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Deficiency of voltage-gated proton channel Hv1 attenuates streptozotocin-induced β-cell damage
Biochemical and Biophysical Research Communications xxx (2018) 1e6
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Deficiency of voltage-gated proton channel Hv1 attenuates streptozotocin-induced b-cell damage Xudong Wang a, 1, Wang Xi a, 1, Jiwei Qin a, Jili Lv a, Yuzhou Wang b, Tianhao Zhang a, Shu Jie Li a, * a
Department of Biophysics, School of Physical Science, The Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, Tianjin 300071, PR China Laboratory Animal Center, College of Life Sciences, Nankai University, Tianjin 300071, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 March 2018 Accepted 13 March 2018 Available online xxx
Reactive oxygen species (ROS) impairs pancreatic b-cells and plays an important role in development of diabetes. Streptozotocin (STZ) can lead to b-cell dysfunction via inducing ROS production. The voltagegated proton channel Hv1 contributes a majority of the charge compensation required for ROS production. Here, we investigated the effects of Hv1 on STZ-induced b-cell damage. We found that deficiency of Hv1 obviously inhibits STZ-induced glucose intolerance in mice, and prevents the decrease in bcell mass and pancreatic insulin content from STZ-treatment. Further studies showed that loss of Hv1 significantly attenuates STZ-induced b-cell damage and ROS production in pancreatic b-cells. Our results suggest that Hv1 might contribute to development of diabetes through producing ROS. © 2018 Published by Elsevier Inc.
Keywords: Voltage-gated proton channel Hv1 Pancreatic b-cells ROS Damage Diabetes
1. Introduction Diabetes is a serious metabolic disease resulting from absolute or relative insulin deficiency. As insulin-producing cells, the dysfunction of pancreatic b-cells is considered as a major factor contribute to type 1 diabetes (T1D) and type 2 diabetes (T2D). T1D is a cytotoxic T cell-mediated antigen-specific process, leading to increased cytokine and reactive oxygen species (ROS) production and destruction of b-cells [1]. T2D is mainly caused by the insufficient insulin to overcome insulin resistance, finally resulting in hyperglycemia [2]. Under high glucose conditions, a persistent state of excessive production of ROS has been proposed to be a contributor to the development of complications and further destruction of b-cells [3,4]. Streptozotocin (STZ) has been usually used as a reagent which induces diabetes in rodents and to study the mechanism of the development of diabetes [5]. STZ-treatment can induce direct b-cells destruction and the indirect b-cells destruction from T-cell-dependent immune reaction [6], both two
* Corresponding author. Department of Biophysics, The Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, 94 Weijin Road, Nankai District, Tianjin 300071, PR China. E-mail address: [email protected] (S.J. Li). 1 These authors contributed equally to this work.
ways generate ROS which may facilitate the destruction [7]. The voltage-gated proton channel Hv1 has been demonstrated to contribute to ROS production cooperated with NADPH oxidase, an enzyme which moves electrons across membranes, in many cell types [8]. In previous study [9], we found that Hv1 is expressed in pancreatic islet b-cells and regulates insulin secretion, but the role of Hv1 in the ROS production and in the development of diabetes remains to be determined. In the present study, we used Hv1 knockout mice and INS1(832/13) cell line to investigate the role of Hv1 in STZ-treated bcell destruction. We demonstrated that Hv1-deficiency protects the b-cells from STZ damage through decreasing ROS production. 2. Materials and methods 2.1. Animals and treatments Mice bearing a targeted disruption in the VSOP/Hv1 (VSOP/ Hv1/, backcrossed eight times) were kindly provided by Dr. Y. Okamura (School of Medicine, Osaka University), as previously described [10]. WT mice (VSOP/Hv1þ/þ) were of the same genetic background (C57BL/6J). Animals were kept in a pathogen-free facility under a 12-h light-dark cycle with access to water and a standard mouse diet (Lillico Biotechnology). Genotyping was
https://doi.org/10.1016/j.bbrc.2018.03.092 0006-291X/© 2018 Published by Elsevier Inc.
Please cite this article in press as: X. Wang, et al., Deficiency of voltage-gated proton channel Hv1 attenuates streptozotocin-induced b-cell damage, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.092
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X. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e6
performed by PCR as described by Ramsey et al. [11]. Experiments were performed with 2 month-old male mice, unless indicated otherwise. All animal husbandry and experiments were approved by and performed in accordance with guidelines from the Animal Research Committee of Nankai University.
determined as a ratio of total insulin-positive area to total pancreatic area. Proliferation was determined as a ratio of total Ki67-positive area to total pancreatic area. Apoptosis was determined as a ratio of total TUNEL-positive area to total pancreatic area by DeadEnd™ Colorimetric TUNEL System (Promege, USA), according to the manufacturer's protocol.
2.2. Isolation of pancreatic islets 2.8. Cell survival rate Pancreatic islets were isolated according to the collagenase digestion method described by Lacy and Kostianovsky [12], with slight modifications. Krebs-Ringer bicarbonate HEPES (KRBH) buffer (in mM: 135 NaCl, 3.6 KCl, 5 NaHCO3, 0.5 NaH2PO4, 0.5 MgCl2, 1.5 CaCl2, 10 HEPES, pH 7.4) was used for islet isolation. And the isolated islets were cultured overnight in RPMI 1640 (GIBCO) containing 10% fetal bovine serum (FBS) in a humidified 5% CO2 atmosphere at 37 C before handpicking for experiments.
The survival rates of cell line were measured by MTT assay as described previously [13]. Briefly, cells were plated in 96-well plates at a concentration of 5 104 cells/ml at 100 ml per well in RPMI 1640 medium. The next day, the medium in 96-well plates was replaced by the KRBH buffer. 3-(4,5-Dimethyl-thiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay was performed after the cells were kept in culture at 37 C for 30 min.
2.3. Cell culture
2.9. Insulin determinations
Pancreatic islet b-cell line, INS-1 (832/13) cells were obtained from Dr. Hans Hohmeier (Duke University) and grown in RPMI 1640 medium (GIBCO) supplemented with 10% FBS, 2 mM glutamate, 1 mM sodium pyruvate and 55 mM b-mercaptoethanol in a humidified 5% CO2 atmosphere at 37 C.
Total insulin content in isolated islets and INS-1 (832/13) cells were extracted with acidic ethanol and determined using rat/ mouse insulin ELISA (Mercodia, Uppsala), according to the manufacturer's protocol. 2.10. Statistical analysis
2.4. siRNA silencing To down-regulate Hv1 expression level in INS 1(832/13) cells, the sequences of the small interfering RNA (siRNA) targeting the Hv1 gene 50 -CTACAAGAAATGGGAGAAT-30 and the scramble sense sequence 50 -TTCTCCGAACGTGTCACGT-30 , which were obtained from Ribobio (Guangzhou, China), were used, as described previously [9].
All statistics were performed using SPSS 20.0 software. Measurement data were represented as mean ± SEM. Comparison of the mean between groups was performed by t-test. P values < 0.05 were considered significant. 3. Results 3.1. Deficiency of Hv1 attenuates STZ-induced b-cell damage
2.5. Measurement of ROS Total ROS production in isolated islets and INS-1 (832/13) cells was determined by dichlorofluorescein diacetate assay (DCF). Islets and cell line were homogenized in 100 ml KRBH buffer and incubated with 50 mmol/l DCF at 37 C 30 min. Fluorescence was measured by excitation (490 nm) and emission (525 nm) spectra with a fluorescent microscope (OLYMPUS). 2.6. STZ-treatment of mice and cell line To induce diabetes, WT, Hv1þ/ and Hv1/ mice were injected with STZ (50 mg/kg body weight i.p. for 5 consecutive days). Additionally, some WT, Hv1þ/ and Hv1/mice were treated with saline as vehicle controls. Blood glucose levels were measured once every two days with fasting 6 h after the final injection of STZ. At the day 9, an i.p. glucose tolerance test (IPGTT) was performed using an i.p. injection of glucose at 2 g/kg body weight after 6 h fasting. Blood glucose was analyzed at 0, 15, 30, 60, and 120 min after introducing glucose. Diabetic hyperglycemia was defined as a fasting blood glucose concentration >11.1 mmol/l for two or more consecutive tests. The cell line was incubated in medium containing 0.5 mmol/l STZ. 2.7. Immunohistochemistry, b-cell mass, proliferation and apoptosis Pancreases from Hv1/ and WT mice after finally injected by STZ for 4 days were fixed in 4% paraformaldehyde for 4 h, embedded in paraffin, and cut into 5 mm sections. Immunohistochemistry was carried out with anti-mouse insulin monoclonal antibody (dilution 1:200, Abcom). Relative b-cell mass was
STZ is a natural compound which could destruct b-cells and cause hyperglycemia and glucose intolerance, so the STZ-induced diabetic mouse model is widely used to inspect protective properties of the proteins against b-cell death [14]. To investigate the effect of Hv1 on STZ-treated b-cell damage, we injected WT, Hv1þ/ and Hv1/ mice with STZ (50 mg/kg body weight for consecutive 5 days). On fifth day after STZ injection, the blood glucose levels were increased both in WT and Hv1/ mice (Fig. 1A), but the WT mice appeared to more serious glucose intolerance compared to Hv1/ mice (Fig. 1B). In WT islets, the functional b-cell mass was greatly decreased, whereas, in Hv1/ mice, the b-cell mass appeared to be protected from STZ treatment (Fig. 1C, left panel). The pancreatic b-cell masses in both Hv1þ/ and Hv1/ mice analyzed from immunohistochemistry were obviously higher than that of WT after STZtreatment five days (Fig. 1C, right panel). Under a normal condition (without STZ), the pancreatic insulin contents of Hv1þ/ and Hv1/ mice were lower than that of WT mice (data not shown). However, after STZ-treatment, the pancreatic insulin content of WT mice was significantly lower than that of both Hv1þ/ and Hv1/ mice (Fig. 1D). Our results demonstrated that loss of Hv1 attenuates STZ-induced b-cell damage. 3.2. Deficiency of Hv1 prevents pancreatic b-cell apoptosis from STZ-treatment To detect whether the loss of Hv1 prevents b-cell death from STZ-treatment, we measured b-cell apoptosis by TUNEL staining. Compared with WT mice, the apoptosis of b-cells in Hv1/ mice was markedly reduced by 70% after STZ-treatment (Fig. 2A), while
Please cite this article in press as: X. Wang, et al., Deficiency of voltage-gated proton channel Hv1 attenuates streptozotocin-induced b-cell damage, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.092
X. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e6
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Fig. 1. Hv1-deficiency protects b-cell destruction from STZ-treatment. A: Basal blood glucose levels of Hv1/ and WT mice after fasting 6 h without and with multiple-low-dose STZ injection for five days (nþ/þ ¼ 8, n/ ¼ 12). Data are mean ± SEM. B: Blood glucose levels measured in whole blood following i.p. injection of glucose (2 g/kg body weight) in Hv1/ and WT mice after STZ treatment (nþ/þ ¼ 8; n/ ¼ 9). Data are means ± SEM. C: Representative images of immunohistochemical analysis of STZ-treated 2 month-old WT, Hv1þ/ and H/ mouse islets using anti-insulin antibodies (left panel). Insulin-positive cells within islets were stained brown. Scale bar, 50 mm. b-cell masses of STZ-treated 2 month-old WT, Hv1þ/ and Hv1/ mice were analyzed based on immunostaining of pancreatic sections (n ¼ 6 per genotype) (right panel). b-cell masses were determined as a ratio of total insulin (b-cell)-positive area to total pancreatic area. Twenty to thirty sections per pancreas were analyzed. Data are presented as mean ± SEM. **p < 0.01, vs. WT. D: Pancreatic insulin contents of STZ-treated WT, Hv1þ/ and Hv1/ mice (n ¼ 8 per genotype). Data are means ± SEM. **p < 0.01, vs. WT.
