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Interaction of sugars and amino acids in intestinal transfer
PRELIMINARY NOTES
281
FORSYTH,Arch. Dis. Child., 4 ° (1965) 251. A. M. BONGIOVANNI AND A. W. ROOT, New Engl. J. Med., 268 (1963) 1283. A. M. BONGIOVANNI AND A. W. ROOT, New Engl. J. Med., 268 (1963) 1342. A. M. BONGIOVANNI AND A. W. ROOT, New Engl. J. Med., 268 (1963) 1391. W. L. GARDINER AND ]~. C. HORNING, Biochim. Biophys. Acta, 115 (1966) 524 . K. BIRCHALL AND E. L. MITCHELL, Steroids, 6 (1965) 427 . R. RYHAGE, Anal. Chem., 36 (1964) 759. }{. EOTHERBY, Biochem. J., 69 (1958) 596. M. OKADA, D. K. FUKUSHIMA AND T. F. GALLAGHER,J. Biol. Chem., 234 (1959) 1688. H. HIRSCHMANN AND F. B. HIRSCHMANN, J. Biol. Chem., 187 (195 o) 137. W. R. EBERLEIN, J. Clin. Endocr., 25 (1965) IIOI. J. W. REYNOLDS, Proc. Soc. Exptl. Biol. Med., 113 (1963) 980. I~. FOTHERBY, A. COLAS, S. M. ATHERDEN AND G. F. MARRIAN, Biochem. dr., 66 (1957) 664. A. M. BONGIOVANNI, J. Clin. Invest., 41 (1962) 2086. J. W. REYNOLDS, Steroids, 3 (1964) 77. A. COLAS, W. L. HEINRICHS AND H. J. TATUM, Steroids, 3 (1964) 417 .
3 D . M . CATHRO, K . BIRCI-IALL, F. L. MITCHELL AND C. C.
4 5 6 7 8 9 Io II 12 13 14 15 16 17 18
Received May 26th, 1966 Revised manuscript received September I2th, 1966 Bioehim. Biophys. Aeta, 13o (1966) 278-281
B B A 21 I6I
Interaction of sugars and amino acids in intestinal transfer NEWEY AND SMYTI-I1 showed that there is an interaction between sugar and amino acid transfer in the rat intestine and this was confirmed by SAUNDERS AND ISSELBACHER 2 in the rat, and ALVARADO3 in the hamster. BINGHAM, ~qEWEY AND SMYTH4 concluded that those sugars which are actively transferred but not metabolised, e.g., galactose, ~-methyl glucoside and 3-0-methyl glucose, inhibit amino acid transfer. In the present paper further consideration is given to the problem of galactose inhibition and to the interaction of glucose and amino acids. Everted sacs of rat small intestine were used as described previously 1. Proline or methionine (7-5 mM) was initially present in the mucosal fluid and sugars in either TABLE I EFFECT OF GALACTOSE ON AMINO ACID TRANSFER in vitro 7.5 mM prolin~ or methionine was present initially in the mucosal fluid, and galactose present in the mucosal or serosal fluids as indicated. The n u m b e r of experiments is shown in parentheses. E x p e r i m e n t a l period, 3 ° miD.
Condition
Transfer (izmoles ~ S.E.)
