The effect of quinolinic acid on the content and distribution of hepatic metabolites

ABCHIWS

OF BIOCHEMISTBY

AND

BIOPHYSICS

The Effect of Quinolinic

164,

%%-601

(1974)

Acid on the Content

of Hepatic 0. S. SPYDERVOLD,

Department

of Biochemistry,

Biochemistry,

and Distribution

Metabolites

l NAJMA ZAHEER-BAQUER, AND A. L. GREENBAUM

PATRICIA

MCLEAN,

University College London, London WClE 6BT, and the Courtauld Middlesex Hospital Medical School, London WlP 5PR, England

Znstitute of

Received March 4, 1974 The effect of quinolinic acid treatment on the hepatic metabolite profile and the flux of glucose through the alternative pathways of metabolism have been measured, and the distribution of metabolites between the cytosolic and mitochondrial compartments has been calculated. Marked increases of the total-cell poiycarboxylic anions were found and these were, in order of magnitude: malate, citrate, isocitrate, aspartate, 2-oxoglutarate, and glutamate. Calculation of the compartmented values suggested that the major increase was in the mitochondrial compartment: cytosolic glutamate, 2-oxoglutarate, and oxaloacetate were decreased and only aspartate increased in this compartment. The changes of the mitochondrial/cytosolic anion ratio was most marked, 69fold, in the case of 2-oxoglutarate. It is suggested that inhibition of transport of 2-oxoglutarate by quinolinic acid could, by blocking the operation of the aspartate shuttle, contribute to the inhibition of gluconeogenesis from lactate. Metabolite and flux data suggest an increase in the rate of lipogenesis in quinolinic acid-treated rats with the decrease of long-chain acyl CoAs, caused by this treatment, being the possible effector for this activation.

Quinolinic acid has been shown to cause an inhibition of gluconeogenesis in the isolated perfused liver, the site of which has been postulated to be at the level of PEPCK2 (l-3). It also causes a massive accumulation of the intermediates of the 2-0x0tricarboxylic acid cycle (citrate, glutarate, malate, and aspartate) in the perfused liver system or in the livers of treated rats whether they had been fed or prestarved before treatment (1,4) although the effect was considerably magnified in the starving animals (4). Recent studies (5-7) have emphasized the importance of an understanding of the intracellular distribution of such metabolites in the interpretation of metabolic control phenomena, such as crossover

plots, and it seemed relevant in the present context to examine the effect of quinolinic acid on the distribution of metabolites between the cytosol and mitochondria with particular reference to malate, oxaloacetate, aspartate, glutamate, and 2-oxoglutarate all of which could play a significant role in gluconeogenesis and in the operation of the malate-aspartate shuttle (8). It has also been reported (1) that the quinolinic acid inhibition of gluconeogenesis could be reversed by the administration of Mn2+. In view of the fact that quinolinic acid readily chelates Mn2+ and that PEPCK is a Mn2+-activated enzyme, the possibility exists that the inhibition of this enzyme is related to the sequestration of the metal ion, and it was therefore appro‘Present address: Department of Medical Bio- priate to make measurements of the activity of other pathways dependent on the chemistry, University of Oslo, Oslo, Norway. *Abbreviation: PEPCK = GTP:oxaloacetate car- presence of Mn2+, such as lipogenesis, and boxy-lyase (transphosphorylating) (EC 4.1.1.32). of the flux of carbons through glycolysis 590 Copyright All riplhts

Q 1074 by Academic Press, of reproducticm in any f
Inc. reerved.

QUINOLINATE

AND METABOLITE

and the pentose phosphate pathway, to determine whether the changes also occurred in these related systems. MATERIALS

AND

METHODS

Animals. Adult male rats (body weight 160-180 g) of the Wistar strain were used and were maintained on stock diet No. 41B. The animals were starved 16 hr and then injected intraperitoneally with either saline or sodium quinolinate (50 mg/lOO g body weight). They were killed 1 hr later by cervical dislocation and the livers rapidly removed either for freeze-clamping or the preparation of tissue slices or homogenates. Metabolite assays and flux measurements. Metabolites were measured on neutralized perchloric acid extracts by established procedures (9, IO). Xylulose 5-phosphate was estimated by the method of Kauffman et al. (11). The procedures for the preparation of slices and the estimation of radioactive fluxes have been previously described (12) except that total lipids were extracted by the method of Bligh and Dyer (13). Details of the incubation procedures are included with the appropriate Tables. Values are reported as the means l SEM. RESULTS

