STIMULATORY EFFECT BY INSULIN ON INCORPORATION OF 32P RADIOACTIVE INORGANIC PHOSPHATE INTO INTRACELLULAR INORGANIC PHOSPHATE ADENINE NUCLEOTIDES AND GUANINE NUCLEOTIDES OF INTACT ISOLATED RAT DIAPHRAGM

The aim of these investigations has been to contribute to an understanding of the mechanism of action of insulin in promoting increased turn over of32P inorganic phosphate into ATP and other mononucleotides in the rat diaphragm. During incubation of the intact rat diaphragm in Krebs Ringer bicarbonate medium without addition of substrate, no effect of insulin on the content of adenine nucleotides and guanine nucleotides including cyclic 3′,5′ AMP and cyclic 3′,5′ GMP has been observed. When32P radioactive inorganic phosphate was present in the medium, insulin increased the specific activity of intracellular inorganic phosphate and promoted an increased incorporation of32P into ATP and GTP. The effect on ADP was less pronounced. It has been shown that these effects of insulin depend upon the ionic composition of the medium and on ion membrane-transport. The insulin-promoted increase of specific activity of intracellular inorganic phosphate and of increased32P labelling of ATP (and GTP) was to a great extent inhibited by ouabain. During incubation in media where sodium chloride had been iso-osmotically replaced by potassium chloride or choline chloride, the above mentioned effects by insulin were abolished. The results indicate that the increased labelling of ATP (and GTP) promoted by insulin can be primarily attributed to events occuring at the cell membrane. The increased specific activity of intracellular inorganic phosphate in the presence of insulin can be explained by increased uptake of32P inorganic phosphate from the medium. Apparently the increased32P labelling of ATP (and GTP) occur as a secondary consequence. It is suggested that this insulin effect is primarily concerned with an effect on membrane enzymes, particularly the Na+, K+, Mg2+ activated ATP-ase. © 1969 Springer-Verlag.