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Biliopancreatic limb plays an important role in metabolic improvement after duodenal–jejunal bypass in a rat model of diabetes
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Biliopancreatic limb plays an important role in metabolic improvement after duodenal–jejunal bypass in a rat model of diabetes Tomohiro Miyachi, MD, PhD,a Munenori Nagao, MD, PhD,a Chikashi Shibata, MD, PhD,b Yoshiro Kitahara, PhD,c Naoki Tanaka, MD, PhD,a Kazuhiro Watanabe, MD, PhD,a Takahiro Tsuchiya, MD,a Fuyuhiko Motoi, MD, PhD,a Takeshi Naitoh, MD, PhD, FACS,a and Michiaki Unno, MD, PhD,a Sendai and Kanagawa, Japan
Background and aims. Roux-en-Y gastric bypass improves glucose metabolism in clinical practice, and duodenal–jejunal bypass (DJB), an experimental bypass procedure, also improves metabolism in animals. However, the mechanism remains controversial; especially, the role of the biliopancreatic limb (BP-limb) remains unclear. Our aim was to examine the importance of the function of the BP-limb after DJB using a novel operative model. Methods. Otsuka Long-Evans Tokushima Fatty rats with diabetes were divided into the following groups: DJB with a short alimentary limb (A-limb) and long BP-limb (B-DJB group), DJB with jejunectomy (J-DJB group) in which the entire length of the jejunum used for the BP-limb of the B-DJB group was excised; and a sham operation group. Glucose tolerance, plasma bile acid levels, and the gut microbiota were assessed postoperatively. Results. Glucose tolerance was improved and weight gain was suppressed after surgery in the B-DJB group. In contrast, these effects were cancelled in the J-DJB group. The plasma levels of bile acids in the B-DJB group were greater than those in other groups. The analysis of gut microbiota showed distinct differences between the B-DJB and other groups; especially, the relative abundance of genus Bifidobacterium was much higher in the B-DJB group. Conclusion. The BP-limb played an important role in the control of weight gain, glucose tolerance, and increased plasma bile acid levels after DJB in this rat model of type 2 diabetes mellitus. Plasma bile acids and gut microbiota may be involved in these processes. (Surgery 2016;j:j-j.) From the Department of Surgery,a Tohoku University Graduate School of Medicine; Department of Surgery,b Tohoku Pharmaceutical University Hospital, Sendai; and cInstitute for Innovation, Ajinomoto Co., Inc., Kanagawa, Japan
BARIATRIC SURGERY has emerged as the most effective method for sustained weight loss in obese patients; it induces a long-term improvement in patients having diabetes mellitus (DM) and its comorbidities
and improves overall survival.1-3 After the operation, improved glucose metabolism is noticeable even before weight loss occurs, suggesting a metabolism-improving mechanism independent
Transcript profiling: Takeshi Naitoh (same as above).
interpretation of the data. N. Tanaka and K. Watanabe assisted in the design of the study and interpretation of the data. T. Tsuchiya assisted in the design of the study and provided technical assistance for the study. F. Motoi and M. Unno assisted in the design of the study.
This study was supported by the grant-in-aid for scientific research from Japan Society for the Promotion of Science and by the grant from the Ajinomoto Co., Inc.. The study was conducted in collaboration with Ajinomoto Co., Inc., which was responsible for the analysis of plasma hormones, gut microbiota, and plasma metabolome analysis. T. Miyachi participated in the design of the study, data collection, statistical analysis, and drafting of the manuscript. T. Naitoh participated in supervision and provided oversight when the manuscript was being drafted. M. Nagao and C. Shibata gave significant advice regarding protocol development and drafting of the manuscript. Y. Kitahara contributed to the design of the study, data collection, statistical analysis, and
Accepted for publication November 27, 2015. Reprint requests: Takeshi Naitoh, MD, PhD, FACS, Department of Surgery, Tohoku University Graduate School of Medicine, 1-1, Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2015.11.027
SURGERY 1
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of weight loss. Many studies have been conducted to elucidate this mechanism to enable its application to the medical treatment of diabetes. However, the underlying mechanism remains unclear. Roux-en-Y gastric bypass (RYGB) is one of the most commonly performed bariatric surgical procedures. Duodenal–jejunal bypass (DJB) is an experimental procedure that was developed to investigate the underlying mechanisms responsible for the metabolic benefits after RYGB.4 In an experimental DJB model, because the whole stomach is preserved, we can focus on only the mechanism of bypassing the alimentary tract excluding the restrictive effect. The schema of DJB is shown in Fig 1. The small intestine in DJB consists of 3 parts in terms of its contents: food in the alimentary limb (A-limb), bile/pancreatic juice in the biliopancreatic limb (BP-limb), and a mixture of food and bile/pancreatic juice in the common channel. The A-limb is also called the Roux limb in RYGB. In surgical bypass procedures, such as RYGB or DJB, changes in the secretion of intestinal hormones in the distal ileum and the role of the A-limb have been reported. In the distal ileum, secretion of gastrointestinal hormones such as glucagon-like peptide-1 (GLP-1), peptide YY (PYY), and fibroblast growth factor (FGF)-19 are increased.5,6 Regarding the A-limb, effects such as activation of nutrient sensing, enhancement of glucose transporter 1-mediated basolateral glucose uptake and utilization, and reduction of the sodium–glucose transporter 1 and taste receptors, have been reported.4,7-9 In contrast, the role of the BP-limb in glycolipid metabolism has been scarcely examined. Recently, clinical trials have reported a relation between the BP-limb length and body-weight loss after RYGB. Nergaard et al10 reported better weight loss using RYGB with a long BP-limb. However, another study reported no correlation.11 We focused our study on the role of the BP-limb by using a rat model of DJB with various lengths of A- and BP-limbs. The aim of our study was to investigate the effect of the BP-limb on metabolic improvement and body weight (BW) loss and to elucidate the effects on bile acids, gut microbiota, and intestinal hormones. Clarifying these issues will contribute to a better understanding of the underlying mechanism of RYGB and DJB. MATERIALS AND METHODS The experimental protocol was approved by the Ethics Committee for Animal Research of the Tohoku University.
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Fig 1. Duodenal–jejunal bypass. A-limb, Alimentary limb; BP-limb, biliopancreatic limb; CC, common channel.
Rats. We used male Otsuka Long-Evans Tokushima Fatty (OLETF) rats as a model for type 2 DM.12-14 These rats lacked cholecystokinin-A receptors and develop obesity and hyperglycemia with hyperinsulinemia after overeating.15 We obtained 4- to 6-week-old male OLETF rats from Japan SLC, Inc. (Shizuoka, Japan). All rats were single-housed under specific pathogen-free conditions and fed normal food (Labo MR Stock composed of 15.2% of kilocalories from fat, 32.6% from protein, and 52.3% from carbohydrates [CHO]; Nosan Corporation, Yokohama, Japan) ad libitum. Study protocols. In this study, we performed 2 protocols to evaluate the effect of different BP-limb length on glucose metabolism in the first series of experiments and the effect of BP-limb resection in the second series. The scheme of each experimental protocol is shown in Figs 2, A, and 3, A. In the first series of experiments, we evaluated only weight gain, food intake, and blood glucose levels in the meal tolerance test 8 weeks after the operation. In the second series, we additionally performed pyruvate acid and insulin tolerance tests 10 and 11 weeks after the operation, respectively. Tissue, blood, and fecal matter were collected 12 weeks after the operation at necropsy in the second series. Surgical preparation and perioperative care. At 19 weeks of age, a meal tolerance test was performed preoperatively, and rats without a diabetic pattern were not used for further analysis. Surgery was performed 1 week after the meal tolerance test.
