Activation and mitochondrial translocation of protein kinase Cdelta are necessary for insulin stimulation of pyruvate dehydrogenase complex activity in muscle and liver cells.
In L6 skeletal muscle cells and immortalized hepatocytes, insulin induced a 2-fold increase in the activity of the pyruvate dehydrogenase (PDH) complex. This effect was almost completely blocked by the protein kinase C (PKC) delta inhibitor Rottlerin and by PKCdelta antisense oligonucleotides. At variance, overexpression of wild-type PKCdelta or of an active PKCdelta mutant induced PDH complex activity in both L6 and liver cells. Insulin stimulation of the activity of the PDH complex was accompanied by a 2.5-fold increase in PDH phosphatases 1 and 2 (PDP1/2) activity with no change in the activity of PDH kinase. PKCdelta antisense blocked insulin activation of PDP1/2, the same as with PDH. In insulin-exposed cells, PDP1/2 activation was paralleled by activation and mitochondrial translocation of PKCdelta, as revealed by cell subfractionation and confocal microscopy studies. The mitochondrial translocation of PKCdelta, like its activation, was prevented by Rottlerin. In extracts from insulin-stimulated cells, PKCdelta co-precipitated with PDP1/2. PKCdelta also bound to PDP1/2 in overlay blots, suggesting that direct PKCdelta-PDP interaction may occur in vivo as well. In intact cells, insulin exposure determined PDP1/2 phosphorylation, which was specifically prevented by PKCdelta antisense. PKCdelta also phosphorylated PDP in vitro, followed by PDP1/2 activation. Thus, in muscle and liver cells, insulin causes activation and mitochondrial translocation of PKCdelta, accompanied by PDP phosphorylation and activation. These events are necessary for insulin activation of the PDH complex in these cells.
Caruso
M
M
Dipartimento di Biologia e Patologia Cellulare e Molecolare and Centro di Endocrinologia ed Oncologia Sperimentale del CNR, Federico II University of Naples, 80131 Naples, Italy.
Maitan
M A
MA
Bifulco
G
G
Miele
C
C
Vigliotta
G
G
Oriente
F
F
Formisano
P
P
Beguinot
F
F
D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008856:Microscopy, Fluorescence; D018482:Muscle, Skeletal / Q000201:enzymology; D009132:Muscles / Q000166:cytology / Q000201:enzymology*; D009154:Mutation; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism*; D051745:Protein Kinase C-delta; D021381:Protein Transport; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D051381:Rats; D013997:Time Factors; D014162:Transfection
D004791:Enzyme Inhibitors; D007328:Insulin; D007527:Isoenzymes; D020033:Protein Isoforms; D011768:Pyruvate Dehydrogenase Complex; C497260:Prkcd protein, rat; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta