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

Garwood CJ, Ratcliffe LE, Morgan SV, Simpson JE, Owens H, Vazquez-Villaseñor I, Heath PR, Romero IA, Ince PG, Wharton SB - Mol Brain (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Sheffield Institute for Translational Neuroscience, Department of Neuroscience, The University of Sheffield, 385a Glossop Road, Sheffield, S10 2HQ, UK.

ABSTRACT

Background: The 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).

Results: We 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.

Discussion: IIS 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.

No MeSH data available.


Related in: MedlinePlus

Schematic of the insulin/IGF1R signalling pathway. A simplified representation of the insulin/IGF1 signalling pathway depicting the downstream activation of Akt and p44-42 MAPK through binding of insulin or IGF1 to their respective receptors. Furthermore insulin can bind to IGF1R and IGF1 to IR and IR α-subunits/ β-subunits can form heterodimers with IGF1R α-subunits /β-subunits adding further complexity at the level of the receptors. There are also numerous downstream feedback loops within this and other signalling pathways which act to regulate signalling through this pathway at multiple levels
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Fig1: Schematic of the insulin/IGF1R signalling pathway. A simplified representation of the insulin/IGF1 signalling pathway depicting the downstream activation of Akt and p44-42 MAPK through binding of insulin or IGF1 to their respective receptors. Furthermore insulin can bind to IGF1R and IGF1 to IR and IR α-subunits/ β-subunits can form heterodimers with IGF1R α-subunits /β-subunits adding further complexity at the level of the receptors. There are also numerous downstream feedback loops within this and other signalling pathways which act to regulate signalling through this pathway at multiple levels

Mentions: Insulin acts through the insulin receptor (IR), a heterotetrameric receptor tyrosine kinase (RTK) composed of two extracellular alpha sub-units, which have ligand-binding activity, and two transmembrane beta subunits that possess tyrosine kinase activity. Similarly the IGF1 receptor (IGF1R) is a RTK with a transmembrane complex which is identical to IR. Both receptors show a high degree of homology; 84 % in the tyrosine kinase domain and 45–65 % in the ligand binding domain [22]. As such both ligands can bind and activate both RTKs and therefore the interaction of these two pathways is important. Binding of insulin or IGF1 triggers autophosphorylation of tyrosine residues within the beta subunit which, in turn, leads to the recruitment of adaptor proteins, namely insulin receptor substrate (IRS)1 and IRS2, as well as Gab1 and Dos [23]. A schematic representation of IIS is shown in Fig. 1.Fig. 1


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

Garwood CJ, Ratcliffe LE, Morgan SV, Simpson JE, Owens H, Vazquez-Villaseñor I, Heath PR, Romero IA, Ince PG, Wharton SB - Mol Brain (2015)

Schematic of the insulin/IGF1R signalling pathway. A simplified representation of the insulin/IGF1 signalling pathway depicting the downstream activation of Akt and p44-42 MAPK through binding of insulin or IGF1 to their respective receptors. Furthermore insulin can bind to IGF1R and IGF1 to IR and IR α-subunits/ β-subunits can form heterodimers with IGF1R α-subunits /β-subunits adding further complexity at the level of the receptors. There are also numerous downstream feedback loops within this and other signalling pathways which act to regulate signalling through this pathway at multiple levels
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4546315&req=5

Fig1: Schematic of the insulin/IGF1R signalling pathway. A simplified representation of the insulin/IGF1 signalling pathway depicting the downstream activation of Akt and p44-42 MAPK through binding of insulin or IGF1 to their respective receptors. Furthermore insulin can bind to IGF1R and IGF1 to IR and IR α-subunits/ β-subunits can form heterodimers with IGF1R α-subunits /β-subunits adding further complexity at the level of the receptors. There are also numerous downstream feedback loops within this and other signalling pathways which act to regulate signalling through this pathway at multiple levels
Mentions: Insulin acts through the insulin receptor (IR), a heterotetrameric receptor tyrosine kinase (RTK) composed of two extracellular alpha sub-units, which have ligand-binding activity, and two transmembrane beta subunits that possess tyrosine kinase activity. Similarly the IGF1 receptor (IGF1R) is a RTK with a transmembrane complex which is identical to IR. Both receptors show a high degree of homology; 84 % in the tyrosine kinase domain and 45–65 % in the ligand binding domain [22]. As such both ligands can bind and activate both RTKs and therefore the interaction of these two pathways is important. Binding of insulin or IGF1 triggers autophosphorylation of tyrosine residues within the beta subunit which, in turn, leads to the recruitment of adaptor proteins, namely insulin receptor substrate (IRS)1 and IRS2, as well as Gab1 and Dos [23]. A schematic representation of IIS is shown in Fig. 1.Fig. 1

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Sheffield Institute for Translational Neuroscience, Department of Neuroscience, The University of Sheffield, 385a Glossop Road, Sheffield, S10 2HQ, UK.

ABSTRACT

Background: The 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).

Results: We 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.

Discussion: IIS 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.

No MeSH data available.


Related in: MedlinePlus