From the
We measured gluconeogenesis (GNG) in rats by mass isotopomer
distribution analysis, which allows enrichment of the true biosynthetic
precursor pool (hepatic cytosolic triose phosphates) to be determined.
Fractional GNG from infused [3-
As pointed out by Krebs et al.(1) , measurement
of gluconeogenesis (GNG)
If flux through GNG could be measured
and if access to hepatic triose-P labeling were achieved, a number of
questions might be answered concerning the integration of biochemical
fluxes within the mammalian liver in response to changes in substrate
availability. An area of great interest in this regard is the
phenomenon of autoregulation, or intrahepatic metabolic adjustments to
substrate loads, proposed by Soskin and Levine (15) many years
ago. Several studies in humans and experimental animals have affirmed
the importance of hepatic autoregulation to gluconeogenic substrate
loads. Infusion of glycerol at 1.5 mg/kg/min to fasted humans does not
increase hepatic glucose production (HGP) from basal rates of
approximately 2 mg/kg/min(16) . Infusions of lactate or glycerol
also do not alter HGP, even when glucose, insulin, growth hormone,
glucagon and cortisol concentrations are held constant(17) .
Similar results have been reported in other
species(18, 19, 20) . The mechanisms allowing
the normal liver to absorb large substrate loads for GNG without
altering HGP are not understood, however, nor is the response of the
liver to progressive glycogen depletion (the fed-to-fasted transition)
well understood. In particular, the mechanism by which GNG from amino
acids is reduced, thereby minimizing nitrogen losses, is not known. Is
this due to reduced entry of precursors for GNG into the triose-P
precursor pool, more efficient channeling of triose-P into GNG, or
both?
In this study, we use the MIDA technique to measure GNG. We
also present an isotope dilution method for measuring the flux through
the hepatic triose-P pool or the rate of appearance of triose-P (Ra
triose-p) and from this the fate of triose-P flux under varied
conditions. The assumptions of the GNG and Ra triose-P techniques are
tested, and the hepatic metabolic response to substrate loads (fructose
or glucose) and progressive fasting are examined.
Male Sprague-Dawley rats weighing 250-350 g were used.
Animals were housed in individual cages and maintained on a 12-h
light/12-h. dark cycle. Rats were fed adlibitum (Purina® rat chow). Some animals underwent progressive fasts
(removal of food at 6:00 a.m.), while other animals were infused with
intravenous substrates (fructose at 10-15 mg/kg/min or glucose at
15-30 mg/kg/min) after a variable period of fasting (Fig. 2). [3-
Glucose and glycerol were isolated from deproteinized plasma
by ion-exchange chromatography (23) and then derivatized with
acetic anhydride in pyridine(25) . Liver glycogen was
precipitated from liver by the method of Good et al.(26) and then was hydrolyzed in 6 N HCl (110
°C, 2 h) prior to neutralization and derivatization. Plasma very
low density lipoprotein was isolated by ultracentrifugation, and the
glycerol remaining after transmethylation of fatty acids in methanolic
HCl (13, 27) was derivatized as described above.
Acetaminophen-glucuronate (GlcA) was isolated from urine by high
performance liquid chromatography(23) . The GlcA moiety was
transmethylated in methanolic HCl (28) prior to acetylation with
acetic anhydride in pyridine.
GC/MS analyses were with an HP model 5971 instrument (Hewlett
Packard, Palo Alto, CA). For lactate, selected ion monitoring (SIM) and
electron impact ionization were used. Lactate was analyzed as the
heptafluorobutyrl n-propylamide derivative (m/z 327 and 328) as described elsewhere(29) . For glucose,
chemical ionization with methane gas and GC/MS was used. A 60-m DB17
column (0.25 mm inner diameter; 0.25 µm film thickness; J and W
Scientific, Folsom, CA) was used in a temperature-programmed mode
starting at 150 °C, rising at 40 °C/min to 270 °C. Glucose
was derivatized with acetic anhydride in pyridine, and the tetraacetate
fragment (m/z 331-333) was analyzed using SIM.
For analysis of undifferentiated three-carbon fragments of glucose, the
isolated glucose was reduced to sorbitol using sodium borohydride in
anhydrous dimethyl sulfoxide (30) and then peracetylated with
acetic anhydride/pyridine. Carbons 1-3 are indistinguishable from
carbons 4-6 in the triose fragment in the mass
spectrum(31) . The fragment at m/z 217-219, representing glucose carbons 1-3 and
4-6, was analyzed by EI with SIM. For comparison of the carbon
1-3 to the carbon 4-6 moieties of glucose, in order to
assess triose-P isotopic equilibrium, the aldonitrile, pentaacetate
derivative was synthesized as described by Guo et
al.(32) . Fragments at m/z 187-189
(representing carbons 3-6) and m/z 243-245 (carbons 1-4) were analyzed by SIM. The
difference between these fragments represents carbons 5-6 versus carbons 1-2, where most or all
The principle of MIDA and its application for measuring
biosynthesis of a variety of polymers have been discussed in detail
elsewhere(11, 12, 13, 14, 33) .
In brief, the technique consists of quantifying mass isotopomer
fractional abundances in an intact polymer or fragment that contains
two or more repeats of a precursor monomeric subunit. A stable
isotope-labeled substrate that is capable of perturbing the isotope
abundances of the monomeric precursor pool is administered, and the
mass isotopomeric fractional abundances in the polymer after labeling
are compared with the natural fractional abundance (base line) values.
The pattern or distribution of mass isotopomeric excesses (
Ra glucose was calculated from the
dilution of blood [
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy where I is the isotopic tracer infusion rate. Contribution of
carbon from precursors (glycerol, lactate) to triose-P was calculated
by comparison of their respective isotopic enrichments, each expressed
in comparable terms (i.e. enrichment of plasma precursors was
corrected by a standard curve to represent the proportion of labeled to
total molecules present). Fractional GNG (the fraction of blood glucose
that came from the GNG pathway) was calculated by MIDA. The ratio of
double-labeled to single-labeled glucose molecules, after subtraction
of the frequency of each species due to natural abundance molecules,
reveals p and the asymptotic isotope enrichment of new glucose
molecules (A
On-line formulae not verified for accuracy
Absolute GNG (the chemical flux through the pathway from
triose-P to plasma glucose) was calculated from f multiplied
times the turnover rate of plasma glucose.
On-line formulae not verified for accuracy
The portion of HGP not due to GNG, which reflects the
contribution from hepatic glycogenolysis under postabsorptive
conditions, was calculated from the difference between Ra glucose and
absolute GNG.
On-line formulae not verified for accuracy
During infusions of intravenous glucose, the measured rate of
exogenous glucose infusion was subtracted from Ra glucose to calculate
HGP and non-GNG HGP. Fractional UDP
On-line formulae not verified for accuracy
Endogenous Ra triose-P was calculated as the difference between
Ra triose-P and the rate of direct entry of exogenous fructose and/or
labeled glycerol into the hepatic triose-P pool (Fig. 1).
On-line formulae not verified for accuracy
Correction was necessary if any direct conversion of fructose to
glucose without passage through the triose-P pool was documented (see
below). It should be noted that recycling of label after leaving the
triose-P pool (e.g. by entry into plasma lactate and return of
the [
On-line formulae not verified for accuracy where plasma glycerol enrichment was expressed in the same manner
as p, as the proportion of labeled molecules, by use of
standard curves (see above). [
On-line formulae not verified for accuracy
A similar calculation was used for
On-line formulae not verified for accuracy
Two peaks with identical mass spectra were observed when
derivatized glucose standards were analyzed by GC/MS (not shown).
