LEUCINE CONTENT OF ISONITROGENOUS

PROTEIN SOURCES POSITIVELY INFLUENCES

BODY COMPOSITION AND MUSCLE MASS IN
RATS

Dr. Layne E. Norton

ISSN Symposium 2010

Division of Nutritional Sciences

University of Illinois at Urbana-Champaign
 Introduction

• High protein diets and athletes

– Increase muscle protein synthesis (MPS)
– Positive body composition impacts
– Large amounts of anecdotal evidence for efficacy
– More functional muscle and strength may transfer to better
performance

• Raises questions about guidelines for protein intake in athletes

 Traditional protein recommendations
• Daily requirements to meet short term nitrogen balance

• Protein quality assessed by meeting requirements for limiting

amino acids

• Substrate concepts: direct amino acid oxidation (DAAO)

Rapid rise in plasma

amino acid concentration
stimulates oxidation
 Dietary protein recommendations

• How do we asses optimal protein intakes in athletes

for enhancing muscle & strength?

• Leucine may be a possible indicator of protein

‘quantity and quality’
 Leucine stimulates muscle protein synthesis

Garlick. J. Nutr. 135: 1553S, 2005

 Leucine and muscle protein synthesis

Insulin
[AMP]
IRS-1 PI3-K Akt/PKB AMPK

cell
mTOR [leucine]
membrane

4E-BP1
S6K
 Oral leucine increases MPS
Leucine Threshold

Crozier et al. J. Nutr. 135: 376, 2005

 Leucine threshold

• Approximately a doubling of plasma leucine required

to stimulate MPS

• Current protein recommendations do not account for

these metabolic actions of leucine
 Research Question

• Does leucine regulate MPS in complete meals

containing isonitrogenous protein sources?
 Experiment 1:
MEALS
Goal:
Breakfast: 4g Determine MPS meal responses in animals adapted to diets
Lunch: ad lib with different protein sources that vary in leucine content
Dinner: ad lib

Assigned treatment groups

matched on weight1
Treatment CHO PRO FAT Leucine2
Food-Deprived (adapted on whey) 54% 16% 30% 0mg
Wheat (Lysine supplemented) 54% 16% 30% 46mg
Soy 54% 16% 30% 54mg
Egg 54% 16% 30% 60mg
Male SD Whey 54% 16% 30% 74mg
Rats @
Sacrifice @ ~ 300g; fasted or 90
240-260g
min post-prandially.

2 day meal Day 1 Day 14

training
1
n=9-10 per group
2
Leucine content of breakfast meal
 Comparison of diet amino acid compositions with NRC
Amino Acid
requirements
Wheat Diet Soy Diet
1,2
Egg Diet Whey Diet NRC
Requirement

g/kg diet

Leucine 11.5 13.6 14.9 18.5 1.8

Lysine 15.4 10.7 11.0 15.4 1.1

Methionine/Cysteine 6.5 4.4 13.9 7.6 2.3

Threonine 4.4 6.5 7.6 10.9 1.8

Tryptophan 2.2 2.0 2.7 2.7 0.5

1
Table 2-2 from Nutrient Requirements of Laboratory Animals Fourth Revised Edition .
2
Values calculated for 300g rat at maintenance.
 Plasma Leucine and Muscle Protein Synthesis

74mg 74mg
60mg 60mg

47mg 54mg
0mg
0mg 47mg 54mg

Values represent means ± SEM; Means not sharing a common letter are different, P <0.05.
1
Leucine and S6K/4E-BP1

[leucine]

mTOR

S6K 4E-BP1

Protein Synthesis
 Translation Factor Activation

Values represent means ± SEM; Means not sharing a common letter are different, P <0.05.
1
 Other Plasma Measurements

• No EAA was significantly below baseline in any fed

group

• Insulin/glucose increased by feeding except in

animals fed soy
 Summary

• A ‘threshold’ amount of dietary leucine is required to

increase plasma leucine and initiate MPS

• Egg and whey but not wheat and soy were able to
stimulate MPS at a small breakfast meal

• Leucine content, not limiting amino acids, predicted

MPS and translation factor activation
 Research Question

• Are long term body composition and muscle weight

outcomes different in animals fed isonitrogenous &
isocaloric diets with different leucine contents?
 Experiment 2
MEALS1
Goal:
Breakfast: 4g Examine long term body composition and muscle outcomes in
Lunch: 4g animals fed different protein sources

Dinner: 6g

All animals placed on

Treatment CHO PRO FAT Leucine2
adaptation diet (Wheat) Wheat (Lysine supplemented) 54% 16% 30% 161mg
Soy 54% 16% 30% 189mg
Egg 54% 16% 30% 210mg
Whey 54% 16% 30% 259mg

Male SD
Rats @
250-270g Sacrifice @ ~ 350-400g

2 day meal Day 1 Day 84: Final time point

training Day 7: Animals assigned Day 21: First time point
to treatment groups based
1
80% of ad libitum intake on weight

2
Daily Leucine Intake
 Leucine distribution
  Breakfast Lunch Dinner Total intake

Diet Groups Leucine intake (mg)

Wheat 46 46 69 161

Soy 54 54 76 184

Egg 60 60 90 210

Whey 74 74 111 259

Bodyweight Results
 2 week MPS and signaling results

1
Values expressed as means ± SEM, n=7-8. Labeled means without a common letter differ (P<0.05).
 2 week muscle weight and body composition

1
Values expressed as means ± SEM, n=6-8. Labeled means without a common letter differ (P<0.05).
11 week muscle protein synthesis
 11 week muscle weight and body composition

1
Values expressed as means ± SEM, n=13. Labeled means without a common letter differ (P<0.05).
 Akt and AMPK signaling

1
Values expressed as means ± SEM, n=8-10. Labeled means without a common letter differ (P<0.05).
 Leucine and muscle protein synthesis

Insulin
[AMP]
IRS-1 PI3-K Akt/PKB AMPK

cell
mTOR [leucine]
membrane

4E-BP1
S6K
 Summary

• Changes in post prandial plasma leucine predict MPS

and changes in muscle weight/body composition

• Stimulation of MPS may cause nutrient partitioning

• Leu content of protein sources predicts protein

quality as related to changes in body composition and
muscle mass
 Research Question

• Examine if protein/leucine distribution makes a

difference in long term body composition and muscle
weights
Protein Distribution
 Experiment 3
MEALS1 Goal:
Breakfast: 4g Determine if long term differences in protein and leucine
Lunch: 4g can cause different muscle weight and MPS outcomes
Dinner: 6g

All animals placed on

Treatment CHO PRO FAT Leucine2
adaptation diet (Wheat) Wheat (adaptation) 54% 16% 30% 161mg
ED-Whey 54% 16% 30% 259mg
UD-Whey 54% 16% 30% 259mg

Male SD
Rats @
250-270g Sacrifice @ ~ 350-400g

2 day meal Day 1 Day 84: Final time point

training Day 7: Animals assigned
to treatment groups based
1
80% of ad libitum intake on weight

2
Daily Leucine Intake
 Protein and Leucine Distribution
  Breakfast Lunch Dinner Total intake

Diet Groups Protein intake (g)

ED-Whey1 0.68 0.68 1.02 2.38

UD-Whey2 0.35 0.35 1.68 2.38

  Breakfast Lunch Dinner Total intake

Diet Groups Leucine intake (mg)

ED-Whey1 74 74 111 259

UD-Whey2   37 37 185 259

1
Protein constituted 16% of diet energy.
2
Whey protein unevenly distributed as 8%, 8%, and 27.5% of energy across meals.
 11 week muscle protein synthesis

1
Values expressed as means ± SEM, n=8-13. Labeled means without a common letter differ (P<0.05).
 11 week tissue weights

1
Values expressed as means ± SEM, n=8-13. Labeled means without a common letter differ (P<0.05).
 Summary

• Protein/leucine distribution is important to optimize

MPS and muscle mass

• Uneven distribution of protein/leucine changes where

lean tissue is deposited
 Conclusions
• Leucine content of complete meals predicts
postprandial MPS outcomes

• Leucine content and distribution impacts long term

body composition and muscle mass

• Current protein recommendations of protein quantity

and quality do not encapsulate the benefits of
different protein sources for optimal muscle mass
 Practical Applications

• Recommendations for maximizing anabolism

– Consumption of multiple meals containing leucine rich

protein sources to reach leucine threshold in plasma

• Approx 2-3g leucine per meal in humans

– Distribute protein/leucine relatively evenly

– A brief word on meal frequency…

 MPS (%/day)
• Our lab has characterized
7.5
the time course of MPS in *
*
7
response to a complete meal †
6.5

• MPS time course was 5.5

*different from baseline P<0.05; †p<0.10
complete at 3 hours but 5
0 45 90 135 180 300
plasma amino acids were Time (minutes)

still elevated.
Plasma Leucine (µM)
300
* * * *
• MPS decreasing though 250

plasma leu is elevated 200

*
150

100
*different from baseline P<0.05
• MPS is ‘refractory’ 50

0
0 45 90 135 180 300
Time (minutes)
• Recent data demonstrates consumption of Leu,
CHO, or Leu + CHO prolongs MPS meal response

• May be beneficial to consume meals every 4-6 hours

with Leu/BCAA alone or in combination with CHO
in between meals to optimize MPS

• Further research required to clarify optimal meal

frequency
 Thank you!
Layman Lab
Chris Moulton My wife, Isabel
Gabe Wilson Family
Dee Walker Friends
Suzanne Devkota
Others
Jacob Wilson
Advisors
Jeremy Loenneke
Dr. Don Layman
Diego Brana
Dr. Peter Garlick Dr. Dominic D’Agastino
Dr. Tracy Anthony Ben Esgro

Special thanks to the ISSN!