db mice

Accepted Manuscript Protective effects of SGLT2 inhibitor luseogliflozin on pancreatic β-cells in obese type 2 diabetic db/db mice Seizo Okauchi, Masashi Shimoda, Atsushi Obata, Tomohiko Kimura, Hidenori Hirukawa, Kenji Kohara, Tomoatsu Mune, Kohei Kaku, Hideaki Kaneto PII:

S0006-291X(15)30809-3

DOI:

10.1016/j.bbrc.2015.10.109

Reference:

YBBRC 34793

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 19 October 2015 Accepted Date: 20 October 2015

Please cite this article as: S. Okauchi, M. Shimoda, A. Obata, T. Kimura, H. Hirukawa, K. Kohara, T. Mune, K. Kaku, H. Kaneto, Protective effects of SGLT2 inhibitor luseogliflozin on pancreatic β-cells in obese type 2 diabetic db/db mice, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.10.109. 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.

ACCEPTED MANUSCRIPT Manuscript category: Original Article

Protective effects of SGLT2 inhibitor luseogliflozin

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on pancreatic β-cells in obese type 2 diabetic db/db mice Seizo Okauchi, Masashi Shimoda, Atsushi Obata, Tomohiko Kimura,

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Hidenori Hirukawa, Kenji Kohara, Tomoatsu Mune, Kohei Kaku, Hideaki Kaneto

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Division of Diabetes, Endocrinology and Metabolism, Kawasaki Medical School

Corresponding author: Seizo Okauchi, Division of Diabetes, Endocrinology and Metabolism, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192, Japan

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Tel: 81-86-462-1111, ext. 27512, Fax: 81-86-464-1046 E-mail: [email protected]

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Short running title: Effects of SGLT2 inhibitor on β-cells

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Word count: 4471 words

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Abstract

It is well known that Sodium-Glucose Co-transporter 2 (SGLT2) inhibitors, new

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hypoglycemic agents, improve glycemic control by increasing urine glucose excretion, but it remained unclear how they exert protective effects on pancreatic -cells. In this study, we examined the effects of SGLT2 inhibitor luseogliflozin on -cell function and mass using

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obese type 2 diabetic db/db mice. Ten-week-old male diabetic db/db mice were treated with luseogliflozin 0.0025% or 0.01% in chow (Luse 0.0025% or Luse 0.01%) or vehicle (control)

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for 4 weeks. Urinary glucose excretion was increased in Luse groups (0.0025% and 0.01%) compared to control mice 3 days after the intervention. Fasting blood glucose levels were significantly lower in mice treated with Luse compared to control mice. Fasting serum insulin concentrations were significantly higher in mice treated with Luse compared to control mice.

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Triglyceride levels tended to be lower in Luse groups compared to control mice. In immunohistochemical study using pancreas tissues, -cell mass was larger in Luse groups compared to control group which was due to the increase of -cell proliferation and decrease

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of -cell apoptosis. Furthermore, in gene analysis using isolated islets, insulin 1, insulin 2, MafA, PDX-1 and GLUT2 gene expression levels were significantly higher in Luse groups

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compared to control group. In contrast, expression levels of fibrosis-related gene such as TGF, fibronectin, collagen I and collagen III were significantly lower in Luse groups. In conclusion, SGLT2 inhibitor luseogliflozin ameliorates glycemic control and thus exerts protective effects on pancreatic -cell mass and function.

Key words: pancreatic β-cells, SGLT2 inhibitor, β-cell mass and function

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1. Introduction

The number of subjects with type 2 diabetes has been markedly increasing all over the

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world, and the main characteristics of the disease are insulin resistance in insulin target tissues (e.g liver, adipocyte and skeletal muscle) and insufficient insulin secretion from pancreatic -cells. When -cells are chronically exposed to hyperglycemia, -cell function further

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deteriorates [1-5]. After exposure to chronic hyperglycemia, insulin biosynthesis and

secretion are gradually decreased which is often accompanied by decrease and/or inactivation

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of insulin gene transcription factors [4-7]. Such phenomena are well known as -cell glucose toxicity. However, such -cell dysfunction is ameliorated by an appropriate intervention with some anti-diabetes agent including insulin, incretin-related medicine and peroxisome proliferator-activated receptor γ (PPAR  agonist [8-14].

