Effect of Insulin and Adrenaline on the Fe Transferrin Uptake of Lactating Mouse Mammary Gland Cells D. A. Moutafchiev and L. M. Sirakov Department of Biochemistry, Medico-biological Institute, Medical Academy, Sofia, Bulgaria
The results of insulin action (0.4 IU per mouse) are demonstrated in intact animals only. This action leads to a higher uptake of 59Fe. rabbit transferrin of isolated cells from lactating mouse mammary gland. It is suggested that most inactive transferrin receptors in the cell membrane are incorporated by the hormone action or some new receptors are synthesized. On the contrary, adrenaline in a dose 0.5 ug per animal demonstrated an opposite effect — a lower uptake of 59Fe. human transferrin from lactating mouse mammary gland. This is probably due to a redistribution of some part (about 28 %) of the iron. Instead of flowing to the mammary gland it flows towards other organs for overcoming the stress situation. An alternative explanation could be the inhibition of endogenous insulin secretion by adrenaline. From our data it follows that insulin and adrenaline have an antagonistic effect on regulation of Fe transport in lactating mouse mammary gland. Key words Mammary Gland — Insulin — Adrenaline — 59 Fe. Transferrin Uptake
Introduction Insulin is an anabolic hormone which is necessary for the growth and differentiation of mammary gland cells {Ichinose and Nandi 1966). This was also proved by experiments in vivo {Freeman and Topper 1977). Turkington, Brew, Vanaman and Hill( 1968) found that for the biosynthesis of galactosyltransferase and lactalbumin in the mouse mammary gland explants the presence of insulin, hydrocortisone and prolactin is necessary. Apostolova, Sirakov and Barth (1976) demonstrated insulin receptors in a crude membrane fraction from lactating white mice. Insulin stimulated iron uptake from the epididymal fat cells through a rearrangement of the transferrin receptors from the inner membrane surface to the outer {Davis, Corvera and Czech 1986).
Horm. metab. Res. 24 (1992) 420 - 423 © GeorgThieme Verlag Stuttgart-New York
Adrenaline release during stress plays an adapting role in extreme situation by elevating glucose level in blood. The effect of adrenaline on Fe transport from blood to mouse mammary gland is unknown. The receptors for Fe. transferrin are found in plasma membranes from lactating rabbit mammary gland cells {Moutafchiev, Shisheva and Sirakov 1983). The anabolic effect of insulin and antistress effect of adrenaline draw our attention to its possible influence on the uptake of transferrin iron from the lactating mouse mammary gland cells. Materials and Methods Isolation of transferrin from rabbit serum, its labelling with59Fe.ascorbinate (from Akademie der Wissenschaften, Germany) and a procedure of isolation of lactating mouse mammary gland cells were described earlier (Moutafchiev, Shisheva and Sirakov 1983). Human transferrin was purchased from "Serva", Germany. All other reagents were of analytical research grade from "Fluka" (Switzerland) and "Merck" (Germany). Insulin-Semilente MC was from "NOVO", Copenhagen, Denmark, with concentration 40 IU/ml. Adrenaline was from "Sigma", USA. White mouse strain H from 7 to 12 day after delivery was used. Mothers and litters were kept together for lactation stimulation. The dose of insulin of 0.4 IU was used as suggested by Baldwin and Martin (1968). 16 hours after injection (during that time the animals were given 5 % sucrose solution) mice were killed and the glands were removed by excision. Isolated cells were incubated with 59Fe. rabbit transferrin at 37 °C for 45 min {Moutafchiev, Shisheva and Sirakov 1983). In in vitro experiments cells were incubated with different doses of insulin diluted in Krebs-Ringer Buffer pH 7.4, at 37 °C for 30 min. The buffer contained: NaCl 0.125 mmol/1, KC1 5 mmol/1, KH2PO3 1.25 mmol/1, MgSO4 7H 2O 1.2 mmol/1, NaHCO3 3.8 mmol/1, NaH2PO4-2H 2O 9 mmol/1. The incubation medium contained the same buffer with the addition of 2% bovine serum albumin (V fraction, from Koch-Light, England). After incubation the cells were treated for 45 min at 37 °C with59Fe.transferrin and the reaction was stopped with ice-cold 0.9% NaCl. The cells sedimented after centrifugation and were counted in gamma counter Rack Gamma II 1270, LKB, Finland. Mice were injected i.m. with adrenaline 0.5 ug and after 5 min were killed. Control mice were injected with 0.1 ml of saline and further treated by the same way. Vitality of the cells, checked by Trypan-blau (Hunter and Greenwood 1962), was about 90%. Cells were counted in a Burker camera. The results were calculated by the variation analysis and the Student-Fisher test.
