(Received for publication, September 8, 1995)
From the
To determine whether the source of carbon for the indirect
pathway of hepatic glycogen synthesis differs between the periportal
and pericentral zones, we studied seven 24-h-fasted conscious rats
given a constant 2-h intraduodenal infusion of glucose, 40% labeled
with [U-C]glucose (99%
C enriched),
to raise and maintain plasma glucose concentration at
10
mM. Glycogen, glutamate, aspartate, and alanine were
selectively sampled from the periportal and pericentral zones of the
liver by the dual-digitonin pulse technique and analyzed by
C-NMR for positional isotopomer distribution and by gas
chromatography-mass spectrometry for mass isotopomer distribution.
Plasma glucose mass isotopomer distribution was determined from gas
chromatography-mass spectrometry. The isotopomer distribution indicates
that there was no significant difference between the zones with respect
to 1) percent direct flux of glucose into the glycogen (periportal, 34
± 4; pericentral, 38 ± 4), 2) extent of
oxaloacetate/fumarate equilibration (periportal, 0.54±.01;,
pericentral, 0.53 ± 0.01), 3) dilution of tracer in oxaloacetate
(periportal, 0.64 ± 0.07;, pericentral, 0.64 ± 0.07), or
4) inflow of pyruvate versus tricarboxylic acid cycle flux
(periportal, 0.70 ± 0.20; pericentral, 0.68 ± 0.16).
Positional isotopomer populations, determined from the
C-
C splitting in C3 and C4 of
periportal and pericentral glycogen, were indistinguishable, indicating
no significant differences in the source of the 3-carbon precursors for
hepatic glycogen synthesis by the indirect pathway. In conclusion,
glucose metabolism is the same in the periportal and pericentral zones
with regard to 1) the relative flux of carbon via the direct/indirect
pathways, 2) the source of the 3-carbon precursor used in the indirect
pathway of glycogen synthesis, and 3) the flux of the 3-carbon
precursors through the tricarboxylic acid cycle.
Liver parenchyma display a heterogenous spatial distribution of
activity of those enzymes associated with gluconeogenesis and
glycolysis, as determined by microdissection and microhistochemical
assays(1) . In vitro, liver perfusion studies showed
that glycogen was deposited only in the periportal zone when
gluconeogenic precursors were perfused and that glycogen was deposited
only in the pericentral zone when glucose was perfused(2) .
Based on these observations, it has been proposed that hepatocytes in
the periportal zone are gluconeogenic and that pericentral hepatocytes
are glycolytic (3) . If so, this has important implications for
interpretation of data obtained from virtually all in vivo based studies of liver metabolism. In a recent study, we
administered [1-C]glucose to awake unstressed
rats and selectively sampled periportal and pericentral glycogen by the
dual-digitonin pulse technique(4, 5) , and we found no
difference between the periportal and pericentral zones in the relative
percentages of the direct
(Glc
Glc-6-P
Glc-1-P
UDP-glc
glycogen) and indirect
(3-carbon units
glycogen) pathways of glycogen
repletion(4) .
However, it is still possible that the source of the 3-carbon precursors for liver glycogen may differ between the zones. Even though the relative contributions for the direct and indirect pathway were similar between the two populations of hepatocytes, it is still possible that the three carbon units for the indirect pathway of glycogen synthesis in one population of hepatocytes was derived by intracellular glycolysis while the other population of hepatocytes derive their three carbon units for glycogenesis from extrahepatic sources. This question carries additional weight in view of our recent study in awake triple-catheterized dogs, in which we found that during an intraduodenal glucose infusion net hepatic glucose uptake could account for all of the glycogen synthesized by the liver from both the direct and indirect pathways(6) . These results imply that the liver is capable of undergoing substantial glycolytic and gluconeogenic flux simultaneously, raising the question as to whether or not, under these conditions, some hepatocytes are mostly glycolytic and others are mostly gluconeogenic or whether substrate cycling between glycolysis and gluconeogenesis is occurring to an equal extent within all hepatocytes.
The purpose of this study was to
assess whether there are differences in the source of carbon for
glycogen synthesized via the indirect pathway in the periportal and
pericentral zones in vivo. Hepatic glycogen stores in awake,
unstressed rats were repleted after a 24-h fast with a constant
intraduodenal glucose infusion enriched with
[U-C]glucose. Periportal and pericentral cytosol
were then selectively sampled by using the dual-digitonin-pulse
technique, and the distribution of positional and mass isotopomers of
glycogen, alanine, aspartate, and glutamate were ascertained by
C-NMR and GC-MS (
)spectroscopy. Any difference
in the source of carbon for the indirect pathway of glycogen repletion
in the periportal and pericentral zones can be determined from
differences in the isotopomer distribution of glycogen and of the amino
acids that equilibrate with metabolites of the tricarboxylic acid
cycle. From mass isotopomer analysis, the relative proportions of the
direct to indirect flux of glucose in glycogen can be determined. In
addition, the tricarboxylic acid cycle parameters of pyruvate
carboxylase to citrate synthase flux and the magnitude of
oxaloacetate/fumarate equilibration can be calculated. From analysis of
the
C-NMR spectra, the positional isotopomers of glycogen,
and hence the relative flux of labeled to unlabeled 3-carbon precursors (i.e. glycerol and free fatty acids relative to pyruvate), can
be evaluated. The data derived from the
C-
C J coupling between the C3
and C4 carbons of glycogen is unique in that the
C-
C splitting pattern will be very
different if the source of the three carbon units for the indirect
pathway is derived from intracellular glycolysis versus extrahepatic sources since the C3 and C4 carbons reflect the
contribution from two different pyruvate molecules. This type of
positional isotopomer analysis is unique and the most comprehensive way
of addressing this question since it reflects the
C
labeling in adjacent carbons within the same glucose molecule as
opposed to examining the
C labeling distribution within a
population of glucose molecules. This type of analysis was not possible
in our previous study since only [1-
C]glucose
was used and does not lead to a comparable array of isotopomeric
species, which provide a richer wealth of informational content.
The percent of
glycogen derived from the flux of [U-C]glucose
via the direct pathway was calculated by a modification of an equation
derived for use of [U-13C]Glc as a
tracer(14, 15) ,
On-line formulae not verified for accuracy
where P is proportional to the number of
glucose molecules with n (n = 1-6)
C atoms, and F is the dilution factor of
enrichment in C
intermediates due to entry of unlabeled
metabolites into the indirect pathway.
Alternatively, the percent
direct pathway was calculated by a modification of equations previously
derived for use of [1-C]Glc as
tracer(11) ,
On-line formulae not verified for accuracy
where Gly represents the
corrected enrichment of glycogen with
[U-
C]glucosyl units, and Glc
is the enrichment of portal vein glucose with
[U-
C]Glc. The correction factor, C.F.,
adjusts for the time for the infusion to reach isotopic steady state in
plasma [U-
C]Glc. The MPE of
[U-
C]glycogen is corrected for basal glycogen
stores with no [U-
C]glucosyl units,
On-line formulae not verified for accuracy
where Gly equals the m+6
MPE of glycogen determined by GC-MS, [Gly]
is the
final glycogen concentration, and [Gly]
is the
basal glycogen concentration.
The probability that two C-labeled 3-C precursors combine to form
C
-glycogen by the indirect pathway,
P
, was calculated as follows:
On-line formulae not verified for accuracy
where MPE is the m+3 MPE
liver alanine, and P
is the m+3 MPE of
glycogen(15) .
Calculation of the relative C
isotopic distribution from the NMR signal intensity of overlapping
resonances was calculated as previously derived(16) .
The results are expressed as means ± S.E. Comparisons between groups were made by the paired student's t test.
From the isotopomer frequency in the mass spectrum of
periportal and pericentral liver glycogen, plasma glucose, and infusate (Table 1), the MPE of each mass isotopomer was calculated (Table 2). During the basal period, plasma glucose concentration
was 6.5 ± 0.1 mM and rose to a plateau of 9.9 ±
0.1 mM by 30 min of infusion. Plasma glucose m+6 MPE
([U-C]Glc) plateaued at 28.2 ± 2.0 by
60 min. The MPE of plasma glucose with one to three
C
atoms continued to rise throughout the study, indicative of some
hepatic glucose output from gluconeogenesis despite hyperglycemia. To
ensure selective recovery of the glycogen from the periportal and
pericentral zones, we limited exposure of the liver to digitonin. For
maximum recovery of glycogen without sacrificing selectivity, we found
a 20-s pulse of digitonin at a concentration of 5 mg/ml to be optimal,
as described in our earlier study(4) . In that study, we varied
digitonin concentration and pulse durations to minimize contamination
between the zones as determined from the enzyme activity of glutamine
synthetase, which occurs only in the pericentral zone(9) .
