AMPK activity and isoform protein expression are similar in muscle of obese subjects with and without type 2 diabetes

Kurt Højlund,2,* Kirsty J. Mustard,3,* Peter Stæhr,2 D. Grahame Hardie,3 Henning Beck-Nielsen,2 Erik A. Richter,1 and Jørgen F. P. Wojtaszewski1

1Department of Human Physiology, Copenhagen Muscle Research Centre, Institute of Exercise and Sport Sciences, University of Copenhagen, DK-2100 Copenhagen; 2Diabetes Research Centre, University of Southern Denmark and Department of Endocrinology, Odense University Hospital, DK-5000 Odense, Denmark; and 3Division of Molecular Physiology, School of Life Sciences, Wellcome Trust Biocentre, Dundee University, Dundee, DD1 5EH Scotland, United Kingdom

Submitted 14 July 2003 ; accepted in final form 2 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acute or chronic activation of AMP-activated protein kinase (AMPK) increases insulin sensitivity. Conversely, reduced expression and/or function of AMPK might play a role in insulin resistance in type 2 diabetes. Thus protein expression of the seven subunit isoforms of AMPK and activities and/or phosphorylation of AMPK and acetyl-CoA carboxylase-{beta} (ACC{beta}) was measured in skeletal muscle from obese type 2 diabetic and well-matched control subjects during euglycemic-hyperinsulinemic clamps. Protein expression of all AMPK subunit isoforms ({alpha}1, {alpha}2, {beta}1, {beta}2, {gamma}1, {gamma}2, and {gamma}3) in muscle of obese type 2 diabetic subjects was similar to that of control subjects. In addition, {alpha}1- and {alpha}2-associated activities of AMPK, phosphorylation of {alpha}-AMPK subunits at Thr172, and phosphorylation of ACC{beta} at Ser221 showed no difference between the two groups and were not regulated by physiological concentrations of insulin. These data suggest that impaired insulin action on glycogen synthesis and lipid oxidation in skeletal muscle of obese type 2 diabetic subjects is unlikely to involve changes in AMPK expression and activity.

adenosine 5'-monophosphate-activated protein kinase (EC 2.7.1.109); acetyl-CoA carboxylase-{beta} (EC 6.4.1.2); glycogen synthase (EC 2.4.1.11); protein phosphorylation


5'-AMP-ACTIVATED PROTEIN KINASE (AMPK) is a signal intermediate in the metabolic regulation in mammalian cells. In skeletal muscle, AMPK has been implicated in the regulation of lipid oxidation, glucose transport, and glycogen synthase activity and, in liver, AMPK activation might lead to inhibition of gluconeogenesis, glycolysis, lipogenesis, and cholesterol formation (16, 17, 23, 36). On this background, AMPK has been suggested to play a role in the pathogenesis of type 2 diabetes, as well as to be a potential target for drug treatment of type 2 diabetes. Support for this concept comes from studies showing that acute and chronic treatment of insulin-resistant rodent models with the AMPK-activating agent 5-aminoimidazole-4-carboxamide ribofuranoside (AICAR) improves glucose homeostasis and insulin sensitivity (5, 9, 15, 21, 32).

AMPK is an {alpha}{beta}{gamma} heterotrimer that is activated by low cellular energy status, such as decreases in both the ATP/AMP ratio and the phosphocreatine content (16, 17, 23). Several isoforms of both the catalytic ({alpha}1, {alpha}2) and the two regulatory subunits ({beta}1, {beta}2, {gamma}1, {gamma}2, and {gamma}3) have been identified in mammalian cells (7, 33, 34). Protein expression of AMPK subunit isoforms is changed in response to exercise training (8, 25, 30), which might contribute to the metabolic alterations induced by exercise training, e.g., enhanced peripheral insulin sensitivity. Mutations in {gamma}2- and {gamma}3-subunit isoforms cause glycogen storage disease in human heart (1) and glycogen accumulation in pig muscle (27), respectively, implying that these subunits have a role in glycogen synthesis. In addition, the whole body {alpha}2-deficient mouse is insulin resistant, possibly due to enhanced sympathetic nervous activity (35). Thus evidence exists to suggest that altered AMPK activity caused by changes in expression or function of different AMPK subunit isoforms might modulate the metabolic profile of specific tissues and might influence whole body metabolism.

