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Am J Physiol Endocrinol Metab 290: E251-E257, 2006. First published August 23, 2005; doi:10.1152/ajpendo.00096.2005
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Chronic rosiglitazone treatment restores AMPK{alpha}2 activity in insulin-resistant rat skeletal muscle

Sarah J. Lessard,1 Zhi-Ping Chen,2 Matthew J. Watt,1 Michael Hashem,1 Julianne J. Reid,1 Mark A. Febbraio,1 Bruce E. Kemp,2,3 and John A. Hawley1

1School of Medical Sciences, Royal Melbourne Institute of Technology University; 2St. Vincent's Institute and Department of Medicine, University of Melbourne, Melbourne; and 3Commonwealth Scientific and Industrial Research Organisation Health Sciences & Nutrition, Parkville, Victoria, Australia

Submitted 3 March 2005 ; accepted in final form 12 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Rosiglitazone (RSG) is an insulin-sensitizing thiazolidinedione (TZD) that exerts peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma})-dependent and -independent effects. We tested the hypothesis that part of the insulin-sensitizing effect of RSG is mediated through the action of AMP-activated protein kinase (AMPK). First, we determined the effect of acute (30–60 min) incubation of L6 myotubes with RSG on AMPK regulation and palmitate oxidation. Compared with control (DMSO), 200 µM RSG increased (P < 0.05) AMPK{alpha}1 activity and phosphorylation of AMPK (Thr172). In addition, acetyl-CoA carboxylase (Ser218) phosphorylation and palmitate oxidation were increased (P < 0.05) in these cells. To investigate the effects of chronic RSG treatment on AMPK regulation in skeletal muscle in vivo, obese Zucker rats were randomly allocated into two experimental groups: control and RSG. Lean Zucker rats were treated with vehicle and acted as a control group for obese Zucker rats. Rats were dosed daily for 6 wk with either vehicle (0.5% carboxymethylcellulose, 100 µl/100 g body mass), or 3 mg/kg RSG. AMPK{alpha}1 activity was similar in muscle from lean and obese animals and was unaffected by RSG treatment. AMPK{alpha}2 activity was ~25% lower in obese vs. lean animals (P < 0.05) but was normalized to control values after RSG treatment. ACC phosphorylation was decreased with obesity (P < 0.05) but restored to the level of lean controls with RSG treatment. Our data demonstrate that RSG restores AMPK signaling in skeletal muscle of insulin-resistant obese Zucker rats.

lipids; peroxisome proliferator-activated receptor-{gamma}; thiazolidinedione; Zucker rats; adenosine monophosphate-activated protein kinase-{alpha}2

SKELETAL MUSCLE INSULIN RESISTANCE is a common state associated with inactivity, aging, genetic predisposition, and environmental factors and is a hallmark feature of a variety of disease states, including obesity, hyperlipidemia, hypertension, and type 2 diabetes (31). Rosiglitazone (RSG) is a member of the thiazolidinedione (TZD) class of oral antidiabetic agents that improve insulin sensitivity in a range of insulin-resistant states (15, 18). TZDs are peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) agonists that, upon activation by fatty acids (FA) or FA-derived compounds, bind to responsive elements located in the promoter regions of many genes and modulate their transcriptive activities (1, 32). Although PPAR{gamma} plays a role in the TZD-induced insulin sensitization, metabolic responses to TZDs can be dissociated from PPAR{gamma}-induced gene transcription (4, 14, 29). Furthermore, we (23) have recently reported that RSG improves glucose tolerance by mechanisms other than a reduction of FA accumulation within skeletal muscle. Hence, it is likely that there exists PPAR{gamma}-independent mechanisms by which TZDs improve insulin sensitivity.

The AMP-activated protein kinase (AMPK) is a cellular energy sensor that regulates glucose and lipid metabolism by phosphorylating key regulatory enzymes (21, 39). AMPK activation causes many metabolic changes that would be beneficial to individuals with type 2 diabetes and the metabolic syndrome, including increased glucose uptake and metabolism by muscle, decreased glucose production by the liver and decreased synthesis, and increased oxidation of FA (16, 39). As TZDs increase insulin-stimulated glucose uptake and utilization and decrease circulating levels of free FA and triglycerides (for reviews, see Refs. 26 and 32), it is reasonable that a component of the insulin-sensitizing effect of these drugs could be mediated through the action of the AMPK pathway. In this regard, Fryer et al. (13) reported that acute incubation of H-2Kb muscle cells with RSG increased the AMP/ATP ratio and activated both {alpha}1- and {alpha}2-containing AMPK isoforms, which lead to a marked increase in the phosphorylation of acetyl-CoA carboxylase (ACC). Furthermore, AMPK has been reported to phosphorylate insulin receptor substrate-1 at Ser789 and enhance p85 docking, which is consistent with improving insulin signaling (19).