the proliferation rate of b-cell showed no significant difference among WT, Hv1þ/ and Hv1/ mice (Fig. 2B). These results showed that deficiency of Hv1 prevents pancreatic b-cell apoptosis from STZ-treatment. 3.3. Knockdown of Hv1 prevents b-cell damage from STZ-treatment To further examine the effect of Hv1 on the apoptosis of b-cells, we also measured the survival rate of INS-1 (832/13) b-cells treated with STZ. As shown in Fig. 3A, the cell survival rates of INS-1 (832/ 13) cells transfected with Hv1-targeting siRNA (siRNA) and scramble siRNA (control) were 25 and 10% by STZ-treatment for 24 h, respectively. There were no differences between the proliferation rates of INS-1 (832/13) b-cells treated Hv1-targeting siRNA (siRNA) and scramble siRNA (control) (Fig. 3B), indicating that knockdown of Hv1 decreases b-cell apoptosis treated with STZ.
destruction, we used the fluorescence probe H2DCF-DA to detect the ROS production in INS-1 (832/13) b-cells and pancreatic islets. As shown in Fig. 4A, ROS in Hv1-scilenced INS-1 (832/13) b-cells transfected with Hv1-targeting siRNA (siRNA) was decreased by 49% compare with the control in the presence of 0.5 mM STZ, which indicates that Hv1-deficiency decreases ROS production. PMA (phorbol myristate acetate) as an Hv1 activator has been extensively used for Hv1 function studies [11,15]. As shown in Fig. 4B, in the presence of 5 mM PMA, the ROS production in INS-1 (832/13) b-cells transfected with Hv1-targeting siRNA (siRNA) was reduced by 26%, compared with the control. While, knockout of Hv1 decreased ROS production to 54% in isolated islets from mice compared to isolated islets from WT mice (Fig. 4C). These data elucidate that Hv1-deficiency decreases ROS production in b-cells. 4. Discussion
3.4. Deficiency of Hv1 decreases STZ- or PMA-stimulated ROS production in b-cells Hv1 plays an important role to sustain production of ROS in neutrophil and macrophage [11,15]. STZ can impair b-cells and lead to diabetes in connection with the ROS production in b-cells. To illuminate the mechanism of Hv1 on STZ-induced b-cell
In the present study, we demonstrate for the first time that Hv1deficiency protects b-cells from the STZ-induced damage. In vivo, STZ-treated Hv1/ mice show a weaker glucose intolerance compared to STZ-treated WT mice. In vitro, Hv1-deficiency decreases STZ-induced b-cell apoptosis. In accord with these, the bcell mass and pancreatic insulin contents in both Hv1þ/ and Hv1/
Please cite this article in press as: X. Wang, et al., Deficiency of voltage-gated proton channel Hv1 attenuates streptozotocin-induced b-cell damage, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.092
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Fig. 2. Hv1-deficiency decreases b-cell apoptosis after multiple-low-dose STZ-treatment. A: Representative images of histological TUNEL-staining of WT, Hv1þ/ and Hv1/ mouse islets treated with/without STZ (left panel). Scale bar, 50 mm. %TUNEL-positive cells of 2 month-old WT, Hv1þ/ and Hv1/ mice based on immunostaining of pancreatic sections before and after STZ treatment (n ¼ 6 per genotype). %TUNEL-positive cells were determined as a ratio of total TUNEL-positive area to total pancreatic area. Twenty to thirty sections per pancreas were analyzed (right panel). Data are mean ± SEM. **p < 0.01, vs. WT. B: %Ki67-positive cells of 2 month-old WT, Hv1þ/ and Hv1/ mouse islets based on immunostaining of pancreatic sections treated with/without STZ (n ¼ 6 per genotype). %Ki67positive cells were determined as a ratio of total Ki67-positive area to total pancreatic area. Twenty to thirty sections per pancreas were analyzed. Data are mean ± SEM. **p < 0.01, vs. WT.
