AMPK activator Metformin found to alleviate accelerated aging defects in Progeria cells: Hypothesis substantiated linking AMPK with aging and HIV-1

"Hutchinson-Gilford Progeria Syndrome" by The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. Scaffidi P, Gordon L, Misteli T; https://commons.wikimedia.org/wiki/File:HIV-budding-Color.jpg#/media/File:HIV-budding-Color.jpg. "HIV-budding-Color" by Photo Credit: C. Goldsmith. Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus.

AMPK activator Metformin found to alleviate accelerated aging defects in Progeria cells: Hypothesis substantiated linking AMPK with aging and HIV-1

A recent study published online in the Journal npj Aging and Mechanisms of Disease (part of the Nature Partner Journals series) in November of 2016 provided startling evidence that metformin, a widely-prescribed anti-diabetic drug derived from the plant Galega officinalis that has been shown to increase the lifespan and healthspan of several organisms, decreased the expression of progerin (a toxic protein that leads to accelerated cellular aging defects) and alleviated pathological defects in cells derived Hutchinson–Gilford progeria syndrome (HGPS) patients [1].  Interestingly, metformin also decreased the expression of the gene splicing factor SRSF1, a protein that has been previously shown to promote the use of a cryptic splice site in the LMNA gene, increasing the expression of the toxic protein progerin that leads to the accelerated aging phenotype observed in HGPS [1].  This study provides direct support and substantiates a hypothesis published in 2014, in which I proposed for the first time that AMPK activators including metformin will improve accelerated aging defects in HGPS by decreasing the levels of SRSF1, thus reducing progerin production via modulation of alternative splicing [2]. 

Interestingly, several chemically distinct compounds that have recently been shown to improve accelerated cellular aging defects in HGPS, including rapamycin, methylene blue, sulforaphane, all-trans retinoic acid, MG132, oltipraz, and vitamin D have each been shown to activate AMPK, similar to metformin (see below).  Metformin has also recently been shown to beneficially alter gene splicing in cells taken from patients with the genetic disorder myotonic dystrophy type I (DMI) in an AMPK-dependent manner.  Additionally, metformin beneficially altered gene splicing in diabetic patients who were taking metformin but who did not have DM1 [3].  The confirmation of my 2014 hypothesis via the npj Aging and Mechanisms of Disease study that metformin indeed decreases SRSF1 and improves accelerated aging defects in HGPS provides a powerful indication that AMPK activation represents an “indirect yet common mechanism of action” linking the therapeutic effects of chemically distinct compounds in HGPS.  Furthermore, as explained below, SRSF1 has been shown to prevent the reactivation of latent HIV-1 viral reservoirs and many chemically distinct compounds, including MG132, have been shown to promote reactivation of latent HIV-1 in immune cells (facilitating detection and destruction of the virus), implicating the novel proposition that latent HIV-1 reactivation is critically dependent on AMPK activation, a proposal that I published for the first time in 2015 [4]. 

HGPS is a rare genetic disorder caused by the faulty splicing of a gene called the LMNA gene, producing large amounts of a mutant protein known as progerin [5].  Progerin accumulation at a very early age in HGPS patients leads to distortions in the shape of the nucleus and aberrations in mechanisms that occur in the nucleus, leading to characteristic symptoms of accelerating aging such as thinning of the hair, wrinkling of the skin, and eventual cardiovascular disease [5].  Interestingly, normal humans produce the same toxic protein progerin via use of the same cryptic splice site in the LMNA gene as progeria patients, just at much lower levels that increase with age [6].  Recent evidence has also shown that inhibition of the splicing factor SRSF1 leads to a reduction in progerin at both the mRNA and protein levels (thus altering the LMNA pre-mRNA splicing ratio) and SRSF1 activity promotes the faulty splicing of genes involved in the maintenance of the vascular system in normal humans (e.g. VEGF, tissue factor, endoglin), leading to accelerated endothelial cell senescence [4,7,8].

In the npj Aging and Mechanisms of Disease study, 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 [1]. 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 [1].          

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 Lmna^G609G/G609G mouse primary fibroblasts (HGPS mouse model) and decreasing both lamin A and progerin mRNA expression in primary HGPS fibroblasts [1]. 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 [1].  

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 [1].  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 [1].  

Again, this study provides compelling evidence and substantiates my hypothesis published in 2014 that proposed for the first time that AMPK activators including metformin will ameliorate accelerated aging defects in cells derived from HGPS patients by decreasing the levels of SRSF1, thus reducing progerin production via modulation of alternative splicing [2]. However, the results from the npj Aging and Mechanisms of Disease study also provides further support for a novel proposal published for the first time in 2015 in which I proposed that a decrease in the splicing activities of SRSF1 by chemically distinct AMPK activators will also lead to the reactivation of latent HIV-1 viral reservoirs [4].  Known as the “shock and kill” approach, this method is an active area among HIV-1 cure researchers and involves reactivating (i.e. “shock”) a T cell (or another immune cell) that harbors dormant HIV-1, hence reactivating the virus itself and thus inducing destruction of the T cell along with the virus or enhancing recognition and destruction of the virus-infected T cell by the immune system (i.e. “kill”) [4]. 

Interestingly, as a preponderance of evidence has convincingly shown that metformin’s primary mechanism of action is via AMPK activation, AMPK is also critical for the activation of T cells and the mounting of an effective immune response to eliminate viruses, bacteria, and cancer cells [9-11].  Strikingly, the same compounds that have been used to induce a “shock” to initiate the creation of human life/oocyte activation (i.e. ionomycin and A23187) have also been used in combination with other compounds as positive controls to initiate a “shock” to facilitate CD4⁺ T cell activation and thus reactivate dormant HIV-1  [9,12]. Calcium ionophores including ionomycin have also been used to induce a “shock” to activate cytotoxic CD8⁺ T cells, a T cell subset that is critical for the destruction of viruses such as HIV-1 and cancer cells. Metformin and AMPK activation has also been shown to promote the formation of long-lived cytotoxic CD8⁺ memory T cells [10,11,13].

Indeed, studies have shown that efficient reactivation of latent HIV-1 involves a reduction in the splicing of the HIV-1 genome by the splicing factor SRSF1, an upregulation in the activity of the splicing-associated protein p32 (an endogenous inhibitor of SRSF1 that is critical for efficient mitochondrial functionality and oxidative phosphorylation), the production of unspliced HIV-1 mRNA (also known as HIV-1 Gag), and the processing of Gag into the HIV-1 p24 antigen, an antigen that is an endpoint that is frequently measured to determine if efficient reactivation of latent HIV-1 by a candidate compound was successful [14,15,16].  Interestingly, the activity of the splicing factor SRSF1 is also downregulated during activation of T cells not infected with HIV-1 [17].

Because metformin, a well-studied AMPK activator, has been shown to reduce the levels of the splicing factor SRSF1 and thus ameliorate aberrant alternative splicing in HGPS cells and because AMPK activation is critical for T cell activation (and thus latent HIV-1 reactivation) and SRSF1 impedes efficient reactivation of latent HIV-1, it would be expected that compounds that both improve accelerated aging defects in HGPS and reactivate latent HIV-1 would also induce AMPK activation.  Indeed, a recent study has demonstrated that metformin, when combined with the protein kinase C modulator bryostatin, induced reactivation of latent HIV-1 in a monocytic cell line in an AMPK-dependent manner.  Bryostatin was also shown to induce phosphorylation and activation of AMPK in that study, implying that bryostatin is an indirect AMPK activator as well [18]. Furthermore, the calcium ionophores ionomycin and A23187, both of which activate AMPK and induce human oocyte activation, are often combined with phorbol 12-myristate 13-acetate (PMA) and are extremely efficient in promoting T cell activation-induced latent HIV-1 reactivation [9,12,19,20].

The compound MG132, a proteasome inhibitor, has also recently been shown in preliminary studies to reduce the levels of the toxic protein progerin via the induction of autophagy and also to reduce progerin production by decreasing the levels of the splicing factor SRSF1, thus beneficially altering splicing of the LMNA gene in HGPS [21,22].  In a separate study, MG132, either alone or in combination with the vitamin A metabolite all-trans retinoic acid, led to a decrease in progerin levels in HGPS cells via the induction of autophagy [23]. MG132 has also been shown to activate AMPK and significantly induce HIV-1 reactivation in two latent HIV-1 primary human CD4+ T cell models that mimic central and effector memory T cells (two memory T cell subsets that are known reservoirs for latent HIV-1) [24,25].

Interestingly, autophagic induction has been shown to be critical for both the removal of the toxic protein progerin in HGPS cells by compounds including MG132 and all-trans retinoic as well as T cell activation. Indeed, autophagy is essential for and upregulated on T cell activation and AMPK activation significantly increases mitochondrial biogenesis, activates ULK1 to induce autophagy, and promotes activation the master antioxidant transcription factor Nrf2 [26-29]. Because the AMPK activators metformin and MG132 have been shown to inhibit SRSF1 and beneficially alter gene splicing in HGPS cells and because AMPK activation is critical for T cell activation, autophagic induction, mitochondrial biogenesis/functionality, and promotes Nrf2 activation, chemically distinct compounds that have been demonstrated to reduce progerin levels and/or ameliorate accelerated aging defects in HGPS cells would be expected to share a common mechanism of AMPK activation.

Indeed, preclinical studies using the macrolide rapamycin in progeria cells indicated that rapamycin corrected cellular aging defects by inducing the degradation of progerin by activating autophagy [30]. Rapamycin was also recently found to potently activate AMPK in vivo in normal old mice as well as induce autophagy and mitochondrial biogenesis [31]. The induction of ULK1-dependent autophagy by rapamycin was also shown to be significantly decreased when the splicing factor p32 (an endogenous inhibitor of SRSF1) was inhibited, indicating that p32 activity is critical for rapamycin-induced autophagy [32].  Because p32 is critical for rapamycin-induced autophagy by ULK1 and because rapamycin, similar to metformin, activates AMPK and AMPK induces autophagy by phosphorylating and activating ULK1, the beneficial effects of rapamycin in progeria likely involves AMPK-mediated alteration of gene splicing as well as AMPK-mediated induction of autophagy.  Both metformin and rapamycin have also been shown to increase the formation of CD8+ memory T cells and rapamycin has been shown to enhance the immune response to viral infections, indicating that rapamycin-induced AMPK activation represents a central node in ameliorating accelerated aging defects in HGPS cells and improving T cell responses to viral pathogens [10,33-35].

Other compounds that have been shown to reduce the levels of progerin and/or improve accelerated aging defects in HGPS cells via autophagic induction, including all-trans retinoic acid and the Nrf2 activator sulforaphane, have also been shown to activate AMPK [36-39].  As AMPK activates PGC-1a, a key transcription factor that promotes mitochondrial functionality/biogenesis and mitochondrial dysfunction characterizes HGPS cells, methylene blue has been shown to correct mitochondrial functioning in HGPS fibroblasts, increase PGC-1a levels, and ameliorate the characteristic nuclear distortion and blebbing observed in HGPS [40,41]. Expectedly, methylene blue has also been shown in independent studies to induce macroautophagy and activate AMPK in vitro and in vivo [42,43].  

Interestingly, AMPK activation has also been shown to phosphorylate and induce nuclear retention of Nrf2, a master regulator of the antioxidant response, thus enhancing Nrf2 activity [28,29].  Strikingly, a recent study demonstrated that the transcriptional activity of Nrf2 is impaired in HGPS patient cells, leading to an increase in chronic oxidative stress.  The reactivation of Nrf2 in HGPS patient cells by the Nrf2 activator oltipraz reversed nuclear aging defects and also restored the in vivo viability of HGPS patient-derived mesenchymal stem cells (MSCs) that were implanted into animal models [44].  Similar to metformin, all-trans retinoic acid, MG132, rapamycin, and methylene blue, oltipraz and/or its metabolites also induce activation of AMPK, increase expression of genes that encode proteins involved in mitochondrial fuel oxidation, increase mitochondria DNA content and oxygen consumption rate, reduce cellular reactive oxygen species (ROS) production, activate LKB1 (an upstream activator of AMPK), and increase the AMP/ATP ratio (an indication of cellular stress induction) [45-49].  Additionally, similar to MG132, which reactivates latent HIV-1 but inhibits active replication of HIV-1, several studies have shown that oltipraz and/or its metabolites inhibit replication of HIV-1, indicating that oltipraz-induced AMPK activation likely also induces immuno-modulatory effects [25,50-52].

Lastly, a recent study demonstrated that 1α,25-dihydroxyvitamin D3 (1,25D), the most potent metabolite of vitamin D, profoundly improved nuclear morphology, significantly reduced DNA damage, improved cellular proliferation, delayed premature cellular senescence, and dramatically reduced progerin production in HGPS patient cells through the promotion of vitamin D receptor (VDR) signaling [53].  Indeed, 1,25D has been shown to activate AMPK in vivo as well as alter gene splicing in cancer cells [54,55].  1,25D also plays a critical role in immune system regulation, as evidenced by an increase in activated CD4+ T cells in HIV-1 patients administered 1,25D in a placebo-controlled randomized study [56].  VDR signaling plays an integral role in T cell activation, with T cell receptor triggering inducing an upregulation of PLC-γ1 (a protein critical for T cell activation) that is dependent on 1,25D and expression of the VDR [4,57].  Interestingly, as PMA (a positive control extensively used in latent HIV-1 reactivation studies) has been demonstrated to enhance 1,25D-induced promoter binding activity of the VDR, Kitano et al. demonstrated that 1,25D, PMA/TPA, and tumor necrosis factor (TNF) stimulated HIV-1 proviral activation to similar levels in a cell line latently-infected with a monocytotropic strain of HIV-1JR-FL [4,58,59].

In conclusion, the results from the npj Aging and Mechanisms of Disease study demonstrating that metformin decreases the expression of both progerin and the splicing factor SRSF1 and alleviates pathological defects in HGPS patient-derived cells provides direct support and substantiates a hypothesis published in 2014 in which I proposed for the first time that AMPK activators including metformin will ameliorate accelerated aging defects in cells derived from HGPS patients by decreasing the levels of SRSF1, thus reducing progerin production via modulation of alternative splicing [2]. Because AMPK activation is critical for T cell activation, increased SRSF1 activity impedes T cell activation and latent HIV-1 reactivation, and the endogenous SRSF1 inhibitor p32 is upregulated on HIV-1 reactivation, the results from the npj Aging and Mechanisms of Disease study also strongly support a hypothesis published in 2015 in which I proposed for the first time that inhibition of SRSF1 by AMPK activators will promote the induction of latent HIV-1 reactivation, facilitating detection and destruction of the virus [4].  Indeed, p32 has been shown to be essential for ULK-1 mediated autophagic induction by rapamycin, a drug that improves immune system responses to viral infections and ameliorates accelerated aging defects in HGPS.  Additionally, the calcium ionophores ionomycin and A23187, both of which have been shown to activate AMPK and are used in a combinatorial fashion as positive controls to reactive latent HIV-1, also induce human oocyte activation, leading to the birth of healthy children. As AMPK activation is also essential for oocyte meiotic resumption, AMPK activation connects amelioration of accelerated aging defects in HGPS not only with latent HIV-1 reactivation, but also with oocyte activation, a process without which there can be no human life.  Moreover, phosphorylated/activated AMPK (pAMPK) has recently been discovered for the first time in human sperm, localized along the tail and across the entire acrosome in the head of the sperm [60].  Because the acrosome reaction is critical for oocyte penetration and fertilization and because compounds that increase intracellular levels of calcium, including vitamin D and A23187, have been shown to induce the acrosome reaction in human sperm and activate AMPK, AMPK activation is likely also essential for the induction of the acrosome reaction in human sperm, a process that is indispensable for the creation of all human life outside of a clinical setting [61,62].  That the symptoms of accelerated aging associated with HGPS, reactivation of latent HIV-1, oocyte activation, and the acrosome reaction in sperm is connected by common pathway, AMPK activation, is no less than astounding. As evidence continues to support and substantiate this connection, a paradigm shift in assessment of disease pathology and the practice of medicine is inevitable.      

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References:

1. 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

2. 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.

3. Laustriat D, Gide J, Barrault L, et al. In Vitro and In Vivo Modulation of Alternative Splicing by the Biguanide Metformin. Mol Ther Nucleic Acids. 2015 Nov 3;4:e262.

4. Finley J. Reactivation of latently infected HIV-1 viral reservoirs and correction of aberrant alternative splicing in the LMNA gene via AMPK activation: Common mechanism of action linking HIV-1 latency and Hutchinson-Gilford progeria syndrome. Med Hypotheses. 2015 Sep;85(3):320-32.

5. Ullrich NJ, Gordon LB. Hutchinson-Gilford progeria syndrome. Handb Clin Neurol. 2015;132:249-64.

6. McClintock D, Ratner D, Lokuge M, et al. The mutant form of lamin A that causes Hutchinson-Gilford progeria is a biomarker of cellular aging in human skin. PLoS One. 2007 Dec 5;2(12):e1269.

7. Lopez-Mejia IC, Vautrot V, De Toledo M, et al. A conserved splicing mechanism of the LMNA gene controls premature aging. Hum Mol Genet. 2011 Dec 1;20(23):4540-55.

8. Blanco FJ, Bernabéu C. The Splicing Factor SRSF1 as a Marker for Endothelial Senescence. Front Physiol. 2012 Mar 28;3:54.

9. 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.

10. Pearce EL, Walsh MC, Cejas PJ, et al. Enhancing CD8 T-cell memory by modulating fatty acid metabolism. Nature. 2009 Jul 2;460(7251):103-7.

11. Blagih J, Coulombe F, Vincent EE, et al. The energy sensor AMPK regulates T cell metabolic adaptation and effector responses in vivo. Immunity. 2015 Jan 20;42(1):41-54. 

12. Gómez-Gonzalo M, Carretero M, Rullas J et al. The hepatitis B virus X protein induces HIV-1 replication and transcription in synergy with T-cell activation signals: functional roles of NF-kappaB/NF-AT and SP1-binding sites in the HIV-1 long terminal repeat promoter. J Biol Chem. 2001 Sep 21;276(38):35435-43.

13. 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 Apr 10;6(10):7944-58.

14. Berro R, Kehn K, de la Fuente C, et al. Acetylated Tat regulates human immunodeficiency virus type 1 splicing through its interaction with the splicing regulator p32. J Virol. 2006 Apr;80(7):3189-204.

15. 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.

16. Hu M, Crawford SA, Henstridge DC, et al. p32 protein levels are integral to mitochondrial and endoplasmic reticulum morphology, cell metabolism and survival. Biochem J. 2013 Aug 1;453(3):381-91.

17. Moulton VR, Gillooly AR, Tsokos GC. Ubiquitination regulates expression of the serine/arginine-rich splicing factor 1 (SRSF1) in normal and systemic lupus erythematosus (SLE) T cells. J Biol Chem. 2014 Feb 14;289(7):4126-34.

18. Mehla R, Bivalkar-Mehla S, Zhang R, et al. Bryostatin modulates latent HIV-1 infection via PKC and AMPK signaling but inhibits acute infection in a receptor independent manner. PLoS One. 2010 Jun 16;5(6):e11160. 

