Obesity and metabolic syndrome in circadian Clock mutant mice.
ABSTRACT The CLOCK transcription factor is a key component of the molecular circadian clock within pacemaker neurons of the hypothalamic suprachiasmatic nucleus. We found that homozygous Clock mutant mice have a greatly attenuated diurnal feeding rhythm, are hyperphagic and obese, and develop a metabolic syndrome of hyperleptinemia, hyperlipidemia, hepatic steatosis, hyperglycemia, and hypoinsulinemia. Expression of transcripts encoding selected hypothalamic peptides associated with energy balance was attenuated in the Clock mutant mice. These results suggest that the circadian clock gene network plays an important role in mammalian energy balance.
- SourceAvailable from: Pablo Martinez-Lozano Sinues[Show abstract] [Hide abstract]
ABSTRACT: Circadian clocks play a significant role in the correct timing of physiological metabolism, and clock disruption might lead to pathological changes of metabolism. One interesting method to assess the current state of metabolism is metabolomics. Metabolomics tries to capture the entirety of small molecules, i.e. the building blocks of metabolism, in a given matrix, such as blood, saliva or urine. Using mass spectrometric approaches we and others have shown that a significant portion of the human metabolome in saliva and blood exhibits circadian modulation; independent of food intake or sleep/wake rhythms. Recent advances in mass spectrometry techniques have introduced completely non-invasive breathprinting; a method to instantaneously assess small metabolites in human breath. In this proof-of-principle study, we extend these findings about the impact of circadian clocks on metabolomics to exhaled breath. As previously established, our method allows for real-time analysis of a rich matrix during frequent non-invasive sampling. We sampled the breath of three healthy, non-smoking human volunteers in hourly intervals for 24 hours during total sleep deprivation, and found 111 features in the breath of all individuals, 36-49% of which showed significant circadian variation in at least one individual. Our data suggest that real-time mass spectrometric "breathprinting" has high potential to become a useful tool to understand circadian metabolism, and develop new biomarkers to easily and in real-time assess circadian clock phase and function in experimental and clinical settings.PLoS ONE 01/2015; 9(12):e114422. · 3.53 Impact Factor
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ABSTRACT: All domains of life feature diverse molecular clock machineries that synchronize physiological processes to diurnal environmental fluctuations. However, no mechanisms are known to cross-regulate prokaryotic and eukaryotic circadian rhythms in multikingdom ecosystems. Here, we show that the intestinal microbiota, in both mice and humans, exhibits diurnal oscillations that are influenced by feeding rhythms, leading to time-specific compositional and functional profiles over the course of a day. Ablation of host molecular clock components or induction of jet lag leads to aberrant microbiota diurnal fluctuations and dysbiosis, driven by impaired feeding rhythmicity. Consequently, jet-lag-induced dysbiosis in both mice and humans promotes glucose intolerance and obesity that are transferrable to germ-free mice upon fecal transplantation. Together, these findings provide evidence of coordinated metaorganism diurnal rhythmicity and offer a microbiome-dependent mechanism for common metabolic disturbances in humans with aberrant circadian rhythms, such as those documented in shift workers and frequent flyers.Cell. 10/2014; 159(3):514-29.
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ABSTRACT: Chronic alcohol use can result in many pathological effects including alcoholic liver disease (ALD). While alcohol is necessary for the development of ALD, only 20-30% of alcoholics develop alcoholic steatohepatitis (ASH) with progressive liver disease leading to cirrhosis and liver failure (ALD). This suggests that while chronic alcohol consumption is necessary it is not sufficient to induce clinically relevant liver damage in the absence of a secondary risk factor. Studies in rodent models and alcoholic patients show that increased intestinal permeability to microbial products like endotoxin play a critical role in promoting liver inflammation in ALD pathogenesis. Therefore identifying mechanisms of alcohol-induced intestinal permeability is important in identifying mechanisms of ALD and for designing new avenues for therapy. Cyp2e1 is a cytochrome P450 enzyme that metabolizes alcohol has been shown to be upregulated by chronic alcohol use and to be a major source of oxidative stress and liver injury in alcoholics and in animal and in vitro models of chronic alcohol use. Because Cyp2e1 is also expressed in the intestine and is upregulated by chronic alcohol use, we hypothesized it could play a role in alcohol-induced intestinal hyperpermeability. Our in vitro studies with intestinal Caco-2 cells and in mice fed alcohol showed that circadian clock proteins CLOCK and PER2 are required for alcohol-induced permeability. We also showed that alcohol increases Cyp2e1 protein and activity but not mRNA in Caco-2 cells and that an inhibitor of oxidative stress or siRNA knockdown of Cyp2e1 prevents the increase in CLOCK or PER2 proteins and prevents alcohol-induced hyperpermeability. With our collaborators we have also shown that Cyp2e1 knockout mice are resistant to alcohol-induced gut leakiness and liver inflammation. Taken together our data support a novel Cyp2e1-circadian clock protein mechanism for alcohol-induced gut leakiness that could provide new avenues for therapy of ALD. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.Redox biology. 10/2014; 3C:40-46.
