Control of skeletal muscle mitochondria respiration by adenine nucleotides: differential effect of ADP and ATP according to muscle contractile type in pigs.

Skeletal muscle exhibits considerable variation in mitochondrial content among fiber types, but it is less clear whether mitochondria from different fiber types also present specific functional and regulatory properties. The present experiment was undertaken on ten 170-day-old pigs to compare functional properties and control of respiration by adenine nucleotides in mitochondria isolated from predominantly slow-twitch (Rhomboideus (RM)) and fast-twitch (Longissimus (LM)) muscles. Mitochondrial ATP synthesis, respiratory control ratio (RCR) and ADP-stimulated respiration with either complex I or II substrates were significantly higher (25-30%, P<0.05) in RM than in LM mitochondria, whereas no difference was observed for basal respiration. Based on mitochondrial enzyme activities (cytochrome c oxidase [COX], F0F1-ATPase, mitochondrial creatine kinase [mi-CK]), the higher ADP-stimulated respiration rate of RM mitochondria appeared mainly related to a higher maximal oxidative capacity, without any difference in the maximal phosphorylation potential. Mitochondrial K(m) for ADP was similar in RM (4.4+/-0.9 microM) and LM (5.9+/-1.2 microM) muscles (P>0.05) but the inhibitory effect of ATP was more marked in LM (P<0.01). These findings demonstrate that the regulation of mitochondrial respiration by ATP differs according to muscle contractile type and that absolute muscle oxidative capacity not only relies on mitochondrial density but also on mitochondrial functioning per se.

First evidence of uncoupling protein-2 (UCP-2) and -3 (UCP-3) gene expression in piglet skeletal muscle and adipose tissue.

Uncoupling proteins (UCPs) facilitate proton transport inside the mitochondria and decrease the proton gradient, leading to heat production. Until now, the presence of UCP1 or other UCP homologs had not been detected in tissues of pig, a species where evidence for the presence of brown adipose tissue has only been provided in 2-3 month old animals. In the light of the improving knowledge on the UCPs family, we decided to examine both UCP2 and UCP3 mRNA expression in piglet skeletal muscle and adipose tissue. Using RT-PCR we have successfully cloned a partial UCP2 sequence and a complete UCP3 cDNA. UCP3's open reading frame (936bp) shares 90, 89 and 85% similarity with bovine, human and rat UCP3 nucleotide sequences, respectively. In 3-5 day old piglets, these genes are expressed in adipose tissue and in both longissimus thoracis (LT) and rhomboïdeus (RH) muscles, without any effect of muscle metabolic type. This is in good agreement with the measurement of the same membrane potential in mitochondria isolated from both types of muscles. In triiodothyronine-treated piglets, UCP3 mRNA is more expressed in LT than in RH muscle. These genes may be involved in the control of the energy metabolism of the piglet.

Characterisation of oxidative phosphorylation in skeletal muscle mitochondria subpopulations in pig: a study using top-down elasticity analysis.

In skeletal muscle, two mitochondrial populations are present which, on the basis of their localisation, are termed intermyofibrillar and subsarcolemmal mitochondria (IMF and SS, respectively). These two populations have different biochemical characteristics and show different responses to physiological stimuli. In this paper, we characterise the oxidative phosphorylation of SS and IMF using 'top-down' elasticity analysis. We excluded the possibility that their different characteristics can be attributed to a different degree of breakage of the two types of mitochondria due to the different isolation procedures used in their preparation. The higher respiration rate and higher respiratory control ratio shown by IMF compared with those shown by SS are principally due to the higher activities of the reactions involved in substrate oxidation as confirmed by the measurement of cytochrome oxidase activity. There is no difference in the leak of protons across the inner mitochondrial membrane between IMF and SS; a faster rate of ATP synthesis and turnover is driven by the lower membrane potential in SS compared with in IMF.

Perinatal ontogeny of porcine growth hormone receptor gene expression is modulated by thyroid status.

