True Ileal Amino Acid Digestibility and Protein Quality of 15N-Labeled Faba Bean in Healthy Humans.

BACKGROUND: The recommended transition toward more plant-based diets, particularly containing legumes, requires a wider knowledge of plant protein bioavailability. Faba beans are cultivated at different latitudes and are used increasingly in human nutrition. OBJECTIVES: We aimed to assess the nutritional quality of faba bean protein in healthy volunteers equipped with an intestinal tube to implement the ileal 15N balance method. METHODS: Nine volunteers completed the study (7 males, 2 females, aged 33 ± 10 y, BMI: 24.7 ± 2.6 kg/m2). They were equipped with a nasoileal tube. After fasting overnight, they ingested a test meal consisting of cooked mash of dehulled faba bean seeds (20 g protein per serving of approximately 250 g) intrinsically labeled with 15N. Samples of ileal contents, plasma, and urine were collected over an 8-h postprandial period. Undigested nitrogen (N) and amino acids (AAs) were determined using isotopic MS, and subsequently, ileal digestibility and digestible indispensable amino acid score (DIAAS) were calculated. The measurement of postprandial deamination allowed calculation of the net postprandial protein utilization (NPPU). RESULTS: The ileal N digestibility was 84.1% ± 7.7%. Postprandial deamination represented 19.2% ± 3.6% of ingested N, and the NPPU was 64.7% ± 9.7%. The ileal digestibility of individual AAs varied from 85.1% ± 13.7% for histidine to 94.2% ± 3.6% for glutamine + glutamate. The mean AA digestibility was ∼6 percentage points higher than the digestibility of N, reaching 89.8% ± 5.9%, whereas indispensable AA digestibility was 88.0% ± 7.3%. Histidine and tryptophan were the first limiting AAs [DIAAS = 0.77 (calculated by legume-specific N-to-protein conversion factor 5.4); 0.67 (by default factor 6.25)]. Sulfur AAs were limiting to a lesser extent [DIAA ratio = 0.94 (N × 5.4); 0.81 (N × 6.25)]. CONCLUSIONS: Protein ileal digestibility of cooked, dehulled faba beans in humans was moderate (<85%), but that of AAs was close to 90%. Overall protein quality was restricted by the limited histidine and tryptophan content. This trial was registered at clinicaltrials.gov as NCT05047757.

Values for the Digestibility of Pea Protein Isolate or Casein Amino Acids Determined using the Dual Isotope Method Are Not Similar to Those Derived with the Standard Ileal Balance Method in Healthy Volunteers.

BACKGROUND: The measurement of ileal amino acid (AA) digestibility is invasive and inappropriate when applied to vulnerable populations. The dual isotope method has been developed over the past 5 y as an alternative method. OBJECTIVE: The aim of this work was to compare the indispensable amino acid (IAA) digestibility values of 2 different proteins obtained using the dual isotope and the standard ileal balance methods in the same subjects. METHODS: Fifteen healthy adults completed the study. Over 4 h, they ingested 9 successive portions of mashed potatoes containing the test protein (pea protein or casein) labeled intrinsically with 15N and 2H, and a 13C-free AA mixture as a reference for the dual isotope method. Plasma was sampled regularly over the 8-h postprandial period, whereas the ileal digesta was collected continuously via a naso-ileal tube. Isotopic enrichments (15N and 13C) were measured in the digesta for the direct determination of ileal IAA digestibility, whereas plasma enrichments (2H and 13C) were measured to determine IAA digestibility using the dual isotope method. RESULTS: The 4-h repeated meal procedure enabled the almost complete digestion of test proteins at 8 h and the attainment of a plasma isotopic plateau between 2.5 and 4 h. These conditions were necessary to perform the ileal balance and dual isotope methods simultaneously. For pea protein, the mean IAA digestibility was similar between the 2 methods, but significant differences (from 10% to 20%) were observed for individual IAA values. For casein, IAA digestibility was significantly lower with the dual isotope method for all the IAA analyzed. CONCLUSIONS: Under our experimental conditions, the degree of agreement between the dual isotope and ileal balance methods varied among AAs and depended on the protein source. Further research is needed to validate the dual isotope method. This study was registered at clinicaltrials.gov as NCT04072770.