Fig. 3. Knockdown of Hv1 decreases b-cell apoptosis treated with STZ. A: INS-1 (832/13) cells transfected with scramble siRNA (control) or Hv1-targeting siRNA (siRNA) were incubated with 0.5 mM STZ. The survival rates of INS-1 (832/13) cells were measured by MTT assay after 0, 12 and 24 h STZ treatment, respectively. Scale bar, 25 mm. Data are mean ± SEM. *p < 0.05, vs. corresponding control. B: Proliferation rate of INS-1 (832/13) cells transfected with scramble siRNA (control) or Hv1-targeting siRNA (siRNA). Data are mean ± SEM. The result showed that knockdown of Hv1 does not affect the b-cell proliferation.
mice show higher than that of WT mice after STZ-treatment. These results clearly indicate that Hv1-deficiency significantly decreases the toxicity of STZ. Pancreatic b-cell dysfunction is a crucial reason in diabetes
mellitus. As a b-cell cytotoxicity, STZ is routinely experimentally used to induce diabetes via the destruction of b-cells [6]. In the multiple-low-dose STZ diabetes model, diabetes is induced by two main mechanisms [6,16,17]. One is the direct cytotoxic effect of STZ
Please cite this article in press as: X. Wang, et al., Deficiency of voltage-gated proton channel Hv1 attenuates streptozotocin-induced b-cell damage, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.092
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Fig. 4. Hv1-deficiency decreases STZ- or PMA-stimulated ROS production in b-cells. A and B: STZ- or PMA-stimulated ROS production in INS-1 (832/13) cells transfected with scramble siRNA (control) or Hv1-targeting siRNA (siRNA). Data are mean ± SEM (n ¼ 6 per condition). *p < 0.05, vs. control at 0 mM STZ. #p < 0.05, vs. corresponding control. C: PMA-stimulated ROS production in isolated islets from WT and Hv1 / mice. Data are mean ± SEM. *p < 0.05, vs. WT at 0 mM STZ. #p < 0.05, vs. corresponding WT.
on b-cells. STZ enters the b-cells and causes DNA alkylation and ROS production. The ROS can evoke DNA fragmentation. The other is the local infiltration in the pancreatic islets and inflammation by lymphocytes and macrophages, which simulate the immune reaction and ROS production [14]. Although ROS has been implicated in both two mechanisms, the mechanisms of the ROS production during diabetes are still not clear. Because of the low expression of antioxidant enzymes, pancreatic b-cells are sensitive to the cytotoxic action of ROS [18]. NADPH oxidase, as a major producer of ROS, has been tangled with the bcell damage [8]. NADPH oxidase transports electrons across the membrane of the phagosome to reduce molecular oxygen into superoxide (O2) [19]. The sustained production of superoxide requires the movement of a compensating charge, which is the protons flowing across Hv1. Protons channels can provide a compensating charge and convert superoxide anions to H2O2 and eventually HOCl, and two knockout studies indicate that Hv1 proton channel is required for high-level superoxide production [10,11]. Hv1 also contributes to brain damage in cerebral ischemia through regulating NOX-dependent ROS generation in microglia [20]. PMA, whose function is to activate PKC, can also stimulate ROS production [11]. In the present study, we demonstrate that Hv1deficiency decreases the ROS production in b-cells via STZ and PMA stimulation, which reveals how Hv1-deficiency protects bcells from the damage induced by STZ. In conclusion, our study demonstrates that Hv1-deficiency attenuates the intolerant of glucose in the multiple-low-dose STZinduced diabetes in mice. The protective effects are results of decreased ROS production in the islets. Our results implicate that Hv1 might represent a new target for the treatment of diabetes.
Acknowledgements We would like to thank Dr. Y. Okamura (School of Medicine, Osaka University) for providing VSOP/Hv1 KO mice. We would like
to thank Dr. Hans E. Hohmeier (Duke University Medical Center) for providing materials mentioned in the text. This work was supported by National Natural Science Foundation of China (No. 31271464). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.03.092. Conflicts of interest The authors declare that they have no conflict of interest. Author contributions SJL and XDW conceived and designed the study. XDW, WX, JWQ, JL, YZW, THZ and SJL performed the experiments. SJL and XDW wrote the paper. SJL and XDW reviewed and edited the manuscript. All authors were involved in data analysis, read and approved the manuscript. References [1] J.W. Yoon, H.S. Jun, Autoimmune destruction of pancreatic beta cells, Am. J. Therapeut. 12 (2005) 580e591. [2] R.P. Robertson, Beta-cell deterioration during diabetes: what's in the gun? Trends Endocrinol. Metabol. 20 (2009) 388e393. [3] H. Kaneto, J. Fujii, T. Myint, N. Miyazawa, K.N. Islam, Y. Kawasaki, et al., Reducing sugars trigger oxidative modification and apoptosis in pancreatic beta-cells by provoking oxidative stress through the glycation reaction, Biochem. J. 320 (1996) 855e863. [4] Y. Ihara, S. Toyokuni, K. Uchida, H. Odaka, T. Tanaka, H. Ikeda, H. Hiai, Y. Seino, Y. Yamada, Hyperglycemia causes oxidative stress in pancreatic beta-cells of GK rats, a model of type 2 diabetes, Diabetes 48 (1999) 927e932. [5] H. Kolb, Mouse models of insulin-dependent diabetes-Low dose streptozotocin induced diabetes and nonobese diabetic (NOD) mice, Diabetes Metab. Rev. 3 (1987) 751e778. [6] S. Lenzen, The mechanisms of alloxan- and streptozotocin-induced diabetes,
