Gastric Bypass Surgery Reverses Diabetic Phenotypes in Bdnf-Deficient Mice
2016; Elsevier BV; Volume: 186; Issue: 8 Linguagem: Inglês
10.1016/j.ajpath.2016.04.009
ISSN1525-2191
AutoresShujun Jiang, Qinghua Wang, Zan Huang, Anying Song, Yu Peng, Siyuan Hou, Shiying Guo, Weiyun Zhu, Sheng Yan, Zhaoyu Lin, Xiang Gao,
Tópico(s)Gastrointestinal motility and disorders
ResumoDuodenum-jejunum gastric bypass (DJB) has been used to treat morbid diabetic patients. However, neither the suitability among patients nor the mechanisms of this surgical treatment is clear. Previously, we reported a new mouse strain named Timo as type 2 diabetes model caused by brain-derived neurotrophic factor (Bdnf) deficiency. In this study, we found that DJB on Timo mice reversed their metabolic abnormalities without altering the expression of Bdnf. Glucose tolerance and insulin sensitivity were improved greatly, along with reduction of fat accumulation in liver and white adipose tissue. The gut flora population was altered by DJB with increased proportion of Firmicutes and decreased Actinobacteria and Proteobacteria in the ileum after surgery. Systemic inflammation in Timo mice was greatly suppressed with less macrophage infiltration and lower tumor necrosis factor-α levels in liver and white adipose tissue after surgery. Interestingly, the alteration of gut microflora abundance and improved metabolism preceded the inflammation alleviation after DJB surgery. These results suggested that DJB can reverse Bdnf deficiency–associated metabolic abnormality. In addition, the reduced inflammation may not be the initial cause for the DJB-associated metabolic and microbiota alterations. The increased BDNF protein levels in hypothalamus and hippocampus may result from microbiota change after DJB surgery. Duodenum-jejunum gastric bypass (DJB) has been used to treat morbid diabetic patients. However, neither the suitability among patients nor the mechanisms of this surgical treatment is clear. Previously, we reported a new mouse strain named Timo as type 2 diabetes model caused by brain-derived neurotrophic factor (Bdnf) deficiency. In this study, we found that DJB on Timo mice reversed their metabolic abnormalities without altering the expression of Bdnf. Glucose tolerance and insulin sensitivity were improved greatly, along with reduction of fat accumulation in liver and white adipose tissue. The gut flora population was altered by DJB with increased proportion of Firmicutes and decreased Actinobacteria and Proteobacteria in the ileum after surgery. Systemic inflammation in Timo mice was greatly suppressed with less macrophage infiltration and lower tumor necrosis factor-α levels in liver and white adipose tissue after surgery. Interestingly, the alteration of gut microflora abundance and improved metabolism preceded the inflammation alleviation after DJB surgery. These results suggested that DJB can reverse Bdnf deficiency–associated metabolic abnormality. In addition, the reduced inflammation may not be the initial cause for the DJB-associated metabolic and microbiota alterations. The increased BDNF protein levels in hypothalamus and hippocampus may result from microbiota change after DJB surgery. 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Inflammation is necessary for long-term but not short-term high-fat diet-induced insulin resistance.Diabetes. 