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/

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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

 

References:

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2. O'Sullivan RJ, Karlseder J. Telomeres: protecting chromosomes against genome instability. Nat Rev Mol Cell Biol. 2010 Mar;11(3):171-81.  
3. Cohen SB, Graham ME, Lovrecz GO, Bache N, Robinson PJ, Reddel RR. Protein composition of catalytically active human telomerase from immortal cells. Science. 2007 Mar 30;315(5820):1850-3.
4. Cong YS, Wen J, Bacchetti S. The human telomerase catalytic subunit hTERT: organization of the gene and characterization of the promoter. Hum Mol Genet. 1999 Jan;8(1):137-42.
5. Cao K, Blair CD, Faddah DA, et al. Progerin and telomere dysfunction collaborate to trigger cellular senescence in normal human fibroblasts. J Clin Invest. 2011 Jul;121(7):2833-44.
6. Gonzalez-Suarez I, Redwood AB, Perkins SM, et al. Novel roles for A-type lamins in telomere biology and the DNA damage response pathway. EMBO J 2009;28(16):2414–27.
7. Decker ML, Chavez E, Vulto I, Lansdorp PM. Telomere length in Hutchinson-Gilford progeria syndrome. Mech Ageing Dev. 2009 Jun;130(6):377-83.
8. Li Y, Zhou G, Bruno IG, Cooke JP. Telomerase mRNA Reverses Senescence in Progeria Cells. J Am Coll Cardiol. 2017 Aug 8;70(6):804-805.
9. Egesipe AL, Blondel S1, Cicero AL, et al. Metformin decreases progerin expression and alleviates pathological defects of Hutchinson-Gilford progeria syndrome cells. NPJ Aging Mech Dis. 2016 Nov 10;2:16026.
10. Park SK, Shin OS. Metformin alleviates ageing cellular phenotypes in Hutchinson-Gilford progeria syndrome dermal fibroblasts. Exp Dermatol. 2017 Oct;26(10):889-895.
11. 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.
12. P32/gC1qR is indispensable for fetal development and mitochondrial translation: importance of its RNA-binding ability. Nucl Acids Res 2012;40(19):9717–37.
13. Jiao H, Su GQ, Dong W, et al. Chaperone-like protein p32 regulates ULK1 stability and autophagy. Cell Death Differ. 2015 Nov;22(11):1812-23.
14. Cao K, Graziotto JJ, Blair CD, et al. Rapamycin reverses cellular phenotypes and enhances mutant protein clearance in Hutchinson-Gilford progeria syndrome cells. Sci Transl Med. 2011 Jun 29;3(89):89ra58.
15. Chiao YA, Kolwicz SC, Basisty N, et al. Rapamycin transiently induces mitochondrial remodeling to reprogram energy metabolism in old hearts. Aging (Albany NY). 2016 Feb;8(2):314-27.
16. Xiong ZM, Choi JY, Wang K, et al. Methylene blue alleviates nuclear and mitochondrial abnormalities in progeria. Aging Cell. 2016 Apr;15(2):279-90.
17. Diman A, Boros J, Poulain F, et al. Nuclear respiratory factor 1 and endurance exercise promote human telomere transcription. Sci Adv. 2016 Jul 27;2(7):e1600031.
18. Xie L, Li W, Winters A, Yuan F, Jin K, Yang S. Methylene blue induces macroautophagy through 5' adenosine monophosphate-activated protein kinase pathway to protect neurons from serum deprivation. Front Cell Neurosci. 2013 May 3;7:56.
19. Gabriel D, Gordon LB, Djabali K. Temsirolimus Partially Rescues the Hutchinson-Gilford Progeria Cellular Phenotype. PLoS One. 2016 Dec 29;11(12):e0168988.
20. Berro R, Kehn K, de la Fuente C, et al. Acetylated Tat regulates human immunodeficiency virus type 1 splicing through its interaction with the splicing regulator p32. J Virol. 2006 Apr;80(7):3189-204.
21. Mehla R, Bivalkar-Mehla S, Zhang R, et al. Bryostatin modulates latent HIV-1 infection via PKC and AMPK signaling but inhibits acute infection in a receptor independent manner. PLoS One. 2010 Jun 16;5(6):e11160.
22. Expert-Bezançon A, Sureau A, Durosay P, et al. HnRNP A1 and the SR proteins ASF/SF2 and SC35 have antagonistic functions in splicing of beta tropomyosin exon 6B. J Biol Chem 2004 Sep 10;279(37):38249–59.
23. LaBranche H, Dupuis S, Ben-David Y, Bani MR, Wellinger RJ, Chabot B. Telomere elongation by hnRNP A1 and a derivative that interacts with telomeric repeats and telomerase. Nat Genet 1998 Jun;19(2):199–202.
24. Petersen-Mahrt SK, Estmer C, Ohrmalm C, Matthews DA, Russell WC, Akusjärvi G. The splicing factor-associated protein, p32, regulates RNA splicing by inhibiting ASF/SF2 RNA binding and phosphorylation. EMBO J 1999;18(4):1014–24.
25. Caputi M, Mayeda A, Krainer AR, Zahler AM. hnRNP A/B proteins are required for inhibition of HIV-1 pre-mRNA splicing. EMBO J. 1999 Jul 15;18(14):4060-7.
26. Zhu D1, Xu G, Ghandhi S, Hubbard K. Modulation of the expression of p16INK4a and p14ARF by hnRNP A1 and A2 RNA binding proteins: implications for cellular senescence. J Cell Physiol. 2002 Oct;193(1):19-25.
27. Wang H, Han L, Zhao G. hnRNP A1 antagonizes cellular senescence and senescence-associated secretory phenotype via regulation of SIRT1 mRNA stability. Aging Cell. 2016 Sep 9. doi: 10.1111/acel.12511. [Epub ahead of print].
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29. 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.
30. Chiang MC, Nicol CJ, Cheng YC. Resveratrol activation of AMPK-dependent pathways is neuroprotective in human neural stem cells against amyloid-beta-induced inflammation and oxidative stress. Neurochem Int. 2017 Oct 5. pii: S0197-0186(17)30362-5.
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39. 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.
40. Calle-Guisado V, de Llera AH, Martin-Hidalgo D, et al. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility. Asian J Androl. 2017 Nov-Dec;19(6):707-714.
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Metformin shown for the first time to inhibit Legionella infection via AMPK : AMPK links pathogen destruction with Progeria & Human Life Creation

