Quantitative dissociation of glucose transport stimulation and insulin receptor tyrosine kinase activation in isolated adipocytes with a covalent insulin dimer (B29,B29'-suberoyl-insulin).

The covalent insulin dimer B29,B29'-suberoyl-insulin was investigated for its effects on insulin receptor binding, insulin receptor tyrosine kinase activity and glucose transport in isolated adipose cells. The dimer stimulated glucose transport (initial 3-O-methylglucose uptake rate) to the same extent as insulin did (basal rate, 35 +/- 3 pmol/sec/microliter lipid; insulin, 380 +/- 27; B29,B29'-suberoyl-insulin, 369 +/- 24, means +/- S.E.), although at higher concentrations (EC50 1.94 +/- 0.64 nM versus 0.1 +/- 0.02 with insulin). In contrast, the dimer only partially (23%) mimicked insulin's effect on phosphate incorporation into insulin receptors immunoprecipitated after equilibration of cells with [32P]phosphate. Similarly, insulin receptor tyrosine kinase as assessed by receptor autophosphorylation and phosphorylation of the substrate poly-(Glu/Tyr) was not fully activated by treatment of cells with the insulin dimer (31 and 42% of the effect of insulin, respectively) in concentrations which maximally activate glucose transport and give rise to full insulin receptor occupancy (5 X 10(-7) M). Further, the dimer activated the receptor tyrosine kinase in solubilized purified insulin receptor preparations from adipose cells to only 25% of the effect of insulin (EC50 32.0 +/- 16 versus 1.9 +/- 1.0 nM with insulin) in spite of full receptor occupancy. Binding of the dimer to insulin receptors followed single site binding kinetics, indicating that the derivative is unable to induce negative cooperativity of the insulin receptor. It is concluded that a partial phosphorylation of insulin receptors and a submaximal tyrosine kinase activation are sufficient for full stimulation of glucose transport in the adipocyte. Further, it is suggested that negative cooperativity of the insulin receptor and activation of its tyrosine kinase require a similar conformational change of the receptor protein.