New study confirms that Metformin alleviates accelerated aging defects and activates AMPK in Progeria cells: Hypothesis further substantiated

"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.

In line with findings published in November of 2016 demonstrating that the anti-diabetic drug metformin alleviated pathological aging defects in cells derived from Hutchinson–Gilford progeria syndrome (HGPS) patients, a recent study published online in the Journal Experimental Dermatology in February of 2017 also provided startling evidence confirming that metformin alleviated nuclear defects and premature aging phenotypes in fibroblasts derived from HGPS patients [1,2]. The authors demonstrated that metformin delayed cellular senescence, decreased the formation of reactive oxygen species (ROS), decreased the expression of progerin (a toxic protein that leads to accelerated cellular aging defects), and significantly increased the number of cells with normal nuclei [1]. Importantly, metformin treatment also led to a significant increase in the phosphorylation and activation of AMPK in HGPS cells [1]. Interestingly, the study published in November of 2016 in the Journal npj Aging and Mechanisms of Disease (part of the Nature Partner Journals series) also showed that metformin decreased the expression of both progerin and 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, thus increasing the levels of progerin [2].

Both of these studies collectively provides direct support and further 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 cells by decreasing the levels of SRSF1 and activating AMPK, thus reducing progerin production via modulation of alternative splicing [3].

Indeed, 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 [4]. The confirmation of my 2014 hypothesis via the studies published in npj Aging and Mechanisms of Disease and Experimental Dermatology that metformin activates AMPK, decreases SRSF1, and improves accelerated aging defects in HGPS cells 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 [5].

Additionally, AMPK activation is critical for oocyte meiotic resumption and maturation (processes that are essential for efficient oocyte activation) and compounds including the calcium (Ca2+) ionophore ionomycin promote latent HIV-1 reactivation (when combined with the phorbol ester PMA) and induce human oocyte activation, giving rise to normal healthy children (see below). Because oocyte activation is a prerequisite for the creation of all human life and because various compounds such as ionomycin that induce oocyte activation also activate AMPK, it is likely that AMPK activation connects latent HIV-1 reactivation, alleviation of accelerated aging defects in HGPS cells, and the creation of all human life, a hypothesis that I published for the first time in 2016 [6].

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 [7]. 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 [7]. 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 [8]. 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 [5,9,10].

In the Experimental Dermatology study, Park et al. initially characterized the cellular phenotypes of primary dermal fibroblasts derived from HGPS patients of different ages and showed increased staining of senescence-associated beta-galactosidase (SA-β-gal, an indicator of cellular senescence) and increased levels of mitochondrial superoxide in HG8 cells (from an 8 year old patient) compared to HG3 (3 year old) and HG5 (5 year old) cells [1]. All cells expressed the toxic protein progerin, superoxide dismutase 2 (SOD2, a mitochondrial antioxidant enzyme) was highest in normal fibroblasts and lowest in HG8 cells, and cellular proliferation rate slowed at an earlier time in HGPS cells compared to normal fibroblasts [1].

Utilizing HG8 cells (which demonstrated the highest levels of senescence), the authors also elucidated the effects of metformin (2mM), rapamycin (200nM), or a combination of both drugs on the nuclear phenotype of HGPS cells. Rapamycin significantly decreased the number of nuclei with abnormal morphology and metformin treatment also led to a significant increase in the number of cells with normal nuclei compared to control-treated cells [1]. Metformin also reduced senescence in HGPS cells (i.e. reduction in SA-β-gal staining) and co-treatment with rapamycin and metformin led to an approximately 34.2 % inhibition of senescence, with similar results observed in HG3 and HG8 cells [1]. Metformin, rapamycin, or co-treatment with both compounds led to a significant reduction in the number of cells containing more than 20 γ-H2AX foci (a marker of DNA damage) in HG8, HG3, and HG5 cells, indicating that metformin increases the efficiency of DNA repair in HGPS cells [1].

Metformin also exerted antioxidant effects in HGPS cells, as evidenced by a significant decrease in ROS production and mitochondrial superoxide formation compared to control cells as well as an upregulation of SOD2 mRNA expression in aged BALB/c mice (>18 months old) [1]. Most importantly, metformin treatment at 2 and 20mM reduced progerin protein expression by approximately 20 % and 60 %, respectively, compared to mock-treated cells and increased the presence of normal nuclear phenotypes in HGPS cells [1]. Metformin treatment also significantly increased the phosphorylation and activation of AMPK in HGPS cells. Furthermore, western blot analysis indicated that rapamycin increased AMPK activation as well. Curiously, treatment of aged mice with metformin significantly increased the proliferation of immune cells (i.e. splenocytes) in response to IL-2 and LPS [1]. As described below, such results may be explained via activation of AMPK, as rapamycin has recently been shown to activate AMPK in vivo in normal aged mice and efficient T cell activation is critically dependent on AMPK activation.  

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 activating AMPK and decreasing the levels of SRSF1, thus reducing progerin production via modulation of alternative splicing [3]. However, the results from the npj Aging and Mechanisms of Disease and Experimental Dermatologystudies 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 [5]. 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”) [5].

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 [6,11,12]. 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 [6,13]. 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 [11,12,14].

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 [15-17]. Interestingly, the activity of the splicing factor SRSF1 is also downregulated during activation of T cells not infected with HIV-1 [18].

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 [19]. 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 [6,13,20,21].          

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 [22,23]. 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 [24]. 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) [25,26].

Interestingly, autophagy 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 of the master antioxidant transcription factor Nrf2 [27-30]. 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, autophagy 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 [31]. Rapamycin was also recently found to potently activate AMPK in vivo in normal old mice as well as induce autophagy and mitochondrial biogenesis [32]. The induction of ULK1-dependent autophagy by rapamycin was also shown to be significantly decreased when the splicing-associated protein p32 (an endogenous inhibitor of SRSF1) was inhibited, indicating that p32 activity is critical for rapamycin-induced autophagy [33]. 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 [11,34-36].

Other compounds that have been shown to reduce the levels of progerin and/or improve accelerated aging defects in HGPS cells via autophagy induction, including all-trans retinoic acid and the Nrf2 activator sulforaphane, have also been shown to activate AMPK [37-40]. 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 [41,42]. Expectedly, methylene blue has also been shown in independent studies to induce macroautophagy and activate AMPK in vitro and in vivo [43,44].

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 [29,30]. 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 [45]. 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 ROS production, activate LKB1 (an upstream activator of AMPK), and increase the AMP/ATP ratio (an indication of cellular stress induction) [46-50]. 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 [26,51-53].

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 [54]. Indeed, 1,25D has been shown to activate AMPK in vivo as well as alter gene splicing in cancer cells [55,56]. 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 [57]. 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 [5,58]. 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 [5,59,60]..

In conclusion, the results from the npj Aging and Mechanisms of Diseaseand Experimental Dermatology studies demonstrating that metformin activates AMPK, 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 [3]. 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 these two new studies 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 [5]. Indeed, p32 has been shown to be essential for ULK-1 mediated autophagy induction by rapamycin, a drug that improves immune system responses to viral infections and ameliorates accelerated aging defects in HGPS cells. 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 reactivate 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 and maturation, 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 [6]. 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 [61]. 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 [62,63]. That the symptoms of accelerated aging associated with HGPS, reactivation of latent HIV-1, oocyte activation, and the acrosome reaction in human 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 etiology and the practice of medicine is inevitable.    

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