From the Research Division, Joslin Diabetes Center; Department of Medicine, Brigham and Women's Hospital; and Harvard Medical School, Boston, Massachusetts.
Address correspondence and reprint requests to Laurie J. Goodyear, PhD, Research Division, Joslin Diabetes Center, 1 Joslin Place, Boston, MA 02215. E-mail: laurie.goodyear{at}joslin.harvard.edu .
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ABSTRACT |
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INTRODUCTION |
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Insulin is a potent stimulator of glucose transport in skeletal muscle. Part of the mechanism by which insulin increases glucose transport in vivo involves enhanced blood flow and glucose delivery to the muscle, a process mediated by the release of NO from the endothelium (9,10,11). The acute administration of the NOS inhibitors NG-monomethyl-L-arginine (L-NMMA) or NG-nitro-L-arginine methyl ester (L-NAME) results in the development of marked insulin resistance, hypertension, and/or hyperglycemia (9,12,13). NOS blockade decreases blood flow to skeletal muscle and impairs insulin-mediated glucose disposal during a hyperinsulinemic-euglycemic clamp in vivo (9,12). In contrast to the effects of NOS inhibition in vivo, NOS inhibitors fail to affect insulin-stimulated glucose transport in isolated muscles incubated using in vitro preparations (6,8,9); this suggests that hemodynamic factors are needed to fully amplify the increase in insulin-stimulated glucose transport in skeletal muscle.
5'AMP-activated protein kinase (AMPK) has recently emerged as a
putative regulator of multiple metabolic processes in skeletal muscle,
including fatty acid and carbohydrate metabolism
(14,15,16,17).
We
(18,19)
and others
(20,21)
have provided evidence that AMPK is an intermediary in the signaling cascade
leading to contraction-stimulated glucose transport in skeletal muscle. AMPK
is a heterotrimer consisting of three subunits, , ß, and
.
The
subunit contains the kinase domain and contributes to the
ATP-binding site (22). Of the
two isoforms of the
subunits,
1 AMPK is widely expressed in
liver, pancreas, adipose tissue, and skeletal muscle, whereas
2 AMPK
catalytic isoform is predominantly expressed in skeletal muscle
(23,24).
Interestingly, AMPK co-immunoprecipitates with eNOS in rat cardiac muscle, and
presently there are data showing that AMPK can phosphorylate eNOS in an vitro
assay (25). However, it is
unknown whether there is an interaction between NO and AMPK signaling to
stimulate glucose transport in skeletal muscle.
In the current study, we determined if the signaling mechanism leading to
NO-stimulated glucose transport is similar to, or distinct from, the signaling
mechanisms leading to insulin- and contraction-stimulated glucose transport in
rat skeletal muscle. Our results demonstrate that NO signaling to glucose
transport is independent of the mechanisms through which insulin and muscle
contraction increase transport. Furthermore, our data demonstrate that
NO-stimulated glucose uptake is associated with an activation of the 1
catalytic subunit of AMPK.
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RESEARCH DESIGN AND METHODS |
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Materials. SNP, wortmannin, L-NAME, LY-83583, D-glucose, mannitol, and pyruvic acid were purchased from Sigma Chemical (St. Louis, MO). L-NMMA was purchased from Calbiochem (San Diego, CA), and 2-deoxy-D-[1,2-3H]glucose and D-[14C]mannitol were purchased from New England Nuclear (Boston, MA).
Muscle incubations. Rats were fed ad libitum before muscle
isolation, and experiments commenced between 9:00 and 10:00 A.M.. Animals were
killed by decapitation, and the EDL muscles were rapidly dissected. Both ends
of each muscle were tied with suture (silk 4-0) and mounted on an incubation
apparatus as previously described
(18). The buffers were
continuously gassed with 95% O2:5% CO2. Muscles were
preincubated in 6 ml Krebs-Ringer bicarbonate buffer (KRBB) containing 2
mmol/l pyruvate at 37°C for 50 min in the presence or absence of SNP (1,
5, 10, or 20 mmol/l) or insulin (50 mU/ml). For contraction treatment, muscles
were stimulated during the last 10 min of the 50-min incubation period (train
rate 2/min, train duration 10 s, rate 100 pulses/s, duration 0.1 ms, 100 V).
