BMC Molecular and Cell
Biology

Sato et al. BMC Molecular and Cell Biology(2024) 25:1
https://doi.org/10.1186/s12860-023-00497-yOpen Access

RESEARCH

Loss of Dec1 inhibits alcohol-induced hepatic
lipid accumulation and circadian rhythm
disorderFuyuki Sato1,2*, Ujjal K. Bhawal3,4, Kosuke Oikawa2 and Yasuteru Muragaki2
Abstract
Chronic alcohol exposure increases liver damage such as lipid accumulation and hepatitis, resulting in hepatic
cirrhosis. Chronic alcohol intake is known to disturb circadian rhythms in humans and animals. DEC1, a basic helixloop-helix transcription factor, plays an important role in the circadian rhythm, inflammation, immune responses,
and tumor progression. We have previously shown that Dec1 deficiency inhibits stresses such as periodontal
inflammation and perivascular fibrosis of the heart. However, the significance of Dec1 deficiency in chronic alcohol
consumption remains unclear. In the present study, we investigated whether the biological stress caused bychronic alcohol intake is inhibited in Dec1 knockout mice. We treated control and Dec1 knockout mice for threemonths by providing free access to 10% alcohol. The Dec1 knockout mice consumed more alcohol than control
mice, however, we observed severe hepatic lipid accumulation and circadian rhythm disturbance in control mice.
In contrast, Dec1 knockout mice exhibited little effect on these outcomes. We also investigated the expression ofperoxisome proliferator-activated receptors (PPARs) and AMP-activated protein kinase (AMPK), which are involvedin the regulation of fatty acid metabolism. Immunohistochemical analysis revealed increases of phosphorylation
AMPK and PPARa but decreases PPARg in Dec1 knockout mice compared to that in control mice. This indicatesa molecular basis for the inhibition of hepatic lipid accumulation in alcohol-treated Dec1 knockout mice. These
results suggest a novel function of Dec1 in alcohol-induced hepatic lipid accumulation and circadian rhythm
disorders.
Keywords Dec1, Alcohol, Immunohistochemistry, AMPK, PPARs*Correspondence:
Fuyuki Sato
fsatoDEC1DEC2@yahoo.co.jp
1
Department of Diagnostic Pathology, Shizuoka Cancer Center,
Sunto-gun 411-8777, Japan2
Department of Pathology, Wakayama Medical University School of
Medicine, Wakayama 641- 8509, Japan
3
Research Institute of Oral Science, Nihon University School of Dentistry
at Matsudo, Chiba 271-8587, Japan
4
Center for Global Health Research , Saveetha Medical College andHospitals , Saveetha Institute of Medical and Technical Sciences, Saveetha
University, Chennai 600077, India

Background
Chronic alcohol intake causes hepatitis, fibrosis, andfatty liver, and promotes hepatocellular carcinoma [1–3].
Several molecular mechanisms involving hepatic lipid
metabolism have been reported [1, 2]. AMP-activatedprotein kinase (AMPK) is a serine/threonine protein
kinase whose activation requires phosphorylation at
Thr172 [4]. AMPK regulates glucose and lipid metabolism [1, 5]. Chronic alcohol exposure inhibits AMPK
activity, leading to steatosis and liver injury [6–8]. Treatment with an AMPK activator, 5-aminoimidazole4-carboxamide-1-b-D-ribofuranoside (AICAR), inhibits© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Sato et al. BMC Molecular and Cell Biology(2024) 25:1

alcohol-induced fatty lipid accumulation in the liver [6,9]. In contrast, peroxisome proliferator-activated receptors (PPARs) are nuclear receptors associated with fatty
liver diseases [1]. PPARa is abundantly expressed in theliver, heart, kidney, and gastrointestinal tract; PPARb/d
are ubiquitously expressed, but dominantly expressed in
the skeletal muscle, skin, and adipose tissue; and PPARg
is highly expressed in the adipose tissue and has low
expression in the liver [10]. However, increased PPARg
expression has been observed in fatty liver and chronic
alcohol intake [11–13].
The circadian rhythm is an important factor to maintain health. Disturbances in the circadian rhythm can
induce various diseases, such as sleep disorders, cancer, and metabolic syndrome [14–17]. The circadianrhythm is predominantly regulated by clock genes, suchas CLOCK, brain and muscle Arnt-like protein (BMAL)
1, period (PER) 1/2/3, cryptochrome (CRY) 1/2, and differentiated embryonic chondrocyte gene (DEC) 1/2 (alsoknown as BHLHE40/41) [16, 17]. Additionally, chronic
alcohol treatment disrupts circadian rhythms and locomotor activity in mice [18, 19]. Furthermore, alcohol
consumption in humans increases the amplitude of clock
genes such as CLOCK, BMAL1, PER1, CRY1, and CRY2[20]. These reports suggest that chronic alcohol exposure disturbs circadian rhythms by inducing abnormalities in the clock genes. DEC1 is a basic helix-loop-helix
transcription factor that regulates tissue differentiation,
cell proliferation, inflammation, tumor progression, andthe circadian rhythm [16, 17, 21–23]. DEC1 negatively
regulates phosphorylated AMPK (pAMPK) throughliver kinase B1 (LKB1) by binding to the E-box site of
the LKB1 promoter [24]. DEC1 also negatively regulates
PPARg by binding to the C/EBP site of its promoter [25,26]. However, the pathogenesis of the alcohol-induced
damage caused by Dec1 remains unclear. Thus, in thisstudy, we examined the role of Dec1 in chronic alcohol
exposure.

Materials and methods
Animals

Animal experiments were conducted according to protocols approved by the Animal Care and Use Committee of Wakayama Medical University (approval number
660). For the experiments, mice were instantaneously
sacrificed by cervical spine dislocation, and the liver tissue was removed, fixed in 4% paraformaldehyde solution, and embedded in paraffin. Whole Dec1 deletion in
Dec1 knockout (KO) mice with a C57BL/6 background
was performed as previously described [21, 27]. Eightto-nine-week-old C57BL/6 wild-type (WT) and Dec1
KO female mice were housed under 12:12 h light:dark(lights on at 8:00 a.m., ZT0; lights off at 8:00 p.m., ZT12)
conditions, as previously described [21, 27]. Ethanol wasPage 2 of 7

diluted with water to a final concentration of 10%. Mice
were allowed free access to 10% ethanol for 3 months(WT, n = 9; Dec1 KO, n = 9). The amount of alcohol
consumed was calculated from the amount of alcohol
remaining in the bottle every ten days. Statistical analyses
were performed using Student’s t-test. Mouse livers were
sampled at ZT2 and subjected to immunohistochemistry.
Activity recording

Mouse locomotor activity was monitored using Supermex (Muromachi Kikai, Tokyo, Japan) (WT, n = 6;
Dec1 KO, n = 6), and data were recorded as previously
described [22]. Activity tests were conducted for at least
40 days.
Immunohistochemistry

Immunohistochemistry was performed using a Discovery Auto-Stainer with automated protocols (Ventana Medical Systems, Inc., Tucson, AZ, USA; Roche,
Mannheim, Germany; Ventana Discovery XT, NexES version 10.6 software, DAB-Map kit 760 − 124), as previously
described [22, 28]. The pAMPK antibody (rabbit monoclonal, 40H9, 2535, and 1:300) was purchased from Cell
Signaling Technology, Inc. (MA, USA). Total AMPK antibody (mouse monoclonal, D-6, sc-74,461, 1:50), PPARg
antibody (mouse monoclonal, E-8, sc-7273, 1:50), andPPARa antibody (mouse monoclonal, H-2, sc-398,394,
1:50) were purchased from Santa Cruz Biotechnology
Inc (TX, USA). We have previously clarified the specificimmunostaining for pAMPK and total AMPK antibodies
in the liver and heart [24]. We performed negative control staining and confirmed that no primary antibodies
were detected in tissues without secondary antibodies.
Statistical analyses

Statistical analyses were performed using a Student’s
t-test in Excel software.

Results
Dec1 KO mice consumed more alcohol than the WT mice

We compared the amount of 10% alcohol consumed by
Dec1 KO and WT mice under free-access conditions.
Subsequently, we calculated the amount of alcohol consumed every month for three months. The data demonstrated that Dec1 KO mice consumed more alcohol than
WT mice at all time points (Fig. 1).
Dec1 KO mice maintained circadian rhythm when
consuming alcohol

Next, we examined the spontaneous locomotor activityof Dec1 KO and WT mice with or without 10% alcohol
consumption using SuperMex. The mice were fed underlight/dark (LD) conditions for six days and then shiftedto constant dark (DD) conditions. As shown in Fig. 2, theSato et al. BMC Molecular and Cell Biology(2024) 25:1

Page 3 of 7

locomotor activity of the WT mice that did not consume
alcohol exhibited a phase shift after shifting to the DD
condition. Alcohol consumption in WT mice decreased
locomotor activity and did not result in a phase shift
after DD (Fig. 2). In contrast, the locomotor activities ofDec1 KO mice were lower than those of WT mice without alcohol consumption and were minimally affected byalcohol consumption.
Lipid accumulation induced by drinking 10% alcohol was
inhibited in Dec1 KO mice via pAMPK, PPARa, and PPARg

Fig. 1 Dec1 KO mice consumed more alcohol than WT mice. Drinking
quantities of 10% alcohol in Dec1 KO and WT mice every month for 3
months. Drinking quantities in nine independent samples were evaluatedby the liquid amounts remaining in the bottle. The raw data are shownin the Supplementary File. Each value represents the mean + SE (bars) ofnine independent samples. 0-1 M: amount from start to 1 month, 1-2 M:
amount from 1 to 2 month, 2-3 M: amount from 2 to 3 month. *P < 0.01,
based on the t-test

Next, we analyzed the livers of WT and Dec1 KO micetreated with 10% alcohol for three months. The livers of
WT mice showed severe congestion and fat accumulationin response to alcohol consumption, whereas those of
Dec1 KO mice showed little change (Fig. 3A and B). Lipid
generation in Dec1 KO mice treated with 10% alcohol
was approximately 14-fold lower than that in WT mice
(Fig. 3C). The livers were quickly extracted and fixed in
4% paraformaldehyde solution. We observed significant
lipid degeneration in hepatic cells of WT mice. In contrast, all Dec1 KO mice showed little effect. We believe

Fig. 2 Dec1 KO mice had little change in locomotor activity with 10% alcohol. Representative locomotor activity records of WT and Dec1 KO mice withor without 10% alcohol. WT: WT mice without 10% alcohol, Dec1 KO: Dec1 KO mice without 10% alcohol, WT with 10% EtOH: WT mice with 10% alcohol,
Dec1 KO with EtOH: Dec1 KO mice with 10% alcohol. LD: 12 h of light and dark cycles, DD: constant darkness. Black squares indicate the periods of darkness. DD started on day 7

Sato et al. BMC Molecular and Cell Biology(2024) 25:1

Page 4 of 7

Fig. 3 Dec1 KO mice had inhibited fatty acid degeneration induced by 10% alcohol. (A) Macroscopic features of two independent WT (WT1 and WT2)
and Dec1 KO (Dec1 KO1 and Dec1 KO2) livers after 10% alcohol consumption. WT mice show severe hepatic congestion, but Dec1 KO mice show littlechange. (B) Microscopy revealed severe lipid degeneration and congestion in the livers of WT mice, whereas Dec1 KO mice were minimally affected. Top
panel, 200 x magnification. Bottom panel, 400 x magnification. Arrows show lipid degeneration. Dotted arrows show central vein. Scale bars, 100 µM. (C)Average numbers of lipid degeneration locations in three independent samples. Lipid degeneration around the central vein was counted in ten microscopic fields under 400 x magnification. *P < 0.01, based on the t-test. The raw data are shown in the Supplementary Filethat degeneration was no affected by sample preparation.
Furthermore, we investigated the expression of pAMPK,PPARa and PPARg, which are involved in the regulationof fatty acid metabolism, by immunohistochemistry. We
observed increased expression of pAMPK in Dec1 KO

mice, but the expression levels of total AMPK remained
almost the same between WT and Dec1 KO mice (Fig. 4).
Moreover, the nuclear amounts of PPARa were higher
and PPARg were lower in Dec1 KO mice compared tothose in WT mice.

Sato et al. BMC Molecular and Cell Biology(2024) 25:1

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Fig. 4 pAMPK, PPARa and PPARg immunoreactivities in the liver of WT and Dec1 KO mice with 10% alcohol consumption. Dec1 KO mice exhibited higher
phosphorylated AMPK (pAMPK), but had little impact on total AMPK. Dec1 KO mice had higher nuclear expression of PPARa compared to that in WT mice;
PPARg expression in Dec1 KO mice was lower. 400 x magnification. Scale bars, 100 µM

Discussion
We have previously shown that Dec1 KO inhibits periodontal inflammation and perivascular fibrosis in theoral mucosa and heart [21, 27, 29]. Thus, Dec1 KO may
provide protection against various stressors. However, it
was unclear whether Dec1 KO inhibits the influence ofstress associated with chronic alcohol exposure on drinking. We revealed that chronic alcohol intake induced adisturbance in the circadian rhythm and lipid accumulation in WT mice, but these abnormal phenotypes did

not occur in Dec1 KO mice. Surprisingly, Dec1 KO mice
consumed more alcohol than WT mice. These results
suggest that the abnormalities do not depend on theamount of alcohol consumed. We propose three hypotheses regarding the mechanism underlying the improvement in lipid accumulation in Dec1 KO mice. First, Dec1
deficiency functionally protected against stress inducedby 10% alcohol intake. Ren et al. showed that cardiacspecific Dec1 knockdown inhibits atrial inflammationand fibrosis [30]. Therefore, Dec1 deficiency may inhibit

Sato et al. BMC Molecular and Cell Biology(2024) 25:1

of chronic alcohol consumption. Second, Dec1 deficiency
may eliminate ingested alcohol via urine. Third, Dec1
deficiency may increase the absorption of ingested alcohol. We have previously reported that Dec1 deficiency
restores age-related metabolic imbalance by inducing theexpression of liver fibroblast growth factor 21 (FGF21)[29]. Therefore, Dec1 deficiency might protect against or
ameliorate stress.
The induction of pAMPK has been shown to inhibit
liver damage by alcohol [1, 5, 6, 8]. We have previously
demonstrated that DEC1 negatively regulates pAMPK
via LKB1 [24]. Thus, Dec1 deficiency may suppress lipid
accumulation and liver damage by inducing the expression of LKB1 and pAMPK.
It has also been reported that chronic alcohol exposure
increases PPARg expression, but decreases PPARa [5,11–13]. Other researchers showed that Dec1 negatively
regulates PPARg [25, 26]. However, our results revealed
that Dec1deficiency inhibited PPARg expression. Dec1
function differs for various types of stress [16]. We
speculate that Dec1 promotes PPARg expression underchronic alcohol exposure, although detailed mechanisms
are unknown. Dec1 may down-regulate PPARa but upregulate PPARg under chronic exposure, suggestingthat Dec1 may regulate target PPARs through different
mechanisms. Further studies are needed to elucidate therole of Dec1 deficiency in alcohol absorption and elimination. No clear changes were observed in food intake,body weight, or fat amounts in WT and Dec1 KO withor without chronic alcohol intake. Many previous studies showed that only chronic alcohol intake affected lipid
metabolism and circadian rhythm [3, 18, 19, 31, 32]. Our
results agree with these findings, and we believe that lipid
degeneration and circadian rhythm disorders depend on
chronic alcohol intake but not on excessive food intake.
Oil-Red O staining is useful method for determining
hepatic lipid accumulation, although researchers must
prepare frozen sections using raw tissues. We plan to use
this method in the future.
Disturbance of the circadian rhythm induces abnormalities in the expression of clock genes, sleep, andmetabolism, resulting in depression, dementia, metabolic
syndrome, type2 diabetes, and cancer [17, 22, 33–36]. We
showed that chronic alcohol intake disturbed the circadian rhythm of WT mice, but had little effect on Dec1
KO mice. As previously mentioned, circadian disorderin Dec1 KO mice may be inhibited by liver absorption
and elimination of alcohol. Further studies are required
to elucidate the detailed molecular mechanisms by which
Dec1 deficiency inhibits circadian disorders under stressful conditions.

Page 6 of 7

Conclusions
We conclude that Dec1 deficiency inhibits lipid accumulation and circadian disorders caused by chronic alcohol consumption. These findings may contribute to thedevelopment of novel treatment strategies for chronic
alcohol-induced damage.
Abbreviations
Dec1	Differentiated embryonic chondrocyte gene 1
H&E	Hematoxylin and eosin
AMPK	AMP-activated protein kinase

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12860-023-00497-y.
Supplementary Material 1
Supplementary Material 2
Acknowledgements
We would like to thank Editage (www.editage.jp) for English language editing.
Author contributions
FS performed the experiments and prepared drafts and figures. UKB, KO, andYM edited the manuscript. All the authors have read and approved the final
version of the manuscript.
Funding
This study was supported by JSPS KAKENHI (grant number 16K09624).
Data availability
All the data analyzed in this study are included in the article. Raw data for thequantification of alcohol consumption and lipid degeneration are available in
the Supplementary file.

Declarations
Ethical approval
The animal studies were approved by the Animal Care and Use Committeeof Wakayama Medical University (approval number 660). All methods were
performed in accordance with the guidelines of Wakayama Medical University,
Basel declaration (https://animalresearchtomorrow.org/en) and ARRIVE(https://arriveguidelines.org).
Consent for publication
Not applicable.
Competing interests
The authors declare no conflicts of interest.
Received: 28 June 2023 / Accepted: 18 December 2023

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