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”. 

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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.

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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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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8. 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.
9. 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.
10. Krieger G, Shkolnik D, Miller G, Fromm H. Reactive Oxygen Species Tune Root Tropic Responses. Plant Physiol. 2016 Oct;172(2):1209-1220.
11. Plieth C, Trewavas AJ. Reorientation of seedlings in the earth's gravitational field induces cytosolic calcium transients. Plant Physiol. 2002 Jun;129(2):786-96.
12. Denninger P, Bleckmann A, Lausser A, et al. Male-female communication triggers calcium signatures during fertilization in Arabidopsis. Nat Commun. 2014 Aug 22;5:4645.
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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44. Deemeh MR, Tavalaee M, Nasr-Esfahani MH. Health of children born through artificial oocyte activation: a pilot study. Reprod Sci. 2015 Mar;22(3):322-8.
45. Tamás P, Hawley SA, Clarke RG, et al. Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes. J Exp Med. 2006 Jul 10;203(7):1665-70.
46. Spina CA, Anderson J, Archin NM, et al. An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients. PLoS Pathog 2013;9(12):e1003834.
47. 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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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; https://www.ncbi.nlm.nih.gov/pubmed/25216752 
5. Hardie DG. AMPK: a target for drugs and natural products with effects on both diabetes and cancer. Diabetes. 2013 Jul;62(7):2164-72.
6. 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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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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25. LaRosa C, Downs SM. Stress stimulates AMP-activated protein kinase and meiotic resumption in mouse oocytes. Biol Reprod. 2006 Mar;74(3):585-92.
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28. 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
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.
30. 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.
31. Fernandez-Garcia MD, Meertens L, Bonazzi M, Cossart P, Arenzana-Seisdedos F, Amara A. Appraising the roles of CBLL1 and the ubiquitin/proteasome system for flavivirus entry and replication. J Virol. 2011 Mar;85(6):2980-9.
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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.