Metformin shown for the first time to inhibit Dengue virus in human liver cells via AMPK: AMPK links Viral Eradication with Oocytes, Sperm, & Progeria

A study recently published in the journal PLoS Pathogens in April of 2017 provided startling evidence demonstrating for the first time that the commonly-prescribed anti-diabetic drug metformin exerted significant antiviral effects in dengue virus-infected human liver cells that was dependent on activation of the master metabolic regulator AMPK [59]. The authors showed that an in increase HMG-CoA reductase (HMGCR) activity, a target of AMPK, was associated with dengue virus (DENV)-infected cells, AMPK activation was reduced in DENV-infected cells at 12 and 24 hours post infection (hpi), and metformin significantly decreased the number of infected cells, viral yield, and viral genome copies, leading the authors to conclude that metformin-induced AMPK activation generates a strong antiviral effect against DENV [59]. Interestingly, as discussed below, recent efforts funded by the U.S. and British governments, the Bill & Melinda Gates Foundation, and the Google health spin-off Verily have sought to decrease the spread of dengue and Zika viruses through the coordinated release of female and/or male mosquitoes (called Aedes aegypti) that were purposely infected with a bacterium that inhibits the mosquito’s ability to transmit the two viruses to humans [1]. Studies have shown that this bacterium, called Wolbachia, enhances the mosquito’s immune response by increasing the levels of reactive oxygen species (ROS), thus enhancing inhibition of dengue virus replication [30]. Because AMPK is activated by cellular stress (e.g. ROS increase, intracellular calcium [Ca2+] increase, AMP/ATP ratio increase, etc.), has been found in Aedes aegypti (Ae. aegypti), and AMPK activation by stress-inducing compounds (e.g. resveratrol) increased average life span and enhanced the immune response in Ae. aegypti in an AMPK-dependent manner, the recent finding that metformin also inhibits DENV replication in human cells in an AMPK-dependent manner provides compelling evidence that the anti-viral effects of AMPK activation likely crosses species boundaries [31]. Additionally, metformin has recently been shown to beneficially alter gene splicing, activate AMPK, and ameliorate accelerated aging defects in cells derived from Hutchison-Gilford progeria syndrome patients (HGPS), as I first hypothesized and published in 2014 [60-62]. AMPK activation also promotes oocyte meiotic induction and maturation (in preparation for oocyte activation) and AMPK has recently been found localized across the entire acrosome in human spermatozoa [53,55]. The induction of cellular stress (e.g. ROS, intracellular Ca2+, and/or AMP/ATP ratio increase) also promotes oocyte meiotic induction/maturation, oocyte activation, and the acrosome reaction in human sperm, processes critical for the creation of all human life [53,56,63]. As further discussed below, such interconnectedness implicates AMPK as a central mediator in the promotion of lifespan and healthspan, amelioration of pathological aging, the mounting of an effective immune response, and the creation of all human life.   

Transmission of DENV by mosquito vectors including Ae. aegypti may lead to febrile illness (known as dengue fever) in humans characterized by fluid retention, respiratory distress, and/or organ impairment [59]. There are four DENV serotypes (DENV 1-4) and several structural (capsid (C), membrane (M), and envelope (E)) and nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are encoded by the DENV genome. Interestingly, lipids and the formation of replication complexes (i.e. membranous compartments) have been shown to play important roles in the DENV replication cycle whereas the viral protein NS4A is directly associated with DENV-associated membrane rearrangement [59]. The authors first showed that AMPK activation was reduced in DENV-infected Huh7 cells (serotype 2 and 4, 2/4) at 12 and 24 hpi compared to mock-treated cells. Importantly, metformin enhanced AMPK activation in DENV 2/4-infected cells compared to mock/vehicle-treated cells, reduced NS3 levels compared to vehicle-treated cells, and reduced the levels of the viral structural proteins E and prM. DENV 2/4 infection also increased the activity of HMGCR (a rate-controlling enzyme of the cholesterol biosynthetic pathway) in vehicle-treated cells compared to mock-infected cells, whereas metformin disturbed the co-localization between HMGCR and NS4A or NS3, disrupted replicative complex integrity, and decreased the levels of the viral proteins NS4A and NS3. Furthermore, metformin also led to a reduction in the levels of DENV dsRNA, indicating that metformin exerted a strong antiviral effect against DENV [59].

