The FASEB Journal article fj.15-282475. Published online December 23, 2015.
The FASEB Journal • Research Communication
Circadian clocks govern calorie restriction–mediated
life span extension through BMAL1- and
IGF-1-dependent mechanisms
Sonal A. Patel, Amol Chaudhari, Richa Gupta, Nikkhil Velingkaar, and Roman V. Kondratov1
Department of Biological, Geological, and Environmental Sciences, and Center for Gene Regulation in
Health and Diseases, Cleveland State University, Cleveland, Ohio, USA
ABSTRACT Calorie restriction (CR) increases longevity
in many species by unknown mechanisms. The circadian
clock was proposed as a potential mediator of CR. De-
ficiency of the core component of the circadian clock-
—transcriptional factor BMAL1 (brain and muscle ARNT
[aryl hydrocarbon receptor nuclear translocator]-like pro-
tein 1)—results in accelerated aging. Here we investigated
the role of BMAL1 in mechanisms of CR. The 30% CR diet
increased the life span of wild-type (WT) mice by 20%
compared to mice on an ad libitum (AL) diet but failed to
increase life span of Bmal12/2 mice. BMAL1 deficiency
impaired CR-mediated changes in the plasma levels of IGF-
1 and insulin. We detected a statistically significantly re-
duction of IGF-1 in CR vs. AL by 50 to 70% in WT mice at
several daily time points tested, while in Bmal12/2 the re-
duction was not significant. Insulin levels in WT were re-
duced by 5 to 9%, while Bmal12/2 induced it by 10 to 35% at
all time points tested. CR up-regulated the daily average
expression of Bmal1 (by 150%) and its downstream target
genes Periods (by 470% for Per1 and by 130% for Per2). We
propose that BMAL1 is an important mediator of CR,
and activation of BMAL1 might link CR mechanisms
with biologic clocks.—Patel, S. A., Chaudhari, A., Gupta,
R., Velingkaar, N., Kondratov, R. V. Circadian clocks govern
calorie restriction–mediated life span extension through
BMAL1- and IGF-1-dependent mechanisms. FASEB J.
30, 000–000 (2016). www.fasebj.org
Key Words: aging • gene expression • glucose • insulin • tran-
scription • food anticipation
Calorie restriction (CR) is a robust intervention that in-
creases longevity across different species, including mam-
mals (1–4). The precise molecular mechanisms of CR are
unknown, and multiple theories have been put forward to
explain CR-mediated effects on life span and health. Several
physiologic systems—such as the mammalian target of
rapamycin (mTOR) signaling pathway, the insulin/IGF-1
signaling pathways, and the sirtuin-controlled pathway—are
Abbreviations: AL, ad libitum; BMAL1, brain and muscle
ARNT (aryl hydrocarbon receptor nuclear translocator)-like
protein 1; CR, calorie restriction; mTORC1, mammalian tar-
get of rapamycin complex 1; NAMPT, nicotinamide phos-
phoribosyl transferase; NPAS2, neuronal PAS domain protein
2; Sirt1, sirtuin (silent mating type information regulation 2
homolog) 1; WT, wild-type; ZT, Zeitgeber time
0892-6638/16/0030-0001 © FASEB
affected by CR in animals and are considered to be po-
tential mediators of CR (5–7). Activation of the NAD-
dependent protein deacetylase sirtuin (silent mating type
information regulation 2 homolog) 1 (SIRT1) is necessary
for the full benefits of CR (8–11). Indeed, several behav-
ioral and physiologic changes induced by CR in wild-type
(WT) mice are impaired in SIRT1-null mice: these animals
do not demonstrate an increase in daily activity (12). CR has
different effects on several metabolic parameters in WT and
SIRT1-null mice (13, 14). Finally, there is no increase in the
life span of SIRT1-null mice on CR (14).
SIRT1 regulates the activity of many transcription fac-
tors. The helix–loop–helix transcription factor BMAL1
(brain and muscle ARNT [aryl hydrocarbon receptor nu-
clear translocator]-like protein 1) is one of the direct tar-
gets of SIRT1 (15). BMAL1 is a component of the circadian
clock mechanism (16). The circadian clock is an internal
timekeeping system that generates daily rhythms in physi-
ology, metabolism, and behavior (17–20). BMAL1 also has
other physiologic functions, such as control of metabolism,
glucose homeostasis, antioxidant defense, immune system,
and memory formation (21–23). Some of these functions
might be linked to the role of BMAL1 as a component of
the circadian clock; however, some of them cannot be
explained through BMAL1 function in the clock mecha-
nism only. Indeed, BMAL1 deficiency in mice leads to dra-
matically reduced life span and development of accelerated
