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Relationship between rate of gluconeogenesis and content of nicotinamide adenine dinucleotide in renal cortex
Life Sciences, Vol. 29, pp. 1195-1202 Printed in the U.S.A.
Pergamon Pres
RELATIONSHIP BETWEEN RATE OF GLUCONEOGENESIS AND CONTENT OF NICOTINAMIDE ADENINE DINUCLEOTIDE IN RENAL CORTEX S. Y. Lise Ou, Stephen A. Kempson and Thomas P. Dousa Mayo Clinic and Foundation, Department of Physiology, Division of Nephrology, Rochester, MN 55901
and
(Received in final form July 6, 1981) Summary Gluconeogenesis in rat renal cortex was measured using tissue slices incubated with or without appropriate substrates. Immediately after incubation the tissue slices were snap-frozen and the content of oxidized nicotinamide adenine dinucleotide (NAD +) was determined. Incubation with 10 mM a-ketoglutarate or L-glutamate led to enhanced glucose production and an increase in tissue content of NAD +. Quinolinate and 3-mercaptopicolinate inhibited the rate of gluconeogenesis from L-glutamate and ~-ketoglutarate respectively, and decreased the tissue levels of NAD +. The enhanced rate of gluconeogenesis was associated with an increase of NAD+ in the cytosol fraction (105 x g supernatant) but not in the particnlate fraction (105 x g pellet) of renal cortex homogenate. Present results indicate that NAD+ content changes in parallel with the rate of gluconeogenesis in renal cortical tissue. In our recent studies we observed that nicotinamide adeninine dinucleotide (NAD) I inhibits sodium-dependent uptake of phosphate (Pi) by renal brush border membrane (BBM) in vitro (I), and that increased in NAD content of renal cortical tissue in response to injection of nicotinamide in vivo is associated with a decreased rate of BBM uptake of Pi and with phosphaturia (I). Based on these studies we proposed a hypothesis that NAD, in particular the oxidized form (NAD+), in cytoplasm could regulate transport of Pi across BBM in renal proximal tubules (1,2). Although diverse metabolic processes occur in the cytoplasm of renal tubular cells, gluconeogenesis appears to be of particular importance for determining the level of cytoplasmic NAD+ (3,4). Since NADH is oxidized to NAD+ at the glyceraldehyde-3 phosphate dehydrogenase (GADP) step of gluconeogenesis (Fig. I), an increase of the rate of gluconeogenesis by supplying substrates which enter this pathway prior to the pbosphoenolpyruvate carboxy-kinase (PEP-CK) step (Fig. I) may be predicted to increase the level of cytoplasmic NAD+ (3,4). Recent studies have shown that both gluconeogenesis per s__ee(5) as well as key gluconeogenic enzymes (6) are
I Abbreviations used in this paper: NAD, nicotinamide adenine dinucleotide; NAD +, oxidized form of NAD; Pi, phosphate; PEP-CK, phosphoenolpyruvate carboxy kinase; GAPD, glyceraldehyde-3-phosphate dehydrogense; KRB, Krebs Ringer phosphate buffer; BBM, brush border membrane. located uniquely in proximal tubules but not in glomeruli or in the more distal tubular segments of the nephron. 0024-3025/81/121195-08502,00/0 Copyright (c) 1981 Pergamon Press Ltd.
1196
Gluconeogenesis
and NAD + in Kidney
Vol. 29, No.
12, 1981
It is of notable interest that many hormonal and metabolic stimuli of ~luconeogenesis in proximal tubules including parathyroid hormone, starvation, acidosis, and glucocorticoids (2,3), are also known to cause phosphaturia bv inhibition of Pi reabsorption across proximal tubular BBM. Therefore, it is very important to establish whether an increased rate of gluconeogenesis indeed causes an increase in tissue NAD +. While the increase in tissue NAD + after stimulation of gluconeogenesis may be expected (Fig. I), no direct evidence for this phenomenon is available. In the present study we address this question by investigating the relationship between the rate of gluconeogenesis in rat renal cortical slices and the content of NAD + in the same slices. The results of these experiments indeed indicate that the rate of gluconeogenesis may influence the content of NAD + in renal cortical tissue.
r
FRUCTOSEI~ISPHOSPHATE
FRUCTOSE
GLYCERALDIEHYDE3-P
"~ GLYCEROL
1.3-BISPHOSPHOGLYCERATE
t
GDP+CO2
'"O,..OE"OERO ......... GTP
CYTOSOL
7L.CT.TE
....,'
CO
'
PYRUVATE
~DRION v
"--GLUTAMATE
[oo,,o ...... ]
FIG.
I
Schematic outline of gluconeogenic pathway . Note oxidation of NADH to NAD + at the GAPD step in cytoplasm, points of entry of ~-ketoglutarate and L-glutamate, and points of action ( ~ ) of inhibitors (in square brackets). Methods Adult male Sprague-Dawley rats weighing 200-250 g were allowed free access to food and drinking water. They were stunned, the kidneys removed and decapsulated and immediately chilled in Krebs-Ringer phosphate buffer (KRB). The kidneys from several animals were pooled together and slices of cortex were randomly distributed to incubation flasks for simultaneous incubations with and without added substrates and inhibitors.
Vol. 29, No.
