Maurice Arnal

Age-related changes in protein synthesis measured in vivo in rat liver and gastrocnemius muscle

This study analyses in detail the effects of ageing on gastrocnemius muscle and liver protein synthesis measured in vivo at three ages, 1.5 months (young), 12 months (adult) and 24 months (old) in Sprague-Dawley rats. Comparing adult and old rats, muscle protein synthesis was decreased in old rats when expressed per unit of RNA and per day (translational efficiency), was unchanged when expressed in absolute terms and increased when expressed in fractional terms as a result of protein loss due to muscle atrophy. In the liver, only translational efficiency tended to decrease in old rats compared to adult rats. It is concluded that the decline in protein turnover described in vitro is consistent with a decrease in translational efficiency, but that absolute synthesis rates are maintained during ageing. Muscle atrophy is unlikely to result from alterations in protein synthesis pathways.

Assessment of in vivo protein synthesis in lamb tissues with [3H]valine flooding doses

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.

The effect of a high dose of 3-hydroxy-3-methylbutyrate on protein metabolism in growing lambs

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.

Influence of dietary leucine content on the activities of branched-chain amino acid aminotransferase ( EC 2.6.1.42) and branched-chain α-keto acid dehydrogenase ( EC 1.2.4.4) complex in tissues of preruminant lambs

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.

Variations through the day of hepatic and muscular cathepsin A (carboxypeptidase A; EC 3.4.12.2), C (dipeptidyl peptidase; EC 3.4.14.1) and D (endopeptidase D; EC 3.4.23.5) activities and free amino acids of blood in rats: influence of feeding schedule.

1. Growing rats were fed either ad lib. or with six (equal) meals offered every 4 h (from 10.00 hours). Rats of each group were killed at intervals of 4 h beginning at 11.00 hours. Activities of cathepsin A (carboxypeptidase A; EC 3.4.12.2), C (dipeptidyl peptidase; EC 3.4.14.1) and D (endopeptidase D EC 3.4.23.5) were measured in liver and muscle homogenates and free amino acids in blood were determined. 2. In the rats fed ad lib. activities of carboxypeptidase A and endopeptidase D in liver and muscle showed significant variation, with maximum activity in the light period. In general, meal-feeding only caused minor differences in cathepsin activities; although significant differences occurred for carboxypeptidase A. For the later enzyme a peak in activity occurred in the dark as well as in the light period. 3. Irrespective of the feeding schedule, the lower concentration of free essential amino acids of blood occurred generally during the night period. With the controlled-feeding schedule there is an increase of essential amino acids and a slight decrease of non-essentail amino acids of blood.

Brief Fasting Decreases Protein Synthesis in the Brain of Adult Rats

The influence of starvation on protein synthesis in the adult rat brain was studied in vivo by an intravenous injection of a flooding dose of unlabeled valine including a tracer dose ofL-[3,4(n)-3H]valine. Brief starvation (24 hours) induced a 20% decline in fractional and absolute rates of brain protein synthesis. This decline resulted from a 20% decrease in the efficiency of protein synthesis (μg protein synthesized per day per μg RNA) whereas the capacity for protein synthesis (μg RNA per mg protein) was maintained. Prolonged starvation (5 days) was marked by no further significant changes in the fractional rate, absolute rate and efficiency of protein synthesis, whereas the capacity for protein synthesis cecreased slightly. The relative contribution of brain to wholebody body protein synthesis increased during fasting, and neither the protein nor the RNA brain content did change during the experiment. These results clearly indicate that brain proteins are spared in response to brief and prolonged food deprivation, and that brain protein synthesis is very sensitive to short-term fasting.

Despite similar rates of alanine release, fasting and diabetes affect de novo alanine synthesis differently