aging, a phenotype that is unique for BMAL1 deficiency and
is not observed in other clock mutants (24).
BMAL1 and its downstream transcriptional targets con-
trol signaling pathways implicated in the CR-mediated in-
crease in longevity. BMAL1 regulates antioxidant defense of
the organism because BMAL1 deficiency is associated with
oxidative stress and development of degenerative diseases
(23, 25). Interestingly, BMAL1 is a target of SIRT1, and in
turn, BMAL1 may regulate SIRT1 activity through the
control of transcription of nicotinamide phosphoribosyl
transferase (NAMPT), a rate-limiting enzyme in NAD
biosynthesis; thus, BMAL1 and SIRT1 form a feedback
1
Correspondence: Department of Biological, Geological,
and Environmental Sciences, and Center for Gene Regulation
in Health and Diseases, Cleveland State University, 2121 Euclid
Ave., Cleveland, OH 44115. E-mail: r.kondratov@csuohio.edu
doi: 10.1096/fj.15-282475
This article includes supplemental data. Please visit http://
www.fasebj.org to obtain this information.
1
loop (26–28). BMAL1 is also involved in the regulation of
mTOR signaling (29–31). We hypothesized that BMAL1
may be a part of the mechanism facilitating the beneficial
effects of CR in mammals. We studied the effects of CR in
Bmal12/2 mice and observed that CR did not increase
longevity of these animals, which suggests that BMAL1 is
an important component of the CR mechanisms, there-
fore linking biologic clocks with CR in mammals.
MATERIALS AND METHODS
Animals
All mice were of C57B6/J background. WT mice on this back-
ground demonstrated strong beneficial response to CR; 30% CR
is one of the most commonly used regimens for longevity ex-
periments (32, 33). Animals were maintained on a 12:12 light:
dark cycle with lights on at 7:00 AM and were fed an 18% protein
rodent diet (Harlan Industries, Indianapolis, IN, USA). The ad
libitum (AL) group had unrestricted access to food. CR was started
at 3 mo of age; during the first week of CR, animals received a 10%
reduction compared to their AL intake and during the second
week a 20% reduction; the 30% reduction was started on wk 3 and
continued till the end of the experiments. Animals were on 30%
CR for 2 mo before tissue collection. For the CR group, food was
provided at Zeitgeber time (ZT) 14 (2 h after light was off) for
longevity experiments, tissue collection, and behavioral experi-
ments. All groups had unrestricted access to water. The CR-adjusted
group excluded those that died during the first 40 d of CR. In-
cage locomotor activity was studied with an in-cage photobeam
activity system (San Diego Instruments, San Diego, CA, USA).
Total daily activity for each animal was normalized and set as 24
arbitrary units. The normalization of locomotor activity was
necessary because there is a significant difference in the total
level of activity between different mice. For every mouse, loco-
motor activity was recorded for 3 consecutive days. Five male
mice of both genotypes were analyzed for AL and CR feeding; all
mice were 5 mo of age. All tissue collection experiments were
performed for 5 mo old WT and Bmal12/2 male mice. All animal
studies were conducted in accordance with the regulations of the
Committee on Animal Care and Use (Cleveland State University).
Statistical analysis
At least 3 animals for every time point, for each feeding type, and
for each genotype were used for all experiments. Data are shown
as averages 6 SD. SPSS Statistics 20 (IBM, Armonk, NY, USA) and
GraphPad Prism 5.04 (GraphPad Software, La Jolla, CA, USA)
software were used for statistical analysis. Effects of genotype
(Bmal12/2 vs. WT), feeding type (AL vs. CR), and time of day
were tested for significance. To assess the effects that genotype,
feeding, and time of the day on plasma glucose, insulin, and IGF-1
levels and for IGF-1 protein and mRNA levels in the liver, analysis
was performed by 3-way ANOVA followed by post hoc analysis with
Bonferroni correction. Two-way repeated-measures ANOVA fol-
lowed by post hoc analysis using Bonferroni correction was used for
analysis of the effect of genotype, feeding, and time of the day on
daily locomotion. Two-way repeated ANOVA followed by post hoc
analysis using Bonferroni correction was used for analysis of the
effect of feeding and time of the day on circadian clock gene
expression. The Student’s t test was used for analysis in other
cases. Thirty-six Bmal12/2 mice (both genders) were on 30% CR,
