Catecholamine stimulation of hepatic gluconeogenesis at the site between pyruvate and phosphoenolpyruvate

Phosphoenolpyruvate carboxykinase has been implicated by Rognstad (Rognstad, R. (1979) J. Biol. Chem. 254, 1875-1878) as the rate-limiting step for gluconeogenesis from lactate on the basis of a linear Dixon plot (reciprocal rate of gluconeogenesis versus concentration of inhibitor, mercaptopicolinate). We have confirmed this result with isolated hepatocytes incubated in the absence, but not the presence, of bovine serum albumin. Nonlinear plots are likely the result of mercaptopicolinate binding to the albumin. Both norepinephrine and dibutyryl cyclic AMP decreased the slopes and intercepts of the Dixon plots, but a linear relationship was still obtained. When aminooxyacetate inhibited transaminase reactions sufficiently to depress gluconeogenesis, the resulting mercaptopicolinate inhibition plot was still linear in the presence or absence of norepinephrine. Thus, linearity in the Dixon plot does not assure that the enzyme at the site of inhibition is the rate-limiting step for a pathway. Flux through phosphoenolpyruvate carboxykinase does not appear to be hormonally regulated by changes in oxalacetate concentration since this compound was unchanged by norepinephrine or dibutyryl cyclic AMP. Ca2+ enhanced norepinephrine stimulation of gluconeogenesis from asparagine and glutamine and of ureogenesis from glutamine, indicating both mitochondrial and cytosolic sites of action for this hormone. The effects of catecholamines and cyclic AMP were most clearly distinguished by their influence on glutamate concentration when glutamine was the substrate. Dibutyryl cyclic AMP increased, but norepinephrine decreased glutamate. It is possible that decreased glutamate concentration is a reflection of a catecholamine-directed oxidation of mitochondrial NADPH.