G proteins and regulation of pyruvate dehydrogenase activity by insulin in human circulating lymphocytes

Pertussis toxin (PT) catalyzes ADP-ribosylation of G protein α subunits, thus preventing their role as transducers of external signals targeting metabolic pathways. In vitro, in human circulating lymphocytes insulin at physiological concentrations (5 μU/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 ( 5μU/ml), PT (1-2 μg/ml or PT-9K, a mutated PT void of catalytic activity (1-10 μg/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 < 0.001); PT-9K instead has no effect on basal or on insulin-stimulated PDH activity. ADP-ribosylation of a plasma membrane component with apparent molecular mass (42 kDa) comparable to that of the Gi (inhibitory) protein A subunit takes place in cells exposed to PT but not in those exposed to PT-9K. In human circulating lymphocytes Gi proteins or Gi protein-like components appear to be involved in preserving basal PDH activity as well as in the mechanism by which insulin exerts its control over PDH.