AMPK activators Metformin & CHIR99021 improve gut bacteria in humans, Fragile X, & promote inner ear, dental pulp, & cancer stem cell differentiation

CC BY 2.5 (http://creativecommons.org/licenses/by/2.5)], via Wikimedia Commons; By Peter Saxon (Own work) [CC BY-SA 4.0 (http://creativecommons.org/licenses/by-sa/4.0)], via Wikimedia Commons; Rocky Mountain Laboratories, NIAID, NIH [Public domain]

A recent study published online in the journal Nature Medicine in May of 2017 presented startling evidence that the AMPK activator metformin, which has also recently been shown to alleviate accelerated aging defects in cells derived from Hutchinson-Gilford progeria (HGPS) patients, exerted a significant beneficial effect on the gut microbiome in humans in a randomized double-blind placebo controlled study [1-4]. In treatment-naïve patients with Type 2 diabetes (placebo: n = 18 or 1,700 mg/d of metformin: n = 22), a significant decrease in hemoglobin A1c (HbA1c) levels and fasting blood glucose was observed only in metformin-treated patients during the 4-month study period [1]. HbA1c levels and fasting blood glucose were also significantly reduced in a subset of placebo-treated patients that were switched to metformin after 6 months of treatment. Interestingly, whole-genome shotgun sequencing of fecal samples indicated that metformin treatment for 2 and 4 months significantly altered the relative abundance of 81 and 86 bacterial strains, respectively, with an increase in Bifidobacterium and Akkermansia muciniphila [1]. Metformin also directly promoted the growth of Bifidobacterium adolescentis and A. muciniphila in vitro, both of which have been associated with improved metabolic features in mice [1].

Importantly, fecal samples from humans that had been treated with metformin for 4 months and transferred to germ-free mice (via oral gavage) fed a high-fat diet improved glucose tolerance in mice compared to mice that received fecal samples from humans before treatment with metformin [1]. Intriguingly, metformin treatment was also linked to gene enrichment for bacterial environmental responses, including metabolism of the short-chain fatty acids and AMPK activators butyrate and propionate [1,5]. Fecal propionate and butyrate concentrations were significantly increased after 4 months of metformin treatment in men compared to the placebo group, indicating that metformin also beneficially modulates bacterial secondary metabolite production by inducing a bacterial stress response [1]. Moreover, several environmental stressors, including heat shock/stress, which activates AMPK in human cells, also promotes the production of various bacterial secondary metabolites, indicating that the mechanism of action by which metformin promotes an increase in beneficial human gut bacteria and release of bacterial secondary metabolites is via the induction of a stress response in bacteria [6,7].

Furthermore, the beneficial effects of the induction of a cellular stress response likely crosses species boundaries, as increases in calcium (Ca2+) and reactivate oxygen species (ROS) (mediators of cellular stress induction) also promotes seed germination, root gravitropism, and fertilization in plants [8-13]. Additionally, an increase in the AMP(ADP)/ATP ratio, intracellular Ca2+ increases, or an increase in the levels of ROS have been shown to activate the master metabolic regulator AMPK and promote the differentiation of embryonic, adult, and cancer stem cells [14-18]. Metformin and butyrate have also been shown to synergistically activate AMPK and decrease the cancer stem cell-like population in breast cancer cells, butyrate has been shown to induce pancreatic cancer stem cell differentiation, and metformin induces glioma stem cell differentiation and elimination in an AMPK-dependent manner, indicating that cellular stress-induced AMPK activation is a critical mediator linking cancer, embryonic, and adult stem cell differentiation, as proposed in my recent publication linking cancer stem cell differentiation and/or apoptosis with latent HIV-1 reactivation [19-21].

Indeed, a recent study published in the journal Cell Reports by researchers from Harvard Medical School and MIT showed that the glycogen synthase kinase 3β (GSK3β) inhibitor CHIR99021 (CHIR) and the histone deacetylase (HDAC) inhibitor valproic acid (VPA), both of which activate AMPK, significantly expanded cochlear supporting cells (i.e. “inner ear stem cells”) that expressed and maintained the epithelial stem cell marker Lgr5 [22-24]. Treatment with CHIR and VPA also led to the differentiation of Lgr5-expressing cells into hair cells in high yield, providing additional evidence that AMPK activation promotes differentiation of adult stem cells including inner ear stem cells, possibly leading to treatments for hearing loss [24]. Interestingly, the authors demonstrated in a previous study that CHIR and VPA also promoted the multilineage differentiation of Lgr5+ intestinal stem cells into mature enterocytes, goblet cells and Paneth cells [25]. AMPK activation has also been shown to improve gut epithelial differentiation and metformin increases goblet and Paneth cell differentiation from intestinal epithelial cells, further indicating that AMPK activation likely represents a common mechanism of action linking structurally dissimilar compounds that enhance inner ear and intestinal stem cell maintenance and differentiation [26,27].  