19. 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.

20. Fogarty S, Hawley SA, Green KA, Saner N, Mustard KJ, Hardie DG. Calmodulin-dependent protein kinase kinase-beta activates AMPK without forming a stable complex: synergistic effects of Ca2+ and AMP. Biochem J. 2010 Jan 27;426(1):109-18.

21. Harhouri: Efficient progerin clearance through autophagy induction and SRSF-1 downregulation in Hutchinson-Gilford Progeria Syndrome. Orphanet Journal of Rare Diseases 2015 10(Suppl 2):O9.

22. http://www.abstractsonline.com/Plan/ViewAbstract.aspx?sKey=8faca3f9-1d38-414c-8cbf-768476a45b61&cKey=fafcb8e0-bbeb-498b-b8f5-cdf7f7686ec6&mKey=cabdedda-497c-457e-8481-34a866ab3681, last accessed November 28, 2016.

23. Pellegrini C, Columbaro M, Capanni C, et al. All-trans retinoic acid and rapamycin normalize Hutchinson Gilford progeria fibroblast phenotype. Oncotarget. 2015 Oct 6;6(30):29914-28.

24. Jiang S, Park DW, Gao Y, et al. Participation of proteasome-ubiquitin protein degradation in autophagy and the activation of AMP-activated protein kinase. Cell Signal. 2015 Feb 26. pii: S0898-6568(15)00070-4.

25. Miller LK, Kobayashi Y, Chen CC, Russnak TA, Ron Y, Dougherty JP. Proteasome inhibitors act as bifunctional antagonists of human immunodeficiency virus type 1 latency and replication. Retrovirology. 2013 Oct 24;10:120.

26. Hubbard VM, Valdor R, Patel B, Singh R, Cuervo AM, Macian F. Macroautophagy regulates energy metabolism during effector T cell activation. J Immunol. 2010 Dec 15;185(12):7349-57.

27. 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.

28. Mo C, Wang L, Zhang J, et al. The crosstalk between Nrf2 and AMPK signal pathways is important for the anti-inflammatory effect of berberine in LPS-stimulated macrophages and endotoxin-shocked mice. Antioxid Redox Signal. 2014 Feb 1;20(4):574-88.

29. Joo MS, Kim WD, Lee KY, Kim JH, Koo JH, Kim SG. AMPK facilitates nuclear accumulation of Nrf2 by phosphorylating at serine 550. Mol Cell Biol. 2016 May 9. pii: MCB.00118-16. [Epub ahead of print].

30. Cao K, Graziotto JJ, Blair CD, et al. Rapamycin reverses cellular phenotypes and enhances mutant protein clearance in Hutchinson-Gilford progeria syndrome cells. Sci Transl Med. 2011 Jun 29;3(89):89ra58.

31. 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.

32. Jiao H, Su GQ2, Dong W, et al. Chaperone-like protein p32 regulates ULK1 stability and autophagy. Cell Death Differ. 2015 Apr 29.

33. Araki K, Turner AP, Shaffer VO. mTOR regulates memory CD8 T-cell differentiation. Nature. 2009 Jul 2;460(7251):108-12.

34. Mannick JB, Del Giudice G, Lattanzi M, et al. mTOR inhibition improves immune function in the elderly. Sci Transl Med. 2014 Dec 24;6(268):268ra179.

35. Keating R, Hertz T, Wehenkel M, et al. The kinase mTOR modulates the antibody response to provide cross-protective immunity to lethal infection with influenza virus. Nat Immunol. 2013 Dec;14(12):1266-76.

36. Pellegrini C, Columbaro M, Capanni C, et al. All-trans retinoic acid and rapamycin normalize Hutchinson Gilford progeria fibroblast phenotype. Oncotarget. 2015 Oct 6;6(30):29914-28.

37. Gabriel D, Roedl D, Gordon LB, Djabali K. Sulforaphane enhances progerin clearance in Hutchinson-Gilford progeria fibroblasts. Aging Cell. 2015 Feb;14(1):78-91.

38. Kim YM, Kim JH, Park SW, Kim HJ, Chang KC. Retinoic acid inhibits tissue factor and HMGB1 via modulation of AMPK activity in TNF-α activated endothelial cells and LPS-injected mice. Atherosclerosis. 2015 Aug;241(2):615-23.

39. Lee JH, Jeong JK, Park SY. Sulforaphane-induced autophagy flux prevents prion protein-mediated neurotoxicity through AMPK pathway. Neuroscience. 2014 Oct 10;278:31-9.

40. Xiong ZM, Choi JY, Wang K, et al. Methylene blue alleviates nuclear and mitochondrial abnormalities in progeria. Aging Cell. 2015 Dec 14. doi: 10.1111/acel.12434.

41. 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.

42. Atamna H, Atamna W, Al-Eyd G, Shanower G, Dhahbi JM. Combined activation of the energy and cellular-defense pathways may explain the potent anti-senescence activity of methylene blue. Redox Biol. 2015 Dec;6:426-35. doi: 10.1016/j.redox.2015.09.004.

43. Xie L, Li W, Winters A, Yuan F, Jin K, Yang S. Methylene blue induces macroautophagy through 5' adenosine monophosphate-activated protein kinase pathway to protect neurons from serum deprivation. Front Cell Neurosci. 2013 May 3;7:56.

44. Kubben N, Zhang W, Wang L, et al. Repression of the Antioxidant NRF2 Pathway in Premature Aging. Cell. 2016 Jun 2;165(6):1361-74. doi: 10.1016/j.cell.2016.05.017.

45. 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. doi: 10.1111/bph.12057.

46. Bae EJ, Yang YM, Kim JW, Kim SG. Identification of a novel class of dithiolethiones that prevent hepatic insulin resistance via the adenosine monophosphate-activated protein kinase-p70 ribosomal S6 kinase-1 pathway. Hepatology. 2007 Sep;46(3):730-9.

47. Shin SM, Kim SG. Inhibition of arachidonic acid and iron-induced mitochondrial dysfunction and apoptosis by oltipraz and novel 1,2-dithiole-3-thione congeners. Mol Pharmacol. 2009 Jan;75(1):242-53. doi: 10.1124/mol.108.051128.

48. Kwon YN, Shin SM, Cho IJ, Kim SG. Oxidized metabolites of oltipraz exert cytoprotective effects against arachidonic acid through AMP-activated protein kinase-dependent cellular antioxidant effect and mitochondrial protection. Drug Metab Dispos. 2009 Jun;37(6):1187-97.

49. Hwahng SH, Ki SH, Bae EJ, Kim HE, Kim SG. Role of adenosine monophosphate-activated protein kinase-p70 ribosomal S6 kinase-1 pathway in repression of liver X receptor-alpha-dependent lipogenic gene induction and hepatic steatosis by a novel class of dithiolethiones. Hepatology. 2009 Jun;49(6):1913-25.

50. Prochaska HJ, Fernandes CL, Pantoja RM, Chavan SJ. Inhibition of human immunodeficiency virus type 1 long terminal repeat-driven transcription by an in vivo metabolite of oltipraz: implications for antiretroviral therapy. Biochem Biophys Res Commun. 1996 Apr 25;221(3):548-53.

51. Prochaska HJ, Bornmann WG, Baron P, Polsky B. Inhibition of human immunodeficiency virus type 1 replication by 7-methyl-6,8-bis(methylthio)pyrrolo[1,2-a]pyrazine, an in vivo metabolite of oltipraz. Mol Pharmacol. 1995 Jul;48(1):15-20.

52. Prochaska HJ, Chavan SJ, Baron P, Polsky B. Oltipraz, a novel inhibitor of human immunodeficiency virus type 1 (HIV-1) replication. J Cell Biochem Suppl. 1995;22:117-25.

53. Kreienkamp R, Croke M, Neumann MA, et al. Vitamin D receptor signaling improves Hutchinson-Gilford progeria syndrome cellular phenotypes. Oncotarget. 2016 Apr 27. doi: 10.18632/oncotarget.9065.

54. Swami S, Krishnan AV, Williams J, et al. Vitamin D mitigates the adverse effects of obesity on breast cancer in mice. Endocr Relat Cancer. 2016 Apr;23(4):251-64.

55. Cristobo I, Larriba MJ, de los Ríos V, García F, Muñoz A, Casal JI. Proteomic analysis of 1α,25-dihydroxyvitamin D3 action on human colon cancer cells reveals a link to splicing regulation. J Proteomics. 2011 Dec 21;75(2):384-97.

56. Bang U, Kolte L, Hitz M, et al. Correlation of increases in 1,25-dihydroxyvitamin D during vitamin D therapy with activation of CD4+ T lymphocytes in HIV-1-infected males. HIV Clin Trials. 2012 May-Jun;13(3):162-70.

57. von Essen MR, Kongsbak M, Schjerling P, Olgaard K, Odum N, Geisler C. Vitamin D controls T cell antigen receptor signaling and activation of human T cells. Nat Immunol. 2010 Apr;11(4):344-9.

58. Jiang Y, Fleet JC. Effect of phorbol 12-myristate 13-acetate activated signaling pathways on 1α, 25 dihydroxyvitamin D3 regulated human 25-hydroxyvitamin D3 24-hydroxylase gene expression in differentiated Caco-2 cells. J Cell Biochem. 2012 May;113(5):1599-607.

59. Kitano K, Rivas CI, Baldwin GC, Vera JC, Golde DW. Tumor necrosis factor-dependent production of human immunodeficiency virus 1 in chronically infected HL-60 cells. Blood. 1993 Nov 1;82(9):2742-8.

60. 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].

61. 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.

