Leucine supplementation of a low-protein meal increases skeletal muscle and visceral tissue protein synthesis in neonatal pigs by stimulating mTOR-dependent translation initiation

Protein synthesis and eukaryotic initiation factor (eIF) activation are increased in skeletal muscle of neonatal pigs parenterally infused with amino acids. Leucine appears to be the most effective single amino acid to trigger these effects. To examine the response to enteral leucine supplementation, overnight food-deprived 5-d-old pigs were gavage fed at 0 and 60 min a: 1) low-protein diet (LP); 2) LP supplemented with leucine (LP+L) to equal leucine in the high-protein diet (HP); or 3) HP diet. Diets were isocaloric and equal in lactose. Fractional protein synthesis rates and translation initiation control mechanisms were examined in skeletal muscles and visceral tissues 90 min after feeding. Protein synthesis rates in longissimus dorsi, gastrocnemius, and masseter muscles, heart, jejunum, kidney, and pancreas, but not liver, were greater in the LP+L group compared with the LP group and did not differ from the HP group. Feeding LP+L and HP diets compared with the LP diet increased phosphorylation of mammalian target of rapamycin (mTOR), 4E-binding protein 1, ribosomal protein S6 kinase-1, and eIF4G and formation of the active eIF4E·eIF4G complex in longissimus dorsi muscle. In all tissues except liver, activation of mTOR effectors increased in pigs fed LP+L and HP vs. LP diets. Our results suggest that leucine supplementation of a low-protein meal stimulates protein synthesis in muscle and most visceral tissues to a rate similar to that achieved by feeding a high-protein meal and this stimulation involves activation of mTOR downstream effectors.     

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.     

Protein digestion in weanling pigs: effect of dietary protein source

The digestion and absorption of dietary protein was measured in weanling pigs (5 wk of age). Diets containing corn and either dried skim milk (DSM), soybean meal (SBM) or corn gluten meal (CGM) were fed for 7 d, and the contents of the stomach and six segments of the small intestine were collected. Nitrogen digestibility increased linearly from the proximal duodenum to the distal ileum, and was highest for DSM, intermediate for SBM and lowest for CGM diets at the distal ileum. The content of free amino acids in digesta increased 8-fold (SBM and CGM) to 12-fold (DSM) between the stomach and the proximal duodenum and reached a maximum concentration in the distal jejunum. Digestion of DSM was more proximal than was that of SBM. Although there was some accumulation of small peptides in the duodenum with a subsequent decrease in the jejunum, the molecular weight profiles of the soluble proteins were relatively constant throughout the small intestine. Protein solubility and the rate of proteolysis in the stomach and upper small intestine were the primary factors that limited the digestion of SBM and CGM.     

Leucine regulates translation initiation of protein synthesis in skeletal muscle after exercise

High-performance physical activity and postexercise recovery lead to significant changes in amino acid and protein metabolism in skeletal muscle. Central to these changes is an increase in the metabolism of the BCAA leucine. During exercise, muscle protein synthesis decreases together with a net increase in protein degradation and stimulation of BCAA oxidation. The decrease in protein synthesis is associated with inhibition of translation initiation factors 4E and 4G and ribosomal protein S6 under regulatory controls of intracellular insulin signaling and leucine concentrations. BCAA oxidation increases through activation of the branched-chain alpha-keto acid dehydrogenase (BCKDH). BCKDH activity increases with exercise, reducing plasma and intracellular leucine concentrations. After exercise, recovery of muscle protein synthesis requires dietary protein or BCAA to increase tissue levels of leucine in order to release the inhibition of the initiation factor 4 complex through activation of the protein kinase mammalian target of rapamycin (mTOR). Leucine's effect on mTOR is synergistic with insulin via the phosphoinositol 3-kinase signaling pathway. Together, insulin and leucine allow skeletal muscle to coordinate protein synthesis with physiological state and dietary intake.     

