Abstract
1. The effects of the protein phosphatase inhibitors okadaic acid and microcystin LR on the regulation by insulin of pyruvate dehydrogenase and acetyl-CoA carboxylase have been studied in rat epididymal fat-pads and isolated cells. These inhibitors both completely blocked the phosphatase activity (against phosphorylase a) present in extracts of epididymal fat-pads, with half-maximal effects in the nanomolar range. 2. Okadaic acid treatment of pads and cells lowered the activity of acetyl-CoA carboxylase assayed in tissue extracts, both before and after treatment of the extracts with the activator, citrate. Further, okadaic acid treatment abolished the 2-3-fold difference in activity observed between extracts from control and insulin-treated tissues, assayed without prior treatment with citrate. 3. Incubation of pads with [32P]Pi, sufficient to label the intracellular pool of ATP, demonstrated that okadaic acid increased the overall phosphorylation of acetyl-CoA carboxylase on a number of distinct sites, as judged by two-dimensional mapping of tryptic peptides. These included the 'I-peptide' [Brownsey & Denton (1982) Biochem. J. 202, 77-86], the phosphorylation of which may be associated with the stimulation of the activity of the enzyme by insulin, as well as inhibitory phosphorylation sites. 4. Incubation with 1 microM-okadaic acid had no effect on the basal level of active pyruvate dehydrogenase apparent after tissue extraction, but abolished the 2-3-fold increase in this parameter which was elicited by insulin in the absence of okadaic acid. However, okadaic acid treatment did not affect the persistent increase in active pyruvate dehydrogenase levels which was apparent in mitochondria subsequently isolated from insulin-treated pads and re-incubated with an oxidizable substrate. It is concluded that the effects of okadaic acid are exerted through changes in metabolite concentrations rather than some direct action on the signalling pathway whereby insulin stimulates pyruvate dehydrogenase. 5. Microcystin LR did not mimic the effects of okadaic acid on intact cells and pads described above.