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WITHDRAWN: Chelating Zinc decreased islet amyloidal polypeptide deposition in beta cell in vivo and in vitro
Accepted Manuscript Title: Chelating Zinc decreased islet amyloidal polypeptide deposition in beta cell in vivo and in vitro Authors: He Tian, Man-Li Zhong, Zhong-Yan Shan, Wei-Ping Teng, Zhi-Hong Chi, Jing-Wei Xie, Tao Wang, Juan Tian, Na Xin, Zhan-You Wang PII: DOI: Reference:
S0040-8166(16)30260-9 http://dx.doi.org/doi:10.1016/j.tice.2017.01.002 YTICE 1068
To appear in:
Tissue and Cell
Received date: Revised date: Accepted date:
10-11-2016 9-1-2017 10-1-2017
Please cite this article as: Tian, He, Zhong, Man-Li, Shan, Zhong-Yan, Teng, WeiPing, Chi, Zhi-Hong, Xie, Jing-Wei, Wang, Tao, Tian, Juan, Xin, Na, Wang, Zhan-You, Chelating Zinc decreased islet amyloidal polypeptide deposition in beta cell in vivo and in vitro.Tissue and Cell http://dx.doi.org/10.1016/j.tice.2017.01.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chelating Zinc decreased islet amyloidal polypeptide deposition in beta cell in vivo and in vitro
He Tiana,c,Man-Li Zhonga,Zhong-Yan Shanb,Wei-Ping Tengb, Zhi-Hong Chia,Jing-Wei Xiea, Tao Wanga, Juan Tiana, Na Xina, Zhan-You Wanga,b﹡
a
Department of Pathophysiology, China Medical University, Shenyang, China
b
Key Laboratory of Endocrine Diseases of Liaoning Province, China Medical
University, Shenyang, China c
Department of Histology and Embryology, Jinzhou Medical University, Jinzhou,
China
* To whom correspondence should be addressed: Zhan-You Wang, Ph.D. Department of Pathophysiology China Medical University No. 77. Puhe Road Shenyang, 110819, P. R. China Tel: +86-24-23256666 5521 E-mail: [email protected]
Highlights The pathological deposition of islet amyloidal polypeptide (IAPP) could be an important factor in the survival of beta cells and in insulin secretion. Synthesis and metabolism of IAPP is closely related to zinc, and the abnormal accumulation of zinc in islet beta cells could be an important factor in amyloid deposition. In this manuscript, we delineated that zinc chelators markedly reduced the expression levels of IAPP and increased the levels of insulin in hIAPP-INS-1 cells and hIAPP mice. The present data indicate that zinc chelator is able to reduce the expression of IAPP, regulating the homeostasis of zinc may be become a new strategy in diabetes mellitus therapy.
Abstract The pathological deposition of islet amyloidal polypeptide (IAPP) could be an important factor in the survival of beta cells and in insulin secretion. The synthesis and metabolism of IAPP is closely related to zinc, and the abnormal accumulation of zinc in islet beta cells could be an important factor in amyloid deposition. Regulating the homeostasis of zinc in diabetes mellitus (DM) patients and maintaining the dynamic balance of the generation and degradation of IAPP, could become a new strategy in DM therapy. To verify the effects of zinc on IAPP and the mechanisms by which zinc controls IAPP deposition, human(h)IAPP transgenic mice and hIAPP transfection insulin-producing beta cell lines (INS-1) were used in this study. Clioquinol (CQ) and TPEN (zinc chelators) were either given to the mice directly or injected into the cells. By means of immunofluorescence double-staining and the western blot analysis, we showed that CQ treatment in hIAPP mice markedly reduced the expression levels of IAPP and increased the levels of insulin. In hIAPP-INS-1 cells treated with TPEN, the expression of IAPP was significantly lower than that of the control group, but the insulin level was higher. Collectively, the present data indicate that a zinc chelator is able to reduce the expression of IAPP in hIAPP transgenic mice and hIAPP-INS-1 cells.
Key words: IAPP; zinc chelator; hIAPP transgenic mice; hIAPP transfected cell
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1.Introduction The major pathophysiologic processes of type 2 diabetes include insulin resistance and decreased beta cell function, the latter of which includes loss of beta cell mass and its dysfunction[1]. Zinc is an essential trace element that plays a critical role in many metabolic disease states including diabetes and cancer[2]. Recent studies have highlighted zinc’s dynamic role as a “cellular second messenger” in the control of insulin signaling and glucose homeostasis[3]. Zinc ions play a role in insulin-induced glucose transport and glycemic control[4].Changes of zinc concentrations in the cytosol and organelles may contribute to insulin responsiveness and thus promote insulin resistance. However, questions on how zinc ions are regulated to cell signaling are largely unknown. IAPP, also known as amylin, is a polypeptide composed of 37 amino acids that is produced and secreted by pancreatic beta cells[5,6]. Recent studies demonstrated the deposition of islet amyloidal polypeptide (IAPP) in the pancreatic islet in the autopsy material of 90% of type 2 DM patients, accompanied by a significant reduction in the number of beta cells[7]. IAPP can control appetite, inhibit gastric emptying, inhibit insulin secretion, and perform other physiological functions [8]. Several studies have shown that a large number of amyloid deposits were found in the islets of Langerhans (islets) in transgenic mice that expressed human (h)IAPP[9,10]. The increase of IAPP deposition is regarded as a crucial event for insulin resistance and hyperglycemia. Deposition of IAPP can also induce pancreatic beta cells apoptosis in the occurrence and development of type 2 DM[11,12]. These studies have demonstrated that deposition of IAPP contributes to the progression of the type 2 diabetes process. Zinc ion is closely related to the synthesis and metabolism of IAPP[13,14]. In beta cells, precursor (prepro) IAPP is processed by prohormone convertase 2 (PC2), prohormone convertase 1/3 (PC1/3), and carboxypeptidase E (CPE) to form mature IAPP[15-17]. The IAPP monomer does not form amyloid deposition, but the fibrous aggregates form the amyloid deposition[18,19]. It has been reported that the zinc ion could bind to the His18 of IAPP (18th histidine residue), so that the adjacent IAPP monomer is crosslinked, eventually forming fibrous IAPP aggregates[20]. These phenomena prompted the consideration that the imbalance in zinc ion metabolism in islet beta cells might be an important factor in amyloid deposition. Regulating the homeostasis of zinc ion in DM patients and maintaining the dynamic balance of generation and degradation of IAPP might become the new strategy in DM therapy. In this 2
