Eur. J. Biochem. 88,467.-473 (1978)
Alanine Metabolism in Rat Liver Mitochondria Peter DIETERLE, Fritz BRAWAND, Ulrich K. MOSER, and Paul WALTER Department of Biochemistry, University of Basel (Received January 27, 1978)
In the presence of 2-oxoglutarate, bicarbonate, Pi and ATP, rat liver mitochondria were found to metabolize alanine. The main products were glutamate, aspartate, malate and citrate. Pyruvate did not accumulate but reached a low steady-state concentration. Addition of octanoate caused a strong stimulation of alanine metabolism and a concomitant decrease in pyruvate concentration. No inhibition of alanine metabolism was obtained in the presence of inhibitors of mitochondrial pyruvate transport, a-cyano-4-hydroxy-cinnamate and a-cyano-cinnamate, showing that alanine was not transaminated by a contamination of the mitochondria by cytosolic alanine aminotransferase. In the presence of L-cycloserine, alanine metabolism in mitochondria was strongly inhibited indicating that in contrast to earlier reports L-cycloserine can enter the mitochondria. In isolated liver cells, addition of a-cyano-cinnamate caused a strong pyruvate accumulation and a nearly complete inhibition of gluconeogenesis with serine as substrate, whereas with alanine no pyruvate increase and a weak inhibition of gluconeogenesis was observed. From a comparison of the employed substrate concentrations and of the observed rates of alanine transamination in intact mitochondria with those under physiological conditions in cellular systems, it is concluded that cellular conversion of alanine to pyruvate most likely occurs to a great extent within the mitochondria as has been proposed earlier by DeRosa and Swick (1975) J . Biol. Chem. 250,7961 - 7967.
Several research groups [I - 51 have demonstrated the existence of at least two alanine aminotransferases in rat liver. In the most recent publication, DeRosa and Swick [5] have reported a total activity of 34 pmol . min-' . g liver-' of which 10- 18% were found in the mitochondrial matrix space whereas most of the rest was cytosolic. The same research group reported for the purified pig mitochondrial enzyme a K , value for alanine of 1.9 mM [5] and for a crude enzyme from rat liver mitochondria a K, value of about 12 mM [4]. The corresponding K, value for alanine of the cytosolic enzyme of rat liver was shown to amount to 42 mM by Swick et al. [4] and to 34 mM by Hopper and Segal[3]. Since the intracellular alanine concentration in rat liver amounts to about 1.5 mM [6,7], DeRosa and Swick [ 5 ] proposed that the main function of the mitochondrial alanine aminotransferase in vivo is to form pyruvate from alanine whereas the cytosolic enzyme may mainly be Enzymes. Alanine aminotransferase or L-alanine :2-oxoglutarate aminotransferase (EC 2.6.1.2); pyruvate carboxylase (EC 6.4.1.1); pyruvate dehydrogenase or rnultienzyme complex of pyruvate dehydrogenase or pyruvate :lipoate oxidoreductase (acceptor-acetylating) (EC 1.2.4.1).
involved in the reverse reaction. If this proposal is correct, the activity of the mitochondrial alanine aminotransferase should be high enough to convert all the alanine needed for gluconeogenesis. This seems to be the case as the mitochondrial enzyme activity measured by DeRosa and Swick of 3.4-6.2pmol . min-' . g liver-' is several-fold higher than the maximal conversion rates of 10 mM alanine to glucose which for perfused livers of fasted rats were reported to amount to 0.9-1.3 pmol . min-' . g liver-' [7,8]. Further support for the proposal of DeRosa and Swick comes from the work of Mendes-Mourao et al. [9] who found with liver cells that a-cyano-4-hydroxycinnamate, an inhibitor of mitochondrial pyruvate transport, exerted only a weak inhibition on gluconeogenesis from alanine whereas glucose production from serine and lactate was inhibited by 78 and 92% respectively. In this paper, the conversion of alanine to pyruvate and subsequently to the products of pyruvate metabolism in intact mitochondria was investigated. The results provide additional support for the proposed role of the mitochondrial alanine aminotransferase in gluconeogenesis. Some of the results were presented at a recent meeting [lo].
Alanine Metabolism in Rat Liver Mitochondria
468
MATERIALS AND METHODS Rat liver mitochondria were isolated according to Johnson and Lardy [ l l ] in 0.25 M mannitol/ 0.07 M sucrose. Mitochondria were suspended in the same medium at a concentration of 1 g original liver weight per ml. The basic incubation mixture in all experiments was: 4 mM ATP, 10 mM MgS04, 12 mM potassium phosphate buffer pH 7.4, 18 mM KHC03, 6.7 mM triethanolamine buffer pH 7.4 and substrates as indicated in the legends. Mannitol/sucrose was added to make the reaction mixture isotonic. All samples were gassed with a mixture of C02 ( 5 % ) and Oz (95%): The final volume was 3 ml. Mitochondria were incubated in 25-ml stoppered conical flasks in a shaking water bath at 37 "C. After an equilibration time of 2.5 min, the reactions were started by addition of 0.5 ml of the mitochondria1 suspension. The reactions were stopped by adding 1 m11.25 M perchloric acid. The samples were neutralized to pH 3.5 with potassium phosphate and the precipitated salt was centrifuged off. The protein and perchlorate precipitates were rinsed with water, centrifuged off and the washing solutions were combined with the respective supernatant samples. Parenchymal cells from rat liver were prepared by a slight modification (121 of the methods of Berry and Friend [13] and Seglen [14,15]. The incubation medium consisted of 5.4 mM KC1, 0.8 mM MgS04, 0.8 mM NaHzP04, 25 mM NatIC03 and 1.3 m M CaCI2. Final volume was 3.0ml containing 5-10 . lo6 cells. Incubations were carried out in 25-ml conical flasks which were gassed with 0 2 and C02 (95/5, v/v), stoppered and shaken at 37 "C for the times indicated. The reactions were stopped by the same method as described for the mitochondria. Pyruvate, citrate, malate, 2-oxoglutarate were measured as described previously [16]. Standard enzymatic assays were used for the determination of alanine [I 71, glutamate [18], aspartate [19] and glucose [201. Male albino Wistar rats from the Swiss Vitamine Institute in Basel were used. The average weight was 180 - 230 g. Enzymes and coenzymes were purchased from Boehringer Mannheim GmbH (Mannheim, Germany), ~-[l-'~C]alanine was from New England Nuclear Chemicals (Dreieich, Germany) and cc-cyano4-hydroxy-cinnamate was from Ega Chemie (Steinheim, Germany). a-Cyano-cinnamate was a gift of Dr Halestrap (Bristol). L-Cycloserine was prepared by A. Furst (F. Hoffmann-La Roche, Basel). All other reagents were of the highest purity commercially obtainable. The results with mitochondria are expressed on the basis of 60 mg mitochondrial protein which is about equivalent to 1 g wet weight of liver [21]. For
l6
t
2o 2 0
-
._ 16
-
7c
i /
/
/ /
a
0
E
l
3
6 9 Tlme of incubation (rnin) Time
12
Fig. 1. Tinie tlcyridw?c,c,ofnzitoc.hondria/ ulaflrric metabolism. Mitochondria were incubated in the basic medium. The concentration of added alanine and 2-oxoglutarate was 4 mM. 16 mg mitochondrial protein were present in each incubation. (W) Akdnine used; (0) glutamate found; (A) aspartate found; (0) malate found; (A) citrate found; ( 0 )pyruvate found
liver cells, the results are expressed per g of wet weight liver corresponding to 125 . lo6 cells [12]. All results are averages of two incubations with the same mitochondrial or cellular preparation.
