In line with recent evidence demonstrating for the first time that metformin improved accelerated aging defects, activated AMPK, and beneficially altered gene splicing in cells derived from Hutchinson-Gilford progeria syndrome (HGPS) patients and exerted significant antiviral effects in dengue virus-infected human liver cells in an AMPK-dependent manner, a study published in the journal Human Molecular Genetics in January of 2017 showed for the first time that metformin corrected mitochondrial dysfunction in human fetal cells taken from aborted fetuses that had been diagnosed with Down syndrome (DS) [1-4]. Metformin induced the expression and activity of PGC-1α (a master regulator of mitochondrial biogenesis) and the PGC-1α target genes NRF-1 and TFAM, enhanced oxygen consumption and ATP production, reversed mitochondrial fragmentation, and increased the expression of the mitochondrial fusion genes OPA1 and MFN2, leading to enhanced mitochondrial activity and strongly promoting mitochondrial biogenesis. Recent studies have also shown that the polyphenols resveratrol and epigallocatechin-3-gallate (EGCG) restored mitochondrial functionality and improved hippocampal neural progenitor cell proliferation in a mouse model of DS in an AMPK-dependent manner, while EGCG significantly reversed cognitive deficits in human DS patients [5,6].
Interestingly, as metformin inhibits dengue virus replication in human liver cells in an AMPK-dependent manner, both EGCG and resveratrol have been shown to increase the lifespan of the mosquito Aedes aegypti (a vector for dengue and Zika viruses), with resveratrol also boosting the immune response and activating AMPK in Aedes aegypti [7]. Recent evidence also demonstrated that resveratrol inhibits dengue virus replication in human liver cells, inhibits emtricitabine-resistant HIV-1, and reactivates latent HIV-1, facilitating immune system detection and destruction [8-10]. EGCG has also shown anti-HIV-1 activity [11]. Although structurally dissimilar, metformin, EGCG, and resveratrol each activate AMPK and AMPK activation links reversal of accelerated aging defects in HGPS with rescue of mitochondrial function in Down syndrome, inhibition of dengue virus and HIV-1 replication, and reactivation of latent HIV-1. Such an interconnectedness, as further explained below, provides substantial support for my initial proposal and publications that AMPK activation links HGPS, latent HIV-1 reactivation, Down syndrome (manuscript in press) and the beginnings of all human life (via oocyte activation and the acrosome reaction) [12-14].
Down syndrome (DS) is caused by either a partial or full trisomy of chromosome 21 and is associated with impairments in cognition, learning and memory, and disorders of the immune system [1]. Human and animal studies strongly suggest that mitochondrial dysfunction is associated with pathogenic features of DS and mitochondrial abnormalities have been found in all DS cells analyzed in culture to date, including neurons, astrocytes, and lymphocytes [1]. Indeed, in DS human fetal fibroblasts, PGC-1α, a master regulator of mitochondrial biogenesis, is down regulated at both the mRNA and protein levels and AMPK phosphorylation of the PGC-1α protein is essential for PGC-1α-dependent induction of the PGC-1α promoter [1].
In the Human Molecular Genetics study, Izzo et al. initially observed that DS human fetal fibroblasts (DS-HFFs) displayed a 40%-50% reduction in PGC-1α mRNA and protein levels compared to non-trisomic counterparts (N-HFFs). DS-HFFs treated with metformin however led to a significant increase in PGC-1α at both the mRNA and protein levels compared to untreated control cells, an increase in the mRNA levels of NRF-1 and TFAM (PGC-1α target genes critical in promoting mitochondrial biogenesis), and an increase in mtDNA content [1]. Additionally, compared to N-HFFs, DS-HFFs were characterized by a decrease in basal oxygen consumption rate (OCR, ~55% inhibition), a decrease in OCR related to ATP production (~58% inhibition), and a reduction in maximal respiratory capacity (~58% inhibition). DS-HFFs treated with metformin increased basal OCR, OCR related to ATP production, ATP concentration, mitochondrial membrane potential, and maximal respiratory capacity, thus restoring mitochondrial respiration in DS-HFFs [1].
