Molecular and Cellular Biochemistry 236: 123–131, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.
123
Oral treatment with vanadium of Zucker fatty rats activates muscle glycogen synthesis and insulin-stimulated protein phosphatase-1 activity Sabina Semiz and John H. McNeill Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada Received 6 March 2001; accepted 1 March 2002
Abstract Since the glucose-lowering effects of vanadium could be related to increased muscle glycogen synthesis, we examined the in vivo effects of vanadium and insulin treatment on glycogen synthase (GS) activation in Zucker fatty rats. The GS fractional activity (GSFA), protein phosphatase-1 (PP1), and glycogen synthase kinase-3 (GSK-3) activity were determined in fatty and lean rats following treatment with bis(maltolato)oxovanadium(IV) (BMOV) for 3 weeks (0.2 mmol/kg/day) administered in drinking water. Skeletal muscle was freeze-clamped before or following an insulin injection (5 U/kg i.v.). In both lean and fatty rats, muscle GSFA was significantly increased at 15 min following insulin stimulation. Vanadium treatment resulted in decreased insulin levels and improved insulin sensitivity in the fatty rats. Interestingly, this treatment stimulated muscle GSFA by 2-fold (p < 0.05) and increased insulin-stimulated PP1 activity by 77% (p < 0.05) in the fatty rats as compared to untreated rats. Insulin resistance, vanadium and insulin in vivo treatment did not affect muscle GSK-3β activity in either fatty or lean rats. Therefore, an impaired insulin sensitivity in the Zucker fatty rats was improved following vanadium treatment, resulting in an enhanced muscle glucose metabolism through increased GS and insulin-stimulated PP1 activity. (Mol Cell Biochem 236: 123–131, 2002) Key words: insulin resistance, skeletal muscle, serine/threonine kinases and phosphatases
Introduction Current evidence indicates that impaired insulin-stimulated glycogen synthesis is a generalized abnormality resulting in impaired insulin-regulated glucose metabolism in skeletal muscle of diabetic patients. Several conflicting abnormalities in the activation of glycogen synthase (GS) in muscle from patient with type 2 diabetes have been reported [1–3]. Defects in activating GS have been observed in insulin resistance in type 2 diabetes, implicating defects in the signaling pathway upstream to the enzyme [4, 5]. Glycogen synthase is regulated by both covalent phosphorylation/dephosphorylation and allosteric modifications [6]. Insulin acts by reducing and increasing the activities of a specific kinase and phosphatase, respectively [7], whereas
glucose-6-phosphate (G6P) allosterically activates a less active phosphorylated form of GS (G6P-dependent form). Phosphorylation (inactivation) of GS is catalyzed in vivo by a minimum of six different protein kinases acting on nine serine residues [8, 9], while the reverse reaction (activation) is catalyzed by a glycogen-associated form of protein phosphatase-1 (PP-1G) [7]. Most of the phosphate released from GS in response to insulin stimulation is removed from two serine residues, sites 3a and 3b [10]. The protein kinase which is most active in phosphorylating these serine residues in GS is glycogen synthase kinase-3 (GSK-3) [11, 12], resulting in inhibition of GS. GSK-3 is constitutively active in unstimulated cells and results from several groups suggest that GSK-3 activity is repressed upon cell stimulation with insulin [13–16]. However,
Address for offprints: J.H. McNeill, Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall, Vancouver, B.C. V6T 1Z3, Canada (E-mail:
[email protected])
124 many of the studies on GSK-3 were performed in vitro and relatively little is known about the physiological regulation of this enzyme in the intact animal in vivo, where numerous influences that are not readily reproducible in vitro may be brought to bear on the insulin receptor. The first study on GSK-3 activity following insulin stimulation in vivo was performed recently, and it has been shown that insulin decreases GSK-3 activity by approximately 40% in Wistar rat skeletal muscle [17]. However, insulin may also activate GS via PP1 [18], which is capable of dephosphorylating all serine residues in GS [19]. In addition to GS, PP1 is also able to inhibit glycogen phosphorylase and phosphorylase kinase activities by dephosphorylation of these enzymes and hence coordinate glycogen synthesis and degradation [18]. PP1 activity is stimulated by insulin in skeletal muscle in vitro [7, 20] and the activation can occur over short periods of time [21]. Recently, the activation of PP1 by insulin in vitro in rat adipocytes, another insulin-dependent tissue, was also reported [22]. In addition, Ortmeyer found an increase in liver PP1 activity following insulin administration in vivo [23]. However, the role of PP1 in the regulation of glycogen synthesis by insulin has not been yet investigated in vivo in skeletal muscle in experimental type 2 diabetes. Therefore, in order to investigate the regulation of muscle glycogen synthesis in an animal model associated with insulin resistance, we measured the activities of GS, GSK-3, and PP1 in skeletal muscle of the Zucker fatty rat, a model which resembles the prediabetic state of type 2 diabetes since animals are hyperinsulinemic, normoglycemic, and insulin resistant, before and after treatment with vanadium and/or insulin. Our interest in treatment of diabetes with vanadium started more than 15 years ago, following the finding in our laboratory that vanadium exhibits insulin-like effects after an oral administration in diabetic animals [24]. This was confirmed by findings from other laboratories that oral vanadium treatment normalizes blood glucose level in both type 1 and type 2 diabetic rats [25–28]. Since the improvement in insulin-stimulated glucose uptake appears to be mediated primarily through increased non-oxidative glucose disposal [29] and increased glycogen synthesis [30], it was of interest to us to study if in vivo vanadium treatment affected the activity of GS, GSK-3, and PP1 in skeletal muscle of Zucker rats.
Materials and methods Materials Materials were obtained from the following sources: regular beef/pork insulin (Iletin) from Eli Lilly; bis(maltolato)oxovanadium(IV) (BMOV) from Dr. Chris Orvig, Department of Chem-
istry, University of British Columbia, Vancouver, B.C., Canada; phospho-GSK-3 substrate peptide from Department of Biochemistry and Microbiology, University of Victoria, Victoria, B.C., Canada; glycogen synthase peptide 2 (Ala 21) from Upstate Biotechnology (Lake Placid, NY, USA); anti-GSK-3β antibodies from Transduction Laboratories (Lexington, KY, USA); glucose-6-phosphate (G6P), phenylmethylsulfonyl fluoride (PMSF), benzamidine, leupeptin, pepstatin A, aprotinin, antipain, and trypsin inhibitor from Sigma-Aldrich Canada (Oakville, Ontario, Canada); okadaic acid from Calbiochem (La Jolla, CA, USA); uridine 5′-diphosphate (UDP)-[U-14C] glucose and [γ-32P]ATP from NEN Life Science Products (Boston, MA, USA).
Treatment and maintenance of the animals Rats were housed on an alternating 12-h light and dark cycle and were allowed free access to food (Lab Diet 5001), water and treatment solution. Treated rats received bis(maltolato)oxovanadium(IV) (BMOV) in drinking water. Body weight and food and fluid intake were measured daily in all animals. The dose of vanadium was calculated on the basis of concentration of the compound in treatment solution, amount of consumed fluid, and body weight. Blood was collected from all rats once every week from the tail vein following a 5-h fasting period and centrifuged (Beckman Allegra 21R) at 20000 × g for 20 min at 4°C. The plasma was stored at –70°C until analyzed. Plasma glucose was determined by the Beckman Glucose Analyzer 2 and glucose oxidase kits (Beckman Instruments, Galway, Ireland), while plasma insulin was determined using a radioimmunoassay kit (Linco Research, St. Charles, MO, USA).
Experimental design For the time-course study, 13 male Zucker fatty and 13 lean rats were obtained at 17–18 weeks of age from the breeding colony in the Department of Physiology at the University of British Columbia, Vancouver, B.C., Canada. Animals were fasted overnight (16 h) and then anesthetized with 100 mg/kg pentobarbital (i.p.). Skeletal muscle from hind legs was removed and freeze-clamped before and 2, 5, and 15 min after insulin injection (5 IU/kg), powdered under liquid nitrogen, and stored at –70°C for further analysis. In this study the timedependent activation of both GS and GSK-3 by insulin was determined. In the vanadium treatment study, 50 male Zucker rats were obtained at 11–12 weeks of age from the same breeding colony. Twenty-five fatty (fa/fa) and 25 lean (Fa/–) littermates were randomly divided into treated and untreated groups: Lean (L, n = 12), Lean Treated (LT, n = 13), Fatty (F, n = 12), and Fatty
125 Treated (FT, n = 13). Treated animals received BMOV for 3 weeks at a final dose of 0.2 mmol/kg/day, administered in drinking water. At the end of treatment period, animals were fasted overnight (16 h) and then anesthetized with 100 mg/kg pentobarbital (i.p.). The skeletal muscle was removed and freezeclamped before and 5 or 15 min following insulin injection (5 U/kg), powdered under liquid nitrogen, and stored at – 70°C prior to further preparation of the muscle extracts.
Calculation of insulin sensitivity The index of insulin sensitivity was calculated using the formula (100/square root of [fasting plasma glucose × fasting plasma insulin] × [mean glucose × mean insulin during OGTT]) recently developed by Matsuda and DeFronzo [31].
dures were carried out according to the recommendation of the manufacturer. PP1 activity was measured in the presence of 3 nM okadaic acid, since this compound at low concentration is a potent and selective inhibitor of PP2A [38].
Statistical analysis All results are expressed as means ± S.E.M. The data were analyzed using Repeated Measures ANOVA followed by the Newman–Keuls test, with p < 0.05 considered as the level of statistical significance.
Results Time-course study
Determination of GSK-3β activity Skeletal muscle extracts were prepared using a previously published procedure [32, 33] as modified in our laboratory [34]. The muscle homogenate was used for GSK-3β immunoprecipitation assay [34]. The kinase assay was performed on pelleted beads or Mono S fractions (10 µL) using a method [35], which was modified as described previously [34]. The GSK-3 substrate phosphopeptide is a specific substrate for GSK-3. GS peptide-2 (Ala 21) in which the priming serine at the site 4 had been replaced by alanine was used as a negative control and the activity obtained using this control peptide was subtracted for the calculation of the final GSK-3 activity.
Determination of glycogen synthase activity Glycogen synthase activity was measured by the method of Ortmeyer et al. [36] with some modifications [34]. Enzyme activity is expressed as nmol of glucose incorporated into glycogen per min per mg of protein. GS activity was determined using uridine diphosphate(UDP)-[U14C] glucose, in the absence (for active GS form) and presence (for total GS activity) of 10 mM glucose-6-phosphate (G6P). Glycogen synthase fractional activity (GSFA) is expressed as the percentage of active to total GS activity.
