Romain Tessier

Lara Ribeiro-Parenti

Ouafa Bruneau

Nadezda Khodorova

Jean- Baptiste Cavin

André Bado

Dalila Azzout-Marniche

Juliane Calvez

Maude Le Gall

Ph.D Claire Gaudichon
email: claire.gaudichon@agroparistech.fr

Effect of different bariatric surgeries on dietary protein bioavailability in rats

Keywords: Gastric Bypass, Sleeve gastrectomy, Protein digestibility, Protein retention, 15 N test meal , bilio-pancreatic limb, FSR, fractional synthesis rate, GC, gas chromatography, HFD, high fat diet, IRMS, isotopic ratio mass spectrometry, MS, mass spectrometry, NAFLD, non-alcoholic fat liver disease, RFD, real fecal digestibility, RYGB, Roux-en-Y gastric bypass, VSG, vertical sleeve gastrectomy

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), Rouxen-Y gastric bypass (RYGB). The control group was composed of pair-fed, sham-operated rats (Sham). Two weeks after surgery, rats were fed a 15 N protein meal. Protein bioavailability was assessed by determination of 15 N recovery in the gastrointestinal tract and organs 6 h after the meal. Fractional protein synthesis rate (FSR) was assessed using a flooding dose of 13 C valine.

Weight loss was the highest in RYGB and the lowest in Sham. Surprisingly, RYGB (95.6 ± 0.7%) improved protein digestibility (P=0.045) compared to Sham (93.5 ± 0.5%) and VSG (93.8 ± 0.6%). In contrast, 15 N 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 15 N distribution along the small intestinal tissue suggests that dietary nitrogen was considerably retained in the remodeled mucosa of RYGB compared to 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 and Notheworthy

Using a sensitive 15 N meal test, we found that gastric bypass slightly improved protein digestibility compared to 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 3 nitrogen by the intestinal mucosa that was hypertrophied. This study provides new insight on 52

the digestive and metabolic fate of dietary protein in different models of bariatric surgery in 53 rats. 54 Downloaded from www.physiology.org/journal/ajpgi at INRA Institut National de la Recherche Agronomique (138.102.192.062) on September 3, 2019.

Introduction

Bariatric surgery is one of the most efficient treatments of morbid obesity, inducing sustainable weight loss [START_REF] Brethauer | Systematic review of sleeve gastrectomy as staging and primary bariatric procedure[END_REF][START_REF] Georgiadou | Efficacy and safety of laparoscopic mini gastric bypass. A systematic review[END_REF][START_REF] O'brien | Systematic Review of Medium-Term Weight Loss after Bariatric Operations[END_REF] and improvement of associated comorbidities [START_REF] Colquitt | Surgery for weight loss in adults[END_REF].Historically, literature distinguished two kinds of bariatric surgeries: purely restrictive versus restrictive and malabsorptive. Food intake restriction was supposed to be induced by a reduction of stomach size, whereas malabsorption may result from the derivation of the proximal small intestine from the alimentary tract, therefore theoretically reducing nutrient absorption. The purely restrictive vertical sleeve gastrectomy (VSG) and the restrictive and malabsorptive Roux-en-Y gastric bypass (RYGB) are the most performed bariatric surgeries worldwide (4). VSG and RYGB procedures limit the gastric volume and thereby reduce food intake during a single meal but this mechanical restriction cannot fully explain the reported reduction of energy intake as subjects could compensate by calorie dense food or multiple meals. The stomach is also a main source of hormones and peptides such as the orexigenic hormone, ghrelin. The latter has reported to be reduced after VSG [START_REF] Alamuddin | Changes in Fasting and Prandial Gut and Adiposity Hormones Following Vertical Sleeve Gastrectomy or Roux-en-Y-Gastric Bypass: an 18-Month Prospective Study[END_REF][START_REF] Kalinowski | Ghrelin, leptin, and glycemic control after sleeve gastrectomy versus Roux-en-Y gastric bypass-results of a randomized clinical trial[END_REF] which can contribute to the anorexigenic effects of this procedure. RYGB has also been associated with reduced ghrelin [START_REF] Cummings | Plasma Ghrelin Levels after Diet-Induced Weight Loss or Gastric Bypass Surgery[END_REF] but this remains disputed [START_REF] Barazzoni | Gastric bypass does not normalize obesity-related changes in ghrelin profile and leads to higher acylated ghrelin fraction[END_REF]. Moreover, RYGB was associated with increases in glucagon-like peptide-1 (GLP-1) and peptide YY (PYY that contribute to reduction of food intake [START_REF] Pucci | Mechanisms underlying the weight loss effects of RYGB and SG: similar, yet different[END_REF]. Additionally, as the stomach secretes hydrochloric acid and pepsinogen that are implicated in the early steps of protein digestion, VSG and RYGB could also lead to a protein maldigestion.

RYGB has been shown to induce nutrient malabsorption [START_REF] Jammu | A 7-Year Clinical Audit of 1107 Cases Comparing Sleeve Gastrectomy, Roux-En-Y Gastric Bypass, and Mini-Gastric Bypass, to Determine an Effective and Safe Bariatric and Metabolic Procedure[END_REF][START_REF] Odstrcil | The contribution of malabsorption to the reduction in net energy absorption after long-limb Roux-en-Y gastric bypass[END_REF] associated with the length of the bypassed limb. Whereas carbohydrate and lipid malabsorption may contribute to the positive effects of bariatric surgery, protein malabsorption [START_REF] Verger | Nutritional and Protein Deficiencies in the Short Term following Both Gastric Bypass and Gastric Banding[END_REF][START_REF] Faintuch | Severe Protein-Calorie Malnutrition after Bariatric Procedures[END_REF] can be the cause of a large range of clinical consequences, from severe undernutrition leading to parenteral nutrition to death [START_REF] Grover | Protein Energy Malnutrition[END_REF]. Low albumin levels and excessive weight loss are signs of protein wasting after bariatric surgery [START_REF] Fromentin | The postprandial use of dietary amino acids as an energy substrate is delayed Downloaded from www.physiology.org/journal[END_REF]. It has been shown that severe protein malnutrition occurred in 4.7% of patients who underwent RYGB surgery [START_REF] Faintuch | Severe Protein-Calorie Malnutrition after Bariatric Procedures[END_REF]. Comment citer ce document : Tessier, R., Ribeiro-Parenti, L., Bruneau, O., Khodorova, N., Cavin, Bado, Azzout-Marniche, D., Calvez, J., Le Gall, Gaudichon, C. (Auteur de correspondance) (2019). Effect of different bariatric surgeries on dietary protein bioavailability in rats. American Journal of Physiology. Gastrointestinal and Liver Physiology, 317, G592-G601. , DOI : 10.1152/ajpgi.00142.2019

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The degree of protein malabsorption after bariatric surgery has not been well established.

Using intrinsically 13 C labeled casein, Bosjen-Møller et al. [START_REF] Bojsen-Møller | Accelerated protein digestion and amino acid absorption after Roux-en-Y gastric bypass[END_REF] showed accelerated blood appearance of dietary amino acids in patients operated for RYGB. In the absence of any digesta collection, it is not known whether this paradoxical effect is only due to a faster digestion kinetic or also to a higher protein bioavailability. In contrast, other studies reported a higher fecal output of nitrogen in rats after RYGB and biliopancreatic diversion [START_REF] Cavin | Malabsorption and intestinal adaptation after one anastomosis gastric bypass compared with Roux-en-Y gastric bypass in rats[END_REF][START_REF] Nadreau | Effects of the biliopancreatic diversion on energy balance in the rat[END_REF] and in humans after RYGB [START_REF] Odstrcil | The contribution of malabsorption to the reduction in net energy absorption after long-limb Roux-en-Y gastric bypass[END_REF]. Overall, none of these studies determined the real digestibility of dietary proteins and their postprandial retention in organs.

This study aimed to characterize digestive and metabolic protein bioavailability by using a test meal containing 15 N labeled proteins. For this purpose, VSG and RYGB rats were compared with pair-fed Sham rats for weight loss, energy intake, body composition, protein real digestibility, dietary protein retention in tissues and protein synthesis rate.

Materials and Methods

Animal and diets

All animal studies comply with the ARRIVE guidelines. Comment citer ce document : Tessier, R., Ribeiro-Parenti, L., Bruneau, O., Khodorova, N., Cavin, Bado, Azzout-Marniche, D., Calvez, J., Le Gall, Gaudichon, C. (Auteur de correspondance) (2019). Effect of different bariatric surgeries on dietary protein bioavailability in rats. American Journal of Physiology. Gastrointestinal and Liver Physiology, 317, G592-G601. , DOI : 10.1152/ajpgi.00142.2019 6 After 4 months of HFD, animals were randomly divided into 3 groups: Roux-en-Y gastric bypass (RYGB, n = 18), vertical sleeve gastrectomy (VSG, n = 7) and sham-operated (Sham, n = 16). Initially, two different Sham procedures were performed to match with sleeve and gastric bypasses but rats were then pooled together without any differences in principal criteria. The rats were fasted the night before surgery. Anesthesia was induced and maintained by gaseous inhalation of isoflurane (Vetflurane, Virbac, France). Procedures were carried out as previously described [START_REF] Arapis | Remodeling of the Residual Gastric Mucosa after Roux-En-Y Gastric Bypass or Vertical Sleeve Gastrectomy in Diet-Induced Obese Rats[END_REF][START_REF] Cavin | Differences Downloaded from www.physiology.org/journal[END_REF][START_REF] Cavin | Malabsorption and intestinal adaptation after one anastomosis gastric bypass compared with Roux-en-Y gastric bypass in rats[END_REF][START_REF] Duboc | Roux-en-Y Gastric-Bypass and sleeve gastrectomy induces specific shifts of the gut microbiota without altering the metabolism of bile acids in the intestinal lumen[END_REF]52). After laparotomy, the stomach was isolated outside of the abdominal cavity. Gastric connections to the spleen and liver were severed along the greater curvature, and the suspensory ligament supporting the upper fundus was dissociated. The surgical procedures are depicted in Fig. 1.

VSG

The non-glandular part of the stomach was removed by application of an ETS-Flex 35 mm staple line (Ethicon, Issy les Moulineaux, France). Then, the procedure was to resect 80% of gastric fundus stomach by application of a second ETS-Flex 35 mm staple line, leaving a thin gastric tube in continuity with the esophagus.

RYGB

The non-glandular part of the stomach (forestomach) was resected using an ETS-Flex 35 mm staple line (Ethicon, Issy les Moulineaux, France). A second staple line (Proximate TX30V 30 mm, Ethicon, Issy les Moulineaux, France), parallel to the first one, was applied to delimit a gastric pouch that represented 20% of the initial stomach size while preserving the arterial and venous supply. The jejunum was transected 20 cm after the pylorus. The alimentary limb was anastomosed to the gastric pouch and the biliopancreatic limb was anastomosed 15 cm distally to gastrojejunal anastomosis. Sutures were made by a 7-0 Prolene wire (Ethicon, Issy les Moulineaux, France). To simulate surgery the stomach was pinched with an unarmed staple gun, and for bypass sham-operated rats the jejunum was incised and stitched immediately after.

Sham

All operations ended with 4-0 vicryl and 3-0 vicryl (Ethicon, Issy les Moulineaux, France) sutures to sew up the abdominal wall and the skin, respectively.

Post-surgery care

Animals received an analgesic (Xylocaine, Astra, 10 mg/kg) and an antibiotic (Penicillin, 20 000 units/kg, PanPharma, Boulogne Billancourt, France) after surgery. They were placed in individual cages with a reversed light/dark cycle with lights on from 8.00 P.M. to 8.00 A.M.

For two days following the surgery, rats were injected subcutaneously with 12 ml/day of an isotonic polyionic solution (Bionolyte G5, Baxter, Guyancourt, France). Between day 3 and day 10, they were refed a hydrated standard diet ad libitum (Supplemental Table 1). Sham animals were pair-fed with operated animals.

Habituation to consumption of a single test meal

From day 11 to day 19, rats were accustomed to rapid consumption of a single meal in the morning. For this purpose they received 4 g of standard diet between 8.30 A.M. and 9.00 A.M., followed by free access to the diet between noon and 6.30 P.M. Between those two periods rats were fasted with free access to water. Sham rats were pair-fed with operated rats. On day 19, rats were fed a single test meal of the same composition as the standard diet, but in which proteins were intrinsically labeled with 15 N, as described previously [START_REF] Lacroix | Heat Markers and Quality Indexes of Industrially Heat-Treated [ 15 N] Milk Protein Measured in Rats[END_REF].

