cells Review AICAr, a Widely Used AMPK Activator with Important AMPK-Independent Effects: A Systematic Review Dora Višnji´c 1,2,*, Hrvoje Lali´c 1,2 , Vilma Dembitz 1,2 , Barbara Tomi´c 1,2 and Tomislav Smoljo 1,2 1 Laboratory of Cell Biology, Croatian Institute for Brain Research, University of Zagreb School of Medicine, 10000 Zagreb, Croatia; [email protected] (H.L.); [email protected] (V.D.); [email protected] (B.T.); [email protected] (T.S.) 2 Department of Physiology, University of Zagreb School of Medicine, 10000 Zagreb, Croatia * Correspondence: [email protected] Abstract: 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAr) has been one of the most com- monly used pharmacological modulators of AMPK activity. The majority of early studies on the role of AMPK, both in the physiological regulation of metabolism and in cancer pathogenesis, were based solely on the use of AICAr as an AMPK-activator. Even with more complex models of AMPK downregulation and knockout being introduced, AICAr remained a regular starting point for many studies focusing on AMPK biology. However, there is an increasing number of studies showing that numerous AICAr effects, previously attributed to AMPK activation, are in fact AMPK-independent. This review aims to give an overview of the present knowledge on AMPK-dependent and AMPK- independent effects of AICAr on metabolism, hypoxia, exercise, nucleotide synthesis, and cancer, calling for caution in the interpretation of AICAr-based studies in the context of understanding AMPK signaling pathway. Citation: Višnji´c,D.; Lali´c,H.; Dembitz, V.; Tomi´c,B.; Smoljo, T. Keywords: AICAr; AMPK; metabolism; acadesine; exercise; cell cycle; purine; pyrimidine; can- AICAr, a Widely Used AMPK cer; leukemia Activator with Important AMPK-Independent Effects: A Systematic Review. Cells 2021, 10, 1095. https://doi.org/10.3390/ 1. Introduction cells10051095 In 1995, 5-amino-4-imidazolecarboxamide (AICA) ribonucleoside or riboside was first proposed to be used as the activator of AMP-kinase (AMPK) in intact cells or, in other words, Academic Editor: Yury Ladilov to play the same role that phorbol esters had in dissecting signaling pathways regulated by protein kinase C [1]. The study by Corton et al. was based on previous work showing that Received: 15 March 2021 administration of the AICA riboside to intact cells causes AICA ribonucleotide or ribotide Accepted: 1 May 2021 Published: 4 May 2021 to accumulate inside the cell [2], and a study by Sullivan et al. [3] demonstrated that AICA ribotide mimicked the effect of AMP on allosteric activation of rat liver AMPK. Since then, the compound that has been widely used as an AMPK-agonist was an exogenous Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in dephosphorylated AICA riboside that should be properly abbreviated AICAr. However, in published maps and institutional affil- more than 1700 articles that can be retrieved from PubMed on AMPK and AICA riboside, iations. AICAr is often abbreviated as AICAR, although the AICAR acronym should be reserved for AICA ribotide or the phosphorylated form that is a physiological, endogenous precursor in de novo purine synthesis [4]. The nomenclature is additionally complicated because the other name used for the endogenous substance or AICAR is ZMP [5]. Moreover, the search of the literature reveals the common use of acadesine instead of AICAr [4,6–19]. Copyright: © 2021 by the authors. As shown in Figure1, AICAR or ZMP is an AMP analog. As a cell-permeable Licensee MDPI, Basel, Switzerland. This article is an open access article nucleotide, AICAr enters the cells through adenosine transporters [20] and becomes phos- distributed under the terms and phorylated by adenosine kinase into AICAR [21]. AICAR or ZMP activates AMPK but conditions of the Creative Commons it is 40- to 50- fold less potent than AMP in AMPK activation and accumulates in high Attribution (CC BY) license (https:// concentrations in the cytoplasm [1], so that it was always likely that AICAr may have creativecommons.org/licenses/by/ several AMPK-independent effects. Similar to AMP, AICAR binds to the γ subunit of 172 4.0/). AMPK, allosterically activates the enzyme, stimulates phosphorylation at Thr by liver Cells 2021, 10, 1095. https://doi.org/10.3390/cells10051095 https://www.mdpi.com/journal/cells Cells 2021Cells, 102021, x ,FOR10, 1095 PEER REVIEW 2 of 172 of 17 tissueskinase or cells B1 (LKB1),is to detect and the protects level of against pThr172 pThr AMPK172 bydephosphorylation Western blot in lysates [22,23 ].upon Therefore, AICAr treat- ment.the most common method to test for AICAr-mediated activation of AMPK in particular tissuesIn this or article, cells iswe to will detect give the a brief level overvi of pThrew172 ofAMPK the present by Western knowledge blot on in lysatesAMPK-dependent upon andAICAr AMPK-independent treatment. effects of AICAr. FigureFigure 1. 1.AICArAICAr structure structure and mechanism.mechanism. 2. AICAr,In thisMetabolism article, we and will Diabetes give a brief overview of the present knowledge on AMPK- dependentSince 1995, and AICAr AMPK-independent has been a valuable effects oftool AICAr. to identify metabolic pathways associated with2. AMPK AICAr, activation Metabolism in and various Diabetes tissues and cells in vivo and in vitro. In liver cells, AICAr was foundSince to 1995, inhibit AICAr fatty has acid been and a valuable cholesterol tool tosynthesis identify metabolic[24], to increase pathways fatty associated acid oxida- tionwith [25], AMPK and to activation inhibit gluconeogene in various tissuessis by and decreasing cells in vivo transcriptionand in vitro. of In PEPCK liver cells, [26]. In skeletalAICAr muscle was found cells, to AICAr inhibit fattywas acidfound and to cholesterol activate glycogen synthesis [phosphorylase24], to increase fatty and acidincrease glycogenolysisoxidation [25 ],[27], and stimulate to inhibit gluconeogenesis fatty acid oxidation by decreasing and glucose transcription uptake of [28],PEPCK and[26 inhibit]. proteinIn skeletal synthesis muscle [29]. cells, All AICAr of these was studies found to su activatepported glycogen the role phosphorylase of AMPK as and a central increase meta- bolicglycogenolysis hub of the cell [27 ],that stimulate is activated fatty acid whenever oxidation the and ratio glucose of AMP/ATP uptake [28 is], high and inhibitand which actsprotein to increase synthesis catabolic [29]. and All ofinhibit these anabolic studies supported processes the(Figure role of2). AMPK as a central metabolic hub of the cell that is activated whenever the ratio of AMP/ATP is high and which acts to increase catabolic and inhibit anabolic processes (Figure2). 2 Cells 2021, 10, x FOR PEERCells REVIEW2021, 10, 1095 3 of 17 3 of 17 Figure 2.FigureA schematic 2. A schematic presentation presentation of molecular of molecular mechanisms mech ofanisms AICAr of Akt/PKB, AICAr Akt/PKB, Akt/protein Akt/protein kinase B; ki- PDK1, 3- phosphoinositidenase B; PDK1, dependent 3-phosphoinositide protein kinase-1; dependent PI3K, phosphatidylinositol protein kinase-1; 3-kinase; PI3K, phosphatidylinositol PIP3, phosphatidylinositol 3-ki- (3,4,5)- trisphosphate;nase; PIP3, IRS, insulin phosphatidylinositol receptor substrate; (3,4,5)-trisphosphate; TSC1/2, tuberous sclerosis IRS, complex insulin 1receptor protein /substrate; tuberous sclerosisTSC1/2, complex tu- 2 protein; Rheb,berous Ras sclerosis homolog complex enriched 1 inprotein the brain; / tuberous mTOR, mechanisticsclerosis complex target of2 rapamycin;protein; Rheb, mTORC1, Ras homolog mTOR complex en- 1; mTORC2,riched mTOR in complex the brain; 2; Rictor,mTOR, rapamycin-insensitive mechanistic target of companion rapamycin; of mTOR; mTORC1, PRAS40, mTOR proline-rich complex Akt1; substrate 40; Raptor, regulatory-associatedmTORC2, mTOR complex protein of2; mTOR;Rictor, AMPK,rapamycin-in adenosinesensitive monophosphate companion (AMP)-activated of mTOR; PRAS40, protein proline- kinase; p53, tumor suppressorrich Akt substrate protein p53; 40; S6K1, Raptor, p70 S6regulatory-associ Kinase 1; OXPHOS,ated oxidative protein phosphorylation;of mTOR; AMPK, 4E-BP1, adenosine eukaryotic mono- translation initiationphosphate factor 4E binding (AMP)-activated protein 1; GLUT4, protei glucosen kinase; transporter p53, tumor 4. suppressor protein p53; S6K1, p70 S6 Ki- nase 1; OXPHOS, oxidative phosphorylation; 4E-BP1, eukaryotic translation initiation factor 4E binding protein 1; GLUT4,However, glucose investigators transporter soon4. realized that ZMP in millimolar concentrations could exert many other effects, including modulation of other AMP-sensitive enzymes, like However, investigatorsglycogen phosphorylase soon realized [ 30that], andZMP fructose-1-6-biphosphatase, in millimolar concentrations a rate-controlling could exert many enzyme in other effects, includinggluconeogenesis, modulation whichof other is likelyAMP-sensitive to explain enzymes, why the like effects glycogen of AICAr phosphorylase on gluconeogenesis [30], and fructose-1-6-biphosphatase,in the liver are AMPK-independent a rate-controlling [ 31enzyme]. The bestin gluconeogenesis, way to assess the which role is of likely AMPK in the to explain why theeffects effects of AICArof AICArin vivoon gluconeocould begenesis provided in the by AMPKliver are knockout AMPK-independent mice. As shown [31]. in Table1, The best way to assessdata obtainedthe role of by AMPK transgenic in the mice effects models of AICAr revealed in vivo that could AICAr-mediated