(a) MEFs were serum starved for 4 h, pretreated with vehicle control or ebselen (10 μM, 40 min), left unstimulated or stimulated with insulin (10 min, 5 min), CM-H₂DCF-DA (10 μM, 10 min) added and DCF fluorescence assessed; arbitrary units (AU) are shown. (b–e) MEFs were serum starved, stimulated with insulin and processed for immunoblot analysis with antibodies to the Y1162/Y1163 phosphorylated IR β-subunit (p-IRβ), Y612 phosphorylated IRS-1 (p-IRS-1), Ser-473 phosphorylated Akt (p-Akt), or the phosphorylated MAPK ERK1/2 (p-ERK1/2) and then reprobed as indicated. Where indicated cells were pretreated with ebselen (10 μM, 40 min) or DPI (100 μM, 30 min). In b and d p-Akt or p-IRβ were quantified and normalised for Akt and IRβ respectively. (f) Gpx1 +/+ MEFs were serum starved and then stimulated with H₂O₂ for 5 min, lysed in the presence of 50 mM N-ethylmaleimide (NEM) to irreversibly alkylate free Cys and prevent the oxidation of Cys post lysis and processed for immunoblot analysis. Indicated are the reduced and oxidized (inactive) forms of PTEN. (g–h) Gpx1−/− and +/+ MEFs were serum starved, pre-incubated with vehicle control or DPI, stimulated, lysed in the presence of NEM and processed for immunoblot analysis. Results shown are representative of three independents experiments. (i) Gpx1−/− and +/+ MEFs were serum starved and then stimulated with insulin. Cells were lysed and oxidised PTPs labelled with the sulfhydryl reactive probe BPP-biotin and subjected to streptavidin-sepharose pull downs and processed for immunoblot analysis. Results shown in a, b & d are means ± SE

8–10 week old Gpx1−/− and +/+ (a) male and (b) female mice were fed a HFD or standard chow diet for 12 weeks. Mice were fasted either for 6 h and blood glucose and plasma insulin levels measured, or 4 h and ITTs performed (0.65 mU insulin/g body weight). Areas under ITT curves were determined. (c–g) 8–10 week old Gpx1−/− and +/+ male mice were fed a HFD for 12 weeks and hyperinsulinemic-euglycemic clamps performed on weight-matched mice. (d) Glucose infusion and disappearance rates were determined and (e) whole body glucose production determined by subtracting the glucose infusion rate from the glucose appearance rate. (f) Livers were harvested at the end of the clamp and processed for quantitative (ΔΔCt) real time PCR to measure the expression of the gluconeogenic genes G6pc (+/+ n=8, −/− n=7), Pck1 (+/+ n=6, −/− n=5) and Fbp1 (+/+ n=9, −/− n=8). (g) The uptake of glucose into white and red gastrocnemius (Gastroc) and diaphragm skeletal muscles, heart and white adipose tissue (epididymal; WAT) was assessed by administrating 2-deoxy-[1-¹⁴C]glucose at the end of the clamp. Results shown in a–g are means ± SE.

8–10 week old Gpx1−/− and +/+ male mice were fed a HFD for 12 weeks, fasted for 4 h, injected with saline or insulin (0.5 mU/g, intraperitoneal) and gastrocnemius muscle extracted, homogenised and insulin signaling in individual mice assessed by immunoblot analysis and quantified by densitometry. Results shown are means ± SE.

(a) Gpx1−/− and +/+ myoblasts were differentiated into myotubes, serum starved, pretreated with vehicle control or ebselen (10 μM, 40 min) and then either left unstimulated or stimulated with 1 nM insulin for 5 min before the addition of CM-H₂DCF-DA (10 μM, 10 min) and the quantification of DCF fluorescence. (b) Serum starved Gpx1 −/− and +/+ myotubes were stimulated with 1 nM insulin for the indicated times and processed for immunoblot analysis. Results shown are representative of three independent experiments. p-Akt was quantified by densitometric analysis and normalised for total Akt. (c) C2C12 cells transduced with control or Gpx1 shRNA lentiviral particles were serum starved for 6 h, pre-treated with vehicle control or ebselen and stimulated with 1 nM insulin for 10 min. Medium was then replenished and cells collected at the indicated times for immunoblot analysis. (d) Gpx1−/− and +/+ myotubes were serum starved in medium with no glucose for 4 h, stimulated with insulin for 30 min, medium removed and incubated with 2-Deoxy-D-[2,6-3H]glucose (1 μCi/mL) and incorporated radioactivity measured after 10 min. Results are means ± SD from sixtuplicate stimulations and are representative of two independent experiments. (e) Serum starved Gpx1−/− and +/+ myotubes were stimulated with 1 nM insulin for the indicated times, IRS-1 immunoprecipitated and the association of the p85 subunit of PI3K monitored by immunoblot analysis. Results shown are representative of three independents experiments. (f) Serum starved myotubes were stimulated with 50 nM insulin for 10 min, IRS-1 immunoprecipitated and associated PI3K activity monitored and quantified as described in the methods; results are means ± SD from triplicate immunoprecipitations and are representative of two independent experiments. Insert: representative ³²P-phosphatidylinositol 3-phosphate (PI3P) generation by the immunoprecipitated PI3K. (g) Serum starved Gpx1−/− and +/+ myotubes were stimulated with 1 nM insulin for the indicated times, lysed in the presence of NEM and processed for immunoblot analysis with antibodies to PTEN. Indicated are the reduced and oxidized (inactive) forms of PTEN. Results shown are representative of three independents experiments.

8–10 week old Gpx1−/− and +/+ male mice were fed a HFD for 12 weeks and either left untreated or administered NAC for seven days (10 mg/L in drinking water). (a, c) H₂O₂ levels in liver, gastrocnemius muscle or blood plasma from fasted and insulin stimulated mice were determined using Amplex Red and normalised to the sample protein content or volume. (b) GSH/GSSG ratios were determined in muscle extracts. (d) Mice were fasted, injected with saline or insulin and gastrocnemius muscle extracted, homogenised and p-Akt signaling in individual mice assessed by immunoblot analysis and quantified by densitometry. (e) Insulin sensitivity in untreated −/− versus +/+ mice, or mice administered NAC was determined by performing ITTs (0.65 mU/g). Areas under ITT curves were determined; for clarity ITT curves for NAC treated +/+ mice are not shown. The corresponding fasted blood glucose levels and body weights determined before and after NAC treatment are also shown. Results in a–e are means ± SE.

8–10 week old (a & c) male and (b) female mice were fed a HFD, or where indicated a standard chow diet, for 12 weeks. Mice were weighed and epididymal, infrarenal and brown adipose (BAT) tissues extracted and weighed. In c epididymal fat tissues were processed for immunohistochemistry (hematoxylin and eosin staining). The cross-sectional diameters of adipocytes (100 cells per mouse) were determined using Image J software (NIH). Represenative micrographs and the quantified results are shown. (d) Gpx1−/− and +/+ MEFs were differentiated into adipocytes and stained with oil red-O to monitor lipid content. Representative micrographs and quantified results are shown. (e) 8–10 week old Gpx1−/− (n = 6) and +/+ (n = 6) male littermates were fed a HFD for 12 weeks and energy expenditure during the light and dark cycles, ambulatory movement (x and y axes) and daily food intake monitored over 48 h. (f) 8–10 week old Gpx1−/− (n = 6) and +/+ (n = 6) male littermates were fed a HFD for 12 weeks and gas exchange data used to calculate the respiratory exchange ratio (RER = VO₂/VCO₂). Carbohydrate versus fat oxidation rates were calculated and expressed as Joules/min. (g) 8–10 week old Gpx1−/− and +/+ male littermates were fed a HFD for 12 weeks, starved for 4 h, injected with saline or insulin (2h, 0.75 mU/g, intraperitoneal) and diaphragm or gastrocnemius muscle extracted and processed for real time PCR (ΔΔCt) to measure pdk4 expression. Results are a-g means ± SE.

Chow fed 10–12 week old mice were starved overnight (12 h) and (a) ITTs (0.25 mU/g) performed, or otherwise (b–c) treadmill exercised (30 min running at 16 m/min at a 5° incline) and ITTs performed (b) immediately after or (c) after 1 h of rest. (d) Gastrocnemius muscle from exercised and 1 h rested mice injected with saline or insulin for 30 min was rapidly excised and processed for immunoblot analysis; results were quantified by densitometric analysis. (e) Mice from c were subsequently administered NAC for seven days (10 mg/L in drinking water), exercised, rested for 1 h and ITTs repeated. (f) Areas under ITT curves from c and e were determined. Results in a–f are means ± SE.