Effects of leucine and its metabolite β-hydroxy-β-methylbutyrate on human skeletal muscle protein metabolism

Abstract

Maintenance of skeletal muscle mass is contingent upon the dynamic equilibrium (fasted losses-fed gains) in protein turnover. Of all nutrients, the single amino acid leucine (Leu) possesses the most marked anabolic characteristics in acting as a trigger element for the initiation of protein synthesis. While the mechanisms by which Leu is 'sensed' have been the subject of great scrutiny, as a branched-chain amino acid, Leu can be catabolized within muscle, thus posing the possibility that metabolites of Leu could be involved in mediating the anabolic effect(s) of Leu. Our objective was to measure muscle protein anabolism in response to Leu and its metabolite HMB. Using [1,2-(13)C2]Leu and [(2)H5]phenylalanine tracers, and GC-MS/GC-C-IRMS we studied the effect of HMB or Leu alone on MPS (by tracer incorporation into myofibrils), and for HMB we also measured muscle proteolysis (by arteriovenous (A-V) dilution). Orally consumed 3.42 g free-acid (FA-HMB) HMB (providing 2.42 g of pure HMB) exhibited rapid bioavailability in plasma and muscle and, similarly to 3.42 g Leu, stimulated muscle protein synthesis (MPS; HMB +70% vs. Leu +110%). While HMB and Leu both increased anabolic signalling (mechanistic target of rapamycin; mTOR), this was more pronounced with Leu (i.e. p70S6K1 signalling 90 min vs. 30 min for HMB). HMB consumption also attenuated muscle protein breakdown (MPB; -57%) in an insulin-independent manner. We conclude that exogenous HMB induces acute muscle anabolism (increased MPS and reduced MPB) albeit perhaps via distinct, and/or additional mechanism(s) to Leu.

Figure 1. Gene expression of HPD

Upper panel, mouse tissues; lower panel, human skeletal muscle, with standard RT-PCR temperature gradient program shown for lower panel.

Figure 2

Study designs for assessing the anabolic effects of HMB (A) and Leu (B)

Figure 3. Plasma HMB (A), Leu (B) and insulin (C) in response to oral HMB (open circle) or Leu (filled circle) consumption

Left y-axis represents scale for HMB group, right y-axis represents scale for Leu group. Letters indicate statistical significance (P < 0.05): a = different from respective basal; b = different between groups at equivalent time-point. Data are presented as means ± SEM.

Figure 4. Intramuscular concentrations of HMB (A), EAA (B) and Leu (C) in response to oral HMB (open circle) or Leu (filled circle) consumption

Dashed line in A indicates intramuscular HMB concentration was below detection limit. Letters indicate statistical significance (P < 0.05): a = different from respective basal; b = different between groups at equivalent time point. Data are presented as means ± SEM.

Figure 5. Myofibrillar FSR in response to oral HMB (open circle) or Leu (filled circle) consumption (A) and leg proteolysis in response to HMB consumption (B)

Letters indicate statistical significance (P < 0.05): a = different from respective basal (a P < 0.05; aa P < 0.01). Data are presented as means ± SEM.

Figure 6. Beclin 1 (A), Mafbx (B), MuRF 1 (C), Calpain 1 (D), Cathepsin L (E) in response to oral HMB consumption

Data are presented as mean ± SEM.

Figure 7. AKTSer473 (A), p70S6K1Thr389 (B), 4EBP1Ser65/Thr70 (C), 4EBP1Thr37/46 (D), eIF2αSer51 (E), eEF2Thr56 (F) in response to oral HMB (open circle) or Leu (filled circle) consumption

Statistical notations: a = different from respective basal; (P < 0.05), b = different between groups at equivalent time-point (P < 0.05), c = different from respective basals under both conditions (P < 0.05). Data are presented as means ± SEM.