Endocrine Abstracts

Type 2 diabetes (T2D) affects 1 in 15 people in the U.K, ischaemic damage is predominant and caused by hypoxia. The physiological response to hypoxia is an increase in the transcription factor hypoxia inducible factor (HIF)-1α, which results in reduced oxygen consuming. Previously we have demonstrated that the diabetic heart fails to respond to hypoxia and the aim of this work was to determine if a similar effect was seen in the kidney. HEK293T cells were incubated for 8h in MEM media with or without 0.5 mM long-chain fatty acids (LCFA). After this initial incubation, media was replenished flasks were split between normoxiaand the hypoxic (2% O₂) for 16h (n = 4 for each condition). qRT-PCR was performed on HIF1α-mediated genes: VEGF-A, GLUT-1, CA9, NDRG1, and EGLN1. Gene expression was normalised to the geomean of housekeeping genes; b-actin, RPL13A and HRPT1. To assess cell viability, a separate 96 well plate was incubated and at 16h alamarBlue was added for another 4h, and absorbance read at 570 nm and 600 nm. In control HEK293T cells 16h of hypoxia significantly (P < 0.05) induced gene expression in all 5 of our target genes, compared to normoxia with between 30 and 70% increase in gene expression. Interestingly, cells incubated with LCFAs failed to increase gene expression in hypoxia, with no significant difference in any of the 5 genes when compared to normoxia. Incubation with LCFAs led to as 54% decrease in cell viability in hypoxia compared to hypoxic controls. Hypoxia significantly increases the expression of HIF1α-mediated genes: VEGF-A, GLUT-1, NDRG1, EGLN1, and CA9 in HEK293T cells. These data suggest that LCFAs blunt the normal HIF-1α response in hypoxia, preventing vital adaptation and cell survival. Thereby demonstrating that LCFAs, in T2D, could be responsible for increased damage during ischemia and increased mortality from renal complications.