AMP-Activated Protein Kinase: a New -Cell Glucose Sensor? Regulation by Amino Acids and Calcium Ions

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AMP-Activated Protein Kinase: A New ␤-Cell Glucose Sensor? Regulation by Amino Acids and Calcium Ions Isabelle Leclerc and Guy A. Rutter

Stimulation of AMP-activated protein kinase (AMPK) in ␣-subunit in response to a decrease in cellular energy skeletal muscle and liver is seen as an exciting prospect charge and a fall in ATP/AMP ratios (4). for the treatment of type 2 diabetes. However, we have Regulation of AMPK phosphorylation in mammalian recently demonstrated that changes in AMPK activity cells is complex (5). Binding of AMP to a regulator accompany the exposure of pancreatic islet ␤-cells to ␥-subunit appears to render the complex more susceptible elevated glucose concentrations and may be involved in to phosphorylation and activation by upstream AMPK the activation of insulin secretion. Here, we discuss this kinases (AMPKKs). Very recent data have suggested that hypothesis and explore the potential role of changes in AMPK activity in the actions of other secretagogues. the putative mammalian AMPKK may be a homolog of the Amino acids decreased AMPK activity in MIN6 ␤-cells S. cerevisiae Snf1 (sucrose nonfermenting factor) kinases leu < Elmp1p, Tos3p, or Pak1 (6,7). Subsequently, mammalian ؍ with an order of potency for inhibition: arg leu ؉ glu < glucose, which was closely correlated LKB1 (derived as a code name for the Peutz-Jeghers ؍ gln By syndrome causative gene [8] and also termed STK11, for .(0.76 ؍ with the stimulation of insulin release (r2 ؉ contrast, increases in intracellular Ca2 concentration serine/threonine kinase 11 [9]) has been shown to phos- provoked by cell depolarization with KCl activated phorylate AMPK in mammalian cells and to mediate the AMPK in the face of increased free intracellular ATP effects of AICAR (5-aminoimidazole-4-carboxamide ribo- concentrations. Elevation of intracellular cAMP levels side) (10–12). However, other closer homologs of the with isobutylmethyxanthine or forskolin had no effect on AMPK activity. We conclude that metabolizable yeast Snf1 kinases, including calmodulin kinase 1 kinase amino acids regulate AMPK in the ␤-cell via increases in (CaMK1K), also exist in mammalian cells. The latter the cytosolic ATP/AMP ratio and via phosphorylation by enzyme phosphorylates AMPK in vitro albeit much more ,the upstream kinase LKB1. Intracellular Ca2؉ ions may poorly than it phosphorylates its usual substrate, CaMK1 activate AMPK by calmodulin kinase 1 kinase–mediated and has therefore been considered to play a minor if any phosphorylation. The latter may act as a novel feedback role in the regulation of AMPK in mammals (13). However, mechanism to inhibit excessive insulin secretion under it is now clear that LKB1 is not a “dedicated” AMPK kinase some circumstances. Diabetes 53 (Suppl. 3):S67–S74, but also phosphorylates and regulates several members of 2004 a wider “AMPK family” (14) whose regulation by nutrients is not yet well understood. Consequences of AMPK activation for intracellular Enzymology of AMP-activated protein kinase. AMP- metabolism. Activation of AMPK is associated with the activated protein kinase (AMPK) is a multimeric serine/ phosphorylation of enzymes involved in ATP-consuming ␣ ␤ threonine protein kinase comprising - (catalytic), - processes, such as fatty acid (acetyl-CoA carboxylase) and ␥ (scaffold), and - (regulatory) subunits (1–3). The enzyme cholesterol (hydroxymethylglutaryl-CoA dehydrogenase) is activated by phosphorylation on threonine-172 of the biosynthesis, and the consequent activation of mitochondrial fatty acid oxidation (15). In this way, regulation of AMPK ensures that cellular ATP is spared during times of From the Henry Wellcome Laboratories for Integrated Cell Signalling and nutrient deprivation. As such, the enzyme has aptly been Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, U.K. described as the “fuel gauge” of mammalian cells (1). Address correspondence and reprint requests to Professor Guy A. Rutter, Thus, in most cell types, basal AMPK phosphorylation and Henry Wellcome Laboratories for Integrated Cell Signalling and Department activity are low, increasing only during metabolic stress of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, BS8 1TD Bristol, U.K. E-mail: [email protected]. (e.g., during exercise in muscle) (16,17) or after the Received for publication 8 March 2004 and accepted in revised form 21 May activation of certain receptors, including those for leptin 2004. (18) and adiponectin (ACRP30) (19). However, and as This article is based on a presentation at a symposium. The symposium and the publication of this article were made possible by an unrestricted educa- discussed below, AMPK activity is regulated somewhat tional grant from Servier. differently and exerts distinct effects on the metabolism in AICAR, 5-aminoimidazole-4-carboxamide riboside; AMPK, AMP-activated pancreatic ␤-cells compared with other tissues. protein kinase; AMPKK, AMPK kinase; [ATP]cyt, free cytosolic ATP concentration; CaMK1K, calmodulin-dependent kinase 1 kinase; CREB, Ca2ϩ/cAMP- AMPK in type 2 diabetes. AMPK is increasingly consid- response element binding protein; DMEM, Dulbecco’s modiﬁed Eagle’s ered a promising target for drug treatment in type 2 medium; IBMX, isobutylmethylxanthine; KHB, Kreb’s-HEPES-bicarbonate buffered medium; MOI, multiplicity of infection; PKA, protein kinase A. diabetes (4). Activation of the enzyme in the liver or © 2004 by the American Diabetes Association. skeletal muscle with the cell-permeant AMP analog AICAR

DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 S67 AMP-ACTIVATED PROTEIN KINASE IN THE ␤-CELL is associated with diminished gluconeogenesis (20,21) and mmol/l glucose) conditions in clonal INS-1 (42) and MIN6 with enhanced glucose uptake (20,22,23), respectively, and (43–46) ␤-cells as well as in isolated rodent and human thus potentially with improved glucose homeostasis in islets (44,47), and is chiefly attributable to the activity of type 2 diabetic patients. Correspondingly, treatment of the ␣1 subunit (43). AMPK activity is rapidly (within obese diabetic rats with AICAR lowers blood glucose minutes) decreased by elevations in glucose concentration levels (24). Moreover, metformin, used as an antidiabetic over the physiological range, concomitant with decreases agent for Ͼ50 years (25), has recently been shown to be an in the phosphorylation of the ␣-subunit at Thr-172 inhibitor of respiratory chain complex 1 (26), which exerts (42,44,47,48). Suggesting that changes in AMPK activity some of its effects through an inhibition of mitochondrial contribute to the regulation of insulin secretion, clamping oxidative metabolism and activation of AMPK (20,27). AMPK activity at the elevated levels apparent at low Moreover, the thiazolidinedione class of antidiabetic glucose concentrations by the expression of constitutively drugs, usually considered to act principally by binding to active AMPK␣ subunits, the use of AICAR, or use of the the nuclear peroxisome proliferator–activator receptor-␥ drug metformin (47) suppresses glucose-stimulated insulin class of nuclear transcription factors (28), are also able to secretion from MIN6 cells and islets (44–46) as well as increase intracellular AMP levels and stimulate AMPK from INS1 ␤-cells (42,49). Conversely, a dominant-negative activity (29). At present, the importance of this effect for form of AMPK stimulates insulin release at low glucose the antidiabetic effects of these agents is unknown. concentrations (44–46). The effect on ␤-cell AMPK activity By inhibiting the expression of lipogenic genes in the of the thiazolidinedione class of antidiabetic agents, which liver (30,31), the activation of AMPK may also contribute has been reported to activate the enzyme in other cell to the antisteatotic effects of metformin (32). Indeed, types (see above), has not been reported. hepatic expression of the lipogenic transcription factor The effects of AMPK activation by AICAR or metformin sterol regulatory element binding protein-1 (SREBP-1c) is appear to involve the inhibition of glucose oxidation reduced by metformin (20), decreasing overall liver tri- (44,49) as well as direct effects on secretory vesicle glyceride and sterol synthesis. Other targets of AMPK in recruitment to the plasma membrane (45) and a diminu- the liver include the key transcription factor hepatocyte tion of tolbutamide but not KCl-induced Ca2ϩ influx (44). nuclear factor (HNF)-4 (33), which is associated with However, the actions of inactive (dominant-negative) maturity-onset diabetes of the young-1 (MODY1) (34) and AMPK do not involve the closure of KATP channels or any is destabilized by AMPK-mediated phosphorylation (35). increase in intracellular Ca2ϩ concentration (44), indicat- The consequent downregulation of apolipoprotein expres- ing that increases in glucose concentration regulate the sion (33) may then reduce hepatic lipid export and thereby latter through AMPK-independent mechanisms. Hence, improve blood lipid profiles. inhibition of AMPK activity by glucose is a necessary, but While