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Late life metformin treatment limits cell survival and shortens lifespan by triggering an aging-associated failure ...

metformin atp exhaustion ampk dietary restriction pka mitochondria

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#1 Engadin

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Posted 28 August 2020 - 08:51 PM








F U L L   T I T L E :   Late life metformin treatment limits cell survival and shortens lifespan by triggering an aging-associated failure of energy metabolism.




O P E N   A C C E S S   S O U R C E :   bioRxiv









The diabetes drug metformin is to be clinically tested in aged humans to achieve health span extension, but little is known about responses of old non-diabetic individuals to this drug. By in vitro and in vivo tests we found that metformin shortens life span and limits cell survival when provided in late life, contrary to its positive early life effects. Mechanistically, metformin exacerbates aging-associated mitochondrial dysfunction towards respiratory failure, aggravated by the inability of old cells to upregulate glycolysis in response to metformin, leading to ATP exhaustion. The beneficial dietary restriction effect of metformin on lipid reserves is abrogated in old animals, contributing to metabolic failure, while ectopic stabilization of cellular ATP levels alleviates late life metformin toxicity in vitro and in vivo. The toxicity is also suspended in nematodes carrying diabetes-like insulin receptor insufficiency and showing prolonged resilience to metabolic stress induced by metformin. In sum, we uncovered an alarming metabolic decay triggered by metformin in late life which may limit its benefits for non-diabetic elderly patients. Novel regulators of life extension by metformin are also presented.
   Late life metformin treatment limits cell survival and shortens lifespan.
   Metformin exacerbates aging-associated mitochondrial dysfunction causing fatal ATP exhaustion.
   Old cells fail to upregulate glycolysis as a compensatory response to metformin.
   The dietary restriction (DR) mimetic response to metformin is abrogated in old animals.
   PKA and not AMPK pathway instigates the early life DR response to metformin.
   Stabilization of cellular ATP levels alleviates late life metformin toxicity in vitro and in vivo.
Metformin is among the most frequently prescribed drugs worldwide and it is used to facilitate glucose catabolism in patients with impaired insulin signaling (diabetes type 2) (Salani et al., 2014). Metformin is thought to act by inhibiting mitochondrial respiration (Wheaton et al., 2014). Recently, metformin was tested for additional physiological effects and found to extend life span in animal models ranging from nematodes to mice (Martin-Montalvo et al., 2013; Onken and Driscoll, 2010). Extensive clinical use in humans enabled the collection and analysis of data on the longevity of human diabetes patients treated with metformin. The metformin-exposed diabetes cohort was found to be longer lived than untreated healthy subjects (Bannister et al., 2014), in line with potential life-prolonging properties of metformin.
Type 2 diabetes is an aging-associated disorder and many patients start metformin treatment in late life. Based on survival analysis of diabetes patients, it was proposed that life prolonging effects of metformin may extend also to metabolic-healthy elderly individuals. Considering moderate metformin side effects in diabetes and the potential healthy aging benefits, metformin has emerged as an attractive candidate to be clinically tested as the first prospective anti-aging drug in humans. A short term trial administering metformin to aged (≥65 years old) pre-diabetic humans for a period of 6 weeks had recently been completed and the data was reported (Kulkarni et al., 2018). While effects on pathways such as TOR and immune response were observed, no obvious physiological changes were detected due to short duration of the treatment, leaving long-term effects of metformin on aged healthy humans an open question. This question is however critical because non-diabetic elderly humans are anticipated to be the first recipients of the putative health span extension treatment with metformin.
Via literature research of animal studies providing evidence of longevity modulation by metformin, we discovered that the pro-survival effect of this drug was mostly studied in young animals or animals exposed to metformin from young adulthood. The few studies performed in older animals (56-60 week old mice, average lifespan 96 weeks; 8 day old nematodes, average lifespan 14-21 days), either failed to detect life extension by metformin (Alfaras et al., 2017; Anisimov et al., 2011) or revealed toxicity that was partially attributed to the metformin overdose (Cabreiro et al., 2013; Martin-Montalvo et al., 2013; Thangthaeng et al., 2017). Strikingly, a dose of 50mM metformin which triggered strongest lifespan extension in a seminal study performed in young C. elegans, was moderately toxic when given to middle aged (adulthood day 8) nematodes (Cabreiro et al., 2013; Onken and Driscoll, 2010). We thus came to a conclusion that the benefits and safety of metformin administration to old non-insulin resistant individuals were not sufficiently investigated, contrary to responses of diabetic patients.
Here we used genetic tests, metabolic measurements, stress reporter assays and omics analyses to detect age-specific effects of metformin in C. elegans and human primary cells. We found that metformin treatment initiated in late life shortens life span and limits cell survival by aggravating aging associated mitochondrial dysfunction towards respiratory failure. In addition to mitochondrial distortion, old cells failed to enhance the use of glycolysis in response to metformin leading to persistent ATP exhaustion. We found that interventions stabilizing cellular ATP levels, such as ATP repletion and TOR inhibitor rapamycin, alleviate late life metformin toxicity in vitro and in vivo. We also discovered that early age metformin treatment instigates a range of stress and metabolic adaptations which likely underlay longevity extension by metformin. Importantly, the induction of these favorable responses was strongly impaired in late life. Particularly, we show that early but not late life metformin treatment induces a lipid turnover response similar to dietary restriction (DR). We also found that this early life DR mimetic phenotype is instigated by the triglyceride lipolysis pathway regulated by the protein kinase A and not by AMP activated protein kinase (AMPK) pathway, as suggested previously. Subsequently, we showed that metformin treatment restricted to early adulthood is sufficient for life extension, strengthening the key role of early life stress and metabolic adaptations in longevity benefits of metformin. Finally, we demonstrate that daf-2(e1370) mutants, carrying diabetes-like insufficiency of the C. elegans insulin receptor, are resilient to late life metformin toxicity in comparison to age-matched wild type controls, due to improved capacity to sustain ATP synthesis during old age metformin exposure. Collectively, we uncovered an alarming capability of metformin to induce metabolic failure in non-insulin resistant old subjects which may limit its benefits for non-diabetic elderly humans.
Late life metformin treatment is detrimental for longevity
To address the outcomes of metformin treatment at different age, we treated young adult (3 days old, day 1 of adulthood), adult at the age of reproduction decline (day 4 of adulthood), middle aged (day 8 of adulthood) and old (day 10 of adulthood) wild type C. elegans worms with different doses of metformin – 10mM, 25mM and 50mM. 50mM metformin is the common dose used to induce lifespan extension in C. elegans while 10mM is the lowest dose linked to reproducible life extension in this model in previous reports (Cabreiro et al., 2013; Onken and Driscoll, 2010; Pryor et al., 2019). We found that metformin treatment started at young age (days 1 and 4 of adulthood) extended lifespan of nematodes at all doses used (Figure 1A and Figure S1A). Within treatment initiated on day 8 of adulthood, the doses of 50mM and 25mM metformin reduced median lifespan but extended maximal lifespan consistent with previous observations (Cabreiro et al., 2013) while 10mM dose was longevity-extending with no detrimental effects (Figure S1B). Strikingly, on day 10 of adulthood metformin was toxic at all doses used with a large proportion of drug-exposed animals dying within first 24 hours of treatment (Figure 1B). Our first experiments in nematodes thus revealed an evident age-dependent decrease in metformin tolerance which culminated in late life toxicity of all metformin doses tested, indicating possible safety risks of late life metformin administration.







