Mitochondrial and peroxisomal in elasmobranchs

fatty acid oxidation

C. D. MOYES, L. T. BUCK, AND P. W. HOCHACHKA of Zoology, University of British Columbia, Vancouver, British Columbia V6T 2A9; Department and Bamfield Marine Station, Bamfield, British Columbia VOR lB0, Canada

MOYES, C. D., L. T. BUCK, AND P. W. HOCHACHKA. Mitochondrial and peroxisomal fatty acid oxidation in elasmobranchs. Am. J. Physiol. 258 (Regulatory Integrative Comp. Physiol. 27): R756-R762, 1990.-In heart and red muscle of dogfish (Squalus acanthias), the maximal activities of the fatty acid catabolizing enzyme carnitine palmitoyltransferase (CPT) are (5% the rate in the same tissues of teleosts (carp, Cyprinus carpio; trout, Salmo gairdneri). CPT activities in these tissues of hagfish (Eptatretus stouti) are -10% the rate in teleosts. However, the maximal activities of the ,&oxidation enzyme ,& hydroxyacyl-CoA dehydrogenase (HOAD) in dogfish red muscle and heart are similar to these tissues in the other species. This paradox prompted a more detailed study on the capacity of mitochondria from dogfish cardiac and red skeletal muscles to utilize fatty acids, possibly by a CPT-independent pathway. Free fatty acids were not oxidized by mitochondria from red muscle (hexanoate, octanoate, decanoate, and palmitate) or from heart (octanoate, palmitate). Neither hyposmotic incubation nor addition of 5 mM ATP could stimulate oxidation of octanoate or palmitate in either preparation, suggesting that these tissues have little capacity to oxidize fatty acids by a carnitine-independent pathway. Palmitoyl carnitine oxidation was detectable at very low rates in these mitochondria only with hyposmotic incubation. Octanoyl carnitine was oxidized at greater rates than palmitoyl carnitine, 10% the rate of pyruvate in both tissues, suggesting that medium-chain fatty acids could be physiologically relevant fuels in elasmobranchs if available to heart and red muscle. One potential source of medium-chain fatty acids is hepatic peroxisomal ,&oxidation, which occurs in dogfish liver at maximal activities similar to carp and trout liver. However, based on relative rates of oxidation, it is likely that dogfish heart and red muscle metabolism are fueled primarily by carbohydrate and ketone bodies.

fish; lipid metabolism; acanthias; ,&oxidation




AEROBIC MUSCLE activity in vertebrates is usually supported primarily by lipid oxidation, often after an early phase of carbohydrate oxidation. The advantage of this strategy appears related to storage capacities of triglyceride vs. glycogen and the need to spare carbohydrate for tissues that rely primarily on glucose, such as the brain (13). With few exceptions, such as flight muscle of Prodenia moths (31), carnitine palmitoyltransferase (CPT) is required for oxidation of long-chain fatty acids by extrahepatic (heart, skeletal muscle) mitochondria (Fig. 1). Consequently, when Zammit and Newsholme (33) reported that extrahepatic tissues of elasmobranchs lack detectable CPT, attention was focused on possible




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metabolic adaptations that may be used to bypass this step in lipid oxidation. As an alternative to direct utilization of fatty acids, it was suggested that the heart and red muscle of elasmobranchs may rely on liver-generated ketone bodies as a “lipid” source (33). Support for this hypothesis is the observation by Driedzic and Hart (8) that elasmobranch hearts, unlike teleost hearts, are able to utilize ketone bodies as fuel for work. Although elasmobranch heart and skeletal muscle lack CPT, they do have the ,&oxidation enzyme 3-hydroxyacyl-CoA dehydrogenase (HOAD) (20). These observations present the possibility that elasmobranch muscle may utilize fatty acids by an alternate pathway. Several possibilities are apparent. Fatty acids could be oxidized directly by a carnitine-independent pathway (Fig. I), such as occurs in mammalian liver and heart (10). Alternately, fatty acid oxidation may be poised toward fatty acids of short and medium chain lengths, a capacity that would not necessarily be reflected by CPT activity. Oxidation of fatty acids by the p-oxi .dation pathway was long thought the domai .n of the mitochondrion. Lazarow and DeDuve (16) described a peroxisomal ,& oxidation pathway in rat liver, which occurs at maximal activities that approach the mitochondrial rate (17). The peroxisomal pathway is unlike the mitochondrial pathway in that CPT is not involved in long-chain fatty acid oxidation because fatty acids are apparently transported as CoA esters (2). Also, long-chain fatty acids are not oxidized completely, which results in the production of medium-chain fatty acids (16). At present there is no concensus on the relative importance of the peroxisomal and mitochondrial pathways of ,&oxidation in the generation of acetyl CoA (18). The inducibility of the peroxisomal pathway in response to metabolic stimuli (nature and quantity of dietary lipid, 26; food deprivation, 14; cold, 24) suggests the relative importance of the two pathways in mammalian tissues is maleable. Fish naturally experience a tremendous range in the factors that are known to affect peroxisomal abundance in mammals. The fatty acids that are particularly effective in causing proliferation of peroxisomal ,&oxidation in rats (long chain, unsaturated; 26) are abundant in the marine food chain (27). A potential role for peroxisomal ,&oxidation in facilitating the CPT-deficient mitochondrial pathway in elasmobranchs is obvious. In the present study we investigate the capacities of dogfish heart and red muscle mitochondria to oxidize various metabolic fuels. The capacities of elasmobranch