12, 1981
Gluconeogenesis
and NAD + in Kidney
1197
Gluconeogenesis was ~etermined basically bv the method described by Klahr (7). Cortical tissue slices (0.5 mm thickness) were prepared by Stadie-Riggs hand-operated tissue slicer and were distributed to incubation flasks containing KRB of the following composition: 127.2 mM NaC1; 4.0 mM KC1; I ~M CaC12; 1.2 mM MKC12; 2.h mM NaH2PO h (pH 7.4). Slices were first preincubated for 60 minutes at 37°C in order to deplete the tissue of endogenous gluconeogenic substrates. The slices (60-80 mg wet weight) were then transferred into 10 ml eresh KRB in Erlenmeyer flasks with or without added substrates and inhibitors as indicated in the Results. In control incubations without substrate, sodium chloride was added in equimolar concentration. Slices were then incubated at 37°C for 60 min in a Dubnoff metabolic shaker bath while 1004 oxygen was continuously bubbled through individual flasks. At the beginning and at the end of the incubation period samples of the incubation medium were taken for determination of glucose content. At the end of the incubation the slices were blotted dry, frozen rapidly in liquid N 2 and a percholoric acid extract was prepared as described previously (I) for determination of NAD + and ATP. In studies on the subcellular distribution of NAD +, fractionation was performed as described for liver tissue (8,9) and also used in our previous study on the kidney (I). Briefly, at the end of incubation slices were quickly removed from incubation medium and a I0~ (wt/vol) homogenate was prepared using 5 passes of a motor-driven glass-teflon homogenizer. The homogenization medium was 0.25 M sucrose, 5 mM Tris-HCl (pH 7.4) containing 50 mM nicotinamide to inhibit enzvmatic degradation of NAD + (8,9), Homogenate was then ultracentrifuged for 60 min at 105 x g, the resulting supernatant (cytosol fraction) and pellet (particulate fraction) were separated and extracted immediately with TCA (8). After centrifuging the TCA extracts to remove protein precipitate, the TCA was removed by several washes with three volumes of water-saturated ether (8). The pH of the washed extracts was adjusted to neutrality and the NAD + content was determined the same day. All steps in the fractionation and extraction procedure were performed at 0 ° 4oc. The assay for NAD + was based on the interconversion of lactate and pyruvate catalyzed by lactate dehydrogenase and requiring NAD (1,10). The final volume of the reaction mixture was 2.0 ml and contained 0.025 mM lactate, 5 mM hydrazine sulfate, 0 . 1 M glycine-NaOH (pH 10.0) and up to 0.1 ml of the acid extract or standard NAD + solution. The reaction was started by the addition of 0.005 ml of lactate dehydrogenase (3000 U/ml). The change in fluorescence was measured on an Aminco-Bowman spectrophotofluorometer with excitation wavelength 340 nm and emission wavelength 470 nm. The increase in fluorescence, after incubation for 30 min at 37 ° C, was linear in the concentration range 1-20 nmoles of NAD +. ATP content of cortical slices was determined from fluorimetric measurement of NADPH using the hexokinase and glucose-6-phosphate dehydrogenase method (1,11). The final reaction mixture (total volume 2.0 ml) contained 10 mM D-glucose, 5 u M NADP, 1.5 mM MgCI2, 0.6 mM EDTA, 12.5 mM triethanolamine HCI (pH 8.0) and up to 0.1 ml of the acid extract or standard ATP solution. The reaction was started by the addition of 0.01 ml of glucose-6-phosphate dehydrogenase (125 U/ml) and 0.03 ml of hexokinase (132 U/ml). After 15 min at 37 ° the increase in fluorescence (excitation at 365 nm, emission at 460 nm) was proportional to the amount of ATP in the range 0-15 nmoles. Samples of the incubation medium were deproteinized by the addition of perchloric aeid (final concentration 0.6 N), the extracts were neutralized with KOH, centrifuged and the glueose content of the clear supernatants was determined as described and used elsewhere (12,13).
I]98
Gluconeogenesis and NAD + in Kidney
Vol. 29, No.
12, 1981
All analyses were run in duplicates or triplicates, and all incubations and analyses (with or without added substrates or inhibitors) were carried out in parallel. The results were evaluated statistically by Student's t-test for group or paired comparisons as specified in the Results. Values of P ) 0.05 were considered not significant (NS). Results Renal cortical slices were incubated with and without addition of e-ketoglutarate or L-glutamate, renal gluconeogenic substrates (13,14), Incubation with G-ketoglutarate caused a marked increase in the glucose production and at the end of the incubation Deriod the tissue content of NAD + was significantly higher than in controls (Table I). Likewise, incubation with Lglutamate increased glucose accumulation, although to a lesser degree, and at the end of the incubation with L-glutamate the NAD + content in the slices was also significantly higher compared to controls (Table II). In contrast, no significant differences were found in levels of ATP measured in the same tissue extracts as NAD + (Tables I and II). TABLE I Stimulation of renal cortical gluconeo~enesis bv ~-ketoglutarate, and inhibition by 3-mercaptopicolinate; effect on tissue level of NAD +
Additions
(n)*
A.
None (control)
(5)
B.
10 mM -ketoglutarate
(5)
10 mM -ketoglutarate + 0.1 mM 3-mercaptopicolinate
Glucose formation (Anmol/K wet weight) 158.8 +
37.1t
1276.2 + 164.g
NAD + (nmol/g wet weight)
ATP (nmol/g wet weight)
142.8 + 23.8
546.7 +
6.97
208.0 + 22.4
581.4 + 99.3
180.3 + 26.6
539.7 + 85.0
C.
