ApoE KO mice (C57BL/6J-ApoEtm1Unc) were purchased from Charles River Laboratories and housed (two animals per cage for all experiments) under controlled ambient conditions and 12 h light and dark cycle. The animals were given free access to water and standard diet (MF; Oriental Yeast Co., Ltd.) and maintained at 25 °C.

We used streptozotocin (STZ) to induce hyperglycemia based on our previous report [7]. ApoE KO mice were intraperitoneally injected with STZ (FUJIFILM Wako Pure Chemical Corporation, Japan) (50 mg/kg/day) for 5 consecutive days at 8 weeks of age. Blood glucose levels were measured every other day at 9 weeks of age. When mice showed apparent hyperglycemia superior to 300 mg/dL at fed status more than twice continuously until 10 weeks of age, ApoE KO mice were divided into three groups: the first group was a normoglycemic group without injecting STZ (non-DM group, n = 10). In the second group, mice were injected with STZ and treated with 0.5% carboxymethyl cellulose (CMC) (control group, n = 12). In the third group, mice were injected with STZ and treated with imeglimin (200 mg/kg, twice daily oral gavage, purchased from Sumitomo Pharma) as we previously reported (imeglimin group, n = 12) [20]. We observed the mice in the three groups from 10 to 18 weeks of age. Body weight and food intake were measured during the experiment period.

The study was approved by the animal use committee of Kawasaki Medical School (No. 23-001) and conducted in compliance with the animal use guidelines of Kawasaki Medical School.

Blood samples were collected from tail vein. Blood glucose levels were measured using a glucometer (Glutest Mint; Sanwa Kagaku Kenkyusho Co, Ltd, Japan). Blood glucose levels were measured with one sample for each measurement in principle, because it was known that the accuracy of glucometer we used was quite high. However, when blood glucose levels were obviously strange due to some reason such as insufficient blood collection, we measured blood glucose levels again and confirmed the accuracy of the levels. Plasma total cholesterol, triglyceride, LDL-cholesterol and HDL-cholesterol were measured enzymatically using the Wako LabAssay, L type Wako (Wako Pure Chemical Industries, Japan). Urine was collected using metabolic cage at 18 weeks of age, and urinary 8-OHdG levels were measured using ELISA kit (Japan Institute for the Control of Aging, NIKKEN SEIL Co, Ltd, Japan).

Inflammation and macrophage makers were assessed using mRNA extracted from the abdominal aorta. Total RNA extraction was performed using a RNeasy lipid tissue mini kit (QIAGEN, Valencia, CA) according to the manufacturers’ instructions. cDNA was produced from mRNA using TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA). Quantitative RT-PCR was conducted using a Step One Plus Real-Time PCR system (Applied Biosystems). To quantify gene expression, the 2−ΔCT was calculated using β-actin as an internal control. Primer sequences used for real time PCR are presented in Table 1.

Under anesthesia, PBS was perfused from left ventricle and then animals were killed and heart and aorta were dissected. Sudan IV (Wako: 192-04392) staining was conducted for aortic arch. Adventitial fat tissue was removed and aorta was dissected longitudinally. The image analysis software NIH Image (version 1.61; http://rsbweb.nih.gov/ij/) was used to calculate the ratio of the plaque lesion to the total aortic arch area.

Isolated thoracic aorta was fixed overnight with formalin at 4 °C. Tissue was routinely processed for paraffin embedding and 4 μm sections of thoracic aorta were cut and mounted on silanized slides and were stained by Hematoxylin Eosin (HE), alpha-smooth muscle actin (α-SMA) (NB300-978, RRID:AB_2273628) and CD68 (ab125212, RRID:AB_10975465, 1:500).

All data were analyzed and expressed as the mean ± standard error of the mean. Differences between two groups were tested for statistical significance using Student’s t-test. p values less than 0.05 were considered to indicate a statistically significant difference.