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BIOCHEMICAL SOCIETY TRANSACTIONS
ranges from 7 to 8 (Davenport, 1971). Under such conditions wheat bran and wholemeal bread, though rich in all three metals, will only release small quantities of these nutrients for absorption because of the physicochemical properties of the fibre. It is possible that the presence of wheat bran and similar fibre-containing foods in the diet will also restrict the availability of trace metals from other sources. As James et al. (1978) have suggested with regard to calcium, there is a need to investigate such deleterious effects of a high-fibre diet in view of the increasing medical and commercial encouragement for the use of bran in the diet. AOAC (1975) Official Methods ofdnulysis, 12th ed., p. 461, Association of Official Analytical Chemists, Washington Burkitt, D. P. & Trowell, H. C. (1975) Refined Carbohydrate Foods and Disease Academic Press, London and New York Davenport, H. W. (1971) Physiology ofthe Digestive Tract, pp. 180-181, Year Book Medical Publishers, Chicago James, W. P. T., Branch, W. J. & Southgate, D. A. T. (1978) Lancet i, 638-639 Prasad, A. S.,Halsted, J. A. & Nadirni, M. (1960) Am. J . Med. 31, 532-546 Reinhold, J. G., Faradji, B., Abadi, P. & Isrnail-Beigi, F. (1976) in Truce Elements in Human Health and Disease (Prasad, A. S., ed.), vol. 1, pp. 163-180, Academic Press, London and New York Southgate, D. A. T. (1973) Proc. Nutr. SOC.32, 131-136 Van Soest, P. J. (1963) J. Assoc. Of, Agric. Chem. 50, 50-55
The Effect of Fructose on Potassium Ion Movements across the Cell Membrane in Liver LEIF SESTOFT, MORTEN FOLKE, LARS 0. KRISTENSEN, STEEN GAMMELTOFT and PAUL BARTELS Institute of Medical Physiology, Department A , University of Copenhagen, Panum Institute, D K 2200 Copenhagen, Denmark, and Huidme Hospital, D K 2930 Klampenborg, Denmark
The accumulation of L-glycerol 3-phosphate in perfused liver during glycerol uptake is accompanied by an increased intracellular electrical negativity (hyperpolarization), which appears to be related to, albeit not quantitatively explained by, an uptake of K+, which is stoicheiometric with the uptake of Pi at pH7.4 (Sestoft et al., 1977). Thus the uptake of K + seems to be secondary to the metabolically induced uptake of Pi. The selective uptake of K+ against its concentration gradient may be accomplished by an increased activity of (Na++K+)-dependent ATPase* or by a decreased permeability for K+. Fructose metabolism is accompanied by an uptake of Pi (Sestoft, 1974), but with this substrate a biphasic movement of K+ is induced: an initial release of K+ followed by a more lasting net uptake (Folke, 1974). The experiments presented here were performed with non-recirculating rat liver perfusion and with isolated hepatocytes in order to investigate the dependence of fructose-induced cation exchange on concomitant transport of Pi, and to estimate whether fructose induces changes in K+ permeability or changes only in the unidirectional influx [as an expression of changes in the (Na+ K+)-dependent ATPase activity]. Fig. 1 shows that perfusion with l0mM-fructose in a medium with 2 . 4 m ~ - Pinduces , a large initial release of K+,which amounts to 0.7pmol/min per g of liver at its maximum and which lasts for l0min. In the following 30min perfusion there is a net K + uptake which attains a maximum rate in the period from 10 to 20min after the addition of fructose. During the whole 40min period with fructose perfusion, there is a net uptake of
+
* Abbreviation : (Na++K+)-dependentATPase, (Na++K+)-dependent adenosine triphosphatase. 1979
579th MEETING, LONDON
.z
205
1.0
:
5gc
0.5
.9 g b s O
2:
L
2 b
O
3 8 t -0.5 4a
c
Y
d"
-1 0
-10
0
10
20
30
40
50
60
Time (min)
Fig. 1. Rates of net movement of K + (w) and P i (0) between liver and perfusate during non-recirculating perfusion of rat livers (n = 4) at 37°C with Krebs-Ringer bicarbonate bufler containing albumin and bovine erythrocytes The concentrations of Pi and K+ were 2.4 and 4.8 mM respectively. In the period depicted by the bar, 10mM-fructosewas present in the medium. Negative values denote hepatic uptake rates. The values are given+ S.E.M. (vertical bars). The control period before addition of fructose lasted for 35min. ----, K+ movements in fructose-metabolizing livers perfused with a medium containing no Pi (n = 3).