Pipecolate and Taurine are Rat Urinary Biomarkers for Lysine and Threonine Deficiencies.

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

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.

Role of liver AMPK and GCN2 kinases in the control of postprandial protein metabolism in response to mid-term high or low protein intake in mice.

PURPOSE: Protein synthesis and proteolysis are known to be controlled through mammalian target of rapamycin, AMP-activated kinase (AMPK) and general control non-derepressible 2 (GCN2) pathways, depending on the nutritional condition. This study aimed at investigating the contribution of liver AMPK and GCN2 on the adaptation to high variations in protein intake. METHODS: To evaluate the answer of protein pathways to high- or low-protein diet, male wild-type mice and genetically modified mice from C57BL/6 background with liver-specific AMPK- or GCN2-knockout were fed from day 25 diets differing in their protein level as energy: LP (5%), NP (14%) and HP (54%). Two hours after a 1 g test meal, protein synthesis rate was measured after a 13C valine flooding dose. The gene expression of key enzymes involved in proteolysis and GNC2 signaling pathway were quantified. RESULTS: The HP diet but not the LP diet was associated with a decrease in fractional synthesis rate by 29% in the liver compared to NP diet. The expression of mRNA encoding ubiquitin and Cathepsin D was not sensitive to the protein content. The deletion of AMPK or GCN2 in the liver did not affect nor protein synthesis rates and neither proteolysis markers in the liver or in the muscle, whatever the protein intake. In the postprandial state, protein level alters protein synthesis in the liver but not in the muscle. CONCLUSIONS: Taken together, these results suggest that liver AMPK and GCN2 are not involved in this adaptation to high- and low-protein diet observed in the postprandial period.

The True Amino Acid Digestibility of 15N-Labelled Sunflower Biscuits Determined with Ileal Balance and Dual Isotope Methods in Healthy Humans.

BACKGROUND: Sunflower is a promising protein source but data on amino acid (AA) digestibility are lacking in humans. Classically, the determination of AA digestibility requires ileal digesta sampling. The dual isotope method is minimally invasive but has not been compared to the conventional approach. OBJECTIVES: This study aimed to determine the true ileal digestibility of sunflower AAs in healthy volunteers who ate biscuits containing 15nitrogen (N) protein isolate, in comparison with the dual isotope method. METHODS: Twelve healthy volunteers (men and women; 40.4 ± 10.5 years old; BMI, 23.7 ± 2.9 kg/m2) were equipped with a naso-ileal tube. For 4 hours, they consumed 9 repeated meals comprising 15N-sunflower protein biscuits together with 13carbon (C)-AAs, carried either in chocolate (SUN + Ch; n = 7) or apple puree (SUN + P; n = 5). Ileal digesta and blood were sampled throughout 8 hours after ingestion of the first meal. The 15N and 13C AA enrichments were measured in digesta to determine ileal digestibility directly and in plasma to determine lysine and threonine digestibility using the dual isotope method. Differences between methods and between vector groups were analyzed using paired and unpaired t-tests, respectively. RESULTS: The ileal digestibility of sunflower indispensable AAs (IAA) was 89% ± 5.3%, with threonine and lysine having the lowest digestibility. In the SUN + Ch meal, IAA digestibility was 3% below that of SUN + P (P < 0.05). The mean free 13C-AA ileal digestibility was 98.1% ± 0.9%. No matter which matrix was used to carry 13C-AAs, plasma 15N and 13C-AA kinetics displayed a 1-hour offset. Digestibility obtained with the dual isotope method (70.4% ± 6.0% for threonine and 75.9% ± 22.3% for lysine) was below the target values. CONCLUSIONS: The ileal digestibility of IAAs from a sunflower isolate incorporated in a biscuit was close to 90% in healthy adults. Under our experimental conditions, the dual isotope method provided lower values than the usual method. Further protocol developments are needed to validate the equivalence between both methods. This trial was registered at clinicaltrials.gov as NCT04024605.

