redox control of β-oxidation in rat liver mitochondria

Eur. J. Biochem. 220, 671-681 (1994) 0 FEBS 1994

Redox control of P-oxidation in rat liver mitochondria Simon EATON', Douglas M. TURNBULL' and Kim BARTLETT'.3

* Department of Clinical Neuroscience, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, England Department of Child Health, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, England

Department of Clinical Biochemistry, Human Metabolism and Diabetes Research Centre, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, England

(Received November 23, 1993) - EJB 93 1741/1

Coupled rat liver mitochondria were incubated with [U-i4C]hexadecanoate and carnitine which resulted in the formation of acyl-, 2-enoyl- and 3-hydroxyacyl-CoA and carnitine esters. The production of 2-enoyl-CoA and 3-hydroxyacyl-CoA esters was associated with a significant lowering of the NAD'NADH ratio, in contrast to rat muscle mitochondria [Eaton, S., Bhuiyan, A. K. M. J., Kler, R. S., Turnbull, D. M. & Bartlett, K. (1993) Biochem. J. 289, 161-1721, suggesting that control by the respiratory chain is important under normal conditions. When NAD'NADH ratios were held low by succinate-induced reverse electron flow, 3-enoyl-CoA esters were also detected, probably formed by the action of 3,2-enoyl-CoA isomerase. Measurement of the flux of P-oxidation at different osmolalities showed that flux was strongly dependent on osmolality changes in the physiological range. Measurement of the CoA and carnitine esters resulting from incubations made at different osmolalities showed that there was an increase in the amounts of the saturated acyl-CoA esters with respect to 2-enoyl-CoA and 3-hydroxyacyl-CoA esters, consistent with control by the electron-transfer flavoprotein-ubiquinone segment [Halestrap, A. P. & Dunlop, J. L. (1986) Biochem. J. 239, 559-5651. This however could not be the only factor operating as indicated by the continued presence of 2-enoyl-CoA and 3-hydroxyacyl-CoA esters at high osmolalities.

Mitochondria1 P-oxidation is linked to the respiratory chain at two stages ; the 3-hydroxyacyl-CoA dehydrogenases to complex I via NAD'DJADH and the acyl-CoA dehydrogenases to ubiquinone via electron-transfer flavoprotein (ETF) and its oxidoreductase (ETF: QO). Inhibition of respiratory-chain activity at the level of complex I leads to diminished P-oxidation flux and the accumulation of 3-hydroxyacyl-CoA and 2-enoyl-CoA and carnitine esters (Bremer and Wojtczak, 1972; Stanley and Tubbs, 1974, 1975; Lopez- Cardozo et al., 1978; Latipaa et al., 1986; Watmough et al., 1989) which may lead to further inhibition of flux at the level of the acyl-CoA dehydrogenases (Davidson and Schulz, 1982; Powell et al., 1987). Similarly deficiency of ETF or ETF: QO leads to diminished P-oxidation flux and an accumulation of long-chain saturated acyl-CoA and and acylcarnitine esters (Singh Kler et al., 1991). ETF-semiquinone, the partially reduced form of ETF, can be produced when the coenzyme Q pool is excessively reduced (Frerman, 1987) and is a potent inhibitor of the acyl-CoA dehydrogenase (Beckmann et al., 1981), so that the activity of the acyl-CoA dehydrogenases may be.responsive to the redox state of the coenzyme Q pool. Little work has been carried out on the importance of this inhibition in the intact mitochondrion.

Correspondence to K . Bartlett, Room 4090, Department of Child Health, Medical School, University of Newcastle upon Tyne, Fram- Iington Place, Newcastle upon Tyne, England NE2 4HH

Fax: +44 91 222 6222. Abbreviations. CPT, carnitine palmitoyl transferase ; ETF,

electron transfer flavoprotein ; ETF : QO, electron transfer flavoprotein: coenzyme Q oxidoreductase.

Kunz (1988) suggested that the redox state at the ETF/ ETF:QO stage may be important in control in intact mitochondria and this work has recently been extended by a pre- liminary application of control theory which showed that flux control was distributed according to conditions but included control by the ETF-ETF: QO and 3-hydroxyacyl-CoA dehydrogenase-complex I segments (Kunz, 1991).

