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 hepatocyte’s 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 student’s 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
cell’s 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 student’s 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.