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Energy-dependent reduction of pyruvate to lactate by intact isolated parenchymal cells from rat liver
Vol. 44, No. 6, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ENERGY-DEPENDENT REDUCTION OF PYRUVATE TO LACTATE BY INTACT ISOLATED PARENCHYMAL CELLS FROM RAT LIVER
Michael N. Berry Department of Pathology and Cardiovascular Research Institute, University of California Medical Centerp San Francisco, Calif. 94122
Received J u l y 1 4 , 1971 Summary. Morphologically intact isolated parenchymal cells obtained from the livers of fasted rats formed lactate from added pyruvate at a linear rate. Reducing equlvalents necessary for this lactate production were derived from the oxidation of a portion of the added pyruvate. Lactate formation was greatly reduced by anaerobiosls and by inhlbltors of electron transport, uncoupling agents or ollgomycln. These results indicate that the transfer of reducing equivalents from mitochondrla to cytoplasm in intact liver cells is an energy-coupled process and provide evidence that energy-llnked reversed electron transport is of physiological significance in animal tissues.
For a number of years there has been conslderable debate concerning the possible physiological significance of the energy-dependent reduction of NAD+ that can be demonstrated with mitochondrlal preparations (1p2).
A major
obstacle in evaluatlng the metabolic role of this phenomenon has been the lack of suitable preparations for examining energy-linked reductlve processes in intact cells.
Recently D it has proved possible to obtain, from rat liver,
preparations of morphologically intact isolated parenchymal cells~ which form glucose and lactate from added pyruvate at rates comparable to those observed in the perfused llver (3).
The availability of Such preparations has provided an
opportunity to examine the contribution made by energy-coupled reactions to hepatic pyruvate reduction.
The experiments reported here indicate thatl in
intact liver cells t pyruvate reduction to lactate, coupled to pyruvate oxldatlon, is an energy-requlrlng process~ sensitive to recognized inhlbitors of electron flux and energy-transductlon. Materials and Methods.
Ollgomycln, atractyloslde, dicumarol D and antimycin
were obtained from Sigma, and rotenone from S. B. Penlck.
1449
Pentachlorophanol
Vol. 44, No. 6, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
was obtained from Aldrlch, and collagenase and hyaluronidase for isolated cell preparation from Worthington and Sigmap respectively.
Enzymes and cofactorsj
used for analytical technlques~ were products of Boehringer. Preparations of morphologically intact isolated parenchymal cells were obtained by the method of Berry and Friend (3) from the livers of male SpragueDawley rats, 300-400 gl fasted for 18 hr to deplete hepatic glycogen.
The
cells were washed 3 times with a balanced-sallne medium (4) and incubated at 37".
Incubations were carried out in a Gilson respirometer in 4 ml of medlmn
containing 0.14 M sodium chlorlde~ 5.4 mH potassium chloride, 0 . 8 m M m a g n ~ s i u m sulfate~ 2 mH calcium chlorlde and 10 mM sodium phosphate buffer I pH 7.4. Sodium pyruvate was present at an initial concentration of lOmM. Oxygen uptake was measured manometrically.
Specific enzymatic techniques
were used to measure intermediary metabolites as follows: malate and fumarate (6); D-B-hydroxybutyrate
pyruvate (5); lactate t
and acetoacetate (7); glucose (8);
ATP (9)~ 14CO2 was measured by liquid scintillation spectrometry.
Metabolic
rates are expressed as ~moles/g fresh weight isolated liver cells/min.
The
value for the wet weight of cells was derived by measuring the dry weight (3) and using an appropriate factor. Results.
Liver cells from animals fasted 18 hr metabolized added pyruvate
at a mean rate of 4 . 1 1 % 0.12 ~moles/g/mln (n = 20).
Glucose was formed at
0.59 ~ 0.02 ~moles/g/min and lactate produced at 1.36 ± 0.08 ~moles/g/mln. Hencel about 38Z of the added pyruvate must have been oxldized~ thereby providing reducing equivalents for gluconeogenesis and lactate formation (i0,ii) (Fig. 1).
Additional reducing power may have been derived from the mitochon-
drlal oxidation of endogenous substrates~ presuaably fatty acids (11-13).
Less
than 1 ~mole of lactate or glucose was produced in the absence of added pyruvate~ during an incubation period of 40 mln. The rate of pyruvate reduction to lactate was linear with time until the lactate:pyruvate ratio approached the physiologlcal range (between 5 and 20 to 1 (14)).
