Structural Biochemistry/AMPK

AMP-activated protein kinase, or AMPK, is a well-known energy sensor, capable of regulating pathways in order to increase the amount of ATP in the cell. However, recent studies have revealed that AMPK is also a general stress sensor that plays an important role in maintaining intracellular homeostasis during times of stress.

AMPK Structure
AMPK is an enzyme that can be found in the majority of eukaryotic organisms, such as yeast, plants, and humans. Mammalian AMPK is a “heterotrimeric complex comprised of a catalytic subunit (α-subunit) and two regulatory subunits (β- and γ- subunits).” There are multiple isoforms of each subunit, which are encoded by specific genes that are distributed across several chromosomes.

Subunits of AMPK
The α-subunit of AMPK contains multiple domains: the catalytic domain (located at the N-terminus), the C-terminal domain, and the autoinhibitory domain. The C-terminal domain allows for the interaction with the β- and γ-subunits. The catalytic domain, also known as the kinase domain, contains the residue Thr172. This residue is crucial for AMPK activation: the phosphorylation of Thr172 allows for the activation of AMPK. Although researchers agree that Thr172 is an important location for AMPK, there is still further debate as to what the effects are of AMP binding. Originally, it was proposed that “AMP promoted AMPK activation by facilitating the phosphorylation of AMPK at Thr172 by upstream kinases.” However, recent research has suggested that “AMP binding also inhibits the dephosphorylation of Thr172.”

The β-subunit of AMPK also contains multiple domains. These include a α- and γ- binding domain located at the C-terminus and a glycogen-binding domain located at the N-terminus. The domain located at the C-terminus allows for stabilization of the interactions between the other two AMPK subunits, while the domain at the N-terminus targets AMPK to glycogen. This targeting is achieved by a carbohydrate-binding module (CBM) that is attached to the N-terminal region of the β-subunit.

The γ-subunit of AMPK has four domains, all of which are cystathionine β-synthase (CBS) domains. These domains coordinate to bind AMP, ADP and ATP, allowing the AMPK γ-subunit to “regulate AMPK activation through allosteric structural changes in the catalytic α-subunits, modulating phosphorylation by upstream kinases and regulating the dephosphorylation of Thr172.”

Regulation of AMPK by AMP/ATP Ratios
AMPK holds a significant role in regulating cellular energy status. AMPK’s activation occurs when the intracellular AMP level increases; this activation takes place at the γ-subunit of AMPK, where AMP can bind. Subsequently, AMPK undergoes an allosteric structural change, allowing for a large increase in AMPK activity. However, as mentioned in the AMPK Structure section, there are multiple isoforms of the AMPK subunits. The different isoforms of the subunits, specifically the α- and γ-subunits, affect the extent of allosteric activation AMPK by AMP; it is found that the “greatest activation by AMP occurs in complexes containing the α2 and γ2 isoforms; however, only weak activation occurs in complexes with the γ3 isoforms.”

In recent studies using crystal structures of mammalian AMPK, it was found that ADP can bind to a AXP (AMP/ADP/ATP)-binding sites in the γ-subunit, protecting AMPK from dephosphorylation and consequently allowing for more activity. Furthermore, AMP allows for AMPK regulation because upon AMP’s binding to the γ-subunit, it has been observed that the phosphorylation of Thr172 by upstream kinases has been enhanced, while the dephosphorylation of Thr172 by various protein phosphatases has been inhibited.

Regulation of AMPK by Upstream Kinases
Although AMPK is regulated via allosteric regulation, as explained in the previous subsection, AMPK is also subject to regulation by upstream kinases. Two upstream kinases of AMPK include liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ). In vitro, purified recombinant LKB1 complex phosphorylate the Thr172 on the α-subunit of AMPK. However, unlike many kinases, LKB1 is not activated by phophorylation; instead, it is regulated by “relocation from the nucleus to the cytoplasm upon binding to STRAD (Ste20-related adaptor) and MO25 (mouse protein 25).” The revelation of CaMKKβ as an upstream kinase of AMPK was due to the research on LKB1-deficient cells. In contrast to AMPK activation by LKB1, AMPK activation by CaMKKβ depends on the increase of intracellular Ca2+. This activation usually occurs in the absence of an increase of AMP.

Other Pathways in AMPK Activity Regulation
As mentioned at the end of the Regulation of AMPK by AMP/ATP Ratios subsection, the dephosphorylation of Thr172 can be caused by various protein phosphatases; two such protein phosphatases are PP2A and PP2C. As such, PP2A and PP2C can dephosphorylate AMPK and therefore inhibit its activation. A mechanism that has been proposed for regulating AMPK activity includes the concept that AMPK can be modulated by “ubiquitin-dependent proteasome degradation mediated by CIDEA (cell-death-inducing DNA fragmentation factor-α-like effector A).” This idea is supported by the discovery of a CIDEA-binding site located on the AMPK β-subunit. Another concept supported by other evidence expresses that “AMPK β-myristoylation plays a key role in the full phosphorylation and activation of AMPK by upstream kinases in response to metabolic stress signals.”

AMPK Activators
Researchers have taken a large interest in discovering and researching various AMPK activators due to AMPK’s involvement in disease prevention and treatment. As of today, many activators, including direct and indirect activators, have been reported.

Activation of AMPK in Physiological and Pathological Conditions
In several physiological and pathological conditions, AMPK is involved in modulating numerous targets to allow the body to adapt to these conditions. Several examples of these conditions include hypoxia, caloric restriction (CR) and physiological exercise.

AMPK is Activated by Hypoxia
Hypoxia is a situation in which the body, or a region of the body, is deprived of adequate oxygen supply. This can occur under physiological conditions, such as strenuous physical exercise or at locations that have less oxygen (i.e. high altitudes), as well as pathological conditions such as “anaemia, hypoventilation and pumonary fibrosis.” Hypoxia is dangerous, mainly because complete oxygen depletion causes cell death. However, cells are able to survive in reduced oxygen situations by activating adaptive processes through what is called hypoxia-inducing factor-1 (HIF). HIF holds the central role in the adaptation to oxygen-reduced conditions.

Although the mechanisms involved in AMPK activation under hypoxia appear somewhat complicated, generally, it can be said that these mechanisms are dependent on the extent of hypoxia. For example, researchers have determined that reactive oxygen species (ROS) and reactive nitrogen species (RNS) are significant in AMPK activation during non-severe hypoxia. In fact, under hypoxic conditions, intracellular AMP/ATP levels remain constant and unchanged, while ROS production, which is conducted via the electron transport chain in the mitochondria, is significantly increased. A different mechanism leading to the activation of AMPK is the reduced ATP production; this reduction occurs because of the “inhibition of β-oxidation under hypoxic conditions.” There are many other mechanisms: peroxynitrite (ONOO-) activates AMPK in endothelial cells, while mitochondrial RNS is required by metformin for the activation of AMPK.

AMPK is Activated by Physical Exercise
Although researchers have determined that AMPK activation is increased during physical exercise, the specific mechanism of the beneficial effects of AMPK activation by exercise are not yet clear. However, several findings prove that there must be benefits of AMPK activation by exercise. AMPK activation increases fatty acid oxidation and glucose uptake in rat muscles. Furthermore, the activation of AMPK by 5-amino-4-imidazolecarboxamide riboside (AICAR) imitates the effect of training and also improves exercise performance in mice. Such evidence shows that AMPK activation during exercise hold benefits. AMPK also holds a role in muscle fibre type change regulation. Recently, using “skeletal-muscle-specific AMPKβ1/AMPKβ2-knockout mice,” a researcher has “demonstrated that AMPK is required in maintaining mitochondrial content and glucose uptake during exercise.”

Natural Activators of AMPK
There are several physiological hormones and natural plant compounds that have been found capable of activating AMPK. One such molecule is leptin, a hormone secreted by adipocytes. Researchers have reported that both acute and chronic leptin treatments “increase AMPK expression and/or phosphorylation in rodent skeletal muscles.” This discovery suggests a significant mechanism by which leptin regulates energy stores. Another hormone produced by adipocytes, called adiponectin, has cardioprotective effects which is believed to function through a mechanism due to AMPK activation. Two natural compounds found to involved with AMPK activation are resveratrol and berberine. Resveratrol is found in red grapes, capable of extending lifespan. Such an ability is believed to be mediated by AMPK. Berberine, found in Berberis, can increase AMPK activity. Although these hormones and natural compounds are important in the activation of AMPK, the specific mechanism by which these substances use to activate AMPK are still being researched.

Chemical Activators of AMPK
According to Shaobin Wang, Ping Song, and Ming-Hui Zou, “the most widely used chemical activator of AMPK in research is AICAR.” AICAR, mentioned in the AMPK is Activated by Physical Exercise subsection, is 5-amino-4-imidazolecarboxamide riboside, which is a chemical that is converted to monophosphate AICAR (ZMP) in the cell. ZMP is capable of imitating the effect of AMP in activating AMPK. Another strong AMPK activator is called 2-deoxy-D-glucose, or 2-DG. This molecule is a glucose analog, capable of blocking glucose utilization to copy the effect of caloric restriction (CR). Finally, another AMPK activator used in clinical settings to treat Type 2 diabetes is metformin.

AMPK Inhibitors
The importance of researching AMPK inhibitors correlates with the reasons for researching AMPK activators – in order to improve the understanding, prevention and treatment of various diseases.

AMPK is Inhibited by High Glucose and Glycogen
Although recent research has suggested that activated AMPK is beneficial for the whole body’s energy homeostasis, AMPK activation is inhibited under high glucose levels. In fact, in animal models, glucose induces acute hyperglycaemia, as well as a decrease in AMPK activation levels in rat muscle and liver. However, the exact mechanism by which high glucose levels inhibits AMPK activation has yet to be determined. Similarly, high glycogen conditions can repress the AMPK activation in animal models: AMPK activation in rat skeletal muscle is decreased under such conditions. It is proposed that glycogen inhibits AMPK activation by binding to the AMPK glycogen-binding domain found in the β-subunit of AMPK.

AMPK is Inhibited by Lipid Overload
Researchers have observed that mice with a high-fat diet have “impaired AMPK phosphorylation and protein expression in multiple tissues and organs, including skeletal muscle, heart, liver and hypothalamus.” Other mice on a palmitate-enriched diet showed the enhancement of PP2A-mediated dephosphorylation of AMPK, which would subsequently inhibit the activation of AMPK.

AMPK is Inhibited by Amino Acids
Research has revealed that a high-protein diet decreases AMPK activity, specifically in the hypothalamus. Consequently, neuropeptide Y (NYP) is inhibited, and food uptake is reduced. Similarly, other cells, specifically C2C12 cells, treated with leucine show a reduction in AMPK phosphorylation, while mammalian TOR (mTOR) activation is enhanced. These findings, as well as others, suggest that theAMPK/mTOR pathway is involved in the coordination of amino acid metabolism.

Pharmacological AMPK Inhibitor
A compound called Compound C is widely used as an AMPK inhibitor. Compound C is a “cell-permeant pyrrazolopyrimidine compound.” However, when using Compound C, researchers much consider the fact that Compound C will target other protein kinases as well. Some protein kinases effected by Compound C include extracellular-signal-regulated kinase 1 (ERK8), phosphorylase kinase (PHK), and Ephrin A2 receptor (Ephrin-A2).

AMPK is an Energy Stress Sensor and Modulator
Both limited nutrition and excessive nutrition can cause cellular stress. AMPK is strongly activated when energy in the body has depleted greatly and inhibited in conditions of over-nutrition. Thus, AMPK plays a key role in sensing and regulating nutrient stress – AMPK is significant for maintaining intracellular energy homeostasis.

AMPK activation by Nutrient Depletion
Nutrient depletion, occurring both in physiological and pathological conditions, leads to an increase in intracellular AMP levels; this increase then directly activates AMPK. Caloric reduction (CR), an activator of AMPK, is believed to benefit human health by extending lifespan. CR is a “20-40% reduction in caloric intake without compromising daily nutritional needs.” Furthermore, although the mechanisms have not been completely discerned, several mechanisms have been proposed. In fact, one such proposal suggests that CR modulates AMPK through ROS-mediated AMPK activation.

AMPK Modulates Multiple Downstream Pathways in Metabolic Regulation
AMPK regulates energy balance at multiple organs by targeting specific substrates. For example, activated AMPK inactivates acetyl-CoA carboxylase (ACC), consequently inhibiting the synthesis of fatty acids and the promotion of mitochondrial β-oxidation; AMPK also inhibits 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase to reduce cholesterol synthesis. Another function of AMPK is to promote the initiation of autophagy. Autophagy is “a major protective mechanism that allows cells to survive in response to multiple stress conditions, especially nutrient depletion.” Although these are only a few of the pathways that AMPK regulates, the existence of so many different functions prove that AMPK is involved in multiple pathways that are involved in maintaining intracellular homeostasis.

AMPK and the Oxidative Stress Response
Oxidative stress is “an imbalance between the levels of pro-oxidants and antioxidants”; it can cause macromolecular damage and disruption of redox signaling and controls. Typically, macromolecular damage due to oxidative stress is caused by free radicals, such as O2-, NO and OH-. In recent studies, AMPK has been suggested as an oxidative stress sensor and redox regulator.

AMPK is Activated by Intracellular Oxidative Species
As mentioned in the AMPK is Activated by Hypoxia subsection, reactive oxygen species, or ROS are involved in the activation of AMPK. This ROS-induced activation is believed to be significant for the beneficial effects of many medicines. Furthermore, it has been found that ROS has been found to provide protection to rat hearts from ischaemic injury in experiments with sevoflurane due to the mechanism’s dependency on ROS-activated AMPK. Other oxidative species capable of activating AMPK includes hydrogen peroxide and ONOO- through various mechanisms.

AMPK’s ability to act as a redox sensor and consequently be activated by increased amounts of ROS/RNS also allows AMPK to be essential in maintaining intracellular redox status. This control occurs through AMPK’s inhibition of oxidant production by NADPH oxidases and mitochondria, or AMPK’s promotion of expression of antioxidant enzymes.

AMPK in Cell Proliferation
AMPK regulates cell proliferation through its control of several pathways that are significant for cell growth and division. After being activated, AMPK suppresses cell growth and division by controlling specific processes that are required for cell proliferation: AMPK can “suppress mTOR signaling by growth factors and amino acids to control protein synthesis.” Other studies express that AMPK can control CDKIs, which would be a more direct method of affecting cell growth control. For example, AMPK has been shown to control mouse vascular smooth muscle cell (mVSMC) proliferation by “modulating p27KiP1 expression through p52 NF-кB-2-dependent Skp2 (S-phase kinase-associated protein 2) regulation.”