Optimal protein Optimal protein intake and meal intake and meal

frequency to support frequency to support maximal protein maximal protein

synthesis and muscle synthesis and muscle mass.mass.Layne Norton, B.S.Layne Norton, B.S.

Division of Nutritional Division of Nutritional SciencesSciences

University of IllinoisUniversity of Illinois

OverviewOverview

BackgroundBackground Determining optimal protein intakeDetermining optimal protein intake Optimal Frequency Optimal Frequency Refractory PhenomenonRefractory Phenomenon Future ResearchFuture Research

BackgroundBackground High protein diets High protein diets

are popular amongst are popular amongst athletes and athletes and bodybuildersbodybuilders

Purported to have Purported to have various beneficial various beneficial effectseffects

Increase muscle massIncrease muscle mass Decrease bodyfatDecrease bodyfat Improve exercise Improve exercise

performance and performance and recoveryrecovery

Current RDA is 0.8g/kg for protein Current RDA is 0.8g/kg for protein intake.intake.

Current recommendations are based on Current recommendations are based on based on minimum needs to achieve based on minimum needs to achieve short-term nitrogen balanceshort-term nitrogen balance Often do not control for protein distribution, Often do not control for protein distribution,

protein source, and are often not applicable protein source, and are often not applicable to meal feeding.to meal feeding.

Empirical evidence suggests many Empirical evidence suggests many trainers and coaches frequently trainers and coaches frequently recommend amounts that are 2-4g/kg or recommend amounts that are 2-4g/kg or even greater.even greater.

How do we define optimal protein How do we define optimal protein intakes to maximize muscle mass?intakes to maximize muscle mass?

BackgroundBackground

Determining Optimal Determining Optimal IntakeIntake

Determine optimal protein intake at individual Determine optimal protein intake at individual meals to maximize protein synthesis (MPS)meals to maximize protein synthesis (MPS)

Determine optimal frequency of meal intakeDetermine optimal frequency of meal intake

These variables will dictate total protein These variables will dictate total protein intakeintake Specific meal recommendations are likely more Specific meal recommendations are likely more

beneficial than daily recommendations beneficial than daily recommendations MPS is regulated from meal to meal, not day to day.MPS is regulated from meal to meal, not day to day.

Leucine in regulation of protein Leucine in regulation of protein synthesissynthesis

Leucine (leu) has been shown to stimulate Leucine (leu) has been shown to stimulate protein synthesis and translation initiation protein synthesis and translation initiation to the same extent as a complete meal and to the same extent as a complete meal and is likely the major amino acid responsible is likely the major amino acid responsible for the anabolic effects of a meal (1).for the anabolic effects of a meal (1).

mTOR (+)

[Leucine]

p70S6K (+) 4E-BP1-eIF4E

eIF4E

eIF4G (+)

eIF4E-eIF4G (+)

Protein Synthesis (+)

Possible that activating Possible that activating and maximizing MPS is and maximizing MPS is dependant upon dependant upon achieving a specific achieving a specific post-prandial post-prandial concentration of leu in concentration of leu in the plasma. the plasma.

A certain amount of A certain amount of dietary leu is required dietary leu is required to increase plasma leu to increase plasma leu levels and activate levels and activate mTOR signaling and mTOR signaling and MPS.MPS.

What level of dietary leu What level of dietary leu at a meal is required to at a meal is required to maximize this response?maximize this response?

0

20

40

60

80

100

120

140

160

Control 10% Protein

WheatWhey

Pla

sma L

eu

cin

e

(µM

)

*

0

1

2

3

4

5

6

7

8

Control 10% Protein

WheatWhey

FS

R

(%/d

ay)

*unpublished data

*

Padden-Jones et al. Padden-Jones et al. showed that showed that administration of an administration of an essential amino acid essential amino acid (EAA) solution containing (EAA) solution containing 2.8g leu increased MPS 2.8g leu increased MPS by ~60% in adult humans by ~60% in adult humans (2).(2).

Tipton et al. demonstrated Tipton et al. demonstrated that amino acid solutions that amino acid solutions containing 4.4g and 8.3g containing 4.4g and 8.3g leu stimulate MPS leu stimulate MPS similarly (50-70% similarly (50-70% increase) in adults (3).increase) in adults (3). It is likely ~3-4g leucine It is likely ~3-4g leucine

(~0.045-0.06 g/kg) will (~0.045-0.06 g/kg) will maximize MPS at a meal in maximize MPS at a meal in adults.adults.

