In vivo effects of insulin and bis(maltolato)- oxovanadium (IV) on PKB activity in the skeletal muscle and liver of diabetic rats

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1 Molecular and Cellular Biochemistry 223: , Kluwer Academic Publishers. Printed in the Netherlands. 147 In vivo effects of insulin and bis(maltolato)- oxovanadium (IV) on PKB activity in the skeletal muscle and liver of diabetic rats Lucy Marzban, 1 Sanjay Bhanot, 2 John H. McNeill 1 1 Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, BC; 2 Kinetek Pharmaceuticals Inc., Vancouver, BC, Canada Received 30 January 2001; accepted 27 April 2001 Abstract In this study, the in vivo effects of insulin and chronic treatment with bis(maltolato)oxovanadium (IV) (BMOV) on protein kinase B (PKB) activity were examined in the liver and skeletal muscle from two animal models of diabetes, the STZ-diabetic Wistar rat and the fatty Zucker rat. Animals were treated with BMOV in the drinking water ( mg/ml) for 3 (or 8) weeks and sacrificed with or without insulin injection. Insulin (5 U/kg, i.v.) increased PKBα activity more than 10-fold and PKBβ activity more than 3-fold in both animal models. Despite the development of insulin resistance, insulin-induced activation of PKBα was not impaired in the STZ-diabetic rats up to 9 weeks of diabetes, excluding a role for PKBα in the development of insulin resistance in type 1 diabetes. Insulin-induced PKBα activity was markedly reduced in the skeletal muscle of fatty Zucker rats as compared to lean littermates (fatty: 7-fold vs. lean: 14-fold). In contrast, a significant increase in insulin-stimulated PKBα activity was observed in the liver of fatty Zucker rats (fatty: 15.7-fold vs. lean: 7.6-fold). Chronic treatment with BMOV normalized plasma glucose levels in STZ-diabetic rats and decreased plasma insulin levels in fatty Zucker rats but did not have any effect on basal or insulin-induced PKBα and PKBβ activities. In conclusion (i) in STZ-diabetic rats PKB activity was normal up to 9 weeks of diabetes; (ii) in fatty Zucker rats insulin-induced activation of PKBα (but not PKBβ) was markedly altered in both tissues; (iii) changes in PKBα activity were tissue specific; (iv) the glucoregulatory effects of BMOV were independent of PKB activity. (Mol Cell Biochem 223: , 2001) Key words: bis(maltolato)oxovanadium (IV), fatty Zucker rat, insulin resistance, insulin signaling, streptozotocin-induced diabetes Introduction Insulin is the main metabolic hormone involved in glucose homeostasis. It increases glucose uptake into muscle and adipose tissue and promotes the synthesis of glycogen, lipids, and proteins while inhibiting their degradation and decreasing hepatic glucose production [1, 2]. Insulin resistance is a characteristic associated with both type 1 and type 2 diabetes mellitus [3 5]. Resistance to insulin action at the level of liver results in an increase in hepatic glucose production, while in the skeletal muscle it causes a decrease in glucose uptake [3], the rate-limiting step for glucose utilization [5, 6]. In contrast to type 2 diabetes, insulin resistance in type 1 seems to be a secondary phenomenon associated with chronic hyperglycemia [4, 5]. In spite of numerous studies in the past decade, the molecular mechanism causing insulin resistance is still not well understood. During the recent years much attention has been focused on early and intermediate steps in the insulin-signaling cascade. However, whether these signaling defects are causative in the development of insulin resistance or secondary to the metabolic disorders associated with diabetes are yet to be investigated. Several studies have demonstrated that most of the metabolic actions of insulin are mediated by phosphat- Present address: S. Bhanot, ISIS Pharmaceuticals Inc., Carlsbad, CA, USA Address for offprints: J.H. McNeill, Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, BC, Canada V6T 1Z3

2 148 idylinositol 3-kinase (PI3-K) [7 11], implying a possible role for PI3-K-dependent protein kinases in the development of insulin resistance. Accumulating evidence has implicated the importance of protein kinase B (PKB), a downstream enzyme of PI3-K, in mediating several metabolic actions of insulin including glucose uptake, glycogen synthesis and glycolysis [12 14]. Vanadium compounds are known to have insulin mimetic/ enhancing effects both in vitro and in vivo. Many studies have shown that vanadium compounds decrease plasma glucose levels in human subjects and different animal models of type 1 diabetes [15 17]. In type 2 diabetes and experimental models of insulin resistance, vanadium lowers plasma insulin levels and improves insulin sensitivity [18 21]. Hence, these compounds are believed to be potential candidates for oral therapy in both type 1 and type 2 diabetes. However, the mechanism(s) by which vanadium produces its in vivo anti-diabetic effects is still under investigation. Vanadium compounds are known to potentiate protein tyrosine phosphorylation through inhibition of protein tyrosine phosphatases [15]. Furthermore, the effects of vanadium are shown to involve both insulin receptor dependent and independent mechanism(s) [15, 22, 23]. The rationale for the present study stems from the notion that defects in protein kinases downstream of the insulin receptor may contribute to insulin resistance and that treatment with bis(maltolato)oxovanadium (IV) (BMOV) may restore these defects by bypassing some steps in insulin signaling. BMOV is an organic form of vanadium; its advantages over the inorganic compounds