ORIGINAL ARTICLE
Energy restriction only slightly influences protein metabolism
in obese rats, whatever the level of protein and its source
in the diet
L Chevalier1,2, C Bos1,2, D Azzout-Marniche1,2, G Fromentin1,2, L Mosoni3,4, N Hafnaoui3,4, J Piedcoq1,2, D Tome´1,2and C Gaudichon1,2
BACKGROUND: High protein (HP) diets during energy restriction have been studied extensively regarding their ability to reduce body fat and preserve lean body mass, but little is known about their effects on protein metabolism in lean tissues. OBJECTIVE: To determine the effects of energy restriction and protein intake on protein anabolism and catabolism in rats. METHODS: For 5 weeks, 56 male Wistar rats were fed an obesity induction (OI) diet . They were then subjected to a 40% energy restriction using the OI diet or a balanced HP diet for 3 weeks, whereas a control group was fed the OI diet ad libitum (n¼ 8 per group). HP-restricted rats were divided into five groups differing only in terms of their protein source: total milk proteins, casein (C), whey (W), a mix of 50% C and W, and soy (n¼ 8). The animals were then killed in the postprandial state and their body composition was determined. Protein synthesis rates were determined in the liver, gastrocnemius and kidney using a subcutaneous13C valine flooding dose. mRNA levels were measured for key enzymes involved in the three proteolysis pathways.
RESULTS: Energy restriction, but not diet composition, impacted weight loss and adiposity, whereas lean tissue mass (except in the kidney) was not influenced by diet composition. Levels of neoglucogenic amino acids tended to fall under energy restriction (Po0.06) but this was reversed by a high level of protein. The postprandial protein synthesis rates in different organs were similar in all groups. By contrast, mRNA levels encoding proteolytic enzymes rose under energy restriction in the muscle and kidney, but this was counteracted by a HP level.
CONCLUSIONS: In adult obese rats, energy restriction but not diet composition affected fat pads and had little impact on protein metabolism, despite marked effects on proteolysis in the kidney and muscle.
International Journal of Obesity (2013) 37, 263 -- 271; doi:10.1038/ijo.2012.19; published online 21 February 2012 Keywords: high protein diet; energy restriction; body composition; protein metabolism
INTRODUCTION
Weight management strategies are designed to reduce body fat while causing no major reduction in lean tissue. The different strategies proposed1,2involve varying levels of energy restriction and modifications to the energy nutrient content of diets, such as limiting the consumption of fat or carbohydrate (CHO) and increasing the protein content.3,4Proteins have been the focus of particular study because of their satiating effect that might both reduce energy intake5 and increase subject compliance with a diet.6,7 In addition, a protein-rich diet may minimize the loss of lean body mass that is observed under a low-calorie diet.8,9The type of protein source may also exert an influence, as reported in rodent models.10,11
The effects of energy restriction on anabolic and catabolic fluxes are poorly understood and the findings available, which differ regarding numerous parameters (model, duration, degree of restrictions), are not comparable. In humans, one study recently showed that muscle protein synthesis was decreased after moderate energy restriction.12Other data have been obtained in rats and revealed contrasting effects of energy restriction on muscle protein anabolism, depending the type of muscle (soleus,
gastrocnemius, plantaris, tibialis anterior) and of proteins (sarco-plasmic or mitochondrial).13 -- 15In addition, protein anabolism has not been investigated in other organs such as the liver or kidney, whereas it has been observed under ad libitum conditions that liver and kidney protein synthesis were modulated by the protein level in the diet, unlike synthesis in muscle.16,17 Studies of the effect of energy restriction on proteolysis are scarce and, unlike those on protein synthesis, concerned the liver18,19 but neither the muscle nor kidney. It thus seems necessary to clarify the consequences of energy restriction on protein fluxes as a function of the composition of the diet and particularly its protein content. The purpose of this study was to better understand the effects of the macronutrient composition of the diet, including the protein source, on body composition and tissue protein metabo-lism during energy restriction in rats.
MATERIALS AND METHODS Animals
All experiments were carried out in accordance with the guidelines of the French Committee for Animal Care and the European Convention on
Received 7 September 2011; revised 15 November 2011; accepted 19 December 2011; published online 21 February 2012
1
INRA, CRNH-IdF, UMR914 Nutrition Physiology and Ingestive Behavior, AgroParisTech, Paris, France;2
AgroParisTech, CRNH-IdF, UMR914 Nutrition Physiology and Ingestive Behavior, Paris, France;3
INRA, UMR 1019 Nutrition Humaine, Saint Gene`s Champanelle, France and4
Univ Clermont 1, UFR Me´decine, UMR1019 Nutrition Humaine, Clermont-Ferrand, France. Correspondence: Dr C Gaudichon, UMR 914 PNCA, UMR914 INRA-AgroParisTech Nutrition Physiology and Ingestive Behavior, AgroParisTech, 16 rue Claude Bernard, Paris F-75005, France.
