«Nutrients 2015, 7, 4689-4704; doi:10.3390/nu7064689 OPEN ACCESS nutrients ISSN 2072-6643 Article Sesamin Ameliorates ...»
Nutrients 2015, 7, 4689-4704; doi:10.3390/nu7064689
Sesamin Ameliorates Advanced Glycation End
Products-Induced Pancreatic β-Cell Dysfunction and Apoptosis
Xiang Kong 1,2,†, Guo-Dong Wang 3,†, Ming-Zhe Ma 4,†, Ru-Yuan Deng 1, Li-Qun Guo 2,
Jun-Xiu Zhang 2, Jie-Ren Yang 2,* and Qing Su 1,* Department of Endocrinology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China; E-Mails: email@example.com (X.K.);
firstname.lastname@example.org (R.-Y.D.) Department of Pharmacology, Wannan Medical College, Wuhu 241002, China;
E-Mails: email@example.com (L.-Q.G.); firstname.lastname@example.org (J.-X.Z.) Department of Pharmacy, Wannan Medical College, Wuhu 241002, China;
E-Mail: email@example.com Department of General Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China; E-Mail: firstname.lastname@example.org † These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: email@example.com (J.-R.Y.);
firstname.lastname@example.org (Q.S.); Tel.: +86-553-3932464 (J.-R.Y.); +86-21-25077530 (Q.S.).
Received: 21 April 2015 / Accepted: 1 June 2015 / Published: 9 June 2015 Abstract: Advanced glycation end products (AGEs), the direct modulators of β-cells, have been shown to cause insulin-producing β-cell dysfunction and apoptosis through increase of intracellular reactive oxygen species (ROS) production. Sesamin has been demonstrated to possess antioxidative activity. This study was designed to investigate whether sesamin protects against AGEs-evoked β-cell damage via its antioxidant property. The effects of sesamin were examined in C57BL/6J mice and MIN6 cell line. In in vivo studies, mice were intraperitoneally injected with AGEs (120 mg/kg) and orally treated with sesamin (160 mg/kg) for four weeks. Intraperitoneal glucose tolerance and insulin releasing tests were performed. Insulin content, ROS generation and β-cell apoptosis in pancreatic islets were also measured. In in vitro studies, MIN6 cells were pretreated with sesamin (50 or 100 μM) and then exposed to AGEs (200 mg/L) for 24 h. Insulin secretion, β-cell death, ROS production as well as expression and activity of NADPH oxidase were determined. Sesamin treatment obviously ameliorated AGE-induced β-cell dysfunction and apoptosis both in vivo Nutrients 2015, 7 4690 and in vitro. These effects were associated with decreased ROS production, down-regulated expression of p67phox and p22phox, and reduced NADPH oxidase activity. These results suggest that sesamin protects β-cells from damage caused by AGEs through suppressing NADPH oxidase-mediated oxidative stress.
Keywords: sesamin; advanced glycation end products; MIN6 cell; reactive oxygen species; apoptosis
1. Introduction Diabetes mellitus (DM) is characterized by hyperglycemia and is becoming a public health problem of considerable magnitude. Advanced glycation end products (AGEs) are generated from nonenzymatic glycoxidation of proteins and lipids, and the formation of AGEs is greatly accelerated by prolonged hyperglycaemia in patients with DM [1–3]. Increasing evidence indicates that the accumulation of AGEs play a critical role in the pathogenesis of diabetic complications [4,5]. Interestingly, recent studies have pointed out that AGEs directly cause insulin-producing β-cell dysfunction and apoptosis in vivo, and contribute to the development of DM [6–8]. Furthermore, the AGE-induced β-cell damage has been demonstrated to be associated with the increase in intracellular reactive oxygen species (ROS) production in our previous studies [9,10] and other reports [7,11,12].
Sesamin is a natural lignan found in sesame oil and seeds, and exerts a variety of pharmacological properties. Both clinical and experimental studies have illustrated the lipid-lowering, antioxidative and antihypertensive effects of sesamin [13–18]. A recent study has shown that sesamin ameliorates the insulin resistance in KK-Ay mice, a kind of type 2 DM model . However, to the best of our knowledge, no studies have been conducted on the effects of sesamin on AGE-induced β-cell lesion.
