(Received for publication, January 3, 1996; and in revised form, February 28, 1996)
From the
Rates of cycling between glucose and glucose 6-phosphate and between glucose and pyruvate, and the effects of these cycles on glucose metabolism, were compared in hepatocytes isolated from fasted normal or streptozotocin-induced diabetic rats. In diabetic hepatocytes the rate of glucose phosphorylation was 30% lower than that in normal hepatocytes, and there was a doubling of the rate of glucose/glucose 6-phosphate cycling. In addition, the rate of glycolysis was 60% lower in diabetic hepatocytes. This inhibition of glycolysis and stimulation of glucose/glucose 6-phosphate cycling appeared to be a consequence of the elevated rates of endogenous fatty acid oxidation observed in diabetic hepatocytes. The proportion of glycolytically derived pyruvate that was recycled to glucose was more than doubled in hepatocytes from diabetic rats compared with normal animals. This increase also appeared to be linked to the high rates of endogenous fatty acid oxidation in diabetic cells. As a consequence of the increased rates of both these cycles, 85% of all glucose molecules taken up by diabetic hepatocytes were recycled to glucose, compared with only 50% in normal hepatocytes. Glucose cycling is therefore likely to make a substantial contribution to the hyperglycemia of diabetes.
Insulin-dependent diabetes is characterized by elevated blood
glucose levels, the result of perturbations of glucose uptake and
metabolism in both the liver and extrahepatic tissues. In addition to
an increase in gluconeogenic activity in liver, a suppression of
hepatic glycolysis contributes to the elevation of blood glucose
levels(1, 2) . There is also evidence (3, 4, 5, 6, 7) that the
decline in net glycolytic flux in diabetes may be associated with an
increase in glucose/glucose 6-phosphate (G/G6P) ()cycling,
whereby glucose is taken up by the liver and phosphorylated, but the
glucose 6-phosphate (G6P) formed is subsequently dephosphorylated and
returned to the circulation.
Recently we have demonstrated a glucose/pyruvate (G/P) cycle in hepatocytes from normal rats, brought about through the concomitant operation of glycolysis and gluconeogenesis(8) . Because the activity of this cycle increased with glucose concentration(8) , it seemed possible that a G/P cycle might also contribute to hepatic glucose output in diabetes. We have examined this possibility in this study and have also determined the contribution of the G/G6P cycle to hepatic glucose output in diabetes. Fatty acid oxidation stimulates both G/G6P and G/P cycling in hepatocytes from normal rats(9) . As livers from diabetic rats exhibit elevated rates of endogenous fatty acid oxidation (10) , we also investigated whether an increase in glucose cycling may be associated with the high rates of fatty acid oxidation in hepatocytes from diabetic rats.
Radiolabeled products of glucose metabolism (lactate, pyruvate,
amino acids, and water) were separated from glucose by ion exchange
chromatography(9, 16) . Radiolabeled glycogen was
measured as described previously(17) . Rates of glucose
phosphorylation were measured as the sum of H
O
released from [2-
H]glucose plus the amount of
tritiated glycogen formed(9) . The tritiated glycogen
measurement was included to decrease the error resulting from
incomplete equilibration between glucose 6-phosphate (G6P) and fructose
6-phosphate(16, 18) . Glycolytic rates were measured
using [6-
H]glucose and were determined from the
sum of tritium recovered in water, lactate, pyruvate, and amino
acids(9) . The rate of G/G6P cycling was calculated from the
difference between the rates of glucose phosphorylation and total
[
H]glucose metabolism (measured as the sum of the
rate of glycolysis and the rate of incorporation of tritium, derived
from [6-
H]glucose, into glycogen)(9) .
