Effects of addition of a dipeptidyl peptidase IV inhibitor to metformin on sirolimus-induced diabetes mellitus

Accepted Manuscript Effects of addition of a dipeptidyl peptidase IV inhibitor to metformin on sirolimusinduced diabetes mellitus Long Jin, Sun Woo Lim, Jian Jin, Byung Ha Chung, Chul Woo Yang PII:

S1931-5244(16)00104-3

DOI:

10.1016/j.trsl.2016.03.012

Reference:

TRSL 1032

To appear in:

Translational Research

Received Date: 27 December 2015 Revised Date:

9 March 2016

Accepted Date: 15 March 2016

Please cite this article as: Jin L, Lim SW, Jin J, Chung BH, Yang CW, Effects of addition of a dipeptidyl peptidase IV inhibitor to metformin on sirolimus-induced diabetes mellitus, Translational Research (2016), doi: 10.1016/j.trsl.2016.03.012. 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.

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Effects of addition of a dipeptidyl peptidase IV inhibitor to metformin on sirolimus-induced diabetes mellitus LONG JIN a, b, SUN WOO LIM a, b, JIAN JIN a, b, BYUNG HA CHUNG a, b, c CHUL WOO YANG a, b, c

Convergent Research Consortium for Immunologic disease

b

c

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a

Transplant research center

Division of Nephrology, Department of Internal Medicine

Korea.

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Corresponding author: Chul Woo Yang, MD,

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Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of

Department of Internal Medicine, Seoul St. Mary’s Hospital 505 Banpo-Dong, Seocho-Ku, 137-040, Seoul, Korea.

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Fax: +82-2-536-0323, Phone: +82-2-2258-6037，E-mail: [email protected]

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Running title: DPP IV inhibitor in diabetes mellitus

Abbreviations: CNI=calcineurin inhibitors; NODAT=new-onset diabetes after transplantation; PTDM=post-transplant diabetes mellitus; SRL=sirolimus; DPP IV=dipeptidyl peptidase IV;

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DM=Diabetes mellitus; LC=LC15-0444; MET=metformin; IPGTT=intraperitoneal glucose tolerance test; ITT=insulin tolerance test; GSIS=glucose-stimulated insulin secretion; EXD=Exendin-4; ROS=reactive oxygen species; 8-OHDG=8-hydroxy-2’-deoxyguanosine

Keywords: DPP IV inhibitor; LC15-0444; Metformin; NODAT; Sirolimus; Mitochondria

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ABSTRACT The guideline for the management of new-onset diabetes after transplantation recommends metformin (MET) as a first-line drug, and addition of a second-line drug is

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needed to better control of hyperglycemia. We tested the effect of addition of a dipeptidyl peptidase IV (DPP IV) inhibitor to MET on sirolimus (SRL)-induced diabetes mellitus (DM). In animal model of SRL-induced DM, MET treatment

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improved pancreatic islet function (blood glucose level and insulin secretion) and attenuated oxidative stress and apoptotic cell death. Addition of a DPP IV inhibitor to

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MET improved these parameters more than MET alone. An in vitro study showed that SRL treatment increased pancreas beta cell death and production of reactive oxygen species (ROS), and pretreatment of ROS inhibitor or p38MAPK inhibitor effectively decreased SRL-induced islet cell death. Exendin-4 (EXD), a substrate of DPP IV, or

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MET significantly improved cell viability and decreased ROS production compared with SRL treatment, and combined treatment with the two drugs improved both parameters. At the subcellular level, impaired mitochondrial respiration by SRL were

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partially improved by MET or EXD, and much improved further after addition of EXD to MET. Our data suggest that addition of a DPP IV inhibitor to MET decreases SRL-

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induced oxidative stress and improves mitochondrial respiration. This finding provides a rationale for the combined use of a DPP IV inhibitor and MET in treating SRLinduced DM

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INTRODUCTION New-onset diabetes after transplantation (NODAT) is a serious complication that can adversely affect the survival of the transplant recipient and graft after solid organ

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transplantation (1). The causes of NODAT are multifactorial (2), but the use of an immunosuppressive regimens is a major contribute to the risk factors for NODAT. Among these drugs, high-dose steroids and calcineurin inhibitors (CNIs) are well-known causes of (3-5).

Sirolimus

(SRL)

was

initially

regarded

as

a

nondiabetogenic

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NODAT

immunosuppressant, but a clinical study has shown that switching from a CNI to SRL can

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cause or further aggravate NODAT, and experimental study has shown that SRL itself causes diabetes mellitus (DM) by impairing insulin secretion or by directly injuring pancreatic islet beta cells (6, 7).

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The guideline for NODAT management is based on type 2 DM (8). In the guideline, metformin (MET) is recommended as a first-line drug for type 2 DM, and addition of a second-line drug is suggested to achieve better control of hyperglycemia. Among these

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second-line drugs, dipeptidyl peptidase IV (DPP IV) inhibitors have recently gained considerable interest for the treatment of type 2 DM and NODAT (9). In addition to providing

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excellent glucose control, DPP IV inhibitors may have pleiotropic effects, such as antiinflammatory, antiapoptotic, and immunomodulatory actions. These protective effects of DPP IV inhibitors have been studied in models of various renal injuries (10-12), DM (13, 14), hepatic impairment (15), and cardiovascular disease (16, 17). Using a well-known animal model, we recently demonstrated that DPP IV inhibitors protect against tacrolimus-induced pancreatic islet and renal injury through their antiapoptotic and antioxidative actions (18, 19).

ACCEPTED MANUSCRIPT Considering these findings, we tested whether addition of DPP IV inhibitors to MET would protect against SRL-induced pancreatic islet injury. First, we evaluated whether a DPP IV inhibitor would have a protective effect in an experimental model of SRL-induced DM.

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Second, we observed whether a DPP IV inhibitor would have a direct protective effect on pancreatic islet cell viability and production of reactive oxygen species (ROS). Third, we evaluated the effects of a DPP IV inhibitor on mitochondrial function by measuring

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mitochondrial respiration. The results of our study demonstrate that DPP IV inhibitors protect against SRL-induced pancreatic islet cell injury and provide a rationale for the addition of a

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DPP IV inhibitor to MET in the treatment of SRL-induced DM in clinical practice.