The ontogeny of growth hormone receptors (GHR) represents a critical stage in growth and metabolism. We have investigated the perinatal ontogeny of hepatic and skeletal muscle GHR gene expression in piglets, and its modulation by GH and thyroid hormones. Test piglets were rendered hypothyroid in late gestation by feeding the sow a high-glucosinolate rapeseed meal. Plasma and tissue samples were obtained from test and control piglets at various ages between 80 days of fetal life (80F) and 2 days postnatally. Plasma hormone levels were determined by radioimmunoassay and GHR mRNA by RNase protection assays. In controls, plasma thyroxine (T4) and 3,5,3'-triiodothyronine (T3) levels increased between 80F and birth and the early postnatal period was characterized by a marked surge in plasma T3. Test piglets were hypothyroid at 110F with total T4, total T3 and free T3 levels being reduced by 28, 53 and 33% respectively. By contrast, the postnatal increase in T3 was more marked in test than in control animals. Plasma GH levels decreased over the perinatal period and there was no effect of treatment. Hepatic GHR mRNA was at the lower limit of detection at 80F but by 110F was expressed in both groups of animals. However, fetal hypothyroidism at 110F resulted in a marked 70% decrease in hepatic GHR mRNA (p < 0.01). The higher postnatal rise in T3 in test piglets was accompanied by a recovery of hepatic GHR mRNA levels. By contrast with liver, skeletal muscle (longissimus dorsi) expressed high levels of GHR mRNA at 80F and hypothyroidism induced a 68% increase in GHR mRNA (p < 0.001). The present results suggest that thyroid hormones may modulate the perinatal ontogeny of GHR gene expression, in addition to other hormonal factors, and that this modulation is tissue-specific.

Dietary coconut oil affects more lipoprotein lipase activity than the mitochondria oxidative capacities in muscles of preruminant calves.

The presence of coconut oil in a milk replacer stimulates the growth rate of calves, suggesting a better oxidation of fatty acid in muscles. Because dietary fatty acid composition influences carnitine palmitoyltransferase I (CPT I) activity in rat muscles, this study was designed to examine the effects of a milk replacer containing either tallow (TA) or coconut oil (CO) on fatty acid utilization and oxidation and on the characteristics of intermyofibrillar (IM) and subsarcolemmal (SS) mitochondria in the heart and skeletal muscles of preruminant calves. Feeding CO did not affect palmitate oxidation rate by whole homogenates, but induced higher palmitate oxidation by IM mitochondria (+37%, P < 0.05). CPT I activity did not significantly differ between the two groups of calves. Heart and longissimus thoracis muscle of calves fed CO had higher lipoprotein lipase activity (+27% and 58%, respectively; P < 0.05) but showed no differences in fatty acid binding protein content or activity of oxidative enzymes. Whatever the muscle and the diet, IM mitochondria had higher respiration rates and enzyme activities than those of SS mitochondria (P < 0.05). Furthermore, CPT I activity of the heart was 28-fold less sensitive to malonyl-coenzyme A inhibition in IM mitochondria than in SS mitochondria. In conclusion, dietary CO marginally affected the activity of the two mitochondrial populations and the oxidative activity of muscles in the preruminant calf. In addition, this study showed that differences between IM and SS mitochondria in the heart and muscles were higher in calves than in other species studied so far.

Effect of thyroid status in the perinatal period on oxidative capacities and mitochondrial respiration in porcine liver and skeletal muscle.

