Can a Valve Become Resistant to Insulin? - Influence of Type 2 Diabetes Mimicking Conditions on Valvular Interstitial Cells

Objectives: Degenerative aortic valve disease (DAVD) is not only a passive wear process, it is rather an active cellular process that is significantly affected by various co-morbidities. A detrimental impact of type 2 diabetes (T2D) has been suggested by recent trials. However, it is unknown whether hyperglycemia and insulin resistance, two hallmarks of T2D, influence aortic valve remodeling. We aimed at developing a reproducible in vitro model of T2D in valvular interstitial cells (VICs) to study cellular mechanisms of DAVD in the context of T2D.

Methods: Primary ovine VICs were cultured either in low glucose (1 g/L) or high glucose (4.5 g/L) DMEM to mimic hyperglycemic conditions in vitro. Insulin resistance was induced by chronic treatment with insulin. To trigger degenerative processes, cells were additionally treated with β-glycerophosphate and CaCl₂. After 5 days, cells were cultured for 4 hour in fasting medium without FCS. Afterwards the insulin cascade was activated with an acute insulin stimulus. The insulin response was quantified by Western blot analyses of AKT (Ser473) phosphorylation. VICs without the last acute insulin stimulus served as controls for the basal state of AKT phosphorylation. Independent experiments using VICs from 6 individual sheep were performed and statistically evaluated by one-way ANOVA.

Results: Phosphorylation of AKT increased significantly after acute insulin stimulation (1- vs. 0.025-fold of untreated VICs, p < 0.0001), representing the physiologic insulin sensitivity of VICs. After repeated insulin treatments, phosphorylation of AKT in response to acute insulin stimulation was significantly inhibited, demonstrating insulin resistance of treated VICs (0.495 vs. 1, p < 0.0001). A pro-degenerative treatment did not influence the phosphorylation of AKT under control conditions (1.046 vs. 1) or in VICs resistant to insulin (0.437 vs. 0.495). Additional hyperglycemic conditions led to comparable effects on the insulin responsiveness of VICs in all treatment modalities.

Conclusion: The presented results demonstrate for the first time that primary VICs are sensitive to insulin. More importantly, continuous elevated insulin levels lead to a reduced intracellular insulin signaling, turning the presented protocol to an in vitro model of T2DM in DAVD. The strength of the developed insulin resistance seems to be independent from the glucose concentration and degenerative conditions used in cell culture.