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. 2016 Mar;7(1):68-78.
doi: 10.1002/jcsm.12032. Epub 2015 May 14.

Conversion of leucine to β-hydroxy-β-methylbutyrate by α-keto isocaproate dioxygenase is required for a potent stimulation of protein synthesis in L6 rat myotubes

María D Girón  1 , José D Vílchez  1 , Rafael Salto  1 , Manuel Manzano  2 , Natalia Sevillano  1 , Nefertiti Campos  2 , Josep M Argilés  3 , Ricardo Rueda  2 , José M López-Pedrosa  2
Affiliations

Conversion of leucine to β-hydroxy-β-methylbutyrate by α-keto isocaproate dioxygenase is required for a potent stimulation of protein synthesis in L6 rat myotubes

María D Girón et al. J Cachexia Sarcopenia Muscle. 2016 Mar.

Abstract

Background: L-Leu and its metabolite β-hydroxy-β-methylbutyrate (HMB) stimulate muscle protein synthesis enhancing the phosphorylation of proteins that regulate anabolic signalling pathways. Alterations in these pathways are observed in many catabolic diseases, and HMB and L-Leu have proven their anabolic effects in in vivo and in vitro models. The aim of this study was to compare the anabolic effects of L-Leu and HMB in myotubes grown in the absence of any catabolic stimuli.

Methods: Studies were conducted in vitro using rat L6 myotubes under normal growth conditions (non-involving L-Leu-deprived conditions). Protein synthesis and mechanistic target of rapamycin signalling pathway were determined.

Results: Only HMB was able to increase protein synthesis through a mechanism that involves the phosphorylation of the mechanistic target of rapamycin as well as its downstream elements, pS6 kinase, 4E binding protein-1, and eIF4E. HMB was significantly more effective than L-Leu in promoting these effects through an activation of protein kinase B/Akt. Because the conversion of L-Leu to HMB is limited in muscle, L6 cells were transfected with a plasmid that codes for α-keto isocaproate dioxygenase, the key enzyme involved in the catabolic conversion of α-keto isocaproate into HMB. In these transfected cells, L-Leu was able to promote protein synthesis and mechanistic target of rapamycin regulated pathway activation equally to HMB. Additionally, these effects of leucine were reverted to a normal state by mesotrione, a specific inhibitor of α-keto isocaproate dioxygenase.

Conclusion: Our results suggest that HMB is an active L-Leu metabolite able to maximize protein synthesis in skeletal muscle under conditions, in which no amino acid deprivation occurred. It may be proposed that supplementation with HMB may be very useful to stimulate protein synthesis in wasting conditions associated with chronic diseases, such as cancer or chronic heart failure.

Keywords: Protein synthesis; Skeletal muscle; mTOR, leucine; β‐Hydroxy‐β‐methylbutyrate.

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Figures

Figure 1
Figure 1
Effects of L‐Leu and HMB on protein synthesis and mTOR activation in L6 myotubes. Protein synthesis was measured following 2 h of 10 mM L‐Leu, 50 μM HMB, or 50 nM insulin supplementation in growth medium (A) and in the absence or presence of PD98059 and LY294002 (B). Inhibitors were added 30 min prior to the L‐Leu or HMB addition. Obtained data were expressed as dpm/µg of proteins (n = 5). Results are expressed as mean ± SEM. a P < 0.05, when compared with untreated control cells; b P < 0.05 compared with leucine treated cells; c P < 0.05 compared with HMB treated cells.
Figure 2
Figure 2
Regulation of mTOR signalling by 10 mM L‐Leu and 50 μM HMB in L6 myotubes. Phosphorylation of PKB/Akt (A), mTOR (B) S6K1 (C), 4E‐BP1 (D), and eIF4E (E) was assayed by western blot. Results are expressed as mean ± SEM (n = 4). a P < 0.05, when compared with untreated control cells; b P < 0.05 compared with leucine treated cells.
Figure 3
Figure 3
Expression profile of the rat KICD. (A) Expression of KICD in L6 myotubes (a) western blot corresponding to 20 µg of proteins from liver, L6 cells (L6) and L6 cells transfected with pKICD plasmid (L6‐t). Protein loading amounts were normalized using a GAPDH antibody. (b) RT‐PCR of KICD from total RNA of non‐transfected and transfected L6 myotubes is shown using β‐actin as reference gene. (B) Expression of KICD in rat tissues. (a) Western blot corresponding to 20 µg of proteins from the liver (L), kidney (K), brain (B), soleus muscle (M), or diaphragm (D) probed with an anti‐4HPPD antibody. Arrows in the figure mark the expected size band for the rat KICD and rat GADPH. (b) RT‐PCR of KICD from total RNA of the same tissues is shown using β‐actin as a reference gene.
Figure 4
Figure 4
Effects of L‐Leu and HMB on protein synthesis and mTOR phosphorylation in KICD L6 transfected myotubes. (A) Protein synthesis rates in the presence of 10 mM D‐Leu, 10 mM L‐Leu, 50 μM HMB, or 50 nM insulin. Data were computed as dpm/µg of proteins (n = 8). (B) Phosphorylation of mTOR by 10 mM D‐Leu, 10 mM L‐Leu, or 50 μM HMB. (C) Phosphorylation of mTOR and protein synthesis by 10 mM L‐Leu and 50 μM HMB in (D) the absence and presence of 1 μM mesotrione. Results are expressed as mean ± SEM (n = 4). a P < 0.05, when compared with untreated control transfected cells; b P < 0.05, when compared with mesotrione treated control transfected cells.
Figure 5
Figure 5
Upstream and downstream signalling by L‐Leu or HMB in KICD transfected L6 myotubes. Phosphorylation of PKB/Akt (A), S6K1 (B), 4E‐BP1 (C), and eIF4E (D) by 10 mM L‐Leu and 50 μM HMB was assayed by western‐Blot. Results are expressed as mean ± SEM (n = 4). a P < 0.05, when compared with untreated control transfected cells.
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