Obesity and Metabolic Syndrome
in Circadian Clock Mutant Mice
Fred W. Turek,1,3Corinne Joshu,3,4* Akira Kohsaka,3,4*
Emily Lin,3,4* Ganka Ivanova,2,4Erin McDearmon,3,5
Aaron Laposky,3Sue Losee-Olson,3Amy Easton,3
Dalan R. Jensen,6Robert H. Eckel,6Joseph S. Takahashi,1,3,5
The CLOCK transcription factor is a key component of the molecular circadian
clock within pacemaker neurons of the hypothalamic suprachiasmatic nucleus.
We found that homozygous Clock mutant mice have a greatly attenuated
diurnal feeding rhythm, are hyperphagic and obese, and develop a metabolic
syndrome of hyperleptinemia, hyperlipidemia, hepatic steatosis, hyperglycemia,
and hypoinsulinemia. Expression of transcripts encoding selected hypothalamic
peptides associated with energy balance was attenuated in the Clock mutant
mice. These results suggest that the circadian clock gene network plays an
important role in mammalian energy balance.
Major components of energy homeostasis,
including the sleep-wake cycle, thermogenesis,
feeding, and glucose and lipid metabolism, are
subjected to circadian regulation that synchro-
nizes energy intake and expenditure with
changes in the external environment imposed
by the rising and setting of the sun. The neural
hour cycles in these behavioral and physiolog-
clock genes can regulate circadian rhythmicity
in vitro in other central as well as peripheral
tissues, including those involved in nutrient
homeostasis (e.g., mediobasal hypothalamus,
liver, muscle, and pancreas), indicates that cir-
cadian and metabolic processes are linked at
multiple levels (4–9). The recent finding that
changes in the ratio of oxidized to reduced
nicotinamide adenine dinucleotide phosphate
control the transcriptional activity of the basic
helix-loop-helix (bHLH) protein NPAS2—a
homolog of a primary circadian gene, Clock—
suggests that cell redox may couple the
expression of metabolic and circadian genes
(10). Miceharboringa mutationinClock show
profound changes in circadian rhythmicity
(11) and offer an experimental genetic model
to analyze the link among circadian gene
networks, behavior, and metabolism in vivo.
Positional cloning and transgenic rescue of
normal circadian phenotype identified Clock
as a member of the bHLH Per-Arnt-Sim
(PAS) transcription factor family (12, 13). Rel-
ative to wild-type mice, the most pronounced
alteration in circadian phenotype in Clock
mutants is a 1-hour increase in the free-
running rhythm of locomotor activity in het-
erozygous mice in constant darkness (DD)
and a 3- to 4-hour increase (i.e., period 0 27
to 28 hours in DD) in circadian period in
homozygous mice, which is often followed
by a total breakdown of circadian rhyth-
micity (i.e., arrhythmicity) after a few weeks
Although previous studies that used run-
ning wheel behavior as a marker of locomotor
activity did not reveal major differences
between homozygous Clock mutant and
wild-type mice maintained on a light-dark
(LD) cycle, use of infrared beam crossing
to monitor total activity revealed a signif-
icant increase in activity during the light
phase and a change in the temporal pattern
of total activity during the dark phase (Fig.
1A) (14). Inparticular,wild-type miceshowed
two pronounced peaks of activity—one
occurring after lights off, the other before
lights on—whereas these peaks were atten-
uated in Clock mutant mice. Surprisingly,
despite there being a clear (but dampened)
diurnal rhythm in locomotor activity in Clock
mutant mice (Fig. 1B), the diurnal rhythm
in food intake was severely altered in these
1DepartmentofNeurologyand2Department of Medicine,
Feinberg School of Medicine,
robiology and Physiology, Northwestern University,
Evanston, IL 60208, USA.
Healthcare (ENH) Research Institute, Evanston, IL
60208, USA.5Howard Hughes Medical Institute, Chevy
Chase, MD 20815, USA.
University of Colorado at Denver and Health Sciences
Center, Aurora, CO 80045, USA.
3Department of Neu-
6Department of Medicine,
*These authors contributed equally to this work.
.To whom correspondence should be addressed.
