Contribution of mitochondria and peroxisomes to palmitate oxidation in rat and bovine tissues

Comparative Biochemistry and Physiology Part B 121 (1998) 185 – 194

Contribution of mitochondria and peroxisomes to palmitate oxidation in rat and bovine tissues Ce´cile Piot a, Jacques H. Veerkamp b, Dominique Bauchart a, Jean-Franc¸ois Hocquette a,* a

INRA, Laboratoire Croissance et Me´tabolismes des Herbi6ores, Centre de Recherches de Clermont-Ferrand/Theix, 63122 Saint-Gene`s Champanelle, France b Department of Biochemistry, Uni6ersity of Nijmegen, Nijmegen, The Netherlands Received 6 May 1998; received in revised form 20 July 1998; accepted 13 August 1998

Abstract Total and peroxisomal palmitate oxidation capacities and mitochondrial enzyme activities were compared in tissues from growing rats, preruminant calves and 15-month-old bulls. Total palmitate oxidation rates were 1.9 – 5.2-fold higher in rat than in bovine tissues and 1.7-fold higher in the heart and muscles from calves than from growing bulls. The peroxisomal contribution to palmitate oxidation was similar between rats and bovines (i.e. calves and bulls) in liver (35 – 51%), heart (26%) but not in muscles (1493% in rats vs 3394.5% in bovines, PB 0.05). Mitochondrial enzyme activities were 1.8 – 4.8-fold higher in rat than in bovine tissues but the citrate synthase to cytochrome-c oxidase ratio was the highest in the liver (17 – 38), intermediate in the heart and muscles from calves and rats (6–10) and the lowest in heart and muscles from bulls (2 – 3, P B 0.05). In all tissues and animal groups, palmitate oxidation rates were similar per unit cytochrome-c oxidase activity, but not always per unit citrate synthase activity. Therefore, differences in mitochondrial contents (as between rats and bovines) or in mitochondrial characteristics (as between liver and muscles) relate to the differences in palmitate oxidation capacity. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Fatty acid oxidation; Mitochondrial enzyme activity; Liver; Heart; Skeletal muscles; Rats; Bovines

1. Introduction The resting and maximal rates of oxygen consumption have been measured in vivo in many animal species. These studies showed that the metabolic rate of the whole animal varies with body mass to the power of 0.75 in animals ranging in size from mouse to elephant. Consequently, the metabolic rate expressed per kg body mass decreases with increasing body size since it varies with body mass to the power of – 0.25 [29]. Oxygen is mainly consumed within mitochondria and so differences between species are probably related to the total mass of mitochondria in the metabolically most active organs in which nutrients (especially fatty acids) are * Corresponding author. Tel.: + 33 4 73624253; fax: + 33 4 73624639; e-mail: [email protected]

oxidized. Therefore, the in vitro capacity of palmitate oxidation per g tissue wet weight was 2.4–6.8-fold lower in the human liver, heart and skeletal muscles than in the corresponding tissues of the rat [10,38]. Likewise, in vitro rate of palmitate oxidation in the liver was 3.2-fold lower in the bovine than in the rat [13] and 3-fold lower in the swine than in the rat [1]. Fatty acid catabolism in the liver, the heart, and the muscles occurs mainly in the mitochondrial matrix via the b-oxidation pathway to produce molecules of acetyl-CoA, which may themselves be completely catabolized via the Krebs cycle reactions. However, fatty acids can also be partly broken down in the peroxisomes which differ from mitochondria in their specificity for fatty acids. These organelles both oxidize medium- and long-chain fatty acids but have a limited ability to oxidize fatty acids shorter than octanoate

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[6,18]. In addition, peroxisomal b-oxidation is not complete in contrast to mitochondrial b-oxidation [18,22]. Several physiological or molecular mechanisms are probably involved in the differences between species in their ability to oxidize fatty acids according to animal size [29]. Firstly, these differences may be simply related to huge differences in the relative sizes of the metabolically most active organs (for instance, liver and muscles) which catabolize fatty acids at different rates [29]. Secondly, as a general trend, the volume density of mitochondria in muscles is higher in small than in large animals [21,29] and the efficiency of mitochondrial oxidative phosphorylation differs between small and large species [7,25]. Thirdly, the relative contribution of peroxisomal b-oxidation to total fatty acid oxidation may differ between species: for instance, it seems to be lower in rat than in cow liver [13]. The differences can depend also (1) on the physiological age of animals [11,38]; (2) on nutritional factors, especially the nature of the available energy-yielding substrates, which differs markedly between non-ruminant and ruminant animals [23] and (3) on hormonal factors such as thyroid hormones, which stimulate mitochondriogenesis [5]. Since fatty acid catabolism is incomplete in whole homogenates, the in vitro capacity of tissues to oxidize fatty acids in mitochondria and peroxisomes has to be determined by quantifying the production of both CO2 and acid-soluble intermediates [38]. No data using this approach are available in farm animals except from studies in porcine tissues [1,41,42] and in the liver of cows [13]. Consequently, the objective of this investigation was to study the relative contribution of mitochondria and peroxisomes to fatty acid oxidation capacity in bovine liver, heart and muscles in comparison with that of rat tissues. We measured the oxidation rates of [1-14C]palmitate and the activities of mitochondrial marker enzymes such as citrate synthase and cytochrome-c oxidase in tissue homogenates. However, since it is difficult to compare rats to cattle at similar physiological age (weaning and puberty occur at 3 and 4 – 5 weeks of age, respectively in rats but at 3 – 5 and 8 – 12 months of age, respectively in cattle), a group of growing rats was compared to groups of preruminant calves and growing bulls which had a lower or a higher physiological age, respectively, than the rats.

rotenone, L-carnitine, L-malate, and coenzyme A were purchased from Sigma (St. Louis, MO, USA). Others reagents were from Merck (Darmstadt, Germany).

2.2. Animals and diets The experiments were performed using eight male Wistar rats of approximately 6 weeks of age, five 5-week-old preruminant cross-bred Holstein–Friesian male calves and four 15-month-old Salers (n=2) or Limousin (n= 2) male growing bulls. From weaning at 21 days of age to slaughter, rats were fed a standard laboratory chow containing (on a dry matter basis), 22% protein, 4% fat, 4% cellulose, 52% carbohydrates, 6% minerals and 2% vitamin mixture and were killed by cervical dislocation. Preruminant calves were fed a conventional milk replacer containing 16% dry matter which was composed of 68% spray-dried skim milk powder (i.e. 22.8% weight protein), 23.0% tallow, 6.8% corn starch, and 2.2% vitamin and mineral mixture (Univor 22, Bridel Retiers SA, 35134 Rennes, France). The total lipid and fatty acid contents of the milk powder amounted to 24.1 and 22.0% dry matter, respectively. Calves were fed according to the recommendations of Toullec [35] to allow an average daily weight gain of 1 kg. Calf tissues were taken under general anesthesia before slaughter. Bulls were fed with a mixed diet composed of (on dry matter basis) 80% silage, 14.7% concentrate, and 5.3% hay according to a feeding pattern designed to allow an average daily gain of 1536 g. Animals were slaughtered by stunning (captive-blot pistol) and exsanguination. All animals were not starved before slaughter: rats had free access to food during all nights, preruminant calves were fed milk replacer every 3 h during the night for 3 days before slaughter and bulls were allowed to eat forage regularly all the time. The major fatty acids of the diets were saturated (palmitate) and unsaturated long-chain fatty acids (oleate for calves and linoleate for rats). In bulls, polyunsaturated fatty acids of the diet were mainly converted to saturated fatty acids and various isomers of monoenoic fatty acids during the digestion of food by the microorganisms present in the rumen. At slaughter, the average body weight of rats, calves and bulls were 180–200 g, 68 and 615 kg, respectively.

2. Materials and methods

2.3. Tissue samples 2.1. Chemicals [1-14C]palmitic acid was obtained from Amersham International (Bucks, UK). ATP, NAD + , and cytochrome-c were supplied by Boehringer-Mannheim, (Meylan, France). Antimycin A, acetyl-Coenzyme A, fatty acid-free bovine serum albumin, palmitic acid,

Samples of liver, heart and skeletal muscles of the carcass (rectus abdominis and longissimus thoracis for the bovine groups and quadriceps muscle for the rat group) were taken at the time of slaughter, rapidly excised with scissors and immediately cooled in ice-cold buffer consisting of 0.25 M sucrose, 2 mM Na2-EDTA

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and 10 mM Tris– HCl (pH 7.4). Whole tissue homogenates (5% w/v) were prepared in the same buffer by hand homogenization using a glass – glass homogenizer. Two pestles with different diameters were used (intervening space 0.050 and 0.075 mm). Relative proportions of type I, IIA and IIB fibers are approximately 34/30/40% and 20/22/50% in rectus abdominis and longissimus thoracis muscles of cattle [34] whereas the rat quadriceps is a mixture of different muscle fibers with various metabolic types [24]. Therefore, we compare our results in rat quadriceps to those in bovine rectus abdominis which is oxido-glycolytic.

2.4. Assay for palmitate oxidation Palmitate oxidation rates were measured in tissue homogenates as previously described [38]. Briefly, palmitate oxidation was performed in a total volume of 0.5 ml containing 25 ml (liver) or 100 ml (heart or skeletal muscles) homogenate in 25 mM sucrose, 75 mM Tris–HCl (pH 7.4), 10 mM K2HPO4, 5 mM MgCl2 and 1 mM Na2-EDTA supplemented with 1 mM NAD + , 5 mM ATP, 25 mM cytochrome-c, 0.1 mM coenzyme A, 0.5 mM L-malate and 0.5 mM L-carnitine. Peroxisomal palmitate oxidation was determined in the presence of inhibitors of mitochondrial oxidation, i.e. antimycin A and rotenone (73 and 10 mM final concentrations, respectively). All assays were performed under conditions which were optimal with respect to time, concentration of palmitate and of tissue material as previously described [13,38] and were made in triplicate. Flasks were preincubated for 5 min at 37°C before addition of 100 ml of 600 mM [1-14C]palmitate bound to albumin in a 5:1 molar ratio. Specific activity usually averaged 1.5–1.7 mCi mmol − 1. Incubation was carried out for 30 min at 37°C with agitation and stopped by 0.2 ml of 3 M perchloric acid. The released 14CO2 was trapped in 0.3 ml ethanolamine/ethylene glycol (1:2 v/v) and measured by liquid scintillation counting in 5 ml of Ready Safe (Beckman Instruments, Fullerton, USA). After 90 min at 4°C, the acid incubation mixture was centrifuged for 5 min at 10000 ×g and 150 ml supernatant containing 14C-labelled perchloric acid-soluble products was assayed for radioactivity by liquid scintillation. Palmitate oxidation rates were calculated from the sum of 14CO2 and 14C-labelled perchloric acid-soluble products and were expressed in nmol of palmitate per min per g tissue wet weight.

2.5. Analytical techniques Protein content of homogenates was determined according to Lowry et al. [19] with bovine serum albumin as standard. Citrate synthase activity in sonicated homogenates was determined by measuring the rate of initial reaction at 412 nm by means of the DTNB

187

[5.5%-dithiobis(2-nitrobenzoate)] method as previously described [30]. The reaction mixture contained 0.2 mM DTNB, 50 mM acetyl-CoA, 100 mM Tris–HCl (pH 8.1), 100 mM oxaloacetate and sample in a total volume of 1 ml. The reaction was carried out at 25°C and initiated by the addition of oxaloacetate. Cytochrome-c oxidase activity was assayed in freeze-thawed and sonicated homogenates at 25°C according to Smith and Conrad [32] with 90 mM reduced cytochrome-c as substrate and 50 mM potassium phosphate (pH 7.4). The velocity was calculated from V= k.[S], in which the first order constant k is determined in the assay and [S] is set at 90 mM. For citrate synthase and cytochrome-c oxidase activities, 1 unit of enzyme is defined as the amount which, under assay conditions, catalyzes the liberation of 1 mmol of coenzyme A, or the oxidation of 1 mmol of cytochrome-c, respectively, per min at 25°C. Specific activity is expressed as units per g of tissue wet weight.

2.6. Statistical analyses Analysis of variance of the data was made using the general linear models procedure (GLM) of SAS [28]. For citrate synthase and cytochrome-c oxidase activities and oxidation rates, the effects tested in the model included group (G) of animals (rats, preruminant calves, growing bulls), animal (A) tested within the animal group and tissue (T). The group factor was tested against animals within groups. The residual mean square was used as the error term for other effects. Comparisons among tissues within a group were analysed using the Student’s t-test for paired data. Comparisons among groups per tissue were made using the Student’s t-test for unpaired data.

