CELL BIOCHEMISTRY AND FUNCTION
VOL.
8: 237-241 (1990)
Effects of Vanadate on 6-Phosphofructo 2-Kinase Activity and Fructose 2,6-Bisphosphate Levels in Cultured Rat Hepatocytes MONTSERRAT MIRALPEIX, NORBERT R. KATZt AND RAMON BARTRONS Unitat de Bioquimica, Departament de Ciencies Fisiolhgiques Humanes i de la Nutricib, Universitat de Barcelona, Zona Universiraria de Bellvitge, 08907 L'Hospitalet, Spain and YMedizinische Klinik der Universitat Freiburg, Hugstetrer Strape 55, 0-7800 Freiburg, Federal Republic of Germany.
The presence of vanadate in primary cultures of rat hepatocytes produced a significant increase in the concentration of fructose 2,6-bisphosphate and in the activity of 6-phosphofructo 2-kinase. Compared with insulin, vanadate had a more potent action on the metabolite increase, but a similar effect on the 6-phosphofructo 2-kinase activity. Both the insulin- and the vanadate-dependent enhancements of 6-phosphofructo 2-kinase were inhibited by cycloheximide which specifically blocks protein synthesis on the translational level, suggesting that the increase of the enzyme activity was due to induction rather than to a change in the catalytic activity. K E Y WORDS -
Fructose 2,6-bisphosphate; 6-phosphofructo 2-kinase; vanadate; cultured rat hepatocytes.
INTRODUCTION
A number of physiologic and metabolic effects of hibitor of fructose 1,6-bisphosphatase, has a major vanadium compounds have been reported, includ- role in the regulation of hepatic carbohydrate ing their insulin-like actions on isolated cells as well metabolism. Its concentration is determined by as on the intact animal (for review see references 1, nutritionally and hormonally induced changes in the activity of the bifunctional enzyme 6-phospho2--5j. 2-kinase/fructose 2,6-bisphosphatase In intact cells, it has been shown that vanadate fructo stimulates glucose transport and oxidation in responsible for its synthesis and degradation.".' adipocytes6-8 and enhances glycogen synthesis in The regulation of this enzyme is a complex function adipocytes,' hepatocytes8 and m ~ s c l e .Further~ of the influence both of substrates and effectors as more, vanadate ions elicit a mitogenic response in well as its covalent modification via phosphorylahuman fibroblasts and in 3T3 and 3T6 cell lines;' tion/dephosph~rylation.'~~' In addition, the longthey interact synergistically with growth factors.' term regulation of the total enzymatic activity is In the intact animal, it has been demonstrated that dependent on the rates of synthesis and degradathe oral administration of vanadate to diabetic rats tion of the enzyme." Recently, it has been demonnormalized the high blood glucose concentra- strated that the low PFK-2 activity found in ti or^,^.^.^ prevented the decline in cardiac perfor- starvation or diabetes"-" results from a diminmance due to diabetes2 and doubled the basal rates ished enzyme content, probably as a consequence of glucose uptake both in liver and in m ~ s c l e . ~of a decreased translational efficiency of PFK-2 Recently, we have shown that vanadate treatment mRNA and (or) increased turnover of the PFK-2 of diabetic rats normalized the diminished hepatic protein, since the mRNA for hepatic PFK-2 did not levels of glycogen and fructose 2,6-bisphosphate, change in these experimental condition^.'^ Refeedand the low hepatic enzyme activities of glucokin- ing and insulin administration caused coordinated ase and 6-phosphofructo 2-kinase (PFK-2j.5 increases of the bifunctional enzyme and its Fructose 2,6-bisphosphate, the most potent m R S A.' activator of 6-phosphofructo 1-kinase and inThe present work was undertaken to compare the effects of insulin and vanadate, as insulinmimetic agent, on the long-term regulation of Addressee for correspondence: R. Bartrons, Unitat de BioquiPFK-2 activity and fructose 2,6-bisphosphate conmica. Facultat d'Odontologia, Universitat de Barcelona, Zona centration using primary hepatocyte cultures. Universitaria de Bellvitge, 08907 L'Hospitalet, Spain.
'
'
0263-6484/90/040237-05 $05.00 $> 1990 by John Wiley & Sons, Ltd
238 MATERIALS AND METHODS Materials
All chemicals were reagent grade, collagenase was from Worthington, medium 199 and neonatal calf serum from Boehringer Mannheim, streptomycin sulfate, penicillin, cycloheximide and sodium orthovanadate (Na,VO,) from Sigma, and bovine serum albumin, bovine insulin and dexamethasone from Serva. Enzymes and biochemical reagents were from either Boehringer Mannheim or Merck. Tissue-culture dishes (diameter 6 cm) were obtained from Falcon.
