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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:2191-2199, September 2004

Nutrient-Gene Interactions

Restriction of Dietary Energy and Protein Induces Molecular Changes in Young Porcine Skeletal Muscles1

Nuno da Costa2, Christine McGillivray, Qianfan Bai, Jeffrey D. Wood*, Gary Evans{dagger} and Kin-Chow Chang

Molecular Medicine Laboratory, Institute of Comparative Medicine, University of Glasgow Veterinary School, G61 1QH, Scotland, UK; * Division of Farm Animal Science, University of Bristol Veterinary School, Langford, Bristol, BS40 5DU, UK; and {dagger} Sygen International plc, Department of Pathology, University of Cambridge, Cambridge, CB2 1QP, UK

2To whom correspondence should be addressed. E-mail: nadc1a{at}udcf.gla.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Little is known about the molecular changes in response to dietary restriction (energy and/or protein) in young growing skeletal muscles. To profile such changes and to gain insights into the signaling molecules that could mediate the diet effects, a dedicated porcine skeletal muscle cDNA-microarray approach was used to characterize differential muscle gene expression between conventionally fed and diet-restricted (20% less protein and 7% less energy) growing pigs, reared from 9 to 21 wk of age. In both red and white muscles, diet restriction resulted in the accumulation of significantly more intramuscular fat, and in the increased expression of genes involved in substrate (protein, glycogen, and lipid) turnover, in translation and mitochondrial function, and in raising glycolytic and oxidative phosphorylation potentials. The unexpected increase in intramuscular lipids in diet-restricted growing pigs could have important health implications for restricted diets in childhood. Despite reduced circulating insulin, more genes, including several novel growth modulatory genes, had higher expression levels, indicating that the cellular response to dietary restriction is an active process. One such responsive gene, P311, was most highly expressed in striated muscles and had a differentiation-dependent increase of expression in murine C2C12 cells, suggesting a role in differentiation/postdifferentiation phenotype determination.

KEY WORDS: • dietary restriction • protein restriction • microarray • skeletal muscle • porcine

Dietary restriction, in particular energy restriction (ER),3 has been extensively shown to reduce the degenerative effects of aging and to extend life expectancy in laboratory rodents (1–5). The biological effects of ER include improved antioxidant defenses (6), reduced oxidative damage (7,8), increased protein turnover, and increased fatty acid metabolism (8). The insulin/insulin-like growth factor type 1 (IGF-1) signaling axis appears to be involved, at least in part, in mediating some of the beneficial effects of dietary restriction, in particular longevity (4,9–11). Selective inactivation of the IGF-1/insulin receptor in vivo was recently shown to increase lifespan in mice by 18–26% (12,13). By contrast, there is evidence to suggest that dietary restriction imposed during fetal development or on early newborns could have deleterious consequences in later life, such as reduced glucose tolerance, type 2 diabetes, and reduced lifespan (14,15). At present, it is not clear whether the beneficial effects of a restricted dietary regimen apply to all stages of life. Equally, the dose and compositional effects of dietary restriction remain poorly understood.