Combined data sets

% I

ncr

ease

fro

m

con

trols

0102030405060708090

100

Controls 2.8g Leu 4.4g Leu 8.3g Leu

Padden-JonesTipton

Leucine content of various protein Leucine content of various protein sourcessources

Protein SourceLeu % of total

protein)Amount of protein from source to reach 3-4g

Leucine Amount of food source required

Whey Protein Isolate 12.0% 25-33g 27-36g

Milk Protein Isolate 9.8% 31-41g 34-46g

Casein 9.3% 32-43g variable depending upon casein powder type

Egg 8.6% 35-47g 280-376g or approx 4.5-6.5 large whole eggs

Fish 8.1% 38-50g 158-208g

Beef 8.0% 38-51g 126-170g

Pork 8.0% 38-51g 133-179g

Chicken 7.5% 41-54g 132-174g

Wheat 6.8% 44-59g 440-590g or 15-20 slices of bread

RecapRecap

MPS is maximized by an oral MPS is maximized by an oral administration of 3-4g (0.045g-administration of 3-4g (0.045g-0.06g/kg) leu in adults and elderly. 0.06g/kg) leu in adults and elderly.

Key Questions: Key Questions: How long does the MPS response last?How long does the MPS response last? When can this response be stimulated When can this response be stimulated

again?again?

Optimal FrequencyOptimal Frequency

The duration of MPS in response to a The duration of MPS in response to a purified leu or EAA solution has been purified leu or EAA solution has been previously characterized and lasts previously characterized and lasts approximately 2 hours in rats and humans approximately 2 hours in rats and humans (4,5).(4,5).

Bohe et al. infused EAA for 6 hours but this Bohe et al. infused EAA for 6 hours but this only produced an MPS response lasting 2 only produced an MPS response lasting 2 hours though plasma amino acids remained hours though plasma amino acids remained elevated for the entire duration of the elevated for the entire duration of the experiment. experiment.

Our lab has Our lab has characterized the time characterized the time course of MPS in rats course of MPS in rats fed a complete meal fed a complete meal containing 20, 50, and containing 20, 50, and 30% of energy from 30% of energy from protein, carbohydrates, protein, carbohydrates, and lipids respectively and lipids respectively (6).(6).

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

MPS decreasing though MPS decreasing though plasma leu remains plasma leu remains elevated.elevated. 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)

The Refractory The Refractory PhenomenonPhenomenon

MPS becomes ‘refractory’ to MPS becomes ‘refractory’ to constant elevations in leu.constant elevations in leu.

Perhaps leu availability stimulates Perhaps leu availability stimulates MPS but sustained elevations in MPS but sustained elevations in plasma leu are insufficient to plasma leu are insufficient to maintain elevated MPS. maintain elevated MPS.

Why are constant elevations in Why are constant elevations in plasma leu unable to produce plasma leu unable to produce sustained increases in MPS?sustained increases in MPS?

Possible explanations?Possible explanations? Reduced availability of intramuscular leu?Reduced availability of intramuscular leu?

150

200

250

300

350

400

0 45 90 135 180 300

*

* **

Does not explain refractory nature of MPS.

mm

ol/

kg

wet

tiss

ue

Time (minutes)

Intramuscular Leucine

Possible Explanations?Possible Explanations? Possible that translation Possible that translation

factor activation is factor activation is reduced?reduced?

Phosphorylation of 4E-Phosphorylation of 4E-BP1 and p70S6K were BP1 and p70S6K were closely associated with closely associated with plasma leu plasma leu concentrations.concentrations.

Translation factor Translation factor activation remained activation remained elevated at 180 minutes elevated at 180 minutes but MPS had returned to but MPS had returned to baseline. baseline.

Does not explain Does not explain refractory phenomenon.refractory phenomenon.

0.2

0.25

0.3

0.35

0.4

0.45

0 45 90 135 180 300

Gam

ma f

orm

/tota

l

4E-BP1 Phosphorylation

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 45 90 135 180 300Gam

ma +

Beta

form

s/to

tal p70S6K Phosphorylation

**

* *

*

**

*

*

*different from baseline P<0.05

*different from baseline P<0.05

Time (minutes)

Time (minutes)

Possible Explanations?Possible Explanations?

Reduced eIF4E*eIF4G binding?Reduced eIF4E*eIF4G binding?

Time (minutes)

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 45 90 135 180 300

‡‡

*

*

eIF

4G

bou

nd

to e

IF4

E/t

ota

l 4

E eIF4E*eIF4G Binding

Unlikely to be responsible for MPS becoming refractory

Possible Explanations?Possible Explanations?

Reductions in other plasma EAA?Reductions in other plasma EAA?

