Daisy Goncalves

PhD Aude Barataud

MS Filipe De Vadder

PhD Jennifer Vinera

MS Carine Zitoun

AD Adeline Duchampt

Dr Gilles Mithieux
email: gilles.mithieux@univ-lyon1.fr

Bile bioavailability and gastric bypass BILE ROUTING MODIFICATION REPRODUCES KEY FEATURES OF GASTRIC BYPASS IN RAT

Bile bioavailability

INTRODUCTION

The last decades have seen an alarming worldwide increase in the prevalence of obesity and its associated diseases, particularly type 2 diabetes, which currently affects hundreds of millions of people. [START_REF] Chen | The worldwide epidemiology of type 2 diabetes mellituspresent and future perspectives[END_REF] Gastric bypass surgery (GBP) has emerged as an effective treatment for morbid obese diabetic patients since it leads to a rapid diabetes remission, suggested from some observational studies to be unrelated to weight loss. [START_REF] Rubino | Metabolic surgery to treat type 2 diabetes: clinical outcomes and mechanisms of action[END_REF][START_REF] Thaler | Minireview: Hormonal and metabolic mechanisms of diabetes remission after gastrointestinal surgery[END_REF] Patients also report a loss of hunger sensation and a disinterest in fatty food, likely to be helpful to the later loss of body weight. [START_REF] Schultes | Hedonic hunger is increased in severely obese patients and is reduced after gastric bypass surgery[END_REF][START_REF] Mathes | Food selection and taste changes in humans after Roux-en-Y gastric bypass surgery: a direct-measures approach[END_REF][START_REF] Stefater | All bariatric surgeries are not created equal: insights from mechanistic comparisons[END_REF] However, the mechanisms by which GBP induces these beneficial effects on glucose homeostasis and food behavior remain largely unclear.

Among the disorders characteristic of type 2 diabetes, an increase in hepatic glucose production (HGP) is considered to be a major cause of insulin resistance and hyperglycemia. [START_REF] Trinh | Perturbation of fuel homeostasis caused by overexpression of the glucose-6-phosphatase catalytic subunit in liver of normal rats[END_REF][START_REF] Clore | Glucose-6-phosphatase flux in vitro is increased in type 2 diabetes[END_REF] Diverging from this dogma, intestinal gluconeogenesis (IGN) has been shown to induce beneficial effects on glucose and energy homeostasis. Indeed, glucose released by IGN is detected by a portal glucose sensor [START_REF] Delaere | The role of sodium-coupled glucose co-transporter 3 in the satiety effect of portal glucose sensing[END_REF] that initiates a gut-brain neural circuit inducing satiety [START_REF] Duraffourd | Mu-opioid receptors and dietary protein stimulate a gut-brain neural circuitry limiting food intake[END_REF][START_REF] Mithieux | Portal sensing of intestinal gluconeogenesis is a mechanistic link in the diminution of food intake induced by diet protein[END_REF] and an increased inhibition of HGP by insulin. [START_REF] Pillot | Protein feeding promotes redistribution of endogenous glucose production to the kidney and potentiates its suppression by insulin[END_REF] In models of GBP, an induction of IGN with in parallel a decrease in HGP has been reported in rodents [START_REF] Troy | Intestinal gluconeogenesis is a key factor for early metabolic changes after gastric bypass but not after gastric lap-band in mice[END_REF][START_REF] Sun | Duodenal-jejunal bypass surgery up-regulates the expression of the hepatic insulin signaling proteins and the key regulatory enzymes of intestinal gluconeogenesis in diabetic Goto-Kakizaki rats[END_REF][START_REF] Paranjape | Improvement in hepatic insulin sensitivity after Roux-en-Y gastric bypass in a rat model of obesity is partially mediated via hypothalamic insulin action[END_REF] and humans. [START_REF] Hayes | Is intestinal gluconeogenesis a key factor in the early changes in glucose homeostasis following gastric bypass?[END_REF][START_REF] Mithieux | Comment about intestinal gluconeogenesis after gastric bypass in human in relation with the paper by[END_REF][START_REF] Immonen | Effect of bariatric surgery on liver glucose metabolism in morbidly obese diabetic and non-diabetic patients[END_REF] Even if these opposite regulations could both explain the improvements in energy and glucose metabolism observed after GBP, the underlying regulatory mechanisms remain to be understood.

Bile acids have emerged as key metabolic regulators, which might account for several anti-diabetic effects. Indeed, they regulate insulin secretion in β-cells [START_REF] Seyer | Hepatic glucose sensing is required to preserve β cell glucose competence[END_REF][START_REF] Düfer | Bile acids acutely stimulate insulin secretion of mouse β-cells via farnesoid X receptor activation and K(ATP) channel inhibition[END_REF][START_REF] Renga | The bile acid sensor FXR regulates insulin transcription and secretion[END_REF] and increase energy expenditure in the brown-adipose tissue and skeletal muscle. [START_REF] Watanabe | Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation[END_REF] Interestingly, they have also been reported to inhibit gluconeogenesis either directly [START_REF] De Fabiani | Coordinated control of cholesterol catabolism to bile acids and of gluconeogenesis via a novel mechanism of transcription regulation linked to the fastedto-fed cycle[END_REF] or through the activation of farnesoid X receptor (FXR)/small heterodimer partner (SHP) pathway. [START_REF] Yamagata | Bile acids regulate gluconeogenic gene expression via small heterodimer partner-mediated repression of hepatocyte nuclear factor 4 and Foxo1[END_REF][START_REF] Ma | Farnesoid X receptor is essential for normal glucose homeostasis[END_REF] Thus, an attractive hypothesis to explain the benefits of GBP is related to the blood versus intestinal bioavailability of bile after the surgery. Indeed, plasma bile acids are elevated after surgery, [START_REF] Patti | Serum bile acids are higher in humans with prior gastric bypass: potential contribution to improved glucose and lipid metabolism[END_REF][START_REF] Pournaras | The role of bile after Roux-en-Y gastric bypass in promoting weight loss and improving glycaemic control[END_REF][START_REF] Simonen | Conjugated bile acids associate with altered rates of glucose and lipid oxidation after Roux-en-Y gastric bypass[END_REF][START_REF] Kohli | Weight loss induced by Roux-en-Y gastric bypass but not laparoscopic adjustable gastric banding increases circulating bile acids[END_REF] whereas the alimentary limb is devoid of bile. Moreover, the benefits of GBP are lost when the bile bioavailability is restored in the digestive tract. [START_REF] Rudnicki | Proximal jejunal and biliary effects on the enteroinsular axis[END_REF] Thus, bile Goncalves et al. 2014 Bile bioavailability and gastric bypass acids might exert a double beneficial role after GBP: decreasing HGP due to their elevated plasma concentration and increasing IGN through their absence in the alimentary limb.

To test the role of the modification of bile routing in the benefits of GBP, we set up original bile diversions in the mid-jejunum or mid-ileum in rats to mimic the modified bile delivery in the gut that occurs in GBP (Fig. 1a). First, we tested whether the only modification of bile routing could reproduce the increase in plasma bile acids observed after GBP. Secondly, we assessed the role of bile in the opposite regulation of gluconeogenesis gene expression taking place in the liver and the intestine after GBP and the involvement of the FXR/SHP pathway in these regulations. Thirdly, we considered the implication of the bile routing modification in the metabolic improvements in GBP by evaluating glucose homeostasis in lean and obese rats. Finally, an unexpected observation led us to highlight a considerable role of bile bioavailability in the change of food preference occurring after GBP.

METHODS

Animals

All procedures were performed in accordance with the principles and guidelines established by the European Convention for the Protection of Laboratory Animals. (incorporated into A04 at 5% (wt/wt) -Sigma). Studies on obese rats were performed after 8 weeks of HFHS feeding.

