Isoform-Specific Regulation of 5' AMP-Activated Protein Kinase in Skeletal Muscle From Obese Zucker (fa/fa) Rats in Response to Contraction

Brian R. Barnes1, Jeffrey W. Ryder1, Tatiana L. Steiler1, Lee G.D. Fryer2, David Carling2, and Juleen R. Zierath1

1 Department of Clinical Physiology and Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden
2 MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London, U.K.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glucose transport can be activated in skeletal muscle in response to insulin via activation of phosphoinositide (PI) 3-kinase and in response to contractions or hypoxia, presumably via activation of 5' AMP-activated protein kinase (AMPK). We determined the effects of insulin and muscle contraction/hypoxia on PI 3-kinase, AMPK, and glucose transport activity in epitrochlearis skeletal muscle from insulin-resistant Zucker (fa/ fa) rats. Insulin-stimulated glucose transport in isolated skeletal muscle was reduced 47% in obese versus lean rats, with a parallel 42% reduction in tyrosine-associated PI 3-kinase activity. Contraction and hypoxia elicited normal responses for glucose transport in skeletal muscle from insulin-resistant obese rats. Isoform-specific AMPK activity was measured in skeletal muscle in response to insulin, contraction, or hypoxia. Contraction increased AMPK{alpha}1 activity 2.3-fold in lean rats, whereas no effect was noted in obese rats. Hypoxia increased AMPK{alpha}1 activity to a similar extent (more than sixfold) in lean and obese rats. Regardless of genotype, contraction, and hypoxia, each increased AMPK{alpha}2 activity more than fivefold, whereas insulin did not alter either AMPK{alpha}1 or -{alpha}2 activity in skeletal muscle. In conclusion, obesity-related insulin resistance is associated with an isoform-specific impairment in AMPK{alpha}1 in response to contraction. However, this impairment does not appear to affect contraction-stimulated glucose transport. Activation of AMPK{alpha}2 in response to muscle contraction/ exercise is associated with a parallel and normal increase in glucose transport in insulin-resistant skeletal muscle.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During recent years, major advances have been made in the understanding of the molecular mechanisms of insulin action (1), bringing the goal of identifying defects in peripheral tissues that lead to insulin resistance closer. In people with type 2 diabetes, impaired whole-body insulin-mediated glucose uptake is associated with defects in glucose transport in skeletal muscle (26). This most likely occurs from defects in postreceptor insulin signal transduction at the level of insulin receptor substrate-1 and phosphoinositide (PI) 3-kinase (69) as well as defects in trafficking and/or function of GLUT4 (10,11), the major insulin-regulated GLUT isoform expressed in skeletal muscle (12). In skeletal muscle from insulin-resistant diabetic rodents, glucose transport defects are not observed in response to contraction, a stimulus known to increase glucose transport by an insulin-independent mechanism (1315). This implies that a greater understanding of mechanisms involved in the regulation of insulin-independent glucose transport may lead to the identification of molecular targets for pharmacological therapy to improve glucose homeostasis. Activation of this insulin-independent pathway may circumvent defects in insulin action on glucose transport that are observed in skeletal muscle from people with type 2 diabetes.

AMP-activated protein kinase (AMPK) has emerged as one potential therapeutic molecular target that plays a role in mediating insulin-independent glucose transport (1618). Much of the evidence supporting the role of AMPK in the regulation of glucose transport comes from studies using 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR), a cell-permeable activator of AMPK. Treatment of rat skeletal muscle with AICAR is associated with parallel increases in AMPK activity and glucose transport via a PI 3-kinase-independent pathway (19,20). Consistent with this, activation of AMPK in response to multiple stress stimuli, including hypoxia, osmotic shock, and mitochondrial uncoupling, is correlated with a parallel increase in glucose transport activity in skeletal muscle (21). Direct evidence for a role of AMPK in the regulation of glucose transport in skeletal muscle has emerged from studies in transgenic mice (17). Transgenic overexpression of a dominant-inhibitory mutant of AMPK in skeletal muscle completely blocked the ability of hypoxia or AICAR to activate glucose uptake while only partially reducing contraction-stimulated glucose uptake (17). Furthermore, adenoviral-mediated expression of a dominant-negative form of the catalytic ({alpha}) subunit of AMPK in a skeletal muscle cell line blocked the stimulation of glucose transport by both AICAR and hyperosmotic stress, but was without effect on either insulin- or phorbol-ester-stimulated transport (18). Thus, AMPK-dependent and -independent pathways contribute to the regulation of glucose uptake in skeletal muscle.

