help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sekar, N.
Right arrow Articles by Shechter, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sekar, N.
Right arrow Articles by Shechter, Y.
Endocrinology Vol. 140, No. 3 1125-1131
Copyright © 1999 by The Endocrine Society

ARTICLES

Independent Signal-Transduction Pathways for Vanadate and for Insulin in the Activation of Glycogen Synthase and Glycogenesis in Rat Adipocytes1

Natesampillai Sekar, Jinping Li, Zehibin He, Dov Gefel and Yoram Shechter2

Department of Biological Chemistry (N.S., J.L., Z.H., Y.S.), The Weizmann Institute of Science, Rehovot 76100, Israel; and Department of Medicine-C (D.G.), Barzilai Medical Center, Ashkelon 78306, Israel

Address all correspondence and requests for reprints to: Yoram Shechter, Department of Biological Chemistry, Weizmann Institute of Science, Rehovot-76100, Israel.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The activating effect of vanadate on glycogenesis and on glycogen synthase (uridine diphosphate-glucose-glycogen glucosyl transferase) activity was studied in rat adipocytes and compared with that of insulin. Using several approaches and specific blockers, we found that vanadate and insulin resemble each other, in the activation of glycogen synthase, in several aspects: both require nonarrested protein phosphatase 1 activity; they are equally suppressed by conditions that elevate cAMP-levels; and both depend on the activation of phosphatidylinositol-3 kinase. The basic differences between them are as follows: 1) vanadate promotes glycogenesis through the activation of a cytosolic protein tyrosine kinase, in an insulin-receptor-independent manner; 2) vanadate elevates glucose-6-phosphate (G-6-P) to a higher level than insulin; 3) vanadate-activated glycogenesis is accompanied by an increase in the cellular content of immunoreactive glycogen synthase, an effect less noticeable with insulin; 4) adipose glucose-6-phosphatase is inhibited by vanadate (dose for 50% inhibition, IC50 = 7 ± 0.7 µM) but not by insulin.

We have concluded that insulin and vanadate activate glycogenesis through a phosphatidylinositol-3 kinase and dephosphorylation-dependent mechanism. Vanadate, however, uses a receptor-independent pathway and is superior to insulin in elevating the level of G-6-P, a key metabolite for activating glycogen synthase. This is attributed to the combined effect of vanadate in enhancing glucose entry and in inhibiting dephosphorylation of endogenously formed G-6-P. The latter effect is not exerted by insulin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VANADIUM SALTS mimic most of the biological effects of insulin, both in vitro and in vivo (Refs. 1, 2, 3, 4, 5, 6, 7 and reviewed in Refs. 8, 9, 10, 11). In 1984, Tamura et al. (12) demonstrated that vanadate activates glycogen synthesis in rat adipocytes, as well. It was initially assumed that vanadate, by virtue of its potency in inhibiting protein phosphotyrosine phosphatases (13), activates the insulin receptor by simply increasing its phosphorylation on tyrosine moieties. In vanadate-treated tissues, however, and in rat adipocytes, the insulin receptor undergoes little phosphorylation on tyrosine moieties (14, 15, 16, 17, 18). In addition, the receptor blocker quercetin fails to inhibit the biological effects of vanadate (19). A cytosolic nonreceptor protein tyrosine kinase (CytPTK) of 53 kDa has been identified (20, 21), which is markedly activated by vanadate in intact adipocytes and in a cell-free experimental system (20, 21, 22). CytPTK is strongly inhibited by staurosporine (inhibitory dose 50, IC50 = 1–2 nM), a weak inhibitor of the insulin-receptor-tyrosine-kinase (InsRTK) (IC50 = 1–2 µM). Based on the inhibitory efficacies of staurosporine in rat adipocytes, we have concluded that CytPTK participates in the activating effects of vanadate on glucose metabolism but not in the activation of hexose transport or in the antilipolytic effect of vanadate (20, 21, 22).

In this study, then, we evaluate the mode(s), the site(s), and the mechanism(s) by which vanadate activates glycogenesis in rat adipocytes. In particular, we have analyzed points of similarity and dissimilarity in activation of glycogenesis by insulin and vanadate.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
D-(U-14C)glucose was purchased from New England Nuclear (Boston, MA), and uridine diphosphate (U-14C)glucose from Amersham (Aylesbury, Buckinghamshire, UK). Collagenase type-1 (134 U/mg) was obtained from Worthington Biochemical Corp. (Freehold, NJ). Okadaic acid, calyculin A, dibutyryl cAMP, isoproterenol, and glycogen were purchased from Sigma Chemical Co. (St. Louis, MO). Antiglycogen synthase antibody (chicken IgY) was kindly provided by Dr. John C. Lawrence (University of Virginia, School of Medicine, Charlottesville, VA). Antichicken IgY-horseradish peroxidase (HRP)-labeled secondary antibody was obtained from Promega Corp. (Madison, WI). Porcine insulin was obtained from Eli Lilly & Co. (Indianapolis, IN). Krebs-Ringer bicarbonate (KRB) buffer (pH 7.4) contained 110 mM NaCl, 25 mM NaHC03, 5 mM KCl, 1.2 mM KH2PO4, 1.3 mM CaCl2, and 1.3 mM MgSO4.

