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Endocrinology Vol. 148, No. 9 4393-4399
Copyright © 2007 by The Endocrine Society

Role of Glycogen Synthase Kinase-3{alpha} in Insulin Action in Cultured Human Skeletal Muscle Cells

Theodore P. Ciaraldi, Svetlana E. Nikoulina, Rahil A. Bandukwala, Leslie Carter and Robert R. Henry

Veterans Affairs San Diego Healthcare System, San Diego, California 92161; and Department of Medicine, University of California, San Diego, La Jolla, California 92093

Address all correspondence and requests for reprints to: Robert R. Henry, M.D., Veterans Affairs San Diego Healthcare System, 3350 La Jolla Village Drive, San Diego, California 92161. E-mail: rrhenry{at}vapop.ucsd.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
An association between glycogen synthase kinase-3 (GSK3) in skeletal muscle and insulin resistance has been demonstrated in type 2 diabetic patients. In addition, inhibition of GSK3 improves insulin action. The aim of the present study was to elucidate the role of the {alpha}-isoform of GSK3 in insulin resistance in human skeletal muscle cells from nondiabetic subjects maintained in culture. Transfection of muscle cells with specific antisense oligonucleotides resulted in a 30–50% decrease of GSK3{alpha} protein expression (P < 0.05). Whereas neither the basal fractional velocity of glycogen synthase (GS FV) (an indicator of the activation state of the enzyme) nor glucose uptake (GU) were altered, reducing GSK3{alpha} expression resulted in increases in insulin stimulation of both GS FV and GU. GSK3{alpha} overexpression (60–100% increase over control) did not alter basal GS FV or GU but impaired insulin stimulation of both responses. Knockdown of GSK{alpha} also led to an increase in insulin receptor substrate-1 protein expression but did not alter insulin stimulation of pS473-Akt phosphorylation. However, GSK3{alpha} overexpression impaired insulin action on pS473-Akt. In summary, we concluded the following: 1) modulation of GSK3{alpha} expression has no effect on basal GU and glycogen synthase activities; 2) reduction of GSK3{alpha} expression results in improvements in insulin action; and 3) elevation of GSK3{alpha} in human skeletal muscle cells can induce insulin resistance for several responses. We conclude that GSK3{alpha} is an important regulator of muscle insulin action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLYCOGEN SYNTHASE KINASE-3 (GSK3) is a serine/threonine kinase with a proline-directed substrate preference. Although originally discovered due to its ability to phosphorylate and inhibit glycogen synthase (GS) (1), numerous other substrates for GSK3 have been identified including protein phosphatase inhibitor 2, ATP-citrate lyase, tau protein, ß-catenin, c-Myc, c-Jun, cAMP response element-binding protein, CCAAT/enhancer-binding protein-{alpha}, and initiation factor eukaryotic initiation factor (reviewed in Ref. 2). It has also been reported that GSK3 can phosphorylate the insulin receptor substrate (IRS)-1, thereby possibly playing a role in regulation of insulin action (3).

Two isoforms of GSK3, {alpha}-isoform and ß-isoform, have been identified in humans; encoded by two distinct genes located on chromosomes 19q13.1–2 and 3q13.3-q21, respectively (4). The {alpha}-isoform is a 51- to 53-kDa polypeptide, whereas the ß-isoform is a 47-kDa polypeptide with 85% amino acid homology to GSK3{alpha}. GSK3 is active in the resting state and is inhibited by hormones such as insulin, endothelial growth factor, and platelet-derived growth factor (2). Inactivation due to acute insulin exposure occurs through phosphorylation of Ser21 on GSK3{alpha} and Ser9 on GSK3ß.

An association between skeletal muscle GSK3 and insulin resistance has been demonstrated in type 2 diabetes and animal models of insulin resistance (5, 6, 7). We found the expression and activity of both GSK3 isoforms to be elevated in skeletal muscle from a group of poorly controlled type 2 diabetic patients, showing an inverse relationship to insulin action measured during a hyperinsulinemic/euglycemic clamp (6). Others have shown skeletal muscle GSK3 expression to be normal in diabetic subjects (8). In addition, chemical inhibition of GSK3 leads to improvements in glucose uptake, glycogen synthesis, and insulin action (7, 9, 10, 11, 12, 13), both in vivo and in vitro. However, the use of such inhibitors does not permit discrimination between the roles of the {alpha}- and ß-isoforms of GSK3 in metabolic regulation.

Evidence exists to suggest that GSK3{alpha} and -ß are not functionally interchangeable. Knockout of the GSK3ß gene in mice proves to be lethal in the embryonic stage with no rescue by an intact GSK3{alpha} isoform, leading to the idea that the ß-isoform may play an important role in cell growth and differentiation (14). Studies from our laboratory regarding insulin regulation of phosphorylation and activity of GSK3 isoforms in human skeletal muscle indicated that the {alpha}-isoform was more insulin responsive and had a stronger association with parameters of insulin resistance (6). In the present study, we therefore chose to specifically modulate the expression of GSK3{alpha} in human skeletal muscle cells to evaluate its role in regulation of glucose metabolism. In so doing, we have been able to demonstrate that knockdown of GSK3{alpha} leads to improvements in insulin action. Conversely, we found that overexpression of GSK3{alpha} generates a state of impaired insulin responsiveness.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
Skeletal muscle cell cultures were established from muscle tissue obtained by needle biopsy samples of the vastus lateralis. Muscle biopsy samples were obtained from a total of 21 nondiabetic subjects (15 males, six females), 45 ± 3 yr of age. On average the subjects were overweight (body mass index 29.6 ± 1.4 kg/m2), with normal fasting glucose (5. 3 ± 0.2 mM), insulin (73 ± 7 pM), and hemoglobin A1c (5.8 ± 0.1%) levels. Different subjects were used for the knockdown and overexpression studies. There were no differences in the clinical characteristics within subject groups for the different sets of studies. None of the subjects had a family history of type 2 diabetes. This qualifier may be important because it has been reported that skeletal muscle cells from first-degree relatives of type 2 diabetic subjects have impaired insulin action (15). The Committee on Human Investigation of the University of California, San Diego, approved the experimental protocol. Informed written consent was obtained from all subjects after explanation of the protocol.

Materials
L-[14C] glucose, [3H]2-deoxyglucose, and [14C]uridine diphosphate (UDP)-glucose, were obtained from DuPont NEN Life Science Products (Boston, MA). Cell culture materials were purchased from Irvine Scientific (Irvine, CA) except for skeletal muscle basal medium, which was obtained from Clonetics Corp. (San Diego, CA). Fetal bovine serum was obtained from Gemini Bio-Products (Woodland, CA). BSA (fraction V) was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Glycogen, 2-deoxyglucose, glucose-6-phosphate (G-6-P), leupeptin, aprotinin, sodium fluoride, sodium pyrophosphate, sodium orthovanadate, phenylmethylsulfonyl fluoride (PMSF), dithiothreitol, and other reagents and chemicals were purchased from Sigma (St. Louis, MO). Protein assay reagents and electrophoresis chemicals were purchased from Bio-Rad Laboratories (Hercules, CA). Anti-GSK-3/Shaggy protein kinase family (51/46 kDa) mouse monoclonal IgG, purified GSK3{alpha} and -ß from rabbit skeletal muscle, and antirat C-terminal IRS-1 rabbit polyclonal IgG were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-pS473-Akt was purchased from Cell Signaling Technology (Beverly, MA) whereas anti-Akt1/2 was from Santa Cruz Biotechnology (Santa Cruz, CA). Antirabbit IgG complexed to horseradish peroxidase and Hyperfilm were from Amersham, Inc. (Arlington Heights, IL). SuperBlock and the SuperSignal chemiluminescent substrate kit were obtained from Pierce (Rockford, IL). An affinity-purified chicken polyclonal antibody against GS was a gift from Dr. J. C. Lawrence, Jr. (University of Virginia, Charlottesville, VA). Transfection reagents used were PerFect Lipids Pfx-6 (Invitrogen, Carlsbad, CA) and Effectene (QIAGEN, Valencia, CA).

Full-length human GSK3{alpha} in pMT2 vector was a kind gift of Dr. J. Woodgett (Toronto, Canada) (16). Antisense oligodinucleotides (ODN) against GSK3{alpha} were identified from a screen of 78 human ODNs performed in T-24 cells (gift from Isis Pharmaceuticals, Carlsbad, CA). ODNs were synthesized as full phosphorothioate chimeric oligonucleotides with 2'-O-methoxyethyl groups on bases 1–5 and 6–20 (17). The antisense ODN used in the current studies had the sequence: AGCCAATGACACCATACCTT. A scrambled ODN was a mixed sequence of the antisense ODN.

Cell culture and transfection
The method of culturing skeletal muscle cells from biopsy samples has been described in detail previously (18, 19). Briefly, satellite cells were obtained by tryptic digestion of muscle biopsy material. Cells were propagated in culture by modifications of the methods described by Blau and Webster (20) and Sarabia et al. (21). When cells attained approximately 80% confluency, they were transferred to differentiation media. After 48 h of differentiation, cells were placed in OptiMEM and treated with lipid transfection reagent (pFx-6), empty vector (pMT2), or varying amounts of GSK3{alpha} RNA in pMT2 for 6–8 h. Cells were then washed and cultured for an additional 48 h in MEM containing glutamine and 2% fetal bovine serum before analysis. Media were changed every 48 h. For down-regulation of GSK3{alpha}, differentiating cells were placed in OptiMEM and treated with scrambled or antisense ODN for 6 h, washed, and then cultured an additional 60 h before protein extraction or assay of glucose uptake. Cells from each individual were used after a single passage.

Protein extraction
After treatment, fully differentiated cells were washed in serum-free {alpha}-MEM containing 0.1% BSA and incubated for 15' at 37 C in the presence or absence of insulin (33 nM). After this treatment, separate samples of cells in 6-well plates were rapidly washed with 4 C PBS and then lysed in extraction buffer: 20 mM Tris-HCl, 145 mM NaCl, 10% glycerol, 5 mM EDTA, 1% Triton X-100, 0.5% Nonidet P-40, 200 µM sodium orthovanadate, 200 µM PMSF, 1 µM leupeptin, 10 µg/ml aprotinin, 100 mM NaF, and 40 mM NaPP. Cells were scraped from the plates and solubilized over 30 min at 4 C with vortexing. Nonsolubilized material was removed by centrifugation at 14,000 x g (10 min, 4 C). Protein concentration was determined according to the method of Bradford (22) and cell extracts were stored at –70 C until further analysis.

Before GS assay, cells from 6-well plates were first incubated in the absence or presence of insulin (33 nM for 30 min at 37 C), washed with PBS, and scraped into 0.3 ml of extraction buffer: 50 mM HEPES, 10 mM EDTA, 100 mM NaF, 5 mM dithiothreitol, 1 µM leupeptin, 1 µM pepstatin, and 200 µM PMSF (pH 7.5).

Electrophoresis and Western blotting
Protein extracts were size fractionated on 7.5 or 10% polyacrylamide gels according to standard procedures (23). Proteins were transferred to nitrocellulose, blocked, and incubated with antibody to the particular protein of interest. Bands were visualized using secondary antibody tagged with horseradish peroxidase followed by enhanced chemiluminescence reaction. Quantification of band intensity was performed using an Alpha Innotech multiimage light cabinet and ChemiImager4000 software (version 4.04; San Leandro, CA). Results were obtained in integrated density value units per 10 µg total protein. Final values were expressed as percentage of the value in control cells for each individual.

Glucose uptake activity
At the same time as protein extraction, glucose uptake was measured in parallel plates using a previously described protocol (18). Briefly, cells were washed in serum-free media and incubated ± insulin (33 nM) for 60–90 min at 37 C in a 5% CO2 incubator. The insulin effect was maximal by 60 min and stable beyond 90 min. Uptake of the nonmetabolized analog 2-deoxyglucose (final concentration 0.01 mM) was measured over 10 min at room temperature (18). An aliquot of the suspension was removed for protein analysis. The uptake of L-glucose is used to correct each sample for the contribution of diffusion. All measures were performed in triplicate for each condition in each individual set of cells.

Assay of glycogen synthase activity
The activity of GS was measured as described in detail previously (19). Briefly, cells were washed and incubated in serum-free media for 30 min at 37 C before insulin (33 nM) was added; cells were washed and harvested after an additional 30 min incubation. GS activity was determined in duplicate for each set of cells in diluted supernatants at a physiological concentration of substrate (0.3 mM UDP-glucose) and expressed as nanomoles of UDP-glucose incorporated into glycogen per minute per milligram of total protein and as fractional velocity (FV = the ratio of activity assayed at 0.1 mM G-6-P/activity at 10 mM G-6-P).

Statistical analysis
Statistical significance was evaluated with a paired student t test and/or Wilcoxon signed rank test using GraphPad Prism (version 4.0; San Diego, CA). Significance was accepted at the P < 0.05 level. All data and calculated results are expressed as the mean ± SEM. Results are expressed as absolute values or normalized against the appropriate nonspecific (control) treatment. For a given manipulation, muscle cells from each subject served as its own control. Effects of gene manipulation were compared with the complete nonspecific condition; transfection reagent + scrambled ODN for knockdown and transfection reagent + empty vector for overexpression, for each individual set of cells, referred to throughout the manuscript as control. Due to limitations in cell availability, not all determinations were performed for each individual. The number of independent determinations is indicated in legends to the figures and tables. It should be noted again that different sets of subjects were used for the knockdown and overexpression studies. There were no significant differences in control activities between the two sets of subjects (Table 1Go).


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  		TABLE 1. Effects of manipulating GSK3{alpha} expression on activities of GS and glucose uptake

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Down-regulation of GSK3{alpha}
Transfection of human skeletal muscle cells with antisense ODN to GSK3{alpha} resulted in a selective reduction in GSK3{alpha} protein expression (Fig. 1Go). Antisense ODN against GSK3{alpha} had no effect on GSK3ß protein content (Fig. 1Go). GS protein expression was also unaltered by GSK3{alpha} AS-ODN (83 ± 12% of control, P = ns). Scrambled (control) ODN was without significant effect on either GSK3{alpha} or GSK3ß expression. GSK3 activity is also influenced by serine phosphorylation (2) as well as expression. GSK3{alpha} knockdown had no significant effect on basal serine phosphorylation of either GSK3{alpha} (S21) or -ß (S9).


Figure 1
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  		FIG. 1. Down-regulation of GSK3{alpha} in human skeletal muscle cells. A, Representative Western blot for total GSK3 in cells treated with control (cont; scrambled) or antisense (AS) ODN (60 nM) as described in Materials and Methods. B, Quantification of Western blots for GSK3{alpha} and GSK3ß protein expression in muscle cells. Results expressed as percent of control (scrambled ODN) treated cells for each individual. Results depicted as mean + SEM, n = 8. *, P < 0.05 vs. control.

 
Effect of GSK3{alpha} knockdown on glucose metabolism and insulin action
The total activity of glycogen synthase, measured at a saturating concentration (10 mM) of the allosteric regulator G-6-P, is essentially independent of the phosphorylation state of the enzyme (reviewed in Ref. 24). This activity was unaffected by transfection with control or GSK3{alpha} antisense ODN (Table 1Go). The FV of GS (activity at 0.1 mM G-6-P/activity at 10 mM G-6-P) is an indicator of the activation state of the enzyme. It is this property that is decreased by serine phosphorylation of the protein and increased by insulin (24). Knockdown of GSK3{alpha} had no impact on basal FV (Table 1Go). However, insulin-stimulated GS FV was elevated after reduction of GSK3{alpha} expression (Table 1Go). The absolute magnitude of the incremental insulin response in GS FV was increased 2-fold by antisense ODN 1 (Fig. 2AGo). Thus, down regulation of GSK3{alpha} improved insulin responsiveness of GS.


Figure 2
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  		FIG. 2. Effect of GSK3{alpha} down-regulation or overexpression on insulin responsiveness of GS and glucose uptake. Results are expressed as the absolute insulin increment in GS FV (A) or glucose uptake (B) calculated for cells from each subject. Results are average + SEM. Knockdown: n = 8 for GS and n = 6 for glucose uptake. Overexpression: n = 8 each for GS and glucose uptake. *, P < 0.05 vs. paired control.

 
Transfection of muscle cells with antisense-ODN had no effect on basal glucose uptake, compared with control ODN (Table 1Go). Maximally insulin-stimulated glucose uptake was elevated after antisense-ODN (Table 1Go). The absolute insulin-induced increment in glucose uptake, determined for each individual set of cells, was greater in antisense ODN-treated cells (Fig. 2BGo). Down-regulation of GSK3{alpha} resulted in a 2- to 3-fold increase in the magnitude of the absolute insulin response for glucose uptake.

Overexpression of GSK3{alpha}
Transfection of human skeletal muscle cells with a vector containing GSK3{alpha} resulted in an increased expression of GSK3{alpha} protein (Fig. 3Go). The response was dose dependent and specific for the {alpha}-isoform. The appropriate control, including empty vector (pMT2), had no significant effect on expression of either GSK3 isoform (Fig. 3Go). Basal (no added insulin) serine phosphorylation of either GSK3 isoform was unaltered by GSK3{alpha} overexpression.


Figure 3
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  		FIG. 3. Overexpression of GSK3{alpha} in human muscle cells. Differentiating myotubes were treated with lipid transfection reagent and empty vector [control (cont)] or varying amounts of GSK3{alpha} DNA in pMT2 (micrograms of GSK3{alpha}) as described in Materials and Methods. A, Representative Western blot for total GSK3. B, Quantification of Western blots for GSK3{alpha} and GSK3ß. Results expressed as percent of control cells for each individual, average + SEM, n = 7. *, P < 0.05 vs. control.

 
Effect of GSK3{alpha} overexpression on glycogen synthase activity
Total GS activity was unaltered by transfection with either control vector or GSK3{alpha} (Table 1Go). GSK3{alpha} overexpression had no effect on the basal GS FV (Table 1Go). Whereas no statistically significant effect of GSK3{alpha} overexpression was seen on insulin-stimulated FV (Table 1Go), the absolute insulin-induced incremental change calculated for each individual was reduced by GSK3{alpha} overexpression (Fig. 2AGo).

Effect of GSK3{alpha} overexpression on glucose uptake
Transfection with GSK3{alpha} had no effect on basal glucose uptake, compared with control (Table 1Go). Insulin acutely stimulated glucose uptake in both ND and T2D cells (Table 1Go); this effect was decreased in GSK3{alpha} overexpressing cells and, in fact, was no longer statistically significant. Thus GSK3{alpha} overexpression results in insulin resistance for stimulation of glucose uptake (Fig. 2BGo).

Effect of manipulation of GSK3{alpha} on insulin signaling
Because the major consequence of changes in GSK3{alpha} protein expression in the current studies is on insulin responsiveness (Fig. 2Go), the effects of these manipulations on the expression of the key insulin signaling molecules IRS-1 and Akt 1/2 were evaluated. None of the transfection protocols themselves had any impact on IRS-1 protein content of muscle cells. Similar to what was observed from treatment with GSK3 inhibitors (9), knockdown of GSK3{alpha} led to a significant increase in IRS-1 expression (Fig. 4Go) In contrast, overexpression of GSK3{alpha} did not result in significant changes in the expression of IRS-1 (Fig. 4Go). Neither manipulation influenced Akt 1/2 protein expression (Fig. 4Go).


Figure 4
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  		FIG. 4. Insulin signaling protein expression after manipulation of GSK3{alpha} expression. Protein extracts were Western blotted for IRS-1 and Akt 1/2. A, Representative Western blots, duplicate cell samples from a single subject. B, Quantification of blots. Results presented as percent of expression in control (cont) cells for each individual. Results are mean + SEM. For IRS-1 n = 10 for knockdown (KD), n = 8 overexpression (OE). For Akt 1/2, n = 9 for knockdown, n = 6 for overexpression. *, P < 0.05 vs. control.

 
The effects of varying GSK3{alpha} expression on insulin signaling was studied at the level of the phosphorylation of serine-473 in Akt, a commonly accepted indicator of insulin signaling. Results in Fig. 5Go are presented as the extent of S473 phosphorylation normalized to the amount of total Akt 1/2 protein in the same sample. Neither up- nor down-regulation of GSK3{alpha} expression had a significant effect on basal phosphorylation. Acute (15 min) insulin exposure resulted in stimulation of S473 phosphorylation, similar to what we reported previously (25). The insulin response in GSK3{alpha} knockdown cells was unaltered: stimulated pS473-Akt/Akt was 109 ± 9% of the control value. However, insulin stimulation of phosphorylation was reduced with GSK3{alpha} overexpression to 62 ± 14% (P < 0.05) of control. This determination was made at the same maximally stimulating insulin level (33 nM) in which we found effects of GSK3{alpha} manipulation on hormone responsiveness for GS FV and glucose uptake.


Figure 5
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  		FIG. 5. Effect of manipulation of GSK3{alpha} expression on Akt phosphorylation. Proteins extracted after 15 min incubation in the absence or presence of insulin (Ins; 33 nM). pS473-Akt determined by Western blotting. Blots were stripped and reprobed for Akt-1/2. Results were normalized against the amount of Akt for each condition, average + SEM. Open bars, control (n = 8); striped bars, knockdown (n = 6); solid bars, overexpression (n = 5). *, P < 0.05 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Whereas GSK3 was first identified through its ability to phosphorylate and inhibit glycogen synthase, the enzyme has been demonstrated to be involved in many cellular processes, including differentiation, transcription, translation, cytoskeletal organization, tumor genesis, cell cycle progression, and the development of neurological diseases (26). Multiple lines of evidence suggest that GSK3 can play a major role in the regulation of glucose metabolism and insulin action. GSK3 has been shown to be elevated in the skeletal muscle of poorly controlled type 2 diabetic subjects (6) as well as animal models of insulin resistance (5, 7). It should be noted that the relationship between GSK3 expression and insulin action is a quantitative one because reports in diabetic humans noted a normal ability of insulin to reduce GSK3 activity (6, 8).

Lithium, which inhibits GSK3 by competing for the cofactor Mg+2, increases both glucose metabolism and insulin action in experimental systems (27, 28). Recently more selective inhibitors of GSK3 have been synthesized, which are ATP competitive (29) or substrate competitive (30). These compounds have frequently been found to be insulin mimetic, increasing GS activity, glycogen synthesis, and glucose uptake in a number of cell lines (9, 29, 30) and improve glucose disposal in insulin-resistant animals (7, 13, 31). Such specific inhibitors have proven useful in studying the role of GKS3 but lack the ability to distinguish between the contributions of the {alpha}- and ß-isoforms. This fact is important because, whereas GSK3{alpha} and GSK3ß are similar in many ways [97% sequence identity in the kinase domain (2)], they are products of different genes and not fully functionally interchangeable.

Although the great majority of studies on GSK3 isoforms have focused on GSK3ß, due in part to the availability of specific reagents (2), we chose to initially target GSK3{alpha} in evaluating the involvement of GSK3 in the development of insulin resistance. There were several reasons for this choice. In our previous in vivo study skeletal muscle GSK3{alpha} showed a greater reduction in activity, and increase in serine phosphorylation, after insulin infusion in humans, compared with GSK3ß (6). The correlations between the activity and expression of GSK3 for measures of insulin action were strongest for GSK3{alpha} (5). Previously Wojtazsewski et al. (32) concluded that GSK3{alpha} had a role in the response of skeletal muscle GS activity to meal-associated hyperinsulinemia in humans, whereas GSK3ß did not. Meanwhile, overexpression of wild-type GSK3ß in 3T3-L1 adipocytes reduced GS activity but did not change the fold stimulation in response to insulin (33). Taken together, these results suggest that GSK3{alpha} could be the predominant isoform for the regulation and mediation of insulin action.

This postulate was tested in cultured human skeletal muscle cells. An advantage of this system is that we (18) and others (34) have shown that this system maintains many of the metabolic properties of intact skeletal muscle, including insulin responsiveness of GS (18, 34) and GSK3 (9, 35). However, culturing muscle cells under controlled conditions may obscure some of the features of the type 2 diabetic phenotype that are acquired from the hyperglycemic, hyperinsulinemic environment present in vivo. A further benefit of cultured human skeletal muscle cells is that they are amenable to transfection and have been used by multiple investigators to probe the role of specific genes in the control of muscle metabolism and insulin action (36, 37, 38). The ability to compare the impact of such manipulations with the in vivo diabetic metabolic phenotype represents an advantage of this experimental system.

Comparison of the results from our previous work on global chemical inhibition of GSK3 in human muscle cells (9) with the effects of targeted knockdown of GSK3{alpha} should provide insights into specific roles of GSK3{alpha}. Several caveats should be considered when making such comparisons. One relates to possible dose effects. We previously attained an approximately 90% reduction in total GSK3 activity by use of selective inhibitors (9). Because GSK3{alpha} is the less abundant form in human muscle cells, 38 ± 2% of total GSK3 protein, a 50% reduction in expression may be estimated to cause an approximately 20% reduction in total enzyme activity. Another concern is that whereas chemical inhibitors affect GSK3 throughout the cell, it is not known whether GSK3{alpha} knockdown alters the subcellular distribution of GSK3 isoforms, which could influence access to specific substrates. With those concerns in mind, specific GSK3{alpha} knockdown and general GSK3 inhibition have several common effects. One is that both interventions increased insulin stimulation of GS FV and glucose uptake. Another is that both led to an up-regulation of IRS-1 protein expression, suggesting that a normal cellular content of GSK3{alpha} can suppress both IRS-1 expression and insulin action. Differences are also apparent in that chronic GSK3 inhibition elevated glucose uptake and GS activities, even in the absence of insulin, whereas GSK3{alpha} knockdown had no such effect, suggesting that changing both GSK3{alpha} and -ß activity influenced basal activities, whereas GSK3{alpha} is linked to the insulin response. This would be consistent with the results from Zucker diabetic fatty rats, in which inhibition of GSK3 enhanced GLUT4 translocation in isolated skeletal muscles (10).

Conversely, the hypothesis generated from our previous in vivo studies and the work of multiple investigators with GSK3 inhibitors would predict that a relative up-regulation of GSK3 would contribute to insulin resistance and other aspects of the diabetic metabolic phenotype. Indeed, the phenotype in muscle cells resulting from overexpression of GSK3{alpha} does have several features that are present in muscle from type 2 diabetic subjects, compared with nondiabetics: reduced insulin stimulation of GS FV (39) and glucose uptake (40) and normal expression of IRS-1 (41). There are also differences. It is interesting to note that the commonly observed diabetes-related defects in basal glucose uptake in skeletal muscle (40) are lacking after GSK3{alpha} overexpression, implicating involvement of kinases besides GSK3{alpha}, possibly including GSK3ß, in the control of this basal activity. Thus, a relative overexpression of GSK3{alpha} in muscle cells induces some, but not all, of the features of type 2 diabetes in muscle.

GSK3 could contribute to the development of insulin resistance in several ways. The previously demonstrated ability of GSK3 to phosphorylate IRS-1 and interfere with its ability to be tyrosine phosphorylated by the insulin receptor (3) could lead to impaired insulin action in the presence of normal IRS-1 protein expression, as was the case with GSK3{alpha} overexpression. Conversely, down-regulation of GSK3{alpha} could serve to improve insulin signaling by reducing serine phosphorylation of IRS-1. In addition, hyperphosphorylation of IRS-1 on serine resides increases proteosomal-mediated degradation of IRS-1 (reviewed in Ref. 42). GSK3-mediated phosphorylation of IRS-1 could be contributing to this response so that with GSK3{alpha} knockdown turnover of IRS-1 is reduced and steady-state levels of the protein increase. These conjectures need to be tested directly.

Whereas the ability of GSK3 to phosphorylate IRS-1 has been proposed as crucial for the generation of insulin resistance (43), the impact of this phosphorylation on downstream insulin signaling events in human skeletal muscle is unknown. We selected serine-473 phosphorylation of Akt as the signal step distal to IRS-1 for further study and found that knockdown of GSK3{alpha} did not alter insulin stimulation of S473-Akt phosphorylation, even as responsiveness of GU and GSFV were augmented. This result is consistent with two studies using different GSK3 inhibitors (9, 11) in which Akt phosphorylation was also not effected. However, overexpression of GSK3{alpha} did result in impaired maximal insulin action on S473-Akt phosphorylation as well as for glucose uptake and glycogen synthase. The difference between these two outcomes is suggestive of a threshold effect, that a relative excess of GSK3{alpha} is required to impede signaling at the level of S473-Akt phosphorylation. If the same behavior occurs for T308-Akt phosphorylation, the other insulin-responsive event is unknown.

In summary, the current results, in which the major effects of manipulation of GSK3{alpha} expression were on insulin-stimulated events, offer evidence to support the idea that although similar in structure, GSK3{alpha} and GSK3ß may have differing roles within the skeletal muscle cell. Whereas GSK3{alpha} is involved with insulin-stimulated glucose metabolism, GSK3ß may be the isoform most responsible for basal energy expenditure, cell growth, and differentiation. Further studies are needed to elucidate the specific roles of GSK3ß because the proposed distinction need not be absolute because muscle-specific overexpression of GSK3ß results in male transgenic mice that are glucose intolerant and display reduced IRS-1 protein levels (44). Targeting the specific GSK3 isoform most closely involved with insulin regulation of metabolism represents an approach worthy of further investigation.


   Footnotes
 
This work was supported by Grant RO1-DK-258291 from the National Institutes of Health (to R.R.H.); a grant from the Medical Research Service, Department of Veterans Affairs, and Veterans Affairs San Diego Healthcare System; a mentor-based fellowship award from the American Diabetes Association (to R.R.H.); and Grant M01 RR-00827 in support of the General Clinical Research Center from the General Clinical Research Branch, Division of Research Sources, National Institutes of Health.

Disclosure Statement: T.P.C., S.E.N., R.A.B., and L.C. have nothing to declare. R.R.H. is a member of the Board of Directors, consults for, and has stock in Isis Pharmaceuticals.

First Published Online June 14, 2007

Abbreviations: FV, Fractional velocity; G-6-P, glucose-6-phosphate; GS FV, fractional velocity of glycogen synthase; GSK3, glycogen synthase kinase-3; GU, glucose uptake; IRS, insulin receptor substrate; ODN, oligodinucleotide; PMSF, phenylmethylsulfonyl fluoride; UDP; uridine diphosphate.

Received July 12, 2006.

Accepted for publication June 5, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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