In the present study, we determined the effects of acute RSG administration in L6 muscle cells and chronic RSG treatment in skeletal muscle from obese, glucose-intolerant Zucker rats on AMPK regulation and its downstream phosphorylation target, ACC. We found that RSG treatment normalizes AMPK{alpha}2 activity in the skeletal muscle of obese Zucker rats compared with their lean littermates.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Female obese Zucker and age-matched lean rats were obtained from Monash Animal Services (Victoria, Australia) at 19 wk of age. Rats were housed under controlled light (12:12-h light-dark cycle) at an ambient temperature of 21°C. Animals had free access to standard rat chow and water, except when overnight fasting was required for blood measurements. All procedures were approved by the Royal Melbourne Institute of Technology University Animal Ethics Committee. Obese rats were randomly divided into two experimental groups: control (OB CON, n = 9) and RSG treated (OB RSG, n = 9). The lean rats (LN CON, n = 9) were treated with vehicle and acted as a control group for the OB CON rats. The rats were dosed daily, for 6 wk by oral gavage with either vehicle, which consisted of 0.5% carboxymethylcellulose (CMC; 100 µl/100 g body mass), or 3 mg/kg RSG (GlaxoSmithKline, Stevenage, UK) suspended in an equal volume of CMC. After the 6-wk experimental period, rats were fasted overnight and euthanized by CO2 asphyxia, followed by exsanguination. The time interval between CO2 administration and muscle harvesting was ~1–2 min, and the interval between the last RSG treatment and tissue collection was ~24 h. The red gastrocnemius muscles were rapidly dissected from the rat hindlimbs, snap-frozen in liquid nitrogen, and stored at –80°C for later analysis.

Muscle metabolite and adenine nucleotide contents. Red gastrocnemius muscle was freeze-dried, powdered, and cleaned of all visible connective tissue and blood under magnification. An ~2-mg aliquot was then taken and extracted in 0.5 M HClO4 (1 mM EDTA) and neutralized with 2.2 M KHCO3. Muscle ATP, phosphocreatine (PCr), creatine, and lactate were measured, and muscle ADP and AMP were calculated as described for cells. Muscle for glycogen analysis was extracted in 2 M HCl and neutralized with 0.67 M NaOH, and glycogen content was determined by fluorometric techniques (27).

AMPK activity and AMPK subunit protein expression. Approximately 80 mg of wet muscle were homogenized in buffer A (50 mM Tris·HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 50 mM NaF, 5 mM Na pyrophosphate, 10% glycerol, 1% Triton X-100, 10 mg/ml trypsin inhibitor, 2 mg/ml aprotinin, 1 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride). The muscle homogenate was then centrifuged at 20,000 g for 25 min, and the supernatant was collected. The supernatant was aliquoted for determination of 1) protein concentration (Pierce, Rockford, IL), 2) immunoprecipitation (AMPK{alpha}1 and AMPK{alpha}2), 3) ACC affinity purification, and 4) protein expression by Western blotting [uncoupling protein-3 (UCP3)]. Aliquots were stored at –80°C until use, and immunoprecipitates were used to determine protein content and enzyme activity (AMPK{alpha}1 and AMPK{alpha}2, described below).

Immunoprecipitation. Approximately 5 mg of protein from the supernatants were incubated with AMPK{alpha}1 or AMPK{alpha}2 antibody-bound protein A-Sepharose beads (6 MB; Amersham Biosciences, Uppsala, Sweden) for 2 h at 4°C. The polyclonal antipeptide antibodies to AMPK{alpha}1 and AMPK{alpha}2 were raised to nonconserved regions of the AMPK{alpha}1 (rat 231–251) and AMPK{alpha}2 isoforms (rat 351–366). Immunocomplexes were washed with PBS and suspended in 50 mM Tris (pH 7.5) buffer for the AMPK activity assay (9). The AMPK activities in the immune complexes were measured in the presence of 200 µM AMP. Activities were calculated as picomoles of phosphate incorporated into the serine-alanine-methionine-serine (SAMS) peptide per minute per milligram of protein subjected to immunoprecipitation (pmol·min–1·mg–1). ACC was affinity purified by incubating the post-AMPK immunoprecipitation supernatants in streptavidin-Sepharose high-performance beads (Amersham Biosciences) for 1 h at 4°C.

Western blotting. The affinity-purified ACC fraction was electrophoresed on 7.5% SDS-PAGE and detected by immunoblotting with anti-phospho-Ser218-ACC polyclonal antibody (8). The blots were then stripped (50 mM Tris, 2% SDS, 115 mM {beta}-mercaptoethanol) at 55°C for 20 min, blocked, and incubated with horseradish peroxidase-conjugated streptavidin (Amersham Pharmacia Biotech UK, Little Chalfont, UK) to determine total ACC. Aliquots of immunoprecipitated AMPK{alpha}1 and AMPK{alpha}2 proteins were electrophoresed on 10% SDS-PAGE and detected by immunoblotting with anti-phospho-Thr172 antibody and raised against AMPK{alpha} peptide (KDGEFLRpTSCGSPNY) as described previously (10). The membranes were then stripped (as described above), washed in PBS, blocked for 60 min in 5% skim milk powder-PBS, and reprobed with antibodies specific for the AMPK{alpha}1 and AMPK{alpha}2 isoforms. Aliquots containing 80 µg of total protein were electrophoresed on 15% SDS-PAGE and detected by immunoblotting with anti-UCP3 antibody (Alpha Diagnostic, San Antonio, TX).