Indeed, 24 hour treatment of DENV2/4-infected Huh7 cells with metformin led to a reduction in the amount of infected cells, decreased the viral yield up to one logarithm, and reduced NS1 secretion up to 90%. Metformin also led to a dose-dependent reduction in viral genome copies (up to 0.7 logarithm for DENV2 and 1.5 logarithm for DENV4) compared to non-treated cells [59]. Additionally, treatment of DENV-infected cells with the AMPK inhibitor compound C (CC) increased viral infection compared to non-treated cells and CC-induced AMPK inhibition increased viral genome copies up to a half logarithm in DENV 2- and up to 0.7 logarithm in DENV 4-infected cells, indicating that metformin’s potent antiviral effects against DENV infection and replication is dependent on AMPK activation [59].     

Metformin’s AMPK-dependent antiviral effects against DENV in human liver cells likely represent a common mechanism that crosses species boundaries to effectuate viral eradication. As noted above, current efforts by organizations including the Bill and Melinda Gates foundation and the Google health spin-off Verily has focused on reducing transmission of dengue and Zika viruses by infecting the mosquito Ae. aegypti with the bacterium Wolbachia, leading to inhibition of viral replication. As explained below, inhibition of DENV replication in both mosquitoes and in human cells is likely orchestrated by stress-induced AMPK activation.

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 Ae. 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 Ae. aegypti and activation of AMPK in Ae. aegypti by compounds that induce cellular stress leads to an enhanced immune response and increased longevity in Ae. aegypti (see below), implicating the provocative assertion that inhibition of dengue and Zika virus replication via Wolbachia infection occurs as a result of cellular stress-induced activation of AMPK [12].

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 autophagy 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 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 Ca2+ 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].

A recent study by Pan et al. showed that infection of Ae. Aegypti with Wolbachia (wAlbB strain) 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 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, metformin 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 virus 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]. Also, as discussed above, metformin has recently been shown to beneficially alter gene splicing, activate AMPK, and ameliorate accelerated aging defects in cells derived from Hutchison-Gilford progeria syndrome patients (HGPS) and significantly inhibit dengue virus infection and replication in human liver cells [59,60-62]. 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 induce 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) [8,52,64].

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 metformin, 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.

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

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61. 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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63. 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.
64. 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.

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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AMPK links beneficial responses to the “Fever Effect” in Autism with Progeria, HIV-1 latency, & Oocyte activation: The "Shock and Live" approach

Hey Blogger Fam, check out my latest post when you have a minute. The post basically explains why some children with Autism get better temporarily (i.e. improvement in speech, decrease in repetitive behaviors) when they get a fever.  Also known as the “fever effect”, this is basically another rendition of the “shock and live” approach, in which the induction of mild stress to a cell, in this case heat stress, leads to the activation of the master metabolic regulator AMPK and a beneficial cellular response.  The child’s behavior improves because mild heat stress or a “shock” to neurons of the brain causes those brain cells to do what they were designed to do that much better.  The broccoli sprout compound sulforaphane, which activates AMPK, also activates the heat shock response and has shown efficacious results in patients with autism and also improves accelerated aging defects in skin cells taken from progeria patients.  How is this possible? Is the correct functioning of neurons related to slowing or reversing accelerated aging defects? Indeed it is.  Heat stress, which has been shown in independent studies to activate AMPK, is yet an additional term that equates to “what doesn’t kill you makes you stronger”.  Heat stress, hyperthermia, or the activation of heat shock proteins have been shown to promote T cell functionality, wake up dormant HIV-1 viruses so that the immune system can detect and kill it, promote the maturation of oocytes in preparation for artificial oocyte activation or activation by sperm, promote the induction of the acrosome reaction in sperm (a process necessary for oocyte penetration), promote “activation” and killing of cancer stem cells, promote the develop of embryonic stem cells into cells that will eventually make up the body of the baby, and enhance learning and memory in the brain.  The fancy term to describe the process that enhances learning and memory is called long-term potentiation, which basically involves a strengthening in connections between neurons if they are continuously challenged. Heat stress, an intellectually stimulating environment, exercise, and countless naturally-occurring compounds all enhance long-term potentiation, learning and memory, and activate AMPK. Interestingly, high-frequency stimulation and deep brain stimulation also activate AMPK in neurons and improve neurological symptoms, indicating that mild transient electrical pulses are another form of stress.  Lastly, a stark example of how heat stress and learning and memory in neurons are connected to the creation of human life involves a heat-sensitive channel called TRPV3. Mild heat stress and a compound from oregano (called carvacrol) activates this channel.  This channel is also present in the brain and in oocytes.  If you knock this channel out in the brain, long-term potentiation and learning and memory are impaired.  However, if you activate this channel on oocytes with the oregano compound, the oocyte will become activated as if it had been fertilized by sperm (called parthenogenesis).  Again, heat stress and compounds that induce mild stress promotes a compensatory and beneficial response from a cell. This response is orchestrated by AMPK activation and results in a “shock to live” (e.g. oocyte maturation/activation, stem cell “activation”, learning and memory in the brain, sperm capacitation/acrosome reaction) or a “shock to kill” (e.g. latent HIV-1 reactivation, cancer stem cell “activation” and/or cell death).  
      

