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

• 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)

Traditional protein recommendations

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

cell membrane

Insulin

IRS-1 PI3-K Akt/PKB

mTOR

S6K 4E-BP1

[leucine]

AMPK

[AMP]

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

Oral leucine increases MPS

Leucine Threshold

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:

Male SDRats @

240-260g

2 day meal training

Day 1 Day 14

Assigned treatment groups matched on weight1

Sacrifice @ ~ 300g; fasted or 90 min post-prandially.

MEALS

Breakfast: 4g

Lunch: ad lib

Dinner: ad lib

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

Whey 54% 16% 30% 74mg

1n=9-10 per group

2Leucine content of breakfast meal

Goal:Determine MPS meal responses in animals adapted to diets with different protein sources that vary in leucine content

Comparison of diet amino acid compositions with NRC requirements1,2

Amino Acid Wheat Diet Soy Diet 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

1Table 2-2 from Nutrient Requirements of Laboratory Animals Fourth Revised Edition .2Values calculated for 300g rat at maintenance.

Plasma Leucine and Muscle Protein Synthesis

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

0mg

60mg

47mg 54mg

74mg

0mg

60mg

47mg 54mg

74mg

Leucine and S6K/4E-BP1

mTOR

S6K 4E-BP1

[leucine]

Protein Synthesis

Translation Factor Activation

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

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

Male SDRats @

250-270g

2 day meal training

Day 1 Day 84: Final time point

All animals placed on adaptation diet (Wheat)

Sacrifice @ ~ 350-400g

MEALS1

Breakfast: 4g

Lunch: 4g

Dinner: 6g

180% of ad libitum intake

2Daily Leucine Intake

Treatment CHO PRO FAT Leucine2

Wheat (Lysine supplemented) 54% 16% 30% 161mg

Soy 54% 16% 30% 189mg

Egg 54% 16% 30% 210mg

Whey 54% 16% 30% 259mg

Day 7: Animals assignedto treatment groups based on weight

Day 21: First time point

Goal:Examine long term body composition and muscle outcomes in animals fed different protein sources

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

1Values expressed as means ± SEM, n=7-8. Labeled means without a common letter differ (P<0.05).

2 week muscle weight and body composition

1Values 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

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

Akt and AMPK signaling

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

Leucine and muscle protein synthesis

cell membrane

Insulin

IRS-1 PI3-K Akt/PKB

mTOR

S6K 4E-BP1

[leucine]

AMPK

[AMP]

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

Male SDRats @

250-270g

2 day meal training

Day 1 Day 84: Final time point

All animals placed on adaptation diet (Wheat)

Sacrifice @ ~ 350-400g

MEALS1

Breakfast: 4g

Lunch: 4g

Dinner: 6g

180% of ad libitum intake

2Daily Leucine Intake

Treatment CHO PRO FAT Leucine2

Wheat (adaptation) 54% 16% 30% 161mg

ED-Whey 54% 16% 30% 259mg

UD-Whey 54% 16% 30% 259mg

Day 7: Animals assignedto treatment groups based on weight

Goal:Determine if long term differences in protein and leucine can cause different muscle weight and MPS outcomes

Protein and Leucine Distribution

  Breakfast Lunch Dinner Total intake

Diet Groups Leucine intake (mg)

ED-Whey1 74 74 111 259

UD-Whey2   37 37 185 259

  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

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

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

11 week tissue weights

1Values 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…

• Our lab has characterized the time course of MPS in response to a complete meal

• MPS time course was complete at 3 hours but plasma amino acids were still elevated.

• MPS decreasing though plasma leu is elevated

• MPS is ‘refractory’0

50

100

150

200

250

300

0 45 90 135 180 300

Plasma Leucine (µM)

5

5.5

6

6.5

7

7.5

0 45 90 135 180 300

MPS (%/day)

* * * *

*

**

†

*different from baseline P<0.05; †p<0.10

*different from baseline P<0.05

Time (minutes)

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 LabChris MoultonGabe WilsonDee WalkerSuzanne Devkota

AdvisorsDr. Don LaymanDr. Peter Garlick Dr. Tracy Anthony

My wife, Isabel Family Friends

OthersJacob WilsonJeremy Loenneke

Diego BranaDr. Dominic

D’AgastinoBen Esgro

Special thanks to the ISSN!