Introduction
Diabetes has become an epidemic disease in the twenty-first century. The International Diabetes Federation estimated that the global diabetes prevalence was 285 million in 2010.
1 Moreover, over 4 million people die due to complications associated with the disease every year. Although initiatives should be pursued to prevent the onset of type 2 diabetes, effective treatment must be available for people who develop type 2 diabetes.
2 Traditional treatment modalities for diabetes do not satisfactorily control the disease or its complications,
3 and current emerging treatments remain in the exploratory or extension phase. Therefore, discovering new treatments has been the focus of considerable medical research. Roux-en-Y gastric bypass (RYGB) is a novel approach among the available bariatric surgery procedures and has been utilized in type 2 diabetes with a good outcome,
4,
5 but the therapeutic mechanisms have remained unclear, and various hypotheses have been debated.
6‐
9 Surgical indication has been restricted, and the surgical efficacy is not completely satisfactory.
10 Therefore, studying the mechanism through which RYGB alleviates type 2 diabetes is a critical theoretical and clinical research endeavor. Our study explored the therapeutic mechanism of RYGB in diabetes using an animal model. In this study, we investigated the impact of RYGB on not only diabetic rats but also healthy rats. By analyzing the similarities and differences in the impact of RYGB on diabetic rats and healthy rats, we found that RYGB promoted the cellular proliferation of islets and improved the function of beta cells, ultimately leading to insulin secretion enhancement.
Materials and Methods
Experimental Animals
Five-week-old male Wistar and Goto-Kakizaki rats were purchased from SLAC Laboratory Animals Co. Ltd. (Shanghai, China). Wistar rats were chosen as the healthy rat model, and Goto-Kakizaki rats were chosen as the non-obese type 2 diabetic rat model. Goto-Kakizaki rats are spontaneous diabetic rats that were developed by the selective inbreeding of Wistar rats.
11,
12 All rats were treated according to the approved protocols of the Tsinghua University Animal Care and Use Committee. Wistar rats (
n = 20) were randomly assigned to a control cohort of healthy rats (WT,
n = 10) or an operation cohort of healthy rats (WTO,
n = 10). Goto-Kakizaki rats (
n = 20) were also randomly assigned to a control cohort of type 2 diabetic rats (GK,
n = 10) or an operation cohort of type 2 diabetic rats (GKO,
n = 10). After the rats had adapted to the environment for 1 week, the operation cohorts underwent RYGB.
13‐
15 The rats were anesthetized with 1 % pentobarbital sodium (30 mg/kg) through intraperitoneal injection. The RYGB procedure included several key steps. (1) Approximately 20 % of the original gastric volume was preserved. (2) The distal jejunum was connected via an end-to-end anastomosis to the preserved stomach after the jejunum was transected 8 cm distal to the ligament of Treitz. (3) The proximal jejunum was connected via an end-to-side anastomosis to the jejunum 10 cm distal to the gastrojejunal anastomosis.
Pancreatic Islet Isolation and Beta Cell Culture
1.
Pancreatic islet isolation: The rats were dissected 4 weeks after RYGB, and the islets were isolated. First, the pancreaticobiliary duct was retrograde intubated and clamped. Next, 10 ml collagenase V was infused in the duct. Then, the pancreas was resected and digested for 25 min. After the digestion was terminated, single islets were isolated under a stereomicroscope.
16
2.
Beta cell culture: The islets were collected in a centrifuge tube, and a 3-ml trypsin solution was added. Then, the centrifuge tube was gently shaken for 3 min. Next, 1 ml of media was added to the tube. The centrifuge tube was centrifuged at 500 rpm for 5 min. Then, the supernatant was removed. Finally, 2 ml of culture medium was added to the centrifuge tube. The medium containing 60–70 % beta cells was transferred to a dish, and the cells were cultured in an incubator.
Measurement of Blood Glucose, Food Intake, and Body Weight
At 4 weeks after RYGB, the rats were fasted for 6 h, their blood glucose was measured by the electrochemical method, and their body weight was measured using an electronic balance. The 24-h spontaneous food intake of rats was measured on three consecutive days using an electronic balance 4 weeks after RYGB.