Thirty minutes before being euthanized, rats were injected in the lateral tail vein with 150 µmol/100 g of body weight of 1-13 C-Valine (Eurisotop, Saint Aubin, France) under gaseous anesthesia. Six hours after the meal, rats were injected intraperitoneally with a lethal dose of pentobarbital sodium (100 mg/kg) and exsanguinated. Whole blood was collected and plasma was isolated and stored at -20°C after centrifugation. Different intestinal segments were identified depending on the surgery procedure: stomach, jejunum, ileum, caecum, and colon. The feces emitted during the postprandial period were pooled with colon content. For

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RYGB the biliopancreatic, alimentary and common limbs were collected (Fig. 1). The ileum was defined as the 10 cm before the caecum to recover sufficient amount of samples, although ileum might be shorter accordingly to Vdoviaková et al. [START_REF] Vdoviaková | Surgical Anatomy of the Gastrointestinal Tract and Its Vasculature in the Laboratory Rat[END_REF] . Segments were rinsed with NaCl solution (9‰), and the contents were collected in their entirety, weighed and stored at -20°C. A sample of each intestinal wall was collected and immediately frozen in liquid nitrogen. Liver, kidney, extensor digitorum longus muscle and skin were also sampled, weighed, frozen in liquid nitrogen and stored at -80°C.

Measurement of food intake, body weight and whole body composition

Body weight and food intake were assessed daily. Whole body composition was measured 7 days before surgery and 15 days after with an Echo Medical systems EchoMRI 900 (Whole Body Composition Analyzers, Houston, USA). For technical reasons related to the device, body composition was assessed before surgery in only 18 animals.

Analytical methods

Protein bioavailability was assessed by following the 15 N recovery in digestive contents and organs. Digestive contents were freeze-dried. Intestine, kidney, liver, muscle and skin were cold-grinded with liquid nitrogen and freeze-dried. Plasma protein extraction was performed by precipitation with 5-sulfosalicylic acid (100%).

Nitrogen percentage and 15 N enrichment in digestive samples, organs and plasma proteins were determined by an elemental analyzer (Vario Micro Cube, Elementar, Lyon, France) coupled with isotopic ratio mass spectrometry (IRMS) (Isoprime, GV Instrument, Manchester, UK). Atropine (Thermo Electron, Milano, Italy) and L-glutamic acid (USGS41, Sigma Aldrich, Saint-Louis, USA) were used as elemental and isotopic standards, respectively.

In vivo protein synthesis rate in liver, kidney, skin and muscle was assessed using the 13 Cvaline flooding dose method, as described previously [START_REF] Chevalier | Highprotein diets differentially modulate protein content and protein synthesis in visceral and peripheral tissues in rats[END_REF][START_REF] Oberli | 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[END_REF]. After protein precipitation of the different tissues, amino acids and protein bound fractions were separated. Free amino acids were derivatized with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide mixed with Comment citer ce document : Tessier, R., Ribeiro-Parenti, L., Bruneau, O., Khodorova, N., Cavin, Bado, Azzout-Marniche, D., Calvez, J., Le Gall, Gaudichon, C. (Auteur de correspondance) (2019). Effect of different bariatric surgeries on dietary protein bioavailability in rats. American Journal of Physiology. Gastrointestinal and Liver Physiology, 317, G592-G601. , DOI : 10.1152/ajpgi.00142.2019

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1% tert-butyldimethylchlorosilane and acetonitrile (Sigma Aldrich, St Louis, USA). 13 C-valine enrichment in free amino acids was analyzed by gas chromatography (GC 6890N, Agilent Technologies, Les Ulis, France) coupled to mass spectrometry (MS 5973N, Agilent Technologies, Les Ulis, France) (GC-MS) with electron impact ionization and selected ion monitoring (ions m/z 288 and 289). After hydrolysis, protein bound amino acids were derivatized with propanol and acetyl chloride (Sigma Aldrich, St Louis, USA). 13 C-valine enrichment in proteins was analyzed by GC-combustion-IRMS (Isoprime, GV Instrument, Manchester, UK).

Histology

Intestinal segments were fixed overnight in formalin and embedded in paraffin. Three µm blank slides were cut from each block to perform hematoxylin phloxine saffron (HPS) staining. Each slide was scanned with an Aperio ScanScope® CS System (Leica Microsystemes SAS, Nanterre, France).

Calculations

The exogenous nitrogen (N exo ) in each tissue was determined as follows:

N = N × APE APE
where N exo is exogenous nitrogen (mmol), N tot is amount of nitrogen in the sample (mmol), and APE s/m is enrichment excess in the sample (s) or the meal (m). (APE s/m = AP s/m -natural enrichment). Natural enrichment values were determined in previous studies for digesta [START_REF] Fromentin | The postprandial use of dietary amino acids as an energy substrate is delayed Downloaded from www.physiology.org/journal[END_REF] and tissues [START_REF] Poupin | The Nature of the Dietary Protein Impacts the Tissue-to-Diet 15N Discrimination Factors in Laboratory Rats[END_REF] in rats after adaptation to a milk protein diet.

Recovery of dietary nitrogen in plasma protein was related to the plasma volume in the rat (3.5% of body weight) [START_REF]Removal of blood from laboratory mammals and birds: First Report of the BVA/FRAME/RSPCA/UFAW Joint Working Group on Refinement[END_REF].

As amino acids and peptides are absorbed massively in the duodenum and jejunum, dietary N losses were determined from the N recovery in the distal parts of the intestine (ileum and Comment citer ce document : Tessier, R., Ribeiro-Parenti, L., Bruneau, O., Khodorova, N., Cavin, Bado, Azzout-Marniche, D., Calvez, J., Le Gall, Gaudichon, C. (Auteur de correspondance) (2019). Effect of different bariatric surgeries on dietary protein bioavailability in rats. American Journal of Physiology. Gastrointestinal and Liver Physiology, 317, G592-G601. , DOI : 10.1152/ajpgi.00142.2019 10 large intestine, including feces). As digestion was almost complete after 6 h according to this study and previous ones [START_REF] Lacroix | Heat Markers and Quality Indexes of Industrially Heat-Treated [ 15 N] Milk Protein Measured in Rats[END_REF][START_REF] Oberli | 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[END_REF], the losses due to residual N in the stomach and intestine were considered as negligible. Accordingly, the real fecal protein digestibility (RFD, % of nitrogen ingested) was calculated as follows:

RFD = N -(N + N + N ) N × 100
where N ing is the amount of nitrogen ingested (mmol).

The fractional protein synthesis rate (FSR, in %/d) in each organ was calculated as follows:

FSR = E -E E × t × 24 × 60 × 100
where E free valine and E protein-bound valine are the 13 C valine enrichments in the free and proteinbound amino acids, respectively. E basal is the value of the non-enriched leucine in the same sample, as a surrogate of basal valine enrichment [START_REF] Boutry | Rapeseed and milk protein exhibit a similar overall nutritional value but marked difference in postprandial regional nitrogen utilization in rats[END_REF]. t inc is the time of incorporation of 13 Cvaline (in min) between the injection and euthanasia (30 min in average). The factors 24 (h) and 60 (min) were used to calculate the value in % per day [START_REF] Chevalier | Highprotein diets differentially modulate protein content and protein synthesis in visceral and peripheral tissues in rats[END_REF].

The absolute synthesis rate (ASR, g/d) was calculated as:

ASR = FSR × P
where P content is the protein content of the tissue (P content = %N x 6.25).

Statistical analysis

All results are expressed as means ± standard errors of the mean (SEM). Follow-up data were analyzed in a mixed model with group as a fixed factor and time as a repeated factor, using SAS 9.1 (SAS Institute, Cary, NC, USA). Otherwise, differences between groups were tested using a one-way ANOVA with the group as a factor and post-hoc Bonferroni tests for

Results

Follow up

Survival rate

Out of forty two rats, one died before surgery, without any explanation. For Sham, the survival rate was 81.3% (13/16). Deaths occurred during anesthesia, sham surgery or the first hours following surgery. The survival rate for RYGB was 50 % (9/18), due to the complexity of surgical procedures in the context of the learning curve of a new surgeon. All deaths occurred during the early post-surgery period. The survival rate of VSG rats was 100% (7/7).

Food intake, weight and body composition

During the first two days, animals did not receive any solid food (Figure 2A), and they all lost weight (Fig. 2B). When refeeding began on day 3, rats rapidly increased their energy intake.

After the beginning of refeeding, rats continued to lose weight for two days. Operated rats lost between 7 and 9 % of their pre-operative weight. Sham tended to loose less weight than operated animals during this period (5.3 ± 0.2%). From day 5, all groups stopped losing weight; VSG regained weight whereas RYGB and pair-fed Sham did not. When habituation to consumption of a single test meal in the morning started at day 10, energy intake dropped for all groups. Subsequently, food consumption increased between day 11 and day 18, from 196.6 kJ ± 4.2 kJ/d (47.4 ± 1.8 kcal/d) to 255.2 ± 12.5 kJ/d (61.0 ± 3.2 kcal/d). Whereas all operated rats lost weight after the beginning of the protocol, Sham rats maintained their weight (about 1% loss in 9 days). The total mass loss was 44 ± 3 g in Sham, 64 ± 5 g in VSG and 73 ± 8 g in RYGB, with a significant effect of the group (P = 0.002), and a significant difference between Sham and RYGB (P = 0.002) and a trend for a difference between Sham and VSG (P=0.07). When pooled together, operated rats lost more weight than Sham rats (P = 0.001). The longitudinal follow up of body composition (Supplemental Table 2, https://figshare.com/articles/Bariatric_surgery/8964152) could be carried out in only some rats, leading to an insufficient number of observations. There was significantly (P = 0.02) more fat mass loss in RYGB (-48 ± 5 g) than in Sham rats (-27 ± 3 g). The fat mass loss observed in VSG (lossed 43 ± 5 g) did not differ from the two other groups. The average lean mass loss was -27 ± 4 g, and we could not detect any differences between groups.

Dietary protein bioavailability

Digestibility

To assess dietary protein digestibility, dietary nitrogen in the lumen of the gastrointestinal tract was measured 6 h after ingestion of the test meal (Table 1). Most of the unabsorbed exogenous nitrogen was found in the caecum and the colon lumens. In the stomach, the small intestine, the ileum and the colon lumens there were no differences between groups. In the caecum, significantly less dietary nitrogen was recovered in RYGB rats than in VSG and Sham rats. Consequently, there was a significant effect of the group on RFD (P = 0.045), digestibility being higher in RYGB than in Sham (P = 0.049).

Dietary nitrogen retention in tissues

To evaluate the metabolic bioavailability, we measured 15 N retention in the blood and different tissues (Fig. 3). There was an effect of the group (P = 0.037) on dietary nitrogen found in plasma protein (Figure 3A). It was similar in Sham (9.6 ± 0.4% of N ingested) and VSG (10.4 ± 1.5% of N ingested) but was lower in RYGB (6.7 ± 1.2%).

There was an effect of the group (P = 0.017) on liver weight, which was about 2 g higher in Sham than in the bariatric surgery groups (data not shown). As a consequence, the group influenced dietary nitrogen recovery in the liver (P = 0.008), with a lower value in the two operated groups than in Sham (Figure 3B). Independently of the liver weight, there was also an effect of the group on dietary N retained per 100 mg of liver (P=0.01), that was 0.057 ± Comment citer ce document : Tessier, R., Ribeiro-Parenti, L., Bruneau, O., Khodorova, N., Cavin, Bado, Azzout-Marniche, D., Calvez, J., Le Gall, Gaudichon, C. (Auteur de correspondance) (2019). Effect of different bariatric surgeries on dietary protein bioavailability in rats. American Journal of Physiology. Gastrointestinal and Liver Physiology, 317, G592-G601. , DOI : 10.1152/ajpgi.00142.2019 14 0.002 % in Sham, 0.045 ± 0.009 % in VSG and 0.039 ± 0.006 % in RYGB, with a significant difference between Sham and RYGB (P=0.001, data not shown).