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In order to obtain good glycemic control and prevent a variety of diabetic complications, many kinds of anti-diabetic agents have been developed and are used in clinical medicine. Among them, sodium-glucose cotransporter 2 (SGLT2) inhibitors are drawing attention in

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clinical medicine as well as basic research area [15-22]. SGLT2 inhibitors function in an insulin-independent manner and lower blood glucose levels by enhancing urinary glucose

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excretion. SGLT2 is mainly present in the S1 segment of the proximal renal tubules and functions for about 90% of the total renal glucose reabsorption. In addition, it was shown that SGLT2 expression in the kidney was increased under diabetic conditions [23]. In this study, we administered SGLT2 inhibitor luseogliflozin to obese diabetic C57BL/KsJ-db/db mice and examined how luseogliflozin could exert protective effects on pancreatic -cells.

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2. Materials and Methods 2.1. Compound Luseogliflozin was synthesized by Medical Chemistry Laboratories in Taisho

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Pharmaceutical Co., Ltd.

2.2. Animals

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We used 10-week-old male C57/BsJ-db/db mice which were purchased (Clea, Tokyo, Japan). They were housed two to three animals per cage in all experiments under controlled

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ambient conditions and a 14:10 h light/dark cycle with lights on at 7 a.m. Animals were given free access to drinking water and food and were maintained at 25°C. These were divided into 3 groups which were treated with luseogliflozin 0.0025% in chow (Luse 0.0025%), luseogliflozin 0.01% (Luse 0.01%) or vehicle (0.05% carboxymethylcellulose, oral) for 4

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weeks (n=10 for each group). This study was approved by the Animal Use Committee of Kawasaki Medical School (No. 14-088) and was conducted in compliance with the Animal Use Guidelines of the Kawasaki Medical School. Body weight and food intake were

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monitored weekly from 10-14 weeks of age.

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2.3. Measurement of biochemical markers Blood samples were collected from the tail vein. Blood glucose levels were measured immediately using a commercially available enzyme electrode method. The concentration of plasma insulin (Insulin ELISA kit; Morinaga Institute of Biological Science, Yokohama, Japan), Plasma TG and plasma NEFA concentrations were measured enzymatically using the Triglyceride E-Test Wako and NEFA C-Test Wako (Wako Pure Chemical Industries, Osaka, Japan).

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2.4. Islet Isolation Islets were isolated after clamping the common bile duct at its entrance to the duodenum,

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2 ml of HBSS (Hanks’ Balanced Salts Solution; Sigma;H1387-10×1L) containing 1.2825mg of collagenase (sigma;C9263-500MG) per ml was injected into the duct. The swollen

pancreas was surgically removed and incubated at 37°C for 15 min. Thereafter, 12 ml of cold

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HBSS containing 9% newborn calf serum (NCS) was added to stop the digestion reaction at shaking 20 times and standing for 5 min on ice. Digested pancreas was dispersed by pipetting

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and rinsed twice with 9 ml of ice-cold HBSS. The digested tissue was re-suspended in cold RPMI medium containing 10% fetal bovine serum (FBS). Islets were manually picked under a dissection microscope using a pipette.

2.5. Measurement of triglyceride content and insulin content in pancreatic islets

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Isolated islets were incubated with RPMI medium (Sigma) containing 10% fetal calf serum overnight at 37°C in 5% CO2. On the following day, size-matched pancreatic islets were collected. The TG content and insulin content in the pancreatic islets were measured as

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previously reported12.

2.6. Glucose-stimulated insulin secretion from isolated pancreatic islets Size-matched pancreatic islets were prepared (5 pancreatic islets/tube) and pre-incubated in KRB-HEPES buffer. The supernatant was replaced with either 3 or 16.7 mmol/l glucose solution, and the mixture was incubated for an additional 60 min. The supernatant was recovered and stored at -80°C until use.

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ACCEPTED MANUSCRIPT 2.7. Oral glucose tolerance test (OGTT) After overnight fasting, 1g/kg glucose solution was orally administered at a volume of 10ml/kg. Blood samples were collected from the tail vein before and 15, 30, 60 and 120 min

measured.

