Biochem. J. (2006) 394, 465–473 (Printed in Great Britain)
465
doi:10.1042/BJ20051174
Effects of insulin and cytosolic redox state on glucose production pathways
in the isolated perfused mouse liver measured by integrated 2 H and 13 C NMR
Natasha HAUSLER*, Jeffrey BROWNING*, Matthew MERRITT*, Charles STOREY*, Angela MILDE*, F. Mark H. JEFFREY*,
A. Dean SHERRY*†, Craig R. MALLOY* and Shawn C. BURGESS*1
*Advanced Imaging Research Center, University of Texas Southwestern Medical Center, 5801 Forest Park Road, Dallas, TX 75235-9085, U.S.A., and †Department of Chemistry,
University of Texas at Dallas, Dallas, TX 75083-0688, U.S.A.
A great deal is known about hepatic glucose production and its response to a variety of factors such as redox state, substrate supply
and hormonal control, but the effects of these parameters on the
flux through biochemical pathways which integrate to control
glucose production are less clear. A combination of 13 C and
[2 H]water tracers and NMR isotopomer analysis were used to
investigate metabolic fluxes in response to altered cytosolic redox
state and insulin. In livers isolated from fed mice and perfused with
a mixture of substrates including lactate/pyruvate (10:1, w/w),
hepatic glucose production had substantial contributions from
glycogen, PEP (phosphoenolpyruvate) and glycerol. Inversion
of the lactate/pyruvate ratio (1:10, w/w) resulted in a surprising
decrease in the contribution from glycogen and an increase in that
from PEP to glucose production. A change in the lactate/pyruvate
ratio from 10:1 to 1:10 also stimulated flux through the tricarboxylic acid cycle (2-fold), while leaving oxygen consumption
and overall glucose output unchanged. When lactate and pyruvate
INTRODUCTION
The rate of glucose production by the liver is sensitive to cellular
redox state [1], which in turn is altered by a number of factors,
including fasting [2], ethanol ingestion [3], exercise [4], hormones
[5,6], and systemic illness [7]. Cytosolic oxidation–reduction
reactions occur in GNG (gluconeogenesis) at two sites. One is the
conversion of glycerol into glycerol 3-phosphate, followed by oxidation to dihydroxyacetone phosphate, catalysed by glycerol3-phosphate dehydrogenase. This results in production of one
NADH molecule. The other is conversion of 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate by GAPDH
(glyceraldehyde-3-phosphate dehydrogenase). This step consumes 1 equiv. of NADH. Hence, a high NADH/NAD+ ratio
typical of normal physiological conditions might be expected to
drive GNG from PEP (phosphoenolpyruvate) and inhibit GNG
from glycerol. This prediction is consistent with conventional
thinking that the origin of most gluconeogenic carbon comes
from the level of activity of the tricarboxylic acid cycle. Further
evidence for a physiologically significant role of cytosolic redox
in control of GNG comes from the observation that glucose
production is maximal when the ratio of lactate to pyruvate is ≈ 10
and decreases as levels of pyruvate increase [1]. An important role
of glycerol in GNG has also been postulated in Type II diabetes
[8]. Wolfe and colleagues [9] found that GNG from glycerol
contributed more than 20 % to plasma glucose in humans after
were eliminated from the perfusion medium, both gluconeogenesis and tricarboxylic-acid-cycle flux were dramatically lower.
Insulin lowered glucose production by inhibiting glycogenolysis
at both low and high doses, but only at high levels of insulin
did gluconeogenesis or tricarboxylic-acid-cycle flux tend towards
lower values (P < 0.1). Our data demonstrate that, in the isolated
mouse liver, substrate availability and cellular redox state have
a dramatic impact on liver metabolism in both the tricarboxylic
acid cycle and gluconeogenesis. The tight correlation of these
two pathways under multiple conditions suggest that interventions
which increase or decrease hepatic tricarboxylic-acid-cycle flux
will have a concomitant effect on gluconeogenesis and vice versa.
Key words: 2 H and 13 C nuclear magnetic resonance (NMR),
gluconeogenesis, glycogenolysis, liver, redox state, tricarboxylic
acid cycle.
prolonged fasting, whereas we found that GNG from glycerol
was also increased among HIV-positive subjects with lipoatrophy
[10]. Together, these observations suggest that the contribution of
glycerol to glucose production may be significant under some
conditions and that this contribution may be controlled to
some extent by cytosolic redox.
Hepatic GLY (glycogenolysis) and GNG are also influenced by
circulating levels of insulin via direct binding to insulin receptors
[11] and secondary substrate moderation due to insulin’s action
on peripheral tissue [12]. It is well known that hepatic GLY is
inhibited by insulin [13], but the direct effect of insulin on hepatic
GNG is less clear. Among early in vitro rat liver studies, insulin was shown to suppress GNG [14], whereas other studies
found that insulin had no effect on GNG [15]. Even in more
recent literature, the mechanism of insulin action on glucose
production is not clear. In normal human subjects, Gastaldelli and
co-workers showed that, after an overnight fast, GNG decreased
significantly in the presence of insulin [16]. However, in fasted
conscious dogs, there was a non-significant decrease in GNG
during a 3 h portal-venous infusion of insulin [17]. Insulin
influences hepatic GNG by powerfully regulating the transcription
of important gluconeogenic enzymes [18,19], but the effect of
enzyme expression on glucose production may not be immediate,
since the mRNA half-life of many gluconeogenic enzymes is close
to 1 h [20] and the enzyme itself takes several hours to degrade
[21]. On the other hand, the supply of gluconeogenic substrates to
Abbreviations used: ACAC, acetoacetate; BHB, β-hydroxybutyrate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLY, glycogenolysis;
GNG, gluconeogenesis; MAG, monoacetone glucose (1,2-O -isopropylidene-α-D-glucofuranose); OAA, oxaloacetate; PEP, phosphoenolpyruvate; PEPCK,
phosphenolpyruvate carboxykinase; PK, pyruvate kinase.
1
To whom correspondence should be addressed (email [email protected]).
c 2006 Biochemical Society
466
N. Hausler and others
the liver has an immediate impact on glucose homoeostasis by increasing GNG [22] and decreasing GLY [23]. Thus an indirect
action of insulin on substrate supply from the periphery becomes
a more appealing mechanism to explain changes in hepatic GNG
[12].
Although the fractional contributions of glycogen, glycerol,
carboxylic acids (particularly pyruvate and lactate) and amino
acids to glucose production may vary widely, intricate connectivities among reactions and compartments result in uninterrupted
production of glucose at a rate tightly coupled to peripheral
utilization of glucose. Extensive information is available about
oxidation–reduction mechanisms in individual reactions of glucose production, and it is likely that redox state is a significant
factor in balancing fluxes among multiple reaction pathways.
However, remarkably little is known about the influence of redox
state on the fractions of glucose derived from glycerol, the tricarboxylic acid cycle and glycogen.
NMR and stable-isotope-tracer methods may now be used
to obtain quantitative data on the sources of glucose carbon
atoms in gluconeogenic tissues. In the present study the impact
of simple manipulation of cytosolic redox and insulin on the
sources of hepatic glucose was evaluated under conditions where
glycogen, glycerol and other gluconeogenic precursors were not
limiting. Glucose production and oxygen consumption by the
isolated perfused liver was insensitive to the ratio of lactate
and pyruvate in the perfusate, whereas the relative contributions
of GLY and GNG from PEP (GNGPEP ) were dramatically altered by a shift in cytosolic redox state. Yet, the contribution
of glycerol to glucose production (GNGglycerol ) was unaffected
by redox state. At near normal physiological concentrations,
insulin had no effect on GNGPEP , but it dramatically reduced
GLY in the presence of lactate and pyruvate. In the absence
of lactate and pyruvate, insulin’s affect on GLY was abolished.
Under hyperinsulinaemic levels of insulin, GLY was inhibited
and GNGPEP and tricarboxylic-acid-cycle flux also tended to
lower values. Tricarboxylic-acid-cycle flux and GNGPEP were
dramatically suppressed when lactate and pyruvate were excluded
from the perfusion medium. Our data demonstrate that substrate
availability and cellular redox state have a striking impact on liver
metabolism by altering fluxes through the tricarboxylic acid cycle
and GNG. The close correlation between hepatic tricarboxylicacid-cycle flux and GNG suggests that interventions in one
pathway will have a parallel effect on the other.
MATERIALS AND METHODS
Chemicals
[U-13 C3 ]Propionate (99 %) and [2 H]water (99 %) were purchased
from Cambridge Isotopes (Andover, MA, U.S.A.). DSC-18 solidphase extraction gel and other common chemicals were purchased
from Sigma (St. Louis, MO, U.S.A.) unless otherwise noted.
and perfused with a buffered medium described below. Once
perfusion was initiated, the livers were dissected and suspended in
a perfusion apparatus where temperature was maintained at 37 ◦C
with a temperature-controlled water jacket. Efferent and afferent
pO2 was measured using a blood gas analyser (Instrumentation
Laboratory, Lexington, MA, U.S.A.) to determine oxygen consumption [24]. Hepatic viability was evaluated by oxygen
consumption and visual inspection. Approx. 25 % of livers were
rejected because they did not maintain a pO2 gradient of
100 mmHg (1 mmHg = 133.3 Pa) or if their visual colouring
changed during the experiment. During insulin experiments,
one of six livers was rejected as an outlier, by a Grubb’s test,
because of a high rate of GLY.
Each liver was perfused at a rate of 10 ml/min with a
non-recirculating media made of a modified Krebs–Henseleit
buffer containing 118 mM NaCl, 5 mM KCl, 1.2 mM MgSO4 ,
1.25 mM CaCl2 , 0.2 mM sodium octanoate, 0.25 mM glycerol,
0.5 mM sodium [U-13 C3 ]propionate, 3 % 2 H2 O (Cambridge
Isotopes) and saturated with O2 /CO2 (19:1), pH 7.4. The perfusion
medium also contained either (a) 1.5 mM lactate and 0.15 mM
pyruvate, (b) 0.15 mM lactate and 1.5 mM pyruvate, (c) no lactate
and pyruvate, (d) 0.81 ng/ml insulin plus 1.5 mM lactate and
0.15 mM pyruvate, (e) 0.81 ng/ml insulin alone, or (f) 48.6 ng/
ml insulin plus 1.5 mM lactate and 0.15 mM pyruvate. Livers
were perfused for 1 h, and effluent was collected from every
15 min to measure glucose production and O2 consumption
and to determine 2 H and 13 C enrichment patterns. Glucose
was determined using a standard hexokinase/glucose-6-phosphate
dehydrogenase coupled assay [25] to evaluate glucose output
in units of µmol · min−1 · g wet weight−1 . ACAC (acetoacetate)
and BHB (β-hydroxybutyrate) were measured by the method of
Williamson et al. [26]. The rate of ACAC and BHB production
were summed to represent the rate of ketogenesis.
Glucose isolation and conversion into MAG [monoacetone glucose
(1,2-O -isopropylidene-α-D-glucofuranose)]
Effluent medium was dried on a rotary evaporator. The resulting
salt was washed with 3 × 10 ml of methanol/water (95:5, v/v)
to extract glucose. The washes were filtered to remove residual
salts, and methanol was removed by rotary evaporation. After
drying, 4 ml of deionized water was added to the material and
it was applied to an ion-exchange column containing 15 ml of
cation- (Dowex 50WX8-200) and 15 ml of anion- (Amberlite
IRA-67) exchange resin (Sigma–Aldrich, St. Louis, MO, U.S.A.)
for purification. The overall yield of glucose taken through this
extraction process was ≈ 80 %. The 1 H-NMR spectrum of glucose
is complicated by poor chemical-shift dispersion and the presence
of two anomers in solution, whereas a simple derivative of
glucose, MAG, has excellent NMR properties [27]. Thus all glucose samples isolated from perfusate were converted into MAG
and purified on DSC-18 columns (Supelco, Bellefonte, PA,
U.S.A.) prior to NMR analysis as previously described [28,29].
Experimental design
The protocol was approved by the Institutional Animal Care and
Use Committee at University of Texas Southwestern Medical
Center. Livers from female C57Bl/6 mice fed ad libitum prior
to experimentation and ranging in weight from 18 to 22 g were
isolated for perfusion. Each mouse was injected with heparin
(0.05 ml, intraperitoneal) 15 min prior to a 0.1 ml intraperitoneal
injection of anesthetic [ketamine (Fort Dodge Animal Health)/
xylazine (Boehringer Ingelheim), 85:15, w/w]. Once anesthetized,
a midline laparotomy was performed to expose the hepatic portal
vein and the livers were isolated and dissected using a previously
described technique [24]. Briefly, the portal vein was cannulated
c 2006 Biochemical Society
NMR methods
MAG was dissolved in 160 µl of HPLC-grade acetonitrile with 5–
10 µl of water and transferred to a 3-mm-diameter NMR tube. 2 HNMR spectra were collected with an actively shielded 600 MHz
Varian INOVA spectrometer and a 3 mm broadband probe tuned
to 92 MHz as previously described [28]. Spectra were averaged
over 4 h with the sample temperature held at 50 ◦C. A 20 µl
portion of [2 H]acetonitrile was added to the MAG sample for
locking purposes, and 13 C NMR spectra were collected with the
spectrometer and probe tuned to 150 MHz as previously described
[28]. A 50◦ pulsewidth, 1.5 s acquisition time and no further delay
Redox and glucose production pathways
467
in which OAA is converted into pyruvate, and then reconverted
into OAA. In the liver, PEPCK/PK (pyruvate kinase) is the likely
pathway, but again, malic enzyme or other pathways may also
make a contribution. To the extent that other pathways convert
tricarboxylic-acid-cycle intermediates to pyruvate, v6 will be
an overestimation of flux through PEPCK and v5 will be an
overestimation of flux through PK. These relationships have been
described in detail elsewhere [28].
Statistics
All results are expressed as means +
− S.D. Comparisons between
fluxes were made using a Student’s two-tailed t test, where
P < 0.05 was considered significant.
RESULTS
Scheme 1
tracers
Metabolic pathways probed by the combination of 2 H and
13
C
Hepatic glucose production (v 1) has contributions from v 2 (glycogenolysis, GLY), v 3 (GNG
from glycerol, GNGglycerol ), v 4 (GNG from phosphoenolpyruvate, GNGpep ) as determined by 2 H
NMR of perfusate glucose. PEPCK flux is presumed to be the major constituent of total efflux
from the hepatic tricarboxylic acid cycle, represented by v 6. Pyruvate cycling, v 5, denotes
pathways such as PK or the malic enzyme which regenerate pyruvate rather than contributing to
GNG. Tricarboxylic-acid-cycle (TCA Cycle) flux is shown as v 7.
time were found to provide the highest sensitivity for MAG 13 C
spectra.
Metabolic-flux determination
Glucose released from the liver becomes enriched with 2 H at different sites depending on its synthetic origin. 2 H-NMR spectroscopy was used to determine the relative 2 H enrichments in the H2,
H5, and H6s resonances of MAG. The fraction of effluent glucose
from each source was calculated as follows [27,28,30,31]:
Fraction from glycogen = (H2 − H5)/H2
Fraction from glycerol = (H5 − H6s)/H2
Fraction from PEP = H6s/H2
The fractional contribution of glycogen, glycerol and PEP to
glucose production was multiplied by the measured glucose
production rate to determine the absolute flux from each
source [28]. Relative fluxes in the tricarboxylic acid cycle were
determined by 13 C NMR analysis of MAG. Briefly, [U-13 C3 ]propionate is used to enrich tricarboxylic-acid-cycle intermediates,
including OAA (oxaloacetate), which serves as a substrate for
GNG. The 13 C labelling pattern in OAA (and therefore effluent
glucose) reflects fluxes associated with the tricarboxylic acid
cycle. Specifically, the C2 resonance in the 13 C spectrum of effluent glucose (after conversion into MAG) was used to determine
anaplerosis, pyruvate cycling and GNG from PEP relative to
tricarboxylic-acid-cycle flux [32]. Normalizing this latter relative
flux with the absolute flux, GNGPEP , determined from glucose production and 2 H NMR results in absolute fluxes for all of the pathways shown in Scheme 1 [28]. It is important to note that v6
in Scheme 1 is determined from total anaplerosis, which must
match total tricarboxylic-acid-cycle efflux. In the liver this flux
is assumed to be predominately through PEPCK (phosphenolpyruvate carboxykinase), but may also have a contribution from
other efflux pathways such as malic enzyme (malate dehydrogenase), for instance. Thus v6 represents a maximal estimation
of PEPCK flux. Pyruvate cycling, v5, represents the futile cycle
Steady-state enrichment of effluent glucose
In initial studies, effluent perfusate was collected in 15 min blocks
(0–15, 16–30, 31–45 and 46–60 min), and the relative resonance
areas in the 2 H- and 13 C-NMR spectra of MAG from each sample
were compared. As there were no significant differences in spectra
of samples collected over the last three time blocks, all subsequent
metabolic measurements were made on effluent collected and
combined over the period 16–60 min.
Oxygen consumption and glucose production
Glucose output and oxygen consumption for the five experimental
conditions are summarized in Table 1. There was no significant
difference in either variable for the 1:10 or the 10:1 (w/w) lactate/
pyruvate (control) groups, but removal of both lactate and pyruvate from the perfusate in the absence of insulin resulted in
decreased glucose production (31–37 %) without a corresponding decrease in oxygen consumption. Addition of a near-physiological concentration of insulin (0.81 ng/ml) to livers perfused
with 10:1 lacate/pyruvate resulted in a 37 % decrease in glucose
production without a corresponding change in O2 consumption.
Removal of both lactate and pyruvate from the perfusate in the
presence of insulin resulted in a further significant decrease in both
glucose production and O2 consumption. Increasing insulin to a
hyperinsulinaemic concentration (48.6 ng/ml) further decreased
hepatic glucose production to 50 % of that of the control group.
Sources of glucose
Typical 2 H-NMR spectra of MAG derived from glucose produced
for a selection of the conditions are shown in Figure 1. The peak
area ratio H5/H2 in the 2 H spectrum of MAG (Figure 1B) derived glucose produced by a representative liver perfused with
lactate and pyruvate in a 10:1 ratio (considered normal physiologically) was 0.80. This indicates that 20 % of the glucose produced by liver under physiological redox conditions comes from
glycogen, whereas the remainder is gluconeogenic: 35 %
from PEP and 45 % from glycerol. The contribution of glycogen to glucose production was quite different when the lactate/
pyruvate ratio was reversed to 1:10. Under these conditions, the
contribution from glycogen decreased to near zero (as indicated
by an H5/H2 ratio ≈ 1 in the spectrum of Figure 1A), whereas
the contribution from PEP increased dramatically from 35 to
60 % (Table 1). Interestingly, the contribution of glycerol to glucose in this experiment tended towards a lower value, but
this did not reach statistical significance. Addition of insulin
to the perfusion medium at physiological redox conditions
(lactate/pyruvate = 10:1) produced effects similar to those
c 2006 Biochemical Society
468
Table 1
N. Hausler and others
Relative fluxes obtained from NMR analysis in isolated perfused livers
Fluxes v 1–v 7 are illustrated in Scheme 1. Livers from fed mice were perfused with non-recirculating media containing various concentrations of lactate (Lac), pyruvate (Pyr) and insulin.
Flux (µmol · min−1 · g wet weight−1 ) (mean +
− S.D.)
[Insulin] (ng/ml) . . . None
Lac/Pyr . . . 10:1
Glucose production (v 1)
O2 consumption
Data from 2 H NMR: fractional sources of effluent
glucose
Glycogen (v 2/v 1)
Glycerol (v 3/v 1)
PEP (v 4/v 1)
Data from 13 C NMR: fluxes associated with the
tricarboxylic acid cycle relative to citrate synthase
PEPCK pathway† (v6/v7)
Pyruvate cycling (v5/v7)
Gluconeogenesis from PEP (v 4/v 7)
0.81
48.6
1:10
None
10:1
None
10:1
0.93 +
− 0.16
2.4 +
− 0.43
n=7
1.0 +
− 0.20
2.4 +
− 0.56
n=6
0.69 +
− 0.17*
2.3 +
− 0.10
n=5
0.59 +
− 0.21*
2.3 +
− 0.18
n=5
0.32 +
− 0.12*
1.8 +
− 0.16*
n=5
0.43 +
− 0.28*
2.3 +
− 0.30
n=4
0.20 +
−6
0.45 +
− 0.10
0.35 +
− 0.07
n=7
0.04 +
− 12*
0.36 +
− 0.10
0.60 +
− 0.08*
n=6
0.46 +
− 8*
0.46 +
− 0.12
0.09 +
− 0.07*
n=5
0+
− 0.06*
0.55 +
− 0.17
0.45 +
− 0.16
n=5
0.49 +
− 0.15*
0.44 +
− 0.22
0.08 +
− 0.08*
n=5
0.02 +
− 0.06*
0.64 +
− 0.10*
0.34 +
− 0.05
n=4
6.3 +
− 0.84
3.1 +
− 0.66
3.2 +
− 0.56
n=7
5.4 +
− 0.81
2.3 +
− 0.49*
3.1 +
− 0.34
n=6
5.0 +
− 2.13
2.5 +
− 1.07
2.5 +
− 1.06
n=3
6.0 +
− 0.29
3.0 +
− 0.17
3.0 +
− 0.24
n=5
6.2 +
− 0.41
3.1 +
− 0.35
3.1 +
− 1.51
n=5
5.7 +
− 0.41
3.0 +
− 0.33
2.7 +
− 0.45*
n=4
* Significantly different from 10:1 lactate/pyruvate group (column 1), P < 0.05.
† Also includes contributions from other efflux pathways such as that involving malic enzyme.
produced by a switch in redox. In this case the H5/H2 ratio was
close to 1, indicating again that little glucose was derived from
glycogen, while the difference was made up nearly equally
from glycerol (55 %) and PEP (45 %; see Table 1). When both
pyruvate and lactate were absent from the perfusion medium in
either the presence or absence of insulin, the fractional contribution of PEP to glucose decreased precipitously, whereas the
fractional contribution of glycerol increased to make up the difference (Table 1). The contribution of glycerol to glucose did not
differ dramatically among the six perfusion conditions, although
it was significantly increased under the high-insulin conditions.
Given these data and the overall glucose production rates in
Table 1, the absolute flux of glucose coming from each pathway
was evaluated (Table 2). Here, glucose production from tricarboxylic-acid-cycle intermediates (v4, in units of µmol · min−1 · g
wet weight−1 ) is represented by GNGPEP (in triose units),
GNGglycerol represents the flux from glycerol to glucose (v3, in
triose units), whereas GLY represents flux from glycogen to
glucose (v2, in hexose units). A switch from physiological redox
(10:1 lactate/pyruvate) to a highly oxidized state (1:10 lactate/
pyruvate) resulted in an approx. 5-fold decrease in GLY, an approx. 2-fold increase in GNGPEP , and no change in GNGglycerol . Removal of both lactate and pyruvate from the perfusate resulted in a
slight but significant increase in GLY, a near complete impairment
of GNGPEP and again no change in GNGglycerol compared with
normal physiological redox levels (10:1 lactate/pyruvate). When
propionate, the only other gluconeogenic precursor present in the
perfusate, was also removed from the perfusate, GNG from PEP
was essentially zero, as reported by the near absence of signal in
H6S of the 2 H-NMR spectrum of MAG (results not shown).
Insulin also had a dramatic effect on GLY in the perfused mouse
liver. As shown in Table 2, 0.81 ng/ml insulin abolished GLY in
fed livers, reducing it from 0.21 µmol · min−1 g wet weight−1
to essentially zero at normal physiological redox levels. This
amount of insulin had no effect on gluconeogenic fluxes from
either GNGPEP or GNGglycerol . Despite the powerful inhibition of
GLY by insulin in livers perfused with 10:1 lactate/pyruvate,
when lactate and pyruvate were excluded from the perfusion
c 2006 Biochemical Society
medium, GLY returned to normal. Since insulin’s direct influence
on gluconeogenic flux is uncertain, we also perfused livers
with a hyperinsulinaemic concentration (48.6 ng/ml). Under these
conditions, GLY was suppressed, and flux through GNGPEP was
also suppressed by approx. 2-fold (P < 0.1).
Tricarboxylic acid cycle and related pathways
To assess the relationship between the tricarboxylic acid cycle and
GNGPEP , the perfusion media also contained [U-13 C]propionate
and the resulting glucose isotopomers were evaluated by 13 C
NMR [32]. A typical 13 C spectrum of MAG derived from effluent
glucose is shown in Figure 2. The insets show expanded C2
resonances of MAG derived under three different physiological
perfusion conditions. The individual C2 multiplet components
(D12, D23, Q) were deconvoluted and those areas were used
to perform a 13 C isotopomer analysis of biochemical fluxes into
and out of the tricarboxylic acid cycle [32]. Differences between
multiplets from perfusion with high lactate (Figure 2B) versus low
lactate (Figure 2A) were relatively minor, with a slight increase
noted in the D23 component relative to the central singlet (S) and
a slight decrease in the D12 component relative to the quartet (Q).
Removal of both lactate and pyruvate from the perfusion medium
resulted in low 13 C enrichment of glucose (MAG) as illustrated
by the low signal-to-noise in the C2 resonance (Figure 2C).
As 13 C was derived from exogenous [U-13 C]propionate in these
experiments, the low 13 C enrichment in this sample is consistent
with a low contribution of GNGPEP as is also demonstrated by 2 H
NMR (Figure 1C).
Flux through PEPCK (v6/v7; also includes flux through the
malic enzyme), pyruvate cycling (v5/v7; includes PK and malic
enzyme) and gluconeogenic flux from PEP (v4/v7), all relative
to tricarboxylic-acid-cycle flux, were determined from 13 C-NMR
data [32] and values are shown in Table 1. Interestingly, these
values are relatively independent of the ratio of lactate to pyruvate
in the perfusate and even of the absence of lactate and pyruvate.
Since GNG from PEP (v4/v7, estimated by 13 C NMR) is related
to the fraction of glucose production derived from PEP (v4/1,
Redox and glucose production pathways
469
Since octanoate is known to stimulate considerable ketogenesis,
even in livers from fed animals [33], we measured ketone
production in a group of livers perfused with 10:1 lactate/pyruvate
and without lactate and pyruvate (Figure 3). We found that total
ketone production was substantial under both of these conditions,
but removing the lactate and pyruvate from the perfusate increased
ketone production by about 20 % over 30 min.
DISCUSSION
Figure 1 Typical 2 H spectra of MAG from isolated perfused mouse livers
under varying redox conditions in the presence and absence of insulin
Proton-decoupled 2 H-NMR spectra of MAG derived from glucose are shown. Glucose was
produced by mouse livers supplied with 0.2 mM octanoate, 0.25 mM glycerol, 0.5 mM sodium
[U-13 C3 ]propionate, and 3 % 2 H2 O plus (A) 0.15 mM lactate/1.5 mM pyruvate, (B) 1.5 mM
lactate/0.15 mM pyruvate, or (C) 1.5 mM lactate/0.15 mM pyruvate plus 0.81 ng/ml insulin,
and (D) no lactate, pyruvate or insulin.
estimated by 2 H NMR), the two measurements were normalized to
each other and glucose production was used to convert the relative
fluxes into absolute fluxes [28]. Those data are summarized in
Table 2.
Significant changes in absolute flux were detected in response
both to changes in redox and to the removal of lactate and
pyruvate. Switching lactate/pyruvate from 10:1 to 1:10 resulted
in almost a 2-fold increase in flux through PEPCK (v6, Table 2),
whereas complete removal of lactate and pyruvate from the
perfusate decreased PEPCK flux to almost zero. Neither PEPCK
nor pyruvate cycling (v5, Table 2) fluxes were affected by addition
of 0.81 ng/ml insulin to livers perfused with 10:1 lactate/pyruvate,
but removal of lactate and pyruvate from livers perfused with
0.81 ng/ml insulin caused a dramatic decrease in flux through
PEPCK, pyruvate cycling and the tricarboxylic acid cycle (v7,
Table 2). When the insulin concentration was increased to
48.6 ng/ml, PEPCK, pyruvate cycling and tricarboxylic-acidcycle fluxes decreased 2-fold (P < 0.1).
The isolated perfused liver has been used to study hepatic
metabolism with regard to GNG, glycogen and protein turnover
[34–36] and the influence of fatty acids and hormones on
hepatic glucose production [1,2,5,37,38]. Relative flux values
through various pathways in isolated perfused rat livers have been
measured using a combination of stable isotope tracers and NMR
or MS [32,39]. Absolute fluxes through the tricarboxylic acid
cycle and associated pathways have been measured by an elegant
combination of mass-isotopomer-distribution analysis (‘MIDA’)
and substrate balance across the perfused rat liver [40] or by
using a rigorous stoichiometric analysis of metabolite uptake and
production [41]. More recently we used the isolated perfused
mouse liver in conjunction with NMR spectroscopy to study
glucose and energy metabolism in the isolated perfused mouse
liver from liver specific PEPCK-null mice [24]. The primary goal
of the present study was to evaluate the influence of variable redox
state, substrate supply and insulin on glucose production, sources
of glucose, O2 consumption and energy homoeostasis in livers
from freely fed mice using a combination of stable isotope tracers
and NMR spectroscopy [24,27,28,32,42].
The power of NMR isotopomer analysis for examining liver
metabolism is that the sources of glucose production and metabolic fluxes associated with the tricarboxylic acid cycle can
be measure simultaneously. Applications of such measurements
to questions associated with diabetes and obesity are obvious.
However, developing a better understanding of hepatic energy
homoeostasis is equally important, because both NADH and ATP
are required for glucose synthesis and these energy sources are met
largely by oxidation of fats. It has been shown that hepatic oxygen
consumption and fatty acid oxidation tend to parallel GNG in the
isolated perfused rat liver [37,43]. Similarly, the link between
GNG and energy homoeostasis has also been demonstrated
by the development of hepatic steatosis in mice having liverspecific omission of PEPCK [24]. We recently showed that the
lack of gluconeogenic activity and lipid accumulation in these
livers are related to each other, in part, by decreased fat oxidation in the tricarboxylic acid cycle [24]. The relationship
between glucose metabolism and energy homoeostasis may be
facilitated by cellular redox state (the NAD+ /NADH ratio) [2]. For
example, fatty acid oxidation enhances glucose production and
also increases the ratio of NADH/NAD+ [37]. Also, manipulating
the hepatic NADH/NAD+ ratio with redox pairs such as lactate/
pyruvate dramatically influences glucose production [1]. The
present study demonstrates that the cytosolic redox state influences hepatic glucose metabolism at multiple levels, most surprisingly at GLY but also at PK (as a part of pyruvate cycling)
and the flux from pyruvate to PEP.
Effects of changes in cytosolic redox state on liver metabolism
The redox state of the hepatocyte is the sum, over time,
of NADH-producing pathways (β-oxidation, glycolysis and
the tricarboxylic acid cycle) balanced by pathways that
utilize NADH (oxidative phosphorylation and GNG). The
lactate/pyruvate ratio is related to the cytosolic redox state via the
c 2006 Biochemical Society
470
Table 2
N. Hausler and others
Absolute fluxes determined from NMR data and the rate of glucose production
Fluxes v 1–v 7 are illustrated in Scheme 1. Livers from fed mice were perfused with non-recirculating medium containing various concentrations of lactate (Lac), pyruvate (Pyr) and insulin.
Absolute flux (µmol · min−1 · g wet weight−1 )
[Insulin] (ng/ml) . . .
Lac/Pyr . . .
Flux of substrate to glucose
GLY (v 2)
GNGglycerol (v 3)
GNGPEP (v 4)
None
0.81
48.6
1:10
None
10:1
None
10:1
10:1
0.21 +
− 0.05
0.84 +
− 0.16
0.60 +
− 0.23
n=7
0.03 +
− 0.12*
0.75 +
− 0.32
1.2 +
− 0.23*
n=6
0.33 +
− 0.14
0.63 +
− 0.16
0.18 +
− 0.08*
n=3
0.0 +
− 0.03*
0.67 +
− 0.36
0.51 +
− 0.17
n=5
0.16 +
− 0.07*
0.27 +
− 0.20*
0.08 +
− 0.04*
n=5
0.01 +
− 0.03*
0.53 +
− 0.32
0.30 +
− 0.20†
n=4
2.1 +
− 0.41*
0.91 +
− 0.19*
0.40 +
− 0.08*
n=6
0.34 +
− 0.07*
0.17 +
− 0.04*
0.06 +
− 0.01*
n=3
1.0 +
− 0.33
0.50 +
− 0.17
0.17 +
− 0.06
n=5
0.17 +
− 0.41*
0.09 +
− 0.04*
0.03 +
− 0.01*
n=5
0.61 +
− 0.39†
0.31 +
− 0.18†
0.11 +
− 0.07†
n=4
Fluxes associated with the tricarboxylic acid cycle
1.2 +
PEPCK pathway‡ (v 6)
− 0.49
0.59 +
Pyruvate cycling (v 5)
− 0.286
0.19 +
Tricarboxylic acid cycle (v 7)
− 0.08
n=7
* Significantly different from 10:1 lactate/pyruvate group (column 1), P < 0.05.
† P < 0.1 versus 10:1 lactate/pyruvate group (column 1).
‡ Also includes contributions from other efflux pathways such as that involving malic enzyme.
Figure 3 Ketone production with and without lactate/pyruvate (Lac/Pyr) by
livers isolated from fed mice and perfused with 0.2 mM octanoate, 0.25 mM
glycerol and 0.5 mM [U-13 C]propionate
Ketone production represents the sum of ACAC and BHB (n = 3).
Figure 2
Typical 13 C-NMR spectra of MAG derived from glucose
The insets show expanded views of the C2 resonance of MAG under the three conditions.
(A) 1:10 lactate/pyruvate; (B) 10:1 lactate/pyruvate; (C) no lactate or pyruvate. The areas of the
multiplet components (D12, D23 and Q) labelled in (B) were used to evaluate the relative fluxes
summarized in the bottom panel of Table 1 and the absolute fluxes summarized in Table 2.
NAD+ /NADH-dependent lactate dehydrogenase reaction and it
is generally accepted that the relative concentrations of cytosolic
NAD+ and NADH are sensitive to intracellular lactate and
pyruvate. When the lactate/pyruvate ratio in the perfusate was
set to 10:1 (considered the normal physiological ratio in vivo),
the isolated liver behaved similarly, but generally with flux
c 2006 Biochemical Society
values on the low side compared with the ‘in vivo’ liver. In
comparison, glucose output from the isolated perfused mouse liver
was equivalent to 69 µmol · min−1 · kg−1 (based on a 20 g mouse),
lower than the in vivo measurement of 114 µmol · min−1 · kg−1 in
the whole mouse [44]. Tricarboxylic-acid-cycle flux in the
in vivo rat liver has been estimated by dynamic 13 C enrichment of
−1
glutamate from [2-13 C]ethanol to be 0.33 +
− 0.09 µmol · min · g
wet weight−1 [45], about 40 % higher than what we measured in
the isolated perfused liver.
Reversal of the lactate/pyruvate ratio to 1:10 presumably
alters cytosolic redox levels, and we found that this results
in a dramatic increase in tricarboxylic-acid-cycle flux, PEPCK
flux and GNGPEP , and suppressed GLY. There are a limited
number of biochemical reactions in the glycolytic/gluconeogenic
pathway that are directly influenced by the NADH/NAD+ ratio.
One is the reaction catalysed by GAPDH, a near-equilibrium
Redox and glucose production pathways
reaction that requires NAD+ for glycolysis and NADH for
GNG. During starvation, the cytosolic ratio of NADH/
NAD+ increases, shifting the GAPDH equilibrium toward GNG
[2]. GNG is maximal in isolated hepatocytes at a lactate/
pyruvate ratio of 10:1 and decreases at both lower and higher
lactate levels (but with fixed pyruvate) [1]. On the basis of thermodynamics, an increase in cytosolic NAD+ (high pyruvate) should
favour formation of 3-phosphoglycerate from glyceraldehyde
3-phosphate (GAPDH reaction) and hence favour glycolysis
[2]. To avoid this, mitochondrial NADH must be transported
to the cytosol to support GNG; thus glucose production from
pyruvate is maximal only when fatty acids are available for
ample production of mitochondrial NADH [38,43]. In the present
study, high pyruvate resulted in a nearly 2-fold increase in
tricarboxylic-acid-cycle flux (Table 2), consistent with increased
mitochondrial NADH generation to support GNG. Stimulation
of tricarboxylic-acid-cycle flux under an oxidized redox state
is also consistent with standard teachings about regulation
of tricarboxylic-acid-cycle flux by NADH/NAD+ levels [46].
Additionally, high pyruvate is associated with high levels of
acetyl-CoA, a regulatory stimulator of both tricarboxylic-acidcycle activity and pyruvate carboxylation. Thus high pyruvate
(low lactate) enhances anaplerosis and flux through PEPCK,
consistent with our observation of a 2-fold increase in flux through
GNGPEP . A small increase in pyruvate cycling was also observed
at high pyruvate levels in the present study. To our knowledge,
the influence of redox on pyruvate cycling has not been reported,
but it has been noted that PK activity is diminished under highly
reducing conditions (such as starvation) in order to spare PEP for
GNG [1].
In the present study, total glucose production did not differ
between the 10:1 and 1:10 lactate/pyruvate groups, yet the source
of glucose shifted from GLY to GNG with high pyruvate. It is not
immediately clear how a change in cytosolic redox state mediated
by lactate/pyruvate might affect GLY, but high pyruvate stimulated GNG, and that in itself may result in suppressed GLY through
autoregulation [47]. Nevertheless, it has been noted that the action
of some hormones on hepatic metabolism may be associated
with changes in cellular redox. For instance, some data indicate
that glucagon-stimulated hepatic glucose release correlates
with an increase in cytosolic NADH/NAD+ [5], while others
have reported the opposite [38]. Similarly, noradrenaline (norepinephrine) results in stimulation of GLY and an increase in
NADH/NAD+ in the perfused rat liver [6]. The present study
shows that the absolute activities of pathways such as GLY, GNG,
pyruvate cycling, PEPCK and the tricarboxylic acid cycle are
all sensitive to changes in cytosolic redox state and substrate
supply, but that the glycerol contribution to glucose production is
relatively insensitive.
Effects of substrate deficiency on metabolism in the isolated
perfused mouse liver
GNG from tricarboxylic-acid-cycle intermediates is almost
completely impaired in mouse livers perfused without lactate or
pyruvate. Residual GNG from PEP may be due to propionate
metabolism, since it decreases to near zero when propionate is
also removed (results not shown). Decreased GNG during
substrate deficiency is expected, but less obvious are the reasons
why removal of both pyruvate and lactate dramatically impairs
tricarboxylic-acid-cycle flux in both the presence and absence of
insulin (Table 2). This observation, however, agrees with results
reported by DeBeer et al. [48], who used an independent method
to calculate tricarboxylic-acid-cycle flux in rat livers perfused
with octanoate in the absence of gluconeogenic precursors and
471
found that tricarboxylic-acid-cycle flux was suppressed by more
than 2-fold. The cause of this is unknown, but may reflect a
decreased energy requirement when the liver is not synthesizing
glucose. Decreased NADH oxidation could result in an inhibition
of the forward reactions of the tricarboxylic acid cycle, similar to
observations in the PEPCK-knockout mouse, where both GNG
and tricarboxylic-acid-cycle flux were found to be impaired
[24]. An important observation in these experiments is that
tricarboxylic-acid-cycle flux appears to be linked more closely to
GNG from PEP than total hepatic energy generation as indicated
by oxygen consumption. Whether this is a consequence of using
octanoate in a perfused liver or is also true in vivo is not known.
However, a very similar linear correlation between tricarboxylicacid-cycle flux and total glucose production has been noted in
isolated hepatocytes exposed to palmitate [49], suggesting that it
is not simply an artifact of octanoate metabolism.
Interestingly, we found that oxygen consumption was relatively
insensitive to changes in redox levels or lack of gluconeogenic
substrates (Table 2). The reason for this is not entirely clear,
but may also be related to the use of octanoate in the perfusion
medium. Unlike long-chain fatty acids, octanoate undergoes
unregulated oxidation, resulting in high rates of ketogenesis,
even in livers from fed animals. McGarry and Foster [33]
showed that most acetyl-CoA was used for ketogenesis rather
than oxidation in the tricarboxylic acid cycle in livers isolated
from fed rats when they were perfused with octanoate. We
also found that mouse livers perfused with octanoate released
ketones at a high rate compared with tricarboxylic-acid-cycle flux
(Figure 3). Under these conditions it has been shown that ketone
metabolism is responsible most of the oxygen consumption [48].
Taken together, these observations suggest a tight interaction between GNG and tricarboxylic-acid-cycle flux, whereas other
pathways of energy production, such as β-oxidation, are, per se,
less influential.
Effects of insulin on metabolism in the isolated perfused
mouse liver
Insulin is involved in the regulation of glucose production by
modulating the delivery of substrates from peripheral tissues,
by modifying enzyme expression at various steps of GNG,
or by inhibiting GLY. Early studies [14,50] suggested that insulin’s
direct action on hepatic GNG was important for controlling
hepatic glucose production. Other studies revealed a correlation
between insulin action on the peripheral tissue and substrate
supply for hepatic GNG [12], and suggested that insulin’s direct
effect on the liver was to inhibit GLY [13] rather than GNG.
We performed experiments using insulin at near-normal and
at hyperinsulinaemic concentrations. Since glucagon was not
included in the perfusion medium, we expected that even a low
concentration of insulin would alter hepatic glucose metabolism.
We found that the major impact of insulin on glucose metabolism
in the isolated liver was to inhibit GLY at both physiological
and hyperinsulinaemic concentrations of insulin. GNG was not
altered during the course of the low-insulin perfusion experiment,
but we cannot rule out an effect at high concentrations. We
found a strong trend (P < 0.1) for decreased flux through GNGPEP ,
PEPCK, pyruvate cycling and tricarboxylic-acid-cycle flux when
a high concentration of insulin was used. These results suggest
that it may be possible for insulin to exert control over hepatic
glucose metabolism beyond GLY under some conditions, but it
remains unknown whether this is relevant in vivo. Interestingly,
we found that the inhibition of GLY by insulin was reversed
when gluconeogenic substrates were completely absent. Although
the combination of high insulin and low substrate availability
c 2006 Biochemical Society
472
N. Hausler and others
is physiologically unlikely, these data suggest that insulin does
not override the natural tendency for autoregulation of glucose
production in liver.
Conclusions
Application of 13 C and 2 H isotopomer analysis to the isolated
mouse liver provided an interesting perspective of the effects of
cytosolic redox and insulin on hepatic fluxes. Both insulin and a
more highly oxidized cytosol (as defined by low lactate and high
pyruvate) inhibit GLY, albeit by different mechanisms. Insulin
appears to directly alter GLY in a pronounced way, whereas it has
little effect on downstream events (GNG, pyruvate cycling and
tricarboxylic-acid-cycle flux), consistent with previous reports
[11,13]. Conversely, a switch in cytosolic redox from high to low
also yielded a significant decrease in GLY, plus an increase
in tricarboxylic-acid-cycle flux, pyruvate-to-PEP flux, pyruvatecycling flux, and GNGPEP . However, the liver maintained a constant rate of total glucose production by levelling GLY in response
to increased GNGPEP . Tricarboxylic-acid-cycle flux appears to
be very sensitive to gluconeogenic flux under all the conditions
studied here, increasing during high GNG and decreasing when
GNG is decreased by the absence of substrates. This insight lends
itself to a variety of physiological conditions where hepatic GNG
or energy homoeostasis, or both, are abnormal.
These studies were supported by the National Institutes of Health (RR02584, U24-DK59632
and HL-34557) and an American Diabetes Association Junior Faculty Award (1-05-JF-05)
to S. C. B. Excellent technical assistance was provided by Erin Smith and Zheng Yan.
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Received 19 July 2005/3 November 2005; accepted 17 November 2005
Published as BJ Immediate Publication 17 November 2005, doi:10.1042/BJ20051174
c 2006 Biochemical Society