Activation by Vanadate of Glycolysis in Hepatocytes From Diabetic Rats JOAN E. RODRIGUEZ-GIL, ANNA AND JOAN J. GUINOVART
GOMEZ-FOIX, CRISTINA FILLAT, FATIMA BOSCH,
In hepatocytes from starved streptozocin-induced diabetic rats, vanadate increases the glycolytic flux because it raises the levels of fructose-2,6bisphosphate (Fru-2,6-P2), the main regulatory metabolite of this pathway. This effect of vanadate on Fru-2,6-P2 levels is time and dose dependent, and it remains in cells incubated in a calcium-depleted medium. Vanadate is also able to counteract the decrease on Fru-2,6-P2 levels produced by glucagon, colforsin, or exogenous cAMP. However, vanadate does not modify 6-phosphofructo-2-kinase and pyruvate kinase activities, but it does counteract the inactivation of these enzymes induced by glucagon. Likewise, Fru-2,6-P2ase activity is also not affected by vanadate. In addition, vanadate is able to increase the production of both lactate and CO2 in hepatocytes from streptozocin-induced diabetic rats incubated in the presence of glucose in the medium. Vanadate behaves as a glycolytic effector in these cells, and this effect may be related to its ability to normalize blood glucose levels in diabetic animals. Diabetes 40:1355-59,1991
I
n the last few years, an increased interest has developed regarding the effects of vanadate on carbohydrate metabolism (1-3). This compound has been reported to exert insulinlike actions and has been considered an important probe in studies on the mechanism of insulin action (4). Vanadate, when administered orally to diabetic rats, normalizes blood glucose concentrations and hepatic fructose2,6-bisphosphate (Fru-2,6-P2) levels, prevents the decline in
From the Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Autonomous University of Barcelona; and the Department of Biochemistry and Physiology, School of Chemistry, University of Barcelona, Barcelona, Spain. Address correspondence and reprint requests to Joan E. Rodriguez-Gil, Department de Bioquimica i Biologia Molecular, Facultat de Veterinaria, Universitat Autdnoma de Barcelona, 08193 Bellaterra (Barcelona), Spain. Received for publication 9 November 1990 and accepted in revised form 3 May 1991.
DIABETES, VOL. 40, OCTOBER 1991
cardiac performance due to diabetes, and also mimics insulin actions by normalizing tissue responsiveness (5-13). In isolated hepatocytes from healthy (14) or diabetic (15) rats, we have shown that vanadate acts contrary to insulin regarding vanadate's action on the regulatory enzymes of glycogen metabolism. Vanadate produces the inactivation and phosphorylation of glycogen synthase and the activation of glycogen phosphorylase, effects opposite to those of insulin and similar to the effects of the glycogenolytic hormones. However, we have also reported that vanadate increases Fru-2,6-P2 levels in isolated hepatocytes from nondiabetic rats and counteracts the effects of glucagon on Fru2,6-P2 concentration (16), cAMP levels, and cAMP-dependent protein kinase activity (17). Therefore, vanadate exerts insulinlike effects on the glycolytic pathway in hepatocytes from nondiabetic rats. Because these effects of vanadate on glycolytic/gluconeogenic flux and glycogen metabolism were difficult to reconcile with vanadate's effects in vivo in diabetic rats, it was necessary to study the effects of vanadate in hepatocytes isolated from diabetic rats. Our results show that vanadate in these cells activates glycolysis, indicating that this effect may contribute to the capacity of vanadate to normalize glycemia when administered orally to diabetic rats.
RESEARCH DESIGN AND METHODS Male Sprague-Dawley rats (190-220 g body wt, Autonomous Univ. of Barcelona Colony) were used. Diabetes was induced by a single injection of 60 mg/kg body wt i.v. streptozocin (STZ) in 10 mM sodium citrate and 0.9% NaCI, pH 4.5. Animals were used 5-10 days after STZ injection. Diabetic rats had mean ± SE blood glucose levels of 17.8 ± 0.7 mM after STZ injection. Body weight decreased from 203 ± 6 to 175 ± 3 g after treatment. Cells were isolated and prepared from 24-h-starved rats as described in ref. 18. Cells were finally resuspended in Krebs bicarbonate buffer (pH 7.4) pregassed with O2/CO2 (19:1 vol/vol). Where stated, Ca2+ was omitted from this medium, and 1 mM ethyleneglycol-bis-(p-aminoethyl ether)
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A/,A/,A/',A/'-tetraacetic acid (EGTA) was added. Portions ( 4 5 x 106 cells/ml) were incubated in stoppered vials at 37°C with continuous shaking. Where stated, cells were added with 16 mM lactate/4 mM pyruvate and preincubated 30 min before the addition of effectors or with 20 mM glucose added 20 min before treatment. The yield of hepatocytes from an individual liver was 250-350 x 106 cells. One gram of cells (wet wt) corresponds to ~7 x 107 cells. The viability of the cells was determined by the trypan blue exclusion (19). Cell preparations with a viability 5 mM were not used because they decrease intracellular ATP levels (14). These effects appeared to be independent on exogenous substrates, because we also observed an increase on Fru2,6-P2 concentrations by vanadate either in cells preincubated for 20 min in a Krebs-Ringer medium free of glucose or any other substrate or in cells preincubated in a medium containing 20 mM glucose (data not shown). Depletion of calcium did not affect vanadate action on Fru2,6-P2 levels. When cells incubated in a Krebs-Ringer medium lacking calcium chloride and in the presence of 1 mM EGTA were treated with 2 mM vanadate, an increase in Fru2,6-P2 levels was observed (Fig. 1 A). On a percentage basis, the increase over the basal values after 5 min of incubation was similar to that observed in the presence of calcium. The concentration of vanadate producing half-maximal effect was unchanged in these conditions (Fig. 18). Because the effect of vanadate on Fru-2,6-P2 concentrations was opposite to that reported for glucagon, we next studied whether vanadate could counteract the effects of the hormone. Treatment of cells from diabetic animals with 10 nM glucagon provoked a significant decrease in Fru-2,6P2 levels (Fig. 2A). However, when cells were incubated with both 10 nM glucagon and 2 mM vanadate, the effect of the hormone was abolished. This counteractive effect of vanadate could be produced either with or before adenylate cy-
1 2
5
[VANADATE] (MM)
FIG. 1 . Effects of vanadate on fructose-2,6bisphosphate concentrations. Hepatocytes were resuspended in Krebs-Ringer medium ( • ) or in calcium-depleted Krebs-Ringer medium with 1 m M EGTA added ( D ) . T h e n , cells were preincubated 30 min in presence of 16 m M lactate/4 m M pyruvate before treatment. A: cells exposed to 2 m M vanadate for the indicated times. Values are m e a n s ± SE for 8 independent experiments. B: cells treated for 5 min with different concentrations of vanadate. Values are means ± SE for 5 independent experiments. *P < 0.05, **P =s 0 . 0 1 , vs. control by paired Student's t test.
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FIG. 3. Effects of vanadate on 6-phosphofructo-2-kinase activity. Hepatocytes were preincubated for 30 min in Krebs-Ringer medium with 16 mM lactate/4 mM pyruvate added. Then, cells were incubated with 2 M vanadate (A), 10 nM giucagon ( • ) , or 2 mM vanadate plus 10 nM giucagon ( • ) . O, Baseline. Values are means ± SE for 5 experiments.
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3). This agrees with the fact that, in hepatocytes from healthy rats, vanadate alone does not modify cAMP intracellular levels but blocks the increase in the cyclic nucleotide induced by giucagon (17). Vanadate also did not modify Fru-2,6P2ase activity (2.0 ± 0.2 nmol • min" 1 • g~1 after 12 min of incubation with 2 mM vanadate vs. 2.2 ± 0.2 nmol • min" 1 • g" 1 in control cells). Because pyruvate kinase is another key enzyme in the regulation of glycolysis, we next studied the ability of vanadate to affect this enzyme. Treatment of hepatocytes with 2 mM vanadate for different times did not change basal pyruvate kinase activity. When cells were incubated with 10
FIG. 2. Effects of vanadate on decreases in fructose-2,6-bisphosphate induced by giucagon, colforsin, and exogenous cAMP. Hepatocytes were preincubated for 30 min in Krebs-Ringer medium with 16 mM lactate/4 mM pyruvate added. Then, cells were treated for indicated times with 10 nM giucagon (D) or 10 nM giucagon plus 2 mM vanadate ( • ; A), 0.1 mM colforsin (A) or 0.1 mM colforsin plus 2 mM vanadate (A; B), or 0.1 mM exogenous cAMP (O) or 0.1 mM exogenous cAMP plus 2 mM vanadate ( • ; C). Values are means ± SE for 5 experiments.
clase or with cAMP phosphodiesterase. To gain more insight into this mechanism, we studied the effects of vanadate when added together with 0.1 mM colforsin, which activates adenylate cyclase by a non-receptor-mediated mechanism. As observed in Fig. 28, vanadate also counteracted the decrease on Fru-2,6-P2 levels induced by colforsin, indicating that vanadate action should implicate a postreceptor mechanism. In addition, when cells were incubated with 2 mM vanadate plus 0.1 mM exogenous cAMP, a complete reversion of cAMP action on Fru-2,6-P2 was observed (Fig. 2C). Therefore, vanadate action seems to be exerted at a postadenylate cyclase step. Vanadate-stimulated increase of Fru-2,6-P2 could be the result of an activation of the enzyme responsible for the synthesis of the metabolite. However, vanadate did not modify PFK-2 activity, although it was able to counteract the inactivation of the enzyme caused by 10 nM giucagon (Fig.
DIABETES, VOL. 40, OCTOBER 1991
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O
2
*
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*
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FIG. 4. Counteractive effects of vanadate on decreases in pyruvate kinase activity induced by giucagon. Prot., protein. Hepatocytes were preincubated for 20 min in Krebs-Ringer medium containing 10 mM glucose. Then, cells were incubated at times indicated with 2 mM vanadate (A), 10 nM giucagon ( • ) , or 2 mM vanadate and 10 nM giucagon together ( • ) . O, Baseline. Values are means ± SE for 4 different experiments.
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TABLE 1 Effects of vanadate on glucose-6-phosphate (G6P) levels and '4CO2 production G6P (nmol/g)
Control Vanadate
16 mM lactate/ 4 mM pyruvate
20 mM glucose
CO2 production (ixmol of glucose oxidized • 60 min- 1 • g' 1 )
12 ± 2 4 ± 2*
28 ± 3 18 ± 2*
0.08 ± 0.02 0.31 ± 0.01*
Values are means ± SE for 4 experiments. Hepatocytes were preincubated for 30 min with 16 mM lactate/4 mM pyruvate or 20 min with 20 mM glucose, then treated for 10 min with 2 mM vanadate to determine G6P levels. To measure 14CO2 production, cells were incubated for 60 min with 20 mM [U-'4C]glucose with or without 2 mM vanadate. *P < 0.01 vs. control by paired Student's t test.
nM glucagon, a marked decrease in pyruvate kinase activity was observed. This effect was significantly counteracted when the cells were treated with 2 mM vanadate together with 10 nM glucagon (Fig. 4). Table 1 shows that vanadate was able to decrease G6P intracellular levels either in cells preincubated with 20 mM glucose or with 16 mM lactate/4 mM pyruvate. As described above, under these conditions, vanadate increased Fru-2,6P2 levels. Therefore, this effect cannot be produced by a "push" resulting from an increase in G6P concentrations. To investigate the fate of glucose, we next studied the effects of vanadate on L-lactate and CO2 production when hepatocytes from starved rats were incubated in the presence of 20 mM glucose. Fig. 5 shows that vanadate clearly stimulated lactate production. Concomitantly, an increase (~4-fold) in 14CO2 production from 20 mM [U-14C]glucose was observed (Table 1).
termined in nondiabetic hepatocytes (2.2 ± 0.1 vs. 5.2 ± 0.3 nmol/g) (16). Incubation with vanadate provoked an approximately twofold increase in Fru-2,6-P2 concentration, reaching levels slightly below those from nondiabetic cells (16). The effect of vanadate on Fru-2,6-P2 concentration is not due to a "push" effect related to an increase in G6P levels, which would result in a greater substrate availability to PFK2. In fact, G6P levels are decreased after incubation with vanadate. Although vanadate inhibition of both PFK-2 (26) and Fru-2,6-P2ase (27) activities in vitro has been described, our results show that these activities are not altered in a permanent way in hepatocytes after incubation with vanadate. Because PFK-2/Fru-2,6-P2ase in the liver is regulated by phospho-/dephosphorylation mechanisms (25), our results seem to indicate that vanadate does not modify Fru2,6-P2 levels through these mechanisms. However, vanadate is able to counteract glucagon effects on PFK-2 and pyruvate kinase activities. These effects are probably due to the counteraction by vanadate of the rise in cAMP levels and, as a consequence, the activation of cAMP-dependent protein kinase induced by glucagon (2,17). The results obtained when cells from nondiabetic (16) or diabetic rats were treated with colforsin or exogenous cAMP in the presence of vanadate suggest that this compound acts, at this level, on a postadenylate cyclase step. The results obtained with L-lactate and CO2 production from glucose indicate that the rise in Fru-2,6-P2 levels induced by vanadate is related to an increase in glucose utilization in hepatocytes from diabetic rats. These insulinlike effects of vanadate on glycolytic flux are produced simul-
DISCUSSION
The results presented in this article show that, in hepatocytes isolated from diabetic rats, vanadate is able to activate glycolysis. Glycolytic flux is very diminished in hepatocytes from diabetic rats compared with cells from healthy animals (24). Although in hepatocytes obtained from diabetic animals, incubation with vanadate stimulated almost 1.5-fold lactate production, and ~4-fold glucose oxidation, the resulting values were still below those previously determined in control cells from healthy rats under the same conditions (16). In these cells when they were incubated with 15 mM glucose, L-lactate production was 38 ± 2 ixmol • g~1 • 90 min""1 (16). On the contrary, in hepatocytes from diabetic animals, L-lactate production from 20 mM glucose was only 12 ± 1 fimol • g~1 • 90 min- 1 after incubation with 2 mM vanadate. Likewise, 14CO2 production in normal control cells was 0.46 ± 0.05 |ximol/g after 60 min (16), whereas in cells from diabetic rats, a value of 0.31 ± 0.01 |xmol/g after 60 min of incubation with vanadate was obtained. The stimulation of glycolysis by vanadate is probably the result of an increase in the intracellular levels of Fru-2,6-P2, the most important regulatory metabolite of the Fru-6-P/Fru1,6-P2 cycle (25). The basal levels of Fru-2,6-P2 in hepatocytes from diabetic rats incubated in the presence of 16 mM lactate/4 mM pyruvate are much lower than those de-
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FIG. 5. Effects of vanadate on L-lactate production. Hepatocytes were incubated in presence of 20 mM glucose in absence (O) or presence ( • ) of 2 mM vanadate. Values are means ± SE for 5 experiments.
