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H. Kashihara, et al. 1
Original article
Duodenal-jejunal bypass improves diabetes and liver steatosis via enhanced glucagon-like peptide-1 elicited by bile acids1
Hideya Kashihara, Mitsuo Shimada, Nobuhiro Kurita, Hirohiko Sato, Kozo Yoshikawa, Jun Higashijima, Motoya Chikakiyo, Masaaki Nishi, Chie Takasu Department of Surgery, Institute of Health Biosciences, University of Tokushima 3-18-15 Kuramoto-cho, Tokushima, Tokushima 770-8503, Japan Running head: Effects of duodenal-jejunal bypass
Word count: Abstract, 247; Text, 2,919
Figures and Tables: 5
Correspondence and reprint requests: Hideya Kashihara, MD Department of Surgery, Institute of Health Biosciences, University of Tokushima 3-18-15 Kuramoto-cho, Tokushima, Tokushima 770-8503, Japan Phone: +81-88-633-7137 Fax: +81-88-631-9698 E-mail: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jgh.12690 This article is protected by copyright. All rights reserved.
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Abstract
Background and Aim: Bariatric surgery not only elicits weight loss, but also rapidly resolves diabetes. However, the mechanisms remain unclear. The present study investigates how diabetes and liver steatosis are improved after duodenal-jejunal bypass (DJB) compared with a glucagon-like peptide-1 (GLP-1) analog and correlations between bile acids and GLP-1 secretion. Methods: We initially determined the effects of bile acids on GLP-1 in vitro and then assigned 12 male 16-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats to groups that underwent DJB, a sham operation, or were treated with the GLP-1 receptor
agonist, liraglutide (n = 4 each). Blood glucose, insulin, GLP-1, serum bile acids, liver steatosis and the number of GLP-1 positive cells (L cells) in the small intestine and colon were investigated in the three groups at eight weeks postoperatively. Results: Levels of GLP-1mRNA were upregulated and GLP-1 secretion increased in cells incubated with bile acids in vitro. Weight gain was suppressed more in the DJB than in the sham group in vivo. Diabetes was more improved and GLP-1 levels were significantly higher in the DJB than in the sham group. Serum bile acids were significantly increased, the number of L cells in the ileum was upregulated compared with the sham group, and liver steatosis was significantly improved in the DJB compared with the other two groups. Conclusions: Duodenal-jejunal bypass might improve diabetes and liver steatosis by enhancing GLP-1 secretion through increasing serum bile acids and the proliferation of L cells in the ileum, compared with liraglutide.
Key words: bariatric surgery, liraglutide, incretin, gastrointestinal hormone
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Introduction The number of patients with type 2 diabetes in Japan has increased from 30,000 during
the 1970s to about 7,000,000 today. Type 2 diabetes is associated with high rates of morbidity and mortality. Therefore, treatment for type 2 diabetes is an important public health issue in Japan. Obesity can cause various kinds of complications such as type 2 diabetes mellitus,
hypertension, and hyperlipidemia. Bariatric surgery improves not only obesity, but also obesity-related complications. A gastric bypass or biliopancreatic diversion resolves type 2 diabetes in 80-90% of morbidly obese patients1-3. Duodenal-jejunal bypass (DJB) is a type of metabolic surgery in which the distal
jejunum is connected to the duodenum and biliopancreatic juices are directed to the ileum. Several studies have found obvious and early improvements in glucose homeostasis after DJB4-6. Two theories have been proposed to explain why type 2 diabetes improves after DJB. The hindgut hypothesis suggests that the rapid transit of nutrients and biliopancreatic juices to the distal bowel improves glucose metabolism by stimulating
the
secretion
of
glucagon-like
peptide-1
(GLP-1)
and
other
appetite-suppressing gut peptides. The foregut hypothesis suggests that an unknown factor promotes insulin resistance and type 2 diabetes mellitus7. The improvement in type 2 diabetes after a duodenal-jejunal bypass (DJB) might be
associated with an increase in GLP-1 secretion from L cells in the ileum8. Enteral carbohydrates and fats stimulate the secretion of GLP-1, a 30 amino-acid peptide by intestinal L cells9. The incretin hormones GLP-1 and gastric inhibitory polypeptide are
responsible for up to 70% of post-prandial insulin secretion9,10. Regarding the correlation between GLP-1 and bile acids, the cell-surface bile acid
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H. Kashihara, et al. 4
receptor G-protein coupled receptor 1 (TGR5) is apparently critical for regulating intestinal GLP-1 secretion in vivo11 and bile acids enhance GLP-1 secretion via TGR5 in the murine enteroendocrine STC-1 cell line12. Liraglutide is a human GLP-1 analog with 97% homology to native GLP-113.
Liraglutide causes mean reductions in levels of A1C, fasting blood glucose and body weight of 0.8-1.5%, 1.4-3.4 mmol/L and 1.2-3.0 kg, respectively, in patients with type 2 diabetes14-17. However, the difference between the effects of bariatric surgery and the GLP-1 analog on diabetes and weight loss remains unclear. The GLP-1 receptor in hepatocytes reportedly contributes to the reduction of
triglycerides and free fatty acids, resulting in improvements in hepatocyte steatosis32.
However, the effect of bariatric surgery and the GLP-1 analog on liver steatosis remains unclear.
We previously found that DJB improves insulin resistance in Sprague-Dawley rats by
increasing bile acids and consequently enhancing GLP-1 secretion18. The present study compares the mechanisms of improved diabetes and liver steatosis
between DJB and liraglutide administration, and investigates correlations between bile acids and GLP-1 secretion.
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Methods
Cell culture The murine enteroendocrine cell line IEC-619 (Summit Pharmaceuticals International,
Tokyo, Japan) (1 × 104 cells/dish) was incubated for seven days in Dulbecco’s modified
Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 70 μg/mL of penicillin, and 100 μg/mL of streptomycin at 37°C in a humidified incubator under a 5% CO2 atmosphere. After three washes with Hank’s balanced salt solution (HBSS), the cells were incubated for 180 min at 37°C in HBSS containing PBS without (PBS group) or with 30 μM of cholic (CA group) or lithocholic (LCA group) acids (Sigma Chemical Co., St. Louis, MO, USA). The conditioned medium was collected and GLP-1 concentrations were determined using AKMGP-011 GLP-1 ELISA kits (Shibayagi, Gunma, Japan).