Cell preparation and bioassay
Rat adipocytes were prepared from fat pads of male Wistar rats (150- 200 g) by collagenase digestion, as described by Rodbell (23). Cell preparations showed more that 95% viability by Tryphan blue exclusion, at least 3 h after digestion.

Assay of glycogen synthase activity
Glycogen synthase (UDP-glucose-glycogen-glucosyltransferase) activity was assayed by the method of Thomas et al. (24). Freshly prepared rat adipocytes [~20% cell suspension in KRB buffer (pH 7.4), 0.7% BSA] were incubated with or without vanadate (30 min) or insulin (15 min) at 37 C. After incubation, cells were washed twice with KRB buffer without BSA and were homogenized in buffer containing 50 mM Tris-HCl (pH 7.8), 100 mM KF, and 5 mM EDTA. After centrifugation at 10,000 x g for 20 min, to remove the fat cake and pelleted debris, homogenates were assayed for the incorporation of UDP-(U-14C)glucose (6.7 mM) into glycogen for 20 min at 30 C in assay buffer containing 50 mM Tris-HCl (pH 7.8), 20 mM EDTA, 25 mM KF, and 10 mg/ml glycogen. The assay was carried out in the presence or absence of 10 mM glucose-6-phosphate (+ or - G-6-P). Results were expressed as the ratio of glycogen synthase activity observed in the absence (i.e. activation in situ) or presence (i.e. total activity) of G-6-P.

Assay of glycogen synthesis (glycogenesis)
Glycogen synthesis in adipocytes was analyzed essentially as described in Refs. 25, 26 . Briefly, freshly prepared rat adipocytes in KRB buffer were pretreated with different blockers (as indicated in the figure legends) and then supplemented with 17 nM insulin or 1 mM sodium metavanadate for 30 min at 37 C. Cells were then incubated with 2 mM D-(U-14C)-glucose, and glycogenesis was performed for an additional 30 min at 37 C. Potassium hydroxide (KOH, 60%) was then added, and the cells were heated in a boiling water bath for 20 min. The solubilized cell extract was transferred to glass tubes containing 7.5 mg glycogen. Ice-cold ethanol (66%) was added to precipitate the glycogen. After centrifugation, the pellet was resuspended in 1 ml water, reprecipitated with ethanol, and centrifuged. The pellet was dissolved in water, and the amount of (U-14C)-glucose incorporated into glycogen was counted.

Immunoblot analysis
Immunoblot analysis was used to quantitate the cellular protein content of glycogen synthase. Isolated rat adipocytes were incubated with insulin or vanadate for 1 h and washed twice at 4 C with a buffer containing 20 mM HEPES (pH 7.4), 250 mM sucrose, 1 mM EDTA, and protease inhibitors (including 5 µg/ml leupeptin, 1 µg/ml pepstatin, and 5 µg/ml aprotinin). The cells were homogenized using the same buffer. The homogenate was centrifuged at 10,000 x g for 15 min to remove the fat cake and pelleted debris, and the supernatant was used for immunoblotting. Equal amounts of protein (25 µg) were solubilized in Laemeli sample buffer (27) and resolved by SDS-PAGE containing 7.5% polyacrylamide. The proteins were then electrophoretically transferred to nitrocellulose filters and incubated with antibody specific for glycogen synthase, followed by antichicken-IgY-HRP-labeled systems. Immunoprecipitate of glycogen synthase protein was detected by enhanced chemiluminescence on hyperfilm ECL after autoradiography. The intensity of glycogen synthase protein was quantitated by scanning densitometry (Bio-Rad Laboratories, Inc., Hercules, CA). The amount of protein used (25 µg/lane) was chosen, based on a titration experiment indicating that this amount produces a signal that lies in the linear range for our immunoblot procedure.

Glucose-6-phosphatase (Glc-6-Pase) activity
Glc-6-Pase activity in adipose cell lysate was estimated according to Refs. 28, 29 . Briefly, adipocytes were washed in phosphate-free buffer and homogenized in 0.25 M sucrose. An assay was performed for 10 min. at 25 C in 0.1 ml of 50 mM Tris-cacodylate buffer (pH 6.5) containing adipose cell homogenate (10 µg protein) and 20 mM G-6-P. The assay was terminated with 0.9 ml stopping solution containing 1 M H2SO4, 5% ammonium molybdate, 1% SDS, and 1% ascorbic acid. The intensity of absorbance at 820 nm was determined after incubation at 37 C for 45 min. Results are expressed as nanomoles of inorganic phosphate formed per minute per milligram of protein.

Estimation of G-6-P
Intracellular levels of G-6-P were determined as described by Lang and Michal (30). Rat adipocytes were incubated for 30 min at 37 C, in the presence or absence of vanadate or insulin, in KRB buffer (pH 7.4) containing 0.7% BSA. Cells were then washed and homogenized. The homogenates were precipitated using 10% perchloric acid. After centrifugation, the supernatant was adjusted to pH 3.5 with potassium carbonate solution (5 mM). G-6-P was quantitated enzymatically using G-6-P dehydrogenase and B-nicotinamide adenine dinucleotide phosphate, reduced form, NADPH.

