For pair feeding, mice were fed 2.7g of HFD containing tofogliflozin

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1 Materials and Methods Pair Feeding Experiment For pair feeding, mice were fed 2.7g of HFD containing tofogliflozin (0.005%), which is average daily food intake of mice fed control HFD ad libitum at week 7 after tofoglifozin treatment. The mice were housed individually and the cages were changed each day to remove the food thrown on the floor of the cage. These mice were also housed under a 12-h light-dark cycle and the daily food was placed in the animal cages before the start of the dark cycle (35). Measurement of Adipocyte Size Epididymal white adipose tissue was routinely processed for paraffin embedding, and 4-μm sections were cut and mounted on silanized slides. The adipose tissue sections were stained with hematoxylin and eosin, and the total adipocyte area was manually traced and analyzed using the Image J software. The white adipocyte area was measured in 150 or more cells per mouse in each group, in accordance with a previously described method but with slight modifications (36,37). Urinary Collection and Urinary Glucose Measurement Animals were placed in individual metabolic cages for the collection of urine samples and had ad libitum access to water and food. Urine samples were frozen at -20 C until the measurement of the glucose concentrations. Urinary glucose was assayed using an enzymatic method (Wako Pure Chemical Industries Ltd., Osaka, Japan). 1

2 Dual X-Ray Absorptiometry (DEXA) Before this measurement, the mice were anesthetized with pentobarbital (1 mg/kg). Whole-body measurements were conducted using a Lunar PIXI Mus2 Densitometer (Lunar Corp., Madison, WI) as previously reported but with slight modifications (38). Analysis of the Respiratory Quotient (RQ) Oxygen consumption was measured every 3 minutes for 24 h in mice using an O 2 /CO 2 metabolism measurement device (Model MK-5000; Muromachikikai, Tokyo, Japan), as previously reported (38-43). The RQ was calculated using the following equation based on the volume of O 2 consumed and the volume of CO 2 produced: RQ = vco 2 /vo 2. Computed Tomography (CT) To determine the fat volume and skeletal muscle volume, CT was performed in mice pair-fed for 6 weeks (Hitachi Aroka). The bone volume and bone mineral density were also measured using the manufacturer s software. HOMA-R HOMA-R was calculated by multiplying the fasting blood glucose and serum insulin values. Animals were denied access to food for 24 h (NC) or for 48 h (HFD) before the blood sample collection. 2

3 Clinical Chemistry Assays Clinical chemistry (ALP, T-Bil, BUN, Cre, ip, Ca, Na, K, Cl) assays were performed at Chugai Pharmaceutical Co., Ltd. using enzymatic methods. The serum transaminase levels were measured with the transaminase C-II test kit (Wako Pure Chemical Industries Ltd., Osaka, Japan). Serum free fatty acid (Wako Pure Chemical Industries Ltd., Osaka, Japan) and serum glycerol levels (Cayman Chemical Company, USA) were also assayed using enzymatic methods in accordance with the manufacturer s instructions. The serum ketone body levels were measured using a ketometer N (Sanwa Kagaku Kenkyusho Co., Ltd., Japan). Hct was measured using i-stat (FUSO Pharmaceutical Industries, Ltd.) Serum Adiponectin and Leptin Measurements Samples were collected during the period when the mice had access to water and food ad libitum. Serum adiponectin levels were determined using a mouse adiponectin enzyme-linked immunosorbent assay kit (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan), and the serum leptin levels were determined using a mouse leptin enzyme-linked immunosorbent assay kit (Morinaga Institute of Biological Science Inc.). Serum Glucagon Measurements Samples were collected at 16 h after the administration of CMC or CMC containing 10 mg/kg of tofogliflozin. A protease inhibitor (Roche life science) was placed in 3

4 tubes on ice and the samples were immediately placed in the tubes, mixed well and allowed to clot, and the serum was separated from the clot by centrifugation. The serum glucagon levels were determined using a mouse glucagon enzyme-linked immunosorbent assay kit (Mercodia Glucagon ELISA) immediately after the samples were collected, in accordance with the manufacturer s instructions. ( RNA Preparation and TaqMan PCR Total RNA was extracted from various tissues with TRIzol reagent (Invitrogen), in accordance with the manufacturer s instructions. After treatment with RNase-Free DNase (Qiagen) to remove genomic DNA, cdna was synthesized with MultiScribe Reverse-Transcriptase (Applied Biosystems, Foster City, CA). The reverse transcription mixture was amplified with specific primers using an ABI Prism 7000 sequence detector equipped with a thermocycler. The probes for cyclophilin, Pepck, Tnf-α, Mcp-1, Il-6, Cpt-1α, Pparα, Pgc1α, Acadm, Acadl, Hmgcs2, and Aqp9 were purchased from Applied Biosystems (Foster City, CA). The primers for cyclophilin and Scd-1, Fasn, Dgat1, and Dgat2 were purchased from Invitrogen. The relative expression levels were compared by normalization to the expression levels of cyclophilin. The primer sequences that were used are shown in Supplemental Table 4. Western-Blot Analysis Tissues were excised and homogenized in ice-cold buffer A (25 mm Tris-HCl [ph7.4], 10 mm sodium orthovanadate, 10 mm sodium pyrophosphate, 100 mm sodium 4

5 fluoride, 10 mm EDTA, 10 mm EGTA, and 1 mm phenylmethylsulfonyl fluoride). Protein concentration of each sample was measured by BCA protein assay reagent (Thermo Fisher Scientific) according to the manufacture s instruction. The sample buffer for analysis under reducing conditions was composed of 3% SDS, 50 mm Tris-HCl (ph6.8), 5% 2-mercaptoethanol, and 10% glycerol. Samples were mixed with 5 sample buffer, heated at 95 C for 5 min for heat denaturation. 15µg of protein was applied to each lane and separated on polyacrylamide gels, and transferred to a Hybond-P polyvinylidene difluoride transfer membrane (Amersham Biosciences). Bands were detected with ECL detection reagents (Amersham Biosciences). Each membrane was cut at 50 kda so as to detect each protein and its internal control (actin). Image J software was used for the measurements of the density of each band. Antibody information is provided in the antibody table. Measurements of the tissue TG and Glycogen Contents To determine the TG contents in the liver and the skeletal muscle, tissue homogenates were extracted with 3:2 (vol/vol) hexan and isopropyl alcohol and the extracts were shaken for 10 min. Then, after centrifugation at rpm for 5 min, the organic layer was collected. The extraction was conducted twice, and the collected samples were dried and resuspended in 1:1 (vol/vol) NP-40 and dioxan. The measurements of the triglyceride and glycogen contents were conducted using Triglyceride C-II Wako (Wako Pure Chemical Industries Ltd., Osaka, Japan) and the Glycogen Assay kit (Wako Pure Chemical Industries Ltd., Osaka, Japan), 5

6 respectively, in accordance with the manufacturer s instructions. Measurements of the Systolic Blood Pressure and Heart Rate Softron BP -98A-L was used to measure the systolic blood pressure and heart rate according to the manufacturer s instructions. The measurements were conducted after the mice had been denied access to food for 16 h. Supplemental Figure Legends Supplemental Figure 1. Outline of all the experiments performed in this study. Supplemental Figure 2. Time-course of changes in the serum drug concentration and urinary glucose excretion rate after the final tofogliflozin administration. Nine-week-old male C57BL/6 mice were fed NC (open bars) or an HFD (filled bars) containing tofogliflozin at 0.015%, a higher concentration than 0.005%, for one week. After one week, the respective meals were administered without tofogliflozin. Blood samples were collected at 0, 24, 48, and 72 h after the switch to meals not containing tofogliflozin. Urine collection was conducted for 12 h during the dark cycle from each of the time-points shown in the figure (n = 4). 6

7 Supplemental Figure 3. Glucose intolerance and insulin resistance after tofogliflozin treatment in mice fed an HFD. (A, B, C) Blood glucose levels, AUC, and serum insulin levels during an OGTT in mice not treated (solid line, open bar) or treated with 0.005% tofogliflozin (dotted line, filled bar) (n = 8). (D) HOMA-R in mice not treated (open bars) or treated with 0.005% tofogliflozin (filled bars) (n = 8). (E, F) Blood glucose and serum insulin levels (n = 14 16). (G, H) G6Pase and Pepck mrna expression levels in the liver (n = 6). (I, J) Serum NEFA and ketone body levels (n = 7 8). (K) RQ during the dark cycle and light cycle (n = 6). (L, M, N, O, P, Q) Liver mrna expression levels (n = 8). Values are the means ± SEM of data obtained from the analysis of each group. *P < **P < Supplemental Figure 4. Serum T-Chol and TG levels after 8 weeks of pair-feeding. The serum T-Chol (left panel) and TG (right panel) levels after the denial of access to food for 16 h in mice not treated (open bars) or treated with 0.005% tofogliflozin (filled bars) (n = 8). Supplemental Figure 5. Systolic blood pressure and heart rate after 8 weeks of pair-feeding. The systolic blood pressure and heart rate after 16 h of the denial of access to food in mice not treated (open bars) or treated with 0.005% tofogliflozin (filled bars) (n = 8). Supplemental Figure 6. Bone volume and bone mineral density after 6 weeks of pair-feeding. The 7

8 bone volume (left panel) and the bone mineral density (right panel) in mice not treated (open bars) or treated with 0.005% tofogliflozin (filled bars) (n = 12-13). Supplemental Figure 7. AUC of OGTT conducted in the pair-fed mice (n = 8-9). Supplemental Figure 8. Skeletal muscle TG contents after 8 weeks of pair-feeding. The TG contents of the skeletal muscle in mice not treated (open bars) or treated with 0.005% tofogliflozin (filled bars) (n = 6-8). 8

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