Effect of long-term insulin exposure on insulin binding in Tetrahymena pyriformis

Previous studies have indicated that Tetrahymena pyriformis can bind the vertebrate hormone insulin. Conventional microscopic studies were conducted to determine the effect of acute and long-term (LTE) insulin exposure on insulin binding. Stock cultures included cells never exposed to insulin and cultures grown in medium containing 6 mg/ml insulin. Logarithmic cultures were exposed to porcine insulin concentrations of 0 and 6 mg/ml for 1 h (insulin treated, (IT)) after 0, 48 h, 1, 3, or 6 months of LTE insulin exposure. 24 h after the 1 h insulin treatment, the cells were fixed, exposed to porcine insulin (antigen), processed immunocytochemically using a primary antibody to porcine insulin and a secondary antibody immunocytochemistry kit, and examined for staining intensity by video image analysis. Morphological observations confirm that T. pyriformis does bind insulin whether or not the cells have had prior exposure to insulin. IT increases insulin binding (up-regulation) in previously unexposed cells (control, P < 0.01) and produces a further amplification in cells having prior acute exposure (48 h) to insulin (P < 0.01). However, LTE exposure to insulin (1, 3 and 6 months) caused a decrease in insulin binding (down-regulation) after IT (P < 0.01) such that LTE-IT cells were not different from control cells following 1, 3 or 6 months of chronic insulin exposure to insulin. Staining intensity was not different between IT cells and cells cultured with insulin throughout the six month study. Results suggest that insulin binding sites of T. pyriformis are subject to regulatory processes similar to those of metazoans.