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Please cite this article in press as: X. Wang, et al., Deficiency of voltage-gated proton channel Hv1 attenuates streptozotocin-induced b-cell damage, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.092
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Deficiency of voltage-gated proton channel Hv1 attenuates streptozotocin-induced b-cell damage Xudong Wang a, 1, Wang Xi a, 1, Jiwei Qin a, Jili Lv a, Yuzhou Wang b, Tianhao Zhang a, Shu Jie Li a, * a
Department of Biophysics, School of Physical Science, The Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, Tianjin 300071, PR China Laboratory Animal Center, College of Life Sciences, Nankai University, Tianjin 300071, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 March 2018 Accepted 13 March 2018 Available online xxx
Reactive oxygen species (ROS) impairs pancreatic b-cells and plays an important role in development of diabetes. Streptozotocin (STZ) can lead to b-cell dysfunction via inducing ROS production. The voltagegated proton channel Hv1 contributes a majority of the charge compensation required for ROS production. Here, we investigated the effects of Hv1 on STZ-induced b-cell damage. We found that deficiency of Hv1 obviously inhibits STZ-induced glucose intolerance in mice, and prevents the decrease in bcell mass and pancreatic insulin content from STZ-treatment. Further studies showed that loss of Hv1 significantly attenuates STZ-induced b-cell damage and ROS production in pancreatic b-cells. Our results suggest that Hv1 might contribute to development of diabetes through producing ROS. © 2018 Published by Elsevier Inc.
Keywords: Voltage-gated proton channel Hv1 Pancreatic b-cells ROS Damage Diabetes
1. Introduction Diabetes is a serious metabolic disease resulting from absolute or relative insulin deficiency. As insulin-producing cells, the dysfunction of pancreatic b-cells is considered as a major factor contribute to type 1 diabetes (T1D) and type 2 diabetes (T2D). T1D is a cytotoxic T cell-mediated antigen-specific process, leading to increased cytokine and reactive oxygen species (ROS) production and destruction of b-cells [1]. T2D is mainly caused by the insufficient insulin to overcome insulin resistance, finally resulting in hyperglycemia [2]. Under high glucose conditions, a persistent state of excessive production of ROS has been proposed to be a contributor to the development of complications and further destruction of b-cells [3,4]. Streptozotocin (STZ) has been usually used as a reagent which induces diabetes in rodents and to study the mechanism of the development of diabetes [5]. STZ-treatment can induce direct b-cells destruction and the indirect b-cells destruction from T-cell-dependent immune reaction [6], both two
* Corresponding author. Department of Biophysics, The Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, 94 Weijin Road, Nankai District, Tianjin 300071, PR China. E-mail address: [email protected] (S.J. Li). 1 These authors contributed equally to this work.
ways generate ROS which may facilitate the destruction [7]. The voltage-gated proton channel Hv1 has been demonstrated to contribute to ROS production cooperated with NADPH oxidase, an enzyme which moves electrons across membranes, in many cell types [8]. In previous study [9], we found that Hv1 is expressed in pancreatic islet b-cells and regulates insulin secretion, but the role of Hv1 in the ROS production and in the development of diabetes remains to be determined. In the present study, we used Hv1 knockout mice and INS1(832/13) cell line to investigate the role of Hv1 in STZ-treated bcell destruction. We demonstrated that Hv1-deficiency protects the b-cells from STZ damage through decreasing ROS production. 2. Materials and methods 2.1. Animals and treatments Mice bearing a targeted disruption in the VSOP/Hv1 (VSOP/ Hv1/, backcrossed eight times) were kindly provided by Dr. Y. Okamura (School of Medicine, Osaka University), as previously described [10]. WT mice (VSOP/Hv1þ/þ) were of the same genetic background (C57BL/6J). Animals were kept in a pathogen-free facility under a 12-h light-dark cycle with access to water and a standard mouse diet (Lillico Biotechnology). Genotyping was
https://doi.org/10.1016/j.bbrc.2018.03.092 0006-291X/© 2018 Published by Elsevier Inc.
Please cite this article in press as: X. Wang, et al., Deficiency of voltage-gated proton channel Hv1 attenuates streptozotocin-induced b-cell damage, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.092
2
X. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e6
performed by PCR as described by Ramsey et al. [11]. Experiments were performed with 2 month-old male mice, unless indicated otherwise. All animal husbandry and experiments were approved by and performed in accordance with guidelines from the Animal Research Committee of Nankai University.
determined as a ratio of total insulin-positive area to total pancreatic area. Proliferation was determined as a ratio of total Ki67-positive area to total pancreatic area. Apoptosis was determined as a ratio of total TUNEL-positive area to total pancreatic area by DeadEnd™ Colorimetric TUNEL System (Promege, USA), according to the manufacturer's protocol.
