Sex Differences in Substrate Metabolism During Endurance Exercise

1
Sex Differences in Substrate Metabolism During
Endurance Exercise

• Michaela Devries-Aboud (BSc Honours U of G)
• PhD Candidate
• McMaster University

2
Outline

• Introduction
• Sex, menstrual cycle and estrogen effects on fuel
  selection during endurance exercise
• CHO
• Lipid
• Protein
• Gene expression, protein content and enzyme
  activity

3
Things to Consider when Conducting Sex Difference
Studies

• Matching subjects
• VO2peak/kgFFM/min
• Training history
• Dietary intake
• Anaerobic threshold
• Menstrual cycle phase

4
Things to Consider when Conducting Sex Difference
Studies

• Training Status
• Endurance training influences substrate use
  during exercise
• Training
• fat oxidation and CHO oxidation
• How to match subjects
• Recruit subjects with similar training regimes
• Match subjects based on VO2peak/kgFFM/min

Carter, AJP, 2001 Phillips, JAP, 1996
5
Things to Consider when Conducting Sex Difference
Studies

• Habitual Dietary Intake
• High fat diet increases fat oxidation and
  decreases CHO oxidation during exercise
• Stellingwerff, AJP, 2005 Burke, MSSE, 2002
• High CHO diet increases CHO oxidation during
  endurance exercise
• Tarnopolsky, JAP, 1995 Andrews, JAP, 2003
• How do we control for this?
• Diet records

6
Habitual Dietary Intake(N 47 females N 49
males)
7
Things to Consider when Conducting Sex Difference
Studies

• Anaerobic Threshold
• Whole body substrate oxidation from RER not valid
  above AT
• Untrained AT 66 of VO2peak
• Pritzlaff-Roy, JAP, 2001
• Trained AT 80 of VO2peak
• Phillips, JAP, 1993
• How do we control for this
• Recreationally active tested at 65 VO2peak

8
Things to Consider When Conducting Sex Difference
Studies

• Menstrual cycle phase can also influence fuel
  selection during endurance exercise
• Follicular phase (FP)
• Before ovulation, low estrogen, high
  estrogenprogesterone
• Luteal phase (LP)
• After ovulation, high estrogen, low
  estrogenprogesterone
• LP
• Lower RER
• Lower glucose Ra and Rd
• Lower muscle glycogen utilization
• How do we control for this?
• Monitor menstrual cycle for 3 months prior to
  study
• Ovulation test kits

Devries, AJP, 2006 Campbell, AJP, 2001 Deon,
AJP, 2002
9
Introduction

• Women outperform men during ultra-endurance
  events
• ?quicker at 90km events
• ??at 42.4 km events
• ?quicker at 10 km events
• Women can sustain higher VO2peak longer
• Due to differences in estrogen?

Speechly, D Med Sci Sports Exerc, 1996
10
Relative Contribution of Fuels Used During 90 min
Exercise Bout at 65 VO2peak
Romijn, JAP, 2000 Romijn, AJP, 1993 van Loon,J
Physiol, 2001
11
Sex Differences in Fuel Utilization During
Exercise

• RER lower in women vs men
• Froberg and Pedersen, 1984 Blatchford,1985
  Tarnopolsky, 1990 Phillips, 1993 Tarnopolsky,
  1995 Tarnopolsky, 1997 Horton, 1998
  Friedlander, 1998 McKenzie, 2000 Rennie, 2000
  Carter, 2000 Lowther, 2000 Lamont, 2001
  Melanson, 2002 Jeukendrup, 2005 Roepstorff,
  2006 Devries, 2006 Devries, 2007
• No difference in RER between sexes
• Costill, 1979 Romijn, 2000 Friedmann and
  Kindermann, 1989 Davis, 2000 Roepstorff, 2002
  Steffansen, 2002.

12
Sex Differences in Fuel Utilization During
Exercise

• What does a lower RER mean?
• RER VCO2/VO2
• RER 0.70 energy derived from lipid
• RER 1.0 energy derived from CHO
• Lower RER in women means women rely more on lipid
  during moderate intensity endurance exercise

13
Meta-analysis of 22 Studies Investigating the
Effect of Sex on Fuel Selection During Endurance
Exercise
P Value lt 0.001 lt 0.01 lt 0.01
lt 0.05
14
CHO Metabolism

• Effect of sex and menstrual cycle phase on CHO
  metabolism during endurance exercise
• Muscle glycogen utilization
• Glucose Ra, Rd and MCR

15
Sex Differences in Muscle Glycogen Utilization

• Matched for training
• 15.5 km run
• No differences in resting glycogen
• ?25 greater muscle glycogen use during exercise

Muscle Glycogen
Tarnopolsky, JAP, 1990
16
Sex Differences in Muscle Glycogen Utilization

• No difference resting muscle glycogen
• LP lower PG, MG and total glycogen utilization
  vs FP
• LP lower PG utilization vs men
• No difference between FP and men

Devries, AJP, 2006
17
Sex Differences in Muscle Glycogen Utilization

• Other studies have shown no effect of sex on
  muscle glycogen utilization
• WHY?
• Difference seen between men and LP women
• Most studies test women in FP only
• Mode of exercise (cycling vs running)

Roepstorff, AJP, 2002 Tarnopolsky, JAP, 1995
Tarnopolsky, JAP, 1990 Devries, AJP, 2006
18
Sex Differences in Glucose Ra, Rd and MCR

• Glucose Ra glucose rate of appearance into the
  blood
• Glucose Rd glucose rate of disappearance from
  the blood
• MCR metabolic clearance rate
• Glucose Rd/(glucoset1 glucoset2)/2
• Stable isotope infusion
• Labelled glucose, FFA or amino acid
• Infused into subject at a known rate
• Based on the ratio of labelledunlabelled
  compound can determine Ra, Rd and MCR

19
Sex Differences in Glucose Ra, Rd and MCR

• LP and FP lower glucose Ra, Rd and MCR vs men
• LP lower glucose Ra vs FP at t 90 min BUT no
  difference when last 30 min of exercise averaged

Devries, AJP, 2006
20
Sex Differences in Glucose Ra, Rd and MCR

• Carter MF
• Horton LP
• Carter Women lower MCR _at_ 75 and 90 min
• Horton Women lower glucose Ra and Rd

Carter, AJP, 2001 Horton, JAP, 2006
21
CHO Summary

• Women lower RER less reliance on CHO vs men
• Specifically
• Lower glucose Ra and Rd
• Lower muscle glycogen utilization
• Dependent on type of exercise and menstrual cycle
  phase
• Luteal phase
• Lower muscle glycogen utilization

22
Lipid Metabolism

• Effect of sex and menstrual cycle phase on lipid
  metabolism during endurance exercise
• Plasma FFA
• IMCL
• Lipoprotein-derived TG

23
Sex Differences in Whole Body Lipolysis

• Glycerol marker of TG breakdown
• 90 min cycling _at_ 60 VO2peak
• Women Higher glycerol Ra and Rd vs men
• Suggests increased rate of lipolysis in women

Carter, AJP, 2001, Mittendorfer, AJP, 2002
24
Sex Differences in Plasma FFA Oxidation During
Endurance Exercise

• No effect of sex on plasma FFA oxidation during
  endurance exercise at various intensities
• 25 Romijn, JAP, 2000
• 45 Burguera, AJP, 2000 Friedlander, JAP, 1999
• 65 Romijn, JAP, 2000 Friedlander, JAP, 1999
• 85 Romijn, JAP, 2000

25
Sex Differences in Plasma FFA Oxidation During
Endurance Exercise

• However
• Although not significant women oxidized 47 more
  plasma FFA than men during the last 60 min of a
  90 min ride at 60 VO2peak

Roepstorff, AJP, 2002
26
Sex Differences in IMCL

• Women IMCL
• Forsberg, Clin Sci 1991 Roepstorff, AJP, 2001
  Steffensen, AJP,2002 Roepstorff, AJP, 2006
  Tarnopolsky, AJP, 2006 Devries, AJP, 2007

IMCL
Mitochondria
27
IMCL Utilization During Endurance Exercise

• Use of IMCL during endurance exercise is
  controversial
• Limitations of traditional methods
• Biochemical extraction contamination by EMCL
• ORO contamination by EMCL
• Stable isotopes not a direct measurement
• New methods
• EM Can look at individual lipid characteristics
• 1H-MRS Large sampling area, low CV

28
IMCL Utilization During Endurance Exercise

• IMCL usage during endurance exercise
• Krssak, 2000 Roepstorff, 2006 Roepstorff,
  2002 Schrauwen-Hinderling, 2003 van Loon, 2003
  White, 2003 Zehnder, 2005 Stellingwerff, 2007
  Devries, 2007
• No IMCL usage during endurance exercise
• Bergman, 1999 Guo, 2000 Kiens, 1993 Wendling,
  1996
• Studies showing no usage all used biochemical
  extraction technique
• 1H-MRS consistently shows IMCL use during
  moderate intensity exercise
• Good correlation between 1H-MRS and EM
• Howald, JAP, 2002

29
Sex Differences in IMCL Utilization During
Endurance Exercise

• ?lt ?
• ?dont use IMCL during endurance exercise
• At all training levels
• Correlation between IMCL and IMCL utilization
• Biochemical extraction method

Steffensen, AJP, 2002
30
Sex Differences in IMCL Utilization During
Endurance Exercise

• ?lt ?
• ?dont use IMCL during endurance exercise
• Biochemical extraction method
• BUT 28 of lipid oxidized unaccounted for in men

Roepstorff, AJP, 2002
31
Sex Differences in IMCL Utilization During
Endurance Exercise

• Lipid Area Density
• ?lt?(P 0.03)
• post exercise (P 0.01)
• No sex difference in net utilization

IMCL Area Density
Devries, AJP, 2007
32
Sex Differences in IMCL Utilization During
Endurance Exercise
IMCL size (um2)

• IMCL size
• ??
• IMCL/um2
• ?higher IMCL/um2 (P 0.002)
• IMCL/um2 post exercise (P 0.03)

IMCL/um2
Devries, AJP, 2007
33
Sex Differences in IMCL Utilization During
Endurance Exercise
Lipid Touching Mitochondria

• lipid touching mitochondria
• Mitochondria furnace of the cell
• Increased proximity may indicate increased lipid
  use and/or increased capacity to use lipid
• ?higher touching post exercise (P 0.03)

Devries, AJP, 2007
34
Lipoprotein-derived TG Use During Endurance
Exercise

• Under normal dietary conditions
  lipoprotein-derived TG contribute 10 of total
  fat oxidation during endurance exercise
• When fasted contribution is even less 5-10 of
  total fat oxidation
• Contribution increases after consumption of a
  mixed meal

Helge, J Physiol, 2001 van Loon, JAP, 2004
35
Sex Differences in Lipoprotein-derived TG
Oxidation During Endurance Exercise

• No sex studies during exercise
• Fasted or following a mixed meal
• Women Greater uptake of serum TG across the leg
• Future research

Horton, AJP, 2002
36
Lipid Summary

• Women whole body lipolysis
• Site of increased lipid oxidation
• Plasma FFA ?
• IMCL ?
• 1H-MRS sex study with exercise duration gt 90 min
• Lipoprotein-derived TG ?
• IMCL characteristics
• ? IMCL/area, not lipid size

37
Estrogen Supplementation and Substrate Use During
Exercise

• Animal Studies
• Improved endurance exercise performance
• Spared muscle and liver glycogen
• Higher FFA oxidation

Campbell, AJP, 2002 Kendrick, AJP, 1987
Kendrick, JAP, 1991
38
Estrogen Supplementation and Substrate Use During
Exercise

• 72h E2 to amenorrheic women (100ug/d)
• Treadmill run at 65 VO2peak for 90 min followed
  by run to exhaustion at 85 VO2peak
• Results
• Increased FFA
• Decreased glucose Ra and Rd during exercise
• No effect on glycerol Ra and Rd
• No change in CHO oxidation, fat oxidation or RER
• No effect on performance

Ruby, MSSE, 1997
39
Estrogen Supplementation and Substrate Use During
Exercise

• 11d E2, men, low dose (100-300ug/d)
• 90 min cycling _at_ 60 VO2peak
• No effect on
• Performance
• Glycogen use
• Trend
• Increased fat oxidation and decreased CHO
  oxidation

