Effect of long-term insulin exposure on insulin binding in Tetrahymena pyriformis

Tissue & Cell, 1995 27 (5) 585-589 © 1995 Pearson Professional Ltd.

Effect of long-term insulin exposure on insulin binding in Tetrahymena

pyriformis

G. K. Christopher, C. A. Sundermann

Abstract. Previous studies have indicated that Tetrahymena pyriformis can bind the vertebrate hormone insulin,. Conventional microscopic studies were conducted to determine the effect of acute and long-term (LTE) insulin exposure on insulin binding. Stock cultures included cells never exposed to insulin and cultures grown in medium containing 6 mg/ml insulin. Logarithmic cultures were exposed to porcine insulin concentrations of 0 and 6 mg/ml for 1 h (insulin treated, (IT)) after 0, 48 h, 1, 3, or 6 months of LTE insulin exposure. 24 h after the 1 h insulin treatment, the cells were fixed, exposed to porcine insulin (antigen), processed immunocytochemically using a primary antibody to porcine insulin and a secondary antibody immunecytochemistry kit, and examined for staining intensity by video image analysis. Morphological observations confirm that T. pyriformis does bind insulin whether or not the cells have had prior exposure to insulin. IT increases insulin binding (up-regulation) in previously unexposed cells (control, P<0.01) and produces a further amplification in cells having prior acute exposure (48 h) to insulin (P<0.01). However, LTE exposure to insulin (1, 3 and 6 months) caused a decrease in insulin binding (down-regulation) after IT (P <0.01) such that LTE-IT cells were not different from control cells following 1, 3 or 6 months of chronic insulin exposure to insulin. Staining intensity was not different between IT cells and cells cultured with insulin throughout the six month study. Results suggest that insulin binding sites of T. pyriformis are subject to regulatory processes similar to those of metazoans. Keywords" Hormonalimprinting, insulin, Tetrahymenapyriformis

Introduction The ciliated protozoan Tetrahymenapyriformis can be easily axenically cultured and has become a widely used model eukaryotic cell. T. pyriformis specifically binds the vertebrate hormone insulin (Kovfics and Csaba, 1990) which then causes alterations in glucose (Csaba and Lantos, 1975) and amino acid (Fulop and Csaba, 1990) uptake, glycogen metabolism and concentrations of second messengers (Kovfics, 1986). Primary exposure Department of Zoology and Wildlife Sciences, Auburn University, AL 36849, USA Received 28 February 1995 Accepted 12 June 1995 Correspondence to: Gayle K. Christopher/C.A. Sundermann, 101 Cary Hall, Auburn University, AL 36849, USA. Tel: (334) 844-3929; Fax: (334) 844-4065; e-mail [email protected].

to insulin results in an increased hormone binding capacity upon subsequent exposure (Christopher and Sundermann, 1992; Csaba, 1980, 1984). This insulin inducible phenomenon is termed hormonal imprinting (Csaba, 1980, 1984). Primary interaction with insulin is thought to result in a gene level event as well as strengthen existing membrane structures resulting in amplification of binding capacity (Csaba 1984). The hormonal imprint can be transferred to unexposed cells by a released 'transfer factor' present in the growth medium (Csaba and Kov/tcs, 1987). This factor may be a protozoan insulin (de Pablo et al., 1986) and could represent a protozoan growth factor necessary for growth and maintenance of cultures (Wheatley et al., 1993). The hormonal imprint has been detected in 500 subsequent generations after initial exposure (Csaba 585

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et al., 1982). Amplified membrane insulin binding sites are subject to regulation with longer exposure to insulin (up to 48 h) causing a decrease in insulin binding in subsequent generations as compared to the optimum 1 h exposure (Csaba and K6hidai, 1986). Ability to imprint also varies with the age of the culture (K6hidai et al., 1986). The objective of the present study was to investigate the effect of LTE insulin exposure (1, 3 and 6 months) on insulin binding capacity of T. pyriformis as compared to an acute exposure of 48 h.