62. Blomberg Jensen M, Bjerrum PJ, Jessen TE, et al. Vitamin D is positively associated with sperm motility and increases intracellular calcium in human spermatozoa. Hum Reprod. 2011 Jun;26(6):1307-17.

AMPK identified for the first time in Human Sperm: The “Shock and Live” approach links the creation of all human life with Progeria & HIV-1

AMPK identified for the first time in Human Sperm: The “Shock and Live” approach links the creation of all human life with Progeria & HIV-1

https://www.linkedin.com/pulse/ampk-identified-first-time-human-sperm-shock-live-approach-finley?articleId=6199709017560928257#comments-6199709017560928257&trk=prof-post 

Sperm cell showing AMPK, highlighted in green, localized across the sperm tail and at the entire acrosome in the sperm head. Adapted from: See reference [1].

In line with recent findings demonstrating that cellular stress-induced activation of the master metabolic regulator AMPK is critical for oocyte meiotic resumption and maturation (processes that are essential for subsequent oocyte activation), T cell activation, and extension of lifespan, a recent study published in September of 2016 identified for the first time that AMPK is present in the whole human spermatozoa (i.e. sperm). AMPK localized along the tail, the midpiece, and at the entire acrosome, with inhibition of AMPK significantly decreasing sperm motility in all motility parameters analyzed [1].  Given that an increase in intracellular calcium levels, which activate AMPK, is critical for oocyte activation, sperm acrosome reaction, and T cell activation, and that several compounds that improve accelerated cellular aging defects in Hutchinson-Gilford progeria syndrome also promote oocyte meiotic induction, induce the acrosome reaction in human sperm, and activate AMPK, the results from this ground-breaking study provide compelling evidence that AMPK likely occupies a central node facilitating the creation of all human life, the potential eradication of HIV-1, and the amelioration of symptoms associated with accelerated aging.          

Similar to oocyte activation, which is indispensable for the creation of all human life, the sperm acrosome reaction is also indispensable for the creation of all human life outside of a clinical setting (human oocyte fertilization and embryonic development can be achieved with sperm that have not undergone the acrosome reaction via in vitro fertilization techniques such as ICSI) [2]. The sperm acrosome reaction, an exocytotic process that is indispensable for oocyte penetration and fertilization, is characterized by the release of contents (e.g. proteolytic enzymes) of the acrosome (a cap-like structure residing in the anterior portion of the sperm head) that facilitates penetration of the outer protective coat of the egg, thus allowing fusion of sperm and egg cell membranes [3].  Interestingly, as an increase in intracellular calcium, similar to oocyte activation, is critical for induction of the acrosome reaction, physiological induction of the acrosome reaction by compounds found in the vicinity of the oocyte (e.g. progesterone) or pharmacological inducers (e.g. the calcium ionophore A23187) of the acrosome reaction each share a common mechanism of promoting intracellular calcium increases in sperm [3,4].

Because an intracellular calcium increase plays a pivotal role in both oocyte activation and induction of the acrosome reaction and because AMPK activation has also been shown to promote oocyte meiotic resumption, maturation, and likely oocyte activation, the acrosome reaction in sperm would also be predicted to be critically dependent on AMPK activation [4].  Indeed, Calle-Guisado et al. identified AMPK for the first time in human spermatozoa (i.e. mature motile sperm), with localization of AMPK at the entire acrosome, midpiece, and tail [5].  The authors used semen obtained from healthy human donors by masturbation and spermatozoa motility was analyzed using motility parameters including curvilinear velocity (VCL), average path velocity (VAP), straight-line velocity (VSL), percent of motile and progressive spermatozoa, amplitude of lateral head movement (ALH), beat cross of flagellum frequency (BCF), and linearity (LIN) [5].  Using both immunoflourescence and Western blotting techniques, the authors initially demonstrated that AMPK protein levels were highly expressed in freshly ejaculated human sperm, with localization of AMPK at the entire acrosome, the midpiece, and along the tail sperm.  Strikingly, using the same techniques along with antibodies to detect the phosphorylated/activated form of AMPK (pThr172-AMPK), the authors also showed that activated AMPK is also located along human spermatozoa, with prominent staining at the most apical portion of the acrosome and at the sperm tail, providing compelling evidence that activation of AMPK likely plays a critical role in the induction of the acrosome reaction and in facilitation of sperm motility [5].  Indeed, separation of sperm fraction into low and high motility sperm for each donor revealed that activated AMPK is predominant and present with higher intensity in highly motile sperm compared to low motility sperm. Additionally, after 20 hours of treatment with compound C, a pharmacological inhibitor of AMPK, the authors noted a significant decrease (65%) in both the percent of motile sperm present and the percentage of sperm with progressive motility (67% decrease).  20 hours of treatment with compound C also led to a significant decrease in the coefficients of BCF (56% reduction), ALH (45% reduction), and LIN (60% reduction) as well as a statistically significant reduction (approximately 50%) in all sperm velocity parameters tested (VCL, VAP, and VSL) [5].

Interestingly, published at around the same time as the study by Calle-Guisado et al., a study published by Aparicio et al. also identified AMPK in human spermatozoa as well as several functionally active autophagy-related proteins, indicating that autophagy may play a crucial role in cell survival and motility [6].  Interestingly, AMPK has been shown to be a critical regulator of autophagy via phosphorylating and activating ULK1, a key initiator of autophagic induction [7].  Autophagy is an evolutionarily conserved process that maintains cellular homeostasis through targeted degradation of damaged or long-lived intracellular proteins and organelles. Aparicio et al. showed that the autophagic proteins LC3, Atg5, Atg16, Beclin 1, and p62 were present in human spermatozoa as well as AMPKα 1/2 and mTOR, two critical regulators of cellular metabolism [6].  Activation of autophagy significantly increased spermatozoa viability and motility whereas autophagy inhibition decreased viability, motility, and the intracellular concentrations of calcium and ATP.  A majority (72%) of freshly incubated sperm cells were stained for LC3 (protein widely used to study autophagic induction) in the acrosome, further indicating that activated AMPK, which also demonstrates prominent staining in the acrosome, is likely critical for the induction of the acrosome reaction [5,6].  Curiously, the ratio of LC3-II/LC3-I (indicator of autophagic induction) was increased by both chloroquine, an autophagy inhibitor that activated AMPK in sperm, and rapamycin, an autophagy activator and mTOR inhibitor that has also been shown to increase the lifespan of several model organisms [6,8]. As the authors also demonstrated that rapamycin increased progressive motility and the percentage of rapid spermatozoa, whereas Calle-Guisado et al. showed that activated AMPK showed prominent staining at the sperm acrosome and tail, it would be expected that rapamycin would also activate AMPK [5,6]. Indeed, two recent studies published in 2016 revealed for the first time that rapamycin potently induced AMPK activation in normal elderly mice in vivo [9,10].  Because rapamycin and AMPK activation have been shown to promote autophagy by activating ULK1, the results from these two studies detecting AMPK for the first time in human spermatozoa provides compelling evidence that AMPK activation is likely a critical determinant for enhancing motility and promoting the acrosome reaction in sperm [7,9].   

As noted above, the induction of the acrosome reaction and oocyte activation are both critically dependent on an increase in intracellular calcium levels.  Interestingly, the acrosome reaction and oocyte activation also share many of the same intracellular signaling mediators, including phospholipase C (PLC), diacylglycerol (DAG), and  protein kinase C (PKC) [3,4].  Because AMPK activation is essential for oocyte meiotic induction, maturation, and likely oocyte activation, and because activated AMPK displays prominent staining at the sperm acrosome and is critical for sperm motility, it would be expected that compounds that induce the acrosome reaction in sperm will also induce oocyte activation and activate AMPK.  Indeed, calcium ionophores including A23187 have been shown to induce the acrosome reaction in human spermatozoa, activate AMPK, and induce human oocyte activation, leading to the birth of normal healthy children [11-13]. Interestingly, reactive oxygen species (ROS)/oxidative stress, which also activates AMPK, has been shown to induce the acrosome reaction in human sperm and the free radical-generating agent menadione has been shown to induce AMPK-dependent meiotic resumption in cumulus-enclosed and denuded mouse oocytes via promotion of oxidative stress, indicating that induction of cellular stress (i.e. “Shock”) via increases in the levels of ROS or intracellular calcium likely induces AMPK-mediated oocyte meiotic induction/maturation and oocyte activation as well as induction of the acrosome reaction in sperm (i.e. “Live”) [11,14,15].  

Furthermore, as the induction of cellular stress (e.g. intracellular calcium increases, ROS, etc.) leads to oocyte activation and the acrosome reaction in sperm, the exposure of HIV-1 latently infected monocyte or lymphocyte cell lines to hydrogen peroxide (H2O2)/ROS has also been shown to reactivate HIV-1 and the calcium ionophore A23187 has been shown reactivate latent HIV-1 as well [16,17]. Additionally, because AMPK plays a critical role in T cell activation (and thus reactivation of latent HIV-1 that resides in T cells) and because knockdown of AMPK or the upstream AMPK kinase CaMKK2 significantly inhibits HIV-1 replication, cellular stress-induced AMPK activation appears to also link oocyte activation, sperm acrosome reaction, and reactivation of latent HIV-1 (facilitating immune system detection and destruction of the virus) [18,19].

Lastly, cellular stress-induced AMPK activation likely also links oocyte meiotic induction, oocyte activation, sperm acrosome reaction, and amelioration of accelerated cellular aging defects associated with the genetic disorder Hutchinson-Gilford progeria syndrome (HGPS).  Strikingly, 1α,25-dihydroxyvitamin D3, the most potent metabolite of vitamin D, has been shown to delay premature senescence and significantly improve accelerated aging in patient-derived HGPS cells, activate AMPK, and induce the acrosome reaction in human sperm [20-22].  MG132, a proteasome inhibitor, has also been shown to improve symptoms of accelerated aging in HGPS fibroblasts, activate AMPK, reactivate latent HIV-1, and induce oocyte meiotic resumption [23-26].  Additionally, methylene blue and retinoic acid, both of which activate AMPK, have been shown to ameliorate accelerated cellular aging defects in HGPS fibroblasts, with methylene blue also inducing oocyte meiotic induction and retinoic acid enhancing maturation and activation of both mouse and human oocytes [23,27-34].

The results from the aforementioned studies engenders a provocative and novel proposition that oocyte activation, a prerequisite for the creation of all human life, and the acrosome reaction in sperm share a common mechanism of AMPK activation that is promoted by the induction of cellular stress (e.g. ROS, intracellular calcium increase, AMP/ADP:ATP ratio increase, etc).  As first proposed in my most recent publication, this cellular stress-induced activation of AMPK also links oocyte activation and reactivation of latently infected HIV-1 reservoirs, facilitating detection and destruction of the virus by the immune system [4].  Additionally, AMPK-activating compounds (e.g. vitamin D, MG132, methylene blue) that have been shown to induce oocyte meiotic resumption and the acrosome reaction in sperm also ameliorate accelerated cellular aging defects in HGPS, a genetic disorder characterized by excessive accumulation of a toxic protein (called progerin) that is also produced in much smaller quantities by normal humans [35].  Also, as noted in my most previous LinkedIn Post, H2O2/ROS induced by the bacterium Wolbachia in a mosquito strain that serves as a vector for dengue and Zika viruses has also been shown to enhance antiviral immune responses in those mosquitoes, decreasing dengue virus replication and transmission [36].  Because AMPK activation in that mosquito strain by compounds that increase cellular stress leads to an increase in lifespan and enhancement of immune responses and because H2O2, ROS, or intracellular calcium increases have been shown to induce oocyte activation, the acrosome reaction in sperm, and reactivate latent HIV-1, the beneficial effects of cellular stress-induced AMPK activation may not only link the creation of all human life with HGPS and latent HIV-1 reactivation, but may indeed represent a unifying theme that crosses species boundaries.        

  References:

1. 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]

2. Gianaroli L, Magli MC, Ferraretti AP, et al. Birefringence characteristics in sperm heads allow for the selection of reacted spermatozoa for intracytoplasmic sperm injection. Fertil Steril. 2010 Feb;93(3):807-13.

3. Darszon A, Nishigaki T, Beltran C, Treviño CL. Calcium channels in the development, maturation, and function of spermatozoa. Physiol Rev. 2011 Oct;91(4):1305-55.

4. 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.

5. 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].

6. Aparicio IM, Espino J, Bejarano I, et al. Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility. Sci Rep. 2016 Sep 16;6:33647.

7. Kim J, Kundu M, Viollet B, Guan KL. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol. 2011 Feb;13(2):132-41.

8. Harrison DE, Strong R, Sharp ZD, et al. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice.  Nature. 2009 Jul 16;460(7253):392-5.

9. 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.

10. Lesniewski LA, Seals DR, Walker AE, et al. Dietary rapamycin supplementation reverses age-related vascular dysfunction and oxidative stress, while modulating nutrient-sensing, cell cycle, and senescence pathways. Aging Cell. 2016 Sep 22. doi: 10.1111/acel.12524. [Epub ahead of print].

11. 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.

12. Fogarty S, Hawley SA, Green KA, Saner N, Mustard KJ, Hardie DG. Calmodulin-dependent protein kinase kinase-beta activates AMPK without forming a stable complex: synergistic effects of Ca2+ and AMP. Biochem J. 2010 Jan 27;426(1):109-18.

13. Ebner T, Montag M, Oocyte Activation Study Group, et al. Live birth after artificial oocyte activation using a ready-to-use ionophore: a prospective multicentre study. Reprod Biomed Online. 2015 Apr;30(4):359-65.

14. Sundararaman A, Amirtham U, Rangarajan A. Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation. J Biol Chem. 2016 Jul 8;291(28):14410-29.

15. LaRosa C, Downs SM. Stress stimulates AMP-activated protein kinase and meiotic resumption in mouse oocytes. Biol Reprod. 2006 Mar;74(3):585-92.

16. Piette J, Legrand-Poels S. HIV-1 reactivation after an oxidative stress mediated by different reactive oxygen species. Chem Biol Interact. 1994 Jun;91(2-3):79-89.

17. Fujinaga K, Zhong Q, Nakaya T, et al. Extracellular Nef protein regulates productive HIV-1 infection from latency. J Immunol. 1995 Dec 1;155(11):5289-98.

18. 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.

19. Zhou H, Xu M, Huang Q, Gates AT, et al. Genome-scale RNAi screen for host factors required for HIV replication. Cell Host Microbe. 2008 Nov 13;4(5):495-504.

20. Kreienkamp R, Croke M, Neumann MA, et al. Vitamin D receptor signaling improves Hutchinson-Gilford progeria syndrome cellular phenotypes. Oncotarget. 2016 Apr 27. doi: 10.18632/oncotarget.9065.

21. Swami S, Krishnan AV, Williams J, et al.. Vitamin D mitigates the adverse effects of obesity on breast cancer in mice. Endocr Relat Cancer. 2016 Apr;23(4):251-64.

22. Blomberg Jensen M, Bjerrum PJ, Jessen TE, et al. Vitamin D is positively associated with sperm motility and increases intracellular calcium in human spermatozoa. Hum Reprod. 2011 Jun;26(6):1307-17.
23. Pellegrini C, Columbaro M, Capanni C, et al. All-trans retinoic acid and rapamycin normalize Hutchinson Gilford progeria fibroblast phenotype. Oncotarget. 2015 Oct 6;6(30):29914-28.

24. Jiang S, Park DW, Gao Y, et al. Participation of proteasome-ubiquitin protein degradation in autophagy and the activation of AMP-activated protein kinase. Cell Signal. 2015 Jun;27(6):1186-97.

25. Huo LJ, Fan HY, Zhong ZS, Chen DY, Schatten H, Sun QY. Ubiquitin-proteasome pathway modulates mouse oocyte meiotic maturation and fertilization via regulation of MAPK cascade and cyclin B1 degradation. Mech Dev. 2004 Oct;121(10):1275-87.

26. Miller LK, Kobayashi Y, Chen CC, Russnak TA, Ron Y, Dougherty JP. Proteasome inhibitors act as bifunctional antagonists of human immunodeficiency virus type 1 latency and replication. Retrovirology. 2013 Oct 24;10:120.

27. Xiong ZM, Choi JY, Wang K, et al. Methylene blue alleviates nuclear and mitochondrial abnormalities in progeria. Aging Cell. 2016 Apr;15(2):279-90.

28. Shin SY, Kim TH, Wu H, Choi YH, Kim SG. SIRT1 activation by methylene blue, a repurposed drug, leads to AMPK-mediated inhibition of steatosis and steatohepatitis. Eur J Pharmacol. 2014 Mar 15;727:115-24.

29. Downs SM, Humpherson PG, Leese HJ. Meiotic induction in cumulus cell-enclosed mouse oocytes: involvement of the pentose phosphate pathway. Biol Reprod. 1998 Apr;58(4):1084-94.

30. Kim YM, Kim JH, Park SW, Kim HJ, Chang KC. Retinoic acid inhibits tissue factor and HMGB1 via modulation of AMPK activity in TNF-α activated endothelial cells and LPS-injected mice. Atherosclerosis. 2015 Aug;241(2):615-23.

31. Nasiri E, Mahmoudi R, Bahadori MH, Amiri I. The Effect of Retinoic Acid on in vitro Maturation and Fertilization Rate of Mouse Germinal Vesicle Stage Oocytes. Cell J. 2011 Spring;13(1):19-24.

32. Tahaei LS, Eimani H, Yazdi PE, Ebrahimi B, Fathi R. Effects of retinoic acid on maturation of immature mouse oocytes in the presence and absence of a granulosa cell co-culture system. J Assist Reprod Genet. 2011 Jun;28(6):553-8.

33. Pauli SA, Session DR, Shang W, et al. Analysis of follicular fluid retinoids in women undergoing in vitro fertilization: retinoic acid influences embryo quality and is reduced in women with endometriosis. Reprod Sci. 2013 Sep;20(9):1116-24.

34. Best MW, Wu J, Pauli SA, et al. A role for retinoids in human oocyte fertilization: regulation of connexin 43 by retinoic acid in cumulus granulosa cells. Mol Hum Reprod. 2015 Jun;21(6):527-34.   

35. 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.

36. https://www.linkedin.com/pulse/probiotic-activates-ampk-mosquitoes-inhibiting-zika-dengue-finley?trk=pulse_spock-articles

 

“Probiotic” activates AMPK in mosquitoes, inhibiting Zika and Dengue viruses: The Shock and Live Approach links Viruses, Oocytes, Sperm, & Progeria

“Probiotic” activates AMPK in mosquitoes, inhibiting Zika and Dengue viruses: The Shock and Live Approach links Viruses, Oocytes, Sperm, & Progeria

https://www.linkedin.com/pulse/probiotic-activates-ampk-mosquitoes-inhibiting-zika-dengue-finley?trk=pulse_spock-articles

  

Reports last week from the Australia-based non-profit organization Eliminate Dengue provided details of an ongoing initiative to decrease the spread of dengue and Zika viruses through the coordinated release of male and female mosquitoes (called Aedes aegypti) that were purposely infected with a bacterium that inhibits the mosquito’s ability to transmit the two viruses to humans [1].  Set to launch in Colombia and Brazil early next year, the control campaigns will be funded with $18 million from donors including the U.S. and British governments as well as the Bill & Melinda Gates Foundation.  Interestingly, the Google health spin-off Verily also recently announced a similar initiative to prevent the spread of dengue and Zika virus transmission by using the same bacterium to infect male mosquitoes only. Infected male mosquitoes are unable to reproduce with normal/uninfected female mosquitoes [1,2].    

Interestingly, in addition to significantly inhibiting the transmission of the two viruses by Aedes aegypti to humans, the bacterium, called Wolbachia, also occurs naturally in many insects and is transmitted successfully to offspring by females after an infected female mates with an infected male, or an infected female mates with a normal/uninfected male [3].  However, a normal/uninfected female is only able to produce viable offspring with a normal/uninfected male, leading to a significant reproductive advantage for Wolbachia-infected females and thus propagation of Wolbachia throughout mosquitoes in the wild. Additionally, only female mosquitoes bite and require a blood meal to promote egg production, further highlighting the potential of reducing dengue and Zika virus transmission to humans via Wolbachia-infected female mosquitoes.

Although the method through which Wolbachia is transmitted to mosquito offspring has been well studied, how Wolbachia infection leads to a decrease in dengue and Zika virus replication in, and transmission by, mosquitoes is heavily debated.  Interestingly, although Wolbachia does not naturally occur in Aedes aegypti, Wolbachia appears to impart beneficial immunomodulatory effects in these mosquitoes not unlike that of certain beneficial bacteria that normally colonize the human gut.  Such bacteria, also known as “probiotics”, are typified by the genera Lactobacillus and Bifidobacterium and have been shown to significantly enhance natural and acquired immunity, induce reactive oxygen species (ROS) generation and the upregulation of Nrf2-dependent cytoprotective genes, produce short chain fatty acids (SCFAs) that promote T cell differentiation, and produce antimicrobial peptides that exhibit bactericidal activity [4-7].  Interestingly, the induction of cellular stress via ROS generation has been shown to activate the master metabolic regulator AMPK and AMPK activation has been shown to be critical for T cell activation and the mounting of an effective immune response in vivo to viral and bacterial infections [8-10].  Indeed, acetate, propionate, and butyrate, three SCFAs abundantly produced by probiotic bacteria, each activate AMPK, indicating that the immunomodulatory effects of probiotic bacteria are dependant on cellular stress-induced activation of AMPK [11]. Furthermore, AMPK has recently been discovered in Aedes aegypti and activation of AMPK in Aedes aegypti by compounds that induce cellular stress leads to an enhanced immune response and increased longevity in Aedes aegypti (see below), implicating the provocative assertion that inhibition of dengue and Zika virus replication via Wolbachia infection occurs as result of cellular stress-induced activation of AMPK [12].

As noted above, Wolbachia, which are found in reproductive tissues of arthropods, often engage in a mutualistic relationship with its host. Indeed, Wolbachia has been shown to induce resistance to RNA viral infections in the model organism Drosophila melanogaster [13]. D. melanogaster (also known as the fruit fly) has been studied extensively in biological research and activation of AMPK has been shown to extend lifespan as well as slow aging in D. melanogaster through autophagic induction via upregulation of Atg1 (ULK1 in mammals), reduction of insulin-like peptide levels in the brain, and an increase in 4E-BP [14,15]. 

Interestingly, insulin-like peptides and hyperphosphorylation of 4E-BP (i.e. inactivation) is positively associated with target of rapamycin (TOR) activation and egg development in Aedes aegypti (Ae. Aegypti) [16]. Mammalian target of rapamycin (mTORC1/TORC1) is a protein kinase that plays a critical role in promoting mRNA translation and is the primary target of rapamycin, a macrolide drug that extends lifespan in several model organisms including normal genetically heterogeneous mice and D. melanogaster [17,18].  Rapamycin was shown to delay yolk deposition in control Ae. aegypti eggs and inhibit insulin-mediated 4E-BP (a negative regulator of translation) phosphorylation. Silencing of 4E-BP also led to a reduction in lifespan of adult female mosquitoes, mirroring results obtained via AMPK activation in D. melanogaster [15,16,19]. Strikingly, in addition to delaying egg maturation in Ae. aegypti, rapamycin has also been shown to dramatically increase the number of Wolbachia in D. melanogaster oocytes via TORC1 inhibition, whereas somatic hyperactivation of TORC1 decreases Wolbachia titer in oocytes [20]. Interestingly, ablation of insulin-producing cells in the D. melanogaster brain abolished the yeast-induced reduction of Wolbachia in oocytes, mirroring previous results showing that AMPK-induced lifespan extension in D. melanogaster is associated with a decrease in insulin-like peptides in the brain [15,20].    

Because rapamycin, similar to AMPK activation by diverse compounds, induces autophagy, inhibits mTOR/TORC1, promotes mitochondrial biogenesis, and extends  lifespan in several model organisms, it would expected that rapamycin would also induce activation of AMPK, likely via the induction of cellular stress (i.e. increase in the levels of ROS, intracellular calcium increase, increase in the AMP:ADP/ATP ratio, etc). Indeed, two recent publications clearly demonstrated that rapamycin potently induced the activation of AMPK in vivo in normal elderly mice, along with an upregulation of ULK1 (mammalian orthologue of Atg1 critical for the induction of autophagy) and PGC-1a (a transcription factor essential for promoting mitochondrial biogenesis) [21,22].  As rapamycin has been shown to increase the number of Wolbachia in D. melanogaster oocytes, delay egg maturation and inhibit insulin-mediated 4E-BP phosphorylation in Ae. aegypti, and increase lifespan in D. melanogaster, the recent finding that activation of AMPK in Ae. aegypti also increases lifespan and improves the immune response (see below) further bolsters the notion that activation of AMPK via the induction of cellular stress likely promotes lifespan extension, immune system enhancement, and Wolbachia  propagation in oocytes in both D. melanogaster and Ae. aegypti.  Moreover, because certain bacteria (e.g. Lactobacillus) that colonize the human gut induces beneficial immune responses by inducing cellular stress (e.g. increased ROS levels) and also produce compounds that activate AMPK, the inhibition of dengue and Zika virus replication in, and transmission by, Ae. aegypti is likely the result of a Wolbachia-induced cellular stress response, leading to the up regulation of anti-viral mechanisms that are likely modulated by AMPK.

Indeed, a recent study by Wong et al. demonstrated that ROS/oxidative stress is positively correlated with Wolbachia-mediated antiviral protection in D. melanogaster [23]. The authors of the study observed that H2O2 (hydrogen peroxide) was increased 1.25- to 2- fold in flies that harbored protective strains of Wolbachia (e.g. wMelCS, wRi, wAu) compared to Wolbachia-free controls. Interestingly, flies with a null mutation in Cu/Zn SOD (an antioxidant enzyme) exhibited elevated endogenous levels of oxidative stress that mimicked Wolbachia-induced oxidative stress.  In these Wolbachia-free Cu/Zn SOD mutant flies, a 70% survival rate was observed at 4 days post-infection after Drosophila C virus infection compared to a less than 10% survival rate for non-mutant flies, indicating that elevated levels of oxidative stress induced by protective Wolbachia strains confers a survival advantage by decreasing susceptibility to viral infection via activation of signaling pathways that potentiate anti-viral immune responses [23].

As noted above, ROS/oxidative stress has been shown to activate AMPK and AMPK activation in both D. melanogaster and Ae. Aegypti leads to an increase in lifespan [8,12,15].  Interestingly, Sykiotis et al. showed that oxidants/oxidative stress promote lifespan extension in D. melanogaster males by activating the Nrf2 (CncC) pathway, a master antioxidant transcription factor that induces the expression of several antioxidant genes including NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) [24].  Curiously, Sykiotis et al. also observed that the synthetic dithiolthione oltipraz, which has been shown to activate AMPK, inhibit HIV-1 replication, and reverse accelerating aging defects in Hutchinson-Gilford progeria syndrome (see below), also induced Nrf2 signaling in flies [24-27].  AMPK has also been shown to increase the transcriptional activity of Nrf2 and increase Nrf2 nuclear retention via phosphorylation, suggesting that AMPK may represent a central node in ROS/oxidative stress-induced immune and anti-viral responses [28,29].

Indeed, a recent study by Pan et al. showed that infection of Ae. Aegypti with Wolbachia led to a ROS/oxidative stress-induced upregulation of genes associated with immunity and reduction-oxidation, thus enhancing inhibition of dengue virus replication [30]. Several gene transcripts related to the immunity and redox/stress/mitochondrion group were upregulated in Wolbachia-infected Ae. aegypti females before blood feeding and after infection with dengue virus (DENV) serotype 2, including the antimicrobial peptides cecropin D (CECD) and defensin C (DEFC) as well as the antioxidants glutathione peroxidase, CuZnSOD, MnSOD, and glutathione peroxidase [30].  A significant increase in the levels of H2O2 and in the transcript abundance of both NADPH oxidase M (NOXM) and dual oxidase 2 (DUOX2), two enzymes that generate ROS, were also observed in Wolbachia-infected mosquitoes compared to control mosquitoes.  Interestingly, the expression of several Toll pathway (a pathway that mediates the production of antioxidants and antimicrobial peptides) marker genes as well as the antimicrobial peptides CECD and DEFC were increased by the addition of H2O2 in a sugar solution given to female control/uninfected mosquitoes [30].  This effect was also mirrored in Wolbachia-infected female mosquitoes, wherein silencing of NOXM and DUOX2 deactivated the Toll pathway and suppressed the expression of CECD and DEFC, indicating that Wolbachia-induced ROS is required for activation of the Toll pathway and induction of antimicrobial peptides.  Importantly, individual or double RNAi-induced knockdown of DEFC and CECD significantly increased viral titers of DENV serotype 2 in Wolbachia-infected mosquitoes compared to controls, providing further evidence that Wolbachia-induced ROS leads to a beneficial cellular response characterized by antimicrobial-mediated inhibition of viral replication [30].

Because Wolbachia-induced ROS/oxidative stress leads to beneficial anti-viral cellular responses in both D. melanogaster and Ae. aegypti and because AMPK activation, which increases Nrf2 transcriptional activity, leads to increased lifespan in D. melanogaster, it would be expected that cellular stress-induced AMPK activation in Ae. aegypti would also lead to an increase in lifespan as well as an enhanced immune response.  Indeed, Nunes et al. showed that autophagy, immune system activation, and the average lifespan of Ae. Aegypti (a vector for dengue as well as chikungunya and Zika virus) is increased by feeding mosquitoes several plant-based polyphenols [31].  Interestingly, administration of resveratrol (derived from grapes), quercetin (found in many fruits and vegetables), epigallocathechin-3-gallate (EGCG, found in green tea), and genistein (found in soybeans) each increased average lifespan for male and female mosquitoes compared to controls.  Insects fed resveratrol also displayed higher levels of AMPK phosphorylation/activation compared to controls and compound C, a pharmacological inhibitor of AMPK, blocked the suppression of triglyceride (TG) content induced by resveratrol [31]. Resveratrol treatment also led to immunomodulatory effects, with reduced bacterial populations for female mosquitoes compared to controls, an effect that was mimicked by AICAR (a prototypical AMPK activator) but inhibited by compound C.  Interestingly, resveratrol only slightly decreased bacterial populations in vitro, indicating that the effects of resveratrol are indirect and likely associated with activation of the immune response in mosquitoes. Autophagy was also stimulated in resveratrol- and AICAR-fed mosquitoes which was abolished by silencing of AMPK [31].  

Interestingly, each of the polyphenols used in that study (resveratrol, quercetin, epigallocathechin-3-gallate, and genistein) have each been shown to induce cellular stress (e.g. ROS generation), similar to Wolbachia-induced ROS generation, in a number of mammalian cells in addition to activating AMPK [32-35].  As AMPK is activated by a number of different compounds (polyphenols, bacterial metabolites, etc.) and methodologies (e.g. electrical stimulation) that induce cellular stress, it is likely that Wolbachia-induced inhibition of dengue and Zika virus replication in and transmission by Ae. Aegypti is also modulated by AMPK, a master metabolic regulator that increases lifespan and improves immune and antioxidant responses in another Wolbachia-infected insect, D. melanogaster [36,37].  Although Nunes et al. did not determine if the polyphenols tested inhibited dengue virus replication in or transmission by Ae. Aegypti, recent studies have shown that in mammalian cells quercetin and analogs of resveratrol exhibit significant inhibitory activities against dengue virus type 2 [38,39].  EGCG has also recently been shown to inhibit Zika virus entry in Vero E6 cells (albeit at higher concentrations), indicating that cellular stress-induced activation of AMPK may represent a common mechanism of action that spans species boundaries to prevent the replication and/or transmission of dengue and Zika viruses [40].  Additionally, salidroside (derived from the plant Rhodiola rosea) and curcumin (derived from the plant Curcuma longa) have both been shown to exhibit anti-dengue virus activity in vitro and induce AMPK activation in vivo, providing further evidence that many structurally diverse compounds likely share a common mechanism of stress-induced AMPK activation to effectuate antiviral responses [41-44]. 

Remarkably, cellular stress-induced activation of AMPK (i.e. “Shock”) appears to link the antiviral effects mediated by a Wolbachia-induced increase in ROS production in A. aegypti with the amelioration of accelerated cellular aging defects in the genetic disorder Hutchinson-Gilford progeria syndrome (HGPS), the reactivation of latent HIV-1 in infected T cells (to facilitate immune system detection and viral destruction), oocyte meiotic resumption, and the acrosome reaction in sperm (i.e. “Live”).

Indeed, the proteasome inhibitor MG132 has recently been shown to inhibit progerin (the toxic protein that causes accelerated cellular aging defects) in fibroblasts derived from HGPS patients, inhibit dengue, West Nile, and Yellow fever viruses, and activate AMPK [45-47]. 1α,25-dihydroxyvitamin D3, the most potent metabolite of vitamin D, has also been shown to delay premature senescence and significantly improve accelerated aging in patient-derived HGPS cells, reduce dengue virus infection in human myelomonocyte and hepatic cell lines, and activate AMPK [48-50].  Interestingly, nuclear aging defects in HGPS patient-derived cells were also reversed via activation of the antioxidant transcription factor Nrf2 by oltipraz. Oltipraz has also been shown to activate AMPK, inhibit HIV-1 replication, and induced Nrf2 signaling in D. melanogaster [24-27]. 

Furthermore, just as Wolbachia-induced ROS/hydrogen peroxide (H2O2) generation (i.e. “Shock”) leads to a compensatory upregulation of antimicrobial peptides and an enhanced antiviral immune response in Ae. Aegypti (i.e. “Live”), the exposure of HIV-1 latently infected monocyte or lymphocyte cell lines to H2O2 has also been shown to reactivate HIV-1 [51]. Additionally, ROS induces activation of AMPK and AMPK plays a critical role in T cell activation (and thus reactivation of latent HIV-1 that resides in T cell cells) [8,52].

AMPK activation has also been shown to play a critical role in the initiation of oocyte meiotic resumption and maturation (in preparation for oocyte activation) and the free radical-generating agent menadione (i.e. “Shock”) has been shown to induce AMPK-dependent meiotic resumption in cumulus-enclosed and denuded mouse oocytes (i.e. “Live”) via promotion of oxidative stress [53].  Lastly, ROS also plays a critical role in the induction of the acrosome reaction in sperm, a process that facilitates oocyte penetration through release of hydrolytic enzymes and is indispensable, along with oocyte activation, for the creation of all human life outside of a clinical setting [54].  Strikingly, AMPK has recently been found for the first time to be localized across the entire acrosome in human sperm and both H2O2/ROS and vitamin D (i.e. “Shock) have been shown to induce the acrosome reaction in human sperm (i.e. “Live”), providing compelling evidence that cellular stress-induced AMPK activation is critical for the induction of the acrosome reaction in human sperm and the creation of human life [55-57].

The antiviral effects mediated by Wolbachia-induced ROS generation paints a clear yet provocative portrait that cellular-stress mediated activation of AMPK represents a common mechanism of action that spans species boundaries, beneficially modulating cellular processes that are involved in the immune response, fertilization, and aging itself.  Indeed, structurally distinct compounds including MG132 and vitamin D have been shown to exert potent antiviral effects against dengue virus and significantly improve accelerated aging defects in HGPS.  More tellingly, H2O2/ROS/oxidative stress has been shown to reactivate latent HIV-1, promote oocyte meiotic resumption, and induce the acrosome reaction in human sperm.  Because AMPK is critical for oocyte meiotic resumption (and hence oocyte activation) and AMPK has recently been found for the first time to be localized across the entire acrosome in human sperm, AMPK activation may indeed be critical for the creation of all human life, as originally proposed in my recent publication [58].  Perhaps beyond perplexing on first glance, Wolbachia, mosquitoes, and the creation of all human life are likely connected.

References

1. http://www.reuters.com/article/us-health-dengue-mosquitoes-idUSKCN12Q1PE

2. https://www.bloomberg.com/news/articles/2016-10-06/alphabet-s-verily-joins-zika-fight-with-sterile-mosquito-lab

3. Werren JH. Biology of Wolbachia. Annu Rev Entomol. 1997;42:587-609.

4. Castillo NA, Perdigón G, de Moreno de Leblanc A. Oral administration of a probiotic Lactobacillus modulates cytokine production and TLR expression improving the immune response against Salmonella enterica serovar Typhimurium infection in mice. BMC Microbiol. 2011 Aug 3;11:177.

5. Gill HS, Rutherfurd KJ, Prasad J, Gopal PK. Enhancement of natural and acquired immunity by Lactobacillus rhamnosus (HN001), Lactobacillus acidophilus (HN017) and Bifidobacterium lactis (HN019). Br J Nutr. 2000 Feb;83(2):167-76.

6. Jones RM, Desai C, Darby TM, et al. Lactobacilli Modulate Epithelial Cytoprotection through the Nrf2 Pathway. Cell Rep. 2015 Aug 25;12(8):1217-25.

7. Park J, Kim M, Kang SG, et al. Short-chain fatty acids induce both effector and regulatory T cells by suppression of histone deacetylases and regulation of the mTOR-S6K pathway. Mucosal Immunol. 2015 Jan;8(1):80-93.

8. Mungai PT, Waypa GB, Jairaman A, et al. Hypoxia triggers AMPK activation through reactive oxygen species-mediated activation of calcium release-activated calcium channels. Mol Cell Biol. 2011 Sep;31(17):3531-45.

9. 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;203(7):1665–70.

10. Blagih J, Coulombe F, Vincent EE, et al. The energy sensor AMPK regulates T cell metabolic adaptation and effector responses in vivo. Immunity. 2015 Jan 20;42(1):41-54.

11. Elamin EE, Masclee AA, Dekker J, Pieters HJ, Jonkers DM. Short-chain fatty acids activate AMP-activated protein kinase and ameliorate ethanol-induced intestinal barrier dysfunction in Caco-2 cell monolayers. J Nutr. 2013 Dec;143(12):1872-81.

12. Nunes RD, Ventura-Martins G, Moretti DM, et al. Polyphenol-Rich Diets Exacerbate AMPK-Mediated Autophagy, Decreasing Proliferation of Mosquito Midgut Microbiota, and Extending Vector Lifespan. PLoS Negl Trop Dis. 2016 Oct 12;10(10):e0005034.

13. Teixeira L, Ferreira A, Ashburner M. The bacterial symbiont Wolbachia induces resistance to RNA viral infections in Drosophila melanogaster. PLoS Biol. 2008 Dec 23;6(12):e2.

14. Yang S, Long LH, Li D, et al. β-Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP-activated protein kinase-dependent increase in autophagy. Aging Cell. 2015 Dec;14(6):1024-33.

15. Ulgherait M, Rana A, Rera M, Graniel J, Walker DW. AMPK modulates tissue and organismal aging in a non-cell-autonomous manner. Cell Rep. 2014 Sep 25;8(6):1767-80.

16. Gulia-Nuss M, Robertson AE, Brown MR, Strand MR. Insulin-like peptides and the target of rapamycin pathway coordinately regulate blood digestion and egg maturation in the mosquito Aedes aegypti. PLoS One. 2011;6(5):e20401.

17. Harrison DE, Strong R, Sharp ZD, et al. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature. 2009 Jul 16;460(7253):392-5.

18. Sun X, Wheeler CT, Yolitz J, et al. A mitochondrial ATP synthase subunit interacts with TOR signaling to modulate protein homeostasis and lifespan in Drosophila. Cell Rep. 2014 Sep 25;8(6):1781-92.

19. Roy SG, Raikhel AS. Nutritional and hormonal regulation of the TOR effector 4E-binding protein (4E-BP) in the mosquito Aedes aegypti. FASEB J. 2012 Mar;26(3):1334-42.

20. Serbus LR, White PM, Silva JP, et al. The impact of host diet on Wolbachia titer in Drosophila. PLoS Pathog. 2015 Mar 31;11(3):e1004777.

21. 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.

22. Lesniewski LA, Seals DR, Walker AE, et al. Dietary rapamycin supplementation reverses age-related vascular dysfunction and oxidative stress, while modulating nutrient-sensing, cell cycle, and senescence pathways. Aging Cell. 2016 Sep 22. doi: 10.1111/acel.12524. [Epub ahead of print].

23. Wong ZS, Brownlie JC, Johnson KN. Oxidative stress correlates with Wolbachia-mediated antiviral protection in Wolbachia-Drosophila associations. Appl Environ Microbiol. 2015 May 1;81(9):3001-5.

24. Sykiotis GP, Bohmann D. Keap1/Nrf2 signaling regulates oxidative stress tolerance and lifespan in Drosophila. Dev Cell. 2008 Jan;14(1):76-85.

25. Prochaska HJ, Fernandes CL, Pantoja RM, Chavan SJ. Inhibition of human immunodeficiency virus type 1 long terminal repeat-driven transcription by an in vivo metabolite of oltipraz: implications for antiretroviral therapy. Biochem Biophys Res Commun. 1996 Apr 25;221(3):548-53.

26. Kubben N, Zhang W2, Wang L, et al. Repression of the Antioxidant NRF2 Pathway in Premature Aging. Cell. 2016 Jun 2;165(6):1361-74.

27. 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. doi: 10.1111/bph.12057.

28. Mo C, Wang L, Zhang J, et al. The crosstalk between Nrf2 and AMPK signal pathways is important for the anti-inflammatory effect of berberine in LPS-stimulated macrophages and endotoxin-shocked mice. Antioxid Redox Signal. 2014 Feb 1;20(4):574-88.

29. Joo MS, Kim WD, Lee KY, Kim JH, Koo JH, Kim SG. AMPK Facilitates Nuclear Accumulation of Nrf2 by Phosphorylating at Serine 550. Mol Cell Biol. 2016 Jun 29;36(14):1931-42.

30. Pan X, Zhou G, Wu J, et al. Wolbachia induces reactive oxygen species (ROS)-dependent activation of the Toll pathway to control dengue virus in the mosquito Aedes aegypti. Proc Natl Acad Sci U S A. 2012 Jan 3;109(1):E23-31.

31. Nunes RD, Ventura-Martins G, Moretti DM, et al. Polyphenol-Rich Diets Exacerbate AMPK-Mediated Autophagy, Decreasing Proliferation of Mosquito Midgut Microbiota, and Extending Vector Lifespan. PLoS Negl Trop Dis. 2016 Oct 12;10(10):e0005034. 

32. Li GX, Chen YK, Hou Z, et al. Pro-oxidative activities and dose-response relationship of (-)-epigallocatechin-3-gallate in the inhibition of lung cancer cell growth: a comparative study in vivo and in vitro. Carcinogenesis. 2010 May;31(5):902-10.

33. Chang YF, Chi CW, Wang JJ. Reactive oxygen species production is involved in quercetin-induced apoptosis in human hepatoma cells. Nutr Cancer. 2006;55(2):201-9.

34. Miki H, Uehara N, Kimura A, et al. Resveratrol induces apoptosis via ROS-triggered autophagy in human colon cancer cells. Int J Oncol. 2012 Apr;40(4):1020-8.

35. Ullah MF, Ahmad A, Zubair H, et al. Soy isoflavone genistein induces cell death in breast cancer cells through mobilization of endogenous copper ions and generation of reactive oxygen species. Mol Nutr Food Res. 2011 Apr;55(4):553-9.

36. Matsuyama S, Moriuchi M1, Suico MA, et al. Mild electrical stimulation increases stress resistance and suppresses fat accumulation via activation of LKB1-AMPK signaling pathway in C. elegans. PLoS One. 2014 Dec 9;9(12):e114690.

37. Dutra HL, Rocha MN, Dias FB, Mansur SB, Caragata EP, Moreira LA. Wolbachia Blocks Currently Circulating Zika Virus Isolates in Brazilian Aedes aegypti Mosquitoes. Cell Host Microbe. 2016 Jun 8;19(6):771-4.

38. Zandi K, Teoh BT, Sam SS, Wong PF, Mustafa MR, Abubakar S. Antiviral activity of four types of bioflavonoid against dengue virus type-2. Virol J. 2011 Dec 28;8:560.

39. Han YS, Penthala NR2, Oliveira M, et al. Identification of resveratrol analogs as potent anti-dengue agents using a cell-based assay. J Med Virol. 2016 Aug 10. doi: 10.1002/jmv.24660. [Epub ahead of print].

40. Carneiro BM, Batista MN, Braga AC, Nogueira ML, Rahal P. The green tea molecule EGCG inhibits Zika virus entry. Virology. 2016 Sep;496:215-8.

41. Padilla-S L, Rodríguez A, Gonzales MM, Gallego-G JC, Castaño-O JC. Inhibitory effects of curcumin on dengue virus type 2-infected cells in vitro. Arch Virol. 2014 Mar;159(3):573-9.

42. Sharma N, Mishra KP, Ganju L. Salidroside exhibits anti-dengue virus activity by upregulating host innate immune factors. Arch Virol. 2016 Dec;161(12):3331-3344.

43. Chang X, Zhang K, Zhou R, et al. Cardioprotective effects of salidroside on myocardial ischemia-reperfusion injury in coronary artery occlusion-induced rats and Langendorff-perfused rat hearts. Int J Cardiol. 2016 Jul 15;215:532-44.

44. Ejaz A, Wu D, Kwan P, Meydani M. Curcumin inhibits adipogenesis in 3T3-L1 adipocytes and angiogenesis and obesity in C57/BL mice.

45.  Pellegrini C, Columbaro M, Capanni C, et al. All-trans retinoic acid and rapamycin normalize Hutchinson Gilford progeria fibroblast phenotype. Oncotarget. 2015 Oct 6;6(30):29914-28.

46. 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.

47.  Jiang S, Park DW, Gao Y, et al. Participation of proteasome-ubiquitin protein degradation in autophagy and the activation of AMP-activated protein kinase. Cell Signal. 2015 Jun;27(6):1186-97.

48. Kreienkamp R, Croke M, Neumann MA, et al. Vitamin D receptor signaling improves Hutchinson-Gilford progeria syndrome cellular phenotypes. Oncotarget. 2016 Apr 27. doi: 10.18632/oncotarget.9065.

49. Puerta-Guardo H, Medina F, De la Cruz Hernández SI, Rosales VH, Ludert JE, del Angel RM. The 1α,25-dihydroxy-vitamin D3 reduces dengue virus infection in human myelomonocyte (U937) and hepatic (Huh-7) cell lines and cytokine production in the infected monocytes. Antiviral Res. 2012 Apr;94(1):57-61.

50.  Swami S, Krishnan AV, Williams J, et al.. Vitamin D mitigates the adverse effects of obesity on breast cancer in mice. Endocr Relat Cancer. 2016 Apr;23(4):251-64.

51. Piette J, Legrand-Poels S. HIV-1 reactivation after an oxidative stress mediated by different reactive oxygen species. Chem Biol Interact. 1994 Jun;91(2-3):79-89.

52. 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.

53. LaRosa C, Downs SM. Stress stimulates AMP-activated protein kinase and meiotic resumption in mouse oocytes. Biol Reprod. 2006 Mar;74(3):585-92.

54. Patrat C, Serres C, Jouannet P. The acrosome reaction in human spermatozoa. Biol Cell. 2000 Jul;92(3-4):255-66.

55. 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].

56. 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.

57. Blomberg Jensen M, Bjerrum PJ, Jessen TE, et al. Vitamin D is positively associated with sperm motility and increases intracellular calcium in human spermatozoa. Hum Reprod. 2011 Jun;26(6):1307-17.

58. 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.