CELL BIOCHEMISTRY AND FUNCTION
VOL 8:
11-17 (1990)
Long-Term Adaptive Response to Dietary Protein of Hexose Monophosphate Shunt Dehydrogenases in Rat Kidney Tubules JUAN PERAGON,FERMIN ARANDA, LETICIA GARCIA-SALGUERO, ALBERTO M. VARGAS AND JOSE A. LUPIANEZ Departamento de Bioquimica y Biologia Molecular, Universidad de Granada, Granada, Spain.
We have studied the effects of several different macronutrients on the kinetic behaviour of rat renal glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). Rats were meal-fed with high-carbohydratellow-protein, high-proteinllow-carbohydrateand high-fat diets. High-protein increased renal G6PDH and 6PDGH activities by 66 per cent and 70 per cent respectively, without significantly changing the K,,, values of either and each Hexose monophosphate dehydrogenase activity increased steadily, reaching a significant difference on day 4.A rise in carbohydrate or fat in the diets, produced no significant change in either the activity or the kinetic parameters, V,,, and K,,, of the two dehydrogenases. In addition, the administration of a high-protein diet for 8 days significantly increased both the pentose phosphate pathway flux (92-6 per cent) and the kidney weight (35 per cent), whereas no significant changes in these parameters were found when the animals were treated with the other diets. Our results suggest that an increase in the leveis of dietary protein induces a rise in the intracellular levels of these enzymes. The possible role of this metabolic pathway in the kidneys under these nutritional conditions is also discussed. KEY
WORDS-Glucose 6-phosphate dehydrogenase; 6-phosphogluconate dehydrogenase; isolated kidney tubules; dietary regulation; kinetic analysis.
INTRODUCTION The role of the hexose monophosphate shunt has been widely studied in various different kinds of living cells, although the tendency has been to concentrate on the liver and its cellular capacity for adaptive response to several types of hormonal and dietary stimuli. It is well established that the activity of rat liver hexose monophosphate dehydrogenases, glucose 6-phosphate dehydrogenase [G6PDH (EC 1.1.1.49)] and 6phosphogluconate dehydrogenase [6PGDH (EC 1.1.1.44)], the rate-limiting enzymes of this metabolic pathway, change according to the metabolic, hormonal and nutritional states of the animal. Several studies have shown that when lipogenesis is stimulated in animals that have been fasted and refed on a high-carbohydrate diet hepatic *Addressee for correspondence: Dr Jose A. Lupianez, Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias, Universidad de Granada, Avenida Fuentenueva sln. 18001, Granada, Spain. Telephone No. 07-3458 202212 Ext. 250. 0263-6484/90/010011-07$05.00 01990 by John Wiley & Sons, Ltd.
G6PDH activity increases 10-30-f0ld,~*'while in animals which have been fasted alone or fasted and subsequently refed on a high-fat diet this enzyme activity drops s i g n i f i ~ a n t l y In . ~ ~addi~~~ tion, the inclusion of protein in the diet of normal rats is essential for the induction of lipogenic These G6PDH inductionlrepression enzymes. phenomena in response to different nutritional states are due primarily to changes in the rate of enzyme s y n t h e s i ~ . ~ * ~ . ' ~ Nevertheless, little information on this metabolic route in kidney cells is available. Sochor er al. l 1 have shown that a significant increase takes place in the renal pentose phosphate pathway flux with experimental diabetes, as a consequence of the renal hypertrophy associated with this pathological condition, and that this also applies to periods of cellular growth. The exact significance and regulatory mechanisms of the hexose monophosphate shunt in kidney cells remains, however, uncertain. The aim of this study has been to make an initial approach towards determining whether the 578*9
12 renal pentose phosphate pathway is able to adapt itself to different nutritional conditions. We have used for this purpose high-protein, highcarbohydrate, high-fat and standard diets and measured the effects of such diets on the pentose phosphate cycle dehydrogenases from isolated rat kidney tubules. Our results indicate the existence of a long-term adaptive mechanism of the two hexose monophosphate dehydroge.nases in response to the ingestion of large amounts of aminoacids.
MATERIALS AND METHODS
Chemicals Fatty-acid-free bovine serum albumin and collagenase from Clostridium histolyticum (type IV) came from the Sigma Chemical Co. (U.S.A.). NADP, glucose 6-phosphate and 6phosphogluconate were bought from Boehringer (Manheim, Germany). Radioactive reagents ([ 114C]-glucose and [6-'4C]-glucose) were supplied by Amersham International (Amersham, Bucks, U.K.). All chemicals were reagent grade.
Animals and Diets All experiments were performed on male Wistar rats initially weighing 140-150 g. The animals were maintained under controlled temperature, (22 rf: 2") and lighting (08:00 to 20:Wh) conditions. The rats were adapted to a standard diet (64 per cent carbohydrate, 20-5 per cent protein, 9 per cent olive oil) for at least 10 days. Afterwards, they were separated into four groups, three of which were fed ad libitum on one of the different experimental diets: high-protein/ low-carbohydrate (6 per cent carbohydrate, 79 per cent protein, 8-5 per cent olive oil), HP/LCH; high-carbohydrate/low-protein (79-5 per cent carbohydrate, 6 per cent protein, 8.5 per cent olive oil), HCH/LP; and high-fat (40.5 per cent carbohydrate, 15 per cent protein, 8 per cent olive oil, 15 per cent butter), HF; the fourth was kept on the standard diet as control. All the groups had free access to water. The animals were kept under these nutritional conditions for 8 days. The experimental assays were performed at 0, 1 , 4 and 8 days. The energy values for these experimental diets were similar, ranging from 1751-4 to 1795.2 kJ/100g) diet.
J . PERAG6N E T A L .
For sampling for metabolic studies, the rats were anaesthetized with sodium pentobarbital (50mgkg-') and maintained at 37°C on a warming plate. Blood was withdrawn in heparinized syringes from the aorta. It was deproteinized with cold erchloric acid (0.6M) for glucose'* and urea' determination.
I:
Isolation of Tubule Suspensionfrom Renal Cortex. Isolated rat kidney tubules were prepared by collagenase digestion as described e l ~ e w h e r e ' ~ ~ ' ~ with some modifications. Briefly, four to six rats were killed by cervical dislocation. The kidneys were removed and placed in ice-cold KrebsHenseleit solution (KHS) pH 7.4, previously gassed with 95 per cent 0 2 / 5 per cent C 0 2 for 30 min. After decapsulation, slices of kidney cortex were obtained with a Stadie Riggs microtome. The slices were minced and the mixture was poured into a siliconized Erlenmeyer flask which contained collagenase (2 mg ml-') bovine albumin (5 mgml-') in a medium of fresh KHS. After digestion for 30min at 37"C, the tissue suspension was filtered to remove the collagen fibres and washed four times with KHS to eliminate the collagenase. No slow-speed centrifugation steps were employed. After the samples had sat for about 2min, the supernatant was aspirated and the tubules were washed by the addition of KHS containing 5 mg ml-' albumin. For flux studies, the tubular suspensions were incubated for 10min at 37°C in siliconized Erlenmeyer flasks with KHS at a final volume of 4ml containing 1 per cent albumin, 6 m glucose ~ as substrate and 1pCi of labelled substrate. The final concentration of tubular suspension was 2-5mg of renal tubules ml-' of Krebs-Henseleit solution. The conversion of specifically labelled glucose into14C02 by kidney tubules was measured in the presence of [ l-'4C]-glucose or [6-14C]-glucose. The activity of the pentose phosphate pathway is p n by the difference in14C02 yields from [ 1- 4C]-glucose and [6-14C]glucose.' Results are expressed in terms of nmol per mg dry tissue weight for 15min.
Enzyme Assays The tubular suspensions (150 mg mi-') were homogenized in a medium containing Tris (10 mM), EDTA ( l m ~ and ) NADP ( O - l m ~ ) ad,
13
DIETARY INFLUENCE ON PPC RENAL DEHYDROGENASES
justed to pH 7.6. The homogenate was centri- Statistical Analysis fuged at 30-OOOg for 30min at 4°C and the All values are reported as means 2 S.E.M. supernatant was used to determine enzyme statistical significance was determined using stuactivities. Glucose 6-phosphate dehydrogenase dent's t-test. was assayed at 25"C, as described by Lupihiiez et al. , I 6 with the following modifications: G6PDH activity was corrected for 6-phosphogluconate RESULTS AND DISCUSSION dehydrogenase activity; the G6PDH assay was carried out in a medium containing, 0-15ml of The effects of several different nutritional condisupernatant, 5 0 m ~Tris-HC1, p H 7-4, 5 m ~tions on kinetic parameters, metabolite concenEDTA, 0.6 mM NADP and G6P in a total volume tration in blood and pentose phosphate pathway of 1ml (the concentrations of glucose 6- activity are shown in Table 1. Arterial glycemia phosphate were from 0.005 mM to 2 mM); 6PGDH was significantly lower with the high-protein diet was measured in the same medium; the concen- than with the control diet, which reflects a trations of 6PG were from 0-005mM to 2 mM, one considerable absorption of this metabolite by mUnit activity being defined as the amount of peripheral tissues, whereas glucose was released by the liver and kidney,'* amino acids being the enzyme required to reduce 1 nmol NADP min-' main substrates for gluconeogenesis. This extenat 25°C. Protein was measured according to the method sive catabolism of aminoacids resulted in a described by Bradford. l7 The protein concentra- significant increase in blood urea. Furthermore, the high protein diet increased the kidney weight tion in the assay solution was corrected to 0-5m Enzyme activity is expressed as mUnitsmg- . by 35 per cent after 8 days of treatment. This The activity ratio is defined as the relationship increase allowed us to calculate that renal between the enzyme activity at subsaturating hypertrophy adds 46.5 mg day-' g-' of kidney. substrate concentration (0.05 mM) and maximum The high protein diet also augmented renal pentose phosphate pathway activity dramatically velocity .
8.
Table 1. Effects of different nutritional conditions on kidney and blood parameters and renal pentose phosphate pathway activity ~~~
-
Diet HP/LCH 8d
HCH/LP 8d
HF
187.8 f 1.3 (8) 225.2 f 1.2 (8) 1.88 f 0.08 (8) 0.83 f 0.06 (8)
188.1 f 1.4 (6) 235.2 f 2.1 (6) 2.58 f 0.07' (6) 1.10 f 0.05" (6)
188.9 I 2 . 1 (6) 224.9 t 2.0 (6) 1.91 t 0.10 (6) 0.85 f 0.08 (6)
187.6 f 2.0 (6) 227.4 2 1.9 (6) 1.92 f 0.18 (6) 0-84 f 0.12 (6)
6.50 f 0.35 (8) 4-22 t 0-41 (8) 1.35 f 0.11 (5)
5.10 f 0.28+ ( 6) 15.39 f 0.79* ( 6) 2.60 t 0.20" (5)
6.43 f 0.48 (6) ND
6.51 f 0.42 (6) ND
1.50 2 0.09 (4)
1.45 k 0.05 (4)
Control
Initial body weight (8) Final body weight (g) Kidney weight (g) Relative kidney weight (g 100g)-' body weight) Blood glucose ( m M) Blood urea
(mM)
Pentose phospate pathway activity (nmol mg-' dry wt of tubules (15'-')
8d
HP/LCH = high-proteinflow-carbohydrate;HCHLP = high-carbohydrateAow-protein;HF = high-fat diets. Results are means k S.E.M. for the number of observations in brackets. Significant differences between control and diets: *p < 0-005; ' p < 0.05. ND, not determined.
14
J . PERAG6N E T A L .
2( c
1c
A
0.L
*
u.4
2 T
20
10
I
I I
I
1
2
SUBSTRATE CONCENTRATION (mM) Figure 1 . Effect of dietary protein on the kinetics of renal glucose 6-phosphate dehydrogenase (upper panel) and 6-phosphogluconate dehydrogenase (bottom panel) as a function of the length of treatment. Isolated kidney tubules were used in the activity assays of both enzymes as described in the Materials and Methods section. Results are means k S.E.M. of two to 13 experiments. Control (O), 1 day H P (0), 4 days H P (B)and 8 days H P (0). Inset graphs show the double reciprocal plots of the kinetic data.
15
DIETARY INFLUENCE O N PPC RENAL DEHYDROGENASES
(92.6 per cent), whereas no significant change5 were found in the flux through this cycle with the other diets One way of determining the regulatory properties of enzymes is by examining their kinetic properties. We have therefore studied the effects of several nutritional conditions on the kinetic behaviour of renal G6PDH and 6PGDH. The effects of an excess of dietary protein are shown in Figure 1. The kinetic curves of both dehydrogenases are always hyperbolic, with no evidence of co-operativity. This was confirmed by Hill's plots, which gave interaction coefficients ( h ) of 1-07 k 0.03. This kineticbehaviour agrees with almost all the results described elsewhere for other tissues, with the exception of those reported by Luzzatto and Afolayan," who obtained a sigmoid curve for intracellular G6PDH activity in human erythrocytes, although these results could not be confirmed later with the isolated enzyme.20 A significant and gradual increase in the initial velocity for both enzymes throughout the length of the saturation curves was present from day 4. No changes were found, however, at day 1 (or at day 2, although results not shown). Similar K,,, values of G6PDH and 6PGDH for glucose 6-phosphate and 6-phosphogluconate were found derived from the double reciprocal plots (insets of Figure 1). There were no changes in the saturation curves for either enzyme during the entire experimental period when the animals were fed on high-carbohydrateAow-protein or high-fat diets (results not shown).
Since double-reciprocal plots tend to emphasize the values obtained at low substrate concentrations, where the degree of error is likely to be greatest,'l the data from these experiments were analysed by a simple least-square fitting of the untransformed data to a rectangular hyperbola." The kinetic data for G6PDH and 6PGDH are shown in Tables 2 and 3 respectively. No significant changes in enzyme activity nor in any other kinetic parameter were found for these renal dehydrogenases in the animals fed on either the high-carbohydrate or the high-fat diet, diets directly related to variations in the intracellular level of these enzymes in hepatic cells.- These results agree with those of Yagil et al. ,23 who have shown that G6PDH from the kidneys of C57BI mice did not change after seven days on either a fat-free or a high-fat diet. Our results demonstrate, however, that a high-protein diet does regulate renal G6PDH and 6PGDH activities. The V,,, of both dehydrogenases in animals fed on this diet increased steadily throughout the experimental period and was significant from day 4 onwards; the highest values reached were almost 1-75times the control values. No significant changes were found in the K , values. This kinetic behaviour confirms that the activity ratio of both enzymes remains unmodified under this nutritional situation, suggesting that the stimulation of the enzyme activity is probably due to an increase in the intracellular levels of both hexose monophosphate dehydrogenases and not to any polymerization process,24 as polymerization requires a shorter
Table 2. Evolution of the kinetic parameters of renal glucose 6-phosphate dehydrogenase under different nutritional conditions. Days of treafment
Diet*
HPiLCH HCH/LP HF
Kinetic parameters' Km (w) V,,, ( m u mg-' protein) Activity ratio ( VO.OcJVmax) Km (PM) V,,, ( m u mg-' protein) Activity ratio ( VO.&',ax) Km ( w ) V,,, ( m u mg-' protein) Activity ratio ( V0.05/V,ax)
Control
25-90 f 3.09 14-44 f 0.60 0-67 f 0.03 23.95 f 2.47 14.03 k 0.59 0.72 f 0.03 25.90 f 3.09 14.44 f 0.60 0.67 f 0.03
(8)' (8) (8) (8) (8) (8) (8) (8) (8)
i
4
8
23.40 i 5-42 (6) 13-65 i 1-20 (6) 0.65 i 0.06 (6) 28-92 f 2.95 (4) 17-09 i 1.64 (4) 0.66 5 0.06 (4) 25.50 f 0.82 (4) 13-25 i 0.57 (4) 0-73 i 0-02 (4)
29.10 f 4.82 (4) 19.81 f 1.11 (4)$ 0.75 i 0.08 (4) 24.09 i 3-48 (2) 15.30 i 0-81 (2) 0.68 i 0.04 (2) 32.11 f 6-44 (4) 14.60 i 1-17 (4) 0.66 f 0.05 (4)
27-59 f 3.03 ( 5 ) 23.93 i 2.22 (5)'l 0-74 f 0.09 ( 5 ) 32.33 i 7.77 (3) 15.16 i 0-14 (3) 0.61 2 0.01 (3) 30-30 f 5.78 (4) 14.95 k 0.78 (4) 0.58 f 0.03 (4)
*HP/LCH = high-protein/low-carbohydrate; HCH/LP = high-carbohydrateilow-protein; H F = high-fat diets. 'Kinetic parameters were determined from a simple least-square hyperbola described by the Michaelis-Menten equation.". Vn 0 5 , velocity at 0.05 mM of substrate. v,,,, maximum velocity. *Results are means f S.E.M. with the number of experiments in bracket>. Significant differences between control and diet treatment are expressed as ' p < 0401, Ib < 0-OOO5.
16
I. PERAG6N E T A L .
Table 3. Evolution of the kinetic parameters of renal 6-phosphogluconate dehydrogenase under different nutritional conditions.
