Targeting the AMP-Regulated Kinase Family to Treat Diabetes: a Research Update

Review

Targeting the AMP-regulated kinase family to treat diabetes: a research update

Gao Sun1 & Guy A Rutter†1

AMP-regulated protein kinase (AMPK), an intracellular sensor of energy change, is regulated by AMP:ATP (and ADP:ATP) ratios and is an important regulator of glucose homeostasis in mammals.

AMPK is emerging as an important regulator of insulin secretion, insulin action and feeding behavior.

AMPK is responsible for some but not all of the actions of antidiabetic drugs including metformin and thiazolidinediones. Points Practice New mouse knockout models shed light on the roles of the enzymes in different tissues.

The roles of the separate AMPK catalytic isoforms a1 and a2, as well as of the upstream kinases LKB1 and CaMKK, are frequently distinct.

AMPK and related kinases including per-arnt sim kinase represent exciting potential targets for new therapeutic drugs.

Summary AMP-activated protein kinase (AMPK) has long been recognized as a master energy sensor. Activation of AMPK in response to metabolic stress preserves energy stores by switching on catabolic pathways, whilst its inhibition consumes the energy by switching on anabolic pathways. Over the past 10–15 years, much attention has been focused on the role of AMPK in mammalian metabolism, and particularly in diabetes. As a consequence, AMPK has emerged as much more than a simple energy regulator and is now recognized as a kinase involved in controlling numerous cellular processes, including cell growth, apoptosis, autophagy and polarity. Using different in vitro and in vivo tools, AMPK has also been found to play important roles in different glucose-sensing organs and to serve as a key regulator of glucose homeostasis in mammals. Perhaps most importantly, AMPK appears to be the major target for several antidiabetic drugs. Here, we review recent advances in the field and particularly those emerging from the generation of tissue‑specific knockout and transgenic mice.

Diabetes & AMP-activated protein kinase obesity-associated diseases including Type 2 Increases in the prevalence of obesity owing diabetes (T2D), with higher risks in certain eth- to sedentary lifestyles and the intake of high- nic groups [1,2]. With T2D almost reaching pan- calorie food have led to a remarkable and par- demic proportions, novel and effective therapies allel increase in the number of patients with targeting this disease are urgently needed.

1Section of Cell Biology, Division of Diabetes, Endocrinology & Metabolism, Department of Medicine, Faculty of Medicine, Imperial College London, Exhibition Road, London, SW7 2AZ, UK †Author for correspondence: [email protected]

Type 2 diabetes normally begins with blunted regulatory b subunit (b1 or b2), which is required responses of peripheral tissues to insulin action for the binding of other subunits [25], and an with higher fasting blood glucose due to abnor- AMP-binding g subunit (g1, g2 or g3) [26]. Each mally regulated liver glucose production and of the separate subunits and isoforms is encoded moderately impaired glucose tolerance due to by an individual gene [24,27]. The a subunit con- the failure of skeletal muscle to take up glucose tains a N‑terminal Ser/Thr kinase domain, an from the bloodstream [3–8]. This insulin resistance auto-inhibitory sequence (AIS) and a C‑terminal leads, by still unknown mechanisms, to hyperin- b‑subunit-binding domain [28–30]. The kinase sulinemia wherein hyperplastic and hypermorphic domain includes the critical phosphorylation site, pancreatic b cells hyper-secrete insulin to compen- Thr172, which is indispensible for the activation sate. At this stage, hyperinsulinemia is still able to of AMPK by upstream kinases including liver control blood glucose within the normal physi- kinase B1 (LKB1; STK 11), a tumor suppressor ological range (3.6–5.8 mM in humans). It is only and Ca2+/calmodulin-dependent protein kinase when b‑cell function deteriorates and/or b‑cell (CaMKKb) [31–35]. AMPK can be inactivated mass falls that glucose intolerance and finally by phosphatases including PP2A and PP2C [36], the onset of frank T2D occurs with its attendant although recent studies suggest that a phospha- complications including failure of other impor- tase complex containing the catalytic subunit of tant organs such as the kidneys (nephropathy), protein phosphatase-1 and regulatory subunit R6 eyes (retinopathy) and heart (cardiovascular dis- might be involved in glucose-regulated AMPK ease) [5–11]. Consequently, different pharmaceuti- inactivation [37]. Single site mutation at Thr172 cal approaches have been developed that aim to to aspartic acid (D) reduces AMPK activity by either reduce insulin resistance (e.g., metformin, almost twofold. Conversely, a T172D mutation thiazolidinediones) or to enhance insulin produc- generates a mutated AMPK resistant to dephos- tion by b cells (e.g., sulphonylureas, glucagon-like phorylation [38]. The (autoinhibitory sequence) peptide‑1 analogs), hence reducing blood glucose AIS domain appears to exert an auto-inhibitory and delaying or preventing the onset of frank effect on AMPK activity, since truncated AMPK T2D [12,13]. a subunits lacking this domain have an almost AMP-activated protein kinase (AMPK), a phy- fourfold higher AMPK activity than those with an logenetically conserved serine/threonine protein intact AIS [29]. Two isoforms of AMPK catalytic kinase [14], has recently emerged as an interest- a subunit, a1 and a2, encoded by two distinct ing potential drug target for treating diabetes [15]. genes (PRKAA1 and PRKAA2), exist and share Indeed, several front-line antidiabetic drugs such high similarities at the N‑terminal of the subunit, as metformin and thiazolidinediones appear to and both are capable of being phosphorylated by act at least in large part through activating AMPK upstream kinases at the Thr172 site [24]. The dis- in the liver and adipose tissues [13,16,17]. As an tribution of a1 and a2 subunits is distinct in both energy sensor, AMPK and the signaling path- the subcellular, cellular and whole-body level, ways upstream and downstream of this enzyme with a2 present in both the cytosol and nucleus, have been intensively studied for the past decade, suggesting a role in controlling transcription and indicating that AMPK might play important gene expression. a2-containing complexes are roles in modulating glucose and insulin sensing predominately found in the skeletal muscle and in organs such as skeletal muscle, liver, brain, adi- heart, whilst a1 complexes, present chiefly in the pose tissues and pancreas [18–21]. However, recent cytosol, are found in most of the other chief meta- exciting studies, particularly those describing bolic organs such as the liver, endocrine pancreas tissue-specific AMPK knockout mice developed and adipose tissues [39,40]. with LoxP-Cre technology [22] have implied that The regulatory b subunit consists of a glyco- AMPK activity is regulated differently in differ- gen-binding domain (GBD) and a- and g‑sub- ent organs in response to glucose or insulin, and unit-binding domains [41,42]. Recently, much that the activation of AMPK may exert different attention has been focused on the functions of downstream effects in different tissues. the b subunit and the observations that: binding of a1→6-linked branches of glycogen at the AMPK structure, isoforms, tissue GBD during glycogen depletion (e.g., caused by distribution & subcellular localization muscle contraction) appears to be necessary for Mammalian AMPK exists as a heterotrimer con- dephosphorylation of glycogen synthase at site 2, sisting of a catalytic a subunit (a1 or a2) [23,24], a promoting glycogen resynthesis [43]; and the

