Extracellular phosphate requirement for insulin action on isolated rat hepatocytes

Isolated rat hepatocytes were prepared in KHB buffer, pH 7.4; were centrifuged and washed twice in KHB buffer containing various amounts of phosphate and calcium; and were incubated at 30 degrees in the presence of tracer [2,3-14C]succinate and a 0.5 mM concentration of each of the 20 natural amino acids. Hepatocytes washed and incubated in KHB buffer containing less than 0.1 mM phosphate failed to show any insulin stimulation of [2,3-14C]succinate oxidation or protein incorporation of tracer carbons. The absence or presence of extracellular phosphate did not alter the specific activity of 32P-adenine nucleotides; they remained the same in the presence or absence of insulin. The maximal insulin stimulatory effect on succinate oxidation and tracer incorporation into protein was observed in the presence of 1.18 mM phosphate and 1.9 mM calcium ion. The lack of external phosphate did not prevent the stimulation of succinate oxidation by either glucagon on epinephrine, whereas removal of calcium from the medium abolished their hormonal effects. The lack of medium calcium also prevented the insulin stimulation of succinate oxidation and protein synthesis. Our data indicate that a diminished insulin responsiveness in hypophosphatemic patients may be due to the insensitivity of mitochondria to insulin in the hypophosphatemic state.