Selective zonal cell permeabilization was further verified by light
microscopy from liver following a 20-s pulse of digitonin (5 mg/ml) at
20 ml/min(4) . Enzyme activities of glutamine synthetase and
lactate dehydrogenase recovered in the eluant following the digitonin
pulse were similar to that found previously, reflecting selective
sampling of the pericentral and periportal zones(4) . Recovery
of glycogen from each zone was a few percent of the total hepatic
glycogen content. We estimate that
70% of the total liver glycogen
was synthesized during the 2-h infusion protocol(4) .
Using
the mass isotopomer distribution (Table 2) in the glycogen
recovered from the different fractions and , it is possible
to calculate the percent direct flux of glucose into glycogen and to
test the hypothesis that liver carbohydrate metabolism is spatially
heterogenous. We found no significant difference in the percent direct
flux of glucose into glycogen within either the periportal (34 ±
4) or pericentral (38 ± 4) zones or the residual glycogen (41
± 1, p = ns). The probability that two C-labeled 3-C precursors combine to form
[U-
C]glycogen by the indirect pathway can be
determined from the MPE of triple-labeled glycogen and alanine (Table 2, ) and was calculated to reduce the estimate
of the percent direct pathway by only
1%. From the enzyme
profiles, we estimate <10% cross-contamination of cytosol from the
two zones. Allowing for 20% cross-contamination (i.e. that the
calculated percent direct flux represents a mix of 80% glycogen from
the upstream zone and 20% from the downstream zone), we calculate that
this will decrease our estimate of the direct flux in the periportal
zone to 32% and increase the direct flux in the pericentral zone to
41%. Because of hepatic glucose production during the study, the plasma
glucose has mass isotopomers in addition to the m+6 of the
infusate. By definition, the plasma glucose m+1
m+3
isotopomers are derived via flux of glucose through the indirect
pathway. We, therefore, have not made any corrections in the observed
mass isotopomer distribution to account for the increase in the
enrichment of the m+1
m+3 isotopomers in the glycogen
from direct flux of plasma glucose enriched in isotopomers other than
m+6. However, if we take the extreme case in which all glycogen in
the pericentral zone is synthesized via the direct pathway and account
for the observed isotopomer pattern in plasma glucose, the direct flux
of plasma glucose into pericentral glycogen will decrease the m+1
m+3 enrichment from 15.5 to 13.5 (15.5 -
(6.9/30.2)*(2.6+3.6+2.5)) and increase the calculated percent
direct by only 3%, from 38 to 41%. The combined effect of 20%
contamination and 100% direct flux of labeled glucose increases the
estimate of the direct pathway in the pericentral zone to 45%. Thus,
over half of the glycogen in both zones is synthesized by way of the
indirect pathway, and the differential between the zones in the direct
pathway is at most only
13%.
These results are in qualitative
and quantitative agreement with those of our earlier study (i.e. direct flux was approximately the same in the two zones:
periportal, 29%; pericentral, 35%) as determined from a comparison of
[1-C]glycogen enrichment with plasma
[1-
C]glucose enrichment(4) . However,
there is a subtle distinction in the interpretation of the percent
direct flux calculated by these two methods. Using
[1-
C]glucose as infusate, we calculate the
fraction of glycogen synthesized directly from plasma glucose, the
remainder of the glycogen synthesized by flux through the indirect
pathway is inclusive of all carbon sources. In contrast, only glycogen
that originated from plasma glucose is considered when a dilution
factor of 1 is used in to calculate the percent direct
flux from the m+1
m+6 mass isotopomer distribution of
glycogen synthesized from [U-
C]glucose.
Therefore, these values represent the maximum contribution of the
direct pathway.
Inclusion of the dilution factor, F, in allows for entry of unlabeled 3-carbon precursors
(primarily, flux of unlabeled glycerol into dihydroxyacetone-P (DHAP)
at the level of triose isomerase), and refines the estimate of the
direct pathway by this method(17, 18) . The magnitude
of the factor, F, in can be estimated by
comparing the calculation of direct flux from the mass isotopomer
distribution within glycogen () with a calculation based on
the ratio of m+6 enrichment in glycogen with that of plasma
glucose (). Using , and parameters determined
previously (4) under similar experimental conditions (basal
glycogen concentration, 0.57 g/100 g of wet liver; glycogen synthesis
rate, 0.61 µmol/g of liver/min; steady state correction factor,
0.79), we estimate the direct flux to be 36% in the periportal
zone and
41% in the pericentral zone. The agreement between the
two methods of calculating percent direct flux indicates that the
dilution of C-3 labeled isotopomers is minor and similar in both zones
(periportal, 1.3; pericentral, 1.3; p = ns). The
dilution by unlabeled carbon we have determined is approximately half
that calculated by Katz and Lee (17) using a similar
experimental design. However, they make no allowance for correction of
basal glycogen stores (which they determined were negligible) or for
the time necessary to reach isotopic steady state (which from our
experience is
0.8-0.9). Using a steady-state correction
factor of 0.8 and the data presented by Katz and Lee(17) , we
calculate a value of 62 percent direct pathway and a value of 1.4 for
the dilution of the 3-carbon pool by non-glucose carbon in their
experiment. Additional dilution of label can occur as the result of
flux of labeled precursor through the tricarboxylic acid cycle, E, and can be calculated from the isotopomer distribution in
the liver glycogen using an equation derived by Katz and
Lee(17, 18) ,
On-line formulae not verified for accuracy
where P is proportional to the number of
glucose molecules that have n (n = 1-3)
C atoms. Comparable to the value of 1.4 determined by Katz
and Lee(17) , we calculate E to be 1.6 in both the
periportal and pericentral zones. The overall dilution, T (the
product of E and F), is then
1.8 in both zones.
This is similar to a value of 1.9 for the overall total dilution of
labeled pyruvate into glycogen using the data of Katz and
Lee(17) , if we incorporate a steady-state correction factor of
0.8 as above. Thus, in either the periportal or pericentral zones of
the glycogen derived via the indirect pathway, approximately 44% of the
carbon was from sources other than the infused glucose.
Flux through
the tricarboxylic acid cycle results in randomization of label and
dilution from equilibration of tricarboxylic acid intermediates with
other unlabeled metabolic pools. Using a model of the tricarboxylic
acid cycle derived to follow the distribution of mass isotopomers in
glycogen originating from [U-C]glucose, we can
calculate the relative rates of pyruvate carboxylase versus citrate synthase (y) and hence determine the extent of
dilution of tracer in OAA (R)(14) . Under the
conditions of glycogen repletion following a fast, there was no
significant periportal/pericentral heterogeneity of glycogen metabolism
with respect to any of these tricarboxylic acid parameters: pyruvate
carboxylase versus tricarboxylic acid cycle flux (y;
periportal, 0.70 ± 0.20; pericentral, 0.68 ± 0.16; p = ns), dilution of label in the conversion of pyruvate to
OAA (R; periportal, 0.64 ± 0.07; pericentral, 0.64
± 0.07; p = ns), and extent of OAA/fumarate
equilibration (periportal, 0.54 ± 0.01; pericentral, 0.53
± 0.01, p = ns) (Fig. 1). We calculate
similar values for these parameters for the residual liver glycogen
following digitonin treatment (y = 0.58 ± 0.10, R = 0.65 ± 0.05, and OAA/fumarate equilibration
= 0.58 ± 0.01; p = ns compared to
periportal or pericentral glycogen). Equilibration of tricarboxylic
acid intermediates (OAA with aspartate and
-KG with glutamine)
with other unlabeled pools as a source of isotopic dilution can be
determined from analysis of the isotopic enrichment in alanine,
aspartate, and glutamate. We found no significant differences between
the two zones in the enrichment of intermediates in equilibration with
the tricarboxylic acid intermediates (Table 2). Therefore, there
was no appreciable heterogeniety in the flux of substrates into the
tricarboxylic acid cycle between the periportal and pericentral zones.
Figure 1:
Pathways of glycogen synthesis and
tricarboxylic acid cycle parameters in periportal and pericentral
hepatic zones. Fraction of glycogen synthesized by the indirect pathway (ind), degree to which equilibration of oxaloacetate to
fumarate is complete (O/F), and pyruvate carboxylase to
tricarboxylic acid cycle flux (P/T) were calculated from the
mass isotopomer distribution of periportal and pericentral glycogen (Table 2) and a model of the tricarboxylic acid cycle developed
to account for mass-isotopomer distribution in glycogen derived from
[U-C]glucose via the indirect
pathway(14) . Glycogen was selectively isolated by the
dual-digitonin-pulse technique (4, 5) from livers of
rats following a 2-h intraduodenal infusion of
[U-
C]glucose.
An index of the relative flux of unlabeled glycerol into DHAP
relative to triose isomerization can be determined from the enrichment
of glycogen C1C3 relative to enrichment of C4
C6. Early
studies of gluconeogenesis using either
C-labeled CO
or glycerol demonstrated asymmetric distribution of label in
glycogen, suggesting incomplete equilibration of label at the
triose-isomerase step(19, 20) . From the NMR spectra,
relative enrichment of
C in C1
C3 and in
C4[arrow]C6 was assessed(16) . No significant
differences were seen in the
C enrichment of glycogen
C1[arrow]C3 (derived from DHAP) compared to
C4[arrow]C6 (derived from glyceraldehyde-3-P, GAP) in the
glycogen isolated from the periportal or pericentral zones
(C1
C3/C4[arrow]C6: 1.2 ± 0.04, 1.1 ±
0.01, respectively, p = ns). Therefore, distribution of
label into fructose 1,6-diphosphate from DHAP and glyceraldehyde-3-P
under these experimental conditions is dominated by triose isomerase
equilibration rather than by flux of unlabeled glycerol in both the
periportal and pericentral zones.
Further evidence of the similarity
of the carbon source for the indirect pathway can be seen in the C-NMR spectrum of glycogen. In the
C-NMR
spectra of hepatic glycogen following an infusion of
[U-
C]glucose, multiple isotopomers are observed
due to coupling of magnetic spin between adjacent
C,
making it possible to distinguish labeled atoms that have 0, 1, or 2
C as nearest neighbors. Positional isotopomers of
C-labeled glycogen can thus be quantified by analysis of
the splitting patterns in the
C-NMR spectra (Table 3). The splitting pattern in C-3 and C-4 of periportal and
pericentral glycogen will differ if there are relative differences in
the number of labeled 3-carbon precursors combining to form glycogen. Fig. 2is typical of the splitting pattern seen in the
C-NMR spectra of
-C-3 and
-,
-C-4 of
periportal and pericentral glycogen. The triplet at C-3 is the
superposition of a triplet due to glycogen labeled at C-2, C-3, and
C-4, with a singlet from molecules labeled at C-3 with no label at
either C-2 or C-3. The doublet at C-3 is the resonance due to glycogen
molecules with label at C-2 and C-3 but not C-4, and with label at C-3
and C-4 but not C-2. The doublet is due to isotopomers derived solely
via the indirect pathway, whereas the triplet resonance (minus the
singlet) is due to glycogen derived mainly from the direct pathway.
Interpretation of the multiplet structure of C-4 is analogous to that
of C-3. The similarity of the splitting pattern (doublet to triplet) at
C-3 (periportal, 0.53 ± 0.9; pericentral, 0.41 ± 0.6; p = ns) and C-4 (periportal, 0.33 ± 0.4;
pericentral, 0.29 ± 0.3, p = ns) indicates that
the glycogen derived from the indirect pathway has an equivalent
distribution of the C3 and C4 positional isotopomers in the two zones.
Figure 2:
Proton-decoupled C NMR (500
MHz) spectra and schematic of positional isotopomers of glycogen
isolated from the periportal and pericentral hepatic zones. Glycogen
was selectively isolated by the dual-digitonin-pulse technique (4, 5) from livers of rats following a 2-h
intraduodenal infusion of [U-
C]glucose. A
doublet at C3 or C4 (J = 37.2 and 40.5 Hz,
respectively) indicates glycogen with one
C atoms vicinal
to C3 or C4, whereas a triplet at C3 or C4 (J = 75.1
and 40.5 Hz, respectively) indicates glycogen with two
C
atoms vicinal to C3 or C4.
In conclusion, we found no isotopic evidence in support of hepatic heterogeneity with respect to either the amount or the source of the 3-carbon precursors used to synthesize glycogen via the indirect pathway. Furthermore, there was no difference between the periportal and pericentral zones with regard to the flux of the 3-carbon precursors through the tricarboxylic acid cycle.