Surgical procedures

Rats were anesthetized with 2% isoflurane. The extremity of a catheter (PE10, Fine-Bore Polyethylene Tubing, Smiths Medical) was inserted in the bile duct, upstream of pancreatic ducts,

Goncalves et al. 2014

Bile bioavailability and gastric bypass pushed towards the liver on 1 cm and secured with sewing thread and biological glue (3M Vetbond, Centravet). According to the group studied, the other extremity of the catheter (SIL-C30, Phymepfor intestine re-insertion) was re-inserted into the mid-jejunum (about 15-20 cm downstream the pylorus), the mid-ileum (about 15-20 cm upstream of the caecum) or in a mesenteric vein, and fixed with sewing thread (only for intestinal re-insertion) and biological glue (Fig. 1a andS1a). For portal denervation, a gauze compress moistened with 80 µl of a capsaicin solution (10 mg/mL in saline,

DMSO and Tween at a ratio of 8:1:1 vol/vol/vol) was applied around the portal vein for 15 min during the mid-jejunum bile diversion. A sham-operated group, which only underwent a laparotomy, was studied in parallel.

Body weight, food intake and food preference

After surgery, rats were individually housed with food and water ad libitum. Body weight and food intake were monitored daily during 15 days. To evaluate food preference, a choice between standard and HFHS diet was offered in the period of 11 to 15 day after surgery.

Insulin and glucose tolerance tests

Seven days after surgery, rats were fasted 6 hours and received an intraperitoneal injection of insulin (0.5 U/kg body weight). An intraperitoneal glucose test tolerance (1 g/kg body weight) was performed on rats fasted for 16 hours, 10 days after surgery. Blood was withdrawn from the tail vein at 0, 15, 30, 45, 60 and 90 minutes after injection for glucose and/or insulin assessment. Blood glucose was measured using an Accu-Chek Go glucometer (Roche Diagnostics) and insulin was quantified using an ELISA kit (Mercodia).

Tissue sampling and metabolic studies

Thirteen days after surgery, 6 hours-fasted rats were euthanized by pentobarbital intraperitoneal injection. The intestine was rapidly sampled as previously described. [START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF] The liver was removed and Bile bioavailability and gastric bypass frozen using tongs previously chilled in liquid N 2 . Blood was withdrawn from the heart and collected in EDTA. Total bile acid was assessed using Diazyme kit. G6Pase activity was assayed under maximal velocity conditions. Proteins were immunoblotted using antibodies against G6PC, [START_REF] Rajas | The glucose-6 phosphatase gene is expressed in human and rat small intestine: regulation of expression in fasted and diabetic rats[END_REF] FXR (1/500 Abcam), β-actin (1/1,000 Cell Signaling) and GAPDH (1/10,000 Cell Signaling). Total RNAs were isolated from tissues with TRIzol reagent (Invitrogen). Reverse transcription and real-time PCR were performed using sequence-specific primers described in supplementary table 1.

RESULTS

Bile diversions improve glucose homeostasis in lean rats.

We first studied the metabolic effects of bile diversions in rats fed a standard diet. First, bile-diverted rats showed a moderate decrease in body weight consecutive to surgery. However, this was transient since they recovered their basal body weight from the 8 th day and exhibited no difference with shamoperated rats 9 days after surgery (Fig. 1b). Food intake in bile-diverted rats was transiently reduced during the first 6 days after surgery and then re-increased to reach a plateau of daily food intake not different from sham-operated rats (Fig. 1c). Insulin and glucose tolerance tests were performed at a time where there was no more difference in body weight and food intake among the groups. Insulin tolerance was significantly enhanced in bile-diverted rats compared with sham-operated rats (Fig. 1d). Similarly, bile-diverted rats exhibited an improvement in glucose tolerance (Fig. 1e). This was associated to an increase in insulin secretion, which could be involved in the improvement in glucose tolerance (Fig. 1f).

Blood and intestinal changes in bile bioavailability are responsible for glucose metabolism improvements after bile diversions.

To determine if the modification of bile routing per se could reproduce the increase in plasma bile acids observed after GBP, we measured bile acid concentration in the peripheral blood circulation 13 days after bile diversions. Interestingly, when bile was derived either in the mid-jejunum or in the

Goncalves et al. 2014

Bile bioavailability and gastric bypass mid-ileum, a rise in plasma bile acid concentration was observed (Fig. 2a). This was in line with the enhancement in insulin secretion observed in bile-diverted rats (Fig. 1f), since bile acids are known as activators of insulin secretion. [START_REF] Seyer | Hepatic glucose sensing is required to preserve β cell glucose competence[END_REF][START_REF] Düfer | Bile acids acutely stimulate insulin secretion of mouse β-cells via farnesoid X receptor activation and K(ATP) channel inhibition[END_REF][START_REF] Renga | The bile acid sensor FXR regulates insulin transcription and secretion[END_REF] To assess the causal role of the change in blood and intestinal bile bioavailability in the improvement in glucose metabolism observed after bile diversions, we submitted mid-jejunum bile-diverted rats to a cholestyramine-enriched diet. Cholestyramine binds bile acids within the gastrointestinal tract and prevents their reabsorption. As expected, no increase in plasma bile acids was observed in midjejunum bile-diverted rats fed a cholestyramine-enriched diet (Fig. 2a). Interestingly, bile-diverted rats exhibited no improvement in glucose tolerance and insulin secretion compared to shamoperated rats fed a cholestyramine-enriched diet (Fig. 2b-c). These data highlight a causal role of plasma bile acids and modified bile bioavailability in the metabolic improvements associated to bile diversions.

An increase in the enterohepatic circulation of bile acids is frequently proposed to explain the elevated plasma bile acid concentration after GBP. [START_REF] Pournaras | The role of bile after Roux-en-Y gastric bypass in promoting weight loss and improving glycaemic control[END_REF][START_REF] Jansen | Alterations of hormonally active fibroblast growth factors after Roux-en-Y gastric bypass surgery[END_REF] To test this hypothesis, we evaluated the expression of bile acid transporters at the site of bile re-insertion in the intestine and in the liver of bile-diverted rats fed a standard-diet. First, the mRNA level of the α-subunit of the organic solute transporter (OSTα), responsible for bile acid import from enterocytes to blood, was increased in the portion of gut where the bile bioavailability was restored in bile-diverted rats (Fig. 2d). In the liver, the mRNA expression of sodium-taurocholate co-transporting polypeptide (NTCP), responsible for hepatic bile acid import from portal blood was drastically reduced, whereas that of OSTα, which accounts for the bile acid transport from hepatocytes to systemic circulation, was significantly upregulated for the two diversions. However, no difference in mRNA abundance of bile salt export pump (BSEP), responsible for bile acid secretion into the bile duct, was observed among the groups (Fig. 2e). These data suggest an induction of bile acid reabsorption in the ileum with an opposite decrease of reabsorption in the liver after bile diversion, which could both account for the increase in the plasma bile acid concentration observed.

Goncalves et al. 2014 Bile bioavailability and gastric bypass

Bile diversions down-regulate hepatic glucose production and induce intestinal gluconeogenesis.

A decrease in HGP and an increase in IGN are key features associated with the improvements in metabolic control in rodents [START_REF] Troy | Intestinal gluconeogenesis is a key factor for early metabolic changes after gastric bypass but not after gastric lap-band in mice[END_REF][START_REF] Sun | Duodenal-jejunal bypass surgery up-regulates the expression of the hepatic insulin signaling proteins and the key regulatory enzymes of intestinal gluconeogenesis in diabetic Goto-Kakizaki rats[END_REF][START_REF] Paranjape | Improvement in hepatic insulin sensitivity after Roux-en-Y gastric bypass in a rat model of obesity is partially mediated via hypothalamic insulin action[END_REF] and humans. [START_REF] Hayes | Is intestinal gluconeogenesis a key factor in the early changes in glucose homeostasis following gastric bypass?[END_REF][START_REF] Mithieux | Comment about intestinal gluconeogenesis after gastric bypass in human in relation with the paper by[END_REF][START_REF] Immonen | Effect of bariatric surgery on liver glucose metabolism in morbidly obese diabetic and non-diabetic patients[END_REF] Thus, we analyzed gluconeogenesis gene expression in the liver and the intestine of bile-diverted rats fed a standard diet. First, both mRNA and protein levels of the catalytic subunit of glucose-6-phosphatase (G6PC) were markedly decreased in the liver of bile-diverted rats (Fig. 3a). Likewise, bile-diverted rats exhibited a substantial reduction of hepatic glucose-6-phosphatase (G6Pase) activity (Fig. 3b). Relatively to the mid-jejunum diversion,

G6Pase activity was increased in the duodenum and in the proximal jejunum, i.e. the portion of gut devoid of bile. On the contrary, G6Pase activity was markedly decreased in the distal jejunum and in the ileum, i.e. the gut section where the bile bioavailability was restored (Fig. 3c). For the bile diversion in the mid-ileum, we obtained comparable results with an increase in G6Pase activity upstream of the site of bile re-insertion and a decrease downstream (Fig. 3d).

