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Stimulation of renal gluconeogenesis by inhibition of the sodium pump
Biochimica et Biophysica Acta, 304 (1973) 142-160
,~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 27044 S T I M U L A T I O N OF R E N A L GLUCONEOGENESIS BY I N H I B I T I O N OF THE S O D I U M PUMP
DAGMAR FRIEDRICHS and WILHELM SCHONER lnstitut fiir Physiologische Chemie der Universitiit Gb'ttingen und lnstitut fiir Biochemie und Endokrinologie, Fachbereich Veteriniirmedizin, Universitiit Giessen, Giessen (Germany)*
(Received September 25th, 1972)
SUMMARY 1. Ouabain (0.1-4 m M ) stimulates glucose formation by isolated kidney tubules and kidney cortex slices from pyruvate, lactate, propionate and fructose by 10-40 %. A stimulation of renal gluconeogenesis from pyruvate, lactate and propionate to a similar degree as obtained with ouabain is also observed in the presence of L-epinephrine (0.03-30 # M ) or N6-2-O-dibutyryl adenosine Y,5'-monophosphate (dibutyrylcyclic A M P ) 1 mM). The diuretics ethacrynic acid (0.25 nM-2.5/~M) and furosemide (0.01-10/~g/ml) also stimulate renal gluconeogenesis from pyruvate by 10-12 'Yo. 2. Ouabain increased Z4CO 2 fixation from the substrates pyruvate, lactate and fructose. Glycolysis remained unaffected by ouabain. 3. In contrast to L-epinephrine, not stimulation but inhibition of gluconeogenesis was observed in the presence of ouabain with glutamate and dicarbonic acids as substrates. A different metabolite profile was obtained in the presence of ouabain and L-epinephrine or dibutyryl cyclic AMP. The stimulatory action of ouabain on renal gluconeogenesis was additive to the stimulatory action of L-epinephrine, dibutyryl cyclic A M P or acetoacetate. 4. Ouabain did not change the activities of adenylate cyclase and phosphodiesterase in the kidney cortex. 5. Ouabain led to an increase in intracellular N a + content, tissue K ÷ concentration being unchanged. 6. High extracellular K + concentrations caused an inhibition of gluconeogenesis in the controls, which was completely prevented by ouabain, although tissue K ÷ contents increased to the same extent in the presence and in the absence of ouabain. 7. Ouabain led to an inhibition of oxygen uptake and to a reduction of 14CO2formation from [2-14C]pyruvate and [1-14C]palmitate in the absence and presence of 1 m M carnitine, as well as to increased tissue levels of malate, lactate and ct-ketoglutarate. Tissue A T P concentration remained unchanged or increased at high extracellular K + concentrations in the presence of ouabain. Abbreviation: dibutyryl cyclic AMP, N6-2-O-dibutyryl adenosine 3',5'-ruonophosphate. * Address for reprint requests: Institut fiir Biochimie und Endocrinologie, D-63 Giessen, Frankfurter Str. 112, Germany.
GLUCONEOGENESIS AND SODIUM PUMP
143
8. It is concluded, that ouabain exerts its stimulatory action on renal gluconeogenesis by inhibition of the sodium pump. Regulation of gluconeogenesis via changes of intracellular cation concentrations is excluded. It is assumed that inhibition of the Na + pump induces a higher energy state of the cell, which in turn favours energyrequiring synthetic processes.
INTRODUCTION Ouabain, a specific inhibitor of the Na + pump of the cell membrane ~, z, is known to exert insulin-like effects in muscle 3- 5 and adipose tissue 6- 8, with respect to its effects on sugar transport 3- 6, 8, glucose metabolism 3, 4, 6, 8 and antilipolytic action 6, 7. The effects of ouabain on transport phenomena and metabolism in muscle and adipose tissue can be mimicked by omitting K + from the incubation medium. Ho et al. 7 were able to relate the antilipolytic action of ouabain or lack of K +, respectively, to a decreased adenylate cyclase activity in adipose tissue homogenates either prepared after preincubation of the tissue with ouabain or in the absence of K +. Decreased levels of cyclic AMP (adenosine 3',5'-monophosphate) in adipose tissue were also measured after incubation of the tissue in the presence of insulin by Butcher et al. 9,10 and by Manganiello et al.11. Phosphodiesterase activity in adipose tissue homogenates was reported to be uninfluenced by ouabain v. In spite of similarities in the action of ouabain and insulin, different effects on glucose uptake and lactate production in the presence of insulin and ouabain have been reported by Ciausen 4 in rat hemidiaphragm. The present work is concerned with the stimulatory action of ouabain on renal gluconeogenesis, an observation first made by Weiss et al. Iz, which points to interactions between the sodium pump and metabolism in the kidney also. Since gluconeogenesis in liver 13and kidney ~4 is known to be under hormonal control, and in the light of the observed influence of ouabain on adenylate cyclase in adipose tissue, it appeared conceivable that, in contrast to adipose tissue, ouabain might induce a stimulation of renal gluconeogenesis by increasing the levels of cyclic AMP in the kidney, as against its inhibitory effect on adipose tissue adenylate cyclase. A dualistic action upon cyclic AMP levels, depending on the tissue, has also been reported for prostaglandins by Butcher and Baird15; prostaglandin E 1 leading to decreased intracellular cyclic AMP levels in isolated fat cells, but to increased cyclic AMP levels in lung, spleen, diaphragm and kidney. MATERIALS AND METHODS Animals
Male Sprague-Dawley rats weighing 200-250 g were used. The animals were kept on Altromin laboratory chow (Altrogge/Lage/Lippe) and starved 24 h before experiments. Tissue preparation
Rats were anaesthetized with ether and the kidneys pre perfused in situ with ice-cold incubation buffer, pH 7.4, to remove blood. The kidneys were excised, decapsulated and the cortex dissected from the medulla with a Stadie-Riggs tissue slicer. Kidney tubules were then prepared by collagenase treatment of the tissue according
144
D. FRIEDRICHS, W. SCHONER
to Burg and Orloff 16 as modified by Guder et al. ~7. The protein content of the final tubule suspension used for incubations was about 10-12 mg/ml. For experiments with kidney cortex slices the kidneys were not preperfused.
Incubation procedure Incubations were carried out in a Warburg apparatus at 37 °C in a final volume of 1.5 ml with a shaking frequency of 110 rev./min. In experiments with isolated kidney tubules, about 2-5 mg of protein were used per incubation vessel; in experiments with kidney cortex slices about 30 mg wet weight were used. In most experiments Krebs-Henseleit bicarbonate buffer 18, p H 7.4, was used, in some experiments K r e b s de Gasquet buffer ~9, p H 7.4. In experiments where the extracellular concentration of K + or Na + was varied, Krebs-Henseleit buffer was modified by isotonic substitution of Na + by K + or choline. The incubation samples were gassed for 10 rain with either a mixture containing 95 ~ O2 and 5 ~ CO2 when Krebs-Henseleit buffer was used, or 100 ~o O2 in experiments with the phosphate buffer. Total incubation time was 60 min in most experiments. Incubations were usually stopped by adding 0.15 ml of 3 M HCIO4, except in experiments in which electrolytes were measured, in which case trichloroacetic acid extracts were prepared.
Preparation of additions Solutions of additions were always prepared on the day of the experiment. N6-2-O-dibutyryl adenosine 3',5'-monophosphate (dibutyryl cyclic A M P ) was stored as 0.01 M solution at - 15 °C and the dilution needed made on the day of the experiment. Ouabain, substrate and dibutyryl cyclic A M P were added as aqueous neutral solutions. L-Epinephrine solution was prepared according to Joel z°. Palmitate was added as an albumin complex. 0.01 M palmitic acid solution was prepared by heating the aqueous solution to about 60 °C and adjusting the p H to 7-8 with K O H . While still warm this solution was added to an equal volume of 10 ~ bovine serum albumin with vigorous stirring. Oligomycin was dissolved in 80 ~ ethanol.
Determinations For the estimation of metabolite concentrations in experiments with isolated kidney tubules, at the end of the incubation period 0.15 ml of ice-cold 3 M HCIO 4 was added to the incubation vessels containing incubation medium plus kidney tubules. All following procedures were carried out at 0 °C. After centrifugation of the HCIO4 extracts, the supernatants were neutralized with solid K H C O 3 and, after removal of the perchlorate by centrifugation, the supernatants were used for the determination of metabolite concentrations. The measured metabolite levels in the tubule experiment therefore represent the sum of the concentration of a particular metabolite in the medium and tubules. For estimations of tissue metabolite levels in slice experiments, the incubations were stopped in an ice-NaC1 bath and the slices quickly taken out and immersed in liquid nitrogen, the slices of three incubations being pooled. The frozen tissue was then homogenized 1 " 10 (w/v) with cold 4 ~o~ HCIO4 in an UltraTurrax homogenizer. The neutralized supernatants of the HC104 extracts were used for metabolite concentration estimations. The incubation medium was deproteinized with 0.15 ml 3 M HCIO~ and glucose, lactate and pyruvate measured. Glucose was determined by the glucose oxidase/peroxidase method 21, and other metabolites by
GLUCONEOGENESIS AND SODIUM PUMP
145
standard enzymic procedures adapted for fluorometric assay, using a Perkin-Elmer MPF 2A fuorescence spectrophotometer. Pyruvate was measured according to Biicher et al. 22, malate, c~-ketoglutarate, ATP and ADP according to Williamson and Corkey z3, lactate by a method analogous to that for the determination of malate according to Williamson and Corkey 23. 3-Phosphoglycerate and phosphoenolpyruvate were measured according to Czock and Eckert 24. Oxygen uptake was measured by the Warburg manometric technique. ~4CO2 fixation was measured according to L'Age et al. 25. 1 4 C 0 2 formation was measured by trapping the 14CO2 formed during the incubation period with 0.2 ml 4 M N a O H on filter rolls in the central well of the Warburg vessels. After termination of the incubations by the addition of HC104, a subsequent incubation for 1 h was performed to assure complete absorption of the x4CO2. The filters were counted in 10 ml ethanol-toluene-PPO-POPOP scintillator in a Packard liquid scintillation spectrometer, Model 3320 or 3380. Na + and K + concentrations were estimated in the trichloroacetic acid extracts by flame photometry. Tissue cation content was corrected for extracellular space, wnich was determined in each incubation sample with [14C]inulin and was found to amount to 25-30 ~o of the tissue wet weight in accordance with results reported by Hillman et al. 26 for kidney tubules. Adenylate cyclase and phosphodiesterase activities were determined according to Ho et al. 7, the radioactive cyclic [14C]AM P was separated from [14C]ATP by paper chromatography 27. Tissue levels of cyclic AMP were measured according to Gilman 2a, using a test combination from Boehringer, Mannheim.
