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.

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

L’obésité est une épidémie mondiale qui touche plus de 400 millions d’adultes présentant de graves comorbidités. La recherche de nouveaux traitements pour diminuer ses conséquences négatives est nécessaire. Les orexines (orexine-A (OxA) et orexine-B (OxB)) sont des neuropeptides hypothalamiques qui interviennent dans différents processus physiologiques, notamment le maintien des états de veille/sommeil, l’homéostasie énergétique, les mécanismes de récompense, la cognition et l’analgésie. Le but de cette étude est d’étudier les conséquences d’un traitement chronique à l’OxA dans des modèles de souris sensibles ou résistantes à l’obésité. Cette étude a été réalisée chez la souris C57BL/6 femelles âgées de 8 semaines. Un groupe de 60 souris a reçu le régime glucido-lipidiques (HL), dont la répartition de l’énergie en % pour les différents nutriments est la suivante (P20, G50 (amidon 50 %, sucrose 50 %) et L30 (25 % huile de soja, 75 % saindoux)). La mesure de la composition corporelle après 3 semaines de régime HL a permis d’identifier 16 souris sensibles au régime HL (OP) et 16 souris résistantes (OR). Chaque groupe a été divisés en 2 sous-groupes : la moitié de chaque groupe recevant des injections d’OxA pendant 9 semaines (8 souris OP, 8 souris OP + OxA, 8 souris OR et 8 souris OR + OxA). En parallèle un groupe de 12 souris contrôles a reçu un régime standard (P20 Soja) dont la répartition de l’énergie en % pour les différents nutriments protéines (P), glucides (G) et lipides (L) est la suivante : P20, G70 (80 % amidon, 20 % sucrose) et L10 (100 % huile de soja). Nous avons fait le suivi du poids des animaux pendant 12 semaines, réalisé des tests de tolérance au glucose, mesuré la prise alimentaire, la dépense énergétique, ainsi que l’expression dans l’hypothalamus de plusieurs neuromédiateurs et les récepteurs à l’orexine. Nos résultats montrent que le traitement des souris par l’OxA réduit la prise du poids des souris OP. Cette diminution de la prise de poids est associée à une diminution de la masse grasse, de la prise alimentaire, d’une réduction de la stéatose hépatique, d’une réduction de la glycémie et d’une augmentation du quotient respiratoire. Nous observons également au niveau de l’hypothalamus, lequel est impliqué dans le contrôle de la prise alimentaire, une diminution de l’expression des récepteurs aux orexines (OX1R & OX2R) ainsi qu’une régulation de l’augmentation induite par le régime HL chez les souris obèses, des neuropeptides AgRP, NPY, POMC et CART, impliqués dans la prise alimentaire. Nous avions précédemment démontré, chez des souris soumises à un régime standard, que les injections d’OxA avaient peu d’effet sur les dépenses énergétiques. Cependant, chez les souris OP l’administration d’OxA réduit la prise de poids en régulant la prise alimentaire et en améliorant le métabolisme énergétique et en modulant l’expression au niveau de l’hypothalamus de certains neuropeptides impliqués dans la prise alimentaire. Ces résultats suggèrent que l’OxA pourrait réduire l’obésité non seulement en améliorant le métabolisme énergétique mais également en agissant directement via le système nerveux central.

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%.

