INSULIN-RECEPTORS IN XENOPUS-LAEVIS LIVER AND FORELIMB REGENERATES AND THE EFFECTS OF LOCAL INSULIN DEPRIVATION ON REGENERATION

As forelimb regeneration in Xenopus laevis is mainly a cell proliferat ive event which results in a spike-shaped appendage, we set out to exa mine the possibility that insulin is a growth-promoting factor in this process. The objectives were 1) to detect the presence of insulin rec eptors (IRs) in the Liver (a specific target organ for insulin) and IR s in the forelimb regenerates of X. laevis, 2) to determine whether th e receptor is similar to IRs identified in other organisms, and 3) to absorb insulin locally by implanting anti-insulin antibody-soaked hydr olyzed polyacrylamide beads into regenerating forelimb outgrowths in o rder to assess the effects of insulin deprivation on regeneration. The results show that IRs are present in Xenopus liver plasma membranes ( XLPM) as well as in plasma membranes of 21 day forelimb regenerates. I nsulin binding to this receptor is time-dependent and specific, as unl abeled bovine insulin competes with radioiodinated insulin for binding to XLPM more effectively than insulin-like growth factor-I, guinea pi g insulin, or glucagon. Scatchard analysis of insulin binding to XLPM describes a two binding site receptor possessing a low affinity (0.16 nM(-1)), high capacity (3.2 +/- 0.9 pM/mg) binding site and a high aff inity (2.7 nM(-1)), low capacity (0.5 +/- 0.3 pM/mg) binding site. The holoreceptor has a molecular mass of 380 kDa. The reduced receptor ha s subunits of 130 kDa and 95 kDa. The 95 kDa subunit undergoes autopho sphorylation following insulin stimulation. Implantation of hydrolyzed polyacrylamide beads, saturated with anti-insulin antibody, into rege nerating Xenopus forelimbs significantly impeded development of the re generates and, therefore, demonstrates that insulin is required for gr owth of Xenopus foreLimb regenerates. (C) 1995 Wiley-Liss, Inc.