Materials
Na214CO3, [1-14C] -, [2-14C]pyruvate, [U-14C]glucose, [8-14C]ATP, cyclic [U-14C]AMP and [carboxyl-lgC]inulin were obtained from the Radiochemical Centre Amersham, [1-14C]palmitic acid from N.E.N. Collagenase isolated from Clostridium histolyticum, N6-2-O-dibutyryl cyclic AMP disodium succinate, the test combinations for estimation of glucose and cyclic AMP, as well as all enzymes used for metabolite determinations were purchased from Boehringer, Mannheim. L-Epinephrine bitartrate was obtained from Calbiochem., ouabain from Merck AG., Darmstadt, and ethacrynic acid from Merck Sharp and Dohme Research Lab., Rahway, N. J. Sodium L-lactate was purchased from Roth, Germany, L-malic acid and oligomycin A from Serva, Heidelberg. All other substances used were analytical grade and obtained from Merck AG., Darmstadt. RESULTS
Effects of ouabain, L-epinephrine, dibutyryl cyclic AMP, ethacrynic acid and furosemide on glucose formation by isolated kidney tubules Gluconeogenesis from pyruvate by isolated rat kidney tubules is stimulated by 10-30 ~o by ouabain in a concentration range from 0.1 mM to 4 mM. Similarly, a 20-40 ~o stimulation of gluconeogenesis from pyruvate could be obtained by the presence of 0.03-30 #M L-epinephrine. With 0.25 nM-2.5 pM ethacrynic acid, and also with 0.01-10 pg/ml furosemide, we found only a 10-12 ~o stimulation of glucose formation from pyruvate, in contrast to results of Ftilgraff et al. 29. Higher concentrations of the above listed agents were inhibitory for gluconeogenesis. As is obvious from Table I, depending on the substrate used, there are differences in the effects on
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T A B L E 11 E F F E C T S O F 2.5 m M A C E T O A C E T A T E A N D 0.1 m M O U A B A I N ON G L U C O N E O G E NESIS F R O M 2.5 m M L-LACTATE BY K I D N E Y C O R T E X SLICES Additions
Glucose f o r m e d (btmoles/h per a wet wt)
(1) (2) (3) (4)
25.48 :k0.93 36.24:k0.82 30.0610.50 42.17:k1.15
None Acetoacetate Ouabain Ouabain + acetoacetate * Pvs2
Pvs3
(6) (6) (5) (4)*
P < 0.001 P < 0.01 P < 0.001
< 0.001
gluconeogenesis exerted by ouabain, by dibutyryl cyclic AMP and by L-epinephrine, the action of which is known to be mediated through increased cyclic AMP levels. Ouabain stimulates gluconeogenesis from the substrates pyruvate, lactate, propionate and fructose, whereas gluconeogenesis from the dicarbonic acids and from glutamate is inhibited by 20-40 ~o. Contrary to the action of ouabain, we found a 20-47 ~o stimulation of gluconeogenesis in the presence of 0.3/zM L-epinephrine or 1 mM dibutyryl cyclic AMP from all substrates listed with the exception of fructose. With fructose as the substrate we found a stimulation of gluconeogenesis by ouabain, but an inhibition in the presence of L-epinephrine or dibutyryl cyclic AMP. The latter result is in agreement with results of Guder et al. 17. As is further evident from Table I, the stimulatory effect of ouabain on renal gluconeogenesis is additive to the stimulatory action of dibutyryl cyclic AMP and L-epinephrine, and also to the stimulatory effect of acetoacetate with 2.5 mM lactate as substrate (Table II). The inhibitory action of ouabain on gluconeogenesis from glutamate and dicarbonic acids could be due to inhibition of a Na +-dependent transport system for these substrates. In favour of this assumption is the experiment with propionate as substrate. This compound is converted intramitochondrially to malate via succinyl-CoA. If transport of dicar-
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boxylic acids across the cell membranes is thus avoided, a stimulation of gluconeogenesis by ouabain is obtained.
Time course of ouabain action Fig. 1 shows that the stimulation of renal gluconeogenesis from pyruvate by ouabain is a short-term effect, which was significant after 25 rain of incubation in the presence of ouabain. There is an increased pyruvate utilization in the presence of ouabain. Influence of ouabain on pyruvate carboxylation and glycolysis Since the previously observed increase in glucose formation in the presence of ouabain might be due to either a stimulation of one of the rate-limiting steps in gluconeogenesis or simply to an inhibition of glycolysis, experiments were undertaken to differentiate between these two possibilities. An inhibition of glycolysis under the influence of ouabain has been reported for various tissues 3°-33. We estimated the ratio of lactate formed to glucose formed in experiments with fructose as the substrate, which may be taken as an indication for the flux in the direction of gluconeogenesis or glycolysis, respectively (Table III). Under conditions which favour glycolysis, such as incubation of the tissue in a Nz atmosphere or at high extracellular K + concentrations, the ratio of lactate formed to glucose formed was raised. It remained, however uninfluenced by ouabain under all conditions. Likewise ~aCO 2 formation from [U-t4C]glucose was unchanged in the presence of ouabain. However, t 4COz fixation as well as glucose formation from the substrates pyruvate, lactate and fructose were increased by ouabain (Table IV). On the basis of these findings, a simple inhibition of glycolysis as the cause for the stimulation of renal gluconeogenesis by ouabain seems excluded. The data point to an involvement of pyruvate carboxylation in the action of ouabain on renal gluconeogenesis. T A B L E IV INFLUENCE OF OUABAIN
Substrate (2.5 mM)
ON
14CO2 F I X A T I O N
BY I S O L A T E D
KIDNEY
TUBULES
Glucose formation (l~moles/h per mo protein)
~4CO z fixation (epm/h per m9 protein)
Control
Ouabain (l raM)
Control
Pyruvate
0 . 2 1 4 ± 0 . 0 0 7 (5)
0 . 2 4 5 ± 0 . 0 0 5 (4) P < 0.02
9402_200 (5)
1 9 7 9 ± 37 (4) P < 0.001
Lactate
0 . 2 3 9 ± 0 . 0 0 8 (3)
0 . 2 9 6 ± 0 . 0 0 3 (4) P < 0.001
1 5 4 0 ~ 2 7 4 (3)
3 7 4 5 ± 5 2 2 (3) P = 0.02
Fructose
0 . 4 9 9 ± 0 . 0 1 7 (9)
0 . 6 1 3 ± 0 . 0 2 0 (8) P < 0.001
485±
Ouabain (l raM)
37 (4)
1 0 2 0 ± 94 (8) P < 0.01
Activities of adenylate cyclase and phosphodiesterase and intracellular levels of cyclic A M P in kidney cortex after preincubation with ouabain Since the "insulin-like" action of ouabain on epinephrine-stimulated lipolysis in adipose tissue could be correlated by Ho et al. v with a decreased adenylate cyclase
150
D. F R I E D R I C H S , W. S C H O N E R
TABLE V INFLUENCE OF OUABAIN ON THE ACTIVITIES OF ADENYLATE P H O D I E S T E R A S E A N D T I S S U E LEVELS O F 3 ' , 5 ' - C Y C L I C A M P
C Y C L A S E , PHOS-
For the d e t e r m i n a t i o n o f adenyl cyclase a n d p h o s p h o d i e s t e r a s e activities, kidney cortex slices were p r e - i n c u b a t e d at 37 °C for 1 h in the presence or absence o f 1 m M o u a b a i n in a modified K r e b s Henseleit buffer, p H 7.4, c o n t a i n i n g 50 m M N a + a n d 100 m M K +. T h e pooled tissue o f three inc u b a t i o n s was t h e n h o m o g e n i z e d with 4 vol. o f 0.05 M T r i s - H C l , p H 7.4. Adenylate cyclase was tested using the 2 000 × y - l0 m i n sediment, p h o s p h o d i e s t e r a s e using the 50 000 × g . l0 m i n s u p e r n a t a n t . In experiments where tissue levels o f cyclic A M P were m e a s u r e d , i n c u b a t i o n s were carried o u t for 5 min. T h e tissue was t h e n i m m e r s e d in liquid N2 a n d tissue extracts prepared according to Gilm a n 28. l0 m M pyruvate was used as the substrate in all experiments.
Control Glucose formed ( ~ m o l e s / h per g wet wt)
Ouabain (1 mM)
16.94J:
A d e n y l a t e cyclase activity (pmoles cyclic A M P f o r m e d / l 0 m i n per m g protein) P h o s p h o d i e s t e r a s e activity (pmoles cyclic A M P hydrolyzed/10 m i n per m g protein)
39.34± P < 0.001
1.37 (11)
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4547
29538
16288
(3)
31330
0.77 5:
Tissue levels o f cyclic A M P ( p m o l e s / m g wet wt)
0.07 (6)
1.86 (13)
~ 293
(5)
±8369
(3)
0.79 ~
0.06 (6)
activity after preincubation of the tissue in the presence of ouabain, we also tested the influence of ouabain on the adenylate cyclase-cyclic AMP system in kidney cortex (Table V). In contrast to the results of rio et aL 7, ouabain had no effect on adenylate cyclase, phosphodiesterase or tissue levels of cyclic AMP in kidney cortex. Effects o f N a + and K + on renal gluconeogenesis Since the " i n s u l i n - l i k e " effect o f o u a b a i n in muscle 3- s a n d a d i p o s e tissue 6-8
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GLUCONEOGENESIS AND SODIUM PUMP
151
T A B L E VI INFLUENCE OF EXTRACELLULAR Na + CONCENTRATION (LEFT PART) A N D K + CONCENTRATION (RIGHT PART) ON THE EFFECT OF OUABAIN ON RENAL GLUCONEOGENESIS Isolated kidney tubules were incubated for 1 h with 10 m M pyruvate in the absence or presence o f 1 m M o u a b a i n at various N a + or K + concentrations in the incubation medium. One of the two cations was held constant at the concentration indicated in brackets. The data are calculated as percentages of glucose formation in the presence of ouabain relative to 100 ~ glucose formation of controls without ouabain.
Na ÷ ( m M ) [25 m M K + ]
~ o f control (1 m M ouabain)
K + (mM) [154 m M Na + ]
°/ooo f control (l m M ouabain)
10 30 50 70 110
123 113 134" 140" 150"
0 3 6 12
82* 104 122" 144"
* Difference from control statistically significant.
could be mimicked by omitting K + from the incubation medium, we investigated the influence of the cations N a ÷ and K ÷ on renal gluconeogenesis in more detail. As shown in Fig. 2, optimal rates of gluconeogenesis from glutamate, the main substrate for renal gluconeogenesis in vivo, are only obtained in the presence of physiological concentrations of Na ÷ and K +. However, similar curves were also obtained with 10 1000
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800
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600
Ouoboin
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Control
200 i 68
I 70
I 7.2
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Fig. 3. p H - D e p e n d e n c e o f o u a b a i n action on gluconeogenesis by isolated kidney tubules from 10 m M pyruvate. Concentration o f ouabain: 1 m M . Each point represents the mean value ~ S . E . M . from 6 incubations.
