Inhibition of Glycogen Synthase Kinase 3 Improves Insulin Action and Glucose Metabolism 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. Diabetes 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 serine/ man skeletal muscle cells with either INH resulted in a threonine kinase originally discovered because dose-dependent activation of glycogen synthase (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 growth factor, and platelet-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 phosphatidylinositol (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 protein 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 kinases and phosphatases 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-phosphate. 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 receptor 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 enzymes 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, insulin receptor 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 insulin resistance 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 phosphorylation (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).