The Protein Status of Rats Affects the Rewarding Value of Meals Due to their Protein Content

The Protein Status of Rats Affects the Rewarding Value of Meals Due to their Protein Content Abstract Background Protein status is controlled by the brain, which modulates feeding behavior to prevent protein deficiency. Objective This study tested in rats whether protein status modulates feeding behavior through brain reward pathways. Methods Experiments were conducted in male Wistar rats (mean ± SD weight; 230 ± 16 g). In experiment 1, rats adapted for 2 wk to a low-protein (LP; 6% of energy) or a normal-protein (NP; 14% of energy) diet were offered a choice between 3 cups containing high-protein (HP; 50% of energy), NP, or LP feed; their intake was measured for 24 h. In 2 other experiments, the rats were adapted for 2 wk to NP and either HP or LP diets and received, after overnight feed deprivation, a calibrated HP, NP, or LP meal daily. After the meal, on the last day, rats were killed and body composition and blood protein, triglycerides, gut neuropeptides, and hormones were determined. In the brain, neuropeptide mRNAs in the hypothalamus and c-Fos protein and opioid and dopaminergic receptor mRNAs in the nucleus accumbens (NAcc) were measured. Results Rats fed an LP compared with an NP diet had 7% lower body weight, significantly higher protein intake in a choice experiment (mean ± SD: 30.5% ± 0.05% compared with 20.5% ± 0.05% of energy), higher feed-deprived blood ghrelin, lower postmeal blood leptin, and higher neuropeptide Y (Npy) and corticotropin-releasing hormone (Crh) mRNA expression in the hypothalamus. In contrast to NP, rats fed an LP diet showed postmeal c-Fos protein expression in the NAcc, which was significantly different between meals, with LP < NP < HP. In contrast, in rats adapted to an HP diet compared with an NP diet, energy intake was lower; and in the NAcc, meal-induced c-Fos protein expression was 20% lower, and mRNA expression was 17% higher for dopamine receptor 2 (Drd2) receptors and 38% lower for κ opioid receptor (Oprk1) receptors. Conclusion A protein-restricted diet induced a reward system–driven appetite for protein, whereas a protein-rich diet reduced the meal-induced activation of reward pathways and lowered energy intake in male rats. protein, satiety, reward, accumbens nucleus, food intake Introduction The maintenance of normal physiologic functions and survival in animals and humans requires a continuous supply of amino acids to tissues to support protein synthesis and other amino acid–dependent metabolic processes. These phenomena depend on a daily intake of an adequate quantity of protein from a diverse array of foods. Protein status, characterized by amino acid sufficiency in the body to support metabolic functions, is tightly and continuously controlled and modulates food motivation, food choice, or food aversion to prevent or counteract protein deficiency (1, 2). Animals learn to detect and avoid very-low-protein diets, protein-deficient diets, or indispensable amino acid–deficient diets (3–5). After food or protein deprivation, a specific appetite for protein and a preference for protein-rich foods have been observed in rodents (6, 7). Animals offered a marginally protein-deficient diet tend to meet their target protein needs with different strategies, including food choice and, under some conditions, a slight increase in food intake (8–10). There have also been observations of a higher appetite for protein-rich foods after protein-restricted diets in humans (11, 12), as well as a preference for foods associated with a protein-rich flavor after a low-protein preload (13). In contrast, high-protein diets are usually reported to decrease food intake in both animals and humans, related to a reported satiating effect of proteins through activation of anorexigenic pathways (1, 14–18). The present study, which used a rat model, aimed to evaluate how protein status (characterized as generous, normal, or deficient) influences the activation of brain regions associated with the control of food intake and reward system, the hypothalamus and the nucleus accumbens (NAcc), respectively, in association with the protein content of a single meal. It is hypothesized that, under conditions of protein deficiency, the activation of the NAcc is positively related to the meal protein content to restore protein status. Indeed, many studies have shown the importance of NAcc, a component of the ventral striatum in basal ganglia circuits, in the reward system. This system is involved in actions that prioritize behavior and promote the continuation of ongoing actions that increase behaviors leading to the procurement and consumption of the reward (positive reinforcement) and in direct future behavioral actions (19). In a first experiment, rats were adapted to either a normal-protein (NP) or low-protein (LP) diet and were then offered a choice between 3 feeds containing NP, high-protein (HP), or LP contents. In 2 other experiments, after habituation to NP, HP, or LP diets, rats were offered, before being killed, a calibrated meal of LP, NP, or HP feeds. Rats were characterized by measuring blood concentrations of protein, TGs, gut neuropeptides [ghrelin, gastric inhibitory polypeptide (GIP), glucagon-like peptide 1 (GLP-1), and peptide YY (PYY)], and metabolic hormones (leptin, insulin, and glucagon). Brain neuropeptide [neuropeptide Y (NPY), proopiomelanocortin, agouti-related peptide, cocaine- and amphetamine-regulated transcript], corticotropin-releasing hormone (CRH), and melanocortin receptor (MC4R) mRNA expressions were determined in the hypothalamus. Total neuronal activation measured by the detection of c-Fos–positive nuclei and changes in mRNA expression of opioid and dopaminergic receptors were determined in the NAcc. Methods Animals and diets Adult male Wistar rats (Envigo) with initial mean ± SD weight of 230 ± 16 g were housed in individual cages at 22 ± 2°C under a 12-h reverse light-dark cycle (lights on at 2100). All of the experimental procedures were approved by the Regional Animal Care and Ethical Committee (approval no. 12/173) and conformed to the European legislation on the use of laboratory animals. Feeds were a modified version of the AIN-93M diet (Table 1) (20, 21). All feeds were moistened with a 1:1 ratio of powder to water for HP and a 1.5:1 ratio for NP and LP feeds to achieve pellets of sufficient consistency and to minimize spillage. Food intake was determined by the difference in food-cup weight before and after each experimental period, corrected for spillage, the amount of added water, and evaporation. All rats had free access to water throughout the experimental period. All of the rats were acclimated for 1 wk to the housing conditions before starting the feeding conditions. TABLE 1 Composition of HP, NP, and LP feeds1 HP NP LP Protein, % of energy 55 14 6 Carbohydrates, % of energy 35 76 84 Fat, % of energy 10 10 10 Milk protein, g/kg 530 140 60 Corn starch, g/kg 287 622 691 Sucrose, g/kg 46 100 111 Soybean oil, g/kg 40 40 40 Salts and minerals, g/kg 35 35 35 Vitamins, g/kg 10 10 10 α-Cellulose, g/kg 50 50 50 Choline, g/kg 2.3 2.3 2.3 HP NP LP Protein, % of energy 55 14 6 Carbohydrates, % of energy 35 76 84 Fat, % of energy 10 10 10 Milk protein, g/kg 530 140 60 Corn starch, g/kg 287 622 691 Sucrose, g/kg 46 100 111 Soybean oil, g/kg 40 40 40 Salts and minerals, g/kg 35 35 35 Vitamins, g/kg 10 10 10 α-Cellulose, g/kg 50 50 50 Choline, g/kg 2.3 2.3 2.3 1Feeds were a modified version of the AIN-93M (20, 21). HP, high-protein; LP, low-protein; NP, normal-protein. View Large TABLE 1 Composition of HP, NP, and LP feeds1 HP NP LP Protein, % of energy 55 14 6 Carbohydrates, % of energy 35 76 84 Fat, % of energy 10 10 10 Milk protein, g/kg 530 140 60 Corn starch, g/kg 287 622 691 Sucrose, g/kg 46 100 111 Soybean oil, g/kg 40 40 40 Salts and minerals, g/kg 35 35 35 Vitamins, g/kg 10 10 10 α-Cellulose, g/kg 50 50 50 Choline, g/kg 2.3 2.3 2.3 HP NP LP Protein, % of energy 55 14 6 Carbohydrates, % of energy 35 76 84 Fat, % of energy 10 10 10 Milk protein, g/kg 530 140 60 Corn starch, g/kg 287 622 691 Sucrose, g/kg 46 100 111 Soybean oil, g/kg 40 40 40 Salts and minerals, g/kg 35 35 35 Vitamins, g/kg 10 10 10 α-Cellulose, g/kg 50 50 50 Choline, g/kg 2.3 2.3 2.3 1Feeds were a modified version of the AIN-93M (20, 21). HP, high-protein; LP, low-protein; NP, normal-protein. View Large Experimental design Rat groups were characterized by diet (e.g., HP, NP, or LP) and, when appropriate, the experimental meal (HP, NP, or LP) expressed as diet/meal (e.g., NP/HP describes rats adapted to the NP diet that received an HP meal). Three experiments were performed. Experiment 1 (food choice in NP- compared with LP-diet–fed rats) After acclimation, rats (n = 16) were divided into 2 groups (n = 8/group) with ad libitum access to either the NP or the LP feed for 2 wk. On the last day, rats were offered a choice between 3 cups containing the HP, NP, or LP feed, and the intake of each feed was measured for 24 h. Experiment 2 (NP compared with LP diets) In experiment 2a, after 7 d of acclimation, rats (n = 48) were divided into 2 groups (n = 24/group) with ad libitum access for 1 wk to either the NP or LP feed during their dark period (Figure 1). Each group was acclimated during the third week to a daily feeding pattern consisting of 30-min access from 0900 to 0930 to a 6-g (43.8-kJ) calibrated NP or LP meal, and then from 1100 to 1800 to ad libitum access to the same NP or LP feed, resulting in the diet/meal NP/NP and LP/LP groups, respectively. On the last day, each group (NP/NP and LP/LP) was divided into 4 subgroups (n = 6/group), which each received a 6-g calibrated LP, NP, or HP feed meal or no meal, resulting in the diet/meal NP/LP, NP/NP, NP/HP, or NP/feed-deprived (15-h feed-deprived) and LP/LP, LP/NP, LP/HP, or LP/feed-deprived (15-h feed-deprived) groups, respectively. RT-PCR analysis of receptors in the hypothalamus and NAcc, tissue and blood sampling, and body-composition measurements were performed. FIGURE 1 View largeDownload slide Experimental design of experiment 2. After 1 wk of acclimation to laboratory animal housing conditions and 1 wk of diet acclimation, rats were accustomed to receiving their food according to a pattern that consisted of a calibrated meal of 6 g (43.8 kJ) for 30 min between 0900 and 0930, free access to food between 1100 and 1800, and feed deprivation between 1800 and 0900 the next day. FIGURE 1 View largeDownload slide Experimental design of experiment 2. After 1 wk of acclimation to laboratory animal housing conditions and 1 wk of diet acclimation, rats were accustomed to receiving their food according to a pattern that consisted of a calibrated meal of 6 g (43.8 kJ) for 30 min between 0900 and 0930, free access to food between 1100 and 1800, and feed deprivation between 1800 and 0900 the next day. In experiment 2b, the same experimental design was followed for immunofluorescence detection of c-Fos protein expression in the NAcc, but with 24 rats divided into 2 groups (n = 12/group; NP/NP and LP/LP) and subsequently divided into 3 subgroups (n = 4/subgroup; NP/LP, NP/NP, NP/HP and LP/LP, LP/NP, LP/HP); the feed-deprived subgroup was not considered. Experiment 3 (NP compared with HP diets) After 7 d of acclimation, rats (n = 18) were divided into 3 groups (n = 6/group). Rats had ad libitum access during their dark period to the NP or the HP feed, or the NP-feed diet pair-fed for energy to the HP-feed diet (NPpfHP), for 2 wk. During the last week, after overnight feed deprivation, rats received a 6-g (43.8 kJ) calibrated NP- or HP-feed meal (NP/NP, HP/HP, and NPpfHP/NP groups) for 30 min from 0900 to 0930. On the last day, the rats were killed 90 min after the beginning of the calibrated meal. Rats were then treated for RT-PCR analysis of receptors in the NAcc. The same experimental design, but with 4 rats per group (n = 12), was followed for immunofluorescence detection of c-Fos protein expression in the NAcc. In experiments 2 and 3, rats trained for 1 wk to receive between 0900 and 0930, after overnight feed deprivation, a 6-g calibrated meal consumed the entire test meals. Immunofluorescence detection of c-Fos protein expression in the NAcc Rats from experiment 2b were killed at 1100 with a lethal intraperitoneal injection of pentobarbital sodium (100 mg/kg; Ceva Santé Animale) 90 min after the beginning of their experimental meal. A detailed description of brain preparations was previously described (20). Twenty-micrometer sections were obtained with the use of a cryostat (CM1520; Leica) in the region of the NAcc (+2.7 to +0.48-mm bregma; every fifth slice was collected), identified by using the Paxinos and Watson stereotaxic atlas (22) (The Rat Brain in Stereotaxic Coordinates, fourth edition, Paxinos & Watson). Slices were mounted on microscope slides SuperFrost+ slides (Dutscher) and stored at –80°C. Slices were treated as previously described (20) with some modifications: we incubated slices with rabbit anti-c-Fos antibody (primary antibody, 1:5000, Ab-5; Calbiochem) for 48 h at 4°C. Slices were then rinsed 3 times for 10 min in PBS solution and then incubated in the PBS/Triton X-100/BSA solution containing Alexa-Fluor 488 (secondary antibody, 1:200; Molecular Probes) for 2 h at room temperature. Finally, slices were washed 3 times in PBS solution and mounted by using a medium containing 4′,6-diamidino-2-phenylindole (Vector Laboratories). The NAcc was identified by using a stereotaxic atlas, and the number of c-Fos immunopositive cells was evaluated in a minimum of 4 consecutive slices per rat. The mean labeling of c-Fos–immunopositive cells was calculated for each rat, and the mean number of labels determined for each rat group. RT-PCR analysis of receptors in the hypothalamus and NAcc Rats from experiment 2a were anesthetized 90 min after receiving the experimental meal with an intraperitoneal injection of sodium pentobarbital (50 mg/kg; Ceva Santé Animale) and then decapitated. The brain was harvested, and the hypothalamus was immediately extracted from the fresh brain by making an incision medial to the piriform lobes caudal to the optic chiasma and anterior to the cerebral crus to a depth of 2 ± 3 mm and was placed directly in TRIzol reagent (Invitrogen), frozen in liquid nitrogen, and stored at –80°C. The brain was then placed in sterile 0.1-M PBS solution and kept at –20°C for 30 min. A 4- to 6-mm slice of brain (measured with square calipers) was cut from the beginning of the olfactory bulb (+2.70 to +0.48-mm bregma). A piece of this slice containing the NAcc area was removed with a scalpel, immediately placed in 1 mL Trizol (Invitrogen) and stored at –80°C. Total RNA was extracted from the hypothalamus and NAcc samples by homogenization as previously described (23). Sequences of primers are given in Supplemental Table 1. Gene expression was determined by using the 2−ΔCt formula, where ΔCt = Ct target gene Ct 18S. Tissue sampling, body composition, and blood analysis Rats from experiment 2a were anesthetized 90 min after ingestion of the calibrated meal, if applicable, with an intraperitoneal injection of sodium pentobarbital (50 mg/kg; Ceva Santé Animale) and decapitated. Tissues and organs (liver, spleen, kidneys, brain, heart, skin, subcutaneous, retroperitoneal, epididymal, and mesenteric adipose tissues) were collected, blotted dry, and weighed to the nearest 0.01 g. Blood samples were collected on EDTA and immediately centrifuged (1500 × g, 4°C), and plasma aliquots were prepared for biochemical or hormonal analysis, after the addition of protease inhibitor (Roche), and stored at −70°C until being assayed. All of the plasma biochemical analyses (albumin, lactate, cholesterol, HDL cholesterol, TGs, FFAs, glycerol, and β-hydroxybutyrate) were performed on an Olympus AU 400 robot specifically calibrated for rat assays (Centre d'Explorations Fonctionnelles Intégrées). Plasma insulin was detected by using enzyme-linked immunoassays (Mercodia Rat Insulin). Plasma glucagon, leptin, GIP, GLP-1, and ghrelin concentrations were assessed by Luminex (Bio-Plex Pro rat standard; Bio-Rad). Blood glucose was determined immediately after sampling with a blood glucose meter (Accu Check Go; Roche). Liver TG concentrations were determined by using an enzymatic assay (TG–TIGS; Randox). Statistical analysis Data are presented as means ± SDs. Statistical analyses were performed by using SAS (version 9.1), and a generalized linear model was applied. The effects of the diets and the meals were tested by 2-factor ANOVA with interaction; and when diet, meal, or diet × meal was significant, pairwise comparisons between rat groups characterized by diet or diet and meal (diet/meal) were performed with post hoc Tukey test for multiple comparisons. Differences were considered significant at P < 0.05. Results Food intake, body weight and composition, and food choice In experiment 1, rats adapted for 2 wk to the NP or the LP diet were subjected during 24 h to a choice between 3 cups with LP, NP, or HP feed (Figure 2, Table 2). Rats previously fed the NP diet showed a significant preference for the NP meal compared with the HP meal, with no choice difference between LP and HP meals. However, rats previously habituated to the LP diet and offered the same range of meals showed no choice difference between the NP and HP meals but had a significant preference for both the NP and HP meals compared with the LP meal. The combination of choices between the LP, NP, and HP meals led to the same total energy intake for rats previously adapted to the LP diet compared with the NP diet (Table 2). Indeed, rats fed an LP diet consumed, during the choice experiment, a significantly higher amount of energy as protein and a significantly lower amount of energy as carbohydrate, whereas no difference was observed in the amount of energy from fat because fat content was the same in the 3 feeds. FIGURE 2 View largeDownload slide Twenty-four-hour food choice between LP, NP, and HP feeds in rats previously adapted for 15 d to an NP or an LP diet (experiment 1; n = 8/group). Values are means ± SDs. Labeled means without a common letter differ, P < 0.05. HP, high-protein; LP, low-protein; NP, normal-protein. FIGURE 2 View largeDownload slide Twenty-four-hour food choice between LP, NP, and HP feeds in rats previously adapted for 15 d to an NP or an LP diet (experiment 1; n = 8/group). Values are means ± SDs. Labeled means without a common letter differ, P < 0.05. HP, high-protein; LP, low-protein; NP, normal-protein. TABLE 2 Energy intake and macronutrient composition of the diet, during the 24-h food choice between LP, NP, and HP feeds in rats previously adapted for 15 d to the NP or the LP diet (experiment 1)1 15-d diet adaptation NP diet LP diet Diet effect, P Total energy intake during the 24-h choice, kJ/100 g body weight 106.0 ± 16.65 97.1 ± 12.00 NS Macronutrient composition of the 24-h choice, % of energy  Protein2 20.5 ± 0.05a 30.5 ± 0.05b 0.02  Carbohydrate2 69.5 ± 0.06b 59.5 ± 0.06a 0.02  Fat3 10.0 ± 0.00 10.0 ± 0.00 15-d diet adaptation NP diet LP diet Diet effect, P Total energy intake during the 24-h choice, kJ/100 g body weight 106.0 ± 16.65 97.1 ± 12.00 NS Macronutrient composition of the 24-h choice, % of energy  Protein2 20.5 ± 0.05a 30.5 ± 0.05b 0.02  Carbohydrate2 69.5 ± 0.06b 59.5 ± 0.06a 0.02  Fat3 10.0 ± 0.00 10.0 ± 0.00 1Values are means ± SDs, n = 8/group. Labeled means without a common letter differ, P < 0.05. LP, low-protein; NP, normal-protein. 