Insulin-ricin B chain conjugate has enhanced biological activity in insulin-insensitive cells.

Abstract

Insulin-ricin B chain conjugate, a hybrid molecule consisting of insulin covalently linked to the binding chain of ricin, was tested for insulin-like biological activity in HTC and H35 rat hepatoma cells, rat adipocytes, rat thymocytes, and human fibroblasts. In H35 cells and adipocytes, cells that have abundant insulin receptors and are very sensitive to insulin (ED50 of 30 pM and 50 pM, respectively), the conjugate had 5% the biological activity of native insulin (ED50 of 500 pM and 1000 pM, respectively). Since the insulin portion of the conjugate has 5% the potency of native insulin in binding to the insulin receptor, these observations suggested that (in these cells) the conjugate was acting via the insulin receptor. Moreover lactose and galactose, potent inhibitors of ricin binding to its receptor, had no effect on the action of the conjugate on H35 cells. In contrast, in thymocytes, HTC cells, and fibroblasts cells that have relatively few insulin receptors and require high concentrations of insulin to elicit biological actions (ED50 of 10 nM, 20 nM, and 1 nM, respectively), the conjugate had more biological activity than was predicted on the basis of its ability to bind to the insulin receptor. In addition, in these three insulin-insensitive cell types, the activity of the conjugate was inhibited by either lactose or galactose. Thus, these observations indicate that in cells which are relatively insensitive to insulin, the biological effects of the conjugate require the interaction of the ricin B chain moiety with the ricin receptor. In addition, in fibroblasts, the activity of the conjugate was inhibited by the addition of a monoclonal antibody to the insulin receptor which inhibits the response of fibroblasts to insulin. These data suggest, therefore, that in insulin-insensitive cells the binding of the ricin B chain moiety to its receptor enhances the interaction of the insulin portion of the conjugate with the insulin receptor.