Loss of Dec1 inhibits alcohol-induced hepatic lipid accumulation and circadian rhythm disorder

doi:10.21203/rs.3.rs-2993395/v1

Download PDF

Research Article

Loss of Dec1 inhibits alcohol-induced hepatic lipid accumulation and circadian rhythm disorder

https://doi.org/10.21203/rs.3.rs-2993395/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

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 BHLH 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 exposure remains unclear. In the present study, we investigated whether biological stress caused by chronic alcohol intake is inhibited in Dec1 knockout mice. We treated control and Dec1 knockout mice with 10% alcohol for three months by chronic free intake. The Dec1 knockout mice consumed more alcohol than control mice, but we observed severe hepatic lipid accumulation and disturbance of the circadian rhythm in control mice. In contrast, Dec1 knockout mice showed little effect. Furthermore, we investigated the expressions of peroxisome proliferator-activated receptors (PPARs) and AMP-activated protein kinase (AMPK), which are involved in the regulation of fatty acid metabolism. Immunohistochemical analysis revealed increases of phosphorylation AMPK and PPARa but decreases PPARg in Dec1 knockout mice compared to control mice. This indicates a molecular basis for the inhibition of hepatic lipid accumulation in alcohol-treated Dec1 knockout mice. These results suggest a novel function for Dec1 in alcohol-induced hepatic lipid accumulation and circadian rhythm disorders.

Dec1

alcohol

immunohistochemistry

AMPK

PPARs

Chronic alcohol intake causes hepatitis, fibrosis, and fatty liver, and promotes hepatocellular carcinoma (Ceni et al. 2014; Purohit et al. 2009; Ghosh et al. 2018). Several molecular mechanisms involving hepatic lipid metabolism have been reported (Ceni et al. 2014; Purohit et al. 2009). AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase whose activation requires phosphorylation at Thr172 (Hawley et al. 2003). AMPK regulates glucose and lipid metabolism (Ceni et al. 2014; Rogers et al. 2008). Chronic alcohol exposure inhibits AMPK activity, leading to steatosis and liver injury (Tomita et al. 2005; You et al. 2004; Lu et al. 2021). Treatment with an AMPK activator, 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR), inhibits alcohol-induced fatty lipid accumulation in liver (Tomita et al. 2005; García-Villafranca et al. 2008). In contrast, peroxisome proliferator-activated receptors (PPARs) are nuclear receptors associated with fatty liver diseases (Ceni et al. 2014). PPARa is abundantly expressed in liver, heart, kidney and gastrointestinal tract; PPARb/d are ubiquitously expressed, but dominantly expressed in skeletal muscle, skin and lipid tissue; and PPARg is highly expressed in lipid tissue and has low expression in the liver (Wang et al. 2020). However, increased expression of PPARg expression has been observed in fatty liver and chronic alcohol treatment (Oliva et al. 2008; Bedoucha et al. 2001; Li et al. 2021).

The circadian rhythm is an important phenomenon for maintaining healthy living. Disturbances in the circadian rhythm can induce various diseases such as sleep disorders, cancer, and metabolic syndrome (Gekakis et al. 1998; Turek et al. 2005; Sato et al. 2016; Sato et al. 2018). Circadian rhythms are predominantly regulated by clock genes, such as 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 (also known as BHLHE40/41) (Sato et al. 2016; Sato et al. 2018). Additionally, chronic alcohol treatment disrupts circadian rhythms and locomotor activity in mice (Gaucher et al. 2019; Logan et al. 2010). Furthermore, alcohol consumption in humans increases the amplitude of clock genes such as CLOCK, BMAL1, PER1, CRY1 and CRY2 (Swanson et al. 2016). These reports suggest that chronic alcohol exposure disturbs circadian rhythms by inducing abnormalities in clock genes. Dec1 is a basic helix-loop-helix transcription factor that regulates tissue differentiation, cell proliferation, inflammation, tumor progression, and the circadian rhythm (Sato et al. 2016; Sato et al. 2018; Sato et al. 2019; Le et al. 2019; Wang et al. 2021). Dec1 negatively regulates phosphorylated AMPK (pAMPK) through liver kinase B1 (LKB1) by binding to the E-box site of the LKB1 promoter (Sato et al. 2015). Dec1 also negatively regulates PPARg by binding to the C/EBP site of its promoter (Park et al. 2012; Yun et al. 2002). However, the pathogenesis of the alcohol-induced damage caused by Dec1 remains unclear. Thus, we examined the role of Dec1 in chronic alcohol exposure in this study.

Animals. This animal experiment was performed according to protocols approved by Animal Care and Use Committee of the Wakayama Medical University (approve number 660). For experiments, mice were instantaneously sacrificed with cervical spine fracture dislocation, took out liver, fixed in 4% paraformaldehyde solution, and embedded in paraffin. Whole Dec1 deletion in Dec1 knockout (KO) mice with a C57BL/6 background were performed as previously described (Le et al. 2019; Zhang et al. 2018). Eight-to-nine-week-old C57BL/6 wild-type (WT) and Dec1 KO female mice were housed under 12 h light (lights on at 8:00 a.m.; ZT0) and dark (lights off at 8:00 p.m.; ZT12) conditions, as previously described (Le et al. 2019; Zhang et al. 2018). The ethanol was diluted with water to final concentration of 10%. Mice were allowed free access to 10% ethanol and maintained 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 a 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 (Sato et al. 2019). 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 ver 10.6 software, DAB-Map kit 760 − 124), as previously described (Sato et al. 2019; Sato et al. 2022). The pAMPK antibody (rabbit monoclonal, 40H9, 2535, 1:300) was purchased from Cell Signaling Technology, Inc. (MA, USA). Total AMPK antibody (mouse monoclonal, D-6, sc-74461, 1:50), PPARg antibody (mouse monoclonal, E-8, sc-7273, 1:50) and PPARa antibody (mouse monoclonal, H-2, sc-398394, 1:50) were purchased from Santa Cruz Biotechnology Inc (TX, USA). We previously clarified specific immunostaining of pAMPK and total AMPK antibodies in livers and hearts (Sato et al. 2015). We performed negative control staining and confirmed all primary antibodies without secondary antibodies were no staining in tissues.

Dec1 KO mice drank more alcohol than the WT mice. We compared the amount of 10% alcohol consumed by Dec1 KO and WT mice under free-choice conditions. Subsequently, we calculated the amount of alcohol consumed every month for three months. The data indicates that Dec1 KO mice consumed more alcohol than WT mice at all points (Fig. 1).

Dec1 KO mice maintained circadian rhythm by drinking alcohol. Next, we examined the spontaneous locomotor activity of Dec1 KO and WT mice with or without 10% alcohol drinking using SuperMex. The mice were first fed under light/dark (LD) conditions for 6 days and then shifted to constant dark (DD) conditions. As shown in Fig. 2, the locomotor activity of WT mice that without alcohol exhibited a phase shift after the shift to DD conditions. WT mice with alcohol drinking decreased locomotor activities and did not show phase shift after DD (Fig. 2). In contrast, the locomotor activities of Dec1 KO mice decreased compared to those of WT mice without alcohol drinking, and was minimally affected by alcohol drinking.

Dec1 KO mice inhibits lipid accumulation induced by drinking 10% alcohol via pAMPK, PPARa, and PPARg. Next, we analyzed the livers of WT and Dec1 KO mice treated with 10% alcohol for three months. The livers of WT mice showed severe congestion and fat accumulation with alcohol drinking, whereas those of Dec1 KO mice showed little effect (Fig. 3A and B). Lipid generation in Dec1 KO mice treated with 10% alcohol was about 14-fold reduction compared to WT mice (Fig. 3C). We quickly extracted livers and fixed in 4% paraformaldehyde solution. We could observe a significant lipid degeneration in hepatic cells of WT mice. On the other hand, all Dec1 KO mice had little affected. We think the degeneration does not depend on sample preparation. Furthermore, we investigated the expression of pAMPK, PPARa and PPARg (which are involved in the regulation of fatty acid metabolism) by immunohistochemistry. We observed increased expression of pAMPK in Dec1 KO mice, but the expression levels of total AMPK remained almost unchanged between the WT and Dec1 KO mice (Fig. 4). Moreover, the nuclear amounts of PPARa was increased and PPARg was decreased in Dec1 KO mice.

We have previously shown that Dec1 KO inhibits periodontal inflammation and perivascular fibrosis in the oral mucosa and heart (Le et al. 2019; Zhang et al. 2018; Fujita et al. 2016). Thus, Dec1 KO may provide protection against various types of stressors. However, it is unclear whether Dec1 KO inhibits the influence of stress associated with chronic alcohol exposure on drinking. We revealed that chronic alcohol intake induced a disturbance in the circadian rhythm and lipid accumulation in WT mice, but these abnormal phenotypes were greatly improved in Dec1 KO mice. Surprisingly, Dec1 KO mice consumed more alcohol than WT mice. These results suggest that the abnormalities do not depend on the amount consumed. We propose three hypotheses regarding the mechanism underlying the improvement in lipid accumulation in Dec1 KO mice. First, Dec1 deficiency functionally protects against stress induced by 10% alcohol intake. We previously reported that Dec1 KO inhibits chronic inflammation and fibrosis in the oral mucosa and heart (Le et al. 2019; Zhang et al. 2018; Fujita et al. 2016). In addition, Ren et al. (2022) showed that cardiac-specific Dec1 knockdown inhibits atrial inflammation and fibrosis (Ren et al. 2022). Therefore, Dec1 deficiency may inhibit chronic alcohol consumption. Second, Dec1 deficiency may eliminate ingested alcohol from the urine. Third, Dec1 deficiency may increase the absorption of ingested alcohol. We previously reported that Dec1 deficiency restores age-related metabolic imbalance by inducing the expression of liver fibroblast growth factor 21 (FGF21) (Fujita et al. 2016). Therefore, Dec1 deficiency may protect against or ameliorate stress.

It has been proven that the induction of pAMPK inhibits liver damage by alcohol (Ceni et al. 2014; Rogers et al. 2008; Tomita et al. 2005; Lu et al. 2021). We previously demonstrated that Dec1 negatively regulates pAMPK via LKB1 (Sato et al. 2015). Thus, Dec1 deficiency may suppress lipid accumulation and liver damage by inducing LKB1 and pAMPK expression.

It has also been reported that chronic alcohol exposure increases PPARg expression, but decreases PPARa (Rogers et al. 2008; Oliva et al. 2008; Bedoucha et al. 2001; Li et al. 2021). Other researchers showed that Dec1 negatively regulates PPARg (Park et al. 2012; Yun et al. 2002). However, our results revealed that Dec1deficiency inhibited PPARg expression. Dec1 function differs in various kinds of stresses (Sato et al. 2016). We speculate Dec1 promote PPARg expression under chronic alcohol exposure, although detailed mechanisms are unknown. Dec1 may down-regulate PPARa but up-regulate PPARg under chronic exposure, suggesting that Dec1 may regulate their PPARs through different mechanisms. Further studies are needed to clarify the role of Dec1 deficiency in alcohol absorption and elimination. No obvious change was visually observed food intake amounts, body weight and fat amounts in WT and Dec1 KO with or without chronic alcohol intake. Many articles showed only chronic alcohol intake affect lipid metabolism and circadian rhythm (Ghosh et al. 2018; Carson et al. 1996; Gaucher et al. 2019; Logan et al. 2010; Kudo et al. 2009). We agree with these findings and think the lipid degeneration and circadian rhythm disorder depends on chronic alcohol intake but not food excessive intake in this study. Oil-red staining is one of the useful methods for hepatic lipid accumulation, although researcher need to prepare frozen section using raw tissues. We think it for future plan.

Disturbance of the circadian rhythm induces abnormalities in the expression of clock genes, sleep, and metabolism, resulting in depression, dementia, metabolic syndrome, type2 diabetes, and cancer (Sato et al. 2018; Sato et al. 2019; Tasali et al. 2008; Copinschi et al. 2000; Karlsson et al. 2001; Scheer et al. 2009). 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 disorder in Dec1 KO mice may be inhibited by liver absorption and elimination. Further studies are required to clarify the detailed molecular mechanisms by which Dec1 deficiency inhibits circadian disorders under stress.

We conclude that Dec1 deficiency inhibits lipid accumulation and circadian disorders caused by chronic alcohol exposure. These findings may contribute to the development of new treatment strategies for chronic alcohol-induced damage.

Dec1: Differentiated embryonic chondrocyte gene 1; H&E: Hematoxylin and eosin; AMPK: AMP-activated protein kinase

Ethical Approval

Animal studies was approved by Animal Care and Use Committee of the Wakayama Medical University (approve number 660).

Competing interests

The authors declare no conflicts of interest.

Authors’ contribution

FS performed the experiments and prepared drafts and figures. UKB, KO, and YM edited the manuscript. All the authors have read and approved the final version of the manuscript.

Funding

This work was supported by JSPS KAKENHI grant number 16K09624 (F.S.).

Bedoucha M, Atzpodien E, Boelsterli UA (2001) Diabetic KKAy mice exhibit increased hepatic PPARgamma1 gene expression and develop hepatic steatosis upon chronic treatment with antidiabetic thiazolidinediones. J Hepatol 35: 17–23
Carson EJ, Pruett SB (1996) Development and characterization of a binge
drinking model in mice for evaluation of the immunological effects of ethanol. Alcohol
Clin Exp Res 20: 132–138
Ceni E, Mello T, Galli A (2014) Pathogenesis of alcoholic liver disease: Role of oxidative metabolism. World J Gastroenterol 20: 17756–17772
Copinschi G, Spiegel K, Leproult R, Van Cauter E (2000) Pathophysiology of human circadian rhythms. Novartis Found Symp 227: 143–157
Fujita Y, Makishima M, Bhawal UK (2016) Differentiated embryo chondrocyte 1 (Dec1) is a novel negative regulator of hepatic fibroblast growth factor 21 (Fgf21) in aging mice. Biochem Biophys Res Commun 469: 477–482
García-Villafranca J, Guillén A, Castro J (2008) Ethanol consumption impairs regulation of fatty acid metabolism by decreasing the activity of AMP-activated protein kinase in rat liver. Biochimie 90: 460–466
Gaucher J, Kinouchi K, Ceglia N, Montellier E, Peleg S, Greco CM, Schmidt A, Forne I, Masri S, Baldi P, Imhof A, Sassone-Corsi P (2019) Distinct metabolic adaptation of liver circadian pathways to acute and chronic patterns of alcohol intake. Proc Natl Acad Sci U S A 116: 25250–25259
Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, King DP, Takahashi JS, Weitz CJ (1998) Role of the clock protein in the mammalian circadian mechanism. Science 280: 1564–1569
Ghosh Dastidar S, Warner JB, Warner DR, McClain CJ, Kirpich IA (2018) Rodent models of alcoholic liver disease: Role of binge ethanol administration. Biomolecules 8:3
Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, Mäkelä TP, Alessi DR, Hardie DG (2003) Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade. J Biol 2: 28
Karlsson B, Knutsson A, Lindahl B (2001) Is there an association between shift work and having a metabolic syndrome? Results from a population based study of 27,485 people. Occup Environ Med 58: 747–752
Kudo T, Tamagawa T, Shibata S (2009). Effect of chronic ethanol exposure on the
liver of clock-mutant mice. J Circadian Rhythms 7: 4
Le HT, Sato F, Kohsaka A, Bhawal UK, Nakao T, Muragaki Y, Nakata M (2019) Dec1 deficiency suppresses cardiac perivascular fibrosis induced by transverse aortic constriction. Int J Mol Sci 20: 4967
Li C, Li J, Loreno EG, Miriyala S, Panchatcharam M, Lu X, Sun H (2021) Chronic low-dose alcohol consumption attenuates post-ischemic inflammation via PPARγ in mice. Int J Mol Sci 22: 5121
Logan RW, Seggio JA, Robinson SL, Richard GR, Rosenwasser AM (2010) Circadian wheel-running activity during withdrawal from chronic intermittent ethanol exposure in mice. Alcohol 44: 239–244
Lu X, Xuan W, Li J, Yao H, Huang C, Li J (2021) AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy. Autophagy 17: 3622–3643
Oliva J, French BA, Li J, Bardag-Gorce F, Fu P, French SW (2008) Sirt1 is involved in energy metabolism: The role of chronic ethanol feeding and resveratrol. Exp Mol Pathol 85: 155–159
Park YK, Park H (2012) Differentiated embryo chondrocyte 1 (DEC1) represses PPARγ2 gene through interacting with CCAAT/enhancer binding protein β (C/EBPβ). Mol Cells 33: 575–581
Purohit V, Gao B, Song BJ (2009) Molecular mechanisms of alcoholic fatty liver. Alcohol Clin Exp Res 33: 191–205
Ren KW, Yu XH, Gu YH, Xie X, Wang Y, Wang SH, Li HH, Bi HL (2022) Cardiac-specific knockdown of Bhlhe40 attenuates angiotensin II (Ang II)-induced atrial fibrillation in mice. Front Cardiovasc Med 9: 957903
Rogers CQ, Ajmo JM, You M (2008) Adiponectin and alcoholic fatty liver disease. IUBMB Life 60: 790–797
Sato F, Kohsaka A, Bhawal UK, Muragaki Y (2018) Potential roles of Dec and Bmal1 genes in interconnecting circadian clock and energy metabolism. Int J Mol Sci 19: 781
Sato F, Bhawal UK, Yoshimura T, Muragaki Y (2016) DEC1 and DEC2 crosstalk between circadian rhythm and tumor progression. J Cancer 7:153–159
Sato F, Otsuka T, Kohsaka A, Le HT, Bhawal UK, Muragaki Y (2019) Smad3 suppresses epithelial cell migration and proliferation via the clock gene Dec1, which negatively regulates the expression of clock genes Dec2 and Per1. Am J Pathol 189: 773–783
Sato F, Muragaki Y, Zhang Y (2015) DEC1 negatively regulates AMPK activity via LKB1. Biochem Biophys Res Commun 467: 711–716
Sato F, Bhawal UK, Osaki S, Sugiyama N, Oikawa K, Muragaki Y (2022) Differential immunohistochemical expression of DEC1, CK‑1ε, and CD44 in oral atypical squamous epithelium and carcinoma in situ. Mol Med Rep 25: 159
Scheer FA, Hilton MF, Mantzoros CS, Shea S (2009) Adverse metabolic and cardiovascular consequences of circadian misalignment. ProcNatl Acad Sci U S A 106: 4453–4458
Swanson GR, Gorenz A, Shaikh M, Desai V, Kaminsky T, Van Den Berg J, Murphy T, Raeisi S, Fogg L, Vitaterna MH, Forsyth C, Turek F, Burgess HJ, Keshavarzian A (2016) Night workers with circadian misalignment are susceptible to alcohol-induced intestinal hyperpermeability with social drinking. Am J Physiol Gastrointest Liver Physiol 311: G192-201
Tasali E, Leproult R, Ehrmann DA, Van Cauter E (2008) Slow-wave sleep and the risk of type 2 diabetes in humans. Proc Natl Acad Sci U S A 105: 1044–1049
Tomita K, Tamiya G, Ando S, Kitamura N, Koizumi H, Kato S, Horie Y, Kaneko T, Azuma T, Nagata H, Ishii H, Hibi T (2005) AICAR, an AMPK activator, has protective effects on alcohol-induced fatty liver in rats. Alcohol Clin Exp Res 29: 240s-245s
Turek FW, Joshu C, Kohsaka A, Lin E, Ivanova G, McDearmon E, Laposky A, Losee-Olson S, Easton A, Jensen DR, Eckel RH, Takahashi JS, Bass J (2005) Obesity and metabolic syndrome in circadian clock mutant mice. Science 308: 1043–1045
You M, Matsumoto M, Pacold CM, Cho WK, Crabb DW (2004) The role of AMP-activated protein kinase in the action of ethanol in the liver. Gastroenterology 127: 1798–1808
Yun Z, Maecker HL, Johnson RS, Giaccia AJ (2002) Inhibition of PPAR gamma 2 gene expression by the HIF-1-regulated gene Dec1/Stra13: A mechanism for regulation of adipogenesis by hypoxia. Dev Cell 2: 331–341
Wang X, Sato F, Tanimoto K, Rajeshwaran N, Thangavelu L, Makishima M, Bhawal UK (2021) The Potential Roles of Dec1 and Dec2 in Periodontal Inflammation. Int J Mol Sci 22: 10349
Wang Y, Nakajima T, Gonzalez FJ, Tanaka N (2020) PPARs as metabolic regulators in the liver: Lessons from liver-specific PPAR-null mice. Int J Mol Sci 21: 2061
Zhang F, Suzuki M, Kim IS, Kobayashi R, Hamada N, Sato F, Bhawal UK (2018) Transcription factor Dec1 is required for maximal experimentally induced periodontal inflammation. J Periodontal Res 53: 883–893

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Loss of Dec1 inhibits alcohol-induced hepatic lipid accumulation and circadian rhythm disorder

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Results

Discussion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1