Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion

Isabelle Leclerc,1 Wolfram W. Woltersdorf,1 Gabriela da Silva Xavier,1 Rebecca L. Rowe,1 Sarah E. Cross,2 Greg S. Korbutt,3 Ray V. Rajotte,3 Richard Smith,2 and Guy A. Rutter1

1Henry Wellcome Laboratories for Integrated Cell Signalling and Department of Biochemistry, School of Medical Sciences, University of Bristol, BS8 1TD Bristol; 2Academic Renal Unit, Southmead Hospital, University of Bristol, BS10 5NB Bristol, United Kingdom; and 3Department of Surgery, Surgical-Medical Research Institute, University of Alberta, Edmonton, Canada T6G 2N8

Submitted 21 November 2003 ; accepted in final form 3 February 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Metformin, a drug widely used in the treatment of type 2 diabetes, has recently been shown to act on skeletal muscle and liver in part through the activation of AMP-activated protein kinase (AMPK). Whether metformin or the satiety factor leptin, which also stimulates AMPK in muscle, regulates this enzyme in pancreatic islets is unknown. We have recently shown that forced increases in AMPK activity inhibit insulin secretion from MIN6 cells (da Silva Xavier G, Leclerc I, Varadi A, Tsuboi T, Moule SK, and Rutter GA. Biochem J 371: 761–774, 2003). Here, we explore whether 1) glucose, metformin, or leptin regulates AMPK activity in isolated islets from rodent and human and 2) whether changes in AMPK activity modulate insulin secretion from human islets. Increases in glucose concentration from 0 to 3 and from 3 to 17 mM inhibited AMPK activity in primary islets from mouse, rat, and human, confirming previous findings in insulinoma cells. Incubation with metformin (0.2–1 mM) activated AMPK in both human islets and MIN6 {beta}-cells in parallel with an inhibition of insulin secretion, whereas leptin (10–100 nM) was without effect in MIN6 cells. These studies demonstrate that AMPK activity is subject to regulation by both glucose and metformin in pancreatic islets and clonal {beta}-cells. The inhibitory effects of metformin on insulin secretion may therefore need to be considered with respect to the use of this drug for the treatment of type 2 diabetes.

5'-adenosine monophosphate-activated protein kinase; human islets of Langerhans; MIN6 cells


5'-AMP-ACTIVATED PROTEIN KINASE (AMPK) is a multisubstrate, heterotrimeric serine/threonine protein kinase consisting of one catalytic {alpha}-subunit and two regulatory {beta}- and {gamma}-subunits (10, 47). AMPK activity is regulated allosterically by AMP and through reversible phosphorylation at Thr172 of the {alpha}-subunit (16–18, 22, 56) by the upstream kinase LKB1 [derived as a code name for the Peutz-Jeghers syndrome-causative gene (3), also termed STK11 (for serine/threonine kinase 11) (24)]. AMPK is a sensor of cellular energy charge that is activated by ATP depletion and the consequent increase in intracellular AMP (26). AMPK activation results in the inhibition of ATP-consuming pathways such as fatty acid and cholesterol biosynthesis by phosphorylation of acetyl-CoA carboxylase (ACC) and hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase) (5) respectively, and promotes ATP production by stimulating fatty acid oxidation (26).

Metformin is a widely used antidiabetic agent whose cellular mechanism of action was, until recently, obscure. However, Zhou et al. (58) showed that metformin activates AMPK in rat hepatocytes and skeletal muscle, an effect that may account for the effect of metformin on increased muscle glucose transport, decreased hepatic glucose output, and beneficial blood lipid profile (12). Metformin action on insulin release in vitro is more controversial, however (33, 44). In vivo, metformin decreases plasma insulin levels in diabetic subjects (12) and in patients with polycystic ovary syndrome (37). Up to now, these actions of metformin have generally been explained by an increase in peripheral insulin sensitivity and, hence, a decrease in blood glucose levels rather than a direct inhibition of pancreatic insulin release.

Leptin is secreted by adipocytes and stimulates fatty acid oxidation (38) and glucose uptake into muscle cells (25, 34) and prevents accumulation of lipids in nonadipose tissues (50). AMPK has recently been identified as the principal mediator of the effects of leptin on fatty acid metabolism in skeletal muscle (35), although this does not seem to be the case in heart muscle (1). In pancreatic islet {beta}-cells, the effects of leptin are controversial, since it has been shown to have no effect, to inhibit, or to stimulate insulin secretion (28).

AMPK is now considered as a potentially interesting pharmacological target for the treatment of type 2 diabetes (41), since activation of the enzyme has been shown to decrease gluconeogenesis and to increase muscle glucose transport, both in vitro (19, 29, 52) and in vivo (23). Although we (8) and others (43) have previously shown that AMPK activity is regulated by glucose in clonal pancreatic {beta}-cell lines, nothing is known about the regulation of this enzyme by glucose, or by metformin or leptin, in intact rodent or human islets of Langerhans. In this report, we tested the hypothesis that AMPK activity is regulated by these agents in primary islets and is involved in the regulation of insulin release. We show that glucose inhibits AMPK activity in islets isolated from three distinct species, namely mouse, rat, and human. In contrast, metformin stimulates AMPK activity and inhibits glucose-stimulated, but not KCl-induced, insulin secretion in both human islets and MIN6 {beta}-cells. Leptin, however, has no effect on AMPK activity or insulin secretion in MIN6 cells or rat islets.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Materials. Collagenase (type V), Histopaque-1077, -1083, and -1119 solutions, and metformin were from Sigma. Mouse recombinant leptin was from Sigma and Calbiochem. 5-Aminoimidazole-4-carboxamide riboside (AICAR) was from Toronto Research Chemicals (Toronto, ON, Canada).

Animals. Wild-type CD-I mice (20–25 g) and Wistar rats (150–200 g) were used for islet isolation and killed by cervical dislocation immediately before the islet isolation procedure (see Cell culture and islet isolation). All animal procedures were in accordance with the British Home Office Animals (Scientific Procedures) Act, 1986.

Antibodies. Guinea pig anti-insulin antibody was from Dako. Sheep anti-AMPK-{alpha}1 and -{alpha}2, and rabbit anti-AMPK-{beta}1/2 antibodies were kindly provided by Dr. D. Carling (MRC Clinical Sciences, London, UK). Sheep anti-phospho-AMPK (Thr172) antibody was a generous gift of Prof. D. G. Hardie (Dept. of Biochemistry, University of Dundee, Scotland, UK). Rabbit anti-phospho-ACC (Ser79) antibody was purchased from Upstate Biochemicals (Lake Placid, NY).

Cell culture and islet isolation. MIN6 cells were used between passages 18 and 30 and grown in Dulbecco's modified Eagle's medium (DMEM) containing 25 mM glucose and supplemented with 15% heat-inactivated fetal calf serum (FCS), 4 mM L-glutamine, 100 µM 2-mercaptoethanol, 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere at 37°C with 5% CO2 unless specified otherwise. Mice and rats were killed by cervical dislocation, and collagenase [0.5 mg/ml in Hanks' balanced salt solution (HBSS)] was injected into the pancreatic duct (2.5 ml/mouse, 8 ml/rat). The distended pancreata were then incubated in a shaking water bath at 37°C in 0.5 mg/ml collagenase in HBSS for 10–20 min, and the islets were recovered by Histopaque density gradient centrifugation. Rodent islets were >=85% pure as assessed by dithizone staining and were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FCS and antibiotics. Human islets were obtained from cadaveric donors' pancreata as described (45). Unless otherwise stated, human islets were of >75% purity, with most of the remaining cellular contamination amylase positive, and were cultured in DMEM containing 25 mM glucose supplemented with 15% FCS and antibiotics for 1–7 days before experiments.

Adenoviruses. Adenoviruses encoding for enhanced green fluorescent protein (eGFP) only, hereafter named pAd-GFP (null), constitutively active AMPK (pAd-AMPK-CA), and dominant negative AMPK (pAd-AMPK-DN), have been described elsewhere (9). AMPK adenoviruses also express eGFP under a distinct cytomegalovirus promoter. Islets were infected at a multiplicity of infection of 100 viral particles/cell.

Immunohistochemistry. Paraffin-embedded rat pancreas slice sections on glass slides were dewaxed, rehydrated, and incubated for 6 min in 10% H2O2 to block endogenous peroxidase activity. The sections were then first incubated for 15 min in 3% BSA to block nonspecific binding and then for 30 min with guinea pig anti-insulin (1:1,000) or sheep anti-AMPK ({alpha}1, 1:500; {alpha}2, 1:100), washed three times in PBS, and incubated for 20 min with anti-guinea pig or anti-sheep biotinylated secondary antibodies (1:200) and stained using the avidin-biotin complex (ABC) method with peroxidase and diaminobenzidine (DAB) as the chromagen (Vector Laboratories, Burlingame, CA). Dissociated human islet cells (~50% pure) were double-stained using the ABC-DAB method for AMPK staining (as above) and FITC-conjugated secondary antibodies for insulin (1:200).

AMPK activity assay. MIN6 cells were cultured in 12-well plates in experimental conditions, washed twice in ice-cold PBS, and scraped into 200 µl of ice-cold lysis buffer [in mM: 50 Tris·HCl (pH 7.4, 4°C), 250 sucrose, 50 NaF, 1 Na pyrophosphate, 1 EDTA, 1 EGTA, 1 DTT, 0.1 benzamidine, and 0.1 PMSF, 5 µg/ml soybean trypsin inhibitor, and 1% (vol/vol) Triton X-100]. Extracts were centrifuged (13,000 g, 5 min, 4°C), and protein concentration was determined using a bicinchoninic acid protein assay reagent from Pierce. AMPK activity was determined using 5 µg of whole extract and the synthetic peptide SAMS (HMRSAMSGLHLVKRR) as substrate (8). Islets were cultured in experimental conditions, and batches of 100 islets were lysed in 25 µl of ice-cold lysis buffer and centrifuged as above. Results are expressed in picomoles of 32P incorporated per microgram of protein per minute (pmol·µg–1·min–1) or as a percentage of control conditions.

Western blot analysis. MIN6 cells or mouse islets were cultured and lysed as for AMPK activity. Fifty micrograms of whole cellular extracts were denatured for 5 min at 100°C in 2% SDS and 5% {beta}-mercaptoethanol, resolved by 10% SDS-PAGE, and transferred to PVDF membranes before immunoblotting, as described in Ref. 31. Sheep anti-phospho-AMPK antibody was used at a dilution of 1:500, rabbit anti-phospho-ACC antibody was used at 1:125 dilution, and rabbit anti-AMPK-{beta}1/2 was used at 1:5,000 dilution. Intensities were measured by digital scanning of gels and quantified using ImageJ (ImageJ@list.nih.gov).

Insulin secretion assay. MIN6 cells seeded in 12-well plates and preincubated as indicated in the figure legends were then incubated for 30 min in 1 ml of Krebs-HEPES-bicarbonate (KHB) solution [in mM: 130 NaCl, 3.6 KCl, 1.5 CaCl2, 0.5 MgSO4, 0.5 KH2PO4, 2 NaHCO3, 10 HEPES,and 0.1% (wt/vol) BSA, pH 7.4] at 37°C containing the indicated glucose concentration in the presence or absence of other additions as given. Human and rat islets were either left uninfected or infected with null, AMPK-CA, or AMPK-DN adenoviruses, incubated for 48 h in RPMI 1640, and then divided in groups of five islets per condition and incubated for 20 min in 1 ml of KHB solution at the indicated glucose concentrations. Total insulin content was extracted into 1 ml of acid-ethanol-Triton solution [1.5% (vol/vol) HCl, 75% (vol/vol) ethanol, 0.1% (vol/vol) Triton X-100]. Secreted insulin and total insulin were measured using radioimmunoassay by competition with 125I-labeled insulin (Linco Research, St. Charles, MO).

Statistics. Data are given as means ± SE of at least three independent experiments. Comparisons between means were performed with Student's t-test for paired data by use of Microsoft Excel software.


    RESULTS
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 MATERIALS AND METHODS
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AMPK-{alpha} subunits are expressed at low levels in the endocrine pancreas. Immunostaining for the catalytic AMPK-{alpha}1 and -{alpha}2 subunits was performed on whole rat pancreata sections (Fig. 1A) and on dissociated human islet cells (Fig. 1B). For the whole rat pancreata staining, consecutives slices were examined. The first slices (Fig. 1A, left) were stained with anti-insulin antibody to localize the islets of Langerhans, and the second slices (Fig. 1A, right) were stained with anti-AMPK antibodies directed against the {alpha}1- or {alpha}2-subunits as indicated. Although staining for each isoform was evident in islets of Langerhans, the level of expression of AMPK subunits was lower than in the surrounding exocrine tissue or than in red blood cells (black arrows). Staining for AMPK-{beta}1/2 subunits in mouse pancreas slices showed the same, relatively low level of expression in islets compared with exocrine tissue or red blood cells (not shown). Similarly, in dissociated human islet cells (Fig. 1B) costained with anti-insulin and anti-AMPK-{alpha}1 or -{alpha}2, insulin-positive cells (in green; Fig. 1B, left) were less intensely stained for AMPK (in brown; Fig. 1B, right) than the other cell types, which were mainly amylase positive (45).



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Fig. 1. 5'-AMP-activated protein kinase (AMPK)-{alpha} subunit expression in whole rat pancreas and in dissociated human islets. A: immunohistochemistry for AMPK-{alpha}1 and -{alpha}2 subunits and insulin was performed on consecutive rat pancreata slices (see MATERIALS AND METHODS). Left: anti-insulin staining for islet localization; right: anti-AMPK-{alpha}1 and -{alpha}2 subunits staining as indicated. Black arrows indicate red blood cells. B: human dissociated islet cells were costained with anti-insulin (left, green signal) and anti-AMPK-{alpha}1 or -{alpha}2 subunits (right, brown signal), as described in MATERIALS AND METHODS. Scale bars, 50 µm (A) or 30 µm (B). Note that the fields selected in B were enriched in cells displaying strong staining for AMPK and likely to correspond to acinar (amylase-positive) cells in the preparation used.

 
AMPK activity is inhibited by glucose in intact rodent and human islets of Langerhans. To determine whether AMPK activity in primary islets was subject to regulation by changes in cellular energy status, as previously demonstrated in insulinoma cells (9, 43), we examined the activity of the enzyme at different extracellular glucose concentrations. Rat islets were incubated for 60 min (Fig. 2A) or overnight (16 h; Fig. 2B) in the presence of 0, 3, or 17 mM glucose or 17 mM glucose plus the cell-permeant activator AICAR (4 mM) (7), as indicated. Mouse islets (Fig. 2, C and E) were incubated overnight in 3 or 17 mM glucose, and human islets (Fig. 2D) were incubated overnight in 0, 3, or 17 mM glucose or 17 mM glucose plus 4 mM AICAR. AMPK activity decreased as the glucose concentration increased over the physiological range from 0 to 17 mM, and incubation with AICAR reactivated AMPK in the presence of high glucose concentration. Figure 2E shows that inhibition of AMPK activity in mouse islets with increased glucose concentration from 3 to 17 mM was due to dephosphorylation of the AMPK-{alpha} subunit.



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Fig. 2. Glucose regulates AMPK activity and phosphorylation in intact rodent and human islets. Rat islets were incubated either for 1 h in Krebs-HEPES-bicarbonate (KHB; A) or overnight (16 h) in DMEM (B) containing indicated concentrations of glucose and 5-aminoimidazole-4-carboxamide riboside (AICAR). Mouse (C and E) and human (D) islets were incubated overnight in indicated concentrations of glucose and AICAR. Cell lysis, AMPK activity assay, and immunoblotting were performed as described in MATERIALS AND METHODS. AMPK activity (A-D) was measured against the SAMS peptide. AMPK phosphorylation (E) was assessed using anti-phospho-AMPK (Thr172) antibody, and total AMPK content was assessed using anti-AMPK-{beta}1/2 antibody. Data represent means ± SE of >=4 independent experiments.

 
AMPK overexpression inhibits insulin secretion from human pancreatic islets. Our previous work in MIN6 cells (9, 48) has shown that the forced activation of AMPK inhibits insulin secretion. To determine whether this phenomenon also obtains in primary human islets, the latter were infected with adenoviruses encoding eGFP only (null virus), AMPK-CA (the primary sequences of AMPK-{alpha}1 and -{alpha}2 are identical over the region used here, amino acids 1–312) or AMPK-DN (Fig. 3, B and C). Because confocal microscopy revealed that cells in the central core of large (>150 µm) mouse (Fig. 3A, bottom) or human (not shown) islets were infected (i.e., expressed eGFP) poorly (13), we performed all subsequent adenoviral experiments on islets of <100 µm in diameter to achieve 60% infection efficiency or better (Fig. 3A, top).



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Fig. 3. Forced changes of AMPK activity modulate insulin secretion in human islets. A: mouse islets were infected with enhanced green fluorescent protein (pAd-GFP; null), at a multiplicity of infection of 100 for 48 h in RPMI 1640 containing 11 mM glucose. Note that efficient infection of cells was apparent only in smaller islets (<100 µm in diameter; top). B and C: human islets of <100 µm in diameter were infected with indicated adenoviruses as in A before measurements of AMPK activity (B) or insulin secretion (C), as described in MATERIALS AND METHODS. CA, constitutively active; DN, dominant negative. Data in B and C represent means ± SE of 3 separate experiments. *P < 0.05 for effects of AMPK viruses compared with null virus.

 
Figure 3B shows AMPK activity in uninfected human islets or in islets infected with null, AMPK-CA and AMPK-DN adenoviruses. No difference in AMPK activity was apparent after adenoviral infection with the null virus compared with noninfected islets. In contrast, infection with AMPK-CA increased measured AMPK activity by ~25% (n = 3 separate experiments, P < 0.05) although the infection with AMPK-DN decreased it by ~10% (n = 3, P < 0.05). Figure 3C shows the secretion of insulin from human islets after adenoviral infection with null, AMPK-CA, or AMPK-DN adenoviruses. The overexpression of AMPK-CA decreased insulin release, whereas the overexpression of AMPK-DN increased insulin secretion from the islets. It should be noted that the more substantial activation of insulin secretion (~25%; Fig. 3C) compared with the inhibition of AMPK (~10%; Fig. 3B) likely reflects the facts 1) that the virus efficiently infects only ~60% of cells even within the small (<100 µm) islets used (Fig. 3A), corresponding to the most glucose-responsive {beta}-cells at the islet periphery (13) and 2) that AMPK activity is already partially inhibited at the glucose concentration employed (11 mM; Leclerc I, unpublished results).

Metformin stimulates AMPK phosphorylation and inhibits glucose-stimulated insulin secretion from pancreatic MIN6 {beta}-cells and human islets. Because metformin has been shown to activate AMPK in other tissues (35, 58), we explored the regulation of AMPK activity by this agent in MIN6 {beta}-cells and human islets. After a 16-h incubation, metformin increased the activity of AMPK in MIN6 cells in a dose-dependent manner at both 3 and 17 mM glucose (Fig. 4A). The metformin doses used were higher than steady-state plasma levels of metformin (2), but studies in rat have shown that tissue levels are severalfold higher than in plasma (53), in part due to the substantial accumulation of the drug by mitochondria (see DISCUSSION). Furthermore, it is recognized that metformin actions in vitro require high doses and are slow in onset due to low rates of plasma membrane transport (2, 58). Correspondingly, a shorter incubation period of 60 min with metformin did not increase AMPK activity, whereas the cell-permeant AMP analog AICAR increased it approximately twofold under these conditions (Fig. 4B).



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Fig. 4. Metformin stimulates AMPK activity and AMPK and acetyl-CoA carboxylase (ACC) phosphorylation and inhibits insulin secretion in MIN6 cells. MIN6 cells were cultured for 16 h (A, C, and D) in DMEM or 60 min in KHB (B) containing indicated concentrations of glucose, metformin, and AICAR before AMPK activity measurements (A and B), immunoblotting (C), or insulin secretion assays (D) as described in MATERIALS AND METHODS. See RESULTS for quantitation of immunoblots (C). D: results represent means ± SE of >=3 separate experiments: ##P < 0.005 for effect of 17 mM glucose; *P < 0.05 and **P < 0.005 for effect of 1 mM metformin or AICAR on insulin secretion. Note that basal release of insulin (100%) corresponded to ~0.2% of total cellular insulin content/30 min.

 
The stimulation of AMPK activity by metformin was accompanied by increased phosphorylation of the AMPK-{alpha} subunits on Thr172 as well as increased phosphorylation of the downstream target ACC on Ser79 (Fig. 4C), with no changes in the total amount of either protein as assessed by immunoblotting using antibodies to nonphosphorylated epitopes of either protein (Leclerc I, unpublished data). Thus quantitation of data from two separate experiments revealed an increase in the amount of AMPK phosphorylated at Thr172 in the presence of metformin of 59 and 66% at 3 and 17 mM glucose, respectively. In separate experiments, phospho-ACC (Ser79) immunoreactivity in extracts of cells incubated at 17 mM glucose was 72.4 ± 1.1% of that measured in cells maintained at 3 mM glucose (means ± SE; n = 3 experiments, P < 0.001), in line with previous results (9). Whereas 1 mM metformin had no significant effect on phospho-ACC levels at 3 mM glucose, the presence of the drug increased ACC phosphorylation at Ser79 to 118 ± 10% of basal values (n = 3 experiments, P < 0.05) at 17 mM glucose.

Because activation of AMPK via the use of AICAR or by overexpression of a constitutively active form of the kinase inhibits glucose-stimulated insulin secretion in MIN6 cells (9), we postulated that metformin may also reduce secretion. As shown in Fig. 4D, incubation of MIN6 cells with 1 mM metformin for 16 h blunted insulin secretion compared with control cells at both 3 and 17 mM glucose. Metformin also stimulated AMPK activity and inhibited insulin secretion from human islets, as shown in Fig. 5, A and B, respectively.



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Fig. 5. Metformin activates AMPK and inhibits insulin secretion from human islets. Human islets were cultured for 16 h at indicated concentrations of glucose and 1 mM metformin before assay of insulin during a subsequent 30-min incubation (see MATERIALS AND METHODS). Cell lysis, AMPK activity (A), and insulin secretion (B and C) were assayed as described in MATERIALS AND METHODS. Data were obtained from 3 (A and B) or a single (C) islet preparation. ##P < 0.005 for effect of glucose; **P < 0.005 for effect of metformin. Where indicated, KCl was present at 30 mM.

 
To determine whether the loss of responsiveness to glucose caused by metformin may be due to a general decrease in the viability of cell or islet preparations, we examined the impact of the drug on the nutrient-independent stimulation of secretion elicited by a depolarizing concentration of KCl. As an argument against any loss of cell viability as the underlying cause, metformin had no impact on the stimulation of secretion provoked by 30 mM KCl from either MIN6 cells (Fig. 4D) or human islets (Fig. 5C).

Leptin does not activate AMPK activity in MIN6 cells. Mouse recombinant leptin failed to stimulate AMPK activity in MIN6 {beta}-cells at either 3 or 17 mM glucose concentrations (Table 1).


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Table 1. Effects of leptin on AMPK activity in MIN6 pancreatic {beta}-cells

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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AMPK activity is regulated by glucose in primary rodent and human islets. It is now well established that glucose inhibits AMPK activity in several insulinoma {beta}-cell lines (8, 43). A concern, however, has been that the regulation of the enzyme may be a feature peculiar to immortalized, clonal {beta}-cells. Indeed, AMPK activity is inhibited by glucose in mhAT3f hepatoma cells (30) and by insulin in Fao hepatoma cells (54) but is completely insensitive to these agents in primary rat or mouse hepatocytes (14, 30, 55) (Leclerc I and Kahn A, unpublished observations). Here, we examined this question in four distinct insulin-secreting cell preparations. First, we used the relatively well-differentiated {beta}-cell line MIN6 (36) to assess the regulation and role of changes in AMPK activity in {beta}- vs. other islet cell types. Second, human islets were employed 1) because these could be obtained in large quantities, facilitating biochemical measurements of changes in AMPK activity by direct phosphotransfer (SAMS peptide) assay and 2) because they allowed the assessment of the role of AMPK activity changes in islets from humans. Third, primary rat or mouse islets, which could be isolated in much smaller numbers than human islets but were of more reproducible quality (i.e., glucose responsiveness), allowed a large series of experiments to be performed on both AMPK activity and insulin secretion. In most respects, the regulation and role of AMPK in each preparation were essentially similar, demonstrating the conservation of this mechanism in three different mammalian species. However, we did note that absolute levels of AMPK catalytic subunits, as assessed by immunocytochemical or biochemical assay, were somewhat higher in the MIN6 cell line than in either human or rodent islets (Leclerc I, unpublished data).

The present study thus demonstrates for the first time that AMPK activity is regulated by glucose, over the physiological range of concentrations, in primary rat and human islets of Langerhans (Fig. 2). It should be stressed that the demonstration of these changes required close attention to several aspects of the islet isolation and incubation protocols. First, any contamination of fresh rat islet preparations with exocrine tissue tended to markedly increase the measured AMPK activity, which was not suppressible by glucose. This is consistent with the higher levels of the enzyme in the exocrine tissue (Fig. 1). However, maintenance of islets in culture for >=3 days before the assay was found to overcome this problem, presumably by allowing time for contaminating both exocrine tissue and the hypoxic central cores of large islets (51), to necrose (26).

Our previous studies (9, 48) suggest that inhibition of AMPK may play an important role in the activation of insulin secretion at elevated glucose concentrations. The present work shows that changes in AMPK activity are also likely to modulate insulin secretion from intact human islets. Thus adenoviral overexpression of AMPK-CA and AMPK-DN inhibits and stimulates, respectively, insulin release from human islets (Fig. 3C). What mechanisms may underlie the effect of AMPK activation to inhibit insulin secretion? Increases in glucose concentration are believed to activate insulin release by enhancing ATP synthesis (27) and closing ATP-sensitive K+ (KATP) channels (4). Subsequent depolarization of the plasma membrane (21) and the opening of voltage-sensitive (L-type) Ca2+ channels (42) cause insulin-containing vesicles to fuse at the plasma membrane (40). In addition, KATP channel-independent "amplifying" effects may also contribute to the stimulation of release independently of changes in intracellular Ca2+ concentration (20). In MIN6 cells, AMPK-CA appears to inhibit insulin release by interfering with both glucose metabolism (9) and the recruitment of insulin-containing vesicles to the plasma membrane (48). Future studies will be required to determine whether similar mechanisms are operative in primary {beta}-cells and islets.

Metformin activates AMPK in islets. Metformin is a drug widely used to treat type 2 diabetes. Although its precise molecular mechanism of action has remained elusive for decades (41), an action to inhibit complex I of the respiratory chain and, hence, mitochondrial respiration and ATP synthesis, has been proposed (41). Although metformin is generally considered to have no direct effect on the pancreatic islet {beta}-cell, an early report demonstrated a dose-related inhibition of glucose-stimulated insulin secretion and insulin biosynthesis by biguanides (44). Moreover, basal and glucose-stimulated insulin plasma levels are consistently decreased in metformin-treated patients (11), an observation usually attributed to the increase in peripheral insulin sensitivity (41). Here, we show that metformin activates AMPK activity in MIN6 cells and human islets of Langerhans and inhibits insulin release. It is likely that metformin, which is positively charged at physiological pH, needs to accumulate inside mitochondria as much as 1,000-fold (39) to exert these actions. Thus, although relatively high concentrations of metformin were used in the present studies, much lower concentrations, close to those likely to be found in blood plasma (2), may be sufficient to activate islet cell AMPK and inhibit insulin secretion after more extended incubations.

AMPK activity is insensitive to leptin in {beta}-cells. The reported effects of leptin (57) on the pancreatic {beta}-cell are complex and controversial (28). Because leptin has recently been shown to activate AMPK in skeletal muscle (35), it seemed important here to determine the effects of this hormone on AMPK activity and insulin secretion in pancreatic {beta}-cells. We did not, however, detect any significant increase in AMPK activity in response to leptin after incubation of MIN6 cells at either low (3 mM) or high (17 mM) glucose (Table 1) or in rat islets incubated overnight in the presence of 10 nM leptin (not shown). These results are reminiscent of the reported absence of leptin-mediated increase in AMPK activity in isolated working rat hearts (1). These data are consistent with the view that leptin signaling might stimulate AMPK activity only in the presence of the {gamma}3-subunit, which appears to be expressed exclusively in skeletal muscle (1, 6).

In conclusion, extensive clinical studies (15) have demonstrated that metformin treatment leads to a reduction of diabetes-related end points, diabetes-related death, and all-cause mortality compared with conventional therapy. Furthermore, metformin can delay the onset of overt diabetes in the Zucker diabetic fatty rat by increasing insulin sensitivity (46). In contrast, the present and previous (8, 9) data suggest that sustained activation of AMPK in the pancreatic {beta}-cell may be detrimental for the maintenance of {beta}-cell phenotype. Correspondingly, a significant decline of {beta}-cell function with time in type 2 diabetes (49) and a better preservation of {beta}-cell function with diet alone (32) have both been reported. Together, these observations suggest that the impact of metformin on insulin secretion should be considered in selecting an appropriate therapy for individual diabetics. The present findings also suggest that drugs able to activate AMPK by stimulating leptin signaling, a mechanism apparently absent in {beta}-cells but operative in skeletal muscle (1), may be of greater value for the treatment of type 2 diabetes than those that act through less specific mechanisms.


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This work was supported by The Wellcome Trust (UK), the Canadian Institutes for Health Research, and the Alberta Heritage Foundation for Medical Research. Funding for human islet isolation was provided by donations from the Bristol and Weston branches of Diabetes UK and the North Bristol NHS Trust. I. Leclerc and G. A. Rutter thank the Wellcome Trust for an Advanced Fellowship and a Research Leave Fellowship, respectively.


    FOOTNOTES
 

Address for reprint requests and other correspondence: I. Leclerc, Dept. of Biochemistry, School of Medical Sciences, Univ. of Bristol, BS8 1TD Bristol, UK (E-mail: i.leclerc{at}bristol.ac.uk).

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.


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 ABSTRACT
 MATERIALS AND METHODS
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 REFERENCES
 

  1. Atkinson LL, Fischer MA, and Lopaschuk GD. Leptin activates cardiac fatty acid oxidation independent of changes in the AMP-activated protein kinase-acetyl-CoA carboxylase-malonyl-CoA axis. J Biol Chem 277: 29424–29430, 2002.[Abstract/Free Full Text]
  2. Bailey CJ and Turner RC. Metformin. N Engl J Med 334: 574–579, 1996.[Free Full Text]
  3. Boudeau J, Sapkota G, and Alessi DR. LKB1, a protein kinase regulating cell proliferation and polarity. FEBS Lett 546: 159–165, 2003.[CrossRef][ISI][Medline]
  4. Bryan J and Aguilar-Bryan L. The ABCs of ATP-sensitive potassium channels: more pieces of the puzzle. Curr Opin Cell Biol 9: 553–559, 1997.[CrossRef][ISI][Medline]
  5. Carling D, Zammit VA, and Hardie DG. A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis. FEBS Lett 223: 217–222, 1987.[CrossRef][ISI][Medline]
  6. Cheung PCF, Salt IP, Davies SP, 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.[CrossRef][ISI][Medline]
  7. Corton JM, Gillespie JG, Hawley SA, and Hardie DG. 5-Aminoimidazole-4-carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells? Eur J Biochem 229: 558–565, 1995.[Abstract]
  8. Da Silva Xavier G, Leclerc I, Salt IP, Doiron B, Hardie DG, Kahn A, and Rutter GA. Role of AMP-activated protein kinase in the regulation by glucose of islet beta cell gene expression. Proc Natl Acad Sci USA 97: 4023–4028, 2000.[Abstract/Free Full Text]
  9. Da Silva Xavier G, Leclerc I, Varadi A, Tsuboi T, Moule SK, and Rutter GA. Role for AMP-activated protein kinase in glucose-stimulated insulin secretion and preproinsulin gene expression. Biochem J 371: 761–774, 2003.[CrossRef][ISI][Medline]
  10. Davies SP, Hawley SA, Woods A, Carling D, Haystead TA, and Hardie DG. Purification of the AMP-activated protein kinase on ATP-gamma-sepharose and analysis of its subunit structure. Eur J Biochem 223: 351–357, 1994.[Abstract]
  11. DeFronzo RA. Pharmacologic therapy for type 2 diabetes mellitus. Ann Intern Med 131: 281–303, 1999.[Abstract/Free Full Text]
  12. DeFronzo RA and Goodman AM. Efficacy of metformin in patients with non-insulin-dependent diabetes mellitus. The Multicenter Metformin Study Group. N Engl J Med 333: 541–549, 1995.[Abstract/Free Full Text]
  13. Diraison F, Parton L, Ferre P, Foufelle F, Briscoe CP, Leclerc I, and Rutter GA. Over-expression of sterol regulatory element binding protein-1c in rat pancreatic islets induces lipogenesis and decreases glucose-stimulated insulin release: modulation by 5-aminoimidazole-4-carboxamide ribonucleoside. Biochem J 378: 769–778, 2004.
  14. Foretz M, Carling D, Guichard C, Ferre P, and Foufelle F. AMP-activated protein kinase inhibits the glucose-activated expression of fatty acid synthase gene in rat hepatocytes. J Biol Chem 273: 14767–14771, 1998.[Abstract/Free Full Text]
  15. Group UKPDS. Effect of intensive blood-glucose control with metformin on complications in overweight patients with type 2 diabetes (UK Prospective Diabetes Study 34). Lancet 352: 854–865, 1998.[CrossRef][ISI][Medline]
  16. Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, Makela TP, Alessi DR, and Hardie DG. Complexes between the LKB1 tumor suppressor, STRADalpha/beta and MO25alpha/beta are upstream kinases in the AMP-activated protein kinase cascade. J Biol 2: 28, 2003.[CrossRef][Medline]
  17. Hawley SA, Davison M, Woods A, Davies SP, Beri RK, Carling D, and Hardie DG. Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase. J Biol Chem 271: 27879–27887, 1996.[Abstract/Free Full Text]
  18. Hawley SA, Selbert MA, Goldstein EG, Edelman AM, Carling D, and Hardie DG. 5'-AMP activates the AMP-activated protein kinase cascade, and Ca2+/calmodulin activates the calmodulin-dependent protein kinase I cascade, via three independent mechanisms. J Biol Chem 270: 27186–27191, 1995.[Abstract/Free Full Text]
  19. Hayashi T, Hirshman MF, Kurth EJ, Winder WW, and Goodyear LJ. Evidence for 5' AMP-activated protein kinase mediation of the effect of muscle contraction on glucose transport. Diabetes 47: 1369–1373, 1998.[Abstract]
  20. Henquin JC. Triggering and amplifying pathways of regulation of insulin secretion by glucose. Diabetes 49: 1751–1760, 2000.[Abstract]
  21. Henquin JC and Meissner HP. Significance of ionic fluxes and changes in membrane potential for stimulus-secretion coupling in pancreatic B-cells. Experientia 40: 1043–1052, 1984.[ISI][Medline]
  22. Hong SP, Leiper FC, Woods A, Carling D, and Carlson M. Activation of yeast Snf1 and mammalian AMP-activated protein kinase by upstream kinases. Proc Natl Acad Sci USA 100: 8839–8843, 2003.[Abstract/Free Full Text]
  23. 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]
  24. Jenne DE, Reimann H, Nezu J, Friedel W, Loff S, Jeschke R, Muller O, Back W, and Zimmer M. Peutz-Jeghers syndrome is caused by mutations in a novel serine threonine kinase. Nat Genet 18: 38–43, 1998.[ISI][Medline]
  25. Kamohara S, Burcelin R, Halaas JL, Friedman JM, and Charron MJ. Acute stimulation of glucose metabolism in mice by leptin treatment. Nature 389: 374–377, 1997.[CrossRef][ISI][Medline]
  26. 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.[CrossRef][ISI][Medline]
  27. Kennedy HJ, Pouli AE, Ainscow EK, Jouaville LS, Rizzuto R, and Rutter GA. Glucose generates sub-plasma membrane ATP microdomains in single islet beta-cells. Potential role for strategically located mitochondria. J Biol Chem 274: 13281–13291, 1999.[Abstract/Free Full Text]
  28. Kieffer TJ and Habener JF. The adipoinsular axis: effects of leptin on pancreatic {beta}-cells. Am J Physiol Endocrinol Metab 278: E1–E14, 2000.[Abstract/Free Full Text]
  29. Kurth-Kraczek EJ, Hirshman MF, Goodyear LJ, and Winder WW. 5' AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle. Diabetes 48: 1667–1671, 1999.[Abstract]
  30. Leclerc I, da Silva Xavier G, and Rutter GA. AMP- and stress-activated protein kinases: key regulators of glucose-dependent gene transcription in mammalian cells? Prog Nucleic Acid Res Mol Biol 71: 69–90, 2002.[ISI][Medline]
  31. Leclerc I, Lenzner C, Gourdon L, Vaulont S, Kahn A, and Viollet B. Hepatocyte nuclear factor 4alpha involved in maturity-onset diabetes of the young (MODY1) is a novel target of AMP-activated protein kinase. Diabetes 50: 1515–1521, 2001.[Abstract/Free Full Text]
  32. Levy J, Atkinson AB, Bell PM, McCance DR, and Hadden DR. Beta-cell deterioration determines the onset and rate of progression of secondary dietary failure in type 2 diabetes mellitus: the 10-year follow-up of the Belfast Diet Study. Diabet Med 15: 290–296, 1998.[CrossRef][ISI][Medline]
  33. Lupi R, Marchetti P, Giannarelli R, Coppelli A, Tellini C, Del Guerra S, Lorenzetti M, Carmellini M, Mosca F, and Navalesi R. Effects of glibenclamide and metformin (alone or in combination) on insulin release from isolated human pancreatic islets. Acta Diabetol 34: 46–48, 1997.[CrossRef][ISI][Medline]
  34. Minokoshi Y, Haque MS, and Shimazu T. Microinjection of leptin into the ventromedial hypothalamus increases glucose uptake in peripheral tissues in rats. Diabetes 48: 287–291, 1999.[Abstract/Free Full Text]
  35. Minokoshi Y, Kim YB, Peroni OD, Fryer LG, Muller C, Carling D, and Kahn BB. Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415: 339–343, 2002.[CrossRef][ISI][Medline]
  36. Miyazaki J, Araki K, Yamato E, Ikegami H, Asano T, Shibasaki Y, Oka Y, and Yamamura K. Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms. Endocrinology 127: 126–132, 1990.[Abstract]
  37. Moghetti P, Castello R, Negri C, Tosi F, Perrone F, Caputo M, Zanolin E, and Muggeo M. Metformin effects on clinical features, endocrine and metabolic profiles, and insulin sensitivity in polycystic ovary syndrome: a randomized, double-blind, placebo-controlled 6-month trial, followed by open, long-term clinical evaluation. J Clin Endocrinol Metab 85: 139–146, 2000.[Abstract/Free Full Text]
  38. Muoio DM, Dohm GL, Fiedorek FT Jr, Tapscott EB, Coleman RA, and Dohn GL. Leptin directly alters lipid partitioning in skeletal muscle. Diabetes 46: 1360–1363, 1997.[Abstract]
  39. Owen MR, Doran E, and Halestrap AP. Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochem J 348: 607–614, 2000.[CrossRef][ISI][Medline]
  40. Rutter GA. Nutrient-secretion coupling in the pancreatic islet beta-cell: recent advances. Mol Aspects Med 22: 247–284, 2001.[CrossRef][Medline]
  41. Rutter GA, da Silva Xavier G, and Leclerc I. Roles of 5'-AMP-activated protein kinase (AMPK) in mammalian glucose homoeostasis. Biochem J 375: 1–16, 2003.[CrossRef][ISI][Medline]
  42. Safayhi H, Haase H, Kramer U, Bihlmayer A, Roenfeldt M, Ammon HP, Froschmayr M, Cassidy TN, Morano I, Ahlijanian MK, and Striessnig J. L-type calcium channels in insulin-secreting cells: biochemical characterization and phosphorylation in RINm5F cells. Mol Endocrinol 11: 619–629, 1997.[Abstract/Free Full Text]
  43. Salt IP, Johnson G, Ashcroft SJ, and Hardie DG. AMP-activated protein kinase is activated by low glucose in cell lines derived from pancreatic beta cells, and may regulate insulin release. Biochem J 335: 533–539, 1998.[ISI][Medline]
  44. Schatz H, Katsilambros N, Nierle C, and Pfeiffer EE. The effect of biguanides on secretion and biosynthesis of insulin in isolated pancreatic islets of rats. Diabetologia 8: 402–407, 1972.[ISI][Medline]
  45. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, and Rajotte RV. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343: 230–238, 2000.[Abstract/Free Full Text]
  46. Sreenan S, Sturis J, Pugh W, Burant CF, and Polonsky KS. Prevention of hyperglycemia in the Zucker diabetic fatty rat by treatment with metformin or troglitazone. Am J Physiol Endocrinol Metab 271: E742–E747, 1996.[Abstract/Free Full Text]
  47. Stapleton D, Gao G, Michell BJ, Widmer J, Mitchelhill K, Teh T, House CM, Witters LA, and Kemp BE. Mammalian 5'-AMP-activated protein kinase non-catalytic subunits are homologs of proteins that interact with yeast Snf1 protein kinase. J Biol Chem 269: 29343–29346, 1994.[Abstract/Free Full Text]
  48. Tsuboi T, DaSilva Xavier G, Leclerc I, and Rutter GA. 5' AMP-activated protein kinase controls insulin-containing secretory vesicle dynamics. J Biol Chem 278: 52042–52051, 2003.[Abstract/Free Full Text]
  49. Turner RC. The UK Prospective Diabetes Study. A review. Diabetes Care 21, Suppl 3: C35–C38, 1998.
  50. Unger RH, Zhou YT, and Orci L. Regulation of fatty acid homeostasis in cells: novel role of leptin. Proc Natl Acad Sci USA 96: 2327–2332, 1999.[Abstract/Free Full Text]
  51. Vasir B, Aiello LP, Yoon KH, Quickel RR, Bonner-Weir S, and Weir GC. Hypoxia induces vascular endothelial growth factor gene and protein expression in cultured rat islet cells. Diabetes 47: 1894–1903, 1998.[Abstract]
  52. Vincent MF, Marangos PJ, Gruber HE, and Van den Berghe G. Inhibition by AICA riboside of gluconeogenesis in isolated rat hepatocytes. Diabetes 40: 1259–1266, 1991.[Abstract]
  53. Wilcock C, Wyre ND, and Bailey CJ. Subcellular distribution of metformin in rat liver. J Pharm Pharmacol 43: 442–444, 1991.[ISI][Medline]
  54. 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]
  55. Woods A, Azzout-Marniche D, Foretz M, Stein SC, Lemarchand P, Ferre P, Foufelle F, and Carling D. Characterization of the role of AMP-activated protein kinase in the regulation of glucose-activated gene expression using constitutively active and dominant negative forms of the kinase. Mol Cell Biol 20: 6704–6711, 2000.[Abstract/Free Full Text]
  56. Woods A, Johnstone SR, Dickerson K, Leiper FC, Fryer LG, Neumann D, Schlattner U, Wallimann T, Carlson M, and Carling D. LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr Biol 13: 2004–2008, 2003.[CrossRef][ISI][Medline]
  57. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, and Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 372: 425–432, 1994.[CrossRef][ISI][Medline]
  58. Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, Wu M, Ventre J, Doebber T, Fujii N, Musi N, Hirshman MF, Goodyear LJ, and Moller DE. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 108: 1167–1174, 2001.[Abstract/Free Full Text]