Protein snacks (or loads)

In the short term dietary protein appears to be a strong appetite inhibitor and reduces food intake in subsequent meals beyond that which can be accounted for by its energy content alone, both in rats⁽Reference Bensaid, Tome and Gietzen^3, Reference Trigazis, Orttmann and Anderson⁹⁾ and in man⁽Reference Porrini, Santangelo and Crovetti¹⁰⁾. Moreover in man⁽Reference Marmonier, Chapelot and Louis-Sylvestre¹¹⁾ and in rats⁽Reference Burton-Freeman, Gietzen and Schneeman¹²⁾, consumption of a HP snack delayed the request for the following meal (inter-meal interval).

In rats⁽Reference Bensaid, Tome and Gietzen³⁾ and man⁽Reference Porrini, Santangelo and Crovetti¹⁰⁾, protein is currently believed to present the greatest appetite-suppressing effect of the three macronutrients. In these human studies ingestion of a HP load induces a difference in the decrease in the total meal or daily food intake in comparison with carbohydrate⁽Reference Bertenshaw, Lluch and Yeomans¹³⁾ or lipid loads⁽Reference Porrini, Santangelo and Crovetti¹⁰⁾ or both⁽Reference Poppitt, McCormack and Buffenstein^2, Reference Latner and Schwartz⁴⁾. However, the commonly acknowledged hierarchy proteins>carbohydrates>lipids with respect to satiety is not always observed in rats⁽Reference Geliebter, Liang and Van Itallie¹⁴⁾, or in human subjects⁽Reference Marmonier, Chapelot and Fantino^15, Reference Raben, Agerholm-Larsen and Flint¹⁶⁾.

Many of these discrepancies have been shown to originate from the physiological status of the subjects, the duration of eating and the method of nutrient administration (meaning oral, intra-gastric or intravenous) and the nature of the load⁽Reference Reid and Hetherington¹⁷⁾. Nowadays, special attention must be given to the characteristics of the protein load: structure, texture, volume, energy density and palatability can influence the satiation or satiety induced by it. We must also take care in the selection of the subjects according to their physiological status and function (such as BMI) in conjunction with their cognitive restraint and disinhibition scores.

It has been claimed that beverages elicit weaker appetitive and/or dietary responses than solid foods⁽Reference Mattes¹⁸⁾. Leidy has suggested that differences in satiety and food intake between protein and other macronutrients are only observed when protein is consumed as a solid and not liquid. Leidy et al. ⁽Reference Leidy, Bales-Voelker and Harris¹⁹⁾ showed that a protein-rich beverage consumed as a breakfast meal leads to weaker appetitive and dietary responses v. a protein-rich solid breakfast meal in adolescents. Martens et al. ⁽Reference Martens, Lemmens and Born²⁰⁾ also investigated the differences in appetite profile and physiological parameters after consumption of a single-macronutrient, subject-specific, HP meal in liquefied v. solid form, controlled for energy density, weight and volume in ten lean male subjects. They observed a stronger suppression of hunger and desire to eat with the solid protein than liquefied protein. However, the hypothesis that energy consumed from solid-protein foods evokes a greater satiety response and suppresses energy intake at a subsequent meal compared with liquid foods is still unresolved. The studies of Akhavan et al. ⁽Reference Akhavan, Luhovyy and Anderson²¹⁾ tested the hypothesis that liquid energy is less satiating than solid energy by using pure macronutrients and similar tastes, ingredients, volumes and matrices to eliminate the confounding factors of taste, texture, smell, structure and familiarity of foods. Akhavan et al. ⁽Reference Akhavan, Luhovyy and Anderson²¹⁾ showed that macronutrient composition is more important than the physical state of foods in determining subjective appetite. In respect to this question, in a previous human study⁽Reference Potier, Fromentin and Lesdema²²⁾ we compared the effect on satiety of different liquid preloads consisting only of protein, fat or carbohydrate in very controlled conditions; the preloads had the same volume, energy content, energy density, viscosity and palatability, since it had been shown that all these properties of food can affect food intake. Our results did not confirm the highest satiety effect of proteins, maybe because we used liquid, novel and unusual foods as preloads.