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METHODS Animal care and drug use. The experiment protocol (CUMC-2012-0117-02) was approved by the Animal Care and Use Committee of the Catholic University of Korea, and all

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procedures performed in this study were in accordance with ethical guidelines for animal studies. Eight-week-old male Sprague Dawley rats (Charles River Technology, Seoul, Korea) that initially weighed 220–230 g were housed in cages (Nalge Co., Rochester, NY) in a

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controlled-temperature and -light environment at the Catholic University of Korea’s animal care facility. The rats received a low-salt diet (0.05% sodium, Teklad Premier, Madison, WI).

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SRL (Wyeth-Ayerst Research, Princeton, NJ, USA) was diluted in Tween 80 (10%), N, Ndimethylacetamide (20%), and polyethylene glycol 400 (70%), to a final concentration of 0.3 mg/kg. The DPP IV inhibitor LC15-0444 was kindly supplied by LG Life Sciences (Seoul, Korea), and was diluted in drinking water to a final concentration of 5 mg/kg. MET (D

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ongwha Pharmaceutical Co., Seoul, Korea) was diluted in drinking water to a final

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concentration of 250 mg/kg.

Experimental design. The study was designed to determine whether the combination of

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LC15-0444 and MET would result in less glycemia than would LC15-0444 or MET administered alone in rats with SRL-induced DM. A total of 45 male Sprague Dawley rats were randomized to five groups each containing nine rats. The rats were treated with SRL (0.3 mg/kg/day, subcutaneous) and LC15-0444 (5 mg/kg/day, oral gavage) and/or MET (250 mg/kg/day, oral gavage) for a total of 6 weeks. SRL were given for the entire 6 weeks and LC15-0444 and/or MET were additionally treated during the last 3 weeks. Vehicle (VH) group rats received a daily subcutaneous injection of olive oil (1mg/kg/day) for 6 weeks. MET dose (250mg/kg) was determined based on preliminary animal studies that two doses of

ACCEPTED MANUSCRIPT 100 mg/kg and 200 mg/kg are effective in glucose control (20-22) and doses more than 600 mg/kg/day shows clinical signs of toxicity (23). The dose of LC15-0444 was chosen based on preliminary studies (24-26). There is no information about the therapeutic doses of LC15-

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0444 in the setting of SRL-induced pancreatic islet dysfunction in rats. We previously tested two different doses (5mg/kg and 15mg/kg) and found that 5mg/kg was but 15mg/kg was not effective in controlling hyperglycemia. Therefore, we chose 5 mg/kg as the optimal dose of

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LC15-0444. Before they started the drug treatment, the rats were pair fed, and body weight was measured every day thereafter. Pancreatic islet function was measured with an

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intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) 6 weeks later. Before sacrifice, animals were housed individually in metabolic cages (Tecniplast, Buguggiate, Italy) for a 24-h urine collection. On the next day, the animals were anesthetized with Zoletil 50 (10 mg/kg, intraperitoneal; Virbac Laboratories, Carros, France) and Rompun

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(15 mg/kg, intraperitoneal; Bayer, Leverkusen, Germany), and blood samples and pancreas tissue were obtained for further analysis. Serum creatinine concentration was measured using an autoanalyzer (Coulter-STKS, Coulter Electronics). The whole-blood SRL concentration

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was measured using a microparticle enzyme immunoassay (Abbott Diagnostics, Abbott Park, IL). Insulin immunohistochemistry and glucose-stimulated insulin secretion (GSIS) were also

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examined. Oxidative stress and apoptosis were measured by immunohistochemistry.

Preservation of pancreatic tissue. Pancreases tissue was preserved by in vivo perfusion through the abdominal aorta. The animals were perfused with 0.01 mol/L phosphate-buffered saline to flush blood from the tissues. Dissected pancreases were immersed in periodatelysine-2% paraformaldehyde solution and embedded in paraffin for further histologic observation.

ACCEPTED MANUSCRIPT In vitro study. As shown in our previous study, the protective effects of DPP IV inhibitors are associated with activation of glucagon-like peptide 1 (GLP-1) (18). To investigate the exact role of the combined treatment with a DPP IV inhibitor and MET on SRL-induced

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pancreatic beta cell injury, we applied the GLP-1 analog exendin-4 (EXD) (0.01nM; SigmaAldrich, St Louis, MO) and MET (10ng/mL) to INS-1 cells, which was donated by Dr. Yoon (Catholic University of Korea, Seoul, Korea). To verify the relationship between ROS and

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apoptosis, we further evaluated the effect of ROS inhibitor N-acetyl-L-cysteine (NAC) (1mM; Sigma-Aldrich, St Louis, MO) and P38 MAPK inhibitor (SB203580) (1uM; Sigma-Aldrich,

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St Louis, MO) on SRL-induced apoptosis. The INS-1 cell line is the most frequently used colony-forming cell model for pancreatic beta cell research (27). We assessed apoptosis, oxidative stress, and mitochondrial function in INS-1 cells. INS-1 cells were cultured in RPMI 1640 medium (Wisent, Saint-Bruno, QC, Canada) supplemented with 11.1 mM sodium pyruvate, 10 mM HEPES, 10% fetal bovine serum (FBS; Wisent), 2 mM L-glutamine,

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50 µM β-mercaptoethanol, 100 U/mL penicillin, and 100 mg/mL streptomycin (except for FBS, all were from Sigma-Aldrich). The cells were incubated at 37°C in a humidified

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atmosphere of 5% CO2 and 95% air for 24 h and subcultured to 70–80% confluence. After 24 h, the culture medium was changed to serum-free medium containing SRL (20 µg/mL), EXD

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(Sigma; 0.01 nM), and/or MET (10 ng/mL). The cells were used for experiments at 12 and 24 h after the change to serum-free medium. All in vitro experiments were conducted at least three times. The experiments were performed with individual samples from separate experiments and not using different wells from the same culture plate.

Measurement of pancreatic islet function and plasma GLP-1 levels. The IPGTT was performed at the end of the 6-week treatment period, as previously described (28, 29), and the

ACCEPTED MANUSCRIPT area under the curve of glucose (AUCg) was calculated by trapezoidal estimation from the values obtained in the IPGTT. The ITT was also performed at the end of the 6-week treatment period. Briefly, after 4 h of fasting, tail blood samples were obtained and glucose

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concentrations were measured using a glucose analyzer (Accu-Check, Roche Diagnostics, Basel, Switzerland). The rats were then injected intraperitoneally with insulin. Doses of 0.75 units/kg of Humulin R (Eli Lilly, Indianapolis, IN, USA) were used, and blood glucose

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concentration was measured at 30, 60, 90 and 120 min after injection. The AUCg was calculated by trapezoidal estimation from the values obtained in the ITT. The levels of active

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GLP-1 in the plasma were measured using a commercially available ELISA kit (Millipore Corporation, Billerica, MA). The plasma insulin level was measured in duplicate by an enzyme-linked immunosorbent assay kit (Millipore Corporation, St. Charles, MO, USA). Hemoglobin A1c (HbA1c) level was measured using the HemoCue C-Glucose Analyzer

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(HemoCue AB, Ängelholm, Sweden) and the DCA 2000+HbA1c kit (Bayer, Elkhart, IN).

