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Inhibitory effect of sugars on amino acid accumulation by slices of rat kidney cortex
PRELIMINARY NOTES
567
PN 1 1 8 0
Inhibitory effect of sugars on amino acid accumulation by slices of rat kidney cortex Accumulation of amino acids against a concentration gradient has been demonstrated in slices of the rat kidney cortex, and some factors influencing this phenomenon have been investigated t,2. In view of the occurrence of aminoaciduria as a manifestation of galactose toxicity in patients with congenital galaetosemia a as well as in rats fed a high galaetose dieO, experiments in vitro were undertaken to ascertain the effect of D-galactose on amino acid accumulation in the kidney slice. This communication reports the finding that not only D-galactose but also D-glucose and D-fructose decrease the ability of the kidney-slice cells to accumulate amino acids against a concentration gradient. TABLE I THE EFFECT OF D-GLUCOSE, D-GALACTOSE AND D-FRUCTOSE ON THE ACCUMULATION OF AMINO ACIDS BY RAT-KIDNEY-CORTEX SLICES 3 slices w e i g h i n g a t o t a l of 8 o - l o o m g were i n c u b a t e d a e r o b i c a l l y (95 % 0 2 - 5 % CO2) for 90 m i n in 2 m l K r e b s - R i n g e r b i c a r b o n a t e buffer (pH 7-4) a t 37 °. S uga r c o n c e n t r a t i o n Was 16.8 m M (300 m g %). Ratio of intracellular to incubation-medium amino acid concentration* Sugar
None D-Glucose D-Galactose D- Fructose
o~-Aminoisobulyrate** (0.07 ram)***
Glycine** (0.03 ram)
Cycloleucine** (0.08 m M)
L-Valine** (o.2z ram)
z-Lysine (0.20 ram)
L-Phenylalanine (0.28 ram)
L-Histidine (o.t8 mM)
5.58 3.76 3.45 3.05
8.93 7.18 6.83 5.98
4.59 2.44 2.08 2.49
3 .08 2-43 2.37 2.14
3-43 3.32 3.46 3.27
2.97 2.98 2.91 3.08
2.77 2.81 2.92 2.78
* Values r e p r e s e n t t h e a v e r a g e s of from 3-5 d e t e r m i n a t i o n s . ** Differences of t h e c o n t r o l r a t i o s of t h e s e a m i n o a c i ds differ s i g n i f i c a n t l y from t h e r a t i o s o b t a i n e d in t h e p r e s e n c e of t h e s u g a r s as d e t e r m i n e d b y t h e s t u d e n t " t " test. *** I n i t i a l m e d i u m a m i n o acid c o n c e n t r a t i o n .
The technique previously described j for the assessment of the intracellular accumulation of 14C-labeled amino acids by slices of rat kidney cortex incubated in K r e b s - R i n g e r bicarbonate buffer (pH 7.4) was employed here except that [14Clinulin was used to estimate extracellular fluid volume instead of [14C~sucrose 5. The accumulation ratio of tile amino acids shown in Table I is based on the ratio of counts/min/ml intraeellular water to counts/min/ml of incubation medium. All sugars were obtained commercially except 3-deoxy-D-glucose, which was synthesized by Dr. J. PRATT of the National Institutes of Health, Bethesda. a-Amino [I-14C]isobutyric acid (3.39 mC per mmole) was purchased from Isotope Specialties Co., Burbank, Calif., [I-14Clphenylalanine (0.69 mC/mrnole) and [carboxy-14C]cycloleucine (2.6 mC per mmole) 6 from New England Nuclear Co., uniformly labelled L-[z4C]lysine (1.62 mC per mmole) from Nuclear Chicago, [2-14C~glycine (5.69 mC/mmole), uniformly labelled L-[14C~histidine (I.O9 mC/mmole) and L- [laelvaline (o.97 me/mmole) from Volk Radiochemical Co., Chicago. The results of these experiments are shown in Table I. 3oo mg % (16.8 mM) galaetose, fructose and glucose inhibits the accumulation of a-aminoisobutyric acid, B i o c h i l n . B i o p h y s . A c l a , 65 (1962) 5 6 7 - 5 6 8
568
PRELIMINARY NOTES
glycine, cycloleucine and L-valine but does not affect the uptake of L-histidine, L-lysine or L-phenylalanine. Incubation of greater than 30 min duration was required to demonstrate inhibition. The 9o-min incubation values in Table I represent an effect on the accumulation at equilibrium for this system 1. D-Glucose was not inhibitory at 5.6 raM, a physiological level, but exerted an effect at 11.2 mM. D-Galactose and D-fructose caused inhibition similar to that seen in Table I even at 5.6 m M (Ioomg %), the lowest concentration thus far tested. The following sugars at 16.8 m M concentration did not inhibit uptake of ~-aminoisobutyric acid: raffinose, sucrose, D-xylose, D-ribose, 3-O-methylglucose, 2-deoxyglucose and 3-deoxyglucose. It appears that the inhibiting effect of sugars is not manifested in all tissues. Fructose caused inhibition of cycloleucine accumulation by intestinal segments but did not alter uptake of ~-aminoisobutyric acid b y the isolated hemidiaphragm. The mechanism of the glucose-, galactose- and fructose-induced depression of amino acid accumulation is under investigation. The phenomenon cannot be explained b y any osmotic effect of these sugars, since equal concentrations of several other sugars do not decrease amino acid uptake. Experiments have revealed that no alteration in total tissue water or extracellular fluid space occurs in the presence of these sugars. There appears to be some specificity both for the amino acids affected and for the sugars causing the effect. Inhibition is observed in the presence of sugars capable of extensive metabolic alteration by the kidney sliceL The latter fact, in addition to the lack of an effect in the first 3o min of incubation, suggests that a metabolite m a y be responsible. It is noteworthy that aminoaciduria in man often accompanies not only the galactosuria of congenital galactosemia but also the glucosuria of the Fanconi syndrome s and diabetes mellitus 9 as well as the fructosuria in hereditary fructose intolerance 1°. The rat-kidney-cortex slice m a y be a useful model system for studying the association of aminoacidurias and melliturias.
The Clinical Endocrinology Branch and Metabolic Diseases Branch, National Institute of Arthritis and Metabolic Diseases and Metabolism Service, National Cancer Institute, National Institutes of Health, Bethesda, Md. (U.S.A.) L. L. A. 4 L. 5 L. 6 H. 7 R. s C. 9 C. 10 Z.
STANTON SEGAL SAMUEL THIER MAURICE F o x LEON ROSENBERG
E. ROSENBERG, A. BLAIR AND S. SEGAL, Biochim. Biophys. dcta, 54 (1961) 2265. E. ROSENBERG, S. DOWNING AND S. SEGAL, J. Biol. Chem., 237 (1962) 2265. HOLZEL, G. M. KOMROWER AND V. K. \VILSON, Brit. Med. J., I (1952) 194. E. ROSENBERG, A. WEINBERG AND S. SEGAL, Biochim. Biophys. dcta, 48 (1961) 500. E. ROSENBERG, S. DOWNING AND S. SEGAL, Am. J. Physiol., 202 (1962) 800. AKEDO AND H. N. CHRISTENSEN, J. Biol. Chem., 237 (1962) 113. B. FLINN, B. LEBOEUF AND G. F. CAHILL, Am. J. Physiol., 200 (1961) 508. E. DENT, Biochem..J., 41 (1947) 240. H. GRAY AND E. K. ILLING, J. Endocrinol., 8 (1951) 44. L. DORMANDY AND R. J. PORTER, Lancet, I (1961) 1189.
Received September I7th, 1962 Biochim. Biophys. dcta, 65 (1962) 567-568
567
PN 1 1 8 0
Inhibitory effect of sugars on amino acid accumulation by slices of rat kidney cortex Accumulation of amino acids against a concentration gradient has been demonstrated in slices of the rat kidney cortex, and some factors influencing this phenomenon have been investigated t,2. In view of the occurrence of aminoaciduria as a manifestation of galactose toxicity in patients with congenital galaetosemia a as well as in rats fed a high galaetose dieO, experiments in vitro were undertaken to ascertain the effect of D-galactose on amino acid accumulation in the kidney slice. This communication reports the finding that not only D-galactose but also D-glucose and D-fructose decrease the ability of the kidney-slice cells to accumulate amino acids against a concentration gradient. TABLE I THE EFFECT OF D-GLUCOSE, D-GALACTOSE AND D-FRUCTOSE ON THE ACCUMULATION OF AMINO ACIDS BY RAT-KIDNEY-CORTEX SLICES 3 slices w e i g h i n g a t o t a l of 8 o - l o o m g were i n c u b a t e d a e r o b i c a l l y (95 % 0 2 - 5 % CO2) for 90 m i n in 2 m l K r e b s - R i n g e r b i c a r b o n a t e buffer (pH 7-4) a t 37 °. S uga r c o n c e n t r a t i o n Was 16.8 m M (300 m g %). Ratio of intracellular to incubation-medium amino acid concentration* Sugar
None D-Glucose D-Galactose D- Fructose
o~-Aminoisobulyrate** (0.07 ram)***
Glycine** (0.03 ram)
Cycloleucine** (0.08 m M)
L-Valine** (o.2z ram)
z-Lysine (0.20 ram)
L-Phenylalanine (0.28 ram)
L-Histidine (o.t8 mM)
5.58 3.76 3.45 3.05
8.93 7.18 6.83 5.98
4.59 2.44 2.08 2.49
3 .08 2-43 2.37 2.14
3-43 3.32 3.46 3.27
2.97 2.98 2.91 3.08
2.77 2.81 2.92 2.78
* Values r e p r e s e n t t h e a v e r a g e s of from 3-5 d e t e r m i n a t i o n s . ** Differences of t h e c o n t r o l r a t i o s of t h e s e a m i n o a c i ds differ s i g n i f i c a n t l y from t h e r a t i o s o b t a i n e d in t h e p r e s e n c e of t h e s u g a r s as d e t e r m i n e d b y t h e s t u d e n t " t " test. *** I n i t i a l m e d i u m a m i n o acid c o n c e n t r a t i o n .
