Skeletal muscle protein turnover in young male pigs

A 6-h continuous infusion of L-[U-14C]tyrosine was used to estimate fractional protein synthesis rates (FSR) in the longissimus dorsi (LD), semitendinosus (ST) and brachialis (BR) muscles of intact male pigs weighing 22 or 45 kg. The FSR was approximately 20% lower for pigs at 45 kg (4.2 vs. 5.2%/d, 5.2 vs. 6.4%/d and 5.1 vs. 6.4%/d for the LD, ST and BR muscles, respectively) compared with pigs at 22 kg. Fractional protein accretion or growth rates (FGR) were estimated over a 2-wk period. At 22 kg, FGR for the LD, ST and BR were 0.7, 2.4 and 1.7%/d, respectively; at 45 kg, FGR for these muscles were 1.7, 1.9 and 0.7%/d. Fractional protein breakdown rates (FBR) derived by difference (FBR = FSR - FGR) were 44, 16 and 8% lower for the LD, ST and BR muscles, respectively, at 45 kg compared with those at 22 kg. Therefore, it is suggested that muscle growth rate is modulated by alterations in FBR. By using the fractional rates found in these muscles to determine total-muscle protein synthesized or degraded in pigs at 22 and 45 kg, the proportion of protein retained was approximately 28% of that synthesized by the pigs at each weight. Since individual skeletal muscles of the pigs differed in protein turnover rates during postnatal growth and development, selection of a muscle(s) for turnover rates in growth studies is critical.     

Insulin dependent and independent actions of dietary protein on in vitro protein synthesis in skeletal muscle of rats.

Insulin dependency of the effect of dietary protein on in vitro protein synthesis in skeletal muscle was studied in rats. Re-feeding fasted rats with an adequate protein diet caused an increase in protein synthetic activity of skeletal muscle within only 5 hours with a concomitant increase in concentration of polyribosomes. During re-feeding of a protein-free diet muscle protein synthesis increased significantly but it was somewhat lower than in rats fed an adequate protein diet. The concentration of insulin in plasma increased promptly and markedly when the rats were re-fed either of these diets. However, the degree of increase was slightly less in rats fed a protein-containing meal. Re-feeding normal rats with egg albumin alone, after fasting 1 day, produced a marked increase in protein synthetic activity of skeletal muscle with a parallel increase in proportion of polyribosomes in the tissue. The stimulatory action of dietary protein on muscle protein synthesis was not abolished even in streptozotocin-diabetic rats. However, there was no appreciable change in ribosomal profile in the diabetic rats. These results indicate that the function of dietary protein in regulating muscle protein synthesis is not mediated entirely by insulin.     

In vitro Na+,K+-ATPase (EC 3.6.1.3)-dependent respiration and protein synthesis in skeletal muscle of pigs fed at three dietary protein levels

1. Eighteen pigs were offered diets containing 130, 170 or 210 g protein/kg with three barrows and three gifts per diet from 20 to 60 kg live weight. Oxygen consumption, Na+,K+-ATPase (EC 3.6.1.3)-dependent and -independent respiration and protein synthesis were measured in vitro in intercostal and sartorius muscle preparations from these pigs. 2. Increasing dietary protein concentration increased (P less than 0.01) daily gain and dissectible muscle in carcass. 3. O2 consumption and Na+,K+-ATPase-dependent respiration of the intercostal and sartorius muscles increased linearly (P less than 0.01) with increase in dietary protein concentration. The requirement for the support of the transport of Na+ and K+ across the cell membrane in these muscles, on average, accounted for 22-25% of the O2 consumption. 4. Synthesis rate (mg/g per d) of protein in the sartorius muscle increased (P less than 0.05) from 3.05 to 5.07 and increased (P less than 0.1) from 2.57 to 4.06 in the intercostal muscle as dietary protein increased from 130 to 210 g/kg diet. 5. Regression of Na+,K+-ATPase-dependent respiration against protein synthesis in each of intercostal and sartorius muscles showed a linear relation, an attestation of a close link between productive processes and auxiliary energy expenditure.     