To further strengthen the causal role of bile in the above hepatic and intestinal changes in gluconeogenesis, we studied an additional model of bile diversion directly in a mesenteric vein (Fig. S1a). As expected, there was a marked increase in plasma bile acids, comparable to that observed in intestinal diversions (Fig. S1b). It is noteworthy that the changes in hepatic bile acid transporter expression were also comparable to those observed in both intestinal diversions (Fig. S1c). Similarly, there was a dramatic suppression of G6PC mRNA and protein expression (Fig. 3a) and of G6Pase enzymatic activity (Fig. 3b) in the liver of mesenteric vein-diverted rats. Moreover, in the absence of bile into the whole gut lumen resulting from the diversion of bile in a mesenteric vein, G6Pase activity showed a 1.5 to 2.5-fold increase in the entire intestine compared to sham-operated rats (Fig. 3e). The data strongly suggest a causal role of bile in the changes in hepatic and intestinal gluconeogenesis taking place after intestinal bile re-routing.

To determine whether IGN has a causal role in the metabolic improvements consecutive to bile diversions, we performed a specific denervation of portal nervous afferents with capsaicin in mid-Bile bioavailability and gastric bypass jejunum bile-diverted rats. The enhancement of glucose tolerance and insulin secretion after bile diversion was maintained in capsaicin-treated rats (Fig. S2a-b). Moreover, bile-diverted rats with or without portal deafferentiation showed similar regulation of hepatic and intestinal G6Pase expression (Fig. S2c-e). These data indicate, firstly, that the metabolic improvements associated to bile diversions in the rat are independent of the phenomenon of portal glucose sensing, and secondly, that manipulation of bile enterohepatic cycling regulates hepatic gluconeogenesis gene expression independently of a gut-brain communication.

The regulation of intestinal gluconeogenesis but not hepatic glucose production depends on the FXR/SHP pathway in bile-diverted rats.

We next evaluated whether the opposite regulations of HGP and IGN subsequent to bile diversions could be mediated through FXR/SHP signaling. Indeed, bile acids bind to and activate FXR, inducing the transcription of its target gene SHP, itself blunting G6Pase gene transcription. [START_REF] Yamagata | Bile acids regulate gluconeogenic gene expression via small heterodimer partner-mediated repression of hepatocyte nuclear factor 4 and Foxo1[END_REF] At the hepatic level, both mRNA and protein levels of FXR were decreased for all diversions (Fig. 4a). Moreover, SHP mRNA abundance was also reduced in the liver of bile-diverted rats compared to sham-operated rats (Fig. 4b). This was not in agreement with an activation of the FXR/SHP pathway, which could account for the suppression of hepatic G6Pase gene expression. In the intestine completely devoid of bile, resulting from the bile diversion in a mesenteric vein, we showed a down-regulation of FXR and a remarkable decrease in SHP mRNA level along the whole intestine (Fig 4c-d). For the diversion of bile in the mid-ileum, FXR and SHP mRNA were both down-regulated in the absence of bile, i.e. upstream of the site of bile re-insertion. It is noteworthy that these regulations were inversed downstream of the bile re-insertion site (Fig 4e-f). These data suggest that the modulation of IGN (increased in the absence of bile and decreased in the presence of bile) could derive from a regulation of the FXR/SHP pathway, whereas that of HGP should proceed through a mechanism independent of FXR/SHP.

Bile diversions improve glucose control in rats fed a diet inducing obesity. Bile bioavailability and gastric bypass

To determine whether the modification of bile routing could improve metabolic disorders associated with diet-induced obesity as GBP does, we performed bile diversions in rats fed a high-fat highsucrose (HFHS) diet. First, unlike bile-diverted rats fed a standard diet, bile-diverted rats fed a HFHS diet showed a lasting reduction of their food intake immediately after the surgery (Fig. 5a), promoting a continuous body weight loss (Fig. 5b). In order to obviate the role of food intake decrease and body weight loss in the glucose metabolism effects of bile diversions, a group of shamoperated pair-fed with bile-diverted rats was studied in parallel. Expectedly, a similar loss of body weight was observed in bile-diverted and sham-operated pair-fed rats (Fig. 5b). However, there was no significant effect on insulin tolerance or glucose tolerance in sham-operated pair-fed rats. On the contrary, bile-diverted rats exhibited improved insulin and glucose tolerance compared to both sham-operated rats and sham-operated pair-fed rats (Fig. 5c-d). These data indicate that there is a proper effect of intestinal bile re-routing to improve glucose control under diet-induced obesity conditions, independently of food intake and body weight loss.

Bile diversions decrease appetite for fatty food.

Faced with the drastic and lasting decrease in food intake of bile-diverted rats fed a HFHS diet, compared to the moderate and transient effect in bile-diverted rats fed a standard diet, we investigated whether this would be the result of a disinterest to fatty food as previously described in GBP-patients. [START_REF] Mathes | Food selection and taste changes in humans after Roux-en-Y gastric bypass surgery: a direct-measures approach[END_REF] First, we proposed a choice between standard and HFHS diet to rats previously fed a HFHS diet 11 days after the bile diversion surgery. Spectacularly, bile-diverted rats ate immediately and almost exclusively the standard diet whereas the daily food ratio of sham and sham pair-fed rats was composed of 60% of HFHS diet on average (Fig. 6a). Next, we performed a reverse experiment in bile-diverted rats previously fed a standard diet. Sham-operated rats chose at a level of 70-80% the HFHS diet to compose their daily food intake, which was in line with the well-known preference for fatty food in the rat. On the contrary, once they were given a choice between standard and HFHS diet, mid-jejunum bile-diverted rats ate significantly less the HFHS diet compared to sham-operated

Goncalves et al. 2014

Bile bioavailability and gastric bypass rats. Over the next days, bile-diverted rats continued to decrease their consumption of the HFHS diet to adopt almost exclusively the standard diet from the 3 rd day (Fig. 6b). These data strongly suggest that intestinal bile re-routing per se could be responsible for the disinterest in fatty food frequently encountered in GBP patients.

DISCUSSION

The rapid and weight-independent resolution of type 2 diabetes after GBP has urged the scientific community to better understand the physiological mechanisms underlying this procedure. Here, we performed bile diversions in rats in order to investigate the role of the bile routing modification in the metabolic improvements of GBP. As GBP, bile diversions lead to an altered bile delivery in the gut and an increase in plasma bile acids. Spectacularly, bile diversions enhance glucose control independently of weight loss and food intake both in lean and obese rats.

It is noteworthy that, combining mid-jejunum bile diversion with a cholestyramine-enriched diet, we ascertained that the metabolic benefits associated to bile diversions causally depend on the change in bile bioavailability in the intestine and in the blood circulation. This experiment also highlights that the presence of undigested food (without bile) in the proximal intestine, which might constitute a powerful mechanism, is not involved in the beneficial outcomes of bile diversions. Interestingly, bile diversions in the mid-jejunum or in the mid-ileum led to the same improvement in glucose metabolism and level of plasma bile acids. This suggests that the metabolic benefits of bile diversion are not proportional to the length of the gut segment devoid of bile. This observation is of interest in the context of the current debate relating to the length of the respective limbs to adopt in GBP.