Diet*
HPiLCH
Kinetic parameterst
Control
Km ( W ) V,,, ( m u mg-' protein)
62.83 f 2.16 13.73 f 0.44 0.45 f 0.01 58.37 f 1.73 13.80 f 0.55 0.47 f 0.02 61.05 f 3.55 14.31 f 0.66 0.45 f 0.02
Activity ratio (Vo.os/Vm.J HCWLP HF
Km ( w )
V,,,,, ( m u mg-' protein) Activity ratio ( Vo.os/Vmax) Km (w) V,,, ( m u mg-' protein) Activity ratio ( Vo.05/Vmax)
Days of treatment 2 (13)* (13) (13) (9) (9) (9) (6) (6) (6)
60.82 f 6.53 14.65 i 0.48 0.45 f 0.01 53-25 f 2.93 13.56 i 1.21 0.48 f 0.04 57.81 f 4.10 12.93 5 0.55 0.47 k 0.02
(4) (4) (4) (4) (4) (4) (4) (4) (4)
8
4
66.25 f 1.98 20.12 f 1.68 0.43 f 0.03 65.09 f 4-25 15.01 f 0.04 0-41 f 0.01 66.09 k 5.07 16.00 f 1.66 0.42 f 0.04
(4) (4)§ (4) (2) (2) (2) (6) (6) (6)
67.02 f 4.99 23.32 f 0.65 0.40 f 0.01 52.09 f 3.44 14.14 f 0.36 0.49 f 0.01 53.20 f 10.41 16.06 f 0.11 0.45 f 0-01
(5) (5)l' (5) (2) (2) (2) (2) (2) (2)
*HP/LCH = high-proteinilow-carbohydrate; HCHiLP = high-carbohydrateilow-protein; H F = high-fat diets. 'Kinetic parameters were determined from a simple least-square hyperbola described by the Michaelis-Menten equation." V,.,,, velocity at 0.05 mM of substrate. V,,,, maximum velocity. *Results are means ? S.E.M. with the number of experiments in brackets. Significant differences between control and diet treatment are expressed as s p < 0-0005, $ < O ~ O O O 1 . ~
time than protein induction and we found no significant change after 1 (results not shown) or even 2 days of high protein ingestion There may be several reasons for the enhancement of renal pentose phosphate pathway activity by dietary protein: (i) an excess of dietary protein causes renal hypertrophy, stimulation in cellular growth and protein for all of which an increase in nucleic acid metabolism and thus nucleotide synthesis is necessary; (ii) the excess in aminoacid which are not used up in protein synthesis might be metabolized through acetylCoA via phosphoenolpyruvate carboxykinase, pyruvate kinase and pyruvate dehydrogenase into fatty acid and cholesterol to be incorporated into the new cell membrane^;',^ (iii) and finally, the generation of oxygen free radicals, such as hydrogen peroxide, as a consequence of high aminoacid oxidation, especially through renal L-aminoacid oxidases, requires an overproduction of NADPH to prevent severe and irreversible cell damage.20 To the best of our knowledge this is the first report demonstrating a dietary protein regulation of renal G6PDH and 6PGDH. The precise nature of these changes, however, remains as yet unclear and we are continuing with our research into the subject. ACKNOWLEDGEMENTS Publication No. 133 from 'Drugs, Environmental Toxics and Cellular Metabolism Research
Group'. The authors are grateful to Dr P. Hortelano for her suggestions and critical reading of the manuscript and to Dr J. Trout for his revision of the English text. J. P. is PFPI of the Consejeria de Educacion y Ciencia de la Junta d e Andalucia, Spain. This study has been supported by a grant from the CAICYT, Project No 1139181, Spain.
REFERENCES 1. Eggleston, L. V. and Krebs, H. A. (1974). Regulation of the pentose phosphate cycle. Biochem. J . , 138,42S435. 2 . Clarke, S. D., Romsos, D. R. and Leveille, G . A. (1977). Influence of dietary fatty acids on liver and adipose tissues lipogenesis and on liver metabolites in meal-fed rats. J . Nutr., 107, 1277-1287. 3. Nace, C. S., Szepesi, B. and Michaelis, D. E. (1979). Regulation of glucose 6-phosphate dehydrogenase and malic enzyme in liver and adipose tissue: effect of dietary trilinolein level in starved-refed and ad libitum fed rats. J . Nutr., 109, 1094-1102. 4. Back, D. W . , Sohal, P. S. and Angel, J . F. (1985). Effects of diet and selected hormones on the activities of hepatic malic enzyme and glucose 6-phosphate dehydrogenase in infant, prematurely weaned rate. J . Nutr., 115, 625-632. 5. Tomlinson, J . E., Nakayama, R. and Holten, D. (1988). Repression of pentose phosphate pathway dehydrogenase synthesis and mRNA by dietary fat in rats. J . Nutr., 118, 40-15. 6. Miksicek, R. J. and Towle, H. C. (1982). Changes in the rates of synthesis and messenger RNA levels of hepatic glucose 6-phosphate and 6-phosphogluconate dehydrogenases following induction by diet or thyroid hormone. J . B i d . Chem., 257, 11829-1835.
