Model-Based Hypothesis Testing of Key Mechanisms in Initial Phase of Insulin Signaling

Type 2 diabetes is characterized by insulin resistance of target organs, which is due to impaired insulin signal transduction. The skeleton of signaling mediators that provide for normal insulin action has been established. However, the detailed kinetics, and their mechanistic generation, remain incompletely understood. We measured time-courses in primary human adipocytes for the short-term phosphorylation dynamics of the insulin receptor (IR) and the IR substrate-1 in response to a step increase in insulin concentration. Both proteins exhibited a rapid transient overshoot in tyrosine phosphorylation, reaching maximum within 1 min, followed by an intermediate steady-state level after approximately 10 min. We used model-based hypothesis testing to evaluate three mechanistic explanations for this behavior: (A) phosphorylation and dephosphorylation of IR at the plasma membrane only; (B) the additional possibility for IR endocytosis; (C) the alternative additional possibility of feedback signals to IR from downstream intermediates. We concluded that (A) is not a satisfactory explanation; that (B) may serve as an explanation only if both internalization, dephosphorylation, and subsequent recycling are permitted; and that (C) is acceptable. These mechanistic insights cannot be obtained by mere inspection of the datasets, and they are rejections and thus stronger and more final conclusions than ordinary model predictions.Author Summary: Insulin is a central player in maintaining energy balance in our bodies and in type 2 diabetes, where the effect of insulin on its target tissues is diminished. Insulin acts on cells by binding to specific insulin receptors (IRs) at the cell surface. This triggers a series of events, including attachment of phosphate to IR, activation of downstream proteins that eventually mediate the signal to specific targets in the cell, and internalization of IR to the inner cytosolic part of the cell. The importance, time relations, and interactions between these events are not fully understood. We have collected experimental time-series and developed a novel analysis method based on mathematical modeling to gain insights into these initial aspects of how insulin controls cells. The main conclusion is that either IR internalization and the subsequent recycling back to the cell surface or feedbacks from downstream proteins (or both) must be significantly active during the first few minutes of insulin action. These conclusions could not have been reached from the experimental data through conventional biological reasoning, and this work thus illustrates the power of modeling to improve our understanding of biological systems.