romp. ~iochem. Physiol. Vol. SOA, No. 1, pp. 41-42, 1985 Printed in Great Britain

0300-9629185 $3.00 + 0.00 0 1985 Pergamon Press Ltd

IMMUNOLOGICAL EVIDENCE OF THE INDUCED INSULIN RECEPTOR IN TETRAHYMENA *

P. KOV~CS, G. CSABA and EDIT BOHDANECZKY Department of Biology, Semmelweis University of Medicine, H-1445, Budapest, Hungary

(Received 6 April 1984)

Abstract-1. Comparative studies on binding of rabbit serum (IgG) antibodies against intact and insulin-pretreated Tetrahymena cells revealed an increased binding affinity of the latter to rat hepato- cellular insulin receptors.

2. The experiments demonstrate that presence of insulin is a prerequisite to the formation of Tetrahvmena’s hormone recenters. which seems to differ little if at all between species representing differeit levels of phylogenesi’s.

INTRODUCIION

The unicellular Tetrahymena, although it does not itself possess a hormonal system, binds certain hor- mones of higher vertebrates and interacts with these (Csaba, 1980, 1981). Since a unicellular organism may not possess preformed receptors for all verte- brate hormones hitherto studied, it has been postu- lated that such receptors could arise under hormonal influence from certain subunits of the dynamic mem- brane (Csaba, 1980). The receptor structures so formed persist long, to judge from the experimental fact that the first interaction with the hormone (hor- monal imprinting) accounts for increased binding on subsequent exposure(s). As yet only indirect evidence has been presented in support of this implication (Csaba, 1980; Csaba et al., 1982a,b). Direct evidence of the necessity of hormonal presence for receptor formation has emerged from the present experiments with hyperimmune sera prepared in rabbits by im- munization with insulin-pretreated and intact Tetra- hymena cells.

MATERIALS AND METHODS

White New Zealand rabbits were immunized subcuta- neously with Tetrahymena pyriformis GL cells (106/ml) treated or not treated with 10m6 M insulin (Insulin Semilente MC, Novo, Denmark) for 24 hr in the logarithmic phase of growth. The first immunizing dose was combined with Freund adjuvant. Booster doses were administered at l-week intervals, using Tetrahymena cells suspended in Losina solution. One day after the 4th booster dose, blood samples were withdrawn from the ear vein to determine the antibody titer by the immobilizing effect on Tetruhymena cells. The rabbits were then killed by bleeding, the IgG fraction of the blood serum was isolated by precipitation with (NH&SO, and purified on a DEAE+zellulose column. Part of the purified antibody was labeled with fluorescein isothiocyanate (FITC, BDH, England) and part was used for direct treatment.

Monolayer cultures were prepared in H-tubes from neo- natal rat (Wistar CFY) hepatocytes isolated by tryp- sinization. The culture medium, Parker 199 + 15% FCS, was exchanged every other day. At 6 days of culturing the

*Supported by the Scientific Research Council, Ministry of Health, Hungary, 16/ I-04/432/Cs.

monolayers were fixed in 4% formalin (in PBS) and incubated in a moist chamber in the presence of 1 mg/ml antibody to intact (TAB) or insulin-treated Tetrahymena (ItTAB). After incubation the cells were washed in two changes of PBS. Subsequently part of the pre-exposed cultures was treated with FITC-labeled insulin and part with FITC-labeled rabbit antibody to rat hepatocellular insulin receptors (FITC-RAB) (Csaba et al., 1984). The protein concentration and FITC/protein ratio of the FITC-labeled insulin and FITC-RAB were 0.5 mg/ml, 0.12; and 0.4 mg/ ml, 1.8 respectively. Incubation in the presence of the FITC-labeled materials also lasted 1 hr, and was followed by three washes in PBS and one rinse in distilled water.

In another experiment rat hepatocyte monolayer cultures prepared as above were treated on the 5th day of culturing with low6 M insulin for 4 hr, returned to plain medium for 24 hr, fixed as above and incubated in the presence of FITC-labeled TAB or ItTAB for 1 hr (both labeled materi- als contained 0.4 mg/ml protein; the FITC/protein ratio was assessed as 1.78 and I .90 for TAB and ItTAB respectively). After incubation the cultures were washed in three changes of PBS and rinsed in distilled water. An appropriate control series was set up without insulin treatment.