Current dietary studies have focused on elucidating the molecular mechanisms linked to the aging process, which requires the use of mature-to-old animals, primarily laboratory rodents (6,8,16). Relatively little is known about the molecular response to dietary restriction in young growing animals, even less in large animal models. In farm animals, the transient use of restricted feeding in the young to induce subsequent compensatory growth is an important husbandry tool. It is relevant to note that the tissue that exhibits the highest postnatal growth rate is skeletal muscle (17). Our understanding of the underlying molecular mechanisms responsible for compensatory growth is limited; the nature of involvement of IGF-1 and other growth factors is not clear (18,19). We applied phenotypically divergent skeletal muscles (longissimus dorsi and psoas) of young growing pigs to a purpose-built porcine skeletal muscle cDNA microarray to profile the molecular changes and gain insights into the signaling molecules that mediate the effects of dietary restriction, including candidate genes that could be involved in compensatory growth.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Animal production. Entire male Duroc-based pigs (n = 48), from the same commercial line, were reared (from 9 wk of age) for 12 wk in a commercial piggery in 2 separate batches. Pigs were housed in groups of 12 with transponder-controlled individual feeding troughs. Pigs were randomly assigned to 1 of 2 pelleted diets (Dalgety Agriculture): a conventional (C) diet [200 g crude protein/kg, 11.4 g lysine/kg, 31.6 g lipid/kg, 14 MJ digestible energy (DE)/kg]; and a low protein and energy (LPE) diet (160 g crude protein/kg, 6.8 g lysine/kg, 28.7 g lipid/kg, 13 MJ DE/kg). Although the same ingredients were used to constitute both diets (main ingredients were barley, wheat, soybean meal, and corn gluten), the LPE diet contained 20% less protein and 7% less energy than the C diet. The oil content and fatty acid composition of the 2 diets were identical: 500 mg of C18:2 (linoleic acid)/g fatty acids and 48 mg of C18:3 ({alpha}-linoleic acid)/g fatty acids. Pigs were fed twice daily at a level estimated to be at 90% of ad libitum intake and weighed each week. After the 12-wk fattening period, pigs were transported to the University of Bristol licensed abattoir, electrically stunned, and slaughtered by severing the blood vessels of the neck. Approximately 200 mg samples of white longissimus dorsi (LD) and red psoas major muscles were removed, frozen in dry ice, and stored at –80°C for subsequent mRNA extraction. The backfat thickness (P2) was recorded at a point 6.5 cm from the dorsal midline at the level of the last rib using an intrascope (SFK), ~30 min after slaughter.

    Murine C2C12 myoblasts. C2C12 cells (CRL-1772) were grown in 75 cm2 flasks in proliferation medium (PM) (10% fetal bovine serum in DMEM). When the cells became 50% confluent, differentiation was initiated with the use of differentiation medium (DF) (2% horse serum in DMEM). C2C12 myoblasts/myotubes were harvested at different time points for mRNA extraction and cDNA conversion (20).

    Fatty acid and insulin measurements. Lipid was extracted from LD and psoas muscles using chloroform:methanol (2:1) as previously described (21). Total lipid (marbling fat) was separated on silicic acid columns (Jones Chromatography) into neutral lipid and phospholipid. FAMEs were prepared using diazomethane and analyzed by GLC (22). Fatty acids in both fractions were quantified using 21:0 as an internal standard. Plasma insulin concentrations were determined by RIA (125I RIA kit, ICN Pharmaceuticals).

    Microarray experiments. Two cohybridization experiments to assess the effects of diet on the LD and psoas muscle were conducted (C diet LD vs. LPE diet LD, and C diet psoas vs. LPE diet psoas). Four duplicate porcine microarray chips were used for each muscle experiment. Each cDNA clone, in turn, was spotted twice on each chip. Details of the design and construction of the porcine skeletal muscle cDNA microarray were previously described (23). Briefly, the microarray comprises 5500 cDNA clones, selected from 2 developmentally stage-specific porcine skeletal muscle cDNA libraries.

Unlike laboratory strains of inbred mice, pigs from the same breed are not genetically identical. To minimize differences due to genetic variability among individual pigs, each cyanine-dye (Cy-dye)–labeled target was derived from pooled LD or psoas mRNA, taken from muscles of 6 individual pigs fed the C or LPE diet. Details of mRNA isolation, Cy-dye labeling by reverse transcription, hybridization, scanning, data capturing using ImaGene 4.0 (BioDiscovery), and data analysis, which included an intensity-dependent (LOWESS) normalization step using GeneSpring 6.0 (Silicon Genetics), were described previously (23). Clones not previously identified were sequenced with T7 and T3 primers using an ABI BigDye Terminator v3.0 PCR-based sequencing kit (Applied Biosystems). Performa DTR gel filtration cartridges (EDGE Biosystems) were used for purification of PCR products before loading onto the ABI PRISM 3100 Genetic Analyzer. All sequence data were subjected to BLAST (Basic Local Alignment Search Tool) searches for gene identification by sequence similarity.