Changes in osmolality cause changes in mitochondria1 matrix volume (Quinlan et al., 1983) and respiratory-chain activity (Halestrap and Dunlop, 1986). There is however a more marked effect of osmolality on p-oxidation flux (Osmundsen and Bremer, 1976; Otto and Ontko, 1982; Halestrap and Dunlop, 1986) and a locus between ETF and the coenzyme Q pool has been proposed as the site of this control (Halestrap and Dunlop, 1986). Such control may be important physiologically, for example in the hormonal control of Boxidation by glucagon (Halestrap, 1989; Tosh et al., 1988).

Our aim in this work was to extend the studies of Wat- mough et al. (1989), in which uncoupled mitochondria with an inhibited Krebs cycle were used, to a more physiological setting with concomitant NAD+/NADH measurements and using a substrate of higher specific activity. The effects of succinate, carnitine and osmolality on the CoA and carnitine esters resulting from P-oxidation were also explored, further- ing our previous work on the relationship between P-oxidation and the respiratory chain in rat muscle (Eaton et al., 1993a), human muscle (Watmough et al., 1990) and fibroblast (Singh Kler et al., 1991) mitochondria.

672

EXPERIMENTAL PROCEDURES Mate r i a 1 s

Sources of most materials were as previously described (Eaton et al., 1993a). 2P-Dinitropheno1, oligomycin, rotenone and valinomycin were from Sigma Chemical Co. Malo- nic acid was from B. D. H. Triethanolamine and tetradecanal were from Fluka. trans-Hexadec-2-enoic acid was from K and K Laboratories. [U-14C]Hexadecanoate was from Amer- sham International.

Synthesis of CoA and carnitine esters Acyl-CoA standards were synthesised as described by

Watmough et al., (1989) or by the mixed-anhydride method of Bernert and Sprecher (1 977). Carnitine esters were synthesised as described by Bhuiyan et al. (1987) or were gifts from Dr M. Pourfarzam, Department of Child Health, Uni- versity of Newcastle-upon-tyne.

Synthesis of trans-hexadec-3-enoic acid trans-Hexadec-3-enoic acid was synthesised by the

method of Boxer and Linstead (1931) as modified by Stoffel and Ecker (1969). To 0.92 g malonic acid were added 3 ml triethanolamine and 1.9 g tetradecanal, the mixture purged with N2, sealed and stirred for 2 h at room temperature fol- lowed by 10 h at 90°C. The mixture was cooled and added to 100 ml 4 M H,SO,. The product was extracted three times with 100 ml dry diethyl ether, the ether extracts washed with water and back-extracted to 1 M KOH. The aqueous solution was reacidified with 1 M HC1, and the product again extracted with dry diethyl ether. The ether extracts were dried over anhydrous Na,SO, and the ether removed at reduced pressure. The product was recrystallised once from hexane, and the identity of the product was confirmed by gas chroma- tography mass spectrometry of the methyl ester (data not shown).

Preparation of mitochondrial fractions Mitochondria were prepared from the livers of 18-h-

starved male Wistar rats (200-25Og) as described by Watmough et al. (1989). Mitochondria1 protein was determined by the modified Lowry method of Peterson (1977) using Boehringer Precimat standards.

Radiochemical incubation conditions Incubations with [U-'4C]hexadecanoate were carried out

in 110mM KC1, 1OmM Hepes, 5 mM MgC12, 2.5 mM KH,PO,, 1 mM EGTA, 100 pM CoA, 5 mM ATP, 0.5 mM carnitine, 0.2 mg/ml cytochrome c, pH 7.4, with 2-4 mg liver mitochondrial protein. All incubations were carried out in a shaking water bath at 37°C. After 2 min incubation, reactions were initiated with 60 nmol [U-14C]hexadecanoate (specific activity 60 pCilpmo1) as a 5 : 1 complex with BSA, so that the final BSA concentration in the incubations was 0.82 mg/ml. These were the standard incubation conditions used and were varied as described.

All incubations were quenched with 200 pi glacial acetic acid. Total acid-soluble radioactivity was measured as described by Watmough et al. (1988), and acyl-CoA and acyl- carnitine ester fractions separated and measured by radio- HPLC as described by Singh Kler et al. (1991) using a Hyp-

ersil 50DS cartridge (Technicol) for analysis of CoA esters. Hypersil 5MOS or Techsphere hexyl columns (HPLC Tech- nology) were used for the analysis of acyl-carnitines.

Measurement of mitochondrial NAD' and NADH concentrations

Incubations were carried out with unlabelled hexadecanoate as described for the radiochemical experiments, and NAD' and NADH measured as described previously (Eaton et al., 1993a).

Osmometry

Osmolality of solutions was determined by the freezing- point-depression method using a Roebling osmometer, and the osmolality of solutions varied by altering the KCl concentration. The osmolality of the standard incubation conditions was 254 mosm.

RESULTS /?-Oxidation flux from [U-14C]hexadecanoate

Flux measured as total acid-soluble radioactivity was linear over the time courses examined (20.9 nmol acetyl units . min-' . mg protein-') and was stimulated by approximately 60% by 40 pM dinitrophenol plus 1 pg/ml oligomycin, and diminished by approximately 30% by 5 mM succinate. The presence of either 1 mM ADP or 2 mM (total) carnitine had no effect on flux (18.7 nmol . min-' . mg protein-' and 20.7 nmol . min-' . mg protein-', respectively).

CoA esters generated by P-oxidation of [U-14C]hexadecanoate

The CoA ester intermediates generated in a time course under standard conditions, and with various additions, are shown in Fig. 1, and typical chromatograms are shown in Fig. 2. In the standard time courses, hexadecanoyl-CoA was formed quickly, reaching approximately 5 -6 nmol/mg protein within 1 min, indicating that acyl-CoA synthetase was not rate-limiting. Tetradecanoyl-, dodecanoyl- and decanoyl- chain-shortened CoA esters were quickly formed in high amounts, and additionally, 2-enoyl-CoA and 3-hydroxyacyl- CoA intermediates were detected at chain lengths down to dodecanoyl-CoA. Their amounts were low in comparison to the corresponding saturated acyl-CoA esters. Most of the CoA esters detected did not reach steady-state concentrations over the time course studied, even though a linear production of acid-soluble radioactivity suggests that steady-state conditions were prevailing. The presence of acetyl-CoA through- out the time course indicates that its rate of disposal to ketone bodies, the citric acid cycle or to acetyl-carnitine may be important (Quant et al., 1989). Similar patterns of intermediates were found in incubations made in the presence of 1 mM ADP (data not shown).

The presence of 40 pM 2,4-dinitrophenol plus 1 pg/ml oligomycin, after 2 inin of incubation, gave a similar pattern of CoA esters, but with lower amounts of 2-enoyl-CoA and 3-hydroxyacyl-CoA esters relative to the corresponding saturated acyl-CoA esters than in incubations made under coupled conditions. In the presence of increased (2 mM total) carnitine concentrations, decreased quantities of many of the

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Fig. 1. The generation of acyl-CoA esters by rat liver mitochondria incubated with [U-'4]hexadecanoate. Rat liver mitochondria were incubated with 60 pM [U-'"Clhexadecanoate under various incubation conditions, and CoA esters analysed by radio-HPLC, as described in the Experimental Procedures. Succinate (succ), 2,4-dinitrophenol (DNP) and carnitine (carn) were added as indicated (2 mM carnitine refers to the final carnitine concentration). CoA esters are indicated with the following notation : Pam (hexadecanoyl), 2-enoylPam (2- hexadecenoyl), 3-hydroxyPam (3-hydroxyhexadecanoyl), Myr (tetradecanoyl), 2-enoylMyr (2-tetradecenoyl), 3-hydroxyMyr (3-hydroxytetradecanoyl), Lau (dodecanoyl), 2-enoylLau (2-dodecenoyl), 3-hydroxyLau (3-hydroxydodecanoyl), Dee (decanoyl), Oct (octanoyl).

CoA esters were detected, presumably reflecting their export from the mitochondrion as carnitine esters (Fig. 1).

The presence of 5 mM succinate led to the accumulation of large amounts of hexadec-2-enoyl-CoA and 3-hydroxyhexadecanoyl-CoA esters, and in addition, an unidentified ra- dioactive compound with a retention time slightly less than that of hexadec-2-enoyl-CoA (Fig. 2B), and a similar compound with a retention time sightly less than that of tetradec- 2-enoyl-CoA (Fig. 2B). The ultraviolet absorption spectra of these components were those of saturated acyl-CoA esters (data not shown). Therefore they could not be cis-2-enoyl- CoA or trans-2-enoyl-CoA esters. Calculation of the specific activity of one of these peaks (peak 11) demonstrated that it had the same specific activity as the starting substrate, i.e. that it had 16 carbon atoms. trans-Hexadec-3-enoic and trans-hexadec-2-enoic acids and the corresponding CoA ester were synthesised as described in Experimental Pro- cedures. trans-Hexadec-3-enoic and trans-hexadec-2-enoic acids were separable by GCMS as their methyl esters (data not shown), and by HPLC as their CoA esters (Fig. 2C), and truns-hexadec-3-enoyl-CoA had the same retention time as the unknown peak. This, together with the spectral and specific activity data, suggests that the unknown peak is hexa-

dec-3-enoyl-CoA, although the stereochemical configuration is unknown. The amount of hexadec-3-enoyl-CoA formed was 0.35 nmol/mg protein and that of tetradec-3-enoyl-CoA was 0.07 nmol/mg protein.

Carnitine esters generated by /?-oxidation of [U-'4C]hexadecanoate

The carnitine esters generated in the incubations described above are shown in Fig. 3. Under standard conditions, hexadecanoyl-carnitine was formed rapidly within 1 min, indicating that CPTI (carnitine palmitoyl transferase I) was not rate limiting. Large amounts of a range of chain-shortened saturated acyl-carnitine esters were detected (C,-C,,) after 3 min incubation. Acetyl-carnitine was also present, although at low concentrations (0.49 nmol/mg protein; approximately 1 % of total acid-soluble products), consistent with ketone bodies and citric acid cycle intermediates being the main products of P-oxidation in rat liver mitochondria. 3-Hydroxyacyl-carnitine and 2-enoyl-carnitine esters were also formed, although C,,-hydroxyacyl-carnitine could not be separated from C,,-,-enoyl carnitine except at a

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Fig. 2. Chromatograms of CoA esters generated by rat liver mitochondria incubated with [U-'*C]hexadecanoate. (A) and (B) Typical radio-HPLC chromatograms of CoA esters arising from the incubation of rat liver mitochondria with 60 pM [U-'4C]hexadecanoate in the (A) absence and (B) presence of 5 mM succinate, and (C) A,,, chromatogram of synthetic CoA esters. The identity of the peaks is as follows; (1) acetyl-, (2) 3-hydroxydodecanoyl-, (3) dodec-2-enoyl-, (4) 3-hydroxytetradecanoyl-, ( 5 ) dodecanoyl-, (6) tetradec-3-enoy1, (7) tetradec-2-enoyl-, (8) 3-hydroxyhexadecanoyI-, (9) tetradecanoyl-, (10) unknown, (11) hexadec-3-enoyl-, (12) hexadec-2-enoyl-, (1 3) hexadecanoyl. Conditions of incubation and analysis were as described in Experimental Procedures.

chain length of Cr,. The amounts of these esters detected were low (about 10% of the saturated acylcarnitines, lower than the corresponding ratio in the CoA ester fraction).

In the presence of dinitrophenol and oligomycin, increased amounts of saturated acyl-carnitines were detected at all chain lengths. The addition of 5 mM succinate caused a marked accumulation of 3-hydroxyhexadecanoyl-carnitine and hexadecenoyl-carnitine. If hexadec-3-enoyl-carnitine

675

was formed, then it is likely that it would chromatogram with hexadec-2-enoyl-carnitine as the resolution of acyl-carnitine esters by HPLC is poorer than that of CoA esters. Increasing carnitine concentration to 2 mM, as expected, increased the proportion of acyl groups appearing as acyl-carnitine esters, so that all the saturated acyl-carnitines (C,-C,,) were detected. Incubations made in the presence of 2 mM carnitine also increased the amount of acetyl-carnitine formed, although it still formed only a minor end product.

NAD' and NADH concentrations during a pulse of P-oxidation

Due to the accumulation of 2-enoyl-CoA and 3-hydroxyacyl-CoA esters resulting from incubation with hexadecanoate, the role of the redox state of the nicotinamide-nucleotide pool in the formation of these intermediates was examined directly. The results are shown in Fig. 4. It can be seen that rat liver mitochondria as isolated were very reduced, probably due to the anaerobicity of such a concentrated mitochondrial suspension. This reduction disappeared within the 2-min prior incubation time, and on addition of substrate, NADH levels quickly rose and those of NAD' fell to give steady-state levels of about 30% reduction of the total pool during a pulse of hexadecanoate oxidation, compared to the rotenone-inhibited levels. This steady 30% reduction and the subsequent turnover of the NAD'NADH pool via the activity of complex I is probably important for the control of P-oxidation under these conditions.

b-Oxidation flux at varying osmolalities

P-Oxidation flux, as total acid-soluble radioactivity, was measured at various osmolalities between 125 and 450 mosm. The results are shown in Fig. 5. P-Oxidation flux was markedly dependent on osmolality. At 250 mosm, the flux was 32 nmol acetyl units . min-' . mg protein-'. When the osmolality was decreased, P-oxidation flux increased so that at 125 mosm, there was an increase in flux to 53 nmol . min-' . mg protein-', a 60% increase over the flux at 250 mosm. An increase in osmolality to 438 mosm decreased flux dramatically, to 6 nmol . min-' . mg protein-', 18% of the flux at 250 mosm. In the presence of valinomycin (1 nM) flux was increased to 44nmol . min-' . mg protein-'. The change in flux was greatest over the range 200-300 mosm. Such results are in agreement with those obtained radio- chemically with [ l-'4C]hexadecanoyl-carnitine (Osmundsen and Bremer, 1976), by the rates of ketogenesis (Otto and Ontko, 1982), and polarographically with hexadecanoyl-carnitine by Halestrap and Dunlop (1986).

CoA and carnitine esters resulting from P-oxidation at various osmolalities

The CoA esters generated by incubation of rat liver mitochondria with [U-'4C]hexadecanoate at three different osmolalities are shown in Table 1 and the corresponding carnitine esters shown in Table 2. The amounts of the saturated acyl- CoA esters increased with osmolality in the range 125- 325 mosm, accounted for as hexadecanoyl-CoA in the range 250- 325 mosm. The corresponding 2-enoyl-CoA and 3-hydroxyacyl-CoA esters remained at relatively steady levels, although there was a slight decrease in the range 250 and

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Fig. 3. The generation of acyl-carnitine esters by rat liver mitochondria incubated with [U-'"C]hexadecanoate. Rat liver mitochondria were incubated with 60 pM [U-'4C]hexadecanoate under various incubation conditions, and carnitine esters analysed by radio-HPLC, as described in Experimental Procedures. The labelling of the axes and notation of the carnitine esters are as in Fig. 1.

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Fig. 4. Intramitochondrial concentrations of NAD' and NADH during p-oxidation of hexadecanoate. Rat liver mitochondria were incubated with 60 pM hexadecanoate and NAD' and NADH measured by HPLC as described in Experimental Procedures. The amounts of NADH (A) and NAD' (B) are shown in the absence (W) or presence (0) of rotenone.

325 mosm. This finding is consistent with rate limitation at the ETF-coenzyme Q segment (Halestrap and Dunlop, 1986) although if this were the only factor operating then lower amounts of 2-enoyl-CoA and 3-hydroxyacyl-CoA would be expected at the higher osmolalities due to the greatly decreased flux.

The changes in the carnitine esters reflected that of the CoA esters. Significant amounts of hexadecanoyl-carnitine were found in all the incubation conditions, suggesting that CPTI activity is not responsible for the changes seen, even though [KCl] has been reported to influence CPT activity in rat liver mitochondria (Saggerson, 1982). At high flux rates (i.e. low osmolality), CPTI may become rate limiting, as diminished quantities of hexadecanoyl-carnitine were found. Saturated acyl-carnitines were found at all chain lengths apart from butyryl, although shorter chain lengths were not present under iso-osmolar or hyper-osmolar conditions. The proportion of saturated acyl-carnitine esters to the corresponding 2-enoyl-carnitine and 3-hydroxyacyl-carnitine esters greatly increased with osmolality, although 2-enoyl-carnitine esters were still found at 325 mosm.

DISCUSSION The rates of P-oxidation observed in the present study

are similar to those reported by Watmough et al., (1989). Furthermore the effects of dinitrophenol and succinate in increasing and lowering P-oxidation flux, respectively, have

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Fig. 5. The effect of varying osmolality on 8-oxidation flux. Rat liver mitochondria were incubated with [U-'4C]hexadecanoate in incubation medium of varying osmolality and acid soluble radioactivity measured as described in Experimental Procedures.

