Saki Shimamoto

The effects of intraperitoneal clenbuterol injection on protein degradation and myostatin expression differ between the sartorius and pectoral muscles of neonatal chicks

The purpose of this study was to investigate the effects of injection of the β2-adrenergic receptor agonist clenbuterol on the skeletal muscles of neonatal chicks (Gallus gallus domesticus). One-day-old chicks were randomly divided into four groups and given a single intraperitoneal injection of clenbuterol (0.01, 0.1, or 1mg/kg) or phosphate-buffered saline. Twenty-four hours after the injection, the sartorius muscles (which consist of both slow- and fast-twitch fibers) of chicks that received 0.01 or 0.1mg/kg clenbuterol were significantly heavier than those of controls, while there were no between-group differences in the weight of the pectoral muscles, which consist of only fast-twitch fibers. Muscle free N(t)-methylhistidine, regarded as an index of myofibrillar proteolysis, was decreased in the sartorius muscle of the clenbuterol-injected chicks, while it was not affected in the pectoral muscles. In the sartorius muscle of the clenbuterol-injected chicks, myostatin and atrogin-1/MAFbx mRNA expressions were decreased, while insulin-like growth factor-I was unaffected. These observations suggested, in 1-day-old chicks, clenbuterol might increase mass of the sartorius muscle by decreasing myostatin gene expression and protein degradation.

Clenbuterol changes phosphorylated FOXO1 localization and decreases protein degradation in the sartorius muscle of neonatal chicks

To investigate the intracellular signaling mechanisms by which clenbuterol reduces muscle protein degradation, we examined the phosphorylation level and intracellular localization of FOXO1 in the sartorius muscle of neonatal chicks. One-day-old chicks were given a single intraperitoneal injection of clenbuterol (0.1 mg/kg body weight). Three hours after injection, AKT protein was phosphorylated in the sartorius muscle by clenbuterol injection. Coincidentally, clenbuterol increased cytosolic level of phosphorylated FOXO1 protein, while it decreased nuclear level of FOXO1 protein in the sartorius muscle. Furthermore, clenbuterol decreased the expression of mRNAs for muscle-specific ubiquitin ligases (atrogin-1/MAFbx and MuRF1) in the sartorius muscle accompanied by decreased plasma 3-methylhistidine concentration, an index of muscle protein degradation, at 3 h after injection. These results suggested that, in the sartorius muscle of the chicks, clenbuterol changed the intracellular localization of phosphorylated FOXO1, and consequently decreased protein degradation via suppressing the expression of genes encoding muscle-specific ubiquitin ligases. Graphical abstract

Single injection of the β2-adrenergic receptor agonist, clenbuterol, into newly hatched chicks alters abdominal fat pad mass in growing birds.

Excessive energy is stored in white adipose tissue as triacylglycerols in birds as well as in mammals. Although β2-adrenergic receptor agonists reduce adipose tissue mass in birds, the underlying mechanism remains unclear. The aim of the current study was to examine the effects of a single intraperitoneal injection of the β2-adrenergic receptor agonist, clenbuterol, on the abdominal fat pad tissue development. Thirty-three chicks at 1-day-old were given a single intraperitoneal injection of clenbuterol (0.1mg/kg body weight) or phosphate-buffered saline. At 2 weeks post-dose, the weight of the abdominal fat tissue was decreased in the clenbuterol-injected chicks, and small adipocyte-like cells were observed in the abdominal fat pad tissue of the clenbuterol-injected chicks. Then, the expression of mRNAs encoding genes related to avian adipogenesis was examined in the abdominal fat pat tissue. The expression of mRNAs encoding Krüppel-like zinc finger transcription factor 5 (KLF-5), KLF-15, and zinc finger protein 423 in the abdominal fat pad tissue of the clenbuterol-injected chicks was significantly lower (P<0.05) than that of the control chicks, while the expression of mRNA encoding peroxisome proliferator-activated receptor-gamma was not affected. In addition, both mRNA expression (P<0.05) and enzymatic activity (P<0.05) of fatty acid synthase (FAS) were decreased in the abdominal fat pad tissue of the clenbuterol-injected chicks, while clenbuterol injection did not affect FAS activity in liver. These results suggested that a single injection with clenbuterol into newly hatched chicks reduces their abdominal fat pad mass possibly via disrupting adipocyte development during later growth stages.

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.

β1- and β2-adrenergic receptor stimulation differ in their effects on PGC-1α and atrogin-1/MAFbx gene expression in chick skeletal muscle

Adrenaline changes expression of the genes encoding peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α), which is known as a regulator of muscle size, and atrogin-1/muscle atrophy F-box (MAFbx), which is a muscle-specific ubiquitin ligase. However, the subtype of β-adrenergic receptor (β-AR) involved in regulating these genes in skeletal muscle is not yet well defined. In this study, the effects of intraperitoneal injection of adrenaline and three β1-3-AR selective agonists on chick skeletal muscle metabolism were examined, to evaluate the functions of β-AR subtypes. Adrenaline decreased atrogin-1/MAFbx mRNA levels accompanied by an increase in PGC-1α mRNA and protein levels. However, among the three selective agonists, only the β1-AR agonist, dobutamine, increased PGC-1α mRNA and protein levels, while the β2-AR agonist, clenbuterol, suppressed atrogin-1/MAFbx mRNA levels. In addition, preinjection of the β1-AR antagonist, acebutolol, and the β2-AR antagonist, butoxamine, inhibited the adrenaline-induced increase in PGC-1α mRNA levels and the decrease in atrogin-1/MAFbx mRNA levels, respectively. Compared with adrenaline administration, the β3-AR agonist, BRL37344, decreased PGC-1α mRNA levels and increased atrogin-1/MAFbx mRNA levels. These results suggest that, in chick skeletal muscle, PGC-1α is induced via the β1-AR, while atrogin-1/MAFbx is suppressed via the β2-AR.

Gene expression pattern of glucose transporters in the skeletal muscles of newly hatched chicks

The gene expression pattern of the glucose transporters (GLUT1, GLUT3, GLUT8, and GLUT12) among pectoralis major and minor, biceps femoris, and sartorius muscles from newly hatched chicks was examined. GLUT1 mRNA level was higher in pectoralis major muscle than in the other muscles. Phosphorylated AKT level was also high in the same muscle, suggesting a relationship between AKT and GLUT1 expression.

Single injection of clenbuterol into newly hatched chicks decreases abdominal fat pad weight in growing broiler chickens

The aim of the current study was to examine the effects of clenbuterol injection into newly hatched chicks on both the abdominal fat pad tissue weight and the skeletal muscle weight during subsequent growth. Twenty-seven 1-day-old chicks were divided into two groups, receiving either a single intraperitoneal (i.p.) injection of clenbuterol (0.1 mg/kg body weight) or phosphate-buffered saline (PBS). Body weight gain, feed intake and feed conversion ratio were not affected by clenbuterol injection during the 5-week experimental period, while the abdominal fat pad tissue weight of the clenbuterol-injected chicks was lower than that of the control chicks at 5 weeks post-injection. Plasma non-esterified fatty acid concentrations were significantly increased in the clenbuterol-injected chicks, while plasma triacylglycerol concentrations did not differ. Additionally, the enzymatic activity of fatty acid synthase was lower in the liver of the clenbuterol-injected chicks. Conversely, the skeletal muscle weights were not affected by clenbuterol injection. These results suggest that a single clenbuterol injection into 1-day-old chicks decreases the abdominal fat pad tissue weight, but may not affect skeletal muscle weights during growth.

The β2-adrenergic receptor is involved in differences in the protein degradation level of the pectoral muscle between fast- and slow-growing chicks during the neonatal period

The aim of this study was to investigate whether β2-AR mRNA expression is involved in either atrogin-1/MAFbx mRNA expression or protein degradation in chicken skeletal muscle by comparing fast- and slow-growing chicks during the neonatal period. Based on their body weight gain from 1 to 5 days of age, 5-day-old chicks (Gallus gallus domestics) were divided into a slow-growing and a fast-growing group, the mean weight gains of which were 6.3 ± 1.3 g/day and 11.3 ± 0.9 g/day, respectively. The ratio of pectoral muscle weight to total body weight was higher in the fast-growing group of chicks than in the slow-growing group. In addition, the plasma 3-methylhistidine concentration, an index of protein degradation in skeletal muscle, was significantly lower in the fast-growing than in the slow-growing chicks. The mRNA expression of β2-AR, which we previously found is involved in decreasing muscle protein degradation by suppression atrogin-1/MAFbx mRNA expression, was significantly higher in the pectoral muscle of the fast-growing group compared with that of the slow-growing group. Concordantly, lower mRNA expression of atrogin-1/MAFbx was observed in the pectoral muscle of the fast-growing chicks. However, in the sartorius muscle, which is a muscle in the thigh, the ratio of the muscle weight to total body weight was not significantly different between the two groups of chicks at 5 days of age. In addition, there was no significant difference in the mRNA expressions of β2-AR and atrogin-1/MAFbx in the sartorius muscle between these two groups. These results suggest that β2-AR expression levels might be physiologically significant in the control of protein degradation in the pectoral muscle of neonatal chicks.

Effects of first exogenous nutrients on the mRNA levels of atrogin-1/MAFbx and GLUT1 in the skeletal muscles of newly hatched chicks

The aim of this study was to examine the effects of first exogenous nutrients on the mRNA levels of muscle atrophy F-box (atrogin-1/MAFbx) and glucose transporters (GLUTs) in the skeletal muscles of newly hatched chicks with no feed experience. In experiment 1, newly hatched chicks had free access to feed or were fasted for the first 24h. The chicks having free access to feed for the first 24h increased their body weight and had decreased atrogin-1/MAFbx mRNA levels in their sartorius and pectoralis major muscles compared with the fasted chicks. In experiment 2, newly hatched chicks received a single feed via intubation into the crop. Three hours after intubation, levels of atrogin-1/MAFbx mRNA in the sartorius muscle were decreased whereas the plasma insulin concentration and phosphorylated AKT levels in the sartorius muscle were increased. In addition, the mRNA levels of GLUT1 and GLUT8 were increased in the sartorius muscle after the intubation. However, in the pectoralis major muscle, AKT phosphorylation and levels of atrogin-1/MAFbx, GLUT1 and GLUT8 mRNA were not affected 3h after intubation. The first exogenous nutrients increased the level of phosphorylated AKT in the sartorius muscle of newly hatched chicks, possibly because of the decrease in atrogin-1/MAFbx mRNA levels. Furthermore, the sartorius muscle in newly hatched chicks appeared to be more susceptible to the first feed compared with the pectoralis major muscle.