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DIETARY INFLUENCE ON PPC RENAL DEHYDROGENASES
7. Frizt, R. S. and Kletzien, R. F. (1987). Regulation of glucose 6-phosphate dehydrogenase by diet and thyroid hormone. Mol. Cell. Endocrinol., 51, 13-17. 8. Gibson, D. M., Lyons, R. T., Scott, D . F. and Muto, Y. (1972). Synthesis and degradation of the lipogenic enzymes of rat liver. Adv. Enzyme Regul., 10, 187-204. 9. Herzberg, G. R. (1983). The influence of dietary fatty acid composition on lipogenesis. Adv. Nutr. Res., 5 , 221-253. 10. Kletzien, R. F., Prostko, C. R., Stumpo, D. J., McClung, K. and Dreher, K. L. (1985). Molecular cloning of DNA sequences complementary to rat liver glucose 6-phosphate dehydrogenase mRNA. Nutritional regulation of mRNA levels. J . Biol. Chem., 260, 5621-5624. 11. Sochor, M., Kunjara, S., Greenbaum, A. L. and McLean, P. (1986). Renal hypertrophy in experimental diabetes. Effect of diabetes on the pathways of glucose metabolism: differential response in adult and immature rats. Biochem. J . , 234, 573-577. 12. Bergmeyer, H. U., Bernt, E., Schmidt, F. and Stork, H. (1974). Glucose. Determination with hexokinase and glucose 6-phosphate dehydrogenase. In: Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.). Academic Press: New York, pp. 11961201. 13. Gutmann, 1. and Bergmeyer, H. U. (1974). Determination of urea with glutamate dehydrogenase as indicator enzyme. In: Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.). Academic Press: New York, pp. 1794-1797. 14. Garcia-Salguero, L. and Lupianez, J . A. (1988). Metabolic adaptation of the renal carbohydrate metabolism. I. Effects of starvation on the gluconeogenic and glycolytic fluxes in the proximal and distal renal tubules. Mol. Cell. Biochem., 83, 167-178. 15. Garcia-Salguero, L., Matinez-L6pez, M. Amores, M. V., Hortelano, P. and Lupiadez, J. A. (1988): Metabolic behaviour in isolated populations of proximal and distal rat renal tubules. Chemosphere, 17, 1049-1056. 16. Lupianez, J. A., Adroher. F. J., Vargas, A. M. and Osuna, A. (1987). Differential behavyour of glucose 6-phosphate dehydrogenase in two morphological forms of Trypanosoma cruzi. In!. J . Biochem., 19, 1085-1089. 17. Bradford, M. (1976). A rapid and sensitive method for
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the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248-254. Garcia-Salguero, L. and Lupiaiiez, J. A. (1989). Metabolic adaptation of the renal carbohydrate metabolism. 111. Effects of highprotein diet on the gluconeogenic and glycolytic fluxes in the proximal and distal renal tubules. Mol. Cell. Biochem., 90, 99-110. Luzzatto, L., and Afolayan, A. (1971). Genetic variants of human erythrocyte glucose 6-phosphate dehydrogenase. I. Regulation of activity by oxidized and reduced nicotinamide-adenin dinucleotide phosphate. Biochemistry, 10, 415-419. Kirkman, H. N. and Gaetani, G. F. (1986). Regulation of glucose 6-phosphate dehydrogenase in human erythrocytes. J. BioI. Chem., 261,403M038. Fersht, A. (1985). Enzyme Structure and Mechanism, 2nd edn. Freeman: San Francisco. Dowd, J. E. and Riggs, D. S. (1965). A comparison of estimates of Michaelis-Menten kinetic constants from various linear transformations. J . Biol. Chem., 240, 86S869. Yagil, G., Shimron, F. and Hizi, A. (1974). On the mechanism of glucose 6-phosphate dehydrogenase regulation in mouse liver. 1. Characterization of the system. Eur. J . Biochem., 45, 189200. Martins, R. N., Stokes, G. B. and Masters, C. L. (1985). Regulation of the multiple molecular forms of rat liver glucose 6-phosphate dehydrogenase by insulin and dietary restriction. Biochem. Biophys. Res. Comrnun., 127, 136142. Smith, A. H. (1933). Nutrition. Ann. Rev. Biochem., 2, 299-316. Madwig, P. and Abraham, S. (1970). Enzyme activities during development of some organs of the rat. J . Nutr., 110, 100-104. Didier, R., Remtsy, C., Dernignt, C. and Fafournoux, P. (1985). Hepatic proliferation of mitochondria in response to a high protein diet. Nurr. Res., 5 , 109>1102.
Received in revised form 24 July 1989 Accepted 20 September 1989
VOL 8:
11-17 (1990)
Long-Term Adaptive Response to Dietary Protein of Hexose Monophosphate Shunt Dehydrogenases in Rat Kidney Tubules JUAN PERAGON,FERMIN ARANDA, LETICIA GARCIA-SALGUERO, ALBERTO M. VARGAS AND JOSE A. LUPIANEZ Departamento de Bioquimica y Biologia Molecular, Universidad de Granada, Granada, Spain.
We have studied the effects of several different macronutrients on the kinetic behaviour of rat renal glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). Rats were meal-fed with high-carbohydratellow-protein, high-proteinllow-carbohydrateand high-fat diets. High-protein increased renal G6PDH and 6PDGH activities by 66 per cent and 70 per cent respectively, without significantly changing the K,,, values of either and each Hexose monophosphate dehydrogenase activity increased steadily, reaching a significant difference on day 4.A rise in carbohydrate or fat in the diets, produced no significant change in either the activity or the kinetic parameters, V,,, and K,,, of the two dehydrogenases. In addition, the administration of a high-protein diet for 8 days significantly increased both the pentose phosphate pathway flux (92-6 per cent) and the kidney weight (35 per cent), whereas no significant changes in these parameters were found when the animals were treated with the other diets. Our results suggest that an increase in the leveis of dietary protein induces a rise in the intracellular levels of these enzymes. The possible role of this metabolic pathway in the kidneys under these nutritional conditions is also discussed. KEY
WORDS-Glucose 6-phosphate dehydrogenase; 6-phosphogluconate dehydrogenase; isolated kidney tubules; dietary regulation; kinetic analysis.
INTRODUCTION The role of the hexose monophosphate shunt has been widely studied in various different kinds of living cells, although the tendency has been to concentrate on the liver and its cellular capacity for adaptive response to several types of hormonal and dietary stimuli. It is well established that the activity of rat liver hexose monophosphate dehydrogenases, glucose 6-phosphate dehydrogenase [G6PDH (EC 1.1.1.49)] and 6phosphogluconate dehydrogenase [6PGDH (EC 1.1.1.44)], the rate-limiting enzymes of this metabolic pathway, change according to the metabolic, hormonal and nutritional states of the animal. Several studies have shown that when lipogenesis is stimulated in animals that have been fasted and refed on a high-carbohydrate diet hepatic *Addressee for correspondence: Dr Jose A. Lupianez, Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias, Universidad de Granada, Avenida Fuentenueva sln. 18001, Granada, Spain. Telephone No. 07-3458 202212 Ext. 250. 0263-6484/90/010011-07$05.00 01990 by John Wiley & Sons, Ltd.
