Research Division, Joslin Diabetes Center, and Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02215
Submitted 25 July 2003 ; accepted in final form 17 September 2003
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ABSTRACT |
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adenosine 5'-monophosphate-activated protein kinase; AMPK3 short form; AMPK
3 long form; cystathionine
-synthase domain
Although the two catalytic subunit isoforms of AMPK have been the major focus of AMPK research over the last 10 years, there is now increasing interest in understanding the roles of the
- and
-subunits in AMPK function. Three
-isoforms have been identified,
1,
2, and
3. Northern blot analysis of human tissues reveals that
1 is widely distributed, whereas AMPK
2 is also widely distributed but has very high expression levels in heart (11, 31). Interestingly,
3 is almost exclusively expressed in the skeletal muscle of humans (11, 31). The divergent expression patterns of the
isoforms among tissues suggest that the different isoforms have tissue-specific functions (11, 44). In comparing the human
1,
2, and
3 amino acid sequences, the NH2 termini have significant variations in length and identity, suggesting that this region may play an important role in conferring isoform specificity either by targeting different downstream molecules or by responding to different upstream stimuli. However, specific functions of the NH2-terminal region have not been identified for all three
-subunits, and there are no known protein motifs in this region. In contrast, the COOH-terminal region contains four consecutive cystathionine
-synthase (CBS) domains that are highly conserved in all
isoforms (11, 41). These CBS domains occupy approximately half of the
-subunit, suggesting that these domains are also critical for
-subunit function (7). Indeed, mutations of CBS domains in a number of proteins, including the CBS domains of the
-subunit of AMPK, are associated with various disease states (4, 8, 20, 28, 31). In skeletal muscle and heart, mutations of the CBS domains in
2 and
3 have been shown to be associated with alterations in glycogen metabolism (3, 4, 12, 31).
In recent years, skeletal muscle has become a major focus of the AMPK field because AMPK may be involved in the regulation of glucose uptake, glycogen metabolism, fatty acid oxidation, and gene transcription in this tissue (5, 21, 23, 25, 34, 36, 43, 47, 49). Although mutations in -subunit CBS domains, including
3, lead to pathological conditions, very little is known about the mechanisms by which the
-isoforms may regulate AMPK activity, cellular metabolism, and tissue phenotype. There have been discrepancies in reports of tissue distributions of
3 mRNA and protein (11, 31), as well as conflicting reports of the translation initiation sites of the human AMPK
3 gene (11, 31). In this study we have cloned the full-length mouse AMPK
3 gene and, by determining its translation initiation sites, have revealed that there are two splice variants of
3. We have also characterized the AMPK
3 isoforms and investigated the effects of overexpressing the wild-type AMPK
3 subunit in vivo on AMPK activity and glycogen metabolism in skeletal muscle.
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METHODS |
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Animals. Female (8 wk) ICR mice (30 g) were purchased from Taconic. 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 National Institutes of Health guidelines.
Human muscle. The experiment using a human muscle lysate study with human subjects was approved by the Ethical Committee at the Karolinska (35).
Cloning of mouse AMPK3 cDNA. Total RNA was extracted from 100 mg of mouse skeletal muscle using Tri Reagent (Molecular Research Center). Total RNA (5 µg) was subjected to the oligo(dT) primer reverse transcription (RT) reaction (Invitrogen), and the cDNA products were used as a source for polymerase chain reaction (PCR) templates. The primers were designed on the basis of conserved regions of pig and human AMPK
3: sense, 5'-tgctgagtccaccggg-3'; and antisense, 5'-agcagggctgagcacc-3'. The PCR product was subcloned into pCR 2.1-TOPO vector (Invitrogen) and sequenced. To obtain the upstream extension of 5'-end and the downstream extension of 3'-end of RT-PCR product, 5'- and 3'-rapid amplification of cDNA ends (RACE) was performed using a Marathon-Ready cDNA kit (Clontech). The antisense primer for 5'-RACE, 5'-agccatggcatcataacaggtgtgttcc-3', and sense primer for 3'-RACE, 5'-tctggagggagttctctcctgccagc-3', were designed on the basis of the DNA sequence of RT-PCR product as described above. 5'-RACE was performed between the anchor sense primer AP1 (Clontech) and the 5'-RACE antisense primer. The resulting PCR products were reamplified by using the nested sense primer AP2 (Clontech) and the same reverse primer, and the PCR products were cloned into pCR 2.1-TOPO vector for cloning and sequencing. 3'-RACE was performed between the 3'-RACE sense primer and anchor antisense primer AP1, and the resulting PCR products were reamplified by using the same sense primer and nested antisense primer AP2. The PCR products were also cloned into pCR 2.1-TOPO vector and further sequenced.