2.2. Isolation of pancreatic islets 2.8. Cell survival rate Pancreatic islets were isolated according to the collagenase digestion method described by Lacy and Kostianovsky [12], with slight modifications. Krebs-Ringer bicarbonate HEPES (KRBH) buffer (in mM: 135 NaCl, 3.6 KCl, 5 NaHCO3, 0.5 NaH2PO4, 0.5 MgCl2, 1.5 CaCl2, 10 HEPES, pH 7.4) was used for islet isolation. And the isolated islets were cultured overnight in RPMI 1640 (GIBCO) containing 10% fetal bovine serum (FBS) in a humidified 5% CO2 atmosphere at 37 C before handpicking for experiments.
The survival rates of cell line were measured by MTT assay as described previously [13]. Briefly, cells were plated in 96-well plates at a concentration of 5 104 cells/ml at 100 ml per well in RPMI 1640 medium. The next day, the medium in 96-well plates was replaced by the KRBH buffer. 3-(4,5-Dimethyl-thiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay was performed after the cells were kept in culture at 37 C for 30 min.
2.3. Cell culture
2.9. Insulin determinations
Pancreatic islet b-cell line, INS-1 (832/13) cells were obtained from Dr. Hans Hohmeier (Duke University) and grown in RPMI 1640 medium (GIBCO) supplemented with 10% FBS, 2 mM glutamate, 1 mM sodium pyruvate and 55 mM b-mercaptoethanol in a humidified 5% CO2 atmosphere at 37 C.
Total insulin content in isolated islets and INS-1 (832/13) cells were extracted with acidic ethanol and determined using rat/ mouse insulin ELISA (Mercodia, Uppsala), according to the manufacturer's protocol. 2.10. Statistical analysis
2.4. siRNA silencing To down-regulate Hv1 expression level in INS 1(832/13) cells, the sequences of the small interfering RNA (siRNA) targeting the Hv1 gene 50 -CTACAAGAAATGGGAGAAT-30 and the scramble sense sequence 50 -TTCTCCGAACGTGTCACGT-30 , which were obtained from Ribobio (Guangzhou, China), were used, as described previously [9].
All statistics were performed using SPSS 20.0 software. Measurement data were represented as mean ± SEM. Comparison of the mean between groups was performed by t-test. P values < 0.05 were considered significant. 3. Results 3.1. Deficiency of Hv1 attenuates STZ-induced b-cell damage
2.5. Measurement of ROS Total ROS production in isolated islets and INS-1 (832/13) cells was determined by dichlorofluorescein diacetate assay (DCF). Islets and cell line were homogenized in 100 ml KRBH buffer and incubated with 50 mmol/l DCF at 37 C 30 min. Fluorescence was measured by excitation (490 nm) and emission (525 nm) spectra with a fluorescent microscope (OLYMPUS). 2.6. STZ-treatment of mice and cell line To induce diabetes, WT, Hv1þ/ and Hv1/ mice were injected with STZ (50 mg/kg body weight i.p. for 5 consecutive days). Additionally, some WT, Hv1þ/ and Hv1/mice were treated with saline as vehicle controls. Blood glucose levels were measured once every two days with fasting 6 h after the final injection of STZ. At the day 9, an i.p. glucose tolerance test (IPGTT) was performed using an i.p. injection of glucose at 2 g/kg body weight after 6 h fasting. Blood glucose was analyzed at 0, 15, 30, 60, and 120 min after introducing glucose. Diabetic hyperglycemia was defined as a fasting blood glucose concentration >11.1 mmol/l for two or more consecutive tests. The cell line was incubated in medium containing 0.5 mmol/l STZ. 2.7. Immunohistochemistry, b-cell mass, proliferation and apoptosis Pancreases from Hv1/ and WT mice after finally injected by STZ for 4 days were fixed in 4% paraformaldehyde for 4 h, embedded in paraffin, and cut into 5 mm sections. Immunohistochemistry was carried out with anti-mouse insulin monoclonal antibody (dilution 1:200, Abcom). Relative b-cell mass was
STZ is a natural compound which could destruct b-cells and cause hyperglycemia and glucose intolerance, so the STZ-induced diabetic mouse model is widely used to inspect protective properties of the proteins against b-cell death [14]. To investigate the effect of Hv1 on STZ-treated b-cell damage, we injected WT, Hv1þ/ and Hv1/ mice with STZ (50 mg/kg body weight for consecutive 5 days). On fifth day after STZ injection, the blood glucose levels were increased both in WT and Hv1/ mice (Fig. 1A), but the WT mice appeared to more serious glucose intolerance compared to Hv1/ mice (Fig. 1B). In WT islets, the functional b-cell mass was greatly decreased, whereas, in Hv1/ mice, the b-cell mass appeared to be protected from STZ treatment (Fig. 1C, left panel). The pancreatic b-cell masses in both Hv1þ/ and Hv1/ mice analyzed from immunohistochemistry were obviously higher than that of WT after STZtreatment five days (Fig. 1C, right panel). Under a normal condition (without STZ), the pancreatic insulin contents of Hv1þ/ and Hv1/ mice were lower than that of WT mice (data not shown). However, after STZ-treatment, the pancreatic insulin content of WT mice was significantly lower than that of both Hv1þ/ and Hv1/ mice (Fig. 1D). Our results demonstrated that loss of Hv1 attenuates STZ-induced b-cell damage. 3.2. Deficiency of Hv1 prevents pancreatic b-cell apoptosis from STZ-treatment To detect whether the loss of Hv1 prevents b-cell death from STZ-treatment, we measured b-cell apoptosis by TUNEL staining. Compared with WT mice, the apoptosis of b-cells in Hv1/ mice was markedly reduced by 70% after STZ-treatment (Fig. 2A), while
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Fig. 1. Hv1-deficiency protects b-cell destruction from STZ-treatment. A: Basal blood glucose levels of Hv1/ and WT mice after fasting 6 h without and with multiple-low-dose STZ injection for five days (nþ/þ ¼ 8, n/ ¼ 12). Data are mean ± SEM. B: Blood glucose levels measured in whole blood following i.p. injection of glucose (2 g/kg body weight) in Hv1/ and WT mice after STZ treatment (nþ/þ ¼ 8; n/ ¼ 9). Data are means ± SEM. C: Representative images of immunohistochemical analysis of STZ-treated 2 month-old WT, Hv1þ/ and H/ mouse islets using anti-insulin antibodies (left panel). Insulin-positive cells within islets were stained brown. Scale bar, 50 mm. b-cell masses of STZ-treated 2 month-old WT, Hv1þ/ and Hv1/ mice were analyzed based on immunostaining of pancreatic sections (n ¼ 6 per genotype) (right panel). b-cell masses were determined as a ratio of total insulin (b-cell)-positive area to total pancreatic area. Twenty to thirty sections per pancreas were analyzed. Data are presented as mean ± SEM. **p < 0.01, vs. WT. D: Pancreatic insulin contents of STZ-treated WT, Hv1þ/ and Hv1/ mice (n ¼ 8 per genotype). Data are means ± SEM. **p < 0.01, vs. WT.
the proliferation rate of b-cell showed no significant difference among WT, Hv1þ/ and Hv1/ mice (Fig. 2B). These results showed that deficiency of Hv1 prevents pancreatic b-cell apoptosis from STZ-treatment. 3.3. Knockdown of Hv1 prevents b-cell damage from STZ-treatment To further examine the effect of Hv1 on the apoptosis of b-cells, we also measured the survival rate of INS-1 (832/13) b-cells treated with STZ. As shown in Fig. 3A, the cell survival rates of INS-1 (832/ 13) cells transfected with Hv1-targeting siRNA (siRNA) and scramble siRNA (control) were 25 and 10% by STZ-treatment for 24 h, respectively. There were no differences between the proliferation rates of INS-1 (832/13) b-cells treated Hv1-targeting siRNA (siRNA) and scramble siRNA (control) (Fig. 3B), indicating that knockdown of Hv1 decreases b-cell apoptosis treated with STZ.
destruction, we used the fluorescence probe H2DCF-DA to detect the ROS production in INS-1 (832/13) b-cells and pancreatic islets. As shown in Fig. 4A, ROS in Hv1-scilenced INS-1 (832/13) b-cells transfected with Hv1-targeting siRNA (siRNA) was decreased by 49% compare with the control in the presence of 0.5 mM STZ, which indicates that Hv1-deficiency decreases ROS production. PMA (phorbol myristate acetate) as an Hv1 activator has been extensively used for Hv1 function studies [11,15]. As shown in Fig. 4B, in the presence of 5 mM PMA, the ROS production in INS-1 (832/13) b-cells transfected with Hv1-targeting siRNA (siRNA) was reduced by 26%, compared with the control. While, knockout of Hv1 decreased ROS production to 54% in isolated islets from mice compared to isolated islets from WT mice (Fig. 4C). These data elucidate that Hv1-deficiency decreases ROS production in b-cells. 4. Discussion
3.4. Deficiency of Hv1 decreases STZ- or PMA-stimulated ROS production in b-cells Hv1 plays an important role to sustain production of ROS in neutrophil and macrophage [11,15]. STZ can impair b-cells and lead to diabetes in connection with the ROS production in b-cells. To illuminate the mechanism of Hv1 on STZ-induced b-cell
In the present study, we demonstrate for the first time that Hv1deficiency protects b-cells from the STZ-induced damage. In vivo, STZ-treated Hv1/ mice show a weaker glucose intolerance compared to STZ-treated WT mice. In vitro, Hv1-deficiency decreases STZ-induced b-cell apoptosis. In accord with these, the bcell mass and pancreatic insulin contents in both Hv1þ/ and Hv1/
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Fig. 2. Hv1-deficiency decreases b-cell apoptosis after multiple-low-dose STZ-treatment. A: Representative images of histological TUNEL-staining of WT, Hv1þ/ and Hv1/ mouse islets treated with/without STZ (left panel). Scale bar, 50 mm. %TUNEL-positive cells of 2 month-old WT, Hv1þ/ and Hv1/ mice based on immunostaining of pancreatic sections before and after STZ treatment (n ¼ 6 per genotype). %TUNEL-positive cells were determined as a ratio of total TUNEL-positive area to total pancreatic area. Twenty to thirty sections per pancreas were analyzed (right panel). Data are mean ± SEM. **p < 0.01, vs. WT. B: %Ki67-positive cells of 2 month-old WT, Hv1þ/ and Hv1/ mouse islets based on immunostaining of pancreatic sections treated with/without STZ (n ¼ 6 per genotype). %Ki67positive cells were determined as a ratio of total Ki67-positive area to total pancreatic area. Twenty to thirty sections per pancreas were analyzed. Data are mean ± SEM. **p < 0.01, vs. WT.
