Citation: Experimental & Molecular Medicine (2016) 48. e224; doi:10.1038/emm.2016.16
Published online 1 April 2016
AMPK activators: mechanisms of action and physiological activities
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Joungmok Kim 1. Goowon Yang 2. Yeji Kim 2. Jin Kim 2 and Joohun Ha 3
- 1 Depatment of Oral Biochemistry and Molecular Biology, School of Dentistry, Kyung Hee University, Seoul, Korea
- 2 Department of Biomedical Science, Graduate School, Kyung Hee University, Seoul, Korea
- 3 Department of Biochemistry and Molecular Biology, Graduate School, Kyung Hee University, Seoul, Korea
Correspondence: Professor J Ha, Department of Biochemistry and Molecular Biology, Graduate School, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Korea. E-mail: hajh@khu.ac.kr
Received 1 December 2015; Revised 28 December 2015; Accepted 29 December 2015
AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis, which coordinates metabolic pathways and thus balances nutrient supply with energy demand. Because of the favorable physiological outcomes of AMPK activation on metabolism, AMPK has been considered to be an important therapeutic target for controlling human diseases including metabolic syndrome and cancer. Thus, activators of AMPK may have potential as novel therapeutics for these diseases. In this review, we provide a comprehensive summary of both indirect and direct AMPK activators and their modes of action in relation to the structure of AMPK. We discuss the functional differences among isoform-specific AMPK complexes and their significance regarding the development of novel AMPK activators and the potential for combining different AMPK activators in the treatment of human disease.
Introduction
As a cellular energy sensor, AMP-activated protein kinase (AMPK) is activated in response to a variety of conditions that deplete cellular energy levels, such as nutrient starvation (especially glucose), hypoxia and exposure to toxins that inhibit the mitochondrial respiratory chain complex. 1. 2 AMPK is a serine / threonine protein kinase complex consisting of a catalytic α -subunit ( α 1 and α 2), a scaffolding β-subunit (β1 and β2) and a regulatory γ -subunit ( γ 1, γ 2 and γ 3; Figure 1 ). Ubiquitous expression of AMPK α 1-, β1- and γ 1-subunits in many tissues makes the α 1β1 γ 1 complex a reference for AMPK assays to identify AMPK activators. However, given the unique functions and / or subcellular (or tissue)-specific distribution of the distinct AMPK complex, 3. 4. 5 referencing screening to the α 1β1 γ 1 complex may present a limited range of the physiology of AMPK. In line with this notion, increasing evidence shows that inactivating mutations and genetic deletion of specific isoforms produce tissue-specific physiological results. 6. 7. 8 Mutations in the AMPK γ 2 subunit have frequently been observed in human cardiomyopathies, and deletion of the AMPK α 2 subunit, but not α 1, has been shown to decrease infarct volume in mouse models of stroke.
Functional domains of AMP-activated protein kinase (AMPK) subunits. The mammalian α 1 / α 2 and β1 / β2 isoforms are very similar, and their characteristic features are shown. AMPK α subunits: KD, kinase domain containing Thr-172 for the activation by upstream kinases; AID, autoinhibitory domain; two α -RIM, regulatory subunit interacting motifs triggering the conformational changes in response to AMP binding to the AMPK γ subunit; α -CTD, C-terminal domain binding to the β-subunit. AMPKβ subunit: CBM, carbohydrate-binding module, in which Ser108 is important for the action of some direct AMPK activators, such as thienopyridone (A-769662) and salicylate; β-CTD, C-terminal domain containing α -subunit-binding site and immediately followed by the domain for γ -subunit interaction. AMPK γ subunit: three γ -subunit isoforms have variable N-terminal domains (NTDs); four CBS, cystathione-β-synthases domain, which forms two Bateman domains that create four adenosine nucleotide-binding sites (Sites 1–4). Site 2 appears to be always empty and Site 4 to have a tightly bound AMP, whereas Sites 1 and 3 represent the regulatory sites that bind AMP, ADP or ATP in competition.
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Allosteric activation of AMPK by AMP
The first class of direct AMPK activators is small molecules that mimic cellular AMP. These molecules trigger a conformational change in the AMPK complex that allows further activation by phosphorylation of Thr-172 in the AMPK α subunit. 9. 10 The molecular mechanism underlying allosteric activation of AMPK by AMP binding has been demonstrated by several recent studies of the three-dimensional structure of AMPK. 11. 12. 13 This crystal structure has shown the importance of cystathionine-β-synthase domain repeats within the AMPK γ subunit in the molecular mechanism by which AMPK is activated in response to cellular adenosine nucleotides (AMP, ADP or ATP). Four consecutive cystathionine-β-synthase domains in the AMPK γ subunit provide four potential adenine nucleotide-binding sites. These sites are numbered Sites 1–4, according to the number of the cystathionine-β-synthase domain repeat carrying a conserved aspartate residue involved in ligand binding. 11. 14. 15 In the mammalian AMPK γ 1 subunit, Site 2 appears to be always empty and Site 4 to have a tightly bound AMP molecule, whereas Sites 1 and 3 represent the regulatory sites that bind AMP, ADP or ATP, which compete for binding. 16 AMP binding to Site 1 appears to cause allosteric activation, whereas binding of AMP or ADP to Site 3 appears to modulate the phosphorylation state of Thr172. 13 Although cellular ADP levels are higher than those of AMP, a recent study has shown that AMP is a bona fide activator that enhances LKB1-dependent Thr 172 phosphorylation in vivo. 17 AMP binding to the AMPK γ subunit serves as an important regulatory feature of the conformational switch that activates the AMPK complex. The catalytic AMPK α subunit contains an N-terminal kinase domain (KD) immediately followed by an autoinhibitory domain (AID). The three-dimensional structure shows that the AID interacts with the small and large lobes of the KD and causes AMPK to be maintained in an inactive conformation. Once AMP binds to the AMPK γ subunit, the α -RIM (regulatory subunit-interacting motif) between the KD / AID and a globular C-terminal domain of the AMPK α subunit interact with one of the regulatory adenosine nucleotides on the AMPK γ subunit in a manner akin to two arms wrapping around the adenosine. These conformation changes release and expose the KD of AMPK α from its AID to activate the AMPK complex.
Regulation of AMPK activity by upstream kinases
Physiological AMPK activation involves phosphorylation of Thr-172 within the activation loop of the KD in the AMPK α catalytic subunit. Two upstream kinases, LKB1 18 and CaMKKβ (Ca 2 + / calmodulin-dependent protein kinase β), 19 have been extensively documented to phosphorylate Thr-172 of the AMPK α subunit. Notably, there are lines of evidence showing that the LKB1-dependent AMPK α phosphorylation at Thr172 is greatly enhanced by the binding of AMP to the AMPK γ -subunit, and, at the same time, the AMP-binding inhibits dephosphorylation of this activating phosphorylation by protein phosphatases, such as PP2A and PP2C in vitro. 20. 21 Interestingly, the effect of AMP on Thr172 phosphorylation of the AMPK α -subunit appears to be dependent on N ‐ terminal myristoylation of the β-subunit, although the underlying mechanism remains to be demonstrated. 22 In contrast to the LKB1 complex, another upstream AMPK kinase, CaMKKβ, can activate AMPK in response to increases in cellular Ca 2 + without any significant change in ATP / ADP / AMP levels. Treatments that deplete cellular ATP do not effectively activate AMPK in LKB1-negative tumors because the basal activity of CaMKKβ is too low to affect the phosphorylation status of AMPK α Thr172, although the increase in AMP due to ATP depletion makes the AMPK α -subunit a better substrate for CaMKKβ. However, these treatments can cause AMPK activation under conditions that elevate intracellular Ca 2 +. These data indicate that the phosphorylation / dephosphorylation equilibrium at Thr-172 on the AMPK α -subunit involves AMP binding to the AMPK γ subunit and N-terminal modification of the AMPK β-subunit, adding another a level of complexity to the AMPK activation mechanism.
Physiological functions of AMPK
As its name suggests, AMPK has a key role in maintaining the balance between anabolic and catabolic programs for cellular homeostasis in response to metabolic stress. 23. 24. 25. 26. 27. 28 Given the functional attributes of AMPK in glucose / lipid homeostasis, body weight, food intake, insulin signaling and mitochondrial biogenesis, AMPK is considered to be a major therapeutic target for the treatment of metabolic diseases including type 2 diabetes and obesity. 29. 30
A number of studies have shed light on the role of AMPK in tumorigenesis. 31 An initial report connecting AMPK to cancer biology described the discovery of the tumor suppressor LKB1 as a major AMPK upstream kinase. 32 Genetic mutations of the LKB1 gene are responsible for inherited Peutz-Jeghers syndrome, which is characterized by the development of hamartomatous polyps in the intestine. 33 Since then, a number of in vitro and in vivo studies have suggested that AMPK indeed mediates the tumor-suppressor effects of LKB1. This is supported by findings that drugs that are capable of activating AMPK (metformin, phenformin, A-769662) delay the onset of tumorigenesis in in vivo models. 34. 35 Much effort has been made to understand the molecular mechanisms underlying the antitumorigenic functions of AMPK. These studies have shown that mTORC1 36. 37 and RNA polymerase I transcription factor TIF-1A, 38 both of which are required for rapidly proliferating cells, are under the control of AMPK. In addition, AMPK activation has been shown to cause G1 cell cycle arrest, which is associated with activation of p53, followed by induction of the cell cycle inhibitor protein, p21. 39. 40 Similarly, AMPK has been shown to cause cell cycle arrest by inducing the phosphorylation and concomitant stabilization of the cyclin-dependent kinase inhibitor p27 kip1 in response to metabolic stress. 41 A recent study has described an additional layer of p53–AMPK–mTORC1 regulation via the p53-repsonsive gene products Sestrin1 / 2. 42 However, it should be noted that AMPK might protect tumor cells against the action of cytotoxic agents, nutrient limitation and hypoxia, once the tumors are established. Therefore, AMPK activators might be deleterious in the treatment of cancer.
Another important aspect of AMPK biology is the role of AMPK in autophagy, a lysosome-dependent catabolic program that maintains cellular homeostasis. 43. 44. 45. 46 A number of studies have demonstrated that AMPK has important roles in autophagy regulation by directly phosphorylating two autophagy-initiating regulators: a protein kinase complex ULK1 (Unc-51-like autophagy-activating kinase) 47. 48 and a lipid kinase complex PI3KC3 / VPS34 (phosphatidylinositol 3-kinase, catalytic subunit type 3; also known as VPS34). 49 A number of reports have demonstrated the metabolic significance of autophagy in glycogenolysis (glycophagy) 50 and lipolysis (lipophagy) 51 and even in regulating adipose mass as well as differentiation in vivo. 52 In this regard, elucidating the molecular connection between AMPK and autophagy will provide a novel avenue to expand the functional network of AMPK in cellular homeostasis, including metabolism.
Given these functional attributes, as summarized in Figure 2. much effort has been made to develop robust AMPK assays and to identify AMPK modulators to provide therapies for a variety of human diseases. 53. 54. 55. 56 In this review, we present a comprehensive summary of both indirect and direct AMPK activators and their modes of action in relation to the structure of AMPK, and discuss the implications of AMPK as a therapeutic target.
A summary of the physiological roles of AMP-activated protein kinase (AMPK).
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