Injection of authentic
Rats were given intravenous infusions of sodium
[3-
The fractional contribution to GNG from plasma
glycerol can also be calculated from the
[2-
HGP was 14.3 ± 1.4 mg/kg/min 6 h after food was
removed and fell to 10.3 ± 0.5 mg/kg/min at 10-11 h and
7.6 ± 0.5 mg/kg/min at 24 h (Fig. 4A). Food is
absent from the stomachs of these adlibitum fed rats
within 4-5 h of the start of fasting, so Ra glucose represents
HGP under these conditions. Concurrent with the fall in HGP was an
increase in f, from 51.3 ± 7.5 to 78.9 ± 8.1 and
to 87.0 ± 1.5% at 6, 11, and 24 h of fasting, respectively (p < 0.05 for 11 and 24 h versus 6 h values, Fig. 4B). Absolute GNG was unchanged at 7.0 ±
0.7, 8.0 ± 0.6, and 6.9 ± 0.3 mg/kg/min (Fig. 4C), whereas non-GNG HGP fell significantly from
7.3 ± 1.8 to 2.6 ± 0.9 and 1.1 ± 0.2 mg/kg/min at
the three time points, respectively (p < 0.05 for 11 and 24
h versus 6 h values, Fig. 4D). Thus, the fall
in HGP during progressive 24-h fasting was entirely due to decreased
non-GNG HGP, representing glycogenolysis, whereas GNG remained
constant.
Next, the effect of substrate loads on fractional and
absolute GNG was studied. Rats were infused intravenously with fructose
at 10-15 mg/kg/min after a preceding 24-h fast. HGP increased by
80-149% (I), from 8.6 ± 0.9 to 15.5 ±
1.3 mg/kg/min at fructose 10 mg/kg/min (p < 0.01 versus base line) and from 6.9 ± 1.5 to 17.2 ± 3.0
mg/kg/min at fructose 15 mg/kg/min (p < 0.01). The infusion
rate of [
Ra
triose-P increased markedly and in a graded fashion during intravenous
infusions of fructose in 20-h fasted rats (I). Base-line
values of 7.0 ± 0.4 mg/kg/min increased to 13.6 ± 0.4
mg/kg/min at fructose 10 mg/kg/min (p < 0.01 versus base line) and from 6.6 ± 0.6 to 16.4 ± 0.7
mg/kg/min at fructose 15 mg/kg/min (p < 0.01 versus base line, p < 0.05 versus fructose 10
mg/kg/min). The increases in Ra triose-P were 94-148% relative to
base-line values, for fructose 10 and 15 mg/kg/min, respectively.
The input from noninfused sources of hepatic triose-P (endogenous Ra
triose-P) was also calculated by subtracting from Ra triose-P the
exogenous infusion rate of labeled glycerol and the rate of unlabeled
fructose entry into triose-P. Direct conversion of fructose to glucose
had to be subtracted from the fructose infusion rate to determine the
rate at which exogenous fructose entered the hepatic triose-P pool.
Endogenous Ra triose-P calculated in this manner fell markedly during
fructose infusions (I), from 6.0 ± 0.4 to 1.9
± 0.4 mg/kg/min (p < 0.01) at fructose 10 mg/kg/min
and from 5.6 ± 0.6 to -0.6 ± 0.7 mg/kg/min (p < 0.01) at fructose 15 mg/kg/min (the negative flux rate is not
significantly different from zero).
The efficiency of conversion of
hepatic triose-P flux to glucose remained high during fructose
infusions (I). Absolute GNG accounted for 80-100% of
triose-P disposal under conditions of 24-h fasting, and subsequent
intravenous fructose infusions did not alter the fractional disposal of
triose-P flux into GNG.
Comparison of plasma glycerol enrichments to p demonstrated that circulating glycerol provided 38-44%
of triose-P subunits at base line, compared with 20-23% during
fructose administration (I). Plasma glycerol flux (Ra
glycerol) did not decrease during fructose infusion. The proportion of
plasma glycerol flux entering GNG remained >85% during fructose
infusions (not shown). The reduced fractional contribution from plasma
glycerol to p therefore reflects an increased input from other
sources (fructose) rather than reduced input from glycerol.
Administration of intravenous glucose at 15-30
mg/kg/min had a very different effect than intravenous fructose on GNG,
reducing f to 23 and 13%, respectively (I). Ra
glucose increased during infusions of exogenous glucose, so that
absolute GNG was 4.3 ± 0.6 and 5.0 ± 0.6 mg/kg/min, while
HGP was calculated to be 3.6 ± 1.2 and 6.3 ± 2.0
mg/kg/min. These values were significantly reduced from base-line
fasted values (p < 0.01). Plasma glycerol flux was reduced
by 29-35%, from 2.86 ± 0.12 to 1.85 ± 0.14
mg/kg/min and from 2.58 ± 0.11 to 1.83 ± 0.25 mg/kg/min,
respectively, (p < 0.05 for each) during intravenous
glucose infusions at 15 and 30 mg/kg/min, respectively. The proportion
of triose-P subunits derived from glycerol was also nonsignificantly
reduced during cold glucose infusions (I). The proportion
of plasma glycerol flux entering plasma glucose was also significantly
reduced (to 66 and 71%) compared with fasted or intravenous
fructose-infused animals (p < 0.05).
At glucose 15
mg/kg/min, Ra triose-P decreased from 8.6 ± 0.4 to 6.8 ±
0.4 mg/kg/min (p < 0.05 versus base line, p < 0.01 versus intravenous fructose 15 mg/kg/min). At
glucose 30 mg/kg/min, Ra triose-P fell from 8.5 ± 0.7 to 7.0
± 0.3 mg/kg/min (p < 0.05 mg base line, p < 0.01 versus fructose 15 mg/kg/min). Thus,
intravenous glucose infusions consistently increased hepatic
triose-P enrichments, resulting in a lower Ra triose-P (I). Plasma glycerol flux was significantly reduced by
intravenous glucose (see above). The reduction in plasma glycerol flux
could account for 56 and 50% of the reduced Ra triose-P at glucose 15
and 30 mg/kg/min, respectively.
Intravenous glucose also reduced the
efficiency of triose-P disposal into plasma glucose. The fraction of
triose-P flux not entering glucose increased from 19.1 ± 1.3 to
36.3 ± 8.0% (p < 0.01) at intravenous glucose 15
mg/kg/min and to 30.2 ± 1.9% (p < 0.01) at intravenous
glucose 30 mg/kg/min (I).
The quantitative accuracy of the Ra triose-P method is
dependent upon the assumption that all of the infused
[
After 11-24 h of fasting, the recovery of
The high recovery of
The question of recycling of label was also
considered. The enrichment of plasma lactate during infusions of
[2-
We evaluated experimental reproducibility of estimates by two
strategies.
The calculated values for p and f were
compared in
We also applied the MIDA approach to secreted
acetaminophen-GlcA, which has been used as a noninvasive index of
hepatic UDP-glucose labeling by us (23, 28, 34) and others(41, 42) .
GlcA should have a similar labeling pattern as plasma glucose if
glucuronidation occurs in the liver from a shared pool of hexose
phosphates. The latter assumption has been
controversial(23, 41, 42) . MIDA provides an
interesting way to approach this question, since the enrichment of two
end products might differ for physiologic reasons (contributions from
unlabeled glycogen, galactose, etc.), but the molecules of each
product, newly synthesized from the GNG pathway, should derive from the
same triose-P pool. The enrichment of the triose-P pools feeding the
two end products can be measured by MIDA. We compared p in
GlcA to blood glucose under conditions of 24 h fasting (Fig. 6).
The ratio of GlcA/glucose for p was 0.996 ± 0.026, with r
These results
exclude double
We have applied the MIDA technique to the long standing
problem of measuring GNG. The results can be discussed in terms of
their methodologic and their metabolic implications. With regard to
methodology, MIDA has previously been used by us for measurement of
polymers composed of larger numbers of repeating monomeric subunits
(11-14, 25, 27, 33, 40). Others (43, 44, 45) have also applied probability
analysis to the precursor-product relationship for long chain polymers.
Measurement of GNG presented some unique methodologic challenges.
Glucose contains the smallest number of monomeric subunits possible for
application of combinatorial analysis (n = 2). In
addition, the possibility that the precursor pool might not be well
mixed (i.e. that there could be isotopic disequilibrium
between DHAP and glyceraldehyde 3-phosphate (Fig. 1)) could in
theory render the binomial or multinomial model inapplicable in this
system. Other potentially confounding factors such as the generation of
double-labeled triose subunits by reincorporation of
The potential methodologic problems in application of
MIDA to GNG were addressed. Based on both theoretical and experimental
results, we conclude that isotopic disequilibrium within the triose-P
pool is not a serious problem under most conditions because, first,
even an isotopic gradient of 2:1 has only a relatively minor effect on
calculated GNG (overestimation of f by about 12-13%, see
``Appendix'') and, second, much less disequilibrium (1.17:1)
is in fact present in the triose-P pool when tested experimentally (). Moreover, double-labeled triose-P do not occur
sufficiently frequently to have a significant quantitative impact and
error sensitivity analysis revealed acceptably low levels of error
amplification in derived parameters ifp is
maintained at 0.08 or greater (``Appendix''). We have also
calculated the effect of inconstancy of p in space, e.g. extrahepatic GNG.
With regard
to the Ra triose-P technique for measuring flux through this
intrahepatic metabolic pool, the first requirement was to establish the
validity of the technique. An important metabolic assumption of the Ra
triose-P measurement is that the infused
[
Several metabolic results of interest emerged, based on
this methodologic foundation. The mechanism by which HGP falls during
the first 24 h of a fast was determined. There was no effect on
absolute GNG, but a 90% reduction in non-GNG HGP (Fig. 4). The
latter presumably reflects reduced glycogen to glucose flux as liver
glycogen becomes depleted. A similar sequence has been reported in
humans, based on in vivo NMR estimates of hepatic
glycogenolysis(46) . The reduction of non-GNG HGP became
apparent as early as the interval from the 6th to the 11th h of
fasting. GNG was maintained constant during a fast despite a reduction
in Ra triose-P, by two mechanisms. First, the efficiency at which
triose-P flux was disposed into plasma glucose increased. After 6 h,
3.0 mg/kg/min of triose-P flux did not enter GNG, while this value fell
to 0.7 mg/kg/min at 24 h of fasting. In addition, the contribution from
plasma glycerol to hepatic triose-P increased significantly, thereby
preventing a further fall in Ra triose-P while sparing amino acids and
other limited sources of precursors for GNG. The liver thereby becomes
more efficient in terms of both the fate and the sources of hepatic
triose as liver glycogen becomes depleted.
A quantitative result
with interesting implications was that intravenous fructose slightly
but consistently increased non-GNG HGP in glycogen-depleted
rats (I). This finding is consistent with the report of
Gopher et al.(47) . They analyzed plasma glucose by NMR
after administration of [U-
Our
results also suggest (I) that flux into the hepatic
triose-P pool is regulated in a feedback manner in response to
variations in exogenous inputs, much as Ra of plasma glucose is
regulated in response to exogenous glucose infusions. Most impressive
was the effect of loads of exogenous gluconeogenic precursors to almost
completely suppress endogenous Ra triose-P. A dose-response
relationship between exogenous load and suppression of Ra triose-P was
also apparent (I). Since >80% of triose-P flux enters
plasma glucose under these conditions, the reduction in endogenous Ra
triose-P by 4.1-6.2 mg/kg/min during fructose infusions prevented
Ra glucose from rising by an additional 4-5 mg/kg/min. The
mechanism by which one source of triose-P reduces input from other
sources cannot be stated at present. Feedback inhibition by a triose-P
or related metabolite on phosphoenolpyruvate carboxykinase or
phosphofructosekinase, or activation of pyruvate kinase or pyruvate
dehydrogenase(49, 50) could be responsible. Since
phosphoenolpyruvate carboxykinase is often considered to be a
regulatory step in GNG(51, 52) , the role of altered
phosphoenolpyruvate carboxykinase activity in the reduced Ra triose-P
is of great interest.
Our observation that HGP increased during
infusions of fructose differs from the lack of increase in HGP observed
by others (16, 17, 18) during infusions of
gluconeogenic substrates. There are several possible explanations for
the difference, in addition to species differences. The rats in our
studies were previously fasted so there was little or no glycogenolysis
to suppress, which is one potential mechanism of intrahepatic
adaptation to substrate loads(17, 53) . Moreover, the
substrate loads that we administered (10-15 mg/kg/min) were
greater than basal HGP (7 mg/kg/min), so that even complete suppression
of endogenous Ra triose-P and other endogenous sources of HGP could not
fully compensate for the exogenous load. Finally, the low insulin state
in these 24-h fasted animals may have limited channeling of GNG into
liver glycogen(54, 55, 56) . It will be
necessary to perform studies in liver glycogen replete rats at
substrate loads lower than HGP to address the efficiency and mechanism
of other hepatic adaptations besides changes in triose-P input under
these conditions.
The lack of an increase in Ra triose-P during
intravenous glucose infusions (I) was somewhat surprising,
in view of the role of the indirect pathway for hepatic glycogen
synthesis from glucose(23, 57, 58) . About 50%
of the reduced triose-P flux was attributed to reduced contribution
from plasma glycerol due to a decrease in plasma glycerol flux, but the
explanation for the remaining 50% was not determined. Several
possibilities might be considered. Increases in plasma glucose and
insulin concentrations could reduce the contribution from glycogen to
hepatic triose-P, although the amount of liver glycogen in these 24-h
fasted rats is quite low. Intravenous glucose infusion could direct
some 3-carbon metabolites away from the triose-P pool, by activating
pyruvate dehydrogenase (49) or pyruvate kinase (19, 59).
Alternatively, liver glycogen deposition by the indirect pathway during
glucose infusions may not involve an increase in carbon flux into the
triose-P pool, but a redirection of GNG flux away from plasma glucose
and into glycogen (54, 55, 56) along with a
replacement of endogenous sources of triose-P by glucose.