334 Diabetes Manage. (2011) 1(3) future science group Targeting the AMP-regulated kinase family to treat diabetes: a research update Review b1 subunit is critical for AMPK activation by the injection of the AMP analog 5‑aminoimidazole- thienopyridone drug A769662 [44,45]. The g sub- 4-carboxamide-1-b-d-ribonucleoside (AICAR), unit of AMPK has two Bateman domains formed an AMPK activator, or single-leg arterial infusion by four tandem cystathionine b‑synthase motifs of AICAR increased AMPK activity and glucose 1–4, three of which are essential for AMP/ATP uptake [55–57]. These changes were also associ- binding [46]. Recently, the resolution of the crystal ated with increased hexokinase II transcription structure of the abg core complex containing the in both red and white muscles [55,56], suggesting C‑terminal domain of a1 and b2 with full length that AMPK stimulation might serve as an exer- g1 confirmed that among these three motifs in cise mimetic to enhance glucose uptake. Global the g subunit, two of them are interchangeable deletion of AMPK a2 (AMPK a2-/- mice), but between AMP and ATP binding and the other not a1, abolished AICAR- but not contraction- one tightly occupied by AMP [47]. induced glucose uptake in skeletal muscle, prob- High AMP:ATP ratios activate AMPK either ably reflecting activation ofa 1 activity in skeletal allosterically [48] or by rendering AMPK a better muscle during muscle contraction [57]. Using a substrate for upstream kinases [49] and a poorer transgenic mouse model carrying an inactivating substrate for protein phosphatases [36]. Owing Lys45 to Arg mutation in the AMPK a2 subunit to the action of adenylate kinase, the AMP:ATP (AMPK a2KD), Mu and colleagues demon- ratio in cells varies approximately as the square strated that reduction of AMPK activity in these of the ADP:ATP ratio, which makes the former mice completely blocked AICAR-induced glu- ratio a very sensitive indicator of cellular energy cose uptake in extensor digitorum longus muscle changes. Therefore, any activities that deplete but only partially reduced contraction-induced ATP such as hypoxia or glucose deprivation are glucose uptake [58]. Some years later, the groups expected to activate AMPK [14,36,50]. of Laurie Goodyear and Lynis Dohm [59,60] confirmed that decreases in AMPK a2 activity Examining the role of AMPK in abolished AICAR, but not contraction- or exer- different tissues cise-induced glucose uptake and translocation to AMPK in muscle the plasma membrane of the glucose transporter The ability of skeletal muscle to take up glucose GLUT4. Reduced maximal exercise capacity and is essential to maintain normal glucose homeo- glycogen accumulation [61] with a concomitant stasis. Aberrant glucose uptake due to insensi- decrease in hexokinase II protein levels [61,62] tivity to insulin has been found in T2D [51,52]. were also observed, using transgenic mice over- Excessive glucose in the bloodstream promotes expressing inactive AMPK a2 subunits selectively insulin secretion from pancreatic b cells, which in muscle (AMPK a2iTg mice). triggers the PI3K/Akt-AS160-Rab GTPase sig- Very recently, the GBD of AMPK b subunit naling pathway in the skeletal muscle cells to has been shown to be necessary for glycogen transport glucose across the cell membrane by replenishment during muscle contraction [43]. It is delivering glucose transporter 4 (GLUT4) onto therefore reasonable to suspect that glucose uptake the cell surface. After glucose is phosphorylated might also be dependent on glycogen binding. by hexokinase, it is later converted into glycogen However, AMPK b2 whole-body knockout mice by glycogen synthase and stored in the muscle with reduced AMPK activity displayed reduced for use during energy depletion [51]. In contrast AICAR-induced, but not contraction-induced, to this insulin-dependent glucose uptake signal- muscle glucose uptake [63]. Moreover, no effect ing pathway, exercise has also been reported to of muscle glucose uptake was seen in AMPK b1 mediate glucose uptake into the muscle cells knockout mice [44], suggesting, in the short term, using an insulin-independent pathway [53]. that glucose uptake during muscle contraction is probably not modulated by glycogen binding to Does AMPK mediate contraction-induced AMPK b subunits. glucose uptake in muscle? The AMPK g3 subunit is specifically expressed The activation of AMPK that is observed in skele in muscle cells and mutation of this subunit tal muscle in parallel to increased glucose uptake (R225Q) leads to downregulation of AMPK in response to exercise or metformin adminis- a activity. Using this mouse model (AMPK tration to Type 2 diabetic patients suggests that g3R225Q), Yu and colleagues confirmed that AMPK might play a role in controlling glucose both basal and AICAR-stimulated glucose uptake [54]. Supporting this view, subcutaneous uptake in skeletal muscle were significantly