Cell culture. L6 myoblasts were maintained at 37°C (95% O2-5% CO2) on 60-mm collagen-coated plastic dishes in {alpha}-modified Eagle's medium ({alpha}-MEM) containing 10% fetal bovine serum (FBS) culture medium and 1% penicillin-streptomycin and 5 mM glucose. Differentiation was induced by switching to medium containing 2% horse serum when the myoblasts were ~90% confluent. Experimental treatments were started after 4 days, by which time nearly all of the myoblasts had fused to form myotubes.

Experimental protocol. To determine the effect of RSG treatment on AMPK activity, AMPK Thr172 phosphorylation, and ACC Ser218 phophorylation, L6 myotubes were incubated with 0 (DMSO), 5, 50, or 200 µM RSG for 30 min at 37°C. After incubation, cells were rinsed three times with PBS and lysed with 200 µl of buffer A. Lysates were immediately frozen in liquid N2 and kept at –80°C until further analysis. AMPK{alpha}1 and AMPK{alpha}2 subunits and ACC{beta} protein were isolated by immunoprecipitation and affinity chromatography, respectively, as described above. AMPK activity was determined using the SAMS peptide assay, and protein expression and phosphorylation were determined as described above.

Palmitate oxidation. To examine the effects of RSG on fat oxidation, cells were grown in 60-mm plates and incubated for 1 h in the absence (DMSO, n = 6) or presence of 5 µM (n = 6) or 200 µM (n = 6) RSG in 3 ml of medium consisting of {alpha}-MEM, 2% BSA, and 0.5 mM [14C]palmitate (Amersham Biosciences, Castle Hill, Australia). After the incubation period, the reaction was stopped, and 2 ml of medium were added to an equal volume of 1 M acetic acid, and the released 14CO2 was collected in benzothonium hydroxide. The cells were rinsed twice with PBS, methanol was added to the plate, and cells were scraped for subsequent analysis of the acid-soluble metabolite (ASM) fraction. The 14C content of both fractions was determined using scinitillation counting, and the oxidation rates (CO2 + ASM) were calculated.

Metabolite analysis. L6 myotubes were incubated with DMSO (vehicle), 200 µM RSG, 500 µM dinitrophenol (DNP), or 200 µM 5-aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside (AICAR) for 30 min. The medium was removed, and the cells were rinsed three times with PBS, lysed with 100 µl of 0.5 M HClO4 (1 mM EDTA), and neutralized with 2.2 M KHCO3. This extract was used for the determination of adenosine triphosphate (ATP), PCr, creatine, and lactate by enzymatic fluorometric assays (2). Free ADP and AMP concentrations were calculated with the assumption of equilibrium of the adenylate kinase and creatine kinase reactions (12). Free ADP was calculated using the measured ATP, creatine, and PCr values, an estimated H+ concentration (30), and the creatine kinase equilibrium constant of 1.66 x 109. Free AMP concentration was calculated from the estimated free ADP and the measured ATP with the adenylate kinase constant of 1.05.

Statistical analysis. Results are presented as means ± SE. Statistical analysis of skeletal muscle measurements was performed using an unpaired Student's t-test. Analysis of cell culture experiments was undertaken using one-way ANOVA. A P value of <0.05 was considered significant. When ANOVA revealed significant differences, a Neuman-Keuls post hoc test was used to locate such differences.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of acute incubation of muscle cells with RSG. Table 1 displays the AMP/ATP ratio of L6 myotubes after incubation with DMSO (control), 200 µM RSG, 500 µM DNP, or 200 µM AICAR for 30 min. Incubation with DNP caused a 2.4-fold increase in the AMP/ATP ratio (P = 0.02 vs. control). No change in AMP/ATP was observed after RSG or AICAR treatment.


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  		Table 1. AMP (free)/ATP ratios from L6 myotubes treated with rosiglitazone, AICAR, or DNP

 
Figure 1 shows the effect of varying concentrations of RSG on the activity of AMPK{alpha}1 (Fig 1A) and Thr172 phosphorylation of AMPK{alpha}1 (Fig. 1B), whereas Fig. 2 displays the ACC Ser218 phosphorylation (Fig. 2A) and palmitate oxidation (Fig. 2B) in L6 myotubes after acute incubation. The dose-response trend of increasing AMPK{alpha}1 activity was significant at a dose of 200 µM RSG (P < 0.05) and corresponded with similar increases in Thr172 phosphorylation of the AMPK{alpha}1 isoform (Fig. 1B). Increased AMPK{alpha}1 activity in L6 myotubes at the highest dose of RSG was also associated with a threefold increase in the ACC Ser218 phosphorylation compared with control (0 vs. 200 µM RSG, P < 0.05; Fig. 2A) and a significant increase in palmitate oxidation (0 vs. 200 µM RSG, 391 ± 20 vs. 455 ± 19 pmol/h, respectively; Fig. 2B). AMPK{alpha}2 activity in L6 myotubes was undetectable.