Moreover, a recently published study in the journal Scientific Reports in January of 2017 demonstrated that topical administration of GSK3β inhibitors including the AMPK activator CHIR led to the mobilization of resident mesenchymal stem cells in the tooth pulp that had been exposed via the drilling of holes in mice molars [28]. GSK3β inhibitor-induced stem cell mobilization promoted a natural process of reparative dentin (also spelled dentine) formation that completely restored dentin, leading the authors to conclude that stimulation of mesenchymal stem cell mobilization and differentiation into odontoblast-like cells may represent a novel approach to clinical tooth restoration [28]. AMPK activation has previously been shown to promote osteogenic (i.e. bone forming) differentiation of human adipose tissue-derived mesenchymal stem cells and metformin induces osteoblastic differentiation of human induced pluripotent stem cell-derived mesenchymal stem cells in an AMPK-dependent manner, providing further evidence that structurally diverse compounds including metformin and CHIR that promote adult stem cell differentiation likely do so via a common mechanism of AMPK activation [29,30].

The induction of cellular stress and AMPK activation may also link beneficial modulation of the gut microbiome in humans not only with adult stem cell maintenance and differentiation, but also with the amelioration of pathologies associated with neurological disorders. A study recently published in the journal Clinical Genetics in April of 2017 demonstrated for the first time that metformin consistently improved behavior in several patients diagnosed with Fragile X Syndrome (FXS), a genetic disorder characterized by intellectual disability and significant deficits in neurological function and cognitive development [31]. An improvement in behavior was documented in the Aberrant Behavior Checklist (ABC) for all cases, as evidenced by consistent improvements (i.e. lower scores compared to pre-metformin treatment) in social avoidance, irritability, hyperactivity, and social unresponsiveness as well as improvements in language and conversational skills reported by familial caretakers [31].

Also, metformin has been shown to rescue and restore memory deficits in a Drosophila model of FXS and a recently published study (2017) demonstrated that metformin corrected social novelty impairment, reduced testicular weight, decreased repetitive grooming, rescued excessive long-term depression and dendritic spine abnormalities, restored excitatory synaptic transmission, and acutely activated AMPK in hippocampal pyramidal neurons in an FXS mouse model [32,33]. Interestingly, GSK3β inhibitors including the AMPK activator CHIR have been shown to rescue deficits in long-term potentiation at medial perforant path-dentate granule cells synapses in an FXS mouse model, indicating that cellular stress-induced AMPK activation by metformin and CHIR links the beneficial effects of those compounds in phenomena as disparate as stem cell differentiation, FXS, and long-term potentiation, hypotheses that I initially proposed in 2017 [34-36].

Lastly, as butyrate has been shown to reactivate latent HIV-1, facilitating immune system detection and virus destruction, and metformin when combined with bryostatin-1 (which also activates AMPK) promotes latent HIV-1 reactivation, cellular stress-induced AMPK activation likely also links beneficial modulation of human gut bacteria with latent HIV-1 reactivation [37-39].

AMPK activation also promotes oocyte meiotic induction and maturation (processes that are critical for efficient oocyte activation) and AMPK has recently been found localized across the entire acrosome in human spermatozoa [40-42]. The induction of cellular stress (e.g. increases in ROS, intracellular Ca2+, and/or AMP(ADP)/ATP ratio increase), which activates AMPK, also promotes oocyte meiotic induction/maturation, oocyte activation, and the acrosome reaction in human sperm, processes critical for the creation of all human life [41,43,44]. Indeed, the calcium ionophore ionomycin, which activates AMPK, is commonly used to promote latent HIV-1 reactivation and is extensively used to activate human oocytes, creating normal healthy children [44-46]. Such evidence indicates and further substantiates the novel and provocative assertion that AMPK activation links the amelioration of pathological cellular defects in FXS and Hutchinson-Gilford progeria syndrome with the gut microbiota, HIV-1 latency, adult and cancer stem cells, learning and memory, and the creation of all human life [4,35,36,39,40,47].