Measurement of pancreatic beta cell area. A minimum of 20 fields per section were

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assessed using a color image analyzer (TDI Scope EyeTM version 3.0 for Window; Olympus, Tokyo, Japan). Briefly, captured images from immunohistochemical staining of insulin were

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quantified using the Polygon program by measuring the pancreas area seen to contain insulinpositive areas besides vacuoles at <200× magnification. Histopathological analysis was performed on randomly selected fields of pancreas sections by a pathologist who was blinded to the identity of the treatment groups.

GSIS. Pancreatic islets were isolated from male Sprague Dawley rats (250-300g) using collagenase digestion as described previously (30, 31). The islets were preincubated in RPMI

ACCEPTED MANUSCRIPT 1640 medium containing 10% FBS and 100 U/mL penicillin at 37°C for 24 h. The isolated islets were then incubated with SRL (90 ng/mL) for 12 h, followed by treatment with EXD (100 ng/mL), MET (165 ng/mL) and combined treatment with EXD and MET sequentially

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for a further 12 h. This was followed by analysis of insulin secretion. After the islets had been cultured with SRL (90 ng/mL), EXD (100 ng/mL), or MET (165 ng/mL) for 24 h, the islets were collected, and groups of 20 islets were then incubated for 1 h in RPMI 1640 containing

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16.7 mM glucose. At the end of the incubation period, the islets were pelleted by centrifugation, and the supernatant fluid was sampled to measure the insulin secretion using

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an immunosorbent assay kit (Millipore Corporation, St. Charles, MO, USA).

Immunohistochemistry. Immunohistochemistry was performed to assess oxidative stress markers, antioxidative stress-related molecules, and apoptosis using the methods described

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previously (28). The oxidative stress markers 8-hydroxy-2′-deoxyguanosine (8-OHDG) and anti-4-hydroxy-2-hexenal (4-HHE) were detected by incubating 4-µm tissue sections for 12 h with specific antibodies against 8-OHDG and 4-HHE (both from JaICA, Shizuoka, Japan) at

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4°C. Antioxidative stress-related molecules such as manganese superoxide dismutase

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(MnSOD) and catalase were also detected by incubating 4-µm tissue sections with primary anti-MnSOD antibody and anti-catalase antibody, respectively (both from Abcam, Cambridge, MA) at 4°C for 12 h. The most representative apoptotic marker caspase-3 was detected by incubating 4-µm tissue sections with specific antibodies against active caspase-3 (Millipore, Billerica, MA) at 4°C for 12 h, and apoptosis was identified in the tissue sections using the ApopTag In Situ Apoptosis Detection Kit (Millipore). The number of terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end labeling (TUNEL)-positive cells was counted in 20 different fields in each section at 200× magnification.

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Measurement of serum 8-OHDG level. Oxidative DNA damage was assessed by measuring the level of the DNA adduct 8-OHDG in serum using a competitive enzyme-linked

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immunosorbent assay (Cell Biolabs, San Diego, CA).

Measurement of cell viability. INS-1 cells were seeded into 96-well plates at a density of 3 4

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× 10 cells/well and preincubated for 24 h in an incubator at 37°C. Cell viability was assayed using a Cell Counting Kit-8 (CCK-8) assay kit (Dojin Laboratories, Kumamoto, Japan)

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according to the manufacturer’s protocol (32).

Flow cytometry. Flow cytometry was performed to assess apoptosis and ROS production. 5

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INS-1 cells were seeded into 12-well plates at a density of 2.5 × 10 cells/well and preincubated for 24 h at 37°C in an incubator. The cells were then incubated in 100 µL of binding buffer containing 5 µL of FITC–annexin V (BD Pharmingen, San Diego, CA) at

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25°C in dark conditions for 15 min. Binding buffer (400 µL) was added to dilute the samples before analysis using a flow cytometer (BD, Biosciences, San Diego, CA). The annexin V-

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positive cells were considered apoptotic. The levels of intracellular ROS and mitochondrial ROS were measured using CM-H2DCFDA and Mito-Sox, respectively (Molecular Probes, Eugene, OR) according to the manufacturer’s instructions. Briefly, INS-1 cells were seeded 5

into 12-well plates at a density of 2.5 × 10 cells/well and preincubated for 24 h at 37°C in an incubator. The cells were treated with serum-free medium containing 10 µM CM-H2DCFDA or 5 mM Mito-Sox at 37°C in an incubator for 1 h, after which the cells were washed and resuspended in phosphate-buffered saline for analysis by flow cytometry (BD, Biosciences,

ACCEPTED MANUSCRIPT San Diego, CA). For each analysis, 10,000 events were recorded (33, 34).

Transmission electron microscopy. Processing for electron microscopic observation was

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performed as previously described (35). Using an image analyzer, the number and areas of mitochondria per cell were measured in 20 random pancreatic beta cells (TDI Scope Eye

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version 3.0 for Windows, Olympus, Tokyo, Japan).

Mitochondrial function. Mitochondrial function was assessed by measuring the oxygen

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consumption rate (OCR), which was determined using a Seahorse XF24 analyzer (Seahorse Bioscience, North Billerica, MA) as previously reported by Cantu et al (36). INS-1 cells were 5

plated on XF24 microplates (Seahorse Bioscience) at 5 × 10 cells/well in RPMI 1640 and kept at 37°C in a 5% CO2 humidified atmosphere overnight. Cells were treated with serum-

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free medium containing SRL (20 µg/mL) and then incubated with EXD (0.01 nM) and/or MET (10 ng/mL) for 6 h. Different parameters of respiration rates were calculated by

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subtracting the average respiration rates before and after the addition of the electron transport inhibitors (1.0 µM oligomycin, 0.25 µM cyanide-p-trifluoromethoxyphenylhydrazone

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[FCCP], and 0.5 µM antimycin-A [AA]). The following parameters were quantified. Basal respiration was calculated as baseline respiration minus AA-dependent respiration. ATPlinked respiration was calculated as baseline respiration minus oligo-dependent respiration. Proton leakage was calculated as oligo-dependent respiration minus AA-dependent respiration. Reserve capacity was calculated as AA-dependent respiration minus FCCPdependent respiration. The values were quantified for each individual well and then averaged for each condition.

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Statistics. Data are expressed as means ± SE. Groups were compared using one-way analysis of variance with the Bonferroni correction for comparisons. The level of significance was

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regarded as P<0.05 and we confirmed that all of our measurements are normally distributed using the Shapiro-Wilk test (P-value is greater than 0.05) by SPSS statistics.

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RESULT Addition of a DPP IV inhibitor to MET effectively controls hyperglycemia in an experimental model of SRL-induced DM. After 3 weeks of SRL treatment, 24-h water

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intake and urine volume of the rats were significantly increased. However, addition of or independent treatment with LC15-0444 or MET suppressed the elevation of water intake and urine excretion. SRL treatment slowed the rate of increase in body weight. Neither LC15-

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0444 or MET alone, nor the combined use of a DPP IV inhibitor and MET significantly recovered these changes (Table 1). The IPGTT was used to assess the basal metabolism of

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plasma glucose concentration. SRL treatment significantly increased the AUCg, whereas MET or LC15-0444 treatment attenuated the increase in AUCg induced by SRL. More importantly, addition of LC15-0444 to MET was more effective in attenuating the AUCg

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compared with LC15-0444 or MET alone (Table 2).

Addition of a DPP IV inhibitor to MET improves insulin resistance and increases plasma GLP-1 and insulin levels in an experimental model of SRL-induced DM. The

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plasma GLP-1 levels in the SRL group was lower than the VH group but addition of LC150444 to MET significantly increased the active GLP-1 levels compare with independent use

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of MET or LC15-0444 treatment (Figure 1, A; P<0.05). SRL significantly decreased plasma insulin level, but addition of LC15-0444 to MET recovered plasma insulin level (Figure1, B; P<0.05). The ITT was performed to examine insulin resistance induced by SRL. Rats treated with SRL exhibited significant elevation in the AUCg. Neither LC15-0444 nor MET significantly suppressed this elevation, but addition of LC15-0444 to MET decreased the AUCg (Figure 1, C and D; P<0.05).

ACCEPTED MANUSCRIPT Addition of a DPP IV inhibitor to MET improves pancreatic islet function. Pancreatic beta cell area was evaluated using immunohistochemistry. The SRL group had smaller islets and a lower intensity of insulin staining within islets compared with the VH group (SRL,

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11.3± 1.1 um2 vs. VH, 17.1± 1.7 um2, P< 0.05, Figure 2 A and B). Although the shrinkage of islets induced by SRL was not prevented by LC15-0444 or MET treatment alone, addition of LC15-0444 to MET rescued the shrinkage of islets (17.0 ± 1.7um2, Figure 2 A and B). To

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evaluate the direct effects of the two drugs, we treated primary rat isolated islets with SRL, EXD, and MET in the culture setting used to evaluate GSIS. As expected, SRL significantly

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decreased glucose-stimulated insulin secretion. Addition of EXD to MET produced a significantly higher insulin level compared with EXD or MET treatment alone and recovered the decreased insulin level (Figure 2, C; P<0.05).

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Addition of a DPP IV inhibitor to MET suppresses SRL-induced oxidative stress. Accumulated evidence indicates that SRL treatment increases oxidative stress in vivo and in vitro (37-39). In this study, immunohistochemistry was performed to evaluate oxidative stress

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by detecting the levels of 8-OHdG and 4-HHE. SRL treatment significantly increased the production of 8-OHdG and 4-HHE, and this effect could be suppressed by independent

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addition of LC15-0444 or MET. Moreover, addition of LC15-0444 to MET was more effective in reducing the levels of 8-OHdG and 4-HHE than was each drug alone. (Figure 3, Figure 4 B and C; P<0.05). 8-OHdG was also detected in the serum in our animal model. Interestingly, only addition of LC15-0444 to MET significantly suppressed the elevation of serum 8-OHdG level (Figure 4 A; P<0.05). MnSOD and catalase functions, which are involved in the elimination of ROS, were detected to assess the oxidative stress. As expected, SRL treatment led to downregulation of MnSOD and catalase, and this effect was recovered by addition of LC15-0444 to MET (Figure 3, Figure 4 D and E; P<0.05).

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Addition of a DPP IV inhibitor to MET suppresses SRL-induced apoptosis in pancreatic islets. Cell apoptosis induced by SRL may lead directly to islet injury (40). In this study,

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TUNEL was used to determine whether DPP IV inhibitors and MET are protective of the cells in islets. Apoptotic cells showed shrinkage and condensation and fragmentation of the nucleus (Figure 5 A). The number of TUNEL-positive cells in islets was significantly higher in the SRL group compared with the VH group. However, the increase in the number of

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TUNEL-positive cells was prevented by LC15-0444 or MET alone, and in combination. The

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combination was more effective in preventing the increase in the number of TUNEL-positive cells compared with use of either alone (Figure 5, B; P<0.05). Caspase-3 is considered a crucial component of the cell death pathways (18). In the SRL-treated group, the active form of caspase-3 was significantly upregulated. Consistent with the TUNEL results, either of the two drugs alone and addition of LC15-0444 to MET reduced the expression of activated

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caspase-3. More importantly, addition of LC15-0444 to MET was more effective than the use

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of each drug alone (Figure 5, C; P<0.05).

Addition of a DPP IV inhibitor to MET decreases SRL-induced ROS production in INS-

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1 cells. Oxidative stress has been linked to beta cell dysfunction and death, which give rise to type 2 DM. ROS scavenging ability has been reported to improve beta cell health (41). H2DCFDA was used as a probe to evaluate changes in intracellular ROS by flow cytometry. After the 15-h incubation, 8.3% of SRL-treated cells stained positive for H2-DCFDA. Combined treatment with MET and EXD decreased the percentage of H2-DCFDA-stained cells to 1.8%. Quantification showed that the combined treatment with MET and EXD significantly decreased intracellular ROS production compared with SRL alone (Figure 6, A and B; P<0.05). Mitochondria are an important source of ROS, which contribute to

ACCEPTED MANUSCRIPT mitochondrial damage and underlie oxidative damage (42). Mitochondrial ROS production was measured using Mito-Sox staining. ROS level was significantly decreased by the combined treatment of MET and EXD compared with SRL alone: 8.4% of SRL-treated cells

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and 3.5% of SRL-treated cells treated with the combination of MET and EXD stained for Mito-Sox (Figure 6, C and D; P<0.05). The results indicated that combined treatment with

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the two drugs protected against SRL-induced ROS production.