RESULTS The time dependence of alanine metabolism in rat liver mitochondria in the presence of 2-oxoglutarate, bicarbonate, ATP and inorganic phosphate is shown in Fig. 1. Glutamate, citrate, malate and aspartate but not pyruvate increased with time during 12 min. In the absence of 2-oxoglutarate, no alanine utilization could be observed (not shown) indicating that transamination of alanine to pyruvate by alanine aminotransferase is the first step. Pyruvate did not accumulate but reached a steady-state concentration of 0.2-0.3 mM which corresponds to about its level in the liver cell [21,22]. As is known from earlier experiments with similarly low pyruvate concentrations [22,23], about equal amounts of pyruvate are carboxy-
P. Dieterle, F. Hrawand, U. K. Moser, and P. Walter
469
Table 1. Effict Of'arsenite and vctanoafr on ilir nwrahoii,sm of pyruvate and aianine The basic medium was used (see Materials and Methods ). Each incubation (3 ml) contained 14.5 mg mitochondria1 protein. In all experiments with alanine, 4 m M 2-oxoglutarate was also present. Negative values represent amounts used, positive values amounts found. n.d. = not determined Additions
Metabolites -.
~-
pyruvate
. ~
~
alanine
~~~
malate
citrate
aspartate
~
-~
~~~
-
glutamate
2-oxoglutaratc
< 0.2 < 0.2
~ < 0.2 < 0.2 < 0.2
n.d. n.d. n.d.
pmol 60 mg protein-' . 10 min-' ~
10 mM Pyruvate 10 mM Pyruvate + 0.4 m M arsenite 10 mM Pyruvate 0.8 m M octanoate 10 m M Pyruvate 0.4 mM arsenite 0.8 inM octanoate 4 m M L-Alanine 4 mM L-Alanine 0.4 mM arsenite 4 mM L-Alanine 0.8 mM octanoate 4 m M i.-Alanine + 0.4 mM arsenite 0.8 mM octanoate
+
63.2 - 0.4 - 59.5 -
+ +
-
+ +
+
30.2 2.9 5.3 1.7
+
~~~~
< 0.2
14.5 5.8 0.8 6.6
< 0.2
-
10.7 13.2 0.8 16.9
< 0.2
10.7 6.6 16.9
1.7 0.4 2.1
8.3 2.5 13.2
-
13.6
4.1
7.0
1.7
9.5
-
+
~
17.4 0.2 17.4
lated to oxyloacetate and oxidized to acetyl-CoA. It was furthermore shown that part of the oxaloacetate formed is either metabolized to citrate by citrate synthase, to malate by malate dehydrogenase or to aspartate by transamination with glutamate [23 - 271. In the experiment in Fig. 1, similar pathways of pyruvate metabolism can be anticipated. However, it should be noted that not all the malate, citrate and aspartate found at the end of the incubation are derived solely from pyruvate but were in part also formed from the added 2-oxoglutarate. The sum of aspartate plus glutamate was as expected equivalent to the amount of alanine used in the experiment of Fig. 1. In Table 1, an experiment is shown in which pyruvate conversion to acetyl-CoA was inhibited by addition of arsenite. Because pyruvate carboxylase requires acetyl-CoA as an allosteric activator [28], octanoate had to be added to provide sufficient acetylCoA [29]. Arsenite also inhibits the oxidation of 2oxoglutarate. Therefore, in the presence of arsenite and octanoate, all the citrate, malate and aspartate formed must originate from pyruvate carboxylation. This was confirmed in the experiment of Table 1 where the sum of pyruvate malate aspartate citrate (14.9 pmol) was about equal to the amount of alanine used (13.6 pmol). The small difference of 1.3 pmol is most likely the result of an incomplete inhibition by arsenite. As in the experiment of Fig. 1, the sums of glutamate plus aspartate should correspond to the amount of alanine used. However, in the incubations with arsenite, some amino equivalents were not accounted for. It may be that under these conditions some glutamate was deaminated to 2oxoglutarate. In the experiments with alanine, pyruvate was continuously formed by transmination. Upon addi-
+
_
< 0.2
10.7 0.6 18.2
~
2.1
_
~~~
< 0.2 < 0.2 < 0.2
~~
n.d. - 27.7 - 1.2 - 31.8 -
15.6
tion of octanoate, the total pyruvate metabolized (equal to alanine used - pyruvate found) increased by about 100"/,from7.8- 15.2 pmol . 60 mgprotein-' . 10 inin-'. At the same time, a drop of the pyruvate concentration in the incubation medium from 0.23 0.1 3 mM was observed (Table 1, see also Table 2). More direct evidence on the effect of octanoate comes from the experiment with ~,-[l-'~C]alanine in Table 2. 14C02 is split off either in the conversion of pyruvate to acetyl-CoA or in the two decarboxylation steps of the sequence isocitrate- 2-oxoglutarate - succinylCoA. The results of Table 2 show that in the absence of octanoate, 8.7 pmol alanine . 60 mg protein-' . 8 min-' were metabolized. In the presence of octanoate, maximally 0.2 pino1 of alanine were decarboxylated and therefore at least 95% or 12.5 pmol of the pyruvate formed from alanine were carboxylated. It is therefore obvious that octanoate strongly stimulated the carboxylation of pyruvate. When in the presence of arsenite and octanoate increasing amounts of alanine and 2-oxoglutarate were added, a corresponding acceleration of alanine conversion was observed (Fig.2). It should be noted that at the physiological concentration of about 1 1.5 mM alanine and 2-oxoglutarate approximately 0.6-0.8 pmol of alanine . min-' . 60 mg protein-' whichisequivalent to0.6-0.8 pmol . min-' . gliver-' was converted to gluconeogenic precursors. Since the greater part of the alanine aminotransferase is cytosolic it could be argued that a cytosolic contamination of the mitochondria by this enzyme was responsible for the transamination of alanine in our incubations. The experiments in Table 3 clearly show that the transamination occurred inside the mitochondria because the inhibitors of pyruvate transport 4-hydroxy-a-cyano-cinnamate and a-cyano-cinnamate
Alanine Metabolism in Rat Liver Mitochondria
470
Table 2. Effect of octanoate on I4CO2production from ~ - [ l - ' ~ C ] a l a n i n e All incubations were carried out in the basic medium including 4 mM 2-oxoglutarate. 1 pCi of ~-[l-'~C]alanine and 17 mg mitochondrial protein were added to each incubation. After the incubations perichloric acid was added and the I4CO2 was trapped in hyamine as described earlier [47]. Negative values represent amounts used, positive values amounts found Metabolites
Additions
pyruvate 4 min
alanine 8 min
14c02
4 min
8 min
4 min
8 min
pmol . 60 mg protein-' ~.
~~
4 mM ~-[I-'~C]Alanine
1.6
1.6
- 5.6
-
8.7
0.6
2.3
4 mM ~ - [ l - ~ ~ C ] A l a n+ i n0.8 e mM octanoate
0.2
0.3
- 7.3
-
13.0
0.1
0.2
Table 3. Effect of inhibitors ofpyruvate transport on the metabolism of pyruvate and alanine in mitochondria For conditions see Table 1. 13.5 mg of mitochondrial protein were added to each incubation
I
'.;c 10.0 W
c
P Q
Additions
r
Metabolites
z5-
E 2
pyruvate
alanine
0
5.0-
pmol . 60 mg protein-'
a ul,
.-0
malate aspartate + citrate + glutamate 10 min-'
c
n
2 2.5-
f 04
0
21 3
2
3
4
[L-alanine] and [2-oxoglutarate] (mM)
Fig. 2. Mitochonclriul metabolism of ulmine in the presence ofarsenite and octanoate at varying alanine concentrations. 2-Oxoglutarate was added at the same concentration as alanine. Mitochondria were incubated in the basic medium. Incubation time was 7 min. 17 mg of mitochondrial protein were present in each incubation. (m) Alanine used; (0) glutamate found; (A) aspartate found; (A) citrate found; (0)malate found; (0)pyruvate found; (+) represents the sum of malate, citrate, aspartate and pyruvate
[30] had very little effect on alanine metabolism whereas the conversion of added pyruvate was completely inhibited. By using a-cyano-cinnamate in whole liver cells (Fig. 3), we could confirm the findings of MendesMourao et al. [9] that gluconeogenesis from serine was inhibited by 86 % whereas glucose production
10 mM Pyruvate - 69.6 10 mM Pyruvate + 0.1 mM a-cyano- 3.5 cinnamate 10 mM Pyruvate +lmM 4-hydroxy-acyano- 4.4 cinnamate 4 mM Alanine 2.2 4 mM Alanine + 0.1 mM a-cyanocinnamate 2.2 4 mM Alanine +lmM 4-hydroxy-acyano-cinnamate 2.2
n.d.
31.9
n.d.
n.d.
1.7
n.d.
n.d. - 17.7
1.7 18.2
n.d. 15.1
- 16.0
19.9
13.7
- 15.5
19.5
13.3
from alanine was affected by only 23%. If the rates of gluconeogenesis are corrected for endogenous glucose formation, no inhibition was observed with alanine as compared to 89% with serine. In addition we found that in the presence of alanine the inhibitor caused no increase in pyruvate whereas with serine a 5-fold accumulation of pyruvate occurred. These results support the concept that there is enough mitochondrial alanine aminotransferase activity for the conversion of alanine to glucose in liver cells. The effects of L-cycloserine (an inhibitor of some transaminating enzymes [31]) on mitochondrial meta-
P. Dieterle, F. Brawand, U. K. Moser, and P. Walter
411
transferase was much more sensitive towards L-CYC~Oserine than aspartate aminotransferase because with 5 mM of the inhibitor only alanine formation was inhibited whereas aspartate production was not decreased. DISCUSSION
0'
I
I
,
I
The purpose of this paper is to provide additional evidence for the proposal of DeRosa and Swick [5] that transamination of alanine to pyruvate predominantly takes place within the mitochondria and not in the cytosol. The results show that between 11.7 pmol alanine . min-' .60 mg protein were metabolized when alanine and 2-oxoglutarate were added at 4 mM each. Addition of octanoate caused a strong stimulation of alanine utilization and a concomitant decrease of the pyruvate concentration. Despite the lower pyruvate level, an increase in the rate of pyruvate carboxylation was observed. This stimulation is most likely the result of an increase of the ATP :ADP ratio [23,24] or (and) of the increased concentration of acetyl-CoA [29]. It is furthermore known from the isolated pyruvate carboxylase that acetyl-CoA not only increases V but under certain conditions also lowers the K , for pyruvate [ 3 2 ] .The increase in acetylCoA may therefore also be responsible for the observed decrease in pyruvate concentration. This mitochondrial system may serve as an experimental model for the situation observed with isolated livers perfused with alanine where also a lowering of the pyruvate concentration with a concomitant increase in the rate of gluconeogenesis was observed in the presence of either fatty acids or glucagon [7,33]. In support of the concept of an intramitochondrial metabolism of alanine, a-cyano-cinnamate had little effect on alanine conversion in isolated mitochondria or cells. However, addition of L-cycloserine to the mitochondrial incubations caused a strong inhibition of alanine aminotransferase but not of aspartate aminotransferase. This inhibitory effect was unexpected because Williamson et al. [34] had stated (without giving results) that cycloserine did not penetrate mitochondria. The finding that only alanine aminotransferase was inhibited by cycloserine is in agreement with work by Azarkh et al. [31] who showed that in liver the sensitivity of alanine aminotransferase towards L-cycloserine was more than 100-times higher than that of aspartate aminotransferase. It therefore appears from our results that L-cycloserine enters the mitochondria in concentrations high enough to inhibit alanine aminotransferase but not aspartate amino transferase. In order to evaluate the physiological importance of the reported findings, the cellular concentrations of 2-oxoglutarate and alanine have to be considered. For 2-oxoglutarate, liver contents between 250 and
'
30 c
L
.-
cn
3
a" '
O
0
0
j
0.2
0.1
[ a-Cyano-cinnarnic
0.3 acid] (rnM)
0.4
0.5
Fig. 3. Effec,/s of'a-cq.ario-cinnamic acid on ~lueoneogenesis/l.omakanine and serine in isolazed liver cells. 6.7 . lo6 parenchymal cells from rat liver were incubated in the medium described under Methods. cr-Cyano-cinnamic acid was added at the beginning, whereas addition of the substrates serine and alanine occurred after 20 min of incubation. The reactions were stopped after 80 min. Glucose and pyruvate were measured as described under Methods. (0) Control; (B) substrate 5 mM alanine; (0)substrate 5 mM serine
Table 4 Efject of r-cycioseritze on nztrumitochondrral alanme aminotranyferase and aspartate nminotramjerase Foi conditions see Table 1 16 5 mg of mitochondria1 protein were added to each incubation Additions
substrates
Metabolites L-cycloserine alanine glutamate aspartate mM
pmol . 60 mg protein-' . I 0 min-'
4 mM L-Glutamate + 4 mM pyruvate 5
4 mM ~-Alanine
+ 4mM
2-oxoglutarate
4.0 0.7
-
14.9 12.0
9.8 10.5
-
-
7.3
6.2
0.7
0.5
- 0.4
< 0.2
< 0.2