https://www.linkedin.com/pulse/ampk-activators-metformin-chir99021-improve-gut-bacteria-finley

References

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6. Yoon V, Nodwell JR. Activating secondary metabolism with stress and chemicals. J Ind Microbiol Biotechnol. 2014 Feb;41(2):415-24.
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13. Duan Q, Kita D, Johnson EA, et al. Reactive oxygen species mediate pollen tube rupture to release sperm for fertilization in Arabidopsis. Nat Commun. 2014;5:3129.
14. Hardie DG. AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function. Genes Dev. 2011 Sep 15;25(18):1895-908.
15. Sook SH, Lee HJ, Kim JH, et al. Reactive oxygen species-mediated activation of AMP-activated protein kinase and c-Jun N-terminal kinase plays a critical role in beta-sitosterol-induced apoptosis in multiple myeloma U266 cells. Phytother Res. 2014 Mar;28(3):387-94.
16. Ji AR, Ku SY, Cho MS, et al. Reactive oxygen species enhance differentiation of human embryonic stem cells into mesendodermal lineage. Exp Mol Med. 2010 Mar 31;42(3):175-86.
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18. Wee S, Niklasson M2, Marinescu VD, et al. Selective calcium sensitivity in immature glioma cancer stem cells. PLoS One. 2014 Dec 22;9(12):e115698.
19. Lee KM, Lee M, Lee J, et al. Enhanced anti-tumor activity and cytotoxic effect on cancer stem cell population of metformin-butyrate compared with metformin HCl in breast cancer. Oncotarget. 2016 Jun 21;7(25):38500-38512.
20. Lang D, Mascarenhas JB, Powell SK, Halegoua J, Nelson M, Ruggeri BA. PAX6 is expressed in pancreatic adenocarcinoma and is downregulated during induction of terminal differentiation. Mol Carcinog 2008;47(2):148–56.
21. Sato A, Sunayama J, Okada M, et al. Glioma-initiating cell elimination by metformin activation of FOXO3 via AMPK. Stem Cells Transl Med 2012;1(11):811–24.
22. Suzuki T, Bridges D, Nakada D, et al. Inhibition of AMPK catabolic action by GSK3. Mol Cell. 2013 May 9;50(3):407-19.
23. Avery LB, Bumpus NN. Valproic acid is a novel activator of AMP-activated protein kinase and decreases liver mass, hepatic fat accumulation, and serum glucose in obese mice. Mol Pharmacol. 2014 Jan;85(1):1-10.
24. McLean WJ, Yin X, Lu L, et al. Clonal Expansion of Lgr5-Positive Cells from Mammalian Cochlea and High-Purity Generation of Sensory Hair Cells. Cell Rep. 2017 Feb 21;18(8):1917-1929.
25. Yin X, Farin HF, van Es JH, et al. Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny. Nat Methods. 2014 Jan;11(1):106-12.
26. Sun X, Yang Q, Rogers CJ, Du M, Zhu MJ. AMPK improves gut epithelial differentiation and barrier function via regulating Cdx2 expression. Cell Death Differ. 2017 May;24(5):819-831.
27. Xue Y, Zhang H, Sun X, Zhu MJ. Metformin Improves Ileal Epithelial Barrier Function in Interleukin-10 Deficient Mice. PLoS One. 2016 Dec 21;11(12):e0168670.
28. Neves VC, Babb R, Chandrasekaran D, Sharpe PT. Promotion of natural tooth repair by small molecule GSK3 antagonists. Sci Rep. 2017 Jan 9;7:39654.
29. Kim EK, Lim S, Park JM, et al. Human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by AMP-activated protein kinase. J Cell Physiol. 2012 Apr;227(4):1680-7.
30. Wang P, Ma T, Guo D, et al. Metformin Induces Osteoblastic Differentiation of Human Induced Pluripotent Stem Cell-derived Mesenchymal Stem Cells. J Tissue Eng Regen Med. 2017 May 11. doi: 10.1002/term.2470. [Epub ahead of print].
31. Dy ABC, Tassone F, Eldeeb M, Salcedo-Arellano MJ, Tartaglia N, Hagerman R. Metformin as Targeted Treatment in Fragile X Syndrome. Clin Genet. 2017 Apr 24. doi: 10.1111/cge.13039. [Epub ahead of print].
32. Monyak RE, Emerson D, Schoenfeld BP, et al. Insulin signaling misregulation underlies circadian and cognitive deficits in a Drosophila fragile X model. Mol Psychiatry. 2017 Aug;22(8):1140-1148.
33. Gantois I, Khoutorsky A, Popic J, et al. Metformin ameliorates core deficits in a Fragile X syndrome mouse model. Nat Med. 2017 Jun;23(6):674-677.
34. Franklin AV, King MK, Palomo V, Martinez A, McMahon LL, Jope RS. Glycogen synthase kinase-3 inhibitors reverse deficits in long-term potentiation and cognition in fragile X mice. Biol Psychiatry. 2014 Feb 1;75(3):198-206.
35. Finley J. Elimination of cancer stem cells and reactivation of latent HIV-1 via AMPK activation: Common mechanism of action linking inhibition of tumorigenesis and the potential eradication of HIV-1. Med Hypotheses. 2017 Jul;104:133-146.
36. Finley J. Facilitation of hippocampal long-term potentiation and reactivation of latent HIV-1 via AMPK activation: Common mechanism of action linking learning, memory, and the potential eradication of HIV-1. Med Hypotheses. Manuscript submitted.
37. Imai K, Ochiai K, Okamoto T. Reactivation of latent HIV-1 infection by the periodontopathic bacterium Porphyromonas gingivalis involves histone modification. J Immunol 2009;182(6):3688–95.
38. Mehla R, Bivalkar-Mehla S, Zhang R, et al. Bryostatin modulates latent HIV-1 infection via PKC and AMPK signaling but inhibits acute infection in a receptor independent manner. PLoS ONE 2010;5(6):e11160.
39. 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.
40. Finley J. Oocyte activation and latent HIV-1 reactivation: AMPK as a common mechanism of action linking the beginnings of life and the potential eradication of HIV-1. Med Hypotheses. 2016 Aug;93:34-47.
41. LaRosa C, Downs SM. Stress stimulates AMP-activated protein kinase and meiotic resumption in mouse oocytes. Biol Reprod. 2006 Mar;74(3):585-92.
42. Calle-Guisado V, de Llera AH, Martin-Hidalgo D, et al. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility. Asian J Androl. 2016 Sep 27. doi: 10.4103/1008-682X.185848. [Epub ahead of print].
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47. Finley J. AMPK activation as a common mechanism of action linking the effects of diverse compounds that ameliorate accelerated cellular aging defects in Hutchinson-Gilford progeria syndrome. Med Hypotheses. Manuscript submitted.

