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Institut National de la Recherche Agronomique, Unité Mixte de Recherches sur le Veau et le Porc, 35590 Saint-Gilles, France
3 Correspondence:
Domaine de la Prise (phone: 33-2-23-48-57-52; fax: 33-2-23-48-50-80; E-mail:
gondret{at}st-gilles.rennes.inra.fr).
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Abstract |
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Key Words: Adipocytes • Fats • Lipogenesis • Pigs • Skeletal Muscle • Transcription Factors
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Intramuscular adipose tissue, the last fat depot to develop, may respond to dietary conditions in a different manner to other fat sites. Furthermore, muscle ability for lipid utilization must be considered. This study aimed to investigate the cellularity of adipocytes, lipogenesis and energy metabolism, and expression level of SREBP-1 in pig muscle in relation to diet-induced variations in i.m. fat content.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). Pigs in the second regimen (FR) received the same diet at 75% of the spontaneous daily feed consumption of their C littermates, calculated on a BW basis, in order to increase their age at slaughter by about 30 d (Quiniou et al., 1995). In the third regimen (PR), pigs were maintained on low protein and energy intakes, through the distribution of a blend of the standard diet and of a low-protein diet (Table 1), in order to obtain similar daily weight gains in FR and PR pigs, and similar carcass composition in PR and C pigs (Lebret et al., 2001). The energy and protein allowances of PR pigs were estimated from the energy intake of C pigs and daily growth rate of FR pigs, respectively, assuming that the energy requirements for maintenance were 1 MJ ME per kg BW0.60 (Noblet et al., 1999) and that the lysine requirement was 21 g/kg of BW gain (Noblet and Quiniou, 1999). Since calculated requirements increased slower for lysine than for energy, the proportion of the standard diet to the low-protein diet was therefore decreased progressively in the PR regimen, from the beginning up to the end of the experimental phase. Feeding regimen for FR and PR pigs were calculated and modified weekly within blocks of three littermates. Animals were reared and slaughtered in accordance with French standard guidelines for humane care and use of animals in research.
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10 g) were taken in the middle of each piece of muscle to further ensure that external adipose tissues were not included. They were immediately frozen in liquid nitrogen. For histological analysis, muscle samples were collected within 45 min after slaughter and prepared following fiber orientation. They were then restrained on flat sticks and frozen in isopentane cooled by liquid nitrogen. All samples were stored at -80°C until analysis.
Muscle Fat Content
Total lipid content was determined after extraction with chloroform/methanol (Folch et al., 1957) and expressed as grams per 100 g of fresh tissue.
Cellularity of Muscle Adipocytes
Cellularity of i.m. adipose tissue was investigated in 10-µm cross sections and cut with a cryostat (2800 Frigocut Reichert-Jung, Francheville, France), as previously described (Gondret et al., 1998). Briefly, five serial sections (10 µm) were taken at 40-µm intervals from each muscle sample and fixed for 10 min in phosphate buffer (100 mM Na2HPO4, 100 mM NaH2PO4, pH 7.4) containing 2.5% glutaraldehyde. Sections were rinsed three times in phosphate buffer, stained for 4 min in isopropanol containing 0.5% Oil red O, quickly rinsed in distilled water, and stained for 20 s in an aqueous solution of Kristallviolet. In muscle, adipocytes are either clustered along myofiber fasciculi or alone among muscle fibers (Gondret et al., 1998). For each sample, clustered and isolated adipocytes were numbered on the whole of the five sections using a projection microscope (Visopan Reichert, Vienna, Austria). The rare visible cells that displayed a diameter smaller than 10 µm were not considered. For the particular case of border cells, we only counted adipocytes that exhibited more than half of a portion in a particular section. The total area of each cut-section was measured using a programmable planimeter (Hitachi Siko, Tokyo, Japan). Results were then expressed as the number of adipocytes per centimeter squared of section (mean of the five determinations for each sample). Individual areas of all the adipocytes (excepted border cells) identified on the five sections were carefully reproduced on transparency sheets using the projection microscope and digitized by a color camera. A macroprogram was developed to measure individual areas using an image analysis system (Optimas 6.5, Media Cybernetics, Silver-Spring, MD). Results corresponding to the mean of the determinations undertaken on the five sections of each sample were expressed as diameter (µm) of clustered or isolated adipocytes.
