1 Institute for Adult Disease, Asahi Life Foundation, Tokyo 116; 2 Department of Internal Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113; 3 Department of Internal Medicine, University of Tohoku, Sendai 980-8575; and 4 Fourth Department of Internal Medicine, Saitama Medical School, Moroyama, Saitama, Japan
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ABSTRACT |
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5-Aminoimidazole-4-carboxamide
ribonucleoside (AICAR) reportedly activates AMP-activated protein
kinase (AMPK) and stimulates glucose uptake by skeletal muscle cells.
In this study, we investigated the role of AMPK in AICAR-induced
glucose uptake by 3T3-L1 adipocytes and rat soleus muscle cells by
overexpressing wild-type and dominant negative forms of the AMPK2
subunit by use of adenovirus-mediated gene transfer. Overexpression of
the dominant negative mutant had no effect on AICAR-induced glucose
transport in adipocytes, although AMPK activation was almost completely
abolished. This suggests that AICAR-induced glucose uptake by 3T3-L1
adipocytes is independent of AMPK activation. By contrast,
overexpression of the dominant negative AMPK
2 mutant in muscle
markedly suppressed both AICAR-induced glucose uptake and AMPK
activation, although insulin-induced uptake was unaffected.
Overexpression of the wild-type AMPK
2 subunit significantly
increased AMPK activity in muscle but did not enhance glucose uptake.
Thus, although AMPK activation may not, by itself, be sufficient to
increase glucose transport, it appears essential for AICAR-induced
glucose uptake in muscle.
AMP-activated protein kinase; 5-aminoimidazole-4-carboxamide ribonucleoside; exercise
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INTRODUCTION |
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IN SKELETAL MUSCLE, BOTH INSULIN and exercise stimulate glucose transport by inducing translocation of GLUT4 to the cell surface, although the respective transduction pathways differ somewhat (2, 9, 18, 29). In that regard, AMP-activated protein kinase (AMPK) was recently implicated in exercise-induced, insulin-independent glucose transport. This enzyme is a well known serine/threonine kinase that phosphorylates acetyl-CoA carboxylase (ACC), thereby reducing its activity (5, 8, 20, 22, 31). Downregulation of ACC, in turn, suppresses the activity of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase I (CPT I), resulting in activation of CPT I and fatty acid oxidation. However, the role of AMPK in exercise-induced glucose transport is still unclear; indeed, the AMPK substrate mediating glucose transport is not yet known.
AMPK exists as a heterotrimer composed of a catalytic -subunit, two
isoforms of which occur in skeletal muscle (
1 and
2), and two
regulatory subunits (
and
) (30). In response to
elevation of the AMP/ATP ratio, perhaps as a result of exercise or
hypoxia, Thr172 of the
-subunit is phosphorylated
by AMPK kinase (AMPKK) (10). Some evidence suggests that
only the
2-subunit is phosphorylated (7, 17, 30), but
substitution of Thr172 with Ala (T172A) abolishes the
kinase activity of the AMPK heterotrimer containing either isoform
(3, 27).
5-Aminoimidazole-4-carboxamide
ribonucleoside (AICAR) is an adenosine analog taken up by
muscle and phosphorylated to form 5-aminoimidazole-4-carboxamide-1--D-ribofuranosyl-5'-monophosphate (ZMP),
which stimulates AMPK activity (14, 28) and glucose transport (1, 10, 11, 16, 33) in skeletal muscle and has
therefore been used to study exercise-induced, insulin-independent glucose uptake. Although the effect of AICAR on glucose uptake is
presumed to be mediated largely by changes in AMPK activity, there is
no evidence that AICAR does not also stimulate activation of other
molecules as well. Furthermore, it was recently reported that, whereas
AICAR- and hypoxia-induced glucose uptake are completely abolished in
the skeletal muscle of transgenic mice overexpressing kinase-dead
AMPK, exercise-induced glucose uptake is only partially inhibited
(17). Clearly, much remains to be learned about the molecular mechanism underlying exercise- and AICAR-induced glucose uptake. In the present study, we confirmed that AICAR stimulates glucose uptake into both rat soleus muscle and 3T3-L1 adipocytes. In
addition, by use of adenovirus-mediated gene transfer, wild-type AMPK
-subunit and a T172A dominant negative mutant were overexpressed to
further investigate the role of AMPK in basal and AICAR-induced glucose transport.
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MATERIALS AND METHODS |
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Antibodies.
An antibody against the AMPK subunit was prepared by
immunizing rabbits with a GST-rat AMPK
2 subunit fusion protein. The antibodies raised against AMPK
were then affinity purified, as previously described (24).
Cell culture. 3T3-L1 fibroblasts were maintained in DMEM supplemented with 10% donor calf serum (Life Technologies) under a 10% CO2 atmosphere at 37°C. Two days after the fibroblasts reached confluence, differentiation was induced by incubating the cells for 48 h in DMEM supplemented with 10% fetal bovine serum, 0.5 mmol/l IBMX, and 4 mg/ml dexamethasone. Thereafter they were maintained in DMEM supplemented with 10% fetal bovine serum for an additional 4-10 days; once >90% of cells expressed the adipocyte phenotype, the cells were used for experimentation.
Generation of recombinant adenovirus.
cDNAs encoding rat AMPK1 and -
2 subunits were produced by
PCR using a rat embryo cDNA library. A cDNA encoding the T172A AMPK
2
point mutant was produced by PCR with mutated primers, as reported
previously (27). All AMPK constructs were designated to
contain a c-myc tag at the NH2 terminus.
Recombinant adenoviruses used to express wild-type and T172A
1- and
2-subunits were constructed by homologous recombination of the
expression cosmid cassettes containing the corresponding cDNAs and the
parental virus genome, as described previously (13). The
amplified adenoviruses were purified and concentrated using cesium
chloride ultracentrifugation. The resultant viruses were then dialyzed
into phosphate-buffered saline containing 10% glycerol.
Gene transfer to 3T3-L1 adipocytes and rat skeletal muscle. For gene transfer into 3T3-L1 adipocytes, cells were incubated for 6 h at 37°C in DMEM containing recombinant adenovirus, after which the virus-containing medium was replaced with normal growth medium. Experiments were performed 2 days later.
For transfer into skeletal muscle, 4-wk-old male Sprague-Dawley rats (Tokyo Experimental Animals, Tokyo, Japan) were anesthetized with pentobarbital sodium (60 mg/kg body wt ip), after which the fur was shaved from the lateral portion of both hindlimbs, a 5-mm incision was made in the skin, and 100 µl of the adenoviral vector (1.3 × 1010 pfu/ml) were injected intramuscularly. Each animal received the wild-type or T172A AMPKAMPK assay.
