Am J Physiol Endocrinol Metab 315: E848–E858, 2018.

First published July 10, 2018; doi:10.1152/ajpendo.00072.2018.

RESEARCH ARTICLE Translational Physiology

Mechanisms of sleep deprivation-induced hepatic steatosis and insulin

resistance in mice
Fumika Shigiyama,1 X Naoki Kumashiro,1 Yousuke Tsuneoka,2 Hiroyuki Igarashi,1 Fukumi Yoshikawa,1
Saori Kakehi,3,4 Hiromasa Funato,2 and Takahisa Hirose1
1
Division of Diabetes, Metabolism, and Endocrinology, Department of Medicine, Toho University Graduate School of
Medicine, Tokyo, Japan; 2Department of Anatomy, Toho University Graduate School of Medicine, Tokyo, Japan;
3
Department of Metabolism and Endocrinology, Juntendo University Graduate School of Medicine, Tokyo, Japan; and
4
Sportology Center, Juntendo University Graduate School of Medicine, Tokyo, Japan
Submitted 22 February 2018; accepted in final form 28 June 2018

Shigiyama F, Kumashiro N, Tsuneoka Y, Igarashi H,
Yoshikawa F, Kakehi S, Funato H, Hirose T. Mechanisms of sleep
deprivation-induced hepatic steatosis and insulin resistance in mice.
Am J Physiol Endocrinol Metab 315: E848 –E858, 2018. First pub-
lished July 10, 2018; doi:10.1152/ajpendo.00072.2018.—Sleep depri-
vation is associated with increased risk for type 2 diabetes mellitus.
However, the underlying mechanisms of sleep deprivation-induced
glucose intolerance remain elusive. The aim of this study was to
investigate the mechanisms of sleep deprivation-induced glucose
intolerance in mice with a special focus on the liver. We established
a mouse model of sleep deprivation-induced glucose intolerance using
C57BL/6J male mice. A single 6-h sleep deprivation by the gentle
handling method under fasting condition induced glucose intolerance.
Hepatic glucose production assessed by a pyruvate challenge test was
significantly increased, as was hepatic triglyceride content (by 67.9%)
in the sleep deprivation group, compared with freely sleeping control
mice. Metabolome and microarray analyses were used to evaluate
hepatic metabolites and gene expression levels and to determine the
molecular mechanisms of sleep deprivation-induced hepatic steatosis.
Hepatic metabolites, such as acetyl coenzyme A, 3␤-hydroxybutyric
acid, and certain acylcarnitines, were significantly increased in the
sleep deprivation group, suggesting increased lipid oxidation in the
liver. In contrast, fasted sleep-deprived mice showed that hepatic gene
expression levels of elongation of very long chain fatty acids-like 3,
lipin 1, perilipin 4, perilipin 5, and acyl-CoA thioesterase 1, which are
known to play lipogenic roles, were 2.7, 4.5, 3.7, 2.9, and 2.8 times,
respectively, those of the fasted sleeping control group, as assessed by
quantitative RT-PCR. Sleep deprivation-induced hepatic steatosis and
hepatic insulin resistance seem to be mediated through upregulation of
hepatic lipogenic enzymes.
hepatic insulin resistance; hepatic steatosis; sleep deprivation
INTRODUCTION
Sleep is one of the most important lifestyle components. The
quality and quantity of sleep correlate with physical and mental
health and homeostasis (43). Sleep disorders are also closely
associated with serious complications, such as hypertension
and type 2 diabetes mellitus (25). In this regard, several clinical
and experimental studies have demonstrated the association
between sleep disorders and glucose intolerance or insulin
resistance (3, 59). In a rodent model, it was reported that sleep
disorders were associated with increased food intake (16, 48)
and reduced whole-body energy expenditure (43). However, in
the above studies, it was not clear whether glucose intolerance
was due to the changes in food intake or energy expenditure or
to the sleep deprivation itself. Thus, the exact underlying
mechanisms remain undetermined.
In the present study, we adopted the single 6-h sleep depri-
vation technique by the gentle handling method (19, 47).
Especially in this study, to mimic the metabolic disorders seen
in lifestyle disturbances in humans, mice were fed a high-fat
diet and provided with sucrose water for 2 wk, and then we
compared the effects of sleep deprivation in fasting and move-
ment-restricted C57BL/6J mice and freely sleeping (accord-
ingly noneating and nonmoving) mice. Thus, the model al-
lowed for the assessment of the effects of sleep deprivation on
glucose intolerance independent of hyperphagia and behavioral
patterns. In addition, we assessed the animals immediately
after a single 6-h sleep deprivation so as to avoid any effects of
deep sleep recovery. Our model is different from the mouse
model of genetic mutation or ablation of molecular clock genes
and thus allowed us to assess the physiological effects of sleep
deprivation. Considering that metabolic disorders occur with
lifestyle disturbances, it is important to assess the effect of
sleep deprivation without genetic modification.
The pathogenesis of type 2 diabetes mellitus is associated
with hepatic and peripheral muscle insulin resistance caused by
ectopic lipid accumulation in the liver and skeletal muscle (50,
56). In mammals, it is also reported that the liver encompasses
a biological circadian clock system (11, 19). In this regard, the
liver plays an important physiological role in energy metabo-
lism, the diurnal processing of nutrients (57, 60). Thus, it
seems important to investigate the underlying mechanisms of
sleep deprivation-induced glucose intolerance with special fo-
cus on the liver. In the present study, hepatic metabolome and
microarray analyses were performed to identify the molecular
mechanisms of sleep deprivation-induced hepatic insulin resis-
tance and hepatic steatosis.
MATERIALS AND METHODS

Animals. Male C57BL/6J mice (CLEA Japan Inc., Tokyo, Japan)

Address for reprint requests and other correspondence: N. Kumashiro, aged 13 wk were used in all experiments. They were individually
6-11-1 Omori-Nishi, Ota-ku, Tokyo, Japan 143-8541 (e-mail: naoki. housed in plastic cages under a 12-h light/12-h dark cycle (lights on
kumashiro@med.toho-u.ac.jp). at 0800) at controlled ambient temperature (24 ⫾ 1°C) and were

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 HEPATIC STEATOSIS AND INSULIN RESISTANCE BY SLEEP DISORDER
provided with food and water ad libitum. During the 14-day habitu-
ation period, mice received a high-fat diet (D12451, Research Diets,
Inc., New Brunswick, NJ) and 5% sucrose water. All procedures
described in this study were approved by the Institutional Animal
Care and Use Committee of Toho University School of Medicine.
Sleep deprivation and fasting protocol. Mice were visually moni-
tored continuously for signs of sleep for 6 h from 0800 to 1400 on the
day of the experiment. To avoid any stress, handling or touching of
mice was minimized. For mice of the sleep deprivation group, they
were physically handled or gently touched once they started to close
their eyes and recline. On the other hand, mice of the control group
were allowed to sleep undisrupted. Unless otherwise stated, the
procedure of sleep deprivation was applied only once per animal in
this study. Mice of both groups were fasted during the 6-h sleep
deprivation period and also maintained in small individual plastic
cages (Natsume Seisakusho Co., Tokyo, Japan) to limit their physical
activity during the sleep deprivation period. Tissue and blood sam-
pling, intraperitoneal glucose tolerance test (ipGTT), and pyruvate
challenge test were performed immediately after the 6-h sleep depri-
vation period. For the chronic assessment, 6 h of sleep deprivation
was conducted daily over 5 consecutive days under the same labora-
tory conditions, and then ipGTT was performed at the end of the 5-day
sleep deprivation period. For the recovery sleep assessment, after a
single 6-h sleep deprivation period, the mice of the recovery group
were released into the usual plastic cage and stayed ad libitum
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E849
overnight. After an overnight, they slept in the small plastic cage
while fasting for 6 h, and then ipGTT or tissue sampling was
performed.
Tissue sampling. Tissues were immediately dissected out at the end
of the sleep deprivation period, wrapped in aluminum foil, flash-
frozen in liquid nitrogen, and preserved in a ⫺80°C refrigerator.
Laboratory tests. Blood glucose concentrations were measured by
a portable glucose meter using Accu-Chek Aviva Nano (Roche, Basel,
Switzerland). Plasma insulin was measured using an ELISA kit
(Morinaga, Kanagawa, Japan). Glucagon concentration was measured
by a radioimmunoassay kit (Euro-Diagnostica AB, Malmö, Sweden).
Corticosterone and adrenocorticotropic hormone (ACTH) were mea-
sured by electrochemiluminescence immunoassay kit (Roche Diag-
nostic K.K., Tokyo, Japan).
ipGTT and pyruvate challenge test. At the end of the sleep
deprivation and fasted period for 6 h, 20% glucose (2.0 g/kg body wt)
or pyruvate (1.5 g/kg body wt) was injected intraperitoneally for
ipGTT or for the pyruvate challenge test, respectively, as described
previously (26, 28, 61). Blood samples were collected from the tail
vein at the indicated time on the figures, and plasma glucose and
insulin levels were measured with the portable glucose meter and
ELISA kit, respectively, as described above.
Hepatic triglyceride assay. Hepatic triglyceride content was deter-
mined by using the triglyceride assay kit (Triglyceride E-test WAKO,
Tokyo, Japan) and a method adapted as described previously (26, 27,
Fig. 1. A and B: intraperitoneal glucose tolerance test (ipGTT).
The 6-h sleep deprivation was induced by the gentle handling
method. A: plasma glucose level at 60 min was significantly
higher in mice of the sleep deprivation group (n ⫽ 8) than the
control group (n ⫽ 8). B: area under the curve (AUC) of
glucose calculated for individual mice and averaged for the two
groups. The AUC of glucose was significantly higher in the
sleep deprivation group. C and D: ipGTT of the chronic model
with 6 h of sleep deprivation for 5 days. C: six-hours of sleep
deprivation was induced by the gentle handling method and
repeated for 5 days. Plasma glucose concentrations at 0 and 60
min were significantly lower in the sleep deprivation group
(n ⫽ 8) than the control group (n ⫽ 8). D: AUC of plasma
glucose concentration during ipGTT was comparable between
the two groups (n ⫽ 8 per group). E and F: ipGTT after 24-h
recovery period, including sleep recovery. E: ipGTT was per-
formed after 24-h recovery period, including sleep recovery
after 6 h of sleep deprivation. In detail, mice of the recovery
group were released into the plastic cage and stayed ad libitum
overnight. Then, they were fasted in the small plastic cage and
slept freely for 6 h, and then ipGTT was performed. Fasting
plasma glucose was significantly higher in the recovery group
than the sleep deprivation group (n ⫽ 8 per group). F: recovery
group had a smaller increase in plasma glucose compared with
the sleep deprivation group, specifically significant at 120 min
(n ⫽ 8 per group). Data are means ⫾ SD. Differences among
groups were assessed by two-way ANOVA followed by the
Tukey post hoc (in A, C, E, and F) or by the unpaired t-test (in
B and D). *P ⬍ 0.05.
E850 HEPATIC STEATOSIS AND INSULIN RESISTANCE BY SLEEP DISORDER

gene ontology analyses were performed by the KURABO Bio-Med-

A 150
ical Department using the Affymetrix GeneChip Mouse Gene 2.0 ST
Array (Affymetrix, Inc., Santa Clara, CA). Genes with a more than
Δblood glucose (mg/dL) * twofold change in their expression levels (sleep deprivation vs.
100 * control) (P ⬍ 0.05) were selected as differentially expressed genes. P
values and fold changes between the two groups were calculated using
the Moderated t-test. A heat map with hierarchical clustering was
50 constructed for the genes that showed different expression levels in
the sleep deprivation and control groups. Gene expression data of each
sample were analyzed using hierarchical clustering, and each color in
0 the heat map indicates gene expression intensity (Log2). In this
0 30 60 analysis, red color reflects much higher than the average standardized
expression level, whereas blue color represents lower than average
expression. Hierarchical clustering analysis was performed using
-50 Time (min) GeneSpring version 14 software (Agilent Technologies, Santa Clara,
B 5000 *
AUC Δblood glucose

4000

3000

2000

1000

0
Control Sleep deprivation
Fig. 2. Pyruvate challenge test. Blood glucose concentrations were measured
following intraperitoneal pyruvate injection (1.5 g/kg) after 6 h of fasting (n ⫽
8 per group). A: Delta (⌬, change in) blood glucose, as an index of extent of
gluconeogenesis. B: area under the curve (AUC) of delta glucose was calcu-
lated for the individual mouse and averaged for each group. Data are
means ⫾ SD. Differences among groups were assessed by two-way ANOVA
followed by the Tukey post hoc test. *P ⬍ 0.05.

29). Briefly, around 100 mg of liver tissues were used to extract liver
triglycerides. Tissues were homogenized in 2:1 chloroform/methanol
on cold ice, and lipids were extracted by shaking for 3– 4 h at room
temperature. After the addition of sulfuric acid to 100 mM, the
samples were vortexed and centrifuged to separate the solution phase.
The organic phase was collected and used for analysis of triglyceride
content.
Oil red O staining. Mice were deeply anesthetized with sodium
pentobarbital (15 mg/kg ip) and then perfused transcardially with 4%
paraformaldehyde in 0.01 M phosphate᎑buffered saline (PBS) (pH
7.4). The livers were removed immediately and postfixed in 4%
paraformaldehyde/PBS at 4°C overnight, followed by cryoprotection
in 30% (wt/vol) sucrose in PBS. The tissue blocks were embedded in
Surgipath (FSC22, Leica Biosystems, Wetzlar, Germany), and then
stored at ⫺80°C until cryosectioning. Livers were cryosectioned into
10-␮m thick sections and mounted on MAS-coated slides. The sec-
tions were stained with 0.3% Oil red O solution, counterstained with
hematoxylin, and coverslipped with aqueous mountant.
Measurement of liver metabolites. Metabolome analysis was con-
ducted by the Dual Scan package of Human Metabolome Technolo-
gies Inc. (Tsuruoka, Japan) using capillary electrophoresis time-of-
flight mass spectrometry and liquid chromatography time-of-flight
mass spectrometry, based on the methods described in detail previ-
ously (37, 40). The migration time, retention time, and mass-to-charge
ratio were compared with authentic standards to identify the metab-
olites in the liver tissue samples. In addition to the quantification of Fig. 3. Plasma corticosterone, glucagon, and adrenocorticotropic hormone
the metabolites, their peak areas were compared with those of authen- (ACTH) levels after 6 h of sleep deprivation. Plasma corticosterone (A),
tic standards. glucagon (B), and ACTH (C) were measured in both groups (n ⫽ 8 per group).
Gene expression microarray and data analysis. To determine the Data are means ⫾ SD. Differences among groups were assessed by the
genetic and signaling pathways involved in the liver, microarray and unpaired t-test. *P ⬍ 0.05.

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 HEPATIC STEATOSIS AND INSULIN RESISTANCE BY SLEEP DISORDER
*
2 = Control group
= Sleep deprivation group
Relative mRNA expression
1 groups (Fig. 1D). We also evaluated the effect of sleep recov-
0 insulin sensitivity, the pyruvate challenge test was performed.
G6Pase FBP1 PC PEPCK1
Fig. 4. Comparison of mRNA expression levels of four hepatic gluconeogen-
esis-related genes between the control and sleep deprivation groups (n ⫽ 7 per
group). The average mRNA expression level of the control group was set as 1.
Data are means ⫾ SD. Differences among groups were assessed by the
unpaired t-test. *P ⬍ 0.05. FBP1, fructose-1,6-bisphosphatase; G6Pase, glu-
cose-6-phosphatase; PC, pyruvate carboxylase; PEPCK1, phosphoenolpyru-
vate carboxykinase 1.
CA). The gene expression values on the heat map were normalized by
the RMA-Sketch method. A complete set of microarray data was
deposited into the Gene Expression Omnibus repository (https://
www.ncbi.nlm.nih.gov/geo/) under accession number GSE92913.
Gene ontology analysis was performed using Fisher’s exact test.
Real-time polymerase chain reaction. Total RNA was extracted
from flash-frozen ~15 mg liver tissue using the RNeasy mini kit
(Qiagen, Tokyo, Japan). RNA was reverse-transcribed into cDNA
using the QuantiTect Reverse Transcription kit (Qiagen, Hilden,
Germany). The abundance of transcripts was assessed by real-time
polymerase chain reaction (PCR) on an Applied Biosystems 7500 Fast
Real-Time PCR System (Thermo Fisher Diagnostics, Tokyo, Japan)
with a Fast SYBR Green Master Mix (Thermo Fisher Diagnostics).
The expression level of each gene of interest was normalized for the
efficiency of amplification with TATA box binding protein mRNA as
the invariant control, as determined by a standard curve.
Statistical analysis. All data are expressed as means ⫾ SD unless
otherwise indicated. Differences among groups were assessed by
two-way ANOVA followed by the Tukey post hoc test (for ipGTT
and pyruvate challenge test), and by the unpaired t-test for other
parameters. P values ⬍0.05 were defined as statistically significant.
Statistical analyses were performed with PRISM.7 software (Graph-
Pad Software, La Jolla, CA).
RESULTS
Single 6-h sleep deprivation episode induces glucose intol-
erance and hepatic insulin resistance. To evaluate the effect of
sleep deprivation on glucose metabolism, each mouse under-
went ipGTT after the sleep deprivation period. Figure 1A
shows a significant increase in plasma glucose level in the
sleep deprivation group after only one session of 6 h of sleep
deprivation. The area under the curve of glucose in ipGTT was
significantly higher in the sleep deprivation group (Fig. 1B),
despite the lack of increase in body weight (26.3 ⫾ 2.4 vs.
25.9 ⫾ 2.5 g, control vs. sleep deprivation, respectively). There
was no difference in plasma insulin level at 60 min of ipGTT
between the two groups (0.29 ⫾ 0.3 vs. 0.54 ⫾ 0.4 ␮U/ml,
control vs. sleep deprivation group, respectively, P ⫽ 0.18).
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E851
We further examined the effects of 6 h of sleep deprivation
conducted daily over 5 consecutive days under the same
laboratory conditions. ipGTT was performed at the end of the
5-day sleep deprivation period. The results of the latter tests
showed significantly lower plasma glucose concentrations at
times 0 and 60 min of ipGTT in the sleep deprivation group
(Fig. 1C). However, in these tests, the area under the curve of
plasma glucose concentration was comparable between the two
ery on glucose tolerance and found that a 24-h recovery period,
including sleep recovery, improved the impaired glucose tol-
erance (Fig. 1, E and F).
Next, we investigated the mechanisms of sleep deprivation-
induced glucose intolerance with a special focus on the liver.
To assess hepatic gluconeogenic capacity that reflects hepatic
Sleep deprivation was associated with significant increase in
glucose level after pyruvate loading compared with the control
group (Fig. 2, A and B). This result was supported by the
finding of significantly higher plasma concentrations of corti-
costerone and glucagon, which increase hepatic glucose pro-
duction, in the sleep deprivation group compared with the
control group (Fig. 3, A and B). We also assessed plasma
ACTH, which is usually released from the pituitary gland and
stimulates corticosterone production from the adrenal glands.
Plasma ACTH level tended to be higher in the sleep depriva-
tion group than the control group (Fig. 3C), albeit insignifi-
cantly. Furthermore, hepatic mRNA expression level of glu-
cose 6-phosphatase, which is one of the gluconeogenic en-
zymes, was significantly higher in the sleep deprivation group
than the control group (Fig. 4).
Sleep deprivation induces hepatic steatosis accompanied by
increased hepatic lipid oxidation and lipogenesis. Since he-
patic insulin resistance is associated with hepatic steatosis (27,
62), we assessed hepatic triglyceride content. Interestingly, a
single 6-h sleep deprivation increased hepatic triglyceride con-
tent by 67.9% compared with the control group (Fig. 5, P ⬍
0.05). To confirm the increase in hepatic triglyceride level, a
histological examination of liver tissue was conducted using
Oil red O staining. Consistent with the above results of bio-
chemical analysis, larger numbers and areas of lipid droplets
were observed in the sleep deprivation group than the control
40
*
Hepatic triglyceride content
30
(mg / g-liver)
20
10
0
Control Sleep deprivation
Fig. 5. Hepatic triglyceride content in the control and sleep deprivation groups
(n ⫽ 7 per group). Data are means ⫾ SD. Differences among groups were
assessed by the unpaired t-test. *P ⬍ 0.05.
E852 HEPATIC STEATOSIS AND INSULIN RESISTANCE BY SLEEP DISORDER

group (Fig. 6). We also evaluated the effect of sleep recovery
on hepatic lipid accumulation and found that only a 24-h
recovery period, including sleep recovery, improved hepatic
lipid accumulation (14.4 ⫾ 7.6 vs. 29.2 ⫾ 9.0 mg/g liver for
recovery group vs. sleep deprivation group, respectively).
To determine the underlying mechanism of hepatic steatosis,
metabolome analysis was performed using frozen liver tissues.
Interestingly, 3-hydroxybutyric acid, acetyl coenzyme A
(CoA), and acylcarnitines were significantly increased in mice
of the sleep deprivation group compared with those of the
control group (Fig. 7, A–C). We also assessed hepatic mRNA
expression levels using microarray analysis and RT-PCR. A
total of 34,474 genes were screened by microarray analysis
using liver samples. Of the entire group, the expression levels
of 58 genes were significantly different between the sleep
deprivation group and control group (Fig. 8). Next, we per-
formed gene ontology analysis to estimate multiplicity between
gene ontology functional classes of the gene sets and genes that
were differentially expressed genes on microarray analysis.
The results showed that genes involved in the lipid metabolic
process, steroid metabolic process, and regulation of fatty acid
metabolic process were significantly different between the two
groups (Table 1). Furthermore, quantitative RT-PCR con-
firmed overexpression of hepatic genes known to be involved
in promoting lipogenesis, such as elongation of very long chain
fatty acids-like 3 (Elovl3), lipin 1 (Lpin1), perilipin 4 (Plin4),
perilipin 5 (Plin5), and acyl-CoA thioesterase 1 (Acot1) (Fig.
9A). On the other hand, the same method showed downregu-
lation of the genes known to be involved in the sterol regula-
tory element-binding protein-1c (SREBP1c), another lipogenic
enzyme. With regard to the genes that promote lipid oxidation,
ATP-binding cassette subfamily D member 2 (ABCD2) was
significantly lower, whereas acetyl-CoA carboxylase 2 was
higher in the sleep deprivation group compared with the
control group (Fig. 9B).
DISCUSSION
In this study, 6 h of sleep deprivation was associated with
the development of glucose intolerance and increased hepatic
glucose production, suggestive of hepatic insulin resistance.
Interestingly, this was also accompanied by hepatic steatosis
without weight gain. Comprehensive analysis using metabo-
lome suggested that sleep deprivation-induced hepatic steatosis
was mediated by increased lipid oxidation in the liver. In
contrast, the expression levels of certain hepatic lipogenic

A B

C D

Fig. 6. Oil red O staining of liver sections.

A–C: control group. D–F: sleep deprivation
group. n ⫽ 3 per group; scale bar ⫽ 100 ␮m.

E F

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 HEPATIC STEATOSIS AND INSULIN RESISTANCE BY SLEEP DISORDER
A 0.03
3-Hydroxybutyric acid
(Relative area)
0.02
0.01
0.00
Control Sleep deprivation
B 0.0006
(Relative area)
Acetyl CoA
0.0004
0.0002
0.0000
Control Sleep deprivation
genes were significantly increased in the fasted sleep-deprived
mice compared with the control sleeping mice, as assessed by
microarray and quantitative RT-PCR. Taken together, our data
suggest that the mechanisms of sleep deprivation-induced he-
patic steatosis and insulin resistance include, at least in part,
overexpression of certain lipogenic genes, such as Elovl3.
Several rodent sleep deprivation models have already been
described (5, 14, 16 –19, 34, 38, 43, 47, 48, 53, 66). With
regard to the model selection, we considered it was important
to assess the effect of sleep deprivation without genetic mod-
ification, considering that metabolic disorders occur with life-
style disturbances. Ferrell and Chiang (19) favored gentle
stimulation with physical contact as a technique of circadian
system disturbance or sleep disruption rather than genetic
mutation or ablation of molecular clock genes in mice. In the
present study, we also simultaneously fasted mice of both the
sleep deprivation and control groups and restricted their phys-
ical activity at comparable levels to assess the direct effects of
sleep deprivation on glucose intolerance independent of hy-
perphagia and behavioral pattern. Then, body weight was not
increased, but glucose intolerance was induced by the applied
technique of sleep deprivation. Our model is also different
from the chronic sleep deprivation model (3) and has been used
to evaluate the direct and immediate effects of sleep depriva-
tion on metabolism in vivo, avoiding the effects of sleep
recovery (2, 14) and chronic compensatory metabolic changes
(4). In this study, we also tested the chronic model, but body
weight tended to decrease in the sleep deprivation group
compared with the control group (27.7 vs. 30.2 g, sleep
deprivation vs. control, n ⫽ 8 vs. 8, P ⫽ 0.054, respectively).
Such body weight reduction in the sleep deprived mice may be
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E853
C
Number of Carbon
Fig. 7. Results of metabolome analysis. A:
3-hydroxybutyric acid. B: acetyl CoA. C:
acylcarnitines. The levels of 3-hydroxybu-
tyric acid, acetyl-CoA, and some acylcar-
nitines were significantly higher in the sleep
deprivation group compared with control
group (n ⫽ 7 per group). Data are
means ⫾ SD of the relative area calculated
as described in M ETHODS . Differences
among groups were assessed by the unpaired
t-test. *P ⬍ 0.05.
= Control group
= Sleep deprivation group
Number of Double bond
due to severe physical stress caused by continuous sleep
deprivation, and it may explain the significant decrease in
plasma glucose concentration. Based on these observations, we
did not perform experiments with chronic sleep deprivation for
more than 5 days. Furthermore, in this study, to mimic the
metabolic disorders in humans associated with lifestyle distur-
bances, we provided mice with a high-fat diet and sucrose
water for 2 wk during the habituation period.
In our model, the 6 h of sleep deprivation significantly
increased hepatic triglyceride content and induced hepatic
insulin resistance. Hepatic gluconeogenesis in vivo was
significantly increased in the sleep deprived mice, as as-
sessed by the pyruvate challenge test and, consistently,
increased glucose 6-phosphatase gene expression, as con-
firmed by RT-PCR. In this regard, previous studies reported
that sleep restriction or fragmentation leads to insulin resis-
tance in rodents (4, 14, 66) and humans (13, 15, 22, 35, 46,
58). In addition, Hsieh et al. (24) reported the association of
short sleep duration with fatty liver in human. It has also
been demonstrated that several genetic variants of the clock
genes correlate with hepatic steatosis (32). In general, he-
patic steatosis is considered to induce hepatic insulin resistance
(27, 62). Thus, sleep deprivation seems to induce hepatic
steatosis, and this, in turn, increases hepatic glucose produc-
tion, reflecting hepatic insulin resistance, at least in part, finally
leading to glucose intolerance.
To determine the underlying molecular mechanisms, we
performed metabolome and microarray analyses. Hepatic
metabolome analysis showed significantly higher levels of
acetyl-CoA, 3-hydroxybutyric acid, and acylcarnitine in the
sleep deprived mice. Acetyl-CoA is known to catalyze the
E854 HEPATIC STEATOSIS AND INSULIN RESISTANCE BY SLEEP DISORDER

01.SD
02.SD
03.SD
05.SD
06.SD
10.SD
13.SD
04.CT
07.CT
08.CT
09.CT
11.CT
12.CT
14.CT
Lpin1
Elovl3
Mfsd2a
St3gal5
Gm19958 Edv Gm3579
Gm19958 Edv Gm3579
Gm19958 Edv Gm3579
Gm19958 Edv Gm3579
E330011021Rik
Erbb4
Lgr5
Dsg1c
Abcd2
Pparg
Sult1c2
1700040L02Rik
Cxcl1
Clec2h
Bcl6
Sc4mol
Per3

Fig. 8. Differentially expressed genes in the Gsap

Car1
control and sleep deprivation groups (n ⫽ 7 Fzd8
per group), assessed by microarray analysis. Sort1
Cdkn1a
Gene expression data of each sample were Ddit4
analyzed using hierarchical clustering. Each Gm15889
Tmc7
color in the heat map indicates the gene Gm19958 Edv Gm3579
expression intensity (Log2). The colors are Gm19958 Edv Gm3579

derived from color range (1). Each fold- Scara5

E030018B12Rik
change score (Log2) between the control Mir344d-2 mmu-mir-344d-2
group and sleep deprivation group is also Fam107a
Cyp2b10
colored. The colors are located on the right Plin4
side of the heat map. The colors were derived Fgf21 LOC100862558
Cidec
from color range (2). Arntl
Srebf1
Apol7a
Ugt2b35
Acot1
Lyve1
Slc45a3
Tbc1d8
Lipg
Fam35a
Slc35g1
LOC100862287 Fkbp5
Ppp1r3c
Cyp39a1
Cyp17a1
Insig2
Plin5
Igfbp1
Ankhd1 Eif4ebp3

Color range(1) Color range(2)

Gene Expression Intensity (Log2) Fold Change (Log2)
CT=Control group
SD=Sleep deprivation group

gluconeogenic function of pyruvate carboxylase (44, 50). In to increased hepatic glucose production. With regard to the
our model, pyruvate carboxylase was not increased at the gene increased levels of hepatic 3␤-hydroxybutyric acid, a ketone
expression level, but its gluconeogenic function was likely body, Chikahisa et al. (10) also reported that 6 h of sleep
increased by the increased hepatic acetyl-CoA content, leading deprivation in mice increased plasma ketone body levels. They

Table 1. Distribution of gene ontology biological process groups for genes that showed significant difference in their
expression levels
Count in Selection Count in Total
Accession GO Term (Numerator) (Denominator) P Value

GO:0006629 lipid metabolic process 13 1032 0.0050

GO:0015074 DNA integration 3 8 0.0050
GO:0004523/GO:0004524 RNA-DNA hybrid ribonuclease activity 3 9 0.0050
GO:0007169 transmembrane receptor protein, tyrosine kinase signaling pathway 7 295 0.0203
GO:0032868 response to insulin 6 196 0.0203
GO:0008202 steroid metabolic process 6 229 0.0351
GO:0019217/GO:0006632 regulation of fatty acid metabolic process 4 64 0.0351
Data are results of gene ontology (GO) analysis for genes of the sleep deprivation group/control group. GO Term criteria is P value of the Fisher exact test
of ⬍0.05.

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 HEPATIC STEATOSIS AND INSULIN RESISTANCE BY SLEEP DISORDER
= Control group
* *
A = Sleep deprivation group
*
Relative mRNA expression
5
* *
4
3
*
2
1
0 (8). The results also described a potential functional crosstalk
SRBP1c Elovl3 Lpin1 Plin4 Plin5 Acot1 FAS
B higher expression levels of Elovl3 and lower expression levels
*
Relative mRNA expression
3
2
* glucocorticoid concentration, leading to upregulation of Elovl3
1 loss on the hypothalamic pituitary adrenal axis (23, 33, 36, 54).
0 volved in lipolysis and lipogenesis and contribute to hepatic
ABCD2 PPARα ACC1 ACC2 ACO
Fig. 9. Comparison of hepatic mRNA expression levels of genes involved in
lipogenesis and lipid oxidation between the control and sleep deprivation
groups (n ⫽ 7 per group). A: lipogenesis-related genes. B: lipid oxidation-
related genes. The average of the control group was set as 1. Data are
means ⫾ SD. Differences among groups were assessed by the unpaired t-test.
*P ⬍ 0.05. ABCD2, ATP-binding cassette subfamily D member 2; ACC1/2,
acetyl-CoA carboxylase 1/2; ACO, acyl-CoA oxidase; Acot1, acyl-CoA thio-
esterase 1; Elovl3, elongation of very long chain fatty acids-like 3; FAS, fatty
acid synthase; Lpin1, lipin 1; Plin4/5, perilipin 4/5; PPAR␣, peroxisome
proliferator-activated receptor ␣; SREBP1c, sterol regulatory element-binding
protein-1c.
suggested that ketone body was associated with sleep/wake
regulation in the brain. However, the effects of changes in
hepatic ketone bodies on brain sleep homeostasis or vice versa
are not well established. Some studies reported the association
of sleep curtailment with increased acylcarnitine levels in
humans (6, 12, 63, 65). Increased acylcarnitine level is
considered to be a sign of mitochondrial dysfunction and a
marker of altered metabolic processes (63). Altered mito-
chondrial parameters are thought to be linked to insulin
resistance (45, 63). Our group has also reported that mito-
chondrial dysfunction is associated with hepatic insulin
resistance in nonalcoholic fatty liver disease in humans (55).
In addition, increased acylcarnitine levels may play a causal
role in the development of insulin resistance through their
proinflammatory effects (49).
Using microarray data, the results of gene ontology analysis
suggested that sleep deprivation significantly affects the met-
abolic processes of lipid metabolism, steroids, and regulation
of fatty acid. We also used quantitative RT-PCR to examine
the upregulated genes involved in lipid metabolism. Interest-
ingly, sleep deprivation was associated with significant de-
crease in the well-known SREBP1c, a master regulator of
lipogenesis. In this regard, circadian rhythm is known to
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E855
regulate numerous hepatic genes (41), including SREBP1c,
fatty acid synthase, and acetyl-CoA carboxylase 1 (7, 41), but
these do not seem to be involved in sleep deprivation-induced
hepatic steatosis based on the results of the present study.
Although it is reported that SREBP1c activity is modulated by
nutrients and circadian rhythm (21, 31), the mechanisms of
decreased SREBP1c expression by sleep deprivation need to be
further investigated. In contrast, Elovl3, whose expression was
significantly altered by sleep deprivation, is considered to be
strongly linked to circadian rhythm (1). Other studies also
reported that hepatic Elovl3 mRNA expression exhibited a
diurnal rhythm and was induced by exposure to glucocorticoids
between Elovl3 and ABCD2 (8). Interestingly, significantly
higher plasma corticosterone concentrations and significantly
of ABCD2 hepatic mRNA were noted in the sleep deprived
mice in the present study compared with the control. These
results suggest that sleep deprivation seems to increase plasma
and downregulation of ABCD2. To date, some animal and
human studies have reported a direct stimulatory effect of sleep
In addition, Patel et al. (42) suggested previously that elevated
glucocorticoid levels modulate hepatic gene expressions in-
steatosis. Thus, the high corticosterone levels could be respon-
sible, at least in part, for the sleep deprivation-induced lipid
accumulation in the liver. We are currently engaged in research
on loss of function using metyrapone to suppress glucocorti-
coid production in our sleep deprivation model. This research
is designed to address the question of whether lipid accumu-
lation is abolished when sleep deprivation is uncoupled with
stress-induced glucocorticoid levels.
In addition to Elovl3, our results detected upregulation of
Lpin1, Plin4, Plin5, and Acot1, suggesting their potential
involvement in the sleep deprivation-induced hepatic steatosis
(20, 30, 39). Sengupta et al. (53) also reported that sleep
restriction induced lipogenesis metabolic switches in the rat
liver. Of note, because our sleep deprived mice were fasted,
any decrease in the expression of lipogenic genes and increase
in the expression of lipid oxidation-related genes could be
caused by fasting, but the increased expression levels of
lipogenic genes, such as Elovl3, Lpin1, Plin4, Plin5, and
Acot1, or decreased expression levels of lipid oxidation-related
genes, such as ABCD2, could be caused by sleep deprivation.
Combining the metabolome and microarray data, there
seems to be an increase in lipid oxidation and lipogenesis in
our acute sleep deprivation model. In this regard, a previous
review suggested that patients with fatty liver exhibit both
increased triglyceride synthesis and increased ␤-oxidation
(51). In addition, Bugianesi et al. (9) reported that hepatic lipid
content, as assessed in liver biopsies, correlated significantly
with lipid oxidation in patients with nonalcoholic fatty liver
disease. Thus, lipid accumulation in the liver seems to be
associated with increased hepatic lipid oxidation. Steatosis
could result from increased delivery of circulating fatty acids to
the liver (64); part of the excess fatty acids would be oxidized,
and the remainder could be esterified to triglyceride in the
liver.
E856 HEPATIC STEATOSIS AND INSULIN RESISTANCE BY SLEEP DISORDER
The present study has several limitations. First, hepatic
steatosis was evaluated in this study by increased hepatic
triglycerides content. Because sometimes hepatic triglycerides
become inert storage and hepatic insulin-sensitive fatty liver
exists in humans (55), future studies should assess other lipid
species, such as diacylglycerol and ceramide, which have been
implicated in mediating insulin resistance (27, 52). Second, no
causality can be ascertained due to the lack of gain or loss of
function analysis. Therefore, intervention studies designed to
prevent sleep deprivation-induced hepatic steatosis and insulin
resistance should be performed in the future. Third, our pro-
tocol of sleep deprivation included sleep deprivation and pos-
sibly stress. Finally, the asleep/awake status was monitored
visually. Similar studies are needed in which the sleep/wake
cycle is monitored by electroencephalography, electrooculog-
raphy, and electromyography.
In conclusion, the molecular mechanisms of hepatic steato-
sis and hepatic insulin resistance induced by sleep deprivation
were investigated comprehensively using metabolome and mi-
croarray analyses in our mouse model. A single session of 6 h
of sleep deprivation was associated with the development of
hepatic steatosis and hepatic insulin resistance under fasting
condition without weight gain. Sleep deprivation-induced he-
patic steatosis and hepatic insulin resistance are probably
mediated through upregulation of hepatic lipogenic enzymes.
ACKNOWLEDGMENTS
We thank Azusa Fujimura, Sachiko Mizuno, Hikari Taka, and Naoko Kaga
for the excellent technical assistance.
GRANTS
This research was supported by the Japan Society for the Promotion of
Science KAKENHI (Grant nos. 25893260, 26702032, and JP17H02180) and
by a research grant from Kanae Foundation for the Promotion of Medical
Science.
DISCLAIMERS
The funding agencies and corporations had no role in study design, data
collection and analysis, decision to publish, or writing of the manuscript.
DISCLOSURES
T. Hirose received research funds from AstraZeneca; Boehringer Ingelheim
Pharmaceuticals, Inc.; Astellas Pharma Inc.; Ono Pharmaceutical Co., Ltd.;
Novo Nordisk Inc; Sanofi-Aventis Deutschland GmbH; Daiichi-Sankyo Co.,
Ltd.; Eli Lilly Japan K.K.; Takeda Pharmaceutical Company Limited; Mit-
subishi Tanabe Pharma Corporation; Dainippon Sumitomo Pharma Co., Ltd.;
Kissei Pharmaceutical Co., Ltd.; and Johnson & Johnson and received lecture
fees from Sanofi-Aventis Deutschland GmbH; Eli Lilly Japan K.K., Novo
Nordisk Inc; Takeda Pharmaceutical Company Limited; Daiichi-Sankyo Co.,
Ltd.; Mitsubishi Tanabe Pharma Corporation; Merck & Co., Inc,; Dainippon
Sumitomo Pharma Co., Ltd.; Novartis Pharma K.K.; Kissei Pharmaceutical
Co., Ltd.; Boehringer Ingelheim Pharmaceuticals, Inc.; Ono Pharmaceutical
Co., Ltd.; and AstraZeneca. The authors declare no conflicts of interest in
relation to the work described in this manuscript.
AUTHOR CONTRIBUTIONS
F.S., N.K., and H.F. conceived and designed research; F.S., N.K., H.I., F.Y.,
and S.K. performed experiments; F.S. and N.K. analyzed data; F.S., N.K.,
Y.T., H.F., and T.H. interpreted results of experiments; F.S. and N.K. prepared
figures; F.S. and N.K. drafted manuscript; F.S. and N.K. edited and revised
manuscript; F.S., N.K., Y.T., H.I., F.Y., S.K., H.F., and T.H. approved final
version of manuscript.
The authors are fully responsible for all the content and editorial decisions,
were involved in all stages of the study and manuscript development, and
approved the final version of the manuscript. N.K. is the guarantor of this work
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and, as such, has full access to all study data and takes responsibility for the
integrity of the data and accuracy of data analysis.
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