Much is known about the effects of long-term changes in dietary intake on protein metabolism, but less is known about the acute response to meals. Rates of protein synthesis in tissues of growing mice are highly sensitive to the immediate intake of food (Funabiki et al. 1986), and muscle protein synthesis in rodents is particularly sensitive to food intake (Preedy and Garlick 1986, Yoshizawa et al. 1995a); however, it is not clear whether changes in muscle protein breakdown occur in vivo in response to food intake. The study of polysome profiles offers a rapid and sensitive index of the state of protein synthesis in individual tissues (Kikuchi et al. 1986). Therefore, the activity of protein synthesis can be evaluated by determining polysome profiles. In contrast, there are no studies on postprandial changes in myofibrillar protein degradation, because no methods exist to detect acute changes in this process. Urinary N-methylhistidine (3-methylhistidine; MeHis) is as an index of myofibrillar protein degradation because MeHis is localized in myofibrillar protein and is not reused for protein synthesis (Young and Munro 1978). However, measurement of urinary excretion of MeHis does not detect acute changes because the minimum collection unit of urinary MeHis is usually 1 d. We have shown that plasma MeHis concentration is also an index of myofibrillar protein degradation (Nagasawa et al. 1996). We suggested that plasma MeHis concentration could reflect acute changes in myofibrillar degradation (Nagasawa et al. 1996). In the present study, we have used plasma MeHis concentration to estimate acute changes in myofibrillar protein degradation after refeeding.

With the onset of refeeding, the serum concentrations of substrates such as glucose and amino acids and the circulating triiodothyronine (T₃) levels increase in rats (Jepson et al. 1988). These factors and others have been implicated in the regulation of protein metabolism. It is clear from the literature concerning both in vivo and in vitro studies that the insulin concentration strongly influences muscle protein metabolism (Baillie and Garlick 1992, Fuller and Sugden 1986, Garlick et al. 1983 and 1988, Kadowaki et al. 1985, Nagasawa et al. 1982, Sugden and Fuller 1991); thus we also measured the concentration of plasma insulin to investigate the role of insulin in mediating the immediate response of protein metabolism to food intake.

MATERIALS AND METHODS

Table 1. Composition of basal diet [View Table]

The muscle polysomes were suspended in buffer B (buffer A without 2-mercaptoethanol), and the suspensions, containing 2.0 A260 units, were analyzed on a linear 0.5-1.5 mol/L sucrose gradient containing buffer B. Centrifugation was carried out in a Hitachi RPS-40T rotor (Hitachi Koki) at 176,000 × g for 90 min. The absorbance at 254 nm was continuously monitored. The areas under the curves for polysomes larger than dimers and for monomers and dimers were measured. The percentage of ribosomes in the form of polysomes was defined as: [polysome area/(polysome area + monomer and dimer area)] × 100.

RESULTS

Postprandial plasma MeHis concentration was the highest in mice subjected to 18 h of food deprivation (Fig. 3A). It immediately decreased to 0.5 µmol/L during refeeding and then remained at an even lower level until 6 h after refeeding. Plasma MeHis concentration increased to the prefeeding level by 12 h after refeeding.

The concentration of free MeHis in muscle was decreased by refeeding, but it returned to the food-deprived level by 6 h after refeeding (Fig. 3B). The magnitude of the change in muscle MeHis concentration was much smaller than that in plasma.

DISCUSSION

The rate of protein synthesis in muscle is acutely sensitive to the intake of food. In mice starved for 18 h, protein synthesis in muscle was stimulated by refeeding a complete diet for 1 h (Yoshizawa et al. 1995a). This increase in muscle protein synthesis in the early phase after refeeding was at the translational level (Yoshizawa et al. 1995a), because the size spectrum of polysomes, which is a useful indicator of translational activities (Monier and Le Marchand-Brustel 1982), shifted to heavier polysomes. In the present study, the proportion of heavier polysomes was increased by refeeding and was reduced to the basal level by 6 h after refeeding (Fig. 2A). We therefore concluded that protein synthesis in muscle was very rapidly stimulated and then reduced in response to food intake.

A number of studies have demonstrated that insulin was the key factor mediating the response of muscle protein synthesis to feeding and food deprivation (Baillie and Garlick 1992, Garlick et al. 1983, Garlick and Grant 1988, Preedy and Garlick 1986). Insulin has been reported to increase muscle protein synthesis exclusively by enhancing initiation (Monier and Le Marchand-Brustel 1982). In isolated soleus muscle of mice, the rate of peptide-chain elongation increased in the presence of insulin (Yoshizawa et al. 1995b). In the present study, both the proportion of heavier polysomes in muscle (Fig. 2A) and plasma insulin concentration (Fig. 2B) reached a maximum level within 1 h and returned to their basal levels at approximately the same time after refeeding commenced. This relationship suggests that insulin may play a role in mediating the acute stimulation of muscle protein synthesis by food intake.

We have shown that a change in plasma MeHis concentration represents a change in degradation of myofibrillar protein and have demonstrated the possibility of evaluating an acute change in myofibrillar protein degradation even after a short period (Nagasawa et al. 1996). However, if the decreased plasma MeHis concentration is due to an increased free MeHis pool in the muscle cells, plasma MeHis concentration is not a true index of myofibrillar protein degradation after refeeding. Because the change in the free MeHis concentration in muscle (size of MeHis pool in muscle cells, Fig. 3B) was similar to that in plasma (Fig. 3A), it is likely that the plasma MeHis concentration can be used as an index of myofibrillar protein degradation during the short time periods used in this study.

Plasma MeHis concentration was decreased immediately after refeeding (Fig. 3A). Because lower MeHis concentration in plasma was indicative of a slower rate of myofibrillar protein degradation (Nagasawa et al. 1996), the present observation strongly suggests that refeeding depresses myofibrillar protein degradation. It has been shown that food deprivation increases myofibrillar protein degradation (Kadowaki et al. 1985 and 1989, Li and Wassner 1984, Lowell et al. 1986), but its acute effects on protein degradation after food deprivation are unclear. We found that the change in myofibrillar protein degradation was very rapid because it decreased during the period of food consumption. Plasma MeHis concentration had fallen by 80% at 3 h after refeeding. The reduction in concentration may have been due to an increase in blood flow, fluid intake or kidney function. However, the reduction was very large and is not easily explained by these altered physiological conditions. Mortimore et al. (1983) have indicated that proteolysis of hepatocytes is inhibited by 80% after refeeding in mice. Thus, changes in protein metabolism in mice may be large.

Postprandial insulin concentration was very high (Fig. 2B) as a result of the high blood glucose concentration after refeeding. We have shown that insulin reduces the rate of myofibrillar protein degradation in diabetic rats (Kadowaki et al. 1985, Nagasawa et al. 1982). It is therefore quite plausible that the decrease in muscle protein degradation was mediated by the rise in plasma insulin concentration as a result of food intake. One action of insulin is a reduction of the number of autophagosomes in the cell (Rannels et al. 1975). However, the mechanisms by which insulin affects protein degradation are still unclear. In contrast to our work, Li and Wassner (1984) and Lowell et al. (1986) found that insulin did not depress myofibrillar protein degradation. Furthermore, because myofibrillar protein degradation, as reflected by plasma MeHis concentration, was at a depressed level at 6 h after refeeding, whereas at this time point protein synthesis (polysome size) had returned to the prefeeding level, it is unlikely that only insulin regulates myofibrillar protein degradation after refeeding. Branched-chain amino acids regulate muscle protein degradation (Tischler et al. 1982). Glucocorticoids stimulate protein degradation through an ATP-ubiquitin-dependent proteolytic system during food deprivation (Wing and Goldberg 1993). Therefore, amino acids produced from dietary protein and/or other hormonal factors may also regulate myofibrillar protein degradation. Further studies are required to determine the factors regulating muscle protein degradation after refeeding.

In conclusion, the response of protein metabolism to food intake involves a decrease in muscle protein breakdown, accompanied by an increase in protein synthesis. The ability of skeletal muscle to use nutrients more effectively by stimulating protein synthesis and reducing protein degradation may cause the accelerated rate of protein accretion in skeletal muscle during the immediate postprandial period.

ACKNOWLEDGMENTS