Cloning of mouse genomic AMPK3. An AMPK
3L cDNA fragment (246822) was used as a probe to screen the mouse genomic DNA library (IncyteGenomics). The
3-containing clones were confirmed by Southern Blotting using the same probe. The genomic organization was identified by comparing cDNA and genomic sequences.
Muscle processing. Muscles were dissected and snap frozen in liquid nitrogen. The pulverized samples were weighed and Polytron homogenized (Brinkmann 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 dithiothreitol (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 assay.
Cell culture. L6 myoblast cells (1 x 10 6 cells; gift from Dr. Amira Klip, University of Toronto, Toronto, ON, Canada) were plated on 60-mm dishes and grown in -MEM with 10% fetal bovine serum. Each plasmid DNA (7 µg) was introduced into cells with Lipofectamine 2000 reagent (Invitrogen). Cells were then cultured for another 48 h and then harvested with ice-cold AMPK lysis buffer.
Immunoblotting. Protein (20 µg) from cells and muscle lysates was separated by 8% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked in Tris-buffered saline with 0.05% Tween 20 (TBST) and 5% nonfat milk for 1 h at room temperature. The membranes were incubated overnight at 4°C with the appropriate primary antibodies. Bound primary antibodies were detected with anti-rabbit (Amersham) or mouse immunoglobulinhorseradish peroxidase-linked whole antibody (Transduction Laboratories, Lexington, KY). The membranes were washed with TBST and then incubated with enhanced chemiluminescence reagents (Perkin-Elmer) and exposed to film. Bands were visualized and quantified using Image-Quant software (Molecular Dynamics).
mRNA levels of AMPK3L and AMPK
3S. The mRNA levels were determined on the basis of PCR. cDNA was synthesized from mouse quadriceps muscle mRNA by reverse transcription, adaptor-ligated double-strand cDNA was used as a template (Clontech), and the adaptor sequence (5'-ccatcctaatacgactcactatagggc-3') attached to the 5'-end of cDNA including both
3S and
3L cDNA was used as the sense primer. The sequence 5'-agccatggcatcataacaggtgtgttcc-3', conserved in both
3L and
3S, was used as the antisense primer. The two resulting products from the unsaturated PCR reaction were separated by 1% agarose gel and confirmed by DNA sequencing.
Immunoprecipitation. Muscle lysates (200 µg) were incubated with anti-AMPK1/2 or -AMPK
antibody-bound protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. The immunocomplex was washed with lysis buffer and boiled in Laemmli's buffer. The samples were separated by 8% SDS-PAGE, transferred to nitrocellulose membranes, and then immunoblotted with appropriate antibodies.
AMPK activity assay and glycogen content. AMPK activity assay was performed as described by Musi et al. (35). Briefly, muscle lysates were immunoprecipitated with specific antibodies to the 1 and
2 catalytic subunits of AMPK and protein A/G beads. Immunoprecipitates were assayed for AMPK activity in assay buffer containing 0.2 mM AMP, 0.2 mM ATP (2 µCi [
-32P]ATP), and 0.2 mM synthetic AMPK substrate peptide (SAMS) with the sequence HMRSAMSGLHLVKRR for 20 min at 30°C (14, 35). For glycogen measurements, a muscle piece was weighed and hydrolyzed 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, St. Louis, MO).
