Metformin

The cells were cultured in six-well plates and treated with BPTES (SelleckChem, Houston, TX, USA) or metformin (MP Biomedicals, Solon, OH, USA) for 48 h. After trypsinization and fixation, the cells were prepared for flow cytometry. For apoptosis assays, the cells were stained with PI and annexin V using the FITC Annexin V Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA). For cell cycle assays, the cells were stained with a PI PI/Triton X-100 staining solution containing 0.1% (v/v) Triton X-100 (EM Science, Darmstadt, Germany), 0.2 mg/ml DNAse-free RNAse A (Thermo Scientific), and 20 mg/ml PI (Roche, Basel, Switzerland) for 15 minutes at 37 °C. Flow cytometry was performed on a BD FACSCalibur analyzer (BD Biosciences). The data were interpreted using the FlowJo software.

Human neuroblastoma SK-N-BE(2) and SH-SY5Y cells (ATCC, Manassas, VA) were maintained in complete culture medium (Dulbecco's Modified Eagle's Medium, DMEM + 10% fetal bovine serum, FBS; Atlanta Biologicals, Lawrenceville, GA) at 37°C in a humidified incubator with 5% CO₂. Stock solution of metformin (MP Biomedicals, Solon, OH) was freshly prepared in sterile triple distilled water before each experiment. Cells treated with equal volume of vehicle were used as control.

Fat Free (FF) mice were generated by mating homozygous Lox-stop-Lox-ROSA-DTA mice to those expressing adiponectin-Cre. BAT-deficient mice were generated by mating homozygous Lox-stop-Lox-ROSA-DTA mice to those expressing UCP1-Cre. Although no gender differences exist in phenotype, male mice were exclusively used. Adiponectin-/-, leptin -/- mice were purchased from Jackson laboratory. Adiponectin/Leptin double knock-out (DKO) mice were created by mating Adiponectin-/- and Leptin +/- animals.
Metformin was purchased from MP Biomedicals (Santa Ana, California) and dissolved in mouse drinking water (2 g/L) for an equivalent dose of 300 mg/kg per day, based on estimates that mice drink 1.5 mL/10 g body weight per day [44 (link)]. Fresh drinking water with Metformin was changed daily for 3 months.

HEK293/TLR4 cells were plated into 96-well plates. Next day, cells were pretreated with different concentrations of metformin (MP Biomedicals, CA) for 24 or 48 h and then incubated with or without 1 μg/ml lipopolysaccharide (LPS, Sigma-Aldrich, MO) for additional 24 h. The concentration of secreted CXCL8 in the cell culture media was determined using ELISA assay.
The high binding half-area 96-well plates (Corning, NY) were coated with 1 μg/ml antihuman IL-8 coating antibody (Invitrogen, MD) overnight. The plates were then washed with washing buffer (1.47 mM KH₂PO4 and 8.32 mM K₂HPO4, 0.05% Tween 20, pH 7.4) and blocked with assay buffer (13.69 mM NaCl, 7.69 mM Na₂HPO4, 1.15 mM K₂HPO4, and 2.68 mM KCl, 0.5% bovine serum albumin, 0.05% Tween 20, pH 7.4).
The cell culture media samples, IL-8 standards and antihuman IL-8 antibodies conjugated with biotin, were added to the ELISA plates and incubated for 2 h. Wells were washed and incubated with streptavidin-HRP solution for 30 min. The plates were then washed, and 1-Step™ Ultra TMB-ELISA (Thermo Scientific, MA) substrate was added to the plates and incubated in dark for 30 min. The HRP reaction was stopped by sulfuric acid, and absorbance was measured at 450 nm and 650 nm. CXCL8 concentration in samples was calculated and managed via Prism software (GraphPad Software, CA).

Cells were exposed for a 24 h period to varying drug concentrations (as indicated in their respective citations below) while control cells were exposed to the corresponding DMSO (CAS 67-68-5, Sigma-Aldrich, D8418) dilution: metformin (glucophage; CAS 1115-70-4, 15169101, MP Biomedicals, stock 3 M in PBS), genistein (CAS 446-72-0, 10005167100 Cayman Chemicals, stock 100 mM in DMSO), haloperidol (haldol; CAS 52-86-8, 15369690 MP Biomedicals, stock 50 mM in DMSO). For acyl-carnitine measurements, the medium was supplemented with 1 mM L-carnitine (CAS 6645-46-1, Sigma-Aldrich, C0283). Each experiment was performed in three biological repeats.