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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
* Unité Mixte de Recherches Systèmes d’Elevage Nutrition Animale et Humaine, Institut National de la Recherche Agronomique, 35590 Saint Gilles, France;
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Unité Mixte de Recherches Génétique Animale, Institut National de la Recherche Agronomique, 35000 Rennes, France;
and
Station de Génétique Quantitative, Institut National de la Recherche Agronomique, 78352 Jouyen-Josas, France; and
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# Département Physiologie Animale et Système d’Elevage, Institut National de la Recherche Agronomique, 35590 Saint-Gilles, France
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: adipocyte • fatty acid binding protein • intramuscular fat • meat quality • myosin • pig
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). This i.m. fat content varies widely between muscles and pig genotypes (Sellier, 1998); particularly, the i.m. fat content is less in conventional European pigs than in Duroc and Meishan pigs. Increasing i.m. fat content in valuable low-fat muscles by selective breeding remains a difficult task because of unfavorable genetic correlations with other production traits (Hovenier et al., 1992). Quantitative trait loci influencing i.m. fat content have been identified in pigs (de Koning et al., 1999). The existence of a major gene for i.m. fat accretion has been postulated in both Meishan (Janss et al., 1997) and Duroc breeds (Sanchez et al., 2002). However, the underlying mechanisms have not been elucidated yet. Metabolic pathways in both myofibers and i.m. adipocytes could theoretically contribute to variation in i.m. fat level (Kelley and Goodpaster, 2001; Gondret et al., 2004). Thus, studies have elucidated several candidate genes, such as the adipocyte fatty-acid binding protein [(FABP)-4] and heart fatty-acid binding protein (FABP-3), involved in intracellular targeting of fatty acids (Gerbens et al., 1998, 1999); the diacylglycerol acyltransferase 1 (DGAT1), which controls triglyceride storage (Nonneman and Rohrer, 2002; Thaller et al., 2003), or the malic enzyme (MEZ; i.e., the main supplier of energy cofactor for lipogenesis; Mourot and Kouba, 1998). In contrast, studies have failed to provide evidence for a strict association between muscle oxidative capacity and i.m. fat content (Leseigneur-Meynier and Gandemer, 1991), despite the fact that i.m. fat content is nearly double as oxidative as glycolytic fibers (Malenfant et al., 2001). To determine the mechanisms underlying variations in i.m. fat content in pigs, we examined adipocyte cellularity, lipogenic capacity, FABP expressions, oxidative metabolism, and myosin heavy-chain (MyHC) polymorphism in pigs exhibiting either a high or a low i.m. fat content in the LM.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The experiment was conducted in accordance with national regulations for human care and use of animals in research. Licenses, procedures, and holding facilities were approved by the French Veterinary Services (certificate of authorization of experiment on living animals no. 35-22 delivered by the French Department of Agriculture to F. Gondret).
The pigs originated from a French selection program devoted to test the existence of a major gene involved in determining i.m. fat content (Sanchez et al., 2002) and were produced at the experimental farm of Le Magneraud (Institut National de la Recherche Agronomique, Surgères, France). At 70 kg of BW, biopsies were performed in LM at the level of the last rib (Talmant et al., 1989). The actual biopsy lasted <1 s, and approximately 1 g of LM was immediately frozen in liquid nitrogen and stored at –75°C until determination of the i.m. lipid content. Twelve F2 Large White x Duroc barrows were then chosen from different litters having either a low (1.15 ± 0.14%, n = 6, LF) or a high (2.82 ± 0.38%, n = 6, HF) i.m. fat content.
The pigs were provided free access to a standard diet (Table 1) and water until slaughter at about 107 kg of BW. Pigs were transported (duration of transport = ~3 h) to the experimental slaughterhouse (Unité Mixte de Recherches Systèmes d’Elevage Nutrition Animale et Humaine, Institut National de la Recherche Agronomique, Saint-Gilles, France) in the afternoon and were slaughtered by electrical stunning and exsanguination after an overnight fast.