As with all foods, consumption of those containing proteins will generate valuable oro-sensory information due to their organoleptic properties; the latter are probably influenced by the presence of proteins in the food but are not specific. Indeed, mixtures containing proteins will have different final food textures when conditions of food processing (conditions of heating, for instance) are modified. Oro-sensory properties of foods containing proteins will be used by animals and man in learning processes such as conditioned food satiety⁽Reference Fromentin, Feurte and Nicolaidis²³⁾. These learning processes are likely to be involved in the satiety effect of dietary proteins. After several times of eating foods containing proteins, the subject will learn to associate the food oro-sensory properties with the pre- and post-ingestive effects of eating such a food. This has been shown in rats by Bensaid et al. ⁽Reference Bensaid, Tome and Gietzen³⁾. It was shown that 2 d were necessary before a significant decrease was seen in the energy intake of the next protein meal, which means that as soon as the second load day, rats have learned to associate the sensory properties of the protein load with its post-ingestive consequences. In contrast, in human subjects only a few studies have shown that repeated consumption of the same food over a few days constitutes a learning phase for the effects on appetite of this food, which then determines food intake during the post-training test⁽Reference Booth, Lee and McAleavey^24–Reference Yeomans, Weinberg and James²⁶⁾. In all the articles the effect of repeated consumption on short-term food intake was studied using loads varying in energy density. However, the effect of protein was not determined.

Finally, protein type can affect satiety in rats (for instance, yeast proteins)⁽Reference Faipoux, Tome and Bensaid²⁷⁾ and in human subjects (whey or soya protein v. albumin)⁽Reference Anderson, Tecimer and Shah²⁸⁾. According to Veldhorst et al. ⁽Reference Veldhorst, Nieuwenhuizen and Hochstenbach-Waelen²⁹⁾, the protein type effect could be more efficient when the protein level in the diet is not too high: differences in appetite ratings between types of protein appear when certain amino acids are above or below particular threshold values (10 and 25 % of the protein:energy ratio, respectively). However, the effect of the nature of the proteins was not present in all experiments. Indeed, various proteins have been used in other experiments described in the literature, and it is still not clear if the nature of the protein can significantly affect the feeding response studied (egg albumin, casein, soya or whey or pea proteins in human subjects)⁽Reference Lang, Bellisle and Alamowitch^30, Reference Lang, Bellisle and Oppert³¹⁾. The results observed with biochemically very different proteins strongly suggest that the satiating effect of protein is primarily a very general property that does not depend on such specific characteristics of one or another protein⁽Reference Bensaid, Tome and Gietzen³⁾. Nevertheless, if protein type can influence the satiating effect, the mechanisms involved remain unclear. For instance, it could be related to a slowdown of the gastric emptying, an increase in brain amino acids, the presence of specific peptides or the presence of certain specific amino acids (see below). However, in such studies the dietary protein used to formulate the food has to be well equilibrated in its essential amino acid composition. Otherwise, eating a protein- or amino acid-imbalanced diet induces a depression in food intake induced by a conditioned food aversion⁽Reference Fromentin, Feurte and Nicolaidis^32, Reference Gietzen, Hao and Anthony³³⁾.

Protein diets

The consumption of a HP diet quickly induces a strong and immediate depression in food intake followed by a progressive but not complete return to the level of energy intake of the control diet in animals⁽Reference Jean, Rome and Mathe¹⁾. Harper & Peters studied this phenomenon in rats by using diets whose protein content ranged from 5 to 75 %⁽Reference Harper and Peters³⁴⁾. The reduction of food intake occurred when the protein content of the diet was greater than 40 % (as protein:energy ratio) and was even more pronounced as it rose higher.