(5)
4~9.0 +
71.2
P-value** A<
~B
0.005
~ O.02g
NS
B<
~C
0.02
~ 0.01
NS
A<
~C
0.005
NS
NS
*number of experiments tmean + SEM **for significance of differences between the three experimental conditions (paired t-test) The increase in glucose production by incubation with ~-ketoglutarate was significantly inhibited by inclusion of 0.1 mM mercaptopicolinate (15) in the incubation medium. This inhibition of gluconeogenesis was associated with significant lowering of NAD + compared to incubation with ~-ketoglutarate alone; no significant differences were found in levels of ATP (Table I). When L-glutamate was used as a substrate, inclusion of 0.5 mM quinolinate (13) into the incubation mixture inhibited the rate of glucose production to levels in controls (no added substrate); the tissue content of NAD +, but not the content of ATP, was significantly lower than in preparations incubated with Lglutamate alone (Table II).
Vol. 29, No.
12, 1981
Gluconeogenesis
and NAD+ in Kidney
1199
Table II Stimulation of Renal Cortical Gluconeogenesis by L-glutamate and Inhibition by Quinolinate; Effect on Tissue Level of NAD +
Additions
(n)*
Glucose formation ( Anmol/g wet weight)
NAD +
ATP
(nmol/g wet weight)
(nmol/g wet weight)
A.
none (control)
(4)
153.5 Z 47.4#
162.2 Z 15.0
539 Z 95
B.
10 mM L-glutamate
(4)
605.2 + 89.4
244.5 + 21.9
546 + 86
C.
10 mM (4) L-glutamate + 0.5 mM quinolinate
60.2 + 60.2
205.0 + 14.7
613 + 83
P-value** A ~
> B
( 0.05
( 0.02
NS
B.~/
> C
( 0.05
( 0.02
NS
A <
> C
NS
NS
NS
* number of experiments # mean + SEM ** significance of differences (paired t-test)
between the three experimental
conditions
Finally, the effect of the increasing rate of gluconeogenesis on NAD + in cytosolic and particulate fractions of renal cortical slices was examined. After incubation with L-glutamate, a portion of the tissue slices was taken for determination of whole tissue NAD + and another portion was fractionated and analyzed as described in the Methods. As in the experiment described in Table II, in the presence of 10 mM L-glutamate, glucose production markedly (6 x) increased and also total tissue NAD + content was significantly higher than in controls without substrate. In cytosolic fractions of homogenate the content of NAD + was more than 3 x higher compare~ to the corresponding particulate fraction (Fig. 2). In preparations from slices incubated with L-glutamate the content of NAD + in cytosol was significantly higher compared to controls, while no significant difference was found between NAD + contents in particulate fractions (Fig. 2). Discussion Since several phosphaturic stimuli are known to stimulate renal cortical gluconeogenesis (3), it was of major interest to establish whether an increase in the rate of renal gluconeogenesis leads to a parallel change in NAD + content in the renal cortex. As a first approach we changed the rate of gluconeogenesis simply by increasing the supply of gluconeogenic substrates which enter this pathway from the mitochondrial compartment (Fig. I). Therefore, both substrates enter the pathway prior to the initial PEP-CK step of gluconeogenesis ~nd also prior to the GAPD step, where NADH is oxidized (Fig. I). Also, both inhibitors used in our studies block renal gluconeogenesis by direct action on PEP-CK and/or precedin~ enzyme reactions (13,15).
1200
Gluconeogenesis
and NAD + in Kidney
I
I
-
12, 1981
10 s x g pellet
Cytosol 200
Vol. 29, No.
I
~ ~zz/
,//, ,/// ///,
NAD*
(, moll
,///
g w wt)
~"/4 ///~
100 r//. ///. -/// ///, ,z/¢ ///, ,//,
0
L-Glu
0
L-Glu
FIG. 2 Effect of increased rate of gluconeogenesis on NAD + content in cytosol (left panel) and in particulate fraction (105 x ~ pellet) (right panel) of homogenate (for details see Methods and Results). Each bar denotes mean + SEM of 6 incubations. Open bars: fractions from control slices incubated without (0) added substrate; shaded bars: fractions from slices incubated with 10 mM L-glutamate (L-GIu). * denotes value siKnificantly (P ( 0.01; group t-test) higher than control (0). Both in control tissue and tissue incubated with L-glutamate the NAD + content in cytoso] ~ significantly (P ( 0. '01; group t-test) higher than in the pellet.
~ccording to current evidence, 3-mercapcopicolinate inhibits gluconeogenesis by direct inhibition of PEP-CK (15) and quinolinate, in addition to inhibition of PEP-CK, also inhibits deamination of L-glutamate to ~-ketoglutarate (13). In any case, both compounds inhibit gluconeogenesis at the steps prior to NAD + generation (Fig. I). Present observations, taken together, are thus compatible with the interpretation that when the rate of ~luconeogenesis is increased (by increased availability of substrates) or decreased (by inhibition at the steps prior to formation of phosphenolpyruvate) there is an increase or decrease in parallel in the tissue NAD + level, most likely by changing the rate of oxidation of NADH to NAD + at the GADP step. The absence of changes in levels of ATP documents that the rate of gluconeogenesis may markedly change without detectable depletion of tissue ATP, which is consumed in this metabolic pathway, and also that changes in NAD + are relatively specific for this nucleotide. In renal cortex, as in other tissues (8,9), the bulk of the cellular pool of NAD ÷ is present in cytoplasm. An observation that the increased rate of gluconeogenesis was associated with an increase in NAD + only in cytosol (Fig. 2) is compatible with the observation that in rat kidney gluconeogenesis is almost completely located in cytosol (16). In the context of the proposed regulation of BBM transport of Pi (1,2,3), it is important that the level of NAD + is changed in the cellular compartment that is in direct contact with BBM.
