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Am J Physiol Endocrinol Metab 291: E557-E565, 2006. First published April 25, 2006; doi:10.1152/ajpendo.00073.2006
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Muscle-specific overexpression of wild type and R225Q mutant AMP-activated protein kinase {gamma}3-subunit differentially regulates glycogen accumulation

Haiyan Yu, Michael F. Hirshman, Nobuharu Fujii, Jason M. Pomerleau, Lauren E. Peter, and Laurie J. Goodyear

Research Division, Joslin Diabetes Center, and Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts

Submitted 10 February 2006 ; accepted in final form 17 April 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
AMP-activated protein kinase (AMPK) is a heterotrimeric complex that works as an energy sensor to integrate nutritional and hormonal signals. The naturally occurring R225Q mutation in the {gamma}3-subunit in pigs is associated with abnormally high glycogen content in skeletal muscle. Becauses skeletal muscle accounts for most of the body's glucose uptake, and {gamma}3 is specifically expressed in skeletal muscle, it is important to understand the underlying mechanism of this mutation in regulating glucose and glycogen metabolism. Using skeletal muscle-specific transgenic mice overexpressing wild type {gamma}3 (WT{gamma}3) and R225Q mutant {gamma}3 (MUT{gamma}3), we show that both WT{gamma}3 and MUT{gamma}3 mice have 1.5- to 2-fold increases in muscle glycogen content. In WT{gamma}3 mice, increased glycogen content was associated with elevated total glycogen synthase activity and reduced glycogen phosphorylase activity, whereas alterations in activities of these enzymes could not explain elevated glycogen in MUT{gamma}3 mice. Basal, 5-aminoimidazole- AICAR- and phenformin-stimulated AMPK{alpha}2 isoform-specific activities were decreased only in MUT{gamma}3 mice. Basal rates of 2-DG glucose uptake were decreased in both WT{gamma}3 and MUT{gamma}3 mice. However, AICAR- and phenformin-stimulated 2-DG glucose uptake were blunted only in MUT{gamma}3 mice. In conclusion, expression of either wild type or mutant {gamma}3-subunit of AMPK results in increased glycogen concentrations in muscle, but the mechanisms underlying this alteration appear to be different. Furthermore, mutation of the {gamma}3-subunit is associated with decreases in AMPK{alpha}2 isoform-specific activity and impairment in AICAR- and phenformin-stimulated skeletal muscle glucose uptake.

THE 5'-AMP-ACTIVATED PROTEIN KINASE (AMPK) functions to regulate energy levels within individual cells (21). More recently, AMPK has been shown to play a wider role in energy metabolism through the regulation of whole body metabolic processes. In skeletal muscle, pharmacological activation of AMPK by 4-carboxamide-1-beta-D-ribofuranoside (AICAR) increases glucose uptake and fatty acid oxidation (25, 39, 42), and inactivation of AMPK in skeletal muscle of transgenic or knockout mice abolishes AICAR-induced glucose uptake (16, 29, 44). AMPK activation in peripheral tissues has been implicated in diminishing adiposity (48), and AMPK activation has also been implicated in increasing food intake through neuronal signaling in the hypothalamus (2, 34, 41). Disruption of any of these AMPK-regulated metabolic systems could accentuate the pathogenesis of type 2 diabetes (28, 30, 31, 42). In fact, the antidiabetic drug metformin increases AMPK activity in skeletal muscles from both rats (56) and humans (45), an effect that is associated with increased glucose disposal. Thus fully understanding the function of AMPK may reveal potential new treatments for diabetes.

AMPK is a heterotrimeric complex consisting of a catalytic subunit ({alpha}) and two regulatory subunits (beta and {gamma}). The {alpha}-subunit has two isoforms, {alpha}1 and {alpha}2, both of which require phosphorylation of Thr172 for activity (10, 12, 23, 43). The beta-subunit, which has two isoforms, beta1 and beta2, acts as a scaffold for the binding of the {alpha}- and {gamma}-subunits (54) and has been proposed to be involved in the regulation of glycogen metabolism on the basis of the presence of a putative glycogen-binding domain in its structure (27, 46).