RNA isolation and quantitative real time RT-PCR Total RNA was extracted using the RNeasy Mini kit (Qiagen, Valencia, CA, USA)
and reverse transcribed with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time reverse transcription (RT)-PCR proceeded using a 7500 Real-Time PCR system with the TaqMan Gene Expression Assay-on-Demand and TaqMan Universal Master Mix
(Applied Biosystems). We assayed levels of glucagon-like peptide-1 (GLP-1; Rn00562293_m1) (Applied Biosystems) and the control gene was TaqMan Rat GAPDH endogenous control (GAPDH; 4352338E; Applied Biosystems). The thermocycling conditions comprised 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C, and 1 min at 65°C. Data were analyzed using Applied Biosystems Prism 7500
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Sequence Detection System ver. 1.3.1.
Animals
Male 16-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats (Japan SLC
Inc., Hamamatsu, Japan) were housed in a room under controlled temperature, humidity and a 12:12-h artificial light-dark cycle. This study proceeded in compliance with the Division for Animal Research Resources, Institute of Health Biosciences, and the University of Tokushima. The Animal Care and Use Committee of the University of Tokushima approved the experiments and procedures.
Duodenal-jejunal bypass surgery, sham operation, and liraglutide administration The OLETF rats were randomly assigned to groups (n = 4 each) that underwent a
duodenal-jejunal bypass (DJB), a sham operation (sham), or treatment with liraglutide. The rats were fasted overnight and intraoperatively anesthetized with 2-3% isoflurane and air/oxygen before undergoing DJB as described by Rubino et al.4. The duodenum was separated and the stump was closed using a 5-0 absorbable suture. The jejunum was interrupted at 10 cm from the ligament of Treitz and the distal side of the jejunum was connected to the duodenum using a 5-0 absorbable suture. The biliopancreatic limb carrying the biliopancreatic juices was reconnected to the jejunum at 15 cm distal from the duodenojejunal anastomosis using a 5-0 absorbable suture. The anterior abdominal
wall of the sham group was incised at the midline and then the incision was closed using 3-0 absorbable sutures. The liraglutide group was subcutaneously administered with 200 μg/kg liraglutide (Novo Nordisk, Bagvaerd, Denmark) every 24 h for eight weeks. All groups were given free access to a methionine- and choline-deficient (MCD),
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H. Kashihara, et al. 7
high fat (HF) diet and tap water for eight weeks. Body weight and fasting blood glucose were measured before and 1, 2, 3, and 8 weeks after surgery. The oral glucose tolerance test (OGTT) and blood analysis proceeded at post-operative week (POW) 8 and then all rats were immediately sacrificed.
Blood analysis Blood samples were obtained from the heart after a 24-h fast at POW 8. Blood samples
were separated by centrifugation and plasma was frozen at –80°C for subsequent measurement of plasma total cholesterol, triglycerides, free fatty acids, AST, ALT, ALP, LDH, and bile acids. These parameters were measured at Shikoku-Chuken (Kagawa,
Japan), a company that specializes in chemical analysis.
Oral glucose tolerance test (OGTT) The OGTT was applied at POW 8. A 50% glucose solution (1 g/kg) was orally
administered and glucose levels were measured in tail-vein blood samples using a Medi
Safe GR-102 Mini glucometer (Terumo, Tokyo, Japan) 0, 15, 30, 60, and 120 minutes later. Blood was simultaneously collected into Eppendorf tubes (for GLP-1; containing aprotinin and EDTA-2Na.) from the tail vein at the same time points and then plasma separated by centrifugation was stored at -80°C. Plasma insulin levels were determined using an AKRIN-010H rat insulin ELISA kit (Shibayagi, Gunma, Japan) and plasma total GLP-1 levels were assayed using a GLP-1 ELISA kit (AKMGP-011) (Shibayagi,
Gunma, Japan).
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GLP-1 assays Samples were incubated in antibody for 2 h in solid phase wells coated with
anti-GLP-1, and then washed. Biotin-labeled anti-GLP-1 antibody was added to the
wells and incubated for 2 h, followed by adipine-bound peroxidase for 30 min. After
washing, peroxidase in the wells was reacted with the coloring reagent 3,3’, 5,5’-tetramethylbenzidine (TMB). The products were measured at a wavelength of 450 nm.
Immunohistochemistry Specimens (1 × 1 cm) of all layers in the roux, biliopancreatic, common limb, and
colon were harvested from the DJB group at sacrifice. Specimens (1 × 1 cm) of all layers in the proximal, middle, and distal small intestine and colon were harvested from the sham and the liraglutide groups. All specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4-μm-thick sections that were deparaffinized in
xylene and rehydrated in a graded series of ethanol. The rehydrated sections were heated at 120°C in an autoclave in 10 mM sodium citrate (pH 6.0) for 10 min, cooled to room temperature, and then incubated in 3.0% hydrogen peroxide in methanol for 20 min to block endogenous peroxidase activity. The slides were then incubated for 1 h at room temperature in 5% bovine serum albumin in phosphate-buffered saline (PBS), then overnight at 4°C with sc-57166 mouse monoclonal anti-GLP-1 antibody (Santa Cruz Biotech, Santa Cruz, CA, USA) diluted 1:100 in PBS. Reactions were developed using the avidin-biotin immunoperoxidase technique. The sections were washed and incubated with the ChemMate envision kit/HRP second antibody (Dako Corporation, Glostrup, Denmark) for 60 min at 37°C. The peroxidase reaction was visualized using
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H. Kashihara, et al. 9
diaminobenzidine as the chromogen. The sections were counterstained with hematoxylin, dehydrated with ethanol, immersed in xylene and enclosed in synthetic resin. Cells were considered positive when cytoplasmic staining was evident. The number of GLP-1 positive cells per villus in the small intestine and colon was counted. The immunohistochemical findings were reviewed in a blinded fashion.
Evaluation of liver fibrosis Livers from sacrificed rats were fixed in 10% buffered formalin and embedded in
paraffin. A single pathologist scored the severity of histological changes in livers
stained with Azan and hematoxylin-eosin in a blinded fashion and evaluated the histopathological features of the liver using Brunt’s grading. Significant lesions including steatosis, ballooning, and intra-acinar and portal inflammation were graded as mild (1+, 66%)34.
Statistical analysis Data were statistically analyzed using either an unpaired Student’s t test or a one-way
ANOVA with Bonferroni’s post hoc test. Differences were considered significant when p < 0.05. The results are expressed as means ± standard error of the mean (SEM). All statistical data were generated using StatView ver. 5.0 for Window (SAS Institute Inc., Cary, NC, USA).
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Results
Effect of bile acids on GLP-1 in vitro We initially examined whether bile acids affect GLP-1 mRNA in IEC-6 cells and
found significantly higher levels of GLP-1 mRNA in the cells incubated with LCA than with PBS or CA (p < 0.05) (Fig. 1a). We then assessed the effects of bile acids on
GLP-1 secretion in these cells. The concentration of GLP-1 was significantly higher in the medium from the LCA than from the PBS and CA groups (p < 0.05; Fig. 1b). In summary, bile acids (LCA) upregulated GLP-1 mRNA and increased the secretion of GLP-1 from IEC-6 cells into the medium in vitro.
Effect of DJB on diabetes in vivo All rats were weighed at POW 0, 1, 2, 3, and 8. Body weight gain was significantly
suppressed in the DJB group compared with the sham group at POW 2, 3, and 8 (p < 0.05), but did not significantly differ between the DJB and liraglutide groups at POW 8 (Fig. 2a). Fasting blood glucose at POW 8 was significantly lower in the DJB group than in the sham group (p < 0.05; Fig. 2b). The results of the OGTT at POW 8 showed that glucose intolerance was similarly
improved in the DJB and liraglutide groups compared with the sham group (p < 0.05; Fig. 2c and d). More GLP-1 was secreted at 15 and 30 min in the DJB and liraglutide groups than in the sham group (p < 0.05; Fig. 2e). The amount of GLP-1 changed more rapidly in response to the orally administered glucose in the DJB group than in the other
two groups. Levels of bile acids were significantly higher in the DJB group than in the other two groups (p < 0.05; Fig. 3a). Levels of total cholesterol, triglycerides, and free fatty acids were lower in the DJB group than in the sham group (p < 0.05) and did not
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significantly differ between the DJB and liraglutide groups (Fig. 3b-d). Liver function tests showed that levels of AST and LDH were lower in the DJB group
than in the other two groups, whereas levels of ALT and ALP did not significantly differ among the three groups (Fig. 3e-h). We immunohistochemically investigated the number of L cells in the proximal, middle,
and distal segments of the small intestine and colon in the three groups to determine whether the three procedures affected GLP-1-positive L cells. Figure 4a shows GLP-1 positive cells from rat villi. More L cells were identified in the distal segment of the DJB group than the other two groups (p < 0.05; Fig. 4b). More L cells were identified in the distal segment of the DJB group than in any other segments (proximal, middle, distal, and colon) in the sham and liraglutide groups (data not shown). We pathologically assessed H&E and Azan staining in livers harvested at POW 8 to
determine whether DJB and liraglutide can improve liver steatosis. Liver inflammation (grade) was significantly improved in the DJB group compared with liraglutide and sham groups (0 vs. 0.75 ± 0.5 and 2.5 ± 0.6, respectively; Fig. 5a and b). Therefore, liver fibrosis (staging) significantly improved in the DJB and liraglutide groups compared with the sham group (0.25 ± 0.vs. 0.50 ± 0.96 and 3.0 ± 1.5, respectively; Fig. 5c and d).
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Discussion We investigated how DJB improves type 2 diabetes in obese diabetic rats compared
with an injected GLP-1 analog. Weight gain was suppressed and diabetes improved with an increase in GLP-1 in the DJB, compared with the sham and liraglutide groups. Furthermore, levels of serum bile acids and the numbers of L cells in distal segments
were significantly higher in the DJB group than in the sham and liraglutide groups. Thus, DJB might improve type 2 diabetes via enhanced GLP-1 secretion through ileal bile acid absorption and increasing the number of ileal L cells. Reports indicate that GLP-1 reduces glucotoxicity by improving islet function and
insulin sensitivity, and reducing hepatic gluconeogenesis20,21. Furthermore, GLP-1 has the potential to elicit glucose-stimulated insulin secretion, increase insulin synthesis,
stimulate β-cell proliferation, and prevent β-cell apoptosis20,22. Improvements in type 2
diabetes after DJB, Roux-en-Y gastric bypass (RYGB), and ileal transposition (IT) are thought to be associated with an increase in GLP-1 secretion from L cells in the ileum8,23,24. The common feature of these three procedures is the rapid exposure of the
ileum to bile and nutrients. Thomas et al. reported that the cell-surface bile acid receptor TGR5 is critical for regulating intestinal GLP-1 secretion in vivo25. This receptor binds to secondary bile acids such as deoxycholic and lithocholic acids that are formed from
cholic and chenodeoxycholic acid, respectively. TGR5 signaling in enteroendocrine L-cells induces GLP-1 secretion, thus improving liver and pancreatic function and enhancing glucose tolerance in obese mice. Katsuma et al. demonstrated that bile acid enhances GLP-1 secretion via TGR5 in the murine enteroendocrine cell line STC-112. The present study found that LCA (secondary bile acid) promoted more GLP-1
secretion than CA (primary bile acid). Furthermore, levels of serum bile acids were
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H. Kashihara, et al. 13
significantly higher in the DJB group than in the sham and liraglutide groups. Both the
RYGB and IT procedures have resulted in increased levels of serum bile acids26,27. In addition, serum bile acids positively correlated with GLP-1 in patients after a gastric
bypass23. The number of L cells was significantly increased in the distal limb (ileum) of
the DJB group compared with that of the sham and liraglutide group. Thus, DJB might increase GLP-1 secretion through (secondary) bile acid absorption and increasing L cell proliferation in the ileum. Sato et al. reported that cholecystoileostomy with a ligated common bile duct seemed
to inhibit interdigestive and postprandial upper gut contractions in association with an increase in levels of plasma Peptide YY (PYY)28. That report did not indicate a correlation between cholecystoileostomy and increased GLP-1. However, their findings might have been associated with GLP-1 secretion because both GLP-1 and PYY are released from L cells in the ileum. Both GLP-1 and PYY released from the ileum are reportedly mediators of upper gut motility inhibition by the perfusion of nutrients into the ileum29,30. In addition, serum bile acids increase energy expenditure in mouse brown
adipose tissues, thus preventing obesity and insulin resistance31. Increased levels of
serum bile acids and GLP-1 might have contributed to the suppressed weight gain in the present study. Gupta et al. reported that the GLP-1 receptor in hepatocytes contributes to reductions
in triglycerides and free fatty acids, resulting in improved hepatocyte steatosis32.
Another possibility is that lowered levels of free fatty acids are the result of increased leptin-induced intracellular fatty oxidation33. There seems to be a cross-reaction of native GLP-1 as well as liraglutide in GLP-1
ELISA kit (AKMGP-011). However, there are no data to suggest that this ELISA kit
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measures native GLP-1 as well as liraglutide. The dose of liraglutide was determined
with reference to Jamie E. et al.35. In this report, the injection of 200 μg/kg liraglutide daily for four weeks improved insulin sensitivity and reduced lipid accumulation in the
liver. This dose of liraglutide was sufficient for the rats, because the maximum dose for humans is 900 μg/day based on a body weight of 60 kg (15μg/kg). The effects of
liraglutide in terms of retarding weight gain and improving diabetes were comparable to the effects of DJB. The bile diversion model in which a catheter is inserted into the common bile duct and
its distal end is anchored to the mid-distal jejunum causes an increase in serum bile acids and reduces hepatic steatosis due to decreasing liver ER stress36. A DJB might improve liver steatosis via the synergistic effects of GLP-1 and bile acids compared with liraglutide. We concluded that DJB results in less weight gain and might improve type 2 diabetes
and liver steatosis. Such improvement might be related to enhanced GLP-1 secretion via increased levels of bile acids and L-cell proliferation in the ileum compared with liraglutide.
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Acknowledgments We are grateful to the staff at the Department of Surgery, Institute of Health
Biosciences, University of Tokushima, for important technical contributions.
Conflict of interest statement Hideya Kashihara and other co-authors have no conflicts of interest to declare.
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Figure legends
Figure 1. Bile acids promote GLP-1 mRNA and GLP-1 secretion in IEC-6 cells in vitro.
Bile acids promote GLP-1 mRNA in IEC-6 cells in vitro (a). PBS vs. LCA and CA vs. LCA; p < 0.05. Values are expressed as means ± SEM. (one-way ANOVA with Bonferroni’s post hoc test). Bile acids promote GLP-1 secretion in IEC-6 cells in vitro (b). PBS vs. LCA and CA vs. LCA; p < 0.05. Values are expressed as means ± SEM. (one-way ANOVA with Bonferroni’s post hoc test). PBS; Phosphate buffered saline, CA; Cholic acids, LCA; Lithocholic acids
Figure 2. Body weight (a), fasting blood glucose (b), blood glucose (c), insulin (d) and GLP-1 (e) in DJB, sham and liraglutide groups.
*DJB vs. sham; †liraglutide vs. sham; p < 0.05. Values are expressed as means ± SEM.
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(one-way ANOVA with Bonferroni’s post hoc test).
Figure 3. Bile acids (a), total cholesterol (b), triglyceride(c), free fatty acids (d), AST (e), ALT (f), ALP (g) and LDH (h) at postoperative 8 weeks in DJB, sham and liraglutide
group. DJB vs. *sham and †liraglutide; p < 0.05. Values are expressed as means ± SEM. (one-way ANOVA with Bonferroni’s post hoc test).
Figure 4. Representative immunohistochemical images (a) and number of L cells in distal segment (b) at postoperative week 8 in DJB, sham and liraglutide groups. DJB vs. sham and liraglutide, p < 0.05. Values are expressed as means ± SEM. (one-way ANOVA with Bonferroni’s post hoc test).
Figure 5. Representative images of H&E (a) and grades (b) at postoperative week 8 in DJB, liraglutide and sham groups. DJB vs. liraglutide, DJB vs. sham and liraglutide vs. sham, p < 0.05 (one-way ANOVA with Bonferroni’s post hoc test). Representative images of Azan staining (c) and stages (d) at postoperative week 8 in
DJB, liraglutide and sham groups. DJB vs. sham and liraglutide vs. sham, p < 0.05 (one-way ANOVA with Bonferroni’s post hoc test).
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Original article
Duodenal-jejunal bypass improves diabetes and liver steatosis via enhanced glucagon-like peptide-1 elicited by bile acids1
Hideya Kashihara, Mitsuo Shimada, Nobuhiro Kurita, Hirohiko Sato, Kozo Yoshikawa, Jun Higashijima, Motoya Chikakiyo, Masaaki Nishi, Chie Takasu Department of Surgery, Institute of Health Biosciences, University of Tokushima 3-18-15 Kuramoto-cho, Tokushima, Tokushima 770-8503, Japan Running head: Effects of duodenal-jejunal bypass
Word count: Abstract, 247; Text, 2,919
Figures and Tables: 5
Correspondence and reprint requests: Hideya Kashihara, MD Department of Surgery, Institute of Health Biosciences, University of Tokushima 3-18-15 Kuramoto-cho, Tokushima, Tokushima 770-8503, Japan Phone: +81-88-633-7137 Fax: +81-88-631-9698 E-mail: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jgh.12690 This article is protected by copyright. All rights reserved.
