Researchs
Effects of Metformin on Aging-Induced Mitochondrial Dysfunction and Longevity: An Animal Model Study
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Introduction
Ageing is a complex biological process characterised by a gradual decline in cellular and physiological functions (1-3). This process is associated with increased susceptibility to disease and increased risk of mortality. Among the mechanisms underlying ageing, mitochondrial dysfunction, which plays a critical role in energy production and redox balance, is a major contributor. Mitochondrial dysfunction is recognised as one of the major determinants of ageing and results in impaired cellular energy metabolism, increased reactive oxygen species (ROS) and an inability to clear damaged organelles. These mechanisms play an important role in the pathogenesis of age-related diseases (4). Metformin, in addition to being a widely used anti-diabetic drug, is attracting attention for its anti-aging effects due to its properties such as increasing mitochondrial efficiency via AMP-activated protein kinase (AMPK) and reducing oxidative stress (5).
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Materials and Methods
Materials and Methods
Materials and Methods
Materials and Methods
Study design and ethical approval
Study design and ethical approval
This experimental study was designed according to ARRIVE guidelines and approved by Istanbul Medipol University Animal Experiments Ethics Committee. All experimental procedures were performed in accordance with national and international guidelines for the care and use of laboratory animals.
This experimental study was designed according to ARRIVE guidelines and approved by Istanbul Medipol University Animal Experiments Ethics Committee. All experimental procedures were performed in accordance with national and international guidelines for the care and use of laboratory animals.
Animal Model
Animal Model
Species and Breed
C57BL/6 mice, a well-characterised model for studying ageing and longevity.
Age Groups
Young group: 3-month-old mice at the beginning of the study.Old group: 18-month-old mice at the beginning of the study.
Sample Size
Total of 60 mice, equally divided among experimental groups (n=10 per group).
Housing Conditions
12-hour light/dark cycle.Temperature maintained at 22 ± 2°C.Standard diet and ad libitum access to water.
Species and Breed
C57BL/6 mice, a well-characterised model for studying ageing and longevity.
Age Groups
Young group: 3-month-old mice at the beginning of the study.Old group: 18-month-old mice at the beginning of the study.
Sample Size
Total of 60 mice, equally divided among experimental groups (n=10 per group).
Housing Conditions
12-hour light/dark cycle.Temperature maintained at 22 ± 2°C.Standard diet and ad libitum access to water.
Experimental Groups
Experimental Groups
Control (Young)
Standard diet, no intervention.
Control (Elderly)
Standard diet, no intervention.
Metformin (Elderly)
Standard diet plus 0.1% (w/w) metformin supplementation for 6 months.
High Dose Metformin (Elderly)
Standard diet with 1% (w/w) metformin supplementation for 6 months.
Control (Young)
Standard diet, no intervention.
Control (Elderly)
Standard diet, no intervention.
Metformin (ElderlyTotal of 60 mice, equally divided among experimental groups (n=10 per group).
Housing Conditions
12-hour light/dark cycle.Temperature maintained at 22 ± 2°C.Standard diet and ad libitum access to water.
Intervention
Intervention
Metformin Administration
Based on the literature (7), metformin was administered by mixing with standard diet to reach the desired concentration.
Duration:
Continuous dietary supplementation was administered for a total of 6 months.
Metformin Administration
Based on the literature (7), metformin was administered by mixing with standard diet to reach the desired concentration.
Duration:
Continuous dietary supplementation was administered for a total of 6 months.
Outcome Measures
Outcome Measures
Primary Outcome
Survival: To be assessed by Kaplan-Meier survival analysis.
Secondary Outcomes
Primary Outcome
Survival: To be assessed by Kaplan-Meier survival analysis.
Secondary Outcomes
Mitochondrial Function
Mitochondrial Function
ATP Production
ATP levels will be quantitatively measured using luciferase-based ATP measurement kits to assess cellular energy metabolism.
Mitochondrial Membrane Potential
JC-1 fluorescent dye will be used to assess mitochondrial membrane integrity and energy production capacity.
ATP Production
ATP levels will be quantitatively measured using luciferase-based ATP measurement kits to assess cellular energy metabolism.
Mitochondrial Membrane Potential
JC-1 fluorescent dye will be used to assess mitochondrial membrane integrity and energy production capacity.
Oxidative Stress
Oxidative Stress
Reactive Oxygen Species (ROS)
Cellular oxidative stress levels will be measured using fluorescence microscopy with dihydroethidium (DHE) dye.
Lipid Peroxidation
Lipid peroxidation levels will be evaluated by measuring malondialdehyde (MDA) levels. High performance liquid chromatography (HPLC) method will be used to provide high sensitivity and specificity in this measurement.
Reactive Oxygen Species (ROS)
Cellular oxidative stress levels will be measured using fluorescence microscopy with dihydroethidium (DHE) dye.
