Being a multi-substrate serine/threonine kinase, AMPK provides binding sites for an array of regulatory nucleotides. The primary physiological manifestation of activated AMPK is to redirect metabolism towards increased catabolism and decreased anabolism through the phosphorylation of key proteins in response to metabolic stress. This metabolic reprogramming is initiated in order to ensure the replenishment of intracellular ATP levels back to normal (12, 13).Studies using AMPKα1 and α2 knockout mouse models by Violett et al., revealed that glucose homoeostasis was unaltered in AMPKα1^−/− mice whereas, high plasma glucose levels and low plasma insulin concentrations were observed in AMPKα2^−/− mice although the insulin secretion was not altered in both types of mice. Furthermore, the team also reported that the AMPKα2 catalytic subunit modulates the activity of the autonomous nervous system in vivo ( 24).

Accumulation of glucose, fatty acids, and amino acids have been reported to suppress AMPK and eventually lead to insulin resistance, while stimulation of AMPK activity improved blood glucose levels, suggesting that development of AMPK activators might potentially function as anti-diabetic drugs (25–27). Metabolic inflexibility and insulin resistance present in Type 2 diabetes mellitus and obesity develop as a result of dysfunctional mitochondria in the muscles (28, 29).

Activation of AMPK in skeletal muscles induces peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) and up-regulates mitochondrial genes (30). These observations suggest that AMPK activators might have a beneficial effect in combating metabolic disorders. Studies also reveal a negative correlation between inflammation and AMPK. The proinflammatory cytokine, tumor necrosis factor α (TNFα), suppresses the phosphorylation of AMPK and increases the expression of protein phosphatase 2C (PP2C) in the skeletal muscles thereby inducing insulin resistance (31).

Interestingly, AMPK has been reported to play a key role in the development of Alzheimer’s disease (AD), a progressive neurodegenerative disease. One of the hallmark features of AD includes the aberrations in β-amyloid metabolism. Previous studies have revealed that AMPK activation is closely linked with the aberrant processing of β-amyloid protein precursor (AβPP). Further, AMPK signaling controls β-amyloid metabolism via the suppression of Glycogen synthase kinase 3β (GSK-3β) (32). Another independent study indicates that AMPK hyperphosphorylation occurs in the brains of mice that have experimental AD, as well as human patients suffering from AD (33, 34). Further studies demonstrated that resveratrol lowers extracellular accumulation of β-amyloid peptides through the activation of AMPK. This study also demonstrated that the inhibition of mammalian target of rapamycin complex 1 (mTORC1) mediated by the activation of AMPK induces autophagy and lysosomal degradation of the β-amyloid peptide (35).

AMPK is also activated under conditions of cardiac ischemia and studies indicate that the intrinsic activation of AMPK protects the heart from injuries induced due to the ischemia (36, 37). Treatment with AMPK activators such as 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) and metformin brought about a significant decrease in contractile dysfunction, apoptosis, and fibrosis in canine heart failure models (38).

AMPK activation has been extensively investigated as a potential therapeutic target in combating different types of cancer. AMPK phenotypically functions as a tumor suppressor by resisting pro-tumorigenic metabolic processes and directly inducing cell-cycle arrest in cancer cells. Cyclooxygenase 2 (COX-2) plays an important role in cancer stemness. Interestingly, AMPK also acts as a COX-2 inhibitor in cancers of the breast and colon (39). AMPK activation is critical in alleviating metabolic and energetic stresses associated with tumor progression. Besides, AMPK activation triggers the onset of multiple cell death mechanisms. It influences the cell cycle checkpoints, autophagy, mitophagy, and apoptosis. AMPK promotes autophagy and mitophagy by activating UNC-51-like kinase 1 (ULK1) and death-associated protein 1 (DAP1) respectively (40, 41). AMPK initiates the apoptotic program via the activation of p53, p21, and p27. It manifests cell cycle arrest via the inhibition of HUR and the concomitant activation of Cyclins A, B1, and D1 (42). Previous reports indicate that metformin, an AMPK activator down-regulates c-MYC in an AMPK-dependent manner in breast cancer cell lines (43).There are several other reports highlighting the significance of AMPK in cancer chemoprevention (44–50). Studies using MT 63–78, a specific and potent direct AMPK activator have revealed that AMPK activation induces mitotic arrest and apoptosis in androgen-sensitive and castration-resistant prostate cancer via mTORC1 blockade and the suppression of de novo lipogenesis (51). Previous studies have documented that treatment of hepatocellular carcinoma cells with the AMPK activators, AICAR and metformin, significantly inhibited their proliferation, and induced cell cycle arrest at the G1-S phase (52). Zou et al. demonstrated that metformin-induced activation of AMPK down-regulated the expression of segment polarity protein dishevelled homolog3 (DVL3), a key oncoprotein that activates the Wnt/β-catenin signaling pathway (53). Down-regulation of DVL3 reduced the levels of β-catenin and its downstream targets, cyclin D1 and c-Myc, resulting in suppression of cell proliferation. AMPK activation has also been reported to induce p53-dependent apoptotic effects in breast cancer cells (9),and inhibit the metastatic potential of melanoma cells by modulating the ERK signaling pathway and reducing the levels of the COX-2 protein (54). It also induces autophagic and apoptotic cell death through AMPK/JNK signaling (55).Vara-Ciruelos et al. have demonstrated the tumor suppressor role of AMPK-α1 by utilizing T-cell-specific knock-outs of Pten and Prkaa1 gene encoding AMPK-α1. In these models, absence of Pten and Prkaa1genes promoted lymphoma development at an early age and the tumors were significantly more aggressive (56).Previous literature also highlights the impacts of AMPK activation in augmenting the chemosensitizing efficacy of natural and synthetic compounds in combination with conventional chemotherapeutics (57–64). A list of various pharmacological activators of AMPK and their implication in different types of cancer is included in Table 1 .

AMPK influences glucose transporter (GLUT)-mediated trafficking of glucose across the plasma membrane. In addition, it promotes cellular glucose uptake by enhancing the mRNA expression of the genes encoding GLUT4 and hexokinase 2 and by mediating the translocation of GLUT4-containing intracellular vesicles across the plasma membrane. Previous studies have reported the role of AMPK in GLUT1/4 vesicle trafficking via the activation of thioredoxin-interacting protein (TXNIP) and Tre-2/Bub2/Cdc16 (TBC) domain family member 1 (TBC1D1) respectively (99). Once glucose is internalized, it is converted to glucose-6-phosphate by the action of hexokinases. Glucose-6-phosphate is further channelized to glycolysis, glycogen synthesis and pentose phosphate pathway. Of all the glucose metabolism pathways, AMPK plays an indispensable role in directly inhibiting aerobic glycolysis and glycogen synthesis (100). AMPK stimulates glycolytic flux through direct phosphorylation of PFKFB2 and PFKFB3 isoforms of the enzyme 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase (101). AMPK phosphorylation also culminates in the inhibition of gluconeogenesis-related enzymes such as glucose-6-phosphatase(G6Pase) and phosphoenol pyruvate carboxykinase (PEPCK) (102). The activation of AMPK impedes glycogen synthesis and activates the rate of glycogen breakdown by promoting the phosphorylation of glycogen phosphorylase (GP).Notably, AMPK activation also enhances insulin sensitivity, inhibits hepatic glucose production in the liver, stimulates glucose uptake in the skeletal muscles, and weakens proinflammatory changes (103).

The conversion of acetyl-CoA to malonyl-CoA is the rate-limiting step in the de novo synthesis of fatty acids. AMPK was initially identified as a kinase that phosphorylates and inhibits acetyl CoA carboxylase (ACC), the enzyme involved in the conversion of acetyl-CoA to malonyl-CoA (104). AMPK activation leads to the inhibitory phosphorylation of sterol regulatory element-binding protein 1c (SREBP1c). SREBP1cis responsible for enhancing the production of lipogenic enzymes, such as, acetyl-CoA carboxylase 1 (ACC1) and fatty acid (FA) synthase. Further, AMPK negatively regulates the de novo synthesis of cholesterol and triglycerides, and promotes β-oxidation of fatty acids. Additionally, AMPK negatively regulates the first committed step in triglyceride (TG) synthesis that is catalyzed by the enzyme, glycerol-3-phosphate acyltransferase (GPAT). AMPK- mediated inhibition of cholesterol synthesis is induced by phosphorylation of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR). Both HMGCR and hormone-sensitive lipase (HSL) enzymes, which are involved in the synthesis of cholesterol and prevention of lipolysis, respectively, are inhibited as a consequence of AMPK activation (105). The role of AMPK in accelerating lipid catabolism is equally important. AMPK facilitates transportation of fatty acids into the mitochondria, where they are subjected to β-oxidation by the enzyme carnitine palmitoyltransferase-1 (CPT-1) (106, 107). Activation of AMPK accelerates the oxidation of fatty acids by inhibiting malonyl Co A and heightens the activity of CPT-1 enzyme (108).

AMPK exercises its inhibitory effects on protein synthesis mainly through the inhibition of mTOR (12). mTOR is a serine/threonine protein kinase that regulates two distinct down-stream catalytic protein complexes, namely, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (109). mTORC1 plays an important role in the growth of cancer cells and in cell division. The major down-stream targets of mTOR that are involved in the translation machinery include p70S6 kinase (p70S6K) and 4E-binding protein (4E-BP). It is well known that eIF4G is a binding site on eIF4E, which is essential for translation initiation (110). However, when 4E-BP is phosphorylated by mTOR, it can no longer bind eIF4E. Various stress factors inhibit mTORC1 activity, which results in the phosphorylation of 4E-BP and in turn, prevention of the translation initiation factor eIF4E (111). AMPK indirectly causes blockade of cap-dependent protein translation by inhibiting mTORC1 expression. This is achieved by phosphorylating tuberous sclerosis complex 2 (TSC2) and regulatory- associated protein of mTOR (RPTOR). Upon inhibition of mTORC1, 4EBP1 gets activated and a simultaneous decrease in the levels of p70S6K is observed. Consequently, the initiation of cap-dependent translation of proteins is halted. Besides, AMPK-mediated activation of eukaryotic elongation factor 2 kinase(eEF2K)also blocks protein synthesis (112). Furthermore, AMPK retards ribosomal RNA synthesis via the inhibitory phosphorylation of transcription initiation factor 1A (TIF-1A). The translational elongation is blocked by AMPK through the phosphorylation of eEF2K, which inhibits eEF2.

Mitochondrial biogenesis replaces fresh mitochondria once the damaged mitochondria are eliminated via autophagic degradation. The process of mitochondrial biogenesis is vital for energy production and cellular response under nutrient-limiting conditions. Interestingly, the substrates for mitochondrial metabolism are generated as part of the autophagic clearance program. Previous research suggests that AMPK regulates mitochondrial biogenesis by regulating peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) (23). It is one of the cofactors which facilitate transcription of mitochondrial genes. AMPK influences PGC1α either by phosphorylating it directly or deacetylating it in a Sirtuin (silent mating type information regulation 2 homolog) 1(SIRT1)-dependent manner. Besides, AMPK-dependent increase in NAD⁺/NADH ratio or high levels of nicotinamide phosphoribosyl transferase(NAMPT) expression also lead to an augmented expression of PGC1α (113). AMPK-mediated mitochondrial biogenesis also accelerates the rates of ATP production. This is achieved by increasing the replication of mitochondrial DNA as well as the expression of many nuclear-encoded mitochondrial proteins via the SIRT 1/PGC-1α axis (114). Shen et al., have reported that the steroid hormone, ouabain, induces the simultaneous activation of AMPK and Src pathways in A549 and MCF7 cells and inhibits the mitochondrial OXPHOS in the cancer cells (115).

Figure 3 summarizes the effects of AMPK on the modulation of cancer cell metabolism, which collectively contributes to cell death, cell growth-arrest, inhibition of tumorigenesis and tumor progression.

Some of the recent studies throw light on the anti-cancer effects of pharmacological inhibitors of AMPK. Dorsomorphin or Compound C, the only known AMPK-specific inhibitor augmented the anti-cancer effects of Aspirin against HER-2-positive breast cancer in an AMPK-independent manner, by regulating lipid metabolism mediated by c-myc (127). Another study identified BAY-3827 as a novel AMPK inhibitor. The compound also inhibited ribosomal 6 kinase (RSK) family proteins.BAY-3827 displayed excellent anti-proliferative effects against androgen-dependent prostate cancer cell lines by blocking HMGCR, fatty acid synthase (FASN) and PFKFB2, all of which are strongly up-regulated by androgen treatment (128). Thus, it would be safe to say that AMPK is a potential molecular target for cancer therapy employing chemoprevention and chemosensitization approaches, irrespective of its individual pro- or anti-tumorigenic/neoplastic effects.