Fig. 3. Knockdown of Hv1 decreases b-cell apoptosis treated with STZ. A: INS-1 (832/13) cells transfected with scramble siRNA (control) or Hv1-targeting siRNA (siRNA) were incubated with 0.5 mM STZ. The survival rates of INS-1 (832/13) cells were measured by MTT assay after 0, 12 and 24 h STZ treatment, respectively. Scale bar, 25 mm. Data are mean ± SEM. *p < 0.05, vs. corresponding control. B: Proliferation rate of INS-1 (832/13) cells transfected with scramble siRNA (control) or Hv1-targeting siRNA (siRNA). Data are mean ± SEM. The result showed that knockdown of Hv1 does not affect the b-cell proliferation.
mice show higher than that of WT mice after STZ-treatment. These results clearly indicate that Hv1-deficiency significantly decreases the toxicity of STZ. Pancreatic b-cell dysfunction is a crucial reason in diabetes
mellitus. As a b-cell cytotoxicity, STZ is routinely experimentally used to induce diabetes via the destruction of b-cells [6]. In the multiple-low-dose STZ diabetes model, diabetes is induced by two main mechanisms [6,16,17]. One is the direct cytotoxic effect of STZ
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Fig. 4. Hv1-deficiency decreases STZ- or PMA-stimulated ROS production in b-cells. A and B: STZ- or PMA-stimulated ROS production in INS-1 (832/13) cells transfected with scramble siRNA (control) or Hv1-targeting siRNA (siRNA). Data are mean ± SEM (n ¼ 6 per condition). *p < 0.05, vs. control at 0 mM STZ. #p < 0.05, vs. corresponding control. C: PMA-stimulated ROS production in isolated islets from WT and Hv1 / mice. Data are mean ± SEM. *p < 0.05, vs. WT at 0 mM STZ. #p < 0.05, vs. corresponding WT.
on b-cells. STZ enters the b-cells and causes DNA alkylation and ROS production. The ROS can evoke DNA fragmentation. The other is the local infiltration in the pancreatic islets and inflammation by lymphocytes and macrophages, which simulate the immune reaction and ROS production [14]. Although ROS has been implicated in both two mechanisms, the mechanisms of the ROS production during diabetes are still not clear. Because of the low expression of antioxidant enzymes, pancreatic b-cells are sensitive to the cytotoxic action of ROS [18]. NADPH oxidase, as a major producer of ROS, has been tangled with the bcell damage [8]. NADPH oxidase transports electrons across the membrane of the phagosome to reduce molecular oxygen into superoxide (O2) [19]. The sustained production of superoxide requires the movement of a compensating charge, which is the protons flowing across Hv1. Protons channels can provide a compensating charge and convert superoxide anions to H2O2 and eventually HOCl, and two knockout studies indicate that Hv1 proton channel is required for high-level superoxide production [10,11]. Hv1 also contributes to brain damage in cerebral ischemia through regulating NOX-dependent ROS generation in microglia [20]. PMA, whose function is to activate PKC, can also stimulate ROS production [11]. In the present study, we demonstrate that Hv1deficiency decreases the ROS production in b-cells via STZ and PMA stimulation, which reveals how Hv1-deficiency protects bcells from the damage induced by STZ. In conclusion, our study demonstrates that Hv1-deficiency attenuates the intolerant of glucose in the multiple-low-dose STZinduced diabetes in mice. The protective effects are results of decreased ROS production in the islets. Our results implicate that Hv1 might represent a new target for the treatment of diabetes.
Acknowledgements We would like to thank Dr. Y. Okamura (School of Medicine, Osaka University) for providing VSOP/Hv1 KO mice. We would like
to thank Dr. Hans E. Hohmeier (Duke University Medical Center) for providing materials mentioned in the text. This work was supported by National Natural Science Foundation of China (No. 31271464). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.03.092. Conflicts of interest The authors declare that they have no conflict of interest. Author contributions SJL and XDW conceived and designed the study. XDW, WX, JWQ, JL, YZW, THZ and SJL performed the experiments. SJL and XDW wrote the paper. SJL and XDW reviewed and edited the manuscript. All authors were involved in data analysis, read and approved the manuscript. References [1] J.W. Yoon, H.S. Jun, Autoimmune destruction of pancreatic beta cells, Am. J. Therapeut. 12 (2005) 580e591. [2] R.P. Robertson, Beta-cell deterioration during diabetes: what's in the gun? Trends Endocrinol. Metabol. 20 (2009) 388e393. [3] H. Kaneto, J. Fujii, T. Myint, N. Miyazawa, K.N. Islam, Y. Kawasaki, et al., Reducing sugars trigger oxidative modification and apoptosis in pancreatic beta-cells by provoking oxidative stress through the glycation reaction, Biochem. J. 320 (1996) 855e863. [4] Y. Ihara, S. Toyokuni, K. Uchida, H. Odaka, T. Tanaka, H. Ikeda, H. Hiai, Y. Seino, Y. Yamada, Hyperglycemia causes oxidative stress in pancreatic beta-cells of GK rats, a model of type 2 diabetes, Diabetes 48 (1999) 927e932. [5] H. Kolb, Mouse models of insulin-dependent diabetes-Low dose streptozotocin induced diabetes and nonobese diabetic (NOD) mice, Diabetes Metab. Rev. 3 (1987) 751e778. [6] S. Lenzen, The mechanisms of alloxan- and streptozotocin-induced diabetes,
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