bolism are shown in Table 4. In the experiment with alanine and 2-oxoglutarate, 0.5 mM L-cycloserine almost completely inhibited the mitochondrial alanine aminotransferase. The results of the incubations with pyruvate and glutamate show that the alanine amino-
412
2800 pmol . g dry weight-' have been reported [22, 35 -441. In recent years, various fractionating procedures have made it possible to determine the approximate compartmentation of various metabolites in the liver cell. For 2-oxoglutarate, concentrations for the mitochondria between 0.5 and 3.2mM and for the cytosol between 0.1 and 1.2mM have been found [22,41- 441. The mitochondriallcytosolic gradients varied between 1.8- 19.3 depending on the fractionating method employed and the physiological state of the cells [41-451. For alanine, cellular contents of 0.46-2.3 pmol . g liver wet weight-' have been measured under various metabolic conditions [6,7,37, 39,461. Tischler et al. [45] furthermore reported a small mitochondrial/cytosolic gradient of 1.5, however, no statistical evaluation and no information on the alanine concentrations in the' two compartments were given. The K, values of alanine aminotransferase in pig liver are 0.42 mM for 2-oxoglutarate and 1.9 mM for alanine [ 5 ] . If these values are also. valid for tlic rat liver enzyme, the conclusion can be drawn that the physiological concentrations of the two substrates are in the range of the K, values. At about physiological concentrations (1 1.5 mM) of 2-oxoglutarate and alanine, we obtained rates of alanine transamination of 0.6 - 0.8 pmol . min-' . 60 mg protein-' (Fig. 2) in our incubations with isolated mitochondria. For comparison, the perfusion experiments of Malette et al. [7] with liver from fed rats and ['4C]alanine are of special interest. At a concentration of 0.45 niM alanine in the perfusate (corresponding to the physiological plasma level in the fed animal), an intracellular alanine concentration of 1.7 mM and an incorporation rate of 3.5 pmol of [I4C]alanine into glucose . h-' . 100 g body weight-' was observed. On the basis of 5 g liver per 100 g rat, this rate is equivalent to 0.01 pmol alanine . min-' . g liver-'. After addition of glucagon, the intracellular alanine concentration dropped by about 30 % whereas the rate augmented to 0.03 and the total alanine utilization was 0.07 pmol alanine . min-l . g liver-'. At non-physiological concentrations of 9 mM alanine, the intracellular concentration amounted to more than 17 mM and the rate of ahnine incorporation was increased to 0.2 pmol . min-' . g liver-'. In perfused livers of fasted animals, higher rates of gluconeogenesis of up to 0.65 pmol of glucose . min-l . g liver-' [8] with 10 mM alanine have been reported. To our knowledge nobody has measured gluconeogenesis at physiological levels of alanine in liver of fasted rats. It is interesting to note, however, that the intracellular level of alanine in the liver of fasted animals is about 3-times lower than in the fed state [6]. From these results it can be concluded that our rates observed with isolated mitochondria are more than sufficient to cover the physiological requirement of the transamination rate of alanine to pyruvate.
Alanine Metabolism in Rat Liver Mitochondria Thiswork was supported by grants of the Swiss National Science Foundation. The excellent technical assistance of Miss L. Lehmann and Miss S. Studer is gratefully acknowledged.
REFERENCES 1 . Katunuma, N., Mikumo, K . , Matsuda, M. & Okada, M. (1962) J . Vitaminol. (Kyoto) 8,68-73. 2. Pakeda, Y., Ichihara, A,, Tanioka, H. & Inoue, H. (1964) J. Biol. Chem. 23Y, 3590-3596. 3. Hopper, S. & Segal, H. L. (1964) Arch. Biochem. Biophys. 105, 501 - 505. 4. Swick, R. W., Barnstein, P. L. & Stange, J. L. (1965) J . Biol. Chem. 240,3334- 3340. 5. DeRosa, G. & Swick, R . W. (1975) J . B i d . Chem. 250, 7961 7967. 6. Williamson, D. H., Lopes-Vieira, 0. & Walker, B. (1967) Biochem. J . 104,497- 502. 7. Mallette, L. E., Exton, J. H. &Park, C. R. (1969) J . B i d . Chem. 244, 5713-5723. 8. Ross, B. D., Hems, R. & Krebs, H. A. (1967) Biochem. J . 102, 942 - 951. 9. Mendes-Mourao, J., Halestrap, A. P., Crisp, D. M. & Pogson, C. I. (1975) FEBS Lett. 53, 29-32. 10. Dieterle, P., Brawand, F., Moser, U. & Walter, P. (1977) Abstr. Commun. 11th M w t . Fed. Eur. Biochem. Soc. A 1-31051. 31. Johnson, D. & Lardy, H. A. (1967) Methods Enzymol. 10, 94-96. 12. Walter, P., Nyfeler, F., Fasel, P., von Glutz, G., Moser, U. K. & DeSagana, M. R. (1976) in Use of Isolated Liver Cells and Kidney Tubules in Metabolic Studies (Tager, J. M., Soling, M. D. & Williamson, J. R., eds) pp. 167- 174, North Holland, Amsterdam. 13. Berry, M. N. & Friend, D . S. (1969) J . Cell. Biol. 43, 506-520. 14. Seglen, P. 0. (1972) Exp. Cell Res. 74, 450-454. 15. Seglen, P. 0. (1973) Exp. Cell Res. 76, 25-30. 16. Walter, P.&Stucki, J. W.(1970) Eur.J. Biochem. 12,508-519. 17. Bergmeyer, H. U. (1974) Methoden der Enzymatischen Analysen, pp. 1724- 1727, Verlag Chemie, Weinheim. 18. Bergmeyer, H. U. (1974) Methoden der Enzymatischen Analysen, pp. 1749- 1753, Verlag Chemie, Weinheim. 19. Bergmeyer, H. U . (1974) Methoden der Enzymatischen Analysen, pp. 1741 - 1745, Verlag Chemie, Weinheim. 20. Bergmeyer, H. U . (1974) Methoden der Eniymatischen Analysen, pp. 1241- 1246, Verlag Chemie, Weinheim. 21. Hohorst, M . S., Kreutz, F. H. & Biicher, T. (1959) Biochem. Z. 332, 18 - 46. 22. Siess, A . E., Brocks, D. G., Lattke, H. K. & Wieland, 0. H. (1977) Biochem. J . 166,225 - 235. 23. Walter, P. & Stucki, J. W . (1971) in Regulation oj' Gluconeogenesis (Soling, H. D. & Willms, B., eds) pp. 50- 62, ThiemeVerlag, Stuttgart. 24. Walter, P., Morikofer, S. & Brawand, F. (1974) in Regulation o/ Hepatic Metabolism, Alfred Benzon Symposium (Lundquist, F.&Tygstrup, F.,eds)vol. 6,pp. 79-91, Munksgaard, Copenhagen. 25. Walter, P., Paetkau, V. & Ldrdy, H. A. (1966) J . B i d . Chem. 241, 2523 - 2532. 26. Stucki, J. W. &Walter, P. (1972) Eur. J . Biochem. 30, 60-72. 27. Walter, P. (1976) in Gluconeogenesis, i t s Regulation in Mammalian Species (Hanson, R. W. & Mehlman, M. A,, eds) pp. 239-265, Wiley, New York. 28. Utter, M . F. & Keech, D. B. (1963) J . Bid. Chem. 238, 26032608. 29. Von Glutz, G. &Walter, P. (1976) FEBS Lett. 72, 299-303. 30. Halestrap, A . P. & Denton, R. M. (1975) Biochem. J . 148,97106.