At a lactate~pyruvate ratio of 18:1 net pyruvate conversion to
1450
Vol. 44, No. 6, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
r
D-~-Hydroxybut),rote Acetoacetate I
0.35
0.42
0.56
0.62
0.86
0.85
Lactate
038
0.74
5.53
IZI
I
I
18.0
19.1
I
I
I
I
I
20
30
40
50
60
40" m w o
E =L
30
m > o
O
20
O
J
~0
9
10
Time (rain.)
Fig. i. Time course of p y r u v a t e reduction to glucose and lactate by isolated liver cells. D-S-hydroxybutyrate:acetoacetate and lactate: pyruvate ratios are recorded for the corresponding times at the top of the figure.
lactate ceased and lactate uptake commenced.
The lactate:pyruvate ratio then
gradually decreased as gluconeogenesis continued (Fig. i).
In contrast to
these alterations in the isctate:pyruvate ratio, the D-B-hydroxybutyrate: acetoacetate ratio changed much less, increasing only to about twice its inltlal value of 0.35 as 2.6 umoles of ketone bodies accumulated during the incubation period (Fig. I). This observation led to the question of whether, under certain conditions, transfer of reducing equivalents from mitochondria to cytoplasm could be inhibited, and acetoacetate reduction favored over pyruvate reduction.
This
was tested by examining the effects of agents known to affect mitochondrlal metabollsm.
Four types of agent were chosen for study, namely:
inhibitors
of electron transport (rotenone (15)~ antlmycln (16)), an inhibitor of energy transductlon (ollgomycln (17)), an inhibitor of adenine nucleotlde translocation (atractyloslde
(18)) and uncoupling agents (dlcuBarol (19), penta-
145i
Vol. 44, No. 6, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
chlorophenol (20), 2,4-dinitrophenol (21)).
The effects of anaerobiosis were
also studied. Table I.
Effects of inhibitors on mitochondrial metabolism and pyruvate reduction in isolated liver cells.
D-B-
14CO2 Treatment
02 uptake ~moles
Lactate found Bmoles
14CO 2 Aceto2_f~C_Py acetate found ~moles Bmoles
f~m i- C-Py ~moles
Malate hydroxy- and butyrate fumarate found found m~moles ~moles
None
24.5
8.4
11.4
2.4
2.3
0.87
Atractyloside
15.8
8.0
7.6
1.5
2.0
1.0
-
Oligomycin
8.3
2.6
4.8
0.I
L5
2.4
65
Ro tenone
5.6
2.9
I. 2
0.05
0.20
i. 7
65
Antimycin
4.8
2.0
4.1
0.05
0.38
2.0
205
2 ~4-Dinitrophenol
27.1
3.1
11.3
.
Dicumarol
30.0
2.7
13.7
-
7.6
0.33
-
PCP
27.2
2.7
11.1
2.3
6.7
0.60
93
Oligomycin ,PCP
27.2
2.7
11.2
2.5
5.3
1.9
-
100% N 2
-
2.0
1.4
-
0.14
1.5
176
100% N2,acetoacetate
-
2.0
10.0
-
29.4
14.4
-
Rotenone, acetoacetate
4.8
2.1
9.3
0.3
25.2
19.4
-
Antimycin, acetoace tare
4.0
1.9
10.8
-
28.5
14.9
-
Oligomy cin, acetoace tat e
8.0
2.6
10.9
0.1
28.5
14.6
-
18.0
2.9
.
.
.
.
26600
5.6
2.9
.
.
.
.
36000
12.1
18.4
.
.
.
.
.
7.1
12.2
.
.
.
.
.
29.0
13.6
.
.
.
.
.
Ro tenone ,succinate Roteno ne ,malate Oligomycln, ethanol Ro tenone, ethanol Dicumaro 1, ethanol
.
.
75
.
Incubation time, 40 rain; Final vol., 4 ml; Temp., 37°C; Substrates, I0 mM; Atractyloside, 15 ~ ; Ollgomycin, 3 ~M; Eotenone, 15 ~M; AntlmycinD 12.5 ~M; 2,4-Dinitrophenoi, dicumarolt pentachlorophenol (PCP)t 125 ~M. The results, derived from several experiments, are expressed on the basis of a cell wet weight of 219 rag, which was the aver a age cell mass for these experiments.
1452
Vol. 44, No. 6, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Anaerobic incubation of the cells and all the agents tested except atractyloside strongly inhibited pyruvate reduction to lactate (Table I).
Lactate
formation could be restored by addition of ethanol to the medium, indicating the site of inhibition to be within the mitochondrial compartment.
