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Metformin induces a dietary restriction-like state and the oxidative stress response to extend C. elegans Healthspan via AMPK, LKB1, and SKN-1.

Onken B, Driscoll M - PLoS ONE (2010)

Bottom Line: Energy sensor AMPK and AMPK-activating kinase LKB1, which are activated in mammals by metformin treatment, are essential for health benefits in C. elegans, suggesting that metformin engages a metabolic loop conserved across phyla.We also show that the conserved oxidative stress-responsive transcription factor SKN-1/Nrf2 is essential for metformin healthspan benefits in C. elegans, a mechanistic requirement not previously described in mammals. skn-1, which functions in nematode sensory neurons to promote DR longevity benefits and in intestines for oxidative stress resistance lifespan benefits, must be expressed in both neurons and intestines for metformin-promoted healthspan extension, supporting that metformin improves healthy middle-life aging by activating both DR and antioxidant defense longevity pathways.In addition to defining molecular players operative in metformin healthspan benefits, our data suggest that metformin may be a plausible pharmacological intervention to promote healthy human aging.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey, United States of America.

ABSTRACT
Metformin, a biguanide drug commonly used to treat type-2 diabetes, has been noted to extend healthspan of nondiabetic mice, but this outcome, and the molecular mechanisms that underlie it, have received relatively little experimental attention. To develop a genetic model for study of biguanide effects on healthspan, we investigated metformin impact on aging Caenorhabditis elegans. We found that metformin increases nematode healthspan, slowing lipofuscin accumulation, extending median lifespan, and prolonging youthful locomotory ability in a dose-dependent manner. Genetic data suggest that metformin acts through a mechanism similar to that operative in eating-impaired dietary restriction (DR) mutants, but independent of the insulin signaling pathway. Energy sensor AMPK and AMPK-activating kinase LKB1, which are activated in mammals by metformin treatment, are essential for health benefits in C. elegans, suggesting that metformin engages a metabolic loop conserved across phyla. We also show that the conserved oxidative stress-responsive transcription factor SKN-1/Nrf2 is essential for metformin healthspan benefits in C. elegans, a mechanistic requirement not previously described in mammals. skn-1, which functions in nematode sensory neurons to promote DR longevity benefits and in intestines for oxidative stress resistance lifespan benefits, must be expressed in both neurons and intestines for metformin-promoted healthspan extension, supporting that metformin improves healthy middle-life aging by activating both DR and antioxidant defense longevity pathways. In addition to defining molecular players operative in metformin healthspan benefits, our data suggest that metformin may be a plausible pharmacological intervention to promote healthy human aging.