In a recent study of the AMPK system in muscle of nonobese type 2 diabetic subjects (28), exercise-induced {alpha}2-associated AMPK activity and protein expression of the {alpha}1-, {alpha}2-, and {beta}1-subunits were found to be normal. Furthermore, metformin treatment was reported to increase basal {alpha}2-associated AMPK activity in muscle of nonobese type 2 diabetic subjects (29). These findings argue against functional defects of the AMPK system in diabetic muscle, at least in the fasting and exercised states. However, we (18) have recently demonstrated that the mechanism responsible for impaired insulin-induced nonoxidative glucose metabolism and impaired activation of glycogen synthase in muscle of obese subjects with type 2 diabetes is likely to involve a hyperphosphorylation of glycogen synthase at the NH2-terminal sites Ser7 and Ser10 (18). AICAR treatment of isolated skeletal muscles and human myoblast in vitro leads to inactivation of glycogen synthase (2, 14, 39), most likely induced by AMPK-mediated phosphorylation of glycogen synthase at Ser7 (6), which primes the protein for subsequent phosphorylation at Ser10 by casein kinase I (10, 11). Thus the possibility exists that AMPK is involved in the hyperphosphorylation of glycogen synthase that occurs during insulin stimulation in obese type 2 diabetic subjects.

Another consistent metabolic feature of insulin resistance in type 2 diabetes is the failure of insulin to suppress lipid oxidation in skeletal muscle (22, 26). Although not consistent (4), an inhibitory effect of insulin on AMPK activity has been reported in hepatoma cells and in rat cardiac and skeletal muscle (3, 13, 37, 38), and this appears to involve decreased phosphorylation of Thr172 in {alpha}-AMPK (3). This effect has been proposed to mediate activation of acetyl-CoA carboxylase and, hence, suppression of lipid oxidation by insulin (13, 38). Thus, despite normal activity of AMPK in the fasting and exercised states in muscle of nonobese type 2 diabetic subjects (28), there are data that could indicate that changes in the expression or function of AMPK subunit isoforms other than {alpha}1, {alpha}2, and {beta}1 could be involved in the failure of insulin to activate glycogen synthase and to suppress lipid oxidation in the muscle of type 2 diabetic subjects.

To explore these possibilities, we measured basal AMPK subunit expression of all of the seven recognized subunit isoforms ({alpha}1, {alpha}2, {beta}1, {beta}2, {gamma}1, {gamma}2, and {gamma}3) and the effect of insulin on AMPK activity in resting skeletal muscle of obese type 2 diabetic subjects and well-matched healthy control subjects.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Study subjects. Ten obese subjects with type 2 diabetes and 10 healthy, obese control male subjects, matched according to age (51.1 ± 2.0 vs. 50.4 ± 1.6 yr) and body mass index (29.6 ± 0.9 vs. 31.1 ± 1.1 kg/m2), participated in the study (18). Subjects with type 2 diabetes were treated either by diet alone or by diet in combination with sulfonylurea or metformin, which was withdrawn 1 wk before the study. The patients were all negative for antibodies to glutamic acid decarboxylase 65 (GAD65) and without signs of diabetic retinopathy, nephropathy, neuropathy, or macrovascular complications. The control subjects had normal glucose tolerance and no family history of diabetes. All subjects had normal results on screening blood tests of hepatic and renal function. All subjects were instructed to refrain from strenuous physical activity for a period of 48 h before the experiment. Informed consent was obtained from all subjects before participation. The study was approved by the local ethics committee and was performed in accord with the Helsinki Declaration.

Study design. All study subjects were admitted to the Diabetes Research Centre at Odense University Hospital (Odense, Denmark). After an overnight fast, all subjects underwent a euglycemic-hyperinsulinemic clamp (4 h of insulin infusion, 40 mU·m–2·min–1), as described in detail previously (20). In type 2 diabetic subjects, plasma glucose was allowed to decline to ~5.5 mmol/l before glucose infusion was initiated. Total glucose disposal rates (GDR) were calculated using Steele's non-steady-state equations adapted for labeled glucose infusates (20). The distribution volume of glucose was taken as 200 ml/kg body wt and the pool fraction as 0.65. The studies were combined with indirect calorimetry by using the flow-through canopy gas analyzer system (Deltatrac; Datex, Helsinki, Finland). Rates of glucose oxidation were calculated from Frayn's equation (12). Nonoxidative glucose metabolism was calculated as the difference between GDR and glucose oxidation. Plasma glucose and glucagon and serum insulin, C-peptide, and free fatty acids (FFA) were measured as described previously (19). Muscle biopsies were obtained from the vastus lateralis muscle before and after the insulin infusion period with a modified Bergström needle by suction under local anesthesia [10–15 ml of lidocaine 2% (20 mg/ml) injected subcutaneously]. Muscle samples were immediately blotted free of blood, fat, and connective tissue and were frozen in liquid nitrogen within 10–15 s.

AMPK {alpha}-isoform-specific activity. Muscle lysates were prepared by homogenization of muscle tissue (1:20, wt/vol) in a buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 20 mM Na-pyrophosphate, 20 mM {beta}-glycerophosphate, 10 mM NaF, 2 mM Na-orthovanadate, 2 mM EDTA, 1% Nonidet P-40, 10% glycerol, 2 mM PMSF, 1 mM MgCl2, 1 mM CaCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 3 mM benzamidine. Homogenates were rotated end over end for 1 h at 4°C and then cleared by centrifugation at 17.500 g at 4°C for 1 h. Protein content in the supernatants was measured by the bicinchoninic acid method (Pierce, Rockford, IL). AMPK {alpha}-isoform-specific activity was measured in immunoprecipitates from 200 µg of muscle lysate protein by use of anti-{alpha}1 or anti-{alpha}2 antibodies (40). A p81-filter paper assay, using SAMS peptide (HMRSAMSGLHLVKRR) (200 µM) as substrate was used to measure AMPK activity in the presence of saturated AMP concentration (0.2 mM) (40).

{alpha}-AMPK and acetyl-CoA carboxylase-{beta} phosphorylation. The phosphorylation of the {alpha}-subunits (Thr172) and acetyl-CoA carboxylase-{beta} (ACC{beta}; Ser221) was evaluated by Western blotting using phosphospecific antibodies from Cell Signaling Technology and Upstate Biotechnology, respectively. The ACC-phosphospecific antibody is raised against a peptide corresponding to the sequence in rat ACC{alpha} containing the Ser79 phosphorylation site, but the antibody also recognized the human ACC{beta} when phosphorylated, most likely at the corresponding Ser221. For the detection of {alpha}-AMPK phosphorylation (Thr172), muscle lysate protein was subjected to SDS-PAGE (7.5% Criterion gradient gel; Bio-Rad Laboratories, Richmond, CA), followed by semi-dry transfer to PVDF membranes (Immobilon Transfer Membrane, Millipore, Glostrup, DK). Immunoreactive bands were visualized with enhanced chemiluminescense (ECL-plus, Amersham Biosciences, Little Chalfont, UK) and detected and quantified using a charge-coupled device-image sensor and 1D software (Kodak Image Station, E440CF, Kodak, Glostrup, DK).

AMPK isoform expression. Muscle lysates were prepared as described (8). Western blotting for the AMPK subunit isoforms was performed as described previously (8) except in the case of {gamma}1. In this latter case, a "pan"-{gamma} antibody was generated in sheep to the peptide CRAAPLWDSKKQSFVG (residues 69–83 of rat {gamma}1, a sequence highly conserved in human {gamma}2 and {gamma}3), coupled to keyhole limpet hemocyanin, and affinity purified as described previously (41).

Calculations and statistical analysis. Control samples were added to all activity assays and loaded on all gels in duplicates, and assay-to-assay variation was accounted for by expressing data relative to these samples. Data calculation and statistical analysis were performed using the SigmaStat for MS Windows version 2.0 software. Data are presented as means ± SE. Two-way ANOVA analysis for repeated measures and Student's t-test for unpaired data were used as appropriate to detect any significant differences. Significance was accepted at the P < 0.05 level.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Metabolic characteristics. The metabolic profile of the subjects and data on glucose metabolism during basal conditions as well as during the clamp were recently published in a paper describing the phosphorylation profile of muscle glycogen synthase (18). Briefly, fasting levels of glycosylated hemoglobin, plasma glucose, serum insulin, and C-peptide were significantly higher in the diabetic group, whereas fasting levels of serum FFA did not differ between the groups (18). During the insulin-stimulated steady-state period (the 210- to 240-min period), euglycemia at a plasma glucose concentration of ~5.5 mmol/l and physiological hyperinsulinemia at a serum insulin concentration of ~400 pmol/l were obtained in both groups. In this period, serum FFA concentrations were significantly higher in the diabetic group vs. the control group (18).

