Life Sciences, Vol. 29, pp. 1641-1648 Printed in the U.S.A.
Pergamon Press
EFFECT OF REDUCEDATMOSPHERIC PRESSUREAND FASTING ON TRANSFERRINSYNTHESIS IN THE RAT Michaele E. Gardiner and E.H. Morgan
Department of Physiology University of Western Australia Nedlands, W.A. 6009 (Received in final form August 13, 1981)
Summary The concentrations of transferrin and albumin in the blood serum and microsomal fraction of the l i v e r and the incorporation of [14C] leucine into the proteins were measured in rats which were fasted while exposed to ambient atmospheric pressure or to a pressure of one-half atmosphere. The rates of protein synthesis were estimated in a relative manner from the ratio of 14C incorporation into the two proteins and in an absolute manner using the l i v e r free 14C and ]eucine concentrations to measure the specific a c t i v i t y of the precursor pool. Fasting at ambient pressure was accompanied by a decrease in the serum and microsomal concentrations of transferrin but not of albumin and by a marked decrease in the relative and absolute synthesis rates of transferrin. By contrast, fasting at reduced ambient pressure was associated with an increase in the serum transferrin concentration and in the relative and absolute rates of synthesis of the protein. I t is concluded that fasting in the rat produces a much greater decrease in the rate of synthesis of transferrin than of albumin and that exposure to reduced ambient pressure stimulates transferrin synthesis but not albumin synthesis. The plasma concentration of transferrin changes in a predictable way during the l i f e cycle and in disease states in humans and under various experimental conditions in laboratory animals (1). The most important factor affecting plasma transferrin level appears to be the iron status. Iron deficiency anaemia and other states associated with relative iron deficiency (pregnancy, infancy) are accompanied by an increase and iron overload by a decrease in the plasma level. The most l i k e l y cause of the observed changes is alteration in rate of synthesis of the protein. Hence, i t is generally believed that the major factor which regulates transferrin synthesis is the status of the iron stores, particularly the amount of storage iron in the l i v e r which is the main site of transferrin synthesis ( l ) . Experimental evidence in favour of this has been obtained in the rat (2-4)~ However, in the rat and rabbit the plasma concentration of transferrin is also increased in haemolytic anaemia and other conditions in which the iron stores are not diminished (5). Also, in the rat i t has been shown that exposure to reduced ambient pressure elevates the plasma transferrin level (6). Since l i v e r slices from rats which have been exposed to low ambient pressure incorporate more radioactive leucine into transferrin than slices from control rats (7) i t is l i k e l y that the increase in plasma concentration is due to accelerated synthesis by the l i v e r . The present experiments were aimed at investigating whether exposure of 0024-3205/81/161641-08502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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rats to reduced atmospheric pressure affects the rate of transferrin synthesis in vivo. Food intake is greatly reduced during the f i r s t few days of such treatment (8). Hence, i t was decided to control the effects of variable food intake by fasting the animals during exposure to the reduced pressure and, also, control rats maintained at normal atmospheric pressure. I t was found that fasting had a profound effect on transferrin synthesis. The synthesis of albumin was studied in addition to that of transferrin in order to obtain results for another plasma protein which could be compared with those of transferrin. This should enable the distinction to be made between a specific effect on transf e r r i n synthesis and a non-specific effect which would be expected to affect the synthesis of transferrin and albumin in a similar manner. Materials and Methods Materials - Radioactive leucine, [U-lhC]-L-leucine, specific a c t i v i t y 300 mCi per mmole was obtained from The Radiochemical Centre, Amersham, England. Rat serum transferrin and serum albumin were purified as described previously (7). Human transferrin was purchased from Behringwerke, Marburg-Lahn, Germany. Antisera against these proteins were produced in rabbits by the intramuscular injection of 3-4 doses at 2-week intervals of I-2 mg of the proteins in l ml 0.15 M NaCl emulsified with l ml complete Freund's adjuvant. The rabbits were bled two weeks after the last injection of antigen. The antisera were examined by immunoelectrophoresis against rat and human serum (9). Each antiserum showed only one arc when used with the appropriate serum. Animals - Male rats of an inbred Wistar strain, weighing 230-260 g, were used. Fasted rats were deprived of food but were given free access to drinking water. The animals exposed to reduced ambient pressure were maintained in a decompression chamber at a pressure equivalent to an altitude of 5,500 m. They were fasted but given drinking water while in the chamber. Control rats were fed a diet of laboratory rat cubes. Estimation of transferrin and albumin synthesis - The rates of synthesis of the proteins were estimated as described previously (lO) using a method based on the incorporation of [14C]-leucine into l i v e r microsomal transferrin and albumin and total l i v e r p r o t e i n 16 min after intravenous injection of the labelled amino acid. Incorporation of the label into transferrin, albumin and total l i v e r protein was measured in a l l ' o f the rats. The results were expressed as [lhC]leucine incorporation into the proteins in dpm per g l i v e r . This provided a means of estimating r e l a t i v e synthesis rates of the two proteins by determining the ratio of the incorporation of the label into transferrin with that into albumin. In addition, in another experiment animals were treated for 3 days, the concentration of protein-free leucine and protein-free 14C in the l i v e r were measured and the rates of synthesis of transferrin and albumin were calculated in absolute terms, mg/h/g l i v e r , by the method used in e a r l i e r work (lO). Analytical methods - Transferrin and albumin in serum or in extracts of l i v e r m~crosomes (lO) were estimated by radial i~unodiffusion ( l l ) . Serum iron concentration was measured by the method prescribed by the International Society of Haematology (12). Liver non-haem iron was determined by the method of Kaldor (13). The haematocrit was determined by the microhaematocrit method. The concentrations of free amino acids were measured in the supernatant solutions obtained by precipitating serum or l i v e r proteins with 5% sulphosalicylic acid. The measurements were made in a LKB Aminoacid Analyser (Model 3201) using a column (0.9 x 54 cm) of sulphonic acid resin (Biorad, Biocal Corp., Richmond, California). Norleucine was added as an internal standard. Radioactivity was measured in a Nuclear-Chicago l i q u i d s c i n t i l l a t i o n counter. The samples containing 14C were dissolved in NCS reagent (Nuclear-Chicago Corp.) and transferred to counting v i a l s with lO ml toluene s c i n t i l l a t o r solution (14).
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Transferrin Synthesis in Hypoxia and Fasting
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Correction of quenching was made by the channels ratio method. Results The effect of fasting at normal ambient pressure and at reduced ambient pressure on serum transferrin and albumin concentrations and haematocrit is shown in Fig. I . No significant differences in mean haematocrit or serum albumin values were observed between the groups of rats exposed to the different pressures. However, both haematocrit and albumin concentration increased s l i g h t l y and to approximately the same degree during the f i r s t two days of the two types of treatment. In the case of serum transferrin the treatments produced markedly different results. Fasting at normal ambient pressure was associated with a progressive f a l l in transferrin concentration but fasting at reduced pressure was accompanied by a rise. The difference between the mean transferrin values of the two treatment groups was highly significant (P < O.OOl) on each day of observation. After three days of fasting at low ambient pressure the mean serum transferrin concentration was 52 per cent greater than that of nonfasted control rats at normal pressure whereas the mean haematocrit value was only I I per cent greater than in the controls.
E ~8
60 ~-~
4
i~c 50 '~
~2
~. I I
I 2
L 1
J 3
I 2
I 3
0oys
FIG. l Changes in the serum transferrin and albumin concentrations and the haematocrit of control rats (zero days) and rats fasted for one, two or three days at normal ambient pressure (¢ =) or at one-half atmospheric pressure (o o). Each point is the mean of 5 or 6 animals. The vertical bars represent ± one standard error. Fasting was associated with a decrease in the weight of the l i v e r . This was r e l a t i v e l y greater in the rats at normal than in those at reduced pressure (Table I ) . One day of fasting at either pressure produced an increase in the concentration of non-haem iron in the l i v e r , but on the second and third day the values were not s i g n i f i c a n t l y different from those of non-fasted control rats (Table I ) . However, the total amount of non-haem iron in the l i v e r s of these rats was reduced below that of the controls; mean values of approximately 570 and 610 ~g were observed with the rats fasted at normal and reduced pressure, respectively, compared with 730 ~g in the controls. The serum iron concentration f e l l s i g n i f i c a n t l y (P < 0.05) in the rats fasted at atmospheric pressure and to a much greater degree in the animals exposed to reduced pressure (Table I ) .
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Transferrin Synthesis in Hypoxia and Fasting
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TABLE I Body and L i v e r Weights, L i v e r Non-haem Iron Concentration and Serum I r o n Concentration i n Control Rats and Rats Fasted f o r I , 2 or 3 days at Normal Atmospheric Pressure or at a Pressure o f Half an Atmosphere. Each value i s the mean (± S.E.) o f the i n d i c a t e d number (n) of r a t s . Treatment
Days
Control
n
Body weight (g)
Liver w e i g h t (g)
6
252 ± 5
I 0 . 0 ± 0.3
L i v e r non-haem i r o n (~g/g) 73 ±
Serum i r o n (~g/ml)
8
2.4 ± 0.30
Fasting at normal pressure
1 2 3
5 6 6
233 ± 6 232 ± 6 242 ± 7
6.9 ± 0.7 5.8 ± 0.2 5.8 ± 0.2
122 ± 19 88 ± 7 99 ± I I
2.07± 0.18 1.81± 0.23
Fasting at low pressure
1 2 3
5 5 6
232 ± 6 233 ± 7 226 ± 4
8.2 ± 0.3 7.6 ± 0.5 6.8 ± 0.2
123 ± 12 83 ± 6 90 ± 7
1.74± 0.15 0.82± 0.09
The r e s u l t s f o r the c o n c e n t r a t i o n s of t r a n s f e r r i n and albumin e x t r a c t e d from the microsomal f r a c t i o n of the l i v e r are presented i n Fig. 2. Fasting at normal pressure led to a l a r g e decrease i n microsomal t r a n s f e r r i n but no change in microsomal albumin when the r e s u l t s were expressed as mg p r o t e i n per g wet weight of l i v e r . Fasting during exposure to reduced pressure had the opposite effect, little change i n the c o n c e n t r a t i o n of microsomal t r a n s f e r r i n but a marked f a l l i n microsomal albumin. When the r e s u l t s were c a l c u l a t e d i n terms of the t o t a l l i v e r content of the p r o t e i n s i n the microsomal f r a c t i o n there was no s i g n i f i c a n t d i f f e r e n c e between the values f o r albumin of the rats fasted at e i t h e r pressure. However, w i t h t r a n s f e r r i n there was a marked decrease during the f i r s t day of f a s t i n g , w i t h l i t t l e subsequent change, and the degree of decrease was much g r e a t e r in the rats at normal pressure than in those at reduced pressure. TRANSFERRIN
ALBO/e4N
0
o5
04
o3
03f
o,2
O'2
OI
iI 2I
i 3
I
I
I
i
2
3
Oa~
FIG. 2 Changes i n the c o n c e n t r a t i o n of t r a n s f e r r i n and albumin in the microsomal f r a c t i o n of the l i v e r of c o n t r o l r a t s (zero days) and r a t s fasted f o r one, two or three days at normal atmospheric pressure (e e) or at o n e - h a l f atmospheric pressure (o o). Each p o i n t is the mean of 5 or 6 animals. The v e r t i c a l bars represent ± one standard e r r o r .
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Transferrin Synthesis in Hypoxia and Fasting
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The r e l a t i v e synthesis rate of t r a n s f e r r i n to albumin was expressed as the r a t i o of [14C]-leucine incorporated i n t o microsomal t r a n s f e r r i n to t h a t incorporated i n t o microsomal albumin (Fig. 3). This r a t i o f e l l to approximately 50 per cent of the control value w i t h i n one day of commencing f a s t i n g at normal pressure and t h e r e a f t e r rose s l i g h t l y but remained much lower than in the cont r o l r a t s . By c o n t r a s t , in the r a t s fasted at reduced pressure the r e l a t i v e synthesis rate of t r a n s f e r r i n increased moderately and p r o g r e s s i v e l y during the treatment period. I t may be noted t h a t r a t t r a n s f e r r i n contains 4.5 per cent less leucine than r a t albumin ( I 0 ) , a d i f f e r e n c e which should be taken i n t o account when i n t e r p r e t i n g the r e s u l t s in Fig. 3. O3
O ¸I
i
i
J
i
2
3
{)o)~
FIG. 3 R e l a t i v e synthesis rates of t r a n s f e r r i n and albumin in the l i v e r of control r a t s (zero days) and of rats fasted f o r one, two and three days at one atmospheric pressure (o ~), or at o n e - h a l f atmospheric pressure (o o). Each point i s the mean of 5 or 6 animals. The v e r t i c a l bars represent _+ one standard e r r o r .
TABLE I I Rates of Synthesis of T r a n s f e r r i n and Albumin in the L i v e r In Vivo by Control Rats and Rats Fasted f o r 3 days at Normal Ambient Pressure or at a Pressure of Half an Atmosphere. The values are given as the mean (_+ S.E.) from the indicated number (n) of r a t s . Treatment Transferrin mean SE
Synthesis Rate (mg/total l i v e r / h ) Albumin t P mean SE
Control
6
1.20
_+0.13
Fasting at normal pressure
7
0.336
_+0.041
6.8
Fasting at low pressure
4
1.122
±0.102
0.4
t
P
6.90
±I.00
<0.01
2.90
_+0.447
3.9 <0.01
NS
3.67
±0.544
2.4
0.02-0.05
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These results indicate that, r e l a t i v e to the rates of synthesis of albumin, the synthesis of transferrin was much greater in the rats at reduced pressure than in those at ambient pressure. This was confirmed by the measurements of absolute synthesis rates of the proteins (Table I I ) . Three days of fasting at normal pressure produced a r e l a t i v e l y much greater f a l l in the rate of transf e r r i n synthesis than in that of albumin. However, in the rats exposed to reduced pressure the rate of transferrin synthesis was not s i g n i f i c a n t l y different from that of the non-fasted control rats, while albumin synthesis rate was sign i f i c a n t l y lower than that of the control (P < 0.05).
Discussion Several investigators have shown that fasting produces a decrease in the rate of protein synthesis in the l i v e r (19-21) associated with a decrease in the amount of RNA and number of ribosomes per cell and disaggregation of polyribosomes (22-24). Decreased l i v e r protein synthesis has also been observed in rats exposed to hypoxic conditions. This has been attributed to a reduction of food intake and, hence, to the same mechanisms as those which operate during fasting (25,26), although a reduction in the concentration of ATP in l i v e r cells during hypoxia may also contribute (27). The present work shows that fasting and hypoxia do not necessarily have the same effects on the synthesis of different proteins by the l i v e r . Thus, fasting produced a greater degree of decrease in the synthesis of transferrin than of albumin, accompanied by a proportionately larger f a l l in the concentration of transferrin in the l i v e r microsomes and serum. Exposure to reduced ambient pressure was able to overcome the effects of fasting on transferrin synthesis and concentrations but not those on albumin. The results confirm for the intact animal e a r l i e r ones obtained by in v i t r o incubation of rat l i v e r slices (7). In the previous experiments i t was shown that slices from rats exposed to reduced ambient pressure incorporated more [14C]-leucine intro transferrin than slices from control rats and slices from fasted rats incorporated less of the label than those from non-fasted rats. The major difference between the in vivo and the in v i t r o results is that, in vivo, fasting had a r e l a t i v e l y greater effect on transferrin than on albumin synthesis, whereas, in v i t r o , there was a similar decrease in the incorporation of radioactive leucine into both proteins. The conclusion that fasting had a greater effect on transferrin than on albumin synthesis is supported by the observed changes in the concentration of proteins in the serum and the microsomes of the l i v e r (Figs. l and 2). Indeed, the serum albumin concentration increased s l i g h t l y but to a lesser degree than the haematocrit, suggesting that the changes in serum albumin were a composite of a reduction in plasma volume, probably due to decreased f l u i d intake by the fasted rats and a decrease in the rate of synthesis of the protein. The observations that fasting at normal ambient pressure produced a greater reduction in the rate of synthesis of transferrin than of albumin and that exposure to reduced ambient pressure while fasting markedly stimulated transferrin synthesis r e l a t i v e to that of albumin show that these two influences exert d i f f erential effects on the synthesis of the two proteins. Their effects cannot be due to changes which would have non-specific or general effect on the protein synthesis machinery, such as changes in amino acid supply, c e l l u l a r content of RNA or ATP or degree of aggregation of ribosomes. Two factors which have been postulated to s p e c i f i c a l l y affect transferrin synthesis in the l i v e r are the l i v e r iron stores (2-4) and the r e l a t i v e balance between oxygen supply and requirement (6). The results for l i v e r non-haem iron (Table I) do not appear to support the f i r s t of these proposals. There was no significant difference in these storage iron values between rats fasted at ambient or at reduced
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Transferrin Synthesis in Hypoxia and Fasting
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pressure, although there were marked differences in the rate of t r a n s f e r r i n synthesis (Table I I , Fig. 3). However, i t is possible that l i v e r storage iron is compartmentalized and that the iron in only one compartment influences transf e r r i n synthesis. I f this were only a small f r a c t i o n of total storage iron or i f changes in the active compartment were accompanied by changes in the opposite d i r e c t i o n in the iron content of other compartments then the results f o r total non-haem iron could easily be misinterpreted. Exposure to reduced ambient pressure was associated with a greater reduction in serum iron concentration than that found in the rats at normal pressure (Table I ) . Possibly this change was accompanied by a reduction in the rate of hepatic uptake of iron from plasma and a reduction in the size of the t r a n s i t iron pool of hepatocytes.
The hypothesis that transferrin synthesis is specifically affected by oxygen supply receives more support from the present results than does the one based on storage iron. Thus, i t has been shown that fasting lowers metabolic rate and oxygen consumption by the animal (28). This could explain the relatively greater reduction of transferrin synthesis than of albumin synthesis in the rats fasted at one atmospheric pressure. Similarly, according to this hypothesis, the effects of reduced ambient pressure are attributable to a reduction in the supply of oxygen to the tissues. The changes in oxygen supply and ~emand may act directly on the cells which synthesize transferrin or they may exert their effect in an indirect manner such as by a hormonal mechanism which is responsive to oxygen balance. One possibility is erythropoietin, the secretion of which is known to decrease during fasting and to increase under hypoxic conditions (29). However, there is no evidence that i t exerts any direct effect on transferrin synthesis. Further work is required to establish whether oxygen balance, directly or indirectly, does exert an effect on transferrin synthesis or whether the observed changes are due to regulation of synthesis by a particular fraction of the storage iron.
Acknowledgements This work was supported by a grant from the Australian Research Grants Committee.
References I. 2. 3. 4. 5. 6. 7. 8. 9. I0. If.. 12. 13.
E.H. MORGAN, in Iron in Biochemistry and Medicine, p. 30-71, (A. Jacobs and M. Worwood, eds.), Academic Press, London and New York, (1974). M. AWAI and E.H. BROWN,J. Lab.Clin.Med. 61, p. 363-396, (1963). A.G. MORTON, S.M. HAMILTON, D.B. RAMSDENan---dA.S. TAVILL, in Plasma Protein Turnover, p. 157-167, (A.S. McFarlane, R. Bianchi and G. Mariani, eds.), Macmillan, London, (1975). A.G. MORTONand O.S. TAVILL, Brit.J.Haematol. 36, p. 383-394, (1977). E.H. MORGAN, Quart.J.Exp.Physiol. 46, p. 220-2~, (1961). E.H. MORGAN, Quart.J.Exp.Physiol. ~ , p. 59-65, (1962). E.H. MORGAN,J.Bio1.Chem. 244, p 7~T93-4199, (lg69). D.D. SCHNAKENBERG, L.F. KRABILL and P.C. WEISER, J.Nutrit. lOl, p. 787-796, (1971). J.J. SCHEIDEGGER, Intern.Arch.Allergy 7, p. 103-110, (1955). E.H. MORGANand T. PETERS, J.Biol.Chem. 246, p. 3500-3507, (1971). G. I~ANCINI, A.O. CARBONARAand J.F. HEREMANS, Immunochemistry 2, p. 235-254, (1965). International Committee for Standardization in Haematology, Brit.J.Haematol. 20, p. 451-453, (1971). I. Y~ALDOR,Aust.J.Exp.Bio1.Med.Sci. 3_22, p. 795-805, (1954).
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14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
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R.J. HERBERG, Anal.Chem. 32, p. 42-49, (1960). E. PICON-REATEGUI, G.R. FRYERS, N.I. BERLIN and J.H. LAWRENCE, Am.J.Physiol 172, p. 33-36, (1953). H. GLAUMANNand J.L.E. ERICSSON, J . C e l l . B i o l . 47, p. 555-547, (1970). T. PETERS, B. FLEISCHER and S. FLEISCHER, J.Biol.Chem. 246, p. 240-244, (1971). E.H. MORGANand T. PETERS, J.Bio1.Chem. 246, p. 3508-3511, (1971). J.B. MARSH, Am.J.Physiol. 201, p. 55-57,--~-961). H.A. ROTHSCHILD, M. ORATZ, J. ~IONGELLI and S.S. SCHREIBER, J . C l i n . l n v e s t . 47, p. 2591-2599, (1968). T-~. PETERS and J.C. PETERS, J.Biol.Chem. 247, p. 3583-3863, (1972). S.H. WILSON and M.Bo HOAGLAND, Biochem.J. 103, p. 556-566, (1967). C.O. ENWONWU, R. STAMBAUGHand L. SPREEBUY, J . N u t r i t . I01, p. 337-345, (1971). T.T. HAYASHI and D. KAZMIEROWSKI, Biochemistry I I , p. 2371-2378, (1972). M.I. SURKS, Am.J.Physiol. 218, p. 842-844, (1970-T. M.I. SURKS and M. BERKOWITZ, Am.J.Physiol. 220, p. 1606-1609, (1971). A.P. SANDERS, D.M. HALE and A.T. MILLER, Am.J.Physiol. 209, p. 443-446, (1965). S. MORGULIS, Fasting and Undernutrition, E.P. Dutton & Co., New York, (1923). W. FRIED, L.K.F. PLZAK, L.O. JACOBSONand E. GOLDWASSER, Proc. Soc. Exp. Biol.Med. 94, p. 237-241, (1957).