Impact of Hyperinsulinemia and Hyperglycemia on Valvular Interstitial Cells - A Link between Calcific Aortic Valve Disease and Type 2 Diabetes

Objectives: Despite the given association of calcific aortic valve disease (CAVD) and type 2 diabetes (T2D) in epidemiologic studies, little is known about insulin action in aortic valve tissue. To improve the understanding of diabetes-induced pathways in heart valves, we developed an in vitro model mimicking two common metabolic conditions of T2D, hyperinsulinemia and hyperglycemia, and examined the insulin responsiveness and glucose uptake of valvular interstitial cells (VIC).

Methods: Primary ovine aortic VIC were cultured with chronic insulin exposure (100 nM) either under physiological glucose concentration (100 mg/dL, NG) or under hyperglycemic conditions (450 mg/dL, HG) to simulate a diabetic environment. After 6 days of treatment, insulin response was determined after acute insulin stimulus by Western blot analysis for phosphorylated GSK-3α/β. Furthermore, glucose uptake of VIC was quantified and gene expression of different glucose transporters (GLUT) was analyzed. Repetitive experiments were performed using VIC from 6 individual sheep.

Results: After acute insulin stimulation phosphorylation of GSK-3α/β was strongly increased (p < 0.0001) demonstrating a high insulin sensitivity of VIC. This insulin response could be impaired by hyperinsulinemia, represented by a decreased phosphorylation of GSK-3α (p < 0.0004). In contrast, phosphorylation of GSK-3β remained stable under chronic insulin exposure. To evaluate the influence of insulin on the glucose metabolism, a glucose uptake assay was performed. Glucose uptake was significantly increased in cells treated with chronic insulin exposure under NG conditions compared with untreated cells (p < 0.027). In contrast, VIC cultured under HG had an impaired glucose uptake (p < 0.008) and the insulin effect was lost. Additionally, we identified GLUT-1 as main GLUT type by gene expression analysis, whereby GLUT-2 and GLUT-4 were not detectable. However, GLUT-1 expression was independent from hyperglycemia and hyperinsulinemia, both on mRNA and on protein level.

Conclusion: Aortic VIC show insulin sensitivity by impaired GSK-3α phosphorylation and an increased glucose uptake due to hyperinsulinemia. Thus, we show that the valves' main cell type is highly susceptible for diabetic conditions, suggesting an underestimated role of VIC in CAVD caused by T2D. This in vitro model may serve for further investigations on signaling pathways altered by T2D in the context of CAVD.