Detection of insulin receptors in newt liver and forelimb regenerates and the effects of local insulin deprivation on epimorphic regeneration

Abstract

Previous in vivo and in vitro studies indicate that insulin is required in adult newt forelimb regeneration. The objectives of the current study were 1) to detect insulin receptors in the liver (a classical target organ for insulin) and once verified, detection of insulin receptors in the adult newt forelimb regenerate; and 2) to determine whether locally implanting insulin antibody-soaked hydrolyzed polyacrylamide beads (hypa beads) into a regenerating forelimb blastema would affect its growth and/or differentiation. The results show that insulin receptors are detectable in the plasma membranes of newt liver and forelimb regenerates. Radioiodinated bovine insulin binding is time-dependent and specific; unlabeled bovine insulin competes with labeled insulin for binding to NLPM more effectively than does insulin-like growth factor-I, guinea pig insulin, and glucagon. The newt hepatic insulin receptor binds insulin with high affinity (1.1 nM-1) and low capacity (63 +/- 8 fmoles/mg). The size of the alpha subunit of the newt insulin receptor is 130 kDA and that of the beta subunit is 95 kDa. The beta subunits undergo insulin-stimulated phosphorylation in response to insulin. An autoantibody against the human insulin receptor recognizes the newt receptor protein. Insulin receptors are also detectable in 15 and 20 day newt forelimb regenerates. Specific immunogold labelling of the receptor-bound antibody appears to be restricted to the cellular processes of the regenerate. Implanting hypa beads soaked with purified insulin antibody into regenerating adult newt forelimbs results in abnormal growth and differentiation of the regenerates, confirming that insulin plays an essential role in adult newt forelimb regeneration.