.~/lucosal fluid
Serosal fluid
Methionine
Proline
-28 mM galactose --
--+ 222 mM galactose + 222 mM sorbose 222 mM galactose 222 mM galactose
33.2 15.o 30.0 32.0 13.4 29.2
39.1 ± 1.3 (13) 24.6 ± 0.6 (6) 36.6 ~ 1.9 (7) 36.0 ± 1.9 (7) 17. 5 ~ 2.8 (7) 39.3 i 2.0 (7)
-
28 mM galactose 28 mM galactose + 5" IO-4 M phlorrhizin
± ± ~ ± ± ~k
1.2 (22) 0. 5 (6) 0.9 (6) 1.6 (7) 0-7 (7) 1.5 (6)
Biochim. Biophys. Acta, I3o (1966) 281-284
282
PRELIMINARY
NOTES
the mucosal or serosal fluids. The in vivo experiments were carried out by the method of SHEEF AND SMYTH 5, and IO mM amino acid with or without 30 mM sugar was circulated through the lumen of the intestine. The results with galactose in vitro are shown in Table I. Sorbose, an inert sugar 4, has been included for comparison. When present in the mucosal fluid galactose inhibited proline and methionine in confirmation of earlier results. Galactose present in both the mucosal and serosal fluids also caused inhibition, which was overcome by the presence of 5" lO-4 M phlorrhizin in the mucosal fluid. Galactose present in a high concentration in the serosal fluid caused little, if any, inhibition. These results show that the presence of galactose on both sides of the luminal membrane does not in itself cause inhibition of amino acid transfer, but that inhibition is related to movement of galactose across the luminal membrane. Since 222 mM serosal galactose did not inhibit, it appears also that a high concentration of galactose within the cells is not the cause of the inhibition. This conclusion is based on the assumption that galactose initially present in the serosal fluid can enter the epithelial cells, and this assumption is supported by the results in Table I I in which other sugars are used. This shows that the presence of glucose or mannose in the serosal fluid stimulates amino acid transfer, and hence these hexoses must be able to enter the cell from the serosal fluid. These results are analogous to those of D U E R D O T H et al3 in which it was shown that certain hexoses initially present in the serosal fluid stimulate fluid transfer. Since other hexoses can enter the cell from the serosal side it is likely that galactose can do so. TABLE
II
t~FFECT OF VARIOUS SUGARS ON METHIONINE AND PROLINE TRANSFER
in vitro
M e t h i o n i n e o r p r o l i n e w a s p r e s e n t i n i t i a l l y i n a c o n c e n t r a t i o n of 7-5 m M i n t h e m u c o s a l f l u i d . T h e s u g a r s w e r e p r e s e n t i n e i t h e r t h e m u c o s a l o r s e r o s a l f l u i d s a s s h o w n . T h e n u m b e r of e x p e r i m e n t s is s h o w n i n p a r e n t h e s e s .
Condition
Transfer (#moles ± S.E.)
Mueosal fluid
Serosal fluid
Methionine
Proline
---
-222 m M g l u c o s e
33.2 ± 1.2 (22) 5 0 . 8 ~ 2 . I (12)
39.1 -~ 1.3 (13) 6 1 . o ~ 3.8 (IO)
--
222 mM mannose
5o.1 ± 4.1
61,o i
28 m M g l u c o s e
222 mM glucose
3 7 , i ~- 1.2 ( i i )
28 m M g l u c o s e
222 mM mannose
36.7 i
1.6
(6)
--
28 m M m a n n o s e
222 m M m a n n o s e
4 6 . 5 ~- 3.0
(6)
--
(6)
2.6
(6)
--
Table I I also shows that glucose on the serosal side stimulates methionine transfer and this contrasts with the finding of DAWSON, NEWEY AND SMYTH7 that glucose initially present in the mucosal fluid does not stimulate methionine transfer. Furthermore, mucosal glucose but not mucosal mannose prevents the stimulation by serosal glucose or serosal mannose. The explanation may be that glucose or mannose in the serosal fluid supplies energy for transfer, while glucose in the mucosal fluid not only supplies energy but also needs energy for its own transfer. Mannose thus differs from glucose in that if present in the mucosal fluid it does not use an energy-requiring transfer mechanism, but it resembles glucose in that if present in Biochm. Biophys. Acta, 13o (1966) 2 8 1 - 2 8 4
283
P R E L I M I N A R Y NOTES
the serosal fluid it can supply energy for transfer. These results are in keeping with the view of •EWEY AND SMYTH1 about competition for energy b y transfer processes in the cell. One fact not satisfactorily explained is that glucose in the mucosal fluid is able to stimulate proline and glycine transfer 7. This raises possibilities that the energy requirements for methionine transfer are in some way different from those of proline and glycine transfer. In discussing the interaction of sugars and amino acids the hypothesis of TABLE
III
EFFECT OF GALACTOSE ON AMINO ACID TRANSFER in vivo M e t h i o n i n e or p r o l i n e ( i o m M ) w a s p r e s e n t t o g e t h e r w i t h 3 ° m M h e x o s e as s h o w n . E x p e r i m e n t a l p e r i o d , 3 ° miD. T h e n u m b e r of e x p e r i m e n t s is s h o w n i n p a r e n t h e s e s .
