H. Segner et al., BINDING AND BIOACTIVITY OF INSULIN IN PRIMARY CULTURES OF CARP (CYPRINUS-CARPIO) HEPATOCYTES, Fish physiology and biochemistry, 11(1-6), 1993, pp. 411-420
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.