Effects of short-term fasting on the Akt-mediated pathway involved in protein metabolism in chicken skeletal muscle.

In the present study, we show that short-term (4 h) fasting significantly decreased the levels of protein synthesis-related factors such as the plasma insulin concentration, skeletal muscle pAkt, and pS6 levels in 2-wk-old chickens (P < 0.05). An intravenous injection of insulin significantly elevated the contents of pAkt and p-S6 in the skeletal muscle (P < 0.01). These findings suggest that decreasing the plasma insulin causes the downregulation of the Akt/S6 pathway in chicken skeletal muscle under short-term fasting conditions. However, protein synthesis was not significantly affected by short-term fasting. In addition, no significant change was observed in the levels of proteolysis-related factors such as plasma Nτ-methylhistidine, phosphorylated forkhead box class O, and muscle ring finger-1 during 4-h fasting, indicating that short-term fasting does not induce skeletal muscle proteolysis in chickens. Interestingly, atrogin-1 expression significantly increased after 2-h fasting (P < 0.05), and insulin injection significantly reversed the fasting-induced atrogin-1 expression in chicken skeletal muscle (P < 0.01). Collectively, these findings suggest that short-term fasting downregulates the insulin-stimulated Akt/S6 pathway but does not significantly affect protein synthesis and proteolysis in chicken skeletal muscle, and that atrogin-1 expression is upregulated in a FOXO1-independent manners.

Effects of fasting and refeeding on structures of the intestinal villi and epithelial cells in White Leghorn hens.

1. The fine structural alterations of villi and epithelial cells in each part of the small intestine were investigated in layer-type hens fasted for 12 h to 20 d or refed for one day after each fasting period. 2. Within the first 24-h-fasting, villi of the duodenum showed a remarkable reduction in height and those of the jejunum revealed a gradual decrease; such a significant reduction of the villus height was not obtained in the ileum. After 36-h-fasting, villus height in each part gradually decreased with days of fasting. 3. All intestinal villus heights increased after only 1-d-refeeding of various kinds of diets following 3-, 10-, or 20-d-fasting. The duodenum especially rapidly recovered even after long-term fasting of 20 d but the ileum showed very slow recovery, suggesting that the ileum seems to be inactive in absorptive function. 4. These variable alterations of villus height in the proximal intestine suggest that the higher intestinal absorptive ability is under the normal feeding, the more rapidly villus height is influenced by nutritional conditions. 5. Cell area and cell mitosis decreased after fasting, the latter showing a marked reduction. However, in spite of a remarkable decrease of cell mitosis in the proximal intestine after fasting, refeeding activated cell renewal and it soon reached control levels, demonstrating that the villus height mainly varied with the numbers of epithelial cells. 6. In the epithelial cells of the proximal intestine in chickens fasted for 20 d, large lysosomal autophagous vacuoles including mitochondria and dense bodies were observed. These were reduced in size by refeeding for only one day, suggesting that fasting may cause intracellular digestion through lysosomal autophagy. 7. These results lead to the conclusion that long-term for force moulting is possible, that a high protein and high energy diet can be fed immediately after fasting and that a cell undergoing lysosomal autophagy in normal chickens indicates undernutrition.

Function of the NH2-terminal domain of the regulatory light chain on the regulation of smooth muscle myosin.

The role of the NH2-terminal domain of the 20,000-dalton light chain on the regulatory function of smooth muscle myosin was studied by exchanging it in myosin with various mutant forms. The 10 S to 6 S conformational transition as well as the thick filament formation were significantly influenced by the deletion or substitution of the amino acid residues at the NH2-terminal side of the phosphorylation site (Ser19). Whereas the deletion of Ser1-Thr10 did not significantly affect these functions, further deletion of Lys11-Arg16 completely abolished the formation of 10 S conformation and induced thick filament formation. Among the residues in this region, Arg13 and Arg16 were most important for these functions since substitution of these residues by Glu or Ala significantly altered these functions. Similar substitutions of Lys11 and Lys12 also stabilized the 6 S conformation and thick filament formation but less effectively. While the 6 S conformation was stabilized, the deletion of NH2-terminal residues did not activate the actin-activated ATPase activity. This suggests that stabilization of the 6 S conformation is not directly coupled with activation of actomyosin ATPase activity but rather a more defined conformational change around the phosphorylation site is necessary for activation. Such a change also influences the 6 S to 10 S conformation and, therefore, the filament formation. To support this notion, substitution of Lys11 and Lys12 by Glu-Glu inhibited the phosphorylation-induced activation of actomyosin ATPase activity.

Involvement of the C-terminal residues of the 20,000-dalton light chain of myosin on the regulation of smooth muscle actomyosin.

The segment of smooth muscle regulatory light chain essential for the phosphorylation dependent activation of actomyosin motor activity and the binding of myosin heavy chain was identified. The C-terminal domain of the 20-kDa light chain, which is less conserved than the rest of the polypeptide among various muscle types, was mutated by either deletion or substitution of amino acid residues and the mutant light chains were then incorporated into myosin by subunit exchange. Deletion of Lys149-Ala166 markedly reduced the affinity of the light chain for the heavy chain, whereas the C-terminal five residues, Lys167-Asp171, did not contribute to the binding affinity. Deletion of Lys149-Phe158 abolished the phosphorylation-dependent activation of actomyosin ATPase activity as well as superprecipitation activity. These results suggest that the C-terminal domain of the regulatory light chain is critical for transmitting the change in the conformation of the regulatory light chain induced by phosphorylation at Ser19 to the heavy chain.

Mutagenesis of the phosphorylation site (serine 19) of smooth muscle myosin regulatory light chain and its effects on the properties of myosin.

A full-length cDNA of smooth muscle regulatory light chain was obtained and the recombinant regulatory light chain was expressed in an Escherichia coli expression system. The recombinant regulatory light chain was introduced into myosin or HMM using a subunit exchange strategy [Morita, J., Takashi, R., & Ikebe, M. (1991) Biochemistry 30, 9539-9545]. The recombinant wild-type regulatory light chain exhibited the same biological properties as the natural isolate, i.e., phosphorylation at Ser-19 by myosin light-chain kinase and phosphorylation-activated actomyosin ATPase activity. To clarify whether or not the activation of the ATPase by phosphorylation is simply due to the introduction of negative charge, we produced three mutant light chains. Two of them contain Ser-19 substituted by either Asp or Ala and the third contains Asp substituted for both Thr-18 and Ser-19. Incorporation of the Asp mutant partially activated actomyosin ATPase activity but the activation level was significantly lower than that by phosphorylation. The Asp/Asp mutant further activated actomyosin ATPase activity. On the other hand, the Ala mutant did not affect the ATPase activity. Incorporation of Asp mutant slightly affected the 10S-6S conformational transition and filament formation of myosin. The Asp/Asp mutant more significantly affected the 10S-6S conformational transition and filament formation of myosin. These results suggested that the activation of smooth muscle myosin requires the introduction of negative charge in the defined spacial position. Using Ser-19 deficient mutants, the effects of Thr-18 phosphorylation on myosin function was also studied. Actin-activated ATPase activity of myosin was significantly activated by phosphorylation of Thr-18.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of electrical stimulation on the rheological properties of rabbit skeletal muscle.

The effect of electrical stimulation on the rheological properties of rabbit skeletal muscle after death was investigated. The extensibility of electrically stimulated psoas muscles decreased more rapidly than that of non-stimulated muscles. For raw non-stimulated longissimus thoracis muscles excised from the carcasses immediately after slaughter, the penetration force required was greatest 24 h after slaughter and then decreased slightly after 168 h. The corresponding force for stimulated longissimus thoracis muscles increased to a maximum in 12 h and then decreased to values less than non-stimulated muscles. However, in the case of raw longissimus thoracis muscles whichhad been attached to the skeleton until measurement, there was no significant difference in penetration force between stimulated and non-stimulated muscles. In cooked muscles, electrical stimulation resulted in lower penetration forces at 24 h post mortem, but on further storage the differences decreased.