Metformin target AMPK shown for the first time to be required for Long-Term Memory Formation: Hypothesis Substantiated

 By Davidboyashi - Own work, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=48165329

A recently published study in the journal iScience in October of 2018 demonstrated for the first time that metabolic plasticity induced by AMPK activation is required for long-term potentiation (LTP) in the CA1 region of the hippocampus in vitro in mouse neurons as well as long-term memory formation in vivo in mice [1]. These results provide direct support and substantiate my previous hypothesis in which I first proposed that activation of AMPK will promote LTP specifically in the CA1 region of the hippocampus and promote memory formation in vivo [2]. AMPK activation is considered a primary mechanism through which the anti-diabetic drug metformin and numerous naturally-occurring compounds exert their therapeutic effects [3]. Learning and memory are generally considered the behavioral correlates of long-term potentiation (LTP), a form of synaptic plasticity associated with a persistent and long-lasting increase in synaptic strength in response to repetitive neuronal stimulation [2]. It is the Schaffer collateral-CA1 excitatory synapse (i.e. CA1 synapses) that has generated increased interest in recent years due to accumulating evidence that high frequency stimulation of CA1 synapses leads to a long–lasting increase in synaptic strength that underlies learning and memory [2]. In the iScience study, Marinangeli et al. initially demonstrated that synaptic activation of primary mouse neurons with a combination of bicuculline and 4-aminopyridine rapidly activated AMPK. AMPK activation was dependent on glutamate receptor activation, as the NMDA and AMPA receptor inhibitors MK-801 and NBQX, respectively, significantly reduced AMPK activation [1].

Inhibition of AMPK by compound C or a kinase-dead dominant-negative AMPK construct also significantly decreased ATP levels and the upregulation of glycolysis and mitochondrial respiration, indicating that AMPK is critical for maintaining neuronal energy levels in response to synaptic activation [1]. Interestingly, AMPK inhibition also significantly reduced the expression of the immediate-early genes Arc, cFos, and Egrl (important for learning and memory), indicating that AMPK activation is required for the expression of these genes following synaptic activation. Importantly, inhibition of AMPK by compound C severely impaired LTP in the CA1 region of the hippocampus induced by electrical theta burst stimulation [1]. The authors also determined if AMPK activation is necessary for long-term memory retention in vivo in mice. Bilateral injection of compound C in the hippocampus before inhibitory avoidance training significantly blocked long-term memory tested at 24 hours which persisted after retesting at 6 days, providing compelling evidence that AMPK activation is critical for CA1 LTP in vitro and long-term memory formation in vivo [1].

As noted above, I first proposed in April of 2018 that AMPK activation would promote LTP specifically in area CA1 of the hippocampus and enhance learning and memory in vivo [2]. Indeed, the authors of the iScience study performed precise experiments that verified and substantiated my hypothesis, as follows: “Knockdown or pharmacological inhibition of both AMPK catalytic subunits (AMPKα1 and AMPKα2) in hippocampal neurons (e.g. hippocampal CA1 pyramidal neurons) would be conducted to determine if AMPK activation is essential for the induction, expression, and/or maintenance of LTP in vitro or the facilitation of learning and memory in vivo.” [2]. I also proposed in this paper that cellular stress-induced AMPK activation links CA1 LTP with the reactivation of latent HIV-1, facilitating immune system detection and potential destruction of the virus [2]. Intriguingly, the iScience study showed that AMPK increased the expression of the immediate-early gene Egr1 whereas the AMPK activator resveratrol reactivates latent HIV-1 via upregulation of Egr-1 [3,4]. Egr-1 was shown to be downregulated during viral latency in HIV-1 infected ACH-2 cells and treatment with resveratrol caused viral reactivation as indicated by a dose-dependent increase in viral p24 expression, suggesting that AMPK activation may indeed facilitate reactivation and destruction of the virus [2,4].

The iScience study also showed that AMPK activation increased the expression of the immediate-early gene Arc. Arc plays a critical role in memory formation and has recently been shown to be derived from a transposable element, DNA sequences first described by Nobel laureate Barbara McClintock that comprise nearly half of the human genome and are able to transpose or move from one genomic location to another [5,6]. I also recently proposed for the first time that AMPK activation would promote beneficial activation and transposition of transposable elements (also known as “jumping genes”) located in the human brain, human sperm, and in human oocytes [6]. Indeed, the transposable element L1 is present in the hippocampus of the human brain and contributes to memory formation in vivo in mice [7,8]. Additionally, metformin promotes AMPK-dependent telomerase activation (critical for telomere maintenance) and induces activation of the endonuclease RAG1 (promotes DNA cleavage and transposition) via AMPK [9,10]. Similar to Arc, both RAG1 and telomerase are derived from transposable elements, providing further evidence that AMPK links learning and memory with potential HIV-1 eradication and transposable element activation and mobilization [2,6,11].

https://www.linkedin.com/pulse/metformin-target-ampk-shown-first-time-required-long-term-finley/

References:

1.  Marinangeli C, Didier S, Ahmed T, et al. AMP-Activated Protein Kinase Is Essential for the Maintenance of Energy Levels during Synaptic Activation. iScience. 2018 Oct 12;9:1-13. doi: 10.1016/j.isci.2018.10.006. [Epub ahead of print].
2. Finley J. Facilitation of hippocampal long-term potentiation and reactivation of latent HIV-1 via AMPK activation: Common mechanism of action linking learning, memory, and the potential eradication of HIV-1. Med Hypotheses. 2018 Jul;116:61-73.
3. Hardie DG. AMPK: a target for drugs and natural products with effects on both diabetes and cancer. Diabetes 2013;62(7):2164–72.
4. Krishnan V, Zeichner SL. Host cell gene expression during human immunodeficiency virus type 1 latency and reactivation and effects of targeting genes that are differentially expressed in viral latency. J Virol 2004;78(17):9458–73.
5. Pastuzyn ED, Day CE, Kearns RB, et al. The Neuronal Gene Arc Encodes a Repurposed Retrotransposon Gag Protein that Mediates Intercellular RNA Transfer. Cell. 2018 Jan 11;172(1-2):275-288.e18.
6. Finley J. Transposable elements, placental development, and oocyte activation: Cellular stress and AMPK links jumping genes with the creation of human life. Med Hypotheses. 2018 Sep;118:44-54.
7. Coufal NG, Garcia-Perez JL, Peng GE, et al. L1 retrotransposition in human neural progenitor cells. Nature 2009;460(7259):1127–31.
8. Bachiller S, Del-Pozo-Martín Y, Carrión ÁM. L1 retrotransposition alters the hippocampal genomic landscape enabling memory formation. Brain Behav Immun 2017;64:65–70.
9. Karnewar S, Neeli PK, Panuganti D, et al. Metformin regulates mitochondrial biogenesis and senescence through AMPK mediated H3K79 methylation: relevance in age-associated vascular dysfunction. Biochim Biophys Acta 2018;1864(4 Pt A):1115–28.
10. Um JH, Brown AL, Singh SK, et al. Metabolic sensor AMPK directly phosphorylates RAG1 protein and regulates V(D)J recombination. Proc Natl Acad Sci USA 2013;110(24):9873–8.
11. Lander ES, Linton LM, Birren B, et al. Initial sequencing and analysis of the human genome. Nature 2001;409(6822):860–921.

