Alpha-lipoic acid as a directly binding activator of the insulin receptor: protection from hepatocyte apoptosis.
ABSTRACT Alpha-lipoic acid has cytoprotective potential which has previously been explained by its antioxidant properties. The aim of this study was to assess LA-induced-specific cytoprotective signalling pathways in hepatocytes.
Apoptosis of rat hepatocytes was induced by actinomycinD/TNF-alpha. Caspase-3-like activity was determined by a fluorometric; LDH by an enzymatic assay; and phosphorylation of the insulin receptor, Akt, and Bad by Western blot (after immunoprecipitation). Protein kinase and insulin receptor activities were measured by in vitro phosphorylation. Computer modeling studies were performed by using the program GRID.
Alpha-lipoic acid decreased actinomycinD/TNF-alpha-induced apoptosis, as did the antioxidants Trolox and N-acetylcysteine. The activation of PI3-kinase/Akt involving phosphorlyation of Bad markedly contributed to the cytoprotective action of alpha-lipoic acid. Alpha-lipoic acid but not other antioxidants protected against actinomycinD/TNF-alpha-induced apoptosis via phosphorylation of the insulin receptor. Computer modeling studies revealed a direct binding site for alpha-lipoic acid at the tyrosine kinase domain of the insulin receptor, suggesting a stabilizing function in loop A that is involved in ATP binding. Treatment of immunoprecipitated insulin receptor with LA induced substrate phosphorylation.
Alpha-lipoic acid mediates its antiapoptotic action via activation of the insulin receptor/PI3-kinase/Akt pathway. We show for the first time a direct binding site for alpha-lipoic acid at the insulin receptor tyrosine kinase domain, which might make alpha-lipoic acid a model substance for the development of insulin mimetics.
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ABSTRACT: Activation of caspases is required in Fas receptor mediated apoptosis. Maintenance of a reducing environment inside the cell has been suggested to be necessary for caspase activity during apoptosis. We explored the possibility to potentiate Fas mediated killing of tumor cells by alpha-lipoic acid (LA), a redox-active drug and nutrient that is intracellularly reduced to a potent reductant dihydrolipoic acid. Treatment of cells with 100 microM LA for 72 h markedly potentiated Fas-mediated apoptosis of leukemic Jurkat cells but not that of peripheral blood lymphocytes from healthy humans. In Jurkat, Fas activation was followed by rapid loss of cell thiols, decreased mitochondrial membrane potential, increased [Ca2+]i and increased PKC activity; all these responses were potentiated in LA pretreated cells. PKCdelta played an important role in mediating the effect of LA on Fas-mediated cell death. In response to Fas activation LA treatment potentiated caspase 3 activation by over 100%. The ability of LA to potentiate Fas mediated killing of leukemic cells was abrogated by a caspase 3 inhibitor suggesting that increased caspase 3 activity in LA-treated Fas-activated cells played an important role in potentiating cell death. This work provides first evidence showing that inducible caspase 3 activity may be pharmacologically up-regulated by reducing agents such as dihydrolipoic acid.Cell Death and Differentiation 06/1999; 6(5):481-91. · 8.37 Impact Factor
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ABSTRACT: The tyrosine kinase domain of the insulin receptor is subject to autoinhibition in the unphosphorylated basal state via steric interactions involving the activation loop. A mutation in the activation loop designed to relieve autoinhibition, Asp-1161 --> Ala, substantially increases the ability of the unphosphorylated kinase to bind ATP. The crystal structure of this mutant in complex with an ATP analog has been determined at 2.4-A resolution. The structure shows that the active site is unobstructed, but the end of the activation loop is disordered and therefore the binding site for peptide substrates is not fully formed. In addition, Phe-1151 of the protein kinase-conserved DFG motif, at the beginning of the activation loop, hinders closure of the catalytic cleft and proper positioning of alpha-helix C for catalysis. These results, together with viscometric kinetic measurements, suggest that peptide substrate binding induces a reconfiguration of the unphosphorylated activation loop prior to the catalytic step. The crystallographic and solution studies provide new insights into the mechanism by which the activation loop controls phosphoryl transfer as catalyzed by the insulin receptor.Journal of Biological Chemistry 03/2001; 276(13):10049-55. · 4.65 Impact Factor