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2.8. Intraperitoneal insulin tolerance test (IPITT)

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after the glucose administration, and blood glucose levels, plasma insulin levels were

After 4 h fast, insulin (2 units/kg body weight) was injected intraperitoneally. Blood

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samples were collected from the tail vein before and 30, 60, 90, 120 min after insulin administration, and blood glucose levels were measured.

2.9. Immunofluorescence

According to the previously established method [12], the sections were incubated with a

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mixture of primary antibodies: mouse anti-glucagon antibody and rabbit anti-insulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Pancreatic islets proliferation was

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identified by staining sections with rabbit anti-Ki-67 polyclonal antibody (Abcam, Cambridge, MA, USA). To evaluate apoptotic cell death in pancreatic islets, a TUNEL assay was

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performed using a DeadEnd Fluorometric TUNEL System (DeadEnd; Promega, Madison, WI, USA).

2.10. Morphometric analysis The microscopy was used for image acquisition, and the image analysis software NIH Image (version 1.61; http://rsbweb.nih.gov/ij/) was used to calculate the pancreas area and islet area. Using a total of 15 sections (3 sections from three different areas of the pancreas)

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ACCEPTED MANUSCRIPT for each group of mice, -cell mass was estimated via the following formula: cell mass (mg) = average of islet area per section / average of pancreas area per section × weight of pancreas × -cell ratio (average of -cell number / cell number in islet). Observations were made using

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a minimum of 50 islets, and when quantified, the - and -cells were expressed as a percentage of the total number of islet cells.

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2.11. Real-Time PCR

RNA samples from the isolated islets were extracted using a RNeasy Mini Kit (Cat. No.

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74106, QIAGEN). The primers were designed using Primer Express, based on mRNA sequences downloaded from the GenBank nucleotide database. A reaction mixture was prepared by combining 1 μl of sample, 1μl of 50 nM primers, 12.5 μl of Sybr Green PCR Master Mix (Applied Biosystems) solution, and 10.5 μl of diluent solution. Dissociation curve analysis was performed in all experiments to determine the dissociation temperature, and the

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size of the PCR products was confirmed using agarose gel electrophoresis. To quantify gene expression, the 2-ΔCT was calculated using 18S rRNA as an internal control, and it was

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compared with the mRNA value for each control group of mice.

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2.12. Statistical analysis

All data are shown as mean ± S.E. The comparison between 3 groups was performed using ANOVA and the multiple comparison was performed using the Tukey-Kramer method. And p< 0.05 was regarded as significant. Statistical analysis was performed using JMP9.

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3. Results 3.1. Alteration of metabolic parameters after 4 weeks of treatment with luseogliflozin in obese type 2 diabetic db/db mice

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At the baseline (10 weeks of age), there was no difference in body weight, food intake, fasting and non-fasting blood glucose levels among 3 groups: vehicle (control), luseogliflozin 0.0025% in chow (Luse 0.0025%), luseogliflozin 0.01% (Luse 0.01%) (Fig. 1A-D). Body

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weight was larger in Luse 0.0025% and Luse 0.01% groups compared to control group (Fig. 1A), although there was no difference in food intake among the 3 groups after 4 weeks of

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treatment (Fig. 1B). Fasting blood glucose levels in Luse 0.0025% and Luse 0.01% groups were significantly lower compared to those in control group (Fig. 1C). Non-fasting blood glucose levels in Luse 0.0025% and Luse 0.01% groups were also significantly lower compared to control group (Fig. 1D).

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Similarly, at the baseline, there was no difference in fasting serum insulin, adiponectin, triglyceride and NEFA levels among the 3 groups (control, Luse 0.0025% and Luse 0.01% groups) (Fig. 1E-G). Fasting serum insulin levels at 12 and 14 weeks of age in Luse 0.0025%

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and Luse 0.01% groups were significantly lower compared to control group (Fig. 1E). There was no difference in adiponectin, triglyceride and NEFA levels 2 and 4 weeks after the

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treatment among the 3 groups (Figs. 1F, G, H). Next, we examined the effects of such treatment on urine volume and urinary glucose volume. As shown in Fig. 1I, 3 days after the treatment with luseogliflozin, urine volume was increased. However, urine volume was decreased after 2 and 4 weeks of treatment which was presumably due to the amelioration of glycemic control. Similar results were obtained with urinal glucose volume. As shown in Fig. 1J, 3 days after the treatment with luseogliflozin, urinary glucose volume was increased. However, urinary glucose volume was decreased after