152
D. F R I E D R I C H S , W. S C H O N E R
mM pyruvate as the substrate. From the data in Table VI it is evident that the effect of ouabain on renal gluconeogenesis from pyruvate is highly dependent on the concentration of the cations Na + and K + in the incubation medium. In contrast to the results obtained in muscle 3 - 5 and adipose tissue 6- 8, omitting K + from theincubation medium did not mimic the stimulatory effect of ouabain on renal gluconeogenesis from pyruvate, but led to an inhibition of gluconeogenesis. Also, the effect of L-epinephrine on renal gluconeogenesis was somewhat dependent on the extracellular concentrations of Na + and K +, but not to such an extent as was the case with ouabain. The effect of ouabain on renal gluconeogenesis from pyruvate is moreover pH dependent (Fig. 3). Ouabain stimulates gluconeogenesis only at pH values above 7.2, whereas at pH values lower than 7.2 gluconeogenesis from pyruvate is inhibited in the presence of ouabain. In further experiments we changed the extracellular concentrations of Na + and K +, leaving the sum of the two cations constant, since Bihler and Sawh 34 and Adam and Haynes 35 showed that changes in osmolarity p e r s e can affect sugar transport 34 and ~4CO2 fixation 35. Under these conditions the gluconeogenesis in the controls was decreased by 80-90 % if the extracellular K + concentration was raised to 150 mM (Fig. 4). The inhibition of gluconeogenesis at high extracellular K + concentrations (up to 100 mM K +) was completely prevented in the presence of ouabain, thus increasing the magnitude of the stimulatory effect of ouabain on gluconeogenesis from pyruvate. However the decrease of gluconeogenesis at high extracellular K + concentrations could not be prevented in the presence of L-epinephrine. These results seemed to suggest that the observed changes in the rate of gluconeogenesis under these conditions might be due to changes in the intracellular Na + and K + levels. Tissue concentrations of Na + and K + in the absence and presence of ouabain were therefore determined with 6 mM and 100 mM extracellular K + concentration. The results of these experiments are presented in Table VII. Tissue levels of Na + were increased in the presence of ouabain. However tissue K + concentrations remained unaltered by ouabain. Surprisingly, with 100 mM extracellular K + concentrations the intracellular K + content was increased about 3-fold even in the presence of ouabain. From these c A
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0 150
150
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60 90
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120 30
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60 90
30 120
0 150
Fig. 4. Influence of the ratio N a + / K ÷ on gluconeogenesis by isolated kidney tubules from 10 m M pyruvate in the presence of L-epinephrine (A) and ouabain (B). Each point represents the mean value ± S . E . M . from 3 incubations.
GLUCONEOGENESIS
153
AND SODIUM PUMP
T A B L E VII INFLUENCE OF OUABAIN ON TISSUE LEVELS OF Na + AND K + K i d n e y cortex slices were used. Substrate: 10 m M pyruvate. Cation concn (raM) in the incubation medium
Tissue cation content (~val/g wet wt)
K+
Control
Na +
K+
Na + Ouabain (1 m M )
Control
Ouabain (I raM)
6
154
35.65-- 3.83 (6)
35.20~-2.87 (6)
185.96~23.04 (6)
319.21~22.21 (6) P < 0.02
100
50
105.59±13.32 (6)
112.54±6.60 (6)
2 0 2 . 4 8 ± 1 5 . 5 2 (6)
259.66±18.15 (4) P < 0.05
results it appears rather unlikely that the stimulation of glucose formation and 14CO2 fixation from pyruvate by ouabain is mediated by a stimulation of pyruvate carboxylase through a decrease of intracellular K + concentration, as was discussed by Weiss et al. ~2.
Influence of ouabain on the energy state of the cell Since the Na + p u m p is known to utilize about 30-40 ~o of the energy production of the kidney cell 36, it seemed necessary to gain information about the energy state of the cell under our conditions. The results of these studies are presented in Table VIII. Oxygen consumption was reduced in the presence of ouabain at 6 m M extracellular K + concentration in accordance with results reported by others 3 ~' 36-3s Raising the extracellular K + concentration to 100 m M led to a stimulation of oxygen uptake even in the presence of ouabain. The levels of A T P measured in the whole cell were elevated in the presence of ouabain at 100 m M extracellular K + concentration. A D P levels were unaffected by ouabain. The ratio of net C 3 carbon units removed to glucose formed, which at values higher than 2 gives a measure for the portion of pyruvate which is not accounted for as glucose or lactate and which is presumably oxidized, is increased by high extracellular K + concentrations. In the presence of ouabain this ratio is decreased and is almost unaffected by high extracellular K + concentrations. This indicated that, in the presence of ouabain, pyruvate is preferentially channelled towards gluconeogenesis rather than towards oxidation. F r o m Table IX it is evident that ouabain stimulates gluconeogenesis from pyruvate even in the presence of oligomycin. This well-known inhibitor of oxidative phosphorylation 39 leads to an increase of A D P levels in the mitochondrial matrix 4°. Concomitantly, a decrease of pyruvate carboxylation occurs, as demonstrated by Walter and Stucki 4° in rat liver mitochondria. A D P is also known to inhibit purified pyruvate carboxylase from chicken liver, as was reported by Keech and Utter 41. 14CO2 formation from [2-14C]pyruvate and [l-14C]palmitate in the absence and presence of carnitine was decreased in the presence of ouabain (Table X). The decreased 14CO 2 formation from [2-14C]pyruvate might be interpreted as anindication for a reduced activity of the citric acid cycle under the influence of ouabain. Similarly ~4CO2 formation from [l-~4C]palmitate may be taken to reflect citrate cycle flux, if one can assume that the
154
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GLUCONEOGENESIS AND SODIU M PU M P
155
TABLE IX I N F L U E N C E OF OUABAIN AND OLIGOMYCIN ON GLUCONEOGENESIS BY KIDNEY CORTEX SLlCES F R O M 10 mM PYRUVATE Additions
Glucose f o r m e d
(pmoles/h per O wet wt) (1) (2) (3) (4)
none Ouabain Oligomycin Oligomycin 4-ouabain
(0.1 mM) (1/~g/ml) (1/zg/ml) (0.1 raM)
30.96±1.52 37.344-1.40 17.68±1.08 22.00± 1.26
(4) (4) (8) (8)*
P < 0.05 P < 0.001 P < 0.002
* P v s 3 < 0.05.
transport of long-chain fatty acids through the kidney cell membrane is not impaired by ouabain. These findings reveal that, under the influence of ouabain, apart from the reduced respiration, the citric acid cycle activity is apparently also reduced. The assumption of reduced citric acid cycle activity in the presence of ouabain is further supported by the increased tissue malate levels (Table XI) and ct-ketoglutarate levels in experiments with lactate as substrate (data not shown) under the influence of ouabain. The increased tissue malate and lactate levels in the presence of ouabain (Table XI) furthermore indicate a more reduced state of the cell under the influence of ouabain, as would be expected under conditions of diminished respiration and citric acid cycle activity. Contrary to the results obtained with ouabain, oxygen uptake and t4CO2 production from [1-14C]palmitate were unaffected by L-epinephrine, oxygen uptake being also uninfluenced by dibutyryl cyclic-AMP (data not shown). x4C02 formation from [1-14C]pyruvate, which can be taken as an estimate of pyruTABLE X INFLUENCE OF 1 mM OUABAIN ON 14C02 FORMATION FROM [I-14C]PYRUVATE, [2-14C]PYRUVATE AND [I-14ClPALMITATE The experiments with [~4Clpyruvate were carried out with isolated kidney tubules, the experiments with [~4C]palmitate with kidney cortex slices. The data for glucose formation are calculated as /~moles/h per mg protein for the tubule experiments and as #moles/h per g wet wt for the slice experiments. 14C0z formation is indicated as cpm/h per mg protein and as cpm/min per g wet wt, respectively. Substrate
Concn
(raM)
14C02 formation
Glucose formation Control
Ouabain
Control
(1 mM)
Ouabain
(1 mM)
[l-14C]Pyruvate 10
0.8524-0.014 (4)
0.959±0.029 (4) P < 0.02
64680±5492 (4)
58933 --3060 (4) N.S.
[2-14C]Pyruvate 10
0.846:k0.009 (4)
0.9104-0.002 (3) P < 0,002
347204-2255 (4)
25672±2159 (4) P < 0.05
[l-14C]Palmitate 0.5
31154- 218 (18)
2103± 214 (24) P ,< 0.01
[1-~4C]Palmitate 0.5 4-DL-carnitine 1
3640± 298 (13)
28624- 201 (12) P < 0.05
156
D. FRIEDRICHS, W. SCHONER
TABLE Xl TISSUES METABOLITE LEVELS UNDER THE INFLUENCE OF OUABAIN AND DIBUTYRYL CYCLIC AMP Isolated kidney tubules were incubated for 1 h in the presence of 1 mM ouabain or 1 mM dibutyryl cyclic AMP or the combination of both, in the presence of 2.5 mM propionate as the substrate. Values are calculated as nmoles/mg protein.
Control Lactate Pyruvate Malate Phosphoenolpyruvate 3-Phosphoglycerate ~-Ketoglutarate
Ouabain
Dibutyryl o'clic AMP
Ouabain-c-cyclic dibutyryl AMP
68.15 ± 3.84 ( 15)
109.08±11.73 (10) 54.04~:1.29 (9) 53.73±0.88 (8) P < 0.001 P < 0.02 P < 0.02 2.49 ±0.15 ( 13) 2.46~ 0.22 (8) 2.36±0.13 (9) 1.78±0.03 (8) P < 0.01 9.44± 0.83 (10) 3.82±0.25 (9) 6.088±0.29 (8) 5.00 ±0.20 (15) P < 0.001 P < 0.01 P < 0.01 0.74_-40.09 (10) 0.61± 0.03 (8) 1.61-z0.22(9) 0.78±0.03 (8) P < 0.05 P < 0.05
31.06± 1.52 (12)
27.43± 2.17 (7)
33.60±0.96 (9)
0.77 ± 0.10 (12)
0.80± 0.14 (7)
0.61 ±0.06 (9)
33.28~0.78 (8) 0.86±0.06 (8)
vate dehydrogenase activity, was not significantly altered by o u a b a i n u n d e r our conditions. DISCUSSION
Relation between the stimulation of renal #luconeogenesis by ouabain, ~&rosemide and ethacrynic acid and the adenylate o'clase-cyclic A M P system The present paper demonstrates that, in kidney cortex, i n h i b i t i o n of the N a + p u m p u n d e r certain c o n d i t i o n s can induce a stimulation of renal gluconeogenesis to a similar extent as can be observed in the presence of L-epinephrine or dibutyryl cyclic A M P . C o n t r a r y to L-epinephrine however, o u a b a i n exerts its stimulatory effect on renal gluconeogenesis by a m e c h a n i s m differing from the cyclic A M P system. S u p p o r t for this a s s u m p t i o n comes (a) from the d e m o n s t r a t i o n of the additive effects o f L-epinephrine a n d o u a b a i n o n renal gluconeogenesis (Table I), (b) from the d e m o n stration of different patterns of tissue metabolites in the presence of o u a b a i n or cyclic A M P (Table XI), a n d (c) from the lack of any effect of o u a b a i n on the adenylate cyclase or phosphodiesterase activities of kidney cortex (Table V). A p a r t from o u a b a i n , the diuretics furosemide a n d ethacrynic acid also stimulate gluconeogenesis in kidney cortex from various substrates, as has been reported by Fiilgraff et al. 29 a n d as is d e m o n s t r a t e d by us in this paper. Furosemide leads to a s t i m u l a t i o n of renal gluconeogenesis, although renal cortical adenylate cyclase remains uninfluenced by furosemide 42. Rat kidney ATPase is inhibited by furosemide according to Park et aL 43, but is uninfluenced according to Yoshida a n d Metcoff 44. Ethacrynic acid stimulates renal gluconeogenesis at very low concentrations (0.3 n M I /~M) according to Ftilgraff eta/. 29 and to us. At these c o n c e n t r a t i o n s ethacrynic acid does n o t affect ( N a + - K + )-activated ATPase 43. 45 - 47 or renal adenylate cyclase 41.