The {gamma}-subunits are encoded by three genes, {gamma}1, {gamma}2, and {gamma}3, all of which contain four cystathionine beta-synthase (CBS) domains. CBS domains (also referred to as Bateman domains) are sequence motifs originally identified in a search for internal sequence duplications (7, 32). Studies have shown that CBS domains work in tandem to bind AMP and ATP (11, 50). CBS domains have been identified in many diverse proteins, and mutations in CBS domains can cause several hereditary diseases in humans (9, 22, 32, 33, 35–37, 47). In the heart, several mutations in the CBS domains of the {gamma}2-subunit of AMPK have been found to be associated with Wolff-Parkinson-White (WPW) syndrome. This condition is characterized by electrophysiological abnormalities, particularly preexcitation and cardiac hypertrophy (3, 8, 18, 19). Transgenic mice harboring a comparable mutation in the CBS domain of the {gamma}2-subunit of AMPK (N488I) have a highly elevated glycogen concentration in the heart and develop ventricular hypertrophy and arrhythmia (4).

The {gamma}3 regulatory subunit of AMPK is exclusively expressed in skeletal muscle (11, 38, 55). A mutation of arginine to glutamine at position 225 (R225Q) in the CBS domain of the {gamma}3-subunit is associated with increased glycogen content in skeletal muscle of the Hampshire pig (1, 40). This R225Q mutation in AMPK{gamma}3 is equivalent to the R302Q in AMPK{gamma}2 that is associated with WPW syndrome in the heart and the D444N mutation in the CBS protein that causes homocystinuria (35). The linkage of these equivalent mutations with multiple diseases makes it important to understand the functional and metabolic consequences of the {gamma}3 R225Q mutation in skeletal muscle. In the present study, we used a muscle-specific promoter to generate transgenic mouse lines overexpressing wild type-{gamma}3 (WT{gamma}3) and R225Q mutant-{gamma}3 (MUT{gamma}3). We studied the effects of expressing these transgenes on whole body glucose disposal, AMPK activation, and glucose and glycogen metabolism in skeletal muscle.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. [{gamma}-32P]ATP was obtained from PerkinElmer (Boston, MA). Affinity-purified polyclonal antibody against AMPK{gamma}3 was raised against mouse AMPK{gamma}3 peptide NH2-CLVDETQHLLGV-COOH ({gamma}3L, 457–467) (55). Polyclonal antibodies against AMPK{alpha}1 and -{alpha}2 were produced against the peptide sequence 341–360 of AMPK{alpha}1 and 491–505 of AMPK{alpha}2 (15), anti-AMPK{alpha}1/2 antibody was raised against the peptide sequence 2–16 of AMPK{alpha}1/2 (AEKQKHDGRVKIGHY), which is conserved in AMPK{alpha}1 and -{alpha}2 isoforms. Anti-AMPKbeta antibody was purchased from Upstate Biochemical, (Lake Placid, NY). Anti-phospho-AMPK{alpha} antibody was from Cell Signaling Technology, (Beverly, MA). Polyclonal anti-Flag antibody was from Sigma (St. Louis, MO). Anti-phospho-glycogen synthase antibody was from Oncogene Research Products (San Diego, CA). Anti-glycogen synthase antibody was a gift from Dr. John C. Lawrence, Jr. (University of Virginia).

Generation of WT{gamma}3 and MUT{gamma}3 transgenic mice. cDNAs encoding mouse Flag-tagged full length AMPK{gamma}3L or R225Q AMPK{gamma}3L were subcloned into an expression vector containing muscle creatine kinase promoter/enhancer and SV40 polyadenylation sequences surrounding the insertion (a gift from C. Ronald Kahn; Joslin Diabetes Center, Boston, MA). The KpnI and SacI partial digestion fragment from the plasmid was injected into FVB mouse oocytes. Transgenic offspring were identified by PCR and Western blot. At least two independent lines were obtained for both WT{gamma}3 and MUT{gamma}3, and each had similar phenotypes. Both male and female mice at 8–10 wk of age were used to determine AMPK activity, glycogen synthase activity, glycogen phosphorylase activity, and glycogen content, because no differences between sexes were observed. Eight- to ten-week-old male mice were used for insulin measurements, 8- to 10-wk-old female mice were used for glucose uptake experiments, and 22-wk-old female mice were used for the glucose, insulin, and AICAR tolerance tests.

Animals. Mice were housed at 22°C with a 12:12-h light-dark cycle and were given standard laboratory chow diet and water ad libitum. Protocols for animal use were reviewed and approved by the Institutional Animal Care and Use Committee of the Joslin Diabetes Center and were in accordance with guidelines from the National Institutes of Health.