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decreased [64]. By contrast, activation of AMPK results from experiments using STO-609 that in skeletal muscle achieved by expression of an direct inhibition of AMPK by this compound active form of AMPK where Arg70 is mutated to has also been reported [71]. A mouse model in Gln in the g1 subunit in mouse skeletal muscle which CaMKKb is specifically deleted in mus- increased glycogen accumulation [62]. cle is needed to fully understand the role of this kinase in muscle glucose uptake. Does AMPK modulate insulin-stimulated Thus, results from various AMPK inactive or glucose uptake? knockout mouse models seem to argue against a Recently, AS160/TBC1D4, activated in response role for AMPK in mediating contraction- or exer- to stimulation of the PI3K/Akt/PKB pathway cise-stimulated glucose uptake and suggest that was reported to be phosphorylated by AMPK alternative signaling pathways might be respon- through activation of a2b2g1 subunits [65,66]. sible for contraction-induced glucose uptake This leads to the binding of phosphorylated [57–59,61,64]. As shown in Figure 1(2), the difference AS160 (inactive) to 14–3–3 proteins and proof contraction-induced glucose uptake displayed motes conversion of less active GDP-bound Rab in muscle in mLKB1 KO versus AMPK a2KD to more active GTP-bound Rab, which then mice implies that other AMPK-related protein releases GLUT4 from vesicles to the plasma mem- kinase(s) might be involved in regulation. On the brane [67]. Another AS160 paralog, TBC1D1, is other hand, the emergence of the ability of AMPK also phosphorylated by purified AMPK and has to phosphorylate AS160 and TBC1D1 connects been reported to modulate glucose transport AMPK to insulin-dependent glucose uptake and [67]. Therefore, the evidence for involvement of sheds light on the role of AMPK in muscle glucose AS160 in GLUT4 translocation seems to link uptake and whole-body glucose disposal [67]. AMPK to insulin-dependent glucose uptake. AMPK in the liver Regulation of glucose uptake by kinases One of the major adverse effects of obesity-induced downstream of LKB1 insulin resistance is elevated fasting hyperglyce- Interestingly, muscle-specific knockout of mia accompanied by increased accumulation of LKB1 (mLKB1 KO) reduced AMPK a2, but lipid in the liver [72]. Correspondingly, drugs tar- not a1 activity and blunted both contraction- geting liver glucose output offer powerful diabe- and AICAR-induced glucose uptake [68,69]. tes therapies. Indeed, metformin reduces glucose By contrast, AMPK a2KD or a2iTg [59–61] levels in T2D patients by reducing liver glucose only partially affected, or had no effect, on production [73]. Since AMPK is strongly acti- contraction-induced glucose uptake. These vated by metformin in hepatocytes and AICAR findings suggest other LKB1 downstream infusion reduces glucose output in Zucker obese AMPK-related kinases are probably involved. rats, it has been reasonable to assume, at least Indeed, targeting of the AMPK-related kinase until recently, that AMPK regulates liver glucose and LKB1 substrate SNARK by overexpression production [73,74]. of mutant SNARK, RNA silencing in C2C12 muscle cells or using skeletal muscle from AMPK in regulating hepatic glucose output +/- whole-body SNARK mice led to reduced Foretz and colleagues [75] first demonstrated contraction-induced glucose uptake and also that short-term activation of AMPK, achieved dephosphorylation of AS160 [68]. By contrast, by injecting streptozotozin-induced diabetic or a further potential upstream kinase of AMPK, ob/ob mice with an adenovirus encoding a consti- CaMKKb [35] has recently been suggested to tutively active form of AMPK a2, led to reduced control glucose uptake in contraction-stimu- blood glucose levels with increased gluconeogenic lated mouse skeletal muscle (Figure 1(2)). Using gene expression, for example phosphoenolpyru- STO‑609, a CaMKK inhibitor, Jensen and col- vate carboxykinase (PEPCK) and glucose 6-phos- leagues demonstrated reduced electrical stimu- phase (G6Pase) in the liver. However, liver from lated 2‑deoxyglucose uptake in skeletal muscle. mice globally deleted for AMPK b1 subunit, with However, STO-609 did not have any effects on a more than 50% reduction of AMPK activity in glucose uptake in muscles from AMPK a2KD the liver, displayed normal glucose output arguing mice, suggesting an AMPK-dependent effect of against the involvement of AMPK in regulating CaMKKb on glucose uptake [70]. However, the hepatic glucose production [44]. More recently, caveat must be borne in mind when interpreting global AMPK a1 and liver-specific AMPK a2

336 Diabetes Manage. (2011) 1(3) future science group Targeting the AMP-regulated kinase family to treat diabetes: a research update Review knockout mice (AMPKa1a2LS-/-) were generated kinase 2 (SIK2) and MARK2 have been sug- and, similar to the findings in AMPK b1-null gested to phosphorylate CRTC2, facilitating its mice [44], the latter mice showed comparable binding to 14–3–3 proteins and sequestration in glucose levels and gluconeogenesis-related gene the cytosol [78]. As shown in Figure 1(1), deletion expression to wild-type controls [76]. By contrast, of LKB1, possibly through the loss of SIK2 and liver-specific LKB1 knockout mice displayed MARK2 function, leads to dephosphorylation phenotypes similar to ob/ob mice with elevated of CRTC2. This, in turn, relocates CRTC2 to blood glucose levels and impaired glucose toler- the nucleus where it binds CREB and facilitates ance (Figure 1(1)) [77]. This was accompanied by CREB-dependent PGC1-a transcription to pro- decreased phosphorylation of TORC2 (CRTC2) mote the subsequent expression of gluconeogenic and increased expression of gluconeogenic genes, genes such as PEPCK and G6Pase. including peroxisome proliferator-activated receptor‑g coactivator 1‑a, G6Pase and PEPCK, AMPK in liver lipotoxicity and that suggests kinases downstream of LKB1, Excessive lipid accumulation in the liver (so- other than AMPK, might be involved in medi- called ‘fatty liver’) is one of the complications ating the effects of LKB1. Indeed, salt-inducible of T2D and results from decreased fatty acid

SIK2? LKB1 SNARK? MARK2?

(1) (2) (4)

AMPK

(3) (5)

Glucose output Glucose uptake Insulin secretion Glucose output

Blood glucose ↓

Figure 1. LKB1 and AMP-activated protein kinase regulation of whole-body glucose metabolism. (1) LKB1 inhibits gluconeogenesis in the liver, probably through phosphorylation and activation of SIK2. (2) LKB1 inhibits glucose uptake into the skeletal muscle via phosphorylation of SNARK. AMPK (3) and LKB1 (4) play roles in regulating insulin secretion from pancreatic b cells. Note that AMPK activation is likely to exert negative effects in the short term but may have positive consequences in the longer term, for example through the regulation of apoptosis and related pathways. LKB1 action may be mediated via an LKB1-MARK2 signaling pathway. (5) Central AMPK activation stimulates hepatic glucose output. AMPK: AMP-activated protein kinase; LKB1: Liver kinase B1; SIK: Salt-inducible kinase.

future science group www.futuremedicine.com 337 Review Sun & Rutter

oxidation and increased lipogenesis [72]. The factor‑4, AMPK may exert a direct effect on the consequent ‘lipotoxicity’ is then thought to lead regulation of lipogenic genes including liver‑type to insulin resistance probably by affecting the pyruvate kinase [19]. insulin receptor substrate-1/2 PI3K-Akt-GSK3 signaling pathway, hence elevating hepatic glu- AMPK in the endocrine pancreas cose output [79,80]. In addition to regulating The endocrine pancreas, and especially pancre- gluconeogenic gene expression and glucose out- atic b cells of the islets of Langerhans, plays a put, AMPK has also been reported to control central role in controlling glucose homeostasis via lipid deposition in the liver by inhibiting acetyl- fluctuations in insulin output. Failed first phase CoA (ACC) activity via phosphorylation of ACC insulin secretion in response to glucose stimula- and decreases in malonyl-CoA content. These tion, and reduced b cell, are thus required for changes subsequently decrease fatty acid syn- the appearance of overt T2D [5–11]. AMPK has thesis and increase fatty acid oxidation to reduce recently emerged as a key regulator of insulin triglyceride storage [81,82]. Activation of AMPK secretion in the minute-to-minute timescale and achieved, for example, by infusion of AICAR b‑cell mass (i.e., survival and proliferation path- into obese Zucker rats, reduced the glycerol ways) more chronically [19]. turnover rate [74]. A similar effect was seen in hepatocytes treated with metformin or in mice AMPK regulates b‑cell insulin secretion overexpressing constitutively active AMPK a2 & gene expression (AMPK a2 CA) in the liver by injecting adeno- The role of AMPK in controlling insulin secre- virus carrying AMPK a2 CA through the penis tion from pancreatic b cells was first addressed by vein; in the latter case fatty acid oxidation was Salt and colleagues in a pharmacological study on increased whilst there was a decrease in lipo- a tumoral b‑cell line and rodent islets [85]. The genic gene expression, in other words in FAS and authors demonstrated that AICAR-treated INS-1 transcription factors related to lipogenesis such rat b cells displayed increased basal (at 3 mmol/l as SREBP‑1 and ChREBP [73,75]. Conversely, glucose) but inhibited glucose (16.7 mmol/l) mice with liver-specific deletion of AMPK a2 stimulated insulin secretion. Similar results were displayed increased plasma triglyceride levels, also obtained by the group of Van de Casteele and suggesting AMPK a2 is an important regulator Pipeleers [86], and by our own group [87], using of fat metabolism in the liver [83]. On the other MIN6 mouse b cells and primary rat islets. The hand, ablation of LKB1 in the liver reduced latter studies showed that AMPK activation by ACC phosphorylation and led to increased FAS AICAR reduced glucose-stimulated insulin secre- and SREBP-1 gene expression [77]. However, tion, preproinsulin promoter activity and insu- since lipogenic gene expression was not assessed lin gene expression [86–88]. Metformin was later in mice deleted for both catalytic AMPK iso- shown to activate AMPK in MIN6 b cells and forms, specifically in the liver, it is still unknown primary human islets [89]. Increases in AMPK whether the effects of LKB1 on lipogenic gene activity, achieved by treatment with metformin, expression are via AMPK [77]. were also associated with reduced glucose-stimu- AMP-activated protein kinase was suggested lated insulin secretion to near basal levels [89,90]. to regulate liver glucose output in early research These results were further confirmed by Wheeler’s studies using AICAR and metformin. However, group showing an almost 50% decrease of glucose- since AICAR is a mimetic of ZMP and metfor- stimulated insulin secretion from isolated rat islets min acts on respiratory complex I, the effects of treated with AICAR and metformin overnight these drugs on liver metabolism might be due to [91]. However, recent investigations of the involve- a secondary effect of a change of AMP (ZMP)/ ment of AMPK in controlling insulin secretion ATP levels rather than AMPK itself [84]. With the by Philipson’s group found that AICAR treat- advent of liver-specific LKB1 or AMPK double ment, rather than reducing glucose-stimulated catalytic isoform knockout mice, more evidence insulin secretion from mouse islets, increased it has been provided to suggest that other AMPK- [90]. Similarly, studies by Birnbaum and Newgard related kinase lying downstream of LKB1, such indicated that AICAR and phenformin (a more as SIK2 or Mark2, might regulate glucose pro- potent analog of metformin), exerted no effect duction and gluconeogenic gene expression on glucose-stimulated insulin secretion in MIN6 (Figure 1) [1]. Finally, through the phosphoryla- b cells and primary mouse islets [92,93]. These tion and destabilization of hepatocyte nuclear divergent results highlight the limitations of

338 Diabetes Manage. (2011) 1(3) future science group Targeting the AMP-regulated kinase family to treat diabetes: a research update Review using pharmacological approaches in the study increased basal insulin release and insulin gene of AMPK. AICAR mimics the effect of AMP on expression [87]. The effects of AMPK activation AMPK activation, and metformin and phenfor- were later shown to reflect reduced insulin vesicle min have also been reported to activate AMPK movement in MIN6 cells infected with AMPK by inhibiting complex I of the respiratory chain, CA-expressing virus (Figure 2(1)) [96]. leading to an increase in cellular AMP:ATP ratio; the impact on other AMPK-independent AMPK regulates b‑cell apoptosis processes and the signaling pathways is likely to Apart from insulin secretion and insulin gene be significant[94] . To avoid such complications, a expression, AMPK has also been reported by AMPK CA (a1312T172D) or a dominant-negative several groups to exert pro-apoptotic effects in form of the enzyme (DN, a2 D157A) [95] were b cells. Physiologically, this is expected to lower used in later studies. Whereas overexpression of b‑cell mass and total insulin output per pan- AMPK CA in MIN6 b cells and rat islets reduced creas [86,97–100]. Prolonged activation of AMPK glucose-stimulated insulin secretion and insulin in MIN6 cells and purified mouse islet b cells, gene expression, AMPK DN-expressing viruses achieved by 24 or 48 h exposure to AICAR or

Insulin

K+

Glucose

Ca2+ GLUT2 1 LKB1 AMPK

ATP/AMP↑ Pyr

Figure 2. Likely mechanisms through which LKB1 and AMP-activated protein kinase regulate insulin secretion and polarity in pancreatic b cells. In pancreatic b cells, membrane glucose transporters (GLUT2) transport glucose across the plasma membrane. Glucose is metabolized via glycolysis to form pyruvate, which enters the citrate cycle in mitochondria to generate ATP. Increased ATP triggers closure of KATP channels, depolarization and opening of voltage-gated Ca2+ channels. Influx of Ca2+ promotes insulin granules to move to the cell surface prior to insulin release events. (1) Increased ATP:AMP ratios inhibit AMPK activity and increase the number of docked insulin granules beneath the plasma membrane. This is probably achieved through an inhibitory effect of AMPK on kinesin light chain‑1 phosphorylation. (2) LKB1 inhibits glucose-stimulated insulin secretion in part by lowering GLUT2 expression. (3) LKB1 affects pancreatic b‑cell polarity by reorganizing microtubules, actin filaments and tight junctions. AMPK: AMP-activated protein kinase; GLUT2: Glucose transporter 2; LKB1: Liver kinase B1.

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metformin, led to an increase in the number of data [105], consistent with earlier studies [87,96], apoptotic cells assessed by DNA fragmentation revealed increased granule number beneath the [86,97,98,101,102]. This change was proposed to be plasma membrane and enhanced insulin release due to increased oxygen radical formation and (Figure 2) [1]. These important apparent discrep- mitochondrial dysfunction. The latter was then ancies are likely to reflect differences in the pro- proposed to activate JNK and caspase 3‑depen- tocols used for the culture of islets (e.g., glucose dent death pathways, eventually leading to the concentrations), and the selection of islets for onset of apoptosis [86,99]. Using an adenovirus studies (size, ‘condition’). A definitive answer is to express AMPK CA, we confirmed that acti- likely to come only from studies using perfused vation of AMPK in MIN6 cells and dissociated pancreas (the most physiological ex vivo prepa- mouse islet b cells significantly increased levels ration for the study of insulin secretion), more of cleaved-caspase 3, a key marker of apoptosis b‑cell selective knockout, achieved, for example, [103]. Conversely, AMPK DN virus-infected cells using RIP-Cre or Pdx1-CreER deleter strains showed decreased levels of active caspase 3 after [109,110], or a more complete understanding of cytokine treatment [103]. Correspondingly, in pan- the effects of hypothalamic AMPK deletion on creatic b cells isolated from AMPK a2-deficient insulin secretion. mice, AICAR-induced apoptosis was significantly In addition to the effects of modulation of reduced [101]. AMPK on insulin secretion, we have also demonstrated that pancreatic b cells from AMPK b‑cell AMPK & LKB1 regulate whole-body dKO mice are smaller than those from control glucose homeostasis in mice islets [105] and show enhanced levels of apoptosis A first attempt to identify the role of AMPK and autophagy [Sun G, Marchetti P, Tooze S, Leclerc I, specifically in pancreatic b cells in controlling Rutter GA, Unpublished Data]. These observations whole-body glucose metabolism was achieved by would appear to argue against the involvement our group using streptozotocin-induced diabetic of mTOR signaling pathways, which have been mice, which were transplanted with islets infected widely believed to lie downstream of AMPK, with AMPK CA or DN viruses. Mice receiving controlling cell growth in many other cell types AMPK CA-infected islets demonstrated poorer [111]. Further examination of isolated islets using glycemic index over 20 days post-transplanta- transmission electron microscopy demonstrated tion and poorer glucose tolerance, whereas those increased apoptosis with enlarged mitochondria, receiving AMPK DN-infected islets had improved indicating the importance of the presence of glycemic control and better glucose tolerance [104]. AMPK for cell survival [Sun G, Marchetti P, Tooze S, More recent studies by our group [105] and that of Leclerc I, Rutter GA, Unpublished Data]. Perhaps the Michael Ashford [106] using RIP2-Cre transgenic most surprising discovery from three recent mice to delete both AMPK a1 and a2 subunits in studies on mice with pancreatic b‑cell LKB1 pancreatic b cells and a subpopulation of ‘RIP2 deletion using RIP2-Cre (our group) (bLKB1 neurons’ in the brain revealed severe glucose KO) [112] or Pdx1-CreER [113,114] transgenes is intolerance. In the ‘double knockout’ mice insu- the extent to which these mice fail to phenocopy lin secretion in vivo was sharply reduced [105,106], bAMPK dKO mice. Thus, mice lacking LKB1 despite increased insulin sensitivity of peripheral in b cells display increased insulin secretion tissues [105]. It seems likely that this reflects cen- and improved glucose tolerance largely due to tral deletion of AMPK, since our further studies enlarged b‑cell mass and insulin synthesis. This revealed that stereotactic injection of AMPK DN is associated with dephosphorylation of MARK2 into the hypothalamus of rats decreased glucose and upregulation of mTOR signaling pathways, output, presumably reflecting increased insulin suggesting the involvement of LKB1–MARK2– sensitivity [107,108]. However, as shown in Figure 1 mTOR signaling pathways in controlling b‑cell [3], although both studies reported that AMPK mass (Figure 1(4)). deletion abolishes insulin secretion in vivo, Of the 12 other AMPK-related kinases [115], glucose-stimulated insulin secretion from islets Snf-related kinase, NUAK1/2 and MARK1–3 in vitro revealed important differences between were found to be highly expressed in pancreatic the two studies. Thus, insulin secretion from islets at the mRNA level [112]. Only a very low double AMPK (a1, a2; dKO) mouse islets was level of expression of SIK1/2 was apparent whilst reduced and b cells were hyperpoloarized in the BRSK1/2 (also-called SAD‑A/B in the central study by Beall et al. [106]. By contrast our own nervous system) mRNA was undetectable [112].

340 Diabetes Manage. (2011) 1(3) future science group Targeting the AMP-regulated kinase family to treat diabetes: a research update Review