GLUCONEOGENES1S AND SODIUM PUMP
! 57
Thus it appears that the stimulation of renal gluconeogenesis by the diuretics furosemide and ethacrynic acid is probably unrelated to adenylate cyclase activity and also not mediated by an influence on ( N a + - K +)-activated ATPase. Relation between stimulation o f renal gluconeogenesis by ouabain and changes in the extraeellular and intracellular concentrations o f Na + and K + Because the stimulatory effect of ouabain on renal gluconeogenesis from pyrurate is most probably not mediated by alterations of the cyclic A M P system, it must be assumed that the stimulation occurs as a consequence of the inhibition of the sodium pump. The sodium p u m p may influence renal metabolism via changes of the intracellular concentrations of Na + and K + or via the energy requirement. Weiss et al.a 2 assumed that ouabain may stimulate renal pyruvate carboxylase by decreasing intramitochondrial K + concentrations. His assumptions were based upon the finding that pyruvate carboxylase from liver is inhibited by K + concentrations higher than 8 0 m M 12,48. The increased 14COz fixation form pyruvate lactate and fructose (Table IV) confirm the data of Weiss et al. lz and point to an influence on intramitochondrial pyruvate carboxylation, provided that glycolysis is uninfluenced, as is likely from our data (Table III and IV). Contrary to the assumption of Weiss et al. ~2 however, the intracellular K + content of kidney cortex slices remained unaltered after 1 h of incubation with ouabain (Table VII). This lack of K + decrease, which is in accordance with the findings of Jones and Landon 31 for rat kidney cortex slices, but is in contrast to those of Letarte et al. 8 for isolated mouse fat cells, is not due to any ineffectiveness of the specific action of ouabain. The increase in tissue Na + content indicates an inhibition of the N a + pump. Although measurements of cation concentrations for the whole cell may not exclude the occurrence of a shift of K + between different intracellular compartment, it seems to us unlikely that the stimulatory action of ouabain on renal gluconeogenesis from pyruvate is the consequence of a decrease of intramitochondrial K + content (which in turn would de-inhibit mitochondrial pyruvate carboxylase). Support for this conclusion comes also from the finding that ouabain stimulates renal gluconeogenesis in the presence of high extracellular K + concentrations which lead to about a 3-fold increase in the intracellular K + content (Table VII). Since tissue Na + concentrations were increased by ouabain, possible effects of Na + on cellular metabolism, which might result in an increased gluconeogenesis form pyruvate, lactate and fructose, have to be considered. Na + was reported to have no influence on pyruvate carboxylase 4s, but to inhibit glycolysis in brain slices 49. However, as already pointed out, we have no evidence for any inhibition of glycolysis by ouabain under our conditions. Influence o f ouabain on energy levels, respiration and the citric acid cycle The influence on mitochondrial processes such as respiration and pyruvate carboxylation which we observed in the presence of ouabain (Tables IV and V I I I ) must be an indirect one, since Blond and Whittam 36 found no influence of ouabain on the respiration of isolated kidney mitochondria. The change of the redox state of the cell towards a more reduced state, which is indicated by increased levels of malate and lactate in the presence of ouabain in our experiments (Table XI), is in line with the results of van Rossum s°, who observed in slices of seagull nasal glands, which actively transport NaC1 in a respiration-dependent manner, an increased reduction
158
D. FRIEDRICHS, W. SCHONER
of nicotinamide nucleotides and cytochrome b in the presence of ouabain. Van Rossum s o assumed that ouabain induces a transition of the mitochondria in the slices from a state approaching the active State 3 towards the resting State 4. He further assumes that the inhibition of respiration is a result of limitation of phosphate acceptor. Similarly, Blond and Whittam 36 conclude from their studies with kidney cortex homogenate and isolated kidney cortex mitochondria that the rate of respiration of kidney mitochondria is controlled by the concentration of A D P or orthophosphate, the formation of which is determined by energy-utilizing processes, including ion transport. The importance of A D P and A T P for the regulation of other mitochondrial processes apart from respiration is also evident from the work of Stucki and coworkers40.5t and of Patel and Hanson 52, using isolated liver mitochondria 4°, 51 and adipose tissue mitochondria 52, respectively. These authors found a stimulation of pyruvate carboxylation in isolated mitochondria by ATP and an inhibition by ADP. The regulation of pyruvate carboxylase by the A T P / A D P ratio within the mitochondrial matrix as proposed by Stucki and co-workers 4°' 5 t is well in agreement with the results which Keech and Utter 4t obtained with purified chicken liver pyruvate carboxylase. Moreover, Walter and Stucki 4° could demonstrate that, with isolated liver mitochondria under conditions which led to elevated A D P levels in the mitochondrial matrix, as was particularly pronounced in the presence of oligomycin, pyruvate carboxylation was strongly inhibited. In our more complex system we also found an inhibition of gluconeogenesis in the presence of oligomycin, which was significantly less if ouabain was present in addition to the oligomycin (Table IX). The decreased 14COz production from [2-t4C]pyruvate in the presence of ouabain points to a reduction of citric acid cycle activity under the influence of ouabain. It seems likely that the reduction of citric acid cycle activity by ouabain occurs at a step between malate dehydrogenase and succinate thiokinase, because gluconeogenesis from propionate is stimulated by ouabain. Thus citrate synthase, isocitrate dehydrogenase and ~-ketoglutarate dehydrogenase remain as possible control points in the citric acid cycle. La Noue e t al. 53 found in isolated rat heart mitochondria a reduction of flux through the citric acid cycle of 75-80 ~o in the respiratory state 4 relative to state 3, which was associated with increased levels of N A D H , high ATP levels and an accumulation of ~-ketoglutarate. In our studies in the intact cell we also found in the presence of ouabain (a) significant higher ATP concentrations under conditions with high extracellular K + concentrations (Table VIII), but not with 6 m M K +, (b) a decrease of ~4COz production fi om [2-14C]pyruvate and [1-I 4C]palmitate (Table X): (c) an accumulation of ~-ketoglutarate; and (d) a more reduced redox state of the cell, as is evident from the increase in malate and lactate contents (Table XI). Thus the conclusions La Noue et al. 53 arrive at from data might well be valid for the interpretation of oul results. According to their La Noue e t al. s3 a feed-back control from the electron transport chain to the citric acid cycle is mediated by the phosphorylation state of the adenine and guanine nucleotides. Changes of these two factors secondarily influence the intramitochondrial concentrations of oxaloacetate, acetyl-CoA and succinyI-CoA, which are direct regulators of citrate synthase and ~-ketoglutarate dehydrogenase activities 53,54.
GLUCONEOGENES1S A N D S O D I U M P U M P
159
CONCLUSION
Thus, the present work provides some evidence for the regulation of renal gluconeogenesis in the intact cell by the sodium pump, which is probably not mediated by the adenylate cyclase-cyclic AMP system. Likewise, the cations K ÷ and Na ÷ do not seem to play a regulatory role under these conditions. The tentative conclusion is drawn, that, in kidney tissues, inhibition of the sodium pump might induce a higher energy state of the cell, which in turn favours energy-requiring synthetic processes. A C K N O W L E DG MENT
We gratefully appreciate the skilful technical assistance of Miss G. Schott and Miss A. Boie. This work was in part supported by the D.F.G. (Scho 139/1-6) and partially by means of the S.F.B. 33. The Stiftung Volkswagenwerk provided, in GSttingen, the MPF 2A Perkin-Elmer fluorimeter. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Schatzmann, PI. J. (1953) Heir. Physiol. Acta 11,346-354 Whittam, R. and Wheeler, K. P. (1970) Annu. Rev. Physiol. 32, 21-60 Clausen, T. (1965) Biochim. Biophys. Acta 109, 164-171 Clausen, T. (1966) Biochim. Biophys. Acta 120, 361-368 Bihler, I. (1968)Biochim. Biophys. Acta 163, 4 0 1 4 1 0 Ho, R. J. and Jeanrenaud, B. (1967) Biochim. Biophys. Acta 144, 61-73 Ho, R. J., Jeanrenaud, B., Posternak, Th. and Renold, A. E. (1967) Biochim. Biophys. Acta 144, 74-82 Letarte, J., Jeanrenaud, B. and Renold, A. E. (1969) Biochim. Biophys. Acta 183, 357-365 Butcher, R. W., Baird, C. E. and Sutherland, E. W. (1968) J. Biol. Chem. 243, 1705-1712 Butcher, R. W., Sneyd, J. G. T., Park, C. R. and Sutherland, E. W. (1966) J. Biol. Chem. 241, 1651-1653 Manganiello, V: C., Murad, F. and Vaughan, M. (1971) J. Biol. Chem. 246, 2195-2202 Weiss, G., Ohly, B. and Seubert, W. (1971) in Regulation o f Gluconeogenesis (SOling, H. D. and Willms, B., eds), pp. 29-41, Thieme Verlag, Stuttgart Exton, J. H. and. Park, C. R. (1969) J. Biol. Chem. 244, 1424-1433 Rasmussen, H. and. Nagata, N. (1970) Biochim. Biophys. Acta 215, 17-28 Butcher, R. W. and Baird, C. E. (1968) J. Biol. Chem. 243, 1713-1717 Burg, M. B. and Orloff, J. (1962) Am. J. PhysioL 203, 327-330 Guder, W., Wiesner, W., Stukowski, B. and Wieland, O. (1971) Hoppe-Seyler's Z. PhysioL Chem. 352, 1319-1328 Krebs, H. A. and Henseleit, K. (1932) Hoppe-Seyler's Z. Physiol. Chem. 210, 33-66 Krebs, H. A. and de Gasquet, P. (1964) Biochem. J. 90, 149-151 Joel, C. D. (1966) J. Biol. Chem. 241,814-821 Bergmeyer, H.-U. and Bernt, E. (1962) in Methoden der enzymatischen Analyse (Bergrneyer, H.-U., ed.) pp. 123-130, Verlag Chemie, Weinheim Biicher, Th., Czock, R., Lamprecht, W. and Latzko, E. (1962) in Methodcn der enzymatischen Analyse (Bermeyer, H.-U., ed. ), pp. 253-259, Verlag Chemie, Weinheim Williamson, J. R. and Corkey, B. E. (1969) in Methods Enzymol. 13, 435-513 Czock, R. and Eckert, L. (1962) in Methoden der enzymatischen Analyse (Bergmeyer, H.-U., ed.), pp. 229-233, Verlag Chemie, Weinheim L'Age, M., Henning, H. V., Ohly, B. and Seubert, W. (1968) Biochem. Biophys. Res. Commun, 31, 241-246 Hillman, R. E., Albrecht, 1. and Rosenberg, L. E. (1968) Biochem. Biophys. Acta 150, 528 530 Dousa, T. and Rychlik, J. (1970) Biochim. Biophys. Acta 204, 1-9
