Measurement of CPT-1 activity

The CPT-1 activity assay was performed as described by Pawel and Bremer⁵⁷. In brief, mitochondria of adipose tissue or adipocytes were first isolated using the commercial isolation kits form Abcam (ab110170 and ab110168; Cambridge, UK) according to the protocols. Then 200 μg of mitochondrial protein was added to the assay medium (20 mM HEPES, 75 mM KCl, 2 mM KCN, 1% fat free BSA, 70 μM palmitoyl-CoA, 0.25 mM L-[³H]carnitine) with or without different levels of malonyl-CoA. Samples were incubated at 37 °C for 3 min. And then stopped by the addition of 0.5 ml of ice-cold perchloric acid. After mitochondrial centrifuged, the mitochondrial pellet was then resuspended in ddH₂O and extracted with 600 μL of butanol 300 μL of the butanol-phase was counted by liquid scintillation.