T cell activation in the TME is governed by various metabolic pathways such as, aerobic glycolysis, amino acid metabolism, glutaminolysis, and de novo fatty acid synthesis. The LKB1-AMPK signaling pathway stimulates catabolic pathways and ATP generation which in turn, facilitate metabolic reprogramming in T cells. AMPK enhances glutaminolysis and mitochondrial OXPHOS which aids in T cell survival. AMPK activation promotes fatty acid oxidation (FAO). The AMPK-mTOR axis is responsible for the generation of memory T cells (T_mem)and adaptation of effector T cells (T_eff) to nutritional stress (135). The inhibition of glycolysis and concomitant activation of FAO and oxidative phosphorylation directs regulatory T cells (T_regs) to undergo metabolic reprogramming which culminates in T_regs-mediated immunosuppression and tumor progression. On the contrary, cytotoxic CD8⁺ T cells play a crucial role in tumor immunosurveillance and elicit anti-tumor immune response by secreting interferon (IFN)-γ and granzyme B (GZB) (136).Rao et al., have demonstrated that AMPK regulates protein phosphatase activity, which controls survival and function of CD8⁺ T cells, thereby enhancing their role in tumor immunosurveillance (95). Besides, AMPK is indispensable for the sustained long-term proliferation of T cells and the survival of effector/memory T cell populations (137). In particular, AMPK promotes the accumulation of effector/memory T cells in competitive homeostatic proliferation settings (138). AMPK is found to be activated in CD8⁺ T cells during primary immune response. It also helps effector T cells (T_eff) in their metabolic adaptation in response to reduced glucose availability. T_eff cells actively engage in glutamine-dependent oxidative phosphorylation (OXPHOS) to maintain ATP concentrations and cell viability under low glucose conditions. Further, AMPKα1-deficient T_eff cells display reduced mitochondrial bioenergetics and cellular ATP in response to glucose limitation (139). Notably, cytotoxic T cells (CD8⁺ T cells) detect abnormal tumor antigens expressed on cancer cells and target them for destruction. AMPKα-1-mediated inhibition of mTORC1 activity in cytotoxic T lymphocytes (CTLs) is required for CD8⁺ T-cell memory (140). Previous studies have revealed that inactivation of both AMPKα1 and α2 coupled with Kirsten rat sarcoma virus (KRAS) activation promotes tumorigenesis and decreases the infiltration of CD8⁺/CD4⁺ T cells (141). Braverman et al., have reported that increasing AMPK activity orchestrates oxidative metabolism, proliferation, and in vitro recovery of human CD4⁺ T cells. They also suggest AMPK as a potential candidate for improving the yield of more functional T cells for CAR-T cell therapy (142). The metabolic fitness of T cells is pivotal for antitumor immunity. A recent report suggests that AMPK is essential for the sustained long-term proliferation of T cells and the survival of effector/memory T cell sub-populations (143). However, under conditions of restricted nutritional supply, T cells acquire an exhausted phenotype inside the immune-suppressive tumor milieu. T cell exhaustion culminates in the loss of effector activities and changes in T cell signaling and is characterized by reduced rates of glycolysis and OXPHOS and the onset of mitochondrial dysfunction (135).

Tumor-associated macrophages (TAMs) are present in abundance across various types of malignant tumors and promote tumor angiogenesis, extravasation and metastasis (144).Macrophages are responsible for driving anti-tumor immunity via phagocytosis and antigen presentation. Macrophages exist in two distinct phenotypes namely, the ‘classically’ activated, tumoricidal phenotype, M1φ, and the ‘alternatively’ activated pro-tumorigenic phenotype, M2φ (145).Some documented results indicate that AMPK inhibition leads to an up-surge in LPS-induced macrophage inflammatory function (141). Previous reports also state that AMPK prevents the polarization of monocyte-derived macrophages towards the M2φ subtype (146).Chiang et al., reported that metformin-mediated AMPK activation suppresses the skewing of macrophage towards M2φ subtype in breast cancer cells (147). The metabolic control of macrophage polarization relies partially upon glycolysis. A shift towards increased glycolytic rates leads to the activation of M1φ and is governed by the activation of mTOR through the Akt-HIF-1α pathway. Conversely, M2φ selectively utilize FAO, instead of aerobic glycolysis, to meet the energy requirements of OXPHOS. While HIF-1α and NF-κB favour the M1φ, PGC1β, peroxisome proliferator-activated receptors (PPAR) and STAT6 skew the balance towards M2φ. AMPK influences M1/M2φ polar mitochondrial biogenesis of macrophages by deacetylating proteins such as, SIRT1 with NAD⁺, and suppressing HIF-1α and NF-κB (1).

Previous reports also suggest that AMPK is involved in the molecular crosstalk between macrophages and cytokines. For instance, studies on the effect of macrophage stimulation using anti-inflammatory cytokines causes rapid phosphorylation of AMPK as opposed to the inactivation of AMPK upon the stimulation using pro-inflammatory LPS stimulus. AMPK directs signaling pathways in macrophages in a manner that suppresses pro-inflammatory responses and promotes macrophage polarization to an anti-inflammatory functional phenotype (146). AMPKα silencing elevated the mRNA expression levels of LPS-induced TNF-α, IL-6, and cyclooxygenase-2. Similarly, transfection of dominant negative AMPKα1 gene increased TNF-α and IL-6 expression levels and down-regulated IL-10 expression in macrophages, upon LPS stimulation (147).Another recent study revealed that AMPK inactivation potentiates the development of LPS-induced inflammatory injury (148). In the TME, AMPK has been reported to suppress the secretion of various pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, interleukins-1/2/8, MCP-1, IFN-γ and chemokines such as RANTES, CCL 1/2/5/10/11 (148).Previous studies indicate that stimulation of wild type macrophages using anti-inflammatory cytokine,IL-10 results in rapid activation of AMPK, phosphoinositide 3-kinase(PI3K) and mTORC1 (149).AMPK is known to inhibit the functions of pro-inflammatory molecules such as IL-1 β. Previous reports suggest that AMPK activation inhibited IL-1-stimulated CXCL10 secretion, via the down-regulation of the MKK4/JNK and IKK/IκBα/NF-κB signaling axis (150).

Myeloid-derived suppressor cells (MDSC) are an immunosuppressive class of immune cells that are pathologically activated in various tumor types. MDSCs promote tumor progression and enhance tumor cell survival, angiogenesis, invasion, metastasis and production of immunosuppressive cytokines such as IL-10 and TGF-β. Recent studies indicate that splenic MDSCs impede T cell response in a ROS-dependent manner, whereas, tumor-infiltrating MDSCs inhibit anti-CD3/28-stimulated response through nitric oxide (NO) production and secretion of Arginase1 (Arg1). MDSCs mediate the addition of nitrate groups to chemokines thereby, blocking CD8⁺ T cells from entering into the tumor site (151). AMPK block the expansion and activation of MDSCs. It curtails the function of MDSCs via inhibition of JAK-STAT, NF-κB, C/EBPβ, CHOP, and HIF-1α pathways that are crucial for the development and migration of MDSCs (152–154).

The influence of AMPK on various immune checkpoints is yet another remarkable event in the alteration of the TME (116). Immune checkpoint molecules are co-stimulatory cell surface receptors that are expressed on the surface of several immune cells, which bind with their corresponding ligand molecules and in turn, prevent an immune attack against self-antigens. However, cancer cells use this feature of immune checkpoints to evade immune attacks. This confers a survival advantage to the cancer cells. Immunotherapy drugs aim at inhibiting these immune checkpoints rendering cancer cells susceptible to immune attacks (155).Blocking of the programmed cell death 1 (PD-1) receptor is currently being used as a first-line treatment option against lung cancer. A recent report indicates that GSK3β-mediated inhibition of glycogen synthase down-regulatesPD-1 expression levels on CD8⁺ T cells in B16F10, murine melanoma cells (136).Several reports also suggest the role of AMPK as an immune checkpoint inhibitor. AMPK activation causes phosphorylation of PD-L1 on Ser²⁸³ and disrupts its interaction with chemokine like factor (CKLF)-like MARVEL transmembrane domain containing 4 (CMTM4), which leads to the degradation of PD-1 ligand (PD-L1). AMPK also potentiates ER-associated degradation of PD-L1 (156, 157). Dai et al., have reported that, in syngeneic mouse tumor models, AMPK agonists enhance the efficacy of anti-CTLA-4 immunotherapy and improve the overall survival rate (156). Besides, reports also suggest the potential use of AMPK activators in combination with anti-VEGF/PD-1 agents as a dual-targeted therapy against ovarian cancer (116). A recent report by Pokhrel et al. states that AMPK drives anti-tumor immunity by blocking PD-1 expression via the HMGCR/p38 MAPK/GSK3β signaling pathway (136). They also report on the synergic antitumor effect of AMPK activators with anti-PD-1 antibodies, anti-CTLA-4 antibodies, or HMGCR inhibitors in murine tumor models (158). Another study has documented the AMPK-mediated blockade of PD-1 through a reduction of tumor hypoxia (159). Taken together, these findings highlight the central role of AMPK in the inhibition of major immune checkpoints. The current knowledgebase can be further expanded by evaluating the therapeutic efficacy of immunotherapy drugs in combination with AMPK inhibitors against multiple cancer types. Figure 4 represents AMPK-mediated modulation of various components of the TME and the signalling pathways associated with it.