M. MIRALPEIX E T A L .
Portions of the resulting supernatants were incubated for 10 min at 30°C in the presence of 50 mM Tris, 5 mM ATP, 7 mM MgCI,, 100 m M KCI, 1 mM Pi, 5 mM fructose-6-P and 17.5 mM glucose-6-P, adjusted to pH 8.5. Lactate dehydrogenase was assayed according to Bergmeyer et al. One unit of activity is defined as the amount of enzyme that transforms 1 pmol of substrate min-’ at 30°C. Fructose 2,6-bisphosphate was extracted and measured as described in reference 18. DNA was estimated according to GiHhorn-Katz and Katz.” The statistical evaluation was performed by Student’s ttest.
’’
RESULTS AND DISCUSSION Cell Culture
Hepatocytes were isolated from fed or 48 h fasted adult female Wistar rats (180-2OOg) by a recirculating collagenase perfusion in situ. Nonparenchymal cells and debris were removed by several washings and sedimentation of hepatocytes at 30 g for 2 min. The basic medium 199 contained glucose (5.5 mM), bovine serum albumin (2 g 1- ’), penicillin (60 mg 1- ’), streptomycin sulfate (120 mg 1-I), 4-(2-hydroxyethyl)-l-piperazineethanesulfonate (Hepes, 10 mM) and NaHCO, (18 mM) as described previo~sly.’~ For the first 4 h medium was supplemented with 4per cent newborn calf serum, 5 mM insulin and 50 mM dexamethasone. After the first medium change (3 ml per dish), serum was omitted and the cells were cultured with 0.5 n M insulin and 5 nM dexamethasone. Incubation was performed at 36.5”C and 5 per cent CO, humidified air. Enzyme induction was initiated by addition of vanadate and/or high insulin concentrations to the hepatocytes after the medium change at 24 h. During the following days the medium was replaced every 24 h. The effect of cycloheximide (10 ,UM)on the enzyme induction was tested between 48 h and 72 h of culture. The incubation was terminated by rapidly aspirating the medium and immersing the dishes in liquid N,.
Under basic culture conditions, it was found that during the first 24 h the concentration of fructose 2,6-bisphosphate increased from 0-4 nmol to 1.1 nmol mg-’ DNA and, in the following 48 h of culture the metabolite levels decreased moderately (1.1 nmol mg-‘ DNA at 24 h to 0.85 at 72 h) (Figure 1). The effect of insulin and vanadate on the fructose 2,6-bisphosphate levels was studied after the medium change at 24 h. The addition of 100 nM insulin prevented the decrease of the metabolite concentration and maintained the levels around 1.2 nmol mg-’ DNA in the period studied (Figure 1). The statistically significant increase of fructose 2,6-bisphosphate levels in the presence of
t
15uM
I 0 4
24
48
72
Time of culture ( h )
Analytical Procedures Figure 1.
Time course of the vanadate-dependent and insulin-
6-phosphofructo 2-kinase was determined as de- dependent enhancement of fructose 2,6-bisphosphate (Fructose scribed by Bartrons et a1.16 using the enzyme assay 2,6-P,) levels in primary cultures of hepatocytes. Hepatocytes for the ‘total’ form at pH 8-5, that represents the were isolated from 48 h fasted rats. After 24 h culture under the medium was changed: vanaVmaXof the enzyme. Cells of single dishes were basic culture conditions (0) date (A) and insulin (U) were added as indicated (arrow). Cells scraped off and homogenized in ice-cold 20 mM Pi, were collected at different times (0,4,24,48 and 72 h). Values are 10 mM EDTA, 100 mM KF, 1 mM dithiothreitol, means 4 S.E.M. for four to 12 determinations from three to six pH 7.1 and centrifuged at 3000 g for 5 min at 0°C. experiments.
239
EFFECTS OF VANADATE IN CULTURED RAT HEPATOCYTES
Table 1. Effect of vanadate and insulin on fructose 2,6-bisphosphate levels and on 6-phosphofructo 2-kinase activity. ~
Addition
None (6) Insulin (6) Vanadate (4) Insulin + vanadate (3)
~
~
~
~
~~
Fructose 2,6-bisphosphate (nmol mg-' DNA)
6-phosphofructo 2-kinase (LJg-' DNA)
0.85 f 0.05 1.21 rt 0.05* 2.64 k 0-09* 2.60 i 0.30*
0.57 5 0.06 1.05 0.05* 0.92 f 0.077 0.88 rt 0.10$
+
Hepatocytes were isolated from 48 h fasted rats. After 24 h culture under basic conditions, the medium was changed and vanadate (15 p ~ and/or ) insulin (100 nM) were added as indicated. Cells were collected after 72 h culture. Values are means S.E.M. for the number of experiments with duplicates indicated in parentheses. Significant differences from cultures without addition are indicated: * p < 0.001; t p < 0.01 ; $ p < 0.05.
+
100nM insulin at 72 h of culture, versus basic conditions (Table I), seems to be due to the decrease in the control values rather than to an increase produced by insulin. Addition of 5 ,UM vanadate resulted in a large and significant increase of the fructose 2,6-bisphosphate concentration in the period studied (1.1 nmol mg-' DNA at 24 h under basic conditions to 2 nmol mg-' DNA at 72 h in the presence of vanadate; p ;0.001), and this effect was almost linear with time for at least 48 h (Figure 1). Furthermore, enhancement of vanadate concentration to 15 ,UM resulted in an additional increase of the metabolite, compared with 5 p~ vanadate (Figure 1 and Table 1). Simultaneous addition of 100 nM insulin and 15 pM vanadate during 48 h did not produce additive effects on the metabolite concentration (Table 1). As shown in Figure 2, the effect of vanadate was dose-dependent, but at concentrations of 50 ,UM or higher (data not shown) the survival of the cultures was impaired and the levels of fructose 2,6bisphosphate strongly decreased. This suggested that vanadate was toxic for hepatocytes at these doses as has been previously reported in isolated cells.20 Therefore, it was important to determine conditions that would allow the cells to be grown for prolonged periods of time in the presence of vanadate. With 15 p~ vanadate the morphology and the overall protein synthesis of the cells was unaffected during the per;od studied. The effectsOf and vanadate On the regu1ation of the total PFK-2 were studied during 48 h of culture beginning " u 24 h after the isolation of the hepatocytes (Table 1). The presence of 100 nM ins*lin resulted in a significant increase of PFK-2 activity to lSo per cent during 48 h. Addition Of 15 pM vanadate also produced a significant enhan-
cement of the enzyme activity to 160 per cent within 48 h. Insulin- and vanadate-dependent enhancements of PFK-2 activity were not additive (Table 1). The insulin- as well as the vanadatedependent increases of the total PFK-2 were inhibited by cycloheximide, which specifically blocks protein synthesis at the translational level (Table 2). The inhibition suggests that the insulin-
Q
z
o,
\E
5
E K
'9
2
't
Figure 2. Effect of vanadate on fructose 2.6-bisphosphate concentration in primary cultures of hepatocytes. Hepatocytes were isolated from fed rats and were cultured for 24 h under basic culture conditions as described in Materials and Methods. After this time, the medium was changed and different concentrations of vanadate were added. Cells were collected after 60 h of culture. Values are means & S.E.M. for two to four determinations from two different cell batches corresponding to two representative experiments.
240
M. MIRALPEIX ETAL. Table 2. Inhibition of vanadate- and insulin-dependent enhancement of 6phosphofructo 2-kinase activity by cycloheximide. ~
Addition
6-phosphofructo 2-kinase (U g - I DNA) Control Cycloheximide
~~~~
Lactate dehydrogenase (U mg-' DNA) Control Cycloheximide ~
None Insulin Vanadate
0.61 5 0.04 1.14 0.08 0-92 i 0.07
0.50 i 0-11 0.69 f O.ll* 0.47 f 0.10*
48.9 i 1.1 63.0 & 2.1 59.6 i 1.4
53.1 f 1.6 66.2 & 5 4 62.8 2.2
Hepatocytes were isolated from 48 h fasted rats. After 24 h culture under basic conditions, the medium was changed and 100 nM insulin o r 15 p~ vanadate were added. The effect of 10 p~ cycloheximide was tested between 48 h and 72 h. Hepatocytes were collected after 72 h culture. Values are means i S.E.M. for six determinations from three experiments. Significant differences from cultures without cycloheximide are indicated: * p < 0.01.