Using the dual isotope method to assess cecal amino acid absorption of goat whey protein in rats, a pilot study.

Measurement of ileal amino acids (AA) bioavailability is recommended to evaluate protein quality. A dual isotope tracer method, based on plasma isotopic enrichment ratios, has been proposed to determine true digestibility in humans. In a pilot study, we aimed to evaluate whether this method could be implemented in rats to determine AA bioavailability based on isotopic enrichment ratios measured in cecal digesta or plasma samples. Goat milk proteins were intrinsically labeled with 15N and 2H. Wistar rats were fed a meal containing the doubly labeled goat whey proteins and a tracer dose of 13C-spirulina. Blood samples were collected 0, 1 h and 3 h after meal ingestion from the tail vein. The rats were euthanized 4 h (n = 6) or 6 h (n = 6) after meal to collect plasma and intestinal contents. True orocecal protein digestibility and AA bioavailability were assessed by means of 15N and 2H enrichment in cecum content and compared with absorption indexes determined at the plasma or cecum level using isotopic ratios. Plasma kinetics of isotopic enrichment could not be completed due to the limited quantity of plasma obtained with sequential blood collection. However, the absorption indexes determined from cecal 15N or 2H/13C ratios gave coherent values with true orocecal AA bioavailability. This dual isotope approach with measurements of isotopic ratios in digestive content could be an interesting strategy to determine true AA bioavailability in ileal digesta of rats.

Moderate adiposity levels counteract protein metabolism modifications associated with aging in rats.

PURPOSE: Physiological parameters such as adiposity and age are likely to influence protein digestion and utilization. The aim of this study was to evaluate the combined effects of age and adiposity on casein protein and amino acid true digestibility and its postprandial utilization in rats. METHODS: Four groups were included (n = 7/8): 2 months/normal adiposity, 2 months/high adiposity, 11 months/normal adiposity and 11 months/high adiposity. Rats were given a calibrated meal containing 15N-labeled casein (Ingredia, Arras, France) and were euthanized 6 h later. Digestive contents were collected to assess protein and amino acid digestibilities. 15N enrichments were measured in plasma and urine to determine total body deamination. Fractional protein synthesis rate (FSR) was determined in different organs using a flooding dose of 13C valine. RESULTS: Nitrogen and amino acid true digestibility of casein was around 95-96% depending on the group and was increased by 1% in high adiposity rats (P = 0.04). Higher adiposity levels counteracted the increase in total body deamination (P = 0.03) that was associated with older age. Significant effects of age (P = 0.006) and adiposity (P = 0.002) were observed in the muscle FSR, with age decreasing it and adiposity increasing it. CONCLUSION: This study revealed that a higher level of adiposity resulted in a slight increase in protein and individual amino acid true digestibility values and seemed to compensate for the metabolic postprandial protein alterations observed at older age.

Multi-criteria assessment of pea protein quality in rats: a comparison between casein, gluten and pea protein alone or supplemented with methionine.

The objective of this study was to assess the nutritional quality of pea protein isolate in rats and to evaluate the impact of methionine (Met) supplementation. Several protein diets were studied: pea protein, casein, gluten, pea protein-gluten combination and pea protein supplemented with Met. Study 1: Young male Wistar rats (n 8/group) were fed the test diets ad libitum for 28 d. The protein efficiency ratio (PER) was measured. Study 2: Adult male Wistar rats (n 9/group) were fed the test diets for 10 d. A protein-free diet group was used to determine endogenous losses of N. The rats were placed in metabolism cages for 3 d to assess N balance, true faecal N digestibility and to calculate the Protein Digestible-Corrected Amino Acid Score (PDCAAS). They were then given a calibrated meal and euthanised 6 h later for collection of digestive contents. The true caecal amino acid (AA) digestibility was determined, and the Digestible Indispensable Amino Acid Score (DIAAS) was calculated. Met supplementation increased the PER of pea protein (2·52 v. 1·14, P < 0·001) up to the PER of casein (2·55). Mean true caecal AA digestibility was 94 % for pea protein. The DIAAS was 0·88 for pea protein and 1·10 with Met supplementation, 1·29 for casein and 0·25 for gluten. Pea protein was highly digestible in rats under our experimental conditions, and Met supplementation enabled generation of a mixture that had a protein quality that was not different from that of casein.

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.

Tissue-specific effect of colitis on protein synthesis in mice: impact of the dietary protein content.

PURPOSE: Inflammatory bowel diseases are associated with an increase in the whole-body protein turnover, thus possibly requiring an additional supply of dietary proteins. Our aim was to evaluate whether increasing dietary protein content could alleviate protein metabolism alterations in the injured splanchnic and peripheral tissues during colitis and spontaneous mucosal healing. METHODS: Mice with acute chemically induced colitis received either a normal protein (P14, 14% as energy), a moderately (P30, 30%) and a very high-protein (P53, 55%) diets. At different times after the challenge, protein synthesis rate was determined in tissues using a flooding dose of 13C valine. RESULTS: Colon, liver and spleen protein synthesis rates were significantly increased after colitis induction, while being decreased in the caecum, kidneys and muscle. Contrastingly to the two other diets, P30 diet consumption allowed faster recovery of the animals, and this coincided with a rapid resaturation of the initial protein synthesis in the colon. In the other tissues studied, the high-protein diets show different effects depending on the dietary protein content consumed and on the examined tissues, with a general trend of P53 in lowering anabolism rates. CONCLUSION: This study highlights the severe impact of acute colonic inflammation on protein metabolism in different organs. In addition, dietary protein content modulated the recovery of the initial protein synthesis rate in the various tissues following colitis induction. P30 diet consumption notably showed a better ability to alleviate protein metabolism perturbations induced by colitis, that may explain its documented beneficial effect on colon mucosal healing.

15N and ²H Intrinsic Labeling Demonstrate That Real Digestibility in Rats of Proteins and Amino Acids from Sunflower Protein Isolate Is Almost as High as That of Goat Whey.

BACKGROUND: In the context of developing plant protein sources for humans, sunflower is a good candidate in its form as an oilseed coproduct. OBJECTIVES: We aimed to compare the real digestibility in rats of a sunflower isolate to that of goat whey protein. We also studied the efficiency of 15N and 2H intrinsic labeling in this assessment. METHODS: Sunflower seeds and goat milk were labeled with 15N and 2H. Male Wistar rats (10 wk old) were fed a meal containing 12% of either sunflower isolate (n = 8) or whey (n = 8). Six hours after meal ingestion, protein and amino acid digestibility were assessed by measuring nitrogen, hydrogen, and amino acids in the digesta, as well as isotope enrichments in the bulk and individual amino acids. The differences between groups and isotopes were respectively tested with an unpaired and a paired t test. RESULTS: Protein isolate purity was 87% for whey and 94% for sunflower. 2H and 15N enrichments were, respectively, 0.12 atom % (AP) and 1.06 AP in sunflower isolate and 0.18 AP and 0.95 AP in whey. Fecal 15N protein digestibility was 97.2 ± 0.2% for whey and 95.1 ± 0.5% for sunflower isolate. The use of 2H resulted in a lower digestibility estimate than 15N for whey (96.9 ± 0.2%, P < 0.05) and sunflower (94.2 ± 0.5%, P < 0.01). For both isotopes, protein digestibility was about 2% higher for whey than for sunflower isolate. Mean 15N amino acid caecal digestibility was 97.5 ± 0.2% for whey and 96.3 ± 0.2% for sunflower isolate. The values obtained with 15N and 2H resulted in significant differences ranging from -0.1% to 3.5%. The DIAAS was >1.0 for whey and 0.84 for sunflower (lysine). CONCLUSIONS: The protein and amino acid digestibility of sunflower isolate was high but its DIAAS reflected a moderate lysine imbalance. Despite slight differences with 15N, deuterium produced comparable results, making it suitable for in vivo digestion studies.

Blood 15N:13C Enrichment Ratios Are Proportional to the Ingested Quantity of Protein with the Dual-Tracer Approach for Determining Amino Acid Bioavailability in Humans.

BACKGROUND: Assessment of amino acid bioavailability is of key importance for the evaluation of protein quality; however, measuring ileal digestibility of dietary proteins in humans is challenging. Therefore, a less-invasive dual stable isotope tracer approach was developed. OBJECTIVE: We aimed to test the assumption that the 15N:13C enrichment ratio in the blood increases proportionally to the quantity ingested by applying different quantities of 15N test protein. METHODS: In a crossover design, 10 healthy adults were given a semi-liquid mixed meal containing 25 g (low protein) or 50 g (high protein) of 15N-labeled milk protein concentrate simultaneous with 0.4 g of highly 13C-enriched spirulina. The meal was distributed over multiple small portions, frequently provided every 20 min during a period of 160 min. For several amino acids, the blood 15N- related to 13C-isotopic enrichment ratio was determined at t = 0, 30, 60, 90, 120, 180, 240, 300, and 360 min and differences between the 2 meals were compared using paired analyses. RESULTS: No differences in 13C AUC for each of the measured amino acids in serum was observed when ingesting a low- or high-protein meal, whereas 15N AUC of amino acids was ∼2 times larger on the high-protein meal (P < 0.001). Doubling the intake of 15N-labeled amino acids increased the 15N:13C ratio by a factor of 2.04 ± 0.445 for lysine and a factor between 1.8 and 2.2 for other analyzed amino acids, with only phenylalanine (2.26), methionine (2.48), and tryptophan (3.02) outside this range. CONCLUSIONS: The amino acid 15N:13C enrichment ratio in the peripheral circulation increased proportionally to the quantity of 15N-labeled milk protein ingested, especially for lysine, in healthy adults. However, when using 15N-labeled protein, correction for, e.g., α-carbon 15N atom transamination is advised for determination of bioavailability of individual amino acids. This trial was registered at www.clinicaltrials.gov as NCT02966704.

Effect of different bariatric surgeries on dietary protein bioavailability in rats.

Bariatric surgery may induce protein malabsorption, although data are scarce. This study aims at evaluating dietary protein bioavailability after different bariatric surgeries in rats. Diet-induced obese Wistar rats were operated for vertical sleeve gastrectomy (VSG) or Roux-en-Y gastric bypass (RYGB). The control group was composed of pair-fed, sham-operated rats (Sham). Two weeks after surgery, rats were fed a 15N protein meal. Protein bioavailability was assessed by determination of 15N recovery in the gastrointestinal tract and organs 6 h after the meal. Fractional protein synthesis rate (FSR) was assessed using a flooding dose of 13C valine. Weight loss was the highest in RYGB rats and the lowest in Sham rats. Surprisingly, RYGB (95.6 ± 0.7%) improved protein digestibility (P = 0.045) compared with Sham (93.5 ± 0.5%) and VSG (93.8 ± 0.6%). In contrast, 15N retained in the liver (P = 0.001) and plasma protein (P = 0.037) was lower than in Sham, with a similar trend in muscle (P = 0.052). FSR was little altered by bariatric surgery, except for a decrease in the kidney of RYGB (P = 0.02). The 15N distribution along the small intestinal tissue suggests that dietary nitrogen was considerably retained in the remodeled mucosa of RYGB compared with Sham. This study revealed that in contrast to VSG, RYGB slightly improved protein digestibility but altered peripheral protein bioavailability. This effect may be ascribed to a higher uptake of dietary amino acids by the remodeled intestine.NEW & NOTEWORTHY Using a sensitive 15N meal test, we found that gastric bypass slightly improved protein digestibility compared with sleeve gastrectomy or control but, in contrast, lowered protein retention in the liver and muscles. This paradox can be due to a higher uptake of dietary nitrogen by the intestinal mucosa that was hypertrophied. This study provides new insight on the digestive and metabolic fate of dietary protein in different models of bariatric surgery in rats.

High Pancreatic Amylase Expression Promotes Adiposity in Obesity-Prone Carbohydrate-Sensitive Rats.

BACKGROUND: We have reported large differences in adiposity (fat mass/body weight) gain between rats fed a low-fat, high-starch diet, leading to their classification into carbohydrate "sensitive" and "resistant" rats. In sensitive animals, fat accumulates in visceral adipose tissues, leading to the suggestion that this form of obesity could be responsible for rapid development of metabolic syndrome. OBJECTIVE: We investigated whether increased amylase secretion by the pancreas and accelerated starch degradation in the intestine could be responsible for this phenotype. METHOD: Thirty-two male Wistar rats (7-wk-old) were fed a purified low-fat (10%), high-carbohydrate diet for 6 wk, in which most of the carbohydrate (64% by energy) was provided as corn starch. Meal tolerance tests of the Starch diet were performed to measure glucose and insulin responses to meal ingestion. Indirect calorimetry combined with use of 13C-labelled dietary starch was used to assess meal-induced changes in whole body and starch-derived glucose oxidation. Real-time polymerase chain reaction was used to assess mRNA expression in pancreas, liver, white and brown adipose tissues, and intestine. Amylase activity was measured in the duodenum, jejunum, and ileum contents. ANOVA and regression analyses were used for statistical comparisons. RESULTS: "Resistant" and "sensitive" rats were separated according to adiposity gain during the study (1.73% ± 0.20% compared with 4.35% ± 0.36%). Breath recovery of 13CO2 from 13C-labelled dietary starch was higher in "sensitive" rats, indicating a larger increase in whole body glucose oxidation and, conversely, a larger decrease in lipid oxidation. Amylase mRNA expression in pancreas, and amylase activity in jejunum, were also higher in sensitive rats. CONCLUSION: Differences in digestion of starch can promote visceral fat accumulation in rats when fed a low-fat, high-starch diet. This mechanism may have important implications in human obesity.

Time course of fractional gluconeogenesis after meat ingestion in healthy adults: a D2O study.

In the postprandial state, glucose homeostasis is challenged by macronutrient intake, including proteins that trigger insulin secretion and provide glucose precursors. However, little is known about the postprandial response of gluconeogenesis to a protein meal. We aimed to quantify the evolution of fractional gluconeogenesis after a meat meal. Thirteen healthy subjects received oral doses of D2O. After fasting overnight, they ingested a steak (120 g). Glycemia, insulinemia, and 2H enrichments in glucose and plasma water were measured for 8 h after the meal. Fractional gluconeogenesis was assessed using the average method. Glucose was stable for 5 h and then decreased. There was a slight increase of insulin 1 h after the meal. 2H enrichment in the carbon 5 position of glucose (C5) increased after 2 h, whereas it decreased in plasma water. Consequently, fractional gluconeogenesis increased from 68.2 ± 7.2% before the meal to 75.5 ± 5.8% 8 h after the meal, the latter corresponding to 22 h without a glucose supply. These values are consistent with the exhaustion of glycogen stores after 24 h but represent the highest among values in the literature. The impact of methodological conditions is discussed.

Metabolic markers of protein maldigestion after a 15N test meal in minipigs with pancreatic exocrine insufficiency.

The effect of pancreatic exocrine insufficiency (PEI) on protein malabsorption is little documented, partly due to methodological barriers. We aimed to validate biomarkers of protein malabsorption using a 15N test meal in a minipig model of PEI. Six pancreatic duct-ligated minipigs were used as a model of PEI and four nonoperated animals as a control. All animals were equipped with an ileocecal reentrant cannula. Minipigs were given a test meal containing [15N]casein. The PEI animals repeated the test three times, in the absence of any pancreatic enzymes, or after pancreatic substitution at two levels [ A or B: 7,500 or 75,000 (lipase) and 388 or 3881 (protease) FIP U]. Ileal chyme, urine, and blood were collected postprandially. Nitrogen and 15N were measured in digestive and metabolic pools. We obtained a gradient of ileal protein digestibility from 29 ± 11% in PEI to 89 ± 6% in the controls and a dose- dependent response of enzymes. Insulin and gastric inhibitory polypeptide secretions were decreased by PEI, an effect that was counteracted with the enzymes at level B. The total recovery of 15N in urinary urea and plasma proteins was 14 ± 5.1% in the control group and decreased to 5.5 ± 2.1% by PEI. It was dose dependently restored by the treatment. Both 15N recovery in plasma and urine were correlated to protein digestibility. We confirm that the 15N transfer in those pools is a sensitive marker of protein malabsorption. Nevertheless, an optimization of the test meal conditions would be necessary in the view of implementing a clinical test. NEW & NOTEWORTHY We designed an intervention study to create a gradient of ileal protein digestibility in minipigs with pancreatic exocrine insufficiency and to validate reliable metabolic markers using a 15N oral meal test. 15N recovery in plasma proteins and to a higher extent in urine was sensitive to protein malabsorption. This test is minimally invasive and could be used to reveal protein malabsorption in patients.

Adaptation to a high-protein diet progressively increases the postprandial accumulation of carbon skeletons from dietary amino acids in rats.

We aimed to determine whether oxidative pathways adapt to the overproduction of carbon skeletons resulting from the progressive activation of amino acid (AA) deamination and ureagenesis under a high-protein (HP) diet. Ninety-four male Wistar rats, of which 54 were implanted with a permanent jugular catheter, were fed a normal protein diet for 1 wk and were then switched to an HP diet for 1, 3, 6, or 14 days. On the experimental day, they were given their meal containing a mixture of 20 U-[15N]-[13C] AA, whose metabolic fate was followed for 4 h. Gastric emptying tended to be slower during the first 3 days of adaptation. 15N excretion in urine increased progressively during the first 6 days, reaching 29% of ingested protein. 13CO2 excretion was maximal, as early as the first day, and represented only 16% of the ingested proteins. Consequently, the amount of carbon skeletons remaining in the metabolic pools 4 h after the meal ingestion progressively increased to 42% of the deaminated dietary AA after 6 days of HP diet. In contrast, 13C enrichment of plasma glucose tended to increase from 1 to 14 days of the HP diet. We conclude that there is no oxidative adaptation in the early postprandial period to an excess of carbon skeletons resulting from AA deamination in HP diets. This leads to an increase in the postprandial accumulation of carbon skeletons throughout the adaptation to an HP diet, which can contribute to the sustainable satiating effect of this diet.

Compared with Raw Bovine Meat, Boiling but Not Grilling, Barbecuing, or Roasting Decreases Protein Digestibility without Any Major Consequences for Intestinal Mucosa in Rats, although the Daily Ingestion of Bovine Meat Induces Histologic Modifications in the Colon.

BACKGROUND: Cooking may impair meat protein digestibility. When undigested proteins are fermented by the colon microbiota, they can generate compounds that potentially are harmful to the mucosa. OBJECTIVES: This study addressed the effects of typical cooking processes and the amount of bovine meat intake on the quantity of undigested proteins entering the colon, as well as their effects on the intestinal mucosa. METHODS: Male Wistar rats (n = 88) aged 8 wk were fed 11 different diets containing protein as 20% of energy. In 10 diets, bovine meat proteins represented 5% [low-meat diet (LMD)] or 15% [high-meat diet (HMD)] of energy, with the rest as total milk proteins. Meat was raw or cooked according to 4 processes (boiled, barbecued, grilled, or roasted). A meat-free diet contained only milk proteins. After 3 wk, rats ingested a (15)N-labeled meat meal and were killed 6 h later after receiving a (13)C-valine injection. Meat protein digestibility was determined from (15)N enrichments in intestinal contents. Cecal short- and branched-chain fatty acids and hydrogen sulfide were measured. Intestinal tissues were used for the assessment of protein synthesis rates, inflammation, and histopathology. RESULTS: Meat protein digestibility was lower in rats fed boiled meat (94.5% ± 0.281%) than in the other 4 groups (97.5% ± 0.0581%, P < 0.001). Cecal and colonic bacterial metabolites, inflammation indicators, and protein synthesis rates were not affected by cooking processes. The meat protein amount had a significant effect on cecal protein synthesis rates (LMD > HMD) and on myeloperoxidase activity in the proximal colon (HMD > LMD), but not on other outcomes. The ingestion of bovine meat, whatever the cooking process and the intake amount, resulted in discrete histologic modifications of the colon (epithelium abrasion, excessive mucus secretion, and inflammation). CONCLUSIONS: Boiling bovine meat at a high temperature (100°C) for a long time (3 h) moderately lowered protein digestibility compared with raw meat and other cooking processes, but did not affect cecal bacterial metabolites related to protein fermentation. The daily ingestion of raw or cooked bovine meat had no marked effect on intestinal tissues, despite some slight histologic modifications on distal colon.

High True Ileal Digestibility but Not Postprandial Utilization of Nitrogen from Bovine Meat Protein in Humans Is Moderately Decreased by High-Temperature, Long-Duration Cooking.

BACKGROUND: Meat protein digestibility can be impaired because of indigestible protein aggregates that form during cooking. When the aggregates are subsequently fermented by the microbiota, they can generate potentially harmful compounds for the colonic mucosa. OBJECTIVE: This study evaluated the quantity of bovine meat protein escaping digestion in the human small intestine and the metabolic fate of exogenous nitrogen, depending on cooking processes. METHODS: Sixteen volunteers (5 women and 11 men; aged 28 ± 8 y) were equipped with a double lumen intestinal tube positioned at the ileal level. They received a test meal exclusively composed of 120 g of intrinsically (15)N-labeled bovine meat, cooked either at 55°C for 5 min (n = 8) or at 90°C for 30 min (n = 8). Ileal effluents and blood and urine samples were collected over an 8-h period after the meal ingestion, and (15)N enrichments were measured to assess the digestibility of meat proteins and the transfer of dietary nitrogen into the metabolic pools. RESULTS: Proteins tended to be less digestible for the meat cooked at 90°C for 30 min than at 55°C for 5 min (90.1% ± 2.1% vs. 94.1% ± 0.7% of ingested N; P = 0.08). However, the particle number and size in ileal digesta did not differ between groups. The appearance of variable amounts of intact fibers was observed by microscopy. The kinetics of (15)N appearance in plasma proteins, amino acids, and urea were similar between groups. The amount of exogenous nitrogen lost through deamination did not differ between groups (21.2% ± 0.8% of ingested N). CONCLUSIONS: Cooking bovine meat at a high temperature for a long time can moderately decrease protein digestibility compared with cooking at a lower temperature for a short time and does not affect postprandial exogenous protein metabolism in young adults. The study was registered at www.clinicaltrials.gov as NCT01685307.