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Protein kinase CĪµ modulates insulin receptor localization and trafficking in mouse embryonic fibroblasts.

Pedersen DJ, Diakanastasis B, Stƶckli J, Schmitz-Peiffer C - PLoS ONE (2013)

Bottom Line: This was in part due to reduced insulin uptake by hepatocytes and insulin clearance, which enhanced insulin availability.PKCε(-/-) MEFs exhibited reduced insulin uptake which was associated with decreased insulin receptor phosphorylation, while downstream signalling through IRS-1 and Akt was unaffected.These alterations in insulin receptor trafficking were associated with reduced expression of CEACAM1, a receptor substrate previously shown to modulate insulin clearance.

View Article: PubMed Central - PubMed

Affiliation: Diabetes and Obesity Program, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.

ABSTRACT
We have previously shown that deletion of protein kinase C epsilon (PKCĪµ) in mice results in protection against glucose intolerance caused by a high fat diet. This was in part due to reduced insulin uptake by hepatocytes and insulin clearance, which enhanced insulin availability. Here we employed mouse embryonic fibroblasts (MEFs) derived from wildtype (WT) and PKCĪµ-deficient (PKCĪµ(-/-)) mice to examine this mechanistically. PKCĪµ(-/-) MEFs exhibited reduced insulin uptake which was associated with decreased insulin receptor phosphorylation, while downstream signalling through IRS-1 and Akt was unaffected. Cellular fractionation demonstrated that PKCĪµ deletion changed the localization of the insulin receptor, a greater proportion of which co-fractionated with flotillin-1, a marker of membrane microdomains. Insulin stimulation resulted in redistribution of the receptor in WT cells, while this was markedly reduced in PKCĪµ(-/-) cells. These alterations in insulin receptor trafficking were associated with reduced expression of CEACAM1, a receptor substrate previously shown to modulate insulin clearance. Virally-mediated reconstitution of PKCĪµ in MEFs increased CEACAM1 expression and partly restored the sensitivity of the receptor to insulin-stimulated redistribution. These data indicate that PKCĪµ can affect insulin uptake in MEFs through promotion of receptor-mediated endocytosis, and that this may be mediated by regulation of CEACAM1 expression.

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Insulin signalling in MEFs after reconstitution of PKCĪµ.MEFs were infected with recombinant adenoviruses and stimulated with insulin as in Fig. 4. Lysates were analysed by SDS-PAGE and immunoblotting for phospho-Y1162/1163 (A), phospho-Y612 (B) and phospho-S473 (C) as for Fig. 2.
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pone-0058046-g005: Insulin signalling in MEFs after reconstitution of PKCĪµ.MEFs were infected with recombinant adenoviruses and stimulated with insulin as in Fig. 4. Lysates were analysed by SDS-PAGE and immunoblotting for phospho-Y1162/1163 (A), phospho-Y612 (B) and phospho-S473 (C) as for Fig. 2.

Mentions: In contrast to these effects on insulin receptor redistribution, restoration of PKCĪµ in MEFs did not restore insulin receptor phosphorylation to levels observed in WT cells (Fig. 5A) and did not affect downstream signalling at the level of IRS-1 or Akt (Fig. 5B,C).


Protein kinase CĪµ modulates insulin receptor localization and trafficking in mouse embryonic fibroblasts.

Pedersen DJ, Diakanastasis B, Stƶckli J, Schmitz-Peiffer C - PLoS ONE (2013)

Insulin signalling in MEFs after reconstitution of PKCĪµ.MEFs were infected with recombinant adenoviruses and stimulated with insulin as in Fig. 4. Lysates were analysed by SDS-PAGE and immunoblotting for phospho-Y1162/1163 (A), phospho-Y612 (B) and phospho-S473 (C) as for Fig. 2.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585804&req=5

pone-0058046-g005: Insulin signalling in MEFs after reconstitution of PKCĪµ.MEFs were infected with recombinant adenoviruses and stimulated with insulin as in Fig. 4. Lysates were analysed by SDS-PAGE and immunoblotting for phospho-Y1162/1163 (A), phospho-Y612 (B) and phospho-S473 (C) as for Fig. 2.
Mentions: In contrast to these effects on insulin receptor redistribution, restoration of PKCĪµ in MEFs did not restore insulin receptor phosphorylation to levels observed in WT cells (Fig. 5A) and did not affect downstream signalling at the level of IRS-1 or Akt (Fig. 5B,C).

Bottom Line: This was in part due to reduced insulin uptake by hepatocytes and insulin clearance, which enhanced insulin availability.PKCε(-/-) MEFs exhibited reduced insulin uptake which was associated with decreased insulin receptor phosphorylation, while downstream signalling through IRS-1 and Akt was unaffected.These alterations in insulin receptor trafficking were associated with reduced expression of CEACAM1, a receptor substrate previously shown to modulate insulin clearance.

View Article: PubMed Central - PubMed

Affiliation: Diabetes and Obesity Program, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.

ABSTRACT
We have previously shown that deletion of protein kinase C epsilon (PKCĪµ) in mice results in protection against glucose intolerance caused by a high fat diet. This was in part due to reduced insulin uptake by hepatocytes and insulin clearance, which enhanced insulin availability. Here we employed mouse embryonic fibroblasts (MEFs) derived from wildtype (WT) and PKCĪµ-deficient (PKCĪµ(-/-)) mice to examine this mechanistically. PKCĪµ(-/-) MEFs exhibited reduced insulin uptake which was associated with decreased insulin receptor phosphorylation, while downstream signalling through IRS-1 and Akt was unaffected. Cellular fractionation demonstrated that PKCĪµ deletion changed the localization of the insulin receptor, a greater proportion of which co-fractionated with flotillin-1, a marker of membrane microdomains. Insulin stimulation resulted in redistribution of the receptor in WT cells, while this was markedly reduced in PKCĪµ(-/-) cells. These alterations in insulin receptor trafficking were associated with reduced expression of CEACAM1, a receptor substrate previously shown to modulate insulin clearance. Virally-mediated reconstitution of PKCĪµ in MEFs increased CEACAM1 expression and partly restored the sensitivity of the receptor to insulin-stimulated redistribution. These data indicate that PKCĪµ can affect insulin uptake in MEFs through promotion of receptor-mediated endocytosis, and that this may be mediated by regulation of CEACAM1 expression.

Show MeSH
Related in: MedlinePlus