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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
* Laboratory for Animal Nutrition and Animal Product Quality, Department of Animal Production, Ghent University, Proefhoevestraat 10, 9090 Melle, Belgium; and
and
Laboratory of Livestock Physiology, Immunology and Genetics, KULeuven, Kasteelpark 30, 3001 Heverlee, Belgium
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Key Words: growth • insulin-like growth factor-II • muscle • pig • proteolytic and lipolytic enzymes
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). A 3-fold increase in IGF-II mRNA expression in skeletal muscle was observed during postnatal growth because of the abrogation of a repressor. The IGF system is well known to be very important in the regulation of muscle growth and development (Florini et al., 1996). Moreover, IGF-II appears to function as an autocrine or paracrine factor in stimulating both proliferation and differentiation of muscle cells (Florini et al., 1991). The IGF-II mutation increases the percentage of lean muscle mass in pigs at the expense of fat, with apparently no effect on birth weight and ADG (Nezer et al., 1999). However, the effects of the IGF-II genotype on muscle growth and the role of protein degradation therein are unknown.
Protein degradation is mediated by a wide variety of proteolytic systems. Calpains (CALP), proteasomal enzymes, and lysosomal enzymes are responsible for the bulk of protein turnover (Ueda et al., 1998). The lysosomal enzymes consist of cysteine proteinases [e.g, cathepsins (CATH)], and are involved in intracellular nonselective protein degradation (Matsuishi and Okitani, 2003). Calpains play an important role in muscle cell differentiation and skeletal muscle protein degradation (Huang and Forsberg, 1998) and consist of several enzymes, of which the Ca2+-dependent proteases, µ-CALP and m-CALP, and their inhibitor, calpastatin (CAST), are the most studied. In addition, µ-CALP is involved in the regulation of myogenesis (Moyen et al., 2004), and the m-CALP:CAST ratio may play an important role in the degradation of membrane-skeleton proteins during myoblast and satellite cell fusion (Dourdin et al., 1999).
The aim of the current study was to investigate the effects of age, muscle type, and IGF-II genotype in pigs on muscle proteolytic and lipolytic enzyme activities during the early postmortem period.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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The experiment was approved by the Ethical Committee of Kuleuven (Belgium).
The boars originated from 2 dam lines of Rattlerow-Seghers (Lebbeke, Belgium) based on Landrace and Large White. The boars were the progeny of 9 sires that were heterozygous for IGF-II (Apat and Gpat alleles). The Apat boars inherited the IGF-II mutation, which is related to the greater muscle mass vs. the Gpat boars, which carried the paternal wild-type allele. Because of the paternal imprinting of the IGF-II gene (Nezer et al., 1999), the genotype of the mother was not taken into consideration. However, only offspring of homozygous sows were used to allow determination of the paternal allele of interest.
At approximately 4, 8, 16, or 26 wk of age, a total of 81 boars were slaughtered. Litters, based on availability, were randomly selected at birth to deliver boars for slaughter, with an equal distribution of the IGF-II genotypes for the different age groups. These ages were chosen according to important changes in growth. The numbers of boars per genotype and per line are shown in Table 1. Piglets were weaned at 4 wk of age. From 3 to 5 wk of age, the boars were fed a prestarter diet followed by a starter diet until 8 wk of age. At approximately 8 wk of age, the boars were moved to the finishing barns and given a growing-finishing diet. Boars were fed ad libitum until slaughter and were housed in a group.
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Boars were slaughtered in age groups of 6 animals, with an equal distribution of the IGF-II genotypes. Boars in the 4- and 8-wk age groups were slaughtered in the slaughterhouse of our department. The boars in the 16- and 26-wk age groups were slaughtered in a small private slaughterhouse (Verstuyft, Nevele, Belgium). In both slaughterhouses, boars were bled after electrical stunning, except that halothane anesthesia was used in 3 animals at 4 wk. Within 45 min after stunning, the Longissimus thoracis et lumborum (LTL), Semimembranosus (SM), and Triceps brachii (TB) muscles of the left carcass side were removed and weighed. Samples were kept at 2°C until arrival at the institute and subsequently were stored at –80°C. Sampling and storage of the samples were always done in a consistent manner. Determination of the µ-CALP, m-CALP, and CAST activities was done within 14 d of storage at –80°C. Afterward, samples were kept at –20°C until analyses of the lysosomal and lipolytic enzyme activities. The enzyme activities measured in these muscle samples taken early postmortem were assumed to reflect enzyme activities that represent in vivo conditions.
Determination of µ-CALP, m-CALP, and CAST Activities
Activities of µ-CALP, m-CALP, and CAST were determined in LTL, SM, and TB muscle samples according to the procedure of Etherington et al. (1987), with some modifications. Frozen muscle tissue (3 g) was homogenized in 27 mL of cold (2°C) 50 mM phosphate buffer that contained 4 mM EDTA, 4 mM dithiothreitol, and 0.04% ascorbic acid using an UltraTurrax homogenizer (IKA Werke, Staufen, Germany) at 13,000 rpm. The pH was adjusted to 7.5 with HCl (3 N) or NaOH (3 N), and the homogenate was kept for 15 min at room temperature. The homogenate was centrifuged at 3,500 x g for 15 min at room temperature.
Sodium chloride was added to an aliquot of the extract, corresponding to 2.0 g of tissue, to a final concentration of 500 mM. This NaCl-treated aliquot was loaded on a self-made, 2-mL, phenyl Sepharose glass column (Amersham Biosciences, Diegem, Belgium), which was equilibrated with 20 mM Tris, pH 7.5, containing 2 mM EDTA, 1 mM dithiothreitol, and 500 mM NaCl at room temperature. Calpains were bound to the column, whereas CAST was collected in the effluent. Thereafter, the CALP were allowed to elute with 14 mL of a salt-free 20-mM Tris buffer at pH 7.5. This eluate was poured over a 0.5-mL DEAE Sephacel ion-exchange column (Pharmacia Biotech, Diegem, Belgium), which was equilibrated with the 20-mM Tris buffer, and the CALP were bound to the column. In a first step, µ-CALP was completely eluted using 2.8 mL of Tris buffer (pH 7.5) containing 200 mM NaCl, followed by the elution of m-CALP using 2.8 mL of a 500-mM, NaCl-containing Tris buffer.
The activity of µ-CALP was measured by incubating 1 mL of the µ-CALP solution for 1 h at 20°C with 1.50 mL of a 1% casein solution at pH 7.5 (Fluka, Bornem, Belgium) containing 100 mM Tris, pH 7.5, 8.33 mM Ca2+, and 3.33 mM dithiothreitol. The reaction was stopped by adding 0.50 mL of 15% (vol/vol) trichloroacetic acid, followed by mixing and filtration (filter paper No. 597, Schleicher & Schuell, Dassel, Germany). Finally, the filtrate was assayed for tryptophan (Messineo and Musarra, 1972), measuring absorbance at 515 nm vs. the blank.
Activities of m-CALP and CAST were determined by mixing 0.6 mL of the m-CALP fraction with 1.50 mL of the casein solution at pH 7.5, followed by mixing with either 0, 0.2, or 0.4 mL of the CAST fraction and adjusting with 500 mM NaCl-Tris buffer to a final volume of 2.5 mL. Further steps in the determination of m-CALP were the same as for µ-CALP. The 3 m-CALP activity values were used in a linear regression equation to calculate m-CALP (intercept) and CAST activity (slope x total volume of the CAST fraction).
The µ-CALP, m-CALP, and CAST enzyme activities are expressed as units per gram of muscle. One unit was defined as the hydrolysis of 1 µg of casein/min at 20°C and pH 7.5.
