Adechian Et Al Nutr (2012) Leu and Energy Restriction

8112019 Adechian Et Al Nutr (2012) Leu and Energy Restriction

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Basic nutritional investigation

Spreading intake of a leucine-rich fast protein in energy-restricted overweight

rats does not improve protein mass

Solange Adechian PhD a b Didier R emond PhD ab Claire Gaudichon PhD c Corinne Pouyet MS a bDominique Dardevet PhD ab Laurent Mosoni PhD ab

a INRA UMR 1019 Nutrition Humaine Saint Genes Champanelle Franceb Universit e Clermont 1 UFR M edecine UMR 1019 Nutrition Humaine Clermont-Ferrand Francec INRA UMR 0914 UMR INRAINA-PGAgroParisTechPhysiologie de la Nutrition et du Comportement Alimentaire Alimentation Humaine Centre de Recherche de Paris

Paris France

a r t i c l e i n f o

Article history

Received 14 June 2011

Accepted 29 August 2011

Keywords

Milk protein fractions

Body composition

Protein metabolism

Wistar rats

a b s t r a c t

Objective Energy restriction decreases fat mass and fat-free mass Our aim was to prevent the latter

using type and timing of protein nutrition as tools

Methods Young male Wistar rats were given a high-energy diet for 5 wk and then energy

restricted and fed a high-protein diet containing caseins milk-soluble proteins (MSP) or a casein ndash

MSP mixture (n frac14 9 per group) as the only source of protein for 3 wk Food intake was spread over

12 h whereas in a previous experiment rats consumed their daily ration within 2 to 3 h Weight

and food intake were recorded The body composition was measured by dual-energy x-ray

absorptiometry before and after energy restriction After 3 wk the hind-limb muscles the kidney

intestine liver and spleen weights metabolic plasma parameters and the liver and extensor

digitorum longus muscle protein synthesis rates were measured in the postprandial state

Results The food intake was similar in all groups Energy restriction induced a signi1047297

cant decreasein body weight and fat mass (P lt 0001) and stopped the slow growth of lean body mass with no

differences between groups Among all tissues a signi1047297cant effect was detected only for the

intestine (P frac14 00012) with a higher weight in the casein group Postprandial liver and muscle

protein synthesis rates were not different between groups

Conclusion When using a high-protein diet spread over 12 h the nature of the protein intake has

no in1047298uence on the sparing of lean body mass during energy restriction in young overweight rats

2012 Elsevier Inc All rights reserved

Introduction

Obesity has reached epidemic proportions more than 1

billion adults are overweight and at least 300 million are clini-

cally obese [1] Obesity is a risk factor for cardiovascular diseases

and diabetes and contributes strongly to the global burden of the

associated health costs Obese individuals seeking weight loss

often use restrictive diets which lead to a decrease in adiposity

but also to a loss of fat-free mass [2] This loss of fat-free mass

and in particular muscle mass should be prevented because

muscle is an emergency store of amino acids that can be used

during stresses allowing an organism to maintain homeostasis

This loss can be limited by including a suf 1047297cient amount of

protein in the energy-restricted diet [3] Previously we compared

the capacity of caseins (slowly digested milk proteins) with that

of milk-soluble proteins (MSP rapidly digested leucine-rich

proteins) to maintain lean body mass in overweight energy-

restricted rats [4] In the present study we investigated

whether the timing of the intake of these proteins could have an

in1047298uence on the sparing of lean body mass

Indeed in our previous experiment [4] rats consumed their

daily ration within 2 to 3 h We found that although the regu-

lations of liver and muscle protein metabolisms were not the

same the 1047297nal nitrogen balance (and thus whole-body protein

mass) was not different between groups In that experiment

postabsorptive muscle protein synthesis rates were higher in the

casein-fed group than in the MSP-fed groups [4] Given the

results obtained in test-meal studies in humans [5ndash7] we

postulated that the muscle protein balance (ie protein synthesis

This project was supported by French National Research Agency and by the

Centre National Interprofessionnel de lrsquoEconomie Laitiere

Corresponding author Tel thorn334-7362-4827 fax thorn334-7362-4755

E-mail address laurentmosoniclermontinrafr (L Mosoni)

0899-9007$ - see front matter 2012 Elsevier Inc All rights reserveddoi101016jnut201108020

Contents lists available at ScienceDirect

Nutrition

j o u r n a l h o m e p a g e w w w n u t r i t i o n j r n l c o m

Nutrition 28 (2012) 566ndash571



minus proteolysis) was probably lower in the casein-fed group

than in the MSP-fed group in the postprandial state and that the

reverse occurred in the postabsorptive state so that the overall

24-h balance was the same To expand on these recent 1047297ndings

we postulated that by spreading protein intake over 12 h a high

postprandial muscle protein balance could be maintained in the

MSP-fed group by its high leucine content a key amino acid for

protein synthesis stimulation [8] and by limiting the duration of the postabsorptive period to limit postabsorptive muscle losses

