Anti-aging drug Rapamycin shares common mechanism of action with Metformin and Artemisinin: Connection between AMPK, Progeria, and HIV-1 reactivation

"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.

Here's another LinkedIn post (see below) I just published about a recent article that adds even more evidence to the notion that diseases that we think are completely unconnected are actually linked by a common mechanism and many natural products and some drugs are able to effectively treat or eradicate diseases including Hutchinson-Gilford progeria syndrome, HIV-1 latency, malaria, autoimmune disorders, cancer, and even slow the aging process itself by beneficially activating a single p...athway. The drug, called rapamycin, is well known throughout the aging research community and has been shown to extend the lifespan and healthspan of basically every organism it’s been tested in. It's also been shown to inhibit accelerated cellular aging defects in progeria, similar to the broccoli compound sulforaphane, the vitamin A metabolite retinoic acid, and methylene blue. Just like the aforementioned compounds, rapamycin, in this study, was shown to be a potent activator of the master metabolic regulator AMPK, very much similar to the diabetes drug metformin and the anti-malarial and Noble Prize-winning drug artemisinin. Interestingly, rapamycin and methylene blue also inhibits the growth of the parasite that causes malaria. Again, the connections among these diseases are striking, especially considering that most of these compounds exert their beneficial effects by indirectly activating one pathway (in which I theorized about and published in a few of my papers). If this turns out to be true......:-)

Anti-aging drug Rapamycin shares common mechanism of action with Metformin and Artemisinin: Connection between AMPK, Progeria, and HIV-1 reactivation

Just as a recent study demonstrated that the anti-malarial drug artemisinin (see last post) shares a common mechanism of action of AMPK activation with the diabetes drug metformin, a recent study published online in February of 2016 by researchers at the University of Washington in the journal Aging (Albany NY) clearly demonstrated that rapamycin, a drug that is used to prevent rejection in organ transplantation that also extends the median and maximal lifespan of several animal models even when fed late in life (e.g. in male and female mice), significantly activated AMPK in vivo (i.e. in a living organism) in hearts from normal elderly mice, indicating that AMPK activation may indeed link pleiotropic effects on aging, HIV-1, and gene splicing exerted by compounds as chemically distinct as rapamycin, metformin, artemisinin, methylene blue, sulforaphane, retinoic acid, and possibly many others [1-3].

Rapamycin (also known as sirolimus) is a macrolide drug originally derived from the bacterium Streptomyces hygroscopicus found on Easter Island (Chilean island in the southeastern Pacific Ocean) that is often used as an immunosuppressant to prevent rejection in organ transplantation [4].  However, rapamycin  has also demonstrated immune enhancing/stimulating properties (discussed below) as well as anti-proliferative properties within the context of viral challenges and cancer metastasis, respectively, indicating that the beneficial effects of rapamycin are likely disease- and cell-type specific [4,5].  The efficacy of rapamycin administration is often attributed to its inhibition of a cellular protein complex known as mammalian target of rapamycin complex 1 (mTORC1).  In response to environmental or nutritional stimuli (e.g. amino acids, growth factors, cellular/energy stress), mTORC1 plays a critical role in the promotion of cellular growth by inhibiting catabolic processes such as autophagy (intracellular degradation system) and promoting anabolic processes such as mRNA translation and lipid biosynthesis [6 ].

Interestingly, in the Aging (Albany NY) study, the authors found that within just two weeks of treatment of a dose of rapamycin (2.24 mg/kg/day) initially described by the National Institute on Aging Interventions Testing Program (a multi-institutional study designed to test the effectiveness of various compounds to extend lifespan in mice) lead to a reversal of cardiac aging phenotypes in normal aged mice compared to control mice, as evidenced by an improvement in diastolic function, a lower heart weight normalized to body weight (HW/BW), and a reversal of cardiac hypertrophy at all time points.  Expectedly, rapamycin also inhibited the phosphorylation of a downstream target of mTORC1, ribosomal protein S6, a protein thought to be involved in pressure-overload induced cardiac hypertrophy [1].