Given the fact that bile acids have emerged as positive metabolic regulators, the increase in plasma bile acids in the peripheral blood observed after GBP is frequently proposed to explain the antidiabetic effects of the surgery. [START_REF] Pournaras | The role of bile after Roux-en-Y gastric bypass in promoting weight loss and improving glycaemic control[END_REF][START_REF] Jansen | Alterations of hormonally active fibroblast growth factors after Roux-en-Y gastric bypass surgery[END_REF][START_REF] Lefebvre | Role of bile acids and bile acid receptors in metabolic regulation[END_REF] Our study provides new insight into the mechanisms by which GBP leads to an increase in plasma bile acids. Indeed, we demonstrate that the only modification of bile delivery in the gut promotes a modulation of bile acid transporter expression in the liver and in Bile bioavailability and gastric bypass the intestine in favor of a bile acid increase in the peripheral blood. In accordance with this finding, Mencarelli et al. correlated an increase in plasma bile acids with a decrease in both NTCP expression and bile acid concentration in the liver after ileal transposition, a bariatric procedure based on the manipulation of bile acid entero-hepatic cycling. [START_REF] Mencarelli | Dissociation of intestinal and hepatic activities of FXR and LXRα supports metabolic effects of terminal ileum interposition in rodents[END_REF] Thus, targeting the expression of bile acid transporters could be an attractive strategy to increase bile acid concentration in the blood and consequently potentiate their action in metabolic tissues, e.g. in the liver.

It is noteworthy that the only modification of bile routing reproduces the beneficial effects of GBP on the hepatic function. Indeed, we show a drastic reduction of G6Pase expression in the liver of bilediverted rats. It must be noted that these hepatic changes could account for the improvements in glucose control observed, since it has been demonstrated that inhibiting gluconeogenesis gene expression specifically in the liver is sufficient per se to normalize glucose control in obese and diabetic mouse. [START_REF] Sloop | Specific reduction of hepatic glucose 6-phosphate transporter-1 ameliorates diabetes while avoiding complications of glycogen storage disease[END_REF][START_REF] Gómez-Valadés | Pck1 gene silencing in the liver improves glycemia control, insulin sensitivity, and dyslipidemia in db/db mice[END_REF] Thus, the improvements in hepatic glucose metabolism could be a major key of the GBP benefits, which could be dependent on the bile routing manipulation by itself. Besides, changes in bile routing could also improve lipid metabolism in the liver. Indeed, Kohli et al., who used a similar technique of bile diversion, showed a decrease in hepatic steatosis associated with a reduction of endoplasmic reticulum stress, both of which could concur to the improvement in hepatic metabolism and consequently systemic glucose control. [START_REF] Kohli | A surgical model in male obese rats uncovers protective effects of bile acids post-bariatric surgery[END_REF] Owing to its beneficial effects on glucose and energy homeostasis, the activation of IGN has emerged as a potential strategy to prevent or treat metabolic diseases. Recently, IGN has been shown to be induced by dietary protein [START_REF] Duraffourd | Mu-opioid receptors and dietary protein stimulate a gut-brain neural circuitry limiting food intake[END_REF] and soluble fiber [START_REF] Vadder | Microbiota-generated metabolites promote metabolic benefits via gut-brain neural circuits[END_REF] and to account for the metabolic benefits associated with both types of nutrients, via a portal glucose signaling to the brain. Similarly, IGN was associated with the metabolic improvements deriving from a model of GBP in mice. [START_REF] Troy | Intestinal gluconeogenesis is a key factor for early metabolic changes after gastric bypass but not after gastric lap-band in mice[END_REF] Here, we highlight bile acids as direct negative regulators of IGN. Thus, in GBP, the removal of bile in the alimentary limb could result in an up-regulation of IGN in this portion. However, the capsaicin experiments in bilediverted rats pointed out that a portal-brain communication is required neither in the systemic metabolic improvements nor in the decrease in HGP associated with bile diversions. We thus

Goncalves et al. 2014 Bile bioavailability and gastric bypass

speculate that the marked inhibition of HGP is sufficient per se to promote the metabolic improvements associated to bile diversion, masking the putative benefits associated to the enhancement of IGN.

The regulation of hepatic glucose metabolism by bile acids has been suggested to be dependent on the activation of the FXR/SHP signaling. [START_REF] Yamagata | Bile acids regulate gluconeogenic gene expression via small heterodimer partner-mediated repression of hepatocyte nuclear factor 4 and Foxo1[END_REF][START_REF] Ma | Farnesoid X receptor is essential for normal glucose homeostasis[END_REF] However, this is a controversial issue since a divergent study suggested, on the contrary, that the impact of bile acids on HGP was independent of FXR. [START_REF] De Fabiani | Coordinated control of cholesterol catabolism to bile acids and of gluconeogenesis via a novel mechanism of transcription regulation linked to the fastedto-fed cycle[END_REF] In the work herein, the FXR/SHP pathway was strongly down regulated in the liver after bile diversions.

It is likely that the decrease in hepatic bile acid reabsorption, linked to the down-regulation of NTCP and the up-regulation of OSTα, could lead to a reduction in bile acid content in the hepatocytes and therefore to an inhibition of the FXR/SHP signaling. This rationale is also supported by the concomitant down-regulation of the target genes of FXR and reduced bile acid concentration taking place in the liver after ileal transposition. [START_REF] Mencarelli | Dissociation of intestinal and hepatic activities of FXR and LXRα supports metabolic effects of terminal ileum interposition in rodents[END_REF] Changes in HGP after bile diversions in rats would thus not be dependent on an effect of bile acids via the activation of the FXR pathway. Conversely, the data herein suggest a key role of the FXR/SHP pathway in the regulation of IGN gene expression.

Interestingly, IGN and portal glucose sensing were suggested to be involved in the beneficial outcomes of a model of GBP in mice. [START_REF] Troy | Intestinal gluconeogenesis is a key factor for early metabolic changes after gastric bypass but not after gastric lap-band in mice[END_REF] This could be related to the recent observation that FXR signaling was required for the benefits on glucose and energy metabolism occurring after sleeve gastrectomy, a weight-loss procedure associated with an increase in plasma bile acids despite it does not involve an intestinal bypassing, in mice. [START_REF] Myronovych | Vertical sleeve gastrectomy reduces hepatic steatosis while increasing serum bile acids in a weight-loss-independent manner[END_REF][START_REF] Ryan | FXR is a molecular target for the effects of vertical sleeve gastrectomy[END_REF] A final key finding linked to bile diversion was the marked decrease in preference for fatty food. GBP patients generally adopt healthier dieting and increase their intake of vegetables, an outcome linked to their decreased appetite for high-calorie food. [START_REF] Kenler | Changes in eating behavior after horizontal gastroplasty and Roux-en-Y gastric bypass[END_REF][START_REF] Olbers | Body composition, dietary intake, and energy expenditure after laparoscopic Roux-en-Y gastric bypass and laparoscopic vertical banded gastroplasty: a randomized clinical trial[END_REF][START_REF] Ernst | Differential changes in dietary habits after gastric bypass versus gastric banding operations[END_REF] Different explanations involving taste detection, hedonic and reward systems have been proposed to be responsible for changes in food preference after GBP but the corresponding mechanisms are largely unclear. [START_REF] Miras | Bariatric surgery and taste: novel mechanisms of weight loss[END_REF][START_REF] Bueter | Alterations of sucrose preference after Roux-en-Y gastric bypass[END_REF][START_REF] Roux | Gastric bypass reduces fat intake and preference[END_REF] A part of these behavioral changes could also be attributed to the lifestyle changes recommended to patients during the post-operative period, involving calorie restriction. The strength of the experiments here is the Bile bioavailability and gastric bypass lack of this type of bias. Bile-diverted rats exhibited a disinterest to high-calorie food while this diet was offered ad libitum after surgery. It is noteworthy that these food preference changes take place without size reduction of the stomach. This allows us to firmly ascertain that bile routing modification leads to a profound change in food preference regardless of nutritional or surgical intervention. Our results also contradict the hypothesis that altered food choices after GBP could be explained by aversive symptoms taking place after the consumption of HFHS food, like dumping syndrome or vomiting. Indeed, bile-diverted rats immediately avoid the HFHS diet, even after being exclusively fed a standard diet (Fig. 5a-6b). Therefore, we here highlight an unexpected role of bile bioavailability as a key player in the mechanisms of regulation of food preference. This warrants further studies to better understand the regulatory mechanisms behind this effect, which could lead to the development of innovative food behavior therapies to reduce body weight and combat obesity.