Condition
-+ 3°mM galactose + 3°mM sorbose + 3° i n M g l u c o s e
Transfer (t~moles 4- S.E.) Methionine
Proline
359 343 338 339
319 3 lo 318 358
± 7.5 (7) -- 9.0 (6) i 11.7 (5) ± 13.4 (6)
± lO.6 (8) i 6.6 (7) ± 8.1 (6) -- 8.1 (6)
ALVARADO~ must be considered, that hexoses can exert an allosteric effect on the amino acid carrier. There are, however, some difficulties about explaining the results by allosteric effects. In the first place it would have to be assumed that the effect of glucose was exerted only on the transfer of methionine and not on the transfer of proline and glycine, although it is known that proline and glycine can use the methionine carrier s, and that galactose inhibits all three amino acidsm,4. Another difficulty arises from the results of the in vivo experiments in Table I I I which show that galactose does not inhibit the transfer of amino acid, although inhibition would be expected if galactose exerted the allosteric effect suggested by ALVARADO. These experiments would, however, fit in with the hypothesis about competition for energy for transfer. In vivo blood glucose is available to supply energy to the epithelial cells, and this could account for the absence of competition between transfer mechanisms for cellular energy. I t is also possible that other common requirements of sugar and amino acid transfer are optimally maintained under in vivo conditions. We are indebted to the Medical Research Council and to John W y e t h and Brother for financial support, and to Miss A. S. BEAL and Miss J. ANDERSON for valuable technical assistance.
Department of Physiology and Medical Research Council Group on Intestinal A bsorption, University of She~eld, She~eld (Great Britain)
JANET
K. BINGHAM H. I~EWEY D. H. SMYTH
i H . N E w E Y AND D. H . SMYTH, Nature, 202 (1964) 400. 2 S. J. SAUNDERS AND K. J. ISSELBACHER, Biochim. Biophys. Acta, lO2 (1965) 397.
Biochim. Biophys. Acta, 13o (1966) 2 8 1 - 2 8 4
284
PRELIMINARY NOTES
F. ALVARADO,Science, 151 (1966) iOlO. J. K. BINGHAM, H. NEWEY AND D. H. SMYTH, Biochim. Biophys. :4cta, 12o (1966) 314 . M. F. SHEFF AND D. H. SMYTH, J. Physiol. London, 128 (1955) 67P. J. K. DUERDOTH, H. NEWEY, P. A. SANFORD AND D. H. SMYTH, J. Physiol. London, 176 (1965) 23P. 7 A. G. DAWSON, H. NEWEY AND D. H. SMYTH,J. Physiol. London, 179 (1965) 56P. 8 H. NEwEY AND D. H. SMYTH, J. Physiol. London, 17o (1964) 328.
3 4 5 6
Received
August
5th, 1966
Biochim. Biophys. Acta, 13o (1966) 281-284
281
FORSYTH,Arch. Dis. Child., 4 ° (1965) 251. A. M. BONGIOVANNI AND A. W. ROOT, New Engl. J. Med., 268 (1963) 1283. A. M. BONGIOVANNI AND A. W. ROOT, New Engl. J. Med., 268 (1963) 1342. A. M. BONGIOVANNI AND A. W. ROOT, New Engl. J. Med., 268 (1963) 1391. W. L. GARDINER AND ]~. C. HORNING, Biochim. Biophys. Acta, 115 (1966) 524 . K. BIRCHALL AND E. L. MITCHELL, Steroids, 6 (1965) 427 . R. RYHAGE, Anal. Chem., 36 (1964) 759. }{. EOTHERBY, Biochem. J., 69 (1958) 596. M. OKADA, D. K. FUKUSHIMA AND T. F. GALLAGHER,J. Biol. Chem., 234 (1959) 1688. H. HIRSCHMANN AND F. B. HIRSCHMANN, J. Biol. Chem., 187 (195 o) 137. W. R. EBERLEIN, J. Clin. Endocr., 25 (1965) IIOI. J. W. REYNOLDS, Proc. Soc. Exptl. Biol. Med., 113 (1963) 980. I~. FOTHERBY, A. COLAS, S. M. ATHERDEN AND G. F. MARRIAN, Biochem. dr., 66 (1957) 664. A. M. BONGIOVANNI, J. Clin. Invest., 41 (1962) 2086. J. W. REYNOLDS, Steroids, 3 (1964) 77. A. COLAS, W. L. HEINRICHS AND H. J. TATUM, Steroids, 3 (1964) 417 .
