J. Prugnaud

Effect of glucocorticoid excess on skeletal muscle and heart protein synthesis in adult and old rats

This study was carried out to analyse glucocorticoid-induced muscle wasting and subsequent recovery in adult (6-8 months) and old (18-24 months) rats because the increased incidence of various disease states results in hypersecretion of glucocorticoids in ageing. Adult and old rats received dexamethasone in their drinking water for 5 or 6 d and were then allowed to recover for 3 or 7 d. As dexamethasone decreased food intake, all groups were pair-fed to dexamethasone-treated old rats (i.e. the group that had the lowest food intake). At the end of the treatment, adult and old rats showed significant increases in blood glucose and plasma insulin concentrations. This increase disappeared during the recovery period. Protein synthesis of different muscles was assessed in vivo by a flooding dose of [13C]valine injected subcutaneously 50 min before slaughter. Dexamethasone induced a significant decrease in protein synthesis in fast-twitch glycolytic and oxidative glycolytic muscles (gastrocnemius, tibialis anterior, extensor digitorum longus). The treatment affected mostly ribosomal efficiency. Adult dexamethasone-treated rats showed an increase in protein synthesis compared with their pair-fed controls during the recovery period whereas old rats did not. Dexamethasone also significantly decreased protein synthesis in the predominantly oxidative soleus muscle but only in old rats, and increased protein synthesis in the heart of adult but not of old rats. Thus, in skeletal muscle, the catabolic effect of dexamethasone is maintained or amplified during ageing whereas the anabolic effect in heart is depressed. These results are consistent with muscle atrophy occurring with ageing.

Digestion of milk, fish and soya-bean protein in the preruminant calf: flow of digesta, apparent digestibility at the end of the ileum and amino acid composition of ileal digesta

1. Digesta were collected from eleven preruminant calves fitted with re-entrant (four calves in Expt 1 and three in Expt 2) or single cannulas (four calves in Expt 1) in the terminal ileum. Collection periods lasted 24 h (Expt 1) or 96 h (Expt 2). 2. Two milk-substitutes (fish and soya bean) and a control diet were given to the calves. In the control diet, protein was entirely provided by skim-milk powder. In the other two diets, protein was provided mainly by a partially hydrolysed white-fish protein concentrate or a soya-bean protein concentrate prepared by extracting soya-bean meal with hot aqueous ethanol. 3. In Expt 1, flow rates of fresh matter, dry matter, nitrogen and ash exhibited two maxima between 6 and 8 h after the morning meal and between 4 and 6 h (control and soya-bean diets) or 6 and 8 h (fish diet) after the evening meal. Minimum pH values were observed at times of maximum flow rate. Variations observed in the flow rates and pH values were larger with fish and especially soya-bean diets than with the control diet. 4. The apparent digestibility of the three diets in the terminal ileum was significantly higher in Expt 2 than in Expt 1: for N, the values were 0.92, 0.83 and 0.75 (Expt 1), and 0.94, 0.87 and 0.88 (Expt 2) with the control, fish and soya-bean diets respectively. 5. The amount of N apparently absorbed in the terminal ileum represented 90-96% of the amount that disappeared from the whole digestive tract in Expt 1 and 95-99% in Expt 2. 6. In Expt 1 the amino acid (AA) composition of digesta changed little with the flow rate when the calves were given the control diet (from 158 to 179 g glutamic acid/kg AA). With the fish and soya-bean diets the AA composition was similar to that observed with the control diet when the flow rate was minimum, but differences became apparent as the flow rate increased (281 and 161 g glutamic acid/kg AA for the soya-bean and control diets respectively with maximum flow rate). In Expt 2, the mean compositions of the digesta were very similar to the means obtained in Expt 1. 7. Different comparisons with dietary, endogenous and bacterial proteins indicated that for the three diets a common mixture containing approximately 65% endogenous and 35% bacterial proteins reached the terminal ileum.(ABSTRACT TRUNCATED AT 400 WORDS)

Assessment of threonine metabolism in vivo by gas chromatography/mass spectrometry and stable isotope infusion.

The fractional contributions (FC) of threonine to glycine and 2-ketobutyrate (KB) fluxes in fed pigs have been assessed by the constant infusion of L-[1-13C]-threonine. The analysis of the enantiomeric purity of labeled threonine by gas chromatography/mass spectrometric (GC/MS) analysis is reported as the N-TFA isopropyl ester derivative. The commercially available [1-13C]threonine comprised 98.7% of the L-enantiomer, enriched at 99 atom percentage excess (APE), and 1.3% of L-allo-threonine contaminant, also enriched at 99 APE. The enantiomeric purity of threonine in plasma of pigs infused for 10 h with [1-13C]threonine showed that the L-allo contaminant did not accumulate. The t-butyl dimethylsilyl derivatives of threonine, glycine, and 2-aminobutyrate (ABA) were used to measure the enrichment of these compounds in plasma and liver samples by GC/MS/selected ion monitoring analysis. Analyses were performed on between 1 and 5 nmol of each amino acid extracted from biological fluids and a 1:10 split injection. GC/MS parameters were assessed with standards at similar quantities and found to be satisfactory; e.g., injection of 1-10 nmol of glycine did not significantly alter the slope and the precision of the standard curve. The coefficient of variation of enrichment determination was less than 10% for standards enriched at 0.4 APE or more and biological samples enriched at 0.6 APE or greater. Within-animal coefficients of variation for four plasma samples obtained at equal intervals between 8 and 10 h of [1-13C]threonine infusion were 4, 21, and 24% for threonine, ABA, and glycine, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential Effects of Cooked Beans and Cooked Lentils on Protein Metabolism in Intestine and Muscle in Growing Rats

Aims: The effect of diets based on cooked beans or lentils on protein metabolism in intestines and muscles was studied in rats. Method: The cooked seeds were used as the unique protein source in balanced diets (containing 229 and 190 g of crude protein per kg dry matter) fed to young growing rats for 20 days. Their effects were compared with those of the control casein diet in pair-fed rats. Protein synthesis rates in small and large intestines and in gastrocnemius and soleus muscles were determined in vivo, in a fed state, by the flooding dose method, using 13C-valine. Results: In the small and large intestine tissues of the legume fed groups, protein, RNA relative masses (mg·100 g BM–1) and protein synthesis rates (FSR and ASR) were higher than in the control rats (p < 0.05). In gastrocnemius and soleus muscles,protein and RNA contents (in mg) and protein synthesis rates were significantly (p < 0.05) lower in the legume-fed groups than in the control rats. Conclusion: The chronic intake of cooked legumes increased protein synthesis rates in intestinal tissues and decreased them in muscles. This effect was greater for beans than for lentils in the large intestine and in gastrocnemius muscle.

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]