G7021

To perform the GTT and ITT assays, the animals were transferred to clean cages and fasted for 6 h from 08:00 h (Zeitgeber Time 0) to 14:00 h (Zeitgeber Time 6) before the tests. A total of 1.5 mg glucose/g body weight (G7021, Sigma-Aldrich, St. Louis, MO, USA) for GTT, and 0.75 UI of insulin/kg body weight (Actrapid, Novo Nordisk, Bagsværd, Denmark) were injected intraperitoneally (i.p.) for GTT and ITT, respectively. Blood samples were collected from the tail vein of each mouse by gently massaging fourfold prior the injection (0 min) and at 30-, 60-, and 120-min post-injection. Glucose levels were measured using a glucometer (Glucocard SM, Menarini, Florence, Italy). GTTs were performed at weeks 7, 14, and 21, and ITTs at weeks 8, 15, and 22 of the nutritional intervention.

The rht-PA producing cell line (CHO TF 70R) was obtained from Pharmacia & Upjohn S.A. (Sweden) (a kind gift of Torsten Björlig). The culture medium SFM4CHO (HyClone, free of glucose and glutamine) was used. This medium was supplemented with: 20 mM glucose (G7021, Sigma, USA) and 6 mM glutamate (G8415, Sigma, USA) for batch culture, and 10 mM glucose and 6 mM glutamate for continuous culture.

Human renal tubular epithelial HK-2 cells and HEK-293T cells were provided by the Cell Bank of Chinese Academy Sciences (Shanghai, China). HK-2 cells were treated with normal glucose (NG, 5.5 mM), high glucose (HG, 30.5 mM) or the osmotic control high mannitol (HM, 5.5 mM glucose and 25 mM mannitol) for 24, 48 or 72 h (22 (link)), which were used for subsequent analysis of HG-induced cell injury. Glucose (G7021) and mannitol (M4125) were obtained from Sigma (St. Louis, MO, USA). For 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or propranolol treatment, cells were treated with AICAR (S1802, Selleck, Houston, TX, USA) at 1 mM or propranolol (S4076, Selleck) at 100 µM.

Six single-gene knockout strains were grown overnight along with 1 wild-type (positive control) and 1 media control (negative control) for a total of 8 samples with shaking at 37°C and 250 rpm in a 96-deep well plate. Samples are tested in singlicate (N = 1) for the initial screen. Then, if the sample presents >25% increase in thermogenesis, it was tested again (N ≥ 3). For all experiments, cells were grown in Luria–Bertani media (Sigma Aldrich 71753–6) supplemented with 2% glucose (Sigma Aldrich G7021). Following overnight incubation, OD₆₀₀ (by Abs₆₀₀, absorbance at 600 nm) was measured in a clear bottom 96-well plate, and the culture was diluted to a final OD₆₀₀ of 0.005 in 10 mL growth media in a 20 mL glass ampoule (TA Instruments). The ampoule was then sealed and placed inside the TAM Air Calorimeter for 4 to 5 h for heat flow measurement. After all samples had reached their peak, they were removed from the calorimeter and again measured for their OD₆₀₀. See S2 Note for more details.

Primary keratinocytes were isolated from the skin of 6–8-week-old mice as previously described (Supplementary ref. 1). Briefly, mice (C57/BL6) were sacrificed (by cervical dislocation) and the hair shaved from their backs. The skin was then sterilized by immersion in a beaker of 10% betadine for 2 min, in a beaker of 70% ethanol for 1 min and then in sterile Dulbecco’s phosphate (PBS) for 1 min. The skin samples were treated with trypsin for 2 h at 33 °C to separate the epidermis from the dermis. Keratinocytes were cultured in F12 (21765-037)–DMEM (41965-062), medium, both from Thermo Fisher, supplemented with hydrocortisone (0.5 µg/ml), epidermal growth factor (EGF) and insulin (5 µg/ml), all from Sigma. The medium was changed three times a week. When the cultures reached 70–80% confluence the keratinocytes were incubated with two concentrations of d-glucose (G7021-Sigma) (5 mM + 20 mM mannitol (for osmolality)) or 25 mM for 45 min and then used for the various experiments.

Male CONV-R Swiss Webster mice of 10–13 weeks of age were weighed, MRI scanned and randomized in a weight-matched manner into two groups (N = 12–13 mice/group). Mice were injected i.p. with 25 mg/kg 3-hydroxydecanoate (Larodan #14–1003) dissolved in 5% v/v DMSO (Sigma #D5879) in saline (Mediq Danmark #B230553) for seven days. The vehicle group received 5% v/v DMSO in saline. Based on the 3-hydroxydecanoate (or vehicle) stock solution, the dosing volume was 5.32 μL/g body weight, resulting in a dose of 0.29 mg/g body weight DMSO. On day seven, after the last 3-hydroxydecanote or vehicle dosing, the mice were fasted for 4 h, followed by an ipGTT. The mice were dosed i.p. with 2 mg/kg glucose (Sigma #G7021) and blood glucose was measured using Bayer Contour XT glucometers (Bayer #84030970) and strips (Bayer #84030881). Tail bleedings for glucose and insulin measurements were taken 30 min before glucose dosing, and thereafter blood glucose was measured right before dosing (time point 0) and 15, 30, 60, 90 and 120 min after glucose dosing. Blood samples were centrifuged (5,000xg for 5 min) and separated serum was snap-frozen and stored at −80°C until insulin measurements were performed. Finally, the mice were scanned using the Bruker minispec MRI (Bruker) and euthanized.

Neonatal rat ventricular cardiomyocytes were isolated from 0- to 3-d old Sprague-Dawley rat neonates as described previously19 (link). After tissue digestion, cells were prepared for 1 h to remove non-myocytes and then plated on gelatinized dishes and cultured overnight in Dulbecco’s modified essential medium (DMEM;GIBCO, 11965–084) with 10% bovine serum. The following day, the cells were washed in phosphate-buffered saline and cultured in DMEM containing 100 Mm of 5-bromo 2′-deoxy-uridine (BrdU; Sigma, B5002). For high glucose studies, cardiomyocytes were cultured for 72 h in glucose free DMEM (GIBCO, 11966) supplemented with 5.5, or 11,22,33 mmol/l of glucose (Sigma, G7021). The osmolarities of all media were made equal to 33 mM by adding different amounts of mannitol (Sigma, M9647). All media contained 100 units/ml penicillin and streptomycin (Sigma, P4333). The study protocol was approved by the Ethics Committee of the Fourth Military Medical University. All experimental procedures used in the study were carried out in accordance with the approved guidelines by the Institutional Review Board of the Fourth Military Medical University.