When added, the inhibitors wortmannin (100 nmol/l), LY-83583 (10 µmol/l),
and L-NMMA (0.1 mmol/l) were present throughout the entire incubation and
present 30 min before stimulation. In preliminary experiments,
dose-dependent inhibition of L-NMMA on NOS activity was measured in EDL muscle
using the method previously described by Roy et al.
(9). Altogether, 1 µmol/l
L-NMMA inhibited NOS activity
50%, 10 µmol/l inhibited NOS activity
85%, and 0.1 mmol/l L-NMMA (the dose we used in the current experiments)
inhibited NOS activity
90%. Then, muscles were immediately used for the
measurement of 2-deoxyglucose uptake or were immediately frozen in liquid
nitrogen and subsequently analyzed for the measurement of ATP, creatine
phosphate, and glycogen concentrations and isoform-specific AMPK activity.
2-Deoxyglucose uptake. The 2-deoxyglucose uptake was measured in 2 ml KRBB containing 1 mmol/l 2-deoxy-D[1,2-3H]glucose (1.5 µCi/ml) and 7 mmol/l D-[14C]mannitol (0.45 µCi/ml) (New England Nuclear, Boston, MA) at 30°C for 10 min. SNP, insulin, wortmannin, LY83583, and L-NMMA were added to the buffer if they were present during the previous incubation period. Muscles were processed, radioactivity was determined by liquid scintillation counting for dual labels, and 2-deoxyglucose uptake was calculated as previously described (26).
Assays for muscle enzymes and metabolites. To measure ATP and creatine phosphate concentrations, frozen muscles were homogenized in HClO4 in an ethanol-solid CO2 bath and centrifuged at 14,000g for 10 min at -5°C. The supernatant of the homogenates was neutralized with a solution of 2N KOH, 0.4 mol/l imidazole, and 0.4 mol/l KCl and then centrifuged at 14,000g at -9°C. The supernatant was collected and analyzed enzymatically for ATP and creatine phosphate (27).
For measurement of muscle glycogen, muscles were dissolved in 30% KOH and 5% Na2SO4 at 70°C for 15 min. Glycogen was then precipitated by mixing with 3 x volume of absolute alcohol and stored overnight at -20°C. The precipitates were collected by centrifugation at 13,000g for 5 min. The glycogen was hydrolyzed in 6N H2SO4 at 100°C for 45 min and cooled. Samples were neutralized with 1N NaOH and glucose was measured using the glucose (HK) reagent (Sigma Chemical).
For the measurement of isoform-specific AMPK activity, muscles were
homogenized in ice-cold lysis buffer (1:100, wt/vol) containing 20 mmol/l
Tris-HCl (pH 7.4), 1% Triton X-100, 50 mmol/l NaCl, 250 mmol/l sucrose, 50
mmol/l NaF, 5 mmol/l sodium pyrophosphate, 2 mmol/l dithiothreitol, 4 mg/l
leupeptin, 50 mg/l trypsin inhibitor, 0.1 mmol/l benzamidine, and 0.5 mmol/l
phenylmethylsulfonyl fluoride, and centrifuged at 14,000g for 20 min
at 4°C. The supernatants (200 µg protein) were immunoprecipitated with
isoform-specific antibodies to the 1 or
2 catalytic subunits of
AMPK and protein A/G beads. These are anti-peptide antibodies made to the
amino acid sequences DFYLATSPPDSFLDDHHLTR (339-358) of
1 and
MDDSAMHIPPGLKPH (352-366) of
2. Immunoprecipitates were washed twice in
both lysis buffer and in wash buffer (240 mmol/l HEPES and 480 mmol/l NaCl).
Kinase reactions were performed in 40 mmol/l HEPES (pH 7.0), 0.2 mmol/l SAMS
peptide (synthetic substrate for AMPK), 0.2 mmol/l AMP, 80 mmol/l NaCl, 0.8
mmol/l dithiothreitol, 5 mmol/l MgCl2, 0.2 mmol/l ATP (2 µCi
[
-32P]ATP), and in a final volume of 40 µl for 20 min at
30°C (28). At the end of
the reaction, a 20-µl aliquot was removed and spotted on What-man P81
paper. The papers were washed six times in 1% phosphoric acid and once with
acetone. 32P incorporation was quantitated with a scintillation
counter, and kinase activity was expressed as fold increases compared with
basal samples.
In situ contraction studies. Rats were fed ad libitum before muscle isolation. Animals were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg body wt). The sciatic nerve was dissected free and connected to subminiature electrodes (Harvard Apparatus, South Natick, MA). A pin under the patella tendon fixed the knee of the leg, and a 100-g weight was attached by a wire around the Achilles' tendon. The sciatic nerve was stimulated for two 5-min periods (train rate 1/s, train duration 500 ms, rate 100 pulses/s, duration 0.1 ms, 3-10 V) separated by 1 min of rest. Immediately after stimulation, EDL muscles were dissected and incubated in 3 ml KRBB containing 8 mmol/l mannitol for 20 min in the absence of presence of L-NMMA. After this incubation period, 2-deoxyglucose uptake was measured as previously described.
Treadmill exercise studies in L-NAME-treated animals. Rats were accustomed to a rodent treadmill (Quinton Instruments, Seattle, WA) for 5 min/day for 2 days before the experiment. L-NAME was added to the drinking water (1 mg/ml), and the water was changed daily for 2 days before the experiment. Systolic blood pressure was measured by a volume-oscillometric method (UR-5000; Ueda, Tokyo) before and during the 2-day L-NAME treatment. Rats ran on a rodent treadmill with a 10% incline for 1 h at 0.7 mph. Animals were killed immediately after exercise, and both the soleus and EDL muscles were rapidly dissected and mounted on the incubation apparatus. The muscles were incubated for 20 min in KRBB containing 8 mmol/l D-mannitol at 30°C, and 2-deoxyglucose uptake was measured as previously described.
Statistical analysis. Data are means ± SE. The effect of SNP on 2-deoxyglucose uptake, ATP, creatine phosphate, and isoform-specific AMPK activity was compared by a one-way analysis of variance with Fisher's protected least significant difference test. For comparison of two means, an unpaired Student's t test was performed.
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RESULTS |
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SNP stimulates glucose uptake through an insulin-independent pathway in
isolated EDL muscles. To determine whether the combination of SNP and a
maximally effective dose of insulin has additive effects on skeletal muscle
glucose uptake, isolated EDL muscles were incubated in KRBB in the absence or
presence of insulin and/or 10 mmol/l SNP.
Figure 2A shows that
the combination of SNP and insulin had nearly full additive effects on
2-deoxyglucose uptake. To determine whether SNP and insulin stimulate glucose
uptake by different signaling mechanisms, isolated muscles were incubated in
the presence or absence of the phosphatidylinositol 3-kinase (PI3K) inhibitor,
wortmannin (100 nmol/l), before stimulation and throughout the remainder of
the study (Fig. 3). As we
(18,19)
and others
(29,30)
have previously observed, wortmannin completely inhibited insulin-stimulated
glucose uptake and had no effect on contraction-stimulated uptake. In
contrast, wortmannin only partially (35%) decreased SNP-stimulated
glucose uptake. These findings suggest that at least part of the mechanism by
which SNP and insulin stimulate glucose uptake is distinct.
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To test this hypothesis further, we used the guanylate cyclase inhibitor
LY-83583 that inhibits SNP-stimulated 2-deoxyglucose uptake in isolated
muscles, as previously reported
(7). Consequently, 10 µmol/l
LY-83583 blocked SNP-stimulated 2-deoxyglucose uptake in the isolated EDL
muscles, whereas the inhibitor did not block contraction-stimulated
2-deoxyglucose uptake (data not shown). However, we found that LY-83583 had
nonspecific effects on skeletal muscle glucose uptake. In the presence of a
low dose of the inhibitor (10 µmol/l), basal 2-deoxyglucose uptake
increased twofold compared with the vehicle. In contrast, higher doses of the
inhibitor (50-250 µmol/l) induced muscle stiffness and
dose-dependently decreased basal rates of 2-deoxyglucose uptake. LY-83483 at
10 µmol/l had no effect on the development of tension during contraction,
whereas higher concentrations altered resting tension and significantly
impaired the ability to develop tension during contraction (data not shown).
Therefore, we do not believe that LY-83583 can be used to determine the role
of NO in the regulation of contraction-stimulated glucose uptake in isolated
muscles.
Effect of SNP on ATP, creatine phosphate, and glycogen concentrations
and 1 and
2 AMPK activities in isolated EDL muscles. One
report suggests that high concentrations of SNP decrease ATP and creatine
phosphate concentrations in EDL muscles
(6). We hypothesized that under
these conditions, SNP may also increase AMPK activity, which is activated in
response to cellular fuel depletion. SNP treatment of the muscles at
concentrations ranging from 1-10 mmol/l did not alter ATP, creatine phosphate,
or glycogen concentrations (Table
1). In contrast to the lack of effect of SNP on these muscle
metabolities, SNP significantly increased
1 AMPK activity.
Interestingly, SNP had no effect on
2 AMPK activity, whereas the
contraction-stimulated increase in both
1 and
2 AMPK activity
was significantly greater than that observed with SNP
(Fig. 4). These data suggest
distinct regulatory mechanisms leading to an increase in isoform-specific AMPK
activity in rat skeletal muscle.
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Effect of L-NMMA on 2-deoxyglucose uptake. To determine whether the NOS inhibitor L-NMMA affects basal- and contraction-stimulated glucose uptake, we first used isolated soleus and EDL muscles that were preincubated and contracted in KRBB in the absence or presence of 0.1 mmol/l L-NMMA. Contraction increased 2-deoxyglucose uptake 2.9-fold above basal in the soleus muscles and 3.8-fold above basal in the EDL muscles. L-NMMA treatment in vitro had no effect on basal or contractionstimulated 2-deoxyglucose uptake in either the soleus or EDL muscles (Fig. 5).
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Then, we contracted (or sham treated) EDL muscles in situ via sciatic nerve stimulation, removed the muscles, and incubated them in KRBB in the absence or presence of 0.1 mmol/l L-NMMA. As shown in Fig. 6, and similar to the results obtained in vitro, L-NMMA had no effect on basal- or contraction-stimulated 2-deoxyglucose uptake in the EDL muscles.
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Effect of treadmill running on 2-deoxyglucose uptake in L-NAMEtreated animals. The administration of L-NAME via the drinking water for 2 days resulted in a significant increase in systolic blood pressure from 104 ± 2 mmHg before L-NAME treatment to 132 ± 3 mmHg after 2 days of treatment (P < 0.001). L-NAME treatment had no effect on exercise tolerance, as all animals were able to complete the 60-min exercise task. Isolated soleus muscles from L-NAMEtreated rats had lower basal 2-deoxyglucose uptake compared with untreated animals (Fig. 7). In contrast, 2 days of L-NAME treatment did not affect exercisestimulated 2-deoxyglucose uptake in the soleus muscles.
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DISCUSSION |
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The administration of L-NMMA in vivo results in the development of marked insulin resistance (9,12,13) and hyperglycemia (13), suggesting an important role for NO in muscle glucose metabolism. Interestingly, these effects of NOS inhibition on insulin-mediated glucose uptake in vivo are not observed when isolated skeletal muscles are incubated with NOS inhibitors and insulin in vitro (6,8,9,35). These findings, combined with our results showing additive effects of SNP plus insulin and the partial inhibition using wortmannin, imply that NO augments insulin's effects systemically, most likely not at the level of skeletal muscle fibers.
Previous studies assessing the role of NO in exercise/contractionstimulated glucose uptake have resulted in contrasting conclusions (5,6,8). In one report, hindlimb muscles were contracted in situ via electrical stimulation of the sciatic nerve, and the EDL muscles were isolated and used for measurement of glucose transport in the presence or absence of the NOS inhibitor L-NMMA (6). Under these conditions, the NOS inhibitor was shown to fully block contraction-stimulated glucose transport. In addition, another report showed that blocking NOS by adding L-NAME to the drinking water of rats prevented GLUT4 translocation to skeletal muscle sarcolemma and glucose transport in response to acute exercise (5). In contrast, when epitrochlearis muscles were contracted in vitro in the absence or presence of L-NMMA and glucose transport was measured, NOS inhibition had no effect on glucose transport (8). In the current study, three independent experiments were performed to assess this problem: 1) hindlimb muscles were contracted in vivo via electrical stimulation of the sciatic nerve, and then isolated EDL muscles were used for measurement of glucose uptake in the presence or absence of L-NMMA (similar to the methods of Balon and Nadler [6]); 2) isolated incubated EDL muscles were used for measurement of contraction-stimulated glucose uptake in the presence or absence of L-NMMA (similar to the methods of Etgen et al. [8]); and 3) L-NAMEtreated rats performed running exercise for 1 h, and isolated soleus muscles were used for measurement of glucose uptake. For all three experiments, the NOS inhibitors failed to affect exercise or contraction-stimulated 2-deoxyglucose uptake. Furthermore, the combination of contraction and SNP had additive effects on glucose uptake. Therefore, our data are consistent with the conclusion that NO is not involved in the signaling pathway leading to contraction-stimulated glucose uptake in skeletal muscle.
We
(18,19)
and others
(20,21)
have hypothesized that AMPK is an essential intermediary in the signaling
cascade leading to contraction-stimulated glucose transport in skeletal
muscle. AMPK activity is increased when cells sense low fuel, acting as a
"fuel gauge" and functioning to increase ATP generation under
conditions of increased energy expenditure
(19,36).
Because one report suggested that incubation of skeletal muscle with a high
concentration of SNP induces a significant decrease in ATP and creatine
phosphate concentrations (6),
we hypothesized that SNP would increase AMPK activity in skeletal muscle and
that this might be part of the mechanism leading to the activation of glucose
uptake. The 2 catalytic subunit of AMPK is highly expressed in skeletal
muscle
(23,24),
and in contrast to electrical stimulation, which activates both
1 and
2 isoforms, only the
2 isoform has been reported to be increased
by physical exercise in rats and humans
(37,38).
Surprisingly, a wide range of SNP concentrations did not significantly alter
ATP, creatine phosphate, or glycogen concentrations in the incubated muscles,
but these results were consistent with the lack of activation of the
2
catalytic subunit of AMPK. The lack of
2 AMPK activation with SNP is
also consistent with distinct signaling mechanisms leading to contraction- and
NO-stimulated glucose uptake.
Despite the lack of 2 activation and the lack of changes in ATP and
creatine phosphate with SNP treatment,
1 AMPK activity was
significantly increased in the incubated EDL muscles. These observations
suggest that there are distinct mechanisms for the regulation of
1 and
2 AMPK activity in skeletal muscle. Furthermore, these observations
demonstrate that these two catalytic isoforms may have different sensitivities
to ATP and creatine phosphate in the intact muscle. In future studies, it will
be interesting to determine if cytokines activate
1 AMPK in skeletal
muscle, because cytokines are thought to modulate muscle glucose transport by
increasing NO production
(39).
In summary, our NOS-inhibitor data suggest that NO is not involved in the
signaling pathway leading to contraction-stimulated glucose uptake in skeletal
muscle and that SNP increases skeletal muscle glucose uptake through a
mechanism that is distinct from the insulin- and contraction-signaling
pathways. These observations suggest that there is a third signaling pathway
that enhances glucose uptake in skeletal muscle. Furthermore, our data
demonstrate that NO-stimulated glucose uptake is associated with an activation
of the 1 catalytic subunit of AMPK.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication February 4, 2000 and accepted in revised form October 24, 2000
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REFERENCES |
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