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28 Gilman, A. G. (1970) Proc. Natl. Acad. Sci. U.S. 67, 305-312 29 Ffilgraff, G. Nfinemann, H. and Sudhoff, D. (1972) Naunyn-Schmiedeber#s Arch. Pharmakol. 273, 86-98 30 Whittam, R. and Ager, M. E. (1965) Biochem. J. 97, 214-227 31 Jones, V. D. and Landon, E. J. (1967) Biochem. Pharmacol. 16, 2163-2169 32 Poole, D. T., Butcher, T. C. and Williams, M. E. (1971) J. Membrane Biol. 261-276 33 Handler, J. S., Preston, A. S. and Rogulski, J. (1968) J. Biol. Chem. 243, 1376-1383 34 Bihler, 1. and Sawh, P. C. (1971) Biochim. Biophys. Acta 241, 302-309 35 Adam, P. A. J. and Haynes, Jr. ,R. C. (1969) J. Biol. Chem. 244, 6444-6450 36 Blond, D. M. and Whittam, R. (1964) Biochem. J. 92, 158-167 37 Van Rossum, G. D. V., Gosalvez, M., Galeotti, T. and Morris, H. P. (1971) Biochim. Biophys. Acta, 245, 263-276 38 Van Rossum, G. D. V. (1966) Biochim. Biophys. Acta 126, 338-349 39 Beechy, R. B., Robertson, A. M., Halloway, T. and Knight, [. G. (1967) Biochemistry 6, 38673879 40 Walter, P. and Stucki, J. W. (1970) Eur. J. Biochem. 12, 508-519 41 Keech, D. B. and Utter, M. F. (1963) J. Biol. Chem. 238, 2603-2614 42 Jakobs, K. H., Schultz, K. and Schultz, G. (1971) Naunyn-Schmiedebergs Arch. Pharmakol. 273, 248-266 43 Park, C. W., Oh, J. S., and Moon, J. C. (1971) Soul Uidae Chapdin 12, 166 44 Yoshida, T. and Metcoff, J. (1972) Fed. Proc. 31,331 45 Proverbio, F., Robinson, J. W. C. and Whittembury, G. (1970) Biochim. Biophys. Acta 211, 327-336 46 Nechay, B. R., Palmer, R. F., Chinoy, D. A. and Posey, V. A. (1967) J. Pharmacol. Exp. Ther. 157, 599-617 47 Duggan, D. E. and Noll, R. M. (1965) Arch. Biochem. Biophys, 109, 388-396 48 McClure, W. R., Lardy, H. A. and Kneifel, H. P. (1971) J. Biol. Chem. 246, 3569-3578 49 Utter, M. F. (1950) J. Biol. Chem. 185, 499-517 50 Van Rossum, G. D. V. (1968) Biochim. Biophys. Aeta 153, 124-131 51 Stucki, J. W., Brawand, F. and Walter, P. (1972) Eur. J. Biochim. 27, 181 191 52 Patel, M. S. and Hanson, R. W. (1970) J. Biol. Chem. 245, 1302 1310 53 La Noue, K. F., Bryla, J. and Williamson, J. R. (1972) Y. Biol. Chem. 247, 667-679 54 Garland, P. B. (1964) Biochem. J. 92, 10C-12C
,~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 27044 S T I M U L A T I O N OF R E N A L GLUCONEOGENESIS BY I N H I B I T I O N OF THE S O D I U M PUMP
DAGMAR FRIEDRICHS and WILHELM SCHONER lnstitut fiir Physiologische Chemie der Universitiit Gb'ttingen und lnstitut fiir Biochemie und Endokrinologie, Fachbereich Veteriniirmedizin, Universitiit Giessen, Giessen (Germany)*
(Received September 25th, 1972)
SUMMARY 1. Ouabain (0.1-4 m M ) stimulates glucose formation by isolated kidney tubules and kidney cortex slices from pyruvate, lactate, propionate and fructose by 10-40 %. A stimulation of renal gluconeogenesis from pyruvate, lactate and propionate to a similar degree as obtained with ouabain is also observed in the presence of L-epinephrine (0.03-30 # M ) or N6-2-O-dibutyryl adenosine Y,5'-monophosphate (dibutyrylcyclic A M P ) 1 mM). The diuretics ethacrynic acid (0.25 nM-2.5/~M) and furosemide (0.01-10/~g/ml) also stimulate renal gluconeogenesis from pyruvate by 10-12 'Yo. 2. Ouabain increased Z4CO 2 fixation from the substrates pyruvate, lactate and fructose. Glycolysis remained unaffected by ouabain. 3. In contrast to L-epinephrine, not stimulation but inhibition of gluconeogenesis was observed in the presence of ouabain with glutamate and dicarbonic acids as substrates. A different metabolite profile was obtained in the presence of ouabain and L-epinephrine or dibutyryl cyclic AMP. The stimulatory action of ouabain on renal gluconeogenesis was additive to the stimulatory action of L-epinephrine, dibutyryl cyclic A M P or acetoacetate. 4. Ouabain did not change the activities of adenylate cyclase and phosphodiesterase in the kidney cortex. 5. Ouabain led to an increase in intracellular N a + content, tissue K ÷ concentration being unchanged. 6. High extracellular K + concentrations caused an inhibition of gluconeogenesis in the controls, which was completely prevented by ouabain, although tissue K ÷ contents increased to the same extent in the presence and in the absence of ouabain. 7. Ouabain led to an inhibition of oxygen uptake and to a reduction of 14CO2formation from [2-14C]pyruvate and [1-14C]palmitate in the absence and presence of 1 m M carnitine, as well as to increased tissue levels of malate, lactate and ct-ketoglutarate. Tissue A T P concentration remained unchanged or increased at high extracellular K + concentrations in the presence of ouabain. Abbreviation: dibutyryl cyclic AMP, N6-2-O-dibutyryl adenosine 3',5'-ruonophosphate. * Address for reprint requests: Institut fiir Biochimie und Endocrinologie, D-63 Giessen, Frankfurter Str. 112, Germany.
GLUCONEOGENESIS AND SODIUM PUMP
143
8. It is concluded, that ouabain exerts its stimulatory action on renal gluconeogenesis by inhibition of the sodium pump. Regulation of gluconeogenesis via changes of intracellular cation concentrations is excluded. It is assumed that inhibition of the Na + pump induces a higher energy state of the cell, which in turn favours energyrequiring synthetic processes.
INTRODUCTION Ouabain, a specific inhibitor of the Na + pump of the cell membrane ~, z, is known to exert insulin-like effects in muscle 3- 5 and adipose tissue 6- 8, with respect to its effects on sugar transport 3- 6, 8, glucose metabolism 3, 4, 6, 8 and antilipolytic action 6, 7. The effects of ouabain on transport phenomena and metabolism in muscle and adipose tissue can be mimicked by omitting K + from the incubation medium. Ho et al. 7 were able to relate the antilipolytic action of ouabain or lack of K +, respectively, to a decreased adenylate cyclase activity in adipose tissue homogenates either prepared after preincubation of the tissue with ouabain or in the absence of K +. Decreased levels of cyclic AMP (adenosine 3',5'-monophosphate) in adipose tissue were also measured after incubation of the tissue in the presence of insulin by Butcher et al. 9,10 and by Manganiello et al.11. Phosphodiesterase activity in adipose tissue homogenates was reported to be uninfluenced by ouabain v. In spite of similarities in the action of ouabain and insulin, different effects on glucose uptake and lactate production in the presence of insulin and ouabain have been reported by Ciausen 4 in rat hemidiaphragm. The present work is concerned with the stimulatory action of ouabain on renal gluconeogenesis, an observation first made by Weiss et al. Iz, which points to interactions between the sodium pump and metabolism in the kidney also. Since gluconeogenesis in liver 13and kidney ~4 is known to be under hormonal control, and in the light of the observed influence of ouabain on adenylate cyclase in adipose tissue, it appeared conceivable that, in contrast to adipose tissue, ouabain might induce a stimulation of renal gluconeogenesis by increasing the levels of cyclic AMP in the kidney, as against its inhibitory effect on adipose tissue adenylate cyclase. A dualistic action upon cyclic AMP levels, depending on the tissue, has also been reported for prostaglandins by Butcher and Baird15; prostaglandin E 1 leading to decreased intracellular cyclic AMP levels in isolated fat cells, but to increased cyclic AMP levels in lung, spleen, diaphragm and kidney. MATERIALS AND METHODS Animals
Male Sprague-Dawley rats weighing 200-250 g were used. The animals were kept on Altromin laboratory chow (Altrogge/Lage/Lippe) and starved 24 h before experiments. Tissue preparation
Rats were anaesthetized with ether and the kidneys pre perfused in situ with ice-cold incubation buffer, pH 7.4, to remove blood. The kidneys were excised, decapsulated and the cortex dissected from the medulla with a Stadie-Riggs tissue slicer. Kidney tubules were then prepared by collagenase treatment of the tissue according
144
D. FRIEDRICHS, W. SCHONER
to Burg and Orloff 16 as modified by Guder et al. ~7. The protein content of the final tubule suspension used for incubations was about 10-12 mg/ml. For experiments with kidney cortex slices the kidneys were not preperfused.
Incubation procedure Incubations were carried out in a Warburg apparatus at 37 °C in a final volume of 1.5 ml with a shaking frequency of 110 rev./min. In experiments with isolated kidney tubules, about 2-5 mg of protein were used per incubation vessel; in experiments with kidney cortex slices about 30 mg wet weight were used. In most experiments Krebs-Henseleit bicarbonate buffer 18, p H 7.4, was used, in some experiments K r e b s de Gasquet buffer ~9, p H 7.4. In experiments where the extracellular concentration of K + or Na + was varied, Krebs-Henseleit buffer was modified by isotonic substitution of Na + by K + or choline. The incubation samples were gassed for 10 rain with either a mixture containing 95 ~ O2 and 5 ~ CO2 when Krebs-Henseleit buffer was used, or 100 ~o O2 in experiments with the phosphate buffer. Total incubation time was 60 min in most experiments. Incubations were usually stopped by adding 0.15 ml of 3 M HCIO4, except in experiments in which electrolytes were measured, in which case trichloroacetic acid extracts were prepared.
Preparation of additions Solutions of additions were always prepared on the day of the experiment. N6-2-O-dibutyryl adenosine 3',5'-monophosphate (dibutyryl cyclic A M P ) was stored as 0.01 M solution at - 15 °C and the dilution needed made on the day of the experiment. Ouabain, substrate and dibutyryl cyclic A M P were added as aqueous neutral solutions. L-Epinephrine solution was prepared according to Joel z°. Palmitate was added as an albumin complex. 0.01 M palmitic acid solution was prepared by heating the aqueous solution to about 60 °C and adjusting the p H to 7-8 with K O H . While still warm this solution was added to an equal volume of 10 ~ bovine serum albumin with vigorous stirring. Oligomycin was dissolved in 80 ~ ethanol.