New study shows AMPK activator MG132 rescues Progeria cells, protects against Microgravity, & inhibits Cancer Stem Cells, HIV, Dengue, & Malaria

Goldsmith Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus [Public domain], via Wikimedia Commons; CC BY 2.5 (http://creativecommons.org/licenses/by/2.5)], via Wikimedia Commons; Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. Scaffidi P, Gordon L, Misteli T. PLoS Biology Vol. 3/11/2005, e395; By NASA [Public domain], via Wikimedia Commons; By Thomas Splettstoesser (www.scistyle.com) [CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons.

A recent study published online in the journal EMBO Molecular Medicine in July of 2017 strikingly demonstrated that the proteasome inhibitor and AMPK activator MG132 alleviated accelerated aging defects in cells derived from children with the genetic disorder Hutchinson-Gilford progeria syndrome(HGPS) by inducing autophagic degradation of progerin, the toxic protein responsible for accelerated aging defects in HGPS cells [1]. MG132 also beneficially altered splicing of the LMNA gene (a gene that is aberrantly spliced to produce large amounts of progerin instead of the normal lamin A gene product) by decreasing the gene splicing factor SRSF1 but increasing the splicing factor SRSF5 [1]. Interestingly, progerin was shown to accumulate in structures known as promyelocytic nuclear bodies (PML-NBs) in the nucleus of HGPS cells and local injection of MG132 into a progeria mouse model also led to a reduction in the levels of SRSF1 and progerin [1]. Intriguingly, normal humans also produce progerin via the same aberrant gene splicing method as do children with HGPS, just at much lower levels that increase with age [2].