P. Dieterle, F. Brawand, U. K . Moser, and P. Walter 31. Azarkh, R. M., Brdunstein, A. E., Paskhina, T . S. &Ting-Seng, H. (1960) Bioclzemistr,y (Moscow),25, 741 -748. 32. Seufert, D., Herlemann, E. M., Albrecht, E. & Seubert, W. (1 971) Hoppe-Se$er’.y Z. Physiol. Chem. 352, 459-478. 33. Williamson, J. R., Browning, E. T. & Scholz, R. (1969) J . Biol. Chem. 244,4607-4616. 34. Williamson, J. R., Meijer, A. J. &Ohkawa, K. (1974) inRcJgulation of Hepatic Metabolism (Lundquist, F. & Tygstrup, N., eds) pp. 457-479, Munksgaard, Copenhagen. 35. Williamson, J. R., Browning, E. T., Thurman, R. G. & Scholz, R. (1 969) J . Bid. Chem. 244, SO55 - 5064. 36. Blair, J. R., Cook, D. E. & Lardy, H. A. (1973) J . B i d . Cherrz. 248, 3608-3614. 37. Ui, M., Claus, T. H., Exton, J. H. & Park, C. R. (1973) J . B i d . Chem. 248,5344-5349. 38. Ui, M., Exton, J. H. & Park, C. R. (1973) J . Bid. Chern. 248, 5350-5359. 39. Parrilia, R., Jimenez, M.-I. & Ayuso-Parrilla, M. S. (1975) Eur. J . Biochem. 56, 375-383. 40. Parrilla, R., Jimenez, M.-I. & Ayuso-Parrilla, M. S. (1976) Arch. Biochern. Biophys. 174, 1 - 12.
473 41. Williamson, J. R. (1976) in Use ~fIsulateriLiverC~,ll.sunt/Kit/~ic,y Tubules in Metaholic Sfudies (Tager, J. M., SBling, H. D. & Williamson, J . R., eds) pp. 79-95, North Holland, Amsterdam. 42. Soboll, S., Scholz, R., Freisl, M., Elbers, R. & Heldt, H. W. (1976) in Use of’ Isolated Liver Cells and K i d n q Tuhulc~sin Me/aholic Sfudies (Tager, J. M., Soling, H. D. & Williamson, J. R., eds) pp. 29-40, North HollandiAmerican Elsevier, Amsterdam, Oxford and New York. 43. Zuurendonk, P. F., Akerboom, T. P. M. & Tager, J. M. (1976) in Use of Isolated Liver Cells and Kidney Tubules in Mrtuholic Studies (Tager, J . M., Soling, H. D. & Williamson, J. R., eds) pp. 17- 27, North-Holland, Amsterdam. 44. Sies, H., Akerboom, P. M. & Tager, J. M . (1977) Eur. J . Biochcm. 72, 301 - 307. 45. Tischler, M. E., Hecht, P. & Williamson, J. R. (1977) Arch. Biochem. Biophys. 181, 278 -292. 46. Schimassek, H. & Gerok, W. (1965) Hoppr-Scyler’s Z. Phy.sioI. Chem. 343,407-415. 47. Mehlman, M. A., Walter, P. & Lardy, H. A. (1967) J . B i d . Chern. 242,4594 4602. ~
P. Dieterle, F. Brawand, U. K . Moser, and P. Walter, Biochemisches Institut der Universitat Basel. Vesalianum, Vesalgasse 1, CH-4051 Basel, Switzerland
Alanine Metabolism in Rat Liver Mitochondria Peter DIETERLE, Fritz BRAWAND, Ulrich K. MOSER, and Paul WALTER Department of Biochemistry, University of Basel (Received January 27, 1978)
In the presence of 2-oxoglutarate, bicarbonate, Pi and ATP, rat liver mitochondria were found to metabolize alanine. The main products were glutamate, aspartate, malate and citrate. Pyruvate did not accumulate but reached a low steady-state concentration. Addition of octanoate caused a strong stimulation of alanine metabolism and a concomitant decrease in pyruvate concentration. No inhibition of alanine metabolism was obtained in the presence of inhibitors of mitochondrial pyruvate transport, a-cyano-4-hydroxy-cinnamate and a-cyano-cinnamate, showing that alanine was not transaminated by a contamination of the mitochondria by cytosolic alanine aminotransferase. In the presence of L-cycloserine, alanine metabolism in mitochondria was strongly inhibited indicating that in contrast to earlier reports L-cycloserine can enter the mitochondria. In isolated liver cells, addition of a-cyano-cinnamate caused a strong pyruvate accumulation and a nearly complete inhibition of gluconeogenesis with serine as substrate, whereas with alanine no pyruvate increase and a weak inhibition of gluconeogenesis was observed. From a comparison of the employed substrate concentrations and of the observed rates of alanine transamination in intact mitochondria with those under physiological conditions in cellular systems, it is concluded that cellular conversion of alanine to pyruvate most likely occurs to a great extent within the mitochondria as has been proposed earlier by DeRosa and Swick (1975) J . Biol. Chem. 250,7961 - 7967.
Several research groups [I - 51 have demonstrated the existence of at least two alanine aminotransferases in rat liver. In the most recent publication, DeRosa and Swick [5] have reported a total activity of 34 pmol . min-' . g liver-' of which 10- 18% were found in the mitochondrial matrix space whereas most of the rest was cytosolic. The same research group reported for the purified pig mitochondrial enzyme a K , value for alanine of 1.9 mM [5] and for a crude enzyme from rat liver mitochondria a K, value of about 12 mM [4]. The corresponding K, value for alanine of the cytosolic enzyme of rat liver was shown to amount to 42 mM by Swick et al. [4] and to 34 mM by Hopper and Segal[3]. Since the intracellular alanine concentration in rat liver amounts to about 1.5 mM [6,7], DeRosa and Swick [ 5 ] proposed that the main function of the mitochondrial alanine aminotransferase in vivo is to form pyruvate from alanine whereas the cytosolic enzyme may mainly be Enzymes. Alanine aminotransferase or L-alanine :2-oxoglutarate aminotransferase (EC 2.6.1.2); pyruvate carboxylase (EC 6.4.1.1); pyruvate dehydrogenase or rnultienzyme complex of pyruvate dehydrogenase or pyruvate :lipoate oxidoreductase (acceptor-acetylating) (EC 1.2.4.1).
involved in the reverse reaction. If this proposal is correct, the activity of the mitochondrial alanine aminotransferase should be high enough to convert all the alanine needed for gluconeogenesis. This seems to be the case as the mitochondrial enzyme activity measured by DeRosa and Swick of 3.4-6.2pmol . min-' . g liver-' is several-fold higher than the maximal conversion rates of 10 mM alanine to glucose which for perfused livers of fasted rats were reported to amount to 0.9-1.3 pmol . min-' . g liver-' [7,8]. Further support for the proposal of DeRosa and Swick comes from the work of Mendes-Mourao et al. [9] who found with liver cells that a-cyano-4-hydroxycinnamate, an inhibitor of mitochondrial pyruvate transport, exerted only a weak inhibition on gluconeogenesis from alanine whereas glucose production from serine and lactate was inhibited by 78 and 92% respectively. In this paper, the conversion of alanine to pyruvate and subsequently to the products of pyruvate metabolism in intact mitochondria was investigated. The results provide additional support for the proposed role of the mitochondrial alanine aminotransferase in gluconeogenesis. Some of the results were presented at a recent meeting [lo].
