Metformin improves healthspan and lifespan in mice methods
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Metformin mimics calorie restriction transcriptome
We have previously associated the induction of similar transcriptome to animals under CR in liver tissue, even in a 8-week treatment. To test whether metformin shifts the physiology of ad lib -fed mice towards that of animals on CR after 30 weeks of treatment, we performed a genome-wide microarray analysis on liver and muscle tissues of three groups of mice ad lib -fed with SD, 0.1% metformin or CR.
Preservation of mitochondrial function by metformin
Our microarray and quantitative PCR analyses showed increased expression of several mitochondrial genes in the livers of metformin-treated mice. However, metformin lowered oxygen consumption in MEFs, suggesting that these alterations in mitochondrial bioenergetic function may occur in cell cultures. The administration of this anti-diabetic drug did not alter several markers of mitochondrial content in both MEFs and the liver of treated mice ( ), while causing only moderate increase, if any, in the expression of several subunits in the ETC complexes ( ). Among key enzymes of the tricarboxylic acid cycle and ETC that were assayed, complex I activity was significantly lower in metformin-treated MEFs ( ). In contrast, mice treated with metformin had a remarkable increase in hepatic complex I activity ( ). The activity for complexes III and IV was significantly reduced by metformin in MEFs...
Preservation of mitochondrial function by metformin
The activation of the SKN-1/Nrf2-dependent antioxidant response is required for the beneficial effects of metformin in Caenorhabditis elegans. There was a trend towards reduction in superoxide production in mitochondrial complexes in the livers of metformin-treated mice ( p =0.09, t -test two tailed) ( ). When divided by the total activity of mitochondrial complexes I-II to III, the proportion of superoxide leakage was significantly decreased by metformin ( ), indicating greater efficiency of mitochondrial complexes in transferring electrons to their expected acceptors in the ETC. Moreover, the marked reduction in the amount of lysine-4-hydroxynonenal-modified proteins and 8-iso-PGF2α, a marker of lipid peroxidation, was consistent with decreased oxidative stress damage in the liver of metformin-treated mice ( ). To investigate whether metformin activates the Nrf2-dependen...
Methods Summary
Animal procedures, housing and diets were in accordance with the guidelines issued by the Intramural Research Program of the National Institutes of Health protocol number 352TGB2013. Male C57BL/6 mice at either 6 months of age (for 0.1% metformin and CR) or 11 months of age (for 1% metformin dose) were purchased from the National Institute on Aging Aged Rodent Colony from Charles Rivers. The C57BL/6 mice were maintained on a standard purified mouse diet (AIN-93G) until they reached one year of age when the treatments started. The CR animals were subjected to a lifelong restriction on AIN-93G diet, starting at 27 weeks of age, with a daily food allotment of 60% of that eaten by the ad lib animals. Besides CR animals, the C57BL/6 groups in this study were fed the standard AIN-93G diet (SD) or AIN-93G plus 0.1% metformin or 1% metformin ad lib for the remainder of their lives. Pure metfo...
Faecal analysis
Total energy in faeces was determined in 0.6±0.04 g of fecal samples in a 1281 Oxygen Bomb Calorimeter (Parr Instrument, Moline, IL) according to the manufacturer's protocol. ( n = 3-5 per group; age = 66 weeks; diet = 12 weeks).
Metabolic Clamps
Mouse metabolic rate was assessed by indirect calorimetry in open-circuit oxymax chambers using the Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments, Columbus, OH). Mice were housed singly with water and food available ad lib, except for CR group, and maintained at 20-22 °C under a 12:12-h light-dark cycle (light period 0600-1800). All mice were acclimatized to monitoring cages for 3-6 h prior to recording. Sample air was passed through an oxygen sensor for determination of oxygen content. Oxygen consumption was determined by measuring oxygen concentration in air entering the chamber compared with air leaving the chamber. The sensor was calibrated against a standard gas mix containing defined quantities of oxygen, carbon dioxide and nitrogen. Constant airflow (0.6 L/min) was drawn through the chamber and monitored by a mass-sensitive flow...
Physical performance tests
Results from rotarod, treadmill and open-field are presented as follows: Time to fall from an accelerating rotarod (4-40 r.p.m. over 5 min); total distance ran in the treadmill test until exhaustion; and the speed of the animals in the open-field. Rotarod: Mice were given a habituation trial on day 1 where they were placed on the rotarod at a constant speed (4 rpm) and had to remain on the rotarod for 1 min. Results shown are the average of 3 trials per mouse, measuring time to fall from an accelerating rotarod (4-40 rpm over 5 min). The maximum trial length was 5 min, and there was a 30-min rest period between each trial ( n = 16 per group; age = 95 weeks; diet = 41 weeks). For the treadmill test, mice were required to exercise on the treadmill until exhaustion. The treadmill was horizontal (0° incline) and mice ran in groups of 6. Subjects were habituated at a const...
Oxygen consumption
Oxygen consumption in MEFs was measured using the Seahorse 24XF instrument (Seahorse Biosciences, North Billerica, MA). In brief, cells were seeded into Seahorse tissue culture plates and treated without or with 1 mM metformin for 16 h. Then, the medium was changed and the assay was run. A detailed explanation of the protocol is included in.
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Evidence quotes (8)
We have previously associated the induction of similar transcriptome to animals under CR in liver tissue, even in a 8-week treatment. To test whether metformin shifts the physiology of ad lib -fed mice towards that of animals on CR after 30 weeks of treatment, we performed a genome-wide microarray analysis on liver and muscle tissues of three groups of mice ad lib -fed with SD, 0.1% metformin or CR.
Our microarray and quantitative PCR analyses showed increased expression of several mitochondrial genes in the livers of metformin-treated mice. However, metformin lowered oxygen consumption in MEFs, suggesting that these alterations in mitochondrial bioenergetic function may occur in cell cultures. The administration of this anti-diabetic drug did not alter several markers of mitochondrial content in both MEFs and the liver of treated mice ( ), while causing only moderate increase, if any, in the expression of several subunits in the ETC complexes ( ). Among key enzymes of the tricarboxylic acid cycle and ETC that were assayed, complex I activity was significantly lower in metformin-treated MEFs ( ). In contrast, mice treated with metformin had a remarkable increase in hepatic complex I activity ( ). The activity for complexes III and IV was significantly reduced by metformin in MEFs, but remained unchanged in liver lysates of metformin-treated mice. The differential effects of metformin in vitro and in vivo may be due to the hepatocytes adapting to long-term inhibition of complex I activity. Interestingly, and in agreement with our findings, preservation of mitochondrial compl...
The activation of the SKN-1/Nrf2-dependent antioxidant response is required for the beneficial effects of metformin in Caenorhabditis elegans. There was a trend towards reduction in superoxide production in mitochondrial complexes in the livers of metformin-treated mice ( p =0.09, t -test two tailed) ( ). When divided by the total activity of mitochondrial complexes I-II to III, the proportion of superoxide leakage was significantly decreased by metformin ( ), indicating greater efficiency of mitochondrial complexes in transferring electrons to their expected acceptors in the ETC. Moreover, the marked reduction in the amount of lysine-4-hydroxynonenal-modified proteins and 8-iso-PGF2α, a marker of lipid peroxidation, was consistent with decreased oxidative stress damage in the liver of metformin-treated mice ( ). To investigate whether metformin activates the Nrf2-dependent antioxidant response, HepG2 cells stably expressing a Nrf2-responsive Antioxidant Response Element (ARE) luciferase reporter construct were treated with increasing concentrations of metformin for 16 h ( ). The increase in Nrf2/ARE reporter activity occurred with an ED 50 of ~1.5 mM metformin witho...