0 1990 the American



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muscle mitochondria to oxidize fatty acids is determined using fatty acyl carnitines and free fatty acids, each of various chain lengths. We compared the activities of several of the key enzymes involved in peroxisomal and mitochondrial fatty acid oxidation in liver, heart, and red muscle of selected species of teleosts (common carp, rainbow trout), chondrichthians (dogfish, ratfish), and agnathans (hagfish). MATERIALS



Animals All fish were held at seasonal temperatures (IO-15°C) in continuously flowing water without feeding (except trout). Dogfish (Squalus acanthias) were caught by hook and line and held for 2-10 days at either Bamfield Marine Station or at the Department of Fisheries and Oceans, West Vancouver. Ratfish (Hydrolagus colliei) were caught by otter trawl and used within l-3 days of capture at Bamfield Marine Station. Hagfish (Eptatretus stouti) were caught by baited trap and held 2-7 days at Bamfield Marine station or the University of British Columbia. Carp (Cyprinus carpio) were purchased from Latek Enterprises (Ruskin, BC) and held 2-5 days at the University of British Columbia. Trout (Salmo gairdneri) were purchased from Lakeland Trout Farms (Langley, BC) and held on a maintenance ration at the University of British Colimbia. Preparation

of Tissues for Enzyme Assays

Heart, red muscle, and liver were sampled from animals that were killed by double pith (dogfish) or decapitation. Single (dogfish, carp, trout) or two to four pooled (hagfish, ratfish) ventricles were taken for each homogenization. Lateral red muscle was taken from the tail region of dogfish (near the second dorsal fin), near the carnitine







tty acyl


Fatty acyl






1. Carnitine-dependent and carnitine-independent fatty acid oxidation by mitochondria. Upper pathway is carnitine-dependent route. Fatty acid is activated into its CoA ester by fatty-acyl-CoA synthase located on outer membrane (enzyme 1). Outer carnitine palmitoyltransferase (CPT; enzyme 3) catalyzes production of fatty acyl carnitine. It has been generally accepted that outer CPT is located on outer side of inner mitochondrial membrane (solid line), but more recent evidence suggests it is present on inner side of outer mitochondrial membrane (broken line; see Ref. 22). After transport of fatty acyl carnitine into mitochondrion by acylcarnitine-carnitine exchanger (enzyme 4), inner CPT (enzyme 5) regenerates acyl CoA, which enters ,Boxidation pathway. Lower pathway is carnitine-independent route. Fatty acids enter matrix where they are activated into acyl CoA esters by fatty-acyl-CoA synthase (enzyme 2; adapted from Ref. 5). FIG.