Alanine Metabolism in Rat Liver Mitochondria
468
MATERIALS AND METHODS Rat liver mitochondria were isolated according to Johnson and Lardy [ l l ] in 0.25 M mannitol/ 0.07 M sucrose. Mitochondria were suspended in the same medium at a concentration of 1 g original liver weight per ml. The basic incubation mixture in all experiments was: 4 mM ATP, 10 mM MgS04, 12 mM potassium phosphate buffer pH 7.4, 18 mM KHC03, 6.7 mM triethanolamine buffer pH 7.4 and substrates as indicated in the legends. Mannitol/sucrose was added to make the reaction mixture isotonic. All samples were gassed with a mixture of C02 ( 5 % ) and Oz (95%): The final volume was 3 ml. Mitochondria were incubated in 25-ml stoppered conical flasks in a shaking water bath at 37 "C. After an equilibration time of 2.5 min, the reactions were started by addition of 0.5 ml of the mitochondria1 suspension. The reactions were stopped by adding 1 m11.25 M perchloric acid. The samples were neutralized to pH 3.5 with potassium phosphate and the precipitated salt was centrifuged off. The protein and perchlorate precipitates were rinsed with water, centrifuged off and the washing solutions were combined with the respective supernatant samples. Parenchymal cells from rat liver were prepared by a slight modification (121 of the methods of Berry and Friend [13] and Seglen [14,15]. The incubation medium consisted of 5.4 mM KC1, 0.8 mM MgS04, 0.8 mM NaHzP04, 25 mM NatIC03 and 1.3 m M CaCI2. Final volume was 3.0ml containing 5-10 . lo6 cells. Incubations were carried out in 25-ml conical flasks which were gassed with 0 2 and C02 (95/5, v/v), stoppered and shaken at 37 "C for the times indicated. The reactions were stopped by the same method as described for the mitochondria. Pyruvate, citrate, malate, 2-oxoglutarate were measured as described previously [16]. Standard enzymatic assays were used for the determination of alanine [I 71, glutamate [18], aspartate [19] and glucose [201. Male albino Wistar rats from the Swiss Vitamine Institute in Basel were used. The average weight was 180 - 230 g. Enzymes and coenzymes were purchased from Boehringer Mannheim GmbH (Mannheim, Germany), ~-[l-'~C]alanine was from New England Nuclear Chemicals (Dreieich, Germany) and cc-cyano4-hydroxy-cinnamate was from Ega Chemie (Steinheim, Germany). a-Cyano-cinnamate was a gift of Dr Halestrap (Bristol). L-Cycloserine was prepared by A. Furst (F. Hoffmann-La Roche, Basel). All other reagents were of the highest purity commercially obtainable. The results with mitochondria are expressed on the basis of 60 mg mitochondrial protein which is about equivalent to 1 g wet weight of liver [21]. For
l6
t
2o 2 0
-
._ 16
-
7c
i /
/
/ /
a
0
E
l
3
6 9 Tlme of incubation (rnin) Time
12
Fig. 1. Tinie tlcyridw?c,c,ofnzitoc.hondria/ ulaflrric metabolism. Mitochondria were incubated in the basic medium. The concentration of added alanine and 2-oxoglutarate was 4 mM. 16 mg mitochondrial protein were present in each incubation. (W) Akdnine used; (0) glutamate found; (A) aspartate found; (0) malate found; (A) citrate found; ( 0 )pyruvate found
liver cells, the results are expressed per g of wet weight liver corresponding to 125 . lo6 cells [12]. All results are averages of two incubations with the same mitochondrial or cellular preparation.
RESULTS The time dependence of alanine metabolism in rat liver mitochondria in the presence of 2-oxoglutarate, bicarbonate, ATP and inorganic phosphate is shown in Fig. 1. Glutamate, citrate, malate and aspartate but not pyruvate increased with time during 12 min. In the absence of 2-oxoglutarate, no alanine utilization could be observed (not shown) indicating that transamination of alanine to pyruvate by alanine aminotransferase is the first step. Pyruvate did not accumulate but reached a steady-state concentration of 0.2-0.3 mM which corresponds to about its level in the liver cell [21,22]. As is known from earlier experiments with similarly low pyruvate concentrations [22,23], about equal amounts of pyruvate are carboxy-
P. Dieterle, F. Hrawand, U. K. Moser, and P. Walter
469
Table 1. Effict Of'arsenite and vctanoafr on ilir nwrahoii,sm of pyruvate and aianine The basic medium was used (see Materials and Methods ). Each incubation (3 ml) contained 14.5 mg mitochondria1 protein. In all experiments with alanine, 4 m M 2-oxoglutarate was also present. Negative values represent amounts used, positive values amounts found. n.d. = not determined Additions
Metabolites -.
~-
pyruvate
. ~
~
alanine
~~~
malate
citrate
aspartate
~
-~
~~~
-
glutamate
2-oxoglutaratc
< 0.2 < 0.2
~ < 0.2 < 0.2 < 0.2
n.d. n.d. n.d.