CC-BY-SA-3.0 http://creativecommons.org/licenses/by-sa/3.0/)], via Wikimedia Commons; By CDC/ Dr. Barry S. Fields [Public domain], via Wikimedia Commons

In line with recent evidence demonstrating that the AMPK activator metformin inhibits Zika virus, Dengue virus, and malaria parasite replication (see below), a study published in The Journal of Immunology in December of 2017 showed for the first time that the anti-diabetic drug metformin significantly suppressed the growth of the bacterium Legionella pneumophila (L. pneumophila) in immune cells derived from both mice and humans by activating AMPK [1]. L. pneumophila is a Gram-negative bacterium that is typically found in water-associated environments and may contaminate hot water tanks and air-conditioning units for large buildings and is the causative agent of Legionnaires' disease [1,2]. Legionnaires' disease is a form of atypical pneumonia characterized by fever, cough, and shortness of breath and is usually acquired by inhalation of small air-borne water droplets [2]. According to the Centers for Disease Control and Prevention (CDC), “the bacterium is named after a 1976 outbreak, during which some people who went to a Philadelphia convention of the American Legion suffered from a new type of pneumonia (lung infection) that became known as Legionnaires’ disease.” [3]. Interestingly, the authors of the study observed that metformin increased the production of mitochondrial reactive oxygen species (ROS) in L. pneumophila-infected immune cells and that inhibition of both AMPK activation and ROS production negated metformin-mediated growth suppression of L. pneumophila [1]. Most importantly, metformin significantly reduced bacterial number, activated AMPK, and increased ROS in the lungs of infected mice, thus improving survival and indicating that metformin inhibits L. pneumophila replication in vivo in an AMPK-dependent manner. Metformin also increased mitochondrial ROS in uninfected immune cells, suggesting that cellular stress induced AMPK activation (via increases in ROS, calcium[Ca2+], and/or an AMP/ATP ratio increase, etc) is critical for mounting an effective immune response to bacteria, viruses, and other pathogens [1].

Strikingly, metformin activates AMPK and alleviates accelerating aging defects in cells from children with the genetic disorder Hutchinson-Gilford progeria syndrome (HGPS), promotes the differentiation and/or apoptosis of cancer stem cells in an AMPK-dependent manner, and destabilizes the latent HIV-1 reservoir in chronically-infected HIV-1 patients (facilitating virus elimination and potentially contributing to an HIV-1 cure). Also, because AMPK is critical for oocyte maturation and bacteria-derived antibiotics (e.g. ionomycin, A23187) that activate AMPK are used extensively to activate human oocytes to create normal, healthy babies, it is likely that stress-induced AMPK activation (e.g. via ROS, intracellular Ca2+, and/or AMP/ATP ratio increase, etc.) represents a common mechanism linking pathogen elimination with HGPS, caner stem cell elimination, HIV-1 eradication, and the creation of all human life, as I originally proposed in several recent publications (see below) [4-7].

As noted above, in addition to inhibition of L. pneumophila replication, metformin also potently inhibited ZIKV replication in HUVECs and AMPK activation has recently been found to exert significant antiviral effects against Rift Valley Fever virus as well as multiple arbovirus family members including the Flavivirus Kunjin virus, the Togavirus Sindbis virus, and the Rhabdovirus Vesicular stomatitis virus [8,9]. Interestingly, as both ZIKV and dengue virus (DENV) are transmitted by the same mosquito vector, a study recently published in the journal PLoS Pathogens in April of 2017 demonstrated for the first time that metformin exerted significant antiviral effects in DENV-infected human liver cells that was dependent on activation of AMPK [10]. The authors showed that an increase in HMG-CoA reductase (HMGCR) activity, a target of AMPK, was associated with DENV-infected cells, AMPK activation was reduced in DENV-infected cells at 12 and 24 hours post infection (hpi), and metformin significantly decreased the number of infected cells, viral yield, and viral genome copies, leading the authors to conclude that metformin-induced AMPK activation generates a strong antiviral effect against DENV [10].

Recent efforts funded by the U.S. and British governments, the Bill & Melinda Gates Foundation, and the Google health spin-off Verily have sought to decrease the spread of dengue and Zika viruses through the coordinated release of female and/or male mosquitoes (called Aedes aegypti) that were purposely infected with a bacterium that inhibits the mosquito’s ability to transmit the two viruses to humans [11,12]. Studies have shown that this bacterium, called Wolbachia, enhances the mosquito’s immune response by increasing the levels of reactive oxygen species (ROS), thus enhancing inhibition of DENV replication [13]. Because AMPK is activated by cellular stress (e.g. ROS increase, intracellular calcium [Ca2+] increase, AMP/ATP ratio increase, etc.), has been found in Aedes aegypti (Ae. aegypti), and AMPK activation by stress-inducing compounds (e.g. resveratrol) increased average life span and enhanced the immune response in Ae. aegypti in an AMPK-dependent manner, the recent finding that metformin also inhibits DENV replication in human cells in an AMPK-dependent manner provides compelling evidence that the anti-viral and antimicrobial effects of AMPK activation likely crosses species boundaries [14].

Moreover, a study published in the journal Cell Reports in September of 2016 by researchers from the Massachusetts Institute of Technology (MIT) and the University of Lisbon also showed for the first time that metformin and other AMPK activators significantly reduced parasite load in human liver cells of different species of Plasmodium, a protozoan parasite that is the etiological agent of malaria [15]. Importantly, the authors also showed that AMPK activation inhibits growth and replication of different Plasmodium spp. (species) and AMPK activators as well as dietary restriction, which activates AMPK, reduces Plasmodium berghei (malaria-causing species in rodents often used as a model for the study of human malaria) infection in mice [15]. The AMPK-activating compounds salicylate and A769662 also reduced P. berghei and P. falciparum (malaria parasite that infects humans) merozoite formation (infectious parasites generated through replication in erythrocytes) in vitro while salicylate decreased parasitaemia in mice in vivo [16].

Cellular stress-induced AMPK activation has also been shown to exert antiviral effects against HIV-1. AMPK activation and several AMPK-activating compounds, including EGCG, curcumin, tanshinone II A (derived from the plant Salvia miltiorrhiza), bryostatin-1 (isolated from the marine organism Bugula neritina), and resveratrol (found in grapes and in the plant Polygonum cuspidatum) have been shown to exhibit antiviral activity in vitro against HIV-1 [17-21].

An active area among HIV-1 cure researchers, known as the “shock and kill” approach, involves reactivating (i.e. “shock”) a T cell (or another immune cell) that harbors dormant HIV-1, hence reactivating the virus itself and thus inducing destruction of the T cell along with the virus or enhancing recognition and destruction of the virus-infected T cell by the immune system (i.e. “kill”) [5]. Strikingly, AMPK is also critical for the activation of T cells and the mounting of an effective immune response to eliminate viruses, bacteria, and cancer cells [6,22,23]. A recent study demonstrated that metformin, when combined with the protein kinase C modulator bryostatin, induced reactivation of latent HIV-1 in a monocytic cell line in an AMPK-dependent manner. Bryostatin was also shown to induce phosphorylation and activation of AMPK in that study, implying that bryostatin is an indirect AMPK activator as well [20]. Furthermore, the calcium ionophores ionomycin and A23187, both of which activate AMPK and induce human oocyte activation, are often combined with phorbol 12-myristate 13-acetate (PMA) and are extremely efficient in promoting T cell activation-induced latent HIV-1 reactivation [24-26].

Perhaps most convincingly, at the International AIDS Society’s (IAS) HIV Cure and Cancer Forum held in Paris, France in July of 2017, researchers from the University of Hawaii demonstrated for the first time that metformin decreased the percentage of CD4+ T cells expressing the immune checkpoint receptors PD-1, TIGIT, and TIM-3 in HIV-1 patients, receptors that are positively associated with T cells that harbor latent HIV-1. Metformin also destabilized the latent viral reservoir in chronically-infected HIV-1 patients, indicating that metformin may indeed contribute to HIV-1 eradication by inducing an AMPK-mediated reactivation of latent HIV-1, as I initially proposed in 2015 and 2016 [5,6,27-31].

Also, stress-induced AMPK activation likely links latent HIV-1 reactivation with alleviation of accelerated aging defects in cells derived from children with the genetic disorder Hutchinson-Gilford progeria syndrome (HGPS). Studies have shown that efficient reactivation of latent HIV-1 involves a reduction in the splicing of the HIV-1 genome by the gene splicing factor SRSF1 [32-34]. Accelerated cellular aging-like phenotypes in HGPS are primarily linked to aberrant splicing of the LMNA gene, leading to the over production of a toxic protein called progerin [4]. Evidence has also shown that inhibition of the splicing factor SRSF1 leads to a reduction in progerin at both the mRNA and protein levels [5,35].