New Harvard Study shows for the first time that meditation upregulates genes in the AMPK pathway similar to metformin

A new study published in 2018 by researchers at Harvard Medical School showed for the first time that a collection of meditative techniques that led to the relaxation response (RR) in human patients significantly upregulated genes in the AMPK signaling pathway [1]. The RR elicitation routine included diaphragmatic breathing, body scan, mantra repetition, mindfulness meditation, and passively ignoring intrusive thoughts [1]. An upregulation of genes in the AMPK signaling pathway was detected via blood samples taken from patients in which total RNA was isolated from peripheral blood mononuclear cells (PBMCs) [1].

This study has an interesting connection to a recently published study in 2018 in which a retreat that included meditation led to an increase in the RNA-binding protein HnRNPA1 [2]. I first hypothesized and proposed that metformin and AMPK activation would beneficially modulate the activity of HnRNPA1 [3,4] . Indeed, the well-studied AMPK activator resveratrol has recently been shown to upregulate HnRNPA1 [5]. Additionally, HnRNPA1 binds to telomerase and plays a critical role in telomere maintenance, promotes latent HIV-1 reactivation (facilitating immune system detection and virus destruction), and is necessary for the transposition or “jumping” of “jumping genes” in human cells [6-9]. Metformin has also been shown to activate human telomerase (hTERT) in an AMPK-dependent manner and reduce cellular makers associated with latent HIV-1 in infected patients [10-12]. This evidence lends substantial support to several hypotheses that I originally proposed linking metformin and AMPK activation with telomerase activation, “jumping genes” in the brain (important for learning and memory), virus destruction, aging deceleration, human life creation, and even consciousness itself [3,4,13-17]. Strikingly, AMPK activation may also play a critical role in the beneficial effects of meditation on the human brain. 

Meditation has been shown to increase brain gamma waves, brain gray matter density, and beneficial transcriptome changes in energy metabolism [18-20]. Interestingly, the Dalai Lama has also described meditation as “hard work”, indicating that meditation is an active process that challenges or slightly stresses the brain, leading to upregulation of genes in the AMPK signaling pathway as shown in the Harvard study [1]. This “mental stressor or challenge” is very much similar to challenging or stressing the human body with exercise, which is well-known to induce AMPK activation, leading to several beneficial effects [21]. The “mental challenge of meditation” is also analogous to the stress or challenge placed on the brain of animals when exposed to a stimulating environment, leading to an enhancement of learning and memory (i.e. long-term potentiation) and the “jumping of genes” [15,16]. Additionally, methodologies and neurotransmitters that are critical for inducing long-term potentiation (e.g. glutamate, high-frequency stimulation) activate AMPK in neurons and nearly every anesthetic used clinically to induce and maintain general anesthesia, including propofol, activates AMPK and excites the brain in low doses (called paradoxical excitation) [15,16]. Nearly every neurotransmitter that plays a critical role in wakefulness, arousal, and cognition also activates AMPK, supporting my original hypothesis that AMPK activation likely plays a central role in promoting consciousness itself [16].

Source: By ISAF Headquarters Public Affairs Office (originally posted to Flickr as 100410-F-7713A-002) [CC BY 2.0 (https://creativecommons.org/licenses/by/2.0)], via Wikimedia Commons; By Anatomist90 [CC BY-SA 3.0 (https://creativecommons.org/licenses/by-sa/3.0) or GFDL (http://www.gnu.org/copyleft/fdl.html)], from Wikimedia Commons

References:

1.  Bhasin MK, Denninger JW, Huffman JC, et al. Specific Transcriptome Changes Associated with Blood Pressure Reduction in Hypertensive Patients After Relaxation Response Training. J Altern Complement Med. 2018 May;24(5):486-504.
2. Conklin QA, King BG, Zanesco AP, et al. Insight meditation and telomere biology: The effects of intensive retreat and the moderating role of personality. Brain Behav Immun. 2018 May;70:233-245.
3. Finley J. Alteration of splice site selection in the LMNA gene and inhibition of progerin production via AMPK activation. Med Hypotheses. 2014 Nov;83(5):580-7.
4. Finley J. Reactivation of latently infected HIV-1 viral reservoirs and correction of aberrant alternative splicing in the LMNA gene via AMPK activation: Common mechanism of action linking HIV-1 latency and Hutchinson-Gilford progeria syndrome. Med Hypotheses. 2015 Sep;85(3):320-32.
5. Moshiri A, Puppo M, Rossi M, Gherzi R, Briata P. Resveratrol limits epithelial to mesenchymal transition through modulation of KHSRP/hnRNPA1-dependent alternative splicing in mammary gland cells. Biochim Biophys Acta. 2017 Mar;1860(3):291-298.
6. Ford LP, Wright WE, Shay JW. A model for heterogeneous nuclear ribonucleoproteins in telomere and telomerase regulation. Oncogene. 2002 Jan 21;21(4):580-3.
7. Madsen JM, Stoltzfus CM. An exonic splicing silencer downstream of the splice site A2 is required for efficient human immunodeficiency virus type 1 replication. J Virol 2005;79(16):10478–86.
8. Goodier JL, Zhang L, Vetter MR, Kazazian Jr. HH. LINE-1 ORF1 protein localizes in stress granules with other RNA-binding proteins, including components of RNA interference RNA-induced silencing complex. Mol Cell Biol 2007;27(18):6469–83.
9. Pedersen I, Fung L, Guzman H, et al. miR-128-induced LINE-1 restriction is dependent on down-regulation of hnRNPA1. bioRxiv 195560; https://doi.org/10.1101/195560.
10. Karnewar S, Neeli PK, Panuganti D, et al. Metformin regulates mitochondrial biogenesis and senescence through AMPK mediated H3K79 methylation: relevance in age-associated vascular dysfunction. Biochim Biophys Acta 2018;1864(4 Pt A):1115–28.
11. Chew GM, Chow DC, Souza SA, et al. Impact of adjunctive metformin therapy on T cell exhaustion and viral persistence in a clinical trial of HIV-infected adults on suppressive ART. J Virus Eradication 2017;3(Suppl. 1):6–19.
12. Chew GM. AAA http://viruseradication.com/abstract-details.php?abstract_id=1188.
13. Finley J. Oocyte activation and latent HIV-1 reactivation: AMPK as a common mechanism of action linking the beginnings of life and the potential eradication of HIV-1. Med Hypotheses. 2016 Aug;93:34-47.
14. Finley J. Elimination of cancer stem cells and reactivation of latent HIV-1 via AMPK activation: Common mechanism of action linking inhibition of tumorigenesis and the potential eradication of HIV-1. Med Hypotheses. 2017 Jul;104:133-146.
15. Finley J. Facilitation of hippocampal long-term potentiation and reactivation of latent HIV-1 via AMPK activation: Common mechanism of action linking learning, memory, and the potential eradication of HIV-1. Med Hypotheses. 2018 Jul;116:61-73.
16. Finley J. Transposable elements, placental development, and oocyte activation: Cellular stress and AMPK links jumping genes with the creation of human life. Med Hypotheses. 2018 Sep;118:44-54.
17. Finley J. Cellular stress and AMPK activation as a common mechanism of action linking the effects of metformin and diverse compounds that alleviate accelerated aging defects in Hutchinson-Gilford progeria syndrome. Med Hypotheses. 2018 Sep;118:151-162.
18. Lutz A, Greischar LL, Rawlings NB, Ricard M, Davidson RJ. Long-term meditators self-induce high-amplitude gamma synchrony during mental practice. Proc Natl Acad Sci U S A. 2004 Nov 16;101(46):16369-73.
19. Hölzel BK, Carmody J, Vangel M, et al. Mindfulness practice leads to increases in regional brain gray matter density. Psychiatry Res. 2011 Jan 30;191(1):36-43.
20. Bhasin MK, Dusek JA, Chang BH, et al. Relaxation response induces temporal transcriptome changes in energy metabolism, insulin secretion and inflammatory pathways. PLoS One. 2013 May 1;8(5):e62817.
21. Richter EA, Ruderman NB. AMPK and the biochemistry of exercise: implications for human health and disease. Biochem J. 2009 Mar 1;418(2):261-75.

Metformin shares common mechanism with nearly every Anesthesia drug: AMPK links Consciousness with Jumping Genes & the Creation of Human Life

By ISAF Headquarters Public Affairs Office (originally posted to Flickr as 100410-F-7713A-002) [CC BY 2.0 (https://creativecommons.org/licenses/by/2.0)], via Wikimedia Commons; By Anatomist90 [CC BY-SA 3.0 (https://creativecommons.org/licenses/by-sa/3.0) or GFDL (http://www.gnu.org/copyleft/fdl.html)], from Wikimedia Commons
  

A recently published study in the journal PLoS One in May of 2018 demonstrated that the anesthetic drug propofol significantly increased intracellular calcium (Ca2+) levels, induced a burst of reactive oxygen species (ROS), and activated the master metabolic regulator AMPK in C2C12 cells [18]. Similar results were also obtained in a recent study published in April of 2018, wherein propofol also increased intracellular Ca2+ levels and activated AMPK in HeLa cells [105]. AMPK is an evolutionarily conserved protein that increases lifespan and healthspan in several model organisms [34]. Activation of AMPK is also the primary mechanism of action of the anti-diabetic drug metformin, a compound that has displayed wide-raging efficacy in multiple disparate disease states, including cancer, dementia, depression, frailty-related diseases, and cardiovascular diseases [34,106]. Interestingly, propofol is considered one of the most popular and widely-used intravenous anesthetic drugs in modern medicine to induce and maintain general anesthesia in humans [107]. Curiously, a recent study published in the journal Current Biology in June of 2018 by researchers from the University of Michigan demonstrated that the compound carbachol reversed anesthesia induced by the inhaled anesthetic sevoflurane and restored wake-like behavior and level of consciousness in rats [27]. Carbachol is a compound that binds to and stimulates acetylcholine receptors in the brain but also activates AMPK in human cells, similar to both metformin and propofol [27,108].

Each of these studies substantiates several novel proposals in a recently published paper I authored in June of 2018 in which I proposed for the first time that cellular stress-induced AMPK activation links consciousness and accelerated emergence from anesthesia with paradoxical excitation, hippocampal long-term potentiation (essential for learning and memory), alleviation of accelerated cellular aging in Hutchinson-Gilford progeria syndrome, oocyte activation and the sperm acrosome reaction (prerequisites for human life creation), and transposable element (i.e. “jumping genes”)-mediated promotion of learning, memory, and the creation of human life [1-6].

As further explained below, nearly every neurotransmitter that plays a critical role in promoting wakefulness, arousal, and consciousness activates AMPK (glutamate, acetylcholine, orexin-A, histamine, norepinephrine, dopamine, and serotonin) [7-17]. Several drugs that are commonly used to induce and maintain general anesthesia also activate AMPK in low doses (propofol, sevoflurane, isoflurane, ketamine, dexmedetomidine, and midazolam) [18-23]. Also, several compounds that have recently been shown to promote accelerated emergence from anesthesia also activate AMPK (carbachol, orexin-A, histamine, dopamine, dopamine D1 receptor agonists, nicotine, caffeine, and forskolin) [9-11,13,24-33].

AMPK, an evolutionarily conserved kinase that is activated by the induction of cellular stress (i.e. increases in intracellular reactive oxygen species [ROS], calcium [Ca2+], and/or an AMP(ADP)/ATP ratio increase), increases lifespan and healthspan in several model organisms (yeast, worms, flies, mice, etc.) [34]. In my prior publication, I first proposed that cellular stress-induced AMPK activation is critical for facilitation of hippocampal long-term potentiation (LTP), considered a cellular correlate for learning and memory [5]. Indeed, AMPK has been found localized in hippocampal CA1 pyramidal neurons and glutamate, NMDA, potassium chloride, and high frequency stimulation have been shown to induce AMPK activation in cortical and hippocampal neurons [7,35,36]. Although an increase in Ca2+ levels is critical for neuronal activation and LTP induction, inhibition of ROS significantly inhibits hippocampal CA1 LTP, indicating that cellular stress-induced AMPK activation may play a pivotal role in neuronal excitation [37-40].

In my most recent publication, I noted that forskolin activates both AMPK and the transposable element syncytin-1 (necessary for human placental formation), increases human oocyte fertilization rates when combined with the AMPK activator cilostamide, and promotes chemically-induced LTP in hippocampal slices [6,26,41-44]. Transposable elements (TEs) are found in human oocytes, human sperm, and in human neural progenitor cells within the hippocampus [45-48]. TEs are also activated and can be induced to transpose or “jump” from one genomic location to another by increases in Ca2+ or ROS [49-51]. Exercise was shown to enhance LINE-1 (L1) retrotransposition (a TE of the retrotransposon class) in the dentate gyrus of the hippocampus in mice and L1 expression and retrotransposition in the adult mouse hippocampus was reported to enable long-term memory formation [52,53]. Because forskolin and caffeine, both of which activate AMPK, have recently been shown to promote accelerated emergence from anesthesia in rats and caffeine activates both mouse oocytes (models for human oocytes) and TEs, I proposed that cellular stress-induced AMPK activation may represent a common mechanism linking consciousness with learning, memory, and the creation of human life [25,26,33,54,55].

A primary cellular target of hypnotic agents (e.g. propofol) used for the induction and maintenance of general anesthesia is the GABAA receptor [66]. The GABAA receptor is located throughout the brain (cortex, thalamus, brain stem, and striatum) and binding of propofol post-synaptically to GABAA receptors enhances neural inhibition by the primary inhibitory neurotransmitter GABA, contributing to a loss of consciousness [66]. Interestingly, the GABAA receptor antagonist bicuculline, which reverses propofol anesthesia, activates AMPK in mouse cortical neurons via Ca2+ influx and flumazenil (a GABAA receptor antagonist) induces preconditioning by increasing the levels of ROS [56-58].  Basheer et al. as well as researchers from the University of Pennsylvania showed that AMPK is activated during extended periods of wakefulness but is inhibited during sleep in the basal forebrain and cerebral cortex of rats and mice [59,60]. Decreases in AMPK activation during sleep were also associated with increases in ATP, which would decrease AMPK activation as increases in the AMP(ADP)/ATP ratio activates AMPK [34,59]. Creatine, which also activates AMPK, decreased total sleep time, NREM sleep, and NREM delta activity significantly in rats [61,62]. Combined use of the anesthetic agents ketamine and xylazine in rats also led to an ATP increase that positively and significantly correlated with EEG delta activity [63]. However, the sedative and α2-receptor agonist clonidine activates AMPK in mice and xylazine, an analog of clonidine, activates AMPK in the rat cerebral cortex, hippocampus, thalamus, and cerebellum, provocatively indicating that low-dose anesthetic administration may actually promote wakefulness, arousal, and consciousness through activation of AMPK [64,65].

Low dose anesthetic-induced AMPK activation may also explain the phenomenon of paradoxical excitation. Curiously, low doses of nearly every anesthetic drug have been shown to induce paradoxical excitation [66]. As the name implies, before inducing unconsciousness, general anesthetic administration may result in a temporary increase in neuronal excitation, characterized by an increase in beta activity on the electroencephalogram (EEG) and eccentric body movements [66,109]. Because AMPK is activated by cellular stress induction (ROS, Ca2+, AMP(ADP)/ATP ratio increase) and because ROS and Ca2+ increases are critical for activation of pyramidal neurons, it is likely that many anesthetics induce rapid neuronal activation and paradoxical excitation in low doses by promoting cellular stress-induced AMPK activation [34,37-40]. Indeed, propofol, one of the most commonly-used anesthetics to induce and maintain general anesthesia, activates AMPK via an increase in ROS and Ca2+, promotes hippocampal neural stem cell differentiation, and promotes neuronal viability [67-69]. Sevoflurane, a commonly-used inhaled anesthetic, activates AMPK via an increase in ROS, increases Ca2+ levels in mouse brain cells, and enhances memory in rats at low doses [70-72]. Ketamine also activates Ca2+ channels in rat cortical neurons, increases ROS levels in the brain of rats, enhances hippocampal CA1 LTP in rats, and also functions as an antidepressant by activating AMPK in the rat hippocampus in vivo [73-76]. Prominent beta activity on the EEG has also been observed just before return of consciousness in healthy adult volunteers anaesthetized with propofol or sevoflurane (similar to paradoxical excitation), suggesting that the decrease of an anesthetic to a low, stimulatory level after removal of anesthesia may explain the increase in beta activity just before return of consciousness as well as during paradoxical excitation [6,66,77]. Hence, low dose anesthetic-induced AMPK activation may potentially accelerate emergence from anesthesia as well as promote beneficial arousal in disorders of consciousness (e.g. minimally conscious state, persistent vegetative state, coma, etc.) [6].

As noted above, nearly every neurotransmitter that plays a critical role in promoting wakefulness, arousal, and consciousness activates AMPK (glutamate, acetylcholine, orexin, histamine, norepinephrine, dopamine, and serotonin) and commonly used drugs that induce and maintain general anesthesia also activate AMPK in low doses (propofol, sevoflurane, isoflurane, ketamine, dexmedetomidine, and midazolam) [7-23]. Compounds that have recently been shown to accelerate emergence from anesthesia also activate AMPK (carbachol, orexin-A, histamine, dopamine, dopamine D1 receptor agonists, nicotine, caffeine, and forskolin) [9-11,13,24-33]. Additionally, a recent study by Hambrecht-Wiedbusch et al. strikingly demonstrated that although sub-anesthetic doses of ketamine increased anesthetic depth and induced burst suppression during isoflurane anesthesia, ketamine paradoxically accelerated recovery of consciousness in rats [78]. Such evidence supports the notion that while larger doses of anesthetics are effective at inducing loss of consciousness, low-dose anesthetic administration may facilitate rapid, cellular stress-induced neuronal activation that is mediated by AMPK activation [6].