Interestingly, Snf-related kinase, NUAK and inhibition of AMPK led to decreased glucagon MARK have been implicated in the control of secretion with blunted low glucose-stimulated 2+ cell polarity and growth in other cell types [116]. intracellular Ca oscillations. By contrast, acti- Studies of these kinases in pancreatic b cells, for vation of AMPK specifically in pancreatica cells example using b‑cell-specific knockout mouse in intact islets by pre-proglucagon promoter- models, may reveal the possible function of these driven AMPK CA expression demonstrated the kinases in controlling pancreatic b‑cell function stimulatory effect of AMPK on glucagon secre- and growth downstream of LKB1. tion [120]. Thus, these observations indicate that In summary, careful studies are still needed to AMPK is a critical regulator of glucagon release distinguish between the effects of AMPK dele- from the a cell, and is likely to be involved in tion in the brain and in b cells, and to determine counter-regulatory responses to hypoglycemia. the involvement of other AMPK-related kinases Key remaining questions are thus whether in the control of insulin secretion. regulation of glucagon secretion via AMPK- dependent pathways will affect total glucose AMPK regulates glucagon secretion & gene metabolism in the long term? Which isoforms of expression in pancreatic a cells AMPK catalytic subunits are involved? What are Glucagon, secreted by pancreatic a cells in the mechanisms through which AMPK exerts response to hypoglycemia [117], is a critical diametrically opposite effects on hormone secre- counter-regulatory hormone to insulin and tion in a cells (stimulation) versus b cells (inhi- thus in the maintenance of glucose homeostasis bition)? Studies with AMPK inactivated specifi- in mammals. Glucagon acts on liver glucagon cally in pancreatic a cells will be needed to fully receptors to promote hepatic glucose produc- address these questions. tion, and downregulation of glucagon signaling, achieved for example by whole-body dele- AMPK in the ventromedial hypothalamus tion of glucagon receptors (GCR-/- mice), leads Hypothalamic AMPK in regulating food intake to severe hypoglycemia during fasting [118]. On The basomedial hypothalamic area of the the other hand, both recurrent hypoglycemia brain is a key compartment for energy sensing in Type 1 diabetic patients and insulin treat- and satiety regulation. Hypothalamic AMPK ment-induced hypoglycemia in Type 2 diabetic activity is regulated by feeding status and vari- patients are associated with hyposecretion of ous hormones and nutrients including insulin, glucagon. Both AMPK a1 and a2 subunits are ghrelin, leptin, adiponectin and cannabinoids, expressed in pancreatic a cells [112]. However, and these changes have been suggested to control little information has been obtained to date bodyweight and food intake [121–126]. Increases regarding the role of AMPK in pancreatic a in AMPK activity, achieved by expressing cells. Interestingly, silencing of Pas (per-arnt- AMPK CA in the mediobasal hypothalamus sim) domain-containing protein kinase (PASK), (including arcuate and paraventricular areas) which is distantly related to AMPK, in mouse or intracerebroventricular injection of AICAR, aTC1–9 cells leads to increased AMPK a2 gene increased food intake and bodyweight, concomi- expression accompanied by increased glucagon tant with increased expression of the orexigenic secretion and pre-proglucagon gene expression. peptides neuropeptide and agouti-related protein Conversely, overexpression of PASK in aTC1–9 (AgRP) [127]. Conversely, introduction of adeno- cells or human islets activates glucagon release, viral AMPK DN or compound C decreased the suggesting that PASK might regulate glucagon expression of both peptides and lowered food signaling partially through AMPK [119]. Recent intake [127]. It was proposed that two intracel- approaches to regulate AMPK activity in the lular signaling pathways might be involved in mouse aTC 1–9 cell line have included using the satiety-controlling effects of AMPK. One AMPK activators, such as metformin, phen- was through hypothalamic lipid metabolism, formin or A769662 [120], or viruses carrying that is, AMPK-mediated inhibition of ACC AMPKa2 CA [120]. In the same study AMPK carboxylase and malonyl-CoA activation lead- was inhibited in a cells using compound C or ing to enhanced carnitine palmitoyltransferase a virus carrying AMPK a1 DN. These studies (CPT)‑1 activity [128]. An alternative mTOR- revealed a potentially important role for AMPK dependent signaling pathway in the mediobasal in regulating glucagon release. Thus, activation hypothalamus was recently suggested to mediate of AMPK increased glucagon secretion whilst AMPK-regulated increase in food intake. Here,