Effect of increasing levels of dietary fish oil on tissue lipids and prostaglandin synthesis in the rat

The effects of increasing levels of n-3 polyunsaturated fatty acids in fish oil on the composition of phospholipids in specific tissues and prostanoid biosynthesis important to vascular integrity were studied. Rats were fed menhaden oil at 5, 10 and 20 weight percent corresponding to 1, 2 and 4 percent dietary eicosapentaenoic acid (EPA). The control diets contained hydrogenated coconut oil and both diets included 2% safflower oil in order to be adequate in essential fatty acids. Significant differences in tissue selectivity for 20:5 and 22:6 were observed for platelet and aorta phospholipids. While platelets avidly took up 20:5 primarily at the expense of 20:4 n-6, 22:6 did not accumulate proprotionally in platelets even at the 20 percent dietary level. Alternatively, aorta phospholipids took up equal quantities of both 20:5 and 22:6 again at the expense of 20:4.

Prostanoid biosynthesis by these tissues was significantly and selectively altered by dietary treatments. Whereas serum thromboxane levels were decreased by 50% in the group receiving 5% fish oil, prostacyclin production by isolated aorta segments from the same group was not correspondingly reduced. In contrast, the higher dietary levels: i.e. 10 and 20 percent fish oil, did not elicit corresponding reductions in thromboxane levels whereas prostacyclin production by isolated aorta tissue was increasingly affected and reached a 50% decrease at 20 percent dietary fish oil. These experiments demonstrated significant and perhaps important sensitivities by different tissues to dietary n-3 PUFA. In spite of these effects, the experimental animals did not exhibit a significant alteration in tail bleeding times, platelet aggregability or platelet sensitivity to the agonists ADP and collagen relative to control animals. Whole blood viscosity was however, lower in the group fed 20 percent fish oil.     

Prebiotics affect nutrient digestibility but not faecal ammonia in dogs fed increased dietary protein levels

An increased protein content and less digestible protein sources in the diet can induce bad faecal odour. The present study investigated the effect of adding prebiotics to dog diets enriched with animal-derived protein sources on apparent digestibilities and faecal ammonia concentration. In three subsequent periods eight healthy beagle dogs were fed a commercial dog diet that was gradually supplemented by up to 50 % with meat and bone meal (MBM), greaves meal (GM) or poultry meal (PM) respectively. Afterwards, 3 % fructo-oligosaccharides or 3 % isomalto-oligosaccharides were substituted for 3 % of the total diet. Supplementation with animal-derived protein sources did not decrease the apparent N digestibility significantly but oligosaccharides did. On the other hand the bacterial N content (% DM) in the faeces was highest in the oligosaccharide groups followed by the protein-supplemented groups and lowest in the control groups. When the apparent N digestibility was corrected for bacterial N no significant differences were noted anymore except for the GM group where the corrected N digestibility was still lower after oligosaccharide supplementation. The amount of faecal ammonia was significantly increased by supplementing with protein or oligosaccharides in the MBM and GM groups but not in the PM group. When apparent N digestibility is interpreted, a correction for bacterial N should be taken into account, especially when prebiotics are added to the diet. Oligosaccharides did not reduce the faecal ammonia concentrations as expected.     

Activities of branched-chain amino acid--degrading enzymes in liver from rats fed different dietary levels of protein

The relationships among dietary protein intake, plasma branched-chain amino acid (BCAA) and keto acid (BCKA) concentrations, and liver BCAA-degrading enzyme activities were investigated in rats fed, for 5 h/d for 2, 6 or 9 d, diets containing from 0 to 50% casein. Plasma, liver and muscle BCAA concentrations were proportional to protein intake over the entire range tested; plasma BCKA concentration, however, was proportional only in the range from 0 to 20% casein, after which a plateau was reached. By d 2, liver cytosolic BCAA aminotransferase activity had increased in rats fed 50% casein; by d 9, activity had increased in rats fed 0 or 5% casein as well. Liver mitochondrial BCAA aminotransferase activity was unresponsive to dietary treatment. Basal liver BCKA dehydrogenase activity and the percent active complex were proportional to protein intake on d 2 and 6. On d 2, total BCKA dehydrogenase activity was the same in all groups; by d 6, total activity had increased in rats fed 30 or 50% casein. We conclude that although the adaptive changes in BCAA-degrading enzyme activities are small, they are sufficient to compensate for excessively high or low protein intakes, so that tolerable concentrations of BCAA and BCKA are maintained.     

High dietary manganese lowers heart magnesium in pigs fed a low-magnesium diet

Young pigs were fed a diet moderately high or low in manganese (Mn) (0.95 +/- 0.10 mmol Mn/kg, n = 8 or 0.040 +/- 0.003 mmol Mn/kg, n = 6) and deficient in magnesium (Mg) (4.1 mmol Mg/kg) for 5 wk. All eight pigs consuming the high Mn diet died following convulsive seizures, whereas only two of six died in the group fed low Mn. In an attempt to determine the cause of death, a subsequent study examined the interactive effect of deficient dietary Mg and Mn on the tissue distribution of Mg and Mn. Pigs were individually fed, for 5 wk, diets that contained: 4.1 mmol Mg/kg and 36.0 micromol Mn/kg, 4.1 mmol Mg/kg and 0.91 mmol Mn/kg, 4.1 mmol Mg/kg and 0.91 mmol Mn/kg with added ultratrace minerals, or 41.1 mmol Mg/kg and 0. 91 mmol Mn/kg, and ultratrace minerals. Liver and skeletal muscle Mn concentrations were significantly elevated by increased dietary Mn. Increased dietary Mn did not affect heart Mn, but heart Mg concentrations were significantly depressed by high, as compared to low, dietary Mn (38.7 +/- 3.3 vs. 32.7 +/- 2.6 mmol Mg/kg). These data suggest high dietary Mn may exacerbate Mg deficiency in heart muscle and thus may be a complicating factor in the deaths observed in Mg-deficient pigs.     

Feeding meals containing soy or whey protein after exercise stimulates protein synthesis and translation initiation in the skeletal muscle of male rats

The purpose of this investigation was to compare the early response of skeletal muscle protein synthesis and translation initiation following the ingestion of different protein sources after endurance exercise. Treadmill-acclimated rats were designated as either nonexercised controls (NEX) or treadmill exercised for 2 h at 26 m/min (approximately 75% VO2max) and then fed either carbohydrate only (EC), carbohydrate plus soy protein (ES), or carbohydrate plus whey protein (EW). One hour after exercise, serum insulin concentrations in EC, ES, and EW were greater than in NEX (P<0.05); the concentration in EW was greater than in EC, with that in ES intermediate. Serum concentrations of branched-chain amino acids in ES and EW were higher than in EC, but serum leucine and isoleucine in EW were higher than in ES (P<0.05). Nevertheless, both ES and EW promoted the fractional rate of skeletal muscle protein synthesis significantly more than EC. Likewise, compared with EC, both ES and EW increased formation of the mRNA cap binding complex eIF4F and stimulated phosphorylation of the translational repressor, 4E-BP1, the 70kD ribosomal protein S6 kinase (S6K1), and the mammalian target of rapamycin (mTOR) kinase at serine 2448. On the other hand, phosphorylation of S6K1 and mTOR was greater in EW than in ES (P<0.05). In conclusion, general protein synthesis and the mRNA cap binding step are promoted comparably by soy protein and whey protein in the skeletal muscle of exercised rats. Furthermore, the data suggest that mTOR signaling in skeletal muscle is acutely responsive to physiological variations in dietary amino acids.     

Turnover of pancreas phospholipids in rats fed inadequate dietary protein

Effect of feeding rice diets with and without lysine and threonine supplementation has been studied on phospholipid turnover of rat pancreas and compared to rats fed casein diet at 20% protein level. Rice diet without amino acid supplementation significantly reduced the levels of pancreas PC, PE, PI, PS and Sph and increased that of total cholesterol and TG. Incorporation of palmitate-1-14C, labelled choline, labelled methionine and NaH2(32)PO4 into PC and of palmitate-1-14C into TG was significantly reduced in rats fed unsupplemented rice diet. Incorporation of NaH2(32)PO4 into pancreas PI, DPI, TPI, PA, LPA and into IP, IP2', IP3 and GPI was significantly reduced in rats fed unsupplemented rice diet.     

Protein and energy utilization in germ-free and conventional chicks given diets containing different levels of dietary protein

The present study was done to clarify the relationship between the amount of dietary protein given to, and the gut microflora of, the host. Day-old chicks were given diets containing three concentrations of dietary protein (50, 200 and 400 g/kg) for 14 d. Body-weight gain, food consumption, body consumption, and protein and energy utilization were measured. There was no difference in body-weight gain and food consumption between germ-free (GF) and conventional (CV) chicks, but food conversion efficiency (g body-weight gained/g food consumed) was significantly higher in GF than in CV chicks. Little difference was found in protein retention (g protein retained/14 d), but protein retention rate (g protein retained/g protein consumed) tended to be higher in GF chicks, particularly those given the diet with the lowest protein. The presence of micro-organisms improved metabolizable energy (ME) values of the diets, but not all of the digested energy in CV chicks was utilized for growth. Therefore there was little difference in energy retention (kJ energy retained/14 d) between environments, although energy retention rate (kJ energy retained/kJ ME consumed) was significantly lower in CV chicks. The amount of body fat in GF chicks was higher than that in CV chicks, especially in those fed on the low-protein diet. It is suggested that although the gut microflora may have beneficial effects on the digestion of dietary energy components, they may have detrimental effects on utilization of ME by their hosts, because chicks harbouring a gut microflora seem to have higher energy requirements for maintenance.     

A deficiency or excess of dietary threonine reduces protein synthesis in jejunum and skeletal muscle of young pigs

Dietary threonine imbalance is known to reduce the growth of the small intestine, liver, and skeletal muscle in young animals, but the underlying mechanism is largely unknown. Using the pig model, this study was conducted to test the hypothesis that either a deficiency or an excess of dietary threonine impairs protein synthesis in these tissues. Young pigs (25 d of age) were fed diets containing 0.37, 0.74 (current NRC requirement) or 1.11% true ileal digestible threonine (TIDT) (n = 6/diet). Pigs receiving the 0.74 and 1.11% TIDT diets were pair-fed with the same amount of feed as pigs receiving the 0.37% TIDT diet. After a 14-d dietary treatment, the fractional synthesis rate (FSR) of protein in tissues was measured using a flooding dose of l-phenylalanine plus L-[ring-(2)H(5)]phenylalanine. The results indicated that the FSR of protein in liver was reduced (P < 0.05) in pigs fed the 0.37% TIDT diet compared with pigs fed the 0.74 or 1.11% TIDT diet, and did not differ between pigs fed the 0.74 and 1.11% TIDT diets. The FSR of protein in longissimus muscle, jejunal mucosa, and mucins was reduced (P < 0.05) in pigs fed the 0.37 or 1.11% TIDT diet compared with pigs fed the 0.74% TIDT diet. The absolute synthesis rate of protein in the jejunal mucosa and muscle was also reduced (P < 0.01) in pigs fed the 0.37 and 1.11% TIDT diets compared with the controls. The absolute synthesis rate of hepatic protein was lower (P < 0.01) in pigs fed the 0.37% TIDT diets when compared with pigs fed the 0.74% TIDT diet. Protein synthesis in skeletal muscle as well as jejunal mucosa and mucins was reduced to a greater extent than that in liver in response to an imbalance of dietary threonine. Collectively, these results indicate that either an excess or a deficiency of dietary threonine decreases protein synthesis in rapidly growing tissues of young pigs. The findings provide a mechanism for the low growth performance of animals fed a threonine-imbalanced diet.     

Muscle fractional protein synthesis is higher in Iberian than in Landrace growing pigs fed adequate or lysine-deficient diets

Protein deposition in Iberian pigs is low and the reasons for this are unknown. We investigated differences in protein synthesis rate in tissues of 30 Iberian and Landrace gilts fed 2 diets with adequate amino acid composition containing 160 or 120 g crude protein (CP)/kg, or a lysine-deficient diet (containing 120 or 160 g CP/kg for Iberian and Landrace pigs, respectively). Pigs were infused with a flooding dose of phenylalanine (15% as [(2)H(5)]-phenylalanine). Blood samples were taken from 12 to 40 min after the start of infusion, and samples from longissimus dorsi (ld), biceps femoris (bf), and semimembranosus (sm) muscles, liver, and duodenum were taken after slaughter. Body weights (BW) were 22.9 +/- 0.37 and 27.1 +/- 0.64 kg for Iberian and Landrace pigs, respectively. Iberian pigs fed the adequate diets had higher muscle fractional protein synthesis rates (FSR, %/d) than Landrace pigs. The FSR were 7.9 +/- 0.34 vs. 6.3 +/- 0.29%/d; 8.3 +/- 0.36 vs. 6.3 +/- 0.21%/d, and 7.7 +/- 0.23 vs. 6.4 +/- 0.36%/d for ld, bf, and sm muscles in Iberian and Landrace breeds, respectively (P < 0.01). However, muscles were between 20 and 32% smaller in the Iberian pigs (P < 0.01). Dietary protein level did not affect muscle FSR or size in either breed. Lysine deficiency reduced muscle FSR (46-49%, P < 0.001). Visceral tissues had greater relative weights in Iberian pigs (P < 0.001) with no breed differences in FSR. These findings might explain the low efficiency of protein and energy utilization by Iberian pigs compared with conventional pig breeds.     

Postprandial lipoprotein composition in pigs fed diets differing in type and amount of dietary fat.

To determine the effects of diet on postprandial lipoprotein composition, growing pigs were fed diets containing 20 or 40% of energy as soybean oil, tallow or a 50:50 blend of soybean oil and tallow. At the end of wk 6, a blood sample was drawn from pigs fasted for 12 h. Pigs were then fed, and blood samples were drawn 1 and 4 h later. In LDL, concentrations of free and total cholesterol were greater in pigs fed 40% of energy as fat than in pigs fed 20% of energy as fat (P less than 0.02). Pigs fasted for 12 h had lesser concentrations of triacylglycerol and greater concentrations of phospholipid in LDL and HDL than did pigs fasted for 1 and 4 h (P less than 0.05). In HDL, total cholesterol and phospholipid concentrations were greater in pigs fed 40% of energy as fat than in pigs fed 20% of energy as fat (P less than 0.01). A greater concentration of triacylglycerol was found in VLDL of pigs fed 40% of energy as fat than in pigs fed 20% of energy as fat (P less than 0.01). Amount of dietary fat had a greater effect than did type of dietary fat on composition of lipoproteins from postprandial pigs.     

Protein synthesis in liver, muscle, and brain of rats fed a high tyrosine-low protein diet.

Effects of feeding rats a high tyrosine-low protein diet on protein synthesis in liver, muscle, and brain were investigated both in vitro and in vivo. Tissue tyrosine concentrations of rats consuming the high tyrosine diet for 7 days were substantially elevated; this was accompanied by severe growth retardation. In the livers of rats fed the high tyrosine diet for 6 days, polysomal profiles showed a shift toward heavier ribosomal aggregates, while in muscle and brain, extensive disaggregation of polysomes occurred. In an in vitro amino acid incorporating system, the activities of both the microsomal and pH 5 enzyme fractions isolated from the livers of the high tyrosine animals that had been fed the diet for 6 weeks were elevated. On the other hand, the capacity of muscle or brain ribosomal preparations to incorporate [14-C] leucine was much reduced. Similar results were obtained in a study of [14-C] leucine incorporation in vivo in which rats were force-fed two meals and killed at various times after the last feeding. In rats fed the high tyrosine diet, incorporation of leucine into liver increased progressively; this was accompanied by a gradual decrease in leucine incorporation into muscle. In contrast, leucine incorporation into brain was immediately suppressed. In view of the apparently paradoxical effect of a high tyrosine load on protein synthesis in the liver, rates of the anabolic and catabolic phases of protein turnover in animals fed a high tyrosine diet were determined from radioactivity measurements made after pulse labeling them with [14-C] bicarbonate. Results indicated that the rates of both synthesis and degradation of liver proteins were elevated over control values. Differences in the effects of a toxic load of tyrosine on protein synthesis in the tissues examined could be the consequence of altered metabolic or hormonal balance as a result of nutritional stress.