(Received for publication, February 17, 1995; and in revised form, June 7, 1995 )
From the
Mass isotopomer distribution analysis allows studying the
synthesis of polymeric biomolecules from N,
C-, or
H-labeled monomeric units in the
presence of unlabeled polymer. The mass isotopomer distribution of the
polymer allows calculation of (i) the enrichment of the monomer and
(ii) the dilution of the newly synthesized polymer by unlabeled
polymer. We tested the conditions of validity of mass isotopomer
distribution analysis of glucose labeled from
[U-
C
]lactate,
[U-
C
]glycerol, and
[2-
C]glycerol to calculate the fraction of
glucose production derived from gluconeogenesis. Experiments were
conducted in perfused rat livers, live rats, and live monkeys. In all
cases, [
C]glycerol yielded labeling patterns of
glucose that are incompatible with glucose being formed from a single
pool of triose phosphates of constant enrichment. We show evidence that
variations in the enrichment of triose phosphates result from (i) the
large fractional decrease in physiological glycerol concentration in a
single pass through the liver and (ii) the release of unlabeled
glycerol by the liver, presumably via lipase activity. This zonation of
glycerol metabolism in liver results in the calculation of
artifactually low contributions of gluconeogenesis to glucose
production when the latter is labeled from
[
C]glycerol. In contrast,
[U-
C
]lactate appears to be a
suitable tracer for mass isotopomer distribution analysis of
gluconeogenesis in vivo, but not in the perfused liver.
In
other perfusion experiments with
[H
]glycerol, we showed that the rat
liver releases glycerol molecules containing one to four
H
atoms. This indicates the operation of a substrate cycle between
extracellular glycerol and liver triose phosphates, where
H
is lost in the reversible reactions catalyzed by
-glycerophosphate
dehydrogenase, triose-phosphate isomerase, and glycolytic enzymes. This
substrate cycle presumably involves
-glycerophosphate hydrolysis.
Mass isotopomer distribution analysis (MIDA) ()is a
powerful tool to study the synthesis of polymeric molecules from the
condensation of identical
N-,
C-, or
H-labeled subunits (1, 2, 3, 4, 5, 6, 7, 8, 9) .
The polymer is characterized by a mass isotopomer distribution (MID),
which can be predicted from the isotopic enrichment of the precursor
subunit (p), using probability analysis and multinomial
expansion. Conversely, the pattern of excess isotopomer (
)frequencies in a polymer allows calculation of p and of the fraction of polymer molecules that are newly
synthesized (f) from the labeled precursor. Then, 1 - f represents unlabeled polymer molecules which entered the
sampling site, thus diluting the labeling of the newly synthesized
pool. MIDA, originally conceived by Strong et al.(1) ,
has been extensively developed by the groups of
Hellerstein(2, 3, 4, 5) ,
Kelleher(6, 7) , and Lee (8, 9) to
study fatty acid and cholesterol synthesis from
C
substrates and
H
O.
MIDA can, in principle, be applied to very long polymers such as proteins or to simple dimers. Glucose can be considered as a dimer formed from the condensation of two triose subunits whose labeling patterns should be, under most conditions, identical because of extensive equilibration via triose-phosphate (TP) isomerase(10) . Investigation of gluconeogenesis (GNG) by MIDA is an attractive possibility since it should not be subjected to artifacts of isotope exchange, which lead to underestimations of rates of glucose production (11, 12, 13) .
The main substrates of in vivo GNG are the gluconeogenic amino acids of proteins and
glycerol derived from lipolysis. The Cori cycle uses the GNG pathway
but does not contribute new glucose to the body's economy. GNG
from proteins and the Cori cycle pass through three C interconverted intermediates, i.e. lactate, pyruvate,
and alanine.
We recently (14) infused
[U-C
]glycerol to 60-h fasted humans
and assayed the MID of plasma glucose. The contribution of GNG to
glucose production, calculated from the MID of glucose, was much lower
than what could be expected to occur in 60-h starved humans. In these
subjects, glycogenolysis could contribute only a minuscule fraction of
glucose production. It was hypothesized (14) that this apparent
dilution results from variations in the MPE of triose phosphates (TP)
across the liver lobule. Such variation could result from a large
decrease in the glycerol concentration across the lobule. In the
present study, we investigated in perfused rat livers and in live rats
and monkeys, the mechanism that results in this unexpected labeling of
glucose from [U-
C
]glycerol. We also
tested the validity of [U-
C
]lactate
as a tracer of GNG. Our data show that physiological zonation of
glycerol metabolism in liver results from a major decrease in substrate
concentration across the lobule. Zonation of glycerol metabolism
results in gradients of enrichment of TP, which explain the observed
MID of glucose. In addition, we found evidence for the operation of a
substrate cycle between extracellular glycerol and liver TP.