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Fig 2. (A) Scheme of experimental protocols of the first series of experiments. (B) Body weight after the operation. (C) Glucose excursion curve in meal tolerance test 8 weeks after the operation. Data are expressed as mean ± standard error of the mean; n = 7–8 rats per group. *P < .05, **P < .01 versus values in the sham group (Dunnett’s test or Dunnett’s C test after Levene’s test). AL, Alimentary limb; BPL, biliopancreatic limb; MTT, meal tolerance test; W.O., weeks old.
In the first series of experiments, the rats were divided randomly into 5 groups (n = 7 or 8 in each group; Table): A sham group, an AL3-BPL3 group with short length of both limbs (A-limb, 3 cm; BPlimb, 3 cm), an AL3-BPL30 group with a long BPlimb and a short A-limb (A-limb, 3 cm; BP-limb, 30 cm), an AL30-BPL3 group with a short BPlimb and a long A-limb (A-limb, 30 cm; BP-limb, 3 cm), and an AL30-BPL30 group with long Aand BP-limbs and a short common channel (Alimb, 30 cm; BP-limb, 30 cm). Because the entire small intestine was approximately 91 cm in our rats, the length of common channel was estimated approximately as 85, 58, and 31 cm in the AL3BPL3, AL3-BPL30/AL30-BPL3, and AL30-BPL30 groups, respectively. A limb length of 30 cm was selected because it is approximately one-third of the total length of the small intestine. Surgery was performed after overnight fasting. The operative procedures for DJB in rats were performed according to our previous report.14 Briefly, the duodenum was divided immediately distal to the pylorus, and the distal end was closed. The jejunum was transected distal to the duodenal–jejunal junction and the distal end of the jejunum was anastomosed end-to-end to the proximal end of the duodenum. The proximal end of the jejunum was anastomosed end-to-side to the jejunum. In the sham group, the duodenum close to the pylorus and the jejunum 3 cm distal from the Treitz ligament, were transected and
reanastomosed. Water was given ad libitum from the day of surgery and rat food was given from the day after the operation. To elucidate the role of the BP-limb in DJBinduced improvement of glucose metabolism, we prepared a second series of experiments using different rats. The rats were divided into 3 groups (n = 6 per group): a sham group, a B-DJB group with a long BP-limb (A-limb, 3 cm; BP-limb, 30 cm), and a J-DJB group with an excision of the 30 cm length of jejunum that was equivalent to the BP-limb in the B-DJB group (Fig 3, B). All operative procedures and postoperative care in the second set of experiments were performed in a similar way as in the first series of experiments. Meal, pyruvate acid, and insulin tolerance tests. All tolerance tests were performed after overnight fasting. Meal tolerance tests were done as described previously.16 Rats were administered a liquid mixed meal (Vanilla Ensure H containing 1.5 kcal/mL with 28% of kilocalories from fat, 15% from protein, and 57% from CHO; Abbott Japan Co., Chiba, Japan) by oral gavage at a dose of 1.2 g CHO/5.8 mL/8.76 kcal/kg BW. Blood samples were drawn from the tail vein before and at 15 30, 60, and 120 minutes after administration of the liquid meal. The blood glucose concentrations were measured with a glucometer (Ascensia Breeze; Bayer, Osaka, Japan). We measured the plasma concentrations of total bile acids as well in the second series of experiments. For pyruvate
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Fig 3. (A) Experimental protocols of the second series of experiments. (B) Surgical model for each group. (C) Body weight after the operation. Data are expressed as mean values ± standard error of the mean; n = 6 rats per group. *P < .05, **P < .01, ***P < .001 versus values in the sham group; yyP < .01, yyyP < .001 versus values in the J-DJB group (1-way analysis of variance, followed by Tukey’s post hoc test). A-limb, Alimentary limb; BP-limb, biliopancreatic limb; CC, common channel; DJB, duodenal–jejunal bypass; MTT, meal tolerance test; ITT, insulin tolerance test; PTT, pyruvate acid tolerance test; W.O., weeks old.
Table. Length of each limb in 4 groups in the first series of experiments
AL3-BPL3 AL3-BPL30 AL30-BPL3 AL30-BPL30
Alimentary limb
Biliopancreatic limb
3 3 30 30
3 30 3 30
Common channel (cm) z z z z
85 58 58 31
The length of common channel was approximately calculated from the length of total jejunoileum (91 ± 0.9 cm, mean ± standard error of the mean) measured in tissue sampling.
acid tolerance tests, rats were injected subcutaneously with sodium pyruvate solution (2 g/kg BW), and blood glucose levels were measured. For insulin tolerance tests, rats were injected intraperitoneally with insulin (0.5 U/kg BW). Tissue, blood, and fecal matter collection. Twelve weeks after the operation in the second series of experiments, all rats were made to fast overnight, and then were administered Vanilla Ensure H by oral gavage at the same dose as for the meal tolerance test and were humanely killed 1
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hour later. We collected tissue, blood, and fecal samples after meal loading, because the key factor in DJB might be a change in the diet flow. Blood samples were withdrawn from the inferior vena cava and collected in ice-chilled tubes containing ethylenediaminetetraacetic acid and dipeptidyl peptidase-IV inhibitor. We weighed the retroperitoneal fat and liver, and liver was stored at 608C. Fecal samples collected from the sigmoid colon were added to 99% ethanol and stored at 308C. Plasma assays. Plasma concentrations of total bile acids in the meal tolerance test were examined using the Total Bile Acids Test Kit (Wako, Osaka, Japan). An electrochemiluminescence assay was used to measure the levels of insulin, glucagon, active GLP-1, total PYY, and FGF15 in the plasma samples harvested at necropsy. FGF15 is a homolog of human FGF19, which is secreted from the ileum and induces insulin-independent glucose lowering.17,18 We also measured plasma triglyceride levels using Spotchem II (Arkray Inc., Kyoto, Japan). Measurement of hepatic triglycerides. Hepatic triglycerides were extracted from the harvested liver tissue using the chloroform–methanol method of Folch et al19 and measured enzymatically using a commercial kit (Wako, Tokyo, Japan). Metabolome analysis. Metabolites in plasma samples harvested at necropsy were analyzed using a relative quantitation method by capillary electrophoresistime-of-flight mass spectrometry (CE-TOFMS) and liquid chromatography TOFMS using the Agilent CE-TOFMS system and the Agilent 1200 Series RRLC System SL (Agilent Technologies, Inc., Santa Clara, CA) in Human Metabolome Technologies, Inc. (Yamagata, Japan), as previously described.20 Data are expressed relative to the mean value in the sham group. Metaanalysis of 16S rRNA gene sequences of gut microbiota. Bacterial DNA was extracted from the fecal samples using the FastDNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA) and the FastPrep instrument. For metaanalysis of the 16S rRNA gene sequences, we used a next generation sequencer (Miseq Illumina, Inc., San Diego, CA), as previously reported.21 The data was analyzed using Mothur (available from: www.mothur.org/ wiki/Mothur_manual) and METAGENassist (available from: www.metagenassist.ca/METAGENassist/ faces/Home.jsp).22 Data analysis. All data are presented as mean values ± standard error of the mean. All statistical analyses were performed using the JMP version 10.0.0 statistical software package (SAS