also been reported previously (Bremer and Wojtczak, 1972). However the formation of 2-enoyl-CoA and 3-hydroxyacyl- CoA esters from [U-'4C]hexadecanoate is different to previous studies of liver mitochondria. These either used insensi- tive methods (Bremer and Wojtczak, 1972; Stanley and Tubbs, 1975 ; Lopez-Cardozo et al., 1978) or low-specific- activity substrate with uncoupled mitochondria in the presence of the citrate-cycle inhibitor fluorocitrate (Watmough et al., 1989). The present work extends that of Watmough et al. (1989) to a more physiological situation i.e. coupled mitochondria, and uses substrate of higher specific activity to increase sensitivity. Since incubations were made in the presence of 5 mM ATP and since there is mitochondrial ATPase activity it is probable that respiratory conditions closer to state 3 than state 4 were prevailing. This was confirmed by the lack of effect of the addition of 1 mM ADP on either flux or intermediates. Hexadecanoyl-CoA is known to be an inhibitor of the mitochondrial adenine-nucleotide translocase (Shug et al., 1971 ; Lerner et al., 1972). However, these inhi- bitions were observed in the absence of albumin or other external proteins, and the incubations described here were carried out in the presence of 0.82 mg/ml BSA (added with the substrate). To further examine a possible inhibition of the translocase, we carried out incubations made in the presence of an additional 1 mg/ml BSA. Flux was 30% diminished in the presence of additional BSA, and slightly lower amounts of 2-enoyl-CoA and 3-hydroxyacyl-CoA esters were observed (data not shown). This latter lowering of 2-enoyl-CoA and 3-hydroxyacyl-CoA esters by BSA was secondary to reduced /3-oxidation flux, and not due to relieving inhibition of the translocase, as relieving inhibition would be expected to give increased rather than diminished flux. The slowing of p-oxidation flux by an additional 1 mg/ml BSA indicates that the flux obtained here is probably faster than that in vivo; an additional in vivo control would be present at the level of CPTI.

The failure of other workers to detect 2-enoyl and 3- hydroxacyl esters may be due to the measurement of carnitine esters, or free acids after alkaline hydrolysis, rather than intact CoA esters directly, as discussed previously for skeletal-muscle mitochondria (Eaton et al., 1993a). This is con-

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Table 1. Acyl-CoA esters detected during incubation of rat liver mitochondria with [U-'4C]hexadecanoate at different osmolalities. Rat liver mitochondria were incubated for 2 min in medium of varying osmolality, then 60 pM hexadecanoate was added. Reactions were quenched after 1.5 min and CoA esters extracted and analysed. Notation of CoA esters is as described in the legend to Fig. 1.

Osmo- Amount ester generated of iality

Pam 2-enoyl-Pam 3-hydroxy-Pam Myr 2-enoyl-Myr 3-hydroxy-Myr Lau 2-enoyl-Lau 3-hydroxy-Lau acetyl

mosm nmol/mg protein

125 8.46 0.23 0.27 0.31 0.04 0.04 0.12 0.02 0.05 0 250 5.16 0.30 0.25 0.91 0.05 0.06 0.36 0.05 0.05 0.45 325 12.57 0.12 0.26 0.57 0.02 0.03 0.13 0.01 0 0

Table 2. Acyl-carnitine esters detected during incubation of rat liver mitochondria with [U-'4C]hexadecanoate at different osmolalities. Rat liver mitochondria were incubated for 2 min in medium of varying osmolality, and 60 pM hexadecanoate was added. Reactions were quenched after 1.5 min and carnitine esters extracted and analysed. Notation of carnitine esters is as described in the legend to Fig. 1, with the addition of Hex for hexanoyl-carnitine.

Osmo- Carnitine ester generated lality

Pam 2-enoyl-Pam 3-hydroxy-Pam Myr 2-enoyl-Myr Lau 2-enoyl-Lau Dec 2-enoyl-Dec Oct Hex acetyl

mosm nmoVmg protein

125 0.66 0.09 0.11 0.37 0.30 0.26 0.27 0.61 0.32 0.73 0.83 3.21 250 2.07 0.04 0.06 1.71 0.10 0.90 0.05 0.79 0.07 0.46 0.44 4.11 325 4.89 0.12 0 1.78 0 0.67 0 0.38 0 0 0 0.21

firmed by the finding that the ratio of 2-enoy1/3-hydroxyacyl esters :saturated esters is higher in the CoA than in the carnitine ester fraction, probably due to the substrate specificity of CPT (Mahadevan et al., 1970; Al-Arif and Blecher, 1971). Recently, Jin et al., (1992) reported production of 2-enoyl- and 3-hydroxy-fatty acids by coupled rat liver mitochondria. However, they did not measure CoA and carnitine esters, neither did they measure flux or the intramitochondrial redox state.

