Binding and bioactivity of insulin in primary cultures of carp (Cyprinus carpio) hepatocytes |
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Authors: | Helmut Segner Ralf Böhm Werner Kloas |
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Institution: | (1) Zoology II, University of Karlsruhe, Kaiserstr. 12, D-W-7500 Karlsruhe, Germany;(2) Present address: Environmental Research Center, Section for Environmental Chemistry and Ecotoxicology, Permoserstr. 15, D-0-7050 Leipzig, Germany |
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Abstract: | Isolated carp hepatocytes cultured in serum-free, chemically defined medium were used to investigate within the same cell
preparation characteristics of the binding of insulin as well as effects of insulin on cellular metabolism. The binding of
human 125I]-insulin to carp hepatocytes was studied in kinetic, saturation and displacement experiments. A dependency of insulin binding
on the collagenase used for cell isolation was demonstrated. Insulin binding decreased during the first 12h of culture but
remained constant during the following 12h. The kinetic experiments revealed that 125I]-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 (Kd1 = 5.5 pM) and low capacity (Bmax1 = 0.14 fmol/mg protein or 77 binding sites/cell) and one with low affinity (Kd2 = 2.4 nM) and high capacity (Bmax2 = 17.6 fmol/mg protein or 9623 binding sites/cell). In competition experiments, 312 pM 125I]-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 was 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.
dedicated to Prof. Dr. W. Hanke on the occasion of his 65th birthday. |
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Keywords: | carp liver cell culture insulin insulin binding insulin bioactivity |
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