seems to have been confirmcd by Jczek and Garlid [6,?] who found that thermogenin transports various monovalent anions which can
substitute for fatty acids in actuating thermogeninAbbr~sihms: dy/. transmcmbranc
electric potential difrcrcncc; Atr,
atractylate; USA. bovine serum albumin; CAtr. carboayatractylate;
DNP. 2,4-dinitrophenol;
EDTA,
cthylencdiaminc-N,~V,WN’-tctraacetic
acid; EGTA.
cthylcnc glycol-his@aminoethyl
cthcr)rV,IV,N’,N’-tctraacetic
acid; FCCP, p-trifluoromcthoxycarbonylcyanide phcnylhydrazonc;
MOPS, morpholinopropanc
sulphonate:
PCB-,
phcnyldicarbaundccaboranc;
SDS. sodium dodccyl sulfalc;
TPP+. tctraphcnyl
phosphonium
C’orrqwmkm-e r~r/dress:Y.P. Skttlnchcv, Department
oT Biocncrgctics, A.N. Bclozcrsky
lnsthu1c oTPhysico-Chemical
Biology. Moscow
State University,
Moscow
119899, USSR
Heat
L mitochondria; Submitochondrial particle
linked H--conducting activity. The anion efficiency was
shown to increase with the increase in the length of
hydrocarbon chain of the anion.
Thermogenin is absent from tissue other than brown
fat, but nevertheless fatty acids can uncouple in these
other tissues (for reviw, see [S]). We assumed [2,5] that
in these cases the role of the fatty acid anion porter is
performed by ATP/ADP antiporter, a protein which is
very similar to thermogenin in amino acid sequence,
domain structure, and some other properties [8.9]. It
was suggested that the adenine nucleotide anion-translocnting machinery of the antiporter can transport hy
drophobic anions without the involvement ATP’(ADP”-)-specific gate [2,5]. In agreement with this hypothesis, the following observations were made by our
group. (1) The ATP/ADP antiporter inhibitors (CAtr.
Atr, bongkrekic acid and pyridoxal 5-phosphate) and
its substrate (ADP anion) rpecifica!!y inhibit fatty acidinduced uncoupling in liver and skeletal muscle mitochondria, while having no effect on FCCP-induced
uncoupling [2-4]. (2) CAtr-sensitive uncoupling is inherent not only to fatty acids but also to other hy&ophobic
monovalent anions such as dodecyl sulfate and cholate
[IO]. (3) The burst in heat production during arousal of
the hibernating ground squirrels is accompanied by (i)
appearance of CAtr-sensitive uncoupling in liver, heart
and skeletal muscle mitochondria and (ii) an increase of
the free fatty acid concentration in tissues and isolated
mitochondria [l l-121.
The observation (1) was recently confirmed and extended by Schonfeld 1131who showed that the uncou-
51
Volume 295. number 12.3
FEBS LETTERS
December I99 I
pling activity of e Patty acid wries in different tissues in
proportion to the ATP/ADP antiporter content (heart
> kidney > iiver).
In this paper, we compared the uncoupling effect of
fatty acids in heart muscle mitochondria and inside-out
submitochondrial particles.
2. MATERIALS
AND METHODS
hlitochondri;: were isolated from rabbit or heart muwle. Chilled
muscle tissur. previously scparalcd ti-om 1;lt and tendons. wcx minced
and then passed throu_rh u staicless-steel press with holes oi about I
nm in diamtcr.
The tissue was then homogcnizcd using a Teflon pcstlc in a gl;~ss
(Pvrcs) homogcnircr Ibr about 3 min. The tissuciisolation medium
radio WIS I :8.
After the first ccntril’ugalion (IO min. 600~s). the supcrnalant was
decanted and filtcrcd through 3 layers of g;~uzc. The filtrate was
cermifugcd (IO min. I2 000x&. the pcllct resuspcndcd in I ml of ~hc
isolation medium (250 mM sucrose, IO mM MOPS. I mM EDTA. pH
7.4) supplemcntcd with BSA (3 m&ml). and then medium wilhoul
BSA was added. The final mitochondrial prccipitalc (IO min,
I:! OOOxg)was suspended in isolation medium with BSA. The mitochondrial suspension (iO-90 mg/ml) was stored on ice.
Inside-out AS-type submitochondrial particles wcrc prcparcd according to Kollar and Vinogradov [l4].
In the majorhy or cxpcrimcnts. the incubation medium contained
250 m&l sucrose. IO mM MOPS. ;lnd 0.5 mM EGTA. .aH 7.4. Glutamate (5 mbl) and malatc (5 mill) or succinatc (5 mM) wre used as
substrates. I PM rotcnone was added IO the medium wh:n succinatc
oxidation ws studied.
Oxygen consuniption was rrcordcd with a Clark-type oxygen clcctrode and LP-7E polarograph. The incubation misturc was stirred
with glass-covcrcd magnetic stirring bar. The concentration of mitochondrial protein WIS I mg/ml (glutamate plus ~nelt~tc)or 0.4 m$ml
(succinatc); the temperature was 26°C.
The sycthaic pcnetraling ions (TPP‘ and PCB-) wcrc used as dy
probes [l5]. The penetrating ion concentration in solution was
measured with TPP’ and PCB-- sensitive electrodes [IG]. Oligomycin.
blOPS. palmitic acid. palmitcgl-CoA. CAtr. Atr and Dug’ acid-l’rcc
BSA wcrc from Sigma; EDTA, EGTA, ADP, GDP, rotcnonc. palmitoyl-L-carnitinc and DNP rrom Scrva; malats from Fluka; FCCP
rrom Bochringcr.
3. RESULTS AND DISCUSSION
As seen in Fig. I, curves a-c, CAtr was strongly
inhibitory for respiration of rat heart mitochondria,
stimuiated by paimirare in the presence of OiigOt@ii.
The inhibition was relcascd by DNP. ADP (curve c),
Atr (curve d), palmitoyl CoA and bongkrekic acid (not
shown) were also inhibitory but their effects were not
as pronounced as that of CAtr. Moreover, CAtr added
after Atr failed to inhibit the palmitate-stimulated respiration as strongly as without Atr (curve d). CAtr-sensitive stimulation of respiration could also be induced by
SDS but at a concentration higher than that of palmitate (curves e and f). Pnlmitoyl carnitine could not substitute for either palmitatc or CAtr (not shown).
Stimulation of the mitochoncirial respiration by palmitate was accompanied by a dty decrcasc which was
completely or partially reversed by CAtr or bongkrekic
acid, rcspectivcly (Fig. 2).
52
Fig. I. Efrccls of CAtr. Air and SDS on 111~respiration or heart
mitochondria. Incubation mixlurc: 0.25 A1sucrose. IO mM MOPS.0.5
mbl EGTA. 5 mXl p.lutamatc, 5 mM :na!atc, oligomycin (I ,@ml).
BSA (0.2 mgml). pH 7.4. 27°C. Additions. mite. a-d. rat heart mitochol:dria (I mg protein/ml): c.l: rabbit heart mitochondria (I mg
protein/ml): Palm. 30~1kf palmitic acid; 2pXl CAtr; 5p.M Air; 40,uM
AD!? 90 phi SDS: 5 PM DNP (a-d) ;III~ 8 PM DNP (c,f). Figures
above cuwcs. respiration ralcs (nmol 0: x min-’ x mg prolcin-‘).
Inside-out beef heart submitochondrial particles were
also found to respond to the p;llmitate addition by a
lowering of 4,~. However, in this case CAtr did not
SUppXSS ihC action Of pLliXitZte.
On tile other
hand:
bongkrekic acid suppressed the palmitate effect. Again,
as in mitochondria, subsequent addition of an artificial
protonophore
(FCCP) caused a dly decrease (Fig. 3).
Palmitoyl CoA proved to decrease the palmitate effect
in both rnitochondria and submitochondrial pnrticles
(not shown).
The data presented above clearly indicate that membrane sidedncss is critical for CAtr inhibition of palmiLate-induced uncoupling in heart muscle mitochondria
and their inside-out particles, CAtr effectively abolishes
the uncoupling in the milochondria but not in the particles. On the other hand, some inhibiting effects of bongkrckic acid and palmitoyl-CoA were seen in both mitochondria and particlcs.
Volume 295, number 1,2.3
FEBS
December 1991
LETTERS
6
Palm
OS25
0125
s 0.25,
zc
c
&
c.5
%c
7
%
L!zi
0.25
0.5
1.0
Fig. 2. Efl’ccls oTCArr and bongkrckic acid (BA) on palmitawinduced dl dccrcasc in mitochondrin. Incubation mixture: 0.25 M sucrose. 10 mhl
MOPS. 0.5 mM EGTA. IO mM succinatc. I pM rownonc. oligo:llyc;:r (2,@ml).
BSA (0.4 m/ml).
l.SpM TPP’. pH 7.4. 26°C. Additions. mite.
rat hoart mimchondria (0.8 m_cprolcin/ml); I phi C&r: 20 @I DNP: 5 PM B& 30 phi (A) and 20 @l (B) palmitic acid.
Just these relationships could be predicted assuming
that it is the ATPlADP antiporter that mediates the
uncoupling effect of free fatty acids. It is known that the
CAtr and bongkrekate binding sites of the antiporter
arc localized on outer and inner surfaces of the inner
3.0
-I
succinate
2
3.0
1
s 2.0.
z
I!%
l.O0.5
Fig. 3. Efrccts of bongkrekic acid. CAtr and FCCP on palmilatcinduced dy dccrcasc in subnlitochondrial parliclcs. lncubalion mixture: 0.25 M sucrose, IO mM MOPS, 0.5 mM EGTA, and submilocbondrial parliclcs (0.45 mg protcinlml); I PM rolenone. oligomycin
(4ggh1l),
I5 AM PCB-, p1-I 7.3, 26°C. Additions, 5 mM succinalc. I
,uM CA& BA. 8 PM bongkrckic ilcid; palm. 30 @l palmilic acid; I
/rM FCCP.
mitochondrial
membrane. respectively, whereas sites
for palmitoyl-CoA are on both membrane surfaces. It
is also found that CAtr cannot penetrate through the
membrane whereas bongkrekic acid can (for review see
[W.
Thus comparison of heart mitochondria and particles
gives one more piccc of evidence that the ATP/A.DP
antiporter is somehow involved in the fatty acid uncoupling.
The results described for heart mitochondria confirmed our previous observations made with liver and
skeletal muscle mitochondria [2-4], i.e. among the specific inhibitors of the antiporter, CAtr is the most potent
agent for suppressing fatty acid-induced uncoupling,
whereas ADP, Atr. palmitoyl CoA and bongkrekic acid
are less effective. Atr added before CAtr prevents, at
least for some time, complete inhibition of the palmitate-stimulated respiration by CAtr (see also [2]). It
should be emphasized that the Atr concentration used
was quite sufficient to completely inhibit oxidative
phosphorylation.
A probable explanation for this fact
is t!~at a!! !!re tested inhibitors combine with the interface-localized, substrate-specific antiporter gates rather
than with the non-specific anion-translocating
mechanism inlaid in the membrane core. Apparently, modification of gate decelerates the translocator mechanism and, as a result, inhibits the fatty acid anion-translocation rate. This deceleration may vary depending on
the kind of inhibitor.
nckrro~~lc~l~or~orfs:
The authors arc very gralcful lo Prof. A.D. Vinogradov for providing us with a preparation oTsubmilochondrial particlcs and to Sigma-Tay Ibr financial support.
REFERENCES
[I] W;dwr, A. and Gulknccht. J. (1984) J. Mcmbr. Biol. 77.355-261.
53
Volume 295, number
123
FEBS LETTERS
Andrcyev. A.Yu.. Bondareva, T.O.. Dedukhova. V.I., Mokhova.
E.N.. Skulachev. V.P.. Tsofina, L.M.. Volkov. N.I. and Vygodina. T.V. (19S9) Eur. J. Biochem. 182. 585-592.
[31 Andrevcv. A.Yu.. Volkov. N.I.. Mokhova. E.N. and Skulachev,
V.P. (1987) Biol. .Membrany 4,474-178 (Russ.).
PI Andrcyev. A.Yu.. Bondarcva. T.O.. Dcdukhova. V.I.. Mokhova,
E.N.. Skulachev. V.P. and Volkov. N.I. (1987) FEBS Lett. 226.
265-269.
PI Skulachcv. V.P. (1988) Mcmbranc Bioenergetics. Springer. Berlin.
Garlid, K.D. (1990) Biochim. Biophys. Acta 1018. 151-154.
it; Jezck. P. and Garlid. K.D. (1990) J. Biol. Chem. 265. 1930319311.
Bl Aquila, H.. Link, T.A. and Klingcnberg. M. (1985) EMBO J. 4.
2369-2376.
PI Bouilland. F.. Weissenbach. J. and Ricquier. D. (1986) J. Biol.
Chem. 261, 1487-1490.
PI
54
December 1991
[IO] Brustovctsky. N.N.. Dedukhova. V.I.. Yegorova. M.N., Mokhow E.N. and Skulachcv. V.P. (1990) FEBS Lctt. 272, 187-189.
[I I] Brustovclsky. N.N., Amerkhilnov, Z.G., Ycgorova, M.N., Mokhova. E.N. and Skulachcv. V.P. (1990) FEES Leti. 272, 190-192.
[I21 Brustovcrsky. N.N.. Amcrkhanov. Z.G., Yegorova. M.N.. MOkhova. E.N. and Skulaclw. V.P. (1991) Biokhimiya 56, 947-953
(Russ.).
II 31 Schonfeld. P. (1990) FEBS Lett. 264, 246-24R.
.-.
[l4] Kotlar, A.B. and Vinogradov, A.D. (1990) Biochim. Biophys.
Acta 101% 151-158.
[IS] Grinius. L.L.. Jasaibs. A.A.. Kadziauskas, J.P., Libcrman, E.A.,
Skulachcv. V.P., Topali, V.P. and Vladimirova. %l.A. (1970) Biochim. Biophys. Acla 2lG, l-12.
[I61 Kamo. N.. Muratsuga, Y., Hongoh, R. and Kobalake, Y. (1979)
J. Membr. Biol. 49, 105-121.