Addition of a DPP IV inhibitor to MET increases cell viability and attenuates cell

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apoptosis in INS-1 cells incubated with SRL. To study the direct effects of MET and LC15-0444, INS-1 cells were incubated with SRL with or without the GLP-1 analog EXD and MET, and cell viability was determined using CCK-8. The viability of INS-1 cells was significantly greater in cells treated with EXD and MET compared with SRL alone (Figure 7,

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A). To examine whether direct treatment with MET and EXD affects apoptotic events associated with SRL-induced cellular damage, apoptosis was measured by flow cytometry of FITC–annexin V binding, which is a sensitive probe used for the identification of cells

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undergoing apoptosis. Cells were incubated with serum-free medium containing SRL (20 µg/mL) and then treated with MET (10 ng/mL) and/or EXD (0.01 nM). After the 16-h

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incubation, apoptosis did not differ between cells treated with MET or EXD alone or in combination compared with those treated with DMSO. In cells treated with SRL alone, 21.3% stained for annexin V. This percentage decreased to 10.3% in cells treated with MET and EXD combined with SRL. The average percentage of FITC–annexin V cell binding showed that significantly fewer cells stained for annexin V in the cells treated with the combined treatment of MET and EXD compared with SRL alone (Figure 7, B and C; P<0.05). These results indicate that addition of EXD to MET protected against SRL-induced cellular damage.

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SRL-induced ROS production is casually associated with apoptosis. To verify the relationship between oxidative stress and cell injury, we further evaluated the effect of ROS

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inhibitor (NAC) or p38MAPK inhibitor on SRL-induced apoptosis. The results showed that Annexin V-stained islet cells with SRL treatment (20.8%) were singnificantly decreased with pretreatment with ROS inhibitor (NAC) (8.3%)or p38MAPK inhibitor (10.6%). The decrease

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of apoptotic cells by NAC or p38MAPK inhibitor was comparable to that of combination treatment of MET and EXD (7.3%) (Figure 8). This finding suggest that oxidative stress is

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causually related to the SRL-induced cell injury.

Addition of a DPP IV inhibitor to MET improves mitochondrial ultrastructure and

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function. Our findings implied that addition of a DPP IV inhibitor to MET protects mitochondrial function, which is strongly associated with ROS production and cell apoptosis. To evaluate the function of mitochondria, electron microscopy was used to detect the

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ultrastructure and to quantify the number of mitochondria. In the islet cells obtained in our

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animal model, SRL treatment decreased both the average mitochondrial area and number. No matter independent use of LC15-0444 or MET could not protect the decrease. Only the addition of LC15-0444 to MET restored the average mitochondrial area and number (Figure 9 A, B and C; P<0.05). Electron microscopy also showed that the number of insulin granules was significantly reduced by SRL treatment. More importantly, only the addition of LC150444 to MET was effective in attenuating this reduction in granule number, which confirmed that addition of a DPP IV inhibitor to MET protected islet function after SRL treatment (Figure 9 A and D ; P<0.05). We also measured mitochondrial respiration as an indicator of

ACCEPTED MANUSCRIPT delicate balance between ROS production and consumption. To reveal exact effect of LC150444 and MET in mitochondrial we assessed oxygen consumption rates (OCR) using extracellular flux analysis. 6 h was chosen due to the observation that this time course

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supported with minimal cell death. The OCR trace graph showed that the combined treatment of MET and EXD produced a higher OCR than SRL alone (Figure 10, A). Compared with SRL alone, the combined treatment of MET and EXD significantly increased basal

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respiration, ATP-linked respiration, maximal respiration, and reserve capacity (Figure10, B; P<0.05). These data suggest that addition of EXD to MET may protect against SRL-induced

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mitochondrial dysfunction.

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DISCUSSION We tested whether addition of a DPP IV inhibitor to MET would have beneficial effect on the control of hyperglycemia in SRL-induced DM. In vivo and in vitro studies showed that

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addition of a DPP IV inhibitor to MET improved glycemic control and insulin secretion further compared with each drug alone. These changes were accompanied by improved pancreatic beta cell function and oxidative stress. These findings suggest that addition of a

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DPP IV inhibitor to MET provides beneficial effects in controlling hyperglycemia by protecting SRL-induced pancreatic islet injury. This finding provides a rationale for the use of

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combined treatment with DPP IV inhibitor and MET for NODAT caused by SRL.

It is known that the pathogenesis of SRL-induced DM involves impaired insulin signaling or direct pancreatic islet cell injury (43). Therefore, the ideal antidiabetic drugs to treat SRL-

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induced DM should work by improving impaired insulin signaling and protecting against islet cell injury. We tested the combination of MET and a DPP IV inhibitor after considering previous reports showing that MET improves the insulin signaling pathway by activating

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AMP-activated protein kinase (44) and that DPP IV inhibitors can protect against pancreatic

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islet cell injury caused by diverse injuries (9), including tacrolimus-induced renal and pancreatic islet injuries (18, 19). Thus, we hypothesized that the combination of MET and a DPP IV inhibitor would provide better control of SRL-induced DM compared with each drug alone. In this study, we evaluated the effects of MET and/or a DPP IV inhibitor on the control of hyperglycemia in rats with SRL-induced DM. We measured IPGTT and found that addition of a DPP IV inhibitor to MET significantly improved parameters compared with each drug alone (Table2). This finding confirms that combination of DPP IV inhibitor and MET provides better control of hyperglycemia in SRL-induced DM.