3. Results

3.1. Palmitate oxidation rate in bo6ine and rat tissues Rates of total and peroxisomal oxidation of palmitate (Fig. 1) differed significantly among the three groups of animals (rats, preruminant calves and growing bulls, PB 0.0001) and among the three studied tissues (liver, heart and skeletal muscle, PB 0.0001). However, differences observed among tissues depended on the species since the statistical analysis of the data revealed an interaction between the animal group and the tested tissues (PB 0.025, Fig. 1). The highest values of total and peroxisomal oxidation rates of palmitate were observed in the liver (from 245 to 499 and from 65 to 250 nmol min − 1 g − 1 tissue wet weight, respectively). Liver and heart from rats showed 2-fold higher total and peroxisomal palmitate oxidation rates than the corresponding tissues from calves and growing bulls

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Fig. 1. Palmitate oxidation rates in homogenates of bovine or rat liver, heart and skeletal muscles (rectus abdominis for bovine, quadriceps for rats). Total and peroxisomal palmitate oxidation rates were measured in whole homogenates. Palmitate:albumine ratio was 5:1 and palmitate concentration was 120 mM. Peroxisomal oxidation rates were determined in the presence of antimycin A, 73 mM and rotenone, 10 mM. Mitochondrial oxidation rates were calculated as the difference between total and peroxisomal oxidation rates. Data are means 9 S.E. for 4–8 animals per group. Oxidation rates are expressed in nmol of palmitate oxidized to 14CO2 and 14C-labelled perchloric acid-soluble products per min per g tissue. Significant effects of group (tested against animals within groups), of tissue and of group ×tissue interaction are observed for total oxidation rate (P B0.0001, PB 0.0001 and PB 0.025, respectively). Significant effects of group (tested against animals within groups), of tissue and of group ×tissue interaction are observed for peroxisomal oxidation rate (all PB 0.0001). A, B, C, D Means of total oxidation with differents superscript letters are significantly different (PB 0.05). a, b, c, d e, f, g Means of peroxisomal oxidation with different superscript letters were significantly different (P B 0.05). * PB0.05, ** PB 0.01, difference in total oxidation rates between rats and cattle (calves and growing bulls).

(P B 0.02, Fig. 1). In the quadriceps muscle from rats, total palmitate oxidation rate was much higher (PB 0.004, Fig. 1) than in the rectus abdominis muscle from both preruminant calves and growing bulls. A significant difference in the peroxisomal oxidation rate was only observed between rat quadriceps and rectus abdominis from growing bulls (× 3.7; PB 0.005, Fig. 1). Total oxidation rates were about 2-fold higher both in the heart and in the rectus abdominis from preruminant calves than from growing bulls (P B 0.01; Fig. 1). Moreover, peroxisomal oxidation rate was 1.4-fold higher in the heart from preruminant calves than from growing bulls (PB0.04). On the contrary, the peroxisomal oxidation rate of palmitate was 1.7-fold higher in the liver of growing bulls than of preruminant calves (PB 0.04) whereas the total oxidation rate was similar between both groups of bovine animals (Fig. 1). The contribution of peroxisomal oxidation to total oxidation rate of palmitate was clearly different among tissues from rats (Table 1). It was the highest in the liver but the lowest in muscles. On the contrary, the contribution of peroxisomal oxidation to total oxidation rate of palmitate did not significantly differ between liver, heart and muscle of bovine animals. Consequently, the contribution of peroxisomal oxidation significantly differed in skeletal muscles between rats (149 3%) and cattle, i.e. calves and bulls together, (33 9 4.5%, PB0.05).

In addition, the contribution of 14CO2 production to total palmitate oxidation rate, which depends on citric acid cycle activity in the system, may differ between rats and cattle. In the heart homogenate of rats, this contribution was indeed significantly higher than those of preruminant calves and growing bulls (27.9 vs 4.0 and 7.7%, respectively, PB 0.001). On the contrary, the contribution of 14CO2 production was similar in homogenates of liver and skeletal muscles from all groups (data not shown).