In the
presence of rotenoneD antimycin, oligomycin, or under anaerobic conditions, D-8-hydroxybutyrate accumulated.
Hence, intramitochondrial redox reactions
were taking place even though transfer of reducing equivalents to the cytoplasmic compartment was prevented. Table I also demonstrates that in the presence of inhibitors there was no correlation between the rates of respiration and the rates of lactate formation. Rotenone, antimycin or oligomycin inhibited cell respiration and pyruvate oxidation, whereas uncoupling agents stimulated oxygen uptake.
On the other
hand, atractyloside inhibited respiration more than 35% but had only minimal effects on pyruvate reduction.
It was found by measurement of the release
of 14C02 from 1-14C-pyruvate that the inhibition of pyruvate oxidation to acetyl CoA~ brought about by anaerobiosis, rotenone, antimycin or oligomycin, could be restored by addition of 10 mM acetoacetate to the incubation medium (Table I).
This promoted a coupled reaction whereby the oxidation of two
molecules of pyruvate to acetyl CoA and subsequent formation of one molecule of acetoacetate was accompanied by the reduction of two molecules of acetoacetate to D-~-hydroxybutyrate.
Despite this restoration of pyruvate oxida-
tion, no transfer of reducing equivalents to the Cytoplasmic compartment took place t all the hydrogen generated being accounted for by accmnulation of D-Bhydroxybutyrate.
Some D-B-hydroxybutyrate was also formed from reduction of
acetoacetate by endogenous substrates (Table I). The presence of acetoacetate failed to restore oxidation of acetyl CoA through the tricarboxylic acid cycle, as measured by formation of 14C02 from 2-14C-pyruvate.
In contrast, the inhibition of acetyl CoA oxidation and
oxygen uptake induced by oligomycin could be completely overcome by inclusion of an uncoupling agent in the incubation medium (Table I).
1453
Again, the restore-
Vol. 44, No. 6, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
tion of pyruvate oxidation was not accompanied by lactate formation. The failure of transfer of reducing equivalents from mitochondria to cytoplasm was not a consequence of a lack of malate for participation in shuttle mechanisms (10,22,23), since accmmulation of malate by cells exposed to anaerobiosis or inhibitors was no less than by cells incubated under control conditions (Table I).
Added malate or succinate was also ineffective in
restoring pyruvate reduction by cells exposed to inhibitors even though the latter substrate strongly stimulated oxygen uptake (Table I). The effects of dicumarol on pyruvate reduction, respiration and steadystate levels of ATP are shown in Fig. 2.
Similar observations were made
with pentachlorophenol and 2,4-dinitrophenol.
Pyruvate reduction was much
more sensitive to variation in the concentration of uncoupler than was respiration.
Thus, oxygen uptake was stimulated to the same extent over a
twentyfold range of dicumarol concentration, whereas the inhibition of pyruvate reduction was manifest only at concentrations of uncoupling agent which caused a pronounced fall in ATP levels in the isolated cells. 12or
I00
40
20
....
~ .c._tat e
1.25 [Dicumerol]
2.5
(MxlO 4)
F i g . 2. E f f e c t s o f d i c u m a r o l c o n c e n t r a t i o n ATP levels in isolated liver cells.
1454
on p y r t r v a t e m e t a b o l i s m and
Vol. 44, No. 6, 1971
Discussion.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The experiments reported here indicate that pyruvate is readily
metabolized by isolated parenchymal cells derived from the rats.
llvers of fasted
A portion of the added substrate is oxidized and, as in kidney slices
(I0) or perfused liver (ii), some of the reducing equivalents generated are utilized in glucose formation and for the reduction of pyruvate to lactate. Some reducing power is also probably derived from the oxidation of endogenous fatty acids, but, since in liver cells from fasted animals glycogen is totally depleted, it is possible to use the formation of lactate as a measure of the transfer of reducing equivalents from mltochondria to cytoplasm (i0). The inhibition of
lactate formation by anaerobiosis, rotenone or antlmycln
indicates that electron flux along the respiratory chain is necessary for the transfer process.
The inhibitory action of ollgomycin and uncoupling agents
demonstrates its energy-dependency.
Although oligomycln blocks pyruvate
reduction, it does not prevent acetoacetate reduction.
A possible explanation
for this is that the reduction of acetoacetate by oligomycln-treated cells D incubated with pyruvate, is a consequence of a non-energy-dependent reaction mediated through the coupling of malate and D-8-hydroxybutyrate dehydrogenases (24), or possibly through the direct coupling of pyruvate and D-8-hydroxybutyrate dehydrogenases.
