Feeding enhances fibronectin adherence of quiescent lymphocytes through non-canonical insulin signalling.

Nutritional availability during fasting and refeeding affects the temporal redistribution of lymphoid and myeloid immune cells among the circulating and tissue-resident pools. Conversely, nutritional imbalance and impaired glucose metabolism are associated with chronic inflammation, aberrant immunity and anomalous leukocyte trafficking. Despite being exposed to periodic alterations in blood insulin levels upon fasting and feeding, studies exploring the physiological effects of these hormonal changes on quiescent immune cell function and trafficking are scanty. Here, we report that oral glucose load in mice and healthy men enhances the adherence of circulating peripheral blood mononuclear cells (PBMCs) and lymphocytes to fibronectin. Adherence to fibronectin is also observed upon regular intake of breakfast following overnight fasting in healthy subjects. This glucose load-induced phenomenon is abrogated in streptozotocin-injected mice that lack insulin. Intra-vital microscopy in mice demonstrated that oral glucose feeding enhances the homing of PBMCs to injured blood vessels in vivo. Furthermore, employing flow cytometry, Western blotting and adhesion assays for PBMCs and Jurkat-T cells, we elucidate that insulin enhances fibronectin adherence of quiescent lymphocytes through non-canonical signalling involving insulin-like growth factor-1 receptor (IGF-1R) autophosphorylation, phospholipase C gamma-1 (PLCγ-1) Tyr783 phosphorylation and inside-out activation of ß-integrins respectively. Our findings uncover the physiological relevance of post-prandial insulin spikes in regulating the adherence and trafficking of circulating quiescent T-cells through fibronectin-integrin interaction.

In-silico identification of Tyr232 in AMPKα2 as a dephosphorylation site for the protein tyrosine phosphatase PTP-PEST.

The AMP-activated protein kinase (AMPK) is known to be activated by the protein tyrosine phosphatase non-receptor type 12 (PTP-PEST) under hypoxic conditions. This activation is mediated by tyrosine dephosphorylation of the AMPKα subunit. However, the identity of the phosphotyrosine residues that PTP-PEST dephosphorylates remains unknown. In this study, we first predicted the structure of the complex of the AMPKα2 subunit and PTP-PEST catalytic domain using bioinformatics tools and further confirmed the stability of the complex using molecular dynamics simulations. Evaluation of the protein-protein interfaces indicated that residue Tyr232 is the most likely dephosphorylation site on AMPKα2. In addition, we explored the effect of phosphorylation of PTP-PEST residue Tyr64 on the stability of the complex. Phosphorylation of the highly conserved Tyr64, an interface residue, enhances the stability of the complex via the rearrangement of a network of electrostatic interactions in conjunction with conformational changes in the catalytic WPD loop. We generated a phosphomimetic (PTP-PEST-Y64D) mutant and used co-immunoprecipitation to study the effect of PTP-PEST phosphorylation on AMPKα2 binding. The mutant exhibited an increased affinity for AMPKα2 and corroborated the in-silico predictions. Together, our findings present a plausible structural basis of AMPK regulation by PTP-PEST and show how phosphorylation of PTP-PEST affects its interaction with AMPKα2.

The protein tyrosine phosphatase PTP-PEST mediates hypoxia-induced endothelial autophagy and angiogenesis via AMPK activation.

Global and endothelial loss of PTP-PEST (also known as PTPN12) is associated with impaired cardiovascular development and embryonic lethality. Although hypoxia is implicated in vascular remodelling and angiogenesis, its effect on PTP-PEST remains unexplored. Here we report that hypoxia (1% oxygen) increases protein levels and catalytic activity of PTP-PEST in primary endothelial cells. Immunoprecipitation followed by mass spectrometry revealed that α subunits of AMPK (α1 and α2, encoded by PRKAA1 and PRKAA2, respectively) interact with PTP-PEST under normoxia but not in hypoxia. Co-immunoprecipitation experiments confirmed this observation and determined that AMPK α subunits interact with the catalytic domain of PTP-PEST. Knockdown of PTP-PEST abrogated hypoxia-mediated tyrosine dephosphorylation and activation of AMPK (Thr172 phosphorylation). Absence of PTP-PEST also blocked hypoxia-induced autophagy (LC3 degradation and puncta formation), which was rescued by the AMPK activator metformin (500 µM). Because endothelial autophagy is a prerequisite for angiogenesis, knockdown of PTP-PEST also attenuated endothelial cell migration and capillary tube formation, with autophagy inducer rapamycin (200 nM) rescuing angiogenesis. In conclusion, this work identifies for the first time that PTP-PEST is a regulator of hypoxia-induced AMPK activation and endothelial autophagy to promote angiogenesis.