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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
adenosine 5'-monophosphate-activated protein kinase (EC 2.7.1.109); acetyl-CoA carboxylase- (EC 6.4.1.2); glycogen synthase (EC 2.4.1.11); protein phosphorylation
AMPK is an 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 (
1,
2) and the two regulatory subunits (
1,
2,
1,
2, and
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
2- and
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
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 2-associated AMPK activity and protein expression of the
1-,
2-, and
1-subunits were found to be normal. Furthermore, metformin treatment was reported to increase basal
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 -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
1,
2, and
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 (1,
2,
1,
2,
1,
2, and
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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·m2·min1), 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 [1015 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 1015 s.
AMPK -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
-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
-isoform-specific activity was measured in immunoprecipitates from 200 µg of muscle lysate protein by use of anti-
1 or anti-
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).
-AMPK and acetyl-CoA carboxylase-
phosphorylation. The phosphorylation of the
-subunits (Thr172) and acetyl-CoA carboxylase-
(ACC
; 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
containing the Ser79 phosphorylation site, but the antibody also recognized the human ACC
when phosphorylated, most likely at the corresponding Ser221. For the detection of
-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 1. In this latter case, a "pan"-
antibody was generated in sheep to the peptide CRAAPLWDSKKQSFVG (residues 6983 of rat
1, a sequence highly conserved in human
2 and
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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%).
|
AMPK activity and -AMPK Thr172 and ACC
Ser221 phosphorylation. AMPK activity was measured in an isoformspecific assay after immunoprecipitation of either of the two
-isoforms. Neither
1- nor
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,
-Thr172 phosphorylation was similar and not affected by insulin in either of the two groups (Fig. 2). Phosphorylation of ACC
on Ser221 has previously been used as an indicator of endogenous AMPK activity. As judged by Western blotting, the ACC
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
phosphorylation was observed in control samples (added to all assays), representing muscle from the resting and contracted conditions, respectively.
|
|
AMPK subunit protein expression. We measured the protein content of all known AMPK subunit isoforms expressed in human muscle 1,
2,
1,
2,
1,
2, and
3. In muscle biopsies from the basal state, all seven subunit isoforms were expressed to similar levels in the two groups (Fig. 3).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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
) might be intact and not regulated by insulin. However, studies of human muscle strips in vitro showed normal regulation of AMPK and ACC
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 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 |
---|
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 |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|