Biochem. J. (2011) 438, 283–289 (Printed in Great Britain)
283
doi:10.1042/BJ20110263
ATF4 deficiency protects mice from high-carbohydrate-diet-induced liver
steatosis
Houkai LI, Qingshu MENG, Fei XIAO, Shanghai CHEN, Ying DU, Junjie YU, Chunxia WANG and Feifan GUO1
Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, the Graduate School of the
Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, People’s Republic of China
Chronic feeding of HCD (high-carbohydrate diet) is one of
the major contributors to the prevailing of metabolic diseases.
ATF4 (activating transcription factor 4) has been shown to
play an important role in the regulation of glucose metabolism
and obesity development; however, it is unclear how ATF4 − / −
mice respond to HCD. In the present study, we show that
8 weeks of HCD results in significant higher accumulation of
TAGs (triacylglycerols) in livers and impairment in glucose
tolerance in ATF4 + / + mice, but not in ATF4 − / − mice, compared
with those on a normal diet. Meanwhile, energy expenditure is
further enhanced by HCD in ATF4 − / − mice. Moreover, we show
that ATF4 deficiency suppresses HCD-induced SCD1 (stearoyl-
CoA desaturase 1) expression, furthermore, oral supplementation
of the main product of SCD1 oleate (18:1) increases TAG
accumulation in livers of ATF4 − / − mice. Taken together, these
results suggest that ATF4 deficiency is protective for HCDinduced hepatic steatosis and impairment of glucose tolerance and
insulin sensitivity. Furthermore, the resistance to hepatic steatosis
is at least in part due to suppression of SCD1 expression under
HCD.
INTRODUCTION
In the present study, we aimed to investigate the role that ATF4
plays under HCD. We found that ATF4 − / − mice are protected
from HCD-induced hepatic steatosis and impairment in glucose
tolerance and insulin sensitivity. ATF4 − / − mice show significantly
lower expression and activity of SCD1 (stearoyl-CoA desaturase
1) under HCD, compared with ATF4 + / + mice. Moreover, we
found that overexpression of ATF4 up-regulates SCD1 expression
in vivo, and dietary supplementation of the SCD1 product oleate
(C18:1 ) increases TAG (triacylglycerol) accumulation in the livers
of ATF4 − / − mice under HCD. Taken together, these results
indicate that ATF4 − / − mice are resistant to HCD-induced hepatic
steatosis, at least partially due to the suppression of SCD1
expression.
Nutritional status and dietary macronutrient composition play
important roles in the development of obesity or its related
metabolic diseases such as NAFLD (non-alcoholic fatty liver
disease), insulin resistance and diabetes mellitus. Several lines
of evidence indicate that genetic background can interact
with environmental factors including diet in determining
predisposition to metabolic diseases [1–3]. Such nutrient–gene
interaction, on the one hand, signifies the complexity of the aetiology of metabolic diseases; on the other hand, it highlights the
significance of investigating the responsiveness to specific diets
such as HFD (high-fat diet) or HCD (high-carbohydrate diet),
under various genetic backgrounds.
ATF4 (activating transcription factor 4), also known as CREB2
(cAMP-response-element-binding protein 2) [4–7], belongs to the
family of basic zipper-containing proteins. ATF4 has been shown
to be involved in the regulation of various processes including
long-term memory [8,9], osteoblast differentiation [10], glucose
and lipid metabolism [11,12] and redox homoeostasis [13].
ATF4 − / − mice are small in body size and have a low fat content on
ND (normal diet) compared with ATF4 + / + counterparts, which
are attributed to higher lipolysis in WAT (white adipose tissue)
and energy expenditure [12]. Recently, it has been reported that
ATF4 − / − mice are resistant to HFD-induced obesity and liver
steatosis [11,14]. Although excessive dietary fat consumption is
one of the major causes of hepatic steatosis [15,16], it is well
recognized that dietary carbohydrates are also deleterious in the
pathogenesis of hepatic steatosis in humans [17]. Little is known,
however, about the impacts of ATF4 deficiency on lipid and
glucose metabolism under HCD.
Key words: activating transcription factor 4 (ATF4), glucose
tolerance, hepatic steatosis, high-carbohydrate diet (HCD),
insulin sensitivity, stearoyl-CoA desaturase 1 (SCD1).
EXPERIMENTAL
Materials
Heterozygous (ATF4 + / − ) mice on a 129 SV background were
kindly provided by Dr Tim Townes (Department of Biochemistry
and Molecular Genetics, University of Alabama, Birmingham,
AL, U.S.A.), Dr Douglas Cavener (Department of Biology,
Pennsylvania State University, University Park, PA, U.S.A.) and
Dr Bob Paulson (College of Agricultural Sciences, Pennsylvania
State University, University Park, PA, U.S.A.). ATF4 + / − mice
were bred to produce homozygous (ATF4 − / − ) and wild-type
(ATF4 + / + ) mice, and genotyping was performed as described
previously [18]. Six-week-old male ATF4 + / + or ATF4 − / − mice
were fed ad libitum for 8 weeks continuously either on a
HCD or ND (Research Diet). The composition of the HCD
and ND is presented in Table 1. All mice were maintained
Abbreviations used: ATF4, activating transcription factor 4; BAT, brown adipose tissue; CREB, cAMP-response-element-binding protein; CNS, central
nervous system; CPT1α, carnitine palmitoyltransferase 1α; GTT, glucose tolerance test; HCD, high-carbohydrate diet; H&E, haematoxylin & eosin; HFD,
high-fat diet; ITT, insulin tolerance test; NAFLD, non-alcoholic fatty liver disease; ND, normal diet; PPARα, peroxisome-proliferator-activated receptor α;
RER, respiratory exchange ratio; RT–PCR, reverse transcription–PCR; SCD1, stearoyl-CoA desaturase 1; TAG, triacylglycerol; WAT, white adipose tissue.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2011 Biochemical Society
284
Table 1
H. Li and others
as Oil Red O staining with OCTTM (optimal cutting temperature)
embedding, as described previously [21].
Composition of the experimental diets
(a) Percentage composition
ND
Protein
Carbohydrate
Fat
Total
kcal/g
Measurement of TAG in liver and serum
HCD
g%
kcal%
g%
kcal%
20.3
66
5
20.8
67.7
11.5
100
20.3
71
0
22.2
77.8
0
100
3.9
3.65
(b)
Ingredient
g
kcal
g
kcal
Casein, 30 mesh
DL-Methionine
Corn starch
Sucrose
Cellulose, BW200
Corn oil
Mineral mix S10001
Vitamin mix V10001
Choline bitartrate
Total
200
3
150
500
50
50
35
10
2
1000
800
12
600
2000
0
450
0
40
0
3902
200
3
0
700
50
0
35
10
2
1000
800
12
0
2800
0
0
0
40
0
3652
on a 12 h light/12 h dark cycle at 25 ◦ C with free access to
water. At the end of the experiment, mice were killed by CO2
inhalation. All animal experimental procedures were approved by
the Institutional Animal Care and Use Committee of the Institute
for Nutritional Sciences. The recombinant adenoviruses used
for ATF4 expression was generated using the AdEasyTM Vector
System (Qbiogene). The ATF4 plasmid [19] was kindly provided
by Dr Zaiqing Yang (College of Life Science and Technology,
Huazhong Agricultural University, Wuhan, China). Adenoviruses
were purified by ultracentrifugation in cesium chloride gradient
and then quantified. Viruses were diluted in PBS and administered
at a dose of 108 pfu (plaque-forming units) for each mouse through
intravenous injection of tail vein.
Hepatic lipids were extracted with chloroform–methanol (2:1)
according to the method of Folch et al. [20]. Hepatic and serum
TAG were measured with a TAG kit according to manufacturer’s
instructions (Wako).
RNA isolation and relative quantitative RT–PCR (reverse
transcription–PCR)
Total RNA was prepared from frozen tissues with TRIzol®
reagent (Invitrogen). RNA (2 μg) was reversely transcribed with
a random primer (Invitrogen) and MMLV (Moloney murine
leukaemia virus) Reverse Transcriptase (Invitrogen). Quantitative
amplification by PCR was carried out using SYBR Green I Master
Mix reagent on an ABI 7900 system (Applied Biosystems).
GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used
as an internal control for each gene of interest. The sequences of
primers used in the present study are available upon request.
Western blotting
Whole-cell lysates from frozen tissues were isolated using RIPA
(radioimmunoprecipitation assay) lysis buffer (150 mM Tris/HCl,
pH 7.4, 50 mM NaCl, 1 % Nonidet P40 and 0.1 % Tween 20).
Primary antibodies against ATF4 (Santa Cruz Biotechnology)
and SCD1 (Santa Cruz Biotechnology) were incubated overnight
at 4 ◦ C and visualized by ECL® Plus (GE Healthcare). Band
intensities were measured using Quantity One software (Bio-Rad
Laboratories) and normalized to actin.
Statistical analysis
All results are expressed as means +
− S.E.M., unless indicated
otherwise. Significant differences were assessed either by twotailed Student’s t test or one-way ANOVA followed by the
SNK (Student–Newman–Keuls) test. P < 0.05 was considered
statistically significant.
RESULTS
GTT (glucose tolerance test) and ITT (insulin tolerance test)
ATF4 deficiency protects mice from HCD-induced hepatic steatosis
After mice were fed HCD or control diet for 8 weeks, GTT and
ITT were conducted. Specifically, in the GTT, overnight-fasted
mice were intraperitoneally injected with 2 g/kg glucose. Blood
glucose was measured at 0, 15, 30, 60 and 120 min after glucose
injection. In the ITT, 4 h-fasted mice were intraperitoneally
injected with 0.75 unit/kg human insulin (Novo Nordisk) and
the blood glucose levels were measured at the same intervals as
in the GTT.
First, the expression of ATF4 in livers from ATF4 + / + and
ATF4 − / − mice was measured using RT–PCR. The results showed
that the expression of ATF4 mRNA in livers of ATF4 − / − mice
was almost undetectable (Figure 1A), indicating the knockdown
of the ATF4 gene in liver of ATF4 − / − mice. It is well established
that chronic HCD leads to NAFLD [22]. To test whether ATF4
deficiency can prevent mice from HCD-induced hepatic steatosis,
we fed both ATF4 + / + and ATF4 − / − mice with HCD continuously
for 8 weeks. Eight weeks of HCD feeding led to significant fat
accumulation in the livers of ATF4 + / + , but not ATF4 − / − , mice
compared with those under ND (Figures 1B and 1C). Consistent
with these changes, a significant increased liver weight index
was also observed in ATF4 + / + mice (Figure 1D). Moreover, the
contents of hepatic and serum TAG were much higher in ATF4 + / +
mice than those of ATF4 − / − mice under HCD, despite a moderate
increase of hepatic TAG in ATF4 − / − mice (Figures 1E and 1F).
Determination of fatty acid composition in livers
Fatty acids in livers were extracted with chloroform–methanol
(2:1) according to the method of Folch et al. [20]. After the
extraction, the lipids’ residues were methylated with methanol in
0.5 M NaOH and incubated at 60 ◦ C for 1 h, and the methylation
was catalysed using 14 % BF3 for another 40 min at 80 ◦ C. The
resultant products containing total fatty acids were dissolved using
hexane for subsequent analysis by GC.
Histological analysis of tissues
Liver samples were fixed with 4 % (w/v) paraformaldehyde
overnight and stained with H&E (haematoxylin & eosin), as well
c The Authors Journal compilation c 2011 Biochemical Society
ATF4 − / − mice show much lower body fat content and higher
energy expenditure under HCD
Although body weight was not increased in ATF4 + / + or ATF4 − / −
mice, fat content was significantly increased by HCD in both
strains of mice as detected by NMR, with ATF4 + / + mice showing
ATF4 deficiency protects from HCD-induced liver steatosis
Figure 1
285
ATF4 deficiency protects mice from HCD-induced hepatic steatosis
+/+
ATF4
and ATF4 − / − mice were fed HCD or ND for 8 weeks. (A) The knockdown of ATF4 in liver tissues from ATF4 − / − mice measured by relative quantitative RT–PCR; (B, C) representative
photomicrographs of liver stained by H&E or Oil Red O (magnification ×200); (D) liver weight; (E) hepatic TAG (TG); and (F) serum TAG. Results are means +
− S.E.M. for six mice of each group.
*Indicates P < 0.05 as compared with ATF4 + / + -ND group, # indicates P < 0.05 as compared with ATF4 + / + -HCD group, & indicates P < 0.05 as compared with ATF4 − / − -ND group. BW, body
weight.
a much higher proportion of body fat than ATF4 − / − mice,
compared with those fed ND (Figures 2A and 2B). Our previous
study indicated that the daily food intake was higher in ATF4 − / −
mice compared with ATF4 + / + mice [12]. To determine whether
the lower levels of fat content in ATF4 − / − mice under HCD was
due to a decrease in food intake, we compared the daily food
intake between the two strains of mice under HCD. Although
the absolute amount of daily food intake was lower in ATF4 − / −
mice, it was higher when body weight disparity was taken
into consideration, compared with ATF4 + / + mice (Figure 2C).
We have shown previously that ATF4 − / − mice exhibit much
higher energy expenditure than ATF4 + / + mice [12], we therefore
also compared the energy expenditure with indirect calorimetry
between the two strains of mice under HCD. We found that oxygen
consumption (VO2 ) was significantly enhanced under HCD in
ATF4 − / − mice, compared with ATF4 + / + mice (Figures 2D and
2E). Moreover, the RER (respiratory exchange ratio) was also
lower in ATF4 − / − mice (Figures 2F and 2G).
ATF4 − / − mice show improved glucose tolerance and insulin
sensitivity under HCD
It has been demonstrated that ATF4 − / − mice are hypoglycaemic
on normal chow diet [11]; however, it is unknown whether glucose
homoeostasis is influenced by ATF4 deficiency under HCD. Although the blood glucose levels were comparable between the two
strains of mice under either HCD or ND at the fed stage, insulin
levels were much higher in ATF4 + / + mice by HCD (Figures 3A
and 3B). Furthermore, overnight fasted blood glucose was much
lower, but of no difference in levels of fasted insulin was observed
in ATF4 − / − mice, compared with ATF4 + / + mice, under HCD
(Figures 3C and 3D). To further determine the impact of ATF4 deficiency on glucose metabolism under HCD, GTTs and ITTs were
conducted by intraperitoneal injection of glucose (2 g/kg) and insulin (0.75 unit/kg) respectively. As expected, HCD-feeding resulted in significant impairment in glucose tolerance and clearance
in ATF4 + / + mice, but not in ATF4 − / − mice (Figures 3E and 3F).
ATF4 deficiency suppresses HCD-induced SCD1 expression in livers
To gain further insight into the underlying mechanisms of the
protection from HCD-induced liver steatosis in ATF4 − / − mice,
we analysed the expression of genes involved in lipogenesis
and fatty acid oxidation in the livers of both strains of mice
under HCD. mRNA levels of most of the lipogenic genes
were significantly induced by HCD including ACC (acetyl
CoA carboxylase), SCD1, FAS (fatty acid synthase) and GPAT
(glycerol-3-phosphate acyltransferase) in the livers of both strains
of mice (Figure 4A). By contrast, the levels of Scd1 mRNA
and protein were significantly lower in the livers of ATF4 − / −
mice, compared with ATF4 + / + mice, under either HCD or
ND (Figures 4A and 4B). Furthermore, the mRNA and protein
levels of CPT1α (carnitine palmitoyltransferase 1α), the ratelimiting enzyme in fatty acid β-oxidation, showed no difference
in the livers of both strains of mice under either HCD or ND
(Figures 4C and 4D). mRNA levels of the transcription factor
PPARα (peroxisome-proliferator-activated receptor α), however,
were decreased by HCD in the livers of ATF4 + / + mice, but not
in ATF4 − / − mice, compared with those under ND (Figure 4C),
whereas levels of PPARα protein were not different in the livers
among each group (Figure 4D). We did not see any difference in
the expression levels of genes involved in fatty acid transportation
in the livers of both strains of mice under HCD (results not shown).
Oral supplementation of oleate increases TAG accumulation in the
livers of ATF4 − / − mice under HC diet
Given the fact that ATF4 deficiency significantly suppressed
SCD1 expression in the liver under HCD or ND, we hypothesized
that ATF4 was able to regulate SCD1 expression. To test this
hypothesis, we overexpressed ATF4 in the livers of wild-type
mice by injecting with Ad-ATF4 vector. As predicted, 7 days
after injection, we found that SCD1 expression protein levels
were significantly increased in the livers by Ad-ATF4 vector
compared with those infected with control vector (Ad-BK)
c The Authors Journal compilation c 2011 Biochemical Society
286
H. Li and others
Figure 2
ATF4 deficiency prevents HCD-induced adiposity and increases energy expenditure
+/+
ATF4
and ATF4 − / − mice were fed HCD or ND for 8 weeks. (A) Body weight; (B) fat content; (C) food intake; (D, E) oxygen consumption; and (F, G) respiratory exchange ratio. Results are
means +
S.E.M.
for six mice of each group. *Indicates P < 0.05 as compared with ATF4 + / + -ND group, # indicates P < 0.05 as compared with ATF4 + / + -HCD group, & indicates P < 0.05 as
−
compared with ATF4 + / + -ND group. BW, body weight.
Figure 3
ATF4 deficiency improves glucose tolerance and insulin sensitivity under HCD
+/+
ATF4
and ATF4 − / − mice were fed HCD or ND for 8 weeks. GTTs and ITTs were preformed as described in the Experimental section. (A, B) Fed stage blood glucose and insulin levels; (C, D)
+/+
-ND group, # indicates P <
fasting blood glucose and insulin levels; (E) GTT; and (F) ITT. Results are means +
− S.E.M. for six mice of each group. *Indicates P < 0.05 as compared with ATF4
0.05 as compared with ATF4 + / + -HCD group.
(Figure 5A). Consistent with a regulatory role of ATF4 on SCD1
expression (Figures 4A and 4B), we found that ATF4 − / − mice
exhibited a lower ratio of C18:1n − 9 /C18:0 , but a comparable ratio of
C18:1n − 7 /C18:0 in the livers, when compared with ATF4 + / + mice
c The Authors Journal compilation c 2011 Biochemical Society
(Figure 5B). Since C18:1n − 9 , rather than C18:1n − 7 , is the main
product of SCD1 and the ratio of C18:1n − 9 /C18:0 is used as an
index of SCD1 activity [12,23], the low ratio of C18:1n − 9 /C18:0
indicated that the activity of SCD1 enzyme in ATF4 − / − mice
ATF4 deficiency protects from HCD-induced liver steatosis
Figure 4
287
ATF4 deficiency suppresses SCD1 expression in the livers triggered by HCD
+/+
ATF4
and ATF4 − / − mice were fed HCD or ND for 8 weeks. (A) Expression of lipogenic genes; (B) SCD1 protein; and (C, D) expression of genes and proteins involved in fatty acid β-oxidation.
+/+
-ND group or as indicated, # indicates P < 0.05 as compared with ATF4 + / + -HCD group,
Results are means +
− S.E.M. for six mice of each group. *Indicates P < 0.05 as compared with ATF4
& indicates P < 0.05 as compared with ATF4 − / − -ND group.
Figure 5
Oral supplementation of oleate increased TAG accumulation in ATF4 − / − mice under HCD
(A) SCD1 protein levels examined in wild-type mice infected with ATF4 by adenovirus or control vector (Ad-BK). Results are means +
− S.E.M. for six mice of each group. *Indicates P < 0.05 as
compared with Ad-BK group; (B) ratio of C18:1n − 9 /C18:0 in the livers of ATF4 + / + and ATF4 − / − mice detected with GC; (C–E) representative photomicrographs of liver stained with Oil Red O from
mice (magnification ×100), liver weight and TAG (TG) content following oral supplementation with oleate (10 ml/kg body weight) continuously for 10 days in both ATF4 + / + and ATF4 − / − mice
after 3 weeks of HCD feeding. (B–E) Results are means +
− S.E.M. for six mice of each group. *Indicates P < 0.05 as compared with control group.
was also inhibited. Miyazaki et al. [24] have shown previously
that dietary supplementation of triolein (a TAG formed from
the unsaturated fatty acid oleate) can increase hepatic TAG
in mice with a liver-specific deletion of SCD1. To determine
whether the suppressed expression of SCD1 in the livers of
ATF4 − / − mice under HCD contributed to the amelioration of
hepatic lipid accumulation, ATF4 + / + or ATF4 − / − mice that
had been fed HCD for 3 weeks, were orally supplemented with
triolein at a dose of 10 ml/kg daily continuously for 10 days.
As expected, we found that triolein supplementation increased
hepatic lipid accumulation and liver weight in ATF4 − / − mice
(Figures 5C–5E).
c The Authors Journal compilation c 2011 Biochemical Society
288
H. Li and others
DISCUSSION
In the present study, we compare the responses to HCD between
ATF4 + / + and ATF4 − / − mice. Our results show that ATF4
deficiency protects mice from HCD-induced hepatic steatosis
and improves glucose tolerance. We also demonstrate that ATF4
deficiency is resistant to HCD-induced SCD1 expression in the
liver, which, at least partially, accounts for the resistance to HCDinduced hepatic steatosis in ATF4 − / − mice.
Humans with consumption of high-glycaemic index foods,
such as high-fructose or -sucrose diets, are predisposed to
obesity and insulin resistance, as well as higher hepatic TAG
accumulation [22,25]. It is interesting that ATF4 − / − mice show
increased lipolysis in WAT and higher energy expenditure under
ND [12], and are also resistant to age- and diet-related obesity
development, as well as hepatic steatosis induced by chronic HFDfeeding [11]. Our current data provide important information for
the understanding of the physiological functions of ATF4 in the
context of different nutrient compositions, as ATF4 deficiency
protects mice from HCD-induced hepatic steatosis and insulin
resistance. As shown previously [26], we did not observe any
difference in body weight increase of both strains of mice under
HCD, though ATF4 + / + mice showed a higher proportion of fat
content to body weight. The increased energy expenditure in
ATF4 − / − mice was further magnified by HCD. Consistent with
these changes, the value of RER in ATF4 − / − mice was lower than
ATF4 + / + mice under HCD or ND, suggesting a tendency of using
fat as the fuel source. Since relatively low energy expenditure and
high RER are important contributors to body weight increase [27],
the low fat content in ATF4 − / − mice under HCD may be caused by
higher energy expenditure and fat-oriented energy supply through
unknown mechanisms. Since the CNS (central nervous system) is
critical in regulation of peripheral energy homoeostasis [28],
it is possible that the higher energy expenditure in ATF4 − / − mice
is due to a central effect of ATF4 deficiency in the CNS. Another
interesting point is that energy expenditure is higher in ATF4 − / −
mice under HFD [11], suggesting that the enhancement in energy
expenditure by ATF4 deficiency is independent of dietary nutrient
composition in mice.
The prevalence of obesity and its related metabolic syndrome
in developed countries highlights the deleterious consequences of
excessive dietary fat intake [29]. Nevertheless, the awareness of
reducing dietary fat intake derives a relatively higher percentage
of energy from carbohydrates such as sucrose or fructose in
processed food, while high sucrose feeding enhances hepatic
lipogenesis and adiposity [30]. In the present study, we only
observed obvious hepatic steatosis in ATF4 + / + mice, with
increased expression of lipogenic genes in the liver. The only
difference in the changes of lipogenic genes in the livers of both
strains of mice is the expression of SCD1, which is not induced
in ATF4 − / − mice by HCD. As we did not see any difference in
the expression of CPT1α, which encodes the rate-limiting enzyme
CPT1α for β-oxidation of fatty acids, in the livers of both strains
of mice under HCD, we hypothesized that the resistance to
HCD-induced hepatic steatosis in ATF4 − / − mice is due to a
suppressed expression of SCD1. Mice with global deletion of
SCD1 are resistant to HFD- and genetic-induced obesity [31,32],
whereas hepatic-specific knockdown of SCD1 derives protection
from HCD-, but not HFD-, induced liver steatosis [24]. The
hypothesis that repression of SCD1 by ATF4 deficiency may
at least in part account for resistance to HCD-induced hepatic
steatosis is confirmed by the fact that overexpression of ATF4
by adenoviruses increases SCD1 expression in vivo and oral
supplement of oleate (C18:1 ), the major product of SCD1, increases
hepatic TAG accumulation in HCD-fed ATF4 − / − mice.
c The Authors Journal compilation c 2011 Biochemical Society
However, it remains unclear how ATF4 regulates SCD1
expression. Since it has been shown that the SCD1 promoter
region contains the CRE (cAMP-response element) site, and
knockdown of CREB with siRNA (small interfering RNA) can
down-regulate the expression of SCD1 gene [33], we speculated
that ATF4 may regulate SCD1 expression through direct binding
to its promoter region. On the other hand, the expression of
transcription factors for SCD1 showed no difference in the livers
of both strains of mice under HCD, including SREBP-1 (sterolregulatory-element-binding protein-1), ChREBP (carbohydrateresponsive element-binding protein) and LXRα (liver X receptor
α) (results not shown), which suggest the involvement of other
transcription factors in mediating suppression of SCD1 by ATF4
deficiency. Therefore future investigation will be required on
how ATF4 regulates SCD1 expression. Additionally, given the
fact that ATF4 deficiency results in resistance to both HFD- and
HCD-induced hepatic steatosis, it is also likely that other tissues
such as WAT or BAT (brown adipose tissue) may co-ordinate the
resistance to hepatic steatosis because a higher lipolysis in WAT
and UCP1 expression in BAT are observed in ATF4 − / − mice [12].
As a result, further investigations are needed to distinguish the
relative contribution of ATF4 in each tissue, such as liver, WAT,
BAT and brain to HCD-induced hepatic steatosis. Currently, we
are generating tissue-specific knockdown of ATF4 mice, which
will help us to uncover the specific functions of ATF4 in different
tissues (including liver, WAT, BAT and brain) for resistance to
diet-induced hepatic steatosis.
Insulin resistance is critical for the development of NAFLD
[34]. It has been demonstrated that ATF4 plays an important
role in glucose metabolism and regulation of insulin sensitivity
[11,14]. In the present study, our data showed that ATF4 deficiency
improves glucose tolerance and insulin sensitivity under HCD,
which may also contribute to the resistance to HCD-induced
hepatic steatosis. Nevertheless, it should be noted that some of
the present results are different from what has been reported
previously [11,14]. For instance, in the study by Seo et al.
[11] ATF4 − / − mice are hypoglycaemic either during the fasted
or the fed state, and glucose tolerance is also increased on
chow diet, whereas only fasted hypoglycaemia was observed in
our present study. In another report, the authors demonstrate
that ATF4 inhibits insulin secretion and therefore decreases
insulin sensitivity in peripheral tissues [14]. We consider that
the differences may be caused by the following reasons: (i) the
genetic background of experimental mice was different in our
study and in their study, i.e. the genetic background of wildtype and mutant mice we used is 129 SV, while that in their
experiments is C57BL6/J, and (ii) the composition of standard
chow diets between their study and our study is not completely
the same, i.e. the fat content is different.
In summary, our results indicate that ATF4 deficiency protects
mice from HCD-induced hepatic steatosis and improves glucose
and insulin tolerance. Moreover, our results also provide
evidence that the resistance to HCD-induced hepatic steatosis
in ATF4 − / − mice is partially due to the suppressed expression of
SCD1.
AUTHOR CONTRIBUTION
Houkai Li and Qingshu Meng performed all of the Western blotting, RT–PCR and animal
experiments. Fei Xiao and Junjie Yu helped in performing GTTs and ITTs. Shanghai Chen
was responsible for providing ATF4 + / + and ATF4 − / − mice. Ying Du constructed and
provided the adenovirus plasmid for overexpression of ATF4. Chunxia Wang performed
biochemical analysis. Houkai Li and Feifan Guo designed the experiments and wrote the
manuscript. All the authors contributed to and approved the final manuscript.
ATF4 deficiency protects from HCD-induced liver steatosis
FUNDING
This work was supported by grants from the Ministry of Science and Technology of
China [973 Program 2009CB919001 and 2010CB912502], the National Natural Science
Foundation [grant numbers 30871208 and 30890043], the Chief Scientist Program
of Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences
[grant number SIBS2008006], the Science and Technology Commission of Shanghai
Municipality [grant number 08DJ1400601], 2010 Key Program of Clinical Research Center,
Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, the Chinese
Academy of Sciences [grant number CRC2010005], and the Key Program of Shanghai
Scientific and Technological Innovation Action Plan [grant number 10JC1416900]. F.G.
was also supported by the One Hundred Talents Program of the Chinese Academy
of Sciences and Pujiang Talents Program of Shanghai Municipality [grant number
08PJ1410700]. H.L. was supported by a China Postdoctoral Science Foundation-funded
project and the K.C. Wong Education Foundation, Hong Kong.
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Received 23 February 2011/26 May 2011; accepted 7 June 2011
Published as BJ Immediate Publication 7 June 2011, doi:10.1042/BJ20110263
c The Authors Journal compilation c 2011 Biochemical Society