This would be the equivalent of four meals a day in humans

whereas in our previous experiment it was more equivalent to

one meal a day

Thus in the present experiment young male Wistar rats were

fed ad libitum for 5 wk with a high-energy diet [9] and then

energy restricted and fed a high-protein diet containing caseins

MSP or a caseinndashMSP mixture for 3 wk Food intake was spread

over 12 h four equal meals were distributed by an automatic

delivery system We used dual-energy x-ray absorptiometry

(DEXA) to assess the changes in lean body mass and we

measured muscle mass and in vivo postprandial protein

synthesis rates

Materials and methods

Animals and diets

This study was performed in accordance with current legislation on animal

experimentation in France Male Wistar rats (n frac14 27 Harlan Gannat France)

weighing 3250 13 g (mean standard error) were housed individually in

cages under controlled environmental conditions (temperature 20 1C

humidity 50 5) with a 12-h inverse lightdark cycle (light on at 1700 h) and

free access to tap water On arrival rats were acclimated to the animal facilities

for 5 d and fed ad libitum with commercial laboratory pellets (UAR 04 UAR

Villemoisson sur Orge France) Then animals were fed ad libitum a high-fat

high-sucrose semiliquid diet (Table 1) during 5 wk We showed previously that

this diet increases animal fat mass [9] Rats were then randomly divided into

three groups each group was fed with a restricted amount of a diet containing

caseins (n frac14 9) MSP (n frac14 9)or a mixture ofcasein andMSP (n frac14 95050 ww)asthe only source of protein (Table 1) for 3 wk Our aim was to provide restricted

rats with 60 of their usual spontaneous energy intake (estimated at 355 kJd in

a separate experiment) and to make sure that energy-restricted animals did not

consume their food in one rapid meal (meal feeding at 0500 0800 1100 and

1400 h with removal of food at 1700 h)However because of thechange in their

environment (automatic delivery system change from a semiliquid diet to a dry

powder) animals consumed less than expected (overall only 78 of what was

offered to them) and progressively increased their intake during the 3 wk of

feeding Food intake was recorded during the entire experiment by measuring

the dry matter intake daily

Measurements of in vivo tissue protein synthesis rates

Tracer injection and tissue collection

After 3 wk of restrictiontissue protein synthesisrateswere measured in vivo

using the 1047298ooding dose method [10] These measurements were performed in

the fed state 4 to 7 h after the beginning of the dark period The fed state was

con1047297rmed by measuring the dry matter content in the stomach It was positive

and similar in the casein (21 03 g) and MSP (22 04 g) groups (means SE)

but in the mixture group two rats were not used for protein synthesis

measurements because their dry matter content in the stomach was low It was

correct for the other mixture-fed rats (17 02 g) Twenty minutes before

euthanasia each rat was injected in a lateral tail vein under a light gaseous

anesthesia (iso1047298urane Baxter Mauripas France) with a 1047298ooding dose of valine

(150 mmol100 g of body weight) to 1047298ood the precursor pools with 50 of L-

[1-13C] valine (99 Cambridge Isotope Laboratories Andover MA USA) The

reliability of valine as tracer has been checked previously [11] Then anesthesia

was induced by an intraperitoneal injection of pentobarbital sodium (Sano1047297

Libourne France) before euthanasia by exsanguination (abdominal aorta) Blood

was rapidly collected in heparinized tubes and centrifuged at 3000 g at thorn4C

for10 min Plasmawas collected andkept frozenin liquid nitrogen beforestoring

at 80C until further analysis The liver and extensor digitorum longus muscle

were excised quickly chilled on ice to stop the tracerincorporation (the liver was

cut into small pieces rinsed in cold saline [NaCl 9 gL] solution to remove the

blood and wiped) weighed frozen in liquid nitrogen 3 to 5 min after exsan-

guination and stored at 20C until analysis The small intestine was emptied

rinsed with cold trichloroacetic acid (012 molL) dried and weighted The

epididymal and renal fat pads were carefully removed and weighed The spleen

kidneys and gastrocnemius tibialis anterior and soleus muscles of both hind

legs were also weighed The stomach content was removed and dried and its

weight was measured The liver and muscle free and protein bound valine

concentrations were determined as described previously [9]

Calculations

The in vivo fractional synthesis rates (FSR percentage per day) of tissue

proteins were calculated as described previously [9] FSR frac14 100 (EP EN)(EA t )

where t is the incorporation time (expressed in days) and EP and EA are the 13C

enrichments of protein-bound valineand of free valine respectively at theend of

the incorporation time The incorporation time was measured for each rat from

the time of injection to the time of exsanguination and averaged (204 24 min

mean standard error) EN is an estimation of the natural 13C enrichment of

protein-bound valine It was determined in three rats that were notinjectedwith

the 1047298ooding dose EP EN and EA were expressed in atom percentages The

absolute synthesis rate was calculated from the product of FSR and the proteincontent of the tissue and expressed in milligrams or grams per day

