BACKGROUND:Inadequate protein intake in early life is associated with both growth retardation and a higher risk of metabolic syndrome in later life. OBJECTIVE:This study aimed to evaluate the effects on energy metabolism of protein or indispensable amino acids (IAAs; lysine, threonine, and methionine) supplementation following a deficiency in growing rats. METHODS:Sixty Wistar Han male rats were fed a control (20% protein by energy content), protein-deficient (P5, 5% protein by energy content), or lysine, threonine, or methionine-deficient diet (L25, T25, and M25, respectively; 25% of the IAA requirement) for 3 wk and thereafter were supplemented for 3 wk with the deficient IAA or protein to reach 100% of the IAA (T100, L100, and M100, respectively) or protein requirement (P20). Body weight (BW) and relative food intake (rFI) were measured daily. Body composition, nasoanal length (NAL), energy expenditure, and plasma fibroblast growth factor 21 were measured at the end of the deficiency and supplementation phases. Data were analyzed using 1-way or mixed-model ANOVA and Bonferonni tests for multiple comparisons. RESULTS:All deficient diets induced growth retardation [lower BW, lean body mass (LBM), and NAL], with threonine deficiency having the most severe effect (60% lower BW of control; P < 0.001). Supplementation induced a resumption of growth, but BW and LBM remained lower (15%-35% for BW and 69%- 84% for LBM of control; P < 0.001). Despite increased rFI, no excess adiposity was observed postsupplementation in P20 and T100 groups, likely due to increased energy expenditure (P < 0.001). In L100 and M100 groups, rFI increased (by 35% and 30%, respectively; P < 0.001) without a corresponding rise in energy expenditure. Fibroblast growth factor 21 was inversely associated with the protein and IAA statuses during both deficiency and supplementation (P < 0.001). CONCLUSIONS:These findings highlight the distinct roles of individual IAAs in growth and metabolic recovery and suggest that targeted IAA supplementation may improve nutritional interventions.

Roux-en-Y Gastric Bypass may be associated with an alteration of protein bioavailability in relation to intestinal remodeling. Our study aimed to test this hypothesis by Roux-en-Y Gastric Bypass. Diet-induced obese rats underwent Roux-en-Y Gastric Bypass surgery (RYGB rats) while a Sham-operated control group was used. All rats received a 15N-labeled protein meal 1 or 3 months after surgery and were euthanized 6h later. Protein digestibility, 15N recovered in organs and urea pool, fractional protein synthesis rate, and intestinal morphometry were assessed. Protein digestibility was similar in all groups (94.2±0.3%). The small intestine was hypertrophied in RYGB rats 1 month after surgery, weighing 9.1±0.2g vs. 7.0±0.3g in Sham rats (P = 0.003). Villus height and crypt depth were increased in the alimentary limb and ileum of RYGB rats. However, Roux-en-Y Gastric Bypass had no impact on the fractional synthesis rate. In the gastrointestinal tract, 15N retention only differed in the ileal mucosa and was higher in RYGB rats at 1 month (0.48±0.2% vs. 0.3±0.09%, P = 0.03). 15N recovery from the liver, muscle, and skin was lower in RYGB rats at 1 month. 15N recovery from urinary and plasma urea was higher in RYGB rats at both times, resulting in increased total deamination (13.2±0.9% vs. 10.1±0.5%, P<0.01). This study showed that Roux-en-Y Gastric Bypass did not affect protein digestibility. Dietary nitrogen sequestration was transitorily and moderately diminished in several organs. This was associated with a sustained elevation of postprandial deamination after Roux-en-Y Gastric Bypass, whose mechanisms merit further studies.

Dietary proteins are energy macronutrients providing nitrogen, amino acids (AA), and energy. AAs are the main nitrogen-containing compounds in the body and are the precursors for the synthesis of body proteins and of several other AA-derived molecules. Among the 20 AAs included in protein sequence, 9 are classified as “nutritionally essential” or “indispensable” AA (IAA) because they cannot be synthesized in the body and must be provided by the diet. IAAs are limiting components for protein synthesis. An adequate intake of protein is required to support growth, maintenance, body functions, health and survival. Official definition of protein requirement is based on nitrogen balance. Protein quality is related to the capacity of protein to provide an adequate quantity of nitrogen and of each of the 9 IAAs for the different physiological situations in humans. Protein source is considered high quality for humans when the protein is readily digested, simultaneously providing an adequate quantity of nitrogen and of each of the 9 IAAs to maintain an adequate metabolic AA pool. The most accurate assessment of protein quality of foods for humans is through metabolic studies that measure nitrogen balance. The protein quality score is the ratio of the content of each IAA in the food and in a reference profile. This score corresponds to the calculated composition of a protein which, when meeting protein requirements, simultaneously meets the requirements of each of the 9 IAAs. AA scores as predictors of protein quality must be adjusted for protein and AA availability.