Vol. 29, No. 12, 1981
Gluconeogenesis and NAD + in Kidney
1201
The limitations in interpretation of the present results should be realized. The content of NAP + in tissue slices preparation was lower than that measured in snap-frozen samples of kidney (I); this difference may be due to lesser oxygen supply to cells which are not perfused by blood. The rate of glucose production as well as levels of NAD + and of ATP were measured only in undivided renal cortical slices and it is not certain to which degree the observed changes reflect processes located specifically in the proximal tubular cells. Likewise, the rate of gluconeogenesis and activities of gluconeogenic enzymes although limited to proximal tubules (5,6) are not equally distributed in various subsegments, and the same could apply to the content and metabolism of NAD +. The above mentioned limitations in design of the present experiments also likely accounts for the lack of strict quantitative correlation between changes in the rate of gluconeogenesis and NAD + levels in the renal cortex. Exact localization, quantification and causality of the relationship between gluconeogenesis and NAD + generation in different specific subsegments of proximal tubules should be established in future studies. However, since the proximal tubules constitute about 50% of renal cortical mass in the rat kidney (17), it is very likely that the presence and directionality of changes in cortical slices reflect processes in proximal tubules rather than in other structures of kidney cortex. While in the present study, the rate of gluconeogenesis was changed by supply of substrate and/or by inhibitors, our most recent observations indicate that similar relationship between NAD + and gluconeogenesis also exist in vivo. Administration of glucocorticoids (18), or metabolic acidosis induced by ammonium chloride load (19) caused in parallel an increase in the rate of renal gluconeogenesis and enhancement of NAD + content in renal tissue. In conclusion, the present results provide to our knowledge the first evidence that by changing the rate of gluconeogenesis in renal cortex,.tissue NAD + content changes in a parallel manner. In general terms, our findings are in support of the hypothesis that phosphaturic stimuli decrease renal Pi reabsorption by increasing renal gluconeogenesis in proximal tubules, leading to increased cytoplasmic NAP + which inhibits Na+-dependent Pi transport across the luminal BBM (1,2,3). Acknowledgments This work was supported by USPHS grants AM-16105 and AM-19715, grantin-aid (MHA-36) from the Minnesota Heart Association and by funds from the Mayo Foundation. S. A. Kempson's current address is Renal-Electrolyte Division, University of Pittsburgh, School of Medicine, Pittsburgh, PA. We thank Bonnie Becker for excellent secretarial assistance. We are indebted to Dr. N. DiTullio from Smith, Kline and French Laboratories, Philadelphia, PA, for the supply of 3-mercaptopicolinate. Reprint requests should be directed to Dr. T. P. Dousa° References I. 2.
3. 4.
S. A. KEMPSON, G. COLON-OTERO, S. Y. L. OU, S. T. TURNER, and T. P. DOUSA, J. Clin. Invest. 67:1347-1360 (1981). T. P. DOUSA, and S. A. KEMPSON, Urolithiasis I Clinical and Basic Research (L. H. Smith, W. E. Robertson, and B. Finlayson, eds.) p. 741-745, Plenum Press, New York (1981). T. P. DOUSA, and S. A. KEMPSON, Mineral Electrolyte Metabolism, (in press) (1981). C. I. POGSON, I. D. LONGSHAW, A. ROOBOL, S. A. SMITH, AND G. A. O. ALLEYNE, Gluconeogenesis t Its Regulation in Mammalian Species
1202
5. 6. 7. 8. 9. 10.
11. 12.
13. 14.
15. 16. 17. 18. 19.
Gluconeogenesis and NAD + in Kidney
Vol. 29, No. 12, 1981
(R. W. Hanson and M. A. Mehlman, eds.) p. 335-368, Wiley, New York (1976). A. MALEQUE, H. ENDOU, C. KOSEKI, and F. SAKAI, FEBS Lett. 116 154-156 (1980). H. B. BURCH, R. G. NARINS, C. CHU, S. FAGIOLI, S. CHOI, W. McCARTHY, and O. H. LOWRY, Am. J. Physiol. 235 F2~6-F253 (1978). S. KLAHR, Methods in Pharmacology (M. Martinez-Maldonado, ed.) vol. 4B, p. 325-349, Plenum Press, New York (1978). P. GREENGARD, G. P. QUINN, and M. B. REID, J. Biol. Chem. 239 1887-1892 (1964). R. L. BLAKE and E. KUN, Methods Enzymol. 18 113-123 (1971). M. KLINGENBERG, Methods of Enzymatic Analysis (H. U. Bergmeyer, ed.) 2nd edition, vol. 4, p. 2045-2059, Academic Press, New York (1974). P. GREENGARD, Methods of Enzymatic Analysis (H. U. Bergmeyer, ed.) p. 551-558, Academic Press, New York (1963). H. U. BERGMEYER, E. BERNT, F. SCHMIDT, and H. STORK, Methods of Enzymatic Analysis (H. U. Bergmeyer, ed.) 2nd edition, vol. 3, p. 1196-1201, Academic Press, New York (1974). S. KLAHR, and A. C. SCHOOLWERTH, Biochim. Biophys. Acta 279 157-162 (1972). J. J. COHEN, and D. E. K A Y , The Kidney (B. M. Brenner and F. C. Rector, eds.) 2nd edition, vol. I, p. 144-248, W. B. Saunders, Philadelphia (1981). N. W. DiTULLIO, C. E. BERKOFF, B. BLANK, V. KOSTOS, E. J. STACK, and H. L. SAUNDERS, Biochem. J. 138 387-394 (1974). M. WATFORD, P. VINAY, G. LEUMIEUX and A. GORGOUX, Canad. J. Biochem. 58 440-445 (1980). W. PFALLER, and M. RITTINGER, Int. J. Biochem. 12 17-22 (1980). T. P. DOUSA, Abstr. 5th Int. Workshop on Phosphate and Other Minerals, New York, NY, September, 1981, (in press) (1981). S. A. KEMPSON, T~e Physiologist, 24:(in press) (1981).