Measurements of body weight and body composition

Body weightwas recorded three times a week Whole-body composition was

measured in vivo on days 0 and 16 of the energy-restriction period using DEXA

(QDR-4500A Hologic Inc Waltham MA USA) after a calibration for small

animals Rats were anesthetized by an intraperitoneal injection of a combination

of Vetranquil 05 (0250mL500 g of live body weight Sano1047297) andImalgen1000

(0376 mL500 g of live body weight Merial Lyon France) and scanned on

a prone position

Plasma assays

Plasma insulin was analyzed using a commercial radioimmunoassay kit

(LINCO Research Labodia France) Other plasma measurements were performedusing commercial kits from Horiba ABX (Montpellier France)

Plasma amino acids were puri1047297ed by ion-exchange chromatography after

protein precipitation 500 mL of plasma was added to 125 mL of sulfosalicylic acid

solution (1 molL in ethanol with thioglycolate 05 molL) that had previously

completely evaporated Norleucine was added as an internal standard Samples

were incubated on ice for 1 h and centrifuged at 3500 g for 1 h at 4C An

aliquot (250 mL) of the supernatant was combined with 125 mL of 01 molL

lithium acetate buffer pH 22 Amino acid concentrations were determined by

ion-exchange chromatography (Bio-Tek Instruments ARL St Quentin Yvelines

France) using postcolumn derivation with Ninhydrin

Statistical analysis

Results were analyzed by analysis of variance or repeated measures analysis

of variance If the analysis of variance F test was signi1047297cant post hoc Tukey tests

were performed to compare means (SAS SAS Institute Cary NC USA) Differ-

ences between means were considered statistically at P lt 005 Results areexpressed as mean standard error

Table 1

Diet composition

Ingredient High fathigh

sucrose

Casein MSP Mixture

L -Cystine (gkg diet) 12

Total milk proteins (gkg diet) 1790

Casein (gkg diet) d 3942 d 1970

MSP (gkg diet) d d 4130 2065

Starch (gkg diet) 689 3250 3320 3290

Sucrose (gkg diet) 4232 d d d

Lactose (gkg diet) d 311 d 153

Lard (gkg diet) 2028 d d d

Soybean oil (gkg diet) 274 1205 1 258 1230

AIN-93M mineral mix (gkg diet) 350 583 583 583

AIN-963M vitamin mix (gkg diet) 100 167 167 167

Cellulose (gkg diet) 500 500 500 500

Choline bitartrate (gkg diet) 25 42 42 42

Protein ( energy) 14 37 37 37

Lipid ( energy) 45 28 28 28

Carbohydrate ( energy) 41 35 35 35

Metab oliza ble energy (MJ kg diet) 2 04 1 75 1 80 17 8MSP milk-soluble proteins

S Adechian et al Nutrition 28 (2012) 566 ndash571 567





protein synthesis rates expressed in fractional rates or absolute

rates were notdifferent among groups (P gt 005 Table 3) Muscle

protein synthesis rates tended to be lower in the mixture group

(P frac14 00984) probably in response to a slightly lower food intake

on the day of the measurement (Table 3)

Liver

At the end of the energy-restriction period in the post-prandial state liver weight and total protein mass were not

different in the MSP and mixture groups (P gt 005 Table 3)

These parameters were higher in the casein group but the

difference was not always signi1047297cant (casein versus MSP group

for liver weight P frac14 00231 casein versus mixture group for total

protein P frac14 00217) Protein concentration and fractional and

absolute synthesis rates were not different among groups (P gt

005 Table 3)

Plasma metabolic parameters

Glucose insulin cholesterol and triacylglycerol plasma

concentrations were not different among groups (P gt 005

Table 4) Only plasma urea content was signi1047297cantly lower in thecasein group than in the MSP group with an intermediary value

in the mixture group (P frac14 00143 Table 4)

Among the postprandial plasma amino acid values few were

signi1047297cantly different among groups (Fig 3) Only tyrosine

(greater in the casein than in the MSP group P frac14 00082) leucine

(lower in the casein and mixture groups than in the MSP group

P frac14 00005) proline (highest in the casein group and higher in

the mixture group than in the MSP group P frac14 00001) and the

sum of glutamine and glutamic acid (higher in the casein than in

the MSP group P frac14 00490) were signi1047297cantly different among

groups These differences are in accordance with differences in

the amino acid contents of caseins and MSP but there were

similar differences in the amino acid composition that did not

induce differences in the plasma values

Discussion

Our aim was to compare the capacity of different milk protein

fractions to minimize the loss of leanbody mass induced by energy

restriction in previously overfed male Wistar rats Caseins (slowly

digested dietary proteins) MSP (rapidly digested leucine-rich

proteins) and a mixture of both (caseinsMSP 5050 ww) were

compared Previously we showed that when food was consumed

within 2 to 3 hday despite a positive effect on postabsorptive

muscle protein synthesis rates and no difference in muscle prote-

olysis caseins were unable to promote greater body protein

retention than MSP in energy-restricted overweight rats [4] It is

likely that the positive effect observed in the postabsorptive state

with caseins was counterbalanced by a lower anabolic effect in the

postprandial state with MSP The present study was designed to

optimize the timing of food intake with the hope of detecting

a difference between caseins and MSP thus the food intake was

spread over 12 hd We postulated that this feeding pattern would

favor MSP by limiting postabsorptive muscle protein losses butmaintaining suf 1047297cient postprandial muscle protein gainbecause of

the high leucine content of these proteins

In our overweight rat model a loss of lean body mass in

response to energy restriction was unlikely 4 d of complete

fasting in adult rats (with signi1047297cant fat stores as in young

overweight rats) did not induce any change in muscle mass

although the liver protein mass was markedly decreased [12]

Ten days without food in the same animals had no additional

effect on the liver protein mass and decreased the muscle protein

mass by only 19 [13] Three weeks of total protein depletion had

similar effects on muscle mass (15) and total body protein

(19) in old rats [14] Previously we showed that during energy

restriction using a high-protein diet nitrogen balance was never

negative in young overweight rats [4] Recently in a comparableexperiment (young overweight rats) Eller and Reimer [15]

observed by DEXA a loss of lean body mass in response to

energy restriction but their protein diet content was low (12 of

energy) and thus protein intake was restricted In the present

experiment DEXA measurements showed that energy restric-

tion only stopped the slow growth of lean body mass This is due

to the high-protein content of our diet (18 of energy) when

energy is restricted including a suf 1047297cient amount of protein in

the diet contributes to the preservation of fat-free mass [3]

Our aim was to see if any additional bene1047297t could be found by

changing the nature and the timing of the protein intake

However we did not detect any signi1047297cant difference in the

evolution of lean body mass during energy restriction among the

groups This result is consistent with our previous experiment

[4] It is also consistent with the study by Eller and Reimer [15]

after 4 wk of energy restriction no signi1047297cant difference was

found between the casein- and MSP-fed groups but a greater

lean body mass was found in rats fed skim milk than in those fed

MSP Skim milk is a complex nutrient containing micronutrients

such as vitamin D calcium phosphorus proteins with w80

caseins and w20 MSP various other immunoproteins and

lactose In our experiment we tested a 5050 mixture of caseins

and MSP and the evolution of lean body mass was no better in

this group than in the other groups It suggests that the positive

effect of skim milk observed by Eller and Reimer [15] was caused

by another nutrient than caseins or MSP

Table 3

Effect of caseins MSP or a mixed feeding on postprandial muscle and liver protein metabolism during energy restriction

EDL muscle Liver

Casein MSP Mixture Casein MSP Mixture

Tissue weight (mg or g) 176 3 161 6 166 4 118 04a 100 05b 106 03ab

Protein weight (mg or g) 33 1 31 1 31 1 22 01a 19 01ab 18 01b

Protein concentration (mgg) 1 85 4 192 6 185 4 187 8 190 3 170 8

FSR (d)y 39 02 42 03 34 02 62 4 64 2 66 2

ASR (mgd or gd)y 13 01 13 01 10 01 14 01 13 01 12 01

ASR absolute synthesis rate EDL extensor digitorum longus FSR fractional synthesis rate MSP milk-soluble proteins

See Table 2 At the end of the restriction period (day 21) in vivo fed-state protein synthesis rates were measured using a 1047298ooding dose of L-[1-13C] valine Values are

presented as mean SE When a signi1047297cant effect was detected by analysis of variance means were compared values with different superscript letters were

signi1047297cantly different

Values for the muscle are in milligrams and those for the liver are in gramsy For these parameters two rats in the mixture group were excluded because their food intake was too low




When examining tissue mass the only tissue where a signif-

icant difference among groups could be found was the intestine

In this tissue the 1047297nal weight was greater in the casein group

than in the other groups This is a ldquorobustrdquo observation because itwas also observed in our previous experiment [4] It seems that

caseins stimulate intestine growth Such an effect was also

observed in reared rat pups [16] The mechanism of this

phenomenon remains to be elucidated but it is certainly a local

effect caused by bioactive peptides or involving casein digestion

Postprandial muscle protein synthesis rates tended to be

higher in the MSP group than in the casein group but the

difference was not signi1047297cant Because of its rapid digestion and

its high leucine content a greater stimulation of muscle protein

synthesis rates in the postprandial state by MSP than by caseins

can be expected [17] Indeed it seems established that post-

prandial muscle protein synthesis rates vary in relation to the

amount of ingested leucine [17] because leucine can have

a speci1047297c effect on muscle protein synthesis through the acti-

vation of mammalian target of rapamycin-dependent signaling

pathways [8]The fact that our rats were trained to consume their

meals over 12 h probably attenuated the difference Muscle

protein synthesis rates tended to be lower in the mixture-fed

group which likely was caused by the slightly lower food

intake in this group on the day of the measurement as re1047298ected

by the amount of dry matter found in the stomach

Liver protein synthesis rates measured in the fed state were

unchanged by the type of protein ingested during energy

restriction It was also the case in our previous experiment in the

postabsorptive state [4] The liver is a key organ in the regulation

of postprandial protein metabolism It is a major site of amino

acid catabolism and considerable quantities of absorbed amino

acids are extracted from the portal vein these amino acids are

made partly available from the liver in other forms notablyproteins or peptides [18] The liver can be seen as a buffer pre-

venting rapid variations in peripheral plasma amino acid

concentrations The main difference between caseins and MSP is

that caseins are slowly absorbed whereas MSP are quickly

digested and induce rapid variations in portal amino acid

concentrations This could have promoted differences in the liver

protein metabolism responses However the fact that in the

present experiment the food intake was spread over 12 h prob-

ably attenuated the differences between caseins and MSP To our

knowledge no similar studies have investigated the liver protein

metabolism response to caseins or MSP in particular during

energy restriction

We detected few metabolic differences among the groups All

signi1047297cant differences observed in the plasma amino acidconcentrations were consistent with the differences in the amino

acid composition of the proteins Glucose insulin cholesterol

and triacylglycerol concentrations were not different among the

groups Regarding glucose and insulin these observations were

consistent with the results obtained by Eller and Reimer [15]

However MSP have been shown to have a lowering effect on

fatty acid synthesis in the liver [19] and on plasma cholesterol

concentrations [20] compared with caseins in normally fed

young rats The discrepancy observed may be due to the fact that

our rats were energy restricted which already decreases

cholesterol and triacylglycerol concentrations (see Adechian

et al [9] for comparative values) We also detected a signi1047297cantly

lower urea concentration in the casein group compared with the

MSP group This result suggests lower postprandial amino acid

oxidation in the casein group and is consistent with results

observed in short-term normally fed humans [5ndash7] However we

showed previously that per day there is no difference in amino

acid oxidation among these groups [4] Thus this difference in

the postprandial state must be compensated for in the post-

absorptive state and in any case does not lead in the end to

a difference in body composition

Amino acids

0

50

100

150

200

250

300

Tyr Hist Phe Arg Try Isol S Asp Ser Gly Leu Thre

micromoles l

0

200

400

600

800

Prol Lys Ala S Glu

Casein

MSP

Mix

Fig 3 Postprandial plasma amino acid concentrations in casein- MSP- and mixture-fed rats See Figure 1 Values for postprandial plasma amino acid concentrations (mmoles

per liter mean SE) are reported A signi1047297cant difference among groups was detected tyrosine casein gt MSP leucine casein frac14 mix lt MSP proline casein gt mix gt MSP

sum of glutamine and glutamic acid casein gt MSP Plasma valine content was not reported because it was used for the 1047298ooding dose and values were very high in all groupsMSP milk-soluble proteins S Asp sum of asparagine and aspartic acid S Glu sum of glutamine and glutamic acid

Table 4

Effect of casein MSP or a mixed feeding on postprandial plasma metabolic

parameters during energy restriction

Casein MSP Mixture

Urea (mmolL) 100 05a 130 06b 120 08ab

Glucose (mM) 126 03 132 03 126 04

Insulin (ngmL) 49 06 39 06 42 09

Cholesterol (mM) 12 01 14 01 16 01

HDL (mM) 055 004 070 003 057 005Triacylglycerols (mM) 15 02 11 01 11 02

HDL high-density protein MSP milk-soluble proteins

See Table 2 At day 21 of restriction plasma urea insulin glucose total choles-

terol HDL cholesterol and triacylglycerol levels were measured in the fed state

Values are presented as mean SE When a signi1047297cant effect was detected by

analysis of variance means were compared values with different superscript

letters were signi1047297cantly different

For HDL analysis of variance detected a signi1047297cant effect but no signi1047297cant

difference was found between groups by the Tukey test

S Adechian et al Nutrition 28 (2012) 566 ndash571570



Conclusion

Despite the high leucine content of MSP and the shortness of

the postabsorptive period there was no difference in the body

protein mass evolution among the groups Thus when protein

intake is high the nature andthe timingof proteinintake have no

in1047298uence on lean body mass changes during energy restriction

Acknowledgments

The authors thank Christian Lafarge and Philippe Lhoste for

animal managementand Corinne Pouyet andEstelle Pujosfor mass

spectrometric measurements

References

[1] World Health Organization Obesity preventing and managing the globalepidemic Report of a WHO consultation WHO technical report 2000series 894256 Geneva World Health Organization 2000

[2] Chaston TB Dixon JB OrsquoBrien PE Changes in fat-free mass duringsigni1047297cant weight loss a systematic review Int J Obes (Lond) 200731

743ndash

50[3] Mahon AK Flynn MG Stewart LK McFarlin BK Iglay HB Mattes RD et alProtein intake during energy restriction effects on body composition andmarkers of metabolic and cardiovascular health in postmenopausalwomen J Am Coll Nutr 200726182ndash9

[4] Adechian S Remond D Gaudichon C Dardevet D Mosoni L Thenature of the ingested protein has no effect on lean body mass duringenergy restriction in overweight rats Obesity (Silver Spring) 2011191137ndash44

[5] Boirie Y Dangin M Gachon P Vasson MP Maubois JL Beaufrere B Slow andfast dietary proteins differently modulate postprandial protein accretionProc Natl Acad Sci U S A 19979414930ndash5

[6] Lacroix M Bos C Leonil J Airinei G Luengo C Dare S et al Compared withcasein or total milk protein digestion of milk soluble proteins is too rapidto sustain the anabolic postprandial amino acid requirement Am J ClinNutr 2006841070ndash9

[7] Dangin M Guillet C Garcia-Rodenas C Gachon P Bouteloup-Demange CReiffers-Magnani K et al The rate of protein digestion affects protein gaindifferently during aging in humans J Physiol 2003549635ndash44

[8] Balage M Dardevet D Long-term effects of leucine supplementation onbody composition Curr Opin Clin Nutr Metab Care 201013265ndash70

[9] Adechian S Giardina S Remond D Papet I Buonocore D Gaudichon C et alExcessive energy intake does not modify fed-state tissue protein synthesisrates in adult rats Obesity (Silver Spring) 2009171348ndash55

[10] Garlick PJ McNurlan MA Preedy VR A rapid and convenient technique for

measuring the rate of protein synthesis in tissues by injection of Biochem J1980192719ndash23[11] Attaix D Manghebati A Grizard J Arnal M Assessment of in vivo

protein synthesis in lamb tissues with Biochim Biophys Acta 1986882389ndash97

[12] Mosoni L Malmezat T Valluy MC Houlier ML Mirand PP Muscle and liverprotein synthesis adapt ef 1047297ciently to food deprivation and refeeding in 12-month-old rats J Nutr 1996126516ndash22

[13] Mosoni L Malmezat T Valluy MC Houlier ML Attaix D Mirand PP Lowerrecovery of muscle protein lost during starvation in old rats despitea stimulation of protein synthesis Am J Physiol Endocrinol Metab1999277E608ndash16

[14] Carter WJ Lynch ME Effect of clenbuterol on recovery of muscle mass andcarcass protein content following dietary protein depletion in young andold rats J Gerontol 199449B162ndash8

[15] Eller LK Reimer RA A high calcium skim milk powder diet results ina lower fat mass in male energy-restricted obese rats more than a lowcalcium casein or soy protein diet J Nutr 20101401234ndash41

[16] Yajima T Kanno T Katoku Y Kuwata T Gut hypertrophy in response to theratios of casein and whey protein in milk formulas in arti1047297cially reared ratpups Biol Neonate 199874314ndash22

[17] Rieu I Balage M Sornet C Debras E Ripes S Rochon-Bonhomme C et alIncreased availability of leucine with leucine-rich whey proteins improvespostprandial muscle protein synthesis in aging rats Nutrition 200723323ndash31

[18] Connell A Calder AG Anderson SE Lobley GE Hepatic protein synthesis inthe sheep effect of intake as monitored by use of stable-isotopendashlabelledglycine leucine and phenylalanine Br J Nutr 199777255ndash71

[19] Morifuji M Sakai K Sanbongi C Sugiura K Dietary whey protein down-regulates fatty acid synthesis in the liver but upregulates it in skeletalmuscle of exercise-trained rats Nutrition 2005211052ndash8

[20] Zhang X Beynen AC Lowering effect of dietary milk-whey protein v caseinon plasma and liver cholesterol concentrations in rats Br J Nutr199370139ndash46




minus proteolysis) was probably lower in the casein-fed group

than in the MSP-fed group in the postprandial state and that the

reverse occurred in the postabsorptive state so that the overall

24-h balance was the same To expand on these recent 1047297ndings

we postulated that by spreading protein intake over 12 h a high

postprandial muscle protein balance could be maintained in the

MSP-fed group by its high leucine content a key amino acid for

protein synthesis stimulation [8] and by limiting the duration of the postabsorptive period to limit postabsorptive muscle losses