Deficiencies in the AMPK signaling pathway have been proposed to result in insulin insensitivity and type 2 diabetes (22) and obesity (23). Activation of AMPK by AICAR may be efficacious in the regulation of glucose transport in skeletal muscle from obese insulin-resistant Zucker rats or ob/ob mice (24,25). To fully validate AMPK as a target for pharmacological intervention to improve glucose homeostasis in obese insulin-resistant or type 2 diabetic subjects, additional studies are warranted. For example, isoform-specific responses of AMPK to more common metabolic stressors have not been fully established in skeletal muscle from animal models of type 2 diabetes. Furthermore, because contraction and hypoxia recruit AMPK-dependent and -independent pathways in the regulation of glucose transport (17), it is not clear whether either of these stimuli elicit a normal response toward AMPK in insulin-resistant models, where it would be advantageous to increase glucose uptake by insulin-independent mechanisms. Thus, the aim of this study was to examine the effects of contraction or hypoxia on isoform-specific activation of AMPK in skeletal muscle from Zucker (fa/fa) rats, an obese animal model of insulin resistance.


    RESEARCH DESIGN AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Obese male Zucker (fa/fa) rats (aged 11–12 weeks) and lean littermates were purchased from Charles River (Uppsala, Sweden) and housed at the animal facility at the Karolinska Institute for 4 weeks before use. Rats were maintained on a 12-h light-dark cycle and given free access to standard rodent chow and water. All studies we performed on rats in the overnight-fasted state. The regional animal ethical committee approved all experimental procedures.

Glucose tolerance test.
Glucose (2 g/kg body wt) was administered by intraperitoneal injection. Blood samples were obtained via the tail vein before and 15, 30, 60, and 120 min after glucose injection. Blood glucose levels were measured using a One Touch Basic glucose meter (Lifescan, Milpitas, CA).

Muscle incubations.
All incubation media were prepared from a stock solution of Krebs-Henseleit bicarbonate buffer (KHBB) supplemented with 5 mmol/l HEPES and 0.1% BSA (radioimmunoassay grade). Rats were anesthetized with sodium pentobarbital (60 mg/kg body wt), and epitrochlearis muscles were carefully isolated for in vitro incubation. The viability of the incubated muscle preparation from large rats has been previously established by measuring high-energy phosphate levels of epitrochlearis muscle from adult rats that are of similar weight to the animals used in our study (26). Muscles were preincubated at 30°C for 40 min in KHBB containing 5 mmol/l glucose and 15 mmol/l mannitol, without (basal) or with insulin (60 nmol/l) for either 6 min (PI 3-kinase activity) or 40 min (glucose transport activity). Unless otherwise stated, all media were continuously gassed with 95% O2/5% CO2. After preincubation, muscles were immediately frozen between aluminum tongs cooled to the temperature of liquid nitrogen or further incubated to assess glucose transport activity.

In vitro muscle contraction.
Epitrochlearis muscles were placed in a temperature-controlled (30°C) incubation chamber and immersed in 4 ml preincubation media without or with insulin as described above. Muscles were positioned between two platinum electrodes. The distal tendon was mounted to the bottom of the incubation chamber. The proximal tendon was connected to an isometric force transducer (Harvard Apparatus, South Natick, MA), and resting tension was adjusted to 0.5 g. Electrical stimulation (contraction group only) was applied during the final 10 min of the 40-min preincubation period. Basal and insulin-stimulated muscles were treated as described above minus the application of electrical stimulation. Muscle contraction was induced via electrical stimulation. Muscles were stimulated at 100 Hz (0.2-ms pulse duration, 20 V), at a rate of one 0.2-s contraction every 2 s for 10 min, as previously described (27). Muscles were frozen immediately for AMPK or PI 3-kinase activity measurements or further incubated for the assessment of glucose transport activity.

Hypoxia incubations.
Epitrochlearis muscles were preincubated at 30°C for 80 min in KHBB containing 5 mmol/l glucose and 15 mmol/l mannitol, under a gas phase of 95% O2/5% CO2 (basal) or 95% N2/5% CO2 (hypoxia) (28). Muscles were frozen immediately for AMPK activity or further incubated for the assessment of glucose transport activity.

Glucose transport activity.
After preincubation, muscles were incubated at 30°C for 10 min in glucose-free KHBB containing 20 mmol/l mannitol. When effects of hypoxia were studied, muscles were incubated for 15 min in KHBB containing 2 mmol/l pyruvate and 18 mmol/l mannitol under a gas phase of 95% O2/5% CO2. Thereafter, muscles were transferred to vials containing 1 mmol/l 2-deoxy-[1,2,3H]glucose (2.5 µCi/ml) and 19 mmol/l [14C]mannitol (0.7 µCi/ml) without or with insulin and incubated for 15 min. The rate of 2-deoxyglucose uptake is linear between 5 and 120 min in rat epitrochlearis muscle (29). Glucose transport activity is expressed as micromoles per milliliter of intracellular water per hour (29).

