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Inhibition of Synthase 3 Improves Action and Glucose in Human Skeletal Muscle Svetlana E. Nikoulina,1,2 Theodore P. Ciaraldi,1,2 Sunder Mudaliar,1,2 Leslie Carter,1,2 Kirk Johnson,3 and Robert R. Henry1,2

Glycogen synthase kinase (GSK)-3 has been implicated on insulin action, using mechanisms that differ from and in the regulation of multiple cellular physiological pro- are additive to those of insulin. 51:2190–2198, cesses in skeletal muscle. Selective cell-permeable re- 2002 versible inhibitors (INHs) of GSK-3 (CT98014 and CHIR98023 [Chiron, Emeryville, CA] and LiCl) were used to evaluate the role of GSK-3 in controlling glucose metabolism. Acute treatment (30 min) of cultured hu- lycogen synthase kinase (GSK)-3 is a / man skeletal muscle cells with either INH resulted in a kinase originally discovered because dose-dependent activation of (GS) of its ability to phosphorylate and inhibit gly- ϳ ␮ with a maximally effective concentration of 2 mol/l. cogen synthase (GS) (1). Human GSK-3 exists The maximal acute effect of either INH on GS (103 ؎ G as two isoforms, ␣ and ␤, encoded by two distinct genes, 25% stimulation over basal) was greater than the max- -imal insulin response (48 ؎ 9%, P < 0.05 vs. INH); LiCl located on chromosomes 19q13.1-2 and 3q13.3-q21, respec was as effective as insulin. The GSK-3 inhibitor effect, tively (2). GSK-3 is constitutively active in resting cells and like that of insulin, was on the activation state (frac- is inhibited by several hormones such as insulin, endothe- tional velocity [FV]) of GS. Cotreatment of muscle cells lial , and -derived growth factor. with submaximal doses of INH and insulin resulted in an Insulin has been demonstrated to cause inactivation of .(additive effect on GS FV (103 ؎ 10% stimulation, P < GSK-3, both in vivo (3,4) and in several cell types (5–7 0.05 vs. either agent alone). Glucose incorporation into The mechanism of inactivation is associated with phos- glycogen was also acutely stimulated by INH. While phorylation of the specific serine residues Ser21 and Ser9 prolonged (6–24 h) insulin exposure led to desensitiza- in GSK-3 isoforms ␣ and ␤, respectively. Insulin deactiva- tion of GS, INH continued to activate GS FV for at least tion of GSK-3 is (PI) 3-kinase–depen- 24 h. Insulin and LiCl acutely activated glucose uptake, whereas INH stimulation of glucose uptake required dent and probably occurs through kinase B/Akt more prolonged exposure, starting at 6 h and continuing (8,9), properties shared with insulin stimulation of GS. to 24 h. Chronic (4-day) treatment with INH increased GS activity is regulated by allosteric and covalent (phos- .(both basal (154 ؎ 32% of control) and insulin-stimu- phorylation/dephosphorylation) mechanisms (10–12 lated (219 ؎ 74%) glucose uptake. Upregulation of Whereas a number of and can act on uptake activity occurred without any change in total GS, GSK-3 plays an important role (13–15). GSK-3 phos- cellular GLUT1 or GLUT4 protein content. Yet the same phorylates GS on three specific residues. This phosphory- chronic treatment resulted in a 65 ؎ 6% decrease in lation causes deactivation of GS and decreases its affinity ؎ GSK-3 protein and a parallel decrease (61 11%) in to allosteric activation by glucose-6-. In addi- GSK-3 total activity. Together with the INH-induced tion, it has been reported that GSK-3 can also phosphory- increase in insulin-stimulated glucose uptake, there was an ϳ3.5-fold increase (P < 0.05) in insulin receptor late insulin substrate (IRS)-1, a key early substrate (IRS)-1 protein abundance. Despite upregula- molecule in insulin-signaling cascades (16), suggesting the tion of IRS-1, maximal insulin stimulation of Akt phos- potential for involvement of GSK-3 in multiple stages of phorylation was unaltered by INH treatment. The insulin action. results suggest that selective inhibition of GSK-3 has an Lithium ion (Li) has been found to cause relatively impact on both GS and glucose uptake, including effects specific inhibition of GSK-3 (15,17) and has been reported to have insulin-like effects on glucose metabolism, includ- ing increases in glucose uptake, activation of GS activity, From the 1Veterans Affairs San Diego Healthcare System, La Jolla, California; and stimulation of glycogen synthesis in skin, muscle, and the 2Department of Medicine, University of California, San Diego, La Jolla, fat cells. However, Li can also inhibit myo-inositol-1- California; and 3Chiron Corporation, Emeryville, California. Address correspondence and reprint requests to R.R. Henry, MD, VA San monophosphatase (18), 1,6 bisphosphatase, and other Diego Health Care System (9111G), 3350 La Jolla Village Dr., San Diego, CA involved in regulation of glucose metabolism 92161. E-mail: [email protected]. Received for publication 22 May 2001 and accepted in revised form 27 (19–21). Several other agents have also been reported to March 2002. inhibit GSK-3 activity: Ro 31-8220, valproic acid, iodotu- DTT, dithiothreitol; FV, fractional velocity; GS, glycogen synthase; GSK, bercidin, staurosporin, sangivemycin, and hymenialdisine glycogen synthase kinase; IA, inactive analog; INH, inhibitor; IRS, substrate; MEM, minimal essential medium; PI, phosphatidylinositol; (22). Like lithium, some of these inhibitors have been PMSF, phenylmethylsulfonyl fluoride. reported to exhibit antidiabetic effects (valproate) or to