Expression Profiling
All GeneChips were processed at the CapitalBio Corporation (Beijing). For gene expression analyses, a total of 600 (60 islets/rat,
n = 10) freshly isolated islets were collected from each cohort. First, total RNA was extracted from each cohort sample using Trizol reagent. Then, the RNA concentration was estimated by measuring the absorbance at 260 nm. The RNA quality was verified by electrophoresis on ethidium bromide-stained 1.5 % agarose gels and A260 nm/A280 nm ratios >1.8. A total of 500 ng of total RNA was synthesized as the transcription cRNA probe in vitro using the BioArray
™ High Yield
™ RNA Transcript Labeling Synthesis Kit (Affymetrix) according to the manufacturer’s instructions. Next, hybridizations were set up. cDNA targets labeled with Cy5 (experimental) and Cy3 (reference) were combined and hybridized to microarrays for 14 to 18 h (overnight) at 42 °C. Following hybridization and washing, the membranes were scanned using a Storm 840 Scanner (Molecular Dynamics).
17 Differentially expressed genes were selected based on an analysis of the fold change. The selection criteria for differentially expressed genes included a log ratio ≥2.0 for an upregulated gene and a log ratio ≤0.5 for a downregulated gene. The molecular function and biological processes of differentially expressed genes were analyzed using Molecule Annotation System 3.0 (MAS 3.0). The Gene Chip Rat Genome 230 2.0 Array was purchased from Affymetrix (USA).
Measurement of Hormone Levels
At 4 weeks after RYGB, hormone levels were measured at room temperature using ELISA kits and a multilabel reader (EnVision, PerkinElmer). After the rats were fasted for 6 h, blood was collected from the orbital venous plexus. The tubes for blood samples of glucagon-like peptide 1 (GLP-1) contained 10 μl dipeptidyl dipeptidase IV inhibitor. After blood samples stood for 30 min, the tubes were centrifuged to obtain the serum, and the hormone levels were measured. Rat insulin, ghrelin, and active GLP-1 (7–36) ELISA kits were purchased from Millipore (Billerica MA, USA).
Measurement of Intracellular Calcium and Insulin Concentration
At 4 weeks after RYGB, the fluorescence intensities of beta cells were measured using confocal microscopy (Zeiss 710 META, GER).
18 The cells were seeded in a dish 1 day before the experiment. After loading the cells with 2 μmol/L fluo-4/AM and 2 μmol/L FITC in RPMI-1640 for 30 min, they were washed three times with Hanks solution. Then, the cells were bathed in RPMI-1640 with 8 mmol/L glucose solution, and measurements were obtained. Beta cells showed red fluorescence in the FITC channel and green fluorescence in the fluo-4/AM channel. The Ca
2+ and insulin fluorescence intensities were recorded at 20-s intervals 400 s after fluorescence intensity stabilization.
F
0 was the initial value of fluorescence intensity after fluorescence intensity stabilization. Changes in the Ca
2+ and insulin fluorescence intensity (Δ
F) were calculated using the following equation: Δ
F =
F
n −
F
n − 1. Axio Vision Rel.4.7 software was used to analyze the images of the cells.
Electrophysiology of Calcium Channels
At 4 weeks after RYGB, whole-cell patch-clamping was performed at room temperature using an EPC-10 patch-clamp amplifier and PatchMaster software (HEKA, Germany). Beta cells grown in vitro were perfused with a standard external solution containing the following (in millimoles per liter): 120 NaCl, 3
d-glucose, 1 MgCl
2, 4 CsCl, 20 TEA.CL, 2.5 CaCl
2, and 10 Hepes, with the osmolarity adjusted to approximately 310 mOsm (pH 7.4). This solution was further supplemented with 1 μmol/L tetrodotoxin and 100 μmol/L DIDS. The patch pipettes were pulled from borosilicate glass and fire polished. The pipette resistance ranged between 3 and 5 MΩ after they were filled with the intracellular solution for voltage-dependent calcium channels (VDCCs) to deplete calcium stores and induce stored operated calcium (SOC) influx. The intracellular solution contained the following (in millimoles per liter): 10 Hepes, 125 CsCl, 1 MgCl
2. 10 EGTA (pH 7.2) , and 2 μmol/L TG. In all whole-cell experiments, the recording began with a series of resistance values below 20 MΩ. Cells with a capacitance of >5 PF and a diameter of 11–12 μm were most likely to be pancreatic beta cells.