DIABETES, VOL. 40, OCTOBER 1991
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taneously with some anti-insulin, glucagonlike effects of vanadate on glycogen metabolism. In this regard, it has also been described that vanadate, both in hepatocytes from nondiabetic (14) or diabetic rats (15), inactivates and phosphorylates glycogen synthase by a calcium-independent mechanism, whereas vanadate activates glycogen phosphorylase in a calcium-dependent manner (14,15). All of these results indicate that vanadate acts on hepatocyte glucose metabolism by at least four different mechanisms: 1) A calcium- and cAMP-independent mechanism responsible for the rise in Fru-2,6-P2 levels. 2) A second mechanism acting on a post-adenylate cyclase step that is able to counteract glucagon and other cAMP-mediated effects. 3) Another calcium-independent mechanism that provokes antiinsulin effects on glycogen synthase activity. This action is probably related to an activation of a calcium- and cAMPindependent protein kinase activity (17). 4) Finally, a calcium-dependent mechanism that is responsible for the effects of vanadate on glycogen phosphorylase activity. In conclusion, our results prove that vanadate acts in a direct manner on glucose metabolism in rat hepatocytes. This action provokes a simultaneous stimulation of the glycogenolytic and glycolytic pathways, and as a result, vanadate activates glucose utilization in these cells either from nondiabetic or diabetic rats. Vanadate's effects on glycolytic flux in isolated hepatocytes are in agreement with those described after treatment in vivo with vanadate (5-7,9-11). Therefore, our results support the concept that the ability of vanadate to stimulate liver glycolysis contributes to the mechanism by which vanadate normalizes glycemia when administered orally to diabetic rats.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
ACKNOWLEDGMENTS
This study was supported by Grant 89/490 from the Fondo de Investigaciones Sanitarias de la Seguridad Social, Spain. C.F. and J.E.R.-G. were recipients of Fellowships from the Programa de Formacion de Personal Investigador (Ministry of Education, Spain). We thank Anna Vilalta and Catalina Relaiio for skilled technical assistance.
20.
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23.
REFERENCES 1. Shechter Y, Amir S, Meyerovitch J: The insulin-like effects of vanadate: possible implications to the future care of diabetes. Diabetes Nutr Metab 1:1-5, 1988 2. Bosch F, G6mez-Foix AM, Ariiio J, Guinovart JJ: Vanadate effects on phosphorylation-dephosphorylation systems involved in glycogen metabolism. Adv Prot Phosphatases 4:351-62, 1987 3. Gresser MJ, Tracey AS, Stankiewicz PJ: The interaction of vanadate with tyrosine kinases and phosphatases. Adv Prot Phosphatases 4:35-57, 1987 4. Shechter Y: Insulin-mimetic effects of vanadate: possible implications for future treatment of diabetes. Diabetes 39:1-5, 1990 5. Heyliger CE, Tahiliani AG, McNeill JH: Effect of vanadate on elevated
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24.
25. 26.
27.
blood glucose and depressed cardiac performance of diabetic rats. Science 227:1474-77, 1985 Meyerovitch J, Farfel Z, Sack J, Shechter Y: Oral administration of vanadate normalizes blood glucose levels in streptozotocin-treated rats. J 8/0/ Chem 262:6658-62, 1987 Gil J, Miralpeix M, Carreras J, Bartrons R: Insulinlike effects of vanadate on glucokinase activity and fructose 2,6-bisphosphate levels in the liver of diabetic rats. J Biol Chem 263:1868-71, 1988 Blondel 0, Bailbe D, Portha B: "In vivo" insulin resistance in streptozotocin-diabetic rats: evidence for reversal following oral vanadate treatment. Diabetologia 32:185-90, 1989 Challis RAJ, Leighton B, Lozeman FJ, Budohoski L, Newsholme EA: Effects of chronic administration of vanadate to the rat on the sensitivity of glycolysis and glycogen synthesis in skeletal muscle to insulin. Biochem Pharmacol 36:357'-61, 1987 Bendayan M, Gingras D: Effects of vanadate administration on blood glucose and insulin levels as well as on the exocrine pancreatic function in streptozotocin-treated rats. Diabetologia 32:561-67, 1989 Bollen M. Miralpeix M, Ventura F, Toth B, Bartrons R, Stalmans W: Oral administration of vanadate to streptozotocin-diabetic rats restores the glucose-induced activation of glycogen synthase. Biochem J 267:26971, 1990 Brichard SM, Pottier AM, Henquin JC: Long term improvement of glucose homeostasis by vanadate in obese hyperinsulinemic fa/fa rats. Endocrinology 125:2510-16, 1989 Brichard SM, Okitolonda W, Henquin JC: Long term improvement of glucose homeostasis by vanadate treatment in diabetic rats. Endocrinology 123:2048-52. 