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ACCEPTED MANUSCRIPT 2 and 4 weeks of treatment which was also presumably due to the amelioration of glycemic control. Next, to examine the effects of the treatment with luseogliflozin on insulin secretory

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capacity, we performed orally glucose tolerance test. As shown in Figs. 2A and 2B, after 4 weeks of treatment with luseogliflozin, blood glucose levels were lower in Luse groups

compared to those in control group, and serum insulin levels were higher in Luse groups

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compared to control group. Next, to further evaluate insulin secretory capacity, we isolated islets and examined glucose-stimulated insulin secretion. As shown in Fig. 2C, insulin

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secretion was increased in response to 16.7 mmol/l glucose in all groups (control group, 4.73-fold increase; Luse 0.0025% group, 6.71-fold increase; Luse 0.01% group, 11.75-fold increase). There was significant difference between control and Luse 0.01% group in insulin secretion after glucose load (p<0.05). In addition, after 4 weeks of intervention, islet insulin contents in the mice treated with luseogliflozin were higher compared to control group (Fig.

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2D). There was no difference in TG content among the 3 groups (Fig. 2E). Next to examine the effects of anti-diabetic treatment with luseogliflozin on insulin resistance, we

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performed intraperitoneal insulin tolerance test (IPITT). Insulin sensitivity in the mice treated with

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luseogliflozin was significantly improved as compared with untreated group (Fig. 2F).

3.2. Alteration of  -cell morphology after 4 weeks of treatment with luseogliflozin in diabetic db/db mice

To further examine the effects of the treatment with luseogliflozin on -cells, we employed morphological examination of pancreatic islets after 4 weeks of the treatments. Double immunostaining with antibodies against insulin and glucagon was carried out. It is known that pancreatic -cells and -cells in mice under normal conditions are usually located

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ACCEPTED MANUSCRIPT in the central and peripheral area of islets, respectively. As shown in Fig. 3A, however, such localization was disarranged under diabetic conditions; in untreated db/db mice, substantial number of -cells were observed in the center of the islets. Furthermore, the disarrangement

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of islet cell localization was ameliorated by luseogliflozin treatment. As shown in Fig. 3B, -cell mass was increased by luseogliflozin treatment. There was no difference in -cell mass among the 3 groups. Next, to explore how -cell mass was preserved by such treatment, we

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evaluated the effects of the treatment on -cell proliferation and apoptosis. Histological

sections of the pancreas were stained by antibody specific for Ki67 and the proportion of

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antibody-positive cells was estimated. As shown in Fig. 3C, D, the number of Ki67-positive -cells was increased after lusegliflozin treatment. In addition, to evaluate the ratio of apoptosis in -cells, we performed TUNEL staining. As shown in Fig. 3E, the number of TUNEL-positive -cells was decreased after lusegliflozin treatment.

diabetic db/db mice

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3.3. Alteration of gene expression profiles after 4 weeks of treatment with luseogliflozin in

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To examine how insulin content was recovered by such treatment, we evaluated insulin mRNA expression levels. Expression levels of insulin 1 and insulin 2 mRNA were increased

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by lusegliflozin treatment (Fig. 4A). Next, we evaluated expression levels of various insulin gene transcription factors such as MafA and PDX-1 both of which are very important to maintain mature -cell function and insulin gene transcription. MafA and PDX-1 levels were significantly higher in Luse groups compared to those in control group (Fig. 4B). Expression level of Glut2 which is very important for glucose-stimulated insulin secretion was also preserved by luseogliflozin treatment (Fig. 4C). Next, to examine how -cell mass was preserved by such treatment, we evaluated gene

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ACCEPTED MANUSCRIPT expression profiles related with -cell mass, proliferation and apoptosis. As shown in Fig. 4D, caspase 3 mRNA expression was down-regulated by luseogliflozin treatment, although there was no difference in Bcl2 expression levels among the 3 groups. Also, we evaluated gene

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expression related with lipid synthesis, but there was no difference in Fas and SREBP-1c expression among the 3 groups (Fig. 4E). In addition, we evaluated expression levels of

fibrosis-related factors. As shown in Fig. 4F, TGF, fibronectin, collagen I and collagen III

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mRNA levels were significantly lower in luseogliflozin group compared to control group.