This estimates the amount of glucose that was phosphorylated but was
not further metabolized either through the glycolytic pathway or to
glycogen. Measurements of glycolysis with
[6-
H]glucose were invariably higher than the
accumulation of glycolytic products (lactate, pyruvate, amino acids,
and CO
), which was estimated using
[U-
C]glucose. This discrepancy is caused by
recycling of glycolytic products back to glucose (G/P cycle) (19, 20, 21, 22) . Tritium is
recovered as
H
O in incubations of hepatocytes
with [6-
H]glucose when pyruvate, derived from
[6-
H]glucose, is oxidized or carboxylated in the
mitochondria. However, label will be lost from
[U-
C]glucose as
CO
only
through the mitochondrial metabolism of pyruvate. Thus, the rate of G/P
cycling can be calculated from the difference between rates of
glycolysis (measured with [6-
H]glucose) and the
accumulation of glycolytic products (measured with
[U-
C]glucose)(19) . Rates of glucose
phosphorylation, glycolysis, and cycling, which were linear over the
60-min standard incubation period, are expressed as µmol of glucose
equivalents
min
(g, wet
weight)
. In all experiments there was a greater than
95% recovery of isotope. Statistical analyses were carried out using
Student's t test for unpaired data.
Figure 1:
Lactate and pyruvate
production in normal and diabetic hepatocytes. Hepatocytes from fasted
normal () or diabetic rats (
) were incubated with 80 mM glucose for up to 2 h. Lactate and pyruvate were measured as
described under ``Experimental Procedures.'' Results show
means ± S.E. of at least 4
experiments.
The observation that glycolysis occurred in
diabetic hepatocytes was confirmed by the use of
[6-H]glucose in the incubation mixture.
Glycolytic flux in diabetic cells was only 40% of that recorded in
hepatocytes from normal rats (Table 1). One factor that
contributed to the decrease in glycolysis in diabetic cells was a
diminished rate of G6P formation. Estimates of the rate of glucose
phosphorylation (from [2-
H]glucose metabolism)
revealed that the rate of synthesis of G6P was only about two-thirds of
that measured in incubations with control hepatocytes. In diabetic
cells, more than 50% of G6P formed was recycled to glucose, whereas
only 25% of G6P was dephosphorylated in control hepatocytes (Table 2). Therefore, the depression of glycolytic flux in
diabetic hepatocytes was due not only to a decrease in the rate of G6P
production but also because a greater proportion of G6P formed was
reconverted to glucose, without undergoing glycolysis.
During the
60-min incubation period, rates of glycogen synthesis were lower in
diabetic cells than in normal cells, and the accumulation of glycolytic
products in diabetic cells was less than 20% of that in normal cells (Table 1). In incubations with normal hepatocytes, 70% of the
label derived from [U-C]glucose metabolism was
recovered in lactate and pyruvate, with
CO
production representing a further 25% of labeled glucose
metabolism. In contrast,
CO
was the main
product of [U-
C]glucose metabolism in
incubations with diabetic cells. In cells from both normal and diabetic
rats, there was a large discrepancy between the rates of glycolysis and
accumulation of glycolytic products (Table 1). Such differences
have been taken to indicate cycling of glycolytically derived pyruvate
back to glucose (19) . In normal hepatocytes about 30% of the
total
H-glycolytic flux could not be accounted for as
C-labeled products and therefore represented G/P cycling (Table 2). In diabetic animals, about 70% of glycolytically
derived pyruvate was recycled to glucose.
The ATP requirement for
glucose cycling can be calculated by assuming that 1 mol of ATP is
cleaved per mol of glucose phosphorylated; 2 mol of ATP are produced
per mol of glucose glycolyzed to lactate and pyruvate, whereas 6 mol
are required for the formation of 1 mol of glucose from pyruvate or
lactate. In diabetic hepatocytes, the ATP requirements for the observed
rates of glycolysis and glucose cycling were 2.33
µmolmin
(g, wet
weight)
, whereas normal cells turned over ATP for
this process at a rate of 1.22
µmol
min
(g, wet
weight)
.
Evidence
for such an interaction was obtained from experiments in which fatty
acid oxidation was inhibited with DL-2-bromopalmitate (23) . In the presence of the inhibitor, endogenous ketone body
production was markedly decreased in diabetic cells to a rate of 0.14
± 0.07 µmolmin
(g, wet
weight)
, n = 3. However, the
inhibitor had no effect on the low rate of ketone body production in
normal liver cells (0.06 ± 0.01
µmol
min
(g, wet
weight)
, n = 5), suggesting that
glucose rather than fatty acid may be the main precursor for ketone
body synthesis in these cells. In diabetic hepatocytes, the
accumulation of [
C]lactate and
[
C]pyruvate was 10-fold higher in the presence
of bromopalmitate (Table 1), but the inhibitor induced only a
small increase in net [
C]lactate and
[
C]pyruvate production in normal liver cells. A
similar increase in the rate of [
C]lactate and
[
C]pyruvate accumulation was noted in diabetic
cells when fatty acid oxidation was inhibited with 2-tetradecylglycidic
acid (24) (results not shown). The addition of bromopalmitate
had no effect on the rate of G/G6P cycling in normal hepatocytes but
induced a substantial decline in the proportion of G6P that was
recycled to glucose in diabetic liver cells (Table 2). In
hepatocytes from normal rats, bromopalmitate had no effect on the rate
of glycolysis, in contrast to diabetic cells where flux through the
pathway was doubled (Table 1). The agent stimulated the
accumulation of glycolytic products by about 25% in normal cells but
induced a 3-4-fold increase in diabetic hepatocytes.
Bromopalmitate had no effect on the activity of the G/P cycle in normal
cells (Table 2). In diabetic cells however, the inhibitor reduced
the proportion of glycolytically derived pyruvate that was recycled to
glucose, although there was an overall increase in the absolute rate of
G/P cycling due to the substantial stimulation of glycolysis.
It is possible that the increase in the activity of the G/G6P cycle that we observed in diabetic hepatocytes relates solely to the change in the relative activities of glucokinase and glucose-6-phosphatase. However, we have previously reported an increase in the rate of G/G6P cycling when palmitate was added to suspensions of normal hepatocytes incubated with glucose(9) . This suggests that the elevated rates of endogenous fatty acid oxidation observed in diabetic hepatocytes may also contribute to the stimulation of G/G6P cycling in these cells. The observation that G/G6P cycling could be reduced in diabetic cells by the inclusion of the inhibitor of carnitine palmitoyltransferase, bromopalmitate, supports this hypothesis. It also implies that the effect of fatty acid is mediated by a product of its metabolism in the mitochondria. In keeping with this, Lickley et al. (3) observed an increase in G/G6P cycling that paralleled a rise in levels of plasma free fatty acid when alloxan diabetic dogs were treated with both somatostatin and glucagon. The molecular mechanism of the effect of fatty acid on the G/G6P cycle is unresolved.
In order for pyruvate to enter the
mitochondria via the pyruvate translocator, there must be an exchange
with a counterion(25) . A number of anions can participate in
this exchange, but the ketone bodies 3-hydroxybutyrate and particularly
acetoacetate are the most effective in stimulating pyruvate
uptake(25) . It is possible, therefore, that the stimulation of
pyruvate uptake into the mitochondria by fatty acid is partly the
result of the provision of these products of fatty acid oxidation.
Indeed, the stimulation of gluconeogenesis from lactate by fatty acid
may be mediated via this mechanism(26) . In both normal and
diabetic cells, most of the pyruvate that enters the mitochondria is
recycled to glucose, only 20-30% is oxidized to CO.
This predominance of the carboxylation and gluconeogenic pathway over
the oxidation pathway is likely to reflect the effects of fasting and
diabetes on pyruvate dehydrogenase(28, 29, 30) and pyruvate carboxylase(28, 30) .
Although the phenomenon of glucose
cycling is well recognized, its physiological significance is unclear.
It has been argued that it may play a role in the regulation of flux
through metabolic pathways(33, 34) , although the
mechanism of this regulation has not been elucidated. We have shown
that the maintenance of lactate concentrations in the incubation medium
by normal hepatocytes is a function of glucose
concentration(17, 35, 36) , a process that
involves glucose cycling. In the absence of glucose cycling it is
possible that hepatic gluconeogenesis could almost completely deplete
blood lactate and, therefore, pyruvate. On the other hand, under
conditions of high rates of glucose utilization, cycling of lactate to
glucose may help to maintain glucose steady states. Alternatively, in
diabetic animals, glucose cycling may be a mechanism to compensate for
a depression in other thermogenic processes (37, 38, 39, 40, 41) as the
ATP turnover due to glucose cycling in diabetic hepatocytes is double
that in normal cells and may account for the previously unexplained
higher rates of O consumption in diabetic
hepatocytes(31) .