Diabetes mellitus was induced in 8-wk-old C57BL/6 mice via a single intraperitoneal injection of streptozotocin (STZ) (150 mg/kg body weight; STZ; Wako, Japan) 5–7 d before transplantation. Blood glucose was monitored by sampling via the tail vein and analyzed using an Accu-Chek monitor (Roche Diagnostics, Japan). Diabetes mellitus was considered to be established when 2 consecutive blood glucose measurements exceeded 450 mg/dL.
Streptozotocin (stz)
Manufactured by Fujifilm
Sourced in Japan
The STZ is a lab equipment product by Fujifilm. It is a compact and durable device designed for laboratory use. The STZ features precise temperature control and advanced monitoring capabilities to support various experimental and analytical procedures.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Insulin humulin, by Eli Lilly (1 mentions)
One touch ultra view, by Johnson & Johnson (1 mentions)
Pdgfr β, by Santa Cruz Biotechnology (1 mentions)
Aicar, by Fujifilm (1 mentions)
2 deoxyglucose 2 dg, by Tokyo Chemical Industry (1 mentions)
Roswell park memorial institute (rpmi), by Nissui Pharmaceutical (1 mentions)
Nicotinamide, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Nissui Pharmaceutical (1 mentions)
Fetal bovine serum (fbs), by Biosera (1 mentions)
Canagliflozin, by Mitsubishi (1 mentions)
Onetouch ultra, by Johnson & Johnson (1 mentions)
Labassay triglyceride, by Fujifilm (1 mentions)
Surebeads protein g magnetic beads, by Bio-Rad (1 mentions)
Oxiselect advanced glycation end product age competitive elisa kit, by Cell Biolabs (1 mentions)
Cholesterol e test wako, by Fujifilm (1 mentions)
Mannitol, by Fujifilm (1 mentions)
Super fix rapid fixative solution, by Kurabo (1 mentions)
Benoxil, by Santen (1 mentions)
Glucometer, by Johnson & Johnson (1 mentions)
40 protocols using streptozotocin (stz)
1
Diabetic Mouse Model Generation
2
Gestational diabetes induction in mice
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STZ (75 mg/kg; FUJIFILM Wako Pure Chemical Corporation; Osaka, Japan, administered on two consecutive days) dissolved in 20 mM citrate buffer (CB; pH 4.7) or CB alone was intraperitoneally (i.p.) administered to virgin female mice at 14 weeks of age (CB-virgin, n = 9; STZ-virgin, n = 7; Fig 1A ). Additionally, female mice similarly treated with CB or STZ were mated with males one week after i.p. administration: CB-pregnant (n = 9) and STZ-pregnant (n = 6). The day of observing the vaginal plug was designated as gestational day 0.5 (GD0.5).
Blood glucose levels (BGL) and body weight were measured during pregnancy every two days. BGL was assessed in tail vein blood using a Medisafe Fit (TERUMO Co., Ltd., Tokyo, Japan). At GD18.5, pregnant mice underwent a glucose tolerance test (GTT). Briefly, all mice were fasted for 6 hours from 8 am to 2 pm, followed by an intraperitoneal injection of a 0.2 g/mL glucose solution (D(+)-Glucose, FUJIFILM Wako Pure Chemical Co.) dissolved in phosphate-buffered saline (PBS) at a dose of 7.5 μL/g body weight. BGLs were measured before (0 min) and at 15, 30, 60, and 120 min after glucose injection. Blood samples collected at 0, 15, and 30 min post-injection were supplemented with 500 KIU/mL aprotinin (FUJIFILM Wako Pure Chemical Co.) and 10 U/mL heparin sodium (Mochida Pharmaceutical Co., Ltd.; Tokyo, Japan) to measure plasma C-peptide concentration.
Blood glucose levels (BGL) and body weight were measured during pregnancy every two days. BGL was assessed in tail vein blood using a Medisafe Fit (TERUMO Co., Ltd., Tokyo, Japan). At GD18.5, pregnant mice underwent a glucose tolerance test (GTT). Briefly, all mice were fasted for 6 hours from 8 am to 2 pm, followed by an intraperitoneal injection of a 0.2 g/mL glucose solution (D(+)-Glucose, FUJIFILM Wako Pure Chemical Co.) dissolved in phosphate-buffered saline (PBS) at a dose of 7.5 μL/g body weight. BGLs were measured before (0 min) and at 15, 30, 60, and 120 min after glucose injection. Blood samples collected at 0, 15, and 30 min post-injection were supplemented with 500 KIU/mL aprotinin (FUJIFILM Wako Pure Chemical Co.) and 10 U/mL heparin sodium (Mochida Pharmaceutical Co., Ltd.; Tokyo, Japan) to measure plasma C-peptide concentration.
3
Streptozotocin-Induced Diabetes in Mice
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Mice were fasted for 4 hours before STZ injection and then administered STZ (115 mg/kg, Wako, Osaka, Japan) in saline by intraperitoneal injection on day 0. Blood glucose concentrations and body weights were measured four times prior to the cell injection to confirm the basal levels of glucose and development of STZ-induced diabetes mellitus: 7 and 3 days before, and 5 and 7 days after STZ injection. Blood was sampled from the right facial vein between 9:00 am and 11:00 am. Drinking water was replaced with a 10% glucose solution (Otsuka, Tokyo, Japan) overnight to avoid hypoglycemia in mice the day after STZ injection. Before cell transplantation, mice with blood glucose concentrations between 220 and 500 mg/dL on days 5 and 7 were considered to have STZ-induced diabetes mellitus.
4
Evaluating GLUT4 Translocation and Glucose Tolerance
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DS20060511 (Fig. 1a , molecular formula C30H32O6·C4H11N, molecular weight 561.71) was obtained from the Medicinal Chemistry Research Laboratories, Daiichi Sankyo Co., Ltd., Japan. The concentrations and doses of DS20060511 used for the in vitro and in vivo experiments in this study were selected based on the results of preliminary concentration- and dose-finding experiments, including the GLUT4 translocation assay in L6-GLUT4myc myotubes and GTT, respectively. 2-Deoxy-D-[l,2-3H]-glucose ([3H]-2-DG, ART0103A) and D-[1-12C]-mannitol ([12C]-mannitol, ARC0127A) were purchased from American Radiolabeled Chemicals, St. Louis, MO. Streptozotocin was purchased from FUJIFILM Wako Pure Chemical, Osaka, Japan. Insulin (Humulin) used in the in vivo experiments was purchased from Eli Lilly, Indianapolis, IN. Insulin solution human (I9278) used in the in vitro experiments was purchased from Sigma–Aldrich, St. Louis, MO.
5
Streptozotocin-Induced Hyperglycemia in Rats
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The rats were divided into two groups. One group received an intraperitoneal injection of physiological saline solution (PSS) to serve as the normoglycemic (NG) group and the other received an intraperitoneal injection of 60 mg/kg streptozotocin (STZ; Wako, Osaka, Japan) to serve as the HG group. Plasma glucose levels were measured 4 days after intraperitoneal injection using One Touch Ultra View (Johnson & Johnson, Tokyo, Japan). Rats with a plasma glucose level of less than 250 mg/dl in the HG group were excluded from analysis. Plasma glucose levels were also measured 1 and 2 weeks after intravitreal injection.
6
Canagliflozin Regulates AMPK and Cell Cycle
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Canagliflozin was provided by Mitsubishi Tanabe Pharma Corporation (Osaka, Japan). Streptozotocin and AICAR were purchased from Wako (Osaka, japan). Nicotinamide was purchased from SIGMA (St. Lewis, MO, USA). 2-Deoxyglucose (2-DG) was purchased from Tokyo Chemical Industries (Tokyo, Japan). Compound C was purchased from Calbiochem (CAS 866405-64-3). The cell culture reagents RPMI and DMEM were purchased from Nissui (Tokyo, Japan). Fetal bovine serum (FBS) was obtained from Biosera (Kansas City, MO, USA).
Antibodies were obtained from Cell Signaling Technology (pACC-11818S, ACC-3662S, pAMPK-4188S, AMPK-5832S, Cyclin D1-2922S, p-p70-9234S, p-70-2708S), R&D (PDGFRβ) and Santa Cruz (actin sc-47778 F1417, Pin-1sc46660 B0917).
Antibodies were obtained from Cell Signaling Technology (pACC-11818S, ACC-3662S, pAMPK-4188S, AMPK-5832S, Cyclin D1-2922S, p-p70-9234S, p-70-2708S), R&D (PDGFRβ) and Santa Cruz (actin sc-47778 F1417, Pin-1sc46660 B0917).