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4. Discussion In this study, we showed that SGLT2 inhibitor luseogliflozin exert protective effects on pancreatic β-cells in obese type 2 diabetic db/db mice. Insulin biosynthesis and secretion were

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markedly increased by luseogliflozin (Fig. 2). In concomitant with such phenomena,

expression levels of insulin mRNA expression levels were up-regulated by luseogliflozin (Fig. 3). We think that the increased expression of insulin mRNA levels explains the increased

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insulin biosynthesis and secretion. In addition, the increased expression of GLUT2 could explain, at least in part, the augmentation of glucose-stimulated insulin secretion. It is known

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that MafA and PDX-1 plays crucial role as an insulin gene transcription factor and functions for maintenance of insulin biosynthesis and secretion [4]. We think that the reduction of MafA and PDX-1 expression in untreated db/db mice explains the decrease of insulin content and secretion and that the increased MafA and PDX-1 expression after the treatment with

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luseogliflozin led to the restoration of insulin content and secretion. In general, SGLT2 inhibitors are thought to decrease body weight, but in the present experiment body weight was larger in luseogliflozin-treated mice compared to control mice,

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although there was no difference in food intake (Fig. 1). Since glycemic control is extremly poor in diabetic db/db mice, we assume that the amelioration of glycemic control with

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luseogliflozin led to the increase of body weight. In other words, we think that the suppression of body weight increase presumably due to very poor glycemic control was mitigated and consequently body weight was increased in this study. In addition, we showed that the influence of luseogliflozin treatment on β-cell mass (Fig. 4). Expression level of Bcl2 was increased and caspase 3 expression was decreased by luseogliflozin (Fig. 4). Since Bcl2 function as an anti-apoptotic factor and caspase 3 functions as an pro-apoptotic factor, we assume that the alteration of such gene expression explains, at

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ACCEPTED MANUSCRIPT least in part, the reason why luseogliflozin preserved -cell mass. Indeed, the number of Ki67 positive -cells was increased after luseogliflozin treatment (Fig. 3D), indicating that luseogliflozin contributed to the augmentation of -cell proliferation. In addition, the number

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of TUNEL-positive -cells was decreased after luseogliflozin treatment (Fig. 3E), indicating that luseogliflozin contributed to the reduction of -cell apoptosis. Taken together, it is likely that luseogliflozin treatment increased -cell mass through the augmentation of both -cell

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proliferation and reduction of -cell apoptosis.

In this study, we showed that an SGLT2 inhibitor luseogliflozin was useful to ameliorate

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glycemic control in diabetic db/db mice which led to protection of β-cells. It was previously reported that glucose lowing with another SGLT2 inhibitor dapagliflozin reduced the decline of pancreatic function and disruption of normal islet morphology in female Zucker diabetic fatty (ZDF) rats24. Take our present data and the above previous report together, the similar results obtained in different studies would strengthen the idea that the therapy with SGLT2

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inhibitors are useful to ameliorate glycemic control and protect -cells in diabetic animals. Taken together, in obese type 2 diabetic db/db mice, SGLT2 inhibitor luseogliflozin increases insulin biosynthesis and secretion accompanied by the increased expression of

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various -cell-related important factors and increases -cell mass through the augmentation of

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-cell proliferation and the reduction of -cell apoptosis. Therefore, it is likely that SGLT2 inhibitor would be promising for the protection of -cells against glucose toxicity which is often observed in type 2 diabetes. It is noted, however, in practical medicine we should be careful in several points when we use them. For example, SGLT2 inhibitors could lead to dehydration and/or urinary tract infection especially in subjects of advanced age.

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Acknowledgements We thank Shiori Ikeda, Yuka Nogami, and Saeko Moriuchi for their assistance. Abstracts

(Boston, 2015).

Disclosure

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of this report were presented at the American Diabetes Association's 75th Scientific Sessions

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KK has been an advisor to, received honoraria for lectures from, and received

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scholarship grants from Novo Nordisk Pharma, Sanwa Kagaku Kenkyusho, Takeda, Taisho Pharmaceutical Co., Ltd, MSD, Kowa, Sumitomo Dainippon Pharma, Novartis, Mitsubishi Tanabe Pharma, AstraZeneca, Nippon Boehringer Ingelheim Co., Ltd, Chugai, Daiichi Sankyo, and Sanofi. HK has received honoraria for lectures and received scholarship grants from Sanofi, Novo Nordisk, Lilly, Boehringer Ingelheim, MSD, Takeda, Ono Pharma, Daiichi

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Sankyo, Sumitomo Dainippon Pharma, Mitsubishi Tanabe Pharma, Pfizer, Kissei Pharma,

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AstraZeneca, Astellas, Novartis, Kowa, Chugai and Taisho Pharma.

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[3] P.A. Halban, K.S. Polonsky, D.W. Bowden, et al. β-cell failure in type 2 diabetes: postulated mechanisms and prospects for prevention and treatment. Diabetes Care 37 (2014) 1751-1758. [4] H. Kaneto, T.A. Matsuoka. Role of pancreatic transcription factors in maintenance of

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Figure Legends Fig. 1. Metabolic variables in C57BLKsJ-db/db mice which were treated with luseogliflozin 0.0025% in chow (Luse 0.0025%) (open triangle), luseogliflozin 0.01% (Luse 0.01%) (open

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circle) or vehicle (0.05% carboxymethylcellulose, oral) (close circle) for 4 weeks. (A) body weight, (B) food intake, (C) fasting blood glucose levels, (D) non-fasting blood glucose levels, (E) fasting insulin levels, (F) fasting adiponectin levels body weight, (G) fasting triglyceride

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levels, (H) fasting NEFA levels. Data are presented as mean±SE. n=10-12 for each group. *: p<0.05 vs vehicle group, †: p<0.05 vs before treatment. Urine volume (I) and urinary glucose

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excretion (J) in C57BLKsJ-db/db mice which were treated with luseogliflozin 0.0025% in chow (open triangle), luseogliflozin 0.01% (open circle) or vehicle (close circle) for 4 weeks. Left panel, before and 3 days after the treatment; right panel, 0-4 weeks after the treatment.

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Data are presented as mean±SE. n=4 for each group.

Fig. 2. Blood glucose levels (A) and serum insulin levels (B) after oral glucose glucose tolerance test in C57BLKsJ-db/db mice which were treated with luseogliflozin 0.0025% in

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chow (Luse 0.0025%) (open triangle), luseogliflozin 0.01% (Luse 0.01%) (open circle) or vehicle (close circle) for 4 weeks. Data are presented as mean±SE. n=6-8 for each group. *:

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p<0.05 vs vehicle group. (C) Glucose-stimulated insulin secretion (GSIS) in C57BLKsJ-db/db mice which were treated with luseogliflozin 0.0025% in chow (Luse 0.0025%), luseogliflozin 0.01% (Luse 0.01%) or vehicle for 4 weeks. GSIS was assessed by using low and high glucose concentrations (3 mmol/l and 16.7 mmol/l). n=5 for each group. *: p<0.05. (D) Insulin content and (E) TG content in islets of C57BLKsJ-db/db mice after 4 weeks of treatment with luseogliflozin. Data are presented as mean±SE. n=5 for each group. *: p<0.05. (F) Insulin sensitivity was assessed by an intraperitoneal insulin tolerance test (IPITT) in

18

ACCEPTED MANUSCRIPT C57BLKsJ-db/db mice which were treated with luseogliflozin 0.0025% in chow (Luse 0.0025%) (open triangle), luseogliflozin 0.01% (Luse 0.01%) (open circle) or vehicle (close circle) for 4 weeks. Left panel; Results of the insulin tolerance test are expressed as a

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percentage relative to the basal blood glucose concentration before insulin administration. Right panel; Area under the curve (AUC) (0-120min) in IPITT. Data are presented as

SC

mean±SE. n=4 for each group. *: p<0.05 vs vehicle group.

Fig. 3. Pancreatic -cell morphology after 4 weeks of treatment with luseogliflozin in diabetic

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db/db mice. (A) Representative immunofluorescence of insulin (green) and glucagon (red) in pancreatic islet tissue sections. Scale bars: 100 m. (B) Volume of - and -cells after 4 weeks of treatment with luseogliflozin. Data are presented as mean±SE. *: p<0.05. (C) Representative immunofluorescence of Ki67 (red) and insulin (green) in pancreatic islet tissue sections. Scale bars: 100 m. (D) Ratio of Ki67-positive -cells. (E) Ratio of

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TUNEL-positive -cells. Data are presented as mean±SE. *: p<0.05.