Although they do not have a nervous system, plants produce nearly every neurotransmitter that promotes wakefulness, arousal, and consciousness in humans, including glutamate, acetylcholine, histamine, norepinephrine, dopamine, and serotonin [79-82]. The production of these neurotransmitters in plants is often associated with the induction of cellular stress (i.e. via wounding, osmotic stress, etc.) and partly serves as a defense mechanism [79-82]. Fungal infection of certain rice cultivars for example increases the production of serotonin, which suppresses leaf damage and reduces biotic stress [83]. ROS and Ca2+ also play critical roles in the production of secondary metabolites, compounds that plants produce partly for the purpose of self defense [84,85]. Interestingly, several abiotic stressors including nutrient deficiency, salt, osmotic, oxidative, and ER stress activates autophagy in Arabidopsis in a SnRK1-dependent manner. SnRK1 is the plant ortholog of AMPK [86]. Such evidence suggests that a mechanism of cellular stress-induced AMPK activation by neurotransmitters may have been evolutionarily conserved to promote neuronal activation in the human brain.

Indeed, the well-studied AMPK activator metformin activates AMPK in hippocampal neurons in vivo and enhances neurogenesis in the subventricular zone and the subgranular zone of the dentate gyrus, indicating that metformin may enhance brain repair and recovery of consciousness in disorders of consciousness [24,87,88]. Metformin also alleviates accelerated cellular aging defects and activates AMPK in Hutchinson-Gilford progeria syndrome (HGPS), a genetic disorder characterized by an accelerated aging phenotype caused by faulty splicing of the LMNA gene that also occurs in normal human cells at low levels [1,89,90]. Interestingly, temsirolimus (an analog of the macrolide rapamycin), alleviates accelerated aging defects in HGPS cells but increases the levels of ROS in both normal and HGPS cells within the first hour of treatment [91]. Metformin also activates the telomere-lengthening enzyme telomerase (which is derived from a transposable element) in an AMPK-dependent manner [92]. Cellular stress and AMPK activation also promotes oocyte maturation (precedes and is critical for oocyte activation), the acrosome reaction in human sperm (necessary for oocyte penetration and fertilization), and human placental development [26,93-95]. Forskolin and caffeine also induce the acrosome reaction in human sperm [96,110].

Lastly, increases in ROS, Ca2+, and AMPK activation are also critical for T cell activation and hence latent HIV-1 reactivation, a method currently pursued by HIV-1 cure researchers to reactivate dormant HIV-1 residing in T cells to facilitate virus detection and destruction by the immune system (called the “shock and kill” approach) [5,97-101]. Strikingly, forskolin reactivates latent HIV-1 in human U1 cells, a myelo-monocytic cell line used as a model for HIV-1 latency [102]. Early data has also demonstrated that metformin destabilized the latent HIV-1 reservoir in patients chronically infected with HIV-1 and significantly reduced cellular markers positively associated with T cells latently infected with HIV-1 [103,104]. Such evidence provides a compelling indication that cellular stress-induced AMPK activation links transposable elements and alleviation of accelerated cellular aging with potential HIV-1 eradication, consciousness, and the creation of human life, all hypotheses that I originally proposed [1-6].

https://www.linkedin.com/pulse/metformin-shares-common-mechanism-nearly-every-drug-ampk-finley/

References

1. Finley J. Alteration of splice site selection in the LMNA gene and inhibition of progerin production via AMPK activation. Med Hypotheses. 2014 Nov;83(5):580-7.
2. Finley J. Reactivation of latently infected HIV-1 viral reservoirs and correction of aberrant alternative splicing in the LMNA gene via AMPK activation: Common mechanism of action linking HIV-1 latency and Hutchinson-Gilford progeria syndrome. Med Hypotheses. 2015 Sep;85(3):320-32.
3. Finley J. Oocyte activation and latent HIV-1 reactivation: AMPK as a common mechanism of action linking the beginnings of life and the potential eradication of HIV-1. Med Hypotheses. 2016 Aug;93:34-47.
4. Finley J. Elimination of cancer stem cells and reactivation of latent HIV-1 via AMPK activation: Common mechanism of action linking inhibition of tumorigenesis and the potential eradication of HIV-1. Med Hypotheses. 2017 Jul;104:133-146.
5. Finley J. Facilitation of hippocampal long-term potentiation and reactivation of latent HIV-1 via AMPK activation: Common mechanism of action linking learning, memory, and the potential eradication of HIV-1. Med Hypotheses. 2018 Jul;116:61-73.
6. Finley J. Transposable elements, placental development, and oocyte activation: Cellular stress and AMPK links jumping genes with the creation of human life. Med Hypotheses. 2018.
7. Terunuma M, Vargas KJ, Wilkins ME, et al. Prolonged activation of NMDA receptors promotes dephosphorylation and alters postendocytic sorting of GABAB receptors. Proc. Natl. Acad. Sci. U.S.A. 2010;107(31):13918–23.
8. Zhao M, Sun L, Yu XJ, et al. Acetylcholine mediates AMPK-dependent autophagic cytoprotection in H9c2 cells during hypoxia/reoxygenation injury. Cell Physiol Biochem. 2013;32(3):601-13.
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Metformin & AMPK Link Nobel Prize-winning Telomeres & Jumping Genes with Learning, HIV, & the Creation of Human Life

Nobel Prize winners, from left to right: Elizabeth Blackburn (discovered telomerase), Barbara McClintock (discovered “jumping genes”), and Françoise Barré-Sinoussi (discovered HIV). By Science History Institute, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=30112731; By Smithsonian Institution/Science Service; Restored by Adam Cuerden - Flickr: Barbara McClintock (1902-1992), Public Domain, https://commons.wikimedia.org/w/index.php?curid=25629182; By Prolineserver (talk) - Own work, GFDL 1.2, https://commons.wikimedia.org/w/index.php?curid=5395403

A recently published study in the journal Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease in 2018 demonstrated for the first time that chronic treatment with the anti-diabetic drug metformin activated human telomerase in human aortic endothelial cells (HAECs) and significantly delayed endothelial senescence in an AMPK-dependent manner [11]. Telomeres are specialized regions of repetitive nucleotide sequences located at the ends of eukaryotic chromosomes that protect chromosomal ends from deterioration [63]. However, continuous cell division leads to telomere shortening, impeding the replenishment of tissues and triggering cellular senescence (i.e. cells cease to divide). Although human telomeres shorten with age, telomeres may be lengthened by the enzyme telomerase [64].

