Modulation of muscle insulin resistance by selective inhibition of GSK-3 in Zucker diabetic fatty rats

Erik J. Henriksen1, Tyson R. Kinnick1, Mary K. Teachey1, Matthew P. O'Keefe1, David Ring2, Kirk W. Johnson2, and Stephen D. Harrison2

1 Muscle Metabolism Laboratory, Department of Physiology, University of Arizona College of Medicine, Tucson, Arizona 85721; and 2 Chiron Corporation, Emeryville, California 94608


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A role for elevated glycogen synthase kinase-3 (GSK-3) activity in the multifactorial etiology of insulin resistance is now emerging. However, the utility of specific GSK-3 inhibition in modulating insulin resistance of skeletal muscle glucose transport is not yet fully understood. Therefore, we assessed the effects of novel, selective organic inhibitors of GSK-3 (CT-98014 and CT-98023) on glucose transport in insulin-resistant muscles of Zucker diabetic fatty (ZDF) rats. Incubation of type IIb epitrochlearis and type I soleus muscles from ZDF rats with CT-98014 increased glycogen synthase activity (49 and 50%, respectively, P < 0.05) but did not alter basal glucose transport (2-deoxyglucose uptake). In contrast, CT-98014 significantly increased the stimulatory effects of both submaximal and maximal insulin concentrations in epitrochlearis (37 and 24%) and soleus (43 and 26%), and these effects were associated with increased cell-surface GLUT4 protein. Lithium enhanced glycogen synthase activity and both basal and insulin-stimulated glucose transport in muscles from ZDF rats. Acute oral administration (2 × 30 mg/kg) of CT-98023 to ZDF rats caused elevations in GSK-3 inhibitor concentrations in plasma and muscle. The glucose and insulin responses during a subsequent oral glucose tolerance test were reduced by 26 and 34%, respectively, in the GSK-3 inhibitor-treated animals. Thirty minutes after the final GSK-3 inhibitor treatment, insulin-stimulated glucose transport was significantly enhanced in epitrochlearis (57%) and soleus (43%). Two hours after the final treatment, insulin-mediated glucose transport was still significantly elevated (26%) only in the soleus. These results indicate that specific inhibition of GSK-3 enhances insulin action on glucose transport in skeletal muscle of the insulin-resistant ZDF rat. This unique approach may hold promise as a pharmacological treatment against insulin resistance of skeletal muscle glucose disposal.

glycogen synthase kinase-3; type 2 diabetes; glucose transport; epitrochlearis; soleus; cell surface glucose transporter-4; lithium


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INSULIN RESISTANCE OF SKELETAL MUSCLE glucose transport and metabolism is considered to be one of the primary defects underlying the development of glucose intolerance and type 2 diabetes (reviewed in Ref. 38). The insulin resistance and the accompanying hyperinsulinemia are closely associated with a number of additional atherogenic risk factors, including hypertension, dyslipidemia, and central obesity, collectively referred to as "syndrome X" (25) or the "insulin resistance syndrome" (9). Therefore, developing strategies to overcome the insulin resistance of skeletal muscle glucose transport is an important step in treating type 2 diabetes.

Glycogen synthase kinase-3 (GSK-3), a serine/threonine kinase that consists of highly homologous alpha - and beta -isoforms (36), functions to phosphorylate and inactivate glycogen synthase (GS) (24, 28, 37). GSK-3 activity can be acutely inactivated by insulin signaling through insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI 3-kinase), and ultimately via the action of protein kinase B (Akt) to phosphorylate specific serine residues on the enzyme (7). An additional substrate of GSK-3 is IRS-1, and phosphorylation of IRS-1 on serine/threonine residues leads to impairment of insulin signaling (10). Therefore, GSK-3 can be a negative modulator of insulin action on GS and, potentially, on glucose transport activity.

Although the etiology of skeletal muscle insulin resistance is multifactorial (38), recent evidence supports a role of elevated GSK-3 as a contributing factor in this pathophysiological state (2, 11, 19, 21, 22). GSK-3 is elevated in tissues of insulin-resistant obese rodent models (2, 11) and in skeletal muscle of obese humans (2) and type 2 diabetic humans (22). Moreover, the elevation in GSK-3 protein in skeletal muscle of type 2 diabetic subjects is negatively correlated with both insulin-stimulated skeletal muscle GS activity and whole body glucose disposal (22). Finally, lithium ions, a noncompetitive and relatively nonselective inhibitor of GSK-3 with a Ki in the millimolar range (8, 16, 18, 32), can enhance both insulin-independent and insulin-dependent glucose transport in skeletal muscle from insulin-sensitive rats (34). Taken collectively, these findings are consistent with GSK-3 as a potential target of inhibition for the enhancement of insulin-stimulated glucose transport in insulin-resistant skeletal muscle.