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Fig. 4. Expression levels of insulin, insulin gene transcription factors (PDX-1 anf MafA) and glucose transporter 2 (Glut2) in C57BLKsJ-db/db mice which were treated with luseogliflozin

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0.0025% in chow (Luse 0.0025%), luseogliflozin 0.01% (Luse 0.01%) or vehicle for 4 weeks. (A) insulin 1, insulin 2, (B) MafA, Pdx-1, (C) glut2, ZnT8. Data are presented as mean±SE. *: p<0.05. Expression levels of apoptosis-related factors (caspase 3, bcl2) (D) and lipid synthesis-related factors (Fas, SREBP-1c) (E) in C57BLKsJ-db/db mice after 4 weeks of treatment with lusegliflozin. (F) Expression levels of fibrosis-related factors in C57BLKsJ-db/db mice after 4 weeks of treatment with luseogliflozin. Data are presented as mean±SE. *: p<0.05.

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ACCEPTED MANUSCRIPT

Body weight

(g)

(g/day) 10.0

Food intake

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50.0

RI PT

B

SC

A

Fig. 1

9.0

45.0

*† *†

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40.0

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35.0

25.0 10W

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30.0

12W

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8.0 7.0 6.0 5.0 4.0 3.0 2.0

9-10W

10-11W

11-12W

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13-14w

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D Fasting blood glucose levels

RI PT

C

Fig. 1

Non-fasting blood glucose levels

(mg/dl)

†

†

800

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300

SC

(mg/dl)

700

250

600

*†

100

*†

400 300

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*† 50 0 10W

12W

500

EP

150

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200

*†

14W

* *

200

*

*

*

*

100 0 10w

11w

12w

13w

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(ng/ml)

Fasting adiponectin levels

SC

Fasting insulin levels

RI PT

F

E

Fig. 1

(μg/ml)

7.00

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20.00 18.00

6.00

†

5.00

TE D

†

4.00

EP

3.00

1.00 0.00 10W

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2.00

12W

16.00 14.00 12.00 10.00

8.00 6.00 4.00 2.00 0.00

14W

10W

12W

14W

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H

SC

(mEq/l)

Fasting NEFA levels

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(mg/dl)

Fasting triglyceride levels

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G

Fig. 1

1.80

250.0

1.60

200.0

1.40

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1.20

150.0

*

50.0 0.0 10W

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EP

100.0

12W

1.00 0.80 0.60 0.40 0.20 0.00

14W

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Fig. 1

I

Urine volume

SC

Urine volume (ml/8h)

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(ml/8h)

3.00

2.50

2.50

2.00

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2.00

1.00 0.50 0.00 9W before treatment

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EP

1.50

3 3day days after treatment

1.50 1.00 0.50 0.00 9W

11W

13W

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RI PT

J

Fig. 1

Urinary glucose excretion

Urinary glucose excretion

(mg/ 8h)

SC

(mg/ 8h)

350.0

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350.00

300.0

300.00

250.0

250.00

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200.00 150.00

50.00 0.00 9W before treatment

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100.00

33day days after treatment

200.0 150.0 100.0 50.0 0.0 9W

11W

13W

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A

Fig. 2

Blood glucose levels in glucose tolerance test

(mg/dl)

RI PT

B Serum insulin levels in glucose tolerance test

(ng/ml) 3

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SC

1200

1000

2

600

* *

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*

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800

*

*

*

200

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1

*

* 0

0 0

15

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120

(min)

0

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RI PT

C

Fig. 2

3 mM glucose

16.7 mM glucose

(ng/ml/islet)

*

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SC

(ng/ml/islet)

3

TE D

3

EP

2

0 V vehicle

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1

Lus 0.0025 Luse 0.0025

Lus 0.01 Luse 0.01

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0 V vehicle

Lus 0.0025 Luse 0.0025

Lus 0.01 Luse 0.01

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D

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E

Fig. 2

Islet TG content

SC

Islet insulin content (ng/islet)

(ng/islet)