After first serum-starving the cells for 3 h in serum-free
DMEM, and then preincubating them for 1 h in Krebs-Ringer-HEPES buffer, 3T3-L1 adipocytes were stimulated by incubating them for 60 min
in Krebs-Ringer- HEPES buffer containing 2 mmol/l AICAR. Alternatively,
intact soleus muscles were incubated for 60 min in 2 ml of
Krebs-Henseleit bicarbonate (KHB) buffer supplemented with 8 mmol/l
glucose, 32 mmol/l mannitol, and 0.1% bovine serum albumin in 20-ml
flasks in a shaking water bath at 35°C. Thereafter, the muscles were
incubated for 60 min in KHB buffer, with or without 2 mmol/l AICAR,
during which the flasks were gassed continuously with 95%
O2-5% CO2. In both preparations, AICAR
stimulation was stopped by freezing with liquid nitrogen. The 3T3-L1
adipocytes and muscles were then lysed in 10 vol/wt of buffer
A [50 mmol/l Tris · HCl (pH 7.5), 50 mmol/l NaF, 5 mmol/l
sodium pyrophosphate, 1 mmol/l EDTA, 1 mmol/l dithiothreitol (DTT), 0.1 mmol/l phenylmethylsulfonyl fluoride, and 10% glycerol] containing
1% Triton X-100; the insoluble material was removed by centrifugation,
and the supernatants were collected. Aliquots of supernatant containing
equal amounts of protein were incubated with anti-myc tag or
anti-AMPK subunit antibody. The resultant immune complexes were
precipitated with protein A Sepharose (Pharmacia Biotech), after which
they were washed twice with buffer A and then twice with
buffer B [50 mmol/l HEPES (pH 7.5), 1 mmol/l EDTA, 1 mmol/l
DTT, and 10% glycerol]. The AMPK activity in the immunoprecipitates
was assessed as a function of phosphorylation of the SAMS peptide
(5, 27, 31). Assay reagents {40 mmol/l HEPES (pH 7.0),
200 µmol/l SAMS peptide, 200 µmol/l AMP, 80 mmol/l NaCl, 0.8 mmol/l
EDTA, 0.8 mmol/l DTT, 8% glycerol, and 200 µmol/l
[
-32P]ATP} were added directly to the
immunoprecipitate, and the mixture was incubated for 15 min at 30°C
with shaking. Aliquots were then removed and spotted onto circle
filters (P81 Whatman), which were then washed three times with 1%
H3PO4 and once with acetone, air dried, and
counted using a Molecular Imager (Bio-Rad).
Immunoprecipitation and immunoblotting.
The supernatants from adipocyte and muscle lysates, prepared as
described above, were immunoprecipitated with anti-myc or anti-AMPK subunit antibody. The immunoprecipitates were then boiled
in Laemmli sample buffer containing 100 mmol/l DTT. SDS-PAGE and
Western blotting were performed as described previously
(23), with anti-AMPK
subunit antibody as a probe.
Assay of glucose transport. Rat soleus muscles were isolated and incubated for 30 min in KHB buffer, with or without AICAR (2 mmol/l) or human insulin (2 mU/ml, Novolin R; Novo Nordisk). The muscles were then rinsed for 10 min at 29°C in 2 ml KHB buffer containing 40 mmol/l mannitol and 0.1% BSA and then incubated for 20 min at 29°C in 1.5 ml of KHB buffer containing 8 mmol/l 2-deoxy-D-[1,2-3H(N)]glucose (2-DG) (2.25 mCi/ml), 32 mmol/l [14C]mannitol (0.3 mCi/ml), 2 mmol/l sodium pyruvate, and 0.1% BSA. AICAR or insulin was present throughout the wash and the glucose uptake incubation. After the incubation, muscles were rapidly blotted, weighed, and solubilized in 1 ml of Soluene 350 (Packard). Radioactivity in the resultant samples was counted using a liquid scintillation counter. 2-DG uptake rates were corrected for extracellular trapping with mannitol counts (19).
Glucose transport in 3T3-L1 adipocytes was performed as described previously (23). ![]() |
RESULTS |
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Characterization of anti-AMPK antibody and overexpression of
wild-type and T172A AMPK
2 subunit in 3T3-L1 adipocytes.
Immunoblot analysis of lysates from 3T3-L1 adipocytes
overexpressing myc-tagged wild-type
1- or
2-subunit or
the T172A
1- or
2-mutant, by use of anti-myc tag
antibody as a probe, yielded bands at ~64 kDa, which corresponds to
the overexpressed
1- and
2-proteins; no band was observed in
control cells overexpressing GFP, however (Fig.
1, A and B,
top). The antibody raised against the full-length
2-subunit also recognized the
1-subunit, although with only about
one-half of the efficiency (Fig. 1A, bottom). Thus the weak bands obtained from GFP-expressing cells were considered to be endogenously expressed
1- and
2-subunits (Fig. 1,
A and B, bottom). The intensity of the
bands corresponding to the overexpressed AMPK
1 or -
2 subunit were
approximately fivefold stronger than those corresponding to the
endogenous subunits (Fig. 1, A and B,
bottom).
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The effect of AMPK overexpression on AICAR-induced AMPK activity in
3T3-L1 adipocytes.
Stimulating 3T3-L1 adipocytes overexpressing wild-type AMPK1 or
-
2 with 2 mmol/l AICAR increased the kinase activity present in the
anti-myc antibody immunoprecipitates by ~50 or 80% , respectively (Fig. 2, A and
C). The basal kinase activity in immunoprecipitates from
cells expressing the T172A
1- or
2-mutant was much lower; in
fact, it was not different from control, and AICAR elicited no increase
in kinase activity.
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Glucose transport in 3T3-L1 adipocytes overexpressing AMPK.
AICAR stimulation increased 2-DG uptake approximately twofold in
3T3-L1 adipocytes expressing GFP, confirming earlier observations (25). Overexpression of neither the wild-type nor the
T172A form of AMPK1 or -
2 had any effect on basal or
AICAR-induced glucose uptake (Fig. 3),
which was somewhat surprising because AMPK activity was clearly
diminished in cells expressing a T172A mutant (Fig. 2, B
and D).
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Overexpression of the wild-type 2-subunit or the T172A mutant in
soleus muscle.
SDS-PAGE and immunoblotting were used to determine the level of
adenovirus-mediated overexpression of wild-type AMPK
2 or the T172A
2-mutant in rat soleus muscle. AMPK
2 was detected as a band at a
slightly greater molecular weight than the endogenous subunit (Fig.
4A). The levels of expression
of the overexpressed wild-type and T172A forms were comparable to the
level of endogenous AMPK
. Moreover, compared with the levels seen in
control cells, overexpression of either wild-type
2- or the T172A
2-mutant resulted in a 50% reduction in the level of endogenous
AMPK
subunit detected (Fig. 4A). The glycogen levels in
rats overexpressing GFP, wild-type, or T172A AMPK
2 were 75.8 ± 8.5, 70.2 ± 12.5, and 73.2 ± 13.1 µmol/g, respectively,
and did not differ significantly.
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The effect of AMPK overexpression on AICAR-induced AMPK activity.
Overexpression of the wild-type 2-subunit in rat soleus muscle
increased basal and AICAR-induced AMPK activity by 30 and 40%,
respectively, compared with overexpression of GFP (Fig. 4B). Stimulation with 2 mmol/l AICAR increased by ~50% the level of activity present in AMPK immunoprecipitates from muscle overexpressing either GFP or wild-type AMPK
2. Overexpression of the T172A
2-mutant had no significant effect on basal AMPK activity but
completely blocked the AICAR-induced AMPK activation. Thus the T172A
2-mutant functioned as a dominant negative form in both 3T3-L1 cells
and rat soleus muscle.
Glucose uptake by rat soleus muscle overexpressing AMPK.
Uptake of 2-DG into isolated soleus muscle was increased twofold
by AICAR stimulation (Fig.
5A). Overexpression of
wild-type 2 had no significant effect on basal or AICAR-induced 2-DG
uptake, whereas overexpression of the T172A
2-mutant slightly but
significantly suppressed the basal 2-DG uptake and abolished the
AICAR-induced increase in uptake. Insulin-stimulated 2-DG uptake, by
contrast, was unaffected by overexpression of the T172A
2-mutant
(Fig. 5B).
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DISCUSSION |
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Skeletal muscle takes up glucose in response to insulin stimulation and contraction (e.g., during exercise). The insulin-induced response is mediated via a pathway in which the insulin receptor (IR), insulin receptor substrates (IRS), and phosphatidylinositol (PI) 3-kinase activation are essential components (4, 12, 15). In contrast, exercise-induced glucose uptake by muscle is insulin independent and does not require PI 3-kinase activation (21). Instead, it appears that AMPK is involved in exercise-induced glucose uptake, because AICAR, an activator of AMPK, stimulates glucose uptake in a manner similar to muscle contraction.
AMPK, which exists as a heterotrimer composed of -,
-, and
-subunits, is activated by increases in the AMP/ATP ratio. The
-subunit, two isoforms of which (
1 and
2) have been
identified, possesses the catalytic activity. Expression of the
1
subunit appears to be ubiquitous, whereas the
2-subunit appears to
be expressed only in heart, liver, and skeletal muscle
(26). Although skeletal muscle contains both isoforms, it
may be that only the
2-subunit is phosphorylated, and thus
activated, by AMPKK during exercise (7, 30). In this
study, we constructed recombinant adenoviruses to express wild-type
AMPK
1 or -
2 or a T172A point mutant, which, consistent with
earlier findings, lacked kinase activity and was therefore considered
to function as a dominant negative mutant.
AICAR increased AMPK activity twofold in 3T3-L1 adipocytes
overexpressing GFP (control cells). Although expression of wild-type AMPK1 or -
2 was increased fivefold in overexpressing cells, basal
and AICAR-induced AMPK activities were similar to those in the control
cells. This is very likely because the
-subunit is capable of
catalytic activity only when complexed with the
- and
-subunits
(3, 27). That the availability of the regulatory subunits
was a key determinant of AMPK activity means that one cannot determine
whether changes in AMPK activity increase glucose uptake by 3T3-L1
adipocytes by overexpressing the
-subunit alone. On the other hand,
whereas AICAR-induced AMPK activation was abolished by overexpression
of the T172A
1- or
2-mutant, AICAR-induced 2-DG uptake was
unaffected, making it very likely that AICAR stimulation increases
glucose transport in 3T3-L1 adipocytes via a mechanism independent of
AMPK activity.
Consistent with earlier findings (3, 17, 32),
overexpression of AMPK2 in skeletal muscle was associated with
significantly diminished levels of the endogenous
-subunit. This
likely reflects the fact that the
-subunit is unstable unless
complexed with the
- and
-subunits. Although the endogenous
-subunit was downregulated, overexpression of AMPK
2 significantly
increased both basal and AICAR-stimulated AMPK activities. Still,
despite the increased AMPK activity, 2-DG uptake was not enhanced in
muscle overexpressing the wild-type subunit; apparently, AMPK
activation alone is not sufficient to induce increases in glucose
transport. Overexpression of a molecule upstream of AMPK, such as
AMPKK, which has yet to be isolated, would enable one to address this
issue more conclusively. In contrast, when AICAR-induced AMPK
activation was abolished by overexpression of the T172A mutant,
AICAR-induced 2-DG uptake was also markedly inhibited, which is
consistent with the findings obtained using kinase-dead AMPK
2
transgenic mice (17). Thus, although AMPK activation may
not, by itself, be sufficient for increased glucose transport, it does
appear to be a necessary component of the pathway leading from AICAR
stimulation to glucose uptake.
In summary, we found that AMPK activation is essential for AICAR-induced glucose transport in skeletal muscle, but not in 3T3-L1 adipocytes. In other words, AICAR appears to stimulate one or more molecules other than AMPK in adipocytes, which likewise leads to increased glucose transport. Our findings also show that, even in rat muscle, AICAR-induced glucose transport is dependent on actions taking place in addition to AMPK activation. Further investigation of the events occurring downstream of AMPK activation, and/or in other transduction pathways, will be required before a complete understanding of the mechanism of AICAR- and exercise-induced glucose transport is achieved.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Asano, Dept. of Internal Medicine, Graduate School of Medicine, Univ. of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan (E-mail: asano-tky{at}umin.ac.jp).
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.
First published February 19, 2002;10.1152/ajpendo.00455.2001
Received 11 October 2001; accepted in final form 12 February 2002.
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