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International Inc., Cary, NC). In the first series of experiments, to compare the results between the sham and other groups, Dunnett’s test or Dunnett’s C test was used after Levene’s test. In the second series of experiments, 1-way analysis of variance, followed by Tukey’s post hoc test was used. RESULTS Effects of A- and BP-limbs on weight gain and glucose metabolism. Weight gain after the operation was suppressed significantly in the 2 groups with the 30-cm BP-limbs (AL3-BPL30 and AL30BPL30 groups) compared with the sham group, and the blood glucose levels in those 2 groups were also reduced significantly throughout the meal tolerance test (Fig 2, B and C). In the AL30-BPL3 group with the long A-limb and short BP-limb, the postoperative weight gain did not differ from that in the sham group, and blood glucose levels were significantly lower only at the 30-minute point. The weight gain and blood glucose levels in the AL3-BPL3 group did not differ from those in the sham group. Thus, obvious improvement of glucose metabolism was observed in the 2 groups with the 30-cm BP-limb. No intergroup differences were observed in food intake 2 and 9 weeks after the operation (data not shown). Effects of resection of BP-limb on glucose metabolism. Postoperative weight gain was suppressed significantly in the B-DJB group in comparison with the sham group (15.3 ± 6.3 vs 65.7 ± 5.8 g; Fig 3, C). However, the effect of weight gain suppression was canceled in the J-DJB group, suggesting that the BP-limb has a role in suppression of the expected weight gain. No intergroup differences were observed in food intake (data not shown). The length of the small intestine measured during tissue sampling was 91 ± 0.9 cm, and the length of the common channel in the groups that underwent DJB was 58.6 ± 1.0 cm. No intergroup differences were observed in those lengths. The J-DJB group had equivalent lengths of the A-limb and common channel responsible for digestion and absorption to that of the B-DJB group. During the meal tolerance test, the B-DJB group had the lowest peak postprandial blood glucose concentrations, and the area under the curve in the B-DJB group was reduced significantly compared with that in the J-DJB and sham groups (Fig 4, A and B). In the insulin tolerance test, during the 15–60 minutes after the intraperitoneal administration of insulin, the blood glucose level relative to that of the baseline was significantly less in the B-DJB group than
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Fig 4. (A) Glucose excursion curve. (B) Area under the curve for the meal tolerance test. (C) Blood glucose levels in insulin tolerance test. (D) Pyruvate acid tolerance test. Tests were performed after 8, 10, and 11 weeks after the operation respectively. Data are expressed as mean values ± standard error of the mean; n = 6 rats per group. *P < .05, **P < .01, ***P < .001 versus values in the sham group; yP < .05, yyP < .01, yyyP < .001; versus values in the J-DJB group (1-way analysis of variance, followed by Tukey’s post hoc test). B-DJB, Duodenal–jejunal bypass with a short alimentary limb and long biliopancreatic limb; J-DJB, duodenal–jejunal bypass with jejunectomy.
that in the sham group. The J-DJB group showed significantly lower values than the sham group only at the 60-minute point, but there was no difference between the J-DJB and sham groups for 0–45 minutes (Fig 4, C). In the pyruvate acid tolerance test performed to evaluate gluconeogenesis in the liver, the blood glucose level relative to that of the baseline remained low in the B-DJB group and was significantly lower at 30 and 45 minutes compared with levels in the sham group and at the 120-minute point compared with that in the J-DJB and sham groups. The J-DJB group tended to have slightly lower values than the sham group, but significant differences were not observed (Fig 4, D). These results suggest improvement in insulin sensitivity and gluconeogenesis suppression in the B-DJB group was reversed in the J-DJB group.
Effects of DJB and resection of BP-limb on triglyceride content in the liver, postprandial levels of hormones, and bile acids. No differences were detected in the plasma triglyceride levels among these groups. However, in the B-DJB group, the triglyceride content in the liver and the retroperitoneal adipose tissue weight per BW were significantly lower than those in the J-DJB and sham groups (Fig 5). The changes observed in the B-DJB group were reversed in the J-DJB group. No differences were observed in plasma levels of insulin, glucagon, GLP-1, PYY, or FGF15 (Fig 6). During the meal tolerance test performed 8 weeks after the operation, plasma concentrations of total bile acids remained high in the B-DJB group. This group had the largest area under the curve, which significantly differed from that of the J-DJB groups (Fig 7, A and B). To investigate changes in
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Fig 5. (A) Plasma levels of triglycerides and (B) triglyceride content in the liver. (C) Weight of the retroperitoneal fat per body weight. Samples were harvested 12 weeks after the operation and 1 hour after administration of a liquid meal (Ensure H). Data are expressed as mean values ± standard error of the mean; n = 6 rats per group. **P < .01, ***P < .001 versus values in the sham group; yyyP < .001 versus values in the J-DJB group; N.S., not significant (1way analysis of variance, followed by Turkey’s post hoc test). B-DJB, Duodenal–jejunal bypass with a short alimentary limb and long biliopancreatic limb; J-DJB, duodenal–jejunal bypass with jejunectomy.
metabolite levels comprehensively, we performed a metabolome analysis using plasma samples harvested 12 weeks after the operation. We found the concentrations of various bile acids increased in the B-DJB group. In particular, taurineconjugated cholic acid, chenodeoxycholic acid (CDCA), glycine-conjugated and taurineconjugated CDCA, glycine-conjugated deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), taurine-conjugated UDCA, and hyodeoxycholic acid levels were significantly greater than those in other groups. Additionally, cholic acid and glycine-conjugated cholic acid levels were significantly greater than those in the J-DJB group (Fig 7, C). Any change in the composition of plasma bile acids is unknown because the absolute amounts of metabolites were not measured in this analysis. Effect of DJB on gut microbiota. In principal component analysis of gut microbiota in the
sigmoid colon with metaanalysis of 16S rRNA gene sequences, the distribution in the B-DJB group profoundly differed from that in other groups (Fig 8, A). The relative abundance of genuses Clostridium, Turicibacter, and Bacteroides in the B-DJB group were lower, and those of Bifidobacterium and Olsenella were much higher, compared with those of the other groups (Fig 8, B). DISCUSSION This is the first study directly assessing the role of the BP-limb using the BP-limb resection model. The results of the present study represent new findings about the BP-limb that may help to elucidate the mechanism of the metabolic benefits observed after RYGB or DJB. The results of the first series of experiments suggested that the BP-limb might have an important role in the suppression of weight gain and improvement of glucose metabolism. In the second series of experiments,
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Fig 6. Plasma hormone levels. Samples were harvested 12 weeks after the operation and 1 hour after administration of a liquid meal (Ensure H). Data are expressed as mean values ± standard error of the mean; n = 6 rats per group. N.S., not significant (1-way analysis of variance, followed by Tukey’s post hoc test). B-DJB, Duodenal–jejunal bypass with a short alimentary limb and long biliopancreatic limb; FGF15, fibroblast growth factor 15; GLP-1, glucagon-like peptide-1; J-DJB, duodenal–jejunal bypass with jejunectomy; PYY, peptide YY.
