As described above, livers, epididymal fat depots, and jejunal segments were excised from the animals immediately after sacrifice and stored at − 80 °C until analysis. Tissue protein concentrations of the three tissues were first determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA) method according to manufacturer’s instruction. TNF-α and MCP-1 levels were subsequently determined in tissue homogenates with enzyme-linked immunosorbent assay (ELISA) kits (AB100785 and AB100778; Abcam, Cambridge, UK) according to manufacturer’s instructions. Absorbance was read using a Spectramax 340PC384 microplate reader at 450 nm (Molecular Devices, CA, USA).