Determination of protein phosphatase-1 activity Skeletal muscle extracts were prepared using the method of Foulkes and Jefferson [37], modified as described previously [34]. Protein phosphatase-1 activity was determined using serine/threonine phosphatase assay kits (Upstate Biotechnology, Lake Placid, NY, USA). Assay conditions and proce-
General characteristics of the Zucker rats at 17–18 weeks of age are shown in Table 1. Results of this time-course study demonstrated that lean rats responded to insulin with a timedependent significant decrease (p < 0.05) in glucose levels at 5 and 15 min. In the insulin resistant Zucker fatty rats, glucose levels did not decline even 15 min after insulin injection. Determination of the GS fractional activity (data not shown) in skeletal muscle of lean rats demonstrated an increase (30%) in enzyme activity at 15 min after insulin stimulation (p < 0.05). Surprisingly, in skeletal muscle from hyperinsulinemic fatty rats GS activity was also increased by 25% following insulin injection (p < 0.05). There was no significant difference in GS activity between Zucker fatty and lean rats. Furthermore, there was no difference in basal GSK-3β activity between fatty and lean rats and insulin injection did not also affect the activity of this enzyme (data not shown). Table 1. Characteristics of Zucker fatty rats at 17–18 weeks of age before and following i.v. injection of 5 IU insulin Group Number Weight (g)
Plasma glucose (mM)
Plasma insulin (ng/ml)
L0 L2 L5 L15 F0 F2 F5 F15
8.84 8.46 7.02 3.70 11.3 11.6 10.8 13.6
0.78 1774 664 256 9.84 1681 1480 693
3 4 3 3 3 4 3 3
364 379 378 375 556 560 535 531
± ± ± ± ± ± ± ±
18* 9* 21* 18* 22 13 4 9
± ± ± ± ± ± ± ±
0.23 0.27 0.19* 0.37*# 0.83 0.81 1.4 4.9
± ± ± ± ± ± ± ±
0.11* 422 162 87 1.27*# 731 859 261
Data are means ± S.E.M. L = lean, F = fatty. Numbers in group indicate time after insulin injection. Weight: *All lean groups weighed significantly less than all fatty groups (p < 0.05). Plasma glucose: *vs. lean (L0,L2) rats and #vs. all other groups (p < 0.05). Plasma insulin: *vs. all insulin-injected groups and #vs. lean (L0) rats (t-test, p = 0.00038).
126 Table 2. Characteristics of Zucker fatty rats at 15–16 weeks of age following vanadium treatment and insulin injection Group Number Weight (g)
Plasma glucose (mM)
Plasma insulin (ng/ml)
L0 L5 L15 F0 F5 F15 LT 0 LT 5 LT 1 5 FT0 FT5 FT15
8.7 6.0 3.8 10.5 11.2 8.0 7.4 6.1 3.5 10.6 9.1 8.7
0.91 1474 429 11.1 3418 861 0.85 2873 656 8.33 3394 1086
4 3 5 4 4 4 4 6 3 4 5 4
369 364 362 552 560 545 338 359 345 525 522 496
± ± ± ± ± ± ± ± ± ± ± ±
11* 5* 15* 15 11 11 11* 9* 10* 18 11 25
± ± ± ± ± ± ± ± ± ± ± ±
0.65 0.92 0.23* 0.43 1.53 0.96 0.24 0.31 0.13# 0.34 0.55 1.81
± ± ± ± ± ± ± ± ± ± ± ±
0.07*# 100 80 0.94*& 958 50** 0.07*# 1383 286** 0.85* 617 137**
Data are means ± S.E.M. L = lean, F = fatty, T = treated with vanadium. Numbers in group indicate time after insulin injection. Weight: *All lean groups weighed significantly less than all fatty groups (p < 0.05). Plasma glucose: *vs. lean (L0) and all fatty rats, and #vs. vanadium-treated lean (LT0) and all fatty animals (p < 0.05). Plasma insulin: *vs. all insulin-injected groups, #vs. fatty (F0, FT0), **vs. 5-min insulin-injected groups, and &vs. all other groups (p < 0.05).