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  		Fig. 1. Activation of AMP-activated protein kinase (AMPK) by phosporylation in rosiglitazone (RSG)-treated L6 myotubes. A: AMPK activity measured using the SAMS peptide assay of AMPK{alpha}1 protein isolated by immunoprecipitation from L6 myotubes incubated with 0 (DMSO), 5, 50, or 200 µM RSG for 30 min. B: ratio of AMPK{alpha}1 protein phosphorylated at Thr172 (pT172) to total levels of AMPK{alpha}1 protein, as determined by Western blotting (representative blots shown) in L6 myotubes incubated with increasing concentrations of RSG. *P < 0.05 vs. 0 µM RSG.

 


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  		Fig. 2. Increased acetyl-CoA carboxylase (ACC) phosphorylation and palmitate oxidation in RSG-treated L6 myotubes. A: ratio of ACC protein phosphorylated at Ser79 to total ACC protein (pACC/total ACC) in L6 myotubes treated with 0 (DMSO), 5, 50, or 200 µM RSG for 30 min. B: rate of [14C]palmitate oxidation in L6 myotubes treated with 0, 5, or 200 µM RSG for 1 h. *P < 0.05 vs. 0 µM RSG.

 
Effects of chronic RSG treatment in skeletal muscle. Table 2 displays the resting nucleotide and metabolite concentrations for the three groups under investigation. There were no differences in ATP, AMP, or ADP concentrations between muscle from LN CON or OB CON animals or OB RSG muscle. Consequently, there were no changes in the AMP/ATP ratio for any group (Table 2). Resting concentrations of PCr, creatine, and lactate were also similar between groups. However, glycogen content was elevated in muscle from obese compared with lean animals (P < 0.05), and this remained high in RSG-treated rats.


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  		Table 2. Resting skeletal muscle nucleotide and metabolite concentrations from LN CON, OB CON, and OB RSG rats

 
The activities of the AMPK{alpha}1 and AMPK{alpha}2 isoforms, along with the ACC Ser218 phosphorylation from skeletal muscle of animals treated for 6 wk with RSG, are shown in Fig. 3. The activity of AMPK{alpha}1 was similar in lean and obese animals and was unaffected by 6 wk of RSG treatment (Fig. 3A). By contrast, the activity of AMPK{alpha}2 was twofold lower in obese than in lean animals (7.45 ± 1.0 vs. 15.7 ± 2.2 pmol·mg–1·min–1, P < 0.05) but was restored to LN CON values after RSG treatment (OB RSG, 14.2 ± 1.5 pmol·mg–1·min–1; Fig. 3B). The observed changes in AMPK{alpha}2 activity were most likely due to corresponding changes in protein expression rather than to changes in phosphorylation of the protein at the Thr172 residue, which were similar in all groups (data not shown). Altered AMPK{alpha}2 activity with obesity and after RSG treatment paralleled changes in the phosphorylation state of its target protein, ACC. The ratio of phosphorylated (p-)ACC to total ACC protein (p-ACC/total ACC) was significantly decreased in muscle from obese animals (P < 0.05) but was restored to LN CON levels after RSG treatment (Fig. 3C). AMPK{alpha}1 protein expression tended to be higher (P = 0.06) in muscle from OB CON than from LN CON animals but was significantly decreased after RSG treatment (P < 0.05, OB CON vs. OB RSG; Fig. 4A). There were reciprocal changes in the protein expression of the AMPK{alpha}2 isoform, which was lower in muscle from obese animals but restored to LN CON values after chronic RSG treatment (Fig. 4B).



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  		Fig. 3. Increased skeletal muscle AMPK{alpha}2 activity in obese rats after chronic RSG treatment. Activity of AMPK{alpha}1 (A) and AMPK{alpha}2 (B) isoforms in muscle taken from lean (LN CON), obese (OB CON), and RSG-treated (OB RSG) rats. Activity was measured using the SAMS peptide assay in protein isolated by immunoprecipitation of muscle lysates. C: pACC/total ACC ratio from the skeletal muscle of LN CON, OB CON, and OB RSG rats. *P < 0.05 vs. LN CON; #P < 0.05 vs. OB CON.

 


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  		Fig. 4. Isoform-specific changes in AMPK protein with obesity and RSG treatment. Mean levels [arbitrary units (AU), n = 8] of AMPK{alpha}1 (A) and AMPK{alpha}2 (B) protein from LN CON, OB CON, and OB RSG rats. Proteins were isolated from skeletal muscle lysates by immunoprecipitation and quantified using Western blot analysis and densitometry. *P < 0.05 vs. LN CON; #P < 0.05 vs. OB CON.

