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Acute hyperketonaemia alters T-cell-related cytokine gene expression within stimulated peripheral blood mononuclear cells following prolonged exercise

  • David M. ShawEmail author
  • Fabrice Merien
  • Andrea Braakhuis
  • Lauren Keaney
  • Deborah K. Dulson
Original Article

Abstract

Purpose

We investigated the effect of the racemic β-hydroxybutyrate precursor, R,S-1,3-butanediol (BD), on T-cell-related cytokine gene expression within stimulated peripheral blood mononuclear cells (PBMC) following prolonged, strenuous exercise.

Methods

A repeated-measures, randomised, crossover study was conducted in nine healthy, trained male cyclists (age, 26.7 ± 5.2 years; VO2peak, 63.9 ± 2.5 mL kg−1 min−1). Participants ingested 0.35 g kg−1 of BD or placebo 30 min before and 60 min during 85 min of steady-state (SS) exercise, which preceded a ~ 30 min time-trial (TT) (7 kJ kg−1). Blood samples were collected at pre-supplement, pre-exercise, post-SS, post-TT and 1-h post-TT. Whole blood cultures were stimulated with Staphylococcal enterotoxin B (SEB) for 24 h to determine T-cell-related interleukin (IL)-4, IL-10 and interferon (IFN)-γ mRNA expression within isolated PBMCs in vitro.

Results

Serum cortisol, total circulating leukocyte and lymphocyte, and T-cell subset concentrations were similar between trials during exercise and recovery (all p > 0.05). BD ingestion increased T-cell-related IFN-γ mRNA expression compared with placebo throughout exercise and recovery (p = 0.011); however, IL-4 and IL-10 mRNA expression and the IFN-γ/IL-4 mRNA expression ratio were unaltered (all p > 0.05).

Conclusion

Acute hyperketonaemia appears to transiently amplify the initiation of the pro-inflammatory T-cell-related IFN-γ response to an immune challenge in vitro during and following prolonged, strenuous exercise; suggesting enhanced type-1 T-cell immunity at the gene level.

Keywords

Ketone supplement Nutritional ketosis Immune Lymphocyte Type-1/type-2 Interleukin Interferon 

Abbreviations

AcAc

Acetoacetate

ANOVA

Analysis of variance

β2-MG

β2-Microglobulin

BD

R,S-1,3-Butanediol

CD

Cluster of differentiation

cDNA

Complementary deoxyribonucleic acid

CO2

Carbon dioxide

D-βHB

D-beta-hydroxybutyrate

ES

Effect size

FBS

Foetal bovine serum

HR

Heart rate

IFN

Interferon

IL

Interleukin

K2EDTA

Dipotassium ethylenediamine tetra-acetic acid

KB

Ketone body

kJ

Kilojoules

mRNA

Messenger ribonucleic acid

PBMC

Peripheral blood mononuclear cell

PBS

Phosphate-buffered saline

PLA

Placebo

RT-PCR

Reverse transcriptase polymerase chain reaction

SEB

Staphylococcal enterotoxin B

SS

Steady-state

T-cell

T-lymphocyte

TT

Time-trial

URTS

Upper respiratory tract symptom

VO2max

Maximal oxygen uptake

VO2peak

Peak oxygen uptake

VT2

Second ventilatory threshold

Wmax

Maximal wattage

Notes

Acknowledgements

The authors would like to thank the participants for their effort, cooperation and humour.

Author contributions

The study was designed by DS, FM, AB and DD; data were collected by DS; data interpretation and manuscript preparation were undertaken by DS, FM, AB, LK and DD. All authors approved the final version of the paper.

Compliance with ethical standards

Conflict of interest

The authors do not have any conflicts of interest.

Supplementary material

421_2019_4263_MOESM1_ESM.docx (235 kb)
Supplementary file1 (DOCX 236 kb)

References

  1. Agarwal SK, Marshall GD (1998) Glucocorticoid-induced type 1/type 2 cytokine alterations in humans: a model for stress-related immune dysfunction. J Interf Cytokine Res 18:1059–1068.  https://doi.org/10.1089/jir.1998.18.1059 CrossRefGoogle Scholar
  2. Ardawi MSM, Newsholme EA (1984) Metabolism of ketone bodies, oleate and glucose in lymphocytes of the rat. Biochem J 221:255–260.  https://doi.org/10.1042/bj2210255 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Beaver WL, Wasserman K, Whipp BJ (1986) A new method for detecting anaerobic threshold by gas exchange. J Appl Physiol 60:2020–2027CrossRefGoogle Scholar
  4. Bermon S, Castell LM, Calder PC et al (2017) Consensus statement: immunonutrition and exercise. Exerc Immunol Rev 23:8–50PubMedGoogle Scholar
  5. Bishop NC, Walker GJ, Bowley LA et al (2005) Lymphocyte responses to influenza and tetanus toxoid in vitro following intensive exercise and carbohydrate ingestion on consecutive days. J Appl Physiol 99:1327–1335.  https://doi.org/10.1152/japplphysiol.00038.2005 CrossRefPubMedGoogle Scholar
  6. Boeuf P, Vigan-Womas I, Jublot D et al (2005) CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use. BMC Immunol.  https://doi.org/10.1186/1471-2172-6-5 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Campbell JP, Riddell NE, Burns VE et al (2009) Acute exercise mobilises CD8+ T lymphocytes exhibiting an effector-memory phenotype. Brain Behav Immun 23:767–775CrossRefGoogle Scholar
  8. Choi YW, Kotzin B, Herron L et al (1989) Interaction of Staphylococcus aureus toxin “superantigens” with human T cells. Proc Natl Acad Sci 86:8941–8945.  https://doi.org/10.1073/pnas.86.22.8941 CrossRefPubMedGoogle Scholar
  9. Clifford T, Wood MJ, Stocks P et al (2017) T-regulatory cells exhibit a biphasic response to prolonged endurance exercise in humans. Eur J Appl Physiol 117:1727–1737.  https://doi.org/10.1007/s00421-017-3667-0 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Cox PJ, Kirk T, Ashmore T et al (2016) Nutritional ketosis alters fuel preference and thereby endurance performance in athletes. Cell Metab 24:256–268.  https://doi.org/10.1016/j.cmet.2016.07.010 CrossRefPubMedGoogle Scholar
  11. Curi R, Williams JF, Newsholme EA (1989) Formation of ketone-bodies by resting lymphocytes. Int J Biochem 21:1133–1136CrossRefGoogle Scholar
  12. Davison G, Kehaya C, Diment BC, Walsh NP (2015) Carbohydrate supplementation does not blunt the prolonged exercise-induced reduction of in vivo immunity. Eur J Nutr 55:1583–1593.  https://doi.org/10.1007/s00394-015-0977-z CrossRefPubMedGoogle Scholar
  13. Dill DB, Costill DL (1974) Calculation of percentage changes in volumes of blood, plasma, and red cells in dehydration. J Appl Physiol 37:247–248CrossRefGoogle Scholar
  14. Diment BC, Fortes MB, Edwards JP et al (2015) Exercise intensity and duration effects on in vivo immunity. Med Sci Sport Exerc 47:1390–1398.  https://doi.org/10.1249/MSS.0000000000000562 CrossRefGoogle Scholar
  15. Elenkov IJ (2004) Glucocorticoids and the Th1/Th2 balance. Ann N Y Acad Sci 1024:138–146.  https://doi.org/10.1196/annals.1321.010/full CrossRefPubMedGoogle Scholar
  16. Evans M, Egan B (2018) Intermittent running and cognitive performance after ketone ester ingestion. Med Sci Sports Exerc 50:2330–2338.  https://doi.org/10.1249/MSS.0000000000001700 CrossRefPubMedGoogle Scholar
  17. Evans M, McSwiney FT, Brady AJ, Egan B (2019) No benefit of ingestion of a ketone monoester supplement on 10-km running performance. Med Sci Sports Exerc.  https://doi.org/10.1249/MSS.0000000000002065 CrossRefPubMedGoogle Scholar
  18. Fabbri M (2003) T lymphocytes. Int J Biochem Cell Biol 35:1004–1008.  https://doi.org/10.1016/S1357-2725(03)00037-2 CrossRefPubMedGoogle Scholar
  19. Fox CJ, Hammerman PS, Thompson CB (2005) Fuel feeds function: energy metabolism and the T-cell response. Nat Rev Immunol 5:844–852.  https://doi.org/10.1038/nri1710 CrossRefPubMedGoogle Scholar
  20. Gjevestad GO, Holven KB, Ulven SM (2015) Effects of exercise on gene expression of inflammatory markers in human peripheral blood cells: a systematic review. Curr Cardiovasc Risk Rep 9:34.  https://doi.org/10.1007/s12170-015-0463-4 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Gleeson M, Bishop NC (2005) The T cell and NK cell immune response to exercise. Ann Transpl 10:44–49Google Scholar
  22. Green KJ, Croaker SJ, Rowbottom DG (2003) Carbohydrate supplementation and exercise-induced changes in T-lymphocyte function. J Appl Physiol 95:1216–1223.  https://doi.org/10.1152/japplphysiol.00179.2003 CrossRefPubMedGoogle Scholar
  23. Grievink HW, Luisman T, Kluft C et al (2016) Comparison of three isolation techniques for human peripheral blood mononuclear cells: cell recovery and viability, population composition, and cell functionality. Biopreserv Biobank 14:410–415.  https://doi.org/10.1089/bio.2015.0104 CrossRefPubMedGoogle Scholar
  24. Hermann C, von Aulock S, Graf K, Hartung T (2003) A model of human whole blood lymphokine release for in vitro and ex vivo use. J Immunol Methods 275:69–79.  https://doi.org/10.1016/S0022-1759(03)00003-6 CrossRefPubMedGoogle Scholar
  25. Holdsworth DA, Cox PJ, Kirk T et al (2017) A Ketone ester drink increases postexercise muscle glycogen synthesis in humans. Med Sci Sports Exerc 49:1789–1795.  https://doi.org/10.1249/MSS.0000000000001292 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Hopkins WG (2006) Spreadsheets for analysis of controlled trials, with adjustment for a predictor. Sportscience 10:46–50Google Scholar
  27. Hopkins WG, Marshall SW, Batterham AM, Hanin J (2009) Progressive statistics for studies in sports medicine and exercise science. Med Sci Sports Exerc 41:3–13.  https://doi.org/10.1249/MSS.0b013e31818cb278 CrossRefPubMedGoogle Scholar
  28. Keaney LC, Kilding AE, Merien F, Dulson DK (2019) Keeping athletes healthy at the 2020 Tokyo summer games: considerations and illness prevention strategies. Front Physiol.  https://doi.org/10.3389/fphys.2019.00426 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Kidd P (2003) Th1/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Altern Med Rev 8:223–246PubMedGoogle Scholar
  30. Lancaster GI (2006) Methods of assessing immune function. In: Gleeson M (ed) Immune function in sport and exercise. Elsevier, UK, pp 45–65CrossRefGoogle Scholar
  31. Lancaster GI, Halson SL, Khan Q et al (2004) Effects of acute exhaustive exercise and chronic exercise training on type 1 and type 2 T lymphocytes. Exerc Immunol Rev 10:91–106PubMedGoogle Scholar
  32. Lancaster GI, Khan Q, Drysdale PT et al (2005) Effect of prolonged exercise and carbohydrate ingestion on type 1 and type 2 T lymphocyte distribution and intracellular cytokine production in humans. J Appl Physiol 98:565–571.  https://doi.org/10.1152/japplphysiol.00754.2004 CrossRefPubMedGoogle Scholar
  33. LaVoy EC, Hussain M, Reed J et al (2017) T-cell redeployment and intracellular cytokine expression following exercise: effects of exercise intensity and cytomegalovirus infection. Physiol Rep 5:e13070.  https://doi.org/10.14814/phy2.13070 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Leckey JJ, Ross ML, Quod M et al (2017) Ketone diester ingestion impairs time-trial performance in professional cyclists. Front Physiol 8:806.  https://doi.org/10.3389/fphys.2017.00806 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Li H, Llera A, Malchiodi EL, Mariuzza RA (1999) The structural basis of T cell activation by superantigens. Annu Rev Immunol 17:435–466.  https://doi.org/10.1146/annurev.immunol.17.1.435 CrossRefPubMedGoogle Scholar
  36. Maciolek JA, Pasternak JA, Wilson HL (2014) Metabolism of activated T lymphocytes. Curr Opin Immunol 27:60–74.  https://doi.org/10.1016/j.coi.2014.01.006 CrossRefPubMedGoogle Scholar
  37. Natarajan K, Li H, Mariuzza RA, Margulies DH (1999) MHC class I molecules, structure and function. Rev Immunogenet 1:32–46PubMedGoogle Scholar
  38. Neudorf H, Durrer C, Myette-Cote E et al (2019) Oral ketone supplementation acutely increases markers of nlrp3 inflammasome activation in human monocytes. Mol Nutr Food Res.  https://doi.org/10.1002/mnfr.201801171 CrossRefPubMedGoogle Scholar
  39. Newsholme P, Curi R, Gordon S, Newsholme EA (1986) Metabolism of glucose, glutamine, long-chain fatty acids and ketone bodies by murine macrophages. Biochem J 239:121–125CrossRefGoogle Scholar
  40. O’Malley T, Myette-Cote E, Durrer C, Little JP (2017) Nutritional ketone salts increase fat oxidation but impair high-intensity exercise performance in healthy adult males. Appl Physiol Nutr Metab 42:1031–1035.  https://doi.org/10.1139/apnm-2016-0641 CrossRefPubMedGoogle Scholar
  41. O'Rourke AM, Rider CC (1989) Glucose, glutamine and ketone body utilisation by resting and concanavalin A activated rat splenic lymphocytes. Biochim Biophys Acta 1010:342–345CrossRefGoogle Scholar
  42. Palmer CS, Ostrowski M, Balderson B et al (2015) Glucose metabolism regulates T cell activation, differentiation, and functions. Front Immunol.  https://doi.org/10.3389/fimmu.2015.00001 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Peake JM, Neubauer O, Walsh NP, Simpson RJ (2016) Recovery of the immune system after exercise. J Appl Physiol 122:1077–1087.  https://doi.org/10.1152/japplphysiol.00622.2016 CrossRefPubMedGoogle Scholar
  44. Piccirillo CA (2008) Regulatory T cells in health and disease. Cytokine 43:395–401.  https://doi.org/10.1016/j.cyto.2008.07.469 CrossRefPubMedGoogle Scholar
  45. Poffé C, Ramaekers M, Van Thienen R, Hespel P (2019) Ketone ester supplementation blunts overreaching symptoms during endurance training overload. J Physiol.  https://doi.org/10.1113/JP277831 CrossRefPubMedGoogle Scholar
  46. Raysmith BP, Drew MK (2016) Performance success or failure is influenced by weeks lost to injury and illness in elite Australian track and field athletes: a 5-year prospective study. J Sci Med Sport 19:778–783.  https://doi.org/10.1016/j.jsams.2015.12.515 CrossRefPubMedGoogle Scholar
  47. Rodger S, Plews D, Laursen P, Driller M (2017) Oral β-hydroxybutyrate salt fails to improve 4-minute cycling performance following submaximal exercise. J Sci Cycl 6:26–31Google Scholar
  48. Rooney BV, Bigley AB, LaVoy EC et al (2018) Lymphocytes and monocytes egress peripheral blood within minutes after cessation of steady state exercise: a detailed temporal analysis of leukocyte extravasation. Physiol Behav 194:260–267.  https://doi.org/10.1016/j.physbeh.2018.06.008 CrossRefPubMedGoogle Scholar
  49. Salicru AN, Sams CF, Marshall GD (2007) Cooperative effects of corticosteroids and catecholamines upon immune deviation of the type-1/type-2 cytokine balance in favor of type-2 expression in human peripheral blood mononuclear cells. Brain Behav Immun 21:913–920.  https://doi.org/10.1016/j.bbi.2007.02.006 CrossRefPubMedGoogle Scholar
  50. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108.  https://doi.org/10.1038/nprot.2008.73 CrossRefPubMedPubMedCentralGoogle Scholar
  51. Shaw DM, Merien F, Braakhuis A, Dulson D (2018) T-cells and their cytokine production: the anti-inflammatory and immunosuppressive effects of strenuous exercise. Cytokine 104:136–142.  https://doi.org/10.1016/j.cyto.2017.10.001 CrossRefPubMedGoogle Scholar
  52. Shaw DM, Merien F, Braakhuis A et al (2019) The Effect of 1,3-butanediol on cycling time-trial performance. Int J Sport Nutr Exerc Metab. 29:466–473.  https://doi.org/10.1123/ijsnem.2018-0284 CrossRefPubMedGoogle Scholar
  53. Steensberg A, Toft AD, Bruunsgaard H et al (2001) Strenuous exercise decreases the percentage of type 1 T cells in the circulation. J Appl Physiol 91:1708–1712CrossRefGoogle Scholar
  54. Svendsen IS, Gleeson M, Haugen TA, Tønnessen E (2015) Effect of an intense period of competition on race performance and self-reported illness in elite cross-country skiers. Scand J Med Sci Sports 25:846–853.  https://doi.org/10.1111/sms.12452 CrossRefPubMedGoogle Scholar
  55. Vandoorne T, De Smet S, Ramaekers M et al (2017) Intake of a ketone ester drink during recovery from exercise promotes mTORC1 signalling but not glycogen resynthesis in human muscle. Front Physiol 8:310.  https://doi.org/10.3389/fphys.2017.00310/full CrossRefPubMedPubMedCentralGoogle Scholar
  56. Youm Y-H, Nguyen KY, Grant RW et al (2015) The ketone metabolite β-hydroxybutyrate blocks NLRP3 inflammasome–mediated inflammatory disease. Nat Med 21:263–269.  https://doi.org/10.1038/nm.3804 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Sports Performance Research Institute New Zealand (SPRINZ)Auckland University of TechnologyAucklandNew Zealand
  2. 2.AUT Roche Diagnostics Laboratory, School of ScienceAuckland University of TechnologyAucklandNew Zealand
  3. 3.Faculty of Medical and Health SciencesUniversity of AucklandAucklandNew Zealand

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