    Quantitative real-time RT-PCR. TaqMan quantitative real-time RT-PCR (Applied Biosystems) was performed on selected porcine and murine genes: myosin heavy chain (MyHC) 2a, MyHC 2b, ß-actin, {alpha}-actin, phytanoyl-CoA hydroxylase, {Delta}-9 desaturase (acc. no. Z97186), cbl-b, muscle creatine kinase, a novel tumor necrosis factor (TNF) member gene (kc2725), and P311. Table 1provides details of primers and probes of genes not previously given. Sequence details of primers, TaqMan probes, and quantification protocol, based on the use of relative standard curve, were as previously described (23,24). Four pools of cDNA samples were used as templates (C diet LD vs. LPE diet LD, and C diet psoas vs. LPE diet psoas); each pool comprised equal amounts of cDNAs from 6 individual pigs of 1 muscle type (LD or psoas) and 1 diet regimen (C or LPE diet). With the exception of 2 individuals, the same pigs were used for cDNA template generation and Cy-dye target labeling for microarray hybridization. A reference cDNA panel, comprising a number of different tissue templates (LD muscle, psoas, heart, uterus, brain, liver, and spleen) of a 7-wk-old pig, was used in tissue profiling.


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  		TABLE 1 Oligonucleotides and TaqMan fluorogenic probes used in real-time RT-PCR on LD and psoas muscle samples from pigs fed conventional or a low protein and energy diets1

 
    Statistical analysis. Growth performance data were analyzed by general linear models procedures (SAS procedure GLM, SAS Institute) using a model that included the fixed effects of trial and diet. The standard error of the difference between 2 sample means was calculated using the error mean square from the ANOVA and the mean sample size per batch. Differences between pair-wise combinations of the least-square means were tested for significance (P < 0.05).

For the microarray analysis, a moderate set of filtering conditions on GeneSpring 6.0 (normalized ratio of at least 1.5 between diets, and 1-sample t test P-values not > 0.1) was used to estimate the total number of genes in each muscle affected by dietary changes. To identify individual genes that were expressed differentially between groups, relatively stringent filtering was performed (normalized ratio of at least 1.5 between diets, and 1-sample t test P-values not > 0.05) to find the top 5% of differences in expression.

Real-time RT-PCR data were analyzed by dividing each of the 3 replicate values for each gene by the mean value for ß-actin in that assay. Differences among ratios were tested in the general linear models (GLM) procedure of SAS with the model ratio = muscle x diet interaction. All models were statistically significant and the significance of differences between diets in the same muscle type was assessed by t tests with the lsmeans cl and pdiff options in the GLM procedure.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Growth performance of pigs fed the LPE diet. The 24 pigs fed the LPE diet had significantly slower growth and greater intramuscular lipid concentrations in both LD and psoas muscles (Table 2). The accumulated intramuscular fat was distributed within and between fibers (data not shown). However, the increase in intramuscular lipids was not accompanied by a significant difference in P2. In pigs fed the LPE diet, only the LD muscle had a significantly greater membrane phospholipid concentration (Table 2).


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  		TABLE 2 Growth rate, back fat thickness and muscle lipid concentration in pigs fed conventional or low protein and energy diets1, 2

 
    Elevated gene expression in muscles from pigs fed the LPE diet. A general but clear finding of the diet microarray experiments for both LD and psoas muscles was that more genes had higher expression levels in pigs fed the LPE diet than in those fed the C diet by a ratio of at least 2:1. Under moderate filtering conditions, 35 cDNA clones were more highly expressed in the LD muscle of pigs fed the LPE diet, compared with only 11 clones identified as more highly expressed in the same muscle type of pigs fed the C diet. Similarly, 85 cDNA clones were more highly expressed in the psoas muscle of pigs fed the LPE diet compared with only 41 more highly expressed clones in the same muscle type derived from pigs fed the C diet.

    Raised catabolic and anabolic potential in muscles from pigs fed the LPE diet. A list of more highly expressed genes, derived from stringent filtering conditions, common to both muscle types in pigs fed the LPE diet are summarized in Table 3. A few genes were identified more than once on the microarray. The LPE diet increased the expression of genes involved in the breakdown of glycogen (glycogen phosphorylase), fatty acids (e.g., phytanoyl-CoA hydroxylase), as well as proteins (e.g., ubiquitin E3 ligase) (Table 3). Elevated ATP generating potentials accompanied the catabolic trend toward increased substrate breakdown. Several key genes involved in glycolysis (e.g., ß-enolase), oxidative phosphorylation (e.g., cytochrome oxidases), and ATP synthesis (e.g., muscle creatine kinase) had higher expression levels (Table 3). The expression of genes involved in translation (ribosomal proteins), sarcomeric structure (e.g., MyHC 2a), and post-translational transport (vacuolar protein sorting) were also elevated (Table 3) in muscles of pigs fed the LPE diet, thereby providing evidence of increased translational capacity in parallel with raised potentials for substrate breakdown and ATP generation.


