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/

References

1. Cheng F, Ramos da Silva S, Huang IC, Jung JU, Gao SJ. Suppression of Zika virus infection and replication in endothelial cells and astrocytes by PKA inhibitor PKI 14-22. J Virol. 2017 Dec 6. pii: JVI.02019-17. doi: 10.1128/JVI.02019-17. [Epub ahead of print].
2. Richard AS, Shim BS, Kwon YC, et al. AXL-dependent infection of human fetal endothelial cells distinguishes Zika virus from other pathogenic flaviviruses. Proc Natl Acad Sci U S A. 2017 Feb 21;114(8):2024-2029.
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. 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.
6. 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.
7. Moser TS, Schieffer D, Cherry S. AMP-activated kinase restricts Rift Valley fever virus infection by inhibiting fatty acid synthesis. PLoS Pathog. 2012;8(4):e1002661.
8. Carneiro BM, Batista MN, Braga AC, Nogueira ML, Rahal P. The green tea molecule EGCG inhibits Zika virus entry. Virology. 2016 Sep;496:215-8.
9. Mounce BC, Cesaro T, Carrau L, Vallet T, Vignuzzi M. Curcumin inhibits Zika and chikungunya virus infection by inhibiting cell binding. Antiviral Res. 2017 Jun;142:148-157.
10. Merino-Ramos T, Jiménez de Oya N, Saiz JC, Martín-Acebes MA. Antiviral activity of nordihydroguaiaretic acid and its derivative tetra-O-methyl nordihydroguaiaretic acid against West Nile virus and Zika virus. Antimicrob Agents Chemother. 2017 May 15. pii: AAC.00376-17.
11. Sze A, Olagnier D, Hadj SB, et al. Sophoraflavenone G Restricts Dengue and Zika Virus Infection via RNA Polymerase Interference. Viruses. 2017 Oct 3;9(10). pii: E287.
12. Huang H, Falgout B, Takeda K, Yamada KM, Dhawan S. Nrf2-dependent induction of innate host defense via heme oxygenase-1 inhibits Zika virus replication. Virology. 2017 Mar;503:1-5.
13. Wong G, He S, Siragam V, et al. Antiviral activity of quercetin-3-β-O-D-glucoside against Zika virus infection. Virol Sin. 2017 Sep 5. doi: 10.1007/s12250-017-4057-9. [Epub ahead of print].
14. Li C, Zhu X, Ji X, et al. Chloroquine, a FDA-approved Drug, Prevents Zika Virus Infection and its Associated Congenital Microcephaly in Mice. EBioMedicine. 2017 Oct;24:189-194.
15. Kim I, He YY. Targeting the AMP-Activated Protein Kinase for Cancer Prevention and Therapy. Front Oncol. 2013 Jul 15;3:175.
16. Zhang H, Li Y, Hu J, et al. Effect of Creosote Bush-Derived NDGA on Expression of Genes Involved in Lipid Metabolism in Liver of High-Fructose Fed Rats: Relevance to NDGA Amelioration of Hypertriglyceridemia and Hepatic Steatosis. PLoS One. 2015 Sep 22;10(9):e0138203.
17. Yang X, Yang J, Xu C, et al. Antidiabetic effects of flavonoids from Sophora flavescens EtOAc extract in type 2 diabetic KK-ay mice. J Ethnopharmacol. 2015 Aug 2;171:161-70.
18. Park EJ, Kim YM, Chang KC. Hemin Reduces HMGB1 Release by UVB in an AMPK/HO-1-dependent Pathway in Human Keratinocytes HaCaT Cells. Arch Med Res. 2017 Jul;48(5):423-431.
19. Spears LD, Tran AV, Qin CY, et al. Chloroquine increases phosphorylation of AMPK and Akt in myotubes. Heliyon. 2016 Mar;2(3):e00083.
20. Soto-Acosta R, Bautista-Carbajal P, Cervantes-Salazar M, Angel-Ambrocio AH, Del Angel RM. DENV up-regulates the HMG-CoA reductase activity through the impairment of AMPK phosphorylation: A potential antiviral target. PLoS Pathog. 2017 Apr 6;13(4):e1006257.
21. http://www.reuters.com/article/us-health-dengue-mosquitoes-idUSKCN12Q1PE
22. https://www.bloomberg.com/news/articles/2016-10-06/alphabet-s-verily-joins-zika-fight-with-sterile-mosquito-lab
23. Pan X, Zhou G, Wu J, et al. Wolbachia induces reactive oxygen species (ROS)-dependent activation of the Toll pathway to control dengue virus in the mosquito Aedes aegypti. Proc Natl Acad Sci U S A. 2012 Jan 3;109(1):E23-31.
24. Nunes RD, Ventura-Martins G, Moretti DM, et al. Polyphenol-Rich Diets Exacerbate AMPK-Mediated Autophagy, Decreasing Proliferation of Mosquito Midgut Microbiota, and Extending Vector Lifespan. PLoS Negl Trop Dis. 2016 Oct 12;10(10):e0005034.
25. Ruivo MT, Vera IM, Sales-Dias J, et al. Host AMPK Is a Modulator of Plasmodium Liver Infection. Cell Rep. 2016 Sep 6;16(10):2539-45.
26. Mancio-Silva L, Slavic K, Grilo Ruivo MT, et al. Nutrient sensing modulates malaria parasite virulence. Nature. 2017 Jul 13;547(7662):213-216.
27. Zhang HS, Wu TC, Sang WW, Ruan Z. EGCG inhibits Tat-induced LTR transactivation: role of Nrf2, AKT, AMPK signaling pathway. Life Sci. 2012 May 22;90(19-20):747-54.
28. Zhang HS, Ruan Z, Sang WW. HDAC1/NFκB pathway is involved in curcumin inhibiting of Tat-mediated long terminal repeat transactivation. J Cell Physiol. 2011 Dec;226(12):3385-91.
29. Zhang HS, Chen XY, Wu TC, Zhang FJ. Tanshinone II A inhibits tat-induced HIV-1 transactivation through redox-regulated AMPK/Nampt pathway. J Cell Physiol. 2014 Sep;229(9):1193-201.
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.
31. Zhang HS, Wu MR. SIRT1 regulates Tat-induced HIV-1 transactivation through activating AMP-activated protein kinase. Virus Res. 2009 Dec;146(1-2):51-7.
32. Pearce EL, Walsh MC, Cejas PJ, et al. Enhancing CD8 T-cell memory by modulating fatty acid metabolism. Nature. 2009 Jul 2;460(7251):103-7.
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.   

Antibiotics produced by Bacteria activate Human Oocytes, creating Healthy Babies: AMPK links the Creation of Human Life with HIV, Progeria, & Cancer

CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/)], via Wikimedia Commons;By De Wood, Pooley, USDA, ARS, EMU. [Public domain], via Wikimedia Commons 


A recent study published online in the journal Fertility and Sterility in September of 2017 systematically reviewed for the first time evidence for the effect of two compounds, ionomycin and A23187 (also known as calcimycin), on fertilization rates and pregnancy outcomes in infertile couples undergoing an in vitro fertilization procedure known as intracytoplasmic sperm injection (ICSI) [1]. ICSI involves the direct deposition of sperm into the oocyte cytoplasm, which typically leads to high rates of fertilization. However, fertilization failure despite repeated ICSI is likely caused by a failure of the oocyte to activate [1]. Physiological oocyte activation is accomplished by the delivery of a sperm-borne oocyte activating factor called phospholipase C zeta (PLCζ). PLCζ activates human oocytes by inducing an intracellular signaling cascade that ultimately results in increased calcium (Ca2+) oscillations in the oocyte, which drives oocyte activation to completion [1]. As oocyte activation is an indispensable prerequisite for the creation of all human life, every human being alive today and any human being that has ever lived began their existence as an activated oocyte [2]. Ionomycin and A23187 increase the levels of intracellular Ca2+ and are thus commonly known as Ca2+ ionophores [1]. The authors of the Fertility and Sterility study showed that over a total of 1,521 ICSI cycles, calcium ionophores including ionomycin and A23187 led to a statistically significant improvement in fertilization, cleavage, blastulation, implantation rates, overall pregnancy, and live-birth rates [1]. Ionomycin and A23187 have also been shown in several independent studies to effectively induce human oocyte activation, leading to the birth of normal, healthy children [3,4].

Strikingly, as described further below, both ionomycin and A23187 are antibiotics that are naturally produced by certain species within the bacterial genus Streptomyces [5,6]. Other structurally distinct compounds and methods have also been shown to induce human oocyte activation, including ethanol, puromycin (an antibiotic and protein synthesis inhibitor produced by Streptomyces alboniger), as well as mechanical manipulation and electrical stimulation, both of with have been reported to result in the creation of normal children [7-11]. Interestingly, as mouse oocytes are considered models for human oocytes, ionomycin, A23187, anisomycin (an antibiotic and protein synthesis inhibitor produced by Streptomyces griseolus), mycophenolic acid (an immunosuppressant produced by the fungus Penicillium brevicompactum), cycloheximide (a protein synthesis inhibitor produced by Streptomyces griseus), carvacrol (a secondary plant metabolite produced by Origanum vulgare{oregano}), and phorbol 12-myristate 13-acetate (PMA, a secondary plant metabolite produced by Croton tiglium) each induce activation of mouse oocytes [12-22]. Ionomycin, A23187, PMA, and reactive oxygen species (ROS) also induce the acrosome reaction in human sperm, a process characterized by the release of hydrolytic enzymes from the head of sperm which is necessary for oocyte penetration and thus indispensable for the creation of all human life outside of a clinical setting (ICSI bypasses the need for oocyte penetration) [23,24]. Additionally, although an over-production of ROS, similar to Ca2+, may lead to deleterious effects including cell death/apoptosis, low levels of ROS have been shown to act as signaling molecules and ROS is significantly increased on or immediately following mouse oocyte activation [25,26].

Furthermore, the master metabolic regulator AMPK is critical for oocyte meiotic resumption and maturation (a process that precedes and is essential for oocyte activation), is found located across the entire acrosome in the head of human sperm, and is activated by increases in ROS and Ca2+ [27-29]. Ionomycin, A23187, ethanol, puromycin, mechanical force, electrical stimulation, anisomycin, mycophenolic acid, carvacrol, and PMA also induce AMPK activation, indicating that a common mechanism of action links chemically distinct compounds with the creation of human life [30-39]. This common mechanism of action likely centers on the induction of cellular stress, mediated by indirect increases in intracellular Ca2+, ROS, and/or the AMP(ADP)/ATP ratio, etc. as I originally proposed in 2016 [40]. Because the bacterial-derived antibiotics ionomycin and A23187 induce both the acrosome reaction in human sperm and human oocyte activation, producing normal, healthy children, it can be said that “non-human organisms have the power to create human life or the power to end life.” As explained below, the beneficial effects of cellular stress induction (i.e. a “shock”) crosses species boundaries and may indeed play a role in facilitating natural selection, a process that underlies and drives evolution.

A number of bacterial species residing within the genus Streptomyces have proven to be extremely important and medicinally valuable as approximately 70% of clinically useful antibiotics are derived from Streptomyces [41]. The antibiotics ionomycin and A23187 are naturally produced by Streptomyces conglobatus and Streptomyces chartreusensis, respectively [5,6]. Other important examples include the antibiotic tetracycline (produced by Streptomyces aureofaciens), the immunosuppressant rapamycin (produced by Streptomyces hygroscopicus), and the anti-helminthic avermectins (produced by Streptomyces avermitilis) [42]. Many soil and aquatic-dwelling species of Streptomyces can be found in harsh environments and are characterized by a unique life cycle, including spore germination followed by vegetative mycelium production, aerial hyphae formation, sporulation (i.e. spore formation), and antibiotic production [43,44]. Curiously, just as cellular stress induction leads to the creation of human life and other beneficial effects in human cells (see below), stress induction also promotes the induction of aerial hyphae formation, sporulation, and antibiotic production in many Streptomyces species (spp.). Indeed, a decrease in the levels of ATP and bacterial growth is associated with sporulation, aerial hyphae formation, and antibiotic production [42,45]. A reduction in glucose/nutritional deprivation, the preferred sugar/carbon source for many Streptomyces spp., also significantly increases antibiotic production [46]. An increase in intracellular ROS and Ca2+ is associated with spore germination, aerial hyphae formation, and antibiotic production [47-49]. Other cellular stressors, including heat shock and ethanol, also significantly increase antibiotic production, provocatively indicating that the effects of cellular stress crosses species boundaries, enhancing bacterial survival and facilitating the creation of human life [50,51].

The beneficial effects of low-level cellular stress induction also extends to plants, as many plants produce secondary metabolites partly for the purpose of self-defense, analogous to antibiotics. Similar to the harsh, stressful environments often inhabited by Streptomyces spp., the Great Basin Bristlecone Pine (Pinus Longaeva), considered the oldest living non-clonal organism on the planet ( >5000 years old), thrives in an exceptionally harsh environment, characterized by increased elevations and exposure to UV radiation, nutritionally-deprived soils, harsh temperatures, and mechanical stress due to wind variances, leading early researchers to conclude that it’s longevity is intimately associated with adversity [52-54]. Conversely, Pinus Longaeva species that are located in less stressful environments (i.e. lower elevations) are strongly associated with younger age classes (<875 years) [55].  Similarly, the Creosote bush (Larrea tridentate), considered one of the oldest living clonal organisms on the planet (>11,000 years old), also thrives in harsh environments including the Mohave Desert [56]. AMPK, which increases lifespan and healthspan in several model organisms, is the primary sensor of cellular stress in eukaryotic organisms (e.g. plants and humans) and the plant AMPK orthologue SnRK1 as well as Ca2+ and ROS are critical for seed germination, fertilization, root gravitropism, and secondary metabolite production [57-64]. The secondary plant metabolites PMA (which activates mouse oocytes and promotes the acrosome reaction in human sperm) and artemisinin (an anti-malarial drug) both activate AMPK and the antibiotic A23187 also increases production of the secondary metabolite resveratrol in grape cell cultures, again indicating that exposure to low-level stressors may promote extension of lifespan and initiate the creation of human life [17,23,39,65,66].

Organismal exposure to beneficial levels of stress may also play a critical role in evolution. As first noted by Charles Darwin, evolution is driven by natural selection, a process characterized by environmentally-induced phenotypic changes that may lead to inheritable survival and reproductive advantages [67]. From “On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life”, Darwin explained that “if there be, owing to the high geometrical powers of increase of each species, at some age, season, or year, a severe struggle for life, and this certainly cannot be disputed;……But if variations useful to any organic being do occur, assuredly individuals thus characterised will have the best chance of being preserved in the struggle for life;” [67].  This “struggle for life” Darwin spoke of is embodied by selective pressures which may be abiotic (i.e. light, wind, temperature, etc.) or biotic (predation, disease, competition, etc.) [68,69]. As alluded to above, such selective pressures are indeed sources of cellular stress, sensed by both prokaryotes and eukaryotes, that induce beneficial responses (at appropriate levels), leading to the acquisition of phenotypes conducive for continued survival. Both biotic (e.g. infection) and abiotic (e.g. heat) stressors/selective pressures activate AMPK (which is evolutionarily conserved among eukaryotes) in human cells [70,71]. A phenomenon often cited as an example of natural selection on a readily observable timescale is the development of bacterial resistance to antibiotics, resulting in problematic mutant strains that may be life-threatening for some individuals (i.e. the elderly and immunocompromised) [72]. Intriguingly, lethal levels of bactericidal antibiotics have been shown to kill microorganisms via the induction of ROS, sub-lethal levels of bactericidal antibiotics however increase mutagenesis and bacterial resistance via induction of lower levels of ROS, and heat as well as nutritional stress increase bacterial resistance to antibiotics, providing compelling evidence that continuous exposure to low levels of stress likely plays a significant role in natural selection and evolution [73-75].

Moreover, gravity itself likely functions as a cellular stressor/selective pressure that has influenced the development of organisms on Earth since the emergence of the very first lifeform. Gravity exerts its effects on living organisms via the application of force, which is experienced by human cells in the form of mechanical loading or stress [76]. The application of force or a mechanical load has recently been shown to activate AMPK and simulated microgravity (i.e. hind limb unloading in mice) significantly decreases AMPK activation [77,78]. Spaceflight also inhibits the activation of T cells (immune cells essential for adaptive immunity), whereas the application of force and AMPK activation promote T cell activation [79-81]. Interestingly, spaceflight has recently been shown to decrease the levels of the master antioxidant transcription factor Nrf2 and the heat shock-inducible protein HSP90a but increase the levels of the growth-promoting kinase mTOR in mice [82]. AMPK however inhibits mTOR but increases the phosphorylation, nuclear retention, and transcriptional activity of Nrf2 [57,83,84]. Also, HSP90 interacts with and maintains AMPK activity and HSP90 is necessary for progesterone-induced human sperm acrosome reaction [85,86]. Interestingly, rapamycin, an immunosuppressant produced by Streptomyces hygroscopicus, extends lifespan in genetically heterogeneous mice, activates AMPK in vivo in normal aged mice, and increases human sperm motility [42,87,88]. Simulated microgravity via the use of NASA-designed rotating wall vessels (RWVs) however drastically reduces rapamycin production (~90%) whereas the antibiotic gentamycin increases rapamycin production by Streptomyces hygroscopicus, providing further evidence that cellular stress, in the form of mechanical loading induced by gravity, is essential for development, function, and survival of Earth-bound organisms [89,90].

The induction of cellular stress also links seemingly dissimilar physiological and pathological states with the activation of AMPK. As discussed above, both ionomycin and ROS activate AMPK and promote oocyte meiotic resumption, a process that is AMPK-dependent and is essential for efficient oocyte activation [27,30,91]. ROS is also critical for ovulation, PMA and ionomycin both activate mouse oocytes, and ionomycin is extensively used during ICSI procedures, creating normal healthy children, suggesting that cellular stress-induced AMPK activation is also essential for oocyte activation [3,4,12,17,92]. The activation of oocytes and T cells share strikingly similar intracellular signaling mechanisms (e.g. PLC-PIP2-DAG-PKC-IP3-Ca2+) and ionomycin combined with PMA are extremely effective in activating T cells and are often used as positive controls in HIV-1 latency reversal studies [93-95]. Reactivating latent/dormant HIV-1 in CD4+ T cells, potentially facilitating immune system detection and virus destruction, is currently being pursued as a method for the potential eradication of HIV-1 (called the “shock and kill” approach) [96]. Similar to oocyte activation, both Ca2+ and ROS are critical for T cell activation (and hence latent HIV-1 reactivation) and other cellular stress-inducing compounds, including NDGA derived from the Creosote bush, butyrate derived from bacteria, as well as ROS and HSP90 have been shown to reactivate latent HIV-1 [26,93,94,97-101]. Interestingly,  AMPK inhibition leads to cell death on T cell activation, knockdown of AMPK significantly decreases HIV-1 replication, and metformin (a well-studied AMPK activator derived from the French Lilac plant) increases butyrate production in human diabetic patients [81,102,103]. Perhaps most convincingly, early preliminary data showed that metformin significantly reduced several markers preferentially associated with cells latently infected with HIV-1 (e.g. PD-1, TIGIT, TIM-3) and also destabilized the latent HIV-1 reservoir in chronically-infected HIV patients, indicating that cellular-stress induced AMPK activation likely links the creation of human life with the potential eradication of HIV-1, as I originally proposed in 2016 [40,104,105].

AMPK activation may also link the disparate disease states of HIV-1 latency and Hutchinson-Gilford progeria syndrome (HGPS). HGPS is a genetic disorder caused by aberrant alternative splicing of the LMNA gene, generating a toxic protein called progerin that induces an accelerated aging phenotype and premature death at approximately 14 years of age [106]. Excessive activity of the gene splicing factor SRSF1 has been shown to prevent reactivation of latent HIV-1 and contribute to aberrant splicing of the LMNA gene in HGPS [107-109]. Metformin however has recently been shown to ameliorate the accelerated aging phenotype in cells derived from children with HGPS by reducing the levels of both SRSF1 and progerin and activating AMPK, as I first proposed in 2014 [110-112]. Interestingly, both Ca2+ and ROS induce autophagy (a process of disposing of damaged/toxic proteins and organelles) and rapamycin, which activates AMPK in vivo and increases intracellular Ca2+ levels, improves accelerated aging in progeria cells by inducing autophagic degradation of progerin [87,113-116]. Temsirolimus, an analog of rapamycin, also alleviated accelerated aging defects in progeria cells but also increased the levels of ROS and superoxide within the first hour of treatment [117]. Such evidence strongly suggests that cellular stress-induced AMPK activation links the reversal of HIV-1 latency and alleviation of accelerated cellular aging defects in HGPS.

Cellular stress-induced AMPK activation also links the potential elimination of cancer stem cells (CSCs) with HIV-1 latency reversal and viral eradication. CSCs, which are largely resistant to chemoradiation therapy, are a subpopulation of cancer cells that exhibit characteristics similar to embryonic stem cells (ESCs), including self-renewal, multi-lineage differentiation, & the ability to initiate tumorigenesis [118,119]. Mechanisms that sustain quiescence & promote self-renewal in adult stem cells (ASCs) & CSCs likely also function to maintain latency of HIV-1 in CD4+ memory T cells. Indeed, HIV-1 has been found to establish long-lasting latency in a recently discovered subset of CD4+ T cells that exhibit stem cell-like properties known as T memory stem (TSCM) cells and increases in Ca2+, ROS, and AMPK activation have been shown to promote T cell activation and ESC, ASC, and CSC differentiation [119,120].  Additionally, A23187 and PMA have been shown to promote CSC differentiation (causing CSCs to become more susceptible to chemoradiation) and metformin induces CSC differentiation and/or apoptosis in an AMPK-dependent manner in the deadliest of cancers, including glioblastoma and pancreatic cancer, providing support for my publication in 2017 in which I first proposed that CSC differentiation and/or apoptosis and HIV-1 latency reversal/viral eradication may be linked by cellular stress-induced AMPK activation [119,121-124].

In conclusion, the ability of non-human organisms including certain Streptomyces spp. to initiate the creation of human life is predicated on the induction of cellular stress, mediated by increases in intracellular ROS, Ca2+, AMP(ADP)/ATP ratio increase, etc.  The beneficial effects of transient cellular stress induction, which may be likened to selective pressures, crosses species boundaries and may indeed play a role in facilitating natural selection, a process that underlies and drives evolution, as evidenced by stress-induced increases in antibiotic production by Streptomyces spp. and stress-induced mutagenesis and antibiotic resistance in various bacterial strains. Because AMPK, a primary sensor of cellular stress in eukaryotic cells that increases lifespan and healthspan, plays a critical role in oocyte meiotic resumption/maturation, T cell activation, and stem cell differentiation, the creation of human life, the potential eradication of HIV-1, amelioration of accelerated aging in HGPS cells, and CSC differentiation/apoptosis are likely linked by a “Shock to Live”, or a “Shock to Kill”. 

https://www.linkedin.com/pulse/antibiotics-produced-bacteria-activate-human-oocytes-creating-finley/

References:

1. Murugesu S, Saso S, Jones BP, et al. Does the use of calcium ionophore during artificial oocyte activation demonstrate an effect on pregnancy rate? A meta-analysis. Fertil Steril. 2017 Sep;108(3):468-482.e3.
2. Tesarik J, Sousa M, Testart J. Human oocyte activation after intracytoplasmic sperm injection. Hum Reprod. 1994 Mar;9(3):511-8.
3. 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.
4. Eftekhar M, Janati S, Rahsepar M, Aflatoonian A. Effect of oocyte activation with calcium ionophore on ICSI outcomes in teratospermia: A randomized clinical trial. Iran J Reprod Med. 2013 Nov;11(11):875-82.
5. 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.
6. Reed PW, Lardy HA. A23187: a divalent cation ionophore. J Biol Chem. 1972 Nov 10;247(21):6970-7.
7. Zhang Z, Wang T, Hao Y, et al. Effects of trehalose vitrification and artificial oocyte activation on the development competence of human immature oocytes. Cryobiology. 2017 Feb;74:43-49.
8. De Sutter P, Dozortsev D, Cieslak J, Wolf G, Verlinsky Y, Dyban A. Parthenogenetic activation of human oocytes by puromycin. J Assist Reprod Genet. 1992 Aug;9(4):328-37.
9. Sankaran L, Pogell BM. Biosynthesis of puromycin in Streptomyces alboniger: regulation and properties of O-demethylpuromycin O-methyltransferase. Antimicrob Agents Chemother. 1975 Dec;8(6):721-32.
10. Tesarik J, Rienzi L, Ubaldi F, Mendoza C, Greco E. Use of a modified intracytoplasmic sperm injection technique to overcome sperm-borne and oocyte-borne oocyte activation failures. Fertil Steril. 2002 Sep;78(3):619-24.
11. Mansour R, Fahmy I, Tawab NA, et al. Electrical activation of oocytes after intracytoplasmic sperm injection: a controlled randomized study. Fertil Steril. 2009 Jan;91(1):133-9.
12. Nikiforaki D, Vanden Meerschaut F, de Roo C, et al. Effect of two assisted oocyte activation protocols used to overcome fertilization failure on the activation potential and calcium releasing pattern. Fertil Steril. 2016 Mar;105(3):798-806.e2.
13. Liu H1, Zhang J, Krey LC, Grifo JA. In-vitro development of mouse zygotes following reconstruction by sequential transfer of germinal vesicles and haploid pronuclei. Hum Reprod. 2000 Sep;15(9):1997-2002.
14. Downs SM. Stimulation of parthenogenesis in mouse ovarian follicles by inhibitors of inosine monophosphate dehydrogenase. Biol Reprod. 1990 Sep;43(3):427-36.
15. Siracusa G, Whittingham DG, Molinaro M, Vivarelli E. Parthenogenetic activation of mouse oocytes induced by inhibitors of protein synthesis. J Embryol Exp Morphol. 1978 Feb;43:157-66.
16. Carvacho I, Lee HC, Fissore RA, Clapham DE. TRPV3 channels mediate strontium-induced mouse-egg activation. Cell Rep. 2013 Dec 12;5(5):1375-86.
17. Colonna R, Tatone C, Malgaroli A, Eusebi F, Mangia F. Effects of protein kinase C stimulation and free Ca2+ rise in mammalian egg activation. Gamete Res. 1989 Oct;24(2):171-83.
18. Tang Z, Xing F, Chen D, et al. In vivo toxicological evaluation of Anisomycin. Toxicol Lett. 2012 Jan 5;208(1):1-11.
19. Regueira TB, Kildegaard KR, Hansen BG, Mortensen UH, Hertweck C, Nielsen J. Molecular basis for mycophenolic acid biosynthesis in Penicillium brevicompactum. Appl Environ Microbiol. 2011 May;77(9):3035-43.
20. K'ominek LA. Cycloheximide production by Streptomyces griseus: control mechanisms of cycloheximide biosynthesis. Antimicrob Agents Chemother. 1975 Jun;7(6):856-6.
21. Ultee A, Slump RA, Steging G, Smid EJ. Antimicrobial activity of carvacrol toward Bacillus cereus on rice. J Food Prot. 2000 May;63(5):620-4.
22. Pal PK, Nandi MK, Singh NK. Detoxification of Croton tiglium L. seeds by Ayurvedic process of Śodhana. Anc Sci Life. 2014 Jan;33(3):157-61.
23. Rotem R, Paz GF, Homonnai ZT, et al. Ca(2+)-independent induction of acrosome reaction by protein kinase C in human sperm. Endocrinology. 1992 Nov;131(5):2235-43.
24. 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.
25. Görlach A, Bertram K, Hudecova S, Krizanova O. Calcium and ROS: A mutual interplay. Redox Biol. 2015 Dec;6:260-71.
26. Nasr-Esfahani MM, Johnson MH. The origin of reactive oxygen species in mouse embryos cultured in vitro. Development. 1991 Oct;113(2):551-60.
27. LaRosa C, Downs SM. Stress stimulates AMP-activated protein kinase and meiotic resumption in mouse oocytes. Biol Reprod. 2006 Mar;74(3):585-92.
28. 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.
29. Mungai PT, Waypa GB, Jairaman A, et al. Hypoxia triggers AMPK activation through reactive oxygen species-mediated activation of calcium release-activated calcium channels. Mol Cell Biol. 2011 Sep;31(17):3531-45.
30. Tamas 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;203(7):1665–70.
31. Hawley SA, Pan DA, Mustard KJ, et al. Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase.  Cell Metab. 2005 Jul;2(1):9-19.
32. Nammi S, Roufogalis BD. Light-to-moderate ethanol feeding augments AMPK-α phosphorylation and attenuates SREBP-1 expression in the liver of rats. J Pharm Pharm Sci. 2013;16(2):342-51.
33. Koh W, Jeong SJ, Lee HJ, et al. Melatonin promotes puromycin-induced apoptosis with activation of caspase-3 and 5'-adenosine monophosphate-activated kinase-alpha in human leukemia HL-60 cells. J Pineal Res. 2011 May;50(4):367-73.
34. Bays JL, Campbell HK, Heidema C, Sebbagh M, DeMali KA. Linking E-cadherin mechanotransduction to cell metabolism through force-mediated activation of AMPK. Nat Cell Biol. 2017 Jun;19(6):724-731.
35. Hutber CA, Hardie DG, Winder WW. Electrical stimulation inactivates muscle acetyl-CoA carboxylase and increases AMP-activated protein kinase. Am J Physiol. 1997 Feb;272(2 Pt 1):E262-6.
36. Ohsaka Y, Nishino H, Nomura Y. Induction of phospho-Thr-172 AMPK in 3T3-L1 adipocytes exposed to cold or treated with anisomycin, mithramycin A, and ionic compounds. Cryo Letters. 2010 May-Jun;31(3):218-29.
37. Fernández-Ramos AA, Marchetti-Laurent C, Poindessous V, et al. A comprehensive characterization of the impact of mycophenolic acid on the metabolism of Jurkat T cells. Sci Rep. 2017 Sep 5;7(1):10550.
38. Kim E, Choi Y, Jang J, Park T. Carvacrol Protects against Hepatic Steatosis in Mice Fed a High-Fat Diet by Enhancing SIRT1-AMPK Signaling. Evid Based Complement Alternat Med. 2013;2013:290104.
39. Zogovic N, Tovilovic-Kovacevic G, Misirkic-Marjanovic M, et al. Coordinated activation of AMP-activated protein kinase, extracellular signal-regulated kinase, and autophagy regulates phorbol myristate acetate-induced differentiation of SH-SY5Y neuroblastoma cells. J Neurochem. 2015 Apr;133(2):223-32.
40. 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.
41. Kitani S1, Miyamoto KT, Takamatsu S, et al. Avenolide, a Streptomyces hormone controlling antibiotic production in Streptomyces avermitilis. Proc Natl Acad Sci U S A. 2011 Sep 27;108(39):16410-5.
42. Challis GL, Hopwood DA. Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species. Proc Natl Acad Sci U S A. 2003 Nov 25;100 Suppl 2:14555-61.
43. Yagüe P, López-García MT, Rioseras B, Sánchez J, Manteca A. Pre-sporulation stages of Streptomyces differentiation: state-of-the-art and future perspectives. FEMS Microbiol Lett. 2013 May;342(2):79-88.
44. Seipke RF, Kaltenpoth M, Hutchings MI. Streptomyces as symbionts: an emerging and widespread theme? FEMS Microbiol Rev. 2012 Jul;36(4):862-76.
45. Meng L, Li M, Yang SH, Kim TJ, Suh JW. Intracellular ATP levels affect secondary metabolite production in Streptomyces spp. Biosci Biotechnol Biochem. 2011;75(8):1576-81.
46. Sánchez S, Chávez A, Forero A, et al. Carbon source regulation of antibiotic production. J Antibiot (Tokyo). 2010 Aug;63(8):442-59.
47. Wang SL, Fan KQ, Yang X, Lin ZX, Xu XP, Yang KQ. CabC, an EF-hand calcium-binding protein, is involved in Ca2+-mediated regulation of spore germination and aerial hypha formation in Streptomyces coelicolor. J Bacteriol. 2008 Jun;190(11):4061-8.
48. Wang D, Wei L, Zhang Y, Zhang M, Gu S. Physicochemical and microbial responses of Streptomyces natalensis HW-2 to fungal elicitor. Appl Microbiol Biotechnol. 2017 Jul 28. doi: 10.1007/s00253-017-8440-0. [Epub ahead of print].
49. Wei ZH, Bai L, Deng Z, Zhong JJ. Enhanced production of validamycin A by H2O2-induced reactive oxygen species in fermentation of Streptomyces hygroscopicus 5008. Bioresour Technol. 2011 Jan;102(2):1783-7.
50. Doull JL, Ayer SW, Singh AK, Thibault P. Production of a novel polyketide antibiotic, jadomycin B, by Streptomyces venezuelae following heat shock. J Antibiot (Tokyo). 1993 May;46(5):869-71.
51. Zhou WW1, Ma B, Tang YJ, Zhong JJ, Zheng X. Enhancement of validamycin A production by addition of ethanol in fermentation of Streptomyces hygroscopicus 5008. Bioresour Technol. 2012 Jun;114:616-21.
52. Flanary BE, Kletetschka G. Analysis of telomere length and telomerase activity in tree species of various lifespans, and with age in the bristlecone pine Pinus longaeva. Rejuvenation Res. 2006 Spring;9(1):61-3.
53. R. S. BeasleyJ. O. Klemmedson. Recognizing site adversity and drought-sensitive trees in stands of bristlecone pine (Pinus longaeva). January 1973, Volume 27, Issue 1, pp 141–146. doi: 10.1007/BF02862228.
54. Schulman E. Longevity under Adversity in Conifers. Science. 1954 Mar 26;119(3091):396-9.
55. Hiebert, R. D.; Hamrick, J. L. 1984. An ecological study of bristlecone pine (Pinus longaeva) in Utah and eastern Nevada. The Great Basin Naturalist. 44(3): 487-494.
56. Jorquera MA, Shaharoona B, Nadeem SM, de la Luz Mora M, Crowley DE. Plant growth-promoting rhizobacteria associated with ancient clones of creosote bush (Larrea tridentata). Microb Ecol. 2012 Nov;64(4):1008-17.
57. Salminen A, Kaarniranta K. AMP-activated protein kinase (AMPK) controls the aging process via an integrated signaling network. Ageing Res Rev. 2012 Apr;11(2):230-41.
58. Leymarie J, Vitkauskaité G, Hoang HH, et al. Role of reactive oxygen species in the regulation of Arabidopsis seed dormancy. Plant Cell Physiol. 2012 Jan;53(1):96-106.
59. Pang X, Halaly T, Crane O, et al.  Involvement of calcium signalling in dormancy release of grape buds. J Exp Bot. 2007;58(12):3249-62.
60. Duan Q, Kita D, Johnson EA, et al. Reactive oxygen species mediate pollen tube rupture to release sperm for fertilization in Arabidopsis. Nat Commun. 2014;5:3129.
61. Gao XQ, Liu CZ, Li DD, et al. The Arabidopsis KINβγ Subunit of the SnRK1 Complex Regulates Pollen Hydration on the Stigma by Mediating the Level of Reactive Oxygen Species in Pollen. PLoS Genet. 2016 Jul 29;12(7):e1006228.
62. Joo JH, Bae YS, Lee JS. Role of auxin-induced reactive oxygen species in root gravitropism. Plant Physiol. 2001 Jul;126(3):1055-60.
63. Lee JS, Mulkey TJ, Evans ML. Reversible loss of gravitropic sensitivity in maize roots after tip application of calcium chelators. Science. 1983 Jun 24;220(4604):1375-6.
64. Zhao J, Davis LC, Verpoorte R. Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv. 2005 Jun;23(4):283-333.
65. Wang H, Sharma L, Lu J, Finch P, Fletcher S, Prochownik EV. Structurally diverse c-Myc inhibitors share a common mechanism of action involving ATP depletion. Oncotarget. 2015 Jun 30;6(18):15857-70.
66. K.V. Kiselev, O.A. Shumakova, A.Y. Manyakhin, A.N. Mazeika. Influence of calcium influx induced by the calcium ionophore, A23187, on resveratrol content and the expression of CDPK and STS genes in the cell cultures of Vitis amurensis. Plant Growth Regulation. December 2012, Volume 68, Issue 3, pp 371–381. DOI: 10.1007/s10725-012-9725-z.
67. Darwin, Charles (1859). On the origin of species by means of natural selection, or the preservation of favoured races in the struggle for life. London: Murray. [1st ed.].
68. Sobral M, Veiga T, Domínguez P, Guitián JA, Guitián P, Guitián JM. Selective Pressures Explain Differences in Flower Color among Gentiana lutea Populations. PLoS One. 2015 Jul 14;10(7):e0132522.
69. Lefebvre V, Kiani SP, Durand-Tardif M. A focus on natural variation for abiotic constraints response in the model species Arabidopsis thaliana. Int J Mol Sci. 2009 Aug 13;10(8):3547-82.
70. Moser TS, Schieffer D, Cherry S. AMP-activated kinase restricts Rift Valley fever virus infection by inhibiting fatty acid synthesis. PLoS Pathog. 2012;8(4):e1002661.
71. Goto A, Egawa T, Sakon I, et al. Heat stress acutely activates insulin-independent glucose transport and 5'-AMP-activated protein kinase prior to an increase in HSP72 protein in rat skeletal muscle. Physiol Rep. 2015 Nov;3(11). pii: e12601.
72. Maclean RC, Hall AR, Perron GG, Buckling A. The evolution of antibiotic resistance: insight into the roles of molecular mechanisms of resistance and treatment context. Discov Med. 2010 Aug;10(51):112-8.
73. Dwyer DJ, Belenky PA, Yang JH, et al. Antibiotics induce redox-related physiological alterations as part of their lethality. Proc Natl Acad Sci U S A. 2014 May 20;111(20):E2100-9.
74. Kohanski MA, DePristo MA, Collins JJ. Sublethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis. Mol Cell. 2010 Feb 12;37(3):311-20.
75. Poole K. Bacterial stress responses as determinants of antimicrobial resistance. J Antimicrob Chemother. 2012 Sep;67(9):2069-89.
76. Ito M, Arakawa T, Okayama M, Shitara A, Mizoguchi I, Takuma T. Gravity loading induces adenosine triphosphate release and phosphorylation of extracellular signal-regulated kinases in human periodontal ligament cells. J Investig Clin Dent. 2014 Nov;5(4):266-74.
77. Bays JL, Campbell HK, Heidema C, Sebbagh M, DeMali KA. Linking E-cadherin mechanotransduction to cell metabolism through force-mediated activation of AMPK. Nat Cell Biol. 2017 Jun;19(6):724-731.
78. Zhong G, Li Y, Li H, et al. Simulated Microgravity and Recovery-Induced Remodeling of the Left and Right Ventricle. Front Physiol. 2016 Jun 29;7:274.
79. Chang TT, Walther I, Li CF, et al. The Rel/NF-jB pathway and transcription of immediate early genes in T cell activation are inhibited by microgravity. J Leukoc Biol 2012;92(6):1133–45.
80. Li YC, Chen BM, Wu PC, et al. Cutting Edge: mechanical forces acting on T cells immobilized via the TCR complex can trigger TCR signaling. J Immunol 2010;184(11):5959–63.
81. Ouyang Z, Wang X, Meng Q, et al. Suppression of adenosine monophosphate-activated protein kinase selectively triggers apoptosis in activated T cells and ameliorates immune diseases. Biochem Biophys Res Commun. 2017 May 27;487(2):223-229.
82. Blaber EA, Pecaut MJ, Jonscher KR. Spaceflight Activates Autophagy Programs and the Proteasome in Mouse Liver. Int J Mol Sci. 2017 Sep 27;18(10). pii: E2062.
83. Mo C, Wang L, Zhang J, et al. The crosstalk between Nrf2 and AMPK signal pathways is important for the anti-inflammatory effect of berberine in LPS-stimulated macrophages and endotoxin-shocked mice. Antioxid Redox Signal. 2014 Feb 1;20(4):574-88.
84. Joo MS, Kim WD, Lee KY, Kim JH, Koo JH, Kim SG. AMPK facilitates nuclear accumulation of Nrf2 by phosphorylating at serine 550. Mol Cell Biol. 2016 Jun 29;36(14):1931-42.
85. Zhang L, Yi Y, Guo Q, et al. Hsp90 interacts with AMPK and mediates acetyl-CoA carboxylase phosphorylation. Cell Signal. 2012 Apr;24(4):859-65.
86. Sagare-Patil V, Bhilawadikar R, Galvankar M, Zaveri K, Hinduja I, Modi D. Progesterone requires heat shock protein 90 (HSP90) in human sperm to regulate motility and acrosome reaction. J Assist Reprod Genet. 2017 Apr;34(4):495-503.
87. Lesniewski LA, Seals DR4, Walker AE, et al. Dietary rapamycin supplementation reverses age-related vascular dysfunction and oxidative stress, while modulating nutrient-sensing, cell cycle, and senescence pathways. Aging Cell. 2017 Feb;16(1):17-26.
88. Aparicio IM, Espino J, Bejarano I, et al. Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility. Sci Rep. 2016 Sep 16;6:33647.
89. Fang A, Pierson DL, Mishra SK, Demain AL. Growth of Steptomyces hygroscopicus in rotating-wall bioreactor under simulated microgravity inhibits rapamycin production. Appl Microbiol Biotechnol. 2000 Jul;54(1):33-6.
90. Cheng YR, Huang J, Qiang H, Lin WL, Demain AL. Mutagenesis of the rapamycin producer Streptomyces hygroscopicus FC904. J Antibiot (Tokyo). 2001 Nov;54(11):967-72.
91. Heindryckx B, Lierman S, Combelles CM, Cuvelier CA, Gerris J, De Sutter P. Aberrant spindle structures responsible for recurrent human metaphase I oocyte arrest with attempts to induce meiosis artificially. Hum Reprod. 2011 Apr;26(4):791-800.
92. Shkolnik K, Tadmor A, Ben-Dor S, Nevo N, Galiani D, Dekel N. Reactive oxygen species are indispensable in ovulation. Proc Natl Acad Sci U S A. 2011 Jan 25;108(4):1462-7.
93. Amdani SN, Jones C, Coward K. Phospholipase C zeta (PLC ζ): oocyte activation and clinical links to male factor infertility. Adv Biol Regul 2013;53(3):292–308.
94. Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol 2009;27:591–619.
95. 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.
96. Marsden MD, Zack JA. HIV/AIDS eradication. Bioorg Med Chem Lett 2013;23(14):4003–10.
97. Sena LA, Li S, Jairaman A, et al. Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling. Immunity. 2013 Feb 21;38(2):225-36.
98. Barquero AA, Dávola ME, Riva DA, Mersich SE, Alché LE. Naturally occurring compounds elicit HIV-1 replication in chronically infected promonocytic cells. Biomed Res Int. 2014;2014:989101.
99. Imai K, Ochiai K, Okamoto T. Reactivation of latent HIV-1 infection by the periodontopathic bacterium Porphyromonas gingivalis involves histone modification. J Immunol. 2009 Mar 15;182(6):3688-95.
100. Piette J, Legrand-Poels S. HIV-1 reactivation after an oxidative stress mediated by different reactive oxygen species. Chem Biol Interact. 1994 Jun;91(2-3):79-89.
101. Anderson I, Low JS, Weston S, et al. Heat shock protein 90 controls HIV-1 reactivation from latency. Proc Natl Acad Sci U S A. 2014 Apr 15;111(15):E1528-37.
102. Zhou H, Xu M, Huang Q, et al. Genome-scale RNAi screen for host factors required for HIV replication. Cell Host Microbe. 2008 Nov 13;4(5):495-504.
103. Wu H, Esteve E, Tremaroli V, et al. Metformin alters the gut microbiome of individuals with treatment-naive type 2 diabetes, contributing to the therapeutic effects of the drug. Nat Med. 2017 Jul;23(7):850-858.
104. 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.
105. http://viruseradication.com/supplement-details/Abstracts_of_the_IAS_HIV_Cure_and_Cancer_Forum_2017/
106. Ullrich NJ, Gordon LB. Hutchinson-Gilford progeria syndrome. Handb Clin Neurol. 2015;132:249-64.
107. 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;80(7):3189–204.
108. Paz S, Lu ML, Takata H, Trautmann L, Caputi M. SRSF1 RNA Recognition Motifs Are Strong Inhibitors of HIV-1 Replication. J Virol. 2015 Jun;89(12):6275-86.
109. 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.
110. Park SK, Shin OS. Metformin alleviates ageing cellular phenotypes in Hutchinson-Gilford progeria syndrome dermal fibroblasts. Exp Dermatol. 2017 Oct;26(10):889-895.
111. 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.
112. 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.
113. Decuypere JP, Welkenhuyzen K, Luyten T, et al. Ins(1,4,5)P3 receptor-mediated Ca2+ signaling and autophagy induction are interrelated. Autophagy. 2011 Dec;7(12):1472-89.
114. Cao Y, Fang Y, Cai J, et al. ROS functions as an upstream trigger for autophagy to drive hematopoietic stem cell differentiation. Hematology. 2016 Dec;21(10):613-618.
115. Decuypere JP, Kindt D, Luyten T, et al. mTOR-Controlled Autophagy Requires Intracellular Ca(2+) Signaling. PLoS One. 2013;8(4):e61020.
116. 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.
117. Gabriel D, Gordon LB, Djabali K. Temsirolimus Partially Rescues the Hutchinson-Gilford Progeria Cellular Phenotype. PLoS One. 2016 Dec 29;11(12):e0168988.
118. Moncharmont C, Levy A, Gilormini M, et al. Targeting a cornerstone of radiation resistance: cancer stem cell. Cancer Lett 2012;322(2):139–47.
119. 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.
120. Buzon MJ, Sun H, Li C, et al. HIV-1 persistence in CD4+ T cells with stem cell-like properties. Nat Med 2014;20(2):139–42.
121. Wee S, Niklasson M, Marinescu VD, et al. Selective calcium sensitivity in immature glioma cancer stem cells. PLoS One. 2014 Dec 22;9(12):e115698.
122. Hayun M, Okun E, Hayun R, Gafter U, et al. Synergistic effect of AS101 and Bryostatin-1 on myeloid leukemia cell differentiation in vitro and in an animal model. Leukemia 2007;21(7):1504–13.
123. Sato A, Sunayama J, Okada M, et al. Glioma-initiating cell elimination by metformin activation of FOXO3 via AMPK. Stem Cells Transl Med 2012;1(11):811–24.
124. Fasih A, Elbaz HA, Hüttemann M, Konski AA, Zielske SP. Radiosensitization of pancreatic cancer cells by metformin through the AMPK pathway. Radiat Res 2014;182(1):50–9.

AMPK activator metformin shown for the first time to destabilize latent HIV-1 reservoir in chronic HIV-1 patients & lower immune checkpoint PD-1

Goldsmith Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus [Public domain], via Wikimedia Commons; CC BY 2.5 (http://creativecommons.org/licenses/by/2.5)], via Wikimedia Commons; By NASA [Public domain], via Wikimedia

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 the anti-diabetic drug and AMPK activator metformin not only decreased the percentage of CD4+ T cells expressing the immune checkpoint receptors PD-1, TIGIT, and TIM-3 but also destabilized the viral reservoir in chronically-infected HIV-1 patients, indicating that metformin may indeed contribute to HIV-1 eradication by reactivating CD4+ T cells latently-infected with HIV-1 [1-3]. Current antiretroviral drugs selectively targets only replicating viruses capable of inducing viral gene expression in activated CD4+ T cells [4]. However, a method known as the “shock and kill” approach in HIV-1 cure research involves the reactivation of a small subset of primarily CD4+ memory T cells latently infected with HIV-1 (dormant) with various agents/drugs (i.e. “shock”) and is thought to facilitate virus-induced cell death or immune recognition and destruction of the virus (i.e. “kill”) [4]. Immune checkpoint receptors including PD-1 are considered markers of T cell exhaustion and have also previously been found to be positively associated with T cells that harbor latent HIV-1 [5,6].

As the inhibition of PD-1/PD-L1 significantly enhances CD8+ T cell-mediated immunological responses to both viruses and cancer cells, the finding that metformin decreases PD-1 and destabilizes the latent viral reservoir in chronically-infected HIV-1 patients provides additional evidence and supports several recent publications in which I proposed for the first time that AMPK, which is critical for T cell activation, links the reactivation of latent HIV-1 with the differentiation and/or apoptosis of cancer stem cells and the amelioration of accelerated cellular aging defects in Hutchinson-Gilford progeria syndrome (HGPS) [4,7-10]. Because metformin has recently been shown to improve accelerated aging defects in cells derived from HGPS patients, decrease PD-1 expression on T cells, and potently induce cancer stem cell differentiation and/or apoptosis (e.g. glioblastoma), AMPK activation may indeed lead to the inhibition of tumorigenesis, viral eradication, and mitigation of accelerated aging [1,11-13].
    
At the IAS HIV Cure and Cancer Forum, Chew et al. presented data from an open label, 24 week clinical trial in which metformin (500 mg increasing to 1000 mg at week 4) was administered to 12 HIV-1 positive patients who were on antiretroviral therapy for greater than a year with plasma HIV RNA less than 50 copies/ml [1]. A non-metformin treated observational arm was also included. Compared to the observational arm, metformin-treated patients exhibited a significant 24 week decease in the change of single positive PD-1+, dual expressing TIGIT+PD-1+, and triple expressing TIGIT+PD-1+TIM-3+ CD4 T cells [1]. Strikingly, although integrated HIV DNA (an indicator of HIV-1 latency) in T cells remained remarkably stable in the 3 subjects in the observation arm, a variance in integrated HIV DNA was observed in metformin-treated patients, indicating that the viral reservoir had been destabilized. Interestingly, the authors also noted a statistically significant decrease in CD32a in metformin-treated patients [1]. CD32a is a surface receptor protein in immune cells that has recently been demonstrated as a reliable marker of CD4+ T cell reservoirs in HIV-1 infected patients harboring replication-competent proviruses, providing compelling evidence that metformin-induced AMPK activation may promote viral eradication by inducing reactivation of latent HIV-1, as evidenced by reductions in PD-1, TIGIT, TIM-3, and CD32a, all markers of CD4+ T cell latent HIV-1 reservoirs [1,14].

In addition to inducing latent HIV-1 reactivation, metformin-induced AMPK activation may also mitigate T cell exhaustion and enhance the immunological response of cytotoxic CD8+ T cells to viruses and cancer cells. Indeed, PD-L1, a ligand that binds to the PD-1 receptor on T cells, has been found on breast and colon cancer stem cells and metformin, AICAR (an AMPK activator), and rapamycin (a macrolide that activates AMPK in vivo) have each been shown to decrease PD-L1 expression on human lung cancer cells [15-18]. Intriguingly, metformin has also been shown to improve intratumoral T cell function and tumor clearance in mice by potentiating PD-1 blockade and significantly increasing the number of activated CD8+ T cells [19].

