Skip to main content
NCBI home page
HAL-INSERM logoLink to HAL-INSERM
. Author manuscript; available in PMC: 2018 Mar 1.
Published in final edited form as: Ann Plast Surg. 2015 Dec;262(6):1006–1015. doi: 10.1097/SLA.0000000000001121

Bile Routing Modification Reproduces Key Features of Gastric Bypass in Rat

Daisy Goncalves 1,2, Aude Barataud 2,1, Filipe De Vadder 2,1, Jennifer Vinera 2,1, Carine Zitoun 2,1, Adeline Duchampt 2,1, Gilles Mithieux 2,1,*
PMCID: PMC5678047  PMID: 25575265

STRUCTURED ABSTRACT

Objective

To evaluate the role of bile routing modification on the beneficial effects of gastric bypass surgery on glucose and energy metabolism.

Summary background data

Gastric bypass surgery (GBP) promotes early improvements in glucose and energy homeostasis in obese diabetic patients. A suggested mechanism associates a decrease in hepatic glucose production (HGP) to an enhanced intestinal gluconeogenesis (IGN). Moreover, plasma bile acids are elevated after GBP and bile acids are inhibitors of gluconeogenesis.

Methods

In male Sprague-Dawley rats, we performed bile diversions from the bile duct to the mid-jejunum or the mid-ileum to match the modified bile delivery in the gut occurring in GBP. Body weight, food intake, glucose tolerance, insulin sensitivity and food preference were analyzed. The expression of gluconeogenesis genes was evaluated in both the liver and the intestine.

Results

Bile diversions mimicking GBP promote an increase in plasma bile acids and a marked improvement in glucose control. Bile bioavailability modification is causal since a bile acid sequestrant suppresses the beneficial effects of bile diversions on glucose control. In agreement with the inhibitory role of bile acids on gluconeogenesis, bile diversions promote a blunting in HGP, whereas IGN is increased in the gut segments devoid of bile. In rats fed a high fat-high sucrose diet, bile diversions improve glucose control and dramatically decrease food intake due to an acquired disinterest in fatty food.

Conclusion

This study shows that bile routing modification is a key mechanistic feature in the beneficial outcomes of GBP.

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.1 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.2,3 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.4–6 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.7,8 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 sensor9 that initiates a gut-brain neural circuit inducing satiety10,11 and an increased inhibition of HGP by insulin.12 In models of GBP, an induction of IGN with in parallel a decrease in HGP has been reported in rodents13–15 and humans.16–18 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-cells19–21 and increase energy expenditure in the brown-adipose tissue and skeletal muscle.22 Interestingly, they have also been reported to inhibit gluconeogenesis either directly23 or through the activation of farnesoid X receptor (FXR)/small heterodimer partner (SHP) pathway.24,25 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,26–29 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.30 Thus, bile 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.

Figure 1. Effects of bile diversions on body weight, food intake and glucose homeostasis in standard-fed rats.

Figure 1

Open in a new tab

(a) Schematic representations of bile diversions. (b) Evolution of body weight and (c) daily food intake of rats after bile diversion in the mid-jejunum, in the mid-ileum or sham-operated rats fed a standard diet. (d) Insulin and (e) glucose tolerance tests were performed respectively 7 and 10 days after surgery. (f) Insulin plasma levels were determined during the glucose tolerance test. Data are expressed as mean ± SEM; n=4–22 rats per group; *p<0.05, **p<0.01 vs sham-operated group (One-way ANOVA followed by Tukey’s post-hoc test).

METHODS

Animals

All procedures were performed in accordance with the principles and guidelines established by the European Convention for the Protection of Laboratory Animals. Our regional animal care committee approved all experiments. Male Sprague-Dawley rats (Charles River Laboratories, France), weighing about 250–275g, were housed in a climate-control room (22 ± 2°C), subjected to a 12 hour light/dark cycle, with free access to water and standard (A04 - SAFE, France), high-fat high-sucrose (HFHS- 36.1% fat, 35% carbohydrates, 19.8% proteins – INRA, France) or cholestyramine-enriched diet (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, 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, Phymep - for 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 and S1a). 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.31 The liver was removed and frozen using tongs previously chilled in liquid N2. 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,32 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 8th day and exhibited no difference with sham-operated 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 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.19–21

Figure 2. Implication of changes in bile bioavailability on glucose metabolism improvements and enterohepatic circulation after bile diversions.