Regulatory thermogenesis is reduced in newborn piglets which have been made hypothyroid during late gestation by giving the sow a high glucosinolate rapeseed diet (test animals). Thereafter, the progressive increase in thermogenic capacity parallels the development of a marked postnatal hyperthyroid state. To explain these effects of thyroid hormones at the tissue and mitochondrial levels, we have examined both liver and skeletal muscle to determine possible underlying changes in (i) tissue oxidative capacities (cytochrome oxidase (CO) activity), between 80 d of gestation and 48 h after birth, and (ii) mitochondrial content and respiratory capacities at 24 h of life. In control piglets, CO activity increased sharply during late gestation and the first 2 d of life in liver and rhomboideus (RH) muscle (P < 0.01), whereas only a prenatal increase was observed in longissimus dorsi (LD) muscle. Test fetuses were hypothyroid and had lower CO activities than controls during late gestation in RH muscle (P < 0.06, at 110 d of gestation; P < 0.08, at birth) and in liver (P < 0.001, at birth). The postnatal increase in CO activity in RH muscle and liver was higher (P < 0.05) in test than in control piglets, and as a result the difference between the 2 groups was not significant by 24-48 h of life. There was no effect of treatment on LD muscle. At 24 h, hyperthyroid test piglets had lower amounts of mitochondrial proteins than controls (P < 0.05) in all three tissues, possibly reflecting reduced mitochondrial protein synthesis during fetal life and suggesting that high postnatal T3 levels did not bring about major increases in protein synthesis within 24 h. However, test piglets exhibited higher rates of mitochondrial respiration than controls in liver and RH muscle, as shown by increases in State III and FCCP-stimulated respirations (P < 0.05), and mitochondrial CO and creatine kinase activities (P < 0.05). In RH muscle, both subsarcolemmal and intermyofibrillar mitochondria showed the same trends. No changes were observed in LD muscle. Our results describe for the first time the effect of thyroid hormones on perinatal oxidative capacities and neonatal mitochondrial respiration in liver and skeletal muscle of the pig, through both the short-term regulation of mitochondrial respiration and the long-term control of mitochondrial biogenesis. The differential sensitivity of LD and RH muscles to thyroid hormones is discussed.

Postnatal changes in mitochondrial protein mass and respiration in skeletal muscle from the newborn pig.

Quantitative and functional changes occurring in mitochondria were studied in pig skeletal muscle between birth and 5 days of life. Postnatal changes were followed separately on intermyofibrillar and subsarcolemmal mitochondria isolated from rhomboïdeus (RH) and longissimus dorsi (LD) muscles. The integrity and purity of the isolated mitochondria was checked by electron microscopic observations. The mass of mitochondrial protein was not different between muscles at birth. It increased tremendously during the first 5 days of life, by 49% in LD (P < 0.001) and 93% in RH (P < 0.001) muscle and was 30% higher in RH than in LD muscle at 5 days of life (P < 0.05). Mitochondria isolated from RH muscle exhibited 30% higher oxidative and phosphorylative capacities than those from LD muscle at 5 days of life (P < 0.05). Intermyofibrillar (IM) mitochondria had high respiration rate, enzyme activities and coupling parameters (respiratory control ratio, phosphorus-oxygen ratio) from birth. Subsarcolemmal (SS) mitochondria were less active than IM mitochondria; their respiration rate and enzyme activities were 60% lower (P < 0.01) and increased with age, particularly in LD muscle (P < 0.05). Short-term cold exposure had no effect on mitochondrial mass and activity. These results suggest that muscle mitochondria are functional from birth and are changing primarily quantitatively. SS and IM mitochondria exhibit specific changes that are probably involved in the postnatal acquisition of skeletal muscle oxidative metabolism.

Metabolic changes associated with sustained 48-hr shivering thermogenesis in the newborn pig.

Metabolic changes associated with sustained 48-hr shivering thermogenesis were studied in piglets maintained at 34 (thermoneutrality) or 25 degrees C (cold) between 6 and 54 hr of life. Despite their high shivering activity and elevated heat production, cold-exposed piglets exhibited a slightly lower rectal temperature than thermoneutral animals (-1.1 degrees C; P < 0.01) at the end of the treatment. The enhancement of heat production and shivering activity were associated with a decrease in muscle glycogen (-47%; P < 0.05) and total lipid content (-23%; P < 0.05), a reduction of blood lactate levels (P < 0.05) and an enhancement of muscle cytochrome oxidase activity (+20%; P < 0.05) which suggests that muscle oxidative potential was increased by cold exposure. Potential for capturing lipids (lipoprotein lipase activity) was also higher in the red rhomboideus muscle (+71%; P < 0.01) and lower in adipose tissue (-58%; P < 0.01) of the cold-exposed piglets. Measurements performed at the mitochondrial level show no changes in rhomboideus muscle, but respiratory capacities (state IV and FCCP-stimulated respiration) and intermyofibrillar mitochondria oxidative and phosphorylative (creatine kinase activity) capacities were enhanced in longissimus dorsi muscle (P < 0.05). These changes may contribute to provide muscles with nonlimiting amount of readily oxidable substrates and ATP necessary for shivering thermogenesis. A rise in plasma norepinephrine levels was also observed during the second day of cold exposure (P < 0.05).