Determination of CATH B+L, and CATH H Activities
Determination of the CATH B+L and CATH H was done in LTL, SM, and TB according to Claeys et al. (2001) and Toldra and Etherington (1988), respectively. Muscle tissue (3 g) was homogenized with an UltraTurrax at 13,000 rpm in 27 mL of a cold (2°C) 0.1 M citric acid buffer at pH 5.0 for CATH B+L and a cold (2°C) 0.1 M phosphate buffer at pH 6.8 for CATH H, with both buffers containing 0.2% Triton X-100 (Sigma-Aldrich, Bornem, Belgium). The homogenate was centrifuged at 4,000 x g for 15 min. The volume of the supernatant was measured, and the supernatant was subsequently filtered (filter paper No. 597, Schleicher & Schuell) and kept at 2°C.
The activity of CATH B+L was determined fluorimetrically (Turner model 112, Turner, Wijnegem, Belgium) using 1 µM of the substrate N-carbobenzoxy-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin (Z-CBZ-Phe-Arg-NHMec, Sigma-Aldrich) at pH 5.5 and 37°C. The activity of CATH H was determined using 0.04 mM of the substrate N-CBZ-L-arginine-7-amido-4-methylcoumarin (N-CBZ-L-Arg-NHMEC, Sigma-Aldrich) at pH 6.8 and 37°C, and the release of 7-amido-4-methylcoumarin was continuously monitored on a recorder (Ankersmit RA 8 recorder, Ankersmit, Brussels, Belgium).
The CATH B+L and CATH H enzyme activities are expressed as units per 100 g of muscle. One unit was defined as the release of 1 nmol of 7-amido-4-methylcoumarin/min at 37°C, and at pH 5.0 and pH 6.8 for CATH B+L and CATH H, respectively. Two incubations per extract were performed for each enzyme assay.
Acid Lipase and Phospholipase Assays
The assays were performed as described by Motilva and Toldra (1993) with slight modifications. The enzymes were extracted with a cold (2°C) 0.1 M citric acid buffer at pH 5.0, containing 1 mM EDTA and 0.2% Triton X-100. The activity was determined fluorimetrically (Turner model 112) against 25 µM 4-methylum-belliferyloleate (Sigma-Aldrich) at 25°C from a methyl-umbelliferyloleate stock solution of 20 mM dissolved in methoxyethanol and stored at –20°C. For the total acid lipase assay, the stock buffer contained 0.1 M citric acid buffer at pH 5.0 and 0.05 M Triton X-100, with the addition of 150 mM NaF for measuring acid phospholipase (PHOSLIP). Acid lipase (LIP) activity was calculated as the difference between the total LIP and PHOS-LIP activities. The release of methylumbelliferon was continuously monitored on a recorder (Ankersmit RA 8 recorder). Background activity was measured each day of analysis and was subtracted from the activities measured.
Acid lipase and PHOSLIP activities are expressed as units per gram of muscle. One unit was defined as nanomoles of methylumbelliferon released per min at 25°C and pH 5.0. Two incubations per extract were performed for each enzyme assay. Because a relatively large effect of the incubation buffer solution was noticed, the observed values were transformed to standard normal values (by subtracting the mean and dividing by the SD) for each series of analyses done with the same buffer stock solution.
Statistical Analyses
The IGF-II genotype and line number as fixed factors and age as a covariate were used in a univariate GLM to determine differences in muscle weights. All enzyme activities were analyzed with a univariate GLM, with IGF-II genotype, muscle type, and age group as fixed factors, along with their 2-way interactions. When muscle type or age group was significant, the means were separated with Tukey’s test. In case of significant 2-way interactions, means for a factor were compared within the other factor. In a preliminary analysis, line appeared not to be significant and was therefore not included in the model. Statistical analyses were done using SPSS 12.0 for Windows (SPSS, Inc., Chicago, IL).