At 180 minutes most EAA were still At 180 minutes most EAA were still elevated above baseline levels.elevated above baseline levels.

Bohe et al. maintained elevated EAA Bohe et al. maintained elevated EAA during an infusion for 6 hours but could during an infusion for 6 hours but could not overcome the refractory not overcome the refractory phenomenon.phenomenon.

Possible Explanations?Possible Explanations? Insulin?Insulin? Similar time Similar time

course as MPS.course as MPS. Similar time Similar time

course to MPS in course to MPS in other studies (4,5).other studies (4,5).

Cause or Cause or coincidence?coincidence? Insulin exerts Insulin exerts

stimulatory effects stimulatory effects on MPS through on MPS through mTOR pathway.mTOR pathway.

60

80

100

120

140

160

180

0 45 90 135 180 300

Insulin

Time (minutes)

Insu

lin

(p

mol/

L) *

*

Possible Explanations?Possible Explanations? At the moment the cause At the moment the cause

of the refractory of the refractory phenomenon to constant phenomenon to constant elevations in amino acids elevations in amino acids is unknown but may is unknown but may involve insulin.involve insulin.

Likely downstream of the Likely downstream of the mTOR pathway.mTOR pathway.

Possible that changes in Possible that changes in leu concentrations are leu concentrations are more important than more important than absolute leu absolute leu concentrations?concentrations?

Possible explanations?Possible explanations?

Paddon-Jones et al. demonstrated MPS Paddon-Jones et al. demonstrated MPS could be improved by taking an EAA could be improved by taking an EAA supplement containing 2.8g leu in supplement containing 2.8g leu in between meals consumed every 5 hours between meals consumed every 5 hours compared to an unsupplemented group compared to an unsupplemented group consuming the same meals (7). consuming the same meals (7).

Possible that a free amino acid Possible that a free amino acid supplement is able to increase plasma leu supplement is able to increase plasma leu concentrations greater than a meal alone concentrations greater than a meal alone and improve MPS. and improve MPS.

Optimal Meal Optimal Meal Frequency?Frequency?

Unlikely that another meal will stimulate Unlikely that another meal will stimulate MPS while it is refractory.MPS while it is refractory.

Possible that plasma leu levels may need to Possible that plasma leu levels may need to fall before MPS can be stimulated again. fall before MPS can be stimulated again.

Unlikely that consuming small amounts of Unlikely that consuming small amounts of protein over many meals will produce protein over many meals will produce sustained elevations in MPS.sustained elevations in MPS.

It is likely better to consume larger doses of It is likely better to consume larger doses of protein to maximize the MPS response and protein to maximize the MPS response and spread these doses apart by at least 4-6 spread these doses apart by at least 4-6 hours. hours.

Consuming an EAA/leu supplement in Consuming an EAA/leu supplement in between meals may help optimize MPS.between meals may help optimize MPS.

General General recommendations?recommendations?

Example: 200 lb male athlete/bodybuilderExample: 200 lb male athlete/bodybuilder 5 meals per day (one meal every 4-6 hours)5 meals per day (one meal every 4-6 hours) Goal: 4g/leu per meal (0.045g leu/kg Goal: 4g/leu per meal (0.045g leu/kg

BW/meal)BW/meal) Meal protein sources: Meal protein sources:

2 meals: whey (33g protein at each meal)2 meals: whey (33g protein at each meal) 2 meals: chicken (54g protein at each meal)2 meals: chicken (54g protein at each meal) 1 meal: beef (51g protein)1 meal: beef (51g protein)

Total protein intake: 225g/dayTotal protein intake: 225g/day 3-4g leu supplement consumed between 3-4g leu supplement consumed between

meals may optimize MPS response.meals may optimize MPS response.

Other ConsiderationsOther Considerations

AgeAge Digestion rates of protein sourcesDigestion rates of protein sources Total calorie intakeTotal calorie intake Training intensity and durationTraining intensity and duration Protein degradationProtein degradation Countless variables will make Countless variables will make

pinpointing specific numbers very pinpointing specific numbers very difficult. difficult.

Future researchFuture research Determine when MPS can be stimulated Determine when MPS can be stimulated

again after a complete meal.again after a complete meal. Further examine the role of insulin in Further examine the role of insulin in

maintaining MPS after a meal. maintaining MPS after a meal. Determine if an oral dose of BCAAs Determine if an oral dose of BCAAs

producing supraphysiological producing supraphysiological concentrations of plasma leu can concentrations of plasma leu can overcome the refractory response of MPS.overcome the refractory response of MPS.