dorsal fin of carp and trout, and in hagfish, at the level of the liver, just below the skin. Red muscle from ratfish was sampled from the muscle pads of the pectoral fins. Enzyme assays were performed on either tissue homogenates or crude peroxisomal fractions. Tissue homogenates were prepared by Polytron dispersal of tissues in six volumes of buffer A [ 1 mM EDTA, 1% Triton X100, and 20 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) at pH 7.41 followed by two 10-s bursts of sonication (Kontes Ultrasonic Cell Disruptor). The homogenates were centrifuged at 4°C for 10 min at 12,000 g. The aqueous supernatant fraction was pipetted from between the sedimented particulate matter and surface lipid and then used for enzyme assays. Tissues used for preparation of crude peroxisomal fractions were dispersed in 10 volumes of buffer using a Potter-Elvejhem homogenizer. Buffer B [(in mM) 150 KCl, 500 sucrose, 1 EDTA, 20 HEPES, at pH 7.21 was used for dogfish, ratfish, and hagfish tissues. Buffer C [(in mM) 150 KCI, 1 EDTA, 20 HEPES, at pH 7.21 was used for teleost (carp, trout) tissues. Homogenates were centrifuged 5 min at 3,000 g to sediment unbroken tissue and heavy mitochondria. Supernatants were collected and centrifuged 20 min at 18,000 g to make a peroxisomeenriched pellet. The pellet was resuspended and sonicated in a small volume of buffer A and used for enzyme assays. Enzyme Assays All assays were performed at 15°C using either a PyeUnicam spectrophotometer model SPl-800 or SP6-550 at the appropriate wavelength. Preliminary studies were performed to establish that substrate concentrations were saturating. Peroxisomal ,&oxidation was assayed according to Lazarow and DeDuve (16) and Neat et al. (23). Peroxisomeenriched fractions were obtained by differential centrifugation as described above. A l-ml cuvette contained (in mM) 0.025 FAD, 0.1 CoA, 0.2 NAD, as well as 0.1% Triton X-100, 20 mM HEPES (pH 7.2), 100 PM palmitoy1 CoA (omitted for control). In this assay peroxisomal ,&oxidation is distinguished from the mitochondrial pathway by solubilization with Triton X-100. In ,&oxidation by intact mitochondria, electrons accepted at the fatty-acyl-CoA dehydrogenase step are donated to the electron transport system. In the solubilized preparation, the electron transport system is disintegrated and the lack of electron acceptors causes the dehydrogenase to be inhibited. The peroxisomal pathway is not affected by solubilization because electrons accepted by fatty-acylCoA oxidase are transferred directly to molecular oxygen. Solubilization clearly blocked the mitochondrial pathway in this study because NADH production was not detected in the fractions prepared from heart and muscle, tissues that possess high levels of mitochondria but low levels of peroxisomes. NADH is produced in the HOAD reaction of the peroxisomal enzyme complex. The rate of appearance of NADH was monitored at 340 nm. Whole tissue activities were adjusted for peroxisomal yield, using catalase as a peroxisomal marker. The assay for CPT included (in mM) 0.2 5,5’-

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dithiobis(2-nitrobenzoic acid), 20 tris(hydroxymethyl)aminomethane.HCl (pH 8.0), and 0.1 palmitoyl CoA. CPT activity, initiated by the addition of 2.5 mM Lcarnitine, was monitored as the change in absorbance at 412 nm. In tissues with low CPT activity relative to background thioesterase activity (dogfish and hagfish tissues, livers of other species), assays were performed with the reference cell containing the complete assay mix with L-carnitine omitted (thioesterase activity). In tissues with low background thioesterase activity relative t.o CPT (teleost extrahepatic tissues), the rate before addition of carnitine was subtracted from that after the addition of carnitine. The assay for catalase activity is described in detail by Aebi (1). The enzyme fraction (homogenate or peroxisome-enriched pellet) was diluted with 20 mM potassium phosphate buffer (pH 7.0) to give a 0.02-0.06 unit decrease in absorbance (240 nm) in 30 s. The reference cuvette contained 2 ml diluted enzyme with 1 ml potassium phosphate buffer (20 mM, pH 7.0). To initiate catalase activity, 1 ml of hydrogen peroxide (30 mM in potassium phosphate buffer) was added to the sample cuvette that also contained 2 ml diluted enzyme. The rate is expressed as k/g wet wt, where k is the rate constant [2.3/30 s x log absorbance at 0 s/absorbance at 30 s UL/Am)] (1). HOAD activity was assayed using tissue homogenates by following the rate of change in absorbance at 340 nm of a cuvette containing acetoacetyl CoA (0.1 mM in heart and muscle preparations, 0.2 mM in liver) and 0.15 mM NADH in 20 mM imidazole at pH 7.0. Mitochondrial

Studies on Dogfish Red Muscle and Heart



assay. After l-2 min of incubation with 0.1 mM malate and 0.5-0.8 mM ADP, the rate of oxygen consumption decreased to a slow linear rate, and saturating amounts of substrates were added to the suspension. In some experiments 5 mM ATP was added simultaneously with 0.5 mM ADP. State 3 is the rate of oxygen consumption in the presence of ADP and substrate. The respiratory control ratio, (RCR) is the ratio of state 3 to state 4, which is the rate of oxygen consumption after all ADP is phosphorylated. RESULTS

CPT and HOAD The activities of enzymes involved in fatty acid oxidation by mitochondria are summarized in Table 1. In the teleosts, CPT and HOAD activities were similar in heart, red muscle, and liver. In the dogfish and hagfish, HOAD and CPT activities were generally higher in liver than in other tissues. If one considers the relative activities of these enzymes as indexes of the capacity for fat oxidation in extrahepatic tissues of different species, conflicting conclusions would arise from the activities of HOAD vs. CPT. What is most striking in the interspecies comparisons is the general similarity of HOAD activities in heart and muscle of each species, with dramatic differences in CPT activity. The activities of CPT in heart and red muscle of hagfish were -10% that in the teleosts, and CPT was not detectable in these dogfish tissues. The observations reported in the present study for heart and muscle and previous studies dealing specifically with heart (30) prompted a more detailed investigation into possible carnitine-independent pathways for fatty acid oxidation.

Dogfish red muscle and cardiac muscle mitochondria were isolated using the following medium (in mM): 500 Mitochondrial Studies sucrose, 150 KCl, 10 EDTA, 5 MgC12, as well as 0.1% bovine serum albumin (BSA) in 20 mM HEPES (pH 7.2) The goals of the mitochondrial studies were to quantify at 20°C. Muscle tissue was minced with scissors and the capacity of heart and red muscle mitochondria to homogenized using a Potter-Elvejhem tissue grinder. The homogenate was centrifuged for 5 min at 1,400 g. TABLE 1. Maximal activities of mitochondrial enzymes The supernatant was poured through three layers of 3-hydroxyacyl-CoA dehydrogenase and carnitine cheesecloth and centrifuged for 5 min at 7,650 g. The palmitoyltransferase pellet was resuspended in isolation medium minus BSA CPT n HOAD n and recentrifuged for 5 min at 7,650 g. The resultant pellet was resuspended in the same medium to -5 mg Carp 6 Liver 1.420.06 10 0.39t0.05 mitochondrial protein/ml. Mitochondrial protein was de0.17t0.04 6 Heart 2.3-1-0.09 10 termined by the biuret method, using 10% deoxycholate 0.32t0.05 6 Red muscle 1.6t0.04 10 to solubilize the mitochondrial protein. Trout Mitochondrial oxygen consumption was monitored 2.9t0.6 11 0.30t0.03 6 Liver 7.2t0.6 9 0.28t0.02 5 using a Clark-type electrode in a glass cell at 15°C. One Heart 8.1kO.6 11 0.51kO.04 6 Red muscle volume of mitochondrial suspension was added to nine Dogfish volumes of reaction medium containing (in mM) 150 7.9t0.5 6 0.09t0.02 3 Liver KCl, 350 urea, 175 trimethylamine oxide, 300 sucrose, 1.6tO.l 6 -co.010 3 Heart and 5 NaZHP04, as well as 0.1% BSA, and 20 mM 2.9t0.2 6 30, n = 5. ND, not detected above rate with malate alone. * All substrates were assayed in presence of 0.1 mM malate. l

3. Mitochondrial oxidation of fatty acids, ketone bodies, and pyruvate by mitochondria from dogfish heart TABLE




Malate (0.1 mM)* tu-Ketoglutarate (0.1 mM) Pyruvate (5 mM) ,B-Hydroxybutyrate (5 mM) +tu-ketoglutarate (0.1 mM) Acetoacetate (5 mM) +cu-ketoglutarate (0.1 mM) Acetyl-DL-carnitine (3 mM) Octanoyl-DL-carnitine (400 PM) Palmitoyl-DL-carnitine (100 PM) Octanoate (100, 500 PM) +5 mM ATP Palmitate (24, 100 PM) +5 mM ATP

4.8t0.4 21.4t1.6 91.6t4.5 40.2t3.2 58.1t7.5 7.4rt0.6 48.3t4.5 7.0t0.4 6.5kO.7

10 10

Values are means protein; n, no. of mg/ml). Respiratory was 22.0 t 1.2, n = * All substrate were