7
Gestational Diabetes Mellitus and LMWH
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Female rats in estrus were allowed to cohabitate for one night with male rats at a 2:1 ratio in order to facilitate conception; female rats with detectable sperm in their vaginal smear were considered pregnant (day 0). The 30 pregnant rats were randomized into three groups (n=10/group): Normal control (NC) group, GDM group and GDM + LMWH group. GDM was induced in animals via intraperitoneally injecting a single dose of streptozotocin (45 mg/kg; Wako Pure Chemical Industries, Ltd.), which had been freshly prepared. Blood glucose levels of all rats were detected with a glucometer (Onetouch, Ultra, Johnson & Johnson). Rats exhibiting a glucose concentration of >16.7 mmol/l for 3 days after the injection were used in this study. NC animals were instead injected with 1 ml sodium citrate buffer. On gestational day 5, rats in the GDM + LMWH group were injected subcutaneously with LMWH (600 IU/kg/d) to establish an LMWH intervention model, while other groups were administered an equal volume of 0.9% saline. On day 16 of pregnancy, animals were sacrificed and the placentas were removed for western blotting, reverse transcription-quantitative (RT-q)PCR, transmission electron microscopy (TEM) and immunohistochemistry (IHC).
8
Diabetic Rat Metabolic Biomarkers
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Streptozotocin (STZ), diethyl ether, sodium citrate buffer solution (pH 4.5), Cholesterol E-test Wako, and LabAssay™ Triglyceride were purchased from FUJIFILM Wako Pure Chemical (Tokyo, Japan）. Heparin (10,000 units/10 mL) was purchased from Mochida Pharmaceutical (Tokyo, Japan). Pierce™ BCA protein assay kit was purchased from Thermo Fisher Scientific (MA, USA). The Rat C-Reactive Protein ELISA kit was purchased from Bioscience (CA, USA). The creatinine calibrator, DZ072B-CAL, was purchased from Diazyme (CA, USA). OxiSelect™ Advanced Glycation End Product (AGE) Competitive ELISA Kit was obtained from Cell Biolabs (Danvers, MA, USA). SureBeads™ Protein G Magnetic Beads were purchased from Bio-Rad (Hercules, CA, USA).
9
Ocular Pharmacokinetics in Diabetic Rats
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NIL was purchased from Tokyo Chemical Industry Co., Ltd. (Saitama, Japan). BAC was obtained from Kanto Chemical Co., Inc. (Tokyo, Japan), and 0.4% Benoxil, 0.5% phenylephrine and 0.5% tropicamide were provided by Santen Pharmaceutical Co., Ltd. (Osaka, Japan). HPβCD and MC were kindly donated by Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan) and Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan), respectively. SUPER FIX™ rapid fixative solution was purchased from Kurabo Industries, Ltd. (Osaka, Japan), and the ELISA Insulin Kit was obtained from Morinaga Institute of Biological Science, Inc. (Kanagawa, Japan). Mannitol and streptozotocin were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals used were of the highest purity commercially available. Wistar rats aged 7 weeks were provided by Kiwa Laboratory Animals Co., Ltd. (Wakayama, Japan). STZ rats were prepared by injecting Wistar rats intraperitoneally with 100 mg/kg streptozotocin twice on two consecutive days, and then housing them for two weeks after the last injection before use in this study. All experiments were performed in accordance with the ARVO resolution on the use of animals in research, and were approved by the Kindai University Faculty of Pharmacy Committee Guidelines for the Care of Laboratory Animals (project identification code KAPS-25-003, 1 April 2013).
10
Streptozotocin-Induced Hyperglycemia Model
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Single i.p. administration of physiological saline solution (PSS) or 60 mg/kg streptozotocin (STZ; Wako Pure Chemical Industries, Ltd., Osaka, Japan) was carried out for the normoglycemic (NG) rats or the hyperglycemic (HG) rats, respectively. The plasma glucose levels were measured using a glucometer (Johnson & Johnson, Tokyo, Japan) 4 days after intraperitoneal injection. We only included the individuals as the HG group when plasma glucose exceeded 250 mg/dl. The plasma glucose levels of HG groups were 441.5±71.3, 435.0±32.3, and 432.1±73.3 mg/dl, immunoblot analysis, immunohistochemical analysis, and morphometric analysis studies, respectively.
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