significant weight gain suppression and greater improvement in glucose metabolism were found in the B-DJB group, which is consistent with the results of the first series. The triglyceride content in the liver and the retroperitoneal adipose tissue weight in the B-DJB group also significantly decreased. However, the J-DJB group in which the entire jejunum corresponding with the BPlimb of the B-DJB group was removed, showed no significant suppression of weight gain. Furthermore, no improvements were observed in the
blood glucose levels during the meal tolerance and pyruvate acid tolerance tests, triglyceride content in the liver, or retroperitoneal adipose tissue weight. The point is that elimination of the BPlimb invalidates the improvement effects of DJB. These results suggest that the changes observed in the B-DJB group are associated closely with the BPlimb, rather than a decrease in digestive and absorptive capacities owing to shortening of the small intestine (as in short bowel syndrome). In addition, an increase in plasma concentrations of
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Fig 7. (A) Change in the plasma total bile acid levels and (B) area under the curve in the meal tolerance test performed 8 weeks after the operation. (C) Ratio of arbitrary units of plasma bile acids compared with the mean in the sham group on the liquid chromatography time-of-flight mass spectrometry analysis. Samples were harvested 12 weeks after the operation. The change in composition of plasma bile acids is unknown, because the absolute amounts of metabolites were not measured in this analysis. Data are expressed as mean values ± standard error of the mean; n = 6 rats per group. (A) **P < .01 versus values in the sham group; yyP < .01 versus values in the J-DJB group; (B and C) *P < .05, **P < .01, ***P < .001 (1-way analysis of variance followed by Tukey’s post hoc test). B-DJB, Duodenal–jejunal bypass with a short alimentary limb and long biliopancreatic limb; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; G-, glycine-conjugated; HDCA, hyodeoxycholic acid; J-DJB, duodenal–jejunal bypass with jejunectomy; T-, taurineconjugated; UDCA, ursodeoxycholic acid.
bile acids and changes in the gut microbiota were found in the B-DJB group; these factors might have affected the results obtained from this group. Such direct effects of the BP-limb on postoperative weight change, glycolipid metabolism, and changes in plasma bile acid levels have not been reported before. In the clinical setting, minigastric bypass, the recently emerged procedure involving the BP-limb but not the A-limb, shows metabolic
benefits comparable with RYGB.23 This may indicate the importance of the BP-limb. Our data provide a theoretical basis for the effects of the BP-limb on metabolic improvement and BW loss. Bile acids are important in the modulation of metabolism. Bile salts act as endogenous ligands for the G-protein-coupled bile acid receptor (TGR5) and nuclear farnesoid X receptor (FXR). Activation of these receptors leads to
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Fig 8. Results of met-analysis of 16S rRNA gene sequences of the gut microbiota in the sigmoid colon. Samples were harvested 12 weeks after the operation. (A) Principal component analysis and (B) composition of the microbiota (genus); n = 6 rats per group. B-DJB, duodenal–jejunal bypass with a short alimentary limb and long biliopancreatic limb; J-DJB, duodenal–jejunal bypass with jejunectomy; PC, principal component.
enhancement of energy expenditure in skeletal muscles and brown adipose tissues, and to regulation of de novo lipogenesis, gluconeogenesis, and insulin sensitivity in the liver.24-28 In various bile acids, CDCA and DCA have potent FXR and TGR5 agonistic activities.29 Although UDCA has very weak agonistic FXR and TGR5 activities.29,30 However, UDCA acts as a molecular chaperone. UDCA has been reported to improve glucose metabolism and insulin sensitivity by mitigating endoplasmic reticulum stress in an obesity mouse model and in obese patients.31,32 In this study, increased plasma levels of various bile acids in the B-DJB group were observed, suggesting the metabolism-improving effects work through these mechanisms. The increase of plasma bile acid levels after RYGB, vertical sleeve gastrectomy, and ileal transposition has been confirmed in humans and rodents, and leads to metabolic improvement
through the activation of bile acid signals.33-35 No similar reports have been available for DJB to date, and this is the first report confirming the increased plasma bile acid level after DJB. Although the shortened enterohepatic circulation of bile acids has been hypothesized to contribute to an increase in bile acid concentrations,36 no detailed examination or analysis has been reported to date on the absorption from the digestive tract or bile acid synthesis in the liver. Approximately 30% of bile acids secreted to the duodenum are reabsorbed passively in the jejunum.37 There is a possibility that passive reabsorption of bile acids in the jejunum, which is equivalent to the BP-limb, may change, leading to the modification in the enterohepatic circulation in the DJB model. Recently, the gut microbiota has also attracted a lot of attention. The gut microbiota affects host energy metabolism and bile acid composition; it is involved in the pathogenesis of obesity, diabetes,
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and hepatocellular carcinoma; and it also alters upon bariatric surgery.38-40 In the present study, the B-DJB group showed a profound change in gut microbiota, including a marked increase in Bifidobacterium and a decrease in Clostridium. This change does not contradict previous reports about the microbiota after metabolic surgery.40,41 Regarding the previous reports, the relative abundance of Clostridium is higher in type 2 DM patients and that of Bifidobacterium decreases in obese patients.42,43 Bifidobacterium produce lactate and acetate, and these short fatty acids suppress glucose incorporation in adipocytes and regulate energy metabolism through the sympathetic neuronal network.44,45 The gut microbiota also affects bile acid homeostasis by bile acid conversion or decomposition. The changed microbiota in the B-DJB group may contribute to the metabolic improvement and also be involved in the change in the composition of plasma bile acids. The changes in amounts and preferences in food intake and alteration of passing routes for nutrients, bile acids, and pancreatic juice are reported as factors involved in postoperative changes in the gut microbiota. However, the details remain unclear.46 In this study, all groups were fed the same diet in comparable quantities. Thus, the observed changes in the gut microbiota are attributable to differences in host factors. Our study has some limitations and leaves some questions unanswered. We measured plasma concentrations of gut hormones only at 60 minutes after the meal load, but not during a fasting state. We did not examine potential concentration changes throughout this period. Thus, the involvement of these hormones in the phenomena observed in each model could not be evaluated. The exact timing of changes in plasma bile acid concentrations, glucose metabolism, and gut microbiota induced after the operation was not examined in this study. Because we did not analyze the detailed interrelations between various parameters, future studies should examine such correlations. Furthermore, our operative models have limitations. The ratios of the length of the A-limb to that of the BP-limb used in this study were unusual with respect to clinical practice. We cannot elucidate the correlation between the length of the BP-limb and the effects on metabolic improvement or BW loss in this all-BP-limb resection model. We also need to investigate the role of the BP-limb using other models with different limb lengths or with different intestine lengths for resection and need to confirm our results in a nonobese DM or diet-induced obesity rodent model.
Although additional studies are needed, the results we present herein reveal for the first time the role of the BP-limb in bariatric surgery. In conclusion, the BP-limb plays a vital role in controlling weight gain and glucose tolerance after DJB, and plasma bile acids and the gut microbiota may be involved in these processes in a rat model of type 2 DM. We believe these findings will contribute to the elucidation of the mechanism of bariatric surgery. The authors thank Ms. Emiko Shibuya for her technical assistance.