2Calculated from the macronutrient composition of feeds (Table 1) and the 24-h intake of each of the 3 feeds during the choice experiment. 3The 3 feeds had the same 10% of energy from fat content. View Large TABLE 2 Energy intake and macronutrient composition of the diet, during the 24-h food choice between LP, NP, and HP feeds in rats previously adapted for 15 d to the NP or the LP diet (experiment 1)1 15-d diet adaptation NP diet LP diet Diet effect, P Total energy intake during the 24-h choice, kJ/100 g body weight 106.0 ± 16.65 97.1 ± 12.00 NS Macronutrient composition of the 24-h choice, % of energy  Protein2 20.5 ± 0.05a 30.5 ± 0.05b 0.02  Carbohydrate2 69.5 ± 0.06b 59.5 ± 0.06a 0.02  Fat3 10.0 ± 0.00 10.0 ± 0.00 15-d diet adaptation NP diet LP diet Diet effect, P Total energy intake during the 24-h choice, kJ/100 g body weight 106.0 ± 16.65 97.1 ± 12.00 NS Macronutrient composition of the 24-h choice, % of energy  Protein2 20.5 ± 0.05a 30.5 ± 0.05b 0.02  Carbohydrate2 69.5 ± 0.06b 59.5 ± 0.06a 0.02  Fat3 10.0 ± 0.00 10.0 ± 0.00 1Values are means ± SDs, n = 8/group. Labeled means without a common letter differ, P < 0.05. LP, low-protein; NP, normal-protein. 2Calculated from the macronutrient composition of feeds (Table 1) and the 24-h intake of each of the 3 feeds during the choice experiment. 3The 3 feeds had the same 10% of energy from fat content. View Large In experiments 2a and 2b, rats fed the LP-diet feeding pattern for 2 wk showed no significant difference in daily energy intake compared with those fed the NP diet, but they had lower body weight gain and lower lean and fat mass than rats in the NP diet group (Table 3). In addition, in experiment 3, rats fed an HP diet for 2 wk had a lower daily energy intake and lower body weight gain than those fed an NP diet, whereas their body weight gain was not different than that of NPpfHP diet group (Table 3). TABLE 3 Daily energy intake, body weight gain, and final fat and lean mass in the different rat groups adapted to LP, NP, or HP diets for 2 wk1 Diet groups n/Group Energy intake, kJ/d Body weight gain, g/d Final fat mass, g Final lean mass, g Experiment 2a  NP 24 335 ± 36.8 4.1 ± 0.64b 35.8 ± 3.72b 265 ± 7.0b  LP 24 305 ± 33.4 0.8 ± 0.15a 23.1 ± 2.36a 200 ± 4.3a  Diet effect, P NS 0.0001 0.005 0.0002 Experiment 2b  NP 12 308 ± 18.9 3.0 ± 0.59b — —  LP 12 310 ± 32.2 1.1 ± 0.83a — —  Diet effect, P NS 0.001 Experiment 3  NP 6 381 ± 24.8b 6.5 ± 0.46b — —  HP 6 312 ± 23.4a 5.4 ± 0.29a — —  NPpfHP 6 302 ± 24.8a 5.4 ± 0.23a — —  Diet effect, P 0.003 0.03 Diet groups n/Group Energy intake, kJ/d Body weight gain, g/d Final fat mass, g Final lean mass, g Experiment 2a  NP 24 335 ± 36.8 4.1 ± 0.64b 35.8 ± 3.72b 265 ± 7.0b  LP 24 305 ± 33.4 0.8 ± 0.15a 23.1 ± 2.36a 200 ± 4.3a  Diet effect, P NS 0.0001 0.005 0.0002 Experiment 2b  NP 12 308 ± 18.9 3.0 ± 0.59b — —  LP 12 310 ± 32.2 1.1 ± 0.83a — —  Diet effect, P NS 0.001 Experiment 3  NP 6 381 ± 24.8b 6.5 ± 0.46b — —  HP 6 312 ± 23.4a 5.4 ± 0.29a — —  NPpfHP 6 302 ± 24.8a 5.4 ± 0.23a — —  Diet effect, P 0.003 0.03 1Values are means ± SDs. Within an experiment, labeled means without a common letter differ, P < 0.05. HP, high-protein. LP, low-protein; NP, normal-protein; NPpfHP, normal-protein diet pair-fed for energy to the high-protein diet. View Large TABLE 3 Daily energy intake, body weight gain, and final fat and lean mass in the different rat groups adapted to LP, NP, or HP diets for 2 wk1 Diet groups n/Group Energy intake, kJ/d Body weight gain, g/d Final fat mass, g Final lean mass, g Experiment 2a  NP 24 335 ± 36.8 4.1 ± 0.64b 35.8 ± 3.72b 265 ± 7.0b  LP 24 305 ± 33.4 0.8 ± 0.15a 23.1 ± 2.36a 200 ± 4.3a  Diet effect, P NS 0.0001 0.005 0.0002 Experiment 2b  NP 12 308 ± 18.9 3.0 ± 0.59b — —  LP 12 310 ± 32.2 1.1 ± 0.83a — —  Diet effect, P NS 0.001 Experiment 3  NP 6 381 ± 24.8b 6.5 ± 0.46b — —  HP 6 312 ± 23.4a 5.4 ± 0.29a — —  NPpfHP 6 302 ± 24.8a 5.4 ± 0.23a — —  Diet effect, P 0.003 0.03 Diet groups n/Group Energy intake, kJ/d Body weight gain, g/d Final fat mass, g Final lean mass, g Experiment 2a  NP 24 335 ± 36.8 4.1 ± 0.64b 35.8 ± 3.72b 265 ± 7.0b  LP 24 305 ± 33.4 0.8 ± 0.15a 23.1 ± 2.36a 200 ± 4.3a  Diet effect, P NS 0.0001 0.005 0.0002 Experiment 2b  NP 12 308 ± 18.9 3.0 ± 0.59b — —  LP 12 310 ± 32.2 1.1 ± 0.83a — —  Diet effect, P NS 0.001 Experiment 3  NP 6 381 ± 24.8b 6.5 ± 0.46b — —  HP 6 312 ± 23.4a 5.4 ± 0.29a — —  NPpfHP 6 302 ± 24.8a 5.4 ± 0.23a — —  Diet effect, P 0.003 0.03 1Values are means ± SDs. Within an experiment, labeled means without a common letter differ, P < 0.05. HP, high-protein. LP, low-protein; NP, normal-protein; NPpfHP, normal-protein diet pair-fed for energy to the high-protein diet. View Large Neuronal activation in the NAcc measured by c-Fos immunolabeling Neural activation was determined by measuring c-Fos protein 90 min after receiving the calibrated 6-g meal in rats on the last day from experiment 2b (NP compared with LP diet) and experiment 3 (NP compared with HP diet) (Figure 3). FIGURE 3 View largeDownload slide c-Fos–positive neurons in the NAcc 90 min after the beginning of the 6-g calibrated meal in rats. (A) Rats fed for 15 d an NP or an LP diet received on the last day an LP, NP, or HP meal (experiment 2b; n = 12/group). (B) Rats fed for 15 d a diet/meal feeding pattern of NP/NP, NPpfHP/NP, or HP/HP (experiment 3; n = 6/group). Values are means ± SDs. Within an experiment, labeled means without a common letter differ, P < 0.05. HP, high-protein; LP, low-protein; NP, normal-protein; NPpfHP, normal-protein diet pair-fed for energy to the high-protein diet. FIGURE 3 View largeDownload slide c-Fos–positive neurons in the NAcc 90 min after the beginning of the 6-g calibrated meal in rats. (A) Rats fed for 15 d an NP or an LP diet received on the last day an LP, NP, or HP meal (experiment 2b; n = 12/group). (B) Rats fed for 15 d a diet/meal feeding pattern of NP/NP, NPpfHP/NP, or HP/HP (experiment 3; n = 6/group). Values are means ± SDs. Within an experiment, labeled means without a common letter differ, P < 0.05. HP, high-protein; LP, low-protein; NP, normal-protein; NPpfHP, normal-protein diet pair-fed for energy to the high-protein diet. In experiment 2b, in rats fed an adequate protein-content diet (NP) there was no difference in NAcc activation in rats that received an LP, NP, or HP meal (NP/LP, NP/NP, or NP/HP groups) (Figure 3A, Supplemental Figure 1). However, in the protein-deficient group (LP diet), NAcc neural activation was significantly different between meals, with LP/LP lower than LP/NP lower than LP/HP, and a lower NAcc activation was observed in LP/LP rats than in all other rat groups. In contrast, as reported in Figure 3B (experiment 3), there was a lower number of c-Fos–positive neurons in response to an HP meal in rats habituated to an HP diet for 2 wk (HP/HP) in comparison to the response to an NP meal in rats habituated to either an NP diet (NP/NP; P = 0.03) or to an NPpfHP diet (NPpfHP/NP; P < 0.05), with no difference between the NP/NP and the NPpfHP/NP groups (P = 0.06). Neuropeptide and neuronal receptor mRNA expression in the hypothalamus and in the NAcc In rats from experiment 2a, the mRNA expression of different neuropeptides and MC4R was measured in the hypothalamus in NP and LP rat groups in the feed-deprived state or 90 min after the NP, LP, or HP meal (Table 4). There was an overall significant diet effect for hypothalamic Npy and Crh expression, which was significantly higher in LP rats compared with NP-diet–fed rats, whereas no meal effect could be observed. No difference in the expression of other neuropeptides or MC4R was found between LP and NP diets, and no meal effect between the different groups was observed. TABLE 4 Neuropeptide mRNA expression in the hypothalamus in the different rat groups 90 min after the meal (experiment 2a)1 Diet/meal groups Npy Pomc Agrp Cartpt Crh Mc4r NP/feed-deprived 2.8 ± 1.31 3.5 ± 0.54 6.4 ± 2.77 1.2 ± 1.48 1.5 ± 0.72 4.9 ± 1.84 NP/LP 2.7 ± 0.49 7.8 ± 2.47 9.4 ± 2.78 2.0 ± 1.25 2.2 ± 0.51 4.7 ± 0.62 NP/NP 1.9 ± 0.38 4.8 ± 0.35 6.3 ± 1.73 0.9 ± 0.15 1.1 ± 0.38 3.5 ± 0.78 NP/HP 2.6 ± 0.82 4.9 ± 1.14 8.6 ± 4.33 1.7 ± 1.18 1.2 ± 0.30 4.2 ± 1.83 LP/feed-deprived 3.1 ± 0.59 4.3 ± 0.59 6.7 ± 2.33 1.1 ± 0.32 1.6 ± 0.64 4.0 ± 1.46 LP/LP 3.1 ± 0.94 4.6 ± 1.49 9.0 ± 5.54 1.7 ± 1.23 2.2 ± 0.68 4.2 ± 1.06 LP/NP 2.8 ± 0.63 4.8 ± 1.02 6.4 ± 1.16 1.4 ± 0.77 1.9 ± 0.91 5.7 ± 3.09 LP/HP 2.9 ± 0.45 4.1 ± 1.08 7.4 ± 2.85 1.5 ± 0.71 2.2 ± 0.79 4.9 ± 1.76 Diet effect, P 0.038 NS NS NS 0.024 NS Meal effect, P NS 0.0012 NS NS NS NS Diet × meal effect, P NS 0.0004 NS NS 0.02 NS Diet/meal groups Npy Pomc Agrp Cartpt Crh Mc4r NP/feed-deprived 2.8 ± 1.31 3.5 ± 0.54 6.4 ± 2.77 1.2 ± 1.48 1.5 ± 0.72 4.9 ± 1.84 NP/LP 2.7 ± 0.49 7.8 ± 2.47 9.4 ± 2.78 2.0 ± 1.25 2.2 ± 0.51 4.7 ± 0.62 NP/NP 1.9 ± 0.38 4.8 ± 0.35 6.3 ± 1.73 0.9 ± 0.15 1.1 ± 0.38 3.5 ± 0.78 NP/HP 2.6 ± 0.82 4.9 ± 1.14 8.6 ± 4.33 1.7 ± 1.18 1.2 ± 0.30 4.2 ± 1.83 LP/feed-deprived 3.1 ± 0.59 4.3 ± 0.59 6.7 ± 2.33 1.1 ± 0.32 1.6 ± 0.64 4.0 ± 1.46 LP/LP 3.1 ± 0.94 4.6 ± 1.49 9.0 ± 5.54 1.7 ± 1.23 2.2 ± 0.68 4.2 ± 1.06 LP/NP 2.8 ± 0.63 4.8 ± 1.02 6.4 ± 1.16 1.4 ± 0.77 1.9 ± 0.91 5.7 ± 3.09 LP/HP 2.9 ± 0.45 4.1 ± 1.08 7.4 ± 2.85 1.5 ± 0.71 2.2 ± 0.79 4.9 ± 1.76 Diet effect, P 0.038 NS NS NS 0.024 NS Meal effect, P NS 0.0012 NS NS NS NS Diet × meal effect, P NS 0.0004 NS NS 0.02 NS 1Values are mean ± SD arbitrary units, n = 6/group. Agrp, agouti-related peptide; Cartpt, prepropeptide cocaine- and amphetamine-regulated transcript; Crh, corticotropin-releasing hormone; HP, high-protein; LP, low-protein; Mc4r, melanocortin receptor; NP, normal-protein; Npy, neuropeptide Y; Pomc, pro-opiomelanocortin. View Large TABLE 4 Neuropeptide mRNA expression in the hypothalamus in the different rat groups 90 min after the meal (experiment 2a)1 Diet/meal groups Npy Pomc Agrp Cartpt Crh Mc4r NP/feed-deprived 2.8 ± 1.31 3.5 ± 0.54 6.4 ± 2.77 1.2 ± 1.48 1.5 ± 0.72 4.9 ± 1.84 NP/LP 2.7 ± 0.49 7.8 ± 2.47 9.4 ± 2.78 2.0 ± 1.25 2.2 ± 0.51 4.7 ± 0.62 NP/NP 1.9 ± 0.38 4.8 ± 0.35 6.3 ± 1.73 0.9 ± 0.15 1.1 ± 0.38 3.5 ± 0.78 NP/HP 2.6 ± 0.82 4.9 ± 1.14 8.6 ± 4.33 1.7 ± 1.18 1.2 ± 0.30 4.2 ± 1.83 LP/feed-deprived 3.1 ± 0.59 4.3 ± 0.59 6.7 ± 2.33 1.1 ± 0.32 1.6 ± 0.64 4.0 ± 1.46 LP/LP 3.1 ± 0.94 4.6 ± 1.49 9.0 ± 5.54 1.7 ± 1.23 2.2 ± 0.68 4.2 ± 1.06 LP/NP 2.8 ± 0.63 4.8 ± 1.02 6.4 ± 1.16 1.4 ± 0.77 1.9 ± 0.91 5.7 ± 3.09 LP/HP 2.9 ± 0.45 4.1 ± 1.08 7.4 ± 2.85 1.5 ± 0.71 2.2 ± 0.79 4.9 ± 1.76 Diet effect, P 0.038 NS NS NS 0.024 NS Meal effect, P NS 0.0012 NS NS NS NS Diet × meal effect, P NS 0.0004 NS NS 0.02 NS Diet/meal groups Npy Pomc Agrp Cartpt Crh Mc4r NP/feed-deprived 2.8 ± 1.31 3.5 ± 0.54 6.4 ± 2.77 1.2 ± 1.48 1.5 ± 0.72 4.9 ± 1.84 NP/LP 2.7 ± 0.49 7.8 ± 2.47 9.4 ± 2.78 2.0 ± 1.25 2.2 ± 0.51 4.7 ± 0.62 NP/NP 1.9 ± 0.38 4.8 ± 0.35 6.3 ± 1.73 0.9 ± 0.15 1.1 ± 0.38 3.5 ± 0.78 NP/HP 2.6 ± 0.82 4.9 ± 1.14 8.6 ± 4.33 1.7 ± 1.18 1.2 ± 0.30 4.2 ± 1.83 LP/feed-deprived 3.1 ± 0.59 4.3 ± 0.59 6.7 ± 2.33 1.1 ± 0.32 1.6 ± 0.64 4.0 ± 1.46 LP/LP 3.1 ± 0.94 4.6 ± 1.49 9.0 ± 5.54 1.7 ± 1.23 2.2 ± 0.68 4.2 ± 1.06 LP/NP 2.8 ± 0.63 4.8 ± 1.02 6.4 ± 1.16 1.4 ± 0.77 1.9 ± 0.91 5.7 ± 3.09 LP/HP 2.9 ± 0.45 4.1 ± 1.08 7.4 ± 2.85 1.5 ± 0.71 2.2 ± 0.79 4.9 ± 1.76 Diet effect, P 0.038 NS NS NS 0.024 NS Meal effect, P NS 0.0012 NS NS NS NS Diet × meal effect, P NS 0.0004 NS NS 0.02 NS 1Values are mean ± SD arbitrary units, n = 6/group. Agrp, agouti-related peptide; Cartpt, prepropeptide cocaine- and amphetamine-regulated transcript; Crh, corticotropin-releasing hormone; HP, high-protein; LP, low-protein; Mc4r, melanocortin receptor; NP, normal-protein; Npy, neuropeptide Y; Pomc, pro-opiomelanocortin. View Large The expression of mRNA coding for different neuronal receptors was also measured in the NAcc of rats from experiments 2a and 3 (Table 5). The expression of mRNA coding for dopamine and opioid receptors in the NAcc showed no difference between the LP and NP diets and there was no meal effect between the different rat groups from experiment 2a, except for a difference for μ opioid receptor (Oprm1) between NP/feed-deprived and NP/LP rats. In rats from experiment 3 adapted to an HP diet for 15 d and that received an HP meal (HP/HP group), 1) the dopamine receptor (Drd) 2 mRNA level in response to the meal was significantly higher (P < 0.05) compared with the NP/NP and the NPpfHP/NP groups, 2) the δ opioid receptor (Oprd1) mRNA level was significantly lower in response to the HP meal compared with the NPpfHP/NP group (P = 0.04) but was not different than that in the NP/NP group, and 3) the κ opioid receptor (Oprk1) mRNA level was significantly lower compared with the NP/NP group (P = 0.04), although not different than that in the NPpfHP/NP group. TABLE 5 Neuronal receptor mRNA expression in the NAcc in the different rat groups 90 min after the meal1 Diet/meal groups Drd2 Drd3 Oprd1 Oprk1 Oprm1 Experiment 2a  NP/feed-deprived 1.7 ± 0.36 1.8 ± 0.15 7.2 ± 0.59 2.7 ± 0.19 2.6 ± 0.34a  NP/LP 1.9 ± 0.31 1.7 ± 0.06 7.3 ± 0.91 3.5 ± 0.89 3.9 ± 0.49b  NP/NP 1.5 ± 0.15 1.6 ± 0.11 6.2 ± 0.69 2.7 ± 0.15 2.2 ± 0.18a  NP/HP 1.4 ± 0.31 2.2 ± 0.29 5.3 ± 1.09 2.7 ± 0.39 2.6 ± 0.48a  LP/feed-deprived 1.3 ± 0.17 1.7 ± 0.15 5.2 ± 0.59 2.6 ± 0.39 2.3 ± 0.15  LP/LP 1.4 ± 0.18 1.9 ± 0.22 5.3 ± 0.64 2.2 ± 0.32 2.6 ± 0.47  LP/NP 1.6 ± 0.26 1.7 ± 0.19 5.6 ± 0.63 2.7 ± 0.48 2.5 ± 0.34  LP/HP 1.7 ± 0.49 1.7 ± 0.09 5.3 ± 1.09 2.6 ± 0.27 2.4 ± 0.26  Diet effect, P NS NS NS NS NS  Meal effect, P NS NS NS NS NS  Diet × meal effect, P NS NS NS NS 0.05 Experiment 3  NP/NP 1.0 ± 0.34a 4.3 ± 1.40 0.2 ± 0.01a 4.2 ± 1.10b 0.9 ± 0.15  NPpfHP/NP 0.8 ± 0.28a 5.7 ± 1.40 0.4 ± 0.09b 2.3 ± 0.16a,b 0.8 ± 0.16  HP/HP 1.7 ± 0.07b 3.8 ± 0.87 0.2 ± 0.04a 1.6 ± 0.15a 0.9 ± 0.21  Diet effect, P 0.05 NS 0.04 0.04 NS Diet/meal groups Drd2 Drd3 Oprd1 Oprk1 Oprm1 Experiment 2a  NP/feed-deprived 1.7 ± 0.36 1.8 ± 0.15 7.2 ± 0.59 2.7 ± 0.19 2.6 ± 0.34a  NP/LP 1.9 ± 0.31 1.7 ± 0.06 7.3 ± 0.91 3.5 ± 0.89 3.9 ± 0.49b  NP/NP 1.5 ± 0.15 1.6 ± 0.11 6.2 ± 0.69 2.7 ± 0.15 2.2 ± 0.18a  NP/HP 1.4 ± 0.31 2.2 ± 0.29 5.3 ± 1.09 2.7 ± 0.39 2.6 ± 0.48a  LP/feed-deprived 1.3 ± 0.17 1.7 ± 0.15 5.2 ± 0.59 2.6 ± 0.39 2.3 ± 0.15  LP/LP 1.4 ± 0.18 1.9 ± 0.22 5.3 ± 0.64 2.2 ± 0.32 2.6 ± 0.47  LP/NP 1.6 ± 0.26 1.7 ± 0.19 5.6 ± 0.63 2.7 ± 0.48 2.5 ± 0.34  LP/HP 1.7 ± 0.49 1.7 ± 0.09 5.3 ± 1.09 2.6 ± 0.27 2.4 ± 0.26  Diet effect, P NS NS NS NS NS  Meal effect, P NS NS NS NS NS  Diet × meal effect, P NS NS NS NS 0.05 Experiment 3  NP/NP 1.0 ± 0.34a 4.3 ± 1.40 0.2 ± 0.01a 4.2 ± 1.10b 0.9 ± 0.15  NPpfHP/NP 0.8 ± 0.28a 5.7 ± 1.40 0.4 ± 0.09b 2.3 ± 0.16a,b 0.8 ± 0.16  HP/HP 1.7 ± 0.07b 3.8 ± 0.87 