Porcine insulin

The method of ex vivo tissue culture mainly referred to the previous study [16 (link)]. The fresh soleus muscle and liver tissue samples were obtained by 16G SuperCore™ semi-automatic biopsy needle (Argon medical device Inc., Plano, TX, USA) to make equivalent column size specimens. The size of each specimen was 1.2 (diameter) × 20 (length) mm; the weight was approximately 25 mg. For treating three different concentrations of insulin, three specimens were obtained from each tissue. During sampling, the specimens were immersed in cold Krebs–Henseleit bicarbonate buffer (KHB; pH 7.35, 10 mM HEPES, 24 mM NaHCO₃, 114 mM NaCl, 5 mM KCl, 1 mM MgCl₂, and 2.2 mM CaCl₂) or DMEM medium (Sigma-Aldrich) without glucose for muscle or liver specimens, respectively. The muscle specimens were then transferred into fresh pre-warmed KHB containing 3 mM glucose, 7 mM mannitol, and 0.1% (w/v) bovine serum albumin (BSA) in the absence (0 nM) and presence (10 or 100 nM) of porcine insulin (Sigma-Aldrich) and incubated at 37 °C for 30 min. After incubation, the specimens were washed with PBS twice and stored at − 80 °C until analysis. For liver specimens, the buffer was substituted with DMEM medium for KHB and the procedure was the same as described above.

Mice were fasted overnight and weighed. The tail was nicked with a fresh razor blade by horizontal cut of the tip and a OneTouch Ultramini glucometer was used to measure baseline blood glucose after overnight fasting. 1.0 unit per kg body weight of diluted porcine insulin (Sigma-Aldrich, St. Louis, MO, United States, I5523) was subsequently injected into the intraperitoneal cavity. Blood glucose was sampled from the tail of each mouse by gently massaging a small drop of blood onto the glucometer strip at 0 (baseline), 15, 30, 60, and 90 min following insulin injection (29 (link)).