In vivo effects of insulin and vanadium treatment on glycogen synthase (GS), glycogen synthase kinase-3 (GSK-3) and pro- tein phosphatase-1 (PP1) activity ...
Molecular and Cellular Biochemistry 231: 23–35, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.
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Effects of diabetes, vanadium, and insulin on glycogen synthase activation in Wistar rats Sabina Semiz,1 Chris Orvig2 and John H. McNeill1 1
Faculty of Pharmaceutical Sciences; 2Department of Chemistry, The University of British Columbia, Vancouver, BC, Canada
Received 27 April 2001; accepted 19 October 2001
Abstract In vivo effects of insulin and vanadium treatment on glycogen synthase (GS), glycogen synthase kinase-3 (GSK-3) and protein phosphatase-1 (PP1) activity were determined in Wistar rats with streptozotocin (STZ)-induced diabetes. The skeletal muscle was freeze-clamped before or following an insulin injection (5 U/kg i.v.). Diabetes, vanadium, and insulin in vivo treatment did not affect muscle GSK-3β activity as compared to controls. Following insulin stimulation in 4-week STZ-diabetic rats muscle GS fractional activity (GSFA) was increased 3 fold (p < 0.05), while in 7-week diabetic rats it remained unchanged, suggesting development of insulin resistance in longer term diabetes. Muscle PP1 activity was increased in diabetic rats and returned to normal after vanadium treatment, while muscle GSFA remained unchanged. Therefore, it is possible that PP1 is involved in the regulation of some other cellular events of vanadium (other than regulation of glycogen synthesis). The lack of effect of vanadium treatment in stimulating glycogen synthesis in skeletal muscle suggests the involvement of other metabolic pathways in the observed glucoregulatory effect of vanadium. (Mol Cell Biochem 231: 23–35, 2002) Key words: streptozotocin-induced diabetes, insulin resistance, glycogen synthesis, serine/threonine kinases and phosphatases
Introduction Glycogen synthesis, notably in skeletal muscle, contributes importantly to the regulation of blood glucose by insulin. Insulin promotes the transport of glucose by inducing the translocation of the GLUT4 transporter to the cellular plasma membrane [1] and stimulates glucose deposition into glycogen (non-oxidative glucose metabolism) through the activation of glycogen synthase (GS). Previous studies have shown both decreased [2] and normal [3] total GS activity in muscle from Type 1 diabetic patients, while fractional GS activity (± G6P activity ratio) was decreased in these patients [3]. Most of the phosphate released from GS in response to insulin stimulation is removed from two serine residues, sites 3a and 3b [4, 5]. The protein kinase, which is most active in phosphorylating these serine residues in GS, is glycogen synthase kinase-3 (GSK-3) [6]. Molecular cloning revealed the existence in mammals of two highly related proteins termed
GSK-3α (the first one purified) and GSK-3β [7]. GSK-3β has been found to be predominant isoform in the human myoblasts [8]. Since GSK-3 phosphorylates and inactivates GS, its down-regulation by insulin will lead to GS activation and increased glycogen synthesis. Results from several groups suggest that GSK-3 activity is repressed upon cell stimulation with insulin [9–11]. However, 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, where numerous influences that are not readily reproducible in vitro may be important. The first in vivo study on GSK3 activity after insulin stimulation was performed recently, and it showed that insulin decreased GSK-3 activity by approximately 40% in Wistar rat skeletal muscle [12]. It is important to keep in mind that inhibition of GSK-3 alone cannot account for the regulation of GS activity, since this enzyme does not phosphorylate GS at site 2, which is
Address for offprints: J.H. McNeill, Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall, Vancouver, BC, Canada, V6T 1Z3
24 dephosphorylated in response to insulin [13]. It also remains entirely possible that insulin activates the glycogen-associated form of protein phosphatase-1 (PP1G), which is capable of dephosphorylating all sites on GS [13]. Several in vitro studies using skeletal muscle demonstrated insulin-induced activation of PP1 [14, 15]. However, the role of PP1 in the regulation of glycogen synthesis by insulin has not yet been investigated in vivo in skeletal muscle of animal models of diabetes. Our laboratory was the first to report that after oral administration to diabetic rats vanadium exhibited insulin-like effects [16]. This was confirmed by results from other laboratories and it was found that in both animal models of Type 1 and Type 2 diabetes oral vanadium treatment normalized blood glucose levels [17–19]. An improvement in insulinstimulated glucose uptake, mediated primarily through increased non-oxidative glucose disposal [20] and increased glycogen synthesis [21, 22] has been reported. In the present study we have measured the activities of GS, GSK-3, and PP1 in skeletal muscle of STZ-diabetic Wistar rats, both before and following treatment with insulin and vanadium in vivo. Since the results from our preliminary studies performed on STZ-diabetic rats demonstrated development of insulin resistance in long-term diabetes, we also studied how the duration of diabetes affected the muscle activities of these three enzymes. In order to improve the effectiveness of vanadium in treatment of diabetes, we have developed a new organic vanadium compound, bis(ethylmaltolato)oxovanadium(IV)(BEOV) and compared its glucoregulatory effect to the previously developed analogous compound, bis(maltolato)oxovanadium(IV)(BMOV).
Materials and methods Materials Materials were obtained from the following sources: regular pork insulin (Iletin) from Eli Lilly; BMOV from Dr. Chris Orvig, Department of Chemistry, University of British Columbia, Vancouver, BC, Canada; BEOV from Kinetek Pharmaceuticals Inc., Vancouver, BC, Canada; protein G-Sepharose from Amersham Pharmacia Biotech; phospho-GSK-3 substrate peptide from Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada; glycogen synthase peptide 2 (Ala 21) from Upstate Biotechnology Inc. (Lake Placid, NY, USA); anti-GSK-3β antibodies from Transduction Laboratories (Lexington, KY, USA); goat anti-mouse IgG conjugated with horse radish peroxidase from Bio-Rad; dithiothreitol, β-mercaptoethanol, phenylmethylsulfonil fluoride (PMSF), benzamidine, leupeptin, pepstatin A, aprotinin, antipain, and trypsin inhibitor from Sigma-Aldrich Canada
Ltd. (Oakville, ON, Canada); uridine 5′-diphosphate (UDP)[U-14C] glucose and [γ-32P]ATP from NEN Life Science Products, Inc. (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 either BMOV or BEOV in drinking water. Body weight and food and fluid intake were measured daily in all animals. The blood from all rats was collected once every week from the tail vein following a period of 5-h fasting 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 using a Beckman Glucose Analyzer 2 and glucose oxidase kits (Beckman Instruments Inc., Galway, Ireland), while plasma insulin was determined by radioimmunoassay kit (Linco Research Inc., St. Charles, MO, USA).
Experimental design Two time-course studies on GSK-3β activity were performed using Wistar rats with either 4- or 7-week streptozotocin (STZ)-induced diabetes. For the first time-course study 48 Wistar rats (Animal Care Centre, South Campus, the University of British Columbia, Vancouver, BC, Canada), weighing 190–220 g (7–8 weeks of age) were used. In 24 rats diabetes was induced by i.v. injection of STZ (60 mg/kg), while 24 age-matched control rats were injected with saline. Diabetes was confirmed by glucose analysis (plasma glucose level > 13 mM) in the blood by Beckman Glucose Analyzer 2 at 72 h after STZ injection. Four weeks after induction of diabetes, skeletal muscle was collected before and 1, 2, 5, and 15 min after insulin injection (5 U/kg i.v.). For the second similar time-course study on GS and GSK-3β activity skeletal muscle was removed from Wistar control and STZ-diabetic rats 7 weeks after induction of diabetes. The muscle was collected before and 2, 5, and 15 min following an insulin injection (5 U/kg i.v.). For the short-term diabetes study, 88 male Wistar rats, weighing 190–220 g, were used. The rats were divided into two groups. One group received a single tail vein injection of STZ (60 mg/kg) and served as the diabetic group. The other group was injected with saline and served as the age-matched controls. The control and diabetic groups were then further subdivided into untreated control (C, n = 22), control-treated (CT, n = 22), untreated diabetic (D, n = 22), and diabetictreated (DT, n = 22) groups. Treatment was started 7 days
25 post-STZ injection. Animals were treated with BMOV for 3 weeks (final dose of 0.3–0.4 mmol/kg/day administered in drinking water). The dose of vanadium was calculated on the basis of concentration of the compound in the treatment solution, amount of consumed fluid, and body weight. Skeletal muscle was collected before and 5 or 15 min after insulin injection (5 U/kg i.v.). For the initial long-term (7-week) diabetes study, 142 male Wistar rats of similar body weight were used and 78 were made diabetic by STZ injection (60 mg/kg i.v.). The control and diabetic groups were then further subdivided into C (n = 32), CT (n = 32), D (n = 38), and DT (n = 40) groups. Animals were treated with BEOV for 6 weeks (final dose of 0.3– 0.4 mmol/kg/day administered in drinking water). Similar to all previous studies, at the end of treatment rats were fasted overnight (16 h) and then anesthetized by pentobarbital (100 mg/kg i.p.). Skeletal muscle was quickly removed from hind legs (fast- and slow-twitch muscle) before and 2, 5 or 15 min after insulin injection (5 U/kg), freeze-clamped, powdered under liquid nitrogen, and stored at –70°C before further processing. For the second long-term (9-week) diabetes study (Mono S chromatography study), 60 male Wistar rats (Charles River, Montreal, Quebec, Canada) of similar body weight were used. In 30 rats diabetes was induced by STZ injection (60 mg/kg i.v.), while 30 age-matched control rats were injected with saline. The rats were then randomly divided into four groups of 15 animals: C, CT, D, and DT. A week after STZ injection, the treated groups received BMOV in the drinking water at a final dose of 0.3–0.4 mmol/kg/day for 8 weeks. At the end of treatment the rats were fasted overnight, anesthetized, and skeletal muscle was removed quickly before and 15 min after insulin injection (5 U/kg i.v.). Muscle extracts were prepared as described previously [23]. Protein concentration in all muscle homogenates was determined by the Bradford method using reagents purchased from BioRad (Hercules, CA, USA).
Mono S chromatography Chromatographic fractionation of crude muscle extracts was performed by a fast protein liquid chromatography (FPLC), following a modified method described previously [24, 25]. Cation exchange chromatography was carried out at 4°C on a Mono S HR 5/5 system using a Pharmacia LKB Biotechnology FPLC. The muscle extracts containing 10 mg of protein were applied at a flow rate of 1 ml/min to Mono S column equilibrated with a buffer A (pH 7.0) containing 20 mM HEPES and 1 mM dithiothreitol (DTT). After a 10mL wash, elution was performed with an 18-mL gradient from 0–800 mM NaCl in buffer A. Fractions (0.25 mL) were collected and stored at –70°C for subsequent assays.