Determinations For the estimation of metabolite concentrations in experiments with isolated kidney tubules, at the end of the incubation period 0.15 ml of ice-cold 3 M HCIO 4 was added to the incubation vessels containing incubation medium plus kidney tubules. All following procedures were carried out at 0 °C. After centrifugation of the HCIO4 extracts, the supernatants were neutralized with solid K H C O 3 and, after removal of the perchlorate by centrifugation, the supernatants were used for the determination of metabolite concentrations. The measured metabolite levels in the tubule experiment therefore represent the sum of the concentration of a particular metabolite in the medium and tubules. For estimations of tissue metabolite levels in slice experiments, the incubations were stopped in an ice-NaC1 bath and the slices quickly taken out and immersed in liquid nitrogen, the slices of three incubations being pooled. The frozen tissue was then homogenized 1 " 10 (w/v) with cold 4 ~o~ HCIO4 in an UltraTurrax homogenizer. The neutralized supernatants of the HC104 extracts were used for metabolite concentration estimations. The incubation medium was deproteinized with 0.15 ml 3 M HCIO~ and glucose, lactate and pyruvate measured. Glucose was determined by the glucose oxidase/peroxidase method 21, and other metabolites by
GLUCONEOGENESIS AND SODIUM PUMP
145
standard enzymic procedures adapted for fluorometric assay, using a Perkin-Elmer MPF 2A fuorescence spectrophotometer. Pyruvate was measured according to Biicher et al. 22, malate, c~-ketoglutarate, ATP and ADP according to Williamson and Corkey z3, lactate by a method analogous to that for the determination of malate according to Williamson and Corkey 23. 3-Phosphoglycerate and phosphoenolpyruvate were measured according to Czock and Eckert 24. Oxygen uptake was measured by the Warburg manometric technique. ~4CO2 fixation was measured according to L'Age et al. 25. 1 4 C 0 2 formation was measured by trapping the 14CO2 formed during the incubation period with 0.2 ml 4 M N a O H on filter rolls in the central well of the Warburg vessels. After termination of the incubations by the addition of HC104, a subsequent incubation for 1 h was performed to assure complete absorption of the x4CO2. The filters were counted in 10 ml ethanol-toluene-PPO-POPOP scintillator in a Packard liquid scintillation spectrometer, Model 3320 or 3380. Na + and K + concentrations were estimated in the trichloroacetic acid extracts by flame photometry. Tissue cation content was corrected for extracellular space, wnich was determined in each incubation sample with [14C]inulin and was found to amount to 25-30 ~o of the tissue wet weight in accordance with results reported by Hillman et al. 26 for kidney tubules. Adenylate cyclase and phosphodiesterase activities were determined according to Ho et al. 7, the radioactive cyclic [14C]AM P was separated from [14C]ATP by paper chromatography 27. Tissue levels of cyclic AMP were measured according to Gilman 2a, using a test combination from Boehringer, Mannheim.
Materials
Na214CO3, [1-14C] -, [2-14C]pyruvate, [U-14C]glucose, [8-14C]ATP, cyclic [U-14C]AMP and [carboxyl-lgC]inulin were obtained from the Radiochemical Centre Amersham, [1-14C]palmitic acid from N.E.N. Collagenase isolated from Clostridium histolyticum, N6-2-O-dibutyryl cyclic AMP disodium succinate, the test combinations for estimation of glucose and cyclic AMP, as well as all enzymes used for metabolite determinations were purchased from Boehringer, Mannheim. L-Epinephrine bitartrate was obtained from Calbiochem., ouabain from Merck AG., Darmstadt, and ethacrynic acid from Merck Sharp and Dohme Research Lab., Rahway, N. J. Sodium L-lactate was purchased from Roth, Germany, L-malic acid and oligomycin A from Serva, Heidelberg. All other substances used were analytical grade and obtained from Merck AG., Darmstadt. RESULTS
Effects of ouabain, L-epinephrine, dibutyryl cyclic AMP, ethacrynic acid and furosemide on glucose formation by isolated kidney tubules Gluconeogenesis from pyruvate by isolated rat kidney tubules is stimulated by 10-30 ~o by ouabain in a concentration range from 0.1 mM to 4 mM. Similarly, a 20-40 ~o stimulation of gluconeogenesis from pyruvate could be obtained by the presence of 0.03-30 #M L-epinephrine. With 0.25 nM-2.5 pM ethacrynic acid, and also with 0.01-10 pg/ml furosemide, we found only a 10-12 ~o stimulation of glucose formation from pyruvate, in contrast to results of Ftilgraff et al. 29. Higher concentrations of the above listed agents were inhibitory for gluconeogenesis. As is obvious from Table I, depending on the substrate used, there are differences in the effects on
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GLUCONEOGENESIS AND SODIUM PUMP
147
T A B L E 11 E F F E C T S O F 2.5 m M A C E T O A C E T A T E A N D 0.1 m M O U A B A I N ON G L U C O N E O G E NESIS F R O M 2.5 m M L-LACTATE BY K I D N E Y C O R T E X SLICES Additions
Glucose f o r m e d (btmoles/h per a wet wt)
(1) (2) (3) (4)
25.48 :k0.93 36.24:k0.82 30.0610.50 42.17:k1.15
None Acetoacetate Ouabain Ouabain + acetoacetate * Pvs2
Pvs3
(6) (6) (5) (4)*
P < 0.001 P < 0.01 P < 0.001
< 0.001
gluconeogenesis exerted by ouabain, by dibutyryl cyclic AMP and by L-epinephrine, the action of which is known to be mediated through increased cyclic AMP levels. Ouabain stimulates gluconeogenesis from the substrates pyruvate, lactate, propionate and fructose, whereas gluconeogenesis from the dicarbonic acids and from glutamate is inhibited by 20-40 ~o. Contrary to the action of ouabain, we found a 20-47 ~o stimulation of gluconeogenesis in the presence of 0.3/zM L-epinephrine or 1 mM dibutyryl cyclic AMP from all substrates listed with the exception of fructose. With fructose as the substrate we found a stimulation of gluconeogenesis by ouabain, but an inhibition in the presence of L-epinephrine or dibutyryl cyclic AMP. The latter result is in agreement with results of Guder et al. 17. As is further evident from Table I, the stimulatory effect of ouabain on renal gluconeogenesis is additive to the stimulatory action of dibutyryl cyclic AMP and L-epinephrine, and also to the stimulatory effect of acetoacetate with 2.5 mM lactate as substrate (Table II). The inhibitory action of ouabain on gluconeogenesis from glutamate and dicarbonic acids could be due to inhibition of a Na +-dependent transport system for these substrates. In favour of this assumption is the experiment with propionate as substrate. This compound is converted intramitochondrially to malate via succinyl-CoA. If transport of dicar-
2000
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boxylic acids across the cell membranes is thus avoided, a stimulation of gluconeogenesis by ouabain is obtained.
Time course of ouabain action Fig. 1 shows that the stimulation of renal gluconeogenesis from pyruvate by ouabain is a short-term effect, which was significant after 25 rain of incubation in the presence of ouabain. There is an increased pyruvate utilization in the presence of ouabain. Influence of ouabain on pyruvate carboxylation and glycolysis Since the previously observed increase in glucose formation in the presence of ouabain might be due to either a stimulation of one of the rate-limiting steps in gluconeogenesis or simply to an inhibition of glycolysis, experiments were undertaken to differentiate between these two possibilities. An inhibition of glycolysis under the influence of ouabain has been reported for various tissues 3°-33. We estimated the ratio of lactate formed to glucose formed in experiments with fructose as the substrate, which may be taken as an indication for the flux in the direction of gluconeogenesis or glycolysis, respectively (Table III). Under conditions which favour glycolysis, such as incubation of the tissue in a Nz atmosphere or at high extracellular K + concentrations, the ratio of lactate formed to glucose formed was raised. It remained, however uninfluenced by ouabain under all conditions. Likewise ~aCO 2 formation from [U-t4C]glucose was unchanged in the presence of ouabain. However, t 4COz fixation as well as glucose formation from the substrates pyruvate, lactate and fructose were increased by ouabain (Table IV). On the basis of these findings, a simple inhibition of glycolysis as the cause for the stimulation of renal gluconeogenesis by ouabain seems excluded. The data point to an involvement of pyruvate carboxylation in the action of ouabain on renal gluconeogenesis. T A B L E IV INFLUENCE OF OUABAIN
Substrate (2.5 mM)
ON
14CO2 F I X A T I O N
BY I S O L A T E D
KIDNEY
TUBULES
Glucose formation (l~moles/h per mo protein)
~4CO z fixation (epm/h per m9 protein)
Control
Ouabain (l raM)
Control
Pyruvate
0 . 2 1 4 ± 0 . 0 0 7 (5)
0 . 2 4 5 ± 0 . 0 0 5 (4) P < 0.02
9402_200 (5)
1 9 7 9 ± 37 (4) P < 0.001
Lactate
0 . 2 3 9 ± 0 . 0 0 8 (3)
0 . 2 9 6 ± 0 . 0 0 3 (4) P < 0.001
1 5 4 0 ~ 2 7 4 (3)
3 7 4 5 ± 5 2 2 (3) P = 0.02
Fructose
0 . 4 9 9 ± 0 . 0 1 7 (9)
0 . 6 1 3 ± 0 . 0 2 0 (8) P < 0.001
485±
Ouabain (l raM)
37 (4)
1 0 2 0 ± 94 (8) P < 0.01
Activities of adenylate cyclase and phosphodiesterase and intracellular levels of cyclic A M P in kidney cortex after preincubation with ouabain Since the "insulin-like" action of ouabain on epinephrine-stimulated lipolysis in adipose tissue could be correlated by Ho et al. v with a decreased adenylate cyclase
150
D. F R I E D R I C H S , W. S C H O N E R
TABLE V INFLUENCE OF OUABAIN ON THE ACTIVITIES OF ADENYLATE P H O D I E S T E R A S E A N D T I S S U E LEVELS O F 3 ' , 5 ' - C Y C L I C A M P
C Y C L A S E , PHOS-
For the d e t e r m i n a t i o n o f adenyl cyclase a n d p h o s p h o d i e s t e r a s e activities, kidney cortex slices were p r e - i n c u b a t e d at 37 °C for 1 h in the presence or absence o f 1 m M o u a b a i n in a modified K r e b s Henseleit buffer, p H 7.4, c o n t a i n i n g 50 m M N a + a n d 100 m M K +. T h e pooled tissue o f three inc u b a t i o n s was t h e n h o m o g e n i z e d with 4 vol. o f 0.05 M T r i s - H C l , p H 7.4. Adenylate cyclase was tested using the 2 000 × y - l0 m i n sediment, p h o s p h o d i e s t e r a s e using the 50 000 × g . l0 m i n s u p e r n a t a n t . In experiments where tissue levels o f cyclic A M P were m e a s u r e d , i n c u b a t i o n s were carried o u t for 5 min. T h e tissue was t h e n i m m e r s e d in liquid N2 a n d tissue extracts prepared according to Gilm a n 28. l0 m M pyruvate was used as the substrate in all experiments.