Materials and methods Cultures Original cultures of T. pyriformis were obtained from the American Type Culture Collection (#30327, Simon strain, Phenoset A, amicronucleate) and maintained axenically in Medium 357 (5.0 g proteose peptone, 5.0 g tryptone, 0.2 g K2HPO4, 1 1 distilled water, adjust pH to 7.2 before autoclaving) at 28°C. Stock cultures were those grown in the absence of insulin (control) or in medium containing 6 mg/ml insulin for either acute (48 h) or LTE exposure (1, 3 or 6 months). These cultures were transferred weekly to fresh medium without or with insulin. After 0, 1, 3, and 6 months, cultures from both media treatments were inoculated to undergo insulin treatment (IT) or serve as untreated control. Insulin treatment IT was accomplished by the method of Kovfics and Csaba (1990). Briefly, cells from logarithmic cultures (48 h from transfer) in centrifuge tubes were exposed to 0 or 6 mg/ml porcine insulin (Sigma Chemical Co., St. Louis, MO) in Medium 357 for 1 h at 28°C, washed in Medium 357, and returned to plain Medium 357 and incubated at 28°C. Preparation for immunocytochcmistry After 24 h at 28°C, cells were pelleted and fixed in 4% formalin (pH7.2) for 5min. The fixed cells (2.5-3.0 × 105 cells/ml) were washed by centrifugation and resuspended three times in phosphate buffered saline (PBS), incubated for 1 h with 0.2 mg/ml porcine insulin in PBS at 37°C, washed three times in 0.01 M phosphate buffer (pH 7.4), dropped onto slides and air dried. Immunocytochemistry Immunocytochemistry was accomplished using a primary antibody (Ab 1) to porcine insulin made in guinea pig and a secondary antibody-avidin-biotin-peroxidase kit (Sigma Chemical Co., St. Louis, MO). The previously published staining procedure (Christopher and Sundermann, 1992) consisted of a quenching step followed by an incubation with blocking reagent to prevent nonspecific binding of the Ab 1. Slides were then exposed to the Ab 1 for 1 h in a humidified chamber at 37°C. One slide from each treatment was not exposed to Ab ~.

After a 1 h incubation with the secondary antibody (Ab 2, rabbit anti-guinea pig) coupled to biotin, slides were treated with avidin-coupled peroxidase. A color reaction was produced using 3-amino-9-ethylcarbazole (AEC) as chromogen and hematoxylin as a counterstain. Except after the blocking reagent (no wash) and during the color development stages (H20 wash), slides were washed in Koplin jars containing PBS between each step in the procedure. Coverslips were then mounted on slides using an aqueous mounting medium. Ab 1 was deleted in one slide from each insulin concentration as a control for the activity of endogenous peroxidases and non-specific binding. Light microscopy and analysis Stained slides were viewed and analyzed for staining intensity with an inverted microscope and a video image analysis program using an absolute grey scale (JAVA 3.1, Jandel Scientific, Corte Madera, CA). Stained cells produced lower numbers (lower light transmittance) than background and heavy staining produced lower numbers than light staining. Fifty observations of cellular staining intensity were taken per slide (treatment). The mean of these was corrected for background staining by dividing by the mean background intensity of that slide to produce a positive number. The corrected measures of staining intensity for each slide were subjected to MEANS analysis using SAS (SAS Institute Inc., Cary, NC) and a randomized block design with trial as the block. Data from four trials were included in the analysis.

Results Increased insulin binding occurred in IT cells as evidenced by an increased staining intensity in an IT cell (Fig. 1B) as compared to a control cell (Fig. 1A). Cells that were exposed to insulin for 48 h (acute exposure) had similar staining to IT cells (Fig. 1C), while IT produced a further amplification of insulin binding in cells that had prior acute exposure to insulin (Fig. 1D). Staining of LTE cells (Fig. 1E) appeared similar to IT and AE cells at 1, 3 & 6 months. However, LTE-IT cells (Fig. 1F) had decreased staining intensity and appeared similar to control cells. Video image analysis of immunostained cells confirmed the results obtained by visual inspection in both acute and LTE exposure studies. IT increased insulin binding (up-regulation) in previously unexposed cells (control, P < 0.01 ) and produced a further amplification in cells having prior acute exposure (48h) to insulin (P<0.01) (Fig. 2A). However, LTE exposure to insulin (1, 3 and 6 months) caused a decrease in insulin binding (down-regulation) after IT (P < 0.01 ) such that LTE-IT cells were not different from control cells at 1, 3 and 6 months (Figs. 2B-D, respectively). IT of LTE cells resulted in decreased staining

LONG-TERM INSULIN EXPOSURE, T E T R A H Y M E N A

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587

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Q Fig. 1 Photomicrographs of A. control; B. insulin treated (IT); C. acute exposure (AE); D. AE-IT; E. long-term exposure (LTE, 1 mouth), and F. LTE-IT immnnostained cells. Notice cilia (arrow), oral area (arrow head) and nucleus (n). Bar = 10 gm.

intensity (P < 0.01). Staining intensity was not different between IT cells and cells cultured with insulin either acutely or chronically at 0, 1, 3 or 6 months.

Discussion Observations confirm previous reports (Christopher and Sundermann, 1992; Csaba, 1980, 1984; Csaba and Lantos, 1975) that T. pyriformis does bind insulin whether or not the cells have had prior exposure to insulin. Insulin binding capacity of cells appears to be dependent on IT concentration (Christopher and Sundermann, 1992) and can be inhibited by the addition of unlabeled insulin (Christopher and Sundermann, 1992; Kovfics and Csaba, 1990). IT increases insulin

binding (up-regulation) in previously unexposed cells and produces a further amplification in cells having prior acute exposure (48 h) to insulin. This is consistent with results from a previous report (Csaba and K6hidai, 1986) where optimum exposure time was determined to be 1 h. However, LTE exposure to insulin caused a decrease in insulin binding (down-regulation) after IT, such that LTE-IT cells were not different from control cells at 1, 3 and 6 months. In a previous study (Csaba and K6hidai, 1986) down-regulation was seen after as little as 3 to 6 h of insulin exposure, a trend that was reversed if the cells were transferred to plain medium for 24 or 48 h. In that study, down-regulation persisted if the cells remained in insulin containing media, but the study period only lasted 48 h. LTE exposed cells in the present study had an increased insulin binding capacity, compared to unexposed control cells, regard-

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CHRISTOPHER AND SUNDERMANN

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Fig. 2 A.Light transmittance for control, insulin treated (IT); acutely insulin-exposed(AE) and AE-IT immunostained cells in the presence and absence of Ab1. Because stained cells transmitted less light and had lower absolute intensity readings on an absolute grey scale, lower numbers indicate a higher degree of staining. Values are means for trials (n = 4) with the mean obtained from each trial consisting of the mean of 50 observations corrected for background. Bars with different letters differ statistically. (P < .01, S.E. for control, IT, AE, and AE-IT = + 0.010). B-D. Light transmittance for control, insulin treated (IT); long-term insulin-exposed(LTE, i, 3, and 6 months, Fig. 2B-D, respectively)and LTE-IT immunostained cells in the presence and absence of Ab~. (For Fig. 2B, a P b, P < .05, a P c, P < .01, b P c, P < .01, S.E. for control, IT, LTE, and LTE-IT = _+0.024). (For Fig. 2C, a P b, P < .07, a Pc, P < .01, b pc, P < .01, S.E. for control, IT, LTE, and LTE-IT = ± 0.023). ( For Fig. 2D, P< .01, S.E. for control, IT, LTE, and LTE-IT = _+0.020). less o f the length of insulin exposure. However, insulin t r e a t m e n t o f LTE cells resulted in decreased staining intensity. Perhaps the m e c h a n i s m s involved in regulating b i n d i n g capacity are m o r e sensitive or up-regulated in L T E cells. Staining intensity was n o t different between I T cells a n d cells cultured with insulin either acutely or chronically at 0, 1, 3 or 6 m o n t h s . Acute exposure consisted

of 48 h while chronic exposure consisted of weekly transfers to i n s u l i n - c o n t a i n i n g m e d i a for 1, 3, or 6 m o n t h s . Conceivably, insulin in the m e d i u m could have degraded or have been phagocytosed in the time between the weekly transfers such that cells formed late in the week experienced a p r i m a r y exposure to insulin with the next transfer to i n s u l i n - c o n t a i n i n g media. Therefore, these cells m i g h t be considered n o n - i m p r i n t e d at the

LONG-TERMINSULINEXPOSURE, TETRAHYMENA 589

time of transfer except that new cells result by fission of existing imprinted cells and the imprint can be transferred to un-exposed cells by a 'transfer factor' in the media (Csaba and Kov~tcs, 1987; Csaba et al., 1982). In addition, because the imprint information is not equally divided between sister cells (Csaba et al., 1989) a great variability in insulin binding might be expected in insulin-exposed cells. By visual inspection the variability in staining intensity (insulin binding) seemed to be greater among control cells than those that had been exposed to insulin for any length of time (unpublished observation). Apparently, insulin exposure not only amplifies but equalizes the insulin binding capacity of T. pyriformis cells. Insulin receptors of m a m m a l s are subject to the processes of up- and down-regulation, involving internalization and recycling (Carpentier, 1989). In T. pyriformis, inhibition of endocytosis, membrane recycling and lysosomal activity inhibits hormone binding and imprint development, suggesting that membrane circulation is involved in the regulation of insulin binding sites (Kov~ics and Csaba, 1988). Cilia are necessary for hormonal imprinting but deciliation does not abolish the imprint (Darvas et al., 1988). Previous studies (Christopher and Sundermann, 1992; Csaba and Lantos, 1975; Csaba et al., 1989) and the present study indicate that the hormone binding sites of T. pyriformis are

subject to regulation in response to hormone exposure. The type of regulation depends on hormone concentration (Christopher and Sundermann, 1992; Csaba et al., 1982) the length of primary exposure (Csaba and K6hidai, 1986) and age of the culture (KOhidai et al., 1986). Insulin-like substances have been isolated from T. pyriformis (de Pablo et al., 1986) and insulin treatment produces measurable changes in phagocytosis and cellular metabolism (Csaba, 1980; Csaba and Lantos, 1975; Ft~16p and Csaba, 1990; Kovfics, 1986) and m a y be necessary for growth of the culture (Wheatley et al., 1993). Perhaps insulin functions in T. pyriformis as a regulator of feeding behavior and metabolism just as its action in metazoans is metabolic. In this study, we report the effects of acute and LTE exposure to insulin on insulin binding capacity of T. pyriformis and suggest that insulin binding sites are subject to regulatory processes similar to those seen in metazoans. ACKNOWLEDGMENTS We thank D r Bob Kemppainen of the Auburn University School of Veterinary Medicine for the use of the video image analysis system. This work was supported by a grant from the National Science Foundation (DCB-9018047).

REFERENCES Carpentier, J.L. 1989. The cell biology of the insulin receptor. Diabetologia, 32, 627-635. Christopher, G.K. and Sundermann, C.A. 1992. Conventional and confocal microscopic studies of insulin receptor induction in Tetrahymenapyriformis. Exp. Cell Res., 201,477-484. Csaba, G. 1980. Phylogeny and ontogeny of hormone receptors: the selection theory of receptor formation and hormonal imprinting. Biol. Rev., 55, 47-63. Csaba, G. 1984. The present state in the phylogeny and ontogeny of hormone receptors. Horm. Metabol. Res., 16, 329-335. Csaba, G. and Lantos, T. 1975. Effect of insulin on glucose uptake in protozoa. Experientia, 31, 1097-1098. Csaba, G. and K6hidai, L. 1986. Modelling the insulin receptor in the Tetrahymena.Time dependence of receptor formation, downregulation and imprinting. Acta Protozool., 25(3), 331-338. Csaba, G. and Kovfics,P. 1987. Transmission of hormonal imprinting in Tetrahymenacultures by intercellular communication. Z. Naturforsch, 42c, 932-934. Csaba, G., Kovfics, P. and L~tszl6, V. 1989. Discrepancy in hormone binding and information transfer between sister cells of Tetrahymena clones. Cell. Molec. Biol., 35(5), 511-514. Csaba, G., N6meth, G. and Vargha, P. 1982. Influence of hormone concentration and time factor on development of receptor memory in a unicellular (Tetrahymena)model system. Comp. Biochem. Physiol., 73B(2), 357-360. Darvas, Z., Nozawa, Y. and Csaba, G. 1988. Cell-to-cell transmission of hormonal imprinting in deciliated and regenerated Tetrahymena.Cytobios. 54, 49-51.

de Pablo, F., Lesniak, M.A., Hernandez, E.R., et al. 1986. Extracts of protozoa contain materials that react specificallyin the immunoassay for guinea pig insulin. Horm. Metabol. Res., 18, 82-87. Ft~16p, A.K. and Csaba, G. 1990. Effect of insulin imprinting on the 3H-amino acid uptake of the Tetrahymena.Acta Physiol. Hung., 75(4), 261-265. K6hidai, L., Thomka, M. and Csaba, G. 1986. Age of the cell culture: A factor influencing hormonal imprinting of Tetrahymena. Acta Microbiol. Hung., 33(4), 295-300. Kov~cs, P. 1986. The mechanism of receptor development as implied from hormone imprinting studies on unicellular organisms. Horm. Metab. Res., 42, 770-775. Kov/tcs, P. and Csaba, G. 1988. Effect of inhibition of endocytosis, recycling and lysosomal activity on the insulin binding capacity and imprintability of Tetrahymena.Acta Physiol. Hung., 71 (2), 315 322. Kovfics, P. and Csaba, G. 1990. Evidence of the receptor nature of the binding sites induced in Tetrahymenaby insulin treatment. A quantitative cytofluorimetrictechnique for the study of binding kinetics. Cell Biochem. Funct., 8, 49-56. Wheatley, D.N., Christensen, S.T., Schousboe, P. and Rasmussen, L. 1993. Signalling in cell growth and death: adequate nutrition alone may not be sufficient for ciliates. Cell Biol. Internatl., 17(9), 817-82.