3.2. Mitochondrial enzyme acti6ities in bo6ine and rat tissues Citrate synthase and cytochrome-c oxidase activities differed markedly among the three groups of animals (PB 0.004) and among the tissues (PB 0.0001; Figs. 2 and 3). However, differences among the tissues depended on the group of animals since a significant interaction between the animal group and tissue was observed (PB 0.002). Important differences were detected between rat and bovine tissues (Figs. 2 and 3). Thus, citrate synthase activity of liver, heart and skeletal muscle from rats was 2–3-fold higher than of the corresponding tissues from both preruminant calves and growing bulls (PB 0.02). Moreover cytochrome-c oxidase activities of the liver and the skeletal muscle from rats were 4.0–4.8-fold higher than of the corre-

7.19c

38.1ab 64.0a 1.89ab

Cytochrome-c oxidase/citrate synthase ratio

Total oxidation rate Per unit citrate synthase Per unit cytochrome-c oxidase 10.2c 1.60ab

6.80c

0.57b

92.3c

14.0c

Muscle

98.7a 3.56ae

25.5a

0.76b

31.4de

28.6bc

Liver

7.05bc 0.85d

9.64c

1.76a

210b

21.8bc

Heart

7.67bc 1.53bd

6.53c

0.18c

34.7de

27.7bc

Muscle

Preruminant calves (n =5)

59.1a 3.58a

17.1b

0.59b

33.9d

49.3ab

Liver

2.67d

0.07c

24.4e

38.6b

Muscle

2.47d 3.92b 1.24bcd 1.54bce

2.07d

0.78b

379a

26.7b

Heart

Growing bulls (n = 4)

11.949 0.461

3.469

0.220

52.13

4.83

SEM

0.32 0.18

0.08

0.0003

0.02

0.08

G

0.28 0.46

0.05

0.06

0.08

0.10

A

0.0001 0.0001

0.0001

0.0001

0.0001

0.0001

T

Statistical effect of: (PB)

0.35 0.17

0.06

0.005

0.003

0.0006

G×T

Data are means values of 4–8 animals per group. Standard error of the means (S.E.M.) is the square root of the residual mean squares/number of observations per treatment and it is calculated for four animals. Citrate synthase and cytochrome-c oxidase activities were determined spectrophotometrically in whole homogenates, expressed in units g−1 and units mg−1 of total protein, respectively. Total and peroxisomal palmitate oxidation rates were measured in whole homogenates as described in Fig. 1. Values are expressed in nmol of palmitate oxidized to 14CO2 and 14 C-labelled perchloric acid-soluble products per min (per unit enzyme activity). Muscles correspond to quadriceps muscle for the rats and to rectus abdominis for the groups of preruminant calves and growing bulls. G, A, T, G×T mean significant effects of animal group (G) tested against animals within group, of animal (A), of tissue (T) and of the group×tissue interaction. a, b, c, d, e Means in the same row with different superscript letters are significantly different (PB0.05).

4.76b 0.86d

2.80a

2.37a

Cytochrome-c oxidase activity Per mg of total protein

573a

26.9b

51.4a

Contribution of peroxisomal oxidation to total oxidation rate (%) 76.0c

Heart

Liver

Tissue

Citrate synthase activity Per g of total protein

Rats (n =5–8)

Group

Table 1 Contribution of peroxisomal oxidation, mitochondrial enzyme activities relative to total protein contents, and total palmitate oxidation rates relative to mitochondrial enzyme activities in tissue whole homogenates from rats, preruminant calves and growing bulls

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Fig. 2. Citrate synthase activity in bovine or rat liver, heart and skeletal muscle. Citrate synthase activity was assayed spectrophotometrically in a 10000× g supernatant of sonicated whole tissue homogenates. Results are expressed in units per g tissue wet weight. Data are means 9S.E. for 4–8 animals per group. Significant effects of group (tested against animals within groups), of animal, of tissue and of the group×tissue interaction are observed for citrate synthase activity (PB 0.004, PB 0.01, P B0.0001 and P B 0.0006, respectively). a, b, c Means with different superscript letters are significantly different (P B0.05). * PB 0.02 and ** P B0.0002.