The availability of indispensable amino acids (IAA) modulates protein turnover. More particularly AAI deficiency reduces protein synthesis while the consequence on proteolysis remains unclear. The present study aims to evaluate the specific response of both protein synthesis and proteolysis to a diet restricted on one strictly indispensable IAA, either lysine or threonine Sixty-four growing rats were divided into 8 groups (n = 8/group). They were fed for 3 weeks isocaloric diets composed with different levels of lysine or threonine (L or T), 15, 25, 40, 60, 75, 100 or 170% of the theoretical lysine/threonine requirements. At the end of the experiment, rats were injected with valine13C and tissues and biological fluids were collected for gene expression measurement and blood amino acids (AA). Protein synthesis rate (Fractional and Absolute rate synthesis, ie FSR, ASR) were determined in liver and muscle. Statistical analysis was done by 1- or 2-factor ANOVA, when data were repeated. Severe (L/T15, L/T25) and moderate (T40) lysine or threonine deficiency resulted in a decrease in body weight gain due to a decrease in lean body mass. Severe restriction (L15, T15, T25) decreased the muscle FSR whereas no effect was observed in the liver. When the rate of protein synthesis was expressed per tissue, the ASR was decreased by severe restriction of lysine and threonine in liver and muscle and by moderate threonine deficiency (T40, T60, T75) in muscle. In liver, no effect of lysine and threonine on proteolysis was observed. In muscle, only severe lysine (L15) deficiency increased proteolysis. Dietary lysine deficiency induced a decrease in lysine concentration in the portal vein and in the vena cava whereas for threonine deficiency, all AAIs except threonine were decreased in the portal vein and vena cava. These results indicate that the decreased protein synthesis is the primary mechanism involved in decreased lean body mass in response to the severe deficiency in a single AAI. Deficiency of a single AAI reproduce the effect of the low protein diet on protein synthesis. Lysine and threonine deficiency differently affect for a part protein turnover probably in relation with the tissue where they are metabolized. This study was funded by the doctoral school ABIES and AlimH-INRAE department.

Amino acids are involved in energy homeostasis, just as are carbohydrates and lipids. Therefore, mechanisms controlling protein intake should operate independently and in combination with systems controlling overall energy intake to coordinate appropriate metabolic and behavioral responses. The objective of this study was to quantify the respective roles of dietary protein and carbohydrate levels on energy balance, plasma fibroblast growth factor 21 (FGF21) and insulin growth factor 1 (IGF-1) concentrations, and hypothalamic neurotransmitters (POMC, NPY, AgRP, and CART). In a simplified geometric framework, 7-wk-old male Wistar rats were fed 12 diets containing 3%-30% protein for 3 wk, in which carbohydrates accounted for 30%-75% of the carbohydrate and fat part of the diet. As a result of this study, most of the studied parameters (body composition, energy expenditure, plasma FGF21 and IGF-1 concentrations, and Pomc/Agrp ratio) responded mainly to the protein content and to a lesser extent to the carbohydrate content in the diet.NEW & NOTEWORTHY As mechanisms controlling protein intake can operate independently and in combination with those controlling energy intakes, we investigated the metabolic and behavioral effects of the protein-carbohydrate interaction. With a simplified geometric framework, we showed that body composition, energy balance, plasma FGF21 and IGF-1 concentrations, and hypothalamic Pomc/Agrp ratio were primarily responsive to protein content and, to a lesser extent, to carbohydrate content of the diet.

Omnivores are able to correctly select adequate amounts of macronutrients from natural foods as well as purified macronutrients. In the rat model, the selected protein levels are often well above the requirements estimated from the nitrogen balance. These high intake levels were initially interpreted as reflecting poor control of protein intake, but the selected levels were later found to be precisely controlled for changes in dietary protein quality and adjusted for cold, exercise, pregnancy, lactation, age, etc. and therefore met physiological requirements. Several authors have also suggested that instead of a given level of protein intake, rodents regulate a ratio of protein to dietary carbohydrates in order to achieve metabolic benefits such as reduced insulin levels, improved blood glucose control, and, in the long term, reduced weight and fat gain. The objective of this review was to analyze the most significant results of studies carried out on rats and mice since the beginning of the 20th century, to consider what these results can bring us to interpret the current causes of the obesity pandemic and to anticipate the possible consequences of policies aimed at reducing the contribution of animal proteins in the human diet.

