Cell Signal. 2014 Jun;26(6):1355-68.

A novel insulin receptor-signaling platform and its link to insulin resistance and type 2 diabetes.

Farah Alghamdi, Merry Guo, Samar Abdulkhalek, Nicola Crawford, Schammim Ray Amith, Myron R. Szewczuk.

Department of Biomedical & Molecular Sciences, Queen’s University, Kingston, Ontario K7L 3N6, Canada

 

Abstract

Insulin-induced insulin receptor (IR) tyrosine kinase activation and insulin cell survival responses have been reported to be under the regulation of a membrane associated mammalian neuraminidase-1 (Neu1). The molecular mechanism(s) behind this process is unknown. Here, we uncover a novel Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in alliance with neuromedin B G-protein coupled receptor (GPCR), which is essential for insulin-induced IR activation and cellular signaling. Neu1, MMP-9 and neuromedin B GPCR form a complex with IRβ subunit on the cell surface. Oseltamivir phosphate (Tamiflu®), anti-Neu1 antibodies, broad range MMP inhibitors piperazine and galardin (GM6001), MMP-9 specific inhibitor (MMP-9i), and GPCR neuromedin B specific antagonist BIM-23127 dose-dependently inhibited Neu1 activity associated with insulin stimulated rat hepatoma cells (HTCs) that overly express human IRs (HTC-IR). Tamiflu, anti-Neu1 antibodies and MMP-9i attenuated phosphorylation of IRβ and insulin receptor substrate-1 (IRS1) associated with insulin-stimulated cells. Olanzapine, an antipsychotic agent associated with insulin resistance, induced Neu3 sialidase activity in WG544 or 1140F01 human sialidosis fibroblast cells genetically defective in Neu1. Neu3 antagonist 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) and anti-Neu3 antibodies inhibited sialidase activity associated with olanzapine treated murine Neu4 knockout macrophage cells. Olanzapine attenuated phosphorylation of IGF-R and IRS1 associated with insulin-stimulated human wild-type fibroblast cells. Our findings identify a novel insulin receptor-signaling platform that is critically essential for insulin-induced IRβ tyrosine kinase activation and cellular signaling. Olanzapine-induced Neu3 sialidase activity attenuated insulin-induced IGF-R and IRS1 phosphorylation contributing to insulin resistance.

doi:10.1016/j.cellsig.2014.02.015

PMID: 24583283

 

Supplement

It is well understood that modification of receptor glycosylation is a key process connecting ligand binding and receptor activation, however the mechanisms controlling this process are unknown. For the insulin receptor (IR), insulin binding has been reported to rapidly induce the interaction of IR with neuraminidase-1 sialidase (Neu1) (1). It is believed that Neu1 specifically hydrolyzes α-2,3 sialic acid residues from the heavily glycosylated IR, a process which removes steric hindrance from the receptor and facilitates the association of IR subunits and subsequent receptor activation (1-3).

Recently, we found that Neu1 desialylation of the epidermal growth factor receptor (EGFR) is critical for receptor dimerization and activation. It was discovered that Neu1 activity is also dependent on neuromedin B G protein-coupled receptor (GPCR) Gαi-signaling and matrix metalloproteinase-9 (MMP9) activation (4). Since IRs belong to the same family of receptor tyrosine kinases as EGFR, we initially asked whether insulin-induced IR activation requires a similar GPCR-MMP9-Neu1 signaling platform.

Using a rat hepatoma cell line overly expressing human IR (HTC-IR), we performed a live cell sialidase assay to detect the effect of insulin stimulation on the sialidase enzyme activity of HTC-IR cells. Indeed, insulin stimulation of live HTC-IR cells dose-dependently induced sialidase activity. It was not surprising that this insulin- induced sialidase activity was completely blocked by specific Neu1 inhibitors, MMP9 inhibitors, and neuromedin B GPCR (NMBR) inhibitors, in a dose-dependent manner.