To measure the kinetics of hepatic glycerol
uptake, eight rat livers were perfused each with four sequential 10-min
plateaus of increasing [H
]glycerol
concentration, to cover the range 0.1-3.5 mM. The
perfusate also contained 1 mM lactate. The concentration and
MID of glycerol were measured in effluent perfusate sampled at the end
of each plateau. In some experiments, the concentration of pyruvate was
assayed at 10 min, before switching to labeled tracers.
To measure
the loss of H from
[
H
]glycerol, we perfused six livers
with 300 ml of recirculating perfusate containing 1 mM lactate
and [
H
]glycerol at initial
concentrations of 0.2, 0.4, and 0.8 mM. Perfusate was sampled
every 30 s for 10 min to measure the concentration and the MID of
glycerol.
Female Maccaca mulatta monkeys were anesthetized with halothane and
infused with [U-C
]lactate (7 and 10
µmol
min
kg
)
or [U-
C
]glycerol (2 µmol
min
kg
). Arterial blood
samples were taken at 3, 4, and 5 h. Samples of liver were then
freeze-clamped.
where M represents the mol
fraction of the nth mass isotopomer of the subscripted
compound, and f is the fraction of glucose produced by GNG.
For n > 0, M
= 0. The parameter n ranges from 0 to 6 since up
to six
C atoms are incorporated in glucose molecules. Note
that M
and M
= 0 for i > 3. Isotopic equilibrium between DHAP and GAP is assumed. We
used non-linear parameter estimation techniques (20) to
determine the MID of TP and f values that best fit the
measured MID of glucose.
Hellerstein et al.(2, 3) calculate f for a polymer, labeled from an M monomeric precursor, by monitoring the excess
labeling of the polymer at two masses, M
and M
. A similar calculation can be derived from a
simplification of . When
[U-
C
]glycerol is infused, the MID of
glucose includes mostly M
, M
,
and M
. Thus, the TP are mostly M
and M
. The explicit expressions for M
and M
of glucose are given
by and .
Assuming isotopic equilibrium of GAP and DHAP, i.e.M = M
= M
, and become and .
Using the approximation M = (1 - M
), the
ratio of excess mass isotopomer in the product is shown by ,
or using the notation of Hellerstein and Neese (p = M; (5) ), by .
The use of [U-C]glycerol greatly
simplifies the technique of Hellerstein et al. because of the
low natural abundance at M
and M
glucose. All MIDs here are corrected for natural
abundance(18) . For this case, the construction of a
theoretical standard curve (2, 3) for the particular
glucose derivative used is unnecessary. Therefore, the enrichment of
the triose phosphate pool can be calculated directly by solving for p in terms of M
/M
. Using the calculated
value of p, the fraction of glucose production derived from
GNG, i.e.f, is calculated by solving or for f.
Assuming there are two pools of labeled TP, the relative
abundances of M and M
mass
isotopomers of glucose assuming isotopic equilibrium between DHAP and
GAP are given by and ,
where the subscripts 1 and 2 refer to the fractional glucose
contributions from labeled TP pools 1 and 2. Then, has
four unknowns, f and f
(the
fractional contributions of each pool of TP to GNG) and M
and M
(their
enrichments).
The TP enrichment calculated using MIDA under the assumption of
one labeled pool does not reflect the arithmetic average of the
enrichments of the two pools (f + f
). With no further information, one cannot
determine the enrichments of these two pools and thus the fractional
contribution of GNG to glucose. This holds true whenever there are more
than two labeled TP pools or, more likely, when a gradient of
enrichment exists between the periportal and perivenous regions of the
liver lobule.
The total amount (µg atom) of C in a
population of glucose molecules with a given MID is given by .
The right side of this equation represents the loss of substrate by metabolism expressed as a Michaelis-Menten reaction rate, as shown by ,
where K is a constant. J
(z), the maximum rate, may vary with
position. In the following analysis, let us assume steady-state
conditions and that J
decreases linearly with
length along the lobule, as shown by ,
where J is the initial rate.
When the constant
is zero, the reaction rate does not change
along the lobule. Using the dimensionless terms:
= C(z)/C
,
= z/L,
= K
/C
,
= J
LS
/QC
,
we can express the model as shown by .
Upon solution using separation of variables, we find
where 0
1 and 0
1. If we
consider
(
), for
> 0, then is
quadratic and its solution is given by .
The solution for = 0 is given by .
We can solve for using measured outlet concentrations
(
= 1) by rearrangement of , as shown by .
We can calculate the relative flux along the lobule using the dimensionless form of , which is shown in .
Fig. 1A (solidsymbols)
shows the kinetics of [H
]glycerol
uptake by perfused livers from 2-day starved rats. A Lineweaver-Burk
plot of the data, for influent concentrations ranging from 0.1 to 1.2
mM (r = 0.99), yields K
and V
of 0.78 mM and 2.1
µmol
min
(g, wet
weight)
, respectively. The V
is similar to that reported in (22) and (23) .
The K
, which is in the range of the K
of glycerol uptake from dog plasma (1.7
mM; (24) ), is much greater than the K
of glycerol kinase in rat liver
(3-10 µM; Refs. 22, 25, and 26). This confirms that
glycerol uptake by the liver is diffusion-limited(23) . Fig. 1A also shows (opensymbols) the
fractional uptake of glycerol; 85-90% of physiological influent
concentrations (0.05-0.2 mM) was taken up in a single
passage through the liver.
Figure 1:
Kinetics of
[H
]glycerol uptake by the perfused
rat liver. PanelA, rate of uptake (solid
circles) and percent uptake in a single passage through the liver (opentriangles). The continuousline is the Michaelis-Menten curve calculated from the Lineweaver-Burk
plot of the uptake for influent concentrations of 0.1-1.2
mM. V
= 2.1 µmol
min
(g, wet weight)
and K
= 0.78 mM. PanelB, production of unlabeled glycerol (solidsquares) and MPE of M
glycerol
isotopomer in effluent perfusate (versus 94% in influent
perfusate).
In the same experiments, we found that
the MPE of [H
]glycerol decreased in a
single passage through the liver. Fig. 1B (opensymbols) shows the M
MPE of effluent
glycerol. Note that this decrease in MPE occurs mostly in the
physiological range of plasma glycerol concentrations. Fig. 1B (closedsymbols) also shows
the absolute release of unlabeled glycerol. This release was erratic
and independent of inflowing glycerol concentration. At high inflowing
[
H
]glycerol concentrations, the
release of unlabeled glycerol was not measurable with precision.
Similar decreases in total MPE were observed in experiments with
[2-
C] and
[U-
C
]glycerol (not shown).
In
addition, for influent concentrations of
[H
]glycerol of 0.4-1.0
mM, effluent glycerol contained a greater MPE of M
to M
isotopomers than the
influent perfusate. This could not be accounted for by the natural
enrichment of released unlabeled glycerol. The production of M
to M
isotopomers could not
be detected in experiments with influent
[
H
]glycerol concentrations lower than
0.4 mM and higher than 1.0 mM because of (i) the near
complete exhaustion of the labeled substrate, and (ii) its low isotopic
dilution, respectively. The release of M
to M
glycerol isotopomers corresponds to 7-10%
of the uptake of M
glycerol. The relative
proportions of the M
to M
isotopomers were 3 (M
), 5 (M
), 32 (M
), and 100 (M
).
To better characterize the loss of H from [
H
]glycerol, we
perfused livers with a large volume (300 ml) of recirculating perfusate
containing 1 mM lactate and various concentrations of
[
H
]glycerol. Fig. 2shows the
accumulation of M
to M
glycerol isotopomers (M
> M
> M
> M
) when the recirculating perfusate contained
initially 0.8 mM [
H
]glycerol. Similar MIDs of
perfusate glycerol were obtained when the initial
[
H
]glycerol concentration was 0.4 and
0.2 mM (not shown). Total glycerol concentration decreased
from 0.6 to 0.4 mM and from 0.2 to 0.05 mM,
respectively.
Figure 2:
Loss of H from
[
H
]glycerol in the perfused rat
liver. A liver was perfused with 300 ml of recirculating perfusate
containing [
H
]glycerol at an initial
concentration of 0.8 mM. The decrease in total glycerol
concentration is shown by the brokenline. Perfusate
glycerol was progressively enriched with M
to M
isotopomers identified in the
caption.
Table 1shows the MID of glucose isolated from
non-recirculating effluent liver perfusates, rat plasma, and monkey
plasma. Glucose was labeled from
[U-C
]lactate,
[U-
C
]pyruvate,
[U-
C
]glycerol, or
[2-
C]glycerol. In many cases, lactate and
glycerol were infused together (with alternate labeling) to simulate
the physiological supply of gluconeogenic substrates. All data of Table 1are corrected for natural
C enrichment by a
technique that takes into account the skew of natural enrichment in
multiply
C-labeled molecules(18) .
In livers
perfused with [U-C
]glycerol (Table 1, rows 2, 4, 8, and 9), the main labeled glucose
isotopomers were M
and M
,
with much lower abundances of the other labeled isotopomers. This was
expected since glycerol is a very direct precursor of glucose. Only a
small fraction of [U-
C
]glycerol was
processed in the CAC, with loss of
C, before generating M
, M
, M
,
and M
glucose. In contrast, with
[U-
C
]lactate (rows 1 and 3), the MID
of glucose was more evenly distributed since all the label from the
substrate passes through oxaloacetate and PEP. This results in losses
of label in the CAC and in the PEP
pyruvate
oxaloacetate
cycle. In livers perfused with unlabeled lactate and
[2-
C]glycerol (rows 10, 11), only M
- and M
-labeled glucose
isotopomers were detected, as expected.
Since the isolated livers
were taken from 2-day starved rats, they were essentially
glycogen-depleted(27) , and all glucose production must have
been derived from GNG. Parameter f, which quantitates the
contribution of GNG to glucose production ( and ), should have been 100% in all cases (Table 1, rows
1-11). In fact, f ranged from 36 to 92%. When the
influent concentration of
[U-C
]glycerol was increased from 0.1
to 0.5 and to 1.5 mM, f increased from 75 to 85 and
to 92%, respectively (Table 1, rows 2, 8, and 9). When glucose
was labeled from [U-
C
]lactate, f was 54% and 36%, in the absence and presence of 0.2 mM octanoate, respectively (rows 1 and 3). Also, when uniform
labeling of lactate and glycerol was alternated, substantially
different values of f were calculated (compare f in
rows 1 and 2 and in rows 3 and 4). In similar perfusions conducted with
4 mM glucose in addition to 1 mM lactate and 0.1
mM [U-
C
]glycerol, M
and M
glucose isotopomers
were the only two for which MPE could be precisely measured (rows 9 and
10) corresponding to f of 3% and 1%. This was expected since
unlabeled glucose was added to the perfusate to simulate the production
of glucose from unlabeled precursors.
In 2-day fasted rats infused
with lactate and glycerol (Table 1, rows 14 and 15), f was 97% and 75% when lactate or glycerol were uniformly labeled,
respectively. When [U-C
]lactate was
infused, no label was detected in plasma glycerol, and the final MID of
plasma lactate was 0.41 ± 0.06 (M
), 0.67
± 0.10 (M
), and 9.69 ± 1.14% (M
, n = 6). When
[U-
C
]glycerol was infused, the MPE
of plasma glycerol was stable between 3 and 5 h, with final level of
15.3 ± 1.8% (n = 6). In the same rats, lactate
became labeled with final MID of 0.82 ± 0.10 (M
), 0.53 ± 0.08 (M
),
and 1.82 ± 0.21% (M
).
So, of all the
above cases where f should have been 100% (rows 1-11,
14, and 15), only in 2-day starved rats infused with
[U-C
]lactate did f approach
this value (row 14). In overnight-fasted monkeys (rows 13 and 14), f was also greater when
[U-
C
]lactate versus [U-
C
]glycerol was infused (80%
and 48%, respectively). So, for in vivo experiments in rats
and monkeys, f for
[U-
C
]glycerol was 3/4 and 3/5 that
for [U-
C
]lactate.
In perfused
livers, the MID of glucose labeled from
[U-C
]glycerol or
[2-
C]glycerol yielded similar values for f (Table 1, rows 2 and 10) when the substrates were used at
their maximal MPE. However, when [2-
C]glycerol
was used at 30% MPE, which is closer to what would occur in in vivo experiments, values of f were erratic (not shown). This
is ascribed to the sensitivity of the M
/M
ratio when the M
enrichment is low (about 1.6%) compared to
natural enrichment at M
(2%). This was the case,
although permethylglucose has the lowest natural enrichment MID of all
common glucose derivatives. For M
to M
glucose isotopomers, natural enrichment is
practically zero, which increases the precision of measurements. So, to
optimize the measurement of f, using the MID of glucose, we
recommend (i) using uniformly labeled rather than singly labeled
substrates and (ii) conducting GC-MS analysis of permethylglucose under
ammonia chemical ionization(17) .
Péroni et al.(28) reported
that, in rat livers perfused with 0.5 mM
[2-C]glycerol (15% MPE) and 0.7 mM lactate, the MID of effluent glucose yielded a f of 91 to
100%, which is substantially higher than the 85% value of f we
obtained using 99% MPE [2-
C]glycerol (Table 1, row 11). Péroni et al. had analyzed the MID of glucose by electron ionization GC-MS of
the aldonitrile pentaacetate derivative. To test for differences in
analytical techniques, we reanalyzed the MID of glucose in our
perfusions with 0.1 and 0.5 mM
[2-
C]glycerol (Table 1, rows 10 and 11),
using the same derivative as Péroni et
al.. Whether the assays were conducted under electron ionization (m/z 212-214, 314-316, 225-227) or
ammonia chemical ionization (m/z 405-407), we obtained
the same values of f as with permethylglucose (84-85%).
We cannot fully explain why the data of Péroni et al. differ from ours. This may result from their use of
higher than physiological concentrations of glycerol and/or from
differences in techniques for correcting the measured MID of glucose
for natural enrichment at M
and M
: standard curves with commercial M
and M
glucoses (28) versus correction for skew of natural
enrichment(18) . A small overestimation of the M
/M
ratio would substantially
increase f.
Some investigators correct the measured MID of
multiply C-labeled compounds by simple subtraction of the
MID of an unlabeled standard(29) . Rosenblatt et al. have shown (19) that the natural MID of compounds is
skewed with increasing
C labeling. To illustrate the
impact of this skew, consider the data shown on row 7 of Table 1(MID of glucose in livers perfused with 1 mM
[U-
C
]pyruvate + 0.1 mM glycerol). Taking the skew of natural MID into
consideration(18) , we calculate a f of 72.4 ±
4.6%. If the raw data were corrected for natural MID as in (29) , f would be decreased to 63.7 ± 4.5% (p = 0.1). Thus, a proper correction for natural MID is
required for precise calculation of f.
Table 2shows,
for most of the same experiments as Table 1, the measured MID of
liver PEP and the calculated MID of the TP (). When
[U-C
]glycerol is the labeled
substrate, the MIDs of PEP and of the TP are very different. When
[U-
C
]lactate is the labeled tracer,
the distributions are quite similar, but significant differences
remain, particularly between the MPEs of M
isotopomers. In addition, the total labeling of TP is much higher
than that of PEP when [U-
C
]glycerol
(but not [U-
C
]lactate) is the
labeled substrate (Table 2, last column).
Table 3shows
the balance of substrates and C in four series of perfused
liver experiments where we had reciprocal and uniform labeling of
lactate and glycerol (corresponding to rows 1-4 of Table 1). Balances are calculated by combining data from
experiments with reciprocal labeling conditions. Note the very
different absolute and fractional uptakes of lactate and glycerol. In
these perfusions conducted with only lactate and glycerol, only 1/4 of
the total uptake of these substrates was converted to glucose (Table 3, row F).
Although lipases have been described in liver(30, 31) , to the best of our review of the literature, this is the first report of the release of glycerol by the intact liver. This has implications for in vivo studies of glycerol metabolism, which usually assume that liver is the main site of utilization of glycerol released by lipolysis. In studies to be published elsewhere, we confirmed that the liver of live dogs releases glycerol.
In livers perfused with
[H
]glycerol, the production of
glycerol isotopomers with fewer than 5 deuterium atoms (Fig. 2)
strongly suggests that a substrate cycle operates between extracellular
glycerol and tissue TP. Cycling between
[
H
]glycerol and DHAP forms M
glycerol. Cycling between
[
H
]glycerol and GAP forms M
glycerol because the TP isomerase reaction
removes specifically the R hydrogen on C-1 of
DHAP(31) . Cycling between
[
H
]glycerol and glycolytic
intermediates between GAP and PEP forms M
glycerol. Finally, cycling between
[
H
]glycerol and PEP that has gone
through the PEP
pyruvate
oxaloacetate
PEP cycle
forms M
and probably M glycerol. As
evidence for cycling between liver TP and oxaloacetate is the
production of M
, M
, M
, and M
glucose in perfused
livers and animals infused with
[U-
C
]glycerol (Table 1, rows
2, 4, 8, and 9).
While -glycerophosphate dehydrogenase and TP
isomerase are reversible enzymes, glycerol kinase is irreversible. So
-glycerophosphate hydrolysis is probably catalyzed by some
phosphatase. The rate of release of M
to M
glycerol isotopomers, which corresponds to
7-10% of the rate of uptake of M
glycerol,
probably underestimates what would be the corresponding rate of cycling
of non-deuterated glycerol. Since the energy of a carbon-deuterium bond
is greater than that of a carbon-protium bond, one can expect sizable
isotope effects at the
-glycerophosphate dehydrogenase and TP
isomerase steps. Indeed a 2.9 kinetic isotope effect has been described
for the isomerization of (R)-[1-
H]DHAP,
compared to the non-deuterated compound(32) . Additionally, in vivo studies showed the discrimination of liver TP
isomerase against [1-
H]GAP(33) .
This
study points to two mechanisms for glycerol release from liver. The
release of unlabeled glycerol in livers perfused with
[H
]-,
[U-
C
]-, or
[2-
C]glycerol results probably from lipase
activity and appears independent of inflowing glycerol concentration (Fig. 1B, solidsymbols). The release
of M
to M
glycerol in livers
perfused with [
H
]glycerol (Fig. 2) can only be explained by a substrate cycle operating
between extracellular glycerol, tissue TP, and lower glycolytic
intermediates. This cycle, which probably involves a phosphatase, may
contribute to the regulation of
-glycerophosphate concentration in
liver cells. The phosphatase may allow production of glycerol via
glycolysis or GNG. This question, which is out of the scope of the
present study, will be investigated separately.
Table 3shows that in livers perfused with 1 mM lactate and 0.1 mM glycerol, in the absence and presence of 0.2 mM octanoate, only 1/4 of the lactate + glycerol uptake was converted to glucose (row F). Thus, 3/4 of the gluconeogenic substrates taken up must have been used for energy production, even in the presence of octanoate.
In Table 3, the calculations of the
loss of label between [U-C
]lactate
and glucose assume that the percent
C from
[U-
C
]glycerol uptake found in
glucose equals the percent conversion of glycerol to glucose (row H).
This is a legitimate assumption since the conversion of glycerol to
glucose is direct without carbon exchange. Rows H and J of Table 3show the percent of the uptake of
C of each
substrate recovered in glucose. This recovery is much larger for
[U-
C
]glycerol than for
[U-
C
]lactate. This is because (i) a
large fraction of lactate uptake is oxidized to generate ATP and (ii)
the conversion of [U-
C
]lactate to
glucose involves losses of label in the CAC and pyruvate cycles. The
last row of Table 3shows the percentages of
C from
the glucose-bound [U-
C
]lactate
molecules that were recovered in glucose. The unrecovered percentages
(74% and 80%) were lost by isotopic exchanges. Using Hetenyi's
nomenclature(34) , these numbers would correspond to dilution
factors of 3.8 and 4.9, respectively.