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Abstract
Background and Aim: Bariatric surgery not only elicits weight loss, but also rapidly resolves diabetes. However, the mechanisms remain unclear. The present study investigates how diabetes and liver steatosis are improved after duodenal-jejunal bypass (DJB) compared with a glucagon-like peptide-1 (GLP-1) analog and correlations between bile acids and GLP-1 secretion. Methods: We initially determined the effects of bile acids on GLP-1 in vitro and then assigned 12 male 16-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats to groups that underwent DJB, a sham operation, or were treated with the GLP-1 receptor
agonist, liraglutide (n = 4 each). Blood glucose, insulin, GLP-1, serum bile acids, liver steatosis and the number of GLP-1 positive cells (L cells) in the small intestine and colon were investigated in the three groups at eight weeks postoperatively. Results: Levels of GLP-1mRNA were upregulated and GLP-1 secretion increased in cells incubated with bile acids in vitro. Weight gain was suppressed more in the DJB than in the sham group in vivo. Diabetes was more improved and GLP-1 levels were significantly higher in the DJB than in the sham group. Serum bile acids were significantly increased, the number of L cells in the ileum was upregulated compared with the sham group, and liver steatosis was significantly improved in the DJB compared with the other two groups. Conclusions: Duodenal-jejunal bypass might improve diabetes and liver steatosis by enhancing GLP-1 secretion through increasing serum bile acids and the proliferation of L cells in the ileum, compared with liraglutide.
Key words: bariatric surgery, liraglutide, incretin, gastrointestinal hormone
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Introduction The number of patients with type 2 diabetes in Japan has increased from 30,000 during
the 1970s to about 7,000,000 today. Type 2 diabetes is associated with high rates of morbidity and mortality. Therefore, treatment for type 2 diabetes is an important public health issue in Japan. Obesity can cause various kinds of complications such as type 2 diabetes mellitus,
hypertension, and hyperlipidemia. Bariatric surgery improves not only obesity, but also obesity-related complications. A gastric bypass or biliopancreatic diversion resolves type 2 diabetes in 80-90% of morbidly obese patients1-3. Duodenal-jejunal bypass (DJB) is a type of metabolic surgery in which the distal
jejunum is connected to the duodenum and biliopancreatic juices are directed to the ileum. Several studies have found obvious and early improvements in glucose homeostasis after DJB4-6. Two theories have been proposed to explain why type 2 diabetes improves after DJB. The hindgut hypothesis suggests that the rapid transit of nutrients and biliopancreatic juices to the distal bowel improves glucose metabolism by stimulating
the
secretion
of
glucagon-like
peptide-1
(GLP-1)
and
other
appetite-suppressing gut peptides. The foregut hypothesis suggests that an unknown factor promotes insulin resistance and type 2 diabetes mellitus7. The improvement in type 2 diabetes after a duodenal-jejunal bypass (DJB) might be
associated with an increase in GLP-1 secretion from L cells in the ileum8. Enteral carbohydrates and fats stimulate the secretion of GLP-1, a 30 amino-acid peptide by intestinal L cells9. The incretin hormones GLP-1 and gastric inhibitory polypeptide are
responsible for up to 70% of post-prandial insulin secretion9,10. Regarding the correlation between GLP-1 and bile acids, the cell-surface bile acid
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receptor G-protein coupled receptor 1 (TGR5) is apparently critical for regulating intestinal GLP-1 secretion in vivo11 and bile acids enhance GLP-1 secretion via TGR5 in the murine enteroendocrine STC-1 cell line12. Liraglutide is a human GLP-1 analog with 97% homology to native GLP-113.
Liraglutide causes mean reductions in levels of A1C, fasting blood glucose and body weight of 0.8-1.5%, 1.4-3.4 mmol/L and 1.2-3.0 kg, respectively, in patients with type 2 diabetes14-17. However, the difference between the effects of bariatric surgery and the GLP-1 analog on diabetes and weight loss remains unclear. The GLP-1 receptor in hepatocytes reportedly contributes to the reduction of
triglycerides and free fatty acids, resulting in improvements in hepatocyte steatosis32.
However, the effect of bariatric surgery and the GLP-1 analog on liver steatosis remains unclear.
We previously found that DJB improves insulin resistance in Sprague-Dawley rats by
increasing bile acids and consequently enhancing GLP-1 secretion18. The present study compares the mechanisms of improved diabetes and liver steatosis
between DJB and liraglutide administration, and investigates correlations between bile acids and GLP-1 secretion.
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Methods
Cell culture The murine enteroendocrine cell line IEC-619 (Summit Pharmaceuticals International,
Tokyo, Japan) (1 × 104 cells/dish) was incubated for seven days in Dulbecco’s modified
Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 70 μg/mL of penicillin, and 100 μg/mL of streptomycin at 37°C in a humidified incubator under a 5% CO2 atmosphere. After three washes with Hank’s balanced salt solution (HBSS), the cells were incubated for 180 min at 37°C in HBSS containing PBS without (PBS group) or with 30 μM of cholic (CA group) or lithocholic (LCA group) acids (Sigma Chemical Co., St. Louis, MO, USA). The conditioned medium was collected and GLP-1 concentrations were determined using AKMGP-011 GLP-1 ELISA kits (Shibayagi, Gunma, Japan).
RNA isolation and quantitative real time RT-PCR Total RNA was extracted using the RNeasy Mini kit (Qiagen, Valencia, CA, USA)
and reverse transcribed with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time reverse transcription (RT)-PCR proceeded using a 7500 Real-Time PCR system with the TaqMan Gene Expression Assay-on-Demand and TaqMan Universal Master Mix
(Applied Biosystems). We assayed levels of glucagon-like peptide-1 (GLP-1; Rn00562293_m1) (Applied Biosystems) and the control gene was TaqMan Rat GAPDH endogenous control (GAPDH; 4352338E; Applied Biosystems). The thermocycling conditions comprised 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C, and 1 min at 65°C. Data were analyzed using Applied Biosystems Prism 7500
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Sequence Detection System ver. 1.3.1.
Animals
Male 16-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats (Japan SLC
Inc., Hamamatsu, Japan) were housed in a room under controlled temperature, humidity and a 12:12-h artificial light-dark cycle. This study proceeded in compliance with the Division for Animal Research Resources, Institute of Health Biosciences, and the University of Tokushima. The Animal Care and Use Committee of the University of Tokushima approved the experiments and procedures.
Duodenal-jejunal bypass surgery, sham operation, and liraglutide administration The OLETF rats were randomly assigned to groups (n = 4 each) that underwent a
duodenal-jejunal bypass (DJB), a sham operation (sham), or treatment with liraglutide. The rats were fasted overnight and intraoperatively anesthetized with 2-3% isoflurane and air/oxygen before undergoing DJB as described by Rubino et al.4. The duodenum was separated and the stump was closed using a 5-0 absorbable suture. The jejunum was interrupted at 10 cm from the ligament of Treitz and the distal side of the jejunum was connected to the duodenum using a 5-0 absorbable suture. The biliopancreatic limb carrying the biliopancreatic juices was reconnected to the jejunum at 15 cm distal from the duodenojejunal anastomosis using a 5-0 absorbable suture. The anterior abdominal
wall of the sham group was incised at the midline and then the incision was closed using 3-0 absorbable sutures. The liraglutide group was subcutaneously administered with 200 μg/kg liraglutide (Novo Nordisk, Bagvaerd, Denmark) every 24 h for eight weeks. All groups were given free access to a methionine- and choline-deficient (MCD),
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high fat (HF) diet and tap water for eight weeks. Body weight and fasting blood glucose were measured before and 1, 2, 3, and 8 weeks after surgery. The oral glucose tolerance test (OGTT) and blood analysis proceeded at post-operative week (POW) 8 and then all rats were immediately sacrificed.
Blood analysis Blood samples were obtained from the heart after a 24-h fast at POW 8. Blood samples
were separated by centrifugation and plasma was frozen at –80°C for subsequent measurement of plasma total cholesterol, triglycerides, free fatty acids, AST, ALT, ALP, LDH, and bile acids. These parameters were measured at Shikoku-Chuken (Kagawa,
Japan), a company that specializes in chemical analysis.
Oral glucose tolerance test (OGTT) The OGTT was applied at POW 8. A 50% glucose solution (1 g/kg) was orally
administered and glucose levels were measured in tail-vein blood samples using a Medi
Safe GR-102 Mini glucometer (Terumo, Tokyo, Japan) 0, 15, 30, 60, and 120 minutes later. Blood was simultaneously collected into Eppendorf tubes (for GLP-1; containing aprotinin and EDTA-2Na.) from the tail vein at the same time points and then plasma separated by centrifugation was stored at -80°C. Plasma insulin levels were determined using an AKRIN-010H rat insulin ELISA kit (Shibayagi, Gunma, Japan) and plasma total GLP-1 levels were assayed using a GLP-1 ELISA kit (AKMGP-011) (Shibayagi,
Gunma, Japan).
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GLP-1 assays Samples were incubated in antibody for 2 h in solid phase wells coated with
anti-GLP-1, and then washed. Biotin-labeled anti-GLP-1 antibody was added to the
wells and incubated for 2 h, followed by adipine-bound peroxidase for 30 min. After
washing, peroxidase in the wells was reacted with the coloring reagent 3,3’, 5,5’-tetramethylbenzidine (TMB). The products were measured at a wavelength of 450 nm.
Immunohistochemistry Specimens (1 × 1 cm) of all layers in the roux, biliopancreatic, common limb, and
colon were harvested from the DJB group at sacrifice. Specimens (1 × 1 cm) of all layers in the proximal, middle, and distal small intestine and colon were harvested from the sham and the liraglutide groups. All specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4-μm-thick sections that were deparaffinized in
xylene and rehydrated in a graded series of ethanol. The rehydrated sections were heated at 120°C in an autoclave in 10 mM sodium citrate (pH 6.0) for 10 min, cooled to room temperature, and then incubated in 3.0% hydrogen peroxide in methanol for 20 min to block endogenous peroxidase activity. The slides were then incubated for 1 h at room temperature in 5% bovine serum albumin in phosphate-buffered saline (PBS), then overnight at 4°C with sc-57166 mouse monoclonal anti-GLP-1 antibody (Santa Cruz Biotech, Santa Cruz, CA, USA) diluted 1:100 in PBS. Reactions were developed using the avidin-biotin immunoperoxidase technique. The sections were washed and incubated with the ChemMate envision kit/HRP second antibody (Dako Corporation, Glostrup, Denmark) for 60 min at 37°C. The peroxidase reaction was visualized using
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diaminobenzidine as the chromogen. The sections were counterstained with hematoxylin, dehydrated with ethanol, immersed in xylene and enclosed in synthetic resin. Cells were considered positive when cytoplasmic staining was evident. The number of GLP-1 positive cells per villus in the small intestine and colon was counted. The immunohistochemical findings were reviewed in a blinded fashion.
Evaluation of liver fibrosis Livers from sacrificed rats were fixed in 10% buffered formalin and embedded in
paraffin. A single pathologist scored the severity of histological changes in livers
stained with Azan and hematoxylin-eosin in a blinded fashion and evaluated the histopathological features of the liver using Brunt’s grading. Significant lesions including steatosis, ballooning, and intra-acinar and portal inflammation were graded as mild (1+, 66%)34.
Statistical analysis Data were statistically analyzed using either an unpaired Student’s t test or a one-way
ANOVA with Bonferroni’s post hoc test. Differences were considered significant when p < 0.05. The results are expressed as means ± standard error of the mean (SEM). All statistical data were generated using StatView ver. 5.0 for Window (SAS Institute Inc., Cary, NC, USA).
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Results
Effect of bile acids on GLP-1 in vitro We initially examined whether bile acids affect GLP-1 mRNA in IEC-6 cells and
found significantly higher levels of GLP-1 mRNA in the cells incubated with LCA than with PBS or CA (p < 0.05) (Fig. 1a). We then assessed the effects of bile acids on
GLP-1 secretion in these cells. The concentration of GLP-1 was significantly higher in the medium from the LCA than from the PBS and CA groups (p < 0.05; Fig. 1b). In summary, bile acids (LCA) upregulated GLP-1 mRNA and increased the secretion of GLP-1 from IEC-6 cells into the medium in vitro.
Effect of DJB on diabetes in vivo All rats were weighed at POW 0, 1, 2, 3, and 8. Body weight gain was significantly
suppressed in the DJB group compared with the sham group at POW 2, 3, and 8 (p < 0.05), but did not significantly differ between the DJB and liraglutide groups at POW 8 (Fig. 2a). Fasting blood glucose at POW 8 was significantly lower in the DJB group than in the sham group (p < 0.05; Fig. 2b). The results of the OGTT at POW 8 showed that glucose intolerance was similarly
improved in the DJB and liraglutide groups compared with the sham group (p < 0.05; Fig. 2c and d). More GLP-1 was secreted at 15 and 30 min in the DJB and liraglutide groups than in the sham group (p < 0.05; Fig. 2e). The amount of GLP-1 changed more rapidly in response to the orally administered glucose in the DJB group than in the other
two groups. Levels of bile acids were significantly higher in the DJB group than in the other two groups (p < 0.05; Fig. 3a). Levels of total cholesterol, triglycerides, and free fatty acids were lower in the DJB group than in the sham group (p < 0.05) and did not
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significantly differ between the DJB and liraglutide groups (Fig. 3b-d). Liver function tests showed that levels of AST and LDH were lower in the DJB group
than in the other two groups, whereas levels of ALT and ALP did not significantly differ among the three groups (Fig. 3e-h). We immunohistochemically investigated the number of L cells in the proximal, middle,
and distal segments of the small intestine and colon in the three groups to determine whether the three procedures affected GLP-1-positive L cells. Figure 4a shows GLP-1 positive cells from rat villi. More L cells were identified in the distal segment of the DJB group than the other two groups (p < 0.05; Fig. 4b). More L cells were identified in the distal segment of the DJB group than in any other segments (proximal, middle, distal, and colon) in the sham and liraglutide groups (data not shown). We pathologically assessed H&E and Azan staining in livers harvested at POW 8 to
determine whether DJB and liraglutide can improve liver steatosis. Liver inflammation (grade) was significantly improved in the DJB group compared with liraglutide and sham groups (0 vs. 0.75 ± 0.5 and 2.5 ± 0.6, respectively; Fig. 5a and b). Therefore, liver fibrosis (staging) significantly improved in the DJB and liraglutide groups compared with the sham group (0.25 ± 0.vs. 0.50 ± 0.96 and 3.0 ± 1.5, respectively; Fig. 5c and d).
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Discussion We investigated how DJB improves type 2 diabetes in obese diabetic rats compared
with an injected GLP-1 analog. Weight gain was suppressed and diabetes improved with an increase in GLP-1 in the DJB, compared with the sham and liraglutide groups. Furthermore, levels of serum bile acids and the numbers of L cells in distal segments
were significantly higher in the DJB group than in the sham and liraglutide groups. Thus, DJB might improve type 2 diabetes via enhanced GLP-1 secretion through ileal bile acid absorption and increasing the number of ileal L cells. Reports indicate that GLP-1 reduces glucotoxicity by improving islet function and
insulin sensitivity, and reducing hepatic gluconeogenesis20,21. Furthermore, GLP-1 has the potential to elicit glucose-stimulated insulin secretion, increase insulin synthesis,
stimulate β-cell proliferation, and prevent β-cell apoptosis20,22. Improvements in type 2
diabetes after DJB, Roux-en-Y gastric bypass (RYGB), and ileal transposition (IT) are thought to be associated with an increase in GLP-1 secretion from L cells in the ileum8,23,24. The common feature of these three procedures is the rapid exposure of the
ileum to bile and nutrients. Thomas et al. reported that the cell-surface bile acid receptor TGR5 is critical for regulating intestinal GLP-1 secretion in vivo25. This receptor binds to secondary bile acids such as deoxycholic and lithocholic acids that are formed from
cholic and chenodeoxycholic acid, respectively. TGR5 signaling in enteroendocrine L-cells induces GLP-1 secretion, thus improving liver and pancreatic function and enhancing glucose tolerance in obese mice. Katsuma et al. demonstrated that bile acid enhances GLP-1 secretion via TGR5 in the murine enteroendocrine cell line STC-112. The present study found that LCA (secondary bile acid) promoted more GLP-1
secretion than CA (primary bile acid). Furthermore, levels of serum bile acids were
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significantly higher in the DJB group than in the sham and liraglutide groups. Both the
RYGB and IT procedures have resulted in increased levels of serum bile acids26,27. In addition, serum bile acids positively correlated with GLP-1 in patients after a gastric
bypass23. The number of L cells was significantly increased in the distal limb (ileum) of
the DJB group compared with that of the sham and liraglutide group. Thus, DJB might increase GLP-1 secretion through (secondary) bile acid absorption and increasing L cell proliferation in the ileum. Sato et al. reported that cholecystoileostomy with a ligated common bile duct seemed
to inhibit interdigestive and postprandial upper gut contractions in association with an increase in levels of plasma Peptide YY (PYY)28. That report did not indicate a correlation between cholecystoileostomy and increased GLP-1. However, their findings might have been associated with GLP-1 secretion because both GLP-1 and PYY are released from L cells in the ileum. Both GLP-1 and PYY released from the ileum are reportedly mediators of upper gut motility inhibition by the perfusion of nutrients into the ileum29,30. In addition, serum bile acids increase energy expenditure in mouse brown
adipose tissues, thus preventing obesity and insulin resistance31. Increased levels of
serum bile acids and GLP-1 might have contributed to the suppressed weight gain in the present study. Gupta et al. reported that the GLP-1 receptor in hepatocytes contributes to reductions
in triglycerides and free fatty acids, resulting in improved hepatocyte steatosis32.
Another possibility is that lowered levels of free fatty acids are the result of increased leptin-induced intracellular fatty oxidation33. There seems to be a cross-reaction of native GLP-1 as well as liraglutide in GLP-1
ELISA kit (AKMGP-011). However, there are no data to suggest that this ELISA kit
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measures native GLP-1 as well as liraglutide. The dose of liraglutide was determined
with reference to Jamie E. et al.35. In this report, the injection of 200 μg/kg liraglutide daily for four weeks improved insulin sensitivity and reduced lipid accumulation in the
liver. This dose of liraglutide was sufficient for the rats, because the maximum dose for humans is 900 μg/day based on a body weight of 60 kg (15μg/kg). The effects of
liraglutide in terms of retarding weight gain and improving diabetes were comparable to the effects of DJB. The bile diversion model in which a catheter is inserted into the common bile duct and
its distal end is anchored to the mid-distal jejunum causes an increase in serum bile acids and reduces hepatic steatosis due to decreasing liver ER stress36. A DJB might improve liver steatosis via the synergistic effects of GLP-1 and bile acids compared with liraglutide. We concluded that DJB results in less weight gain and might improve type 2 diabetes
and liver steatosis. Such improvement might be related to enhanced GLP-1 secretion via increased levels of bile acids and L-cell proliferation in the ileum compared with liraglutide.
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Acknowledgments We are grateful to the staff at the Department of Surgery, Institute of Health
Biosciences, University of Tokushima, for important technical contributions.
Conflict of interest statement Hideya Kashihara and other co-authors have no conflicts of interest to declare.
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21. Abu-Hamadah R, Rabiee A, Meneilly GS, et al. Clinical review: The extrapancreatic effects of glucagon-like peptide-1 and related peptides. J Clin Endocrinol Metab 2009; 94: 1843-52. 22. Salehi M, Aulinger BA, D’Alessio DA. Targeting beta-cell mass in type 2 diabetes: promise and limitations of new drugs based on incretins. Endor Rev 2008; 29: 367-79. 23. Patriti A, Facchiano E, Annetti C, et al. Early improvement of glucose tolerance after ileal transposition in a non-obese type 2 diabetes rat model. Obes Surg. 2005 Oct; 15(9):1258-64. 24. Suzuki S, Ramos EJ, Goncalves CG, et al. Changes in GI hormones and their effect on gastric emptying and transit times after Roux-en-Y gastric bypass in rat model. Surgery. 2005; 138:283–90. 25. Charles Thomas, Antimo Gioiello, Lilia Noriega, et al. TGR5-Mediated Bile Acid Sensing Controls Glucose Homeostasis. Cell Metab. 2009 Sep; 10(3):167-77. 26. Rohit Kohli, Michelle Kirby, Kenneth DR Setchell, et al. Intestinal adaptation after ileal interposition surgery increases bile acid recycling and protects against obesity related co-morbidities. Am J Physiol Gastrointest Liver Physiol. 2010 Sep; 299(3). 27. Patti, M.E., Houten, S.M., Bianco, A.C. , 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 Sep; 17(9):1671-7. 28. Sato M, Shibata C, Kikuchi D, et al. Effects of biliary and pancreatic juice diversion into the ileum on gastrointestinal motility and gut hormone secretion in conscious dogs. Surgery. 2010 Nov; 148(5):1012-9. 29. Van Citters GW, Lin HC. Ileal brake: neuropeptidergic control of intestinal transit. Curr Gastroenterol Rep 2006; 8: 367-73. 30. Holst JJ. The physiology of glucagon-like peptide 1. Physiol Rev 2007; 87: 1409-39 31. Watanabe M, Houten SM, Mataki C, et al. Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation. Nature. 2006 Jan 26; 439(7075):484-9. Epub 2006 Jan 8. 32. Gupta NA, Mells J, Dunham RM, et al. Glucagon-like peptide-1 receptor is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway. Hepatology. 2010 May; 51(5):1584-92. 33. Shimabukuro M, Koyama K, Chen G, et al. Direct antidiabetic effect of leptin
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through triglyceride depletion of tissues. Proc Natl Acad Sci U S A. 1997 Apr 29; 94(9):4637-41. 34. Brunt EM, Janney CG, Di Bisceglie AM, et al. Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol. 1999;94(9):2467–2674. 35. Jamie E. Mells, Ping P. Fu, Shvetank Sharma, et al. Glp-1 analog, liraglutide, ameliorates hepatic steatosis and cardiac hypertrophy in C57BL/6J mice fed a Western diet. Am J Physiol Gastrointest Liver Physiol 2012;302:G225–G235. 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 Jul;154(7):2341-51.
Figure legends
Figure 1. Bile acids promote GLP-1 mRNA and GLP-1 secretion in IEC-6 cells in vitro.
Bile acids promote GLP-1 mRNA in IEC-6 cells in vitro (a). PBS vs. LCA and CA vs. LCA; p < 0.05. Values are expressed as means ± SEM. (one-way ANOVA with Bonferroni’s post hoc test). Bile acids promote GLP-1 secretion in IEC-6 cells in vitro (b). PBS vs. LCA and CA vs. LCA; p < 0.05. Values are expressed as means ± SEM. (one-way ANOVA with Bonferroni’s post hoc test). PBS; Phosphate buffered saline, CA; Cholic acids, LCA; Lithocholic acids
Figure 2. Body weight (a), fasting blood glucose (b), blood glucose (c), insulin (d) and GLP-1 (e) in DJB, sham and liraglutide groups.
*DJB vs. sham; †liraglutide vs. sham; p < 0.05. Values are expressed as means ± SEM.
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(one-way ANOVA with Bonferroni’s post hoc test).
Figure 3. Bile acids (a), total cholesterol (b), triglyceride(c), free fatty acids (d), AST (e), ALT (f), ALP (g) and LDH (h) at postoperative 8 weeks in DJB, sham and liraglutide
group. DJB vs. *sham and †liraglutide; p < 0.05. Values are expressed as means ± SEM. (one-way ANOVA with Bonferroni’s post hoc test).
Figure 4. Representative immunohistochemical images (a) and number of L cells in distal segment (b) at postoperative week 8 in DJB, sham and liraglutide groups. DJB vs. sham and liraglutide, p < 0.05. Values are expressed as means ± SEM. (one-way ANOVA with Bonferroni’s post hoc test).
Figure 5. Representative images of H&E (a) and grades (b) at postoperative week 8 in DJB, liraglutide and sham groups. DJB vs. liraglutide, DJB vs. sham and liraglutide vs. sham, p < 0.05 (one-way ANOVA with Bonferroni’s post hoc test). Representative images of Azan staining (c) and stages (d) at postoperative week 8 in
DJB, liraglutide and sham groups. DJB vs. sham and liraglutide vs. sham, p < 0.05 (one-way ANOVA with Bonferroni’s post hoc test).
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JGH_12690_F1
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JGH_12690_F2c-F2e
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JGH_12690_F5a-F5b
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JGH_12690_F5c-F5d
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