Prolonged exposure of mouse and human podocytes to insulin induces insulin resistance through lysosomal and proteasomal degradation of the insulin receptor

Aims/hypothesis Podocytes are insulin-responsive cells of the glomerular filtration barrier and are key in preventing albuminuria, a hallmark feature of diabetic nephropathy. While there is evidence that a loss of insulin signalling to podocytes is detrimental, the molecular mechanisms underpinning the development of podocyte insulin resistance in diabetes remain unclear. Thus, we aimed to further investigate podocyte insulin responses early in the context of diabetic nephropathy. Methods Conditionally immortalised human and mouse podocyte cell lines and glomeruli isolated from db/db DBA/2J mice were studied. Podocyte insulin responses were investigated with western blotting, cellular glucose uptake assays and automated fluorescent imaging of the actin cytoskeleton. Quantitative (q) RT-PCR was employed to investigate changes in mRNA. Human cell lines stably overproducing the insulin receptor (IR) and nephrin were also generated, using lentiviral constructs. Results Podocytes exposed to a diabetic environment (high glucose, high insulin and the proinflammatory cytokines TNF-alpha and IL-6) become insulin resistant with respect to glucose uptake and activation of phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling. These podocytes lose expression of the IR as a direct consequence of prolonged exposure to high insulin concentrations, which causes an increase in IR protein degradation via a proteasome-dependent and bafilomycin-sensitive pathway. Reintroducing the IR into insulin-resistant human podocytes rescues upstream phosphorylation events, but not glucose uptake. Stable expression of nephrin is also required for the insulin-stimulated glucose uptake response in podocytes and for efficient insulin-stimulated remodelling of the actin cytoskeleton. Conclusions/interpretation Together, these results suggest that IR degradation, caused by high levels of insulin, drives early podocyte insulin resistance, and that both the IR and nephrin are required for full insulin sensitivity of this cell. This could be highly relevant for the development of nephropathy in individuals with type 2 diabetes, who are commonly hyperinsulinaemic in the early phases of their disease.