Alternatively, reduction of acetoacetate in the
presence of pyruvate may be brought about by energy-llnked but ollgomyclninsensitive reactions (25).
Atractyloside, although inhibiting respiration
more than 35%, has minimal effects on pyruvate reduction.
Thus, the
reductlve process is not dependent on ATP translocatlon (18).
In fact D the
ATP level in cells exposed to atractyloslde is only 69% of that of controls, indicating a lack of close correlation between cell ATP concentrations and the rate of pyruvate reduction. The utilization of energy to drive redox processes has been repeatedly demonstrated with isolated particulate preparations.
Well established examples are
the reversed electron flow associated with the reduction of NAD by succlnate (1) and the energy-requlring reductions of NAD (26) or NADP (27) by NAD-llnked
1455
Vol. 44, No. 6, 1971
substrates.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The demonstration of energy-dependent reduction of pyruvate,
coupled to pyruvate or endogenous subs,rate oxidation, by intact isolated liver cells now provides evidence that energy-coupled reversed electron transport is not merely an artifact associated with broken cell preparations. Rather, it appears likely that in the intact cell energy-coupled redox reactions play a key role in driving reductive synthesis and in regulating metabolic flux.
Acknowledgements. This work was supported in part by USPHS Grant AM 12284. The technical assistance of Mrs. Ann Friend and Mrs. Gwen Childress is gratefully acknowledged. Reference s 1. 2. 3. 4. 5. 6.
Chance, B., and Hollunger, G., J. Biol. Chem., 236, 1577 (1961). Ernster, L., and Lee, C.-P., Ann. R~v. Biochem., 33, 729 (1964). Berry, M.N. and Friend, D.S., J. Cell Biol., 43, 506 (1969). Hanks, J.H., and Wallace, R.E., Proc. Soc. Exp. Biol. Med., 71, 196 (1969). Holzer, A., and Holldorf, A., Biochem. Z., 329, 292 (1957). Hohorst, H.J., in Methods of Enz~matlc Analysis, H.U. Bergmeyer (ed.), Academic Press, New York, 1963, p. 266. 7. Williamson, D.H., Mellanby, J., and Krebs, H.A., Biochem. J., 82, 90 (1962). 8. Stein, M.W., in Methods of Enzymatic Analysis, H.U. Bergmeyer (ed.), Academic Press, New York, 1963, p. 117. 9. Lamprecht, W., and Trautschold, I., in Methods of Enzymatic Analyslst H.U. Bergmeyer (ed.), Academic Press, New York, 1963, p. 543. 10. Krebs, H.A., Gascoyne, T., and Not,on, B.M., Biochem. J., 102, 275 (1967). ii0 Ross, B.D., Hems, R., and Krebs, H.A., Biochem. J., i0.2, 942 (1967). 12. Fritz, I.B., Physiol. Rev., 41, 52 (1961). 13. Menahan, L.A., and Wieland, O., Europ. J. Biochem., 9, 182 (1969). 14. Huckabee, W.E., J. Clin. Invest., 37, 244 (1958). 15. Ernster, L., Dallner, G., and Azzone, G.F., J. Biol. Chem., 238, i124 (1963). 16. Estabrook, R.W., Biochim. Biophys. Acta m 60, 236 (1962). 17. Lardy, H.A., Johnson, D., and McMurray, W.C., Arch. Biochem. Biophys. 78, 587 (1958). 18. Held,, H.W., Jacobs, H., and Klingenberg, M., Biochem. Biophys. Res. Commun., 18, 174 (1965)o 19. Chance, B., Williams, G.R., and Hollunger, G., J. Biol. Chem., 238, 439 (1963). 20. Weinbach, E.C., J. Biol. Chem., 210, 545 (1954). 21. Loom,s, W.F., and Lipmann, F., J. Biol. Chem., 113, 807 (1948). 22. Borst, P., Biochlm. Biophys. Acta, 57, 270 (1962). 23. Lardy, H.A., Paetkau, V., and Walter, P., Proc. Soc. Nat. Acad. Sci., 53, 1410 (1965). 24. Hoberman, H.D., and Prosky, L., J. Biol. Chem., 242, 3944 (1967). 25. Ernster, L., Proc. Internat. Congr. Biochem. 5th, Moscow, 1961, 5, i15 (1963). 26. Tager, J.M., Biochim. Biophys. Acre, 77, 258 (1963). 27. Klingenberg, M., and Slenczka, W., Biochem. Z., 331, 486 (1959).
1456
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ENERGY-DEPENDENT REDUCTION OF PYRUVATE TO LACTATE BY INTACT ISOLATED PARENCHYMAL CELLS FROM RAT LIVER
Michael N. Berry Department of Pathology and Cardiovascular Research Institute, University of California Medical Centerp San Francisco, Calif. 94122
Received J u l y 1 4 , 1971 Summary. Morphologically intact isolated parenchymal cells obtained from the livers of fasted rats formed lactate from added pyruvate at a linear rate. Reducing equlvalents necessary for this lactate production were derived from the oxidation of a portion of the added pyruvate. Lactate formation was greatly reduced by anaerobiosls and by inhlbltors of electron transport, uncoupling agents or ollgomycln. These results indicate that the transfer of reducing equivalents from mitochondrla to cytoplasm in intact liver cells is an energy-coupled process and provide evidence that energy-llnked reversed electron transport is of physiological significance in animal tissues.
For a number of years there has been conslderable debate concerning the possible physiological significance of the energy-dependent reduction of NAD+ that can be demonstrated with mitochondrlal preparations (1p2).
A major
obstacle in evaluatlng the metabolic role of this phenomenon has been the lack of suitable preparations for examining energy-linked reductlve processes in intact cells.
Recently D it has proved possible to obtain, from rat liver,
preparations of morphologically intact isolated parenchymal cells~ which form glucose and lactate from added pyruvate at rates comparable to those observed in the perfused llver (3).
The availability of Such preparations has provided an
opportunity to examine the contribution made by energy-coupled reactions to hepatic pyruvate reduction.
The experiments reported here indicate thatl in
intact liver cells t pyruvate reduction to lactate, coupled to pyruvate oxldatlon, is an energy-requlrlng process~ sensitive to recognized inhlbitors of electron flux and energy-transductlon. Materials and Methods.
Ollgomycln, atractyloslde, dicumarol D and antimycin
were obtained from Sigma, and rotenone from S. B. Penlck.
1449
Pentachlorophanol
Vol. 44, No. 6, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
was obtained from Aldrlch, and collagenase and hyaluronidase for isolated cell preparation from Worthington and Sigmap respectively.
Enzymes and cofactorsj
used for analytical technlques~ were products of Boehringer. Preparations of morphologically intact isolated parenchymal cells were obtained by the method of Berry and Friend (3) from the livers of male SpragueDawley rats, 300-400 gl fasted for 18 hr to deplete hepatic glycogen.
The
cells were washed 3 times with a balanced-sallne medium (4) and incubated at 37".
Incubations were carried out in a Gilson respirometer in 4 ml of medlmn
containing 0.14 M sodium chlorlde~ 5.4 mH potassium chloride, 0 . 8 m M m a g n ~ s i u m sulfate~ 2 mH calcium chlorlde and 10 mM sodium phosphate buffer I pH 7.4. Sodium pyruvate was present at an initial concentration of lOmM. Oxygen uptake was measured manometrically.
Specific enzymatic techniques
were used to measure intermediary metabolites as follows: malate and fumarate (6); D-B-hydroxybutyrate
pyruvate (5); lactate t
and acetoacetate (7); glucose (8);
ATP (9)~ 14CO2 was measured by liquid scintillation spectrometry.
Metabolic
rates are expressed as ~moles/g fresh weight isolated liver cells/min.
The
value for the wet weight of cells was derived by measuring the dry weight (3) and using an appropriate factor. Results.
Liver cells from animals fasted 18 hr metabolized added pyruvate
at a mean rate of 4 . 1 1 % 0.12 ~moles/g/mln (n = 20).
Glucose was formed at
0.59 ~ 0.02 ~moles/g/min and lactate produced at 1.36 ± 0.08 ~moles/g/mln. Hencel about 38Z of the added pyruvate must have been oxldized~ thereby providing reducing equivalents for gluconeogenesis and lactate formation (i0,ii) (Fig. 1).
Additional reducing power may have been derived from the mitochon-
drlal oxidation of endogenous substrates~ presuaably fatty acids (11-13).
Less
than 1 ~mole of lactate or glucose was produced in the absence of added pyruvate~ during an incubation period of 40 mln. The rate of pyruvate reduction to lactate was linear with time until the lactate:pyruvate ratio approached the physiologlcal range (between 5 and 20 to 1 (14)).
At a lactate~pyruvate ratio of 18:1 net pyruvate conversion to
1450
Vol. 44, No. 6, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
r
D-~-Hydroxybut),rote Acetoacetate I
0.35
0.42
0.56
0.62
0.86
0.85
Lactate
038
0.74
5.53
IZI
I
I
18.0
19.1
I
I
I
I
I
20
30
40
50
60
40" m w o
E =L
30
m > o
O
20
O
J
~0
9
10
Time (rain.)
Fig. i. Time course of p y r u v a t e reduction to glucose and lactate by isolated liver cells. D-S-hydroxybutyrate:acetoacetate and lactate: pyruvate ratios are recorded for the corresponding times at the top of the figure.
lactate ceased and lactate uptake commenced.
The lactate:pyruvate ratio then
gradually decreased as gluconeogenesis continued (Fig. i).
In contrast to
these alterations in the isctate:pyruvate ratio, the D-B-hydroxybutyrate: acetoacetate ratio changed much less, increasing only to about twice its inltlal value of 0.35 as 2.6 umoles of ketone bodies accumulated during the incubation period (Fig. I). This observation led to the question of whether, under certain conditions, transfer of reducing equivalents from mitochondria to cytoplasm could be inhibited, and acetoacetate reduction favored over pyruvate reduction.
This
was tested by examining the effects of agents known to affect mitochondrlal metabollsm.
Four types of agent were chosen for study, namely:
inhibitors
of electron transport (rotenone (15)~ antlmycln (16)), an inhibitor of energy transductlon (ollgomycln (17)), an inhibitor of adenine nucleotlde translocation (atractyloslde
(18)) and uncoupling agents (dlcuBarol (19), penta-
145i
Vol. 44, No. 6, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
chlorophenol (20), 2,4-dinitrophenol (21)).
The effects of anaerobiosis were
also studied. Table I.
Effects of inhibitors on mitochondrial metabolism and pyruvate reduction in isolated liver cells.
D-B-
14CO2 Treatment
02 uptake ~moles
Lactate found Bmoles
14CO 2 Aceto2_f~C_Py acetate found ~moles Bmoles
f~m i- C-Py ~moles
Malate hydroxy- and butyrate fumarate found found m~moles ~moles
None
24.5
8.4
11.4
2.4
2.3
0.87
Atractyloside
15.8
8.0
7.6
1.5
2.0
1.0
-
Oligomycin
8.3
2.6
4.8
0.I
L5
2.4
65
Ro tenone
5.6
2.9
I. 2
0.05
0.20
i. 7
65
Antimycin
4.8
2.0
4.1
0.05
0.38
2.0
205
2 ~4-Dinitrophenol
27.1
3.1
11.3
.
Dicumarol
30.0
2.7
13.7
-
7.6
0.33
-
PCP
27.2
2.7
11.1
2.3
6.7
0.60
93
Oligomycin ,PCP
27.2
2.7
11.2
2.5
5.3
1.9
-
100% N 2
-
2.0
1.4
-
0.14
1.5
176
100% N2,acetoacetate
-
2.0
10.0
-
29.4
14.4
-
Rotenone, acetoacetate
4.8
2.1
9.3
0.3
25.2
19.4
-
Antimycin, acetoace tare
4.0
1.9
10.8
-
28.5
14.9
-
Oligomy cin, acetoace tat e
8.0
2.6
10.9
0.1
28.5
14.6
-
18.0
2.9
.
.
.
.
26600
5.6
2.9
.
.
.
.
36000
12.1
18.4
.
.
.
.
.
7.1
12.2
.
.
.
.
.
29.0
13.6
.
.
.
.
.
Ro tenone ,succinate Roteno ne ,malate Oligomycln, ethanol Ro tenone, ethanol Dicumaro 1, ethanol
.
.
75
.
Incubation time, 40 rain; Final vol., 4 ml; Temp., 37°C; Substrates, I0 mM; Atractyloside, 15 ~ ; Ollgomycin, 3 ~M; Eotenone, 15 ~M; AntlmycinD 12.5 ~M; 2,4-Dinitrophenoi, dicumarolt pentachlorophenol (PCP)t 125 ~M. The results, derived from several experiments, are expressed on the basis of a cell wet weight of 219 rag, which was the aver a age cell mass for these experiments.
1452
Vol. 44, No. 6, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Anaerobic incubation of the cells and all the agents tested except atractyloside strongly inhibited pyruvate reduction to lactate (Table I).
Lactate
formation could be restored by addition of ethanol to the medium, indicating the site of inhibition to be within the mitochondrial compartment.
In the
presence of rotenoneD antimycin, oligomycin, or under anaerobic conditions, D-8-hydroxybutyrate accumulated.
Hence, intramitochondrial redox reactions
were taking place even though transfer of reducing equivalents to the cytoplasmic compartment was prevented. Table I also demonstrates that in the presence of inhibitors there was no correlation between the rates of respiration and the rates of lactate formation. Rotenone, antimycin or oligomycin inhibited cell respiration and pyruvate oxidation, whereas uncoupling agents stimulated oxygen uptake.
On the other
hand, atractyloside inhibited respiration more than 35% but had only minimal effects on pyruvate reduction.
It was found by measurement of the release
of 14C02 from 1-14C-pyruvate that the inhibition of pyruvate oxidation to acetyl CoA~ brought about by anaerobiosis, rotenone, antimycin or oligomycin, could be restored by addition of 10 mM acetoacetate to the incubation medium (Table I).
This promoted a coupled reaction whereby the oxidation of two
molecules of pyruvate to acetyl CoA and subsequent formation of one molecule of acetoacetate was accompanied by the reduction of two molecules of acetoacetate to D-~-hydroxybutyrate.
Despite this restoration of pyruvate oxida-
tion, no transfer of reducing equivalents to the Cytoplasmic compartment took place t all the hydrogen generated being accounted for by accmnulation of D-Bhydroxybutyrate.
Some D-B-hydroxybutyrate was also formed from reduction of
acetoacetate by endogenous substrates (Table I). The presence of acetoacetate failed to restore oxidation of acetyl CoA through the tricarboxylic acid cycle, as measured by formation of 14C02 from 2-14C-pyruvate.
In contrast, the inhibition of acetyl CoA oxidation and
oxygen uptake induced by oligomycin could be completely overcome by inclusion of an uncoupling agent in the incubation medium (Table I).
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Again, the restore-
Vol. 44, No. 6, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
tion of pyruvate oxidation was not accompanied by lactate formation. The failure of transfer of reducing equivalents from mitochondria to cytoplasm was not a consequence of a lack of malate for participation in shuttle mechanisms (10,22,23), since accmmulation of malate by cells exposed to anaerobiosis or inhibitors was no less than by cells incubated under control conditions (Table I).
Added malate or succinate was also ineffective in
restoring pyruvate reduction by cells exposed to inhibitors even though the latter substrate strongly stimulated oxygen uptake (Table I). The effects of dicumarol on pyruvate reduction, respiration and steadystate levels of ATP are shown in Fig. 2.
Similar observations were made
with pentachlorophenol and 2,4-dinitrophenol.
Pyruvate reduction was much
more sensitive to variation in the concentration of uncoupler than was respiration.
Thus, oxygen uptake was stimulated to the same extent over a
twentyfold range of dicumarol concentration, whereas the inhibition of pyruvate reduction was manifest only at concentrations of uncoupling agent which caused a pronounced fall in ATP levels in the isolated cells. 12or
I00
40
20
....
~ .c._tat e
1.25 [Dicumerol]
2.5
(MxlO 4)
F i g . 2. E f f e c t s o f d i c u m a r o l c o n c e n t r a t i o n ATP levels in isolated liver cells.
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on p y r t r v a t e m e t a b o l i s m and
Vol. 44, No. 6, 1971
Discussion.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The experiments reported here indicate that pyruvate is readily
metabolized by isolated parenchymal cells derived from the rats.
llvers of fasted
A portion of the added substrate is oxidized and, as in kidney slices
(I0) or perfused liver (ii), some of the reducing equivalents generated are utilized in glucose formation and for the reduction of pyruvate to lactate. Some reducing power is also probably derived from the oxidation of endogenous fatty acids, but, since in liver cells from fasted animals glycogen is totally depleted, it is possible to use the formation of lactate as a measure of the transfer of reducing equivalents from mltochondria to cytoplasm (i0). The inhibition of
lactate formation by anaerobiosis, rotenone or antlmycln
indicates that electron flux along the respiratory chain is necessary for the transfer process.
The inhibitory action of ollgomycin and uncoupling agents
demonstrates its energy-dependency.
Although oligomycln blocks pyruvate
reduction, it does not prevent acetoacetate reduction.
A possible explanation
for this is that the reduction of acetoacetate by oligomycln-treated cells D incubated with pyruvate, is a consequence of a non-energy-dependent reaction mediated through the coupling of malate and D-8-hydroxybutyrate dehydrogenases (24), or possibly through the direct coupling of pyruvate and D-8-hydroxybutyrate dehydrogenases.
Alternatively, reduction of acetoacetate in the
presence of pyruvate may be brought about by energy-llnked but ollgomyclninsensitive reactions (25).
Atractyloside, although inhibiting respiration
more than 35%, has minimal effects on pyruvate reduction.
Thus, the
reductlve process is not dependent on ATP translocatlon (18).
In fact D the
ATP level in cells exposed to atractyloslde is only 69% of that of controls, indicating a lack of close correlation between cell ATP concentrations and the rate of pyruvate reduction. The utilization of energy to drive redox processes has been repeatedly demonstrated with isolated particulate preparations.
Well established examples are
the reversed electron flow associated with the reduction of NAD by succlnate (1) and the energy-requlring reductions of NAD (26) or NADP (27) by NAD-llnked
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Vol. 44, No. 6, 1971
substrates.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The demonstration of energy-dependent reduction of pyruvate,
coupled to pyruvate or endogenous subs,rate oxidation, by intact isolated liver cells now provides evidence that energy-coupled reversed electron transport is not merely an artifact associated with broken cell preparations. Rather, it appears likely that in the intact cell energy-coupled redox reactions play a key role in driving reductive synthesis and in regulating metabolic flux.
Acknowledgements. This work was supported in part by USPHS Grant AM 12284. The technical assistance of Mrs. Ann Friend and Mrs. Gwen Childress is gratefully acknowledged. Reference s 1. 2. 3. 4. 5. 6.
Chance, B., and Hollunger, G., J. Biol. Chem., 236, 1577 (1961). Ernster, L., and Lee, C.-P., Ann. R~v. Biochem., 33, 729 (1964). Berry, M.N. and Friend, D.S., J. Cell Biol., 43, 506 (1969). Hanks, J.H., and Wallace, R.E., Proc. Soc. Exp. Biol. Med., 71, 196 (1969). Holzer, A., and Holldorf, A., Biochem. Z., 329, 292 (1957). Hohorst, H.J., in Methods of Enz~matlc Analysis, H.U. Bergmeyer (ed.), Academic Press, New York, 1963, p. 266. 7. Williamson, D.H., Mellanby, J., and Krebs, H.A., Biochem. J., 82, 90 (1962). 8. Stein, M.W., in Methods of Enzymatic Analysis, H.U. Bergmeyer (ed.), Academic Press, New York, 1963, p. 117. 9. Lamprecht, W., and Trautschold, I., in Methods of Enzymatic Analyslst H.U. Bergmeyer (ed.), Academic Press, New York, 1963, p. 543. 10. Krebs, H.A., Gascoyne, T., and Not,on, B.M., Biochem. J., 102, 275 (1967). ii0 Ross, B.D., Hems, R., and Krebs, H.A., Biochem. J., i0.2, 942 (1967). 12. Fritz, I.B., Physiol. Rev., 41, 52 (1961). 13. Menahan, L.A., and Wieland, O., Europ. J. Biochem., 9, 182 (1969). 14. Huckabee, W.E., J. Clin. Invest., 37, 244 (1958). 15. Ernster, L., Dallner, G., and Azzone, G.F., J. Biol. Chem., 238, i124 (1963). 16. Estabrook, R.W., Biochim. Biophys. Acta m 60, 236 (1962). 17. Lardy, H.A., Johnson, D., and McMurray, W.C., Arch. Biochem. Biophys. 78, 587 (1958). 18. Held,, H.W., Jacobs, H., and Klingenberg, M., Biochem. Biophys. Res. Commun., 18, 174 (1965)o 19. Chance, B., Williams, G.R., and Hollunger, G., J. Biol. Chem., 238, 439 (1963). 20. Weinbach, E.C., J. Biol. Chem., 210, 545 (1954). 21. Loom,s, W.F., and Lipmann, F., J. Biol. Chem., 113, 807 (1948). 22. Borst, P., Biochlm. Biophys. Acta, 57, 270 (1962). 23. Lardy, H.A., Paetkau, V., and Walter, P., Proc. Soc. Nat. Acad. Sci., 53, 1410 (1965). 24. Hoberman, H.D., and Prosky, L., J. Biol. Chem., 242, 3944 (1967). 25. Ernster, L., Proc. Internat. Congr. Biochem. 5th, Moscow, 1961, 5, i15 (1963). 26. Tager, J.M., Biochim. Biophys. Acre, 77, 258 (1963). 27. Klingenberg, M., and Slenczka, W., Biochem. Z., 331, 486 (1959).
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