Taking into consideration the depression in food intake and the induced active avoidance, it has been claimed that eating a HP diet probably induces a conditioned food aversion⁽Reference Tews, Repa and Harper³⁵⁾. The respective roles of conditioned food aversion, satiety and palatability in the depression of food intake induced by a HP diet were studied by analysing the behavioural responses in comparison with a normal-protein (NP) diet⁽Reference Bensaid, Tome and L'Heureux-Bourdon^36–Reference Ropelle, Pauli and Fernandes³⁹⁾. Conditioned food aversion is an acquired mechanism allowing a subject to avoid consuming a food when the post-ingestive consequences are remembered as harmful. Conditioned food aversion requires that the subject links one or more oro-sensory characteristics of the food to unpleasant post-ingestive consequences. Our own experiments⁽Reference Bensaid, Tome and L'Heureux-Bourdon^36, Reference L'Heureux-Bouron, Tome and Bensaid³⁷⁾ showed that only behavioural and food intake parameters were disturbed during day 1 when an animal ate the HP diet, and that most parameters returned to baseline values as soon as day 2 of the HP diet. Rats adapted to the HP diet did not acquire a conditioned food aversion but exhibited satiety, and a normal behavioural satiety sequence. Similar to ours, other studies⁽Reference Kinzig, Hargrave and Hyun^38, Reference Ropelle, Pauli and Fernandes³⁹⁾ did not confirm a conditioned food aversion hypothesis.

Also, it has been shown that different protein sources may differently affect food intake in a context of HP diets in rats⁽Reference Pichon, Potier and Tome⁴⁰⁾. Whatever the protein type used in the HP diet, these diets decrease average energy intake more than the NP diets. This effect is modulated by the ratio between carbohydrate and fat and by the protein type (total milk protein, whey protein or β-lactoglobulin)⁽Reference Pichon, Potier and Tome⁴⁰⁾. For example, whey-derived protein sources, and particularly β-lactoglobulin, reduce food intake, body-weight gain and the adiposity index more than total milk protein. However, we also observed that biochemically very different proteins such as total milk and soya protein induced no differences in depression of energy intake⁽Reference Pichon⁴¹⁾. As in the case of protein snacks, this result suggests that the satiating effect of protein used in a HP diet is primarily a very general property that does not depend on specific characteristics of one or another protein, which in contrast could explain the modulation of food intake observed when different types of proteins are used.

It has been suggested that the sensing of protein ingestion by animals might be linked to the l-glutamate (free+protein bound) content in foods⁽Reference Kondoh, Mallick and Torii⁴²⁾. This might provide a reasonable index of protein ingestion because l-glutamate is the most abundant amino acid in almost all dietary proteins. Nevertheless, 15 d of eating a NP diet (total milk protein, protein:energy of 14 %) enriched or not in glutamate (2 %) did not induce a decrease in food intake or in weight gain⁽Reference Boutry, Bos and Matsumoto⁴³⁾. Glutamic acid is well tolerated by the rat in amounts as high as 5 %, and probably up to 10 %, in diets with low to moderate protein content⁽Reference Harper, Benevenga and Wohlhueter⁴⁴⁾.

The poor palatability of HP diets has been documented⁽Reference McArthur, Kelly and Gietzen⁴⁵⁾, but with respect to protein intake its relative importance remains unclear. It is possible that the appetite-suppressing effect of dietary protein is partially induced by poor palatability. In order to study the role of oro-sensorial factors, food intake was measured after modifying the composition of the HP diet, meaning the type of proteins or carbohydrates present⁽Reference L'Heureux-Bouron, Tome and Bensaid⁴⁶⁾. However, we were unable to modify the depression of food intake induced by a HP diet. Taken together, our experiments indicate that the overall behavioural response more probably originated from an initial lower palatability of the food combined with an enhanced satiety effect of the HP diet and a delay required for the metabolic adaptation.