The cultures were dried and examined for intensity of nuclear and cytoplasmic fluorescence in a Zeiss Fluoval cytofluorimeter connected with an HP 41 calculator, which was programmed to determine mean values for the groups, standard deviation and significance of difference from the control (by r test and variance analysis). Twenty cells were assayed for fluorescence in each group, and three replica assays were performed for determination of the mean values.

RESULTS AND DISCUSSION

Hepatocytes already possess insulin receptors at birth (Blazquez et al., 1976). The IgG formed to insulin-treated Tetruhymena cells (ItTAB) inhibited the binding of FITC-labeled insulin (Fig. 1) to the hepatocellular membrane more efficiently than did the antibody to intact Tetruhymena (TAB). Similar observations were made with the FITC-labeled anti- body to the insulin receptor, whose binding configur- ation corresponds to that of insulin and whose bind- ing to the insulin receptor of Tetruhymena was sub- stantiated earlier (Csaba et al., 1984). Pretreatment of Tetruhymenu with insulin obviously enhanced the binding of antibodies having affinity for the liver insulin receptor. Similar conclusions emerged from

41

42 P. Kovics et al.

ii? 0 Nucleus q Cytoplasm

FITC-insulin FITC-RAB

Fig. 1. Binding of FITC-insulin and FITC-RAB to rat hepatocytes treated after fixation with rabbit I&i against intact (TAB) and insulin-nretreated Tetrahvmena cells (ItTAB)‘. s L Significance between

P < 0.01.

2 ,a0 [3 Maclaus; q Cyto~losm

z

f 140 6 b 2 120

B

60 Insulin- trwted

TAB and ItTAB:

FITC-TAB FITC- ItTAB

Fig. 2. Binding of FITC-TAB and FITC-ItTAB to rat hepatocytes treated with insulin in uiuo before fixation.

Significance to control: s = P < 0.01; y = P i 0.05.

the second experiment (Fig. 2), in which the hepa- tocyte culture was treated with insulin in unfixed state to enhance insulin receptor formation (hormonal imprinting). The insulin-pretreated hepatocytes bound less TAB but considerably more ItTAB than the untreated control hepatocytes, indicating that amplification of the hepatocellular membrane recep-

tors with insulin accounted for an increase in hepa- tocellular binding capacity for antibodies to insulin- amplified Tetrahymena.

The formation of hormone (insulin) receptors in the membrane of the Tetrahymena was provoked by the hormone (insulin) itself, judging from the specific binding of antibodies against the receptor so formed to rat hepatocellular membrane. Thus presence of the hormone is a fundamental prerequisite of receptor formation and, as implied previously (Ginsberg et al., 1977; Muggeo et al., 1979a, b; Csaba et al., 1984) the hormone receptors are fairly stable structures which differ little, if at all, between species representing different levels of phylogenesis.

REFERENCES

Blazquez E., Rubalcava B., Montesano R., Orci L. and Unger R. H. (1976) Development of insulin and glucagon binding and the adenylate cyclase response in liver mem- branes of the prenatal, postnatal, and adult rat: evidence of glucagon “resistance”. Endocrinology 98, 1014-1023.

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. (1981) Ontogeny and Phylogeny of Hormone Receptors. Karger, Basel.

Csaba G., Ntmeth G., Juvancz I. and Vargha P. (1982a) Involvement of selection and amplification mechanisms in hormone receptor development in a unicellular model system. Biosystems 15, 59-63.

Csaba G., Ntmeth G. and Vargha P. (1982b) Development and persistence of receptor “memory” in a unicellular model system. Exp. Cell. Biol. SO, 291-294.

Csaba G., Kovacs P. and Inczeti-Gonda A (1984) Insulin binding sites induced in the Tetrahymena by rat liver receptor antibody. Z. Naturforsch. 39c, 183- 185.

Ginsberg B. H., Kahn C. R. and Roth J. (1977) The insulin receptor of the turkey erythrocyte: similarity to mam- malian insulin receptors. Endocrinology 100, 520-525.

Muggeo M., Ginsberg B. H., Roth J., Neville G. M., Meyts P. de and Kahn C. R. (1979a) The insulin receptor in vertebrates is functionally more conserved during evo- lution than the insulin itself. Endocrinology 104, 1393-1402.

Muggeo M., Obberghen E., Kahn C. R. von, Roth J., Ginsberg B. H., Meyts P. de, Emdin S. 0. and Falkmer S. (1979b) The insulin receptor and insulin of the Atlantic hagtish. Diabetes 28, 175-181.