Figure 2

Open in a new tab

(a) Plasma bile acids were quantified 13 days after bile diversions in rats fed a standard or cholestyramine-enriched diet. (b) Evolution of glucose and (c) insulin plasma levels during a glucose tolerance test performed 10 days after surgery in rats fed a cholestyramine-enriched diet. (d) Relative mRNA level of the α-subunit of the organic solute transporter (Ostα) in the distal ileum of mid-ileum bile diverted rats. (e) Hepatic mRNA level of sodium-taurocholate cotransporting polypeptide (Ntcp), Ostα and bile salt export pump (Bsep). The mRNA levels are expressed as a ratio relative to the ribosomal protein l19 (Rpl19) mRNA level. Data are expressed as mean + or ± SEM; n=4–17 rats per group; *p<0.05, **p<0.01, ***p<0.001 vs sham-operated rats fed a standard diet (One and two-way ANOVA followed by respectively Tukey’s and Bonferroni post-hoc tests for (a), t test for (b) and (c), one-way ANOVA followed by Tukey’s post-hoc test for (d) and (e)).

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 mid-jejunum 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 sham-operated 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.27,33 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 up-regulated 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.

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 rodents13–15 and humans.16–18 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).

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

Figure 3

Open in a new tab

(a) Relative mRNA level and western blot of the catalytic subunit of glucose-6-phospotase (G6PC) and (b) activity of glucose-6-phosphatase (G6Pase) in the liver. (c) Intestinal G6Pase activity of rats with bile diversion in the mid-jejunum, (d) in the mid-ileum and (e) in a mesenteric vein. The site of bile reinsertion is indicated by a grey arrow for the mid-jejunum and the mid-ileum bile diversions. Data were obtained 13 days after bile diversions in standard-fed rats in the post absorptive state and are expressed as mean + SEM; n=5–7 rats per group; *p<0.05, **p<0.01, ***p<0.001 vs sham-operated group (One-way ANOVA followed by Tukey’s post-hoc test for the liver analyzes and t test for the studies in the intestine).

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-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.24 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.

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

Figure 4

Open in a new tab

(a) Relative mRNA level and western blot of FXR in the liver. (b) Quantification of hepatic Shp mRNA level. (c–d) Relative mRNA levels of Fxr and Shp in the intestine of rats with bile diversion in a mesenteric vein and (e–f) in the mid-ileum. The site of bile reinsertion is indicated by a grey arrow for the mid-ileum bile diversion. Data were obtained 13 days after surgery in standard-fed rats in the post absorptive state and are expressed as mean + SEM; n=4–7 rats per group; *p<0.05, **p<0.01, ***p<0.001 vs sham-operated group (One-way ANOVA followed by Tukey’s post-hoc test for the studies in the liver and t test for the quantification in the intestine).

Bile diversions improve glucose control in rats fed a diet inducing obesity

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 high-sucrose (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 sham-operated 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.

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

Figure 5

Open in a new tab

(a) Evolution of daily food intake and (b) body weight of bile-diverted rats fed a HFHS diet. (c) Insulin and (d) glucose tolerance tests were performed respectively 7 and 10 days after surgery. Data are expressed as mean ± SEM; n=4–7 rats per group; **p<0.01, ***p<0.001 bile-diverted groups vs sham-operated group; §§p<0.01 sham-operated pair-fed group vs sham-operated group; $$p<0.01 vs mid-ileum group; ##p<0.01 mid-jejunum group vs sham-operated and sham-operated pair-fed groups (One-way ANOVA followed by Tukey’s post-hoc test).

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.5 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 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 3rd 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.

Figure 6. Food preference in bile-diverted rats.

Figure 6

Open in a new tab

(a) Food preference was assessed 11 to 15 days after surgery in rats previously fed a HFHS diet or (b) 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 (One-way 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).

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 anti-diabetic effects of the surgery.27,33,34 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 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.35 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 bile-diverted 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.36,37 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.38

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 protein10 and soluble fiber39 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.13 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 bile-diverted 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 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.24,25 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.23 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.35 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.13 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.40,41

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.42–44 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.45–47 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 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,27,38,41 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.