The recent finding that MG132-induced proteasome inhibition also results in a rapid activation of the master metabolic regulator AMPK (a kinase that increases lifespan and healthspan in several model organisms) and AMPK-dependent autophagy stimulation via the induction of cellular stress (i.e. reactive oxygen species [ROS] generation) further substantiates my hypothesis published in 2014 in which I was first to propose that AMPK activation by structurally diverse compounds (e.g. metformin, resveratrol, etc.) will lead to alleviation of accelerated aging defects in HGPS cells by decreasing the gene splicing factor SRSF1, thus beneficially altering splicing of the LMNA gene, as well as decreasing progerin levels by AMPK-induced autophagy [3,4,33].

Indeed, metformin, which induces cellular stress by mildly inhibiting complex I of the electron transport chain (thus increasing the AMP/ATP ratio) has also recently been shown to reduce the levels of SRSF1 and progerin and activate AMPK in HGPS cells [5-7]. Platelet-derived growth factor BB (PDGF-BB) also increases intracellular calcium (Ca2+) and ROS levels (mediators of cellular stress induction), activates AMPK, and increases SRSF5 in HGPS cells, thus altering splice site selection and beneficially increasing the lamin A/progerin ratio, providing compelling evidence that cellular stress-induced AMPK activation indeed represents a common mechanism of action for gene splicing- and autophagy-induced reductions of progerin in HGPS cells [8-10].

Interestingly, SRSF1 (also known as ASF/SF2) and PML-NBs also inhibit latent HIV-1 reactivation (preventing immune system detection and virus eradication) and SRSF5 (also known as SRp40) increases the abundance and translation of unspliced HIV-1 RNA, which is necessary for latent HIV-1 reactivation [11-13]. As AMPK promotes both latent HIV-1 reactivation and prevents HIV-1 transactivation, MG132 has been shown to reactivate latent HIV-1 and inhibit HIV-1 replication, substantiating my hypothesis published in 2015 in which I first proposed that AMPK activation links correction of aberrant alternative splicing in HGPS cells with reactivation of latent HIV-1 by compounds including MG132, metformin, and resveratrol [14-18].

Additionally, as the induction of cellular stress (e.g. intracellular Ca2+ increase, ROS generation, AMP/ATP ratio increase, etc.) activates AMPK, reactivates latent HIV-1, and leads to the differentiation and/or apoptosis of cancer stem cells in an AMPK-dependent manner, MG132 has also recently been found to induce apoptosis in glioma cancer stem cells, substantiating my most recent publication (2017) in which I propose for the first time that AMPK activation links reactivation of latent HIV-1 with cancer stem cell differentiation and/or apoptosis by diverse compounds that induce cellular stress including metformin and MG132 [5,19-23].

MG132 also induces mouse oocyte meiotic resumption (a process orchestrated by cellular stress-induced AMPK activation and is critical for efficient oocyte activation), delays in vitro oocyte aging, promotes embryonic development from aged oocytes after in vitro fertilization procedures, and alleviates deleterious effects associated with simulated microgravity, further supporting my hypotheses in 2016 and 2017 in which I first proposed that cellular stress-induced AMPK activation links oocyte activation (and hence the beginning of all human life) with latent HIV-1 reactivation and that AMPK activation will improve the activation of T cells in simulated microgravity/spaceflight (which is dependent on intracellular increases in Ca2+ and ROS) [23-28].

As AMPK activators including metformin and MG132 also inhibit dengue virus replication and malaria parasite growth, the aforementioned studies strongly suggests the novel observation that AMPK activation represents a common mechanism of action linking chemically distinct compounds and the effects of those compounds in diseases and phenomena as seemingly disparate as HGPS, HIV-1, microgravity/spaceflight, cancer stem cells, dengue fever, and malaria [29-32]. 

https://www.linkedin.com/pulse/new-study-shows-ampk-activator-mg132-rescues-progeria-finley?published=t

References

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29. Soto-Acosta R, Bautista-Carbajal P, Cervantes-Salazar M, Angel-Ambrocio AH, Del Angel RM. DENV up-regulates the HMG-CoA reductase activity through the impairment of AMPK phosphorylation: A potential antiviral target. PLoS Pathog. 2017 Apr 6;13(4):e1006257.
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