In the basal state, GDR were significantly higher, whereas in the insulin-stimulated state GDR were significantly lower in the diabetic group compared with the control group (Table 1). Indirect calorimetry data showed no significant differences in glucose oxidation, nonoxidative glucose metabolism (glucose storage), or lipid oxidation in the basal state between the groups. During the insulin-stimulated state, both glucose oxidation and glucose storage were significantly lower, whereas lipid oxidation was significantly higher in type 2 diabetic subjects compared with control subjects (Table 1). The reduction in insulin-stimulated GDR in type 2 diabetic subjects was primarily accounted for by impaired glucose storage (75%).


View this table:
[in this window]
[in a new window]
 
Table 1. Euglycemic hyperinsulinemic clamp data

 

AMPK activity and {alpha}-AMPK Thr172 and ACC{beta} Ser221 phosphorylation. AMPK activity was measured in an isoformspecific assay after immunoprecipitation of either of the two {alpha}-isoforms. Neither {alpha}1- nor {alpha}2-associated activities displayed any regulation with insulin, and both were similar in magnitude between control and type 2 diabetic subjects (Fig. 1). In support of these in vitro measured activities, {alpha}-Thr172 phosphorylation was similar and not affected by insulin in either of the two groups (Fig. 2). Phosphorylation of ACC{beta} on Ser221 has previously been used as an indicator of endogenous AMPK activity. As judged by Western blotting, the ACC{beta} phosphorylation was also similar at rest and not affected by insulin in the two groups (Fig. 2). Several observations indicate that these findings are based on reliable assay conditions and usable muscle lysate samples. First, the absolute AMPK activities measured fell within the linear range of the assay and were similar to the levels previously reported by us and others using immunopurified AMPK from human muscle. Second, although AMPK activity or phosphorylation was not affected by insulin, the activity/phosphorylation of several other enzymes (measured in the same lysate) was regulated by insulin (18). Finally, the expected increase in AMPK activity/phosphorylation and ACC{beta} phosphorylation was observed in control samples (added to all assays), representing muscle from the resting and contracted conditions, respectively.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 1. AMPK activity associated with {alpha}1- (A) and {alpha}2- (B) AMPK subunits. Activities were measured in an in vitro assay using saturated AMP concentrations. Data are given as pmol phosphate incorporated into SAMS peptide·min–1·mg muscle lysate protein–1 used for immunoprecipitation. T2DM, type 2 diabetes mellitus. Open bars, basal conditions; solid bars, data obtained at end of steady-state period of euglycemic hyperinsulinemic clamp. All data are expressed as means ± SE (n = 10).

 


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2. Thr172 phosphorylation on {alpha}-AMPK (A) and Ser221 phosphorylation of acetyl-CoA carboxylase-{beta} (ACC{beta}, B) were measured by Western blotting. Data are given as arbitrary scanning units. Open bars, basal conditions; solid bars, data obtained at end of steady-state period of euglycemic hyperinsulinemic clamp. Insets: representative immunoblots. Bas and Ins, basal and insulin-treated conditions. Specific bands were observed at 63 kDa for Thr172 phosphorylated {alpha}-AMPK and at ~260 kDa for Ser221 phosphorylated ACC{beta}. All data are expressed as means ± SE (n = 10).

 

AMPK subunit protein expression. We measured the protein content of all known AMPK subunit isoforms expressed in human muscle {alpha}1, {alpha}2, {beta}1, {beta}2, {gamma}1, {gamma}2, and {gamma}3. In muscle biopsies from the basal state, all seven subunit isoforms were expressed to similar levels in the two groups (Fig. 3).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3. Protein expression of AMPK subunit isoforms measured by Western blotting by use of muscle lysates from biopsies obtained during basal conditions. Data (means ± SE) are given as arbitrary scanning units (n = 10). The protein level in type 2 diabetic subjects was normalized to the level observed in the control subjects. Open bars, control subjects; solid bars, type 2 diabetic subjects. Specific bands were observed at (kDa) 63- {alpha}1, 63- {alpha}2, 34- {beta}1, 30- {beta}2, 35- {gamma}1, 63- {gamma}2, and 55- {gamma}3.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Because of the apparent influence of AMPK on a variety of metabolic processes dysregulated in type 2 diabetic subjects, AMPK has been hypothesized to play a role in the pathogenesis of insulin resistance in patients with type 2 diabetes (16, 17, 36). In the present study, we found that muscle from obese type 2 diabetic subjects had a level of protein expression of all seven AMPK subunit isoforms that was similar to that of healthy but obese control subjects. We also observed similar AMPK activities associated with both the {alpha}1- and {alpha}2-catalytic isoforms, a similar degree of phosphorylation of Thr172 in the {alpha}-AMPK isoforms, and a similar phosphorylation of the AMPK target site on ACC{beta} (Ser221). This suggests that AMPK function is intact in muscle of obese type 2 diabetic subjects, at least in the fasted and insulin-stimulated states. These observations extend those previously reported in nonobese exercise-tolerant type 2 diabetic subjects (28).