Even if several studies, conducted in human subjects fed low-energy diets, have shown that using one or two HP meal replacements per d might lead to a decrease in energy intake and body weight⁽Reference Ashley, St Jeor and Perumean-Chaney^47, Reference Noakes, Foster and Keogh⁴⁸⁾, the long-term consequences of the consumption of protein meals in non-restricted subjects remain unknown.

The usefulness of protein diets may be questioned in relation to weight loss and maintenance of lean body mass in two different situations: that of energy restriction and that of ad libitum feeding. The effect of diet composition on weight loss during energy restriction has been widely studied and the additional effect of macronutrient composition above energy restriction was not clearly demonstrated. The absence of any effect of diet composition in human subjects (apart from energy restriction), such as the protein:carbohydrate ratio, has been reported by several authors⁽Reference Sacks, Bray and Carey^49, Reference Gilbert, Bendsen and Tremblay⁵⁰⁾, although not all⁽Reference Treyzon, Chen and Hong^51, Reference Layman, Evans and Erickson⁵²⁾. The loss of lean body mass accompanying energy restriction is a major obstacle to successful long-term dieting. One important suggestion relative to increasing dietary protein during energy restriction is that it might prevent a loss of lean body mass⁽Reference Chaston, Dixon and O'Brien⁵³⁾. In rats, eating a HP diet ad libitum mainly induced a reduction in the fat mass of rats but also a higher ratio of lean:fat mass⁽Reference Jean, Rome and Mathe¹⁾. In human subjects only a few studies have been carried out in order to analyse the long-term effects on body-weight gain and adiposity of eating a diet rich in protein ad libitum. Using two isoenergetic diets in overweight subjects, Weigle et al. ⁽Reference Weigle, Breen and Matthys⁵⁴⁾ showed that an increase in dietary protein from 15 to 30 % of energy at a constant carbohydrate intake resulted in rapid losses of weight and body fat due to a sustained decrease in appetite and energy intake.

Detection of protein and amino acids during digestion and control of food intake by feedback signals

Amino acid detection occurs as early as within the oral cavity. Indeed, several amino acids taste sweet, bitter or umami to man and are attractive to rodents and other animals⁽Reference Beauchamp⁵⁵⁾. Taste receptors (namely T1R1 and T1R3 heterodimers) are present on the tongue and detect most of the twenty amino acids. Some, such as glutamate, can be detected specifically through the means of metabotropic glutamate receptors (mGluR1 and mGluR4), while others, such as glycine, taste sweet to man. There are different sources of l-glutamate in food: either protein-bound or in free form. When glutamate is protein-bound, it is tasteless and does not provide an umami taste to food. l-Glutamate is present as a free amino acid in various food products such as seaweed, soya sauce, fermented beans, an extract made from beef meat, aged cheeses, cured ham and tomatoes. Thus, it is unlikely that free glutamate is a major taste marker of dietary protein to the individual⁽Reference Kondoh, Mallick and Torii^42, Reference Beauchamp⁵⁵⁾.

Satiation feedback signals originating from the stomach are the result of volumetric signals produced by mechanoreceptors⁽Reference Powley and Phillips⁵⁶⁾. The initial increase in gastric volume subsequent to the ingestion of dietary protein is probably due to increased gastric secretions and increased water intake⁽Reference L'Heureux-Bouron, Tome and Bensaid⁴⁶⁾. As explained below, dietary proteins and amino acids are detected within the duodenum and this detection delays gastric emptying⁽Reference Ma, Stevens and Cukier⁵⁷⁾, hence prolonging gastric distension satiation signals. As proposed by Janssen et al. ⁽Reference Janssen, Vanden Berghe and Verschueren⁵⁸⁾, gastric emptying is a key mediator of hunger satiation and satiety. Thus, signals originating from the stomach are likely to contribute to satiation signalling provoked by protein intake.

A strong contributor to the effect of protein and amino acids on food intake is the upper intestine. There is evidence that protein and protein digestion products, such as amino acids and oligopeptides, are detected within the lumen of the duodenum. While a first proposed mechanism for this detection is linked to protein and amino acid absorption and processing by enterocytes⁽Reference Raybould, Glatzle and Freeman⁵⁹⁾, other groups have shown that nutrient-specific receptors exist on the apical side of enterocyte and enteroendocrine cells, being similar to either lingual taste receptors⁽Reference Lindemann⁶⁰⁾ or functional oligopeptide transporters⁽Reference Liou, Chavez and Espero^61, Reference Darcel, Liou and Tome⁶²⁾. Ultimately, amino acid and oligopeptide detection in the intestinal wall is dependent upon the release of cholecystokinin (CCK) by enteroendocrine cells⁽Reference Conigrave, Quinn and Brown⁶³⁾. Duodenal CCK then increases the firing rate of vagus nerve afferents that extend terminals to the close vicinity of the brush border, and that convey information to the nucleus of the solitary tract (NTS) in the brainstem. The implication of this detection on food intake seems to be restricted to short-term food intake control⁽Reference Raybould⁶⁴⁾.

Within the lower intestine, the ileal brake is a feedback mechanism that results in the inhibition of proximal gastrointestinal motility and secretion. Animal and human studies have shown that activation of it by local nutrient perfusions increases feelings of satiety and reduces ad libitum food intake⁽Reference Maljaars, Peters and Mela⁶⁵⁾. These results point to a potential role for the ileal brake in the regulation of digestion, exerting a direct or indirect impact on eating behaviour and satiety. Ileal protein infusions in both human subjects and animals activate the ileal brake⁽Reference Meyer, Hlinka and Tabrizi⁶⁶⁾. It is notably mediated by peptide YY (PYY), which is released by L cells located in the mucosa of the ileum. According to Moran & Dailey⁽Reference Moran and Dailey⁶⁷⁾, the pattern of secretion in plasma can remain elevated for up to 6 h following meal termination. This long-lasting pattern of release suggests roles for this mediator that extend beyond the meal that originally stimulated its release. According to Batterham et al. ⁽Reference Batterham, Heffron and Kapoor⁶⁸⁾, a HP diet intake induced the greatest release of PYY and the most pronounced satiety in normal-weight and obese human subjects. A long-term augmentation of dietary protein in mice induced elevated plasma PYY levels, and reduced food intake and adiposity. In order to determine directly the role of PYY in mediating the satiating effects of protein, Batterham et al. ⁽Reference Batterham, Heffron and Kapoor⁶⁸⁾ generated PYY-null mice that were selectively resistant to the satiating and weight-reducing effects of protein; these animals developed marked obesity that was reversed by exogenous PYY treatment.

In addition to CCK and PYY, a wide range of intestinal mediators have been described to be linked to dietary protein intake, as reviewed by Karhunen et al. ⁽Reference Karhunen, Juvonen and Huotari⁶⁹⁾. Ghrelin, an orexigenic peptide, is decreased after consumption of a HP breakfast in lean subjects⁽Reference Blom, Lluch and Stafleu⁷⁰⁾. This result was confirmed by Bowen et al. ⁽Reference Bowen, Noakes and Clifton⁷¹⁾ in lean and overweight subjects but not by Westerterp's studies (reviewed in Veldhorst et al. ⁽Reference Veldhorst, Smeets and Soenen⁷²⁾). It is also possible that HP diet but not HP meal intake influences ghrelin responses⁽Reference Smeets, Soenen and Luscombe-Marsh⁷³⁾. Also, compared with other macronutrients, dietary protein is also a very strong stimulus for gastric inhibitory polypeptide⁽Reference Hall, Millward and Long⁷⁴⁾ and glucagon-like peptide-1 (GLP-1) release by the small intestine⁽Reference Bowen, Noakes and Clifton⁷¹⁾. Since GLP-1 receptors are expressed in vagal afferent neurons it is likely that GLP-1 acts on vagal afferent terminals in close vicinity to the enteroendocrine L cells⁽Reference Moran and Dailey⁶⁷⁾. Complex cooperative interplay might occur between ghrelin and CCK as described by de Lartigue et al. ⁽Reference de Lartigue, Lur and Dimaline⁷⁵⁾, and also between CCK, gastric inhibitory polypeptide and GLP-1, suggesting critical synergistic effects⁽Reference Rehfeld⁷⁶⁾.