Lipid Peroxidation
Lipid peroxidation levels will be evaluated by measuring malondialdehyde (MDA) levels. High performance liquid chromatography (HPLC) method will be used to provide high sensitivity and specificity in this measurement.
Gene Expression
Gene Expression
PGC-1α and Nrf2
will be analysed by qPCR for mitochondrial biogenesis and antioxidant response. Expression levels of mitochondrial biogenesis (PGC-1α) and antioxidant response (Nrf2) markers will be analysed by quantitative polymerase chain reaction (qPCR).
PGC-1α and Nrf2
will be analysed by qPCR for mitochondrial biogenesis and antioxidant response. Expression levels of mitochondrial biogenesis (PGC-1α) and antioxidant response (Nrf2) markers will be analysed by quantitative polymerase chain reaction (qPCR).
Inflammation
Inflammation
Serum IL-6 and TNF-α levels
IL-6 and TNF-α levels, which are markers of systemic inflammation, will be measured using enzyme-linked immunosorbent assay (ELISA) method.
Serum IL-6 and TNF-α levels
IL-6 and TNF-α levels, which are markers of systemic inflammation, will be measured using enzyme-linked immunosorbent assay (ELISA) method.
Physical Health Indicators
Physical Health Indicators
Body Weight
In order to evaluate the effect of metformin on body weight, the weights of all mice will be measured monthly with a precision balance.
Exercise Capacity
A rotarod test will be performed to assess muscle strength and coordination capacity. The residence time of the mice on the rotarod will be measured and the effects of metformin on physical performance will be analysed based on these data.
Body Weight
In order to evaluate the effect of metformin on body weight, the weights of all mice will be measured monthly with a precision balance.
Exercise Capacity
A rotarod test will be performed to assess muscle strength and coordination capacity. The residence time of the mice on the rotarod will be measured and the effects of metformin on physical performance will be analysed based on these data.
Sample Collection and Analysis
Sample Collection and Analysis
At the end of the study, liver, skeletal muscle and brain tissues will be removed from mice for biochemical and histological analyses. Immediately after surgical removal, the tissues will be washed with cold physiological saline solution and frozen with liquid nitrogen and stored at -80°C for biochemical analyses. For the evaluation of mitochondrial ultrastructure, transmission electron microscopy (TEM) will be used as part of histological analysis. Mitochondrial membrane integrity, cristae structure and general mitochondrial morphology will be evaluated in TEM analyses. For TEM analyses, tissues will be fixed with 2.5% glutaraldehyde and 1% osmium tetroxide, followed by dehydration with ethanol and propylene oxide, respectively. After dehydration, tissues will be embedded with epoxy resin, ultrathin sections will be taken and stained with uranyl acetate and lead citrate.
At the end of the study, liver, skeletal muscle and brain tissues will be removed from mice for biochemical and histological analyses. Immediately after surgical removal, the tissues will be washed with cold physiological saline solution and frozen with liquid nitrogen and stored at -80°C for biochemical analyses. For the evaluation of mitochondrial ultrastructure, transmission electron microscopy (TEM) will be used as part of histological analysis. Mitochondrial membrane integrity, cristae structure and general mitochondrial morphology will be evaluated in TEM analyses. For TEM analyses, tissues will be fixed with 2.5% glutaraldehyde and 1% osmium tetroxide, followed by dehydration with ethanol and propylene oxide, respectively. After dehydration, tissues will be embedded with epoxy resin, ultrathin sections will be taken and stained with uranyl acetate and lead citrate.
Statistical Analysis
Statistical Analysis
Statistical analysis of the study was performed using different methods to assess the primary and secondary outcome measures. In survival analysis, the Kaplan-Meier method was applied to assess differences in survival between groups and statistical significance was tested by the log-rank test. One-way analysis of variance (ANOVA) was used to compare parameters such as mitochondrial function, oxidative stress markers (ATP production, reactive oxygen species, lipid peroxidation) and inflammatory cytokine levels (IL-6, TNF-α) between groups. In cases where significant differences were found, Bonferroni correction was applied to determine which groups were different. Pearson correlation analysis was performed to examine the relationship between mitochondrial markers (e.g., PGC-1α and Nrf2 expression) and survival.
All statistical analyses were performed in accordance with appropriate assumptions depending on whether the data were normally distributed. In cases where the data were not normally distributed, nonparametric tests (e.g. Kruskal-Wallis test) were applied to confirm the results. In statistical analyses, p<0.05 was accepted as significance criterion and all analyses were performed using SPSS (IBM SPSS Statistics for Windows, Version 25.0) software.
Statistical analysis of the study was performed using different methods to assess the primary and secondary outcome measures. In survival analysis, the Kaplan-Meier method was applied to assess differences in survival between groups and statistical significance was tested by the log-rank test. One-way analysis of variance (ANOVA) was used to compare parameters such as mitochondrial function, oxidative stress markers (ATP production, reactive oxygen species, lipid peroxidation) and inflammatory cytokine levels (IL-6, TNF-α) between groups. In cases where significant differences were found, Bonferroni correction was applied to determine which groups were different. Pearson correlation analysis was performed to examine the relationship between mitochondrial markers (e.g., PGC-1α and Nrf2 expression) and survival.
All statistical analyses were performed in accordance with appropriate assumptions depending on whether the data were normally distributed. In cases where the data were not normally distributed, nonparametric tests (e.g. Kruskal-Wallis test) were applied to confirm the results. In statistical analyses, p<0.05 was accepted as significance criterion and all analyses were performed using SPSS (IBM SPSS Statistics for Windows, Version 25.0) software.
Expected Impact
Expected Impact
This study aims to elucidate the mechanisms by which metformin extends lifespan by reducing age-related mitochondrial dysfunction and oxidative stress. The results of this study will not only contribute to the development of strategies for the treatment of age-related diseases, but will also provide a new perspective for translational research into healthspan extension. This study may be an important step in understanding the fundamental mechanisms of ageing biology through the regulation of mitochondrial function and may provide a scientific basis for the development of anti-aging therapeutic approaches.
This study aims to elucidate the mechanisms by which metformin extends lifespan by reducing age-related mitochondrial dysfunction and oxidative stress. The results of this study will not only contribute to the development of strategies for the treatment of age-related diseases, but will also provide a new perspective for translational research into healthspan extension. This study may be an important step in understanding the fundamental mechanisms of ageing biology through the regulation of mitochondrial function and may provide a scientific basis for the development of anti-aging therapeutic approaches.
References
López-Otín C., Blasco M.A., Partridge L., Serrano M., Kroemer G. The Hallmarks of Aging. Cell. 2013;153:1194–1217.
Clemente J.C., Ursell L.K., Parfrey L.W., Knight R. The impact of the gut microbiota on human health: An integrative view. Cell. 2012;148:1258–1270.
Bana B., Cabreiro F. The Microbiome and Aging. Annu. Rev. Genet. 2019;53:239–261.
Braidy N, Liu Y. NAD+ therapy in age-related degenerative disorders: A benefit/risk analysis. Exp Gerontol. 2020;132:110831.
Zhou G, Myers R, Li Y, et al. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest. 2001;108(8):1167-1174.
Zhu X, Shen W, Liu Z, et al. Effect of Metformin on Cardiac Metabolism and Longevity in Aged Female Mice. Front Cell Dev Biol. 2021;8:626011.
Martin-Montalvo A, Mercken EM, Mitchell SJ, et al. Metformin improves healthspan and lifespan in mice. Nat Commun. 2013;4:2192.
López-Otín C., Blasco M.A., Partridge L., Serrano M., Kroemer G. The Hallmarks of Aging. Cell. 2013;153:1194–1217.
Clemente J.C., Ursell L.K., Parfrey L.W., Knight R. The impact of the gut microbiota on human health: An integrative view. Cell. 2012;148:1258–1270.
Bana B., Cabreiro F. The Microbiome and Aging. Annu. Rev. Genet. 2019;53:239–261.
Braidy N, Liu Y. NAD+ therapy in age-related degenerative disorders: A benefit/risk analysis. Exp Gerontol. 2020;132:110831.
Zhou G, Myers R, Li Y, et al. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest. 2001;108(8):1167-1174.
Zhu X, Shen W, Liu Z, et al. Effect of Metformin on Cardiac Metabolism and Longevity in Aged Female Mice. Front Cell Dev Biol. 2021;8:626011.
Martin-Montalvo A, Mercken EM, Mitchell SJ, et al. Metformin improves healthspan and lifespan in mice. Nat Commun. 2013;4:2192.
López-Otín C., Blasco M.A., Partridge L., Serrano M., Kroemer G. The Hallmarks of Aging. Cell. 2013;153:1194–1217.
Clemente J.C., Ursell L.K., Parfrey L.W., Knight R. The impact of the gut microbiota on human health: An integrative view. Cell. 2012;148:1258–1270.
Bana B., Cabreiro F. The Microbiome and Aging. Annu. Rev. Genet. 2019;53:239–261.
Braidy N, Liu Y. NAD+ therapy in age-related degenerative disorders: A benefit/risk analysis. Exp Gerontol. 2020;132:110831.
Zhou G, Myers R, Li Y, et al. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest. 2001;108(8):1167-1174.
Zhu X, Shen W, Liu Z, et al. Effect of Metformin on Cardiac Metabolism and Longevity in Aged Female Mice. Front Cell Dev Biol. 2021;8:626011.
Martin-Montalvo A, Mercken EM, Mitchell SJ, et al. Metformin improves healthspan and lifespan in mice. Nat Commun. 2013;4:2192.
Metformin
Metformin
Researchs
Effects of Metformin on Aging-Induced Mitochondrial Dysfunction and Longevity: An Animal Model Study