Pertussis toxin (PT) catalyzes ADP-ribosylation of G protein alpha subunits, thus preventing their role as transducers of external signals targeting metabolic pathways. In vitro, in human circulating lymphocytes insulin at physiological concentrations (5 microU/ml) determines sharp activation of pyruvate dehydrogenase (PDH), the rate limiting enzyme in glucose oxidative breakdown. This study evaluates whether the above-described effects of insulin over PDH are mediated through G proteins. Human circulating lymphocytes (six samples from different donors) were exposed to insulin (5 microU/ml), PT (1-2 micrograms/ml) or PT-9K, a mutated PT void of catalytic activity (1-10 micrograms/ml), and to insulin in combination with the two toxins, and then assessed for PDH activity. Plasma membranes from cells incubated with and without PT or PT-9K were subjected to ADP-ribosylation in the presence of [32P] NAD+ and activated PT. In circulating lymphocytes exposed to PT alone, or in combination with insulin, PDH activity falls significantly below basal values (P