Abstract
Background: Insulin signalling contributes to diverse cellular activities including protein synthesis, proliferation and cell survival. Insulin resistance describes the inability of cells to activate the insulin signalling pathway effectively; leading to pathological effects in multiple organ systems including the kidney. In diabetic kidney disease, there is progressive glomerular dysfunction and recent studies have demonstrated that the kidney podocyte is a direct target for insulin action. In this study we defined the literature-based insulin receptor (INSR) interactome and utilised an unbiased proteomic approach to examine INSR interactors in podocytes. Methods: Human podocytes expressing the INSR were characterised under basal and insulin resistant conditions. The INSR was isolated by whole cell immunoprecipitation following a time course stimulation of 2, 7, and 15 minutes with of 100nM insulin. The resulting INSR complexes were analysed by label-free mass spectrometry (MS) to detect protein interactors. Results: We identified 27 known, direct INSR interactors in addition to novel interactors including doublecortin domain-containing protein 2 (DCDC2). The interaction of DCDC2 with the INSR was confirmed by immunoprecipitation and immunofluorescence, and under insulin resistant conditions, DCDC2 had increased association with the INSR. siRNA knockdown of DCDC2 in podocytes resulted in cell morphological change and altered INSR localisation. Conclusion: This study provides insight into the complexity of INSR interactors in podocytes and highlights DCDC2 as a novel INSR binding protein. Involvement of this novel interactor in insulin signalling and podocyte biology may explain how insulin resistance alters morphology and integrity of the glomerular filtration barrier.
Highlights
Insulin plays a key role in energy homeostasis and insulin signalling contributes to diverse cellular activities including protein synthesis, proliferation and cell survival[1]
In this study we defined an in silico interaction network for the insulin receptor (INSR) across multiple tissues and focussed on the glomerular podocyte to identify novel INSR interactors
Insulin signalling is key for normal podocyte function[11] and it is disrupted in DKD2
Summary
Insulin plays a key role in energy homeostasis and insulin signalling contributes to diverse cellular activities including protein synthesis, proliferation and cell survival[1]. Abnormal physiological conditions and disease processes can interfere with the insulin signalling pathway leading to complete or partial loss of downstream effects This state is known as insulin resistance[2], which describes the inability of cells to activate the insulin signalling pathway effectively leading to pathological effects in multiple organ systems including muscle, liver, and kidneys[3,4]. Insulin resistance describes the inability of cells to activate the insulin signalling pathway effectively; leading to pathological effects in multiple organ systems including the kidney. Conclusion: This study provides insight into the complexity of INSR interactors in podocytes and highlights DCDC2 as a novel INSR binding protein Involvement of this novel interactor in insulin signalling and podocyte biology may explain how insulin resistance alters morphology and integrity of the glomerular filtration barrier
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