0.2 ± 0.04a 1.6 ± 0.15a 0.9 ± 0.21  Diet effect, P 0.05 NS 0.04 0.04 NS 1Values are mean ± SD arbitrary units, n = 6/group. Within an experiment, labeled means without a common letter differ, P < 0.05. Drd2, dopamine receptor 2; Drd3, dopamine receptor 3; HP, high-protein; LP, low-protein; NAcc, nucleus accumbens; NP, normal-protein; NPpfHP, normal-protein diet pair-fed for energy to the high-protein diet; Oprd1, δ opioid receptor; Oprk1, κ opioid receptor; Oprm1, μ opioid receptor. View Large TABLE 5 Neuronal receptor mRNA expression in the NAcc in the different rat groups 90 min after the meal1 Diet/meal groups Drd2 Drd3 Oprd1 Oprk1 Oprm1 Experiment 2a  NP/feed-deprived 1.7 ± 0.36 1.8 ± 0.15 7.2 ± 0.59 2.7 ± 0.19 2.6 ± 0.34a  NP/LP 1.9 ± 0.31 1.7 ± 0.06 7.3 ± 0.91 3.5 ± 0.89 3.9 ± 0.49b  NP/NP 1.5 ± 0.15 1.6 ± 0.11 6.2 ± 0.69 2.7 ± 0.15 2.2 ± 0.18a  NP/HP 1.4 ± 0.31 2.2 ± 0.29 5.3 ± 1.09 2.7 ± 0.39 2.6 ± 0.48a  LP/feed-deprived 1.3 ± 0.17 1.7 ± 0.15 5.2 ± 0.59 2.6 ± 0.39 2.3 ± 0.15  LP/LP 1.4 ± 0.18 1.9 ± 0.22 5.3 ± 0.64 2.2 ± 0.32 2.6 ± 0.47  LP/NP 1.6 ± 0.26 1.7 ± 0.19 5.6 ± 0.63 2.7 ± 0.48 2.5 ± 0.34  LP/HP 1.7 ± 0.49 1.7 ± 0.09 5.3 ± 1.09 2.6 ± 0.27 2.4 ± 0.26  Diet effect, P NS NS NS NS NS  Meal effect, P NS NS NS NS NS  Diet × meal effect, P NS NS NS NS 0.05 Experiment 3  NP/NP 1.0 ± 0.34a 4.3 ± 1.40 0.2 ± 0.01a 4.2 ± 1.10b 0.9 ± 0.15  NPpfHP/NP 0.8 ± 0.28a 5.7 ± 1.40 0.4 ± 0.09b 2.3 ± 0.16a,b 0.8 ± 0.16  HP/HP 1.7 ± 0.07b 3.8 ± 0.87 0.2 ± 0.04a 1.6 ± 0.15a 0.9 ± 0.21  Diet effect, P 0.05 NS 0.04 0.04 NS Diet/meal groups Drd2 Drd3 Oprd1 Oprk1 Oprm1 Experiment 2a  NP/feed-deprived 1.7 ± 0.36 1.8 ± 0.15 7.2 ± 0.59 2.7 ± 0.19 2.6 ± 0.34a  NP/LP 1.9 ± 0.31 1.7 ± 0.06 7.3 ± 0.91 3.5 ± 0.89 3.9 ± 0.49b  NP/NP 1.5 ± 0.15 1.6 ± 0.11 6.2 ± 0.69 2.7 ± 0.15 2.2 ± 0.18a  NP/HP 1.4 ± 0.31 2.2 ± 0.29 5.3 ± 1.09 2.7 ± 0.39 2.6 ± 0.48a  LP/feed-deprived 1.3 ± 0.17 1.7 ± 0.15 5.2 ± 0.59 2.6 ± 0.39 2.3 ± 0.15  LP/LP 1.4 ± 0.18 1.9 ± 0.22 5.3 ± 0.64 2.2 ± 0.32 2.6 ± 0.47  LP/NP 1.6 ± 0.26 1.7 ± 0.19 5.6 ± 0.63 2.7 ± 0.48 2.5 ± 0.34  LP/HP 1.7 ± 0.49 1.7 ± 0.09 5.3 ± 1.09 2.6 ± 0.27 2.4 ± 0.26  Diet effect, P NS NS NS NS NS  Meal effect, P NS NS NS NS NS  Diet × meal effect, P NS NS NS NS 0.05 Experiment 3  NP/NP 1.0 ± 0.34a 4.3 ± 1.40 0.2 ± 0.01a 4.2 ± 1.10b 0.9 ± 0.15  NPpfHP/NP 0.8 ± 0.28a 5.7 ± 1.40 0.4 ± 0.09b 2.3 ± 0.16a,b 0.8 ± 0.16  HP/HP 1.7 ± 0.07b 3.8 ± 0.87 0.2 ± 0.04a 1.6 ± 0.15a 0.9 ± 0.21  Diet effect, P 0.05 NS 0.04 0.04 NS 1Values are mean ± SD arbitrary units, n = 6/group. Within an experiment, labeled means without a common letter differ, P < 0.05. Drd2, dopamine receptor 2; Drd3, dopamine receptor 3; HP, high-protein; LP, low-protein; NAcc, nucleus accumbens; NP, normal-protein; NPpfHP, normal-protein diet pair-fed for energy to the high-protein diet; Oprd1, δ opioid receptor; Oprk1, κ opioid receptor; Oprm1, μ opioid receptor. View Large Plasma hormones, gut neuropeptides, TGs, and proteins Plasma hormones and gut neuropeptides were measured in the feed-deprived state and 90 min after the meal in rats from experiment 2. Rats fed the LP diet compared with those fed the NP diet showed significantly lower leptin concentrations in relation to their lower body fat mass and significantly higher fasting ghrelin, whereas ghrelin secretion was suppressed by the different meals (LP, NP, and HP) in both NP- and LP-fed rats compared with the feed-deprived condition (Figure 4). FIGURE 4 View largeDownload slide Plasma concentrations of leptin (A) and ghrelin (B) in rats fed for 15 d an NP or an LP diet on the last day in the feed-deprived state or 90 min after a calibrated NP, LP, or HP meal (experiment 2a; n = 24/group). Values are means ± SDs. Labeled means without a common letter differ, P < 0.05. HP, high-protein; LP, low-protein; NP, normal-protein. FIGURE 4 View largeDownload slide Plasma concentrations of leptin (A) and ghrelin (B) in rats fed for 15 d an NP or an LP diet on the last day in the feed-deprived state or 90 min after a calibrated NP, LP, or HP meal (experiment 2a; n = 24/group). Values are means ± SDs. Labeled means without a common letter differ, P < 0.05. HP, high-protein; LP, low-protein; NP, normal-protein. Plasma concentrations of gut neuropeptides, hormones, total protein, and TGs (Tables 6 and 7) were measured in rats from experiment 2a in the feed-deprived state or 90 min after the beginning of the different meals. Measurement with only 1 blood sampling at 90 min after the beginning of the 6-g meal, due to the experimental design, did not allow us to capture all of the potential differences in dynamics, although some differences could be observed between groups. Insulin was lower in the LP/feed-deprived group than in the NP/feed-deprived group. When measured 90 min after the meal, insulin was lower and glucagon was higher in the NP/HP group than in both NP/LP and NP/NP groups (Table 6). For GLP-1, a difference was found between NP/feed-deprived and NP/HP groups, although no difference in GLP-1 secretion could be observed for the other groups (Table 6). For both NP and LP diets, a significant diet and meal effect was observed for fed-state plasma GIP concentration (Table 6). As expected, no differences were found in plasma PYY concentrations (Table 7), plasma protein was higher in the NP- than in LP-diet group, and TG content in plasma was significantly lower in LP/feed-deprived rats (Table 7). TABLE 6 Plasma concentrations of insulin, glucagon, GLP-1, GIP, and PYY in the different rat groups in the feed-deprived state or 90 min after the meal (experiment 2a)1 Insulin, µg/L Glucagon, µg/L GIP, pg/mL GLP-1, pg/mL PYY, pg/mL Diet/meal groups  NP/feed-deprived 3.0 ± 0.10b 28.7 ± 3.00a 25 ± 1.9a 113 ± 25.3a ND  NP/LP 3.1 ± 0.20b 28.1 ± 1.30a 435 ± 27.4c 191 ± 25.5a,b 31.9 ± 4.50b  NP/NP 3.4 ± 0.40b 27.1 ± 4.00a 318 ± 29.9c 156 ± 29.4a,b 26 ± 5.30a,b  NP/HP 2.2 ± 0.20a 41.6 ± 7.80b 151 ± 27.3b 219 ± 26.0b 34.2 ± 5.60b  LP/feed-deprived 2.3 ± 0.40a 18.9 ± 1.70a 37 ± 4.9a 183 ± 13.2a,b ND  LP/LP 2.9 ± 0.50a,b 25.1 ± 1.20a 316 ± 29.8c 196 ± 7.8a,b 16.7 ± 2.00a  LP/NP 2.7 ± 0.20a,b 31.6 ± 2.20a,b 220 ± 29.9b.c 241 ± 16.2b 28.0 ± 4.50a,b  LP/HP 2.0 ± 0.60a 38.0 ± 7.00b 118 ± 29.5b 240 ± 16.5b 38.9 ± 2.90b Diet effect, P NS NS 0.036 0.008 NS Meal effect, P  LP meal/NP meal NS NS 0.0145 NS NS  NP meal/HP meal 0.0294 0.0107 0.0007 NS NS  HP meal/LP meal NS <0.0001 <0.0001 NS NS Insulin, µg/L Glucagon, µg/L GIP, pg/mL GLP-1, pg/mL PYY, pg/mL Diet/meal groups  NP/feed-deprived 3.0 ± 0.10b 28.7 ± 3.00a 25 ± 1.9a 113 ± 25.3a ND  NP/LP 3.1 ± 0.20b 28.1 ± 1.30a 435 ± 27.4c 191 ± 25.5a,b 31.9 ± 4.50b  NP/NP 3.4 ± 0.40b 27.1 ± 4.00a 318 ± 29.9c 156 ± 29.4a,b 26 ± 5.30a,b  NP/HP 2.2 ± 0.20a 41.6 ± 7.80b 151 ± 27.3b 219 ± 26.0b 34.2 ± 5.60b  LP/feed-deprived 2.3 ± 0.40a 18.9 ± 1.70a 37 ± 4.9a 183 ± 13.2a,b ND  LP/LP 2.9 ± 0.50a,b 25.1 ± 1.20a 316 ± 29.8c 196 ± 7.8a,b 16.7 ± 2.00a  LP/NP 2.7 ± 0.20a,b 31.6 ± 2.20a,b 220 ± 29.9b.c 241 ± 16.2b 28.0 ± 4.50a,b  LP/HP 2.0 ± 0.60a 38.0 ± 7.00b 118 ± 29.5b 240 ± 16.5b 38.9 ± 2.90b Diet effect, P NS NS 0.036 0.008 NS Meal effect, P  LP meal/NP meal NS NS 0.0145 NS NS  NP meal/HP meal 0.0294 0.0107 0.0007 NS NS  HP meal/LP meal NS <0.0001 <0.0001 NS NS 1Values are means ± SDs, n = 6. Labeled means without a common letter differ, P < 0.05. GIP, gastric inhibitory polypeptide; GLP-1, glucagon-like peptide 1; HP, high-protein; LP, low-protein; ND, not detected; NP, normal-protein: PYY, peptide YY. View Large TABLE 6 Plasma concentrations of insulin, glucagon, GLP-1, GIP, and PYY in the different rat groups in the feed-deprived state or 90 min after the meal (experiment 2a)1 Insulin, µg/L Glucagon, µg/L GIP, pg/mL GLP-1, pg/mL PYY, pg/mL Diet/meal groups  NP/feed-deprived 3.0 ± 0.10b 28.7 ± 3.00a 25 ± 1.9a 113 ± 25.3a ND  NP/LP 3.1 ± 0.20b 28.1 ± 1.30a 435 ± 27.4c 191 ± 25.5a,b 31.9 ± 4.50b  NP/NP 3.4 ± 0.40b 27.1 ± 4.00a 318 ± 29.9c 156 ± 29.4a,b 26 ± 5.30a,b  NP/HP 2.2 ± 0.20a 41.6 ± 7.80b 151 ± 27.3b 219 ± 26.0b 34.2 ± 5.60b  LP/feed-deprived 2.3 ± 0.40a 18.9 ± 1.70a 37 ± 4.9a 183 ± 13.2a,b ND  LP/LP 2.9 ± 0.50a,b 25.1 ± 1.20a 316 ± 29.8c 196 ± 7.8a,b 16.7 ± 2.00a  LP/NP 2.7 ± 0.20a,b 31.6 ± 2.20a,b 220 ± 29.9b.c 241 ± 16.2b 28.0 ± 4.50a,b  LP/HP 2.0 ± 0.60a 38.0 ± 7.00b 118 ± 29.5b 240 ± 16.5b 38.9 ± 2.90b Diet effect, P NS NS 0.036 0.008 NS Meal effect, P  LP meal/NP meal NS NS 0.0145 NS NS  NP meal/HP meal 0.0294 0.0107 0.0007 NS NS  HP meal/LP meal NS <0.0001 <0.0001 NS NS Insulin, µg/L Glucagon, µg/L GIP, pg/mL GLP-1, pg/mL PYY, pg/mL Diet/meal groups  NP/feed-deprived 3.0 ± 0.10b 28.7 ± 3.00a 25 ± 1.9a 113 ± 25.3a ND  NP/LP 3.1 ± 0.20b 28.1 ± 1.30a 435 ± 27.4c 191 ± 25.5a,b 31.9 ± 4.50b  NP/NP 3.4 ± 0.40b 27.1 ± 4.00a 318 ± 29.9c 156 ± 29.4a,b 26 ± 5.30a,b  NP/HP 2.2 ± 0.20a 41.6 ± 7.80b 151 ± 27.3b 219 ± 26.0b 34.2 ± 5.60b  LP/feed-deprived 2.3 ± 0.40a 18.9 ± 1.70a 37 ± 4.9a 183 ± 13.2a,b ND  LP/LP 2.9 ± 0.50a,b 25.1 ± 1.20a 316 ± 29.8c 196 ± 7.8a,b 16.7 ± 2.00a  LP/NP 2.7 ± 0.20a,b 31.6 ± 2.20a,b 220 ± 29.9b.c 241 ± 16.2b 28.0 ± 4.50a,b  LP/HP 2.0 ± 0.60a 38.0 ± 7.00b 118 ± 29.5b 240 ± 16.5b 38.9 ± 2.90b Diet effect, P NS NS 0.036 0.008 NS Meal effect, P  LP meal/NP meal NS NS 0.0145 NS NS  NP meal/HP meal 0.0294 0.0107 0.0007 NS NS  HP meal/LP meal NS <0.0001 <0.0001 NS NS 1Values are means ± SDs, n = 6. Labeled means without a common letter differ, P < 0.05. GIP, gastric inhibitory polypeptide; GLP-1, glucagon-like peptide 1; HP, high-protein; LP, low-protein; ND, not detected; NP, normal-protein: PYY, peptide YY. View Large TABLE 7 Plasma total protein and TG concentrations in rats adapted to NP or LP diets (experiment 2a)1 Diet/meal groups Plasma protein, g/L Plasma TGs, mmol/L NP/feed-deprived 53.2 ± 3.05b 1.2 ± 0.41b NP/LP 49.9 ± 2.31b 1.1 ± 0.35b NP/NP 50.1 ± 1.34b 1.5 ± 0.81b NP/HP 52.2 ± 2.40b 1.0 ± 0.45b LP/feed-deprived 43.9 ± 5.86a 0.5 ± 0.13a LP/LP 42.7 ± 5.35a 1.2 ± 0.54b LP/LP 41.2 ± 2.44a 1.2 ± 0.44b LP/HP 42.6 ± 7.85a 0.8 ± 0.15b Diet effect, P 0.05 0.0001 Combined groups  NP groups 51.3 ± 1.14 1.2 ± 0.11  LP groups 42.6 ± 1.11* 0.9 ± 0.09* Diet/meal groups Plasma protein, g/L Plasma TGs, mmol/L NP/feed-deprived 53.2 ± 3.05b 1.2 ± 0.41b NP/LP 49.9 ± 2.31b 1.1 ± 0.35b NP/NP 50.1 ± 1.34b 1.5 ± 0.81b NP/HP 52.2 ± 2.40b 1.0 ± 0.45b LP/feed-deprived 43.9 ± 5.86a 0.5 ± 0.13a LP/LP 42.7 ± 5.35a 1.2 ± 0.54b LP/LP 41.2 ± 2.44a 1.2 ± 0.44b LP/HP 42.6 ± 7.85a 0.8 ± 0.15b Diet effect, P 0.05 0.0001 Combined groups  NP groups 51.3 ± 1.14 1.2 ± 0.11  LP groups 42.6 ± 1.11* 0.9 ± 0.09* 1Values are means ± SDs, n = 6. Labeled means without a common letter differ, P < 0.05. *Different from NP, P < 0.5. HP, high-protein; LP, low-protein; NP, normal-protein. View Large TABLE 7 Plasma total protein and TG concentrations in rats adapted to NP or LP diets (experiment 2a)1 Diet/meal groups Plasma protein, g/L Plasma TGs, mmol/L NP/feed-deprived 53.2 ± 3.05b 1.2 ± 0.41b NP/LP 49.9 ± 2.31b 1.1 ± 0.35b NP/NP 50.1 ± 1.34b 1.5 ± 0.81b NP/HP 52.2 ± 2.40b 1.0 ± 0.45b LP/feed-deprived 43.9 ± 5.86a 0.5 ± 0.13a LP/LP 42.7 ± 5.35a 1.2 ± 0.54b LP/LP 41.2 ± 2.44a 1.2 ± 0.44b LP/HP 42.6 ± 7.85a 0.8 ± 0.15b Diet effect, P 0.05 0.0001 Combined groups  NP groups 51.3 ± 1.14 1.2 ± 0.11  LP groups 42.6 ± 1.11* 0.9 ± 0.09* Diet/meal groups Plasma protein, g/L Plasma TGs, mmol/L NP/feed-deprived 53.2 ± 3.05b 1.2 ± 0.41b NP/LP 49.9 ± 2.31b 1.1 ± 0.35b NP/NP 50.1 ± 1.34b 1.5 ± 0.81b NP/HP 52.2 ± 2.40b 1.0 ± 0.45b LP/feed-deprived 43.9 ± 5.86a 0.5 ± 0.13a LP/LP 42.7 ± 5.35a 1.2 ± 0.54b LP/LP 41.2 ± 2.44a 1.2 ± 0.44b LP/HP 42.6 ± 7.85a 0.8 ± 0.15b Diet effect, P 0.05 0.0001 Combined groups  NP groups 51.3 ± 1.14 1.2 ± 0.11  LP groups 42.6 ± 1.11* 0.9 ± 0.09* 1Values are means ± SDs, n = 6. Labeled means without a common letter differ, P < 0.05. *Different from NP, P < 0.5. HP, high-protein; LP, low-protein; NP, normal-protein. View Large Discussion This study evaluated in rats the relation between protein status, gut neuropeptides (ghrelin, GIP, GLP-1, and PYY), and metabolic hormones (leptin, insulin, and glucagon) involved in feeding control and markers of hypothalamus and NAcc activity, 2 key brain regions involved in food intake control and in reward system activation, respectively. Three different nutritional situations were explored over a period of 15 d, including an NP diet (control), an HP diet, and an LP diet. Feeds were prepared by an exchange between protein and carbohydrate with a constant level of 10% fat to keep energy density unchanged. Both NP and HP diets were associated with a protein-sufficient status, whereas rats fed the LP diet compared with those fed the NP diet did not show any difference in energy intake but had a protein-deficient status as evidenced by lower plasma protein concentration, body weight gain, fat mass, and lean mass. The results showed that both preferences for protein-containing foods and neuronal activation of NAcc by a protein-containing meal were sensitive to protein status. As indicated by c-Fos, NAcc activation was not different between LP, NP, or HP meals in a context of protein sufficiency (NP and HP diet), whereas it was significantly different between meals, with LP lower than NP lower than HP, in a context of protein deficiency (LP diet) and that correlated with a preference for protein-containing feed. This could be partly related to blood concentrations of protein, ghrelin, and leptin, as measured in this study, and of plasma free amino acids, which, although not determined in this study, were previously discussed among peripheral signals of protein deficiency (3–5, 24). It was previously shown that animals fed a protein-deficient diet try to increase their protein intake by a preference for protein-rich feeds or, under some conditions, by an increase in food intake. In the present study, the food-choice experiment confirmed a preference for protein-containing food in protein-deficient rats and the observed differences in preferences between protein-deficient and protein-sufficient rats suggest that appetite for protein induced by a protein-deficient status may take precedence over neophobia. In contrast, the present results in rats did not show an increase in food intake with the LP diet, as previously observed in mice fed an LP diet, which showed both increased energy intake and adiposity and also decreased food efficiency and increased energy expenditure to partly compensate for the increased energy intake (10). Energy expenditure was not measured in the present study, but the results indicated that because rats fed the LP diet had the same energy intake as NP-fed rats but lower body weight, lean, and fat mass, food efficiency was also lower, and energy expenditure should be higher, compared with NP rats, as observed in mice (10). The differences probably originate from species differences between rat and mice and indicate that rats are more resistant than mice to overconsumption induced by a protein-deficient diet, although the results of the choice experiment confirmed that, both in mice and rats the protein-deficient diet induced an appetite for protein (8, 9). An important result was that the appetite for protein induced in rats fed a protein-deficient diet correlated with the neuronal activation of the NAcc that was significantly and positively related to the protein content of the meal when rats were offered calibrated meals with different protein contents. In other words, the number of c-Fos–immunopositive neurons in the NAcc was low in protein-deficient rats offered an LP meal, significantly higher in rats offered an NP meal, and even higher in rats offered an HP meal. Interestingly, in LP rats compared with NP rats, the feed-deprived concentration of ghrelin was higher, the feed-deprived concentration of insulin was lower, and the feed-deprived and fed concentrations of leptin were lower. In addition, a higher expression of the orexigenic neuropeptide NPY and of the stress-related neuropeptide CRH was observed in the hypothalamus, which is in line with an overall increased motivation for food and for protein in protein-deficient animals (Figure 4, Table 3). This corresponds to the need to increase protein intake to compensate for protein deficiency in LP-fed animals. Indeed, orexigenic ghrelin notably increases food intake reward with central NPY and opioid signaling as the necessary mediators (25–27). In contrast, leptin contributes to decreasing the food reward by a modulation of dopamine-signaling pathways (28), and this process is downregulated in LP-fed animals characterized by lower fat tissue and lower blood leptin concentrations. These results concur with the idea that protein deficiency induces a specific appetite for protein, as observed both in animals (8, 13) and in humans (11–13). Although the present results do not allow us to identify which (sub)nuclei within the hypothalamus may be affected and suggest that the conclusions must be made carefully, these results agree with the assumption that protein deficiency induces an appetite for protein associated with a cascade of events from the periphery to the brain that could involve plasma free amino