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. 2018 Mar;68(2):165-174.
doi: 10.1007/s12576-016-0520-x. Epub 2017 Jan 12.

Effects of beta-hydroxy-beta-methylbutyrate (HMB) on the expression of ubiquitin ligases, protein synthesis pathways and contractile function in extensor digitorum longus (EDL) of fed and fasting rats

Frederico Gerlinger-Romero  1   2 , Lucas Guimarães-Ferreira  3   4 , Caio Yogi Yonamine  3 , Rafael Barrera Salgueiro  3 , Maria Tereza Nunes  3
Affiliations

Effects of beta-hydroxy-beta-methylbutyrate (HMB) on the expression of ubiquitin ligases, protein synthesis pathways and contractile function in extensor digitorum longus (EDL) of fed and fasting rats

Frederico Gerlinger-Romero et al. J Physiol Sci. 2018 Mar.

Abstract

Beta-hydroxy-beta-methylbutyrate (HMB), a leucine metabolite, enhances the gain of skeletal muscle mass by increasing protein synthesis or attenuating protein degradation or both. The aims of this study were to investigate the effect of HMB on molecular factors controlling skeletal muscle protein synthesis and degradation, as well as muscle contractile function, in fed and fasted conditions. Wistar rats were supplied daily with HMB (320 mg/kg body weight diluted in NaCl-0.9%) or vehicle only (control) by gavage for 28 days. After this period, some of the animals were subjected to a 24-h fasting, while others remained in the fed condition. The EDL muscle was then removed, weighed and used to evaluate the genes and proteins involved in protein synthesis (AKT/4E-BP1/S6) and degradation (Fbxo32 and Trim63). A sub-set of rats were used to measure in vivo muscle contractile function. HMB supplementation increased AKT phosphorylation during fasting (three-fold). In the fed condition, no differences were detected in atrogenes expression between control and HMB supplemented group; however, HMB supplementation did attenuate the fasting-induced increase in their expression levels. Fasting animals receiving HMB showed improved sustained tetanic contraction times (one-fold) and an increased muscle to tibia length ratio (1.3-fold), without any cross-sectional area changes. These results suggest that HMB supplementation under fasting conditions increases AKT phosphorylation and attenuates the increased of atrogenes expression, followed by a functional improvement and gain of skeletal muscle weight, suggesting that HMB protects skeletal muscle against the deleterious effects of fasting.

Keywords: EDL; HMB; Muscle contraction; Protein degradation; Protein synthesis.

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Conflict of interest statement

The authors declare they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Experimental design
Fig. 2
Fig. 2
Myogram representative images. Isotonic contractions were analyzed and are shown in Fig. 6 and time parameters (TTP, HRT, LRT) in Table 1. Tetanic contractions were analyzed and shown in Fig. 7 and times of sustained isometric contraction (TSI) in Table 1
Fig. 3
Fig. 3
Weight of EDL muscle; in white Fed:Control and Fasting:Control and in black Fed:HMB and Fasting:HMB; values are mean ± SEM (n = 9–14 animals/group); asterisk vs. Fed:HMB; Control (p < 0.0001) and Fasting:HMB (p < 0.001); effect of supplementation (p < 0.05); effect of fasting (p < 0.0001) and interaction: supplementation and fasting (p < 0.001)
Fig. 4
Fig. 4
Real-time PCR analysis of Fbxo32 and Trim63 mRNA expression, respectively, in EDL muscle normalized to Gapdh. Data are expressed as mean ± SEM (n = 8–10 animals/group), in arbitrary units (AU). In white Fed:Control and Fasting:Control and in black Fed:HMB and Fasting:HMB; double asterisks vs. Fed (p < 0.001); in (a) asterisk vs. Fed:Control and HMB (p < 0.05) and in (b) asterisk vs. Fed:Control (p < 0.05)
Fig. 5
Fig. 5
Western blotting analysis of AKT, 4EBP1 and S6; content of phosphorylation/total (a, b, c) for each protein, respectively. Quantitative representation obtained by densitometric analysis is shown. Data are expressed as mean ± SEM (n = 6–9 animals/group), in arbitrary units (AU). in white Fed:Control and Fasting:Control and in black Fed:HMB and Fasting:HMB. Asterisk effect of supplementation (p = 0.0316) (a); hash interaction supplementation and fasting (p = 0.0281) and asterisk effect of fasting (p = 0.0329) (b)
Fig. 6
Fig. 6
Fiber cross-sectional area (CSA) analysis of EDL muscle. In white Fed:Control and Fasting:Control and in black Fed:HMB and Fasting:HMB. a Quantitative analysis of the CSA in μm2, measuring the circumference of no less than 800 adjacent fibers per animal (n = 5 animals/group); b showing the distribution of CSAs
Fig. 7
Fig. 7
Data of maximum skeletal muscle strength production in EDL. Muscle twitch force was determined at 1 Hz and tetanic force was at 100 Hz electrical stimulation frequency. a Muscle twitch force; b muscle tetanic force; c specific muscle twitch force normalized per CSA (fed vs. fasting, p = 0.07); d muscle tetanic force normalized per CSA. Results are expressed as mean ± SEM (n = 6 animals/group); in white Fed:Control and Fasting:Control and in black Fed:HMB and Fasting:HMB
Fig. 8
Fig. 8
Resistance to acute fatigue in contracting EDL muscle. Successive tetanic contractions were evocked at 100 Hz each 10 s of interval. a Muscle tension during successive tetanic contractions; b resistance to fadigue index was calculated as area under the curve to each animal. In white Fed:Control and Fasting:Control and in black Fed:HMB and Fasting:HMB. Data are expressed as mean ± SEM (n = 6 animals/group)
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