This would be the equivalent of four meals a day in humans

whereas in our previous experiment it was more equivalent to

one meal a day

Thus in the present experiment young male Wistar rats were

fed ad libitum for 5 wk with a high-energy diet [9] and then

energy restricted and fed a high-protein diet containing caseins

MSP or a caseinndashMSP mixture for 3 wk Food intake was spread

over 12 h four equal meals were distributed by an automatic

delivery system We used dual-energy x-ray absorptiometry

(DEXA) to assess the changes in lean body mass and we

measured muscle mass and in vivo postprandial protein

synthesis rates

Materials and methods

Animals and diets

This study was performed in accordance with current legislation on animal

experimentation in France Male Wistar rats (n frac14 27 Harlan Gannat France)

weighing 3250 13 g (mean standard error) were housed individually in

cages under controlled environmental conditions (temperature 20 1C

humidity 50 5) with a 12-h inverse lightdark cycle (light on at 1700 h) and

free access to tap water On arrival rats were acclimated to the animal facilities

for 5 d and fed ad libitum with commercial laboratory pellets (UAR 04 UAR

Villemoisson sur Orge France) Then animals were fed ad libitum a high-fat

high-sucrose semiliquid diet (Table 1) during 5 wk We showed previously that

this diet increases animal fat mass [9] Rats were then randomly divided into

three groups each group was fed with a restricted amount of a diet containing

caseins (n frac14 9) MSP (n frac14 9)or a mixture ofcasein andMSP (n frac14 95050 ww)asthe only source of protein (Table 1) for 3 wk Our aim was to provide restricted

rats with 60 of their usual spontaneous energy intake (estimated at 355 kJd in

a separate experiment) and to make sure that energy-restricted animals did not

consume their food in one rapid meal (meal feeding at 0500 0800 1100 and

1400 h with removal of food at 1700 h)However because of thechange in their

environment (automatic delivery system change from a semiliquid diet to a dry

powder) animals consumed less than expected (overall only 78 of what was

offered to them) and progressively increased their intake during the 3 wk of

feeding Food intake was recorded during the entire experiment by measuring

the dry matter intake daily

Measurements of in vivo tissue protein synthesis rates

Tracer injection and tissue collection

After 3 wk of restrictiontissue protein synthesisrateswere measured in vivo

using the 1047298ooding dose method [10] These measurements were performed in

the fed state 4 to 7 h after the beginning of the dark period The fed state was

con1047297rmed by measuring the dry matter content in the stomach It was positive

and similar in the casein (21 03 g) and MSP (22 04 g) groups (means SE)

but in the mixture group two rats were not used for protein synthesis

measurements because their dry matter content in the stomach was low It was

correct for the other mixture-fed rats (17 02 g) Twenty minutes before

euthanasia each rat was injected in a lateral tail vein under a light gaseous

anesthesia (iso1047298urane Baxter Mauripas France) with a 1047298ooding dose of valine

(150 mmol100 g of body weight) to 1047298ood the precursor pools with 50 of L-

[1-13C] valine (99 Cambridge Isotope Laboratories Andover MA USA) The

reliability of valine as tracer has been checked previously [11] Then anesthesia

was induced by an intraperitoneal injection of pentobarbital sodium (Sano1047297

Libourne France) before euthanasia by exsanguination (abdominal aorta) Blood

was rapidly collected in heparinized tubes and centrifuged at 3000 g at thorn4C

for10 min Plasmawas collected andkept frozenin liquid nitrogen beforestoring

at 80C until further analysis The liver and extensor digitorum longus muscle

were excised quickly chilled on ice to stop the tracerincorporation (the liver was

cut into small pieces rinsed in cold saline [NaCl 9 gL] solution to remove the

blood and wiped) weighed frozen in liquid nitrogen 3 to 5 min after exsan-

guination and stored at 20C until analysis The small intestine was emptied

rinsed with cold trichloroacetic acid (012 molL) dried and weighted The

epididymal and renal fat pads were carefully removed and weighed The spleen

kidneys and gastrocnemius tibialis anterior and soleus muscles of both hind

legs were also weighed The stomach content was removed and dried and its

weight was measured The liver and muscle free and protein bound valine

concentrations were determined as described previously [9]

Calculations

The in vivo fractional synthesis rates (FSR percentage per day) of tissue

proteins were calculated as described previously [9] FSR frac14 100 (EP EN)(EA t )

where t is the incorporation time (expressed in days) and EP and EA are the 13C

enrichments of protein-bound valineand of free valine respectively at theend of

the incorporation time The incorporation time was measured for each rat from

the time of injection to the time of exsanguination and averaged (204 24 min

mean standard error) EN is an estimation of the natural 13C enrichment of

protein-bound valine It was determined in three rats that were notinjectedwith

the 1047298ooding dose EP EN and EA were expressed in atom percentages The

absolute synthesis rate was calculated from the product of FSR and the proteincontent of the tissue and expressed in milligrams or grams per day

Measurements of body weight and body composition

Body weightwas recorded three times a week Whole-body composition was

measured in vivo on days 0 and 16 of the energy-restriction period using DEXA

(QDR-4500A Hologic Inc Waltham MA USA) after a calibration for small

animals Rats were anesthetized by an intraperitoneal injection of a combination

of Vetranquil 05 (0250mL500 g of live body weight Sano1047297) andImalgen1000

(0376 mL500 g of live body weight Merial Lyon France) and scanned on

a prone position

Plasma assays

Plasma insulin was analyzed using a commercial radioimmunoassay kit

(LINCO Research Labodia France) Other plasma measurements were performedusing commercial kits from Horiba ABX (Montpellier France)

Plasma amino acids were puri1047297ed by ion-exchange chromatography after

protein precipitation 500 mL of plasma was added to 125 mL of sulfosalicylic acid

solution (1 molL in ethanol with thioglycolate 05 molL) that had previously

completely evaporated Norleucine was added as an internal standard Samples

were incubated on ice for 1 h and centrifuged at 3500 g for 1 h at 4C An

aliquot (250 mL) of the supernatant was combined with 125 mL of 01 molL

lithium acetate buffer pH 22 Amino acid concentrations were determined by

ion-exchange chromatography (Bio-Tek Instruments ARL St Quentin Yvelines

France) using postcolumn derivation with Ninhydrin

Statistical analysis

Results were analyzed by analysis of variance or repeated measures analysis

of variance If the analysis of variance F test was signi1047297cant post hoc Tukey tests

were performed to compare means (SAS SAS Institute Cary NC USA) Differ-

ences between means were considered statistically at P lt 005 Results areexpressed as mean standard error

Table 1

Diet composition

Ingredient High fathigh

sucrose

Casein MSP Mixture

L -Cystine (gkg diet) 12

Total milk proteins (gkg diet) 1790

Casein (gkg diet) d 3942 d 1970

MSP (gkg diet) d d 4130 2065

Starch (gkg diet) 689 3250 3320 3290

Sucrose (gkg diet) 4232 d d d

Lactose (gkg diet) d 311 d 153

Lard (gkg diet) 2028 d d d

Soybean oil (gkg diet) 274 1205 1 258 1230

AIN-93M mineral mix (gkg diet) 350 583 583 583

AIN-963M vitamin mix (gkg diet) 100 167 167 167

Cellulose (gkg diet) 500 500 500 500

Choline bitartrate (gkg diet) 25 42 42 42

Protein ( energy) 14 37 37 37

Lipid ( energy) 45 28 28 28

Carbohydrate ( energy) 41 35 35 35

Metab oliza ble energy (MJ kg diet) 2 04 1 75 1 80 17 8MSP milk-soluble proteins











Liver








005 Table 3)


















Discussion
























































Table 3


EDL muscle Liver





FSR (d)y 39 02 42 03 34 02 62 4 64 2 66 2

ASR (mgd or gd)y 13 01 13 01 10 01 14 01 13 01 12 01



















































energy restriction























Amino acids

0

50

100

150

200

250

300


micromoles l

0

200

400

600

800

Prol Lys Ala S Glu

Casein

MSP

Mix




Table 4



Casein MSP Mixture

Urea (mmolL) 100 05a 130 06b 120 08ab

Glucose (mM) 126 03 132 03 126 04

Insulin (ngmL) 49 06 39 06 42 09

Cholesterol (mM) 12 01 14 01 16 01













Conclusion






Acknowledgments




References



743ndash






























Liver








005 Table 3)


















Discussion
























































Table 3


EDL muscle Liver





FSR (d)y 39 02 42 03 34 02 62 4 64 2 66 2

ASR (mgd or gd)y 13 01 13 01 10 01 14 01 13 01 12 01



















































energy restriction























Amino acids

0

50

100

150

200

250

300


micromoles l

0

200

400

600

800

Prol Lys Ala S Glu

Casein

MSP

Mix




Table 4



Casein MSP Mixture

Urea (mmolL) 100 05a 130 06b 120 08ab

Glucose (mM) 126 03 132 03 126 04

Insulin (ngmL) 49 06 39 06 42 09

Cholesterol (mM) 12 01 14 01 16 01













Conclusion






Acknowledgments




References



743ndash




























Liver








005 Table 3)


















Discussion
























































Table 3


EDL muscle Liver





FSR (d)y 39 02 42 03 34 02 62 4 64 2 66 2

ASR (mgd or gd)y 13 01 13 01 10 01 14 01 13 01 12 01



















































energy restriction























Amino acids

0

50

100

150

200

250

300


micromoles l

0

200

400

600

800

Prol Lys Ala S Glu

Casein

MSP

Mix




Table 4



Casein MSP Mixture

Urea (mmolL) 100 05a 130 06b 120 08ab

Glucose (mM) 126 03 132 03 126 04

Insulin (ngmL) 49 06 39 06 42 09

Cholesterol (mM) 12 01 14 01 16 01













Conclusion






Acknowledgments




References



743ndash

































































energy restriction























Amino acids

0

50

100

150

200

250

300


micromoles l

0

200

400

600

800

Prol Lys Ala S Glu

Casein

MSP

Mix




Table 4



Casein MSP Mixture

Urea (mmolL) 100 05a 130 06b 120 08ab

Glucose (mM) 126 03 132 03 126 04

Insulin (ngmL) 49 06 39 06 42 09

Cholesterol (mM) 12 01 14 01 16 01













Conclusion






Acknowledgments




References



743ndash























Conclusion






Acknowledgments




References



743ndash





















Documents

Adechian Et Al Nutr (2012) Leu and Energy Restriction