18 Bmal12/2 mice (both genders) were on the AL diet, 27 WT
mice (both genders) were on 30% CR, and 41 WT mice (both
genders) were on the AL diet. If mice needed to be killed,
2 Vol. 30 April 2016 The FASEB Journal x www.fasebj.org
according to veterinarian recommendation as a result of mor-
bidity or severe pathology, these mice were counted as alive be-
fore the day of death and as missing after the day of death.
Because the treatment was started with mice 3 mo of age (90 d),
we excluded from consideration all animals that had died
before the beginning of the treatment. For the CR-adjusted
group, animals that had died during first 40 d after the start of
CR were excluded from analysis. The log rank test was used
for analysis of longevity experiments. P , 0.05 was considered
to be a statistically significant difference.
Analysis of protein expression
RIPA buffer with Protease/Phosphatase Inhibitor Cocktail (#5872;
Cell Signaling Technology, Danvers, MA, USA) was used for tissue
extract preparations. Total protein concentration was de-
termined using the Bio-Rad DC protein assay kit (Bio-Rad,
Hercules, CA, USA) according to the manufacturer’s protocol.
Staining of the same membranes for b-actin was used for nor-
malization. Anti-IGF-1 ab 36532 (Abcam, Cambridge, MA, USA)
and anti-b-actin (Sigma-Aldrich, St. Louis, MO, USA) antibodies
were used for Western blot analysis.
Analysis of mRNA expression
Total RNA was isolated using TriZol reagent (Invitrogen,
Carlsbad, CA, USA) according to the manufacturer’s protocol.
RNA quantification was performed using quantitative real-time
PCR with SYBR Green mix (Bio-Rad); relative mRNA abundance
was calculated using the comparative dCt method with ribosomal
18S rRNA as standard. The following primers were used for the
analysis of expression of the following: 18S rRNA, F 59-GCT TAA
TTT GAC TCA ACA CGG GA-39; 18S rRNA R, 59-AGC TAT CAA
TCT GTC AAT CCT GTC-39; Per1, F 59-AGG TGG CTT TCG TGT
TGG-39; Per1, R 59-CAA TCG ATG GAT CTG CTC TGA-39;
Bmal1, F 59-CCA AGA AAG TAT GGA CAC AGA CAA A-39;
Bmal1, R 59-GCA TTC TTG ATC CTT CCT TGG T-39; Per2, F 59-
AAT CTT CCA ACA CTC ACC CC-39; Per2, R 59-AAT CTT CCA
ACA CTC ACC CC-39; Clock, F 59-CCT TCT GCC GTA GCC CTA
GT-39; Clock, R 59-CCC ATA AGG ATC CCC AGG CA-39.
Measurement of plasma IGF-1, insulin, and glucose levels
Plasma samples were collected at 6 time points (ZT2, ZT6, ZT10,
ZT14, ZT18, and ZT22) from 3 animals per time point. Plasma
IGF-1 level was determined using the RayBio Mouse IGF-1 ELISA
kit (RayBiotech, Norcross, GA, USA) according to the manu-
facturer’s protocol. Sandwich ELISA with 2 different anti-
bodies and streptavidin–biotin horseradish peroxidase system
with manufacturer-provided TMB substrate was used for detection
of both IGF-1 and insulin. The detection limit for IGF-1 mea-
surement is 4 pg/ml and for insulin is 5 U/ml. Blood glucose level
was measured by True Test result kit by Nipro Diagnostics (Fort
Lauderdale, FL, USA), according to the manufacturer’s protocol.
The detectable range of glucose is 20 to 600 mg/dl.
RESULTS
Bmal12/2 mice have normal behavioral response to
30% CR
WT mice subjected to CR demonstrate robust increase in
locomotion around the time of feeding (food anticipation)
(34). We investigated whether Bmal12/2 mice have defects
PATEL ET AL.
in CR-induced food anticipation. We monitored in cage
locomotion for 3 d. Results of behavioral experiments are
presented in Fig. 1. The 3-way interaction of genotype 3
diet 3 time was statistically significant (F = 2.688, P = 0.015;
Supplemental Table 1). For both WT and Bmal12/2
mice, the increase in locomotion at ZT12 and ZT13
was statistically significant (P , 0.05, post hoc analysis
using Bonferroni correction), which was an indication
of food anticipation. Thus, BMAL1 is not necessary for
the CR-mediated increase in locomotion before the
feeding time.
BMAL1 is necessary for life span extension on
30% CR
WT and Bmal12/2 mice were subjected to CR starting at 3
mo of age. For longevity experiments, food for CR groups
was provided at ZT14 (2 h after light was off, which is a
normal physiologic feeding time for mice). At this age,
Bmal12/2 mice do not demonstrate any signs of accelera-
ted aging; they are indistinguishable from WT mice by
gross appearance and body weight. At the start of the ex-
periment, average body weights were as follows: WT males,
27.3 6 2.1 g; Bmal12/2 males, 26.4 6 3.3 g; WT females,