MIDA of glucose formed in the
presence of a C-labeled gluconeogenic precursor allows, in
principle, calculation of parameter f, i.e. the
fraction of glucose production derived from GNG. The (1 - f) unlabeled fraction of glucose production derives presumably
from preformed glycogen. In all experiments reported in rows
1-11, 14, and 15 of Table 1, f should have been
100% since the liver donors and the live rats had been starved for 2
days and thus depleted of liver glycogen(27) . Except for the
infusion of [U-
C
]lactate in live
rats (row 14), the lower than 100% f values would seem to
indicate the production of unlabeled glucose by the livers. Even more
incompatible with the MID theory is the variation of f in alternate
labeling experiments (compare f in rows 1 and 2 and in rows 3
and 4 of Table 1). Let us now show how these contradictory data
can be ascribed to variations in the MPE of TP across the lobule.
The concept of metabolic zonation (35) is based on variations in enzymatic activities in hepatocytes across the liver lobule. Striking examples of enzymatic zonation are the distributions of the enzymes of urea and glutamine metabolism(36) . Although there is no information on the zonation of glycerol kinase, other gluconeogenic enzymes are present at higher activities in periportal compared to pericentral hepatocytes(37) .
Although
variations in the activities of many enzymes across the lobule are well
established, our data suggest that such variations are not necessary to
induce metabolic zonation if (i) a substantial decrease in substrate
concentration occurs across the lobule and (ii) the range of substrate
concentration is close to or below the Kof the first limiting enzyme metabolizing the substrate. The K
of glycerol kinase for glycerol is
3-10 µM in rat
liver(22, 25, 26) , which is much lower than
the 0.78 mMK
for glycerol
uptake in the perfused rat liver (Fig. 1A). Therefore
glycerol uptake is transport-limited. and allow calculating the simulated profiles of relative
glycerol concentrations (C/C
) and
relative glycerol flux (J/J
) across the
liver lobule for different inflowing concentrations. When parameter
= 0, J
is constant across the
lobule. Fig. 3(A and B) shows these profiles
calculated if one assumes no zonation of glycerol kinase activity. Fig. 4(A and B) shows the corresponding
profiles if one assumes a linear decrease to zero of glycerol kinase
activity from periportal to pericentral cells (
= 1). Note
that for the low 0.1 mM initial glycerol concentration, C/C
and J/J
decrease by the same percentage (80%) across the lobule. This is
because the range of glycerol concentration is much lower than the K
for glycerol uptake. The curvature of
these profiles is greater when one assumes a linear decrease in
glycerol kinase activity across the lobule. At physiological influent
glycerol concentration (0.1 mM), the decrease in glycerol
concentration across the lobule, in a range much lower than the K
for glycerol uptake, results in marked
variations in the simulated relative flux of label into the cells (Fig. 3B and 4B).
Figure 3: Simulation of relative concentration of glycerol (panel A) and relative flux of glycerol (panel B) across the lobule. A constant glycerol kinase kinase activity is assumed. The simulations are run for three initial glycerol concentrations (0.1, 0.5, and 1.5 mM).
Figure 4: Simulation of relative concentration of glycerol (panel A) and relative flux of glycerol (panel B) across the lobule. The activity of glycerol kinase is assumed to decrease linearly to zero from the periportal to the pericentral area of the liver lobule. The simulations are run for three initial glycerol concentrations (0.1, 0.5, and 1.5 mM).
Let us turn to perfusions
with [U-C
]lactate and unlabeled
glycerol (Table 1, rows 1 and 3). The influent and effluent
concentrations of [U-
C
]lactate were
1.0 and 0.70 mM, respectively. The influent pyruvate
concentration was zero, while its effluent concentration was about 4
µMi.e. much lower than the 400 µMK
of pyruvate carboxylase for
pyruvate(38) . We cannot assess the profile of pyruvate
concentration across the lobule. Because of the high activity of liver
lactate dehydrogenase, it is likely that the intracellular
concentration of pyruvate (i) increased from zero to some unknown level
in the periportal area and (ii) decreased further down the lobule as
lactate concentration decreased by 30%. So the rate of pyruvate
carboxylation decreased by at least 30% between some periportal cells
and pericentral cells. The decrease in the rate of pyruvate
carboxylation might even be greater than 30%, given that the activity
of pyruvate carboxylase is greater in periportal than in pericentral
cells(37) .
In vivo, there is always some pyruvate
in plasma. So, to check whether the low f calculated from
perfusions with [U-C
]lactate (Table 1, rows 1 and 3) resulted from the absence of pyruvate in
the influent, we perfused some livers with 1 mM
[U-
C
]lactate + 0.2 mM [U-
C
]pyruvate + 0.1 mM glycerol in the absence and presence of 0.2 mM octanoate (Table 1, rows 5 and 6). The calculated f was still low
(62% and 87%). In these experiments, 86% and 95% of the influent
[U-
C
]pyruvate was taken up in a
single passage through the liver. Finally, to insure a large supply of
[U-
C
]pyruvate to all areas of the
liver lobule, we perfused livers with 1 mM
[U-
C
]pyruvate + 0.1 mM glycerol (Table 1, row 7). Contrary to our expectations, f was only 72%. Thus, in all liver perfusions with labeled
lactate and/or pyruvate, unrealistic low values of f were
calculated from the MID of glucose.
The variations of f when octanoate is added to the perfusate (Table 1, rows 3,
4, and 6) are difficult to explain. In the presence of
[U-C
]lactate, f is
decreased by octanoate (compare rows 1 and 3 of Table 1). In
contrast, in the presence of unlabeled lactate +
[U-
C
]glycerol or in the presence of
[U-
C
]lactate +
[U-
C
]pyruvate, f is
increased by octanoate (compare rows 2 versus 4 and rows 5 versus 6 of Table 1). We have no explanation for these
effects of octanoate. Still, somehow octanoate must affect the profile
of triose phosphate labeling across the liver lobule.
The above data lead us to conclude that, in livers perfused under the conditions of rows 1-11 of Table 1, the MIDs of TP and of synthesized glucose vary across the lobule. Therefore, the measured MIDs of effluent glucose and of liver PEP are composites, and not necessarily averages, of the MIDs of these compounds in cells across the lobule. Let us illustrate this point by two numerical examples.
Fig. 5shows the theoretical combination of two equal pools of
glucose made from two homogeneous pools of TP labeled from
[U-C
]glycerol. The TP are only
labeled in M
isotopomers with enrichments p
and p
of 20% and 5%,
respectively. Such conditions reflect the periportal and the
pericentral cells of a liver perfused with
[U-
C
]glycerol. Since each pool is
homogeneous, f
= f
= 100%. This condition and impose in each
primary pool the M
and M
MID
of glucose shown on Fig. 5. The combination of these two pools
results in a measured MID of glucose from which one computes () erroneous values for f (73.5%) and of the M
MPE of TP (17%). Although the two pools of
glucose were of equal size, the combined M
/M
ratio was not the average
of the individual ratios.
Figure 5:
Combination of two pools of glucose of
equal size made from triose phosphates labeled from
[U-C
]glycerol with a M
MPE (p) of 20% (pool 1) and 5% (pool
2). Each primary pool is constrained with a f of 100%. The
combined pool (which would be actually measured in liver effluent) had
a MID of glucose from which an artifactual f of 73.5% is
calculated.
Similar calculations can be made (Fig. 6) for 20 equal pools of glucose made from 20 pools of TP
whose M MPE decrease exponentially along the
lobule. The equation of decay is M
(pool i)
= 100
exp(-0.095
(i - 1)).
The rate constant 0.095 results in a decrease in the MPE of the TP to
15% of its original value between the 1st and the 20th pool. We
observed a similar decrease in the
[
H
]glycerol MPE in perfused livers (Fig. 1B). For each pool of TP, f =
100%. Fig. 6shows the enrichments of M
TP, M
glucose, and M
glucose
along a liver lobule divided into 20 compartments. Again, using , one computes impossible values for f (76%
instead of 100%) and of p, the integrated M
MPE of TP (60% instead of 45%). Although a 100% M
MPE of TP would not occur in vivo, this simulation
reflects the consequence of such exponential decrease from any initial
MPE. This simulation also shows that the M
/M
ratio in glucose varies
over a large range with p, i.e. the M
MPE of TP.
Figure 6:
Simulation of the decrease in the M MPE of triose phosphates across the liver
lobule. The simulation assumes 20 equal pools of glucose made from 20
pools of TP whose M
MPE decreases exponentially
from the periportal to the pericentral area. The equation of decay is M
(pool i) = 100
exp(-0.095
(i - 1)).
The only experiment where MIDA of glucose yields a
plausible f (97%) is in the case of 2-day starved rats infused
with unlabeled glycerol and
[U-C
]lactate (Table 1, row
14). Such high f must result from practically constant
enrichment of TP in the gluconeogenic cells. Note that the MID of liver
PEP in these experiments (Table 2) is still different from the
calculated MID of the TP. This may result from a different MID of PEP
in non-gluconeogenic pericentral hepatocytes. Also, the f with
[U-
C
]lactate is much higher in live
rats than in perfused rat livers. This may result from the
equilibration of the MPEs of plasma lactate and pyruvate, via red cell
metabolism, before and during passage through the liver. In contrast,
in isolated livers perfused with
[U-
C
]lactate in red cell-free
medium, the first periportal cells are probably not in contact with
[U-
C
]pyruvate. Also, the rate of
perfusion of red cell-free perfusate (4-5 ml
g
min
) is higher than the rate of blood perfusion in vivo (1 ml
g
min
). The
shorter transit time of labeled substrates through isolated livers and
the absence of red cells do not allow equilibration of the MPE of
lactate and pyruvate when only one of these two substrates is infused
in the non-recirculating medium. We realize that, in isolated livers, f is lower than in vivo even when livers are perfused
with a mixture of [U-
C
]lactate
+ [U-
C
]pyruvate (Table 1,
rows 5 and 6). The marked increase in the
[lactate]/[pyruvate] ratio between influent and
effluent perfusates (from 5 to 23) reflects the lack of equilibration
of lactate and pyruvate concentrations in these perfusions. In
contrast, with labeled glycerol where the problem of equilibration of
the two partners of a redox couple does not arise, the perfused liver
data and the in vivo data are very compatible.
In 18-h
starved monkeys infused with
[U-C
]lactate, f was 80% (Table 1, row 16). This probably reflects a 20% contribution of
preformed liver glycogen to glucose production. Although less than what
was reported in 14-h fasted humans (23-42%; (39) ) using
the incorporation of
H from
H
O on
C-2 and C-6 of glucose, this percentage is reasonable for a small
animal with a high metabolic rate. A similar value of f (82%)
was reported by Lee et al.(40) in 24-h starved rats
infused intragastrically with
[2,3-
C
]lactate.
The calculated
total labeling of TP was much higher than the measured total labeling
of PEP when [U-C
]glycerol (but not
[U-
C
]lactate) was the labeled
substrate (Table 2, last column). This, in itself, does not
reflect zonation of TP labeling from
[U-
C
]glycerol but different degrees
of isotopic equilibrium between TP and PEP. Consider, for example, the
pair of alternate labeling experiments whose data are presented in Table 2(rows 1 and 2) and in Table 3(first column of
data). Roughly, in these experiments, glycerol and lactate contribute
equally to glucose production (Table 3, rows K and L and first
data column). The ratio (total
C in TP)/(total
C in PEP) depends on the degree of reversibility of the
steps between TP and PEP. When
[U-
C
]glycerol +
[
C]lactate are used, the ratio ranges from
(no reversibility) to 1.0 (reversibility at infinite velocity).
With [
C]glycerol +
[U-
C
]lactate, the corresponding
range of the ratio is 0.5-1.0. A ratio of 4.7 with unlabeled
lactate + [U-
C
]glycerol (Table 2, row 2) shows reversibility between TP and PEP. A ratio
slightly greater than 1.0 with
[U-
C
]lactate + unlabeled
glycerol (Table 2, row 1) reflects probably (i) the effect of
some errors in the many numbers that make up this ratio and/or (ii)
heterogeneity of PEP labeling across the liver lobule (e.g. PEP in pericentral non-gluconeogenic hepatocytes may be less
labeled than in periportal gluconeogenic hepatocytes).
In the in
vivo experiments with
[U-C
]glycerol (Table 1, rows
15 and 17), p was 3/4 and 6/10 those of the corresponding
experiments with [U-
C
]lactate (rows
14 and 16). This confirms the unsuitability of labeled glycerol for
assessing the contribution of GNG to glucose production(14) .
Our data contrast with those of Hellerstein's group(41) ,
who reported similar values for f (87-90%) in 48-h
starved rats infused with [3-
C]lactate,
[1-
C]lactate, and
[2-
C]glycerol. We found very different f when using lactate versus glycerol tracers (Table 1, rows 14-17). Elsewhere(42) , they report
that, in 24-h starved rats infused with
[2-
C]glycerol, f was 86%. However, the
glycogen content of the rats' livers (<5 mg
g
) could not supply the missing 14% of the glucose
turnover (7 mg
min
kg
) for more than 2-3 h.
We draw the
following conclusions from our study. First, our data identify and
confirm limitations of MIDA for measuring the contribution of GNG to
glucose production. In this study conducted in animal models, and in
our previous study conducted in humans(14) ,
[C]glycerol yields artifactually low
contributions of GNG to glucose production. This is ascribed to
metabolic zonation of glycerol metabolism across the liver lobule. This
zonation does not necessarily require variations in glycerol kinase
activity, but can be mostly ascribed to (i) the large decrease in
glycerol concentration across the lobule and (ii) the low range of
glycerol concentrations compared to the K
of glycerol uptake by the liver. Second, our data suggest
that [U-
C
]lactate is a suitable
substrate for MIDA of GNG in vivo. This is not the case for
perfused livers, presumably because of disequilibrium of labeling
between lactate and pyruvate across the lobule, resulting in marked
variations in the MPE of TP. Third, measurements of the MID of glucose
are more precise with uniformly labeled than with singly labeled
C substrates. In the latter case, M
/M
ratios in glucose are
very sensitive to a small error in the fairly high background
correction at M
. Fourth and last, correction of
measured MID for natural enrichment should take into account (i) the
variability of measured natural MID compared to theoretical values (18) and (ii) the skew of natural
C enrichment in
multiply labeled compounds(18, 19) .