A recent study published online in the Journal npj Aging and Mechanisms of Disease in November of 2016 provided startling evidence that metformin decreased the expression of progerin and SRSF1 and alleviated pathological defects in cells derived from HGPS patients [36]. Another study published online in the Journal Experimental Dermatology in February of 2017 confirmed that metformin alleviated nuclear defects and premature aging phenotypes and activated AMPK in fibroblasts derived from HGPS patients, substantiating my original hypotheses from 2014 and 2015 proposing that AMPK activators including metformin would improve accelerated aging defects in HGPS cells by inhibiting SRSF1 and activating AMPK [4,5,37]. Temsirolimus, an analog of the macrolide rapamycin (which activates AMPK in vivo), also partially rescued the HGPS cellular phenotype but significantly increased the levels of ROS and superoxide within the first hour of treatment, providing further indication that the induction of cellular stress and subsequent AMPK activation links virus and pathogen elimination with alleviation of accelerated cellular aging defects in HGPS [38,39].

Furthermore, ROS and calcium are well-studied mediators of cellular stress-induced differentiation of embryonic and adult stem cells, AMPK has recently been shown to be essential for mouse embryonic stem cell differentiation, and metformin targets and promotes differentiation and/or apoptosis of cancer stem cells in the deadliest of cancers in an AMPK-dependent manner, including glioblastoma and pancreatic cancer [7]. Such evidence strongly suggests that cellular stress-induced AMPK activation by compounds including metformin links pathogen and virus elimination with HGPS and cancer stem cell differentiation and/or apoptosis, a hypothesis that I first proposed in 2017 [7].

Lastly, AMPK activation also promotes oocyte meiotic induction and maturation (processes that are critical for efficient oocyte activation) and AMPK has recently been found localized across the entire acrosome in human spermatozoa [6,40,41].  The induction of cellular stress (e.g. increases in ROS, intracellular calcium, and/or AMP/ATP ratio increase), which activates AMPK, also promotes oocyte meiotic induction/maturation, oocyte activation, and the acrosome reaction in human sperm, processes critical for the creation of all human life [40,42,43]. Indeed, oocyte activation is indispensable for the creation of all human life and the bacteria-derived calcium ionophore ionomycin, which activates AMPK, is commonly used to promote latent HIV-1 reactivation and is extensively used to activate human oocytes, creating normal healthy children (i.e. the “shock and live” approach) [43-47]. Such evidence indicates and further substantiates the novel and provocative assertion that AMPK activation links L. pneumophila inhibition and pathogen elimination with the amelioration of accelerated aging defects in HGPS cells, HIV-1 latency and replication, adult and cancer stem cells, and the creation of all human life [1,4-7].

https://www.linkedin.com/pulse/metformin-shown-first-time-inhibit-legionella-infection-finley/

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20. Mehla R, Bivalkar-Mehla S, Zhang R, et al. Bryostatin modulates latent HIV-1 infection via PKC and AMPK signaling but inhibits acute infection in a receptor independent manner. PLoS One. 2010 Jun 16;5(6):e11160.
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24. Gómez-Gonzalo M, Carretero M, Rullas J et al. The hepatitis B virus X protein induces HIV-1 replication and transcription in synergy with T-cell activation signals: functional roles of NF-kappaB/NF-AT and SP1-binding sites in the HIV-1 long terminal repeat promoter. J Biol Chem. 2001 Sep 21;276(38):35435-43.
25. Tamás P, Hawley SA, Clarke RG, et al. Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes. J Exp Med. 2006 Jul 10;203(7):1665-70.
26. Fogarty S, Hawley SA, Green KA, Saner N, Mustard KJ, Hardie DG. Calmodulin-dependent protein kinase kinase-beta activates AMPK without forming a stable complex: synergistic effects of Ca2+ and AMP. Biochem J. 2010 Jan 27;426(1):109-18.
27. G.M. Chew, D.C. Chow, S.A. Souza, 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.  Journal of Virus Eradication 2017; 3 (Supplement 1): 6–19.
28. http://viruseradication.com/supplement-details/Abstracts_of_the_IAS_HIV_Cure_and_Cancer_Forum_2017/
29. http://www.iasociety.org/HIV-Programmes/Towards-an-HIV-Cure/Events/HIV-Cure-Cancer-Forum
30. Fromentin R, Bakeman W, Lawani MB, et al. CD4+ T Cells Expressing PD-1, TIGIT and LAG-3 Contribute to HIV Persistence during ART. PLoS Pathog. 2016 Jul 14;12(7):e1005761.
31. Chew GM, Fujita T, Webb GM, et al. TIGIT Marks Exhausted T Cells, Correlates with Disease Progression, and Serves as a Target for Immune Restoration in HIV and SIV Infection. PLoS Pathog. 2016 Jan 7;12(1):e1005349.
32. Berro R, Kehn K, de la Fuente C, et al. Acetylated Tat regulates human immunodeficiency virus type 1 splicing through its interaction with the splicing regulator p32. J Virol. 2006 Apr;80(7):3189-204.
33. Spina CA, Anderson J, Archin NM, et al. An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients. PLoS Pathog. 2013;9(12):e1003834.
34. Hu M, Crawford SA, Henstridge DC, et al. p32 protein levels are integral to mitochondrial and endoplasmic reticulum morphology, cell metabolism and survival. Biochem J. 2013 Aug 1;453(3):381-91.
35. Lopez-Mejia IC, Vautrot V, De Toledo M, et al. A conserved splicing mechanism of the LMNA gene controls premature aging. Hum Mol Genet. 2011 Dec 1;20(23):4540-55.
36. Egesipe AL, Blondel S, Cicero AL, et al. Metformin decreases progerin expression and alleviates pathological defects of Hutchinson-Gilford progeria syndrome cells. NPJ Aging Mech Dis. 2016 Nov 10;2:16026.
37. Park SK, Shin OS. Metformin Alleviates Ageing Cellular Phenotypes in Hutchinson-Gilford Progeria Syndrome Dermal Fibroblasts. Exp Dermatol. 2017 Oct;26(10):889-895.
38. Gabriel D, Gordon LB, Djabali K. Temsirolimus Partially Rescues the Hutchinson-Gilford Progeria Cellular Phenotype. PLoS One. 2016 Dec 29;11(12):e0168988.
39. Chiao YA, Kolwicz SC, Basisty N, et al. Rapamycin transiently induces mitochondrial remodeling to reprogram energy metabolism in old hearts. Aging (Albany NY). 2016 Feb;8(2):314-27.
40. LaRosa C, Downs SM. Stress stimulates AMP-activated protein kinase and meiotic resumption in mouse oocytes. Biol Reprod. 2006 Mar;74(3):585-92.
41. Calle-Guisado V, de Llera AH, Martin-Hidalgo D, et al. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility. Asian J Androl. 2016 Sep 27. doi: 10.4103/1008-682X.185848. [Epub ahead of print].
42. de Lamirande E, Tsai C, Harakat A, Gagnon C. Involvement of reactive oxygen species in human sperm arcosome reaction induced by A23187, lysophosphatidylcholine, and biological fluid ultrafiltrates. J Androl. 1998 Sep-Oct;19(5):585-94.
43. Tesarik J, Sousa M, Testart J. Human oocyte activation after intracytoplasmic sperm injection. Hum Reprod. 1994 Mar;9(3):511-8.
44. Deemeh MR, Tavalaee M, Nasr-Esfahani MH. Health of children born through artificial oocyte activation: a pilot study. Reprod Sci. 2015 Mar;22(3):322-8.
45. Tamás P, Hawley SA, Clarke RG, et al. Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes. J Exp Med. 2006 Jul 10;203(7):1665-70.
46. Spina CA, Anderson J, Archin NM, et al. An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients. PLoS Pathog 2013;9(12):e1003834.
47. Liu WC, Slusarchyk DS, Astle G, Trejo WH, Brown WE, Meyers E. Ionomycin, a new polyether antibiotic. J Antibiot (Tokyo). 1978 Sep;31(9):815-9.    

Metformin shown for the first time to inhibit Zika virus in human umbilical cells: AMPK links virus destruction with Progeria, HIV & Cancer stem cells

By Jim Gathany [Public domain], via Wikimedia Commons; CC-BY-SA-3.0 http://creativecommons.org/licenses/by-sa/3.0/)], via Wikimedia Commons

A study published in the Journal of Virology in December of 2017 by researchers from the University of Southern California showed for the first time that AMPK activators including the anti-diabetic drug metformin caused an approximately 60% to 80% decrease in virus production from human umbilical vein endothelial cells (HUVECs) infected with Zika virus (ZIKV) [1]. ZIKV has been causally linked to microcephaly (head circumference smaller than normal due to abnormal brain development) and has been shown to efficiently infect HUVECs, which directly contact the fetal blood stream [2]. Metformin-induced inhibition of ZIKV replication in HUVECs may thus represent a powerful, safe, and economically viable option to treat and/or prevent conditions associated with ZIKV infection. Interestingly, as further explained below, metformin and AMPK have recently been shown to exert antiviral and anti-parasitic effects against dengue virus (which is transmitted by the same mosquito vector as ZIKV) and different Plasmodium species (the etiological agent of malaria), respectively.

Additionally, metformin activates AMPK and alleviates accelerating aging defects in cells from children with the genetic disorder Hutchinson-Gilford progeria syndrome (HGPS), promotes the differentiation and/or apoptosis of cancer stem cells in an AMPK-dependent manner, and destabilizes the latent HIV-1 reservoir in chronically-infected HIV-1 patients (facilitating virus elimination and potentially contributing to an HIV-1 cure). Also, because AMPK is critical for oocyte maturation and bacteria-derived antibiotics (e.g. ionomycin, A23187) that activate AMPK are used extensively to activate human oocytes to create normal, healthy babies, it is likely that stress-induced AMPK activation (e.g. via reactive oxygen species, intracellular calcium, and/or AMP/ATP ratio increase, etc.) represents a common mechanism linking pathogen elimination with HGPS, caner stem cell elimination, HIV-1 eradication, and the creation of all human life, as I originally proposed in several recent publications (see below) [3-6].   

As noted above, the AMPK activators metformin and AICAR potently inhibited ZIKV replication in HUVECs [1]. Although the authors unexplainably found that compound C (an AMPK inhibitor) also inhibited ZIKV replication, AMPK activation has recently been found to exert significant antiviral effects against Rift Valley Fever virus as well as multiple arbovirus family members including the Flavivirus Kunjin virus, the Togavirus Sindbis virus, and the Rhabdovirus Vesicular stomatitis virus [1,7]. Indeed, several AMPK-activating compounds have also recently demonstrated antiviral effects against ZIKV infection and replication. For example, EGCG (found in green tea) inhibited ZIKV entry in Vero E6 cells, curcumin (derived from the plant Curcuma longa) inhibited ZIKV replication in HeLa cells, NDGA (derived from the plant Larrea tridentate) reduced viral yield in Vero cells infected with a ZIKV strain isolated from a human patient, sophoraflavenone G (isolated from the plant Sophora Flavecens) inhibits ZIKV replication in A549 cells, hemin (an iron-containing porphyrin) significantly inhibited ZIKV replication in primary human monocyte-derived macrophages, quercetin (found in a variety of plants) exerted antiviral activity against ZIKV in both tissue culture and knockout mice, and chloroquine (an anti-malarial compound) inhibited ZIKV infection in vitro and protected fetal mice from ZIKV-induced microcephaly [8-14]. Similar to metformin, each of the aforementioned compounds or the plant extracts from which they are derived activates AMPK in vivo and/or in vitro [15-19].

Interestingly, as both ZIKV and dengue virus (DENV) are transmitted by the same mosquito vector, a study recently published in the journal PLoS Pathogens in April of 2017 demonstrated for the first time that metformin exerted significant antiviral effects in DENV-infected human liver cells that was dependent on activation of the master metabolic regulator AMPK [20]. The authors showed that an increase in HMG-CoA reductase (HMGCR) activity, a target of AMPK, was associated with DENV-infected cells, AMPK activation was reduced in DENV-infected cells at 12 and 24 hours post infection (hpi), and metformin significantly decreased the number of infected cells, viral yield, and viral genome copies, leading the authors to conclude that metformin-induced AMPK activation generates a strong antiviral effect against DENV [20]. Recent efforts funded by the U.S. and British governments, the Bill & Melinda Gates Foundation, and the Google health spin-off Verily have sought to decrease the spread of dengue and Zika viruses through the coordinated release of female and/or male mosquitoes (called Aedes aegypti) that were purposely infected with a bacterium that inhibits the mosquito’s ability to transmit the two viruses to humans [21,22]. Studies have shown that this bacterium, called Wolbachia, enhances the mosquito’s immune response by increasing the levels of reactive oxygen species (ROS), thus enhancing inhibition of DENV replication [23]. Because AMPK is activated by cellular stress (e.g. ROS increase, intracellular calcium [Ca2+] increase, AMP/ATP ratio increase, etc.), has been found in Aedes aegypti (Ae. aegypti), and AMPK activation by stress-inducing compounds (e.g. resveratrol) increased average life span and enhanced the immune response in Ae. aegypti in an AMPK-dependent manner, the recent finding that metformin also inhibits DENV replication in human cells in an AMPK-dependent manner provides compelling evidence that the anti-viral and antimicrobial effects of AMPK activation likely crosses species boundaries [24]. 

Moreover, a study published in the journal Cell Reports in September of 2016 by researchers from the Massachusetts Institute of Technology (MIT) and the University of Lisbon also showed for the first time that metformin and other AMPK activators significantly reduced parasite load in human liver cells of different species of Plasmodium, a protozoan parasite that is the etiological agent of malaria [25]. Importantly, the authors also showed that AMPK activation inhibits growth and replication of different Plasmodium spp. (species) and AMPK activators as well as dietary restriction, which activates AMPK, reduces Plasmodium berghei (malaria-causing species in rodents often used as a model for the study of human malaria) infection in mice [25]. The AMPK-activating compounds salicylate and A769662 also reduced P. berghei and P. falciparum (malaria parasite that infects humans) merozoite formation (infectious parasites generated through replication in erythrocytes) in vitro while salicylate decreased parasitaemia in mice in vivo [26].

Cellular stress-induced AMPK activation has also been shown to exert antiviral effects against HIV-1. AMPK activation and several AMPK-activating compounds, including EGCG, curcumin, tanshinone II A (derived from the plant Salvia miltiorrhiza), byrostatin-1 (isolated from the marine organism Bugula neritina), and resveratrol (found in grapes and in the plant Polygonum cuspidatum) have been shown to exhibit antiviral activity in vitro against HIV-1 [27-31].