Supplementary Material

1

Supplemental table 1: List of sequence-specific primer used for q-PCR

Supplemental data 1: Effect of the bile diversion in a mesenteric vein on the enterohepatic circulation.

(a) Schematic representation of the bile diversion in a mesenteric vein. (b) Plasma bile acids were quantified 13 days after mesenteric vein-bile diversion in rats fed a standard diet. (c) Hepatic mRNA level of Ntcp, Ostα and Bsep are expressed as a ratio relative to the Rpl19 mRNA level. Data are expressed as mean + SEM; n=4–6 rats per group *p<0.05, ***p<0.001 vs sham-operated rats fed a standard diet (t test).

Supplemental data 2: Effects of portal deafferentiation on glucose metabolism in bile-diverted rats.

(a) Glucose tolerance test was performed 10 days after mid jejunum bile diversion with or without capsaicin treatment. (b) Insulin plasma levels were determined during the glucose tolerance test. (c) Relative mRNA level of G6pc and (d) G6Pase activity in the liver. (e) Intestinal G6Pase activity. The site of bile reinsertion is indicated by a grey arrow for the mid jejunum bile diversion. Data were obtained 13 days after surgeries in standard-diet fed rats in the post-absorptive state and are expressed as mean + or ± SEM; n=5–11 rats per groups; *p<0.05, **p<0.01, ***p<0.001 vs sham-operated group (One-way ANOVA followed by Tukey’s post-hoc test).

Acknowledgments

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 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).

The work was supported by an academic grant from the “Société Francophone du diabète”.

Sources of support:

« Agence Nationale de la Recherche » (ANR11-BSV1-016-01) and « Société Francophone du Diabète » (2012).

Footnotes

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.

