Contrarily to whey and high protein diets, dietary free leucine supplementation cannot reverse the lack of recovery of muscle mass after prolonged immobilization during ageing

Abstract

During ageing, immobilization periods increase and are partially responsible of sarcopaenia by inducing a muscle atrophy which is hardly recovered from. Immobilization-induced atrophy is due to an increase of muscle apoptotic and proteolytic processes and decreased protein synthesis. Moreover, previous data suggested that the lack of muscle mass recovery might be due to a defect in protein synthesis response during rehabilitation. This study was conducted to explore protein synthesis during reloading and leucine supplementation effect as a nutritional strategy for muscle recovery. Old rats (22–24 months old) were subjected to unilateral hindlimb casting for 8 days (I8) and allowed to recover for 10–40 days (R10–R40). They were fed a casein (±leucine) diet during the recovery. Immobilized gastrocnemius muscles atrophied by 20%, and did not recover even at R40. Amount of polyubiquitinated conjugates and chymotrypsin- and trypsin-like activities of the 26S proteasome increased. These changes paralleled an ‘anabolic resistance' of the protein synthesis at the postprandial state (decrease of protein synthesis, P-S6 and P-4E-BP1). During the recovery, proteasome activities remained elevated until R10 before complete normalization and protein synthesis was slightly increased. With free leucine supplementation during recovery, if proteasome activities were normalized earlier and protein synthesis was higher during the whole recovery, it nevertheless failed in muscle mass gain. This discrepancy could be due to a ‘desynchronization' between the leucine signal and the availability of amino acids coming from casein digestion. Thus, when supplemented with leucine-rich proteins (i.e. whey) and high protein diets, animals partially recovered the muscle mass loss.

Figure 1. Food intake (A) and body weight (B) of casted and non-casted PF rats

Food intake and body weight of casted and non-casted rats are similar during the whole experiment between the CON and the LEU groups and the STANDARD diet group. IM: immobilization period. Data are means ± SEM.

Figure 2. Ubiquitin–proteasome-dependent proteolysis in immobilized gastrocnemius muscles before and after the free leucine supplementation

A, accumulation of polyubiquitinated polyconjugates was assessed on 25 μg of proteins by immunoblotting using an antibody that recognizes polyubiquitin chains. B and C, the chymotrypsin-like activity (B) and trypsin-like activity (C) of the proteasome were measured by using the fluorogenic substrate succinyl-LLVY-AMC and Boc-LRR-AMC as indicated in Methods. Data are expressed in relative fluorescence units (RFU μg⁻¹ min⁻¹). D, phospho-FoxO3a/FoxO3a ratio traduces an anti-proteolytic potential. FoxO3a and its phosphorylated form phospho-FoxO3a (Ser253) were determined using appropriate antibodies on 50 μg of proteins. IM: immobilization period; I0: before immobilization; I8: 8 days of casting; R10 to R40: 10 to 40 days of recovery. Data are means ± SEM. As no effect of the meal was observed, postabsortive and postprandial values are pooled. *P < 0.05, vs. I0. Data are means ± SEM.

Figure 3. Muscle protein synthesis and protein pathway in immobilized gastrocnemius muscles before and after the free leucine supplementation

A, muscle protein synthesis at I0 and I8 in the postabsorptive and the postprandial states for each time is expressed as the absolute synthesis rate (ASR), i.e. the amount of proteins synthesized in mg day⁻¹. B and C, ASR during immobilization and recovery in the postabsorptive (B) and the postprandial states (C). D and E, protein S6 phosphorylation in gastrocnemius for the CON group (D) and the LEU group (E) in the postabsorptive and the postprandial states. The amount of protein S6 phosphorylated was assessed by immunoblotting on 30 μg of proteins. F and G, amount of protein 4EBP1 expressed in arbitrary units as the ratio γ form/total forms in the CON group (F) and LEU group (G). IM: immobilization period; I0: before immobilization; I8: 8 days of casting; R10 to R40: 10 to 40 days of recovery; PA: postabsorptive state; PP: postprandial state. *P < 0.05, PA vs. PP. Data are means ± SEM.

Figure 4. Muscle mass of gastrocnemius after immobilization and after free leucine supplementation

In immobilized muscles, muscle mass decreased after an 8 day immobilization period. The nutritional supplementation started after cast removal but muscle mass was not different between the CON group and the LEU group at R40 and never returned to its pre-immobilization value. IM: immobilization period; I0: before immobilization; I8: 8 days of casting; R10 to R40: 10 to 40 days of recovery. *P < 0.05, vs. I0. No significant differences between CON and LEU groups were recorded. Data are means ± SEM.

Figure 5. Effect of the whey and high protein diets on muscle mass gain, muscle protein synthesis and amino acid concentrations

A, muscle mass gain after the nutritional intervention is presented as the gain versus I8, in mg of muscle. Four diets were tested during the recovery period: a CON diet (13% casein), a LEU diet (13% casein + 4.45% Leucine), a WHEY diet (13% whey, i.e. leucine-rich and fast digested protein diet), and a HIGH PROTEIN diet (13% casein + 13% whey). *P < 0.05, vs. I8, Student's t test. Data are means ± SEM. B, muscle protein synthesis is expressed as the percentage of the absolute synthesis rate (ASR), i.e. the amount of proteins synthesized in mg day⁻¹, at I8. C, plasma amino acid concentrations expressed in μmol l⁻¹ were determined on animals fed each experimental diet at R20 and R40. I8: 8 days of casting; R20 and R40: 10 and 40 days of recovery; LEU: leucine. *P < 0.05, vs. CON and LEU; †P < 0.05, vs. I8.