3 D . M . CATHRO, K . BIRCI-IALL, F. L. MITCHELL AND C. C.
4 5 6 7 8 9 Io II 12 13 14 15 16 17 18
Received May 26th, 1966 Revised manuscript received September I2th, 1966 Bioehim. Biophys. Aeta, 13o (1966) 278-281
B B A 21 I6I
Interaction of sugars and amino acids in intestinal transfer NEWEY AND SMYTI-I1 showed that there is an interaction between sugar and amino acid transfer in the rat intestine and this was confirmed by SAUNDERS AND ISSELBACHER 2 in the rat, and ALVARADO3 in the hamster. BINGHAM, ~qEWEY AND SMYTH4 concluded that those sugars which are actively transferred but not metabolised, e.g., galactose, ~-methyl glucoside and 3-0-methyl glucose, inhibit amino acid transfer. In the present paper further consideration is given to the problem of galactose inhibition and to the interaction of glucose and amino acids. Everted sacs of rat small intestine were used as described previously 1. Proline or methionine (7-5 mM) was initially present in the mucosal fluid and sugars in either TABLE I EFFECT OF GALACTOSE ON AMINO ACID TRANSFER in vitro 7.5 mM prolin~ or methionine was present initially in the mucosal fluid, and galactose present in the mucosal or serosal fluids as indicated. The n u m b e r of experiments is shown in parentheses. E x p e r i m e n t a l period, 3 ° miD.
Condition
Transfer (izmoles ~ S.E.)
.~/lucosal fluid
Serosal fluid
Methionine
Proline
-28 mM galactose --
--+ 222 mM galactose + 222 mM sorbose 222 mM galactose 222 mM galactose
33.2 15.o 30.0 32.0 13.4 29.2
39.1 ± 1.3 (13) 24.6 ± 0.6 (6) 36.6 ~ 1.9 (7) 36.0 ± 1.9 (7) 17. 5 ~ 2.8 (7) 39.3 i 2.0 (7)
-
28 mM galactose 28 mM galactose + 5" IO-4 M phlorrhizin
± ± ~ ± ± ~k
1.2 (22) 0. 5 (6) 0.9 (6) 1.6 (7) 0-7 (7) 1.5 (6)
Biochim. Biophys. Acta, I3o (1966) 281-284
282
PRELIMINARY
NOTES
the mucosal or serosal fluids. The in vivo experiments were carried out by the method of SHEEF AND SMYTH 5, and IO mM amino acid with or without 30 mM sugar was circulated through the lumen of the intestine. The results with galactose in vitro are shown in Table I. Sorbose, an inert sugar 4, has been included for comparison. When present in the mucosal fluid galactose inhibited proline and methionine in confirmation of earlier results. Galactose present in both the mucosal and serosal fluids also caused inhibition, which was overcome by the presence of 5" lO-4 M phlorrhizin in the mucosal fluid. Galactose present in a high concentration in the serosal fluid caused little, if any, inhibition. These results show that the presence of galactose on both sides of the luminal membrane does not in itself cause inhibition of amino acid transfer, but that inhibition is related to movement of galactose across the luminal membrane. Since 222 mM serosal galactose did not inhibit, it appears also that a high concentration of galactose within the cells is not the cause of the inhibition. This conclusion is based on the assumption that galactose initially present in the serosal fluid can enter the epithelial cells, and this assumption is supported by the results in Table I I in which other sugars are used. This shows that the presence of glucose or mannose in the serosal fluid stimulates amino acid transfer, and hence these hexoses must be able to enter the cell from the serosal fluid. These results are analogous to those of D U E R D O T H et al3 in which it was shown that certain hexoses initially present in the serosal fluid stimulate fluid transfer. Since other hexoses can enter the cell from the serosal side it is likely that galactose can do so. TABLE
II
t~FFECT OF VARIOUS SUGARS ON METHIONINE AND PROLINE TRANSFER
in vitro
M e t h i o n i n e o r p r o l i n e w a s p r e s e n t i n i t i a l l y i n a c o n c e n t r a t i o n of 7-5 m M i n t h e m u c o s a l f l u i d . T h e s u g a r s w e r e p r e s e n t i n e i t h e r t h e m u c o s a l o r s e r o s a l f l u i d s a s s h o w n . T h e n u m b e r of e x p e r i m e n t s is s h o w n i n p a r e n t h e s e s .
Condition
Transfer (#moles ± S.E.)