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  		TABLE 3 Genes at least 1.5-fold more highly expressed in pigs fed the low protein and energy diet than in those fed the conventional diet in both the LD and psoas muscles as measured by microarray analysis

 
    Elevation of certain genes in only 1 muscle type in pigs fed the LPE diet. In addition to genes that were more highly expressed in both LD and psoas muscles of pigs fed the LPE diet, certain genes had higher expression levels in only 1 muscle type, possibly reflecting functional differences between white and red muscles (Table 4). Nonetheless, the transcriptional profile of these genes in the respective muscle (Table 4) was consistent with the overall profile of both muscles (Table 3), which was one of increased substrate turnover, translation, and mitochondrial function. For instance, malonyl-CoA decarboxylase, elevated in the psoas muscle of pigs fed the LPE diet, is responsible for the conversion of malonyl-CoA to acetyl-CoA in the oxidation of fatty acids (25). Another example is sarcolipin, which associates with SERCA1 in the sarcoplasmic reticulum and inhibits calcium uptake from the cytosol (26). The elevated expression of sarcolipin in the psoas muscle, possibly leading to raised cytosolic calcium, was consistent with increased oxidative function (Table 4).


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  		TABLE 4 Genes at least 1.5-fold more highly expressed in pigs fed the low protein and energy diet than in those fed the conventional diet for one muscle as measured by microarray analysis

 
    Elevated novel growth-related genes in muscle of pigs fed the LPE diet. In addition to increased substrate breakdown, raised protein turnover, and elevated ATP production potential, a set of growth-related genes was elevated in muscles of pigs fed the LPE diet (cbl-b, LIG-1, ITM2B, a novel TNF superfamily member, maged1 and P311) (Tables 3, and 4). These genes are novel insofar as their functional roles in skeletal muscle have not been determined. In nonmuscle cells, they variously modulated cell growth and induced apoptosis or differentiation. Cbl-b is a ubiquitin E3 ligase that targets the p85 regulatory subunit of phosphatidylinositol 3-kinase to downregulate activated tyrosine kinase–coupled receptors (including insulin and IGF-1 receptors), thereby suppressing T-cell activation (27). It is also a major susceptibility gene for rat type 1 diabetes mellitus (28). LIG-1 is an integral membrane glycoprotein with growth suppression properties on skin epithelial cells (29). ITM2B is another membrane protein that induces apoptosis in T cells by triggering the loss of mitochondrial membrane permeability (30). ITM2B is associated with chondrogenic differentiation (31). Although the function of Maged1 has not been formally described, it is a novel member of the MAGE/necdin gene family whose members were variously shown to promote neuronal differentiation and cell arrest (32). P311 is a novel gene, originally described as a neuronal protein, but found recently to have multiple actions including the induction of myofibroblast phenotype (33,34). The elevated presence of this entire range of genes suggests that growth modulation is actively driven under ER conditions.

    Validation and extension of microarray findings. To validate and extend the microarray findings, real-time quantitative RT-PCR was performed to determine the relative expression levels of a number of representative genes. Because intramuscular lipid concentration was greater in pigs fed the LPE diet, mRNA levels of 2 crucial lipid metabolism enzymes, phytanoyl-CoA hydroxylase (Table 3) and {Delta}-9 desaturase (stearoyl-CoA desaturase) (35,36), not reported on the present microarray, were quantified. Phytanoyl-CoA hydroxylase is an essential enzyme for the conversion of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA in the oxidation of the BCFA, phytanic acid (37). In Refsum disease, the enzyme is deficient, leading to the accumulation of phytanic acid (37). {Delta}-9 Desaturase catalyzes the synthesis of important monounsaturated fatty acids from stearoyl-CoA and palmitoyl-CoA. Muscle creatine kinase, another gene involved in energy metabolism, responsible for the conversion of creatine to creatine phosphate (38), was also selected for real-time RT-PCR analysis. Expression of all 3 genes was elevated in both LD and psoas muscles of pigs fed the LPE diet (Fig. 1).