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To define the possible mechanism of DPP IV inhibitor or MET on control of hyperglycemia, we evaluated the effect of two drugs on both insulin resistance and secretion. Insulin

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resistance using ITT revealed that exogenous insulin administration failed to control SRLinduced hyperglycemia but was effective in animals with combination of a DPP IV inhibitor or MET treatment. This finding suggests that insulin resistance is involved in the

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development of SRL-induced DM and combination of these two drugs improves SRLinduced insulin resistance. It is generally accepted that insulin resistance is a main component

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to SRL-induced hyperglycemia. Therefore, it is expected that SRL induces hyperinsulinemia and combination of a DPP IV inhibitor and MET decreases blood glucose and subsequent decline of plasma insulin concentrations if these two drugs improves insulin resistance. On the contrary, our study showed that SRL treatment decreased plasma insulin level and

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combination of a DPP IV inhibitor and MET increased plasma insulin level. This opposite result seems to be related to the our model which is based on SRL-induced pancreatic islet cell injury. Thus, both insulin resistance and impaired insulin secretion are involved in our

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model and main pathogenesis is reduced insulin secretion. This may explain why SRL treatment decreased plasma insulin level and combination of MET and DPP IV inhibitor

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increased plasma insulin level in our study.

To evaluate the effect of MET or DPP IV inhibitor on insulin secretion, we measured plasma insulin and active GLP-1 levels in experimental groups and evaluated insulin secretion using GSIS in isolated pancreatic islet cells. The results showed that addition of DPP IV inhibitor to MET significantly recovered the plasma active GLP-1 and insulin levels which were decreased in SRL-treated rats (Figure 1A and 1B), and GSIS in isolated pancreatic islet cells also showed increased insulin secretion with the combination of MET and EXD as shown

ACCEPTED MANUSCRIPT figure 2C. This finding suggests that insulin secretion is impaired in SRL-induced islet injury and the combination of MET and a DPP IV inhibitor effectively controls hyperglycemia by increasing insulin secretion. It is well known that islets pre-exposed to SRL significantly

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decrease both the first and second phases of GSIS, and reason for decreased entire secretion is related to the deceased functional islet mass (45). Using immunohistochemistry of insulin, we confirmed that pancreatic islet size is decreased in animals with SRL treatment (Figure

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2A) and the combination of MET and a DPP IV inhibitor restores pancreatic islet size. This finding suggests that decreased functional islet mass by SRL is effectively preserved with

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combination treatment of MET and a DPP IV inhibitor.

To define the protective effect of combination of MET and DPP IV inhibitor against SRLinduced pancreatic islet injury, we focused on the oxidative stress which is known to activate

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various intracellular signaling pathways, which can induce apoptosis or cell overgrowth and lead to organ dysfunction (46). There is increasing evidence of an association between the development of diabetes and increased oxidative stress (47), and SRL-induced DM is

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associated with oxidative stress (37). In this study, we studied SRL-induced oxidative stress in animal model (Figure 3 and 4) and in vitro study (Figure 6), and found that addition of

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DPP IV inhibitor to MET further decreases oxidative stress compared with each drug alone in SRL-treated animals and in vitro study using INS-1 cells. These findings suggest that SRLinduced pancreatic islet injury is associated with oxidative stress (37), and combination of MET and DPP IV inhibitor effectively SRL-induced oxidative stress. This may explain wellcontrolled hyperglycemia by combination of MET and a DPP IV inhibitor in SRL-induced DM. The mechanism of anti-oxidant effects of DPP IV inhibitor or MET are multifactorial. DPP IV inhibitor exerts its effect by mediating GLP-1 (48-50), and activation of GLP-1 receptor signaling protects pancreas islets via amelioration of oxidative DNA damage,

ACCEPTED MANUSCRIPT apoptosis, and ROS (10-13, 51-53). MET also has protective effects on cell viability and ROS production via a variety of mechanisms (54, 55). Taken together, our animal and in vitro study reveals that addition of DPP IV inhibitor to MET provides protection against SRL-

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induced pancreatic islet injury by decreasing oxidative stress.

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In this research, we found that addition of a DPP IV inhibitor to MET decreased oxidative stress caused by SRL, but it was uncertain that decreased oxidative stress is a cause or a result

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of the cell injury suppression. To verify the relationship between oxidative stress and cell injury, we further evaluated the effect of ROS inhibitor (NAC) and p38MAPK inhibitor on SRL-induced apoptosis in INS-1. The 20.8% of islet cells treated with SRL alone was stained for annexin V, and the proportion of stained cells decreased to 10.6% or 8.3% with treatment

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of p38MAPK inhibitor or NAC as shown Figure 8. These results suggest that SRL increases apoptosis through ROS generation in INS-1 and p38 is involved in SRL-induced apoptosis. Therefore, we propose that oxidative stress is causually related to the SRL-induced cell injury,

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and decrease of oxidative stress is responsible for the cell injury suppression

We evaluated further whether mitochondrial ROS contribute to SRL-induced oxidative stress. The mitochondrial respiratory chain is the major source of ROS in most mammalian cells, and excess production of mitochondrial ROS is related to diabetes (56). A previous study reported that sustained and uncontrolled oxidative stress has detrimental effects on mitochondrial function and insulin secretion (57). Mitochondria are susceptible to oxidative damage and are a major source of superoxide. The accumulation of oxidative damage in mitochondria might contribute to mitochondrial dysfunction and cell death in a range of

ACCEPTED MANUSCRIPT degenerative diseases (58). In this study, we evaluated whether addition of a DPP IV inhibitor to MET would protect the structure and function of mitochondria. Electron microscopy showed shrunken mitochondria in the islet cells of SRL-treated rats and that the appearance

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was restored to normal by the addition of a DPP IV inhibitor to MET. Mitochondria function assessed by OCR showed that INS-1 cells treated with MET and EXD had a higher OCR compared with those treated with SRL alone (Figure 10). The combined treatment of MET

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and EXD significantly increased basal respiration, ATP-linked respiration, maximal respiration, and reserve capacity compared with MET or SRL treatment alone. These data

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suggest that the addition of EXD to MET protects against SRL-induced mitochondrial dysfunction.

The results of our study show that both EXD and MET improves mitochondrial function but

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the mechanism of these drugs on mitochondrial function is different. MET acts directly on mitochondria to inhibit complex I-mediated mitochondrial respiration and citric acid cycle functions (59). GLP-1 enhances mitochondrial function in pancreatic beta-cells through the

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activation of several Krebs cycle dehydrogenases (60). EXD also has a stimulatory effect on mitochondrial mass and function, and this effect is accompanied by up regulation of PGC1

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alpha, a key regulator of mitochondrial biogenesis (61). Therefore, it is expected that combination of these two drugs shows an additive or synergistic action on mitochondrial function, and this may explain further improvement of mitochondrial function than each drug alone in our study.