2011; 60: 2474-2483Crossref PubMed Scopus (382) Google Scholar Timo mutant strain was generated in our laboratory as a T2DM animal model induced by Bdnf deficiency, in which a conserved genomic locus of Bdnf is disrupted by a trans gene insertion that decreases brain-derived neurotrophic factor (BDNF) levels to 30% of wild-type mice. Timo mice exhibit insulin resistance, hyperlipidemia, and metabolic inflammation before the onset of obesity and diabetes.21Sha H. Xu J. Tang J. Ding J. Gong J. Ge X. Kong D. Gao X. Disruption of a novel regulatory locus results in decreased Bdnf expression, obesity, and type 2 diabetes in mice.Physiol Genomics. 2007; 31: 252-263Crossref PubMed Scopus (25) Google Scholar However, whether the DJB surgery can reverse the metabolic phenotypes of Timo mutants is not known. In addition, Timo mice may also serve as a good model to investigate how inflammation and microbiota regulate insulin sensitivity and glucose metabolism after DJB surgery. In this study, we demonstrated that DJB surgery improved glycemic homeostasis and insulin sensitivity in Timo mice. We also observed alterations in gut microbiota before reduced inflammation and increased BDNF in Timo mice. Timo mice were generated in our laboratory as described previously,21Sha H. Xu J. Tang J. Ding J. Gong J. Ge X. Kong D. Gao X. Disruption of a novel regulatory locus results in decreased Bdnf expression, obesity, and type 2 diabetes in mice.Physiol Genomics. 2007; 31: 252-263Crossref PubMed Scopus (25) Google Scholar and male C57BL/6J mice were housed in a specific pathogen-free facility under 12-hour light-dark cycle and hydrated with acidified water at the Model Animal Research Center of Nanjing University. Timo mice were fed a standard chow diet ad libitum. Seven-week-old male wild-type C57BL/6J mice were fed a HFD for 17 weeks before surgery or a normal chow diet as control. The Institutional Animal Care and Use Committee of the Model Animal Research Center of Nanjing University approved the experimental protocols. Ten-week-old Timo mice and HFD-induced C57BL/6J mice underwent DJB surgery as previously described.22Woods M. Lan Z. Li J. Wheeler M.B. Wang H. Wang R. Antidiabetic effects of duodenojejunal bypass in an experimental model of diabetes induced by a high-fat diet.Br J Surg. 2011; 98: 686-696Crossref PubMed Scopus (17) Google Scholar Mice were deprived of food overnight before surgery and anesthetized with intraperitoneal 1.25% Avertin (T48402; Sigma-Aldrich, St. Louis, MO). All mice received 100 μL 0.5% carprofen subcutaneously to alleviate discomfort after surgery. Timo mice were deprived of food 16 hours before the oral glucose [D (+)-glucose; Sigma-Aldrich] tolerance test (2 g/kg body weight). Blood glucose concentrations were tested at 0, 15, 30, 60, 90, and 120 minutes after glucose administration. Mice were injected with 0.5 U of insulin for 1 kg body weight after a 6-hour food deprivation for the insulin tolerance test (Novo Nordisk Pharmaceutical Industries, Malov, Denmark), and blood glucose concentrations were measured at 0, 15, 30, 60, 90, and 120 minutes after insulin injection. Blood concentrations were tested using a Breeze 2 Blood Glucose Meter (Bayer HealthCare LLC, Mishawaka, IN). Body weight was measured weekly at the same time point. Blood was collected from Timo mice eye socket veins after overnight food deprivation in heparinized tubes, and samples were stored at room temperature for 30 minutes to separate serum from whole blood. Serum was collected after centrifugation at 3000 × g for 15 minutes. Plasma (10 μL) was used to measure insulin concentrations using a mouse insulin enzyme-linked immunosorbent assay kit (Millipore, Billerica, MA; catalog no. EZRMI-13K), according to the manufacturer's instruction. Plasma concentrations of leptin were determined using a mouse leptin enzyme-linked immunosorbent assay kit (Millipore; catalog no. EZRMI-82K). Plasma BDNF levels were detected using a BDNF enzyme-linked immunosorbent assay kit (Boatman Tech, Shanghai, China). Lipid metabolites and hepatitis concentrations, including total cholesterol, triglycerides, high-density lipoprotein, very-low-density lipoprotein, alanine aminotransferase, and aspartate aminotransferase, in plasma of Timo mice after DJB surgery were quantified using colorimetric assays in a 7020 automatic analyzer (Hatachi High Technology, Tokyo, Japan). Food intake and energy expenditure were measured using a complementary laboratory animal metabolic system. Liver and adipose tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5 mm) were generated using a Leica (Wetzlar, Germany) microtome (RM2155) and stained with hematoxylin and eosin according to standard procedures. Liver frozen sections (10 mm) were stained with Oil Red O to analyze lipid accumulation. High-quality total RNA from liver, white adipose tissue (WAT), brown adipocyte tissue (BAT), and muscle was isolated by extraction with RNAiso plus (9108; TaKaRa, Tokyo, Japan). cDNA was synthesized using a PrimeScript RT reagent Kit with a gDNA Eraser kit (RR047A; TaKaRa). The primers used for F4/80, Cd11b, Cd11c, tumor necrosis factor-α (Tnfa), IL-6 (Il6), IL-1 β (Il1b), and monocyte chemoattractant protein 1 (Mcp1), and 36B4 were as follows: F4/80, 5′-AGTACGATGTGGGGCTTTTG-3′ (forward) and 5′-CCCCATCTGTACATCCCACT-3′ (reverse); Cd11b, 5′-CAGTTCCCAGAGGCTCTCA-3′ (forward) and 5′-GGAGCCATCAATCAAGAAG-3′ (reverse); Cd11c, 5′-ATGGAGCCTCAAGACAGGAC-3′ (forward) and 5′-GGATCTGGGATGCTGAAATC-3′ (reverse); Mcp-1, 5′-CATCCACGTGTTGGCTCA-3′ (forward) and 5′-GATCATCTTGCTGGTGAATGAGT-3′ (reverse); Tnf-α, 5′-CTTCTCATTCCTGCTTGTGG-3′ (forward)and 5′-GGTCTGGGGCATAGAACTGA-3′ (reverse); Il-1β, 5′-AACCTGCTGGTGTGTGACGTTC-3′ (forward) and 5′-AGCACGAGGCTTTTTTGTTGT-3′ (reverse); and Il-6, 5′-CGCTATGAAGTTCCTCTCTGC-3′ (forward) and 5′-CCTCTGTGAAGTCTCCTCTCC-3′ (reverse). Quantitative reverse transcription-PCR for each gene was performed using SYBR Premix Ex Taq (RR420A; TaKaRa) in an ABI 7700 sequence detector (Applied Biosystems, Foster City, CA). The relative abundance of target gene transcripts was normalized to 36B4 expression. Total protein extracts from WAT, liver, hypothalamus, and hippocampus were homogenized in RIPA lysis buffer that contained a 1% protease inhibitor cocktail (Sigma-Aldrich) and a 1% tyrosine phosphatase inhibitor cocktail (Sigma-Aldrich). Immunoblotting was performed as previously described.23Arkan M.C. Hevener A.L. Greten F.R. Maeda S. Li Z.W. Long J.M. Wynshaw-Boris A. Poli G. Olefsky J. Karin M. IKK-beta links inflammation to obesity-induced insulin resistance.Nat Med. 2005; 11: 191-198Crossref PubMed Scopus (1476) Google Scholar Total TNF-α was immunoblotted using a rat anti–TNF-α antibody (BD Biosciences, San Jose, CA; catalog no. 559064). Total BDNF was immunoblotted using a rabbit anti-BDNF antibody (Santa Cruz Biotechnology, Santa Cruz, CA; sc-546). Plasma TNF-α levels were determined using a mouse TNF-α enzyme-linked immunosorbent assay kit (MTA00B; R&D Systems, Minneapolis, MN), according to the manufacturer's protocol. Mouse recombinant TNF-α (10 μg/kg) (GenScript, Nanjing, China; Z02918-100) was continuously injected intraperitoneally into Timo mice for 7 days after DJB surgery. Fecal samples and different parts of the biliopancreatic