Insulin and IGF1 signalling pathways in human astrocytes in vitro and in vivo; characterisation, subcellular localisation and modulation of the receptors

Abstract

BackgroundThe insulin/IGF1 signalling (IIS) pathways are involved in longevity regulation and are dysregulated in neurons in Alzheimer’s disease (AD). We previously showed downregulation in IIS gene expression in astrocytes with AD-neuropathology progression, but IIS in astrocytes remains poorly understood. We therefore examined the IIS pathway in human astrocytes and developed models to reduce IIS at the level of the insulin or the IGF1 receptor (IGF1R).ResultsWe determined IIS was present and functional in human astrocytes by immunoblotting and showed astrocytes express the insulin receptor (IR)-B isoform of Ir. Immunocytochemistry and cell fractionation followed by western blotting revealed the phosphorylation status of insulin receptor substrate (IRS1) affects its subcellular localisation. To validate IRS1 expression patterns observed in culture, expression of key pathway components was assessed on post-mortem AD and control tissue using immunohistochemistry. Insulin signalling was impaired in cultured astrocytes by treatment with insulin + fructose and resulted in decreased IR and Akt phosphorylation (pAkt S473). A monoclonal antibody against IGF1R (MAB391) induced degradation of IGF1R receptor with an associated decrease in downstream pAkt S473. Neither treatment affected cell growth or viability as measured by MTT and Cyquant® assays or GFAP immunoreactivity.DiscussionIIS is functional in astrocytes. IR-B is expressed in astrocytes which differs from the pattern in neurons, and may be important in differential susceptibility of astrocytes and neurons to insulin resistance. The variable presence of IRS1 in the nucleus, dependent on phosphorylation pattern, suggests the function of signalling molecules is not confined to cytoplasmic cascades. Down-regulation of IR and IGF1R, achieved by insulin + fructose and monoclonal antibody treatments, results in decreased downstream signalling, though the lack of effect on viability suggests that astrocytes can compensate for changes in single pathways. Changes in signalling in astrocytes, as well as in neurons, may be important in ageing and neurodegeneration.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0138-6) contains supplementary material, which is available to authorized users.

Brain insulin resistance in type 2 diabetes and Alzheimer disease: concepts and conundrums

“…Astrocytes bind insulin with high affinity 89 and express IRS1, IRS2 and downstream signalling molecules AKT and MAPK. Functional assays have demonstrated activation of these canonical pathways with insulin or IGF1 90–92 . Interestingly, glial insulin receptors are downregulated in response to chronically high levels of insulin whereas neuronal insulin receptors are not 93 .…”

The neuronal insulin receptor in its environment

“…; Garwood et al . ). The two isoforms, when present in the same cell type, display different insulin binding kinetics (Knudsen et al .…”

“…Isoform A of the IR (IR-A), which lacks exon 11 (Fig. 1), predominates in the embryo and is the only isoform detectable in adult neurons, while IR-B, which includes exon 11, constitutes > 90% of total IR in the adult liver, adipose tissue and glia; the skeletal muscle contains comparable amounts of IR-A and IR-B (Joost 1995;Kenner et al 1995;Garwood et al 2015). The two isoforms, when present in the same cell type, display different insulin binding kinetics (Knudsen et al 2011), and downstream effects (Leibiger et al 2001).…”

Identification of conserved proteomic networks in neurodegenerative dementia

“…While MSN, a microglial enriched gene, is involved in cleavage of APP (Darmellah et al, 2012), GFAP and VIM are reactive astrocyte markers (Kamphuis et al, 2012), reinforcing the role of glia in early AD pathogenesis (Dzamba et al, 2016;Efthymiou and Goate, 2017;Garwood et al, 2015;Heneka et al, 2015;Nagele et al, 2004;. DDAH2 is involved in nitric oxide production, a component of the immune response (Lambden et al, 2016).…”