Carnitine Palmitoyltransferase I, Carnitine Palmitoyltransferase II, and Acyl-CoA Oxidase Activities in Atlantic Salmon (Salmo salar) Livar Frøylanda,*, Lise Madsenb, Karen M. Eckhoffa, Øyvind Liea, and Rolf K. Bergeb a
Institute of Nutrition, Directorate of Fisheries, N-5002 Bergen, Norway, and bDepartment of Clinical Biochemistry, University of Bergen, Haukeland Hospital, N-5021 Bergen, Norway
ABSTRACT: Salmon farmers are currently using high-energy feeds containing up to 35% fat; the fish’s capability of fully utilizing these high-energy feeds has received little attention. Carnitine is an essential component in the process of mitochondrial fatty acid oxidation and, with the cooperation of two carnitine palmitoyltransferases (CPT-I and CPT-II) and a carnitine acylcarnitine transporter across the inner mitochondrial membrane, acts as a carrier for acyl groups into the mitochondrial matrix where β-oxidation occurs. However, no reports are available differentiating between CPT-I and CPT-II activities in fish. In order to investigate the potential for fatty acid catabolism, the activities of key enzymes involved in fatty acid oxidation were determined in different tissues from farmed Atlantic salmon (Salmo salar), i.e., acyl-CoA oxidase (ACO) and CPT-I and CPTII. Malonyl-CoA was a potent inhibitor of CPT-I activity not only in red muscle but also in liver, white muscle, and heart. By expressing the enzyme activities per wet tissue, the CPT-I activity of white muscle equaled that of the red muscle, both being >> liver. CPT-II dominated in red muscle whereas the liver and white muscle activities were comparable. ACO activity was high in the liver regardless of how the data were calculated. Based on the CPT-II activity and total palmitoyl-L-carnitine oxidation in white muscle, the white muscle might have a profound role in the overall fatty acid oxidation capacity in fish. Lipids 33, 923–930 (1998).
Fatty acid β-oxidation occurs in two distinct organelles, i.e., peroxisomes and mitochondria. Mitochondria and peroxisomes are vital for sustaining life, but these organelles have evolved with distinct metabolic features. Fatty acid β-oxidation in peroxisomes, like mitochondria, generates acetyl-CoA through successive steps of dehydrogenation, hydration, and thiolytic cleavage. In contrast to mitochondrial β-oxidation, the first dehydrogenation step involves the reduction of O2 to H2O2 by acyl-CoA oxidase, whereas in mitochondria the second dehydrogenation step reduces NAD+ to NADH (1). The *To whom correspondence should be addressed at Institute of Nutrition, Directorate of Fisheries, P.O. Box 185, N-5002 Bergen, Norway. E-mail:
[email protected] Abbreviations: ACO, acyl-CoA oxidase (E.C. 1.3.3.6); BSA, bovine serum albumin; CPT-I, carnitine palmitoyltransferase I (E.C. 2.3.1.21); CPT-II, carnitine palmitoyltransferase II (E.C. 0.0.0.0); TG, triacylglycerol.
Copyright © 1998 by AOCS Press
peroxisomal β-oxidation sequence, which is not coupled to a phosphorylating system, is not inhibited by cyanide (1). Mitochondrial β-oxidation of fatty acids is thought to be essentially complete, whereas this seems not to be the case in peroxisomes (2). Mitochondria are more abundant than peroxisomes in most animal cells and under normal conditions oxidize more than 90% of long-chain fatty acids (3). The processes of lipid catabolism are less well known in fish (4,5), but teleosts, like higher vertebrates, store a considerable proportion of their triacylglycerol (TG) in a discrete abdominal tissue (6) and in adipocytes which are distributed throughout the muscle myosepta (7–9). In addition, a lipase which degrades long-chain TG occurs in red and white muscles of fish (10). During starvation, ketone bodies, but not nonesterified fatty acids, are an important fuel for muscle in elasmobranchs, whereas nonesterified fatty acids, but not ketone bodies, are an important fuel in teleosts (11,12). Both mitochondria and peroxisomes show a broad chain-length specificity of hepatic β-oxidation of fatty acids in fish (13–15). In an Antarctic fish (Notothenia gibberifrons) and another teleost (Myoxocephalus octodecimspinosus), substrate selectivities were broader for peroxisomal β-oxidation than for mitochondrial β-oxidation and the peroxisomal β-oxidation system could account for up to 30 and 50% of total hepatic β-oxidation, respectively (16,17). In addition to the liver, red muscle (18,19) and heart (20) also possess a high capacity for the oxidation of fatty acids, whereas the kidney and particularly the white muscle (21) have a lower ability to oxidize fatty acids. Atlantic salmon fed L-carnitine revealed a higher capacity to β-oxidize [1-14C]palmitic acid in both liver cubes and isolated hepatocytes (22). The muscle metabolic organization of salmonids undergoes seasonal variation (23), and during cold conditions (24–26) and spawning (27) an enhanced capacity for lipid oxidation occurs. In general, the amount of fat in the fish feed is continuously rising, and today a normal commercial salmonid feed contains around 30–35% fat. However, no reports exist as to fish handling these high-fat diets and the effect it will have on fat deposition and/or fatty acid catabolism in different tissues. Another important aspect is that in most cases the capacity to β-oxidize fatty acids in different tissues is expressed per gram wet weight
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and/or per milligram protein (specific activity). The importance of considering tissue size was emphasized elsewhere (28–31); therefore, we have also presented the data per wet tissue. The aim of the present study was to study key enzymes involved in peroxisomal and mitochondrial β-oxidation in different tissues and to differentiate between carnitine palmitoyltransferase-I (CPT-I) and CPT-II activities in Atlantic salmon. MATERIALS AND METHODS Chemicals. [N-Me-14C]L-carnitine was purchased from New England Nuclear (Boston, MA). 2′,7′-Dichlorofluorescein diacetate was obtained from Eastman Kodak Company (Rochester, NY). Palmitoyl-CoA, horseradish peroxidase, Lcarnitine, and other cofactors were from Sigma Chemical Co. (St. Louis, MO). All other chemicals were of reagent grade and obtained from common commercial sources. Fish. Five sexually immature adult Atlantic salmon (Salmo salar) weighing 4183 ± 687 g (69.6 ± 4.3 cm) were collected from Institute of Marine Research, Matre Aquaculture Research Center, Norway). The fish had been kept in net pens in the sea and fed a standard diet containing 29% lipid (Elite Granulat, T. Skretting A/S, Bergen, Norway). The protocol was approved by the Norwegian State Board of Biological Experiments with Living Animals (Bergen, Norway). Preparation of tissue homogenates. When fully anesthetized (methomidate), the fish were killed by a blow to the head; and the liver and heart were removed, weighed, and homogenized with an Ultra-Turrax T25 to 20% (wt/vol) in icecold sucrose solution containing 0.25 M sucrose in 10 mM HEPES buffer and 1 mM EDTA, pH 7.4. Red muscle was sampled from one side of the fish and dissected free from skin, subdermal lipid, and underlying white muscle. White muscle was sampled on the same side directly below the dorsal fin toward the vertebral column. Red and white muscles were weighed and homogenized as described above. The resulting total homogenates were then centrifuged (1880 × g for 10 min at 2°C). The resulting postnuclear fraction (E-fraction) was collected, and some portions were used immediately to determine CPT-I activity. Other aliquots were stored at −80°C until analyzed. Lipid analysis. Tissue lipids were determined in the Efractions by enzymatic colorimetric methods. TG and cholesterol were determined according to Technicon Method no. SA4-0324L90 (32) and Technicon Method no. SA4-0305L90 (33) (Technicon Instruments, Tarrytown, NY), respectively. Phospholipids were measured by the method of bioMérieux (Marcy-l’Etoile, France) (34). Enzyme activities. Fatty acid oxidation was determined in the E-fractions as acid-soluble products using [1-14C]palmitoyl-L-carnitine as substrate (35). All samples were preincubated for 2 min at the different temperatures used before adding the substrate. After incubation for 10 min, oxidation was stopped by addition of 150 µL 1.5 M KOH; 25 µL fatty acid-free bovine serum albumin (BSA) (100 mg/mL) was then added to the suspension in order to bind nonoxidized Lipids, Vol. 33, no. 9 (1998)
substrate. Next, 500 µL of 4 M HClO4 was added to precipitate nonoxidized substrates bound to BSA. The total solution was then centrifuged at 1880 × g for 10 min at 2°C. Aliquots of 500 µL were assayed for radioactivity. The rate-limiting enzyme in peroxisomal β-oxidation, acyl-CoA oxidase (ACO) (E.C. 1.3.3.6), was determined according to Small et al. (36). Oxidation of 2′,7′-dichlorofluorescein diacetate was monitored by following the increase in A502 for 7 min after the samples had been preincubated for 3 min at the different temperatures used. CPT-I (E.C. 2.3.1.21) and CPT-II (E.C. 0.0.0.0) activities were measured essentially as described by Bremer (37). CPT-I is deeply anchored on the outer mitochondrial membrane and loses activity upon exposure to detergents but is stabilized by BSA (38,39). Briefly, the assay medium for CPT-I contained 70 mM KCl, 5 mM KCN, 100 µM palmitoyl-CoA, 10 µL BSA (10 mg/mL), 600 µg tissue protein, and 20 mM HEPES, pH 7.5. The reaction was started with 200 µM [N-Me-14C]L-carnitine (1000 dpm/nmol). When included, malonyl-CoA (5 µM) was added to the assay mixture 15 min prior to the start of the reaction. CPT-II, which is localized on the inner surface of the inner mitochondrial membrane, is readily released by a variety of detergents (38,39). Assay conditions for determining CPT-II activity were identical except that BSA was omitted, 0.01% Triton X-100 was included, and the amounts of tissue protein were 400 µg (white muscle), 40 µg (red muscle), and 100 µg (heart and liver). The reaction was stopped with 1 mL 1 N HCL, and water-saturated 1-butanol was added to extract the product, i.e., palmitoyl-[14C]carnitine. Aliquots were assayed for radioactivity on an LKB Wallac 1219 Rackbeta liquid scintillation counter. All enzyme assays were run in duplicate and performed under conditions where product formation was linear with respect to both the time of incubation and the amount of protein. Protein was determined using the Bio-Rad protein kit (Bio-Rad, Richmond, CA). RESULTS The fatty acid oxidation capacity and enzyme activities increased in all tissues with increasing assay temperatures but were practically not detectable at 50°C. The reason for this is probably that the organelle membranes had disintegrated, and the enzymes to some extent were denatured. The mass of the different tissues is given in Table 1. Lipids and protein content of the E-fractions are shown in Table 2. TABLE 1 Tissue Mass and Percentage of Body Massa Tissue Heart Liver Red muscleb White muscleb a
Mass (g)
Body mass (%)
5.1 ± 1.2 40.3 ± 8.4 209 ± 34 2510 ± 412
0.1 1.0 5.0 60.0
Tissue values are means ± SD, n = 5. Red and white muscle mass in Atlantic salmon were derived from the assumption that they occupy 5 and 60% of the total body mass, respectively (Ref. 64). b
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TABLE 2 Lipids and Protein Content in E-fractions from Various Organsa Organ Heart Liver Red muscle White muscle a
Protein (mg/g tissue)
Triacylglycerol
Cholesterol (µmol/g tissue)
28.7 ± 3.1 94.4 ± 8.5 20.3 ± 1.5 36.1 ± 4.9
3.6 ± 0.8 5.6 ± 1.4 16.8 ± 5.9 16.0 ± 4.8
2.1 ± 0.3 4.9 ± 0.8 1.4 ± 0.2 1.8 ± 0.2
Phospholipids 3.7 ± 0.5 15.4 ± 2.2 3.7 ± 0.4 2.1 ± 0.5
All values are means ± SD, n = 5.
The amount of TG was high in muscle, whereas the liver contained high levels of cholesterol and especially phospholipids. In addition, the protein content of liver was ca. threefold higher than the other tissues.
Figure 1 shows CPT-I activities in different tissues with varying assay temperatures. In panels A–C the CPT-I activity is expressed as nmol/min/mg protein (specific activity), nmol/min/g wet tissue, and nmol/min/wet tissue, respectively. Increased CPT-I activities with increasing temperature with a maximum at 30°C were observed for all tissues, and CPT-I was malonyl-CoA-sensitive. Above 30°C the enzyme activity declined and at 50°C no enzyme activity could be detected. The order of CPT-I activities in different tissues was red muscle >> liver > white muscle. If the results are calculated as nmol/min/g wet tissue, the CPT-I activity in liver equaled that of red muscle, whereas both were much greater than white muscle (panel B). However, by calculating the enzyme activity as nmol/min/wet tissue, a completely different picture emerged (panel C). The total enzyme activity in white muscle equaled that in red muscle and both were much greater than in liver. Figures 2 to 4 are presented in the same manner as Figure 1. Figure 2 shows that CPT-II activity dominated in red muscle when expressed as specific activity, and the CPT-II activities in liver and heart were much greater than that of white muscle at all temperatures (panel A). As for CPT-I, the CPTII activity in liver equaled that of red muscle when calculated as nmol/min/g wet tissue (panel B). The total CPT-II activity dominated in red muscle, whereas no differences were observed in liver and white muscle (panel C). The order of activity of the key enzyme in peroxisomal β-oxidation, i.e., acyl-CoA oxidase (ACO), was liver >> red and white muscle and heart (Fig. 3). Figure 4 shows the fatty acid oxidation measured as acidsoluble products in E-fractions with palmitoyl-L-carnitine as a substrate. The total oxidation of palmitoyl-L-carnitine was highest in red muscle when calculated as nmol/min/mg protein (panel A) or as nmol/min/wet tissue (panel C). If the data are calculated as nmol/min/g wet tissue, the activities in liver equaled that of red muscle (panel B). DISCUSSION
FIG. 1. Activity of carnitine palmitoyltransferase I (CPT-I) in different tissues of Atlantic salmon with varying assay temperatures. The results are expressed per milligram protein (A), per gram wet tissue (B), and per wet tissue (C). The values represent means ± SD (n = 5).
The heart, liver, and especially the red muscle, but not white muscle, are generally accepted as the most important tissues involved in fatty acid oxidation in fish (4,5,20). In the present study we analyzed the rate of key enzymes involved in mitochondrial and peroxisomal β-oxidation of fatty acids in different tissues from Atlantic salmon. Enzyme activities are normally expressed per gram wet mass or per
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FIG. 2. Activity of carnitine palmitoyltransferase II (CPT-II) in different tissues of Atlantic salmon with varying assay temperatures. The results are expressed per milligram protein (A), per gram wet tissue (B), and per wet tissue (C). The values represent means ± SD (n = 5).
milligram protein (specific activity), but we have included the rates per wet tissue, i.e., per organ of fish based on the information given in Table 1. CPT has a wide distribution in the animal kingdom, and the concentration of the enzyme seems to be correlated to the metabolic activity of the animal (40). CPT-I and its sensitivity toward malonyl-CoA, an intermediate in fatty acid synthesis, are believed to regulate hepatic fatty acid oxidation and ketogenesis (41,42). Rodnick and Sidell (43) reported that CPT-I in red muscle of striped bass (Morone saxatilis) was malonylCoA-sensitive, a fact never before shown in Atlantic salmon. No data differentiating CPT-I and CPT-II activities from any fish have been published. Figure 1 shows that CPT-I was sensitive toward malonyl-CoA in all tissues analyzed, indicating
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FIG. 3. Activity of acyl-CoA oxidase (ACO) in different tissues of Atlantic salmon with two assay temperatures. The results are expressed per milligram protein (A), per gram wet tissue (B), and per wet tissue (C). The values represent means ± SD (n = 5). *Denotes not determined. The reason for this was that the assay mixture at 40°C became cloudy and led to an unaccounted for unspecific activity.
that the fatty acid synthesis and fatty acid oxidation are regulated in the same manner as in mammalian tissues. The liver contains about three times more protein as the other tissues (Table 2). This is reflected when the enzyme activities of hepatic CPT-I and CPT-II are expressed per gram wet tissue as the hepatic activities equaled that of red muscle (Figs. 1 and 2). In contrast, when hepatic CPT-I and CPT-II activities are expressed per milligram protein or per wet tissue, they do not reach the level of red muscle (Fig. 1). Another interesting finding was observed when enzyme activities were expressed per wet tissue as the CPT-I activity
CARNITINE PALMITOYLTRANSFERASE ACTIVITIES IN SALMON
FIG. 4. Oxidation of palmitoyl-L-carnitine in different tissues of Atlantic salmon with varying assay temperatures. The results are expressed per milligram protein (A), per gram wet tissue (B), and per wet tissue (C). The values represent means ± SD (n = 5).
of white muscle was of the same magnitude as for red muscle (Fig. 1), whereas the CPT-II activity was much lower and comparable to the liver (Fig. 2). Importantly, CPT-I and CPTII activities are found not only in mitochondria but also in other organelles, e.g., peroxisomes and microsomes (44,45). However, the role of CPT-II in other organelles remains uncertain (46,47), whereas mitochondrial CPT-II is involved in the process of β-oxidation, i.e., generates acyl-CoA from acylcarnitines in the mitochondrial matrix (Fig. 5). By comparing Figure 2 with the oxidation of palmitoyl-Lcarnitine (Fig. 4), which must be converted to acyl-CoA by CPT-II before it can undergo β-oxidation (Fig. 5), this study indicates that CPT-II activity in Atlantic salmon reflects the
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capacity of mitochondrial fatty acid oxidation and involvement of L-carnitine in mitochondrial long-chain fatty acid oxidation. In addition, adding L-carnitine to isolated mitochondria stimulated the oxidation of fatty acids in rainbow trout (Salmo gairdneri) (20), and supplementation of L-carnitine to Atlantic salmon led to an increased fatty acid oxidation capacity (22). Furthermore, accelerated growth and reduced body fat were reported for hatchery-reared sea bass (Dicentrarchus labrax) (48,49) and African catfish (Clarias gariepinus) (50) fed carnitine. The peroxisomal ACO activity dominated in liver regardless of how the results are expressed. Thus this organ has a high capacity for peroxisomal β-oxidation, whereas red and white muscles seem to possess a limited ACO activity (Fig. 3). The rate of key enzymes involved in the fatty acid oxidation system and the interpretation of their possible role in the metabolic pathways depend on how the data are expressed (51). After comparing the rate of palmitoyl-L-carnitine oxidation in red muscle from Atlantic salmon with hepatic fatty acid oxidation of palmitoyl-L-carnitine from other species, evidently this tissue possesses a high capacity to oxidize fat (Table 3). In fish muscle, lipids exist in two forms: neutral lipids (TG) in lipid droplets distributed in cell cytoplasm as local energy stores and polar lipids (phospholipids) serving as major components of the cell membrane. Atlantic salmon stores most of its reserve of lipids (TG) in the muscle instead of the liver (54,55). Histological studies revealed that approximately 40% of white muscle TG was stored in myosepta, whereas the remaining 60% was localized to the perimysium (56–58), which is the connective tissue surrounding a bundle of muscle fibers. Involvement of white muscle in the higher range of sustained cruising speeds of active species was suggested by several authors (59–61). Moreover, the white muscle of pelagic species is functionally and structurally adapted for sustained aerobic activity with relatively abundant mitochondria being preferentially situated close to the source of gas and metabolite exchange (62,63). This is interesting as more than 60% of the total body mass of Atlantic salmon is composed of white muscle (64); thus, the overall metabolic capacities of this tissue might have been underestimated up to now. Currently, salmon feed usually contains 30–35% fat, but with improving technology the amount of fat in salmon feed will most likely increase to 40–45%. Are salmon capable of metabolizing this added dietary fat? Moreover, how will different fat (oil) sources affect fat deposition vs. catabolism, i.e., storage or energy utilization? Results from a relatively limited number of studies performed on mitochondrial β-oxidation in fish suggest that a substrate preference exists (for review see Ref. 5); thus, the importance of selecting the right fat (oil) source with an optimal fatty acid composition for energy utilization becomes evident. In conclusion, based on CPT-II activity and palmitoyl-Lcarnitine oxidation, Atlantic salmon seem to possess a high capacity to utilize fat as an energy source. Mitochondrial βLipids, Vol. 33, no. 9 (1998)
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FIG. 5. Schematic presentation of the carnitine-dependent transfer of activated fatty acids (acylCoA esters) into mitochondria. CPT-I in the outer mitochondrial membrane converts acyl-CoA to acylcarnitine. This is a necessary step as acyl-CoA esters cannot penetrate the inner mitochondrial membrane. The outer membrane also contains the long-chain acyl-CoA synthetase (not shown). Carnitine/acylcarnitine translocase in the inner mitochondrial membrane exchanges acylcarnitine with carnitine. CPT-II on the inner surface of the inner mitochondrial membrane is responsible for the conversion of acylcarnitine to acyl-CoA. Acyl-CoA enters the β-oxidation sequence and leads to formation of acid-soluble products. *Denotes acid-soluble product. See Figures 1 and 2 for abbreviations.
TABLE 3 The Rate of Palmitoyl-L-Carnitine Oxidation Measured as Acid-Soluble Products in Different Speciesa Species Atlantic salmon Hamster Rabbit Rat a
Tissue Red muscle Liver Liver Liver
Palmitoyl-L-carnitine oxidation (nmol/min/mg protein) 0.45b 0.41 1.47 0.22
Values are expressed as means. Determined at 20°C, whereas the other values were measured at 30°C.
b
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Reference 35 52 53
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oxidation dominates in red muscle, but white muscle makes a significant contribution to the overall fatty acid oxidation capacity in fish. ACKNOWLEDGMENTS The authors are obliged to the technical staff at Matre Aquaculture Research Station and to Kari Helland, Kari-Elin Langeland, Svein Krüger, and Kari Williams for excellent technical assistance. This study was supported by The Research Council of Norway (grant no. 1160085/122).
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[Received March 23, 1998; and in final revised form July 16, 1998; revision accepted July 20, 1998]
