J Mol Med DOI 10.1007/s00109-015-1303-1

ORIGINAL ARTICLE

Betacellulin ameliorates hyperglycemia in obese diabetic db/db mice Yoon Sin Oh 1,2 & Seungjin Shin 3 & Hui Ying Li 1,6 & Eun-Young Park 7 & Song Mi Lee 1,6 & Cheol Soo Choi 1,2 & Yong Lim 4 & Hye Seung Jung 5 & Hee-Sook Jun 1,2,6

Received: 10 July 2014 / Revised: 20 April 2015 / Accepted: 20 May 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract We found that administration of a recombinant adenovirus (rAd) expressing betacellulin (BTC) into obese diabetic db/ db mice ameliorated hyperglycemia. Exogenous glucose clearance was significantly improved, and serum insulin levels were significantly higher in rAd-BTC-treated mice than rAd-β-gal-treated control mice. rAd-BTC treatment increased insulin/bromodeoxyuridine double-positive cells in the islets, and islets from rAd-BTC-treated mice exhibited a significant increase in the level of G1-S phase-related cyclins as compared with control mice. In addition, BTC treatment increased messenger RNA (mRNA) and protein levels of these cyclins and cyclin-dependent kinases in MIN-6 cells. BTC treatment induced intracellular Ca2+ levels through phospholipase C-γ1 activation, and upregulated calcineurin B (CnB1) levels as well as calcineurin activity. Upregulation of CnB1 by BTC

Electronic supplementary material The online version of this article (doi:10.1007/s00109-015-1303-1) contains supplementary material, which is available to authorized users.

treatment was observed in isolated islet cells from db/db mice. When treated with CnB1 small interfering RNA (siRNA) in MIN-6 cells and isolated islets, induction of cell cycle regulators by BTC treatment was blocked and consequently reduced BTC-induced cell viability. As well as BTC’s effects on cell survival and insulin secretion, our findings demonstrate a novel pathway by which BTC controls beta-cell regeneration in the obese diabetic condition by regulating G1-S phase cell cycle expression through Ca2+ signaling pathways. Key messages & Administration of BTC to db/db mice results in amelioration of hyperglycemia. & BTC stimulates beta-cell proliferation in db/db mice. & Ca2+ signaling was involved in BTC-induced beta-cell proliferation. & BTC has an anti-apoptotic effect and potentiates glucosestimulated insulin secretion. Keywords Betacellulin . db/db mice . Cell cycle . Calcineurin B1 . Beta cell regeneration

* Hee-Sook Jun [email protected]

Introduction 1

Lee Gil Ya Cancer and Diabetes Institute, Gachon University, 7-45 Songdo-dong, Yeonsu-ku, Incheon, Korea

2

Gachon Medical Research Institute, Gil Hospital, Incheon, Korea

3

Northwestern University, Evanston, IL, USA

4

Department of Microbiology, Chosun University College of Medicine, Chonnam, Korea

5

Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea

6

College of Pharmacy, Gachon University, Incheon, Korea

7

College of Pharmacy, Mokpo National University, Jeonnam, Korea

Type 2 diabetes is characterized by insulin resistance and the reduction of the functional pancreatic beta-cell mass. Although there is an initial compensatory increase of the beta-cell mass in response to insulin resistance, diabetes occurs when the functional beta-cell mass fails to expand sufficiently [1]. Finding ways to preserve or increase the mass of functional beta-cells in diabetic patients is a key strategy in controlling type 2 diabetes in humans [2]. Various nutrients, peptide hormones, and growth factors have been implicated as regulators of the beta-cell mass.

J Mol Med

Previously, we reported that administration of a recombinant adenovirus expressing betacellulin (rAd-BTC), a member of the epidermal growth factor (EGF) family, increases the number of pancreatic beta-cells and restores normoglycemia both chemically induced and spontaneously autoimmune type 1 diabetic mice [3]. Other researchers also reported the effect of BTC on the remission of type 1 diabetic animal models such as streptozotocin-treated mice, alloxan-induced mice, and 90 % pancreatectomized rats [4–6]. However, the effect of BTC on the control of diabetes in type 2 diabetic animal models has not been examined. In this study, we investigated whether BTC treatment could regulate blood glucose levels in a type 2 diabetic animal model and determined the mechanisms involved in BTC-induced beta-cell proliferation and regeneration. We found that administration of BTC to db/db mice, a rodent model of obese type 2 diabetes, resulted in amelioration of hyperglycemia by stimulating in vivo regeneration of beta-cells. Moreover, we demonstrated that Ca2+ signaling-dependent regulation of cyclins and cyclin-dependent kinases (CDKs) was involved in betacell proliferation by BTC treatment.

Methods and materials Materials Recombinant mouse BTC was from R&D Systems (Minneapolis, MN, USA). Polyclonal antibodies against cyclin D2, cyclin A2, CDK2, CDK4, p19, and p27 and monoclonal antibody against cyclin E2 and secondary horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Antibodies against phospho-PLC-γ1 (Tyr-783), phopho-AKT (Ser-473), phopho-ERK1/2, PLC-γ1, AKT, ERK1/2, and Bcl-2 were from Cell Signaling (Danver, MA, USA). Monoclonal antibody against BrdU and polyclonal guinea pig anti-insulin antibody were from Dako (Carpinteria, CA, USA). Polyclonal antibody against Ki67 was from Novus Biologicals (Littleton, CO, USA) All other biochemical reagents were from Sigma (St. Louis, MO, USA) or Invitrogen (Carlsbad, CA, USA). Animals db/db mice (C57BL/KsJ background) were obtained from Jackson Laboratories (Bar Harbor, ME, USA; KRIBB, Daejeon, Korea). Mice with blood glucose levels higher than 400 mg/dl (6–8 weeks of age) were considered to be diabetic. Diabetic db/db mice were intravenously injected with rAdBTC or rAd-β-gal (4×1011 particles/ mice) through the tail vein [3]. C57BL6 mice were obtained from the Orient Bio Inc. (Gyeonggi, Korea). All experiments using mice were approved by the Institutional Animal Care and Use Committee at the Rosalind Franklin University of Medicine and Science and by the animal care committee of the Gachon University.

Cell culture MIN-6 cells were cultured and treated with or without 0.5 nM of BTC, as described previously [7]. Cell viability was checked using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. Isolation of islets from mice Islets of Langerhans were isolated from C57BL6 mice (8–10 weeks old) and db/db mice (6–8 weeks) by the collagenase digestion technique [8]. Healthy islets of appropriate size were hand-picked under a stereomicroscope. The islets were dissociated into single cells by trypsinization. 3

H-thymidine incorporation assay For the measurement of DNA synthesis, 1 μCi/ml of 3H-thymidine (925 GBq/mmol, Amersham) was added to each well for the last 4 h of culture. 3 H-radioactivity was measured in a liquid scintillation counter. Glucose and insulin tolerance tests Glucose and insulin tolerance tests were conducted 8 weeks after rAd-BTC or rAd-βgal treatment. For glucose tolerance tests, mice were fasted overnight and then injected with glucose (2 g/kg body weight, intraperitoneal (i.p.)). For insulin tolerance tests, mice were fasted for 4 h and then injected with insulin (1 U/kg body weight, i.p.). Glucose levels were measured in the tail vein blood at 0, 30, 60, 90, and 120 min after injection. Immunohistochemical analyses At 3, 7, and 60 days after viral injection, pancreatic or liver tissues were fixed in 10 % buffered formalin and embedded in paraffin. The tissues were incubated with anti-insulin, anti-BrdU, or anti-Ki67 antibodies, and fluorescein isothiocyanate-conjugated anti-guinea pig IgG, rhodamine-conjugated anti-mouse IgG, or horseradish peroxidase anti-rabbit IgG was used as secondary antibody. Fluorescence was imaged using a laser scanning confocal microscope (Carl Zeiss Inc.), and peroxidase staining was performed with VIP as a chromogen (Vector Laboratories). Measurement of the beta-cell area and serum insulin Quantitative evaluation of the beta-cell area was performed on insulin-stained sections using a phase contrast microscope (Carl Zeiss) and Axio Vision software (magnification ×100). The ratio of the beta-cell area was calculated by dividing the area of all insulin-positive cells from at least 20 sections/mouse by the total area of the pancreas. Serum insulin was measured using an insulin ultrasensitive enzyme immunoassay (EIA) kit (Alpco Diagnostics, Windham, NH). RT-PCR and quantitative RT-PCR Various tissues were removed from db/db mice treated with rAd-BTC at 7 days after viral injection, and the expression of BTC messenger RNA (mRNA) was analyzed by conventional reverse transcriptase (RT)-PCR followed by agarose gel electrophoresis [3]. As a control, mouse glyceraldehyde-3-phosphate dehydrogenase

