Warning: fopen(/home/virtual/enm-kes/journal/upload/ip_log/ip_log_2024-03.txt): failed to open stream: Permission denied in /home/virtual/lib/view_data.php on line 88 Warning: fwrite() expects parameter 1 to be resource, boolean given in /home/virtual/lib/view_data.php on line 89 Serotonin Regulates De Novo Lipogenesis in Adipose Tissues through Serotonin Receptor 2A
Skip Navigation
Skip to contents

Endocrinol Metab : Endocrinology and Metabolism

clarivate
OPEN ACCESS
SEARCH
Search

Articles

Page Path
HOME > Endocrinol Metab > Volume 35(2); 2020 > Article
Original Article
Serotonin Regulates De Novo Lipogenesis in Adipose Tissues through Serotonin Receptor 2A
Ko Eun Shong1*orcid, Chang-Myung Oh2*orcid, Jun Namkung3, Sangkyu Park4orcid, Hail Kim1,5orcid
Endocrinology and Metabolism 2020;35(2):470-479.
DOI: https://doi.org/10.3803/EnM.2020.35.2.470
Published online: June 30, 2020

1Biomedical Science and Engineering Interdisciplinary Program, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea

2Department of Biomedical Science and Engineering, Gwangju Institute of Science and Technology, Gwangju, Korea

3Department of Biochemistry, Yonsei University Wonju College of Medicine, Wonju, Korea

4Department of Precision Medicine, Yonsei University Wonju College of Medicine, Wonju, Korea

5Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea

Corresponding authors: Sangkyu Park. Department of Precision Medicine, Yonsei University Wonju College of Medicine, 20 Ilsan-ro, Wonju 26426, Korea, Tel: +82-33-741-0154, Fax: +82-33-742-5034, E-mail: skpark00@yonsei.ac.kr.
Hail Kim. Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Korea, Tel: +82-42-350-4243, Fax: +82-42-350-4287, E-mail: hailkim@kaist.edu
* These authors contributed equally to this work.
• Received: November 27, 2019   • Revised: March 4, 2020   • Accepted: April 7, 2020

Copyright © 2020 Korean Endocrine Society

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

  • 8,300 Views
  • 259 Download
  • 21 Web of Science
  • 20 Crossref
  • 20 Scopus
This article has been corrected. See "Erratum: Correction of Figure. Serotonin Regulates De Novo Lipogenesis in Adipose Tissues through Serotonin Receptor 2A" in Volume 35 on page 672.
  • Background
    Obesity is defined as excessive fat mass and is a major cause of many chronic diseases such as diabetes, cardiovascular disease, and cancer. Increasing energy expenditure and regulating adipose tissue metabolism are important targets for the treatment of obesity. Serotonin (5-hydroxytryptophan [5-HT]) is a monoamine metabolite of the essential amino acid tryptophan. Here, we demonstrated that 5-HT in mature adipocytes regulated energy expenditure and lipid metabolism.
  • Methods
    Tryptophan hydroxylase 1 (TPH1) is the rate-limiting enzyme during 5-HT synthesis in non-neural peripheral tissues. We generated adipose tissue-specific Tph1 knockout (Tph1 FKO) mice and adipose tissue-specific serotonin receptor 2A KO (Htr2a FKO) mice and analyzed their phenotypes during high-fat diet (HFD) induced obesity.
  • Results
    Tph1 FKO mice fed HFD exhibited reduced lipid accumulation, increased thermogenesis, and resistance to obesity. In addition, Htr2a FKO mice fed HFD showed reduced lipid accumulation in white adipose tissue and resistance to obesity.
  • Conclusion
    These data suggest that the inhibition of serotonin signaling might be an effective strategy in obesity.
Obesity is a chronic disease resulting from an imbalance between energy ingested and energy expended and has been recognized as a major risk factor for type 2 diabetes mellitus, atherosclerosis, metabolic syndrome, cardiovascular diseases, and various types of cancer [1]. In addition, obesity is associated with higher all-cause mortality [2]. Hence, intensive efforts have been taken to uncover the basic mechanisms of obesity and to discover effective therapeutic targets for the treatment of obesity. However, to date, safe and efficacious therapeutics for obesity remain scarce.
Serotonin (5-hydroxytryptophan [5-HT]) is a monoamine metabolite of the essential amino acid tryptophan. Tryptophan is hydroxylated to 5-HT by tryptophan hydroxylase (TPH). TPH represents the rate-limiting step in the pathway, with the enzyme occurring in two isoforms. TPH1 is expressed in non-neural tissues and TPH2 is localized in the neural tissues [3]. In the next step, 5-HT is catalyzed by L-amino acid decarboxylase to produce serotonin. Since serotonin poorly crosses the blood-brain barrier, central, and peripheral serotonin represent separate signaling systems. Peripheral serotonin is produced in enterochromaffin cells in the gastrointestinal tract, pancreatic β-cells, and adipocytes [4]. Serotonin synthesized in the periphery exerts a wide variety of physiological roles by interacting with serotonin receptors in the control of vasoconstriction, intestinal motility, and glucose and lipid metabolism.
Several studies have reported the possible relationships between serotonin and obesity. In humans, the blood serotonin level and Tph1 expression in the duodenum has been positively correlated with body mass index [5]. The genetic analysis reported that Tph1 and 5-HT(2A) receptor, 5-HT(2B) receptor polymorphisms are associated with the development of obesity [6,7]. Furthermore, serotonin synthesis and serotonin levels were reportedly elevated in the liver and adipose tissue of glucocorticoid-induced insulin-resistant rats [8]. These reports suggest that obesity is associated with increased serotonin concentrations in both serum and adipose tissue.
Recent studies have reported that the inhibition of peripheral serotonin results in resistance to obesity and reduced lipid accumulation in peripheral tissues [911]. This suggests a link between peripheral serotonin and energy balance. However, the metabolic phenotypes of adipose tissue-specific Tph1 and its receptor knockout mice have not been reported. Therefore, in this study, we generated adipose tissue specific Tph1 knockout (Tph1 FKO) mice and adipose tissue-specific serotonin receptor 2A knockout (Htr2a FKO) mice and analyzed their metabolic phenotypes. We observed that the deletion of Tph1 in adipose tissue resulted in a resistance to high-fat diet (HFD) induced obesity, increased thermogenesis, and reduced lipid accumulation in the adipose tissue. Furthermore, Htr2a FKO mice demonstrated resistance to obesity, with reduced lipid accumulation in the adipose tissue. Our studies contribute to the information regarding serotonin function in adipocyte metabolism and indicate that targeting Tph1 and Htr2a in the adipose tissue could be a potential treatment strategy to manage obesity and related metabolic diseases.
Animal experiments
The generation of Tph1-floxed mice, Htr2a-floxed mice, and adiponectin (Adipoq)-Cre mice have previously been reported [1113]. C57BL/6 J mice were purchased from Charles River Japan (Yokohama, Japan). Tph1 FKO mice were crossed with uncoupled protein 1 (Ucp1)-luciferase transgenic mice. The mice were housed in climate-controlled, specific pathogen-free barrier facilities, under a 12-hour light-dark cycle, with chow and water provided ad libitum. Male mice (aged 8 weeks) were fed either a standard chow diet (SCD; 12% fat calories, Research Diets D10001) or an HFD (60% fat calories, Research Diets D12492). In case of the transgenic mice, we compared the data between KO mice and their wild type (WT) littermates. The experimental protocols for this study were approved by the Institutional Animal Care and Use Committee (LML 15–535) at the Korea Advanced Institute of Science and Technology. These experiments were performed unblinded.
Metabolic analysis
The mice were housed individually in a 12-chamber, open-circuit Oxymax/CLAMS (Columbus Instruments Comprehensive Lab Animal Monitoring system) system and the metabolic rate was measured as previously described [11]. After one day of acclimation, each mouse was assessed for 72 hours in the fed state to assess the metabolic rates. The respiratory exchange ratio (RER=VCO2/VO2) and heat production (HP=3.185×VO2+ 1.232×VCO2) were calculated. Fat mass and lean body mass were measured using Minispec time-domain nuclear magnetic resonance analyzer (Bruker Optics, Billerica, MA, USA).
Glucose tolerance test and insulin tolerance test
To perform the glucose tolerance test, the mice were fasted overnight. Then, 2 g/kg D-glucose in phosphate-buffered saline (PBS) was intraperitoneally injected into the mice. For the insulin tolerance test (ITT), 0.75 U/kg human insulin (Humulin R, Lilly, Indianapolis, IN, USA) was intraperitoneally injected into 6 hours fasted mice. Blood samples were collected from the tail vein and glucose concentrations were measured using a Gluco DR plus glucometer (Allmedicus, Anyang, Korea) as previously described [10].
Quantitative real-time polymerase chain reaction analysis
Total RNA extractions from the mice adipose tissues were performed using TRIzol as previously described [14]. After TURBO DNase (Invitrogen, Waltham, MA, USA) treatment, 2 μg of total RNA was used to generate complementary DNA with Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. Quantitative real-time polymerase chain reaction (RT-PCR) was performed with Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) and a ViiA 7 Real-time PCR system (Applied Biosystems). Gene expressions were analyzed using the delta-delta Ct method as previously described [11]. The primer sequences are presented in Table 1.
Cell culture
Murine 3T3-L1 cells (American Type Culture Collection) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum and 100 μg/mL penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37°C. Cells were differentiated and analyzed as previously described [10]. Immunostaining was performed on differentiated day 8.
Histological analysis
Inguinal, epididymal, and interscapular adipose tissues were harvested, fixed in 4% (w/v) paraformaldehyde in PBS and embedded in paraffin. Next, 5-μm-thick tissue sections were deparaffinized, rehydrated, and used for hematoxylin and eosin staining, immunohistochemistry and immunofluorescence [10].
In vivo luciferase assay
We obtained Ucp1-luciferase mice from the laboratory of Dr. Shingo Kajimura (University of California, San Francisco) [15]. Briefly, 150 mg/kg of D-Luciferin potassium salt (GoldBio, St. Louis, MO, USA) was injected into WT and Tph1 FKO with Ucp1-luciferase fed SCD or HFD. Fifteen minutes post-injection, we detected luciferase activity using IVIS Lumina S5, using the Living Image software (PerkinElmer, Waltham, MA, USA) to setup (autoexposure), analyze, and organize data.
Validation of gene expression using nanostring
The correlations of BXD white adipose tissue (WAT; subcutaneous fat) Tph1 mRNA and phenotypes were analyzed by using database (http://www.genenetwork.org/). The comparison of Tph1 mRNA levels and total 2000 phenotypes correlated with Tph1 in the WAT expressed by the volcano plot. The X axis represents Spearman’s Rho, with positive values representing positive correlations and negative values representing negative correlations, and the y-axis representing inversed P values. The gray horizontal solid line on the y-axis are statistically significant with a P value of less than 0.5, while the vertical solid line on the x-axis has a blue or red color with only Rho values greater than 0.5 (right) or less than −0.5 (left). Expressed in color can be viewed as having a positive or negative correlation. Marked in red is the metabolic phenotype. The result was obtained without bias using the whole without excluding one value from the entire BXD data.
Statistics
All values are expressed as the mean±standard error of the mean. Statistical significance was determined by Student’s t test or two-way analysis of variance (ANOVA) with Bonferroni’s post hoc test. Two-way ANOVA was performed in body weight (Figs. 1B, 4B), glucose tolerance test (Fig. 1F, Supplemental Fig. S3B), ITT (Supplemental Figs. S2A, S3C), and energy expenditure assays (Fig. 2A, Supplemental Figs. S1E, S2B). P<0.05 was considered statistically significant.
Serotonin depletion in adipose tissue reduced weight gain and body fat mass in mice fed high-fat diet
To investigate whether serotonin is associated with body weight and composition, we analyzed RNA expression in WAT of the BXD family from GeneNetwork. Tph1 mRNA expression has shown a positive correlation with fat mass and glucose levels and a negative correlation with lean mass (Fig. 1A). We hypothesized that if serotonin increase by TPH1 in WAT has a positive correlation with increased fat mass, serotonin depletion in WAT may reduce fat mass and body weight gain. Previously, we generated inducible Tph1 knockout (adipocyte protein [aP2]-CreERT2+/−/Tph1 flox/flox) mice and reported that serotonin depletion at the adult stage prevents HFD induced obesity [10]. In this study, we investigated the role of peripheral serotonin in mature adipocytes in the basal status as well as overnutrition status. For this, we generated adipocyte-specific Tph1 knockout (Adipoq-Cre+/−/Tph1 flox/flox, Tph1 FKO) mice and analyzed the phenotypes of these mice at young and mature adult stages (Supplemental Fig. S1A, B). The Ap2 gene can be expressed in adipocyte progenitors and other tissues [16,17]. Notably, Adipoq gene expression is more specific in mature adipocytes than the Ap2 gene expression [18]. In young adults (8 weeks of age), Tph1 FKO mice did not show significant difference in body weight compared to WT mice (Fig. 1B). However, inn mature adults (20 weeks of age), Tph1 FKO mice demonstrated lower body weight and fat mass compared to the WT group when fed a HFD (Fig. 1B, Supplemental Fig. S1C, D). These results are consistent with the correlation analysis of the BXD reference group (Fig. 1A).
To explain the result, we measured the energy expenditure in these mice groups. As expected, Tph1 FKO mice with SCD feeding showed increased energy expenditure (increase VO2, VCO2, and HP) compared to WT mice fed SCD (Supplemental Fig. S1E). Under HFD conditions, after 12 weeks on HFD, these differences observed between Tph1 FKO and WT mice are greater than those observed under SCD conditions. Tph1 FKO fed the HFD showed reduced weight gain and lower body fat mass compared to WT mice (Fig. 1B–D). Notably, Tph1 FKO mice fed the HFD reported an improvement in glucose tolerance and insulin resistance when compared to WT mice fed the HFD (Fig. 1F, Supplemental Fig. S2A).
Serotonin depletion in adipocytes increased thermogenesis in brown adipose tissue
Tph1 FKO mice fed the HFD demonstrated increased energy expenditure (Fig. 2A, Supplemental Fig. S2B) compared to WT. To elucidate this phenotype, we generated in vivo reporter system for brown adipose tissue (BAT) activity using Ucp1-luciferase reporter mice [15]. Fig. 2B shows increased luciferase activity in the BAT area of Tph1 FKO and WT mice. Tph1 FKO mice fed the SCD and HFD showed significantly increased luciferase activity compared to WT mice fed the SCD and HFD (Fig. 2C). Then, we analyzed the histologic changes in BAT by aging. At 8 weeks of age, brown adipocytes in Tph1 FKO mice and WT mice appeared identical (Fig. 2D). This is consistent with their body weight at 8 weeks of age (Fig. 1B). As the mice grew older, from 8 to 20 weeks of age, the size of adipose cells and lipid droplets increased. However, Tph1 FKO BAT maintained a similar size of adipose cells at 20 weeks of age, even after 12 weeks of HFD (Fig. 2D).
Serotonin regulated beige adipocytes formation and lipid accumulation in WAT
Previously, we reported that serotonin depletion in the subcutaneous adipose tissue induced beige adipocyte formation in mice fed the HFD [10]. We observed similar changes after depleting serotonin in mature subcutaneous adipose tissue after HFD (Fig. 3A). Interestingly, serotonin depletion also increased beige adipocyte formation in the subcutaneous adipose tissue of mice fed the SCD (Fig. 3B). In visceral adipose tissue, Tph1 FKO mice fed HFD demonstrated a reduced adipose cell size and lipid droplets compared to WT mice fed on the HFD (Fig. 3C, D). Under the SCD conditions, Tph1 FKO mice did not indicate significant differences in the visceral adipose tissue compared to WT mice (Fig. 3C).
Serotonin regulated lipogenesis in adipose tissue through Htr2a
Previously, we identified the role of HTR2A in 3T3-L1 adipocytes [10]. In 3T3-L1 cell lines, HTR2A agonist increased lipogenesis gene expression in differentiated 3T3-L1 adipocytes [10]. Using these in vitro data, we suggested that HTR2A may regulate lipogenesis in WAT. To evaluate this hypothesis, we performed both in vitro and in vivo studies. We observed the lipid accumulation process in the in vitro system. We stained differentiated 3T3-L1 adipocytes with lipid (green, BODIPY 493/503) and serotonin (red, 5-HT). Fig. 4A shows the presence of serotonin and lipid droplets in differentiated 3T3-L1 adipocytes. Interestingly, we observed 5-HT and BODIPY copositive adipocytes (white arrowhead, Fig. 4A). The yellow staining implied that serotonin is related to lipid accumulation. Accordingly, the lipogenic gene and Htr2a gene expressions, in 3T3-L1 adipocytes, were increased during differentiation (Supplemental Fig. S3A).
Next, we generated adipocyte-specific Htr2a knockout (Adipoq-Cre+/−/Htr2a flox/flox, Htr2a FKO) mice. Htr2a FKO mice fed the HFD demonstrated a lower body weight gain and improved glucose tolerance compared to WT mice fed the HFD (Fig. 4B, Supplemental Fig. S3B, C). As expected, the adipose cell size and lipid droplets were decreased not only in the visceral adipose tissue but also in subcutaneous and BAT of Htr2a FKO mice (Fig. 4C, D, Supplemental Fig. S3D, E). The gene expression analysis also showed reduced lipogenic gene expression in Htr2a FKO mice (Fig. 4E). These in vivo data strongly suggested that HTR2A in WAT regulates lipogenesis.
Recent studies have reported that serotonin regulates energy metabolism in peripheral tissues such as the adipose tissue and liver [911,19]. Regarding the HFD condition, HFD increases serotonin levels in the adipose tissue [10]. The inhibition of serotonin synthesis increased energy expenditure and thermogenesis in BAT of mice fed the HFD [9,10]. In the liver, gut-derived serotonin regulates lipid accumulation [11,19]. These data consistently show a strong association between serotonin and lipid metabolism.
In this study, we investigated the role of serotonin in mature adipocytes under basal and overnutrition conditions. When we depleted serotonin levels genetically, the mice demonstrated resistance to obesity, increased energy expenditure, and elevated BAT activity (Figs. 1B–E, 2A–D). The WAT of HFD-fed mice maintained similar sizes of cell and lipid droplets to adipocytes in SCD-fed mice (Fig. 3C), suggesting that high serotonin leads to obesity-prone adipocytes by increasing lipogenesis and reducing thermogenesis.
In terms of energy metabolism, we observed an increased HP in the mice fed SCD as well as HFD (Fig. 2A, Supplemental Fig. S1E). Under low serotonin conditions, UCP1 activity in BAT and beige adipocyte were increased in the subcutaneous adipose tissue of both SCD- and HFD-fed Tph1 FKO mice (Figs. 2C, 2D, 3A, 3B). This implied that basal serotonin in mature adipocytes suppresses thermogenic activity in BAT and beige adipocyte formation in WAT. Furthermore, this finding expands the clinical importance of peripheral serotonin. If serotonin suppresses energy dissipation even under basal conditions, the anti-obesity effect of serotonin inhibition may be observed irrespective of the serotonin levels in adipose tissue. Intriguingly, Tph1 FKO mice demonstrated an increased food intake during both SCD and HFD feeding (Supplemental Figs. S1E, S2B). However, we failed to elucidate the exact underlying mechanism which could explain this behavioral change. The secondary effects could be attributed to the increased energy expenditure or unknown alterations in the lipid-brain axis due to serotonin deficiency. Nonetheless, Tph1 FKO mice demonstrate resistance to obesity despite the increased food intake.
With regard to lipogenesis in WAT, we focused on HTR2A. Several studies, including our group, have shown that HTR2A has a role in lipogenesis in 3T3-L1 cells [10,20]. In this study, we generated Htr2a FKO mice and observed that serotonin depleted WAT demonstrated reduced adipose cell sizes and lipid droplets (Fig. 4C, D), suggesting that HTR2A mediates the lipogenic changes in WAT. Moreover, these changes were only observed in the WAT of HFD-fed Htr2a FKO mice. The WAT of SCD-fed mice was similar to the WAT of WT mice. This implied that elevated serotonin, not basal serotonin, regulates lipogenesis in WAT under overnutrition conditions.
We reported that HTR3 is the responsible receptor that regulates thermogenesis in BAT [10]. Concerning the formation of beige adipocytes in the subcutaneous adipose tissue, we observed several UCP1 positive adipocytes in the subcutaneous adipose tissue of Tph1 FKO mice (Fig. 3B). This implied that the inhibition of serotonin synthesis induced beige adipocyte formation. However, the exact process of beige adipocyte formation after serotonin depletion remains unclear, necessitating further evaluation of the role of serotonin in the fate of pre-adipocyte, UCP1 positive adipocyte recruitment, and activation. Furthermore, we plan to investigate the responsible receptors regulating this change in the subcutaneous adipose tissue.
In conclusion, adipocyte-derived serotonin regulates lipogenesis and thermogenesis in the adipose tissue. Inhibition of serotonin synthesis in mature adipocytes reduced lipid accumulation in visceral WAT, induced beige formation in subcutaneous WAT and increased thermogenesis in BAT. In addition, serotonin increased lipogenesis through HTR2A signaling in visceral adipose tissue. Therefore, HTR2A inhibition, as well as TPH1 inhibitor, might be an effective treatment strategy for obesity, especially in case of visceral obesity.
Acknowledgements
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1A6A3A04010466 to Chang-Myung Oh, 2019M3A9A8066460 to Sangkyu Park, and 2016M3A9B6902871 to Hail Kim). BXD family mice data were taken from GeneNetwork database (www.genenetwork.org).

CONFLICTS OF INTEREST

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

AUTHOR CONTRIBUTIONS

Conceptualization: C.M.O., S.P., H.K. Acquisition, analysis, and interpretation of data: K.E.S., C.M.O., J.N. Drafting the work and revising: K.E.S., C.M.O., J.N., S.P., H.K. Final approval of the manuscript: S.P., H.K.

Fig. 1
Tryptophan hydroxylase 1 (Tph1) knockout (FKO) protects against high-fat diet (HFD)-induced obesity. (A) Volcano plot for mRNA expression of adipose tissue from the BXD strains. (B) Bodyweight curves in wild type (WT) and Tph1 FKO mice fed standard chow diet (SCD) or HFD for 12 weeks. Bodyweight of WT and Tph1 FKO mice were measured weekly from week 4 to week 20; n=5 in each group fed SCD, n=8 in each group fed HFD. (C). Body fat and lean body mass of mice after 12 weeks of HFD feeding. (D, E) Gross appearance and fat mass of visceral, inguinal and brown fat of 20-week-old WT and Tph1 FKO mice (left) fed on HFD. Adipose tissue weight (right); n=7 in each group. (F) Glucose tolerance tests. Blood glucose concentrations were measured at the indicated time points after fasted for 16 hours; n=10 in each group fed HFD. iWAT, inguinal white adipose tissue; eWAT, epidydimal white adipose tissue; BAT, brown adipose tissue. aP<0.05; bP<0.01 indicated significance.
enm-2020-35-2-470f1.jpg
Fig. 2
Tryptophan hydroxylase 1 (Tph1) knockout (FKO) increased energy expenditure. (A) Oxygen (O2) consumption (left), carbon dioxide (CO2) production (middle) and heat production (right) in wild type (WT) and Tph1 FKO mice fed on high-fat diet (HFD) for 12 weeks; n=8 in each group fed HFD. (B, C) In vivo luciferase assay of WT and Tph1 FKO mice. Tph1 FKO mice show increased uncoupled protein 1 (UCP1) activity. (D) Histological analysis of brown adipose tissue (BAT) at the indicated age. Adipose tissue sections were stained with H&E (scale bar, 100 μm). SCD, standard chow diet. aP<0.05; bP<0.01 indicated significance.
enm-2020-35-2-470f2.jpg
Fig. 3
Representative images of adipose tissues of tryptophan hydroxylase 1 (Tph1) knockout (FKO) mice. (A) Histological analysis of iWAT at the indicated age. Adipose tissue was stained with H&E. (B) Immunostaining of UCP1 in iWAT (scale bar, 100 μm). (C) Histological analysis of eWAT at the indicated age. Adipose tissue sections were stained with H&E (scale bar, 100 μm). (D) The adipocyte size was analyzed using ImageJ software (NIH). iWAT, inguinal white adipose tissue; UCP1, uncoupled protein 1; eWAT, epididymal white adipose tissue; SCD, standard chow diet; HFD, high-fat diet; WT, wild type. aP<0.05 indicated significance.
enm-2020-35-2-470f3.jpg
Fig. 4
Inhibition of adipose tissue-specific serotonin receptor 2A (HTR2A) signaling reduces lipid accumulation in white adipose tissue (WAT). (A) Representative images of immunofluorescence staining of differentiated 3T3-L1 adipocytes with BODIPY (green), anti-5-hydroxytryptophan (5-HT) antibody (red), and 4′,6-diamidino-2-phenylindole (DAPI; blue). White arrow indicates BODIPY and 5-HT co-positive cells. (B) Body weight curves in wild type (WT) and Htr2a FKO mice fed standard chow diet (SCD) or high-fat diet (HFD) for 12 weeks. (C) Histological analysis of epididymal white adipose (eWAT) in of WT and Htr2a FKO mice fed SCD or HFD. (D) The adipocyte sizes were analyzed using the ImageJ program. (E) mRNA expression level of fatty acid synthesis (Acaca, Fasn, Scd1), triglyceride synthesis (Dgat1, Dgat2, Gpam, Mogat1), and Plin1. Acaca, acetyl-coA carboxylase alpha; Fasn, fatty acid synthase; Scd1, stearoyl-CoA desaturase 1; Dgat1, diacylglycerol O-acyltransferase 1; Dgat2, diacylglycerol O-acyltransferase 2; Gpam, glycerol-3-phosphate acyltransferase, mitochondrial; Mogat1, monoacylglycerol O-acyltransferase 1; Plin1, perilipin 1. aP<0.05; bP<0.001 indicated significance.
enm-2020-35-2-470f4.jpg
Table 1
List of Primers Used for Real Time Quantitative Polymerase Chain Reaction
Gene Sequence
Actb Forward 5′-GGTACCACCATGTACCCAGG-3′
Reverse 5′-GAAAGGGTGTAAAACGCAGC-3′

Tph1 Forward 5′-ACCATGATTGAAGACAACAAGGAG-3′
Reverse 5′-TCAACTGTTCTCGGCTGATG-3′

Ucp1 Forward 5′-CTTTGCCTCACTCAGGATTGG-3′
Reverse 5′-ACTGCCACACCTCCAGTCATT-3′

Cidea Forward 5′-GCCGTGTTAAGGAATCTGCTG-3′
Reverse 5′-TGCTCTTCTGTATCGCCCAGT-3′

Dio2 Forward 5′-TTGGGGTAGGGAATGTTGGC-3′
Reverse 5′-TCCGTTTCCTCTTTCCGGTG-3′

Pgc1a Forward 5′-GCCCAGGTACGACAGCTATG-3′
Reverse 5′-ACGGCGCTCTTCAATTGCTT-3′

Prdm16 Forward 5′-AGCCCTCGCCCACAACTTGC-3′
Reverse 5′-TGACCCCCGGCTTCCGTTCA-3′

Adipoq Forward 5′-CTCCACCCAAGGGAACTTGT-3′
Reverse 5′-GGACCAAGAAGACCTGCATC-3′

Pparg Forward 5′-GGTGTGATCTTAACTGCCGGA-3′
Reverse 5′-GCCCAAACCTGATGGCATTG-3′

Cd36 Forward 5′-TGGCCAAGCTATTGCGACAT-3′
Reverse 5′-ACACAGCGTAGATAGACCTGC-3′

Dgat1 Forward 5′-GGATCTGAGGTGCCATCGTC-3′
Reverse 5′-ATCAGCATCACCACACACCA-3′

Fasn Forward 5′-AAGCGGTCTGGAAAGCTGAA-3′
Reverse 5′-AGGCTGGGTTGATACCTCCA-3′

Acaca Forward 5′-CAGTAACCTGGTGAAGCTGGA-3′
Reverse 5′-GCCAGACATGCTGGATCTCAT-3′

Hsl Forward 5′-GCAGTGGTGTGTAACTAGGAT-3′
Reverse 5′-CGCTGAGGCTTTGATCTTGC-3′

Atgl Forward 5′-TAGGAGGAATGGCCTACTGAA-3′
Reverse 5′-GGCTGCAATTGATCCTCCTCT-3′

Actb, beta-actin; Tph1, tryptophan hydrxylase 1; Ucp1, uncoupled protein 1; Cidea, cell death-inducing DNA fragmentation factor alpha-like effector A; Dio2, iodothyronine deiodinase 2; Pgc1a, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; Prdm16, PR/SET domain 16; Adipoq, adiponectin; Pparg, peroxisome proliferator-activated receptor gamma; Cd36, cluster of differentiation 36; Dgat1, diacylglycerol O-acyltransferase 1; Fasn, fatty acid synthase; Acaca, acetyl-coA carboxylase alpha; Hsl, hormone sensitive lipase; Atgl, adipose triglyceride lipase.

  • 1. Van Gaal LF, Mertens IL, De Block CE. Mechanisms linking obesity with cardiovascular disease. Nature 2006;444:875–80.ArticlePubMedPDF
  • 2. Flegal KM, Kit BK, Orpana H, Graubard BI. Association of all-cause mortality with overweight and obesity using standard body mass index categories: a systematic review and meta-analysis. JAMA 2013;309:71–82.ArticlePubMedPMC
  • 3. Walther DJ, Peter JU, Bashammakh S, Hortnagl H, Voits M, Fink H, et al. Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 2003;299:76.ArticlePubMed
  • 4. Gershon MD, Tack J. The serotonin signaling system: from basic understanding to drug development for functional GI disorders. Gastroenterology 2007;132:397–414.ArticlePubMed
  • 5. Young RL, Lumsden AL, Martin AM, Schober G, Pezos N, Thazhath SS, et al. Augmented capacity for peripheral serotonin release in human obesity. Int J Obes (Lond) 2018;42:1880–9.ArticlePubMedPDF
  • 6. Li P, Tiwari HK, Lin WY, Allison DB, Chung WK, Leibel RL, et al. Genetic association analysis of 30 genes related to obesity in a European American population. Int J Obes (Lond) 2014;38:724–9.ArticlePubMedPDF
  • 7. Kwak SH, Park BL, Kim H, German MS, Go MJ, Jung HS, et al. Association of variations in TPH1 and HTR2B with gestational weight gain and measures of obesity. Obesity (Silver Spring) 2012;20:233–8.ArticlePubMed
  • 8. Li T, Guo K, Qu W, Han Y, Wang S, Lin M, et al. Important role of 5-hydroxytryptamine in glucocorticoid-induced insulin resistance in liver and intra-abdominal adipose tissue of rats. J Diabetes Investig 2016;7:32–41.ArticlePubMed
  • 9. Crane JD, Palanivel R, Mottillo EP, Bujak AL, Wang H, Ford RJ, et al. Inhibiting peripheral serotonin synthesis reduces obesity and metabolic dysfunction by promoting brown adipose tissue thermogenesis. Nat Med 2015;21:166–72.ArticlePubMedPDF
  • 10. Oh CM, Namkung J, Go Y, Shong KE, Kim K, Kim H, et al. Regulation of systemic energy homeostasis by serotonin in adipose tissues. Nat Commun 2015;6:6794.ArticlePubMedPMCPDF
  • 11. Choi W, Namkung J, Hwang I, Kim H, Lim A, Park HJ, et al. Serotonin signals through a gut-liver axis to regulate hepatic steatosis. Nat Commun 2018;9:4824.ArticlePubMedPMCPDF
  • 12. Yadav VK, Ryu JH, Suda N, Tanaka KF, Gingrich JA, Schutz G, et al. Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum. Cell 2008;135:825–37.ArticlePubMedPMC
  • 13. Eguchi J, Wang X, Yu S, Kershaw EE, Chiu PC, Dushay J, et al. Transcriptional control of adipose lipid handling by IRF4. Cell Metab 2011;13:249–59.ArticlePubMedPMC
  • 14. Rio DC, Ares M Jr, Hannon GJ, Nilsen TW. Purification of RNA using TRIzol (TRI reagent). Cold Spring Harb Protoc 2010 2010:pdb.prot5439..Article
  • 15. Galmozzi A, Sonne SB, Altshuler-Keylin S, Hasegawa Y, Shinoda K, Luijten IHN, et al. ThermoMouse: an in vivo model to identify modulators of UCP1 expression in brown adipose tissue. Cell Rep 2014;9:1584–93.ArticlePubMedPMC
  • 16. Shan T, Liu W, Kuang S. Fatty acid binding protein 4 expression marks a population of adipocyte progenitors in white and brown adipose tissues. FASEB J 2013;27:277–87.ArticlePubMedPMC
  • 17. Lee KY, Russell SJ, Ussar S, Boucher J, Vernochet C, Mori MA, et al. Lessons on conditional gene targeting in mouse adipose tissue. Diabetes 2013;62:864–74.ArticlePubMedPMC
  • 18. Chakrabarti P. Promoting adipose specificity: the adiponectin promoter. Endocrinology 2010;151:2408–10.ArticlePubMedPDF
  • 19. Sumara G, Sumara O, Kim JK, Karsenty G. Gut-derived serotonin is a multifunctional determinant to fasting adaptation. Cell Metab 2012;16:588–600.ArticlePubMedPMC
  • 20. Kinoshita M, Ono K, Horie T, Nagao K, Nishi H, Kuwabara Y, et al. Regulation of adipocyte differentiation by activation of serotonin (5-HT) receptors 5-HT2AR and 5-HT2CR and involvement of microRNA-448-mediated repression of KLF5. Mol Endocrinol 2010;24:1978–87.ArticlePubMedPMCPDF

Figure & Data

References

    Citations

    Citations to this article as recorded by  
    • Gut microbiota dysbiosis and decreased levels of acetic and propionic acid participate in glucocorticoid-induced glycolipid metabolism disorder
      Qin Zhang, Gaopeng Guan, Jie Liu, Wenmu Hu, Ping Jin, Yung-Fu Chang
      mBio.2024;[Epub]     CrossRef
    • The Chain-Mediating Effect of Obesity, Depressive Symptoms on the Association between Dietary Quality and Cardiovascular Disease Risk
      Shuai Zhang, Limei E, Zhonghai Lu, Yingying Yu, Xuebin Yang, Yao Chen, Xiubo Jiang
      Nutrients.2023; 15(3): 629.     CrossRef
    • Metabolic and Molecular Response to High-Fat Diet Differs between Rats with Constitutionally High and Low Serotonin Tone
      Petra Baković, Maja Kesić, Darko Kolarić, Jasminka Štefulj, Lipa Čičin-Šain
      International Journal of Molecular Sciences.2023; 24(3): 2169.     CrossRef
    • Linking serotonin homeostasis to gut function: Nutrition, gut microbiota and beyond
      Lili Jiang, Dandan Han, Youling Hao, Zhuan Song, Zhiyuan Sun, Zhaolai Dai
      Critical Reviews in Food Science and Nutrition.2023; : 1.     CrossRef
    • Serotonin transporter-deficient mice display enhanced adipose tissue inflammation after chronic high-fat diet feeding
      Johannes Hoch, Niklas Burkhard, Shanshan Zhang, Marina Rieder, Timoteo Marchini, Vincent Geest, Krystin Krauel, Timm Zahn, Nicolas Schommer, Muataz Ali Hamad, Carolina Bauer, Nadine Gauchel, Daniela Stallmann, Claus Normann, Dennis Wolf, Rüdiger Eberhard
      Frontiers in Immunology.2023;[Epub]     CrossRef
    • The role of serotonin in prenatal ontogenesis
      Inna I. Evsyukova
      Journal of obstetrics and women's diseases.2023; 72(4): 81.     CrossRef
    • Genome-wide survey and functional analysis reveal TCF21 promotes chicken preadipocyte differentiation by directly upregulating HTR2A
      Xinyang Zhang, Bohan Cheng, Yanyan Ma, Yumeng Liu, Ning Wang, Hui Zhang, Yumao Li, Yuxiang Wang, Peng Luan, Zhiping Cao, Hui Li
      Biochemical and Biophysical Research Communications.2022; 587: 131.     CrossRef
    • Maternal Metabolic State and Fetal Sex and Genotype Modulate Methylation of the Serotonin Receptor Type 2A Gene (HTR2A) in the Human Placenta
      Marina Horvatiček, Maja Perić, Ivona Bečeheli, Marija Klasić, Maja Žutić, Maja Kesić, Gernot Desoye, Sandra Nakić Radoš, Marina Ivanišević, Dubravka Hranilovic, Jasminka Štefulj
      Biomedicines.2022; 10(2): 467.     CrossRef
    • Synthesis and biological evaluation of tyrosine derivatives as peripheral 5HT2A receptor antagonists for nonalcoholic fatty liver disease
      Minhee Kim, Wonil Choi, Jihyeon Yoon, Byung-kwan Jeong, Suvarna H. Pagire, Haushabhau S. Pagire, Jungsun Park, Jung Eun Nam, Chang Joo Oh, Jae-Han Jeon, Seong Soon Kim, Byung Hoi Lee, Jin Sook Song, Myung Ae Bae, In-Kyu Lee, Hail Kim, Jin Hee Ahn
      European Journal of Medicinal Chemistry.2022; 239: 114517.     CrossRef
    • Serotonin in the regulation of systemic energy metabolism
      Joon Ho Moon, Chang‐Myung Oh, Hail Kim
      Journal of Diabetes Investigation.2022; 13(10): 1639.     CrossRef
    • Traumatic brain injury alters the gut-derived serotonergic system and associated peripheral organs
      Natosha M. Mercado, Guanglin Zhang, Zhe Ying, Fernando Gómez-Pinilla
      Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease.2022; 1868(11): 166491.     CrossRef
    • Modulation of adipose tissue metabolism by microbial-derived metabolites
      Wenyun Liu, Ge Yang, Pinyi Liu, Xin Jiang, Ying Xin
      Frontiers in Microbiology.2022;[Epub]     CrossRef
    • Risperidone Exacerbates Glucose Intolerance, Nonalcoholic Fatty Liver Disease, and Renal Impairment in Obese Mice
      Hsiao-Pei Tsai, Po-Hsun Hou, Frank-Chiahung Mao, Chia-Chia Chang, Wei-Cheng Yang, Ching-Feng Wu, Huei-Jyuan Liao, Tzu-Chun Lin, Lan-Szu Chou, Li-Wei Hsiao, Geng-Ruei Chang
      International Journal of Molecular Sciences.2021; 22(1): 409.     CrossRef
    • Pancreatic Sirtuin 3 Deficiency Promotes Hepatic Steatosis by Enhancing 5-Hydroxytryptamine Synthesis in Mice With Diet-Induced Obesity
      Xing Ming, Arthur C.K. Chung, Dandan Mao, Huanyi Cao, Baoqi Fan, Willy K.K. Wong, Chin Chung Ho, Heung Man Lee, Kristina Schoonjans, Johan Auwerx, Guy A. Rutter, Juliana C.N. Chan, Xiao Yu Tian, Alice P.S. Kong
      Diabetes.2021; 70(1): 119.     CrossRef
    • Peripheral Selective Oxadiazolylphenyl Alanine Derivatives as Tryptophan Hydroxylase 1 Inhibitors for Obesity and Fatty Liver Disease
      Eun Jung Bae, Won Gun Choi, Haushabhau S. Pagire, Suvarna H. Pagire, Saravanan Parameswaran, Jun-Ho Choi, Jihyeon Yoon, Won-il Choi, Ji Hun Lee, Jin Sook Song, Myung Ae Bae, Mijin Kim, Jae-Han Jeon, In-Kyu Lee, Hail Kim, Jin Hee Ahn
      Journal of Medicinal Chemistry.2021; 64(2): 1037.     CrossRef
    • A Systems Biology Approach to Investigating the Interaction between Serotonin Synthesis by Tryptophan Hydroxylase and the Metabolic Homeostasis
      Suhyeon Park, Yumin Kim, Jibeom Lee, Jeong Yun Lee, Hail Kim, Sunjae Lee, Chang-Myung Oh
      International Journal of Molecular Sciences.2021; 22(5): 2452.     CrossRef
    • Metabolic Disturbances in Rat Sublines with Constitutionally Altered Serotonin Homeostasis
      Maja Kesić, Petra Baković, Ranko Stojković, Jasminka Štefulj, Lipa Čičin-Šain
      International Journal of Molecular Sciences.2021; 22(10): 5400.     CrossRef
    • The Mechanism of Secretion and Metabolism of Gut-Derived 5-Hydroxytryptamine
      Ning Liu, Shiqiang Sun, Pengjie Wang, Yanan Sun, Qingjuan Hu, Xiaoyu Wang
      International Journal of Molecular Sciences.2021; 22(15): 7931.     CrossRef
    • Inhibiting serotonin signaling through HTR2B in visceral adipose tissue improves obesity-related insulin resistance
      Won Gun Choi, Wonsuk Choi, Tae Jung Oh, Hye-Na Cha, Inseon Hwang, Yun Kyung Lee, Seung Yeon Lee, Hyemi Shin, Ajin Lim, Dongryeol Ryu, Jae Myoung Suh, So-Young Park, Sung Hee Choi, Hail Kim
      Journal of Clinical Investigation.2021;[Epub]     CrossRef
    • Serotonergic Regulation of Hepatic Energy Metabolism
      Jiwon Park, Wooju Jeong, Chahyeon Yun, Hail Kim, Chang-Myung Oh
      Endocrinology and Metabolism.2021; 36(6): 1151.     CrossRef

    Figure

    Endocrinol Metab : