AMPK activators Metformin & CHIR99021 improve gut bacteria in humans, Fragile X, & promote inner ear, dental pulp, & cancer stem cell differentiation

CC BY 2.5 (http://creativecommons.org/licenses/by/2.5)], via Wikimedia Commons; By Peter Saxon (Own work) [CC BY-SA 4.0 (http://creativecommons.org/licenses/by-sa/4.0)], via Wikimedia Commons; Rocky Mountain Laboratories, NIAID, NIH [Public domain]

A recent study published online in the journal Nature Medicine in May of 2017 presented startling evidence that the AMPK activator metformin, which has also recently been shown to alleviate accelerated aging defects in cells derived from Hutchinson-Gilford progeria (HGPS) patients, exerted a significant beneficial effect on the gut microbiome in humans in a randomized double-blind placebo controlled study [1-4]. In treatment-naïve patients with Type 2 diabetes (placebo: n = 18 or 1,700 mg/d of metformin: n = 22), a significant decrease in hemoglobin A1c (HbA1c) levels and fasting blood glucose was observed only in metformin-treated patients during the 4-month study period [1]. HbA1c levels and fasting blood glucose were also significantly reduced in a subset of placebo-treated patients that were switched to metformin after 6 months of treatment. Interestingly, whole-genome shotgun sequencing of fecal samples indicated that metformin treatment for 2 and 4 months significantly altered the relative abundance of 81 and 86 bacterial strains, respectively, with an increase in Bifidobacterium and Akkermansia muciniphila [1]. Metformin also directly promoted the growth of Bifidobacterium adolescentis and A. muciniphila in vitro, both of which have been associated with improved metabolic features in mice [1].

Importantly, fecal samples from humans that had been treated with metformin for 4 months and transferred to germ-free mice (via oral gavage) fed a high-fat diet improved glucose tolerance in mice compared to mice that received fecal samples from humans before treatment with metformin [1]. Intriguingly, metformin treatment was also linked to gene enrichment for bacterial environmental responses, including metabolism of the short-chain fatty acids and AMPK activators butyrate and propionate [1,5]. Fecal propionate and butyrate concentrations were significantly increased after 4 months of metformin treatment in men compared to the placebo group, indicating that metformin also beneficially modulates bacterial secondary metabolite production by inducing a bacterial stress response [1]. Moreover, several environmental stressors, including heat shock/stress, which activates AMPK in human cells, also promotes the production of various bacterial secondary metabolites, indicating that the mechanism of action by which metformin promotes an increase in beneficial human gut bacteria and release of bacterial secondary metabolites is via the induction of a stress response in bacteria [6,7].

Furthermore, the beneficial effects of the induction of a cellular stress response likely crosses species boundaries, as increases in calcium (Ca2+) and reactivate oxygen species (ROS) (mediators of cellular stress induction) also promotes seed germination, root gravitropism, and fertilization in plants [8-13]. Additionally, an increase in the AMP(ADP)/ATP ratio, intracellular Ca2+ increases, or an increase in the levels of ROS have been shown to activate the master metabolic regulator AMPK and promote the differentiation of embryonic, adult, and cancer stem cells [14-18]. Metformin and butyrate have also been shown to synergistically activate AMPK and decrease the cancer stem cell-like population in breast cancer cells, butyrate has been shown to induce pancreatic cancer stem cell differentiation, and metformin induces glioma stem cell differentiation and elimination in an AMPK-dependent manner, indicating that cellular stress-induced AMPK activation is a critical mediator linking cancer, embryonic, and adult stem cell differentiation, as proposed in my recent publication linking cancer stem cell differentiation and/or apoptosis with latent HIV-1 reactivation [19-21].

Indeed, a recent study published in the journal Cell Reports by researchers from Harvard Medical School and MIT showed that the glycogen synthase kinase 3β (GSK3β) inhibitor CHIR99021 (CHIR) and the histone deacetylase (HDAC) inhibitor valproic acid (VPA), both of which activate AMPK, significantly expanded cochlear supporting cells (i.e. “inner ear stem cells”) that expressed and maintained the epithelial stem cell marker Lgr5 [22-24]. Treatment with CHIR and VPA also led to the differentiation of Lgr5-expressing cells into hair cells in high yield, providing additional evidence that AMPK activation promotes differentiation of adult stem cells including inner ear stem cells, possibly leading to treatments for hearing loss [24]. Interestingly, the authors demonstrated in a previous study that CHIR and VPA also promoted the multilineage differentiation of Lgr5+ intestinal stem cells into mature enterocytes, goblet cells and Paneth cells [25]. AMPK activation has also been shown to improve gut epithelial differentiation and metformin increases goblet and Paneth cell differentiation from intestinal epithelial cells, further indicating that AMPK activation likely represents a common mechanism of action linking structurally dissimilar compounds that enhance inner ear and intestinal stem cell maintenance and differentiation [26,27].  

Moreover, a recently published study in the journal Scientific Reports in January of 2017 demonstrated that topical administration of GSK3β inhibitors including the AMPK activator CHIR led to the mobilization of resident mesenchymal stem cells in the tooth pulp that had been exposed via the drilling of holes in mice molars [28]. GSK3β inhibitor-induced stem cell mobilization promoted a natural process of reparative dentin (also spelled dentine) formation that completely restored dentin, leading the authors to conclude that stimulation of mesenchymal stem cell mobilization and differentiation into odontoblast-like cells may represent a novel approach to clinical tooth restoration [28]. AMPK activation has previously been shown to promote osteogenic (i.e. bone forming) differentiation of human adipose tissue-derived mesenchymal stem cells and metformin induces osteoblastic differentiation of human induced pluripotent stem cell-derived mesenchymal stem cells in an AMPK-dependent manner, providing further evidence that structurally diverse compounds including metformin and CHIR that promote adult stem cell differentiation likely do so via a common mechanism of AMPK activation [29,30].

The induction of cellular stress and AMPK activation may also link beneficial modulation of the gut microbiome in humans not only with adult stem cell maintenance and differentiation, but also with the amelioration of pathologies associated with neurological disorders. A study recently published in the journal Clinical Genetics in April of 2017 demonstrated for the first time that metformin consistently improved behavior in several patients diagnosed with Fragile X Syndrome (FXS), a genetic disorder characterized by intellectual disability and significant deficits in neurological function and cognitive development [31]. An improvement in behavior was documented in the Aberrant Behavior Checklist (ABC) for all cases, as evidenced by consistent improvements (i.e. lower scores compared to pre-metformin treatment) in social avoidance, irritability, hyperactivity, and social unresponsiveness as well as improvements in language and conversational skills reported by familial caretakers [31].

Also, metformin has been shown to rescue and restore memory deficits in a Drosophila model of FXS and a recently published study (2017) demonstrated that metformin corrected social novelty impairment, reduced testicular weight, decreased repetitive grooming, rescued excessive long-term depression and dendritic spine abnormalities, restored excitatory synaptic transmission, and acutely activated AMPK in hippocampal pyramidal neurons in an FXS mouse model [32,33]. Interestingly, GSK3β inhibitors including the AMPK activator CHIR have been shown to rescue deficits in long-term potentiation at medial perforant path-dentate granule cells synapses in an FXS mouse model, indicating that cellular stress-induced AMPK activation by metformin and CHIR links the beneficial effects of those compounds in phenomena as disparate as stem cell differentiation, FXS, and long-term potentiation, hypotheses that I initially proposed in 2017 [34-36].

Lastly, as butyrate has been shown to reactivate latent HIV-1, facilitating immune system detection and virus destruction, and metformin when combined with bryostatin-1 (which also activates AMPK) promotes latent HIV-1 reactivation, cellular stress-induced AMPK activation likely also links beneficial modulation of human gut bacteria with latent HIV-1 reactivation [37-39].

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 [40-42]. 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 [41,43,44]. 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 [44-46]. Such evidence indicates and further substantiates the novel and provocative assertion that AMPK activation links the amelioration of pathological cellular defects in FXS and Hutchinson-Gilford progeria syndrome with the gut microbiota, HIV-1 latency, adult and cancer stem cells, learning and memory, and the creation of all human life [4,35,36,39,40,47].

https://www.linkedin.com/pulse/ampk-activators-metformin-chir99021-improve-gut-bacteria-finley

References

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4. 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.
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6. Yoon V, Nodwell JR. Activating secondary metabolism with stress and chemicals. J Ind Microbiol Biotechnol. 2014 Feb;41(2):415-24.
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13. 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.
14. Hardie DG. AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function. Genes Dev. 2011 Sep 15;25(18):1895-908.
15. Sook SH, Lee HJ, Kim JH, et al. Reactive oxygen species-mediated activation of AMP-activated protein kinase and c-Jun N-terminal kinase plays a critical role in beta-sitosterol-induced apoptosis in multiple myeloma U266 cells. Phytother Res. 2014 Mar;28(3):387-94.
16. Ji AR, Ku SY, Cho MS, et al. Reactive oxygen species enhance differentiation of human embryonic stem cells into mesendodermal lineage. Exp Mol Med. 2010 Mar 31;42(3):175-86.
17. Sun S, Liu Y, Lipsky S, Cho M. Physical manipulation of calcium oscillations facilitates osteodifferentiation of human mesenchymal stem cells. FASEB J. 2007 May;21(7):1472-80.
18. Wee S, Niklasson M2, Marinescu VD, et al. Selective calcium sensitivity in immature glioma cancer stem cells. PLoS One. 2014 Dec 22;9(12):e115698.
19. Lee KM, Lee M, Lee J, et al. Enhanced anti-tumor activity and cytotoxic effect on cancer stem cell population of metformin-butyrate compared with metformin HCl in breast cancer. Oncotarget. 2016 Jun 21;7(25):38500-38512.
20. Lang D, Mascarenhas JB, Powell SK, Halegoua J, Nelson M, Ruggeri BA. PAX6 is expressed in pancreatic adenocarcinoma and is downregulated during induction of terminal differentiation. Mol Carcinog 2008;47(2):148–56.
21. 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.
22. Suzuki T, Bridges D, Nakada D, et al. Inhibition of AMPK catabolic action by GSK3. Mol Cell. 2013 May 9;50(3):407-19.
23. Avery LB, Bumpus NN. Valproic acid is a novel activator of AMP-activated protein kinase and decreases liver mass, hepatic fat accumulation, and serum glucose in obese mice. Mol Pharmacol. 2014 Jan;85(1):1-10.
24. McLean WJ, Yin X, Lu L, et al. Clonal Expansion of Lgr5-Positive Cells from Mammalian Cochlea and High-Purity Generation of Sensory Hair Cells. Cell Rep. 2017 Feb 21;18(8):1917-1929.
25. Yin X, Farin HF, van Es JH, et al. Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny. Nat Methods. 2014 Jan;11(1):106-12.
26. Sun X, Yang Q, Rogers CJ, Du M, Zhu MJ. AMPK improves gut epithelial differentiation and barrier function via regulating Cdx2 expression. Cell Death Differ. 2017 May;24(5):819-831.
27. Xue Y, Zhang H, Sun X, Zhu MJ. Metformin Improves Ileal Epithelial Barrier Function in Interleukin-10 Deficient Mice. PLoS One. 2016 Dec 21;11(12):e0168670.
28. Neves VC, Babb R, Chandrasekaran D, Sharpe PT. Promotion of natural tooth repair by small molecule GSK3 antagonists. Sci Rep. 2017 Jan 9;7:39654.
29. Kim EK, Lim S, Park JM, et al. Human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by AMP-activated protein kinase. J Cell Physiol. 2012 Apr;227(4):1680-7.
30. Wang P, Ma T, Guo D, et al. Metformin Induces Osteoblastic Differentiation of Human Induced Pluripotent Stem Cell-derived Mesenchymal Stem Cells. J Tissue Eng Regen Med. 2017 May 11. doi: 10.1002/term.2470. [Epub ahead of print].
31. Dy ABC, Tassone F, Eldeeb M, Salcedo-Arellano MJ, Tartaglia N, Hagerman R. Metformin as Targeted Treatment in Fragile X Syndrome. Clin Genet. 2017 Apr 24. doi: 10.1111/cge.13039. [Epub ahead of print].
32. Monyak RE, Emerson D, Schoenfeld BP, et al. Insulin signaling misregulation underlies circadian and cognitive deficits in a Drosophila fragile X model. Mol Psychiatry. 2017 Aug;22(8):1140-1148.
33. Gantois I, Khoutorsky A, Popic J, et al. Metformin ameliorates core deficits in a Fragile X syndrome mouse model. Nat Med. 2017 Jun;23(6):674-677.
34. Franklin AV, King MK, Palomo V, Martinez A, McMahon LL, Jope RS. Glycogen synthase kinase-3 inhibitors reverse deficits in long-term potentiation and cognition in fragile X mice. Biol Psychiatry. 2014 Feb 1;75(3):198-206.
35. 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.
36. 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.
37. Imai K, Ochiai K, Okamoto T. Reactivation of latent HIV-1 infection by the periodontopathic bacterium Porphyromonas gingivalis involves histone modification. J Immunol 2009;182(6):3688–95.
38. 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;5(6):e11160.
39. 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.
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. LaRosa C, Downs SM. Stress stimulates AMP-activated protein kinase and meiotic resumption in mouse oocytes. Biol Reprod. 2006 Mar;74(3):585-92.
42. 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].
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47. Finley J. AMPK activation as a common mechanism of action linking the effects of diverse compounds that ameliorate accelerated cellular aging defects in Hutchinson-Gilford progeria syndrome. Med Hypotheses. Manuscript submitted.

New study shows AMPK activator MG132 rescues Progeria cells, protects against Microgravity, & inhibits Cancer Stem Cells, HIV, Dengue, & Malaria

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; 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; By Thomas Splettstoesser (www.scistyle.com) [CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons.

A recent study published online in the journal EMBO Molecular Medicine in July of 2017 strikingly demonstrated that the proteasome inhibitor and AMPK activator MG132 alleviated accelerated aging defects in cells derived from children with the genetic disorder Hutchinson-Gilford progeria syndrome(HGPS) by inducing autophagic degradation of progerin, the toxic protein responsible for accelerated aging defects in HGPS cells [1]. MG132 also beneficially altered splicing of the LMNA gene (a gene that is aberrantly spliced to produce large amounts of progerin instead of the normal lamin A gene product) by decreasing the gene splicing factor SRSF1 but increasing the splicing factor SRSF5 [1]. Interestingly, progerin was shown to accumulate in structures known as promyelocytic nuclear bodies (PML-NBs) in the nucleus of HGPS cells and local injection of MG132 into a progeria mouse model also led to a reduction in the levels of SRSF1 and progerin [1]. Intriguingly, normal humans also produce progerin via the same aberrant gene splicing method as do children with HGPS, just at much lower levels that increase with age [2].

The recent finding that MG132-induced proteasome inhibition also results in a rapid activation of the master metabolic regulator AMPK (a kinase that increases lifespan and healthspan in several model organisms) and AMPK-dependent autophagy stimulation via the induction of cellular stress (i.e. reactive oxygen species [ROS] generation) further substantiates my hypothesis published in 2014 in which I was first to propose that AMPK activation by structurally diverse compounds (e.g. metformin, resveratrol, etc.) will lead to alleviation of accelerated aging defects in HGPS cells by decreasing the gene splicing factor SRSF1, thus beneficially altering splicing of the LMNA gene, as well as decreasing progerin levels by AMPK-induced autophagy [3,4,33].

Indeed, metformin, which induces cellular stress by mildly inhibiting complex I of the electron transport chain (thus increasing the AMP/ATP ratio) has also recently been shown to reduce the levels of SRSF1 and progerin and activate AMPK in HGPS cells [5-7]. Platelet-derived growth factor BB (PDGF-BB) also increases intracellular calcium (Ca2+) and ROS levels (mediators of cellular stress induction), activates AMPK, and increases SRSF5 in HGPS cells, thus altering splice site selection and beneficially increasing the lamin A/progerin ratio, providing compelling evidence that cellular stress-induced AMPK activation indeed represents a common mechanism of action for gene splicing- and autophagy-induced reductions of progerin in HGPS cells [8-10].

Interestingly, SRSF1 (also known as ASF/SF2) and PML-NBs also inhibit latent HIV-1 reactivation (preventing immune system detection and virus eradication) and SRSF5 (also known as SRp40) increases the abundance and translation of unspliced HIV-1 RNA, which is necessary for latent HIV-1 reactivation [11-13]. As AMPK promotes both latent HIV-1 reactivation and prevents HIV-1 transactivation, MG132 has been shown to reactivate latent HIV-1 and inhibit HIV-1 replication, substantiating my hypothesis published in 2015 in which I first proposed that AMPK activation links correction of aberrant alternative splicing in HGPS cells with reactivation of latent HIV-1 by compounds including MG132, metformin, and resveratrol [14-18].

Additionally, as the induction of cellular stress (e.g. intracellular Ca2+ increase, ROS generation, AMP/ATP ratio increase, etc.) activates AMPK, reactivates latent HIV-1, and leads to the differentiation and/or apoptosis of cancer stem cells in an AMPK-dependent manner, MG132 has also recently been found to induce apoptosis in glioma cancer stem cells, substantiating my most recent publication (2017) in which I propose for the first time that AMPK activation links reactivation of latent HIV-1 with cancer stem cell differentiation and/or apoptosis by diverse compounds that induce cellular stress including metformin and MG132 [5,19-23].

MG132 also induces mouse oocyte meiotic resumption (a process orchestrated by cellular stress-induced AMPK activation and is critical for efficient oocyte activation), delays in vitro oocyte aging, promotes embryonic development from aged oocytes after in vitro fertilization procedures, and alleviates deleterious effects associated with simulated microgravity, further supporting my hypotheses in 2016 and 2017 in which I first proposed that cellular stress-induced AMPK activation links oocyte activation (and hence the beginning of all human life) with latent HIV-1 reactivation and that AMPK activation will improve the activation of T cells in simulated microgravity/spaceflight (which is dependent on intracellular increases in Ca2+ and ROS) [23-28].

As AMPK activators including metformin and MG132 also inhibit dengue virus replication and malaria parasite growth, the aforementioned studies strongly suggests the novel observation that AMPK activation represents a common mechanism of action linking chemically distinct compounds and the effects of those compounds in diseases and phenomena as seemingly disparate as HGPS, HIV-1, microgravity/spaceflight, cancer stem cells, dengue fever, and malaria [29-32]. 

https://www.linkedin.com/pulse/new-study-shows-ampk-activator-mg132-rescues-progeria-finley?published=t

References

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19. Sook SH, Lee HJ, Kim JH, et al. Reactive oxygen species-mediated activation of AMP-activated protein kinase and c-Jun N-terminal kinase plays a critical role in beta-sitosterol-induced apoptosis in multiple myeloma U266 cells. Phytother Res. 2014 Mar;28(3):387-94.
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29. 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.
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33. 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.

AMPK & Metformin links Cancer Stem Cells, HIV-1, Progeria, Microgravity, & Spaceflight

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, e395; CC BY 2.5 (http://creativecommons.org/licenses/by/2.5)], via Wikimedia Commons; By NASA [Public domain], via Wikimedia Commons

A recent study published in the journal Oncogene in June of 2017 by researchers from The Johns Hopkins University School of Medicine and Emory University School of Medicine demonstrated that a bioactive molecule derived from Magnolia grandiflora significantly inhibited breast cancer stem cells (also known as tumor-initiating cells) in vivo in mice and also inhibited the expression of the stem cell/pluripotency markers Oct4, Nanog, and Sox2 in breast cancer cells in an AMPK-dependent manner [1]. Interestingly, a recent study published in the journal Oncotarget in May of 2017 showed that metformin activated AMPK and inhibited cell proliferation, clonogenic ability, tumorsphere formation (a characteristic of cancer stem cells), and the cancer stem cell population in chemotherapy-resistant colorectal cancer cells [2]. Metformin has also been shown to promote the differentiation and/or apoptosis of cancer stem cells in vitro and in vivo in an AMPK-dependent manner from the deadliest of cancers, including glioblastoma and pancreatic cancer [3]. Cancer stem cells (CSCs) exhibit several stem cell-like qualities that are characteristic of embryonic or adult stem cells, including self-renewal, differentiation, and the ability to initiate tumorigenesis. CSCs, similar to adult stem cells (ASCs), may also undergo quiescence, a state or period of inactivity or dormancy that contributes to the resistance of CSCs to radiation and/or chemotherapy, potentially allowing CSCs to re-seed tumors at a later time [3]. Intriguingly, HIV-1 has been shown to establish durable and long-stating latency, similar to CSC quiescence, in central CD4+ memory T cells as well as in T memory stem cells, T cell subsets that also display stem-cell like qualities including self renewal and differentiation. Latency establishment by HIV-1 in these T cell subsets prevents immune system detection and virus destruction or virus-induced cell death, analogous to CSC quiescence [3]. Because the use of AMPK activators, including metformin, bryostain-1, JQ1, and resveratrol as single agents or in a combinatorial fashion reactivates latent HIV-1 and induces CSC differentiation and/or apoptosis, a novel observation would logically follow that AMPK activation links the reactivation of latent HIV-1 (known as the “shock and kill” approach in HIV-1 cure research) with the “activation” (i.e. cell-cycle reentry), differentiation, and/or apoptosis of CSCs (which may also be labeled as a “shock and kill” approach) [3].

Indeed, a recently published paper in June of 2017 that I authored (and an additional paper currently under review) elucidates a novel link that connects CSCs with HIV-1 latency, microgravity and spaceflight, Hutchinson-Gilford progeria syndrome (HGPS), oocyte activation, the sperm acrosome reaction, Down syndrome, and hippocampal long-term potentiation [3,4]. This connection is predicated on the induction of cellular stress (mediated by increases in intracellular calcium [Ca2+] levels, reactive oxygen species [ROS] generation, and/or AMP/ATP ratio increases, etc), leading to beneficial cellular responses that are mediated by the master metabolic regulator AMPK [3]. As further explained below, AMPK is activated by a variety of stressors (osmotic, electrical stimulation, chemical, heat stress, etc.) as well as stressors that are diminished in microgravity/spaceflight (e.g. shear stress, force). AMPK also increases lifespan and healthspan in several model organisms (e.g. yeast, worms, flies, and mice) and metformin alleviates accelerated aging defects in cells from children with the genetic disorder HGPS. Because AMPK is also critical for T cell activation and the mounting of an effective immune response, promotes embryonic and adult stem cell differentiation, and metformin enhances bone formation, osteoprotegerin synthesis, and telomere integrity, AMPK activation by structurally diverse compounds that have a proven safety record may indeed provide protection for astronauts embarking on long-term space missions [3,5-7].

In May of 2017, I published for the first time that cellular stress-induced AMPK activation links the differentiation and/or apoptosis of cancer stem cells (CSCs) with the reactivation of latent HIV-1 and alleviation of accelerated aging-like cellular defects resulting from microgravity and spaceflight [3]. ROS and Ca2+ 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 [3]. Interestingly, AMPK is critical for T cell activation and the mounting of an effective immune response in vivo, as AMPK inhibition during T cell activation leads to T cell death [3]. HIV-1 has been shown to establish latency (thus preventing viral eradication) in long-lived T cell subsets that display longer telomeres, including central CD4+ memory T cells and CD4+ T memory stem cells [3]. However, the phorbol ester PMA combined with the calcium ionophore ionomycin is used as a positive control to reactivate latent HIV-1 and both compounds have been shown in independent studies to activate AMPK. Several structurally diverse compounds that also induce reactivation of latent HIV-1 in CD4+ memory T cells (called the “shock and kill” approach in HIV-1 cure research), including metformin combined with bryostatin-1, butyrate, JQ1, etc. also promote the differentiation and/or apoptosis of CSCs, indicating that the cellular states of CSC quiescence and HIV-1 latency are analogous and the induction of a cellular stress response, mediated by AMPK, may lead to both CSC elimination and viral eradication [3].

Additionally, simulated microgravity has been shown to inhibit AMPK activation, transient levels of hypergravity activate AMPK, and shear stress and the application of force have both been shown to activate AMPK, indicating that gravity itself represents a cellular stressor and is essential for efficient T cell activation and the mounting of an effective immune response [3,8,9]. Indeed, the application of force enhances T cell activation and both embryonic stem cell differentiation and T cell activation are significantly inhibited during actual spaceflight [3]. The lack of cellular stress induction (i.e. mechano-transduction) in microgravity/spaceflight may also explain the recent unusual observance of longer telomeres in the flight-based compared to the ground-based subject in the NASA TWINS Study [10]. Central memory T cells and T memory stem cells (TSCMs) are long-lived T cell subsets with longer telomeres and are capable of self-renewal and differentiation into more differentiated effector T cells [11,12]. However, as noted above, cellular stress and subsequent AMPK activation is essential for T cell activation and the mounting of an effective immune response and microgravity/spaceflight minimizes cellular stress induction, leading to an increase in undifferentiated T cell subsets that possess stem-cell like qualities (e.g. longer telomeres), with a consequent deficiency in the response to viruses, bacteria, and pathogens that return to normal levels after re-introduction of cellular stress (i.e. return to Earth’s gravitational field) [3].  

Strikingly, cells that are placed in NASA-developed rotating wall vessel bioreactors (RWVs), devices that are commonly used to mimic microgravity that also reduce shear stress, exhibit stem cell-like behavior (i.e. sphere formation) and reduced differentiation, very much similar to cancer stem cells [13]. A recent study has also shown that multipotent cells treated with strontium chloride, a compound that activates mouse oocytes and has been used to activate human oocytes, producing normal healthy children, induced activation of osteoblastogenesis in RWVs, indicating that cellular stress, mediated by gravity/force (i.e. mechano-transduction), Ca2+, ROS, and/or AMP/ATP ratio increases, etc. links AMPK activation with microgravity/spaceflight, oocyte activation, cancer stem cells, and HGPS (see below) [14-16].

In 2014, I was the first to propose and publish that metformin would alleviate accelerated aging defects in cells derived from patients with the accelerated aging disorder HGPS by promoting cellular stress-induced AMPK activation and beneficially altering the activity of the gene splicing factor SRSF1 (which is dysregulated in HGPS) [17]. This hypothesis was substantiated in 2016 and 2017, with metformin activating AMPK, decreasing the levels of SRSF1, and alleviating accelerated aging defects in HGPS cells [18,19]. In 2015, I was also the first to publish that the gene splicing factor SRSF1 links HGPS and HIV-1 latency, as excessive splicing activity of SRSF1 prevents reactivation of HIV-1, preventing viral cytopathic or cytolytic effects [20]. Because AMPK activation is critical for T cell activation, I also hypothesized that structurally dissimilar compounds that induce latent HIV-1 reactivation actually do so via indirect cellular stress-induced AMPK activation [20]. Intriguingly, a study by researchers at Merck showed that knockdown of AMPK or CaMKK2 (an upstream kinase/activator of AMPK) significantly inhibits HIV-1 replication [21]. Several compounds and methodologies that reactivate latent HIV-1, including heat stress, free-radical generating compounds, resveratrol, and many others have been shown to induce AMPK activation, indicating that cellular stress-induced AMPK activation orchestrates and connects both latent HIV-1 reactivation and HGPS [3,20,25]. Interestingly, temsirolimus, a rapamycin analog, was recently shown to correct accelerated aging defects in HGPS cells but transiently increase the levels of ROS and superoxide anion but decrease VO2 max within the first hour, providing further evidence that cellular stress-induced AMPK activation links HIV-1 latency and HGPS [22]. Intriguingly, rapamycin production by the bacterium Streptomyces hygroscopicus is significantly inhibited (~90%) when the bacterium is exposed to conditions of microgravity (i.e. being placed in a rotating wall vessel bioreactor), indicating that the beneficial effects of cellular stress induction may cross species boundaries, leading to increased production of valuable secondary metabolites by microorganisms (e.g. penicillin, rapamycin, avermectins, etc.) [23].

In 2016, I also first proposed that AMPK activation links oocyte activation, which is a prerequisite for the creation of all human life, with the reactivation of latent HIV-1 reservoirs [24]. The intracellular signaling mechanisms that characterize oocyte activation are incredibly similar to those that characterize latent HIV-1 reactivation in memory CD4+ T cells, and include the PLC-PIP2-IP3-Ca2+ and PLC-PIP2-DAG-PKC pathways. AMPK activation is also critical for oocyte meiotic resumption and maturation, two processes that precede and are critical for efficient oocyte activation [24]. Also, PMA and ionomycin, both of which activate AMPK and are used as positive controls in HIV-1 latency reversal studies, also induce mouse oocyte activation (models for human oocytes) and ionomycin is extensively used during in vitro fertilization procedures to activate human oocytes, producing normal healthy children [24]. Interestingly, MG132 (proteasome inhibitor), methylene blue, menadione (free radical-generating agent), resveratrol analogs, heat stress, and osmotic stress have each been shown to induce mouse oocyte meiotic resumption and activate AMPK [24]. Additionally, AMPK has recently been found localized across the entire acrosome in human spermatozoa and stress-inducing compounds, including ROS, PMA, ionomycin, vitamin D, and capsaicin have each been shown to induce the acrosome reaction in human sperm, an indispensable process for the creation of all human life outside of a clinical setting [3,4,26]. Interestingly, electrical stimulation, puromycin (a protein synthesis inhibitor), and ethanol have also been shown to activate human oocytes and induce AMPK activation, suggesting that cellular stress-induced AMPK activation, mediated by Ca2+, ROS, and AMP/ATP ratio increases, etc. links the creation of all human life (i.e. the “shock and live” approach) with HGPS and latent HIV-1 reactivation (i.e. the “shock and kill” approach) [24].

Also, I have a recently submitted manuscript (2017) currently under review that links cellular stress-induced AMPK activation with hippocampal CA1 long-term potentiation (LTP, a process critical for learning and memory) and HIV-1 latency [4]. Indeed, an increase in intracellular Ca2+ levels is indispensable for the induction of hippocampal CA1 LTP, the inhibition of ROS production or ROS scavengers significantly inhibits hippocampal CA1 LTP, and the primary excitatory neurotransmitter glutamate activates AMPK in neurons and also enhances T cell activation/proliferation [4]. Metformin also activates AMPK in hippocampal CA1 neurons, resveratrol upregulates AMPA receptors in the hippocampus in an AMPK-dependent manner (AMPA receptors are considered critical for the expression of LTP), the phorbol ester PMA enhances hippocampal CA1 LTP, and metformin alleviates Aβ-mediated reductions in hippocampal LTP, indicating that cellular stress-induced AMPK activation links hippocampal CA1 LTP with HIV-1 latency, HGPS, cancer stem cells, oocyte activation, sperm acrosome reaction, and spaceflight [4]. Interestingly, metformin also improves behavioral defects and activates AMPK in a mouse model of Fragile X syndrome, reverses mitochondrial defects in human fibroblasts derived from fetuses with Down syndrome, and inhibition of the Down syndrome gene DYRK1A induces reactivation of latent HIV-1, indicating that cellular stress-induced AMPK activation is also beneficial for and links certain genetic disorders with HIV-1 latency [3,27].

In conclusion, accumulating evidence strongly suggests that the induction of cellular stress, mediated by increases in ROS, intracellular Ca2+, and/or AMP/ATP ratio increases, etc. leads to beneficial cellular responses that are orchestrated by the master metabolic regulator AMPK. As first proposed in my current and forthcoming publications, cellular stress-induced AMPK activation links the therapeutic effects of structurally diverse compounds in seemingly disparate physiological and patho-physiological states, including cancer stem cells, HIV-1 latency, microgravity and spaceflight, Hutchinson-Gilford progeria syndrome, oocyte activation, the sperm acrosome reaction, Down syndrome, Fragile X syndrome, and hippocampal long-term potentiation. As evidence continues to accrue substantiating this novel link, a paradigm shift in the assessment of disease etiology and the practice of medicine is inevitable.

https://www.linkedin.com/pulse/ampk-metformin-links-cell-aging-during-spaceflight-cancer-finley?published=t

 

References:

1. Sengupta S, Nagalingam A, Muniraj N, et al. Activation of tumor suppressor LKB1 by honokiol abrogates cancer stem-like phenotype in breast cancer via inhibition of oncogenic Stat3. Oncogene. 2017 Jun 5. doi: 10.1038/onc.2017.164. [Epub ahead of print].
2. Kim SH, Kim SC, Ku JL. Metformin increases chemo-sensitivity via gene downregulation encoding DNA replication proteins in 5-Fu resistant colorectal cancer cells. Oncotarget. 2017 May 11. doi: 10.18632/oncotarget.17798. [Epub ahead of print].
3. 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 May, In Press, Accepted Manuscript; https://authors.elsevier.com/a/1VBXX15pGbnzkq
4. 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. 
5. Shah M, Kola B, Bataveljic A, et al. AMP-activated protein kinase (AMPK) activation regulates in vitro bone formation and bone mass. Bone. 2010 Aug;47(2):309-19.
6. Mai QG, Zhang ZM, Xu S, et al. Metformin stimulates osteoprotegerin and reduces RANKL expression in osteoblasts and ovariectomized rats. J Cell Biochem. 2011 Oct;112(10):2902-9.
7. 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.
8. 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.
9. Dixit M, Bess E, Fisslthaler B, et al. Shear stress-induced activation of the AMP-activated protein kinase regulates FoxO1a and angiopoietin-2 in endothelial cells. Cardiovasc Res. 2008 Jan;77(1):160-8.
10. https://www.nasa.gov/feature/symphonizing-the-science-nasa-twins-study-team-begins-integrating-results
11. Ahmed R, Roger L, Costa Del Amo P, et al. Human Stem Cell-like Memory T Cells Are Maintained in a State of Dynamic Flux. Cell Rep. 2016 Dec 13;17(11):2811-2818.
12. Graef P, Buchholz VR, Stemberger C, et al. Serial transfer of single-cell-derived immunocompetence reveals stemness of CD8(+) central memory T cells. Immunity. 2014 Jul 17;41(1):116-26.
13. Ingram M, Techy GB, Saroufeem R, et al. Three-dimensional growth patterns of various human tumor cell lines in simulated microgravity of a NASA bioreactor. In Vitro Cell Dev Biol Anim. 1997 Jun;33(6):459-66. 
14. Louis F, Bouleftour W, Rattner A, et al. RhoGTPase stimulation is associated with strontium chloride treatment to counter simulated microgravity-induced changes in multipotent cell commitment. npj Microgravity 3, Article number: 7 (2017).
15. Ma SF, Liu XY, Miao DQ, et al. Parthenogenetic activation of mouse oocytes by strontium chloride: a search for the best conditions. Theriogenology. 2005 Sep 15;64(5):1142-57.
16. Kim JW, Kim SD, Yang SH, Yoon SH, Jung JH, Lim JH. Successful pregnancy after SrCl2 oocyte activation in couples with repeated low fertilization rates following calcium ionophore treatment. Syst Biol Reprod Med. 2014 Jun;60(3):177-82.
17. 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; https://www.ncbi.nlm.nih.gov/pubmed/25216752 
18. Egesipe, Blondel, Cicero, et al. Metformin decreases progerin expression and alleviates pathological defects of Hutchinson–Gilford progeria syndrome cells. npj Aging and Mechanisms of Disease 2, Article number: 16026 (2016).
19. Park SK, Shin OS. Metformin Alleviates Aging Cellular Phenotypes in Hutchinson-Gilford Progeria Syndrome Dermal Fibroblasts. Exp Dermatol. 2017 Feb 13. doi: 10.1111/exd.13323. [Epub ahead of print].
20. 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; https://www.ncbi.nlm.nih.gov/pubmed/26115946
21. 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.
22. Gabriel D, Gordon LB, Djabali K. Temsirolimus Partially Rescues the Hutchinson-Gilford Progeria Cellular Phenotype. PLoS One. 2016 Dec 29;11(12):e0168988.
23. 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.
24. 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; https://www.ncbi.nlm.nih.gov/pubmed/27372854
25. Finley J. AMPK activation as a common mechanism of action linking the effects of diverse compounds that ameliorate accelerated cellular aging defects in Hutchinson-Gilford progeria syndrome. Med Hypotheses. Manuscript submitted.
26. De Toni L, Garolla A, Menegazzo M, et al. Heat Sensing Receptor TRPV1 Is a Mediator of Thermotaxis in Human Spermatozoa. PLoS One. 2016 Dec 16;11(12):e0167622.
27. Gantois I, Khoutorsky A, Popic J, et al. Metformin ameliorates core deficits in a Fragile X syndrome mouse model. Nat Med. 2017 May 15. doi: 10.1038/nm.4335. [Epub ahead of print]. 

Metformin shown for first time to inhibit malaria parasite in human liver cells: AMPK links pathogen destruction, Down syndrome, Fragile X, & Progeria

Vanellus Foto (Own work) [GFDL (http://www.gnu.org/copyleft/fdl.html); "Hutchinson-Gilford Progeria Syndrome" by The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. Scaffidi P, Gordon L, Misteli T. PLoS Biology Vol. 3/11/2005, e395; Peter Saxon (Own work) [CC BY-SA 4.0 (http://creativecommons.org/licenses/by-sa/4.0)], via Wikimedia Commons; Jim Gathany [Public domain], via Wikimedia Commons

In line with recent evidence that demonstrated for the first time that metformin exerted significant antiviral effects in dengue virus-infected human liver cells in an AMPK-dependent manner, 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 [1,2]. 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 [1]. Interestingly, metformin combined with another AMPK activator, bryostatin-1, has been shown to reactivate latent HIV-1 (facilitating immune system detection and virus destruction) while AMPK activation inhibits HIV-1 transactivation (a process necessary for HIV-1 replication), indicating that metformin will likely enhance latent HIV-1 reactivation and inhibit productive HIV-1 replication via AMPK activation, a hypothesis that I first proposed in an upcoming publication [3-5]. Additionally, metformin has recently been shown to beneficially alter gene splicing, activate AMPK, and alleviate accelerated aging defects in cells derived from Hutchison-Gilford progeria syndrome (HGPS) patients, reverse mitochondrial dysfunction in human fetal cells with Down syndrome (DS), restore behavioral and morphological defects in a mouse model of Fragile X syndrome (FXS), and improve behavior in humans with FXS (see below). Such evidence strongly suggests that AMPK activation induced by cellular stress (i.e. AMP/ATP ratio increase, reactive oxygen species generation, and/or intracellular calcium increases, etc.) links the amelioration and/or reversal of pathological cellular defects in HGPS, DS, and FXS with HIV-1 latency and replication, adult and cancer stem cells, learning and memory, and the creation all human life (via oocyte activation/sperm acrosome reaction), hypotheses that I first proposed in several publications and pending manuscripts [5-10].

Malaria is mosquito-borne infectious disease caused by several species of Plasmodium and is characterized by fever and flu-like illness that may lead to coma or death in severe cases [1]. Interestingly, although blood stage parasites cause the symptoms of malaria, malaria infection begins with parasitization of liver cells, marked by invasion and replication of sporozoites (infective agents introduced into the host) via schizogony (asexual reproduction by multiple fission) into thousands of merozoites (new parasites) that typically occurs in human and rodent parasites within 2 weeks and 48 hours, respectively [1].

The authors initially showed that phosphorylation/activation of AMPKα is decreased in Huh7 cells (human liver cells) infected with the rodent parasite Plasmodium berghei (P. berghei) compared to non-infected cells at 18 hours (hr) post-infection. RNAi-induced knockdown of both AMPKα subunits (AMPKα1 & AMPKα2) in P. berghei-infected cells also led to a significant increase in mean size distribution of schizont parasite forms (cells that divide by schizogony to form daughter cells) at 48 hr post-infection [1]. However, Huh7 cells expressing a constitutively active form of the AMPKα1 subunit displayed significantly smaller hepatic schizonts compared to control cells, indicating that AMPK restricts growth of P. berghei in liver cells. Exposure of P. berghei-infected Huh7 cells to the AMPK-activating compounds salicylate, metformin, 2-deoxy-D-glucose, and A769662 led to a significant decrease in schizont size and a dose-dependent reduction of total parasite load for each compound tested [1]. Further testing with salicylate (an ester of salicylic acid; aspirin is a derivative of salicylic acid) revealed similar inhibitory effects on different species of Plasmodium in both mouse and human cells, with salicylate inhibiting parasite development in Hepa1-6 cells (mouse hepatoma cell line) infected with P. yoelii (used to infect mice as a model of human malaria), Huh7 cells and mouse primary hepatocytes infected with P. berghei, and in human donor-derived primary hepatocytes infected with P. falciparum (causes malaria in humans) [1].

Additionally, after maturation of P berghei into the final endstage of hepatic development, characterized by fully mature merozoites and merosome release from the substratum, salicylate treatment reduced merosome size and number at 66 hr and reduced total merosome load by 80% up to 74 hr [1]. Cells treated with salicylate also contained smaller MSP1-positive schizonts (MSP1 is a merozoite surface marker essential for merozoite maturation), indicating that salicylate-induced AMPK activation will reduce the total number of merozoites released into the blood to infect erythrocytes [1]. Indeed, salicylate activated AMPKα in vivo in mouse livers, significantly reduced parasite size compared to control mice (after intradermal injection of sporozoites to mimic a mosquito bite), and reduced the number of infected erythrocytes by 57 % 72 hr after infection. Intriguingly, mice placed on a dietary restriction protocol for 2–3 weeks prior to and during liver-stage infection displayed increased liver AMPK activation, a significant reduction of hepatic schizont size, and a 66% decrease in pre-patent blood stage infection, demonstrating that AMPK activation potently inhibits Plasmodium growth during malaria liver-stage infection and reduces subsequent erythrocyte infection [1].

As noted above, in addition to reducing malaria parasite load in human liver cells,  metformin has also been shown to inhibit dengue virus replication in human liver cells in an AMPK-dependent [1,2]. The AMPK activator resveratrol also inhibits dengue virus replication in human liver cells, inhibits emtricitabine-resistant HIV-1, and reactivates latent HIV-1 (facilitating immune system detection and virus destruction or viral cytopathic effects) [11-13]. Furthermore, a common metabolite of artemisinin and its analogs, a powerful drug considered the standard first-line treatment for malaria caused by P. falciparum, shares a common mechanism of AMPK activation with both metformin and the BRD4 inhibitor JQ1, a compound that has shown exceptional effects in reactivating latent HIV-1, implicating AMPK activation as common mechanism through which structurally distinct compounds exert anti-viral effects against dengue virus, HIV-1 latency and replication, and malaria [14-16]. Moreover, as discussed below, metformin also activates AMPK and alleviates accelerated aging defects in cells from Hutchinson-Gilford progeria syndrome (HGPS) patients, rescues mitochondrial defects in human fetal cells with Down syndrome (DS), and significantly improves behavioral deficits in humans and in a mouse model with the genetic disorder Fragile X syndrome (FXS), providing compelling evidence that AMPK activation induced by cellular stress (i.e. AMP/ATP ratio increase, reactive oxygen species generation, and/or intracellular calcium increases, etc.) links the amelioration and/or reversal of pathological cellular defects in HGPS, DS, and FXS with HIV-1 latency and replication, adult and cancer stem cells, learning and memory, and the creation of all human life (via oocyte activation/sperm acrosome reaction), hypotheses that I first proposed in several publications and pending manuscripts [5-10].

Dy ABC et al. demonstrated for the first time that metformin consistently improved behavior in several patients diagnosed with Fragile X Syndrome (FXS), a genetic disorder characterized by intellectual disability and significant deficits in neurological function and cognitive development [17]. An improvement in behavior was documented in the Aberrant Behavior Checklist (ABC) for all cases, as evidenced by consistent improvements (i.e. lower scores compared to pre-metformin treatment) in social avoidance, irritability, hyperactivity, and social unresponsiveness as well as improvements in language and conversational skills reported by familial caretakers [17]. Metformin has also recently been shown to rescue and restore memory deficits in a Drosophila model of FXS and correct social novelty impairment, reduce testicular weight, decrease repetitive grooming, rescue excessive long-term depression and dendritic spine abnormalities, and alter excitatory synaptic transmission in an FXS mouse model [18,19].

Down syndrome (DS) is caused by either a partial or full trisomy of chromosome 21 and is associated with impairments in cognition, learning and memory, and disorders of the immune system [20]. Human and animal studies strongly suggest that mitochondrial dysfunction is associated with pathogenic features of DS and mitochondrial abnormalities have been found in all DS cells analyzed in culture to date, including neurons, astrocytes, and lymphocytes [20]. Indeed, in DS human fetal fibroblasts, PGC-1α, a master regulator of mitochondrial biogenesis, is down regulated at both the mRNA and protein levels and AMPK phosphorylation of the PGC-1α protein is essential for PGC-1α-dependent induction of the PGC-1α promoter [20].

Izzo et al. observed that DS human fetal fibroblasts (DS-HFFs) displayed a 40%-50% reduction in PGC-1α mRNA and protein levels compared to non-trisomic counterparts (N-HFFs). DS-HFFs treated with metformin however led to a significant increase in PGC-1α at both the mRNA and protein levels compared to untreated control cells, an increase in the mRNA levels of NRF-1 and TFAM (PGC-1α target genes critical in promoting mitochondrial biogenesis), and an increase in mtDNA content [20]. Additionally, compared to N-HFFs, DS-HFFs were characterized by a decrease in basal oxygen consumption rate (OCR, ~55% inhibition), a decrease in OCR related to ATP production (~58% inhibition), and a reduction in maximal respiratory capacity (~58% inhibition). DS-HFFs treated with metformin increased basal OCR, OCR related to ATP production, ATP concentration, mitochondrial membrane potential, and maximal respiratory capacity, thus restoring mitochondrial respiration in DS-HFFs [20].

Furthermore, compared to N-HFFs, DS-HFFs exhibited extensive mitochondrial damage, as evidenced by swollen cristae (~60%-90% of DS-HFFs vs. ~15% of N-HFFs), mitochondria with intra-oedema (~40% of DS-HFFs vs. ~6% of N-HFFs), and an increase in the number of damaged mitochondria (~90% in DS-HFFs vs. ~25% in N-HFFs). Metformin-treated DS-HFFs, compared to untreated cells, displayed narrower cristae with widths that were comparable to N-HFFs cells, fewer damaged mitochondria (~40%), and fewer mitochondria with intra-oedema (~5%) [20]. Interestingly, as mitochondrial fusion also plays an important role in mitochondrial functionality, the mRNA and protein expression of OPA1 and MFN2 (genes that regulate mitochondrial fusion) were significantly down-regulated in DS-HFFs vs. N-HFFs and correlated with extensive mitochondrial fragmentation in DS-HFFs. Treatment of DS-HFFs with metformin significantly increased the mRNA and protein levels of OPA1 and MFN2 and also reduced fragmentation of the mitochondrial network, indicating that metformin induces correction of mitochondrial phenotype by also restoring mitochondrial fusion machinery [20].     

The AMPK activator EGCG also reverses cognitive defects in DS patients, promotes oxidative phosphorylation and mitochondrial biogenesis in lymphoblasts and fibroblasts from DS patients (i.e. increased levels and activity of PGC-1α, NRF-1 and TFAM), and restores oxidative phosphorylation, mitochondrial biogenesis, and improves hippocampal neural progenitor cell proliferation in a DS mouse model (Ts65Dn) in combination with the polyphenol resveratrol (i.e. increase in PGC-1α) in an AMPK-dependent manner [21-23]. Mitochondrial dysfunction is also a prominent feature associated with the accelerated aging disorder Hutchison-Gilford progeria syndrome (HGPS) and metformin has recently been shown to alleviate accelerated aging defects, activate AMPK, and beneficially alter gene splicing in HGPS patient cells, as I first hypothesized and proposed in 2014 and 2015 [6,7].

Egesipe et al. demonstrated, using mesenchymal stem cells (MSCs) derived from HGPS induced pluripotent stem cells (i.e. HGPS MSCs), a significant dose-dependent decrease in SRSF1 mRNA levels after metformin treatment and up to a 40% decrease in SRSF1 protein levels after treatment with 5 mmol/l of metformin (SRSF1 is a gene splicing factor that is upregulated in HGPS cells and causes faulty gene splicing of the LMNA gene leading to increased production of the toxic protein progerin) [24]. A significant decrease was also observed in both lamin A and progerin mRNA expression in HGPS MSCs treated with 5 mmol/l metformin, with progerin mRNA expression and protein levels reduced to levels lower than that of lamin A mRNA expression and protein levels, indicating that metformin-induced inhibition of SRSF1 led to an increase in the lamin A/progerin ratio and thus beneficially altered gene splicing [24].      

The results obtained using HGPS MSCs were also replicated in additional in vitro cell models, with 5 mmol/l of metformin decreasing progerin mRNA expression up to 50% in LmnaG609G/G609G mouse primary fibroblasts (HGPS mouse model) and decreasing both lamin A and progerin mRNA expression in primary HGPS fibroblasts [24]. Interestingly, 5 mmol/l of metformin also decreased progerin mRNA expression in wild-type/normal MSCs that had been incubated with a compound that induces progerin expression, indicating that metformin may also prove beneficial in reducing progerin levels in normal humans [24].

Most importantly, however, treatment of HGPS MSCs with 5 mmol/l of metformin reduced the percentage of abnormal nuclei from 60% pre-treatment to less than 40% after treatment (wild-type MSCs presented less than 20% of abnormal nuclei). The metformin-induced reduction in abnormal nuclei was comparable to the reference treatment tipifarnib (1 μmol/1), a farnesyl-transferase inhibitor [24]. Additionally, as HGPS MSCs are characterized by premature osteogenic differentiation (indicated by increased alkaline phosphatase activity compared to wild-type osteogenic progenitor cells), 5 mmol/l of metformin led to a significant rescue of alkaline phosphatase activity in HGPS osteogenic progenitor cells, comparable to levels found in tipifarnib-treated cells [24].

Park et al. also characterized the cellular phenotypes of primary dermal fibroblasts derived from HGPS patients of different ages and showed increased staining of senescence-associated beta-galactosidase (SA-β-gal, an indicator of cellular senescence) and increased levels of mitochondrial superoxide in HG8 cells (from an 8 year old patient) compared to HG3 (3 year old) and HG5 (5 year old) cells [25]. All cells expressed the toxic protein progerin, superoxide dismutase 2 (SOD2, a mitochondrial antioxidant enzyme) was highest in normal fibroblasts and lowest in HG8 cells, and cellular proliferation rate slowed at an earlier time in HGPS cells compared to normal fibroblasts [25].

Utilizing HG8 cells (which demonstrated the highest levels of senescence), the authors also elucidated the effects of metformin (2mM), rapamycin (200nM), or a combination of both drugs on the nuclear phenotype of HGPS cells. Rapamycin significantly decreased the number of nuclei with abnormal morphology and metformin treatment also led to a significant increase in the number of cells with normal nuclei compared to control-treated cells [25]. Metformin also reduced senescence in HGPS cells (i.e. reduction in SA-β-gal staining) and co-treatment with rapamycin and metformin led to an approximately 34.2 % inhibition of senescence, with similar results observed in HG3 and HG8 cells [25]. Metformin, rapamycin, or co-treatment with both compounds led to a significant reduction in the number of cells containing more than 20 γ-H2AX foci (a marker of DNA damage) in HG8, HG3, and HG5 cells, indicating that metformin increases the efficiency of DNA repair in HGPS cells [25].

Metformin also exerted antioxidant effects in HGPS cells, as evidenced by a significant decrease in ROS production and mitochondrial superoxide formation compared to control cells as well as an upregulation of SOD2 mRNA expression in aged BALB/c mice (>18 months old) [25]. Most importantly, metformin treatment at 2 and 20mM reduced progerin protein expression by approximately 20 % and 60 %, respectively, compared to mock-treated cells and increased the presence of normal nuclear phenotypes in HGPS cells [25]. Metformin treatment also significantly increased the phosphorylation and activation of AMPK in HGPS cells. Furthermore, western blot analysis indicated that rapamycin increased AMPK activation as well.

Strikingly, 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 [8,26,27]. The induction of cellular stress (e.g. increases in reactive oxygen species, 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 [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 indicates and further substantiates the novel and provocative assertion that AMPK activation links the amelioration of pathological cellular defects in FXS, HGPS, and DS with HIV-1 latency and replication, adult and cancer stem cells, learning and memory, and the creation of all human life [5-10].

https://www.linkedin.com/pulse/metformin-shown-first-time-inhibit-malaria-parasite-human-finley

References:

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4. 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.
5. 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 (manuscript in press). 
6. 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.
7. 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.
8. 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.
9. Finley J. AMPK activation as a common mechanism of action linking the effects of diverse compounds that ameliorate accelerated cellular aging defects in Hutchinson-Gilford progeria syndrome (manuscript submitted).
10. 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 (manuscript submitted). 
11. Zainal N, Chang CP, Cheng YL, et al. Resveratrol treatment reveals a novel role for HMGB1 in regulation of the type 1 interferon response in dengue virus infection. Sci Rep. 2017 Feb 20;7:42998.
12. Heredia A, Davis C, Amin MN, et al. Targeting host nucleotide biosynthesis with resveratrol inhibits emtricitabine-resistant HIV-1. AIDS. 2014 Jan 28;28(3):317-23.
13. Pan XY, Zhao W, Zeng XY, et al. Heat Shock Factor 1 Mediates Latent HIV Reactivation. Sci Rep. 2016 May 18;6:26294.
14. 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.
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16. Darcis G, Kula A, Bouchat S, et al. An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression. PLoS Pathog. 2015 Jul 30;11(7):e1005063.
17. Dy ABC, Tassone F, Eldeeb M, Salcedo-Arellano MJ, Tartaglia N, Hagerman R. Metformin as Targeted Treatment in Fragile X Syndrome. Clin Genet. 2017 Apr 24. doi: 10.1111/cge.13039. [Epub ahead of print].
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24. Egesipe, Blondel, Cicero, et al. Metformin decreases progerin expression and alleviates pathological defects of Hutchinson–Gilford progeria syndrome cells. npj Aging and Mechanisms of Disease 2, Article number: 16026 (2016); http://www.nature.com/articles/npjamd201626?WT.feed_name=subjects_drug-discovery
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Metformin shown for the first time to improve behavior in humans with Fragile X Mental Retardation Syndrome: AMPK links Progeria, FXS, & Down syndrome

By Vanellus Foto (Own work) [GFDL (http://www.gnu.org/copyleft/fdl.html) or CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons; "Hutchinson-Gilford Progeria Syndrome" by The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. Scaffidi P, Gordon L, Misteli T; By Peter Saxon (Own work) [CC BY-SA 4.0 (http://creativecommons.org/licenses/by-sa/4.0)], via Wikimedia Commons

A study recently published in the journal Clinical Genetics in April of 2017 by researchers from the University of California, Davis Medical Center and the University of Colorado School of Medicine demonstrated for the first time that metformin consistently improved behavior in several patients diagnosed with Fragile X Syndrome (FXS), a genetic disorder characterized by intellectual disability and significant deficits in neurological function and cognitive development [1]. An improvement in behavior was documented in the Aberrant Behavior Checklist (ABC) for all cases, as evidenced by consistent improvements (i.e. lower scores compared to pre-metformin treatment) in social avoidance, irritability, hyperactivity, and social unresponsiveness (see table below) as well as improvements in language and conversational skills reported by familial caretakers [1]. Interestingly, a study published in 2016 showed that metformin rescued and restored memory deficits in a Drosophila (fly) model of FXS and a soon-to-be published study currently in press also demonstrated that metformin restored behavioral and morphological defects in a mouse model of FXS [2-4].

In 2014 and 2015, I was first to hypothesize and publish that AMPK activation via compounds including metformin would beneficially alter gene splicing and alleviate accelerated aging defects in cells derived from patients with the genetic disorder Hutchinson-Gilford progeria syndrome, which was later substantiated by studies published in 2016 and 2017 (see below). Because AMPK activation has been shown to restore severe mitochondrial impairment in hippocampal progenitor cells from a Down syndrome mouse model and metformin reverses mitochondrial dysfunction in human fetal cells with Down syndrome, the activation of AMPK via the induction of cellular stress (i.e. AMP/ATP ratio increase, reactive oxygen species generation, and/or intracellular calcium increases, etc.) links Fragile X syndrome, Hutchison-Gilford progeria syndrome, and Down syndrome with the therapeutic effects of structurally dissimilar AMPK activators including metformin, resveratrol, and lithium. Indeed, in my recently submitted manuscripts and publications, I initially propose that all three of the aforementioned genetic disorders as well as learning and memory (i.e. hippocampal long-term potentiation), adult and cancer stem cells, and the creation all human life (via oocyte activation/sperm acrosome reaction) are connected to HIV-1 latency via AMPK activation [5-10].

Fragile X syndrome (FXS) is the most common inherited cause of autism spectrum disorder (ASD) and intellectual disability (ID) and results from a trinucelotide expansion-induced hypermethylation and silencing of the Fragile X Mental Retardation 1 gene (FMR1) and resultant silencing of the FMR1 gene product FMR1 protein (FMRP) [1]. Symptomatic behaviors associated with FXS include aggression, self-injurious behaviors, anxiety, and attention deficit hyperactivity disorder (ADHD). In the study, several patients, listed below, were treated clinically with metformin at the Fragile X Treatment and Research Center at the University of California, Davis Medical Center MIND Institute for at least six months along with assessments of medical and behavioral co-morbidities and laboratory studies before and after treatment [1].

Case 1:

Case 1 was a 19 year-old male diagnosed with FXS and ASD at age 5 with a history of hyperphagia, characteristic features of FXS including macroorchidism (abnormally large testicles), and behavioral issues including aggression, hand biting, hyperactivity, ADHD, poor eye contact, and skin picking and scratching [1]. After being placed on several mediations (e.g. minocycline, divalproex, etc.), he was started on metformin 500 mg twice a day (bid) and subsequently experienced significant improvements in hyperphagia, irritability, aggression, communication, and behavior. It was also noted that the patient no longer focuses on food (as he did for many years), now enjoys outdoor activities, and has considerably improved self-esteem [1].    

Case 2:

Case 2 was a 13 year-old boy with FXS who at age 10 had a Leiter IQ of 44 and an Autism Diagnostic Observation Schedule (ADOS) total score of 25 (well into ASD range) [1]. Behavioral symptoms consisted of hyperphagia, severe hyperactivity and impulsivity, significant aggression characterized by kicking and hitting at home and at school, and escalating aggressiveness towards his mother (which led to her becoming depressed), leading to his placement in a residential group home to control his eating and aggression [1]. After starting metformin 500 mg bid at age 12, his mother and care center noted significant improvements in his behavior over the course of a year, including the patient being more calm and patient, negotiating with teachers more, displaying a better understanding when things were explained to him, being more responsive to rewards, and able to follow directions better and work longer with less agitation and aggression [1].

Case 3:

Case 3 was another 19-year-old boy diagnosed at age 3 with FXS and had also been diagnosed with ADHD, ASD, and specific phobias [1].  Behavioral symptoms included impulsive episodic outbursts, persistent hyperphagia, skin pricking, enuresis (inability to control urination), and binge eating to the point of vomiting. After starting metformin 500 mg bid, the patient experienced significant behavioral improvements including a decrease in tantrums, improvement in communication ability, and cessation of head-banging and other self-injurious behavior [1].

Case 4

Case 4 was a 60-year-old female diagnosed with FXS and mild ID currently residing in a group home with a history of morbid obesity along with repetitive behavior, memory deficits, agitation, and anxiety that caused concern among her caregivers [1]. After starting on metformin 500 mg bid, the patient is no longer overeating, has lost 41.3 pounds, and her irritability and social responsiveness has improved (see table below) [1].

Case 5 

Case 5 was a 31-year-old man diagnosed with FXS and ASD with a full scale IQ of 54 on the WISC III. The patient exhibited macroorchidism and also had problems with anxiety and tantrums [1]. After starting metformin 500 mg bid, the patient’s appetite decreased, more language was noted by his speech and language therapist, and he has improved self-initiative. Also, he “will carry out chores such as cleaning his room or cleaning the house without excessive urging or reminders, helps his mom with gardening and is able to coordinate the rake better, has a lot of trivia knowledge, and asks more inquisitive questions. The mother is able to have a discussion with him even about emotional issues, he is able to talk about his grief and to relate his father's death to his excessive smoking and drinking problems, and he seems to be putting together concepts better and having back-and-forth conversations.” [1].

Case 6

Case 6 was originally seen at 10 years of age and exhibited typical characteristics of FXS on physical examination, including soft skin, flat feet, and poor eye contact [1]. DNA testing confirmed a diagnosis of FXS and the patient demonstrated a full scale IQ of 50. The patient also displayed a dramatic increase in anxiety, aversion to people, and intermittent aggression and headaches due to a traumatic episode wherein the patient had to be tasered by police at a hospital due to his aggression [1].  After starting metformin 500 mg bid at age 24, he has not experienced on outburst, “seems happier and more active, has improvement in language, is able to carry out a two way conversation, has become more outgoing socially with less anxiety, and eats without gorging himself.” [1].

Case 7

Case 7 was a 4 year old boy diagnosed with FXS at 14 months and displayed features typical of FXS, including prominent ears, a broad forehead, and flat feet [1]. He had motor and language delays at an early age and the parents self-treated the child with a cannabinoid tincture at age 2 due to staring spells and a lack of language. The parents noted that the tincture led to a decrease in staring spells, increased verbalization, and improved anxiety [1]. “After 6 months of a dose of metformin 50 mg the family felt he has fewer outbursts or tantrums, better attention, less hyperactivity and improvements in his language. Developmental testing after 4 months of treatment at 4.5 years showed expressive language at the 31 month age equivalent.” [1]. 

As demonstrated by these case studies, it appears that metformin significantly improves cognitive and behavioral deficits in the genetic disorder FXS. Additionally, as noted above, metformin has recently been shown to rescue and restore memory deficits in a Drosophila model of FXS and a study in press has shown that metformin corrected social novelty impairment, reduced testicular weight, decreased repetitive grooming, rescued excessive long-term depression and dendritic spine abnormalities, and altered excitatory synaptic transmission in an FXS mouse model, indicating that metformin likely exerts significant beneficial effects in disparate genetic disorders [2-4]. Indeed, metformin has also been shown to correct alternative splicing defects in primary myoblasts derived from patients with myotonic dystrophy type I (DM1) as well as in derivatives of embryonic stem cells that carry the DM1 mutation via activation of AMPK. DM1 is a genetic disorder characterized by muscle wasting that is also caused by the expansion of a trinucleotide repeat, similar to FXS [11]. As further explained below, because metformin activates AMPK and alleviates accelerated aging defects in cells from Hutchinson-Gilford progeria syndrome (HGPS) patients and because metformin also rescues mitochondrial defects in human fetal cells with Down syndrome (DS), AMPK activation induced by cellular stress (i.e. AMP/ATP ratio increase, reactive oxygen species generation, and/or intracellular calcium increases, etc.) links the amelioration and/or reversal of pathological cellular defects in FXS, HGPS, DM1, and DS with HIV-1 latency, adult and cancer stem cells, learning and memory, and the creation all human life (via oocyte activation/sperm acrosome reaction), hypotheses that I first proposed in several publications and pending manuscripts [5-10].

Down syndrome (DS) is caused by either a partial or full trisomy of chromosome 21 and is associated with impairments in cognition, learning and memory, and disorders of the immune system [12]. Human and animal studies strongly suggest that mitochondrial dysfunction is associated with pathogenic features of DS and mitochondrial abnormalities have been found in all DS cells analyzed in culture to date, including neurons, astrocytes, and lymphocytes [12]. Indeed, in DS human fetal fibroblasts, PGC-1α, a master regulator of mitochondrial biogenesis, is down regulated at both the mRNA and protein levels and AMPK phosphorylation of the PGC-1α protein is essential for PGC-1α-dependent induction of the PGC-1α promoter [12].

Izzo et al. initially observed that DS human fetal fibroblasts (DS-HFFs) displayed a 40%-50% reduction in PGC-1α mRNA and protein levels compared to non-trisomic counterparts (N-HFFs). DS-HFFs treated with metformin however led to a significant increase in PGC-1α at both the mRNA and protein levels compared to untreated control cells, an increase in the mRNA levels of NRF-1 and TFAM (PGC-1α target genes critical in promoting mitochondrial biogenesis), and an increase in mtDNA content [12]. Additionally, compared to N-HFFs, DS-HFFs were characterized by a decrease in basal oxygen consumption rate (OCR, ~55% inhibition), a decrease in OCR related to ATP production (~58% inhibition), and a reduction in maximal respiratory capacity (~58% inhibition). DS-HFFs treated with metformin increased basal OCR, OCR related to ATP production, ATP concentration, mitochondrial membrane potential, and maximal respiratory capacity, thus restoring mitochondrial respiration in DS-HFFs [12].

Furthermore, compared to N-HFFs, DS-HFFs exhibited extensive mitochondrial damage, as evidenced by swollen cristae (~60%-90% of DS-HFFs vs. ~15% of N-HFFs), mitochondria with intra-oedema (~40% of DS-HFFs vs. ~6% of N-HFFs), and an increase in the number of damaged mitochondria (~90% in DS-HFFs vs. ~25% in N-HFFs). Metformin-treated DS-HFFs, compared to untreated cells, displayed narrower cristae with widths that were comparable to N-HFFs cells, fewer damaged mitochondria (~40%), and fewer mitochondria with intra-oedema (~5%) [12]. Interestingly, as mitochondrial fusion also plays an important role in mitochondrial functionality, the mRNA and protein expression of OPA1 and MFN2 (genes that regulate mitochondrial fusion) were significantly down-regulated in DS-HFFs vs. N-HFFs and correlated with extensive mitochondrial fragmentation in DS-HFFs. Treatment of DS-HFFs with metformin significantly increased the mRNA and protein levels of OPA1 and MFN2 and also reduced fragmentation of the mitochondrial network, indicating that metformin induces correction of mitochondrial phenotype by also restoring mitochondrial fusion machinery [12].      

The AMPK activator EGCG also reverses cognitive defects in DS patients, promotes oxidative phosphorylation and mitochondrial biogenesis in lymphoblasts and fibroblasts from DS patients (i.e. increased levels and activity of PGC-1α, NRF-1 and TFAM), and restores oxidative phosphorylation, mitochondrial biogenesis, and improves hippocampal neural progenitor cell proliferation in a DS mouse model (Ts65Dn) in combination with the polyphenol resveratrol (i.e. increase in PGC-1α) in an AMPK-dependent manner [13-15]. Mitochondrial dysfunction is also a prominent feature associated with the accelerated aging disorder Hutchison-Gilford progeria syndrome (HGPS) and metformin has recently been shown to alleviate accelerated aging defects, activate AMPK, and beneficially alter gene splicing in HGPS patient cells.

Egesipe et al. initially demonstrated, using mesenchymal stem cells (MSCs) derived from HGPS induced pluripotent stem cells (i.e. HGPS MSCs), a significant dose-dependent decrease in SRSF1 mRNA levels after metformin treatment and up to a 40% decrease in SRSF1 protein levels after treatment with 5 mmol/l of metformin (SRSF1 is a gene splicing factor that is upregulated in HGPS cells and causes faulty gene splicing of the LMNA gene leading to increased production of the toxic protein progerin) [16]. A significant decrease was also observed in both lamin A and progerin mRNA expression in HGPS MSCs treated with 5 mmol/l metformin, with progerin mRNA expression and protein levels reduced to levels lower than that of lamin A mRNA expression and protein levels, indicating that metformin-induced inhibition of SRSF1 led to an increase in the lamin A/progerin ratio and thus beneficially altered gene splicing [16].       

The results obtained using HGPS MSCs were also replicated in additional in vitro cell models, with 5 mmol/l of metformin decreasing progerin mRNA expression up to 50% in LmnaG609G/G609G mouse primary fibroblasts (HGPS mouse model) and decreasing both lamin A and progerin mRNA expression in primary HGPS fibroblasts [16]. Interestingly, 5 mmol/l of metformin also decreased progerin mRNA expression in wild-type/normal MSCs that had been incubated with a compound that induces progerin expression, indicating that metformin may also prove beneficial in reducing progerin levels in normal humans [16].

Most importantly, however, treatment of HGPS MSCs with 5 mmol/l of metformin reduced the percentage of abnormal nuclei from 60% pre-treatment to less than 40% after treatment (wild-type MSCs presented less than 20% of abnormal nuclei). The metformin-induced reduction in abnormal nuclei was comparable to the reference treatment tipifarnib (1 μmol/1), a farnesyl-transferase inhibitor [16]. Additionally, as HGPS MSCs are characterized by premature osteogenic differentiation (indicated by increased alkaline phosphatase activity compared to wild-type osteogenic progenitor cells), 5 mmol/l of metformin led to a significant rescue of alkaline phosphatase activity in HGPS osteogenic progenitor cells, comparable to levels found in tipifarnib-treated cells [16].

Park et al. also characterized the cellular phenotypes of primary dermal fibroblasts derived from HGPS patients of different ages and showed increased staining of senescence-associated beta-galactosidase (SA-β-gal, an indicator of cellular senescence) and increased levels of mitochondrial superoxide in HG8 cells (from an 8 year old patient) compared to HG3 (3 year old) and HG5 (5 year old) cells [17]. All cells expressed the toxic protein progerin, superoxide dismutase 2 (SOD2, a mitochondrial antioxidant enzyme) was highest in normal fibroblasts and lowest in HG8 cells, and cellular proliferation rate slowed at an earlier time in HGPS cells compared to normal fibroblasts [17].

Utilizing HG8 cells (which demonstrated the highest levels of senescence), the authors also elucidated the effects of metformin (2mM), rapamycin (200nM), or a combination of both drugs on the nuclear phenotype of HGPS cells. Rapamycin significantly decreased the number of nuclei with abnormal morphology and metformin treatment also led to a significant increase in the number of cells with normal nuclei compared to control-treated cells [17]. Metformin also reduced senescence in HGPS cells (i.e. reduction in SA-β-gal staining) and co-treatment with rapamycin and metformin led to an approximately 34.2 % inhibition of senescence, with similar results observed in HG3 and HG8 cells [3]. Metformin, rapamycin, or co-treatment with both compounds led to a significant reduction in the number of cells containing more than 20 γ-H2AX foci (a marker of DNA damage) in HG8, HG3, and HG5 cells, indicating that metformin increases the efficiency of DNA repair in HGPS cells [17].

Metformin also exerted antioxidant effects in HGPS cells, as evidenced by a significant decrease in ROS production and mitochondrial superoxide formation compared to control cells as well as an upregulation of SOD2 mRNA expression in aged BALB/c mice (>18 months old) [17]. Most importantly, metformin treatment at 2 and 20mM reduced progerin protein expression by approximately 20 % and 60 %, respectively, compared to mock-treated cells and increased the presence of normal nuclear phenotypes in HGPS cells [17]. Metformin treatment also significantly increased the phosphorylation and activation of AMPK in HGPS cells. Furthermore, western blot analysis indicated that rapamycin increased AMPK activation as well.

Strikingly, metformin has recently been shown to inhibit dengue virus replication in human liver cells in an AMPK-dependent manner, resveratrol (an AMPK activator) has recently been shown to reactivate latent HIV-1, and HIV-1 replication is significantly inhibited via knockdown of AMPK, indicating that AMPK activation leads to both the reactivation of latent viruses (facilitating immune system detection and destruction) as well as inhibition of productive viral replication [18-20]. 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 [7,21,22]. 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 [21,23,24]. 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 [24-26]. Such evidence indicates and further substantiates the novel and provocative assertion that AMPK activation links the amelioration of pathological cellular defects in FXS, HGPS, DS, and DM1 with HIV-1 latency, adult and cancer stem cells, learning and memory, and the creation of all human life [5-10].

https://www.linkedin.com/pulse/metformin-shown-first-time-improve-behavior-humans-fragile-finley

*Table 2 adapted from: Dy ABC, Tassone F, Eldeeb M, Salcedo-Arellano MJ, Tartaglia N, Hagerman R. Metformin as Targeted Treatment in Fragile X Syndrome. Clin Genet. 2017 Apr 24. doi: 10.1111/cge.13039. [Epub ahead of print].

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