Indispensable Amino Acid Digestibility of Moroccan Fava Bean Using the Dual Isotope Method in Healthy Adults.

BACKGROUND: Assessment of protein quality is necessary to satisfy the nutritional needs of populations across the world. In addition to indispensable amino acid (IAAs) composition, protein digestibility is a major component of IAA bioavailability, playing a crucial role in human health and affecting the linear growth of children. OBJECTIVES: This study aimed to evaluate IAA digestibility of fava beans, a legume widely consumed in Morocco using the dual-tracer method. METHODS: 2H-intrinsically labeled Fava beans supplemented with 12 mg/kg BW of 13C spirulina were given to 5 healthy volunteers (3 men and 2 women), aged 25.8 ± 3.3 y, with a mean BMI of 20.0 kg/m2. The meal was spread in small portions and was given hourly throughout 7 h. Blood was sampled at baseline and hourly from 5 to 8 h after meal ingestion. IAA digestibility was evaluated by gas chromatography-combustion-isotope ratio mass spectrometry using the 2H/13C ratio in plasma IAA. Digestible indispensable amino acid ratios (DIAAR) were calculated using the scoring pattern for people older than 3 y. RESULTS: Fava beans had an adequate level of lysine but were limiting in several IAAs, especially methionine. Under our experimental conditions, the average IAA digestibility of fava bean was 61.1% ± 5.2%. Valine had the highest digestibility (68.9% ± 4.3%) and threonine had the lowest (43.7% ± 8.2%). In consequence, the lowest DIAAR was 67% for threonine and only 47% for sulfur amino acids (SAA). CONCLUSIONS: The present study is the first to determine the digestibility of fava bean amino acids in humans. The mean IAA digestibility was moderate, and consequently, we conclude that fava bean provides a limited amount of several IAAs, especially SAA, but adequately for lysine. Preparation and cooking methods of fava beans should be improved to increase digestibility. This study was registered at ClinicalTrials.gov as NCT04866927.

Protein-carbohydrate interaction effects on energy balance, FGF21, IGF-1, and hypothalamic gene expression in rats.

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

Severe protein deficiency induces hepatic expression and systemic level of FGF21 but inhibits its hypothalamic expression in growing rats.

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

Protein and amino acid digestibility of 15N Spirulina in rats.

PURPOSE: Spirulina is often used as dietary supplement for its protein content and quality. However, in vivo data on protein digestibility are lacking. This study aims to determine nitrogen and amino acid digestibility in rats. A secondary objective was to test the effect of sonication prior to ingestion to break cell walls. METHODS: Wistar rats were fed a single test meal containing 15N Spirulina that was either sonicated (n = 11) or not (control, n = 13). Rats were euthanized 6 h after the meal ingestion. Spirulina nitrogen digestibility was measured by assessment of 15N recovery in digestive contents. Amino acid digestibility was measured by quantification of the caecal amino acid content and their 15N enrichment. RESULTS: Real fecal nitrogen digestibility was 86.0 ± 0.7%, without any differences between groups. Mean 15N amino acid caecal digestibility was 82.8 ± 1.3%, and values ranged between 77.9 ± 1.9% for serine and 89.4 ± 1.0% for methionine. No effect of sonication was observed. The most limiting AA was histidine, with a chemical score of 0.98 and a PD-CAAS of 0.84. Lysine was also limiting in a lesser extent. CONCLUSION: The nitrogen and amino acid digestibility of Spirulina is relatively low, and showed no effect of prior sonication. Its amino acid composition is relatively well balanced but not enough to compensate for the poor digestibility.

Low-protein and methionine, high-starch diets increase energy intake and expenditure, increase FGF21, decrease IGF-1, and have little effect on adiposity in mice.

Low-protein diets most often induce increased energy intake in an attempt to increase protein intake to meet protein needs with a risk of accumulation as fat of the excess energy intake. In female adult BALB/c mice, a decrease in dietary casein from 20% to 6% and 3% increased energy intake and slightly increased adiposity, and this response was exacerbated with soy proteins with low methionine content. The effect on fat mass was however limited because total energy expenditure increased to the same extent as energy intake. Lean body mass was preserved in all 6% fed mice and reduced only in 3% casein-fed animals. Insulin response to an oral glucose tolerance test was reduced in soy-fed mice and in low-protein-fed mice. Low-protein diets did not affect uncoupling protein 1 and increased fibroblast growth factor 21 (FGF21) in brown adipose tissue and increased FGF21, fatty acid synthase, and cluster of differentiation 36 in the liver. In the hypothalamus, neuropeptide Y was increased and proopiomelanocortin was decreased only in 3% casein-fed mice. In plasma, when protein was decreased, insulin-like growth factor-1 decreased and FGF21 increased and plasma FGF21 was best described by using a combination of dietary protein level, protein-to-carbohydrate ratio, and protein-to-methionine ratio in the diet. In conclusion, reducing dietary protein and protein quality increases energy intake but also energy expenditure resulting in an only slight increase in adiposity. In this process, FGF21 is probably an important signal that responds to a complex combination of protein restriction, protein quality, and carbohydrate content of the diet.

Fructo-oligosaccharides reduce energy intake but do not affect adiposity in rats fed a low-fat diet but increase energy intake and reduce fat mass in rats fed a high-fat diet.

The ingestion of low or high lipid diets enriched with fructo-oligosaccharide (FOS) affects energy homeostasis. Ingesting protein diets also induces a depression of energy intake and decreases body weight. The goal of this study was to investigate the ability of FOS, combined or not with a high level of protein (P), to affect energy intake and body composition when included in diets containing different levels of lipids (L). We performed two studies of similar design over a period of 5weeks. During the first experiment (exp1), after a 3-week period of adaptation to a normal protein-low fat diet, the rats received one of the following four diets for 5weeks (6 rats per group): (i) normal protein (14% P/E (Energy) low fat (10% L/E) diet, (ii) normal protein, low fat diet supplemented with 10% FOS, (iii) high protein (55%P/E) low fat diet, and (iv) high protein, low fat diet supplemented with 10% FOS. In a second experiment (exp2) after the 3-week period of adaptation to a normal protein-high fat diet, the rats received one of the following 4 diets for 5weeks (6 rats per group): (i) normal protein, high fat diet (35% of fat), (ii) normal protein, high fat diet supplemented with 10% FOS, (iii) high protein high fat diet and (iv) high protein high fat diet supplemented with 10% FOS. In low-fat fed rats, FOS did not affect lean body mass (LBM) and fat mass but the protein level reduced fat mass and tended to reduce adiposity. In high-fat fed rats, FOS did not affect LBM but reduced fat mass and adiposity. No additive or antagonistic effects between FOS and the protein level were observed. FOS reduced energy intake in low-fat fed rats, did not affect energy intake in normal-protein high-fat fed rats but surprisingly, and significantly, increased energy intake in high-protein high-fat fed rats. The results thus showed that FOS added to a high-fat diet reduced body fat and body adiposity.

The satiating effects of eggs or cottage cheese are similar in healthy subjects despite differences in postprandial kinetics.

Studies have reported a better satiating effect of eggs when compared with common cereal-based breakfasts, an effect that can be attributed to their macronutrient composition. Our aim was to compare the satiating power of an omelette and cottage cheese, both being common food snacks with similar nutrient compositions (containing proteins and lipids) but in different food forms. Thirty healthy volunteers participated in a randomized crossover trial. On each test day, the subjects consumed one of the two snacks, both providing 1346 kJ, 26 g protein, 21 g lipids, and 8 g lactose. The elapsed time between the snack and lunch request, their food intake at lunch, and their satiety scores were recorded. In a subgroup of 10 volunteers, blood was sampled to measure plasma metabolites and hormones. The two preloads were similar in terms of the time between the snack and a request for the buffet (167 ± 8 min), energy intake at the buffet (3988 ± 180 kJ) and appetite ratings. Plasma amino acid and urea concentrations indicated a marked delay in kinetic delivery after the eggs compared with the cottage cheese. In contrast, glucose, triglycerides and cholesterol displayed similar profiles after the snack. GIP and insulin secretions increased significantly after the cottage cheese, while glucagon and GLP-1 secretions were delayed with the omelette. We conclude that despite important differences in protein kinetics and their subsequent effects on hormone secretion, eggs and cottage cheese had a similar satiating power. This strongly suggests that with dose of proteins that is compatible to supplement strategies, i.e. 20-30 g, a modulation of protein kinetics is ineffective in increasing satiety.

Beneficial effects of an amino acid mixture on colonic mucosal healing in rats.

BACKGROUND: Mucosal healing (MH) decreases the relapse risk in patients with inflammatory bowel disease, but the role of dietary supplementation in this process has been poorly investigated. Here, we investigated the effect of an amino acid mixture supplement on rat MH. METHODS: Colitis was induced using 5% of dextran sodium sulfate for 6 days. Then, rats received a mixture of threonine (0.50 g/d), methionine (0.31 g/d), and monosodium glutamate (0.57 g/d) or an isonitrogenous amount of alanine (control group). Colons were recovered after colitis induction and after dietary supplementation for measuring colon characteristics, myeloperoxidase, cytokine gene expression, glutathione content, protein synthesis rate, and for histological analysis. Short-chain fatty acids were measured in the colonic content. RESULTS: Colitis induction resulted in anorexia, thickening and shortening of the colon, and ulceration. Colonic cytokine expression and neutrophil infiltration were increased. An increased amount of water and a decreased amount of butyrate, propionate, and acetate were measured in the colonic content. Supplementation with the amino acid mixture coincided with a reduced protein synthesis rate in the colon compatible with the observed increased colonic MH. Mucosal regeneration/re-epithelialization was visible within 3 days after colitis induction at a time when mucosal inflammation was severe. Histological analysis revealed an increased regeneration/re-epithelialization after 10-day supplementation. In contrast, the spontaneous resolution of inflammation was not affected by the supplementation. CONCLUSIONS: Amino acid supplementation ameliorates colonic MH but not mucosal inflammatory status. Our data sustain the use of adjuvant dietary intervention on initiated intestinal MH.

A pilot study for the intrinsic labeling of egg proteins with 15N and 13C.

The aim of this study was to produce intrinsically and uniformly doubly (15)N-(13)C-labeled proteins. These proteins can be used as intrinsic tracers of dietary amino acids, both α-amino groups and carbon skeletons, during postprandial metabolic utilization. Two (Rhodes) laying hens were fed for 16 days with a standard poultry diet supplemented with 0, 0.2% or 0.4% of a mixture of 20 doubly (15)N-(13)C-labeled AAs. A third hen was given a non-enriched diet, as the control. The eggs laid were collected over 24 days, from 3 days before to 4 days after supplementation. The (15)N and (13)C enrichments in proteins from white and yolk were measured by EA-IRMS and GC-C-IRMS for enrichment in individual amino acids. After 10 days of supplementation, the (15)N enrichment reached an isotopic plateau at 1500 to 3000 ‰, depending on the supplementation level, in both white and yolk while the (13)C enrichment was 220 to 650 ‰ in white and was 100 to 250 ‰ in yolk. The (15)N enrichment was similar among the amino acids, except for the aromatic ones in which the enrichment was lower. The δ(13)C values were variable among amino acids in both white and yolk, ranging from 77 ‰ for tyrosine to 555 ‰ for proline with the 0.2 % supplementation level. In conclusion, the incorporation of 0.2 % labeled amino acids in the hen diet allowed us to achieve sufficient enrichment for metabolic studies. However, due to the non-homogeneity of the (13)C labeling, adequate (13)C enrichment of individual amino acids must be considered depending on the investigated metabolic pathway.

The postprandial use of dietary amino acids as an energy substrate is delayed after the deamination process in rats adapted for 2 weeks to a high protein diet.

The aim of this study was to determine the contribution of dietary amino acids (AA) to energy metabolism under high protein (HP) diets, using a double tracer method to follow simultaneously the metabolic fate of α-amino groups and carbon skeletons. Sixty-seven male Wistar rats were fed a normal (NP) or HP diet for 14 days. Fifteen of them were equipped with a permanent catheter. On day 15, after fasting overnight, they received a 4-g meal extrinsically labeled with a mixture of 20 U-[(15)N]-[(13)C] AA. Energy metabolism, dietary AA deamination and oxidation and their transfer to plasma glucose were measured kinetically for 4 h in the catheterized rats. The transfer of dietary AA to liver glycogen was determined at 4 h. The digestive kinetics of dietary AA, their transfer into liver AA and proteins and the liver glycogen content were measured in the 52 other rats that were killed sequentially hourly over a 4-h period. [(15)N] and [(13)C] kinetics in the splanchnic protein pools were perfectly similar. Deamination increased fivefold in HP rats compared to NP rats. In the latter, all deaminated AA were oxidized. In HP rats, the oxidation rate was slower than deamination, so that half of the deaminated AA was non-oxidized within 4 h. Non-oxidized carbon skeletons were poorly sequestrated in glycogen, although there was a significant postprandial production of hepatic glycogen. Our results strongly suggest that excess dietary AA-derived carbon skeletons above the ATP production capacity, are temporarily retained in intermediate metabolic pools until the oxidative capacities of the liver are no longer overwhelmed by an excess of substrates.

Intestinal lysine metabolism is driven by the enteral availability of dietary lysine in piglets fed a bolus meal.

Previous steady-state continuous-feeding studies have shown that the gut mucosa removes substantial amounts of both dietary and systemic amino acids. However, enteral nutrition is often given under non-steady-state conditions as a bolus meal, and this has been shown to influence systemic metabolism. Therefore, our aim was to quantify the relative metabolism of dietary and systemic lysine by the portal-drained viscera (PDV) under non-steady-state conditions after a single bolus meal. Five 28-day-old piglets implanted with arterial, venous, and portal catheters and with an ultrasonic portal flow probe were given an oral bolus feeding of a milk formula containing a trace quantity of intrinsically 15N-labeled soy protein and a continuous intravenous infusion of [U-13C]lysine for 8 h. Total lysine use by the PDV was maximal 1 h after the meal (891 micromol x kg(-1) x h(-1)) and was predominantly of dietary origin (89%), paralleling the enteral delivery of dietary lysine. Intestinal lysine use returned to a low level after 4 h postprandially and was derived exclusively from the arterial supply until 8 h. Cumulative systemic appearance of dietary lysine reached 44 and 80% of the ingested amount 4 and 8 h after the meal, respectively, whereas the PDV first-pass use of dietary lysine was 55 and 32% of the intake for these two periods, respectively. We conclude that the first-pass utilization rate of dietary lysine by the PDV is directly increased by the enteral lysine availability and that it is higher with a bolus than with continuous oral feeding.