and the vanadate-dependent enhancements of the enzyme activity was due to induction rather than to a change in the catalytic activity. The long-term effect of insulin on PFK-2 activity is in line with earlier studies showing that insulin, in cultured rat hepatocytes, activates PFK-2 activity without producing changes in CAMP-dependent protein kinase activity and in enzyme effector levels." Insulin administration to diabetic rats restored the low hepatic PFK-2 activity, suggesting that the main effect of the hormone is on its V,,,, that results probably from an increased enzyme content.' The insulin-dependent induction of PFK-2 can be regarded as a physiological phenomenon, since the high insulin (100 nM) concentration used in hepatocyte cultures is decreased to less than 20 nM during the following 24 h after medium change, being at this concentration not much higher than insulin levels in the portal vein of refed rats after starvation.22 The increase of PFK-2 by vanadate agreed also with previous results obtained in diabetic rats, showing that PFK-2 and glucokinase were induced after vanadate treatment.5 The specificity of the insulin- and the vanadatedependent increases were tested by comparing the PFK-2 activity with the activity of the non-inducible enzyme lactate dehydrogenase. The PFK-2 enzyme activity was significantly increased in the presence of insulin and vanadate, whereas the lactate dehydrogenase activity was essentially unchanged or only slightly enhanced. This slight increase of activity produced by the two agents (insulin and vanadate) was not inhibited by cycloheximide (Table 2). The unspecific phenomenon after prolonged treatment with insulin has been reported previously.' 5 * 1 9 '3"
The results presented in this paper show that vanadate has a more potent action on the increase in fructose 2,6-bisphosphate levels than insulin in primary hepatocyte cultures. However, both agents have a similar action on the increase in the V,,, of the PFK-2. The enhancement of the enzyme activity by insulin and vanadate would produce an increase in the fructose 2,6-bisphosphate concentration (Figure 1 and Table 1) and in the glycolytic f l ~ x . ' ~ ~ 'The ' ~ ~ larger ' effect of vanadate on the metabolite concentration can be due to additional effects on hexose 6-phosphate concentration, increased in isolated hepatocytes treated with ana ad ate,^^ as a consequence of increased hexose uptake3 or hexose pho~phorylation.~ In contrast, insulin did not stimulate liver glucose ~ p t a k e . ' ~ These arguments could explain the differences observed between insulin and vanadate action on fructose 2,6-bisphosphate levels. In conclusion, the results reported here show that vanadate, in cultured rat hepatocytes, increases the concentration of fructose 2,6-bisphosphate more than insulin, but both have a similar effect on PFK-2 activity. The fact that cycloheximide inhibits the vanadate-dependent enhancement of enzyme activity supports the idea that vanadate induces the synthesis of PFK-2 in diabetic rats,5 and argues against an indirect mechanism mediated by an increase in vivo insulin secretion. ACKNOWLEDGEMENTS We would like to thank Dr J. Gil for constructive criticism during the preparation of this manuscript. We would also like to thank Mrs Brigitte Kreisel for excellent technical assistance. M. Miralpeix
EFFECTS OF VANADATE IN CULTURED RAT HEPATOCYTES
was the recipient of a FEBS Summer Fellowship. This work has been supported by the Deutsche Forschungsgemeinschaft (S.F.B. 154), FIS and DGICYT.
REFERENCES I. 2.
3.
4.
5.
6. 7.
8. 9. !0.
11.
Ramasarma, T. and Crane, F. L. (1981). Does vanadium play a role in cellular regulation? Curr. Top. Cell. Regul., 20, 247-301. Heyliger, C. E., Tahilini, A. G. and McNeill, J. H. (1985). Effectof vanadate on elevated blood glucose and depressed cardiac performance of diabetic rats. Science, 227, 1474-1477. Meyerovitch, J., Farfel, Z., Sack, J. and Shechter, Y.(1987). Oral administration of vanadate normalizes blood glucose levels in streptozotocin-treated rats. J. Biol Chem., 262, 6658-6662. Challiss, R. A. J., Leighton, B., Lozeman, F. J., Budottoski, L. and Newsholme, E. A. (1987). Effects of chronic administration of vanadate to the rat on the sensitivity of glycolysis and glycogen synthesis in skeletal muscle to insulin. Biochem. Pharmcol., 36, 357-361. Gil. J., Miralpeix, M., Carreras, J. and Bartrons, R. (1988). Insulin-like effects of vanadate on glucokinase activity and fructose 2,6-bisphosphate levels in the liver of diabetic rats. J . Biol. Chem., 263, 1868-1871. Shechter, Y. and Karlish, S. J. D. (1980). Insulin-like stimulation of glucose oxidation in rat adipocytes by vanadyl (IV) ions. Nature, 284,556-558. Tamura, S., Brown, T. A., Whipple, J. H., Fujita-Yamaguchi, Y., Dubler, R. E., Cheng, K. and Larner, J. (1984). A novel mechanism for the insulin-like effect of vanadate on glycogen synthase in rat adipocytes. J. Biol. Chem., 259, 6650 6658. Tolman, E. L., Barns, E., Burns, M., Pansini, A. and Partridge, R. (1979). Effects of vanadium on glucose metabolism in nitro. Life Sci., 25, 1159-1164. Smith, J. B. (1983). Vanadium ions stimulate DNA synthesis in Swiss mouse 3T3 and 3T6 cells. Proc. Natl. Arad. Sci. U.S.A.,SO, 6 162-6166. Hue, L. and Rider, M. H. (1987). Role of fructose 2,6bisphosphate in the control of glycolysis in mammalian tissues. Biochem. J., 245, 313-324. Pilkis, S. J. and El-Maghrabi, M. R. (1988). Hormonal regulation of hepatic gluconeogenesis and glycolysis. Ann. Rev. Biochem., 57, 755-783.