This study substantiates and confirms several novel proposals in a recently published paper I authored in April of 2018 in which I first proposed that because telomerase is derived from a “jumping gene” (see below for discussion), metformin would activate telomerase via AMPK [6]. My paper also highlights a novel link between hippocampal long-term potentiation (essential for learning and memory), alleviation of accelerated cellular aging in Hutchinson-Gilford progeria syndrome, oocyte activation and the sperm acrosome reaction (prerequisites for human life creation), and transposable element (i.e. “jumping genes”)-mediated promotion of learning, memory, and the creation of human life [1-7]. Indeed, these novel proposals also link several Nobel Prize-winning discoveries, including the discovery of telomerase by Elizabeth Blackburn (photo-left), the discovery of “jumping genes” by Barbara McClintock (photo-middle), and the discovery of HIV by Françoise Barré-Sinoussi (photo-right).
 
The link between such disparate physiological and pathophysiological phenomena is cellular stress-induced modulation of energy metabolism, leading to the activation of the master metabolic regulator AMPK, a kinase that increases lifespan and healthspan in several model organisms [12]. I was the first to propose and publish (2014) that an increase in beneficial levels of cellular stress (e.g. increases in the levels reactive oxygen species [ROS], calcium [Ca2+], and/or an AMP(ADP)/ATP ratio increase, etc.) and activation of AMPK by compounds including metformin would alleviate accelerated cellular aging defects in children diagnosed with the genetic disorder Hutchinson-Gilford progeria syndrome (HGPS), a disease characterized by an accelerated aging phenotype and death at ~14.6 years of age [1]. This hypothesis was substantiated in 2016 and 2017, with metformin activating AMPK in cells taken from HGPS kids and ameliorating accelerated cellular aging defects (e.g. correcting nuclear morphology, decrease in senescence markers, etc.) [8,9]. Additionally, transfection of telomerase been shown to reverse senescence in HGPS cells [65]. As metformin activates telomerase and normal humans make the same toxic protein (called progerin) that leads to accelerated aging in HGPS kids (just at lower amounts that accumulate with age), AMPK activation may also play a significant role in ameliorating diseases associated with physiological aging [10,11].

AMPK also links HGPS with potential virus eradication. I first proposed in 2015 that AMPK activation links alleviation of accelerated aging in HGPS with the potential eradication of HIV-1 via the “shock and kill” approach, a method currently being pursued by HIV cure researchers to possibly eradicate HIV-1 [2,13]. The same gene splicing factor that promotes accelerated aging in HGPS (called SRSF1 or ASF/SF2) also inhibits reactivation of latent HIV-1 (i.e. “shock”), preventing immune system detection and virus destruction (i.e. “kill”) [8,14]. Metformin was shown to slow aging in HGPS cells by decreasing this splicing factor, as I originally predicted in 2014, and several compounds that potently induce latent HIV-1 reactivation in T cells from infected patients, including PMA (a phorbol ester) combined with ionomycin, each activate AMPK [1,8,15-17]. AMPKα1 deletion leads to a decrease in primary T cell responses to bacterial and viral infections in vivo, AMPK knockdown leads to cell death on T cell activation, and metformin has recently been shown to inhibit Zika and Dengue viruses, the malaria parasite, and Legionella pneumophila [18-23]. Intriguingly, early data presented at the International AIDS Conference in 2017 demonstrated that metformin destabilized the latent HIV-1 reservoir in chronically-infected HIV patients and decreased the percentage of CD4+ T cells expressing the immune checkpoint receptors PD-1, TIGIT, and TIM-3, each markers associated with T cells latently infected with HIV-1, indicating that AMPK activation may indeed contribute to a cure for HIV-1 [24,25].

As I first proposed in 2016 and 2017, the induction of cellular stress and AMPK activation also links HGPS and potential HIV-1 eradication with oocyte activation and the sperm acrosome reaction, prerequisites for the creation of human life [3,4]. Increases in both ROS and Ca2+ are critical for T cell activation (and hence latent HIV-1 reactivation) and ROS is transiently increased in HGPS cells when treated with a rapamycin analog to alleviate accelerated aging [26-28]. Stress-induced activation of AMPK by AICAR and other compounds promotes oocyte maturation, which precedes and is essential for efficient oocyte activation [29,30]. Oocyte activation is indispensable for the creation of all human life and PMA and ionomycin, which collectively reactivates latent HIV-1, activates mouse and human oocytes, respectively [31,32]. AMPK is also found in the acrosome of the human sperm head and ionomycin induces the acrosome reaction in human sperm, a process necessary for oocyte penetration and fertilization [33,34]. Ionomycin is also used extensively during fertility procedures to activate human oocytes (i.e. “shock”), creating normal, healthy children (i.e. “live”) [32]. Interestingly, ionomycin is a narrow spectrum antibiotic produced by certain species within the bacterial genus Streptomyces, from which ~70 percent of clinically useful antibiotics are derived [35,36]. Cellular stress, mediated by increases in ROS, Ca2+, and/or an AMP(ADP)/ATP ratio increase, etc. also enhances antibiotic production in many Streptomyces strains, reinforcing the notion that the beneficial effects of cellular stress induction crosses species boundaries [37,38].