AMPK links beneficial responses to the “Fever Effect” in Autism with Progeria, HIV-1 latency, & Oocyte activation: The "Shock and Live" approach

https://www.linkedin.com/pulse/ampk-links-beneficial-responses-fever-effect-autism-progeria-finley?trk=pulse_spock-articles 

In line with recent findings demonstrating that sulforaphane, an isothiocyanate heavily concentrated in broccoli sprouts, significantly ameliorated accelerated aging defects in cells derived from patients diagnosed with Hutchinson-Gilford progeria syndrome (HGPS), a recently completed placebo-controlled, double-blind, randomized trial by researchers from Harvard Medical School, The Johns Hopkins University School of Medicine, and University of Massachusetts Medical School demonstrated that participants diagnosed with moderate to severe Autism Spectrum Disorder (ASD) who received sulforaphane showed substantial and significant improvements in behavior, social interaction, and verbal communication compared to participants assigned to placebo [1,2].  Discontinuation of sulforaphane also led to a reversal of these improvements, mirroring levels obtained before initiation of treatment [2].

Interestingly, parents and clinicians, over the past few decades, have reported noticeable improvements in behavior in children with ASD during or after the onset of febrile illness (i.e. fever).  Also known as the “fever effect”, transient increases in body temperature may positively influence neuronal and synaptic function in the brain of ASD patients, an effect that appears to be at least partially mimicked by sulforaphane. As described below, because sulforaphane and other compounds regulate proteins that are critical for the induction of the heat shock response and because heat shock proteins also play critical roles in the reactivation of latent HIV-1, oocyte maturation, sperm capacitation/acrosome reaction, the promotion of learning and memory in the brain, and differentiation of adult stem cells, the “fever effect” likely represents a “shock” or the induction of cellular stress, leading to the activation of the master metabolic regulator AMPK, resulting in a beneficial cellular stress response. The response to this stressor generates a cell-specific “shock to live” (e.g. oocyte maturation/activation, stem cell differentiation, stimulation of learning and memory, sperm capacitation/acrosome reaction) or a “shock to kill” (e.g. latent HIV-1 reactivation, cancer stem cell differentiation and/or cell death).

ASD, a neuro-developmental disorder that disproportionately affects males, is characterized by repetitive or compulsive behaviors, impairments in social development, and deficiencies in verbal and non-verbal communication that are often observable within the first two years of life.  Causes underlying ASD also appear to be multi-factorial, with interactions between environmental factors and genetics leading to deleterious alterations in neuronal network functionality and immune system regulation [3-5].  As noted above, anecdotes and case reports have indicated that amelioration of symptoms associated with ASD are positively correlated with the onset of febrile illness. A particularly interesting example of the “fever effect” in ASD occurred at New York University’s Bellevue Psychiatric Hospital, in which improvement in concentration and social interactions were noted in children with temperatures between 38.9 to 40.6°C, due to an outbreak of viral upper respiratory tract infections [6-8].  Indeed, a prospective study of 30 children with ASD during and after febrile illness (body temperature greater than or equal to 38.0°C) revealed that compared to afebrile ASD patients, fewer aberrant behaviors for febrile ASD patients were recorded on the Aberrant Behavior Checklist subscales of irritability, hyperactivity, stereotypy (i.e. persistent repetition of an act), and inappropriate speech, indicating a transient enhancement or correction of neuronal functioning in response to fever onset [8].

Because sulforaphane has been shown to transiently induce cellular stress, resulting in the upregulation of the master antioxidant transcription factor Nrf2 and activation of AMPK (which activates Nrf2), febrile illness likely induces a transient induction of cellular stress (i.e. “heat shock”), resulting in a compensatory and beneficial upregulation of cellular factors that enhance or promote neuronal functioning [9,10,11]. Indeed, Singh et al. recently demonstrated in a placebo-controlled, double-blind, randomized trial that patients receiving sulforaphane showed significant behavior improvements as measured by the Aberrant Behavior Checklist and the Social Responsiveness Scale compared to patients receiving placebo [2].  Patients who received sulforaphane also experienced improvements in social interaction, abnormal behavior, and verbal communication on the Clinical Global Impression Improvement Scale [2].

