ucn-1028-c and 12-deoxyphorbolphenylacetate

Glucose transport in skeletal muscle can be stimulated by insulin and also by contractions and hypoxia. Activation of protein kinase C (PKC) stimulates glucose transport in muscle and other insulin-responsive cells. This study was performed to determine if the diacylglycerol (DAG)/phorbol ester-sensitive PKC isoforms participate in insulin and/or hypoxia-stimulated glucose transport in skeletal muscle. The phorbol ester 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA) induced a three- to fourfold increase in glucose transport in rat epitrochlearis muscle. The effects of dPPA on glucose transport and on cell surface GLUT-4 were completely additive to the maximal effects of insulin or hypoxia. Phorbol ester treatment induced 5- to 10-fold increases in phosphorylation of the myristoylated alanine-rich C kinase substrate protein in muscle, whereas insulin and hypoxia had negligible effects. Calphostin C, an inhibitor of DAG-sensitive PKC isoforms, decreased glucose transport stimulation by dPPA but not by insulin or hypoxia. These results provide evidence that activation of DAG/phorbol ester-sensitive PKCs is not involved in the pathways by which either insulin or hypoxia stimulates muscle glucose transport. They also show that activation of this group of PKCs increases glucose transport by a mechanism that is independent of and additive to the effects of insulin or hypoxia.

Topics: Animals; Biological Transport; Enzyme Activation; Enzyme Inhibitors; Glucose; Glucose Transporter Type 4; Hypoxia; Intracellular Signaling Peptides and Proteins; Inulin; Isoenzymes; Kinetics; Male; Membrane Proteins; Monosaccharide Transport Proteins; Muscle Contraction; Muscle Proteins; Muscle, Skeletal; Myristoylated Alanine-Rich C Kinase Substrate; Naphthalenes; Phorbol Esters; Protein Kinase C; Proteins; Rats; Rats, Wistar; Tetradecanoylphorbol Acetate

The human colon cancer cell lines HCT 116 (poorly differentiated) and GEO (well differentiated) express the mitogenic peptide transforming growth factor alpha (TGF-alpha). The secretion of TGF-alpha was enhanced by phorbol 12-myristate 13-acetate (PMA), indicating the possible role of protein kinase C (PKC) in the formation of mature TGF-alpha. Cells were metabolically labeled with 35S-cysteine and the formation of the mature 6 kDa TGF-alpha polypeptide from the 17 kDa pro-TGF-alpha precursor was determined. The conversion of pro-TGF-alpha was complete in 2-4 hr with the HCT 116 cells showing faster kinetics of TGF-alpha formation than GEO cells. HCT 116 cells secreted more TGF-alpha than GEO cells and the rate and extent of formation of TGF-alpha was enhanced by PMA in both cell lines. The expression of several PKC isozymes by HCT 116 and GEO cells was examined by immunoblotting. The expression of all isozymes examined was higher in HCT 116 cells compared with GEO cells. Calphostin C, an inhibitor of PKC, reduced the enzyme activity and significantly inhibited the PMA-induced secretion of TGF-alpha by both cell lines. Two agonists of PKC that act on specific PKC isozymes, thymeleatoxin and 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), stimulated the release of TGF-alpha into the medium to the same extent as PMA. Since dPPA has been reported to stimulate PKC-beta 1 specifically, our results suggest a potential role for PKC-beta in the processing of pro-TGF-alpha by these 2 human colon carcinoma cell lines.

Topics: Colonic Neoplasms; Humans; Isoenzymes; Naphthalenes; Phorbol Esters; Protein Kinase C; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha; Tumor Cells, Cultured