Recently, a class of novel and selective organic inhibitors of GSK-3 has been developed (27) and has shown promise as modulators of GS activity, glucose disposal, and glucose transport activity in diabetic rodent models (27) and in human muscle cells (21). These compounds are substituted aminopyrimidine molecules and act as potent competitive inhibitors (acting at the ATP-binding site) of human GSK-3 (Ki < 10 nM) with >= 500-fold selectivity against 20 other protein kinases (27). The purposes of the present investigation were 1) to compare the in vitro effects of a novel, selective organic inhibitor of GSK-3 (CT-98014) and lithium ions on basal and insulin-stimulated GS activity and glucose transport activity in type I (soleus) and type IIb (epitrochlearis) skeletal muscle of insulin-sensitive lean Zucker (Fa+/-) and insulin-resistant Zucker diabetic fatty (ZDF) rats, the latter being a model of type 2 diabetes; 2) to assess whether any improvement of muscle glucose transport activity due to this GSK-3 inhibitor is associated with enhanced cell-surface GLUT4 protein; and 3) to determine the effect of the acute oral administration of a GSK-3 inhibitor (CT-98023) to ZDF rats on glucose tolerance and skeletal muscle glucose transport activity.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male ZDF/Gmi-fa rats were obtained from Genetic Models (Indianapolis, IN), and lean Zucker rats were purchased from Harlan (Indianapolis, IN) at the age of 8-9 wk and used in the experiments at 10 wk of age. At the time of their use, the ZDF rats weighed 300-340 g, whereas the age-matched lean Zucker rats weighed 180-210 g. Lean animals were maintained on regular lab chow (Purina, St. Louis, MO), whereas ZDF animals had free access to diabetogenic chow (Purina 5008). All procedures were approved by the University of Arizona Animal Use and Care Committee.

In vitro treatments of skeletal muscle. After an overnight fast, animals were deeply anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg), and intact epitrochlearis muscles and strips of soleus muscles (~25 mg) were prepared for in vitro incubation in the unmounted state. Each muscle was incubated for 1 h at 37°C in 3 ml of oxygenated (95% O2-5% CO2) Krebs-Henseleit buffer (KHB) with the NaHCO3 concentration set at 14 mM. This KHB was supplemented with 8 mM glucose, 32 mM mannitol, 0.1% BSA (radioimmunoassay grade, Sigma Chemical, St. Louis, MO), 0.5% dimethyl sulfoxide, and the indicated additions of GSK-3 inhibitor, insulin, or lithium chloride. Thereafter, the muscles were used for the determination of glucose transport activity [as assessed using 2-deoxyglucose (2-DG) uptake] or GS activity as described in Muscle glucose transport activity.

Oral administration of GSK-3 inhibitor. Animals were fasted after 6 PM of the evening before the test. At 8 AM and then again at 11:30 AM, the animals received by gavage either vehicle (1% carboxymethylcellulose sodium/0.1% Tween) or a bolus of GSK-3 inhibitor CT-98023 at a dose of 30 mg/kg body wt. Thirty minutes after the second administration, some animals underwent an oral glucose tolerance test (OGTT). These animals received a 2 g/kg body wt glucose load by gavage. Blood was collected from a small cut at the tip of the tail immediately before and at 30, 60, 90, and 120 min after glucose administration, thoroughly mixed with EDTA (final concentration of 18 mg/ml), and centrifuged at 13,000 g to isolate the plasma. The plasma was stored at -80°C and subsequently assayed for glucose (Sigma Chemical) and insulin by radioimmunoassay (Linco, St. Louis, MO).

Other animals were used for the in vitro assessment of insulin-stimulated glucose transport activity in skeletal muscle. At the indicated times after the final bolus of vehicle or GSK-3 inhibitor, the animals were deeply anesthetized with pentobarbital sodium, and epitrochlearis muscles and soleus strips were prepared. All muscles were then incubated as described for 60 min in KHB in the absence or presence of the indicated concentration of insulin. Thereafter, glucose transport activity was assessed as described in Muscle glucose transport activity. At the time of muscle dissection, samples of plasma and soleus and plantaris muscles were taken and frozen in liquid nitrogen for the determination of GSK-3 inhibitor levels by high-performance liquid chromatography and mass spectroscopy.

GS activity. GS was assayed essentially as described by Thomas et al. (35), with slight modifications (15). Muscles were homogenized in a Duall tube containing 2 ml of ice-cold 50 mM Tris buffer (pH 7.8), 100 mM KF, 10 mM EDTA, 2 mM EGTA, and 2 mM KH2PO4 at 4°C. The homogenates were centrifuged at 13,000 g for 15 min. An aliquot (30 µl) of the supernatant was added to 60 µl of 50 mM Tris buffer (pH 7.8), 20 mM EDTA, 25 mM KF, 10 mg/ml rabbit liver glycogen, and 5 mM UDP-[U-14C]glucose (150 µCi/mmol) and incubated for 20 min at 30°C without glucose 6-phosphate (GSI activity). Total GS activity was assessed in the presence of 5 mM glucose 6-phosphate.

Muscle glucose transport activity. After the initial incubation period, the muscles were rinsed for 10 min at 37°C in 3 ml of oxygenated KHB containing 40 mmol/l mannitol, 0.1% BSA, and any addition present previously. Thereafter, the muscles were transferred to 2 ml of KHB containing 1 mM 2-deoxy-[1,2-3H]glucose (300 mCi/mmol; Sigma), 39 mM [U-14C]mannitol (0.8 mCi/mmol; ICN Radiochemicals, Irvine, CA), 0.1% BSA, and any additions present previously. At the end of this final 20-min incubation period at 37°C, the muscles were removed, trimmed of excess fat and connective tissue, quickly frozen between aluminum blocks cooled in liquid nitrogen, and weighed. The frozen muscles were dissolved in 0.5 ml of 0.5 N NaOH. After the muscles were completely solubilized, 5 ml of scintillation cocktail were added, and the specific intracellular accumulation of 2-DG was determined as described previously (14). This method for assessing glucose transport activity in isolated muscle has been validated (12).

Statistical analysis. All data are presented as means ± SE. The significance of differences between multiple groups was assessed by a factorial ANOVA with a post hoc Fisher's paired least significant difference test (StatView version 5.0, SAS Institute, Cary, NC). Differences between two groups were determined by an unpaired Student's t-test. A P value of <0.05 was considered to be statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In vitro effects of GSK-3 inhibitor CT-98014 or lithium on GS activity. We initially determined the effectiveness of CT-98014 in inhibiting GSK-3 in isolated skeletal muscle preparations by use of an increase in GSI activity as the biomarker. As shown in Fig. 1, 500 nM CT-98014 caused increases of 51 and 48% in GSI activity in the epitrochlearis and soleus muscles, respectively, of the lean Zucker rat. These increases due to CT-98014 were comparable to those elicited by 2 mU/ml insulin in these muscles (47 and 78%). The effects of CT-98014 and insulin on activation of GSI in these muscles of the lean Zucker rats were completely additive. The total activity of GS was not altered in these muscles, due to the interventions (data not shown).


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Fig. 1.   Interactions of insulin and glycogen synthase kinase-3 (GSK-3) inhibitor CT-98014 on glycogen synthase (GSI) activity in skeletal muscle of lean Zucker and Zucker diabetic fatty (ZDF) rats. Epitrochlearis and soleus muscles were incubated with 2 mU/ml insulin and 500 nM CT-98014 for 60 min. Values are means ± SE for 5 muscles per group. *P < 0.05 vs. control group; #P < 0.05 vs. all other groups.

Insulin elicited small, but significant, increases in the GSI activity of epitrochlearis (24%) and soleus (23%) muscles of the ZDF rat, reflecting the insulin-resistant state of these muscles. In contrast, CT-98014 elicited a completely normal activation of GSI in these insulin-resistant muscles (49 and 50%). Moreover, when CT-98014 and insulin were present in combination, the activation of GS was substantially more than could be accounted for by summing the individual effects of the stimuli in the epitrochlearis (0.776 nmol · mg-1 · min-1 vs. theoretical additive value of 0.662) and the soleus (1.31 vs. 1.11). The total activity of GS was again not altered, due to the interventions (data not shown). The total GS activities in the epitrochlearis (1.69 ± 0.04 vs. 1.26 ± 0.05 nmol · mg muscle-1 · min-1) and soleus (2.41 ± 0.06 vs. 1.63 ± 0.05 nmol · mg muscle-1 · min-1) were significantly greater in ZDF rats compared with lean Zucker rats, consistent with previous findings in the obese Zucker rat (30).

The effects of 10 mM lithium on the GSI activity in muscle from lean Zucker and ZDF rats are shown in Fig. 2. Lithium significantly activated GSI in epitrochlearis and soleus muscles of the lean Zucker rat (86 and 38%) and the ZDF rat (71 and 86%). As was the case with CT-98014, when lithium was combined with insulin, there was a completely additive interaction on GSI activation in muscle from the lean Zucker rat and a synergistic interaction on GS activation in epitrochlearis (1.01 vs. theoretical additive value of 0.846) and soleus muscles (1.59 vs. 1.45) of the ZDF rat.


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Fig. 2.   Interactions of insulin and lithium on glycogen synthase (GSI) activity in skeletal muscle of lean Zucker and ZDF rats. Epitrochlearis and soleus muscles were incubated with 2 mU/ml insulin and 10 nM lithium for 60 min. Values are means ± SE for 5 muscles per group. *P < 0.05 vs. control group; #P < 0.05 vs. all other groups.

In vitro effects of GSK-3 inhibitor CT-98014 or lithium on glucose transport activity. CT-98014 (500 nM) had no effect on basal glucose transport or on submaximally or maximally insulin-stimulated glucose transport activity in epitrochlearis and soleus muscle from the lean Zucker rat (Fig. 3). In contrast, 10 mM lithium elicited significant increases in both basal glucose transport and insulin-stimulated glucose transport activity in skeletal muscle from the lean Zucker rats.


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Fig. 3.   Effects of GSK-3 inhibitor CT-98014 or lithium on glucose transport activity in skeletal muscle of lean Zucker rats. Epitrochlearis and soleus muscles were incubated with insulin and 500 nM CT-98014 or 10 mM LiCl for 60 min. Values are means ± SE for 5 muscles per group. Soleus data with CT-98014 are adapted from Ring et al. (27). *P < 0.05 vs. minus lithium group.

CT-98014 at 500 nM also did not cause an activation of basal glucose transport in skeletal muscle of the ZDF rats, whereas 10 mM lithium alone stimulated basal glucose transport in both epitrochlearis and soleus muscles of these insulin-resistant animals (Fig. 4). Importantly, 500 nM CT-98014 caused significant enhancements of glucose transport activity in the presence of submaximally effective (37 and 43%) and maximally effective (24 and 27%) concentrations of insulin in epitrochlearis and soleus (27) muscles of ZDF rats. These significant effects of CT-98014 on insulin-stimulated glucose transport activity in muscle of the ZDF rat were observed at concentrations as low as 100 nM, with no greater maximal effects with concentrations as high as 1 µM (data not shown). Lithium at 10 mM also significantly enhanced glucose transport activity in ZDF muscle incubated with submaximally and maximally effective insulin concentrations, although most of this effect could be attributed to the activation of basal glucose transport (Fig. 4).


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Fig. 4.   Effects of GSK-3 inhibitor CT-98014 or lithium on glucose transport activity in skeletal muscle of ZDF rats. Epitrochlearis and soleus muscles were incubated with 500 nM CT-98014 or 10 mM lithium for 60 min. Values are means ± SE for 5 muscles per group. Soleus data with CT-98014 are adapted from Ring et al. (27). *P < 0.05 vs. minus CT-98014 or minus lithium group.

In vitro effects of GSK-3 inhibitor CT-98014 on cell surface GLUT4. To gain some insight into the molecular mechanism for the effects of CT-98014 on glucose transport, we assessed the level of exofacial GLUT4 protein, using the impermeable photolabel 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzonyl]-1,3-bis(D-mannos-4-yloxy)-2-propylamine (ATB-[2-3H]BMPA) (ATB-[3H]BMPA; Fig. 5). In the absence of insulin, CT-98014 did not alter cell-surface GLUT4 in either epitrochlearis or soleus muscles of the ZDF rat. Insulin caused significant increases in cell-surface GLUT4 in these muscles. Moreover, the insulin-stimulated increases in cell-surface GLUT4 were significantly enhanced by CT-98014 in both the epitrochlearis (29%) and the soleus (21%) muscles of the ZDF rat.


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Fig. 5.   Effect of GSK-3 inhibitor CT-98014 on cell surface GLUT4 in skeletal muscle of ZDF rats. Epitrochlearis and soleus muscles were incubated with 500 nM CT-98014 for 60 min. Values are means ± SE for 5 muscles per group. *P < 0.05 vs. control or plus CT-98014 groups; #P < 0.05 vs. plus insulin group.

Effects of oral administration of GSK-3 inhibitor CT-98023 on glucose tolerance. The acute oral administration of the GSK-3 inhibitor CT-98023 (30 mg/kg body wt) led to substantial increases of the compound in plasma and skeletal muscle of the ZDF rats 30 min after completion of the treatments (Fig. 6), indicating that the metabolic actions of the compound in vivo should not be limited by bioavailability. Thirty minutes after the completion of these treatments, an OGTT was performed, and the responses of plasma glucose and insulin were assessed (Fig. 7). In vehicle-treated ZDF rats, the glucose feeding led to a large increase in plasma glucose at 30 min, and this glucose response was substantially reduced in the GSK-3 inhibitor-treated animals. The total glucose area under the curve (AUC) was 26% less in the GSK-3 inhibitor-treated ZDF rats than in the vehicle-treated control group (Fig. 8). Moreover, the insulin response during the OGTT was also dramatically reduced in the GSK-3 inhibitor-treated group, and the total insulin AUC was 34% less in the GSK-3 inhibitor-treated ZDF rats than in the control group. These decreases in the glucose and insulin responses of the OGTT following the GSK-3 inhibitor treatment were even more pronounced when the incremental AUCs for glucose (13,620 ± 1,216 vs. 6,600 ± 644 mg · dl-1 · min) and insulin (4,290 ± 540 vs. 1,020 ± 205 µU · ml-1 · min) were calculated. The dose of GSK-3 inhibitor used in this in vivo study (30 mg/kg) is maximally effective in enhancing oral glucose tolerance, as a higher dose (48 mg/kg) does not have any further effect (27).


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Fig. 6.   Levels of GSK-3 inhibitor CT-98023 in plasma and skeletal muscle following acute oral administration. Bars represent the level of compound 30 min after the final oral treatment with 30 mg/kg CT-98023. Values are means ± SE for 6 animals per group.



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Fig. 7.   Effect of GSK-3 inhibitor CT-98023 on glucose and insulin responses during an oral glucose tolerance test in ZDF rats. Animals were treated twice with either vehicle (open circle ) or 30 mg/kg body wt CT-98023 (). Values are means ± SE for 6-7 animals per group. *P < 0.05 vs. vehicle-treated control group at same time point.