The tissue content of metabolic intermediates in the livers of control and quinolinic acid-treated rats are presented in Table I together with the calculated values for the redox state of the cytosolic nicotinamide nucleotide couples and ATP/ADP x P, ratio and the mitochondrial NAD+/NADH ratio. Veneziale et al. (1) reported that quinolinate caused increases in the tissue content of malate, oxaloacetate, and aspartate and very marked decreases of phosphoenolpyruvate, 2- and 3-phosphyglycerate. The present data differ in that no evidence was found for a rise of oxaloacetate and the decline of the phosphorylated derivatives was less pronounced. It should be noted, however, that a strict comparison between the present results and those of Veneziale et al. (1) is not entirely valid since the preparations and time of starvation were different, and no glucogenic substrate was presented to the animals in the experiments reported here. A more direct comparison can be made between the present results and those of Williamson et al. (4) who also used rapid freezing techniques to study changes in the livers of intact rats

DISTRIBUTION

591

treated with quinolinic acid. The metabolite profile reported here is more nearly akin to that reported by Williamson et al. (4) for their fed animals than that obtained from rats starved for 48 hr, where the changes were qualitatively similar but quantitatively more marked, although some differences, notably in the redox state of the NAD couples, are apparent. The present results extend the data previously available to include a number of metabolites which permit the calculation of the intracellular distribution of the polycarboxylic acids and also include values for intermediates of the pentose phosphate pathway and of some CoA derivatives. Of the intermediates of the pentose phosphate pathway, only xylulose 5-phosphate is statistically different in the quinolinic acid-treated rats, although it should be noted that there is a considerable increase in the sedoheptulose 7-phosphate content also. These relative changes are important in two contexts. First, the fall in the tissue level of xylulose 5-phosphate is reflected in a change of the cytosolic NADPH/NADP+ couple (calculated from the tissue level of 6-phosphogluconate, xylulose &phosphate, and K,.,. GlcAdehydrog.) which increases from 96 to 120, in line with the more reduced state of the cytosolic NAD+/NADH couple. It may also be observed from Table I that when the NADPH/NADP+,s ratio is calculated from the whole cell content of the reactants of the malic enzyme reaction, ratios are obtained which are widely different from those found with the 6-phosphogluconate dehydrogenase reaction, which points to the fact that the considerable changes in total cell malate are not necessarily related to the changes of malatec, which is the component involved in calculating the redox state from the reactants of the malic enzyme (see below). Second, the tissue levels of xylulose &phosphate and sedoheptulose 7-phosphate change in opposite directions. It is possible to interpret these changes as indicating an increased activity of the transketolase reaction, the ’ The subscripts c and m refer to the cytosolic mitochondrial compartments.

and

592

SPYDERVOLD ET AL.

rate-limiting step in the nonoxidative reac- of formation of W02 from [I-“Clglucose tions of the pentose phosphate pathway, - [6-Wlglucose (a first approximation to particularly in view of the fact that the rate the activity of the pentose phosphate pathTABLE I CONTENT OF METABOLIC INTERMEDUTES IN THE LIVERS OF NORMAL AND QUINOLINIC ACID-TREATED RATSO

Control

juinolinic acid treated

~in&n&acid (o/o)

ATP ADP AMP NAD+ NADP+ Glucose l-phosphate Glucose g-phosphate Fructose 6-phosphate Fructose 1,6-bisphosphate Dihydroxyacetone phosphate Glyceraldehyde phosphate a-Glycerolphosphate 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate Pyruvate Lactate Acetyl CoA Free CoA Long-chain acyl CoA Oxaloacetate Citrate Isocitrate 2-Oxoglutarate Malate NH,+ Glutamate Aspartate Acetoacetate @Hydroxybutyrate 6-Phosphogluconate Xylulose Sphosphate Sedoheptulose 7-phosphate P, NAD+ c [K, lactate dehydrogenase] NADH NAD+ m [K&3-hydroxybutyrate NADH

dehydrogenase]

NAD+ m [K., glutamate dehydrogenase] NADH NADPH c [Keq 6-P-GlcA dehydrogenase] NADP+ NADPH c [K,, malic enzyme] NADP+

2325 zt 45 988 f 44 278 zt 38 636 A 20 58.6 zt 4.21 17.66 + 0.96 271 f 20 70.3 zt 6.8 11.08 + 1.10 9.33 f 0.70 3.43 zt 0.46 166 f 12 338 zt 34 39.4 f 2.09 87.1 zt 6.61 90 zt 6.3 1004 f 64 33.6 + 1.34 122 f 17 106 + 16 4.65 + 0.56 375 f 21 27.9 f 1.31 121 f 6.8 455 + 24 378 + 30 2470 zt 223 918 + 88 86.5 zt 12.0 220 zt 25.0 10.8 + 0.94 22.3 l 0.88 49.6 i 11.64 3679 zt 83 935 11.1 4.90

2219 zt 5.6 1233 zt 84 398 zt 44 694 * 28 52.0 zt 3.01 13.59 f 1.73 260 f 23 64.6 zt 5.81 11.5 zt 2.34 8.26 zt 0.96 3.36 i 0.48 116 + 16.9 253 zt 29 37.5 f 3.40 74.5 f 4.75 82.4 zt 8.0 1285 zt 64 20.7 zt 2.48 88 i 10.5 88 & 9.8 3.27 zt 0.57 920 * 75 52.8 zt 3.16 184 zt 11.0 1141 l 92 523 + 44 3291 * 370 1736 +z110 98 zt 14.9 228 ziz20.0 10.3 zt 0.69 17.1 zt 0.85 65.4 * 12.48 3721 zt 133 676 8.1 7.56

96

120

150

411

95 123’: 143* 109 89 77* 96 92 104 89 98 70* 75 95 86 92 128** 60** 72 84 88 245** 189** 152** 251** 138** 133 189** 113 103 95 77** 132 101

QUINOLINATE

AND METABOLITE

593

DISTBIBUTION

TABLE I-Continued Control

Quirr;~J~acid

Quinc$&acid 6)

ATP ADPXP,

c [Calc. glyceraldehyde 3-P dehydrogenasel 3-phosphoglycerate kinase]

ATP ___ c [Measured] ADP x P, Acetyl CoA lMeasured] Free CoA &Hydroxybutyrate x 2-Oxoglutarate x NH,+ Acetoacetate x Glutamate

813

640

640

484

0.275 (MM1

Williamson et al. (4)

0.235

47 x 10-z

68 x 10-s

63 x 1O-s

100 x 10-B

Fed rats

* P less than 0.05; ** P less than 0.01. n Expressed as nanomoles per gram wet weight of tissue. Values given as means * SEM and represent the means of not less than eight experiments.

way) is unchanged in quinolinic acid-treated rats (Table II). It should be noted, however, that direct comparison of in uiuo and in vitro data assumes that the effect of injected quinolinic acid persists in uitro. Table I also shows significant changes in the levels of the adenine nucleotides and CoA derivatives. Treatment with quinolinic acid has little effect on the ATP content but raises both the ADP and AMP contents. These changes are reflected in the ATP/ADP x P, ratio, calculated either directly from the measured contents of these compounds or from the reactants and combined equilibrium constant of the glyceraldehyde 3-phosphate dehydrogenasel 3-phosphoglycerate kinase reaction. The changes in the CoA derivatives are largely centered on the fall of acetyl CoA although there is also a large, but variable, fall in the free CoA of liver, which is not statistically significant. The most striking differences shown in Table I are, however, the changes in tissue level of the intermediates of the tricarboxylic acid cycle. The huge rises of these compounds after quinolinic acid treatment is entirely in line with similar changes already reported by Veneziale et al. (1) and by Williamson et al. (4). In view of the considerable importance of many of these compounds in metabolic processes, an attempt has been made to calculate the

intracellular distribution of these intermediates. The results of these calculations are shown in Table III. The distribution of oxaloacetate and malute. This has been calculated by two

independent methods (6, 7) since a knowledge of the distribution of these two metabolites is an essential preliminary to further steps in the calculation. The two procedures yield results which are, essentially, in good agreement, except that the Williamson (6) procedure (Table III, Method 2) gives a somewhat lower value for the cytosolic oxaloacetate in quinolinic acid-treated rats. There is little change in the cytosolic content of malate but the mitochondrial content of the two metabolites is increased some 4-5-fold. The agreement is a point of some importance in view of the reported effect of Mn2+ in reversing the quinolinic acid effect (1) since Method 1 uses the equilibration of enzymes subject to metal activation, in particular malic enzyme requires Mn2+, while the second method doe%not. The distribution mate, 2-onoglutarate,

aspartate, glutaand citrate. The cal-

of

culated intracellular distribution of these anions is also given in Table III. This table shows: (1) that aspartate is mainly located in the cytosol and, quantitatively, the larger fraction of the increased aspartate resulting from quinolinic acid treatment

594

SPYDERVOLD ET AL. TABLE II

CONVERSION OF SPECIFICALLY LABELED SUBSTRATESINTO %O, AND “C-LIPID AND QUINOLINIC ACID-TREATED RAT@

Control “CO, Production (ag atoms substrate carbon x g-’ x hr-‘) Activity of pentose phosphate pathway [1-“C]Glucose (14) 2.67 + 0.17 [1-“C]Glucose + Phenazine methosulfate (7) 8.11 f 0.87 [6-l% ]Glucose (14) 1.69 ct 0.11 Activity of pyruvate dehydrogenase [l-l% ]Pyruvate (20 mM) (11) 57.4 f 3.4 [1-“CJPyruvate (5 mM) (5) 21.9 zt 2.3 Activity of tricarboxylic acid cycle [2-“CJPyruvate (11) 17.8 f 0.9 [1-“C]Acetate (11) 15.2 zt 0.8 2- [5-“C]Oxoglutarate + glucose (5) 4.04 A 0.36 (l-“C]Glutamate + glucose (5) 3.88 zt 0.29

BY LIVER SLICES TOM CONTROL

Quinolinic acid treated

Significance

2.68 + 0.26 9.11 f 1.01 1.54 l 0.09 59.8 + 2.7 22.6 + 2.6 16.0 i 0.8 14.2 •t 0.3 4.15 f 0.39 4.12 + 0.27

“C-Lipid formation (Kg atoms substrate carbon x g-’ x hr-I) Activity of lipogenic pathway [l-‘C IGlucose (14) [6-l% IGlucose (14) [l-l% jPyruvate (11) [2-“C jPyruvate (11) (1-“C]Acetate (10) 2- [5-“Cl Oxoglutarate + glucose (5) Ratio of pathways [2-Y!jPyruvate (11) [l-“C JPyruvate

0.93 zt 0.06 1.06 f 0.11 0.34 f 0.03 2.81 l 0.31 2.32 zt 0.19 0.43 zt 0.06

1.16 + 0.06 1.45 f 0.09 0.39 f 0.03 3.67 zt 0.18 3.36 i 0.29 0.35 f 0.05

** **

7.81 * 0.49

9.96 zt 0.69

**

2.46 +z0.10

2.43 i 0.08

* **

“Liver slices (500 mg) were incubated with 4.5 ml Krebs-Ringer bicarbonate medium (gas phase OJCO,, 95/5) containing 0.5 &i “C-labeled substrate. The final substrate concentration was 20 mM except for 2-oxoglutarate and glutamate which were used at 5 mM in the presence of 5 mM glucose as described by Madsen et al. (38, 39). Phenazine methosulfate was present at 0.1 mM final concentration. Pyruvate, 20 and 5 mM final concentrations, were used to approximate to pyruvate in the fully activated and basal states (15). The time of incubation was 1 hr except for experiments with 5 mM pyruvate when 30-min incubation and 206 mg of slices were used to prevent exhaustion of substrate. Values are given as means + SEM; * indicates Pvalues < 0.05, ** Pvalues < 0.01. Where no symbol is given, there are no significant differences. Figures in parentheses are the number of observations.

remains in this compartment, although it should be noted that the mitochondrial aspartate increases 6-7-fold; (2) that, while in control animals the bulk of the glutamate is in the cytosol, quinolinic acid treatment reverses this pattern. Although quinolinic acid only increases the total cell glutamate by about 30%, the mitochondrial component increases by about 900%; (3) the 2-oxoglutarate pattern follows that of glutamate, i.e., a predominantly cyto-

solic location is transformed to one heavily weighted to the intramitochondrial compartment; (4) the approximately even distribution of citrate in control animals is replaced by a pattern in which the citrate is almost exclusively mitochondrial. Thus, the overall effect of quinolinic acid is to cause an accumulation of anions in the mitochondria and to raise the m/c quotient markedly, the greatest increases being for 2-oxoglutarate and citrate.

QUINOLINATE

AND METABOLITE

TABLE III THE INTRACELLULAR DISTRIBUTION OF SOME HEPATIC METABOLITES IN NORMAL AND QUINOLIMC ACID-TREATED RATS’

Control

Quinolinic acid treated

Distribution of malate and oxaloacetate Method 1 (Based on: Malate, Pyruvate, x 6-Phosphogluconate x K,.,.,,,, dewmx. = Rib-5-P x Mali, enzyme ’ Malate, Malate, Malate, m/c gradient Oxaloacetate, Oxaloacetate, m/c gradient

= Malate, 275 181 5.9 6.16 0.04 0.05

- Malate,) 313 828 24 5.02 0.20 0.36

DISTRIBUTION

595

lites in the two compartments, given in Table III, it is possible to construct a crossover analysis of the intermediates of the gluconeogenic pathway; this is presented in Fig. 1. From this figure it is apparent that no crossover occurs at either pyruvate carboxylase or at PEPCK. The values presented here would imply that if it is possible to represent the activity of the translocase system of the mitochondrial membrane by the concentration of the substrates on each side of the membrane, then the concentration of malate on each side of the membrane might be interpreted as a block of translocation under the influence of quinolinic acid in that the cytosolic malate does not follow the increased mitochondrial content. Effect of quinolinic acid on enzymes involved in the calculation procedures. The

effect of quinolinic acid on the activity of a number of enzymes, assumed to be equilib’ rium enzymes in the calculation proceoxaloacetate+, Malat%, and K., Malate dehydrodures, was measured in rats treated with genase quinolinic acid to ensure that the activities were not so modified by the treatment as to Malate, 205 189 Malate, 251 952 disqualify them from use in the calculam/c gradient 11.0 45 tions. In all cases the activities of the Oxaloacetate, 4.59 3.04 enzymes remained unchanged. The enOxaloacetate, 0.05 0.23 zymes investigated were: malate dehydrom/c gradient 0.11 0.68 genase (EC 1.1.1.37), malic enzyme (EC Distribution of aspartate, glutamate, 2-oxoglutarate, 1.1.1.40), isocitrate dehydrogenase (EC and citrate by Method 1 1.1.1.42), glutamate-oxaloacetate transamAspakate, 915 1716 inase (EC 2.6.1.1.), glutamate-pyruvate Aspartate, 3.2 20.6 transaminase (EC 2.6.1.2), and 6-phosphom/c gradient 0.03 0.11 Glutamate, 2174 626 gluconate dehydrogenase. In addition, the Glutamate,,, 296 2665 following enzymes were also measured and m/c gradient 1.2 38 found to be unchanged: phosphofructoki2-Oxoglutarate, 96.8 12.1 nase (EC 2.7.1.11), citrate cleavage en2-Oxoglutarate, 24.2 172 zyme (EC 4.1.3.8), fructose bisphosphatase m/c gradient 2.2 128 (EC 3.1.3.11), transaldolase (EC 2.2.1.2), Citrate, 180 28 transketolase (EC 2.2.1.1.), glyceraldehyde Citrate, 224 738 3-phosphate dehydrogenase (EC 1.2.1.12), m/c gradient 11 238 glucokinase (EC 2.7.1.2), all three isoenOValues given in this table are tissue contents as zymes of hexokinase (EC 2.7.1.1), and nanomoles x g-l. The m/c gradient represents the glucose 6-phosphate dehydrogenase. NAD+ NAD’ Method 2 (Based on: -CC,-----m NADH NADH

ratio of the concentrations in the mitochondrial and cytosolic compartments. For details of Method 1, see Ref. (7); for Method 2, see Ref. (6).

Crossover analysis using compartmented values of hepatic metabolites. Using the

calculated values for the hepatic metabo-

Activity of alternative pathways of glucose utilization and of lipogenesis in quinolinic acid-treated rats. If quinolinic acid

treatment leads to a chelation of metal ions such as Mn2+, required for the activity of acetyl CoA carboxylase (EC 6.4.1.2), and an inhibition of citrate efflux, a source of

596

SPYDERVOLD ET AL. GLUCOSE

PVRUVATE

te

4 GAP

1

NAD*

I:

NADH;--ew--.-q-

$yT:”

t . . . .

PVRUVATE _ &-LACTATE

: CEC

f

. . . . :G.LrT: \’

1 NADPH

. . . . : sG:

LIPID

t2;7:. . . . :O.A*f

:

k

OAA

~UVATE

GLi! . . . .

‘;$DJl

DAA

\ JA

‘?A

?A 2-OG *G,-

m

?!a

=I-

ASP

.&;{I+~)

ACETVL Co*

Y

1 ICT NADH A

II NAD+

MALI

piiJ-

P pig

2

FIG. 1. Relationship of changes in the content of cell metabolites to the pathways of gluconeogenesis and lipogenesis and postulated sites of action of quinolinic acid. The solid black square represents the mitochondrial membrane. Those metabolites enclosed in dots are decreased in content, those enclosed in solid lines are increased in content after treatment with quinolinic acid. Possible loci of quinolinic acid (QA) action are marked by the asterisks.

substrate for acetyl CoA carboxylase, from the mitochondria, it might be anticipated that such treatment would also result in changes in the rate of fatty acid synthesis. Measurements were, therefore, made of the flux of carbons in the pathways of carbohydrate metabolism and through the lipogenie system. The results of these experiments are shown in Table II. The data in this Table show that the rate of lipogenesis from either [1-14C] or [6WZ]glucose, [1-14C]acetate, and [214C]pyruvate is increased, while that from 2- [5- l*C loxoglutarate remains unchanged. The incorporation of [l-“Clpyruvate into lipid is also unchanged. The incorporation of [1-“C]pyruvate into fat must be into the glycerol moiety of the triglycerides and this occurs via the gluconeogenic route. It is therefore noteworthy that this process is unchanged by quinolinic acid treatment. This point is made all the more significant by the observation that, from the ratio of [2-‘“C]pyruvate/ [l-‘“C ]pyruvate incorporation into the total lipid fraction, a differential effect on the route of glycerol and the route to long-chain fatty acids exists. It is also of interest that the ratio of incorpora-

tion of [2-14C]pyruvate/[6-14C]glucose into lipid is unchanged by quinolinic acid treatment. The incorporation of the carbon-6 of glucose into lipid involves the flux through the glycolytic pathway in addition to the common reactions shared with pyruvate. Thus, the coordination between the glycolytic flux and the lipogenic route is clearly maintained, and it is the relative rates of lipogenesis versus gluconeogenesis which are showing, as in so many cases of hormonal and dietary modification, a reciprocal relationship. The most marked stimulation of lipogenesis is observed when acetate is the substrate, when the activity in quinolinic acid-treated rats is 45% greater than in the controls. The increase in lipogenic rate from glucose is of the order of about 30% and it is probable that this is not a sufficiently powerful drain on the NADPH availability to cause a statistically significant rise of the pentose phosphate pathway. If it is calculated that an extra 0.4 I.cmoles of glucose carbon (~0.8 Gmoles of acetate) are incorporated x ggl x hr-’ in the quinolinic acid-treated rats (Table II), then the NADPH requirement could be

QUINOLINATE

AND METABOLITE

met by the oxidation of as little as 0.26 pmoles of glucose in the pentose phosphate pathway and even less if part of the reducing equivalents were supplied via the malic enzyme. This figure, only 10% of the total [l- ‘“C jglucose oxidation, is probably too low to show statistically. No significant changes were found in pyruvate dehydrogenase activity, measured with excess substrate in the fully activated form, or with 5 mm pyruvate, where differences in the active and inactive forms may be revealed (14).

DISTRIBUTION

597

tion of the alternative system. Berry and Kun (18) have suggested that transfer processes between mitochondrial and cytosolic compartments must be considered as potential rate-limiting and regulatory sites for gluconeogenesis. If the activity of the translocase system of the mitochondrial membrane were represented by the concentration of the substrates on each side of the membrane, then it should be reasonable to consider the distribution of the metabolites in terms of the crossover theorem (19, 20). From the calculated mitochondrial and cytosolic DISCUSSION contents of malate, it would appear that, in The inhibitory effect of quinolinic acid quinolinic acid-treated rats, a very considon gluconeogenesis has been attributed to erable increase in the miotchondrial conan inhibition of PEPCK. This conclusion tent is not accompanied by an equivalent was based on an apparent crossover point rise in the cytosol, i.e., that there is a less at this enzyme when whole-cell values for ready transfer of malate from the mitometabolic intermediates were used (l-3). chondria. This block is only partial since Further, Ballard and Hopgood (15) have re- sufficient transfer occurs in the quinolinic ported both an increased synthesis and acid-treated rats to sustain a normal cytodecreased breakdown of PEPCK after solic concentration of this metabolite tryptophan treatment, an observation con- (Table III). On the basis of these calculated sistent with the report by Foster et al. (2) of compartmented values there is no evidence an anomolous high level of this enzyme in for an inhibition of PEPCK, which would liver after treatment of the rat with trypto- require an increase of the cytosolic oxalphan. oacetate in the quinolinic acid-treated rats. The inhibition of malate transport and The present study considers the compartmental values of the relevant metabo- oxidation is clearly associated with delites and suggests an additional explana- creased gluconeogenesis, as shown by intion for the action of quinolinic acid. hibitors such as butylmalonate, fluoromalGluconeogenesis in the intact animal ate, and d-malate all of which have been involves the formation of glucose from two used to study the contribution of this main carbon precursors, lactate and amino system to gluconeogenesis (18, 21, 22). acids, e.g., alanine, with the transfer of Williamson et al. (21) have shown that carbon skeletons in the former case and gluconeogenesis from pyruvate or lactate is carbon skeletons and reducing equivalents inhibited some 50-80% by butylmalonate, in the latter case, from the mitochondrial an inhibitor of malate transfer across the compartment to the cytosol. Two schemes mitochondrial membrane, and it could are generally accepted for such transfers. therefore be expected that a comparison of In the case of lactate the main pathway is the changes in the concentration of methe aspartate shuttle while, for the more tabolites resulting from quinolinic acid or oxidized substrates, malate translocation butylmalonate treatment could throw light serves to transfer both carbons and reduc- on the possible locus of action of quinolinic ing equivalents (8, 16, 17). It has been acid on transport systems. When reducing postulated (18), from studies using fluoro- equivalents are available, then both inhibidicarboxylic acids, that both shuttles are tors cause a rise in total-cell malate, asparoperative, each working close to maximal tate, and citrate and in the mitochondrial capacity, and that, further, neither shuttle malate content. It may be inferred from seems to be able to compensate for inhibithese parallel changes that malate trans-

598

SPYDERVOLD ET AL.

port mechanisms may be altered in both conditions. A point of contrast is seen in the total-cell 2-oxoglutarate, which falls on butylmalonate treatment but rises after quinolinic acid treatment and which, in the latter condition, has a particularly high m/c ratio (changed from 2.3 in control rats to 128 in quinolinic acid-treated rats), which focuses attention on the transport of 2-oxoglutarate, or on an inhibition of the cytosolic formation of 2-oxoglutarate (via an Mn2+-dependent reaction) as a site of action of quinolinic acid. The inhibition of 2-oxoglutarate transfer, as revealed by the calculated compartmented values, may be postulated to have the following consequences which are consistent with the exerimental findings and other calculated values. 1. The low level of cytosolic 2-oxoglutarate acts to limit the rate at which cytosolic oxaloacetate is generated by the transamination of aspartate with 2-oxoglutarate. That such a reduction of transamination does occur is shown (a) by the high level of aspartate which accumulates; (b) by the low level of cytosolic glutamate, possibly due to the depressed transamination; and (c) by a lowered level of cytosolic oxaloacetate, the second product of the transamination reaction. 2. The increased level of mitochondrial 2-oxoglutarate provides one substrate for mitochondrial transamination reactions, which could account for the high level of mitochondrial glutamate. 3. The elevated mitochondrial content of glutamate and oxaloacetate would favor an increased mitochondrial content of aspartate (see Table III). It must be presumed that there is no additional impediment to the transport of aspartate in quinolinic acid-treated rats as calculation shows that the cytosolic aspartate is also raised, and Veneziale et al. (1) reported that tryptophan, which has an action analogous to that of quinolinic acid, raised the blood aspartate. 4. The raised oxaloacetate concentration of the mitochondrial compartment in quinolinic acid-treated rats (control 0.4 PM; quinolinic acid treatment, 2.0 PM) could well serve to prime the tricarboxylic

acid cycle and to raise the concentration of citrate since the K, of hepatic citrate synthase is 2.1 PM (23). On the basis of these considerations, it is proposed that a primarychange after quinolinic acid treatment is a modification of the transport of 2-oxoglutarate across the mitochondrial membrane. The consequent lowering of the cytosolic oxaloacetate concentration from 4.6 PM, already at a critical value with respect to the K, for PEPCK [26 PM (24)], to 3.0 PM could contribute significantly to an inhibition of gluconeogenesis at the level of the PEPCK reaction. Another possible mechanism which would explain the above data would be an increased entry of glutamate into the mitochondria (thus sparking the accumulation of polycarboxylic anions, via the glutamate dehydrogenase reaction, and the decrease of cytosolic 2-oxoglutarate and citrate via isocitrate dehydrogenase) but this is thought to be less likely than that suggested above as the most pronounced effect observed is on 2-oxoglutarate distribution. Other factors which could contribute to the decrease of gluconeogenesis at the site of PEPCK are, firstly, the decrease in the ratio ATP/ADP x Pi (Table I) in quinolinic acid-treated rats which, if paralleled by similar alterations in the GTP/GDP x Pi quotient, would provide less favorable conditions for the synthesis of phosphoenolpyruvate from oxaloacetate and, secondly, the known property of quinolinic acid to act as a chelating agent for divalent metal ions. The reversal of the inhibitory effect of quinolinic acid on gluconeogenesis by stoichiometric amounts of MnZ+ is, indeed, a powerful argument for a primary site of action of quinolinic acid on PEPCK. The possibility that Fe*+, rather than Mn2+, might be the physiologically important cation was suggested by Snoke et al. (3). This is an attractive alternative to Mn2+ since there exists the apparently paradoxical situation that two opposing cytosolic systems are both activated by Mn*+ (and, presumably, inhibited by its removal by chelation), i.e., the key enzymes of gluconeogenesis (PEPCK and fructose bisphosphatase) and the lipogenic enzymes (NADP-linked isocitrate dehy-

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drogenase, malic enzyme, and acetyl CoA carboxylase), and it is difficult to envisage the overall lowering of cytosolic Mn2+ as having opposite effects on manganeseactivated cytosolic enzymes. This does not rule out the possibility of differential degrees of inhibition related to the different K, values for Mn*+ among the gluconeogenie and fatty acid-synthesizing enzymes. That a possible locus of action of Mn2+ is on translocation mechanisms is apparent from the experiments of Harris and Berent (25), which showed a marked accumulation of 2-oxoglutarate in mitochondria exposed to Ca*+ and Mn2+, and of Friedman and Rasmussen (26), who found an increased level of malate, from lactate, in perfused livers after the addition of Mn2+. Thus, the removal of this ion by chelation could be expected to lead to a decrease of anion movement across the mitochondrial membrane. The present calculations imply a marked depression of 2-oxoglutarate movement and a lesser, but still positive, effect on malate efflux. The consequences of such changes in anion movement would be a severe disruption of the aspartate shuttle, which depends on 2-oxoglutarate movement, and a smaller effect on the malate shuttle. It is now well established, by the use of specific inhibitors, that when pyruvate is the gluconeogenic substrate, the efflux of malate is important as the means of carrying both carbon and reducing equivalents from the mitochondria. But, when lactate is the substrate, the reducing equivalents are, of necessity, already located in the cytosol (via lactate dehydrogenase) and the aspartate shuttle is the main route. It is in this latter sequence that 2-oxoglutarate plays such a key role. A site of action of quinolinic acid on the translocation of 2oxoglutarate provides a reasonable explanation of the results of Ui et al. (27) and of Veneziale et al. (28). The former authors found that when the pair lactate and pyruvate (in the physiological ratio of 10: 1) was the substrate presented, i.e., an aspartate shuttle substrate, then tryptophan (precursor of quinolinic acid) caused a 94% inhibition of glucose formation, whereas when pyruvate alone was presented, that is

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a malate shuttle substrate, then the inhibition of glucose formation by quinolinic acid was only 40% (28, 29). The proposed site of action of quinolinic acid on these two pathways is shown in Fig. 1. Another aspect of the present work is the significant increase in the rate of lipogenesis from a variety of substrates (glucose, pyruvate and acetate) found in the quinolinic acid-treated rats. This is likely to be an underestimate of the magnitude of the effect since it is known that certain ATPdependent reactions are decreased in liver slices relative to intact liver (30). The present findings are in accord with the observation that quinolinic acid or tryptophan causes a threefold stimulation of lipogenesis in duo, with acetate as substrate (31). However, in contrast to the results of Sakurai et al. (31), no inhibition of pyruvate dehydrogenase was observed in the present experiments (Table II). The metabolite profile indicates a possible activation of acetyl CoA carboxylase in quinolinic acid-treated rats on the basis of a decrease in the total-cell acetyl CoA accompanied by an increased flux through the lipogenic pathway, acetyl CoA carboxylase being the rate-limiting enzyme of the sequence (32). In examining the metabolite profile for changes in effector molecules for this enzyme, two possibilities present themselves, citrate and long-chain acyl CoAs. The total cell citrate increases in quinolinic acid-treated rats and would, at first sight, appear to be a likely mediator for the activation of fatty acid synthesis, in keeping with the many studies on the role of this compound in the allosteric activation of the carboxylase and with the recent results of Goodridge (33) on the close association between the tissue level of citrate and the rate of lipogenesis. However, the calculated values found in the present study suggest that the cytosolic levels are decreased after quinolinic acid treatment and this would not be in accord with a role in the activation of lipogenesis. It may be noted here that it has been reported that when acetyl CoA carboxylase is measured under the appropriate conditions, e.g., in the presence of phospholipids (34) or in whole tissue homogenates (35),

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the activity of the enzyme is independent of the addition of citrate. Long-chain acyl CoA derivatives appear more likely agents for the acceleration of lipogenesis since they change in a direction consistent with a de-inhibition of the carboxylase and, as Goodridge (36) has shown, can act specifically and reversibly. There remains, however, the apparent anomaly of an increased rate of lipogenesis associated with a decreased cytosolic content of citrate, the precursor of acetyl CoA. An explanation for this may be advanced as follows: since, in quinolinic acid-treated rats, the calculated cytosolic citrate (30 FM) has fallen to approximately half the K, of citrate cleavage enzyme [70 PM (37)], relative to a value of twice the K, found in control rats, it may be anticipated that the effective activity of the cleavage enzyme will be reduced to about one-quarter of the V measured in uitro, i.e., it would be approximately 0.3 units x g-‘, a value still considerably greater than the maximal potential activity of acetyl CoA carboxylase in the tissue (approximately 0.1 units x g- ‘). Thus, in spite of some depletion of available cytosolic citrate, an adequate amount is available to sustain an enhanced rate of lipogenesis. ACKNOWLEDGMENTS This work was supported by grants from the Wellcome Trust, the British Diabetic Association, and the Medical Research Council. O.S.S. was in receipt of a grant from the British Council.

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