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  		FIGURE 1 The effects of diet in pigs fed conventional or low protein and energy diets on the relative expression of specific genes, normalized to ß-actin, in red (psoas) and white (LD) muscles involved in energy metabolism as measured by real time RT-PCR: (A) phytanoyl-CoA hydroxylase, (B) {Delta}-9 desaturase, and (C) muscle creatine kinase. Each of the 4 cDNA muscle pools (psoas LPE, psoas C, LD LPE, LD C) comprised 6 different individual cDNA samples. The error bars are SD, n = 3 replicates within the same experiment. Differences between diets in the relative expression of each gene in each muscle type were assessed by t test (*P < 0.05; **P < 0.01).

 
A cDNA microarray is intrinsically less effective in differentiating between highly homologous members of the same gene family (23). In such a situation, it is even more important to perform real-time RT-PCR to validate the microarray data. The relative expression of 2 representative skeletal MyHC isoforms (fast oxidative-glycolytic 2a and fast glycolytic 2b) was determined (Fig. 2). The LPE diet increased MyHC 2a expression in both the LD (P < 0.05) and psoas (P < 0.01) muscles, consistent with the microarray finding (Table 3). Interestingly, MyHC 2b was elevated in the psoas muscle of pigs fed the LPE diet (P < 0.01) (compared with the psoas muscle in pigs fed the C diet) but lowered in the LD muscle of those fed the LPE diet (P < 0.01) (compared with the LD muscle in pigs fed the C diet). Keeping in mind that both glycolytic and oxidative potentials were elevated in the muscles of pigs fed the LPE diet, this contrast in MyHC 2b expression could be related to the glycolytic (white) phenotype of LD muscle and the oxidative (red) phenotype of psoas muscle. LD muscle has greater scope for oxidative change, whereas psoas muscle has more potential for a glycolytic shift (Fig. 2).



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  		FIGURE 2 The effects of diet in pigs fed conventional or low protein and energy diets on the relative expression of (A) MyHC 2a (oxidative-glycolytic), and (B) MyHC 2b (glycolytic) isoforms, normalized to ß-actin, in red (psoas) and white (LD) muscles as measured by real time RT-PCR. Each of the 4 cDNA muscle pools (psoas LPE, psoas C, LD LPE, LD C) comprised 6 different individual cDNA samples. The error bars are SD, n = 3 replicates within the same experiment. Differences between diets in the relative expression of each gene in each muscle type were assessed by t test (*P < 0.05, **P < 0.01).

 
Three genes (cbl-b, kc2725 and P311) with potential to modulate muscle growth were assessed by real-time RT-PCR. Expression patterns of cbl-b, kc2725 (a novel member of the TNF superfamily), and P311 were similar to the microarray findings (Fig. 3). Occasional discrepancies between microarray and real-time RT-PCR results can occur and can be attributed in part to genetic differences among individual pigs used in the 2 experiments, and to environmental effects (such as individual differences in food intake). Unlike inbred mice, pigs from the same breed are not genetically identical, which inevitably introduces greater experimental variation (23). To assess the relative distribution of each of the 3 putative growth modulatory genes in relation to muscle expression, real-time RT-PCR was performed on a panel of porcine reference cDNAs derived from a range of tissues (Fig. 4). Expression of cbl-b (Fig. 4A) and kc2725 (Fig. 4B) in skeletal muscles was relatively weak in relation to other tissues types. However, P311 had the highest relative expression in striated muscles among the different tissues in the panel (Fig. 4C).



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  		FIGURE 3 The effects of diet in pigs fed conventional or low protein and energy diets on the normalized (ß-actin) relative expression of 3 growth-related genes in red (psoas) and white (LD) muscles: (A) cbl-b, and (B) kc2725 (a novel TNF member), and (C) P311 as measured by real time RT-PCR. Each of the 4 cDNA muscle pools (psoas LPE, psoas C, LD LPE, LD C) comprised 6 different individual cDNA samples. The error bars are SD, n = 3 replicates within the same experiment. Differences between diets in the relative expression of each gene in each muscle type were assessed by t test (**P < 0.01).

 


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  		FIGURE 4 Tissue distribution of (A) cbl-b (B) kc2725 and (C) P311 in a reference tissue cDNA panel derived from a 7-wk-old pig. The relative expression of each gene was assessed by real time RT-PCR. The results are presented as normalized means ± SD, n = 3 replicates within the same experiment.

 
    The relation between circulating insulin and growth modulatory genes. The well-documented increase in lifespan induced by dietary restriction was recently shown to be mediated in part by the downregulation of the insulin/IGF-1 signaling pathway (12,13). It is possible that this pathway plays a key role in muscle growth during dietary restriction. Plasma insulin, measured by RIA, tended to be higher (P = 0.14) in pigs fed the C diet (147.2 ± 18.8 pmol/L, n = 10) than in those fed the LPE diet (106.3 ± 18.8 pmol/L, n = 10) due to wide variations between individuals. The trend for reduced circulating insulin and raised cbl-b expression (Table 3), a potent downregulator of tyrosine kinase-coupled receptor, is consistent with reduced insulin/IGF-1 influence during dietary restriction (39).

    P311 upregulated during muscle differentiation. The functional role of P311 in skeletal muscle is unknown. Current data, although limited, suggest that it could have a wide range of effects on endocrine factors and their receptors, and on transcription factors of myogenesis (33,34). Because it was most highly expressed in skeletal muscle without preference to muscle type (Figs. 3Cand 4C), its relative expression during myogenic differentiation was examined in murine C2C12 muscle cells. In the presence of differentiation medium (DF) to induce myogenesis, as evidenced by increasing myotube formation and skeletal {alpha}-actin expression, there was a progressive rise in P311 expression (Fig. 5), suggesting a possible role for P311 in skeletal muscle differentiation/postdifferentiation phenotype determination.



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  		FIGURE 5 Expression of P311 and {alpha}-actin in C2C12 muscle cells of pigs fed conventional or low protein and energy diets as determined by real-time RT-PCR at 6 developmental time points. The error bars are SD, n = 3 replicates within the same experiment.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
    Findings in relation to other studies. Previous dietary studies tended to focus on the effects of energy restriction on ameliorating the process of aging, in particular its role in the downregulation of stress-response genes and in the upregulation of reactive-oxygen species–scavenging genes (6,8,40). Such studies, by definition, were performed on skeletal muscles of mature mice (8,16), adult rats (6), adult rhesus monkeys (40), and adult humans (41), with dietary regimens that often resulted in weight loss. Energy restriction affects lifespan extension similarly to protein restriction (3,42). Our microarray findings are similar to those of an energy restriction gene profile study on murine skeletal muscles in which dietary restriction increased the expression of glycolytic and mitochondrial oxidative genes, and genes involved in protein turnover (8). Lee et al. (8) also found indications of increased fatty acid synthesis and growth suppression in energy-restricted mice. In our porcine study, the lack of detection of stress-response genes was likely due to their absence in the microarray design. Our results were also consistent with the main finding by White et al. (43) of increased oxidative capacity of all skeletal muscles tested in 6-wk-old pigs fed an energy-restricted diet for 3 wk. Preliminary fiber typing work on LD and psoas muscles indicates that the diet-induced metabolic changes in skeletal muscle have little or no effect on the composition of MyHC fiber type (44).

    Dietary restriction and intramuscular lipid accumulation. One of our main findings is that reduced dietary energy and protein in growing pigs significantly increased intramuscular fat. One possible explanation for this effect is that the low protein content restricted muscle growth, resulting in surplus energy being converted into intramuscular lipids. Previous work on growing pigs fed a carbohydrate-rich diet, in relation to protein, seems to support this suggestion (45). Another possible explanation is that restricted growth from the LPE diet led to fat redistribution, resulting in an increase in intramuscular fat (17). Our microarray results indicate that fatty acid oxidation was one of the main potential sources of fuel in muscle. However, subcutaneous fat was significantly higher in pigs fed the LPE diet (46). These observations seem to support the notion that in addition to raised oxidative potential, lipids were actively redistributed to and/or accumulated in certain tissues of pigs fed the LPE diet, including skeletal muscle. In obese humans, the accumulation of intramuscular (inter- and intrafiber) lipids is associated with insulin resistance and the subsequent development of type 2 diabetes mellitus (DM) (47). Obese individuals with type 2 DM have reduced rates of fatty acid oxidation, and reduced overall oxidative capacity in their muscles. In contrast, the accumulation of intramuscular lipids in porcine muscles after consumption of the LPE diet was accompanied by elevated glycolytic and oxidative potentials. Indeed, endurance-trained athletes have increased levels of intramuscular lipids (48,49). Hence, the accumulation of intramuscular lipids per se may not necessarily be detrimental.