Study in the vanadium-treated Zucker fatty rats In this study Zucker fatty rats at 15–16 weeks of age were used and their characteristics are summarized in Table 2. There was no effect of BMOV treatment on body weight or on daily food and fluid intake (food intake (g): lean, 22 ± 2; fatty, 26 ± 1; lean-treated, 19 ± 1; fatty-treated, 31 ± 2; fluid intake (mL): lean, 42 ± 2; fatty, 47 ± 4; lean-treated, 32 ± 3; fatty-treated, 48 ± 4). Both the fatty and fatty-treated groups weighed significantly more than the lean rats. Vanadium treatment resulted in a decreased insulin level in fatty animals. Accordingly, impaired insulin sensitivity in the Zucker fatty rats was also improved following BMOV treatment (Fig. 1A). There was no difference in basal glucose levels between Zucker lean and fatty rats (Fig. 1B). Following an insulin injection there was a significant decline in glucose levels in both vanadium-treated and untreated lean rats. In the insulinresistant Zucker fatty rats plasma glucose level was not decreased after an insulin injection. Furthermore, vanadium treatment per se or in combination with insulin did not produce an effect on glucose levels in fatty rats. As shown in Fig. 2A, our results demonstrate that there was no difference in muscle GSFA between Zucker lean and fatty rats. This is in accordance with our findings from the Zucker time-course study and also from the studies using Wistar STZ-diabetic rats [34], where no difference in basal GSFA was observed between diabetic and control animals. Following an insulin injection there was an increase in GS activity in lean (2-fold) and surprisingly, in fatty rats (2-fold) as well. This is in agreement with our results from the Zucker
Fig. 1. Study in the Zucker rats using skeletal muscle extracts: (A) An index of insulin sensitivity in Zucker Lean (L), Fatty (F), Lean vanadium-treated (LT), and Fatty vanadium-treated (FT) rats was calculated using the methodology as described by [31], following an oral glucose tolerance test (OGTT). Data are expressed as mean ± S.E.M. for 12–13 individual animals. *p < 0.05 vs. all other groups at termination; #p < 0.05 vs. Lean groups initial; &p < 0.05 Fatty groups initial (t-test, p = 0.0026). (B) Plasma glucose in Zucker Lean (L), Fatty (F), Lean vanadium-treated (LT), and Fatty vanadium-treated (FT) rats before (0), 5, and 15 min following insulin injection (5 U/kg). Data are expressed as mean ± S.E.M. for 3–5 individual animals and each sample was done as a triplicate. *p < 0.05 vs. basal glucose level in lean (L0) and all fatty rats; **p < 0.05 vs. lean vanadium-treated (LT0) and all fatty animals.
time-course study, where an increase in GSFA was also observed after the injection of insulin in both lean and fatty rats. Therefore, it seems that in the Zucker fatty rats, which are insulin-resistant, GS activation by insulin was not defective. An important observation from this study is that vanadium treatment per se resulted in significantly increased GSFA by 2-fold in fatty rats compared to untreated rats, which was not further increased after combined treatment of BMOV
127 skeletal muscle of Zucker lean and fatty rats has shown similar results as observed in our studies in which skeletal muscle from Wistar STZ-diabetic rats were used [34]. There was no significant difference in basal GSK-3β activity between lean and fatty rats. Furthermore, GSK-3β activity was not significantly changed following insulin and/or vanadium treatment in both lean and fatty rats. Results of determination of PP1 activity in the Zucker rat muscle are presented in Fig. 2C. Similar to our findings from studies involving Wistar STZ-diabetic rats [34], there was a significant increase in basal PP1 activity in fatty rats compared to leans. PP1 activity was increased by 76% in the lean rats 15 min following an insulin injection (p < 0.05). In the fatty rats insulin administration resulted in significantly decreased PP1 activity, which was brought to near-lean values at 15 min following insulin injection (p < 0.05). The vanadium treatment per se, like the insulin treatment, also resulted in significantly decreased PP1 activity in the fatty rats compared to untreated rats. However, combined treatment of fatty rats with BMOV and insulin resulted in an increase in PP1 activity, by 77%, compared to basal activity in fatty vanadium-treated animals (p < 0.05).
Discussion
Fig. 2. Study on the regulation of glycogen synthesis in skeletal muscle from the Zucker rats: (A) Glycogen synthase fractional activity, (B) GSK-3β activity, and (C) PP1 activity in skeletal muscle from Zucker Lean (L), Fatty (F), Lean vanadium-treated (LT), Fatty vanadium-treated (FT) rats before (0), 5, and/or 15 min following insulin injection (5 U/kg). Data are expressed as mean ± S.E.M. for 3–5 individual animals and each sample was done as a triplicate. GSFA: *p < 0.05 vs. Lean (L0, LT0) and Fatty (F0) animals; #p < 0.05 vs. Lean insulin-injected (L15) rats. PP1: *p < 0.05 vs. lean (L0) rats; **p < 0.05 vs. fatty (F0) animals, #p < 0.05 vs. vanadium-treated lean (LT0) rats; ##p < 0.05 vs. vanadium-treated fatty (FT0) rats.