The guideline for NODAT management is based on type 2 DM (8). MET is recommended as a first-line drug and second-line drug is needed to achieve optimal glycemic control. In this study, we tested whether a DPP IV inhibitors are recommendable as second-line drug. It is

ACCEPTED MANUSCRIPT well known that combination of DPP IV inhibitors and MET has additive effects. MET enhances the biological effect of GLP-1 by increasing GLP-1 secretion, suppressing activity of DPP IV and up regulating the expression of GLP-1 receptor in pancreatic β-cells (62).

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Conversely, DPP IV inhibitors have a favorable effect on insulin sensitivity in patients with type 2 DM (63). The results of our study adds rationale and evidence for combination of DPP IV inhibitor and MET in point that addition of DPP IV inhibitor to MET further decreases

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SRL-induced oxidative stress compared with each drug alone. Taken together, the results of our study can be translated into clinical practice, and we recommend DPP IV inhibitors as

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second-line drug in SRL-induced DM.

Our animal model seems not to have reached the state of the diabetic considering normal fasting blood sugar (FBS) concentration. The reason for not reach overt diabetic state by SRL

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is related to the low diabetogenic potential. We previously tested three doses of SRL (0.15, 0.3 and 0.6 mg/kg), and found that all three doses of SRL developed DM, but did not increase FBS levels even high dose of SRL (0.6 mg/kg) (37). This finding suggests that SRL-induced

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DM is so mild that it is not diagnosed easily using only the FBS concentration. However, most clinical trials have not routinely included an oral glucose tolerance test (OGTT) to

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determine the exact incidence of glycemic abnormalities in renal transplant recipients who are treated with an mTOR inhibitor (64-67). Therefore, we would expect a higher incidence of glucose metabolism abnormalities in the renal transplant population treated with SRL if OGTT is included in the diagnosis of DM.

In conclusion, addition of a DPP IV inhibitor to MET had protective effects on pancreatic islet injury induced by SRL. This finding suggests that combination of a DPP IV inhibitor and MET may be useful in the treatment of SRL-induced DM in clinical practice.

ACCEPTED MANUSCRIPT

Acknowledgements Conflicts of interest: All authors have read the journal’s policy on disclosure of potential conflicts of interest and have none to declare

for Health & Welfare, Republic of Korea (HI14C3417).

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This study was supported by a grant of the Korean Health Technology R&D Project, Ministry

All authors have read the journal’s authorship agreement, and all named authors have read

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approved the final manuscript

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ACCEPTED MANUSCRIPT TABLE 1.

Basic parameters in each group SRL (n = 9)

SRL+MET (n = 9)

SRL+LC (n = 9)

SRL+ MET+ LC (n = 9)

∆BW (g)

91 ± 5

47 ± 3#

52 ± 2#

52 ± 5#

54 ± 2#

UV (mL)

10 ± 1

26 ± 3#

12 ± 1$

14 ± 2$

17 ± 3$

Water intake (mL)

12 ± 4

25 ± 6#

18 ± 4#,$

18 ± 7#,$

19 ± 7#

Scr (mg/dL)

0.41 ± 0.06

0.36 ± 0.04

0.36 ± 0.02

0.40 ± 0.05

0.38 ± 0.05

HbA1c (%)

3.6 ± 0.1

3.7 ± 0.1

3.7 ± 0.2

3.7 ± 0.2

3.6 ± 0.1

3.2 ± 0.3

2.6 ± 0.2

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SRL con.(ng/mL)

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VH (n = 9)

3.4 ± 0.7

2.5 ± 0.3

Values are means ± SE. n, No. of animals; BW, body weight; UV, urine volume; Scr, serum creatinine;

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SRL con., Sirolimus concentration; HbA1c, Hemoglobin A1c; VH, vehicle; SRL, Sirolimus; LC,

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LC15-0444; MET, metformin. #P < 0.05 vs. VH. $P < 0.05 vs. SRL.

ACCEPTED MANUSCRIPT TABLE 2. Effect of combination treatment of DPP IV inhibitor and MET on control of SRL-induced hyperglycemia using IPGTT 0min

30min

60min

90min

120min

AUCg

VH

92 ± 3

259 ± 10

155 ± 5

134 ± 6

121 ± 4

327 ± 9

SRL

85 ± 4

373 ± 21#

272 ± 11#

198 ± 8#

133 ± 4

476 ± 17#

SRL+MET

88 ± 4

341 ± 16#

243 ± 11#

173 ± 12#

129 ± 7

432 ± 17#

SRL+LC

87 ± 5

351 ± 13#

233 ± 14#

149 ± 10

116 ± 7

417 ± 15#,$

SRL+MET+LC

92 ± 5

271 ± 16$,@,*

193 ± 22$

135 ± 6$

113 ± 4

350 ± 19$,@,*

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Group

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Values are means ± SE. VH, vehicle; SRL, Sirolimus; MET, metformin; LC, LC15-0444. #P < 0.05 vs. VH. $P < 0.05 vs. SRL. @P < 0.05 vs. SRL+ MET. *P < 0.05 vs. SRL+LC.

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FIGURE LEGENDS Fig 1. The effects of combined treatment with metformin (MET) and LC15-0444 (LC) on

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plasma active GLP-1 levels, plasma insulin levels and insulin resistance in experimental model of sirolimus (SRL)-induced DM. (A) Active GLP-1 levels (B) Plasma insulin levels. (C) Insulin tolerance test (ITT). (D) The area under the curve for glucose (AUCg) for ITT Note that all parameters recovered after combined treatment with MET and LC. #P < 0.05 vs.

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VH. $P < 0.05 vs. SRL. @P < 0.05 vs. SRL+ MET. *P < 0.05 vs. SRL+LC.

Fig 2. Effects of sirolimus (SRL) on pancreatic islet size and glucose-stimulated insulin secretion. (A) Representative images of insulin staining of pancreatic sections. (B) Quantitative analysis of islet area. (C) Effects of combined treatment with metformin (MET)

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and the GLP-1 analog exendin-4 (EXD) on glucose-stimulated insulin secretion during sirolimus (SRL)-induced injury. Primary rat isolated islets were treated directly with MET and EXD combined with SRL in the culture setting. Note that all parameters recovered after

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the combined treatment with MET and LC. n = 9 rats per group. #P < 0.05 vs. VH. $P < 0.05

Fig 3. Representative images of 8-OHDG, 4-HHE, MnSOD, and catalase staining in each treatment group. The strong dark nuclear expression of 8-OHDG and 4-HHE in islets was higher in the sirolimus (SRL) group compared with groups treated with LC15-0444 (LC) or metformin (MET) alone, or in combination (treatment groups). In contrast to the pattern seen for 8-OHDG and 4-HHE, the nuclear expression of MnSOD and catalase in islets was weaker in the SRL group compared with the treatment groups. Original magnifications, 400×. n = 9

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Fig 4. Effects of combined treatment with metformin (MET) and LC15-0444 (LC) on

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oxidative stress in pancreatic islets and serum in experimental model of sirolimus (SRL)induced DM. (A) SRL-induced serum 8-hydroxy-2′-deoxyguanosine (8-OHDG) level was reduced by the addition of LC to MET. (B) Quantitative analysis of 8-OHdG in islets. (C)

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Quantitative analysis of anti-4-hydroxy-2-hexenal (4-HHE) in islets. Note that the expression levels of 8-OHDG and 4-HHE were significantly decreased in the combined-treatment group.