Electrophoresis and immunoblotting For further identification of GSK-3β in Mono S column profiles, column fraction immunoblotting was performed as described previously [23]. Aliquots of Mono S column fractions were subjected to SDS polyacrylamide gel electrophoresis (with 10% slab gels) as described by Laemmli [26]. Gel electrophoresis was performed at 10 mA/gel overnight and the proteins were then transferred onto a nitrocellulose membrane at 300 mA for 3 h. Following the transfer completion, the membrane was stained with 1% Ponceau S solution in 5% acetic acid, in order to demonstrate the abundance of the proteins on the membrane. After the Ponceau S staining, a 2-h blocking with 5% skim milk in Tris-buffered saline (100 mM Tris, pH 7.50, 1.25 M NaCl) was performed. The membrane was then incubated with the primary antibodies (mouse IgG monoclonal anti GSK-3β) overnight at 4°C, washed, and incubated with the secondary antibodies (goat anti-mouse IgG conjugated with horse radish peroxidase) for 1 h at room temperature. Enhanced chemiluminescence (ECL) was used as the detection procedure following manufacturer’s instructions. Determination of GSK-3β activity The procedure described previously [27, 28] was modified and used to prepare skeletal muscle extracts. The frozen muscle powder was homogenized in ice-cold MOPS buffer (25 mM, pH 7.2) containing 2 mM EDTA, 5 mM EGTA, 75 mM β-glycerophosphate, 5 µM β-methyl-aspartic acid, 1 mM dithiothreitol (DTT) and various protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 3 mM benzamidine, 10 µM leupeptin, 5 µM pepstatin A, 200 µg/mL trypsin inhibitor, and 10 µg/mL aprotinin). The homogenate was centrifuged at 100,000 × g for 60 min and the clear supernatant was used for GSK-3β immunoprecipitation assay. For this assay, the muscle extracts (1 mg protein) was incubated in total of 350 µL 6% NETF buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM EDTA, 50 mM NaF, and 6% Nonidet P40) with 35 µL of protein G-Sepharose beads, and 5 µL of the anti GSK-3β antibodies. The highly specific monoclonal anti-GSK-3β antibodies were used in this assay. After a 2.5 h incubation period at 4°C, the beads were pelleted by centrifugation at 14,000 × g for 2 min at 4°C, washed twice with 6% NETF, twice with 100 mM Tris (pH 7.4), and then once with 10 mM Tris. The kinase assay was performed using pelleted beads or Mono S fractions (10 µL) and adding 5 µL phospho-GSK-3 substrate peptide (125 µM) or the GSK-3 (Ala-21) control peptide, 30 µL buffer (8 mM MOPS pH 7.4, 0.2 mM EDTA, and 10 mM Mg-acetate), and 50 µM [γ-32P]ATP (specific activity ~ 2000 cpm/pmol). The kinase reaction was allowed to
26 proceed for 30 min at 30°C after which it was stopped by spotting the reaction mixture onto P81 Whatman paper squares. The papers were washed 10 times with 1% orthophosphoric acid, once with acetone, and then counted for radioactivity. The activity obtained using glycogen synthase peptide-2 (Ala 21) as a negative control was subtracted for the calculation of the final GSK-3 activity. Determination of glycogen synthase activity The frozen muscle powder was homogenized in a buffer (4.2% wt/vol) (pH 7.5, 4°C) containing 50 mM tricine, 10 mM EDTA, 100 mM NaF, 0.5 mM DTT, 0.1% β-mercaptoethanol, and 0.5 mM PMSF. The homogenate was centrifuged at 8800 × g for 10 min at 4°C and the resultant supernatant was used for GS assay. GS activity was determined using a modified method described previously [29]. Briefly, the total and glucose-6-phosphate independent (G6Pi) activity of GS were measured after mixing 40 µL of muscle sample with 60 µL of reaction mixture (pH 7.4) containing 50 mM Tris, 20 mM EDTA, 87.5 mM KF, 0.2 mM uridine-5′-diphosphoglucose, 5000 counts per min per nM UDPG-14C, 1% glycogen, and either 15 mM G6P (for total GS activity) or no G6P (for G6Pi GS activity). The enzymatic reaction was allowed to proceed for 6 min at 30°C, after which it was terminated by spotting sample reaction mixture onto paper squares. The papers were washed 3 times with 66% ethanol, and then counted for radioactivity. GS fractional activity (GSFA) is expressed as the percentage of G6Pi (active) GS to total GS activity. Determination of protein phosphatase-1 activity Skeletal muscle extracts were prepared as described previously [30]. Briefly, the frozen muscle powder was homogenized in 3 volumes of buffer (pH 8.3, 4°C) containing 20 mM Tris-HCl, 250 mM sucrose, 4 mM EDTA, 30 mM β-mercaptoethanol, and various protease inhibitors (100 µM PMSF, 1 mM benzamidine, and 10 µg/mL of aprotinin, leupeptin, antipain, trypsin inhibitor and pepstatin-A). The homogenates were centrifuged at 12000 × g for 15 min. The clear supernatants were then filtered on Sephadex G-50 fine columns (Amersham Pharmacia Biotech, Uppsala, Sweden) with eluting buffer containing 50 mM Tris-HCl, 0.1 mM EDTA, 0.1% Brij 35, 30 mM β–mercaptoethanol, and the same protease inhibitors as mentioned above. A fraction containing protein (3.5 mL) was collected and used to measure PP1 activity by serine/threonine phosphatase assay kits (Upstate Biotechnology, Lake Placid, NY, USA). Assay conditions and procedures were followed according to the manufacturer’s recommendation. 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 [31].
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 studies General characteristics of the 4-week and 7-week STZ-diabetic rats are shown in Tables 1 and 3, respectively. Results of the 4-week time-course study demonstrated that STZ-diabetic Wistar rats still responded to insulin stimulation and a time-dependent significant decrease in plasma glucose levels at 5 and 15 min following an insulin injection was observed (diabetic-initial: 26.3 ± 3.1; diabetic-final at 5 min: 11.2 ± 1.9; diabetic-final at 15 min: 5.3 ± 1.6 mmol/L, p < 0.05; control-initial: 5.60 ± 0.67; control-final at 15 min: 2.51 ± 0.28 mmol/L, p < 0.05). In 7-week diabetic rats the lack of effect of insulin administration on plasma glucose levels was accompanied by an impaired insulin-stimulated muscle GS activity (Figs 1A and 1B). In control rat muscle GSFA was significantly increased at 2, 5 and 15 min after insulin stimulation, with the most prominent increase (2-fold) at 15 min (Fig. 1B). The activities of both total and active form of GS were decreased in these 7-week STZ-diabetic rats as compared to controls (Total GS: diabetic-initial (D0), 3.7 ± 0.2; D2, 3.7 ± 0.2; D5, 2.7 ± 0.1; D15, 3.4 ± 0.1 nmol/min/mg; control-initial (C0), 7.6 ± 0.6; C2, 5.1 ± 0.5; C5, 6.7 ± 0.2; C15, 6.9 ± 0.6 nmol/min/mg). However, no significant difference in GSFA was observed between STZ-7 weeks-diabetic and control rats. Similarly, results of both time-course studies showed that there was also no significant difference in basal GSK-3β activity between STZ-diabetic and control rats (data not shown). Furthermore, results of these two time-course studies revealed no difference in GSK-3β activity following insulin stimulation at 2, 5, and 15 min (5 U/kg i.v.) in both control and STZ-diabetic rats and the duration of diabetes did not cause any difference in the GSK-3β response to insulin stimulation. Study in the short-term STZ-diabetic Wistar rats Table 2 summarizes the general characteristics of STZ-4 weeks-diabetic Wistar rats prior to and after vanadium and/ or insulin treatment. The BMOV treatment did not affect body weight gain in either control or diabetic animals. Both diabetic and diabetic-treated groups weighed significantly less than the control rats. Diabetic untreated rats exhibited hyperphagia and polydipsia, and BMOV treatment normalized food
27 Table 1. Characteristics of STZ-4 weeks-diabetic Wistar rats (time-course study) Group
Number
Weight (g)
Plasma glucose (mmol/L)
Plasma insulin (ng/mL)
C0 C1 C2 C5 C15 D0 D1 D2 D5 D15
5 5 4 5 4 5 5 5 4 3
328 ± 6* 347 ± 13* 366 ± 8* 350 ± 11* 370 ± 6* 286 ± 6 293 ± 4 285 ± 8 289 ± 12 296 ± 4
5.59 ± 0.67 6.09 ± 0.87 6.11 ± 0.59 4.97 ± 0.36 2.51 ± 0.28* 26.3 ± 3.11* 21.1 ± 2.68 19.9 ± 4.63 11.2 ± 1.88# 5.30 ± 1.69#
1.28 ± 0.56* 3879 ± 565 3676 ± 214 456 ± 23 # 168 ± 26 # 0.41 ± 0.18* 3237 ± 410 2352 ± 268 222 ± 34 # 144 ± 12 #
Data are the mean ± S.E.M. Weight: *All control groups weighed significantly more than all diabetic groups (p < 0.05); Plasma glucose: *vs. all control and # vs. diabetic (D0, D1, D2) groups (p < 0.05); Plasma insulin: *vs. all insulin-injected and #vs. all 1- and 2-min insulin-injected groups (p < 0.05).
and fluid intake in these animals (food intake (g): control, 32 ± 0.3; diabetic, 63 ± 1; control-treated, 29 ± 1; diabetic-treated, 30 ± 1; fluid intake (mL): control, 65 ± 1; diabetic, 323 ± 7; control-treated, 40 ± 2; diabetic-treated, 52 ± 4). A significant decrease in the plasma glucose levels in diabetic rats was shown following an insulin and vanadium treatment (Fig. 2A). Three-week BMOV-treated animals were euglycemic at the time of sacrifice. Similar to results from time-course studies (Fig. 1B), basal GSFA in rat skeletal muscle was not significantly different between control and STZ-diabetic rats (Fig. 2B). The activity of total form of GS was not also significantly different between control and diabetic animals (C0, 4.6 ± 0.2; C15, 4.2 ± 0.2; CT0, 4.1 ± 0.04; CT15, 3.8 ± 0.6; D0, 4.0 ± 0.2; D15, 3.5 ± 0.2; DT0, 4.5 ± 0.1; DT15, 3.8 ± 0.6 nmol/min/mg). Following an insulin injection, GSFA was significantly increased by 2- and 2.5-fold in control and STZ-diabetic rats, respectively, compared to the basal GS activity (p < 0.05). However, vanadium treatment did not affect either basal or insulin-stimulated GSFA in either control or diabetic rats. Diabetes, insulin, and vanadium treatment did not affect GSK-3β activity in rat skeletal muscle as compared to controls. Measurement of PP-1 activity in skeletal muscle of Wistar control rats showed significantly increased enzyme activity (2-fold) at 5 min following insulin injection (p < 0.05) as compared to the basal activity (Fig. 2D). Surprisingly, basal PP1 activity was significantly increased 3-fold in STZ-diabetic rats compared to controls (p < 0.05). Following insulin administration to diabetic rats PP1 activity significantly declined to near-normal values. Similarly, vanadium treatment of diabetic rats also resulted in a significant decline in PP1 activity to near-control values. Fig. 1. Time-course study in long-term STZ-diabetic rats: (A) Plasma glucose, (B) muscle GS fractional activity and (C) muscle GSK3β activity in Wistar control (C, n = 3) and 7-weeks diabetic (D, n = 3) rats before (0) and 2, 5, and 15 min following injection of insulin (5 U/kg i.v.). *p < 0.05 vs. control (C0) rats; **p < 0.05 vs. diabetic (D0) rats.
