BINDING AND BIOACTIVITY OF INSULIN IN PRIMARY CULTURES OF CARP (CYPRINUS-CARPIO) HEPATOCYTES

Isolated carp hepatocytes cultured in serum-free, chemically defined m edium were used to investigate within the same cell preparation charac teristics of the binding of insulin as well as effects of insulin on c ellular metabolism. The binding of human [I-125]-insulin to carp hepat ocytes was studied in kinetic, saturation and displacement experiments . A dependency of insulin binding on the collagenase used for cell iso lation was demonstrated. Insulin binding decreased during the first 12 h of culture but remained constant during the following 12h. The kinet ic experiments revealed that [I-125]-insulin binding reached a steady state within 20-30 min of incubation. The mathematical analysis of the saturation experiments demonstrated the existence of two populations of binding sites, one with high affinity (K(d1) = 5.5 pM) and low capa city (B(max1) = 0.14 fmol/mg protein or 77 binding sites/cell) and one with low affinity (K(d2) = 2.4 nM) and high capacity (B(max2) = 17.6 fmol/mg protein or 9623 binding sites/cell). In competition experiment s, 312 pM [I-125]-insulin was displaced by cold insulin, IGF-I and IGF -II with IC50 values of 2.2, 7.9 and 20.3 nM, respectively. Glucagon w as without effect. Binding of insulin to carp hepatocytes resulted in a significant reduction of glucose release and a significant increase of protein synthesis as of de novo fatty acid synthesis.