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The effect of a high dose of 3-hydroxy-3-methylbutyrate (HMB, a leucine catabolite) on protein metabolism was investigated in growing male lambs fed on hay and concentrate. Concentrate was supplemented with either Ca(HMB)2 (4 g/kg) or Ca(CO3)2 in experimental (HMB) and control groups respectively. Both groups consisted of six 2-month old lambs. Three complementary methods to study protein metabolism were carried out consecutively 2.5 months after beginning the dietary treatment: whole body phenylalanine fluxes, postprandial plasma free amino acid time course and fractional rates of protein synthesis in skeletal muscles. Feeding a high dose of HMB led to a significant increase in some plasma free amino acids compared with controls. Total, oxidative and non-oxidative phenylalanine fluxes were not modified by dietary HMB supplementation. Similarly, an acute infusion of HMB, in the control group, did not change these fluxes. In skeletal muscles, fractional rates of protein synthesis were not affected by long-term dietary supplementation with HMB. Taken together our results showed that administration of a high dose of HMB to lambs was able to modify plasma free amino acid pattern without any effect on whole-body protein turnover and skeletal muscle protein synthesis.

Plasma concentrations of cortisol and glucagon have been measured in preruminant lambs after feeding a meal containing either 2.3 or 10.6 g leucine/100 g dry matter and were found similar. These two hormones are probably not involved in the effects induced by dietary leucine excess.

The effects of excess leucine intake on food intake, branched-chain amino acid and branched-chain alpha-keto acid concentrations in plasma and nitrogen retention were investigated in the preruminant lamb. Lambs were fed leucine in excess in either an adequate protein diet [24% of dry matter (DM)] or a low protein diet (15% DM) for 2 d. Increasing the dietary leucine content of 2.3 to 10.6 or 12.6% DM led to a significant decrease in food intake. This depressing effect was not influenced by dietary protein content. Increasing the dietary leucine content from 2.3 to 6.4% DM in an adequate protein diet for a week did not significantly improve nitrogen retention in the preruminant lamb. Plasma leucine and its alpha-keto acid concentrations increased with leucine intake. Plasma valine and isoleucine concentrations were significantly decreased only in lambs fed the highest excess leucine diet. Surprisingly, a maximal 50% decrease of their plasma alpha-keto acid concentrations occurred even in the group fed the lowest excess leucine diet. Our results might be explained by an inhibition of the rate of transamination of valine and isoleucine by high leucine concentration.

1. Branched-chain amino acid aminotransferase (EC 2.6.1.42; BCAAT) and branched-chain alpha-keto acid dehydrogenase (EC 1.2.4.4; BCKDH) activities were measured preruminant lamb liver, longissimus dorsi muscle, kidney, jejunum and adipose tissue, 2 h after a meal with or without an excess of leucine. 2. Skeletal muscle contained about 70% of the total basal BCAAT activities of the tissues studied whereas liver contained about 60% of the total BCKDH activities of these tissues. 3. BCAAT activities were very low in preruminant lamb tissues. BCKDH was more phosphorylated in tissues of preruminant lambs than in rats, especially in liver. These low catalytic potentialities might contribute to a low rate of branched-chain amino acid catabolism in sheep. 4. Ingestion of an excess of leucine led to an increase in liver and jejunum BCAAT activities and activation of BCKDH in jejunum.

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.

In vivo pancreatic protein synthesis rates were obtained from the uptake of L-[3,4(n)-3H]valine co-injected with a flooding dose of unlabeled valine into 1-, 5-, and 8-wk-old suckling lambs, and 8-wk-old weaned animals. Protein fractional synthesis rate was 184%/d at 1 wk of age and 153%/d in 5-wk-old animals (P greater than 0.05). This lack of developmental change resulted from constant (P greater than 0.05) ribosomal capacities (total RNA/protein ratios) and efficiencies of protein synthesis (synthetic rates relative to RNA). No further alteration for protein fractional synthesis rate (144%/d) occurred in 8-wk-old suckling animals (P greater than 0.05). In contrast, 8-wk-old ruminants exhibited higher protein fractional synthesis rate (244%/d) than 8-wk-old suckling animals, although ribosomal capacity was markedly higher in both 8-wk-old groups than in youngest animals (P less than 0.05). The present findings clearly indicate that in vivo protein synthesis in the developing ovine pancreas depends primarily on age. Potentialities for increased rates of pancreatic protein synthesis, i.e., increases in total RNA content and ribosomal capacity appear between 5 and 8 wk of age in this species. At 8 wk of age, however, when lambs are generally weaned, solid food ingestion resulted in a rise for both fractional and absolute rates of protein synthesis, essentially because ruminants maintained a higher efficiency of protein synthesis than milk-fed animals (P less than 0.005). Finally, there was a relationship between pancreatic protein synthesis and protein intake in only ruminant lambs.