Pi. After cessation of fructose perfusion, there is a large net release of K+ as well as of Pi. In perfusions with no Pi in the media there is an increased and prolonged release of K+, which rises to 1.2,umol/min per g of liver and lasts for 15min (Fig. 1). This release is followed by a net uptake during the following 25min fructose-perfusion period, but the maximum rate of uptake is only 0.2pmol/min per g of liver. Thus, in fructosemetabolizing livers, net K+ movements are not linked with movements of Pi in experiments without Pi in the medium or in the initial phase of experiments with PI in the perfusion medium. In the experiments with 2 . 4 m - P i in the medium, 10m-fructose increases the rate of O2 uptake by 30% after IOmin, whereas in perfusions without Pi in the medium, the rate of O2 uptake is decreased by 30% after lOmin with fructose. Since fructose induces severe depletion of the cellular concentration of ATP, and this effect is augmented in parallel to decreasing concentrations of extracellular PI (Sestoft, 1974; Morris et al., 1978), the initial release of K+ could be a consequence of the lack of ATP for the (Na++K+)-dependent ATPase activity. Therefore the unidirectional influx of' K+ was investigated by measuring 42Kuptake in isolated hepatocytes. Fig. 2 shows that 42K uptake is linear within the initial lOmin period. This uptake is largely determined by the activity of the (Na++K+)-dependent ATPase, since the rate of uptake is decreased to 20% of the control value in experiments where 2 . 5 m ~ ouabain was added (Fig. 2). Fructose ( ] O m ) decreases the rate of 42K uptake in the initial fructose-metabolizing period to 57.5 f3.4% (s.E.,n = 6) of the control value (Fig. 2). Thus the net loss of K+ can be explained by a decreased rate of K+ influx, which may well be the consequence of a decreased activity of the (Na++K+)-dependent ATPase. The increased K+ loss in experiments with no Pi in the perfusion medium may be interpreted as a consequence of extremely low concentrations of ATP due to (1) a decreased rate of O2 uptake and (2) an increased rate of AMP degradation (Morris et al., 1978). After 20min perfusion with fructose the K+ release was reversed to an uptake. In hepatocytes the 42Kinflux, measured as the initial 42Kuptake in hepatocyte preparations that had been exposed to fructose for 20min, was 108% of the control value. Thus the K+ influx was completely restored after 20min of fructose metabolism,
Vol. 7
BIOCHEMICAL SOCIETY TRANSACTIONS
206 U
=
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5 1 -
2
Y
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o
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1
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Time (rnin) Fig. 2. Uptake of 42Kin hepatocytes Isolated hepatocytes were preincubated at a concentration of about 6% (v/v). At time zero 200.~1of the cell suspension was transferred to lOml of a 42K-containing buffer with no substrate (o), lorn-fructose ( 0 ) or 2 . 5 ~ - o u a b a i n( A ) . At the times indicated the cells were separated from the buffer by centrifugation of 200.~1samples through silicone oil followed by counting of the radioactivity in the cell pellets.
although the mean cellular ATP concentration has been shown to be the same after 5 and 20-40min of fructose metabolism (Sestoft, 1974). The restoration of K+ influx may be due to either changed regulatory properties of the (Na++K+)-dependent ATPase or a redistribution of ATP between the mitochondria1 and cytosolic compartments in the same period. In order to estimate the effect of fructose on K+ permeability, hepatocytes were preloaded with 42K during preincubation and the fractional decrease in the radioactivity in the cells was then followed after suspension of the cells in isotope-free medium. The cell radioactivity decreased with a rate constant of 0.693h-' in experiments with lorn-fructose (n = 7) as compared with 0.777 h-' (n = 9) in control experiments without substrate (P< 0.05).The fructose-induced decrease in the rate constant can be explained, at least partly, by the concomitant hyperpolarization which decreases the driving force on the efflux of K+. Thus changes in K+ permeability seem to play no significant role in the fructose-induced K+ movements. However, it remains to be seen whether an increase in K+ permeability is involved during the K+ movement that occurs after a sudden removal of fructose from the perfusate (Fig. 1). Folke, M. (1974) Actu Physiol. Scand. 91, 18A-19A Morris, R. C., Nigon, K. & Reed, E. B. (1978) J. Clin. Invest. 61,209-220 Sestoft, L. (1974) Biochim. Biophys. Actu 343, 1-16 Sestoft, L., Folke, M., Bartels, P. D., Garnrneltoft, S. & Kristensen, L. 0. (1977) Biochim. Biophys. Actu 469, 163-170
1979
BIOCHEMICAL SOCIETY TRANSACTIONS
ranges from 7 to 8 (Davenport, 1971). Under such conditions wheat bran and wholemeal bread, though rich in all three metals, will only release small quantities of these nutrients for absorption because of the physicochemical properties of the fibre. It is possible that the presence of wheat bran and similar fibre-containing foods in the diet will also restrict the availability of trace metals from other sources. As James et al. (1978) have suggested with regard to calcium, there is a need to investigate such deleterious effects of a high-fibre diet in view of the increasing medical and commercial encouragement for the use of bran in the diet. AOAC (1975) Official Methods ofdnulysis, 12th ed., p. 461, Association of Official Analytical Chemists, Washington Burkitt, D. P. & Trowell, H. C. (1975) Refined Carbohydrate Foods and Disease Academic Press, London and New York Davenport, H. W. (1971) Physiology ofthe Digestive Tract, pp. 180-181, Year Book Medical Publishers, Chicago James, W. P. T., Branch, W. J. & Southgate, D. A. T. (1978) Lancet i, 638-639 Prasad, A. S.,Halsted, J. A. & Nadirni, M. (1960) Am. J . Med. 31, 532-546 Reinhold, J. G., Faradji, B., Abadi, P. & Isrnail-Beigi, F. (1976) in Truce Elements in Human Health and Disease (Prasad, A. S., ed.), vol. 1, pp. 163-180, Academic Press, London and New York Southgate, D. A. T. (1973) Proc. Nutr. SOC.32, 131-136 Van Soest, P. J. (1963) J. Assoc. Of, Agric. Chem. 50, 50-55
The Effect of Fructose on Potassium Ion Movements across the Cell Membrane in Liver LEIF SESTOFT, MORTEN FOLKE, LARS 0. KRISTENSEN, STEEN GAMMELTOFT and PAUL BARTELS Institute of Medical Physiology, Department A , University of Copenhagen, Panum Institute, D K 2200 Copenhagen, Denmark, and Huidme Hospital, D K 2930 Klampenborg, Denmark
The accumulation of L-glycerol 3-phosphate in perfused liver during glycerol uptake is accompanied by an increased intracellular electrical negativity (hyperpolarization), which appears to be related to, albeit not quantitatively explained by, an uptake of K+, which is stoicheiometric with the uptake of Pi at pH7.4 (Sestoft et al., 1977). Thus the uptake of K + seems to be secondary to the metabolically induced uptake of Pi. The selective uptake of K+ against its concentration gradient may be accomplished by an increased activity of (Na++K+)-dependent ATPase* or by a decreased permeability for K+. Fructose metabolism is accompanied by an uptake of Pi (Sestoft, 1974), but with this substrate a biphasic movement of K+ is induced: an initial release of K+ followed by a more lasting net uptake (Folke, 1974). The experiments presented here were performed with non-recirculating rat liver perfusion and with isolated hepatocytes in order to investigate the dependence of fructose-induced cation exchange on concomitant transport of Pi, and to estimate whether fructose induces changes in K+ permeability or changes only in the unidirectional influx [as an expression of changes in the (Na+ K+)-dependent ATPase activity]. Fig. 1 shows that perfusion with l0mM-fructose in a medium with 2 . 4 m ~ - Pinduces , a large initial release of K+,which amounts to 0.7pmol/min per g of liver at its maximum and which lasts for l0min. In the following 30min perfusion there is a net K + uptake which attains a maximum rate in the period from 10 to 20min after the addition of fructose. During the whole 40min period with fructose perfusion, there is a net uptake of
+
* Abbreviation : (Na++K+)-dependentATPase, (Na++K+)-dependent adenosine triphosphatase. 1979
579th MEETING, LONDON
.z
205
1.0
:
5gc
0.5
.9 g b s O
2:
L
2 b
O
3 8 t -0.5 4a
c
Y
d"
-1 0
-10
0
10
20
30
40
50
60
Time (min)
Fig. 1. Rates of net movement of K + (w) and P i (0) between liver and perfusate during non-recirculating perfusion of rat livers (n = 4) at 37°C with Krebs-Ringer bicarbonate bufler containing albumin and bovine erythrocytes The concentrations of Pi and K+ were 2.4 and 4.8 mM respectively. In the period depicted by the bar, 10mM-fructosewas present in the medium. Negative values denote hepatic uptake rates. The values are given+ S.E.M. (vertical bars). The control period before addition of fructose lasted for 35min. ----, K+ movements in fructose-metabolizing livers perfused with a medium containing no Pi (n = 3).