Surgery had no effect on dietary nitrogen found in the kidney (Fig. 3C). In the muscle, there was a trend for an effect of surgery (P=0.054), RYGB rats tending to have less exogenous nitrogen than Sham (P=0.07) (Fig. 3D).

Following our observation that the alimentary limb and the ileum of RYGB were hypertrophied compared to the corresponding segments in Sham (jejunum and ileum) (Fig. 4A), the amount of dietary nitrogen in different locations of the intestinal tissue was determined for RYGB and Sham groups (Fig. 4B). Surprisingly, there was as much dietary nitrogen in the bilio-pancreatic limb (BPL) (0.083 ± 0.010% of N ingested) than in the alimentary limb (0.078 ± 0.007% of N ingested). There were no differences between the jejunum of Sham rats and the common limb of RYGB rats. In contrast, there was less nitrogen in the RYGB ileum (0.052 ± 0.004 % of N ingested) than in Sham (0.065 ± 0.005% of N ingested; P = 0.04).

Protein anabolism

The effect of surgery on protein anabolism was assessed by determining the protein synthesis rate in various organs. There was a group effect (P = 0.037) on the FSR in the kidney, with a significant decrease in RYGB compared to Sham and VSG (Table 2). There was also a similar effect on the ASR in the kidney (P=0.017) with a lower value in RYGB than in Sham (P=0.018). There was no effect of surgery on protein anabolism in the liver, muscle and skin, regardless of the parameter, FSR or ASR. Comment citer ce document : Tessier, R., Ribeiro-Parenti, L., Bruneau, O., Khodorova, N., Cavin, Bado, Azzout-Marniche, D., Calvez, J., Le Gall, Gaudichon, C. (Auteur de correspondance) (2019). Effect of different bariatric surgeries on dietary protein bioavailability in rats. American Journal of Physiology. Gastrointestinal and Liver Physiology, 317, G592-G601. , DOI : 10.1152/ajpgi.00142. 2019 15

Discussion

In the present study, we assessed the consequences of different types of bariatric surgery on protein bioavailability in diet-induced obese rats. For this purpose, we used a single 15 N test meal to determine dietary protein digestibility and its postprandial retention in tissues. We observed that bypass surgery improved real protein digestibility, but that less dietary nitrogen was retained in tissues, except for the hypertrophied intestine. According to our study, intestine remodeling could trigger a preferential use of dietary amino acids by the intestinal mucosa at the expense of other organs.

Concerning weight and food intake, our results are in accordance with other studies on the effectiveness of bariatric surgery on weight loss in rat models [START_REF] Grayson | Weight loss by calorie restriction versus bariatric surgery differentially regulates the hypothalamo-pituitary-adrenocortical axis in male rats[END_REF][START_REF] Hao | Vagal Innervation of Intestine Contributes to Weight Loss After Roux-en-Y Gastric Bypass Surgery in Rats[END_REF][START_REF] Lutz | Physiological Mechanisms behind Roux-en-Y Gastric Bypass Surgery[END_REF]. During the first days after surgery, all animals experienced rapid weight loss due to substantial food restriction. After refeeding, RYGB continued to lose weight but stabilized around six days after surgery, whereas VSG tended to regain weight. Weight regain has been reported VSG [START_REF] Mumphrey | Sleeve Gastrectomy Does Not Cause Hypertrophy and Reprogramming of Intestinal Glucose Metabolism in Rats[END_REF], but only after two weeks.

At day 10, we started a specific feeding schedule in order to implement the final 15 N test meal. This training implied reduced access time to food. This resulted in a substantial reduction of food intake -almost halved -and therefore a recurrence of weight loss in operated rats, particularly in RYGB. Surprisingly, Sham rats maintained their weight despite their being pair-fed with operated rats. The observation that operated rats lose more weight than pair-fed Sham rats has already been reported (1). This could be the consequence of intestinal remodeling that has been described after bypass surgeries [START_REF] Cavin | Differences Downloaded from www.physiology.org/journal[END_REF][START_REF] Cavin | Malabsorption and intestinal adaptation after one anastomosis gastric bypass compared with Roux-en-Y gastric bypass in rats[END_REF][START_REF] Hansen | Hypertrophy Dependent Doubling of L-Cells in Roux-en-Y Gastric Bypass Operated Rats[END_REF], resulting in a higher energy demand. Total energy expenditure has consistently been reported to increase by 13% in RYGB rats compared to body weight-matched Sham rats [START_REF] Bueter | Gastric Bypass Increases Energy Expenditure in Rats[END_REF]. Thereby, food intake during the habituation protocol was enough for Sham to limit their weight loss in contrast to operated rats. As previously described [START_REF] Carey | Body Composition and Metabolic Changes Following Bariatric Surgery: Effects on Fat Mass, Lean Mass and Basal Metabolic Rate[END_REF], malabsorptive bariatric surgery leads to a notable loss of fat as well as lean mass. This loss of lean mass is an indication of protein imbalance [START_REF] Jensen | Adult starvation and disease-related malnutrition: A proposal for etiology-based diagnosis in the clinical practice setting from the International Consensus Guideline Committee[END_REF]. In human subjects, protein deficiency after bariatric surgery has been Comment citer ce document : Tessier, R., Ribeiro-Parenti, L., Bruneau, O., Khodorova, N., Cavin, Bado, Azzout-Marniche, D., Calvez, J., Le Gall, Gaudichon, C. (Auteur de correspondance) (2019). Effect of different bariatric surgeries on dietary protein bioavailability in rats. American Journal of Physiology. Gastrointestinal and Liver Physiology, 317, G592-G601. , DOI : 10.1152/ajpgi.00142.2019 16 reported to be more or less prevalent, depending on the judgment criteria (albumin, lean mass loss) [START_REF] Verger | Nutritional and Protein Deficiencies in the Short Term following Both Gastric Bypass and Gastric Banding[END_REF][START_REF] Bloomberg | Nutritional Deficiencies following Bariatric Surgery: What Have We Learned?[END_REF][START_REF] Faintuch | Severe Protein-Calorie Malnutrition after Bariatric Procedures[END_REF][START_REF] Moizé | Protein intake and lean tissue mass retention following bariatric surgery[END_REF][START_REF] Verger | Micronutrient and Protein Deficiencies After Gastric Bypass and Sleeve Gastrectomy: a 1-year Follow-up[END_REF].