Effects of acute asphyxia at birth on subsequent heat production capacity in newborn pigs.

The effect of acute asphyxia at birth on subsequent ability to produce heat was investigated in 30 newborn pigs. A model of experimentally induced asphyxia consisting of the prevention of breathing within the first four minutes of life was used. Blood was sampled from an umbilical artery catheter within the first 75 minutes of life for blood gas, pH, glucose, lactate and catecholamine analysis. After the treatment and 24 hours later, heat production capacity, shivering intensity and rectal temperature were measured 10 degrees C below thermoneutrality. Effects on blood gas parameters were severe but transient whereas alterations in carbohydrate metabolism were maintained during the first 75 minutes (P < 0.05). Acute asphyxia at birth induced only minor alterations of thermoregulatory abilities during the first day of life: rectal temperature was lower one hour after birth (P < 0.05) and the postnatal increase in heat production capacity was less pronounced than in controls. It is suggested that the lower viability usually reported for piglets suffering from asphyxia during delivery is most likely to result from reduced vigour and colostrum intake, as well as altered carbohydrate metabolism early after birth.

Effect of recombinant porcine somatotropin on energy and protein utilization by growing pigs: interaction with capacity for lean tissue growth.

Three litters of four pigs from each of four different groups were used to evaluate the effect of porcine somatotropin (pST) on growth performance, body gain composition, energy and N metabolism, and in vitro cytochrome oxydase (final enzyme of the respiratory chain) activity of tissues. The four groups included boars from a synthetic line (SG1) or the Large White breed (SG2) and barrows from the Large White breed (SG3) or crossbred between Large White and Meishan breeds (SG4). Inherent capacity for daily lean tissue growth (LTG) decreased from SG1 to SG4. Within a litter, one pig was slaughtered and dissected at the beginning of the experiment (55 kg BW) and the three others were fed the same daily supply of protein and amino acids (26 g of lysine/d) but relative daily energy levels were either 113 (without pST: E1/0), 100 (3 mg of pST/d: E2/pST) or 87 (3 mg of pST/d: E3/pST). The 100 energy level corresponded to the ad libitum intake of E2/pST pigs. Two energy and N balances were carried out in respiration chambers during the experimental period. Pigs were slaughtered and dissected at approximately 95 kg BW and composition of gain was estimated using the comparative slaughter technique. In E1/0 pigs, daily BW, lean, and N gain were affected (P less than .01) by group; 566, 471, 374, and 315 g/d of lean tissue gain in SG1, SG2, SG3, and SG4 pigs, respectively. At high ME intake (E2/pST vs E1/0), pST increased daily BW (+14%), lean (+27%), or N (+26%) gain and reduced adipose tissue (-50%) gain, but the pST effect was inversely related to LTG: for N, the improvement was 2.8, 7.1, 7.0, and 11.1 g/d in SG1, SG2, SG3, and SG4 groups, respectively. Energy restriction (E3/pST vs E2/pST) reduced (P less than .001) adipose tissue gain in all groups but did not affect lean tissue or N gains in SG1, SG2, and SG3 pigs. In the pST-treated pigs of the SG4 group, the lean tissue or N gains were reduced (P less than .01) by energy restriction. Energy restriction combined with pST treatment (E3/pST) led to negligible amounts of fat deposited (40 g/d for SG1 + SG2 + SG3 pigs) and a gain:feed ratio higher than 500 g/kg (580 in SG1 pigs). The increased heat production measured in pST-treated pigs was due to its maintenance component: 275 vs 257 kcal of ME.kg BW-.60.d-1 (P less than .01).(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of selection for lean tissue growth on body composition and physiological state of the pig at birth.

The effects of selection for lean tissue growth on the metabolic and physiological state (i.e., level of maturity) of the pig at birth have been examined on newborns from three breeds that markedly differ with respect to birth weight and postnatal muscle growth potential: a primitive Chinese breed (Meishan, MS), a European breed (Large White, LW), and a composite line (CL) highly selected for high rate of gain. Within each breed, 40 pigs from eight litters were used for whole carcass and tissue sampling, blood sampling, and for a fat tolerance test at 2 h of age. The CL pigs were heavier (P < .001) than the LW and MS pigs at birth but exhibited lower percentages of carcass protein, fat, mobilizable fat, and ash than the MS pigs (P < .05). In addition, MS pigs had larger adipose tissue adipocytes than pigs from the two other breeds (P < .001). Despite their 31% higher RNA capacity in longissimus muscle (higher RNA:protein ratio, P < .05), CL pigs exhibited a lower percentage of muscle protein (P < .05) than did MS pigs. Relative liver weight was higher for LW than for CL pigs (P < .05), which had the lowest percentage of liver phospholipids (P < .01). The CL pigs exhibited lower hematocrit (P < .01), glucose (P < .01), albumin (P < .01), cortisol (P < .01), and thyroxine (P < .05) levels than the MS pigs.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of oxygen inhalation at birth on the reduction of early postnatal mortality in pigs.

Asphyxia during delivery is considered a main cause of stillbirth in pigs, but piglets suffering from intermittent asphyxia during delivery are also less viable at birth and less prone to adapt to extrauterine life. In an effort to improve pig viability, one attractive solution would be to increase oxygen supply through oxygen inhalation by the newborn pig. The objective of this study was to test effects of oxygen inhalation immediately after birth on various physiological parameters and piglet survival. The experiment was performed on 252 Piétrain x Large White piglets, half of them reoxygenated immediately after birth. They were maintained during 20 min in a chamber where oxygen concentration was monitored at 40% and were then put back with the sow and the control pigs. Oxygen inhalation affected piglet metabolism. Through stimulation of oxidative metabolism (reduction of circulating levels of lactate) and lowering of the level of postnatal hypothermia (particularly for the lightest pigs), oxygen inhalation increased piglet viability and reduced mortality during the 1st d of life by 75% (2 vs 8%). No additional effects were observed during the following days and overall mortality between birth and weaning at 21 d was reduced from 12 to 8%.

Effect of fat content of colostrum on voluntary colostrum intake and fat utilization in newborn pigs.

The effects of colostral fat level on voluntary colostrum and ME intake were determined in 25 newborn pigs during the first postnatal day. Within a litter, five pigs were obtained before nursing and allotted on the basis of initial body weight (BW) at 2 h of age to one of the five treatments: killed or ad libitum bottle-fed sow colostrum containing 2.5, 5.0, 7.5, or 10.0% of total fat. A total of 24 feedings was provided at 60-min intervals, and pigs were killed 1 h after the final feeding. Total colostrum intake averaged 584.8 +/- 42 g (i.e., 436 g/kg of average BW) with the first two feedings accounting for 19.8% of the total consumption. Colostrum intake decreased linearly (P < .08) by 5.9 g/kg of average BW per 1% increase in the level of fat. However, GE and ME intake increased linearly (P < .01) by 7.65 and 4.09 kcal/kg average BW per 1.0 g/kg of average BW increase in fat intake, respectively. Adipose tissue lipoprotein lipase increased (P < .01) during the first postnatal day. Carcass fat deposition and fat oxidation increased linearly (P < .01) by .36 and .20 g/kg of average BW per 1.0 g/kg of average BW increase in fat intake, respectively. We suggest that increasing the fat content in colostrum has little effect on voluntary colostrum intake, and the practice may be an efficient method for improving the energy supply to newborn pigs.

Utilization of colostral energy by the newborn pig.

Twenty-five newborn pigs were used to evaluate the energy utilization of sow colostrum by pigs maintained for 24 h in respiratory chambers at an environmental temperature of 33 degrees C. Within a litter, five neonatal pigs were obtained before nursing and allotted on the basis of initial body weight (IBW) at 3 h of age to one of the five treatments: killed, fed intragastrically 6, 12, or 18 g of sow colostrum/kg IBW per meal, or fasted. A total of 24 meals at 60-min intervals was provided and pigs were killed 1 h after the last meal. Heat production (HP) was measured by indirect calorimetry and energy retention (ER) was calculated by metabolizable energy (ME)-HP. A balance technique was used to determine nitrogen (N) retention, and comparative slaughter technique (CST) was used to determine fat accretion and glycogen mobilization. The ME intake increased quadratically (P < .01) with the level of colostrum intake. The efficiency of ME for ER was 91 +/- 4%. Nitrogen absorbed was utilized with an efficiency of 89 +/- 3% for N retention, and the estimated energy cost of 1 kcal of protein synthesized was 1.11 +/- .11 kcal. Thermoneutral maintenance ME requirement was low (68.5 kcal.kg avg BW-1 x 24 h-1) because of the low activity of pigs. Fasting heat production (FHP) measured by calorimetry and CST amounted to 56.4 and 60.4 kcal.kg avg BW-1 x 24 h-1, respectively. Estimates of the contribution of glycogen and protein catabolism to FHP were 83.0 and 6.8%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the level of asphyxia during delivery on viability at birth and early postnatal vitality of newborn pigs.

Newborn pigs (n = 117) were used to provide information on the relationships of degree of asphyxia during delivery, viability at birth, and some striking aspects of postnatal vitality including survival, interval between birth and first udder contact and between birth and first suckling, rectal temperature at 24 h of life (RT24), and growth rate over the first 10 d of life. The degree of asphyxia at birth was estimated from cord blood pCO2, pH, and lactate levels. Onset of respiration, heart rate, skin color, and attempts to stand during the first minute after birth were used to estimate the viability score. Neonatal asphyxia, i.e., decreased blood pH and increased blood pCO2 and lactate, was associated with the production of unusually high levels of catecholamines. The degree of asphyxia increased with late position in the birth order (P < .01) and was higher in piglets born posteriorly (P < 0.5). Further, the average blood pCO2 within a litter increased (P < .05) with litter size. The was an inverse relationship between the degree of asphyxia and the viability score (P < .001). Highly viable piglets reached the udder more rapidly (P < .001) and had a higher RT24 (P < .001) than those of low viability. Plasma glucose concentrations increased with blood pCO2 and plasma epinephrine concentrations (P < .001). Neonatal asphyxia reduced postnatal vitality by delaying the first contact with the udder (P < .03) and was associated with a lower RT24 (P < .05), growth rate (P < .001), and survival over 10 d (P < 0.06). These variables, i.e., interval between birth and first udder contact, RT24, and growth rate, were correlated with birth weight (P < .001); RT24 was also shown to decrease (P < .001) with the time taken to reach the udder. Overall, results suggest that piglet suffering from asphyxia during delivery are less viable at birth and less prone to adapt to extrauterine life.

Estimation of genetic trends from 1977 to 1998 of body composition and physiological state of Large White pigs at birth.

Genetic trends for body composition and blood plasma parameters of newborn piglets were estimated through the comparison of two groups of pigs (G77 and G98, respectively) produced by inseminating Large White (LW) sows with semen from LW boars born either in 1977 or in 1998. Random samples of 18 G77 and 19 G98 newborn piglets were used for whole carcass and tissue sampling. Plasma concentrations of glucose, albumin and IGF-1 were determined on 75 G77 and 90 G98 piglets from 18 litters. The G98 piglets had less carcass dry matter, protein and energy (P < 0.01) than their G77 counterparts. When expressed in g/kg birth weight, livers were lighter (P < 0.001) and contained less glycogen (P < 0.01) in G98 piglets, with no difference in the activity of the hepatic glucose-6-phosphatase between G98 and G77 piglets. Concentrations of protein, DNA, RNA in longissimus dorsi muscle were unaffected by selection. Plasma concentrations of glucose (P < 0.05) and IGF-1 (P < 0.01) were lower in G98 than in G77 piglets. On the whole, the results suggest that the improvement in lean growth rate and in sow prolificacy from 1977 to 1998 has resulted in a lower maturity of piglets at birth.

Number of intramuscular adipocytes and fatty acid binding protein-4 content are significant indicators of intramuscular fat level in crossbred Large White x Duroc pigs.

Intramuscular fat content is generally associated with improved sensory quality and better acceptability of fresh pork. However, conclusive evidence is still lacking for the biological mechanisms underlying i.m. fat content variability in pigs. The current study aimed to determine whether variations in i.m. fat content of longissimus muscle are related to i.m. adipocyte cellularity, lipid metabolism, or contractile properties of the whole muscle. To this end, crossbred (Large White x Duroc) pigs exhibiting either a high (2.82 +/- 0.38%, HF) or a low (1.15 +/- 0.14%, LF) lipid content in LM biopsies at 70 kg of BW were further studied at 107 +/- 7 kg of BW. Animals grew at the same rate, but HF pigs at slaughter presented fatter carcasses than LF pigs (P = 0.04). The differences in i.m. fat content between the 2 groups were mostly explained by variation in i.m. adipocyte number (+127% in HF compared with LF groups, P = 0.005). Less difference (+13% in HF compared with LF groups, P = 0.057) was noted in adipocyte diameter, and no significant variation was detected in whole-muscle lipogenic enzyme activities (acetyl-CoA carboxylase, P = 0.9; malic enzyme, P = 0.35; glucose-6-phosphate dehydrogenase, P = 0.75), mRNA levels of sterol-regulatory element binding protein-1 (P = 0.6), or diacylglycerol acyltransferase 1 (P = 0.6). Adipocyte fatty acid binding protein (FABP)-4 protein content in whole LM was 2-fold greater in HF pigs than in LF pigs (P = 0.05), and positive correlation coefficients were found between the FABP-4 protein level and adipocyte number (R2 = 0.47, P = 0.02) and lipid content (R2 = 0.58, P = 0.004). Conversely, there was no difference between groups relative to FABP-3 mRNA (P = 0.46) or protein (P = 0.56) levels, oxidative enzymatic activities (citrate synthase, P = 0.9; beta-hydroxyacyl-CoA dehydrogenase, P = 0.7), mitochondrial (P = 0.5) and peroxisomal (P = 0.12) oxidation rates of oleate, mRNA levels of genes involved in fatty acid oxidation (carnitine-palmitoyl-transferase 1, P = 0.98; peroxisome proliferator-activated receptor delta, P = 0.73) or energy expenditure (uncoupling protein 2, P = 0.92; uncoupling protein 3, P = 0.84), or myosin heavy-chain mRNA proportions (P > 0.49). The current study suggests that FABP-4 protein content may be a valuable marker of lipid accretion in LM and that i.m. fat content and myofiber type composition can be manipulated independently.

Ca2+-activated myosin-ATPases, creatine and adenylate kinases regulate mitochondrial function according to myofibre type in rabbit.

Mitochondrial respiration rates and their regulation by ADP, AMP and creatine, were studied at different free Ca(2+) concentrations (0.1 versus 0.4 microm) on permeabilized fibre bundles of rabbit skeletal muscles differing in their myosin heavy chain profiles. Four fibre bundle types were obtained: pure types I and IIx, and mixed types IIax (approximately 50% IIa and 50% IIx fibres) and IIb+ (60% IIb fibres, plus IIx and IIa). At rest, pure type I fibres displayed a much higher apparent K(m) for ADP (212 microm) than IIx fibres (8 microm). Within the IIax and IIb+ mixed fibre bundle types, two K(ADP)(m) values were observed (70 microm and 5 microm). Comparison between pure IIx and mixed types indicates that the intermediate K(m) of 70 microm most probably corresponds to the mitochondrial affinity for ADP in IIa fibres, the lowest K(m) for ADP (5 microm) corresponding to IIx and IIb types. Activation of mitochondrial creatine and adenylate kinase reactions stimulated mitochondrial respiration only in type I and IIax fibre bundles, indicating an efficient coupling between both kinases and ADP rephosphorylation in type I and, likely, IIa fibres, since no effect was observed in pure IIx fibres. Following Ca(2+)-induced activation of myosin-ATPase, an increase in mitochondrial sensitivity to ADP of 45% and 250% was observed in type IIax and I bundles, respectively, an effect mostly prevented by addition of vanadate, an inhibitor of myosin-ATPase. Ca(2+)-induced activation of myosin-ATPase also prevented the stimulation of respiration rates by creatine and AMP in I and IIax bundles. In addition to differential regulation of mitochondrial respiration and energy transfer systems at rest in I and IIa versus IIx and IIb muscle fibres, our results indicate a regulation of phosphotransfer systems by Ca(2+) via the stimulation of myosin-ATPases in type I and IIa fibres of rabbit muscles.

