(Received for publication, August 10, 1995; and in revised form, November 14, 1995)
From the
Pyruvate has been estimated to enter the citric acid cycle in
islets by carboxylation to the same extent or more than by
decarboxylation. Those estimates were made assuming the dimethyl esters
of [1,4-C]succinate and
[2-3-
C]succinate, incubated with islets at
a concentration of 10 mM, gave the same ratio of
CO
yields as if
[1-
C]acetate and
[2-
C]acetate had been incubated. The labeled
succinates, at 10 mM, but not 1 mM, are now shown to
give ratios higher than the labeled acetates at those concentrations
and therefore higher estimates when related to yields from
[2-
C]glucose and
[6-
C]glucose. Using the labeled acetate ratios
in paired incubations, the rate of pyruvate carboxylation is still
estimated to be about two-thirds the rate of pyruvate decarboxylation.
Participation of the malic enzyme-catalyzed reaction explains the
greater ratio of yields of
CO
from the
succinates at 10 mM than 1 mM and increases in those
ratios on glucose addition and can account for the removal from the
citric acid cycle of oxaloacetate carbon formed in the carboxylation.
Pyruvate carboxylation to oxaloacetate on incubation of islets
with glucose has been estimated to occur to as great an extent or more
than pyruvate decarboxylation(1, 2, 3) . If
correct, that has important implications with regard to islet and
presumably cell metabolism. Since in the turning of the citric
acid cycle the amount of oxaloacetate is unchanged, i.e. oxaloacetate + acetyl-CoA
citrate
2CO
+ oxaloacetate, at least half the carbon of glucose entering
the citric acid cycle would have to leave the cycle in a product or
products other than CO
. The formation of so much
oxaloacetate and hence its product(s) would presumably have a purpose.
The estimates depend in principle upon a comparison of the ratio of
the yields of CO
from
[2-
C]pyruvate and
[3-
C]pyruvate to those from
[1-
C]acetate and
[2-
C]acetate(4) . Since
[2-
C]pyruvate and
[3-
C]pyruvate on decarboxylation yield
[1-
C]acetate and
[2-
C]acetate, respectively, if pyruvate's
metabolism were only via decarboxylation to acetyl-CoA, the ratios from
the labeled pyruvates and acetates should be the same (Fig. 1).
Since [1-
C]acetate is oxidized to
CO
in fewer turns of the citric acid cycle
than [2-
C]acetate, unless the only fate of the
carbons of acetate is to CO
, the yield of
CO
from [1-
C]acetate
will exceed that from [2-
C]acetate. If pyruvate
is carboxylated because of rapid equilibration between oxaloacetate and
fumarate, both labeled pyruvates should yield
[2,3-
C]oxaloacetate (Fig. 1). If
equilibration is complete, the yield of
CO
from both pyruvates would be the same. Therefore, to the extent
there is carboxylation relative to decarboxylation, the ratio of the
yields from the labeled acetates should exceed that from the labeled
pyruvates.
Figure 1:
Formation of CO from carbon
1 and acetyl-CoA from carbons 2 and 3 of pyruvate on its
decarboxylation and carboxylation of pyruvate to oxaloacetate and
equilibration of the oxaloacetate with
fumarate.
MacDonald (2, 3) substituted the yields
of CO
from [2-
C]glucose
and [6-
C]glucose for those from the labeled
pyruvates, since [2-
C]pyruvate and
[3-
C]pyruvate are formed, respectively, via the
Embden-Meyerhof pathway from those labeled glucoses. MacDonald
substituted the yields of
CO
from
[1,4-
C]succinate and
[2,3-
C]succinate for those from the labeled
acetates, since the labeled succinates are formed, respectively, in the
initial turn of the citric acid cycle from
[1-
C]acetate and
[2-
C]acetate(4) . Actually, the dimethyl
esters of the labeled succinates were used, since the esters penetrate
the cell membrane and once inside the cell are hydrolyzed. Ratios of
yields of
CO
from the labeled glucoses were
about 2. Ratios of 4-6 or more, the larger in the presence of
unlabeled glucose, were observed when the labeled succinates were
incubated at 10 mM concentration, resulting in the
quantitation of equal or greater rates of pyruvate carboxylation than
decarboxylation.
Uncertainty exists with regard to the quantitation.
First, the method of Kelleher and Bryan (4) specifies the use
of the paired flask technique, i.e. labeled pyruvate in the
presence of unlabeled acetate or equivalent and labeled acetate in the
presence of unlabeled pyruvate or equivalent incubated under identical
conditions. Second, the metabolism of acetate and succinate are not
equivalent, even though the fate of acetate and succinate tracers will
be identical if incubated under identical conditions. In the citric
acid cycle a quantity of acetate, but not succinate, is oxidized to
CO, i.e. succinate
oxaloacetate +
acetyl-CoA
citrate
2CO
+ succinate. We
have now incubated the
C-labeled glucoses, acetates and
dimethyl succinates with islets using the paired flask technique and
from the ratios of the yields of
CO
quantified
pyruvate carboxylation.
The cups were removed and 2
ml of 5% BaCl were added to each vial. The barium carbonate
that precipitated was collected by filtering under suction the contents
of the vial onto a preweighed filter paper. The barium carbonate that
collected on the paper was washed with CO
-free water, dried
and weighed. The barium carbonates weighed between 96 and 110 mg, about
the theoretical yield from 0.5 mmol of NaHCO
.
The barium
carbonate, still on filter paper, was placed at the bottom of a
wide-mouth bottle containing 5 ml of water and closed with a rubber
stopper from which a scintillation vial containing 2 ml of Hyamine was
suspended. After evacuating air from the bottle through the stopper, 2
ml of 1 N HSO
was injected through the
stopper into the water. The bottle with its contents was kept at 37
°C for 2 h to allow the CO
evolved from the barium
carbonate to be absorbed into the Hyamine. Scintillation fluid was then
added and
C activity assayed in a scintillation counter.
where ACO
is the ratio from labeled
acetate or succinate, Pyr
CO
is the ratio from
labeled glucose, and F, assumed to be 0.8, the ratio of
C from [6-
C]glucose, the equivalent
of [3-
C]pyruvate, in carbon 2 to carbon 3 of
oxaloacetate(1, 2) .
Yields of CO
and the ratios of those
yields in the first two series of experiments are recorded in Table 1. The ratio of the
CO
yield from
[2-
C]glucose to that from
[6-
C]glucose at a glucose concentration of 20
mM was the same in the absence and presence of 1 mM acetate (means of 1.55 and 1.58). The ratios of the yields at 1
mM acetate were also the same in the absence and presence of
20 mM glucose (means of 2.64 and 2.72) and significantly more, p < 0.05 and p < 0.001, respectively, than the
glucose ratios (1.55 and 1.58). The ratios of yields from the
succinates were also significantly more than those from the labeled
glucoses (p < 0.001, except p < 0.05 for 2.35 versus 1.55). The ratio at 1 mM succinate was not
different from the ratio at 1 mM acetate in the absence (2.35 versus 2.64) but was in the presence of glucose (3.46 versus 2.72, p < 0.05). The ratios for succinate
at 1 and 10 mM succinate were more in the presence than
absence of 20 mM glucose (3.46 versus 2.35, p < 0.01, and 4.47 versus 3.05, p < 0.025).
At 10 mM succinate in the presence of glucose the ratio was
significantly more than for acetate (4.47 compared with 2.72, p < 0.01). At 10 mM succinate 3-5 times as much
succinate was oxidized to CO
as at 1 mM succinate.
The increase in the ratio of the yields from succinate on unlabeled
glucose addition was due mostly to a decrease in the yield of
CO
from
[2,3-
C]succinate.
The ratios of 1.55 for
PyrCO
and 2.64 for A
CO
calculate to a pyruvate carboxylation contribution of 43%. Using
2.35 for A
CO
the contribution is 39% and using
3.05 it is 48%. The ratios of 1.58 for Pyr
CO
and 2.72 for A
CO
give a contribution of
43%; with 3.46 it is 49%, and with 4.47 it is 54%.
In the series of
experiments comparing the ratios of yields at 1 and 10 mM acetate, and 10 mM acetate in the presence of glucose,
there were no significant differences among the ratios (Table 2).
The ratio at 1 mM acetate in that series, 2.62 ± 0.24,
was the same as in the first series, 2.64 ± 0.33 (Table 1). In contrast to succinate, increasing acetate's
concentration 10-fold resulted in less than a 2-fold increase in its
oxidation to CO. Furthermore, the addition of glucose had
no effect on the yield of
CO
. Incubations of
fresh islets with 1 mM acetate and 20 mM glucose (Table 3) gave similar ratios to those of cultured islets. The
ratios of 2.45 and 1.65, significantly different at p <
0.01, calculate to a carboxylation contribution of 35%.
In introducing the CO
ratio method
for estimating pyruvate carboxylation activity, Kelleher and Bryan (4) proposed and used in incubations of mitochondria
[1,4-
C]succinate and
[2,3-
C]succinate rather than
[1-
C]acetate and
[2-
C]acetate. They noted that the advantage was
that succinate, unlike acetate, is not volatile and therefore does not
produce large blank values for
CO
as generally
collected. MacDonald (1, 2, 3) used the
succinates because of high blanks he experienced on collecting
CO
formed from the labeled acetates. We
eliminated high blanks by collecting CO
in NaOH and then
precipitating it as BaCO
. Since sodium acetate and barium
acetate are water-soluble, [
C]acetate absorbed
in the NaOH was removed when the BaCO
was collected. The
conditions of islet preparation and incubation we used were similar to
those used by MacDonald(2, 3) . Islets were cultured
by him in medium containing 1, 5, 8, and 20 mM glucose rather
than just 11 mM glucose.
Succinate used in a trace
quantity, so that metabolism is not altered, should in theory be able
to replace acetate. That is evidenced from similar ratios at 1 mM succinate and acetate. However, the addition of succinate as a
substrate can alter metabolism. That is evidenced in the present study
by 1) similar ratios at 1 and 10 mM acetate but not 1 and 10
mM succinate, 2) the greater increase in succinate's
oxidation to CO with the increase in concentration, and 3)
the increase with glucose addition of the ratios with labeled succinate
but not acetate.
Succinate is converted to oxaloacetate in the
citric acid cycle (Fig. 2), and in one turn of the cycle
succinate is regenerated, albeit containing the carbons of the
acetyl-CoA that condensed with the oxaloacetate. Therefore, when
succinate enters the cycle in substrate amounts, at steady state, the
quantity of carbon leaving the cycle must equal the quantity in the
succinate entering, and that amount cannot leave as CO. To
the extent succinate's uptake exceeds the amount of acetyl-CoA
available for condensation, that succinate can only be metabolized in a
segment of the cycle. That segment, the dicarboxylic acid segment, is
from succinate to oxaloacetate, and the amount of carbon leaving the
segment, equal to the amount in the succinate taken up, can only be as
malate and/or aspartate (oxaloacetate and fumarate do not cross the
inner mitochondrial membrane).
Figure 2: Entrance into the citric acid cycle of succinate formed on hydrolysis of dimethyl succinate.
Following the transport of malate
from the mitochondrial matrix to the cytosol,
[1,4-C]malate would yield
[1-
C]pyruvate and
CO
,
catalyzed by malic enzyme. [2,3-
C]Malate would
yield [2,3-
C]pyruvate and no
CO
. Thus, the greater ratio of
CO
yields, coupled with the 3-5-fold
greater oxidation of the succinate to CO
at 10 mM than at 1 mM, with no source of exogenous acetyl-CoA, is
evidence for much of the succinate at 10 mM being metabolized
via the malic enzyme-catalyzed pathway. The increased ratio on glucose
addition can be explained by dilution of labeled pyruvate by unlabeled
pyruvate formed from the glucose. As a result, a smaller amount of
labeled pyruvate formed from labeled succinate would be expected to be
oxidized. The greater reduction in the yield of
CO
from [2,3-
C]succinate than from
[1,4-
C]succinate (Table 1) is in accord
with expectations. Indeed, if none of the labeled pyruvate was
oxidized,
CO
would be formed via the malic
enzyme-catalyzed reaction from [1,4-
C]succinate
but not [2,3-
C]succinate. In two of three
incubations of 10 mM labeled succinate in the presence of
unlabeled glucose, MacDonald (2) detected
CO
formation from [1,4-
C]succinate but not
[2,3-
C]succinate.
MacDonald demonstrated the
presence of malic enzyme in islets(1, 5) . Malaisse et al.(6) , incubating islets with glutamine at a
substrate concentration, 10 mM, reported that a major fraction
of the glutamine, via conversion to -ketoglutarate, left the cycle
as malate, which was converted to pyruvate. However, Malaisse and Sener (7) found similar ratios of yields of
CO
, about 2, on incubating fresh islets with
2.8-16.7 mM [2-
C]glucose and
[6-
C]glucose and with 1 mM [1-
C]acetate and
[2-
C]acetate. This was the reason we incubated
fresh islets by the paired flask technique.
The estimate using the
paired glucose and acetate ratios, that about 40% of pyruvate entering
the citric acid cycle was via carboxylation, is still near to the
estimate of 54% substituting the 10 mM succinate ratios. Thus,
the estimates are relatively insensitive to changes within the range of
the CO
ratios.
[2-
C]Glucose and
[6-
C]glucose are used, rather than
[2-
C]pyruvate and
[3-
C]pyruvate because of pyruvate's
instability. Randomization of carbon 2 of glucose-6-P in the pentose
cycle is assumed not to affect the ratio of the
CO
yields. The relative small contribution of the pentose cycle to
glucose utilization by islets supports that
assumption(2, 7) . If there is significant conversion
of dihydroxyacetone-3-P to glycerol or its derivatives, isotopic
equilibration of the dihydroxyacetone-3-P with glyceraldehyde-3-P is
assumed sufficiently complete so as not to result in an overestimation
of carboxylation. Relative high activity of triose-P isomerase in
islets(8) , a relatively small incorporation of glucose carbon
into lipid via glycerol 3-phosphate(9) , and only slightly less
yields of
CO
from
[6-
C]glucose than
[1-
C]glucose (2) support that
assumption. Equal yields of
H
O from
[2-
H]glucose and
[5-
H]glucose are not evidence for equilibration
of the triose phosphates(2) , since
H
O
is formed in the conversion of [2-
H]glucose-6-P
to fructose-6-P and not in the equilibration, while
H from
[5-
H]glucose not lost to
H
O in the equilibration will be lost in the
conversion of 2-phosphoglycerate to phosphoenolpyruvate.
The
assumption that F = 0.8 in the calculations, i.e. that there is extensive equilibration between oxaloacetate and
fumarate, is supported by high activities of malic dehydrogenase and
fumarase in islet mitochondria (2) and incorporations of about
80% as much C from [3-
C]lactate
into carbons 2 and 5 as carbons 1 and 6 of glucose formed by liver and
kidney(10, 11) . However, in the presence of a pool of
unlabeled succinate formed from dimethyl succinate and a pool of
labeled oxaloacetate formed from labeled pyruvate, isotopic
equilibration of the dicarboxylic acids should be less than in the
absence of the unlabeled succinate pool. An estimate for example of
54%, assuming F = 0.8, decreases to 32% if there is no
equilibration, F = 0. An assumption of course is also
that there are single pools of intermediates, e.g. acetate
enters the same pool of acetyl-CoA as acetyl-CoA formed from pyruvate.
That goes beyond any concern that the islet contains several cell
types, even though
cells predominate.
In conclusion, the
quantitation of pyruvate carboxylation in islets has been examined. It
is estimated to proceed at about two-thirds the rate of pyruvate
decarboxylation. The malate enzyme-catalyzed reaction allows for the
removal from the citric acid cycle of the oxaloacetate formed. That is
in accord with the recent report that when mitochondria from islets
were incubated with [1-C]pyruvate,
C was recovered in the incubation medium mainly in malate,
and when islets were incubated with
[U-
C]succinate
C appeared in
pyruvate and lactate(12) . Cytosolic NADPH would then be
generated for cellular needs. There would then be cycling as the
oxaloacetate, formed by fixation of pyruvate by CO
, is
decarboxylated via malate and pyruvate is reformed (Fig. 3). A
portion of the pyruvate would then be recarboxylated to oxaloacetate
with the remainder decarboxylated to acetyl-CoA and CO
or
reduced to lactate.
Figure 3: Carboxylation of pyruvate, the malic enzyme-catalyzed reaction, and the fate of pyruvate.