Inhibition of intracellular proteolysis by insulin in isolated rat hepatocytes. Possible role of internalized hormone.
The mechanism of insulin's action upon intracellular proteolysis in isolated hepatocytes was studied. At 37 degrees C insulin inhibited intracellular degradation of intracellular proteins in a dose-dependent manner. A maximal 40% inhibition of intracellular proteolysis was achieved at an insulin concentration of 500 ng/ml with a half-maximal inhibition observed at 2.5 ng/ml of insulin. Insulin inhibited intracellular proteolysis both in the presence and in the absence of amino acids in the incubation mixture. Low concentrations of trypsin (10 micrograms/ml) mimicked insulin's effect upon glucose incorporation into glycogen, but not on intracellular proteolysis. Four protease inhibitors (phenylmethylsulfonyl fluoride (0.5 mM), p-nitrophenyl-p-guanidinobenzoate (0.25 mM), p-tosyl-L-arginine methyl ester (1 mM), and N alpha-p-tosyl-L-lysine chloromethyl ketone (1 mM) blocked the stimulatory effect of insulin upon [14C]glucose incorporation into glycogen, but did not affect the inhibitory action of insulin upon intracellular proteolysis. These results suggest that the mechanism of insulin's action upon intracellular proteolysis differs from that involved in stimulation of glycogenesis. Low temperature (15 degrees C) and short time exposure (10 min) of the hepatocytes to insulin eliminated the inhibitory effect of insulin on intracellular proteolysis. Similarly, insulin's effect on intracellular proteolysis was eliminated by dansylcadaverine, a transglutaminase inhibitor that blocked insulin internalization. In contrast, dansylcadaverine had no effect on insulin's ability to stimulate [14C]glucose incorporation into glycogen. These experiments strongly suggest the necessity of insulin internalization for its inhibitory effect on endogenous protein degradation.
D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D003080:Cold Temperature; D004305:Dose-Response Relationship, Drug; D007328:Insulin / Q000378:metabolism*; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D007328:Insulin; D011480:Protease Inhibitors; D011506:Proteins; D014357:Trypsin