*2.3. Blood Sampling*

Approximately 0.5 mL of blood was collected at each timepoint by inserting a 23 G butterfly catheter (SV\*23BLK, Terumo Australia Pty Ltd., Macquarie Park, NSW, Australia) into the injection port of the tail vein catheter. The initial saline-diluted drops of blood were discarded before sample collection. A glucometer was used to immediately measure the blood glucose concentration (Accu-Chek Guide, Roche, BellaVista, Australia). After each sample, the catheter was flushed with 0.5 mL of heparinised saline (5 IU/mL) to prevent clot formation in the catheter and replace blood volume. In the event that su fficient blood could not be collected from the catheter, blood was drawn percutaneously from the lateral saphenous veins, medial saphenous veins or femoral arteries through a butterfly catheter.

All blood samples were collected in 3 mL Eppendorf tubes and allowed to clot at room temperature for 10 min before refrigeration. Refrigerated samples were centrifuged within 4 h of collection using an Eppendorf MiniSpin plus centrifugation at 2000× *g* for 10 min. Approximately 0.2 mL of serum supernatant from each sample was collected and transferred into new 3 mL Eppendorf tubes. These serum samples were then frozen at −80 ◦C.
