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.

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).

[Receptor-mediated endocytosis in the cells of cold-blooded animals. I. Insulin internalization in the hepatocytes of the lamprey and the frog]. / Retseptoroposreduemyi éndotsitoz v kletkakh kholodnokrovnykh. I. Internalizatsiia insulina v gepatotsitakh minogi i liagushki.

A method of preparation of the lamprey and frog isolated hepatocytes is described. The binding of 125I-insulin to the cell receptors and the internalization of insulin--receptor complexes in hepatocytes have been studied at the temperature of species survival. Two classes of binding sites (with Kd equal to 10(-9) and 10(-8) M, resp.) were determined in cells of both species. The molecular weight of the insulin receptor binding subunit estimated in cross-linking experiments appeared 130 kDa in the lamprey and frog, which is in agreement with the data previously obtained for the rat liver receptor. The internalization of bound 125I-insulin in both species examined occurred at the range of temperature within 0 to 20 degrees C. In the lamprey cells the maximum level of the internalized hormone remained equal at all the temperatures studied. In the frog hepatocytes, the level of internalized hormone at 5 and 20 degrees C was higher than at 0 degrees C. The data obtained show that there are no limitation of the insulin--receptor complexes endocytosis, and of the 125I-insulin internalization in hepatocytes of cold-blooded vertebrates at the survival temperature.

[Receptor-mediated endocytosis in the cells of cold-blooded animals. II. The fate of internalized 125I-insulin in the isolated hepatocytes of the lamprey and the frog]. / Retseptoroposreduemyi éndotsitoz v kletkakh kholodnokrovnykh. II. Sud'ba internalizovannogo 125I-insulina v izolirovannykh gepatotsitakh minogi i liagushki.

The 125I-insulin outflow from isolated hepatocytes of the frog and lamprey "loaded" with the labeled hormone has been studied. It is shown that the ligand outflow from the frog cells increased with the increase in the incubation temperature from 0 up to 20 degrees C. The curves of the rest cell radioactivity were reciprocal to those of the radioactivity accumulated in the medium at the corresponding temperatures. At 0.5 and 20 degrees C the degraded 125I-insulin made 5.7 and 17% of the whole hormone accumulated in the medium. In the lamprey hepatocytes, neither accumulation in the incubation medium nor outflow of the radioactivity from cell was seen at all temperatures studied. The intracellular degradation of 125I-insulin in the frog hepatocytes was no more than 7% of the internalized ligand, compared to about 25% in the lamprey cells. The specific binding of 125I-insulin was twice increased in the presence of lysosomal inhibitor chloroquin; contrary to this, no increase was found in the lamprey hepatocytes. The results of experiments on the frog hepatocytes lead us to a conclusion that the processing pathway of internalized insulin in cold-blooded vertebrate cells is similar mainly to that in cells of warm-blooded species, but takes place at lower temperatures and with slower rates. The peculiarities of processing in the lamprey hepatocytes (extralysosomal ligand degradation, the inability to release the internalized ligand and its degradation products) are dependent on a deep transformation of hepatocytes during prespawning migration period.

[Certain biochemical characteristics of an insulin-like substance from the bivalve mollusk Anodonta cygnea]. / Nekotorye biokhimicheskie kharakteristiki insulinopodobnogo veshchestvadvustvorchatogo molliuska Anodonta cygnea.

An insulin-like substance (ILS) was isolated from the visceral organs of the bivalve mollusc Anodonta cygnea by chromatography on a sulfocationite CU-23 and purified by reverse phase liquid chromatography. ILS was shown to be made up to several fractions with Mr ranging from 9 to 20 kDa which have identical amino acid composition but different hydrophobicity and N-terminal amino acids. It was supposed that the heterogeneity of ILS fractions is due to its genetical or posttranslational polymorphism. ILS has a low (0.02%) affinity for the mammalian insulin receptor and a low immune affinity for mammalian insulin and possesses a mitogenic activity which is commensurate with that of the epidermal growth factor. The data obtained suggest that Anodonta cygnea ILS represents a separate branch of a relatively ancient family of insulin-like hormones and growth factors responsible for metabolism and proliferation of invertebrate tissues.

[Glucagon receptor binding and its effect on the cAMP level in isolated rat and chicken hepatocytes]. / Sviazyvanie gliukagona retseptorami i vliianie na uroven' tsAMF v izolirovannykh gepatotsitakh krys i kur.

The binding sites to glucagon with KD 10(-9) M in rat and chicken isolated hepatocytes and with KD 10(-7) M in chicken hepatocytes only where revealed. The first, but not the second binding sites where decreased in their affinity by the adding of GTP nonhydrolyzing analogue Gpp(NH). Glucagon activated the cAMP accumulation in rat and chicken hepatocytes in dose-depended manner. The dose-response curve being plateau after maximum in rats and decreased in chicken. Glucagon inserted an inhibiting effect when occupying chicken-specific sites of binding with KD 10(-7) M. The nature of these binding sites and mechanism of glucagon inhibiting effect on the cAMP accumulation are discussed.

[Glucagon binding by isolated chick hepatocytes]. / Sviazyvanie gliukagona izolirovannymi gepatotsitami kur.

Studies have been made on the binding of 125I-glucagon by isolated chick hepatocytes. It was shown that pH and temperature dependence of the binding does not differ from that in rat hepatocytes. Optimum binding was observed at pH 7.6, the rate of binding being higher at 37 degrees C as compared to that at 20 degrees C, although the binding capacity increased with the decrease in the temperature. Unlabeled glucagon was able to compete with 125I-glucagon at the binding sites. Scatchard plot was found to be curvilinear revealing two classes of the binding sites with Kd values 10(-9) and 10(-7) M at temperatures 20 and 37 degrees C correspondingly. Earlier studies revealed in rats the binding sites of a sole class with Kd value 10(-9) M. Preincubation of cells with native glucagon results in changes of labeled glucagon binding, the effect being proportional to the concentration of native glucagon. Preincubation effect was observed at 37 degrees C, being absent at 20 degrees C; the effect was due to the decrease in the number of both high and low affinity binding sites. The presence of down-regulation of glucagon receptors in chick hepatocytes is suggested.

[Insulin binding by receptors of the plasma membranes of the liver in hens in ontogenesis]. / Sviazyvanie insulina retseptorami plazmaticheskikh membran pecheni kur v ontogeneze.

Specific binding of 125I-insulin to the liver plasma membranes was studied in the chick embryos from the 10th day of incubation on, in chickens and adult fowl. The level of binding was the same in all cases although the insulin concentration of blood increases during ontogenesis, the number of receptors and their affinity to the hormone remaining constant. The data on insulin-receptor interactions in the liver have been compared with the earlier results of the authors obtained on the chick skeletal muscle and erythrocytes.

[Insulin receptor reaction of the lamprey Lampetra fluvialis to hyperinsulinemia induced by administration of the hormone]. / Reaktsiia insulinovykh retseptorov minogi Lampetra fluvialis na giperinsulinemiiu, vyzvannuiu vvedeniem gormona.

Insulin injections to the lamprey Lampetra fluviatilis result in prolonged and significant hyperinsulinemia, but do not decrease the number of receptors in its target cells. These findings were made in studies on the specific binding to 125I-insulin by membrane fractions of the brain and cardiac muscle in migrating and winter (fasting) lampreys. It is also suggested that no regulation of the number of the receptors by hormonal level takes place in the lamprey, which is presumably due to a low rate of internalization of hormone-receptor complexes and to a total decrease in the turnover of the receptors in a pre-spawning period.

[Erythrocyte insulin receptors in the ontogeny of chickens]. / Insulinovye retseptory éritrotsitov v ontogeneze kur.

Specific binding of 125I-insulin by the erythrocytes was studied in the chick embryos, chicken and adult fowl. The binding was shown to decrease during ontogenesis. The maximal level of binding was observed in the chick embryos. Within a month after hatching, it decreased to the level of adult fowl due, mainly, to the decrease in the number of receptors per erythrocyte. This phenomenon can be accounted for both by the increase of insulin content in blood with age and the age changes of the population of erythrocytes. It is proved that during ontogenesis of the domestic fowl the decrease of a portion of immature erythrocytes in the peripheral blood with age accounts for the dynamics of binding observed. At the same time the affinity of hormone receptors increased but the influence of this factor on the binding pattern was not leading.

[Brain insulin receptors in the evolution of vertebrates]. / Insulinovye retseptory golovnogo mozga v évoliutsii pozvonochnykh.

Studies have been made on 125I-insulin binding for brain membranes from cyclostomes (the lamprey Lampetra fluviatilis), fish (pink salmon Oncorhynchus gorbuscha) and mammals (rats). The species studied differed by the level of binding (the highest in the rat and the lowest in the lamprey), which was due mainly to differences in the number of binding sites per membrane protein. Qualitative properties of the receptors in the species studied were found to be very similar. All three types of the receptors were capable of differentiating between the insulins from pig, pink salmon and lamprey, all of them binding porcine insulin more readily than the salmon one and the latter better than the insulin from the lamprey. It means that these insulins reacted not to the species specific properties of the hormone, but to biological activity of the insulin. The data obtained indicate that functionally mature insulin receptor may be found already in the brain of cyclostomes and that in the course of animal evolution from cyclostomes to mammals functional properties of this receptor did not undergo any significant changes.

[Study of insulin-receptor interactions in plasma membrane of rat liver using antibodies against insulin]. / Analiz insulin-retseptornykh vzaimodeistvii v plazmaticheskoi membrane pecheni krys s ispol'zovaniem antitel k insulinu.

Preincubation of rat liver cell plasma membranes with antiinsulin serum (AIS) results in a 3-fold increase of specific binding of [125I]insulin added to AIS-free membranes. The degree of such increase depends on preincubation time and AIS concentration; at a rise of both parameters the dependence curve shown a plateau. The Scatchard plots suggest that preincubation with AIS increases the high and low affinity of binding sites; their number remains thereby unchanged. Using several types of immune and non-immune sera for preincubation the specificity of this effect was established. It was assumed that the antibodies initiate the immunological reaction of the membrane. Study of AIS effect on dissociation of membrane-bound labelled insulins differing in their affinities for the given AIS revealed that the antibodies can interact with the newly formed hormone-receptor complex but cannot affect the receptor within insulin. It is concluded that the intact hepatocyte membrane contains a receptor-bound insulin which is a target of AIS action. The antigen-antibody interaction changes the functional state of occupied and free receptors which bind [125I]insulin. The latter becomes repeatedly involved in this process as a result of destabilization of site-site interactions provoked by antibodies against the hormones.