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(D) Quantitative analysis of manganese superoxide dismutase (MnSOD) in islets. (E) Quantitative analysis of catalase in islets. In contrast to 8-OHDG and 4-HHE, the expression levels of MnSOD and catalase were significantly lower in the SRL group and significantly increased in the combined-treatment group. #P < 0.05 vs. VH. $P < 0.05 vs. SRL. @P < 0.05

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vs. SRL+ MET.

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Fig 5. Effects of combined treatment with metformin (MET) and LC15-0444 (LC) on apoptosis in pancreatic islets in experimental model of sirolimus (SRL)-induced DM. (A)

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Representative immunohistochemistry of the active form of caspase-3 (caspase-3) in islets and in situ TdT-mediated dUTP–biotin nick end labeling (TUNEL) assay to detect apoptosis in pancreatic islets. (B) Analysis to detect apoptosis in pancreatic islets in the experimental groups. Addition of LC to MET significantly reduced the number of TUNEL-positive cells (arrows) compared with LC or MET alone. (C) Quantitative analysis of caspase-3 in islets. Note that the addition of LC to MET significantly reduced active caspase-3 staining compared with LC or MET alone. Original magnifications, 400×. n = 9 rats per group. #P < 0.05 vs. VH. $P < 0.05 vs. SRL. @P < 0.05 vs. SRL+ MET. *P < 0.05 vs. SRL+LC.

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Fig 6. Effects of adding exendin-4 (EXD) to metformin (MET) on SRL-induced ROS production. (A) H2-DCFDA was used as a probe to evaluate intracellular ROS alterations

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using a flow cytometer. Flow cytometry analysis after 15 h of drug treatment. (B) The quantified graph shows that the combined treatment of MET and EXD significantly decreased intracellular ROS production compared with SRL alone. (C) Mitochondrial ROS

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were measured using Mito-Sox staining. Flow cytometry analysis after 15 h of drug treatment. (D) The quantified graph shows that the combined treatment of MET and EXD significantly

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decreased the percentage value compared with SRL alone. #P < 0.05 vs. VH. $P < 0.05 vs. SRL. @P < 0.05 vs. SRL+ MET. *P < 0.05 vs. SRL+EXD.

Fig 7. Effects of combined treatment of metformin (MET) and exendin-4 (EXD) on SRL-

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induced cellular damage in vitro. (A) The viability of INS-1 cells was increased by the combined treatment of MET and EXD compared with SRL alone. (B) Evaluation of apoptosis using FITC–annexin V staining followed by flow cytometry. Representative results of FITC–

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annexin V staining of INS-1 cells treated with SRL first and then with MET and/or EXD. (C)

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Graph showing the mean percentage of annexin V-positive cells. Note that there were fewer annexin V-stained cells in the group treated with MET and EXD compared with the group treated with SRL alone. #P < 0.05 vs. VH. $P < 0.05 vs. SRL. @P < 0.05 vs. SRL+ MET. *P < 0.05 vs. SRL+EXD.

Fig 8. Effects of combined treatment of metformin (MET) and exendin-4 (EXD) on relationship between the ROS and apoptosis. (A) Evaluation of apoptosis using FITC–

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treatment with ROS inhibitor (NAC) or p38MAPK inhibitor significantly blocked SRLinduced apoptotic cell death. #P < 0.05 vs. VH. $P < 0.05 vs. SRL. @P < 0.05 vs. SRL+ MET.

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*P < 0.05 vs. SRL+EXD. !P < 0.05 vs. SRL+MET+EXD.

Fig 9. Transmission electron microscopy. (A) Mitochondrial ultrastructure is disordered, and

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the number of dense core insulin granules is lower in pancreatic beta cells from the sirolimus (SRL) group. By contrast, in the group treated with LC15-0444 (LC) and metformin (MET), the mitochondrial ultrastructure is well developed and there are denser core insulin granule numbers. The scale bar equals 500 nm. White arrows indicate mitochondria. Arrowheads

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indicate insulin granules. (B–D) Quantitative analysis of the mitochondrial area and number of insulin granules in the experimental groups. Note the significantly decreased mitochondrial area and number of insulin granules in the SRL group compared with the VH group. Addition

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of LC to MET significantly increased the mitochondrial area and number of insulin granules

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compared with the SRL group. #P < 0.05 vs. VH. $P < 0.05 vs. SRL. @P < 0.05 vs. SRL+ MET. *P < 0.05 vs. SRL+LC.

Fig 10. Effects of combined treatment of metformin (MET) and exendin-4 (EXD) on SRLinduced mitochondrial function. Cells were treated with serum-free medium containing SRL (20 µg/mL) and then treated with MET (10 ng/mL) and/or EXD (0.01 nM) and incubated for 6 h. (A) Addition of EXD to MET significantly increased basal respiration, ATP-linked respiration, maximal respiration, and reserve capacity compared with SRL alone. (B) A

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AT A GLANCE COMMENTARY Jin L, et al. Background

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The protective role of dipeptidyl peptidase IV (DPP IV) inhibitors against diverse types of injury is being increasingly recognized. This study evaluated whether a DPP IV inhibitor can protect against sirolimus (SRL)-induced pancreatic islet injury.

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Translational Significance

We demonstrate that addition of a DPP IV inhibitor to metformin improved glucose control

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by decreasing oxidative stress in an animal model of SRL-induced diabetes mellitus. At the subcellular level, impaired mitochondrial respiration caused by SRL improved markedly after combined treatment with a DPP IV inhibitor and metformin. This finding may provide a rationale for the clinical use of a DPP IV inhibitor in the treatment of SRL-induced diabetes

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mellitus.