Figure 1. Metformin treatment initiated in late life exerts toxicity and limits survival independently of AMPK and microbial changes.

Wild type (WT, N2 Bristol strain) nematodes were treated with indicated doses of metformin (Met) on day 1 (A) and day 10 (B) of adulthood (AD1 and AD10 respectively), survival was scored daily. WT nematodes were grown on alive and UV-killed HT115 © and OP50 (D) E. coli and treated with 50mM metformin on AD10. Survival was scored daily. WT (E, left) and AMPK deficient (E, right) nematodes were treated with 50mM metformin on AD1 and AD10, survival was scored daily. Significance was measured by log-rank test, n≥100 in all cases, the exact n numbers and statistical values for all panels are presented in Table S1. Each experiment was repeated ≥3 times; one representative result is shown in all cases.
Late life metformin toxicity is independent of microbiome
To understand the mechanism of late life metformin toxicity, we first addressed known pathways regulating lifespan extension by this compound. The pro-longevity effect of metformin in young C. elegans nematodes was previously linked to changes of microbial metabolism induced by this drug (Cabreiro et al., 2013). To test if old age metformin toxicity relied on similar microbiome alterations we treated worms with metformin in the presence of living and UV-killed OP50 (metformin sensitive) and HT115 (metformin-resistant) E. coli strains. Old age metformin toxicity developed regardless of the bacterial viability and/or strain (Figure 1C and D) suggesting that late life metformin intolerance is independent of previously uncovered microbiome changes. Interestingly, the baseline survival of nematodes differed between UV killed OP50 and HT115 diets in line with recently reported dependence of nematode physiological behaviors on the bacterial source (Revtovich et al., 2019).
AMPK is not required for late life toxicity of metformin
Another component essential for young age life-extending effect of metformin is AMP-activated protein kinase (AMPK); particularly metformin failed to promote longevity in nematodes lacking AMPK orthologue AAK-2 (Onken and Driscoll, 2010). In order to probe the requirement of AMPK for old age toxicity of metformin we treated young and old wild type and aak-2(ok524) mutant animals with this drug. Consistent with previous reports, early life metformin treatment was unable to induce lifespan extension in aak-2 deficient worms (Figure 1E); at the same time late life metformin toxicity did develop in mutant animals, indicating that life shortening induced by metformin at old age is not executed by AMPK.
Metformin toxicity is triggered by mitochondrial impairments
One of metformin’s primary functions is to inhibit complex I of the mitochondrial electron transport chain (ETC), affecting mitochondrial membrane potential and ATP production (Andrzejewski et al., 2014; Cameron et al., 2018; Wheaton et al., 2014). Growth inhibition by metformin was previously linked to impaired mitochondrial respiration in nematodes and mammalian cells (Wu et al., 2016). Additionally, the accumulation of damaged and dysfunctional mitochondria, which may enhance the negative impact of ETC complex I inhibitors on cell survival, is one of the best characterized hallmarks of aging (Bratic and Larsson, 2013; Bratic and Trifunovic, 2010; Cellerino and Ori, 2017; Sun et al., 2016; Taylor and Dillin, 2011).
Mitochondrial deterioration comparable to aging occurs prematurely in mutant animals, defective in mitochondrial biogenesis and quality control (Sun et al., 2016; Trifunovic et al., 2004). To test if metformin toxicity during aging (and metformin toxicity in general) is driven by accumulation of mitochondrial impairments, we obtained mutants harboring deficiencies of diverse mitochondrial homeostasis pathways: mitochondrial unfolded protein response (atfs-1(gk3094)) (Nargund et al., 2012), mitochondrial biogenesis (skn-1(zj15)) (Palikaras et al., 2015), mitochondrial respiration (isp-1(qm150)) (Feng et al., 2001) and mitochondrial protein quality control (ubl-5(ok3389)) (Benedetti et al., 2006), and treated these animals with metformin along with wild type counterparts. A combination of metformin with congenital mitochondrial impairments led to an early life onset of metformin toxicity in all mutant backgrounds tested (including normally long-lived isp-1(qm150) mutants) clearly linking metformin intolerance to elevated abundance of dysfunctional mitochondria (Figure 2A-C, Figure S2A). The same effect (premature onset of metformin toxicity at young age) was observed in nematodes and human primary cells incubated with mitochondrial uncoupling agent carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) along with metformin administration (Figure 2D-E and Figure S2B). Of note, congenital mitochondrial respiration defects were recently found to limit the life extending interplay between metformin and the microbiome (Pryor et al., 2019) in line with the key role of mitochondrial integrity in diverse longevity benefits of metformin. To test if mitophagy and/or mitochondrial unfolded protein response (UPR MT) - the protective pathways responding to mitochondrial failure and known to deteriorate during aging (Sun et al., 2016), were directly induced by metformin we measured the abundance of mitochondrial proteins and the expression of hsp-6::gfp transgene (UPR MT reporter) in young and old animals treated with this drug. At both ages metformin administration didn’t lead to either elevated expression of GFP or reduction of mitochondrial protein levels (Figure S3A-C), indicating that mitophagy and UPR MT are not prominently triggered by metformin and likely play no role in immediate cellular adaptation to metformin-induced effects. Of note, lack of UPR MT induction by metformin has previously been reported by an independent group (De Haes et al., 2014). We thus show that the early onset of metformin toxicity in mitochondrial mutants is likely driven by the accumulation of mitochondrial damages prior to metformin administration, comparable to what occurs during aging.
Figure 2. Mitochondrial impairments pre-dispose nematodes and human cells to metformin toxicity.
atfs-1(gk3094) (A), skn-1(zj15) (B) and isp-1(qm150) © nematodes were exposed to 50mM metformin on adulthood day 1 (AD1) along with age-matched WT animals, survival was scored daily. AD1 WT nematodes (D) and early passage (population doubling, PD37) human primary fibroblasts (E) were co-treated with FCCP and metformin (50mM in worms), survival was measured daily in worms and after 24h of metformin treatment in cells; DMSO was used as a vehicle control for FCCP in both cells and worms; in E all values are relative to a respective untreated (no metformin) condition (separately for vehicle and FCCP). For A-D, significance was measured by log-rank test; all n numbers (n≥100 in all cases) and statistical values are presented in Table S1. For E n=3, mean and SEM are presented, two-tailed unpaired t-test was used for the statistical analysis, all statistical values are shown in Table S2; ** p<0,01; *** p<0,001; **** p<0,0001.




Metformin toxicity associates with ATP exhaustion and is alleviated by ATP repletion
To measure direct age-specific effects of metformin on mitochondrial performance and to test the conservation of our findings in humans, we analyzed the impact of metformin treatment on homeostasis of early passage (young) and late passage (old, replicative senescent) primary human skin fibroblasts. The replicative senescence model was chosen because of its high relevance for normal human aging as senescent cells accumulate in aging tissues. In vitro aging was performed according to standard procedures demonstrated to yield cells carrying key hallmarks of aging (Tigges et al., 2014), and aging-associated mitochondrial decline of late passage cells was verified by the Seahorse analysis (Figure 3C-D, Figure S4A and C). We found that, at high doses, metformin was toxic to both young and old cells but old cells (similar to old nematodes) showed a much stronger viability decline and succumbed to toxicity already at lower doses of metformin (Figure 3A-B). The oxygen consumption rate (OCR) measurements demonstrated a significant effect of metformin on basal respiration in young and old cells (Figure 3C, Figure S4A), while inhibition of mitochondrial ATP synthesis by metformin was stronger in old fibroblasts (Figure S4C). In addition, only old cells showed a decrease of maximal respiration in response to metformin (Figure 3D, Figure S4A) consistent with a stronger negative impact of this drug on mitochondria of old cells. Interestingly, the extracellular acidification rate (ECAR) was potently increased in young metformin treated fibroblasts (Figure 3E, Figure S4B) in line with their elevated reliance on glycolysis in response to mitochondrial insufficiency triggered by metformin. This adaptive increase of glycolysis was markedly reduced in old metformin exposed cells (Figure 3E, Figure S4B), depriving these cells, in combination with the stronger mitochondrial hindrance by metformin, of effective pathways of ATP synthesis. Subsequent determination of cellular ATP content indeed showed a stronger decline of ATP levels in old metformin treated cells (Figure 3F). We also detected a stronger distortion of the mitochondrial membrane potential in old compared to young metformin exposed fibroblasts (Figure 3G), in line with a more potent mitochondrial decline observed in old cells via oxygen consumption analysis.
Figure 3. Metformin toxicity associates with loss of mitochondrial homeostasis and ATP exhaustion in human cells and nematodes.
Young (PD36) and old (PD61) human skin fibroblasts were treated with metformin for 15 (F, G) or 20 (A, B) hours; cell survival (A, MTT assay), cell death (B, LDH assay), mitochondrial membrane potential (G, JC-1 assay) and ATP content (F) were measured; all values are relative to the untreated control of a given age. (C–E) Young (PD29) and old (PD61) primary human skin fibroblasts were treated with metformin for 16h, then washed and pre-incubated with media containing high glucose (10mM), glutamine (2mM) and pyruvate (1mM); continuous oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were recorded following injection of oligomycin (1μM), FCCP (3μM), antimycin-A (0,5μM) and rotenone (0,5μM) (shown in Fig S4). The recorded values were normalized to protein content, basal respiration ©, maximal respiration (D) and basal ECAR (E) were quantified. (H) Young (adulthood day 1) and old (adulthood day 10) wild type nematodes were treated with 50mM metformin, whole organism ATP levels were measured after 36h. Pre-senescent (PD44) fibroblasts were treated with metformin for 24h in presence or absence of ATP; ATP content (I) and cell survival (J) were measured; all values are relative to the untreated control (no metformin, no ATP) for each assay. For H n=100, for all other panels n=3, mean and SEM are presented, two-tailed unpaired t-test was used for the statistical analysis, all statistical values are presented in Table S2; * p<0,05; ** p<0,01; *** p<0,001; **** p<0,0001.
Figure S16. Metformin exerts age-specific effects on energy metabolism and instigates a severe energetics failure in late life.
While early life metformin treatment triggers metabolic adaptations, such as elevated glycolysis and the DR-like utilization of lipids, which support the longevity benefits of this drug, late life metformin exposure acts in concert with aging-associated metabolic distortions to trigger a sever metabolic failure culminating in ATP exhaustion incompatible with cell viability. Importantly, the early life DR effect of metformin is executed by the PKA pathway while AMPK likely prevents the untimely lipid expenditure by this pathway to ensure long-term longevity benefits of metformin.

Edited by Engadin, 28 August 2020 - 08:54 PM.

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