acids, enhanced feed-deprived ghrelin, lower leptin, increased hypothalamic Npy and Crh expressions, and augmented activation of the NAcc in response to protein intake. The results also showed that in LP rats, the HP meal compared with the LP meal did not further modify the expression of the genes controlling dopamine and opioid pathways in the NAcc, which play an important role in reward-seeking behavior and reward-related learning (29). In LP rats, pathways other than these transcriptional processes are involved in the control of dopamine activity in the NAcc in response to meals with different protein content. In addition, as previously observed, rats fed the HP diet compared with rats fed the NP diet had a lower daily energy intake in relation to higher satiety and lower body weight gain associated with lower fat mass without a difference in lean mass (3, 30–33). The results showed a significantly lower NAcc neuronal activation in response to the HP meal in rats previously fed an HP diet for 15 d (HP/HP group) in comparison with rats fed a control NP diet that received an NP meal (NP/NP group). This was also observed between HP/HP and NPpfHP/NP groups. The results also showed that, with an adequate protein status (i.e., NP rats), NAcc neuronal activation was not sensitive to the protein content of the meal as shown for NP/NP, NP/LP, and NP/HP rat groups. These observations confirmed a specific decrease of energy intake with the HP-content diet, which is in line with the reported satiating effect of protein (1, 14–18); and the present results further indicate that this is associated with a lower activation of the NAcc. The HP diet resulted in a specific activation of anorexigenic pathways associated with lower activation of the NAcc by the meal and a decreased motivation for food. The NAcc quantification of mRNA coding genes implicated in the reward system showed an increased expression of Drd2 mRNA in rats fed an HP diet compared with NP rats, which is associated with a deficit of addiction-like rewards (34). Overconsumption of palatable food in obese rats was shown to decrease Drd2 mRNA in relation to a state of reward hypofunction (34). The HP meal did not influence the expression of Drd3 receptors implicated in motivational processes in rats, but these receptors are not involved in appetitive value or motivation for food (35, 36). The results also showed differences in the expression of gene coding for Oprd1 and Oprk1, with a downregulation of Oprd1 expression after rat adaptation to the HP diet compared with the NPpfHP diet, but their role in modulating feeding is not fully understood. Although an increase in Oprd1 expression could result in decreased γ-aminobutyric acid release (37, 38) and might reduce food intake, it was also observed that an increase in Oprd1 expression corresponds to increased food consumption (39). Because there was no difference in Oprd1 expression between NP and HP diets, the results show an increased Oprd1 expression in hungry rats (NPpfHP) that could be related to food-seeking behaviors (40). These observations showed that protein status modifies the activity of the brain reward pathway that contributes to the control of food behavior. The observed effects between NP and LP diets on food choice and brain activities are related to the 50% reduction in protein content (14% to 6%, respectively) and not to carbohydrate, because both NP and LP feeds were very high in carbohydrate (76% and 84%, respectively), and this was confirmed in the choice experiment, which showed that rats fed the LP protein-deficient diet, in comparison to the NP diet, did not change the choice for the NP feed, which is high in carbohydrate, and only increased the HP feed at the expense of the LP feed. The status of protein insufficiency (LP diet) was characterized by high feed-deprived ghrelin concentrations, low postmeal leptin concentrations, high hypothalamic Npy and Crh expressions, high sensitivity of dopamine-dependent reward pathways in the NAcc, and high motivation for protein. Under such protein-deficient conditions, activation of NAcc becomes highly sensitive and positively related to the protein content of meals, with an increased motivation for protein when there is a need to increase protein intake to compensate for protein deficiency and reach an adequate protein status (Figure 5). In contrast, nutritional protein sufficiency induced low ghrelin, high leptin, and lower sensitivity of dopamine-dependent reward pathways. In contrast, under conditions of protein sufficiency, with either the NP or HP diets there was no specific motivation for protein, and homeostatic control relates mainly to energy control. This effect is even reinforced with a generous intake of protein, which could explain the slight decrease in energy intake observed with the HP diet compared with the NP diet, creating conditions for no motivation for protein-rich food with a strong inhibition of reward system reactivity. This is in line with the reported satiating effect of protein, and the present observations confirmed a specific decrease of energy intake with the HP diet, not related to the decreased carbohydrate content, because the NPpfHP/NP group did not reproduce the effect of the HP/HP group on NAcc activity, which was not different between the NPpfHP/NP and the NP/NP groups (Figure 3B). However, it is also important to highlight that the precise peripheral and metabolic signals involved in the control of protein intake are probably ubiquitous and redundant in relation to the complexity of amino acid and protein function in the body. The action of the brain reward pathways to control protein intake, prevent protein imbalance, and restore an adequate protein status involves many different metabolic signals induced by the protein content of the diet and is also sensitive to differential weight gain, growth rate, and other body functions related to protein metabolism. FIGURE 5 View largeDownload slide Protein deficiency is associated with a cascade of events from the periphery to the brain that involve enhanced feed-deprived ghrelin, lower leptin, enhanced hypothalamic NPY, and enhanced activity of the reward system in response to protein intake. CRH, corticotropin-releasing hormone; NPY, neuropeptide Y. FIGURE 5 View largeDownload slide Protein deficiency is associated with a cascade of events from the periphery to the brain that involve enhanced feed-deprived ghrelin, lower leptin, enhanced hypothalamic NPY, and enhanced activity of the reward system in response to protein intake. CRH, corticotropin-releasing hormone; NPY, neuropeptide Y. Acknowledgments The authors’ responsibilities were as follows—CC, IR, GF, ND, and DT: designed the research and wrote the manuscript; CC, IR, SB, and JP: conducted the research; CC, IR, SB, and DT: analyzed the data or performed statistical analysis; CC, IR, and DT: had primary responsibility for the final content; and all authors: read and approved the final manuscript. Notes Supported by public funding from AgroParisTech (Paris, France) and Institut National de la Recherche Agronomique (INRA, France). Author disclosures: CC, IR, GF, SB, JP, ND, and DT, no conflicts of interest. Present address for IR: Institute of Food Science Research, Ciencias de la Alimentación (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Madrid, Spain. Present address for SB: Université de Lorraine, Unité de Recherche Animal et Fonctionnalités des Produits Animaux, Vandœuvre-lès-Nancy, France. CC and IR contributed equally to this work. Supplemental Table 1 and Supplemental Figure 1 are available from the “Supplementary data” link in the online posting of the article and from the same link in the online table of contents at https://academic.oup.com/jn/. Abbreviations used: CRH, corticotropin-releasing hormone; DRD, dopamine receptor; GIP, gastric inhibitory polypeptide; GLP-1, glucagon-like peptide 1; HP, high-protein; LP, low-protein; MC4R, melanocortin receptor; NAcc, nucleus accumbens; NP, normal-protein; NPpfHP, normal-protein diet pair-fed for energy to the high-protein diet; NPY, neuropeptide Y; OPRD, δ opioid receptor; PYY, peptide YY. References 1. Journel M , Chaumontet C , Darcel N , Fromentin G , Tome D . Brain responses to high-protein diets . Adv Nutr 2012 ; 3 : 322 – 9 . Google Scholar CrossRef Search ADS PubMed 2. Davidenko O , Darcel N , Fromentin G , Tome D . Control of protein and energy intake—brain mechanisms . Eur J Clin Nutr 2013 ; 67 : 455 – 61 . Google Scholar CrossRef Search ADS PubMed 3. Harper AE , Peters JC . Protein intake, brain amino acid and serotonin concentrations and protein self-selection . J Nutr 1989 ; 119 : 677 – 89 . Google Scholar CrossRef Search ADS PubMed 4. Fromentin G , Nicolaidis S . Rebalancing essential amino acids intake by self-selection in the rat . Br J Nutr 1996 ; 75 : 669 – 82 . Google Scholar CrossRef Search ADS PubMed 5. Gietzen DW , Hao S , Anthony TG . Mechanisms of food intake repression in indispensable amino acid deficiency . Annu Rev Nutr 2007 ; 27 : 63 – 78 . Google Scholar CrossRef Search ADS PubMed 6. Booth DA . Food intake compensation for increase or decrease in the protein content of the diet . Behav Biol 1974 ; 12 : 31 – 40 . Google Scholar CrossRef Search ADS PubMed 7. Gibson EL , Booth DA . Acquired protein appetite in rats: dependence on a protein-specific need state . Experientia 1986 ; 42 : 1003 – 4 . Google Scholar CrossRef Search ADS PubMed 8. Du F , Higginbotham DA , White BD . Food intake, energy balance and serum leptin concentrations in rats fed low-protein diets . J Nutr 2000 ; 130 : 514 – 21 . Google Scholar CrossRef Search ADS PubMed 9. Beaton JR , Feleki V , Stevenson JA . Insulin hyperphagia in rats fed a low-protein diet . Can J Physiol Pharmacol 1965 ; 43 : 225 – 33 . Google Scholar CrossRef Search ADS PubMed 10. Blais A , Chaumontet C , Azzout-Marniche D , Piedcoq J , Fromentin G , Gaudichon C , Tome D , Even PC . Low protein diet-induced hyperphagia and adiposity are modulated through interactions involving thermoregulation, motor activity, and protein quality in mice . Am J Physiol Endocrinol Metab 2018 ; 314 ( 2 ): E139 – 51 . Google Scholar CrossRef Search ADS PubMed 11. Griffioen-Roose S , Mars M , Siebelink E , Finlayson G , Tome D , de Graaf C . Protein status elicits compensatory changes in food intake and food preferences . Am J Clin Nutr 2012 ; 95 : 32 – 8 . Google Scholar CrossRef Search ADS PubMed 12. Griffioen-Roose S , Smeets PA , van den Heuvel E , Boesveldt S , Finlayson G , de Graaf C . Human protein status modulates brain reward responses to food cues . Am J Clin Nutr 2014 ; 100 : 113 – 22 . Google Scholar CrossRef Search ADS PubMed 13. Gibson EL , Wainwright CJ , Booth DA . Disguised protein in lunch after low-protein breakfast conditions food-flavor preferences dependent on recent lack of protein intake . Physiol Behav 1995 ; 58 : 363 – 71 . Google Scholar CrossRef Search ADS PubMed 14. Westerterp-Plantenga MS , Nieuwenhuizen A , Tome D , Soenen S , Westerterp KR . Dietary protein, weight loss, and weight maintenance . Annu Rev Nutr 2009 ; 29 : 21 – 41 . Google Scholar CrossRef Search ADS PubMed 15. McArthur LH , Kelly WF , Gietzen DW , Rogers QR . The role of palatability in the food intake response of rats fed high-protein diets . Appetite 1993 ; 20 : 181 – 96 . Google Scholar CrossRef Search ADS PubMed 16. L'Heureux-Bouron D , Tome D , Bensaid A , Morens C , Lacroix M , Huneau JF , Fromentin G . Preabsorptive factors are not the main determinants of intake depression induced by a high-protein diet in the rat . Physiol Behav 2004 ; 81 : 499 – 504 . Google Scholar CrossRef Search ADS PubMed 17. Morrison CD , Reed SD , Henagan TM . Homeostatic regulation of protein intake: in search of a mechanism . Am J Physiol Regul Integr Comp Physiol 2012 ; 302 : R917 – 28 . Google Scholar CrossRef Search ADS PubMed 18. Fromentin G , Darcel N , Chaumontet C , Marsset-Baglieri A , Nadkarni N , Tome D . Peripheral and central mechanisms involved in the control of food intake by dietary amino acids and proteins . Nutr Res Rev 2012 ; 25 : 29 – 39 . Google Scholar CrossRef Search ADS PubMed 19. Fulton S . Appetite and reward . Front Neuroendocrinol 2010 ; 31 : 85 – 103 . Google Scholar CrossRef Search ADS PubMed 20. Faipoux R , Tome D , Bensaid A , Morens C , Oriol E , Bonnano LM , Fromentin G . Yeast proteins enhance satiety in rats . J Nutr 2006 ; 136 : 2350 – 6 . Google Scholar CrossRef Search ADS PubMed 21. Reeves PG , Nielsen FH , Fahey GC Jr . AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet . J Nutr 1993 ; 123 : 1939 – 51 . Google Scholar CrossRef Search ADS PubMed 22. Paxinos G , Franklin KBJ . The Mouse Brain in Stereotaxic Coordinates . (Elsevier, Ed.) (Fourth Ed.) . Amsterdam : Academic Press , 1998 . 23. Chaumontet C , Even PC , Schwarz J , Simonin-Foucault A , Piedcoq J , Fromentin G , Azzout-Marniche D , Tome D . High dietary protein decreases fat deposition induced by high-fat and high-sucrose diet in rats . Br J Nutr 2015 ; 114 : 1132 – 42 . Google Scholar CrossRef Search ADS PubMed 24. Rogers QR , Leung PM . The influence of amino acids on the neuroregulation of food intake . Fed Proc 1973 ; 32 : 1709 – 19 . Google Scholar PubMed 25. White BD , He B , Dean RG , Martin RJ . Low protein diets increase neuropeptide Y gene expression in the basomedial hypothalamus of rats . J Nutr 1994 ; 124 : 1152 – 60 . Google Scholar CrossRef Search ADS PubMed 26. Skibicka KP , Dickson SL . Ghrelin and food reward: the story of potential underlying substrates . Peptides 2011 ; 32 : 2265 – 73 . Google Scholar CrossRef Search ADS PubMed 27. Dickson SL , Egecioglu E , Landgren S , Skibicka KP , Engel JA , Jerlhag E . The role of the central ghrelin system in reward from food and chemical drugs . Mol Cell Endocrinol 2011 ; 340 : 80 – 7 . Google Scholar CrossRef Search ADS PubMed 28. Pfaffly J , Michaelides M , Wang GJ , Pessin JE , Volkow ND , Thanos PK . Leptin increases striatal dopamine D2 receptor binding in leptin-deficient obese (ob/ob) mice . Synapse 2010 ; 64 : 503 – 10 . Google Scholar CrossRef Search ADS PubMed 29. Arias-Carrion O , Poppel E . Dopamine, learning, and reward-seeking behavior . Acta Neurobiol Exp (Wars) 2007 ; 67 : 481 – 8 . Google Scholar PubMed 30. Jean C , Rome S , Mathe V , Huneau JF , Aattouri N , Fromentin G , Achagiotis CL , Tome D . Metabolic evidence for adaptation to a high protein diet in rats . J Nutr 2001 ; 131 : 91 – 8 . Google Scholar CrossRef Search ADS PubMed 31. Bensaid A , Tome D , L'Heureux-Bourdon D , Even P , Gietzen D , Morens C , Gaudichon C , Larue-Achagiotis C , Fromentin G . A high-protein diet enhances satiety without conditioned taste aversion in the rat . Physiol Behav 2003 ; 78 : 311 – 20 . Google Scholar CrossRef Search ADS PubMed 32. Darcel N , Fromentin G , Raybould HE , Gougis S , Gietzen DW , Tome D . Fos-positive neurons are increased in the nucleus of the solitary tract and decreased in the ventromedial hypothalamus and amygdala by a high-protein diet in rats . J Nutr 2005 ; 135 : 1486 – 90 . Google Scholar CrossRef Search ADS PubMed 33. Faipoux R , Tome D , Gougis S , Darcel N , Fromentin G . Proteins activate satiety-related neuronal pathways in the brainstem and hypothalamus of rats . J Nutr 2008 ; 138 : 1172 – 8 . Google Scholar CrossRef Search ADS PubMed 34. Johnson PM , Kenny PJ . Dopamine D2 receptors in addiction-like reward dysfunction and compulsive eating in obese rats . Nat Neurosci 2010 ; 13 : 635 – 41 . Google Scholar CrossRef Search ADS PubMed 35. Duarte C , Biala G , Le Bihan C , Hamon M , Thiebot MH . Respective roles of dopamine D2 and D3 receptors in food-seeking behaviour in rats . Psychopharmacology 2003 ; 166 : 19 – 32 . Google Scholar CrossRef Search ADS PubMed 36. Le Foll B , Goldberg SR , Sokoloff P . The dopamine D3 receptor and drug dependence: effects on reward or beyond? Neuropharmacology 2005 ; 49 : 525 – 41 . Google Scholar CrossRef Search ADS PubMed 37. Margolis EB , Fields HL , Hjelmstad GO , Mitchell JM . Delta-opioid receptor expression in the ventral tegmental area protects against elevated alcohol consumption . J Neurosci 2008 ; 28 : 12672 – 81 . Google Scholar CrossRef Search ADS PubMed 38. Hack SP , Bagley EE , Chieng BC , Christie MJ . Induction of delta-opioid receptor function in the midbrain after chronic morphine treatment . J Neurosci 2005 ; 25 : 3192 – 8 . Google Scholar CrossRef Search ADS PubMed 39. Zhang M , Kelley AE . Opiate agonists microinjected into the nucleus accumbens enhance sucrose drinking in rats . Psychopharmacology 1997 ; 132 : 350 – 60 . Google Scholar CrossRef Search ADS PubMed 40. Katsuura Y , Taha SA . Modulation of feeding and locomotion through mu and delta opioid receptor signaling in the nucleus accumbens . Neuropeptides 2010 ; 44 : 225 – 32 . Google Scholar CrossRef Search ADS PubMed © 2018 American Society for Nutrition. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Nutrition Oxford University Press

The Protein Status of Rats Affects the Rewarding Value of Meals Due to their Protein Content

Loading next page...
