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Endocrinology Vol. 147, No. 6 2728-2736
Copyright © 2006 by The Endocrine Society

Long-Term Metformin Treatment Stimulates Cardiomyocyte Glucose Transport through an AMP-Activated Protein Kinase-Dependent Reduction in GLUT4 Endocytosis

Jing Yang and Geoffrey D. Holman

Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom

Address all correspondence and requests for reprints to: Dr. Geoffrey D. Holman, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom. E-mail: g.d.holman{at}bath.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Long-term (18 h) metformin treatment of cardiomyocytes increased glucose transport activity 3- to 5-fold, as measured using the phosphorylated sugar 2-deoxy-D-glucose and the nonphosphorylated sugar 3-O-methyl-D-glucose. The affinity for 3-O-methyl-D-glucose transport was not increased by metformin treatment. Total levels of glucose transporter 4 (GLUT4) were not changed by 18-h culture with or without insulin or metformin treatment. GLUT1 levels were elevated after 18 h in culture, but this increase was not altered by insulin or metformin treatment. Metformin-induced stimulation of transport was not inhibited by treatment with wortmannin and was additive with that of insulin. These data suggest that the metformin effect is mediated by a signaling route independent of phosphatidylinositol 3-kinase and Akt. Surprisingly, however, levels of both phospho-AMP-activated protein kinase and phospho-Akt were increased 4- and 3-fold, respectively, after metformin treatment. Chronic treatment with insulin for 18 h led to down-regulation of insulin-stimulated glucose transport. Cotreatment with metformin bypassed this insulin resistance by maintaining high transport levels. These data also indicate an independent point of convergence of metformin and insulin stimuli on GLUT4 regulatory processes. To test the possibility of altered GLUT4 subcellular trafficking, the kinetics of GLUT4 exocytosis and endocytosis were determined. Metformin treatment markedly slowed endocytosis of GLUT4, but exocytosis was not increased. We conclude that metformin treatment leads to a longer residence time of GLUT4 in the plasma membrane due to an AMP-activated protein kinase-dependent reduction in endocytosis. This accounts for metformin’s ability to enhance hexose transport activity above insulin-stimulated and Akt-dependent levels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RECENT CLINICAL studies have revealed that reduced insulin sensitivity is a risk factor for mortality in patients with coronary heart failure (1, 2). In addition, it has been shown that measurements of insulin-stimulated glucose disposal are prognostic markers of coronary heart failure (1, 3). Metformin is used clinically to alleviate insulin resistance (4), but whether such treatment has beneficial effects on heart metabolism has been controversial. Beneficial effects on heart metabolism in humans (5) may be counteracted by adverse effects on blood vessel function (6). In vitro metformin treatment has been found to ameliorate insulin resistance by inducing glucose transporter 4 (GLUT4) translocation in obese Zucker rats (7) and streptozotocin-diabetic rats (8). However, the concentrations of metformin required for in vitro responses on glucose transport activity in isolated tissues such as muscle (9), fat (10), and heart (11) are generally much higher than the therapeutic doses used to treat type 2 diabetes (12).

Metformin produces many diverse changes in intracellular metabolism that result in lowering of blood glucose levels. These include reduction of intestinal absorption of sugars and suppression of hepatic glucose output (4). These changes may occur as a consequence of the accumulation of the drug in mitochondria and the resulting reduction of mitochondrial complex 1 activity (13). Recent advances in the understanding of metformin action have centered on the discovery that metformin leads to activation and increased phosphorylation of AMP-activated protein kinase (AMPK) (14, 15). This activation of AMPK appears to be associated with only small changes in the AMP/ATP ratio and phosphocreatine levels (16). Because the AMPK system acts as an energy-sparing sensor, this kinase is a good candidate as a mediator of metformin action on the enhanced catalysis of glucose uptake and the improved cellular energy status that follows this uptake.