Protein concentration was determined by the method of Bradford (31). All assays were performed in duplicate or triplicate. Each figure or table stands for a representative experiment performed at least 3–5 times with identical results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of glycogen synthase by vanadate in rat adipocytes
To examine the insulin-like effects of vanadate on glycogen synthase, isolated rat adipocytes were incubated with vanadate (30 min) or with insulin (15 min). Cell homogenates were used for assaying activation of glycogen synthase (see experimental section). Vanadate converted glycogen synthase into its active form, as did insulin, reaching 3.5- to 4.2-fold the basal level (Fig. 1Go). Activation was dose dependent and was already observed at 10 µM vanadate. ED50 was at 20 ± 4 µM, and maximal activation at 1 mM vanadate.



View larger version (40K):
[in this window]
[in a new window]
  		Figure 1. Vanadate activates glycogen synthase in rat adipocytes, as does insulin, with half-maximal effect at 20 ± 4 µM. Freshly prepared rat adipocytes in KRB buffer (pH 7.4, 37 C) were divided into plastic vials (0.5 ml per vial, ~3x105 cells) and incubated with the indicated concentrations of vanadate (30 min, 37 C) or insulin (15 min, 37 C). Adipocytes were then homogenized, and the supernatant fractions (0.2 ml) were assayed for the incorporation of UDP[U-14C]glucose into glycogen (experimental section). Results are expressed as the fractional activation of glycogen synthase (I/I + D ratio) and each individual treatment (insulin- or vanadate-activated; n = 4). Maximal (I + D) activation is that obtained by the inclusion of 10 mM G-6-P in the assay.

 
Insulin-independent activation of glycogenesis by vanadate
Vanadate and insulin activated glycogenesis 12- to 15-fold over basal level (Fig. 2Go, left columns). Vanadate-evoked glycogenesis is 4- to 5-fold more sensitive to vanadate, compared with the metalooxide effect in activating lipogenesis (Ref. 4 , and data not shown). To analyze the possible involvement of InsRTK in vanadate-evoked glycogenesis, adipocytes were preincubated with quercetin (30 min), a cell permeable blocker of the InsRTK-transphosphorylation (19), and then were analyzed for the activation of glycogenesis by insulin or vanadate (Fig. 2Go). Quercetin blocked insulin-evoked glycogenesis half-maximally at 80 ± 7 µM and maximally at 300 ± 30 µM (Fig. 2Go). In contrast, inhibition of vanadate-evoked glycogenesis by quercetin was negligible, showing only approximately 20% inhibition at the highest concentration of quercetin used (i.e. at 300 µM, Fig. 2Go).



View larger version (38K):
[in this window]
[in a new window]
  		Figure 2. Quercetin, a blocker of insulin-receptor tyrosine kinase trans-phosphorylation, blocks insulin- but not vanadate-evoked glycogenesis. Adipocytes (0.5 ml/vial, 3x105 cells) were preincubated with the indicated concentrations of quercetin, for 30 min at 37 C, and then with insulin (17 nM) or vanadate (1 mM) for an additional 30 min at 37 C. Cells were then supplemented with 2 mM (U-14C)-glucose, (3000 cpm/nmol, incubated for an additional 30 min at 37 C; and the amount of (U-14C)glucose incorporated into glycogen was determined (experimental section). Insulin and vanadate, at the concentrations used, stimulated glycogenesis maximally. In all experiments, insulin or vanadate stimulated glycogenesis 12- to 15-fold. Basal values represent approximately 150 ± 20 cpm per 3x105 cells/30 min; values for insulin or vanadate are 1800–2200 cpm per 3x105 cells/30 min.

 
Role of nonreceptor CytPTK in vanadate- evoked glycogenesis
Figure 3Go demonstrates the effect of staurosporine, a potent inhibitor of CytPTK, on vanadate and insulin-evoked glycogenesis. Staurosporine arrested the activating effect of vanadate, with IC50 = 0.35 ± 0.03 µM. In contrast, the activating effect of insulin on glycogenesis was appreciably resistant to inhibition by staurosporine, amounting to 5% at 0.2 µM, 10% at 0.5 µM, and 25% at 1 µM staurosporine (Fig. 3Go).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 3. Selective inhibitory effect of staurosporine on vanadate-evoked glycogenesis. Adipocytes (0.5 ml/vial, ~ 3x105 cells) were preincubated for 30 min at 37 C with the indicated concentration of staurosporine, and then with 17 nM insulin or 1 mM vanadate for an additional 30 min at 37 C. Cells were then supplemented with 2 mM [U-14C]glucose (3000 cpm/nmol) and assayed for glycogenesis for 30 min. Results are expressed as percent of maximal activation. Activation of 100% was obtained by 17 nM insulin or 1 mM vanadate in the absence of staurosporine.