decreases in cellular ATP/AMP ratios are likely to not usually a sufficient, stimulus for insulin secretion. explain many of the effects of metformin on AMPK activ- Preservation of ␤-cell function and mass. ␤-Cell mass ity, it should be pointed out that evidence has also been is profoundly decreased in type 1 diabetes, as a result of provided for a nonmetabolic action of the biguanide on immune-mediated destruction (50), and is more modestly AMPK activity (29,36). However, whether such apparent decreased in type 2 diabetes (51), by mechanisms that are effects are actually due to changes in free or compartmen- currently unclear. Activation of AMPK has been reported talized ATP or AMP concentration, which are masked in to enhance ␤-cell death through apoptosis (48,52) via the measurements of total nucleotide concentrations, remains activation of the c-Jun NH2-terminal kinases JNK1/2. How- to be ruled out. ever, these findings contrast with those of other workers AMPK and the pancreatic islet ␤-cell. In light of the (53) who have proposed that by stimulating lipolysis and role of pancreatic ␤-cells as the principal “fuel sensor” for thus the “detoxification” of fatty acids, active AMPK the whole organism, might the functions of AMPK not be decreases ␤-cell death, consistent with an antiapoptotic well suited as the key intracellular nutrient sensor in these role in other cell types (12,54–56), and the absence of cells? As discussed below, recent findings (rev. in 4) apoptosis in cells expressing the activated mutant of suggest this may well be the case. AMPK catalytic subunits in MIN6 cells (44). Secretion of insulin from pancreatic islets is biphasic in Role of AMPK in the secretory response to nonglu- response to an increase in plasma glucose concentration cose secretagogues. Different secretagogues act at vari- (37). The first phase of secretion (likely to correspond to ous levels of the insulin secretory machinery (40). KATP channel–dependent or “triggering pathway”) (38) Depolarizing agents such as KCl, as well as certain posi- involves the rapid release, upon calcium influx, of a tively charged amino acids including arginine, stimulate “readily releasable pool” of secretory vesicles docked at Ca2ϩ influx and the first phase of insulin release but have the plasma membrane (39). The second phase (which may little effect on the second, sustained phase. By contrast, correspond to the KATP channel–independent “amplifying” fuel secretagogues, like glucose or metabolizable amino or “potentiation” pathway) (38) then appears to involve acids such as leucine and glutamine, enhance both phases the priming and/or the translocation to release sites of through a dual action on cellular metabolism and Ca2ϩ more remotely located secretory granules (40). The sec- influx (40). Cyclic AMP-raising agents including GLP-1 ond phase is known to be ATP-dependent (41), likely (57), the phosphodiesterase inhibitor isobutylmethylxan- reflecting an energy requirement for the priming and/or thine (IBMX), and the adenylate cyclase activator forsko- docking of secretory vesicles at sites of exocytosis. lin stimulate both phases of insulin secretion (58) and are In contrast to the situation in liver, muscle, and adipose believed to act principally via PKA. The latter enzyme tissue, AMPK activity is clearly detectable under basal (5.5 appears to phosphorylate voltage-sensitive Ca2ϩ channels

S68 DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 I. LECLERC AND G.A. RUTTER

(59) as well as undeﬁned components of the exocytotic machinery (60). In addition, cAMP may also enhance secretion via a PKA-independent action on the activity of the guanine nucleotide exchange factor Epac/cAMP- GEF-II and intracellular Ca2ϩ mobilization (61). Recent data have suggested that the activation of AMPK may be involved in the regulation by ␤-adreno receptors of adipose tissue lipolysis (62,63), but the impact of cAMP/PKA on AMPK activity in the ␤-cell is unknown. Whereas increases in glucose concentration (42–44, 47,48) inhibit AMPK activity in clonal ␤-cells and islets (see above), the effects of exposure to amino acids or of changing intracellular free Ca2ϩ concentrations (64), both important stimuli for insulin release, have not been ex- plored. Given that both can affect intracellular metabolism and thus ATP and AMP levels (61,65), it is conceivable that changes in AMPK activity may play a role in the insulinotropic effects of each class. Here, we explore these issues using the model MIN6 ␤-cell line (66) to monitor changes in AMPK activity and secretion in response to a variety of secretagogues.

RESEARCH DESIGN AND METHODS Materials. KN-62 was from Calbiochem. AICAR was from Toronto Research Chemicals (Toronto, ON, Canada). MEM Amino Acids Solution (50X) and MEM Non-Essential Amino Acids Solution (100X) were from GIBCO (Invitro- gen; cat. no. 11130051 and 11140050/11140076, respectively; www.invitrogen- FIG. 1. Amino acids inhibit AMPK activity in MIN6 cells. A: MIN6 cells .com) and gave final concentrations of L-amino acids (mmol/l) as follows: gly, were cultured overnight in DMEM containing 3 mmol/l glucose then ala, asn, asp, glu, pro, and ser (0.1); arg (0.82); cys (0.2); His (0.27); Ile and leu incubated 60 min in KHB supplemented with glucose, amino acids, and (0.4); lys (0.5); met (0.1); Thr (0.4); Trp (0.05); Tyr (0.2); and val (0.4). All other AICAR (as indicated) before cell lysis and AMPK assays, as described in RESEARCH DESIGN AND METHODS. Amino acids (Essential and Non- consumables were from Sigma. Essential Amino acids solutions [GIBCO]) were added at a final Antibodies. Sheep anti-phospho AMPK (T172) antibody was a generous gift concentration of 1؋ (see RESEARCH DESIGN AND METHODS). AICAR was of Professor D.G. Hardie (Department of Biochemistry, University of Dundee, added at a final concentration of 1 mmol/l. B: MIN6 cells seeded on U.K.). Rabbit anti-AMPK ␤1/2 antibody was kindly provided by Dr. D. Carling coverslips were infected with cytosolic luciferase-expressing adenovi- (MRC Clinical Sciences, London, U.K.). Rabbit anti-phospho Ca2ϩ/cAMP- rus at an MOI of 30 for 48 h before perifusion with 0, 3, or 17 mmol/l ␮ response element binding protein (CREB) (S133) was from Cell Signaling glucose-containing KHB buffer, supplemented with 5 mol/l luciferin. Amino acids (1؋) were added as indicated. Photon counting was (Hong Kong). performed as described under RESEARCH DESIGN AND METHODS.*P < 0.05, ␤ Cell culture. MIN6 pancreatic -cells were used between passages nos. 18 ***P < 0.0005 for the effect of glucose compared with the correspond- and 35 and grown in Dulbecco’s modified Eagle’s medium (DMEM), containing 0 mmol/l condition. ##P < 0.005, ###P < 0.0005 for the effects of ing 25 mmol/l glucose, supplemented with 15% heat-inactivated fetal calf amino acids compared with the same glucose concentration. Data .serum (FCS), 4 mmol/l L-glutamine, 100 ␮mol/l 2-mercaptoethanol, 100 represent means ؎ SEM of four independent experiments units/ml penicillin, and 100 ␮g/ml streptomycin in a humidified atmosphere at

37°C with 5% CO2 unless specified otherwise. Adenoviruses. Adenoviruses encoding for enhanced green fluorescent pro- RESULTS tein (eGFP) only, hereafter named pAd-GFP (Null), and for cytosolic lucif- Regulation of ␤-cell AMPK by amino acids: correla- erase (44,67) are described elsewhere. MIN6 cells were infected at a tion with insulin release. Amino acids stimulate insulin multiplicity of infection (MOI) of 30 pfu/cell for 24–48 h before experiments. AMPK activity assay. AMPK activity assays in MIN6 cells were performed by secretion through a variety of different mechanisms involv- “SAMS” phospho-transfer assay, as described (47). ing an increase in oxidative metabolism and ATP synthesis Western (immuno-) blot analysis. MIN6 cells were cultured and lysed as (65), direct membrane depolarization (arginine, histidine) for AMPK activity. Ten to 50 ␮g of whole cellular extracts were denatured for (69), or a combination of both effects (70). 5 min at 100°C in 2% SDS and 5% ␤-mercaptoethanol, resolved by 10% To determine whether amino acids may affect the activ- SDS-PAGE and transferred to polyvinylidine fluoride (PVDF) membranes ity of AMPK in ␤-cells, clonal MIN6 cells were incubated before immunoblotting as described (68). Sheep anti-phospho AMPK antibody was used at a dilution of 1:500, rabbit anti-AMPK ␤1/2 was used at 1:5,000 with a mixture of all essential and nonessential amino dilution, and rabbit anti–phospho-CREB antibody was used at 1:1,000 dilution. acids, except for glutamine (Fig. 1). In the absence of Measurements of insulin secretion and luciferase luminescence. Insulin added amino acids, glucose (3 or 17 mmol/l) caused a secretion assays were performed during incubation in Kreb’s-HEPES-bicar- marked suppression of AMPK activity, which was reversed bonate buffered medium (KHB) (140 mmol/l NaCl, 3.6 mmol/l KCl, 0.5 mmol/l by the inclusion of AICAR. These effects were potentiated NaH PO , 0.5 mmol/l MgSO , 2.0 mmol/l NaHCO , 10 mmol/l Hepes [pH 7.4] 2 4 4 3 by amino acids at each glucose concentration in either the 0.1% [wt/vol] bovine serum albumin [BSA], and 1.0 mmol/l CaCl2 equilibrated

with O2/CO2 [95:5 vol/vol]) and containing the indicated glucose concentra- absence or presence of AICAR (Fig. 1A). To determine tions, as described (47). Cytosolic ATP concentrations were measured as whether the effects of the amino acid mixture might be luciferase luminescence in KHB medium without BSA and as described in ref. ascribed, at least in part, to the oxidative metabolism of 67, except that light output was recorded every 1 s and averaged over 20-s amino acids and thus increases in cytosolic free ATP intervals. Statistics. Data are shown as the means Ϯ SEM of the number of observa- concentration ([ATP]cyt), the latter was measured dynam- tions given. Statistical significance was calculated using Student’s t test ically in cell populations transduced with an adenovirus- (Microsoft Excel). expressing recombinant firefly luciferase (64). Assayed at

DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 S69 AMP-ACTIVATED PROTEIN KINASE IN THE ␤-CELL each glucose concentration tested, the amino acid mixture increased apparent intracellular free ATP concentration, as assessed from the increase in cellular luminescence, with the most substantial increases in the presence of 3 mmol/l glucose (Fig. 1B). Importantly, increasing glucose concentrations still inhibited AMPK activity in the presence of this physiological mixture of amino acids, consistent with the likely ability of the sugar to regulate ␤-cell AMPK activity in the context of islets in vivo. We predicted that if changes in AMPK activity are involved in the regulation of insulin secretion in response to amino acids, as well as to glucose (44), then AMPK activity and secretory rate should be inversely correlated (i.e., for a given amino acid or amino acid combination, the highest rates of insulin secretion would be expected when AMPK activity was at its lowest, and vice versa). In agreement with this, AMPK activity was inhibited by L-amino acids with increasing efficiency in the order leu ϭ arg Ͻ gln ϭ gln ϩ leu Յ glucose, while insulin secretion was stimulated in essentially the reverse of this order (Fig. 2A and B). Plotting these two parameters revealed a significant correlation (r2 ϭ 0.768; Fig. 2C). Regulation of AMPK activity by cAMP-dependent PKA. Moule and Denton (62) as well as Yin et al. (63) have provided evidence that AMPK may be a downstream effector of PKA in adipocytes, thus mediating some of the effects of adrenergic agonists on lipolysis in fat tissue. To determine whether increases in PKA activity may influ- ence AMPK activity in ␤-cells, MIN6 cells were incubated at various glucose concentrations and in the presence of either the phosphodiesterase inhibitor, IBMX, or the ade- nyl cyclase activator, forskolin. Whereas either IBMX (Fig. 3B) or forskolin (data not shown) caused substantial increases in the phosphorylation at Ser (133) of CREB, consistent with the expected robust increases in intracellular cAMP concentration and PKA activity, AMPK activity was unaffected under all conditions tested (Fig. 3A). Thus, changes in AMPK activity are unlikely to contribute to the stimulation of insulin secretion by cAMP-raising agents (58). Regulation of ␤-cell AMPK activity by changes in intracellular free Ca2؉ concentration. Depolarization of ␤-cells, leading to increases in cytosolic (71) and consequently mitochondrial (72) free Ca2ϩ concentration, causes a stimulation of oxidative metabolism and conse- FIG. 2. Inhibition of AMPK activity by individual amino acids corre- lates with secretory potency. A: MIN6 cells were incubated for 30 min quent increases in cytosolic and mitochondrial free ATP in KHB buffer supplemented with the indicated concentrations of concentration (61,64,67) (Fig. 4C). We reasoned that these individual amino acids or glucose before cell lysis and measurement of increases in free [ATP] (and presumably decreases in free AMPK activity (RESEARCH DESIGN AND METHODS). B: MIN6 cells were incubated as in A for 30 min, except that KHB buffer was supplemented [AMP], given the equilibrium at myokinase) (73) may lead with 0.1% BSA for static insulin secretion assays as described under to an inhibition of AMPK activity, which may in turn RESEARCH DESIGN AND METHODS. C: Correlation between AMPK activity contribute to the stimulation of secretion under these and secreted insulin. Data are taken from the experiments shown in A and B.*P < 0.05 and **P < 0.005 for the effects of amino acids or -conditions. We therefore measured AMPK activity in cells glucose. Data represent means ؎ SEM of three independent experi incubated at either 0 or 17 mmol/l glucose for various ments. times after the addition of 30 mmol/l KCl (Fig. 4A). Unexpectedly, KCl addition caused clear and highly repro- tion status in intact cells directly by immunoblotting (Fig. ducible increases in AMPK activity at each glucose con- 4B). KCl-induced increases in the phosphorylation of centration and at the three time points tested (Fig. 4A). AMPK␣ at Thr-172 were clearly evident at both 0 and 17 The SAMS peptide used to quantify AMPK activity may mmol/l glucose, consistent with the apparent changes in conceivably also report the activity of the Ca2ϩ-dependent AMPK activity reported by direct phosphotransfer assay protein kinase, CaMK1, since the latter enzyme has a (Fig. 4A). similar substrate recognition motif (13). To assess this We next assessed the role of 1) intracellular [Ca2ϩ] possibility, we examined changes in AMPK phosphoryla- increases and 2) calmodulin function using KN-62, an

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on insulin secretion from isolated islets (but leads to glucose intolerance due to elevated catecholamine levels) (78), inactivation of the ␣1 complex has no effect on either parameter (79). Such findings suggest redundancy in the function of these isoforms in islet ␤-cells. Double knockout of both isoforms selectively in ␤-cells will be required to test this hypothesis. Given that the dose response for glucose toward both AMPK inhibition and the “amplifying” pathway for insulin secretion are similar (left-shifted compared with that for the triggering pathway), an intriguing possibility is that amplification is mediated, at least in part, by AMPK inhibition. Although direct evidence for this is lacking, it is noteworthy that the recruitment of secretory vesicles to the cell surface, likely to be involved in secretory potentiation, is inhibited by active AMPK (45). The molecular mechanisms through which AMPK acts on vesicle recruitment are unresolved but have been postulated (4) to involve the phosphorylation of microtubule motor proteins including conventional kinesin, given the key role of the latter in insulin vesicle movement (80). By showing that elevated glucose concentrations markedly inhibit AMPK activity even in the FIG. 3. Effects of cAMP-raising agents on AMPK activity in MIN6 cells. presence of a depolarizing concentration of KCl (Fig. 4A), the A: Cells were incubated for 20 min in KHB buffer containing the present observations are consistent with, but do not prove, a indicated glucose concentration and 0.1 mmol/l IBMX or 1 ␮mol/l forskolin before cell lysis and AMPK assay by phosphotransfer. B: role for AMPK in the amplification pathway for glucose- MIN6 cells were incubated for 20 min in 3 mmol/l glucose KHB in the stimulated insulin release. presence or absence of 0.1 mmol/l IBMX. Cell lysis and immunoblotting were performed as given in RESEARCH DESIGN AND METHODS. Phospho- Regulation of AMPK by amino acids and calcium ions. CREB was revealed with a specific antibody directed against phosphor- We show here that ␤-cell AMPK is regulated by individual ylated Ser-133. *P < 0.05 for the effect of glucose compared with the amino acids, in good correlation to their insulinotropic corresponding 0 mmol/l condition. Data represent means ؎ SEM of three independent experiments. effect. We also reveal that substantial increases in intracellular free Ca2ϩ ions activate AMPK in the face of 2ϩ inhibitor of both voltage-gated (L-type) Ca channels (74) increases in free cytosolic ATP concentration. This result and calmodulin-dependent kinases (75). While KN-62 was unexpected given that LKB1-mediated phosphoryla- caused only a partial (ϳ50%) decrease in KCl-induced tion of AMPK catalytic subunits is likely to be inhibited ϩ Ca2 increases (results not shown), the activation of under these conditions. The most straightforward inter- AMPK was completely inhibited in the presence of this pretation of these data are that AMPK is phosphorylated drug (Fig. 4D). by CaMK1K under these conditions, consistent with a complete inhibition of AMPK activation but only partial DISCUSSION suppression of KCl-induced Ca2ϩ influx. Activated AMPK Role of AMPK in the regulation of insulin secretion may then contribute to a feedback loop to suppress the by glucose. Recent data from this laboratory (43–45,47) further stimulation of insulin release. and others (42,49) have highlighted the likely role of Conclusions and perspectives: is AMPK in the ␤-cell a AMPK in the regulation of insulin secretion (rev. in 4). help or a hindrance to the development of antidia- These data contrast with earlier reports of the effects of betic therapies? Present thinking in respect to the use of AICAR (76,77). It should be emphasized that the use of AMPK activators focuses on the actions of these com- AICAR is not without complications due to uncertainties pounds on extrapancreatic tissues. Our own studies, de- as to 1) the extent to which the compound is accumulated scribed here and in earlier publications (43–45,47), into cells; 2) the proportion converted into the monophos- indicate that the effects of AMPK activation on the ␤-cell phorylated form (and AMP analog) ZMP, the true activator may also need to be considered. Whereas the effects of of AMPK; 3) potential changes in the concentration of AMPK on ␤-cell survival are still a matter of debate, there endogenous adenine nucleotides (ATP, ADP, and AMP); is a growing consensus that active AMPK reduces insulin and 4) the degree of further phosphorylation to the triply secretion. We suspect that, in the context of the whole phosphorylated ATP analog. The latter phenomenon is animal, this effect on pancreatic function may be masked likely to complicate results through the closure of ATP- by more marked and beneficial effects on glucose metab- sensitive Kϩ channels, with the consequent activation of olism in the liver and skeletal muscle, lowering the depen- insulin secretion, a tendency observed in islets at low but dency on circulating insulin. Nevertheless, drugs that may not stimulatory glucose concentrations (42). Thus, the use be able to selectively regulate different AMPK isoforms— of recombinant approaches, including adenoviruses that is, those that target ␣2 subunits in muscle but are (44,47,49) or of mouse transgenic/knockout models, are ineffective against the ␣1 subunits predominant in ␤-cells likely to provide less ambiguous results. It might be (42,43)—may eventually provide advantages with respect mentioned, however, that whereas inactivation of the ␣2 to AICAR, metformin, or thiazolidinediones (29), which isoform of AMPK catalytic subunit in mice is without effect are not isoform selective. Further studies are therefore

DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 S71 AMP-ACTIVATED PROTEIN KINASE IN THE ␤-CELL

FIG. 4. Effects of increases in intracellular Ca2؉ ions on AMPK activity. A: MIN6 cells were incubated in KHB buffer containing 0 or 17 mmol/l glucose in the presence or absence of 30 mmol/l KCl for 1, 15, or 45 min as indicated. Cell lysis and AMPK activity assays were performed as described in RESEARCH DESIGN AND METHODS. B: MIN6 cells were incubated 45 min as above. Cell lysis and immunoblotting were performed as in RESEARCH DESIGN AND METHODS. The phosphorylation status of AMPK␣ subunits was revealed with sheep anti-PT172 antibody, and total AMPK content was revealed with rabbit anti-AMPK-␤1/2 antibody. C: MIN6 cells seeded on coverslips were infected with cytosolic luciferase-expressing adenovirus at an MOI of 30 for 48 h before perifusion with 0 mmol/l glucose KHB buffer supplemented with 5 ␮mol/l luciferin. KCl (30 mmol/l) was added as indicated. Photon counting was performed as described in RESEARCH DESIGN AND METHODS. D: MIN6 cells were preincubated for1hin the presence or absence of 10 ␮mol/l KN-62 in DMEM then incubated 45 min in 17 mmol/l glucose-containing KHB in the presence or absence of KN-62 (as indicated) before cell lysis and AMPK assay, as described in RESEARCH DESIGN AND METHODS. Data represent the means ؎ SEM of at least three independent experiments. *P < 0.05 and **P < 0.005 for the KCl effect; #P < 0.05 and ##P < 0.005 for the effect of glucose. needed to determine the relative levels of expression of Trust for Advanced and Research Leave Fellowships, each of the AMPK isoforms (␣1, ␣2, ␤1, ␤2, ␥1, ␥2, and ␥3) respectively. in ␤-cells and other islet cell types. The ﬁnding that leptin stimulates AMPK activity in skeletal muscle (18) but not in REFERENCES ␤ -cells (47) may be of particular importance in this respect 1. Hardie DG, Carling D, Carlson M: The AMP-activated/SNF1 protein kinase because activators of the leptin signaling pathway may subfamily: metabolic sensors of the eukaryotic cell? Annu Rev Biochem provide selective stimulation of extrapancreatic AMPK, 67:821–855, 1998 including glucose-sensing neurons of the hypothalamus 2. Carling D: The AM: P-activated protein kinase cascade: a unifying system for energy control. Trends Biochem Sci 29:18–24, 2004 (81,82). Importantly, changes in AMPK in the latter may 3. Kemp BE, Stapleton D, Campbell DJ, Chen ZP, Murthy S, Walter M, Gupta represent a means of regulating satiety and, thus, the A, Adams JJ, Katsis F, van Denderen B, Jennings IG, Iseli T, Michell BJ, obesity underlying much of the recent increase in the Witters LA: AMP-activated protein kinase, super metabolic regulator. incidence of type 2 diabetes (83). Biochem Soc Trans 31:162–168, 2003 4. Rutter GA, da SilvaXavier G, Leclerc I: Roles of 5Ј-AMP-activated protein kinase (AMPK) in mammalian glucose homoeostasis. Biochem J 375:1–16, ACKNOWLEDGMENTS 2003 This study was supported by grants from the Wellcome 5. Hardie DG: Minireview: the AMP-activated protein kinase cascade: the key Trust (Project 062321; Programme 067081/Z/02/Z), Diabe- sensor of cellular energy status. Endocrinology 144:5179–5183 6. Sutherland CM, Hawley SA, McCartney RR, Leech A, Stark MJ, Schmidt tes UK, the Biotechnological and Biological Research MC, Hardie DG: Elm1p is one of three upstream kinases for the Saccha- Council, and the Juvenile Diabetes Research Foundation romyces cerevisiae SNF1 complex. Curr Biol 13:1299–1305, 2003 International. I.L. and G.A.R. are grateful to the Wellcome 7. Hong SP, Leiper FC, Woods A, Carling D, Carlson M: Activation of yeast

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