and insulin. Surprisingly, results of this study showed significantly decreased insulin-stimulated GS activity in lean vanadium-treated rats compared to untreated controls. As shown in Fig. 2B, determination of GSK-3β activity in
In an attempt to evaluate the enzymes involved in insulin signaling which regulate glycogen synthesis in an important insulin-sensitive tissue, we measured the activities of glycogen synthase, glycogen synthase kinase-3, and protein phosphatase-1 in rat skeletal muscle before and after vanadium treatment. In the present study, we used the Zucker fatty rat, which closely resemble the prediabetic stage of type 2 diabetes mellitus, since they are hyperinsulinemic, normoglycemic or mildly hyperglycemic, and insulin-resistant. Previous studies have demonstrated that the predominant defect in diabetes associated with insulin resistance lies in non-oxidative glucose metabolism [1, 2, 39]. Our results show that GS fractional activity was significantly increased following an insulin injection to lean rats. However, in Zucker fatty rats insulin-stimulated GS activity was not defective, suggesting that the available insulin probably overcame any insulin resistance of this enzyme in muscle. Furthermore, our results demonstrated that in fatty rat muscle basal GSFA was not reduced. Similar results were observed in our previous studies with Wistar STZ-diabetic rats. These animals are insulin resistant but no difference in GSFA between diabetic and control rats was demonstrated [34]. Our results are also in accordance with a recent Zucker rat study, in which a lack of significant change in GS activity between skeletal muscle of fatty and lean rats was observed [40]. A possible explanation for a lack of decrease in GS activity in
128 diabetic rats may be that hyperinsulinemia noted in the patients with mild type 2 diabetes compensates for the GS defect [41]. Importantly, our results demonstrated an activation of PP1 by insulin in Zucker lean rats. Similarly, an increase in PP1 activity following in vivo insulin stimulation was demonstrated previously in our studies using Wistar STZ-diabetic rats, representing the first report concerning insulin-stimulated PP1 activity in skeletal muscle in vivo [34]. Furthermore, previous studies demonstrated an insulin-induced increase in PP1 activity in the liver, another insulin-sensitive tissue [23]. Our data indicated increased PP1 activity in muscle from fatty compared to lean rats. This is in accordance with our data concerning PP1 activity in muscle from Wistar STZ-diabetic rat study [34] and a previous report where PP1 activity was found to be increased in the liver of Zucker fatty rats [42]. The increased PP1 activity in both prediabetic and diabetic rats may be a possible explanation for near-normal GS activity in these animals. Since PP1 activates GS, highly activated PP1 may keep GS in an active state even under diabetic conditions. Interestingly, following an insulin injection in Zucker fatty rats, muscle PP1 activity was decreased to near-control values. Previous studies reported a glycogen-induced decrease in PP1 activity [43, 44] and it was postulated that this may be concordant with a feedback inhibition of a stimulatory effect of insulin on GS activity by an elevated muscle glycogen concentration in skeletal muscle of healthy men [45]. Since muscle glycogen content was not measured in our studies, we do not know if there was a difference in the glycogen amount between Zucker fatty and lean rat muscle and how this might affect the GS activation. Opposing results on PP1 activity after insulin injection in lean and fatty rats can also, at least partially, be explained by the fact that in our studies we have measured total PP1 activity. It is possible that targeting of the PP1G form only would provide a more precise determination of the enzyme activity involved in the regulation of glycogen metabolism. Interestingly, a recent study using PP1G/RGL knockout mice demonstrated that although the regulatory-targeting subunit (RGL or GM) has an important role in glycogen synthesis, this enzyme is not essential for insulin activation of GS [46]. Therefore, it would be of interest to study the role of another specific regulatory subunit of PP1 in the activation of GS in skeletal muscle. The role of GSK-3, as an upstream regulator of glycogen synthase activity in insulin signaling and its inhibition by insulin, has been mainly described in vitro [14, 15, 47], where effects of insulin counteracting hormones, such as adrenaline and glucagon, do not occur. In vivo studies reported only about a 40% inhibition of GSK-3 activity by insulin [15, 17]. However, results of our in vivo study demonstrated no difference in muscle GSK-3 activity between Zucker fatty and lean rats. This is in accordance with results from our previous in vivo studies using skeletal muscle from short- and
long-term (insulin-resistant) STZ-diabetic rats, which have demonstrated no significant difference in basal GSK-3β activity between STZ-diabetic and control rats [34]. In support of our studies, a recent in vivo study reported that GSK-3 activity did not change significantly in muscle and liver of diabetic mice, while its activity was significantly increased in the epididymal fat tissue [48], suggesting that the regulation of GSK-3 differs between tissues. Furthermore, our results did not demonstrate an effect of insulin stimulation on muscle GSK-3 activity in either Zucker fatty rats or STZ-diabetic, as well as their corresponding controls. These findings are in agreement with a recent study [49] in which the failure of insulin administration to decrease GSK-3β activity in skeletal muscle of nondiabetic and type 2 diabetic subjects was also demonstrated. Furthermore, it was demonstrated recently that, even in in vitro studies, insulin treatment does not result in more than a 20–25% inhibition of GSK-3 [50]. In addition, Sung et al. demonstrated in hepatic cells that insulin failed to significantly decrease GSK-3β activity at several time-points (2.5– 30 min) following insulin administration [51]. Interestingly, previous studies have shown that sites 3a and 3b in glycogen synthase, phosphorylated by GSK-3, can also be directly phosphorylated by some other currently unidentified protein kinases [52, 53]. Furthermore, Skurat et al. demonstrated recently that GSK-3 action is not essential for GS activation, suggesting involvement of GSK-3-independent mechanisms in the regulation of GS activity [54]. The role of protein kinase C, which can phosphorylate at least two distinct sites within GS, has been suggested in the regulation of GS activity in human and rat skeletal muscle [55, 56]. Therefore, the activation of muscle GS in response to insulin appears complex and probably proceeds through two mechanisms which must operate in concert, namely stimulation of protein phosphatase coupled with an inhibition of currently unidentified specific protein kinase. Vanadium treatment per se or in combination with insulin did not produce an effect on glucose levels in the Zucker fatty rats. This agrees with a previous report where improvement in glucose uptake after vanadyl sulfate treatment was found only in type 2 diabetics, but not in the insulin-resistant obese nondiabetic controls [57]. However, we found that BMOV treatment decreased plasma insulin levels in fatty rats, associated with insulin resistance, suggesting that response to insulin was improved in these animals treated with a more potent organic vanadium compound, as compared to the study [57] in which vanadyl sulfate was used. In support, results from our Zucker rat study demonstrated that an oral BMOV treatment improved sensitivity to insulin in both fatty and lean rats. In our studies the insulin sensitivity in Zucker rats was estimated by calculation of an index of insulin sensitivity recently developed by Matsuda and DeFronzo [31]. This novel estimate provides a reasonable approximation of whole-body insulin sensitivity from oral glucose tolerance testing. In ad-
129 dition, it is highly correlated with the rate of insulin-stimulated glucose disposal during the euglycemic insulin clamp [31], which is considered to be one of the most precise methods available for assessing in vivo insulin action. Similarly to our results, vanadium-enhanced insulin sensitivity has been reported previously [58, 59] and the effect of vanadium was sustained for two weeks after discontinuing treatment [30]. Furthermore, a modest inverse relationship was reported between plasma vanadium and insulin levels, suggesting complementary interactions between these two agents [60]. The data support the postulate that vanadium affects carbohydrate metabolism by its insulin-enhancing properties [60]. Our study represents the first report that vanadium treatment mimics insulin action on PP1 activity in skeletal muscle of Zucker fatty and lean rats. Vanadium treatment normalized basal PP1 activity and restored insulin-stimulated PP1 activity in skeletal muscle of fatty rats. Interestingly, restored PP1 activity was accompanied by GS activation in skeletal muscle of the fatty rats, whose insulin-sensitivity was also improved after vanadium treatment. Regarding post-vanadium GSK-3 activity, similarly to our findings on this enzyme activity after an insulin injection, we have demonstrated that vanadium treatment did not affect GSK-3 activity in skeletal muscle from Zucker fatty and lean rats. In conclusion, the present results demonstrate that vanadium treatment improved an impaired insulin sensitivity in the Zucker fatty rats, resulting in an enhanced muscle glucose metabolism through increased glycogen synthase and insulin-stimulated PP1 activity. Our results indicate that either GSK-3 does not play a role in regulating GS by insulin or it plays a role only in combination with other components to control GS and simply looking at GSK-3 may not show any dramatic effect. However, if coupled to the action of PP1 this relatively small change in the activity level of GSK-3 may be sufficient to control glycogen synthesis in vivo.
Acknowledgements The authors wish to express their thanks to Erika Vera, Violet Yuen, Mary Battell, and Becky Dinesen for technical assistance. This study was funded by a Natural Sciences and Engineering Research Council /TPP grant and also by the Medical Research Council of Canada. SS was the recipient of an Rx and D-Health Research Foundation/MRC studentship.
References 1. Vestergaard H, Lund S, Larsen FS, Bjerrum OJ, Pedersen O: Glycogen synthase and phosphofructokinase protein and mRNA levels in skeletal muscle from insulin-resistant patients with non-insulindependent diabetes mellitus. J Clin Invest 91: 2342–2350, 1993