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Metformin extends median lifespan via an AMPK and LKB1-dependent mechanism.A. Survival curves of AMPK catalytic subunit mutants aak-2(ok524) and aak-2(rr48) raised on metformin plates at 20°C for their entire lives. Median survival for aak-2(ok524) animals on 0 mM, 1 mM, 10 mM, and 50 mM metformin is 17, 17, 15, and 15 days, respectively, and there are no significant differences between any of the survival curves (Log-rank test). Median survival for aak-2(rr48) animals on 0 mM, 1 mM, 10 mM, and 50 mM is 13, 15, 15, and 15 days, respectively, and there are no significant differences between the survival curves. Pooled data from three independent trials for each aak-2 strain show approximately 11% declines with 10 mM and 50 mM metformin treatment in aak-2(ok524) animals, and approximately 6% and 19% declines with 10 mM and 50 mM metformin treatment, respectively, in aak-2(rr48) animals. Survival curves are significantly shifted to the left in aak-2(ok524) animals treated with 10 mM and 50 mM metformin (P = 0.0209 and <0.0001, respectively), and in aak-2(rr48) animals raised on 10 mM and 50 mM metformin (P = 0.0442 and 0.0004, respectively). See Table S1E for additional data. We conclude that AMPK activity is needed for median lifespan extension induced by metformin, and that metformin has detrimental lifespan effects in the absence of AMPK. B. Survival curves of temperature-sensitive lkb-1/par-4 mutants grown on 0 mM and 50 mM metformin plates, maintained at 15°C and shifted to 25°C at the L4 stage. C. elegans par-4 encodes an ortholog of mammalian LKB1, which has been shown to activate AMPK in both mammals and nematodes. Median survival is 9 days for the par-4(it47) mutants grown on both 0 mM and 50 mM metformin, and there is no significant difference between the survival curves by the Log-rank test. Similarly, par-4(it57) animals grown on both 0 mM and 50 mM metformin have a median survival of 11 days, and their survival curves are not significantly different by the Log-rank test. Pooled data from three independent trials using the par-4(it47) strain show no differences in median lifespan for 0 mM vs. 50 mM metformin-treated animals (9 days for both conditions), although the 50 mM metformin survival curve is significantly shifted to the left (P = 0.0500, Log-rank; Table S1F). Pooled data for four independent trials with the par-4(it57) mutants show a decrease in median lifespan (9 days for 50 mM metformin plates vs. 10 days for 0 mM metformin; Table S1F) and a significantly left-shifted survival curve for 50 mM metformin-treated animals (P = 0.0063, Log-rank; Table S1F). Thus, LKB1 is required for metformin to increase median lifespan, and, as is seen in the absence of AMPK, metformin has harmful effects when LKB1 is disrupted.
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pone-0008758-g003: Metformin extends median lifespan via an AMPK and LKB1-dependent mechanism.A. Survival curves of AMPK catalytic subunit mutants aak-2(ok524) and aak-2(rr48) raised on metformin plates at 20°C for their entire lives. Median survival for aak-2(ok524) animals on 0 mM, 1 mM, 10 mM, and 50 mM metformin is 17, 17, 15, and 15 days, respectively, and there are no significant differences between any of the survival curves (Log-rank test). Median survival for aak-2(rr48) animals on 0 mM, 1 mM, 10 mM, and 50 mM is 13, 15, 15, and 15 days, respectively, and there are no significant differences between the survival curves. Pooled data from three independent trials for each aak-2 strain show approximately 11% declines with 10 mM and 50 mM metformin treatment in aak-2(ok524) animals, and approximately 6% and 19% declines with 10 mM and 50 mM metformin treatment, respectively, in aak-2(rr48) animals. Survival curves are significantly shifted to the left in aak-2(ok524) animals treated with 10 mM and 50 mM metformin (P = 0.0209 and <0.0001, respectively), and in aak-2(rr48) animals raised on 10 mM and 50 mM metformin (P = 0.0442 and 0.0004, respectively). See Table S1E for additional data. We conclude that AMPK activity is needed for median lifespan extension induced by metformin, and that metformin has detrimental lifespan effects in the absence of AMPK. B. Survival curves of temperature-sensitive lkb-1/par-4 mutants grown on 0 mM and 50 mM metformin plates, maintained at 15°C and shifted to 25°C at the L4 stage. C. elegans par-4 encodes an ortholog of mammalian LKB1, which has been shown to activate AMPK in both mammals and nematodes. Median survival is 9 days for the par-4(it47) mutants grown on both 0 mM and 50 mM metformin, and there is no significant difference between the survival curves by the Log-rank test. Similarly, par-4(it57) animals grown on both 0 mM and 50 mM metformin have a median survival of 11 days, and their survival curves are not significantly different by the Log-rank test. Pooled data from three independent trials using the par-4(it47) strain show no differences in median lifespan for 0 mM vs. 50 mM metformin-treated animals (9 days for both conditions), although the 50 mM metformin survival curve is significantly shifted to the left (P = 0.0500, Log-rank; Table S1F). Pooled data for four independent trials with the par-4(it57) mutants show a decrease in median lifespan (9 days for 50 mM metformin plates vs. 10 days for 0 mM metformin; Table S1F) and a significantly left-shifted survival curve for 50 mM metformin-treated animals (P = 0.0063, Log-rank; Table S1F). Thus, LKB1 is required for metformin to increase median lifespan, and, as is seen in the absence of AMPK, metformin has harmful effects when LKB1 is disrupted.

