Role of CYP2E1 in ketone-stimulated insulin release in pancreatic B-cells

The role of CYP2E1 in ketone-stimulated insulin release was investigated using isolated pancreatic islets of Langerhans and two mammalian insulin secreting pancreatic beta-cell lines engineered to stably express human CYP2E1 (designated BRIN BD11h2E1 and INS-1h2E1). Isolated rat pancreatic islets were shown to express the CYP2E1 isoform which was inducible by pretreatment of animals with acetone. The cDNA encoded CYP2E1 was expressed and inducible in the engineered cells as shown by Western blotting. The transfected protein was enzymatically active in the heterologous cells as determined by p-nitrophenol hydroxylation rates (0.176 +/- 0.08 vs. 0.341 +/- 0.08 nmol/min/mg microsomal protein in BRIN BD11 control cells and BRIN BD11h2E1 cells respectively, P < 0.001; 0.204 +/- 0.03 vs. 0.633 +/- 0.102 nmol/min/mg microsomal protein in INS-1 and INS-1h2E1, respectively, P < 0.001). Cultivation of CYP2E1 expressing BRIN BD11h2E1 and INS-1h2E1 cells in 40 mM ethanol increased the rate of p-nitrophenol hydroxylation (0.968 +/- 0.09 nmol/min/mg microsomal protein, P < 0.001 and 0.846 +/- 0.103 nmol/min/mg microsomal protein, P < 0.001, respectively) providing further evidence that the heterologous protein is inducible. Cultivation of control cells with ethanol had no observable effect (0.186 +/- 0.05 and 0.195 +/- 0.03 in BRIN BD11 and INS-1, respectively). These cell lines also express NADPH-cytochrome P450 reductase protein which was enzymatically active (0.632 +/- 0.023 in parental BRIN BD11 vs. 0.657 +/- 0.066 without ethanol and 0.824 +/- 0.014 nmol/min/mg microsomal protein with ethanol in BRIN BD11h2E1, P < 0.05; and 1.568 +/- 0.118 in parental INS-1 vs. 1.607 +/- 0.093 without ethanol and 1.805 +/- 0.066 nmol/min/mg microsomal protein with ethanol in INS-1h2E1, P < 0.05) thereby providing a functional cytochrome P450 system. The insulin secretory response of control cell lines and islets was similar to cell lines and islets which had been chemically pretreated, to induce CYP2E1 expression, in response to known nutrient secretagogues. However, insulin output was significantly higher in pretreated islets (1.3-fold, P < 0.05) and CYP2E1 expressing cell lines (BRIN BD11h2E1 2.3-fold, P < 0.001; INS1-1h2E1 1.6-fold, P < 0.001) when stimulated with the ketone 3-hydroxybutyrate than control islets and parental cell lines respectively. Similar acute exposure to acetoacetate enhanced insulin secretion by 1.3-fold (P < 0.05) in pretreated islets, 2.6-fold (P < 0.001) in ethanol pretreated BRIN BD11h2E1 and 1.4-fold (P < 0.001) in ethanol pretreated INS-1h2E1 cells compared to the respective control islets or ethanol pretreated control parental cells. Therefore, these studies highlight a possible role for CYP2E1 in pancreatic cell dysfunction.