The methods used in this study do not distinguish between intra- and extra-mitochondria1 CoA and carnitine esters, and it is possible that some control over P-oxidation flux is exterted at the level of CPTII and the carnitine/ acylcarnitine translocase, both in the case of entry of hexadecanoyl-carnitine, and exit of chain-shortened acyl-carnitine esters. The export of chain-shortened acyl-carnitine esters would additionally free intramitochondrial CoA, and chain- shortened CoA esters could accumulate extra-mito- chondrially due to the action of CPTI in the reverse direction.

Incubations made in the presence of dinitrophenol (40 pM) plus 1 pg/ml oligomycin, i.e. in uncoupled mitochondria without stimulating ATP-ase activity, markedly increased flux and lowered the concentrations of 2-enoyl- CoA and 3-hydroxyacyl-CoA esters compared to the corresponding saturated acyl-CoA esters, probably because in addition to uncoupling mitochondria, dinitrophenol also stimu- lates respiratory-chain activity. However it is difficult to in- terpret these differences as the CoA esters were not in steady state; the increased rate of flux in the presence of dinitrophe- no1 means that higher amounts of CoA esters would be expected. These findings are consistent with some intramitochondrial control of P-oxidation by the respiratory chain at the level of 3-hydroxyacyl-CoA dehydrogenase activity

rather than by the absolute enzyme activities. The latter would predict the acyl-CoA dehydrogenases to have by far the greatest control strength since their measured activities are the lowest of the four steps of P-oxidation (Melde et al., 1991). Such a role of the redox state has been postulated previously (Bremer and Wojtczak, 1972 ; Lopez-Cardozo et al., 1978; Latipaa et al., 1986), although only when respiratory-chain activity was decreased. Kunz (1 991) found significant control to reside with the respiratory chain under all conditions, although it is important to note that the concentration of CoA esters never attained a steady state in our experiments, as found by Stanley and Tubbs (1974, 1975), even though flux measured as acid-soluble radioactivity would suggest steady-state conditions to be prevailing. This finding renders the application of steady-state control theory problematic.

The hypothesis of significant control by the respiratory chain under normal conditions was here confirmed by the direct measurement of NAD' and NADH levels during a pulse of hexadecanoate oxidation. NADH levels rose to, and remained at about 30% reduction, relative to the NAD'/ NADH ratio in the presence of rotenone. This finding is in contrast to the situation in skeletal-muscle mitochondria (Eaton et al., 1993a) in which NADH levels do not rise sig- nificantly during hexadecanoate oxidation, although the pos- sibility of channelling and sub-compartmentation of NAD'/ NADH with complex I (Sumegi and Srere, 1984; Fukushima et al., 1989; Sumegi et al., 1991 ; Eaton et al., 1993a) must be taken into account. Hence there seems to be important inter-tissue variation in the control of mitochondria1 P-oxidation. It is also interesting to note that there seems to be variation in human tissues; control human skeletal-muscle (Jack- son et al., 1992), heart (Eaton, 1992) and fibroblast (Singh

679

Kler et al., 1991) mitochondria produce only saturated intermediates, whereas human liver mitochondria produce significant levels of 2-enoyl-CoA and 3-hydroxyacyl-CoA intermediates (Eaton et al., 1993b).

In a recent report it is suggested that 2-enoyl-CoA-hydratase activity is product inhibited by 3-hydroxyacyl-CoA esters (He et a]., 1992); such a finding is consistent with the observation here of higher amounts of 2-enoyl-CoA esters than 3-hydroxyacyl-CoA esters. However, He et al. (1992) also postulate that such control may be more important at shorter chain lengths. There is no evidence for this in our findings ; no short-chain 3-hydroxyacyl-CoA or 2-enoyl-CoA esters were observed, and such observations carried out on purified enzymes are unlikely to be of significance in vivo since there may be three enoyl-CoA hydratases in human tissues (Jackson et al., 1992). In addition the long-chain activities of enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-oxoacyl-CoA thiolase reside on the same membrane-associated protein (Uchida et a]., 1992 ; Carpenter et al., 1992) and it is probable that the properties of this complex and its relationship to the respiratory chain is important in the control of Boxidation. The control by the respiratory chain may, however, be exaggerated in our experiments due to the high substrate load.

