were under a time-restricted feeding proto-
col (UW), which should be beneficial. Thus, it
would be of considerable interest to explore
the effects of time-restricted feeding and cir-
cadian phase of CR in future studies in non-
human primates.
The benefits of circadian alignment of feed-
ing and fasting cycles are widespread across
species. It increases life span in flies ( 54 ),
protects against metabolic disorders in mice
( 17 , 18 , 55 , 56 ), and promotes health in humans
( 57 , 58 ). We also found that CR promotes
widespread metabolic benefits, which include
lower body weight with reduced fat content.
Furthermore, CR (particularly the longest-
lived group, CR-night) attenuated age-related
changes observed under AL by improving glu-
cose homeostasis, insulin sensitivity, and hor-
monal profiles.
Using circadian gene profiling, we found
that timing of food intake led to complex
genome-wide reprogramming of circadian
gene expression in the liver, which is con-
sistent with other findings ( 11 , 14 , 33 , 59 – 61 ).
This emphasizes the importance of consid-
ering the sampling time, which is often a
snapshot, before concluding whether any in-
tervention increases or decreases the expres-
sion of a gene of interest. If a gene has a
circadian oscillation, then a single snapshot
could lead to the opposite conclusions depend-
ing on the time it was taken, particularly in
classicCRprotocolsresembling our CR-day-2h
group ( 1 , 7 ).
Aging was associated with increased expres-
sion of the genes involved in immune processes
and inflammation and decreased expression of
the genes involved in metabolism and circadian
biology. CR treatment restored or attenuated
many of these age-related changes in gene
expression in a manner similar to results
reported by the de Cabo ( 24 , 34 ) and Sassone-
Corsi ( 14 ) groups. Decreases in circadian ampli-
tude of gene expression with age were reduced
by CR. Overall, feeding time and fasting had
additive effects on CR-mediated life-span ex-
tension. Together, CR and time restriction of
feeding to the nighttime optimally extended
life span and delayed many of the age-related
gene expression changesin immune function,
inflammation, and metabolism. Further studies
are required to determine whether disruption
of circadian sleep/wake cycles by daytime feed-
ing also contributes to the day versus night
differential responses in the liver transcriptome
and life span. Thus, circadian interventions
such as timed feeding can enhance the well-
known life-span benefits of CR.
There are many links between the circa-
dian system and aging ( 20 ). High-amplitude
circadian rhythms correlate with well-being
( 13 , 62 , 63 ), whereas clock dysfunction leads
to metabolic disorders, premature aging, and
reduced life span ( 42 , 64 – 67 ). During normal
aging, rhythms decrease in amplitude and
often exhibit a shift in phase ( 14 , 20 , 68 – 72 ).
In both flies and mice, the effects of dietary
restriction on life span require core circadian
clock genes ( 73 – 75 ). The circadian system reg-
ulates most of the metabolic pathways impli-
cated in longevity ( 3 , 4 , 13 , 22 , 32 , 59 , 76 , 77 ).
Molecules known to function in the regula-
tion of life span by dietary restriction, such as
insulin and insulin-like growth factor 1 (IGF-1),
Sirtuin1 (SIRT1), nicotinamide phosphoribosyl-
transferase (NAMPT), AMP-activated protein
kinase (AMPK), PPARgcoactivator 1 (PGC-1a),
mechanistic target of rapamycin (mTOR), and
glycogen synthase kinase 3b(GSK3b), are all
intricately involved in the molecular mecha-
nisms of circadian clocks ( 77 – 89 ). The master
circadian transcription factors CLOCK and
BMAL1 have direct gene targets in every fun-
damental metabolic pathway in the liver
( 25 , 32 , 90 , 91 ). Because of these direct links
among the pathways involved in aging and
longevity, metabolism, and the circadian clock,
our results demonstrate the importance of
timing of CR and indicate that optimizing the
phase of circadian gene expression may be a
powerful intervention for extending life span.
We used C57BL/6J male mice in this study.
However, there could be strain- and sex-specific
responses worth studying further ( 24 , 92 ), be-
cause, for example, ovarian hormones can
protect females against dietary challenges
that otherwise disrupt circadian rhythms in
males ( 55 ). In future work, both sexes and
multiple genetic backgrounds ( 92 – 94 )should
be used to explore the broader effects of cir-
cadian interventions on aging, and the results
may support the application of circadian-timed
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