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32. Kars M, Yang L, Gregor MF, et al. Tauroursodeoxycholic acid may improve liver and muscle but not adipose tissue insulin sensitivity in obese men and women. Diabetes 2010;59: 1899-905. 33. 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. Obesity (Silver Spring) 2014;22:390-400. 34. Patti ME, 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. Obesity (Silver Spring) 2009;17:1671-7. 35. Kohli R, Kirby M, Setchell KD, et al. Intestinal adaptation after ileal interposition surgery increases bile acid recycling and protects against obesity-related comorbidities. Am J Physiol Gastrointest Liver Physiol 2010;299:G652-60. 36. 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. 37. Dietschy JM. Mechanisms for the intestinal absorption of bile acids. J Lipid Res 1968;9:297-309. 38. Yoshimoto S, Loo TM, Atarashi K, et al. Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome. Nature 2013;499:97-101. 39. Turnbaugh PJ, Ley RE, Mahowald MA, et al. An obesityassociated gut microbiome with increased capacity for energy harvest. Nature 2006;444:1027-31. 40. Kugelberg E. Surgery: Altered gut microbiota trigger weight loss. Nat Rev Endocrinol 2013;9:314. 41. Aron-Wisnewsky J, Clement K. The effects of gastrointestinal surgery on gut microbiota: potential contribution to improved insulin sensitivity. Curr Atheroscler Rep 2014; 16:454. 42. Le Chatelier E, Nielsen T, Qin J, et al. Richness of human gut microbiome correlates with metabolic markers. Nature 2013;500:541-6. 43. Qin J, Li Y, Cai Z, et al. A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature 2012; 490:55-60. 44. Kimura I, Ozawa K, Inoue D, et al. The gut microbiota suppresses insulin-mediated fat accumulation via the shortchain fatty acid receptor GPR43. Nat Commun 2013;4:1829. 45. Inoue D, Kimura I, Wakabayashi M, et al. Short-chain fatty acid receptor GPR41-mediated activation of sympathetic neurons involves synapsin 2b phosphorylation. FEBS Lett 2012;586:1547-54. 46. Liou AP, Paziuk M, Luevano JM Jr, et al. Conserved shifts in the gut microbiota due to gastric bypass reduce host weight and adiposity. Sci Transl Med 2013;5:178ra41.
Biliopancreatic limb plays an important role in metabolic improvement after duodenal–jejunal bypass in a rat model of diabetes Tomohiro Miyachi, MD, PhD,a Munenori Nagao, MD, PhD,a Chikashi Shibata, MD, PhD,b Yoshiro Kitahara, PhD,c Naoki Tanaka, MD, PhD,a Kazuhiro Watanabe, MD, PhD,a Takahiro Tsuchiya, MD,a Fuyuhiko Motoi, MD, PhD,a Takeshi Naitoh, MD, PhD, FACS,a and Michiaki Unno, MD, PhD,a Sendai and Kanagawa, Japan
Background and aims. Roux-en-Y gastric bypass improves glucose metabolism in clinical practice, and duodenal–jejunal bypass (DJB), an experimental bypass procedure, also improves metabolism in animals. However, the mechanism remains controversial; especially, the role of the biliopancreatic limb (BP-limb) remains unclear. Our aim was to examine the importance of the function of the BP-limb after DJB using a novel operative model. Methods. Otsuka Long-Evans Tokushima Fatty rats with diabetes were divided into the following groups: DJB with a short alimentary limb (A-limb) and long BP-limb (B-DJB group), DJB with jejunectomy (J-DJB group) in which the entire length of the jejunum used for the BP-limb of the B-DJB group was excised; and a sham operation group. Glucose tolerance, plasma bile acid levels, and the gut microbiota were assessed postoperatively. Results. Glucose tolerance was improved and weight gain was suppressed after surgery in the B-DJB group. In contrast, these effects were cancelled in the J-DJB group. The plasma levels of bile acids in the B-DJB group were greater than those in other groups. The analysis of gut microbiota showed distinct differences between the B-DJB and other groups; especially, the relative abundance of genus Bifidobacterium was much higher in the B-DJB group. Conclusion. The BP-limb played an important role in the control of weight gain, glucose tolerance, and increased plasma bile acid levels after DJB in this rat model of type 2 diabetes mellitus. Plasma bile acids and gut microbiota may be involved in these processes. (Surgery 2016;j:j-j.) From the Department of Surgery,a Tohoku University Graduate School of Medicine; Department of Surgery,b Tohoku Pharmaceutical University Hospital, Sendai; and cInstitute for Innovation, Ajinomoto Co., Inc., Kanagawa, Japan
BARIATRIC SURGERY has emerged as the most effective method for sustained weight loss in obese patients; it induces a long-term improvement in patients having diabetes mellitus (DM) and its comorbidities
and improves overall survival.1-3 After the operation, improved glucose metabolism is noticeable even before weight loss occurs, suggesting a metabolism-improving mechanism independent
Transcript profiling: Takeshi Naitoh (same as above).
interpretation of the data. N. Tanaka and K. Watanabe assisted in the design of the study and interpretation of the data. T. Tsuchiya assisted in the design of the study and provided technical assistance for the study. F. Motoi and M. Unno assisted in the design of the study.
This study was supported by the grant-in-aid for scientific research from Japan Society for the Promotion of Science and by the grant from the Ajinomoto Co., Inc.. The study was conducted in collaboration with Ajinomoto Co., Inc., which was responsible for the analysis of plasma hormones, gut microbiota, and plasma metabolome analysis. T. Miyachi participated in the design of the study, data collection, statistical analysis, and drafting of the manuscript. T. Naitoh participated in supervision and provided oversight when the manuscript was being drafted. M. Nagao and C. Shibata gave significant advice regarding protocol development and drafting of the manuscript. Y. Kitahara contributed to the design of the study, data collection, statistical analysis, and
Accepted for publication November 27, 2015. Reprint requests: Takeshi Naitoh, MD, PhD, FACS, Department of Surgery, Tohoku University Graduate School of Medicine, 1-1, Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2015.11.027
SURGERY 1
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of weight loss. Many studies have been conducted to elucidate this mechanism to enable its application to the medical treatment of diabetes. However, the underlying mechanism remains unclear. Roux-en-Y gastric bypass (RYGB) is one of the most commonly performed bariatric surgical procedures. Duodenal–jejunal bypass (DJB) is an experimental procedure that was developed to investigate the underlying mechanisms responsible for the metabolic benefits after RYGB.4 In an experimental DJB model, because the whole stomach is preserved, we can focus on only the mechanism of bypassing the alimentary tract excluding the restrictive effect. The schema of DJB is shown in Fig 1. The small intestine in DJB consists of 3 parts in terms of its contents: food in the alimentary limb (A-limb), bile/pancreatic juice in the biliopancreatic limb (BP-limb), and a mixture of food and bile/pancreatic juice in the common channel. The A-limb is also called the Roux limb in RYGB. In surgical bypass procedures, such as RYGB or DJB, changes in the secretion of intestinal hormones in the distal ileum and the role of the A-limb have been reported. In the distal ileum, secretion of gastrointestinal hormones such as glucagon-like peptide-1 (GLP-1), peptide YY (PYY), and fibroblast growth factor (FGF)-19 are increased.5,6 Regarding the A-limb, effects such as activation of nutrient sensing, enhancement of glucose transporter 1-mediated basolateral glucose uptake and utilization, and reduction of the sodium–glucose transporter 1 and taste receptors, have been reported.4,7-9 In contrast, the role of the BP-limb in glycolipid metabolism has been scarcely examined. Recently, clinical trials have reported a relation between the BP-limb length and body-weight loss after RYGB. Nergaard et al10 reported better weight loss using RYGB with a long BP-limb. However, another study reported no correlation.11 We focused our study on the role of the BP-limb by using a rat model of DJB with various lengths of A- and BP-limbs. The aim of our study was to investigate the effect of the BP-limb on metabolic improvement and body weight (BW) loss and to elucidate the effects on bile acids, gut microbiota, and intestinal hormones. Clarifying these issues will contribute to a better understanding of the underlying mechanism of RYGB and DJB. MATERIALS AND METHODS The experimental protocol was approved by the Ethics Committee for Animal Research of the Tohoku University.
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Fig 1. Duodenal–jejunal bypass. A-limb, Alimentary limb; BP-limb, biliopancreatic limb; CC, common channel.