In conclusion, this work provides a novel understanding in the mechanisms by which GBP promotes its rapid metabolic outcomes. Our data strongly suggest that the modification of bile routing per se is able to initiate the beneficial effects of GBP on both glucose control and body weight in a context of obesity, including the disinterest in high calorie food. The fact that this study was conducted only in animals could be a potential limitation regarding to its translational potential. However, it must be emphasized that the nutritional and metabolic effects of bile diversions reported here perfectly match those of GBP, which are well documented in human studies. Implementing other recent studies, [START_REF] Pournaras | The role of bile after Roux-en-Y gastric bypass in promoting weight loss and improving glycaemic control[END_REF][START_REF] Kohli | A surgical model in male obese rats uncovers protective effects of bile acids post-bariatric surgery[END_REF][START_REF] Ryan | FXR is a molecular target for the effects of vertical sleeve gastrectomy[END_REF] this study places the bile routing modification in the center of the metabolic benefits associated to GBP, which could be a first milestone toward the development of future approaches of prevention or treatment of metabolic diseases. a standard diet by proposing to the animal standard and HFHS diet concomitantly. Data are expressed as mean ± SEM; n=4-9 rats per group; *p<0.05, ***p<0.001 vs sham-operated group (Oneway ANOVA followed by Tukey's post-hoc test for the studies in rats previously fed a HFHS diet and t test for rats previously fed a standard diet).

Supplemental table 1:

List of sequence-specific primer used for q-PCR The authors declare no competing financial interests. The work was supported by an academic grant from the "Société Francophone du diabète".

Gene

Financial Disclosure

Figure 1 :

 1 Figure 1: Effects of bile diversions on body weight, food intake and glucose homeostasis in

Figure 2 :

 2 Figure 2: Implication of changes in bile bioavailability on glucose metabolism improvements and

Figure 3 :

 3 Figure 3: Regulation of glucose-6-phosphatase expression in bile-diverted rats.

Figure 4 :

 4 Figure 4: Hepatic and intestinal expression of FXR and SHP in bile-diverted rats.

Figure 5 :

 5 Figure 5: Metabolic effects of bile diversions in HFHS-fed rats.

Figure 6 :

 6 Figure 6: Food preference in bile-diverted rats. (a) Food preference was assessed 11 to 15 days after surgery in rats previously fed a HFHS diet or (b)

  glucose (mg/dL)

Supplemental data 1 :

 1 Figure 1

Supplemental data 2 :

 2 Figure S1

ACKNOWLEDGEMENTS

We thank the members of the "Animalerie Lyon Est Conventionnelle et SPF" for animal care. We are also grateful to Dr Bart Staels and Dr Jean-Marc Vanacker for helpful advising in the course of this 15

Goncalves et al. 2014

Bile bioavailability and gastric bypass work. We also thank the French "Ministère de l'Enseignement Supérieur et de la Recherche (D.G., F.D.V., J.V.), the "Agence Nationale de la Recherche" (A.B.), the "Institut National de la Santé et de la Recherche Médicale" (A.D., C.Z.) and the "Centre National de la Recherche Scientifique" (G.M.) for funding our positions. This work was supported by research grants from the "Agence Nationale de la

Recherche" (ANR11-BSV1-016-01) and the "Société Francophone du Diabète" (exceptional funding-2012).

AUTHOR CONTRIBUTIONS

D.G. conducted and designed experiments, performed data analyses and wrote the manuscript. A.B., F.D.V. and J.V. assisted in experiments and contributed to the interpretation of data. A.D. and C.Z. assisted in surgical procedures. G.M. supervised the project and edited the manuscript.

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.


Tiago Coelho
email: tcoelho@uninova.pt

Rita De Oliveira
email: r.oliveira@lsbu.ac.uk

Tiago Cardoso

Yves Rybarczyk

Body Ownership of Virtual Avatars: An Affordance Approach of Telepresence

Keywords: Avatar, telepresence, ownership, affordances, natural user interface, virtual environment

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Telepresence and Ownership

Telepresence is the feeling of being present in a place where the person is not [1]. This feeling can be achieved while an individual is using a simulator or performing a certain task in a virtual environment such as a game [START_REF] Slater | Depth of presence in virtual environments[END_REF]. Another way this phenomenon can occur is in teleoperation, in which the user remote-controls a robot with a camera that provides the teleoperator with visual feedback from the working space [START_REF] Rybarczyk | Effect of visuo-manual configuration on a telerobot integration into the body schema[END_REF]. Telepresence is a critical mental process as it increases the immersion of the individual within a certain task. Teleoperation and virtual environments are the most common situations in which a feeling of telepresence may occur. One of the most specific cases of telepresence is body ownership, in which the individual is so immerse in the teleoperation task he/she is performing that he/she be-lieves the remote machine is part of him/her [START_REF] Rybarczyk | Sensori-motor appropriation of an artefact: a neuroscientific approach[END_REF]. In an experiment carried out by Sumioka et al. [START_REF] Sumioka | Teleoperated android for mediated communication: body ownership, personality distortion, and minimal human design[END_REF], subjects remote-controlled a human-like machine. They had a first person view over the machine, which replicated every move of the individual. The participant's reactions were gauged by measurement of the skin conductance. The results show that the participants seemed to feel the machine as it was their own body. In addition, the feeling of ownership can also happen in other cases of mediated situations such as in virtual reality, in which the individual believes he/she is the avatar.

The study that revealed for the first time the phenomenon of ownership is the Rubber Hand Illusion [START_REF] Botvinick | Rubber hands 'feel' touch that eyes see[END_REF]. In this experiment, the participant's hand is hidden and a rubber hand is visible in its place. A tactile stimulation is applied simultaneously to both hands. After a while, the individual has the feeling that the fake hand is his/her own. A similar effect has also been observed in virtual reality [START_REF] Yuan | Is the rubber hand illusion induced by immersive virtual reality?[END_REF][START_REF] Tsakiris | Having a body versus moving your body: How agency structures body-ownership[END_REF]. Ehrsson et al. [START_REF] Ehrsson | That's my hand! Activity in premotor cortex reflects feeling of ownership of a limb[END_REF] recorded the brain activity of participants when they were submitted to the same experiment. The results showed a significant activation of the parietal cortex only in the presence of a synchronous and congruent visuo-tactile stimulation of the rubber and the real hand. In addition, a positive correlation between the physiological and ownership questionnaire data confirmed that the participants were considering the rubber hand as their own hand.

It seems that the ownership feeling is not exclusive of the individual limbs and can occur on the entire body. In Petkova and Ehrsson's [START_REF] Petkova | If I were you: perceptual illusion of body swapping[END_REF] experiment, participants wore a head-mounted display and had a first person view over a body-sized mannequin. They received visual and tactile stimulations over the whole body. In that condition, the participants had the feeling that the mannequin's body was their own. The ownership feeling was measured through skin conductance, which can detect psychological or physiological alterations. Authors stress the fact that a human-like representation of the mannequin and a synchronous visuo-tactile stimulation were crucial to trigger the ownership illusion.

A more surprising observation is the fact that body ownership may also occur in a simple situation of tool-use. Studies showed that when individuals manipulate an artifact, they consider it as an extension of their arm [START_REF] Maravita | Tools for the body (schema)[END_REF]. The initial study was performed with non-human primates and their brain activity was measured by electrodes. The results show that some specific bimodal neurons coding for the monkey's hand fire in a similar manner when a stimulus is applied to the hand or close to the tool manipulated by the animal [START_REF] Iriki | Coding of modified body schema during tool use by macaque postcentral neurons[END_REF]. This is clear evidence showing that the artifact seems to be integrated into the primate body schema.

Overall, these experiments showed that ownership occurs in the brain, after integration of multimodal information (vision, touch and proprioception) in order to build a coherent representation of the body.

Methods for Measuring Telepresence

Questionnaires. There are several ways to measure telepresence. One way the evaluation can be done is by questionnaire, in which the user answers a few questions in order to express what they felt during the experiment. This is probably the most popular kind of presence assessment. A significant number of studies on telepresence or ownership ask their participants to fill a questionnaire when the experiment is over [START_REF] Botvinick | Rubber hands 'feel' touch that eyes see[END_REF][START_REF] Ehrsson | That's my hand! Activity in premotor cortex reflects feeling of ownership of a limb[END_REF][START_REF] Petkova | If I were you: perceptual illusion of body swapping[END_REF][START_REF] Maselli | The building blocks of the full body ownership illusion[END_REF].

Questionnaires are mostly used because of the simplicity of their implementation and the large range of possible questions. They are also a very quick and practical way for people to express their feelings, on a numerical scale that allows quantification and comparisons. Nevertheless there are some disadvantages, such as misinterpretation of questions, subjectivity of answers, the scale level number (odd vs. even) or, since it happens after the experiment, participants might forget what they felt. Another disadvantage is the number of questions: if there are too many, the participants may lose interest and answer randomly.

Physiological Parameters. Another way to gauge presence is by physiological parameters such as heart rate, galvanic skin response, electromyography or electroencephalogram. The galvanic skin response (GSR) measures the skin conductance of electricity. The variation of skin conductance occurs by changes in the moisture of the skin. Emotional stimulus triggers the sympathetic nervous system to increase the activity of the sweat glands. Armel and Ramachandran [START_REF] Armel | Projecting sensations to external objects: evidence from skin conductance response[END_REF] measured the skin conductance in their rubber hand illusion experiment. They threatened to harm the rubber hand. If the participant thought that the rubber hand was his/her own, the skin conductance results showed signs of arousal.

The electromyography (EMG) measures the electric activity of activated muscles. The signals can also be used to detect neurologic activity. Slater et al. [START_REF] Slater | Inducing illusory ownership of a virtual body[END_REF] used this technique during a rubber hand illusion experiment carried out in virtual reality. When ownership was achieved, the virtual hand was twisted. The EMG showed the twist induced a motor activity along the participant's real arm.

The electroencephalogram (EEG) directly measures electric activity of the brain in a non-invasive way. González-Franco et al. [START_REF] González-Franco | Virtual Embodiment Elicits a Mu Rhythm ERD When the Virtual Hand is Threatened[END_REF] showed that EEG can be used to assess presence in a virtual environment. They conducted an experiment in which the arm of the avatar was threatened. Results showed that when facing a threat, the participants lowered their motor cortex activity.

Behavioral Assessment: Affordances. In this project, affordances are used as a behavioral assessment to measure telepresence in a virtual environment. Affordances are a concept first suggested in the literature by J.J. Gibson [START_REF] Gibson | The ecological approach to visual perception[END_REF]. An affordance is an action possibility whereby people perceive their environment and the objects within it as possibilities of doing certain actions and not doing other actions. Affordances exist where the characteristics of the object and the characteristics of the person match in a particular way. For instance, most chairs will afford sitting to most adults, but will not afford sitting to a 6-months baby, and might afford standing to someone making a speech. This concept is also applicable to other animal species, like for example a tree can afford nourishment to a giraffe but for a bird it can afford nesting. An affordance is a combination of the physical characteristics of the object and the animal, the knowledge about the object, and the needs to the animal at a particular time. In some cases, the action possibility may be harmful, in which case the animal may choose not to perform the action. For example a knife affords cutting into various surfaces be-cause it has a blade. If someone grasps it by the handle it affords cutting into bread or cut through paper but it also affords injury if grasped by the blade. Another example is about apertures. An aperture will only afford passage if it is wider than the individual. If it is narrower it may afford passage if the individual rotates upon himself [START_REF] Warren | Visual guidance of walking through apertures: body-scaled information for affordances[END_REF]. Affordances are based on experience, in the sense that people learn to perceive the relevant characteristics of the environment and objects within it. This means they will be common to many individuals (e.g., passing through apertures which are large enough) but different from one individual to another (e.g., a rugby player, a gymnast, or a child will fit through different apertures). The link between perception and action which guides people's decision evolves over time through experience [START_REF] De Oliveira | The bidirectional links between decision making, perception, and action[END_REF].

After the initial study by Warren and Whang [START_REF] Warren | Visual guidance of walking through apertures: body-scaled information for affordances[END_REF] testing affordances, other studies have followed which explore and test the notion of affordances, such as [START_REF] Mark | Eyeheight-scaled information about affordances: a study of sitting and stair climbing[END_REF][START_REF] Esteves | Posture-related affordances guide attack in basketball[END_REF]. One crucial finding was that the possibilities for action available to an individual are scaled to the individual's body. This scaling factor is important because it links object properties and individual's dimensions through an invariant value; this means there is a lawful relation underpinning (at least some) affordances. Such lawful relations have been found in various animals. In human participants, this was found in stair climbing where participants deem a stair climbable (without the aid of hands) if the raiser is smaller than 0.88 their leg length [START_REF] Warren | Perceiving affordances: visual guidance of stair climbing[END_REF]. This was also found to be the case in passing through apertures where participants rotate their shoulders over their longitudinal axis if the aperture is smaller than 1.4 the width of their shoulders [START_REF] Warren | Visual guidance of walking through apertures: body-scaled information for affordances[END_REF].

2

Questioning and Prototype Solution

Problematic

The main purpose of this article is to evaluate the effect of an avatar on the feelings of telepresence. Avatars are very common in virtual environments. They are the alteregos of users in the virtual world. The avatar is usually seen from a first or third person perspective. Several aspects can be studied regarding the avatar, such as its dynamics, morphology or physical appearance. In addition, aspects such as the camera perspective or a visuo-motor feedback from hardware might change the ownership feelings about the avatar. The physical user interface is an important parameter to take into account because the control the user has on the avatar influences his/her behavior [START_REF] Meadows | I, avatar[END_REF]. All of these features may influence the way a person feels towards the avatar [START_REF] Castronova | Theory of the Avatar[END_REF]. Here, the avatar's morphology and dynamics were studied when a human user controlled it through a NUI.

The morphology of the avatar is an important feature in achieving body ownership. For instance, Tsakiris and Haggard [START_REF] Tsakiris | The rubber hand illusion revisited: visuotactile integration and self-attribution[END_REF] carried out the rubber hand illusion experiment with a fake hand and with a wooden stick. Their results showed that, with the rubber hand, ownership was easier to achieve than with the wooden stick. Because feelings of ownership happened with the hand, a similar result is expected if applied on a larger scale to a scaled-to-user avatar. This was done by Petkova and Ehrsson [START_REF] Petkova | If I were you: perceptual illusion of body swapping[END_REF] in a study using a camera on a mannequin in the real world.

The dynamics of the avatar may also help to induce the feelings of telepresence and ownership on its user. If there is real-time congruence between the movements of the user and the movements of the avatar, then the feelings of telepresence and ownership should be greater than with incongruent movements. This is supported by the experiment carried out by Kalckert and Ehrsson [START_REF] Kalckert | Moving a rubber hand that feels like your own: a dissociation of ownership and agency[END_REF]. These authors showed that the rubber hand illusion can be induced through a simple visuomotor correlation, without the need for a tactile stimulation as had been used in the original study of Botvinik and Cohen [START_REF] Botvinick | Rubber hands 'feel' touch that eyes see[END_REF].

The study presented here is composed with two experimental conditions. In one of the conditions, the avatars are morphologically proportional (similar) to each participant. It is possible to have a dynamic avatar fully proportional to the user thanks to a full body motion capture. In the other condition, the avatar resembles the first one in how it looks, but it is the same (standard) for every participant. In addition, the movements of this standard avatar do not exactly match the participant's movements, as it only moves sideways and rotates upon itself. Aside from the avatar conditions, there was also a speed condition: fast and slow.

Developed Tool

The ATTAVE (Avatar Telepresence Testing: Affordances in Virtual Environments) is the name of the prototype developed. The ATTAVE is a virtual environment where the avatars exist and where experiments using affordances are performed. This virtual environment will be the same for both avatar conditions. Thus, the only parameters that change in the experiment are the avatar's characteristics. The design of the prototype is based on a study performed by Warren and Whang [START_REF] Warren | Visual guidance of walking through apertures: body-scaled information for affordances[END_REF]. The authors evaluated how people passed through apertures considering the shoulder width of the participant and the ratio between the aperture and their shoulder width. The participants passed through several apertures of various sizes and the degree of their shoulder rotation was recorded. There were two speed conditions: a slow and a fast walking speed.

Results showed that the participants only walked frontally through the aperture when the ratio between the aperture and the shoulders was smaller than 1.4. The present study will be similar to the one performed by Warren and Whang [START_REF] Warren | Visual guidance of walking through apertures: body-scaled information for affordances[END_REF], but will be performed in a virtual environment. By replicating a study performed in the real world, telepresence can be verified if the same behaviors that happen in the real world also happen in the virtual environment. The display of ATTAVE consists of a virtual scenario showing a long treadmill moving towards a visible avatar (and also towards the participant). The avatar resembles a wooden mannequin and is visible from head to knees, as the viewpoint of the participant is 2 m behind the avatar. The treadmill is enclosed on the side by tall walls. On the treadmill itself there are frontal green walls with an aperture on the left, centre or right side of the wall. All surfaces have texture as can be seen in Figure 1. The participants could control the translation and rotation of the avatars by physically moving side to side and rotating their own shoulders. Shoulder rotation proportionally slowed down the treadmill. The participants' task was to avoid collisions and pass through all doors as fast as possible. The software chosen for modeling the avatars was Blender. The final models resembled a wooden mannequin (Figure 2). The software used to create the animation of the (standard) avatar was iPi Recorder and iPi Mocap Studio (iPi Soft). This tool was chosen because it can record the movements captured by the Kinect and associate those movements to the avatar. With this method, an animation was created of a sidestep with a human model performing the action. This was the standard side-step used by the avatar in the standard condition.

Unity 3d was the game engine chosen for designing ATTAVE and running the application. The advantage of Unity 3d is its MonoDevelop IDE (Integrated Development Environment) integrated in the software, which facilitates the scripting part of the game. The scripting was made entirely in Java scripting language for Unity. Another reason that led to the use of Unity 3d in the project was its easy integration with the Kinect, which is made possible thanks to a framework called OpenNI (Open Source Natural Interaction) provided by Primesense.

For the full body motion capture a Kinect NUI was used. This device uses an optic technology that allows detection of the human body thanks to an infrared depth camera. This choice was due to its ease to set up, as it can be ready to use in less than five minutes, and also due to its low cost compared to other equipment of the same category. Another advantage in using this system is the fact that the user does not have to wear a specific suit and, consequently, he/she has full range of movement. In order to provide the participant with audio-visual feedback, a noise and a flash next to the avatar's shoulders were displayed whenever there was a collision with a wall.

When the participant rotated the shoulders, the speed of the treadmill decreased in proportion to the cosine of the angle between the shoulders' axis and the avatar's translation axis, as described by the formula below. current speed = set speed * (1 -0.4 * sin(angle))

In (1), the value of the angle is taken as 0 if the individual is in a frontal position towards the door and 90º if the individual is facing the side walls. The angles are in absolute values from 0º to 90º. The speed decrease with rotation was introduced because it simulates a normal behavior observed in the real world. People usually reduce their locomotion velocity when they have to pass through a narrow aperture. The decreasing value of 0.4, used to calculate the current speed, was based on pilot trials. Moreover, the correlation between rotation and speed was an incentive for participants not to rotate their shoulders unless there was a danger of collision, as the rotation slowed down the treadmill and added to their total time on the task.

Experiment

Participants

Participants were 24 university students (18 male and 6 female, aged between 20 and 28), with normal or corrected-to-normal vision and varied experience in playing video games. Half of the participants performed in two conditions (similar fast, similar slow) and the other half performed in two other conditions (standard fast, standard slow). This was done to enable the study of eventual learning effects under each speed condition. The experiments were approved by the local ethics committee of the Nova University Lisbon.

Setup

The experiment was conducted in a 3 × 3 m area. Participants stood 3 m away from a 75 cm height table. On the table was mounted an off-the-shelf Kinect sensor (Microsoft, for Xbox 360) and an 18" computer screen (1440 × 900 pixel resolution), both connected to a PC. Three small marks on the floor indicated the positions aligned with the three apertures on the display (see Figure 3).

Experimental Design

Participants performed 32 trials for each of 2 speed conditions and for each of 4 sessions. Also, there were 2 avatar conditions; each used in a group of participants. The 32 trials consisted of apertures that showed in the central position with widths gradually increasing relative to the avatar's shoulders from 0.7 to 2.2 and then gradually decreasing from 2.2 to 0.7 (in steps of 0.1). When the avatar passed through each of these apertures, the value corresponding to the angle between the shoulders was recorded. These trials were alternated with 32 dummy trials with apertures of constant size shown in the right and left side positions. These side apertures remained twice the shoulder width of the avatar and were not used for data collection. Every aperture was 10 meters away from the next aperture. The two speed conditions were slow and fast (respectively 5 and 10 Km/h). They were taken from the walking speeds reported by Warren and Whang [START_REF] Warren | Visual guidance of walking through apertures: body-scaled information for affordances[END_REF] and adjusted during pilot testing. The avatar condition consisted of manipulating the morphology and movements of the avatar. In the similar avatar condition the avatar was anatomically proportional to the dimensions of the participant and all segments were animated to mimic the natural movements of the participant. In the standard condition the dimensions of the avatar were standard for all participants and the avatar had only two degrees of freedom: translation sideways and rotation upon itself (see arrows on Figure 3). An animation of a sidestep was implemented on the avatar when the participant performed a sidestep. This animation was recorded with a natural user interface and results from a sidestep performed by a human being.

The ratio between each virtual door and the avatar's shoulders width was the independent variable manipulated. The dependent variable was the angle between the shoulders during the passage through each aperture.

Procedure

The experiment started with participants reading and signing the consent form. Then, the Kinect was calibrated to the participants' movements. Participants were instructed to avoid collisions and complete the task in the shortest possible time, and were informed that shoulder rotation proportionally decreased the speed of the treadmill. In each session, participants completed the increasing-decreasing series in the slow condition followed by the fast condition. Participants were asked to fill in a questionnaire adapted from Witmer et al. [START_REF] Witmer | The factor structure of the Presence questionnaire[END_REF]. Finally, the measure of participants' height and shoulder width was taken. In total, each session lasted about 20 minutes.

Results

Data Analysis. The main dependent variable was the critical ratio after which the participant passed without rotation. Following Warren and Whang [START_REF] Warren | Visual guidance of walking through apertures: body-scaled information for affordances[END_REF], the two values of the critical ratio of shoulder rotation from the increasing-decreasing series were averaged. The critical ratio was that with an angle smaller than 16º and after which all angles were smaller than 16º (one exception was permitted provided the angle was smaller than 40º and the average angles remained smaller than 16º). A critical ratio was calculated for each participant, condition, and session and these were used in the data analysis.

To examine learning effects from session to session, the critical ratios were submitted to a multivariate repeated measures analysis of variance (MANOVA) with the factor session (4 levels), and using the 4 conditions as measures (slow-similar, fastsimilar, slow-standard, fast-standard). Based on the results of this analysis, the averages of the last 3 sessions were used in the remaining analysis.

To examine the effect of conditions on the critical ratios, the individual critical ratios from the last 3 sessions were averaged and submitted to a repeated measures analysis of variance (ANOVA) with factors speed (2 levels: slow and fast) and avatar (2 levels: similar and standard). The same analysis was conducted for the total durations, that is, the time that the participants took to complete the task by going through all the apertures. The same analysis was also used to test the effects on collisions.

To examine how participants felt regarding the experienced environment, the scores for each dimension of the questionnaires were averaged and submitted to a multivariate repeated measures analysis of variance (MANOVA) with factors avatar (2 levels: similar and standard) and session (4 levels) and using the 5 dimensions as measures (realism, possibility, quality, ownership, and self-evaluation).

Finally, Pearson's r was used to test the correlation between critical ratios and the dimension of ownership as measured by the questionnaire.

Learning of Critical Ratios. Overall, there was a significant learning effect on the critical ratios, F(12, 86) = 6.85, p < .001, η 2 = .49. This was reflected in the four conditions: slow-similar, F(3, 33) = 17.74, p < .001, η 2 = .62; fast-similar, F(3, 33) = 3.08, p < .05, η 2 = .22; slow-standard, F(3, 33) = 8.74, p < .001, η 2 = .44; and faststandard, F(3, 33) = 3.07, p = .05, η 2 = .22.

Pairwise comparisons showed significant differences between the first and the last three sessions which did not differ between them (see Figure 4).