J Mol Med

mRNA was amplified using specific primers. Quantitative RTPCR analysis was performed using SYBR Master Mix (Applied Biosystems) using the ABI 7900 Real-Time PCR System (Applied Biosystems). Primers used for RT-PCR and quantitative RT-PCR are listed in Supplementary Table 1. Western blotting Protein from the lysates (35–40 μg) was resolved by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were incubated with specific antibodies and visualized by blotting with horseradish peroxidase-conjugated secondary antibodies. Intracellular Ca2+ level analysis MIN-6 cells were treated with or without BTC for 48 h and harvested with lysis buffer. Intracellular Ca2+ levels were examined using an AU480 chemistry analyzer (Beckman Coulter, Brea, CA, USA). Measurement of calcineurin activity in vitro The ability of BTC to induce calcineurin in vitro was assessed with an assay kit (Calbiochem, San Diego, CA, USA). BTCtreated cells were lysed and incubated with recombinant calcineurin and synthetic RII phosphopeptide as a substrate. We measured absorbance at 620 nm with a microplate reader (Molecular Devices Corp., Menlo Park, CA, USA), which is correlated with released phosphate and reflects calcineurin activity. Glucose-stimulated insulin secretion Islets isolated from db/db mice were plated, and 0.5 nM of BTC was treated for 24 h. Insulin secretion was stimulated by treatment of cells with 3 and 17 mM glucose (GSIS). The amount of insulin released into the supernatant was quantified using an EIA kit (Alpco Diagnostics, Windham, NH, USA). Transfection with siRNA CnB1 small interfering RNAs (siRNAs) were purchased from Bioneer (Daejeon, Korea) as AccuTarget™ genome-wide pre-designed siRNA. The target sequences were as follows: 5′-CUCUCAAU AACUCAGUGUA-3′ and 5′-UACACUGAGUUAUUGA GAG-3′. A synthetic cyclophilin B control pool siRNA was used as a negative control. Cells were plated and transfected with 25 pmol of siRNA and RNAi Max reagent (Invitrogen), according to the manufacturer’s instructions. Statistical analysis Results are expressed as mean±SEM of three separate experiments. Differences between the control group and the treated group were assessed via the Student’s t test. ANOVA followed by Scheffe’s multiple comparison test was used to determine the significance of any differences among more than two groups. P400 mg/dl). Expression of BTC mRNA was detected in pancreas, liver, and kidney in rAd-BTC-treated mice, whereas BTC mRNA was not detected in any of the tissues in rAd-βgal-treated mice (Supplementary Fig. 1). Body weight was significantly higher in the rAd-BTC-treated group than in db/db mice treated with rAd-β-gal (Fig. 1a). Blood glucose levels in rAd-BTC-treated db/db mice gradually decreased by 2 weeks after injection and remained at low levels when the experiment was terminated. In contrast, rAd-β-gal-treated mice remained hyperglycemic (Fig. 1b). When glucose tolerance tests were performed at 8 weeks after the injection, glucose tolerance was improved in rAd-BTCtreated mice compared with rAd-β-gal-treated mice (Fig. 1c, e). Insulin tolerance tests showed that rAdBTC-treated mice had significantly reduced glucose levels as compared with rAd-β-gal-treated mice only at 90 min after insulin injection, but not at any other time point (Fig. 1d). However, the area under the curve in the rAd-BTC-treated mice was slightly but significantly lower than that in the rAd-β-gal-treated mice (Fig. 1f). Increased beta-cell area and insulin levels in rAd-BTCtreated db/db mice To investigate whether the improved glucose tolerance was a result of an increase in the beta-cell area, we compared islet morphology and size in pancreases from healthy lean mice, rAd-β-gal-treated mice, and rAd-BTCtreated mice. Immunostaining with insulin antibody indicated that the rAd-β-gal-treated db/db mice showed a large islet morphology, but deterioration in the beta-cell area relative to that of the lean control. Eight weeks after injection of rAd-BTC, islet size was increased compared with rAd-β-gal treatment (Fig. 2a). Quantification analysis demonstrated that the insulin-positive cell area in rAd-BTC-treated mice was significantly larger than that in rAd-β-gal-treated db/db mice (Fig. 2b) and that this increase could be seen as early as 3 days after rAd-BTC treatment (Supplementary Fig. 2). Serum insulin levels in rAd-BTC-treated db/db mice were significantly higher than those in rAd-β-gal-treated mice (Fig. 2c). In vitro treatment of islets with BTC also significantly increased insulin secretion in response to high glucose compared with islets without BTC treatment (Supplementary Fig. 3). To determine whether BTC has a pro-survival effect on islets, we analyzed the mRNA expression of the pro-survival genes AKT and Bclw and apoptotic gene, Bax. The mRNA expression of AKT and Bcl-w was increased whereas mRNA expression of Bax was decreased in BTC-treated db/db islets (Supplementary Fig. 4a). Moreover, we confirmed survival gene products from islets at

J Mol Med

Fig. 1 Remission of diabetes in db/db diabetic mice after systemic administration of rAd-BTC. a Body weights and b non-fasting blood glucose levels were measured. c Glucose tolerance test and d insulin

tolerance test were performed after rAd-BTC or rAd-β-gal treatment. Area under the curve (AUC) from e glucose tolerance tests and f insulin tolerance tests. *P