2 . Kim YB, Nikoulina SE, Ciaraldi TP, Henry RR, Kahn BB: Normal insulin-dependent activation of Akt/protein kinase B, with diminished activation of phosphoinositide 3-kinase, in muscle in type 2 diabetes. J Clin Invest 104: 733–741, 1999 3 . Damsbo P, Vaag A, Hother-Nielsen O, Beck-Nielsen H: Reduced glycogen synthase activity in skeletal muscle from obese patients with and without type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia 34: 239–245, 1991 4 . Kahn CR: Banting Lecture. Insulin action, diabetogenes, and the cause of type II diabetes. Diabetes 43: 1066–1084, 1994 5 . Kida Y, Esposito-Del Puente A, Bogardus C, Mott DM: Insulin resistance is associated with reduced fasting and insulin-stimulated glycogen synthase phosphatase activity in human skeletal muscle. J Clin Invest 85: 476–481, 1990 6 . Cohen P: In: P.D. Boyer, E.G. Krebs (eds). The Enzymes, Vol. 17. Academic Press, Orlando, FL, 1986, pp 461–479 7 . Dent P, Lavoinne A, Nakielny S, Caudwell FB, Watt P, Cohen P: The molecular mechanism by which insulin stimulates glycogen synthesis in mammalian skeletal muscle. Nature 348: 302–308, 1990 8 . Poulter L, Ang SG, Gibson BW, Williams DH, Holmes CF, Caudwell FB, Pitcher J, Cohen P: Analysis of the in vivo phosphorylation state of rabbit skeletal muscle glycogen synthase by fast-atom-bombardment mass spectrometry. Eur J Biochem 175: 497–510, 1988 9 . Nakielny S, Campbell DG, Cohen P: The molecular mechanism by which adrenalin inhibits glycogen synthesis. Eur J Biochem 199: 713–722, 1991 10. Parker PJ, Caudwell FB, Cohen P: Glycogen synthase from rabbit skeletal muscle; effect of insulin on the state of phosphorylation of the seven phosphoserine residues in vivo. Eur J Biochem 130: 227–234, 1983 11. Embi N, Rylatt DB, Cohen P: Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. Eur J Biochem 107: 519– 527, 1980 12. Wang Y, Roach PJ: Inactivation of rabbit muscle glycogen synthase by glycogen synthase kinase-3. Dominant role of the phosphorylation of Ser-640 (site-3a). J Biol Chem 268: 23876–23880, 1993 13. Ramakrishna S, Benjamin WB: Insulin action rapidly decreases multifunctional protein kinase activity in rat adipose tissue. J Biol Chem 263: 12677–12681, 1988 14. Welsh GI, Proud CG: Glycogen synthase kinase-3 is rapidly inactivated in response to insulin and phosphorylates eukaryotic initiation factor eIF-2B. Biochem J 294: 625–629, 1993 15. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA: Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378: 785–789, 1995 16. Murai H, Okazaki M, Kikuchi A: Tyrosine dephosphorylation of glycogen synthase kinase-3 is involved in its extracellular signaldependent inactivation. FEBS Lett 392: 153–160, 1996 17. Cross DA, Watt PW, Shaw M, van der Kaay J, Downes CP, Holder JC, Cohen P: Insulin activates protein kinase B, inhibits glycogen synthase kinase-3 and activates glycogen synthase by rapamycin-insensitive pathways in skeletal muscle and adipose tissue. FEBS Lett 406: 211–215, 1997 18. Alemany S, Pelech S, Brierley CH, Cohen P: The protein phosphatases involved in cellular regulation. Evidence that dephosphorylation of glycogen phosphorylase and glycogen synthase in the glycogen and microsomal fractions of rat liver are catalysed by the same enzyme: protein phosphatase-1. Eur J Biochem 156: 101–110, 1986
130 19. Lawrence JC Jr, Skurat AV, Roach PJ, Azpiazu I, Manchester J: Glycogen synthase: Activation by insulin and effect of transgenic overexpression in skeletal muscle. Biochem Soc Trans 25: 14– 19, 1997 20. Ragolia L, Begum N: Protein phosphatase-1 and insulin action. Mol Cell Biochem 182: 49–58, 1998 21. Kida Y, Raz I, Maeda R, Nyomba BL, Stone K, Bogardus C, Sommercorn J, Mott DM: Defective insulin response of phosphorylase phosphatase in insulin-resistant humans. J Clin Invest 89: 610–617, 1992 22. Srinivasan M, Patel MS: Glycogen synthase activation in the epididymal adipose tissue from chronic hyperinsulinemic/obese rats. J Nutr Biochem 9: 81–87, 1998 23. Ortmeyer HK: Insulin increases liver protein phosphatase-1 and protein phosphatase-2C activities in lean, young adult rhesus monkeys. Horm Metab Res 30: 705–710, 1998 24. Heyliger CE, Tahiliani AG, McNeill JH: Effect of vanadate on elevated blood glucose and depressed cardiac performance of diabetic rats. Science 227: 1474–1477, 1985 25. Meyerovitch J, Farfel Z, Sack J, Shechter Y: Oral administration of vanadate normalizes blood glucose levels in streptozotocintreated rats. Characterization and mode of action. J Biol Chem 262: 6658–6662, 1987 26. Brichard SM, Pottier AM, Henquin JC: Long term improvement of glucose homeostasis by vanadate in obese hyperinsulinemic fa/ fa rats. Endocrinology 125: 2510–2516, 1989 27. Meyerovitch J, Rothenberg P, Shechter Y, Bonner-Weir S, Kahn CR: Vanadate normalizes hyperglycemia in two mouse models of non-insulin-dependent diabetes mellitus. J Clin Invest 87: 1286– 1294, 1991 28. Yuen VG, Vera E, Battell ML, Li WM, McNeill JH: Acute and chronic oral administration of bis(maltolato)oxovanadium(IV) in Zucker diabetic fatty (ZDF) rats. Diabetes Res Clin Pract 43: 9– 19, 1999 29. Goldfine AB, Simonson DC, Folli F, Patti ME, Kahn CR: Metabolic effects of sodium metavanadate in humans with insulin-dependent and noninsulin-dependent diabetes mellitus in vivo and in vitro studies. J Clin Endocrinol Metab 80: 3311–3320, 1995 30. Cohen N, Halberstam M, Shlimovich P, Chang CJ, Shamoon H, Rossetti L: Oral vanadyl sulfate improves hepatic and peripheral insulin sensitivity in patients with non-insulin-dependent diabetes mellitus. J Clin Invest 95: 2501–2509, 1995 31. Matsuda M, DeFronzo RA: Insulin sensitivity indices obtained from oral glucose tolerance testing: Comparison with the euglycemic insulin clamp. Diabetes Care 22: 1462–1470, 1999 32. Gregory JS, Boulton TG, Sang BC, Cobb MH: An insulin-stimulated ribosomal protein S6 kinase from rabbit liver. J Biol Chem 264: 18397–18401, 1989 33. Pelech SL, Krebs EG: Mitogen-activated S6 kinase is stimulated via protein kinase C-dependent and independent pathways in Swiss 3T3 cells. J Biol Chem 262: 11598–11606, 1987 34. Semiz S, Orvig C, McNeill JH: Effects of diabetes, vanadium, and insulin on glycogen synthase activation in Wistar rats. Mol Cell Biochem 231: 23–25, 2002 35. Bhanot S, Salh BS, Verma S, McNeill JH, Pelech SL: In vivo regulation of protein-serine kinases by insulin in skeletal muscle of fructose-hypertensive rats. Am J Physiol 277: E299–307, 1999 36. Ortmeyer HK, Bodkin NL, Hansen BC: Insulin-mediated glycogen synthase activity in muscle of spontaneously insulin-resistant and diabetic rhesus monkeys. Am J Physiol 265: R552–558, 1993 37. Foulkes JG, Jefferson LS: Protein phosphatase-1 and -2A activities in heart, liver, and skeletal muscle extracts from control and diabetic rats. Diabetes 33: 576–579, 1984
38. Cohen P, Klumpp S, Schelling DL: An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues. FEBS Lett 250: 596–600, 1989 39. Beck-Nielsen H: General characteristics of the insulin resistance syndrome: Prevalence and heritability. European Group for the study of Insulin Resistance (EGIR). Drugs 58: 7–10; discussion 75–82, 1999 40. Bruce CR, Lee JS, Hawley JA: Postexercise muscle glycogen resynthesis in obese insulin-resistant Zucker rats. J Appl Physiol 91: 1512–1519, 2001 41. Beck-Nielsen H, Vaag A, Damsbo P, Handberg A, Nielsen OH, Henriksen JE, Thye-Ronn P: Insulin resistance in skeletal muscles in patients with NIDDM. Diabetes Care 15: 418–429, 1992 42. Lavoie L, Bollen M, Stalmans W, van de Werve G: Increased synthase phosphatase activity is responsible for the super-activation of glycogen synthase in hepatocytes from fasted obese Zucker rats. Endocrinology 129: 2674–2678, 1991 43. Villar-Palasi C: Oligo- and polysaccharide inhibition of muscle transferase D phosphatase. Ann NY Acad Sci 166: 719–730, 1969 44. Mellgren RL, Coulson M: Coordinated feedback regulation of muscle glycogen metabolism: Inhibition of purified phosphorylase phosphatase by glycogen. Biochem Biophys Res Commun 114: 148–154, 1983 45. Laurent D, Hundal RS, Dresner A, Price TB, Vogel SM, Petersen KF, Shulman GI: Mechanism of muscle glycogen autoregulation in humans. Am J Physiol Endocrinol Metab 278: E663–668, 2000 46. Suzuki Y, Lanner C, Kim JH, Vilardo PG, Zhang H, Yang J, Cooper LD, Steele M, Kennedy A, Bock CB, Scrimgeour A, Lawrence JC Jr, DePaoli-Roach AA: Insulin control of glycogen metabolism in knockout mice lacking the muscle-specific protein phosphatase PP1G/RGL. Mol Cell Biol 21: 2683–2694, 2001 47. Borthwick AC, Wells AM, Rochford JJ, Hurel SJ, Turnbull DM, Yeaman SJ: Inhibition of glycogen synthase kinase-3 by insulin in cultured human skeletal muscle myoblasts. Biochem Biophys Res Commun 210: 738–745, 1995 48. Eldar-Finkelman H, Schreyer SA, Shinohara MM, LeBoeuf RC, Krebs EG: Increased glycogen synthase kinase-3 activity in diabetes- and obesity-prone C57BL/6J mice. Diabetes 48: 1662–1666, 1999 49. Nikoulina SE, Ciaraldi TP, Mudaliar S, Mohideen P, Carter L, Henry RR: Potential role of glycogen synthase kinase-3 in skeletal muscle insulin resistance of type 2 diabetes. Diabetes 49: 263–271, 2000 50. Singh LP, Crook ED: The effects of glucose and the hexosamine biosynthesis pathway on glycogen synthase kinase-3 and other protein kinases that regulate glycogen synthase activity. J Investig Med 48: 251–258, 2000 51. Sung CK, Choi WS, Scalia P: Insulin-stimulated glycogen synthesis in cultured hepatoma cells: Differential effects of inhibitors of insulin signaling molecules. J Recept Signal Transduct Res 18: 243– 263, 1998 52. Skurat AV, Roach PJ: Phosphorylation of sites 3a and 3b (Ser640 and Ser644) in the control of rabbit muscle glycogen synthase. J Biol Chem 270: 12491–12497, 1995 53. Skurat AV, Roach PJ: Multiple mechanisms for the phosphorylation of C-terminal regulatory sites in rabbit muscle glycogen synthase expressed in COS cells. Biochem J 313: 45–50, 1996 54. Skurat AV, Dietrich AD, Roach PJ: Glycogen synthase sensitivity to insulin and glucose-6-phosphate is mediated by both NH2- and COOH-terminal phosphorylation sites. Diabetes 49: 1096–1100, 2000 55. Pugazhenthi S, Khandelwal RL: Regulation of glycogen synthase activation in isolated hepatocytes. Mol Cell Biochem 149–150: 95–101, 1995
131 56. Chalfant CE, Ciaraldi TP, Watson JE, Nikoulina S, Henry RR, Cooper DR: Protein kinase Ctheta expression is increased upon differentiation of human skeletal muscle cells: Dysregulation in type 2 diabetic patients and a possible role for protein kinase Ctheta in insulin-stimulated glycogen synthase activity. Endocrinology 141: 2773–2778, 2000 57. Halberstam M, Cohen N, Shlimovich P, Rossetti L, Shamoon H: Oral vanadyl sulfate improves insulin sensitivity in NIDDM but not in obese nondiabetic subjects. Diabetes 45: 659–666, 1996 58. Cam MC, Cros GH, Serrano JJ, Lazaro R, McNeill JH: In vivo
antidiabetic actions of naglivan, an organic vanadyl compound in streptozotocin-induced diabetes. Diabetes Res Clin Pract 20: 111–121, 1993 59. Goldfine AB, Patti ME, Zuberi L, Goldstein BJ, LeBlanc R, Landaker EJ, Jiang ZY, Willsky GR, Kahn CR: Metabolic effects of vanadyl sulfate in humans with non-insulin-dependent diabetes mellitus: in vivo and in vitro studies. Metabolism 49: 400–410, 2000 60. Cam MC, Brownsey RW, McNeill JH: Mechanisms of vanadium action: insulin-mimetic or insulin-enhancing agent? Can J Physiol Pharmacol 78: 829–847, 2000
132