Rats. We used male Otsuka Long-Evans Tokushima Fatty (OLETF) rats as a model for type 2 DM.12-14 These rats lacked cholecystokinin-A receptors and develop obesity and hyperglycemia with hyperinsulinemia after overeating.15 We obtained 4- to 6-week-old male OLETF rats from Japan SLC, Inc. (Shizuoka, Japan). All rats were single-housed under specific pathogen-free conditions and fed normal food (Labo MR Stock composed of 15.2% of kilocalories from fat, 32.6% from protein, and 52.3% from carbohydrates [CHO]; Nosan Corporation, Yokohama, Japan) ad libitum. Study protocols. In this study, we performed 2 protocols to evaluate the effect of different BP-limb length on glucose metabolism in the first series of experiments and the effect of BP-limb resection in the second series. The scheme of each experimental protocol is shown in Figs 2, A, and 3, A. In the first series of experiments, we evaluated only weight gain, food intake, and blood glucose levels in the meal tolerance test 8 weeks after the operation. In the second series, we additionally performed pyruvate acid and insulin tolerance tests 10 and 11 weeks after the operation, respectively. Tissue, blood, and fecal matter were collected 12 weeks after the operation at necropsy in the second series. Surgical preparation and perioperative care. At 19 weeks of age, a meal tolerance test was performed preoperatively, and rats without a diabetic pattern were not used for further analysis. Surgery was performed 1 week after the meal tolerance test.
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Fig 2. (A) Scheme of experimental protocols of the first series of experiments. (B) Body weight after the operation. (C) Glucose excursion curve in meal tolerance test 8 weeks after the operation. Data are expressed as mean ± standard error of the mean; n = 7–8 rats per group. *P < .05, **P < .01 versus values in the sham group (Dunnett’s test or Dunnett’s C test after Levene’s test). AL, Alimentary limb; BPL, biliopancreatic limb; MTT, meal tolerance test; W.O., weeks old.
In the first series of experiments, the rats were divided randomly into 5 groups (n = 7 or 8 in each group; Table): A sham group, an AL3-BPL3 group with short length of both limbs (A-limb, 3 cm; BPlimb, 3 cm), an AL3-BPL30 group with a long BPlimb and a short A-limb (A-limb, 3 cm; BP-limb, 30 cm), an AL30-BPL3 group with a short BPlimb and a long A-limb (A-limb, 30 cm; BP-limb, 3 cm), and an AL30-BPL30 group with long Aand BP-limbs and a short common channel (Alimb, 30 cm; BP-limb, 30 cm). Because the entire small intestine was approximately 91 cm in our rats, the length of common channel was estimated approximately as 85, 58, and 31 cm in the AL3BPL3, AL3-BPL30/AL30-BPL3, and AL30-BPL30 groups, respectively. A limb length of 30 cm was selected because it is approximately one-third of the total length of the small intestine. Surgery was performed after overnight fasting. The operative procedures for DJB in rats were performed according to our previous report.14 Briefly, the duodenum was divided immediately distal to the pylorus, and the distal end was closed. The jejunum was transected distal to the duodenal–jejunal junction and the distal end of the jejunum was anastomosed end-to-end to the proximal end of the duodenum. The proximal end of the jejunum was anastomosed end-to-side to the jejunum. In the sham group, the duodenum close to the pylorus and the jejunum 3 cm distal from the Treitz ligament, were transected and
reanastomosed. Water was given ad libitum from the day of surgery and rat food was given from the day after the operation. To elucidate the role of the BP-limb in DJBinduced improvement of glucose metabolism, we prepared a second series of experiments using different rats. The rats were divided into 3 groups (n = 6 per group): a sham group, a B-DJB group with a long BP-limb (A-limb, 3 cm; BP-limb, 30 cm), and a J-DJB group with an excision of the 30 cm length of jejunum that was equivalent to the BP-limb in the B-DJB group (Fig 3, B). All operative procedures and postoperative care in the second set of experiments were performed in a similar way as in the first series of experiments. Meal, pyruvate acid, and insulin tolerance tests. All tolerance tests were performed after overnight fasting. Meal tolerance tests were done as described previously.16 Rats were administered a liquid mixed meal (Vanilla Ensure H containing 1.5 kcal/mL with 28% of kilocalories from fat, 15% from protein, and 57% from CHO; Abbott Japan Co., Chiba, Japan) by oral gavage at a dose of 1.2 g CHO/5.8 mL/8.76 kcal/kg BW. Blood samples were drawn from the tail vein before and at 15 30, 60, and 120 minutes after administration of the liquid meal. The blood glucose concentrations were measured with a glucometer (Ascensia Breeze; Bayer, Osaka, Japan). We measured the plasma concentrations of total bile acids as well in the second series of experiments. For pyruvate
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Fig 3. (A) Experimental protocols of the second series of experiments. (B) Surgical model for each group. (C) Body weight after the operation. Data are expressed as mean values ± standard error of the mean; n = 6 rats per group. *P < .05, **P < .01, ***P < .001 versus values in the sham group; yyP < .01, yyyP < .001 versus values in the J-DJB group (1-way analysis of variance, followed by Tukey’s post hoc test). A-limb, Alimentary limb; BP-limb, biliopancreatic limb; CC, common channel; DJB, duodenal–jejunal bypass; MTT, meal tolerance test; ITT, insulin tolerance test; PTT, pyruvate acid tolerance test; W.O., weeks old.
Table. Length of each limb in 4 groups in the first series of experiments
AL3-BPL3 AL3-BPL30 AL30-BPL3 AL30-BPL30
Alimentary limb
Biliopancreatic limb
3 3 30 30
3 30 3 30
Common channel (cm) z z z z
85 58 58 31
The length of common channel was approximately calculated from the length of total jejunoileum (91 ± 0.9 cm, mean ± standard error of the mean) measured in tissue sampling.
acid tolerance tests, rats were injected subcutaneously with sodium pyruvate solution (2 g/kg BW), and blood glucose levels were measured. For insulin tolerance tests, rats were injected intraperitoneally with insulin (0.5 U/kg BW). Tissue, blood, and fecal matter collection. Twelve weeks after the operation in the second series of experiments, all rats were made to fast overnight, and then were administered Vanilla Ensure H by oral gavage at the same dose as for the meal tolerance test and were humanely killed 1
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hour later. We collected tissue, blood, and fecal samples after meal loading, because the key factor in DJB might be a change in the diet flow. Blood samples were withdrawn from the inferior vena cava and collected in ice-chilled tubes containing ethylenediaminetetraacetic acid and dipeptidyl peptidase-IV inhibitor. We weighed the retroperitoneal fat and liver, and liver was stored at 608C. Fecal samples collected from the sigmoid colon were added to 99% ethanol and stored at 308C. Plasma assays. Plasma concentrations of total bile acids in the meal tolerance test were examined using the Total Bile Acids Test Kit (Wako, Osaka, Japan). An electrochemiluminescence assay was used to measure the levels of insulin, glucagon, active GLP-1, total PYY, and FGF15 in the plasma samples harvested at necropsy. FGF15 is a homolog of human FGF19, which is secreted from the ileum and induces insulin-independent glucose lowering.17,18 We also measured plasma triglyceride levels using Spotchem II (Arkray Inc., Kyoto, Japan). Measurement of hepatic triglycerides. Hepatic triglycerides were extracted from the harvested liver tissue using the chloroform–methanol method of Folch et al19 and measured enzymatically using a commercial kit (Wako, Tokyo, Japan). Metabolome analysis. Metabolites in plasma samples harvested at necropsy were analyzed using a relative quantitation method by capillary electrophoresistime-of-flight mass spectrometry (CE-TOFMS) and liquid chromatography TOFMS using the Agilent CE-TOFMS system and the Agilent 1200 Series RRLC System SL (Agilent Technologies, Inc., Santa Clara, CA) in Human Metabolome Technologies, Inc. (Yamagata, Japan), as previously described.20 Data are expressed relative to the mean value in the sham group. Metaanalysis of 16S rRNA gene sequences of gut microbiota. Bacterial DNA was extracted from the fecal samples using the FastDNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA) and the FastPrep instrument. For metaanalysis of the 16S rRNA gene sequences, we used a next generation sequencer (Miseq Illumina, Inc., San Diego, CA), as previously reported.21 The data was analyzed using Mothur (available from: www.mothur.org/ wiki/Mothur_manual) and METAGENassist (available from: www.metagenassist.ca/METAGENassist/ faces/Home.jsp).22 Data analysis. All data are presented as mean values ± standard error of the mean. All statistical analyses were performed using the JMP version 10.0.0 statistical software package (SAS
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International Inc., Cary, NC). In the first series of experiments, to compare the results between the sham and other groups, Dunnett’s test or Dunnett’s C test was used after Levene’s test. In the second series of experiments, 1-way analysis of variance, followed by Tukey’s post hoc test was used. RESULTS Effects of A- and BP-limbs on weight gain and glucose metabolism. Weight gain after the operation was suppressed significantly in the 2 groups with the 30-cm BP-limbs (AL3-BPL30 and AL30BPL30 groups) compared with the sham group, and the blood glucose levels in those 2 groups were also reduced significantly throughout the meal tolerance test (Fig 2, B and C). In the AL30-BPL3 group with the long A-limb and short BP-limb, the postoperative weight gain did not differ from that in the sham group, and blood glucose levels were significantly lower only at the 30-minute point. The weight gain and blood glucose levels in the AL3-BPL3 group did not differ from those in the sham group. Thus, obvious improvement of glucose metabolism was observed in the 2 groups with the 30-cm BP-limb. No intergroup differences were observed in food intake 2 and 9 weeks after the operation (data not shown). Effects of resection of BP-limb on glucose metabolism. Postoperative weight gain was suppressed significantly in the B-DJB group in comparison with the sham group (15.3 ± 6.3 vs 65.7 ± 5.8 g; Fig 3, C). However, the effect of weight gain suppression was canceled in the J-DJB group, suggesting that the BP-limb has a role in suppression of the expected weight gain. No intergroup differences were observed in food intake (data not shown). The length of the small intestine measured during tissue sampling was 91 ± 0.9 cm, and the length of the common channel in the groups that underwent DJB was 58.6 ± 1.0 cm. No intergroup differences were observed in those lengths. The J-DJB group had equivalent lengths of the A-limb and common channel responsible for digestion and absorption to that of the B-DJB group. During the meal tolerance test, the B-DJB group had the lowest peak postprandial blood glucose concentrations, and the area under the curve in the B-DJB group was reduced significantly compared with that in the J-DJB and sham groups (Fig 4, A and B). In the insulin tolerance test, during the 15–60 minutes after the intraperitoneal administration of insulin, the blood glucose level relative to that of the baseline was significantly less in the B-DJB group than
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Fig 4. (A) Glucose excursion curve. (B) Area under the curve for the meal tolerance test. (C) Blood glucose levels in insulin tolerance test. (D) Pyruvate acid tolerance test. Tests were performed after 8, 10, and 11 weeks after the operation respectively. Data are expressed as mean values ± standard error of the mean; n = 6 rats per group. *P < .05, **P < .01, ***P < .001 versus values in the sham group; yP < .05, yyP < .01, yyyP < .001; versus values in the J-DJB group (1-way analysis of variance, followed by Tukey’s post hoc test). B-DJB, Duodenal–jejunal bypass with a short alimentary limb and long biliopancreatic limb; J-DJB, duodenal–jejunal bypass with jejunectomy.
that in the sham group. The J-DJB group showed significantly lower values than the sham group only at the 60-minute point, but there was no difference between the J-DJB and sham groups for 0–45 minutes (Fig 4, C). In the pyruvate acid tolerance test performed to evaluate gluconeogenesis in the liver, the blood glucose level relative to that of the baseline remained low in the B-DJB group and was significantly lower at 30 and 45 minutes compared with levels in the sham group and at the 120-minute point compared with that in the J-DJB and sham groups. The J-DJB group tended to have slightly lower values than the sham group, but significant differences were not observed (Fig 4, D). These results suggest improvement in insulin sensitivity and gluconeogenesis suppression in the B-DJB group was reversed in the J-DJB group.
Effects of DJB and resection of BP-limb on triglyceride content in the liver, postprandial levels of hormones, and bile acids. No differences were detected in the plasma triglyceride levels among these groups. However, in the B-DJB group, the triglyceride content in the liver and the retroperitoneal adipose tissue weight per BW were significantly lower than those in the J-DJB and sham groups (Fig 5). The changes observed in the B-DJB group were reversed in the J-DJB group. No differences were observed in plasma levels of insulin, glucagon, GLP-1, PYY, or FGF15 (Fig 6). During the meal tolerance test performed 8 weeks after the operation, plasma concentrations of total bile acids remained high in the B-DJB group. This group had the largest area under the curve, which significantly differed from that of the J-DJB groups (Fig 7, A and B). To investigate changes in
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Fig 5. (A) Plasma levels of triglycerides and (B) triglyceride content in the liver. (C) Weight of the retroperitoneal fat per body weight. Samples were harvested 12 weeks after the operation and 1 hour after administration of a liquid meal (Ensure H). Data are expressed as mean values ± standard error of the mean; n = 6 rats per group. **P < .01, ***P < .001 versus values in the sham group; yyyP < .001 versus values in the J-DJB group; N.S., not significant (1way analysis of variance, followed by Turkey’s post hoc test). B-DJB, Duodenal–jejunal bypass with a short alimentary limb and long biliopancreatic limb; J-DJB, duodenal–jejunal bypass with jejunectomy.
metabolite levels comprehensively, we performed a metabolome analysis using plasma samples harvested 12 weeks after the operation. We found the concentrations of various bile acids increased in the B-DJB group. In particular, taurineconjugated cholic acid, chenodeoxycholic acid (CDCA), glycine-conjugated and taurineconjugated CDCA, glycine-conjugated deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), taurine-conjugated UDCA, and hyodeoxycholic acid levels were significantly greater than those in other groups. Additionally, cholic acid and glycine-conjugated cholic acid levels were significantly greater than those in the J-DJB group (Fig 7, C). Any change in the composition of plasma bile acids is unknown because the absolute amounts of metabolites were not measured in this analysis. Effect of DJB on gut microbiota. In principal component analysis of gut microbiota in the
sigmoid colon with metaanalysis of 16S rRNA gene sequences, the distribution in the B-DJB group profoundly differed from that in other groups (Fig 8, A). The relative abundance of genuses Clostridium, Turicibacter, and Bacteroides in the B-DJB group were lower, and those of Bifidobacterium and Olsenella were much higher, compared with those of the other groups (Fig 8, B). DISCUSSION This is the first study directly assessing the role of the BP-limb using the BP-limb resection model. The results of the present study represent new findings about the BP-limb that may help to elucidate the mechanism of the metabolic benefits observed after RYGB or DJB. The results of the first series of experiments suggested that the BP-limb might have an important role in the suppression of weight gain and improvement of glucose metabolism. In the second series of experiments,
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Fig 6. Plasma hormone levels. Samples were harvested 12 weeks after the operation and 1 hour after administration of a liquid meal (Ensure H). Data are expressed as mean values ± standard error of the mean; n = 6 rats per group. N.S., not significant (1-way analysis of variance, followed by Tukey’s post hoc test). B-DJB, Duodenal–jejunal bypass with a short alimentary limb and long biliopancreatic limb; FGF15, fibroblast growth factor 15; GLP-1, glucagon-like peptide-1; J-DJB, duodenal–jejunal bypass with jejunectomy; PYY, peptide YY.
significant weight gain suppression and greater improvement in glucose metabolism were found in the B-DJB group, which is consistent with the results of the first series. The triglyceride content in the liver and the retroperitoneal adipose tissue weight in the B-DJB group also significantly decreased. However, the J-DJB group in which the entire jejunum corresponding with the BPlimb of the B-DJB group was removed, showed no significant suppression of weight gain. Furthermore, no improvements were observed in the
blood glucose levels during the meal tolerance and pyruvate acid tolerance tests, triglyceride content in the liver, or retroperitoneal adipose tissue weight. The point is that elimination of the BPlimb invalidates the improvement effects of DJB. These results suggest that the changes observed in the B-DJB group are associated closely with the BPlimb, rather than a decrease in digestive and absorptive capacities owing to shortening of the small intestine (as in short bowel syndrome). In addition, an increase in plasma concentrations of
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Fig 7. (A) Change in the plasma total bile acid levels and (B) area under the curve in the meal tolerance test performed 8 weeks after the operation. (C) Ratio of arbitrary units of plasma bile acids compared with the mean in the sham group on the liquid chromatography time-of-flight mass spectrometry analysis. Samples were harvested 12 weeks after the operation. The change in composition of plasma bile acids is unknown, because the absolute amounts of metabolites were not measured in this analysis. Data are expressed as mean values ± standard error of the mean; n = 6 rats per group. (A) **P < .01 versus values in the sham group; yyP < .01 versus values in the J-DJB group; (B and C) *P < .05, **P < .01, ***P < .001 (1-way analysis of variance followed by Tukey’s post hoc test). B-DJB, Duodenal–jejunal bypass with a short alimentary limb and long biliopancreatic limb; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; G-, glycine-conjugated; HDCA, hyodeoxycholic acid; J-DJB, duodenal–jejunal bypass with jejunectomy; T-, taurineconjugated; UDCA, ursodeoxycholic acid.
bile acids and changes in the gut microbiota were found in the B-DJB group; these factors might have affected the results obtained from this group. Such direct effects of the BP-limb on postoperative weight change, glycolipid metabolism, and changes in plasma bile acid levels have not been reported before. In the clinical setting, minigastric bypass, the recently emerged procedure involving the BP-limb but not the A-limb, shows metabolic
benefits comparable with RYGB.23 This may indicate the importance of the BP-limb. Our data provide a theoretical basis for the effects of the BP-limb on metabolic improvement and BW loss. Bile acids are important in the modulation of metabolism. Bile salts act as endogenous ligands for the G-protein-coupled bile acid receptor (TGR5) and nuclear farnesoid X receptor (FXR). Activation of these receptors leads to
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Fig 8. Results of met-analysis of 16S rRNA gene sequences of the gut microbiota in the sigmoid colon. Samples were harvested 12 weeks after the operation. (A) Principal component analysis and (B) composition of the microbiota (genus); n = 6 rats per group. B-DJB, duodenal–jejunal bypass with a short alimentary limb and long biliopancreatic limb; J-DJB, duodenal–jejunal bypass with jejunectomy; PC, principal component.
enhancement of energy expenditure in skeletal muscles and brown adipose tissues, and to regulation of de novo lipogenesis, gluconeogenesis, and insulin sensitivity in the liver.24-28 In various bile acids, CDCA and DCA have potent FXR and TGR5 agonistic activities.29 Although UDCA has very weak agonistic FXR and TGR5 activities.29,30 However, UDCA acts as a molecular chaperone. UDCA has been reported to improve glucose metabolism and insulin sensitivity by mitigating endoplasmic reticulum stress in an obesity mouse model and in obese patients.31,32 In this study, increased plasma levels of various bile acids in the B-DJB group were observed, suggesting the metabolism-improving effects work through these mechanisms. The increase of plasma bile acid levels after RYGB, vertical sleeve gastrectomy, and ileal transposition has been confirmed in humans and rodents, and leads to metabolic improvement
through the activation of bile acid signals.33-35 No similar reports have been available for DJB to date, and this is the first report confirming the increased plasma bile acid level after DJB. Although the shortened enterohepatic circulation of bile acids has been hypothesized to contribute to an increase in bile acid concentrations,36 no detailed examination or analysis has been reported to date on the absorption from the digestive tract or bile acid synthesis in the liver. Approximately 30% of bile acids secreted to the duodenum are reabsorbed passively in the jejunum.37 There is a possibility that passive reabsorption of bile acids in the jejunum, which is equivalent to the BP-limb, may change, leading to the modification in the enterohepatic circulation in the DJB model. Recently, the gut microbiota has also attracted a lot of attention. The gut microbiota affects host energy metabolism and bile acid composition; it is involved in the pathogenesis of obesity, diabetes,
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and hepatocellular carcinoma; and it also alters upon bariatric surgery.38-40 In the present study, the B-DJB group showed a profound change in gut microbiota, including a marked increase in Bifidobacterium and a decrease in Clostridium. This change does not contradict previous reports about the microbiota after metabolic surgery.40,41 Regarding the previous reports, the relative abundance of Clostridium is higher in type 2 DM patients and that of Bifidobacterium decreases in obese patients.42,43 Bifidobacterium produce lactate and acetate, and these short fatty acids suppress glucose incorporation in adipocytes and regulate energy metabolism through the sympathetic neuronal network.44,45 The gut microbiota also affects bile acid homeostasis by bile acid conversion or decomposition. The changed microbiota in the B-DJB group may contribute to the metabolic improvement and also be involved in the change in the composition of plasma bile acids. The changes in amounts and preferences in food intake and alteration of passing routes for nutrients, bile acids, and pancreatic juice are reported as factors involved in postoperative changes in the gut microbiota. However, the details remain unclear.46 In this study, all groups were fed the same diet in comparable quantities. Thus, the observed changes in the gut microbiota are attributable to differences in host factors. Our study has some limitations and leaves some questions unanswered. We measured plasma concentrations of gut hormones only at 60 minutes after the meal load, but not during a fasting state. We did not examine potential concentration changes throughout this period. Thus, the involvement of these hormones in the phenomena observed in each model could not be evaluated. The exact timing of changes in plasma bile acid concentrations, glucose metabolism, and gut microbiota induced after the operation was not examined in this study. Because we did not analyze the detailed interrelations between various parameters, future studies should examine such correlations. Furthermore, our operative models have limitations. The ratios of the length of the A-limb to that of the BP-limb used in this study were unusual with respect to clinical practice. We cannot elucidate the correlation between the length of the BP-limb and the effects on metabolic improvement or BW loss in this all-BP-limb resection model. We also need to investigate the role of the BP-limb using other models with different limb lengths or with different intestine lengths for resection and need to confirm our results in a nonobese DM or diet-induced obesity rodent model.
Although additional studies are needed, the results we present herein reveal for the first time the role of the BP-limb in bariatric surgery. In conclusion, the BP-limb plays a vital role in controlling weight gain and glucose tolerance after DJB, and plasma bile acids and the gut microbiota may be involved in these processes in a rat model of type 2 DM. We believe these findings will contribute to the elucidation of the mechanism of bariatric surgery. The authors thank Ms. Emiko Shibuya for her technical assistance.
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