pmol 60 mg protein-' . 10 min-' ~
10 mM Pyruvate 10 mM Pyruvate + 0.4 m M arsenite 10 mM Pyruvate 0.8 m M octanoate 10 m M Pyruvate 0.4 mM arsenite 0.8 inM octanoate 4 m M L-Alanine 4 mM L-Alanine 0.4 mM arsenite 4 mM L-Alanine 0.8 mM octanoate 4 m M i.-Alanine + 0.4 mM arsenite 0.8 mM octanoate
+
63.2 - 0.4 - 59.5 -
+ +
-
+ +
+
30.2 2.9 5.3 1.7
+
~~~~
< 0.2
14.5 5.8 0.8 6.6
< 0.2
-
10.7 13.2 0.8 16.9
< 0.2
10.7 6.6 16.9
1.7 0.4 2.1
8.3 2.5 13.2
-
13.6
4.1
7.0
1.7
9.5
-
+
~
17.4 0.2 17.4
lated to oxyloacetate and oxidized to acetyl-CoA. It was furthermore shown that part of the oxaloacetate formed is either metabolized to citrate by citrate synthase, to malate by malate dehydrogenase or to aspartate by transamination with glutamate [23 - 271. In the experiment in Fig. 1, similar pathways of pyruvate metabolism can be anticipated. However, it should be noted that not all the malate, citrate and aspartate found at the end of the incubation are derived solely from pyruvate but were in part also formed from the added 2-oxoglutarate. The sum of aspartate plus glutamate was as expected equivalent to the amount of alanine used in the experiment of Fig. 1. In Table 1, an experiment is shown in which pyruvate conversion to acetyl-CoA was inhibited by addition of arsenite. Because pyruvate carboxylase requires acetyl-CoA as an allosteric activator [28], octanoate had to be added to provide sufficient acetylCoA [29]. Arsenite also inhibits the oxidation of 2oxoglutarate. Therefore, in the presence of arsenite and octanoate, all the citrate, malate and aspartate formed must originate from pyruvate carboxylation. This was confirmed in the experiment of Table 1 where the sum of pyruvate malate aspartate citrate (14.9 pmol) was about equal to the amount of alanine used (13.6 pmol). The small difference of 1.3 pmol is most likely the result of an incomplete inhibition by arsenite. As in the experiment of Fig. 1, the sums of glutamate plus aspartate should correspond to the amount of alanine used. However, in the incubations with arsenite, some amino equivalents were not accounted for. It may be that under these conditions some glutamate was deaminated to 2oxoglutarate. In the experiments with alanine, pyruvate was continuously formed by transmination. Upon addi-
+
_
< 0.2
10.7 0.6 18.2
~
2.1
_
~~~
< 0.2 < 0.2 < 0.2
~~
n.d. - 27.7 - 1.2 - 31.8 -
15.6
tion of octanoate, the total pyruvate metabolized (equal to alanine used - pyruvate found) increased by about 100"/,from7.8- 15.2 pmol . 60 mgprotein-' . 10 inin-'. At the same time, a drop of the pyruvate concentration in the incubation medium from 0.23 0.1 3 mM was observed (Table 1, see also Table 2). More direct evidence on the effect of octanoate comes from the experiment with ~,-[l-'~C]alanine in Table 2. 14C02 is split off either in the conversion of pyruvate to acetyl-CoA or in the two decarboxylation steps of the sequence isocitrate- 2-oxoglutarate - succinylCoA. The results of Table 2 show that in the absence of octanoate, 8.7 pmol alanine . 60 mg protein-' . 8 min-' were metabolized. In the presence of octanoate, maximally 0.2 pino1 of alanine were decarboxylated and therefore at least 95% or 12.5 pmol of the pyruvate formed from alanine were carboxylated. It is therefore obvious that octanoate strongly stimulated the carboxylation of pyruvate. When in the presence of arsenite and octanoate increasing amounts of alanine and 2-oxoglutarate were added, a corresponding acceleration of alanine conversion was observed (Fig.2). It should be noted that at the physiological concentration of about 1 1.5 mM alanine and 2-oxoglutarate approximately 0.6-0.8 pmol of alanine . min-' . 60 mg protein-' whichisequivalent to0.6-0.8 pmol . min-' . gliver-' was converted to gluconeogenic precursors. Since the greater part of the alanine aminotransferase is cytosolic it could be argued that a cytosolic contamination of the mitochondria by this enzyme was responsible for the transamination of alanine in our incubations. The experiments in Table 3 clearly show that the transamination occurred inside the mitochondria because the inhibitors of pyruvate transport 4-hydroxy-a-cyano-cinnamate and a-cyano-cinnamate
Alanine Metabolism in Rat Liver Mitochondria
470
Table 2. Effect of octanoate on I4CO2production from ~ - [ l - ' ~ C ] a l a n i n e All incubations were carried out in the basic medium including 4 mM 2-oxoglutarate. 1 pCi of ~-[l-'~C]alanine and 17 mg mitochondrial protein were added to each incubation. After the incubations perichloric acid was added and the I4CO2 was trapped in hyamine as described earlier [47]. Negative values represent amounts used, positive values amounts found Metabolites
Additions
pyruvate 4 min
alanine 8 min
14c02
4 min
8 min
4 min
8 min
pmol . 60 mg protein-' ~.
~~
4 mM ~-[I-'~C]Alanine
1.6
1.6
- 5.6
-
8.7
0.6
2.3
4 mM ~ - [ l - ~ ~ C ] A l a n+ i n0.8 e mM octanoate
0.2
0.3
- 7.3
-
13.0
0.1
0.2
Table 3. Effect of inhibitors ofpyruvate transport on the metabolism of pyruvate and alanine in mitochondria For conditions see Table 1. 13.5 mg of mitochondrial protein were added to each incubation
I
'.;c 10.0 W
c
P Q
Additions
r
Metabolites
z5-
E 2
pyruvate
alanine
0
5.0-
pmol . 60 mg protein-'
a ul,
.-0
malate aspartate + citrate + glutamate 10 min-'
c
n
2 2.5-
f 04
0
21 3
2
3
4
[L-alanine] and [2-oxoglutarate] (mM)
Fig. 2. Mitochonclriul metabolism of ulmine in the presence ofarsenite and octanoate at varying alanine concentrations. 2-Oxoglutarate was added at the same concentration as alanine. Mitochondria were incubated in the basic medium. Incubation time was 7 min. 17 mg of mitochondrial protein were present in each incubation. (m) Alanine used; (0) glutamate found; (A) aspartate found; (A) citrate found; (0)malate found; (0)pyruvate found; (+) represents the sum of malate, citrate, aspartate and pyruvate
[30] had very little effect on alanine metabolism whereas the conversion of added pyruvate was completely inhibited. By using a-cyano-cinnamate in whole liver cells (Fig. 3), we could confirm the findings of MendesMourao et al. [9] that gluconeogenesis from serine was inhibited by 86 % whereas glucose production
10 mM Pyruvate - 69.6 10 mM Pyruvate + 0.1 mM a-cyano- 3.5 cinnamate 10 mM Pyruvate +lmM 4-hydroxy-acyano- 4.4 cinnamate 4 mM Alanine 2.2 4 mM Alanine + 0.1 mM a-cyanocinnamate 2.2 4 mM Alanine +lmM 4-hydroxy-acyano-cinnamate 2.2
n.d.
31.9
n.d.
n.d.
