Experimental animals

Male C57BL/6 mice (four weeks old) were purchased from Guangzhou Animal Center, China. Mice were housed in a temperature-regulated environment (20°C) with a reversed 12:12 hour light-dark cycle. The principles of laboratory animal care were followed and the experiments were approved by the Animal Care Committee, Shenzhen University. After one week of adaptive feeding, mice were divided randomly into normal-chow-diet (NCD) and high-fat-diet (HFD) groups. NCD contained (wt/wt) 4.3% fat, 19.2% protein, and 67.3% carbohydrate. HFD contained (wt/wt) 24% fat, 24% protein, and 41% carbohydrate. Both diets were otherwise identical and manufactured by Jiangsu Medicience Ltd, China. Eight to ten mice of each group were sacrificed at 3d, 1wk, 2wk and 4wk respectively, and body weight was monitored before sacrificing. After intraperitoneal injection of pentobarbital (1%, 50mg/kg), blood samples and hind limb muscles (soleus muscle, extensor digitorum longus muscle and tibialis anterior muscle) were collected. Hind limb muscles were used for giant sarcolemmal vesicles isolation immediately or fixed by 4% paraformaldehyde followed by immunostaining. Blood was immediately centrifuged, and the plasma was separated and stored at -80°C until assayed for blood glucose and lipid profile.