1988 Bosch F, Ariiio J, Gomez-Foix AM, Guinovart JJ: Glycogenolytic noninsulin-like effects of vanadate on rat hepatocyte glycogen synthase and phosphorylase. J Biol Chem 262:218-22, 1987 Rodriguez-Gil JE, Gomez-Foix AM, Ariiio J, Guinovart JJ, Bosch F: Control of glycogen synthase and phosphorylase in hepatocytes from diabetic rats: effects of glucagon, vasopressin, and vanadate. Diabetes 38:79398, 1989 Gomez-Foix AM, Rodriguez-Gil JE, Fillat C, Guinovart JJ, Bosch F: Vanadate raises fructose 2,6-bisphosphate concentrations and activates glycolysis in rat hepatocytes. Biochem J 255:507-12, 1988 Villar-Palasi C, Guinovart JJ, G6mez-Foix AM, Rodriguez-Gil JE, Bosch F: Effects of vanadate on protein kinases in rat hepatocytes. Biochem J 262:563-67, 1989 Massague J, Guinovart JJ: insulin control of rat hepatocyte glycogen synthase and phosphorylase in the absence of glucose. FEBS Lett 82:317-20, 1977 Howard RB, Pesch LA: Respiratory activity of intact, isolated parenchymal cells from rat liver. J Biol Chem 243:3105-109, 1968 Van Schaftingen E, Lederer B, Bartrons R, Hers HG: A kinetic study of pyrophosphate: fructose 6-phosphate phosphotransferase from potato tubers: application to a microassay of fructose 2,6-bisphosphate. Eur J Biochem 129:191-95, 1982 Bartrons R, Hue L, Van Schaftingen E, Hers HG: Hormonal control of fructose 2,6-bisphosphate concentration in isolated rat hepatocytes. Biochem J 214:829-37, 1983 Kuwajima M, Golden S, Katz J, Unger RH, Foster DW, McGarry JD: Active hepatic glycogen synthesis from gluconeogenic precursors despite high tissue levels of fructose 2,6-bisphosphate. J Biol Chem 261:2632-37, 1986 Feliu JE, Hue L, Hers HG: Regulation in vitro and in vivo of adenosine 3' :5'-monophosphate-dependent inactivation of rat liver pyruvate kinase type L. Eur J Biochem 81:609-17, 1977 Gil J, Carreras J, Bartrons R: Effects of diabetes on fructose 2,6-bisphosphate, glucose 1,6-bisphosphate and 6-phosphofructo-2-kinase in rat liver. Biochem Biophys Res Commun 136:498-503, 1986 Hue L, Rider MH: Role of fructose 2,6-bisphosphate in the control of glycolysis in mammalian tissues. Biochem J 245:313-24, 1987 Kountz PD, McCain RW, El-Maghrabi MR, Pilkis SJ: Hepatic 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase: phosphate dependence and effects of other oxyanions. Arch Biochem Biophys 251:104— 13, 1986 Rider MH, Bartrons R, Hue L: Vanadate inhibits liver fructose 2,6-bisphosphatase. Eur J Biochem 190:53-56, 1990
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GOMEZ-FOIX, CRISTINA FILLAT, FATIMA BOSCH,
In hepatocytes from starved streptozocin-induced diabetic rats, vanadate increases the glycolytic flux because it raises the levels of fructose-2,6bisphosphate (Fru-2,6-P2), the main regulatory metabolite of this pathway. This effect of vanadate on Fru-2,6-P2 levels is time and dose dependent, and it remains in cells incubated in a calcium-depleted medium. Vanadate is also able to counteract the decrease on Fru-2,6-P2 levels produced by glucagon, colforsin, or exogenous cAMP. However, vanadate does not modify 6-phosphofructo-2-kinase and pyruvate kinase activities, but it does counteract the inactivation of these enzymes induced by glucagon. Likewise, Fru-2,6-P2ase activity is also not affected by vanadate. In addition, vanadate is able to increase the production of both lactate and CO2 in hepatocytes from streptozocin-induced diabetic rats incubated in the presence of glucose in the medium. Vanadate behaves as a glycolytic effector in these cells, and this effect may be related to its ability to normalize blood glucose levels in diabetic animals. Diabetes 40:1355-59,1991
I
n the last few years, an increased interest has developed regarding the effects of vanadate on carbohydrate metabolism (1-3). This compound has been reported to exert insulinlike actions and has been considered an important probe in studies on the mechanism of insulin action (4). Vanadate, when administered orally to diabetic rats, normalizes blood glucose concentrations and hepatic fructose2,6-bisphosphate (Fru-2,6-P2) levels, prevents the decline in
From the Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Autonomous University of Barcelona; and the Department of Biochemistry and Physiology, School of Chemistry, University of Barcelona, Barcelona, Spain. Address correspondence and reprint requests to Joan E. Rodriguez-Gil, Department de Bioquimica i Biologia Molecular, Facultat de Veterinaria, Universitat Autdnoma de Barcelona, 08193 Bellaterra (Barcelona), Spain. Received for publication 9 November 1990 and accepted in revised form 3 May 1991.