BACKGROUND:The consumption of poor-quality protein increases the risk of essential amino acid (EAA) deficiency, particularly for lysine and threonine. Thus, it is necessary to be able to detect easily EAA deficiency.OBJECTIVES:The purpose of this study was to develop metabolomic approaches to identify specific biomarkers for an EAA deficiency, such as lysine and threonine.METHODS:Three experiments were performed on growing rats. In experiment 1, rats were fed for 3 weeks with lysine (L30), or threonine (T53)-deficient gluten diets, or nondeficient gluten diet (LT100) in comparison with the control diet (milk protein, PLT). In experiments 2a and 2b, rats were fed at different concentrations of lysine (L) or threonine (T) deficiency: L/T15, L/T25, L/T40, L/T60, L/T75, P20, L/T100 and L/T170. Twenty-four-hour urine and blood samples from portal vein and vena cava were analyzed using LC-MS. Data from experiment 1 were analyzed by untargeted metabolomic and Independent Component - Discriminant Analysis (ICDA) and data from experiments 2a and 2b by targeted metabolomic and a quantitative Partial Least- Squares (PLS) regression model. Each metabolite identified as significant by PLS or ICDA was then tested by 1-way ANOVA to evaluate the diet effect. A two-phase linear regression analysis was used to determine lysine and threonine requirements.RESULTS:ICDA and PLS found molecules that discriminated between the different diets. A common metabolite, the pipecolate, was identified in experiments 1 and 2a, confirming that it could be specific to lysine deficiency. Another metabolite, taurine, was found in experiments 1 and 2b, so probably specific to threonine deficiency. Pipecolate or taurine breakpoints obtained give a value closed to the values obtained by growth indicators.CONCLUSIONS:Our results showed that the EAA deficiencies influenced the metabolome. Specific urinary biomarkers identified could be easily applied to detect EAA deficiency and to determine which AA is deficient.

In developing countries, children are exposed to a risk of growth retardations due to their low protein (LP) intake or poor quality of protein sources with unbalanced indispensable amino acid (IAA) composition. However, the specificity of each IAA and the ability of children to catch-up their growth retardation remains unclear. The aim of this study was to assess the supplementation efficiency following a protein or IAA (lysine, threonine, and methionine) deficiencies in growing rats, and to identify the specific IAA deficiency effect. Sixty growing rats were fed by a control (20% of proteins; P20), a LP (5% of proteins; LP) or IAA deficient (25% of the rat's requirement in lysine, threonine, or methionine; L25, T25 and M25) diets for 3w. Thereafter, all rats were supplemented by the control (P20) or a control-equivalent diet containing free AA. Body weight (BW) and food intake were daily recorded. Naso-anal length (NAL), bone mineral density (BMD) and body composition were measured at the end of each period. During the deficiency, IAA as LP diets reduced BW gain from day 2 for LP, L25 and M25 and from day 1 for T25. At the end of the deficiency, BW was reduced by 30% for L25 and M25, 50% for LP and 60% for T25. NAL was also reduced by 9, 18, 25% for L25/M25, LP and T25. At the end of the deficiency, all groups had less lean body mass (LBM), whereas only LP and T25 had a decreased BMD. Furthermore, the fat mass was only decreased for LP and T25 groups. During the supplementation, growth resumes and the weight's gap between each group was reduced, but remains after supplementation. The BW were reduced by 15, 25 and 35% for L25/M25, LP and T25, respectively. For NAL and LBM, the gap slightly reduced too, but the difference remains after the supplementation. Indeed, the NAL was reduced by 5% for L25 and M25, 8% for LP and 10% for T25. All groups had reduced kidney, muscle and carcass weight, and LP and T25 had a reduced liver weight and BMD. The T25 group was the most affected by the deficiency, even more than LP. A single IAA deficiency as LP induced growth retardation, and the LBM is highly affected. A supplementation allows growth resume, but the growth retardation cannot be catch up. The stronger effect observed for threonine deficiency, could be due to a wrong estimation of threonine requirement. This work was supported by CEFIPRA and AlimH -INRAe.