AMPK activity.
Muscles were homogenized in ice-cold lysis buffer containing 50 mmol/l Tris-HCl (pH 7.5), 1 mmol/l EDTA, 1 mmol/l diothiothreitol (DTT), 10% (vol/vol) glycerol, 50 mmol/l NaF, 5 mmol/l Na pyrophosphate, 1 mmol/l benzamidine, 0.1 mmol/l phenylmethylsulfonyl fluoride (PMSF), and 1% (vol/vol) Triton X-100. Muscle homogenates were subjected to centrifugation at 14,000g for 10 min at 4°C. Supernatants were removed and used for determination of protein content using a commercially available kit based on the Bradford method (Bio-Rad, Hercules, CA). Aliquots (400 µg protein) were sequentially immunoprecipitated for 2 h at 4°C with sheep AMPK{alpha}1 and -{alpha}2 antibodies precoupled to protein G-Agarose (5 mg/sample). The antibodies were raised against peptides predicted from the rat AMPK{alpha}1 and -{alpha}2 amino acid sequences, as previously described (30). Immunoprecipitates were washed three times with lysis buffer and twice with 50 mmol/l HEPES (pH 7.5), 10% (vol/vol) glycerol, 1 mmol/l EDTA, and 1 mmol/l DTT. AMPK activity in the immune complex was determined by in vitro phosphorylation of the SAMS (full sequence: HMRSAMSGLHLVKRR) synthetic peptide substrate (30), as previously described (18). Kinase reactions performed in reaction buffer (40 mmol/l HEPES buffer, pH 7.0, 0.2 mmol/l SAMS peptide, 0.2 mmol/l AMP, 80 mmol/l NaCl2, 0.8 mmol/l DTT, 5 mmol/l MgCl2, and 0.2 mmol/l ATP (containing 2 µCi [{gamma}-32P]ATP). Reactions were incubated on a vibrating platform for 60 min at 30°C and terminated by centrifugation (9,000g for 30 s). Immediately thereafter, aliquots were spotted onto phosphocellulose units (Pierce, Rockford, IL). Phosphocellulose units were washed with 1% phosphoric acid according to the manufacturer specifications. Incorporation of [32P] into peptide substrate was measured by liquid scintillation counting.

PI 3-kinase activity.
Epitrochlearis skeletal muscle was homogenized in ice-cold lysis buffer (20 mmol/l Tris, pH 8.0, 137 mmol/l NaCl, 2.7 mmol/l KCl, 1 mmol/l MgCl2, 0.5 mmol/l Na3VO4, 1% Triton X-100, 10% vol/vol glycerol, 10 µg/ml leupeptin, 0.2 mmol/l PMSF, 10 mmol/l NaF, and 10 µg/ml aprotinin). Homogenates were subjected to centrifugation at 8,000g for 10 min at 4°C, and protein content was determined as described above. Supernatants (800 µg) were subjected to immunoprecipitation with a polyclonal anti-phosphotyrosine antibody (Signal Transduction Laboratories, Lexington, KY) coupled to protein A-Sepharose. PI 3-kinase activity was assayed for 20 min at room temperature as described (31). Reaction products were separated by thin-layer chromatography, and quantification of [ 32P] incorporation into PI was determined using a phosphoimager (Bio-Rad Laboratories, Hercules, CA).

Statistical analyses.
Differences between two groups were determined by unpaired t test. Differences between more than two groups were determined by one-way ANOVA followed by the Fisher’s least significant difference post hoc analysis. Significance was accepted at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animal characteristics.
The body weight of 11-week-old obese Zucker rats was significantly greater than that of age-matched lean rats (393 ± 7 vs. 267 ± 3 g; P < 0.05). Epitrochlearis muscle weights were reduced in obese Zucker rats (42.2 ± 1.6 vs. 59.4 ± 2.1 mg, obese versus lean rats; P < 0.05). Obese Zucker rats displayed marked glucose intolerance, with blood glucose levels significantly elevated versus lean rats at all time points measured (P < 0.05) (Fig. 1).



View larger version (13K):
[in this window]
[in a new window]
 
FIG. 1. Glucose tolerance in lean or obese Zucker rats after an intraperitoneal injection of glucose. Blood glucose was measured at 0, 15, 30, 60, and 120 min. Results are means ± SE for lean ({circ}; n = 16) and obese (•; n = 17) rats. P < 0.05 vs. lean rats at all time points.

 
Skeletal muscle glucose uptake.
Skeletal muscle glucose uptake was determined in response to insulin, contraction, or hypoxia. Insulin increased 2-deoxyglucose transport 3.4-fold in isolated skeletal muscle from lean rats (P < 0.05) (Fig. 2). Obese rats exhibited marked skeletal muscle insulin resistance. The absolute rate of insulin-stimulated glucose transport in obese rats was reduced 47% compared with lean controls (1.27 ± 0.08 vs. 0.68 ± 0.06 µmol · ml-1 · h-1; P < 0.05). Rates of contraction- and hypoxia-stimulated glucose transport in skeletal muscle were similar between lean and obese rats (Fig. 2). In lean rats, contraction- and hypoxia-stimulated glucose transport was increased 3.1-fold (P < 0.05) and 3.4-fold (P < 0.05), respectively. In insulin-resistant obese Zucker rats, contraction- and hypoxia-stimulated glucose transport was increased 3.9-fold (P < 0.05) and 4.1-fold (P < 0.05), respectively.



