Ultrasensitive rat insulin elisa

Bodyweight, food intake and fasting glucose level were recorded every month. Blood was collected from tail vein, and plasma was isolated by centrifugation. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (TC), low‐density lipoprotein cholesterol (LDL‐C) and HDL‐cholesterol (HDL‐C) levels were measured with an auto‐analyser (Cobas 6000 c501; Roche Diagnostics). Fasting blood glucose was measured with a One‐Touch Accu‐Chek Glucometer (Roche Diagnostics), and fasting serum insulin was determined using a rat‐specific insulin ELISA kit (Ultrasensitive Rat Insulin ELISA; Mercodia) following the kit instruction.

Serum and pancreatic insulin levels were measured by an enzyme-linked immunosorbent assay (Mercodia, Ultrasensitive Rat Insulin ELISA) as described previously [21 (link), 22 (link), 38 (link)]. Blood samples were collected from the saphenous vein at weeks 7, 11 and 15. At week 15, during OGTT blood was collected at 0, 30 and 120 min for serum insulin level measurements. Blood samples were centrifuged (4500 rpm for 10 min at 4 °C) and kept at −20 °C until the assay was performed. At week 15, 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 Ultraturrax homogenizer and were kept at 4 °C for 24 h. Then pancreas homogenates were centrifuged (900g 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. Insulin ELISA was carried out according to the instructions of the manufacturer from either sera or homogenized pancreatic tissue samples of GK and control rats.

Pancreatic islets were isolated from adult male Wistar rats by using the standard collagenase digestion method [59 (link)]. Islet cells having viability greater than 90% were chosen for the studies. Insulin secretion study was determined in both 1 and 24 h time intervals to evaluate the effect of isolated compounds and its active fraction against pancreatic islet cells. Therefore, the isolated islets (150 cells/mL medium) were incubated with 5% CO₂ at 37 °C in a humidified incubator. Test samples of varying concentrations (25–100 μg/mL) were treated with normal (4 mM glucose) and diabetic (20 mM) conditions, respectively. After incubation, cells were centrifuged at 1500× g for 15 min at 4 °C. The supernatant obtained was subjected to measure insulin secretion according to the manufacturer’s instruction (Mercodia ultrasensitive rat insulin ELISA, Uppsala, Sweden).