Insulin-family peptide-receptor interaction at the early stage of vertebrate evolution.

This is an overview of our studies on insulin and insulin-like growth factor-I (IGF-I) interactions with their own and each other's receptors in the lamprey (Lampetra fluviatilis L.), an extant representative of the ancient vertebrate group of Agnathans as compared to mammal (rat). Lamprey insulin receptor shows species specificity, namely, it binds its own insulin with higher affinity than mammalian hormone. Nevertheless, and unlike mammalian insulin receptor, lamprey receptor discriminates relatively poorly between insulin and IGF-I. Autophosphorylation patterns are identical for both receptors. In contrast, IGF-I receptors in lamprey tissues are very similar to mammalian IGF-I receptors confirming known evolutionary conservatism of IGF receptor system. Presumed common evolutionary origin of insulin and IGF-I receptors and poor ability of lamprey insulin receptor to discriminate between two ligands, implies that lamprey insulin receptor is closer to putative ancestral protein that IGF-I receptor. Contrary to the common belief, ambient temperatures for lampreys (4-15 degrees C) put no constraints on either downregulation of receptors or the endocytosis of hormone-receptor complexes.

Lamprey but not porcine insulin binds with different affinity to lamprey and rat hepatocytes.

The displacement of porcine [125I] insulin bound to rat and lamprey isolated hepatocytes with unlabeled lamprey and porcine insulins was investigated. Binding affinity of lamprey insulin for insulin receptor of rat was similar to that of porcine insulin. In contrast, the binding affinity of lamprey insulin for its own insulin receptor was higher than for a rat receptor. To determine the binding affinity constants of lamprey insulin receptor, the competition binding experiments were carried out on isolated lamprey hepatocytes using lamprey insulin as unlabeled ligand and tracer. The affinity of the same binding sites on lamprey hepatocytes was assessed in similar experiments but employing porcine insulin as unlabeled ligand and tracer. It was found that while Kd of low affinity binding sites on lamprey hepatocytes were similar for lamprey and porcine insulins, the Kd of high affinity binding sites was different: the displacement curve for lamprey insulin being shifted to the left as compared to the curve for porcine insulin. The number of high and low affinity binding sites, calculated independently in Scatchard plots, was equal. We conclude that the high affinity insulin binding sites of lamprey but not of rat hepatocytes reveal some species specificity in ligand-receptor interaction.

Insulin and insulin-like growth factor-I receptors in fish brain.

We investigated insulin and insulin-like growth factor-I (IGF-I) receptor-binding and receptor intrinsic tyrosine kinase activity in the brain of carp (Cyprinus carpio) and trout (Salmo trutta fario). Glycoprotein fractions of semi-purified receptors were prepared by WGA-agarose affinity chromatography. Insulin receptors were found in the brains of both fish species investigated. Carp and trout brain preparations bound, respectively (per 50 micrograms glycoprotein), with 6.0 +/- 1.5% and 8.0 +/- 2.0% of 125I-labeled insulin added to the assay. Insulin binding was specific: much higher quantity of IGF-I (EC50 165 +/- 11 nM for carp and 88.0 +/- 6 nM for trout receptors) than insulin (EC50 0.26 +/- 0.04 nM for carp and 0.25 +/- 0.02 nM for trout) was necessary to displace bound insulin tracer. In preparations of brain receptors, IGF-I binding (52.8 +/- 6.5% in carp brain and 55.0 +/- 13.0% in trout brain) surpassed insulin binding several fold. IGF-I bound to the brain receptors with high affinity (Kd for carp was 0.13 +/- 0.06 nM and for trout 0.22 +/- 0.11 nM) and specificity. Although IGF-I binding could be displaced with insulin, EC50 were 660 +/- 51 nM for carp and 1557 +/- 194 nM for trout. Both ligands stimulated phosphorylation of exogenous substrates in a dose-dependent manner. Carp brain receptors were not significantly different from trout receptors with respect to basal phosphotransferase activities (250.0 +/- 50.0 fm P/mg glycoprotein in carp and 330.0 +/- 120.0 fm P/mg glycoprotein in trout). In both species IGF-I caused higher maximal stimulation (308.0 +/- 36.0% and 270.0 +/- 39%, for carp and trout, respectively) than insulin (250.0 +/- 13.0% and 209.0 +/- 6.0%, for carp and trout, respectively).

Receptor-mediated endocytosis of insulin in lower vertebrates: internalization and intracellular processing of 125I-insulin in isolated hepatocytes of lamprey and frog.

The binding of 125I-insulin to cellular insulin receptors and the internalization of insulin-receptor complexes have been studied in isolated hepatocytes of frog and lamprey. Two classes of binding sites (Kd 10(-9) and 10(-8) M) were found in cells of both species. The molecular weight of the insulin receptor alpha-subunit was 130 kDa in both species. Internalization of bound 125I-insulin in both species was found in the temperature range 0 to 20 degrees. Cells "loaded" with 125I-insulin were used to estimate the fate of the internalized ligand. Release of internalized ligand from frog cells increased at temperatures ranging from 0 to 20 degrees. At 0 degrees the degraded 125I-insulin was 5%, at 5 degrees 7%, and at 20 degrees 17% of total radioactivity accumulated in the medium. In lamprey hepatocytes there was neither radioactivity accumulation in the incubation medium nor release from cells at all temperatures studied. The intracellular degradation of internalized 125I-insulin in frog hepatocytes was much lower than that in lamprey cells. In frog hepatocytes the specific binding of 125I-insulin was increased twofold in the presence of the lysosomal inhibitor chloroquine. In contrast no increase was found in lamprey hepatocytes. In conclusion, the processing pathways of internalized insulin in the cells of ectothermal and endothermal vertebrates are generally similar but in ectothermal animals all events take place at lower temperatures and at lower rates. The peculiarities of insulin processing in lamprey hepatocytes most likely result from the transformation of hepatocytes during the nonfeeding prespawning period.

Insulin receptor downregulation in isolated hepatocytes of river lamprey (Lampetra fluviatilis).

Insulin receptor downregulation in the isolated hepatocytes of lamprey (Lampetra fluviatilis) was studied at the ambient temperature for this species. Preincubation of hepatocytes with 10(-9)-10(-8) M unlabeled insulin decreased insulin binding capacity to 43, 37, and 34% at 4, 15, and 25 degrees, respectively. Preincubation of hepatocytes in the presence of 10(-10) M unlabeled insulin had no effect on the 125I-insulin binding. The maximal decrease in the 125I-insulin binding was reached after 10 min of preincubation and was then maintained at a constant level for 2 hr. Competitive binding assays demonstrated that preincubation with unlabeled insulin resulted in a 45% decline in the number of binding sites. In rat adipocytes and frog hepatocytes used for the comparison, preincubation with insulin caused a 65% decrease in cell-surface receptors, while intracellular (internalized receptors) increased proportionally. In contrast, in the lamprey hepatocytes both cell-surface and intracellular receptors decreased. We conclude that insulin receptors of the lamprey hepatocytes could be down-regulated at the ambient temperature for the species and at the physiological concentrations of insulin (10(-9)-10(-8) M).