The relationship between incorporation of intravenously injected 14C lysine and specific radio-activity of precursor was used to estimate protein synthesis in muscle of growing lambs. The rate of protein synthesis per unit of muscle weight in Supraspinatus and Extensor digitorum longus decreased strongly from one week of age to puberty (10 weeks); afterwards it decreased in supraspinatus and increased slightly in Extensor digitorum longus. The rate of protein synthesis increase in muscle protein weight was constant during the whole experiment (1 week-16 weeks). In preruminant Lambs )1 week-5 weeks) the rate of protein synthesis per unit of muscle weight decreased; however, due to the increase in muscle weight, the rate of protein synthesis in whole muscle remained relatively constant. In order Lambs the rate of protein synthesis in whole muscle decreased. The turnover time of protein increased with age. These results give some explanation on muscular development of Lambs.

Week-old lambs received an intravenous injection of 4.3, 8.5, 12.8 or 17.1 mmol [3H]valine/5 kg body weight, i.e., 3.6-14.4-times the whole-body free valine content. To ensure that protein synthesis measurements in lambs are reliable within a 30-min period, these large amounts of valine must account for at least around 11-times the total free pool of valine. This amounted to 12.8 mmol valine/5 kg body weight. There were no significant variations in plasma insulin and plasma glucagon levels 5, 13 and 30 min after the injection of so much valine. The fractional rates of protein synthesis were determined in tissues of animals receiving either 12.8 or 17.1 mmol valine/5 kg body weight. The rates of protein synthesis in the jejunum (87.5%/day), liver (106.6%/day) and tensor fasciae latae muscle (18.8%/day) of lambs injected with the 12.8 mmol [3H]valine flooding dose, were in the range of data obtained in immature rats. Increasing the flooding amount of valine up to 17.1 mmol/5 kg body weight did not significantly alter protein synthesis rates in the jejunum, liver or skeletal muscle. This suggested that both the flooding-dose method in itself and valine had no effect on in vivo protein synthesis.

1. In Expt 1, fractional synthesis rates (FSR) of tissue protein were measured along the gastrointestinal tract (GIT) of six 1-week-old, milk-fed lambs by using a large amount of L-[3,4(n)-3H]valine. 2. In Expt 2, eighteen lambs were used to determine the fractional growth rate (FGR) of gastrointestinal tissue protein. 3. FSRMinimum (Min) and FSRMaximum (Max) were calculated assuming plasma or tissue homogenate free valine specific radioactivity was representative of the valine precursor pool for protein synthesis. There were no significant differences between FSRMin and FSRMax in any gastrointestinal tissue of lambs used in Expt 1 (P greater than 0.05). FSR gradually and significantly (P less than 0.05) increased from the oesophagus (FSRMax 26.5%/d), reticulo-rumen (30.1%/d), omasum (41.0%/d) and abomasum (56.1%/d) to small intestine (87.5%/d), and then declined significantly (P less than 0.05) towards the caecum (45.2%/d) and the colon (38.4%/d). No significant differences were observed between FSR in the duodenum, jejunum or ileum (P greater than 0.05). 4. FGR ranged from 2.6%/d in the oesophagus to 8.7%/d in the omasum. The ratio, FGR:FSR, which reflected the efficiency of protein deposition, was at a maximum in the stomachs and caecum and at a minimum in the small intestine. 5. The relative contribution of the oesophagus, stomachs, small intestine and large intestine to GIT protein synthesis was 1, 13, 76 and 10% respectively. The GIT accounted for approximately 11.5% of whole-body protein synthesis.