Tarnopolsky, Int J Sports Med, 2001
40
Estrogen Supplementation and Substrate Use During
Exercise

• 8d E2 (1mg/d 2 d and 2mg/d 6d), men
• 90 min cycling _at_ 65 VO2peak
• E2 lowered RER and CHO oxidation and increased
  fat oxidation

Devries, JCEM, 2005
41
Estrogen Supplementation and Substrate Use During
Exercise

• Lowered resting glycogen
• No effect on muscle glycogen utilization

Devries, JCEM, 2005
42
Estrogen Supplementation and Substrate Use During
Exercise

• Devries 1mg/d 2d, 2 mg/d 6 d
• Carter 3mg/d 8d
• Lower glucose Ra and Rd
• Lower MCR (Carter only)

Devries, JCEM, 2005 Carter, JAP, 2001
43
Estrogen Supplementation and Substrate Use During
Exercise

• E2 supplementation to men (3mg/d)
• No effect of E2 on glycerol Ra or Rd

Carter, JAP, 2001
44
Differences between E2 studies
45
Protein Metabolism

• Protein oxidation 5-6 of total substrate
  oxidation during endurance exercise
• Leu oxidation (BCAA) Highly metabolized in
  muscle (others are not)

Hamadeh, JCEM, 2005
46
Sex Differences in Protein Metabolism During
Endurance Exercise

• Men protein oxidation at rest and during
  exercise
• Women non-oxidative leucine disposal
  marker of protein synthesis
• Women leucine flux

Protein oxidation rate (umol/kg/h)
P lt 0.01
McKenzie, AJP, 2000 Phillips, JAP, 1993 Lamont,
JAP, 2001
47
Sex Differences in Protein Oxidation During
Endurance Exercise

• 6 studies directly compared men/women.
• 60 - 90 min _at_ 50 75 VO2peak
• - Women (N 41) 2.1 of total E
• - Men (N 40) 5.5 of total E
• (P 0.02)

Tarnopolsky, JAP, 1990 Phillips, JAP, 1993
Tarnopolsky, JAP, 1995 Tarnopolsky, JAP, 1997
McKenzie, AJP, 2000, Lamont, JAP, 2001
48
Sex Differences in Nitrogen Balance in Endurance
Athletes
Phillips, JAP, 1993
49
Estrogen Supplementation and Protein Metabolism
During Endurance Exercise

• Estrogen
• leucine oxidation at rest and during exercise
  (16, P 0.005)
• Protein balance less negative (20, P 0.01)
• No effect on leucine flux, protein synthesis or
  breakdown
• lipid oxidation (22-44, P 0.02) and
  CHO oxidation (5-16, P 0.04) during exercise

Hamadeh, JCEM, 2005
50
Protein Summary

• Protein oxidation contributes 5 of total
  substrate oxidation during endurance exercise
• ? leu oxidation during exercise and protein
  synthesis (NOLD)
• ?less ve N balance post exercise
• E2 leu oxidation at rest and during exercise
  and less ve N balance post exercise

51
Sex Differences in Gene Expression, Protein
Content and Enzyme Activity

• What at the molecular level is different
• between men and women that can explain
• the observed sex differences in metabolism
• at rest and during endurance exercise?

52
Acetyl Coa
ALB R
ß - oxidation
TFP LCAD
FFA
TG
FATP
CPT 2
HSL
mtGPAT
CD36
CPT 1
FFA
FFA
FABPc
CD36
ALB
FFA
mtGPAT
FABPpm
FFA
HSL
IMCL
Circulation
Myocyte
Adipocyte
53
Function of Various Genes/Proteins

• PPARa
• Regulates genes involved in ß-oxidation
• PPAR?
• Involved in adipocyte differentiation
• SREBP 1
• Involved in cholesterol homeostasis and lipid
  biosynthesis
• LPL
• Hydrolysis of lipoproteins
• AMPK
• (-) FA synthesis and () FA and CHO oxidation