Fig. 1. Altered diurnal rhythms in locomotor activity, feeding, and metabolic rate in Clock mutant
mice. (A) Activity counts over the 24-hour cycle during light (unshaded) and dark (shaded) periods
[wild-type, n 0 5, black line; Clock, n 0 9, blue line]. Inset: Actograms showing locomotor activity over
a 30-day period in representative adult wild-type (top) and Clock mutant (bottom) mice individually
housed in 12:12 LD (at 23-C) and provided food and water ad libitum. Activity bouts were analyzed
using ClockLab software in 6-min intervals across 7 days of recording (selected days are indicated by
red vertical lines to the left of the actograms). (B) Diurnal rhythm of locomotor activity for mice in
(A). Activity counts were accumulated over the 12-hour light and 12-hour dark periods and are
expressed in each period as a percentage of total 24-hour activity (*P G 0.05). Total activity over the
24-hour period was similar between wild-type (WT) and Clock mutant (CL) genotypes. (C) Diurnal
rhythm of food intake. Different groups of adult WT (N 0 7) and Clock mutant (N 0 5) mice were
maintained on a regular diet (10% kcal/fat), and food intake (in grams) was measured during light
and dark periods. Results shown are average food intake during light and dark periods as a percentage
of total food intake (*P G 0.001). (D) Diurnal rhythm of metabolic rate. Metabolic rate was
determined in additional groups of WT (N 0 7) and Clock mutant (N 0 9) mice by indirect
calorimetry under 12:12 LD conditions over a 3-day continuous monitoring period (*P G 0.05).
Results shown are average metabolic rates during the light and dark periods as a percentage of total
metabolic rate. All results shown are expressed as group means T SEM.
R E P O R T S
www.sciencemag.org SCIENCE VOL 30813 MAY 2005
mice (Fig. 1C): Only 53% of the food intake
occurred during the dark phase in Clock
mutant mice, versus 75% in wild-type mice.
In a preliminary analysis, we found that the
diurnal rhythm of food intake was already
attenuated in 3-week-old mice before an
increase in weight (fig. S1). Similarly, the
rhythm in energy expenditure, as measured by
respiratory gas analysis, was attenuated in the
Clock mutant mice (Fig. 1D). Overall there
was a net 10% decrease in energy expenditure
in the mutants.
Clock mutant mice fed either a regular or
high-fat diet showed a significant increase in
energy intake and body weight (Fig. 2, A and
B). In Clock mutant and wild-type adult mice
fed either a control or high-fat diet for a
period of 10 weeks beginning at 6 weeks of
age (Fig. 2C), the increase in body weight was
24% in wild-type and 29% in Clock mutant
mice fed a regular diet, versus 38% in wild-
type and 49% in Clock mutant mice fed a
high-fat diet. Comparison of somatic growth
and solid organ mass did not reveal genotype-
specific differences. Instead, the marked
weight gain in Clock mutants fed a regular
diet was attributable to a 65% increase in lean
mass and a 35% increase in fat mass, whereas
in mutants fed a high-fat diet, the weight gain
was due to a 25% increase in lean mass and a
75% increase in fat mass relative to wild-type
control mice (fig. S2).
Because the Clock mutation could affect
early fetal growth and development, we
analyzed the body weights of Clock and lit-
termate pups throughout the first 8 weeks of
life. Body weights were similar in Clock
mutant and wild-type mice during the first 5
weeks, but by 6 weeks of age Clock mutant
mice were consistently heavier (Fig. 2D),
which suggests that the mutation did not affect
fetal growth or nutrition.
We next investigated whether the Clock
mutation altered the adipose–central nervous
system (CNS) axis that regulates feeding and
energy expenditure. Histological analysis
revealed adipocyte hypertrophy and lipid
engorgement of hepatocytes with prominent
glycogen accumulation (fig. S3A) in Clock
mice fed a high-fat diet relative to wild-type
controls; these are hallmarks of diet-induced
obesity in wild-type mice. At 6 to 7 months of
age, Clock mutant mice also had hyper-
cholesterolemia, hypertriglyceridemia, hyper-
glycemia, and hypoinsulinemia (Table 1). In
addition, serum leptin levels increased dur-
ing the light phase in Clock mutant mice fed
a regular diet; this increase was enhanced in
mice fed a high-fat diet (fig. S3B). These
markers of metabolic dysregulation were not
due to an increase in glucocorticoid pro-
duction, because levels of corticosterone were
lower in the Clock mutant mice across the
24-hour LD cycle (wild-type, 5.5 T 1.4 mg/dl;
Clock mutant, 2.6 T 0.4 mg/dl; P G 0.05).
