Metabolic Functions of the Two Pathways of Oleate β -Oxidation Double Bond Metabolism During the β -Oxidation of Oleic Acid in Rat Heart Mitochondria

Abstract Unsaturated fatty acids with odd-numbered double bonds, e.g. oleic acid, can be degraded by β-oxidation via the isomerase-dependent pathway or the reductase-dependent pathway that differ with respect to the metabolism of the double bond. In an attempt to elucidate the metabolic functions of the two pathways and to determine their contributions to the β-oxidation of unsaturated fatty acids, the degradation of 2-trans,5-cis-tetradecadienoyl-CoA, a metabolite of oleic acid, was studied with rat heart mitochondria. Kinetic measurements of metabolite and cofactor formation demonstrated that more than 80% of oleate β-oxidation occurs via the classical isomerase-dependent pathway whereas the more recently discovered reductase-dependent pathway is the minor pathway. However, the reductase-dependent pathway is indispensable for the degradation of 3,5-cis-tetradecadienoyl-CoA, which is formed from 2-trans,5-cis-tetradecadienoyl-CoA by Δ3,Δ2-enoyl-CoA isomerase, the auxiliary enzyme that is essential for the operation of the major pathway of oleate β-oxidation. The degradation of 3,5-cis-tetradecadienoyl-CoA is limited by the capacity of 2,4-dienoyl-CoA reductase to reduce 2-trans,4-trans-tetradecadienoyl-CoA, which is rapidly formed from its 3,5 isomer by Δ3,5,Δ2,4-dienoyl-CoA isomerase. It is concluded that both pathways are essential for the degradation of unsaturated fatty acids with odd-numbered double bonds inasmuch as the isomerase-dependent pathway facilitates the major flux through β-oxidation and the reductase-dependent pathway prevents the accumulation of an otherwise undegradable metabolite.

4 mitochondria, respectively (7). However, that study relied on the quantification of fatty acid metabolites in intact mitochondria. These metabolites are not true intermediates of β-oxidation but rather products that have leaked from the pathway, especially when functionally compromised mitochondria are involved (8). Hence, it is very doubtful that these values are meaningful estimates of the flux through the reductase-dependent pathway. In fact, when the degradation of 2-trans,5-cis-octadienoyl-CoA, a medium-chain intermediate of linolenic acid metabolism, was studied with a soluble extract of rat liver mitochondria in the presence of NAD + , CoASH, and NADPH, 80% of the metabolite was observed to be degraded via the isomerase-dependent pathway (9). The uncertainty about the contributions of the two pathways to the β-oxidation of long-chain dietary fatty acids prompted this study of the degradation of the oleate metabolite 2-trans,5-cis-tetradecadienoyl-CoA (III) by solubilized rat heart mitochondria.

Experimental Procedures
Materials -CoASH, NAD + , NADH, NADPH, dodecanoyl-CoA, decanoyl-CoA, and acetyl-CoA were purchased from Life Science Resources, Milwaukee, WI. Acyl-CoA oxidase from Arthrobacter species was bought from Boehringer Mannheim. Sep-Pak C 18 cartridges used for concentrating acyl-CoAs and µBondapak C 18 columns (30 cm × 3.9 mm) were purchased from Waters. Sigma was the supplier of most standard biochemicals. Bovine liver enoyl-CoA hydratase (crotonase) (10), recombinant pig liver L-3-hydroxyacyl-CoA dehydrogenase (11), pig heart 3-ketoacyl-CoA thiolase (12), recombinant human peroxisomal enoyl-CoA isomerase (13), rat liver enoyl-CoA isomerase (14), and recombinant rat liver dienoyl-CoA isomerase (15) were purified by published procedures. 2-trans-Dodecenoic acid was synthesized from n-decanal and malonic acid as described in principle by Linestead et al. (16). 5-cis-Tetradecenoic acid was a kind gift from Dr. Howard Sprecher, Ohio State University. Syntheses of Substrates and Metabolites -5-cis-Tetradecenoyl-CoA and 2-trans-dodecenoyl-CoA were synthesized from 5-cis-tetradecenoic acid and 2-trans-dodecenoic acid, respectively, by the mixed anhydride method as described by Fong and Schulz (17). Both products were purified by HPLC. For the synthesis of 2-trans-5-cis-tetradecadienoyl-CoA, a solution of 5 µmol of 5-cis-tetradecenoyl-CoA in 30 ml of 0.1 M KP i (pH 9.0) was saturated with air for 30 min and dehydrogenated by acyl-CoA oxidase at room temperature. The near complete conversion was achieved by the addition of 10 to 20 units of acyl-CoA oxidase in several aliquots over a period of 45 min. The progress of the conversion was monitored by HPLC. When a maximal conversion was achieved as indicated by the disappearance of 5cis-tetradecaenoyl-CoA, the pH of the solution was adjusted to 1.5 with 6 N HCl to terminate the reaction. Precipitated protein was removed by filtering the solution through a 0.22-µm pore size membrane. After adjusting the pH to 4 with 4 N KOH, the solution was concentrated by passing it through a Sep-Pak C 18 cartridge and eluting it with a small volume of methanol, which subsequently was evaporated under reduced pressure. The resultant 2-trans-5-cis-tetradecadienoyl-CoA was purified by HPLC. Fractions containing 2-trans-5-cis-tetradecadienoyl-CoA were combined, concentrated as described above, and finally dissolved in deionized water. The pH of the final preparation was adjusted to 3 ~ 4 and the thioester concentration of this solution was determined spectrophotometrically by quantification of CoASH with Ellman's reagent (18) after cleaving the thioester bond with NH 2 OH at pH 7.0 (17). The concentration of 2-trans,5-cis-tetradecadienoyl-CoA was calculated by subtracting the concentration of 3,5-cis-tetradecadienoyl-CoA from that of 2-trans,5-cis-tetradecadienoyl-CoA plus 3,5cis-tetradecadienoyl-CoA. The concentrations of 2-trans,5-cis-tetradecadienoyl-CoA plus 3,5-cistetradecadienoyl-CoA and of 3,5-cis-tetradecadienoyl-CoA were determined by measuring the absorbance changes at 300 nm due to their conversions in 0.1 M KP i (pH 8.0) to 2,4-tetradecadienoyl-CoA upon additions of 0.1 unit of dienoyl-CoA isomerase plus 0.05 unit of enoyl-CoA isomerase and of 6 0.1 unit of dienoyl-CoA isomerase, respectively. Concentrations of 2,4-dienoyl-CoA were calculated using an extinction coefficient of 28,000 M -1 cm -1 (19).
Preparation of a Solubilized Extract from Rat Heart Mitochondria -Rat heart mitochondria were isolated as described by Chappell and Hansford (20) and stored at -70 0 C. The thawed rat heart mitochondria were suspended in 0.2 M KP i containing 0.5 mM ethylenediaminetetraacetate, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 10 mM benzamidine, and 5mM 2-mercaptoethanol 7 and incubated for 30 min on ice. The mixture was centrifuged at 100,000 x g at 4 0 C and the supernatant was used for metabolic assays. Purification and Analyses of Acyl-CoA Thioesters by HPLC -Acyl-CoA substrates were purified and metabolites were analyzed by reverse-phase HPLC on a Waters µBondapak C 18 column (30 cm x 3.9 mm) attached to a Waters gradient HPLC system. The absorbance of the eluate was monitored at 254 nm. Separation of substrates and metabolites was achieved by washing the µBondapak C 18 column with 50 mM ammonium phosphate (pH 5.5) containing 30% of acetonitrile/water (9:1, v/v) for 20 min and then eluting acyl-CoAs by linearly increasing the organic phase from 30 to 60% in 20 min at a flow rate of 2 ml/min. All samples were cleared of particulate matter by passing them through a 0.22-µm pore size membrane before they were injected into the HPLC system. Diluted samples were concentrated by passing them through Sep-Pak C 18 cartridges and eluting them with small amounts of methanol, which subsequently were removed by evaporation under reduced pressure.

Kinetics of 2-trans,5-cis-Tetradecadienoyl-CoA Degradation -2-trans,5-cis-Tetradecadienoyl-
CoA (III) is an intermediate that is formed during the β-oxidation of oleate and that can be further metabolized by either the isomerase-dependent pathway or the reductase-dependent pathway (see Scheme 1). The kinetics of 2-trans,5-cis-tetradecadienoyl-CoA degradation via these two pathways were studied with rat heart mitochondria because of their minimal contamination with peroxisomes that are estimated to account for less than 3% of cardiac fatty acid β-oxidation (24).
Treatment of rat heart mitochondria with 1% of Triton X-100 yielded a soluble extract that contained all β-oxidation enzymes required for the degradation of 2-trans,5-cis-tetradecadienoyl-9 CoA (III) to decanoyl-CoA (X) and dodecanoyl-CoA (XVII). Since it had previously been determined that Triton X-100 at the applied concentration did not affect the activities of the enzymes of the β-oxidation spiral (25,26), it was only necessary to assess how Triton X-100 affects the activities of enoyl-CoA isomerase, 2,4-dienoyl-CoA reductase, and dienoyl-CoA isomerase. Such test revealed that none of these three auxiliary enzymes was negatively affected by 1% of Triton X-100 (data not shown). When 2-trans,5-cis-tetradecadienoyl-CoA (III) was incubated with an extract of rat heart mitochondria in the presence of 1 mM NAD + and 0.3 mM CoASH but in the absence of NADPH, it was possible to determine rates of β-oxidation via the isomerase-dependent pathway without interference from the reductase-dependent pathway by measuring spectrophotometrically the formation of NADH at 360 nm. The entry into the reductase-dependent pathway was determined separately by measuring at 300 nm the accumulation of 2,4-tetradecadienoyl-CoA (XII) in the absence of any cofactor. The results of these experiments are shown in Fig. 1. Specific activities for the isomerase-dependent pathway are based on initial velocity measurements that were linear during the first two minutes when, on the average, 1.5 moles of NADH were produced per mole of degraded 2-trans,5-cistetradecadienoyl-CoA (III). The conversion of 2-trans,5-cis-tetradecadienoyl-CoA (III) to 2,4tetradecadienoyl-CoA (XII) was measured 30 sec after initiation of the raction when rates were linear. When the flux of 2-trans,5-cis-tetradecadienoyl-CoA (III) through the isomerasedependent pathways is compared with its entry into the reductase-dependent pathway it is obvious that the former pathway is the dominant one and that the ratio of rates for the two pathways does not vary significantly over a considerable range of substrate concentrations (see Fig. 1).
Consequently, the results that were obtained by studying the degradation of 2-trans,5-cis-10 tetradecadienoyl-CoA at one concentration may reflect the situation in intact mitochondria, for which the concentrations of true intermediates are unknown.
In subsequent experiments we analyzed the time-dependent formation of metabolites that accumulate when 20 µM 2-trans,5-cis-tetradecadienoyl-CoA (III) was incubated with an extract of solubilized rat heart mitochondria in the presence of 1mM NAD + , 0.3 mM CoASH, and 0.5 mM NADPH.
Representative HPLC chromatograms are shown in Fig. 2. Product analysis five seconds after initiating the incubation revealed the rapid hydration of 2-trans,5-cis-tetradecadienoyl-CoA (III) to 3-hydroxy-5cis-tetradecenoyl-CoA (IV) (see Fig. 2A). Since the hydration is freely reversible, both intermediates can enter either pathway. In addition, traces of 2-dodecenoyl-CoA (VII) and 2,4-tetradecadienoyl-CoA (XII) were detected. These two metabolites are committed to proceed through the isomerase-dependent pathway and reductase-dependent pathway, respectively. After one minute of incubation all intermediates of the isomerase-dependent pathway with the exception of 3-ketododecanoyl-CoA (IX) were present at detectable levels (see Fig. 2B). Decanoyl-CoA was the final product of this metabolic sequence due to the absence of cofactors that are necessary for its further degradation by β-oxidation.
Entry into the reductase-dependent pathway had also continued as indicated by the formation of more 2,4-tetradecadienoyl-CoA (XII) while dodecanoyl-CoA (XVII) remained undetectable (see Fig.2B).
Five minutes after initiating the incubation, dodecanoyl-CoA (XVII), the end product of the reductasedependent pathway under the prevailing experimental conditions, was present together with its precursor, 2,4-tetradecadienoyl-CoA (XII) (see Fig. 2C). 3,5-Tetradecadienoyl-CoA was difficult to detect because it was insufficiently separated from 2-trans,5-cis-tetradecadienoyl-CoA. However, it is unlikely to accumulate because of the high activity of dienoyl-CoA isomerase in the mitochondrial extract. After a total reaction time of 5 minutes, 2-trans,5-cis-tetradecadienoyl-CoA had been completely metabolized and all intermediates of the isomerase-dependent pathway had been converted by guest on March 23, 2020 http://www.jbc.org/ Downloaded from to decanoyl-CoA (X). The small amount of material marked ∆ 2 -C 12 -CoA was identified as a nonmetabolizable side product that seems to be formed from either 2,5-tetradecadienoyl-CoA (III) or 2,4tetradecadienoyl-CoA (XII) in a time-dependent manner and that was eluted from the reverse-phase column together with ∆ 2 -C 12 -CoA.
The kinetics of 2-trans,5-cis-tetradecadienoyl-CoA degradation and metabolite formation are shown in Fig. 3. Most dramatic was the rapid hydration of 2-trans,5-cis-tetradecadienoyl-CoA to 3-hydroxy-5-cis-tetradecenoyl-CoA. This reaction preceded the slower dehydrogenation of the 3- tetradecadienoyl-CoA into the reductase-dependent pathway was initially quite rapid as indicated by the formation of 2,4-tetradienoyl-CoA but declined as the concentration of 2-trans,5-cistetradecadienoyl-CoA decreased due to its hydration. However, dodecanoyl-CoA, the end product of this pathway, was formed very slowly with the result that only a fraction of its precursor, 2,4tetradecadienoyl-CoA, was converted to the final product during the five-minute incubation period, which was sufficient for the complete conversion of all intermediates of the isomerase-dependent pathway to the final product decanoyl-CoA. Thus, it seems that the NADPH-dependent reduction of 2,4-tetradecadienoyl-CoA restricts the flux through the reductase-dependent pathway.
Degradation of 3,5-Tetradecadienoyl-CoA -3,5-cis-Tetradecadienoyl-CoA is an assumed intermediate of oleate β-oxidation that we did not detect during the characterization of metabolites formed from 2-trans,5-cis-tetradecadienoyl-CoA because it was not separated from its precursor by HPLC. In addition we asked whether 3,5-cis-tetradecadienoyl-CoA could be metabolized via the isomerase-dependent pathway in addition to being degraded by the reductase-dependent pathway. To address these issues, 3,5-cis-tetradecadienoyl-CoA was incubated with an extract of rat heart mitochondria in the presence of NAD + and CoASH and its metabolites were analyzed by HPLC. Since the absence of NADPH prevents degradation via the reductase-dependent pathway, the flux through the isomerase-dependent pathway can be evaluated. As shown in Fig. 4A, 3,5cis-tetradecadienoyl-CoA was rapidly converted to its 2,4-isomer, but did not enter the isomerasedependent pathway to a significant degree. After five minutes of incubation a trace of decanoyl-CoA was detected (data not shown), which could have been formed either via the isomerasedependent pathway as outlined in Scheme 1 or more likely by direct β-oxidation of 2,4tetradecadienoyl-CoA. The rapid degradation of 3,5-cis-tetradecadienoyl-CoA via the reductasedependent pathway was demonstrated by incubating it with an extract of rat heart mitochondria in the presence of all required cofactors including NAD + , CoASH, and NADPH. As shown in Fig.   4B, 3,5-cis-tetradecadienoyl-CoA was rapidly converted to its 2,4 isomer, which was slowly reduced as indicated by the delayed appearance of dodecanoyl-CoA (C 12 -CoA) in the absence of significant amounts of downstream metabolites. This experiment demonstrates that 3,5-cistetradecadienoyl-CoA is only metabolized via the reductase-dependent pathway and additionally 13 confirms the conclusion reached during the first part of this study that the reduction of 2,4tetradecadienoyl-CoA is the rate-limiting reaction in the reductase-dependent pathway.

Effects of NADH and Acetyl-CoA on the β-Oxidation of 2-trans,5-cis-Tetradecadienoyl-CoA -
The metabolic studies described above were carried out with NAD + , CoASH, and NADPH as cofactors but in the absence of NADH and acetyl-CoA that are present in mitochondria. Since NADH and acetyl-CoA may inhibit β-oxidation enzymes and thereby the flux through the pathways, we assessed their effects on the degradation of 2-trans,5-cis-tetradecadienoyl-CoA. For this purpose we determined the formation of decanoyl-CoA (C 10 -CoA) and dodecanoyl-CoA (C 12 -CoA) plus 2,4-tetradecadienoyl-CoA (∆ 2,4 -C 14 -CoA) as a function of the incubation time to measure fluxes through the isomerase-dependent pathway and reductase-dependent pathway, respectively. Shown in Fig. 5 are the results that were obtained when no NADH (Fig. 5A), 0.17 mM NADH (Fig. 5B), or 0.5 mM NADH (Fig. 5C) was included in the incubation mixture in addition to the required cofactors NAD + , CoASH, and NADPH. When the product formation during the first three minutes was evaluated, the presence of NADH at the lower level resulted in slightly lower rates of β-oxidation but did not affect the relative flux through the reductase-dependent pathway of approximately 10%. At the higher NADH concentration, the rate of product formation was further reduced while the relative flux through the reductase-dependent pathway was only slightly increased to approximately 15% of the total. Thus, NADH inhibits β-oxidation without significantly affecting the relative contributions of the two pathways to the degradation of 2trans,5-cis-tetradecadienoyl-CoA. The effect of acetyl-CoA on the operation of the two pathways was also investigated. An increasing substitution of up to 80% of CoASH in the incubation mixture by acetyl-CoA did not affect the rate of 2-trans,5-cis-tetradecadienoyl-CoA β-oxidation nor did it change the contributions of the two pathways to this process (data not shown). Mitochondria from rat heart were used because of their minimal contamination by peroxisomes, which contain a β-oxidation system different from the mitochondrial one. Mitochondria were solubilized with Triton X-100 to obtain a system that, in contrast to intact mitochondria, would permit rate measurements of the individual pathways. The concentration of CoASH was fixed at 0.3 mM because this is its estimated concentration in mitochondria that rapidly oxidize fatty acids (23). The concentrations of NAD + and NADPH were set at 1 mM and 0.5 mM, respectively, because these are saturating concentrations even though they are lower than their estimated intramitochondrial concentrations. When rates of 2-trans,5-cis-tetradecadienoyl-CoA degradation via the isomerase-dependent pathway were compared with rates of its entry into the reductasedependent pathway, the former pathway was estimated to account for more than 85% of the βoxidation of this metabolite of oleic acid. Similar results were obtained when the accumulation of products was determined. Decanoyl-CoA, which is formed via the isomerase-dependent pathway, accounted for 85% of the products formed from 2-trans,5-cis-tetradecadienoyl-CoA. The ratio of pathways. This idea was tested by determining the effects that NADH and acetyl-CoA have on the formation of products via the two pathways. When 15% of the total NADH was in the reduced form, the relative contributions of the two pathways were unchanged even though the total flux through β-oxidation was reduced. An increase of NADH to one-third of the total coenzyme level further reduced the rate of oxidation but only slightly increased the relative contribution of the reductase-dependent pathway from 10% to 15%. Since only 5% of the total NAD + is estimated to be in the reduced state during fatty acid β-oxidation in actively respiring mitochondria (23), it is unlikely that NADH would significantly change the contribution of the reductase-dependent pathway to oleate β-oxidation. The same conclusion was reached with regard to the effect of acetyl-CoA. This product of β-oxidation neither affected the rate of the process nor the contributions of the two pathways even when it comprised 80% of the total CoA content of the system. A major reason for the limited flux through the reductase-dependent pathway is the rapid and dramatic decrease in the concentration of 2-trans,5-cis-tetradecadienoyl-CoA due to its hydration. The consequence is a greatly reduced rate of its isomerization to 3,5-cistetradecadienoyl-CoA, the first metabolite of the reductase-dependent pathway. Together the results of this study lead to the conclusion that the reductase-dependent pathway only makes a minor contribution to the total β-oxidation of oleate. If the reductase-dependent pathway contributes little to the β-oxidation of oleate, what is its metabolic function? In an attempt to answer this question, we studied the degradation of 3,5-cistetradecadienoyl-CoA, the first metabolite of oleate with two conjugated double bonds. Although it was assumed that this oleate intermediate could be metabolized via the reductase-dependent pathway, it was uncertain if it also could be degraded by way of the isomerase-dependent pathway. The results clearly demonstrate that 3,5-cis-tetradecadienoyl-CoA is rapidly converted to 2,4-tetradecadienoyl-CoA, which is reduced by NADPH-dependent 2,4-dienoyl-CoA reductase before being degraded by β-oxidation to dodecanoyl-CoA. Since only a trace of decanoyl-CoA was detected, 3,5-cis-tetradecadienoyl-CoA is not a substrate of the isomerase-dependent pathway nor is its product, 2,4-tetradecadienoyl-CoA, effectively degraded by direct β-oxidation. The first observation agrees with the previous conclusion that 3,5-dienoyl-CoAs cannot be metabolized via the isomerase-dependent pathway (9). This is most likely due to the unfavorable energetics of the 3,5-dienoyl-CoA to 2,5-dienoy-CoA conversion. Surprising was the observation that 2-trans,4trans-tetradecadienoyl-CoA, in contrast to the medium-chain metabolite 2-trans,4-transoctadienoyl-CoA (9), was not directly degraded by β-oxidation. It should be noted that 2-trans,4trans-decadienoyl-CoA but not its 2-trans,4-cis isomer is a substrate, albeit a poor one, of direct βoxidation (19). The most likely reason for the different reactivities of 2,4-tetradecadienoyl-CoA and 2,4-octadienoyl-CoA is the involvement of two different sets of β-oxidation enzymes. 2,4-Octadienoyl-CoA is presumably hydrated by crotonase and the resultant 3-hydroxyoctanoyl-CoA is dehydrogenated by 3-hydroxyacyl-CoA dehydrogenase because both of these enzymes are more active with short-chain and medium-chain substrates than with long-chain ones (17,21,22). In The reductase-dependent pathway is, however, the major pathway for the β-oxidation of unsaturated fatty acids with conjugated double bonds. Such fatty acids, specifically conjugated linoleic acid, are constituents of the human diet because they are formed in ruminants and during the partial hydrogenation of fats. The most common conjugated linoleic acid is 9-cis,11-transoctadecadienoic acid. β-Oxidation of this fatty acid is expected to produce 3-cis,5-transdodecadienoyl-CoA as an intermediate, which can only be degraded by way of the reductasedependent pathway. As previously pointed out (28), β-oxidation of 9-cis,11-trans-octadecadienoic acid also yields 2-trans,5-cis,7-trans-tetradecadienoyl-CoA as an intermediate. This metabolite might be degraded in part via the reductase-dependent pathway that requires the participation of ∆ 3,5,7 ,∆ 2,4,6 -trienoyl-CoA isomerase, which is an inherent activity of dienoyl-CoA isomerase (29). Surprising and interesting was the observed accumulation of 2,4-tetradecadienoyl-CoA during the β-oxidation of either 2-trans,5-cis-tetradecadienoyl-CoA or 3,5-cis-tetradecadienoyl-CoA. This finding prompted the idea that the reaction catalyzed by 2,4-dienoyl-CoA reductase may limit the flux through the pathway even though the entry into this pathway is already restricted by competition with the dominant isomerase-dependent pathway. A previous evaluation of a possible control exerted by 2,4-dienoyl-CoA reductase over the β-oxidation of oleic acid and docosahexaenoic acid in cardiomyocytes came to the conclusion that an increase in the activity of 2,4-dienoyl-CoA reductase in response to the treatment of rats with growth hormone did not result in higher rates of β-oxidation (30). It is possible, however, that isolated and mostly quiescent cardiomyocytes are not suitable for such study because their low energy need severely restricts fatty acid oxidation with the possible result that none of the reactions of β-oxidation is limiting the rate of the energy production.
In summary, the reductase-dependent pathway only makes a minor contribution to the β-oxidation of oleic acid, which is mostly degraded via the classical isomerase-dependent pathway. However, the reductase-dependent pathway is essential for the degradation of 3,5-cis-tetradecadienboyl-CoA, which is formed from the oleate metabolite 2-trans,5-cis-teradecadienoyl-CoA by enoyl-CoA isomerase that functions in the isomerase-dependent pathway. The reductase-dependent pathway is also essential for the β-oxidation of conjugated linoleic acid like 9-cis,10-transoctadecadienoic acid.