24 1
12. Gil, J., Carreras, J. and Bartrons, R. (1986). Effects of diabetes on fructose 2,6-P,, glucose 1,6-P, and 6-phosphofructo 2-kinase in rat liver. Biochem. Biophys. Rex Commun., 136,498-503. 13. Crepin, K. M., Darville, M. I., Hue, L. and Rousseau, G. G. (1988). Starvation or diabetes decreases the content but not the mRNA of 6-phosphofructo 2-kinase in rat liver. FEBS Lett., 221, 136-140. 14. Colosia, A. D., Marker, A. J., Lange, A. J., El-Maghrabi, M. R., Granner, D. K., Tauler, A., Pilkis, J. and Pilkis, S. J. (1988). Induction of rat liver 6-phosphofructo 2-kinase/ Fructose 2,6-bisphosphatase mRNA by refeeding and insulin. J . Biol. Chem., 263, 18669-18677. 15. Katz, N. R. and Giflhorn, S. (1983). Glucose- and insulindependent induction of ATP citrate lyase in primary cultures of rat hepatocytes. Biochem. J., 212, 65-71. 16. Bartrons, R., Hue, L., Van Schaftingen, E. and Hers, H. G. (1983). Hormonal control of fructose 2,6-bisphosphate concentration in isolated rat hepatocytes. Biochem. J., 214, 829-837. 17. Bergmeyer, H. U., Gawehn, V. and Grasse, M. (1974). Methoden der enzymatischen Analyse (Bergmeyer, H. U., ed.) Verlag Chemie: Weinheim, pp. 512-513. 18. Van Schaftingen, E., Lederer, B., Bartrons, R. and Hers, H. G. (1982). A kinetic study of pyrophosphate: fructose 6phosphatephosphotransferase from potato tubers. Eur. J. Biochem., 129, 191-195. 19. Gifiorn-Katz, S. and Katz, N. R. (1986). Carbohydratedependent induction of fatty acid synthase in primary cultures of rat hepatocytes. Eur. 1.Biochem., 159,513-518. 20. Seglen, P. 0. and Gordon, P. B. (1981). Vanadate inhibits protein degradation in isolated rat hepatocytes. J . Biol. Chem., 256, 7699-7701. 21. Miiller, A., Unthan-Fechner, K. and Probst, 1. (1988). Activation of phosphofructokinase 2 by insulin in cultured hepatocytes without accompanying changes of effector levels or CAMP-stimulated protein kinase activity ratios. Eur. J. Biochem., 176,415-420. 22. Balks, H.-J. and Jungermann, K. (1984). Regulation of peripheral insulin/glucagon levels by rat liver. Eur. J . Biochem., 141, 645-650. 23. Miralpeix, M., Gil, J., Rosa, J. L., Carreras, J. and Bartrons, R. (1989). Vanadate counteracts glucagon effects in isolated rat hepatocytes. Life Sci., 44,1491-1497. 24. Bruton, S. D. and Ishida, T. (1965). Effect of insulin and potassium on glucose movement in perfused rat liver. Am. J. Physio/., 209, 1145-1161. Received 14 May 1990 4ccepled 13 June 1990
VOL.
8: 237-241 (1990)
Effects of Vanadate on 6-Phosphofructo 2-Kinase Activity and Fructose 2,6-Bisphosphate Levels in Cultured Rat Hepatocytes MONTSERRAT MIRALPEIX, NORBERT R. KATZt AND RAMON BARTRONS Unitat de Bioquimica, Departament de Ciencies Fisiolhgiques Humanes i de la Nutricib, Universitat de Barcelona, Zona Universiraria de Bellvitge, 08907 L'Hospitalet, Spain and YMedizinische Klinik der Universitat Freiburg, Hugstetrer Strape 55, 0-7800 Freiburg, Federal Republic of Germany.
The presence of vanadate in primary cultures of rat hepatocytes produced a significant increase in the concentration of fructose 2,6-bisphosphate and in the activity of 6-phosphofructo 2-kinase. Compared with insulin, vanadate had a more potent action on the metabolite increase, but a similar effect on the 6-phosphofructo 2-kinase activity. Both the insulin- and the vanadate-dependent enhancements of 6-phosphofructo 2-kinase were inhibited by cycloheximide which specifically blocks protein synthesis on the translational level, suggesting that the increase of the enzyme activity was due to induction rather than to a change in the catalytic activity. K E Y WORDS -
Fructose 2,6-bisphosphate; 6-phosphofructo 2-kinase; vanadate; cultured rat hepatocytes.