study, using INS-1 cells and the hIAPP transgenic mice model, we investigated the contribution of Zinc to deposition of IAPP, and explored the mechanism of zinc-mediated IAPP deposition in vitro and in vivo. 2.Materials and methods 2.1 INS-1 cell culture and treatment Rat INS-1 cells were grown to RM1640 (Invitrogen Technology) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen Technology), 100 IU/mL penicillin, and 100 μg/mL streptomycin at 37°C in humidified 5% CO2 air. Then the cells were infected with LV- hIAPP and LV-scramble (company) at a multiplicity of infection of 20 after 6 days according to the manufacturer’s instructions. 2.2 Exposure to ZnSO4 and Zinc chelator After (72h) transfection, when 80% of the bottom of the buffer solution was covered by cells, ZnSO4 and TPEN were added to the medium. ZnSO4 was added in a double blind manner to the medium with concentrations (10μM,20μM,50μM,70μM,100μM). Another group, a zinc special chelator, N,N,Nʹ,Nʹ-tetrakis(2-pyrid-ylmethyl) ethylenediamine (TPEN,Sigma-Aldrich), was dissolved in phosphate buffer (PBS). TPEN (0.5μM,1μM,2μM,5μM,10μM) was added to the medium by a double-blinding manner. 2.3 Cell viability assessment The extent cultured INS-1 cell death was assessed by using the MTT assay (company) according to the manufacturer’s protocol. 2.4 Animals and drug administration Male hIAPP transgenic mice were used (breeding pairs were obtained from the Jackson Laboratory, West Grove, PA, USA) in this study. Thirty-six 1 month-old mice (20～25g) were housed in cages and fed a high-fat, high-sugar diet and distilled water under a 12-h light/dark cycle at a temperature of 22–25°C. Then they were divided into three groups. One group was given 20 mg/kg/day CQ (5mg/mL dissolved in 0.05% carboxymethylcellulose [CMC]) by gavage once a day for 1 month. Another group was given 10 mg/mL ZnSO4 in the water. The control group was given only the vehicle (0.05% CMC). All animals were handled according to the care and use of medical laboratory animals (Ministry of Health, Peoples Republic of China, 1998) and the guidelines of the laboratory animal ethical standards of China Medical University. 3
The body weight and glucose levels of the mice were monitored for 4 weeks. Blood samples were obtained by tail prick and blood glucose levels were measured using a portable blood glucose meter(Johnson, m211667, USA). 2.5 Co-staining by the immunofluorescence method Sections of pancreas or INS-1 cells were preincubated with normal donkey serum (NDS; 1:20; Jackson Immuno Research Laboratory, West Grove,PA, USA) for 1 h. The sections and cells were separately incubated overnight in primary antibodies: rabbit anti-IAPP (1:100; Sigma-Aldrich) and goat anti-insulin (1:100; Santa Cruz) or rabbit anti-IAPP (1:100; Sigma-Aldrich) and mouse anti-glucagon (1:100; SigmaAldrich). After several rinses, the sections were incubated for 2 h with a mixture of secondary antibodies: Texas Red-conjugated donkey anti-rabbit IgG (1:200, Cell Signaling Technology) and fluorescein (FITC)-conjugated donkey anti-goat IgG (1:200, Cell Signaling Technology). The culture cells were incubated with the same secondary antibodies as the tissue sections. After rinsing with PBS, the sections and culture cells were mounted using an antifading mounting medium and examined under a fluorescence microscope. Images were collected and processed using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA, USA). To assess non-specific staining, sections were incubated with normal sera instead of primary antibodies. 2.6 Western blot analysis Pancreas tissue and INS-1 cells were homogenized in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1.0% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL leupeptin, 1.0 mM sodium orthovanadate, and 1.0 mM sodium fluoride) overnight at 4°C. Homogenates (50 μg of total protein) were boiled and then electrophoresed in 10 or 15% SDS-polyacrylamide gels and transferred onto PVDF membranes (Millipore). Membranes were then incubated with primary antibody of IAPP (1:500, SigmaAldrich), insulin (1:200, Sigma-Aldrich), CPE(1:200, Sigma-Aldrich). To control sample loading and protein transfer, the membranes were stripped and reprobed with anti-GAPDH (1:5000, KC-5G5, Kang Chen, 0811). The levels of proteins were normalized with GAPDH. 2.7 Statistical analysis Data were presented as mean ± SD. Statistical analysis was performed using student t-test or one-way ANOVA. A value of P<0.05 was considered to be statistically significant. 4
3. Results 3.1 Zinc regulates a key enzyme of IAPP secretion in INS-1 cells To demonstrate the critical role in contributing to islet beta cells, we choose the INS-1 cell, which is a rat’s insulin tumor cell line, and it has a hormone secretion capacity similar to that of islet beta cells. Then, to verify that zinc might be involved in IAPP and insulin secretion, we use the INS-1 cells transfected with hIAPP DNA as an in vitro model with exogenously added or chelated zinc. By MTT colorimetric assay, the concentration of ZnSO4 was determined to be 50 μM and TPEN to be 1.0 μM (Fig1A,B). These concentrations did not affect the cell survival rate. Next we examined whether changing of zinc concentration influenced IAPP secretion. We choose an important enzyme (Carboxypeptidase E,CPE) in the IAPP synthesis. The level of CPE in IAPP transfected cells were measured using western blot (Fig.1C,D). Statistical analysis indicated that the exposure to zinc increased the expression of CPE. However, under the TPEN treated group, we found that the CPE level was decreasing dramatically. These findings indicate that Zinc contributed to the IAPP synthesis. Chelating zinc could decrease the level of CPE. 3.2 Chelating zinc upregulates the expression of insulin and attenuates the expression of IAPP In order to verify the role of zinc, we further examined the insulin and IAPP in hIAPP-INS-1 cells. As shown in Fig.2A, with immunofluorescence double-staining, both IAPP and insulin were shown to express in the cytoplasm of hIAPP transfected INS-1 cells. In the cells treated with ZnSO4, the expression of IAPP increased, but the expression of insulin decreased. In cells treated with TPEN, the expression of insulin increased, but the expression of IAPP decreased. The result of the western blot further confirmed the ability of TPEN to improve cell function. The additional zinc ions reduced the secretion of insulin by 37% compared to that of the vehicle (P<0.01) ( Fig.2B,D), but increased IAPP protein by 160% (P<0.01) (Fig.2B,C). In cells treated with TPEN, the expression of insulin was increased by 152% (P<0.01) compared to that of the vehicle (Fig.2B,D), and the expression of IAPP decreased by 50% (P<0.05) compared to that of the vehicle (Fig.2B,C). These findings indicate that zinc is likely the regulating factor for IAPP deposition in INS-1 cells.
