One of the key functions of catabolic metabolism is to maintain high levels of ATP (Adenosine Triphosphate) and cells rapidly respond to any stress that threatens to lower ATP levels by arresting non-essential ATP-utilizing functions and stimulating available ATP-generating pathways. A central player in this system is the AMPK (AMP (Adenosine 5'-Monophosphate)-Activated Protein Kinase). AMP-activated protein kinase (AMPK) is a crucial energy sensor and plays a key role in integration of cellular functions to maintain homeostasis. When AMPK is activated by stimuli like muscle contraction, inflammation, sepsis, metabolic poisoning, exercise, hypoxia, ischemia, heat shock, neuronal necrosis, etc it controls many metabolic processes that include increased Glucose uptake; increased Fatty acid oxidation; decreased Fatty acid, Glycogen, Cholesterol and protein synthesis; and induction of mitochondrial biogenesis (Ref.1 and 2). Full activation of AMPK requires phosphorylation of its activation loop at Thr172 (Threonine-172) of its catalytic Alpha-subunit, by upstream kinases collectively known as AMPKKss (AMPK (AMP-Activated Protein Kinase) Kinases). AMPK is a heterotrimer consisting of catalytic Alpha-subunits and regulatory Beta-subunits and Gamma-subunits-subunits. It is regulated by the cellular AMP (Adenosine 5'-Monophosphate)/ATP ratio and by upstream kinases. The Alpha-subunit contains the Kinase domain, which transfers a phosphate from ATP to the target protein. The Beta-subunits- and Gamma- subunits are considered as regulatory components. All three subunits are required for expression of full activity of the AMPK Enzyme Complex. The Alpha2-isoform and Gamma-subunits-subunits are more sensitive to AMP. The AMPKKss activating AMPK include LKB1/STK11 (Serine/Threonine Kinase-11), MO25/CAB39 (Calcium Binding Protein-39) and STRAD (Ste20 Related Adaptor) (Ref.3 and 4).
AMP-activated protein kinase (AMPK) is well known to be induced by exercise and to mediate important metabolic changes in the skeletal muscle of mammals. The LKB1-STRAD-MO25 or the MO25 or the AMPKK complex phosphorylates and activates the AMPK under exercise and stress conditions and in turnn is activated by AMP, ZMP (AICA Ribotide or 5-Aminoimidazole-4-Carboxamide-1Beta-D-Ribofuranotide) and PKA (cAMP-Dependent Protein Kinase). Inactivation of this complex is the major cause of PJS (Peutz-Jeghers Syndrome). AMPK is activated by 5'-AMP but not cAMP (Cyclic 3',5'-AMP), whereas the converse is true of PKA. However, high levels of AMP under stress conditions like hypoxia, ischemia and heat shock do activate PKA. AMPK is also termed “AMP-activated” and not “AMP-dependent” because (unlike PKA) it has a significant basal activity in the absence of the activating nucleotide. The AMPK system is allosterically inhibited by physiologically relevant concentrations of Creatine Phosphate. Creatine Phosphate is converted back to Creatine by the action of intracellular CPK (Creatine Phosphokinase) in order to stimulate muscle contraction (facilitated by neurotransmitter like Ach (Acetylcholine) and its receptor AchR (Acetylcholine Receptor) at the neuromuscular junctions) (Ref.5 and 6). AMPK stimulates Fatty acid oxidation by inactivating ACC (Acetyl-CoA Carboxylase) and so decreasing the concentration of Malonyl-CoA (Malonyl-Coenzyme-A) which inhibits the entry and the subsequent oxidation of long-chain Fatty acids into the mitochondria. The classical target for AMPK, ACC is known to occur as two isoforms i.e., ACC1, which is expressed in tissues active in Fatty acid synthesis such as liver and adipose tissue, and ACC2, which is expressed in tissues active in Fatty acid oxidation such as skeletal and cardiac muscle. Another vital target of AMPK in muscle is the MCD (Malonyl-CoA Decarboxylase) (Ref.7).
The metabolic responses to exercise or contraction that mediate AMPK activation include the phosphorylation of AICAR (AICA Riboside or (5-Aminoimidazole-4-Carboxamide-1Beta-D-Ribofuranoside)). The non-phosphorylated riboside AICAR, an Adenosine analog and cell-permeable activator of AMPK, is taken up by cells and is phosphorylated by cellular AK (Adenylate Kinase/ Myokinase) to form ZMP, a nucleotide and an analog of AMP that mimic all of the effects of AMP on the AMPK system, including increased phosphorylation and decreased dephosphorylation. Iodotubercidin, an inhibitor of AK, blocks the formation of ZMP from AICAR. ZMP mimics AMP and causes an activation of AMPK in vivo. The AICAR activation of AMPK and MO25 or the AMPKK in muscle regulates the action of ACC (Ref.7). Malonyl-CoA, produced by ACC is exclusively involved with regulation of Fatty acid oxidation, rather than as a precursor for Fatty acid synthesis. AMPK phosphorylate and inactivate ACC at Ser-79 (Serine-79) position in human muscle. MCD is also activated by contraction or by AICAR through AMPK (Ref.8). Malonyl-CoA inhibits Fatty acid oxidation by inhibiting CPT1 (Carnitine Palmitoyl-CoA Acyl Transferase-1) on the outer surface of the inner mitochondrial membrane, thus preventing uptake of long chain fatty acids into mitochondria. ACC localizes to mitochondria, and the Malonyl-CoA it produces is therefore presented directly to CPT1 and consequently plays no role in cytoplasmic Fatty acid synthesis. CPT1 converts Carnitine and Palmitoyl-CoA (Palmitoyl-Coenzme-A) to Palmitoylcarnitine. Palmitoylcarnitine is converted back to Carnitine and Palmitoyl-CoA by CPT2 that in turns forms an intermediate during Beta-Oxidation. Acetyl-CoA (Acetyl-Coenzyme-A) produced during Beta-Oxidation acts as a substrate for ACC and is made available for ACC by ACC by CRAT (Carnitine Acetyltransferase) (Ref.7). Apart from MCD and ACC, activity of GPAT (Glycerol Phosphate Acyl Transferase)/GPAM (Glycerol-3-Phosphate Acyltransferase (Mitochondrial)), which catalyzes the first committed reaction in Triacylglycerol Synthesis and, which like ACC, is phosphorylated and inhibited by AMPK. The changes in ACC2, MCD and GPAT occur in the presence of AICAR, indicating that the exercise induced alterations in these enzymes are AMPK-mediated. AMPK plays a major role in regulating Lipid metabolism in multiple tissues following exercise. The net effect of its activation is to increase Fatty acid oxidation and diminish Glycerolipid Synthesis. Besides these an increase in AMPK activity in muscle caused by Leptin and Isoproterenol is, at least in part, ADR-Alpha (Alpha-Adrenergic Receptor)/G-Proteins mediated. Adiponectin also stimulates this activation. AMPK concurrently increases Fatty acid oxidation and diminishes its esterification in multiple tissues in the post-exercise state (Ref.9).
During exercise, AMPK is viewed as a metabolic switch, primarily in light of its ability to inhibit ACC and stimulate Fatty-acid oxidation and Insulin-independent Glucose transport in skeletal muscle, in order to avoid problems such as Insulin resistance, Obesity and Lipotoxicity. Upon AICAR activation AMPK increases Glucose transport into skeletal and cardiac muscle, independent of Insulin action. A combination of increased GLUT1 (Glucose Transporter-1) and GLUT4 translocation to the plasma membrane and increased transcription of the GLUT4 gene contribute to the increased transport. Insulin acts via InsR (Insulin Receptor) and activates IRSs (Insulin Receptor Substrates), which in turn has an inductive effect on PI3K (Phosphatidylinositde-3 Kinase). The co-localization of TFR (Transferrin Receptor) and GLUT4 is increased by Insulin and decreased by contractions. Activation of AMPK leads to the translocation of GLUT1 and GLUT4 in form of microvesicles. AMPK activates PI3K through MKK3 (Mitogen-Activated Protein Kinase Kinase-3) (Ref.10 and 11). PI3K in turn activates p38MAPK (p38 Mitogen-Activated Protein Kinase) upon IRSs and AMPK stimulation and a combinatorial activation of Akt (v-Akt Murine Thymoma Viral Oncogene Homolog) and p38MAPK by AMPK leads to increased transport of Glucose across cells through active translocation of GLUT1, GLUT4 and TFR. Insight to such AMPK activity is highly beneficial for the treatment of Type-2 Diabetes (Ref.12).
further AMP inhibits the dephosphorylation and inactivation of AMPK by PP2A(Protein Phosphatase-2A Catalytic Subunit) and PP2C (Protein Phosphatase-2C), thereby enhancing Fatty acid oxidation and Glucose uptake. This is also facilitated by hypoglycemic compounds like Metformin and drugs like Rosiglitazone, etc, that act synergistically to AMP in order to activate AMPK at Thr172. AMPK activation switches on anabolic pathways and other processes that consume ATP, while switching on catabolic pathways that generate ATP (Ref.7). Examples of the former include acute inhibition of Lipid biosynthesis by phosphorylation and inactivation of key metabolic enzymes such as ACC (Fatty acid Synthesis), GPAT/GPAM (Triacylglycerol Synthesis), HSL (Hormone Sensitive Lipase) (Lipolysis), FASN (Fatty Acid Synthase) (Fatty Acid Synthesis) and HMGCR (3-Hydroxy-3-Methylglutaryl-Coenzyme-A Reductase) (Steroid/Isoprenoid Biosynthesis). AMPK also inactivates the muscle isoform of GYS (Glycogen Synthase) in order to prevent Glycogen Synthesis. AMPK inhibits CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), a Cl- (Chloride) channel involved in trans-epithelial ion transport and fliud secretion, whose open probability is increased by phosphorylation by PKA, thereby preventing Cystic Fibrosis. AICAR stimulated activation of AMPK also inhibits protein synthesis. Under normal conditions the p70S6K (p70 Ribosomal-S6 Kinase) and eIF4EBP1 (Eukaryotic Translation Initiation Factor-4E-Binding Protein-1), participate in the control of protein synthesis and prevent autophagy. AICAR prevents the activation and phosphorylation of p70S6K and eIF4EBP1 by phosphorylating these targets during mTOR (Mammalian Target of Rapamycin) signaling. AMPK activates TSC (Tuberous Sclerosis) that in turn inhibits the action of mTOR and Raptor activity and ultimately phosphorylates p70S6K and eIF4EBP1 in the Thr389 and Thr37 positions, respectively, leading to the seizure of protein synthesis. Under normal conditions TSC is inhibited by upstream Akt. Therefore, the activation of p70S6K and eIF4EBP1 by AMPK, resulting in the phosphorylation and inactivation of these proteins, provides a novel mechanism for the inhibition of protein synthesis (Ref.9 and 13).
iNOS-derived Nitric Oxide, through its mitochondrial effect at COX (Cytochrome-C Oxidase), transiently decreases the cellular energy charge, a necessary factor for activating AMPK (by AMP-driven phosphorylation). Once active, AMPK activates PFK2 (6-Phosphofructo-2-Kinase), which synthesizes F(2,6)P2 (Fructose-2,6-Bisphosphate), the most potent PFK1 (Phosphofructokinase-1) allosteric effector and F(1,6)P2 (Fructose-1,6-Bisphosphate), from F6P (Fructose-6-Phosphate) resulting in the rapid activation of Glycolysis (Ref.11 and 14). Hypoxia-reoxygenation via the generation of oxidants, likely Nitric Oxide, ONOO- and superoxide anions (like O2-), activates AMPK via a Src (v-Src Avian Sacroma (Schmidt-Ruppin A-2) Viral Oncogene)-mediated, PI3K-dependent pathway. Activation by AMP and Nitric Oxide , O2- and ONOO- increase phosphorylation of PDK-1 (3-Phosphoinositide-Dependent Protein Kinase-1) downstream of PI3K. So, as a consequence, AMPK becomes a downstream target of PI3K and PDK-1. This in turn, promotes mitogen-stimulated Akt activation and Angiogenesis. Angiogenesis plays a critical role in the neo-vascularization that is associated with tumor growth and occlusive vascular diseases. Akt phosphorylates eNOS (Endothelial Nitric-Oxide Synthase) at Ser1177 in hypoxic cells. Direct eNOS phosphorylation by AMPK occurs under conditions of prolonged Hypoxia or ONOO– production or activation by AMP. Also AMPK regulate the coordination of SWI/SNF Complex and PCAF (p300/CBP-Associated Factor)/GCN5 in order to combat stress conditions (Ref.10 and 14).
Energy insufficiency is sensed by the AMP-activated protein kinase (AMPK), a master metabolic regulator that stimulates the catalytic process to enhance energy production. A decline in energy supply and a disruption in bioenergy homeostasis play a critical role in multiple neuropathological conditions including ischemia, stroke, and neurodegenerative diseases including Alzheimer’s disease and traumatic brain injuries (Ref.15).