sponding tissues from both preruminant calves and growing bulls (PB 0.003). However, values of cytochrome-c oxidase activity in the heart only differed significantly between rats and growing bulls (× 4.6, P B 0.01). No significant difference in citrate synthase activity was observed for each tissue between preruminants and growing bulls. On the contrary, cytochrome-c oxidase activities were about 3-fold higher in the heart and in rectus abdominis from preruminants than from growing bulls (PB 0.05) but similar in the liver of these two groups. Similar results were observed when activities of mitochondrial enzymes were expressed per g tissue or mg of total protein in homogenates (Table 1) since protein amounts per g tissue wet weight did not significantly differ between tissues and between groups (128 – 132, 109 – 147, 103 – 127 mg g − 1 tissue weights in rats, preruminant calves and growing bulls, respectively). Finally, the mean ratio of the activities of cytochrome-c oxidase and citrate synthase was 1.5-, 4.6- and 2.4-fold higher in the liver, in the heart and in skeletal muscle from preruminant calves than in the corresponding tissues from growing bulls (P B 0.05, Table 1).

3.3. Relationships between palmitate oxidation rates and enzyme acti6ities When total palmitate oxidation rates were normalized to citrate synthase or to cytochrome-c oxidase activity (Table 1), the observed differences (Fig. 1)

between the three groups of animals (rats, preruminant calves and growing bulls) disappeared. However, palmitate oxidation rate relative to the citrate synthase activity was still higher in the heart of rats and preruminant calves than in the heart of bulls. A similar tendency was observed for skeletal muscle (Table 1). Whatever the group, the oxidation rate per unit of citrate synthase activity was 13-fold higher in the liver than in the heart due to a much higher activity of this enzyme in the heart (PB0.01). Similarly, values were 6.7-fold higher in the liver than in the skeletal muscle (P B 0.03, Table 1). Expressed relative to cytochrome-c oxidase activity, palmitate oxidation rate exhibited smaller differences among tissues (Table 1). Total palmitate oxidation rates (ox, in nmol min − 1 g − 1) were indeed correlated with cytochrome-c oxidase (cox) activity (in units g − 1) in the liver (ox=1.03 cox+201.5; r= 0.77, n= 14, PB0.01), heart (ox=0.38 cox+ 91.0, r=0.77, n= 14, PB 0.01), and skeletal muscle (ox= 1.71 cox -6.1; r= 0.91, n =14, PB 0.01) when data on the three groups of animals were analysed together. In addition, mitochondrial palmitate oxidation rates (mito ox, which was calculated as the difference between rates of total and peroxisomal oxidation) were also correlated with cytochrome-c oxidase (cox) activity for heart (mito ox= 0.29 cox+ 69.0, r= 0.72, n= 14, P B0.01), and skeletal muscle (mito ox= 1.54 cox -9.76; r= 0.92, n= 14, PB 0.01) but not for the liver when data of the three groups of animals were analysed together.

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Fig. 3. Cytochrome-c oxidase activity in bovine or rat liver, heart and skeletal muscle. Cytochrome-c oxidase activity was measured spectrophotometrically in freeze-thawed homogenates at 25°C. Results are expressed in units per g tissue wet weight. Data are means 9S.E. for 4–5 animals per group. Significant effects of group (tested against animals within groups), of animal, of tissue and of the group×tissue interaction are observed for the cytochrome-c oxidase activity (PB 0.004, P B 0.04, P B0.0001 and PB 0.002, respectively). a, b, c Means with differents superscript letters are significantly different (P B 0.05). * PB 0.02 and ** PB 0.0002.

3.4. Oxidati6e metabolism in bo6ine skeletal muscles Small differences in the oxidative metabolism were observed between rectus abdominis and longissimus thoracis (data not shown) considered as oxido-glycolytic and glycolytic muscles, respectively [14]. However, oxidation rates of palmitate were higher in both muscles from preruminant calves than those from growing bulls (30.7 vs 10.6 in rectus abdominis and 24.2 vs 17.3 nmol min − 1 g − 1 tissue in longissimus thoracis, respectively, P B 0.03). On the contrary, peroxisomal oxidation rates in both skeletal muscles did not differ significantly between the two groups of animals (from 3.6 to 8.6 nmol min − 1 g − 1 tissue). No significant differences were observed for both groups in citrate synthase and cytochrome-c oxidase activity and in oxidation rates relative to mitochondrial enzyme activities.

4. Discussion

4.1. Total oxidation rate of palmitate in bo6ine and rat tissues As previously shown for rats [11,12,38], the lowest values of total palmitate oxidation capacity were observed in skeletal muscle whereas the heart and the liver were characterized by a much higher oxidative capacity in both rats and cattle. Our values in the rat quadriceps muscle and liver confirmed previous values [13,37,38]. On the contrary, our values in the rat heart (255 nmol

min − 1 g − 1 tissue) were somewhat lower than those previously published (458–838 nmol min − 1 g − 1 tissue depending on the age of the rats; see Veerkamp and Van Moerkerk [38]. These differences between authors can be explained by tissue sampling. In large mammals (pigs, cattle, humans), and also in rats in our study, only a sample of heart was used, whereas other authors homogenized the entire heart [10,37,38]. Nutrition clearly differed between the three groups of animals we studied (growing rats, 5-week-old calves and 15-month-old growing bulls) since carbohydrates, both long-chain fatty acids and carbohydrates, or both volatile fatty acids are the major digestion end-products for growing rats, preruminant milk-fed calves, and weaned forage-fed cattle respectively [9,14]. Consequently, comparison of the results between calves and cattle has to take into account the effects of both physiological age and nutrition. The higher total oxidation rates of palmitate observed in tissues from rats than those from cattle (from 1.9- to 5.2-fold) were similar or smaller than those previously [10,38] reported between rats and humans (from 2.4- to 6.8-fold) although the difference in body size is much larger between cattle and rats than between humans and rats. The difference in liver oxidative capacity between cattle and rats was greater in the study of Grum et al. [13] than in our study (3.2- vs 2.0-fold) but Grum compared two groups of animals with different physiological ages; i.e. 37-month-old cows and growing rats weighing 250 g.

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The lack of difference in the hepatic palmitate oxidation rate between young calves before weaning and growing bulls were in contrast with the observations in rats, in which the hepatic palmitate oxidation rate decreased after weaning [38]. Similarly, the heart and skeletal muscles showed a lower total palmitate oxidation capacity in growing bulls than in preruminant calves whereas the palmitate oxidation rate was higher in the heart from weaned than from suckling rats [38]. On the contrary, smaller differences were observed in the skeletal muscle of rats [11]. In addition, no differences in the rate of palmitate oxidation were shown in skeletal muscle of human subjects ranging in age from 2 months to 77 years [36]. These differences between rats, bovines and humans may be due to species-specific effects of age or nutrition since both these factors affect palmitate oxidation rates in rat tissues [38,39]. Indeed, both the fatty acid content and composition of the diet regulate the activity and sensitivity of carnitine palmitoyltransferase I to inhibition by malonylCoA in rat tissues [26,27]. The contribution of 14CO2 to total palmitate oxidation rate was higher in homogenates of rat heart (28%) than of bovine heart (4 – 8%) as previously observed between rat and human heart homogenates [10]. This suggests that the capacity of the Krebs cycle relative to that of the b-oxidation pathway may be lower in large mammals such as cattle and humans than in rats in accordance with a lower energetic requirements (per unit time and body mass) of heart from larger animals than from smaller [15,33]. The relative contribution of carbohydrate and fatty acids to muscle oxidation depend on the supply of the different energy-yielding substrates to muscle which differs greatly between single-stomached and ruminant species [23], and also probably between preruminant calves and growing bulls due to huge differences in the end-products of digestion. Thus, the difference observed between rat and bovine muscles are unlikely to be explained by nutritional factors.

However, Grum et al. [13] observed a lower contribution of peroxisomes to total palmitate oxidation (27%) in the liver of fed rats, compared to our results and those of Veerkamp and Van Moerkerk, [38] (52%). This may be related to the different age of the rats used in the study of Grum et al. [13]. Furthermore, Grum et al. [13] did not observe any difference between fed and fasted rats contrary to Veerkamp and Van Moerkerk, [38]. These differences may be linked to the duration of the fast (48 vs 18 h) or, in the case of fed rats, by the amount and the nature of dietary fatty acids [39]. As a consequence of the different results on rat liver, we were unable to confirm the main finding of Grum et al. [13] i.e. to detect any difference in the relative contribution of the peroxisomal oxidation in the liver between cattle and rats. A main finding of our study was a similar peroxisomal contribution in the liver, the heart and skeletal muscles from cattle in contrast to the differences (from 14% in quadriceps muscle to 51% in the liver) between rat tissues (Table 1). This difference between species was comparable to that observed between rats and humans; the relative contribution of peroxisomes being indeed similar among tissues in humans (19–33%) but not in rats [38]. In the heart, the peroxisomal contribution was similar in rats (27%), cattle (22–27%, Table 1) and humans (20% according to Veerkamp and Van Moerkerk [38]) suggesting that the function of the cardiac peroxisomal system is highly conserved among species (rat, human, bovine). By contrast, the activity of the peroxisomal system was more pronounced in skeletal muscles of large species such as cattle (39%, Table 1), or humans (27%) [38] than of small ones such as rats (14%, Table 1) [38]. This may be related to a lower capacity for transport and diffusion of oxygen to mitochondria [40] or to a lower mitochondrial content in tissues of larger species compared with smaller ones [21].

4.2. Contribution of peroxisomes to total palmitate oxidation rate

For all three tissues studied, the higher palmitate oxidation rates in rat than in bovine tissues reflected similar differences in both citrate synthase and cytochrome-c oxidase activities as previously described in the heart for citrate synthase only [4]. Indeed, differences between species or tissues were mostly eliminated when total palmitate oxidation rate was expressed relative to mitochondrial enzyme, especially when relative to cytochrome-c oxidase. In addition, when all data of the three animal groups were analysed together irrespective of the tissue, total palmitate oxidation rates correlated positively with cytochrome-c oxidase activity (r= 0.77; PB 0.01) as previously described for different types of human muscles [36].

The relative contribution of peroxisomes to total palmitate oxidation rate in the bovine liver was higher in adult cows (50%) [13] and in bulls (49%, Table 1) than in preruminant calves (29%, Table 1) indicating an increase of the relative activity of the peroxisomal system during growth. Similar variations with age were observed previously in the rat liver [17,38]. In our study, the peroxisomal contribution was also clearly different between liver, heart and quadriceps muscle of rats, in agreement with previous data in rats [38].

4.3. Differences in mitochondrial enzyme acti6ities between species and between tissues

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Therefore, the differences between species may be simply explained by a higher amount of mitochondria per g of wet tissue in small species such as rats as previously described for muscle [3,21] and for liver [31]. Similarly, higher mitochondrial enzyme activities and higher palmitate oxidation rate in the heart and in skeletal muscle reflect a larger mitochondrial compartment in the cardiac muscle than in the skeletal muscle [11] for both bovines and rats. Despite small differences in the proportion of fiber types, no differences in palmitate oxidation rates or in mitochondrial enzyme activities were detected between bovine rectus abdominis and longissimus thoracis. In a previous study, we showed that mitochondrial isocitrate dehydrogenase activity was similar in these two muscles [14]. On the contrary, the differences between the liver, the heart and skeletal muscles were both quantitative and qualitative (amount and properties of mitochondria respectively) whatever the species: the ratio of cytochrome-c oxidase to citrate synthase activities was much higher in the liver than in the other tissues which suggested that the mitochondrial content and the biochemical properties of mitochondria differ between the liver on the one hand and between the heart and the skeletal muscle on the other. This is in line with the many types of utilisation of fatty acid and other metabolic roles of the liver. The differences between mitochondria of calves and growing bulls were also both quantitative and qualitative: cytochrome-c oxidase activity as well as the cytochrome-c oxidase/citrate synthase ratio were lower in liver, heart and skeletal muscle from growing bulls than from preruminant calves, as was previously shown for the bovine heart between young and old adults [20]. This indicates that the biochemical properties of the mitochondria may change during the life of the animal as previously shown in the rat [2,8,16]. In conclusion, differences in the oxidative capacities among species (rats vs cattle) or between heart and skeletal muscle are mainly due to variations in the amounts of mitochondria as reflected by citrate synthase and cytochrome-c oxidase activities [4,10,11]. Alternatively, the differences between liver and muscles on the one hand and between tissues of the same animal species under different physiological conditions (age, nutrition) on the other may also relate to changes in mitochondrial properties.

Acknowledgements We thank Drs H.T.B Van Moerkerk (Department of Biochemistry, University of Nijmegen) and D. Durand (INRA, Laboratoire Croissance et Me´tabolismes des Herbivores) for helpful discussions. We also thank Nicole Guivier for her skilled technical assistance,

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Robert Jailler and his group for the management of the animals and Roland Jailler and his group for the management of the slaughterhouse.

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