Insulin stimulation of human adipocytes activates the kinase of only a fraction of the insulin receptors

The degree of insulin receptor kinase activation by in situ stimulation was studied in isolated human adipocytes. Although maximal in situ stimulation increased the kinase activity approximate to 10-fold, this activity could again be doubled by subsequent activation in a cell-free system. To investigate how in situ stimulation resulted in incomplete activation, receptors binding or not binding to anti-phosphotyrosine antibody (alpha-PY) were studied separately. Even after maximal insulin stimulation of the cells, similar to 50% of the receptors did not bind to alpha-PY and had low kinase activity. In the cell-free system, however, these receptors reached activity levels similar to the other receptors, suggesting that they were intact and that factors in their cellular environment had prevented their activation. The activity of the alpha-PY-binding receptors could only be slightly increased in the cell-free system, suggesting that almost complete activation had been attained in situ. In situ stimulation with increasing insulin concentrations increased the number of activated receptors rather than their individual activity. We conclude that factors in the in situ environment prevent insulin activation of similar to 50% of the insulin receptors in human adipocytes and might therefore be important regulators of insulin signaling.