20.7 6 1.1 g; and Bmal12/2 females, 20.6 6 1.6 g (Supple-
mental Fig. 1). Animals of both genotypes consumed the
same amount of food (3.4 6 0.31 g for males and 3.1 6 0.23
for females). The beneficial effect of 30% CR on life span
of WT mice was in agreement with the previously pub-
lished data for C57B6 mice; while the experiment is still
in progress, we observed a significant life span extension
in the CR group of WT mice compared to the AL group
(Fig. 2D). The effect of 30% CR on life span of Bmal12/2
mice is shown on Fig. 2A. Because gender might affect
the outcome of CR, we checked the longevity of males (13
mice) and females (23 mice) separately. As evidenced by
data presented in Supplemental Fig. 2, we did not detect
any effect of gender on survival. Previously, we reported
that gender does not affect the life span of Bmal12/2;
therefore, we combined male and female data for longevity
analysis. In contrast to the effects in WT mice, 30% CR did
not extend life span of Bmal12/2 mice (Fig. 2A); moreover,
we observed a reduction in the life span of Bmal12/2 mice.
The reduction in life span of Bmal12/2 on 30% CR was
statistically significant. Average life span of Bmal12/2 mice
on the AL diet was 8 mo but was only 6 mo on the 30% CR
diet. We also noticed that a significant number of animals
died during the early stages of CR, when, most likely,
metabolic changes had to occur in order to permit the
animal to adapt to a novel feeding regimen. About 40% of
Bmal12/2 animals (15 of 36) died within 40 d from the start
of CR, while only 1 animal died in the WT group. Again, we
did not detect any effect of gender: 6 of 13 males and 9 of 23
females died during the adaptation period. When we ex-
cluded from the analysis the Bmal12/2 mice that died
during the adaptation period (shown in Fig. 2A–C as the
CR-adjusted group), we still did not detect any increase in
the life span on CR: the average life span of this group was
about 7.5 mo. The difference in the life span between CR-
adjusted and AL groups was not statistically significant.
Thus, BMAL1 plays an important role in preventing
death during the metabolic adaptation to CR, and BMAL1
CR-MEDIATED LIFE SPAN EXTENSION THROUGH BMAL1
A
6
locomotion, a.u.
WT * AL
4 * CR
2
0
0 4 8 12 16 20 24
food
ZT (hrs)
B
Bmal1-/- AL
4 * * CR
locomotion, a.u.
2
0
0 4 8 12 16 20 24
food
ZT (hrs)
Figure 1. BMAL1 deficiency did not affect CR-mediated increase
in locomotion around the feeding time. In-cage locomotion of
WT (A) and Bmal12/2 (B) male mice. Mice were subjected to
following diets: AL, gray circles, gray dotted lines; 30% CR, black
squares, solid black lines. Each graph represents average normal-
ized activity per hour and SE (3 consecutive days for every animal,
5 mice per group). a.u., arbitrary units of normalized daily loco-
motor activity; total daily activity was set as 24 a.u. Zero and 24 h
time points were double plotted. *Statistically significant increase
in locomotor activity before feeding (food anticipation) for CR
group compared to AL group. Light and dark bars represent light
and dark phase of day. ZT0 is time when light is on and ZT12 is
time when light is off. Food for CR group was provided at ZT14.
is necessary for CR-induced life span extension. Hence,
BMAL1 is a necessary mediator of the beneficial CR effects
on life span.
As expected, 30% CR significantly affected body weight
of both WT and Bmal12/2 mice (Fig. 3A, B; Supplemental
Fig. 1). WT mice demonstrated some reduction in body
weight during the first few weeks of CR, after which their
body weight stabilized and did not change during the
rest of the experiment; the stabilized weight was about
85% of the original weight for males and about 90% of
the original weight for females. In contrast, the body
weight of Bmal12/2 mice demonstrated dramatic reduction
compared to WT mice. Importantly, because the average
food consumption was the same for both genotypes at the
start of the experiment, Bmal12/2 mice actually consumed
more food relative to their body weight (Fig. 4A). Daily food
consumption for WT males and females equaled about
10% of their body weight, while for Bmal12/2 males this was
about 12% and for Bmal12/2 females about 14% of their
body weight. This difference between genotypes was statis-
tically significant. We also monitored water consumption
and found that Bmal12/2 mice on 30% CR consumed
3
 A Bmal1-/- C
90 90 AL
AL CR
Survival (%) 60
CR
60
CR adj
CR adj Bmal1 -/-
30 30

0 0
0 100 200 300 400 0 100 200 300 400

Age (days) Age (days)

B Bmal1-/- D
90 90
Survival (%)

60 60
AL WT AL
30 CR 30
WT 30%CR
CR adj
0 0
0 100 200 300 400 0 200 400 600 800 1000 1200
Age (days) Age (days)

Figure 2. Circadian clock protein BMAL1 is necessary for life span extension in response to CR. A) Kaplan-Meier survival curves
of Bmal12/2 mice on AL (n = 18, gray circles), 30% CR (n = 36, dark gray diamonds), and CR-adjusted (n = 21, black squares)
feeding. Mice that died during first 3 wk of 30% CR were excluded from analysis. Mice of both genders were used. Difference
between survival curves of AL and CR is statistically significant by log rank test; no statistically significant difference between AL
and CR-adjusted groups was observed. B) Kaplan-Meier survival curves of female Bmal12/2 mice on AL (n = 9, gray circles), 30%
CR (n = 23, dark gray diamonds), and CR-adjusted (n = 14 black squares) feeding. Mice that died during first 3 wk of 30% CR
were excluded from analysis. Difference between survival curves of AL and CR is statistically significant according to log rank test;
no statistically significant difference between AL and CR-adjusted groups was observed. C ) Kaplan-Meier survival curves of male
Bmal12/2 mice on AL (n = 9, gray circles), 30% CR (n = 13, dark gray diamonds), and CR-adjusted (n = 9 black squares) feeding.
Mice that died during first 3 wk of 30% CR were excluded from analysis. Difference between survival curves of AL and CR is
statistically significant according to log rank test; no statistically significant difference between AL and CR-adjusted groups was
observed. D) Kaplan-Meier survival curves of WT mice on AL (n = 73, gray circles and gray dotted line) or CR (n = 31; black
squares and solid black line) feeding. Mice of both genders were used. *Difference between survival curves of AL and CR
statistically significant according to log rank test.

about 30% less water than WT (Fig. 4B), whereas there
was no significant difference in water consumption be-
tween the genotypes when mice had constant access to
food. The cause for the reduced water consumption is
unknown; however, the phenomenon may contribute to
the observed difference in body weight.
BMAL1 regulates CR-mediated effects on insulin and
IGF-1 levels
CR significantly affects glucose, insulin, and IGF-1 levels in
WT mice (35, 36); therefore, we next studied these pa-
rameters in Bmal12/2 mice maintained on AL and CR
regimens. Results of statistical analysis are presented in
Supplemental Table 2. Similar to the effect in WT mice, CR
resulted in reduced levels of glucose in the blood of Bmal12/2
mice (Fig. 5A). The reduced levels of plasma glucose were
comparable for both genotypes (the difference was sig-
nificant for the diets but not for the genotypes); we thus
concluded that Bmal12/2 mice had a normal response to
CR for this parameter. At the same time, we found that in
contrast to Bmal12/2 mice maintained on the AL regi-
men, which had reduced levels of insulin, in agreement
with previously published data (37), Bmal12/2 mice
maintained on a CR regimen demonstrated increased
insulin levels (Fig. 5B). Thus, in spite of the reduced
levels of plasma glucose, Bmal12/2 mice had an increased
level of insulin, which may indicate different insulin sen-
sitivity between the genotypes on the CR regimen. Next,
plasma and liver IGF-1 levels were measured. In the AL WT
groups, the level of plasma IGF-1 significantly varied across
the day, with the highest levels at ZT2 to ZT6 and the
lowest levels at ZT14. In Bmal12/2 animals, IGF-1 did not
significantly change across the day (Fig. 5C). In WT mice,
CR resulted in significant reduction in plasma IGF-1 levels
compared to the AL group (Fig. 5C and Supplemental
Table 2). In Bmal12/2 mice, the effect of CR was not sta-
tistically significant (Fig. 5C). Thus, BMAL1 deficiency
resulted in a strong suppression of CR-mediated reduction
in the plasma levels of IGF-1. At the same time, the effect of
CR on the levels of IGF-1 expressed in the liver were
comparable between the genotypes, the difference be-
tween the AL and CR groups was statistically significant at
ZT18 for WT (Fig. 5D); the effect of CR was thus de-
pendent on the time of day. There were also time-of-day-
dependent effects of diet and genotype on IGF-1 mRNA
level in the liver; a statistically significant reduction was
observed on CR in WT mice at ZT18 and ZT22 (Fig. 5E).
CR affects expression of Bmal1 and BMAL1
transcriptional targets
Next we decided to study whether CR has any effects on the
expression and transcriptional activity of BMAL1. BMAL1