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Means and SD for age and BW at slaughter per age group are shown in Table 1
. No differences between IGF-II genotypes were apparent for these traits and also no differences between the genotypes were found for ADG (data not shown). Relative muscle weights were used as an approximate for carcass lean content and were calculated as the percentage proportion of the muscle weight relative to the carcass weight. Because carcass weight of the boars of the younger age groups were not assessed, only data of 26-wk-old boars were available. For this age group, a greater relative weight (P < 0.05) of the LTL and the sum of the 3 muscles in the Apat animals, compared with the Gpat animals, were found (Table 2). The relative weights for SM and TB did not differ for the animals bearing the mutation. Although differences were relatively small in the current study, our data correspond to previous reports that the IGF-II mutation causes an increase in carcass lean content at the expense of fat (our unpublished data), but without an effect on birth weight (Van Laere et al., 2003; our unpublished data) and growth rate (Jeon et al., 1999; Nezer et al., 1999).
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Muscle protein degradation plays an important role in muscle growth because an increase in muscle mass is the result of a positive balance between protein synthesis and protein breakdown. The CALP system is involved in myofibrillar protein turnover (Goll et al., 1992), and CATH are responsible for lysosomal protein degradation (Sentandreu et al., 2002). The ratio of µ-CALP to CAST (µ-CALP:CAST) and m-CALP to CAST (m-CALP:CAST) were proposed as an index of muscle proteolytic potential by Goll et al. (1992). Because the IGF-II mutation affects the percentage of lean muscle mass at the expense of fat, we hypothesized that this mutation could also affect proteolytic and lipolytic enzyme activities.
Significance of the main effects of muscle, genotype, and age group, and the 2-way interactions are shown in Table 3. Means for the IGF-II genotype are shown per age group across the different muscles (LTL, SM, and TB) in Table 4. The activity of CAST was greater (P < 0.001) and m-CALP:CAST less (P < 0.001) in Apat compared with Gpat animals. The other enzyme activities were not affected by the IGF-II genotype. A genotype x age interaction was observed for µ-CALP:CAST (P < 0.05) and CATH B+L (P < 0.001) with varying differences between genotypes at different ages. However, no significant muscle x genotype interaction was found.
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) or in subcutaneous adipose tissue (Gardan et al., 2006). Hence, differences in the percentage of lean mass must arise during postnatal muscle development. Because the number of muscle fibers is determined prenatally, postnatal skeletal muscle growth in pigs occurs through hypertrophy of existing muscle fibers (Wigmore and Stickland, 1983). Satellite cells, which are quiescent mononucleated myogenic cells, facilitate skeletal muscle DNA accretion through proliferation, followed by differentiation and fusion with existing muscle fibers. Insulin-like growth factor-II appears to function as an autocrine–paracrine factor both in stimulating the proliferation and in differentiating satellite cells (Florini et al., 1991; Oksbjerg et al., 2004). Moreover, it is known to suppress protein degradation (Ewton et al., 1987). Furthermore, IGF-II acts as a critical survival factor during the transition from proliferating to differentiating myoblasts and satellite cells. In addition, m-CALP:CAST was shown to play a role in myoblast fusion (Barnoy et al., 1998). Degradation of membrane-skeleton structures by m-CALP (Schollmeyer, 1986; Dourdin et al., 1999) allows destabilization of the membrane and fusion of the myoblast. The balance between m-CALP and CAST seems to be important for normal fusion development (Barnoy et al., 1996); that is, the CAST level diminishes from a high level in proliferating myoblasts to a low level in differentiating myoblasts (Barnoy et al., 1998). These results were obtained in cell culture models, which provide a convenient system for studying factors involved in muscle formation and growth (Barnoy et al., 1998). Given that the CAST activity was greater and the m-CALP:CAST ratio was less in the Apat genotype with the greatest percentage of muscle mass, it is likely that differences in proteolytic enzyme activities are involved in the effect of the IGF-II mutation on muscle hypertrophy.