Loose-coupled subsarcolemmal mitochondria from muscle Rhomboideus in cold-acclimated piglets.

1. Intermyofibrillar (IM) and subsarcolemmal (SM) mitochondria were isolated from rhomboideus (RH) and longissimus dorsi (LD) muscles of cold-acclimated (12 degrees C for 3 weeks) and control (23 degrees C) 8-week-old piglets. 2. Together with measurements of yield of mitochondrial protein and enzyme activities (cytochrome oxydase-CO; creatine kinase--CK), the respiratory rate of isolated mitochondria was followed polarographically in order to determine the respiratory control ratio (RCR) and consequently the tightness of coupling in response to ADP. 3. In control and cold-acclimated piglets, there were more IM than SM (P less than 0.05) and more mitochondria in RH than LD muscle (P less than 0.05). In both muscles, the yield of mitochondria was slightly but not significantly higher after cold acclimation than in controls. 4. In both muscles, IM were tightly coupled and their RCR (congruent to 4.5) were similar in both groups of piglets. RCR values were increased in the presence of bovine serum albumin (BSA). 5. In controls, SM exhibited lower respiration rates than IM (P less than 0.05) and were slightly coupled (RCR congruent to 2). Cold acclimation increases the loose-coupling of SM (P less than 0.05), especially in RH muscle. No changes appeared in the mitochondrial coupling after the addition of BSA. 6. After cold acclimation, CO and CK activities were increased in IM (P less than 0.05) while only CO activity was increased in SM (P less than 0.05). These results support a coupling defect in SM and therefore confirm mitochondrial respiration results.

Carnitine palmitoyltransferase I (CPT I) activity and its regulation by malonyl-CoA are modulated by age and cold exposure in skeletal muscle mitochondria from newborn pigs.

Whole-body lipid utilization is progressively enhanced during the first postnatal day in pigs, especially during cold exposure and muscular shivering thermogenesis. This study was designed to examine early postnatal changes in fatty acid oxidation potential, carnitine palmitoyltransferase I activity and regulation by malonyl-CoA in skeletal muscle mitochondria isolated from newborn and 5-d-old piglets. At 5 d of life, pigs were maintained for a 4-h period in thermoneutral (30 degreesC) or cold (20 degreesC) conditions. Intermyofibrillar and subsarcolemmal mitochondria were isolated from longissimus dorsi and rhomboïdeus muscles. In subsarcolemmal mitochondria, carnitine palmitoyltransferase I activity increased with age (P < 0.01) and was 80% lower (P < 0.001) than in intermyofibrillar mitochondria. Intermyofibrillar mitochondria had high enzyme activities and fatty acid oxidation potential from birth. The fatty acids 16:0, 18:1(n-9) and 18:2(n-6) were oxidized at a higher rate than 18:0 (-37%) and 8:0 (-55%). Sensitivity of carnitine palmitoyltransferase I to malonyl-CoA inhibition and malonyl-CoA levels decreased by 47% (P < 0.05) and 33% (P < 0.01) with age, respectively. After 4 h of cold exposure, sensitivity of carnitine palmitoyltransferase I to malonyl-CoA was unaffected in the rhomboideus and tended to be greater (P < 0.06) in longissimus dorsi muscle. Malonyl-CoA levels were lower (P < 0.05) in the rhomboideus and were unaffected in longissimus dorsi muscle. These results demonstrate that fatty acid oxidation is effective from birth in isolated intermyofibrillar mitochondria. The postnatal enhancement of fatty acid utilization observed in vivo can be explained, at least in part, by a rise in carnitine palmitoyltransferase I activity in subsarcolemmal mitochondria and a modulation of its activity by malonyl-CoA in intermyofibrillar mitochondria.