Administration of glucose had relatively minor effects on Ra
triose-P and on the contribution of plasma glycerol to the triose-P
pool, but it suppressed GNG, HGP, and non-GNG HGP while increasing flux
to glycogen (Tables III and IV). The major consequence of glucose
infusion was therefore on the disposal of triose-P rather than on its
production. Intravenous fructose infusion at a high rate (15 mg/kg/min)
increased non-GNG triose-P disposal to 2.8 mg/kg/min. If this all
represented glycogen synthesis, it could provide about 4 mg of
glycogen/g of liver/h via the ``indirect'' pathway. The
difference between Ra triose-P and absolute GNG may provide an index of
hepatic indirect glycogen synthesis under certain refeeding conditions.
Our measurements clearly demonstrate that the disposal as well as
production of hepatic triose-P adapts to metabolic conditions.
We
observed fractional GNG to be consistently about 90% after a 24-h fast,
leaving 10% unaccounted for. Although we did not determine other
sources of glucose experimentally, some possibilities include turnover
of glycoprotein carbohydrates (galactose, mannose, glucosamine, etc.),
a persistent small contribution from liver glycogen, release of free
glucose from muscle or other glycogen-containing tissues, or input from
gluconeogenic pools to which labeled glycerol or lactate do not have
access.
Finally, the hexose phosphate pool from which
glucuronidation of xenobiotics occurs had been an area of
uncertainty(23, 41, 42) . MIDA provides an
interesting additional tool to address this question. Under conditions
of fasting or fructose infusion, the isotopic enrichment of the
triose-P pool for GlcA was identical to that for blood glucose (Fig. 6). One can infer that glucuronidation and glucose
secretion derive from the same tissue and the same pool (or at least
isotopically well mixed pools) under these conditions. Other end
products can be compared in an analogous fashion, to evaluate
compartmentalization of ostensibly shared precursor pools (e.g. GlcA and glycogen under various refeeding conditions).
In
conclusion, the ability to quantify fluxes into hepatic GNG in vivo opens up a wide variety of metabolic regulatory questions to
experimental analysis. Our results indicate that the liver exerts
control over GNG, the metabolic sources of triose-P, flux into the
triose-P pool, the efficiency of triose-P disposal into GNG, and the
contribution from glycogen to plasma glucose, in response to
alterations in substrate availability. Since MIDA does not involve
radiation exposure, it is also suitable for measurement of GNG and
related parameters in humans.
Plasma metabolite refers to plasma lactate in studies infusing
[3-
Values shown are means ± S.E.M.
Calculations of
Experimental
details are described in the text. The ratio of DHAP/G3P was determined
by analysis of the m/z 242 to the m/z 187 ion abundances from the aldonitrile, pentaacetate derivative
of glucose by GC/MS (24).
On-line formulae not verified for accuracy
-We thank Christina Papageorgopoulos for helpful
discussions and Cici Hyde for preparation of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
C]lactate,
[1-
C]lactate, and
[2-
C]glycerol was 88 ± 2, 89 ± 3,
and 87 ± 2%, respectively, after 48 h of fasting.
[2-
C]Glycerol was the most efficient label and
allowed measurement of rate of appearance of intrahepatic triose
phosphate (Ra triose-P), by dilution. IV fructose (10-15
mg/kg/min) increased absolute GNG by 81-147%. Ra triose-P
increased proportionately, but endogenous Ra triose-P was almost
completely suppressed, suggesting feedback control. Interestingly,
15-17% of fructose was directly converted to glucose without
entering hepatic triose-P. IV glucose reduced GNG and Ra triose-P. 24-h
fasting reduced hepatic glucose production by half, but absolute GNG
was unchanged due to increased fractional GNG (51-87%). Reduced
hepatic glucose production was entirely due to decreased glycogen
input, from 7.3 ± 1.8 to 1.1 ± 0.2 mg/kg/min. Ra triose-P
fell during fasting, but efficiency of triose-P disposal into GNG
increased, maintaining GNG constant. Secreted glucuronyl conjugates and
plasma glucose results correlated closely. In summary, GNG and
intrahepatic triose-P flux can be measured by mass isotopomer
distribution analysis with [2-
C]glycerol.
(
)with isotopes is
complicated by the fact that the metabolic pathway from pyruvate
proceeds through mitochondrial oxalacetate (Fig. 1), which is
exposed to numerous metabolic sources of carbon dilution during an
isotope labeling
experiment(2, 3, 4, 5, 6) . This
is a problem because in order to interpret incorporation from labeled
GNG substrates such as lactate or alanine into glucose or glycogen, it
is necessary to establish the isotope content (specific activity or
enrichment) of the true biosynthetic precursor. For GNG, the true
all-source precursor pool is most accurately considered to be the
triose phosphate (triose-P) pool, consisting of glyceraldehyde
3-phosphate and dihydroxyacetone phosphate (DHAP) (Fig. 1).
Several solutions to the problem of dilution in GNG intermediates have
been proposed, including the [
C]acetate method
developed by Weinman et al.(2) , which has since been
modified by Hetenyi(3) , Katz(4) , and
Kelleher(5) . This approach has recently been applied in humans
(6). The technique remains open to severe
criticisms(7, 8, 9) , however. A stable isotope
method for estimating the dilution of recycled
[
C]glucose in GNG intermediates after passage
through the indirect pathway of glycogen synthesis has been proposed by
Katz and Lee(10) . This approach is attractive but is currently
limited to recycled glucose and cannot address incorporation from
``new'' (nonrecycled) gluconeogenic precursors. Thus, despite
its importance in intermediary metabolism, there currently exists no
satisfactory technique for measuring GNG in vivo.
Figure 1:
Pathways of GNG and hepatic triose-P
metabolism in liver. Abbreviations: AcCoA, acetyl-CoA; OAA, oxalacetate; TCA, tricarboxylic acid cycle; Pyr, pyruvate; PEP, phosphoenolpyruvate; -Glyc-P,
-glycerol phosphate; G-3P,
glyceraldehyde 3-phosphate; Fru-1,6-P
, fructose
1,6-bisphosphate; Glc-6P, glucose 6-phosphate; Glc-1-P, glucose 1-phosphate; GlcUA, glucuronate. At
metabolic steady state, the sum of metabolic inputs into triose-P (solidlines) equals the sum of outflows (dashedlines) equals Ra triose-P.
We (11, 12, 13, 14, 15) recently
described a general technique for measuring the synthesis of biological
polymers in vivo, which we have called mass isotopomer
distribution analysis (MIDA). This technique uses probability logic
with mass spectrometric analysis to calculate the isotopic enrichment
of the true precursor subunits from which a polymer was synthesized. In
principle, this approach can be used for any polymer composed of
repeating monomeric subunits(11, 12) . Once the true
precursor enrichment is determined, calculation of the fractional
synthesis of a polymer is
straightforward(11, 12, 13, 14) . In
principle, the MIDA technique can be used for measurement of GNG and
the isotopic enrichment of the hepatic triose-P pool, if glucose is
considered to be a polymer composed of two triose subunits. Any
carbon-labeled substrate that enters the triose-P precursor pool can
then be used to measure GNG.
C]Lactic acid and
[1-
C]lactic acid were purchased from Isotec,
Inc. (Miamisburg, OH). [2-
C]Glycerol was
purchased from Tracer Technologies (Somerville, MA), Cambridge Isotope
Laboratories (Woburn, MA) and Isotec, Inc. All labeled substrates were
>98% enriched. The [
C]lactates and
[2-
C]glycerol were infused at 0.011 mmol/kg/min
(1 mg/kg/min). During infusions of fructose, the rate of
[2-
C]glycerol infusion was increased to 0.022
mmol/kg/min (2 mg/kg/min). Some animals received concurrent intravenous
infusions of [6-
H]glucose (DuPont NEN) at 10
µCi/h and/or acetaminophen (40 mg/kg/h) as described
previously(23) . Authentic
- and
-anomers of D-glucose pentaacetate for use as standards were purchased
commercially (from Aldrich). Acetaminophen for parenteral
administration was prepared by Dr. L. Tomimatsu of the University of
California San Francisco School of Pharmacy.
Figure 2:
Metabolic infusion protocol in
catheterized rats.
Catheterization of rats
for constant infusions of labeled precursors was as described
previously (23, 24) with the addition of an indwelling
carotid artery catheter for blood sampling during infusions. All
procedures were carried out with the approval of the Office of
Laboratory Animal Care of the University of California at Berkeley.
Water was available adlibitum during periods of
fasting. The carotid artery catheter for blood sampling was placed
contralateral to the jugular venous infusion catheter. Sequential
samples of 0.3-0.5 ml were drawn prior to sacrifice, acid
precipitated, and centrifuged for measurement of blood metabolite
enrichments.
Isolation and Preparation of Metabolites for Mass
Spectrometric Analyses
Gas Chromatography/Mass Spectrometry (GC/MS)
C
from [2-
C]glycerol is likely to be. Glycerol
from plasma was isolated by ion-exchange chromatography, as described
above for glucose, peracetylated with acetic anhydride/pyridine, and
analyzed by SIM as m/z 159 and 160, using chemical
ionization with methane gas. Blood glycerol and lactate enrichments
were calculated by comparison to standard curves, prepared by mixing
labeled and natural abundance materials. Measurement of blood
glucose-specific activities during [
H]glucose
infusions was by liquid scintillation counting and enzymatic glucose
measurements on the eluate collected from the ion-exchange
column(23) . Acetaminophen-GlcA was isolated from urine as
described previously (23) and then transmethylated in
methanolic/HCl and peracetylated in acetic anhydride/pyridine. GC/MS of
the acetylated methyl ester of GlcA was under the same conditions as
for glucose pentaacetate with SIM of m/z 317, which
contains all six carbons of GlcA. All analyses for MIDA calculations
were performed in quadruplicate, with frequent interjection of
base-line measurements (e.g. every fourth sample) throughout
the GC/MS run. When base-line mass isotopomer fractional abundances
were not within ± 0.0050 molar excess of theoretical values
expected based on the elemental composition of the ion analyzed (see
below and ``Appendix''), a run was rejected. In the case of
the m/z 331-333 ions for glucose tetraacetate,
for example (elemental composition
C
H
O
), calculated isotopomeric
fractional abundances are M
(m/z 331) = 0.8396, M
(m/z 332) = 0.1348, and M
( m/z 333) = 0.0256. For the m/z 317-319 ions for methyl, triacetyl-GlcA, (elemental
composition C
H
O
), calculated
fractional isotopomeric abundances are M
(m/z 317) = 0.8489, M
(m/z 318) = 0.1267, and M
(m/z 319) = 0.0244. If persistent
divergence of base-line fractional abundances from expected values was
observed, the instrument was retuned, or the source was cleaned. Also,
samples were preinjected, and volumes were then adjusted to achieve
similar absolute areas for the most abundant ion in all samples, in
order to minimize the possibility of detector nonlinearity or
compromised mass resolution due to spill-over between adjacent masses.
Application of MIDA for Measurement of GNG
fractional abundances) in the polymer reveals the proportion (p) of the biosynthetic precursor units entering the polymer
that were isotopically labeled,
(
)using formulae
derived from the binomial or multinomial expansions (Refs. 11 and 12
and see ``Appendix,'' ). Once p is
established, the precursor-product relationship can be applied for
kinetic analysis or for calculating the fraction (f) of
polymers that were derived from the endogenous biosynthetic pathway
during the labeling period. In the case of GNG (for glucose or GlcA),
the ratio of excess M
(EM
) to excess M
(EM
) fractional abundances
(
)reveals the isotopic abundance of the true precursor
pool (hepatic triose-P, Fig. 1), from which f can
be calculated. For GNG, however, there are several potential problems
that might influence the use of MIDA: 1) the possibility that
CO
reincorporation into oxalacetate (or
C entry through other metabolic pathways) could result in
double-labeled triose-P, which could confound interpretation of
isotopomer ratios in glucose; 2) the possibility of isotopic
disequilibrium between the two triose-P, glyceraldehyde 3-phosphate and
DHAP (Fig. 1), in which case the assumption of a single precursor
enrichment would not be correct; 3) the short chain length of glucose
as a polymer, containing only n = 2 subunits, which
reduces the likelihood of double-labeled glucose molecules being formed
during an isotope-incorporation experiment and thereby could represent
a practical limitation to the method, if analytic precision is
insufficient. These potential problems are discussed below.
Calculations
Metabolite Fluxes, GNG, and HGP
Calculations of
precursor enrichment (p) and fractional GNG are described in
the ``Appendix.''
H]glucose during constant
infusions of [3-
H]glucose and Ra glycerol from
the dilution of blood glycerol during constant infusions of
[2-
C]glycerol.
) synthesized at this value of
p(11, 12) . The actual enrichment of labeled glucose
molecules (EM
) is then compared with the
asymptotic value for new glucose molecules (A
) to calculate fractional GNG (f).
GNG was calculated by
isolation of secreted urinary acetaminophen-GlcA (19, 25, 26) with application of the MIDA
algorithm to fragments of the GlcA derivative analyzed by GC/MS.
Ra Triose-P
The turnover of this intracellular
metabolite pool was determined by use of the isotope dilution
principle. Isotope dilution techniques are based on three central
requirements or assumptions(34) : first, that a labeled
metabolite can be injected into a metabolic pool at a known rate;
second, that the isotopic content (enrichment or specific activity) of
the metabolite in the pool can be measured; and third, that an isotopic
steady state is attained and can be documented in the metabolite pool.
The first requirement can be fulfilled for the hepatic triose-P pool by
administration of [C]glycerol at a constant rate
intravenously, if all infused [
C]glycerol is
taken up by the liver (35, 36) and metabolized via
hepatic triose-P (Fig. 1). Ra triose-P will be over-estimated in
proportion to extrahepatic utilization of labeled glycerol. This
assumption can be tested experimentally (see below). The second
requirement can be fulfilled by application of MIDA on a polymeric end
product derived from the hepatic triose-P pool, such as secreted
glucose or GlcA-conjugates(11, 34, 37) to infer p of the precursor pool. The third requirement is readily
tested by performing serial measurements on the end product. If these
requirements are met, the dilution principle can be applied to the
hepatic triose-P pool.
C]lactate to the hepatic triose-P pool),
will result in proportional underestimation of Ra triose-P (see below).
Isotopic Contribution of Plasma Glycerol to Hepatic
Triose-P
The proportion of the hepatic triose-P pool derived
from plasma glycerol was estimated based on the precursor-product
relationship C]Glycerol Recovery in Plasma Glucose or
Other End Products-The rate of labeled glycerol entry into
plasma glucose was calculated by using a stoichiometric or isotope
balance approach. Absolute GNG was multiplied times the proportion of
the triose-P pool that was labeled (enriched), to determine the flux of
C atoms into glucose.
C recovery in
liver glycogen, secreted GlcA-conjugates, and plasma
triglyceride-glycerol. For glycogen, the final concentration in liver
was multiplied by f and then p; for GlcA, the urinary
excretion rate was multiplied by f and then p; for
triglyceride-glycerol, the estimated(38, 39) triglyceride production rate was multiplied by very low
density lipoprotein-glycerol enrichment.
Non-GNG Triose-P Disposal
The disposal of
triose-P flux into non-glucose pathways was also calculated.
Gas Chromatographic Separation of Glucose Anomers
- and
-anomers of glucose-pentaacetate
confirmed the identity of these two peaks. D-Galactose, D-mannose, D-fructose, and other monosaccharides are
separated from the glucose peaks under these GC conditions. The
existence of dual peaks is useful because they can be used as internal
replicates for quantification of mass isotopomeric abundances (see
below).
Comparison of GNG from
[3-
C]Lactate,
[1-
C]Lactate, and
[2-
C]Glycerol during Fasting
C]lactate or sodium
[1-
C]lactate (60-70 mg/kg/h of the lactate
moiety, or 0.67-0.78 mmol/kg/h) or
[2-
C]glycerol (60-70 mg/kg/h) during fasts
of 36-48-h duration. The value for p generally reached a
fairly stable value within 2-3 h of starting a constant infusion
of [3-
C]lactate or
[2-
C]glycerol (Fig. 3). Blood lactate
enrichments of 0.155 ± 0.019 were associated with p = 0.085 ± 0.014, for a dilution from blood lactate
to the true precursor of 46 ± 6% (). The range of
dilutions was from 31 to 72%. Measurement of f was performed
from [3-
C]lactate (). After
36-48 h of food deprivation, f was 87 ± 2% (n = 22 animals). With [1-
C]lactate, f was 89 ± 3% (n = 4) (not shown).
Figure 3:
Values for EM,
EM
, p and f over time during infusions of
[3-
C]lactate (60 mg/kg/h) or
[2-
C]glycerol (60 mg/kg/h) in 24-h fasted rats.
EM
, EM
and p are higher with labeled
glycerol than with labeled lactate, but calculated values for f are not different from the two labeled
substrates.
With [2-C]glycerol infused at the same rate,
values for p were higher (0.12-0.18 ME), but the values
for f were identical at 88 ± 2% ( and Fig. 3). Mass spectrometric data and their conversion into p and f values in representative individual animals
administered [2-
C]glycerol are shown (). The constancy of fractional abundances for
- and
-anomers of glucose and for consecutive time points and the
reproducibility of derived values for p and f are
clearly apparent when [2-
C]glycerol is used
under these conditions. Because of the superiority of
[2-
C]glycerol for labeling the triose-P
precursor pool, further studies used this labeled substrate for
measuring GNG.
C]glycerol data. Isotope enrichments of
plasma glycerol were about 3 times the calculated triose-P enrichments
(0.440 ± 0.029 versus 0.148 ± 0.008,
respectively). The ratio of triose-P/glycerol enrichments was 34
± 1%, (range of 30-37%) in 48-h fasted rats (). The roughly 3-fold dilution between plasma glycerol and
the hepatic triose phosphate pool indicates that about one-third of the
subunits in the triose-P pool for GNG came directly or indirectly from
circulating glycerol. Ra glucose was 7 mg/kg/min, and GNG provided
approximately 88% of glucose flux (), so the glycerol
contribution to GNG was approximately 2.1 mg/kg/min (7 mg/kg/min
0.88
0.34). Comparison of these values to plasma
glycerol flux is of interest. The average Ra glycerol in these animals
was 2.3 mg/kg/min, so about 90% of glycerol entering the circulation
could be accounted for by entry into blood glucose, under conditions of
36-48 h fasting.
Fed to Fasted Transition
Figure 4:
Effects of progressive fasting on GNG and
related parameters. A, HGP (mg/kg/min); B, f (percent); C, absolute (Abs) GNG (mg/kg/min); D, non-GNG HGP (mg/kg/min); *, p < 0.01 versus 5-6 value; **, p < 0.05 versus 10-11 h value. The metabolic infusion protocol and
measurements are described in the text.
Ra triose-P fell from 9.9 ± 0.9 mg/kg/min at
5-6 h of fasting and 9.3 ± 0.7 mg/kg/min at 10-11 h
to 7.6 ± 0.2 mg/kg/min at 20-24 h (p < 0.05
for 20-24 h value versus other two time points, Fig. 5A). The proportion of the triose-P pool derived
from plasma glycerol increased significantly from 11 to 24 h of
fasting, (24.2 ± 3.2 to 37.3 ± 1.3%) (p <
0.05). Accordingly, the non-glycerol Ra triose-P fell by 37% during
progressive fasting, from 7.5 ± 0.9 to 4.8 ± 0.2
mg/kg/min (p < 0.05). Moreover, the efficiency by which
triose-P flux was converted to plasma glucose (Ra triose-P minus
absolute GNG) also increased between 6 and 24 h of fasting (Fig. 5B), from 70.8 ± 7.3 to 88.0 ± 2.6% (p < 0.05). Thus, at 6 h of fasting, 3.0 ± 0.8
mg/kg/min of triose-P flux was not accounted for as flux into GNG; at
10 h, the value was 1.3 ± 0.6 mg/kg/min (nonsignificantly
different versus 6 h), and at 24 h, all but 0.7 ± 0.3
mg/kg/min of flux into the triose-P pool was directed into plasma
glucose (p < 0.01 versus 6 h) (Fig. 5C).
Figure 5:
Effect of progressive fasting on metabolic
parameters relating to triose-P flux. A, Ra triose-P
(mg/kg/min); B, efficiency of triose-P disposal into glucose
(percent triose-P flux into GNG); C, nongluconeogenic triose-P
disposal (mg/kg/min); *, p < 0.05 versus 5-6
h value;**, p < 0.05 versus 10-11 h
value.
Effect of Substrate Loads
IV Fructose Administration
C]glycerol was doubled during fructose
infusions to maintain p at high values, and in fact p remained fairly constant (not shown). The basal value of f did not change during fructose infusion (84.7 ± 3.0 to 84.4
± 3.2% and 80.7 ± 1.5 to 79.9 ± 3.6% at fructose
10 and 15 mg/kg/min, respectively), while absolute GNG increased by
81-147% (from 7.2 ± 0.5 to 13.0 ± 0.8 mg/kg/min and
from 5.5 ± 1.1 to 13.6 ± 2.1 mg/kg/min, respectively, p < 0.01 versus basal for each). Interestingly,
non-GNG HGP also increased, from 1.4 ± 0.4 to 2.5 ± 0.6
mg/kg/min (nonsignificantly different versus base line) and
from 1.4 ± 0.4 to 3.6 ± 1.0 mg/kg/min (p <
0.05 versus base line). Since these animals are depleted of
glycogen, the most likely explanation for increased non-GNG HGP is
direct conversion of 15-17% of the fructose load to glucose,
without having passed through triose-P and GNG(27) .
IV Glucose Administration
Quantitative Recovery of
C in End
Products of Hepatic Triose-P Metabolism
C]glycerol in fact enters the hepatic triose-P
pool sampled (see above). Although it is generally accepted that the
liver accounts for the great majority of glycerol disposal in
mammals(33, 34) , the quantitative validity of this
assumption needed to be tested under the experimental conditions
present in these studies. The effect of administering different
unlabeled substrates in particular had to be evaluated. We addressed
this difficult question by performing an isotope accounting. The rate
of entry of
C atoms into glucose and other selected end
products of hepatic triose-P metabolism (glycogen, secreted GlcA,
plasma triglyceride-glycerol) was tabulated and compared with the rate
of
C infusion (). The rate of
C
entry into each end product was calculated as the flux from triose-P
into the product (the production rate) multiplied times the fraction of
triose-P labeled with
C (see above). In the case of plasma
glucose, this calculation reduces to absolute GNG times p. For
liver glycogen, it is the glycogen accumulation rate times the fraction
from GNG times p of glycogen, and for GlcA it is the urinary
excretion rate times the fraction from GNG times p of GlcA.
For triglyceride-glycerol, it is the triglyceride production rate
(estimated from literature values, Refs. 38 and 39) times the
enrichment of glycerol isolated from plasma triglycerides. It should be
noted that release from the liver as lactate or other glycolytic end
products was not quantitatively included in this estimate. Also not
included is de novo hepatic lipogenesis, which is
quantitatively minor under most conditions(13, 40) .
C in
these end products was 91.4 ± 6.8 to 95.5 ± 2.7% (). With fructose infusions at 10-15 mg/kg/min,
C recovery was not significantly different
(99.7-102.3%). During intravenous glucose infusions, however,
C recovery was slightly lower (79.7-83.7%), perhaps
reflecting release of glycolytic end products from the liver. The
observation that triose-P enrichments were higher during
intravenous glucose infusions (I) argues against the lower
C recovery being due to a reduction in the rate of
[
C]glycerol entry into the hepatic triose-P pool
during intravenous glucose administration, which would have tended to decrease triose-P enrichments.
C in these end products of triose-P metabolism under
conditions of GNG is direct evidence in support of the central
assumption of this dilution method, namely that the administered label
enters the pool of interest. Based on these results, the Ra triose-P
estimates are unlikely to be overestimated by more than about
5-10%, if at all, due to incomplete isotope entry into the
hepatic triose-P pool.
C]glycerol was 20-25% of the hepatic
triose-P enrichments (not shown). Since lactate contributes about 50%
of the carbon in triose-P (see ) under fasting conditions,
recycled [
C]lactate contributes about
10-12% of triose-P enrichment. Put differently, Ra triose-P might
be underestimated by about 10% due to the reentry of substrate that is
labeled rather than being a source of dilution.
Reproducibility of Estimates
Comparison of
- and
-Anomers of Glucose
- and
-anomers of glucose from all analyses made.
The ratio of their values for p was 1.009 ± 0.056 and
for f was 0.995 ± 0.047 (mean ± S.D.). The
correlation coefficient was also very high (r
= 0.99 for both p and f) for
- versus
-anomers. This degree of reproducibility was
achieved by use of quadruplicate analyses, by preinjection to achieve
similar peak areas in sequential samples, and by paying careful
attention to the closeness of base-line fractional abundances to
expected values (as discussed above).
Comparison of Plasma Glucose to Secreted GlcA
Conjugates
= 0.973. These results support the
analytic reproducibility of the MIDA technique as well as the identity
of (or isotopic equilibrium within) both the triose-P and glucose
6-phosphate precursor pools for glucose and secreted GlcA-conjugates.
Figure 6:
Comparison of values for p between plasma glucose and secreted urinary GlcA (Glc-UA)
(from acetaminophen-GlcA). Line of identity is shown. Ratio of
GlcA/glucose = 0.996 ± 0.026 (r = 0.973).
Evaluation of Potential Deviations from Model
Impact of Isotopic Disequilibrium between Triose
Phosphates on MIDA Calculations
Theoretical Considerations
Use of the binomial
expansion assumes that there exists a single p (proportion of
subunits undergoing combination that are isotopically labeled, Refs.
11-13). The value for p may change over time, but at any
moment it is assumed that there exists a single value for the isotope
enrichment of the precursor subunit. The same applies for the
multinomial expansion; more than two choices are available, but their
probabilities are the same for each trial. In the case of GNG, this
assumption may be called into question because there is not in fact a
single biochemical triose phosphate precursor for GNG but a pair of
precursors (DHAP and glyceraldehyde 3-phosphate) that combine to form
the glucose ``polymer'' (Fig. 1). The assumption of
complete isotopic equilibrium between these two triose phosphates has
not been fully evaluated and may not be justified under all conditions.
Many, but not all, studies have indicated that isotopic equilibrium is
present between the triose phosphates(7, 32) , based on
comparisons of labeling in glucose carbons 1-3 (representing
DHAP) and carbons 4-6 (representing glyceraldehyde-3-phosphate).
It can be shown, however, that divergence from isotopic equilibrium
between DHAP and glyceraldehyde 3-phosphate generally has only a very
minor impact on MIDA calculations for glucose (see
``Appendix''). Unless the isotopic disequilibrium is
substantial (e.g. one triose-P enrichment is less than
40-50% of the other), effect on calculation of p and f is minor ( Fig. 1of the ``Appendix'').
Experimental Testing
To evaluate the presence
or absence of isotopic equilibrium experimentally, we used the GC/MS
technique described by Guo et al.(32) for fragmenting
glucose into carbon 1-4 and carbon 3-6 moities. The
aldonitrile, pentaacetate derivative is analyzed for this purpose.
Under electron-impact ionization, the fragment at m/z 187 represents carbons 3-6 and m/z 242
represents carbons 1-4(24) . Differences in isotopic
enrichments between these two fragments reveal differences in carbons
5-6 versus carbons 1-2 (with
[2-C]glycerol more highly labeling carbons
1-2 and [3-
C]lactate labeling carbons
5-6, if disequilibrium exists). Since
[2-
C]glycerol primarily labels positions C-2 and
C-5 of glucose and secondarily randomizes into C-1 and C-6 in the
fumarase equilibrium, only entering C-3 and C-4 of glucose after
sequential turns of the tricarboxylic acid cycle, comparison of these
two fragments provides a quantitative index of isotopic equilibrium
between DHAP and glyceraldehyde 3-phosphate (represented by carbons
1-2 and carbons 5-6, respectively). First, in order to test
the accuracy of the aldonitrile fragmentation technique, we mixed known
ratios of [1-
C]glucose and
[6-
C]glucose and compared measured with expected
ratios of carbons 3-6/carbons 1-4. The observed results
were very close to expected (ratio of measured/prepared = 0.975
± 0.060, r
= 0.997). Next, the
aldonitrile, pentaacetate derivative of plasma glucose was analyzed by
electron-impact ionization/mass spectometry in fasted and
fructose-infused rats (). The ratio between the carbon
3-6/carbon 1-4 moieties of the glucose molecule was
1.16-1.17. The impact of this degree of disequilibrium on
calculated parameters (p and f) is <1% (0.7% at a
ratio of 1.17). A quantitatively important degree of isotopic
disequilibrium therefore is not present under these conditions.
Consistent with theoretical expectations (Fig. 1), the ratio of
carbons 3-6/carbons 1-4 was slightly less than 1.0
from [
C]lactate (0.93, ), but again
this is not significant in terms of quantitative impact on calculated
parameters.
CO
Reincorporation
and Double-labeling of the Hepatic Triose Phosphate Pool
Theoretical Considerations
The fixation of a CO
by [3-
C]pyruvate
molecule results in double-labeling in the hepatic triose phosphate
precursor pool after randomization of oxalacetate. Combination of
[2-
C]acetyl-CoA (e.g. after
decarboxylation of [3-
C]lactate) with
[3-
C]oxalacetate (e.g. from
carboxylation of [3-
C]pyruvate) could also
result in double-labeled triose phosphates (e.g. derived from
[1,3-
C]oxalacetate), although this route has a
much lower probability of occurring. Other low probability pathways
exist that might result in double-labeled triose subunits (e.g. GlcA to xylulose followed by nonoxidative portion of pentose
phosphate pathway). Doubly-labeled triose subunits would complicate
MIDA, because the assumption of the MIDA technique using
[3-
C]lactate or
[2-
C]glycerol is that double-labeled glucose
molecules represent the combination of two single-labeled precursor
triose-P subunits. Double labeling of an individual triose-P subunit,
if present, would increase the EM
/EM
ratio in
glucose, thereby artifactually increasing the estimated p and
reducing estimated fractional GNG. The
CO
enrichment in hepatic mitochondria under these conditions is
difficult to predict, so the probability of
CO
combining with [
C]pyruvate cannot be
excluded on theoretical grounds. The analytic strategy and calculations
used to address this question are described in the
``Appendix.''
Experimental Testing
In order to test
whether double C-labeling of triose units occurred, we
asked whether the measured M
isotopomers of the
triose fragment of sorbitol (m/z 219) could be
completely accounted for on the basis of natural abundance isotope
distributions combined with incorporation of a single
C-labeled atom or whether double-labeled
[
C]triose units needed to be invoked (see
``Appendix''). Measurements were carried out on 22 plasma
glucose samples after reduction to sorbitol and GC/MS analysis of the m/z 217-219 fragment ion envelope, from rats
infused with [2-
C]glycerol or
[3-
C]lactate (). Enrichments of
EM
in the triose fragment were high (0.0400-0.1500
ME) and the divergence of observed from expected EM
values
was very small (-0.0005 ± 0.0001).
C-labeling of triose units as a significant
process during infusions of [2-
C]glycerol or
[3-
C]lactate. A second experimental strategy to
exclude
CO
reincorporation or other sources of
triose double-labeling was also employed.
[1-
C]Lactate cannot become double-labeled from
the simple
CO
reincorporation/carboxyl group
randomization/decarboxylation sequence or from the pyruvate
decarboxylation/acetyl-CoA recombination sequence, because loss of
either carboxyl group will result in loss of
C. As noted
above, when [1-
C]lactate was infused to 48-h
fasted rats, the value for f was 89 ± 3%, identical to
results from [3-
C]lactate, which is further
evidence against an important role for any artifact due to double
labeling in the triose phosphate pool.
Error-Sensitivity Analysis
Theoretical Considerations
See
``Appendix'' for theoretical considerations.
Experimental Testing
Evidence for experimental
reproducibility of estimates is presented above (comparisons between
and
-anomers of glucose and between glucose and GlcA). The
need for analytic accuracy as well as precision, if MIDA is to be
applied reliably to a short chain (n = 2) polymer like
glucose, must be emphasized.
CO
also had to be considered. Nevertheless,
the widely recognized absence of an alternative method for measuring
GNG(2, 3, 4, 5, 6, 7, 8, 9, 10) and various
advantages of MIDA (that it measures all-source GNG rather than only
PEP-GNG and that the intracellular triose-P enrichment can be
calculated) motivated the attempt to apply MIDA to GNG, as did the
large number of related metabolic questions that might be addressed if
GNG and hepatic triose-P enrichments could be measured in
vivo.
(
)As long as the second
source provides less than about 20% of GNG and the ratio of values for p is 2:1 or closer, effects on calculated p and f are minor. Finally, various technical aspects of the mass
spectrometric analyses can help maintain both precision and accuracy of
quantification. This last point is critical for successful application
of MIDA to an n = 2 polymer like glucose, since the
abundance of the M
-isotopomer will tend to be
lower than with longer chain length polymers and therefore must be
quantified with a high degree of precision and accuracy.
C]glycerol enters the hepatic triose-P pool in
a quantitative fashion (Fig. 1). Isotope recovery studies
demonstrated that, in the prolonged fasted state, about 85-90% of
infused [
C]glycerol could be accounted for as
entry into plasma glucose via the hepatic triose phosphate pool.
Another approximately 1-2% of [
C]glycerol
could be accounted for by secretion of
[
C]glycerol into very low density
lipoprotein-glycerol and about 2-3% as entry into the GlcA moiety
of infused acetaminophen (). Thus, even without
considering possible entry of
C from glycerol into
lactate, acetyl-CoA, or fat (all likely to be minor in 24-h fasted
rats), 90-95% of labeled glycerol was documented to pass through
the hepatic triose-P pool used for GNG. Brief fasting was associated
with a lower recovery of [
C]glycerol in plasma
glucose compared with more prolonged fasting, as might have been
anticipated. Administration of fructose at 10-15 mg/kg/min to
previously fasted rats resulted in 82-90% recovery as plasma
glucose, and >95% total recovery in end products of hepatic triose-P
metabolism. These results all support the expectation that label from
glycerol enters the hepatic triose-P pool in rats in a nearly
quantitative way(35, 36) , here documented to be 90% or
greater.
C]fructose in normal
children and found that at least 50% of fructose was directly converted
to glucose without being broken down to the 3-carbon level (i.e. not proceeding via fructose-1-phosphate aldolase). Our results in
rats given higher rates of intravenous fructose also indicate that some
direct conversion of fructose to glucose occurs, representing roughly
15-17% of the fructose load. Direct conversion may occur by
phosphorylation of fructose 1-phosphate by 1-phosphofructokinase, but
this has not been established. Chandramouli et al.(48) did not find evidence for substantial direct
conversion of fructose to glucose (0-15%, mean 5.1% of the load)
when fructose was administered at much lower doses (0.3 mg/kg/min) to
adult humans. Fructose load, species, age, route of administration, and
other factors may explain different activities of this pathway.
Table: Incorporation from
[3-C]lactate and
[2-
C]glycerol into hepatic triose-P precursor
pool
C]lactate and plasma glycerol in studies
infusing [2-
C]glycerol. Rats were fasted for
36-48 h prior to infusion of labeled substrate. Number of rats
studied in each group given by n. Experimental details are
described in the text.
Table: Primary data and derived GNG parameters
in three rats infused with [2-C]glycerol
Table: Recovery of
[C]glycerol in various end products of hepatic
triose-P metabolism
C-recovery in various end-products are
described in text. VLDL, very low density lipoprotein.
Table: Quantification of isotopic disequilibrium in
triose-P pool under various experimental conditions
Table: 0p4in
Extra M
residual (double
labeled) = labeled (residual) - Expected M
residual.(119)
C or
H does not by
itself make a subunit labeled, by the following definition:
as
the monoisotopic mass + 0; M
is the
monoisotopic mass + 1; etc.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.