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

Pedersen DJ, Diakanastasis B, Stöckli 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 (PKCε) 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 PKCε-deficient (PKCε(-/-)) mice to examine this mechanistically. PKCε(-/-) 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 PKCε 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 PKCε(-/-) 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 PKCε in MEFs increased CEACAM1 expression and partly restored the sensitivity of the receptor to insulin-stimulated redistribution. These data indicate that PKCε 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 receptor subcellular fractionation in WT and PKCε−/− MEFs.A. Cell extracts were separated by centrifugation using a continuous Opti-Prep density gradient. The presence of the markers flotillin1, EEA1 and pan-cadherin were determined in each fraction by immunoblotting and the means from two independent experiments shown. B. WT and PKCε−/− MEFs were serum starved for 2 h and stimulated with 100 nM insulin for 2 or 10 min. Cells were extracted by nitrogen cavitation and fractionated as in A. Insulin receptor localisation was determined by immunoblotting (B) and mean levels in each fraction calculated from densitometry of 2 independent experiments (C).
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pone-0058046-g003: Insulin receptor subcellular fractionation in WT and PKCε−/− MEFs.A. Cell extracts were separated by centrifugation using a continuous Opti-Prep density gradient. The presence of the markers flotillin1, EEA1 and pan-cadherin were determined in each fraction by immunoblotting and the means from two independent experiments shown. B. WT and PKCε−/− MEFs were serum starved for 2 h and stimulated with 100 nM insulin for 2 or 10 min. Cells were extracted by nitrogen cavitation and fractionated as in A. Insulin receptor localisation was determined by immunoblotting (B) and mean levels in each fraction calculated from densitometry of 2 independent experiments (C).

Mentions: We next explored whether the basal and insulin-stimulated cellular localization of the insulin receptor was altered by PKCε deletion. Subcellular fractionation of MEFs was performed by iodixanol density gradient centrifugation and the presence of subcellular marker proteins used to characterize the fractions. Flotillin1, a marker of membrane microdomains, was enriched in fractions 3–4 (Fig. 3A, peak 1). Early endosomal antigen 1 (EEA1), a marker for early endosomes, was most highly enriched within fractions 5–6 (Fig. 3A, peak 2). Pan-cadherin was employed as a general plasma membrane marker and exhibited a relatively broad distribution, with major peaks at fraction 11 as well as fractions 17–19 (Fig. 3A, peak 3). The distribution of these markers was similar in both WT and PKCε−/− MEFs. There was however, a striking difference in localization of the insulin receptor in the basal state (Fig. 3B,C), with the receptor of PKCε−/− MEFs found to be highly enriched in peak 1, coinciding with the flotillin1-enriched fractions of the gradient, compared to a broader distribution observed in WT MEFs. The insulin-induced redistribution of the receptor was also perturbed in PKCε−/− MEFs. Upon insulin treatment, the receptor became relatively enriched in fractions 11–14 from WT cells especially after 10 min, whereas this was not observed in fractions from PKCε−/− cells (Fig. 3B,C). This is consistent with reduced internalization of the insulin receptor in the absence of PKCε.


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

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

Insulin receptor subcellular fractionation in WT and PKCε−/− MEFs.A. Cell extracts were separated by centrifugation using a continuous Opti-Prep density gradient. The presence of the markers flotillin1, EEA1 and pan-cadherin were determined in each fraction by immunoblotting and the means from two independent experiments shown. B. WT and PKCε−/− MEFs were serum starved for 2 h and stimulated with 100 nM insulin for 2 or 10 min. Cells were extracted by nitrogen cavitation and fractionated as in A. Insulin receptor localisation was determined by immunoblotting (B) and mean levels in each fraction calculated from densitometry of 2 independent experiments (C).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585804&req=5

pone-0058046-g003: Insulin receptor subcellular fractionation in WT and PKCε−/− MEFs.A. Cell extracts were separated by centrifugation using a continuous Opti-Prep density gradient. The presence of the markers flotillin1, EEA1 and pan-cadherin were determined in each fraction by immunoblotting and the means from two independent experiments shown. B. WT and PKCε−/− MEFs were serum starved for 2 h and stimulated with 100 nM insulin for 2 or 10 min. Cells were extracted by nitrogen cavitation and fractionated as in A. Insulin receptor localisation was determined by immunoblotting (B) and mean levels in each fraction calculated from densitometry of 2 independent experiments (C).
Mentions: We next explored whether the basal and insulin-stimulated cellular localization of the insulin receptor was altered by PKCε deletion. Subcellular fractionation of MEFs was performed by iodixanol density gradient centrifugation and the presence of subcellular marker proteins used to characterize the fractions. Flotillin1, a marker of membrane microdomains, was enriched in fractions 3–4 (Fig. 3A, peak 1). Early endosomal antigen 1 (EEA1), a marker for early endosomes, was most highly enriched within fractions 5–6 (Fig. 3A, peak 2). Pan-cadherin was employed as a general plasma membrane marker and exhibited a relatively broad distribution, with major peaks at fraction 11 as well as fractions 17–19 (Fig. 3A, peak 3). The distribution of these markers was similar in both WT and PKCε−/− MEFs. There was however, a striking difference in localization of the insulin receptor in the basal state (Fig. 3B,C), with the receptor of PKCε−/− MEFs found to be highly enriched in peak 1, coinciding with the flotillin1-enriched fractions of the gradient, compared to a broader distribution observed in WT MEFs. The insulin-induced redistribution of the receptor was also perturbed in PKCε−/− MEFs. Upon insulin treatment, the receptor became relatively enriched in fractions 11–14 from WT cells especially after 10 min, whereas this was not observed in fractions from PKCε−/− cells (Fig. 3B,C). This is consistent with reduced internalization of the insulin receptor in the absence of PKCε.

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 (PKCε) 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 PKCε-deficient (PKCε(-/-)) mice to examine this mechanistically. PKCε(-/-) 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 PKCε 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 PKCε(-/-) 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 PKCε in MEFs increased CEACAM1 expression and partly restored the sensitivity of the receptor to insulin-stimulated redistribution. These data indicate that PKCε 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