G6PDH activity increases 10-30-f0ld,~*'while in animals which have been fasted alone or fasted and subsequently refed on a high-fat diet this enzyme activity drops s i g n i f i ~ a n t l y In . ~ ~addi~~~ tion, the inclusion of protein in the diet of normal rats is essential for the induction of lipogenic These G6PDH inductionlrepression enzymes. phenomena in response to different nutritional states are due primarily to changes in the rate of enzyme s y n t h e s i ~ . ~ * ~ . ' ~ Nevertheless, little information on this metabolic route in kidney cells is available. Sochor er al. l 1 have shown that a significant increase takes place in the renal pentose phosphate pathway flux with experimental diabetes, as a consequence of the renal hypertrophy associated with this pathological condition, and that this also applies to periods of cellular growth. The exact significance and regulatory mechanisms of the hexose monophosphate shunt in kidney cells remains, however, uncertain. The aim of this study has been to make an initial approach towards determining whether the 578*9
12 renal pentose phosphate pathway is able to adapt itself to different nutritional conditions. We have used for this purpose high-protein, highcarbohydrate, high-fat and standard diets and measured the effects of such diets on the pentose phosphate cycle dehydrogenases from isolated rat kidney tubules. Our results indicate the existence of a long-term adaptive mechanism of the two hexose monophosphate dehydroge.nases in response to the ingestion of large amounts of aminoacids.
MATERIALS AND METHODS
Chemicals Fatty-acid-free bovine serum albumin and collagenase from Clostridium histolyticum (type IV) came from the Sigma Chemical Co. (U.S.A.). NADP, glucose 6-phosphate and 6phosphogluconate were bought from Boehringer (Manheim, Germany). Radioactive reagents ([ 114C]-glucose and [6-'4C]-glucose) were supplied by Amersham International (Amersham, Bucks, U.K.). All chemicals were reagent grade.
Animals and Diets All experiments were performed on male Wistar rats initially weighing 140-150 g. The animals were maintained under controlled temperature, (22 rf: 2") and lighting (08:00 to 20:Wh) conditions. The rats were adapted to a standard diet (64 per cent carbohydrate, 20-5 per cent protein, 9 per cent olive oil) for at least 10 days. Afterwards, they were separated into four groups, three of which were fed ad libitum on one of the different experimental diets: high-protein/ low-carbohydrate (6 per cent carbohydrate, 79 per cent protein, 8-5 per cent olive oil), HP/LCH; high-carbohydrate/low-protein (79-5 per cent carbohydrate, 6 per cent protein, 8.5 per cent olive oil), HCH/LP; and high-fat (40.5 per cent carbohydrate, 15 per cent protein, 8 per cent olive oil, 15 per cent butter), HF; the fourth was kept on the standard diet as control. All the groups had free access to water. The animals were kept under these nutritional conditions for 8 days. The experimental assays were performed at 0, 1 , 4 and 8 days. The energy values for these experimental diets were similar, ranging from 1751-4 to 1795.2 kJ/100g) diet.
J . PERAG6N E T A L .
For sampling for metabolic studies, the rats were anaesthetized with sodium pentobarbital (50mgkg-') and maintained at 37°C on a warming plate. Blood was withdrawn in heparinized syringes from the aorta. It was deproteinized with cold erchloric acid (0.6M) for glucose'* and urea' determination.
I:
Isolation of Tubule Suspensionfrom Renal Cortex. Isolated rat kidney tubules were prepared by collagenase digestion as described e l ~ e w h e r e ' ~ ~ ' ~ with some modifications. Briefly, four to six rats were killed by cervical dislocation. The kidneys were removed and placed in ice-cold KrebsHenseleit solution (KHS) pH 7.4, previously gassed with 95 per cent 0 2 / 5 per cent C 0 2 for 30 min. After decapsulation, slices of kidney cortex were obtained with a Stadie Riggs microtome. The slices were minced and the mixture was poured into a siliconized Erlenmeyer flask which contained collagenase (2 mg ml-') bovine albumin (5 mgml-') in a medium of fresh KHS. After digestion for 30min at 37"C, the tissue suspension was filtered to remove the collagen fibres and washed four times with KHS to eliminate the collagenase. No slow-speed centrifugation steps were employed. After the samples had sat for about 2min, the supernatant was aspirated and the tubules were washed by the addition of KHS containing 5 mg ml-' albumin. For flux studies, the tubular suspensions were incubated for 10min at 37°C in siliconized Erlenmeyer flasks with KHS at a final volume of 4ml containing 1 per cent albumin, 6 m glucose ~ as substrate and 1pCi of labelled substrate. The final concentration of tubular suspension was 2-5mg of renal tubules ml-' of Krebs-Henseleit solution. The conversion of specifically labelled glucose into14C02 by kidney tubules was measured in the presence of [ l-'4C]-glucose or [6-14C]-glucose. The activity of the pentose phosphate pathway is p n by the difference in14C02 yields from [ 1- 4C]-glucose and [6-14C]glucose.' Results are expressed in terms of nmol per mg dry tissue weight for 15min.
Enzyme Assays The tubular suspensions (150 mg mi-') were homogenized in a medium containing Tris (10 mM), EDTA ( l m ~ and ) NADP ( O - l m ~ ) ad,
13
DIETARY INFLUENCE ON PPC RENAL DEHYDROGENASES
justed to pH 7.6. The homogenate was centri- Statistical Analysis fuged at 30-OOOg for 30min at 4°C and the All values are reported as means 2 S.E.M. supernatant was used to determine enzyme statistical significance was determined using stuactivities. Glucose 6-phosphate dehydrogenase dent's t-test. was assayed at 25"C, as described by Lupihiiez et al. , I 6 with the following modifications: G6PDH activity was corrected for 6-phosphogluconate RESULTS AND DISCUSSION dehydrogenase activity; the G6PDH assay was carried out in a medium containing, 0-15ml of The effects of several different nutritional condisupernatant, 5 0 m ~Tris-HC1, p H 7-4, 5 m ~tions on kinetic parameters, metabolite concenEDTA, 0.6 mM NADP and G6P in a total volume tration in blood and pentose phosphate pathway of 1ml (the concentrations of glucose 6- activity are shown in Table 1. Arterial glycemia phosphate were from 0.005 mM to 2 mM); 6PGDH was significantly lower with the high-protein diet was measured in the same medium; the concen- than with the control diet, which reflects a trations of 6PG were from 0-005mM to 2 mM, one considerable absorption of this metabolite by mUnit activity being defined as the amount of peripheral tissues, whereas glucose was released by the liver and kidney,'* amino acids being the enzyme required to reduce 1 nmol NADP min-' main substrates for gluconeogenesis. This extenat 25°C. Protein was measured according to the method sive catabolism of aminoacids resulted in a described by Bradford. l7 The protein concentra- significant increase in blood urea. Furthermore, the high protein diet increased the kidney weight tion in the assay solution was corrected to 0-5m Enzyme activity is expressed as mUnitsmg- . by 35 per cent after 8 days of treatment. This The activity ratio is defined as the relationship increase allowed us to calculate that renal between the enzyme activity at subsaturating hypertrophy adds 46.5 mg day-' g-' of kidney. substrate concentration (0.05 mM) and maximum The high protein diet also augmented renal pentose phosphate pathway activity dramatically velocity .