Animal procedures, housing and diets were in accordance with the guidelines issued by the Intramural Research Program of the National Institutes of Health protocol number 352TGB2013. Male C57BL/6 mice at either 6 months of age (for 0.1% metformin and CR) or 11 months of age (for 1% metformin dose) were purchased from the National Institute on Aging Aged Rodent Colony from Charles Rivers. The C57BL/6 mice were maintained on a standard purified mouse diet (AIN-93G) until they reached one year of age when the treatments started. The CR animals were subjected to a lifelong restriction on AIN-93G diet, starting at 27 weeks of age, with a daily food allotment of 60% of that eaten by the ad lib animals. Besides CR animals, the C57BL/6 groups in this study were fed the standard AIN-93G diet (SD) or AIN-93G plus 0.1% metformin or 1% metformin ad lib for the remainder of their lives. Pure metformin was obtained from Farmhispania (Farmhispania S.A. Barcelona, Spain) and mixed to homogeneity during manufacturing of the diets (Dyets Inc., Bethlehem, PA). For B6C3F1 mice longevity study, male mice (Harlan Breeders; Indianapolis) were randomly assigned to treatment groups at 12 months of age....
Total energy in faeces was determined in 0.6±0.04 g of fecal samples in a 1281 Oxygen Bomb Calorimeter (Parr Instrument, Moline, IL) according to the manufacturer's protocol. ( n = 3-5 per group; age = 66 weeks; diet = 12 weeks).
Mouse metabolic rate was assessed by indirect calorimetry in open-circuit oxymax chambers using the Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments, Columbus, OH). Mice were housed singly with water and food available ad lib, except for CR group, and maintained at 20-22 °C under a 12:12-h light-dark cycle (light period 0600-1800). All mice were acclimatized to monitoring cages for 3-6 h prior to recording. Sample air was passed through an oxygen sensor for determination of oxygen content. Oxygen consumption was determined by measuring oxygen concentration in air entering the chamber compared with air leaving the chamber. The sensor was calibrated against a standard gas mix containing defined quantities of oxygen, carbon dioxide and nitrogen. Constant airflow (0.6 L/min) was drawn through the chamber and monitored by a mass-sensitive flow meter. The concentrations of oxygen and carbon dioxide were monitored at the inlet and outlet of the sealed chambers to calculate oxygen consumption. Measurement in each chamber was recorded for 30 s at 30-min intervals for a total of 60 h. The second dark:light cycle is represented in the plots. M...
Results from rotarod, treadmill and open-field are presented as follows: Time to fall from an accelerating rotarod (4-40 r.p.m. over 5 min); total distance ran in the treadmill test until exhaustion; and the speed of the animals in the open-field. Rotarod: Mice were given a habituation trial on day 1 where they were placed on the rotarod at a constant speed (4 rpm) and had to remain on the rotarod for 1 min. Results shown are the average of 3 trials per mouse, measuring time to fall from an accelerating rotarod (4-40 rpm over 5 min). The maximum trial length was 5 min, and there was a 30-min rest period between each trial ( n = 16 per group; age = 95 weeks; diet = 41 weeks). For the treadmill test, mice were required to exercise on the treadmill until exhaustion. The treadmill was horizontal (0° incline) and mice ran in groups of 6. Subjects were habituated at a constant speed of 4 m.min -1 for 5 min. The following day each mouse was given a trial starting at 7 m.min -1 for 0-3 min, 12 m.min -1 for 3-7 min, 15 m.min -1 for 7-25 min, and 19 m.min -1 for 25 min ( n = 9 per group; age = 73 weeks; diet = 19 weeks). For...
Oxygen consumption in MEFs was measured using the Seahorse 24XF instrument (Seahorse Biosciences, North Billerica, MA). In brief, cells were seeded into Seahorse tissue culture plates and treated without or with 1 mM metformin for 16 h. Then, the medium was changed and the assay was run. A detailed explanation of the protocol is included in.
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