Mueosal fluid
Serosal fluid
Methionine
Proline
---
-222 m M g l u c o s e
33.2 ± 1.2 (22) 5 0 . 8 ~ 2 . I (12)
39.1 -~ 1.3 (13) 6 1 . o ~ 3.8 (IO)
--
222 mM mannose
5o.1 ± 4.1
61,o i
28 m M g l u c o s e
222 mM glucose
3 7 , i ~- 1.2 ( i i )
28 m M g l u c o s e
222 mM mannose
36.7 i
1.6
(6)
--
28 m M m a n n o s e
222 m M m a n n o s e
4 6 . 5 ~- 3.0
(6)
--
(6)
2.6
(6)
--
Table I I also shows that glucose on the serosal side stimulates methionine transfer and this contrasts with the finding of DAWSON, NEWEY AND SMYTH7 that glucose initially present in the mucosal fluid does not stimulate methionine transfer. Furthermore, mucosal glucose but not mucosal mannose prevents the stimulation by serosal glucose or serosal mannose. The explanation may be that glucose or mannose in the serosal fluid supplies energy for transfer, while glucose in the mucosal fluid not only supplies energy but also needs energy for its own transfer. Mannose thus differs from glucose in that if present in the mucosal fluid it does not use an energy-requiring transfer mechanism, but it resembles glucose in that if present in Biochm. Biophys. Acta, 13o (1966) 2 8 1 - 2 8 4
283
P R E L I M I N A R Y NOTES
the serosal fluid it can supply energy for transfer. These results are in keeping with the view of •EWEY AND SMYTH1 about competition for energy b y transfer processes in the cell. One fact not satisfactorily explained is that glucose in the mucosal fluid is able to stimulate proline and glycine transfer 7. This raises possibilities that the energy requirements for methionine transfer are in some way different from those of proline and glycine transfer. In discussing the interaction of sugars and amino acids the hypothesis of TABLE
III
EFFECT OF GALACTOSE ON AMINO ACID TRANSFER in vivo M e t h i o n i n e or p r o l i n e ( i o m M ) w a s p r e s e n t t o g e t h e r w i t h 3 ° m M h e x o s e as s h o w n . E x p e r i m e n t a l p e r i o d , 3 ° miD. T h e n u m b e r of e x p e r i m e n t s is s h o w n i n p a r e n t h e s e s .
Condition
-+ 3°mM galactose + 3°mM sorbose + 3° i n M g l u c o s e
Transfer (t~moles 4- S.E.) Methionine
Proline
359 343 338 339
319 3 lo 318 358
± 7.5 (7) -- 9.0 (6) i 11.7 (5) ± 13.4 (6)
± lO.6 (8) i 6.6 (7) ± 8.1 (6) -- 8.1 (6)
ALVARADO~ must be considered, that hexoses can exert an allosteric effect on the amino acid carrier. There are, however, some difficulties about explaining the results by allosteric effects. In the first place it would have to be assumed that the effect of glucose was exerted only on the transfer of methionine and not on the transfer of proline and glycine, although it is known that proline and glycine can use the methionine carrier s, and that galactose inhibits all three amino acidsm,4. Another difficulty arises from the results of the in vivo experiments in Table I I I which show that galactose does not inhibit the transfer of amino acid, although inhibition would be expected if galactose exerted the allosteric effect suggested by ALVARADO. These experiments would, however, fit in with the hypothesis about competition for energy for transfer. In vivo blood glucose is available to supply energy to the epithelial cells, and this could account for the absence of competition between transfer mechanisms for cellular energy. I t is also possible that other common requirements of sugar and amino acid transfer are optimally maintained under in vivo conditions. We are indebted to the Medical Research Council and to John W y e t h and Brother for financial support, and to Miss A. S. BEAL and Miss J. ANDERSON for valuable technical assistance.
Department of Physiology and Medical Research Council Group on Intestinal A bsorption, University of She~eld, She~eld (Great Britain)
JANET
K. BINGHAM H. I~EWEY D. H. SMYTH
i H . N E w E Y AND D. H . SMYTH, Nature, 202 (1964) 400. 2 S. J. SAUNDERS AND K. J. ISSELBACHER, Biochim. Biophys. Acta, lO2 (1965) 397.
Biochim. Biophys. Acta, 13o (1966) 2 8 1 - 2 8 4
284
PRELIMINARY NOTES
F. ALVARADO,Science, 151 (1966) iOlO. J. K. BINGHAM, H. NEWEY AND D. H. SMYTH, Biochim. Biophys. :4cta, 12o (1966) 314 . M. F. SHEFF AND D. H. SMYTH, J. Physiol. London, 128 (1955) 67P. J. K. DUERDOTH, H. NEWEY, P. A. SANFORD AND D. H. SMYTH, J. Physiol. London, 176 (1965) 23P. 7 A. G. DAWSON, H. NEWEY AND D. H. SMYTH,J. Physiol. London, 179 (1965) 56P. 8 H. NEwEY AND D. H. SMYTH, J. Physiol. London, 17o (1964) 328.
3 4 5 6
Received
August
5th, 1966
Biochim. Biophys. Acta, 13o (1966) 281-284