future science group www.futuremedicine.com 341 Review Sun & Rutter

AMPK was proposed to inhibit phosphoryla- hypothalamus by resistin might regulate b‑cell tion of its downstream target, S6K1 [129,130]. To function and b‑cell growth [133]. As mentioned clarify exactly which areas of the hypothalamus, above, deletion of both AMPK catalytic isoforms and which neuron types, were involved in these using the RIP2-Cre transgene also expressed in effects, Wither’s group generated proopiomela- the hypothalamus [109] led to opposing insulin nocortin and AgRP‑specific AMPK a2 knock- secretion patterns in vivo and in vitro, implicat- out mice. Mice lacking AMPK a2 in proopi- ing the involvement of central AMPK in con- omelanocortin neurons displayed increased food trolling insulin secretion in vivo [105]. Careful intake and became obese, while mice inactivated analysis of which hypothalamic neuronal cell for AMPK a2 in AgRP neurons showed the types and which AMPK isoforms are involved opposite phenotype, with decreased food intake in controlling b‑cell function or mass needs to and bodyweight [131]. Glucose-inhibited neurons be further addressed. isolated from the basomedial hypothalamus responded to decreased glucose concentrations Upstream kinases for AMPK in in a similar way to the treatment of neurons with the hypothalamus AICAR. The stimulatory effects of AICAR on Mice deleted for LKB1 in RIP2-expressing cells these neurons were reversed by the AMPK inhib- (bLKB1 KO), including those in the hypo- itor, compound C. These results indicated that thalamus [109], showed a hypophagic phenotype activation of AMPK in these neurons might lead and decreased bodyweight [112]. Neither b‑cell to increased bodyweight [125]. selective-AMPK double knockout mice (generated using the same RIP2-Cre deleter mice as Hypothalamic AMPK & the regulation of for LKB1 elimination in these cells) or mice in hepatic glucose production which LKB1 was inactivated in the b‑cell of In addition to regulating satiety and bodyweight, adult mice using a regulatable Pdx1 CreERT the hypothalamus has also been implicated in mouse strain, showed any changes in satiety integrating energy signaling to control hepatic or bodyweight. These findings suggest that the glucose production. In light of the reduced glu- observed decrease of food intake observed as a cose output in mice with decreased CPT‑1 lev- result of hypothalamic LKB1 elimination (a els in the hypothalamus [132], it is plausible that consequence of the expression of the RIP2.Cre AMPK, whose phosphorylation increases CPT‑1 transgene in parts of the central nervous system, activity through decreasing ACC and malonyl- as well as in the pancreatic b-cell) was indepen- CoA levels, might also increase hepatic glucose dent of AMPK [105,113,114]. Further studies of production. Most recently, using rats infused into LKB1, or possibly other AMPK-related kinases, the mediobasal hypothalamus with virus carrying in RIP2 neurons are needed to fully understand AMPK DN or compound C, Yang and colleagues the mechanisms by which LKB1 controls satiety demonstrated that inhibition of AMPK in this in RIP2-Cre neurons. brain region reduced glucose production without In summary, AMPK is clearly a key regula- changing peripheral glucose disposal [107]. Thus, tor of energy signals in the hypothalamus, inte- central AMPK might be an important regulator grating signals from peripheral tissues to control of hepatic glucose production (Figure 1(5)). satiety, glucose production and insulin secretion. However, caution needs to be taken when inter- Hypothalamic AMPK in controlling b‑cell preting data from intracerebroventricular infu- function & b‑cell mass sion studies that affect large areas of the medio- As well as modulating hepatic glucose produc- basal hypothalamus. Thus, the identity of the tion, the hypothalamus may be able to control neurons involved, and the role played by AMPK b‑cell function and b‑cell mass by control- in such neurons, remains only partly clarified. ling central hormone secretion such as resistin Total ablation of AMPK using neuron-specific [133]. Thus, intracerebroventricular infusion of Cre transgenes will be a useful tool to answer resistin into rats for 4 weeks led to a remark- these questions. ably elevated first phase insulin secretion, concomitant with increased b‑cell mass. Decreased Conclusion & future perspective AMPK phosphorylation in response to leptin In this article we have focused on recent studies was also alleviated by resistin infusion, implying in which gene inactivation has been used as a that the modulation of AMPK activity in the tool to study the role and regulation of AMPK

342 Diabetes Manage. (2011) 1(3) future science group Targeting the AMP-regulated kinase family to treat diabetes: a research update Review in diabetes-relevant tissues in mice. However, gradually emerging that the effects of metformin it is important to point out two caveats when are probably not, or only partially, mediated via interpreting such studies [1]: AMPK. Moreover, discrepancies between the phe- The possibility exists that changing the notypes of mice inactivated for AMPK or LKB1 expression of a gene might unmask a pathway in the same tissue suggest that other AMPK- whose role in normal physiology is, due to related kinases downstream of LKB1 might be redundancy, usually minimal or zero. Such associated with the control of whole-body glucose redundancies mean that the absence (or mild- metabolism. Notably, in the cases of muscle con- ness) of a phenotype apparent after the inac- traction-induced glucose uptake and hepatic glu- tivation of a particular gene cannot be taken cose production, it seems very likely that SNARK as proof that the gene and gene product in and SIK2 or MARK2 are involved. Likewise, the question are unimportant [2]; discrepancies in the phenotypes of RIP2-AMPK dKO and RIP2-LKB1 KO mice in terms of food That the considerable differences between the intake and insulin secretion also argue against physiology of mice (mass ~25 g; lifespan the universal control of satiety by a simple linear <3 years) versus man (mass ~50–90 kg; ‘LKB1-AMPK’ signaling pathway. Therefore, the lifespan ~75 years) mean that extrapolation of concept of AMPK as a master energy sensor in results from the former to the latter must be different glucose- and insulin-sensing organs may made with caution. need careful re-evaluation. In particular, further Nonetheless, at least for the time being, mice investigation of the role of the other 12 AMPK- do provide the most suitable in vivo system to related kinases [115] using tissue-specific knock- analyze the role of enzymes such as AMPK and out or transgenic mice model may help to under- the role they play in health and in disease settings. stand how these kinases regulate glucose levels. Because AMPK balances ATP generation and Importantly, drugs acting specifically on certain consumption, it is natural to think of it as acting kinases or downstream targets may provide the as a gauge to control energy status [15,50]. Thus, promise of new therapies for T2D. manipulation of AMPK might restore the imbal- ance of energy usage in metabolic disorders such Acknowledgement as T2D where AMPK activities and ATP/AMP The authors thank G Silva Xavier for discussion and review levels can be affected. In fact, the most widely of the manuscript. prescribed antidiabetic drug, metformin, strongly activates AMPK in many insulin-sensitive and Financial & competing interests disclosure glucose-regulating organs, such as the liver, mus- Work in GA Rutter’s laboratory is supported by grants from cle, hypothalamus and pancreas [134,135]. Early the Wellcome Trust, MRC, JDRF, Diabetes UK and the studies using metformin also demonstrated the EU. The authors have no other relevant affiliations or finan- beneficial effects of AMPK in restoring energy cial involvement with any organization or entity with a balance, such as decreasing hepatic glucose out- financial interest in or financial conflict with the subject put, elevating skeletal muscle glucose uptake and matter or materials discussed in the manuscript apart from promoting satiety in the hypothalamus [134]. those disclosed. However, with increased usage of tissue-specific No writing assistance was utilized in the production of AMPK and LKB1 knockout mouse models, it is this manuscript.

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