E-mail: Claire.Gaudichon@agroparistech.fr
Vertebrate Animals Used for Experimentation. Male Wistar rats (n¼ 56, 300 -- 320 g) were purchased from Harlan (Horst, The Netherlands) and housed under controlled environmental conditions (temperature, 12 h dark period starting at 08:30). The rats had free access to water and commercial laboratory chow for 5 days before the start of their dietary adaptation to the experimental diets.
Diets
A total of 56 rats were fed an obesity induction (OI) diet ad libitum for 5 weeks (12% energy as protein, 43% as CHO, 44% as fat) and were then allocated to seven different groups for 3 weeks (n¼ 8 per group). The control group was kept on the same diet ad libitum for 3 weeks. OI-R rats were fed the OI diet with a 45% energy restriction, whereas the other rats were energy restricted with a high protein (HP-R) diet (33% energy as protein, 37% as CHO and 30% as fat). Five different protein sources were tested in HP-R rats: milk proteins (MP), whey (W), casein (C), a mix of C and W in equal proportion (CW) and soy (S). The energy supplied in the restricted group was calculated to provide 55% of the average energy intake observed during the last 2 weeks of induction. Supplementary Information on the study design is available at Int J Obes website (Figure S1). Total MP were the protein source in the IO and IO-R diets (Table 1). In terms of composition, the HP-R diets were representative of the weight-loss diets commonly used in humans. The OI-R and HP-R diets were supplemented with minerals and vitamins in order to prevent any deficiencies due to the energy restriction.
The diets were supplied in a semi-liquid form in order to prevent spillage and improve recording of the quantities ingested. During the last week of the restriction period, rats were habituated to receiving their food following a pattern that consisted of a first period between 08:30 and 09:30 supplying 1/3 of the daily ration, and a second period between 12:00 and 18:00 supplying the remainder of the ration. OI rats were submitted to the same schedule but had free access to food between 12:00 and 18:00. This pattern was adopted so as to accustom the animals for eating a standard meal in its entirely within a period of 1 h on the experimental day.
Experimental protocol
The body weight and food intake of the rats were measured daily during the first 3 days and then every 2 days subsequently. On day 56, the rats were fed a calibrated meal corresponding to 1/3 of their daily ration, 2 h before killing. In order to measure protein fractional synthesis rates (FSR), the animals were injected subcutaneously with 300 mmol kg-- 1 BW of a flooding dose of L-(1-13C)-valine (50 mol%, Cambridge Isotope Labora-tories, Andover, MA, USA) 20 min before killing. The animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg kg1of body weight). After incision of the abdomen, blood from
vena cava was collected in EDTA pre-filled tubes and centrifuged to measure plasma metabolites and hormones. The rats were then killed by rupture of the caudal vena cava and aorta. Under sterile conditions, the liver, kidneys and gastrocnemius muscle were removed, rinsed, weighed and frozen until analysis. The spleen, soleus muscle, intestine and stomach were also weighed. Abdominal fat pads (epididymal, mesenteric and retroperitoneal) were excised and the inguinal subcutaneous fat pad was removed. The carcass was stripped to assess lean body mass. The weight of all these tissues was recorded.
Tissue protein content and biochemical measurements
The tissues were freeze-dried and weighed. Their total N content was assessed by the Dumas method using an elemental analyzer (Euro Elemental Analyser 3000, EuroVector, Milan, Italy) with atropina as the standard.
The total protein content (g) of tissues was determined as P¼ TM %DM %N 6.25/10 000 where TM is the wet tissue mass (g) and DM the dry matter.
Plasma amino-acid concentrations were determined on deproteinized plasma samples using ion exchange chromatography with post-column nynhidrin derivatization (Biotech Instrument, St Quentin-en-Yvelines, France). Insulin and leptin concentrations were analyzed using a rat endocrine panel (RENDO, Linco Research, Saint Charles, MI, USA) on a Bioplex 200 system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Urea concentrations were determined using a commercial kit (Bio-Me´rieux, Marcy l’Etoile, France).
Table 1. Nutrient composition of the experimental diets
Diet name OI OI-R HP-MP-R HP-W-R HP-CW-R HP-C-R HP-S-R Dry matter (g kg1)
Total milk proteinsa 179 175 422.6
Casein isolatea 198 394 Whey isolatea 413 202 Soy isolateb 364 Cornstarch 69 67 327 332 327 325.3 345 Sucrose 423.2 402.3 Lactose 18.3 31 30.8 Lard 202.8 197.3 Soybean oil 27 28 122 125.8 125.5 120.5 131 Minerals mixc 35 58.3 58.3 58.3 58.3 58.3 58.3 Vitamin mixc 10 16.7 16.7 16.7 16.7 16.7 16.7 Cellulose 50 50 50 50 50 50 50 Choline 2.5 4.2 4.2 4.2 4.2 4.2 4.2 L-Cystine 1.2 1.2 Total energy (%) Total protein 12.2 12.2 33 33 33 33 33 Total carbohydrate 43.3 43.3 37 37 37 37 37 Total fat 44.5 44.5 30 30 30 30 30
Dry matter (kcal g1)
Metabolizable energy 4.73 4.63 3.89 3.92 3.84 3.83 3.93
Abbreviations: DM, dry matter; OI, obesity induction diet; OI-R, obesity induction diet modified for energy restriction; HP-C-R, high protein diet containing 100% of protein as casein; HP-CW-R, high protein diet containing 50% of protein as casein and 50% as whey; HP-MP-R, high protein diet containing total milk protein; HP-W-R, high protein diet containing 100% of protein as whey; P-S-R, diet containing 100% of protein as soy protein.aIngredia, France.bNutrinov,
Rennes, FrancecAIN-93M, ICN biochemicals, Cleveland, OH, USA.
Isotopic measurements
The liver, kidney and muscle samples were prepared as previously detailed.17Briefly, the protein fraction was precipitated with 5-sulfosalicylic acid and the free amino acids were extracted from the supernatant on a cation exchange resin. The pellet containing the tissue proteins was rinsed, freeze-dried and then hydrolyzed for 48 h with HCl 6N under N2,and the
amino acids released from the proteins were extracted on cation exchange resin following the same procedure as for the free amino-acid fraction. To determine13C-valine enrichment in free amino acids, amino acids were
derivatized according to the silylation method and the derivative was then analyzed on a gas chromatography mass spectrometer (6890 N, Hewlett-Packard, Palo Alto, CA, USA), as described previously.17 13C-valine
enrichments in protein-bound amino-acid pools were determined after derivatization using the N-acetyl propyl method. Amino acids were first esterified with 1-propanol:HCl 8Mfor 1 h at 110 1C. An acetylation reaction was then performed using a mix of acetone:triethylamine:acetic anhydride (62.5 ml:25 ml:12.5 ml) at 60 1C for 4 min. After evaporating the reagent, the dried samples were diluted in ethyl acetate and then analysed by GC-C-IRMS HP5890/Isoprime, VG Instruments, (Manchester, UK) using a 50 m apolar column (HP5 MS, Hewlett-Packard).
FSR(% per day) of tissue proteins were calculated as FSR¼ Ebound val/ (Efree val t) 100 where Ebound val and Efree val are the protein-bound and free13C-valine enrichments in tissues. Absolute synthesis rates (ASR, g per day) were calculated as ASR¼ FSR P, where P is the tissue total protein content. Total muscle mass was estimated as 45% of body weight.20
Gene expression
Total RNA was extracted from frozen tissue using TRIzol reagents (Invitrogen, Carlsbad, CA, USA). The total RNA concentration was quantified at 260 nm and ethidium bromide staining was used to confirm RNA integrity. The synthesis of first strand complementary DNA (cDNA) was performed on 400 ng RNA, using a reverse transcription kit (Applied Biosystems, Courtaboeuf, Les Ulis, France) with a PTC-200 thermocycler (MJ research, Waltham, MA, USA). Real-time PCR was carried out using the ‘power syber green PCR master mix’ (Applied Biosystems) on a 7300 real-time PCR system (Applied Biosystems). Primers were designed using Oligo Explorer 1.1.0 software (GeneLink, Hawthorne, NY, USA). The sequences of the PCR primers used were: 50-ACACTGGCTCCTCAACCTG-30(forward) and 50-TCCAC CTTGATACCTCCTAAG-30(reverse) for cathepsin D (NM_134334), 50-CGCAC
CCTCTCTGACTACA-30(reverse) and 50-GCCCTCTTTATCCTGGATCT-30(reverse)
for ubiquitin (Ubb, NM_138895), 50-GGAAAACAAACGGGAGTATG-30(forward)
and 50-ACACAACGACGATGGAAAG-30(reverse) for 14 kDa E2 enzyme (M62388),
50-GCTGGAGGAAGAAGATGAAG-30(forward) and 50-GAAGTAGAAGAAGGAG
GTCG-30 (reverse) for m-calpaine (NM_017116), 50-GGGAGCCTGAGAAAC
GGC-30and 50-GGGTCGGGAGTGGGTAATTT-30for 18S.
All PCR reactions were performed as follows: denaturation at 95 1C for 10 min, 40 amplification cycles with each cycle consisted in 15 s at 95 1C followed by 1 min at 60 1C. The cycle threshold (CT) for each sample was
determined at a constant fluorescence threshold line. Ribosomal 18S RNA amplifications were used to account for variability in the initial quantities of cDNA, and inter-plate variations were corrected using an RT calibrator. Gene expression was determined using 2DCtformula, where 2 represents the optimum efficiency of the PCR and DCt¼ Ct target geneCt 18S. PCR
efficiency was determined in each plate using a serial dilution of reverse-transcribed RNA.
Statistics
Data are expressed as means±s.d. The group effect was analyzed using one-way ANOVA (version 9.1; SAS Institute Inc., Cary, NC, USA) and post-hoc Tukey tests were performed for multiple comparisons. The effects of energy restriction and the protein level were analyzed using contrast statements within the model in OI groups and restricted groups, respectively. The effect of the protein source was analyzed in the HP-R groups. For growth curves, data were analyzed using a mixed model with time as the repeated factor. Po0.05 was taken as the criterion for statistical significance.
RESULTS
Food intake, animal growth and body composition
Food intake during the first induction period averaged 94±8 kcal per day and did not differ between the groups (supplementary Information is available on International Journal of Obesity website (Table S1)). During the second period, the non-restricted OI group spontaneously reduced its energy intake by 15% whereas the target level of energy restriction was achieved in all restricted groups, ranging 40% (HP-S-R) to 46% (HP-MP-R) of that recorded during the induction period. Restriction was significantly higher in the HP-MP-R than in the HP-S-R group. When compared with the OI group, energy restriction thus averaged 37%. Protein intake was the lowest in the OI-R group (1.7±0.1 g per day) and the highest in the HP-R groups, with a slight but significant difference between the HP-CW-R (4.1±0.2 g per day) and HP-S-R (4.6±0.3 g per day) groups. In the OI group, the protein intake was 2.6±0.3 g per day.
Body weight was similar in all the groups at the beginning of the experiment (318±11 g), as were the growth curves during the OI period (Figure 1). For greater clarity, only one HP-R group was represented because all the restricted groups followed the same trend in weight loss. The average weight gain was 133.2±27.4 g during the first 5 weeks. During the second period, OI rats continued to gain weight (35±14 g), whereas the restricted OI-R rats lost 13±12 g. At the end of the experiment, the body weight of OI-R rats was 10% lower than that of OI rats (Po0.0001) and did not differ from that of the five HP-restricted groups.
Body composition was influenced by energy restriction, whereas the macronutrient composition of the diets (that is, the protein level or protein source) had no effect (Table 2). The kidney was the only organ in which weight was significantly increased by the protein level. Energy restriction reduced the fat pads, whereas
Figure 1. Growth of rats during the 5-week OI period and during energy restriction for 3 weeks. Mean±s.d. (n¼ 8). For more clarity, only one HP-R group is presented, because of the lack of any difference between the restricted groups. * significantly different from controls (mixed model with repeated measurements, post hoc Tukey test). OI, control rats fed the OI diet ad libitum; OI-R, energy-restricted rats under the OI diet; HP-MP-R, energy energy-restricted rats under the HP diet containing total MP.
lean tissues were only marginally affected by energy restriction. There was no difference in body composition between the different restricted groups whatever the level and nature of the protein content in their diet.
Plasma urea, hormones and amino acids
Plasma metabolites and hormones were measured 2 h after the meal (Table 3). Uremia was 64% higher in HP rats than in OI rats (Po0.0001) but was not influenced by energy restriction. Glycaemia and triglyceride levels were lowered in HP rats but were not influenced by energy restriction, whereas free fatty acids decreased in line with energy restriction and the HP level. Insulinemia and leptinemia (not shown) were modified by neither the energy intake nor the macronutrient manipulation of energy-restricted diets. The protein source had no effect on any of those parameters.
Postprandial concentrations of total, essential and non-essential plasma amino acids did not differ between the groups (Table 3), but a group effect was observed with respect to branched chain amino acids and a trend was seen for neoglucogenic amino acids (alanine, serine, glycine, threonine, aspartate, glutamine). Energy restriction tended to increase neoglucogenic amino-acid concen-trations (OI versus OI-R), whereas the protein level increased branched chain amino acids that were also impacted by the protein source. Individual amino acids were differently affected
(Figure 2). Glutamine and tyrosine were increased by energy restriction. Glycine, alanine, serine and glutamine decreased in HP groups compared with OI-R animals, whereas tryprophane, leucine and isoleucine increased. The protein source influenced several amino acids, reflecting differences in the amino-acid composition of the source protein. Proline was increased in C groups, especially HP-C-R and HP-MP-R, whereas methionine and threonine were lower in the HP-S-R group than in milk protein groups (whatever the protein fraction); leucine and isoleucine were higher in W protein groups (especially HP-W-R and HP-CW-R) and glycine was higher in the HP-S-R group.
Tissue composition, protein synthesis rates and the expression of proteolysis pathway genes
The protein content, FSR and ASR of the liver, gastrocnemius muscle and kidney are presented in Table 4. The findings on all HP-R groups were pooled because of the absence of any effect of the protein source. Energy restriction and the protein level did not influence the protein content and protein synthesis fluxes in these tissues, assessed using either FSR or ASR.
In the liver, the expression of cathepsin D, ubiquitin, E2 enzyme and m-Calpain was not modulated by the diet (Figure 3). By contrast, RNA levels of the genes involved in proteolysis pathways was significantly increased by energy restriction in the kidney and decreased by the protein level under energy restriction diets, for
Table 2. Body composition of rats after energy restriction (R) for 3 weeks
OI OI-R HP-MP-R HP-W-R HP-CW-R HP-C-R HP-S-R Statistics
g P-value
Body weight 479±45 432±22 432±28 440±29 443±33 435±33 438±39 ER: 0.006
Liver 11.7±1.4 11.2±1.0 11.0±1.5 10.9±1.0 10.6±0.9 11.1±0.7 10.9±1.1 NS Kidney 2.22±0.21 2.15±0.13 2.24±0.20 2.36±0.14 2.28±0.16 2.31±0.14 2.46±0.24 PL: 0.01 Intestine 7.4±1.0 6.8±0.6 6.7±0.6 7.16±0.72 6.83±0.81 7.33±0.56 6.93±0.76 NS Gastrocnemius 1.46±0.16 1.42±0.12 1.42±0.12 1.54±0.07 1.49±0.10 1.45±0.10 1.51±0.16 NS Soleus (mg) 271.0±87.6 335.0±17.7 319.8±38.3 347.5±19.1 341.4±19.5 337.5±25.5 357.5±28.7 NS Stripped carcass 197.6±15.3 190.5±8.0 182.5±14.5 194.3±8.3 192.7±13.1 190.6±10.2 193.7±16.8 NS
Inguinal subcutaneous fat pad 26.3±6.1 19.7±5.5 21.4±4.4 21.3±4.1 20.5±4.1 20.8±4.9 20.8±4.9 ER: 0.01
Mesenteric fat pad 13.8±3.8 8.9±2.9 9.9±2.5 8.9±2.1 9.4±1.5 9.0±3.0 9.8±3.6 ER: 0.001
Retroperitoneal fat pad 20.8±6.0 15.5±4.3 16.7±3.9 16.1±4.3 15.0±3.2 15.8±3.2 16.5±3.5 ER: 0.01
Epidydimal fat pad 10.1±2.2 6.9±1.7 7.9±1.4 7.4±2.1 7.0±1.3 7.8±1.9 7.2±1.9 ER: 0.001
Abbreviations: ER, effect of energy restriction, analyzed by contrast statement in OI groups; NS, not significant factor; PL, effect of the protein level in restricted groups, analyzed by contrast statement. Values are means±SD, n¼ 8. Results of one-way ANOVA, with the group as the main factor.
Table 3. Circulating plasma metabolites and insulin in rats in the fed state, 2 h after a calibrated meal, after energy restriction (R) for 3 weeks
OI OI-R HP-MP-R HP-W-R HP-CW-R HP-C-R HP-S-R Statistics
Total AA (mmol l1) 3300±831 3702±445 3483±299 3354±382 3365±463 3336±503 3376±520.4 NS
Indispensable AA (mmol l1) 1025±33 1117±149 1196±154 1234±189 1220±234 1149±233 1084±194 NS
Dispensable AA (mmol l1) 2274±537 2585±344 2287±189 2123±237 2189±273 2145±249 2292±329 ER: 0.06
PL: 0.004
Branched chain AA (mmol l1) 165±89 163±43 254±28 317±64 245±61 301±62 235±48 PL: 0.0001
PS: 0.02
Neoglucogenic AA (mmol l1) 1876±469 2144±236 1828±161 1793±219 1711±253 1730±233 1858±264 ER: 0.06
PL: 0.001
Urea (mmol l1) 6.4±0.7 7.0±0.7 11.0±1.6 10.1±1.6 9.6±1.16 10.7±1.74 10.0±1.57 PL: 0.0001
Glucose (mmol l1) 7.4±0.9 8.0±1.2 7.1±1.2 6.5±0.5 6.5±0.7 6.7±0.4 6.8±0.4 PL: 0.0001
Free fatty acids (mmol l1) 417±154 252±67 213±140 217±97 144±44 193±69 221±129 ER: 0.004
PL: 0.03
Triglycerides (mmol l1) 11.5±3.8 10.5±4.6 4.6±1.8 4.3±1.2 5.5±2.1 5.1±1.9 3.9±0.9 PL: 0.0001
Insuline (pg ml1) 6542±3705 5985±3787 4150±3834 3972±2380 4110±3094 4249±2536 4048±1085 NS
Abbreviations: ER, effect of energy restriction, analyzed by contrast statement in OI groups; NS, not significant factor; PL, effect of the protein level in restricted groups, analyzed by contrast statement; PS, effect of the protein source in HP-R groups, analyzed by ANOVA. Values are means±SD, n¼ 8. Results of one-way ANOVA, with the group as the main factor.
all four enzymes. An effect of the protein source was also observed for ubiquitin, E2 enzyme and M-calpain, with a trend towards a higher level of expression in the MP group compared with Cas and S. In the gastrocnemius muscle, the expression of genes of the proteolysis enzymes (except cathepsin D) tended to increase with energy restriction, whereas the HP level drastically decreased the expression of the four enzymes.
DISCUSSION
This study showed that in rats submitted to an energy restriction of 40 -- 45%, weight loss and body composition were mainly
influenced by energy restriction, whereas the composition of the diet and the protein source had no major effects. The HP level improved circulating glucose and blood lipids whereas energy restriction only lowered free fatty acids. A HP level counteracted the trend towards the increase in circulating neoglucogenic amino acids resulting from energy restriction. Protein synthesis rates were not impacted by either energy restriction or the protein level in the diet, whereas in the kidney and muscle, key enzymes of proteolysis were over-expressed under energy restriction and under-expressed when the protein level increased. However, these modulations of the proteolysis pathways did not impact lean mass loss, whatever the protein content of the diet.
Figure 2. Plasma concentrations of amino acids that were influenced by energy restriction, protein level and/or the protein source. Mean±s.d. (n¼ 8). OI, control rats fed the OI diet ad libitum (black bar); OI-R, energy-restricted rats under the OI diet (white bar); HP-R, energy-restricted rats under the HP diet (grey bars) with protein from different sources (MP; W; CW; C; S). The main statistical effects are indicated on each graph: energy restriction (ER), protein level (PL), protein source (PS).
Our study targeted conditions close to what is frequently performed in human weight loss programs, that is, an intensity of restriction of about 40% and an increase in the protein level (30%) at the expense of CHOs (lowered to 30%). The first induction period was designed to enhance adiposity with a fat high-sucrose diet. We noticed a decrease of hyperphagia after 3 weeks and throughout all the restriction period in the control group. This phenomenon has been reported by others21,22and may be linked to the slowing capacity of energy storage when adipocytes are hyperplasic. Our study clearly showed that energy restriction alone to a level of 40% drove weight loss without there being any effects of the macronutrient composition and protein source. The effect of diet composition on weight loss during energy restriction has been widely studied and the additional effect of macro-nutrient composition above calorie restriction was not clearly demonstrated. The absence of any effect of diet composition in humans (apart from calorie restriction), such as the protein/CHO ratio, has been reported by several authors,6,23 -- 25 although not all.26 -- 29In humans, Treyson et al.30reported that a HP diet failed to enhance weight loss compared with a standard diet but increased fat-mass loss, a result that we confirmed in our model. The benefits to weight management of increasing the protein level have also been highlighted during the weight maintenance period.31 -- 33In animals, a HP diet (50% of protein) under ad libitum conditions has been demonstrated to reduce spontaneous energy intake.20,34,35The discrepancies observed between studies may be explained by the nutritional conditions, such as the level of energy restriction or the macronutrient composition. For instance, in the work of Marsset -- Baglieri where rats were fed CHO-free diets, a 50% protein diet displayed a greater fat-mass loss than a 30% protein diet when animals were energy restricted to 35% but not to 75%.36In contrast, with a lower energy restriction (25%), it was reported that a high-fat diet (45% of total energy) attenuated the weight loss effect of energy restriction compared with a normal-fat diet, and the reduction of visceral adipose depots was detectable early after the beginning of the restriction.
One important suggestion relative to increasing dietary protein during energy restriction is that it might prevent a loss of lean body mass. This was initially proposed in the context of very low-calorie diets where the energy deficit is drastic, because the protein intake is sharply reduced, leading to a massive use of lean body mass to sustain energy requirements.8In humans, a loss of lean body mass always occurs during energy restriction, its amplitude depending on the degree of energy restriction, as reflected by the meta-regression developed by Krieger et al.9
During our study, a 40 -- 45% energy restriction for 3 weeks was not enough to impair lean mass (as represented by the stripped carcass) in rats to a significant extent, although a trend towards a decrease was observed. The body composition measurements were not longitudinal (unlike those used in human studies), and this may have masked a significant lean mass loss. Studying heavier rats (4600 g) that were 30% energy-restricted for 4 weeks, Eller & Reimer11reported an 8% loss of lean body mass, but the statistical significance of this loss was not tested (versus controls or baseline). This result was consistent with that reported by McLean et al.37who did not observe a significant fat-free mass loss in rats after a 40% energy restriction for 2 weeks. A loss of lean body mass was, however, observed in rats when more stringent energy restriction of at least 60% was applied,36but at lower levels it seemed that fat mass alone was sufficient to sustain energy needs.
A lack of lean mass loss was consistent with the absence of any effect of energy restriction on protein synthesis rate in the liver, muscle and kidney, in the fed state. Consequently, the protein content of these organs was similar in all groups. We had previously shown that in rats fed ad libitum, a high level of protein in the diet (30 to 50% of energy) decreased protein FSR in the liver for as long as the protein content remained high.16,17The lack of any effect of diet composition during the present study suggests that this modulation in the liver could not occur in a situation of energy deficit. Because proteolysis gene expression was not decreased in the liver, contrasting with what had previously been observed with HP diets,16,17we therefore concluded that there is no regulation because the overall protein intake was not sufficient to trigger these mechanisms (trough AA signaling in proteolysis pathways). Few data are available on energy restriction and protein synthesis rates, and they all concern muscle and the findings have been controversial, depending on the muscle type as well as physiological and nutritional status. In rats under a 30% energy restriction for 2 weeks, You et al.14reported a decreased mass of plantaris but not soleus muscle, and a lower FSR in both tissues in the postprandial state. In older rats, Zangarelli et al.15 showed that a 40% energy restriction for 5 months produced different effects on protein anabolism, depending on the muscle and protein fraction concerned. In the post-absorptive state, the FSR of mitochondrial proteins was decreased in both muscles, whereas for actin and myosin it remained unchanged in the soleus and increased in the tibialis anterior. Also in the post-aborptive state, Katzeff et al.13reported a reduction in the weight and FSR of the gastrocnemius and soleus muscles, depending on the level of
Table 4. Tissue composition and protein synthesis rates in rats after energy restriction (R) for 3 weeks
Diet name OI OI-R HP-R Statistical effect of the group
Liver
Protein (g per100 g tissue) 19.2±1.4 18.9±3.1 20.4±1.5 NS
FSR (% per day) 69.7±17.5 68.3±16.2 65.7±14.4 NS
ASR (g per day) 1.54±0.38 1.51±0.33 1.45±0.35 NS
Muscle
Protein (g per100 g tissue) 20.2±2.0 21.3±1.5 20.6±1.2 NS
FSR (% per day) 8.8±1.7 7.7±1.3 8.1±2.0 NS
ASR (g per day) 3.51±0.98 3.19±0.74 3.35±0.92 NS
Kidney
Protein (g per 100 g tissue) 17.1±1.8 17.3±2.7 18.3±2.7 NS
FSR (% per day) 73.1±10.7 74.1±12.0 71.4±10.9 NS
ASR (mg per day) 287.1±51.5 278.8±55.2 288.6±55.9 NS
Abbreviations: ASR, absolute synthesis rates; FSR, fractional synthesis rates; HP-R, energy restricted rats under the high protein diet, whatever the protein source (n¼ 35); OI, control rats fed the obesity induction diet ad libitum (n ¼ 8); OI-R, energy restricted rats under the obesity induction diet (n ¼ 8). Values are means±SD. Results of one-way ANOVA. NS: not significant.
energy restriction (25 to 50%), but this finding needs to be considered with caution because it was obtained using a very short tracer incorporation time (only 10 min). In humans, Piasakos et al.12 observed that a moderate energy restriction (20%) decreased the mixed muscle FSR by 19% in the fasted state, although protein intake was maintained at 1.5 g kg-- 1per day. This reduction in the FSR was associated with a decrease in 4E-BP1 phosphorylation, which suggests a decrease of protein synthesis
through the lowering of translation initiation. At the whole body level in humans, protein turnover has been shown to be reduced by 20% after a 50% energy restriction8,38,39, but not under a 25% energy restriction with a protein intake of 1.2 to 1.5 g kg-- 1 per day.40,41These results show that the effect of energy restriction on protein metabolism is strongly dependent on the methodological conditions (fed or fasted state, degree of energy restriction, protein level) and on the model studied (humans or rodents).
Figure 3. Relative expression of genes of the proteolysis pathway (cathepsin D, ubiquitin, E2 enzyme, m-Calpain) in the liver, gastrocnemius muscle and kidney in rats after energy restriction for 3 weeks. Mean±s.d. (n¼ 8). OI, control rats fed the OI diet ad libitum (black bar); OI-R, energy-restricted rats under the OI diet (white bar); HP-R, energy-restricted rats under the HP diet (grey bars) with protein from different sources (MP; W; CW, mix of C and W; C; S). The main statistical effects are indicated on each graph: group, energy restriction (ER), protein level (PL), protein source (PS).
The only dietary effects we were able to observe relative to protein metabolism was the enhancing effect of restriction on kidney proteolysis and the lowering effect of a HP level on muscle proteolysis. However, these effects had no impact on the protein pool in tissues, suggesting that they were compensated over the day or by other phenomena. Few data are available in the literature on tissue proteolysis during energy restriction, and those found generally focused on the effects of long-term caloric restriction aimed at counterbalancing the age effect. In the liver, Scrofano et al.18did not find any effect of caloric restriction on the ubiquitin-dependent pathway in young mice, whereas in old mice, restriction counterbalanced an increase in proteolysis. Nor was the autophagy pathway impacted by caloric restriction in the rat liver.19 In the muscle, variable results have been reported, depending on the muscle, age and model (rat, monkey).42 -- 44 Hepple et al.42showed that proteosome activity in muscle during caloric restriction decreased in line with muscle mass, but Chambon -- Savanovitch et al.45did not report any effect of energy restriction on 3-metylhistidine excretion in old rats. During our study, energy restriction did not cause any loss of muscle mass, and the lack of any effect on the mRNA-encoding key enzymes involved in proteolysis pathways (at least the proteasome pathway) was consistent with the observations made by Hepple et al.42 By contrast, we determined a marked effect of the protein level on the muscle gene expression of these enzymes, whatever the pathway. This could be explained by the fact that protein consumption in the HP-R group was nearly 3-fold that of the OI-R group, and 2-fold that of the OI rats, and was in line with the down-regulation of proteolysis pathways by amino acids in muscle.46,47 To our knowledge, no data are available on the kidney and energy restriction; further studies are needed to understand the biological significance of an increase in proteolysis markers in this organ and to confirm these observations by flux measurements.
Finally, because energy restriction and the protein level in the diet exerted no major effects, it was logical that the protein source had no impact on lean mass. Our initial hypothesis was that under conditions of energy deficit, the source of proteins might limit losses of lean body mass, especially when combining W proteins providing a massive afflux of leucine, which could stimulate protein anabolism, and C to supply amino acids throughout the postprandial anabolic window.48,49 In the literature, differences between C and W proteins, or caseins and S proteins, in sustaining lean mass during energy restriction, were not observed,11,50 although Adechian et al.51reported a higher post-absorptive FSR in 40% restricted rats fed C than in those receiving W proteins, a result that was linked to a higher leucine plasma concentration in the C group. During our study, we also observed differences in amino-acid concentrations, including those of leucine, but without there being any impact on protein anabolism. In the work by Eller & Reiner,11 total MP enabled a better preservation of lean body mass than W proteins. During their study, the protein level was kept at 14% during energy restriction, leading to a sharp reduction in protein intake, unlike our study where the protein level was increased to 30%. It is possible that under conditions of a limited protein intake, proteins may be discriminated in their ability to sustain lean tissues, but it appears that under conditions of moderate energy restriction (40%) and an increased protein intake, the protein source did not affect any of the parameters we measured.
In conclusions, in adult obese rats subjected to 40% energy restriction for 3 weeks, their weight and fat loss were only driven by caloric intake, independently of the macronutrient composition. Lean mass was preserved whatever the protein level, and protein metabolism was only slightly impacted, except for proteolysis pathways in the muscle and kidney, where any postprandial activation by energy restriction was counteracted by a HP level. Finally, when the protein level was high, all protein sources were equivalent in terms of their effects.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
This work was supported by a grant from CNIEL, and the French Agency for Research and Technology (Program PNRA 2006; SURPROL). We are indebted to the CNIEL for its financial support and constructive scientific discussions. We thank Ange´lique Simonin for her assistance with animal care and dissection procedures.
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Supplementary Information accompanies the paper on International Journal of Obesity website (http://www.nature.com/ijo)