4 Vol. 30 April 2016 The FASEB Journal x www.fasebj.org PATEL ET AL.

A 30% CR did not increase the expression of Clock gene (Fig.
(norm. to weight on day 0)
1.0
**
Rel. body weight
0.8
0.6
0 100 200
Days after CR start
B different organisms. Interestingly, it was demonstrated that
(norm. to weight on day 0)
1.2 **
Rel. body weight
1.0
0.8
0.6
0 100 200
Days after CR start
Figure 3. Thirty percent CR affects body weight of WT and
Bmal12/2 mice. A) Changes in body weights of male WT mice
(n = 16, black squares and solid black line) and Bmal12/2 mice
(n = 13, gray circles and gray dotted line). B) Changes in body
weights for female WT (n = 14, black squares and solid black
line) and Bmal12/2 (n = 19, gray circles and gray dotted line)
mice. Mouse weights were normalized to weight of animals at
start of experiment. **Statistically significant difference be-
tween genotypes detected at indicated age range.
is a transcription factor that, in complex with another
transcription factor, CLOCK (or its paralog, neuronal PAS
domain protein 2 [NPAS2], in the CNS and cardiovascular
system), drives the expression of its target genes (38–40).
Analysis of Bmal1 expression in the liver tissue of WT mice
maintained on different diets is presented in Fig. 6A. CR
significantly induced Bmal1 mRNA at several time points
during the day (Supplemental Table 3). Expressions of 2
well-known BMAL1 targets, Per1 and Per2, were also sig-
nificantly induced at several time points in the liver of CR
mice (Fig. 6B, C). The daily average expression of Bmal1,
Per1, and Per2 was increased (Supplemental Fig. 3A). The
increase in daily average expression was also accompanied
by a dramatic increase in the amplitude of rhythms for
Bmal1, Per1, and Per2 (Supplemental Fig. 3B). Interestingly,
A wt
Bmal1-/-
15
* *
(% of body weight)
food intake
10
5 2
0 0
CR-MEDIATED LIFE SPAN EXTENSION THROUGH BMAL1
6D), whereas in contrast, the daily average and amplitude
Bmal1-/- were even reduced on CR (Supplemental Fig. 3).
WT
DISCUSSION
The beneficial effects of CR on longevity have been re-
ported in different organisms, including humans. Differ-
300 400 500 ent physiologic systems were proposed as important
mediators of CR. Biologic clock proteins such as BMAL1
and PER2 have been implicated in the control of aging in
both these proteins are targets of SIRT1 (15, 41). Both
Bmal1- and Sirt1-null mice have significantly reduced life
span (42, 43). We investigated the effect of CR in Bmal12/2
mice. We found that 30% CR did not extend longevity of
Bmal12/2 mice (Fig. 2A–C), while the same intervention
Bmal1-/- significantly increased the life span of WT mice (Fig. 2D).
WT Even more, 30% CR resulted in reduced longevity of
Bmal12/2 mice. One of the known adverse effects of CR is
300 400 500 elevated mortality during the first week of CR: as reported
previously, when CR was applied to mice caught in wilder-
ness, a significant number of animals died during the ad-
aptation period; animals that survived this period lived
longer than the AL-fed group (43). The cause of this dif-
ference in adaptation is unknown; our data demonstrate
that BMAL1 is critically important for metabolic adaptation
to CR. It will be interesting to study whether natural varia-
tions in biologic rhythms of BMAL1 expression and activity
could be responsible for the differential adaptation to CR.
CR does not extend the longevity of Sirt1-null mice (13,
14); thus, it is tempting to speculate that BMAL1 and SIRT1
can be components of the same mechanism that mediates
life span extension by CR. However, we found that several
behavioral (Fig. 1) and physiologic (Fig. 5) changes in-
duced by CR in WT mice can also be detected in Bmal12/2
animals, indicating that BMAL1 is not involved in all ben-
eficial effects of CR. The conclusions on the role of BMAL1
in the beneficial effects of CR on longevity must be taken
with caution because Bmal12/2 mice developed a pre-
mature aging phenotype and have a short life span, which
might contribute to the failure of CR in this model. Im-
portantly, at the starting point of the CR (3 mo of age),
Bmal12/2 mice are very similar with WT mice in terms of
gross appearance, food consumption, and body weight.
Interestingly, treatment with rapamycin increases the life
span of these mice (30), suggesting that longevity in these
mice can be affected by another antiaging intervention.
Figure 4. Daily food and water consumptions
B in WT and Bmal12/2 mice on 30% CR. A)
*
Relative daily food intake of WT (black bars)
and Bmal12/2 (light gray bars) for male and
6
water intake (ml)
female mice on 30% CR. Relative food intake
4 was calculated by dividing daily food con-
AL
sumption by mouse body weight. *Statistically
CR
significant difference between genotypes
(P , 0.05). B) Daily water consumption of
WT and Bmal12/2 mice on AL (light gray
bars) or 30% CR (black bars) feeding.
wt Bmal1-/-
*Difference between genotypes statistically
significant (P , 0.05).
5
 A Plasma Glucose D Liver IGF1

Protein expression, a.u.

2.0
250 a a,d
a
Glucose, mg/dl
a,c 1.5
a,c a,c WT-AL
200 WT-AL
a,c
WT-CR 1.0 WT-CR
Bmal1-/--AL Bmal1-/--AL
150
Bmal1-/-- CR
0.5 Bmal1-/-- CR

100 0.0
ZT2 ZT6 ZT10 ZT14 ZT18 ZT22 ZT2 ZT6 ZT10 ZT14 ZT18 ZT22

B Plasma Insulin E Liver Igf1

c a, b

mRNA expression, a.u.

300
27
a,b,c,d
Insulin, ulU/ml

c,d
b,c,d a,b,c,d c,d WT-AL 200 WT-AL
22 a,c
WT-CR WT-CR
Bmal1-/--AL Bmal1-/--AL
17 100
Bmal1-/-- CR Bmal1-/-- CR

12 0
ZT2 ZT6 ZT10 ZT14 ZT18 ZT22 ZT2 ZT6 ZT10 ZT14 ZT18 ZT22

C Plasma IGF1
1500 a,d b,d
a,b,d
b,d a – WT-AL versus WT-CR, p<0.05
1200 a,d a,d
IGF1, pg/ml

WT-AL b – WT-AL versus Bmal1-/--AL, p<0.05

900 WT-CR c – Bmal1-/--AL versus Bmal1-/--CR, p<0.05
d – WT-CR versus Bmal1-/--CR, p<0.05
600 Bmal1-/--AL
300 Bmal1-/-- CR

0
ZT2 ZT6 ZT10 ZT14 ZT18 ZT22

Figure 5. Circadian clock protein BMAL1 is necessary for reduction in plasma IGF-1 level in response to CR. Daily profiles of (A)
glucose, (B) insulin, and (C ) IGF-1 in plasma of WT and Bmal12/2 mice on AL and CR. D) Daily profiles of liver IGF-1 protein
level of WT and Bmal12/2 mice on AL and CR. E ) Daily profiles of liver IGF-1 mRNA level of WT and Bmal12/2 mice on AL and
CR. Three male mice of each genotype and on each feeding regimen were studied per time point. A–E) WT mice on AL feeding
(black circles, black dashed lines); WT mice on CR feeding (black squares, black solid lines); Bmal12/2 mice on AL feeding (gray
triangles, gray dashed lines); Bmal12/2 mice on CR feeding (gray diamonds, gray solid lines). a) Statistically significant difference
(P , 0.05) between WT mice on AL and CR feeding. b) Statistically significant difference (P , 0.05) between WT and Bmal12/2
mice on AL feeding. c) Statistically significant difference (P , 0.05) between Bmal12/2 mice on AL and CR feeding.
d) Statistically significant difference (P , 0.05) between WT and Bmal12/2 mice on CR feeding.

CR induces dramatic reduction of the plasma levels of
glucose, insulin, and IGF-1 and leads to increased insulin
sensitivity in WT mice (35, 36). We observed that in
Bmal12/2 mice CR resulted in a comparable reduction in
blood glucose level. Thus, while it was demonstrated that
BMAL1 is involved in the control of glucose homeostasis
(21), it is not necessary for the CR-mediated reduction in
plasma glucose. At the same time, we found that CR did not
reduce the plasma level of insulin in Bmal12/2 mice;
moreover, the insulin level in the CR group of Bmal12/2
mice was increased compared to the AL group, which,
together with the data on reduced glucose levels, indicates
reduced insulin sensitivity in Bmal12/2 animals. Increased
insulin sensitivity is implicated in life span extension by CR
(35). As previously reported, BMAL1 regulates insulin se-
cretion by pancreatic islets (37); in agreement with that, we
observed reduced plasma insulin levels in Bmal12/2 mice
maintained on an AL diet compared to the WT AL group
(Fig. 5). This difference in the effect of BMAL1 deficiency
on insulin requires further studies on the role of BMAL1 in
pancreatic islet functions. Thus, BMAL1 is involved in in-
sulin production and most likely in insulin sensitivity under
conditions of CR, which warrants further study of BMAL1
functions. Finally, BMAL1 deficiency led to significant
defects in the regulation of plasma IGF-1. It is well known
that the plasma IGF-1 level is significantly affected by nu-
trients, but molecular mechanisms of reduced level of
plasma IGF-1 on CR are not well studied. We did not ob-
serve significant reductions of circulating IGF-1 in the
blood of Bmal12/2 mice (the difference was significant
only at 1 time point), compared to dramatic reductions in
WT mice (Fig. 5). mRNA expression of Igf1 in the liver was
comparable between genotypes under both AL and CR
conditions, and it was equally suppressed under CR com-
pared to AL in both WT and Bmal12/2 mice. The effect of
CR on the level of the IGF-1 protein in the liver was com-
plicated; CR led to elevated levels of the IGF-1 protein in
the liver of WT animals at ZT14 and ZT18 and to re-
duced levels at ZT6. CR also affected levels of IGF-1
protein in the liver of Bmal12/2 mice; the difference
was significant at ZT18 (Fig. 5). These data suggest that
in WT mice, CR might suppress plasma IGF-1 levels
through control of IGF-1 production and secretion
(a dramatic reduction in plasma IGF-1 has been observed,
while the effect on liver IGF-1 was moderate and at some
time points opposite to the effect on plasma IGF-1), and

6 Vol. 30 April 2016 The FASEB Journal x www.fasebj.org PATEL ET AL.

 A C
5
6

(norm. to AL daily average)

(norm. to AL daily average)
Bmal1 * Per2 *
5 AL * 4
*

mRNA expression
AL
mRNA expression * CR CR
4
3

3 *
2
*
2
*
1
1

0 0
0 4 8 12 16 20 24 2 6 10 14 18 22
Time of the day (hrs.) Time of the day (hrs.)
B D
15 3

(norm. to AL daily average)

*
(norm. to AL daily average)

mRNA expression
Per1 Clock
mRNA expression

12
AL * * 2 AL
9 CR CR
*
6
1
*
3
*

0 0
0 4 8 12 16 20 24 0 4 8 12 16 20 24
Time of the day (hrs.) Time of the day (hrs.)