PCR genotyping of transgenic mice. Tail DNA was prepared by proteinase K digestion using Qiagen kits. Mouse AMPK{gamma}3L and R225Q{gamma}3L were amplified by PCR using primer pairs: forward primer, 5'-ACTACAAGGACGACGATGACAAG-3'; and reverse primer, 5'-AGCCATGGCATCATAACAGGTGTG-3'. The forward primer was designed on the basis of the Flag-tag sequence, and the reverse primer was from AMPK{gamma}3L 571–594 that was conserved in both WT{gamma}3 and R225Q{gamma}3.

Muscle processing. Muscles were dissected and snap-frozen in liquid nitrogen. The samples were pulverized, weighed, and Polytron homogenized (Brinkman Instruments) in ice-cold lysis buffer (1:10, wt/vol) containing 20 mM Tris·HCl (pH 7.4), 1% Triton X-100, 50 mM NaCl, 250 mM sucrose, 50 mM NaF, 5 mM sodium pyrophosphate, 2 mM DTT, 4 mg/l leupeptin, 50 mg/l trypsin inhibitor, 0.1 mM benzamidine, and 0.5 mM PMSF followed by centrifuging at 14,000 g for 20 min at 4°C. Supernatants were removed and used for protein concentration measurements, Western blotting, and AMPK activity.

AMPK activity assay. AMPK activity assay was performed as we have previously described (24). Briefly, muscle lysates were immunoprecipitated with specific antibodies to the {alpha}1 and {alpha}2 catalytic subunits of AMPK or anti-Flag polyclonal antibody (Sigma, St. Louis, MO) and protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoprecipitates were assayed for AMPK activity in assay buffer containing 0.2 mM AMP, 0.2 mM ATP (2 µCi [{gamma}-32P]ATP), and 0.2 mM synthetic AMPK substrate peptide with the sequence HMRSAMSGLHLVKRR for 20 min at 30°C (14).

Glycogen synthase and phosphorylase activities and glycogen contents. Frozen muscle samples were pulverized under liquid N2 and subjected to Polytron homogenization in 19 vol of buffer consisting of 50 mmol/l Tris·HCl (pH 7.8), 100 mmol/l NaF, and 5 mmol/l EDTA. Glycogen synthase activity was determined in the absence or presence of 6.7 mmol/l glucose 6-phosphate (G-6-P) (52), and glycogen phosphorylase activity was determined in the absence or presence of 3 mmol/l 5'-AMP (17). Muscle glycogen concentrations were measured in homogenates after 2 h of hydrolysis in 2 N HCl at 100°C for 2 h followed by neutralization with 2 N NaOH. Glycogen concentrations were then determined by the hexokinase enzymatic method, using the glucose HK reagent (Sigma).

Incubation of isolated muscles and measurement of 2-deoxy-D-glucose uptake. Mice were killed by cervical dislocation, and extensor digitorum longus (EDL) muscles were isolated. Tendons from both ends of the muscle were tied with suture (silk 4-0) and mounted on the incubation apparatus to maintain resting length. Isolated muscles were incubated in Krebs-Ringer bicarbonate buffer (KRB) at 37 °C. The buffers were continuously gassed with 95% O2-5% CO2. The incubation time was 50 min for both AICAR (2 mM) and phenformin (2 mM) and 40 min for insulin (50 mU/ml). After the incubation period, muscles were incubated in 2 ml KRB containing 1 mM 2-deoxy-D-[2,6-3H]glucose (1.5 µCi/ml) and 7 mM D-[14C]mannitol (0.45 µCi/ml) at 30°C for an additional 10 min. Reagents (AICAR, phenformin, and insulin) were added to each buffer if present during the previous incubation period. Transport was stopped by washing the muscle in KRB containing 80 µmol/l cytochalasin B at 4°C, and the muscle was blotted, trimmed, and frozen in liquid N2 and then stored at –80°C. Muscles were weighed and processed as previously described (26).

Glucose, insulin, and AICAR tolerance tests. Glucose (2 g/kg), insulin (0.75 U/kg; Eli Lilly), or AICAR (0.2 mg/kg) was given intraperitoneally at time 0. Tail blood was collected at 0, 20, 40, 60, and 120 min for glucose and AICAR tolerance tests and at 0, 20, 40, and 60 min for the insulin tolerance test. Blood glucose concentrations were determined using a One Touch glucometer (LifeScan, High Wycombe, UK). Mice were fasted overnight before the glucose tolerance test.

Plasma insulin. Insulin levels were measured by ELISA (Crystal Chem) using purified mouse insulin standards.

