Modulation of IRS1 Activity by GSK3β Dictates Insulin Signaling Sensitivity in Podocytes: Implications for Diabetic Kidney Disease

Background: Insulin signaling in kidney cells, in particular podocytes, plays a critical role in kidney homeostasis independent of glycemic levels. As a critical transducer of insulin signaling, GSK3β also acts as a convergent point for myriad pathways implicated in kidney injury. However, its role in DKD remains elusive. Methods: Mouse podocytes were exposed to insulin or a type 2 diabetic milieu, following GSK3β silencing, ectopic expression of a constitutively active GSK3β mutant (S9A), or treatment with tideglusib, an inhibitor of GSK3β. Podocyte injury and insulin signaling were assessed and validated in kidneys from db/db or control mice. Results: Upon insulin stimulation, insulin signaling mediators like Akt and GSK3β, were phosphorylated, associated with increased glucose uptake and expression of GLUT1 and 4. GSK3β silencing sensitized insulin signaling, marked by potentiated induction of p-Akt and p-ERK1/2 and enhanced glucose uptake and GLUT expression. Conversely, S9A de-sensitized insulin signaling and mitigated GLUT induction and glucose uptake. Among the many insulin signaling transducers, IRS1 co-precipitated and interacted with GSK3β. Moreover, in silico analysis indicated that IRS1S332, which is known to negatively regulate IRS1 activity and target IRS1 for proteasomal degradation, resides in the consensus motifs for phosphorylation by GSK3β. Indeed, insulin-induced p-IRS1S332 was suppressed by GSK3β silencing but was enhanced by S9A. Furthermore, in podocytes exposed to a type 2 diabetic milieu, inhibitory p-GSK3βS9 was suppressed, denoting GSK3β hyperactivity. This was associated with enhanced p-IRS1S332. Tideglusib treatment counteracted this effect, re-sensitized insulin signaling, and averted diabetic podocyte injury. In db/db mice, glomerular expression of p-IRS1S332 was augmented in glomerular podocytes. Based on immunoblotting of isolated glomeruli, the expression ratio of p-GSK3βS9/GSK3β in glomeruli was repressed in db/db mice as compared with control mice, denoting GSK3β hyperactivity, and this was negatively correlated with the level of p-IRS1S332. Conclusion: Diabetes-associated GSK3β hyperactivity promotes IRS1 phosphorylation, contributing to desensitization of insulin signaling in podocytes. Therapeutic targeting of GSK3β could re-sensitize insulin signaling in podocytes via regulation of IRS1. Funding: NIDDK Support