 
/lp/ou_press/the-protein-status-of-rats-affects-the-rewarding-value-of-meals-due-to-0RALibq0EE
Publisher
American Society for Nutrition
Copyright
© 2018 American Society for Nutrition.
ISSN
0022-3166
eISSN
1541-6100
D.O.I.
10.1093/jn/nxy060
Publisher site
See Article on Publisher Site

Abstract

Abstract Background Protein status is controlled by the brain, which modulates feeding behavior to prevent protein deficiency. Objective This study tested in rats whether protein status modulates feeding behavior through brain reward pathways. Methods Experiments were conducted in male Wistar rats (mean ± SD weight; 230 ± 16 g). In experiment 1, rats adapted for 2 wk to a low-protein (LP; 6% of energy) or a normal-protein (NP; 14% of energy) diet were offered a choice between 3 cups containing high-protein (HP; 50% of energy), NP, or LP feed; their intake was measured for 24 h. In 2 other experiments, the rats were adapted for 2 wk to NP and either HP or LP diets and received, after overnight feed deprivation, a calibrated HP, NP, or LP meal daily. After the meal, on the last day, rats were killed and body composition and blood protein, triglycerides, gut neuropeptides, and hormones were determined. In the brain, neuropeptide mRNAs in the hypothalamus and c-Fos protein and opioid and dopaminergic receptor mRNAs in the nucleus accumbens (NAcc) were measured. Results Rats fed an LP compared with an NP diet had 7% lower body weight, significantly higher protein intake in a choice experiment (mean ± SD: 30.5% ± 0.05% compared with 20.5% ± 0.05% of energy), higher feed-deprived blood ghrelin, lower postmeal blood leptin, and higher neuropeptide Y (Npy) and corticotropin-releasing hormone (Crh) mRNA expression in the hypothalamus. In contrast to NP, rats fed an LP diet showed postmeal c-Fos protein expression in the NAcc, which was significantly different between meals, with LP < NP < HP. In contrast, in rats adapted to an HP diet compared with an NP diet, energy intake was lower; and in the NAcc, meal-induced c-Fos protein expression was 20% lower, and mRNA expression was 17% higher for dopamine receptor 2 (Drd2) receptors and 38% lower for κ opioid receptor (Oprk1) receptors. Conclusion A protein-restricted diet induced a reward system–driven appetite for protein, whereas a protein-rich diet reduced the meal-induced activation of reward pathways and lowered energy intake in male rats. protein, satiety, reward, accumbens nucleus, food intake Introduction The maintenance of normal physiologic functions and survival in animals and humans requires a continuous supply of amino acids to tissues to support protein synthesis and other amino acid–dependent metabolic processes. These phenomena depend on a daily intake of an adequate quantity of protein from a diverse array of foods. Protein status, characterized by amino acid sufficiency in the body to support metabolic functions, is tightly and continuously controlled and modulates food motivation, food choice, or food aversion to prevent or counteract protein deficiency (1, 2). Animals learn to detect and avoid very-low-protein diets, protein-deficient diets, or indispensable amino acid–deficient diets (3–5). After food or protein deprivation, a specific appetite for protein and a preference for protein-rich foods have been observed in rodents (6, 7). Animals offered a marginally protein-deficient diet tend to meet their target protein needs with different strategies, including food choice and, under some conditions, a slight increase in food intake (8–10). There have also been observations of a higher appetite for protein-rich foods after protein-restricted diets in humans (11, 12), as well as a preference for foods associated with a protein-rich flavor after a low-protein preload (13). In contrast, high-protein diets are usually reported to decrease food intake in both animals and humans, related to a reported satiating effect of proteins through activation of anorexigenic pathways (1, 14–18). The present study, which used a rat model, aimed to evaluate how protein status (characterized as generous, normal, or deficient) influences the activation of brain regions associated with the control of food intake and reward system, the hypothalamus and the nucleus accumbens (NAcc), respectively, in association with the protein content of a single meal. It is hypothesized that, under conditions of protein deficiency, the activation of the NAcc is positively related to the meal protein content to restore protein status. Indeed, many studies have shown the importance of NAcc, a component of the ventral striatum in basal ganglia circuits, in the reward system. This system is involved in actions that prioritize behavior and promote the continuation of ongoing actions that increase behaviors leading to the procurement and consumption of the reward (positive reinforcement) and in direct future behavioral actions (19). In a first experiment, rats were adapted to either a normal-protein (NP) or low-protein (LP) diet and were then offered a choice between 3 feeds containing NP, high-protein (HP), or LP contents. In 2 other experiments, after habituation to NP, HP, or LP diets, rats were offered, before being killed, a calibrated meal of LP, NP, or HP feeds. Rats were characterized by measuring blood concentrations of protein, TGs, gut neuropeptides [ghrelin, gastric inhibitory polypeptide (GIP), glucagon-like peptide 1 (GLP-1), and peptide YY (PYY)], and metabolic hormones (leptin, insulin, and glucagon). Brain neuropeptide [neuropeptide Y (NPY), proopiomelanocortin, agouti-related peptide, cocaine- and amphetamine-regulated transcript], corticotropin-releasing hormone (CRH), and melanocortin receptor (MC4R) mRNA expressions were determined in the hypothalamus. Total neuronal activation measured by the detection of c-Fos–positive nuclei and changes in mRNA expression of opioid and dopaminergic receptors were determined in the NAcc. Methods Animals and diets Adult male Wistar rats (Envigo) with initial mean ± SD weight of 230 ± 16 g were housed in individual cages at 22 ± 2°C under a 12-h reverse light-dark cycle (lights on at 2100). All of the experimental procedures were approved by the Regional Animal Care and Ethical Committee (approval no. 12/173) and conformed to the European legislation on the use of laboratory animals. Feeds were a modified version of the AIN-93M diet (Table 1) (20, 21). All feeds were moistened with a 1:1 ratio of powder to water for HP and a 1.5:1 ratio for NP and LP feeds to achieve pellets of sufficient consistency and to minimize spillage. Food intake was determined by the difference in food-cup weight before and after each experimental period, corrected for spillage, the amount of added water, and evaporation. All rats had free access to water throughout the experimental period. All of the rats were acclimated for 1 wk to the housing conditions before starting the feeding conditions. TABLE 1 Composition of HP, NP, and LP feeds1 HP NP LP Protein, % of energy 55 14 6 Carbohydrates, % of energy 35 76 84 Fat, % of energy 10 10 10 Milk protein, g/kg 530 140 60 Corn starch, g/kg 287 622 691 Sucrose, g/kg 46 100 111 Soybean oil, g/kg 40 40 40 Salts and minerals, g/kg 35 35 35 Vitamins, g/kg 10 10 10 α-Cellulose, g/kg 50 50 50 Choline, g/kg 2.3 2.3 2.3 HP NP LP Protein, % of energy 55 14 6 Carbohydrates, % of energy 35 76 84 Fat, % of energy 10 10 10 Milk protein, g/kg 530 140 60 Corn starch, g/kg 287 622 691 Sucrose, g/kg 46 100 111 Soybean oil, g/kg 40 40 40 Salts and minerals, g/kg 35 35 35 Vitamins, g/kg 10 10 10 α-Cellulose, g/kg 50 50 50 Choline, g/kg 2.3 2.3 2.3 1Feeds were a modified version of the AIN-93M (20, 21). HP, high-protein; LP, low-protein; NP, normal-protein. View Large TABLE 1 Composition of HP, NP, and LP feeds1 HP NP LP Protein, % of energy 55 14 6 Carbohydrates, % of energy 35 76 84 Fat, % of energy 10 10 10 Milk protein, g/kg 530 140 60 Corn starch, g/kg 287 622 691 Sucrose, g/kg 46 100 111 Soybean oil, g/kg 40 40 40 Salts and minerals, g/kg 35 35 35 Vitamins, g/kg 10 10 10 α-Cellulose, g/kg 50 50 50 Choline, g/kg 2.3 2.3 2.3 HP NP LP Protein, % of energy 55 14 6 Carbohydrates, % of energy 35 76 84 Fat, % of energy 10 10 10 Milk protein, g/kg 530 140 60 Corn starch, g/kg 287 622 691 Sucrose, g/kg 46 100 111 Soybean oil, g/kg 40 40 40 Salts and minerals, g/kg 35 35 35 Vitamins, g/kg 10 10 10 α-Cellulose, g/kg 50 50 50 Choline, g/kg 2.3 2.3 2.3 1Feeds were a modified version of the AIN-93M (20, 21). HP, high-protein; LP, low-protein; NP, normal-protein. View Large Experimental design Rat groups were characterized by diet (e.g., HP, NP, or LP) and, when appropriate, the experimental meal (HP, NP, or LP) expressed as diet/meal (e.g., NP/HP describes rats adapted to the NP diet that received an HP meal). Three experiments were performed. Experiment 1 (food choice in NP- compared with LP-diet–fed rats) After acclimation, rats (n = 16) were divided into 2 groups (n = 8/group) with ad libitum access to either the NP or the LP feed for 2 wk. On the last day, rats were offered a choice between 3 cups containing the HP, NP, or LP feed, and the intake of each feed was measured for 24 h. Experiment 2 (NP compared with LP diets) In experiment 2a, after 7 d of acclimation, rats (n = 48) were divided into 2 groups (n = 24/group) with ad libitum access for 1 wk to either the NP or LP feed during their dark period (Figure 1). Each group was acclimated during the third week to a daily feeding pattern consisting of 30-min access from 0900 to 0930 to a 6-g (43.8-kJ) calibrated NP or LP meal, and then from 1100 to 1800 to ad libitum access to the same NP or LP feed, resulting in the diet/meal NP/NP and LP/LP groups, respectively. On the last day, each group (NP/NP and LP/LP) was divided into 4 subgroups (n = 6/group), which each received a 6-g calibrated LP, NP, or HP feed meal or no meal, resulting in the diet/meal NP/LP, NP/NP, NP/HP, or NP/feed-deprived (15-h feed-deprived) and LP/LP, LP/NP, LP/HP, or LP/feed-deprived (15-h feed-deprived) groups, respectively. RT-PCR analysis of receptors in the hypothalamus and NAcc, tissue and blood sampling, and body-composition measurements were performed. FIGURE 1 View largeDownload slide Experimental design of experiment 2. After 1 wk of acclimation to laboratory animal housing conditions and 1 wk of diet acclimation, rats were accustomed to receiving their food according to a pattern that consisted of a calibrated meal of 6 g (43.8 kJ) for 30 min between 0900 and 0930, free access to food between 1100 and 1800, and feed deprivation between 1800 and 0900 the next day. FIGURE 1 View largeDownload slide Experimental design of experiment 2. After 1 wk of acclimation to laboratory animal housing conditions and 1 wk of diet acclimation, rats were accustomed to receiving their food according to a pattern that consisted of a calibrated meal of 6 g (43.8 kJ) for 30 min between 0900 and 0930, free access to food between 1100 and 1800, and feed deprivation between 1800 and 0900 the next day. In experiment 2b, the same experimental design was followed for immunofluorescence detection of c-Fos protein expression in the NAcc, but with 24 rats divided into 2 groups (n = 12/group; NP/NP and LP/LP) and subsequently divided into 3 subgroups (n = 4/subgroup; NP/LP, NP/NP, NP/HP and LP/LP, LP/NP, LP/HP); the feed-deprived subgroup was not considered. Experiment 3 (NP compared with HP diets) After 7 d of acclimation, rats (n = 18) were divided into 3 groups (n = 6/group). Rats had ad libitum access during their dark period to the NP or the HP feed, or the NP-feed diet pair-fed for energy to the HP-feed diet (NPpfHP), for 2 wk. During the last week, after overnight feed deprivation, rats received a 6-g (43.8 kJ) calibrated NP- or HP-feed meal (NP/NP, HP/HP, and NPpfHP/NP groups) for 30 min from 0900 to 0930. On the last day, the rats were killed 90 min after the beginning of the calibrated meal. Rats were then treated for RT-PCR analysis of receptors in the NAcc. The same experimental design, but with 4 rats per group (n = 12), was followed for immunofluorescence detection of c-Fos protein expression in the NAcc. In experiments 2 and 3, rats trained for 1 wk to receive between 0900 and 0930, after overnight feed deprivation, a 6-g calibrated meal consumed the entire test meals. Immunofluorescence detection of c-Fos protein expression in the NAcc Rats from experiment 2b were killed at 1100 with a lethal intraperitoneal injection of pentobarbital sodium (100 mg/kg; Ceva Santé Animale) 90 min after the beginning of their experimental meal. A detailed description of brain preparations was previously described (20). Twenty-micrometer sections were obtained with the use of a cryostat (CM1520; Leica) in the region of the NAcc (+2.7 to +0.48-mm bregma; every fifth slice was collected), identified by using the Paxinos and Watson stereotaxic atlas (22) (The Rat Brain in Stereotaxic Coordinates, fourth edition, Paxinos & Watson). Slices were mounted on microscope slides SuperFrost+ slides (Dutscher) and stored at –80°C. Slices were treated as previously described (20) with some modifications: we incubated slices with rabbit anti-c-Fos antibody (primary antibody, 1:5000, Ab-5; Calbiochem) for 48 h at 4°C. Slices were then rinsed 3 times for 10 min in PBS solution and then incubated in the PBS/Triton X-100/BSA solution containing Alexa-Fluor 488 (secondary antibody, 1:200; Molecular Probes) for 2 h at room temperature. Finally, slices were washed 3 times in PBS solution and mounted by using a medium containing 4′,6-diamidino-2-phenylindole (Vector Laboratories). The NAcc was identified by using a stereotaxic atlas, and the number of c-Fos immunopositive cells was evaluated in a minimum of 4 consecutive slices per rat. The mean labeling of c-Fos–immunopositive cells was calculated for each rat, and the mean number of labels determined for each rat group. RT-PCR analysis of receptors in the hypothalamus and NAcc Rats from experiment 2a were anesthetized 90 min after receiving the experimental meal with an intraperitoneal injection of sodium pentobarbital (50 mg/kg; Ceva Santé Animale) and then decapitated. The brain was harvested, and the hypothalamus was immediately extracted from the fresh brain by making an incision medial to the piriform lobes caudal to the optic chiasma and anterior to the cerebral crus to a depth of 2 ± 3 mm and was placed directly in TRIzol reagent (Invitrogen), frozen in liquid nitrogen, and stored at –80°C. The brain was then placed in sterile 0.1-M PBS solution and kept at –20°C for 30 min. A 4- to 6-mm slice of brain (measured with square calipers) was cut from the beginning of the olfactory bulb (+2.70 to +0.48-mm bregma). A piece of this slice containing the NAcc area was removed with a scalpel, immediately placed in 1 mL Trizol (Invitrogen) and stored at –80°C. Total RNA was extracted from the hypothalamus and NAcc samples by homogenization as previously described (23). Sequences of primers are given in Supplemental Table 1. Gene expression was determined by using the 2−ΔCt formula, where ΔCt = Ct target gene Ct 18S. Tissue sampling, body composition, and blood analysis Rats from experiment 2a were anesthetized 90 min after ingestion of the calibrated meal, if applicable, with an intraperitoneal injection of sodium pentobarbital (50 mg/kg; Ceva Santé Animale) and decapitated. Tissues and organs (liver, spleen, kidneys, brain, heart, skin, subcutaneous, retroperitoneal, epididymal, and mesenteric adipose tissues) were collected, blotted dry, and weighed to the nearest 0.01 g. Blood samples were collected on EDTA and immediately centrifuged (1500 × g, 4°C), and plasma aliquots were prepared for biochemical or hormonal analysis, after the addition of protease inhibitor (Roche), and stored at −70°C until being assayed. All of the plasma biochemical analyses (albumin, lactate, cholesterol, HDL cholesterol, TGs, FFAs, glycerol, and β-hydroxybutyrate) were performed on an Olympus AU 400 robot specifically calibrated for rat assays (Centre d'Explorations Fonctionnelles Intégrées). Plasma insulin was detected by using enzyme-linked immunoassays (Mercodia Rat Insulin). Plasma glucagon, leptin, GIP, GLP-1, and ghrelin concentrations were assessed by Luminex (Bio-Plex Pro rat standard; Bio-Rad). Blood glucose was determined immediately after sampling with a blood glucose meter (Accu Check Go; Roche). Liver TG concentrations were determined by using an enzymatic assay (TG–TIGS; Randox). Statistical analysis Data are presented as means ± SDs. Statistical analyses were performed by using SAS (version 9.1), and a generalized linear model was applied. The effects of the diets and the meals were tested by 2-factor ANOVA with interaction; and when diet, meal, or diet × meal was significant, pairwise comparisons between rat groups characterized by diet or diet and meal (diet/meal) were performed with post hoc Tukey test for multiple comparisons. Differences were considered significant at P < 0.05. Results Food intake, body weight and composition, and food choice In experiment 1, rats adapted for 2 wk to the NP or the LP diet were subjected during 24 h to a choice between 3 cups with LP, NP, or HP feed (Figure 2, Table 2). Rats previously fed the NP diet showed a significant preference for the NP meal compared with the HP meal, with no choice difference between LP and HP meals. However, rats previously habituated to the LP diet and offered the same range of meals showed no choice difference between the NP and HP meals but had a significant preference for both the NP and HP meals compared with the LP meal. The combination of choices between the LP, NP, and HP meals led to the same total energy intake for rats previously adapted to the LP diet compared with the NP diet (Table 2). Indeed, rats fed an LP diet consumed, during the choice experiment, a significantly higher amount of energy as protein and a significantly lower amount of energy as carbohydrate, whereas no difference was observed in the amount of energy from fat because fat content was the same in the 3 feeds. FIGURE 2 View largeDownload slide Twenty-four-hour food choice between LP, NP, and HP feeds in rats previously adapted for 15 d to an NP or an LP diet (experiment 1; n = 8/group). Values are means ± SDs. Labeled means without a common letter differ, P < 0.05. HP, high-protein; LP, low-protein; NP, normal-protein. FIGURE 2 View largeDownload slide Twenty-four-hour food choice between LP, NP, and HP feeds in rats previously adapted for 15 d to an NP or an LP diet (experiment 1; n = 8/group). Values are means ± SDs. Labeled means without a common letter differ, P < 0.05. HP, high-protein; LP, low-protein; NP, normal-protein. TABLE 2 Energy intake and macronutrient composition of the diet, during the 24-h food choice between LP, NP, and HP feeds in rats previously adapted for 15 d to the NP or the LP diet (experiment 1)1 15-d diet adaptation NP diet LP diet Diet effect, P Total energy intake during the 24-h choice, kJ/100 g body weight 106.0 ± 16.65 97.1 ± 12.00 NS Macronutrient composition of the 24-h choice, % of energy  Protein2 20.5 ± 0.05a 30.5 ± 0.05b 0.02  Carbohydrate2 69.5 ± 0.06b 59.5 ± 0.06a 0.02  Fat3 10.0 ± 0.00 10.0 ± 0.00 15-d diet adaptation NP diet LP diet Diet effect, P Total energy intake during the 24-h choice, kJ/100 g body weight 106.0 ± 16.65 97.1 ± 12.00 NS Macronutrient composition of the 24-h choice, % of energy  Protein2 20.5 ± 0.05a 30.5 ± 0.05b 0.02  Carbohydrate2 69.5 ± 0.06b 59.5 ± 0.06a 0.02  Fat3 10.0 ± 0.00 10.0 ± 0.00 1Values are means ± SDs, n = 8/group. Labeled means without a common letter differ, P < 0.05. LP, low-protein; NP, normal-protein. 2Calculated from the macronutrient composition of feeds (Table 1) and the 24-h intake of each of the 3 feeds during the choice experiment. 3The 3 feeds had the same 10% of energy from fat content. View Large TABLE 2 Energy intake and macronutrient composition of the diet, during the 24-h food choice between LP, NP, and HP feeds in rats previously adapted for 15 d to the NP or the LP diet (experiment 1)1 15-d diet adaptation NP diet LP diet Diet effect, P Total energy intake during the 24-h choice, kJ/100 g body weight 106.0 ± 16.65 97.1 ± 12.00 NS Macronutrient composition of the 24-h choice, % of energy  Protein2 20.5 ± 0.05a 30.5 ± 0.05b 0.02  Carbohydrate2 69.5 ± 0.06b 59.5 ± 0.06a 0.02  Fat3 10.0 ± 0.00 10.0 ± 0.00 15-d diet adaptation NP diet LP diet Diet effect, P Total energy intake during the 24-h choice, kJ/100 g body weight 106.0 ± 16.65 97.1 ± 12.00 NS Macronutrient composition of the 24-h choice, % of energy  Protein2 20.5 ± 0.05a 30.5 ± 0.05b 0.02  Carbohydrate2 69.5 ± 0.06b 59.5 ± 0.06a 0.02  Fat3 10.0 ± 0.00 10.0 ± 0.00 1Values are means ± SDs, n = 8/group. Labeled means without a common letter differ, P < 0.05. LP, low-protein; NP, normal-protein. 2Calculated from the macronutrient composition of feeds (Table 1) and the 24-h intake of each of the 3 feeds during the choice experiment. 3The 3 feeds had the same 10% of energy from fat content. View Large In experiments 2a and 2b, rats fed the LP-diet feeding pattern for 2 wk showed no significant difference in daily energy intake compared with those fed the NP diet, but they had lower body weight gain and lower lean and fat mass than rats in the NP diet group (Table 3). In addition, in experiment 3, rats fed an HP diet for 2 wk had a lower daily energy intake and lower body weight gain than those fed an NP diet, whereas their body weight gain was not different than that of NPpfHP diet group (Table 3). TABLE 3 Daily energy intake, body weight gain, and final fat and lean mass in the different rat groups adapted to LP, NP, or HP diets for 2 wk1 Diet groups n/Group Energy intake, kJ/d Body weight gain, g/d Final fat mass, g Final lean mass, g Experiment 2a  NP 24 335 ± 36.8 4.1 ± 0.64b 35.8 ± 3.72b 265 ± 7.0b  LP 24 305 ± 33.4 0.8 ± 0.15a 23.1 ± 2.36a 200 ± 4.3a  Diet effect, P NS 0.0001 0.005 0.0002 Experiment 2b  NP 12 308 ± 18.9 3.0 ± 0.59b — —  LP 12 310 ± 32.2 1.1 ± 0.83a — —  Diet effect, P NS 0.001 Experiment 3  NP 6 381 ± 24.8b 6.5 ± 0.46b — —  HP 6 312 ± 23.4a 5.4 ± 0.29a — —  NPpfHP 6 302 ± 24.8a 5.4 ± 0.23a — —  Diet effect, P 0.003 0.03 Diet groups n/Group Energy intake, kJ/d Body weight gain, g/d Final fat mass, g Final lean mass, g Experiment 2a  NP 24 335 ± 36.8 4.1 ± 0.64b 35.8 ± 3.72b 265 ± 7.0b  LP 24 305 ± 33.4 0.8 ± 0.15a 23.1 ± 2.36a 200 ± 4.3a  Diet effect, P NS 0.0001 0.005 0.0002 Experiment 2b  NP 12 308 ± 18.9 3.0 ± 0.59b — —  LP 12 310 ± 32.2 1.1 ± 0.83a — —  Diet effect, P NS 0.001 Experiment 3  NP 6 381 ± 24.8b 6.5 ± 0.46b — —  HP 6 312 ± 23.4a 5.4 ± 0.29a — —  NPpfHP 6 302 ± 24.8a 5.4 ± 0.23a — —  Diet effect, P 0.003 0.03 1Values are means ± SDs. Within an experiment, labeled means without a common letter differ, P < 0.05. HP, high-protein. LP, low-protein; NP, normal-protein; NPpfHP, normal-protein diet pair-fed for energy to the high-protein diet. View Large TABLE 3 Daily energy intake, body weight gain, and final fat and lean mass in the different rat groups adapted to LP, NP, or HP diets for 2 wk1 Diet groups n/Group Energy intake, kJ/d Body weight gain, g/d Final fat mass, g Final lean mass, g Experiment 2a  NP 24 335 ± 36.8 4.1 ± 0.64b 35.8 ± 3.72b 265 ± 7.0b  LP 24 305 ± 33.4 0.8 ± 0.15a 23.1 ± 2.36a 200 ± 4.3a  Diet effect, P NS 0.0001 0.005 0.0002 Experiment 2b  NP 12 308 ± 18.9 3.0 ± 0.59b — —  LP 12 310 ± 32.2 1.1 ± 0.83a — —  Diet effect, P NS 0.001 Experiment 3  NP 6 381 ± 24.8b 6.5 ± 0.46b — —  HP 6 312 ± 23.4a 5.4 ± 0.29a — —  NPpfHP 6 302 ± 24.8a 5.4 ± 0.23a — —  Diet effect, P 0.003 0.03 Diet groups n/Group Energy intake, kJ/d Body weight gain, g/d Final fat mass, g Final lean mass, g Experiment 2a  NP 24 335 ± 36.8 4.1 ± 0.64b 35.8 ± 3.72b 265 ± 7.0b  LP 24 305 ± 33.4 0.8 ± 0.15a 23.1 ± 2.36a 200 ± 4.3a  Diet effect, P NS 0.0001 0.005 0.0002 Experiment 2b  NP 12 308 ± 18.9 3.0 ± 0.59b — —  LP 12 310 ± 32.2 1.1 ± 0.83a — —  Diet effect, P NS 0.001 Experiment 3  NP 6 381 ± 24.8b 6.5 ± 0.46b — —  HP 6 312 ± 23.4a 5.4 ± 0.29a — —  NPpfHP 6 302 ± 24.8a 5.4 ± 0.23a — —  Diet effect, P 0.003 0.03 1Values are means ± SDs. Within an experiment, labeled means without a common letter differ, P < 0.05. HP, high-protein. LP, low-protein; NP, normal-protein; NPpfHP, normal-protein diet pair-fed for energy to the high-protein diet. View Large Neuronal activation in the NAcc measured by c-Fos immunolabeling Neural activation was determined by measuring c-Fos protein 90 min after receiving the calibrated 6-g meal in rats on the last day from experiment 2b (NP compared with LP diet) and experiment 3 (NP compared with HP diet) (Figure 3). FIGURE 3 View largeDownload slide c-Fos–positive neurons in the NAcc 90 min after the beginning of the 6-g calibrated meal in rats. (A) Rats fed for 15 d an NP or an LP diet received on the last day an LP, NP, or HP meal (experiment 2b; n = 12/group). (B) Rats fed for 15 d a diet/meal feeding pattern of NP/NP, NPpfHP/NP, or HP/HP (experiment 3; n = 6/group). Values are means ± SDs. Within an experiment, labeled means without a common letter differ, P < 0.05. HP, high-protein; LP, low-protein; NP, normal-protein; NPpfHP, normal-protein diet pair-fed for energy to the high-protein diet. FIGURE 3 View largeDownload slide c-Fos–positive neurons in the NAcc 90 min after the beginning of the 6-g calibrated meal in rats. (A) Rats fed for 15 d an NP or an LP diet received on the last day an LP, NP, or HP meal (experiment 2b; n = 12/group). (B) Rats fed for 15 d a diet/meal feeding pattern of NP/NP, NPpfHP/NP, or HP/HP (experiment 3; n = 6/group). Values are means ± SDs. Within an experiment, labeled means without a common letter differ, P < 0.05. HP, high-protein; LP, low-protein; NP, normal-protein; NPpfHP, normal-protein diet pair-fed for energy to the high-protein diet. In experiment 2b, in rats fed an adequate protein-content diet (NP) there was no difference in NAcc activation in rats that received an LP, NP, or HP meal (NP/LP, NP/NP, or NP/HP groups) (Figure 3A, Supplemental Figure 1). However, in the protein-deficient group (LP diet), NAcc neural activation was significantly different between meals, with LP/LP lower than LP/NP lower than LP/HP, and a lower NAcc activation was observed in LP/LP rats than in all other rat groups. In contrast, as reported in Figure 3B (experiment 3), there was a lower number of c-Fos–positive neurons in response to an HP meal in rats habituated to an HP diet for 2 wk (HP/HP) in comparison to the response to an NP meal in rats habituated to either an NP diet (NP/NP; P = 0.03) or to an NPpfHP diet (NPpfHP/NP; P < 0.05), with no difference between the NP/NP and the NPpfHP/NP groups (P = 0.06). Neuropeptide and neuronal receptor mRNA expression in the hypothalamus and in the NAcc In rats from experiment 2a, the mRNA expression of different neuropeptides and MC4R was measured in the hypothalamus in NP and LP rat groups in the feed-deprived state or 90 min after the NP, LP, or HP meal (Table 4). There was an overall significant diet effect for hypothalamic Npy and Crh expression, which was significantly higher in LP rats compared with NP-diet–fed rats, whereas no meal effect could be observed. No difference in the expression of other neuropeptides or MC4R was found between LP and NP diets, and no meal effect between the different groups was observed. TABLE 4 Neuropeptide mRNA expression in the hypothalamus in the different rat groups 90 min after the meal (experiment 2a)1 Diet/meal groups Npy Pomc Agrp Cartpt Crh Mc4r NP/feed-deprived 2.8 ± 1.31 3.5 ± 0.54 6.4 ± 2.77 1.2 ± 1.48 1.5 ± 0.72 4.9 ± 1.84 NP/LP 2.7 ± 0.49 7.8 ± 2.47 9.4 ± 2.78 2.0 ± 1.25 2.2 ± 0.51 4.7 ± 0.62 NP/NP 1.9 ± 0.38 4.8 ± 0.35 6.3 ± 1.73 0.9 ± 0.15 1.1 ± 0.38 3.5 ± 0.78 NP/HP 2.6 ± 0.82 4.9 ± 1.14 8.6 ± 4.33 1.7 ± 1.18 1.2 ± 0.30 4.2 ± 1.83 LP/feed-deprived 3.1 ± 0.59 4.3 ± 0.59 6.7 ± 2.33 1.1 ± 0.32 1.6 ± 0.64 4.0 ± 1.46 LP/LP 3.1 ± 0.94 4.6 ± 1.49 9.0 ± 5.54 1.7 ± 1.23 2.2 ± 0.68 4.2 ± 1.06 LP/NP 2.8 ± 0.63 4.8 ± 1.02 6.4 ± 1.16 1.4 ± 0.77 1.9 ± 0.91 5.7 ± 3.09 LP/HP 2.9 ± 0.45 4.1 ± 1.08 7.4 ± 2.85 1.5 ± 0.71 2.2 ± 0.79 4.9 ± 1.76 Diet effect, P 0.038 NS NS NS 0.024 NS Meal effect, P NS 0.0012 NS NS NS NS Diet × meal effect, P NS 0.0004 NS NS 0.02 NS Diet/meal groups Npy Pomc Agrp Cartpt Crh Mc4r NP/feed-deprived 2.8 ± 1.31 3.5 ± 0.54 6.4 ± 2.77 1.2 ± 1.48 1.5 ± 0.72 4.9 ± 1.84 NP/LP 2.7 ± 0.49 7.8 ± 2.47 9.4 ± 2.78 2.0 ± 1.25 2.2 ± 0.51 4.7 ± 0.62 NP/NP 1.9 ± 0.38 4.8 ± 0.35 6.3 ± 1.73 0.9 ± 0.15 1.1 ± 0.38 3.5 ± 0.78 NP/HP 2.6 ± 0.82 4.9 ± 1.14 8.6 ± 4.33 1.7 ± 1.18 1.2 ± 0.30 4.2 ± 1.83 LP/feed-deprived 3.1 ± 0.59 4.3 ± 0.59 6.7 ± 2.33 1.1 ± 0.32 1.6 ± 0.64 4.0 ± 1.46 LP/LP 3.1 ± 0.94 4.6 ± 1.49 9.0 ± 5.54 1.7 ± 1.23 2.2 ± 0.68 4.2 ± 1.06 LP/NP 2.8 ± 0.63 4.8 ± 1.02 6.4 ± 1.16 1.4 ± 0.77 1.9 ± 0.91 5.7 ± 3.09 LP/HP 2.9 ± 0.45 4.1 ± 1.08 7.4 ± 2.85 1.5 ± 0.71 2.2 ± 0.79 4.9 ± 1.76 Diet effect, P 0.038 NS NS NS 0.024 NS Meal effect, P NS 0.0012 NS NS NS NS Diet × meal effect, P NS 0.0004 NS NS 0.02 NS 1Values are mean ± SD arbitrary units, n = 6/group. Agrp, agouti-related peptide; Cartpt, prepropeptide cocaine- and amphetamine-regulated transcript; Crh, corticotropin-releasing hormone; HP, high-protein; LP, low-protein; Mc4r, melanocortin receptor; NP, normal-protein; Npy, neuropeptide Y; Pomc, pro-opiomelanocortin. View Large TABLE 4 Neuropeptide mRNA expression in the hypothalamus in the different rat groups 90 min after the meal (experiment 2a)1 Diet/meal groups Npy Pomc Agrp Cartpt Crh Mc4r NP/feed-deprived 2.8 ± 1.31 3.5 ± 0.54 6.4 ± 2.77 1.2 ± 1.48 1.5 ± 0.72 4.9 ± 1.84 NP/LP 2.7 ± 0.49 7.8 ± 2.47 9.4 ± 2.78 2.0 ± 1.25 2.2 ± 0.51 4.7 ± 0.62 NP/NP 1.9 ± 0.38 4.8 ± 0.35 6.3 ± 1.73 0.9 ± 0.15 1.1 ± 0.38 3.5 ± 0.78 NP/HP 2.6 ± 0.82 4.9 ± 1.14 8.6 ± 4.33 1.7 ± 1.18 1.2 ± 0.30 4.2 ± 1.83 LP/feed-deprived 3.1 ± 0.59 4.3 ± 0.59 6.7 ± 2.33 1.1 ± 0.32 1.6 ± 0.64 4.0 ± 1.46 LP/LP 3.1 ± 0.94 4.6 ± 1.49 9.0 ± 5.54 1.7 ± 1.23 2.2 ± 0.68 4.2 ± 1.06 LP/NP 2.8 ± 0.63 4.8 ± 1.02 6.4 ± 1.16 1.4 ± 0.77 1.9 ± 0.91 5.7 ± 3.09 LP/HP 2.9 ± 0.45 4.1 ± 1.08 7.4 ± 2.85 1.5 ± 0.71 2.2 ± 0.79 4.9 ± 1.76 Diet effect, P 0.038 NS NS NS 0.024 NS Meal effect, P NS 0.0012 NS NS NS NS Diet × meal effect, P NS 0.0004 NS NS 0.02 NS Diet/meal groups Npy Pomc Agrp Cartpt Crh Mc4r NP/feed-deprived 2.8 ± 1.31 3.5 ± 0.54 6.4 ± 2.77 1.2 ± 1.48 1.5 ± 0.72 4.9 ± 1.84 NP/LP 2.7 ± 0.49 7.8 ± 2.47 9.4 ± 2.78 2.0 ± 1.25 2.2 ± 0.51 4.7 ± 0.62 NP/NP 1.9 ± 0.38 4.8 ± 0.35 6.3 ± 1.73 0.9 ± 0.15 1.1 ± 0.38 3.5 ± 0.78 NP/HP 2.6 ± 0.82 4.9 ± 1.14 8.6 ± 4.33 1.7 ± 1.18 1.2 ± 0.30 4.2 ± 1.83 LP/feed-deprived 3.1 ± 0.59 4.3 ± 0.59 6.7 ± 2.33 1.1 ± 0.32 1.6 ± 0.64 4.0 ± 1.46 LP/LP 3.1 ± 0.94 4.6 ± 1.49 9.0 ± 5.54 1.7 ± 1.23 2.2 ± 0.68 4.2 ± 1.06 LP/NP 2.8 ± 0.63 4.8 ± 1.02 6.4 ± 1.16 1.4 ± 0.77 1.9 ± 0.91 5.7 ± 3.09 LP/HP 2.9 ± 0.45 4.1 ± 1.08 7.4 ± 2.85 1.5 ± 0.71 2.2 ± 0.79 4.9 ± 1.76 Diet effect, P 0.038 NS NS NS 0.024 NS Meal effect, P NS 0.0012 NS NS NS NS Diet × meal effect, P NS 0.0004 NS NS 0.02 NS 1Values are mean ± SD arbitrary units, n = 6/group. Agrp, agouti-related peptide; Cartpt, prepropeptide cocaine- and amphetamine-regulated transcript; Crh, corticotropin-releasing hormone; HP, high-protein; LP, low-protein; Mc4r, melanocortin receptor; NP, normal-protein; Npy, neuropeptide Y; Pomc, pro-opiomelanocortin. View Large The expression of mRNA coding for different neuronal receptors was also measured in the NAcc of rats from experiments 2a and 3 (Table 5). The expression of mRNA coding for dopamine and opioid receptors in the NAcc showed no difference between the LP and NP diets and there was no meal effect between the different rat groups from experiment 2a, except for a difference for μ opioid receptor (Oprm1) between NP/feed-deprived and NP/LP rats. In rats from experiment 3 adapted to an HP diet for 15 d and that received an HP meal (HP/HP group), 1) the dopamine receptor (Drd) 2 mRNA level in response to the meal was significantly higher (P < 0.05) compared with the NP/NP and the NPpfHP/NP groups, 2) the δ opioid receptor (Oprd1) mRNA level was significantly lower in response to the HP meal compared with the NPpfHP/NP group (P = 0.04) but was not different than that in the NP/NP group, and 3) the κ opioid receptor (Oprk1) mRNA level was significantly lower compared with the NP/NP group (P = 0.04), although not different than that in the NPpfHP/NP group. TABLE 5 Neuronal receptor mRNA expression in the NAcc in the different rat groups 90 min after the meal1 Diet/meal groups Drd2 Drd3 Oprd1 Oprk1 Oprm1 Experiment 2a  NP/feed-deprived 1.7 ± 0.36 1.8 ± 0.15 7.2 ± 0.59 2.7 ± 0.19 2.6 ± 0.34a  NP/LP 1.9 ± 0.31 1.7 ± 0.06 7.3 ± 0.91 3.5 ± 0.89 3.9 ± 0.49b  NP/NP 1.5 ± 0.15 1.6 ± 0.11 6.2 ± 0.69 2.7 ± 0.15 2.2 ± 0.18a  NP/HP 1.4 ± 0.31 2.2 ± 0.29 5.3 ± 1.09 2.7 ± 0.39 2.6 ± 0.48a  LP/feed-deprived 1.3 ± 0.17 1.7 ± 0.15 5.2 ± 0.59 2.6 ± 0.39 2.3 ± 0.15  LP/LP 1.4 ± 0.18 1.9 ± 0.22 5.3 ± 0.64 2.2 ± 0.32 2.6 ± 0.47  LP/NP 1.6 ± 0.26 1.7 ± 0.19 5.6 ± 0.63 2.7 ± 0.48 2.5 ± 0.34  LP/HP 1.7 ± 0.49 1.7 ± 0.09 5.3 ± 1.09 2.6 ± 0.27 2.4 ± 0.26  Diet effect, P NS NS NS NS NS  Meal effect, P NS NS NS NS NS  Diet × meal effect, P NS NS NS NS 0.05 Experiment 3  NP/NP 1.0 ± 0.34a 4.3 ± 1.40 0.2 ± 0.01a 4.2 ± 1.10b 0.9 ± 0.15  NPpfHP/NP 0.8 ± 0.28a 5.7 ± 1.40 0.4 ± 0.09b 2.3 ± 0.16a,b 0.8 ± 0.16  HP/HP 1.7 ± 0.07b 3.8 ± 0.87 0.2 ± 0.04a 1.6 ± 0.15a 0.9 ± 0.21  Diet effect, P 0.05 NS 0.04 0.04 NS Diet/meal groups Drd2 Drd3 Oprd1 Oprk1 Oprm1 Experiment 2a  NP/feed-deprived 1.7 ± 0.36 1.8 ± 0.15 7.2 ± 0.59 2.7 ± 0.19 2.6 ± 0.34a  NP/LP 1.9 ± 0.31 1.7 ± 0.06 7.3 ± 0.91 3.5 ± 0.89 3.9 ± 0.49b  NP/NP 1.5 ± 0.15 1.6 ± 0.11 6.2 ± 0.69 2.7 ± 0.15 2.2 ± 0.18a  NP/HP 1.4 ± 0.31 2.2 ± 0.29 5.3 ± 1.09 2.7 ± 0.39 2.6 ± 0.48a  LP/feed-deprived 1.3 ± 0.17 1.7 ± 0.15 5.2 ± 0.59 2.6 ± 0.39 2.3 ± 0.15  LP/LP 1.4 ± 0.18 1.9 ± 0.22 5.3 ± 0.64 2.2 ± 0.32 2.6 ± 0.47  LP/NP 1.6 ± 0.26 1.7 ± 0.19 5.6 ± 0.63 2.7 ± 0.48 2.5 ± 0.34  LP/HP 1.7 ± 0.49 1.7 ± 0.09 5.3 ± 1.09 2.6 ± 0.27 2.4 ± 0.26  Diet effect, P NS NS NS NS NS  Meal effect, P NS NS NS NS NS  Diet × meal effect, P NS NS NS NS 0.05 Experiment 3  NP/NP 1.0 ± 0.34a 4.3 ± 1.40 0.2 ± 0.01a 4.2 ± 1.10b 0.9 ± 0.15  NPpfHP/NP 0.8 ± 0.28a 5.7 ± 