Severely low-protein (LP) diets induce a decrease in body weight and an increase in relative food including intake (FI) in rat. In the liver, changes in anabolic and catabolic protein pathways could transitorily participate to compensate for amino acid (AA) deficiency. The present study investigated these liver and muscle protein metabolic pathways on LP diet fed growing rats. Growing rats were fed for three weeks different diets containing 3–5–8–12–15 or 20% energy from milk protein. Body weight and FI were measured daily. At the end of the experiment, rats were injected with 13C valine and tissues and biological fluids were collected for gene expression measurement, blood AA UPLC analysis and protein synthesis rate determination in liver and muscle. Statistical analysis was done by 1- or 2-factor ANOVA, when data were repeated. P3, P5 and P8% diets resulted in significant growth retardation and significant decrease in lean mass. Severe protein deficiency induced a decrease in the rate of protein synthesis in the liver and muscle. In addition, the results showed activation of the GCN2 pathway, via ATF4-CHOP-TRB3 both in the liver and in the muscle, which suggests the inhibition of the initiation of translation at the level of the binding of the RNAt-Met. Liver proteolytic pathways were up-regulated including the ubiquitin-proteasome, the caspase system and the autophagy. In muscle, both the ubiquitin-proteasome pathway, and autophagy were increased as well as the calpain system. The GCN2 pathway, via ATF4-CHOP-TRB3 was activated in both liver and muscle, confirming the activation of protein degradation by the ubiquitin-proteasome pathways, and autophagy. In portal vein, indispensable AA were lower in severe protein deficient diet whereas in vena cava no difference was observed. Severe protein restriction lowered protein synthesis and activated protein catabolism in both liver and muscle whereas no effect was observed for moderate protein restriction. These results confirm that the liver and muscle play a major role in supplying the body with indispensable AA in response to severe protein restriction. This study was funded by the doctoral school ABIES and AlimH-INRAE department.

BACKGROUND:Under dietary self-selection (DSS), rats ingest 25-30% of energy as protein. This high level appears to be explained by metabolic benefits related to reduced carbohydrate dependence and associated pathologies. However, the mechanisms underlying these choices remain largely misunderstood.OBJECTIVES:The aim was to test the hypothesis that in a DSS model, rats select a protein-to-energy (PE) ratio to maintain the protein-to-carbohydrate (PC) ratio constant and that fibroblast growth factor 21 (FGF21) is involved in this response.METHODS:Adult male Wistar rats were used in 3 experiments. The first was to determine whether the PE ratio was influenced by changes in carbohydrate content. The second was to test whether the PE ratio was defended with a modified DSS model. The third was to determine whether the selected PE ratio was of metabolic interest compared with a standard 15% protein diet. Food intake, body weight, and energy expenditure were measured. After 3 wk, plasma was sampled and rats were killed to determine body composition and gene expression. Statistical analyses were mainly done by ANOVA tests and correlation tests.RESULTS:The selected PE ratio increased from 20% to 35% when the carbohydrate content of the protein-free diet increased from 30% to 75% (R2 = 0.56; P < 10-6). Consequently, the PC ratio was constant (70%) in all groups (P = 0.18). In self-selecting rats, plasma FGF21 concentrations were 3 times lower than in rats fed the 5% protein diet (P < 10-4) and similar to those in rats fed a 30% diet.CONCLUSIONS:This study showed that self-selecting rats established PE ratios larger than those considered sufficient to achieve optimal growth in adult rats (10-15%), and the ratios were highly dependent on carbohydrates, apparently with the aim of maintaining a constant and high PC ratio. This was associated with a minimization of plasma FGF21.