2190 DIABETES, VOL. 51, JULY 2002 S.E. NIKOULINA AND ASSOCIATES promote glycogen formation (iodotubercidin). However, University of California, San Diego. Informed written consent was obtained these agents are not specific for GSK-3 inhibition and also from all subjects after explanation of the protocol. The method of culturing skeletal muscle cells from biopsy samples has inhibit other protein kinases involved in insulin signal been described in detail in several publications (24,25). Satellite cells were transduction and/or glucose metabolism. Consequently, it obtained by trypsin digestion of muscle biopsy material. Cells were propa- has been difficult to confidently ascribe the insulin-like gated in culture by modifications of the methods described by Blau and effects of these agents to GSK-3 inhibition alone. Webster (28) and Sarabia et al. (29). When cells attained ϳ80% confluency, Several recent observations suggest an important role they were transferred to differentiation media. Differentiation into multinucle- ated myotubes was complete within 4 days. Media were changed every 48 h. for GSK-3 in the regulation of GS and possible involvement When cells were treated chronically for 4 days, media with fresh agents were in the development of in type 2 diabetes. changed every other day. Acutely, cells were treated with the selective GSK-3 Overexpression of GSK-3 in 293 cells and 3T3-L1 adipo- inhibitor CHIR 98024 or its IA in serum-free media for 30 min or as indicated cytes results in decreased GS affinity to glucose-6-phos- and, in some cases, together with insulin (33 nmol/l) at the completion of the phate as well as impaired insulin-stimulated IRS-1 differentiation period. GS activity. GS activity was measured as described in detail previously (15,16). In addition, GSK-3 expression is (25,30). Cells from six-well plates were washed with PBS and scraped into 0.3 elevated in skeletal muscle of type 2 diabetic subjects and ml of extraction buffer (50 mmol/l HEPES, 10 mmol/l EDTA, 100 mmol/l NaF, is inversely related to whole-body insulin action (22). 5 mmol/l dithiothreitol [DTT], 1 ␮mol/l leupeptin, 1 ␮mol/l pepstatin, and 200 However, the physiological role of GSK-3 in insulin-sensi- ␮mol/l PMSF, pH 7.5). Protein concentration was determined by the Bradford tive tissues, such as skeletal muscle and especially its role method (31) with BSA as a standard. GS activity was determined at a in regulation of glucose metabolism, remains to be deter- physiological concentration of substrate (0.3 mmol/l UDP glucose) and expressed in nanomoles of UDP glucose incorporated into glycogen per mined. minute per milligram of total protein and as fractional velocity (FV) (the ratio The current experiments used novel, potent, and selec- of activity assayed at 0.1 mmol/l glucose-6-phosphate divided by the activity at tive inhibitors of GSK-3 to study the regulation of glucose 10 mmol/l glucose-6-phosphate). This is believed to be an indicator of changes metabolism and insulin action in the major insulin target in the phosphorylation state of GS in response to insulin (12). tissue—skeletal muscle. These studies were performed in Glycogen synthesis. Glycogen synthesis was determined in differentiated myotubes as D-[14C]glucose incorporation into glycogen during a 1-h incuba- cultured human skeletal muscle cells, a system that tion at 37°C (24). After incubation, cells were rinsed four times with PBS at closely reflects the in vivo metabolic behavior of skeletal 4°C and solubilized with 1N NaOH at 55°C for 1 h. An aliquot (100 ␮l) of the muscle, including impaired insulin activation of GS (23– lysate was removed for protein analysis. The final glycogen pellets were 25). resuspended in 0.5 ml H2O, mixed with scintillation fluid, and radioactivity determined by liquid scintillation counting. Absolute results are expressed as nanomoles glucose converted to glycogen per milligram protein per hour. Assay of GSK-3 activity. Total GSK-3 (␣ and ␤) activity was measured in the RESEARCH DESIGN AND METHODS homogenates against a peptide substrate (RRAAEELDSRAGpSPQL) derived Materials. UDP-[14C]glucose, D-[U-14C]glucose, L-[14C]glucose, and [3H]-2- from the e-subunit of eIF2-(SP). This peptide and control peptide [where the deoxyglucose were obtained from DuPont NEN (Boston, MA). [␥-32P]ATP was Ser(P) is replaced by Ala] were obtained from Chiron. After treatment, cells purchased from ICN Pharmaceuticals (Costa Mesa, CA). Horseradish-conju- were rapidly washed with PBS at 4°C and then lysed in buffer containing 20 gated anti-rabbit and anti-mouse IgG was obtained from Amersham (Arlington mmol/l Tris-HCl, 145 mmol/l NaCl, 10% glycerol, 5 mmol/l EDTA, 1% Triton Heights, IL). SuperSignal Chemiluminescent Substrate was obtained from X-100, 0.5% NP-40, 200 ␮mol/l sodium orthovanadate, 200 ␮mol/l PMSF, 1 Pierce (Rockford, IL). BSA (Fraction V) and glucose-6-phosphate were ␮mol/l