Pi. After cessation of fructose perfusion, there is a large net release of K+ as well as of Pi. In perfusions with no Pi in the media there is an increased and prolonged release of K+, which rises to 1.2,umol/min per g of liver and lasts for 15min (Fig. 1). This release is followed by a net uptake during the following 25min fructose-perfusion period, but the maximum rate of uptake is only 0.2pmol/min per g of liver. Thus, in fructosemetabolizing livers, net K+ movements are not linked with movements of Pi in experiments without Pi in the medium or in the initial phase of experiments with PI in the perfusion medium. In the experiments with 2 . 4 m - P i in the medium, 10m-fructose increases the rate of O2 uptake by 30% after IOmin, whereas in perfusions without Pi in the medium, the rate of O2 uptake is decreased by 30% after lOmin with fructose. Since fructose induces severe depletion of the cellular concentration of ATP, and this effect is augmented in parallel to decreasing concentrations of extracellular PI (Sestoft, 1974; Morris et al., 1978), the initial release of K+ could be a consequence of the lack of ATP for the (Na++K+)-dependent ATPase activity. Therefore the unidirectional influx of' K+ was investigated by measuring 42Kuptake in isolated hepatocytes. Fig. 2 shows that 42K uptake is linear within the initial lOmin period. This uptake is largely determined by the activity of the (Na++K+)-dependent ATPase, since the rate of uptake is decreased to 20% of the control value in experiments where 2 . 5 m ~ ouabain was added (Fig. 2). Fructose ( ] O m ) decreases the rate of 42K uptake in the initial fructose-metabolizing period to 57.5 f3.4% (s.E.,n = 6) of the control value (Fig. 2). Thus the net loss of K+ can be explained by a decreased rate of K+ influx, which may well be the consequence of a decreased activity of the (Na++K+)-dependent ATPase. The increased K+ loss in experiments with no Pi in the perfusion medium may be interpreted as a consequence of extremely low concentrations of ATP due to (1) a decreased rate of O2 uptake and (2) an increased rate of AMP degradation (Morris et al., 1978). After 20min perfusion with fructose the K+ release was reversed to an uptake. In hepatocytes the 42Kinflux, measured as the initial 42Kuptake in hepatocyte preparations that had been exposed to fructose for 20min, was 108% of the control value. Thus the K+ influx was completely restored after 20min of fructose metabolism,
Vol. 7
BIOCHEMICAL SOCIETY TRANSACTIONS
206 U
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Time (rnin) Fig. 2. Uptake of 42Kin hepatocytes Isolated hepatocytes were preincubated at a concentration of about 6% (v/v). At time zero 200.~1of the cell suspension was transferred to lOml of a 42K-containing buffer with no substrate (o), lorn-fructose ( 0 ) or 2 . 5 ~ - o u a b a i n( A ) . At the times indicated the cells were separated from the buffer by centrifugation of 200.~1samples through silicone oil followed by counting of the radioactivity in the cell pellets.
although the mean cellular ATP concentration has been shown to be the same after 5 and 20-40min of fructose metabolism (Sestoft, 1974). The restoration of K+ influx may be due to either changed regulatory properties of the (Na++K+)-dependent ATPase or a redistribution of ATP between the mitochondria1 and cytosolic compartments in the same period. In order to estimate the effect of fructose on K+ permeability, hepatocytes were preloaded with 42K during preincubation and the fractional decrease in the radioactivity in the cells was then followed after suspension of the cells in isotope-free medium. The cell radioactivity decreased with a rate constant of 0.693h-' in experiments with lorn-fructose (n = 7) as compared with 0.777 h-' (n = 9) in control experiments without substrate (P< 0.05).The fructose-induced decrease in the rate constant can be explained, at least partly, by the concomitant hyperpolarization which decreases the driving force on the efflux of K+. Thus changes in K+ permeability seem to play no significant role in the fructose-induced K+ movements. However, it remains to be seen whether an increase in K+ permeability is involved during the K+ movement that occurs after a sudden removal of fructose from the perfusate (Fig. 1). Folke, M. (1974) Actu Physiol. Scand. 91, 18A-19A Morris, R. C., Nigon, K. & Reed, E. B. (1978) J. Clin. Invest. 61,209-220 Sestoft, L. (1974) Biochim. Biophys. Actu 343, 1-16 Sestoft, L., Folke, M., Bartels, P. D., Garnrneltoft, S. & Kristensen, L. 0. (1977) Biochim. Biophys. Actu 469, 163-170
1979