8.
Table 1. Effects of different nutritional conditions on kidney and blood parameters and renal pentose phosphate pathway activity ~~~
-
Diet HP/LCH 8d
HCH/LP 8d
HF
187.8 f 1.3 (8) 225.2 f 1.2 (8) 1.88 f 0.08 (8) 0.83 f 0.06 (8)
188.1 f 1.4 (6) 235.2 f 2.1 (6) 2.58 f 0.07' (6) 1.10 f 0.05" (6)
188.9 I 2 . 1 (6) 224.9 t 2.0 (6) 1.91 t 0.10 (6) 0.85 f 0.08 (6)
187.6 f 2.0 (6) 227.4 2 1.9 (6) 1.92 f 0.18 (6) 0-84 f 0.12 (6)
6.50 f 0.35 (8) 4-22 t 0-41 (8) 1.35 f 0.11 (5)
5.10 f 0.28+ ( 6) 15.39 f 0.79* ( 6) 2.60 t 0.20" (5)
6.43 f 0.48 (6) ND
6.51 f 0.42 (6) ND
1.50 2 0.09 (4)
1.45 k 0.05 (4)
Control
Initial body weight (8) Final body weight (g) Kidney weight (g) Relative kidney weight (g 100g)-' body weight) Blood glucose ( m M) Blood urea
(mM)
Pentose phospate pathway activity (nmol mg-' dry wt of tubules (15'-')
8d
HP/LCH = high-proteinflow-carbohydrate;HCHLP = high-carbohydrateAow-protein;HF = high-fat diets. Results are means k S.E.M. for the number of observations in brackets. Significant differences between control and diets: *p < 0-005; ' p < 0.05. ND, not determined.
14
J . PERAG6N E T A L .
2( c
1c
A
0.L
*
u.4
2 T
20
10
I
I I
I
1
2
SUBSTRATE CONCENTRATION (mM) Figure 1 . Effect of dietary protein on the kinetics of renal glucose 6-phosphate dehydrogenase (upper panel) and 6-phosphogluconate dehydrogenase (bottom panel) as a function of the length of treatment. Isolated kidney tubules were used in the activity assays of both enzymes as described in the Materials and Methods section. Results are means k S.E.M. of two to 13 experiments. Control (O), 1 day H P (0), 4 days H P (B)and 8 days H P (0). Inset graphs show the double reciprocal plots of the kinetic data.
15
DIETARY INFLUENCE O N PPC RENAL DEHYDROGENASES
(92.6 per cent), whereas no significant change5 were found in the flux through this cycle with the other diets One way of determining the regulatory properties of enzymes is by examining their kinetic properties. We have therefore studied the effects of several nutritional conditions on the kinetic behaviour of renal G6PDH and 6PGDH. The effects of an excess of dietary protein are shown in Figure 1. The kinetic curves of both dehydrogenases are always hyperbolic, with no evidence of co-operativity. This was confirmed by Hill's plots, which gave interaction coefficients ( h ) of 1-07 k 0.03. This kineticbehaviour agrees with almost all the results described elsewhere for other tissues, with the exception of those reported by Luzzatto and Afolayan," who obtained a sigmoid curve for intracellular G6PDH activity in human erythrocytes, although these results could not be confirmed later with the isolated enzyme.20 A significant and gradual increase in the initial velocity for both enzymes throughout the length of the saturation curves was present from day 4. No changes were found, however, at day 1 (or at day 2, although results not shown). Similar K,,, values of G6PDH and 6PGDH for glucose 6-phosphate and 6-phosphogluconate were found derived from the double reciprocal plots (insets of Figure 1). There were no changes in the saturation curves for either enzyme during the entire experimental period when the animals were fed on high-carbohydrateAow-protein or high-fat diets (results not shown).