Figure 6. CR increases expression of Bmal1 and BMAL1 transcriptional target genes. mRNA expression of Bmal1 (A), 2 BMAL1
transcriptional target genes; Per1 (B) and Per2 (C ), and Clock gene (D) have been studied in liver of mice subjected to following
feeding regimens: AL, gray circles and gray dotted line; 30% CR, black squares and solid black lines. At least 3 male mice were
used for every time point in both groups. *Statistically significant (P , 0.05) with AL group at this time point. Light and dark bars
represent light and dark phase of day. ZT0 is time when light is on and ZT12 is time when light is off. Food for CR group was
provided at ZT14.

that BMAL1 deficiency leads to impaired control of se-
cretion of the IGF-1 protein. We also cannot exclude the
possibility that CR might affect the turnover rate of
plasma IGF-1. Thus, BMAL1 may be involved in control
of IGF-1 secretion and/or turnover.
We observed that 30% CR results in a significant increase
in the expression of the circadian clock genes Bmal1, Per1,
and Per2. Per1 and Per2 are direct transcriptional targets of
the BMAL1/CLOCK complex (16). Interestingly, we did
not observe induction of Clock gene expression, which
suggests that the effect of CR is not universal for all clock
genes. One of the possible effects of CR is an increase in the
activity of the BMAL1/CLOCK transcriptional complex;
the observed induction of the BMAL1/CLOCK down-
stream targets is in agreement with that. Alternatively, it is
possible that rhythmic feeding increases synchronization
of individual cellular oscillators in a tissue. In this case, we
would expect an increase in the amplitude of expression
without any significant effect on daily average expression,
which is not the case. However, we cannot completely rule
out this interpretation because it is possible that CR has
different effects on clock gene expression during different
times of the day. This selective regulation may occur
through some epigenetic mechanisms that have been re-
cently proposed as regulators of the circadian tran-
scriptome (44). Regardless of the particular mechanism
involved, our data demonstrate that CR significantly in-
creases the expression of BMAL1 target genes, suggesting
that CR may directly affect BMAL1 transcriptional activity,
thus supporting the importance of BMAL1 for the full ef-
fects of CR.
CR leads to increased expression and activation
of BMAL1 through circadian clock–dependent or
–independent mechanisms. CR also activates SIRT1, which
may regulate CLOCK and BMAL1. BMAL1 contributes to
CR-mediated regulation of circulating IGF-1 levels. IGF-1 is
important for CR-induced increase in longevity (35, 36).
Another pathway considered to be critical for CR mech-
anisms is the mTOR complex 1 (mTORC1) signaling
pathway (45). Previously we demonstrated that BMAL1 is a
negative regulator of mTORC1 (30). Therefore, it will be
important to study whether the effects of CR on mTORC1
activity are impaired in Bmal12/2 mice.
Our study also puts forward several important ques-
tions that ought to be addressed. BMAL1 is a compo-
nent of the circadian clock, which was proposed to be a
contributor to the beneficial effects of CR (46). In-
duction of expression of several clock genes argues that
activation of the clock mechanisms might be important
for CR. At the same time, BMAL1 has clock-independent
metabolic functions; thus, the observed induction of
Per1 and Per2 expression can be a biomarker of the in-
creased BMAL1 expression and activity, while other
downstream targets of BMAL1 are functionally impor-
tant for the CR mechanisms. At this stage, it is too early
to discuss if the role of BMAL1 in CR depends on

CR-MEDIATED LIFE SPAN EXTENSION THROUGH BMAL1 7

circadian clock mechanisms or is circadian clock in-
dependent. Future experiments on the effects of CR in
other circadian clock mutants will help address this
question. It is also important to note that although we
clearly observed the role of BMAL1 in CR-mediated
longevity, the effect of CR on clock gene expression was
studied only in the liver, and we do not know if it has the
same effect in other tissues. However, independent of
the outcome of these future studies, our data suggest
that BMAL1 is an important target of CR, and the
BMAL1-controlled pathways ought to be considered for
the understanding of CR mechanisms and for de-
veloping antiaging strategies.
This work was supported by U.S. National Institutes of
Health/National Institute on Aging Grant 1R01AG039547, funds
from the Center for Gene Regulation in Health and Disease
(Cleveland State University) to R.V.K., and a Dissertation Re-
search Award (Cleveland State University) to S.P. The authors
thank J. Holcomb (Department of Mathematics, Cleveland State
University), for help with statistical analysis. The authors also
thank 2 anonymous reviewers for their helpful suggestions.
The authors declare no conflicts of interest.
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Received for publication October 2, 2015.
Accepted for publication December 12, 2015.
9