Mentions: We next took genetic approaches toward identifying molecules that mediate metformin healthspan benefits in physiological context. In mammals, energy sensor AMPK is activated by metformin [20], [21], and metformin-activated AMPK increases glucose uptake in muscle and inhibits gluconeogenesis in hepatocytes [19]. C. elegans aak-2 encodes one of two homologs of the catalytic α subunit of the AMPK heterotrimeric complex, and nematode AAK-2 is activated by AMP, as occurs for mammalian AMPK [55]. Overexpression of the catalytic AMPK α subunit AAK-2 increases C. elegans longevity [55], and aak-2 is required for the increased lifespan of several longevity mutants [56] and for lifespan extension under at least one tested mode of DR induction [34]. To investigate the role of AMPK in metformin-induced extended median lifespan in C. elegans, we tested for metformin effects in the backgrounds of two independent aak-2 alleles: aak-2(ok524), a presumed molecular [55], and aak-2(rr48), which carries a point mutation predicted to disrupt the catalytic activity of the α subunit [57]. As shown in Fig. 3A, no tested metformin concentration significantly increased the median lifespan of either aak-2 mutant (in three repeated trials for each strain; Table S1E). Instead, metformin treatment decreased mid-life viability in aak-2 mutants in a dose-dependent manner (Table S1E) and reduced locomotory ability in both aak-2 strains (Fig. S2A). We conclude that the catalytic AMPK subunit is required for median lifespan extension and locomotory healthspan increase in metformin-treated C. elegans. Moreover, we note that in the absence of healthspan-promoting AMPK activity, metformin confers deleterious consequences on culture survival and swimming prowess. Interestingly, although we did not note statistically significant differences in Nile Red staining (Fig. S2B), we did find lower age pigments in aak-2(ok524) mutants treated with 50 mM metformin vs. controls (Fig. S2C, other allele not tested). This observation suggests age pigment changes might be conferred via an AMPK-independent mechanism.


Metformin induces a dietary restriction-like state and the oxidative stress response to extend C. elegans Healthspan via AMPK, LKB1, and SKN-1.

Onken B, Driscoll M - PLoS ONE (2010)

Metformin extends median lifespan via an AMPK and LKB1-dependent mechanism.A. Survival curves of AMPK catalytic subunit mutants aak-2(ok524) and aak-2(rr48) raised on metformin plates at 20°C for their entire lives. Median survival for aak-2(ok524) animals on 0 mM, 1 mM, 10 mM, and 50 mM metformin is 17, 17, 15, and 15 days, respectively, and there are no significant differences between any of the survival curves (Log-rank test). Median survival for aak-2(rr48) animals on 0 mM, 1 mM, 10 mM, and 50 mM is 13, 15, 15, and 15 days, respectively, and there are no significant differences between the survival curves. Pooled data from three independent trials for each aak-2 strain show approximately 11% declines with 10 mM and 50 mM metformin treatment in aak-2(ok524) animals, and approximately 6% and 19% declines with 10 mM and 50 mM metformin treatment, respectively, in aak-2(rr48) animals. Survival curves are significantly shifted to the left in aak-2(ok524) animals treated with 10 mM and 50 mM metformin (P = 0.0209 and <0.0001, respectively), and in aak-2(rr48) animals raised on 10 mM and 50 mM metformin (P = 0.0442 and 0.0004, respectively). See Table S1E for additional data. We conclude that AMPK activity is needed for median lifespan extension induced by metformin, and that metformin has detrimental lifespan effects in the absence of AMPK. B. Survival curves of temperature-sensitive lkb-1/par-4 mutants grown on 0 mM and 50 mM metformin plates, maintained at 15°C and shifted to 25°C at the L4 stage. C. elegans par-4 encodes an ortholog of mammalian LKB1, which has been shown to activate AMPK in both mammals and nematodes. Median survival is 9 days for the par-4(it47) mutants grown on both 0 mM and 50 mM metformin, and there is no significant difference between the survival curves by the Log-rank test. Similarly, par-4(it57) animals grown on both 0 mM and 50 mM metformin have a median survival of 11 days, and their survival curves are not significantly different by the Log-rank test. Pooled data from three independent trials using the par-4(it47) strain show no differences in median lifespan for 0 mM vs. 50 mM metformin-treated animals (9 days for both conditions), although the 50 mM metformin survival curve is significantly shifted to the left (P = 0.0500, Log-rank; Table S1F). Pooled data for four independent trials with the par-4(it57) mutants show a decrease in median lifespan (9 days for 50 mM metformin plates vs. 10 days for 0 mM metformin; Table S1F) and a significantly left-shifted survival curve for 50 mM metformin-treated animals (P = 0.0063, Log-rank; Table S1F). Thus, LKB1 is required for metformin to increase median lifespan, and, as is seen in the absence of AMPK, metformin has harmful effects when LKB1 is disrupted.
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Related In: Results  -  Collection