If insulin-induced IR activation is dependent on Neu1, we then hypothesized that Neu1 inhibitors should impede IR activation in insulin-treated cells, and thus prevent downstream phosphorylation of both insulin receptor substrate-1 (IRS1) and IRβ. Immunocytochemistry and western blot analyses of insulin-treated WT human fibroblast cells demonstrated that Neu1 inhibitors significantly inhibited detection of phosphorylated IRS1 (pIRS1) and phosphorylated IRβ (pIRβ) compared to the uninhibited controls. Since Neu1 activity is dependent on NMBR signaling and MMP9 activation, we also hypothesized that specific inhibitors of NMBR and MMP9 should also impede IR activation and downstream detection of pIRS1 and pIRβ. Indeed, similar results were found for IR-expressing and insulin-induced cells that were treated with these inhibitors.

Finally, co-localization and co-immunoprecipitation analyses were used to confirm the cell-surface association of IR, NMBR, MMP9, and Neu1. Results indicated that Neu1 forms a complex with IRβ subunits on the cell surface prior to ligand binding, and that catalytic desialylation of IR by Neu1 may be the critical intermediate step involved in IR activation. Detection of co-localized IRβ and MMP9, as well as IRβ and NMBR, validated the predicted signaling model. Together, the intracellular and cell- surface co-localization of the tripartite NMBR-MMP9-Neu1 complex tethered to IRβ represents a novel and essential signaling platform for IR-expressing cells.

Significance: The findings in this report provide novel insights on the current understanding of insulin signaling. Here, we present an innovative signaling platform involved in insulin-induced IR activation, and implicate a new approach in targeting insulin signaling with therapeutic intent.

 

References

  1. Dridi L, Seyrantepe V, Fougerat A, Pan X, Bonneil E, Thibault P, et Positive regulation of insulin signaling by neuraminidase 1. Diabetes. 2013;62(7):2338-46.
  2. Ciaraldi Neuraminidase treatment of isolated rat adipocytes and differential regulation of basal and insulin-stimulated glucose transport. Diabetes. 1989;38(8):951-8.
  3.  Arabkhari M, Bunda S, Wang Y, Wang A, Pshezhetsky AV, Hinek Desialylation of insulin receptors and IGF-1 receptors by neuraminidase-1 controls the net proliferative response of L6 myoblasts to insulin. Glycobiology. 2010;20(5):603-16.
  4.  Gilmour AM, Abdulkhalek S, Cheng TS, Alghamdi F, Jayanth P, O’Shea LK, et A novel epidermal growth factor receptor-signaling platform and its targeted translation in pancreatic cancer. Cell Signal. 2013;25(12):2587-603.

 

Graphical Abstract

MS Fig1MS Fig2

Fig. A novel molecular organizational G-protein-coupled receptor (GPCR)-signaling platform potentiates neuraminidase-1 (Neu1) and matrix metalloproteinase-9 (MMP9) cross talk on the cell surface that is essential for the activation of the insulin receptor β subunit (IRβ) tyrosine kinases and downstream cellular signaling.

Notes: Insulin-binding to insulin receptor α subunits (IRα) potentiates neuromedin B GPCR-signaling and subsequent MMP9 activation to induce Neu1 sialidase. Activated MMP9 is proposed to remove the elastin-binding protein (EBP) from the multienzymatic complex composed of β-galactosidase/Neu1/protective protein cathepsin A (PPCA). Activated Neu1 hydrolyzes α-2,3 sialyl residues of IRβ at the ectodomain to remove steric hindrance and facilitate IRβ subunits association and tyrosine kinase activation.

Citation: Taken in part from (A) Research and Reports in Biochemistry 2013:3 17–30. © 2013 Abdulkhalek et al,    publisher and licensee Dove Medical Press Ltd. This is an Open Access article which permits unrestricted non-commercial use, provided the original work is properly cited, and (B) Cellular Signalling Volume 26, Issue 6, June 2014, Pages 1355–1368 © Alghamdi et al, publisher Elsevier B.V. or its licensors or contributors, Under a Creative Commons license.

Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann