Elsevier

Neuroscience

Volume 143, Issue 1, 17 November 2006, Pages 165-173
Neuroscience

Molecular neuroscience
Endogenous insulin signaling protects cultured neurons from oxygen–glucose deprivation-induced cell death

https://doi.org/10.1016/j.neuroscience.2006.07.055Get rights and content

Abstract

Curiosity surrounding the physiological relevance of neural insulin signaling has gradually developed since the discovery that nervous tissue contains both the hormone and its receptor. Similar to other receptor tyrosine kinases, ligand interaction with the insulin receptor (IR) activates a variety of intracellular signaling pathways, particularly those relevant to cellular survival. Consequently, one explanation for the presence of the insulin pathway in the brain may involve participation in the response to neuronal injury. To investigate this possibility, the present study began by examining the effect of oxygen–glucose deprivation (OGD), a well-characterized in vitro model of ischemia, on ligand-binding, surface expression, and function of the IR in cultured rat neurons that were prepared under serum-free conditions. Reduced insulin-binding was observed following OGD, although surface expression of the receptor was not altered. However, OGD did significantly decrease the ability of insulin to stimulate phosphorylation of the transmembrane IR β-subunit, without affecting protein expression of this subunit. Subsequent experiments focused on the manner in which pharmacologically manipulating IR function affected neuronal viability after OGD. Application of the IR sensitizer metformin moderately improved neuronal viability, while the specific IR tyrosine kinase inhibitor tyrphostin A47 was able to dramatically decrease viability; both compounds acted without affecting IR surface expression. Our study suggests that not only does the IR appear to play an important role in neuronal survival, but also that neurons may actively maintain IRs on the cell surface to compensate for the OGD-induced decrease in the ability of insulin to phosphorylate its receptor.

Section snippets

Primary neuronal cultures

Primary cultured cortical and hippocampal neurons were prepared from Wistar rats at approximately embryonic day 18 in accordance with guidelines for the use of experimental animals established by the Canadian Council on Animal Care. All procedures were approved by the institutional animal care committee, and were designed to minimize both the number of animals employed and their suffering. Briefly, isolated cortices or hippocampi were pooled, dissociated via mechanical trituration, and

IRs are constitutively present at the plasma membrane and are synaptically localized in cultured neurons

Although earlier studies reported that IRs were present in cultured rat neurons (Raizada et al 1982, Boyd and Raizada 1983, Abbott et al 1999), the intracellular versus plasmalemmal expression profile of the receptor had not been examined. To determine the proportion of IRs expressed at the plasma membrane in cultured hippocampal neurons, cell culture ELISAs were performed under both low-permeant (plasmalemmal) and permeant (total) conditions with an antibody recognizing an epitope in the

Discussion

In agreement with earlier studies (Weyhenmeyer et al 1985, Abbott et al 1999), the current immunochemical findings illustrate that the IR is abundant in cultured neurons and may be found throughout both somata and neurites. For the first time, the proportion of IRs present upon the surface of hippocampal neurons was examined, and the receptor was shown to be nearly equally distributed between plasmalemmal and intracellular locations, which is in stark contrast to the pattern observed with

Acknowledgments

The authors would like to thank Edmund Lo for technical contributions, and Dr. Joseph Tauskela for a review of the manuscript and constructive discussion.

References (78)

  • H.Y. Man et al.

    Activation of PI3-kinase is required for AMPA receptor insertion during LTP of mEPSCs in cultured hippocampal neurons

    Neuron

    (2003)
  • E.J. Meuillet et al.

    Metformin modulates insulin receptor signaling in normal and cholesterol-treated human hepatoma cells (HepG2)

    Eur J Pharmacol

    (1999)
  • S. Nagamatsu et al.

    Glucose transporter expression in brain

    J Biol Chem

    (1992)
  • R.A. Palovcik et al.

    Insulin inhibits pyramidal neurons in hippocampal slices

    Brain Res

    (1984)
  • R.V. Rajala et al.

    In vivo regulation of phosphoinositide 3-kinase in retina through light-induced tyrosine phosphorylation of the insulin receptor β-subunit

    J Biol Chem

    (2002)
  • S. Shibata et al.

    Inhibitory action of insulin on suprachiasmatic nucleus neurons in rat hypothalamic slice preparations

    Physiol Behav

    (1986)
  • J.A. Weyhenmeyer et al.

    Light and electron microscopic analysis of insulin binding sites on neurons in dissociated brain cell cultures

    Brain Res Bull

    (1985)
  • M.F. White et al.

    The insulin signaling system

    J Biol Chem

    (1994)
  • M.J. White et al.

    Assessment of neuronal viability with Alamar blue in cortical and granule cell cultures

    J Neurosci Methods

    (1996)
  • J. Xu et al.

    Ethanol impairs insulin-stimulated neuronal survival in the developing brainRole of PTEN phosphatase

    J Biol Chem

    (2003)
  • W. Zhao et al.

    Brain insulin receptors and spatial memoryCorrelated changes in gene expression, tyrosine phosphorylation, and signaling molecules in the hippocampus of water maze trained rats

    J Biol Chem

    (1999)
  • W. Zhao et al.

    Role of insulin and insulin receptor in learning and memory

    Mol Cell Endocrinol

    (2001)
  • M.A. Abbott et al.

    The insulin receptor tyrosine kinase substrate p58/53 and the insulin receptor are components of CNS synapses

    J Neurosci

    (1999)
  • G. Ahmadian et al.

    Tyrosine phosphorylation of GluR2 is required for insulin-stimulated AMPA receptor endocytosis and LTD

    EMBO J

    (2004)
  • N.P. Birch et al.

    Immunoreactive insulin from mouse brain cells in culture and whole rat brain

    Biochem J

    (1984)
  • K. Bouhaddi et al.

    Insulin effect on GABA uptake in astroglial primary cultures

    Neurochem Res

    (1988)
  • F.T. Boyd et al.

    Effects of insulin and tunamycin on neuronal insulin receptors in culture

    Am J Physiol

    (1983)
  • G.J. Brewer et al.

    Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination

    J Neurosci Res

    (1993)
  • T.H. Brown et al.

    Hippocampus

  • B.P. Ceresa et al.

    Inhibition of clathrin-mediated endocytosis selectively attenuates specific insulin receptor signal transduction pathways

    Mol Cell Biol

    (1998)
  • D.W. Clarke et al.

    Insulin is released from rat brain neuronal cells in culture

    J Neurochem

    (1986)
  • J.G. de Diego et al.

    Ligand-induced changes in insulin receptors in cell surface and Golgi fractions of fetal rat liver

    Endocrinology

    (1988)
  • S.M. de la Monte et al.

    Ethanol impairs insulin-stimulated mitochondrial function in cerebellar granule neurons

    Cell Mol Life Sci

    (2001)
  • S.M. de la Monte et al.

    Ethanol inhibits insulin expression and actions in the developing brain

    Cell Mol Life Sci

    (2005)
  • R.J. Douglas et al.

    Neocortex

  • H. Dudek et al.

    Regulation of neuronal survival by the serine-threonine protein kinase Akt

    Science

    (1997)
  • S. El Messari et al.

    Immunocytochemical localization of the insulin-responsive glucose transporter 4 (Glut4) in the rat central nervous system

    J Comp Neurol

    (1998)
  • J. Eng et al.

    Insulin recoverable from tissues

    Diabetes

    (1980)
  • M. Foti et al.

    Insulin and IGF-1 receptor trafficking and signalling

    Novartis Found Symp

    (2004)
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