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3.3 Chelating zinc decreased plasma glucose in hIAPP transgenic mice The in vitro study above with a cellular model indicated a role of zinc in IAPP deposition. We then investigated the zinc influence on the pancreas function in hIAPP transgenic mice. The zinc chelator, CQ, or vehicle control was administrated during one month. Plasma glucose level was used to assess the pancreas function. As shown in fig 4A, the blood glucose continued to rise in the vehicle group. Chelating zinc with CQ markedly reversed the blood glucose level in the hIAPP transgenic mice. These results suggest that zinc indeed participates in insulin secretion following IAPP deposition by hIAPP transgenic mice. 3.4 Chelating zinc reduces IAPP level and increases insulin level in hIAPP transgenic mice IAPP is a major component of islet amyloid deposits and coexists with insulin in islet beta cells (i.e., IAPP is released parallel to insulin).To detect the effects of CQ on the secretion of IAPP and insulin, immunofluorescence double-staining was used to observe the expression of IAPP and insulin in the islets. As shown in Fig.3A, the positive result for IAPP is red and is expressed in the cytoplasm of islet beta cells; the positive result for insulin is green, also expressed in the cytoplasm of islet beta cells. Under CQ treatment, in contrast to the vehicle, the expression of IAPP decreased and the expression of insulin increased. At the same time, to further clarify the sedimentary location of IAPP in the islet cells, glucagon was chosen as a marker for islet alpha cells. The expression of glucagon and IAPP was detected by immunofluorescence double-staining. The results showed IAPP and glucagon were expressed in different parts of the islet—IAPP was positioned in the beta cells, but glucagon was positioned in the alpha cells—CQ affects only the deposition of IAPP, it had no significant effect on the glucagon (Fig.3B). The western blot test was used to further detect the effect of CQ on the expression levels of IAPP and insulin protein. As shown in Fig.4, zinc obviously increased the synthesis of IAPP in the pancreas cells of hIAPP transgenic mice compared to those of the vehicle; the level of IAPP had increased by 149.2% (P<0.01) (Figs. 4B, C).In contrast, the expression of IAPP in pancreas cells of CQ-treated mice had reduced by 53.3% (P< 0.01) (Figs. 4B, C). Because IAPP and insulin coexist in islet beta cells, they exhibited competitive inhibition with each other and the secretion of insulin was 6
also affected by CQ. CQ could improve the function of the islet cells in the pancreas of CQ-treated mice. The amount of insulin increased by 140.7% (P < 0.01) compared to that in the vehicle (Figs. 4B, D) and the level of zinc at a higher than normal concentration reduced the secretion of insulin by 40.4% (P<0.01) compared to that of the vehicle (Figs. 4B, D). 4. Discussion The present study provides the first direct evidence that zinc is a critical contributing factor to regulating IAPP. We found that high concentration of exogenous zinc deteriorates the INS-1 cell function under hIAPP upregulated condition. Moreover, chelating zinc could reduce the IAPP level and increase insulin secretion. We also found that chelating zinc could decrease plasma glucose level and inhibit IAPP deposition in hIAPP transgenic mice. Findings from these in hIAPP transfected INS-1 cells and in hIAPP transgenic mice experiments reveal a novel pathological role of zinc that works on IAPP depositon in type 2 DM. Amyloid deposits were found in 90% of the islets examined in the autopsy material from patients with type 2 DM
[21]
.The amyloid deposits were caused by an
abnormal accumulation of IAPP[22]. Abnormal accumulation of IAPP severely affected the function of islet beta cells, and was closely related to type 2 DM [23,24]. Zinc binding sites are contained in IAPP, a zinc-binding protein[25]. In our study, we first found that a zinc chelator can inhibit the deposition of IAPP and promote the expression of insulin in hIAPP transgenic mice and IAPP transfected INS-1 cells. IAPP is the hormone secreted by islet beta cells, stimulated by glucose and secreted in parallel with insulin. IAPP reaches the body organs through the bloodstream and regulates many bodily functions[26,27]. Islet beta cells are rich with zinc ions. In the secretory vesicles of insulin, every 6 insulin monomers can be combined with 2 zinc ions to form the insulin hexamer[28].Because of insulin resistance in type 2 DM, the secretion of insulin increases accompanied by an increased secretion of IAPP in beta cells. With the release of insulin, zinc ions transient aggregate near the membrane of beta cells. Moreover, patients with type 2 DM often have an abnormal expression of zinc ion transporter gene[29,30]. Zinc ions cannot enter the insulin secretory vesicles and abnormally accumulate in the cytoplasm[31]. Interestingly, recent in vitro studies have shown that zinc ions can combine with IAPP through His18, making a crosslink with adjacent IAPP monomers and eventually forming fibrous IAPP aggregates[32]. In previous studies, it was 7
confirmed that zinc chelator can inhibit the amyloidogenic pathway in the APP/PS1 mouse brain[33], therefore, it was assessed whether the zinc chelator might have a wholesome effect on beta cells. hIAPP transgenic mice were used in this study to determine whether zinc chelator could reduce amyloid deposition in the islets of diabetic patients. The mice were treated with CQ, a zinc chelator. The expression of IAPP and insulin was detected by immunofluorescence double-staining and the western blot analysis. From the results, it was found that CQ can inhibit IAPP deposition and promote the expression of insulin in the islets of hIAPP transgenic mice. The results proved that zinc chelator can reduce amyloid deposition in islet beta cells and improve the symptoms of DM. To further observe the effects of zinc and zinc chelator on IAPP synthesis and secretion, in vitro experiments were performed. ZnSO4 and TPEN were added to hIAPP transfected INS-1 cells (the correct dose concentration was filtered out using the MTT method). The in vitro experiments resulted in data similar to those of in vivo experiments. In hIAPP transfected INS-1 cells, chelating zinc with TPEN reduced the expression of IAPP and increased the secretion of insulin. Our findings were consistent with those of a previous study that confirmed that a low concentration of zinc ion can suppress amyloid deposition[34]. Another in vitro study used immunofluorescence and transmission electron microscopy to observe the effects of zinc on the aggregation of IAPP in beta cells. The results showed that zinc ion in higher than normal concentrations will accelerate abnormal accumulation of IAPP [35]. Mature IAPP was synthesis in islet, carboxypeptidase E(CPE), prohormone convertase (PC)1/3 and prohormone convertase (PC)2 were key enzymes. And IAPP metabolizes through the urinary tract, regulated by metalloproteinase, insulin degrading enzyme (IDE) and other enzymes. If the activity of these enzymes was abnormal, IAPP intermediate increased, compared with mature IAPP, intermediates more easily formed amyloidosis aggregation[17,36]. Studies had confirmed that CPE and IDE were both metalloproteins with zinc-binding sites on the molecule, and zinc ions could affect the activity of IDE[32,37,38]. In our experiment, we had detected the effect of zinc on the expression of CPE, western blot result showed that high concentration of zinc ions increasing the expression of CPE in hIAPP transfected INS-1 cells. However,treated with TPEN, the level of CPE decreased dramatically. Zinc ions may affect the deposition of IAPP by regulating the synthesis of CPE. In summary, the present study has shown that zinc chelator treatment markedly 8
reduces IAPP accumulation in the islet beta cells of hIAPP transgenic mice or hIAPP transfected INS-1 cells. This study was the first in vitro experiment to demonstrate that a zinc chelator could affect the secretion of insulin and IAPP deposition, and could improve the symptoms of DM. These findings were expected to provide information for a new therapeutic strategy of type 2 DM.
Disclosures: The author has no financial conflicts of interests to declare.
Acknowledgments This study was supported by the Natural Science Foundation of China (81170561, 81170775), and the Key Project of China’s National Basic Research Program (973 Program) (2012CB722405).
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polypeptide aggregation to develop anti-amyloidogenic agents for type-2 diabetes. Biochimie. 2011, 93: 793-805. [25] Brender JR, Hartman K, Nanga RP, et al. Role of zinc in human islet amyloid polypeptide aggregation. Journal of the American Chemical Society. 2010, 132(26):8973-8983. [26] Morita S, Ueyama M, Sakagashira S, et al. Protective role of human insulin against the cytotoxicity associated with human mutant S20G islet amyloid polypeptide. Diabetes Investig, 2013, 4(5): 436-444. [27] Westwell-Roper CY, Ehses JA, Verchere CB. Resident macrophages mediate islet amyloid polypeptide-induced islet IL-1β production and β-cell dysfunction. Diabetes. 2014, 63(5): 1698-1711. [28] Park JY, No HS, Ahn YR, et al. Pathologic changes and glucose homeostasis according to expression of human islet amyloid polypeptide in type 2 diabetic patients. Journal of Histochemistry & Cytochemistry. 2010, 58: 731-740. [29] Quraishi I, Collins S, Pestaner JP, et al. Role of zinc and zinc transporters in the molecular pathogenesis of diabetes mellitus. Med Hypotheses, 2005, 65(5):887-892. [30] Foster M, Petocz P, Samman S. Inflammation markers predict zinc transporter gene expression in women with type 2 diabetes mellitus. J Nutr Biochem, 2013, 24(9):1655-1661. [31] Scott LJ, Mohlke KL, Bonnycastle LL, et al. A genome-wide association study of type 2 diabetes in Finns detects multiple susceptibility variants. Science. 2007, 316(5829):1341- 1345. [32] Bellia F, Grasso G. The role of copper (II) and zinc (II) in the degradation of human and murine IAPP by insulin-degrading enzyme. J Mass Spectrom. 2014, 49(4):274-279. [33] Tao Wang, Chun-Yan Wang, Zhong-Yan Shan, et al. Clioquinol Reduces Zinc Accumulation in Neuritic Plaques and Inhibits the Amyloidogenic Pathway in APP/PS1 Transgenic Mouse Brain. Journal of Alzheimer’s Disease, 2012, (29):549–559. [34]
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Fig1. Effects of zinc on the expression of CPE in INS-1cells.(A) cell viability of INS-1cells treated with ZnSO4. (B)cell viability of INS-1cells treated with TPEN. Results showed that if the concentration of ZnSO4 was 50 μM and TPEN to be 1.0 μM, did not affect cell survival rate.(C) Immunoblot analyses of the protein levels of CPE in hIAPP transfected INS-1 cells. GAPDH was used as a loading control. (D) A marked reduction in CPE was detected in TPEN-treated cells. In addition, CPE was significantly increased after zinc sulfate (ZnSO4) treatment.
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Fig2.Expression of islet amyloidal polypeptide (IAPP) and insulin in human (h)IAPP transfected INS-1 cells. (A)Double-immunofluorescence labeling of IAPP and insulin isoforms show that both IAPP and insulin are located in the cytoplasm of INS-1cells. The expression of IAPP was reduced significantly after being treated with TPEN, and TPEN-treated cells had an increase in the expression of insulin compared with that of the vehicle. (Scale bar = 50 um). (B) Immunoblot analyses of the protein levels of IAPP in hIAPP-INS-1 cells. GAPDH was used as a loading control. (C) A marked reduction in IAPP was detected in TPEN-treated cells. In addition, IAPP was significantly increased after zinc sulfate (ZnSO4)treatment.(D) TPEN treatment increased the expression of insulin, but ZnSO4 treatment significantly decreased the level of insulin compared with that of the vehicle mice﹡P < 0.05,﹡﹡P < 0.01.
15
Fig 3. (A)Expression of islet amyloidal polypeptide (IAPP) and insulin in the islets of Langerhans (islets) in human (h)IAPP transgenic mice.Double-immunofluorescence labeling of IAPP and insulin isoforms showing that both IAPP and insulin are located in the beta cells, compared with those of the vehicle, and the expression of IAPP was reduced in the islets of CQ-treated mice, but the expression of insulin was increased. (B) Expression of islet amyloidal polypeptide (IAPP) and glucagon in the islets of Langerhans (islets) in human (h)IAPP transgenic mice. Doubleimmunofluorescence labeling of IAPP and glucagon isoforms showing that IAPP is located in the beta cells, but glucagon is located in the alpha cells. Clioquinol (CQ) or zinc sulfate (ZnSO4) affected only the expression of IAPP. They had no effect on glucagon. (Scale bar = 50 um)
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Fig.4 (A) Effect of zinc on the level of blood glucose in hIAPP transgenic mice. Chelating zinc with CQ markedly reversed blood glucose level in hIAPP transgenic mice.(B) Immunoblot analyses of the protein levels of IAPP and insulin in the transgenic mouse pancreas. GAPDH was used as a loading control. (C) A marked reduction in IAPP was detected in the clioquinol (CQ)-treated mouse pancreas. Also, the level of IAPP protein was significantly increased after zinc sulfate (ZnSO4) treatment. (D) CQ treatment increased the level of insulin,but ZnSO4 significantly reduced the expression level of insulin compared with that in the vehicle mice.﹡﹡ P < 0.01.
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S0040-8166(16)30260-9 http://dx.doi.org/doi:10.1016/j.tice.2017.01.002 YTICE 1068
To appear in:
Tissue and Cell
Received date: Revised date: Accepted date:
10-11-2016 9-1-2017 10-1-2017
Please cite this article as: Tian, He, Zhong, Man-Li, Shan, Zhong-Yan, Teng, WeiPing, Chi, Zhi-Hong, Xie, Jing-Wei, Wang, Tao, Tian, Juan, Xin, Na, Wang, Zhan-You, Chelating Zinc decreased islet amyloidal polypeptide deposition in beta cell in vivo and in vitro.Tissue and Cell http://dx.doi.org/10.1016/j.tice.2017.01.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chelating Zinc decreased islet amyloidal polypeptide deposition in beta cell in vivo and in vitro
He Tiana,c,Man-Li Zhonga,Zhong-Yan Shanb,Wei-Ping Tengb, Zhi-Hong Chia,Jing-Wei Xiea, Tao Wanga, Juan Tiana, Na Xina, Zhan-You Wanga,b﹡
a
Department of Pathophysiology, China Medical University, Shenyang, China
b
Key Laboratory of Endocrine Diseases of Liaoning Province, China Medical
University, Shenyang, China c
Department of Histology and Embryology, Jinzhou Medical University, Jinzhou,
China
* To whom correspondence should be addressed: Zhan-You Wang, Ph.D. Department of Pathophysiology China Medical University No. 77. Puhe Road Shenyang, 110819, P. R. China Tel: +86-24-23256666 5521 E-mail: [email protected]
Highlights The pathological deposition of islet amyloidal polypeptide (IAPP) could be an important factor in the survival of beta cells and in insulin secretion. Synthesis and metabolism of IAPP is closely related to zinc, and the abnormal accumulation of zinc in islet beta cells could be an important factor in amyloid deposition. In this manuscript, we delineated that zinc chelators markedly reduced the expression levels of IAPP and increased the levels of insulin in hIAPP-INS-1 cells and hIAPP mice. The present data indicate that zinc chelator is able to reduce the expression of IAPP, regulating the homeostasis of zinc may be become a new strategy in diabetes mellitus therapy.
Abstract The pathological deposition of islet amyloidal polypeptide (IAPP) could be an important factor in the survival of beta cells and in insulin secretion. The synthesis and metabolism of IAPP is closely related to zinc, and the abnormal accumulation of zinc in islet beta cells could be an important factor in amyloid deposition. Regulating the homeostasis of zinc in diabetes mellitus (DM) patients and maintaining the dynamic balance of the generation and degradation of IAPP, could become a new strategy in DM therapy. To verify the effects of zinc on IAPP and the mechanisms by which zinc controls IAPP deposition, human(h)IAPP transgenic mice and hIAPP transfection insulin-producing beta cell lines (INS-1) were used in this study. Clioquinol (CQ) and TPEN (zinc chelators) were either given to the mice directly or injected into the cells. By means of immunofluorescence double-staining and the western blot analysis, we showed that CQ treatment in hIAPP mice markedly reduced the expression levels of IAPP and increased the levels of insulin. In hIAPP-INS-1 cells treated with TPEN, the expression of IAPP was significantly lower than that of the control group, but the insulin level was higher. Collectively, the present data indicate that a zinc chelator is able to reduce the expression of IAPP in hIAPP transgenic mice and hIAPP-INS-1 cells.
Key words: IAPP; zinc chelator; hIAPP transgenic mice; hIAPP transfected cell
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1.Introduction The major pathophysiologic processes of type 2 diabetes include insulin resistance and decreased beta cell function, the latter of which includes loss of beta cell mass and its dysfunction[1]. Zinc is an essential trace element that plays a critical role in many metabolic disease states including diabetes and cancer[2]. Recent studies have highlighted zinc’s dynamic role as a “cellular second messenger” in the control of insulin signaling and glucose homeostasis[3]. Zinc ions play a role in insulin-induced glucose transport and glycemic control[4].Changes of zinc concentrations in the cytosol and organelles may contribute to insulin responsiveness and thus promote insulin resistance. However, questions on how zinc ions are regulated to cell signaling are largely unknown. IAPP, also known as amylin, is a polypeptide composed of 37 amino acids that is produced and secreted by pancreatic beta cells[5,6]. Recent studies demonstrated the deposition of islet amyloidal polypeptide (IAPP) in the pancreatic islet in the autopsy material of 90% of type 2 DM patients, accompanied by a significant reduction in the number of beta cells[7]. IAPP can control appetite, inhibit gastric emptying, inhibit insulin secretion, and perform other physiological functions [8]. Several studies have shown that a large number of amyloid deposits were found in the islets of Langerhans (islets) in transgenic mice that expressed human (h)IAPP[9,10]. The increase of IAPP deposition is regarded as a crucial event for insulin resistance and hyperglycemia. Deposition of IAPP can also induce pancreatic beta cells apoptosis in the occurrence and development of type 2 DM[11,12]. These studies have demonstrated that deposition of IAPP contributes to the progression of the type 2 diabetes process. Zinc ion is closely related to the synthesis and metabolism of IAPP[13,14]. In beta cells, precursor (prepro) IAPP is processed by prohormone convertase 2 (PC2), prohormone convertase 1/3 (PC1/3), and carboxypeptidase E (CPE) to form mature IAPP[15-17]. The IAPP monomer does not form amyloid deposition, but the fibrous aggregates form the amyloid deposition[18,19]. It has been reported that the zinc ion could bind to the His18 of IAPP (18th histidine residue), so that the adjacent IAPP monomer is crosslinked, eventually forming fibrous IAPP aggregates[20]. These phenomena prompted the consideration that the imbalance in zinc ion metabolism in islet beta cells might be an important factor in amyloid deposition. Regulating the homeostasis of zinc ion in DM patients and maintaining the dynamic balance of generation and degradation of IAPP might become the new strategy in DM therapy. In this 2
study, using INS-1 cells and the hIAPP transgenic mice model, we investigated the contribution of Zinc to deposition of IAPP, and explored the mechanism of zinc-mediated IAPP deposition in vitro and in vivo. 2.Materials and methods 2.1 INS-1 cell culture and treatment Rat INS-1 cells were grown to RM1640 (Invitrogen Technology) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen Technology), 100 IU/mL penicillin, and 100 μg/mL streptomycin at 37°C in humidified 5% CO2 air. Then the cells were infected with LV- hIAPP and LV-scramble (company) at a multiplicity of infection of 20 after 6 days according to the manufacturer’s instructions. 2.2 Exposure to ZnSO4 and Zinc chelator After (72h) transfection, when 80% of the bottom of the buffer solution was covered by cells, ZnSO4 and TPEN were added to the medium. ZnSO4 was added in a double blind manner to the medium with concentrations (10μM,20μM,50μM,70μM,100μM). Another group, a zinc special chelator, N,N,Nʹ,Nʹ-tetrakis(2-pyrid-ylmethyl) ethylenediamine (TPEN,Sigma-Aldrich), was dissolved in phosphate buffer (PBS). TPEN (0.5μM,1μM,2μM,5μM,10μM) was added to the medium by a double-blinding manner. 2.3 Cell viability assessment The extent cultured INS-1 cell death was assessed by using the MTT assay (company) according to the manufacturer’s protocol. 2.4 Animals and drug administration Male hIAPP transgenic mice were used (breeding pairs were obtained from the Jackson Laboratory, West Grove, PA, USA) in this study. Thirty-six 1 month-old mice (20～25g) were housed in cages and fed a high-fat, high-sugar diet and distilled water under a 12-h light/dark cycle at a temperature of 22–25°C. Then they were divided into three groups. One group was given 20 mg/kg/day CQ (5mg/mL dissolved in 0.05% carboxymethylcellulose [CMC]) by gavage once a day for 1 month. Another group was given 10 mg/mL ZnSO4 in the water. The control group was given only the vehicle (0.05% CMC). All animals were handled according to the care and use of medical laboratory animals (Ministry of Health, Peoples Republic of China, 1998) and the guidelines of the laboratory animal ethical standards of China Medical University. 3
The body weight and glucose levels of the mice were monitored for 4 weeks. Blood samples were obtained by tail prick and blood glucose levels were measured using a portable blood glucose meter(Johnson, m211667, USA). 2.5 Co-staining by the immunofluorescence method Sections of pancreas or INS-1 cells were preincubated with normal donkey serum (NDS; 1:20; Jackson Immuno Research Laboratory, West Grove,PA, USA) for 1 h. The sections and cells were separately incubated overnight in primary antibodies: rabbit anti-IAPP (1:100; Sigma-Aldrich) and goat anti-insulin (1:100; Santa Cruz) or rabbit anti-IAPP (1:100; Sigma-Aldrich) and mouse anti-glucagon (1:100; SigmaAldrich). After several rinses, the sections were incubated for 2 h with a mixture of secondary antibodies: Texas Red-conjugated donkey anti-rabbit IgG (1:200, Cell Signaling Technology) and fluorescein (FITC)-conjugated donkey anti-goat IgG (1:200, Cell Signaling Technology). The culture cells were incubated with the same secondary antibodies as the tissue sections. After rinsing with PBS, the sections and culture cells were mounted using an antifading mounting medium and examined under a fluorescence microscope. Images were collected and processed using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA, USA). To assess non-specific staining, sections were incubated with normal sera instead of primary antibodies. 2.6 Western blot analysis Pancreas tissue and INS-1 cells were homogenized in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1.0% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL leupeptin, 1.0 mM sodium orthovanadate, and 1.0 mM sodium fluoride) overnight at 4°C. Homogenates (50 μg of total protein) were boiled and then electrophoresed in 10 or 15% SDS-polyacrylamide gels and transferred onto PVDF membranes (Millipore). Membranes were then incubated with primary antibody of IAPP (1:500, SigmaAldrich), insulin (1:200, Sigma-Aldrich), CPE(1:200, Sigma-Aldrich). To control sample loading and protein transfer, the membranes were stripped and reprobed with anti-GAPDH (1:5000, KC-5G5, Kang Chen, 0811). The levels of proteins were normalized with GAPDH. 2.7 Statistical analysis Data were presented as mean ± SD. Statistical analysis was performed using student t-test or one-way ANOVA. A value of P<0.05 was considered to be statistically significant. 4
3. Results 3.1 Zinc regulates a key enzyme of IAPP secretion in INS-1 cells To demonstrate the critical role in contributing to islet beta cells, we choose the INS-1 cell, which is a rat’s insulin tumor cell line, and it has a hormone secretion capacity similar to that of islet beta cells. Then, to verify that zinc might be involved in IAPP and insulin secretion, we use the INS-1 cells transfected with hIAPP DNA as an in vitro model with exogenously added or chelated zinc. By MTT colorimetric assay, the concentration of ZnSO4 was determined to be 50 μM and TPEN to be 1.0 μM (Fig1A,B). These concentrations did not affect the cell survival rate. Next we examined whether changing of zinc concentration influenced IAPP secretion. We choose an important enzyme (Carboxypeptidase E,CPE) in the IAPP synthesis. The level of CPE in IAPP transfected cells were measured using western blot (Fig.1C,D). Statistical analysis indicated that the exposure to zinc increased the expression of CPE. However, under the TPEN treated group, we found that the CPE level was decreasing dramatically. These findings indicate that Zinc contributed to the IAPP synthesis. Chelating zinc could decrease the level of CPE. 3.2 Chelating zinc upregulates the expression of insulin and attenuates the expression of IAPP In order to verify the role of zinc, we further examined the insulin and IAPP in hIAPP-INS-1 cells. As shown in Fig.2A, with immunofluorescence double-staining, both IAPP and insulin were shown to express in the cytoplasm of hIAPP transfected INS-1 cells. In the cells treated with ZnSO4, the expression of IAPP increased, but the expression of insulin decreased. In cells treated with TPEN, the expression of insulin increased, but the expression of IAPP decreased. The result of the western blot further confirmed the ability of TPEN to improve cell function. The additional zinc ions reduced the secretion of insulin by 37% compared to that of the vehicle (P<0.01) ( Fig.2B,D), but increased IAPP protein by 160% (P<0.01) (Fig.2B,C). In cells treated with TPEN, the expression of insulin was increased by 152% (P<0.01) compared to that of the vehicle (Fig.2B,D), and the expression of IAPP decreased by 50% (P<0.05) compared to that of the vehicle (Fig.2B,C). These findings indicate that zinc is likely the regulating factor for IAPP deposition in INS-1 cells.
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3.3 Chelating zinc decreased plasma glucose in hIAPP transgenic mice The in vitro study above with a cellular model indicated a role of zinc in IAPP deposition. We then investigated the zinc influence on the pancreas function in hIAPP transgenic mice. The zinc chelator, CQ, or vehicle control was administrated during one month. Plasma glucose level was used to assess the pancreas function. As shown in fig 4A, the blood glucose continued to rise in the vehicle group. Chelating zinc with CQ markedly reversed the blood glucose level in the hIAPP transgenic mice. These results suggest that zinc indeed participates in insulin secretion following IAPP deposition by hIAPP transgenic mice. 3.4 Chelating zinc reduces IAPP level and increases insulin level in hIAPP transgenic mice IAPP is a major component of islet amyloid deposits and coexists with insulin in islet beta cells (i.e., IAPP is released parallel to insulin).To detect the effects of CQ on the secretion of IAPP and insulin, immunofluorescence double-staining was used to observe the expression of IAPP and insulin in the islets. As shown in Fig.3A, the positive result for IAPP is red and is expressed in the cytoplasm of islet beta cells; the positive result for insulin is green, also expressed in the cytoplasm of islet beta cells. Under CQ treatment, in contrast to the vehicle, the expression of IAPP decreased and the expression of insulin increased. At the same time, to further clarify the sedimentary location of IAPP in the islet cells, glucagon was chosen as a marker for islet alpha cells. The expression of glucagon and IAPP was detected by immunofluorescence double-staining. The results showed IAPP and glucagon were expressed in different parts of the islet—IAPP was positioned in the beta cells, but glucagon was positioned in the alpha cells—CQ affects only the deposition of IAPP, it had no significant effect on the glucagon (Fig.3B). The western blot test was used to further detect the effect of CQ on the expression levels of IAPP and insulin protein. As shown in Fig.4, zinc obviously increased the synthesis of IAPP in the pancreas cells of hIAPP transgenic mice compared to those of the vehicle; the level of IAPP had increased by 149.2% (P<0.01) (Figs. 4B, C).In contrast, the expression of IAPP in pancreas cells of CQ-treated mice had reduced by 53.3% (P< 0.01) (Figs. 4B, C). Because IAPP and insulin coexist in islet beta cells, they exhibited competitive inhibition with each other and the secretion of insulin was 6
also affected by CQ. CQ could improve the function of the islet cells in the pancreas of CQ-treated mice. The amount of insulin increased by 140.7% (P < 0.01) compared to that in the vehicle (Figs. 4B, D) and the level of zinc at a higher than normal concentration reduced the secretion of insulin by 40.4% (P<0.01) compared to that of the vehicle (Figs. 4B, D). 4. Discussion The present study provides the first direct evidence that zinc is a critical contributing factor to regulating IAPP. We found that high concentration of exogenous zinc deteriorates the INS-1 cell function under hIAPP upregulated condition. Moreover, chelating zinc could reduce the IAPP level and increase insulin secretion. We also found that chelating zinc could decrease plasma glucose level and inhibit IAPP deposition in hIAPP transgenic mice. Findings from these in hIAPP transfected INS-1 cells and in hIAPP transgenic mice experiments reveal a novel pathological role of zinc that works on IAPP depositon in type 2 DM. Amyloid deposits were found in 90% of the islets examined in the autopsy material from patients with type 2 DM
[21]
.The amyloid deposits were caused by an
abnormal accumulation of IAPP[22]. Abnormal accumulation of IAPP severely affected the function of islet beta cells, and was closely related to type 2 DM [23,24]. Zinc binding sites are contained in IAPP, a zinc-binding protein[25]. In our study, we first found that a zinc chelator can inhibit the deposition of IAPP and promote the expression of insulin in hIAPP transgenic mice and IAPP transfected INS-1 cells. IAPP is the hormone secreted by islet beta cells, stimulated by glucose and secreted in parallel with insulin. IAPP reaches the body organs through the bloodstream and regulates many bodily functions[26,27]. Islet beta cells are rich with zinc ions. In the secretory vesicles of insulin, every 6 insulin monomers can be combined with 2 zinc ions to form the insulin hexamer[28].Because of insulin resistance in type 2 DM, the secretion of insulin increases accompanied by an increased secretion of IAPP in beta cells. With the release of insulin, zinc ions transient aggregate near the membrane of beta cells. Moreover, patients with type 2 DM often have an abnormal expression of zinc ion transporter gene[29,30]. Zinc ions cannot enter the insulin secretory vesicles and abnormally accumulate in the cytoplasm[31]. Interestingly, recent in vitro studies have shown that zinc ions can combine with IAPP through His18, making a crosslink with adjacent IAPP monomers and eventually forming fibrous IAPP aggregates[32]. In previous studies, it was 7
confirmed that zinc chelator can inhibit the amyloidogenic pathway in the APP/PS1 mouse brain[33], therefore, it was assessed whether the zinc chelator might have a wholesome effect on beta cells. hIAPP transgenic mice were used in this study to determine whether zinc chelator could reduce amyloid deposition in the islets of diabetic patients. The mice were treated with CQ, a zinc chelator. The expression of IAPP and insulin was detected by immunofluorescence double-staining and the western blot analysis. From the results, it was found that CQ can inhibit IAPP deposition and promote the expression of insulin in the islets of hIAPP transgenic mice. The results proved that zinc chelator can reduce amyloid deposition in islet beta cells and improve the symptoms of DM. To further observe the effects of zinc and zinc chelator on IAPP synthesis and secretion, in vitro experiments were performed. ZnSO4 and TPEN were added to hIAPP transfected INS-1 cells (the correct dose concentration was filtered out using the MTT method). The in vitro experiments resulted in data similar to those of in vivo experiments. In hIAPP transfected INS-1 cells, chelating zinc with TPEN reduced the expression of IAPP and increased the secretion of insulin. Our findings were consistent with those of a previous study that confirmed that a low concentration of zinc ion can suppress amyloid deposition[34]. Another in vitro study used immunofluorescence and transmission electron microscopy to observe the effects of zinc on the aggregation of IAPP in beta cells. The results showed that zinc ion in higher than normal concentrations will accelerate abnormal accumulation of IAPP [35]. Mature IAPP was synthesis in islet, carboxypeptidase E(CPE), prohormone convertase (PC)1/3 and prohormone convertase (PC)2 were key enzymes. And IAPP metabolizes through the urinary tract, regulated by metalloproteinase, insulin degrading enzyme (IDE) and other enzymes. If the activity of these enzymes was abnormal, IAPP intermediate increased, compared with mature IAPP, intermediates more easily formed amyloidosis aggregation[17,36]. Studies had confirmed that CPE and IDE were both metalloproteins with zinc-binding sites on the molecule, and zinc ions could affect the activity of IDE[32,37,38]. In our experiment, we had detected the effect of zinc on the expression of CPE, western blot result showed that high concentration of zinc ions increasing the expression of CPE in hIAPP transfected INS-1 cells. However,treated with TPEN, the level of CPE decreased dramatically. Zinc ions may affect the deposition of IAPP by regulating the synthesis of CPE. In summary, the present study has shown that zinc chelator treatment markedly 8
reduces IAPP accumulation in the islet beta cells of hIAPP transgenic mice or hIAPP transfected INS-1 cells. This study was the first in vitro experiment to demonstrate that a zinc chelator could affect the secretion of insulin and IAPP deposition, and could improve the symptoms of DM. These findings were expected to provide information for a new therapeutic strategy of type 2 DM.
Disclosures: The author has no financial conflicts of interests to declare.
Acknowledgments This study was supported by the Natural Science Foundation of China (81170561, 81170775), and the Key Project of China’s National Basic Research Program (973 Program) (2012CB722405).
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Fig1. Effects of zinc on the expression of CPE in INS-1cells.(A) cell viability of INS-1cells treated with ZnSO4. (B)cell viability of INS-1cells treated with TPEN. Results showed that if the concentration of ZnSO4 was 50 μM and TPEN to be 1.0 μM, did not affect cell survival rate.(C) Immunoblot analyses of the protein levels of CPE in hIAPP transfected INS-1 cells. GAPDH was used as a loading control. (D) A marked reduction in CPE was detected in TPEN-treated cells. In addition, CPE was significantly increased after zinc sulfate (ZnSO4) treatment.
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Fig2.Expression of islet amyloidal polypeptide (IAPP) and insulin in human (h)IAPP transfected INS-1 cells. (A)Double-immunofluorescence labeling of IAPP and insulin isoforms show that both IAPP and insulin are located in the cytoplasm of INS-1cells. The expression of IAPP was reduced significantly after being treated with TPEN, and TPEN-treated cells had an increase in the expression of insulin compared with that of the vehicle. (Scale bar = 50 um). (B) Immunoblot analyses of the protein levels of IAPP in hIAPP-INS-1 cells. GAPDH was used as a loading control. (C) A marked reduction in IAPP was detected in TPEN-treated cells. In addition, IAPP was significantly increased after zinc sulfate (ZnSO4)treatment.(D) TPEN treatment increased the expression of insulin, but ZnSO4 treatment significantly decreased the level of insulin compared with that of the vehicle mice﹡P < 0.05,﹡﹡P < 0.01.
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Fig 3. (A)Expression of islet amyloidal polypeptide (IAPP) and insulin in the islets of Langerhans (islets) in human (h)IAPP transgenic mice.Double-immunofluorescence labeling of IAPP and insulin isoforms showing that both IAPP and insulin are located in the beta cells, compared with those of the vehicle, and the expression of IAPP was reduced in the islets of CQ-treated mice, but the expression of insulin was increased. (B) Expression of islet amyloidal polypeptide (IAPP) and glucagon in the islets of Langerhans (islets) in human (h)IAPP transgenic mice. Doubleimmunofluorescence labeling of IAPP and glucagon isoforms showing that IAPP is located in the beta cells, but glucagon is located in the alpha cells. Clioquinol (CQ) or zinc sulfate (ZnSO4) affected only the expression of IAPP. They had no effect on glucagon. (Scale bar = 50 um)
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Fig.4 (A) Effect of zinc on the level of blood glucose in hIAPP transgenic mice. Chelating zinc with CQ markedly reversed blood glucose level in hIAPP transgenic mice.(B) Immunoblot analyses of the protein levels of IAPP and insulin in the transgenic mouse pancreas. GAPDH was used as a loading control. (C) A marked reduction in IAPP was detected in the clioquinol (CQ)-treated mouse pancreas. Also, the level of IAPP protein was significantly increased after zinc sulfate (ZnSO4) treatment. (D) CQ treatment increased the level of insulin,but ZnSO4 significantly reduced the expression level of insulin compared with that in the vehicle mice.﹡﹡ P < 0.01.
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