Metformin shown for the first time to activate Telomere enzyme Telomerase in human cells via AMPK: Link between Progeria and HIV

Goldsmith Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus [Public domain], via Wikimedia Commons;The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. Scaffidi P, Gordon L, Misteli T. PLoS Biology Vol. 3/11/2005, e39 

A recently published study in the journal Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease in 2018 demonstrated for the first time that chronic treatment with the anti-diabetic drug metformin activated human telomerase reverse transcriptase (hTERT) in human aortic endothelial cells (HAECs) and significantly delayed endothelial senescence in an AMPK-dependent manner [1]. AMPK is activated by the induction of cellular stress, mediated by increases in intracellular calcium (Ca2+), reactive oxygen species (ROS), and/or an AMP(ADP)/ATP ratio increase, etc. [41].  Telomeres are specialized regions of repetitive nucleotide sequences located at the ends of eukaryotic chromosomes that protect chromosomal ends from deterioration [2].  However, continuous cell division leads to telomere shortening, impeding the replenishment of tissues and triggering cellular senescence (i.e. cells cease to divide).  Although human telomeres shorten with age, telomeres may be lengthened by the enzyme telomerase, a ribonucleoprotein that consists of the catalytic subunit hTERT, telomerase RNA, and the nucleolar protein dyskerin [2,3]. hTERT, which is considering limiting for telomerase activity, is a protein that exhibits reverse transcriptase activity and synthesizes telomeric DNA from an RNA template [4]. 

The authors of the study initially demonstrated that both metformin and the AMPK activator AICAR significantly increased hTERT levels and enhanced AMPK activation in human aortic endothelial cells (HAECs) [1].  Importantly, inhibition or knockdown of AMPK with compound C or siAMPKα inhibited the metformin-induced increase in hTERT while metformin failed to reverse siAMPKα-induced senescence in HAECs, indicating that hTERT expression is regulated by AMPK activation in endothelial cells.  Indeed, continuous culturing of HAECs in the presence of metformin significantly increased hTERT protein levels and activity, reduced the expression of the senescence markers p53, p21, p27, and p16, and reduced senescence-associated beta-galactosidase (SA-β-gal, a biomarker of cellular senescence) staining in HAECs, again indicating that metformin delays cellular senescence and increases hTERT levels via AMPK activation [1].  Strikingly, using ApoE-/- mice (which spontaneously develop atherosclerosis and age faster compared to normal mice), the authors also showed that chronic low-dose metformin administration for fourteen months in drinking water enhanced the levels of activated AMPK and Pgc-1α observed in the endothelial layer of the aorta [1].  Metformin also increased the transcript and protein levels of Tert, decreased senescence markers (p16, p21, p27, p53) in the total aortic homogenate, and significantly reduced SA-β-gal staining of aorta compared to untreated ApoE-/- mice, demonstrating that metformin-induced AMPK activation delays vascular aging and protects from age-associated atherosclerosis in ApoE-/- mice [1].

Interestingly, telomere length has been shown to be significantly reduced in cells derived from patients with the accelerated aging disorder Hutchinson-Gilford progeria syndrome (HGPS) and telomere shortening in normal cells that occurs during cellular senescence activates progerin production, a toxic protein that leads to an accelerating aging phenotype in children with HGPS via aberrant alternative splicing of the LMNA gene [5].  Normal lamin A plays a critical role in supporting nuclear architecture and morphology.  Lamin A binding to subtelomeric repeats also localizes telomeres to the nuclear periphery and loss of lamin A leads to defects in telomeric heterochromatin, altered nuclear distribution and shortening of telomeres, inefficient processing of dysfunctional telomeres by non-homologus end joining, and increased genomic instability [6]. Telomere length has been found to be significantly reduced in fibroblasts derived from HGPS patients and a recent study also confirmed that in normal human fibroblasts, progressive telomere damage that occurs during cellular senescence activates progerin production and also leads to extensive changes in alternative splicing of many other genes, highlighting a striking similarity between normal aging and accelerated aging in HGPS patients [5,7]. Indeed, transfection of HGPS fibroblasts with human telomerase (hTERT) mRNA restored cell proliferation, reduced cell loss, extended cellular lifespan, increased telomerase activity and telomere length, and reduced SA-β-gal staining compared to HGPS cells expressing catalytically inactive hTERT mRNA [8].  Because metformin also increases hTERT expression and inhibits senescence in human cells, it is likely that cellular stress-induced AMPK activation, mediated by increases in intracellular calcium (Ca2+), reactive oxygen species (ROS), and/or an AMP(ADP)/ATP ratio increase, etc., represents a central node linking structurally diverse compounds and methodologies that alleviate accelerated aging in HGPS cells.              

As the splicing factor SRSF1 has been shown to increase progerin production by promoting the use of a cryptic splice located in the LMNA gene, metformin was recently shown to significantly reduce the expression of SRSF1 and progerin, activate AMPK, and improve nuclear architecture in HGPS cells, indicating that AMPK activation by metformin beneficially alters gene splicing in HGPS cells by modulating SRSF1, a hypothesis that I first proposed and published in 2014 [9-11].  Also, p32, a splicing-associated protein that is an endogenous inhibitor of SRSF1 and is critical for the maintenance of mitochondrial functionality and oxidative phosphorylation, has also been shown to be essential for rapamycin- or starvation-induced autophagy mediated by ULK1 [12,13].  Because rapamycin, also an AMPK activator in vivo,  improves accelerated aging defects in HGPS cells by reducing progerin levels via induction of autophagy, AMPK activation likely also beneficially modulates the activity of p32, leading to inhibition of SRSF1 splicing activity and enhancement of mitochondrial functionality [14,15].  Moreover, PGC-1α, which is activated by AMPK and metformin and promotes telomere transcription, is downregulated in HGPS cells, leading to significant mitochondrial dysfunction [1,16,17].  Methylene blue, which activates AMPK in vivo, was shown to increase PGC-1α levels, induce progerin solubility, and alleviate accelerated aging defects in HGPS cells [16,18].  Additionally, the rapamycin analog temsirolimus alleviated accelerated aging defects in HGPS cells but transiently increased ROS and superoxide anion levels in both HGPS and normal cells within the first hour of treatment, again indicating that cellular stress-induced AMPK activation represents a common mechanism for inhibiting senescence and ameliorating symptoms associated with accelerated aging [19].    

The inhibition of SRSF1 and the promotion of hTERT expression and telomere transcription by metformin via AMPK activation also link HGPS and telomere integrity with HIV-1 latency.  Increased splicing activity of SRSF1 inhibits reactivation of latent HIV-1 residing in infected immune cells, preventing immune system detection and destruction of the virus [20].  Reactivation of latent HIV-1 (i.e. the “shock and kill” approach) leads to a reduction in SRSF1 but an increase in p32 activity and bryostatin-1 (a PKC modulator) has been shown to reactivate latent HIV-1 via AMPK activation [20,21].  Interestingly, p32 modulation via AMPK activation may also enhance and stabilize the splicing activities of hnRNPA1, a heteroribonuclear protein that associates with p32, antagonizes the splicing function of SRSF1, prevents splicing of the HIV-1 genome (promoting viral reactivation), and participates in the maintenance and preservation of telomeres [22-25].  hnRNPA1 is also decreased in senescent human fibroblasts and antagonizes cellular senescence and the senescence-associated secretory phenotype (SASP) via increasing SIRT1 expression [26,27].  Resveratrol, a plant-derived polyphenol that activates AMPK, increases hnRNPA1 protein expression and SIRT1, a histone deacetylase that plays a role in a number of age related diseases and in the extension of lifespan, is also activated by AMPK [28-30].  Also, T cell activation, an efficient method for reactivating latent HIV-1, is dependent on increases in intracellular Ca2+ and ROS, telomerase is transiently increased on T cell activation, and AMPK knockdown leads to T cell death during in vitro activation [31-34].  Furthermore, resveratrol reactivates latent HIV-1 and preliminary data demonstrated that metformin decreased the percentage of CD4+ T cells expressing PD-1, TIGIT, and TIM-3, each markers associated with T cells latently infected with HIV-1, in chronically-infected HIV-1 patients [35-37].  Such evidence strongly suggests that cellular stress-induced AMPK activation, mediated by increases in intracellular calcium (Ca2+), reactive oxygen species (ROS), and/or an AMP (ADP)/ATP ratio increase, etc. links the alleviation of accelerated cellular aging defects in HGPS with the potential eradication of HIV-1, a hypothesis that I first proposed in 2015 [38].        

 

The evidence presented in the Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease publication further substantiates that AMPK activation represents a central node that connects the therapeutic benefits of chemically distinct compounds in diseases as seemingly dissimilar as HGPS and HIV-1 latency.  Indeed, AMPK activators including metformin increase hTERT expression, promote telomere transcription and integrity, decrease the splicing activity of SRSF1 that increases progerin production but prevents latent HIV-1 reactivation, and potentially beneficially modulates the activity of the SRSF1 inhibitor p32 and the ribonucleoprotein hnRNPA1.  As AMPK activators (e.g. ionomycin) induce human oocyte activation (giving rise to normal healthy children) and AMPK is localized throughout the entire acrosome in human sperm (likely promoting the acrosome reaction), AMPK activation links normal human aging, Progeria, and HIV-1 latency with the creation of all human life (i.e. the “shock and live” approach) [11,38-40]. 

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