Generation of recombinant AMPK3. The coding region of AMPK
3S or AMPK
3L was obtained by PCR with the sense primer spanning the start codon containing a Kozac sequence, with or without a Flag-tag sequence and antisense primer spanning the stop codon. The purified PCR product was subcloned into pCR 2.1-TOPO vector and sequenced. The EcoI fragment containing the AMPK
3 in the pCR 2.1-TOPO vector was excised and subcloned into the pCAGGS vector. Sense primers were as follows:
3S, 5'-gcggccgcgccaccatggacttcttagaacaaggagaaaactcatggc-3'; Flag-
3S, 5'-gcggccgcgccaccatggactacaaggacgacgatgacaagatggacttcttagaacaaggagaaaactcatggc-3';
3L, 5'-gcggccgcgccaccatggagcccgagctggagcacac-3'; and Flag-
3L, gcggccgcgccaccatggactacaaggacgacgatgacaagatggagcccgagctggagcacac-3'; and the antisense primer for
3S, Flag-
3S,
3L, and Flag-
3L was 5'-gaggccgctcaggcgctgagggcatcga-3'.
Expression of AMPK3S and AMPK
3L in tibialis anterior muscle. DNA injection and in vivo electroporation were done by a modification (18) of the method of Aihara and Miyazaki (2). Mice were anesthetized with pentobarbital sodium (90 mg/kg body wt ip), and 100 µg of pCAGGS-AMPK
3S, pCAGGS-AMPK
3L, or an equal amount of empty pCAGGS vector in 25 µl of saline was injected into the tibialis anterior (TA) muscle, using an insulin syringe with a 29-gauge needle. For the control, an equal amount of pCAGGS alone was injected into the opposite leg. With the use of an electric pulse generator, square-wave electrical pulses (200 V/cm) were applied eight times at a rate of 1 pulse/s with each pulse being 20 ms in duration. The electrodes used were a pair of stainless steel needles inserted and fixed 5 mm apart into the TA muscle. Nine days after gene delivery, the muscles were removed and prepared for AMPK activity, Western blotting, and the measurements of glycogen content.
Muscle contraction. pCAGGS-AMPK3S, pCAGGS-AMPK
3L, or an equal amount of empty pCAGGS vector was injected into the TA muscle, followed by electroporation. Nine days later, mice were anesthetized with pentobarbital sodium (90 mg/kg body wt ip), the sciatic nerves of both legs were surgically exposed, and subminiature electrodes were attached to the nerves (22, 39). Hindlimb muscles of one leg were electrically stimulated to contract for 20 min (train rate 1/s, train duration 500 ms, rate 100 pulses/s, duration 0.1 ms, 13 V), using a Grass S88 pulse generator, and the other leg served as a sham-operated control. Immediately after nerve stimulation, mice were killed and tibialis muscles were rapidly dissected and frozen in liquid nitrogen.
Statistical analysis. Data are presented as means ± SE. One-way analysis of variance was used for statistical evaluation. Differences between means were determined using the Student-Newman-Keuls test.
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RESULTS |
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Figure 1C shows the deduced amino acid sequence alignment of mouse 3S and
3L compared with pig
3, the two different human
3 sequences that were reported with different initiation sites, and rat
3 (XM_237293
[GenBank]
). Mouse
3S shows overall 85.5% identity to human (AF214519
[GenBank]
) and pig
3. Mouse
3L is most similar to another reported human
3 gene (NM_017431
[GenBank]
) that contains an altered initiation site and also has 85.5% identity. The four CBS domains are highly conserved, showing 96% identity in all species, whereas the remainder of the NH2-terminal region shows 66% identity. Interestingly, both AMPK
3 splice variants are rich in serine and threonine at the NH2-terminal region; these are potential phosphorylation sites for a number of protein kinases including glycogen synthase kinase 3, protein kinase A, and protein kinase C, based on consensus sequence searches for these kinases using PhosphoBase (http://www.cbs.dtu.dk/databases/PhosphoBase/).
AMPK3 protein expression. To determine the expression pattern of AMPK
3, we generated an antibody against a conserved sequence within COOH termini of mouse
3S and
3L, human
3, and pig
3 (Fig. 1C). Immunoblotting with
3 antibody revealed that AMPK
3 is highly expressed in gastrocnemius muscle, with no detectable signal in heart, brain, lung, liver, kidney, pancreas, spleen, white fat, or brown fat (Fig. 2A). These results are consistent with Northern hybridization data from human tissues, which showed that AMPK
3 is specifically expressed in skeletal muscle (11, 31). Interestingly, white fast-twitch glycolytic skeletal muscle that contains mostly type IIb fibers (white quadriceps and white gastrocnemius) showed higher levels of AMPK
3 expression compared with the red fast-twitch oxidative-glycolytic muscle, which contains primarily type IIa fibers (red quadriceps and red gastrocnemius). AMPK
3 was not detectable in soleus, a muscle that is composed almost exclusively of red slow-twitch oxidative type I fibers. This expression pattern suggests that AMPK
3 plays a unique role in skeletal muscle, especially in the more fast-twitch glycolytic fiber types.
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Immunoblotting of mouse skeletal muscle (quadriceps) and human skeletal muscle with 3 antibody reveals a single band (Fig. 2B). To determine whether
3S or
3L is expressed in mouse and human skeletal muscle, we overexpressed recombinant mouse
3S and
3L in L6 myoblast cells that do not express endogenous AMPK
3. Cell lysates were compared with endogenous
3 from mouse and human muscle lysates. As shown in Fig. 2B, mouse endogenous AMPK
3 had the same molecular size as
3L, suggesting that AMPK
3L is the predominant form in mouse muscle. On the other hand, human AMPK
3 had the same molecular size as
3S, although this finding will need to be confirmed in future studies using overexpressed human
3. The double band on SDS-PAGE of overexpressed
3S suggests that the expressed protein in the cells was partially degraded.
In addition, 3L is more abundant compared with
3S at the mRNA level (Fig. 2C), and this is consistent with Western blotting data showing that
3L is the predominant form in skeletal muscle (Fig. 2B). The
3S variant was detected by PCR (5'-RACE), but protein was not expressed. This may be explained by the following possibilities: 1) the lower
3S mRNA results in very low levels of
3S protein, beyond the detection of the
3 antibody; 2) there may be an inhibitory mechanism at the translational or posttranslational steps that suppresses
3S protein expression; and/or 3)
3S protein is less stable and is more rapidly degraded.
The molecular masses of 3S and
3L in SDS-PAGE were 64 and 67 kDa, respectively, different from their predicted molecular masses of 51 and 54 kDa. The reason for the mobility shift of
3 in a denatured gel is unclear, although interestingly, the same phenomenon has been observed in the AMPK
1 subunit. In SDS-PAGE the molecular mass of AMPK
1 isolated from rat liver is 38 kDa higher than the predicted molecular mass of 30 kDa (50).
Association of 3 subunit with
- and
-subunits. To determine whether the
3 subunit associates with the
catalytic subunit, we used mouse muscle lysates for the immunoprecipitation of AMPK
, using an anti-AMPK
-specific antibody that recognizes both
1 and
2. As shown in Fig. 3A, the anti-AMPK
1/2 antibody immunoprecipitates
1/2 and coprecipitates both the
1 subunit and the
3 subunit from mouse skeletal muscle lysates. There was also concomitant immunodepletion of AMPK
1/2, AMPK
1, and the AMPK
3 subunits from the immunosupernatant. In contrast, control IgG did not pull down any of these proteins, and AMPK
1/2, AMPK
1, and AMPK
3 are preserved in the supernatant. This experiment also confirms that AMPK
3 runs as a 67-kDa band in SDS-PAGE (Fig. 2, A and B, and Fig. 3A). When AMPK
1 was immunoprecipitated in skeletal muscle lysates with the use of a commercially available antibody, AMPK
1 was barely detectable (not shown), and because an antibody for the immunoprecipitation of AMPK
2 was not commercially available, the potential association of
with
3 was determined by expressing exogenous AMPK
1 and
3L in L6 myoblast cells. We chose
3L because this is the endogenous splice isoform of AMPK
3 in mouse skeletal muscle. This experiment revealed that expressed AMPK
3 could be coimmunoprecipitated with the
1 subunit (Fig. 3B).
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Overexpression of AMPK3S and AMPK
3L in mouse TA muscle in vivo. To determine the effects of
3S and
3L on AMPK activity and glycogen metabolism in skeletal muscle, we used a gene transfer/electroporation system to express the two splice variants in mouse TA muscle in vivo. By using this method in preliminary experiments using the LacZ gene, 85.7 ± 2.3% (n = 6) of fibers were determined to express
-galactosidase (18). AMPK
3S and AMPK
3L expression levels were measured at days 7, 9, and 14 after gene transfer/electroporation, and maximal expression occurred at day 9 (
1.5- to 2.5-fold greater than endogenous AMPK
3); thus all subsequent experiments were done using this time point. Figure 4A, top, shows that, as predicted, Flag-tagged AMPK
3L ran at a higher molecular mass compared with Flag-tagged
3S. Figure 4A, middle, shows the degree of overexpression of
3S and
3L compared with endogenous
3 and also confirms the experimental results shown in Fig. 2B, demonstrating that the
3L splice variant is endogenously expressed in mouse skeletal muscle. Figure 4A, bottom, shows that recombinant AMPK
3S and AMPK
3L did not affect endogenous
1 protein levels, suggesting that
3 overexpression does not replace this isoform. Endogenous
2 isoform was not determined because anti-
2 antibodies were not commercially available. In lysates from TA muscle, overexpressed AMPK
3S and AMPK
3L could be coimmunoprecipitated with the
-subunit (Fig. 4B).
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Overexpression of both AMPK3S and AMPK
3L in vivo resulted in a significant increase in AMPK
1 catalytic activity (Fig. 5A, left). The increase in AMPK
1 activity was associated with an increase in AMPK
1 Thr172 phosphorylation, determined by AMPK
1 immunoprecipitation followed by Western blotting using a phosphospecific AMPK
antibody (Fig. 5A, right). The increases in AMPK
1 activity and phosphorylation were not due to an increase in AMPK
1 protein expression (Fig. 5A, right). In contrast, AMPK
3S and AMPK
3L expression in vivo had no effect on AMPK
2 activity, phosphorylation, and expression (Fig. 5B), although the phosphorylation of AMPK
2 in the basal condition was barely detectable. The increase in AMPK
1 activity was not associated with changes in glycogen concentrations in the muscle (Fig. 5C). Consistent with this finding, overexpression of AMPK
3S and AMPK
3L had no effect on glycogen synthase protein expression or the phosphorylation state of glycogen synthase (Fig. 5C). Several other muscle proteins were also not altered by expression of AMPK
3S and AMPK
3L, including GLUT-4, citrate synthase, and phosphoacetyl CoA carboxylase proteins known to be involved in the regulation of glucose uptake, mitochondria biogenesis, and fatty acid oxidation, respectively (data not shown).
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In addition, AMPK3L or AMPK
3S overexpression did not change the susceptibility of AMPK to activation by contraction. As we and others have previously reported (19, 46), in situ contraction resulted in a very modest increase in AMPK
1 activity, and this increase was not different among the three groups (vector control: 33.4 ± 14.8% over basal,
3S: 27.6 ± 14.8%,
3L: 26.7 ± 13.4%, n = 8/group). The increase in AMPK
2 activity in response to in situ contraction was also similar among the three groups (vector control: 116.7 ± 14.5%,
3S: 90.3 ± 23.5%,
3L: 121.8 ± 26.5%, n = 8/group).
3L and
3S expression was confirmed by Western blotting of muscle lysates.
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DISCUSSION |
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Western blot analysis of mouse tissue lysates using a 3-specific antibody clearly showed that AMPK
3 was only detected in skeletal muscle. However, another report has suggested that
3 is mainly distributed in brain and testis, by measuring the proportion of total AMPK activity (11). Although we do not have an explanation for the difference between our work and that of Cheung et al. (11), our data are consistent with Northern blotting results from two independent groups showing
3 mRNA only in skeletal muscle (11, 31). Though
3 is only present in skeletal muscle, we found different levels of expression of
3 in muscle composed of various muscle fiber types. White muscles, which contain predominantly fast-twitch glycolytic (type IIb) fibers, showed the highest levels of
3 expression. The red muscles, which contain predominantly fast-twitch oxidative glycolytic (type IIa) fibers, had lower levels of expression, whereas
3 was undetectable in soleus muscle, which is a slow-twitch oxidative muscle with predominantly type I fibers. The type IIb fibers present predominantly in white muscles are abundant in fasttwitch myosin isoforms and have a more glycolytic metabolic phenotype. These fibers are recruited during high-intensity types of contractions, rapidly providing energy through glycogenolysis. Whether wild-type
3 regulates AMPK activity in a manner that promotes the rapid utilization of glycogen is not known, but this is an interesting hypothesis to test in future studies. The lower levels of
3 expression in the red oxidative fibers provide an explanation for the previous observation that excess glycogen accumulation specifically occurs in white muscles in pigs with the RN mutation in the PRKAG3 gene (R200Q mutation in AMPK
3) (17). Interestingly, incubation of muscles with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) only increases AMPK activity in the highly glycolytic white epitrochlearis muscle but has no effect in the red oxidative (type I) soleus muscle (1, 27), (Hirshman MF, Goodyear LJ, and Hayashi T, unpublished observation). It is not known whether
3 expression is needed for AMPK to respond to AICAR in mouse skeletal muscle.
We successfully overexpressed AMPK3S and AMPK
3L in mouse skeletal muscle by using a gene transfer and electroporation system and found that both splice variants associate with the endogenous AMPK
subunit (Fig. 4B). Using this experimental system, we found that expression of AMPK
3S and AMPK
3L increased AMPK
1 activity and phosphorylation but had no effect on AMPK
2 activity and phosphorylation. The increase in AMPK
1 activity was not due to an increase in AMPK
1 protein, suggesting that the increase in phosphorylation of AMPK
1 is responsible for the increase in AMPK
1 activity. The mechanism by which
3 overexpression increases
1 phosphorylation and activity in vivo is unclear. Different
-subunits have been reported to account for different proportions of total AMPK activity (11), and therefore an exchange between isoforms could lead to a change in AMPK activity. However, endogenous
1 expression was not changed in response to
3 overexpression (Fig. 4A), making this mechanism unlikely to account for the increase in AMPK
1 activity. Another possibility is that
3 could favorably alter the conformation of
1-containing AMPK heterotrimer, which in turn could enhance
1 phosphorylation by upstream signals such as AMPK kinase or retard dephosphorylation by phosphatases. Unfortunately, we are not able to directly compare the amount of
3 subunit associated with
1 and
2, because anti-AMPK
1 and anti-AMPK
2 antibodies have different affinities to
1 and
2 subunits.
There are now multiple examples demonstrating 1- and
2-specific AMPK regulation in response to various perturbations (6, 27, 45). One example is in cardiac hypertrophy, where AMPK
1 activity and expression are increased whereas
2 expression is decreased (45). Exercise training also specifically upregulates AMPK
1 in skeletal muscle (6). Another example is the recent report that obesity-related insulin resistance is associated with a specific impairment in contraction-stimulated
1 activity in skeletal muscle in rats (6). Because we found that
3 overexpression results in an
1-specific activation, in future studies it will be interesting to determine whether altered
3 expression plays a role in the impaired
1 response observed in these insulin resistant animals.
The AMPK3 mutation from arginine to glutamine at position 200 (R200Q) results in muscle glycogen accumulation in the Hampshire pig. In contrast, the valine-to-isoleucine mutation at position 199 in the same species reduces muscle glycogen content (3, 12). The fact that adjacent mutations at V199 and R200 in the CBS domain of the
3 subunit cause opposite effects on muscle glycogen content suggests that structural changes in this part of the CBS domain alter enzyme function. In our study, modest overexpression of wild-type AMPK
3 did not change muscle glycogen concentrations and had no effect on the expression and phosphorylation of glycogen synthase (Fig. 5C), despite significant increases in AMPK
1 activity. Furthermore, despite major differences in the amount of endogenous
3 protein among muscles of different fiber types, we did not observe a significant difference in muscle glycogen concentrations in these various muscles (Yu H, Hirshman MF, and Goodyear LJ, unpublished observation). These results suggest that wild-type AMPK
3 does not directly regulate muscle glycogen concentrations and that the mutations are the cause of the alterations in glycogen metabolism.
In summary, we have cloned the full-length mouse AMPK3 gene and identified two
3 splice variants,
3S and
3L. Both
3S and
3L can associate with the
- and
-subunits of AMPK. These two splice variants appear to be differentially expressed in human and mouse skeletal muscle. Overexpression of either
3S or
3L results in a specific increase in AMPK
1 activity and phosphorylation, but these changes are not associated with alterations in muscle glycogen.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants AR-42338 and AR-45670 and by grants from the American Diabetes Association (to L. J. Goodyear). H. Yu is supported by a mentor-based fellowship awarded to L. J. Goodyear from the American Diabetes Association.
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FOOTNOTES |
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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.
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