Effects of potassium deficiency on growth and protein synthesis in skeletal muscle and the heart of rats

The effects of potassium deficiency on growth, K content and protein synthesis have been compared in 4-13-week-old rats. When maintained on K-deficient fodder (1 mmol/kg) rats ceased to grow within a few days, and the incorporation of [3H]leucine into skeletal muscle protein in vivo was reduced by 28-38%. Pair-feeding experiments showed that this inhibition was not due to reduced energy intake. Following 14 d on K-deficient fodder, there was a further reduction (39-56%) in the incorporation of [3H]leucine into skeletal muscle protein, whereas the incorporation into plasma, heart and liver proteins was not affected. The accumulation of the non-metabolized amino acid alpha-aminoisobutyric acid in the heart and skeletal muscles was not reduced. The inhibitory effect of K deficiency on 3H-labelling of muscle protein was seen following intraperitoneal (10-240 min) as well as intravenous (10 min) injection of [3H]leucine. In addition, the incorporation of [3H]phenylalanine into skeletal muscle protein was reduced in K-depleted animals. Following acute K repletion in vivo leading to complete normalization of muscle K content, the incorporation of [3H]leucine into muscle protein showed no increase within 2 h, but reached 76 and 104% of the control level within 24 and 72 h respectively. This was associated with a rapid initial weight gain, but normal body-weight was not reached until after 7 weeks of K repletion. Following 7 d on K-deficient fodder the inhibition of growth and protein synthesis was closely correlated with the K content of the fodder (1-40 mmol/kg) and significant already at modest reductions in muscle K content. In vitro experiments with soleus muscle showed a linear relationship between the incorporation of [3H]leucine into muscle protein and K content, but the sensitivity to cellular K deficiency induced in vitro was much less pronounced than that induced in vivo. Thus, in soleus and extensor digitorum longus (EDL) muscles prepared from K-deficient rats, the incorporation of [3H]leucine was reduced by 30 and 47% respectively. This defect was completely restored by 24 h K repletion in vivo. It is concluded that in the intact organism protein synthesis and growth are very sensitive to dietary K deficiency and that this can only partly be accounted for by the reduction in cellular K content per se. The observations emphasize the need for adequate K supplies to ensure optimum utilization of food elements for protein synthesis and growth.     

Contribution of liver, skin and skeletal muscle to whole-body protein synthesis in the young lamb

1. Protein fractional synthesis rate (FSR) was measured in some major tissues and in the whole body of six 1-week-old sucking lambs by a large injection of L-[3H]valine. 2. Upper estimates of tissue protein FSR (%/d), assuming that the tissue-homogenate free-valine specific radioactivity defined that of valyl tRNA, were 115.0 in liver, 24.1 in skin, 22.9 in the white M. tensor fasciae latae, 21.6 in the red M. diaphragma and 19.6 in the remainder (exsanguinated whole body without liver and gastrointestinal tract) of lambs. 3. Absolute synthesis rates (ASR) of tissue protein were 17, 19 and 42 g/d in the liver, skin and skeletal muscle respectively, and 112 g/d in the remainder. The ASR of whole-body protein, derived from the tissue values, was 146 g/d, i.e. 33 g/d per kg body-weight. The calculated whole-body protein FSR was 23.9%/d. 4. The relative percentage contribution of liver, skin and skeletal muscle to whole-body protein synthesis was 11.7, 13.1, and 29.0. 5. We concluded that tissue protein FSR in lambs were in exactly the same decreasing order, from visceral tissues to skeletal muscles, as observed in rats. The ovine FSR estimates and the partitioning of protein synthesis between tissues were in the same range as values recently obtained by flooding-dose experiments in immature rats, piglets, and even in chicks. These findings suggest that inter-species differences are rather limited.     

Consequences of severe copper deficiency are independent of dietary carbohydrate in young pigs

The ability of carbohydrates (CHO), such as fructose and sucrose, to aggravate copper deficiency in rats and the recent dietary trends of Western human populations led to the suggestion that the Cu X CHO interaction may be pertinent to public health. This hypothesis was tested with pigs because their cardiovascular and gastrointestinal systems closely resemble those of humans. Weanling pigs were fed a diet containing either 59% sucrose or cornstarch with either deficient (0.8 mg/kg diet) or adequate (6.4 mg/kg) copper for 10 wk. Plasma and tissue copper, the activities of plasma ceruloplasmin ferroxidase and erythrocyte Cu,Zn-superoxide dismutase, hematocrits, and serum cholesterol and triglyceride were all decreased (p less than 0.05) and relative cardiac mass was increased (p less than 0.05) by severe dietary copper deficiency. The type of dietary CHO did not differentially influence the values of these variables. Thus, these data fail to support the hypothesis that the Cu X CHO interaction observed in rats represents a health risk for humans.     

Protection against zinc deficiency by prior excess dietary zinc in young Japanese quail.

Three experiments were designed to determine whether an excess of zinc during the first week of life would afford any protection to young Japanese quail during a subsequent period of zinc deprivation. Day-old birds of both sexes were fed an adequate purified diet containing soybean protein with requirement (25-30 mg/kg of diet) or higher levels of zinc for 1 week. The zinc level was reduced during the second week to the deficient level of 1mg/kg of diet, except for the controls which continued to receive an adequate or higher level of zinc (25, 30 or 75 mg/kg of diet). With this low zinc intake, birds that received an initial level of zinc in excess of requirement grew significantly better (body weight and length of primary wing feathers) than those that initially received the required amount of zinc. Data on zinc content of liver, breast muscle, tibia and whole body retention suggest that bone may store zinc consumed in excess of requirement and that this zinc may be available for utilization during a subsequent period of zinc deprivation in a growing animal that has rapidly remodeling bones.     

Activation of skeletal muscle protein breakdown following consumption of soyabean protein in pigs

Diets with protein of inferior quality may increase protein breakdown in skeletal muscle but the experimental results are inconsistent. To elucidate the relationship, pigs were fed isoenergetic and isonitrogenous diets based on soyabean-protein isolate or casein for 15 weeks, with four to six animals per group. A higher plasma level of urea (2.5-fold the casein group value, P = 0.01), higher urinary N excretion (2.1-fold the casein group value, P = 0.01), a postabsorptive rise in the plasma levels of urea, 3-methylhistidine and isoleucine in soyabean protein-fed pigs suggested recruitment of circulatory amino acids by protein breakdown in peripheral tissues. Significant differences between dietary groups were detected in lysosomal and ATP-dependent proteolytic activities in the semimembranosus muscle of food-deprived pigs. A higher concentration of cathepsin B protein was found, corresponding to a rise in the cathepsin B activity, in response to dietary soyabean protein. Muscle ATP-stimulated proteolytical activity was 1.6-fold the casein group value (P = 0.03). A transient rise in the level of cortisol (2.9-times the casein group value, P = 0.02) occurred in the postprandial phase only in the soyabean group. These data suggest that the inferior quality of dietary soyabean protein induces hormonally-mediated upregulation of muscle protein breakdown for recruitment of circulatory amino acids in a postabsorptive state.     

Intestinal threonine utilization for protein and mucin synthesis is decreased in formula-fed preterm pigs

Threonine is an essential amino acid necessary for synthesis of intestinal (glyco)proteins such as mucin MUC2 to maintain adequate gut barrier function. In premature infants, reduced barrier function may contribute to the development of necrotizing enterocolitis (NEC). Human milk protects against NEC compared with infant formula. Therefore, we hypothesized that formula feeding decreases the MUC2 synthesis rate concomitant with a decrease in intestinal first-pass threonine utilization, predisposing the preterm neonate to NEC. Preterm pigs were delivered by caesarian section and received enteral feeding with formula (FORM; n = 13) or bovine colostrum (COL; n = 6) for 2 d following 48 h of total parenteral nutrition. Pigs received a dual stable isotope tracer infusion of threonine to determine intestinal threonine kinetics. NEC developed in 38% of the FORM pigs, whereas none of the COL pigs were affected (P = 0.13). Intestinal fractional first-pass threonine utilization was lower in FORM pigs (49 ± 2%) than in COL pigs (60 ± 4%) (P = 0.02). In FORM pigs compared with COL pigs, protein synthesis (369 ± 31 mg·kg(-1)·d(-1) vs. 615 ± 54 mg·kg(-1)·d(-1); P = 0.003) and MUC2 synthesis (121 ± 17%/d vs. 184 ± 15%/d; P = 0.02) were lower in the distal small intestine (SI). Our results suggest that formula feeding compared with colostrum feeding in preterm piglets reduces mucosal growth with a concomitant decrease in first-pass splanchnic threonine utilization, protein synthesis, and MUC2 synthesis in the distal SI. Hence, decreased intestinal threonine metabolism and subsequently impaired gut barrier function may predispose the formula-fed infant to developing NEC.     

Leucine excess under conditions of low or compensated aminoacidemia does not change skeletal muscle and whole-body protein synthesis in suckling lambs during postprandial period.

We determined the effect of a 4-h leucine infusion, leading to 15-fold elevated plasma leucine concentrations, on skeletal muscle and whole-body protein synthesis in suckling lambs during the postprandial period. The [3H]phenylalanine large dose method was validated and used to quantify the fractional rates of protein synthesis (Ks in %/d) at the end of the leucine infusion. In the first experiment leucine infusion lowered plasma amino acid concentrations but did not change the Ks, the capacity for protein synthesis (Cs, mg RNA/g protein) or the efficiency of translation [g protein synthesized/(d-g RNA)] in any muscles studied or the whole body. In the second experiment the leucine-induced decreases in plasma amino acid concentrations were compensated by the simultaneous infusion of substantial amounts of amino acids. Again leucine excess did not significantly change Ks, Cs and efficiency of protein synthesis. These results indicated that leucine excess in suckling lambs during the postprandial period lowered aminoacidemia without any change of the protein synthesis rates in skeletal muscles or the whole body.     

Mechanotransduction and the regulation of protein synthesis in skeletal muscle

Repeated bouts of resistance exercise produce an increase in skeletal muscle mass. The accumulation of protein associated with the growth process results from a net increase in protein synthesis relative to breakdown. While the effect of resistance exercise on muscle mass has long been recognized, the mechanisms underlying the link between high-resistance contractions and the regulation of protein synthesis and breakdown are, to date, poorly understood. In the present paper skeletal muscle will be viewed as a mechanosensitive cell type and the possible mechanisms through which mechanically-induced signalling events lead to changes in rates of protein synthesis will be examined.     

The digestion of protein in young pigs and the utilization of dietary methionine

Forty pigs between 23 and 51 d of age were given ad lib. diets containing wheat and one of five protein concentrates: meat meal A, meat meal B, soya-bean meal, milk and lupins (Lupinus augustifolius). Twenty of these pigs were given indigestible markers from 51 to 56 d of age and were killed at 56 d of age. The diets containing meat meals A and B, soya-bean meal and milk contained 2.3 g total methionine/kg and the diet containing lupins contained 2.1 g/kg. A further forty pigs of the same age were given the same diets supplemented with 1 g synthetic methionine/kg. The weight gains and feed conversion ratios of the pigs given the diets containing 2.1-2.3 g methionine and 3.1-3.3 g methionine/kg were not significantly different. The weight gains of the pigs given lupins (2.1 g methionine/kg) were less than those of the pigs given the diets containing 2.3 g methionine/kg. The apparent digestibility of dry matter (DM) and nitrogen was less for the diets containing the meat meals (0.75 and 0.78 respectively) than for those containing the other protein concentrates (0.80 and 0.84). The retention times in the large intestine of the diets containing soya-bean meal and lupins were 965 and 1083 min which were greater than those of the diets containing the other protein concentrates, mean 732 min. The major site of N digestion and absorption for the diet containing milk was the area of the small intestine 25-50% of total length from the pylorus, while for the other protein concentrates the major site was 50-75% of its total length from the pylorus. The digestion and absorption of N in the large intestine was less (3.4%) for the diet containing milk than for those containing the other protein concentrates (7.5-11.3%). The apparent digestibility of the methionine to the ileum for the five diets ranged from 0.74 to 0.86 while the calculated retention of the apparently-absorbed methionine was 1.00. It was suggested that methionine digestibility could be used as an indicator of availability. The calculated retention of apparently absorbed N in the carcass was 0.71 for the pigs given the diet containing milk and 0.51-0.58 for the pigs given the other diets.     

Leucine content of dietary proteins is a determinant of postprandial skeletal muscle protein synthesis in adult rats

ABSTRACT: BACKGROUND: Leucine (Leu) regulates muscle protein synthesis (MPS) producing dose-dependent plasma Leu and MPS responses from free amino acid solutions. This study examined the role of Leu content from dietary proteins in regulation of MPS after complete meals. METHODS: Experiment 1 examined 4 protein sources (wheat, soy, egg, and whey) with different Leu concentrations (6.8, 8.0, 8.8, and 10.9% (w/w), respectively) on the potential to increase plasma Leu, activate translation factors, and stimulate MPS. Male rats (~250 g) were trained for 14 day to eat 3 meals/day consisting of 16/54/30% of energy from protein, carbohydrates and fats. Rats were killed on d14 either before or 90 min after consuming a 4 g breakfast meal. Experiment 2 compared feeding wheat, whey, and wheat + Leu to determine if supplementing the Leu content of the wheat meal would yield similar anabolic responses as whey. RESULTS: In Experiment 1, only whey and egg groups increased post-prandial plasma Leu and stimulated MPS above food-deprived controls. Likewise, greater phosphorylation of p70 S6 kinase 1 (S6K1) and 4E binding protein-1 (4E-BP1) occurred in whey and egg groups versus wheat and soy groups. Experiment 2 demonstrated that supplementing wheat with Leu to equalize the Leu content of the meal also equalized the rates of MPS. CONCLUSION: These findings demonstrate that Leu content is a critical factor for evaluating the quantity and quality of proteins necessary at a meal for stimulation of MPS.     

The diurnal pattern of protein synthesis in human skeletal muscle

The diurnal pattern of protein synthesis in skeletal muscle was studied in relation to the serum concentrations of glucose, insulin and cortisol. Twelve healthy volunteers were given an ordinary hospital diet at 4-hourly intervals. The concentration of insulin showed peaks in response to food intake, and that of cortisol decreased over the day. Muscle biopsies were taken 30 min before the first food intake and 1.5 or 3.5 h after a meal. Irrespective of the time intervals after food intake, the total ribosome concentration, percentage amount of polyribosomes and polyribosome concentrations remained unaltered, indicating that there were no alterations in the capacity for protein synthesis accompanying the feeding schedule. The results suggest that a muscle sample taken in the morning after an overnight fast is representative of the protein synthesis during the day as assessed by the ribosome concentration and the size distribution of ribosomes.     

A moderate excess of dietary lysine lowers plasma and tissue carnitine concentrations in pigs

This study was performed to investigate whether dietary lysine concentration influences the carnitine status of pigs. Therefore, an experiment with twenty young pigs with an average body weight of 21 kg was performed which were fed either a control diet (9.7 g lysine/kg) or a diet with a moderate excess of lysine (16.8 g lysine/kg). Concentrations of all the other amino acids did not differ between the diets. Pigs fed the high-lysine diet had lower concentrations of free and total carnitine in plasma, liver, kidney and skeletal muscle than control pigs (P<0.05). Pigs fed the high-lysine diet moreover had an increased concentration of trimethyllysine (TML), a reduced mRNA abundance of TML dioxygenase and reduced concentrations of gamma-butyrobetaine (BB) in muscle, indicating that the conversion of TML into BB in muscle was impaired. Concentrations of BB, the metabolic precursor of carnitine, in plasma, liver and kidney were also reduced in pigs fed the high-lysine diet while the activity of BB dioxygenase in kidney was not different and that in liver was even increased compared to control pigs (P<0.05). In conclusion, this study shows that a moderate dietary excess of lysine lowers plasma and tissue carnitine concentrations in pigs. Reduced concentrations of BB in liver and kidney suggest that the depressed carnitine status was likely caused by a decreased rate of carnitine synthesis due to a diminished availability of carnitine precursor, probably mainly as a result of an impaired BB formation in muscle.     

Restriction of dietary energy and protein induces molecular changes in young porcine skeletal muscles

Little is known about the molecular changes in response to dietary restriction (energy and/or protein) in young growing skeletal muscles. To profile such changes and to gain insights into the signaling molecules that could mediate the diet effects, a dedicated porcine skeletal muscle cDNA-microarray approach was used to characterize differential muscle gene expression between conventionally fed and diet-restricted (20% less protein and 7% less energy) growing pigs, reared from 9 to 21 wk of age. In both red and white muscles, diet restriction resulted in the accumulation of significantly more intramuscular fat, and in the increased expression of genes involved in substrate (protein, glycogen, and lipid) turnover, in translation and mitochondrial function, and in raising glycolytic and oxidative phosphorylation potentials. The unexpected increase in intramuscular lipids in diet-restricted growing pigs could have important health implications for restricted diets in childhood. Despite reduced circulating insulin, more genes, including several novel growth modulatory genes, had higher expression levels, indicating that the cellular response to dietary restriction is an active process. One such responsive gene, P311, was most highly expressed in striated muscles and had a differentiation-dependent increase of expression in murine C2C12 cells, suggesting a role in differentiation/postdifferentiation phenotype determination.