Effects of low-protein diet and feed restriction on mRNA expression of cationic amino acid transporters in porcine skeletal muscles.

We investigated the effects of a low-protein diet and feed restriction on the mRNA expression of cationic amino acid transporters (CATs) in the longissimus dorsi (LD), rhomboideus (RH), and biceps femoris (BF) muscles of pigs. Eighteen piglets were divided into three groups: a control (CP21%), low-protein diet (LP, CP16%), and feed-restricted diet (FR, CP21%, 76% feed intake of control pigs) groups. The expression levels of CAT-1 in the LD and BF muscles of LP pigs were higher than that of control pigs, whereas that of FR pigs showed no difference. The CAT-2A expression levels in the RH muscle of FR pigs were higher than that of control pigs. The free lysine concentrations in all muscles of LP and FR pigs were lower than that of control pigs. To examine the factors that affect CATs mRNA expression, we evaluated the effects of lysine, arginine, insulin-like growth factor-I, and dexamethasone on the expression of CATs in C2C12 myotubes. CAT-1 expression levels increased in lysine and/or arginine deprivation. We show that CAT-1 and CAT-2A expression levels in skeletal muscles differ in response to dietary treatments and CAT-1 expression in skeletal muscles appears to increase in response to low free lysine concentrations.

Regulation of autophagy in chick myotube cultures: Effect of uncoupling mitochondrial oxidative phosphorylation.

Abstracts: Skeletal muscles have a high demand for ATP, which is met largely through mitochondria oxidative phosphorylation. Autophagy is essential for the maintenance of skeletal muscle mass under catabolic conditions. This study investigated the effect of uncoupling mitochondrial oxidative phosphorylation on autophagy in chicken skeletal muscle. Chick myotubes were incubated with the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at 25 µM for 3h. CCCP prevented the phosphorylation of p70 ribosomal S6 kinase 1 (Thr389), S6 ribosomal protein (Ser240/244), and eukaryotic translation initiation factor 4E-binding protein 1 (Thr37/46), which are the measures of the mechanistic target of rapamycin complex 1 (mTORC1) activity. CCCP significantly increased cytoplasmic and mitochondrial LC3-II content, which act as indices of index for autophagosome formation and mitophagy, respectively, but did not influence the expression of autophagy-related genes LC3B, GABARAPL1, and ATG12. Finally, surface sensing of translation method revealed that protein synthesis, a highly energy consuming process, was significantly decreased upon CCCP treatment. These results indicate that the uncoupling of mitochondrial oxidative phosphorylation stimulates autophagy and inhibits protein synthesis through mTORC1 signaling in chick myotube cultures.

AMP-activated Protein Kinase Activation Suppresses Protein Synthesis and mTORC1 Signaling in Chick Myotube Cultures.

Protein synthesis in skeletal muscle is considered one of the most energy-consuming cellular processes. AMP-activated protein kinase (AMPK) is a metabolic master switch that regulates glucose and lipid metabolism, and it is implicated in protein synthesis control in skeletal muscles. The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of protein metabolism in cells. However, the effect of AMPK activation on protein synthesis and mTORC1 signaling in chicken skeletal muscle remains unclear. Therefore, in this study, we aimed to investigate the effect of 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR), an AMPK activator, on protein synthesis and mTORC1 signaling in chick myotube cultures. The incubation of chick myotubes with AICAR (1 mM) for 3 h led to a significant increase in AMPK (Thr172) phosphorylation. Nonetheless, protein synthesis, measured using the surface sensing of translation method, was significantly decreased by AICAR. In addition, the phosphorylation of p70 ribosomal S6 kinase 1 (S6K1, Thr389), S6 ribosomal protein (Ser240/244), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1, Thr37/46) was significantly reduced by AICAR. These results suggest that AMPK activation suppresses protein synthesis and mTORC1 signaling (through the phosphorylation of S6K1, S6 ribosomal protein, and 4E-BP1) in chick myotubes.

Insulin Stimulation of Protein Synthesis and mTOR Signaling in Chick Myotube Cultures.

Insulin stimulates protein synthesis in skeletal muscles. Protein synthesis is controlled by the mechanistic target of rapamycin (mTOR) signaling in skeletal muscles. This study was conducted to investigate the effect of insulin on protein synthesis and mTOR signaling in chick myotube cultures. Chick myotubes were incubated with insulin (1 µg/ml) for 1 h. Protein synthesis, measured using the surface sensing of translation method, was significantly increased by insulin. The phosphorylation of AKT (Thr308 and Ser473), p70 ribosomal S6 kinase 1 (S6K1, Thr389), S6 ribosomal protein (Ser235/236), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1, Thr37/46) was also significantly increased by insulin. These results suggest that insulin stimulates protein synthesis via mTOR signaling (phosphorylation of AKT, S6K1, S6 ribosomal protein, and 4E-BP1) in chick myotube cultures.

Regulation of Autophagy in Chick Skeletal Muscle: Effect of mTOR Inhibition.

Autophagy in the skeletal muscle increases under catabolic conditions resulting in muscle atrophy. This study investigated the effect of inhibition of mechanistic target of rapamycin (mTOR) on autophagy in chick skeletal muscle. We examined the effects of Torin1, an mTOR inhibitor, on autophagy. Chick myotubes were incubated with Torin1 (100 nM) for 3 h. It was observed that Torin1 inhibited the phosphorylation of AKT (Ser473), p70 ribosomal S6 kinase 1 (S6K1, Thr389), S6 ribosomal protein (Ser235/236), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1, Thr37/46), which are used for measurement of mTOR activity. Torin1 significantly (P< 0.01) increased the LC3-II/LC3-I ratio, an index for autophagosome formation, while it did not influence the expression of autophagy-related genes (LC3B, GABARAPL1, and ATG12). In addition, Torin1 increased atrogin-1/MAFbx (a muscle-specific ubiquitin ligase) mRNA expression. Fasting for 24 h inhibited the phosphorylation of AKT (Ser473), S6K1 (Ther389), S6 ribosomal protein (Ser235/236), and 4E-BP1 (Thr37/46) in chick skeletal muscle and significantly (P<0.01) increased the LC3-II/LC3-I ratio. Fasting also increased GABARAPL1 and atrogin-1/MAFbx mRNA expression but not LC3B or ATG12 mRNA expression. These results indicate that mTOR signaling regulates autophagy and the ubiquitin-proteasome proteolytic pathway in chick skeletal muscle.

Effects of dietary lysine levels and lighting conditions on intramuscular fat accumulation in growing pigs.

This study was conducted to test our hypothesis that intramuscular fat (IMF) accumulation increases in pigs fed on a low lysine diet during the dark period than those fed on the same diet during the light period. Using barrows aged 6 weeks, we monitored whether serum glucose and insulin levels were affected by light conditions. Two diets with different levels of lysine, 0.78% (LL diet) and 1.37% (control diet) were prepared. Eight pigs were fed on the diet during the light period, while the remaining pigs were fed during the dark period. The pigs were fed either the LL diet or the control diet. Although IMF contents of Longissimus dorsi (LD) muscle were higher in the pigs fed on a LL diet (p < .05), the light conditions had no effect. Low dietary lysine caused reduction in serum glucose levels (p < .05) and serum insulin levels (p = .0613). However, they were also unaffected by the lighting conditions. To gain further insights, we determined the messenger RNA levels of insulin receptor, insulin receptor substrate 1, acetyl CoA carboxylase, and fatty acid synthase in LD and Rhomboideus muscles and in the liver.

Regulation of Autophagy in Chick Myotubes: Effects of Insulin, Insulin-Like Growth Factor-I, and Amino Acids.

Autophagy, an intracellular bulk protein degradation system in skeletal muscle, is increased under catabolic conditions resulting in muscle atrophy. This study aimed to investigate the effects of insulin, insulin-like growth factor (IGF)-I, and amino acids on autophagy (LC3-II content and expression of autophagy-related genes) in chick myotubes. Chick myotubes were incubated with insulin (1 µg/ml), IGF-I (100 ng/ml), and amino acids for 3 h. The LC3-II content, an index of autophagosome formation, and mRNA expression of LC3B and GABARAPL1 were significantly decreased by insulin. The LC3-II content, but not mRNA expression of autophagy-related genes, was also significantly decreased by IGF-I. The LC3-II content and LC3B mRNA level were also significantly decreased by amino acids. The mRNA expression of atrogin-1/MAFbx, a muscle-specific ubiquitin ligase, was also significantly decreased by insulin, IGF-I, and amino acids in chick myotubes. These results indicated that insulin, IGF-I, and amino acids regulate autophagy as well as the ubiquitin-proteasome proteolytic pathway in chick myotubes.

Expression of cationic amino acid transporters in pig skeletal muscles during postnatal development.

The cationic amino acid transporter (CAT) protein family transports lysine and arginine in cellular amino acid pools. We hypothesized that CAT expression changes in pig skeletal muscles during rapid pig postnatal development. We aimed to investigate the tissue distribution and changes in the ontogenic expression of CATs in pig skeletal muscles during postnatal development. Six piglets at 1, 12, 26, 45, and 75 days old were selected from six litters, and their longissimus dorsi (LD), biceps femoris (BF), and rhomboideus (RH) muscles, and their stomach, duodenum, jejunum, ileum, colon, liver, kidney, heart, and cerebrum were collected. CAT-1 was expressed in all the 12 tissues investigated. CAT-2 (CAT-2A isoform) expression was highest in the skeletal muscle and liver and lowest in the jejunum, ileum, kidney, and heart. CAT-3 was expressed mainly in the colon and detected in the jejunum, ileum, and cerebrum. The CAT-1 expression was higher in the skeletal muscle of day 1 pigs than in that of older pigs (P < 0.05). The CAT-2 mRNA level was lowest at day 1, but increased with postnatal development (P < 0.05). There was no significant change in CAT-1 expression among the LD, BF, and RH during postnatal development (P > 0.05); however, there was a change in CAT-2 expression. The CAT-2 expression was highest in the LD of 12-, 26-, 45-, and 75-day-old pigs, followed by the BF and RH (P < 0.05). These results suggest that CAT-1 and CAT-2 play different roles in pig skeletal muscles during postnatal development.

Effects of Insulin-Like Growth Factor-I on the Expression of Atrogin-1/MAFbx in Chick Myotube Cultures.

The expression of atrogin-1/MAFbx, a muscle-specific E3 ubiquitin ligase, is increased in catabolic conditions that result in muscle atrophy. The expression of atrogin-1/MAFbx mRNA is also decreased by the insulin-like growth factor-I (IGF-I) in mammalian skeletal muscle cell cultures. This study investigated the effect of IGF-I on the expression of atrogin-1/MAFbx in chicken skeletal muscle cell cultures. Chick myotubes were incubated with IGF-I for 1, 6, or 24 h. Protein content was increased by IGF-I (100 ng/ml) and incubated for 24 h in chick myotubes. The expression of atrogin-1/MAFbx mRNA decreased in the presence of IGF-I (1, 10, and 100 ng/ml) for 6 h in chick myotubes. The expression of the m-calpain large subunit and cathepsin B mRNA was not decreased by IGF-I. Phosphorylation of Akt and FOXO1 increased in the presence of IGF-I (100 ng/ml) for 1 h in chick myotubes. These results indicate that IGF-I suppresses atrogin-1/MAFbx mRNA expression by phosphorylation of Akt and FOXO1, resulting in an increase in muscle growth in chick myotube cultures.

Changes in muscle fiber type and expression of mRNA of myosin heavy chain isoforms in porcine muscle during pre- and postnatal development.

The purpose of this study is to elucidate developmental changes in muscle fiber type in the pig during pre- and postnatal development. For this purpose, we performed a histochemical analysis for myosin adenosine triphosphatase activity to assess muscle fiber type and determined abundances of messenger RNA (mRNA) of myosin heavy chain (MHC) isoforms. Samples of Longissimus dorsi (LD) muscle were taken from fetuses on day 90 of the fetal stage. Further, samples of LD, Rhomboideus and Biceps femoris (B. femoris) muscles were taken from pigs when they were 1, 12, 26, 45 or 75 days old. Expression of MHC 2b mRNA in the LD and the B. femoris muscles rapidly and considerably increased from the late fetal stage to the early postnatal stage and this increase was associated with the development of type 2b fibers at least in the LD muscle. As shown by the rapid and considerable changes in expression of MHC 2b mRNA, it seems that a certain plasticity of muscle fiber type still remains in this developmental stage.

Effect of dexamethasone on the expression of atrogin-1/MAFbx in chick skeletal muscle.

Expression of atrogin-1/MAFbx, a muscle-specific E3 ubiquitin ligase, is high under catabolic conditions, that result in muscle atrophy. Messenger RNA (mRNA) expression of atrogin-1/MAFbx is increased by the glucocorticoid dexamethasone in mammalian skeletal muscle. This study investigated the effects of dexamethasone on expression of atrogin-1/MAFbx in skeletal muscle of neonatal chicks and in chick myotubes. Chicks were given a single intraperitoneal injection of dexamethasone at a concentration of 10 mg/kg body weight. Twenty-four hours after dexamethasone administration, the Pectoralis muscle weight of chicks was decreased. mRNA expression of atrogin-1/MAFbx in skeletal muscle of chicks was significantly increased by dexamethasone administration. Expression of other proteolytic-related genes (20S proteasome C2 subunit, m-calpain large subunit, and cathepsin B) in skeletal muscle of chicks was not increased by dexamethasone administration. Chick myotubes were incubated with dexamethasone (1, 10 or 100 µmol/L) for 6 h. Expression of atrogin-1/MAFbx mRNA in chick myotubes was increased in the presence of all concentrations of dexamethasone. However, expression of other proteolytic-related genes (20S proteasome C2 subunit, m-calpain large subunit and cathepsin B) in chick myotubes was not affected by dexamethasone treatment. These results indicate that dexamethasone enhances atrogin-1/MAFbx expression in chick skeletal muscle, resulting in increased muscle atrophy.

Comparison of atrogin-1/MAFbx mRNA expression in the gizzards of egg- and meat-type chickens.

We examined atrogin-1/MAFbx mRNA expression in the smooth muscle of gizzards from egg- and meat-type chickens. Gizzard weight relative to body weight was significantly lower in the meat-type chickens than in the egg-type at 14 d of age. In contrast, the level of atrogin-1/MAFbx mRNA in the gizzard was significantly higher in the meat-type chickens than in the egg-type chickens. Thus atrogin-1/MAFbx mRNA expression in the smooth muscle of the gizzard was higher in meat-type chickens than in egg-type chickens, in contrast to its expression in the skeletal muscles.

Compensatory growth of C2C12 myotubes induced by the combined effect of lysine sufficiency and modulation of IGF-I and glucocorticoid levels.

We have reported that a change from a lysine-deficient diet to a lysine-sufficient diet induced compensatory growth in rats and pigs. The aim of the present study was to determine whether compensatory growth of C2C12 myotubes occurs only by sufficiency of lysine or also by the synergic effect of sufficiency of lysine and modulation of the levels of insulin-like growth factor-I (IGF-I) and glucocorticoid in a medium. The results provide the first evidence of compensatory growth of C2C12 myotubes induced by sufficiency of a single amino acid in combination with modulation of the levels of IGF-I and glucocorticoid.

Atrogin-1/MAFbx, a muscle-specific ubiquitin ligase, is highly expressed in the smooth muscle of the chicken gizzard.

This study was conducted to investigate the expression of atrogin-1/MAFbx, a muscle-specific ubiquitin ligase, in the smooth muscle of the chicken gizzard. Atrogin-1/MAFbx mRNA expression was detected in the skeletal muscle, heart (cardiac muscle), gizzard (smooth muscle), brain, and liver of chicks, with highest expression in the smooth muscle of the gizzard. The expression of atrogin-1/MAFbx mRNA in the smooth muscle of the gizzard was increased by fasting (24 h), and this increase was reduced by refeeding (2 h). These results indicate that atrogin-1/MAFbx mRNA is highly expressed in the smooth muscle of the chicken gizzard, and that the expression of it is regulated by nutritional conditions.

Effects of low protein diet and low protein diet supplemented with synthetic essential amino acids on meat quality of broiler chickens.

We investigated the effects of a low crude protein (CP) diet and a low CP diet supplemented with synthetic essential amino acids (EAA) on the meat quality of broiler chickens. Twenty-one-day-old chickens were assigned to one of three diets: control, low CP (LCP), or low CP supplemented with EAA (ELCP). The chickens received these diets for 10 days. The shear force value (SFV) and free glutamate content of the Pectoralis major muscle were measured as indicators of the meat toughness and taste. The collagen and crude fat content of the muscle and the cross-sectional area of myofibers were measured to evaluate the effects of the LCP and ELCP diets on meat toughness. The SFV of the ELCP group was 47% lower than that of the control group (P<0.01). However, the LCP diet did not affect the SFV. The collagen and crude fat content were not affected by the dietary treatment. The cross-sectional area was lower in the LCP and ELCP groups (P<0.05) than the control group. The free glutamate content of muscle was not affected by the dietary treatment. Thus, a low CP diet supplemented with EAA is an effective means of producing tender meat.

Effects of dietary low level of threonine and lysine on the accumulation of intramuscular fat in porcine muscle.

We attempted to determine whether shortage of dietary threonine enhances the accumulation of intramuscular fat (IMF) in porcine muscle. Although dietary low levels of lysine enhanced the concentration of IMF in the longissimus dorsi and rhomboideus muscles, dietary low threonine did not. We conclude that the effect of dietary threonine levels on the accumulation of IMF in porcine muscle is negligible.

Nitrogen balance during compensatory growth when changing the levels of dietary lysine from deficiency to sufficiency in growing pigs.

Two experiments were conducted to elucidate the nitrogen (N) balance of pigs exhibiting compensatory growth when changing the dietary lysine levels from deficiency to sufficiency. Experiment 1 elucidated whether pigs exhibited compensatory growth with dietary lysine sufficiency. Twenty 6-week-old males were assigned to one of two treatments: control and LC (lysine and control). Control pigs were fed a control diet throughout the 24-day experimental period, whereas LC pigs were fed a low lysine diet until day 21 of the experiment, followed by the control diet until the end of experiment. The dietary lysine sufficiency treatment induced an 80% increase in the growth rate of LC pigs (P < 0.05). Experiment 2 focused on the N balance of pigs that exhibited compensatory growth with dietary lysine sufficiency. Eighteen 6-week-old males were assigned to one of three treatments: control, LC, and LL (low lysine). LL pigs were fed a low lysine diet throughout the 24-day experimental period. Pigs that exhibited compensatory growth with dietary lysine sufficiency tended to retain a higher amount of N than control pigs (P = 0.10). These finding suggest that the compensatory growth induced in pigs by dietary lysine sufficiency was partly attributable to a higher level of N retention.

The effects of concentrations of lysine in media on differentiation of 3T3-L1 preadipocytes.

Intramuscular fat content is increased by feeding of low lysine diets in pigs. Reduction in dietary lysine intake results in low plasma lysine concentration and low cytosolic lysine concentration in skeletal muscles. From these observations, we hypothesized that low plasma lysine concentration in pigs fed on low lysine diets reduced supply of lysine from blood circulation to preadipocytes, and this limited supply of lysine might promote adipocyte differentiation in porcine muscles. In order to verify the hypothesis, we investigated the effects of low concentrations of lysine in culture medium on differentiation of 3T3-L1 preadipocytes. Low concentration of lysine suppressed lipid accumulation and messenger RNA (mRNA) expression and enzyme activity of fatty acid synthase. mRNA expressions of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) were lower in cells cultured in low lysine medium. On the other hand, mRNA and protein expressions of C/EBPß and C/EBPδ were not inhibited by low concentrations of lysine in culture medium. These results indicate that low lysine concentrations in culture medium inhibit differentiation of 3T3-L1 preadipocytes through inhibiting the mRNA expressions of PPARγ and C/EBPα.

Muscle protein metabolism during compensatory growth with changing dietary lysine levels from deficient to sufficient in growing rats.

Livestock and laboratory animals show compensatory growth when they are fed ad libitum following a period of restriction feeding. Lysine is a major limiting essential amino acid in the diets both for humans and animals. We hypothesized that changing dietary lysine levels from deficient to sufficient induced compensatory growth in young rats. We elucidated the effect of lysine sufficiency on the dynamics of hormones, relevant to muscle protein synthesis and degradation, insulin-like growth factor-I (IGF-I) and corticosterone, and on the expression of proteolytic-related genes in skeletal muscle during compensatory growth. Lysine sufficiency where the dietary lysine level was increased from 0.46% to 1.30% after 2 wk of subjecting the rats to the lower lysine level induced 80% enhancement of growth rate of rats. During compensatory growth with the lysine sufficiency, fractional muscle protein synthesis rates were higher whereas fractional muscle protein degradation rates were lower than those of the control group (p<0.05). After lysine sufficiency, the expression of atrogin-1/MAFbx mRNA was decreased in gastrocnemius muscle (p<0.05). With the lysine sufficiency, serum IGF-I concentration increased (p<0.05) whereas serum corticosterone decreased (p<0.05). These findings suggest that compensatory growth with lysine sufficiency is due to a change of hormone levels before and after changing diets, resulting in incrementation of protein synthesis and suppression of protein degradation of skeletal muscle.

Comparison of proteolytic-related gene expression in the skeletal muscles of layer and broiler chickens.

Previous studies have shown that the muscle protein degradation rates of broiler are lower than those of layer chickens. In this study, we assessed proteolytic-related gene expression in the pectoralis muscle of layer and broiler chickens. The mRNA levels of atrogin-1/MAFbx and proteasome C2 subunit, but not those of ubiquitin, m-calpain large subunit, cathepsin B, or caspase-3, were lower in the skeletal muscle of the broilers than in the layers at 7 and 14 d of age. We infer that the lower muscle protein degradation of broilers than of layers at least partly relates to lower mRNA expression of atrogin-1/MAFbx and proteasome C2 subunit in the skeletal muscle of broilers.