Statistical analysis. Data are presented as means ± SE. Comparison of data was by one-way analysis of variance followed by post hoc analysis using the Student-Newman-Keuls test when experimental comparisons were for three groups without treatment. Comparison of data was by two-way analysis of variance followed by post hoc analysis using the Student-Newman-Keuls test when experimental comparisons were for three groups under both basal and treated conditions (Figs. 3 and 6).


Figure 3
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  		Fig. 3. Basal, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR)-, and phenformin-stimulated AMPK{alpha}1 and AMPK{alpha}2 activity. Isolated extensor digitorum longus (EDL) was treated as indicated, and AMPK{alpha}1 (A) and AMPK{alpha}2 (B) activities were measured. Data are means ± SE; n = 4–10; *P < 0.05 vs. corresponding basal; +P < 0.05 vs. WT within treatment.

 

Figure 6
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  		Fig. 6. Basal and AICAR-, phenformin-, and insulin-stimulated glucose uptake in EDL of WT and transgenic mice. Isolated EDL was incubated in the absence or presence of AICAR, phenformin, or insulin, and 2-deoxyglucose uptake was measured as described in METHODS. Data are means ± SE; n = 4–10. *P < 0.05 vs. corresponding basal; +P < 0.05 vs. WT within treatment; #P < 0.05 vs. WT and WT{gamma}3.

 

   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
General characteristics of the transgenic mice. All experiments were performed using corresponding wild-type (WT) control littermates for the WT{gamma}3 and MUT{gamma}3 transgenic animals. There was no difference between the two WT control groups, and the combination of the WT controls did not change the statistical significance. Therefore, data from the WT controls are combined in all figures. Both WT{gamma}3 and MUT{gamma}3 mice were born at the expected Mendelian frequency and showed normal growth curves and development (data not shown). There were no differences in body weights or blood glucose concentrations between controls, WT{gamma}3, and MUT{gamma}3 mice up to 40 wk of age. Both types of transgenic mice appeared normal by gross inspection. Gross locomotive activity, O2 consumption, CO2 production, food and water consumption, and fat distribution in MUT{gamma}3 mice were not different from controls based on the Comprehensive Laboratory Animal Monitoring System (Columbus Instruments) metabolic profiling (Table 1) and dual-energy X-ray absorptiometry (Lunar PIXImus2 mouse densitometer; General Electric Medical Systems, Madison, WI) scan analysis (data not shown).


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  		Table 1. CLAMS data in fed and fasted 22-wk-old male WT and MUT{gamma}3 mice

 
Tissue expression profile of WT{gamma}3 and MUT{gamma}3 transgenic mice. Transgene expression was assessed by Western blot analysis using an anti-Flag antibody. Flag was not detectable in nonmuscle tissues, such as brain and liver (Fig. 1A), but was expressed in all skeletal muscles, including soleus, tibialis anterior, EDL, gastrocnemius, and cardiac muscle. Transgenic lines with comparable WT{gamma}3 and MUT{gamma}3 in EDL and gastrocnemius were chosen for this study. Expression of WT{gamma}3 and MUT{gamma}3 was ~4.2- and 3.6-fold higher than endogenous AMPK{gamma}3 in WT littermates (Fig. 1E). Because overexpressed WT{gamma}3 and MUT{gamma}3 overlapped with endogenous {gamma}3 on Western blots, it could not be determined whether the endogenous {gamma}3 was replaced by overexpressing WT{gamma}3 or MUT{gamma}3. Both AMPK{alpha}2 (Fig. 1B) and -beta2 protein (Fig. 1C) were significantly elevated in WT{gamma}3 and MUT{gamma}3 mice, whereas AMPK{alpha}1 (Fig. 1D) and -{gamma}1 (data not shown) did not significantly change in either type of transgenic mice. The beta1-protein was not detected in skeletal muscle. The {gamma}2 expression level could not be determined because no adequate antibody was available. Immunoprecipitation with an anti-Flag antibody showed that both WT{gamma}3 and MUT{gamma}3 associated with the {alpha}2- and beta2-subunits (Fig. 1F), whereas association with {alpha}1 was not detectable.


Figure 1
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  		Fig. 1. A: expression of wild type {gamma}3 (WT{gamma}3) and R225Q mutant AMP-activated protein kinase (AMPK){gamma}3 (MUT{gamma}3) in transgenic mice and endogenous {gamma}3 in wild-type (WT) mice. Multiple tissue lysates from transgenic and WT mice were immunoblotted with Flag and {gamma}3 antibodies. B, C, D, and E: expression of AMPK{alpha}2, -beta2, -{alpha}1, and -{gamma}3 subunits in WT and transgenic mice. Muscle lysates from gastrocnemius muscle were immunoblotted with anti-AMPK{alpha}2 or -beta2 antibodies. Data are means ± SE; n = 5–7; *P < 0.05 vs. WT mice. F: association of WT{gamma}3 and MUT{gamma}3 with other AMPK subunits. Muscle lysates from WT{gamma}3 and MUT{gamma}3 mice were immunoprecipitated with anti-Flag antibody followed by immunoblotting with anti-Flag, -AMPK{alpha}1, -AMPK{alpha}2, and -AMPKbeta2 antibody, respectively.

 
AMPK activity in transgenic mice. To determine the effects of the transgenes on AMPK activity, we first assayed AMPK activity in the basal state in gastrocnemius muscles snap-frozen in liquid nitrogen. AMPK{alpha}1 activity in the basal state was not altered in the gastrocnemius muscle of either the WT{gamma}3 or MUT{gamma}3 transgenic mice (Fig. 2A). AMPK{alpha}2 activity was not different in the WT{gamma}3 mice but was significantly lower in the muscles from the MUT{gamma}3 transgenic mice than WT controls (Fig. 2B). The decrease in AMPK{alpha}2 but not -{alpha}1 activity in MUT{gamma}3 mice is consistent with the predominant association of MUT{gamma}3 with the {alpha}2-subunit (Fig. 1D). To directly compare AMPK activity associated with the {gamma}3 transgenes, enzyme activity was measured in anti-Flag immunoprecipitates. Figure 2C shows that MUT{gamma}3-associated AMPK activity is lower than WT{gamma}3-associated activity, consistent with the lower AMPK{alpha}2 activity in the MUT{gamma}3 mice.


Figure 2
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  		Fig. 2. AMPK activity in basal gastrocnemius muscle. Immunoprecipitation with anti-AMPK{alpha}1 (A), -{alpha}2 (B) or Flag (C) antibodies of gastrocnemius lysates from WT, WT{gamma}3, and MUT{gamma}3 mice were assayed for AMPK activity. Data are means ± SE; n = 6–7; *P < 0.05 vs. WT{gamma}3; +P < 0.05 vs. WT and WT{gamma}3. Flag-associated AMPK activity was normalized by the corresponding Flag expression level (C).

 
To study AMPK activity in response to stimuli, isolated EDL muscles were incubated in vitro in the absence or presence of AICAR or phenformin. Phenformin is a higher potency analog of the antidiabetic drug metformin. We (56) have previously shown that metformin increases AMPK activity in skeletal muscle. Similar to results in the gastrocnemius muscle, AMPK{alpha}1 activity did not change in WT{gamma}3 or MUT{gamma}3 mice in EDL muscles incubated in the basal state (Fig. 3A). AICAR incubation significantly increased AMPK{alpha}1 activity, and there were no differences among genotypes. Phenformin also significantly increased AMPK{alpha}1 activity in all groups, with WT{gamma}3 mice having the greatest increase (Fig. 3A). Basal AMPK{alpha}2 activity was significantly reduced in the EDL muscles from the MUT{gamma}3 mice (Fig. 3B), consistent with the in vivo gastrocnemius data. Overexpression of WT{gamma}3 enhanced AICAR- and phenformin-stimulated AMPK{alpha}2 activity, whereas overexpression of MUT{gamma}3 severely blunted AMPK{alpha}2 activity in basal and both treatment states (Fig. 3B).

Increased glycogen concentrations in transgenic mice. Increased muscle glycogen accumulation is the major phenotype associated with the MUT{gamma}3 mutation originally identified in the Hampshire pig (40). Here, we assessed glycogen content in multiple muscles and in the brain as a control. Surprisingly, despite opposing effects on AMPK activity, both the WT{gamma}3 and the MUT{gamma}3 mice had increased muscle glycogen concentrations in the predominantly fast-twitch EDL and tibialis anterior muscles and the mixed fiber type gastrocnemius muscle (Fig. 4). Glycogen concentrations were not altered in brain from either genotype.


Figure 4
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  		Fig. 4. Glycogen content in WT and transgenic mice. Glycogen content was measured as described in METHODS. Data are means ± SE; n = 7. *P < 0.05 vs. WT.

 
Glycogen synthase and glycogen phosphorylase in transgenic mice. To elucidate the mechanism for increased glycogen concentrations in WT{gamma}3 and MUT{gamma}3 mice, we assessed the enzymes involved in glycogen metabolism. Gastrocnemius muscles were used to measure glycogen synthase and glycogen phosphorylase activities, the rate-limiting enzymes for glycogen synthesis and breakdown, respectively. In WT{gamma}3 mice, glycogen synthase protein levels, as assessed by immunoblotting, were increased by 50% (Fig. 5A). Total glycogen synthase activity measured in the presence of 6.7 mM G-6-P was significantly increased (Fig. 5B), whereas the G-6-P-independent form of glycogen synthase activity was unchanged. Interestingly, glycogen phosphorylase activities in the absence and presence of AMP were decreased in the gastrocnemius of the WT{gamma}3 mice (Fig. 5C). These findings suggest that increased total glycogen synthase activity and decreased phosphorylase activities contribute to the increased glycogen content in muscles of WT{gamma}3 mice.


Figure 5
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  		Fig. 5. Glycogen synthase (GS) and glycogen phosphorylase (GP). A: muscle lysates from gastrocnemius was subjected to SDS-PAGE followed by immunoblotting with anti-GS. Data are means ± SE; n = 5. *P < 0.05 vs. WT. B and C: muscle lysates prepared as described in METHODS were assayed for both GS activity in the absence or presence of glucose 6-phosphate (G-6-P) and GP activity in the absence or presence of 5'-AMP; n = 4–6. *P < 0.05 vs. WT. AU, arbitrary units.

 
In contrast to the WT{gamma}3 mice, muscles of MUT{gamma}3 mice displayed unaltered glycogen synthase protein expression (Fig. 5A), a decrease in the G-6-P-independent glycogen synthase activity (I-form; Fig. 5B), and an increase in the active form of glycogen phosphorylase (Fig. 5C). Although these findings appear to favor decreased glycogen accumulation in the muscles of the MUT{gamma}3 mice, it should also be noted that there was a small but significant increase in total glycogen synthase activity, which would favor enhanced glycogen accumulation.

Plasma insulin concentrations from mice in the fed state were not significantly different among groups (WT, 1.6 ± 0.1; WT{gamma}3, 2.1 ± 0.4; MUT{gamma}3, 2.1 ± 0.3 ng/ml, n = 4–8), and thus insulin is unlikely to account for the changes in muscle glycogen content.

Glucose uptake in transgenic mice. Under certain conditions, glucose uptake is the rate-limiting step in the regulation of glycogen synthesis in skeletal muscle. In addition, numerous studies have suggested that AMPK is a positive regulator of glucose uptake (25, 45, 56). To determine the effects of WT{gamma}3 and MUT{gamma}3 transgene expression on glucose uptake, isolated EDL muscles were incubated in the absence or presence of AICAR, phenformin, or insulin. Basal rates of glucose uptake were slightly but significantly reduced in both the WT{gamma}3 and MUT{gamma}3 muscles (Fig. 6). This result is consistent with the findings that high cellular glycogen content decreases glucose uptake (53).

AICAR incubation increased glucose uptake in all groups; however, the increase in MUT{gamma}3 mice was severely blunted compared with WT and WT{gamma}3 mice (Fig. 6). Similar to AICAR, phenformin-stimulated glucose uptake in EDL was markedly suppressed in MUT{gamma}3 mice (Fig. 6). The decrease in both AICAR and phenformin-stimulated glucose uptake in MUT{gamma}3 mice is consistent with the decrease in AMPK{alpha}2 activity (Fig. 3B) but not likely due to elevated glycogen concentrations, because glycogen levels in EDL muscles were increased to similar levels in both WT{gamma}3 and MUT{gamma}3 mice. In contrast to the dramatic impairment of glucose uptake in response to AICAR and phenformin stimulation, insulin-stimulated glucose uptake was normal in MUT{gamma}3 mice (Fig. 6). This is consistent with data suggesting that insulin-stimulated glucose uptake occurs via a signaling mechanism independent of AMPK (25).

AICAR, glucose, and insulin tolerance tests. To determine the effects of muscle-specific WT{gamma}3 and MUT{gamma}3 overexpression on whole body glucose utilization, mice were injected with AICAR, glucose, or insulin, and blood glucose concentrations were determined for up to 120 min. AICAR injection resulted in a significant reduction in blood glucose concentrations in WT control and WT{gamma}3 mice, but the hypoglycemic effect of AICAR was clearly blunted in mice expressing MUT{gamma}3 in skeletal muscle (Fig. 7A). This impaired whole body glucose disposal in MUT{gamma}3 mice in response to AICAR was consistent with decreased muscle glucose uptake in the isolated muscle studies (Fig. 6B). For the glucose tolerance test, the glucose excursion at 20 min was significantly increased in the MUT{gamma}3 mice (Fig. 7B); however, area under the curve was not significantly altered (data not shown). The hypoglycemic effect of insulin injection occurred in parallel in WT, WT{gamma}3, and MUT{gamma}3 transgenic mice, indicating that whole body insulin sensitivity was preserved in WT{gamma}3 and MUT{gamma}3 mice (Fig. 7C).


Figure 7
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  		Fig. 7. AICAR, glucose, and insulin tolerance tests in WT and transgenic mice. AICAR (ATT, 0.2 mg/kg; A), glucose (GTT, 2 g/kg; B), and insulin (ITT, 0.75 U/kg) tolerance tests (C) were performed in WT, WT{gamma}3, and MUT{gamma}3 mice by intraperitoneal injection of one of the agents, as described in METHODS. Blood glucose was measured at the indicated time points. Data are means ± SE; n = 6. AUC1–60, area under the curve between 1 and 60 min. *P < 0.05 vs. WT; +P < 0.05 vs. WT and WT{gamma}3.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Naturally occurring mutations of the {gamma}-subunit of AMPK can result in profound increases in glycogen accumulation that result in pathological changes to skeletal and cardiac muscle (40). In the Hampshire pig, the R225Q mutation of the {gamma}3-isoform alters skeletal muscle glycogen metabolism, resulting in high muscle glycogen and poor meat quality. In humans, mutations in the AMPK{gamma}2 subunit have been associated with familial WPW syndrome and hypertrophic cardiomyopathy (3, 8, 18). Because AMPK is now considered a drug target for the treatment of diabetes and potentially other disorders, an important unsettled question in the AMPK field is whether these mutations in the {gamma}-subunit cause increases or decreases in AMPK catalytic activity. Increases in AMPK activity have been reported in studies using COS7 cells that express the R225Q mutation of {gamma}3 (6) and the equivalent R70Q mutation in {gamma}1 (20). Other reports have shown no change in AMPK activity in CCL13 cells expressing the {gamma}2 mutation (13, 50) or in transgenic mice expressing the R225Q mutation of {gamma}3 (5). Yet other studies (4, 51) have shown that the equivalent R302Q{gamma}2 mutation in heart significantly decreases AMPK activity. In the present study, we were able to directly compare how expression of the R225Q mutant and WT proteins affect AMPK activity in vivo in skeletal muscle by having both transgenes coexpress the same Flag tag. Our data clearly demonstrate that the R225Q{gamma}3 mutation of {gamma}3 significantly reduces AMPK activity under nonstimulated conditions. Further evidence that the R225Q{gamma}3 mutation has a deactivating effect is the data showing significant decreases in AICAR- and phenformin-stimulated AMPK{alpha}2 activity. Our finding that AMPK{alpha}2 but not -{alpha}1 activity is decreased in the MUT{gamma}3 mice, is consistent with the predominant association of MUT{gamma}3 and WT{gamma}3 with this isoform. AMPK{alpha}2 activity (Figs. 2B and 3B) does not parallel the increase in AMPK{alpha}2 expression (Fig. 1B), suggesting that AMPK activation is not necessarily determined by its amount but by modifications, such as interactions with its other regulatory subunits, the AMP-to-ATP ratio, and the phosphorylation state (10, 12, 21). Because glycogen was comparable in the MUT{gamma}3 and WT{gamma}3 transgenic mice, decreased AMPK activity with the R225Q{gamma}3 mutation is not likely due to feedback inhibition that has been associated with high glycogen levels. The mechanism for the decrease in AMPK{alpha}2 activity in MUT{gamma}3 mice is not entirely clear, but the equivalent mutation in {gamma}2 has been shown to disrupt the AMP binding site that is critical for AMPK activation (50). Unchanged basal WT{gamma}3 AMPK{alpha}1 activity and increased glycogen content in WT{gamma}3 transgenic mice are different from our observations with in vivo gene transfer and expression in tibialis anterior muscle (55), and the discrepancy may be due to lower protein expression associated with in vivo expression, different mouse strains, or different gene expression methods.

Activation of AMPK by AICAR enhances muscle glucose uptake (25, 39), and studies (16, 29, 44) using both AMPK{alpha}2 dominant negative mice and AMPK{alpha}2 whole body knockout mice demonstrate that AMPK{alpha}2 activation is essential for AICAR-stimulated glucose uptake in skeletal muscle. Thus impairment of AICAR- or phenformin-stimulated muscle glucose uptake in MUT{gamma}3 mice may be due to reduced AMPK{alpha}2 activity. In addition, our data suggest that normal AMPK{alpha}1 activity is not sufficient to increase glucose uptake with AICAR. These findings demonstrate that the R225Q mutation of {gamma}3 not only impairs AMPK activation but also functionally alters glucose metabolism.

Increased glycogen concentrations were observed in the skeletal muscles of both WT{gamma}3 and MUT{gamma}3 mice. This result is consistent with the findings that glycogen is higher in heart-specific mice expressing both WT and N488I-mutated {gamma}2, although the increase in glycogen was significantly greater in the N488I-mutated {gamma}2 transgenic mice (3). In a previous report by Barnes et al. (6), where the myosin light-chain promoter was used to generate muscle-specific transgenic mice on a C57BL/6J background, expression of the R225Q{gamma}3 transgene resulted in significant increases in skeletal muscle glycogen. In contrast to the present report, glycogen was not significantly altered in WT{gamma}3 transgenic mice in the resting state; however, there was a tendency toward increased concentrations during recovery from exercise (6). In {gamma}3 knockout mice, glycogen content in the fed and fasted conditions were unaltered compared with WT controls, although glycogen resynthesis following exercise was severely impaired (6). Taken together, it appears that overexpression of both WT{gamma}3 and R225Q{gamma}3 result in enhanced glycogen accumulation in skeletal muscle, whereas lack of {gamma}3 impairs the rate at which glycogen is synthesized, at least after exercise. Interestingly, the enhanced glycogen accumulation in R225Q{gamma}3 mutant mice occurs despite an inhibition of basal and stimulated glucose uptake in isolated muscles.

Increases in glycogen synthase activity and decreases in glycogen phosphorylase activity are both likely contributors to the increase in glycogen content in WT{gamma}3 mice. In contrast, the mechanism for the increase in glycogen content in mutant {gamma}3 mice is less clear, but there are many possibilities. One possibility is that the small but statistically significant increase in total glycogen synthase activity results in glycogen accumulation in the MUT{gamma}3 mice (Fig. 5B). It is possible that the R225Q{gamma}3 mutation could alter glycogen accumulation by regulating glycogen debranching enzyme, which is associated with the beta-subunit of AMPK (49). The {gamma}3 mutation alters the function of the AMPKbeta subunit's glycogen-binding domain to associate with glycogen (46). Another possibility is that overexpression of the {gamma}-subunit has a stabilizing effect on the heterotrimer, which in and of itself may permit increased trimer content, leading to greater accumulation of glycogen regardless of AMPK activity. Therefore, it cannot be entirely excluded that the MUT{gamma}3 mice may share a mechanism with the WT{gamma}3 mice, where an increase in {gamma}3 protein contributes to increased glycogen content.

In conclusion, our results suggest that glycogen content increases in both WT{gamma}3 and MUT{gamma}3 mice and that these increases are not due to a single mechanism. In addition, our results demonstrate that the R225Q{gamma}3 mutation of the regulatory subunit of AMPK decreases AMPK{alpha}2 activity and also decreases AICAR- and phenformin-stimulated glucose uptake. These effects cannot be explained by a negative feedback mechanism resulting from elevated muscle glycogen content. Therefore, the {gamma}3-subunit plays a functional role in the AMPK heterotrimer to regulate muscle glycogen metabolism.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases [AR-45670 and AR-42338 (L. J. Goodyear)] and Diabetes Endocrinology Research Center Grant DK-36836 (Joslin Diabetes Center). H. Yu. was supported by a mentor-based fellowship awarded to L. J. Goodyear from the American Diabetes Association.


   ACKNOWLEDGMENTS
 
We express our gratitude to Dr. Lee A. Witters (Dartmouth Medical School) for providing the anti-AMPK{gamma}1 antibody and Dr. John C. Lawrence, Jr. (University of Virginia) for providing the anti-glycogen synthase antibody. We are also grateful to the Specialized Assay Core at Joslin Diabetes Center (5P30-DK-36836) for the plasma insulin assay.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. J Goodyear, Joslin Diabetes Center, One Joslin Place, Boston, MA, 02215 (e-mail: Laurie.Goodyear{at}Joslin.Harvard.edu)

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


   REFERENCES
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