1.40 0.4 ± 0.09b 2.3 ± 0.16a,b 0.8 ± 0.16  HP/HP 1.7 ± 0.07b 3.8 ± 0.87 0.2 ± 0.04a 1.6 ± 0.15a 0.9 ± 0.21  Diet effect, P 0.05 NS 0.04 0.04 NS 1Values are mean ± SD arbitrary units, n = 6/group. Within an experiment, labeled means without a common letter differ, P < 0.05. Drd2, dopamine receptor 2; Drd3, dopamine receptor 3; HP, high-protein; LP, low-protein; NAcc, nucleus accumbens; NP, normal-protein; NPpfHP, normal-protein diet pair-fed for energy to the high-protein diet; Oprd1, δ opioid receptor; Oprk1, κ opioid receptor; Oprm1, μ opioid receptor. View Large TABLE 5 Neuronal receptor mRNA expression in the NAcc in the different rat groups 90 min after the meal1 Diet/meal groups Drd2 Drd3 Oprd1 Oprk1 Oprm1 Experiment 2a  NP/feed-deprived 1.7 ± 0.36 1.8 ± 0.15 7.2 ± 0.59 2.7 ± 0.19 2.6 ± 0.34a  NP/LP 1.9 ± 0.31 1.7 ± 0.06 7.3 ± 0.91 3.5 ± 0.89 3.9 ± 0.49b  NP/NP 1.5 ± 0.15 1.6 ± 0.11 6.2 ± 0.69 2.7 ± 0.15 2.2 ± 0.18a  NP/HP 1.4 ± 0.31 2.2 ± 0.29 5.3 ± 1.09 2.7 ± 0.39 2.6 ± 0.48a  LP/feed-deprived 1.3 ± 0.17 1.7 ± 0.15 5.2 ± 0.59 2.6 ± 0.39 2.3 ± 0.15  LP/LP 1.4 ± 0.18 1.9 ± 0.22 5.3 ± 0.64 2.2 ± 0.32 2.6 ± 0.47  LP/NP 1.6 ± 0.26 1.7 ± 0.19 5.6 ± 0.63 2.7 ± 0.48 2.5 ± 0.34  LP/HP 1.7 ± 0.49 1.7 ± 0.09 5.3 ± 1.09 2.6 ± 0.27 2.4 ± 0.26  Diet effect, P NS NS NS NS NS  Meal effect, P NS NS NS NS NS  Diet × meal effect, P NS NS NS NS 0.05 Experiment 3  NP/NP 1.0 ± 0.34a 4.3 ± 1.40 0.2 ± 0.01a 4.2 ± 1.10b 0.9 ± 0.15  NPpfHP/NP 0.8 ± 0.28a 5.7 ± 1.40 0.4 ± 0.09b 2.3 ± 0.16a,b 0.8 ± 0.16  HP/HP 1.7 ± 0.07b 3.8 ± 0.87 0.2 ± 0.04a 1.6 ± 0.15a 0.9 ± 0.21  Diet effect, P 0.05 NS 0.04 0.04 NS Diet/meal groups Drd2 Drd3 Oprd1 Oprk1 Oprm1 Experiment 2a  NP/feed-deprived 1.7 ± 0.36 1.8 ± 0.15 7.2 ± 0.59 2.7 ± 0.19 2.6 ± 0.34a  NP/LP 1.9 ± 0.31 1.7 ± 0.06 7.3 ± 0.91 3.5 ± 0.89 3.9 ± 0.49b  NP/NP 1.5 ± 0.15 1.6 ± 0.11 6.2 ± 0.69 2.7 ± 0.15 2.2 ± 0.18a  NP/HP 1.4 ± 0.31 2.2 ± 0.29 5.3 ± 1.09 2.7 ± 0.39 2.6 ± 0.48a  LP/feed-deprived 1.3 ± 0.17 1.7 ± 0.15 5.2 ± 0.59 2.6 ± 0.39 2.3 ± 0.15  LP/LP 1.4 ± 0.18 1.9 ± 0.22 5.3 ± 0.64 2.2 ± 0.32 2.6 ± 0.47  LP/NP 1.6 ± 0.26 1.7 ± 0.19 5.6 ± 0.63 2.7 ± 0.48 2.5 ± 0.34  LP/HP 1.7 ± 0.49 1.7 ± 0.09 5.3 ± 1.09 2.6 ± 0.27 2.4 ± 0.26  Diet effect, P NS NS NS NS NS  Meal effect, P NS NS NS NS NS  Diet × meal effect, P NS NS NS NS 0.05 Experiment 3  NP/NP 1.0 ± 0.34a 4.3 ± 1.40 0.2 ± 0.01a 4.2 ± 1.10b 0.9 ± 0.15  NPpfHP/NP 0.8 ± 0.28a 5.7 ± 1.40 0.4 ± 0.09b 2.3 ± 0.16a,b 0.8 ± 0.16  HP/HP 1.7 ± 0.07b 3.8 ± 0.87 0.2 ± 0.04a 1.6 ± 0.15a 0.9 ± 0.21  Diet effect, P 0.05 NS 0.04 0.04 NS 1Values are mean ± SD arbitrary units, n = 6/group. Within an experiment, labeled means without a common letter differ, P < 0.05. Drd2, dopamine receptor 2; Drd3, dopamine receptor 3; HP, high-protein; LP, low-protein; NAcc, nucleus accumbens; NP, normal-protein; NPpfHP, normal-protein diet pair-fed for energy to the high-protein diet; Oprd1, δ opioid receptor; Oprk1, κ opioid receptor; Oprm1, μ opioid receptor. View Large Plasma hormones, gut neuropeptides, TGs, and proteins Plasma hormones and gut neuropeptides were measured in the feed-deprived state and 90 min after the meal in rats from experiment 2. Rats fed the LP diet compared with those fed the NP diet showed significantly lower leptin concentrations in relation to their lower body fat mass and significantly higher fasting ghrelin, whereas ghrelin secretion was suppressed by the different meals (LP, NP, and HP) in both NP- and LP-fed rats compared with the feed-deprived condition (Figure 4). FIGURE 4 View largeDownload slide Plasma concentrations of leptin (A) and ghrelin (B) in rats fed for 15 d an NP or an LP diet on the last day in the feed-deprived state or 90 min after a calibrated NP, LP, or HP meal (experiment 2a; n = 24/group). Values are means ± SDs. Labeled means without a common letter differ, P < 0.05. HP, high-protein; LP, low-protein; NP, normal-protein. FIGURE 4 View largeDownload slide Plasma concentrations of leptin (A) and ghrelin (B) in rats fed for 15 d an NP or an LP diet on the last day in the feed-deprived state or 90 min after a calibrated NP, LP, or HP meal (experiment 2a; n = 24/group). Values are means ± SDs. Labeled means without a common letter differ, P < 0.05. HP, high-protein; LP, low-protein; NP, normal-protein. Plasma concentrations of gut neuropeptides, hormones, total protein, and TGs (Tables 6 and 7) were measured in rats from experiment 2a in the feed-deprived state or 90 min after the beginning of the different meals. Measurement with only 1 blood sampling at 90 min after the beginning of the 6-g meal, due to the experimental design, did not allow us to capture all of the potential differences in dynamics, although some differences could be observed between groups. Insulin was lower in the LP/feed-deprived group than in the NP/feed-deprived group. When measured 90 min after the meal, insulin was lower and glucagon was higher in the NP/HP group than in both NP/LP and NP/NP groups (Table 6). For GLP-1, a difference was found between NP/feed-deprived and NP/HP groups, although no difference in GLP-1 secretion could be observed for the other groups (Table 6). For both NP and LP diets, a significant diet and meal effect was observed for fed-state plasma GIP concentration (Table 6). As expected, no differences were found in plasma PYY concentrations (Table 7), plasma protein was higher in the NP- than in LP-diet group, and TG content in plasma was significantly lower in LP/feed-deprived rats (Table 7). TABLE 6 Plasma concentrations of insulin, glucagon, GLP-1, GIP, and PYY in the different rat groups in the feed-deprived state or 90 min after the meal (experiment 2a)1 Insulin, µg/L Glucagon, µg/L GIP, pg/mL GLP-1, pg/mL PYY, pg/mL Diet/meal groups  NP/feed-deprived 3.0 ± 0.10b 28.7 ± 3.00a 25 ± 1.9a 113 ± 25.3a ND  NP/LP 3.1 ± 0.20b 28.1 ± 1.30a 435 ± 27.4c 191 ± 25.5a,b 31.9 ± 4.50b  NP/NP 3.4 ± 0.40b 27.1 ± 4.00a 318 ± 29.9c 156 ± 29.4a,b 26 ± 5.30a,b  NP/HP 2.2 ± 0.20a 41.6 ± 7.80b 151 ± 27.3b 219 ± 26.0b 34.2 ± 5.60b  LP/feed-deprived 2.3 ± 0.40a 18.9 ± 1.70a 37 ± 4.9a 183 ± 13.2a,b ND  LP/LP 2.9 ± 0.50a,b 25.1 ± 1.20a 316 ± 29.8c 196 ± 7.8a,b 16.7 ± 2.00a  LP/NP 2.7 ± 0.20a,b 31.6 ± 2.20a,b 220 ± 29.9b.c 241 ± 16.2b 28.0 ± 4.50a,b  LP/HP 2.0 ± 0.60a 38.0 ± 7.00b 118 ± 29.5b 240 ± 16.5b 38.9 ± 2.90b Diet effect, P NS NS 0.036 0.008 NS Meal effect, P  LP meal/NP meal NS NS 0.0145 NS NS  NP meal/HP meal 0.0294 0.0107 0.0007 NS NS  HP meal/LP meal NS <0.0001 <0.0001 NS NS Insulin, µg/L Glucagon, µg/L GIP, pg/mL GLP-1, pg/mL PYY, pg/mL Diet/meal groups  NP/feed-deprived 3.0 ± 0.10b 28.7 ± 3.00a 25 ± 1.9a 113 ± 25.3a ND  NP/LP 3.1 ± 0.20b 28.1 ± 1.30a 435 ± 27.4c 191 ± 25.5a,b 31.9 ± 4.50b  NP/NP 3.4 ± 0.40b 27.1 ± 4.00a 318 ± 29.9c 156 ± 29.4a,b 26 ± 5.30a,b  NP/HP 2.2 ± 0.20a 41.6 ± 7.80b 151 ± 27.3b 219 ± 26.0b 34.2 ± 5.60b  LP/feed-deprived 2.3 ± 0.40a 18.9 ± 1.70a 37 ± 4.9a 183 ± 13.2a,b ND  LP/LP 2.9 ± 0.50a,b 25.1 ± 1.20a 316 ± 29.8c 196 ± 7.8a,b 16.7 ± 2.00a  LP/NP 2.7 ± 0.20a,b 31.6 ± 2.20a,b 220 ± 29.9b.c 241 ± 16.2b 28.0 ± 4.50a,b  LP/HP 2.0 ± 0.60a 38.0 ± 7.00b 118 ± 29.5b 240 ± 16.5b 38.9 ± 2.90b Diet effect, P NS NS 0.036 0.008 NS Meal effect, P  LP meal/NP meal NS NS 0.0145 NS NS  NP meal/HP meal 0.0294 0.0107 0.0007 NS NS  HP meal/LP meal NS <0.0001 <0.0001 NS NS 1Values are means ± SDs, n = 6. Labeled means without a common letter differ, P < 0.05. GIP, gastric inhibitory polypeptide; GLP-1, glucagon-like peptide 1; HP, high-protein; LP, low-protein; ND, not detected; NP, normal-protein: PYY, peptide YY. View Large TABLE 6 Plasma concentrations of insulin, glucagon, GLP-1, GIP, and PYY in the different rat groups in the feed-deprived state or 90 min after the meal (experiment 2a)1 Insulin, µg/L Glucagon, µg/L GIP, pg/mL GLP-1, pg/mL PYY, pg/mL Diet/meal groups  NP/feed-deprived 3.0 ± 0.10b 28.7 ± 3.00a 25 ± 1.9a 113 ± 25.3a ND  NP/LP 3.1 ± 0.20b 28.1 ± 1.30a 435 ± 27.4c 191 ± 25.5a,b 31.9 ± 4.50b  NP/NP 3.4 ± 0.40b 27.1 ± 4.00a 318 ± 29.9c 156 ± 29.4a,b 26 ± 5.30a,b  NP/HP 2.2 ± 0.20a 41.6 ± 7.80b 151 ± 27.3b 219 ± 26.0b 34.2 ± 5.60b  LP/feed-deprived 2.3 ± 0.40a 18.9 ± 1.70a 37 ± 4.9a 183 ± 13.2a,b ND  LP/LP 2.9 ± 0.50a,b 25.1 ± 1.20a 316 ± 29.8c 196 ± 7.8a,b 16.7 ± 2.00a  LP/NP 2.7 ± 0.20a,b 31.6 ± 2.20a,b 220 ± 29.9b.c 241 ± 16.2b 28.0 ± 4.50a,b  LP/HP 2.0 ± 0.60a 38.0 ± 7.00b 118 ± 29.5b 240 ± 16.5b 38.9 ± 2.90b Diet effect, P NS NS 0.036 0.008 NS Meal effect, P  LP meal/NP meal NS NS 0.0145 NS NS  NP meal/HP meal 0.0294 0.0107 0.0007 NS NS  HP meal/LP meal NS <0.0001 <0.0001 NS NS Insulin, µg/L Glucagon, µg/L GIP, pg/mL GLP-1, pg/mL PYY, pg/mL Diet/meal groups  NP/feed-deprived 3.0 ± 0.10b 28.7 ± 3.00a 25 ± 1.9a 113 ± 25.3a ND  NP/LP 3.1 ± 0.20b 28.1 ± 1.30a 435 ± 27.4c 191 ± 25.5a,b 31.9 ± 4.50b  NP/NP 3.4 ± 0.40b 27.1 ± 4.00a 318 ± 29.9c 156 ± 29.4a,b 26 ± 5.30a,b  NP/HP 2.2 ± 0.20a 41.6 ± 7.80b 151 ± 27.3b 219 ± 26.0b 34.2 ± 5.60b  LP/feed-deprived 2.3 ± 0.40a 18.9 ± 1.70a 37 ± 4.9a 183 ± 13.2a,b ND  LP/LP 2.9 ± 0.50a,b 25.1 ± 1.20a 316 ± 29.8c 196 ± 7.8a,b 16.7 ± 2.00a  LP/NP 2.7 ± 0.20a,b 31.6 ± 2.20a,b 220 ± 29.9b.c 241 ± 16.2b 28.0 ± 4.50a,b  LP/HP 2.0 ± 0.60a 38.0 ± 7.00b 118 ± 29.5b 240 ± 16.5b 38.9 ± 2.90b Diet effect, P NS NS 0.036 0.008 NS Meal effect, P  LP meal/NP meal NS NS 0.0145 NS NS  NP meal/HP meal 0.0294 0.0107 0.0007 NS NS  HP meal/LP meal NS <0.0001 <0.0001 NS NS 1Values are means ± SDs, n = 6. Labeled means without a common letter differ, P < 0.05. GIP, gastric inhibitory polypeptide; GLP-1, glucagon-like peptide 1; HP, high-protein; LP, low-protein; ND, not detected; NP, normal-protein: PYY, peptide YY. View Large TABLE 7 Plasma total protein and TG concentrations in rats adapted to NP or LP diets (experiment 2a)1 Diet/meal groups Plasma protein, g/L Plasma TGs, mmol/L NP/feed-deprived 53.2 ± 3.05b 1.2 ± 0.41b NP/LP 49.9 ± 2.31b 1.1 ± 0.35b NP/NP 50.1 ± 1.34b 1.5 ± 0.81b NP/HP 52.2 ± 2.40b 1.0 ± 0.45b LP/feed-deprived 43.9 ± 5.86a 0.5 ± 0.13a LP/LP 42.7 ± 5.35a 1.2 ± 0.54b LP/LP 41.2 ± 2.44a 1.2 ± 0.44b LP/HP 42.6 ± 7.85a 0.8 ± 0.15b Diet effect, P 0.05 0.0001 Combined groups  NP groups 51.3 ± 1.14 1.2 ± 0.11  LP groups 42.6 ± 1.11* 0.9 ± 0.09* Diet/meal groups Plasma protein, g/L Plasma TGs, mmol/L NP/feed-deprived 53.2 ± 3.05b 1.2 ± 0.41b NP/LP 49.9 ± 2.31b 1.1 ± 0.35b NP/NP 50.1 ± 1.34b 1.5 ± 0.81b NP/HP 52.2 ± 2.40b 1.0 ± 0.45b LP/feed-deprived 43.9 ± 5.86a 0.5 ± 0.13a LP/LP 42.7 ± 5.35a 1.2 ± 0.54b LP/LP 41.2 ± 2.44a 1.2 ± 0.44b LP/HP 42.6 ± 7.85a 0.8 ± 0.15b Diet effect, P 0.05 0.0001 Combined groups  NP groups 51.3 ± 1.14 1.2 ± 0.11  LP groups 42.6 ± 1.11* 0.9 ± 0.09* 1Values are means ± SDs, n = 6. Labeled means without a common letter differ, P < 0.05. *Different from NP, P < 0.5. HP, high-protein; LP, low-protein; NP, normal-protein. View Large TABLE 7 Plasma total protein and TG concentrations in rats adapted to NP or LP diets (experiment 2a)1 Diet/meal groups Plasma protein, g/L Plasma TGs, mmol/L NP/feed-deprived 53.2 ± 3.05b 1.2 ± 0.41b NP/LP 49.9 ± 2.31b 1.1 ± 0.35b NP/NP 50.1 ± 1.34b 1.5 ± 0.81b NP/HP 52.2 ± 2.40b 1.0 ± 0.45b LP/feed-deprived 43.9 ± 5.86a 0.5 ± 0.13a LP/LP 42.7 ± 5.35a 1.2 ± 0.54b LP/LP 41.2 ± 2.44a 1.2 ± 0.44b LP/HP 42.6 ± 7.85a 0.8 ± 0.15b Diet effect, P 0.05 0.0001 Combined groups  NP groups 51.3 ± 1.14 1.2 ± 0.11  LP groups 42.6 ± 1.11* 0.9 ± 0.09* Diet/meal groups Plasma protein, g/L Plasma TGs, mmol/L NP/feed-deprived 53.2 ± 3.05b 1.2 ± 0.41b NP/LP 49.9 ± 2.31b 1.1 ± 0.35b NP/NP 50.1 ± 1.34b 1.5 ± 0.81b NP/HP 52.2 ± 2.40b 1.0 ± 0.45b LP/feed-deprived 43.9 ± 5.86a 0.5 ± 0.13a LP/LP 42.7 ± 5.35a 1.2 ± 0.54b LP/LP 41.2 ± 2.44a 1.2 ± 0.44b LP/HP 42.6 ± 7.85a 0.8 ± 0.15b Diet effect, P 0.05 0.0001 Combined groups  NP groups 51.3 ± 1.14 1.2 ± 0.11  LP groups 42.6 ± 1.11* 0.9 ± 0.09* 1Values are means ± SDs, n = 6. Labeled means without a common letter differ, P < 0.05. *Different from NP, P < 0.5. HP, high-protein; LP, low-protein; NP, normal-protein. View Large Discussion This study evaluated in rats the relation between protein status, gut neuropeptides (ghrelin, GIP, GLP-1, and PYY), and metabolic hormones (leptin, insulin, and glucagon) involved in feeding control and markers of hypothalamus and NAcc activity, 2 key brain regions involved in food intake control and in reward system activation, respectively. Three different nutritional situations were explored over a period of 15 d, including an NP diet (control), an HP diet, and an LP diet. Feeds were prepared by an exchange between protein and carbohydrate with a constant level of 10% fat to keep energy density unchanged. Both NP and HP diets were associated with a protein-sufficient status, whereas rats fed the LP diet compared with those fed the NP diet did not show any difference in energy intake but had a protein-deficient status as evidenced by lower plasma protein concentration, body weight gain, fat mass, and lean mass. The results showed that both preferences for protein-containing foods and neuronal activation of NAcc by a protein-containing meal were sensitive to protein status. As indicated by c-Fos, NAcc activation was not different between LP, NP, or HP meals in a context of protein sufficiency (NP and HP diet), whereas it was significantly different between meals, with LP lower than NP lower than HP, in a context of protein deficiency (LP diet) and that correlated with a preference for protein-containing feed. This could be partly related to blood concentrations of protein, ghrelin, and leptin, as measured in this study, and of plasma free amino acids, which, although not determined in this study, were previously discussed among peripheral signals of protein deficiency (3–5, 24). It was previously shown that animals fed a protein-deficient diet try to increase their protein intake by a preference for protein-rich feeds or, under some conditions, by an increase in food intake. In the present study, the food-choice experiment confirmed a preference for protein-containing food in protein-deficient rats and the observed differences in preferences between protein-deficient and protein-sufficient rats suggest that appetite for protein induced by a protein-deficient status may take precedence over neophobia. In contrast, the present results in rats did not show an increase in food intake with the LP diet, as previously observed in mice fed an LP diet, which showed both increased energy intake and adiposity and also decreased food efficiency and increased energy expenditure to partly compensate for the increased energy intake (10). Energy expenditure was not measured in the present study, but the results indicated that because rats fed the LP diet had the same energy intake as NP-fed rats but lower body weight, lean, and fat mass, food efficiency was also lower, and energy expenditure should be higher, compared with NP rats, as observed in mice (10). The differences probably originate from species differences between rat and mice and indicate that rats are more resistant than mice to overconsumption induced by a protein-deficient diet, although the results of the choice experiment confirmed that, both in mice and rats the protein-deficient diet induced an appetite for protein (8, 9). An important result was that the appetite for protein induced in rats fed a protein-deficient diet correlated with the neuronal activation of the NAcc that was significantly and positively related to the protein content of the meal when rats were offered calibrated meals with different protein contents. In other words, the number of c-Fos–immunopositive neurons in the NAcc was low in protein-deficient rats offered an LP meal, significantly higher in rats offered an NP meal, and even higher in rats offered an HP meal. Interestingly, in LP rats compared with NP rats, the feed-deprived concentration of ghrelin was higher, the feed-deprived concentration of insulin was lower, and the feed-deprived and fed concentrations of leptin were lower. In addition, a higher expression of the orexigenic neuropeptide NPY and of the stress-related neuropeptide CRH was observed in the hypothalamus, which is in line with an overall increased motivation for food and for protein in protein-deficient animals (Figure 4, Table 3). This corresponds to the need to increase protein intake to compensate for protein deficiency in LP-fed animals. Indeed, orexigenic ghrelin notably increases food intake reward with central NPY and opioid signaling as the necessary mediators (25–27). In contrast, leptin contributes to decreasing the food reward by a modulation of dopamine-signaling pathways (28), and this process is downregulated in LP-fed animals characterized by lower fat tissue and lower blood leptin concentrations. These results concur with the idea that protein deficiency induces a specific appetite for protein, as observed both in animals (8, 13) and in humans (11–13). Although the present results do not allow us to identify which (sub)nuclei within the hypothalamus may be affected and suggest that the conclusions must be made carefully, these results agree with the assumption that protein deficiency induces an appetite for protein associated with a cascade of events from the periphery to