INTRODUCTION
A number of physiologic and metabolic effects of hibitor of fructose 1,6-bisphosphatase, has a major vanadium compounds have been reported, includ- role in the regulation of hepatic carbohydrate ing their insulin-like actions on isolated cells as well metabolism. Its concentration is determined by as on the intact animal (for review see references 1, nutritionally and hormonally induced changes in the activity of the bifunctional enzyme 6-phospho2--5j. 2-kinase/fructose 2,6-bisphosphatase In intact cells, it has been shown that vanadate fructo stimulates glucose transport and oxidation in responsible for its synthesis and degradation.".' adipocytes6-8 and enhances glycogen synthesis in The regulation of this enzyme is a complex function adipocytes,' hepatocytes8 and m ~ s c l e .Further~ of the influence both of substrates and effectors as more, vanadate ions elicit a mitogenic response in well as its covalent modification via phosphorylahuman fibroblasts and in 3T3 and 3T6 cell lines;' tion/dephosph~rylation.'~~' In addition, the longthey interact synergistically with growth factors.' term regulation of the total enzymatic activity is In the intact animal, it has been demonstrated that dependent on the rates of synthesis and degradathe oral administration of vanadate to diabetic rats tion of the enzyme." Recently, it has been demonnormalized the high blood glucose concentra- strated that the low PFK-2 activity found in ti or^,^.^.^ prevented the decline in cardiac perfor- starvation or diabetes"-" results from a diminmance due to diabetes2 and doubled the basal rates ished enzyme content, probably as a consequence of glucose uptake both in liver and in m ~ s c l e . ~of a decreased translational efficiency of PFK-2 Recently, we have shown that vanadate treatment mRNA and (or) increased turnover of the PFK-2 of diabetic rats normalized the diminished hepatic protein, since the mRNA for hepatic PFK-2 did not levels of glycogen and fructose 2,6-bisphosphate, change in these experimental condition^.'^ Refeedand the low hepatic enzyme activities of glucokin- ing and insulin administration caused coordinated ase and 6-phosphofructo 2-kinase (PFK-2j.5 increases of the bifunctional enzyme and its Fructose 2,6-bisphosphate, the most potent m R S A.' activator of 6-phosphofructo 1-kinase and inThe present work was undertaken to compare the effects of insulin and vanadate, as insulinmimetic agent, on the long-term regulation of Addressee for correspondence: R. Bartrons, Unitat de BioquiPFK-2 activity and fructose 2,6-bisphosphate conmica. Facultat d'Odontologia, Universitat de Barcelona, Zona centration using primary hepatocyte cultures. Universitaria de Bellvitge, 08907 L'Hospitalet, Spain.
'
'
0263-6484/90/040237-05 $05.00 $> 1990 by John Wiley & Sons, Ltd
238 MATERIALS AND METHODS Materials
All chemicals were reagent grade, collagenase was from Worthington, medium 199 and neonatal calf serum from Boehringer Mannheim, streptomycin sulfate, penicillin, cycloheximide and sodium orthovanadate (Na,VO,) from Sigma, and bovine serum albumin, bovine insulin and dexamethasone from Serva. Enzymes and biochemical reagents were from either Boehringer Mannheim or Merck. Tissue-culture dishes (diameter 6 cm) were obtained from Falcon.
M. MIRALPEIX E T A L .
Portions of the resulting supernatants were incubated for 10 min at 30°C in the presence of 50 mM Tris, 5 mM ATP, 7 mM MgCI,, 100 m M KCI, 1 mM Pi, 5 mM fructose-6-P and 17.5 mM glucose-6-P, adjusted to pH 8.5. Lactate dehydrogenase was assayed according to Bergmeyer et al. One unit of activity is defined as the amount of enzyme that transforms 1 pmol of substrate min-’ at 30°C. Fructose 2,6-bisphosphate was extracted and measured as described in reference 18. DNA was estimated according to GiHhorn-Katz and Katz.” The statistical evaluation was performed by Student’s ttest.
’’
RESULTS AND DISCUSSION Cell Culture
Hepatocytes were isolated from fed or 48 h fasted adult female Wistar rats (180-2OOg) by a recirculating collagenase perfusion in situ. Nonparenchymal cells and debris were removed by several washings and sedimentation of hepatocytes at 30 g for 2 min. The basic medium 199 contained glucose (5.5 mM), bovine serum albumin (2 g 1- ’), penicillin (60 mg 1- ’), streptomycin sulfate (120 mg 1-I), 4-(2-hydroxyethyl)-l-piperazineethanesulfonate (Hepes, 10 mM) and NaHCO, (18 mM) as described previo~sly.’~ For the first 4 h medium was supplemented with 4per cent newborn calf serum, 5 mM insulin and 50 mM dexamethasone. After the first medium change (3 ml per dish), serum was omitted and the cells were cultured with 0.5 n M insulin and 5 nM dexamethasone. Incubation was performed at 36.5”C and 5 per cent CO, humidified air. Enzyme induction was initiated by addition of vanadate and/or high insulin concentrations to the hepatocytes after the medium change at 24 h. During the following days the medium was replaced every 24 h. The effect of cycloheximide (10 ,UM)on the enzyme induction was tested between 48 h and 72 h of culture. The incubation was terminated by rapidly aspirating the medium and immersing the dishes in liquid N,.