Because sulforaphane induces cellular stress via an increase in stressors including reactive oxygen species (ROS) and because febrile illness represents the induction of cellular stress, the beneficial effects of sulforaphane demonstrated in ASD patients suggests that sulforaphane may also induce cellular stress and a beneficial cellular response by activating or upregulating mediators that promote the heat shock response. Heat shock consists of subjecting a cell to a higher temperature than the ideal body temperature of an organism.  The heat shock response is a cellular response to heat shock that induces the activation of heat shock factor-1 (HSF1), the major regulator of heat shock protein (HSP) transcription, and the upregulation of HSPs that aid in repairing or targeting misfolded proteins for degradation [12].  Interestingly, HSPs also respond to other forms of cellular stress, including ROS and intracellular calcium (Ca2+) increases, both of which also activate AMPK [13-15].  Interestingly, sulforaphane has been shown to induce a significant and rapid HSF1 mediated heat shock response and heat stress has also been shown to increase ROS production, promoting nuclear translocation of Nrf2 and upregulation of Nrf2 target genes [16,17]. Furthermore, as sulforaphane activates AMPK and AMPK activates Nrf2, heat stress has also been shown to activate AMPK, promoting insulin-independent glucose transport in muscle cells and the killing of breast and pancreatic cancer stem cells [18,19].  HSP90 has also been to found to interact with and maintain AMPK activity, providing further evidence that the induction of  heat shock likely leads to activation of AMPK and a beneficial cellular response [53].

Interestingly, as sulforaphane has been shown to activate both AMPK and Nrf2 and significantly ameliorate accelerated aging defects associated with Hutchinson-Gilford progeria syndrome (HGPS) (which is associated with dysfunctional Nrf2 signaling), other AMPK-activating compounds that have demonstrated efficacious results in HGPS may also be expected to induce certain mediators constituting the heat shock response [1].  Indeed, the proteasome inhibitor MG132, the macrolide rapamycin, and vitamin D have each been shown to improve symptoms of accelerated aging in HGPS fibroblasts, activate HSF1 or increase HSP expression, and activate AMPK, indicating that chemically distinct compounds including sulforaphane, MG132, rapamycin, and vitamin D likely induce cellular stress, leading to the activation of AMPK and a beneficial cellular response in diseases as disparate as HGPS and ASD [20-28].

As febrile illness and the induction of the heat shock response have been shown to mitigate irritability, hyperactivity, stereotypy, and inappropriate speech in ASD patients, it would be expected that the application of heat stress or the induction of mediators of the heat shock response would also enhance or improve certain aspects of neuronal functionality.  Indeed, heat stress alone and heat stress preconditioning before diffuse axonal injury led to higher expression levels of HSP70 and a significant improvement in the Morris Water Maze task (a behavioral procedure used to study spatial learning and memory) and long-term potentiation (LTP) in rats compared with diffuse axonal injury alone [29]. LTP is characterized by a “persistent increase in synaptic strength following high-frequency stimulation” and is often studied in pyramidal neurons of the hippocampus, an area of the brain important for learning and memory [30].  Interestingly, neuronal depolarization (i.e. activation) has been shown to activate HSF1, heat shock has been shown to improve synaptic integrity and memory consolidation, and HSF1 agonists (e.g. exercise) have been shown to improve cognition in models of dementia [31]. A recent study by Notenboom et al. also showed that prolonged hyperthermia in rats enhanced hippocampal CA1 long-term potentiation and sprouting of mossy fiber collaterals into the dentate gyrus [32].  Perhaps most proactively, however, is a recent study by Brown et al. demonstrating that high-frequency stimulation-induced LTP was attenuated at CA3-CA1 pyramidal cell synapses in hippocampal slices from Trpv1 and Trpv3 knockout (KO) mice [33].

Interestingly, carvacrol, a monoterpenoid phenol found in the essential oil of Origanum vulgare (oregano), is a potent activator of the transient receptor potential cation channel, subfamily V, member 3 (TRPV3), a nonselective cation channel that functions in vasoregulation and temperature sensation and is activated between the temperatures of 22 and 40 degrees Celsius [34,35].  The TRPV3 channel has also been shown to associate with and form heteromeric channels with the Ca2+-permeable TRPV1 channel, another nonselective cation channel that is also regulated by temperature sensation and is activated by both physical and chemical stimuli, including temperatures greater than 43 degrees Celsius as well as the natural compound capsaicin (an active component of chili peppers from the genus Capsicum) [35-37]. As both carvacrol and capsaicin have been shown to activate AMPK and heat stress has been shown to both enhance hippocampal LTP and activate AMPK, AMPK activation likely represents a common mechanism of action explaining the therapeutic actions of several chemically distinct compounds and methodologies in the promotion of synaptic plasticity and LTP [38,39].  Indeed, a recent study demonstrated that high frequency stimulation of hippocampal neurons in the dentate gyrus activates AMPK and induces early-phase long-term potentiation (E-LTP) in vivo in rats [40]. E-LTP was prevented by pharmacological inhibition of AMPK, highlighting a critical role for AMPK in learning and memory [40]. High frequency deep brain stimulation of the lateral habenula (an area of the brain that plays an important role in emotion, motivation, and reward) has also been shown to activate AMPK, facilitating antidepressant actions in an animal model of tricyclic antidepressant resistance [41].  Resveratrol, a phytoalexin found in grapes and red wine, has also been shown to rapidly increase  protein levels and synaptic accumulation of the AMPA receptor and increase the strength of excitatory synaptic transmission in rat primary neurons via AMPK activation [42]. Because AMPA receptors mediate fast excitatory transmission and are essential for the induction of LTP and synaptic plasticity, AMPK activation likely represents a central node underlying the beneficial effects of heat stress and compounds such as sulforaphane in ameliorating neuro-developmental effects associated ASD.

Interestingly, because knockdown of TRPV3 channels (channels that are activated in response to mild temperature elevations and by compounds including carvacrol) attenuates LTP in hippocampal pyramidal neurons and because TRPV3 channels are also located on oocytes, it would be expected that mild and transient heat stress would enhance or promote oocyte maturation and/or activation.  Indeed, AMPK activation has been consistently shown to play a critical role in the induction of oocyte meiotic resumption and maturation in preparation for oocyte activation and heat stress has also been shown to stimulate oocyte meiotic resumption and maturation in an AMPK-dependent manner [43, 44]. Intriguingly, the proteasome inhibitor MG132 and methylene blue have both been shown to activate AMPK, alleviate accelerated aging defects in fibroblasts derived from Hutchinson-Gilford progeria syndrome (HGPS) patients, and stimulate oocyte meiotic resumption, providing compelling evidence that AMPK activation represents a central node linking the therapeutic effects of chemically distinct compounds that facilitate the creation of human life [20,27,45-48].

Furthermore, Carvacho et al. demonstrated that the TRPV3 channel is differentially expressed in mouse oocytes during maturation and reaches peak density and activity at metaphase II, the stage at which oocytes develop the competency to initiate Ca2+ oscillations in response to fertilization [49].  The authors also showed that strontium chloride (SrCl2), a compound that induces parthenogenetic activation of mammalian oocytes and has resulted in successful term pregnancies and the birth of normal children, promotes Ca2+ oscillations and induces oocyte activation via TRPV3-mediated Sr2+ influx, as TRPV3 deletion in oocytes (TrpV3−/−) failed to respond to Sr2+-induced activation [49,50].  As the TRPV3 channel is Ca2+ permeable and SrCl2-induced oocyte activation is thought to occur via mimicking Ca2+ by sensitizing and potentiating IP3 receptors, the authors also showed that application of the TRPV3 agonist carvacrol in heterozygous oocytes (TrpV3+/−) led to a substantial increase in intracellular Ca2+ levels as well as parthenogenesis in both TrpV3+/− and wild-type oocytes (TrpV3+/+) (as measured by pronuclear formation and cleavage to the 2-cell stage), whereas TRPV3-deficient oocytes failed to respond [49]. Because TRPV3 channels are also critical for hippocampal LTP in the brain and because the TRPV3 channel agonist carvacrol activates AMPK and AMPK activation is essential for the induction of hippocampal LTP, activation of AMPK is likely critical for the creation of all human life (via oocyte activation) and for the processes of learning and memory formation [51].

