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The Journal of Physiological Sciences

, Volume 68, Issue 2, pp 165–174 | Cite as

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-RomeroEmail author
  • Lucas Guimarães-Ferreira
  • Caio Yogi Yonamine
  • Rafael Barrera Salgueiro
  • Maria Tereza Nunes
Original Paper

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

HMB Protein synthesis Protein degradation EDL Muscle contraction 

Abbreviations

AIDS

Acquired immune deficiency syndrome

ANOVA

Analysis of variance

AU

Arbitrary units

AUC

Area under the curve

CSA

Cross-sectional area

DEXA

Dexametasone

ECL

Enhanced chemiluminescence

EDL

Extensor digitorum longus

FOXO

Forkhead box O

HMB

Beta-hydroxy-beta-methylbutyrate

HRT

Half-relaxation time

LRT

Late-relaxation time

mTOR

Mammalian target of rapamycin

PMSF

Phenylmethanesulfonylfluoride

TTP

Time to peak tension

TSI

Time of sustained isometric contraction

Notes

Acknowledgements

F. Gerlinger-Romero and L. Guimarães-Ferreira, for a research fellowship from São Paulo Research Foundation, CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior); C. Yogi Yonamine and R. Barrera Salgueiro, for a research fellowship from FAPESP (Fundação de Amparo Pesquisa do Estado de São Paulo); and M. T. Nunes, for a fellowship from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). The authors would like to thank Dr. John A. Rathmacher from Metabolic Technologies, Inc. HMB-FA was formulated and supplied by Metabolic Technologies, Inc. The late, for Adhemar Pettri Filho.

Compliance with ethical standards

Conflict of interest

The authors declare they have no conflict of interest.

Supplementary material

12576_2016_520_MOESM1_ESM.tif (755 kb)
Supplementary material 1 (TIFF 754 kb)