Pergamon Pres
RELATIONSHIP BETWEEN RATE OF GLUCONEOGENESIS AND CONTENT OF NICOTINAMIDE ADENINE DINUCLEOTIDE IN RENAL CORTEX S. Y. Lise Ou, Stephen A. Kempson and Thomas P. Dousa Mayo Clinic and Foundation, Department of Physiology, Division of Nephrology, Rochester, MN 55901
and
(Received in final form July 6, 1981) Summary Gluconeogenesis in rat renal cortex was measured using tissue slices incubated with or without appropriate substrates. Immediately after incubation the tissue slices were snap-frozen and the content of oxidized nicotinamide adenine dinucleotide (NAD +) was determined. Incubation with 10 mM a-ketoglutarate or L-glutamate led to enhanced glucose production and an increase in tissue content of NAD +. Quinolinate and 3-mercaptopicolinate inhibited the rate of gluconeogenesis from L-glutamate and ~-ketoglutarate respectively, and decreased the tissue levels of NAD +. The enhanced rate of gluconeogenesis was associated with an increase of NAD+ in the cytosol fraction (105 x g supernatant) but not in the particnlate fraction (105 x g pellet) of renal cortex homogenate. Present results indicate that NAD+ content changes in parallel with the rate of gluconeogenesis in renal cortical tissue. In our recent studies we observed that nicotinamide adeninine dinucleotide (NAD) I inhibits sodium-dependent uptake of phosphate (Pi) by renal brush border membrane (BBM) in vitro (I), and that increased in NAD content of renal cortical tissue in response to injection of nicotinamide in vivo is associated with a decreased rate of BBM uptake of Pi and with phosphaturia (I). Based on these studies we proposed a hypothesis that NAD, in particular the oxidized form (NAD+), in cytoplasm could regulate transport of Pi across BBM in renal proximal tubules (1,2). Although diverse metabolic processes occur in the cytoplasm of renal tubular cells, gluconeogenesis appears to be of particular importance for determining the level of cytoplasmic NAD+ (3,4). Since NADH is oxidized to NAD+ at the glyceraldehyde-3 phosphate dehydrogenase (GADP) step of gluconeogenesis (Fig. I), an increase of the rate of gluconeogenesis by supplying substrates which enter this pathway prior to the pbosphoenolpyruvate carboxy-kinase (PEP-CK) step (Fig. I) may be predicted to increase the level of cytoplasmic NAD+ (3,4). Recent studies have shown that both gluconeogenesis per s__ee(5) as well as key gluconeogenic enzymes (6) are
I Abbreviations used in this paper: NAD, nicotinamide adenine dinucleotide; NAD +, oxidized form of NAD; Pi, phosphate; PEP-CK, phosphoenolpyruvate carboxy kinase; GAPD, glyceraldehyde-3-phosphate dehydrogense; KRB, Krebs Ringer phosphate buffer; BBM, brush border membrane. located uniquely in proximal tubules but not in glomeruli or in the more distal tubular segments of the nephron. 0024-3025/81/121195-08502,00/0 Copyright (c) 1981 Pergamon Press Ltd.
1196
Gluconeogenesis
and NAD + in Kidney
Vol. 29, No.
12, 1981
It is of notable interest that many hormonal and metabolic stimuli of ~luconeogenesis in proximal tubules including parathyroid hormone, starvation, acidosis, and glucocorticoids (2,3), are also known to cause phosphaturia bv inhibition of Pi reabsorption across proximal tubular BBM. Therefore, it is very important to establish whether an increased rate of gluconeogenesis indeed causes an increase in tissue NAD +. While the increase in tissue NAD + after stimulation of gluconeogenesis may be expected (Fig. I), no direct evidence for this phenomenon is available. In the present study we address this question by investigating the relationship between the rate of gluconeogenesis in rat renal cortical slices and the content of NAD + in the same slices. The results of these experiments indeed indicate that the rate of gluconeogenesis may influence the content of NAD + in renal cortical tissue.
r
FRUCTOSEI~ISPHOSPHATE
FRUCTOSE
GLYCERALDIEHYDE3-P
"~ GLYCEROL
1.3-BISPHOSPHOGLYCERATE
t
GDP+CO2
'"O,..OE"OERO ......... GTP
CYTOSOL
7L.CT.TE
....,'
CO
'
PYRUVATE
~DRION v
"--GLUTAMATE
[oo,,o ...... ]
FIG.
I
Schematic outline of gluconeogenic pathway . Note oxidation of NADH to NAD + at the GAPD step in cytoplasm, points of entry of ~-ketoglutarate and L-glutamate, and points of action ( ~ ) of inhibitors (in square brackets). Methods Adult male Sprague-Dawley rats weighing 200-250 g were allowed free access to food and drinking water. They were stunned, the kidneys removed and decapsulated and immediately chilled in Krebs-Ringer phosphate buffer (KRB). The kidneys from several animals were pooled together and slices of cortex were randomly distributed to incubation flasks for simultaneous incubations with and without added substrates and inhibitors.
Vol. 29, No.
12, 1981
Gluconeogenesis
and NAD + in Kidney
1197
Gluconeogenesis was ~etermined basically bv the method described by Klahr (7). Cortical tissue slices (0.5 mm thickness) were prepared by Stadie-Riggs hand-operated tissue slicer and were distributed to incubation flasks containing KRB of the following composition: 127.2 mM NaC1; 4.0 mM KC1; I ~M CaC12; 1.2 mM MKC12; 2.h mM NaH2PO h (pH 7.4). Slices were first preincubated for 60 minutes at 37°C in order to deplete the tissue of endogenous gluconeogenic substrates. The slices (60-80 mg wet weight) were then transferred into 10 ml eresh KRB in Erlenmeyer flasks with or without added substrates and inhibitors as indicated in the Results. In control incubations without substrate, sodium chloride was added in equimolar concentration. Slices were then incubated at 37°C for 60 min in a Dubnoff metabolic shaker bath while 1004 oxygen was continuously bubbled through individual flasks. At the beginning and at the end of the incubation period samples of the incubation medium were taken for determination of glucose content. At the end of the incubation the slices were blotted dry, frozen rapidly in liquid N 2 and a percholoric acid extract was prepared as described previously (I) for determination of NAD + and ATP. In studies on the subcellular distribution of NAD +, fractionation was performed as described for liver tissue (8,9) and also used in our previous study on the kidney (I). Briefly, at the end of incubation slices were quickly removed from incubation medium and a I0~ (wt/vol) homogenate was prepared using 5 passes of a motor-driven glass-teflon homogenizer. The homogenization medium was 0.25 M sucrose, 5 mM Tris-HCl (pH 7.4) containing 50 mM nicotinamide to inhibit enzvmatic degradation of NAD + (8,9), Homogenate was then ultracentrifuged for 60 min at 105 x g, the resulting supernatant (cytosol fraction) and pellet (particulate fraction) were separated and extracted immediately with TCA (8). After centrifuging the TCA extracts to remove protein precipitate, the TCA was removed by several washes with three volumes of water-saturated ether (8). The pH of the washed extracts was adjusted to neutrality and the NAD + content was determined the same day. All steps in the fractionation and extraction procedure were performed at 0 ° 4oc. The assay for NAD + was based on the interconversion of lactate and pyruvate catalyzed by lactate dehydrogenase and requiring NAD (1,10). The final volume of the reaction mixture was 2.0 ml and contained 0.025 mM lactate, 5 mM hydrazine sulfate, 0 . 1 M glycine-NaOH (pH 10.0) and up to 0.1 ml of the acid extract or standard NAD + solution. The reaction was started by the addition of 0.005 ml of lactate dehydrogenase (3000 U/ml). The change in fluorescence was measured on an Aminco-Bowman spectrophotofluorometer with excitation wavelength 340 nm and emission wavelength 470 nm. The increase in fluorescence, after incubation for 30 min at 37 ° C, was linear in the concentration range 1-20 nmoles of NAD +. ATP content of cortical slices was determined from fluorimetric measurement of NADPH using the hexokinase and glucose-6-phosphate dehydrogenase method (1,11). The final reaction mixture (total volume 2.0 ml) contained 10 mM D-glucose, 5 u M NADP, 1.5 mM MgCI2, 0.6 mM EDTA, 12.5 mM triethanolamine HCI (pH 8.0) and up to 0.1 ml of the acid extract or standard ATP solution. The reaction was started by the addition of 0.01 ml of glucose-6-phosphate dehydrogenase (125 U/ml) and 0.03 ml of hexokinase (132 U/ml). After 15 min at 37 ° the increase in fluorescence (excitation at 365 nm, emission at 460 nm) was proportional to the amount of ATP in the range 0-15 nmoles. Samples of the incubation medium were deproteinized by the addition of perchloric aeid (final concentration 0.6 N), the extracts were neutralized with KOH, centrifuged and the glueose content of the clear supernatants was determined as described and used elsewhere (12,13).
I]98
Gluconeogenesis and NAD + in Kidney
Vol. 29, No.
12, 1981
All analyses were run in duplicates or triplicates, and all incubations and analyses (with or without added substrates or inhibitors) were carried out in parallel. The results were evaluated statistically by Student's t-test for group or paired comparisons as specified in the Results. Values of P ) 0.05 were considered not significant (NS). Results Renal cortical slices were incubated with and without addition of e-ketoglutarate or L-glutamate, renal gluconeogenic substrates (13,14), Incubation with G-ketoglutarate caused a marked increase in the glucose production and at the end of the incubation Deriod the tissue content of NAD + was significantly higher than in controls (Table I). Likewise, incubation with Lglutamate increased glucose accumulation, although to a lesser degree, and at the end of the incubation with L-glutamate the NAD + content in the slices was also significantly higher compared to controls (Table II). In contrast, no significant differences were found in levels of ATP measured in the same tissue extracts as NAD + (Tables I and II). TABLE I Stimulation of renal cortical gluconeo~enesis bv ~-ketoglutarate, and inhibition by 3-mercaptopicolinate; effect on tissue level of NAD +
Additions
(n)*
A.
None (control)
(5)
B.
10 mM -ketoglutarate
(5)
10 mM -ketoglutarate + 0.1 mM 3-mercaptopicolinate
Glucose formation (Anmol/K wet weight) 158.8 +
37.1t
1276.2 + 164.g
NAD + (nmol/g wet weight)
ATP (nmol/g wet weight)
142.8 + 23.8
546.7 +
6.97
208.0 + 22.4
581.4 + 99.3
180.3 + 26.6
539.7 + 85.0
C.
(5)
4~9.0 +
71.2
P-value** A<
~B
0.005
~ O.02g
NS
B<
~C
0.02
~ 0.01
NS
A<
~C
0.005
NS
NS
*number of experiments tmean + SEM **for significance of differences between the three experimental conditions (paired t-test) The increase in glucose production by incubation with ~-ketoglutarate was significantly inhibited by inclusion of 0.1 mM mercaptopicolinate (15) in the incubation medium. This inhibition of gluconeogenesis was associated with significant lowering of NAD + compared to incubation with ~-ketoglutarate alone; no significant differences were found in levels of ATP (Table I). When L-glutamate was used as a substrate, inclusion of 0.5 mM quinolinate (13) into the incubation mixture inhibited the rate of glucose production to levels in controls (no added substrate); the tissue content of NAD +, but not the content of ATP, was significantly lower than in preparations incubated with Lglutamate alone (Table II).
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Gluconeogenesis
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Table II Stimulation of Renal Cortical Gluconeogenesis by L-glutamate and Inhibition by Quinolinate; Effect on Tissue Level of NAD +
Additions
(n)*
Glucose formation ( Anmol/g wet weight)
NAD +
ATP
(nmol/g wet weight)
(nmol/g wet weight)
A.
none (control)
(4)
153.5 Z 47.4#
162.2 Z 15.0
539 Z 95
B.
10 mM L-glutamate
(4)
605.2 + 89.4
244.5 + 21.9
546 + 86
C.
10 mM (4) L-glutamate + 0.5 mM quinolinate
60.2 + 60.2
205.0 + 14.7
613 + 83
P-value** A ~
> B
( 0.05
( 0.02
NS
B.~/
> C
( 0.05
( 0.02
NS
A <
> C
NS
NS
NS
* number of experiments # mean + SEM ** significance of differences (paired t-test)
between the three experimental
conditions
Finally, the effect of the increasing rate of gluconeogenesis on NAD + in cytosolic and particulate fractions of renal cortical slices was examined. After incubation with L-glutamate, a portion of the tissue slices was taken for determination of whole tissue NAD + and another portion was fractionated and analyzed as described in the Methods. As in the experiment described in Table II, in the presence of 10 mM L-glutamate, glucose production markedly (6 x) increased and also total tissue NAD + content was significantly higher than in controls without substrate. In cytosolic fractions of homogenate the content of NAD + was more than 3 x higher compare~ to the corresponding particulate fraction (Fig. 2). In preparations from slices incubated with L-glutamate the content of NAD + in cytosol was significantly higher compared to controls, while no significant difference was found between NAD + contents in particulate fractions (Fig. 2). Discussion Since several phosphaturic stimuli are known to stimulate renal cortical gluconeogenesis (3), it was of major interest to establish whether an increase in the rate of renal gluconeogenesis leads to a parallel change in NAD + content in the renal cortex. As a first approach we changed the rate of gluconeogenesis simply by increasing the supply of gluconeogenic substrates which enter this pathway from the mitochondrial compartment (Fig. I). Therefore, both substrates enter the pathway prior to the initial PEP-CK step of gluconeogenesis ~nd also prior to the GAPD step, where NADH is oxidized (Fig. I). Also, both inhibitors used in our studies block renal gluconeogenesis by direct action on PEP-CK and/or precedin~ enzyme reactions (13,15).
1200
Gluconeogenesis
and NAD + in Kidney
I
I
-
12, 1981
10 s x g pellet
Cytosol 200
Vol. 29, No.
I
~ ~zz/
,//, ,/// ///,
NAD*
(, moll
,///
g w wt)
~"/4 ///~
100 r//. ///. -/// ///, ,z/¢ ///, ,//,
0
L-Glu
0
L-Glu
FIG. 2 Effect of increased rate of gluconeogenesis on NAD + content in cytosol (left panel) and in particulate fraction (105 x ~ pellet) (right panel) of homogenate (for details see Methods and Results). Each bar denotes mean + SEM of 6 incubations. Open bars: fractions from control slices incubated without (0) added substrate; shaded bars: fractions from slices incubated with 10 mM L-glutamate (L-GIu). * denotes value siKnificantly (P ( 0.01; group t-test) higher than control (0). Both in control tissue and tissue incubated with L-glutamate the NAD + content in cytoso] ~ significantly (P ( 0. '01; group t-test) higher than in the pellet.
~ccording to current evidence, 3-mercapcopicolinate inhibits gluconeogenesis by direct inhibition of PEP-CK (15) and quinolinate, in addition to inhibition of PEP-CK, also inhibits deamination of L-glutamate to ~-ketoglutarate (13). In any case, both compounds inhibit gluconeogenesis at the steps prior to NAD + generation (Fig. I). Present observations, taken together, are thus compatible with the interpretation that when the rate of ~luconeogenesis is increased (by increased availability of substrates) or decreased (by inhibition at the steps prior to formation of phosphenolpyruvate) there is an increase or decrease in parallel in the tissue NAD + level, most likely by changing the rate of oxidation of NADH to NAD + at the GADP step. The absence of changes in levels of ATP documents that the rate of gluconeogenesis may markedly change without detectable depletion of tissue ATP, which is consumed in this metabolic pathway, and also that changes in NAD + are relatively specific for this nucleotide. In renal cortex, as in other tissues (8,9), the bulk of the cellular pool of NAD ÷ is present in cytoplasm. An observation that the increased rate of gluconeogenesis was associated with an increase in NAD + only in cytosol (Fig. 2) is compatible with the observation that in rat kidney gluconeogenesis is almost completely located in cytosol (16). In the context of the proposed regulation of BBM transport of Pi (1,2,3), it is important that the level of NAD + is changed in the cellular compartment that is in direct contact with BBM.