View larger version (16K):
[in this window]
[in a new window]
 
FIG. 2. 2-Deoxyglucose transport activity in epitrochlearis muscle from lean ({square}) and obese ({blacksquare}) Zucker rats incubated in the absence (basal) or presence of insulin, after exposure to in vitro muscle contraction, or after exposure to hypoxia. Values are means ± SE for 6–11 muscles per group. *P < 0.05 vs. basal; {dagger}P < 0.05 vs. lean rats.

 
PI 3-kinase activity.
Phosphotyrosine-associated PI 3-kinase activity was determined in epitrochlearis skeletal muscle from lean and obese rats, in response to insulin or contraction. Insulin increased phosphotyrosine-associated PI 3-kinase activity 2.7-fold in lean rats (P < 0.05) (Fig. 3), with a 42% reduction noted in skeletal muscle from obese rats (P < 0.05 vs. lean). PI 3-kinase activity was not increased after muscle contraction.



View larger version (11K):
[in this window]
[in a new window]
 
FIG. 3. PI 3-kinase activity in epitrochlearis muscles from lean ({square}) or obese ({blacksquare}) Zucker rats incubated in the absence (basal) or presence of insulin or after in vitro muscle contraction. Results are expressed as means ± SE (arbitrary phosphoimager units for five to six muscles per group). *P < 0.05 vs. basal; {dagger} P < 0.05 vs. lean rats.

 
Isoform-specific AMPK activity.
Basal AMPK{alpha}1 (Fig. 4A) and AMPK{alpha}2 (Fig. 4B) activities were similar in skeletal muscle between lean and obese rats during experiments involving insulin and muscle contraction. Regardless of genetic background, insulin was without effect on AMPK{alpha}1 or -{alpha}2 activity. Contraction elicited an increase in activation of both the {alpha}1 and {alpha}2 isoforms in skeletal muscle. In lean rats, AMPK{alpha}1 and -{alpha}2 activity was increased 2.3- and 4.8-fold, respectively (P < 0.05). Interestingly, in skeletal muscle from obese rats, an isoform-specific effect of contraction on AMPK was noted: AMPK{alpha}1 activity was not altered (P = 0.10), whereas AMPK{alpha}2 activity was increased 4.3-fold (P < 0.05). Furthermore, when comparing responses between both genotypes, the contraction-mediated response of AMPK{alpha}1 tended to be reduced in obese rats (P < 0.08). In contrast, contraction-mediated AMPK{alpha}2 activity was comparable between lean and obese rats. In response to hypoxia, AMPK{alpha}1 (Fig. 5A) and -{alpha}2 (Fig. 5B) activity in lean rats was increased 6.6-fold (P < 0.05) and 5.5-fold (P < 0.05), respectively. In obese rats, hypoxia-stimulated AMPK was preserved, with increases in AMPK{alpha}1 and -{alpha}2 activity 8.4-fold (P < 0.05) and 6.5-fold (P < 0.05) over basal values, respectively. Basal AMPK{alpha}1 and -{alpha}2 activity in obese rats was slightly reduced (P < 0.05).



View larger version (20K):
[in this window]
[in a new window]
 
FIG. 4. Insulin- and contraction-stimulated AMPK activity in epitrochlearis muscles from lean ({square}) or obese ({blacksquare}) Zucker rats incubated in the absence (basal) or presence of insulin or after in vitro muscle contraction. AMPK{alpha}1 (A) and AMPK{alpha}2 (B) activity is expressed as means ± SE (cpm · mg protein- 1 · min-1 for six muscles per group). * P < 0.05 vs. basal.

 


View larger version (16K):
[in this window]
[in a new window]
 
FIG. 5. Hypoxia-stimulated AMPK activity in epitrochlearis muscles from lean ({square}) or obese ({blacksquare}) Zucker rats. Muscles were incubated under basal (95% O2/5% CO2) or hypoxic (95% N2/5% CO2) conditions. AMPK{alpha}1 (A) and AMPK{alpha}2 (B) activity is expressed as means ± SE (cpm · mg protein- 1 · min-1 for five to six muscles per group. *P < 0.05 vs. basal; {dagger}P < 0.05 vs. lean rats.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In skeletal muscle, separate and distinct signaling pathways can activate glucose transport. Insulin increases glucose transport via activation of PI 3-kinase (13,14) and possibly via activation of the CAP/TC10 pathway (32,33). However, the latter pathway has not been validated in skeletal muscle. Exercise (34), muscle contraction (35,36), and hypoxia (28,37) increase glucose transport via insulin-independent pathways (1315), presumably via activation of AMPK (17,18). In type 2 diabetic patients, defects in insulin-mediated whole-body glucose uptake are coupled to impairments in glucose transport in skeletal muscle (4,10), which arise from aberrant signal transduction at the level of insulin receptor substrate-1, PI 3-kinase (6,8,9), and GLUT4 translocation (10,38). Since muscle contraction and hypoxia increase glucose transport via an alternative mechanism that bypasses defective insulin signaling (37,39,40), strategies to identify and characterize components of this insulin-independent pathway will potentially reveal novel entry points that can be targeted to increase glucose uptake in insulin-resistant muscle. Recent interest has focused on AMPK, as it is the most distal signaling molecule identified in the insulin-independent regulation of glucose transport. AMPK mediates contraction- and hypoxia-regulated glucose transport in skeletal muscle (17) and has been proposed as a promising target for treatment of altered glucose homeostasis in type 2 diabetes (41). Contraction-stimulated glucose transport in skeletal muscle is normal in severely insulin-resistant obese Zucker rats (39,40); however, effects of contraction and hypoxia on isoform-specific activation of AMPK in skeletal muscle from this animal model have not been determined.

Consistent with earlier studies (39,40), insulin resistance at the level of PI 3-kinase and glucose transport was observed in skeletal muscle from obese Zucker rats, whereas stimulation of AMPK{alpha}2 activity and glucose transport in response to muscle contraction or hypoxia was normal. These findings are also consistent with observations in humans, whereby moderate aerobic exercise elicits an appropriate increase in AMPK{alpha}2 in skeletal muscle from type 2 diabetic subjects (42). Thus, contraction and hypoxia increase AMPK{alpha}2 activity and glucose transport in insulin-resistant skeletal muscle. These studies provide evidence to suggest that exercise- and hypoxia-induced AMPK activity is not impaired in insulin-resistant skeletal muscle. Furthermore, they support the recent observation in cultured skeletal muscle, indicating direct activation of AMPK alone is sufficient for stimulation of glucose uptake via an increase in cell surface expression of GLUTs (18).

An isoform-specific reduction in AMPK{alpha}1 activity in skeletal muscle from obese Zucker rats was observed in response to contraction, but not hypoxia. Contraction increased AMPK{alpha}1 activity in epitrochlearis muscle from lean rats, whereas no effect was observed in obese rats. In contrast, hypoxia-stimulated AMPK{alpha}1 activity was similar between lean and obese rats; however, basal AMPK{alpha}1 activity was slightly reduced. Interestingly, basal AMPK{alpha}1, although not significant, tended to be lower in skeletal muscle from type 2 diabetic subjects (42). A previous study provides evidence that total AMPK activity in calf muscles in response to AICAR infusion was similar between lean and obese Zucker rats (24). However, since total rather than isoform-specific AMPK activity was measured (24), subtle defects in AMPK{alpha}1 activity may have been masked.

An explanation for the differential response of AMPK{alpha}1 in response to contraction versus hypoxia in the present study may be related to the amount of cellular stress applied to the muscle. The hypoxia stimulus is likely to have elicited a greater degree of stress on the muscle (21). Thus, the AMPK{alpha}1 defect may represent reduced sensitivity of kinase to cellular stress. Physiological evidence for this comes from human studies in which AMPK{alpha}1 activity is increased in response to anaerobic (sprint) (43), but not aerobic (endurance) (44,45), cycle ergometry. The contraction protocol used in the present study is more closely related to anaerobic (high-intensity) rather than aerobic (low-intensity) exercise. An alternative possibility is that the lack of a contraction effect on AMPK{alpha}1 in obese muscle may be due to the fact that the contraction protocol was not intense enough, although it did stimulate glucose transport to the same extent. Despite the isoform-specific defect in AMPK{alpha}1 activity in response to contraction, normal glucose transport was achieved in skeletal muscle from insulin-resistant Zucker rats.

Hypoxia increases AMPK activity to a greater degree than in vitro muscle contraction (21). Since only subtle defects in contraction-induced AMPK{alpha}1 activity were observed in obese rats in the present investigation, the observed defect is not likely to have a major impact on the rate of glucose transport in response to muscle contraction. Importantly, full activation of AMPK may not be necessary for complete activation of exercise/contraction-stimulated glucose transport. Transgenic overexpression of a dominant inhibitory mutant of AMPK in skeletal muscle completely blocks the ability of hypoxia or AICAR to activate glucose uptake while only partially (30–40%) reducing contraction-stimulated glucose uptake (17). Although these results (17) confirm a role of AMPK in contraction-induced glucose transport, they also demonstrate that AMPK is only partially responsible for this effect. Thus, AMPK-dependent and -independent pathways contribute to the regulation of glucose uptake in skeletal muscle in response to exercise (17). Consistent with this hypothesis, glucose transport in slow-twitch muscle can be markedly activated in response to contraction, without measurable changes in AMPK activity (46). Collectively, these studies illustrate the complexity in identifying the precise role of AMPK signaling in the regulation of metabolic events and they strongly suggest that additional factors contribute to the regulation of exercise-mediated glucose uptake.

In summary, obesity-related insulin resistance in skeletal muscle is associated with reduced contraction-stimulated AMPK{alpha}1 activity, whereas AMPK{alpha}2 activity is normal. However, when muscles were challenged by cellular hypoxia, activation of both AMPK{alpha}1 and -{alpha}2 were normal. In skeletal muscle from lean animals, hypoxia was more effective than contraction in stimulating AMPK{alpha}1 activity. Thus, the impairment in AMPK{alpha}1 in insulin-resistant muscle may be related to a reduced sensitivity of the enzyme in response to cellular stress. Importantly, contraction-mediated glucose transport is normal, despite impaired AMPK{alpha}1 activity in muscles from obese Zucker rats. Thus, AMPK{alpha}1 does not appear to play a major role in stimulation of glucose transport by muscle contraction. Our results are consistent with the hypothesis that activation of AMPK{alpha}2 in response to muscle contraction/exercise is associated with a parallel and normal increase in glucose transport in insulin-resistant skeletal muscle.


    ACKNOWLEDGMENTS
 
This study was supported by grants from the Swedish Medical Research Council, the Swedish Diabetes Association, the Foundation for Scientific Studies of Diabetology, the Swedish National Center for Research in Sports, Diabetes U.K., the Medical Research Council U.K., and the following research foundations: Karolinska Institutet, Novo-Nordisk, Thurings, Wiberg, Tore Nilson, and the AFA-Insurance Jubilee Foundation for Research in National Diseases.


    FOOTNOTES
 
Address correspondence and reprint requests to Juleen R. Zierath, Department of Clinical Physiology, Section for Integrative Physiology, Karolinska Institute, Von Eulers väg 4 II, SE-171 77 Stockholm, Sweden. E-mail: juleen.zierath{at}fyfa.ki.se.

Received for publication 9 April 2002 and accepted in revised form 5 June 2002.

B.R.B. and J.W.R. contributed equally to this study.

J.R.Z. is employed by Biovitrum.

AICAR, 5-aminoimidazole-4-carboxyamide ribonucleoside; AMPK, AMP-activated protein kinase; DTT, diothiothreitol; KHBB, Krebs-Henseleit bicarbonate buffer; PI, phosphoinositide; PMSF, phenylmethylsulfonyl fluoride.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Virkamäki A, Ueki K, Kahn CR: Protein-protein interactions in insulin signaling and the molecular mechanisms of insulin resistance. J Clin Invest103 :931 –943,1999[Free Full Text]
  2. Dohm GL, Tapscott EB, Pories WJ, Dabbs DJ, Flickinger EG, Meelheim D, Fushiki T, Atkinson SM, Elton CW, Caro JF: An in vitro human skeletal muscle preparation suitable for metabolic studies: decreased insulin stimulation of glucose transport in muscle from morbidly obese and diabetic subjects. J Clin Invest82 :486 –494,1988[Medline]
  3. Andréasson K, Galuska D, Thörne A, Sonnenfeld T, Wallberg-Henriksson H: Decreased insulin-stimulated 3-O-methylglucose transport in in vitro incubated muscle strips from type II diabetic subjects. Acta Physiol Scand142 :255 –260,1991[Medline]
  4. Galuska D, Nolte L, Zierath JR, Wallberg-Henriksson H: Effect of metformin on glucose transport in isolated skeletal muscle obtained from type II diabetic patients and healthy individuals. Diabetologia37 :872 –879,1994
  5. Bonadonna RC, Del Prato S, Bonora E, Saccomani MP, Gulli G, Natali A, Frascerra S, Pecori N, Ferrannini E, Bier D, Cobelli C, DeFronzo RA: Roles of glucose transport and glucose phosphorylation in muscle insulin resistance of NIDDM. Diabetes45 :915 –925,1996[Abstract]
  6. Krook A, Björnholm M, Galuska D, Jiang X-J, Fahlman R, Myers Jr MG, Wallberg-Henriksson H, Zierath JR: Characterization of signal transduction and glucose transport in skeletal muscle from type 2 diabetic patients. Diabetes49 :284 –292,2000[Abstract]
  7. Goodyear LJ, Giorgino F, Sherman LA, Carey J, Smith RJ, Dohm GL: Insulin receptor phosphorylation, insulin receptor substrate-1 phosphorylation and phosphatidylinositol 3-kinase activity are decreased in intact skeletal muscle strips from obese subjects. J Clin Invest95 :2195 –2204,1995[Medline]
  8. Björnholm M, Kawano Y, Lehtihet M, Zierath JR: Insulin receptor substrate-1 phosphorylation and phosphatidylinositol 3-kinase activity are decreased in skeletal muscle from NIDDM subjects following in vivo insulin stimulation. Diabetes46 :524 –527,1997[Abstract]
  9. Kim YB, Nikoulina SE, Ciaraldi TP, Henry RR, Kahn BB: Normal insulin-dependent activation of Akt/protein kinase B, with diminished activation of phosphoinositide 3-kinase, in muscle in type 2 diabetes. J Clin Invest104 :733 –741,1999[Abstract/Free Full Text]
  10. Zierath JR, He L, Guma A, Wahlström E, Klip A, Wallberg-Henriksson H: Insulin action on glucose transport and plasma membrane GLUT4 content in skeletal muscle from patients with NIDDM. Diabetologia39 :1180 –1189,1996[Medline]
  11. Ryder JW, Yang J, Galuska D, Rincón J, Björnholm M, Krook A, Lund S, Pedersen O, Wallberg-Henriksson H, Zierath JR, Holman GD: Use of a novel impermeable biotinylated photolabeling reagent to assess insulin- and hypoxia-stimulated cell surface GLUT4 content in skeletal muscle from type 2 diabetic patients. Diabetes647 –654,2000
  12. Ryder JW, Kawano Y, Chibalin AV, Rincon J, Tsao T-S, Stenbit AE, Combatsiaris T, Yang J, Holman GD, Zierath JR: In vitro analysis of the glucose-transport system in GLUT4-null skeletal muscle. Biochem J342 :321 –328,1999[Medline]
  13. Yeh J-I, Gulve EA, Rameh L, Birnbaum MJ: The effects of wortmannin on rat skeletal muscle. J Biol Chem270 :2107 –2111,1995[Abstract/Free Full Text]
  14. Lund S, Holman GD, Schmitz O, Pedersen O: Contraction stimulates translocation of glucose transporter GLUT4 in skeletal muscle through a mechanism distinct from that of insulin. Proc Natl Acad Sci U S A92 :5817 –5821,1995[Abstract/Free Full Text]
  15. Lee AD, Hansen PA, Holloszy JO: Wortmannin inhibits insulin-stimulated but not contraction-stimulated glucose transport activity in skeletal muscle. FEBS Lett361 :51 –54,1995[Medline]
  16. Winder WW, Holmes BF, Rubink DS, Jensen EB, Chen M, Holloszy JO: Activation of AMP-activated protein kinase increases mitochondrial enzymes in skeletal muscle. J Appl Physiol88 :2219 –2226,2000[Abstract/Free Full Text]
  17. Mu J, Brozinick JTJ, Valladares O, Bucan M, Birnbaum MJ: A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skeletal muscle. Mol Cell7 :1085 –1094,2001[Medline]
  18. Fryer LG, Foufelle F, Barnes K, Baldwin SA, Woods A, Carling D: Characterization of the role of the AMP-activated protein kinase in the stimulation of glucose transport in skeletal muscle cells. Biochem J363 :167 –174,2002[Medline]
  19. Merrill GF, Kurth EJ, Hardie DG, Winder WW: AICA riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle. Am J Physiol273 :E1107 –E1112,1997[Medline]
  20. Bergeron R, Russell III RR, Young LH, Ren J-M, Marcucci M, Lee A, Shulman GI: Effect of AMPK activation on muscle glucose metabolism in conscious rats. Am J Physiol276 :E938 –E944,1999[Medline]
  21. Hayashi T, Hirshman MF, Fujii N, S. A. H, Witters LA, Goodyear LJ: Metabolic stress and altered glucose transport: activation of AMP-activated protein kinase as a unifying coupling mechanism. Diabetes49 :527 –531,1999[Abstract]
  22. Winder WW, Hardie DG: AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes. Am J Physiol277 :E1 –E10,1999[Medline]
  23. Winder WW: Energy-sensing and signaling by AMP-activated protein kinase in skeletal muscle. J Appl Physiol91 :1017 –1028,2001[Abstract/Free Full Text]
  24. Bergeron R, Previs SF, Cline GW, Perret P, Russell RR, 3rd, Young LH, Shulman GI: Effect of 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside infusion on in vivo glucose and lipid metabolism in lean and obese Zucker rats. Diabetes50 :1076 –1082,2001[Abstract/Free Full Text]
  25. Song X, Fiedler M, Galuska D, Ryder J, Fernström M, Chibalin A, Wallberg-Henriksson H, Zierath J: 5-aminoimidazole-4-carboxamide ribonucleoside treatment improves glucose homeostasis in insulin-resistant diabetic (ob/ob) mice. Diabetologia45 :56 –65,2002[Medline]
  26. Gulve EA, Henriksen EJ, Rodnick KJ, Youn JH, Holloszy JO: Glucose transporters and glucose transport in skeletal muscle of 1- to 25-mo-old rats. Am J Physiol264 :E319 –E327,1993[Abstract/Free Full Text]
  27. Ryder JW, Fahlman R, Wallberg-Henriksson H, Alessi DR, Krook A, Zierath JR: Effect of contraction on mitogen-activated protein kinase signal transduction in skeletal muscle: involvement of the mitogen- and stress-activated protein kinase 1. J Biol Chem275 :1457 –1462,2000[Abstract/Free Full Text]
  28. Cartee GD, Douen AG, Ramlal T, Klip A, Holloszy JO: Stimulation of glucose transport in skeletal muscle by hypoxia. J Appl Physiol70 :1593 –1600,1991[Abstract/Free Full Text]
  29. Hansen P, Gulve EA, Gao J, Schluter J, Mueckler MM, Holloszy JO: Kinetics of 2-deoxyglucose transport in skeletal muscle: effects of insulin and contractions. Am J Physiol259 :C30 –C35,1995
  30. Woods A, Salt I, Scott J, Hardie DG, Carling D: The {alpha}1 and {alpha}2 isoforms of the AMP-activated protein kinase have similar activities in rat liver but exhibit differences in substrate specificity in vitro. FEBS Lett397 :347 –351,1996[Medline]
  31. Krook A, Whitehead JP, Dobson SP, Griffiths MR, Ouwens M, Baker C, Hayward AC, Sen SK, Maassen JA, Siddle K, Tavare JM, O’Rahilly S: Two naturally occurring insulin receptor tyrosine kinase domain mutants provide evidence that phosphatidylinositol 3-kinase activation alone is not sufficient for the meditation of insulin’s metabolic and mitogenic effects. J Biol Chem272 :30208 –30214,1997[Abstract/Free Full Text]
  32. Baumann CA, Ribon V, Kanzaki M, Thurmond DC, Mora S, Shigematsu S, Bickel PE, Pessin JE, Saltiel AR: CAP defines a second signalling pathway required for insulin-stimulated glucose transport. Nature407 :147 –148,2000[Medline]
  33. Chiang SH, Baumann CA, Kanzaki M, Thurmond DC, Watson RT, Neudauer CL, Macara IG, Pessin JE, Saltiel AR: Insulin-stimulated GLUT4: translocation requires the CAP-dependent activation of TC10. Nature410 :944 –948,2001[Medline]
  34. Wallberg-Henriksson H, Constable SH, Young DA, Holloszy JO: Glucose transport into rat skeletal muscle: interaction between exercise and insulin. J Appl Physiol65 :909 –913,1988[Abstract/Free Full Text]
  35. Wallberg-Henriksson H, Holloszy JO: Contractile activity increases glucose uptake by muscle in severely diabetic rats. J Appl Physiol57 :1045 –1049,1984[Abstract/Free Full Text]
  36. Wallberg-Henriksson H, Holloszy JO: Activation of glucose transport in diabetic muscle: responses to contraction and insulin. Am J Physiol249 :C233 –C237,1985[Abstract]
  37. Zierath JR, Houseknecht K, Gnudi L, Kahn BB: High-fat feeding impairs insulin-stimulated GLUT4 recruitment in muscle via an early insulin-signaling defect. Diabetes46 :215 –223,1997[Abstract]
  38. Ryder JW, Kawano Y, Galuska D, Fahlman R, Wallberg-Henriksson H, Charron MJ, Zierath JR: Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4-deficient mice. FASEB J13 :2246 –2256,1999[Abstract/Free Full Text]
  39. Brozinick JT Jr, Etgen GJ Jr, Yaspelkis BB 3rd, Ivy JL: Glucose uptake and GLUT-4 protein distribution in skeletal muscle of the obese Zucker rat. Am J Physiol267 :R236 –R243,1994[Abstract/Free Full Text]
  40. Etgen GJ Jr, Wilson CM, Jensen J, Cushman SW, Ivy JL: Glucose transport and cell surface GLUT-4 protein in skeletal muscle of the obese Zucker rat. Am J Physiol271 :E294 –E301,1996[Abstract/Free Full Text]
  41. Moller DE: New drug targets for type 2 diabetes and the metabolic syndrome. Nature414 :821 –827,2001[Medline]
  42. Musi N, Fujii N, Hirshman MF, Ekberg I, Fröberg S, Ljungqvist O, Thorell A, Goodyear LJ: AMP-activated protein kinase (AMPK) is activated in muscle of subjects with type 2 diabetes during exercise. Diabetes50 :921 –927,2001[Abstract/Free Full Text]
  43. Chen Z-P, McConell GK, Michell BJ, Snow RJ, Canny BJ, Kemp BE: AMPK signaling in contracting human skeletal muscle: acetyl-CoA carboxylase and NO synthase phosphorylation. Am J Physiol Endocrinol Metabol279 :E1202 –E1206,2000[Abstract/Free Full Text]
  44. Fujii N, Hayashi T, Hirshman MF, Smith JT, Habinowski SA, Kaijser L, Mu J, Ljungqvust O, Birnbaum MJ, Witters LA, Thorell A, Goodyear LJ: Exercise induces isoform-specific increase in 5' -AMP-activated protein kinase activity in human skeletal muscle. Biochem Biophys Res Comm273 :1150 –1155,2000[Medline]
  45. Wojtaszewski JPF, Nielsen P, Hansen BF, Richter EA, Kiens B: Isoform-specific and exercise intensity-dependent activation of 5'-AMP-activated protein kinase in human skeletal muscle. J Physiol (Lond)528 :1221 –1226,2000
  46. Derave W, Ai H, Ihlemann J, Witters LA, Kristiansen SK, Richter EA, Ploug T: Dissociation of AMP-activated protein kinase activation and glucose transport in contracting slow-twitch muscle. Diabetes49 :1281 –1287,2000[Abstract]