Aiko Ishida

Suppression of myofibrillar proteolysis in chick skeletal muscles by α-ketoisocaproate

Summary.We previously reported that L-leucine suppresses myofibrillar proteolysis in chick skeletal muscles. In the current study, we compared the effects of L- and D-enantiomers of leucine on myofibrillar proteolysis in skeletal muscle of chicks. We also assessed whether leucine itself or its metabolite, α-ketoisocaproate (α-KIC), mediates the effects of leucine. Food-deprived (24 h) chicks were orally administered 225 mg/100 g body weight L-leucine, D-leucine or α-KIC and were sacrificed after 2 h. L-Leucine administration had an obvious inhibitory effect on myofibrillar proteolysis (plasma Nτ-methylhistidine concentration) in chicks while D-leucine and α-KIC were much more effective. We also examined the expression of the proteolytic-related genes (ubiquitin, proteasome, m-calpain and cathepsin B) by real-time PCR of cDNA in chick skeletal muscles. Ubiquitin mRNA expression was decreased by D-leucine and α-KIC but not L-leucine. Proteasome and m-calpain mRNA expressions as well as cathepsin B mRNA expression were likewise decreased by L-leucine, D-leucine and α-KIC. These results indicate that D-leucine and α-KIC suppress proteolytic-related genes, resulting in an decrease in myofibrillar proteolysis while L-leucine is much less effective in skeletal muscle of chicks, may be explain by conversion of D-leucine to α-KIC.

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.

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 orally administered glycine on myofibrillar proteolysis and expression of proteolytic-related genes of skeletal muscle in chicks

Summary.We examined the effects of orally administered glycine on myofibrillar proteolysis in food-deprived chicks. Food-deprived (24 h) chicks were orally administered 57, 113, and 225 mg glycine/100 g body weight and killed after 2 h. The plasma Nτ-methylhistidine concentration, used as myofibrillar proteolysis, was decreased by glycine. We also examined the expression of proteolytic-related genes by real-time PCR of cDNA from chick skeletal muscles. The mRNA expression of atrogin-1/MAFbx, proteasome C2 subunit, m-calpain large subunit, and cathepsin B was decreased by glycine in a dose-dependent manner. The plasma corticosterone concentration was also decreased by glycine, but the plasma insulin concentration was unaffected. These results indicate that orally administered glycine suppresses myofibrillar proteolysis and expression of proteolytic-related genes of skeletal muscle by decreasing the plasma corticosterone concentration in chicks.

Effects of Orally Administrated Amino Acids on Myofibrillar Proteolysis in Chicks

We examined the effects of orally administrated amino acids on myfibrillar proteolysis in food-deprived chicks. Plasma Nτ-methylhistidine concentration, as an index of myofibrillar proteolysis, was decreased by the administration of Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg but not by Asp, Val, Phe, Tyr or His to chicks. Orally administrated Cys was fatal to chicks. These results indicate that oral Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg administration suppressed myofibrillar proteolysis in chicks.

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.

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/EBPβ 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.

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.

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.

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.