All the animal experiments were performed in accordance with the National Institutes of Health guidelines (NIH Publication, revised 2011) and the guidelines of the Animal Care and Use Committee of Renmin Hospital of Wuhan University. The animal studies also follow the ARRIVE guidelines. Male C57/B6J mice (age: 8–10 weeks; body weight: 25.5 ± 2 g) were purchased from the Institute of Laboratory Animal Science at the Chinese Academy of Medical Sciences (Beijing, China) and housed for more than 1 week before experimentation. After that, the mice were grouped according to a random number table and fed a high-fat diet (HFD, 45% kilocalories from fat, Institute of Laboratory Animal Science at the Chinese Academy of Medical Sciences, D12451, composition: protein 20 kcal%, carbohydrate 35 kcal%, and fat 45 kcal%) or a normal diet (ND, 10% kilocalories from fat) for 24 weeks, with only the last 3 weeks including a 21-day treatment with a previously used dose of geniposide (50 mg/kg, 0.2 ml, po) or an equal volume of saline (0.2 ml, po). The blood glucose levels were measured by mandibular puncture blood sampling. At the endpoint, all the mice were sacrificed with an overdose of sodium pentobarbital (200 mg/kg, i.p.) to harvest their heart and to calculate the following ratio: heart weight (HW)/tibia length (TL). To confirm the role of AMPKα in geniposide-mediated cardioprotection, Ampkα2 global knockout mice were used and subjected to HFD or ND for 24 weeks with treatment with geniposide for 3 weeks. The source of Ampkα2 global knockout mice has been described previously [16 (link), 17 (link)]. To verify the hypothesis that Sirt1 is involved in geniposide-mediated cardioprotection, siSirt1 and the siRNA control were delivered to the heart using a nanoparticle transfection reagent (Altogen Biosystems, NV, USA) via 3 injections (once every week) into the tail vein beginning from the initial geniposide treatment (21 weeks after HFD) [18 (link)].
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