leupeptin, 1 ␮mol/l pepstatin, and 10 mg/ml aprotinin, pH 7.5. Kinase purchased from Boehringer Mannheim (Indianapolis, IN). Protein assay assays were performed on the cell homogenates in reaction mixtures that reagents and electrophoresis chemicals were purchased from BioRad Labo- included 3 mg/ml peptide (substrate or control), 110 ␮mol/l [␥-32P]ATP, 16 ratories(Hercules,CA).Glycogen,2-deoxyglucose,pepstatin,leupeptin,phenyl- mmol/l magnesium chloride, 0.1 mg/ml BSA, 1 mmol/l DTT, and 20 mmol/l methylsulfonyl fluoride (PMSF), and other reagents and chemicals were Tris, pH 7.4. Reactions were stopped by spotting 0.03-ml aliquots on What- purchased from Sigma (St. Louis, MO). Anti–GSK-3/Shaggy mann P81 paper squares and immediately transferring to cold 100 mmol/l family (51/46-kDa) mouse monoclonal IgG, purified GSK-3␣ and -␤ from rabbit phosphoric acid. Squares were washed three times with 100 mmol/l phospho- skeletal muscle, and anti-rat COOH-terminal IRS-1 rabbit polyclonal IgG were ric acid and once with acetone and counted in a liquid scintillation counter. purchased from Upstate Biotechnology (Lake Placid, NY). Akt antibody, 32Pi incorporation into the negative control peptide was subtracted from which detects total Akt1, Akt2, and Akt3 isoforms, was obtained from Cell values obtained using substrate peptide. GSK-3 activity was expressed as nCi Signaling Technologies (Beverly, MA). The antibody against the S473 phos- of 32Pi incorporated into phosphopeptide per 30 min by 1 mg protein, phorylated form of Akt was purchased from New England Biolabs (Beverly, measured by the Bradford assay (31). MA). Antibodies against GLUT1 (polyclonal) and GLUT4 were obtained from Glucose uptake assay. Glucose uptake measurements were performed as Biogenesis (Kingston, NH). An affinity-purified chicken polyclonal antibody described previously (32). Serum-free media were added to the cells together against GS was a gift from Dr. J.C. Lawrence, Jr. (University of Virginia, with insulin (0–33 nmol/l), and the cells incubated for 120 min in a 5% CO2 Charlottesville, VA). GSK-3 inhibitors CHIR 98014 and CHIR 98023 were incubator before washing, uptake assay, and solubilization. An aliquot of the obtained from Chiron (Emeryville, CA) and have been described previously suspension was removed for protein analysis using the Bradford method. The ␣ ␤ ϭ (26). They inhibit GSK-3 and - in vitro with Ki 16 nmol/l (98014) and Ki uptake of L-glucose was used to correct each sample for the contribution of ϭ Ն ␮ 4 nmol/l (98023). Ki values are 10 mol/l for more than 20 other kinases, diffusion. This procedure measures both the transport and phosphorylation of including cdc2, Akt, erk, PI 3-kinase, rsk, and InsRTK (26). A structurally glucose. Control studies indicate that transport was the rate-limiting process Ն ␮ related and inactive analog (IA) (Ki against GSK-3 1 mol/l) was also used. under the assay conditions used. Inhibitors (INHs) were diluted in DMSO to obtain a 10 mmol/l concentration Immunoblotting. On the day of the experiment, control and insulin- or (stock). Before being added to the cell cultures, the stock solution was mixed INH-treated cells were washed three times with ice-cold PBS and homoge- with serum-free media to obtain concentrations ranging from 0.5 to 5.0 ␮mol/l. nized in a buffer containing 20 mmol/l Tris-HCl (pH 7.5), 1.5 mmol/l EGTA, 1 In most experiments, the final concentration of DMSO (vehicle) in culture mmol/l EDTA, 10 mmol/l NaF, 1 mmol/l DTT, 0.5 mmol/l sodium orthovana- media was Ͻ0.0005%. This concentration of DMSO was tested in multiple date, 10 ␮g/ml aprotinin, 20 ␮g/ml leupeptin, 0.2 mmol/l PMSF, and 0.5% Triton experiments (n ϭ 20) and did affect glucose metabolism in human skeletal X-100. The levels of GSK-3 isoforms (␣ and ␤, Shaggy protein kinase family) muscle cultures. were determined by Western blotting using monoclonal antibodies raised Human skeletal muscle cell culture. Skeletal muscle cell cultures were against a peptide with a common sequence for ␣ and ␤ GSK-3 isoforms. established from muscle tissue obtained by needle biopsy samples of the Partially purified rabbit GSK-3␣ and/or GSK-3␤ were included on each gel as vastus lateralis. Muscle biopsy samples were obtained from 47 nondiabetic internal standards. Other antibodies are described in RESEARCH DESIGN AND and type 2 diabetic patients. All subjects underwent a 2-h 75-g oral glucose METHODS. Western blotting was performed by the method of Burnette (33). tolerance test. Normal glucose tolerance was defined as a fasting glucose level After SDS-PAGE (7.5 or 10%), were transferred to nitrocellulose Ͻ7.0 mmol/l and 2-h glucose level Ͻ7.8 mmol/l (27). The experimental membranes. Nonspecific binding was reduced by incubation in a blocking protocol was approved by the Committee on Human Investigation of the solution containing Tris-buffered saline, 3% nonfat dry milk, and 0.05%

DIABETES, VOL. 51, JULY 2002 2191 INHIBITION OF GSK-3 IN HUMAN SKELETAL MUSCLE

LiCl. Both INHs acutely activated GS FV in a dose- dependent manner with a similar concentration-response profile (CHIR 98023 shown in Fig. 2A). Maximal effects were observed at 1–2 ␮mol/l for both INHs. This effect was on the activation state (FV) of the because maxi- mal GS activity was unaltered. The effects of the inhibitors were compared with the maximal effects of insulin and LiCl (20 mmol/l). Stimula- tory actions of the INHs were greater than the maximal insulin or LiCl effects (Fig. 2B). The structurally related ϫϾ compound that is inactive (IC50 100 98014 or 98023) against GSK-3 (IA) had no effect on GS activity. Whereas values for GS FV (both basal and insulin-stimulated) in type 2 diabetic subjects were lower (P Ͻ 0.05) than those in the nondiabetic subjects, in agreement with earlier results (25), the relative effects of the INHs were similar in muscle cell cultures from nondiabetic and type 2 diabetic subjects (Fig. 2B). Because the absence or presence of diabetes did not influence the relative effects of INH treatment, data in the two subject groups were combined, although cells from each subject were assayed individu- ally. Possible interactions between GSK-3 inhibition and in- sulin action on GS were investigated by treating cells simultaneously with insulin and CHIR 98023. Cotreatment with submaximal concentrations of CHIR 98023 and insu- lin (10 nmol/l) had a synergistic effect on the GS activation state (Fig. 2C). Maximal insulin action was also augmented by CHIR 98023. CHIR 98023 addition after preincubation with a maximal insulin dose resulted in an amplification of FIG. 1. GSK-3 inhibitory compounds. A: Structures of inhibitors stud- the insulin effect on GS FV (data not shown) without ied. B: Effect of inhibitor (CHIR 98023) and IA on GSK-3 activity. altering total GS activity. Compounds (2 ␮mol/l) were added directly to GSK-3 assay before Kinetics of GS activation. Differences were observed determination of enzyme activity. Results are average ؎ SE for assays between the time course of GS activation by insulin and ؍ performed on extracts prepared from cells of multiple individuals; n .(for IA. *P < 0.0005 vs. control. GSK-3 inhibitors. Insulin rapidly activated GS FV (Fig. 3 5 ؍ for CHIR 98023, and n 10 Stimulation increased for up to 2 h, followed by a return to Tween-20. Anti-mouse or anti-rabbit IgG conjugated with horseradish perox- near-basal activity after 24 h of treatment (Fig. 3). Chron- idase was used as a secondary antibody. Proteins were visualized using the ically insulin-treated cells were also desensitized to sub- SuperSignal Chemiluminescent Substrate and exposed to specific super- sensitive autoradiograph film (Hyperfilm; Amersham). Multiple exposures sequent acute insulin exposure (not shown). Similarly, were performed to verify linearity of the images. The intensity of the bands LiCl also rapidly increased GS FV (30 min), but no effect was quantified by scanning densitometry using NIH Image software, and was seen after prolonged exposure (Fig. 3). A biphasic results were expressed in integrated optical density units per 0.01 mg total effect of CHIR 98023 on GS FV was observed. There was protein (arbitrary units). Statistical analysis. Statistical significance was evaluated with the Student’s rapid activation (0.5–2 h) of a magnitude greater than or t test. Paired analysis was used to determine the effect of the inhibitors in the equal to that of insulin or LiCl, which continued to same culture. All data and calculated results are expressed as the average Ϯ increase until 24 h of treatment and was sustained for 96 h. SE. Significance was accepted at the P Ͻ 0.05 level. The maximal activated GS FV, approximately fivefold that of control activity, could not be stimulated further by RESULTS addition of acute insulin (not shown). Acute activation of GS by GSK-3 inhibitors. Figure 1 Effect of GSK-3 inhibition on glucose uptake. GSK-3 presents the structures of the GSK-3 inhibitor compounds has been proposed to play a role in insulin-regulated used in the current study. Confirmation of GSK-3 inhibi- glucose uptake (34). Acute (2-h) exposure of muscle cells tion is presented in Fig. 1B. Direct addition of CHIR 98023 to a dose of CHIR 98023 that maximally activated GS FV to the GSK-3 assay reduced enzyme activity by 72 Ϯ 7% resulted in an insignificant increase in glucose uptake (Fig. (P Ͻ 0.0005), whereas a structurally related inactive 4A) compared with the ϳ80% stimulation in response to compound (IA) had no effect (1 Ϯ 17% inhibition). Detailed insulin. Interestingly, similar to insulin, LiCl acutely stim- dose-response curves performed with purified kinases ulated glucose uptake (78 Ϯ 16% increase, P Ͻ 0.05). As revealed inhibitory potencies greater than those seen with was the case for GS FV, prolonged exposure to insulin (24 whole-cell extracts (26). h) or LiCl (96 h) led to a desensitization of glucose uptake, To determine whether specific inhibition of GSK-3 with both a return toward basal activity (Fig. 4A) and a acutely controls GS activation in skeletal muscle, we used failure to respond to subsequent acute insulin exposure cell-permeable inhibitors of GSK-3—CHIR 98014 and CHIR (not shown). In contrast, INHs increased glucose uptake 98023 (Chiron) (26), collectively referred to as INH and significantly at 6–24 h after CHIR 98023 treatment (284 Ϯ

2192 DIABETES, VOL. 51, JULY 2002 S.E. NIKOULINA AND ASSOCIATES

FIG. 3. Time course of GS activation by CHIR 98023 (2 ␮mol/l), insulin ؍ nmol/l), or LiCl (20 mmol/l) in human skeletal muscle cultures (n 33) ؎ nondiabetic patients, 2 diabetic patients). Results are averages 2 ;4 SE.