References

  1. 1.
    Aversa Z, Bonetto A, Costelli P, Minero VG, Penna F, Baccino FM, Lucia S, Rossi Fanelli F, Muscaritoli M (2011) β-Hydroxy-β-methylbutyrate (HMB) attenuates muscle and body weight loss in experimental cancer cachexia. Int J Oncol 38:713–720. doi: 10.3892/ijo.2010.885 CrossRefGoogle Scholar
  2. 2.
    Noh KK, Chung KW, Choi YJ, Park MH, Jang EJ, Park CH, Yoon C, Kim ND, Kim MK, Chung HY (2014) Beta-hydroxy beta-methylbutyrate improves dexamethasone-induced muscle atrophy by modulating the muscle degradation pathway in SD rat. PLoS ONE 9:e102947CrossRefGoogle Scholar
  3. 3.
    Zanchi NE, Gerlinger-Romero F, Guimarães-Ferreira L, de Siqueira Filho MA, Felitti V, Lira FS, Seelaender M, Lancha AH (2011) HMB supplementation: clinical and athletic performance-related effects and mechanisms of action. Amino Acids 40:1015–1025CrossRefGoogle Scholar
  4. 4.
    Pinheiro CH, Gerlinger-Romero F, Guimaraes-Ferreira L, de Souza-Jr AL, Vitzel KF, Nachbar RT, Nunes MT, Curi R (2012) Metabolic and functional effects of beta-hydroxy-beta-methylbutyrate (HMB) supplementation in skeletal muscle. Eur J Appl Physiol 112:2531–2537CrossRefGoogle Scholar
  5. 5.
    Kovarik M, Muthny T, Sispera L, Holecek M (2010) Effects of β-hydroxy-β-methylbutyrate treatment in different types of skeletal muscle of intact and septic rats. J Physiol Biochem 66:311–319CrossRefGoogle Scholar
  6. 6.
    Eley HL, Russell ST, Tisdale MJ (2008) Attenuation of depression of muscle protein synthesis induced by lipopolysaccharide, tumor necrosis factor, and angiotensin II by beta-hydroxy-beta-methylbutyrate. Am J Physiol Endocrinol Metab 295:E1409–E1416CrossRefGoogle Scholar
  7. 7.
    Lowery RP, Joy JM, Rathmacher JA, Baier SM, Fuller J Jr, Shelley MC 2nd, Jaeger R, Purpura M, Wilson SM, Wilson JM (2014) Interaction of beta-hydroxy-beta-methylbutyrate free acid (HMB-FA) and adenosine triphosphate (ATP) on muscle mass, strength, and power in resistance trained individuals. J Strength Cond Res 30(7):1843–1854CrossRefGoogle Scholar
  8. 8.
    Robinson EHT, Stout JR, Miramonti AA, Fukuda DH, Wang R, Townsend JR, Mangine GT, Fragala MS, Hoffman JR (2014) High-intensity interval training and beta-hydroxy-beta-methylbutyric free acid improves aerobic power and metabolic thresholds. J Int Soc Sports Nutr 11:16CrossRefGoogle Scholar
  9. 9.
    Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, Picard A, Walsh K, Schiaffino S, Lecker SH, Goldberg AL (2004) Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell 117:399–412CrossRefGoogle Scholar
  10. 10.
    Schiaffino S, Dyar KA, Ciciliot S, Blaauw B, Sandri M (2013) Mechanisms regulating skeletal muscle growth and atrophy. FEBS J 280(17):4294–4314CrossRefGoogle Scholar
  11. 11.
    Fitschen PJ, Wilson GJ, Wilson JM, Wilund KR (2013) Efficacy of beta-hydroxy-beta-methylbutyrate supplementation in elderly and clinical populations. Nutrition 29:29–36CrossRefGoogle Scholar
  12. 12.
    Eley HL, Russell ST, Baxter JH, Mukerji P, Tisdale MJ (2007) Signaling pathways initiated by beta-hydroxy-beta-methylbutyrate to attenuate the depression of protein synthesis in skeletal muscle in response to cachectic stimuli. Am J Physiol Endocrinol Metab 293:E923–E931CrossRefGoogle Scholar
  13. 13.
    Lang CH, Pruznak A, Navaratnarajah M, Rankine KA, Deiter G, Magne H, Offord EA, Breuille D (2013) Chronic alpha-hydroxyisocaproic acid treatment improves muscle recovery after immobilization-induced atrophy. Am J Physiol Endocrinol Metab 305(3):E416–E428CrossRefGoogle Scholar
  14. 14.
    Miramonti AA, Stout JR, Fukuda DH, Robinson EHT, Wang R, La Monica MB, Hoffman JR (2016) Effects of 4 weeks of high-intensity interval training and beta-hydroxy-beta-methylbutyric free acid supplementation on the onset of neuromuscular fatigue. J Strength Cond Res 30:626–634CrossRefGoogle Scholar
  15. 15.
    Russell DM, Atwood HL, Whittaker JS, Itakura T, Walker PM, Mickle DA, Jeejeebhoy KN (1984) The effect of fasting and hypocaloric diets on the functional and metabolic characteristics of rat gastrocnemius muscle. Clin Sci (Lond) 67:185–194CrossRefGoogle Scholar
  16. 16.
    Nishio ML, Madapallimattam AG, Jeejeebhoy KN (1992) Comparison of six methods for force normalization in muscles from malnourished rats. Med Sci Sports Exerc 24:259–264CrossRefGoogle Scholar
  17. 17.
    Wijngaarden MA, Bakker LE, van der Zon GC, t Hoen PA, van Dijk KW, Jazet IM, Pijl H, Guigas B (2014) Regulation of skeletal muscle energy/nutrient-sensing pathways during metabolic adaptation to fasting in healthy humans. Am J Physiol Endocrinol Metab 307:E885–E895CrossRefGoogle Scholar
  18. 18.
    Vendelbo MH, Moller AB, Christensen B, Nellemann B, Clasen BF, Nair KS, Jorgensen JO, Jessen N, Moller N (2014) Fasting increases human skeletal muscle net phenylalanine release and this is associated with decreased mTOR signaling. PLoS ONE 9:e102031CrossRefGoogle Scholar
  19. 19.
    Bodine SC, Baehr LM (2014) Skeletal muscle atrophy and the E3 ubiquitin ligases MuRF1 and MAFbx/atrogin-1. Am J Physiol Endocrinol Metab 307:E469–E484CrossRefGoogle Scholar
  20. 20.
    Foletta VC, White LJ, Larsen AE, Leger B, Russell AP (2011) The role and regulation of MAFbx/atrogin-1 and MuRF1 in skeletal muscle atrophy. Pflug Arch 461:325–335CrossRefGoogle Scholar
  21. 21.
    Gerlinger-Romero F, Guimarães-Ferreira L, Giannocco G, Nunes MT (2011) Chronic supplementation of beta-hydroxy-beta methylbutyrate (HMβ) increases the activity of the GH/IGF-I axis and induces hyperinsulinemia in rats. Growth Horm IGF Res 21:57–62CrossRefGoogle Scholar
  22. 22.
    Busquets S, Alvarez B, Lopez-Soriano FJ, Argiles JM (2002) Branched-chain amino acids: a role in skeletal muscle proteolysis in catabolic states? J Cell Physiol 191:283–289CrossRefGoogle Scholar
  23. 23.
    Harcourt LJ, Holmes AG, Gregorevic P, Schertzer JD, Stupka N, Plant DR, Lynch GS (2005) Interleukin-15 administration improves diaphragm muscle pathology and function in dystrophic mdx mice. Am J Pathol 166:1131–1141CrossRefGoogle Scholar
  24. 24.
    Farrell PA, Fedele MJ, Hernandez J, Fluckey JD, John L, Iii M, Lang CH, Vary TC, Kimball SR, Leonard S et al (1999) Hypertrophy of skeletal muscle in diabetic rats in response to chronic resistance exercise. J Appl Physiol 87(3):1075–1082CrossRefGoogle Scholar
  25. 25.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408CrossRefGoogle Scholar
  26. 26.
    Garfin DE (1990) One-dimensional gel electrophoresis. Methods Enzymol 182:425–441CrossRefGoogle Scholar
  27. 27.
    Yonamine CY, Teixeira SS, Campello RS, Gerlinger-Romero F, Rodrigues CF Jr, Guimaraes-Ferreira L, Machado UF, Nunes MT (2014) Beta hydroxy beta methylbutyrate supplementation impairs peripheral insulin sensitivity in healthy sedentary Wistar rats. Acta Physiol (Oxf) 212:62–74CrossRefGoogle Scholar
  28. 28.
    Bassit RA, Pinheiro CH, Vitzel KF, Sproesser AJ, Silveira LR, Curi R (2010) Effect of short-term creatine supplementation on markers of skeletal muscle damage after strenuous contractile activity. Eur J Appl Physiol 108:945–955CrossRefGoogle Scholar
  29. 29.
    Schiaffino S, Sandri M, Murgia M (2007) Activity-dependent signaling pathways controlling muscle diversity and plasticity. Physiology (Bethesda, Md: 1985) 22:269–278Google Scholar
  30. 30.
    Goldberg AL, Goodman HM (1969) Relationship between cortisone and muscle work in determining muscle size. J Physiol 200:667–675CrossRefGoogle Scholar
  31. 31.
    Nissen S, Sharp R, Ray M, Rathmacher JA, Rice D, Fuller JC, Connelly AS, Abumrad N (1996) Effect of leucine metabolite beta-hydroxy-beta-methylbutyrate on muscle metabolism during resistance-exercise training. J Appl Physiol (Bethesda, Md: 1985) 81:2095–2104CrossRefGoogle Scholar
  32. 32.
    Nissen SL, Abumrad NN (1997) Nutritional role of the leucine metabolite β-hydroxy β-methylbutyrate (HMB). J Nutr Biochem 8:300–311CrossRefGoogle Scholar
  33. 33.
    Whitehouse AS, Tisdale MJ (2001) Downregulation of ubiquitin-dependent proteolysis by eicosapentaenoic acid in acute starvation. Biochem Biophys Res Commun 285:598–602CrossRefGoogle Scholar
  34. 34.
    Lecker SH, Jagoe RT, Gilbert A, Gomes M, Baracos V, Bailey J, Price SR, Mitch WE, Goldberg AL (2004) Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression. FASEB J 18:39–51CrossRefGoogle Scholar
  35. 35.
    Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R, Zlotchenko E, Scrimgeour A, Lawrence JC, Glass DJ, Yancopoulos GD (2001) AKT/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol 3:1014–1019CrossRefGoogle Scholar
  36. 36.
    Townsend JR, Hoffman JR, Gonzalez AM, Jajtner AR, Boone CH, Robinson EH, Mangine GT, Wells AJ, Fragala MS, Fukuda DH, Stout JR (2015) Effects of beta-hydroxy-beta-methylbutyrate free acid ingestion and resistance exercise on the acute endocrine response. Int J Endocrinol 2015:856708CrossRefGoogle Scholar
  37. 37.
    Kornasio R, Riederer I, Butler-Browne G, Mouly V, Uni Z, Halevy O (2009) Beta-hydroxy-beta-methylbutyrate (HMB) stimulates myogenic cell proliferation, differentiation and survival via the MAPK/ERK and PI3K/AKT pathways. Biochim Biophys Acta 1793:755–763CrossRefGoogle Scholar
  38. 38.
    Jagoe RT, Lecker SH, Gomes M, Goldberg AL (2002) Patterns of gene expression in atrophying skeletal muscles: response to food deprivation. FASEB J 16:1697–1712CrossRefGoogle Scholar
  39. 39.
    Bouskila M, Hirshman MF, Jensen J, Goodyear LJ, Sakamoto K (2008) Insulin promotes glycogen synthesis in the absence of GSK3 phosphorylation in skeletal muscle. Am J Physiol Endocrinol Metab 294:E28–E35CrossRefGoogle Scholar
  40. 40.
    Zhao J, Brault JJ, Schild A, Cao P, Sandri M, Schiaffino S, Lecker SH, Goldberg AL (2007) FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell Metab 6:472–483CrossRefGoogle Scholar
  41. 41.
    Aversa Z, Alamdari N, Castillero E, Muscaritoli M, Rossi Fanelli F, Hasselgren PO (2012) β-Hydroxy-β-methylbutyrate (HMB) prevents dexamethasone-induced myotube atrophy. Biochem Biophys Res Commun 423:739–743CrossRefGoogle Scholar
  42. 42.
    Li JB, Goldberg AL (1976) Effects of food deprivation on protein synthesis and degradation in rat skeletal muscles. Am J Physiol 231:441–448CrossRefGoogle Scholar

Copyright information

© The Physiological Society of Japan and Springer Japan 2017

Authors and Affiliations

  1. 1.Department of Physiology and Biophysics, Institute of Biomedical SciencesUniversity of São PauloSão PauloBrazil
  2. 2.Exercise Metabolism Research Group, Department of Sports, Center of Physical Education and SportsFederal University of Espirito SantoVitoriaBrazil
  3. 3.Prédio Biomédicas I-Cidade Universitária-ButantãSão PauloBrazil

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