Studies in the long-term STZ-diabetic Wistar rats We performed two separate studies using skeletal muscle from Wistar rats with a long-term STZ-induced diabetes. In
28 Table 2. Characteristics of STZ-4 weeks-diabetic Wistar rats (BMOV treatment) Group
Number
Weight (g)
Plasma glucose (mmol/L)
Plasma insulin (ng/mL)
C0 C5 C15 D0 D5 D15 CT0 CT5 CT15 DT0 DT5 DT15
7 8 7 7 8 7 7 8 7 7 8 7
399 ± 6* 386 ± 8* 399 ± 10* 295 ± 7 314 ± 9 305 ± 8 367 ± 6* 351 ± 7* 356 ± 10* 329 ± 15 295 ± 12 320 ± 16
7.8 ± 0.3 5.8 ± 0.2 3.0 ± 0.2* 27.5 ± 2.0# 21.0 ± 1.7# 14.9 ± 3.5* 7.6 ± 0.7 5.6 ± 0.2 2.8 ± 0.2* 7.9 ± 0.3 6.1 ± 0.3 3.0 ± 0.2*
0.72 ± 0.17* 524 ± 121 292 ± 31# 0.43 ± 0.17* 749 ± 78 169 ± 49# 1.06 ± 0.36* 864 ± 169 253 ± 29# 0.61 ± 0.16* 886 ± 73 188 ± 27#
Data are the mean ± S.E.M. Weight: *All control groups weighed significantly more than all diabetic groups (p < 0.05); Plasma glucose: *vs. non-insulin treated and #vs. all other groups (p < 0.05); Plasma insulin: *vs. all insulin-injected and #vs. all 5-min insulin-injected groups (p < 0.05).
one study we used 7-week diabetic rats treated with BEOV, while in the other study 9-week diabetic rats were treated with BMOV. The rationale of this experimental design was to study does the longer duration of diabetes result in the de-
velopment of insulin resistance and how does the duration of diabetes affect the activities of GS, GSK-3, and PP1 in rat skeletal muscle. In addition, the glucose-lowering effects of BMOV and BEOV were compared. General characteristics
Fig. 2. Study in the short-term STZ-diabetic rats treated with BMOV: (A) Plasma glucose in Wistar control (C), 4-week diabetic (D), control vanadiumtreated (CT), and diabetic vanadium-treated (DT) rats. Blood was taken from rats before (0) and 15 min following i.v. injection of insulin (5 U/kg). (B) GSFA, (C) GSK-3β activity, and (D) PP1 activity in skeletal muscle from Wistar control (C), diabetic (D), control vanadium-treated (CT), and diabetic vanadium-treated (DT) rats before (0), 5, and/or 15 min after injection of insulin (5 U/kg). Data are expressed as mean ± S.E.M. for 7–8 individual animals and each sample was done as a triplicate. *p < 0.05 vs. non-insulin treated groups; #p < 0.05 vs. all other groups; ♦p < 0.05 vs. control (C0) rats; **p < 0.05 vs. diabetic (D0) rats.
29 of the STZ-7 weeks-diabetic Wistar rats are shown in Table 3. The data regarding body weight, daily food and fluid intake, and plasma insulin levels before and after vanadium and/or insulin treatment were similar to that obtained in the 4-week study. Diabetic untreated rats were hyperphagic and polydipsic, and vanadium treatment normalized food and fluid intake in these animals (food intake (g): control, 30 ± 0.4; diabetic, 61 ± 1; control-treated, 31 ± 1; diabetic-treated, 33 ± 1; fluid intake (mL): control, 72 ± 4; diabetic, 278 ± 8; control-treated, 46 ± 3; diabetic-treated, 67 ± 4). Similar to our time-course study involving long-term STZdiabetic rats, results of this study demonstrated that the basal and insulin-stimulated total GS activities were decreased in diabetic rats as compared to controls (D0, 3.3 ± 0.4; D15, 2.6 ± 0.3; C0, 6.1 ± 0.9; C15, 5.7 ± 1.2 nmol/min/mg). However, similar to the same time-course study, no significant difference in basal GSFA was observed between diabetic and control rats. Effects of insulin on GSFA correlated with the effects on glucose levels (Figs 3A and 3B) in control and diabetic rats. A decrease in glucose levels in control rats following an insulin injection was accompanied by increased GS activity (2-fold) in skeletal muscle (p < 0.05). Interestingly, after 7- or 9-week diabetes, no decline in the plasma glucose level in STZ-diabetic rats was induced by insulin administration (Figs 3A and 4E) and a significantly reduced response in GS activation to insulin was observed. Vanadium treatment in both long-term diabetic studies resulted in a significantly decreased plasma glucose levels in diabetic rats compared to untreated animals, without a significant recovery in the plasma insulin level. However, the treatment with vanadium did not improve the defective insulin-stimulated GS activity in diabetic rat muscle. Determination of PP1 activity in rat skeletal muscle demonstrated, as in the 4-week study, that both diabetes and insulin caused a significant increase (70%) of PP1 activity