limb, the Roux limb, ileum, cecum, colon, and rectum were collected from DJB and sham mice and stored at −80°C for further use. Bacterial genomic DNA was extracted using the QiaAmp DNA stool Mini DNA-Isolation Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Concentrations were measured using the Qubit 2.0 instrument and the QubitdsDNA HS Assay (Life Technologies, Invitrogen Division, Darmstadt, Germany). Primers were used to amplify the 16S rRNA hyper variable region V6 according to the report by Huber et al24Huber J.A. Mark Welch D.B. Morrison H.G. Huse S.M. Neal P.R. Butterfield D.A. Sogin M.L. Microbial population structures in the deep marine biosphere.Science. 2007; 318: 97-100Crossref PubMed Scopus (704) Google Scholar; all primers sequences are as follows: 967F-PP, 5′-CNACGCGAAGAACCTTANC-3′; 967F-UC1, 5′-CAACGCGAAAAACCTTACC-3′; 967F-UC2, 5′-CAACGCGCAGAACCTTACC-3′; 967F-UC3, 5′-ATACGCGARGAACCTTACC-3′; 967F-AQ, 5′-CTAACCGANGAACCTYACC-3′; 1046R, 5′-CGACAGCCATGCANCACCT-3′; 1046R-PP, 5′-CGACAACCATGCANCACC-3′; 1046R-AQ1, 5′-CGACGGCCATGCANCACCT-3′; and 1046R-AQ2, 5′-CGACGACCATGCANCACCT-3′. The generated amplicon libraries were used in the Ion Plus Fragment Library Kit (Life Technologies, Invitrogen Division; catalog no. 4471252) according to the manufacturer's instructions. Library concentrations were measured using the Qubit 2.0 instrument and the Qubit dsDNA HS Assay (Life Technologies, Invitrogen Division). The Ion Torrent Personal Genome Machine Template OT2 200 kit (Life Technologies; catalog no. 4480974) was applied to perform emulsion PCR using the Ion OneTouch 2 system as described in the User Guidelines (Part No. 4469004 Rev. B 07/2011). Amplicon libraries were sequenced on the Ion Torrent Personal Genome Machine system using the Ion Torrent Personal Genome Machine Sequencing 200 Kit v2 (catalog no. 4482006; Life Technologies) according to the manufacturer's protocol. The sphere was loaded to a 316 chip (Life Technologies) according to the method of Sebastian.25Junemann S. Prior K. Szczepanowski R. Harks I. Ehmke B. Goesmann A. Stoye J. Harmsen D. Bacterial community shift in treated periodontitis patients revealed by ion torrent 16S rRNA gene amplicon sequencing.PloS ONE. 2012; 7: e41606Crossref PubMed Scopus (108) Google Scholar ShangHai Biotechnology Corporation (Shanghai, China) analyzed 16S rRNA gene sequences. The relative abundance of bacterial taxonomic groups was compared in all sequences, and a principal coordinate's analysis plot of weighted UniFrac distances was also performed in different groups. Fecal bacteria DNA (100 ng) was amplified by 16rRNA v6-v8 variation region primers (968F primer contains GC clamp: 5′-CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCAGGGGAACGCGAAGAACCTTAC-3′; 1401R primer: CGGTGTGTACAAGACCC) using a 2X Taqplus Master Polymerase mix (Vanzyme; P212-01/02/03), 433-bp amplicon was generated. Eight percent acrylamide gel (acrylamide:bisacrylamide, 37.5:1) with a 35% to 55% (w/v) urea denaturant gradient was run at 80V for 12 hours. The gels were stained by 2 mg/mL silver in Cairns' fixation solution, and 1.5% NaOH that contained 0.4% formaldehyde as developer. The special band was extracted, transformed into Escherichia coli with PMD19-T vector (TaKaRa; catalog no. 6013), sequenced, and compared using BLAST (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov, last accessed March 19, 2016). Statistically significant differences between Timo mice and wild-type littermates are presented as means ± SEM. All data were analyzed using one-way analysis of variance (when three groups are compared) or repeated measurement analysis of variance (for time course data) and unpaired Student's t-test. P < 0.05 was considered significant. Timo mice exhibited obesity, diabetes, and inflammation at 4 weeks of age (Supplemental Figure S1). To investigate whether DJB surgery reversed obesity in Timo mice, we performed DJB surgery on 10-week-old male Timo mice according to Woods et al22Woods M. Lan Z. Li J. Wheeler M.B. Wang H. Wang R. Antidiabetic effects of duodenojejunal bypass in an experimental model of diabetes induced by a high-fat diet.Br J Surg. 2011; 98: 686-696Crossref PubMed Scopus (17) Google Scholar (Supplemental Figure S2). We found that DJB surgery caused a decrease in body weight from 34.99 ± 4 g to 26.59 ± 3.14 g after 1 week in Timo mice (Figure 1, A–C). HFD-induced obese mice, as a positive control, also exhibited body weight loss from 40.88 ± 0.18 g to 31.55 ± 0.79 g 1 week after surgery, and exhibited better control of glucose (Supplemental Figure S3). Decreased body weight in Timo mice directly correlated with reductions in fat mass (Figure 1, D and E). Timo mice consumed fewer calories but exhibited no differences in physical activity from that of sham mice 8 weeks after DJB surgery (Figure 1, F–J, and Supplemental Figure S4). Our data confirm that DJB significantly reduced body weights in Timo mice and reduced food intake. Bariatric surgery is one of the new clinical treatments for severe T2DM.26Kashyap S.R. Gatmaitan P. Brethauer S. Schauer P. Bariatric surgery for type 2 diabetes: weighing the impact for obese patients.Cleve Clin J Med. 2010; 77: 468-476Crossref PubMed Scopus (58) Google Scholar, 27Rubino F. Schauer P.R. Kaplan L.M. Cummings D.E. Metabolic surgery to treat type 2 diabetes: clinical outcomes and mechanisms of action.Annu Rev Med. 2010; 61: 393-411Crossref PubMed Scopus (303) Google Scholar Insulin sensitivity was normalized to the level of wild-type littermates 2 weeks after DJB surgery (Figure 2A). The effect of DJB surgery on insulin sensitivity was maintained for at least 8 weeks (Figure 2, B and C). Plasma insulin concentrations were reduced to normal concentrations in DJB Timo mice compared with sham wild-type littermates 9 weeks after DJB surgery (Figure 2D). The correction of glucose intolerance in Timo mice was maintained for at least 8 weeks after surgery (Figure 2, E and F). Obesity and T2DM are accompanied by leptin resistance because lipid accumulation damages leptin signaling.28Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. Chen H. Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance.J Clin Invest. 2003; 112: 1821-1830Crossref PubMed Scopus (5170) Google Scholar, 29Saltiel A.R. Kahn C.R. Insulin signalling and the regulation of glucose and lipid metabolism.Nature. 2001; 414: 799-806Crossref PubMed Scopus (3910) Google Scholar Lipid accumulation in liver and adipose tissue (WAT and BAT) and enlarged adipocyte size occurred in 8-week-old male Timo mice (Figure 3A). Timo mice also exhibited a tendency for leptin resistance with increased concentrations at 6 weeks (Supplemental Figure S5A). Histologic analyses and Oil Red O staining revealed that fat content decreased significantly in the livers of Timo mice 8 weeks after surgery compared with that of the sham Timo mice (Figure 3B). Hepatitis (alanine aminotransferase) was alleviated 9 weeks after DJB surgery, which contributed to the improvement in lipid metabolism (Figure 3D). We measured serum concentrations of total cholesterol, triglycerides, high-density lipoprotein, and very-low-density lipoprotein to investigate whether the decreased weight of Timo mice 9 weeks after DJB surgery reflected changes in lipid lysis. Total cholesterol and very-low-density lipoprotein decreased postoperatively in male and female Timo mice after DJB surgery (Figure 3C and Supplemental Figure S5B). Leptin concentrations in serum also decreased to normal concentrations in Timo mice 8 weeks after DJB surgery (Figure 3E). Accumulating evidence indicates that bariatric surgery affects the diversity of gut microbiota.13Ryan K.K. Tremaroli V. Clemmensen C. Kovatcheva-Datchary P. Myronovych A. Karns R. Wilson-Perez H.E. Sandoval D.A. Kohli R. Backhed F. Seeley R.J. FXR is a molecular target for the effects of vertical sleeve gastrectomy.Nature. 2014; 509: 183-188Crossref PubMed Scopus (684) Google Scholar, 14Liou A.P. Paziuk M. Luevano Jr., J.M. Machineni S. Turnbaugh P.J. Kaplan L.M. Conserved shifts in the gut microbiota due to gastric bypass reduce host weight and adiposity.Sci Transl Med. 2013; 5: 178ra41Crossref PubMed Scopus (713) Google Scholar, 17Li J.V. Ashrafian H. Bueter M. Kinross J. Sands C. le Roux C.W. Bloom S.R. Darzi A. Athanasiou T. Marchesi J.R. Nicholson J.K. Holmes E. Metabolic surgery profoundly influences gut microbial-host metabolic cross-talk.Gut. 2011; 60: 1214-1223Crossref PubMed Scopus (341) Google Scholar We analyzed fecal bacterial composition 1 week and 2 weeks after DJB surgery using denaturing gradient gel electrophoresis analysis to examine immediate changes in gut microbiota. Fecal bacteria exhibited different dominant populations with lower diversity in DJB mice than in wild-type littermates. Bacterial communities were unstable, which was indicated by large variations in different samples (Figure 4A). Desulfovibrionaceae abundance was much higher in the sham group of Timo mice (Table 1 and Supplemental Figure S6A) 1 week after surgery, Clostridiales abundance decreased (Table 2, Supplemental Figure S6A), and Verrucomicrobiaceae abundance increased (Supplemental Figure S6A and Table 2) in Timo DJB mice 2 weeks after surgery. The predominant gut bacteria community became consistent within the same experimental group 2 weeks after DJB surgery. The increase in Verrucomicrobiaceae abundance was more significant in Timo DJB mice (Table 2, Supplemental Figure S6B).Table 1DGGE Band Analysis for 1 Week from the Feces of Timo Mice and Sham Control Mice after SurgeryNo.DomainPhylumClassOrderFamilyGenusIdentity (%)1-1BacteriaFirmicutesClostridiaClostridialesEubacteriaceaeEubacterium901-2BacteriaVerrucomicrobiaVerrucomicrobiaeVerrucomicrobialesVerrucomicrobiaceaeAkkermansia1001-3BacteriaFirmicutesClostridiaClostridialesLachnospiraceae901-4BacteriaFirmicutesClostridiaClostridialesLachnospiraceaeRoseburia921-5BacteriaFirmicutesClostridiaClostridialesLachnospiraceae881-6BacteriaFirmicutesClostridiaClostridialesLachnospiraceaeRoseburia891-7BacteriaProteobacteriaDeltaproteobacteriaDesulfovibrionalesDesulfovibrionaceaeLawsonia891-8BacteriaFirmicutesClostridiaClostridialesLachnospiraceaeBlautia911-9BacteriaFirmicutesClostridiaClostridialesLachnospiraceaeRoseburia881-10BacteriaFirmicutesClostridiaClostridialesLachnospiraceaeRoseburia951-11BacteriaFirmicutesClostridiaClostridialesLachnospiraceaeRoseburia941-12BacteriaProteobacteriaGammaproteobacteriaEnterobacterialesEnterobacteriaceaeCitrobacter941-13BacteriaVerrucomicrobiaVerrucomicrobiaeVerrucomicrobialesVerrucomicrobiaceaeAkkermansia1001-1
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