 
Figure 5 displays the protein levels of UCP3 in skeletal muscle of lean and obese animals after RSG treatment. UCP3 protein abundance was not different between lean and obese animals. However, RSG treatment resulted in a significant increase in UCP3 protein levels (P < 0.05).



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  		Fig. 5. Uncoupling protein-3 (UCP3) protein expression with RSG treatment. Mean levels (AU, n = 8) of UCP3 protein from LN CON, OB CON, and OB RSG rats. Skeletal muscle protein was quantified using Western blot analysis and densitometry. #P < 0.05 vs. OB CON.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
TZDs are known to improve insulin sensitivity by activating PPAR{gamma} and inducing adipogenesis in adipose tissue (11) but also via PPAR{gamma}-independent mechanisms. Here, we report that RSG increases AMPK, leading to phosphorylation of ACC in vitro and in vivo.

In the present investigation, we observed maximal activation of AMPK in L6 myotubes when treated with 200 µM RSG, due to an increase in the activity of the AMPK{alpha}1 isoform. Moreover, the increased AMPK{alpha}1 activity at the highest dose of RSG was associated with a marked increase in the phosphorylation of ACC and a concomitant increase in palmitate oxidation. Recently, Cha et al. (6) reported that cultured muscle cells from type 2 diabetic patients had lower rates of basal {beta}-oxidation but that this defect was normalized after chronic (4 days) TZD treatment. Accordingly, an AMPK-stimulated increase in lipid oxidation might be one mechanism by which RSG exerts its insulin-sensitizing effect.

Only one previous study has examined the effect of RSG on AMPK activity in skeletal muscle myotubes. Fryer et al. (13) observed that treating muscle cells derived from heterozygous H-2Kb-tsA58-transgenic mice with RSG leads to increases in the AMP/ATP ratio and a concomitant increase in AMPK activity. We, too, observed an increase in AMPK activity in RSG-treated cells. However, we did not detect any difference in the energy charge (AMP/ATP) of the cell, suggesting that RSG's action was exerting an energy charge-independent effect on AMPK. The reasons for the differences in results between the present and previous studies (13), in terms of the AMP/ATP ratio, are not readily apparent. The H-2Kb cell line contains both AMPK{alpha}1 and AMPK{alpha}2 isoforms (13), and we cannot rule out the possibility that this is a more sensitive cell line to treatments that affect energy charge. However, it should be noted that the H-2Kb lines are immortalized cells derived from the simian virus 40 large tumor antigen transgenic mouse (20) that might have some limitations with respect to their physiological significance. To be consistent in the present investigation, we chose to use L6 myotubes derived from rat skeletal muscle so that the acute effects of RSG might be compared with the chronic effects of this treatment regimen in obese Zucker rats.

Previous studies (17, 24, 34, 40) have suggested that altered skeletal muscle AMPK activity may not play a role in obesity and diabetes. However, a recent study (7) found that, although basal AMPK activity was not altered, AMPK activation was impaired in cultured human skeletal muscle from obese type 2 diabetics. There is also evidence that basal AMPK phosphorylation is reduced in the cardiac muscle of both Zucker diabetic fatty rats and ob/ob mice (37) and that this defect is normalized by TZD treatment. Further study is clearly needed to draw general conclusions on the role of skeletal muscle AMPK defects in obesity and diabetes.

To date, no studies have determined the effects of chronic RSG treatment on the responses of AMPK in skeletal muscle or, more importantly, the effect of RSG on skeletal muscle from insulin-resistant animals. Our finding that chronic RSG treatment increased both AMPK{alpha}2 and ACC phosphorylation in obese Zucker rats is of major clinical significance, given the widespread use of TZDs as an antidiabetic drug. The altered AMPK{alpha}2 activity, with obesity and RSG treatment, corresponded to changes in the phosphorylation state of its target protein, ACC, which was decreased with obesity but restored to the level of lean controls after RSG administration. Of interest was the reciprocal response of the AMPK protein levels to the chronic RSG administration. AMPK{alpha}2 protein was reduced in obesity but restored to lean control values after RSG treatment, whereas AMPK{alpha}1 protein abundance was suppressed with treatment. Consistent with our in vitro cellular data, the muscle nucleotide concentrations were unaltered by chronic RSG treatment in vivo (Table 2).

Because hypoxia stimulates AMPK activity and a catecholamine response (3, 15), the combination of euthanasia by CO2 asphyxiation and the elapsed time between tissue collection and freezing might have resulted in an altered intracellular environment that could have contributed to changes in AMPK activity. These factors are potential limitations of the present study. However, we are confident that the observed AMPK activation in the present study was not due to hypoxia for the following reasons. First, muscle metabolites were within the expected ranges for all treatment groups and were similar to those obtained by other groups studying obese Zucker rats (22, 29). Second, the observed changes in AMPK activation were paralleled by changes in AMPK protein levels (Fig. 4) and were not associated with changes in the Thr172 phosphorylation of AMPK. This suggests that the increase in activity was due to chronically altered protein levels rather than to acute changes in cellular energy status. Finally, the time taken to extract muscle was not different between groups; therefore, any differences in AMPK activity are indicative of the treatment.

The improved metabolic action, as a consequence of upregulating AMPK{alpha}2 by RSG, might be due to an increase in fat oxidation, because increased {beta}-oxidation in muscle cells has been shown (28) to enhance insulin-stimulated glucose metabolism and protect against insulin resistance in the face of elevated intramyocellular lipid content. Future studies should determine whether the observed increase in skeletal muscle AMPK activity with chronic RSG treatment is associated with a concomitant increase in fat oxidation in vivo. However, it is also possible that RSG-mediated upregulation of AMPK{alpha}2 might improve metabolic action via alternative mechanisms. Treatment with either RSG (5) or AICAR (35, 36) increases UCP expression in skeletal muscle. In addition, UCP3 overexpressing transgenic mice display enhanced glucose tolerance and palmitate oxidation (38), a phenotype that is compatible with chronic TZD treatment. In the present investigation, we found that UCP3 protein expression was upregulated by chronic RSG treatment, suggesting that this protein may account for part of its metabolic action in skeletal muscle.

Previously, Musi et al. (25) reported that the antidiabetic drug metformin (biguanide) increased AMPK{alpha}2 activity in type 2 diabetic patients after a 10-wk treatment regimen. A significant increase in Thr172 phosphorylation was observed, but there was no significant increase in AMPK{alpha}2 levels in the biopsy samples. The increase in AMPK{alpha}2 activity seen in their study was paralleled by decreases in ACC activity consistent with AMPK mediated phosphorylation. Previously, it has been suggested that metformin and RSG activate AMPK by different pathways (13), with RSG causing an increase in AMP levels. Irrespective of the mechanism of activation, both drugs (metformin, human type 2 diabetics; RSG, obese Zucker rats) enhance AMPK signaling via the {alpha}2-isoform and inactivate ACC.

In conclusion, the results from the present study demonstrate that chronic RSG administration stimulated isoform-specific regulation of the AMPK signaling cascade in skeletal muscle from obese Zucker rats and in myotubes in vitro. The increased basal activity of AMPK{alpha}2 paralleled changes in the phosphorylation state of its target protein, ACC{beta}, and occurred without detectable changes in cellular energy charge. These changes in AMPK{alpha}2 seem likely to be an important component of the insulin-sensitizing effect of RSG that contributes to the improved metabolic action associated with chronic TZD drug administration. Future studies should determine the mechanisms underlying altered transcriptional regulation and/or turnover of AMPK{alpha}2 protein in skeletal muscle from obese Zucker rats and how this is reversed by TZD treatment.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by grants from the National Health and Medical Research Council of Australia (NHMRC), Australian Research Center (ARC), and National Heart Foundation. M. J. Watt is an NHMRC Peter Doherty postdoctoral fellow, M. A. Febbraio is an NHMRC Senior Research Fellow, and B. E. Kemp is an ARC Federation Fellow.


   ACKNOWLEDGMENTS
 
We acknowledge Winnie Lau for technical assistance. We also thank GlaxoSmithKline (Stevenage, UK) for the gift of rosiglitazone.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Hawley, School of Medical Sciences, RMIT University, PO Box 71, Bundoora, Victoria 3083, Australia (e-mail: john.hawley{at}rmit.edu.au)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Auwerx J. PPARgamma, the ultimate thrifty gene. Diabetologia 42: 1033–1049, 1999.[CrossRef][Web of Science][Medline]
  2. Bergmeyer HU. Methods in Enzymatic Analysis. New York: Academic, 1974.
  3. Borovsky V, Herman M, Dunphy G, Caplea A, and Ely D. CO2 asphyxia increases plasma norepinephrine in rats via sympathetic nerves. Am J Physiol Regul Integr Comp Physiol 274: R19–R22, 1998.[Abstract/Free Full Text]
  4. Brunmair B, Gras F, Neschen S, Roden M, Wagner L, Waldhausl W, and Furnsinn C. Direct thiazolidinedione action on isolated rat skeletal muscle fuel handling is independent of peroxisome proliferator-activated receptor-gamma-mediated changes in gene expression. Diabetes 50: 2309–2315, 2001.[Abstract/Free Full Text]
  5. Brunmair B, Gras F, Wagner L, Artwohl M, Zierhut B, Waldhausl W, and Furnsinn C. Expression of uncoupling protein-3 mRNA in rat skeletal muscle is acutely stimulated by thiazolidinediones: an exercise-like effect? Diabetologia 47: 1611–1614, 2004[CrossRef][Medline]
  6. Cha BS, Ciaraldi TP, Park KS, Carter L, Mudaliar SR, and Henry RR. Impaired fatty acid metabolism in type 2 diabetic skeletal muscle cells is reversed by PPAR{gamma} agonists. Am J Physiol Endocrinol Metab 289: E151–E159, 2005.[Abstract/Free Full Text]
  7. Chen MB, McAinch AJ, Macaulay SL, Castelli LA, O'brien PE, Dixon JB, Cameron-Smith D, Kemp BE, and Steinberg GR. Impaired activation of AMP-kinase, and fatty acid oxidation by globular adiponectin in cultured human skeletal muscle from obese type 2 diabetics. J Clin Endocrinol Metab 90: 3665–3672, 2005.[Abstract/Free Full Text]
  8. Chen ZP, McConell GK, Michell BJ, Snow RJ, Canny BJ, and Kemp BE. AMPK signaling in contracting human skeletal muscle: acetyl-CoA carboxylase, and NO synthase phosphorylation. Am J Physiol Endocrinol Metab 279: E1202–E1206, 2000.[Abstract/Free Full Text]
  9. Christopher MJ, Chen ZP, Rantzau C, Kemp BE, and Alford FP. Skeletal muscle basal AMP-activated protein kinase activity is chronically elevated in alloxan-diabetic dogs: impact of exercise. J Appl Physiol 95: 1523–1530, 2003.[Abstract/Free Full Text]
  10. Clark SA, Chen ZP, Murphy KT, Aughey RJ, McKenna MJ, Kemp BE, and Hawley JA. Intensified exercise training does not alter AMPK signaling in human skeletal muscle. Am J Physiol Endocrinol Metab 286: E737–E743, 2004.[Abstract/Free Full Text]
  11. Day C. Thiazolidinediones: a new class of antidiabetic drugs. Diabet Med 16: 179–192, 1999.[CrossRef][Web of Science][Medline]
  12. Dudley GA, Tullson PC, and Terjung RL. Influence of mitochondrial content on the sensitivity of respiratory control. J Biol Chem 262: 9109–9114, 1987.[Abstract/Free Full Text]
  13. Fryer LG, Parbu-Patel A, and Carling D. The anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways. J Biol Chem 277: 25226–25232, 2002.[Abstract/Free Full Text]
  14. Fujiwara T, Yoshioka S, Yoshioka T, Ushiyama I, and Horikoshi H. Characterization of new oral antidiabetic agent CS-045 studies in KK and ob/ob mice and Zucker fatty rats. Diabetes 37: 1549–1558, 1988.[Abstract]
  15. Hallsten K, Virtanen KA, Lonnqvist F, Sipila H, Oksanen A, Viljanen T, Ronnemaa T, Viikari J, Knuuti J, and Nuutila P. Rosiglitazone but not metformin enhances insulin- and exercise-stimulated skeletal muscle glucose uptake in patients with newly diagnosed type 2 diabetes. Diabetes 51: 3479–3485, 2002.[Abstract/Free Full Text]
  16. Hardie DG. Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endocrinology 144: 5179–5183, 2003.[Abstract/Free Full Text]
  17. Hojlund K, Mustard KJ, Staehr P, Hardie DG, Beck-Nielsen H, Richter EA, and Wojtaszewski JF. AMPK activity and isoform protein expression are similar in muscle of obese subjects with and without type 2 diabetes. Am J Physiol Endocrinol Metab 286: E239–E244, 2004.[Abstract/Free Full Text]
  18. Inzucchi SE, Maggs DG, Spollett GR, Page SL, Rife FS, Walton V, and Shulman GI. Efficacy and metabolic effects of metformin and troglitazone in type II diabetes mellitus. N Engl J Med 338: 867–872, 1998.[Abstract/Free Full Text]
  19. Jakobsen SN, Hardie DG, Morrice N, and Tornqvist HE. 5'-AMP-activated protein kinase phosphorylates IRS-1 on Ser-789 in mouse C2C12 myotubes in response to 5-aminoimidazole-4-carboxamide riboside. J Biol Chem 276: 46912–46916, 2001.[Abstract/Free Full Text]
  20. Jat PS, Noble MD, Ataliotis P, Tanaka Y, Yannoutsos N, Larsen L, and Kioussis D. Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse. Proc Natl Acad Sci USA 88: 5096–100, 1991.[Abstract/Free Full Text]
  21. Kemp BE, Stapleton D, Campbell DJ, Chen ZP, Murthy S, Walter M, Gupta A, Adams JJ, Katsis F, van Denderen B, Jennings IG, Iseli T, Michell BJ, and Witters LA. AMP-activated protein kinase, super metabolic regulator. Biochem Soc Trans 31: 162–168, 2003.[Web of Science][Medline]
  22. Klein M, Kaminsky P, Walker PM, Straczek J, Barbe F, Duc M, and Burlet C. Muscle bioenergetics in obese Zucker rats. Am J Physiol Endocrinol Metab 266: E410–E417, 1994.[Abstract/Free Full Text]
  23. Lessard SJ, Lo Giudice SL, Lau W, Reid JJ, Turner N, Febbraio MA, Hawley JA, and Watt MJ. Rosiglitazone enhances glucose tolerance by mechanisms other than reduction of fatty acid accumulation within skeletal muscle. Endocrinology 145: 5665–5670, 2004.[Abstract/Free Full Text]
  24. Musi N, Fujii N, Hirshman MF, Ekberg I, Froberg S, Ljungqvist O, Thorell A, and Goodyear LJ. AMP-activated protein kinase (AMPK) is activated in muscle of subjects with type 2 diabetes during exercise. Diabetes 50: 921–927, 2001.[Abstract/Free Full Text]
  25. Musi N, Hirshman MF, Nygren J, Svanfeldt M, Bavenholm P, Rooyackers O, Zhou G, Williamson JM, Ljunqvist O, Efendic S, Moller DE, Thorell A, and Goodyear LJ. Metformin increases AMP activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes. Diabetes 51: 2074–2081, 2002.[Abstract/Free Full Text]
  26. Olefsky JM. Treatment of insulin resistance with peroxisome proliferator-activated receptor gamma agonists. J Clin Invest 106: 467–472, 2000.[Web of Science][Medline]
  27. Passonneau JV and Lauderdale VR A comparison of three methods of glycogen measurement in tissues. Anal Biochem 60: 405–412, 1974.[CrossRef][Web of Science][Medline]
  28. Perdomo G, Commerford SR, Richard AM, Adams SH, Corkey BE, O'Doherty BE, and Brown NF. Increased {beta}-oxidation in muscle cells enhances insulin-stimulated glucose metabolism and protects against fatty acid-induced insulin resistance despite intramyocellular lipid accumulation. J Biol Chem 279: 27177–27186, 2004.[Abstract/Free Full Text]
  29. Ranganathan S and Kern PA. Thiazolidinediones inhibit lipoprotein lipase activity in adipocytes. J Biol Chem 273: 26117–26122, 1998.[Abstract/Free Full Text]
  30. Sahlin K, Harris RC, Nylind B, and Hultman E. Lactate content and pH in muscle obtained after dynamic exercise. Pflügers Arch 367: 143–149, 1976.[CrossRef][Web of Science][Medline]
  31. Saltiel A. The molecular and physiological basis of insulin resistance: emerging implications for metabolic and cardiovascular diseases. J Clin Invest 106: 163–164, 2000.[Web of Science][Medline]
  32. Saltiel A and Olefsky JM. Thiazolidinediones in the treatment of insulin resistance and type II diabetes. Diabetes 45: 1661–1669, 1996.[Abstract]
  33. Sreenan S, Keck S, Fuller T, Cockburn B, and Burant CF. Effects of troglitazone on substrate storage and utilization in insulin-resistant rats. Am J Physiol Endocrinol Metab 276: E1119–E1129, 1999.
  34. Steinberg GR, Smith AC, Van Denderen BJ, Chen Z, Murthy S, Campbell DJ, Heigenhauser GJ, Dyck DJ, and Kemp BE. AMP-activated protein kinase is not down-regulated in human skeletal muscle of obese females. J Clin Endocrinol Metab 89: 4575–4580, 2004.[Abstract/Free Full Text]
  35. Stoppani J, Hildebrandt AL, Sakamoto K, Cameron-Smith D, Goodyear LJ, and Neufer PD. AMP-activated protein kinase activates transcription of the UCP3 and HKII genes in rat skeletal muscle. Am J Physiol Endocrinol Metab 283: E1239–E1248, 2002.[Abstract/Free Full Text]
  36. Suwa M, Nakano H, and Kumagai S. Effects of chronic AICAR treatment on fiber composition, enzyme activity, UCP3, and PGC-1 in rat muscles. J Appl Physiol 95: 960–968, 2003.[Abstract/Free Full Text]
  37. Wang MY and Unger RH. Role of PP2C in cardiac lipid accumulation in obese rodents and its prevention by troglitazone. Am J Physiol Endocrinol Metab 288: E216–E221, 2005.[Abstract/Free Full Text]
  38. Wang S, Subramaniam A, Cawthorne MA, and Clapham JC. Increased fatty acid oxidation in transgenic mice overexpressing UCP3 in skeletal muscle. Diabetes Obes Metab 5: 295–301, 2003.[CrossRef][Web of Science][Medline]
  39. Winder WW and Hardie DG. AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes. Am J Physiol Endocrinol Metab 277: E1–E10, 1999.[Abstract/Free Full Text]
  40. Wojtaszewski JF, Birk JB, Frosig C, Holten M, Pilegaard H, and Dela F. 5'AMP activated protein kinase expression in human skeletal muscle: effects of strength training and type 2 diabetes. J Physiol 564: 563–573, 2005.[Abstract/Free Full Text]



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