(Received for publication, May 8, 1995; and in revised form, July 10, 1995)
From the
In order to examine metabolic zonation in human liver,
glycerol, which labels carbons 2 and 5 of
glucose-6-P, and [1-C]lactate, which labels
carbons 3 and 4 of glucose-6-P, in the process of gluconeogenesis, were
infused intravenously into healthy subjects who ingested acetaminophen
and had fasted 36 h. Distributions of
C were determined in
glucose in blood and in the glucuronic acid moiety of acetaminophen
glucuronide excreted in urine. Ratios of
C in carbons 2
and 5 to
C in carbons 3 and 4 were significantly higher in
blood glucose than in glucuronide. Since glucose and glucuronic acid
are formed from glucose-6-P in liver without randomization of carbon,
the differences in the ratios indicate that the pool of glucose-6-P in
liver is not homogeneous. The glucuronide sampled glucose-6-P with more
label from lactate than glycerol compared to the glucose-6-P sampled by
the glucose. The apparent explanation is the greater decrease in
glycerol compared with lactate concentration as blood streams from the
periportal to the perivenous zones of the liver lobule. Glucuronidation
is then expressed in humans relatively more in the perivenous than
periportal zones and gluconeogenesis from glycerol more in the
periportal than perivenous zones.
Hepatocytes from the periportal and perivenous zones of livers of animals have been found by in vitro techniques to have differing metabolic capacities(1, 2) . In gluconeogenesis, glucose-6-P formed from gluconeogenic substrates is hydrolyzed to glucose. In glucuronidation, glucuronic acid from UDP-glucuronic acid is conjugated. The UDP-glucuronic acid is formed from glucose-6-P with glucose-1-P and UDP-glucose as intermediates. Thus, both glucose and glucuronic acid are formed from glucose-6-P with carbon skeletons unchanged.
In humans fasted for 12 h and 60 h, the concentration of glycerol, but not lactate, is much lower in hepatic vein than arterial blood(3, 4, 5) . This means that during fasting periportal, as compared to perivenous, cells of human liver are exposed to relatively higher glycerol than lactate concentrations. Since a higher capacity for gluconeogenesis is reported to exist in periportal than perivenous cells of animals and for glucuronidation in perivenous than periportal cells(1, 2) , we postulated that if carbon-labeled lactate and glycerol were administered to humans and metabolic zonation similarly exists in human liver in vivo, more label from the glycerol would be found in glucose than in a glucuronide relative to label from the lactate.
We infused into fasted normal subjects,
given acetaminophen, [2-C]glycerol, which labels
carbons 2 and 5 of glucose-6-P, and
[1-
C]lactate, which labels carbons 3 and 4 of
glucose-6-P (Fig. 1). The ratios of the
C in
carbons 2 and 5 to the
C in carbons 3 and 4 of blood
glucose were compared with the ratios in the acetaminophen glucuronide
excreted in urine.
Figure 1:
Pathways in the
conversion of C from [2-
C]glycerol
(position of label shown by
) and from
[1-
C]lactate (position of label shown by
)
into glucose and glucuronic acid.
Blood samples, each 75 ml, were drawn between 3 and 5 h in the first two subjects and between 5 and 8 h in the other two subjects. Subjects were encouraged during the infusions to drink about 240 ml of water/h. Urine was collected from the first two subjects between 2-3.5 h and 3.5-5 h and in the other two between 5-6.5 h and 6.5-8 h. One ml blood samples were collected at the beginning and end of each infusion for determination of plasma glucose concentration.
The acetaminophen
glucuronide was isolated, reduced to its glucoside, and glucose
isolated from the glucoside as described
previously(7, 8) . Briefly, urine at the time of
collection was taken to pH 4.5 and frozen until processed further. The
urine was concentrated, then brought to its original volume with
methanol, and the precipitate that formed was discarded. The methanol
was evaporated and the resulting concentrate made basic and applied to
a column of AG1-X8 in the acetate form (Bio-Rad). The column was washed
with water and then increasing concentrations of acetic acid. The
fraction eluted containing the glucuronide, identified using
carbazole(7) , was evaporated to dryness. The glucuronide was
reduced with diborane to acetaminophen glucoside and the glucoside
hydrolyzed with -glucosidase. Glucose from the deionized
hydrolysate was isolated as just described for glucose from blood.
A
portion of each glucose from blood and urine was combusted to
CO. The remainder of each glucose was degraded to yield
each of its six carbons as CO
. Each CO
was
assayed for
C specific activity(6, 7) .
Plasma glucose concentrations at the beginning of the
infusions ranged from 3.9 to 5.1 mM. Over the duration of the
infusions concentrations declined by 0.5-0.7 mM. Amounts
of C in glucoses isolated from some blood samples were
small, and therefore degradations of those glucoses were not done.
C was in greatest percentages in carbons 2, 3, 4, and 5 (Table 1) in accord with the pathways followed by the labeled
carbons of the glycerol and lactate (Fig. 1). Recoveries were
good. There was only about one-third as much
C in carbons
2 and 5 as carbons 3 and 4 of glucose from the first subject, AD. This
was the reason for giving 25 µCi rather than 10 µCi of
[2-
C]glycerol to the other three subjects, since
the lower the amount of
C in a carbon, the more likely an
error in the determination of the amount.
C in carbon 2
was more than in carbon 5 (p < 0.001), and
C
in carbon 4 was more than in carbon 3 (p < 0.001). This
result is in accord with glycerol's entrance into the triose-P
pool via dihydroxylacetone-3-P and lactate's via
glyceraldehyde-3-P and incomplete isotopic equilibration of the triose
phosphates. Distributions in the glucose from blood and from
glucuronide in the periods of collection were remarkably similar. The
ratios of
C in carbons 2 and 5 to
C in
carbons 3 and 4 were significantly different in blood glucose and in
glucose from glucuronide (p < 0.005), being higher by an
average of 30% (range from 26 to 37%). Incorporations into carbons 1
and 6 occur in the formation of glucose-6-P from
[2-
C]lactate formed from the
[2-
C]glycerol. Subtraction of the incorporations
of
C from the [2-
C]lactate into
carbons 2 through 5 (10, 11) will give a still greater
difference in the carbons 2 and 5 to carbons 3 and 4 ratios.
The duration of infusion was increased from 5 to 8 h, with
sampling during the last 3 h of infusion, to assure a steady state.
However, just the very similar distributions at different times of
blood and urine collections for each subject provide that assurance.
Since glucose and glucuronic acid are both formed from glucose-6-P
without randomization of carbon, the differences in the distributions,
reflected in the differing C + C
/C
+ C
ratios, mean there was not a homogeneous
pool of glucose-6-P in liver. The glucuronide sampled glucose-6-P in
hepatocytes where there was overall more label from lactate than
glycerol, than was the case for the glucose-6-P the glucose sampled.
The approach we introduce here for testing for metabolic
heterogeneity in liver demonstrates differing metabolism in hepatocytes
in humans in vivo. That conclusion rests on two assumptions.
First, that the distribution of C in blood glucose
reflects distribution in glucose-6-P in liver. Liver is the major
source of blood glucose, but there is a report of a significant
contribution of kidney to glucose production in dogs fasted
overnight(12) . Second, that the distribution of
C
in the glucuronide reflects distribution in glucose-6-P in liver. Liver
is the major site of glucuronidation, although glucuronidation can
occur in other tissues (13) . The present results are in accord
with our previous finding that when
[U-
C]glycerol and acetaminophen are given to
fasted subjects isotopomer patterns are different in blood glucose and
glucuronic acid from urinary acetaminophen glucuronide(8) .
Neese et al. recently reported (14) that rats
infused with [2-C]glycerol and acetaminophen,
and fasted 24 h or infused with fructose, formed glucose and
acetaminophen glucuronide from triose-P of the same enrichment. This
seeming difference from our findings may be related to the difference
in species, a quantity of glycerol rather than a trace amount being
given to the rats, the conversion of glycerol to lactate, and the
effect of the fructose load when that was also given.
A different gradient in the concentrations of glycerol and lactate along the liver lobule (3, 4, 5) is an apparent major reason for the differing distributions we observe. Other factors, such as differences in the activity of glycerokinase along the liver lobule, could play a role. Hence, hepatocytes in the hepatic vein zone were presumably exposed to more label from the lactate than glycerol compared to hepatocytes in the periportal zone. The glucuronide compared to the glucose then sampled glucose-6-P to a greater extent in the hepatic venous than periportal zone. That conclusion is in accord with a higher capacity for glucuronidation found in rat liver in vitro in hepatic venous than periportal zones.