Objective: The aim of this study was to analyze the protein digestibility and postprandial metabolism in rats of milk protein matrices obtained by different industrial processes. Material and methods: The study was conducted on Wistar rats that consumed a meal containing different 15Nlabeled milk proteins. Four milk matrices were tested: native micellar caseins (C1), caseins low in calcium (C2 low Ca2+), a matrix containing a ratio 63:37 of caseins and whey proteins (CW2) and whey proteins alone (W). Blood and urine were collected during the postprandial period and rats were euthanized 6 h after meal intake to collect digestive contents and organs. Results: Orocaecal digestibility values of amino acids ranged between 96.0 +/- 0.2% and 96.6 +/- 0.4% for C1-, C2 low Ca2+- and W-matrices, while this value was significantly lower for CW2 matrix (92.4 +/- 0.5%). More dietary nitrogen was sequestered in the splanchnic area (intestinal mucosa and liver) as well as in plasma proteins after ingestion of W matrix, especially compared to the C1- and C2 low Ca2+-matrices. Peptidomic analysis showed that more milk protein-derived peptides were identified in the caecum of rats after the ingestion of the matrices containing caseins compared to W matrix. Conclusion: We found that demineralization of micellar caseins did not modify its digestibility and postprandial metabolism. The low digestibility of the modified casein-to-whey ratio matrix may be ascribed to a lower accessibility of the protein to digestive enzymes due to changes in the protein structure, while the higher nitrogen splanchnic retention after ingestion of whey was probably due to the fast assimilation of its protein content. Finally, our results showed that industrial processes that modify the structure and/or composition of milk proteins influence protein digestion and utilization.

The objective of this study is to evaluate the effects of a strictly essential amino acid (lysine or threonine; EAA) deficiency on energy metabolism in growing rats. Rats were fed for three weeks severely (15% and 25% of recommendation), moderately (40% and 60%), and adequate (75% and 100%) lysine or threonine-deficient diets. Food intake and body weight were measured daily and indirect calorimetry was performed the week three. At the end of the experimentation, body composition, gene expression, and biochemical analysis were performed. Lysine and threonine deficiency induced a lower body weight gain and an increase in relative food intake. Lysine or threonine deficiency induced liver FGF21 synthesis and plasma release. However, no changes in energy expenditure were observed for lysine deficiency, unlike threonine deficiency, which leads to a decrease in total and resting energy expenditure. Interestingly, threonine severe deficiency, but not lysine deficiency, increase orexigenic and decreases anorexigenic hypothalamic neuropeptides expression, which could explain the higher food intake. Our results show that the deficiency in one EAA, induces a decrease in body weight gain, despite an increased relative food intake, without any increase in energy expenditure despite an induction of FGF21.

Protein synthesis and proteolysis are known to be controlled through mammalian target of rapamycin, AMP-activated kinase (AMPK) and general control non-derepressible 2 (GCN2) pathways, depending on the nutritional condition. This study aimed at investigating the contribution of liver AMPK and GCN2 on the adaptation to high variations in protein intake. To evaluate the answer of protein pathways to high- or low-protein diet, male wild-type mice and genetically modified mice from C57BL/6 background with liver-specific AMPK- or GCN2-knockout were fed from day 25 diets differing in their protein level as energy: LP (5

Female rabbits were injected intraperitoneally (i.p.) with purified flavoglaucin from Aspergillus chevalieri. After 2 h the rabbits were bled and the livers removed for enzyme assays. No changes were found for plasma lactate dehydrogenase (LDH) and its isozymes or liver transketolase (TK) activities. A small but not statistically significant increase was found in plasma glutamate pyruvate transaminase activity. A highly significant increase in plasma glutamic oxaloacetate transaminase activity was observed along with an increase in liver LDH which was, however, not quite significant. The liver also showed some histological evidence of hepatic damage.

The recent Food and Agricultural Organization/World Health Organization/United Nations University expert consultations on protein requirements and quality have emphasized the need for the new Digestible Indispensable Amino Acid Score (DIAAS), as a measure of protein quality. This requires human measurements of the true ileal digestibility of individual indispensable amino acids (IAAs) until the end of the small intestine. Digestibility is measured using standard oro-ileal balance methods, which can only be achieved by an invasive naso-ileal intubation in healthy participants or fistulation at the terminal ileum. Significant efforts have been made over the last 2 decades to develop noninvasive or minimally invasive methods to measure IAA digestibility in humans. The application of intrinsically labeled (with stable isotopes like 13C, 15N, and 2H) dietary proteins has helped in circumventing the invasive oro-ileal balance techniques and allowed the differentiation between endogenous and exogenous protein. The noninvasive indicator amino acid oxidation (IAAO) technique, which is routinely employed to measure IAA requirements, has been modified to estimate metabolic availability (a sum of digestibility and utilization) of IAA in foods, but provides an estimate for a single IAA at a time and is burdensome for participants. The recently developed minimally invasive dual isotope tracer method measures small intestinal digestibility of multiple amino acids at once and is suitable for use in vulnerable groups and disease conditions. However, it remains to be validated against standard oro-ileal balance techniques. This review critically evaluates and compares the currently available stable isotope-based protein quality evaluation methods with a focus on the digestibility and metabolic availability measurements in humans. In view of building a reliable DIAAS database of various protein sources and subsequently supporting protein content claims in food labeling, a re-evaluation and harmonization of the available methods are necessary.

The western dietary pattern is known for its frequent meals rich in saturated fat and protein, resulting in a postprandial state for a large part of the day. Therefore, our aim was to investigate the postprandial glucose and lipid metabolism in response to high (HP) or normal (NP) protein, high-fat hypercaloric diet and to identify early biomarkers of protein intake and hepatic lipid accumulation. In a crossover design, 17 healthy subjects were randomly assigned to consume a HP or NP hypercaloric diet for two weeks. In parallel, a control group (CD; n = 10) consumed a weight-maintaining control diet. Biomarkers of postprandial lipid and glucose metabolism were measured in 24 h urine and in plasma before and following a meal challenge. The metabolic profile of urine but not plasma, showed increased excretion of 13C, carnitine and short chain acyl-carnitines after adaptation to the HP diet. Urinary excretion of decatrienoylcarnitine and octenoylcarnitine increased after adaptation to the NP diet. Our results suggest that the higher excretion of short-chain urinary acyl-carnitines could facilitate the elimination of excess fat of the HP diet and thereby reduce hepatic fat accumulation previously reported, whereas the higher excretion medium-chains acyl-carnitine could be early biomarkers of hepatic lipid accumulation.

En condition de choix alimentaire, les rats ingèrent 25–30 % et jusqu’à 50 % de leurs apports énergétiques sous forme de protéines. Ce niveau d’ingestion élevé semble motivé par des avantages métaboliques à long terme liés à une diminution de la dépendance du métabolisme vis-à-vis des glucides et des pathologies associées. Cependant, les mécanismes qui sous-tendent ces choix restent largement incompris. Le but de l’étude était, d’une part, de démontrer que chez les rats libres de choisir entre un régime purement protéique et un mélange glucido-lipidique sans protéines, le niveau d’ingestion protéique serait plus élevé avec un mélange riche en glucides, et d’autre part, que les rats en libre choix maintiennent des niveaux plasmatiques très réduits de fibroblast growth factor 21 (FGF21), une hépatokine, qui signale le stress métabolique, en particulier un apport insuffisant en protéines. Des rats mâles adultes de la souche Wistar ont été soumis à un choix entre un régime constitué uniquement de protéines de lait et un régime glucido-lipidique contenant 30 %, 45 %, 60 % ou 75 % de glucides. Pour servir de témoins, 2 groupes de rats ont été nourris avec des régimes complets contenant 15 % ou 30 % de protéines, notés 15P et 30P. Le poids corporel et la prise alimentaire ont été mesurés 2 fois par semaine. Après 3 semaines sous ces régimes, des prélèvements de plasma ont été réalisés afin de déterminer leur concentration en FGF21 (test ELISA). À la fin de l’étude, les rats ont été euthanasiés et la composition corporelle déterminée par dissection des organes et tissus. L’analyse statistique a été faite par analyse de variance. Les rats en choix ont moins mangé et ont moins grossi que les témoins 15P. Ils ont augmenté leur niveau d’ingestion protéique de 20 à 35 % lorsque la teneur en glucides du mélange glucido-lipidique augmentait de 30 % à 75 % (R2 = 0,56 ; p < 10−6). Cette évolution a conduit à l’établissement d’un ratio protéines/glucides (∼70 %) identique dans tous les groupes (p = 0,18). Chez tous les rats en choix, les taux plasmatiques de FGF21 était faibles et non influencés par les niveaux d’apports protéiques et glucidiques. Ils étaient comparables aux taux mesurés chez les témoins 30P, et significativement plus faibles que chez les témoins 15P (respectivement 162,93(a), 294,0(a) et 499,3(b) pg/mL ; p = 3,22 × 10−5). Cette étude a montré que les rats en choix ingèrent plus de protéines que le niveau considéré comme suffisant pour atteindre l’équilibre en azote (∼5 %) ou une croissance optimale chez les rats adultes (10–15 %), et que ce niveau d’ingestion dépend fortement de la teneur en glucides du régime alimentaire, avec apparemment pour but de maintenir un ratio protéines/glucides constant et élevé. Un tel ratio en réduisant la dépendance du métabolisme vis-à-vis des glucides pourrait réduire à terme le développement de l’insulinorésistance. Enfin, le fait que les rats en choix et les témoins 30P aient maintenu des niveaux de FGF21 plasmatique inférieurs aux témoins 15P suggère que les apports en protéines de ce régime standard sont insuffisants, et que le maintien de taux de FGF21 bas participe au contrôle du niveau d’ingestion protéique chez les rats en choix.

Protein requirement has been determined at 10%-15% energy. Under dietary self-selection, rats ingest 25%-30% energy as protein and regulate FGF21 (a hormone signaling protein deficiency) to levels lower than those measured with a 15% protein (15P) diet. Our hypothesis is that if a 15P diet was indeed sufficient to ensure protein homeostasis, it is probably a too low protein level to ensure optimal energy homeostasis. Adult male Wistar rats were used in this study. The first objective was to determine the changes in food intake, body composition, and plasma FGF21, IGF-1, and PYY concentrations in rats fed 8P, 15P, 30P, 40P, or 50P diets. The second was to determine whether the FGF21 levels measured in the rats were related to spontaneous protein intake. Rats were fed a 15P diet and then allowed to choose between a protein diet and a protein-free diet. Food intake and body weight were measured throughout the experiments. Body composition was determined at different experimental stages. Plasma samples were collected to measure FGF21, IGF-1, and PYY concentrations. A 15P diet appears to result in higher growth than that observed with the 30P, 40P, and 50P diets. However, the 15P diet probably does not provide optimal progression of body composition owing to a tendency of 15P rats to fix more fat and energy in the body. The variable and higher concentrations of FGF21 in the 15P diet suggest a deficit in protein intake, but this does not appear to be a parameter reflecting the adequacy of protein intake relative to individual protein requirements.NEW & NOTEWORTHY Under dietary self-selection, rats choose to ingest 25%-30% of energy as protein, a value higher than the protein requirement (10%-15%). According to our results, this higher spontaneous intake reflects the fact that rats fed a 15% protein diet, compared with high-protein diets, tend to bind more fat and have higher concentrations of FGF21, a hormone signaling protein deficiency. A 15% protein diet appears to be sufficient for protein homeostasis but not for optimal energy homeostasis.

To study, in young growing rats, the consequences of different levels of dietary protein deficiency on food intake, body weight, body composition, and energy balance and to assess the role of FGF21 in the adaptation to a low protein diet. Thirty-six weanling rats were fed diets containing 3%, 5%, 8%, 12%, 15% and 20% protein for three weeks. Body weight, food intake, energy expenditure and metabolic parameters were followed throughout this period. The very low-protein diets (3% and 5%) induced a large decrease in body weight gain and an increase in energy intake relative to body mass. No gain in fat mass was observed because energy expenditure increased in proportion to energy intake. As expected, Fgf21 expression in the liver and plasma FGF21 increased with low-protein diets, but Fgf21 expression in the hypothalamus decreased. Under low protein diets (3% and 5%), the increase in liver Fgf21 and the decrease of Fgf21 in the hypothalamus induced an increase in energy expenditure and the decrease in the satiety signal responsible for hyperphagia. Our results highlight that when dietary protein decreases below 8%, the liver detects the low protein diet and responds by activating synthesis and secretion of FGF21 in order to activate an endocrine signal that induces metabolic adaptation. The hypothalamus, in comparison, responds to protein deficiency when dietary protein decreases below 5%.