Production of 3-enoyl-CoA esters by rat liver mitochondria has not been directly demonstrated previously, although work by Davidoff and Korn (1965), Bremer and Wojtczak (1972), Stanley and Tubbs (1975), Lopez-Cardozo et al. (1978) and Jin et al. (1992) suggested that such a CoA ester might be formed in situations in which the NAD+/ NADH ratio is held artificially low, either by succinate-induced reversed electron flow (Chance and Hollunger, 1957 ; Ernster and Lee, 1967) or by rotenone inhibition of complex I (Slater, 1967), The 3-enoyl-CoA derivatives probably arise from the action of 3,2-enoyl-CoA isomerase (Stoffel and Ecker, 1969) on trans-2-enoyl-CoA formed by the acyl-CoA dehydrogenases. The stereospecificity of the 3-enoyl-CoA esters could not be determined here, although Jin et al. (1 992) found approximately equal amounts of cis- and trans-acids. It is surprising that such an isomerisation should take place in the reverse direction to that thought of as the physiological direction of the enzyme; Stoffel and Ecker (1969) described the reaction as going essentially to completion. However, these workers studied an easily solubilised mitochondrial 3,2-enoyl-CoA isomerase which is specific for short-chain CoA esters (Palosaari et al., 1990). The long-chain 3,2-enoyl- CoA isomerase recently described (Kilponen et al., 1990) is probably the enzyme activity functional here, and the cata- lytic properties of the enzyme have not been fully investi- gated. The inability to detect 3-hexadecenoyl-CoA under normal incubation conditions, even though significant amounts of 2-enoyl-CoA esters were formed means that further work on the relationship of the long-chain 3,2-enoyl- CoA isomerase to the other enzymes of P-oxidation, espe- cially the trifunctional protein (Uchida et al., 1992), is necessary. Luo et al. (1993) have recently found that an inner- membrane preparation contains little isomerase activity, so that the long-chain isomerase may be a matrix enzyme.

The increase in saturated acyl-CoA and acyl-carnitine esters with increased osmolalities is in keeping with a locus of control between ETF and coenzyme Q, resulting in inhibition of the acyl-CoA dehydrogenases, as suggested by Halestrap and Dunlop (1 986). However, the data presented suggest that this is not the only effect. If the effects of changes in osmolality on a-oxidation were mediated solely at a locus between

ETF and coenzyme Q then only saturated acyl-CoA and carnitine esters would be predicted to accumulate under hyperosmotic conditions. The finding of similar amounts of 2-enoyl-CoA and 3-hydroxyacyl-CoA at varying osmotic strengths, even though the flux varied fourfold, suggests that some degree of control resides at the 3-hydroxyacyl-CoA dehydrogenase or in the complex I to coenzyme Q segment of the respiratory chain. Oxidation of NAD'-linked substrates has been reported to be diminished by hyperosmotic conditions, although not as markedly as hexadecanoyl-carnitine oxidation (Halestrap and Dunlop, 1986). Such an effect could be mediated by NAD+/NADH pool turnover, so that a dual inhibition of both the NAD' and ETF-linked stages by hyperosmotic conditions would explain the pattern of intermediates detected, with the ETF-linked stage inhibited more than the NAD+-linked stage at high osmolalities. However, the data of Otto and Ontko (1982) are not in agreement with this hypothesis as they found that 3-hydroxybutyrate/acetoacetate ratios increased with decreasing osmolality, rather than remaining constant, as would be expected if the NAD+/ NADH ratios were constant. Sub-compartmentation of intramitochondrial NAD' (Sumegi and Srere, 1984; Fukushima et al., 1989) could explain this discrepancy. A recent study has suggested the effect of osmolality on the respiratory chain to be at the level of ubiquinone diffusion within the membrane (Mathai et al., 1993). Further work is necessary to characterise the changes seen, but it is almost certain that the situation is more complex than a single site of inhibition. Osmolality, in causing mitochondrial swelling, would be expected to alter the membrane environment of those enzymes of P-oxidation which are membrane associated (trifunctional protein, CPT, translocase, ETF: QO and possibly long-chain acyl-CoA dehydrogenase) as well as components of the respiratory chain, and alterations in co-factor access are possible.

S. E. was in receipt of a Medical Research Council studentship. Action Research for the Crippled Child and the Muscular Dystrophy Group of Great Britain are gratefully thanked for the provision of equipment and support.

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