Received: 17 July 1991
Accepted: 30 Jan. 1992 after revision
Downloaded by: Universite Laval. Copyrighted material.
Summary
Fe Transferrin Uptake of Mammary Gland Cells
Fig. 2 Binding of 59Fe. rabbit transferrin in dependence of increasing number of cells. Abscissa: number of the cells ( x 106). Added 10.75 9 g 59 Fe. rabbit transferrin calculated as Fe. Ordinate: bound 59Fe in x 10 _9 g. Data from three control ( • ) and treated with 0.4 IU insulin in vivo (O) animals with ±SE.
Downloaded by: Universite Laval. Copyrighted material.
Fig. 1 Influence of insulin in vivo (0.4 IU) in dependence from increasing concentration of 59Fe. rabbit transferrin. Abscissa: increasing concentration of 59Fe. Tf calculated as x 10- 9 g. Ordinate: bound 59Fe. in x 10- 9 g from106cells. Data from three control ( • ) and three treated (O) animals with ±SE.
Horm. metab. Res. 24 (1992) 421
Fig. 3 Data from three experiments in vitro upon increasing concentrations of added insulin in x 10- 9g (abscissa). Ordinate: bound59Fein x 10- 9 g with +SE. The first point of the curve was control ( • ) .
Results Isolated mammary gland cells from insulintreated (0.4 IU) mice bound more labelled 59Fe. rabbit transferrin than cells from control animals (Fig. 1). The Fe-uptake, calculated as ng Fe, was also increased. At constant 59Fe amount (12.66 ng) the binding of Fe. transferrin as a function of the increasing cell number was higher in cells from insulintreated animals than in cells from control ones (p < 0.01, Fig. 2). This shows that when insulin was injected into living mice, it stimulated binding of Fe. transferrin. If insulin was given in vitro in the incubation medium (from 0.1 to 104 ng) for 30 min at 37 °C the results were different (Fig. 3). There was no substantional difference at the range of insulin amounts from 1 to 103 ng. At the highest amount (104 ng) there was a decrease (p < 0.02). Data shown in Figure 1 were presented as a Scatchard plot (1949). There are two receptor sites in control and experimental groups (Fig. 4). The binding capacity and affinity constants are given in Table 1. Data from Table 1 might suggest a negative cooperative effect, i.e. with rising of binding capacity of receptors, the receptor affinity for Fe. transferrin decreases. An adrenaline injection (0.5 ug) in lactating mice brought to a sig-
Fig. 4 Scatchard's plot from data shown in Fig. 1. Abscissa: bound Fe on 106 cells. Ordinate: the ratio bound/free Fe in control ( • ) and treated (O) animals.
Binding capacity (10- 9 gFe) per 10 cells Kaf M- 1
Control I site II site
Experiment I site II site
0.23
1.0
0.55
1.5
2.6 x 10 8
3.2 x 10 6
6.2 x 10 7
4.1 x 10 6
nificant decrease of transferrin iron uptake by isolated mammary cells (Fig. 5). Studies at different amounts of59Fe.transferrin demonstrated that substantial differences were observed at amounts of 0.34 ng (p < 0.01)., 0.90 ng (p < 0.01), and 1.29 ng (p < 0.002) (Fig. 5). Time-dependence of binding is given in Fig. 6, where, again the adrenaline effect is seen with significant differences (p < 0.001) in all points except the first one.
Horm.metab. Res. 24(1992)
D. A. Moutafchiev andL. M. Sirakov
Fig. 5 Binding of Fe from human transferrin from 1.5 x 106 cells of lactating mouse mammary gland of 4 nontreated ( • ) and 8 treated in vivo (O) animals with 0.5 ug adrenaline. Abscissa: added 59Fe x 10- 9 g. Ordinate: bound 59Fein x 10- 9 g with ± SE.
Fig. 7 Scatchard's plot from data shown in Fig. 5. Abscissa: bound 59Fe on 1.5 x 106 cells. Ordinate: ratio of bound-free 59 Fe in control ( • ) and treated (O) animals.
Fig. 6 Dependence of binding of Fe. human transferrin on the time of incubation at 37 °C. Abscissa: time of incubation in minutes. Ordinate: bound 59Fe in x 109 g±SE. Data from 8 treated (O) in vivo with 0.5 ug adrenaline and 4 control ( • ) mice. The number of cells was 1.5 x 10 .
Control I site II site Binding capacity (10- 9 gFe)per 1.58 x 10 6 cells Kaf M-1
1.4
3.07
Experiment I site II site 0.93
1.8
4.92 x 10 7 6.87 x 10 6 1.01 x 10 8 8.7 x 10 6
uptake with a redistribution of transferrin receptors from the intracellular space towards plasma membrane as an answer to insulin action. Such an explanation could not be applied to mammary gland cells with a secretory function. The Scatchard plot (1949) presented in Fig. 7 was drawn from data in Fig. 5. The calculated capacities and affinity constants are presented in Table 2. Here also two types of binding sites are demonstrated for binding of Fe. human transferrin. Discussion In this work we have assumed that cell receptors do not make any difference between labelled and nonlabelled iron atoms bound to transferrin. Therefore a competitive non-labelled transferrin is not used in the experiments. Insulin is responsible for the function and growth of mammary gland {Reithel 1979). Therefore it is useful to study its effect on the transport of such an essential element as iron from blood to milk during lactation. Lactation results in hypoinsulinaemia in rodents {Williamson 1980). Insulin injected in lactating mice causes a higher uptake of transferrin iron from mammary gland cells than in control animals (Figs. 1 and 2). The same effect was found also in epididymal fat cells {Davis, Corvera and Czech 1986). The authors explained this rise of Fe. transferrin
An alternative explanation could be that new receptors are synthesized or activated on the plasma membrane. The mammary gland appears to be much more sensitive to insulin {Jones, Ilic and Williamson 1984) than adipose tissue and muscle, hence it is able to maintain high rates of insulinsensitive processes during lactation despite the prevailing hypoinsulinaemia (Vernon 1989). The mechanism responsible for this enhanced sensitivity to insulin is unknown, but in contrast to the other tissues described previously, the number of insulin receptors of mammary epithelial cells is increased during lactation {Flint 1982; Inagaki and Kohmoto 1982). It is possible that some other processes are involved, such as rising of synthesis of transport RNA {Reithel 1979). As one can see (Table 1) the number of transferrin receptors on the plasma membrane of mammary gland cells is increased and there is a higher uptake of transferrin iron from mammary gland cells in experiments in vivo (Figs. 1 and 2) but not in vitro (Fig. 3). Davis, Corvera and Czech (1986) and this work demonstrated that insulin stimulated at least two different types of cells to accept the transferrin iron through the increase of number of receptors. The behaviour of mammary gland cells in vivo and in vitro suggests that the mechanism of insulin action is still unknown.
Downloaded by: Universite Laval. Copyrighted material.
