98 6 ART for Antiaging
noticed that ART, SNP, or ARG can lead to the upregulation of mitochondrial bio-
markers, such as mitofusin 2, cytochrome c, and COX4, which are coordinated
with their upstream activators, including SIRT1 and PGC-1α. Besides, an activated
phosphorylation form of mTOR, p-mTORSer2448, was also detected upon treatment
by ART, SNP, or ARG (Wang et al. 2014 ).
To follow up the time-course changes of mitochondrial signatures, we exam-
ined the expression dynamics of AMPK, PGC-1α, and cytochrome c in mouse
skeletal muscles treated by ART, SNP, or ARG. When compared with the constitu-
tively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH), we found
that AMPK reaches its maximal level within 3 h and subsequently maintains a sta-
ble-steady level. Differently, PGC-1α and cytochrome c exhibit a time-dependent
feature in their expression, namely short-time treatment allows a relative lower
level, long-time treatment leads to the highest level (Wang et al. 2014 ). These
results indicated that the signal is conveyed from AMPK to PGC-1α, and finally to
cytochrome c rather than vice versa.
To verify whether enhanced mitochondrial protein expressions also ensure the
according increase of their activities, we measured the activities of eNOS and COX4
after treatment of mouse skeletal muscles by ART, SNP, or ARG. Consequently, all
treatments can really lead to the increase of eNOS and COX activities, indicating
that an enhanced expression necessarily confer an increased activity for eNOS and
COX4 (Wang et al. 2015b). Besides, upregulation of SIRT3, a mitochondria-spe-
cific deacetylase, was also monitored after the same treatments as above-mentioned,
which may be considered an indirect hint implying mitochondrial biogenesis.
6.3.2.5 Association of CR Mimetics-Derived NO with Mitochondrial
Biogenesis
The expression data have revealed the induced upregulation of eNOS by CR
mimetics, but direct evidence confirming the elevation of NO levels is still lacking.
So we determined the NO level in skeletal muscles of mice injected by CR mimet-
ics. As results, NO burst was seen after treatment for 6 h although a decline trend
was observed after treatment for 3 days (Fig. 6.3a), addressing that all kinds of
CR mimetics used in this study play their roles upon NO signaling. Furthermore,
we also measured the ATP levels in the skeletal muscles of mice injected by CR
mimetics. The results as depicted in Fig. 6.3b indicated that ATP is increased after
treatment for 6 h, but maintains a steady-state higher level after treatment for
3 days. These results provided support to CR-enhanced mitochondrial functions.
At last, we scrutinized whether the density of mitochondria are changed in
mouse skeletal muscle cells exposed to ART, SNP, ARG, or H 2 O 2. As compared
with one-layer and linear-arrayed mitochondria seen in AL-exposed cells, SNP-
treated cells, or H 2 O 2 -treated cells (Fig. 6.3c–e) show a remarkable mitochondrial
proliferation with multilayer mitochondria, and ART-treated cells or ARG-treated
cells (Fig. 6.3f and g) also possess more mitochondrial layers than AL-exposed
cells after treatment for 6 h.