More importantly, however, was the rapamycin-induced induction of autophagy, mitochondrial biogenesis, and phosphorylation and activation of AMPK in elderly mice hearts. As phosphorylation of ULK1 (a kinase critical for autophagic induction) at serine-757 by mTORC1 inhibits autophagic induction by ULK1, rapamycin transiently increased autophagic flux after 1 week of treatment (returned to control levels at weeks 2 and 10), as evidenced by an increased LC3II/LC3I ratio, an increase in ATG5, and a decrease in ULK1 phosphorylation at serine-757 [1].  Using real-time PCR, the authors also showed that rapamycin treatment transiently increased the expression of the mitochondrial biogenesis marker PCG-1α in the first 2 weeks as well as the protein expression of mitochondrial transcriptional factor A (TFAM) (a transcription factor downstream of PCG-1α) in the 1st and 2nd weeks, with both returning to control levels by week 10 [1].  Because global proteomic analysis revealed a mixture of increased and decreased levels of mitochondrial proteins during autophagic induction and initial mitochondrial biogenesis after the 1st week of treatment, but a general increase in mitochondrial proteins after the 2nd week of treatment (autophagy returned to normal but mitochondrial biogenesis still elevated), rapamycin appears to initially enhance the removal of old mitochondrial components via ULK1-initiated autophagy and replace those components with newly synthesized mitochondria [1].

Most strikingly, however, was the phosphorylation and activation of AMPK in vivo in elderly mice hearts by rapamycin.  Interestingly, the authors demonstrated that rapamycin treatment significantly increased phosphorylation and activation of AMPK during the entire 10 week period at all time points (1, 2, and 10 weeks), with peak levels at week 2 [1].  Because rapamycin-induced phosphorylation and activation of AMPK coincides with and possibly precedes the initial induction of ULK1-mediated autophagy, upregulation of PCG-1α- and TFAM-induced mitochondrial biogenesis, and the reversal of fatty acid oxidation reduction (see study), the inhibition of mTORC1 by rapamycin likely represents a cellular stress response, leading to the activation of AMPK and generating the observed responses in elderly mice hearts.  Indeed, AMPK has been shown in separate studies to phosphorylate and activate ULK1 at serine 317 and serine 777, inhibit mTORC1, increase the levels of PCG-1α and TFAM, enhance mitochondrial biogenesis, and promote fatty acid oxidation [7-10].

The Aging (Albany NY) study provides compelling evidence that the beneficial pleiotropic and lifespan-extending effects of rapamycin are orchestrated by the phosphorylation and activation of AMPK via inhibition of mTORC1.  This mechanism of action is also shared by other chemically distinct compounds that have been shown to activate AMPK, increase mitochondrial biogenesis, and induce autophagy, leading to beneficial effects in diseases as seemingly as disparate as Hutchinson-Gilford progeria syndrome (HGPS), malaria, and HIV-1 latency. 

With respect to HGPS, an accelerated aging disease caused by a dominant negative, gene splicing-mediated mutation leading to the overexpression of the toxic protein progerin, rapamycin has been shown in studies to enhance autophagic degradation of progerin in HGPS fibroblasts, leading to an improvement in nuclear morphology [11,12].  As noted above, AMPK inhibits mTORC1 and phosphorylates and activates ULK1, a critical initiator of autophagic flux.  Because rapamycin also activates AMPK and enhances autophagic flux, autophagic degradation of progerin production in HGPS is likely mediated by rapamycin-induced AMPK activation.  Indeed, sulforaphane, an isothiocyanate derived from cruciferous vegetables, also activates AMPK and has been recently shown to increase autophagic degradation of progerin, decrease levels of ROS, enhance cellular proliferation, improve nuclear morphology, and increase intracellular ATP levels in HGPS fibroblasts, implying a provocative common mechanism of action of AMPK activation between sulforaphane and rapamycin [13,14].  This common mechanism of action appears to extend to the recent findings that both methylene blue and retinoic acid, each shown independently to activate AMPK, also improves nuclear morphology and, in the case of methylene blue, corrects mitochondrial functioning in HGPS fibroblasts [12,15-17].  Interestingly, similar to rapamycin in the Aging (Albany NY) study, methylene blue was shown to not only increase the levels of  PCG-1α in HGPS fibroblasts, but also correct damage to mitochondria in healthy cells [15].  Also, similar to both rapamycin and sulforaphane, methylene blue, in an AMPK-dependent manner, has been shown to induce macroautophagy in vivo in the hippocampus (area of the brain essential for learning and memory) of mice and in mouse hippocampal HT22 cells [18].

Additionally, methylene blue increased the levels of LMNA mRNA (gene involved in the pathology of HGPS) and retinoic acid induced a down regulation in progerin mRNA transcripts, implying that methylene blue and retinoic acid beneficially altered the splicing of the LMNA gene [12,15].  Moreover, metformin, a prototypical AMPK activator that has been shown to exert beneficial effects in numerous disease states as well as improve lifespan and healthspan, has been shown to correct alternative splicing defects in cells derived from myotonic dystrophy type I patients in an AMPK-dependent manner and beneficially alter splicing of the insulin receptor gene in diabetic patients who were taking metformin but who did not have DM1, in typical therapeutic dosages [19].

The interplay of alternative splicing correction and autophagic induction by AMPK activators is also substantiated by the actions of the splicing-associated factor p32.  p32, often noted as a molecular chaperone, is essential for mitochondrial bioenergetics and oxidative phosphorylation, both of which are enhanced by AMPK activation [20].  A recent study also demonstrated that p32 is essential for the induction of autophagic flux by acting as a chaperone for the autophagic initiator ULK1 [21].  Indeed, silencing or depletion of p32 significantly inhibited autophagic flux induced by both rapamycin and starvation, implying a provocative connection between p32, ULK1, AMPK, and rapamycin [21].  Interestingly, SRSF1 (also known as ASF/SF2), a splicing factor that has been implicated in the aberrant splicing of the LMNA gene in HGPS as well as in faulty splicing of genes in normal humans that lead to endothelial senescence, activates mTORC1 but is inhibited by p32 [22-25].  UV radiation has also been shown, in separate studies, to upregulate SRSF1 and induce the production of progerin, while mutation of splice sites in genes have been shown to increase SRSF1 recruitment [26-28].  Strikingly, increased SRSF1 activity has been shown to prevent the reactivation of latent HIV-1 reservoirs while p32 activity has been shown to be increased in latent HIV-1 reactivation, thus inhibiting SRSF1 activity and providing an astounding parallel between HGPS, autophagy, normal aging, and HIV-1 latency [29] (see below).

Interestingly, rapamycin also shares a common mechanism of action of AMPK activation with artemisinin, a powerful anti-malarial drug that has been in use for more than 2000 years. Dihydroartemisinin (a metabolite of an artemisinin derivative) was shown to induce cancer cell differentiation via activation of AMPK, a result that was mimicked by metformin administration in the same study [3].  Dihydroartemisinin has also been shown to inhibit phosphorylation of mTORC1 in rhabdomyosarcoma cells as well as induce autophagy in glioma cells in vitro and in vivo [30,31].   Additionally, artesunate (artemisinin derivative metabolized to dihydroartemisinin) mimicked certain aspects of caloric restriction when given to mice (increased SIRT1, MnSOD, CuZnSOD catalase, glutathione), trigged mitochondrial biogenesis (increased AMPK activation, PCG-1α, SIRT3, CYTC and MNF2), and attenuated telomere attrition [32]. Because the AMPK activators methylene blue, sulforaphane, and retinoic acid have also been shown to be effective in the treatment of malaria (see prior Artemisinin post for references), rapamycin would also be expected to exert at least partial anti-malarial effects. Indeed, in a recent in vitro study, both rapamycin and everolimus (mTOR inhibitor) were associated with complete inhibition of Plasmodium falciparum growth while other studies demonstrated that inhibition of mTOC1 with rapamycin increases parasite clearance in the spleen, reduces parasite accumulation in peripheral tissues (e.g. brain), and prevents experimental cerebral malaria, indicating a common mechanism of action between artemisinin and rapamycin [33,34].

Lastly, activation of AMPK in normal elderly mice demonstrated in the Aging (Albany NY) study implicates the potential utility of rapamycin administration to enhance reactivation of latent HIV-1 reservoirs, facilitating its detection and destruction by the immune system. Interestingly, studies have shown that T cell activation (and thus latent HIV-1 reactivation) is critically dependent on AMPK activation and mitochondrial ATP production, as knockdown of a subunit of AMPK (AMPKα1) leads to a significant reduction in mitochondrial bioenergetics and an inhibition of primary T cell responses in vivo to viral and bacterial infections [35-37].  Additionally, autophagy has also been shown to be essential for and upregulated on T cell activation, as evidenced by a decrease in ATP generation, defective IL-2 and IFN-γ production, and reduced proliferation in Th cells after deletion of Atg7 (a gene critical for macroautophagy) specifically in T cells [38].  Also, the activity of the splicing factor SRSF1, which activates mTOC1, is upregulated during HIV-1 latency, causing excessive HIV-1 mRNA gene splicing and thus reducing the unspliced mRNA necessary for reactivation of the virus [29]. However, viral reactivation was indeed shown to reduce SRSF1 activity and increase the activity of p32, an endogenous inhibitor of SRSF1 that also increases mitochondrial functionality and oxidative phosphorylation [20,29].  Interestingly, rapamycin has also been shown to antagonize the effects of SRSF1 in several independent studies [24,39].

Based on these studies, a common mechanism of AMPK activation as a prerequisite for efficient latent HIV-1 reactivation is clear. Although rapamycin acts as an immunosuppressant in the context of organ transplantation, several studies have shown that both rapamycin and metformin enhance the immune response to viral challenges in both humans and mice, indicating a temporally appropriate facilitation of CD4+ and CD8+ T cell activation and formation of memory CD8+ T cells, thus enhancing the detection and destruction of viruses, intracellular pathogens, and cancer cells by functioning as immunotherapeutics [40-43].  Interestingly, metformin may also act as an immunosuppressant in autoimmune-specific disease contexts, but when combined with bryostatin (a PKC activator that also activates AMPK), enhances the reactivation of cells latently infected with HIV-1 [44,45]. Interestingly, a recent study also demonstrated that when combined with bryostatin, the compound JQ1 fully reactivated latently infected HIV-1 viral reservoirs to the level of positive controls [46].  Although the compound JQ1 is used in latency reversal studies to reactivate latent HIV-1, JQ1 is also being studied as an anti-cancer agent due to its indirect inhibition of the oncoprotein c-Myc.  However, a recent study has demonstrated that JQ1, dihydroartemisinin (metabolite of an artemisinin derivative), and other compounds labeled as c-Myc inhibitors, share a common mechanism of action that involves the induction of cellular stress, followed by the activation of AMPK and subsequent differentiation and/or apoptosis of cancer cells [3].

Again, data from the Aging (Albany NY) combined with the foregoing studies paints an astonishing picture of the interconnectedness of diseases as seemingly disparate as HGPS, HIV-1 latency, normal aging, and malaria. Even more shocking is that AMPK, which is activated by rapamycin, metformin, artemisinin, methylene blue, sulforaphane, and retinoic acid, is critical for T cell activation, enhances ULK1-initated autophagy, beneficially alters gene splicing, inhibits mTORC1, stimulates mitochondrial biogenesis, and improves lifespan and healthspan in several animal model organisms.  If further studies continue to affirm AMPK activation by chemically distinct compounds as a central mechanism of action for the amelioration or eradication of disease, a paradigm shift in the practice of medicine must ensue.

https://www.linkedin.com/pulse/anti-aging-drug-rapamycin-shares-common-mechanism-action-finley?trk=mp-reader-card

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