23%). Moreover, the increase was sustained for up to 96 h of treatment (Fig. 4A). A similar response was seen with CHIR 98014 (Fig. 4B). The inactive compound had no effect on glucose uptake after either acute or prolonged (Fig. 4B) treatment. Although cells chronically treated with insulin did not respond to subsequent acute insulin treatment (not shown), INH-treated cells displayed a fur- ther response to insulin (Fig. 4B). Insulin-stimulated up- take after INH treatment was approximately double that seen in control cells, whereas it was normal in LiCl-treated cells. Thus, sustained exposure to CHIR 98023 and CHIR 98014 resulted in upregulation of both basal and insulin- stimulated glucose uptake in skeletal muscle cells. Regulation of glycogen formation. The effects of GSK-3 inhibitors on the major fate of glucose in skeletal muscle— storage in glycogen—were also evaluated. Insulin and INHs acutely stimulated glucose incorporation into glyco- gen, whereas the inactive analog had no effect (Fig. 5). Similar results were observed in cultured liver cells using other selective inhibitors of GSK-3 (35). The responses to insulin and INHs were comparable. The combination of FIG. 2. Acute effects of insulin and GSK-3 inhibitors on GS FV in human insulin and INHs tended (P Ͻ 0.1) to stimulate glycogen skeletal muscle cells. A: Dose response. Human skeletal muscle cell formation greater than that seen with either agent alone, cultures were incubated for 30 min with a range of concentrations of CHIR 98023 in fusion media at 37°C. Results are means ؎ SE, deter- as might be expected from the effect of insulin on glucose mined in three to five cell cultures obtained from nondiabetic subjects. uptake (Fig. 4A) combined with maximal GS activation GS activity is expressed as FV (GS FV), as described in RESEARCH DESIGN due to INHs. AND METHODS. B: Comparison of effects of agents in muscle cells from diabetic and nondiabetic subjects. Cell cultures were incubated for 30 Effect of GSK-3 inhibitors on protein expression. min in serum-free media (␣-MEM) containing 33 nmol/l insulin, 2.5 Although the acute effects of INHs and LiCl on GS could be ␮mol/l CHIR 98023 or IA, or 20 mmol/l LiCl. Results are means ؎ SE attributed to inhibition of GSK-3 activity, the temporally and type 2 (10–5 ؍ obtained from cell cultures of nondiabetic (n subjects. C: Insulin and CHIR 98023 have additive biphasic nature of the effect of CHIR 98023 on GS FV and (10–5 ؍ diabetic (n effects on GS FV in human muscle cells. GS FV was determined in cells glucose uptake suggests that additional mechanisms could of seven subjects (three nondiabetic patients and four type 2 diabetic patients) after 30 min of treatment with 2.5 ␮mol/l CHIR 98023 alone be involved. Chronic (4-day) exposure of muscle cells to (INH), insulin at a low concentration (IL) (1.7 nmol/l) or high concen- INHs resulted in a dose-dependent decrease (65%, P Ͻ tration (IH) (33 nmol/l) alone, or a combination with 2.5 ␮mol/l 0.05) in the expression of GSK-3 protein (Fig. 6A). Both the ␤ ␣ .CHIR 98023 (INH ؉ I). Results are averages ؎ SE. *P < 0.05 vs paired control; †P < 0.05 vs. paired insulin-treated value for same and isoforms were reduced (Fig. 5B) by 68 and 45%, individual. respectively, after treatment with 2 ␮mol/l CHIR 98023.

DIABETES, VOL. 51, JULY 2002 2193 INHIBITION OF GSK-3 IN HUMAN SKELETAL MUSCLE

FIG. 5. Regulation of glucose incorporation into glycogen in human muscle cells. Cells were acutely treated during the 1-h assay of 14C-glucose incorporation into glycogen with 2 ␮mol/l CHIR 98014 > P* .4 ؍ INH) or IA, or LiCl (20 mmol/l). Results are averages ؎ SE; n) 0.05 vs. no addition.

Because prolonged INH treatment increased insulin action on glucose uptake (Fig. 4B) independent of changes in glucose transporter expression (Fig. 6C), the influence of INH treatment on the expression of several key insulin- signaling proteins was investigated. Chronic treatment of FIG. 4. Effect of GSK-3 inhibitors on basal and insulin-stimulated muscle cells caused a concentration-dependent increase in glucose uptake in human muscle cells. A: Time course of glucose uptake ϳ activation in human muscle cells by CHIR 98023 (2 ␮mol/l), insulin (33 IRS-1 protein expression (Fig. 7A). There was an 3.5-fold nmol/l), or LiCl (20 mmol/l). Results are averages ؎ SE, normalized upregulation of IRS-1 protein after treatment with 2 ␮mol/l against glucose uptake in untreated control cells from the same CHIR 98023 (P Ͻ 0.05, n ϭ 10). Neither the inactive and type (5 ؍ B: Muscle cells from nondiabetic (n .(4 ؍ individual (n -subjects were treated for 4 days with CHIR 98023 compound nor LiCl had any effect on IRS-1 protein expres (4 ؍ diabetic (n 2 (2.5 ␮mol/l), CHIR 98014 (2.5 ␮mol/l), inactive compound (IA, 2.5 sion. Increases in IRS-1 protein were first seen after6hof ␮mol/l), or LiCl (20 mmol/l), as described in detail in RESEARCH DESIGN INH treatment, increasing for 24 h (Fig. 7B) and sustained AND METHODS. After chronic treatment, half of the cell cultures were washed with fresh media and incubated with 33 nmol/l insulin for 2 h. for 96 h (not shown). This time course is consistent with .Results are averages ؎ SE, normalized against the control and basal that seen for CHIR 98023 action on glucose uptake (Fig value for each individual. *P < 0.05 vs. untreated paired control; †P < 0.05 vs. paired basal. 4A). Moreover, the INH effect on IRS-1 was specific because Akt 1/2 protein abundance was unaltered by treatment (Fig. 7A). The consequences of IRS-1 upregula- Neither the inactive compound nor LiCl had any effect. tion on insulin signaling were investigated at the level of Under the same conditions, total GSK-3 activity was insulin stimulation of phosphorylation of Akt on serine reduced by 61 Ϯ 11% (P Ͻ 0.05). The decrease in enzyme 473, an event downstream of IRS-1 and PI 3-kinase (7,8). activity is truly reflective of the decrease in GSK-3 protein Stimulation of Akt phosphorylation at a maximal insulin and not a residual inhibitory effect of INH because cells level was unaltered by chronic treatment with either INHs were extensively washed before extraction and extracts or IA (Fig. 7C): 105 Ϯ 15 and 98 Ϯ 5% of the paired control were diluted during the assay, removing the effect of the response with INH and IA treatment, respectively (n ϭ 4). inhibitors as might be expected, since the nature of the inhibition is ATP competitive and reversible (26). The DISCUSSION results indicated that the effects of the INHs were on GS One approach to elucidating the importance of GSK-3 in activity state because total GS activity was unaltered after insulin action and control of glucose metabolism has been either acute or chronic treatment. GS protein expression the use of LiCl as an inhibitor of GSK-3. To better was also unchanged by chronic INH treatment (Fig. 6A). understand the role of GSK-3 in regulation of glucose The sustained (2- to 4-day) increase in glucose uptake metabolism, we used both LiCl and more selective and could not be explained by an upregulation of glucose potent inhibitors of GSK-3. CHIR 98014 and CHIR 98023 transporters because INH treatment had no effect (Fig. 6C; have 500- to 10,000-fold selectivity for GSK-3 over a large 100 Ϯ 14% of control, n ϭ 4) on total cell GLUT1 protein number of tested kinases (26). For example, CHIR 98014 ϳ expression. In keeping with the lack of effect of chronic has a Ki of 10 nmol/l for GSK-3 (both isoforms with exposure on glucose uptake, neither the inactive analog similar potency) and stimulates GS with an IC50 of 50–100 (101 Ϯ 2%) nor LiCl (87 Ϯ 4%) influenced GLUT1 content. nmol/l (26), orders of magnitude more selective than LiCl A similar lack of change was seen for total cellular GLUT4 (17,34). In addition, both INHs (CHIR 98014 and CHIR protein expression (Fig. 6C) after either INH (96 Ϯ 10% of 98023) appear to have higher affinities and better selectiv- control, n ϭ 4) or inactive analog (98 Ϯ 2%) treatment. ity than several recently described GSK-3 inhibitors (35).

2194 DIABETES, VOL. 51, JULY 2002 S.E. NIKOULINA AND ASSOCIATES

FIG. 7. Influence of chronic INH treatment on IRS-1 and Akt protein expression and Akt phosphorylation. A: Representative Western blot image of IRS-1 and Akt protein in muscle cells treated as described in the legend to Fig. 5. B: Time course of CHIR 98023 (2 ␮mol/l) effect on IRS-1 protein expression. Representative Western blot. C: Serine 473 phosphorylation of Akt in response to acute insulin exposure after chronic treatment of cells as described in Fig. 5.

situation. In the overall time course of INH action on GS, FIG. 6. Influence of chronic INH treatment on GSK-3, GS, GLUT1, and we observed two phases: a rapid activation that was GLUT4 protein expression. A: Representative Western blot image of complete within a half hour and a gradual increase over GSK-3 and GS protein in muscle cells untreated or treated with 0.5, 1, 6–24 h that was sustained even longer. This was opposite or 2 ␮mol/l CHIR 98023 for 4 days in fusion media. Western blotting was performed as described in RESEARCH DESIGN AND METHODS. B: Quan- to the response to insulin, with transient activation of GS titation of GSK-3␣ and GSK-3␤ protein in muscle cells after chronic and then development of desensitization, as has been treatment with 2 ␮mol/l CHIR 98023, 2 ␮mol/l inactive compound (IA), reported by multiple investigators of other cell types (37). six nondiabetic ;10 ؍ or 20 mmol/l LiCl. Results are averages ؎ SE (n patients, four diabetic patients). C: Representative Western blot image The prolonged upregulation of GS activity in response to of GLUT1 and GLUT4 protein in muscle cells untreated or treated with INH treatment involves GS activation because total GS 2 ␮mol/l CHIR 98023 or IA, or LiCl (20 mmol/l) for 4 days in fusion activity and protein expression were unaltered. The pro- media. *P < 0.05 vs. untreated control or cells treated with IA or LiCl. gressive GS activation apparently involves downregulation of GSK-3 protein expression because total activity and Besides stimulating GS, these compounds, collectively protein expression were reduced to the same extent. referred to here as INHs, can augment insulin action on Protein abundance of both isoforms, which are present at glucose uptake into skeletal muscle from insulin-resistant similar amounts in control cells, was decreased in INH- Zucker diabetic fatty rats (36). The experimental system treated cells. The downregulation of GSK-3 proteins ap- used in the current study is cultured human skeletal pears to involve inhibition of the enzyme because the muscle cells, which maintain many of the metabolic prop- response was observed with INHs and not the inactive erties of skeletal muscle, including impaired GS activity in analog. The lack of effect of LiCl on GSK-3 expression may cells from diabetic subjects (24,25). be related to reduced potency over extended times and/or As would be expected, INHs acutely activated GS in selectivity relative to INHs. Control of GSK-3 expression human muscle cells at concentrations far lower than LiCl. appears to be a second mechanism, available to INHs and Other GSK-3 inhibitors have also been demonstrated to not Li, for regulation of GSK-3 activity. increase glycogen synthesis in liver cells (35). The cur- Several laboratories have reported that GS protein ex- rently studied INHs alone were more effective than either pression is normal in the skeletal muscle of type 2 diabetic insulin or LiCl. INHs were able to augment insulin action subjects (30,38). Together with the evidence that GSK-3 on GS, whether added together with or after insulin. The expression is elevated in skeletal muscle of type 2 diabetic increase in the response to a submaximal insulin dose subjects (25), this would suggest that an abnormally low might indicate the ability of INHs to improve insulin GS/GSK-3 ratio is a feature of diabetic skeletal muscle, sensitivity. The maximal phosphorylation and deactivation shifting the balance toward inactivation of GS. This behav- attainable in response to insulin is not enough to fully ior is also present in cultured muscle cells, where the ratio inactivate GSK-3, whereas INHs can directly and more of GS protein to GSK-3 in cells from diabetic subjects completely block enzyme activity. (0.52 Ϯ 0.08, n ϭ 7) is significantly lower than the same Prolonged treatment of muscle cells with INHs was value in nondiabetic cells (1.09 Ϯ 0.12, n ϭ 7, P Ͻ 0.005). tested as a model more closely resembling the therapeutic Agents that can both inhibit and downregulate GSK-3 may

DIABETES, VOL. 51, JULY 2002 2195 INHIBITION OF GSK-3 IN HUMAN SKELETAL MUSCLE have an added therapeutic benefit for downregulation of 3T3-L1 adipocytes (34,47). When seeking possible mecha- GSK-3, whereas not influencing GS expression would have nisms for increased insulin action, we found that chronic the ultimate result of increasing the GS/GSK-3 ratio; such INH treatment increases IRS-1 protein expression with a a change would drive GS and GSK-3 toward a more normal time course similar to that for INH effects on GS activity balance. The specific molecular mechanism(s) by which and glucose uptake. Such an upregulation of IRS-1 could INHs downregulate GSK-3, impaired synthesis, and/or potentiate insulin action, just as overexpression of IRS-1 accelerated degradation are unknown and represent an has been shown to do in cell lines (48). The INH-induced intriguing area for further investigation. upregulation of IRS-1 was not part of a generalized in- Li has been shown in some (34,39,40) but not all (41,15) crease in insulin-signaling molecules because Akt expres- situations to acutely stimulate glucose transport or GLUT4 sion was unaltered. IRS-1 has been identified as an in vivo translocation. This response differs from activation of GS substrate for GSK-3, and, acutely, GSK-3–mediated phos- because it can be blocked with wortmannin (34), indicat- phorylation can impair its function (16). In addition, ing that it occurs at the level of PI 3-kinase activation, hyperphosphorylation of IRS-1 on serine residues induces whereas GS stimulation is not wortmannin sensitive and IRS-1 degradation in proteosomes (49,50). Hence, inhibi- occurs downstream of PI 3-kinase (34,15). Cultured human tion and/or downregulation of GSK-3 could reduce IRS-1 skeletal muscle cells behave much like other cells because phosphorylation and slow its degradation. Alternatively, Li is as effective as insulin in acutely stimulating glucose IRS-1 protein synthesis could increase as a result of GSK-3 uptake. Whether the glucose transport response to Li inhibition and reactivation of the translation initiation involves direct inhibition of GSK-3 is uncertain because factor eIF2B (45). Whatever the mechanism, an increase in INHs have little acute effect. Other mechanisms for Li IRS-1 protein expression in skeletal muscle could augment action on glucose transport have been proposed after insulin action, a further therapeutic benefit beyond activa- ruling out inhibition of myo-inositol-1-monophosphatase tion of GS. This potential insulin-sensitizing action of and accumulation of inositol monophosphates (40). These GSK-3 inhibition might be even more obvious in vivo mechanisms may include effects on Ca2ϩ and 1,6 bisphos- because we have recently demonstrated that muscle cells phatase (19,41). In rat muscles, Li has been shown to from type 2 diabetic subjects express normal amounts of change Ca2ϩ intracellular distribution, which could lead to IRS-1 protein (51), implicating something other than IRS-1 activation of glucose transport (42). The lack of acute INH expression as the major site of impaired insulin signaling effect on glucose uptake in muscle cells is in agreement in vitro. Whatever the effects on insulin signaling, the with studies in isolated muscles from ZDF rats (36), where INH-induced upregulation of IRS-1 expression was not uptake in the absence of insulin is unaltered by short-term found to increase insulin action on Akt phosphorylation. exposure to CHIR 98014. However, the current result was obtained at a maximally Insulin and Li have a similar activation profile on stimulating insulin concentration (33 nmol/l), which might glucose uptake. These agents acutely stimulate glucose obscure effects to improve insulin sensitivity. uptake followed by desensitization. In contrast, the INH The use of highly selective inhibitors has revealed effect on glucose uptake was delayed but caused a gradual several potential mechanisms by which GSK-3 can control and sustained increase. The increases in glucose uptake glucose metabolism and insulin action in skeletal muscle. with INH treatment could not be attributed to changes in In the short term, GSK-3 acts to suppress GS activity and transporter protein expression, indicating that INHs may possibly reduce insulin sensitivity. In the long term, GSK-3 be acting on other aspects of transporter function, such as activity may provide a negative influence on glucose transporter intrinsic activity or localization to the plasma uptake and insulin signaling because inhibition of GSK-3 membrane; these questions remain to be studied. Unlike leads to upregulation of glucose uptake and insulin re- GS, where chronically activated enzymes in INH-treated sponsiveness. What is notable here is both the magnitude cultures could not be stimulated further by insulin, glu- of upregulation with INHs and the delayed kinetics. In cose uptake could still be stimulated by insulin to a level addition, GSK-3 activity appears to be required to sustain approximately twofold greater than that possible in con- GSK-3 expression. Thus, along with expected effects on trol cells. Although partly the result of an elevation of GS, GSK-3 can play roles in other more upstream events in basal uptake, this final response suggests that insulin insulin-signaling pathways. Sustained GSK-3 inhibition action may also be augmented. may represent a useful means of countering the reduced The possibility that regulation of protein expression is levels of muscle glucose uptake and metabolism charac- one mechanism for the chronic effects of GSK-3 inhibitors teristic in type 2 diabetes. is supported by the fact that GSK-3 phosphorylates and Whereas the current work has focused on the involve- activates a number of factors (43–45). GSK-3 ment of GSK-3 in glucose metabolism and insulin action, is also a predominant kinase of translation initiation GSK-3 has been demonstrated to play a number of impor- factor, eIF2B (46), phosphorylation of which causes its tant roles, including control of cell differentiation and inactivation. Thus, inhibition of GSK-3 might reactivate proliferation through the Wnt pathway (52) and regulation protein synthesis at the level of translation. Alternatively, of specific gene transcription (53). Continuous activation the upregulation of glucose uptake by INHs could be of the Wnt pathway, associated with inhibition of GSK-3 indirect—the result of a pull of intracellular glucose and activity, is often linked to tumor development (54). Addi- glucose-6-phosphate into glycogen by activated GS. tionally, a GSK-3␤ knockout in mice is embryonically There have been reports in which inhibition of GSK-3 lethal because of liver degeneration (55). Thus, inhibition has been shown to increase insulin action on glucose of GSK-3 has the potential for broad effects beyond transport in isolated rodent skeletal muscles (26,41) and metabolism. Yet Li has an extended history of therapeutic

2196 DIABETES, VOL. 51, JULY 2002 S.E. NIKOULINA AND ASSOCIATES application, and augmented tumor formation has not been 17. Stambolic V, Ruel L, Woodgett JR: Lithium inhibits glycogen synthase reported in those patients. In fact, evidence suggests that kinase-3 activity and mimics wingless signalling in intact cells. Curr Biol 6:1664–1668, 1996 Li might even have a protective effect against cancer 18. Klein PS, Melton DA: A molecular mechanism for the effect of lithium on development (56). Considerable further study will be development. Proc Natl Acad SciUSA93:8455–8459, 1996 needed to sort out the various roles and regulation of 19. Bosh F, Rodriguez-Gil J, Hatzoglou M, Gomez-Foix A, Hanson R: Lithium GSK-3 in cell metabolism and development. Selective inhibits hepatic and phosphoenolpyruvate carboxykinase inhibitors of GSK-3 may prove to be useful tools in gene expression. J Biol Chem 267:2888–2893, 1992 answering these questions. 20. Rodriguez-Gil JE, Fernandez-Novell JM, Barbera A, Guinovart JJ: Lithium’s effects on rat liver glucose metabolism in vivo. Arch Biochem Biophys 375:377–384, 2000 ACKNOWLEDGMENTS 21. Hers I, Tavare JM, Denton RM: The inhibitors bisindoly- This work was supported by grant Rd-DK-258291 from the maleimide I (GF 109203x) and IX (Ro 31–8220) are potent inhibitors of National Institutes of Health; a grant from the Medical glycogen synthase kinase-3 activity. FEBS Lett 460:433–436, 1999 22. Nikoulina SE, Ciaraldi TP, Mudaliar S, Mohideen P, Carter L, Henry RR: Research Service, Department of Veterans Affairs and VA Potential role of glycogen synthase kinase-3 in skeletal muscle insulin San Diego Healthcare System; a grant from Chiron Corpo- resistance of type 2 diabetes. Diabetes 49:263–271, 2000 ration; and grant M01 RR-00827 in support of the General 23. 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