19 Voltage-dependent currents were corrected for linear leakage and residual capacitance using on-line P/n subtraction. The beta cells were held at −70 mV. For the normalized current recordings, the peak currents were measured at 0 mV. For the detection of SOC influx, voltage ramps of 100 ms spanning a range of −100 to 100 mV were delivered from a holding potential of 0 mV every 2 s over 600–1,000 s. A maximal current of −100 mV was used for the statistical analysis.
20 All voltages were corrected for a liquid-junction potential of 13 mV. Capacitance currents were determined and automatically compensated for using the EPC-10 amplifier. Data were analyzed using IGOR Pro 6.20 (Wavemetrics, USA).
Morphology Techniques
1.
At 4 weeks after RYGB, small samples of fresh pancreatic tissue were collected from the pancreas of each rat. Then, the pancreatic tissue was fixed, embedded in paraffin, and continuously cut into 5-μm sections. After sections that were a certain distance apart were selected, the sections were stained with HE and imaged with light microscopy (IX71-A12FL/PH Olympus, Japan).
2.
At 4 weeks after RYGB, a total of 300 (30 islets/rat,
n = 10) freshly isolated islets were isolated from each cohort and conventionally fixed. Then, the islets were dehydrated in ethanol, embedded in Epon-Araldite, strained with toluidine blue, and cut into ultrathin sections. The samples were observed with transmission electron microscopy (Hitachi 7650, Japan).
21 Image-Pro PluS6.0 software was used to analyze the images.
Reagents
Collagenase V, FITC, trypsin, and Trizol were purchased from Sigma (USA). Fluo-4/AM was purchased from Invitrogen (USA). Thapsigargin was purchased from Merck (GER).
Statistical Analysis
Measurement data were expressed as the mean ± SD, and count data were expressed as percentage differences. The data were analyzed with a repeated-measures one-way ANOVA. P < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 18.0 statistical software.
Discussion
RYGB has a novel therapeutic value for type 2 diabetes. However, the therapeutic mechanism remains unclear. Using an animal model, this study explored the therapeutic mechanism of RYGB in diabetes.
First, an RYGB animal model was established. Then, the therapeutic efficacy of animal model was evaluated. The results confirmed that the model could be applied for exploring the therapeutic mechanism of RYGB.
Next, the therapeutic mechanism of RYGB in diabetes was explored. First, the gene expression profile of islets was analyzed. The gene expression profile can show changes in genome-wide expression. And the physiological dysfunction of islets is the basis of diabetes. Thus, valuable information can be acquired through studying the gene expression profiles of islets. The result showed that after RYGB, a new metabolic environment was present, and the gene expression in islets was changed to adapt to the new metabolic environment. Furthermore, 13 differentially expressed genes that were found were closely related to the RYGB procedure and diabetes metabolism. And the molecular functions and biological process of the genes were mainly related to hormones, calcium signals, and cellular proliferation by gene ontology analysis.
22‐
27 Further research effects of RYGB on hormones, calcium, and cellular morphology could provide more information.
As messengers that transmit information, hormones play an important regulatory role in the physiological processes of the body. Insulin is an important factor that affects the balance of the body’s glucose metabolism. The results showed that RYGB increased serum insulin levels. Furthermore, HOMA-β was used to evaluate the level of insulin secretion.
28 HOMA-β in diabetic rats increased after RYGB. These findings suggested that the underlying therapeutic mechanism of RYGB was enhanced insulin secretion. Previous studies have also reported that RYGB leads to a marked improvement in glucose control, in which gastrointestinal hormones play a role.