 
Effect of wortmannin on vanadate-stimulated glycogenesis
Insulin activates phosphatidylinositol-3 kinase (PI3-kinase), an essential effect (but probably insufficient) to promote all the rapid metabolic effects of insulin, including the activation of glycogen synthesis (32, 33, 34, 35, 36). Similarly, vanadate activates PI3-kinase; and whereas it seems to be required for most of the rapid metabolic effects manifested by vanadate, it is not required for the inhibition of lipolysis (37). Figure 4Go shows the effect of wortmannin, a potent and specific inhibitor of PI3-kinase (32), on vanadate-evoked glycogenesis. Preincubation of rat adipocytes with wortmannin decreased the activating effects of insulin and vanadate on glycogenesis by 90 ± 5% and 95 ± 4%, respectively (Fig. 4Go).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 4. Wortmannin, a blocker of PI3-kinase, arrests vanadate and insulin- evoked glycogenesis. Adipocytes (0.5 ml, ~3x105 cells) were preincubated for 30 min at 37 C in the presence or absence of 100 nM wortmannin. Subsequently, cells were incubated for an additional 30 min with 17 nM insulin or with 1 mM vanadate, then supplemented with 2 mM (U-14C)-glucose and assayed for glycogenesis for 30 min. Results are expressed as percent of maximal response (obtained with 17 nM insulin or 1 mM vanadate).

 
Effect of okadaic acid and calyculin A on vanadate-stimulated glycogen synthesis
Active protein phosphatase 1 (PP1 or PP1-G) is essential for promoting the activation of glycogenesis by insulin (38). To investigate whether PP1 plays a major role in vanadate-evoked glycogenesis, adipocytes were pretreated with calyculin A, a potent inhibitor of PP1 (inhibition constant, Ki = 0.2 nM), or with okadaic acid, a weak inhibitor of PP1 (Ki = 20 nM) and a potent inhibitor of PP2A (Ki = 0.02 nM) (39, 40). Calyculin A and okadaic acid blocked vanadate-evoked glycogenesis, with IC50 values of 20 ± 3 and 350 ± 30 nM, respectively. Also, both inhibitors were equipotent in inhibiting either insulin- or vanadate-induced glycogenesis (Fig. 5Go). Thus, PP1 (but not PP2A) seems to be equally essential to promote glycogenesis by either insulin or vanadate.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 5. Calyculin A (a potent inhibitor of PP1) and okadaic acid (a weak inhibitor of PP1 and a potent inhibitor of PP2A) block vanadate-evoked glycogenesis. Adipocytes were preincubated at 37 C for 30 min with or without the indicated concentrations of calyculin A or okadaic acid. Adipocytes were then incubated for an additional 30 min at 37 C in the presence or absence of vanadate (1 mM) or insulin (17 nM). Glycogenesis was then performed under the experimental conditions specified in the legend for Fig. 2Go.

 
cAMP-mediated inhibition of glycogen synthesis
Glycogen synthesis is decreased upon addition of ß-adrenergic agonists. This effect is caused by elevating cAMP levels, the concomitant activation of PKA, and the conversion of active glycogen synthase into a phosphorylated, less active form (41). To determine the role of intracellular concentration of cAMP on insulin- and vanadate-induced glycogenesis, we preincubated adipocytes with isoproterenol, an activator of adenylate cyclase, for 10 min before the assay of glycogenesis (Fig. 6Go). Isoproterenol decreased both vanadate and insulin-stimulated glycogenesis nearly equally. Half-maximal inhibition was evident at approximately 0.2 µM isoproterenol.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 6. Isoproterenol and dibutyryl-cAMP suppress vanadate and insulin-evoked glycogenesis. Adipocytes were preincubated for 30 min at 37 C with the indicated concentrations of isoproterenol or dibutyryl-cAMP. Cells were then incubated for an additional 30 min at 37 C in the absence and the presence of 17 nM insulin or 1 mM vanadate. Glycogenesis was then performed under the experimental conditions specified in the legend for Fig. 2Go.

 
Addition of dibutyryl cAMP, a cell permeable derivative of this cyclic nucleotide, suppressed insulin- and vanadate-evoked glycogenesis nearly to the same extent. Fifty percent suppression was evident at approximately 200 µM dibutyryl cAMP (Fig. 6Go). Thus, the inhibition observed is cAMP-dependent and can occur at a site downstream to the activation of adenylate cyclase by isoproterenol.

Vanadate increases G-6-P level in rat adipocytes
G-6-P promotes activation of glycogen synthase in a reversible fashion, both in cell-free and in intact cellular systems (42, 43). We therefore examined whether the activating effect of vanadate on glycogenesis relates mechanistically to alteration in the intracellular level of G-6-P. In medium containing 5 mM glucose, pretreatment of adipocytes with insulin increased the G-6-P level from 12.5 ± 2 nmol/1x106 cells (basal level) to 23.5 ± 3; and with vanadate, to 41 ± 4 nmol/1x106 cells (Fig. 7Go). Thus, insulin and vanadate elevated G-6-P levels 1.9- and 3.3-fold, respectively.



View larger version (41K):
[in this window]
[in a new window]
  		Figure 7. Vanadate elevates the level of G-6-P in intact rat adipocytes. Freshly prepared rat adipocytes, in KRB buffer (pH 7.4) 0.7% BSA containing 5 mM glucose, were incubated in the presence or absence of 1 mM vanadate or 17 nM insulin for 30 min. Cells were then washed and homogenized, and the intracellular concentration of G-6-P was determined (experimental procedure). Results are expressed as picomoles of G-6-P per 106 cells (average of three separate determinations for each treatment).