Dear Sir, Diabetes and fasting have long been known to induce a substantial release of alanine from muscle whereas the intramuscular alanine pool remains unchanged [1, 2]. The substantial alterations of alanine metabolism in fasting and diabetes are not completely understood, especially with regard to the contribution of leucine as nitrogen donor in alanine de novo synthesis. Alanine remains an important gluconeogenic precursor in humans [3, 4], although glutamine was recently reported as a major source of carbon for gluconeogenesis in diabetic or postabsorptive humans [5, 6]. For this reason, we recently assessed, by using [15N]leucine, the capacity of skeletal muscle to synthesise alanine de novo through leucine transamination in experimental diabetic rats. Indeed, few studies have addressed either the direct effect of leucine supply on both synthesis (within the muscle) and release of alanine (from the muscle) or the contribution of proteolysis and de novo synthesis to alanine production in muscle. For this purpose, we used 15N/1H NMR combined with GC-MS. We have previously demonstrated that streptozotocin-induced diabetes in growing rats is associated with: 1) an increase in nitrogen exchange between leucine and alanine leading to newly synthesised [15N]alanine; and 2) an increase in total alanine release from muscle originating from both proteolysis and de novo synthesis [7]. In the present letter, we would like to report some data obtained with extensor digitorum longus muscles from fasted rats in order to compare nitrogen exchange between leucine and alanine in 48 h-fasting and in experimental diabetes. We observed that the size of [15N]alanine pool within the muscle was smaller in fasted than in diabetic and control extensor digitorum longus muscles (Table 1). Yet, in order to evaluate the true nitrogen transfer between leucine and alanine, both the labelled intramuscular alanine pool (ML) and the labelled alanine released (RL) at the end of experiment should be taken into account. For this reason, we calculated the sum of these two components under each experimental condition (see Table 1). Surprisingly, nitrogen exchange was less intense in fasted than in diabetic rats. Moreover, it should be pointed out that the intramuscular total alanine pool was severely depressed by 48 h-fasting. This could be related to the extensive alanine utilisation in the whole body during the period of fasting before the superfusion experiment. By contrast, a similar increase in total alanine release from muscle of fasted and streptozotocin-diabetic rats (Table 1) was observed. Even though 48 h-fasting had a greater effect on proteolysis than did streptozotocin-diabetes (as reflected by the measurement of net tyrosine release, e. g. 292 ± 25 vs 186 ± 12 nmol tyrosine × g-1 × 2 h-1), the percentage of alanine release originating from proteolysis was the same in fasted muscle as it is in streptozotocin-diabetic extensor digitorum longus muscles (approximatively 60 %). Consequently, leucine contributed to approximately 40 % of alanine a-amino N released in both control and diabetic rats. Only a concomitant increase in proteolysis and de novo synthesis can explain the increase in alanine released from fasted extensor digitorum longus muscles, as previously reported in experimental diabetes [7]. In conclusion, these results confirm the interest of studying both muscle and medium superfusion compartments to better understand alanine regulation within the muscle of fasted rats. Surprisingly, leucine appears less efficient as a donor of a-amino N for the synthesis of alanine in skeletal muscle from fasted than in streptozotocin-diabetic rat even though leucine transamination has been previously described as being increased to a similar extent by fasting or diabetes [8]. These data confirm that alanine synthesis within the muscle may be limited by the availability of amino group acceptors [2]. Whereas a-ketoglurate is added to the medium of superfusion (3 mmol/l) regardless of the animals conditions, glucose is presumably extensively oxidized in superfused extensor digitorum longus muscles from fasted rats, as reflected by the high pH value (7±7.3) in comparison to fed muscles [9]. Consequently, decreased availability of pyruvate may be a limiting factor in glucose-superfused extensor digitorum longus muscles from fasted rats, explaining a lower rate of alanine synthesis in fasted rats than in diabetic rats.

Contribution of proteolysis and de novo synthesis to alanine production in diabetic rat skeletal muscle : a 15N/1H nuclear magnetic resonance study

Summary To assess the role of leucine as a precursor of alanine α-amino nitrogen in skeletal muscle during diabetes, extensor digitorum longus muscles from control (n = 7 experiments) and streptozotocin-diabetic rats (n = 8 experiments) were isolated and superfused with [15N]leucine (3 mmol/l) in the presence of glucose (10 mmol/l) for 2 h. Muscle perchloric acid extraction was performed at the end of superfusion in order to quantify newly synthesized alanine by 15N/1H nuclear magnetic resonance. Release of [15N]alanine in the superfusion medium was also measured. The pool of newly synthesized [15N]alanine was significantly increased ( ∼ 40 %) in extensor digitorum longus muscles from streptozotocin-diabetic rats. Whereas a significant enhancement of total alanine release from muscle was induced by diabetes (20 %), only a slight increase in [15N]alanine release was detectable under our experimental conditions. Consequently, we conclude that streptozotocin-diabetes in growing rats induces in skeletal muscle: 1) an increase in nitrogen exchange between leucine and alanine leading to newly synthesized [15N]alanine; and 2) an increase of total alanine release from muscle originating from both proteolysis and de novo synthesis. [Diabetologia (1997) 40: 1159–1165]