are greater potency, lower toxicity and improved tolerance [24, 25]. The main goal of this study was to investigate the regulation of PKB in diabetes and examine the effects of chronic treatment with BMOV on PKB activity in vivo. We looked at basal and insulin-induced activation of PKB in the streptozotocin (STZ)-diabetic Wistar rat, an animal model of type 1 diabetes and the fatty Zucker rat, an animal model that presents several characteristics of type 2 diabetes. Our results indicate that insulin-induced PKBα activity was not impaired in the skeletal muscle and liver of STZ-diabetic rats up to 9 weeks of diabetes. In contrast, insulin-induced PKBα activity was significantly altered in both tissues in fatty Zucker rats. Chronic treatment with BMOV normalized plasma glucose levels in STZ-diabetic rats and restored plasma insulin levels in fatty Zucker rats but did not have any effect on PKB activity, implying that the glucoregulatory effects of BMOV are independent of PKB activity in vivo. Materials and methods Anti-PKBα (C-terminal) antibody and HRP conjugated antigoat IgG were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-PKBα (PH domain) antibody, Anti-PKBβ antibody, PKBα specific substrate peptide (RPRAATF) and HRP conjugated anti-sheep IgG were purchased from Upstate Biotechnology Inc. (Lake Placid, NY, USA). Regular beef/ pork insulin (Iletin R) was from Eli Lilly Co. (Indianapolis, IN, USA). Protein G Sepharose beads were from Amersham Pharmacia Biotech. (Oakville, ON, Canada). Aprotinin, leupeptin, pepstatin A, soyabean trypsin inhibitor, benzamidine, phenylmethylsulphonyl fluoride (PMSF), dithiothreitol (DTT), β-methylaspartic acid, β-glycerophosphate, Triton X- 100, camp-dependent protein kinase inhibitor (PKI), streptozotocin, EDTA, EGTA, and MOPS buffer (ph 7.2) were purchased from Sigma (St. Louis, MO, USA). Nitrocellulose membrane and electrophoresis chemicals were from Bio- Rad Laboratories (Hercules, CA, USA). [γ- 32 P]ATP was from Dupont NEN (Boston, MA, USA). The radioimmunoassay kit for rat insulin was obtained from Linco Research Inc. (St. Charles, MO, USA). Wistar rats Male Wistar rats, weighing g were received from Animal Care Center, The University of British Columbia, Vancouver, Canada. The animals were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care. Rats were housed, 1 per cage in the treated groups and 2 per cage in the control groups, on a 12 h light/ 12 h dark schedule and given food and fluid ad libitum in all studies. Rats were randomly divided into 4 groups: control (C), control treated with BMOV (CT), diabetic (D), and diabetic treated with BMOV (DT). Animals in the D and DT groups were made diabetic by a single intravenous injection of streptozotocin (STZ) dissolved in 0.9% saline (60 mg/kg) via the tail vein under halothane anesthesia. Three days post STZ injection, rats with a blood glucose level greater than 14 mm were considered diabetic. Treatment with BMOV was started 1 week after STZ injection. Animals in the BMOVtreated groups received BMOV for 3 weeks (4-week study) or 8 weeks (9-week study) at an initial concentration of 0.5 mg/ ml in the drinking water, which was increased to a maximum concentration of 1 mg/ml or until the animal reached an euglycemic state. The dose of BMOV administration was calculated based on the solution concentration, body weight, and fluid consumption. The mean BMOV doses during the last week of treatment in the control-treated and STZ-diabetictreated groups were 0.26 ± 0.01 mmol/kg/day and 0.41 ± 0.01 mmol/kg/day, respectively. In the 9-week study the mean BMOV doses in the last three weeks of treatment in the control-treated and STZ-diabetic-treated groups were 0.19 ± 0.01 mmol/kg/day and 0.30 ± 0.01 mmol/kg/day, respectively. Plasma glucose and insulin levels were monitored weekly throughout the studies. Blood was collected from the

3 149 tail vein following a 5 h fast, centrifuged (17,500 g, 20 min, 4 C) and plasma was stored at 20 C until assayed. Body weight and food and fluid intake were measured weekly throughout the studies to monitor animals health. At termination, overnight fasted rats were randomly divided into basal and insulin injected subgroups and were anesthetized with pentobarbital (65 mg/kg, i.p.) and sacrificed without insulin (basal state) or at different time points after tail-vein injection with insulin (5 U/kg, i.v.). The chest cavity was opened and blood was collected by cardiac puncture for determination of plasma glucose and insulin levels. Hind limb skeletal muscles from both legs and liver were removed immediately, freeze clamped in liquid nitrogen and stored at 70 C. Zucker rats Age-matched male lean and fatty Zucker rats (14 16 weeks old) were received from the Department of Physiology, The University of British Columbia, Vancouver, Canada. Animals were fasted overnight (16 h) and two oral glucose tolerance tests (OGTT) were performed, one at weeks of age, before the beginning of treatment and one a week before termination. Lean and fatty Zucker rats were randomly divided into four groups: lean (L), lean-treated with BMOV (LT), fatty (F), and fatty-treated with BMOV (FT). BMOV treatment was started at an initial concentration of 0.25 mg/ml in the drinking water and was increased to a maximum concentration of 0.75 mg/ml within the first week of treatment. The mean BMOV dose during the last week of treatment in the lean-treated and fatty-treated rats was 0.19 ± 0.01 mmol/kg/ day. After 3 weeks of BMOV treatment, rats were fasted overnight and animals in each treatment group (L, LT, F, and FT) were divided into basal and insulin injected subgroups and sacrificed without insulin (basal state) or at different time points after insulin injection (5 U/kg, i.v.). Oral glucose tolerance test Zucker rats were fasted overnight (16 h). Blood samples were taken immediately prior to a glucose load via oral gavage (1 g/kg) and at 10, 20, 30, 60 and 90 min after glucose administration. Plasma glucose levels were measured with a Beckman Glucose Analyzer 2 and plasma insulin levels were determined with a double antibody radioimmunoassay by using a kit from Linco Research Inc. Insulin sensitivity indices (ISI) were calculated as previously described [26] by the following formula: K/SQRT((fasted plasma glucose fasted plasma insulin) (mean plasma glucose mean plasma insulin)), K = 100. Tissue extract preparation Liver and muscle tissues were powdered in liquid nitrogen using a mortar and pestle. Approximately 50 mg tissue powder per ml of buffer was homogenized in ice-cold buffer using a polytron (Brinkmann, model: PT 3100) for 2 15 sec at 6000 rpm. Homogenization buffer contained 25 mm MOPS buffer, 5 mm EGTA, 2 mm EDTA, 75 mm β-glycerophosphate, 5 µm β-methylaspartic acid, 5 µm pepstatin A, 3 mm benzamidine, 10 µm leupeptin, 10 µg/ml aprotinin, 200 µg/ ml trypsin inhibitor, 1 mm DTT, 1 mm PMSF, and 0.5% Triton-X100, ph 7.2. Homogenized tissues were then centrifuged at 100,000 g for 1 h at 4 C. Supernatants were filtered through a 53 µm mesh (Nitex Netting nylon monofilament) carefully avoiding fat, and were stored at 70 C until assayed. Protein concentration in the homogenates was measured using the Bradford method. PKB immunoprecipitation assay PKB kinase activity was measured by an immunoprecipitation assay followed by a 32 P kinase assay. Aliquots of protein (2 mg) were diluted with approximately equal volume of 3% NETF buffer containing 100 mm NaCl, 5 mm EDTA, 50 mm Tris-HCl (ph 7.4), 50 mm NaF, and 3% Nonidet P-40, (ph 8.0) and pre-incubated with 3 µg of anti-pkbα-ph domain or anti-pkbβ antibody (from Upstate Biotechnology) for 2 h at 4 C. Forty µl of protein G Sepharose beads in 3% NETF was then added and incubated for 1 h at 4 C. Pellets were washed twice with 3% NETF buffer and twice with K II buffer (1.25 mm β-glycerophosphate, 1.25 mm MOPS (ph 7.2), 0.5 mm EGTA, 2 mm MgCl 2, 5 mm sodium fluoride, 25 µm DTT, 2.5 µm β-methylaspartic acid). Beads were incubated in a reaction mixture containing 10 µl of 0.4 mm Akt/ PKB specific substrate peptide, 5 µl of 200 mm MgCl 2, 3.2 µl of 25 µm camp-dependent protein kinase inhibitor (ph 7.4), 16.8 µl assay dilution buffer, and 10 µl of [γ- 32 P]ATP (specific activity: ~ 3000 Ci/mmol) diluted 1:50 in assay dilution buffer (25 mm β-glycerophosphate, 20 mm MOPS (ph 7.2), 5 mm EGTA, 2 mm EDTA, 20 mm MgCl 2, 0.25 mm DTT, 5 mm β-methylaspartic acid) for 30 min at 30 C. Supernatant (25 µl) was spotted onto 2-cm 2 P81 phosphocellulose papers which were washed 5 times in 1% phosphoric acid, dried and counted for radioactivity. Electrophoresis and immunoblotting Immunoprecipitated samples or aliquots of protein (100 µg) were boiled with 20 µl of 5 Laemmli s sample digestion buffer for 5 min and subjected to 10% SDS-PAGE (16 h,

4 ma/gel) following which the proteins were transferred to nitrocellulose membrane (3 h, 0.3 A). Membranes were blocked with 5% non-fat dry milk in TBS (250 mm NaCl, 25 mm Trizma base, 0.02 N HCl, ph 7.5) for 1.5 h at room temperature, washed with TBS containing 0.5% Tween 20 (TBS-T) and then incubated with anti-pkbα PH-domain (or anti-pkbβ) antibody (1 µg/ml) or anti-pkbα C-terminal antibody (0.4 µg/ml) in TBS-T for 2 h at room temperature. The membranes were washed with TBS-T and incubated for 1 h at room temperature with HRP conjugated anti-sheep IgG antibody (for anti-pkbα PH-domain and anti-pkbβ antibody) or anti-goat IgG antibody (for anti-pkbα C-terminal antibody) diluted 1:10,000 and 1:2,000 in TBS-T, respectively. Washed membranes were placed in a 1:1 solution of chemiluminescence reagents (from Amersham Pharmacia) and exposed to high performance chemiluminescence films. Films were developed and bands were quantified by densitometry. Statistical analyses Values are expressed as the mean ± S.E.M. and n indicates the number of rats in each group. Statistical analyses were performed using a general linear model of ANOVA or oneway ANOVA followed by a Newman-Keuls test as appropriate. Insulin sensitivity indices (ISI) were analyzed using Student s t-test. P < 0.05 was taken as level of significance. Results Time course study At the beginning of the study, a time course experiment was performed by using a separate group of animals to determine the time point of maximum activation of PKBα, in vivo Fig. 1. PKBα activity in basal fasting state or at different time points after insulin injection (5 U/kg, i.v.) in the skeletal muscle and liver homogenates from control and STZ-diabetic Wistar rats (A) and lean and fatty Zucker rats (B). PKBα was immunoprecipitated using anti-pkbα-ph antibody. Immunoprecipitates were then assayed for phosphotransferase activity as described in Materials and methods. Results are presented as the mean ± S.E.M. of 1 assay performed in triplicate for each muscle or liver homogenate (n = 3 5 per group). PKBα activity in the muscle or liver homogenates from control basal rats was taken as 100% in each experiment. *Significantly different from corresponding insulin injected groups; # significantly different from lean group at the same time lean at 15 min significantly different from lean at 5 min (p < 0.05, ANOVA).

5 151 (Fig. 1). Wistar rats were made diabetic with streptozotocin (60 mg/kg) as described in methods. Control and STZ-diabetic Wistar rats (4 weeks of diabetes), as well as lean and fatty Zucker rats (18 20 weeks old) were injected with 5 U/ kg insulin and sacrificed at different time points after insulin injection. Results showed that PKBα was activated by insulin in the skeletal muscle as early as 1 min, with a maximum activation occurring at 5 min post insulin injection in control Wistar rats and at 15 min in lean Zucker rats (Fig. 1). There was no significant difference in basal or insulin-induced PKBα activity in the skeletal muscle between control and STZ-diabetic rats (Fig. 1A). However, insulin-induced activation of PKBα was significantly lower in the skeletal muscle of fatty Zucker rats as compared to lean rats (Fig. 1B). Similar to the results seen in muscle, in the liver of both control and STZ-diabetic Wistar rats, insulin stimulated activation of PKBα after 1 min, and the enzyme activity remained high up to 15 min. Furthermore, there was no significant difference in PKBα activity between control and STZ-diabetic rats (Fig. 1A). Interestingly, insulin-stimulated activation of PKBα was significantly higher in the liver of fatty Zucker rats as compared to lean littermates. Although PKBα activity in the basal state was higher in fatty Zucker rats as compared to lean littermates, this difference was not statistically significant (Fig. 1B). Insulin (5 U/kg, i.v.) increased PKBβ activity about 6-fold and 5-fold in the skeletal muscle of control Wistar rats and Zucker rats respectively, and about 3-fold in the liver of both animal models (Fig. 2). Furthermore, our results did not show any significant difference in basal or insulin-induced PKBβ activity, at the time point of maximum activation, between lean and fatty Zucker rats in either the skeletal muscle or liver (Figs 2C and 2D). PKBα activity in BMOV-treated Wistar rats Four week study Streptozotocin injection resulted in an increase in blood glucose concentration in Wistar rats which was normalized following BMOV treatment (Fig. 3A). BMOV did not have any effect on plasma glucose in control animals. In C, CT and DT groups, the glucose lowering effects of insulin were observed Fig. 2. Basal and insulin-induced PKBβ activity in the skeletal muscle and liver of control Wistar rats (A, B) and lean and fatty Zucker rats (C, D). PKBβ activity in each tissue was measured at the time point of maximum activation (muscle: 5 min, liver: 15 min after insulin injection). Immunoprecipitation assays were performed using anti-pkbβ antibody. Results are given as the mean ± S.E.M. for PKBβ activity in each group (n = 5 per group). Basal PKBβ activity in a pooled muscle or liver homogenate was taken as 100%. The upper panels are the representative Western blots of the immunoprecipitates at the basal state ( ) or after insulin injection (+). *Significantly different from basal state (ANOVA or Student s t-test as appropriate p < 0.05).

6 152 Fig. 3. Plasma glucose levels in different experimental groups at termination (16-h fasted) and calculated insulin sensitivity indices in fatty Zucker rats. Wistar rats (4-week study) (A), Wistar rats (9-week study) (B), and Zucker rats (4-week study) (C). (D) shows insulin sensitivity indices (ISI) in fatty Zucker rats pre-treatment and post-treatment with BMOV. The insulin sensitivity indices were calculated from the results of oral glucose tolerance tests performed at the beginning of the study and after treatment with BMOV, as described in Materials and methods. Results are presented as the mean ± S.E.M. *Significantly different from corresponding group at basal state; # significantly different from corresponding non-treated group (p < 0.05, F-treated significantly different from fatty (p < 0.05, Student s t-test). C control; CT BMOV-treated control; D STZ-diabetic; DT BMOV-treated diabetic; L lean; LT BMOV-treated lean; F fatty; FT BMOV-treated fatty. at 15 min after insulin injection, while plasma glucose levels remained high in untreated diabetic rats up to 15 min, indicating resistance to insulin action in these animals. Treatment with BMOV normalized food and fluid intakes but did not have any effect on body weight in the STZ-diabetic rats (Table 1). PKB activity was measured using an immunoprecipitation assay as described in Materials and methods. Insulin increased PKBα activity with a maximum activation ranging from fold in various groups at 5 min after insulin injection. Three weeks treatment with BMOV ( mg/ml) in the drinking water did not affect basal or insulin-stimulated PKBα activity in the skeletal muscle of control and diabetic Wistar rats. Furthermore, treatment with BMOV did not shift the time point of maximum activation of PKBα (Fig. 4A). Nine week study Observations from our previous studies indicate that insulin resistance becomes more severe in longer periods of diabetes in STZ-diabetic rats. Therefore, to investigate the association between the development of insulin resistance in type 1 diabetes and PKBα activity, we extended the period of diabetes from 4 to 9 weeks. In this experiment PKBα activity was measured at the time point of maximum activation following insulin injection (5 min). Consistent with results from the first part of study, one week after STZ injection plasma glucose levels were significantly higher in STZ-diabetic rats as compared to control rats. After 9 weeks of diabetes body weights and plasma insulin levels were significantly lower in diabetic animals as compared to control rats (Tables 1 and 2). Treatment with BMOV ( mg/ml) for 8 weeks, restored food and fluid intakes, normalized plasma glucose levels and improved body weights in the STZ-diabetic rats (Fig. 3B, Table 1). Following 5 U/kg insulin, PKBα activity was increased about 14-fold in various experimental groups (C, CT, D, and DT). However, prolonging the period of diabetes from 4 to 9 weeks did not affect basal or insulin-induced PKBα activity in the skeletal muscle (Fig. 4B) or liver (data not shown) of STZ-diabetic rats.