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Fig. 8.   Effect of GSK-3 inhibitor CT-98023 on glucose and insulin areas under the curves (AUCs) and the glucose-insulin index in ZDF rats. Values for the AUCs were calculated from the data presented in Fig. 7. The glucose-insulin index is calculated as the product of the glucose AUC and the insulin AUC for each animal. Values are means ± SE for 6-7 animals per group. *P < 0.05 vs. vehicle-treated control group.

The glucose-insulin index is defined as the product of the glucose and insulin AUCs, and a reduction in this value represents indirect evidence of improved in vivo peripheral insulin action (6). After acute treatment with CT-98023, the glucose-insulin index was reduced by 51% compared with the vehicle-treated group, consistent with an increase in whole body insulin sensitivity. This decrease in the glucose-insulin index due to GSK-3 inhibition was also observed when the incremental AUCs were used (58 ± 12 vs. 7 ± 1 units × 106).

Effects of oral administration of GSK-3 inhibitor CT-98023 on muscle glucose transport. To identify a potential cellular locus for the improved peripheral insulin sensitivity, insulin-stimulated glucose transport activity was determined in epitrochlearis and soleus muscles of the ZDF rats at various time points after the acute administration of CT-98023 (Fig. 9). Thirty minutes after the completion of the oral GSK-3 inhibitor treatment, the maximal insulin-mediated increase in glucose transport activity was enhanced in both the epitrochlearis (56%) and soleus (43%) compared with the corresponding control groups. At this same time point, glucose transport activity due to a submaximally effective concentration of insulin (150 µU/ml) was similarly enhanced in these muscles from the CT-98023-treated ZDF rats (data not shown). Two hours after the oral administration of CT-98023, the enhanced action of the maximally effective concentration of insulin was still detectable in the soleus muscle (26%) but not in the epitrochlearis muscle, consistent with the diminished glucose-lowering effect of the treatment at this time point (Fig. 7). The enhanced insulin action on glucose transport activity due to the GSK-3 inhibitor treatment was no longer detectable in the soleus after 4 h.


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Fig. 9.   Insulin-mediated glucose transport activity in epitrochlearis (A) and soleus (B) muscles at various times following acute oral administration of GSK-3 inhibitor CT-98023 in ZDF rats. Values represent the increase above basal due to 5 mU/ml insulin. Values are means ± SE for 5-8 muscles per group. *P < 0.05 vs. vehicle-treated control group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have demonstrated in the present investigation that the selective inhibition of GSK-3 in insulin-resistant skeletal muscle of the ZDF rat was associated with a potentiation of the ability of insulin to stimulate both GS activity (Fig. 1) and glucose transport activity (Fig. 3). These effects of the GSK-3 inhibitor CT-98014 were elicited at concentrations in the nanomolar range, with maximal effects at 500 nM. Interestingly, although the GSK-3 inhibitor CT-98014 clearly activated GS activity in skeletal muscle from the insulin-sensitive lean Zucker rat (Fig. 1), it had absolutely no effect on either basal or insulin-stimulated glucose transport activity in muscle from these lean animals. These results support the idea that an elevation in GSK-3 activity, which exists in muscle of the ZDF rat but not the lean rat (2), is necessary for a cell to respond to GSK-3 inhibition for enhancement of insulin-stimulated glucose transport activity.

These results demonstrating a potentiation of in vitro insulin action on GS and glucose transport in rat muscle by selective GSK-3 inhibition are in agreement with the recent findings of Nikoulina et al. (21), who showed in cultured human myocytes that these same GSK-3 inhibitors, when administered acutely (30 min), can upregulate insulin-stimulated GS activity and, when administered chronically (4 days), can enhance insulin-mediated glucose transport activity. Interestingly, these investigators also demonstrated that chronic treatment of cultured human muscle cells with GSK-3 inhibitor caused a downregulation of GSK-3 protein expression and activity and a large upregulation of IRS-1 protein level. Whether the chronic administration of GSK-3 inhibitor causes similar effects in insulin-resistant muscle from rodent models or humans remains to be determined.

At least part of the beneficial effect of the GSK-3 inhibitor CT-98014 on insulin-stimulated glucose transport activity in skeletal muscle of the ZDF rat was due to an enhancement of GLUT4 protein at the cell surface (Fig. 5). This is in contrast to the finding that the nonspecific GSK-3 inhibitor lithium did not enhance insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes overexpressing GSK-3beta (33). The underlying molecular mechanism for the increased GLUT4 translocation in response to the GSK-3 inhibition in insulin-resistant skeletal muscle was not addressed in the present study. However, it is known that intact activation of PI 3-kinase by tyrosine-phosphorylated IRS-1 is necessary for this insulin-stimulated GLUT4 translocation to take place (reviewed in Ref. 38). Because GSK-3 is known to inhibit IRS-1 action by serine/threonine phosphorylation (10), one possibility for the beneficial effect of the GSK-3 inhibitor is through a disinhibition of IRS-1 functionality, with a subsequent enhancement of PI 3-kinase activity and increased GLUT4 translocation. It is clear that this would be a fruitful area for future investigations.

