neutrophils

Effects of intense physical exercise and glutamine supplementa)on on neutrophil apoptosis Tania Cris)na Pithon-­‐Curi Ins)tute of Physical Ac)vity Sciences and Sports -­‐ Cruzeiro do Sul University São Paulo, Brazil NEUTROPHILS •  Neutrophils are polymorphonuclear cells being the most abundant leukocytes in the human blood •  Neutrophils represent 50 to 60% of total circula)ng leukocytes •  These cells are released from the bone marrow and have a life)me of approximately 10 hours in the blood •  Neutrophils usually die by apoptosis (the programmed cell death) Intensive exercise training and Upper Respiratory Tract Infec)on (URTI) risk Risk of URTI
Above average
Average
Below average
Ma#hews et al 2002 Nieman et al 2011 Low
Moderate
Training volume
Nieman 1994 Med Sci Sports Exerc 26: 128-139
High
Peters & Batemen 1982 Nieman et al 1990 Gleeson et al 1999 Fahlman & Engels 2005 Cunniffe et al 2011 Gleeson et al 2012 It is well known that physical exercise modulates leukocyte func)on EXERCISE AND LEUKOCYTE FUNCTION Several studies have shown that intensive and prolonged exercise impairs neutrophil func)on Nieman DC 2001, Castell L & Newsholme P, 2001 Our group demonstrated that intensive exercise (85% VO2max) induces neutrophil apoptosis in rats Lagranha CJ, Curi R, Pithon-­‐Curi TC et al. Med Sci Sports Exerc, 2004 Apoptosis Described by Kerr & Currie (1972) Programmed cell death Various biochemical and morphological events that lead cells to die AIM •  To inves)gate the occurrence of apoptosis in neutrophils from elite athletes a_er a triathlon compe))on Volunteers and athletes who par)cipated in the study: -­‐ 12 healthy male elite triathletes -­‐ 11 healthy male sedentary volunteers Groups
Body weight (kg) Height (cm)
Body fat (%)
Sedentary
74
174
20
Triathlete
79
177
12
The blood was collected before and a_er compe))on Neutrophils were obtained from blood by using a histopaque gradient TRIATHLON COMPETITION: It was performed in Ubatuba, seaside of São Paulo State, Brazil The Half Ironman consisted of: SWIMMING 2 Km CYCLING 90 Km RUNNING 21 Km The average )me taken by the athletes to complete the compe))on was 4 hours hHp://www.triathlon.org/uploads/features/msj_2747.jpg Occurrence of apoptosis was es)mated by measuring: The percentage of cells with: → DNA Fragmenta)on → Phospha)dylserine Externaliza)on The measurements were carried out by flow cytometry Percentage of Neutrophils with DNA Fragmenta)on * p<0.01
Percentage of Neutrophils with Phospha)dylserine Externaliza)on * p<0.05
CONCLUSION The triathlon compe))on induced neutrophil apoptosis as indicated by DNA fragmenta)on and phospha)dylserine externaliza)on ROS produc)on is well known to be involved in cell death (Cury-­‐Boaventura MF et al, 2008; Fialkow L et al, 2007; Arruda MA et al, 2006; de Melo MP et al, 2004) The produc)on of ROS by neutrophils from athletes and sedentary volunteers was then inves)gated ROS Produc)on by Neutrophils #p<0.01
CONCLUSION Evidence is presented herein that a triathlon compe))on induces neutrophil apoptosis
• 
It is well known that athletes u[lize supplements to increase performance. Among these supplements glutamine is frequently used. hHp://au.bodybuilding.com/store/glutamine.html?&_reques[d=365728 Glutamine Metabolism in Leukocytes Ardawi & Newsholme E -­‐ glutamine metabolism in lymphocytes (Biochem J. 21: 834-­‐42, 1983) Curi R & Newsholme E -­‐ glutamine metabolism in lymphocyte mitochondria (Mol Cell Biochem. 86:71-­‐76, 1989) Newsholme P, Curi R & Newsholme E -­‐ glutamine metabolism in monocytes (Biochem J. 242: 631-­‐6, 1987) Pithon-­‐Curi TC & Curi R -­‐ glutamine metabolism in neutrophils (Am J Physiol. 273: 124-­‐9, 1997) Pithon-­‐Curi TC & Curi R -­‐ glutamine is involved in ROS produc)on by neutrophils (Clin Sci. 103: 403-­‐8, 2002) Pithon-­‐Curi TC, Newsholme P & Curi R -­‐ glutamine delays spontaneous apoptosis in neutrophils (Am J Physiol, 284: C1355-­‐61, 2003) Curi R, et al., JCP, 204:
392-401, 2005.
AIM To inves[gate the protec[ve effect of glutamine on neutrophil death induced by one session of intensive exercise in rats Protocol of Glutamine Supplementation:
Glutamine (1.25 g b.w.)
gavage
Treadmill
10:00 a.m.
8:00 a.m.
Glycogen injection (i.p.)
11:00 a.m
Exercise
85% VO2max.
The control group: 3mL of PBS by gavage
• Rats age: 110 days old (240 g)
12:00 a.m.
End of Exercise
Neutrophil
Collection
Percentage of Neutrophils with Fragmented DNA Not Supplemented
Percentage
20.0
Glutamine
Supplemented
15.0
10.0
5.0
0.0
*
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Sedentary
*
*
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Exercised
Sedentary
P < 0.05 due to glutamine supplementation
Exercised
Neutrophils with Phospha)dylserine Externaliza)on Not Suplemented
Glutamine
Supplemented
70
*
Percentage
60
50
40
30
20
10
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0
Control
Exercised
Control
* P < 0.05 due to glutamine supplementation
Exercised
Neutrophils with Depolarized Mitochondria Not Suplemented
Glutamine
Supplemented
Depolarized
120
100
Percentage
*
Polarized
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60
40
20
0
Control
*
Exercised
Control
P < 0.05 due to glutamine supplementation
Exercised
CONCLUSION Oral glutamine supplementa[on par[ally prevented the exercise-­‐induced apoptosis in rat neutrophils Professors of the Ins)tute of Physical Ac)vity Sciences and Sports: Adriana Cris[na Levada-­‐Pires Maria Fernanda Cury-­‐Boaventura Elaine Hatanaka José Angelo Barela Ana Maria For[ Barela Paulo B. Freitas Júnior Renata Gorjão Sandro Massao Hirabara ACKNOWLEDGEMENTS This study was carried out by: Adriana Cris)na Levada-­‐Pires, PhD Cláudia Jacques Lagranha, PhD Fabiano Pinheiro Peres, MsD Collaborators • 
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Rui Curi André Bacchi Sandro M. Hirabara Cris[na Neves Borges Silva Maria Fernanda Cury-­‐Boaventura Renata Gorjão Elaine Hatanaka Vinícius Caneglari Aline Hirosi César Momeso Heloisa Pinheiro Paula Andrade Paulo R.S. Silva Financial support: FAPESP, CNPq and CAPES