Control Glucose formed ( ~ m o l e s / h per g wet wt)
Ouabain (1 mM)
16.94J:
A d e n y l a t e cyclase activity (pmoles cyclic A M P f o r m e d / l 0 m i n per m g protein) P h o s p h o d i e s t e r a s e activity (pmoles cyclic A M P hydrolyzed/10 m i n per m g protein)
39.34± P < 0.001
1.37 (11)
4144
dz 608
(4)
4547
29538
16288
(3)
31330
0.77 5:
Tissue levels o f cyclic A M P ( p m o l e s / m g wet wt)
0.07 (6)
1.86 (13)
~ 293
(5)
±8369
(3)
0.79 ~
0.06 (6)
activity after preincubation of the tissue in the presence of ouabain, we also tested the influence of ouabain on the adenylate cyclase-cyclic AMP system in kidney cortex (Table V). In contrast to the results of rio et aL 7, ouabain had no effect on adenylate cyclase, phosphodiesterase or tissue levels of cyclic AMP in kidney cortex. Effects o f N a + and K + on renal gluconeogenesis Since the " i n s u l i n - l i k e " effect o f o u a b a i n in muscle 3- s a n d a d i p o s e tissue 6-8
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GLUCONEOGENESIS AND SODIUM PUMP
151
T A B L E VI INFLUENCE OF EXTRACELLULAR Na + CONCENTRATION (LEFT PART) A N D K + CONCENTRATION (RIGHT PART) ON THE EFFECT OF OUABAIN ON RENAL GLUCONEOGENESIS Isolated kidney tubules were incubated for 1 h with 10 m M pyruvate in the absence or presence o f 1 m M o u a b a i n at various N a + or K + concentrations in the incubation medium. One of the two cations was held constant at the concentration indicated in brackets. The data are calculated as percentages of glucose formation in the presence of ouabain relative to 100 ~ glucose formation of controls without ouabain.
Na ÷ ( m M ) [25 m M K + ]
~ o f control (1 m M ouabain)
K + (mM) [154 m M Na + ]
°/ooo f control (l m M ouabain)
10 30 50 70 110
123 113 134" 140" 150"
0 3 6 12
82* 104 122" 144"
* Difference from control statistically significant.
could be mimicked by omitting K + from the incubation medium, we investigated the influence of the cations N a ÷ and K ÷ on renal gluconeogenesis in more detail. As shown in Fig. 2, optimal rates of gluconeogenesis from glutamate, the main substrate for renal gluconeogenesis in vivo, are only obtained in the presence of physiological concentrations of Na ÷ and K +. However, similar curves were also obtained with 10 1000
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600
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200 i 68
I 70
I 7.2
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152
D. F R I E D R I C H S , W. S C H O N E R
mM pyruvate as the substrate. From the data in Table VI it is evident that the effect of ouabain on renal gluconeogenesis from pyruvate is highly dependent on the concentration of the cations Na + and K + in the incubation medium. In contrast to the results obtained in muscle 3 - 5 and adipose tissue 6- 8, omitting K + from theincubation medium did not mimic the stimulatory effect of ouabain on renal gluconeogenesis from pyruvate, but led to an inhibition of gluconeogenesis. Also, the effect of L-epinephrine on renal gluconeogenesis was somewhat dependent on the extracellular concentrations of Na + and K +, but not to such an extent as was the case with ouabain. The effect of ouabain on renal gluconeogenesis from pyruvate is moreover pH dependent (Fig. 3). Ouabain stimulates gluconeogenesis only at pH values above 7.2, whereas at pH values lower than 7.2 gluconeogenesis from pyruvate is inhibited in the presence of ouabain. In further experiments we changed the extracellular concentrations of Na + and K +, leaving the sum of the two cations constant, since Bihler and Sawh 34 and Adam and Haynes 35 showed that changes in osmolarity p e r s e can affect sugar transport 34 and ~4CO2 fixation 35. Under these conditions the gluconeogenesis in the controls was decreased by 80-90 % if the extracellular K + concentration was raised to 150 mM (Fig. 4). The inhibition of gluconeogenesis at high extracellular K + concentrations (up to 100 mM K +) was completely prevented in the presence of ouabain, thus increasing the magnitude of the stimulatory effect of ouabain on gluconeogenesis from pyruvate. However the decrease of gluconeogenesis at high extracellular K + concentrations could not be prevented in the presence of L-epinephrine. These results seemed to suggest that the observed changes in the rate of gluconeogenesis under these conditions might be due to changes in the intracellular Na + and K + levels. Tissue concentrations of Na + and K + in the absence and presence of ouabain were therefore determined with 6 mM and 100 mM extracellular K + concentration. The results of these experiments are presented in Table VII. Tissue levels of Na + were increased in the presence of ouabain. However tissue K + concentrations remained unaltered by ouabain. Surprisingly, with 100 mM extracellular K + concentrations the intracellular K + content was increased about 3-fold even in the presence of ouabain. From these c A
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GLUCONEOGENESIS
153
AND SODIUM PUMP
T A B L E VII INFLUENCE OF OUABAIN ON TISSUE LEVELS OF Na + AND K + K i d n e y cortex slices were used. Substrate: 10 m M pyruvate. Cation concn (raM) in the incubation medium
Tissue cation content (~val/g wet wt)
K+
Control
Na +
K+
Na + Ouabain (1 m M )
Control
Ouabain (I raM)
6
154
35.65-- 3.83 (6)
35.20~-2.87 (6)
185.96~23.04 (6)
319.21~22.21 (6) P < 0.02
100
50
105.59±13.32 (6)
112.54±6.60 (6)
2 0 2 . 4 8 ± 1 5 . 5 2 (6)
259.66±18.15 (4) P < 0.05
results it appears rather unlikely that the stimulation of glucose formation and 14CO2 fixation from pyruvate by ouabain is mediated by a stimulation of pyruvate carboxylase through a decrease of intracellular K + concentration, as was discussed by Weiss et al. ~2.
Influence of ouabain on the energy state of the cell Since the Na + p u m p is known to utilize about 30-40 ~o of the energy production of the kidney cell 36, it seemed necessary to gain information about the energy state of the cell under our conditions. The results of these studies are presented in Table VIII. Oxygen consumption was reduced in the presence of ouabain at 6 m M extracellular K + concentration in accordance with results reported by others 3 ~' 36-3s Raising the extracellular K + concentration to 100 m M led to a stimulation of oxygen uptake even in the presence of ouabain. The levels of A T P measured in the whole cell were elevated in the presence of ouabain at 100 m M extracellular K + concentration. A D P levels were unaffected by ouabain. The ratio of net C 3 carbon units removed to glucose formed, which at values higher than 2 gives a measure for the portion of pyruvate which is not accounted for as glucose or lactate and which is presumably oxidized, is increased by high extracellular K + concentrations. In the presence of ouabain this ratio is decreased and is almost unaffected by high extracellular K + concentrations. This indicated that, in the presence of ouabain, pyruvate is preferentially channelled towards gluconeogenesis rather than towards oxidation. F r o m Table IX it is evident that ouabain stimulates gluconeogenesis from pyruvate even in the presence of oligomycin. This well-known inhibitor of oxidative phosphorylation 39 leads to an increase of A D P levels in the mitochondrial matrix 4°. Concomitantly, a decrease of pyruvate carboxylation occurs, as demonstrated by Walter and Stucki 4° in rat liver mitochondria. A D P is also known to inhibit purified pyruvate carboxylase from chicken liver, as was reported by Keech and Utter 41. 14CO2 formation from [2-14C]pyruvate and [l-14C]palmitate in the absence and presence of carnitine was decreased in the presence of ouabain (Table X). The decreased 14CO 2 formation from [2-14C]pyruvate might be interpreted as anindication for a reduced activity of the citric acid cycle under the influence of ouabain. Similarly ~4CO2 formation from [l-~4C]palmitate may be taken to reflect citrate cycle flux, if one can assume that the
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GLUCONEOGENESIS AND SODIU M PU M P
155
TABLE IX I N F L U E N C E OF OUABAIN AND OLIGOMYCIN ON GLUCONEOGENESIS BY KIDNEY CORTEX SLlCES F R O M 10 mM PYRUVATE Additions
Glucose f o r m e d
(pmoles/h per O wet wt) (1) (2) (3) (4)
none Ouabain Oligomycin Oligomycin 4-ouabain
(0.1 mM) (1/~g/ml) (1/zg/ml) (0.1 raM)
30.96±1.52 37.344-1.40 17.68±1.08 22.00± 1.26
(4) (4) (8) (8)*
P < 0.05 P < 0.001 P < 0.002
* P v s 3 < 0.05.
transport of long-chain fatty acids through the kidney cell membrane is not impaired by ouabain. These findings reveal that, under the influence of ouabain, apart from the reduced respiration, the citric acid cycle activity is apparently also reduced. The assumption of reduced citric acid cycle activity in the presence of ouabain is further supported by the increased tissue malate levels (Table XI) and ct-ketoglutarate levels in experiments with lactate as substrate (data not shown) under the influence of ouabain. The increased tissue malate and lactate levels in the presence of ouabain (Table XI) furthermore indicate a more reduced state of the cell under the influence of ouabain, as would be expected under conditions of diminished respiration and citric acid cycle activity. Contrary to the results obtained with ouabain, oxygen uptake and t4CO2 production from [1-14C]palmitate were unaffected by L-epinephrine, oxygen uptake being also uninfluenced by dibutyryl cyclic-AMP (data not shown). x4C02 formation from [1-14C]pyruvate, which can be taken as an estimate of pyruTABLE X INFLUENCE OF 1 mM OUABAIN ON 14C02 FORMATION FROM [I-14C]PYRUVATE, [2-14C]PYRUVATE AND [I-14ClPALMITATE The experiments with [~4Clpyruvate were carried out with isolated kidney tubules, the experiments with [~4C]palmitate with kidney cortex slices. The data for glucose formation are calculated as /~moles/h per mg protein for the tubule experiments and as #moles/h per g wet wt for the slice experiments. 14C0z formation is indicated as cpm/h per mg protein and as cpm/min per g wet wt, respectively. Substrate
Concn
(raM)
14C02 formation
Glucose formation Control
Ouabain
Control
(1 mM)
Ouabain
(1 mM)
[l-14C]Pyruvate 10
0.8524-0.014 (4)
0.959±0.029 (4) P < 0.02
64680±5492 (4)
58933 --3060 (4) N.S.