Using the 15 N test meal, we aimed at clarifying whether an alteration of protein bioavailability could contribute to the reported protein wasting and loss of lean mass. Nevertheless, due to technical problems in the Eco-MRI follow-up, we could not evidence a lean mass loss after surgery, although it has been shown in rats [START_REF] Shin | Longitudinal Assessment of Food Intake, Fecal Energy Loss, and Energy Expenditure After Roux-en-Y Gastric Bypass Surgery in High-Fat-Fed Obese Rats[END_REF] and humans [START_REF] Carey | Body Composition and Metabolic Changes Following Bariatric Surgery: Effects on Fat Mass, Lean Mass and Basal Metabolic Rate[END_REF]. Digestive bioavailability was established using a standardized procedure to determine oro-fecal digestibility 6 h after the test meal ingestion [START_REF] Boutry | Rapeseed and milk protein exhibit a similar overall nutritional value but marked difference in postprandial regional nitrogen utilization in rats[END_REF][START_REF] Lacroix | Heat Markers and Quality Indexes of Industrially Heat-Treated [ 15 N] Milk Protein Measured in Rats[END_REF][START_REF] Oberli | Lipo-Protein Emulsion Structure in the Diet Affects Protein Digestion Kinetics, Intestinal Mucosa Parameters and Microbiota Composition[END_REF][START_REF] Oberli | 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[END_REF], in the absence of any standardized method to determine ileal digestibility in rats. We measured dietary nitrogen in all the distal segments, including caecum, colon and feces. Amino acids that reach the caecum and colon are metabolized by colonic bacteria, leading to the production of metabolites such as ammonia and indole, which can be absorbed in the colon [START_REF] Davila | Intestinal luminal nitrogen metabolism: Role of the gut microbiota and consequences for the host[END_REF][START_REF] Yao | Review article: insights into colonic protein fermentation, its modulation and potential health implications[END_REF]. This results in a systematic overestimation of protein nitrogen digestibility [START_REF] Darragh | Quantifying the Digestibility of Dietary Protein[END_REF][START_REF] Rutherfurd | Protein Digestibility-Corrected Amino Acid Scores and Digestible Indispensable Amino Acid Scores Differentially Describe Protein Quality in Growing Male Rats[END_REF]. However, this error is acceptable, especially in the case of highly digestible protein, for which the differences between ileal and fecal digestibility have been shown to be of limited extent [START_REF] Rutherfurd | Protein Digestibility-Corrected Amino Acid Scores and Digestible Indispensable Amino Acid Scores Differentially Describe Protein Quality in Growing Male Rats[END_REF].

As expected, most of the dietary nitrogen was found in the caecum due to a mostly complete digestion. Surprisingly, there was less dietary nitrogen in the caecum of RYGB than in VSG and Sham rats. Consequently, RYGB rats showed a significant increase of protein digestibility compared to Sham, while there was no difference for VSG rats compared to Sham. Although there is no comparable data in the literature, this result concurs with a study on patients operated with RYGB (9) that showed a faster blood appearance of amino acids from 13 C casein given as a meal, thus suggesting a faster absorption. In contrast, a diminution of the apparent fecal protein digestibility was reported 5 and 14 months after RYGB in obese patients [START_REF] Odstrcil | The contribution of malabsorption to the reduction in net energy absorption after long-limb Roux-en-Y gastric bypass[END_REF]. Of note in this last study, the reduced absorption of protein was mostly due to a reduced protein intake rather than to protein malabsorption. Other studies reported an increase of fecal protein output in a rat model of biliopancreatic diversion [START_REF] Nadreau | Effects of the biliopancreatic diversion on energy balance in the rat[END_REF] or one anastomosis gastric bypass [START_REF] Cavin | Malabsorption and intestinal adaptation after one anastomosis gastric bypass compared with Roux-en-Y gastric bypass in rats[END_REF]. This seems to be in contradiction with our Comment citer ce document : Tessier, R., Ribeiro-Parenti, L., Bruneau, O., Khodorova, N., Cavin, Bado, Azzout-Marniche, D., Calvez, J., Le Gall, Gaudichon, C. (Auteur de correspondance) (2019). Effect of different bariatric surgeries on dietary protein bioavailability in rats. American Journal of Physiology. Gastrointestinal and Liver Physiology, 317, G592-G601. , DOI : 10.1152/ajpgi.00142.2019 17 results. However, this nitrogen loss may not be the result of lower digestibility of dietary proteins, but could arise from caecal bacterial overgrowth and/or an increase of intestinal cell desquamation. Further characterization of these nitrogen losses would be necessary.

The metabolic bioavailability was assessed by measuring 15 N retention in the blood and different tissues. It was noticeable that in operated rats, the liver weighed less than in Sham rats. A decrease in liver weight has already consistently been shown in rats after VSG [START_REF] Watanabe | Effects of Sleeve Gastrectomy on Nonalcoholic Fatty Liver Disease in an Obese Rat Model[END_REF] or gastric bypass surgery (40). An interesting observation is that the 15 N distribution in the splanchnic tissues was altered differently by surgery. In the liver, there was a clear decrease of dietary N uptake regardless of the surgery. This effect can be partly ascribed to the lower liver weight but when expression per unit of tissue weight, the 15 N retention was consistently lower in RYGB. Moreover, incorporation in plasma proteins was altered only in RYGB. As plasma proteins are mainly composed of exported liver proteins, this means that the early postprandial incorporation of dietary amino acids in liver protein synthesis was altered due to a deficit of precursor amino acids. In contrast, we found no difference in the liver protein synthesis rate, as assayed by 13 C-valine incorporation. This, though, is not inconsistent because it was measured 6 h after the meal, i.e. in the post-absorptive phase. One may suppose that FSR would have been decreased in the bypass groups if measured just after the meal.

In the peripheral organs, we also found a trend for a decreased retention of 15 N in the muscle RYGB. Regarding protein anabolism, we evidenced little effect of surgery, except in the kidney where FSR and ASR were lower in RYGB than in Sham. It is known that obesity is associated with kidney disorders [START_REF] Wang | Association between obesity and kidney disease: A systematic review and meta-analysis[END_REF], and it has already been shown that bariatric surgery and subsequent weight loss result in kidney function improvement [START_REF] Chang | Bariatric surgery is associated with improvement in kidney outcomes[END_REF][START_REF] Currie | Bariatric Surgery and Renal Function[END_REF][START_REF] Neff | The effect of bariatric surgery on renal function and disease: a focus on outcomes and inflammation[END_REF]. Our observation of a lower FSR may have been related to a normalization of some biological parameters such as inflammation proteins.

In the intestinal tissues, 15 N retention could only be measured in RYGB and in Sham animals. Comment citer ce document : Tessier, R., Ribeiro-Parenti, L., Bruneau, O., Khodorova, N., Cavin, Bado, Azzout-Marniche, D., Calvez, J., Le Gall, Gaudichon, C. (Auteur de correspondance) (2019). Effect of different bariatric surgeries on dietary protein bioavailability in rats. American Journal of Physiology. Gastrointestinal and Liver Physiology, 317, G592-G601. , DOI : 10.1152/ajpgi.00142.2019 18 the ileum of RYGB compared to Sham. The latter could be interpreted either as a lower absorption of dietary AA in the distal portion of the ileum, or by a decrease of substrate availability. The second interpretation is more probable considering that protein digestibility did increase in RYGB. Surprisingly, we recovered some dietary nitrogen in the biliopancreatic limb, as much as in the alimentary limb, although stomach chyme is not expected to transit through the BPL. One hypothesis is that 15 N amino acids, coming mainly from the mesenteric arteria, were absorbed by the basolateral side of BPL enterocytes. It is possible that mucosa remodeling enhanced this metabolic pathway. A similar observation has been made for glucose, where GLUT1 has appeared to be over expressed at the basolateral membrane in RYGB rats [START_REF] Cavin | Differences Downloaded from www.physiology.org/journal[END_REF][START_REF] Saeidi | Reprogramming of Intestinal Glucose Metabolism and Glycemic Control in Rats After Gastric Bypass[END_REF]. This has been linked to the hyperplasia of jejunal mucosa, reported in several studies [START_REF] Bueter | Gastric Bypass Increases Energy Expenditure in Rats[END_REF][START_REF] Cavin | Differences Downloaded from www.physiology.org/journal[END_REF][START_REF] Taqi | The influence of nutrients, biliary-pancreatic secretions, and systemic trophic hormones on intestinal adaptation in a Roux-en-Y bypass model[END_REF] including the present one, resulting in an increase of villi height in the common and alimentary limbs (56) as well as mucosal volume and weight [START_REF] Bueter | Gastric Bypass Increases Energy Expenditure in Rats[END_REF]. This trophic effect may be mediated by GLP-2 that has been reported to be over excreted in animal models and in humans [START_REF] Cazzo | GLP-2: a poorly understood mediator enrolled in various bariatric/metabolic surgery-related pathophysiologic mechanisms[END_REF][START_REF] Cazzo | Correlation between pre and postoperative levels of GLP-1/GLP-2 and weight loss after Roux-en-Y Gastric Bypass: a Prospective study[END_REF]. We also observed a hyperplasia in the common limb and even in the ileum (Figure 4). We did not weigh the intestinal mucosa, but the gut weight has been shown to be twofold heavier in gastric bypass than in Sham operated rats [START_REF] Bueter | Gastric Bypass Increases Energy Expenditure in Rats[END_REF][START_REF] Saeidi | Reprogramming of Intestinal Glucose Metabolism and Glycemic Control in Rats After Gastric Bypass[END_REF]. As we found a similar amount of dietary N per g of intestine, we can suppose that the intestine sequestrated twice more dietary N in RYGB than in Sham. If we consider an average 15 N retention of 0.06%/100 mg for the whole intestine and an intestinal weight of 20 g in RYGB and 10 g in Sham as previously reported [START_REF] Bueter | Gastric Bypass Increases Energy Expenditure in Rats[END_REF], this would result in an intestinal dietary N sequestration of 12% in RYGB vs. 6% in Sham rats. This would consistently explain why dietary AA were less present in the peripheral area, whereas digestibility was improved. They were likely preferentially used by the remodeled intestine in RYGB rats.

This study had some limitations, in particular, the number of animals in each group, which was a consequence of the high mortality after RYGB procedure and which resulted in a lack of statistical power. Moreover, we did not provide a complete understanding of dietary protein Comment citer ce document : Tessier, R., Ribeiro-Parenti, L., Bruneau, O., Khodorova, N., Cavin, Bado, Azzout-Marniche, D., Calvez, J., Le Gall, Gaudichon, C. (Auteur de correspondance) (2019). Effect of different bariatric surgeries on dietary protein bioavailability in rats. American Journal of Physiology. Gastrointestinal and Liver Physiology, 317, G592-G601. , DOI : 10.1152/ajpgi.00142.2019 19 metabolism after bariatric surgery due to missing measurements, such as initial body composition, postprandial FSR measurements as well as FSR in the intestine. The latter was not determined because we have chosen an incorporation time of the tracer (30 min) that allowed to study several organs at once but that was too long for a very high turnover tissue such as small intestine mucosa [START_REF] Adegoke | Jejunal mucosal protein synthesis: validation of luminal flooding dose method and effect of luminal osmolarity[END_REF][START_REF] Rajendram | Skeletal muscle and jejunal protein synthesis in normal and ethanol-treated rats: The effect of the nitric oxide synthase inhibitors, L-ω-nitro-L-arginine methyl ester and N(G)-nitro-L-arginine in vivo[END_REF]. This study addressed the short-term consequences of bariatric surgery; it would be useful to study these parameters in the long-term. Indeed, alteration of protein bioavailability could be transitory, considering intestine remodeling and adaptation. It would also be necessary to challenge these results in human studies using convenient and poorly invasive procedures to assess dietary protein bioavailability.

In conclusion, this study provided the first data on dietary protein bioavailability after bariatric surgery in rats. Unexpectedly, we reported a slight improvement of protein digestibility after RYGB but not after VSG. In contrast, RYGB decreased dietary nitrogen retention in the peripheral organs except in the intestine, due to a deficit of amino acid delivery. This strongly suggests a higher uptake of amino acids by the remodeled intestine for its own needs. Comment citer ce document : Tessier, R., Ribeiro-Parenti, L., Bruneau, O., Khodorova, N., Cavin, Bado, Azzout-Marniche, D., Calvez, J., Le Gall, Gaudichon, C. (Auteur de correspondance) (2019). Effect of different bariatric surgeries on dietary protein bioavailability in rats. American Journal of Physiology. Gastrointestinal and Liver Physiology, 317, G592-G601. , DOI : 10.1152/ajpgi.00142.2019

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15 N

 15 recovery was similar in the common limb of RYGB and the jejunum in Sham, but lower in Downloaded from www.physiology.org/journal/ajpgi at INRA Institut National de la Recherche Agronomique (138.102.192.062) on September 3, 2019.

25 Figure Caption Figure 1 :

 25Caption1 Figure Caption

Figure 2 :

 2 Figure 2: Energy intake (A) and weight loss (B) after bariatric surgery. Refeeding begun 3 days after surgery. Habituation to a discontinuous food pattern begun at d10. Sham rats were pair-fed with operated rats. Values are means ± SEM, n = 13 for Sham, n = 7 for VSG, n = 9 for RYGB. Surgery and time effects were tested by a mixed model. Values with different letters at a given day are statistically different (post hoc Bonferroni's test).

Figure 3 :

 3 Figure 3: Exogenous nitrogen recovered in plasma proteins (A), liver (B), kidney (C) and muscle (D) six hours after the meal, expressed in % nitrogen ingested for the whole organ. Values are means ± SEM, n = 12-13 for Sham, n = 5-7 for VSG and n = 8-9 for RYGB. Surgery effect was tested in each organ by a one-way ANOVA and post hoc Bonferroni's test for multiple comparisons. * P<0.05; ** P<0.01.

Figure 4 :

 4 Figure 4: Histology of intestine (A) and dietary nitrogen recovered in gastrointestinal tract tissues expressed in % of nitrogen ingested per 100 mg of fresh weight (B) of Sham and RYGB rats. Values are means ± SEM, n = 5 for Sham and n = 6 for RYGB. The differences between Sham and RYGB were tested in the ileum on one hand and in the jejunum and the common limb on the other hand. * P<0.05

  Figure 1

Figure 2 0

 2 Figure 2

  Figure 3

Figure 4

 4 Figure 4

  They were conducted in compliance with EU directive 2010/63/EU for animal experiments and approved by the Institutional Animal Care and Use Committee (Comité d'éthique en expérimentation animale N° 121) and the Ministry of Higher Education and Research (Ref #02285.03). Male Wistar rats (Janvier Labs, Le Genest-St-Isle, France) (n = 42) weighing 275-280 g were housed in standard environmental conditions (temperature 21-22 °C, 12/12 h light/dark cycle, tap water and food ad libitum). Before surgery, they were fed a high fat diet (HFD) (Altromin, Genestil, Royancourt, France) for 4 months. The composition is detailed in Supplemental Table 1(https://figshare.com/articles/Bariatric_surgery/8964152). The experimental design of the study is detailed in Supplemental Fig. 1 (https://figshare.com/articles/Bariatric_surgery/8964152).

	Surgical Procedures

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	7
	Comment citer ce document :
	Tessier, R., Ribeiro-Parenti, L., Bruneau, O., Khodorova, N., Cavin, Bado, Azzout-Marniche, D.,
	Calvez, J., Le Gall, Gaudichon, C. (Auteur de correspondance) (2019). Effect of different
	bariatric surgeries on dietary protein bioavailability in rats. American Journal of Physiology.
	Gastrointestinal and Liver Physiology, 317, G592-G601. , DOI : 10.1152/ajpgi.00142.2019

  Downloaded from www.physiology.org/journal/ajpgi at INRA Institut National de la Recherche Agronomique (138.102.192.062) on September 3, 2019.

	Comment citer ce document :
	Tessier, R., Ribeiro-Parenti, L., Bruneau, O., Khodorova, N., Cavin, Bado, Azzout-Marniche, D.,
	Calvez, J., Le Gall, Gaudichon, C. (Auteur de correspondance) (2019). Effect of different
	bariatric surgeries on dietary protein bioavailability in rats. American Journal of Physiology.
	Gastrointestinal and Liver Physiology, 317, G592-G601. , DOI : 10.1152/ajpgi.00142.2019

  Downloaded from www.physiology.org/journal/ajpgi at INRA Institut National de la Recherche Agronomique (138.102.192.062) on September 3, 2019.

Table 1 :

 1 Dietary nitrogen recovered in gastrointestinal tract contents (stomach, small intestine, ileum, caecum and colon) depending on the type of bariatric surgery. Values are means ± SEM, n = 13 for Sham, n = 7 for VSG and n = 9 for RYGB. The effect of the group was tested with an ANOVA model and post hoc Bonferroni test for pairwise comparisons. Values with different letters within the same row are statistically different. RFD: real fecal digestibility.

		Sham	VSG	RYGB	Group Effect
			% of N ingested
	Stomach	0.52 ± 0.11	0.10 ± 0.34	0.30 ± 0.15	0.066
	Small intestine	0.69 ± 0.05	0.81 ± 0.21	0.65 ± 0.16	NS
	Ileum	0.32 ± 0.07	0.69 ± 0.32	0.15 ± 0.05	0.078
	Caecum	4.46 ± 0.33 a 4.45 ± 0.54 a 2.42 ± 0.43 b	0.002
	Colon	1.76 ± 0.38	1.07 ± 0.21	1.85 ± 0.35	NS
	RFD (%)	93.5 ± 0.5 a	93.8 ± 0.6 ab	95.6 ± 0.7 b	0.045

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Table 2 :

 2 Fractional synthesis rate and absolute synthesis rate in liver, kidney, muscle and skin, depending on the type of bariatric surgery. Values are means ± SEM, n = 11-12 for Sham, n = 6-7 for VSG and n = 8 for RYGB. The effect of the group was tested with an ANOVA model and post hoc Bonferroni test for pairwise comparisons. Values with different letters within the same row are statistically different.

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C valine Downloaded from www.physiology.org/journal/ajpgi at INRA Institut National de la Recherche Agronomique (138.102.192.062) on September 3, 2019.

Acknowledgements: N/A

Conflict of interest statement: The authors declare no conflict of interest.