1.7
n.d.
n.d. - 17.7
1.7 18.2
n.d. 15.1
- 16.0
19.9
13.7
- 15.5
19.5
13.3
from alanine was affected by only 23%. If the rates of gluconeogenesis are corrected for endogenous glucose formation, no inhibition was observed with alanine as compared to 89% with serine. In addition we found that in the presence of alanine the inhibitor caused no increase in pyruvate whereas with serine a 5-fold accumulation of pyruvate occurred. These results support the concept that there is enough mitochondrial alanine aminotransferase activity for the conversion of alanine to glucose in liver cells. The effects of L-cycloserine (an inhibitor of some transaminating enzymes [31]) on mitochondrial meta-
P. Dieterle, F. Brawand, U. K. Moser, and P. Walter
411
transferase was much more sensitive towards L-CYC~Oserine than aspartate aminotransferase because with 5 mM of the inhibitor only alanine formation was inhibited whereas aspartate production was not decreased. DISCUSSION
0'
I
I
,
I
The purpose of this paper is to provide additional evidence for the proposal of DeRosa and Swick [5] that transamination of alanine to pyruvate predominantly takes place within the mitochondria and not in the cytosol. The results show that between 11.7 pmol alanine . min-' .60 mg protein were metabolized when alanine and 2-oxoglutarate were added at 4 mM each. Addition of octanoate caused a strong stimulation of alanine utilization and a concomitant decrease of the pyruvate concentration. Despite the lower pyruvate level, an increase in the rate of pyruvate carboxylation was observed. This stimulation is most likely the result of an increase of the ATP :ADP ratio [23,24] or (and) of the increased concentration of acetyl-CoA [29]. It is furthermore known from the isolated pyruvate carboxylase that acetyl-CoA not only increases V but under certain conditions also lowers the K , for pyruvate [ 3 2 ] .The increase in acetylCoA may therefore also be responsible for the observed decrease in pyruvate concentration. This mitochondrial system may serve as an experimental model for the situation observed with isolated livers perfused with alanine where also a lowering of the pyruvate concentration with a concomitant increase in the rate of gluconeogenesis was observed in the presence of either fatty acids or glucagon [7,33]. In support of the concept of an intramitochondrial metabolism of alanine, a-cyano-cinnamate had little effect on alanine conversion in isolated mitochondria or cells. However, addition of L-cycloserine to the mitochondrial incubations caused a strong inhibition of alanine aminotransferase but not of aspartate aminotransferase. This inhibitory effect was unexpected because Williamson et al. [34] had stated (without giving results) that cycloserine did not penetrate mitochondria. The finding that only alanine aminotransferase was inhibited by cycloserine is in agreement with work by Azarkh et al. [31] who showed that in liver the sensitivity of alanine aminotransferase towards L-cycloserine was more than 100-times higher than that of aspartate aminotransferase. It therefore appears from our results that L-cycloserine enters the mitochondria in concentrations high enough to inhibit alanine aminotransferase but not aspartate amino transferase. In order to evaluate the physiological importance of the reported findings, the cellular concentrations of 2-oxoglutarate and alanine have to be considered. For 2-oxoglutarate, liver contents between 250 and
'
30 c
L
.-
cn
3
a" '
O
0
0
j
0.2
0.1
[ a-Cyano-cinnarnic
0.3 acid] (rnM)
0.4
0.5
Fig. 3. Effec,/s of'a-cq.ario-cinnamic acid on ~lueoneogenesis/l.omakanine and serine in isolazed liver cells. 6.7 . lo6 parenchymal cells from rat liver were incubated in the medium described under Methods. cr-Cyano-cinnamic acid was added at the beginning, whereas addition of the substrates serine and alanine occurred after 20 min of incubation. The reactions were stopped after 80 min. Glucose and pyruvate were measured as described under Methods. (0) Control; (B) substrate 5 mM alanine; (0)substrate 5 mM serine
Table 4 Efject of r-cycioseritze on nztrumitochondrral alanme aminotranyferase and aspartate nminotramjerase Foi conditions see Table 1 16 5 mg of mitochondria1 protein were added to each incubation Additions
substrates
Metabolites L-cycloserine alanine glutamate aspartate mM
pmol . 60 mg protein-' . I 0 min-'
4 mM L-Glutamate + 4 mM pyruvate 5
4 mM ~-Alanine
+ 4mM
2-oxoglutarate
4.0 0.7
-
14.9 12.0
9.8 10.5
-
-
7.3
6.2
0.7
0.5
- 0.4
< 0.2
< 0.2
bolism are shown in Table 4. In the experiment with alanine and 2-oxoglutarate, 0.5 mM L-cycloserine almost completely inhibited the mitochondrial alanine aminotransferase. The results of the incubations with pyruvate and glutamate show that the alanine amino-
412
2800 pmol . g dry weight-' have been reported [22, 35 -441. In recent years, various fractionating procedures have made it possible to determine the approximate compartmentation of various metabolites in the liver cell. For 2-oxoglutarate, concentrations for the mitochondria between 0.5 and 3.2mM and for the cytosol between 0.1 and 1.2mM have been found [22,41- 441. The mitochondriallcytosolic gradients varied between 1.8- 19.3 depending on the fractionating method employed and the physiological state of the cells [41-451. For alanine, cellular contents of 0.46-2.3 pmol . g liver wet weight-' have been measured under various metabolic conditions [6,7,37, 39,461. Tischler et al. [45] furthermore reported a small mitochondrial/cytosolic gradient of 1.5, however, no statistical evaluation and no information on the alanine concentrations in the' two compartments were given. The K, values of alanine aminotransferase in pig liver are 0.42 mM for 2-oxoglutarate and 1.9 mM for alanine [ 5 ] . If these values are also. valid for tlic rat liver enzyme, the conclusion can be drawn that the physiological concentrations of the two substrates are in the range of the K, values. At about physiological concentrations (1 1.5 mM) of 2-oxoglutarate and alanine, we obtained rates of alanine transamination of 0.6 - 0.8 pmol . min-' . 60 mg protein-' (Fig. 2) in our incubations with isolated mitochondria. For comparison, the perfusion experiments of Malette et al. [7] with liver from fed rats and ['4C]alanine are of special interest. At a concentration of 0.45 niM alanine in the perfusate (corresponding to the physiological plasma level in the fed animal), an intracellular alanine concentration of 1.7 mM and an incorporation rate of 3.5 pmol of [I4C]alanine into glucose . h-' . 100 g body weight-' was observed. On the basis of 5 g liver per 100 g rat, this rate is equivalent to 0.01 pmol alanine . min-' . g liver-'. After addition of glucagon, the intracellular alanine concentration dropped by about 30 % whereas the rate augmented to 0.03 and the total alanine utilization was 0.07 pmol alanine . min-l . g liver-'. At non-physiological concentrations of 9 mM alanine, the intracellular concentration amounted to more than 17 mM and the rate of ahnine incorporation was increased to 0.2 pmol . min-' . g liver-'. In perfused livers of fasted animals, higher rates of gluconeogenesis of up to 0.65 pmol of glucose . min-l . g liver-' [8] with 10 mM alanine have been reported. To our knowledge nobody has measured gluconeogenesis at physiological levels of alanine in liver of fasted rats. It is interesting to note, however, that the intracellular level of alanine in the liver of fasted animals is about 3-times lower than in the fed state [6]. From these results it can be concluded that our rates observed with isolated mitochondria are more than sufficient to cover the physiological requirement of the transamination rate of alanine to pyruvate.
Alanine Metabolism in Rat Liver Mitochondria Thiswork was supported by grants of the Swiss National Science Foundation. The excellent technical assistance of Miss L. Lehmann and Miss S. Studer is gratefully acknowledged.
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P. Dieterle, F. Brawand, U. K . Moser, and P. Walter, Biochemisches Institut der Universitat Basel. Vesalianum, Vesalgasse 1, CH-4051 Basel, Switzerland