Accordingly, the current study was designed to investigate whether sesamin could protect against AGE-induced β-cell dysfunction and apoptosis both in vitro and in vivo, and to further clarify whether the protective effects of sesamin were attributed to its antioxidant properties.
2. Materials and Methods
2.1. Drugs and Reagents All of the reagents were purchased from Sigma unless stated otherwise. Sesamin (94% purity) was provided by Tianyi Lvbao Technology Co. (Wuhu, China). Its structure and invention patent number were described in our previous study . Mouse insulin ELISA Kit was purchased from Shibayagi Co.
(Shibukawa, Japan). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Cell Death Detection ELISAPlus assay kits were purchased from Roche CO. (Indianapolis, USA).
2’,7’-dichlorodihydrofluorescin diacetate (DCFH-DA) and dihydroethidium (DHE) probes were purchased from Beyotime Biotechnology Inc. (Nantong, China). Polyclonal rabbit anti-insulin antibody was purchased from Abcam Inc. (Cat No.: ab63820, Cambridge, UK). Polyclonal rabbit anti-nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits p22phox (Cat No.: sc-20781) and p67phox (Cat No.: sc-15342) antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).
Nutrients 2015, 7 4691 Polyclonal rabbit anti-cleaved caspase 3 (Cat No.: AC033) and poly (ADP-ribose) polymerase (PARP, Cat No.: AP102) antibodies as well as monoclonal mouse anti-tubulin antibody (Cat No.: AT819) were purchased from Beyotime Biotechnology Inc. (Nantong, China).
2.2. Preparation and Validation of AGEs
AGEs were prepared as described by Makita et al. . In brief, 50 mg/mL of bovine serum albumin (BSA) were incubated under sterile condition with 0.1 M of glyceraldehyde in 0.2 M of phosphate buffer (pH 7.4) for seven days. The unincorporated sugar was removed by dialysis. Non-glycated BSA was incubated under the same condition except for the absence of glyceraldehyde as a negative control. The content of AGEs was determined using the standard spectrum λex 370 nm/λem 450 nm with a fluorence microplate reader. The AGE preparation was tested for endotoxin using a limulus amebocyte lysate reagent (Associates of Cape Cod, Inc., East Falmouth, MA, USA) and was confirmed to have an endotoxin level of less than 15 EU/L. Furthermore, the prepared AGEs and BSA were assessed for changes in molecules by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% gel and Coomassie Blue staining.
2.3. Laboratory Rodent Studies
Ten-week-old male C57BL/6J mice were obtained from SLAC Laboratory Animal Co. (Shanghai, China). All of the mice were maintained in specific pathogen-free facilities at the experimental animal center of Xinhua Hospital. All studies were approved by the Institutional Animal Care and Use Committee (IACUC) at the Xinhua Hospital, Shanghai Jiaotong University School of Medicine.
All mice received humane care in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996).
Mice were separated into the following groups (n = 6 each group): BSA control group, AGE model group and sesamin treatment group (AGEs plus sesamin, given sesamin by gavage at the daily dose of 160 mg/kg body weight). BSA or AGEs were administered intraperitoneally daily for 4 weeks with the dosage (120 mg/kg body weight) according to a previous report . Based on previous studies, the dosage of sesamin has the sufficient in vivo antioxidant capacity [13,17]. During the entire period of the experiment, body weight (BW) and food consumption were measured weekly.
2.4. Intraperitoneal Glucose Tolerance Test (GTT), Insulin Releasing Test (IRT) and Intraperitoneal Insulin Tolerance Test (ITT) An intraperitoneal GTT was used to assess glucose tolerance. After fasting overnight, mice were intraperitoneally injected with 10% glucose solution (1.5 mg/g body weight), and glucose levels were determined at 0, 30, 60, 90, and 120 min by a glucometer. For measuring glucose stimulated insulin secretion, blood was collected at 0, 30, and 60 min after glucose loading, and insulin levels were determined using the ELISA Kit.
After 6 h fasting, ITT was performed in mice. Blood samples were collected from tails before and 15, 30, 45, and 60 min after an intraperitoneal injection of human regular insulin (0.75 U/kg).
Glucose levels were measured using a glucometer.
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2.5. Immunofluorescent Staining for Insulin in Mice Pancreatic Islets
A part of the pancreas was fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm sections. For immunofluorescent staining, fixed pancreatic sections were heated for 15 min in boiling 10 mM citrate buffer for antigen retrieval. Sections were subsequently probed with anti-insulin antibody (1:200), followed by incubation with specific secondary antibodies. Nuclear staining was achieved by incubating with DAPI. Sections were photographed by fluorescent microscopy and analyzed using Image J software as described in our previous report . Briefly, mean fluorescence intensity (MFI) of insulin staining, reflecting the insulin content, was quantified in ten randomly selected pancreatic islets of each mouse. Data were pooled to calculate a mean value, and a statistical analysis was applied to compare the results obtained from different experimental groups.
2.6. Assessment of Apoptosis in Mice Pancreatic Islets
The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit was used to evaluate the islet apoptosis. Briefly, 5 μm sections were incubated with TUNEL reagent for 1 h in dark.
After rinsing with PBS, the 3,3’-diaminobenzidine reagent was added. In addition, the positive control includes permeabilization of sections with deoxyribonuclease 1 to induce DNA strand breaks. The slides were mounted and photographed under light microscope. The TUNEL positive cells and total islet cells were counted manually in ten randomly selected islets of each mouse under a 20× objective microscope by an investigator who was blinded to the experiments. The results were adjusted for β cell area in serial sections stained with insulin and used to calculate the TUNEL positive β cells per islets.
2.7. In Situ Detection of ROS Production in Mice Pancreatic Islets
A part of the pancreas was included in O.C.T. embedding medium. Sections of 10 μm were obtained in a freezing microtome. Sections were incubated for 30 min with 10 μM DHE to evaluate pancreatic superoxide anion levels in situ. DHE is oxidized by superoxide anion to yield ethidium, which stains DNA. Sections were photographed and MFI of DHE was quantified in ten randomly selected pancreatic islets of each mouse with Image J software. Data were pooled to calculate a mean value and the results were statistically evaluated.
2.8. Cell Culture and Treatment
The pancreatic MIN6 β-cell line was a gift from the Institute of Endocrinology of Rui Jin Hospital affiliated with the Shanghai Jiao Tong University School of Medicine. As described in our previous study , MIN6 cells (15–30 passages) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS, 50 μM 2-mercaptoethanol, 100 U/mL penicillin, and 100 μg/mL streptomycin.
MIN6 cells were grown to confluence, pretreated with sesamin (50 or 100 μM) for 2 h, and then stimulated with AGEs (200 mg/L) for 24 h.
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2.9. Measurement of Insulin Secretion in MIN6 Cells After treatment, the MIN6 cells were starved in Krebs buffer containing 0.2% bovine BSA with 3 mM glucose for 30 min. The cells were then incubated in Krebs buffer with 3 or 25 mM glucose for 60 min. An aliquot of the buffer was taken and insulin release was measured by ELISA.
2.10. Western Blot Analysis As described in our previous study , equal amounts of proteins were applied to a 10% or 12% SDS-polyacrylamide separating gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). After blocking with 5% skim milk or 1% BSA in Tris buffered saline with Tween 20 (TBST) at room temperature for one hour, the membrane was incubated with the primary antibodies against cleaved caspase 3 (1:500), PARP (1:800), NADPH oxidase subunit p22phox (1:400), or p67phox (1:250) overnight. After washing the membranes three times, the immunoblots were incubated with the appropriated secondary antibodies for 2 h. Antibody-bound proteins were detected by Millipore enhanced chemiluminescence kit. The blots were evaluated by densitometry using Image J software. The intensity of the bands was normalized to that of tubulin.
2.11. Detection of Cell Death Histone/DNA complexes released from the nucleus to the cytosol (DNA fragmentation), a marker for apoptosis, were measured according to the manufacturer’s protocol.
2.12. Evaluation of ROS Production in MIN6 Cells 2’,7’-dichlorodihydrofluorescin diacetate (DCFH-DA) was used to evaluate intracellular ROS in the same treated MIN6 cells. DCFH-DA, a cell-permeant probe, is oxidized to form highly fluorescent DCF in the presence of hydrogen peroxide (H2O2) or low molecular weight peroxides produced by the cells.
After treatment, the MIN6 cells were incubated with 10 μM DCFH-DA for 30 min at 37 °C. After incubation, the cells were washed with PBS twice, trypsinized, resuspended and immediately submitted to flow cytometry analysis.
2.13. Measurement of NADPH Oxidase Activity in MIN6 Cells