An active area among HIV-1 cure researchers, known as the “shock and kill” approach, involves reactivating (i.e. “shock”) a T cell (or another immune cell) that harbors dormant HIV-1, hence reactivating the virus itself and thus inducing destruction of the T cell along with the virus or enhancing recognition and destruction of the virus-infected T cell by the immune system (i.e. “kill”) [4]. Strikingly, AMPK is also critical for the activation of T cells and the mounting of an effective immune response to eliminate viruses, bacteria, and cancer cells [5,32,33]. A recent study demonstrated that metformin, when combined with the protein kinase C modulator bryostatin, induced reactivation of latent HIV-1 in a monocytic cell line in an AMPK-dependent manner. Bryostatin was also shown to induce phosphorylation and activation of AMPK in that study, implying that bryostatin is an indirect AMPK activator as well [30]. Furthermore, the calcium ionophores ionomycin and A23187, both of which activate AMPK and induce human oocyte activation, are often combined with phorbol 12-myristate 13-acetate (PMA) and are extremely efficient in promoting T cell activation-induced latent HIV-1 reactivation [34-36].

Perhaps most convincingly, at the International AIDS Society’s (IAS) HIV Cure and Cancer Forum held in Paris, France in July of 2017, researchers from the University of Hawaii demonstrated for the first time that metformin decreased the percentage of CD4+ T cells expressing the immune checkpoint receptors PD-1, TIGIT, and TIM-3 in HIV-1 patients, receptors that are positively associated with T cells that harbor latent HIV-1. Metformin also destabilized the latent viral reservoir in chronically-infected HIV-1 patients, indicating that metformin may indeed contribute to HIV-1 eradication by inducing an AMPK-mediated reactivation of latent HIV-1, as I initially proposed in 2015 and 2016 [4,5, 37-41].

Also, stress-induced AMPK activation likely also links latent HIV-1 reactivation with alleviation of accelerated aging defects in cells derived from children with the genetic disorder Hutchinson-Gilford progeria syndrome (HGPS). Studies have shown that efficient reactivation of latent HIV-1 involves a reduction in the splicing of the HIV-1 genome by the gene splicing factor SRSF1 [42-44]. Accelerated cellular aging-like phenotypes in HGPS are primarily linked to aberrant splicing of the LMNA gene, leading to the over production of a toxic protein called progerin [3]. Evidence has also shown that inhibition of the splicing factor SRSF1 leads to a reduction in progerin at both the mRNA and protein levels [4,45].

A recent study published online in the Journal npj Aging and Mechanisms of Disease in November of 2016 provided startling evidence that metformin decreased the expression of progerin and SRSF1 and alleviated pathological defects in cells derived from HGPS patients [46]. Another study published online in the Journal Experimental Dermatology in February of 2017 confirmed that metformin alleviated nuclear defects and premature aging phenotypes and activated AMPK in fibroblasts derived from HGPS patients, substantiating my original hypotheses from 2014 and 2015 proposing that AMPK activators including metformin would improve accelerated aging defects in HGPS cells by inhibiting SRSF1 and activating AMPK [3,4,47]. Temsirolimus, an analog of the macrolide rapamycin (which activates AMPK in vivo), also partially rescued the HGPS cellular phenotype but significantly increased the levels of ROS and superoxide within the first hour of treatment, providing further indication that the induction of cellular stress and subsequent AMPK activation links virus and pathogen elimination with alleviation of accelerated cellular aging defects in HGPS [48,49].

Furthermore, ROS and calcium are well-studied mediators of cellular stress-induced differentiation of embryonic and adult stem cells, AMPK has recently been shown to be essential for mouse embryonic stem cell differentiation, and metformin targets and promotes differentiation and/or apoptosis of cancer stem cells in the deadliest of cancers in an AMPK-dependent manner, including glioblastoma and pancreatic cancer [6]. Such evidence strongly suggests that cellular stress-induced AMPK activation by compounds including metformin links pathogen and virus elimination with HGPS and cancer stem cell differentiation and/or apoptosis, a hypothesis that I first proposed in 2017 [6].

Lastly, AMPK activation also promotes oocyte meiotic induction and maturation (processes that are critical for efficient oocyte activation) and AMPK has recently been found localized across the entire acrosome in human spermatozoa [5,50,51]. The induction of cellular stress (e.g. increases in ROS, intracellular calcium, and/or AMP/ATP ratio increase), which activates AMPK, also promotes oocyte meiotic induction/maturation, oocyte activation, and the acrosome reaction in human sperm, processes critical for the creation of all human life [50,52,53]. Indeed, oocyte activation is indispensable for the creation of all human life and the bacteria-derived calcium ionophore ionomycin, which activates AMPK, is commonly used to promote latent HIV-1 reactivation and is extensively used to activate human oocytes, creating normal healthy children (i.e. the “shock and live” approach) [53-57]. Such evidence indicates and further substantiates the novel and provocative assertion that AMPK activation links Zika virus inhibition and pathogen elimination with the amelioration of accelerated aging defects in HGPS cells, HIV-1 latency and replication, adult and cancer stem cells, and the creation of all human life [1,3-6]. 

https://www.linkedin.com/pulse/metformin-shown-first-time-inhibit-zika-virus-human-umbilical-finley/