7 153 Table 1. General characteristics of rats in different treatment groups at termination Body wt Food intake Fluid intake (g) (g/day) (ml/day) Wistar Rats (4-week study) C (n = 21) 396 ± 5 32 ± 1 66 ± 2 CT (n = 21) 365 ± 5 29 ± 1 41 ± 2 D (n = 20) 311 ± 5 a 64 ± 1 a 340 ± 9 a DT (n = 20) 316 ± 10 a 31 ± 2 b 49 ± 4 b Wistar Rats (9-week study) C (n = 9) 486 ± ± 1 54 ± 2 CT (n = 8) 416 ± 12 b 29 ± 1 46 ± 2 D (n = 11) 342 ± 5 a 53 ± 1 a 269 ± 6 a DT (n = 8) 390 ± 17 a,b 30 ± 2 b 49 ± 4 b Zucker Rats (4-week study) L (n = 17) 356 ± 7 22 ± 2 42 ± 2 LT (n = 21) 335 ± ± 1 31 ± 4 F (n = 24) 537 ± 9 c 25 ± 1 48 ± 4 FT (n = 18) 482 ± 13 b,c 23 ± 1 39 ± 3 b Values are the mean ± S.E.M. and represent 16-h fasted levels at termination. a D or DT vs. C; b BMOV-treated group vs. corresponding untreated group; c F or FT vs. L and LT (p < 0.05, ANOVA). C, CT, D and DT denote control, BMOV-treated control, STZ-diabetic and BMOV-treated diabetic rats, respectively. L, LT, F and FT denote lean, BMOV-treated lean, fatty and BMOV-treated fatty rats, respectively. PKBα activity in BMOV-treated Zucker rats Measurement of plasma glucose levels in Zucker rats at the beginning of study (14 16 weeks old) showed that there was not a marked difference in plasma glucose between fatty and lean Zucker rats. Plasma glucose levels in weeks old Zucker rats ranged from 7.3 ± 0.6 to 9.5 ± 0.5 mmol/l (16 h fasted), without any significant difference between lean and fatty Zucker rats (Fig. 3C). However, plasma insulin levels were significantly higher in fatty Zucker rats as compared to their lean controls (Table 2). Insulin injection (5 U/kg) decreased glucose levels after 15 min in lean Zucker rats but did not change the glucose level in fatty Zucker rats, indicating resistance to glucoregulatory effects of insulin (Fig. 3C). After 3 weeks treatment with BMOV (0.75 mg/ml) plasma glucose levels remained unchanged in the BMOVtreated lean and fatty Zucker rats as compared to untreated animals. However, plasma insulin levels in BMOV-treated fatty Zucker rats were reduced to lean values (Table 2), indicating an improvement in insulin sensitivity following BMOV treatment. In addition, insulin sensitivity indices calculated from the OGTT results, showed a 33% increase in insulin sensitivity in BMOV-treated fatty Zucker rats as compared to untreated fatty rats (F: 0.8 ± 0.1 vs. FT: 1.2 ± 0.1, p < 0.05) (Fig. 3D). There was no significant difference in insulin sensitivity index between BMOV-treated and -untreated lean rats (L: 10.9 ± 0.8 vs. LT: 13.0 ± 0.8, p > 0.05). Fig. 4. The effect of BMOV treatment on basal and insulin-stimulated PKBα activity in the skeletal muscle from 16-h fasted STZ-diabetic Wistar rats after 3 weeks (A) and 8 weeks (B) of treatment with BMOV. PKBα activity in a pooled muscle homogenate from control basal rats was taken as 100% in each experiment. Results are presented as the mean ± S.E.M. of 1 experiment performed in triplicate for each muscle homogenate, n = 5 7 per group (3-week study) and n = 3 5 per group (8-week study). *Significantly different from corresponding insulin injected groups (p < 0.05, ANOVA). There was no significant difference in PKBα activity between BMOV-treated and -untreated control or STZ-diabetic Wistar rats (p > 0.05, ANOVA). The lower panels are representative Western blots of PKBα immunoprecipitates (IP), showing the equal amount of immunoprecipitated protein in different groups. C control; CT BMOV-treated control; D STZ-diabetic; DT BMOV-treated diabetic. However, there was a marked difference in insulin sensitivity index between lean and fatty rats due to the severe insulin resistance in fatty rats. Treatment with BMOV resulted in a modest decrease in body weight and fluid intake in fatty Zucker rats but did not have any effect on food intake (Table 1). Although BMOV reduced plasma insulin and increased insulin sensitivity in fatty Zucker rats, it did not restore the

8 154 Table 2. Plasma insulin levels (ng/ml) in different treatment groups C CT D DT Wistar rats (4-week study) Beginning 2.3 ± ± ± ± 0.1 Week 4* 0.6 ± ± ± ± 0.2 Wistar rats (9-week study) Beginning 1.6 ± ± ± ± 0.2 Week 9* 1.0 ± ± ± 0.1 a 0.4 ± 0.1 a L LT F FT Zucker rats (4-week study) Beginning 1.1 ± ± ± 1.1 b 11.9 ± 1.3 b Week 4* 2.5 ± ± ± 0.8 b 3.3 ± 0.7 c Values are the mean ± S.E.M. *Values represent 16-h fasted levels at termination vs. 5-h fasted levels at the beginning of the study; a D or DT vs. corresponding control; b F or FT vs. corresponding lean; c FT vs. F (p < 0.05, ANOVA). C, CT, D and DT denote control, BMOV-treated control, STZ-diabetic and BMOV-treated diabetic rats respectively. L, LT, F and FT denote lean, BMOV-treated lean, fatty and BMOV-treated fatty rats, respectively. - Insulin + Insulin reduced levels of insulin-induced PKBα activity in the skeletal muscle of fatty Zucker rats after 3 weeks of treatment (Fig. 5A). Furthermore, BMOV treatment was unable to normalize the elevated levels of insulin-induced PKBα activity in the liver of fatty Zucker rats (Fig. 5B). Based on the results of densitometry performed on immunoprecipitated PKBα bands, there was no detectable difference in PKBα protein expression between lean and fatty Zucker rats (Figs 5A and 5B). There was not any significant difference in basal or insulin-stimulated PKBβ activity in the skeletal muscle or liver between BMOV-treated and -untreated lean and fatty Zucker rats (data not shown). Discussion Fig. 5. The effect of BMOV treatment on basal and insulin-induced (5 U/ kg, i.v.) PKBα activity in the skeletal muscle (A) and liver (B) of Zucker rats, 5 and 15 min after insulin injection. Male fatty and lean Zucker rats (14 16 weeks old) were treated with BMOV in the drinking water for 3 weeks. Animals were 16-h fasted at termination. PKBα activity in a pooled muscle or liver homogenate from lean rats at basal state was taken as 100%. Results are presented as the mean ± S.E.M. of 1 assay performed in triplicate for each animal, n = 6 10 per group (0 and 5 min), n = 3 4 per group (15 min). *Significantly different from corresponding group at basal state; # significantly different from corresponding lean group (p < 0.05, ANOVA). The lower panels are representative Western blots of PKBα immunoprecipitates (IP), showing equal amount of precipitated protein in different treatment groups. L lean; LT BMOV-treated lean; F fatty; FT BMOV-treated fatty. Many studies have shown that the metabolic effects of insulin such as glucose uptake and glycogen synthesis, both of which are decreased in diabetes [1, 3, 5, 27], are mediated by PI3-K via activation of PKB [8, 12 14], suggesting a possible role for PKB in the development of insulin resistance in diabetes. Although insulin has been shown to activate PKB via a wortmannin sensitive pathway both in vitro and in vivo [13, 28 30], the finding that PKB could also be activated via PI3-K independent pathways [31 33] implies that in vivo regulation of this enzyme may be far more complex. The fact that vanadium compounds are potent glucose lowering agents and can be administered orally to treat diabetes, has stimulated interest in finding the mechanism(s) by which these compounds mediate their glucose lowering effects. Several potential sites in the insulin-signaling pathway have been proposed for the insulin-like effects of vanadium. Recent studies have shown that inorganic vanadium compounds stimulate PKB activity in vitro [30, 34]. Although one can not argue against the importance of in vitro studies, the difference between the results obtained from in vitro and in vivo studies

9 155 has always been an important concern. In the present study, we have investigated the association between PKB activity and diabetes as well as the relationship between the glucose lowering effects of BMOV and PKB activity in two animal models of diabetes in vivo. PKB activity in STZ-diabetic rats STZ-diabetic Wistar rat is an animal model of poorly controlled type 1 diabetes and is characterized by hypoinsulinemia, hyperglycemia and exhibits insulin resistance [16]. Hyperglycemia in this model is due to the lack of insulin in the system. Consistent with the results obtained from other in vitro and in vivo studies [8, 11, 13], our results showed that insulin increased PKBα activity in the skeletal muscle and liver of Wistar rats. There was no significant difference in basal or insulin-induced PKBα activity between control and STZ-diabetic rats in either the skeletal muscle or liver, indicating that PKBα activity was not impaired in these animals. Previously our laboratory had shown that both basal and insulin stimulated IRS-1 associated PI3-K activity were normal in STZ-diabetic rats [35], suggesting that insulin signaling is normal at the level of PI3-K and PKB in STZ-diabetic rats. Furthermore, in spite of the development of insulin resistance, basal and insulin-induced PKBα activity was normal up to 9 weeks of diabetes, indicating that changes in PKBα are not important in the development of insulin resistance in type 1 diabetes. PKB activity in fatty Zucker rats Skeletal muscle Fatty Zucker rat (fa/fa) is an animal model that presents several features of type 2 diabetes and is characterized by marked hyperinsulinemia, insulin resistance, euglycemia or mild hyperglycemia and obesity [36, 37]. Skeletal muscle is the primary site of insulin resistance in type 2 diabetes [1, 3] and accounts for more than 70% of glucose disposal in the body [7]. Most of the glucose that enters muscle cells in response to insulin is deposited as glycogen [27] and hence, a defect in muscle glycogen synthesis is suggested to contribute to insulin resistance [1, 5]. It is well documented that PKB is involved in transmission of stimulatory effects of insulin on glycogen synthesis [7, 11, 14]. As with Wistar rats, in the Zucker rats insulin stimulated PKBα activity in the muscle with a time point similar to that observed in Wistar rats. However, insulin-induced activation of PKBα was markedly reduced in the fatty Zucker rats as compared to their lean littermates, indicating a decrease in response to insulin in these animals. Hence, it seems that there is an association between PKBα activity and insulin resistance in fatty Zucker rats. Results obtained from Western blots showed that the decrease in PKBα activity in fatty rats was not due to its lower protein expression. These results are in agreement with those obtained from our laboratory and others that have shown an impairment in kinase activity at the level of PI3-K [35, 38, 39] and PKB [40, 41] in the skeletal muscle in type 2 diabetes. Measurement of PKBβ activity, the other isoform of PKB which has been shown to be activated by insulin to a lesser degree in the skeletal muscle, ruled out the possibility that changes in PKBβ activity might compensate for the decrease observed in insulin-induced PKBα activity in the fatty Zucker rats. Liver In the liver glucose uptake is not rate-limiting for glucose utilization [5]. However, as with muscle, insulin-induced activation of glycogen synthase in hepatocytes is mediated via the PI3-K/PKB/GSK-3 axis [11]. In contrast to muscle, insulin-induced PKBα activity was significantly increased in the liver of fatty Zucker rats as compared to lean controls, suggesting that (i) the response to insulin was increased in the liver; (ii) changes in PKBα were tissue specific; (iii) PKBα might have different roles in various insulin sensitive tissues. It has also been shown that IRS-1 associated PI3-K activity was decreased in the liver of fatty Zucker rats [38], while at the level of PKB we found a marked increase in enzyme activity in the liver. These observations indicate that there might be a dissociation between PI3-K and PKBα in the liver. Alterations in PKBα activity in the absence of changes in PI3-K activity or vice versa seen in other studies further support this hypothesis [42, 43]. Insulin (5 U/kg, i.v.) activated PKBβ in the liver to a much lesser extent as compared to PKBα. Our results did not show any significant difference in basal or insulin-induced PKBβ activity between lean and fatty Zucker rats. However, a recent study reported an impairment in insulin-stimulated PKBβ activity in the liver of 8-week old female fatty Zucker rats with 10 U/kg insulin [44]. The difference observed between the results of these two studies can be explained by differences in doses of insulin used, and age/gender of rats. The increase in response to insulin observed in the liver of fatty Zucker rats was an interesting and unexpected finding. The importance of this finding is still not clear. Liver is the main site of gluconeogenesis, which contributes to the elevated fasting plasma glucose levels in diabetes [3]. Insulin inhibits gene expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), the two key enzymes in the gluconeogenesis which have been shown to be up regulated in diabetes, via a PI3-K dependent pathway [45, 46]. Although downstream mediators of PI3- K are still not clear, there are several pieces of evidence indicating that inhibitory effects of insulin might be mediated via PKB [45, 47] and support the concept that PKB may

10 156 contribute to the control of net hepatic glucose production. Furthermore, nuclear translocation of PKB leads to the possibility that PKB may be involved in the transcription of at least some of insulin-induced hepatic genes such as PEPCK and G6Pase [48]. Taken together, it can be postulated that an increase in PKBα activity in the liver of fatty Zucker rats may be due to the activation of a compensatory mechanism which results in the stimulation of PKBα, probably via a PI3-K independent pathway, in order to inhibit the elevated levels of gluconeogenesis by suppressing the key enzyme(s) in gluconeogenesis pathway. However, further studies are required to understand the steps following PKB activation, and the key enzymes that may act as substrates for PKB in the liver still remain to be identified. Effects of BMOV-treatment on PKB activity Treatment with BMOV normalized plasma glucose concentrations in STZ-diabetic rats but did not have any effect on basal or insulin-stimulated PKBα activity or its protein expression in the skeletal muscle and liver. In fatty Zucker rats, BMOV restored plasma insulin levels and improved insulin sensitivity but did not restore PKBα activity to normal level in the liver or muscle, neither did it have any effect on PKBβ activity. These results clearly show that, in contrast to the results obtained from in vitro studies with vanadium compounds [30, 34], the glucoregulatory effects of BMOV are not mediated via PKB in vivo. Our previous studies had shown that BMOV was also unable to normalize the reduced level of insulin-induced IRS-1 associated PI3-K activity in the skeletal muscle of fatty Zucker rats [35]. Furthermore, BMOV did not have any effect on glycogen synthase kinase- 3 (GSK-3) activity in the skeletal muscle or liver in vivo (S. Semiz and J.H. McNeill, unpublished data). Glucose homeostasis depends upon a balance between hepatic glucose production and glucose utilization by peripheral tissues. BMOV, at doses that are known to decrease plasma glucose in type 1 diabetes and attenuate plasma insulin in type 2 diabetes, does not affect insulin signaling at the level of PI3-K, PKB or GSK-3, three key enzymes involved in glucose utilization. Hence, one alternative mechanism by which BMOV may mediate its glucose lowering effects would be via inhibition of the enzymes involved in the hepatic glucose production. Indeed, data from our recent studies show that BMOV prevents the increase in PEPCK and G6Pase gene expression in the liver of diabetic rats (L. Marzban and J.H. McNeill, unpublished data). However, further investigation is required to elucidate exactly which pathway(s) are involved in mediating the effects of BMOV. In summary, these results suggest that PKBα may not be important in the development of insulin resistance in type 1 diabetes. In contrast, the increased insulin-stimulated PKBα activity in the liver in type 2 diabetes may have physiological meaning taken in concert with the decreased insulin-induced PKBα activity in the skeletal muscle. BMOV at doses that was able to normalize plasma glucose levels and improve insulin sensitivity did not have any effect on PKBα or PKBβ activity indicating that the glucoregulatory effects of BMOV are independent of PKB in vivo. Acknowledgements This study was supported by an NSERC/TPP grant and by Kinetek Pharmaceuticals Inc. Lucy Marzban is a recipient of the Canadian Institutes of Health Research studentship. Technical support of Ms. Mary Battell, Ms. Violet Yuen, and Ms. Becky Dinesen is gratefully acknowledged. The authors would like to thank Dr. Chris Orvig and Dr. Katherine Thompson, Department of Chemistry, The University of British Columbia, for providing the BMOV. References 1. Kahn CR: Insulin action, diabetogenes, and the cause of type II diabetes. Diabetes 43: , Saltiel AR: Diverse signaling pathways in the cellular actions of insulin. Am J Physiol Endocrinol Metab 270: E375 E385, DeFronzo RA: The triumvirate: β-cell, muscle, liver: A collusion responsible for NIDDM. Diabetes 37: , Yki-Järvinen H, Koivisto VA: Natural course of insulin resistance in type 1 diabetes. N Engl J Med 315: , Yki-Järvinen H, Sahlin K, Ren JM, Koivisto VA: Localization of ratelimiting defect for glucose disposal in skeletal muscle of insulin-resistant type 1 diabetic patients. Diabetes 39: , Ziel FH, Venkatesan N, Davidson MB: Glucose transport is rate limiting for skeletal muscle glucose metabolism in normal and STZ-induced diabetic rats. Diabetes 37: , Hei YJ: Recent progress in insulin signal transduction. J Pharmacol Toxicol Meth 40: , Halse R, Rochford JJ, McCormack JG, Vandenheede JR, Hemmings BA, Yeaman SJ: Control of glycogen synthesis in cultured human muscle cells. J Biol Chem 274: , Yeh JI, Gulve EA, Rameh L, Birnbaum MJ: The effects of wortmannin on rat skeletal muscle. J Biol Chem 270: , Lazar DF, Wiese RJ, Brady MJ, Mastick CC, Waters SB, Yamauchi K, Pessin JE, Cuatrecasas P, Saltiel AR: Mitogen-activated protein kinase kinase inhibition does not block the stimulation of glucose utilization by insulin. J Biol Chem 270: , Lavoie L, Band CJ, Kong M, Bergeron JJM, Posner BI: Regulation of glycogen synthase in rat hepatocytes. J Biol Chem 274: , Kohn AD, Summers SA, Birnbaum MJ, Roth RA: Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J Biol Chem 271: , Turinsky J, Damrau-Abney A: Akt kinases and 2-deoxyglucose uptake in rat skeletal muscles in vivo: Study with insulin and exercise. Am J Physiol Reg Integ Comp Physiol 276: R277 R282, 1999

11 Ueki K, Yamamoto-Honda R, Kaburagi Y, Yamauchi T, Tobe K, Burgering BMT, Coffer PJ, Komuro I, Akanuma Y, Yazaki Y, Kadowaki T: Potential role of protein kinase B in insulin-induced glucose transport, glycogen synthesis, and protein synthesis. J Biol Chem 273: , Sekar N, Li J, Shechter Y: Vanadium salts as insulin substitutes: Mechanisms of action, a scientific and therapeutic tool in diabetes mellitus research. Crit Rev Biochem Mol Biol 31: , Blondel O, Bailbe D, Portha B: In vivo insulin resistance in streptozotocin-diabetic rats - evidence for reversal following oral vanadate treatment. Diabetologia 32: , Ramanadham S, Mongold JJ, Brownsey RW, Cros GH, McNeill JH: Oral vanadyl sulfate in treatment of diabetes mellitus in rats. Am J Physiol Heart Circ Physiol 257: H904 H911, Brichard SM, Pottier AM, Henquin JC: Long term improvement of glucose homeostasis by vanadate in obese hyperinsulinemic fa/fa rats. Endocrinology 125: , 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: , Bhanot S, McNeill JH: Vanadyl sulfate lowers plasma insulin and blood pressure in spontaneously hypertensive rats. Hypertension 24: , Bhanot S, McNeill JH, Bryer-Ash M: Vanadyl sulfate prevents fructose-induced hyperinsulinemia and hypertension in rats. Hypertension 23: , Elberg G, He Z, Li J, Sekar N, Shechter Y: Vanadate activates membranous nonreceptor protein tyrosine kinase in rat adipocytes. Diabetes 46: , Shisheva A, Shechter Y: Role of cytosolic tyrosine kinase in mediating insulin-like actions of vanadate in rat adipocytes. J Biol Chem 268: , Poucheret P, Verma S, Grynpas MD, McNeill JH: Vanadium and diabetes. Mol Cell Biochem 188: 73 80, McNeill JH, Yuen VG, Hoveyda HR, Orvig C: Bis(maltolato)oxovanadium (IV) is a potent insulin mimic. J Med Chem 35: , Matsuda M, DeFronzo RA: Insulin sensitivity indices obtained from oral glucose tolerance testing. Diabetes Care 22: , Shulman GI, Rothman DL, Jue T, Stein P, DeFronzo RA, Shulman RG: Quantitation of muscle glycogen synthesis in normal subjects and subjects with non-insulin-dependent diabetes by 13 C nuclear magnetic resonance spectroscopy. N Engl J Med 322: , Shepherd PR, Nave BT, Rincon J, Haigh RJ, Foulstone E, Proud C, Zierath JR, Siddle K, Wallberg-Henriksson H: Involvement of phosphoinositide 3-kinase in insulin stimulation of MAP-kinase and phosphorylation of protein kinase-b in human skeletal muscle: Implications for glucose metabolism. Diabetologia 40: , Moule SK, Denton RM: Multiple signaling pathways involved in the metabolic effects of insulin. Am J Cardiol 80: 41A 49A, Wijkander J, Holst LS, Rahn T, Resjö S, Castan I, Manganiello V, Belfrage P, Degerman E: Regulation of protein kinase B in rat adipocytes by insulin, vanadate, and peroxovanadate. J Biol Chem 272: , Konishi H, Matsuzaki H, Tanaka M, Ono Y, Tokunaga C, Kuroda S, Kikkawa U: Activation of RAC-protein kinase by heat shock and hyperosmolarity stress through a pathway independent of phosphatidylinositol 3-kinase. Proc Natl Acad Sci USA 93: , Konishi H, Matsuzaki H, Tanaka M, Takemura Y, Kuroda S, Ono Y, Kikkawa U: Activation of protein kinase B (Akt/RAC-protein kinase) by cellular stress and its association with heat shock protein Hsp27. FEBS Lett 410: , Sable CL, Filippa N, Hemmings B, Van Obberghen E: camp stimulates protein kinase B in a wortmannin-insensitive manner. FEBS Lett 409: , Donthi RV, Huisamen B, Lochner A: Effects of vanadate and insulin on glucose transport in isolated adult rat cardiomyocytes. Cardiovasc Drugs Ther 14: , Mohammad A, Bhanot S, McNeill JH: In vivo effects of vanadium in diabetic rats are independent of changes in PI-3 kinase activity in skeletal muscle. Mol Cell Biochem (in press) 36. Ionescu E, Sauter JF, Jeanrenaud B: Abnormal oral glucose tolerance in genetically obese (fa/fa) rats. Am J Physiol Endocrinol Metab 248: E500 E506, Kasiske BL, O Donnell MP, Keane WF: The Zucker rat model of obesity, insulin resistance, hyperlipidemia, and renal injury. Hypertension 19(suppl I): I-110 I-115, Anai M, Funaki M, Ogihara T, Terasaki J, Inukai K, Katagiri H, Fukushima Y, Yazaki Y, Kikuchi M, Oka Y, Asano T: Altered expression levels and impaired steps in the pathway to phosphatidylinositol 3- kinase activation via insulin receptor substrates 1 and 2 in Zucker fatty rats. Diabetes 47: 13 23, Björnholm M, Kawano Y, Lehtihet M, Zierath JR: Insulin receptor substrate-1 phosphorylation and phosphatidylinositol 3-kinase activity in skeletal muscle from NIDDM subjects after in vivo insulin stimulation. Diabetes 46: , Krook A, Kawano Y, Song XM, Efendi S, Roth RA, Wallberg-Henriksson H, Zierath JR: Improved glucose tolerance restores insulinstimulated Akt kinase activity and glucose transport in skeletal muscle from diabetic Goto-Kakizaki rats. Diabetes 46: , Krook A, Roth RA, Jiang XJ, Zierath JR, Wallberg-Henriksson H: Insulin-stimulated Akt kinase activity is reduced in skeletal muscle from NIDDM subjects. Diabetes 47: , 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: , Kurowski TG, Lin Y, Luo Z, Tsichlis PN, Buse MG, Heydrick SJ, Ruderman NB: Hyperglycemia inhibits insulin activation of Akt/protein kinase B but not phosphatidylinositol 3-kinase in rat skeletal muscle. Diabetes 48: , Kim YB, Peroni OD, Franke TF, Kahn BB: Divergent regulation of Akt1 and Akt2 isoforms in insulin target tissues of obese Zucker rats. Diabetes 49: , Schmoll D, Walker KS, Alessi DR, Grempler R, Burchell A, Guo S, Walther R, Unterman TG: Regulation of glucose-6-phosphatase gene expression by protein kinase Bα and the forkhead transcription factor FKHR. J Biol Chem 275: , Sutherland C, Waltner-law M, Gnudi L, Kahn BB, Granner DK: Activation of the Ras mitogen-activated protein kinase-ribosomal protein kinase pathway is not required for the repression of phosphoenolpyruvate carboxykinase gene transcription by insulin. J Biol Chem 273: , Liao J, Barthel A, Nakatani K, Roth RA: Activation of protein kinase B/Akt is sufficient to repress the glucocorticoid and camp induction of phosphoenolpyruvate carboxykinase gene. J Biol Chem 273: , Galetic I, Andjelkovic M, Meier R, Brodbeck D, Park J, Hemmings BA: Mechanism of protein kinase B activation by insulin/insulin-like growth factor-1 revealed by specific inhibitors of phosphoinositide 3- kinase significance for diabetes and cancer. Pharmacol Ther 82: , 1999

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