compared to the basal activity in control rats (Fig. 3D). Furthermore, the basal PP1 activity was significantly increased by 70% in STZ-diabetic rats compared to controls and this is also in agreement with our results from the previous 4-week diabetic rat study. Interestingly, the levels of both GSFA and PP1 activity were higher in older Wistar rats used in the longterm study (Figs 3B and 3D) compared to younger rats from the short-term diabetic study (Figs 2B and 2D). Similar to our previous results, PP1 activity in muscle of long-term diabetic rats was significantly decreased following an insulin injection. However, while in short-term diabetic animals PP1 activity significantly declined only after 5 min (Fig. 2D), in this study the decrease occurred 15 min following insulin administration. Similar findings were observed after vanadium treatment of diabetic rats. While in long-term STZ-diabetic rats, vanadium treatment per se did not produce any significant effect on PP1 activity, combined vanadium and insulin treatment resulted in significantly decreased enzyme activity 15 min following an insulin injection, a response similar to the one observed in untreated rats. However, in both vanadium-treated and untreated rats with short-term diabetes the response of PP1 to insulin stimulation occurred more quickly and was more profound (Fig. 2D). Results of this study demonstrated also that diabetes, vanadium, and insulin in vivo treatment did not produce an effect on GSK-3β activity (Fig. 3C) in skeletal muscle from Wistar rats with long-term STZ-induced diabetes. In order to purify the muscle homogenates and remove the proteins whose presence may decrease specificity of the GSK-3 assay, we performed an additional long-term (9-week) diabetes study, in which muscle GSK-3 was analyzed by cation-exchange chromatography using a Mono S column. This procedure has been shown to separate and specifically measure GSK-3 activity [24]. To produce a column profile that was more representative of each group, muscle extracts from
Table 3. Characteristics of STZ-7 weeks-diabetic Wistar rats (BEOV treatment) Group
Number
Weight (g)
Plasma glucose (mmol/L)
Plasma insulin (ng/mL)
C0 C5 C15 D0 D5 D15 CT0 CT5 CT15 DT0 DT5 DT15
8 8 8 8 10 9 8 8 8 9 7 9
471 ± 15* 480 ± 14* 486 ± 16* 359 ± 14 363 ± 11 358 ± 12 428 ± 17* 408 ± 14* 447 ± 12* 306 ± 11 333 ± 7 338 ± 10
7.3 ± 0.14 6.5 ± 0.37 3.3 ± 0.29 # 17.9 ± 0.12* 20.3 ± 0.95* 18.6 ± 1.22* 7.5 ± 0.38 5.6 ± 0.1 3.0 ± 0.11# 8.6 ± 0.54** 7.3 ± 0.44 3.4 ± 0.14#
1.92 ± 0.41* 1059 ± 175 400 ± 1** 0.53 ± 0.08*# 1008 ± 91 182 ± 7** 1.56 ± 0.32* 999 ± 149 339 ± 27** 0.33 ± 0.05*# 1164 ± 109 291 ± 37**
Data are the mean ± S.E.M. Weight: *All control groups weighed significantly more than all diabetic groups (p < 0.05); Plasma glucose: *vs. all control (C0) and diabetic vanadium-treated, #vs. all non-insulin treated, and **vs. diabetic (D0) rats (p < 0.05); Plasma insulin: *vs. all insulin-injected, #vs. control noninsulin treated (C0, CT0), and **vs. all 5-min insulin-injected groups (p < 0.05).
30
Fig. 3. Study in the long-term STZ-diabetic rats treated with BEOV: (A) Plasma glucose in Wistar control (C), 7-week diabetic (D), control vanadiumtreated (CT), and diabetic vanadium-treated (DT) rats. Blood was taken from rats before (0) and 15 min following i.v. insulin injection (5 U/kg). (B) GSFA, (C) GSK-3β activity, and (D) PP1 activity in skeletal muscle from Wistar control (C), diabetic (D), control vanadium-treated (CT), and diabetic vanadiumtreated (DT) rats before (0), 5, and/or 15 min after injection of insulin (5 U/kg). Data are expressed as mean ± S.E.M. for the number of animals indicated in Table 3. *p < 0.05 vs. non-insulin treated groups; 6p < 0.05 vs. all control and diabetic vanadium-treated groups; #p < 0.05 vs. all other groups; ♦p < 0.05 vs. control (C0) rats; **p < 0.05 vs. diabetic (D0) rats.
Wistar rats in different groups were each pooled separately and applied to the column. The Mono S column fractions were then assayed for GSK-3 activity against specific phosphoGSK-3 substrate peptide. Results from this study revealed two basal peaks of GSK-3 activity in skeletal muscle from Wistar control rats (Fig. 4A). When the same fractions were assayed using the negative control (Ala-21) peptide, no detectable GSK-3 activity was observed. In STZ-diabetic Wistar rats, GSK-3 activity of the first peak, which has been previously shown to contain the alpha isoform of GSK-3 [32], was decreased compared to control rats (Fig. 4B). However, the second peak of GSK-3 activity remained unchanged in diabetic rats. Immunoblotting studies identified GSK-3β in both peaks of GSK-3 activity (Fig. 4C). To further confirm that the changes in both peaks of GSK-3 activity were due to GSK-3β, immunoprecipitation studies were performed on the specific Mono S fractions (number 20–31) using specific antibodies for this isoform of GSK-3. These experiments confirmed no difference in basal GSK-3β activity between STZ-diabetic and control rats, as well as no effect of insulin injection on this enzyme activity in both control and diabetic rats (Fig. 4D). Therefore, although Mono S fractions showed a decrease of the first peak of GSK-3 activity after insulin
treatment, it seems that this effect is only negligible or due to a possible decrease in GSK-3α activity.
Discussion Although the activation of muscle glycogen synthase by insulin was recognized more than 40 years ago [33], the molecular mechanisms of this insulin effect are still unclear. In an attempt to evaluate enzymes involved in insulin signaling that regulates glycogen synthesis in an important insulinsensitive tissue, we measured the activities of GS, GSK-3, and PP-1 in rat skeletal muscle before and after treatment. Treated animals received vanadium chronically (3 or 6 weeks), while insulin was administered acutely at 5 or 15 min before termination. Our laboratory reported earlier that treatment with an organic vanadium compound, BMOV, at a lower dose than was required for vanadyl sulphate effectively reduced plasma glucose, without any overt signs of toxicity [34]. BEOV, an ethyl derivative of BMOV, demonstrated the same characteristics as a glucose-lowering agent as its counterpart. This is the first report to show that treatment with both organic vanadium
31
Fig. 4. Mono S chromatography study in the long-term STZ-diabetic rats treated with BMOV: (A) Mono-S profile of GSK-3 activity in skeletal muscle from control rats and (B) 9-week diabetic rats at 0 (closed circle) and 15 min (open circle) following insulin injection (5 U/kg). Each column run represents a pool of 5 animals. (C) Representative immunoblot of GSK-3β in Mono S fractions 20-31 from skeletal muscle of Wistar control rats. (D) Skeletal muscle homogenates from untreated control (C, n = 5) and diabetic (D, n = 5) as well as insulin-treated rats were collected before (0) and 15 min after i.v. injection of insulin (5 U/kg), and fractionated over a Mono S cation exchange column. The eluted fractions 20–31 (that immunoblotted for GSK-3β) were pooled and immunoprecipitation assays were performed with anti-GSK-3β antibodies. The immunoprecipitates were then assayed for GSK-3 activity as described under ‘Materials and methods’. (E) Plasma glucose in Wistar control (C), diabetic (D), control vanadium-treated (CT), and diabetic vanadium-treated (DT) rats before (0) and 15 min following injection of insulin (5 U/kg). Data are expressed as mean ± S.E.M. *p < 0.05 vs. non-insulin treated groups; 6p < 0.05 vs. all control and diabetic vanadium-treated groups.
compounds, BMOV and BEOV, produced a very similar glucose-lowering effect in STZ-diabetic rats (Figs 2A, 4A and 5E). Vanadium treatment did not result in elevated insulin levels in either short- or long-term STZ-diabetic animals, showing that an increase in insulin levels is not responsible for improved glucose disposal in these rats. Food and fluid intake in diabetic untreated rats was normalized following vanadium treatment. Our laboratory has recently reported that in STZ-diabetic rats insulin-enhancing effects of vanadium were not secondary to a reduced food intake [35].
Results from our studies show that STZ-diabetic rats develop insulin resistance over time. In support, other studies also demonstrated development of insulin resistance in Type 1 diabetes associated with β-cell destruction [36]. Our data indicate that STZ-diabetic rats were not insulin resistant four weeks following an induction of diabetes. However, 3–5 weeks later insulin resistance had developed and there was no decline in plasma glucose level following an insulin injection. These results correlated with muscle GS fractional activity, which was stimulated after an insulin injection in
32 short-term diabetic rats, but stimulation was impaired in longterm diabetes. Accordingly, previous studies have demonstrated that the predominant defect in diabetes associated with insulin resistance lies in non-oxidative glucose metabolism [37–39] and an impaired insulin-stimulated glycogen synthesis in skeletal muscle of obese subjects under clamp conditions was reported [40]. Our data from the present studies in which STZ-diabetic rats were used, showed that in skeletal muscle of the shortterm Type 1 diabetic rats there was no change in either basal or insulin-stimulated GSFA post-vanadium treatment, while in long-term diabetic rats GSFA was surprisingly decreased following the same treatment. A lack of effect of vanadium to modify the insulin stimulation of glycogen synthesis in skeletal muscle was also observed in a recent study done by Goldfine et al. [41]. Interestingly, determination of muscle glycogen content in diabetic animals revealed that vanadium treatment did not have an effect on fasting muscle glycogen [21]. The role of GSK-3, as an upstream regulator of GS activity, in insulin signaling and its inhibition by insulin has been mainly described in vitro [8–10]. Results from our in vivo studies (both short- and long-term diabetes studies) demonstrated that in skeletal muscle there was no significant difference in basal GSK-3β activity between STZ-diabetic and control rats. Furthermore, no effect of insulin stimulation on muscle GSK-3β activity from both control and diabetic Wistar rats was shown. Results of our preliminary time-course studies demonstrated that muscle GSK-3β activity was not also significantly changed at the earlier time points (1, 2, and 5 min) after insulin injection. In support, chromatographic separation of GSK-3 by Mono S column from skeletal muscle extracts and consequent determination of GSK-3β activity by specific immunoprecipitation assay confirmed that both diabetes and insulin did not affect GSK-3β activity. These findings are in agreement with a recent hyperinsulinemiceuglycemic study [42] in which the failure of insulin administration to decrease GSK-3β activity in skeletal muscle of nondiabetic and Type 2 diabetic subjects was also shown. The same study demonstrated only a 30% decrease in activity of the other isoform, GSK-3α, following insulin administration [42]. In the present study we measured the activity of GSK3β only, since our preliminary results demonstrated a similar response of GSK-3α and GSK-3β activities to insulin treatment. In contrast to our studies where a lack of effect of insulin injection on GSK-3β activity was shown, results of another previous study done in vivo demonstrated a decrease in muscle GSK-3 activity (by only 40%) following insulin stimulation [12]. Different results in the analysis of GSK-3 activity could be due to different experimental conditions and a possible difference in assay technique. In the study done by Cross et al. [12] all rats were injected simultaneously with insulin and propranolol. Although by using propranolol, con-
founding effects of counter-regulatory responses following insulin injection may be avoided, this may have also resulted in a more prominent inhibition of GSK-3 than with injection of insulin alone. Propranolol, a β-adrenergic receptor antagonist decreases adrenaline-induced phosphorylation and reactivates GS [43]. The regulation of muscle GS by adrenaline is a β-adrenergic effect, mediated by an elevation of cyclic AMP [44]. Recently, it was demonstrated that increased cyclic AMP stimulates GSK-3 activity by tyrosine phosphorylation [45]. Propranolol, hence, may block this phosphorylation, resulting in inhibition of GSK-3 activity. Consequently, the effects observed in the study [12] may not be representative of the acute inhibition of GSK-3 produced by insulin. 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 [46]. These observations suggest that the regulation of GSK-3 differs between tissues. In addition, results from our recent in vivo study in which Zucker fatty rats were used as an animal model of the prediabetic state of Type 2 diabetes, demonstrated no difference in muscle GSK-3β activity between fatty and lean rats [47]. Interestingly, other results from our laboratory demonstrated that the activity of protein kinase B, which seems to be a direct upstream regulator of GSK-3, also remained unchanged in skeletal muscle from both STZ-diabetic and Zucker fatty rats as compared to controls [48]. Therefore, these findings apparently exclude a role for GSK-3 and PKB in the development of both types of diabetes. Our results also 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. Other reports have shown no more than 50% insulin induced inhibition of GSK-3, mainly in vitro, where effects of insulin counteracting hormones, such as adrenaline and glucagon, will not be seen. However, coupled to the action of the PP1 lying upstream of GS [49], this relatively small change in the activity level of GSK-3 may be sufficient to affect glycogen synthesis in vivo. As expected, we have demonstrated activation of PP1 following insulin injection in Wistar control rats. Surprisingly, our data indicated increased PP1 activity in Wistar STZ-diabetic rats compared to controls. The increased PP1 activity in the muscle of diabetic rats may be a possible explanation for the near-normal GS activity in these rats. Since PP1 activates GS, highly activated PP1 could keep GS in an active state even under diabetic conditions. Interestingly, following insulin injection in STZ-diabetic rats, muscle PP1 activity was decreased to near-control values and a delay in the insulin effect on PP1 activity in rats with long-term diabetes, associated with insulin resistance, was demonstrated. Furthermore, our results also demonstrated that the levels of both
33 GSFA and PP1 activity were higher in older Wistar rats used in the long-term study compared to younger rats from the short-term diabetic study apparently as a result of the rat aging. Our study represents the first report that vanadium treatment mimics insulin action on PP1 activity in skeletal muscle of control and STZ-diabetic rats. It was found that following vanadium treatment per se PP1 activity was significantly decreased in muscle of Wistar rats with short-term STZ-diabetes, while in muscle of insulin-resistant Wistar rats with long-term (7-week) diabetes this effect of vanadium treatment on PP1 activity was absent. Another interesting finding was that while in short-term STZ-diabetic rats vanadium was not additive or synergistic with insulin on PP1 activity, in longterm diabetic rats combined insulin and vanadium treatment resulted in decreased PP1 activity compared to untreated diabetic rats. However, no direct correlation between PP1 and GS activity in muscle of STZ-diabetic rats was observed after vanadium treatment. Hence, it is possible that PP1 is involved in the regulation of some other cellular events of vanadium (other than regulation of glycogen synthesis) or that vanadium affects PP1 activity through mechanisms in diabetic rats that are independent of the known insulin signaling pathways. Since vanadium is a well-known phosphatase inhibitor, it is also possible that vanadium exerts its activity, at least partially, by inhibiting specific phosphatases including PP1. A lack of coherence between the changes in GSFA and changes in GSK-3 and PP1 activities following vanadium treatment suggests a role for other factors, such as a subtype of PP1 (PP1G) or the levels of G6P, in the regulation of the glucose-lowering effect of vanadium. In our studies we have measured total PP1 activity and 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. Furthermore, a recent study done by Sun et al. demonstrated that vanadium treatment fully restored G6P levels, which were reduced in skeletal muscle, liver, and adipose tissue from STZ-diabetic rats [50]. This suggests that vanadium might normalize blood glucose levels by increasing G6P levels in insulin-responsive tissues, and that this initial product in a glucose disposal may be responsible for an improvement in peripheral tissue sensitivity to insulin. Importantly, while a majority of the previous studies on the mechanisms of insulin and vanadium action were done in vitro using cultured cells, our study represents one of the few studies done in vivo. In contrast to in vitro studies, which are performed under controlled conditions, the in vivo studies represent the true complexity of intracellular factors involved in metabolic regulation by insulin and other hormones in an intact animal. In addition to the effect of insulin-counter-regulatory hormones, it is most likely that glucose levels also have
a direct impact on the insulin signaling pathways, further complicating the interpretation of in vivo studies. Bearing that in mind, the discrepancy between results of in vitro and in vivo studies is possible and logical to expect. In conclusion, results of this study indicate that STZ-diabetic rats develop insulin resistance over time. A lack of effect of insulin stimulation on GSK-3β activity in skeletal muscle from control and STZ-diabetic rats suggests that either this enzyme is not a key protein kinase in the regulation of glycogen synthesis by insulin or its inhibition is only transient. PP1 seems to be a more important regulating enzyme in the development of diabetes and in the regulation of GS activity by insulin and vanadium. In skeletal muscle from control Wistar rats following in vivo insulin administration, increased PP1 activity is accompanied by stimulation of GSFA and a consequent decline in plasma glucose levels. Furthermore, vanadium and/or insulin treatment normalizes PP1 activity in diabetic rat muscle. The lack of effect of vanadium treatment to stimulate glycogen synthesis in skeletal muscle may suggest involvement of other metabolic pathways in vanadium-mediated blood glucose disposal. To clarify this and other issues raised in this work, further studies are required.
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 Science Engineering Research Council/TPP grant and Kinetek Pharmaceutical Inc. SS was the recipient of an Rx&D-Health Research Foundation and Medical Research Council of Canada Scholarship. We thank Dr. Roger W. Brownsey for his constructive criticism of the manuscript.
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