GRAPHICAL ABSTRACT

Highlights

• Gut-derived serotonin (GDS) is elevated in both patients and mouse models of ALD.

• Inhibition of GDS synthesis reduces hepatic lipogenesis in ALD mouse models.

• Hepatic ER stress is alleviated by inhibiting GDS synthesis.

• The effects of GDS are mediated through hepatic HTR2A signaling.

INTRODUCTION

Alcoholic liver disease (ALD) is one of the most prevalent liver diseases worldwide [1,2]. Despite the profound socioeconomic burden of ALD, there are limited treatment strategies other than alcohol abstinence in all stages of the disease [3]. ALD encompasses a wide range of hepatic pathologies caused by excessive alcohol intake including steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma [4]. Alcohol intake upregulates lipogenic pathways while down-regulates β-oxidation pathways, leading to hepatic steatosis [4]. Chronic alcohol consumption stimulates sterol regulatory element binding protein 1c (SREBP1c), a master regulator of hepatic lipogenic pathways, which in turn activates its target genes to enhance lipogenesis in hepatocytes [5-7]. Concurrently, decreased peroxisome proliferator activated receptor alpha (PPARα) activity inhibits fatty acid (FA) oxidation, further promoting alcoholic steatosis [8].

ALD begins with steatosis and progresses to hepatitis, fibrosis, and cirrhosis [9,10]. However, the detailed mechanism of ALD progression from steatosis to hepatitis and fibrosis remains to be elucidated. Endoplasmic reticulum (ER) stress has been identified as one of the major contributors to ALD progression [11]. It is also known that the ER stress is increased in ALD patients and ALD animal models [12,13]. Alcohol intake increases the expression of unfolded protein response (UPR) genes such as binding immunoglobulin protein (Bip), glucose regulating protein 94 (Grp94), and CCAAT-enhancer-binding protein homologous protein (Chop) in rodent ALD models [14]. Moreover, since long-chain saturated FAs reduce ER Ca²⁺ stores [15], hepatic steatosis may contribute to decreased ER Ca²⁺ stores, and the reduction of Ca²⁺ concentrations in the ER increases UPR and ER stress because most ER chaperones are Ca²⁺-dependent [16]. In addition, ER stress contributes to disease progression by activating nuclear factor-κB and c-Jun N-terminal kinase (JNK) pathways and by increasing CHOP expression, which plays a key role in hepatocyte apoptosis but not in hepatic steatosis [17].

Serotonin (5-hydroxytryptamine [5-HT]) is a monoamine neurotransmitter that has various functions in central and peripheral tissues. It is synthesized from the essential amino acid tryptophan, and its production is primarily determined by the activity of the rate-limiting enzyme tryptophan hydroxylase (TPH) [18]. There are two isoforms of TPH that are expressed in mutually exclusive tissue patterns, TPH1 in peripheral tissues and TPH2 in neurons of the central and enteric nervous system [19,20]. As 5-HT cannot cross the blood brain barrier, central and peripheral 5-HT systems are functionally separated [21]. The majority of the peripheral 5-HT is synthesized by enterochromaffin cells in the gut [22]. 5-HT exerts its biological action primarily by binding to 5-HT receptors (HTRs), which are membrane bound G-protein coupled receptors (GPCRs) [23-30].

Peripheral 5-HT is known to regulate various biological processes in the liver, such as gluconeogenesis, lipogenesis, inflammation, fibrosis, and liver regeneration [25,30-33]. We have shown that gut-derived 5-HT (gut-derived serotonin [GDS]) increases hepatic lipid accumulation by activating HTR2A [31]. Activation of hepatic 5-HT signaling through HTR2A increases the expression of sterol regulatory element binding transcription factor 1 (Srebf1) and thereby promotes lipogenesis, and increased hepatic lipogenesis and Srebf1 expression are also key features of alcoholic steatosis. However, the direct role of 5-HT in the development and progression of ALD has not been elucidated. Here, we examine the functional role of 5-HT in the development of alcoholic steatohepatitis. Blood 5-HT levels were increased in patients with ALD. Ethanol (EtOH) intake induced the up-regulation of Tph1 expression in the gut and Htr2a expression in the liver. Genetic inhibition of both GDS synthesis and hepatic HTR2A signaling attenuated hepatic steatosis, liver injury, and ER stress in various EtOH diet-fed mouse models. Therefore, GDS induced lipogenesis and ER stress in the liver via hepatic HTR2A in EtOH diet-fed mice.

METHODS

Animals and diets

To generate gut-specific Tph1 knockout (Tph1 GKO) mice, Tph1 flox/flox (Mouse Genome Informatics [MGI]: 6271993) mice were crossed with Villin-Cre (MGI: 2448639) mice [34]. To generate liver-specific Htr2a knockout (Htr2a LKO) and liver-specific Htr2b knockout (Htr2b LKO) mice, Albumin-Cre (MGI: 2176228) mice were crossed with Htr2a flox/flox (MGI: 4946779) mice and Htr2b flox/flox (MGI: 3837400) mice [31]. C57BL/6J mice were purchased from the Charles River Japan (Yokohama, Japan). Mice were housed in climate-controlled, specific pathogen-free barrier facilities under a 12-hour lightdark cycle. For chronic EtOH diet-fed mice, mice were fed Lieber-DeCarli EtOH diet (Dyets Inc., Bethlehem, PA, USA) containing 5% EtOH for 4 weeks and sacrificed ad libitum. For chronic plus binge EtOH diet-fed mice, mice were fed Lieber-DeCarli EtOH diet (Dyets Inc.) containing 5% EtOH for 10 days and the mice were finally administered EtOH (5 g/kg) by oral gavage and sacrificed after 9 hours fasting. Control mice were fed isocaloric Lieber-DeCarli control diet (Dyets Inc.) containing dextrin-maltose (MP Biomedicals, Solon, OH, USA) and 9 g/kg dextrin-maltose was administered by oral gavage instead of EtOH, as previously reported [35]. For acute EtOH binge drinking mice, mice were fed EtOH (5 g/kg) by oral gavage for 3 consecutive days and sacrificed after 9 hours fasting. All animal experiments were complied with relevant ethical regulations. Experimental protocols for this study were approved by the Institutional Animal Care and Use Committee at the Korea Advanced Institute of Science and Technology.

Quantification of human blood 5-HT

All experiments using human participant blood samples were complied with relevant ethical regulations. After previous approval by the Institutional Review Board (IRB) of Severance Hospital (4-2015-0184), and written informed consent by all subjects, 22 living donors for liver transplantation and 44 alcoholic liver disease patients were included in this study. This IRB project is titled “Collection and Management of remnant Samples for Future Biomedical Research of Liver Diseases.” The samples used in this study were leftover samples obtained from patients undergoing routine medical examinations. These patients voluntarily consented to donate their leftover samples for liver disease research. To minimize the potential impact of recent alcohol consumption on blood serotonin levels, only blood samples were used where the patients reported abstaining from alcohol for at least 1 week prior to collection. Laboratory parameters of study participants are given in Supplementary Table 1. Blood samples for 5-HT measurements were drawn from peripheral veins. 5-HT levels were measured in human platelet-poor plasma using ClinRep high performance liquid chromatography kit (Recipe, Munich, Germany) at GreenCross LabCell (Yongin, Korea).

Mouse blood profile analysis

Blood samples were collected by retro-orbital bleeding into serum separation tubes (BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 30 minutes, followed by centrifugation at 1,000 ×g for 15 minutess at 4°C. Enzymatic colorimetric assay kits for total cholesterol (Roche, Basel, Switzerland), triglyceride (TG) (Roche), aspartate aminotransferase (AST) (Roche), and alanine aminotransferase (ALT) (Roche) were used to determine serum levels on a Cobas 8000 modular analyzer (Roche), and serum 5-HT levels were measured by lipid chromatography-tandem mass spectrometry (5500 Qtrap, AB SCIEX, Framingham, MA, USA) at GreenCross LabCell.

Cell culture and treatment

Alpha mouse liver 12 (AML-12) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM): F-12 medium supplemented with 10% fetal bovine serum, 10 μg/mL insulin, 5.5 μg/mL transferrin, 5 ng/mL selenium, 40 ng/mL dexamethasone and antibiotics in humidified atmosphere of 5% CO₂ at 37°C. To test direct effect of 5-HT in EtOH-induced ER stress, AML-12 cells were treated with 100 mM EtOH, 100 nM 5-HT and 10 μM ketanserin in DMEM: F-12 medium for 24 hours. All of reagents in the cell experiments were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Quantitative real time-polymerase chain reaction analysis

Total RNA extractions from frozen liver tissues and sectioned duodenum, jejunum, ileum, and colon of gut were performed using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Complementary DNA was synthesized using 1 μg of total RNA with high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative real time-polymerase chain reaction (PCR) was performed with Fast SYBR Green Master Mix (Applied Biosystems) and a Viia 7 Real-time PCR System (Applied Biosystems) according to the manufacturer’s instructions. Gene expression was relatively quantified based on the ∆∆Ct (threshold cycle) method with the 36B4 gene as a reference gene. The sequences of primers are given in Supplementary Table 2.

Histological analysis

Mouse liver, inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), and gut (duodenum, jejunum, ileum, and colon) were harvested and fixed in 10% neutral buffered formalin solution (Sigma-Aldrich) and embedded in paraffin. The 5 μm-thick tissues sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E). For gut with immunohistochemistry staining, gut sections were deparaffinized, rehydrated and antigen retrieval with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) was applied to the sections. After blocking endogenous peroxidase by Bloxall (#SP-6000, Vector, Newark, VA, USA), sections are incubated with 4% donkey serum in phosphate-buffered saline (PBS). Next, anti-5-HT (diluted 1:200, #20080, Immunostar, Hudson, WI, USA) is adjusted to the sections overnight. The sections were then washed with PBS and incubated with anti-rabbit immunoglobulin G (IgG) antibody (diluted 1:200, #PK-4007, Vector), avidin-biotin complex (ABC) mixture (#PK-4007, Vector), and 3,3´-diaminobenzidine (DAB) (#SK-4100, Vector) in sequence.

Quantification of hepatic triglyceride

Liver tissues were homogenized in 5% NP-40 using FastPrep-24 (MP Biomedicals). To solubilize TG, the homogenates were heated to 95°C for 5 minutes and cooled at 23°C, and repeated. TG Reagent (Sigma-Aldrich) or PBS was added and incubated at 37°C for 30 minutes to hydrolyze TG into glycerol. For the colorimetric assay of hydrolyzed TG levels, samples were incubated with Free Glycerol Reagent (Sigma-Aldrich) at 37°C for 5 minutes. Differences in absorbance at 540 nm between hydrolyzed or non-hydrolyzed TG were quantified using a glycerol standard (Sigma-Aldrich). TG contents were normalized by the protein concentrations of homogenates, which were measured with a bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific, Waltham, MA, USA).

Western blot analysis

Whole-cell lysates were extracted by homogenizing liver tissues in radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific) containing protease inhibitor cocktail solution (GenDEPOT, Katy, TX, USA) and phosphatase inhibitor cocktail (GenDEPOT) using FastPrep-24 (MP Biomedicals). Supernatants were collected following a centrifugation at 15,000 ×g for 15 minutes at 4°C and protein concentrations were measured with a BCA Protein Assay Kit (Thermo Scientific). For membrane and nuclear fractionation, liver tissues were homogenized by fractionation buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES; pH 7.4], 10 mM KCl, 2 mM MgCl₂, 1 mM ethylenediaminetetraacetic acid [EDTA], 1 mM ethyleneglycol-bil-N,N´-tetraacetic acid [EGTA], 1 mM dithiothreitol [DTT], and protease inhibitor cocktail solution) as described previously [36]. After centrifugation at 720 ×g for 5 minutes at 4°C, membrane fractions were obtained from supernatant. And the pellet, containing nuclei, was resuspended in Tris-buffered saline (TBS) with 0.1% sodium dodecyl sulfate (SDS). After fractionation, protein concentrations were measured with a BCA Protein Assay Kit (Thermo Scientific). Next, the protein samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Sigma-Aldrich). After blocking in a 3% bovine serum albumin solution, the membranes were incubated with the following specific antibodies; anti-SREBP1c antibody (diluted 1:450, MABS 1987, Sigma-Aldrich), anti-calnexin (diluted 1:1,000, Cell Signaling #2679, Danvers, MA, USA), anti-LaminB1 (diluted 1:1,000, sc374015, Santa Cruz Biotechnology, Dallas, TX, USA), anti-HTR2A antibody (diluted 1:1,000, ab66049, Abcam, Cambridge, UK), anti-phospho-inositol-requiring enzyme 1α (IRE1α; S724) antibody (diluted 1:1,000, NB100-2323, Novus Biologicals, Centennial, CO, USA), anti-BiP antibody (diluted 1:1,000, Cell Signaling #3177), anti-CHOP antibody (diluted 1:1,000, Cell Signaling #2895), anti-phospho-S6 (diluted 1:1,000, Cell Signaling #2211), anti-S6 (diluted 1:1,000, Cell Signaling #2217), and anti-β-actin antibody (diluted 1:5,000, Cell Signaling #5125). The membranes were then washed with TBST and incubated with anti-rabbit IgG Horseradish peroxidase (HRP)-linked antibody (diluted 1:5,000, Cell Signaling #7074), or anti-mouse IgG HRP-linked antibody (diluted 1:5,000, Cell Signaling #7076). Proteins were visualized by enhanced chemiluminescence according to the manufacturer’s instruction (Sigma-Aldrich).

Statistical analysis

Data are represented as the mean±standard error of the mean. To compare values obtained from two groups, Student’s t-test was performed. A value of P<0.05, P<0.01, or P<0.001 was considered statistically significant.

RESULTS

GDS production is induced by alcohol intake

In order to investigate the possible relationship between 5-HT and ALD in humans, we measured 5-HT concentrations in platelet-poor plasma of ALD patients, the normal range of which is the ng/mL [37]. Plasma 5-HT concentrations were increased in ALD patients compared to healthy control subjects (Fig. 1A). This data motivated us to explore the possible role of 5-HT in alcohol-induced hepatic steatosis. We treated mice with three different EtOH feeding protocols, including acute (5 g/kg of EtOH by oral gavage for 3 consecutive days), chronic plus binge (5% EtOH diet for 10 days with 5 g/kg of EtOH single binge on the last day), and chronic (5% EtOH diet for 4 weeks) EtOH diet [35]. The three EtOH feeding mouse models showed slightly different ALD phenotypes assessed by liver histology, hepatic TG, blood AST, ALT, and lipid profiles (Supplementary Fig. 1). In acute EtOH feeding mice, hepatic steatosis was not observed, but blood levels of AST and ALT were increased. In contrast, in chronic EtOH feeding mice, robust hepatic steatosis was observed, but blood levels of AST and ALT were not increased. In chronic plus binge EtOH feeding mice, hepatic steatosis was observed and AST, ALT, TG, and cholesterol levels were increased.

To determine if EtOH feeding can induce GDS production, we measured serum 5-HT levels, the normal range of which is the μg/mL due to platelet activation [37], and Tph1 expression in the gut of mice fed with these three different EtOH diets. As human ALD patients showed increased blood 5-HT level, serum 5-HT levels were increased in the three EtOH feeding mouse models (Fig. 1B). Also, Tph1 expression was increased in the gut of EtOH diet-fed mice (Fig. 1C). These data suggest that EtOH diet feeding induces GDS production and GDS may contribute to the development of ALD.

GDS induces alcoholic steatosis by activating hepatic HTR2A

To confirm if GDS can mediate alcohol-induced hepatic steatosis, we generated gut-specific Tph1 knockout (Villin-Cre^+/−; Tph1^flox/flox, herein named Tph1 GKO) mice and confirmed the Tph1 GKO mice with expression of Tph1 and synthesis of 5-HT (Supplementary Fig. 2A and B). And then we fed them a chronic EtOH diet. Histological analysis exhibited that hepatic steatosis was ameliorated and hepatic TG content was reduced in Tph1 GKO mice (Fig. 2A and B) without significant change in iWAT and eWAT (Supplementary Fig. 2C). Profiling of mRNA expression of metabolic markers showed that the expressions of genes involved in FA uptake (cluster of differentiation 36 [Cd36], fatty acid binding protein 1 [Fabp1], fatty acid transport protein 2 [Fatp2], and Fatp5), FA oxidation (carnitine palmitoyltransferase 1A [Cpt1a], Ppara, and Pparg coactivator 1 alpha [Pgc1a]), and very low-density lipoprotein (VLDL) secretion (apolipoprotein B [Apob] and microsomal triglyceride transfer protein [Mttp]) pathways were not altered in the liver of Tph1 GKO mice fed a chronic EtOH diet (Supplementary Fig. 2D-F). Intriguingly, although mRNA expression of Srebp1c was not changed in Tph1 GKO mice, protein levels of SREBP1c were markedly decreased in the liver of Tph1 GKO mice (Fig. 2C and D). Moreover, expression levels of its target genes including adenosine triphosphate citrate lyase (Acly), acetyl-CoA carboxylase 1 (Acaca), fatty acid synthase (Fasn), and stearoyl-CoA desaturase 1 (Scd1) were also down-regulated in the liver of Tph1 GKO mice (Fig. 2E). Taken together, inhibiting GDS synthesis alleviates alcoholic steatosis by downregulating lipogenic pathways in the liver.

The biological action of 5-HT is mainly mediated through its binding to HTRs. To identify the HTR that mediates the lipogenic effect of GDS in the liver, we checked the expression profile of hepatic HTR genes. Intriguingly, among the detectable HTR genes in the liver, Htr2a expression was significantly increased by chronic EtOH diet feeding (Fig. 3A). To determine if HTR2A mediates the lipogenic action of GDS in the liver, we generated Htr2a LKO (albumin-Cre^+/−; Htr2a^flox/flox, herein named Htr2a LKO) mice (Supplementary Fig. 3A and B) and induced alcoholic steatosis by feeding a chronic EtOH diet. Hepatic steatosis was robustly reduced in Htr2a LKO mice and hepatic TG concentration was also decreased accordingly (Fig. 3B and C). Furthermore, similar to Tph1 GKO mice, SREBP1c protein level and the expression of its target genes were decreased whereas expression of genes involved in FA uptake, FA oxidation, and VLDL secretion pathways were not affected (Fig. 3D-F and Supplementary Fig. 3C-E). These data indicate that Htr2a LKO mice are protected against alcohol-induced hepatic steatosis and are a phenocopy of Tph1 GKO mice with regard to alcoholic steatosis.

GDS aggravates alcohol-induced ER stress through HTR2A in the liver

ER stress is the major contributor to ALD progression by promoting hepatocyte apoptosis and inflammation [14,38,39]. In order to investigate if GDS signaling through HTR2A regulates hepatic ER stress in chronic EtOH diet-fed mice, ER stress markers were examined in the livers of both Tph1 GKO and Htr2a LKO mice. Interestingly, ER stress markers including phospho-IRE1α, CHOP, and BiP were markedly decreased in the livers of both EtOH diet-fed Tph1 GKO and Htr2a LKO mice (Fig. 4A and B). In addition, mRNA expression of ER stress marker genes including Chop, Bip, X-box binding protein 1-spliced (Xbp1s), Grp94, and activating transcription factor 4 (Atf4) was also decreased in Tph1 GKO and Htr2a LKO mice (Fig. 4C and D).

To determine if 5-HT can directly regulate EtOH-induced ER stress in hepatocytes, AML-12 hepatocytes were treated with EtOH, 5-HT, and/or HTR2A antagonist, ketanserin. Both 5-HT and EtOH increased the levels of phospho-IRE1α, CHOP, and BiP, which were diminished by ketanserin (Fig. 4E). However, 5-HT and EtOH did not synergistically increase ER stress (Fig. 4E) and ketanserin did not diminish EtOH-induced ER stress (data not shown), suggesting 5-HT not only has a secondary effect on ER stress by increasing hepatic steatosis but also could directly induce ER stress to activate IRE1α pathway through HTR2A signaling in hepatocytes. Additionally, 5-HT-induced S6 phosphorylation, a molecular marker of mammalian target of rapamycin complex 1 (mTORC1) signaling, was diminished by ketanserin (Supplementary Fig. 3F), suggesting that blocking HTR2A signaling may affect ER stress by down-regulating protein synthesis. Taken together, inhibition of HTR2A signaling could ameliorate hepatic ER stress in chronic EtOH diet-fed mice.

GDS regulates EtOH-induced ER stress and inflammation in the liver through HTR2A

Among the three mouse models used in this study, inflammation was barely observed in the liver of mice fed with chronic EtOH diet despite the increased lipogenesis and ER stress. In contrast, we could clearly observe inflammation in the liver of mice fed with chronic plus binge EtOH diet. To determine if GDS is involved in the development of alcohol-induced liver damage as well as steatosis, the chronic plus binge EtOH diet model was introduced in Tph1 GKO mice. In chronic plus binge EtOH diet-fed Tph1 GKO mice, hepatic steatosis was improved and hepatic TG concentration was reduced (Fig. 5A and B). ER stress was also decreased in Tph1 GKO mice, similar to the mice fed with chronic EtOH diet (Fig. 5C and D). Intriguingly, gene expressions of hepatic cytokines (tumor necrosis factor-α [Tnfa], interleukin 6 [Il-6], and Il-1b), chemokines (monocyte chemoattractant protein 1 [Mcp-1], macrophage inflammatory protein 1α [Mip-1a], Mip-1b, and Mip-2), neutrophil marker (lymphocyte antigen 6 family member G [Ly6g]), and monocyte/macrophage marker (Cd68) which have been known to be induced by chronic plus binge EtOH feeding [40] were decreased in Tph1 GKO mice (Fig. 5E). Accordingly, blood AST and ALT levels were decreased in Tph1 GKO mice, suggesting that inhibiting GDS synthesis reduces alcohol-induced liver damage (Fig. 5F).

To further investigate if GDS accelerates ALD progression through HTR2A in the liver, we also introduced chronic plus binge EtOH diet to Htr2a LKO mice and confirmed that Htr2a LKO phenocopied Tph1 GKO mice. Hepatic steatosis, ER stress, inflammation, and liver damage induced by chronic plus binge EtOH diet were improved in Htr2a LKO mice (Fig. 6A-F). However, Htr2b LKO mice didn’t show an improved phenotype in hepatic steatosis, ER stress, inflammation, and liver damage which were induced by chronic plus binge EtOH diet (Supplementary Fig. 4). These data indicate that HTR2A selectively mediates the role of GDS in the progression of ALD. Taken together, GDS signaling through HTR2A accelerates ALD progression by inducing steatosis and ER stress in the liver of mice fed with chronic plus binge EtOH diet.

To further investigate if HTR2A signaling is involved in acute EtOH-induced liver injury, we introduced acute EtOH diet in Htr2a LKO mice. Interestingly, blood levels of AST and ALT were decreased in Htr2a LKO mice, although hepatic steatosis was not changed in Htr2a LKO mice fed an acute EtOH diet unlike chronic or chronic plus binge EtOH diet-fed mice model (Supplementary Fig. 5A-C). Furthermore, acute EtOH diet-induced ER stress in the liver was abrogated in Htr2a LKO mice (Supplementary Fig. 5D and E). However, phenotypic differences regarding hepatic steatosis, ER stress, and liver damage were not observed in Htr2b LKO mice fed an acute EtOH diet (Supplementary Fig. 6). Collectively, these data suggest that inhibiting HTR2A signaling protects the liver from acute EtOH-induced injury by reducing ER stress.

DISCUSSION

Peripheral serotonergic signaling is known to be involved in multiple physiological processes in the liver [25,30-33]. However, it is largely unknown whether peripheral 5-HT directly contributes to the development and progression of ALD. Here, we demonstrate that EtOH diet feeding up-regulates gut Tph1 expression and that inhibition of GDS synthesis by Tph1 GKO alleviates alcoholic steatosis and hepatic ER stress. Moreover, Htr2a LKO mice phenocopied Tph1 GKO mice in terms of alcoholic steatosis and hepatic ER stress. In addition, inhibiting HTR2A signaling prevents disease progression by attenuating alcohol-induced liver injury and inflammation in chronic plus binge and acute EtOH diet feeding models. These findings led us to propose GDS as a direct regulator of ALD through a gut-liver endocrine axis.

In the present study, we examined the effects of inhibiting serotonergic signaling pathway through a gut-liver axis in the three EtOH diet-fed mouse models. Both Tph1 GKO and Htr2a LKO mice ameliorated alcoholic steatosis by down-regulating lipogenic pathways (SREBP1c and the expression of its target genes) without affecting other pathways that are responsible for hepatic lipid metabolism, such as FA uptake, FA oxidation, and VLDL secretion. These results were similar to our previous study that investigated the gut TPH1-liver HTR2A axis as a novel pathological pathway for metabolic dysfunction-associated steatotic liver disease [31]. It is necessary to further identify the detailed downstream molecular mechanisms of how hepatic HTR2A signaling regulates lipogenesis through SREBP1c and its downstream target genes.

Our results also highlight the functional potential of the gut TPH1-liver HTR2A signaling pathway in the progression of ALD. ER stress is known as a driver in promoting development of advanced stages of ALD [12]. Both Tph1 GKO and Htr2a LKO mice exhibited decreased ER stress in chronic, chronic plus binge, and acute binge EtOH feeding models. Although the intracellular mechanism by which HTR2A signaling directly induces ER stress is unclear, one possible explanation is that inhibiting HTR2A signaling may decrease the UPR by down-regulating protein synthesis (Supplementary Fig. 3F), which in turn ameliorates ER stress. Additionally, another potential mechanism may exist, as HTR2A is a Gq-coupled receptor that, when activated, releases ER Ca²⁺ into the cytosol via inositol triphosphate (IP₃) and activates protein kinase C through diacylglycerol. Since ER Ca²⁺ depletion is one of the factors inducing ER stress, inhibiting HTR2A signaling may alleviate ER stress by preventing ER Ca²⁺ depletion through both IP₃-mediated Ca²⁺ release and protein kinase C-dependent Ca²⁺ entry [41]. Although detailed mechanism remains to be elucidated, our data suggest that inhibition of enterohepatic signaling pathways mediated by GDS and HTR2A might protect against the progression of ALD [17,42,43]. It would be of interest to test whether inhibiting the gut TPH1-liver HTR2A axis can effectively stop the progression of alcoholic steatosis to the late stage ALD in the future.

Inhibiting peripheral serotonergic signaling pathways is emerging as a new therapeutic target in various diseases [44]. Since ALD has limited pharmacological treatment options until now, hepatic HTR2A antagonism may be a novel therapeutic strategy for ALD.

SUPPLEMENTARY MATERIALS

NOTES

CONFLICTS OF INTEREST

No potential conflict of interest relevant to this article was reported.

AUTHOR CONTRIBUTIONS

Conception or design: I.H., H.K.

Acquisition, analysis, or interpretation of data: all authors.

Drafting the work or revising: I.H., J.E.N., Y.A.M., J.Y.P., H.K.

Final approval of the manuscript: all authors.

FUNDING

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2022-00166199 to Inseon Hwang, NRF002475581G-0003101 to Won Gun Choi, 2021R1C1C1005109 and RS-2023-00222910 to Hyeongseok Kim, NRF-2020M3A9E4038695 to Hail Kim).

Acknowledgements

We thank Dr. Gerard Karsenty (Columbia University) for the Htr2b floxed mice. We also thank all members of iMOD (Integrated laboratory of Metabolism, Obesity, and Diabetes) for their helpful discussions.

Fig. 1.

Blood serotonin levels are increased in alcoholic liver disease. (A) Plasma 5-hydroxytryptamine (5-HT) concentrations of peripheral blood in healthy control subjects (n=22) and alcoholic liver disease patients (n=44). (B) Serum 5-HT concentrations in three ethanol (EtOH) diet-fed mice model (acute, chronic plus binge, and chronic); n=6, six for acute and chronic plus binge EtOH diet mice model, respectively; n=6, five for chronic EtOH diet groups. (C) Relative tryptophan hydroxylase 1 (Tph1) mRNA expression in the duodenum, jejunum, ileum, and colon from three EtOH diet-fed mice model (acute, chronic plus binge, and chronic) as assessed by quantitative real time-polymerase chain reaction; n=6, six for acute and chronic plus binge EtOH diet mice model, respectively; n=6, five for chronic EtOH diet groups. Data are expressed as the mean±standard error of the mean. ^aP<0.05, ^bP<0.01, ^cP<0.001 compared to the corresponding control, based on Student’s t-test between two groups.

Fig. 2.

Gut-derived serotonin induces alcoholic steatosis by activating hepatic lipogenic pathways. Wild-type (WT) and tryptophan hydroxylase 1 (Tph1) gut-specific knockout (GKO) mice were fed with Lieber-DeCarli liquid diet containing 5% ethanol (EtOH) for 4 weeks. (A) Representative liver histology by H&E staining from WT and Tph1 GKO mice. Scale bars, 100 μm. (B) Hepatic triglyceride levels of WT and Tph1 GKO mice; n=5, four for WT and Tph1 GKO mice, respectively. (C) Relative sterol regulatory element binding protein 1c (Srebp1c) mRNA expression in the liver; n=5, four for WT and Tph1 GKO mice, respectively. (D) Western blot analysis of SREBP1c (precursor SREBP1c detected from membrane fraction [pSREBP1c], nuclear SREBP1c detected from nuclear fraction [nSREBP1c]) in the liver from WT and Tph1 GKO mice. (E) Relative mRNA expression of genes involved in lipogenic pathways in the liver; n=5, four for WT and Tph1 GKO mice, respectively. Data are expressed as the mean±standard error of the mean. Acly, adenosine triphosphate citrate lyase; Acaca, acetyl-CoA carboxylase 1; Fasn, fatty acid synthase; Scd1, stearoyl-CoA desaturase 1. ^aP<0.05, ^bP<0.01 compared to the corresponding control, based on Student’s t-test between two groups.

Fig. 3.

5-Hydroxytryptamine (5-HT) receptor 2A (HTR2A) mediates the lipogenic action of gut-derived serotonin in the liver of chronic ethanol (EtOH) diet-fed mice. (A) Relative mRNA expression of indicated HTRs as assessed by quantitative real time-polymerase chain reaction in liver of control and EtOH diet-fed C57BL6/J mice; n=6, five for control and EtOH diet-fed groups, respectively. (B-F) Wild-type (WT) and Htr2a liver-specific knockout (LKO) mice were fed with Lieber-DeCarli liquid diet containing 5% EtOH for 4 weeks. (B) Representative liver histology by H&E staining from WT and Htr2a LKO mice. Scale bars, 100 μm. (C) Hepatic triglyceride levels of WT and Htr2a LKO mice; n=6 per group. (D) Relative sterol regulatory element binding protein 1c (Srebp1c) mRNA expression in the liver; n=6 per group. (E) Western blot analysis of SREBP1c (precursor SREBP1c detected from membrane fraction [pSREBP1c], nuclear SREBP1c detected from nuclear fraction [nSREBP1c]) in the liver from WT and Htr2a LKO mice. (F) Relative mRNA expression of genes involved in lipogenic pathways in the liver; n=6 per group. Data are expressed as the mean±standard error of the mean. Acly, adenosine triphosphate citrate lyase; Acaca, acetyl-CoA carboxylase 1; Fasn, fatty acid synthase; Scd1, stearoyl-CoA desaturase 1. ^aP<0.05, ^bP<0.01 compared to the corresponding control, based on Student’s t-test between two groups.

Fig. 4.

Gut-derived serotonin aggravates ethanol (EtOH)-induced endoplasmic reticulum (ER) stress via signaling through 5-hydroxytryptamine (5-HT) receptor 2A (HTR2A). (A-D) Tryptophan hydroxylase 1 (Tph1) gut-specific knockout (GKO) and the littermate control (A, C) and Htr2a liver-specific knockout (LKO) and littermates control mice (B, D) were fed with Lieber-DeCarli liquid diet containing 5% EtOH for 4 weeks. (A) Western blot analysis of ER stress marker protein (phospho-inositol-requiring enzyme 1α [IRE1α], CCAAT-enhancer-binding protein homologous protein [CHOP], and binding immunoglobulin protein [BiP]) in the liver from wild-type (WT) and Tph1 GKO mice. (B) Western blot analysis of ER stress marker protein (phospho-IRE1α, CHOP, and BiP) in the liver from WT and Htr2a LKO mice. (C) Relative mRNA expression of ER stress marker genes in the liver; n=5, four for WT and Tph1 GKO mice, respectively. (D) Relative mRNA expression of ER stress marker genes in the liver; n=6 per group. (E) Western blot analysis of ER stress marker protein (phospho-IRE1α, CHOP, and BiP) in alpha mouse liver 12 (AML-12) cells treated with 100 mM EtOH, 100 nM 5-HT, and 10 μM ketanserin for 24 hours. Data are expressed as the mean±standard error of the mean. Xbp1u, X-box binding protein 1-unspliced; Xbp1u, X-box binding protein 1-unspliced; Xbp1s, X-box binding protein 1-spliced; Grp94, glucose regulating protein 94; Atf4, activating transcription factor 4. ^aP<0.05 compared to the corresponding control, based on Student’s t-test between two groups.

Fig. 5.

Inhibiting gut-derived serotonin synthesis ameliorates hepatic endoplasmic reticulum (ER) stress and inflammation in chronic plus binge ethanol (EtOH) diet-fed mice. Wild-type (WT) and tryptophan hydroxylase 1 (Tph1) gut-specific knockout (GKO) mice were fed with Lieber-DeCarli liquid diet containing 5% EtOH for 10 days and the mice were administered EtOH (5 g/kg) by oral gavage. (A) Representative liver histology by H&E staining from WT and Tph1 GKO mice. Scale bars, 100 μm. (B) Hepatic triglyceride levels of WT and Tph1 GKO mice; n=6 per group. (C) Western blot analysis of ER stress marker protein (phosphoinositol-requiring enzyme 1α [IRE1α], CCAAT-enhancer-binding protein homologous protein [CHOP], and binding immunoglobulin protein [BiP]) in the liver from WT and Tph1 GKO mice. (D) Relative mRNA expression of ER stress marker genes in the liver; n=6 per group. (E) Relative mRNA expression of genes involved in inflammation in the liver; n=6 per group. (F) Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels of WT and Tph1 GKO mice; n=6 per group. Data are expressed as the mean±standard error of the mean. Xbp1u, X-box binding protein 1-unspliced; Xbp1s, X-box binding protein 1-spliced; Grp94, glucose regulating protein 94; Atf4, activating transcription factor 4; Tnfa, tumor necrosis factor-α; IL, interleukin; Mcp, monocyte chemoattractant protein; Mip-1α, macrophage inflammatory protein 1α; Ly6g, lymphocyte antigen 6 family member G. ^aP<0.05, ^bP<0.01, ^cP<0.001 compared to the corresponding control, based on Student’s t-test between two groups.

Fig. 6.

5-Hydroxytryptamine (5-HT) receptor 2A (Htr2a) liver-specific knockout (LKO) mice exhibit improved hepatic endoplasmic reticulum (ER) stress and inflammation in chronic plus binge ethanol (EtOH) diet-fed mice. Wild-type (WT) and Htr2a LKO mice were fed with Lieber-DeCarli liquid diet containing 5% EtOH for 10 days and the mice were administered EtOH (5 g/kg) by oral gavage. (A) Representative liver histology by H&E staining from WT and Htr2a LKO mice. Scale bars, 100 μm. (B) Hepatic triglyceride levels of WT and Htr2a LKO mice; n=6 per group. (C) Western blot analysis of ER stress marker protein (phosphoinositol-requiring enzyme 1α [IRE1α], CCAAT-enhancer-binding protein homologous protein [CHOP], and binding immunoglobulin protein [BiP]) in the liver from WT and Htr2a LKO mice. (D) Relative mRNA expression of ER stress marker genes in the liver; n=6 per group. (E) Relative mRNA expression of genes involved in inflammation in the liver; n=6 per group. (F) Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels of WT and Htr2a LKO mice; n=6 per group. Data are expressed as the mean±standard error of the mean. Xbp1u, X-box binding protein 1-unspliced; Xbp1s, X-box binding protein 1-spliced; Grp94, glucose regulating protein 94; Atf4, activating transcription factor 4; Tnfa, tumor necrosis factor-α; IL, interleukin; Mcp, monocyte chemoattractant protein; Mip-1α, macrophage inflammatory protein 1α; Ly6g, lymphocyte antigen 6 family member G. ^aP<0.05, ^bP<0.01, ^cP<0.001 compared to the corresponding control, based on Student’s t-test between two groups.

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Hwang I, Nam JE, Choi W, Choi WG, Lee E, Kim H, Moon YA, Park JY, Kim H. Serotonin Regulates Lipogenesis and Endoplasmic Reticulum Stress in Alcoholic Liver Disease. Diabetes Metab J. 2025 Feb 5. doi: 10.4093/dmj.2024.0215. Epub ahead of print.

Received: Apr 26, 2024; Accepted: Sep 21, 2024

DOI: https://doi.org/10.4093/dmj.2024.0215.

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