Co

S*ao

R

fsimtacImttbmbmtthm

c

tftwbbmgcc

t

6

Biochemical and Biophysical Research Communications 285, 537–539 (2001)

doi:10.1006/bbrc.2001.5201, available online at http://www.idealibrary.com on

arnitine Palmitoyl Transferase I and the Controlf b-Oxidation in Heart Mitochondria

imon Eaton,*,1 Kim Bartlett,†,‡ and Patti A. Quant*Unit of Paediatric Surgery, Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, United Kingdom;nd †Department of Child Health and ‡Department of Clinical Biochemistry, Sir James Spence Institutef Child Health, Royal Victoria Infirmary, Newcastle-upon-Tyne, NE1 4LP, United Kingdom

eceived May 29, 2001

malonyl-CoA inhibition and CPT I can be described astcaiimI[9ptm1Itnsbdhls

M

sbwihcHmmbrtdabm

Mitochondrial b-oxidation provides much of theuel requirements of heart and skeletal muscle de-pite the malonyl-CoA concentration greatly exceed-ng the IC50 of carnitine palmitoyl transferase for

alonyl-CoA. To try to explore the relationship be-ween inhibition of carnitine palmitoyl transferase Ictivity and b-oxidation flux, we measured the fluxontrol coefficient of carnitine palmitoyl transferaseover b-oxidation carbon flux in suckling rat heartitochondria. The flux control coefficient was found

o be 0.08 6 0.05 and 50% of carnitine palmitoylransferase I activity could be inhibited before-oxidation flux was affected. These observationsay help to explain the presence of high rates of

-oxidation despite the high concentration ofalonyl-CoA in rat heart; we hypothesize that al-

hough not rate-limiting in vitro, carnitine palmitoylransferase is rate-limiting in vivo because of theigh malonyl-CoA concentration in heart anduscle. © 2001 Academic Press

Key Words: heart; b-oxidation; flux control coeffi-ient; suckling; carnitine palmitoyl transferase I.

Mitochondrial outer membrane carnitine palmitoylransferase I (CPT I; EC 2.3.1.21) catalyses the trans-er of an acyl moiety from a long-chain acyl-CoA estero carnitine to form a long-chain acyl-carnitine ester,hich can then enter the mitochondrion and undergo-oxidation (1). CPT I is a site for regulation of-oxidation flux via the physiological inhibitor,alonyl-CoA, so that b-oxidation is inhibited when

lucose is plentiful. Malonyl-CoA levels in the hepato-yte are such that b-oxidation flux is sensitive tohanges in small changes in CPT I activity caused by

Abbreviations used: CPT I; Mitochondrial outer membrane carni-ine palmitoyl transferase I (EC 2.3.1.21).

1 To whom correspondence should be addressed. Fax: 144 20 7404181. E-mail: s.eaton@ich.ucl.ac.uk.

537

he rate-controlling step in liver (i.e., it has a fluxontrol coefficient close to 1) (2–5). It has been similarlyssumed that CPT I is rate-limiting for b-oxidation fluxn all tissues and preparations. However, the musclesoform of CPT I is very much more sensitive to

alonyl-CoA than the liver isoform (6) such that itsC50 is greatly (;10-fold) exceeded by the approximatemalonyl-CoA] in the heart and in skeletal muscle (7–). Hence, although there is considerable evidence sup-orting a role for modulation of malonyl-CoA levels inhe control of b-oxidation flux in heart and skeletaluscle in a variety of physiological conditions (7, 10–

5), it is difficult to see how b-oxidation proceeds if CPTactivity is rate-limiting for b-oxidation, unless most ofhe malonyl-CoA is intramitochondrial or bound andot available to inhibit CPT I. The aim of the presenttudy was to measure the control exerted by CPT I over-oxidation carbon flux in neonatal rat heart mitochon-ria. We chose to examine this question in suckling rateart mitochondria because CPTI is thought be rate-

imiting for b-oxidation flux at this developmentaltage (16, 17).

ATERIALS AND METHODS

Mitochondria were isolated from the hearts of 11–15 day-old (peakuckling (18)) Wistar rats, 10 hearts per mitochondrial preparationy the method described in (19) except that Percoll centrifugationas found to be unnecessary for rats of this age. Mitochondria were

ntact (.95% as judged by citrate synthase latency) and routinelyad a respiratory control ratio between 2.2 and 3.4. Incubations werearried out at 37°C in a medium containing: 80 mM KCl, 10 mMepes, 5 mM MgCl2, 2.5 mM KH2PO4, 1 mM EGTA, 1 mM ATP, 0.2g/ml cytochrome c, 30 mM creatine phosphate, 20 mM creatine, 0.1g/ml creatine phosphokinase (to maintain state 3.5), and 1.6 mg/ml

ovine serum albumin (BSA), pH 7.2. The measurement of fluxequired the presence of 1 mM carnitine and 1 mM malate in addi-ion. Mitochondria were preincubated for 4 min in the presence ofifferent concentrations of etomoxir-CoA (60–900 nM; synthesiseds in (20)) before measurement of either [U-14C]palmitoyl-CoA-oxidation flux (as in (21), in the presence of 1 mM carnitine and 1M malate) or CPT I activity (as (22), except 40 mg/ml myxothiazol

0006-291X/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

wmto(1l

R

smbcpcoatiMebhvistalsttan

Smtta(ct(

ifpleCifliccvscncaCl0ac

t

iMo

Vol. 285, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

as used instead of antimycin/rotenone and [3H]carnitine was at 1M). Curve fitting, statistical analysis, and obtaining the flux con-

rol coefficient for CPT I over b-oxidation was carried out by meansf linear and nonlinear regression using the program Prism 3Graphpad Software, San Diego, CA) and verified using Enzfitter.05 (Biosoft, Cambridge, UK), using the equation described by Gel-erich et al. (23) for nonlinear regression.

ESULTS AND DISCUSSION

Mitochondria were incubated and maintained intate 3.5 (appropriate for the heart) (24). Malate (1M) was included to generate maximal flux rates of-oxidation by providing a tricarboxylic acid cycle ac-eptor (21) and incubations were carried out in theresence of 1 mM carnitine to approximate the tissueoncentration of carnitine found in the heart at this agef rat (17). At this concentration of carnitine, both livernd muscle isoforms of CPT I are active (17). We regardhese conditions as providing a model of mitochondrian the heart working at a high rate of cardiac output.

alonyl-CoA was absent from the incubations; insteadtomoxir-CoA was used to inhibit of CPT I activity and-oxidation flux because it is a strong irreversible in-ibitor of both liver and muscle CPT that binds co-alently to the active site (25). Although etomoxir-CoAnhibits both liver and muscle isoforms of CPT I, in thistudy we were interested in the relationship betweenotal CPT I activity and b-oxidation flux rather thanttempting to assess the individual contributions of theiver and muscle isoforms. CPT I activity in the ab-ence of inhibitor was 10.01 6 1.17 nmol/min/mg pro-ein (mean 6 SEM, n 5 7) and b-oxidation flux (asotal acid-soluble radioactivity) was 29.93 6 3.01 nmolcetyl units/min/mg protein (equivalent to 3.74 6 0.38mol palmitate oxidised/min/mg protein) (mean 6

FIG. 1. Inhibition of b-oxidation flux as a function of carnitine pasolated and incubated for measurement of b-oxidation flux and carn

ethods. (A) Indicates the experimental data fitted as described by Gf the log–log plot (28).

538

EM, n 5 7). 14CO2 release in the presence of 1 mMalate plus 1 mM carnitine was negligible (,1% of

otal carbon product) as in previous experiments underhese conditions (19, 21). The measurement of CPTctivity was of CPT I rather than CPT I plus CPT II asi) the mitochondria were .95% intact as judged byitrate synthase latency, and (ii) residual activity inhe presence of an excess of etomoxir-CoA was very lowCPT II is not inhibited by etomoxir-CoA; (25)).

In contrast to experiments in liver and brain whichndicate that inhibition of CPT I activity strongly af-ects b-oxidation flux (3–5, 18, 26, 27), we were sur-rised to observe that when the inhibition of CPT I wasess than approximately 50%, there was very littleffect on b-oxidation flux (Fig. 1A). However, whenPT I was inhibited by greater than 50%, flux dimin-

shed abruptly. These data allowed the calculation of aux control coefficient for CPT I in intact mitochondria

n the absence of any inhibitor. The flux control coeffi-ient for CPT I at 0% inhibition of CPT I activity wasalculated as described by (23), and had the numericalalue 0.08 6 0.05 (mean 6 SEM; n 5 7 titrations fromeparate mitochondrial preparations). The curve indi-ated on Fig. 1A represents this fitted curve. Unfortu-ately, this analysis cannot be undertaken with non-ovalent inhibitors such as malonyl-CoA (23). Inddition, we determined the flux control coefficient ofPT I over b-oxidation by linear regression of the log–

og plot (Fig. 1B) (28), with the numerical value 0.02 6.01, although it should be noted that this type ofnalysis is more error-prone than fitting the entireurve (29).Although initially surprising, these results may help

o explain the apparent paradox that the heart is very

itoyl transferase I inhibition. Suckling rat heart mitochondria weree palmitoyl transferase I activity as described under Materials andrich et al. (23). (B) Indicates the same data fitted by linear regression

lmitinelle

active in b-oxidation despite the estimated cytosolic[CbhicctbnbhecrC(

pfiiicol1pbs

A

fo

R

6. Saggerson, E. D., and Carpenter, C. A. (1981) FEBS Lett. 129,

1

1

1

1

1

1

1

1

1

1

2

2

2

2

2

2

2

2

223

3

Vol. 285, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

malonyl-CoA] greatly exceeding the IC50 of malonyl-oA for CPT. In the absence of any inhibitor,-oxidation flux is quite insensitive to CPT I inhibitors,owever in vivo the inhibition of CPT I by malonyl-CoA

s such that b-oxidation flux is responsive to smallhanges in inhibition by changes in malonyl-CoA con-entration. In other words, we hypothesise that al-hough not rate-limiting in vitro with control sharedetween other steps such as NADH recycling (21), car-itine palmitoyl transferase is rate-limiting in vivoecause of the high malonyl-CoA concentration ineart and muscle. This cannot, however, be the onlyxplanation for the apparent discrepancy between theoncentration of malonyl-CoA in the heart and the highate of b-oxidation flux; some of the measured malonyl-oA must be inaccessible to CPTI due to being bound

30) or intramitochondrial (31).Further experiments are necessary to verify this hy-

othesis; namely the determination of the control coef-cient of CPT I over b-oxidation flux in vivo or in an

solated heart preparation and additional experimentsn different ages of rats and at different carnitine con-entrations. However, evidence for the in vivo controlf beta-oxidation flux by modulation of malonyl-CoAevels in cardiac or skeletal muscle is compelling (7,0–15) and the phenomenon described in this paperrovides a potential mechanism whereby control ofeta-oxidation flux can be achieved despite the highensitivity of muscle CPT I to malonyl-CoA inhibition.

CKNOWLEDGMENTS

The British Heart Foundation is gratefully acknowledged for aellowship to S.E. Wolfram Kunz is gratefully thanked for his advicen analysis of the data.

EFERENCES

1. Zammit, V. A. (1999) Prog. Lipid Res. 38, 199–224.2. Guzman, M., and Geelen, M. (1993) Biochim. Biophys. Acta

1167, 227–241.3. Spurway, T. D., Sherratt, H. S. A., Pogson, C. I., and Agius, L.

(1997) Biochem. J. 323, 119–122.4. Drynan, L., Quant, P. A., and Zammit, V. A. (1996) Biochem. J.

317, 791–795.5. Drynan, L., Quant, P. A., and Zammit, V. A. (1996) Biochem. J.

318, 767–770.

539

229–232.7. Awan, M. M., and Saggerson, E. D. (1993) Biochem. J. 295,

61–66.8. McGarry, J. D., Mills, S. E., Long, C. S., and Foster, D. W. (1983)

Biochem. J. 214, 21–28.9. Goodwin, G. W., Ahmad, F., Doenst, T., and Taegtmeyer, H.

(1998) Am. J. Physiol. 274, H1239–H1247.0. Lopaschuk, G. D., Witters, L. A., Itoi, T., Barr, R., and Barr, A.

(1994) J. Biol. Chem. 269, 25871–25878.1. Hamilton, C., and Saggerson, E. D. (2000) Biochem. J. 350,

61–67.2. Saddik, M., Gamble, J., Witters, L. A., and Lopaschuk, G. D.

(1993) J. Biol. Chem. 268, 25836–25845.3. Dyck, J. R. B., Barr, A. J., Barr, R. L., Kolattukudy, P. E., and

Lopaschuk, G. D. (1998) Am. J. Physiol. 275, H2122–H2129.4. Goodwin, G. W., and Taegtmeyer, H. (1999) Am. J. Physiol. 277,

E772–E777.5. Chien, D., Dean, D., Saha, A. K., Flatt, J. P., and Ruderman,

N. B. (2000) Am. J. Physiol. 279, E259–E265.6. Weis, B. C., Esser, V., Foster, D. W., and McGarry, J. D. (1994)

J. Biol. Chem. 269, 18712–18715.7. Brown, N. F., Weis, B. C., Husti, J., Foster, D. W., and McGarry,

J. D. (1995) J. Biol. Chem. 270, 8952–8957.8. Krauss, S., Lascelles, C. V., Zammit, V. A., and Quant, P. A.

(1996) Biochem. J. 319, 427–433.9. Eaton, S., Pourfarzam, M., and Bartlett, K. (1996) Biochem. J.

319, 633–640.0. Bernert, J. T., Jr., and Sprecher, H. (1977) J. Biol. Chem. 252,

6736–6744.1. Eaton, S., Bhuiyan, A. K. M. J., Kler, R. S., Turnbull, D. M., and

Bartlett, K. (1993) Biochem. J. 289, 161–168.2. Grantham, B. D., and Zammit, V. A. (1986) Biochem. J. 239,

485–488.3. Gellerich, F. N., Kunz, W. S., and Bohnensack, R. (1990) FEBS

Lett. 274, 167–170.4. Mildaziene, V., Baniene, R., Nauciene, Z., Marcinkeviciute, A.,

Morkuniene, R., Borutaite, V., Kholodenko, B., and Brown, G. C.(1996) Biochem. J. 320, 329–334.

5. Lilly, K., Chung, C., Kerner, J., Vanrenterghem, R., and Bieber,L. (1992) Biochem. Pharmacol. 43, 353–361.

6. New, K. J., Krauss, S., Elliott, K. R. F., and Quant, P. A. (1999)Eur. J. Biochem. 259, 684–691.

7. Blazquez, C., Sanchez, C., Velasco, G., and Guzman, M. (1998)J. Neurochem. 71, 1597–1606.

8. Fell, D. A. (1992) Biochem. J. 286, 313–330.9. Small, J. R. (1993) Biochem. J. 296, 423–433.0. Dugan, R., Osterlund, B., Drong, R., and Swenson, T. (1987)

Biochem. Biophys. Res. Commun. 147, 234–241.1. Scholte, H. R. (1969) Biochim. Biophys. Acta 178, 137–144.