Thus, the Clock mutant mice developed a
spectrum of tissue and biochemical abnor-
malities that are hallmarks of metabolic
To explore whether the Clock mutation
affects expression of neuropeptides involved
in appetite regulation and energy balance,
we analyzed transcripts corresponding to
selected orexigenic and anorexigenic neu-
ropeptides expressed in the mediobasal hy-
pothalamus (MBH). We studied the orexin
transcript because the orexinergic system is
involved in both feeding and sleep-wake
regulation (15, 16); we also studied the tran-
scripts for ghrelin and CART (cocaine- and
amphetamine-regulated transcript) because
the corresponding genes contain CLOCK-
responsive E-box elements (17, 18). In ad-
circadian clock gene, Per2, which has a
diurnal rhythm of expression in the retro-
chiasmatic area. The expression levels of Per2,
orexin, and ghrelin mRNA were markedly
reduced in Clock mutant mice at virtually all
time points of the 12L:12D cycle (Fig. 3). A
smallbut significant decrease in the expression
level of CART in Clock mutant mice occurred
at the beginning and end of the 12-hour light
phase (Fig. 3).
These broad effects of the Clock gene
mutation on nutrient regulation reveal an
unforeseen role for the circadian clock system
in regulating more than just the timing of food
intake and metabolic processes. The effect of
Fig. 2. Obesity in Clock mutant mice. (A) Average caloric intake over a 10-week period in male WT
and Clock mutant mice. WT and Clock mutant mice were provided ad libitum access to regular (10%
kcal/fat; WT, n 0 8; Clock, n 0 10) or high-fat chow (45% kcal/fat; WT, n 0 7; Clock, n 0 11) for 10
weeks beginning at 6 weeks of age. Weekly food intake was analyzed in the two groups (*P G 0.01).
(B) Body weights for the mice in (A) after the 10-week study (*P G 0.01). (C) Body weights of WT
(open symbols) and Clock mutant (solid symbols) mice over the 10-week study for mice in (A) fed
either regular (circles) or high-fat (squares) diets. (D) Body weight of mice after weaning, from 10
days to 8 weeks of age. Growth curves in WT (open circles) and Clock mutant (solid circles) mice on
regular chow were obtained by weekly weighing. Significant differences did not appear until 6 weeks
of age (*P G 0.05). All values represent group means T SEM.
Table 1. Metabolic parameters in WT and Clock mutant mice. Serum triglyceride, cholesterol, glucose,
insulin, and leptin concentrations were determined in 7- to 8-month-old WT and Clock mutant mice fed
a regular diet ad libitum (n 0 4 to 8 mice per group). For measurement of glucose, insulin, and leptin,
blood was collected at 4-hour intervals over a 24-hour time period via an indwelling catheter (40 ml per
blood sample), and the data were pooled to provide an overall mean (TSEM) value. For triglyceride and
cholesterol measurement, a single blood sample (160 ml) was collected at zeitgeber time 0.
Metabolic parameter WTClockP value
136 T 8
141 T 9
130 T 5
1.7 T 0.3
3.4 T 0.4
164 T 8
163 T 6
161 T 7
1.1 T 0.1
4.6 T 0.3
R E P O R T S
13 MAY 2005VOL 308SCIENCE www.sciencemag.org
the Clock mutation on body weight in mice
fed a regular diet was similar in magnitude to
the effect of a high-fat diet in wild-type mice
(Fig. 2). In addition, when Clock mutant mice
were fed a high-fat diet, the combined effect
of the diet plus mutation led to the most
severe alteration in body weight and markers
of metabolism (Fig. 2). Note that the weight
gainintheClock mutant mice is similar to that
observed in a number of metabolic mutants
Alterations in fuel metabolism in animals
carrying a mutant circadian Clock gene could
emerge from a cascade of neural events
initiated by an alteration in circadian rhythms
under the direct control of the SCN (21), in
particular the feeding rhythm, which is
greatly attenuated in Clock mutant mice.
Thus, the misalignment of food intake and/
or the near-loss of feeding rhythmicity
could create metabolic instabilities that
lead to hyperphagia and associated obesity
and lipid/glucose irregularities. On the
other hand, because circadian clock genes
are also expressed in nearly all CNS and
peripheral tissues, alterations in metabo-
lism could be due to cell-autonomous ef-
fects associated with altered expression of
Clock in CNS feeding centers and/or periph-
eral tissues involved in metabolism (5, 22).
The observation that mRNAs of selected
energy-regulatory peptides are altered in
both diurnality and absolute expression level
in the MBH supports the idea of a molec-
ular coupling between circadian and meta-
bolic transcription networks. These results
are consistent with the recent finding that in
addition to regulating the timing of many
genes, the circadian clock regulates the
absolute expression levels of approximate-
ly 3 to 10% of transcripts (23–25).