Since double-reciprocal plots tend to emphasize the values obtained at low substrate concentrations, where the degree of error is likely to be greatest,'l the data from these experiments were analysed by a simple least-square fitting of the untransformed data to a rectangular hyperbola." The kinetic data for G6PDH and 6PGDH are shown in Tables 2 and 3 respectively. No significant changes in enzyme activity nor in any other kinetic parameter were found for these renal dehydrogenases in the animals fed on either the high-carbohydrate or the high-fat diet, diets directly related to variations in the intracellular level of these enzymes in hepatic cells.- These results agree with those of Yagil et al. ,23 who have shown that G6PDH from the kidneys of C57BI mice did not change after seven days on either a fat-free or a high-fat diet. Our results demonstrate, however, that a high-protein diet does regulate renal G6PDH and 6PGDH activities. The V,,, of both dehydrogenases in animals fed on this diet increased steadily throughout the experimental period and was significant from day 4 onwards; the highest values reached were almost 1-75times the control values. No significant changes were found in the K , values. This kinetic behaviour confirms that the activity ratio of both enzymes remains unmodified under this nutritional situation, suggesting that the stimulation of the enzyme activity is probably due to an increase in the intracellular levels of both hexose monophosphate dehydrogenases and not to any polymerization process,24 as polymerization requires a shorter
Table 2. Evolution of the kinetic parameters of renal glucose 6-phosphate dehydrogenase under different nutritional conditions. Days of treafment
Diet*
HPiLCH HCH/LP HF
Kinetic parameters' Km (w) V,,, ( m u mg-' protein) Activity ratio ( VO.OcJVmax) Km (PM) V,,, ( m u mg-' protein) Activity ratio ( VO.&',ax) Km ( w ) V,,, ( m u mg-' protein) Activity ratio ( V0.05/V,ax)
Control
25-90 f 3.09 14-44 f 0.60 0-67 f 0.03 23.95 f 2.47 14.03 k 0.59 0.72 f 0.03 25.90 f 3.09 14.44 f 0.60 0.67 f 0.03
(8)' (8) (8) (8) (8) (8) (8) (8) (8)
i
4
8
23.40 i 5-42 (6) 13-65 i 1-20 (6) 0.65 i 0.06 (6) 28-92 f 2.95 (4) 17-09 i 1.64 (4) 0.66 5 0.06 (4) 25.50 f 0.82 (4) 13-25 i 0.57 (4) 0-73 i 0-02 (4)
29.10 f 4.82 (4) 19.81 f 1.11 (4)$ 0.75 i 0.08 (4) 24.09 i 3-48 (2) 15.30 i 0-81 (2) 0.68 i 0.04 (2) 32.11 f 6-44 (4) 14.60 i 1-17 (4) 0.66 f 0.05 (4)
27-59 f 3.03 ( 5 ) 23.93 i 2.22 (5)'l 0-74 f 0.09 ( 5 ) 32.33 i 7.77 (3) 15.16 i 0-14 (3) 0.61 2 0.01 (3) 30-30 f 5.78 (4) 14.95 k 0.78 (4) 0.58 f 0.03 (4)
*HP/LCH = high-protein/low-carbohydrate; HCH/LP = high-carbohydrateilow-protein; H F = high-fat diets. 'Kinetic parameters were determined from a simple least-square hyperbola described by the Michaelis-Menten equation.". Vn 0 5 , velocity at 0.05 mM of substrate. v,,,, maximum velocity. *Results are means f S.E.M. with the number of experiments in bracket>. Significant differences between control and diet treatment are expressed as ' p < 0401, Ib < 0-OOO5.
16
I. PERAG6N E T A L .
Table 3. Evolution of the kinetic parameters of renal 6-phosphogluconate dehydrogenase under different nutritional conditions.
Diet*
HPiLCH
Kinetic parameterst
Control
Km ( W ) V,,, ( m u mg-' protein)
62.83 f 2.16 13.73 f 0.44 0.45 f 0.01 58.37 f 1.73 13.80 f 0.55 0.47 f 0.02 61.05 f 3.55 14.31 f 0.66 0.45 f 0.02
Activity ratio (Vo.os/Vm.J HCWLP HF
Km ( w )
V,,,,, ( m u mg-' protein) Activity ratio ( Vo.os/Vmax) Km (w) V,,, ( m u mg-' protein) Activity ratio ( Vo.05/Vmax)
Days of treatment 2 (13)* (13) (13) (9) (9) (9) (6) (6) (6)
60.82 f 6.53 14.65 i 0.48 0.45 f 0.01 53-25 f 2.93 13.56 i 1.21 0.48 f 0.04 57.81 f 4.10 12.93 5 0.55 0.47 k 0.02
(4) (4) (4) (4) (4) (4) (4) (4) (4)
8
4
66.25 f 1.98 20.12 f 1.68 0.43 f 0.03 65.09 f 4-25 15.01 f 0.04 0-41 f 0.01 66.09 k 5.07 16.00 f 1.66 0.42 f 0.04
(4) (4)§ (4) (2) (2) (2) (6) (6) (6)
67.02 f 4.99 23.32 f 0.65 0.40 f 0.01 52.09 f 3.44 14.14 f 0.36 0.49 f 0.01 53.20 f 10.41 16.06 f 0.11 0.45 f 0-01
(5) (5)l' (5) (2) (2) (2) (2) (2) (2)
*HP/LCH = high-proteinilow-carbohydrate; HCHiLP = high-carbohydrateilow-protein; H F = high-fat diets. 'Kinetic parameters were determined from a simple least-square hyperbola described by the Michaelis-Menten equation." V,.,,, velocity at 0.05 mM of substrate. V,,,, maximum velocity. *Results are means ? S.E.M. with the number of experiments in brackets. Significant differences between control and diet treatment are expressed as s p < 0-0005, $ < O ~ O O O 1 . ~
time than protein induction and we found no significant change after 1 (results not shown) or even 2 days of high protein ingestion There may be several reasons for the enhancement of renal pentose phosphate pathway activity by dietary protein: (i) an excess of dietary protein causes renal hypertrophy, stimulation in cellular growth and protein for all of which an increase in nucleic acid metabolism and thus nucleotide synthesis is necessary; (ii) the excess in aminoacid which are not used up in protein synthesis might be metabolized through acetylCoA via phosphoenolpyruvate carboxykinase, pyruvate kinase and pyruvate dehydrogenase into fatty acid and cholesterol to be incorporated into the new cell membrane^;',^ (iii) and finally, the generation of oxygen free radicals, such as hydrogen peroxide, as a consequence of high aminoacid oxidation, especially through renal L-aminoacid oxidases, requires an overproduction of NADPH to prevent severe and irreversible cell damage.20 To the best of our knowledge this is the first report demonstrating a dietary protein regulation of renal G6PDH and 6PGDH. The precise nature of these changes, however, remains as yet unclear and we are continuing with our research into the subject. ACKNOWLEDGEMENTS Publication No. 133 from 'Drugs, Environmental Toxics and Cellular Metabolism Research
Group'. The authors are grateful to Dr P. Hortelano for her suggestions and critical reading of the manuscript and to Dr J. Trout for his revision of the English text. J. P. is PFPI of the Consejeria de Educacion y Ciencia de la Junta d e Andalucia, Spain. This study has been supported by a grant from the CAICYT, Project No 1139181, Spain.
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DIETARY INFLUENCE ON PPC RENAL DEHYDROGENASES
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Received in revised form 24 July 1989 Accepted 20 September 1989