 
Vanadate increases the cellular content of immunoreactive glycogen synthase
It has been previously shown that glucose (5–10 mM, within 30–60 min incubation period), but not insulin (in the absence of glucose), increases the cellular (cytosolic) content of total (I + D form) glycogen synthase protein in rat adipocytes (44). In Fig. 8Go, we have tested the capacity of vanadate to promote this effect, relative to insulin at lower concentrations of glucose (2 mM), using specific antibodies to glycogen synthase (experimental section). Figure 8AGo shows the single protein band corresponding to the 90-kDa glycogen synthase protein; Fig. 8BGo shows a densitometric analysis of this protein. In the presence of 2 mM glucose, vanadate and insulin increased the cellular content of glycogen synthase protein by about 1.4- and 2.1-fold, respectively; thus, the effect of vanadate on this parameter exceeds, by over 2-fold, that of insulin.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 8. Vanadate increases the cellular content of immunoreactive glycogen synthase. Adipocytes were preincubated for 1 h (in the absence of glucose) with and without 17 nM insulin or 1 mM vanadate. A, Total cellular proteins were then solubilized, size-fractionated by SDS-PAGE, immobilized on nitrocellulose, and reacted with antibodies specific for glycogen synthase, followed by labeling with anti(chicken)IgY-HRP; B, relative amounts of glycogen synthase were quantitated by densitometric analysis of auto radiograms.

 
Vanadate inhibits adipose Glc-6-Pase
Glc-6-Pase (D-G-6-P, phosphohydrolase) has been more characterized in gluconeogenic tissues, such as liver. Figure 9AGo (first lane) shows that the homogenate of rat adipocytes contains a fair amount of Glc-6-Pase activity. Vanadate inhibited adipose Glc-6-Pase in a concentration-dependent manner. Half-maximal inhibition was obtained at 7 ± 0.7 µM (Fig. 9BGo). Insulin, added in the assay (Fig. 9AGo) or to the intact adipocytes 30 min before cell homogenization (not shown), had no detectable effect in lowering or elevating adipose Glc-6-Pase activity.



View larger version (30K):
[in this window]
[in a new window]
  		Figure 9. Inhibition of adipose Glc-6-Pase by vanadate. (A) The assay mixture (0.1 ml, in 50 mM Tris-cacodylate, pH 6.5) consisted of 20 mM G-6-P, adipose homogenate (10 µg protein), and was performed for 10 min at 25 C in the absence and the presence of the indicated concentrations of vanadate. The amount of Pi released was determined by a spectroscopic assay (experimental section). Each point is a triplicate determination ± SE. Results are expressed as Pi released/min·mg protein (B) concentration-dependent inhibition of adipose Glc-6-Pase by vanadate. Results are expressed as percent of maximal activity (obtained in the absence of vanadate).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Vanadium therapy very effectively restored the low levels of glycogen synthase and glycogen reserves to normal values; and sensitized glycogen synthase activity, to insulin in tissues of type I diabetic rodents (45, 46, 47, 48, 49). Using the rat-adipose cell in vitro, we have attempted to gain some insight into its mechanism of action. We report here on intense activation of glycogenesis by vanadate (12- to 15-fold of basal level in different experiments), an effect we attribute to the activation of glycogen synthase (Figs. 1Go and 2Go). Of all the metabolic actions of vanadate in rat adipocytes, activation of glycogenesis is the most sensitive. Its half-maximal concentration is evident at 20 ± 4 µM (Fig. 1Go), as opposed to half-maximal concentrations in the range of 100–150 µM for facilitating lipogenesis, activating glucose oxidation, enhancing hexose uptake, and inhibiting isoproterenol-mediated lipolysis (1, 2, 3, 50).

We found that vanadate-evoked glycogenesis shares common denominators with insulin in being equally suppressed by conditions elevating cAMP levels, in its dependence on intact PP1 activity, and (most important) in its requirement for active nonarrested PI3-kinase ( Figs. 4–6GoGoGo). The site of action for the glycogenetic effect of vanadate would then seem to be an early one, preceding the activation of PI3-kinase. Unlike insulin, quercetin (which arrests InsRTK-transphosphorylation) (19), did not block vanadate-induced glycogenesis (Fig. 2Go). This suggested to us, with a high degree of confidence, that this particular metabolic action is independent of receptor activation. On the other hand, staurosporine, which inhibits CytPTK, blocked vanadate-induced (but not insulin-induced) glycogenesis, with a half-maximal effect at 0.35 ± 0.03 µM (Fig. 4Go). Staurosporine also blocked the activation of lipogenesis and glucose oxidation by vanadate (21). Thus, like lipogenesis and glucose oxidation, glycogenesis is mediated through the vanadate-activated staurosporine-sensitive, water-soluble CytPTK; and it differs from the metalooxide effect in activating hexose uptake and in inhibiting lipolysis. These two latter effects are not inhibited at this low staurosporine concentration and are mediated through another nonreceptor vanadate-activated protein tyrosine kinase of a membranous origin (51). It should be noted, however, that quercetin does not arrest insulin-evoked autophosphorylation or the antilipolytic action of insulin in rat adipocytes (19). Although not very likely, it has to be examined, in future studies, whether receptor-autophosphorylation per se is a sufficient condition for activating glycogenesis by insulin.

We now addressed the question as to why activation of glycogen synthase should be 5–7 times more sensitive to vanadate than other rapid metabolic actions triggered by vanadate. Therefore, we searched for an additional event, dependent on vanadate, that particularly encourages glycogen synthesis. We found that vanadate causes a large increase in the cellular content of glycogen-synthase protein (Fig. 8Go, A and B). This effect is exclusively dependent on the presence of glucose in the medium (44). We therefore further postulated that a glucose metabolite is involved in this glycogen synthase-enhancing effect.

G-6-P is an allosteric activator of glycogen synthase. It induces a conformational change that favors dephosphorylation and activation of glycogen synthase by PP1G (43). Liver contains Glc-6-Pase activity that is inhibited by micromolar concentrations of vanadate (52). It was believed that this enzymatic activity is absent in nongluconeogenic tissues such as fat. Recently, however, we found that lipolytic hormones activate glycogenolysis in rat adipocytes (53). Because Glc-6-Pase is the terminal enzyme of glycogenolysis (54), we looked for and found Glc-6-P dephosphorylating activity in this tissue too (Fig. 9AGo). Vanadate inhibited adipose Glc-6-Pase activity, with an IC50 value of 7.0 ± 0.7 µM (Fig. 9BGo). We also examined directly G-6-P levels and found them to be markedly elevated by vanadate (Fig. 7Go). Elevation exceeded that manifested by insulin. We interpreted this as a dual effect of vanadate. Like insulin, it facilitates glucose uptake; but unlike the hormone, it also inhibits adipose Glc-6-Pase, with the net result of higher level of G-6-P and a more efficient activation of glycogen synthase.

In summary, we conclude that vanadate resembles insulin, in activating glycogenesis in rat adipocytes through the very same pathway. This includes activation of PI3-kinase, ser/thr dephosphorylation of glycogen synthase by PP1G, and suppression under experimental conditions elevating cAMP levels. Two fundamental differences, however, do exist: 1) the early (prerequisite) tyrosyl phosphorylating event proceeds through the activation of the cytosolic nonreceptor protein tyrosine kinase, in an InsRTK-independent manner; and 2) vanadate facilitates additional effects by elevating G-6-P levels. These findings can explain the documented proficiency of vanadium therapy to restore to normal levels the low activity glycogen synthase and glycogen reserves in type I diabetic rodents (45, 46, 47, 48, 49). It was also reported that vanadium therapy increased insulin-stimulated glycogen deposition in human type II diabetic patients (55). Preliminary studies in this direction seem to indicate that the cytosol, nonreceptor tyrosine phosphorylating capacity of adipose and muscle tissue of streptozocin-treated rats remained nonmodified after induction of diabetes (manuscript in preparation).


   Acknowledgments
 
We thank Elana Friedman for typing this manuscript and Dr. Sandra Moshonov for editing it.


   Footnotes
 
1 This study was supported, in part, by grants from the Minerva Foundation (Germany), the Rowland Shaefer Contribution to Diabetes Research, the Levine Fund, Teva Pharmaceutical Fund, the Israel Ministry of Health, and the Israel Academy of Sciences and Humanities. Back

2 Incumbent of the C. H. Hollenberg Chair in Metabolic and Diabetes Research, established by the Friends and Associates of Dr. C. H. Hollenberg of Toronto, Canada. Back

Received August 17, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Shechter Y, Karlish SJD 1980 Insulin-like stimulation of glucose oxidation in rat adipocytes by vanadyl(IV) ions. Nature 284:556–558[CrossRef][Medline]
  2. Dubyak GR, Kleinzeller A 1980 The insulin-mimetic effects of vanadate as a (Na+-K+)ATPase inhibitor. J Biol Chem 255:5306–5312[Free Full Text]
  3. Degani H, Gochin M, Karlish SJD, Shechter Y 1981 Electron paramagnetic resonance studies and insulin-like effects of vanadium in rat adipocytes. Biochemistry 20:5795–5799[CrossRef][Medline]
  4. Shechter Y, Ron A 1986 Effect of depletion of phosphate and bicarbonate ions on insulin action in rat adipocytes. J Biol Chem 261:14951–14954[Abstract/Free Full Text]
  5. Meyerovitch J, Farfel Z, Sack Y, Shechter Y 1987 Oral administration of vandaate normalized blood glucose levels in streptozotocin-treated rats: characterization and mode of action. J Biol Chem 262:6658–6662[Abstract/Free Full Text]
  6. Heyliger CE, Tahiliani AG, McNeill JH 1985 Effect of vanadate on elevated blood glucose and depressed cardiac performance of diabetic rats. Science 227:1474–1476[Abstract/Free Full Text]
  7. Miralpeix M, Decaux J, Kahn A, Bartrons R 1991 Vanadate induction of L-type pyruvate kinase mRNA in adult rat hepatocytes in primary culture. Diabetes 40:462–464[Abstract]
  8. Shechter Y 1990 Insulin-mimetic effects of vanadate. Possible implications for future treatment of diabetes. Diabetes 39:1–5[Abstract]
  9. Shechter Y, Li J, Meyerovitch J, Gefel D, Bruck R, Elberg G, Miller DS, Shisheva A 1995 Insulin-like actions of vanadate are mediated in an insulin receptor-independent manner via non-receptor protein tyrosine kinases and protein phosphotyrosine phosphatases. Mol Cell Biochem 153:39–47[CrossRef][Medline]
  10. Brichard MS, Henquin JC 1995 The role of vanadium in the management of diabetes. Trends Pharmacol Sci 16:265–270[CrossRef][Medline]
  11. Sekar N, Li J, Shechter Y 1996 Vanadium salts as insulin substitutes: mechanisms of action, a scientific and therapeutic tool in diabetes mellitus research. Crit Rev Biochem Mol Biol 31:339–359[Medline]
  12. Tamura S, Brown TA, Whipple JH, Yamaguchi YF, Dubler RE, Chen K, 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[Abstract/Free Full Text]
  13. Swarup G, Cohen S, Garbers DL 1982 Inhibition of membrane phospho-tyrosyl-protein phosphatase activity by vanadate. Biochem Biophys Res Commun 107:1104–1109[CrossRef][Medline]
  14. Mooney RA, Bordwell KL, Luhowskyj S, Casnellie JE 1989 The insulin-like effect of vanadate on lipolysis in rat adipocytes is not accompanied by an insulin-like effect on tyrosine phosphorylation. Endocrinology 124:422–429[Abstract]
  15. Strout HV, Vicario PD, Superstein R, Slater EE 1989 The insulin-mimetic effect of vanadate is not correlated with insulin receptor tyrosine kinase activity nor phosphorylation in mouse diaphragm in vivo. Endocrinology 124:1918–1924[Abstract]
  16. Fantus GI, Kadota S, Deragon G, Foster B, Posner BI 1987 Pervanadate [peroxide(s) of vanadate] mimics insulin action in rat adipocytes via activation of the insulin receptor tyrosine kinase. Biochemistry 28:8864–8871
  17. D’Onotrio F, Le MQ, Chiassion JL, Srivastava AK 1994 Activation of mitogen activated protein (MAP) kinases by vanadate is independent of insulin receptor autophosphorylation. FEBS Lett 340:269–275[CrossRef][Medline]
  18. Wilden PA, Broadway D 1995 Combination of insulinomimetic agents H2O2 and vanadate enhances insulin receptor phosphorylation of IRS-1 leading to IRS-1 association with the phosphatidylinositol 3-kinase. J Cell Biochem 58:279–291[CrossRef][Medline]
  19. Shisheva A, Shechter Y 1992 Quercetin selectively inhibits insulin receptor function in vitro and the bioresponse of insulin and insulinomimetic agents in rat adipocytes. Biochemistry 31:8059–8063[CrossRef][Medline]
  20. Shisheva A, Shechter Y 1992 A cytosolic tyrosine kinase in rat dipocytes. FEBS Lett 300:93–96[CrossRef][Medline]
  21. Shisheva A, Shechter Y 1993 Role of cytosolic tyrosine kinase in mediating insulin-like actions of vanadate in rat adipocytes. J Biol Chem 268:6463–6469[Abstract/Free Full Text]
  22. Elberg G, Li J, Shechter Y 1994 Vanadium activates or inhibits receptor and non-receptor protein tyrosine kinases in cell-free experiment, depending on its oxidation state. Possible role of endogenous vanadium in controlling cellular protein tyrosine kinase activity. J Biol Chem 269:9521–9527[Abstract/Free Full Text]
  23. Rodbell M 1964 Metabolism of isolated fat cells. I. Effect of hormones on glucose metabolism and lipolysis. J Biol Chem 239:375–380[Free Full Text]
  24. Thomas JA, Schendler KK, Larner J 1968 A rapid filter paper assay for UDP glucose-glycogen glucosyltransferase, including an improbed biosynthesis of UDP-14C-glucose. Anal Biochem 72:486–499
  25. Lawrence JC, Guinovart JJ, Larner J 1977 Activation of rat adipocyte glycogen synthase by insulin. J Biol Chem 252:444–450[Abstract/Free Full Text]
  26. Hess SL, Suchin CR, Saltiel AR 1991 The specific protein phosphatase inhibitor okadaic acid differentially modulate insulin action. J Cell Biochem 45:374–380[CrossRef][Medline]
  27. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
  28. Ames BN 1966 Assay of inorganic phosphate and phosphatases. Methods Enzymol 8:115–118
  29. Bickerstaff GF, Burchell B 1980 Studies on the purification of glucose 6-phosphatase from rabbit liver microsomal fraction. Biochem Soc Trans 8:389–390[Medline]
  30. Lang G, Michal G 1974 D-glucose-6-phosphate and D-fructose-6-phosphate. In: Bergmeyer HU (ed) Methods of Enzymatic analysis. Academic Press, New York, vol 3:1238–1242
  31. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 46:359–384
  32. Okada T, Kawano Y, Sakakibara T, Hazeki O, Ui M 1994 Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. Studies with selective inhibitor wortmannin. J Biol Chem 269:3568–3573[Abstract/Free Full Text]
  33. Kelly KI, Ruderman NB 1993 Insulin-stimulated phosphatidylinositol 3-kinase association with a 185-kDa tyrosine phosphorylated protein (IRS-1) and localization in a low density membrane vesicle. J Biol Chem 268:4391–4398[Abstract/Free Full Text]
  34. Heller-Harrison RA, Morin MM, Guilherme A, Czech MP 1996 Insulin mediated targeting of phosphatidylinositol 3-kinase to GLUT4-containing vesicles. J Biol Chem 271:10200–10204[Abstract/Free Full Text]
  35. Cross DAE, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA 1995 Inhibition of glycogen synthase kinase-3 by insulin mediated protein kinase B. Nature 378:785–789[CrossRef][Medline]
  36. Burgering BMT, Coffer PJ 1995 Protein kinase B (c-Akt) in phospha-tidylinositol 3-OH kinase signal transduction. Nature 376:599–602[CrossRef][Medline]
  37. Li J, Elberg G, Sekar N, He Z, Shechter Y 1997 Antilipolytic actions of vanadate and insulin in rat adipocytes mediated by distinctly different mechanisms. Endocrinology 138:2274–2279[Abstract/Free Full Text]
  38. Dent P, Lavoinne A, Nakienly S, Caudwell FB, Watt P, Cohen P 1990 The molecular mechanism by which insulin stimulates glycogen synthesis in mammalian skeletal muscle. Nature 348:302–308[CrossRef][Medline]
  39. Cohen P, Holmes CFB, Tsukitani Y 1990 Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem Sci 15:98–102[CrossRef][Medline]
  40. Haystead TAY, Sim ATR, Carling D, Honnor RC, Tsukitani Y, Cohen P, Hardie DG 1989 Effects of tumor promotor okadaic acid on intracellular protein phosphorylation and metabolism. Nature 337:78–81[CrossRef][Medline]
  41. Lawrence JC, James C, Hiken JF 1986 Control of glycogen synthase by insulin and isoproterenol in rat adipocytes: changes in the distribution of phosphate in the synthase subunit in response to insulin and beta-adrenergic receptor activation. J Biol Chem 261:669–677[Abstract/Free Full Text]
  42. Roach PJ 1986 Liver glycogen synthase. In: Boyer PD, Krebs EG (eds) Enzymes, ed 3. Academic Press, New York, vol 17:499–539
  43. Villar-Palasi C 1991 Substrate specific activation by glucose 6-phosphate on the dephosphorylation of muscle glycogen synthase. Biochim Biophys Acta 1244:203–208
  44. Lima BP, Bao S, Garvey WT 1994 Biological actions of insulin are differentially regulated by glucose and insulin in primary cultured adipocytes. Chronic ability to increase glycogen synthase activity. Diabetes 43:53–62[Abstract]
  45. Blondel O, Simon J, Chevalier B, Portha B 1990 Impaired insulin action but normal insulin receptor activity in diabetic rat liver: effect of vanadate. Am J Physiol 258:E459–E467
  46. Gil J, Miralpeix M, Carreras J, Bartrons R 1988 Insulin-like effects of vanadate on glucokinase activity and fructose 2,6 biphosphate levels in the liver of diabetic rats. J Biol Chem 263:1868–1871[Abstract/Free Full Text]
  47. Bollen M, Miralpeix M, Ventura F, Toth B, Bartrons R, Stalmans W 1990 Oral administration of vanadate to streptozotocin-diabetic rats restores the glucose-induced activation of liver glycogen synthase. Biochem J 267:269–271[Medline]
  48. Venkatesan N, Avidan A, Davidson MB 1991 Antidiabetic action of vanadyl in rats independent of in vivo insulin-receptor kinase activity. Diabetes 40:492–498[Abstract]
  49. Rossetti L, Laughlin MR 1989 Correction of chronic hyperglycemia with vanadate, but not with phlorizin, normalized, in vivo glycogen repletion and in vitro glycogen synthase activity in diabetic skeletal muscle. J Clin Invest 84:892–899
  50. Green A 1986 The insulin-like effect of sodium vanadate on adipocyte glucose transport is mediated at a post-insulin-receptor level. Biochem J 238:663–669[Medline]
  51. Elberg G, He Z, Li J, Sekar N, Shechter Y 1997 Vanadate activates membranous nonreceptor protein tyrosine kinase in rat adipocytes. Diabetes 46:1684–1690[Abstract]
  52. Singh J, Nordlie CR, Jorgenson RA 1981 Vanadate, a potent inhibitor of multifunctional glucose-6-phosphatase. Biochim Biophys Acta 678:477–482[Medline]
  53. Sekar N, Li J, He Z, Shechter Y 1997 A novel assay for evaluating glycogenolysis in rat adipocytes and the inability of insulin to antagonize glycogenolysis in this cell type. Biochemistry 36:16206–16211[CrossRef][Medline]
  54. McLean B, Greenbaum AL 1968 In: Dickens F, Whelan WJ, Randle PJ (eds) Handbook of Biochemistry. Academic Press, London and New York, pp 397–425
  55. Cohen N, Halberstam M, Shlimovich P, Chang CJ, Shamoon H, Rossetti L 1995 Oral vanadyl sulfate improves hepatic and peripheral insulin sensitivity in patients with non-insulin-dependent diabetes mellitus. J Clin Invest 95:2501–2509




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sekar, N.
Right arrow Articles by Shechter, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sekar, N.
Right arrow Articles by Shechter, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals