Ethical statement
All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee at the University of Washington, Seattle, under the approved protocol #3051.
Mice
Tsc1fl/fl mice were obtained from David Kwiatkowski at Brigham and Women’s Hostpital (Boston, MA). Ptenfl/fl (#006068) and albumin (Alb)-Cre (#003574) mice were purchased from Jackson Laboratories (Bar Harbor, ME). Ptenfl/fl and Tsc1fl/fl mice have been crossed to AlbCre mice and subsequently intercrossed to generate Tsc1+/+;Ptenfl/fl;AlbCre+ and Tsc1fl/fl;Pten+/+;AlbCre+ [11]. Animals with AlbCre- genotypes from the same litters were used as control mice. Since the transgenic mice originated from different genetic backgrounds, they have undergone multiple generations (>5) of brother-sister mating to minimize any genetic background variation. Only males were used in this study to reduce variability due to gender differences. Animals were housed 5 per cage in a modified SPF facility under 14–10 hour light/dark cycle and were fed normal chow ad libitum.
Exposure
Certified BDE-47 was obtained form Accustandard, Inc. (New Haven, CT, No. BDE-047 N). Five mg of BDE-47 was dissolved in 250 uL of DMSO, and then further diluted into 4.75 mL of corn oil. Placebo consisted of 250 uL of DMSO diluted in 4.75 mL of corn oil. Upon weaning, 3–6 week old mice were randomly assigned to receive BDE-47 (1 mg/kg/day) or placebo for 6 weeks. For each genotype, block randomization with a block size of 4 was used to assign treatment arm. The dose (Monday through Friday) was based on daily weight measurement and the treatment was administered via oral gavage. Animals were monitored daily during treatment and were euthanized within 24 hours of the last exposure following an overnight fast.
Glucose tolerance and insulin sensitivity testing
After four weeks of treatment, mice underwent glucose tolerance testing (GTT), followed exactly one week later by insulin sensitivity testing (IST). For GTT, mice were fasted for sixteen hours and weighed. After sixteen hours, fasting blood glucose was obtained from venous blood via tail nick and measured with the OneTouch blood glucose monitoring system and test strips from LifeScan, Inc. (Milpitas, CA). Mice received an IP injection of glucose (1 mg/g body weight). Blood glucose values were obtained at 15, 30, 60, and 120 minutes. At 30 minutes 50 μl of blood was procured via retro-orbital bleed for insulin assay. Plasma insulin was quantified using Linco Elisa Kits (Millipore, Billerica, MA).
For the IST mice were fasted for four hours and weighed. After fasting, a blood glucose level was obtained at time 0 and then 1 mU/g of insulin was given via IP injection and blood glucose values were obtained at 30, 60, 90 and 120 minutes.
Weight measures
Following treatment, fasted mice were euthanized via anesthetized cervical dislocation. Following death, blood was drawn through cardiac puncture and tissues were harvested. Liver tissue, muscle from the quadriceps, and white adipose tissue were weighed and frozen on liquid nitrogen. Body weight, liver to body weight ratio, and white adipose tissue weight were calculated.
Liver triglyceride analysis
Hepatic triglyceride content was determined following lipid extraction using the Folch method as previously described [11].
BDE-47 plasma levels
Fasting plasma samples from the BDE-47-treated wild-type mice were analyzed for the concentration of the congener using established methods [12].
Immunoblot analysis
Mouse liver tissue was homogenized in ice-cold radioummunoprecipitation (RIPA) buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 10 mM Tris (pH 7.2), 0.025 M β-glycophosphate (pH 7.2), 2 mM EDTA, and 50 mM sodium fluoride) with protease and kinase inhibitors (0.05 mM AEBSF, 10 μg/ml aprotinin, 10 μg/ml pepstatin, 1 mM orthovanadate, 10 μg/ml leupeptin, 1 mM microcystin LR). The protein concentration was measured using the BCA Protein Assay (Pierce, Rockford, IL). Equal amounts of protein were separated by SDS-PAGE, transferred to Immobilin-P membranes (Millipore, Bedford, MA) and blotted with antibodies from the following sources: phospho-S6 Ribosomal Protein (Ser235/236) (#2211), S6 Ribosomal Protein (#2217), phospho-Akt (Ser 473) (#4060), Akt (#9272), Tsc1 (#4906), and Pten (#9552) all from Cell Signaling (Boston, MA). Beta-Actin (#A5441) was purchased from Sigma (St. Louis, MO).
qRT-PCR
Total RNA was extracted from liver tissue using a commercially available RNA extraction kit according to the manufacturer’s protocol (Agilent Technologies, Santa Clara, CA). After spectroscopic quantification, RNA was reverse-transcribed, and cDNA was analyzed by real-time quantitative PCR using SYBR green (600882, Agilent Technologies) master mix. Primers specific for individual genes were purchased from Invitrogen or IDT. Data were normalized to housekeeping genes Gapdh or Actb. Relative amounts of the target gene were calculated using the ΔΔCt formula.
Statistical analyses
Based on our past studies of HFD-induced steatosis [11], a sample size of 6 mice per group has been adequate to detect a difference of 1.25 standard deviations with a confidence level of 5% and statistical power between 80% and 90%. Quantitative data were analyzed by unpaired t test. A p-value less than .05 was considered significant.