Cathepsin, LIP, and PHOSLIP activities were not affected by the IGF-II genotype in the current study. Most of the CATH (e.g., CATH B, L, H, K, and S) are lysosomal cysteine proteinases that are involved in intracellular protein degradation and in cell differentiation and proliferation (Otto and Schirmeister, 1997; Béchet et al., 2005). Lipase and phospholipase are responsible for the release of fatty acids from triacylglycerols and phospholipids, respectively. Acid lipase is important in the energy metabolism of the muscle, and its activity has been linked with myoblast differentiation (Sauro et al., 1985). Cathepsin and (phospho)lipases have also been related to postmortem lipolysis and proteolysis, respectively; for example, in dry-cured ham contributing to flavor development (Toldra and Etherington, 1988; Motilva and Toldra, 1993). Based on the lack of effect of the IGF-II mutation on these enzymes, it is unlikely that the IGF-II genotype would have an effect on flavor development in processed meat.
Effect of Age on Proteolytic and Lipolytic Enzyme Activities
Age had the largest effect of the main factors and was significant (P < 0.001) for all traits except for LIP (Table 3). The muscle x age interaction was also significant (P < 0.05) for all traits except for µ-CALP:CAST and m-CALP:CAST. Means for the IGF-II genotype effect are shown per age group across the 3 muscles (LTL, SM, and TB) in Table 4. However, the age patterns, regardless of the IGF-II genotype, were neither uniform nor linear in time and were difficult to interpret. The µ-CALP activity decreased (P < 0.05) from 4 until 8 wk and increased (P < 0.05) from 16 until 26 wk, but no differences were observed between 8 and 16 wk or between 4 and 26 wk. The m-CALP activity decreased (P < 0.05) from 4 until 16 wk, and remained at the same level thereafter. The CAST activity decreased between 4 and 16 wk, followed by a significant (P < 0.05) but muscle-dependent increase between 16 and 26 wk. Irrespective of muscle, the CATH B+L activity was greater (P < 0.05) at 8 wk compared with the other age groups, which did not differ. The CATH H activity did not alter until the age group of 16 wk, followed by an increase (P < 0.05) in the 26-wk age group. Activity of PHOSLIP was constant until the age group of 16 wk and decreased (P < 0.05) thereafter.
Although there was a clear muscle effect, a similar pattern in proteolytic and phospholipolytic activity with age was found for the 3 muscles. However, it is not possible to give a biological explanation for all the changes in proteolytic and lipolytic enzyme activity that were observed with age. In addition, one must keep in mind that the effect of age group was confounded to some extent by management differences (e.g., dietary changes, weaning at 4 wk of age) that are inevitable in this type of experimental approach. To the best of our knowledge, only limited data are available in the literature for comparison, and none from pig studies. A difference in CALP and CAST activities in sheep between weaning and slaughter age was reported by Ou et al. (1991). A small drop in CATH B activity in adult rats, compared with a constant level during the growth phase, was previously reported (Goldspink and Lewis, 1985).
If increased muscle growth were tightly related to down-regulation of protease activity, then one would expect an increase in proteolytic activity with increasing age, given the decrease in relative rate of protein deposition that normally occurs with age (Wagner et al., 1999). In pig lines differing in muscularity, increased muscle mass is associated with an increased DNA content (Mesires and Doumit, 2002), and the growth rate of skeletal muscle cells is dependent on satellite cell proliferation and protein accretion (Allen et al., 1979). It is generally accepted that the proportion of satellite cells declines with age. The majority of muscle DNA accumulation in pigs occurs between 7 and 21 wk of age (Mesires and Doumit, 2002). This corresponds with the observed increase of m-CALP:CAST or increased proteolytic potential for satellite cell fusion from 4 wk up to 16 wk of age, followed by a decrease for the age group of 26 wk.
The age patterns of the other enzymes were consistent across muscles for CATH and PHOSLIP, but not for LIP. The sharp increase in CATH B+L activity at 8 wk of age, and the increase in CATH H activity and decrease in PHOSLIP activity at slaughter age (26 wk), compared with the other age groups, were most noticeable. However, no reasonable explanation for these changes can be given.
Effect of Muscle on Proteolytic and Lipolytic Enzyme Activities
An effect of muscle (P < 0.05) was found for all traits except for µ-CALP and m-CALP (Table 3). Moreover, the muscle x age interaction was also significant (P < 0.05) for all traits, except for µ-CALP:CAST and m-CALP:CAST. Figure 1 shows µ-CALP, m-CALP, and CAST per muscle and age group. The differences were regardless of the IGF-II genotype. The µ-CALP was greater (P < 0.05) in SM compared with the other 2 muscles at 16 wk. The µ-CALP, m-CALP, and CAST were greater (P < 0.05) in TB compared with LTL and SM at 26 wk. Hence, the µ-CALP and CAST increased faster and to a greater level compared with the 4-wk group in TB. The µ-CALP:CAST was less (P < 0.05) for the TB at 16 wk and the m-CALP:CAST was less (P < 0.05) for the TB at 26 wk compared with the other 2 muscles (Figure 2). Figure 3 shows the CATH B+L and CATH H per muscle and age group. Inconsistent differences between muscles were found for CATH B+L. However, a consistent muscle effect was found for the CATH H activity, with LTL having lower (P < 0.05) values compared with the TB in all age groups. An irregular and different age pattern was also observed for LIP according to muscle (Figure 4). At 16 and 26 wk, LTL had lower (P < 0.05) PHOSLIP values compared with TB and SM.
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showed that protein synthesis is correlated with the content of slow oxidative fibers (type I) but not with the relative proportions of the fast glycolytic (type IIb) to fast oxidative glycolytic fibers (type IIa). Muscles with more slow-twitch fibers, based on the percent area of the muscle, synthesize protein more rapidly than the muscles with more fast-twitch fibers, probably due in part to an increased number of type-I IGF-receptors in slow-twitch muscle fibers (Louveau and Gondret, 2004). Greater CALP and CATH activities were detected in slow-twitch muscles, compared with fast-twitch muscles, in calves (Therkildsen et al., 2002; Béchet et al., 2005), and CAST activity was also found to be greater in slow-twitch muscles in sheep (Whipple and Koohmaraie, 1992; Delgado et al., 2001; Sazili et al., 2005). Our findings correspond with these reports. The proteolytic and lipolytic enzyme activities, except for CATH B+L, in the age group of 26 wk in our study were all greatest in the TB muscle. This muscle contains more slow-twitch fibers compared with the SM and LTL (Laborde et al., 1985). Although greater CATH B and CATH H activities in slow-twitch muscles were reported previously in rats (Goldspink and Lewis, 1985), our results indicated a lower CATH B+L activity in TB.
The effect of muscle on LIP activity was strongly age dependent. Differences in acid lipase activity were related to the content of oxidative fibers in the muscle, with greater and increasing acid lipase activities in TB compared with lower and declining activities in the LTL muscle (Langfort et al., 2003).
In conclusion, differences in proteolytic and lipolytic enzyme activities suggest that the greater percentage muscle mass in the mutant IGF-II genotype may be associated with decreased protein degradation, increased myoblast fusion, or both. However, more information is needed to better understand the effect of the IGF-II genotype on muscle protein synthesis capacity and protein turnover.
Patterns were found in the activities of proteolytic enzymes and PHOSLIP during growth, along with differences between muscles differing in metabolism. Although the evolution with age could not be easily explained, our results are in agreement with previous reports in other species stating that proteolytic and lipolytic enzyme activities are affected by age and muscle type.
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Footnotes |
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2 Corresponding author: Stefaan.DeSmet{at}UGent.be
Received for publication August 23, 2006. Accepted for publication December 14, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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= Semi-membranosus, and 
= Triceps brachii) and age group across IGF-II genotypes. Values with different letters differ between muscles within age group (P < 0.05). One unit (U) is defined as the hydrolysis of 1 µg of casein/ min at 20°C and pH 7.5.
