Limits...
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

Insulin/IGF1 pathway characterisation in astrocytes. Immunoblots of astrocytes cultured in either serum containing or serum deprived media with additional supplementation from either a recombinant human 1 μM insulin or b 11.2nM recombinant human IGF1. Respresentative images from blots probed with antibodies against IRβ, IGF1Rβ, IRS1, IRS2, pAkt, Total Akt, p44/42 MAPK are shown. *α-tubulin was used as a loading control for blots and a representative loading control is shown. Molecular weight markers are indicated (kDa)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Insulin/IGF1 pathway characterisation in astrocytes. Immunoblots of astrocytes cultured in either serum containing or serum deprived media with additional supplementation from either a recombinant human 1 μM insulin or b 11.2nM recombinant human IGF1. Respresentative images from blots probed with antibodies against IRβ, IGF1Rβ, IRS1, IRS2, pAkt, Total Akt, p44/42 MAPK are shown. *α-tubulin was used as a loading control for blots and a representative loading control is shown. Molecular weight markers are indicated (kDa)

Mentions: Cultured human astrocytes expressed the receptors IR and IGF1R, the adaptor proteins IRS1 and IRS2 and the downstream signalling kinases Akt and p44/42 MAPK (ERK1/2) in both the commercially obtained Sciencell astrocytes and astrocytes derived from temporal lobe resections (see Fig. 1 for schematic representation of the insulin/IGF1 signalling pathway). There was no impact of serum deprivation or insulin/IGF1 supplementation on either the pre- or mature receptors (Fig. 3 and Additional file 3: Figure S3). Amounts of the adaptor protein, IRS1, remained consistent although the detected molecular weight increased in the presence of serum or when astrocytes were supplemented with recombinant insulin or IGF1 (Fig. 3 and Additional file 3: Figure S3), indicating a change in the phosphorylation status of the protein; the complex regulation of IIS is achieved through phosphorylation of IRS1 on tyrosine and serine residues. A shift in IRS2 was also observed by immunoblotting in response to stimulation with ligand, indicating that insulin/IGF signalling is mediated by both downstream adaptors proteins, IRS1 and IRS2 (Fig. 3).Fig. 3


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)

Insulin/IGF1 pathway characterisation in astrocytes. Immunoblots of astrocytes cultured in either serum containing or serum deprived media with additional supplementation from either a recombinant human 1 μM insulin or b 11.2nM recombinant human IGF1. Respresentative images from blots probed with antibodies against IRβ, IGF1Rβ, IRS1, IRS2, pAkt, Total Akt, p44/42 MAPK are shown. *α-tubulin was used as a loading control for blots and a representative loading control is shown. Molecular weight markers are indicated (kDa)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Insulin/IGF1 pathway characterisation in astrocytes. Immunoblots of astrocytes cultured in either serum containing or serum deprived media with additional supplementation from either a recombinant human 1 μM insulin or b 11.2nM recombinant human IGF1. Respresentative images from blots probed with antibodies against IRβ, IGF1Rβ, IRS1, IRS2, pAkt, Total Akt, p44/42 MAPK are shown. *α-tubulin was used as a loading control for blots and a representative loading control is shown. Molecular weight markers are indicated (kDa)
Mentions: Cultured human astrocytes expressed the receptors IR and IGF1R, the adaptor proteins IRS1 and IRS2 and the downstream signalling kinases Akt and p44/42 MAPK (ERK1/2) in both the commercially obtained Sciencell astrocytes and astrocytes derived from temporal lobe resections (see Fig. 1 for schematic representation of the insulin/IGF1 signalling pathway). There was no impact of serum deprivation or insulin/IGF1 supplementation on either the pre- or mature receptors (Fig. 3 and Additional file 3: Figure S3). Amounts of the adaptor protein, IRS1, remained consistent although the detected molecular weight increased in the presence of serum or when astrocytes were supplemented with recombinant insulin or IGF1 (Fig. 3 and Additional file 3: Figure S3), indicating a change in the phosphorylation status of the protein; the complex regulation of IIS is achieved through phosphorylation of IRS1 on tyrosine and serine residues. A shift in IRS2 was also observed by immunoblotting in response to stimulation with ligand, indicating that insulin/IGF signalling is mediated by both downstream adaptors proteins, IRS1 and IRS2 (Fig. 3).Fig. 3

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