Effect of inhibitors and activators of tyrosine kinase on insulin imprinting in Tetrahymena.

Primary exposure of Tetrahymena cells to insulin gave rise to hormonal (insulin) imprinting in the offspring generations, as judged from the increase in binding upon reexposure to insulin. Vanadate mimicked the action of insulin, inasmuch as it also induced imprinting for insulin, whereas the other tyrosine kinase activator tested, namely H2O2, had no such effect. However, combined treatment with vanadate+H2O2 + insulin induced a more pronounced imprinting for insulin than either insulin or vanadate on their own. The tyrosine kinase inhibitor genistein, a plant flavonoid, did not change the value for insulin binding significantly relative to the control immediately after exposure, but increased it slightly in the offspring generations after 24 h at high dilution. Upon combination with insulin, 10(-4)M genistein inhibited imprinting by insulin. These experimental observations suggest that there may be a key role for tyrosine kinase activity in the mechanism (development) of imprinting.     

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.     

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 stress and stress hormones on the hormone (insulin) binding of Tetrahymena

The unicellular Tetrahymena pyriformis GL produce, store and secrete vertebrate‐like hormones. In earlier experiments the effect of different stressors on the hormone levels of Tetrahymena was studied and an elevation of these was found. In the present experiments the hormone binding was investigated, using flow cytometric method. FITC‐insulin binding was elevated after concentrated (5, 10, or 20 mg ml^?1) NaCl or 0.01%, 0.1%, or 0.05% formaldehyde treatment, or after thermal stress (37°C). Serotonin given together with NaCl increased and together with formaldehyde decreased the binding. Histamine always decreased the binding and insulin was indifferent. Four hours after osmotic stress, hormone binding significantly decreased and this was not influenced by hormones. However, 4 h after formaldehyde stress the binding elevated and this was further increased by repeated hormone treatments. The results show that the stress in Tetrahymena provokes an activation of its hormonal system (hormone production and binding), which is differently influenced by exogeneously given hormones. Copyright © 2009 John Wiley & Sons, Ltd.     

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 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.     

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.     

Insulin pretreatment (imprinting) produces elevated capacity in the insulin binding of Tetrahymena. Different binding by the cilia of the body and oral field

Gold-labeled insulin is bound first of all to the cilia of the oral field of Tetrahymena. A primary treatment (hormonal imprinting) with insulin increases the binding capacity even after 24h and makes it more sensitive for appearance a week later, within a minute of giving insulin-gold. The food vacuoles contain insulin-gold in pretreated cells or without pretreatment as well, though in imprinted situations the label can be found in pinocytotic vesicles at the bases of cilia in the oral field. Altogether, a functional difference can be observed between the cilia of the oral and non-oral surfaces of Tetrahymena and hormonal imprinting has a specifying effect on the binding of labeled hormone.     

Induction of digoxin-like material production, and the digoxin binding in the unicellular organism Tetrahymena by digitoxin.

Thin layer chromatographic, and laser-confocal microscopic analyses with a monoclonal antibody to digoxin also displaying high affinity to digoxigenin, were used to determine the presence and localization of cardioactive glycosides. Tetrahymena pyriformis was found to possess digitoxigenin-like material, but digoxin, digitoxin, digoxigenin, gitoxin and lanatoside C were not detected. Digitoxin treatment elicited the appearance of a digoxin-like material in the progeny generations. Digoxin was taken up by untreated Tetrahymena, especially strongly 24 h after digitoxin treatment. While the cardenolide was localized in vesicles of the cell body in untreated Tetrahymena, the engulfed digoxin appeared in the epiplasmic layer and also in the cilia after digitoxin pretreatment. Digoxin pretreatment did not increase digoxin uptake. These data indicate that Tetrahymena has: (1) the capacity to discriminate between closely related molecules; (2) the ability to induce digoxin-like material production; and/or (3) enzymes that can effect a digitoxin-digoxin transformation.     

Immunocytochemical verification of the insulin receptor's specificity in the nuclear envelope of Tetrahymena. Comparison with receptors of the plasma membrane

The unicellular Tetrahymena possess hormone receptors in the nuclear envelope similarly to higher rank animals. These receptors bind insulin and their specificity is detectable by monoclonal antibodies developed to insulin. The hormonal (insulin) pretreatment (imprinting) of the cell did not alter the binding capacity of the nuclear membrane, demonstrated by antibody-technique. The specific binding characteristics of the plasma membrane was demonstrated and this was significantly increased following imprinting. In the nucleus of Tetrahymena presence of insulin was not detected by immunocytochemical method.     

Is the saccharide component of the insulin receptor involved in hormonal imprinting?

Hormonal imprinting takes place at the first interaction of a given hormone with the cell (the Tetrahymena in the present case) and accounts for a greater responsiveness to the hormone on re-exposure(s). The Tetrahymena is able to bind insulin and Concanavalin-A (Con-A) as well. Exposure to both ligands—simultaneously or in sequence—enhances the binding of both in the progeny generations. It follows that the lectin, which inhibits insulin binding by direct action, enhances rather than depresses the effect of insulin-induced imprinting.     

Effect of acute zinc deficiency on insulin receptor binding in rat adipocytes

Considerable data have been reported on the relationship between insulin resistance and zinc deficiency. In this study, insulin receptor binding was measured in isolated rat adipocytes. Two assays were carried out at 37°C (binding and internalization) and 16°C (binding) using¹²⁵I insulin 0.05–20 nM. A decreased insulin receptor binding was observed in zinc-deficient rat adipocytes, but we could not make any distinction between the specific zinc depletion effects and the effects of the caloric restriction induced by zinc deficiency.     

Long-Term Maintenance of Tetrahymena Spp.

SYNOPSIS. Simple media for Tetrahymena, using rat gut or soybean as the nutrient source are described. Cultures can be maintained in these media up to one year at room temperature.     

Effect of insulin on the incorporation of3H-inositol into the inositol phospholipids (PI,PIP, PIP2) and Glycosyl-phoshatidylinositols (GPIs) of Tetrahymena pyriformis

The unicellular tetrahymena contains inositol phospholipids (PI, PIP, PIP₂) and GPIs. Treatment with 10^–5M insulin decreases the total³H-inositol incorporation and incorporation into PI. 24 h after 10^–6M insulin treatment there is an elevation of these parameters. Second treatment with 10^–6M insulin doubles and 10^–5M decreases these levels. This means that the effect on phosphoinositide turnover by insulin in Tetrahymena is rather concentration dependent. Inositol incorporation into GPIs is also influenced by insulin.     

Inhibition of insulin receptors by vanadate and ouabain

Insulin binding studies were performed, using cells from 5 non-obese, non-diabetic subjects, on four separate days: 2 were paired control studies to demonstrate precision, and 2 other sets were binding studies in which one incubation solution was a control and the other contained either vanadate, (10(-4) M) or ouabain (10(-4) M). For both substances tracer binding of 125I insulin was reduced significantly, 27% by vanadate and 30% by ouabain. Furthermore, at all points on the binding curve these substances inhibited binding by 18-98%, in a pattern consistent with reduced receptor number. The concentrations of vanadate or ouabain which we used did not change cell volume or inhibit trypan blue dye exclusion, as an index of cell viability. Because vanadate and ouabain inhibit Na+K+ATPase and have largely dissimilar effects on a variety of cell systems, our observations may reflect specific involvement of Na+K+ATPase in binding or closely related processes.     

The binding of diazepam in the mitochondria of Tetrahymena pyriformis as detected by quantitative high resolution autoradiography

Tritiated diazepam accumulates mainly in the mitochondria of the unicellular Tetrahymena. This is the case in both a single (the first encounter) and a repeated (one day or a week after the first) administration of the drug. When imprinting of Tetrahymena by diazepam (the first encounter) is followed a week later by the administration of the labelled drug, the membranes of the vesicles, too, show the appearance of label. Regarding the studies presented here, the unicellular Tetrahymena also contain diazepam receptors in the mitochondria as suggested for cells of higher rank animals.     

Influence of imprinting with A and B chains of insulin on binding and functional changes in tetrahymena

Insulin and its A and B chain increased the quantity of intracellular PAS-positive material (glycogen) in tetrahymena, whereas the combined A+B chains decreased it. Imprinting—previous interaction—with insulin, its A and B chains in themselves and with the A+B chain increased the hormone binding capacity of tetrahymena, but the functional effect of imprinting (storage or breakdown of glycogen) showed a different tendency with insulin and A+B chain on the one hand, and A chain and B chain on the other. Since the imprinting potential of a molecule promotes the induction of receptor formation, the fact remains that both component chains of insulin were able to act as potential imprinters, although the A chain was superior to the B chain in this respect throughout, and combined treatment with the A+B chain ultimately induced the formation of a similar binding site as insulin itself.     

Impact of starvation on hormone binding and hormonal imprinting in Tetrahymena.

Tetrahymena cells maintained (starved) in a physiological salt solution showed a considerable decrease in insulin binding capacity. The cells previously imprinted with insulin showed a comparable relative binding decrease after a similar exposure. This change was reversible by prolonged maintenance in plain nutrient medium after which the binding capacity of the imprinted cells increased appreciably over the control. The cells maintained (starved) in salt solution for 2 h were no longer imprintable with insulin; it follows that prolonged starvation not only reduced the recognition potential, but also extinguished the imprintability of Tetrahymena cells.     

Protein binding to expanded telomere repeats in Tetrahymena thermophila

The ends of eukaryotic chromosomes are protected by DNA-protein structures called telomeres. Telomeric DNA is highly conserved, usually consisting of long tracts of a repeating G-rich sequence. Tetrahymena thermophila telomeric DNA consists of alternating blocks of GGGG and TT sequences (i.e. a G4T2 repeat sequence). We examined the relative importance of the guanine and thymine elements of the repeat sequence in promoting in vitro binding by T. thermophila proteins. We identified single- and, for the first time, double-stranded telomere binding activities from a crude T. thermophila protein extract and tested the binding of these activities to altered telomere repeat sequences. All deletions or substitutions made to the guanine element virtually abolished binding, indicating that four G's are essential for recognition by the binding activity. However, G's alone are not sufficient for efficient binding, as elimination of the thymine element dramatically reduced binding. By contrast, substantial expansion of the thymine element was well tolerated, even though one such change, G4T4, is lethal in vivo. We tested up to a four-fold expansion of the thymine element and found that highly efficient binding was still achieved. These results suggest a minimal recognition sequence for T. thermophila proteins, with the T element providing an important spacer between essential G elements.