Streptozotocin (stz)

Male C57BL/6J mice (7–8 weeks old) were obtained from Jackson Laboratory (Bar Harbor, ME, USA), and housed on a 12/12 h dark-light cycle in a controlled environment (22.5 ± 0.5°C, 50 ± 5% humidity) with access to food and water ad libitum. After at least one week of acclimatisation, animals were randomised into the study by weight. A model of type 1 diabetes was induced with streptozotocin (50–55 mg/kg, daily for 5 days, i.p.; Enzo Life Sciences, Farmingdale, NY, USA); non-diabetic controls received vehicle. Development of diabetes (fasting blood glucose ≥16.7 mmol/l) was confirmed 28 days later, and mice were maintained for eight additional weeks before intravitreal injections. Animals that did not convert to diabetes or that later showed apparent intraocular injection injuries were excluded, but otherwise all animals that consecutively completed the study protocol were included. As far as possible, the ocular outcome assessments described below were performed using a ‘blinded’ study design. The study was approved by the Institutional Animal Care and Use Committee of the University of Oklahoma Health Sciences Center.

HX-1171 (1-O-hexyl-2,3,5-trimethylhydroquinone) was obtained from Biotoxtech Co., Ltd. (Cheongju, Korea), and streptozotocin (STZ) was purchased from Enzo Life Sciences (Farmingdale, NY, USA). HX-1171 was dissolved in ethanol, and STZ was dissolved in culture medium then used for in vitro treatment. The final working concentration of ethanol was 0.1% (v/v). For in vivo experiments, HX-1171 was dissolved in a solvent mixture of 8% (v/v) Tween 80, 5% (w/v) NaCl, and distilled water; STZ was dissolved in 0.1 M citrate buffer (pH 4.5).

The following reagents and antibodies were used in this study: p21 Waf1/Cip1 (12D1) rabbit, p16 INK4A (D7C1M) rabbit, p53 (1C12) mouse (Cell Signaling Technology, Frankfurt, Germany); anti-phospho-IRE1 (S724) rabbit monoclonal antibody (Boster, Germany); human XBP1 antibody (R & D System, Wiesbaden, Germany); anti ATF6 alpha (Rockland, Germany); alexa fluor FITC goat anti-rabbit IgG, alexa fluor TRITC goat anti-rabbit IgG, alexa fluor TRITC goat anti-mouse IgG and alexa fluor FITC goat anti-mouse IgG (Invitrogen, Karlsruhe, Germany). Human coronary artery endothelial cells were purchased from Lonza Germany (Catalog #: CC-2585); EGMTM -2 MV microvascular endothelial cell growth medium-2 bulletkit (Lonza, Köln, Germany); D-(+)-Glucose (Sigma, Darmstadt, Germany); human oxidized low density lipoprotein (oxLDL) (Thermo Fisher Scientific, Dreieich, Germany); vectashield mounting medium with DAPI (Vector Laboratories, Newark, CA, USA); streptozotocin (Enzo Life Sciences, Lörrach, Germany); “high-fat diet” (HFD) experimental food (Western Type Diet, 43% carbohydrates, 15% proteins and 42%, Ssniff, Soest, Germany); accu-chek test strips, accu-check glucometer; albumin fraction V (Carl Roth, Karlsruhe, Germany); rompun 2% (Bayer, Leverkusen, Germany); ketamine 10% (beta-pharm, Augsburg, Germany); chemiluminescent western blot reagents (Merk Millipore, Darmstadt, Germany).

Sprague–Dawley rats weighing 180–200 g (Charles River Laboratories, Shizuoka, Japan) had ad libitum access to tap water and standard rat chow. Diabetes was induced by a single tail vein injection of streptozotocin (STZ, 60 mg/kg body weight; Sigma Chemical, St. Louis, MO, USA) [diabetes mellitus (DM) rats]; the control rats were injected with an equal volume of citrate buffer. Three weeks after STZ injection, a group of DM rats was treated with BFM B1 (50, 100, 200 nmol/kg/day intraperitoneally; Enzo Life Sciences, Ann Arbor, MI, USA). Twenty-four-hour urine and blood samples were collected using a metabolic cage until day 7 morning under feeding condition with free access to water and food, and then under 24-h starvation conditions without food [16 (link)]. On day 8, the rats were anesthetized with pentobarbital (50 mg/kg body weight), and then their kidneys and liver were removed and used for western blotting or immunohistochemistry. Intravenous insulin tolerance tests (ITTs) were performed to assess the degree of insulin resistance, and the extent of insulin resistance was evaluated according to the K index of ITT (KITT) as described previously [16 (link)].
All the procedures were conducted in accordance with the Guidelines for Animal Experiment and Ethics Committee in The University of Tokyo (P10-079, 15-P-134), and Dokkyo Medical University (17-918).