References

  • 1.Chen L, Magliano DJ, Zimmet PZ. The worldwide epidemiology of type 2 diabetes mellitus -present and future perspectives. Nat Rev Endocrinol. 2012;8:228–36. doi: 10.1038/nrendo.2011.183. [DOI] [PubMed] [Google Scholar]
  • 2.Rubino F, Schauer PR, Kaplan LM, et al. Metabolic surgery to treat type 2 diabetes: clinical outcomes and mechanisms of action. Annu Rev Med. 2010;61:393–411. doi: 10.1146/annurev.med.051308.105148. [DOI] [PubMed] [Google Scholar]
  • 3.Thaler JP, Cummings DE. Minireview: Hormonal and metabolic mechanisms of diabetes remission after gastrointestinal surgery. Endocrinology. 2009;150:2518–25. doi: 10.1210/en.2009-0367. [DOI] [PubMed] [Google Scholar]
  • 4.Schultes B, Ernst B, Wilms B, et al. Hedonic hunger is increased in severely obese patients and is reduced after gastric bypass surgery. Am J Clin Nutr. 2010;92:277–83. doi: 10.3945/ajcn.2009.29007. [DOI] [PubMed] [Google Scholar]
  • 5.Mathes CM, Spector AC. Food selection and taste changes in humans after Roux-en-Y gastric bypass surgery: a direct-measures approach. Physiol Behav. 2012;107:476–83. doi: 10.1016/j.physbeh.2012.02.013. [DOI] [PubMed] [Google Scholar]
  • 6.Stefater MA, Wilson-Pérez HE, Chambers AP, et al. All bariatric surgeries are not created equal: insights from mechanistic comparisons. Endocr Rev. 2012;33:595–622. doi: 10.1210/er.2011-1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Trinh KY, O’Doherty RM, Anderson P, et al. Perturbation of fuel homeostasis caused by overexpression of the glucose-6-phosphatase catalytic subunit in liver of normal rats. J Biol Chem. 1998;273:31615–20. doi: 10.1074/jbc.273.47.31615. [DOI] [PubMed] [Google Scholar]
  • 8.Clore JN, Stillman J, Sugerman H. Glucose-6-phosphatase flux in vitro is increased in type 2 diabetes. Diabetes. 2000;49:969–74. doi: 10.2337/diabetes.49.6.969. [DOI] [PubMed] [Google Scholar]
  • 9.Delaere F, Duchampt A, Mounien L, et al. The role of sodium-coupled glucose co-transporter 3 in the satiety effect of portal glucose sensing. Mol Metab. 2012;2:47–53. doi: 10.1016/j.molmet.2012.11.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Duraffourd C, De Vadder F, Goncalves D, et al. Mu-opioid receptors and dietary protein stimulate a gut-brain neural circuitry limiting food intake. Cell. 2012;150:377–88. doi: 10.1016/j.cell.2012.05.039. [DOI] [PubMed] [Google Scholar]
  • 11.Mithieux G, Misery P, Magnan C, et al. Portal sensing of intestinal gluconeogenesis is a mechanistic link in the diminution of food intake induced by diet protein. Cell Metab. 2005;2:321–9. doi: 10.1016/j.cmet.2005.09.010. [DOI] [PubMed] [Google Scholar]
  • 12.Pillot B, Soty M, Gautier-Stein A, et al. Protein feeding promotes redistribution of endogenous glucose production to the kidney and potentiates its suppression by insulin. Endocrinology. 2009;150:616–24. doi: 10.1210/en.2008-0601. [DOI] [PubMed] [Google Scholar]
  • 13.Troy S, Soty M, Ribeiro L, et al. Intestinal gluconeogenesis is a key factor for early metabolic changes after gastric bypass but not after gastric lap-band in mice. Cell Metab. 2008;8:201–11. doi: 10.1016/j.cmet.2008.08.008. [DOI] [PubMed] [Google Scholar]
  • 14.Sun D, Wang K, Yan Z, et al. 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. Obes Surg. 2013;23:1734–42. doi: 10.1007/s11695-013-0985-0. [DOI] [PubMed] [Google Scholar]
  • 15.Paranjape SA, Chan O, Zhu W, et al. Improvement in hepatic insulin sensitivity after Roux-en-Y gastric bypass in a rat model of obesity is partially mediated via hypothalamic insulin action. Diabetologia. 2013;56:2055–8. doi: 10.1007/s00125-013-2952-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Hayes MT, Foo J, Besic V, et al. Is intestinal gluconeogenesis a key factor in the early changes in glucose homeostasis following gastric bypass? Obes Surg. 2011;21:759–62. doi: 10.1007/s11695-011-0380-7. [DOI] [PubMed] [Google Scholar]
  • 17.Mithieux G. Comment about intestinal gluconeogenesis after gastric bypass in human in relation with the paper by Hayes et al., Obes. Surg. 2011. Obes Surg. 2012;22:1923–4. doi: 10.1007/s11695-012-0755-4. [DOI] [PubMed] [Google Scholar]
  • 18.Immonen H, Hannukainen JC, Iozzo P, et al. Effect of bariatric surgery on liver glucose metabolism in morbidly obese diabetic and non-diabetic patients. J Hepatol. 2014;60:377–83. doi: 10.1016/j.jhep.2013.09.012. [DOI] [PubMed] [Google Scholar]
  • 19.Seyer P, Vallois D, Poitry-Yamate C, et al. Hepatic glucose sensing is required to preserve β cell glucose competence. J Clin Invest. 2013;123:1662–76. doi: 10.1172/JCI65538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Düfer M, Hörth K, Wagner R, et al. Bile acids acutely stimulate insulin secretion of mouse β-cells via farnesoid X receptor activation and K(ATP) channel inhibition. Diabetes. 2012;61:1479–89. doi: 10.2337/db11-0815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Renga B, Mencarelli A, Vavassori P, et al. The bile acid sensor FXR regulates insulin transcription and secretion. Biochim Biophys Acta. 2010;1802:363–72. doi: 10.1016/j.bbadis.2010.01.002. [DOI] [PubMed] [Google Scholar]
  • 22.Watanabe M, Houten SM, Mataki C, et al. Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation. Nature. 2006;439:484–9. doi: 10.1038/nature04330. [DOI] [PubMed] [Google Scholar]
  • 23.De Fabiani E, Mitro N, Gilardi F, et al. Coordinated control of cholesterol catabolism to bile acids and of gluconeogenesis via a novel mechanism of transcription regulation linked to the fasted-to-fed cycle. J Biol Chem. 2003;278:39124–32. doi: 10.1074/jbc.M305079200. [DOI] [PubMed] [Google Scholar]
  • 24.Yamagata K, Daitoku H, Shimamoto Y, et al. Bile acids regulate gluconeogenic gene expression via small heterodimer partner-mediated repression of hepatocyte nuclear factor 4 and Foxo1. J Biol Chem. 2004;279:23158–65. doi: 10.1074/jbc.M314322200. [DOI] [PubMed] [Google Scholar]
