Regulation of PTP1B Activity and Insulin Resistance by Cellular Cholesterol

Abstract

Protein Tyrosine phosphatase 1B (PTP1B), is an endoplasmic resident protein and a well-known negative regulator of the insulin receptor, dephosphorylating Tyr-1162, and Tyr-1163, two residues located in the activation loop of the insulin receptor. Mice lacking the PTPN1 gene encoding for PTP1B exhibit increased insulin sensitivity and improved glucose tolerance. Apart from its role in insulin signaling, mice lacking PTP1B show resistance to weight gain on a highfat diet, increased basal metabolic rate, and decreased cholesterol levels. In addition, PTP1B was previously identified in a proteome-wide mapping of cholesterol-interacting proteins in mammalian cells. However, the relationship between PTP1B and cholesterol is still unclear. To better understand the role of cholesterol on PTP1B function and on insulin signaling, we first used an in silico approach to predict cholesterol-binding sites in the 3D structure of the phosphatase and confirmed the binding sites through fluorescence binding studies and mass fingerprinting. We confirmed that the association between PTP1B and cholesterol occurred in both in vitro and in mammalian cells. In an attempt to understand whether cholesterol affects the ability of PTP1B to dephosphorylate substrates, we performed activity assays in various conditions. We observed that cholesterol could reduce and reactivate the reversibly oxidized form of PTP1B in vitro. Treatment of mammalian cells with cholesterol confirmed that excess cholesterol kept PTP1B reduced, and decreased Insulin Receptor phosphorylation and downstream signaling. In vivo results obtained by exposing mice to a high cholesterol diet support a role in the cholesterol-mediated reduction of PTP1B and decreased insulin sensitivity in the liver. We have established an electron tunneling path between the allosteric site and the catalytic cysteine residue and used a redox-sensitive fluorophore to measure electron tunneling in vitro. Hence, our results demonstrate for the first time that cholesterol binds to PTP1B at an allosteric site and reduces the phosphatase to regulate its activity and insulin signaling. Based on these results we propose a novel role for cholesterol in activating enzymes and in the context of insulin resistance.