422
Fe Transferrin Uptake of Mammary Gland Cells
Our data suggest that insulin and adrenaline have opposite effects on the uptake of Fe. transferrin from lactating mammary gland cell. Insulin is known to stimulate protein synthesis in cells. This might explain the higher number of receptor sites in the plasma membrane (Table 1). On the other side, adrenaline inhibits insulin secretion and this might lead to a lower uptake of Fe. transferrin by the cells. With these effects, the hormone may play a significant role for adaptation to stress. It is also possible that they regulate the flow of iron to the mammary gland of lactating mouse. Regoeczi and Hatton (1980) suggested that use of heterologous transferrin is a matter of convenience. They found good correlation between parameters in different animals using heterologous transferrin. Our previous experiments (unpublished data) show that there are two binding sites on the plasma membrane of mouse mammary gland cell for the heterologous transferrin while there is only one site for the homologous one. In Figs. 4 and 7 and Tables 1 and 2 it can be seen that both iron-binding sites of transferrin (from rabbit and human origin) gave its iron to cell receptors. The second iron binding site of both transferrins is a better donor for Fe in experimental and control animals. Thus, the heterologous transferrins transfer its iron to cell receptors in coincidence with hormonal action. The absence of peak in Fig. 6 shows that adrenaline treated cells of mammary gland also keep their capability to absorb iron however with a lower rate than controls. This shape of binding curve probably represents the irreversibility of the iron flow from blood to milk, the problem has already been discussed in detail in our earlier work (Moutafchiev, Shisheva and Sirakov 1983). Our experiments demonstrate new effects of insulin and adrenaline unknown for the lactating mammary gland. They include an opposite effect on iron transport from Fe. transferrin in blood to milk. Acknowledgements This study was supported by grant No. 268 from the State Committee of Science (Bulgaria).
423
References Apostolova, J., L. Sirakov, T. Barth: Insulin receptors in lactating mouse mammary gland. Collection Czechoslov. Chem. Comm. 41:3830-3836(1976) Baldwin, R., R. Martin: Protein and nucleic acid synthesis in rat mammary gland during early lactation. Endocrinology 82: 1209-1216 (1968) Davis, R., S. Corvera, M. Czech: Insulin stimulates cellular iron uptake and causes the redistribution of intracellular transferrin receptors to plasma membrane. J. Biol. Chem. 261:8708-8711 (1986) Flint, D. J.: Insulin binding to rat mammary gland at various stages of cell isolation and purification. Mol. Cell. Endocrinol. 26: 281-294 (1982) Freeman, C, Y. Topper: Progesterone and insulin in the growth and differentiation of mouse mammary epithelium in vivo. Fed. Proa, Fed. Am. Soc. Exp. Biol. 36:925 (1977) Hunter, W., F. Greenwood: Preparation of Iodine-131 labelled human growth hormone of high specific activity. Nature (London) 194: 495-496(1962) Ichinose, R., S. Nandi: Influence of hormones on lobuloalveolar differentiation of mouse mammary glands in vitro. J. Endocrinol. 35: 331-340(1966) Inagaki, Y, K. Kohmoto: Changes in Scatchard plots for insulin binding to mammary epithelial cells from cycling, pregnant and lactating mice. Endocrinology 110: 176-182(1982) Jones, R. G., V. Ilic, D. H. Williamson: Physiological significance of altered insulin metabolism in the conscious rat during lactation. Biochem. J. 220:455-460(1984) Moutafchiev, D., A. Shisheva, L. Sirakov: Binding of transferrin-iron to the plasma membrane of a lactating rabbit mammary gland cell. Int. J. Biochem. 15:755-758 (1983) Regoeczi, E., M. Hatton: Transferrin catabolism in mammalian species of different body sizes. Am. J. Physiol. 238: R306—R310 (1980) Reithel, F: In: Chemical Zoology, vol. XI: 199-228 (1979), Academic Press, Inc., New York Scatchard, J.: The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51:660-675(1949) Turkington, R., K. Brew, T. Vanaman, R. Hill: The hormonal control of lactose synthetase in the developing mouse mammary gland. J. Biol. Chem. 243:3382-3387 (1968) Vernon, R. G.: Endocrine control of metabolic adaptation during lactation. Proc. Nutr. Soc. 48:23 - 32 (1989) Williamson, D. H. .Integration of metabolism in tissues of the lactating rat.FEBSLettersll8:K93-K105(1980)
Requests for reprints should be addressed to: Prof. Dr. L. M. Sirakov, Dr. Sci. Department of Biochemistry Medico-biological Institute, Medical Academy ul. Zdrave 2 1431 Sofia Bulgaria
Downloaded by: Universite Laval. Copyrighted material.
Adrenaline injection in lactating mouse leads to lowering of transferrin iron uptake (Figs. 5 and 6). This could be due to a redistribution of the iron pool among important organs (such as brain, liver, muscle and heart) under the stress influence. The highest drop in Fe uptake by experimental animals was about 28 % (Figs. 5 and 6). In Table 2 it can be seen that the number of receptors decreases and the affinity constantly increases. The mechanism of such action of adrenaline is unknown.