54
Glycogen
Glycogen Synthase Glycogenin
Glycogen Phosphorylase
Glucose
GLUT 4
G-1-P
G-6-P
Acetyl CoA TCA cycle
Pyruvate
Circulation
Myocyte
55
mRNA Content for Genes Involved in CHO Metabolism

• Glycogen Phosphorylase
• Glycogen breakdown
• LP lt FP
• No difference b/w men and women
• GLUT 4
• Exercise and insulin () glucose uptake
• No sex difference

Glycogen Phosphorylase
GLUT 4
Fu, In preparation, 2007
56
mRNA Content for Genes Involved in CHO Metabolism

• Glycogenin
• Primer for glycogen synthesis
• LP gt FP
• No sex difference
• Glycogen synthase I
• Involved in glycogen synthesis
• No effect of menstrual phase or sex

Glycogenin
Glycogen synthase I
Fu, In preparation, 2007
57
Sex and mRNA for Lipid Metabolism

• N 12 men and 12 women, cycle for 90 min at 65
  VO2peak.
• Real time RT-PCR (Taqman probe, ß2-MG).
• mRNA Exercise effect Sex effect (? gt ?)
• TFP
• FABP
• PPAR ?
• PPAR?
• HSL
• VLCAD

Fu, unpublished, 2007
58
Sex and mRNA for Lipid Metabolism

• mRNA Exercise effect Sex effect (? gt ?)
• mtGPAT
• SREBP-1
• FAT
• CPT1
  
• CPT2
• LCAD
  
• PGC1-alpha

LP only



LP only

Fu, unpublished, 2007
59
Sex Differences in mRNA for Lipid Metabolism

• Genes for lipid transport into the cell
• FAT/CD36 NT?lt ?
• FABPpm NT ?lt ?
• FABPc NT ?lt ?
• FATP1 no difference
• No sex difference in trained

Kiens, JAP, 2004
60
Sex Difference in Protein Content for Lipid
Metabolism

• FAT/CD36 50 in?
• Irrespective of training status
• mRNA and protein data suggest that ?can bring FA
  into cell more readily

Kiens, JAP, 2004
61
Sex Difference in mRNA and Enzyme Activity for
Lipid Metabolism

• LPL
• Hydrolysis of lipoproteins
• 160 higher mRNA in ?
• No difference in activity between sexes

Kiens, JAP, 2004
62
Sex Differences in mRNA and Protein Content for
Lipid Metabolism

• HSL
• Hydrolysis of TG
• Vastus lateralis
• No sex difference in mRNA
• protein content in?

Roepstorff, AJP, 2006
63
Sex Differences in Phosphorylation of Enzymes
Related to Lipid Metabolism

• BUT
• HSL activity dependent on phosphorylation at
  several sites
• ?gt?HSL phosphorylation at two sites

Roepstorff, AJP, 2006
64
Sex Differences in AMPK Activity

• AMPK
• (-) FA synthesis
• () glucose and FA oxidation
• ?gt?AMPK phosphorylation during exercise

Roepstorff, J Physiol, 2006
65
Sex and mRNA for Protein Metabolism
BCOAD-kinase mRNA

• Pre and post 90 min exercise, 65VOpeak
• inhibit activation state
• No effect on AST or BCOAD

Men FP LP
Fu, M., in preparation, 2006
66
Molecular Summary

• Lipid metabolism master regulator
• CHO metabolism just along for the ride
• ?have
• Capacity to bring FA into the cell (fat
  transporters)
• mRNA for ß-oxidation (TFP, CPT-1, LCAD, PPARa)
• mRNA for lipid synthesis (mtGPAT, SREBP-1, PPAR?)
• Decreased leu oxidation (BCOAD kinase)
• No sex differences
• LPL

67
Conclusions

• Women rely more on lipid to fuel endurance
  exercise vs men
• mRNA data suggests that lipid metabolism is the
  main regulator
• Future research protein content and enzyme
  activity

68
Acknowledgements

• Dr Mark Tarnopolsky
• Dr Mazen Hamadeh
• Dr Terry Graham
• Dr Stuart Phillips
• Dr Sandy Raha
• Sandy Glover
• Colin McCready
• Members of the Tarnopolsky Lab
• EMRG

• NSERC
• Nestlé Canada
• CIHR

69
Questions?