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pone-0008758-g003: Metformin extends median lifespan via an AMPK and LKB1-dependent mechanism.A. Survival curves of AMPK catalytic subunit mutants aak-2(ok524) and aak-2(rr48) raised on metformin plates at 20°C for their entire lives. Median survival for aak-2(ok524) animals on 0 mM, 1 mM, 10 mM, and 50 mM metformin is 17, 17, 15, and 15 days, respectively, and there are no significant differences between any of the survival curves (Log-rank test). Median survival for aak-2(rr48) animals on 0 mM, 1 mM, 10 mM, and 50 mM is 13, 15, 15, and 15 days, respectively, and there are no significant differences between the survival curves. Pooled data from three independent trials for each aak-2 strain show approximately 11% declines with 10 mM and 50 mM metformin treatment in aak-2(ok524) animals, and approximately 6% and 19% declines with 10 mM and 50 mM metformin treatment, respectively, in aak-2(rr48) animals. Survival curves are significantly shifted to the left in aak-2(ok524) animals treated with 10 mM and 50 mM metformin (P = 0.0209 and <0.0001, respectively), and in aak-2(rr48) animals raised on 10 mM and 50 mM metformin (P = 0.0442 and 0.0004, respectively). See Table S1E for additional data. We conclude that AMPK activity is needed for median lifespan extension induced by metformin, and that metformin has detrimental lifespan effects in the absence of AMPK. B. Survival curves of temperature-sensitive lkb-1/par-4 mutants grown on 0 mM and 50 mM metformin plates, maintained at 15°C and shifted to 25°C at the L4 stage. C. elegans par-4 encodes an ortholog of mammalian LKB1, which has been shown to activate AMPK in both mammals and nematodes. Median survival is 9 days for the par-4(it47) mutants grown on both 0 mM and 50 mM metformin, and there is no significant difference between the survival curves by the Log-rank test. Similarly, par-4(it57) animals grown on both 0 mM and 50 mM metformin have a median survival of 11 days, and their survival curves are not significantly different by the Log-rank test. Pooled data from three independent trials using the par-4(it47) strain show no differences in median lifespan for 0 mM vs. 50 mM metformin-treated animals (9 days for both conditions), although the 50 mM metformin survival curve is significantly shifted to the left (P = 0.0500, Log-rank; Table S1F). Pooled data for four independent trials with the par-4(it57) mutants show a decrease in median lifespan (9 days for 50 mM metformin plates vs. 10 days for 0 mM metformin; Table S1F) and a significantly left-shifted survival curve for 50 mM metformin-treated animals (P = 0.0063, Log-rank; Table S1F). Thus, LKB1 is required for metformin to increase median lifespan, and, as is seen in the absence of AMPK, metformin has harmful effects when LKB1 is disrupted.
Mentions: We next took genetic approaches toward identifying molecules that mediate metformin healthspan benefits in physiological context. In mammals, energy sensor AMPK is activated by metformin [20], [21], and metformin-activated AMPK increases glucose uptake in muscle and inhibits gluconeogenesis in hepatocytes [19]. C. elegans aak-2 encodes one of two homologs of the catalytic α subunit of the AMPK heterotrimeric complex, and nematode AAK-2 is activated by AMP, as occurs for mammalian AMPK [55]. Overexpression of the catalytic AMPK α subunit AAK-2 increases C. elegans longevity [55], and aak-2 is required for the increased lifespan of several longevity mutants [56] and for lifespan extension under at least one tested mode of DR induction [34]. To investigate the role of AMPK in metformin-induced extended median lifespan in C. elegans, we tested for metformin effects in the backgrounds of two independent aak-2 alleles: aak-2(ok524), a presumed molecular [55], and aak-2(rr48), which carries a point mutation predicted to disrupt the catalytic activity of the α subunit [57]. As shown in Fig. 3A, no tested metformin concentration significantly increased the median lifespan of either aak-2 mutant (in three repeated trials for each strain; Table S1E). Instead, metformin treatment decreased mid-life viability in aak-2 mutants in a dose-dependent manner (Table S1E) and reduced locomotory ability in both aak-2 strains (Fig. S2A). We conclude that the catalytic AMPK subunit is required for median lifespan extension and locomotory healthspan increase in metformin-treated C. elegans. Moreover, we note that in the absence of healthspan-promoting AMPK activity, metformin confers deleterious consequences on culture survival and swimming prowess. Interestingly, although we did not note statistically significant differences in Nile Red staining (Fig. S2B), we did find lower age pigments in aak-2(ok524) mutants treated with 50 mM metformin vs. controls (Fig. S2C, other allele not tested). This observation suggests age pigment changes might be conferred via an AMPK-independent mechanism.

Bottom Line: Energy sensor AMPK and AMPK-activating kinase LKB1, which are activated in mammals by metformin treatment, are essential for health benefits in C. elegans, suggesting that metformin engages a metabolic loop conserved across phyla.We also show that the conserved oxidative stress-responsive transcription factor SKN-1/Nrf2 is essential for metformin healthspan benefits in C. elegans, a mechanistic requirement not previously described in mammals. skn-1, which functions in nematode sensory neurons to promote DR longevity benefits and in intestines for oxidative stress resistance lifespan benefits, must be expressed in both neurons and intestines for metformin-promoted healthspan extension, supporting that metformin improves healthy middle-life aging by activating both DR and antioxidant defense longevity pathways.In addition to defining molecular players operative in metformin healthspan benefits, our data suggest that metformin may be a plausible pharmacological intervention to promote healthy human aging.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey, United States of America.

ABSTRACT
Metformin, a biguanide drug commonly used to treat type-2 diabetes, has been noted to extend healthspan of nondiabetic mice, but this outcome, and the molecular mechanisms that underlie it, have received relatively little experimental attention. To develop a genetic model for study of biguanide effects on healthspan, we investigated metformin impact on aging Caenorhabditis elegans. We found that metformin increases nematode healthspan, slowing lipofuscin accumulation, extending median lifespan, and prolonging youthful locomotory ability in a dose-dependent manner. Genetic data suggest that metformin acts through a mechanism similar to that operative in eating-impaired dietary restriction (DR) mutants, but independent of the insulin signaling pathway. Energy sensor AMPK and AMPK-activating kinase LKB1, which are activated in mammals by metformin treatment, are essential for health benefits in C. elegans, suggesting that metformin engages a metabolic loop conserved across phyla. We also show that the conserved oxidative stress-responsive transcription factor SKN-1/Nrf2 is essential for metformin healthspan benefits in C. elegans, a mechanistic requirement not previously described in mammals. skn-1, which functions in nematode sensory neurons to promote DR longevity benefits and in intestines for oxidative stress resistance lifespan benefits, must be expressed in both neurons and intestines for metformin-promoted healthspan extension, supporting that metformin improves healthy middle-life aging by activating both DR and antioxidant defense longevity pathways. In addition to defining molecular players operative in metformin healthspan benefits, our data suggest that metformin may be a plausible pharmacological intervention to promote healthy human aging.

Show MeSH
Related in: MedlinePlus