Furthermore, compared to N-HFFs, DS-HFFs exhibited extensive mitochondrial damage, as evidenced by swollen cristae (~60%-90% of DS-HFFs vs. ~15% of N-HFFs), mitochondria with intra-oedema (~40% of DS-HFFs vs. ~6% of N-HFFs), and an increase in the number of damaged mitochondria (~90% in DS-HFFs vs. ~25% in N-HFFs). Metformin-treated DS-HFFs, compared to untreated cells, displayed narrower cristae with widths that were comparable to N-HFFs cells, fewer damaged mitochondria (~40%), and fewer mitochondria with intra-oedema (~5%) [1]. Interestingly, as mitochondrial fusion also plays an important role in mitochondrial functionality, the mRNA and protein expression of OPA1 and MFN2 (genes that regulate mitochondrial fusion) were significantly down-regulated in DS-HFFs vs. N-HFFs and correlated with extensive mitochondrial fragmentation in DS-HFFs. Treatment of DS-HFFs with metformin significantly increased the mRNA and protein levels of OPA1 and MFN2 and also reduced fragmentation of the mitochondrial network, indicating that metformin induces correction of mitochondrial phenotype by also restoring mitochondrial fusion machinery [1].
As noted above, the AMPK activator EGCG reverses cognitive defects in DS patients, promotes oxidative phosphorylation and mitochondrial biogenesis in lymphoblasts and fibroblasts from DS patients (i.e. increased levels and activity of PGC-1α, NRF-1 and TFAM), and restores oxidative phosphorylation, mitochondrial biogenesis, and improves hippocampal neural progenitor cell proliferation in a DS mouse model (Ts65Dn) in combination with the polyphenol resveratrol (i.e. increase in PGC-1α) in an AMPK-dependent manner [5,6,15]. Mitochondrial dysfunction is also a prominent feature associated with the accelerated aging disorder Hutchison-Gilford progeria syndrome (HGPS) and metformin has recently been shown to alleviate accelerated aging defects, activate AMPK, and beneficially alter gene splicing in HGPS patient cells, as I first hypothesized and published in 2014 and 2015 [12,13].
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 (SRSF1 is a gene splicing factor that is upregulated in HGPS cells and causes faulty gene splicing of the LMNA gene leading to increased production of the toxic protein progerin) [2]. 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 [2].
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 LmnaG609G/G609G mouse primary fibroblasts (HGPS mouse model) and decreasing both lamin A and progerin mRNA expression in primary HGPS fibroblasts [2]. 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 [2].
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 [2]. 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 [2].
Park et al. also 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 [3]. 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 [3].
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 [3]. 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 [3]. 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 [3].
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) [3]. 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 [3]. 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.
As noted previously, metformin-induced AMPK activation also exerts potent antiviral effects. With respect to dengue virus replication, Soto-Acosta et al. first showed that AMPK activation was reduced in DENV-infected Huh7 (human liver) cells (serotype 2 and 4, 2/4) at 12 and 24 hpi compared to mock-treated cells [4]. 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 [4].
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 [4]. 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 [4].
In conclusion, data from recent evidence strongly suggests a compelling and novel assertion that the activation of AMPK via the induction of cellular stress (e.g. intracellular calcium increase, increased reactive oxygen species [ROS] production, and/or AMP/ATP ratio increase, etc.) represents a central node linking the therapeutic effects of structurally diverse compounds (e.g. metformin) with physiological and patho-physiological states, including Down syndrome, HGPS, HIV-1 latency and replication, dengue virus replication, oocyte activation, and sperm acrosome reaction induction. Indeed, AMPK activation is critical for oocyte meiotic resumption and maturation and AMPK has recently been found localized in human spermatozoa across the entire acrosome, indicating that induction of the acrosome reaction likely involves AMPK activation. Because oocyte activation is indispensable for the creation of all human life and AMPK activators including the calcium ionophores ionomycin and A23187 are extensively used to activate human oocytes, creating normal healthy children, AMPK likely connects the amelioration of normal and pathological aging defects with potential viral eradication, Down syndrome, and the creation of all human life.
https://www.linkedin.com/pulse/metformin-shown-first-time-restore-mitochondria-human-finley
References:
- Izzo A, Nitti M, Mollo N, et al. Metformin restores the mitochondrial network and reverses mitochondrial dysfunction in Down syndrome cells. Hum Mol Genet. 2017 Jan 13. pii: ddx016. doi: 10.1093/hmg/ddx016. [Epub ahead of print].
- 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
- Park SK, Shin OS. Metformin Alleviates Aging Cellular Phenotypes in Hutchinson-Gilford Progeria Syndrome Dermal Fibroblasts. Exp Dermatol. 2017 Feb 13. doi: 10.1111/exd.13323. [Epub ahead of print].
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