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. Author manuscript; available in PMC: 2021 Dec 1.
Published in final edited form as: Surg Endosc. 2020 Jun 30;35(6):3104–3114. doi: 10.1007/s00464-020-07741-y

Metabolic effects of duodenojejunal bypass surgery in a rat model of type 1 diabetes

Roman Vangoitsenhoven 1,2, Rickesha Wilson 1, Gautam Sharma 1, Suriya Punchai 1,3, Ricard Corcelles 1,4, Dvir Froylich 1,5, Anny Mulya 6, Philip R Schauer 7, Stacy A Brethauer 8, John P Kirwan 7, Naseer Sangwan 9,10, J Mark Brown 9,11, Ali Aminian 1
PMCID: PMC8633809  NIHMSID: NIHMS1674739  PMID: 32607903

Abstract

BACKGROUND:

Metabolic surgery has beneficial metabolic effects, including remission of type 2 diabetes. We hypothesized that duodenojejunal bypass (DJB) surgery can protect against development of type 1 diabetes (T1D) by enhancing regulation of cellular and molecular pathways that control glucose homeostasis.

METHODS:

BBDP/Wor rats, which are prone to develop spontaneous autoimmune T1D, underwent loop DJB (n=15) or sham (n=15) surgery at a median age of 41 days, before development of diabetes. At T1D diagnosis, a subcutaneous insulin pellet was implanted, oral glucose tolerance test was performed 21 days later, and tissues were collected 25 days after onset of T1D. Pancreas and liver tissues were assessed by histology and RT-qPCR. Fecal microbiota composition was analyzed by 16S V4 sequencing.

RESULTS:

Postoperatively, DJB rats weighed less than sham rats (287.8 vs 329.9 g, P=0.04). In both groups, 14 of 15 rats developed T1D, at similar age of onset (87 days in DJB vs 81 days in sham, P=0.17). There was no difference in oral glucose tolerance, fasting and stimulated plasma insulin and c-peptide levels, and immunohistochemical analysis of insulin positive cells in the pancreas. DJB rats needed 1.3±0.4 insulin implants vs 1.9±0.5 in sham rats (P=0.002). Fasting and glucose stimulated glucagon-like peptide 1 (GLP-1) secretion was elevated after DJB surgery. DJB rats had reduced markers of metabolic stress in liver. After DJB, the fecal microbiome changed significantly, including increases in Akkermansia and Ruminococcus, while the changes were minimal in sham rats.

CONCLUSION:

DJB does not protect against autoimmune T1D in BBDP/Wor rats, but reduces the need for exogenous insulin and facilitates other metabolic benefits including weight loss, increased GLP-1 secretion, reduced hepatic stress, and altered gut microbiome.

Introduction

Metabolic surgery has proven to induce substantial and durable weight loss and improvement of type 2 diabetes (T2D) in the majority of patients [1]. The early, weight-loss independent improvement of T2D after metabolic surgery has been attributed to a combination of increased hepatic insulin sensitivity, increased postprandial secretion of the incretin hormones, in particular Glucagon-like peptide 1 (GLP1) and peptide YY (PYY), and altered bile acid signaling [2]. Several studies suggest an improved intrinsic beta-cell function post-Roux-en-Y gastric bypass (RYGB) [3, 4]. Development of postprandial reactive hypoglycemia, which has been attributed to an exaggerated insulin secretion after RYGB in the long-term [5], also suggests direct effects of bypass surgery on insulin secretion. Furthermore, previous studies in non-obese animal models of metabolic surgery such as rats and pigs reported increased beta-cell function and hyperplasia [4, 6].

While T2D is characterized by a relative beta-cell insufficiency in the setting of insulin resistance, type 1 diabetes (T1D) is caused by auto-immune destruction of beta-cells, resulting in absolute insulin deficiency. Only about 10% of all diabetes diagnoses are T1D, but the incidence is rising [7]. The current treatment strategy for T1D is insulin replacement therapy, which prolongs the life expectancy of T1D patients tremendously [7], but is unable to consistently maintain blood glucose levels within the normal physiological range. As a result, patients suffer from episodes of hypoglycemia as well as hyperglycemia, and T1D remains a leading cause of end-stage renal disease, retinopathy, neuropathy, ketoacidosis, amputation, and cardiovascular disease [8].

The glycemic effects of metabolic surgery are limited in patients with established autoimmune diabetes [9, 10]. However, recent immunotherapy studies suggest that loss of beta-cell function and mass may be halted or even restored when given in the early stages of the disease process [11–13], and a previous study reported that duodenojejunal bypass (DJB) surgery was able to protect pancreatic beta cells from apoptosis, which leads to better glycemic control and delayed onset of diabetes in streptozotocin-induced diabetic rats [14]. Therefore, we hypothesized that metabolic surgery might also have protective effects on beta cells in early T1D and thus prevent or delay diabetes development.

We set out to study changes in diabetes development, glucose control, and beta-cell mass after metabolic surgery in a rat model of autoimmune T1D. Secondly, we aimed to examine underlying mechanisms, including postoperative changes in incretin hormones and the gut microbiome.

Material and Methods

Rat model, diabetes diagnosis and treatment protocol

The experimental set-up is synthetized in Figure 1. The male BioBreeding Diabetes Prone Rats (BBDP/Wor, Biomere, Worcester, MA) were randomized to loop DJB (n=15) or sham surgery (n=15). Rats were fed a standard autoclavable chow diet (Purina 5010 providing 29% of energy needs from protein, 13% from fat, and 58% from carbohydrate). Food intake and body weight was recorded twice a week, and the sham rats were pair-fed to their DJB-littermate by restricting the daily allowance for sham rats to the amount consumed by the DJB.

Figure 1. Experimental set-up.

Figure 1.

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BBDP rats underwent loop DJB (n=15) or sham surgery (n=15) at a median age of 41 days. From age 55 days, the rats were monitored for development of type 1 diabetes (T1D) by checking for glucosuria. Exogenous insulin administration was started as needed. An Oral Glucose Tolerance Test (OGTT) was performed 21 days after development of diabetes. At 25 days after diabetes diagnosis, the rats were euthanized after an overnight fast. Fresh fecal pellets were collected from the cage bottom prior to surgery and on postoperative days 35. See text for details.

The BBDP/Wor rats develop cell-mediated autoimmune destruction of the pancreatic beta cells, resulting in an abrupt onset of T1D. The development of diabetes occurs between 60 and 120 days of age (average 70–80 days). This peripubertal onset of disease is comparable to the peak onset of T1D that is observed in 12- to 14-year-old humans. In the absence of exogenous insulin therapy, rats usually die after 4–7 days. The overall incidence of diabetes in BBDP strains is generally reported as 86%. [15]

Rats in each group were paired based on the exact age at the time of DJB and sham operations. Median age of rats at the time of surgery was 41 days (interquartile range, 35–42). Starting at age 55 days, approximately 14 days after the surgical intervention, the rats were monitored for diabetes development by checking for glucosuria (Multistix SG 10 SG, Siemens) 3 times per week. When glucosuria (1+) was recorded, immediate random blood glucose was measured from tail vein blood using a glucometer (AlphaTRAK, Abbott). A blood glucose level >250 mg/dL was used to confirm the diagnosis of overt diabetes, and exogenous insulin administration was initiated by implanting a subcutaneous sustained release insulin implant (Linplant, LinShin Canada Inc., Canada) following manufacturer’s insertion and dosing instructions to control hyperglycemia and to prevent diabetic ketoacidosis. The implants provide an estimated insulin release of ~2U/24 hour for over 3 weeks [16,17]. During follow-up, when blood glucose levels were in the diabetic range (measured 3 times per week), an additional insulin pellet was re-implanted.

Rats were housed in specific pathogen free (SPF) conditions in individual sterilized cages in the Cleveland Clinic Biological Resources Unit with temperature controlled to 22°C and a 12 h dark/light cycle. All animal experimental procedures were approved by the Cleveland Clinic Institutional Animal Care and Use Committee. The National Institute of Health Animal Use Guidelines were adhered to for this study.

Surgical procedures and perioperative care

To perform single anastomosis (loop) DJB surgery, the stomach was dissected free using blunt forceps and cotton tips, avoiding damage to the pancreas. The proximal duodenum was ligated twice using a silk suture. A loop gastrojejunal anastomosis was made between the anterior wall of stomach and 15 cm distal to the ligament of Treitz with a single layer of continuous stitches (Supplemental Figure 1). For the sham operation, the stomach was similarly exposed and a 5 mm gastrotomy and antimesenteric jejunotomy were made, on similar sites as the DJB group, but incisions were immediately closed.

Postoperatively, the rats were kept nil per os for 24 hours, but received hydration with 50 mL/kg/day saline subcutaneously for the first 48 h. Feeding was resumed 24 h after the procedure with ad libitum liquid diet of Boost (Nestle, Buffalo Grove, IL), and on postoperative day (POD) 3, the sterilized Purina 5010 diet was resumed until the end of the study. Analgesia was maintained with buprenorphine 0.1 mg/kg every 12 h the day of surgery and POD 1 and 2.

Oral glucose tolerance test

An Oral Glucose Tolerance Test (OGTT) was performed after an overnight fast in awake animals 21 days after diagnosis of diabetes. Briefly, fasting blood glucose was determined (by glucometer on tail vein blood). Then, glucose (2 g/kg body weight) was orally gavaged, followed by measurement of glycemia levels at 10, 30, 60, and 120 min. At baseline and 30 min after glucose load, 200 microliters of blood was drawn from the saphenous vein into a tube that was preloaded with EDTA, DPP4-inhibitor and protease inhibitor cocktail for hormonal assay.

Tissue harvesting and plasma analyses

Twenty-five days after diabetes diagnosis, the rats were euthanized by exsanguination under isoflurane anesthesia after an overnight fast. Blood was collected by cardiac puncture and blood samples were centrifuged at 4000 × g for 10 minutes at 4°C and then the plasma was stored at −80°C for subsequent analysis. Liver and full pancreas were excised, cut into 30–50 mg pieces and snap-frozen in liquid nitrogen and stored at −80°C for subsequent analysis.

Plasma hormone levels were determined using a multispot assay for Mouse/Rat Active GLP-1, Insulin, Glucagon Kit (Meso Scale Discovery K15171C), mouse C-peptide (MSD F203V-3) or Mouse/Rat PYY (MSD F206S-3) according to the manufacturer’s instructions.

Extraction of liver lipids and quantification of total plasma and hepatic triglycerides, cholesterol, and cholesterol esters was conducted using enzymatic assays as described previously [18].

RNA extraction and RT-qPCR

Total RNA was extracted from 20 mg of frozen and ground pancreatic and hepatic tissue using commercial guadinium thiocyanate-phenol reagent, Trizol (Life Technologies, Beverly, MA). The RNA concentration and purity were determined by measuring the absorbance at 230, 260, and 280 nm using NanoDrop ND-1000 Spectrophotomoter (Thermo Scientific, Wilmington, DE). Isolated RNA was aliquoted and stored at −80°C. Five hundred ng of complementary DNA (cDNA) was reverse transcribed using an iScript cDNA synthesis kit (BioRad, Hercules, CA) following the manufacturer’s instructions, and cDNA was stored at −20°C. Determination of relative mRNA expression was performed in duplicate as previously described [19]. Rat glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an internal standard for sample normalization; expression was not influenced by experimental conditions. The relative change in mRNA abundance was calculated using the comparative ΔΔCt method. Gene-specific primers for qRT-PCR analysis are listed in Supplemental Table 1.

Histological evaluation pancreas and liver

The pancreas tail (approximately 1/3 of the total pancreas) was cut into 10 µm cryo-sections at a distance of 100 µm. At least 3 non-overlapping sections were stained for insulin (Cell signaling 3014S), Glucagon (Sigma Aldrich G2654) and DAPI nuclear staining. Micrographs were taken on an inverted fluoromicroscope using automated slide scanning and stitching to obtain scans of the full pancreas section with 10× magnification (Leica, Wetzlar, Germany). All photographed sections were analyzed with Leica Application Suite X software (version 3.011.20652). Liver samples were fixed in formalin, paraffin embedded and 10 µm sections were stained with hematoxylin and eosin (H&E). Photomicrographs were taken of a representative region at 5×, 10×, and 20x× magnification using a confocal microscope (Leica, Wetzlar, Germany) and Leica Application Suite X software (version 3.011.20652). Liver images were captured by selecting representative regions of each sample on each slide for both groups. A nonalcoholic fatty liver disease (NAFLD) scoring system for rodents, modified from the nonalcoholic steatohepatitis (NASH) Clinical Research Network, was used to evaluate liver histology [20]. Specifically, steatosis was evaluated for microvesicular steatosis, macrovesicular steatosis, and hypertrophy by a researcher blinded to the experimental conditions

Fecal pellet collection, DNA extraction, sequencing and bioinformatic analysis

Prior to surgery and on POD 35, fecal pellets were collected from the cage bottom 1 h after placing the animals in a clean cage, and were stored at −80°C. Genomic DNA was extracted using a MoBio Power Soil DNA extraction kit (Omega, Norcross, GA). The 16S rRNA V4 region was amplified and sequenced using protocols described in the Earth Microbiome Project (EMP) [21].

Raw 16S amplicon sequence and metadata were demultiplexed using split_libraries_fastq.py script implemented in QIIME1.9.1 [22]. The demultiplexed fastq file was split into sample specific fastq files using split_sequence_file_on_sample_ids.py script from Qiime1.9.1 [22]. Individual fastq files without non-biological nucleotides were processed using the Divisive Amplicon Denoising Algorithm (DADA) pipeline [23]. The output of the dada2 pipeline (feature table of amplicon sequence variants (an ASV table)) was processed for alpha and beta diversity analysis using phyloseq [24], and microbiomeSeq (http://www.github.com/umerijaz/microbiomeSeq) packages in R. Alpha diversity estimates were measured within group categories using estimate-richness function of the phyloseq package4. Multidimensional scaling (MDS, also known as principal coordinate analysis; PCoA) was performed using Bray-Curtis dissimilarity matrix [25] between groups and visualized by using ggplot2 package [26].

Statistical Analysis

Statistical analyses were performed with GraphPadPrism 8.0.2 for Windows (GraphPad Software, San Diego, CA). Normal distribution was confirmed by Kolmogorov-Smirnov test, prior to data comparison between the DJB and sham groups using paired Student’s t-test. Data are presented as mean±SD. Differences were considered significant at P<0.05.

For the fecal microbiota, statistical significance was assed wherever necessary with p-value adjustment for false discovery rate using the Benjamini and Hochberg method. We performed an analysis of variance (ANOVA) among sample categories while measuring α-diversity measures using the plot-anova-diversity function in the microbiomeSeq package (http://www.github.com/umerijaz/microbiomeSeq). Permutational multivariate analysis of variance (PERMANOVA) with 999 permutations was performed on all principal coordinates obtained during PCoA with the ordination function of the microbiomeSeq package.

Results

The body weight evolution throughout the experiment is shown in figure 2A. Preoperatively, the rats weighed 116±25 g. At the time of sacrifice, the DJB rats weighed less than the sham rats (288.2±44.5 g vs 317.5±27.47 g, P=0.03). By experimental design, there were no differences in food intake (Figure 2B).

Figure 2. Body weight and food intake of BBDP rats after DJB or SHAM surgery.

Figure 2.

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Data are presented as mean±SD, * P<0.05.

Of the 15 pairs of DJB-sham rats, 1 rat of a different pair in each group did not develop diabetes by the age of 120 days. Overall, 93% (28/30) of rats developed T1D in this experiment. There was no difference in the time of diabetes onset between DJB and sham rats, when compared by age (Figure 3A) or by POD (47.0±14.0 vs 41.9±6.4, P=0.24, Figure 3B, n=13 pairs of rats that developed diabetes). The need for exogenous insulin was less in DJB rats, as only 5 DJB rats needed a second insulin pellet to avoid overt hyperglycemia, compared to 11 sham rats (Fisher exact P=0.04). DJB rats needed 1.3±0.4 insulin implants vs 1.9±0.5 in sham rats (P=0.002, Figure 3C)

Figure 3. Diabetes development and glucose tolerance after DJB or SHAM surgery.

Figure 3.

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A: Percentage of non-diabetic rats per group, B: development of diabetes (n=13 per group, as 2 unpaired rats did not develop diabetes), C: Number of insulin pellets implanted, D: Blood glucose during oral glucose tolerance test (OGTT) E: Area under the curve for OGTT. Data are presented as mean±SD, * P<0.05, ** P<0.01.

Upon OGTT 21-days after diagnosis of T1D, while rats were on insulin pellets, we recorded similar glycemia levels for DJB and sham rats at baseline, 10, 30, 60 and 120 min after bolus administration (Figure 3D-E). DJB and sham rats had similar insulin levels at baseline and at the 30 min peak (Figure 4A, F). The C-peptide level was undetectable in all animals, except the one rat in each group that never developed T1D (Figure 4B,G). Serum GLP-1 levels were 1.8 fold higher in DJB rats than in sham rats at baseline, and 1.7 fold higher after the oral glucose load (P=0.046 at baseline, and P=0.042 at 30 min during OGTT, Figure 4C, H). There were no significant differences in PYY or glucagon levels between DJB and sham rats, in fasted or stimulated conditions (Figure 4D, E, I, J).

Figure 4. Glucoregulatory hormones after DJB or SHAM surgery.

Figure 4.

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Fasting plasma concentration of insulin (A), C-peptide (B), Glucagon-like-peptide 1 (GLP-1, C), Peptide YY (PYY, D), and glucagon (E). Plasma concentration of insulin (F), C-peptide (G), GLP-1 (H), PYY (I), and glucagon (J) 30 minutes after oral glucose stimulation (OGTT). Data are presented as mean±SD, * P<0.05.

Immunohistological assessment of pancreatic tissue did not show insulin-positive islets cells in the pancreases from animals that had become diabetic, while islets were clearly identifiable in 2 non-diabetic rats (Figure 5A-B). Congruently, insulin (Ins2) mRNA was only detectable in pancreas of non-diabetic rats (Figure 5C). There were no significant differences in glucagon mRNA levels between DJB and sham (Figure 5D).

Figure 5. Pancreatic islets after DJB or SHAM surgery.

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Representative micrograph of pancreas tissue of a DJB rat that did not develop T1D showing abundant insulin-positive beta cells (in yellow) in islets of Langerhans (A), while no islets could be identified in DJB or SHAM rats that developed diabetes (B). Relative mRNA expression of Insulin 2 (Ins2, panel C) and glucagon (Gcg, panel D) in SHAM and DJB rats (no significant differences).

Triglyceride and cholesterol levels were measured in both hepatic tissue and serum. There was no difference in triglyceride content between DJB and sham rats (Figure 6A-B), but the cholesterol content in DJB livers was only half of that in sham livers (P<0.0001, Figure 6C and serum cholesterol was 16% lower in DJB rats compared with sham rats (P=0.038, Figure 6D).

Figure 6. Lipid parameters in liver and serum after DJB or SHAM surgery.

Figure 6.

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Triglycerides (TG, panel A,B) and total cholesterol (panel C,D). Data are presented as mean±SD, * P<0.05, *** P<0.001.

Histological analysis of liver samples was evaluated using the modified NAFLD scoring system for rodents. There was no evidence of hepatic steatosis to suggest NAFLD in either the sham or DJB liver specimens (Supplemental Figure S2), nor could we detect differences in mRNA expression of inflammatory markers including Il1-beta, Tnf-alpha, and Mcp-1 in liver tissue in DJB or sham rats (Figure 7 A-C). However, there was a trend towards reduced expression of Fgf21, a known marker of metabolic dysregulation, in DJB rats (P=0.057 vs sham) and several markers of endoplasmic reticulum stress, including Grp78, Atf4, Xbp1s, and Chop were approximately 50% lower in liver tissue of DJB rats compared to the sham group (Figure 7D-H).

Figure 7. Markers of hepatic inflammation and endoplasmic reticulum stress after DJB or SHAM surgery.

Figure 7.

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Expression of mRNA of interleukin-1 beta (IL-1b, panel A), tumor necrosis factor-alpha (Tnf-a, panel B), macrophage chemoattractant protein-1 (Mcp1, panel C), fibroblast growth factor-21 (Fgf21, panel D), glucose-regulated protein 78kD (Grp78, panel E), activating transcription factor 4 (Atf4, panel F), X-box binding protein 1-spliced (Xbp1s, panel G) and CCAAT-enhancer-binding protein homologous protein (Chop, panel H) relative to glyceraldehyde-3-phosphate dehydrogenase expression in liver tissue after DJB or SHAM surgery. Data are presented as mean±SD, * P<0.05, *** P<0.001.

The fecal microbiota did not differ in terms of alpha diversity between DJB and sham animals. However, there was a significant (PERMANOVA: P<0.05 using Bray–Curtis dissimilarity indices) difference in the beta diversity in postoperative DJB rats compared with preoperative and postoperative sham rats (P=0.001, Figure 8). At the genus level in the taxonomic classification scheme, there were notable changes in the microbiome after both sham and DJB procedures (Figure 9). After DJB intervention, there was a marked increase in Defluviitaleaceae, Enterococcus, Esherichica/Shigella, Provotellaceae, Ruminococcaceae_UCG-005, and Akkermansia, while Alloprevotella, Bilophila, Desulfovibrio, Faecallbaculum, Flavonifractor, Lactobacillus, Parasutterella, Peptococcus, Rikenellaceae, Streptococcus, Oscillibacter, and Tyzzerella all decreased after DJB surgery (Supplemental Figure S3). In sham rats, Esherichica/Shigella also increased after surgery and we noted a decrease in Anaerofustis, Anaerotruncus, Bifidobacterium, and Faecalibaculum. (Supplemental Figure S4).

Figure 8. Fecal microbiota after DJB or SHAM surgery.

Figure 8.

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Beta diversity (panel A), relative abundance at the genus level (panel B).

Figure 9. Fecal microbiota after DJB or SHAM surgery.

Figure 9.

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Relative abundance at the phylum level.

Discussion

This is the first detailed assessment of the effects of metabolic surgery on the development of autoimmune diabetes. We used the BBDP/Wor rat model as it closely resembles the auto-immune destruction of islets, as seen in T1D [15] and the rat model of gastrointestinal surgery, which is well established in our lab [27, 28]. In contrast to our hypothesis, DJB surgery did not prevent or delay development of T1D. The need for exogeneous insulin was lower, potentially through reduced insulin resistance, as there was no evidence of preserved beta cell function or mass in the C-peptide measurements, insulin gene expression or pancreatic histology. Moreover, several other beneficial metabolic effects of metabolic surgery were observed including weight loss, increased GLP-1 secretion, and reduced hepatic ER stress.

These findings fit the observational data in human T1D that underwent metabolic surgery, where effects on glycemic status are modest at best [29–31], but weight loss in severely obese patients with T1D is significant and results in a significant reduction in insulin requirements [9, 32]. Patients with type 2 diabetes and absent or low residual beta-cell function also have a reduced likelihood of diabetes remission [33–35]. However, the results from the current study are somewhat in contrast to the data presented by Breen et al. who reported lowered glucose concentrations immediately after DJB surgery (on PODs 2 and 6) in the BBDP rats that had been diabetic preoperatively [36]. Differences in experimental design, including the focus on “treatment” is clearly different from our “protection” approach, and only reporting the immediate postoperative time point may also help to explain the divergent results.

The current study was not primarily designed to study the effects of metabolic surgery on NAFLD and insulin resistance, hence we did not use a high fat diet. Unsurprisingly, histological evaluation showed normal liver tissue without hepatic steatosis or inflammation in both sham and DJB rats, and there were no differences in hepatic triglyceride content between the two experimental groups. However, the key markers of ER stress were reduced after DJB surgery compared with sham, congruently with previous data after weight loss interventions [37]. We also observed that total cholesterol levels in liver tissue and plasma were reduced after DJB, suggesting that a metabolic benefit in liver metabolism is to be expected after surgically-induced weight loss.

Finally, we compared the fecal microbiota before and after sham and DJB surgery. Changes in the fecal microbiome after sham surgery were very limited, confirming the validity of the sham surgery as a control intervention. The noted increase in Escherichica/Shigella has been observed after most gastrointestinal surgical interventions [38–40]. In contrast, after DJB the beta diversity was significantly different from preoperative samples. Moreover, several of the observed changes in microbiota after DJB surgery have been associated with beneficial outcomes in human and animal studies. For instance, the increase in relative abundance of Gammaproteobacteria and Verrucomicrobia including Akkermansia has been observed in humans [41], and in a key murine study that showed weight loss and a decreased fat mass after transfer of the gut microbiota from RYGB-treated mice to non-operated, germ-free mice [42]. The postoperative decrease of Lactobacilli and Parasuttella is intriguing, as it suggests they may be effects on gut permeability [43] and gut autoimmunity [44].

This study has several limitations. First, as in any animal study, the translatability of our findings to human patients is uncertain, even though the BBDP/Wor rat model has been validated and is a widely used model for autoimmune T1D. Our data should ideally be confirmed in human patients, but a prospective study of metabolic surgery in T1D patients is challenging because of the low number of potential candidates. Moreover, direct and accurate assessment of beta cell mass in humans is still not possible. Second, although we tried to perform surgery at the young age, earlier intervention could have yielded different results. In our experiment, the interval between surgery and diabetes onset was about 40–45 days. One can speculate that a diabetes prevention effect of surgery might occur if surgery would have been done earlier. We were logistically restricted to do the surgery earlier by the allowed shipping age of rats, and the mandatory 2-week quarantine period in our facility before starting the experiment. Third, we chose to study islet mass and function 25 days after diabetes diagnosis, and not immediately after development of T1D. As discussed above, it is possible that earlier after surgery, the DJB intervention may have positive effects on the beta cells. However, if so, such effect was at least not sufficient to delay the clinical diagnosis of diabetes in this study. Forth, the OGTT data were interpreted while the rats had insulin implants which may impact the findings. Fifth, although we assessed liver tissue, other metabolically active organs such as fat and muscle were not studied as they were outside the scope of the current study.

In conclusion, DJB surgery does not protect against autoimmune T1D in BBDP/Wor rats, but reduces the need for exogenous insulin and facilitates other metabolic benefits including weight loss, increased GLP-1 secretion, reduced hepatic stress, and altered gut microbiome.

Supplementary Material

supplement table S1

Supplemental Table S1. Primers used for RT-qPCR

2

Supplemental Figure S1. Schematic of loop (single anastomosis) duodenojejunal bypass (DJB) surgery

In the loop DJB surgery, the pylorus and the first part of duodenum are dissected bluntly using curved forceps, avoiding damage to the pancreas. First part of the duodenum is ligated twice using silk suture. A Loop gastrojejunal anastomosis is made between anterior wall of stomach and 15 cm distal to the ligament of Treitz with a single layer of continuous stitches.

Supplemental Figure S2. Liver histology

Representative micrographs of H&E stained liver sections showed no steatosis or inflammation after SHAM (A) or DJB (B) surgery. Magnification 20×.

Supplemental Figure S3. Significant differences in abundance pre vs post DJB intervention

Supplemental Figure S4. Significant differences in abundance pre vs post sham intervention

Acknowledgements

This study was primarily supported by the SAGES General Research Grant, and in part by grants from the National Institutes of Health including R01 HL122283 (J.M.B.), R01 DK120679 (J.M.B.), P50 AA024333 (J.M.B), and P01 HL147823 (J.M.B.). R.V. was supported by a fellowship of the Fulbright Commission Belgium and a fellowship of the Belgian American Educational Foundation.

Footnotes

Disclosures

The authors have no direct conflicts of interest or financial ties to disclose.

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Associated Data

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

Supplementary Materials

supplement table S1

Supplemental Table S1. Primers used for RT-qPCR

NIHMS1674739-supplement-supplement_table_S1.xlsx (10.1KB, xlsx)
2

Supplemental Figure S1. Schematic of loop (single anastomosis) duodenojejunal bypass (DJB) surgery

In the loop DJB surgery, the pylorus and the first part of duodenum are dissected bluntly using curved forceps, avoiding damage to the pancreas. First part of the duodenum is ligated twice using silk suture. A Loop gastrojejunal anastomosis is made between anterior wall of stomach and 15 cm distal to the ligament of Treitz with a single layer of continuous stitches.

Supplemental Figure S2. Liver histology

Representative micrographs of H&E stained liver sections showed no steatosis or inflammation after SHAM (A) or DJB (B) surgery. Magnification 20×.

Supplemental Figure S3. Significant differences in abundance pre vs post DJB intervention

Supplemental Figure S4. Significant differences in abundance pre vs post sham intervention

NIHMS1674739-supplement-2.pdf (712.2KB, pdf)

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