Adult humans subjected to severe energy restriction for brief periods lost weight and had reduced lipid content in muscle fibers (8,42,50). It is not clear whether this is an age-dependent response to severe dietary restriction. Our study showed that a moderately restricted diet, sufficient to promote weight gain in young growing pigs (puberty starts at ~21 wk of age), could elevate the levels of intramuscular lipids. This finding could have important implications for childhood dieting. For obese children, dietary restriction, contrary to expectation, could inadvertently raise intramuscular lipid, possibly contributing to the risk of type 2 DM development. Clearly, more work is required to examine the effects of dietary restriction on fatty acid metabolism in growing skeletal muscle.

In farm animal production, the ability to raise lipid content within muscle by reduced feeding could be a viable way to modify the quality of the meat products (44). In addition, the ability to upregulate {Delta}-9 desaturase is widely regarded as desirable to increase the relative levels of unsaturated fatty acids in meat (51). Our results show that dietary restriction increases the transcriptional rate of {Delta}-9 desaturase. Work is in progress to determine the enzymatic activity of {Delta}-9 desaturase and the levels of monosaturated fatty acids in muscle in energy-restricted animals.

    Growth modulation and differentiation. The molecular mechanisms involved in growth modulation/differentiation during dietary restriction are not well documented. In this study, we identified a number of genes that appear to have growth modulatory properties, although their effects on muscle cells were not reported. This finding suggests that the cellular response to dietary restriction is an actively driven process. Some of these genes could play pivotal roles in growth suppression and muscle phenotype determination. One such candidate is cbl-b, a RING finger type ubiquitin E3 ligase, which functions as a downregulator of activated tyrosine kinase–coupled receptors that include insulin/IGF-1 receptor, and is a major susceptibility gene for rat type 1 DM (28). The dampening down of the insulin/IGF-1 axis during dietary restriction, via reduced circulating insulin and downregulation of insulin signaling by elevated cbl-b, could be a way of inducing increased insulin sensitivity during subsequent refeeding (52). Another gene, P311, was not only responsive to dietary restriction (Fig. 3C) but was most highly expressed in skeletal muscles (Fig. 4C), even though it was originally found in the brain and smooth muscle (33,34,53). Among a number of potential biological functions recently reported, P311 seems to play a crucial role in myofibroblast transformation of nonmuscle cells (33,34). Its progressive rise in expression during in vitro myogenic differentiation points to its possible importance in differentiation/postdifferentiation skeletal muscle phenotype determination.

In conclusion, we found that a moderately reduced energy and protein diet in growing pigs that led to reduced growth was accompanied by significantly increased intramuscular lipid, and increased potentials for substrate (protein, glycogen, and lipid) turnover, and mitochondrial function. A number of novel growth-regulatory genes were identified; one such gene, P311, was highly expressed in striated muscles and appeared to be involved in differentiation/postdifferentiation phenotype determination.


   FOOTNOTES
 
1 Supported by the Biotechnology and Biological Sciences Research Council. Back

3 Abbreviations used: C, conventional diet; Cy-dye, cyanine dye; DE, digestible energy; DF, differentiation medium; DM, diabetes mellitus; ER, energy restriction; IGF-1, insulin-like growth factor 1; LD, longissimus dorsi; LPE, low protein and energy; MyHC, myosin heavy chain; P2, backfat thickness; TNF, tumor necrosis factor. Back

Manuscript received 6 January 2004. Initial review completed 9 February 2004. Revision accepted 27 May 2004.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

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