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Anush Swaminathan

Circadian clocks orchestrate daily physiology and metabolism. This study measured mitochondrial DNA (mDNA) levels in mouse liver cells throughout a 24-hour period to determine if mitochondrial numbers vary circadianly. mDNA levels peaked between 4-16 hours but did not follow a circadian pattern. Comparing wild type and clock-disrupted mice, mDNA levels did not differ, indicating mitochondrial numbers are independent of the core circadian clock mechanism. While no circadian oscillations were found, mitochondrial proteins and numbers peaked at similar times, suggesting possible daily variations in mitochondrial numbers deserve further study.

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Abstract Circadian clocks are based on a cellular molecular mechanism, which orchestrates our daily physiology and metabolism. In recent years, the relationship between the mammalian clock and various metabolic processes has been elucidated in part through descriptions of daily oscillations in mitochondrial activity. Furthermore, recent work done in our lab has shown variations in mitochondrial protein content. Therefore, we were interested to find whether these changes are accompanied by temporal variations in the cellular mitochondrial number. To address this question, we set out to measure the amount of mitochondrial DNA throughout a mouse hepatocytes circadian period. We purified DNA from wild type mice liver from six different time points, at four hour intervals throughout 24-hours, using the TriReagent Protocol. Quantitative Real Time PCR was then used to amplify certain segments of the extracted DNA targeting four genes (qPCR is traditionally used to target mRNA for gene expression, but the four genes used in our experiment targeted specifically DNA sequences). Two of the genes measured the mDNA whereas the other two were used to target nDNA for normalization purposes, since it is assumed to be constant. The use of the students t-test to compare the similarities of data in two opposing time points (i.e time 0 with 12, 4 with 16, and so on) yielded a statistically significant change in ratio between the mDNA and nDNA at only two time points: between time 4 and 16 with the ratio peaking at time 4. Past work done in our lab had shown that oscillating proteomes peaked within the mitochondria at a similar time (~time 3). Therefore, a correlation between our results of the mitochondrial number and the mitochondrial proteomes can be demonstrated. However, a juxtaposition of a cosine wave on the data led to statistically insignificant similarities, designating our results not circadian in nature (i.e no single peak over the 24 hour period). Next, we wanted to investigate the relationship between the mitochondrial number and the cells core clock mechanism. Since the circadian clock was previously shown to influence mitochondrial function, we wanted to examine its potential influence on mitochondrial copy number. To this effect, we utilized our system to investigate a clock disrupted mouse model, knockout mice which were demonstrated as noticeably arrhythmic. Again, DNA was extracted using the TriReagent Protocol, using qPCR to target the above mentioned genes. This time wild type mice and PER1/2 double-knockout mice were compared, at the same time point. Our results showed a statistically insignificant change in the mDNA to nDNA ratio. Such an analysis was verified using a students t-test. Therefore, we propose that mitochondrial copy number is independent of proper clock function (specifically the PER1/2 proteins) and that the amount of mDNA is not oscillating in the mouse liver throughout the day. However, even though circadian oscillations in mitochondrial numbers were not found, the peaking of the mitochondrial proteomes and the mitochondrial number at similar time points may showcase a possible temporal variation in mitochondrial number throughout the day. Further research is needed to elucidate the exact mechanism that supports the functional changes previously described for mitochondria in mice liver cells.

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Abstract (1)

  • 1. Abstract Circadian clocks are based on a cellular molecular mechanism, which orchestrates our daily physiology and metabolism. In recent years, the relationship between the mammalian clock and various metabolic processes has been elucidated in part through descriptions of daily oscillations in mitochondrial activity. Furthermore, recent work done in our lab has shown variations in mitochondrial protein content. Therefore, we were interested to find whether these changes are accompanied by temporal variations in the cellular mitochondrial number. To address this question, we set out to measure the amount of mitochondrial DNA throughout a mouse hepatocytes circadian period. We purified DNA from wild type mice liver from six different time points, at four hour intervals throughout 24-hours, using the TriReagent Protocol. Quantitative Real Time PCR was then used to amplify certain segments of the extracted DNA targeting four genes (qPCR is traditionally used to target mRNA for gene expression, but the four genes used in our experiment targeted specifically DNA sequences). Two of the genes measured the mDNA whereas the other two were used to target nDNA for normalization purposes, since it is assumed to be constant. The use of the students t-test to compare the similarities of data in two opposing time points (i.e time 0 with 12, 4 with 16, and so on) yielded a statistically significant change in ratio between the mDNA and nDNA at only two time points: between time 4 and 16 with the ratio peaking at time 4. Past work done in our lab had shown that oscillating proteomes peaked within the mitochondria at a similar time (~time 3). Therefore, a correlation between our results of the mitochondrial number and the mitochondrial proteomes can be demonstrated. However, a juxtaposition of a cosine wave on the data led to statistically insignificant similarities, designating our results not circadian in nature (i.e no single peak over the 24 hour period). Next, we wanted to investigate the relationship between the mitochondrial number and the cells core clock mechanism. Since the circadian clock was previously shown to influence mitochondrial function, we wanted to examine its potential influence on mitochondrial copy number. To this effect, we utilized our system to investigate a clock disrupted mouse model, knockout mice which were demonstrated as noticeably arrhythmic. Again, DNA was extracted using the TriReagent Protocol, using qPCR to target the above mentioned genes. This time wild type mice and PER1/2 double-knockout mice were compared, at the same time point. Our results showed a statistically insignificant change in the mDNA to nDNA ratio. Such an analysis was verified using a students t-test. Therefore, we propose that mitochondrial copy number is independent of proper clock function (specifically the PER1/2 proteins) and that the amount of mDNA is not oscillating in the mouse liver throughout the day. However, even though circadian oscillations in mitochondrial numbers were not found, the peaking of the mitochondrial proteomes and the mitochondrial number at similar time points may showcase a possible temporal variation in mitochondrial number throughout the day. Further research is needed to elucidate the exact mechanism that supports the functional changes previously described for mitochondria in mice liver cells.