Biochemical Analyses
The activities of enzymes controlling key steps of lipogenesis (acetyl-CoA carboxylase [ACC]) or providing a reduced equivalent for fatty acid synthesis (glucose-6-phosphate dehydrogenase [G6PDH], and malic enzyme) were measured on muscle homogenates. Details of the analytical procedure were described earlier (Mourot and Kouba, 1998). Briefly, a weighted quantity of muscle (1.5 g) was homogenized in 2.5 mL of 0.25 M saccharose ice-cold buffer. The mixture was centrifuged at 30,000 x g for 40 min at 4°C. The supernatant fraction was collected and used for the enzyme assays. The activity of ACC was determined by the H14CO3- fixation method. In the assay procedure, ACC is maximally activated by citrate (provided in excess) with ATP and glutathione as cofactors; therefore, the activity does not reflect the actual rate in vivo but represents the maximal potential of the enzyme under optimal conditions. Malic enzyme and G6PDH activities were assayed spectrophotometrically at 340 nm absorbance. All activities of the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-producing enzymes were linear for at least 4 min after the substrates had been added. One unit of activity was defined as the amount of enzyme that incorporated 1 nmole of bicarbonate (ACC) or reduced 1 nmol of NADP+ (malic enzyme; G6PDH) per minute. Enzyme activities were expressed as unit per gram of fresh tissue. Considering the great differences in total muscle lipid content between the three feeding groups, the enzyme activities were also expressed as units per milligram of muscle lipids, by dividing the above activity by the quantity of total lipids per gram of muscle sample.
Muscle glycolytic pathway was assayed by determination of lactate dehydrogenase (LDH) activity (Bergmeyer and Bernt, 1974). The activity levels of mitochondria oxidative markers, reflecting fatty acid ß-oxidation (ß-hydroxyacyl-CoA dehydrogenase [HAD]) or mitochondrial density (citrate synthase [CS]) were also investigated (Bass et al., 1969; Srere, 1969). Briefly, about 100 mg of muscle was homogenized and sonicated in 50 volumes (wt/vol) of ice-chilled 0.1 M phosphate buffer (pH 7.5) containing 2 mM EDTA. 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. Enzyme activities were assessed at 30°C by determination of optical density every 25 s for 5 min using an automatic spectrophotometric analyzer (Cobas Mira, Roche, Meylan, France). Activities of LDH and HAD were estimated at 340 nm, after addition of pyruvic acid and NADH or of aceto-acetyl-coA and NADH. One unit of activity was defined as the amount of enzyme that released 1 µm of NADPH per minute. Determination of CS activity was performed at 405 nm and was based on the chemical coupling of coenzyme-A (CoASH), released from acetyl-coenzyme A during the synthesis of citrate, to Ellman’s reagent (dithiobis-nitrobenzoic acid). One unit of CS activity was defined as the amount of enzyme that released 1 µmol of mercaptide ion per minute.
Total Ribonucleic Acid Isolation and Northern-Blot Hybridization
Total RNA was extracted from 500-mg tissue samples frozen in liquid nitrogen using the method of Chomczynski and Sacchi (1987). The RNA concentration was determined spectrophotometrically at 260 nm, and RNA quality was confirmed by observation of ribosomal RNA integrity following electrophoresis and ethidium bromide staining. Twenty micrograms of total RNA were then denatured in formamide and formaldehyde and subsequently separated in 1% formaldehyde-agarose gel. The RNA was then transferred overnight into a Hybond N+ membrane (Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were prehybridized at 42°C for at least 5 h and then hybridized according to Gondret et al. (2001) with labeled probes obtained by random priming in the presence of [
-32P]deoxycytidine triphosphate (RTS labeling, Life Technologies, Rockville, MD). A partial cDNA fragment encoding porcine SREBP-1 was used for probe generation (Gondret et al., 2001). A porcine 18S-probe was used to normalize for loading of RNA samples. Autoradiograms for SREBP-1 were revealed after 6 d, scanned, and quantified with an image processor program (Quantity One, V4, Bio-Rad, Fullerton, CA).
Statistical Analysis
The SAS software (SAS Inst., Inc., Cary, NC) was used in all statistical evaluations. Data were submitted to a two-way ANOVA, with feeding, sex, and their interactions as main effects and litter as covariate. Mean values for the effect of feeding treatments were compared using the pdiff statement of the GLM procedure. Correlation coefficients between total lipid content and histological adipocyte measurements were determined by general Pearson’s correlation. Partial correlation coefficients taking into account main effect of feeding and sex were also calculated using the residues of the ANOVA model.
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Results |
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). The ADG was significantly lower in FR and PR than in C animals, giving rise to a 30 d increase in the age at slaughter for the former groups compared to C pigs. Restricting total feed allowance (FR) led to leaner carcasses than those obtained in C pigs, as lean meat content slightly increased and backfat proportion sharply decreased (Table 2). In contrast, PR and C pigs exhibited similar carcass composition. This was achieved as protein and energy intakes decreased by 0.34 and 0.22, respectively, in the former group with respect to the latter group. Moreover, the lysine/DE ratio (g/Mcal DE) of PR regimen was progressively reduced from 2.50 to 2.10 during the experimental phase, since calculated requirements for lysine increased slower than those for energy during this period. On the opposite, this ratio remained constant in the FR group and similar to that of the C pigs (2.92 g/Mcal DE), since the FR pigs received the same diet than the C pigs but at a restricted level.
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). Feeding-induced variation in adipocyte diameters closely followed that of i.m. fat content. For both categories of adipocytes, FR pigs displayed the smallest adipose cells, whereas PR animals had the largest cells (P < 0.001, Figure 1). Correlation coefficients between i.m. fat content and adipocyte diameters were highly significant (r = 0.81 and r = 0.78, for clustered and isolated adipocytes, respectively, Figure 2). The partial correlation coefficient (taking into account the main effects of feeding and sex) between i.m. fat and the diameter of clustered adipocytes was significant (r = 0.41), whereas that between i.m. fat and the size of isolated adipocytes did not reach significance (r = 0.26). Whatever the groups, isolated adipocytes were far less numerous than adipocytes clustered along myofiber fasciculi (Figure 1). We observed an increased number of clustered adipocytes in the two restricted groups compared to the control group (+70% and +41% for FR and PR groups, respectively, P = 0.07). The pattern of clustered adipocyte numbers was parallel to that of age at slaughter (Figure 1). In contrast, the number of isolated adipocytes did not vary among groups (P = 0.50). Regression analyses between i.m. fat content and adipocyte numbers (Figure 2) were not significant.
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). On the contrary, FR and PR pigs displayed markedly lower activities of malic enzyme (-37% and -22%, respectively, P < 0.05) and G6PDH (-50% and -47%, respectively, P < 0.05) compared to C animals, when data were expressed on a gram per wet tissue basis. Considering the very diverse size of adipocytes between groups, it should be assumed that 1 g of muscle would represent a very diverse number of adipocytes. Therefore, we also corrected the enzyme data for the amount of lipids measured in each sample. When expressing the data on a milligram of lipid basis, the NADPH-producing enzyme activities in FR and C pigs were comparable (P > 0.25), whereas those in the PR group decreased to a considerable extent relative to C (-43% and -59% for malic enzyme and G6PDH, respectively, P < 0.05, Table 3).
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). Activities of CS and HAD (P > 0.05) were independent of feeding status. Consequently, the ratio of LDH to CS activities, indicative of glycolytic to oxidative muscle capacitiy, was depressed (-25%, P < 0.05) in PR pigs compared to the other two groups.
Expression of Sterol Regulatory Element Binding Protein Messenger Ribonucleic Acid
Whatever the feeding treatment, we clearly detected a signal corresponding to porcine SREBP-1 mRNA from whole skeletal muscle total RNA (Figure 3). Sterol regulation element binding protein messenger RNA was equally represented (P = 0.64) in C and PR pigs. Its expression was decreased by 12% in FR pigs compared to the two other groups; however, the difference did not reach significance.
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Discussion |
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At a cellular level, we demonstrated that variation in i.m. fat content was closely related to changes in muscle adipocyte diameters, in accordance with data reported for s.c. adipose tissue either in severely feed-restricted pigs (McNeel and Mersmann, 2000) or in piglets offered a low protein diet (Akanbi and Mersmann, 1996). Significant correlation between average cell size and i.m. lipid content has been also identified in longissimus muscle of growing bovine (Hood and Allen, 1973). In contrast to the current results, other studies have reported significant relationships between i.m. fat content and the estimated number of muscle adipocytes (Hood and Allen, 1973 in bovine; Gondret et al., 1998 in rabbit). The discrepancy between these different results is not explicable at the present time. Clustered adipocytes were more numerous in the older FR and PR pigs than in their 30-d-younger C littermates. These findings suggest the existence of a late adipocyte hyperplasia in pig muscles, as previously noticed in bovine muscles (Hood and Allen, 1973).
Variations in cell size between the different feeding groups may have included modifications of adipocyte capacity for lipogenesis, changes in the rate of fatty acid utilization within myofibers, or may be simply a consequence of different duration in fat accumulation. In the current study, the ACC activity in longissimus muscle of pigs did not respond to feeding manipulations. This result agrees with those reported by Winder et al. (1995) in fasting/refeeding rats and by Gondret et al. (2000) in feed-restricted rabbits compared to animals given ad libitum access to feed. However, ACC has been assessed under optimum buffer (especially concerning cofactors and substrate availability); therefore, the data reflected metabolic capacity and not in vivo actual rate. Furthermore, the isoenzyme of ACC expressed in skeletal muscle has been recently postulated to play an important role in governing fatty acid oxidation during muscle contraction rather than exerting a key control on lipogenesis (Winder et al., 1995). Conversely, malic enzyme and G6PDH are believed to supply the reducing equivalents for fatty acid synthesis (e.g., O’Hea and Leveille, 1969). Studies on porcine isolated adipocytes show that more than 80% of the enzyme activity measured in homogenates could be ascribed to the i.m. adipocyte metabolism (Mourot and Kouba, 1998). The precise delineation of G6PDH activity between lipogenesis in adipocytes and other energy processes within myofibers is more controversial, as very little activity (Glock and McLean, 1954) or substantial amounts (Allen et al., 1967) have also been detected in porcine muscle oxidative fibers. In our study, decreased activity levels for malic enzyme and G6PDH were currently demonstrated in FR pigs compared to C pigs when expressed in a grams of wet tissue basis. Considering that i.m. adipocytes were far numerous in muscle sections from FR pigs compared to C pigs, it should be assumed that the lipogenic capacity of each adipose cell has been actually decreased in response to feed restriction. This suggests that, in a situation of feed (energy) restriction, both substrate availability and cell metabolic capacity would be decreased. However, further investigations combining the isolation of muscle adipocytes, the use of 14C-labeled substrates, and the determination of NADPH concentrations are required. Finally, no differences between groups were noticed for the enzyme data when corrected by the amount of i.m. lipids, supporting the existence of a correlation between the activity levels of the NADPH-producing enzymes and i.m. fat accretion in FR and C pigs. Among the signaling molecules that could be involved in the regulation of the entire process of lipogenesis by feed intake, SREBP-1 is a possible candidate in nonmuscular adipose tissue (Kim et al., 1998) and in the liver (Horton et al., 1998). For the first time, we evidenced significant amounts of SREBP-1 mRNA in the longissimus muscle of pigs slaughtered at 110 kg BW. Previous studies conducted in younger animals (20 to 30 kg BW) failed to detect this transcript (Ding et al., 2000). The hybridization pattern obtained for SREBP-1 in the muscle (i.e., two bands approximately 5 kb in length) is similar to that reported in the porcine leaf fat and s.c. adipose tissue by us and others (Ding et al., 2000; Gondret et al., 2001). Starvation clearly reduces SREBP-1 mRNA concentration in rat adipocytes (Kim et al., 1998). However, the effects of fasting on porcine adipocyte transcripts are generally greater than those observed in chronically feed-restricted pigs (Housekenecht et al., 1998). In addition, McNeel et al. (2000) failed to evidence significant variations in several transcripts of fat anabolism in relation with feed restriction. In agreement with this, our results suggest that SREBP-1 mRNA concentration in muscle homogenates was not altered by a moderate long-term feed restriction. However, some studies have reported the existence of post-translational modifications, such as phosphorylation (Kotzka et al., 2000), which might modulate SREBP-1 activity. The putative role of SREBP-1 in the lipid pathways between adipocytes and myofibers also remains to be clarified.
In the current study, PR pigs exhibited much more i.m. fat and larger adipocytes at 110 kg BW than the C pigs. Thus, net fat accumulation could be simply a function of time, as PR pigs have accumulated fat over a much longer period (30 d) than C animals. However, the fact that PR pigs and FR pigs exhibited large differences in i.m. fat content despite the same age at slaughter provides evidence that the cell metabolic capacity is also involved in i.m. fat variation between PR pigs and C pigs. Surprisingly, we reported decreased activity levels of NADPH-producing enzymes in the former group. Therefore, the mechanisms by which adipocyte hypertrophy and i.m. accretion were enhanced by restricted protein allowance remain questionable. First, dietary protein restriction might be expected to involve a large decrease in the rate of lipolysis in the adipocytes, thus resulting in an imbalance between catabolism and anabolism in favor to fat accretion in PR pigs. However, this has not been previously observed in pigs fed 13 vs 21% protein diets at a constant energy level (Adeola and Young, 1989). Second, protein deficiency has been demonstrated to decrease the muscle respiration rate (Adeola and Young, 1989), thus likely reducing the demand for fatty acid oxidation within the myofibers. However, we have no direct evidence for such a modulation in our study, as neither citrate synthase (tricarboxylic acid cycle) nor HAD (beta-oxidation in mitochondria) activities were substantially modified in PR group compared to C group. However, Morio et al. (2001) recently showed that ex vivo rate of fatty acid oxidation in human muscle was largely independent of the maximal activity levels of the mitochondrial markers. Therefore, measurements of the actual rate of fatty acid oxidation would be helpful to elucidate the different mechanisms involved in adipocyte hypertrophy in response to low protein and energy intakes. In agreement with Karlsson et al. (1993), our results further indicated a lower glycolytic metabolism in the muscle of pigs offered the low-protein diet compared to controls. This effect seems to be specifically due to protein deficiency, since no difference in LDH activity was observed between FR and C pigs.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Footnotes |
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2 The authors gratefully acknowledge J. Mourot for lipogenic investigations and N. Clochefert for expert technical assistance.
Received for publication January 7, 2002. Accepted for publication July 16, 2002.
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Literature Cited |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). Gene 225:89–96.[Medline]
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