[2-14C]Pyruvate 10
0.846:k0.009 (4)
0.9104-0.002 (3) P < 0,002
347204-2255 (4)
25672±2159 (4) P < 0.05
[l-14C]Palmitate 0.5
31154- 218 (18)
2103± 214 (24) P ,< 0.01
[1-~4C]Palmitate 0.5 4-DL-carnitine 1
3640± 298 (13)
28624- 201 (12) P < 0.05
156
D. FRIEDRICHS, W. SCHONER
TABLE Xl TISSUES METABOLITE LEVELS UNDER THE INFLUENCE OF OUABAIN AND DIBUTYRYL CYCLIC AMP Isolated kidney tubules were incubated for 1 h in the presence of 1 mM ouabain or 1 mM dibutyryl cyclic AMP or the combination of both, in the presence of 2.5 mM propionate as the substrate. Values are calculated as nmoles/mg protein.
Control Lactate Pyruvate Malate Phosphoenolpyruvate 3-Phosphoglycerate ~-Ketoglutarate
Ouabain
Dibutyryl o'clic AMP
Ouabain-c-cyclic dibutyryl AMP
68.15 ± 3.84 ( 15)
109.08±11.73 (10) 54.04~:1.29 (9) 53.73±0.88 (8) P < 0.001 P < 0.02 P < 0.02 2.49 ±0.15 ( 13) 2.46~ 0.22 (8) 2.36±0.13 (9) 1.78±0.03 (8) P < 0.01 9.44± 0.83 (10) 3.82±0.25 (9) 6.088±0.29 (8) 5.00 ±0.20 (15) P < 0.001 P < 0.01 P < 0.01 0.74_-40.09 (10) 0.61± 0.03 (8) 1.61-z0.22(9) 0.78±0.03 (8) P < 0.05 P < 0.05
31.06± 1.52 (12)
27.43± 2.17 (7)
33.60±0.96 (9)
0.77 ± 0.10 (12)
0.80± 0.14 (7)
0.61 ±0.06 (9)
33.28~0.78 (8) 0.86±0.06 (8)
vate dehydrogenase activity, was not significantly altered by o u a b a i n u n d e r our conditions. DISCUSSION
Relation between the stimulation of renal #luconeogenesis by ouabain, ~&rosemide and ethacrynic acid and the adenylate o'clase-cyclic A M P system The present paper demonstrates that, in kidney cortex, i n h i b i t i o n of the N a + p u m p u n d e r certain c o n d i t i o n s can induce a stimulation of renal gluconeogenesis to a similar extent as can be observed in the presence of L-epinephrine or dibutyryl cyclic A M P . C o n t r a r y to L-epinephrine however, o u a b a i n exerts its stimulatory effect on renal gluconeogenesis by a m e c h a n i s m differing from the cyclic A M P system. S u p p o r t for this a s s u m p t i o n comes (a) from the d e m o n s t r a t i o n of the additive effects o f L-epinephrine a n d o u a b a i n o n renal gluconeogenesis (Table I), (b) from the d e m o n stration of different patterns of tissue metabolites in the presence of o u a b a i n or cyclic A M P (Table XI), a n d (c) from the lack of any effect of o u a b a i n on the adenylate cyclase or phosphodiesterase activities of kidney cortex (Table V). A p a r t from o u a b a i n , the diuretics furosemide a n d ethacrynic acid also stimulate gluconeogenesis in kidney cortex from various substrates, as has been reported by Fiilgraff et al. 29 a n d as is d e m o n s t r a t e d by us in this paper. Furosemide leads to a s t i m u l a t i o n of renal gluconeogenesis, although renal cortical adenylate cyclase remains uninfluenced by furosemide 42. Rat kidney ATPase is inhibited by furosemide according to Park et aL 43, but is uninfluenced according to Yoshida a n d Metcoff 44. Ethacrynic acid stimulates renal gluconeogenesis at very low concentrations (0.3 n M I /~M) according to Ftilgraff eta/. 29 and to us. At these c o n c e n t r a t i o n s ethacrynic acid does n o t affect ( N a + - K + )-activated ATPase 43. 45 - 47 or renal adenylate cyclase 41.
GLUCONEOGENES1S AND SODIUM PUMP
! 57
Thus it appears that the stimulation of renal gluconeogenesis by the diuretics furosemide and ethacrynic acid is probably unrelated to adenylate cyclase activity and also not mediated by an influence on ( N a + - K +)-activated ATPase. Relation between stimulation o f renal gluconeogenesis by ouabain and changes in the extraeellular and intracellular concentrations o f Na + and K + Because the stimulatory effect of ouabain on renal gluconeogenesis from pyrurate is most probably not mediated by alterations of the cyclic A M P system, it must be assumed that the stimulation occurs as a consequence of the inhibition of the sodium pump. The sodium p u m p may influence renal metabolism via changes of the intracellular concentrations of Na + and K + or via the energy requirement. Weiss et al.a 2 assumed that ouabain may stimulate renal pyruvate carboxylase by decreasing intramitochondrial K + concentrations. His assumptions were based upon the finding that pyruvate carboxylase from liver is inhibited by K + concentrations higher than 8 0 m M 12,48. The increased 14COz fixation form pyruvate lactate and fructose (Table IV) confirm the data of Weiss et al. lz and point to an influence on intramitochondrial pyruvate carboxylation, provided that glycolysis is uninfluenced, as is likely from our data (Table III and IV). Contrary to the assumption of Weiss et al. ~2 however, the intracellular K + content of kidney cortex slices remained unaltered after 1 h of incubation with ouabain (Table VII). This lack of K + decrease, which is in accordance with the findings of Jones and Landon 31 for rat kidney cortex slices, but is in contrast to those of Letarte et al. 8 for isolated mouse fat cells, is not due to any ineffectiveness of the specific action of ouabain. The increase in tissue Na + content indicates an inhibition of the N a + pump. Although measurements of cation concentrations for the whole cell may not exclude the occurrence of a shift of K + between different intracellular compartment, it seems to us unlikely that the stimulatory action of ouabain on renal gluconeogenesis from pyruvate is the consequence of a decrease of intramitochondrial K + content (which in turn would de-inhibit mitochondrial pyruvate carboxylase). Support for this conclusion comes also from the finding that ouabain stimulates renal gluconeogenesis in the presence of high extracellular K + concentrations which lead to about a 3-fold increase in the intracellular K + content (Table VII). Since tissue Na + concentrations were increased by ouabain, possible effects of Na + on cellular metabolism, which might result in an increased gluconeogenesis form pyruvate, lactate and fructose, have to be considered. Na + was reported to have no influence on pyruvate carboxylase 4s, but to inhibit glycolysis in brain slices 49. However, as already pointed out, we have no evidence for any inhibition of glycolysis by ouabain under our conditions. Influence o f ouabain on energy levels, respiration and the citric acid cycle The influence on mitochondrial processes such as respiration and pyruvate carboxylation which we observed in the presence of ouabain (Tables IV and V I I I ) must be an indirect one, since Blond and Whittam 36 found no influence of ouabain on the respiration of isolated kidney mitochondria. The change of the redox state of the cell towards a more reduced state, which is indicated by increased levels of malate and lactate in the presence of ouabain in our experiments (Table XI), is in line with the results of van Rossum s°, who observed in slices of seagull nasal glands, which actively transport NaC1 in a respiration-dependent manner, an increased reduction
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of nicotinamide nucleotides and cytochrome b in the presence of ouabain. Van Rossum s o assumed that ouabain induces a transition of the mitochondria in the slices from a state approaching the active State 3 towards the resting State 4. He further assumes that the inhibition of respiration is a result of limitation of phosphate acceptor. Similarly, Blond and Whittam 36 conclude from their studies with kidney cortex homogenate and isolated kidney cortex mitochondria that the rate of respiration of kidney mitochondria is controlled by the concentration of A D P or orthophosphate, the formation of which is determined by energy-utilizing processes, including ion transport. The importance of A D P and A T P for the regulation of other mitochondrial processes apart from respiration is also evident from the work of Stucki and coworkers40.5t and of Patel and Hanson 52, using isolated liver mitochondria 4°, 51 and adipose tissue mitochondria 52, respectively. These authors found a stimulation of pyruvate carboxylation in isolated mitochondria by ATP and an inhibition by ADP. The regulation of pyruvate carboxylase by the A T P / A D P ratio within the mitochondrial matrix as proposed by Stucki and co-workers 4°' 5 t is well in agreement with the results which Keech and Utter 4t obtained with purified chicken liver pyruvate carboxylase. Moreover, Walter and Stucki 4° could demonstrate that, with isolated liver mitochondria under conditions which led to elevated A D P levels in the mitochondrial matrix, as was particularly pronounced in the presence of oligomycin, pyruvate carboxylation was strongly inhibited. In our more complex system we also found an inhibition of gluconeogenesis in the presence of oligomycin, which was significantly less if ouabain was present in addition to the oligomycin (Table IX). The decreased 14COz production from [2-t4C]pyruvate in the presence of ouabain points to a reduction of citric acid cycle activity under the influence of ouabain. It seems likely that the reduction of citric acid cycle activity by ouabain occurs at a step between malate dehydrogenase and succinate thiokinase, because gluconeogenesis from propionate is stimulated by ouabain. Thus citrate synthase, isocitrate dehydrogenase and ~-ketoglutarate dehydrogenase remain as possible control points in the citric acid cycle. La Noue e t al. 53 found in isolated rat heart mitochondria a reduction of flux through the citric acid cycle of 75-80 ~o in the respiratory state 4 relative to state 3, which was associated with increased levels of N A D H , high ATP levels and an accumulation of ~-ketoglutarate. In our studies in the intact cell we also found in the presence of ouabain (a) significant higher ATP concentrations under conditions with high extracellular K + concentrations (Table VIII), but not with 6 m M K +, (b) a decrease of ~4COz production fi om [2-14C]pyruvate and [1-I 4C]palmitate (Table X): (c) an accumulation of ~-ketoglutarate; and (d) a more reduced redox state of the cell, as is evident from the increase in malate and lactate contents (Table XI). Thus the conclusions La Noue et al. 53 arrive at from data might well be valid for the interpretation of oul results. According to their La Noue e t al. s3 a feed-back control from the electron transport chain to the citric acid cycle is mediated by the phosphorylation state of the adenine and guanine nucleotides. Changes of these two factors secondarily influence the intramitochondrial concentrations of oxaloacetate, acetyl-CoA and succinyI-CoA, which are direct regulators of citrate synthase and ~-ketoglutarate dehydrogenase activities 53,54.
GLUCONEOGENES1S A N D S O D I U M P U M P
159
CONCLUSION
Thus, the present work provides some evidence for the regulation of renal gluconeogenesis in the intact cell by the sodium pump, which is probably not mediated by the adenylate cyclase-cyclic AMP system. Likewise, the cations K ÷ and Na ÷ do not seem to play a regulatory role under these conditions. The tentative conclusion is drawn, that, in kidney tissues, inhibition of the sodium pump might induce a higher energy state of the cell, which in turn favours energy-requiring synthetic processes. A C K N O W L E DG MENT
We gratefully appreciate the skilful technical assistance of Miss G. Schott and Miss A. Boie. This work was in part supported by the D.F.G. (Scho 139/1-6) and partially by means of the S.F.B. 33. The Stiftung Volkswagenwerk provided, in GSttingen, the MPF 2A Perkin-Elmer fluorimeter. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
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