In this study, we addressed whether there are beneficial effects of metformin action on glucose uptake in heart cells and whether long-term treatment with this drug can either reverse or circumvent insulin resistance. Insulin resistance in fat and skeletal muscle cells can be produced at the cellular level by prolonged incubations with insulin in the presence of relatively high glucose levels (17, 18). We show here that the same phenomenon occurs in isolated rat cardiomyocytes. The chronic effects of glucose and insulin are associated with reductions in early insulin-signaling events at the level of tyrosine phosphorylation of the insulin receptor (19, 20) and phosphatidylinositol 3-kinase (PI 3-kinase) activation of Akt (20). We therefore examined whether metformin activation of the AMPK signaling pathway can reverse the changes in Akt signaling. The alternative possibility is that stimulation of the AMPK signaling pathway bypasses the need for signaling via the insulin pathway. Because the insulin signaling pathway leads primarily to an enhanced rate of GLUT4 exocytosis (21, 22), whereas AMPK activation leads primarily to a decrease in GLUT4 endocytosis (23), there would seem to be more potential for a bypass rather than a reversal effect on the stimulation of transport activity. However, there is considerable cross-talk between the Akt and AMPK signaling pathways (24, 25, 26, 27), and the extent to which this cross-talk is transmitted to the GLUT4 exocytosis and endocytosis processes is examined in this study. We show that despite the cross-talk, the stimulatory effects of metformin and insulin on glucose transporter trafficking are independent, additive, and compensatory. These data lead to the hypothesis that the activation of AMPK may compensate for pathophysiological impairments in insulin action and therefore may have beneficial effects on heart metabolism that are of clinical relevance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cardiomyocyte isolation and their maintenance in culture
Cardiomyocytes from adult male Wistar rats (260–280 g) were prepared by collagenase digestion (type II; Worthington Biochemical Corp., Freehold, NJ). The study was approved by our institutional animal care and use committee. Cell suspensions were adjusted to approximately 30% cytocrit in sterile KRH buffer [6 mM KCl, 1 mM Na2HPO4, 0.2 mM NaH2PO4, 1.4 mM MgSO4, 1 mM CaCl2, 128 mM NaCl, and 10 mM HEPES (pH 7.4)] with 0.5% fatty acid-free BSA (Roche, Indianapolis, IN). For each condition, 1-ml aliquots of this cell suspension were added to 30 ml DMEM containing 1% BSA, 100 U penicillin, and 100 µg/ml streptomycin and cultured for 18 h in a 5% CO2 incubator. The DMEM contained 25 mM glucose, which is considered to be important (together with high insulin levels) for inducing insulin resistance. The cells were maintained under basal conditions or were treated with 1 mM metformin or 500 nM insulin. After 18-h culture, cells were washed twice with 20 ml KRH buffer containing 2 mM D-glucose.

Hexose transport activity
One-milliliter aliquots of cardiomyocyte suspensions at 10% cytocrit in 0.5% BSA/KRH buffer were maintained at 37 C and continuously gassed with O2. 2-Deoxy-D-glucose transport assays were initiated by the addition of substrate [100 µM final concentration; containing 0.5 µCi 2-deoxy-D-[3H]glucose (Amersham Biosciences, Arlington Heights, IL)]. Uptake was terminated after 5 min by transferring the cell suspension to microfuge tubes containing 400 µM phloretin in KRH buffer. Background activity was determined by addition of cells to tubes that contained 2-deoxy-D-glucose premixed with 400 µM phloretin. The samples were quickly mixed and immediately centrifuged at 3500 x g for 1 min. The supernatants were removed, and the cells were washed three times with 1 ml KRH buffer containing 400 µM phloretin. Cells were lysed with 1 ml ice-cold 0.1 M NaOH, and aliquots were taken for determination of radioactivity and protein levels. 3-O-Methyl-D-glucose uptake assays were carried out using aliquots of 50 µl cell suspension at 50% cytocrit in 0.5% BSA/KRH buffer. Fifty microliters of radiolabeled sugar (100 µM cold 3-O-methyl-D-glucose containing 0.4 µCi 3-O-[14C]methyl-D-glucose) in KRH buffer was then added. Uptake was allowed to proceed for 10–60 sec, then was terminated by rapidly adding 1 ml KRH buffer containing 400 µM phloretin, followed by two washes with this buffer. Cells were lysed as described above, and radioactivity was counted.

Akt and AMPK phosphorylation
Three-milliliter aliquots of cardiomyocytes (~10% cytocrit) were washed with HES buffer [20 mM HEPES (pH 7.2), 1 mM EDTA, and 255 mM sucrose plus protease inhibitors (antipain, pepstatin A, and leupeptin; each at 1 µg/ml), 100 µM 4-(2-aminoethyl)benzenesulfonyl fluoride, and phosphatase inhibitors (10 mM sodium fluoride, 1 mM NaMO4, 200 µM Na3VO4, 58 nM cypermethrin, 5 µM dephostain, 100 nM okadaic acid, and 10 pM nuclear inhibitor of protein phosphatase-1; Calbiochem, La Jolla, CA)]. The cells were then centrifuged at 624 x gmax for 3 min. The pellets were solubilized with 0.4 ml 1% Triton X-100, 1% deoxycholic acid, and 0.2% sodium dodecyl sulfate (with the protease and phosphatase inhibitors described above) by rotation at 0–4 C for 50 min. The insoluble material was then removed after centrifugation at 17,530 x gmax. Forty to 60 µg solubilized protein were resolved by 8% SDS-PAGE and Western blotted using the following antibodies. Phospho-AMPK-{alpha} (Thr172), Akt1 and Akt2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and AMPK-{alpha} and phospho-Akt (Ser473; Cell Signaling Technology, Beverly, MA). Signals were detected by enhanced chemiluminescence (ECL) or advanced ECL and quantified using an Optichem detector with associated software (UV Products, Upland, CA).