Moreover, as AMPK is critical for T cell activation and AMPK inhibition during T cell activation leads to T cell death, recent studies indicating that AMPK activators decrease PD-1 expression on T cells and enhance CD8+ T cell-mediated immunological responses strongly suggests that AMPK represents a central node linking latent HIV-1 reactivation with T cell-mediated virus and cancer cell elimination (i.e. immunotherapy) [20]. Eikawa et al. for example showed that metformin enabled normal mice (but not T cell-deficient mice) to reject solid tumors by increasing the number of CD8+ tumor-infiltrating lymphocytes (TILs) and protecting them from exhaustion in an AMPK-dependent manner [21]. Perhaps most convincingly, Chamoto et al. demonstrated in a model using mouse colon cancer MC38 cells that PD-1 blockade (using a PD-L1 inhibitor) led to activation of mitochondria in tumor-reactive cytotoxic CD8+ T lymphocytes, evidenced by increased mitochondrial ROS (i.e. superoxide), larger mitochondrial mass, and higher mitochondrial membrane potential [22].

As mitochondria-derived ROS have previously been shown to be critical, if not indispensable for T cell activation, the combination of a PD-L1 inhibitor/mAb with Luperox (a ROS precursor) greatly enhanced the antitumor activity and survival of tumor-bearing mice [22,23]. Interestingly, the mitochondrial uncouplers FCCP and DNP also extended the survival time of PD-L1 mAb-treated animals, which was mitigated by the ROS scavenger MnTBAP [22]. As ROS have been independently shown to induce AMPK activation and AMPK is essential for T cell activation, the PD-L1 mAb alone induced AMPK activation and the combination of mitochondrial uncouplers with the PD-L1 mAb also further enhanced AMPK activation in CD8+ T cells, indicating that AMPK is essential for enhanced CD8+ T cell activation by PD-1 blockade, ROS generators, and mitochondrial uncouplers [4,22]. Indeed, the authors also showed that the AMPK activator A769662 further enhanced the antitumor activity by PD-L1 mAb treatment and improved animal survival. Additionally, oltipraz and bezafibrate, two compounds that also activate AMPK, strongly enhanced tumor-growth suppression and animal-survival by anti–PD-L1 treatment [22,24,25]. 

Such data, combined with the recent findings from the IAS HIV Cure and Cancer forum showing that metformin decreases the percentage of CD4+ T cells expressing PD-1, TIGIT, and TIM-3 and destabilizes the viral reservoir in chronically-infected HIV-1 patients, indicates that AMPK activation likely links the reactivation of latent HIV-1 with the facilitation of CD8+ T cell-mediated virus and cancer cell killing, potentially leading to HIV-1 eradication and cancer stem cell elimination, a hypothesis I initially proposed in May of 2017 [4].

Furthermore, 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 [10,26,27]. The induction of cellular stress (e.g. increases in ROS, intracellular Ca2+, and/or AMP(ADP)/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 [26,28,29]. Indeed, the 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 [29-31]. Such evidence further substantiates the novel and provocative assertion that AMPK activation links the amelioration of pathological cellular defects in Hutchinson-Gilford progeria syndrome with HIV-1 latency, adult and cancer stem cells, learning and memory, and the creation of all human life [4,8-10,32].

[Additional references for Figure above]: The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. Scaffidi P, Gordon L, Misteli T. PLoS Biology Vol. 3/11/2005, e395; By NASA [Public domain], via Wikimedia Commons.

https://www.linkedin.com/pulse/ampk-activator-metformin-shown-first-time-destabilize-finley

References

1. 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.
2. http://viruseradication.com/supplement-details/Abstracts_of_the_IAS_HIV_Cure_and_Cancer_Forum_2017/
3. http://www.iasociety.org/HIV-Programmes/Towards-an-HIV-Cure/Events/HIV-Cure-Cancer-Forum
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. 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.
6. 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.
7. Sakthivel P, Gereke M, Bruder D. Therapeutic intervention in cancer and chronic viral infections: antibody mediated manipulation of PD-1/PD-L1 interaction. Rev Recent Clin Trials. 2012 Feb;7(1):10-23.
8. 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.
9. 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.
10. 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.
11. Park SK, Shin OS. Metformin alleviates ageing cellular phenotypes in Hutchinson-Gilford progeria syndrome dermal fibroblasts. Exp Dermatol. 2017 Feb 13. doi: 10.1111/exd.13323. [Epub ahead of print].
12. 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.
13. Sato A, Sunayama J, Okada M, et al. Glioma-initiating cell elimination by metformin activation of FOXO3 via AMPK. Stem Cells Transl Med 2012;1(11):811–24.
14. Descours B, Petitjean G, López-Zaragoza JL, et al. CD32a is a marker of a CD4 T-cell HIV reservoir harbouring replication-competent proviruses. Nature. 2017 Mar 23;543(7646):564-567.
15. Wu Y, Chen M, Wu P, Chen C, Xu ZP, Gu W. Increased PD-L1 expression in breast and colon cancer stem cells. Clin Exp Pharmacol Physiol. 2017 May;44(5):602-604.
16. Kurimoto R, Iwasawa S, Ebata T, et al. Drug resistance originating from a TGF-β/FGF-2-driven epithelial-to-mesenchymal transition and its reversion in human lung adenocarcinoma cell lines harboring an EGFR mutation. Int J Oncol. 2016 May;48(5):1825-36.
17. Lastwika KJ, Wilson W 3rd, Li QK, et al. Control of PD-L1 Expression by Oncogenic Activation of the AKT-mTOR Pathway in Non-Small Cell Lung Cancer. Cancer Res. 2016 Jan 15;76(2):227-38.
18. 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.
19. Scharping NE, Menk AV, Whetstone RD, Zeng X, Delgoffe GM. Efficacy of PD-1 Blockade Is Potentiated by Metformin-Induced Reduction of Tumor Hypoxia. Cancer Immunol Res. 2017 Jan;5(1):9-16.
20. Rao E, Zhang Y, Zhu G, et al. Deficiency of AMPK in CD8+ T cells suppresses their anti-tumor function by inducing protein phosphatase-mediated cell death. Oncotarget 2015;6(10):7944–58.
21. Eikawa S, Nishida M, Mizukami S, Yamazaki C, Nakayama E, Udono H. Immune-mediated antitumor effect by type 2 diabetes drug, metformin. Proc Natl Acad Sci U S A. 2015 Feb 10;112(6):1809-14.
22. Chamoto K, Chowdhury PS, Kumar A, et al. Mitochondrial activation chemicals synergize with surface receptor PD-1 blockade for T cell-dependent antitumor activity. Proc Natl Acad Sci U S A. 2017 Jan 31;114(5):E761-E770.
23. Sena LA, Li S, Jairaman A, et al. Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling. Immunity. 2013 Feb 21;38(2):225-36.
24. Zhong X, Xiu LL, Wei GH, et al. Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation. Acta Pharmacol Sin. 2011 May;32(5):591-600.
25. Kim TH, Eom JS, Lee CG, Yang YM, Lee YS, Kim SG. An active metabolite of oltipraz (M2) increases mitochondrial fuel oxidation and inhibits lipogenesis in the liver by dually activating AMPK. Br J Pharmacol. 2013 Apr;168(7):1647-61.
26. LaRosa C, Downs SM. Stress stimulates AMP-activated protein kinase and meiotic resumption in mouse oocytes. Biol Reprod. 2006 Mar;74(3):585-92.
27. 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].
28. 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.
29. 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.
30. 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.
31. 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.
32. 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. Manuscript submitted.