Cellular stress induction and AMPK activation also link HGPS, potential HIV-1 eradication, and human life creation with learning and memory, a hypothesis I originally proposed in 2018 [6]. Hippocampal long-term potentiation (LTP) is considered the cellular correlate of learning and memory and AMPK has been found localized in hippocampal CA1 dendrites and is activated in neurons by metformin, AICAR, ionomycin, and glutamate, a neurotransmitter essential for hippocampal LTP induction [39-41]. The glutamate receptors AMPAR and NMDAR are found on and modulate T cell activation, AMPK activation increases synthesis and membrane insertion of AMPARs (critical for LTP expression), PMA enhances hippocampal CA1 LTP, and inhibition of ROS significantly impairs hippocampal CA1 LTP [42-46]. Also, neuronal depolarization decreases the recruitment efficiency of SRSF1 to nascent RNAs and promotes SRSF1 nuclear speckle accumulation [6]. SRSF1, a gene splicing factor that is inhibited by metformin, enhances progerin production in HGPS cells and prevents latent HIV-1 reactivation [2,8]. Metformin also significantly reduces pathology-associated reductions in LTP in animal models in vivo, indicating that learning and memory are linked to HGPS, potential HIV-1 eradication, and human life creation via the induction of beneficial levels of cellular stress [47].

Lastly, cellular stress and AMPK activation also links the activation and mobilization of transposable elements (i.e. “jumping genes”) with telomerase activation, potential HIV-1 eradication, learning and memory, and the creation of human life, a hypothesis I originally proposed in 2018 [6]. Transposable elements (TEs) are DNA sequences first described by Nobel laureate Barbara McClintock that comprise nearly half of the human genome, are able to transpose or move from one genomic location to another, and have played an extensive role in human genome evolution [48-50]. Strikingly, McClintock also described in her Nobel Prize speech that a genome “shock” seemed to promote TE activation and mobilization [50]. As first noted in my recently published paper, this “shock” is the same “shock” that HIV cure researchers are using during the “shock and kill” approach to reactivate latent HIV-1 to potentially effectuate a cure [6]. Indeed, several forms of cellular stress, including heat shock and radiation, have been convincingly shown to activate and enhance TE mobilization in several model organisms and in human cells [51-53]. This same “shock” McClintock referred to, mediated by increases in ROS, Ca2+, and/or an AMP(ADP)/ATP ratio, etc. is also what leads to the creation of human life, as the antibiotic ionomycin activates AMPK, promotes TE activation, and induces human oocyte activation [17,32,54]. LINE-1 (L1), a member of the retrotransposon class of TEs, is active and capable of mobilization in human oocytes, human sperm, and in human neural progenitor cells [55-57]. Inhibition of L1 impairs both oocyte maturation in vitro and long-term memory formation in vivo in mice [58,59]. L1 has also been detected in the human brain and is capable of mobilization in human neurons [57]. As noted above, AMPK is critical for oocyte maturation and metformin promotes hippocampal neurogenesis and spatial memory formation [29,60]. The landmark initial sequencing of the human genome also noted that both telomerase and RAG1 (promotes DNA cleavage and transposition in human cells) are derived from TEs [49]. Because metformin activates both telomerase and RAG1 via AMPK, it is likely that cellular stress-induced AMPK activation facilitates beneficial TE activation and mobilization (i.e. learning and memory associated with L1 mobilization), linking human genome evolution and the creation of human life with hippocampal LTP, HGPS, and potential HIV-1 eradication [61,62].

https://www.linkedin.com/pulse/metformin-ampk-link-nobel-prize-winning-telomeres-jumping-finley/

References 

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Metformin shown for the first time to activate Telomere enzyme Telomerase in human cells via AMPK: Link between Progeria and HIV

Goldsmith Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus [Public domain], via Wikimedia Commons;The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. Scaffidi P, Gordon L, Misteli T. PLoS Biology Vol. 3/11/2005, e39 

A recently published study in the journal Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease in 2018 demonstrated for the first time that chronic treatment with the anti-diabetic drug metformin activated human telomerase reverse transcriptase (hTERT) in human aortic endothelial cells (HAECs) and significantly delayed endothelial senescence in an AMPK-dependent manner [1]. AMPK is activated by the induction of cellular stress, mediated by increases in intracellular calcium (Ca2+), reactive oxygen species (ROS), and/or an AMP(ADP)/ATP ratio increase, etc. [41].  Telomeres are specialized regions of repetitive nucleotide sequences located at the ends of eukaryotic chromosomes that protect chromosomal ends from deterioration [2].  However, continuous cell division leads to telomere shortening, impeding the replenishment of tissues and triggering cellular senescence (i.e. cells cease to divide).  Although human telomeres shorten with age, telomeres may be lengthened by the enzyme telomerase, a ribonucleoprotein that consists of the catalytic subunit hTERT, telomerase RNA, and the nucleolar protein dyskerin [2,3]. hTERT, which is considering limiting for telomerase activity, is a protein that exhibits reverse transcriptase activity and synthesizes telomeric DNA from an RNA template [4]. 

The authors of the study initially demonstrated that both metformin and the AMPK activator AICAR significantly increased hTERT levels and enhanced AMPK activation in human aortic endothelial cells (HAECs) [1].  Importantly, inhibition or knockdown of AMPK with compound C or siAMPKα inhibited the metformin-induced increase in hTERT while metformin failed to reverse siAMPKα-induced senescence in HAECs, indicating that hTERT expression is regulated by AMPK activation in endothelial cells.  Indeed, continuous culturing of HAECs in the presence of metformin significantly increased hTERT protein levels and activity, reduced the expression of the senescence markers p53, p21, p27, and p16, and reduced senescence-associated beta-galactosidase (SA-β-gal, a biomarker of cellular senescence) staining in HAECs, again indicating that metformin delays cellular senescence and increases hTERT levels via AMPK activation [1].  Strikingly, using ApoE-/- mice (which spontaneously develop atherosclerosis and age faster compared to normal mice), the authors also showed that chronic low-dose metformin administration for fourteen months in drinking water enhanced the levels of activated AMPK and Pgc-1α observed in the endothelial layer of the aorta [1].  Metformin also increased the transcript and protein levels of Tert, decreased senescence markers (p16, p21, p27, p53) in the total aortic homogenate, and significantly reduced SA-β-gal staining of aorta compared to untreated ApoE-/- mice, demonstrating that metformin-induced AMPK activation delays vascular aging and protects from age-associated atherosclerosis in ApoE-/- mice [1].

Interestingly, telomere length has been shown to be significantly reduced in cells derived from patients with the accelerated aging disorder Hutchinson-Gilford progeria syndrome (HGPS) and telomere shortening in normal cells that occurs during cellular senescence activates progerin production, a toxic protein that leads to an accelerating aging phenotype in children with HGPS via aberrant alternative splicing of the LMNA gene [5].  Normal lamin A plays a critical role in supporting nuclear architecture and morphology.  Lamin A binding to subtelomeric repeats also localizes telomeres to the nuclear periphery and loss of lamin A leads to defects in telomeric heterochromatin, altered nuclear distribution and shortening of telomeres, inefficient processing of dysfunctional telomeres by non-homologus end joining, and increased genomic instability [6]. Telomere length has been found to be significantly reduced in fibroblasts derived from HGPS patients and a recent study also confirmed that in normal human fibroblasts, progressive telomere damage that occurs during cellular senescence activates progerin production and also leads to extensive changes in alternative splicing of many other genes, highlighting a striking similarity between normal aging and accelerated aging in HGPS patients [5,7]. Indeed, transfection of HGPS fibroblasts with human telomerase (hTERT) mRNA restored cell proliferation, reduced cell loss, extended cellular lifespan, increased telomerase activity and telomere length, and reduced SA-β-gal staining compared to HGPS cells expressing catalytically inactive hTERT mRNA [8].  Because metformin also increases hTERT expression and inhibits senescence in human cells, it is likely that cellular stress-induced AMPK activation, mediated by increases in intracellular calcium (Ca2+), reactive oxygen species (ROS), and/or an AMP(ADP)/ATP ratio increase, etc., represents a central node linking structurally diverse compounds and methodologies that alleviate accelerated aging in HGPS cells.              

As the splicing factor SRSF1 has been shown to increase progerin production by promoting the use of a cryptic splice located in the LMNA gene, metformin was recently shown to significantly reduce the expression of SRSF1 and progerin, activate AMPK, and improve nuclear architecture in HGPS cells, indicating that AMPK activation by metformin beneficially alters gene splicing in HGPS cells by modulating SRSF1, a hypothesis that I first proposed and published in 2014 [9-11].  Also, p32, a splicing-associated protein that is an endogenous inhibitor of SRSF1 and is critical for the maintenance of mitochondrial functionality and oxidative phosphorylation, has also been shown to be essential for rapamycin- or starvation-induced autophagy mediated by ULK1 [12,13].  Because rapamycin, also an AMPK activator in vivo,  improves accelerated aging defects in HGPS cells by reducing progerin levels via induction of autophagy, AMPK activation likely also beneficially modulates the activity of p32, leading to inhibition of SRSF1 splicing activity and enhancement of mitochondrial functionality [14,15].  Moreover, PGC-1α, which is activated by AMPK and metformin and promotes telomere transcription, is downregulated in HGPS cells, leading to significant mitochondrial dysfunction [1,16,17].  Methylene blue, which activates AMPK in vivo, was shown to increase PGC-1α levels, induce progerin solubility, and alleviate accelerated aging defects in HGPS cells [16,18].  Additionally, the rapamycin analog temsirolimus alleviated accelerated aging defects in HGPS cells but transiently increased ROS and superoxide anion levels in both HGPS and normal cells within the first hour of treatment, again indicating that cellular stress-induced AMPK activation represents a common mechanism for inhibiting senescence and ameliorating symptoms associated with accelerated aging [19].    

The inhibition of SRSF1 and the promotion of hTERT expression and telomere transcription by metformin via AMPK activation also link HGPS and telomere integrity with HIV-1 latency.  Increased splicing activity of SRSF1 inhibits reactivation of latent HIV-1 residing in infected immune cells, preventing immune system detection and destruction of the virus [20].  Reactivation of latent HIV-1 (i.e. the “shock and kill” approach) leads to a reduction in SRSF1 but an increase in p32 activity and bryostatin-1 (a PKC modulator) has been shown to reactivate latent HIV-1 via AMPK activation [20,21].  Interestingly, p32 modulation via AMPK activation may also enhance and stabilize the splicing activities of hnRNPA1, a heteroribonuclear protein that associates with p32, antagonizes the splicing function of SRSF1, prevents splicing of the HIV-1 genome (promoting viral reactivation), and participates in the maintenance and preservation of telomeres [22-25].  hnRNPA1 is also decreased in senescent human fibroblasts and antagonizes cellular senescence and the senescence-associated secretory phenotype (SASP) via increasing SIRT1 expression [26,27].  Resveratrol, a plant-derived polyphenol that activates AMPK, increases hnRNPA1 protein expression and SIRT1, a histone deacetylase that plays a role in a number of age related diseases and in the extension of lifespan, is also activated by AMPK [28-30].  Also, T cell activation, an efficient method for reactivating latent HIV-1, is dependent on increases in intracellular Ca2+ and ROS, telomerase is transiently increased on T cell activation, and AMPK knockdown leads to T cell death during in vitro activation [31-34].  Furthermore, resveratrol reactivates latent HIV-1 and preliminary data demonstrated that metformin decreased the percentage of CD4+ T cells expressing PD-1, TIGIT, and TIM-3, each markers associated with T cells latently infected with HIV-1, in chronically-infected HIV-1 patients [35-37].  Such evidence strongly suggests that cellular stress-induced AMPK activation, mediated by increases in intracellular calcium (Ca2+), reactive oxygen species (ROS), and/or an AMP (ADP)/ATP ratio increase, etc. links the alleviation of accelerated cellular aging defects in HGPS with the potential eradication of HIV-1, a hypothesis that I first proposed in 2015 [38].        

 

The evidence presented in the Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease publication further substantiates that AMPK activation represents a central node that connects the therapeutic benefits of chemically distinct compounds in diseases as seemingly dissimilar as HGPS and HIV-1 latency.  Indeed, AMPK activators including metformin increase hTERT expression, promote telomere transcription and integrity, decrease the splicing activity of SRSF1 that increases progerin production but prevents latent HIV-1 reactivation, and potentially beneficially modulates the activity of the SRSF1 inhibitor p32 and the ribonucleoprotein hnRNPA1.  As AMPK activators (e.g. ionomycin) induce human oocyte activation (giving rise to normal healthy children) and AMPK is localized throughout the entire acrosome in human sperm (likely promoting the acrosome reaction), AMPK activation links normal human aging, Progeria, and HIV-1 latency with the creation of all human life (i.e. the “shock and live” approach) [11,38-40].  

https://www.linkedin.com/pulse/metformin-shown-first-time-activate-telomere-enzyme-human-finley/?published=t

 

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