Trafficking of photolabeled GLUT4
A biotin tag was introduced into GLUT4 using a photolabeling procedure (28, 29). For internalization trafficking experiments, cardiomyocytes were first cultured for 18 h with or without 1 mM metformin. Multiple 1-ml aliquots of cell suspension at 10% cytocrit were transferred to 35-mm diameter polystyrene dishes cooled to 18 C to slow transporter recycling. The photolabeling reagent GP15 (28, 30) was added for 2 min at a 500-µM final concentration. Cells were then irradiated at a wavelength of 300–350 nm for 1 min in a Rayonet RPR-100 reactor (Southern New England Ultra Violet Co., Branford, CT). After irradiation, cells for each condition were pooled, transferred to 50-ml tubes, and washed once with 50 ml KRH buffer (pH 6.0) containing 1% BSA and 2 mM glucose to remove excess reagent, then twice with 50 ml KRH buffer (pH 7.4), also containing 1% BSA and 2 mM glucose. For internalization experiments, the washed cells were then redivided into 1-ml aliquots at 10% cytocrit and incubated at 37 C to initiate internalization of the biotin-tagged GLUT4. At the indicated internalization time points, 100 µg/ml neutravidin (Pierce Chemical Co., Rockford, IL) was added for 5 min at 37 C to block those transporters that were still at the cell surface. The maximum signal of GLUT4, which internalized and escaped the surface quenching, was determined in each experiment from an insulin-stimulated sample that was returned to the basal state and was allowed to internalize the biotinylated GLUT4 for 40 min. At specified times of internalization, cells were then washed three times in 25 ml ice-cold KRH buffer. For the comparison of internalization rates, the biotin signal that escaped the surface neutravidin treatment was quantified as described below, and data are presented as a percentage of the maximum internalization.

For exocytosis experiments, cells were incubated in culture and labeled in the presence of insulin. Insulin-stimulated cells were photoaffinity labeled with GP15 and subsequently washed to remove insulin. They were then maintained at 37 C for 40 min to obtain maximal internalization. To begin determination of exocytosis, 90 µg/ml neutravidin was added. The rate of loss of the internal transporters as they became quenched by the surface neutravidin was determined at the indicated time points. To terminate exocytosis, the cells were washed three times in 25 ml ice-cold KRH buffer. The data are presented as a percentage of the maximum internalized biotinylated GLUT4 signal present at the beginning of the time course. The fractional change (f) in GLUT4 internalization with time (t) was curve-fitted to the equation: f = (kex + ken.exp(–t x (ken + kex)))/(ken + kex), and this was used to extract the endocytosis rate constant, ken. A single exponential function, f = exp(–t x kex), was used to calculate the exocytosis rate constant, kex.

Determination of levels of biotinylated GLUT4
After termination of trafficking and the washing steps described above, cardiomyocytes were solubilized in 1% Triton, 1% deoxycholic acid, and 0.2% sodium dodecyl sulfate in PBS (pH 7.2) with protease inhibitors (1 µg/ml antipain, aprotinin, pepstatin, and leupeptin) and 100 µM 4-(2-amino-ethyl)-benzenesulfonyl fluoride for 50 min. Insoluble material was removed by centrifugation at 20,000 x gmax for 20 min at 4 C. The biotinylated transporters from solubilized supernatants were precipitated with 50 µl of a 50% slurry of streptavidin-agarose beads (Pierce Chemical Co.) overnight at 4 C with rotation. The streptavidin-agarose beads were then washed three times with 1 ml PBS buffer containing 1% (wt/vol) Thesit (Sigma-Aldrich Corp., St. Louis, MO), three times with 1 ml PBS buffer containing 0.1% (wt/vol) Thesit, and once with 1 ml PBS buffer only. Bound biotinylated GLUT4 was eluted in 30 µl electrophoresis sample buffer [62.5 mM Tris-HCl (pH 6.7), 2% (wt/vol) sodium dodecyl sulfate, and 10% (vol/vol) glycerol] by heating at 95 C for 30 min. The eluate was collected, and the elution procedure was repeated. The resulting eluates were pooled, 20 mM dithiothreitol was added, and samples were loaded onto 10% SDS-PAGE gels and transferred to nitrocellulose. GLUT4 was detected with a polyclonal GLUT4 antibody and a horseradish peroxidase-linked secondary antibody using advanced ECL reagents. Chemiluminescence was quantified using an Optichem detector with associated software (UV Products).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Metformin treatment of cardiomyocytes increases transport activity of phosphorylated and nonphosphorylated sugar substrates
Transport of 2-deoxy-D-glucose in cardiomyocytes that were maintained in culture for 18 h was stimulated approximately 3- to 4-fold after acute insulin treatment (Fig. 1AGo). If cells were maintained in the continuous presence of insulin throughout the culture period, then the levels of transport activity were increased only slightly above basal levels. These data are consistent with the known effect of chronic insulin on other systems and, in particular, fat cells (17, 20, 31). The effects have been associated with the down-regulation of GLUT4 from the cell surface and with the routing of a high glucose flux through the hexosamine metabolism pathways (18). The down-regulation effect was partially reversible, and if cells were washed and restimulated with insulin, then some insulin resistance remained. However, transport did not increase to the levels that occurred after acute treatment of basal cells with insulin. To assess whether metformin could reverse these insulin resistance effects, cardiomyocytes were cultured in the presence of 1 mM metformin for 18 h (Fig. 1AGo). Under these conditions, the levels of 2-deoxy-D-glucose transport were elevated even in the absence of insulin to concentrations more than 3- to 5-fold higher than normal basal levels (Fig. 1Go, A and B). A 10-fold lower concentration of metformin in was less effective in raising basal transport activity after the 18-h incubation period (Fig. 1BGo). The stimulation of transport activity required a long incubation, because treatment of freshly isolated cardiomyocytes with metformin at 1 mM for 30 min or 3 h did not increase basal transport activity (Fig. 1CGo).


Figure 1
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  		FIG. 1. Metformin treatment of insulin-resistant cardiomyocytes elevates glucose transport activity. A, Insulin resistance was induced in cardiomyocytes by an 18-h treatment with 500 nM insulin in the presence of DMEM containing glucose. After the 18-h culture, the levels of 2-deoxy-D-glucose transport activities in basal (Ba) and acutely insulin-stimulated (In) cells were determined. The presence of high insulin [In(Chronic)] led to low transport activity that was only partially reversed by washing and fresh insulin treatment [In(Restim)]. Cardiomyocytes were treated with 1 mM metformin (Met) as indicated. Results are the mean ± SEM for six experiments. *, P < 0.05 vs. Ba – Met; §, P < 0.05 vs. In – Met; {ddagger}, P < 0.05 vs. Ba + Met; ¶, P < 0.05 vs. In + Met (by paired t test). B, Cardiomyocytes were incubated for 18 h either in the basal state (Ba) or at the indicated concentrations of metformin (Met). The response of basal cells to a subsequent acute insulin treatment was also examined (In). Results are the mean ± SEM of three experiments. C, Cardiomyocytes were incubated for 0.5 or 3 h with 1 mM metformin. Results are the mean ± SEM of three to five separate experiments.

 
Superimposed on the large increase in transport after the 18-h treatment with 1 mM metformin was a substantial acute insulin response. The 18-h chronic insulin treatment in the presence of metformin still led to an impaired acute response to insulin. Therefore, the insulin resistance was still present, but was superimposed on the very high transport activity due to metformin treatment.

To examine whether the changes in transport were associated with changes in the total amounts of either GLUT1 or GLUT4, we blotted for the presence of these proteins in cell lysates. The 18-h culture period resulted in a marked increase, compared with freshly isolated cells, in the total amount of GLUT1 present (Fig. 2AGo, top panels). However, the same increase was observed under all conditions examined. Consequently, neither the insulin resistance effects nor the metformin effect could be attributed to changes in the level of this protein. No changes in total levels of GLUT4 were observed under any of the culture conditions used in this study. The results were quantified over a series of experiments (Table 1Go). Similarly, in rat adipose cells maintained in culture, no changes in GLUT4 were observed (20). Some down-regulation of total cellular GLUT4 was observed in 3T3-L1 cells maintained in culture by some investigators (32), whereas others observed no changes (31, 33). The differences may be attributable to the culture conditions or the clone of 3T3-L1 cells used.


Figure 2
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  		FIG. 2. Effects of metformin on glucose transport activity are independent of transporter levels and substrate phosphorylation. A, Neither insulin resistance nor metformin stimulatory effects on glucose transport activity were due to changes in the total levels of GLUT1 or GLUT4. Total levels of GLUT1 (top panels) and GLUT4 (bottom panels) were measured in freshly isolated cells and after culture for 18 h in the absence (–) or presence (+) of 1 mM metformin (Met). In both cases, cells were treated with insulin acutely (In) during the culture (Ic) or were cultured in the presence of insulin, then washed and restimulated (Rs). B, Transport of the nonphosphorylated substrate, 3-O-methyl-D-glucose, was determined in cardiomyocytes maintained in culture for 18 h in the absence or presence of 1 mM metformin (Ba and Met, respectively). Transport was then determined for basal and metformin-treated cells and after an additional treatment with 30 nM insulin for 30 min (In). Results are the mean ± SEM from four experiments. *, P < 0.05 vs. Ba; §, P < 0.05 vs. Met (by paired t test). C, To determine whether metformin altered the affinity for 3-O-methyl-D-glucose, the inhibition of tracer 2-deoxy-D-glucose was determined in the presence of the indicated concentrations of 3-O-methyl-D-glucose. Results are the mean ± SEM from three experiments.

 

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  		TABLE 1. Cardiomyocyte culture conditions do not significantly alter total levels of GLUT4, AMPK, Akt1, or Akt2 but GLUT1 levels increase

 
Because 2-deoxy-D-glucose was metabolized to its phosphate derivative, and stimulatory effects of AMPK activators had been observed on the phosphorylation step (34), we also examined whether metformin treatment led to elevated transport levels of 3-O-methyl-D-glucose (a hexose analog that is very poorly phosphorylated). Metformin and acute insulin treatment both led to increased uptake of this substrate (by ~4-fold). The effects of metformin and insulin were additive (Fig. 2BGo), producing a stimulation of transport greater than that produced by either treatment alone. An elevation of transport activity at a tracer concentration (as used in this study) could be due to an increase in the affinity of the transport system for the transported sugar. To test this possibility, we measured tracer 2-deoxy-D-glucose transport at a range of competing 3-O-methyl-D-glucose concentrations (Fig. 2CGo). Neither insulin nor metformin treatment led to a change in the affinity for 3-O-methyl-D-glucose. Half-maximal inhibition occurred at approximately 10 mM for both treatments.

Metformin treatment of cardiomyocytes in culture leads to increased levels of Akt and AMPK
We examined whether metformin-induced changes in transport activity were associated with changes in signaling by the AMPK or Akt pathway by blotting for the total cellular levels of these kinases. We examined the extent to which they were activated using phospho-specific antibodies. The total levels of AMPK were not elevated by maintenance of cardiomyocytes in culture for 18 h (Fig. 3Go and Table 1Go). Neither the effects of insulin resistance nor those of metformin treatment were correlated with changes in total AMPK levels. However, when we determined the extent to which AMPK was phosphorylated, we found that the metformin treatment led to a 4.4-fold increase in phosphorylation at threonine 172 (Fig. 3Go, bottom panels) compared with freshly isolated cells (Fig. 3Go, middle panels). No changes in phosphorylated AMPK (compared with fresh cells) were observed in cardiomyocytes maintained in culture under conditions that led to insulin resistance (Fig. 3BGo, bottom panels).


Figure 3
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  		FIG. 3. Activation of AMPK by metformin in cardiomyocytes. A, The total levels of AMPK were unaltered after culture for 18 h in either the absence (–) or presence of 1 mM metformin (Met) or after chronic 18-h treatment with 500 nM insulin (Ic). B, Levels of phospho-threonine 172-AMPK were increased 4.4 ± 0.9-fold by 18-h culture with 1 mM metformin, but were not increased by metformin treatment of freshly isolated cells (top panels) or chronic 18-h treatment with 500 nM insulin (Ic) or after acute insulin stimulation (In) or restimulation after washing away the 500 nM insulin (Rs).

 
The total levels of Akt1 and Akt2 were unaltered by the culture conditions and were the same in fresh cells after metformin treatment and under conditions that induced insulin resistance in glucose transport activity (Fig. 4AGo, top two panels). The results were quantified over a series of experiments (Table 1Go). The effects of insulin, including down-regulation after chronic insulin treatment, were clearly attributable to the changes in Akt phosphorylation and matched well the corresponding changes in glucose transport activity (Fig. 4BGo). The signaling changes in Akt were quite well maintained after the culture period, and the robust stimulation of Akt observed in fresh cells was also observed after the 18-h culture period. Metformin treatment led to an increase in the phosphorylation of Akt at serine 473. This effect was less marked than the changes in phospho-AMPK under the same conditions. The stimulatory effects of insulin on the phosphorylation of Akt were much greater and were maintained in cells that had been cultured in the presence of metformin (Fig. 4BGo).


Figure 4
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  		FIG. 4. Activation of Akt by insulin in cardiomyocytes. A, Total levels of Akt1 and Akt2 were unaltered by either chronic 500-nM insulin treatment (Ic) or 1 mM metformin treatment (Met). B, Levels of Akt phosphorylated on serine 473 were determined in fresh cells (right panels) or after culture for 18 h (left panels) in the absence (–) or presence (+) of 1 mM metformin (Met). Cells were maintained in the basal state (Ba), treated acutely with insulin (In), maintained in culture with 500 nM insulin [In(Chronic)], or treated chronically with insulin, washed, then restimulated [In(Restim)]. Metformin stimulated Akt phosphorylation by 3.1 ± 0.4-fold. The bars are the mean ± SEM from four experiments, and a typical blot for one of these experiments is shown below the bars. The labeling of the experimental conditions is the same for the bars and the blot lanes. *, P < 0.05 vs. Ba (by paired t test).

 
Wortmannin treatment fully inhibited the insulin responses on glucose transport activity (Fig. 5AGo) and Akt phosphorylation (Fig. 5BGo), but did not inhibit the elevation in transport activity or Akt phosphorylation due to metformin treatment. Wortmannin was added after metformin, and we cannot eliminate the possibility that if wortmannin were present throughout the 18 h of metformin treatment then it might have inhibited the stimulation of Akt phosphorylation. Collectively, these data indicate that Akt can be phosphorylated over a long-term metformin treatment, and there may be some cross-talk between the AMPK and Akt signaling pathways. However, the effect of metformin on Akt phosphorylation is unlikely to be primarily responsible for the stimulated glucose transport activity.


Figure 5
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  		FIG. 5. Sensitivity of the metformin response in cardiomyocytes to PI 3-kinase inhibition. A, Cardiomyocytes were maintained in culture for 18 h in the absence or presence of 1 mM metformin (Met). Cells without metformin in the culture were subsequently treated acutely with insulin (In) or insulin plus wortmannin (In + wort). Cells with metformin in the culture were maintained with metformin during a subsequent incubation with wortmannin (Met + wort). The results are the mean ± SEM from three experiments. *, P < 0.05 vs. In (by unpaired t test); NS, not significant vs. Met B. The levels of phosphorylation of Akt on serine 473 were determined under the conditions described in A. Only the large 8-fold rise in p-Akt observed in insulin-treated cells was blocked by wortmannin. Metformin treatment also produced a smaller 3-fold increase in p-Akt. but this was not significantly reversed by subsequent wortmannin treatment. The bars are the mean ± SEM from three experiments, and a representative blot is shown in the top panel. *, P < 0.05 vs. Ia; NS, not significant vs. Met (by unpaired t test)

 
Metformin treatment of cardiomyocytes in culture leads to increased retention time of GLUT4 in plasma membrane
We have previously found that elevated levels of phosphorylated AMPK (due to hypoxia or mitochondrial inhibition) led to increased cell surface GLUT4 levels because of a reduction in the rate of its internalization (23). By contrast, the insulin response was associated with increased phosphorylation of Akt and an associated increase in exocytosis toward the plasma membrane. Because metformin treatment led to increased phosphorylation of both these proteins, we sought to determine whether the transport stimulations were primarily due to reduced endocytosis or increased exocytosis of GLUT4.

We first measured changes in glucose transport activity that followed the removal of an insulin stimulus. The insulin reversal condition was achieved by using a pH 6.0-buffer-wash step to remove insulin from its receptor (35). After 18-h culture under basal conditions, cells were stimulated with insulin. Insulin removal then resulted in a decline in glucose transport activity to the basal level over 40 min (Fig. 6AGo). The rate of reversal of transport activity was reduced by approximately 50% when cells were cultured for 18 h with metformin present.


Figure 6
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  		FIG. 6. Internalization of GLUT4 in cardiomyocytes is reduced by metformin treatment. A, Cardiomyocytes were maintained in culture for 18 h in the absence ({blacksquare}) or presence of 1 mM metformin ({blacktriangleup}). Cells were then stimulated with 5 nM insulin to recruit GLUT4 to the cell surface and washed to remove insulin (In), and the return of transporters to intracellular compartments and to the basal state (Ba) was assessed by measuring the decline in the rate of 2-deoxy-D-glucose transport activity. B, Cardiomyocytes were cultured in the absence ({blacksquare}) or presence ({blacktriangleup}) of 1 mM metformin. Basal cells were stimulated with insulin, whereas metformin-treated cells were maintained with metformin alone, then GLUT4 was biotin tagged with the photolabel GP15 (–A) in an aliquot of cells. The return of biotin-tagged GLUT4 to the basal state in separate aliquots of cells was assessed at the indicated times by first quenching the GLUT4 that remained at the cell surface with avidin (+A) and then precipitating the internalized transporters on streptavidin-agarose beads. Levels of precipitated GLUT4 were determined by Western blotting with a polyclonal GLUT4 antibody (top panel). The data in the plotted time course (bottom panel) are the mean ± SEM from six experiments.

 
To determine whether the reductions in transport activity were directly attributable to internalization of GLUT4, we biotin-tagged the protein with GP15 photolabel. When insulin was removed from cells that had been labeled in the insulin-stimulated state, biotin-tagged GLUT4 escaped avidin addition to the cells with a time course (Fig. 6BGo) that paralleled the decrease in transport activity (Fig. 6AGo). It was mainly the endocytosis rate constant that determined the shape of the internalization curve as the recycling or exocytosis rate is lowered by cutting off insulin signaling. Analysis of the endocytosis rate constant values derived from both transport and labeling data revealed that the endocytosis process was slowed by approximately 50% after metformin treatment (Fig. 7BGo).


Figure 7
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  		FIG. 7. Kinetics of GLUT4 exocytosis and endocytosis in metformin-treated cardiomyocytes. A, Cardiomyocytes were maintained in culture for 18 h in the absence ({blacksquare}) or presence of 1 mM metformin ({blacktriangleup}). Cells were then stimulated with 5 nM insulin to recruit GLUT4 to the cell surface and washed to remove insulin and return transporters to intracellular compartments and the basal state (Ba). Avidin was added to quench those internalized transporters that returned to the cell surface. The rate of loss (top panel) of those biotinylated transporters that escaped this avidin treatment was determined by precipitation on streptavidin agarose and Western blotting using a polyclonal GLUT4 antibody (bottom panel). Results are the mean ± SEM of three separate experiments. B, The internalization data in Fig. 6BGo were subjected to curve fitting to obtain the endocytosis rate constant (ken); the data in panel A were used to obtain the exocytosis rate constant (kex). Metformin leads to a significant slowing of GLUT4 endocytosis without significantly increasing the rate of exocytosis. Data are mean ± SEM from six endocytosis experiments and three exocytosis experiments. *, P < 0.05.

 
To measure exocytosis in cardiomyocytes, the cells were first treated with insulin to bring the transporters to the cell surface. They were then tagged with the GP15 photolabel and incubated in the absence of insulin to internalize the label and return to the basal state. Avidin was added to the cells to quench any GLUT4 that was retranslocated back to the plasma membrane upon stimulation. However, no elevation of the exocytosis rate was observed in cells that had been maintained in culture with metformin, and the rate of exocytosis was similar to the slow exocytosis rate observed in cells in the basal state (Fig. 7AGo). Curve fitting of the GLUT4 blotting data to a simple exponential time function gave the exocytosis rate constants, but no significant increase due to metformin treatment was found (Fig. 7BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Induction of insulin-resistant glucose transport in cardiomyocytes
Recent studies have identified insulin resistance as a risk factor for mortality of patients with heart failure (1, 2). The authors of these studies concluded that therapeutically targeting impaired insulin sensitivity may potentially be beneficial in patients with coronary heart failure. To examine this issue at the cellular level, we used a technique for inducing cellular insulin resistance. This procedure involves long-term treatment with insulin under conditions in which the medium contains high glucose concentrations (17). The use of this technique with other cell systems, such as adipocytes, has shown that there is down-regulation of GLUT4 from the cell surface. This may be an adaptation to the abundant glucose in the medium, but the mechanism has not been resolved (18). This change in glucose uptake has been correlated with changes in early signaling, including reduced tyrosine phosphorylation of the insulin receptor (19, 20) and insulin receptor substrate 1 and consequential reductions in stimulation of Akt coupled to the downstream PI 3-kinase (20). The data presented in this report indicate that the same down-regulation of glucose transport activity occurs in cardiomyocytes. This is evident using both phosphorylatable 2-deoxy-D-glucose and the poorly phosphorylated 3-O-methyl-D-glucose. The changes in glucose transport are not associated with changes in the total levels of GLUT1 or GLUT4 transporter isoforms. The GLUT1 level rises after culture, but metformin treatment does not alter this increase. We have previously found that metformin treatment of adipose cells does not alter GLUT1 trafficking (20) and have therefore only examined the possibility of an alteration in GLUT4 trafficking in this study. However, because GLUT1 is present at relatively high levels, we cannot rule out a contribution of GLUT1 trafficking to the increased hexose transport activity. As is the case in fat cell systems, the changes in insulin resistance are associated with down-regulation of phosphorylation of Akt by approximately 50%, but whether this reduction is sufficient to cause the corresponding decrease in transport activity is unclear, because it is generally assumed that there is an excess of Akt over that required to stimulate glucose transport activity. Glucose transport stimulation by insulin is one of the most sensitive acute responses, and this sensitivity has given rise to the ideas of spare receptor and spare signaling capacities (36).

Metformin treatment of cardiomyocytes circumvents impairments in glucose transport activity
Although cardiomyocyte glucose transport activity is not increased after short-term treatment with 1 mM metformin for up to 3 h, longer treatments do lead to increases in this activity (11). In this study we have shown that very large stimulations of transport activity follow an 18-h treatment with this drug. The concentrations of metformin required for this cellular response are 0.2–1 mM and are much higher than the doses of metformin used clinically. However, if clinical treatment leads to a gradual accumulation of this cationic drug in an acidic cellular compartment such as the outer mitochondrial membrane space, then the concentrations reached in this compartment may be similar to those used in vitro (13). We have addressed whether the stimulatory effect of metformin can reverse the insulin resistance induced by chronic insulin treatment in cell culture through a mechanism that impinges upon the insulin signaling pathway. Our data suggest that this is probably not the case, because the effects of chronic insulin treatment are additive with those of metformin. Although metformin leads to elevated levels of glucose transport activity, all the responses to insulin (including both the acute stimulatory response and the insulin resistance) are still present, but are superimposed on the elevated transport level. We have observed this additivity in the actions of insulin and metformin using both a phosphorylated and a nonphosphorylated sugar, and this suggests that the effects occur at the level of the transporter and not the subsequent hexose phosphorylation step. In addition, we have found that although the insulin responses are sensitive to the PI 3-kinase inhibitor, wortmannin, the responses to metformin are not. These studies indicate that separate signaling pathways are responsible for the responses to insulin and metformin.

The AMPK and Akt signaling pathways that regulate GLUT4 trafficking act independently on endocytosis and exocytosis
To examine the mechanism by which metformin leads to increased glucose transport activity, we measured the levels of total and of phosphorylated Akt and AMPK. The total levels of Akt1, Akt2, and AMPK were not elevated after this treatment. However, the levels of phosphorylation of both Akt and AMPK were increased. The increase in AMPK phosphorylation was slightly more marked and correlated with the large increase in glucose transport activity, but the increased phosphorylation of both kinases was increased by more than 3-fold. We cannot eliminate an involvement of activated Akt in metformin’s action on the stimulation of glucose transport, because the interactions between these two kinases are complex. There is evidence for cross-talk between the Akt and AMPK pathways. Several studies (23, 25, 26) have shown that insulin action via Akt leads to a reduction in AMPK phosphorylation.

The increase in Akt phosphorylation due to metformin action was much less than that due to insulin and appeared to be insufficient to trigger an increase in GLUT4 exocytosis. It may be that a threshold level of Akt activation is required, as has been suggested for the increase in phosphorylation of AS160, a downstream Akt substrate implicated in insulin-stimulated GLUT4 exocytosis (37, 38). However, in some respects the actions of metformin on glucose transport appear separate from those on Akt activation. First, the metformin response on transport is not sensitive to short-term wortmannin treatment; in addition, the responses to metformin are additive with those of insulin. These data are more consistent with an independent point of convergence with GLUT4 trafficking rather than convergence at a single signaling intermediate. The cross-talk between the Akt and AMPK pathways may provide sensitive feedback that dampens excess responses via either pathway, but this cross-talk does not appear to be responsible for the primary signaling events leading to independent action on GLUT4 trafficking.

Either an increase in GLUT4 exocytosis or a reduction in its endocytosis would result in increased transport activity. However, we found that metformin action converges on the GLUT4 internalization process and not on the exocytosis step. These results are consistent with the more marked elevation of AMPK than of Akt phosphorylation. Although Akt phosphorylation is increased, this stimulation is much less than that which occurs in response to insulin and appears to be insufficient to enhance exocytosis in the same manner as that which occurs in response to insulin. A convergence of metformin action at the endocytosis step can account for its additivity to the insulin response. The insulin stimulation of exocytosis via activation of Akt would lead to an increase in surface GLUT4, and this level would be further elevated if these transporters became trapped at the surface because their endocytosis was reduced. The residence time of the transporters at the surface would be increased, and there would therefore be increased flux of glucose into the cell. This blocking of GLUT4 endocytosis would be an energy-conserving step and would be an addition to the previously recognized enhancements in energy economy that follow signaling via the AMPK pathway (39, 40, 41).


   Footnotes
 
This work was supported by the Medical Research Council (United Kingdom) and the British Heart Foundation.

J.Y. and G.D.H. have nothing to declare.

First Published Online March 2, 2006

Abbreviations: AMPK, AMP-activated protein kinase; ECL, enhanced chemiluminescence; GLUT, glucose transporter; PI 3-kinase, phosphatidylinositol 3-kinase.

Received November 14, 2005.

Accepted for publication February 17, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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