©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Suppression of Glycolysis Is Associated with an Increase in Glucose Cycling in Hepatocytes from Diabetic Rats (*)

(Received for publication, January 3, 1996; and in revised form, February 28, 1996)

Debra C. Henly (§) John W. Phillips Michael N. Berry

From the Department of Medical Biochemistry, School of Medicine, The Flinders University of South Australia, G.P.O. Box 2100, Adelaide, South Australia 5001, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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.


EXPERIMENTAL PROCEDURES

Materials

[U-^14C]Glucose and high pressure liquid chromatography-purified [2-^3H]- and [6-^3H]glucose were obtained from DuPont NEN. DL-2-Bromopalmitate, which was bound to defatted albumin (11) , was purchased from Fluka (Switzerland). Streptozotocin, bovine serum albumin, and alpha-cyano-4-hydroxycinnamate (CHC) were purchased from Sigma. All enzymes and cofactors required for the enzymatic determination of metabolites were from Boehringer Mannheim (Germany). Other chemicals were of the highest grade commercially available.

Methods

Diabetes was induced by a single intraperitoneal injection of streptozotocin (100 mg/kg) dissolved in 20 mM citrate buffer, pH 4.2, 48 h prior to cell preparation. Rats were judged to be diabetic if, at the time of hepatocyte isolation, blood glucose concentrations exceeded 20 mM. Both normal and diabetic rats were fasted for 24 h before hepatocyte preparation was commenced. Hepatocytes were isolated as described in (12) by a modification of the method of Berry and Friend(13) . Hepatocytes isolated from normal rats are designated normal hepatocytes or normal liver cells whereas those from diabetic rats are referred to as diabetic liver cells or diabetic hepatocytes. Liver cells (approximately 100 mg wet wt) were incubated in a final volume of 2 ml with 80 mM glucose containing 1 µCi of [2-^3H]-, 1 µCi of [6-^3H]-, or 0.5 µCi of [U-^14C]glucose. This elevated concentration of glucose was used because the K(m) of glucokinase for glucose is raised in in vitro preparations (14) and also to mimic the conditions of severe diabetes. In experiments in which ^14CO(2) generation was measured, duplicate incubations were carried out in sealed vials; perchloric acid was injected through the seal at the end of the incubation period, and ^14CO(2) was collected in phenethylamine (0.5 ml) for subsequent counting. Measurements of glucose, lactate, pyruvate, acetoacetate, and 3-hydroxybutyrate were carried out by standard enzymatic techniques (15) on a Cobas FARA analyzer (Roche, Switzerland). Because CHC absorbs strongly at 340 nm the inhibitor was removed with activated charcoal (4% w/v) before sample analysis. This procedure yielded quantitative recoveries of the analytes reported.

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 ^3H(2)O released from [2-^3H]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-^3H]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 [^3H]glucose metabolism (measured as the sum of the rate of glycolysis and the rate of incorporation of tritium, derived from [6-^3H]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-^3H]glucose were invariably higher than the accumulation of glycolytic products (lactate, pyruvate, amino acids, and CO(2)), which was estimated using [U-^14C]glucose. This discrepancy is caused by recycling of glycolytic products back to glucose (G/P cycle) (19, 20, 21, 22) . Tritium is recovered as ^3H(2)O in incubations of hepatocytes with [6-^3H]glucose when pyruvate, derived from [6-^3H]glucose, is oxidized or carboxylated in the mitochondria. However, label will be lost from [U-^14C]glucose as ^14CO(2) 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-^3H]glucose) and the accumulation of glycolytic products (measured with [U-^14C]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 equivalentsbulletminbullet(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.


RESULTS

Glucose Metabolism in Hepatocytes from Normal and Diabetic Rats

Normal liver cells, incubated with 80 mM glucose, accumulated lactate and pyruvate, which reached a maximum concentration at about 60 min (Fig. 1), after which time the concentration of lactate and pyruvate slowly declined. In contrast, when diabetic hepatocytes were incubated under similar conditions, there was virtually no net accumulation of lactate and pyruvate for up to 2 h. From these experiments it could be inferred that glycolysis was severely inhibited in diabetic liver cells. However, experiments in which hepatocytes were incubated with [U-^14C]glucose revealed that the inhibition of lactate and pyruvate accumulation in diabetic liver cells was not the result of a complete block of the glycolytic pathway, as ^14CO(2) production was ongoing, although the rate of CO(2) formation was about half that observed with normal cells (Table 1).


Figure 1: Lactate and pyruvate production in normal and diabetic hepatocytes. Hepatocytes from fasted normal (circle) 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-^3H]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-^3H]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-^14C]glucose metabolism was recovered in lactate and pyruvate, with ^14CO(2) production representing a further 25% of labeled glucose metabolism. In contrast, ^14CO(2) was the main product of [U-^14C]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 ^3H-glycolytic flux could not be accounted for as ^14C-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 µmolbulletminbullet(g, wet weight), whereas normal cells turned over ATP for this process at a rate of 1.22 µmolbulletminbullet(g, wet weight).

Effect of Fatty Acid Oxidation on Glycolysis

When hepatocytes from normal fasted rats were incubated in the presence of glucose and palmitate, there was initially a suppression of lactate and pyruvate accumulation(9) . This effect was not the result of a complete inhibition of glycolysis as ^3H(2)O formation from [6-^3H]glucose and CO(2) production from [U-^14C]glucose continued. Rather, the inhibition of net lactate and pyruvate production was associated with an increase in both G/G6P and G/P cycling(9) . Once the added fatty acid was depleted, lactate and pyruvate accumulation commenced, and the rates of turnover of both glucose cycles declined. Hepatocytes from diabetic rats synthesized ketone bodies at a rate of 0.78 ± 0.14 µmolbulletminbullet(g, wet weight), n = 15, a rate that was 10-fold greater than that observed in incubations with control cells (0.07 ± 0.005 µmolbulletminbullet(g, wet weight), n = 15). This difference can be attributed to the expanded stores of triacylglycerol in livers of diabetic rats(10) . Therefore, we investigated the possibility that the inhibition of lactate and pyruvate accumulation in hepatocytes from diabetic rats was related to the increased rates of endogenous fatty acid oxidation observed in these cells.

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 µmolbulletminbullet(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 µmolbulletminbullet(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 [^14C]lactate and [^14C]pyruvate was 10-fold higher in the presence of bromopalmitate (Table 1), but the inhibitor induced only a small increase in net [^14C]lactate and [^14C]pyruvate production in normal liver cells. A similar increase in the rate of [^14C]lactate and [^14C]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.

Effect of CHC on Lactate Accumulation

The possible role of the pyruvate translocator (25, 26) in the interaction between fatty acid oxidation, G/P cycling, and lactate and pyruvate accumulation was investigated in experiments in which the translocator was inhibited with CHC(25) . CHC had no effect on the accumulation of glycolytic products in normal cells, but in diabetic cells CHC induced a 3-fold increase in glycolytic product formation, reflecting higher rates of accumulation of [^14C]lactate and [^14C]pyruvate (Table 1). Thus, it was apparent that the suppression of net lactate and pyruvate production by endogenous fatty acid oxidation in diabetic liver cells could be partially overcome by inhibiting the pyruvate translocator with CHC. The decreased production of ^14CO(2) (Table 1) was consistent with the inhibition of pyruvate uptake into the mitochondria when CHC was present. As a consequence of the inhibition of the translocator with CHC, G/P cycling was halved in diabetic cells, in contrast to normal hepatocytes in which the activity of the cycle was unaffected (Table 2). In addition to its effects on glycolysis, CHC induced a small inhibition of glucose phosphorylation (Table 1) that appears to be the result of a direct inhibition of glucokinase by CHC. (^2)


DISCUSSION

G/G6P Cycling in Diabetes

An increase in the rate of cycling between glucose and G6P in the liver may contribute to the development of hyperglycemia in diabetes(3, 4, 5, 6, 7) . We have shown that G/G6P cycling is stimulated in hepatocytes from streptozotocin-treated rats. A repression of glucokinase activity and a consequent decrease in glucose phosphorylation, as well as a stimulation of glucose-6-phosphatase, have been shown to parallel the stimulation of hepatic G/G6P cycling in partially pancreatectomized rats(4) . The decrease in glucose uptake observed in diabetic hepatocytes was considered to be a result of a repression of glucokinase synthesis in response to the fall in the insulin:glucagon ratio(4) . There is no evidence that the transport of glucose into the hepatocytes is compromised in diabetes(27) .

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.

G/P Cycling in Diabetes

Diabetic hepatocytes recycled a greater proportion of glycolytically derived pyruvate to glucose. This accounts for the inhibition of lactate and pyruvate accumulation as almost all the glycolytically derived pyruvate was metabolized intramitochondrially, in contrast to its fate in normal hepatocytes where a substantial portion of pyruvate remained in the cytoplasm to be reduced to lactate. One effect of diabetes, therefore, is a channeling of pyruvate into the mitochondria. Endogenous fatty acid oxidation appears to be associated with this channeling because the absence of lactate and pyruvate accumulation could be overcome with bromopalmitate. This effect of fatty acid could be mimicked in cells from normal rats by the addition of exogenous fatty acid, leading to a similar inhibition of lactate accumulation and stimulation of pyruvate recycling to glucose(9) .

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(2). 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) .

Physiological Significance of Glucose Cycling in Diabetes

Other authors (4, 5, 6, 7) have suggested that there may be an increase in G/G6P cycling in diabetes, but the potential magnitude of this cycling has not been recognized previously. In hepatocytes from diabetic rats, the accumulation of glycolytic products during the 60-min incubation period represented only 15% of the total glucose phosphorylated, since almost all of the glucose taken up into the cells was eventually re-released. In contrast, hepatocytes from normal rats accumulated about 50% of glucose phosphorylated as glycolytic products and glycogen. The rate of flux through the combined G/G6P and G/P cycles is similar to the maximum rate of gluconeogenesis from lactate in diabetic hepatocytes(31) . Therefore, in vivo, glucose cycling is likely to contribute significantly to the development of diabetic hyperglycemia. The pivotal role of endogenous fatty acid oxidation in the stimulation of both G/G6P and G/P cycling has also not been recognized previously. Although it has been reported that antilipolytic agents improve glucose tolerance and stimulate glucose oxidation, a mechanism has not been conclusively shown(32) . One means by which these agents will effect a decrease in plasma glucose concentrations is via a depression of the rate of hepatic glucose cycling.

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(2) consumption in diabetic hepatocytes(31) .


FOOTNOTES

*
This work was supported by grants from the National Health and Medical Research Council of Australia and from the Flinders Medical Centre Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 61-8-2045191; Fax: 61-8-3740139.

(^1)
The abbreviations used are: G/G6P, glucose/glucose 6-phosphate; CHC, alpha-cyano-4-hydroxycinnamate; G6P, glucose 6-phosphate; G/P, glucose/pyruvate.

(^2)
J. W. Phillips, unpublished observation.


ACKNOWLEDGEMENTS

We are grateful for the skilled technical assistance of S. Phillips, E. Williams, R. Norris, D. Doherty, and S. Chiveralls and for the secretarial skills of J. Burton and J. McCulloch. We also thank Drs. D. Clark and A. Grivell for their advice.


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