The present findings, and the observation that nonobese type 2 diabetic subjects displayed normal AMPK activation during exercise (28) and showed increased AMPK activity in response to metformin treatment (29), argue against a functional defect in AMPK and suggest that pharmacological activation of the AMPK system is a feasible and attractive treatment of insulin resistance in type 2 diabetes. The finding that ACC{beta} Ser221 is phosphorylated to a similar extent in muscle of type 2 diabetic and healthy control subjects in both the basal and insulin-stimulated states suggests that any regulation of lipid oxidation by AMPK (via ACC{beta}) might be intact and not regulated by insulin. However, studies of human muscle strips in vitro showed normal regulation of AMPK and ACC{beta} phosphorylation in response to AICAR treatment (24); however, despite this, AICAR-induced glucose transport was impaired, leading to the conclusion that downstream defects might still compromise actions of AMPK.

From a range of studies, it seems that altered AMPK activity might modify the metabolic profile of diabetic animals and might change insulin sensitivity of skeletal muscle (5, 9, 15, 21, 32). The data from our study and others of human skeletal muscle in vivo (28) indicate that type 2 diabetes per se is not associated with changes in AMPK expression or activity, but we cannot exclude the possibility that obesity-related insulin resistance might be associated with changes in AMPK expression. In addition, neither of these studies can exclude a role of altered AMPK function in other tissues in the pathogenesis of type 2 diabetes. From the present data, showing similar activity and expression of AMPK in muscle of type 2 diabetic and control subjects despite different insulin sensitivity, it appears that there is no simple relationship between these variables. This is in line with recent observations showing that lack of expression of the {alpha}2-AMPK isoform in knockout mice does not cause alterations in insulin sensitivity of isolated muscle, but rather causes insulin resistance due to central mechanisms (35).

We have recently published a subset of data from the present study showing that glycogen synthase in muscle from type 2 diabetic subjects becomes hyperphosphorylated on Ser7 and Ser10 in response to insulin stimulation and suggesting that this abnormality is involved in the impaired insulin activation of glycogen synthase found in this group of patients (18). Both biochemical and physiological studies in vitro suggest that AMPK is a glycogen synthase kinase, phosphorylating Ser7 at the NH2 terminus, which decreases glycogen synthase activity (2, 6, 14, 39). In cultured skeletal muscle cells, glucose deprivation has been shown to stimulate both of the catalytic subunit isoforms of AMPK, and this was associated with a significant decrease in the fractional velocity of glycogen synthase activity (14). A regulatory role of AMPK in vivo is suggested from findings in patients with glycogen phosphorylase deficiency (McArdle's disease) in whom the exercise-induced AMPK activation is correlated with a decreased glycogen synthase activity (31). However, the present data suggest that AMPK is not the kinase leading to the dysregulation of glycogen synthase, because AMPK activity in muscle of type 2 diabetic subjects is not increased in the basal state or in response to insulin.

In summary, the present data suggest that changes in AMPK activity or protein expression of AMPK subunit isoforms are not present in muscle of obese type 2 diabetic subjects compared with healthy obese controls. Therefore, AMPK does not appear to be a major contributor to the altered metabolic profile in type 2 diabetes. Importantly, the data also demonstrate that AMPK activity in resting human skeletal muscle is not regulated by physiological concentrations of insulin. Thus failure of insulin to activate glycogen synthesis and to suppress lipid oxidation in muscle of type 2 diabetic subjects is unlikely to involve changes in expression or activity of AMPK.


    ACKNOWLEDGMENTS
 
GRANTS

This study was supported by grants from the Danish National Research Foundation (no. 504-14), by The Media and Grants Secretariat of the Danish Ministry of Culture, by the Danish Diabetes Association, by the Institute of Clinical Research at the University of Southern Denmark, by the Novo Nordisk Foundation, and by a Research & Technological Development Project (QLG1-CT-2001-01488) funded by the European Commission. D. G. Hardie was supported by a Programme Grant from Wellcome Trust, K. J. Mustard by a studentship from the Biotechnology and Biological Sciences Research Council and by Novo Nordisk, and J. F. P. Wojtaszewski by a Hallas Møller fellowship from the Novo Nordisk Foundation.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. F. P. Wojtaszewski, Copenhagen Muscle Research Centre, Dept. of Human Physiology, Institute of Exercise and Sport Sciences, Univ. of Copenhagen, 13 Universitetsparken, Copenhagen DK-2100, Denmark (E-mail: Jwojtaszewski{at}aki.ku.dk).

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.

* These authors contributed equally to this work. Back


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Arad M, Benson DW, Perez-Atayde AR, McKenna WJ, Sparks EA, Kanter RJ, McGarry K, Seidman JG, and Seidman CE. Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy. J Clin Invest 109: 357–362, 2002.[Abstract/Free Full Text]
  2. Aschenbach WG, Hirshman MF, Fujii N, Sakamoto K, Howlett KF, and Goodyear LJ. Effect of AICAR treatment on glycogen metabolism in skeletal muscle. Diabetes 51: 567–573, 2002.[Abstract/Free Full Text]
  3. Beauloye C, Marsin AS, Bertrand L, Krause U, Hardie DG, Vanover-schelde JL, and Hue L. Insulin antagonizes AMP-activated protein kinase activation by ischemia or anoxia in rat hearts, without affecting total adenine nucleotides. FEBS Lett 505: 348–352, 2001.[ISI][Medline]
  4. Bergeron R, Previs SF, Cline GW, Perret P, Russell RR III, Young LH, and Shulman GI. Effect of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside infusion on in vivo glucose and lipid metabolism in lean and obese Zucker rats. Diabetes 50: 1076–1082, 2001.[Abstract/Free Full Text]
  5. Buhl ES, Jessen N, Pold R, Ledet T, Flyvbjerg A, Pedersen SB, Pedersen O, Schmitz O, and Lund S. Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying features of the insulin resistance syndrome. Diabetes 51: 2199–2206, 2002.[Abstract/Free Full Text]
  6. Carling D and Hardie DG. The substrate and sequence specificity of the AMP-activated protein kinase. Phosphorylation of glycogen synthase and phosphorylase kinase. Biochim Biophys Acta 1012: 81–86, 1989.[ISI][Medline]
  7. Cheung CF, Salt IP, Davies A, Hardie DG, and Carling D. Characterization of AMP-activated protein kinase gamma subunit isoforms and their role in AMP binding. Biochem J 346: 659–669, 2000.[ISI][Medline]
  8. Durante PE, Mustard KJ, Park SH, Winder WW, and Hardie DG. Effects of endurance training on activity and expression of AMP-activated protein kinase isoforms in rat muscles. Am J Physiol Endocrinol Metab 283: E178–E186, 2002.[Abstract/Free Full Text]
  9. Fisher JS, Gao J, Han DH, Holloszy JO, and Nolte LA. Activation of AMP kinase enhances sensitivity of muscle glucose transport to insulin. Am J Physiol Endocrinol Metab 282: E18–E23, 2002.[Abstract/Free Full Text]
  10. Flotow H, Graves PR, Wang AQ, Fiol CJ, Roeske RW, and Roach PJ. Phosphate groups as substrate determinants for casein kinase I action. J Biol Chem 265: 14264–14269, 1990.[Abstract/Free Full Text]
  11. Flotow H and Roach PJ. Synergistic phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase and casein kinase I. Implications for hormonal regulation of glycogen synthase. J Biol Chem 264: 9126–9128, 1989.[Abstract/Free Full Text]
  12. Frayn K. Calculation of substrate oxidation rates in vivo from gaseous exchange. J Appl Physiol 55: 628–634, 1983.[Abstract/Free Full Text]
  13. Gamble J and Lopaschuk GD. Insulin inhibition of 5' adenosine monophosphate-activated protein kinase in the heart results in activation of acetyl coenzyme A carboxylase and inhibition of fatty acid oxidation. Metabolism 46: 1270–1274, 1997.[ISI][Medline]
  14. Halse R, Fryer LG, McCormack JG, Carling D, and Yeaman SJ. Regulation of glycogen synthase by glucose and glycogen: a possible role for AMP-activated protein kinase. Diabetes 52: 9–15, 2003.[Abstract/Free Full Text]
  15. Hamilton SR, Stapleton D, O'Donnell JB, Kung JT, Dalal SR, Kemp BE, and Witters LA. An activating mutation in the gamma1 subunit of the AMP-activated protein kinase. FEBS Lett 500: 163–168, 2001.[ISI][Medline]
  16. Hardie DG and Carlson M. The AMP activated protein kinase—fuel gauge of the mammalian cell? Eur J Biochem 246: 259–273, 1997.[Abstract]
  17. Hardie DG and Hawley SA. AMP-activated protein kinase: the energy charge hypothesis revisited. Bioessays 23: 1112–1119, 2001.[ISI][Medline]
  18. Højlund K, Stæhr P, Hansen BF, Green KA, Hardie DG, Richter EA, Beck-Nielsen H, and Wojtaszewski JFP. Increased phosphorylation of skeletal muscle glycogen synthase at NH2-terminal sites during physiological hyperinsulinemia in type 2 diabetes. Diabetes 52: 1393–1402, 2003.[Abstract/Free Full Text]
  19. Højlund K, Wildner-Christensen M, Eshøj O, Skjaerbaek C, Holst JJ, Koldkjaer O, Møller Jensen D, and Beck-Nielsen H. Reference intervals for glucose, {beta}-cell polypeptides, and counterregulatory factors during prolonged fasting. Am J Physiol Endocrinol Metab 280: E50–E58, 2001.[Abstract/Free Full Text]
  20. Hother-Nielsen O, Henriksen JE, Holst JJ, and Beck-Nielsen H. Effects of insulin on glucose turnover rates in vivo: isotope dilution versus constant specific activity technique. Metabolism 45: 82–91, 1996.[ISI][Medline]
  21. Iglesias MA, Ye JM, Frangioudakis G, Saha AK, Tomas E, Ruderman NB, Cooney GJ, and Kraegen EW. AICAR administration causes an apparent enhancement of muscle and liver insulin action in insulin-resistant high-fat-fed rats. Diabetes 51: 2886–2894, 2002.[Abstract/Free Full Text]
  22. Kelley DE and Mandarino LJ. Fuel selection in human skeletal muscle in insulin resistance: a reexamination. Diabetes 49: 677–683, 2000.[Abstract]
  23. Kemp BE, Mitchelhill KI, Stapleton D, Michell BJ, Chen ZP, and Witters LA. Dealing with energy demand: the AMP-activated protein kinase. Trends Biochem Sci 24: 22–25, 1999.[ISI][Medline]
  24. Koistinen H, Galuska D, Chibalin AV, Yang J, Zierath JR, Holman GD, and Wallberg-Henriksson. 5-Amino-imidazole carboxamide riboside increases glucose transport and cell-surface content in skeletal muscle from subjects with type 2 diabetes. Diabetes 52: 1066–1072, 2003.[Abstract/Free Full Text]
  25. Langfort J, Viese M, Ploug T, and Dela F. Time course of GLUT4 and AMPK protein expression in human skeletal muscle during one month of physical training. Scand J Med Sci Sports 12: 1–6, 2002.[ISI][Medline]
  26. Mandarino LJ, Consoli A, Jain A, and Kelley DE. Interaction of carbohydrate and fat fuels in human skeletal muscle: impact of obesity and NIDDM. Am J Physiol Endocrinol Metab 270: E463–E470, 1996.[Abstract/Free Full Text]
  27. Milan D, Jeon JT, Looft C, Amarger V, Robic A, Thelander M, Rogel-Gaillard C, Paul S, Iannuccelli N, Rask L, Ronne H, Lundstrom K, Reinsch N, Gellin J, Kalm E, Roy PL, Chardon P, and Andersson L. A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle. Science 288: 1248–1251, 2000.[Abstract/Free Full Text]
  28. Musi N, Fujii N, Hirshman MF, Ekberg I, Fröberg S, Ljungqvist O, Thorell A, and Goodyear LJ. AMP activated protein kinase is activated in muscle of subjects with type 2 diabetes during exercise. Diabetes 50: 921–927, 2001.[Abstract/Free Full Text]
  29. Musi N, Hirshman MF, Nygren J, Svanfeldt M, Bavenholm P, Rooyackers O, Zhou G, Williamson JM, Ljunqvist O, Efendic S, Moller DE, Thorell A, and Goodyear LJ. Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes. Diabetes 51: 2074–2081, 2002.[Abstract/Free Full Text]
  30. Nielsen JN, Mustard K, Graham D, Pilegaard H, Haiyan Y, Goodyear LJ, Hardie DG, Richter EA, and Wojtaszewski JFP. AMPK actvity and subunit expression in exercise-trained human skeletal muscle. J Appl Physiol 94: 631–641, 2003.[Abstract/Free Full Text]
  31. Nielsen JN, Wojtaszewski JFP, Haller RG, Hardie DG, Kemp BE, Richter EA, and Vissing J. Role of 5'AMP-activated protein kinase in exercise regulation of glucose utilization and glycogen synthase activity in skeletal muscle; insights from patients with McArdles's disease. J Physiol 541: 979–989, 2002.[Abstract/Free Full Text]
  32. Song XM, Fiedler M, Galuska D, Ryder JW, Fernstrom M, Chibalin AV, Wallberg-Henriksson H, and Zierath JR. 5-Aminoimidazole-4-carboxamide ribonucleoside treatment improves glucose homeostasis in insulin-resistant diabetic (ob/ob) mice. Diabetologia 45: 56–65, 2002.[ISI][Medline]
  33. Stapleton D, Mitchelhill KI, Gao G, Widmer J, Michell BJ, Teh T, House CM, Fernandez CS, Cox T, Witters LA, and Kemp BE. Mammalian AMP-activated protein kinase subfamily. J Biol Chem 271: 611–614, 1996.[Abstract/Free Full Text]
  34. Thornton C, Snowden MA, and Carling D. Identification of a novel AMP-activated protein kinase beta subunit isoform that is highly expressed in skeletal muscle. J Biol Chem 273: 12443–12450, 1998.[Abstract/Free Full Text]
  35. Viollet B, Andreelli F, Jorgensen SB, Perrin C, Geloen A, Flamez D, Mu J, Lenzner C, Baud O, Bennoun M, Gomas E, Nicolas G, Wojtaszewski JF, Kahn A, Carling D, Schuit FC, Birnbaum MJ, Richter EA, Burcelin R, and Vaulont S. The AMP-activated protein kinase alpha2 catalytic subunit controls whole-body insulin sensitivity. J Clin Invest 111: 91–98, 2003.[Abstract/Free Full Text]
  36. Winder WW and Hardie DG. AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes. Am J Physiol Endocrinol Metab 277: E1–E10, 1999.[Abstract/Free Full Text]
  37. Winder WW and Holmes BF. Insulin stimulation of glucose uptake fails to decrease palmitate oxidation in muscle if AMPK is activated. J Appl Physiol 89: 2430–2437, 2000.[Abstract/Free Full Text]
  38. Witters LA and Kemp BE. Insulin activation of acetyl-CoA carboxylase accompanied by inhibition of the 5'-AMP-activated protein kinase. J Biol Chem 267: 2864–2867, 1992.[Abstract/Free Full Text]
  39. Wojtaszewski JFP, Jørgensen SB, Hellsten Y, Hardie DG, and Richter EA. Glycogen-dependent effects of 5-aminoimidazole-4-carboxamide (AICA)-riboside on AMP-activated protein kinase and glycogen synthase activities in rat skeletal muscle. Diabetes 51: 284–292, 2002.[Abstract/Free Full Text]
  40. Wojtaszewski JFP, Nielsen P, Hansen BF, Richter EA, and Kiens B. Isoform-specific and exercise intensity-dependent activation of 5'AMP-activated protein kinase in human skeletal muscle. J Physiol 528: 221–226, 2000.[Abstract/Free Full Text]
  41. Woods A, Salt I, Scott J, Hardie DG, and Carling D. The alpha1 and alpha2 isoforms of the AMP-activated protein kinase have similar activities in rat liver but exhibit differences in substrate specificity in vitro. FEBS Lett 397: 347–351, 1996.[ISI][Medline]