We also compared the metabolic effects of lithium, which has been used previously as an inhibitor of GSK-3, with those of the more selective GSK-3 inhibitor CT-98014 (Figs. 1-4). Both lithium (at 10 mM) and CT-98014 (at 500 nM) elicited enhancement of GS activity in muscle from lean Zucker and ZDF rats and displayed an additive interaction with insulin for stimulation of GS activity in muscle from lean animals. This additivity between lithium and insulin for activation of GS in insulin-sensitive cells is consistent with previous studies (3, 6, 23, 33). Moreover, lithium and CT-98014 both displayed a synergistic interaction with insulin for stimulation of GS activity and glucose transport activity in muscle from ZDF rats. However, there were some striking differences between lithum and CT-98014 concerning stimulation of glucose transport activity. Although CT-98014 had no effects on glucose transport activity in the absence of insulin in both the lean Zucker and the ZDF groups or on insulin-stimulated glucose transport activity in the lean group, lithium caused an increase in basal glucose transport in both groups and in insulin-stimulated glucose transport activity in the lean group. Whereas lithium can certainly inhibit GSK-3, this ion obviously has additional effects that must account for its actions on basal and insulin-stimulated glucose transport in insulin-sensitive skeletal muscle and on basal glucose transport activity in insulin-resistant skeletal muscle. Therefore, one must exercise caution in using lithium as an inhibitor of GSK-3 in skeletal muscle studies, as other effects of this ion apart from those on GSK-3 may confound interpretation of the results.

There were some important differences in the glucose transport responses of the epitrochlearis, which consists primarily of type IIb fibers (20, 29), and the soleus, which consists mainly of type I fibers (1), to the GSK-3 inhibitor CT-98014 (Fig. 4). The absolute increase in insulin-stimulated glucose transport activity due to GSK-3 inhibition was 34-37 pmol · mg-1 · 20 min-1 in the epitrochlearis, whereas in the soleus this increase was substantially greater (122-126 pmol · mg-1 · 20 min-1). This greater response to GSK-3 inhibition in the soleus may reflect the fact that type I fibers express a much higher level of insulin-signaling factors (31) and GLUT4 protein (13, 17) than type IIb fibers and therefore may have a higher capacity for insulin signaling once inhibition of one of these factors [e.g., GSK-3 inhibition of IRS-1 (10)] is removed.

The oral administration of GSK-3 inhibitors to the insulin-resistant ZDF rat also elicited improvements in glucose metabolism. The disposal of an oral glucose load by the ZDF rats acutely treated with the GSK-3 inhibitor CT-98023 was markedly enhanced compared with the vehicle-treated animals (Figs. 7 and 8). One potential cellular locus for this increased glucose disposal was skeletal muscle, as insulin-stimulated glucose transport activity in both the epitrochlearis and the soleus was substantially increased at the 30-min time point, when the glucose response during the OGTT was maximally reduced (Fig. 7). Indeed, at the 120-min time point following the final administration of the GSK-3 inhibitor, the enhancement of insulin-stimulated glucose transport activity was no longer observed in the epitrochlearis and was substantially diminished in the soleus (Fig. 9), whereas at this same time point the glucose levels during the OGTT were not different between the GSK-3 inhibitor-treated and the vehicle-treated control animals (Fig. 7). This apparent reversibility of the GSK-3 inhibition is consistent with the observation that removal of the compound from tumor cells causes beta -catenin levels to decrease (Harrison SD, unpublished data).

One additional potential site of action of GSK-3 inhibitors that could contribute to an improvement in whole body glucose homeostasis is the liver (4, 19). Cline et al. (4) have recently reported that treatment of ZDF rats with the selective GSK-3 inhibitor CT-98023 significantly enhances GS activity and glycogen synthesis in the liver. Moreover, Lochhead et al. (19) have demonstrated that the selective reduction of GSK-3 activity in rat hepatoma cells by treatment with lithium or cell-permeable, small-molecular-weight compounds (SB-216763 and SB-415286) (5) is associated with a reduction in the expression of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. Therefore, an enhancement of hepatic glycogen synthesis and a reduction in hepatic glucose output likely contribute to the glucose-lowering effect of GSK-3 inhibitors in rodent models of type 2 diabetes (27).

In summary, we have demonstrated that direct in vitro exposure of insulin-resistant type I and type IIb skeletal muscle from the ZDF rat to a novel, selective organic inhibitor of GSK-3 can potentiate the action of insulin to stimulate GS activity and glucose transport. Although lithium also potentiated insulin-stimulated GS activity and glucose transport in insulin-resistant muscle, it additionally elicited effects on basal glucose transport and on insulin-stimulated glucose transport in muscle from lean Zucker rats that were not affected by the selective GSK-3 inhibitor. The effect of the GSK-3 inhibitor on insulin-stimulated glucose transport in skeletal muscle from the ZDF rat was due, at least in part, to an enhanced cell surface GLUT4 protein level. Moreover, the acute oral administration of a selective GSK-3 inhibitor caused a significant improvement in whole body glucose disposal and insulin sensitivity that was associated with enhanced skeletal muscle glucose transport activity. This novel approach using selective organic GSK-3 inhibitors appears useful as a pharmacological treatment against insulin resistance of skeletal muscle glucose disposal.


    ACKNOWLEDGEMENTS

We thank Melanie B. Schmit, Stephen A. Miller, Thomas Maier, and Julie A. Sloniger for excellent technical assistance.


    FOOTNOTES

The research was supported by a grant from Chiron Corporation (to E. J. Henriksen) and the American Diabetes Association (to E. J. Henriksen).

Address for reprint requests and other correspondence: E. J. Henriksen, Dept. of Physiology, Ina E. Gittings Bldg. #93, Univ. of Arizona, Tucson, AZ 85721-0093 (E-mail: ejhenrik{at}u.arizona.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 7, 2003;10.1152/ajpendo.00346.2002

Received 5 August 2002; accepted in final form 30 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ariano, MA, Armstrong RB, and Edgerton VR. Hindlimb muscle fiber populations of five animals. J Histochem Cytochem 21: 51-55, 1973[ISI][Medline].

2.   Brozinick, JT, Misener EA, Ni B, Ryder JW, and Dohm GL. Impaired insulin signaling through GSK3 in insulin resistant skeletal muscle (Abstract). Diabetes 49, Suppl 1: A326, 2000.

3.   Choi, WS, and Sung CK. Effects of lithium and insulin on glycogen synthesis in L6 myocytes: additive effects on inactivation of glycogen synthase kinase-3. Biochim Biophys Acta 1475: 225-230, 2000[ISI][Medline].

4.   Cline, GW, Johnson K, Regittnig W, Perret P, Tozzo E, Xiao L, Damico C, and Shulman GI. Effects of a novel glycogen synthase kinase-3 inhibitor on insulin-stimulated glucose metabolism in Zucker diabetic fatty (fa/fa) rats. Diabetes 51: 2903-2910, 2002[Abstract/Free Full Text].

5.   Coghlan, MP, Culbert AA, Cross DAE, Corcoran SL, Yates JW, Pearce NJ, Rausch OL, Murphy GL, Carter PS, Cox LR, Mills D., Brown MJ, Haigh D, Ward RW, Smith DG, Murray KJ, Reith AD, and Holder JC. Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem Biol 24: 1-11, 2000.

6.   Cortez, MY, Torgan CE, Brozinick JT, and Ivy JL. Insulin resistance of obese Zucker rats exercise trained at two different intensities. Am J Physiol Endocrinol Metab 261: E613-E619, 1991[Abstract/Free Full Text].

7.   Cross, DA, Alessi DR, Cohen P, Andjelkovich M, and Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378: 785-789, 1995[ISI][Medline].

8.   Davies, SP, Reddy H, Caviano M, and Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95-105, 2000[ISI][Medline].

9.   DeFronzo, RA, and Ferrannini E. Insulin resistance: a multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care 14: 173-194, 1991[Abstract].

10.   Eldar-Finkelman, H, and Krebs EG. Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action. Proc Natl Acad Sci USA 94: 960-9664, 1997.

11.   Eldar-Finkelman, H, Schreyer SA, Shinohara MM, LeBoeuf RC, and Krebs EG. Increased glycogen synthase kinase-3 activity in diabetes- and obesity-prone C57BL/6J mice. Diabetes 48: 1662-1666, 1999[Abstract].

12.   Hansen, PA, Gulve EA, and Holloszy JO. Suitability of 2-deoxyglucose for in vitro measurement of glucose transport activity in skeletal muscle. J Appl Physiol 76: 979-985, 1994[Abstract/Free Full Text].

13.   Henriksen, EJ, Bourey RE, Rodnick KJ, Koranyi L, Permutt MA, and Holloszy JO. Glucose transporter protein content and glucose transport capacity in rat skeletal muscles. Am J Physiol Endocrinol Metab 259: E593-E598, 1990[Abstract/Free Full Text].

14.   Henriksen, EJ, and Halseth AE. Early alterations in soleus GLUT-4, glucose transport, and glycogen in voluntary running rats. J Appl Physiol 76: 1862-1867, 1994[Abstract/Free Full Text].

15.   Henriksen, EJ, Tischler ME, and Johnson DG. Increased response to insulin of glucose metabolism in the six-day unloaded rat soleus muscle. J Biol Chem 261: 10707-10712, 1986[Abstract/Free Full Text].

16.   Hong, M, Chen DC, Klein PS, and Lee VM. Lithium reduces tau phosphorylation by inhibition of glycogen synthase kinase-3. J Biol Chem 272: 25326-25332, 1997[Abstract/Free Full Text].

17.   Kern, M, Wells JA, Stephens JM, Elton CW, Friedman JE, Tapscott EB, Pekala PH, and Dohm GL. Insulin responsiveness in skeletal muscle is determined by glucose transporter (Glut4) protein level. Biochem J 270: 397-400, 1990[ISI][Medline].

18.   Klein, PS, and Melton DA. A molecular mechanism for the effect of lithium on development. Proc Natl Acad Sci USA 93: 8455-8459, 1996[Abstract/Free Full Text].

19.   Lochhead, PA, Coghlan M, Rice SQJ, and Sutherland C. Inhibition of GSK-3 selectively reduces glucose-6-phosphate and phosphoenolpyruvate carboxykinase gene expression. Diabetes 50: 937-946, 2001[Abstract/Free Full Text].

20.   Nesher, R, Karl IE, Kaiser KK, and Kipnis DM. Epitrochlearis muscle. I. Mechanical performance, energetics, and fiber composition. Am J Physiol Endocrinol Metab 239: E454-E460, 1980[Abstract/Free Full Text].

21.   Nikoulina, SE, Cirialdi TP, Mudaliar S, Carter L, Johnson K, and Henry RR. Inhibition of glycogen synthase kinase-3 improves insulin action and glucose metabolism in human skeletal muscle. Diabetes 51: 2190-2198, 2002[Abstract/Free Full Text].

22.   Nikoulina, SE, Cirialdi TP, Mudaliar S, Mohideen P, Carter L, and Henry RR. Potential role of glycogen synthase kinase-3 in skeletal muscle insulin resistance of type 2 diabetes. Diabetes 49: 263-271, 2000[Abstract].

23.   Orena, SJ, Torchia AJ, and Garofalo RS. Inhibition of glycogen synthase kinase-3 stimulates glycogen synthase and glucose transport by distinct mechanisms in 3T3-L1 adipocytes. J Biol Chem 275: 15765-15772, 2000[Abstract/Free Full Text].

24.   Parker, PJ, Caudwell FB, and Cohen P. Glycogen synthase from rabbit skeletal muscle: effect of insulin on the state of phosphorylation of the seven phosphoserine residues in vivo. Eur J Biochem 130: 227-234, 1983[ISI][Medline].

25.   Reaven, GM. Role of insulin resistance in human disease (syndrome X): an expanded definition. Annu Rev Med 44: 121-131, 1993[ISI][Medline].

27.  Ring D, Johnson KW, Henriksen EJ, Nuss JM, Goff D, Kinnick TR, Ma ST, Reeder JW, Samuels I, Slabiak T, Wagman A, Wernette-Hammond ME, and Harrison SD. Selective GSK3 inhibitors potentiate insulin action on glucose transport and utilization in vitro and in vivo. Diabetes. In press.

28.   Roach, PJ. Control of glycogen synthase by hierarchal protein phosphorylation. FASEB J 4: 2961-2968, 1990[Abstract].

29.   Rodnick, KJ, Henriksen EJ, James DE, and Holloszy JO. Exercise training, glucose transporters, and glucose transport in rat skeletal muscles. Am J Physiol Cell Physiol 262: C9-C14, 1992[Abstract/Free Full Text].

30.   Sherman, WM, Katz AL, Cutler CL, Withers RT, and Ivy JL. Glucose transport: locus of muscle insulin resistance in obese Zucker rats. Am J Physiol Endocrinol Metab 255: E374-E382, 1988[Abstract/Free Full Text].

31.   Song, XM, Ryder JW, Kawano Y, Chibalin AV, Krook A, and Zierath JR. Muscle fiber type specificity in insulin signal transduction. Am J Physiol Regul Integr Comp Physiol 277: R1690-R1696, 1999[Abstract/Free Full Text].

32.   Stambolic, V, Ruel L, and Woodgett JR. Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr Biol 6: 1664-1668, 1996[ISI][Medline].

33.   Summers, SA, Kao AW, Kohn AD, Backus GS, Roth RR, Pessin JE, and Birnbaum MJ. The role of glycogen synthase kinase 3beta in insulin-stimulated glucose metabolism. J Biol Chem 274: 17934-17940, 1999[Abstract/Free Full Text].

34.   Tabata, I, Schluter J, Gulve EA, and Holloszy JO. Lithium increases susceptibility of muscle glucose transport to stimulation by various agents. Diabetes 43: 903-907, 1994[Abstract].

35.   Thomas, JA, Schlender KK, and Larner J. A rapid filter paper assay for UDP-glucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose. Anal Biochem 25: 486-499, 1968[ISI][Medline].

36.   Woodgett, JR. Molecular cloning and expression of glycogen synthase kinase-3/factor A. EMBO J 9: 2431-2438, 1990[Abstract].

37.   Zhang, W, DePaoli-Roach AA, and Roach PJ. Mechanisms of multisite phosphorylation and inactivation of rabbit muscle glycogen synthase. Arch Biochem Biophys 304: 219-225, 1993[ISI][Medline].

38.   Zierath, JR, Krook A, and Wallberg-Henriksson H. Insulin action and insulin resistance in human skeletal muscle. Diabetologia 43: 821-835, 2000[ISI][Medline].


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