15 Additionally, researchers have proposed various hypotheses regarding gastrointestinal hormones.
29,
30 This study investigated gastrointestinal hormones, including ghrelin and GLP-1. Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor.
31,
32 Ghrelin can be expressed in alpha cells and beta cells of islets, and it is not only directly involved in insulin secretion but also determines the differentiation of islet cells. GLP-1 is an important and extensively studied incretin.
33,
34 GLP-1 can stimulate insulin secretion and enhance beta cell proliferation. A synthetic analogue of GLP-1 (exendin-4) has been successfully introduced to treat diabetes patients.
35 Our results showed that RYGB promoted the secretion of GLP-1 and decreased ghrelin levels. Based on the effects of RYGB on the cellular morphology and hormone levels, and the production location of ghrelin and GLP-1, we speculated that promoting cellular proliferation and improving cellular function are two of the roles that gastrointestinal hormones play in the therapeutic mechanism of RYGB in diabetes.
And previous researchers have proposed that ghrelin and GLP-1 regulate insulin secretion by affecting the calcium signaling pathway. Intracellular free Ca
2+ not only triggers secretion
36 but is also involved in the regulation of exocytosis.
37,
38 Fluorescence labeling may monitor the continuous changes in concentration. Fluo-4 is a highly specific Ca
2+ fluorescence probe, and changes in its fluorescence intensity can sensitively reflect changes in the concentration of intracellular free Ca
2+. Under appropriate conditions, FITC can bind to insulin. The changes in intracellular fluorescence intensity indirectly indicate the cells’ ability to secrete insulin. Labeling calcium and insulin can effectively reflect the calcium signals and insulin secretion in beta cells. The results showed that RYGB increased the concentration of intracellular free Ca
2+ and insulin secretion, and the change in intracellular free Ca
2+ was closely related to insulin secretion after RYGB. Thus, Ca
2+ signal played an important role in the RYGB effect on diabetes. RYGB caused an increase in the concentration of intracellular free Ca
2+, which promoted insulin secretion. Our group further studied Ca
2+ channels and attempted to elucidate the mechanism through which RYGB increased intracellular free Ca
2+. The intracellular free Ca
2+ concentration is maintained at a low level. Calcium influx across the cell membrane via a Ca
2+ channel increases the Ca
2+ concentration within the cytosol. The Ca
2+ channels in beta cell membranes are mainly VDCCs, and SOC also exists.
39 SOC may be involved in Ca
2+ signal transmission.
40 The result showed that RYGB had little effect on VDCC activity. However, RYGB significantly enhanced SOC activity. The finding not only elucidates the mechanism through which RYGB elevated intracellular free Ca
2+ but also provides new ideas for the drug treatment of diabetes.
Cellular structure and function are intricately related. After RYGB, the number of small islets and secretory vesicles was increased. The results supported that RYGB could promote the differentiation of primitive cells into islets cells, and improve the function of beta cells. RYGB increased the quantity and improved the quality of insulin secretion cells. These morphological changes were the result of hormone adjustment and calcium signal changes. At the same time, these morphological changes were the basis of insulin secretion improvement.
In summary, we hypothesize that RYGB led to the creation of a new metabolic environment and triggered a series of changes, primarily in hormone levels and Ca2+ signaling, to adapt to this new environment. These adjustments further induced the cellular differentiation of primitive cells into islets cells and improved the function of beta cells. RYGB increased the quantity and improved the quality of insulin secretion cells. RYGB ultimately led to enhancing insulin secretion to treat diabetes. The study of the mechanism through which RYGB alleviates type 2 diabetes is a critical theoretical and clinical research endeavor. The study is a novel approach to determining the mechanism of RYGB. There are more parallel and intersecting comparisons between diabetic rats and healthy rats and the operation cohort and control cohort.
However, the animal model cannot be fully compliant with human type 2 diabetes characteristics. And it is difficult to obtain a large number of high-quality islets. The long-term effects of RYGB have not been explored. Thus, our next study will aim to elucidate the mechanism of long-term effects of RYGB on rats.