The technique previously described j for the assessment of the intracellular accumulation of 14C-labeled amino acids by slices of rat kidney cortex incubated in K r e b s - R i n g e r bicarbonate buffer (pH 7.4) was employed here except that [14Clinulin was used to estimate extracellular fluid volume instead of [14C~sucrose 5. The accumulation ratio of tile amino acids shown in Table I is based on the ratio of counts/min/ml intraeellular water to counts/min/ml of incubation medium. All sugars were obtained commercially except 3-deoxy-D-glucose, which was synthesized by Dr. J. PRATT of the National Institutes of Health, Bethesda. a-Amino [I-14C]isobutyric acid (3.39 mC per mmole) was purchased from Isotope Specialties Co., Burbank, Calif., [I-14Clphenylalanine (0.69 mC/mrnole) and [carboxy-14C]cycloleucine (2.6 mC per mmole) 6 from New England Nuclear Co., uniformly labelled L-[z4C]lysine (1.62 mC per mmole) from Nuclear Chicago, [2-14C~glycine (5.69 mC/mmole), uniformly labelled L-[14C~histidine (I.O9 mC/mmole) and L- [laelvaline (o.97 me/mmole) from Volk Radiochemical Co., Chicago. The results of these experiments are shown in Table I. 3oo mg % (16.8 mM) galaetose, fructose and glucose inhibits the accumulation of a-aminoisobutyric acid, B i o c h i l n . B i o p h y s . A c l a , 65 (1962) 5 6 7 - 5 6 8
568
PRELIMINARY NOTES
glycine, cycloleucine and L-valine but does not affect the uptake of L-histidine, L-lysine or L-phenylalanine. Incubation of greater than 30 min duration was required to demonstrate inhibition. The 9o-min incubation values in Table I represent an effect on the accumulation at equilibrium for this system 1. D-Glucose was not inhibitory at 5.6 raM, a physiological level, but exerted an effect at 11.2 mM. D-Galactose and D-fructose caused inhibition similar to that seen in Table I even at 5.6 m M (Ioomg %), the lowest concentration thus far tested. The following sugars at 16.8 m M concentration did not inhibit uptake of ~-aminoisobutyric acid: raffinose, sucrose, D-xylose, D-ribose, 3-O-methylglucose, 2-deoxyglucose and 3-deoxyglucose. It appears that the inhibiting effect of sugars is not manifested in all tissues. Fructose caused inhibition of cycloleucine accumulation by intestinal segments but did not alter uptake of ~-aminoisobutyric acid b y the isolated hemidiaphragm. The mechanism of the glucose-, galactose- and fructose-induced depression of amino acid accumulation is under investigation. The phenomenon cannot be explained b y any osmotic effect of these sugars, since equal concentrations of several other sugars do not decrease amino acid uptake. Experiments have revealed that no alteration in total tissue water or extracellular fluid space occurs in the presence of these sugars. There appears to be some specificity both for the amino acids affected and for the sugars causing the effect. Inhibition is observed in the presence of sugars capable of extensive metabolic alteration by the kidney sliceL The latter fact, in addition to the lack of an effect in the first 3o min of incubation, suggests that a metabolite m a y be responsible. It is noteworthy that aminoaciduria in man often accompanies not only the galactosuria of congenital galactosemia but also the glucosuria of the Fanconi syndrome s and diabetes mellitus 9 as well as the fructosuria in hereditary fructose intolerance 1°. The rat-kidney-cortex slice m a y be a useful model system for studying the association of aminoacidurias and melliturias.
The Clinical Endocrinology Branch and Metabolic Diseases Branch, National Institute of Arthritis and Metabolic Diseases and Metabolism Service, National Cancer Institute, National Institutes of Health, Bethesda, Md. (U.S.A.) L. L. A. 4 L. 5 L. 6 H. 7 R. s C. 9 C. 10 Z.
STANTON SEGAL SAMUEL THIER MAURICE F o x LEON ROSENBERG
E. ROSENBERG, A. BLAIR AND S. SEGAL, Biochim. Biophys. dcta, 54 (1961) 2265. E. ROSENBERG, S. DOWNING AND S. SEGAL, J. Biol. Chem., 237 (1962) 2265. HOLZEL, G. M. KOMROWER AND V. K. \VILSON, Brit. Med. J., I (1952) 194. E. ROSENBERG, A. WEINBERG AND S. SEGAL, Biochim. Biophys. dcta, 48 (1961) 500. E. ROSENBERG, S. DOWNING AND S. SEGAL, Am. J. Physiol., 202 (1962) 800. AKEDO AND H. N. CHRISTENSEN, J. Biol. Chem., 237 (1962) 113. B. FLINN, B. LEBOEUF AND G. F. CAHILL, Am. J. Physiol., 200 (1961) 508. E. DENT, Biochem..J., 41 (1947) 240. H. GRAY AND E. K. ILLING, J. Endocrinol., 8 (1951) 44. L. DORMANDY AND R. J. PORTER, Lancet, I (1961) 1189.
Received September I7th, 1962 Biochim. Biophys. dcta, 65 (1962) 567-568