The participation of hepatic portal vein vagal afferents in protein sensing and signalling to the brain is notably supported by electrophysiological recordings showing that hepatic portal vein perfusion of amino acids activates vagal afferent fibres⁽Reference Darcel, Liou and Tome⁶²⁾. However, hepatic vagal afferents do not seem essential to the peripheral detection of a HP diet⁽Reference Tome, Schwarz and Darcel⁶⁾. Others have proposed the hepatic portal vein as being the critical and necessary site for HP diets to alter food intake⁽Reference Penhoat, Mutel and Correig⁷⁷⁾, but this view seems rather unlikely considering the high level of redundancy present in gut–brain axis satiety signalling⁽Reference Berthoud, Shin and Zheng⁷⁸⁾.

Such generated signals (gut peptides but also circulating amino acids) have a dual mode of action on the central nervous system: (i) at the gastrointestinal tract via the vagus nerve to the brainstem within the NTS (the first central relay for afferent vagal fibres); and (ii) directly at the hypothalamus (arcuate nucleus of the hypothalamus; ARC) and brainstem via the bloodstream (notably through hormones and circulating nutrients acting as mediators) and an indirect transmission through the nervous system which innervates the gastrointestinal tract, namely the vagus nerve and splanchnic nerves (though sub-diaphragmatic vagotomy fails to suppress the depression of food intake induced by the administration of a HP diet in rats⁽Reference L'Heureux-Bouron, Tome and Rampin⁷⁹⁾).

Detection of protein and amino acids, post-absorptive signals and feedback signals controlling food intake

Since the 2000s, many studies in human subjects have been conducted to examine differences in postprandial hormone profiles that could be the cause of satiety induced by proteins. Studies have focused on CCK, GLP-1, amylin, ghrelin, leptin, insulin and glucagon, but very often no clear correlation emerged between these hormones and satiety⁽Reference Bowen, Noakes and Clifton^71, Reference Veldhorst, Smeets and Soenen⁷²⁾. As for individual mediators, studies have shown that ghrelin is primarily blood-borne and alters hypothalamic function, whereas the mode of action of CCK and GLP-1 is, in contrast, primarily vagally mediated⁽Reference Moran and Dailey⁶⁷⁾. Plasma CCK levels are unlikely to be a relevant signal⁽Reference Brenner, Yox and Ritter⁸⁰⁾.

It must be mentioned that an increase in energy expenditure could also be the cause of peripheral signals of satiety induced by proteins. The role of dietary protein in body-weight control could be a direct consequence of thermogenesis. Proteins are thought to produce a greater thermogenic effect than other macronutrients participating in protein-induced satiety. This is due in part to the energetic cost necessary to incorporate each amino acid into protein and for catabolism of the amino acid in excess. In human subjects⁽Reference Latner and Schwartz^4, Reference Westerterp-Plantenga, Nieuwenhuizen and Tome⁵⁾ but not in rats⁽Reference Stepien, Gaudichon and Azzout-Marniche⁸¹⁾, ingestion of proteins stimulates postprandial thermogenesis at a higher level than other macronutrients. Conversely, it has never been shown that an increase in postprandial thermogenesis was the direct cause of satiety induced by the ingestion of food proteins. At most, correlations between dietary protein intakes, an effect on satiety and increased thermogenesis were highlighted. Furthermore, central mechanisms linking the direct effect of increased thermogenesis and postprandial satiety remain to be demonstrated.

