RESUMO
Protein kinase C epsilon (PKCÉ) activation in the liver is proposed to inhibit insulin action through phosphorylation of the insulin receptor. Here, however, we demonstrated that global, but not liver-specific, deletion of PKCÉ in mice protected against diet-induced glucose intolerance and insulin resistance. Furthermore, PKCÉ-dependent alterations in insulin receptor phosphorylation were not detected. Adipose-tissue-specific knockout mice did exhibit improved glucose tolerance, but phosphoproteomics revealed no PKCÉ-dependent effect on the activation of insulin signaling pathways. Altered phosphorylation of adipocyte proteins associated with cell junctions and endosomes was associated with changes in hepatic expression of several genes linked to glucose homeostasis and lipid metabolism. The primary effect of PKCÉ on glucose homeostasis is, therefore, not exerted directly in the liver as currently posited, and PKCÉ activation in this tissue should be interpreted with caution. However, PKCÉ activity in adipose tissue modulates glucose tolerance and is involved in crosstalk with the liver.
Assuntos
Tecido Adiposo/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Fígado/metabolismo , Proteína Quinase C-épsilon/fisiologia , Animais , Dieta Hiperlipídica , Técnicas de Inativação de Genes , Intolerância à Glucose , Resistência à Insulina , Metabolismo dos Lipídeos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase C-épsilon/genéticaRESUMO
AIMS/HYPOTHESIS: An accumulation of ceramides has been implicated in the generation of insulin resistance in skeletal muscle upon an oversupply of fatty acid. Different ceramide species are generated through the actions of ceramide synthases (CerSs), which incorporate specific acyl side chains. We tested whether particular CerS isoforms promoted insulin resistance through the generation of more inhibitory ceramide species, thus representing potential targets for intervention. METHODS: CerS isoforms CerS1, CerS2, CerS4, CerS5 and CerS6 were overexpressed in L6 myotubes using adenovirus, and cells were treated with palmitate and stimulated with insulin. Alternatively, CerS isoforms were knocked down using siRNAs. Sphingolipids were examined by mass spectrometry and tracer incorporation. Phosphorylation of IRS1 and Akt was measured by immunoblotting, while glucose disposal was assessed by measuring GLUT4 translocation and the incorporation of [(14)C]glucose into glycogen. RESULTS: Palmitate treatment increased the levels of several ceramides but reduced the levels of sphingomyelins, while insulin had no effect. The fatty acid also inhibited insulin-stimulated Akt phosphorylation and glycogen synthesis. Overexpression of CerS isoforms increased specific ceramides. Unexpectedly, the overexpression of CerS1 and CerS6 promoted insulin action, while no isoform had inhibitory effects. CerS6 knockdown had effects reciprocal to those of CerS6 overexpression. CONCLUSIONS/INTERPRETATION: Palmitate may increase intracellular ceramide levels through sphingomyelin hydrolysis as well as de novo synthesis, but no particular species were implicated in the generation of insulin resistance. The modulation of ceramides through an alteration of CerS expression does not affect the action of insulin in the same way as ceramide generation by palmitate treatment. Conversely, certain isoforms promote insulin action, indicating the importance of ceramides in cell function.
Assuntos
Ceramidas/metabolismo , Resistência à Insulina , Insulina/metabolismo , Proteínas de Membrana/biossíntese , Músculo Esquelético/metabolismo , Oxirredutases/metabolismo , Palmitatos/farmacologia , Ceramidas/biossíntese , Glucose/metabolismo , Glicogênio/metabolismo , Humanos , Espectrometria de Massas , Fibras Musculares Esqueléticas , Músculo Esquelético/citologia , Palmitatos/metabolismo , Fosforilação , Isoformas de Proteínas/metabolismo , Esfingolipídeos/metabolismoRESUMO
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.