Clues to the effects of the Clock mutation
on energy balance may be derived from the
emerging map of SCN projections to critical
energy centers within the hypothalamus. For
example, SCN projections form synapses
directly on lateral hypothalamic area neurons
that express orexins (26), as well as indirectly
via the subparaventricular nuclei (SPV). Addi-
tional evidence suggests that connections
between the SCN and neurons within the
MBH may have important effects on cell and
molecular functions. Specifically, recent
analyses from several groups have indicated
that the growth hormone agonist ghrelin,
originally discovered as an incretin hormone
within the stomach, may also be produced
within the MBH/SPV (27, 28). Our real-time
polymerase chain reaction (PCR) results pro-
vide further support for expression of ghrelin
within the MBH. Remarkably, we find that
ghrelin mRNA is greatly reduced in the MBH
from Clock mutant mice, which suggests that
signaling from SCN neurons and/or expres-
sion of the Clock gene within the MBH may
play a critical role in transcriptional control
of target genes within the MBH. Similarly,
we found that orexin levels were lower in
Clock mutant than wild-type mice, and the
normal diurnal variation in expression was
Previous transcriptome analysis in the SCN
and liver of Clock mutant mice has uncov-
ered global changes in metabolic pathways,
including those encoding enzymes of gly-
colysis, mitochondrial oxidative phospho-
rylation, and lipid metabolism (23). The
connection between metabolism and circa-
dian rhythmicity is particularly intriguing in
view of the finding that genes involved in
mitochondrial redox metabolism account for
a large fraction of the circadian transcriptome
in most tissues (10). Although these earlier
results indicated that cell redox flux can alter
the molecular circadian core machinery, our
results in Clock mutant mice indicate that
alterations in this molecular clock may alter
cell metabolism as well.
References and Notes
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Handbook of Behavioral Neurobiology (Kluwer
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11. M. H. Vitaterna et al., Science 264, 719 (1994).
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14. Male mice were used in all studies.
15. J. T. Willie, R. M. Chemelli, C. M. Sinton, M. Yanagisawa,
Annu. Rev. Neurosci. 24, 429 (2001).
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26, 132 (2002).
18. N. Kanamoto et al., Endocrinology 145, 4144 (2004).
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20. J. C. Bruning et al., Science 289, 2122 (2000).
21. S. E. la Fleur, A. Kalsbeek, J. Wortel, M. L. Fekkes, R. M.
Buijs, Diabetes 50, 1237 (2001).
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Physiol. 287, R1071 (2004).
29. Supported by NIH grants AG18200, DK02675,
AG11412, HL75029, HL59598, and DK26356. We
thank the Northwestern University Robert H. Lurie
Comprehensive Cancer Center Pathology Core Fac-
ulty for their assistance. All animal procedures were
performed at Northwestern University following
institutional guidelines. J.S.T. is a cofounder of, has
equity in, and is a member of the scientific advisory
board of Hypnion Inc., which develops sleep-related
pharmaceuticals. He is also on the scientific advisory
boards of NutraGenomics Inc. and the Allen Institute
for Brain Science. F.W.T. is a cofounder of and has
equity in Slowave Inc., which develops sleep-related
pharmaceuticals, and is on the board of directors of
the National Sleep Foundation. Additional paid
consulting relationships are disclosed in the SOM.
Supporting Online Material
Materials and Methods
Figs. S1 to S3
Conflicts of Interest Disclosure
16 December 2004; accepted 23 February 2005
Published online 21 April 2005;
Include this information when citing this paper.
Fig. 3. Clock mutant mice display altered
diurnal rhythms and abundances of Per2
mRNA (A) and mRNAs encoding selected
hypothalamic peptides involved in energy
balance (B to D). Real-time PCR was used to
determine transcript levels as they varied
across a 12:12 LD cycle (indicated by bar at
bottom). Values for WT (red line) and Clock
mutant (black line) mice are displayed as
relative abundance (RA; mean T SEM) after
normalization to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) expression levels in
the same sample. Note that for visual clarity
the RA scales vary for the different transcripts
and vary between genotypes for orexin (Orx)
and ghrelin (Ghr). Brains of four WT and four
Clock mutant mice were collected at 4-hour
intervals across the 12:12 LD cycle. At each
time point, genotype comparisons were made
by independent-sample t tests (*P G 0.05).
CART, cocaine- and amphetamine-regulated
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www.sciencemag.orgSCIENCEVOL 30813 MAY 2005