Saline 60 day slow release

Male mice of both ArKO and WT at 3 and 6 months of age were used in these studies, groups included were: untreated WT, untreated ArKO and ArKO implanted 17β-estradiol pellet (E2). Three month-old ArKO mice were implanted with E2 pellet (E2) (0.05 mg in 21 days i.e. 2.5 µg/day, Innovative Research of America, Toledo, OH, USA) for 3 weeks. Six month-old ArKO mice were implanted with E2 pellet (0.15 mg in 60 days, i.e. 2.5 µg/day; Innovative Research of America, Toledo, OH, USA) 6 weeks. In preliminary studies no difference in body mass or glucose tolerance were detected between untreated and placebo pellet (saline 60 day slow release, Innovative Research of America, Toledo, OH, USA) treated six month-old ArKO male mice (Table S1 and Figure S1). A total number of 5–15 mice were used per group and age, details specified on related graphs. After treatment, mice were killed using a lethal dose of anesthetic (Ketamine (300 mg/kg)/Xylazine (30 mg/kg)). Blood was collected by cardiac puncture and serum was separated, and stored at −20°C. Adipose, liver and muscle tissues were removed, weighed, snap frozen and stored at −80°C.

Each ArKO mouse was implanted with a 17β-estradiol pellet (E2; 0.15 mg in 60 days i.e. 2.5 μg/day, for 6-weeks; Innovative Research of America, Toledo, OH, USA). No differences in body mass, organ weights or glucose tolerance were detected between 6 month-old untreated ArKO and littermates implanted with a placebo pellet (saline 60 day slow release; Innovative Research of America, Toledo, OH, USA; see S1 Table and S1 Fig), hence untreated 6 month-old ArKO female mice were used in this study. After treatment, mice were killed using a lethal dose of anesthetic (100mg/ml Ketamine and 20mg/ml Xylazine in PBS). Blood was collected by cardiac puncture and serum was separated, and stored at –20°C. Adipose, liver and muscle tissues were removed, weighed and snap frozen in liquid nitrogen and stored at -80°C.