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Gluconeogenesis and NAD + in Kidney
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The limitations in interpretation of the present results should be realized. The content of NAP + in tissue slices preparation was lower than that measured in snap-frozen samples of kidney (I); this difference may be due to lesser oxygen supply to cells which are not perfused by blood. The rate of glucose production as well as levels of NAD + and of ATP were measured only in undivided renal cortical slices and it is not certain to which degree the observed changes reflect processes located specifically in the proximal tubular cells. Likewise, the rate of gluconeogenesis and activities of gluconeogenic enzymes although limited to proximal tubules (5,6) are not equally distributed in various subsegments, and the same could apply to the content and metabolism of NAD +. The above mentioned limitations in design of the present experiments also likely accounts for the lack of strict quantitative correlation between changes in the rate of gluconeogenesis and NAD + levels in the renal cortex. Exact localization, quantification and causality of the relationship between gluconeogenesis and NAD + generation in different specific subsegments of proximal tubules should be established in future studies. However, since the proximal tubules constitute about 50% of renal cortical mass in the rat kidney (17), it is very likely that the presence and directionality of changes in cortical slices reflect processes in proximal tubules rather than in other structures of kidney cortex. While in the present study, the rate of gluconeogenesis was changed by supply of substrate and/or by inhibitors, our most recent observations indicate that similar relationship between NAD + and gluconeogenesis also exist in vivo. Administration of glucocorticoids (18), or metabolic acidosis induced by ammonium chloride load (19) caused in parallel an increase in the rate of renal gluconeogenesis and enhancement of NAD + content in renal tissue. In conclusion, the present results provide to our knowledge the first evidence that by changing the rate of gluconeogenesis in renal cortex,.tissue NAD + content changes in a parallel manner. In general terms, our findings are in support of the hypothesis that phosphaturic stimuli decrease renal Pi reabsorption by increasing renal gluconeogenesis in proximal tubules, leading to increased cytoplasmic NAP + which inhibits Na+-dependent Pi transport across the luminal BBM (1,2,3). Acknowledgments This work was supported by USPHS grants AM-16105 and AM-19715, grantin-aid (MHA-36) from the Minnesota Heart Association and by funds from the Mayo Foundation. S. A. Kempson's current address is Renal-Electrolyte Division, University of Pittsburgh, School of Medicine, Pittsburgh, PA. We thank Bonnie Becker for excellent secretarial assistance. We are indebted to Dr. N. DiTullio from Smith, Kline and French Laboratories, Philadelphia, PA, for the supply of 3-mercaptopicolinate. Reprint requests should be directed to Dr. T. P. Dousa° References I. 2.
3. 4.
S. A. KEMPSON, G. COLON-OTERO, S. Y. L. OU, S. T. TURNER, and T. P. DOUSA, J. Clin. Invest. 67:1347-1360 (1981). T. P. DOUSA, and S. A. KEMPSON, Urolithiasis I Clinical and Basic Research (L. H. Smith, W. E. Robertson, and B. Finlayson, eds.) p. 741-745, Plenum Press, New York (1981). T. P. DOUSA, and S. A. KEMPSON, Mineral Electrolyte Metabolism, (in press) (1981). C. I. POGSON, I. D. LONGSHAW, A. ROOBOL, S. A. SMITH, AND G. A. O. ALLEYNE, Gluconeogenesis t Its Regulation in Mammalian Species
1202
5. 6. 7. 8. 9. 10.
11. 12.
13. 14.
15. 16. 17. 18. 19.
Gluconeogenesis and NAD + in Kidney
Vol. 29, No. 12, 1981
(R. W. Hanson and M. A. Mehlman, eds.) p. 335-368, Wiley, New York (1976). A. MALEQUE, H. ENDOU, C. KOSEKI, and F. SAKAI, FEBS Lett. 116 154-156 (1980). H. B. BURCH, R. G. NARINS, C. CHU, S. FAGIOLI, S. CHOI, W. McCARTHY, and O. H. LOWRY, Am. J. Physiol. 235 F2~6-F253 (1978). S. KLAHR, Methods in Pharmacology (M. Martinez-Maldonado, ed.) vol. 4B, p. 325-349, Plenum Press, New York (1978). P. GREENGARD, G. P. QUINN, and M. B. REID, J. Biol. Chem. 239 1887-1892 (1964). R. L. BLAKE and E. KUN, Methods Enzymol. 18 113-123 (1971). M. KLINGENBERG, Methods of Enzymatic Analysis (H. U. Bergmeyer, ed.) 2nd edition, vol. 4, p. 2045-2059, Academic Press, New York (1974). P. GREENGARD, Methods of Enzymatic Analysis (H. U. Bergmeyer, ed.) p. 551-558, Academic Press, New York (1963). H. U. BERGMEYER, E. BERNT, F. SCHMIDT, and H. STORK, Methods of Enzymatic Analysis (H. U. Bergmeyer, ed.) 2nd edition, vol. 3, p. 1196-1201, Academic Press, New York (1974). S. KLAHR, and A. C. SCHOOLWERTH, Biochim. Biophys. Acta 279 157-162 (1972). J. J. COHEN, and D. E. K A Y , The Kidney (B. M. Brenner and F. C. Rector, eds.) 2nd edition, vol. I, p. 144-248, W. B. Saunders, Philadelphia (1981). N. W. DiTULLIO, C. E. BERKOFF, B. BLANK, V. KOSTOS, E. J. STACK, and H. L. SAUNDERS, Biochem. J. 138 387-394 (1974). M. WATFORD, P. VINAY, G. LEUMIEUX and A. GORGOUX, Canad. J. Biochem. 58 440-445 (1980). W. PFALLER, and M. RITTINGER, Int. J. Biochem. 12 17-22 (1980). T. P. DOUSA, Abstr. 5th Int. Workshop on Phosphate and Other Minerals, New York, NY, September, 1981, (in press) (1981). S. A. KEMPSON, T~e Physiologist, 24:(in press) (1981).