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Age, BW, and carcass weight were recorded at slaughter. Blood samples (10 mL) were collected on heparin at exsanguination, and plasma was then obtained by centrifugation at 2,500 x g for 10 min at 4°C. Plasma samples were stored at –20°C. Backfat thickness (mean of measurements taken at the third and/or fourth lumbar vertebra and third and/or fourth last rib levels) was measured using a Fat-O-Meater (SFK, Herlev, Denmark).
Within 30 min after slaughter, a piece of LM (third lumbar vertebra level) was carefully excised from the right side of the carcass, avoiding any contamination with subcutaneous adipose tissue, and was immediately processed for determination of the ex vivo oleate oxidation rate. Other samples, oriented following the myofiber longitudinal axis, were placed on flat sticks, frozen in liquid nitrogen, and stored at –75°C until histological and biochemical analyses. A 1-cm thick slice of LM, the last sampling, was minced, freeze-dried, pulverized, and kept at –20°C under vacuum until lipid content determination. The day after slaughter, the weights of dissectible backfat, loin, and ham of the left side of the carcass were recorded.
Hormone Concentrations
Plasma concentrations of insulin were measured by RIA as previously described (Prunier et al., 1993). Concentrations of IGF-I were determined in plasma using a double RIA after acid-ethanol extraction (Louveau and Bonneau, 1996). All samples were analyzed in duplicate within a single assay. The intraassay CV was 6.3% for insulin and 8.8% for IGF-I.
Muscle Lipid Content
Lipids were extracted from freeze-dried muscles using a 17-fold dilution of tissue in 2:1 chloroform/methanol (vol:vol) according to the method outlined by Folch et al. (1957). Lipid content of fresh tissue (g/100 g) was obtained by taking into account the DM content determined from the weight of minced tissues before and after freeze-drying.
Histochemistry
Intramuscular adipocyte characteristics were investigated in 5 LM serial cross-sections (10 µm thick, 40-µm interval) obtained by using a cryostat (2800 Frigo-cut Reichert-Jung, Francheville, France) and stained with oil red O (Gondret and Lebret, 2002). For each sample, all visible adipocytes were counted on the whole of the 5 sections using a projection microscope (Visopan Reichert, Wien, Austria). The rare visible cells that displayed a diameter <10 µm were not considered. For the particular case of border cells, we only counted adipocytes that exhibited more than one-half portion in a particular section.
The total area of each cross-section was measured using a programmable planimeter (Hitachi, Siko, Japan). The results were expressed as the number of adipocytes/cm2 of section (mean of the 5 determinations for each sample). Individual areas of all of the adipocytes, except border cells (total of approximately 100 to 250 adipocytes per section) were determined in each section. The proportion of Sudan Black B positive fibers, which stains intramyocellular lipids, was determined in 3 randomly selected sections of approximately 300 fibers, using a projection microscope (Visopan Reichert) according to Dubowitz (1985).
Lipogenic Enzyme Activities
The activities of enzymes controlling key steps of lipogenesis (acetyl-CoA carboxylase; ACC) or providing reduced NAD phosphate for fatty acid synthesis [MEZ and glucose-6-phosphate dehydrogenase (G6PDH)] were measured on whole-muscle cytosolic fractions with cofactors and excess of substrates. The activity of ACC was determined by the H14CO3– fixation method (Chang et al., 1967), whereas MEZ and G6PDH activities were determined by spectrophotometry within the linear phase of the reaction (Bazin and Ferré, 2001). Activities were defined as the amount of enzyme that incorporated 1 nmol of H14CO3– (ACC) or reduced 1 nmol of NAD phosphate+ (ME, G6PDH) per min/g of fresh tissue.
Fatty Acid Oxidation Rate
Oxidation rates of oleate were determined from 0.3 g of freshly excised muscle samples using [1-14C] oleate as the substrate according to Herpin et al. (2003). Total oleate oxidation was determined in the absence of mitochondrial inhibitors of the respiratory chain, whereas these inhibitors (75.6 µM antimycin A and 10 µM rote-none; Sigma-Aldrich Co., St. Louis, MO) were required to measure peroxisomal oleate oxidation. The difference between total oxidation and peroxisomal oxidation was considered to be mitochondrial oxidation. All assays were performed in triplicate. Oleate oxidation rates were expressed as nanomoles per minute per gram of fresh muscle.