Under basic culture conditions, it was found that during the first 24 h the concentration of fructose 2,6-bisphosphate increased from 0-4 nmol to 1.1 nmol mg-’ DNA and, in the following 48 h of culture the metabolite levels decreased moderately (1.1 nmol mg-‘ DNA at 24 h to 0.85 at 72 h) (Figure 1). The effect of insulin and vanadate on the fructose 2,6-bisphosphate levels was studied after the medium change at 24 h. The addition of 100 nM insulin prevented the decrease of the metabolite concentration and maintained the levels around 1.2 nmol mg-’ DNA in the period studied (Figure 1). The statistically significant increase of fructose 2,6-bisphosphate levels in the presence of
t
15uM
I 0 4
24
48
72
Time of culture ( h )
Analytical Procedures Figure 1.
Time course of the vanadate-dependent and insulin-
6-phosphofructo 2-kinase was determined as de- dependent enhancement of fructose 2,6-bisphosphate (Fructose scribed by Bartrons et a1.16 using the enzyme assay 2,6-P,) levels in primary cultures of hepatocytes. Hepatocytes for the ‘total’ form at pH 8-5, that represents the were isolated from 48 h fasted rats. After 24 h culture under the medium was changed: vanaVmaXof the enzyme. Cells of single dishes were basic culture conditions (0) date (A) and insulin (U) were added as indicated (arrow). Cells scraped off and homogenized in ice-cold 20 mM Pi, were collected at different times (0,4,24,48 and 72 h). Values are 10 mM EDTA, 100 mM KF, 1 mM dithiothreitol, means 4 S.E.M. for four to 12 determinations from three to six pH 7.1 and centrifuged at 3000 g for 5 min at 0°C. experiments.
239
EFFECTS OF VANADATE IN CULTURED RAT HEPATOCYTES
Table 1. Effect of vanadate and insulin on fructose 2,6-bisphosphate levels and on 6-phosphofructo 2-kinase activity. ~
Addition
None (6) Insulin (6) Vanadate (4) Insulin + vanadate (3)
~
~
~
~
~~
Fructose 2,6-bisphosphate (nmol mg-' DNA)
6-phosphofructo 2-kinase (LJg-' DNA)
0.85 f 0.05 1.21 rt 0.05* 2.64 k 0-09* 2.60 i 0.30*
0.57 5 0.06 1.05 0.05* 0.92 f 0.077 0.88 rt 0.10$
+
Hepatocytes were isolated from 48 h fasted rats. After 24 h culture under basic conditions, the medium was changed and vanadate (15 p ~ and/or ) insulin (100 nM) were added as indicated. Cells were collected after 72 h culture. Values are means S.E.M. for the number of experiments with duplicates indicated in parentheses. Significant differences from cultures without addition are indicated: * p < 0.001; t p < 0.01 ; $ p < 0.05.
+
100nM insulin at 72 h of culture, versus basic conditions (Table I), seems to be due to the decrease in the control values rather than to an increase produced by insulin. Addition of 5 ,UM vanadate resulted in a large and significant increase of the fructose 2,6-bisphosphate concentration in the period studied (1.1 nmol mg-' DNA at 24 h under basic conditions to 2 nmol mg-' DNA at 72 h in the presence of vanadate; p ;0.001), and this effect was almost linear with time for at least 48 h (Figure 1). Furthermore, enhancement of vanadate concentration to 15 ,UM resulted in an additional increase of the metabolite, compared with 5 p~ vanadate (Figure 1 and Table 1). Simultaneous addition of 100 nM insulin and 15 pM vanadate during 48 h did not produce additive effects on the metabolite concentration (Table 1). As shown in Figure 2, the effect of vanadate was dose-dependent, but at concentrations of 50 ,UM or higher (data not shown) the survival of the cultures was impaired and the levels of fructose 2,6bisphosphate strongly decreased. This suggested that vanadate was toxic for hepatocytes at these doses as has been previously reported in isolated cells.20 Therefore, it was important to determine conditions that would allow the cells to be grown for prolonged periods of time in the presence of vanadate. With 15 p~ vanadate the morphology and the overall protein synthesis of the cells was unaffected during the per;od studied. The effectsOf and vanadate On the regu1ation of the total PFK-2 were studied during 48 h of culture beginning " u 24 h after the isolation of the hepatocytes (Table 1). The presence of 100 nM ins*lin resulted in a significant increase of PFK-2 activity to lSo per cent during 48 h. Addition Of 15 pM vanadate also produced a significant enhan-
cement of the enzyme activity to 160 per cent within 48 h. Insulin- and vanadate-dependent enhancements of PFK-2 activity were not additive (Table 1). The insulin- as well as the vanadatedependent increases of the total PFK-2 were inhibited by cycloheximide, which specifically blocks protein synthesis at the translational level (Table 2). The inhibition suggests that the insulin-
Q
z
o,
\E
5
E K
'9
2
't
Figure 2. Effect of vanadate on fructose 2.6-bisphosphate concentration in primary cultures of hepatocytes. Hepatocytes were isolated from fed rats and were cultured for 24 h under basic culture conditions as described in Materials and Methods. After this time, the medium was changed and different concentrations of vanadate were added. Cells were collected after 60 h of culture. Values are means & S.E.M. for two to four determinations from two different cell batches corresponding to two representative experiments.