Horm. metab. Res. 24 (1992)
The results of insulin action (0.4 IU per mouse) are demonstrated in intact animals only. This action leads to a higher uptake of 59Fe. rabbit transferrin of isolated cells from lactating mouse mammary gland. It is suggested that most inactive transferrin receptors in the cell membrane are incorporated by the hormone action or some new receptors are synthesized. On the contrary, adrenaline in a dose 0.5 ug per animal demonstrated an opposite effect — a lower uptake of 59Fe. human transferrin from lactating mouse mammary gland. This is probably due to a redistribution of some part (about 28 %) of the iron. Instead of flowing to the mammary gland it flows towards other organs for overcoming the stress situation. An alternative explanation could be the inhibition of endogenous insulin secretion by adrenaline. From our data it follows that insulin and adrenaline have an antagonistic effect on regulation of Fe transport in lactating mouse mammary gland. Key words Mammary Gland — Insulin — Adrenaline — 59 Fe. Transferrin Uptake
Introduction Insulin is an anabolic hormone which is necessary for the growth and differentiation of mammary gland cells {Ichinose and Nandi 1966). This was also proved by experiments in vivo {Freeman and Topper 1977). Turkington, Brew, Vanaman and Hill( 1968) found that for the biosynthesis of galactosyltransferase and lactalbumin in the mouse mammary gland explants the presence of insulin, hydrocortisone and prolactin is necessary. Apostolova, Sirakov and Barth (1976) demonstrated insulin receptors in a crude membrane fraction from lactating white mice. Insulin stimulated iron uptake from the epididymal fat cells through a rearrangement of the transferrin receptors from the inner membrane surface to the outer {Davis, Corvera and Czech 1986).
Horm. metab. Res. 24 (1992) 420 - 423 © GeorgThieme Verlag Stuttgart-New York
Adrenaline release during stress plays an adapting role in extreme situation by elevating glucose level in blood. The effect of adrenaline on Fe transport from blood to mouse mammary gland is unknown. The receptors for Fe. transferrin are found in plasma membranes from lactating rabbit mammary gland cells {Moutafchiev, Shisheva and Sirakov 1983). The anabolic effect of insulin and antistress effect of adrenaline draw our attention to its possible influence on the uptake of transferrin iron from the lactating mouse mammary gland cells. Materials and Methods Isolation of transferrin from rabbit serum, its labelling with59Fe.ascorbinate (from Akademie der Wissenschaften, Germany) and a procedure of isolation of lactating mouse mammary gland cells were described earlier (Moutafchiev, Shisheva and Sirakov 1983). Human transferrin was purchased from "Serva", Germany. All other reagents were of analytical research grade from "Fluka" (Switzerland) and "Merck" (Germany). Insulin-Semilente MC was from "NOVO", Copenhagen, Denmark, with concentration 40 IU/ml. Adrenaline was from "Sigma", USA. White mouse strain H from 7 to 12 day after delivery was used. Mothers and litters were kept together for lactation stimulation. The dose of insulin of 0.4 IU was used as suggested by Baldwin and Martin (1968). 16 hours after injection (during that time the animals were given 5 % sucrose solution) mice were killed and the glands were removed by excision. Isolated cells were incubated with 59Fe. rabbit transferrin at 37 °C for 45 min {Moutafchiev, Shisheva and Sirakov 1983). In in vitro experiments cells were incubated with different doses of insulin diluted in Krebs-Ringer Buffer pH 7.4, at 37 °C for 30 min. The buffer contained: NaCl 0.125 mmol/1, KC1 5 mmol/1, KH2PO3 1.25 mmol/1, MgSO4 7H 2O 1.2 mmol/1, NaHCO3 3.8 mmol/1, NaH2PO4-2H 2O 9 mmol/1. The incubation medium contained the same buffer with the addition of 2% bovine serum albumin (V fraction, from Koch-Light, England). After incubation the cells were treated for 45 min at 37 °C with59Fe.transferrin and the reaction was stopped with ice-cold 0.9% NaCl. The cells sedimented after centrifugation and were counted in gamma counter Rack Gamma II 1270, LKB, Finland. Mice were injected i.m. with adrenaline 0.5 ug and after 5 min were killed. Control mice were injected with 0.1 ml of saline and further treated by the same way. Vitality of the cells, checked by Trypan-blau (Hunter and Greenwood 1962), was about 90%. Cells were counted in a Burker camera. The results were calculated by the variation analysis and the Student-Fisher test.
Received: 17 July 1991
Accepted: 30 Jan. 1992 after revision
Downloaded by: Universite Laval. Copyrighted material.
Summary
Fe Transferrin Uptake of Mammary Gland Cells
Fig. 2 Binding of 59Fe. rabbit transferrin in dependence of increasing number of cells. Abscissa: number of the cells ( x 106). Added 10.75 9 g 59 Fe. rabbit transferrin calculated as Fe. Ordinate: bound 59Fe in x 10 _9 g. Data from three control ( • ) and treated with 0.4 IU insulin in vivo (O) animals with ±SE.
Downloaded by: Universite Laval. Copyrighted material.
Fig. 1 Influence of insulin in vivo (0.4 IU) in dependence from increasing concentration of 59Fe. rabbit transferrin. Abscissa: increasing concentration of 59Fe. Tf calculated as x 10- 9 g. Ordinate: bound 59Fe. in x 10- 9 g from106cells. Data from three control ( • ) and three treated (O) animals with ±SE.
Horm. metab. Res. 24 (1992) 421
Fig. 3 Data from three experiments in vitro upon increasing concentrations of added insulin in x 10- 9g (abscissa). Ordinate: bound59Fein x 10- 9 g with +SE. The first point of the curve was control ( • ) .
Results Isolated mammary gland cells from insulintreated (0.4 IU) mice bound more labelled 59Fe. rabbit transferrin than cells from control animals (Fig. 1). The Fe-uptake, calculated as ng Fe, was also increased. At constant 59Fe amount (12.66 ng) the binding of Fe. transferrin as a function of the increasing cell number was higher in cells from insulintreated animals than in cells from control ones (p < 0.01, Fig. 2). This shows that when insulin was injected into living mice, it stimulated binding of Fe. transferrin. If insulin was given in vitro in the incubation medium (from 0.1 to 104 ng) for 30 min at 37 °C the results were different (Fig. 3). There was no substantional difference at the range of insulin amounts from 1 to 103 ng. At the highest amount (104 ng) there was a decrease (p < 0.02). Data shown in Figure 1 were presented as a Scatchard plot (1949). There are two receptor sites in control and experimental groups (Fig. 4). The binding capacity and affinity constants are given in Table 1. Data from Table 1 might suggest a negative cooperative effect, i.e. with rising of binding capacity of receptors, the receptor affinity for Fe. transferrin decreases. An adrenaline injection (0.5 ug) in lactating mice brought to a sig-
Fig. 4 Scatchard's plot from data shown in Fig. 1. Abscissa: bound Fe on 106 cells. Ordinate: the ratio bound/free Fe in control ( • ) and treated (O) animals.
Binding capacity (10- 9 gFe) per 10 cells Kaf M- 1
Control I site II site
Experiment I site II site
0.23
1.0
0.55
1.5
2.6 x 10 8
3.2 x 10 6
6.2 x 10 7
4.1 x 10 6
nificant decrease of transferrin iron uptake by isolated mammary cells (Fig. 5). Studies at different amounts of59Fe.transferrin demonstrated that substantial differences were observed at amounts of 0.34 ng (p < 0.01)., 0.90 ng (p < 0.01), and 1.29 ng (p < 0.002) (Fig. 5). Time-dependence of binding is given in Fig. 6, where, again the adrenaline effect is seen with significant differences (p < 0.001) in all points except the first one.
Horm.metab. Res. 24(1992)
D. A. Moutafchiev andL. M. Sirakov
Fig. 5 Binding of Fe from human transferrin from 1.5 x 106 cells of lactating mouse mammary gland of 4 nontreated ( • ) and 8 treated in vivo (O) animals with 0.5 ug adrenaline. Abscissa: added 59Fe x 10- 9 g. Ordinate: bound 59Fein x 10- 9 g with ± SE.
Fig. 7 Scatchard's plot from data shown in Fig. 5. Abscissa: bound 59Fe on 1.5 x 106 cells. Ordinate: ratio of bound-free 59 Fe in control ( • ) and treated (O) animals.
Fig. 6 Dependence of binding of Fe. human transferrin on the time of incubation at 37 °C. Abscissa: time of incubation in minutes. Ordinate: bound 59Fe in x 109 g±SE. Data from 8 treated (O) in vivo with 0.5 ug adrenaline and 4 control ( • ) mice. The number of cells was 1.5 x 10 .
Control I site II site Binding capacity (10- 9 gFe)per 1.58 x 10 6 cells Kaf M-1
1.4
3.07
Experiment I site II site 0.93
1.8
4.92 x 10 7 6.87 x 10 6 1.01 x 10 8 8.7 x 10 6
uptake with a redistribution of transferrin receptors from the intracellular space towards plasma membrane as an answer to insulin action. Such an explanation could not be applied to mammary gland cells with a secretory function. The Scatchard plot (1949) presented in Fig. 7 was drawn from data in Fig. 5. The calculated capacities and affinity constants are presented in Table 2. Here also two types of binding sites are demonstrated for binding of Fe. human transferrin. Discussion In this work we have assumed that cell receptors do not make any difference between labelled and nonlabelled iron atoms bound to transferrin. Therefore a competitive non-labelled transferrin is not used in the experiments. Insulin is responsible for the function and growth of mammary gland {Reithel 1979). Therefore it is useful to study its effect on the transport of such an essential element as iron from blood to milk during lactation. Lactation results in hypoinsulinaemia in rodents {Williamson 1980). Insulin injected in lactating mice causes a higher uptake of transferrin iron from mammary gland cells than in control animals (Figs. 1 and 2). The same effect was found also in epididymal fat cells {Davis, Corvera and Czech 1986). The authors explained this rise of Fe. transferrin
An alternative explanation could be that new receptors are synthesized or activated on the plasma membrane. The mammary gland appears to be much more sensitive to insulin {Jones, Ilic and Williamson 1984) than adipose tissue and muscle, hence it is able to maintain high rates of insulinsensitive processes during lactation despite the prevailing hypoinsulinaemia (Vernon 1989). The mechanism responsible for this enhanced sensitivity to insulin is unknown, but in contrast to the other tissues described previously, the number of insulin receptors of mammary epithelial cells is increased during lactation {Flint 1982; Inagaki and Kohmoto 1982). It is possible that some other processes are involved, such as rising of synthesis of transport RNA {Reithel 1979). As one can see (Table 1) the number of transferrin receptors on the plasma membrane of mammary gland cells is increased and there is a higher uptake of transferrin iron from mammary gland cells in experiments in vivo (Figs. 1 and 2) but not in vitro (Fig. 3). Davis, Corvera and Czech (1986) and this work demonstrated that insulin stimulated at least two different types of cells to accept the transferrin iron through the increase of number of receptors. The behaviour of mammary gland cells in vivo and in vitro suggests that the mechanism of insulin action is still unknown.
Downloaded by: Universite Laval. Copyrighted material.
422
Fe Transferrin Uptake of Mammary Gland Cells
Our data suggest that insulin and adrenaline have opposite effects on the uptake of Fe. transferrin from lactating mammary gland cell. Insulin is known to stimulate protein synthesis in cells. This might explain the higher number of receptor sites in the plasma membrane (Table 1). On the other side, adrenaline inhibits insulin secretion and this might lead to a lower uptake of Fe. transferrin by the cells. With these effects, the hormone may play a significant role for adaptation to stress. It is also possible that they regulate the flow of iron to the mammary gland of lactating mouse. Regoeczi and Hatton (1980) suggested that use of heterologous transferrin is a matter of convenience. They found good correlation between parameters in different animals using heterologous transferrin. Our previous experiments (unpublished data) show that there are two binding sites on the plasma membrane of mouse mammary gland cell for the heterologous transferrin while there is only one site for the homologous one. In Figs. 4 and 7 and Tables 1 and 2 it can be seen that both iron-binding sites of transferrin (from rabbit and human origin) gave its iron to cell receptors. The second iron binding site of both transferrins is a better donor for Fe in experimental and control animals. Thus, the heterologous transferrins transfer its iron to cell receptors in coincidence with hormonal action. The absence of peak in Fig. 6 shows that adrenaline treated cells of mammary gland also keep their capability to absorb iron however with a lower rate than controls. This shape of binding curve probably represents the irreversibility of the iron flow from blood to milk, the problem has already been discussed in detail in our earlier work (Moutafchiev, Shisheva and Sirakov 1983). Our experiments demonstrate new effects of insulin and adrenaline unknown for the lactating mammary gland. They include an opposite effect on iron transport from Fe. transferrin in blood to milk. Acknowledgements This study was supported by grant No. 268 from the State Committee of Science (Bulgaria).
423
References Apostolova, J., L. Sirakov, T. Barth: Insulin receptors in lactating mouse mammary gland. Collection Czechoslov. Chem. Comm. 41:3830-3836(1976) Baldwin, R., R. Martin: Protein and nucleic acid synthesis in rat mammary gland during early lactation. Endocrinology 82: 1209-1216 (1968) Davis, R., S. Corvera, M. Czech: Insulin stimulates cellular iron uptake and causes the redistribution of intracellular transferrin receptors to plasma membrane. J. Biol. Chem. 261:8708-8711 (1986) Flint, D. J.: Insulin binding to rat mammary gland at various stages of cell isolation and purification. Mol. Cell. Endocrinol. 26: 281-294 (1982) Freeman, C, Y. Topper: Progesterone and insulin in the growth and differentiation of mouse mammary epithelium in vivo. Fed. Proa, Fed. Am. Soc. Exp. Biol. 36:925 (1977) Hunter, W., F. Greenwood: Preparation of Iodine-131 labelled human growth hormone of high specific activity. Nature (London) 194: 495-496(1962) Ichinose, R., S. Nandi: Influence of hormones on lobuloalveolar differentiation of mouse mammary glands in vitro. J. Endocrinol. 35: 331-340(1966) Inagaki, Y, K. Kohmoto: Changes in Scatchard plots for insulin binding to mammary epithelial cells from cycling, pregnant and lactating mice. Endocrinology 110: 176-182(1982) Jones, R. G., V. Ilic, D. H. Williamson: Physiological significance of altered insulin metabolism in the conscious rat during lactation. Biochem. J. 220:455-460(1984) Moutafchiev, D., A. Shisheva, L. Sirakov: Binding of transferrin-iron to the plasma membrane of a lactating rabbit mammary gland cell. Int. J. Biochem. 15:755-758 (1983) Regoeczi, E., M. Hatton: Transferrin catabolism in mammalian species of different body sizes. Am. J. Physiol. 238: R306—R310 (1980) Reithel, F: In: Chemical Zoology, vol. XI: 199-228 (1979), Academic Press, Inc., New York Scatchard, J.: The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51:660-675(1949) Turkington, R., K. Brew, T. Vanaman, R. Hill: The hormonal control of lactose synthetase in the developing mouse mammary gland. J. Biol. Chem. 243:3382-3387 (1968) Vernon, R. G.: Endocrine control of metabolic adaptation during lactation. Proc. Nutr. Soc. 48:23 - 32 (1989) Williamson, D. H. .Integration of metabolism in tissues of the lactating rat.FEBSLettersll8:K93-K105(1980)
Requests for reprints should be addressed to: Prof. Dr. L. M. Sirakov, Dr. Sci. Department of Biochemistry Medico-biological Institute, Medical Academy ul. Zdrave 2 1431 Sofia Bulgaria
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Adrenaline injection in lactating mouse leads to lowering of transferrin iron uptake (Figs. 5 and 6). This could be due to a redistribution of the iron pool among important organs (such as brain, liver, muscle and heart) under the stress influence. The highest drop in Fe uptake by experimental animals was about 28 % (Figs. 5 and 6). In Table 2 it can be seen that the number of receptors decreases and the affinity constantly increases. The mechanism of such action of adrenaline is unknown.
Horm. metab. Res. 24 (1992)