Oxidative Enzyme Activities
Frozen muscle (about 0.2 g) was homogenized in 50 vol (wt/vol) of ice-chilled 0.1 M phosphate buffer (pH 7.5) containing 2 mM EDTA and sonicated. After centrifugation at 1,700 x g for 15 min at 4°C, the supernatant fraction (soluble enzymes and mitochondrial material) was collected and used for further analyses. The maximal activities of mitochondria oxidative markers, reflecting either fatty acid beta-oxidation (ß-hydroxyacyl-CoA dehydrogenase; HAD) or mitochondrial density (citrate synthase; CS) were determined according to the methods described by Bass et al. (1969) and Srere (1969), respectively. Enzyme activities were assessed at 30°C using an automatic spectrophotometric analyzer (Cobas Mira, Roche, Basel, Switzerland) and expressed as micromoles of degraded substrate per minute per gram of fresh muscle.
Real-Time Reverse Transcription-PCR
Expression of genes involved in fatty acid transport (FABP), lipogenesis (sterol-regulatory element binding protein; SREBP-1), terminal esterification (DGAT1), fatty acid oxidation [carnitine-palmitoyl-transferase 1 (CPT-1); peroxisome proliferator-activated receptor delta (PPAR
)], or energy expenditure (uncoupling proteins; UCP) were investigated by real-time quantitative reverse transcription-PCR (ABI PRISM 7000 SDS thermal cycler; Applied Biosystems, Foster City, CA). Total RNA was extracted from frozen samples according to the method of Chomczynski and Sacchi (1987). Primers were designed using the Primer Express software (Applied Biosystems) based on Sus scrofa sequences (Table 2). Complementary DNA was synthesized from 2 µg of total DNAse-treated RNA in 40 µL of reaction buffer using random primers and murine Moloney leukemia virus reverse transcription, according to the manufacturer’s instructions (Applied Biosystems). Forty cycles of amplification were performed in 25 µL of PCR buffer (SYBRGreen I PCR core reagents, Applied Biosystems) with 5 µL of diluted (4:100) first-strand cDNA reaction and 0.3 µM forward and reverse primers (except 0.5 µM for SREBP-1). Uracil DNA glycosylase (1 U/100 µL; Invitrogen, Cergy Pontoise, France) was used to prevent any contamination from previous PCR. Amplification product specificity was checked by dissociation curve analyses. Assuming that efficiencies of the target genes and 18S are the same, the amount of a specific target, normalized to an endogenous reference and relative to a calibrator (i.e., one sample from the low i.m. fat group) was calculated with the following formula (Pfaffl, 2001):
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where 
CT = (CTgene – CT18S)sample – (CTgene –CT18S)calibrator.
The proportions of each MyHC mRNA for slow-twitch type I and fast-twitch types IIa, IIx, and IIb muscle fibers were determined by reverse transcription-PCR using the TaqMan system (Applied Biosystems) as previously described (Lefaucheur et al., 2004). Briefly, cDNA were synthesized using a MyHC-specific primer common to all MyHC. Then, the real-time PCR was performed on the polymorphic actin-binding site corresponding to loop 2 (Chikuni et al., 2001). Importantly, the forward and reverse primers were identical for all 4 MyHC, thus avoiding any difference in primer annealing efficiencies between MyHC isoforms. The detection of each MyHC was based on 4 specific TaqMan minor groove binder probes labeled with 6-carboxyfluoroscein. For a given sample, the 4 MyHC were measured separately in triplicate within the same plate. Results are expressed as the relative percentage of each MyHC.