Fig. 4. Average critical ratios for the four conditions over the four sessions

Critical Ratios. There was a strong tendency for an effect of critical ratio on avatar, F(1, 11) = 4.62, p = .055, η 2 = .30 (Figure 5), which was caused by participants rotating their shoulders at smaller critical ratios when the avatar was standard than when the avatar was similar (standard M = 1.40, se = 0.05, similar M = 1.57, se = 0.07).

Fig. 5. Effect of the avatar on the critical ratios

There was a significant main effect of critical ratio on speed, F(1, 11) = 5.13, p < .05, η 2 = .57 (Figure 6). This was caused by participants rotating their shoulders at smaller critical ratios in the slow condition compared to the fast condition (slow M = 1.43, se = 0.04; fast M = 1.54, se = 0.05).

Fig 6. Effect of speed on critical ratios

There was no Speed × Avatar interaction, F(1, 11) = 2.51, however it is noteworthy that the effect of speed was more marked when avatars were similar than when avatars were standard (Figure 7).

32).

Collisions. There were no significant main effects of collisions on speed, F(1, 11) = 0.0, avatar, F(1, 11) = 2.19, and no significant interaction effect, F(1, 11) = 0.40. On average there were 2 collisions on each session across conditions. Questionnaire. Overall, there was no significant main effect of avatar, F(2, 7) = 1.83 or session, F(2, 7) = 0.93. However, there was a significant Avatar × Session interac-tion, F(15, 80) = 1.87, p < .05, η 2 = .24. This significant interaction was reflected in three dimensions: realism, F(3, 33) = 4.00, p < .05, η 2 = .27; ownership, F(3, 33) = 3.93, p < .05, η 2 = .26 and self-evaluation, F(3, 33) = 3.17, p < .05, η 2 = .22. This interaction occurred because feelings of realism, ownership and self-evaluation increased in the similar condition and decreased in the standard condition (Figure 8). Ownership and Critical Ratios. Overall, there was a small, positive correlation between feelings of ownership and critical ratios, r = 0.36, n = 96, p < 0.05 indicating that increases in one variable were accompanied by increases in the other variable (Figure 9).

Discussion

The main objective of this research was to study whether the dynamics and the morphology of an avatar would reflect on the feelings of telepresence and ownership of the participant. Also, to know whether affordances were used in a virtual environment the same way they are in a real environment. In order to perform the experiment, a prototype named ATTAVE was developed. ATTAVE possess a virtual environment where the participant's avatar performs the experimental task. The user controlled the avatar through a natural motion capture carried out by a Kinect NUI. There were two avatar conditions to test: one morphologically proportional to the user and that replicated his/her movements; and another with an avatar that was identical for every participant, and exhibited limited mobility, as it could only rotate upon himself and step sideways.

The results show that participants adjusted to the virtual environment after taking their first session. This learning effect is only significant in the first session, and the other three sessions were similar to each other in a same condition. The learning effect that occurred in the first session indicates that participants learned to use appropriate information, provided by the virtual environment, to solve the problem of passing through apertures. It is usual in Human-Machine Interaction (HMI) that users take time to adapt to the artifact. For instance, Peters et al. [START_REF] Peters | Robonaut task learning through teleoperation[END_REF] showed that six trials on a teleoperated robotic arm are necessary to have an accurate representation of the task. On the contrary to a simple tool held in hand, the remote or virtual interaction is not straightforward and involves a specific design of the artifact to be ergonomically adapted to the human user [START_REF] Rybarczyk | Effect of temporal organization of the visuo-locomotor coupling on the predictive steering[END_REF]. After learning, the critical ratios obtained in the virtual environment (1.4 and 1.57) were very similar to those in the real environment (1.4 reported by Warren and Whang [START_REF] Warren | Visual guidance of walking through apertures: body-scaled information for affordances[END_REF]), which demonstrates that people perceive similar body-scaled affordances in the virtual environment as in real environments.

The effect of the different avatars was significant. Participants with the standard avatar had a smaller critical ratio than the participants with the similar avatar (1.4 vs. 1.57). It means that participants who controlled a similar avatar tended to keep a greater safety margin between the body and wall, compared with a standard avatar. This result can suggest a higher feeling of agency for the similar avatar by the individual, who may care about his/her alter-ego and therefore make sure it does not collide with the wall. In contrast, participant seemed less concerned regarding the standard avatar. This interpretation is supported by the analysis of the correlation between the ownership questionnaire and the critical ratios. There was a positive correlation between these two variables, albeit small, which suggests that the rise in the critical ratios is related with an increase in the feelings of ownership.

The higher critical ratios in the similar condition compared to the standard condition could also be explained by distraction because the similar condition involved seeing more of their own movements in the avatar's movements. The amount movements may have led the participants to attend to something other than the shoulders and the apertures. The movements of the standard avatar were much more restricted, which decreased the amount of cues available. In the standard condition, the participant's body controlled the avatar as if it were a simple joystick. Consequently, this condition can be interpreted in terms of a classical remote control of a teleoperated machine. In a study carried out by Moore et al. [START_REF] Moore | Perception of robot passability with direct line of sight and teleoperation[END_REF], the user controlled a robot and supervises the environment by means of a camera on top of the robot. The operator's task was to judge whether or not the robot could pass through apertures of various sizes. The results indicate that the participants judged the robot could pass even when it could not. Authors argued that the results might be influenced by structural and morphological aspects. The same effect could explain the results of the experiment presented here. The fact that the individual underestimates the aperture width could signify that he/she considers the standard avatar as a machine-like character and, consequently, he/she can hardly be engaged in an ownership process with it.

In addition, one aspect that can increase the immersiveness in a virtual environment is the type of interaction between human and machine. If the interaction is done through a classic controller such as a mouse or joystick, the users need to learn the mapping between their own movement and its consequence in the virtual world [START_REF] De Oliveira | Driving skills of young adults with developmental coordination disorder: Regulating speed and coping with distraction[END_REF][START_REF] Wise | Arbitrary associations between antecedents and actions[END_REF]. On the contrary, the mapping is facilitated if the interaction is done through a full motion capture. Research has shown that natural user interfaces, in which users can recognize their own movements in the virtual environment, are more immersive [START_REF] Bruder | Arch-explore: A natural user interface for immersive architectural walkthroughs[END_REF][START_REF] Francese | Wiimote and Kinect: gestural user interfaces add a natural third dimension to HCI[END_REF]. It could explain why the similar avatar condition seems to bring a higher feeling of ownership compared with the standard condition.

Analyzing the effect of speed on the critical ratios, it is observable that in both avatar conditions the critical ratio is larger when the speed is higher. This happens because at higher speeds people leave larger safety margins. In the real world, when an individual is confronted with an aperture, he/she will reduce his/her speed in order to fit through without colliding. In this project, the only way to decrease the speed was by rotating the shoulders, which resulted in a higher critical ratio. The relation between speed and accuracy is well-known in the area of motor control and was described for the first time in Fitts' law [START_REF] Fitts | The information capacity of the human motor system in controlling the amplitude of movement[END_REF]. The fact that the participants of this experiment reproduced this natural motor control adaptation suggests their immersion into the virtual environment (ATTAVE).

Finally, the questionnaire showed that the similar avatar elicited an increasing feeling of ownership and realism over the four sessions. In contrast, the standard avatar caused a decreasing feeling of ownership and realism from session to session. This result means that an agency process in which the participants consider the similar avatar as a natural extension of themselves seems to happen and to increase through the interaction with the avatar. In contrast, with the standard avatar, participants may have become bored due to the avatar's movement not being as diverse as their own. This lack of biological motion could lead the participants to act as if they were controlling a machine instead of a virtual representation of themselves. Overall, consider-

Fig. 1 .Fig. 2 .

 12 Fig. 1. ATTAVE seen from above (this was not the perspective used in the study)

Fig. 3 .

 3 Fig. 3. Physical setup of the experiment

Fig. 7 .

 7 Fig. 7. Effect of speed condition and avatar on the critical ratios in the four sessions (black bars represent the similar avatar and grey lines represent the standard avatar)

Fig. 8 .

 8 Fig. 8. Questionnaires results for the three dimensions over sessions

Fig. 9 .

 9 Fig. 9. Scatter plot and Pearson's r

ing the questionnaire results and the affordances described through the critical ratios, it seems that an avatar with natural movements and tailored the morphology of the user can significantly enhance the feelings of body ownership.