240
M. MIRALPEIX ETAL. Table 2. Inhibition of vanadate- and insulin-dependent enhancement of 6phosphofructo 2-kinase activity by cycloheximide. ~
Addition
6-phosphofructo 2-kinase (U g - I DNA) Control Cycloheximide
~~~~
Lactate dehydrogenase (U mg-' DNA) Control Cycloheximide ~
None Insulin Vanadate
0.61 5 0.04 1.14 0.08 0-92 i 0.07
0.50 i 0-11 0.69 f O.ll* 0.47 f 0.10*
48.9 i 1.1 63.0 & 2.1 59.6 i 1.4
53.1 f 1.6 66.2 & 5 4 62.8 2.2
Hepatocytes were isolated from 48 h fasted rats. After 24 h culture under basic conditions, the medium was changed and 100 nM insulin o r 15 p~ vanadate were added. The effect of 10 p~ cycloheximide was tested between 48 h and 72 h. Hepatocytes were collected after 72 h culture. Values are means i S.E.M. for six determinations from three experiments. Significant differences from cultures without cycloheximide are indicated: * p < 0.01.
and the vanadate-dependent enhancements of the enzyme activity was due to induction rather than to a change in the catalytic activity. The long-term effect of insulin on PFK-2 activity is in line with earlier studies showing that insulin, in cultured rat hepatocytes, activates PFK-2 activity without producing changes in CAMP-dependent protein kinase activity and in enzyme effector levels." Insulin administration to diabetic rats restored the low hepatic PFK-2 activity, suggesting that the main effect of the hormone is on its V,,,, that results probably from an increased enzyme content.' The insulin-dependent induction of PFK-2 can be regarded as a physiological phenomenon, since the high insulin (100 nM) concentration used in hepatocyte cultures is decreased to less than 20 nM during the following 24 h after medium change, being at this concentration not much higher than insulin levels in the portal vein of refed rats after starvation.22 The increase of PFK-2 by vanadate agreed also with previous results obtained in diabetic rats, showing that PFK-2 and glucokinase were induced after vanadate treatment.5 The specificity of the insulin- and the vanadatedependent increases were tested by comparing the PFK-2 activity with the activity of the non-inducible enzyme lactate dehydrogenase. The PFK-2 enzyme activity was significantly increased in the presence of insulin and vanadate, whereas the lactate dehydrogenase activity was essentially unchanged or only slightly enhanced. This slight increase of activity produced by the two agents (insulin and vanadate) was not inhibited by cycloheximide (Table 2). The unspecific phenomenon after prolonged treatment with insulin has been reported previously.' 5 * 1 9 '3"
The results presented in this paper show that vanadate has a more potent action on the increase in fructose 2,6-bisphosphate levels than insulin in primary hepatocyte cultures. However, both agents have a similar action on the increase in the V,,, of the PFK-2. The enhancement of the enzyme activity by insulin and vanadate would produce an increase in the fructose 2,6-bisphosphate concentration (Figure 1 and Table 1) and in the glycolytic f l ~ x . ' ~ ~ 'The ' ~ ~ larger ' effect of vanadate on the metabolite concentration can be due to additional effects on hexose 6-phosphate concentration, increased in isolated hepatocytes treated with ana ad ate,^^ as a consequence of increased hexose uptake3 or hexose pho~phorylation.~ In contrast, insulin did not stimulate liver glucose ~ p t a k e . ' ~ These arguments could explain the differences observed between insulin and vanadate action on fructose 2,6-bisphosphate levels. In conclusion, the results reported here show that vanadate, in cultured rat hepatocytes, increases the concentration of fructose 2,6-bisphosphate more than insulin, but both have a similar effect on PFK-2 activity. The fact that cycloheximide inhibits the vanadate-dependent enhancement of enzyme activity supports the idea that vanadate induces the synthesis of PFK-2 in diabetic rats,5 and argues against an indirect mechanism mediated by an increase in vivo insulin secretion. ACKNOWLEDGEMENTS We would like to thank Dr J. Gil for constructive criticism during the preparation of this manuscript. We would also like to thank Mrs Brigitte Kreisel for excellent technical assistance. M. Miralpeix
EFFECTS OF VANADATE IN CULTURED RAT HEPATOCYTES
was the recipient of a FEBS Summer Fellowship. This work has been supported by the Deutsche Forschungsgemeinschaft (S.F.B. 154), FIS and DGICYT.
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