Western Blotting
Western blot analyses were performed on the whole-muscle cytosolic fraction (Laemmli, 1970; Towbin et al., 1979). Proteins (15 µg) were diluted in Laemmli loading buffer, separated by electrophoresis on a 15% polyacrylamide/0.1% SDS gel for 45 min at 200 V and then electrotransferred onto a poly(vinylidene difluoride) membrane (Amersham Biosciences, Piscataway, NJ) for 1 h at 100 V. The membrane was blocked with PBS-Tween 20 (5% vol/vol) supplemented with 5% nonfat dry milk and incubated for 1 h with porcine antiFABP-3 (1/20,000) or rat antiFABP-4 (1/10,000) polyclonal antibodies, kindly provided by J. H. Veerkamp (Department of Biochemistry, University of Nijmegen, The Netherlands). Horseradish peroxidase-coupled antirabbit IgG was used as the secondary antibody (1/100,000). Immunodetection was performed with the ECL Plus Western Blot detection kit (Amersham Biosciences), and the membranes were scanned on a Storm phosphorimager (Molecular Dynamics, Sunnyvale, CA). Signals were quantified using ImageQuant software (Molecular Dynamics). Membranes were stained with Ponceau working solution (Sigma-Aldrich) to check for good protein transfer and equivalent loading.
Statistical Analyses
The SAS software (SAS Inst. Inc., Cary, NC) was used for all statistical evaluations. Data were analyzed by one-way ANOVA for the main effect of i.m. fat groups (HF vs. LF). The effect of slaughter day was removed from the final model because it did not significantly influence any of the characteristics studied. Differences between means of the HF and LF groups were considered significant at P < 0.05. A probability value <0.10 was discussed as a trend. Overall Pearson correlation coefficients were calculated between i.m. fat content and FABP-4 or FABP-3 expression levels. We also assessed the correlation between FABP-4 content and adipocyte number. Finally, stepwise regression analysis was performed using each MyHC percentage as the dependent variable and the other muscle characteristics as the independent variables. Only variables reaching the 0.15 significance level were retained for entry into the model.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The HF and LF pigs had similar final BW and age at slaughter (Table 3). Plasma insulin concentrations were similar in both groups, whereas plasma IGF-I concentrations tended (P = 0.06) to be lower in HF pigs than in LF pigs. Carcass composition differed between groups; HF pigs exhibited greater backfat thickness (+16%, P = 0.02), greater backfat proportion (+21%, P = 0.04), and a slightly lower proportion of loin (–4%, P = 0.05) than LF pigs.
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Total lipid content in LM at slaughter was 70% greater (P < 0.001) in HF pigs than in LF pigs (Table 4). This increase was parallel to a greater number of i.m. adipocytes/cm2 (+127%, P = 0.005) and a tendency for enlarged adipocytes (+13%, P = 0.057) in HF pigs compared with LF pigs. Considering adipocyte as a sphere, this led to a 49% difference in adipocyte volume between HF and LF pigs (P = 0.072). Positive correlations were also found between LM fat content at slaughter and adipocyte number (R2 = 0.73, P < 0.001) and adipocyte diameter (R2 = 0.47, P = 0.013). Sudan Black positive fibers accounted for 17% of the total analyzed muscle fibers in both LF and HF groups.
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The activities of lipogenic enzymes (ACC, MEZ, and G6PDH), and mRNA levels of genes coding for key metabolic factors involved in the control of lipogenesis (SREBP1) and esterification (DGAT1) did not differ between the 2 groups (P > 0.35; Table 4
).
Intracellular Fatty Acid Transport
Both FABP-3 mRNA expression (P = 0.46) and protein content (P = 0.56) were similar in HF and LF pigs (Table 4). In contrast, we observed a 2-fold greater (P = 0.05) FABP-4 content in HF pigs than in LF pigs and no difference at the mRNA level (P = 0.49, Table 4). A significant positive correlation was also found between FABP-4 protein content and i.m. adipocyte number/cm2 (R2 = 0.47, P = 0.02, Figure 1A). The correlation coefficient between FABP-4 protein content and i.m. fat percentage at slaughter reached 0.58 (P = 0.004, Figure 1B). A stronger correlation between FABP-4 content and i.m. fat level (R2 = 0.78, n = 18, P < 0.001) has been achieved in another experiment with Large White x Duroc backcross pigs (data not shown). This relationship was not observed at the FABP-4 mRNA level. There was no significant relationship at P = 0.05 between LM i.m. fat content and FABP-3 mRNA or protein levels.
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Activities of HAD and CS and mitochondrial oleate oxidation rates were similar in both groups (Table 5). The level of mRNA from key genes involved in the control of lipid oxidation process (CPT-1, PPAR), mitochondrial uncoupling (UCP2, UCP3) and MyHC polymorphism did not differ between groups. Stepwise regression analysis revealed that only FABP-3 predicted MyHC 1 and 2b proportions at P = 0.15. The FABP-3 protein content was positively (R2 = 0.47, P = 0.01) and negatively (R2 = 0.57, P = 0.004) correlated with MyHC 1 and 2b mRNA proportion, respectively (Figure 2). No relationship was found with the other 2 MyHC.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Gondret et al., 1998). Therefore, many muscle intrinsic pathways in both i.m. adipocytes and myofibers could contribute an explanation of the variability of i.m. fat content, such as the balance between lipid anabolic and catabolic pathways, intracellular trafficking of fatty acids supported by FABP, and myofiber energetic metabolism in relation to muscle contraction. However, the current study did not show any relationship between i.m. fat content and whole-muscle lipogenic capacity, FABP-3 expression, MyHC proportions, or various traits related to fatty acid oxidation. Recently, decreased i.m. fat content has been related to overexpression of UCP3 in transgenic mice (Bezaire et al., 2005). Because UCP are thought to play a role in fatty acid handling to facilitate oxidation in muscle (Samec et al., 1998), the absence of variation of UCP2 and UCP3 expressions between HF and LF pigs could be due to the lack of differences observed on oxidative metabolic variables such as enzymatic activities (CS and HAD), mitochondrial and peroxisomal oxidation rates of oleate, and mRNA levels of CPT-1 and PPAR. The fact that the amount of i.m. fat is not related to fiber type composition per se is in accordance with previous observations (Leseigneur-Meynier and Gandemer, 1991; Larzul et al., 1997). Interestingly, the proportion of slow-twitch type I MyHC mRNA (19%) was greater than previously reported in pure Large White pigs (Lefaucheur et al., 2004), which could be explained by the greater percentage of type I fibers encountered in Duroc than in Large White pigs (Fazarinc et al., 1995). Because a permissive action of FABP-3 in delivering fatty acid to mitochondrial oxidation has been shown in various models (Glatz et al., 2003; Haunerland and Spener, 2004), our results, which show a positive relationship between the proportion of MyHC 1 and FABP-3 protein level, are consistent with the fact that slow-twitch type I fibers are more oxidative and use more lipids as fuel substrate than other fiber types. The lack of any difference among groups in FABP-3 does not support QTL analyses, suggesting FAPB-3 as a candidate gene for i.m. fat deposition in Duroc pigs (Gerbens et al., 1998, 1999). Altogether, the present results strongly suggest that the primary mechanisms involved in the regulation of i.m. fat content are not related to fiber type composition and energy catabolism, but would rather reside within the i.m. adipocytes themselves in relation to their environment. The most striking histological difference between LF and HF groups in the current study is the greater number of i.m. adipocytes in the LM of HF than LF pigs with less difference in adipocyte size. Moreover, stepwise regression analysis asserts that differences in i.m. fat content are mostly explained by variation in adipocyte number because no other variable reached the P = 0.05 significance level for entry into the model. These conclusions are also supported by our observations in rhomboideus red muscle, where adipocyte number was increased by 76% in HF pigs without any significant variation of adipocyte diameter (data not shown). Because lipogenesis in LM, assessed as de novo lipogenic enzymes activity and expression of genes involved in the control of lipogenesis (SREBP-1) and triglycerides storage (DGAT1), remained similar in both groups, it is likely that differences between HF and LF pigs in lipid content mainly involved differences in duration of adipocyte hyperplasia.
The lack of difference in DGAT1 mRNA level between groups was quite surprising, because QTL analyses have previously underlined DGAT1 as a candidate gene for i.m. fat deposition in pigs (Nonneman and Rohrer, 2002) and cattle (Thaller et al., 2003). In addition, Roorda et al. (2005) further indicated that overexpression of DGAT1 protein in muscle using DNA electroporation was able to induce intramyocellular triglyceride storage in rat. Posttranscriptional events could first explain the discrepancies between these studies and our data. However, because DGAT1 is expressed in both myocytes (75% of the muscle volume) and adipocytes, it is also possible that basal mRNA level in the myofibers might have masked an induction in i.m. adipocytes.
Interestingly, positive correlation coefficients were found in the current study between FABP-4 level and both adipocyte number and i.m. fat content, whereas correlations with the other variables acquired in this study were not significant. Moore et al. (1991) first suggested that the correlation observed between FABP activity and marbling score in beef muscle could be due to interfascicular adipocyte FABP. More recently, a positive association between i.m. fat content and FABP-4 gene polymorphism (Gerbens et al., 1998, 1999) or FABP-4 gene expression (Wang et al., 2005) has been shown in both Duroc pigs and different bovine breeds. However, Gerbens et al. (2001) failed to find this relationship between i.m. fat content and FABP-4 expression in crossbred Large White x Dutch Landrace pigs. Therefore, it is possible that only pure Duroc (Gerbens et al., 1999) or crossbred Duroc pigs (current study) have the correct allele in segregation. Fatty acid binding protein-4 is known as a late marker of adipogenesis (Spiegelman et al., 1983), and its ectopic expression could also induce a transdifferentiation of existing myoblasts or satellite cells to an adipogenic cell type (Taylor-Jones et al., 2002). However, whether FABP-4 was involved as a primary cause or a consequence of i.m. adipogenesis remains to be elucidated. Alternatively, an interaction between FABP-4 activity and lipolysis intensity cannot be excluded and has been proposed by others. Indeed, Coe et al. (1999) reported an impairment of lipolysis in adipocytes of FABP-4 null mice. Moreover, Shen et al. (1999) suggested that absence of interaction between FABP-4 and hormone sensitive lipase (HSL) led to feedback inhibition of HSL by fatty acids. Therefore, in the current study, a mutation of the FABP-4 gene in HF pigs might have prevented an interaction between FABP-4 and HSL, impairing lipolysis that could lead to a metabolic balance favoring fat accumulation in HF pigs. However, further investigation combining determination of HSL activity, FABP-4 genotyping, and in vitro protein-protein interaction are required to validate this hypothesis. However, the lack of any relationship between FABP-4 protein and mRNA level suggests a posttranscriptional regulation, which would be consistent with such a protein-protein interaction mechanism.
Finally, in addition to difference in i.m. fat content, HF pigs also exhibited fatter carcasses than LF ones. This is consistent with most studies showing a positive genetic correlation (+0.3 on average; Sellier, 1998) between i.m. fat content and fat proportion in the carcass of pigs. However, the Duroc breed has been reported to exhibit a greater i.m. fat content at the same backfat thickness (Wood et al., 2004). In the present experiment, the lower plasma IGF-I concentration of HF pigs compared with LF pigs could at least partly explain increased carcass fatness in the former. Indeed, circulating IGF-I mainly reflects growth hormone action in ad libitum fed animals, which is known to induce a dramatic decrease in the fat mass by lowering insulin action on glucose transport and lipogenesis (Louveau and Bonneau, 2001). Moreover, in Duroc pigs, a positive correlation has been found between i.m. fat content and serum IGF-I concentrations at 8 wk of age but not at slaughter age (Suzuki et al., 2004). Thus, more work is necessary to explain the greater subcutaneous fat depot in HF pigs than in LF pigs through the somatotropic axis and lipid turnover.
The present findings suggest that both the number of adipocytes interspersed between myofiber fasciculi and the level of FABP-4 may be valuable markers of i.m. fat accretion. The absence of any relationship between i.m. fat level and whole-muscle energetic and contractile properties suggests that i.m. fat content and myofiber type composition can be manipulated independently.
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Footnotes |
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2 Corresponding author: Marie.Damon{at}rennes.inra.fr
Received for publication July 19, 2005. Accepted for publication December 20, 2005.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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= low fat (LF) and 
= high fat (HF); n = 6] and i.m. fat content of the LM at slaughter (panel B; 
0.01.