  • 25.Ma K, Saha PK, Chan L, et al. Farnesoid X receptor is essential for normal glucose homeostasis. J Clin Invest. 2006;116:1102–9. doi: 10.1172/JCI25604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Patti M-E, Houten SM, Bianco AC, et al. Serum bile acids are higher in humans with prior gastric bypass: potential contribution to improved glucose and lipid metabolism. Obes Silver Spring Md. 2009;17:1671–7. doi: 10.1038/oby.2009.102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Pournaras DJ, Glicksman C, Vincent RP, et al. The role of bile after Roux-en-Y gastric bypass in promoting weight loss and improving glycaemic control. Endocrinology. 2012;153:3613–9. doi: 10.1210/en.2011-2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Simonen M, Dali-Youcef N, Kaminska D, et al. Conjugated bile acids associate with altered rates of glucose and lipid oxidation after Roux-en-Y gastric bypass. Obes Surg. 2012;22:1473–80. doi: 10.1007/s11695-012-0673-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Kohli R, Bradley D, Setchell KD, et al. Weight loss induced by Roux-en-Y gastric bypass but not laparoscopic adjustable gastric banding increases circulating bile acids. J Clin Endocrinol Metab. 2013;98:E708–12. doi: 10.1210/jc.2012-3736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Rudnicki M, Patel DG, McFadden DW, et al. Proximal jejunal and biliary effects on the enteroinsular axis. Surgery. 1990;107:455–60. [PubMed] [Google Scholar]
  • 31.Mithieux G, Bady I, Gautier A, et al. Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes. Am J Physiol Endocrinol Metab. 2004;286:E370–5. doi: 10.1152/ajpendo.00299.2003. [DOI] [PubMed] [Google Scholar]
  • 32.Rajas F, Bruni N, Montano S, et al. The glucose-6 phosphatase gene is expressed in human and rat small intestine: regulation of expression in fasted and diabetic rats. Gastroenterology. 1999;117:132–9. doi: 10.1016/s0016-5085(99)70559-7. [DOI] [PubMed] [Google Scholar]
  • 33.Jansen PLM, van Werven J, Aarts E, et al. Alterations of hormonally active fibroblast growth factors after Roux-en-Y gastric bypass surgery. Dig Dis Basel Switz. 2011;29:48–51. doi: 10.1159/000324128. [DOI] [PubMed] [Google Scholar]
  • 34.Lefebvre P, Cariou B, Lien F, Kuipers F, Staels B. Role of bile acids and bile acid receptors in metabolic regulation. Physiol Rev. 2009 Jan;89(1):147–91. doi: 10.1152/physrev.00010.2008. [DOI] [PubMed] [Google Scholar]
  • 35.Mencarelli A, Renga B, D’Amore C, et al. Dissociation of intestinal and hepatic activities of FXR and LXRα supports metabolic effects of terminal ileum interposition in rodents. Diabetes. 2013;62:3384–93. doi: 10.2337/db13-0299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Sloop KW, Showalter AD, Cox AL, et al. Specific reduction of hepatic glucose 6-phosphate transporter-1 ameliorates diabetes while avoiding complications of glycogen storage disease. J Biol Chem. 2007;282:19113–21. doi: 10.1074/jbc.M610759200. [DOI] [PubMed] [Google Scholar]
  • 37.Gómez-Valadés AG, Méndez-Lucas A, Vidal-Alabró A, et al. Pck1 gene silencing in the liver improves glycemia control, insulin sensitivity, and dyslipidemia in db/db mice. Diabetes. 2008;57:2199–210. doi: 10.2337/db07-1087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Kohli R, Setchell KD, Kirby M, et al. A surgical model in male obese rats uncovers protective effects of bile acids post-bariatric surgery. Endocrinology. 2013;154:2341–51. doi: 10.1210/en.2012-2069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.De Vadder F, Kovatcheva-Datchary P, Goncalves D, et al. Microbiota-generated metabolites promote metabolic benefits via gut-brain neural circuits. Cell. 2014;156:84–96. doi: 10.1016/j.cell.2013.12.016. [DOI] [PubMed] [Google Scholar]
  • 40.Myronovych A, Kirby M, Ryan KK, et al. Vertical sleeve gastrectomy reduces hepatic steatosis while increasing serum bile acids in a weight-loss-independent manner. Obes Silver Spring Md. 2014;22:390–400. doi: 10.1002/oby.20548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Ryan KK, Tremaroli V, Clemmensen C, et al. FXR is a molecular target for the effects of vertical sleeve gastrectomy. Nature. 2014 doi: 10.1038/nature13135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Kenler HA, Brolin RE, Cody RP. Changes in eating behavior after horizontal gastroplasty and Roux-en-Y gastric bypass. Am J Clin Nutr. 1990;52:87–92. doi: 10.1093/ajcn/52.1.87. [DOI] [PubMed] [Google Scholar]
  • 43.Olbers T, Björkman S, Lindroos A, et al. Body composition, dietary intake, and energy expenditure after laparoscopic Roux-en-Y gastric bypass and laparoscopic vertical banded gastroplasty: a randomized clinical trial. Ann Surg. 2006;244:715–22. doi: 10.1097/01.sla.0000218085.25902.f8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Ernst B, Thurnheer M, Wilms B, et al. Differential changes in dietary habits after gastric bypass versus gastric banding operations. Obes Surg. 2009;19:274–80. doi: 10.1007/s11695-008-9769-3. [DOI] [PubMed] [Google Scholar]
  • 45.Miras AD, le Roux CW. Bariatric surgery and taste: novel mechanisms of weight loss. Curr Opin Gastroenterol. 2010;26:140–5. doi: 10.1097/MOG.0b013e328333e94a. [DOI] [PubMed] [Google Scholar]
  • 46.Bueter M, Miras AD, Chichger H, et al. Alterations of sucrose preference after Roux-en-Y gastric bypass. Physiol Behav. 2011;104:709–21. doi: 10.1016/j.physbeh.2011.07.025. [DOI] [PubMed] [Google Scholar]
  • 47.Le Roux CW, Bueter M, Theis N, et al. Gastric bypass reduces fat intake and preference. Am J Physiol Regul Integr Comp Physiol. 2011;301:R1057–66. doi: 10.1152/ajpregu.00139.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Supplemental table 1: List of sequence-specific primer used for q-PCR

Supplemental data 1: Effect of the bile diversion in a mesenteric vein on the enterohepatic circulation.

(a) Schematic representation of the bile diversion in a mesenteric vein. (b) Plasma bile acids were quantified 13 days after mesenteric vein-bile diversion in rats fed a standard diet. (c) Hepatic mRNA level of Ntcp, Ostα and Bsep are expressed as a ratio relative to the Rpl19 mRNA level. Data are expressed as mean + SEM; n=4–6 rats per group *p<0.05, ***p<0.001 vs sham-operated rats fed a standard diet (t test).

Supplemental data 2: Effects of portal deafferentiation on glucose metabolism in bile-diverted rats.

(a) Glucose tolerance test was performed 10 days after mid jejunum bile diversion with or without capsaicin treatment. (b) Insulin plasma levels were determined during the glucose tolerance test. (c) Relative mRNA level of G6pc and (d) G6Pase activity in the liver. (e) Intestinal G6Pase activity. The site of bile reinsertion is indicated by a grey arrow for the mid jejunum bile diversion. Data were obtained 13 days after surgeries in standard-diet fed rats in the post-absorptive state and are expressed as mean + or ± SEM; n=5–11 rats per groups; *p<0.05, **p<0.01, ***p<0.001 vs sham-operated group (One-way ANOVA followed by Tukey’s post-hoc test).

halms1350737-supplement_1.pdf (692KB, pdf)

RESOURCES