Quantifying Hyperinsulinemia and Pancreatic Insulin

Serum and pancreatic insulin levels were measured by an enzyme immunoassay (Mercodia, Ultrasensitive Rat Insulin ELISA) in order to verify the development of hyperinsulinemia and decreased pancreatic insulin content as a consequence of beta cell damage in impaired glucose tolerance. Insulin ELISA was carried out according to the instructions of the manufacturer from either sera or homogenized pancreatic tissue samples of fructose-fed and control rats. Sera were centrifuged (2000 g for 10 min at 4°C) and kept at -20°C until further investigation. Pancreata were removed, trimmed free of adipose tissue, and weighed. Pancreata were homogenized in 6 mL cold acidified ethanol (0.7 M HCl : ethanol (1 : 3 v/v)) with an Ultra Turrax homogenizer and were kept at 4°C for 24 h. Then, pancreas homogenates were centrifuged (900 g for 15 min at 4°C), and the supernatants were stored at 4°C. The pellet was extracted again with 3 mL acidified ethanol for 24 h at 4°C. The supernatant obtained after centrifugation was pooled with the previous one and kept at -20°C until assayed [34 (link), 35 (link)].