Recombinant irisin

The mouse hepatocyte AML12 cell line (ATCC; Manassas, VA, USA) was cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM; HyClone, Logan, UT, USA) with 10% fetal bovine serum (FBS; HyClone), 1% penicillin/streptomycin (Invitrogen; Carlsbad, CA, USA), ITS supplement (Sigma), and 40 ng/mL dexamethasone. The mouse macrophage RAW264.7 cell line was cultured in DMEM medium with 10% FBS and 1% penicillin/streptomycin. All cells were grown in an incubator maintained at 37 °C with 5% CO₂. Recombinant irisin was purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA) and recombinant human MD2 (rhMD2) was purchased from R&D Systems (Minneapolis, MN, USA). Lipid accumulation in the hepatocytes was observed by Oil Red O staining as described previously [28 (link)].

3T3-L1 mouse preadipocytes were purchased from ATCC (Manassas, VA, United States). For maintenance of preadipocytes, 100 mm culture plates were used. For gene expression analysis and Western blotting the cells were seeded in 6 well plates, whereas 96 well plates were used for cell viability assay and Oil Red O staining. The cells were maintained in Dulbecco modified Eagle medium (DMEM) with 10% FBS (HyClone, Australia) and 1% penicillin/streptomycin (Carlsbad, CA, United States) and incubated at 37°C in 5% CO₂. 3T3-L1 cells were treated with differentiation media (MDI; methylisobutylxanthine, dexamethasone, insulin) to induce adipogenesis as previously described (Huh et al., 2012 (link)). To examine the effect of irisin on adipogenesis, 100 ng/mL Recombinant irisin was treated every other day for 6 days, starting from 2 days before changing to MDI. Recombinant irisin was purchased from Phoenix Pharmaceuticals, Inc. (Burlingame, CA, United States). Plasmid encoding FNDC5 gene and small interfering RNA were also used to examine the effect of genetic manipulation of irisin on adipocyte differentiation, as described below.

Cardiac hypertrophy was established in 4-month-old male C57BL/6J mice using transverse aortic constriction (TAC)^{43 (link)}. The mice were anesthetized and placed in a supine position, and a midline cervical incision was made to expose the trachea. After opening the chest cavity, the transverse aorta was sutured using 7–0 nylon thread, connected using a blunt 27-gauge needle, and then detached. The sham-operated controls underwent the same procedure without aortic ligation. The chest was then sutured, and the animals were ventilated until they could breathe on their own. The mice were intravenously injected with PBS (control) or recombinant irisin (2 μg/kg/week, Phoenix Pharmaceuticals, Inc., USA)^{44 (link)} at 0, 1, 2, and 3 weeks after TAC, and euthanized after 4 weeks. The cardiac tissues were resected for further analysis. All experimental procedures were conducted as per the guidelines for the Animal Care and Use Committee of North Sichuan Medical College, China, and approved by the Medical Ethics Committee.