the brain that could involve plasma free amino acids, enhanced feed-deprived ghrelin, lower leptin, increased hypothalamic Npy and Crh expressions, and augmented activation of the NAcc in response to protein intake. The results also showed that in LP rats, the HP meal compared with the LP meal did not further modify the expression of the genes controlling dopamine and opioid pathways in the NAcc, which play an important role in reward-seeking behavior and reward-related learning (29). In LP rats, pathways other than these transcriptional processes are involved in the control of dopamine activity in the NAcc in response to meals with different protein content. In addition, as previously observed, rats fed the HP diet compared with rats fed the NP diet had a lower daily energy intake in relation to higher satiety and lower body weight gain associated with lower fat mass without a difference in lean mass (3, 30–33). The results showed a significantly lower NAcc neuronal activation in response to the HP meal in rats previously fed an HP diet for 15 d (HP/HP group) in comparison with rats fed a control NP diet that received an NP meal (NP/NP group). This was also observed between HP/HP and NPpfHP/NP groups. The results also showed that, with an adequate protein status (i.e., NP rats), NAcc neuronal activation was not sensitive to the protein content of the meal as shown for NP/NP, NP/LP, and NP/HP rat groups. These observations confirmed a specific decrease of energy intake with the HP-content diet, which is in line with the reported satiating effect of protein (1, 14–18); and the present results further indicate that this is associated with a lower activation of the NAcc. The HP diet resulted in a specific activation of anorexigenic pathways associated with lower activation of the NAcc by the meal and a decreased motivation for food. The NAcc quantification of mRNA coding genes implicated in the reward system showed an increased expression of Drd2 mRNA in rats fed an HP diet compared with NP rats, which is associated with a deficit of addiction-like rewards (34). Overconsumption of palatable food in obese rats was shown to decrease Drd2 mRNA in relation to a state of reward hypofunction (34). The HP meal did not influence the expression of Drd3 receptors implicated in motivational processes in rats, but these receptors are not involved in appetitive value or motivation for food (35, 36). The results also showed differences in the expression of gene coding for Oprd1 and Oprk1, with a downregulation of Oprd1 expression after rat adaptation to the HP diet compared with the NPpfHP diet, but their role in modulating feeding is not fully understood. Although an increase in Oprd1 expression could result in decreased γ-aminobutyric acid release (37, 38) and might reduce food intake, it was also observed that an increase in Oprd1 expression corresponds to increased food consumption (39). Because there was no difference in Oprd1 expression between NP and HP diets, the results show an increased Oprd1 expression in hungry rats (NPpfHP) that could be related to food-seeking behaviors (40). These observations showed that protein status modifies the activity of the brain reward pathway that contributes to the control of food behavior. The observed effects between NP and LP diets on food choice and brain activities are related to the 50% reduction in protein content (14% to 6%, respectively) and not to carbohydrate, because both NP and LP feeds were very high in carbohydrate (76% and 84%, respectively), and this was confirmed in the choice experiment, which showed that rats fed the LP protein-deficient diet, in comparison to the NP diet, did not change the choice for the NP feed, which is high in carbohydrate, and only increased the HP feed at the expense of the LP feed. The status of protein insufficiency (LP diet) was characterized by high feed-deprived ghrelin concentrations, low postmeal leptin concentrations, high hypothalamic Npy and Crh expressions, high sensitivity of dopamine-dependent reward pathways in the NAcc, and high motivation for protein. Under such protein-deficient conditions, activation of NAcc becomes highly sensitive and positively related to the protein content of meals, with an increased motivation for protein when there is a need to increase protein intake to compensate for protein deficiency and reach an adequate protein status (Figure 5). In contrast, nutritional protein sufficiency induced low ghrelin, high leptin, and lower sensitivity of dopamine-dependent reward pathways. In contrast, under conditions of protein sufficiency, with either the NP or HP diets there was no specific motivation for protein, and homeostatic control relates mainly to energy control. This effect is even reinforced with a generous intake of protein, which could explain the slight decrease in energy intake observed with the HP diet compared with the NP diet, creating conditions for no motivation for protein-rich food with a strong inhibition of reward system reactivity. This is in line with the reported satiating effect of protein, and the present observations confirmed a specific decrease of energy intake with the HP diet, not related to the decreased carbohydrate content, because the NPpfHP/NP group did not reproduce the effect of the HP/HP group on NAcc activity, which was not different between the NPpfHP/NP and the NP/NP groups (Figure 3B). However, it is also important to highlight that the precise peripheral and metabolic signals involved in the control of protein intake are probably ubiquitous and redundant in relation to the complexity of amino acid and protein function in the body. The action of the brain reward pathways to control protein intake, prevent protein imbalance, and restore an adequate protein status involves many different metabolic signals induced by the protein content of the diet and is also sensitive to differential weight gain, growth rate, and other body functions related to protein metabolism. FIGURE 5 View largeDownload slide Protein deficiency is associated with a cascade of events from the periphery to the brain that involve enhanced feed-deprived ghrelin, lower leptin, enhanced hypothalamic NPY, and enhanced activity of the reward system in response to protein intake. CRH, corticotropin-releasing hormone; NPY, neuropeptide Y. FIGURE 5 View largeDownload slide Protein deficiency is associated with a cascade of events from the periphery to the brain that involve enhanced feed-deprived ghrelin, lower leptin, enhanced hypothalamic NPY, and enhanced activity of the reward system in response to protein intake. CRH, corticotropin-releasing hormone; NPY, neuropeptide Y. Acknowledgments The authors’ responsibilities were as follows—CC, IR, GF, ND, and DT: designed the research and wrote the manuscript; CC, IR, SB, and JP: conducted the research; CC, IR, SB, and DT: analyzed the data or performed statistical analysis; CC, IR, and DT: had primary responsibility for the final content; and all authors: read and approved the final manuscript. Notes Supported by public funding from AgroParisTech (Paris, France) and Institut National de la Recherche Agronomique (INRA, France). Author disclosures: CC, IR, GF, SB, JP, ND, and DT, no conflicts of interest. Present address for IR: Institute of Food Science Research, Ciencias de la Alimentación (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Madrid, Spain. Present address for SB: Université de Lorraine, Unité de Recherche Animal et Fonctionnalités des Produits Animaux, Vandœuvre-lès-Nancy, France. CC and IR contributed equally to this work. Supplemental Table 1 and Supplemental Figure 1 are available from the “Supplementary data” link in the online posting of the article and from the same link in the online table of contents at https://academic.oup.com/jn/. Abbreviations used: CRH, corticotropin-releasing hormone; DRD, dopamine receptor; GIP, gastric inhibitory polypeptide; GLP-1, glucagon-like peptide 1; HP, high-protein; LP, low-protein; MC4R, melanocortin receptor; NAcc, nucleus accumbens; NP, normal-protein; NPpfHP, normal-protein diet pair-fed for energy to the high-protein diet; NPY, neuropeptide Y; OPRD, δ opioid receptor; PYY, peptide YY. References 1. Journel M , Chaumontet C , Darcel N , Fromentin G , Tome D . Brain responses to high-protein diets . Adv Nutr 2012 ; 3 : 322 – 9 . Google Scholar CrossRef Search ADS PubMed 2. Davidenko O , Darcel N , Fromentin G , Tome D . Control of protein and energy intake—brain mechanisms . Eur J Clin Nutr 2013 ; 67 : 455 – 61 . Google Scholar CrossRef Search ADS PubMed 3. Harper AE , Peters JC . Protein intake, brain amino acid and serotonin concentrations and protein self-selection . J Nutr 1989 ; 119 : 677 – 89 . Google Scholar CrossRef Search ADS PubMed 4. Fromentin G , Nicolaidis S . Rebalancing essential amino acids intake by self-selection in the rat . Br J Nutr 1996 ; 75 : 669 – 82 . Google Scholar CrossRef Search ADS PubMed 5. Gietzen DW , Hao S , Anthony TG . Mechanisms of food intake repression in indispensable amino acid deficiency . Annu Rev Nutr 2007 ; 27 : 63 – 78 . Google Scholar CrossRef Search ADS PubMed 6. Booth DA . Food intake compensation for increase or decrease in the protein content of the diet . Behav Biol 1974 ; 12 : 31 – 40 . Google Scholar CrossRef Search ADS PubMed 7. Gibson EL , Booth DA . Acquired protein appetite in rats: dependence on a protein-specific need state . Experientia 1986 ; 42 : 1003 – 4 . Google Scholar CrossRef Search ADS PubMed 8. Du F , Higginbotham DA , White BD . Food intake, energy balance and serum leptin concentrations in rats fed low-protein diets . J Nutr 2000 ; 130 : 514 – 21 . Google Scholar CrossRef Search ADS PubMed 9. Beaton JR , Feleki V , Stevenson JA . Insulin hyperphagia in rats fed a low-protein diet . Can J Physiol Pharmacol 1965 ; 43 : 225 – 33 . Google Scholar CrossRef Search ADS PubMed 10. Blais A , Chaumontet C , Azzout-Marniche D , Piedcoq J , Fromentin G , Gaudichon C , Tome D , Even PC . Low protein diet-induced hyperphagia and adiposity are modulated through interactions involving thermoregulation, motor activity, and protein quality in mice . Am J Physiol Endocrinol Metab 2018 ; 314 ( 2 ): E139 – 51 . Google Scholar CrossRef Search ADS PubMed 11. Griffioen-Roose S , Mars M , Siebelink E , Finlayson G , Tome D , de Graaf C . Protein status elicits compensatory changes in food intake and food preferences . Am J Clin Nutr 2012 ; 95 : 32 – 8 . Google Scholar CrossRef Search ADS PubMed 12. Griffioen-Roose S , Smeets PA , van den Heuvel E , Boesveldt S , Finlayson G , de Graaf C . Human protein status modulates brain reward responses to food cues . Am J Clin Nutr 2014 ; 100 : 113 – 22 . Google Scholar CrossRef Search ADS PubMed 13. Gibson EL , Wainwright CJ , Booth DA . Disguised protein in lunch after low-protein breakfast conditions food-flavor preferences dependent on recent lack of protein intake . Physiol Behav 1995 ; 58 : 363 – 71 . Google Scholar CrossRef Search ADS PubMed 14. Westerterp-Plantenga MS , Nieuwenhuizen A , Tome D , Soenen S , Westerterp KR . Dietary protein, weight loss, and weight maintenance . Annu Rev Nutr 2009 ; 29 : 21 – 41 . Google Scholar CrossRef Search ADS PubMed 15. McArthur LH , Kelly WF , Gietzen DW , Rogers QR . The role of palatability in the food intake response of rats fed high-protein diets . Appetite 1993 ; 20 : 181 – 96 . Google Scholar CrossRef Search ADS PubMed 16. L'Heureux-Bouron D , Tome D , Bensaid A , Morens C , Lacroix M , Huneau JF , Fromentin G . Preabsorptive factors are not the main determinants of intake depression induced by a high-protein diet in the rat . Physiol Behav 2004 ; 81 : 499 – 504 . Google Scholar CrossRef Search ADS PubMed 17. Morrison CD , Reed SD , Henagan TM . Homeostatic regulation of protein intake: in search of a mechanism . Am J Physiol Regul Integr Comp Physiol 2012 ; 302 : R917 – 28 . Google Scholar CrossRef Search ADS PubMed 18. Fromentin G , Darcel N , Chaumontet C , Marsset-Baglieri A , Nadkarni N , Tome D . Peripheral and central mechanisms involved in the control of food intake by dietary amino acids and proteins . Nutr Res Rev 2012 ; 25 : 29 – 39 . Google Scholar CrossRef Search ADS PubMed 19. Fulton S . Appetite and reward . Front Neuroendocrinol 2010 ; 31 : 85 – 103 . Google Scholar CrossRef Search ADS PubMed 20. Faipoux R , Tome D , Bensaid A , Morens C , Oriol E , Bonnano LM , Fromentin G . Yeast proteins enhance satiety in rats . J Nutr 2006 ; 136 : 2350 – 6 . Google Scholar CrossRef Search ADS PubMed 21. Reeves PG , Nielsen FH , Fahey GC Jr . AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet . J Nutr 1993 ; 123 : 1939 – 51 . Google Scholar CrossRef Search ADS PubMed 22. Paxinos G , Franklin KBJ . The Mouse Brain in Stereotaxic Coordinates . (Elsevier, Ed.) (Fourth Ed.) . Amsterdam : Academic Press , 1998 . 23. Chaumontet C , Even PC , Schwarz J , Simonin-Foucault A , Piedcoq J , Fromentin G , Azzout-Marniche D , Tome D . High dietary protein decreases fat deposition induced by high-fat and high-sucrose diet in rats . Br J Nutr 2015 ; 114 : 1132 – 42 . Google Scholar CrossRef Search ADS PubMed 24. Rogers QR , Leung PM . The influence of amino acids on the neuroregulation of food intake . Fed Proc 1973 ; 32 : 1709 – 19 . Google Scholar PubMed 25. White BD , He B , Dean RG , Martin RJ . Low protein diets increase neuropeptide Y gene expression in the basomedial hypothalamus of rats . J Nutr 1994 ; 124 : 1152 – 60 . Google Scholar CrossRef Search ADS PubMed 26. Skibicka KP , Dickson SL . Ghrelin and food reward: the story of potential underlying substrates . Peptides 2011 ; 32 : 2265 – 73 . Google Scholar CrossRef Search ADS PubMed 27. Dickson SL , Egecioglu E , Landgren S , Skibicka KP , Engel JA , Jerlhag E . The role of the central ghrelin system in reward from food and chemical drugs . Mol Cell Endocrinol 2011 ; 340 : 80 – 7 . Google Scholar CrossRef Search ADS PubMed 28. Pfaffly J , Michaelides M , Wang GJ , Pessin JE , Volkow ND , Thanos PK . Leptin increases striatal dopamine D2 receptor binding in leptin-deficient obese (ob/ob) mice . Synapse 2010 ; 64 : 503 – 10 . Google Scholar CrossRef Search ADS PubMed 29. Arias-Carrion O , Poppel E . Dopamine, learning, and reward-seeking behavior . Acta Neurobiol Exp (Wars) 2007 ; 67 : 481 – 8 . Google Scholar PubMed 30. Jean C , Rome S , Mathe V , Huneau JF , Aattouri N , Fromentin G , Achagiotis CL , Tome D . Metabolic evidence for adaptation to a high protein diet in rats . J Nutr 2001 ; 131 : 91 – 8 . Google Scholar CrossRef Search ADS PubMed 31. Bensaid A , Tome D , L'Heureux-Bourdon D , Even P , Gietzen D , Morens C , Gaudichon C , Larue-Achagiotis C , Fromentin G . A high-protein diet enhances satiety without conditioned taste aversion in the rat . Physiol Behav 2003 ; 78 : 311 – 20 . Google Scholar CrossRef Search ADS PubMed 32. Darcel N , Fromentin G , Raybould HE , Gougis S , Gietzen DW , Tome D . Fos-positive neurons are increased in the nucleus of the solitary tract and decreased in the ventromedial hypothalamus and amygdala by a high-protein diet in rats . J Nutr 2005 ; 135 : 1486 – 90 . Google Scholar CrossRef Search ADS PubMed 33. Faipoux R , Tome D , Gougis S , Darcel N , Fromentin G . Proteins activate satiety-related neuronal pathways in the brainstem and hypothalamus of rats . J Nutr 2008 ; 138 : 1172 – 8 . Google Scholar CrossRef Search ADS PubMed 34. Johnson PM , Kenny PJ . Dopamine D2 receptors in addiction-like reward dysfunction and compulsive eating in obese rats . Nat Neurosci 2010 ; 13 : 635 – 41 . Google Scholar CrossRef Search ADS PubMed 35. Duarte C , Biala G , Le Bihan C , Hamon M , Thiebot MH . Respective roles of dopamine D2 and D3 receptors in food-seeking behaviour in rats . Psychopharmacology 2003 ; 166 : 19 – 32 . Google Scholar CrossRef Search ADS PubMed 36. Le Foll B , Goldberg SR , Sokoloff P . The dopamine D3 receptor and drug dependence: effects on reward or beyond? Neuropharmacology 2005 ; 49 : 525 – 41 . Google Scholar CrossRef Search ADS PubMed 37. Margolis EB , Fields HL , Hjelmstad GO , Mitchell JM . Delta-opioid receptor expression in the ventral tegmental area protects against elevated alcohol consumption . J Neurosci 2008 ; 28 : 12672 – 81 . Google Scholar CrossRef Search ADS PubMed 38. Hack SP , Bagley EE , Chieng BC , Christie MJ . Induction of delta-opioid receptor function in the midbrain after chronic morphine treatment . J Neurosci 2005 ; 25 : 3192 – 8 . Google Scholar CrossRef Search ADS PubMed 39. Zhang M , Kelley AE . Opiate agonists microinjected into the nucleus accumbens enhance sucrose drinking in rats . Psychopharmacology 1997 ; 132 : 350 – 60 . Google Scholar CrossRef Search ADS PubMed 40. Katsuura Y , Taha SA . Modulation of feeding and locomotion through mu and delta opioid receptor signaling in the nucleus accumbens . Neuropeptides 2010 ; 44 : 225 – 32 . Google Scholar CrossRef Search ADS PubMed © 2018 American Society for Nutrition. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Journal of NutritionOxford University Press

Published: Jun 7, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off