Glucometer

Blood glucose was measured at necropsy using a glucometer (Life Scan, Inc., Milpitas, CA). Serum leptin was determined using Mouse Leptin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN). Serum CTX was determined using Mouse C terminal telopeptides of type I collagen (CTX1) ELISA kit (Life Sciences Advanced Technologies, St. Petersburg, FL). ELISA were done according to respective manufacturer’s protocol.

Blood glucose and β-hydroxybutyrate concentrations were quantified in venous blood samples with enzyme-based reagent strips using a Glucometer and a Ketometer (OneTouch Verio, LifeScan Inc., Burnaby, BC, Canada), respectively. Blood samples were obtained by incision of the tail vein with a lance. 2 µL samples were drawn before (t = 0) and at 5, 10, 15, 30, 60, 90, 180, and 240 min after ketone or NaCl administration. Measurements were performed by researchers without knowing the treatment group.

1-methyl-1, 2, 3, 4-tetrahydroisoquinoline (CAS:4965-09-7) was purchased from Nanjing Dolon Biotechnology Co., Ltd. China, Streptozotocin (CAS: 41910012-3), from Bioshop (Burlington, ON, Canada), Yohimbine from Sigma Aldrich (CAS: 65190). Naloxone 0.4 mg/mL (NALOX^® by Rehman Medicines Co.), and Ondansetron 8 mg/4mL (ONSET^® by Pharmedic Pvt Ltd.) were purchased locally. Glucometer (One-touch basic blood glucose monitoring system) (Lifescan, Brussels, Belgium) was used.

Hepatic lipids were extracted according to our previously published method [26 (link)], and analyzed for TG concentrations using a TG assay kit (# 236–60, Sekisui Diagnostics, P.E.I Inc., Canada). Plasma samples were used for determination of non-esterified fatty acids (NEFA) using a kit (# 993–35191, Wako Chemicals Inc., USA). Fasting blood glucose concentrations were measured at the time of sacrifice using a commercially available glucometer (Lifescan Inc. CA, USA) after snipping the tail.

Blood samples were collected from the retro-orbital venous sinus at week 4, 8, 12 and 16 from 12-h fasted rats. Serum was obtained by centrifugation of the blood samples at 4000× g for 10 min at 4 °C. The concentrations of serum TG, total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol (HDL-C), uric acid (UA), activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were measured using commercial assay kits (Biosino Biotechnology and Science Inc., Beijing, China) on an Alcyon 300 automatic analyzer (Alcyon, USA). The MDA levels of the serum were determined with kits purchased from Nanjing Jiancheng Bioengineering Inst. (Nanjing, China). Concentration of fasting blood insulin (FBI) was determined by ELISA kits from the Beijing Sino-uk Institute of Biological Technology (Beijing, China). Fasting blood glucose (FBG) levels were collected from the tail and measured with a glucometer (Life Scan Inc., Milpitas, CA, USA). The homeostasis model of assessment for the insulin resistance index (HOMA-IR) is used to estimate this insulin resistance. It is calculated as follows, HOMA-IR= FBG (mmol/L) × FBI (mU/L)/22.5.

Diabetes was induced using STZ, as previously described48 (link). Briefly, STZ (2-deoxy-2–3-[methyl-3-nitrosoureido]-D-glucopyranose; Sigma, St. Louis, MO) was dissolved in 0.1 M sodium citrate buffer (pH 4.5) and injected intraperitoneally within 15 min of preparation, at a dose of 50 mg/kg/day for 5 consecutive days to produce a beta cell destruction model. Control wild-type and transgenic mice were injected with the citrate buffer vehicle. Blood glucose level was measured in non-fasted animals from tail venous blood using a glucometer (LifeScan, Milpitas, CA). Mice were evaluated every 2 days at 2:00 P.M. and were considered diabetic when blood glucose levels exceeded 250 mg/dl, usually 7 to 9 days after the last STZ injection. All mice were sacrificed by a blind investigator for tissue collection.

The fasting blood glucose levels and body weight levels of the mice were assessed every 4 weeks, and the fasting blood glucose levels were measured using a glucometer (Life Scan, Milpitas, CA, USA). Serum TC, ALT, and AST concentrations were measured using an ADVIA 2400 Chemistry System Analyzer (Siemens, Tarrytown, NY, USA) according to the manufacturer’s instructions.