DIABETES, VOL. 40, OCTOBER 1991
cardiac performance due to diabetes, and also mimics insulin actions by normalizing tissue responsiveness (5-13). In isolated hepatocytes from healthy (14) or diabetic (15) rats, we have shown that vanadate acts contrary to insulin regarding vanadate's action on the regulatory enzymes of glycogen metabolism. Vanadate produces the inactivation and phosphorylation of glycogen synthase and the activation of glycogen phosphorylase, effects opposite to those of insulin and similar to the effects of the glycogenolytic hormones. However, we have also reported that vanadate increases Fru-2,6-P2 levels in isolated hepatocytes from nondiabetic rats and counteracts the effects of glucagon on Fru2,6-P2 concentration (16), cAMP levels, and cAMP-dependent protein kinase activity (17). Therefore, vanadate exerts insulinlike effects on the glycolytic pathway in hepatocytes from nondiabetic rats. Because these effects of vanadate on glycolytic/gluconeogenic flux and glycogen metabolism were difficult to reconcile with vanadate's effects in vivo in diabetic rats, it was necessary to study the effects of vanadate in hepatocytes isolated from diabetic rats. Our results show that vanadate in these cells activates glycolysis, indicating that this effect may contribute to the capacity of vanadate to normalize glycemia when administered orally to diabetic rats.
RESEARCH DESIGN AND METHODS Male Sprague-Dawley rats (190-220 g body wt, Autonomous Univ. of Barcelona Colony) were used. Diabetes was induced by a single injection of 60 mg/kg body wt i.v. streptozocin (STZ) in 10 mM sodium citrate and 0.9% NaCI, pH 4.5. Animals were used 5-10 days after STZ injection. Diabetic rats had mean ± SE blood glucose levels of 17.8 ± 0.7 mM after STZ injection. Body weight decreased from 203 ± 6 to 175 ± 3 g after treatment. Cells were isolated and prepared from 24-h-starved rats as described in ref. 18. Cells were finally resuspended in Krebs bicarbonate buffer (pH 7.4) pregassed with O2/CO2 (19:1 vol/vol). Where stated, Ca2+ was omitted from this medium, and 1 mM ethyleneglycol-bis-(p-aminoethyl ether)
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A/,A/,A/',A/'-tetraacetic acid (EGTA) was added. Portions ( 4 5 x 106 cells/ml) were incubated in stoppered vials at 37°C with continuous shaking. Where stated, cells were added with 16 mM lactate/4 mM pyruvate and preincubated 30 min before the addition of effectors or with 20 mM glucose added 20 min before treatment. The yield of hepatocytes from an individual liver was 250-350 x 106 cells. One gram of cells (wet wt) corresponds to ~7 x 107 cells. The viability of the cells was determined by the trypan blue exclusion (19). Cell preparations with a viability 5 mM were not used because they decrease intracellular ATP levels (14). These effects appeared to be independent on exogenous substrates, because we also observed an increase on Fru2,6-P2 concentrations by vanadate either in cells preincubated for 20 min in a Krebs-Ringer medium free of glucose or any other substrate or in cells preincubated in a medium containing 20 mM glucose (data not shown). Depletion of calcium did not affect vanadate action on Fru2,6-P2 levels. When cells incubated in a Krebs-Ringer medium lacking calcium chloride and in the presence of 1 mM EGTA were treated with 2 mM vanadate, an increase in Fru2,6-P2 levels was observed (Fig. 1 A). On a percentage basis, the increase over the basal values after 5 min of incubation was similar to that observed in the presence of calcium. The concentration of vanadate producing half-maximal effect was unchanged in these conditions (Fig. 18). Because the effect of vanadate on Fru-2,6-P2 concentrations was opposite to that reported for glucagon, we next studied whether vanadate could counteract the effects of the hormone. Treatment of cells from diabetic animals with 10 nM glucagon provoked a significant decrease in Fru-2,6P2 levels (Fig. 2A). However, when cells were incubated with both 10 nM glucagon and 2 mM vanadate, the effect of the hormone was abolished. This counteractive effect of vanadate could be produced either with or before adenylate cy-
1 2
5
[VANADATE] (MM)
FIG. 1 . Effects of vanadate on fructose-2,6bisphosphate concentrations. Hepatocytes were resuspended in Krebs-Ringer medium ( • ) or in calcium-depleted Krebs-Ringer medium with 1 m M EGTA added ( D ) . T h e n , cells were preincubated 30 min in presence of 16 m M lactate/4 m M pyruvate before treatment. A: cells exposed to 2 m M vanadate for the indicated times. Values are m e a n s ± SE for 8 independent experiments. B: cells treated for 5 min with different concentrations of vanadate. Values are means ± SE for 5 independent experiments. *P < 0.05, **P =s 0 . 0 1 , vs. control by paired Student's t test.
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FIG. 3. Effects of vanadate on 6-phosphofructo-2-kinase activity. Hepatocytes were preincubated for 30 min in Krebs-Ringer medium with 16 mM lactate/4 mM pyruvate added. Then, cells were incubated with 2 M vanadate (A), 10 nM giucagon ( • ) , or 2 mM vanadate plus 10 nM giucagon ( • ) . O, Baseline. Values are means ± SE for 5 experiments.
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3). This agrees with the fact that, in hepatocytes from healthy rats, vanadate alone does not modify cAMP intracellular levels but blocks the increase in the cyclic nucleotide induced by giucagon (17). Vanadate also did not modify Fru-2,6P2ase activity (2.0 ± 0.2 nmol • min" 1 • g~1 after 12 min of incubation with 2 mM vanadate vs. 2.2 ± 0.2 nmol • min" 1 • g" 1 in control cells). Because pyruvate kinase is another key enzyme in the regulation of glycolysis, we next studied the ability of vanadate to affect this enzyme. Treatment of hepatocytes with 2 mM vanadate for different times did not change basal pyruvate kinase activity. When cells were incubated with 10
FIG. 2. Effects of vanadate on decreases in fructose-2,6-bisphosphate induced by giucagon, colforsin, and exogenous cAMP. Hepatocytes were preincubated for 30 min in Krebs-Ringer medium with 16 mM lactate/4 mM pyruvate added. Then, cells were treated for indicated times with 10 nM giucagon (D) or 10 nM giucagon plus 2 mM vanadate ( • ; A), 0.1 mM colforsin (A) or 0.1 mM colforsin plus 2 mM vanadate (A; B), or 0.1 mM exogenous cAMP (O) or 0.1 mM exogenous cAMP plus 2 mM vanadate ( • ; C). Values are means ± SE for 5 experiments.
clase or with cAMP phosphodiesterase. To gain more insight into this mechanism, we studied the effects of vanadate when added together with 0.1 mM colforsin, which activates adenylate cyclase by a non-receptor-mediated mechanism. As observed in Fig. 28, vanadate also counteracted the decrease on Fru-2,6-P2 levels induced by colforsin, indicating that vanadate action should implicate a postreceptor mechanism. In addition, when cells were incubated with 2 mM vanadate plus 0.1 mM exogenous cAMP, a complete reversion of cAMP action on Fru-2,6-P2 was observed (Fig. 2C). Therefore, vanadate action seems to be exerted at a postadenylate cyclase step. Vanadate-stimulated increase of Fru-2,6-P2 could be the result of an activation of the enzyme responsible for the synthesis of the metabolite. However, vanadate did not modify PFK-2 activity, although it was able to counteract the inactivation of the enzyme caused by 10 nM giucagon (Fig.
DIABETES, VOL. 40, OCTOBER 1991
-
-
O
2
*
-
*
-
FIG. 4. Counteractive effects of vanadate on decreases in pyruvate kinase activity induced by giucagon. Prot., protein. Hepatocytes were preincubated for 20 min in Krebs-Ringer medium containing 10 mM glucose. Then, cells were incubated at times indicated with 2 mM vanadate (A), 10 nM giucagon ( • ) , or 2 mM vanadate and 10 nM giucagon together ( • ) . O, Baseline. Values are means ± SE for 4 different experiments.
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VANADATE ACTIVATION OF GLYCOLYSIS
TABLE 1 Effects of vanadate on glucose-6-phosphate (G6P) levels and '4CO2 production G6P (nmol/g)
Control Vanadate
16 mM lactate/ 4 mM pyruvate
20 mM glucose
CO2 production (ixmol of glucose oxidized • 60 min- 1 • g' 1 )
12 ± 2 4 ± 2*
28 ± 3 18 ± 2*
0.08 ± 0.02 0.31 ± 0.01*
Values are means ± SE for 4 experiments. Hepatocytes were preincubated for 30 min with 16 mM lactate/4 mM pyruvate or 20 min with 20 mM glucose, then treated for 10 min with 2 mM vanadate to determine G6P levels. To measure 14CO2 production, cells were incubated for 60 min with 20 mM [U-'4C]glucose with or without 2 mM vanadate. *P < 0.01 vs. control by paired Student's t test.
nM glucagon, a marked decrease in pyruvate kinase activity was observed. This effect was significantly counteracted when the cells were treated with 2 mM vanadate together with 10 nM glucagon (Fig. 4). Table 1 shows that vanadate was able to decrease G6P intracellular levels either in cells preincubated with 20 mM glucose or with 16 mM lactate/4 mM pyruvate. As described above, under these conditions, vanadate increased Fru-2,6P2 levels. Therefore, this effect cannot be produced by a "push" resulting from an increase in G6P concentrations. To investigate the fate of glucose, we next studied the effects of vanadate on L-lactate and CO2 production when hepatocytes from starved rats were incubated in the presence of 20 mM glucose. Fig. 5 shows that vanadate clearly stimulated lactate production. Concomitantly, an increase (~4-fold) in 14CO2 production from 20 mM [U-14C]glucose was observed (Table 1).
termined in nondiabetic hepatocytes (2.2 ± 0.1 vs. 5.2 ± 0.3 nmol/g) (16). Incubation with vanadate provoked an approximately twofold increase in Fru-2,6-P2 concentration, reaching levels slightly below those from nondiabetic cells (16). The effect of vanadate on Fru-2,6-P2 concentration is not due to a "push" effect related to an increase in G6P levels, which would result in a greater substrate availability to PFK2. In fact, G6P levels are decreased after incubation with vanadate. Although vanadate inhibition of both PFK-2 (26) and Fru-2,6-P2ase (27) activities in vitro has been described, our results show that these activities are not altered in a permanent way in hepatocytes after incubation with vanadate. Because PFK-2/Fru-2,6-P2ase in the liver is regulated by phospho-/dephosphorylation mechanisms (25), our results seem to indicate that vanadate does not modify Fru2,6-P2 levels through these mechanisms. However, vanadate is able to counteract glucagon effects on PFK-2 and pyruvate kinase activities. These effects are probably due to the counteraction by vanadate of the rise in cAMP levels and, as a consequence, the activation of cAMP-dependent protein kinase induced by glucagon (2,17). The results obtained when cells from nondiabetic (16) or diabetic rats were treated with colforsin or exogenous cAMP in the presence of vanadate suggest that this compound acts, at this level, on a postadenylate cyclase step. The results obtained with L-lactate and CO2 production from glucose indicate that the rise in Fru-2,6-P2 levels induced by vanadate is related to an increase in glucose utilization in hepatocytes from diabetic rats. These insulinlike effects of vanadate on glycolytic flux are produced simul-
DISCUSSION
The results presented in this article show that, in hepatocytes isolated from diabetic rats, vanadate is able to activate glycolysis. Glycolytic flux is very diminished in hepatocytes from diabetic rats compared with cells from healthy animals (24). Although in hepatocytes obtained from diabetic animals, incubation with vanadate stimulated almost 1.5-fold lactate production, and ~4-fold glucose oxidation, the resulting values were still below those previously determined in control cells from healthy rats under the same conditions (16). In these cells when they were incubated with 15 mM glucose, L-lactate production was 38 ± 2 ixmol • g~1 • 90 min""1 (16). On the contrary, in hepatocytes from diabetic animals, L-lactate production from 20 mM glucose was only 12 ± 1 fimol • g~1 • 90 min- 1 after incubation with 2 mM vanadate. Likewise, 14CO2 production in normal control cells was 0.46 ± 0.05 |ximol/g after 60 min (16), whereas in cells from diabetic rats, a value of 0.31 ± 0.01 |xmol/g after 60 min of incubation with vanadate was obtained. The stimulation of glycolysis by vanadate is probably the result of an increase in the intracellular levels of Fru-2,6-P2, the most important regulatory metabolite of the Fru-6-P/Fru1,6-P2 cycle (25). The basal levels of Fru-2,6-P2 in hepatocytes from diabetic rats incubated in the presence of 16 mM lactate/4 mM pyruvate are much lower than those de-
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FIG. 5. Effects of vanadate on L-lactate production. Hepatocytes were incubated in presence of 20 mM glucose in absence (O) or presence ( • ) of 2 mM vanadate. Values are means ± SE for 5 experiments.
DIABETES, VOL. 40, OCTOBER 1991
J.E. RODRIGUEZ-GIL AND ASSOCIATES
taneously with some anti-insulin, glucagonlike effects of vanadate on glycogen metabolism. In this regard, it has also been described that vanadate, both in hepatocytes from nondiabetic (14) or diabetic rats (15), inactivates and phosphorylates glycogen synthase by a calcium-independent mechanism, whereas vanadate activates glycogen phosphorylase in a calcium-dependent manner (14,15). All of these results indicate that vanadate acts on hepatocyte glucose metabolism by at least four different mechanisms: 1) A calcium- and cAMP-independent mechanism responsible for the rise in Fru-2,6-P2 levels. 2) A second mechanism acting on a post-adenylate cyclase step that is able to counteract glucagon and other cAMP-mediated effects. 3) Another calcium-independent mechanism that provokes antiinsulin effects on glycogen synthase activity. This action is probably related to an activation of a calcium- and cAMPindependent protein kinase activity (17). 4) Finally, a calcium-dependent mechanism that is responsible for the effects of vanadate on glycogen phosphorylase activity. In conclusion, our results prove that vanadate acts in a direct manner on glucose metabolism in rat hepatocytes. This action provokes a simultaneous stimulation of the glycogenolytic and glycolytic pathways, and as a result, vanadate activates glucose utilization in these cells either from nondiabetic or diabetic rats. Vanadate's effects on glycolytic flux in isolated hepatocytes are in agreement with those described after treatment in vivo with vanadate (5-7,9-11). Therefore, our results support the concept that the ability of vanadate to stimulate liver glycolysis contributes to the mechanism by which vanadate normalizes glycemia when administered orally to diabetic rats.
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ACKNOWLEDGMENTS
This study was supported by Grant 89/490 from the Fondo de Investigaciones Sanitarias de la Seguridad Social, Spain. C.F. and J.E.R.-G. were recipients of Fellowships from the Programa de Formacion de Personal Investigador (Ministry of Education, Spain). We thank Anna Vilalta and Catalina Relaiio for skilled technical assistance.
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