Différentes études ont rapporté qu’une restriction protéique impacte le métabolisme énergétique de rats en croissance. Une déficience sévère en protéine (3 % ou 5 % du besoin) induit notamment une diminution du gain de poids corporel, une augmentation de la prise alimentaire relative, de la dépense énergétique, ainsi que des taux de fibroblast growth factor 21 (FGF21) plasmatiques. Cependant, l’implication précise de tous ou certains acides aminés (AA) dans ces effets reste inconnue. Cette étude a pour objectif d’évaluer les effets d’une déficience en un AA strictement indispensable (lysine ou thréonine ; AAI) sur le métabolisme énergétique de rats en croissance. Des rats en croissance ont été nourris avec des régimes déficients en lysine ou thréonine pendant trois semaines : sévèrement déficients (P3 L/T15 et P5 L/T25), modérément déficients (P8 L/T40 et P12 L/T60), contrôles (P15 L/T75 et P20 L/T100), excessif (P20 L/T170) ou un contrôle de l’effet des AA libres dans le régime (P3 L/T100). Le poids corporel et la prise alimentaire ont été mesurés quotidiennement, la dépense énergétique a été évaluée par calorimétrie indirecte durant la troisième semaine. À la fin de l’expérimentation, les tissus et fluides biologiques ont été prélevés et la composition corporelle a été analysée. L’analyse statistique a été faite par modèle mixte en mesure répétée et ANOVA à 1 facteur. Les régimes sévèrement déficients en lysine induisent une réduction du gain de poids corporel, dès 3 et 6 jours (pour P3 L15 et P5 L25 respectivement). Pour la thréonine, les régimes sévèrement déficients, ainsi que le régime P8 T40, provoquent une diminution du gain de poids corporel dès 1 et 6 jours. Parallèlement, on observe une augmentation de la prise alimentaire relative (PAR) pour les régimes sévèrement déficients en lysine et thréonine, ainsi que pour les régimes P8 L40/P8 T40. Les régimes déficients en lysine n’influent pas sur la dépense énergétique de repos (DER) et le coût d’activité, et ne sont pas significativement différents des régimes contrôles pour la dépense énergétique totale (DET) et d’activité (DEA). Pour la déficience en thréonine, on observe une diminution de la DET pour le régime P3 T15, confortée par une diminution de la DER, sans différence significative de la DEA par rapport aux régimes contrôles. De plus, le coût d’activité tend à être diminué pour le régime P3 T15. Les déficiences sévères en lysine ou thréonine induisent une augmentation de FGF21 plasmatique. La déficience en un AAI, lysine ou thréonine, induit une diminution du gain de poids corporel chez le rat en croissance, malgré une PAR augmentée, avec un effet plus marqué dans le cas de la thréonine. Comparativement aux régimes contrôles, la déficience en lysine n’impacte pas la dépense énergétique, contrairement à la déficience en thréonine qui entraîne une diminution de la DET et de la DER.

In mice, low protein (LP) diets increase food intake (FI) thereby energy intake, to compensate for protein deficiency, but mice do not gain fat because total energy expenditure (TEE) is also increased. Fibroblast Growth Factor 21 (FGF21), which is expressed in the liver and in the brain, appears as a key player in these effects. The present study hypothesized that both LP diet but also only lysine or threonine deficiency can modulate FGF21 in the liver and in the hypothalamus that in turn is involved in the control of FI and energy expenditure. Growing rats were fed for 3 weeks: i) LP diets containing 3-5-8-12-15 or 20% of milk protein, or ii) the same diets but supplemented with free amino acids at the level of the 20% protein diet except for lysine or threonine leading to lysine or threonine deficient diets. Body weight and FI were measured daily and energy expenditure were measured by indirect calorimetry. At the end of the experiment, rats were euthanized, tissues and biological fluids were removed and frozen, and body composition as well as gene expression and plasma FGF21 were analyzed. Diets with 3 and 5% protein, and diets highly deficient in lysine or threonine (85% and 75%) result in significant growth retardation. LP 3% and 5% induced an increase in relative FI. Surprisingly, an increase in TEE was observed under LP 5% protein, due to an increase in motor activity. Hepatic FGF21 expression is increased at 3 and 5% and strongly decreased at 12, 15 and 20% protein. In contrast, in the hypothalamus, FGF21 expression was significantly lower in LP 3% compared to a 20% protein diet, and for the other LP diets, the values are intermediate. Plasma FGF21 was higher in 3, 5, 8 and 12% protein than in 15 and 20% protein diets. Lysine or threonine deficiency were able to reproduce the effect of LP diet at 3 and 5% whereas at 8%, only the deficiency of threonine was able to reproduce the LP effect. These results showed that hepatic and hypothalamic expression FGF21 are inversely affected by protein deficiency. Such situations of deficiency induced an up-regulation of hepatic expression of FGF21 that increased TEE and a down-regulation of hypothalamic expression of FGF21 that could led to hyperphagia. Interestingly, the levels of FGF21 observed for the 3, 5, 8% protein diets could be due at least to lysine and threonine deficiency. ABIES, AlimH department of INRAe.

The protein requirement is generally defined as the amount necessary to maintain the body's protein pool. However, under free choice conditions, animal models often ingest more protein than required for nitrogen balance (10%–15%). This behavior possibly reflects the search for a high protein-to-carbohydrate ratio (0.6–0.8), inducing metabolic benefits. This indicates that in addition to protein homeostasis, dietary proteins are also involved in energy homeostasis. The mechanisms controlling protein and energy intake are partly independent and in specific conditions, there may be a conflict between the two. Protein density in the human diet has decreased ∼2% since the 1970s and, according to the protein leverage hypothesis, this decrease may be responsible for the increase in energy intake and prevalence of obesity observed today.

AbstractIntroduction:The quality of dietary protein sources became a particularly sensitive issue in the current debates on a rebalancing between animal and vegetable food sources.The ability of a protein to meet the nutritional requirements of essential amino acid (EAA) is the basis for assessing the quality of protein.The objective of this study was to characterize the impact of lysine- and threonine-deficient gluten-based diets on the metabolism of growing rats and to identify molecular markers of these diets.Materials and Methods:Growing rats were fed for 3 weeks with a threonine-supplemented and 70% lysine-deficient gluten diet; a lysine-supplemented and 47% threonine-deficient gluten diet; a gluten diet supplemented in lysine and threonine to meet all the AA requirements, and a control diet with milk protein to meet all the AA requirements.Body weight and food intake were measured daily. At the end of the experiment, tissues and biological fluids were removed. The body composition was analyzed, gene expression measurements involved in protein and lipid metabolism were performed and the urinary metabolome was analyzed by LC-MS. Statistical analysis was done by variance analysis and metabolome analysis by discriminant analysis of independent components.Results:This EAA deficiency does not modify the food intake. Lysine deficiency induces a decrease in body weight gain, and lean body mass, associated with an increased in proteolysis and a decreased in proteosynthesis, a decreased in bone mineral density, and no effect on lipid metabolism.Threonine deficiency induces a decrease in body weight gain, and liver and skin weight, without changes in protein metabolism, bone mineral density, and lipid metabolism. After approval of the deficiency model, the metabolomic analysis performed on urine samples revealed the presence of specific discriminating molecules of the diets and types of proteins.Discussion:EAA deficiency has an impact on the growth, and bone and protein metabolism of growing rats. These deficiency states have resulted in different metabolome profiles that could lead to the identification of specific molecular markers of protein sources and related to EAA deficiencies.

Protein sufficiency is tightly controlled through different sensing and signaling processes that modulate and adapt protein and energy metabolism and feeding behavior to reach and maintain a well-balanced protein status. High-protein diets, often discussed in the context of body weight management, usually activate anorexigenic pathways, leading to higher satiety, decreased food and energy intake, and decreased body weight and adiposity. Diets marginally low in protein (3-8% energy) or marginally deficient in some indispensable amino acid more often activate orexigenic pathways, with higher appetite and a specific appetite for protein, a response that leads to an increase in protein intake to partially compensate for the deficit in protein and amino acid. Diets severely deficient in protein (2-3% energy as protein) usually depress food intake and induce lower weight and lower fat mass and lean tissues that characterize a status of protein deficiency. The control of protein sufficiency involves various peripheral and central signals, including modulation of both metabolic pathways at the periphery as well as central pathways of the control of food and protein intake, including a reward-driven specific sensitivity to the protein content of foods.

Fibroblast Growth Factor 21 (FGF21), a response to metabolic stress, is influenced by the dietary protein content. Previous studies have shown that a protein level below 10% of energy increases FGF21 hepatic secretion in mice, and increases food intake and energy expenditure. However, it has also been shown in vitro that glucose stimulates FGF21 secretion in liver. The objective of this study was to determine the respective roles of dietary protein and carbohydrate contents on FGF21 secretion and associated metabolic responses. 70 male Wistar rats were subjected at one to 12 diets with various milk protein contents (3, 5, 8, 15 and 30% P of energy) and a mixture of carbohydrates and fats in which carbohydrates amounted 30, 45, 60 or 75% of energy. Body weight and energy intake were measured twice a week, and energy expenditure was measured one time after 2 weeks by indirect calorimetry. After 3 weeks, plasma was collected and an ELISA test was used to determine plasma concentration of FGF21. Tissues were dissected and weighed to determine body composition. Pieces of liver were frozen for measuring expression of Fgf21 mRNA by RT-PCR. Statistical analyzes were done by analysis of variance. The decrease in %P in diets increased liver Fgf21 mRNA. Using the 30% P fed group as a reference, Fgf21 mRNA were increased not significantly 3x in 15% P, but significantly 19x in 8% P, 44x in 5% P and 60x in 3% P (P < 0.001). This was related to an increase of plasma FGF21. In contrast, dietary carbohydrate contents did not affect FGF21. In response to the increased of FGF21 secretion, energy intake was increased at 8% and 5% P and was decreased in 3% P fed mice; and energy expenditure was increased in 5% and 3% P. Finally, weight gain was negative at 3% P, and lower in at 8% and 5% P than in 15% and 30% P, consequently to a lean body mass smaller in 3%, 5% and 8% P. Liver expression of Fgf21 mRNA and plasma FGF21 increased sharply in response to the decrease in dietary protein levels confirming the role of FGF21 in signaling protein deficiency and associated metabolic and behavioral responses. The lack of effect of the carbohydrate content of diets suggests that, in vivo, only protein content affects FGF21. At last, it should be noted that despite an apparently optimal growth at 15% P, FGF21 secretion is already higher than at 30% P. The funding of the experiments is provided by UMR PNCA.

Severely low-protein diets (LP) induce behavioral and metabolic changes including a decrease in body weight, an increase in relative food intake (FI) and alterations in hepatic metabolism. During such protein restriction, changes in hepatic anabolic and catabolic protein pathways could transitory participate to compensate for amino acid (AA) deficiency. In the present study, liver expression of gene involved in proteosynthesis and proteolysis pathways, were related to FI, blood AA levels and body composition in rats fed LP diet. Growing rats were fed for three weeks different diets containing 3-5-8-12-15 or 20% energy of milk protein. Body weight and FI were measured daily. At the end of the experiment, tissues and biological fluids were removed for gene expression measurement and blood AA UPLC analysis. Statistical analysis was done by 1- or 2-factor ANOVA, when data were repeated. Despite an increase in relative food intake under P3 and P5% diets, P3, P5 and P8% diets resulted in significant growth retardation compared to other groups. Lean mass was significantly decreased in rats under P3, P5 and P8% compared to P12, P15 and P20% diets, while there was no difference in fat mass between all groups. P3, P5 and P8% diets induced a decrease in essential amino acid concentrations in portal vein, whereas there was no significant difference between groups in veina cava. Severely protein restricted P3% and P5% diets induced an increase in hepatic gene expression involved in proteolysis as calpain 2 and ubiquitin, and an activation of ATF4-CHOP-TRB3 pathway. These results suggested that under severe protein restriction, hepatic protein catabolism became a source of plasma amino acid that could partially compensate for the AA not provided by the diet. These observations confirm that liver plays a major role in the adaptation of the body to dietary protein restriction and highlight that severe dietary protein restriction induced liver protein catabolism by inducing an activation of ATF4-CHOP-TRB3 pathway in order to provide amino acids to body tissues. ABIES, AlimH-INRAE.

Le besoin en protéines est en général défini comme la quantité nécessaire pour le maintien du pool de protéines corporelles, or dans des conditions de libre choix, les modèles animaux ingèrent souvent plus de protéines que nécessaire pour l’équilibre azoté (10 %–15 %). Ce comportement semble refléter la recherche d’un rapport protéines/glucides élevé (0,6–0,8), induisant des bénéfices métaboliques. Cela indique qu’en plus de l’homéostasie des protéines, les protéines alimentaires sont impliquées dans l’homéostasie énergétique. Les mécanismes contrôlant l’apport protéique et énergétique sont en partie indépendants et dans des conditions spécifiques, il peut y avoir un conflit entre les deux. La densité des protéines dans l’alimentation humaine a diminué d’environ 2 % depuis les années 1970 et, selon l’hypothèse de l’effet de levier des protéines, cette diminution pourrait contribuer à l’augmentation de l’apport énergétique et de la prévalence de l’obésité observées aujourd’hui.