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.
Highlights
Insulin and insulin-like growth factor (IGF) signal primarily through the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt and Ras/Mitogen activated protein kinase (MAPK) pathways to affect multiple cellular functions including cell growth, cell survival and cellular metabolism [1]
We demonstrate that IRS1 localisation is dependent on its phosphorylation state and report the development of models for the modification of these pathways; using a combined insulinfructose treatment protocol we impair insulin signalling in these cells, and through the use of an IGF1 receptor (IGF1R) monoclonal antibody we impair Insulin-like growth factor 1 (IGF1) signalling through this pathway
The expression of IRβ was lower in the Sciencell media (Additional file 2: Figure S2b, Sciencell astrocytes) and all subsequent experiments were performed in F10:MEMα media
Summary
Insulin and insulin-like growth factor (IGF) signal primarily through the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt and Ras/Mitogen activated protein kinase (MAPK) pathways to affect multiple cellular functions including cell growth, cell survival and cellular metabolism [1] These complex signalling pathways are increasingly implicated in the pathogenesis of Alzheimer’s disease (AD) and other neurodegenerative diseases including Parkinson’s disease and motor neurone disease [2,3,4]. An insulin resistant state is evident in the brain early in AD progression [5, 6], and a number of epidemiological studies have identified Type-2 diabetes (T2D), in which an insulin resistant state exists, as a risk factor for developing AD [7,8,9] These pathways have been implicated as regulators of longevity [1] and may be important in brain ageing and its interaction with neurodegeneration. We examined the IIS pathway in human astrocytes and developed models to reduce IIS at the level of the insulin or the IGF1 receptor (IGF1R)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