Elucidate differences in MPS responses to Elucidate differences in MPS responses to meals containing different isonitrogenous meals containing different isonitrogenous protein sources with varying leu contents. protein sources with varying leu contents.

ReferencesReferences 1. Norton LE and Layman DK.  Leucine regulates translation initiation of protein 1. Norton LE and Layman DK.  Leucine regulates translation initiation of protein

synthesis in skeletal muscle after exercise. J Nutr. 2006; 136(2):533S-537S. synthesis in skeletal muscle after exercise. J Nutr. 2006; 136(2):533S-537S.  2. Padden-Jones D, Sheffield-Moore M, Zhang XJ, Volpi E, Wolf SE, Aarsland A, 2. Padden-Jones D, Sheffield-Moore M, Zhang XJ, Volpi E, Wolf SE, Aarsland A,

Ferrando AA, Wolfe RR. Amino acid ingestion improves muscle protein synthesis in Ferrando AA, Wolfe RR. Amino acid ingestion improves muscle protein synthesis in the young and elderly. Am J Physiol Endocrinol Metab. 2004 Mar;286(3):E321-8. the young and elderly. Am J Physiol Endocrinol Metab. 2004 Mar;286(3):E321-8.

3. Tipton KD, Ferrando AA, Phillips SM, Doyle Jr, Wolfe RR. Postexercise net protein 3. Tipton KD, Ferrando AA, Phillips SM, Doyle Jr, Wolfe RR. Postexercise net protein synthesis in human muscle from orally administered amino acids. Am J Physiol. 1999 synthesis in human muscle from orally administered amino acids. Am J Physiol. 1999 Apr;276(4 Pt 1):E628-34.Apr;276(4 Pt 1):E628-34.

4. Anthony JC, Lang CH, Crozier SJ, Anthony TG, MacLean DA, Kimball SR, Jefferson 4. Anthony JC, Lang CH, Crozier SJ, Anthony TG, MacLean DA, Kimball SR, Jefferson LS. Contribution of insulin to the translational control of protein synthesis in skeletal LS. Contribution of insulin to the translational control of protein synthesis in skeletal muscle by leucine. Am J Physiol Endocrinol Metab. 2002 May;282(5):E1092-101.muscle by leucine. Am J Physiol Endocrinol Metab. 2002 May;282(5):E1092-101.

5. Bohe J, Low JF, Wolfe RR, Rennie MJ. Latency and duration of stimulation of 5. Bohe J, Low JF, Wolfe RR, Rennie MJ. Latency and duration of stimulation of human muscle protein synthesis during continuous infusion of amino acids. J Physiol. human muscle protein synthesis during continuous infusion of amino acids. J Physiol. 2001 Apr 15;532(Pt 2):575-9.2001 Apr 15;532(Pt 2):575-9.

6. Norton, L.E., Layman, D.K., Garlick, P.J., Brana, D., Anthony, T.G., Zhao, L., 6. Norton, L.E., Layman, D.K., Garlick, P.J., Brana, D., Anthony, T.G., Zhao, L., Devkota, S. and Walker, D.A., (2007) Translational controls of skeletal muscle protein Devkota, S. and Walker, D.A., (2007) Translational controls of skeletal muscle protein synthesis are delayed and prolonged associated with ingestion of a complete meal. synthesis are delayed and prolonged associated with ingestion of a complete meal. 2007 Experimental Biology meeting abstracts [on CD-ROM], Abstract #694.62007 Experimental Biology meeting abstracts [on CD-ROM], Abstract #694.6

7. Paddon-Jones D, Sheffield-Moore M, Aarsland A, Wolfe RR, Ferrando AA.7. Paddon-Jones D, Sheffield-Moore M, Aarsland A, Wolfe RR, Ferrando AA. Exogenous amino acids stimulate human muscle anabolism without interfering with Exogenous amino acids stimulate human muscle anabolism without interfering with

the response to mixed meal ingestion. Am J Physiol Endocrinol Metab. 2005 the response to mixed meal ingestion. Am J Physiol Endocrinol Metab. 2005 Apr;288(4):E761-7. Epub 2004 Nov 30.Apr;288(4):E761-7. Epub 2004 Nov 30.

AcknowledgmentsAcknowledgments

Dr. Don Layman Dr. Don Layman Dee WalkerDee Walker Suzanne DevkotaSuzanne Devkota Chris MoultonChris Moulton Gabe and Jacob WilsonGabe and Jacob Wilson Dr. Tracy AnthonyDr. Tracy Anthony Piwan BunpoPiwan Bunpo My wife, IsabelMy wife, Isabel

Questions?Questions?