*

60

1500

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80

*

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1000

EP

40

0 V vehicle

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20

Lus 0.0025 Luse 0.0025

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0 V vehicle

Lus 0.0025 Luse 0.0025

Lus 0.01 Luse 0.01

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F

Percentage of glucose reduction in insulin tolerance test

Fig. 2

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AUC (0-120 min) in insulin tolerance test

180

SC

*

*

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160

140%

140 120

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60%

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*

*

100 80 60

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100%

40

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vehicle vehicle

Luse 0.0025% Luse 0.0025

Luse Luse0.01% 0.01

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A

Fig. 3

B

vehicle

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Luse 0.01%

0.5

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Luse 0.0025%

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*

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b a

b

a

vehicle

Luse 0.0025

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a

Luse 0.01

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D

E

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C

Fig. 3

Ratio of Ki67-positive b-cells

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SC

vehicle

Ratio of TUNEL-positive b-cells

(%)

*

3

2

EP

Luse 0.0025%

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*

(%)

*

0.16

0.12

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0.08

1

0.04

Luse 0.01% 0

0 V vehicle

Luse 0.0025% 0.01% Luse 0.0025 Luse Luse 0.01

vehicle vehicle

Luse 0.025% 0.01% Luse 0.0025 Luse Luse 0.01

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RI PT

A

insulin 2

SC

insulin 1 *

（×10-1） 7

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（×10-1） 2

Fig. 4

*

*

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5

EP

1

0 V vehicle

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Ratio vs 18S rRNA

6

Luse 0.0025% Luse 0.0025 Luse Luse0.01% 0.01

4 3 2 1 0 V Luse 0.01% vehicle Luse0.0025% 0.0025 Luse Luse 0.01

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B

MafA

（×10-4） 4

（×10-5）

*

2

1

SC

2 1

Pdx-1 *

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0

0

C

Luse 0.0025% Luse 0.0025 Luse Luse0.01% 0.01

GLUT2 *

（×10-5）

V vehicle

Luse 0.0025% Luse 0.01% Luse 0.0025 Luse 0.01

ZnT8 *

（×10-4） 6

EP

5

TE D

V vehicle

4

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Ratio vs 18S rRNA

*

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Ratio vs 18S rRNA

3

Fig. 4

3 2

4

2

1 0

0

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Luse0.0025% 0.0025 Luse Luse 0.01 Luse 0.01%

V vehicle

Luse 0.0025% Luse 0.0025

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D

TGFβ

（×10-4） 2

fibronectin

（×10-4） 10

*

*

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4

SC

6 1

*

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Ratio vs 18S rRNA

8

2 0

0

6

*

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5

*

EP

collagenⅠ

（×10-3）

4 3

vehicle V

Luse Luse0.0025% 0.0025 Luse Luse0.01% 0.01

TE D

V vehicle

Ratio vs 18S rRNA

Fig. 4

Luse 0.0025 Luse Luse 0.01 Luse 0.0025% 0.01%

collagenⅢ * *

（×10-6） 8 7 6 5 4 3

2

2 1

1 0

0 vehicle V

Luse 0.0025 Luse 0.0025%

Luse 0.01 Luse 0.01%

vehicle V

Luse0.0025% 0.0025 Luse Luse 0.01 Luse 0.01%

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5 4

10

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F

Luse Luse0.0025% 0.0025 Luse Luse0.01% 0.01

FAS

AC C

EP

（×10-3） 3

TE D

V vehicle

Ratio vs 18S rRNA

Bcl2

SC

Ratio vs 18S rRNA

（×10-5）

caspase 3 * *

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（×10-5）

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E

Fig. 4

2

V vehicle

Luse Luse0.0025% 0.0025 Luse Luse0.01% 0.01

SREBP-1c

（×10-4） 5 4

3 2

1

1 0

0 vehicle V

Luse0.0025% 0.0025 Luse Luse 0.01 Luse 0.01%

vehicle V

Luse 0.0025 Luse Luse 0.01 Luse 0.0025% 0.01%

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Highlight

1. SGLT2 inhibitor luseogliflozin ameliorates glycemic control in db/db mice. 2. Luseogliflozin increases -cell proliferation and decreases -cell apoptosis.

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3. Luseogliflozin preserves various -cell-specific gene expression.

AC C

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4. Luseogliflozin decreases various fibrosis-related factors in db/db mice.