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30. Mehla R, Bivalkar-Mehla S, Zhang R, et al. Bryostatin modulates latent HIV-1 infection via PKC and AMPK signaling but inhibits acute infection in a receptor independent manner. PLoS One. 2010 Jun 16;5(6):e11160.
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33. Blagih J, Coulombe F, Vincent EE, et al. The energy sensor AMPK regulates T cell metabolic adaptation and effector responses in vivo. Immunity. 2015 Jan 20;42(1):41-54.
34. Gómez-Gonzalo M, Carretero M, Rullas J et al. The hepatitis B virus X protein induces HIV-1 replication and transcription in synergy with T-cell activation signals: functional roles of NF-kappaB/NF-AT and SP1-binding sites in the HIV-1 long terminal repeat promoter. J Biol Chem. 2001 Sep 21;276(38):35435-43.
35. Tamás P, Hawley SA, Clarke RG, et al. Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes. J Exp Med. 2006 Jul 10;203(7):1665-70.
36. Fogarty S, Hawley SA, Green KA, Saner N, Mustard KJ, Hardie DG. Calmodulin-dependent protein kinase kinase-beta activates AMPK without forming a stable complex: synergistic effects of Ca2+ and AMP. Biochem J. 2010 Jan 27;426(1):109-18.
37. G.M. Chew, D.C. Chow, S.A. Souza, 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. Journal of Virus Eradication 2017; 3 (Supplement 1): 6–19.
38. http://viruseradication.com/supplement-details/Abstracts_of_the_IAS_HIV_Cure_and_Cancer_Forum_2017/
39. http://www.iasociety.org/HIV-Programmes/Towards-an-HIV-Cure/Events/HIV-Cure-Cancer-Forum
40. Fromentin R, Bakeman W, Lawani MB, et al. CD4+ T Cells Expressing PD-1, TIGIT and LAG-3 Contribute to HIV Persistence during ART. PLoS Pathog. 2016 Jul 14;12(7):e1005761.
41. Chew GM, Fujita T, Webb GM, et al. TIGIT Marks Exhausted T Cells, Correlates with Disease Progression, and Serves as a Target for Immune Restoration in HIV and SIV Infection. PLoS Pathog. 2016 Jan 7;12(1):e1005349.
42. Berro R, Kehn K, de la Fuente C, et al. Acetylated Tat regulates human immunodeficiency virus type 1 splicing through its interaction with the splicing regulator p32. J Virol. 2006 Apr;80(7):3189-204.
43. Spina CA, Anderson J, Archin NM, et al. An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients. PLoS Pathog. 2013;9(12):e1003834.
44. Hu M, Crawford SA, Henstridge DC, et al. p32 protein levels are integral to mitochondrial and endoplasmic reticulum morphology, cell metabolism and survival. Biochem J. 2013 Aug 1;453(3):381-91.
45. Lopez-Mejia IC, Vautrot V, De Toledo M, et al. A conserved splicing mechanism of the LMNA gene controls premature aging. Hum Mol Genet. 2011 Dec 1;20(23):4540-55.
46. Egesipe AL, Blondel S, Cicero AL, et al. Metformin decreases progerin expression and alleviates pathological defects of Hutchinson-Gilford progeria syndrome cells. NPJ Aging Mech Dis. 2016 Nov 10;2:16026.
47. Park SK, Shin OS. Metformin Alleviates Ageing Cellular Phenotypes in Hutchinson-Gilford Progeria Syndrome Dermal Fibroblasts. Exp Dermatol. 2017 Oct;26(10):889-895.
48. Gabriel D, Gordon LB, Djabali K. Temsirolimus Partially Rescues the Hutchinson-Gilford Progeria Cellular Phenotype. PLoS One. 2016 Dec 29;11(12):e0168988.
49. Chiao YA, Kolwicz SC, Basisty N, et al. Rapamycin transiently induces mitochondrial remodeling to reprogram energy metabolism in old hearts. Aging (Albany NY). 2016 Feb;8(2):314-27.
50. LaRosa C, Downs SM. Stress stimulates AMP-activated protein kinase and meiotic resumption in mouse oocytes. Biol Reprod. 2006 Mar;74(3):585-92.
51. Calle-Guisado V, de Llera AH, Martin-Hidalgo D, et al. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility. Asian J Androl. 2016 Sep 27. doi: 10.4103/1008-682X.185848. [Epub ahead of print].
52. de Lamirande E, Tsai C, Harakat A, Gagnon C. Involvement of reactive oxygen species in human sperm arcosome reaction induced by A23187, lysophosphatidylcholine, and biological fluid ultrafiltrates. J Androl. 1998 Sep-Oct;19(5):585-94.
53. Tesarik J, Sousa M, Testart J. Human oocyte activation after intracytoplasmic sperm injection. Hum Reprod. 1994 Mar;9(3):511-8.
54. Deemeh MR, Tavalaee M, Nasr-Esfahani MH. Health of children born through artificial oocyte activation: a pilot study. Reprod Sci. 2015 Mar;22(3):322-8.
55. Tamás P, Hawley SA, Clarke RG, et al. Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes. J Exp Med. 2006 Jul 10;203(7):1665-70.
56. Spina CA, Anderson J, Archin NM, et al. An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients. PLoS Pathog 2013;9(12):e1003834.
57. Liu WC, Slusarchyk DS, Astle G, Trejo WH, Brown WE, Meyers E. Ionomycin, a new polyether antibiotic. J Antibiot (Tokyo). 1978 Sep;31(9):815-9.