Evidence of the receptor nature of the binding sites induced in Tetrahymena by insulin treatment. A quantitative cytofluorimetric technique for the study of binding kinetics

Tetrahymena pyriformis GL cells pretreated (imprinted) and not pretreated with insulin showed dissimilar quantitative relations of FITC-insulin binding. Displacement of FITC-insulin by unlabelled insulin was considerably less in the control than in the imprinted series. The curve for saturation of the binding sites with FITC-insulin resembled a true saturation curve. The imprinted cells bound considerably more hormone in a shorter time than the control cells at identical levels of exposure. The dissociation of bound hormone from the imprinted cells increased over the control at 23 degrees C, and to a still greater degree at 4 degrees C. The effect of the pH of the medium on the dissociation of bound FITC-insulin also differed between the imprinted and not imprinted cells. Thus the proposed cytofluorimetric assay of binding kinetics demonstrated the actual conditions of receptor activity, and indicated that the induced insulin binding sites of Tetrahymena behaved similarly to 'classical' receptors.     

Effect of glucocorticoids and adrenaline on insulin receptors in plasma membranes and nuclear envelopes of the rat liver

To elucidate the course of regulation of insulin receptors in nuclear envelope and its relationship with insulin receptors in the plasma membrane a comparative study of these receptors in both subcellular fractions was conducted under the influence of the involved cell surface receptor factor. It is found that under adrenalectomy the number of nuclear envelope receptors and degree of their affinity did not increase as this occurs in plasma membrane receptors. Hydrocortisone replacement therapy in these animals lowers the receptor number in the both fractions. Hydrocortisone-induced hypercorticism does not change 125I-insulin binding by nuclear envelope but decreases its binding (accounted for the number of receptors and affinity) by the plasma membrane. Under hyperadrenalinemia the number of receptors decreases in the both subcellular fractions. The results suggest no independent regulation of insulin receptors on the surfaces of nuclei and cells.     

Effect of inhibition of endocytosis, recycling and lysosomal activity on the insulin binding capacity and imprintability of Tetrahymena

Dinitrophenol (DNP), an inhibitor of endocytosis of hormone receptors, Tris, an inhibitor of recycling and chloroquine, an inhibitor of lysosomal degradation, all decreased the binding of insulin and inhibited the development of hormonal imprinting in Tetrahymena. The effects of DNP and Tris seemed to be similar even quantitatively. The effect of chloroquine proved to be somewhat different, it appeared later, was more pronounced after 24 hours and more marked when insulin was also administered. Combined administration of Tris + DNP inhibited the binding of insulin but this inhibition was the one which disappeared most completely after 24 hours and the one where the inhibition of imprinting was the most pronounced. Tris + chloroquine led to severe destruction of the cells. The conclusion has been drawn that the inhibition of membrane circulation inhibits not only the hormone binding but also the development of imprinting in Tetrahymena.     

Cytoplasmic manifestation of the nuclear membrane's hormone binding capacity during cell division

Binding of insulin and thyrotropic hormone (TSH) to the nuclear membrane of Chang liver cells was demonstrated by qualitative and quantitative cytofluorimetry, which failed to substantiate a similar binding affinity for BSA. It appears that in the dividing cell the binding structures (receptors) of the nuclear membrane migrate in the cytoplasm together with the chromosomes by the end of the prophase and become reorganized in the nucleus around the telophase. The fluorescence which indicated binding also appeared in the midbody region during division of the two daughter cells. These experimental observations strongly suggest that, after cell division, only part of the nuclear membrane's receptor complement has to be resynthesized in the daughter cells, because the receptor number required by a single cell is conserved in cytoplasmic membrane details of nuclear membrane origin.     

Cytoplasmic manifestation of the nuclear membrane's hormone binding capacity during cell division

Summary Binding of insulin and thyrotropic hormone (TSH) to the nuclear membrane of Chang liver cells was demonstrated by qualitative and quantitative cytofluorimetry, which failed to substantiate a similar binding affinity for BSA. It appears that in the dividing cell the binding structures (receptors) of the nuclear membrane migrate in the cytoplasm together with the chromosomes by the end of the prophase and become reorganized in the nucleus around the telophase. The fluorescence which indicated binding also appeared in the midbody region during division of the two daughter cells. These experimental observations strongly suggest that, after cell division, only part of the nuclear membrane's receptor complement has to be resynthesized in the daughter cells, because the receptor number required by a single cell is conserved in cytoplasmic membrane details of nuclear membrane origin.     

Effects of extremely low concentrations of hormones on the insulin binding of Tetrahymena

FITC-insulin binding to previously hormone-treated Tetrahymena was studied by flow cytometry and confocal microscopy. Hormones produced by Tetrahymena were chosen for study and the hormone concentrations were administered between 10(-6) and 10(-21)M for 30 min. Endorphin, serotonin and insulin significantly reduced the hormone binding however histamine did not influence it at all. Endorphin, serotonin and insulin were significantly effective down to 10(-18)M and the effect of insulin and endorphin suggest a similar mechanism. The results call attention to the efficacy of very low hormone concentrations, which can influence the hormone content (earlier experiments) and receptor binding capacity (present study) of a unicellular organism. This seems to be very important, as in wild (natural) conditions the dilution of signaling materials secreted by a water-living protozoan is very high. In addition, the results point to the selectivity of response, as not all of the hormones that deeply influence other physiological indices (e.g. histamine) have an effect on insulin content or insulin receptors.     

Effect of starvation on insulin production and insulin binding in Tetrahymena

Tetrahymena pyriformis GL was starved for 24 h and then the immunologically demonstrable insulin content and FITC-insulin binding were measured by flow cytometry and localization was studied by confocal microscopy. The amount of endogeneous insulin as well as FITC insulin binding, was highly significantly elevated. Glucose feeding for 30 min abolished the elevation of FITC-insulin binding. In starved cells, insulin-binding sites disappeared from the surface and FITC-insulin was bound inside the cells, within large food vacuoles. Endogeneous insulin was dispersed in the cytoplasm both in the control and starved cells and food vacuoles did not contain it. The results call attention to the stimulatory effect of starvation on insulin production in Tetrahymena, in parallel with the internal storage of insulin receptors, which points to an autocrine mechanism.     

Comparison of solubilized and purified plasma membrane and nuclear insulin receptors

Prior studies have detected biochemical and immunological differences between insulin receptors in plasma membranes and isolated nuclei. To further investigate these receptors, they were solubilized in Triton X-100 and partially purified by wheat germ agglutinin-agarose chromatography. In these preparations, the nuclear and plasma membrane receptors had very similar pH optima (pH 8.0) and reactivities to a group of polyclonal antireceptor antibodies. Further, both membrane preparations had identical binding activities when labeled insulin was competed for by unlabeled insulin (50% inhibition at 800 pM). Next, nuclear and plasma membranes were solubilized and purified to homogeneity by wheat germ agglutinin-agarose and insulin-agarose chromatography. In both receptors, labeled insulin was covalently cross-linked to a protein of 130 kilodaltons representing the insulin receptor alpha subunit. When preparations of both receptors were incubated with insulin and then adenosine 5'-[gamma-32P]triphosphate, a protein of 95 kilodaltons representing the insulin receptor beta subunit was phosphorylated in a dose-dependent manner. These studies indicate, therefore, that solubilized plasma membrane and nuclear insulin receptors have similar structures and biochemical properties, and they suggest that they are the same (or very similar) proteins.     

Permanence of the cell-to-cell transmission of insulin induced hormonal imprinting

When insulin-treated (imprinted) Chang liver cell cultures were mixed with cultures which did not receive insulin treatment the information of imprinting was transmitted to the cultures which were not in direct contact with insulin. The ability of the cells to transmit imprinting was long lasting and could be detected even after four weeks, when it was nearly of the same degree as at the first measurement. Difference was found between the binding capacity of the receptors of the plasma membrane and those of the nuclear membrane.     

Comparison of the insulin binding, uptake and endogenous insulin content in long- and short-term starvation in Tetrahymena

FITC-insulin binding and endogenous insulin content of Tetrahymena pyriformis, that had been 24 h or 30 min starved, continuously fed or re-fed after starvation was studied by flow cytometry and confocal microscopy. Long starvation elevated both insulin binding and endogenous insulin content of the cells. Short re-feeding after long starvation or short starvation after continuous feeding does not change the situation. Fixed cells also bind FITC-insulin, however, in this case long starvation reduces, and re-feeding after long starvation elevates, the binding, which means that hormone binding by receptors only differs from receptor binding and engulfment (in living cells). The increase of FITC-insulin content in living cells seems to be due to engulfment, rather than by receptor binding. The results point to the unicellular organism's requirement for insulin production and binding in a life-threatening stress situation.     

Effect of femtomolar concentrations of hormones on insulin binding by Tetrahymena, as a function of time

The unicellular ciliate Tetrahymena, contains and binds hormones, characteristic of vertebrates. Earlier experiments demonstrated the effect of extremely low concentrations of hormones. In the present experiments, the effect of various hormones (endorphin, serotonin, histamine, insulin and epidermal growth factor [EGF]) in 10(-15) M, or oxytocin, gonadotropin at 0.001 IU concentrations) on the binding of FITC-insulin was studied by using flow cytometry and confocal microscopy, after 1, 5, 15, 30 and 60 min. Six of the seven hormones promptly decreased the cells' hormone binding capacity, the exception being EGF, and in four cases (endorphin, serotonin, insulin and oxytocin) the reduction was enormous. The decreased binding was durable. However, in the case of endorphin and oxytocin after 30 min, and in the case of serotonin after 60 min the binding returned to the control level. In the case of oxytocin after 60 min, binding significantly surpassed the control level. Histamine returned to the control level after 15 min, but after that the binding became even lower. EGF provoked special behaviour: it increased hormone binding after 30 and 60 min. The results call attention to the extreme sensitivity of Tetrahymena receptors to hormonal inductions and to its quick response ability.     

Non-conventional locations of hormone receptors (binding sites). A review

Recent findings on the noncanonical positions of some well-known extracellular mediators and their receptors are reviewed. Peptide hormones (insulin) and/or their binding sites (cell membrane insulin receptor, nuclear insulin receptor); steroid hormones (corticosteroids and estrogens) and their putative membrane receptors are in the scope of this paper. The possible roles of these unusually located receptors in the intracellular signal propagation and physiological responses are also discussed.     

Insulin binding to liver nuclei from lean and obese mice is altered by dietary fat.

Insulin binding to the plasma membrane is known to be altered by modifying the membrane composition through dietary treatment. As insulin binding receptors are also present on nuclear membrane, this study was undertaken to investigate if specific binding of insulin to the liver nuclei is altered by diet. 8-wk-old female C57 B 6J lean and ob/ob mice were fed semipurified diets containing 20% (w/w) fat of either high or low polyunsaturated-to-saturated (P/S) fatty acid ratio for 4 wk. Liver nuclei were prepared, insulin binding was measured and nuclear phospholipids were isolated for lipid analysis. Insulin binding was highest in nuclei prepared from lean mice fed a high P/S diet. Specific binding of insulin to nuclei prepared from obese mice was also increased by the high P/S diet, but to a lesser extent compared to lean mice. Feeding a high P/S diet increased polyunsaturated fatty acid content of membrane phospholipids from both lean and ob/ob mice. Obese mice were characterized by higher levels of arachidonic acid and lower levels of linoleic acid in phosphatidylcholine. The present study establishes that insulin binding to liver nuclei is increased by feeding a high P/S diet, and that insulin binding to liver nuclei from obese mice is lower than from lean mice.     

Insulin-like growth factors, insulin, and epidermal growth factor cause rapid cytoskeletal reorganization in KB cells. Clarification of the roles of type I insulin-like growth factor receptors and insulin receptors

Insulin-like growth factor (IGF) I (greater than or equal to 10(-10)M, insulin-like growth factor II (greater than or equal to 10(-9) M), insulin (greater than or equal to 10(-9) M, and epidermal growth factor (EGF, greater than or equal to 10(-11) M) caused rapid membrane ruffling in KB cells. The morphological change was observed within 1 min after the addition of these growth factors and was accompanied by microfilament reorganization, but not by microtubule reorganization. IGF-I, IGF-II, and insulin induced morphologically very similar or identical membrane ruffles with the order of potency IGF-I greater than IGF-II greater than insulin, whereas EGF-induced membrane ruffles were morphologically different. KB cells possessed EGF receptors, type I IGF receptors, and insulin receptors, but few or no type II IGF receptors. Monoclonal antibody against type I IGF receptors, which completely inhibited the binding of 125I-IGF-I to the cells but did not inhibit the binding of 125I-insulin, caused marked inhibition of IGF-I (10(-8) M)-stimulated membrane ruffling. IGF-II (10(-8) M)-stimulated membrane ruffling was partially inhibited in the presence of this antibody, but insulin (10(-7) M)-stimulated membrane ruffling was only slightly inhibited. In contrast, monoclonal antibody against insulin receptors blocked insulin (10(-7) M) stimulation, but not IGF-I (10(-8) M) stimulation, of membrane ruffling. Thus, this study provides evidence that IGF-I and insulin act mostly through their own (homologous) receptors and that IGF-II acts by cross-reacting with both type I IGF and insulin (heterologous) receptors in causing rapid alterations in cytoskeletal structure.     

Comparison of Insulin Binding Proteins In Plasma and Nuclear Membranes of Obese and Lean Mouse Liver

Abstract

Plasma membranes obtained from obese (ob/ob) and lean (+/+ or +/ob) mouse livers were chemically crosslinked to [¹²⁵I] -insulin and examined by electrophoresis and autoradiography. The pattern of crosslinked hormone was qualitatively similar in obese and lean plasma membranes. A major insulin binding protein of approximately M 120,000 was observed. Two additional bands were apparent, one which remained near the top of the gel and one about M 90,000. A minor band at approximately M 50,000 was also detected. For each of the insulin binding proteins a reduction in the amount of [¹²⁵I]-insulin bound was observed with obese plasma membranes as compared with lean. For all proteins the insulin binding was specific as determined by competition with unlabeled hormone. In addition to plasma membrane receptors, insulin has also been reported to bind to nuclear membranes. The autoradiographic patterns of gels of [¹²⁵]-insulin bound and crosslinked to nuclear membranes from obese and lean mouse livers indicated the presence of proteins of the same M as those described for plasma membranes. Nuclear membrane proteins bound less insulin than plasma membranes and, again, the obese was decreased relative to the lean. Contamination of the nuclear membrane fraction by plasma membranes was ruled out. Scatchard analyses of [¹²⁵]-insul in bound to plasma and nuclear membranes indicated that the decrease in hormone binding in the obese mouse is a result of a reduction in the absolute number of receptors. The findings presented in this study provide additional support for this conclusion by demonstrating that membranes from obese mice are comprised of the same set of apparently unaltered insulin binding proteins. Further, the presence of similar insulin binding proteins in both nuclear and plasma membranes suggests a physiological relationship between these structures with respect to hormone binding and/or in the mechanism of action of insulin.     

Discrepancy in hormone binding and information transfer between sister cells of Tetrahymena clones

Tetrahymena cells treated with insulin in mass cultures were separated to single-cell clones or one of the "sister-cells" of dividing Tetrahymena (in single-cell culture) was treated with insulin. In both cases the FITC-insulin binding of sister-cells were compared. The insulin imprinting significantly increased the insulin binding of cells. There was also a significant difference between the imprinted and not imprinted sisters as well as between the not imprinted sisters. This demonstrates the existence of a difference (in hormone binding) between sister-cells and justifies that the information of the first hormone treatment (imprinting) is not equally divided between the sister-cells.     

A confocal microscopic evaluation of the effects of insulin imprinting on the binding of Concanavalin A by Tetrahymena pyriformis.

With confocal microscopy it is possible to study the Concanavalin A (Con A) binding characteristics of the surface and interior of a single cell by viewing optical sections. It was observed in Tetrahymena pyriformis that Con A bound both to the plasma membrane and to intracellular structures. Incubation of cells with a competing sugar a-methylmannopyranoside, decreased binding. Hormonal imprinting with insulin resulted in an increase in binding of Con A to the cell surface and a decrease in intracellular binding. It is possible that the intracellular binding sites may migrate to the plasma membrane.     

Role of ATP in specific binding of 125I-insulin to cytoplasmic receptors of the liver and muscle membranes of controls and diabetic rats

A study was made of the action of various concentrations of ATP on insulin ability to bind to the receptors of the liver and muscle membranes in control and streptozocin-induced diabetes animals. Specific binding of 125I-insulin to the receptors of the liver and muscle membranes was shown to rise in animals with streptozocin-induced diabetes as compared to control. This effect was most pronounced in the muscle membranes. Preincubation of the membranes with ATP did not affect insulin binding to the liver and muscle receptors of control animals. However, hormone binding to the liver receptors of diabetic rats was drastically suppressed by ATP (10(-3) M). Less ATP concentrations (10(12) M) produced an additional inhibitory action which was not marked. ATP led to decreased insulin binding to the muscle receptors of diabetic rats only at extremely low concentrations (10(-12) M). The data obtained may be of importance for regulation of membrane phosphorylation in the states characteristic of insulin resistance.     

Insulin stimulates cellular iron uptake and causes the redistribution of intracellular transferrin receptors to the plasma membrane

Insulin stimulates the accumulation of iron by isolated fat cells by increasing the uptake of diferric transferrin. Analysis of the cell-surface binding of diferric 125I-transferrin indicated that insulin caused a 3-fold increase in the cell surface number of transferrin receptors. This result was confirmed by the demonstration that insulin increases the binding of an anti-rat transferrin receptor monoclonal antibody (OX-26) to the surface of fat cells. The basis of this effect of insulin was examined by investigating the number of transferrin receptors in membrane fractions isolated from disrupted fat cells. Two methods were employed. First the binding isotherm of diferric 125I-transferrin to the isolated membranes was studied. Second, the membranes were solubilized with detergent, and the number of transferrin receptors was measured by immunoblotting using the monoclonal antibody OX-26. It was observed that insulin treatment of intact fat cells resulted in an increase in the number of transferrin receptors located in the isolated plasma membrane fraction of the disrupted fat cells. Furthermore, the increase in the number of plasma membrane transferrin receptors was associated with a concomitant decrease in the transferrin receptor number in a low density microsome fraction previously shown to consist of intracellular membranes. This redistribution of transferrin receptors between cellular membrane fractions in response to insulin is remarkably similar to the regulation by insulin of glucose transporters and type II insulin-like growth factor receptors. We conclude that insulin stimulates fat cell iron uptake by a mechanism that may involve the redistribution of transferrin receptors from an internal membrane compartment (low density microsomes) to the cell surface (plasma membrane).