Cl316 243 cl

Young mice used in the experiments were male and female C57Bl/6J purchased from Jackson Laboratories. Old mice were either aged from mice purchased from Jackson or obtained from the National Institute of Aging (NIA). Mice were fed ad libitum either a control ND consisting of 13% fat (5001, Lab Diet) or a HFD of 60% of calories from fat (D12492, Research Diets, Inc.) starting at 6-week of age (young) or 10 months of age (old) for 16 to 24-week duration. Animals were housed in a specific pathogen-free facility with a 12h light/12h dark cycle and given free access to food and water. All animal use was in compliance with the Institute of Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and approved by the University Committee on Use and Care of Animals at the University of Michigan (Animal welfare assurance number A3114-01. For stimulation of lipolysis, mice were injected intraperitoneally with 1 mg/kg of CL-316, 243 (CL; Tocris Bioscience), an ADRB3 selective agonist or saline twice (4 h between each injection). Mice were sacrificed 14 h after the second injection [25 (link), 59 (link)]. Liver, GWAT and IWAT were excised and weighed for each animal. Tissue samples were frozen in liquid nitrogen and stored at –80°C prior to RNA extraction. Adipose tissues were collected and immediately processed for SVF preparation and flow cytometry staining.

10 min after starting temperature monitoring, mice received either the β3-adrenergic receptor (β3-AR) agonist CL316,243 (CL; Tocris Bioscience Bristol, United Kingdom; 20 μg/mouse) or vehicle (phosphate-buffered saline; PBS) by subcutaneous injection (100 µl/mouse).

For β-adrenergic stimulation to induce browning of white adipose tissue (WAT), CL316243 (CL) (Tocris Bioscience) in 0.9% NaCl or vehicle was administered at continuous rates at a dose of 1 µg/g BW/day by subcutaneous implantation of an osmotic pump (ALZET model 1002). After 10 days mice were sacrificed.