Amino acid-induced gluconeogenesis could also prevent a decrease in glycaemia that could contribute to satiety⁽Reference Veldhorst, Westerterp-Plantenga and Westerterp⁸²⁾. The fact that gluconeogenesis in man is thought to remain relatively stable in varying metabolic conditions is still a matter of debate⁽Reference Nuttall, Ngo and Gannon⁸³⁾. However, Velhorst et al. ⁽Reference Veldhorst, Westerterp and Westerterp-Plantenga⁸⁴⁾ has shown that after a HP diet, gluconeogenesis was increased and appetite was lower compared with a NP diet; however, these were unrelated to each other. The glucostatic theory indicates that a drop of plasma glucose level precedes the start of the following meal. However, no one has shown that the flow of plasma glucose induced by the ingestion of a meal rich in protein lasts longer than one that follows a NP meal⁽Reference Marmonier, Chapelot and Fantino¹⁵⁾.

The high plasma concentration of amino acids after the ingestion of a protein diet could be the cause of peripheral signals that would be detected in some specific regions in the hypothalamus⁽Reference Choi, Fletcher and Anderson⁸⁵⁾. Hall et al. ⁽Reference Hall, Millward and Long⁷⁴⁾ explained that their results on the satiating effect of whey protein being greater than that of casein were in agreement with the work of Boirie et al. ⁽Reference Boirie, Dangin and Gachon⁸⁶⁾, originally the conceiver of ‘slow and fast proteins’, and the aminostatic theory⁽Reference Mellinkoff, Frankland and Boyle⁸⁷⁾. These authors concluded that the massive influx of amino acids following ingestion of whey proteins could lead to higher satiating power of these proteins as compared with other, slower, sources of amino acids (such as casein). Finally, the presence of some specific amino acids such as glutamate or leucine could also play a role in the central control of appetite (see below).

The general hypothesis that the specific role of certain amino acid precursors of neurotransmitters would explain the decrease in food intake induced by proteins is still a matter of debate. In particular, the amount of tryptophan (the precursor of serotonin and a mediator known to inhibit appetite⁽Reference Blundell and Latham⁸⁸⁾) was used to demonstrate this phenomenon. The high tryptophan content of α-lactalbumin has been suggested to explain the satiating effect of this protein⁽Reference Veldhorst, Nieuwenhuizen and Hochstenbach-Waelen^29, Reference Nieuwenhuizen, Hochstenbach-Waelen and Veldhorst⁸⁹⁾. Indeed, the ingestion of α-lactalbumin increases the plasma level of tryptophan and the tryptophan:branched-chain amino acids ratio more than other proteins such as gelatin (which is devoid of tryptophan). However, the addition of free tryptophan to gelatin at the same level as that of α-lactalbumin does not affect responses on scales of hunger⁽Reference Veldhorst, Nieuwenhuizen and Hochstenbach-Waelen²⁹⁾. Similarly, tyrosine, a precursor of dopamine, added at a 5 % level in the diet of rats had no influence on the level of intake⁽Reference Bassil, Hwalla and Obeid⁹⁰⁾. Because the ingestion of foods high in protein (and thus l-glutamate) does not lead to appreciable changes in plasma l-glutamate concentrations⁽Reference Kondoh, Tsurugizawa and Torii⁹¹⁾, the body (the brain) is unlikely to monitor protein intake via meal- or diet-related variations in plasma l-glutamate. In the same way, results concerning the addition of histidine, a precursor of histamine, to the diet are conflicting. Chronic ingestion of histidine added at a 5 % level in the diet of rats had no influence on the level of intake⁽Reference Bassil, Hwalla and Obeid⁹⁰⁾. At the same time a 8 d ingestion of histidine added at a 1, 2·5 or 5 % level in the diet of rats decreased food intake⁽Reference Kasaoka, Tsuboyama-Kasaoka and Kawahara⁹²⁾, so the role of central histamine in the depression of food intake has been suggested⁽Reference Mercer⁹³⁾.