Role of SGKs in Glucocorticoid-Mediated Regulation of NHE3

Abstract

mechanisms not fully understood. Since high level of plasma glucose is the main feature of diabetes mellitus, we hypothesized that hyperglycemia induces NPC1L1 expression in diabetic patients via modulation of gene transcription. Therefore, current studies were undertaken to examine the transcriptional regulation of NPC1L1 in human intestinal Caco2 cells in response to glucose. Cells were transiently transfected with different promoter constructs of NPC1L1 gene. Relative promoter activity was measured by luciferase activity normalized to β-galactosidase. The relative abundance of NPC1L1 mRNA was evaluated by real-time PCR. Incubation of Caco2 cells with D-glucose (25 mM, 24 h) significantly increased the NPC1L1 mRNA expression by ~6 fold. In contrast, the removal of D-glucose from the culture medium of Caco2 cells for 24 h significantly decreased the NPC1L1 promoter activity (45.1±2.5 % of control, P<0.001). Consistent with increased mRNA expression, 24 h incubation with different concentrations of glucose (1, 5, 25 mM) significantly increased the promoter activity in a dose dependent manner with a maximum effect at 5 mM concentration (230±10.6 %, P<0.001) compared to control cells treated with 5 mM mannitol. Exposure of Caco2 cells to the non-metabolized form of glucose (3-O-methyl-D-glucopyranose, 5 mM, 24h) had no effect on the promoter activity of NPC1L1 indicating that observed effects are dependent on glucose metabolism. Also, pyruvate showed no effect on NPC1L1 promoter activity suggesting that metabolic products of glycolysis upstream of pyruvate mediate the effects of glucose. Deletion of NPC1L1 promoter construct to -291 nucleotide did not alter glucose-induced stimulation. These data indicated that glucose responsive elements are located in the region between -291 and +56 of NPC1L1 promoter. Conclusion: High levels of glucose stimulate NPC1L1 expression via transcriptional regulation. These data provide mechanistic insights into the increase in NPC1L1 expression observed in diabetesmellitus and may be exploited to improve therapeutic modalities for the associated hypercholesterolemia.