5 10 5 5 10 5 5

5.OkO.6 22.4t1.8 99.8k4.8 41.0t1.9 67.7t5.1 10.4k1.3 50.4t5.6 8.3t0.6 15.1t0.7


7.3t1.1 ND ND ND ND

n 10 10 10

5 5 10


3 3 3

t SE for rates in nmol0. min. mg mitochondrial animals. Albumin concentration was 0.014 mM (1 control ratio for isosmotic conditions (pyruvate) 5. ND, not detected above rate with malate alone. assayed in presence of 0.1 mM malate.




observed because of ATP depletion intramitochondrially is unlikely, because addition of 5 mM ATP, giving ATP/ ADP > 10, did not stimulate oxidation of free fatty acids under isosmotic or hyposmotic conditions. Fatty acyl carnitines. The results observed with fatty acyl carnitines were similar for red muscle and heart mitochondria. When incubated under isosmotic conditions, palmitoyl carnitine was not oxidized at rates greater than the control rate (malate + ADP). Octanoyl carnitine was oxidized at several-fold higher rates than palmitoyl carnitine, when adjusted for the control rates. In red muscle the best acyl carnitine substrate tested was acetyl carnitine, but in heart acetyl carnitine oxidation was lower than octanoyl carnitine, which suggested that the activity of carnitine acetyltransferase was rate limiting in this tissue. Hyposmotic incubation stimulated oxidation of all acyl carnitines, and oxidation of palmitoy1 carnitine became consistently detectable, although the rate observed remained low. Carbohydrate and ketone bodies. Elasmobranch extrahepatic tissues are thought to use glucose or ketone bodies to support metabolism (8, 33). Glucose oxidation requires mitochondrial pyruvate oxidation; pyruvate was oxidized at high rates in both tissues (Table 2). In dogfish heart mitochondria, pyruvate was oxidized at higher rates than the ketone bodies, but in red muscle they were oxidized at similar rates. In the absence of a-ketoglutarate, acetoacetate oxidation occurred at very low rates. Sparking mitochondrial respiration with cw-ketoglutarate stimulated acetoacetate oxidation, presumably by providing a source of succinyl CoA that is needed for activation of acetoacetate into its CoA ester. Addition of aketoglutarate also stimulated ,&hydroxybutyrate oxidation, although high rates of oxygen consumption were observed in the absence of cu-ketoglutarate because of the production of reducing equivalents in the P-hydroxybutyrate dehydrogenase reaction. ,&Hydroxybutyrate was oxidized at slightly higher rates than acetoacetate in the presence of cu-ketoglutarate in both tissues. Peroxisomal @Oxidation Peroxisomal ,&oxidation occurred at similar activities in the liver of each species examined, except hagfish, where it was below detectible limits (Table 4). The rates of peroxisomal P-oxidation in chondrichthians are more impressive when the higher lipid content is taken into consideration. The activities of peroxisomal ,&oxidation in extrahepatic tissues of all species were below detectable levels. Catalase activity in each species was highest in liver, followed by heart and red muscle, each oneseventh to one-fortieth that of liver (Table 4). Interestingly, hagfish liver catalase activity was similar to dogfish liver, yet these tissues had markedly different capacities for peroxisomal ,&oxidation. Also, whereas carp had 50% higher rates of peroxisomal ,&oxidation than trout, catalase activity was M-fold greater in trout. DISCUSSION

Oxidation of glucose and lipid demands mitochondrial oxidation of pyruvate and fatty acids. Long-chain fatty

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TABLE 4. Maximal activities and peroxisomal ,&oxidation PBO Carp Liver Heart Red muscle Trout Liver Heart Red muscle Ratfish Liver Heart Red muscle Dogfish Liver Heart Red muscle Hagfish Liver Heart Lateral muscle 7




of catalase n



0.049t0.002 co.005 co.005

8 3 3

31.3t2.4 2.25t0.2 0.77t0.04

14 12 12

0.033t0.004 co.005 co.005

5 3 3

79.7t7.7 1.45&O. 12 0.87t0.15

14 9 11

0.020t0.002 co.005 co.005

5 3 3





0.038t0.004 co.005 co.005

4 3 3

11.4t1.2 0.16kO.03 0.18t0.05

7 5 6

Mitochondrial and peroxisomal fatty acid oxidation in elasmobranchs.

In heart and red muscle of dogfish (Squalus acanthias), the maximal activities of the fatty acid catabolizing enzyme carnitine palmitoyltransferase (C...
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