Interestingly, recent studies have also demonstrated that knockout of HSF1 in oocytes leads to depletion of HSP90alpha, delayed meiotic resumption, and defective asymmetrical division [52]. As HSP90 has been shown to interact with and maintain AMPK activity and AMPK activation is essential for efficient T cell activation, heat stress or the induction of mediators of the heat shock response would be expected to promote the reactivation of latent HIV-1 in CD4+ memory T cells via low-level T cell activation, a method known as the “shock and kill” approach [51,53]. T cell activation-induced latent HIV-1 reactivation will likely facilitate destruction of the virus through immune-system detection or via virus-induced destruction of the host cell [51]. Indeed, recent studies have demonstrated that T cell activation at fever temperatures (39.5°C) activates HSF1 and HSF1 is essential for T cell proliferation in vitro [54,55]. Also, knockdown of AMPK and CaMKK2 (an upstream activator of AMPK) has been shown to significantly inhibit HIV-1 replication [56]. Strikingly, Roesch et al. showed that HIV-1 replication was increased 2 to 7 fold by culturing primary CD4+ T lymphocytes at a fever-like temperature (39.5°C) and that hyperthermia enhanced HIV-1 reactivation in a model of latently-infected cells in a HSP90-dependent manner [57].  Several recent studies have also demonstrated that HSP90 promotes HIV-1 reactivation from latency in CD4+ T cells by enhancing the activity of host cell several transcription factors (NF-κB, NFAT, and STAT5) that are critical for both T cell activation and HIV-1 reactivation and replication [58,59].  Particularly compelling is a recent study by Pan et al. showing that HSF1 participates in HIV-1 transcription and is essential for latent HIV-1 reactivation by binding to the HIV 5'-long terminal repeat (LTR) to reactivate viral transcription [60].  Overexpression of HSF1 improved HIV transcription whereas knockout of  HSF1 inhibited HIV transcription [60].  Interestingly, in this same study, resveratrol, MG132, and hemin (an iron-containing porphyrin) were also shown to reactivate latent HIV-1 in a T cell line [60]. Similar to resveratrol and MG132, hemin has also been shown to activate AMPK, again placing AMPK activation as a centerpiece facilitating the reactivation of latent HIV-1, oocyte meiotic induction and activation, and learning and memory formation induced by LTP [61].

Activation of mediators of the heat shock response may also play critical roles in other physiological processes that are essential for the creation and the beginnings of human life.  As noted above, in addition to heat stress, HSPs also respond to other forms of cellular stress, including ROS and intracellular Ca2+ increases, both of which activate AMPK [13-15].  Additionally, HSP90 has been found to localize in the neck, midpiece, and tail regions of human sperm and HSP90 inhibition significantly decreases intracellular Ca2+ concentrations during capacitation, a process that is essential for oocyte fertilization [62].  Indeed, an increase in the levels of intracellular Ca2+ is critical for the initiation of the acrosome reaction in sperm, a process that facilitates sperm penetration of the oocyte and is thus indispensable for fertilization.  Interestingly, ROS and vitamin D, both of which activate AMPK, have each been shown to induce the acrosome reaction in sperm, indicating that induction of cellular stress is critical for the promotion of the acrosome reaction in sperm, facilitating the creation of human life [15,28,63,64].  Vitamin D has also been shown to improve accelerated cellular aging defects in progeria, indicating that the creation of human life and the amelioration of symptoms of accelerated aging may depend on the induction of a cellular stress response [22].  Heat stress has also been shown to promote the differentiation of human adult stem cells and AMPK activation has also recently been shown to be critical in embryonic development by facilitating the differentiation of mouse embryonic stem cells into endoderm, again indicating that AMPK activation is critical, if not indispensable, for the creation and beginnings of life [65,66,67].

In conclusion, the evidence presented above strongly supports the provocative implication that the induction of cellular stress, mediated by heat, ROS, intracellular Ca2+ increases, an AMP/ATP ratio increase,  etc. leads to a beneficial compensatory cellular response.  The induction of this cellular response and subsequent activation of AMPK by chemically distinct compounds including sulforaphane, MG132, vitamin D, rapamycin, methylene blue, carvacrol, and likely many others indicates that cellular stress-induced AMPK activation may represent a central node in the amelioration of disease and the creation of human life. Indeed, cell and context-specific AMPK activation may induce a “shock to live” (e.g. oocyte maturation/activation, stem cell differentiation, learning and memory in the brain, sperm capacitation/acrosome reaction) or a “shock to kill” (e.g. latent HIV-1 reactivation, cancer stem cell differentiation and/or cell death).

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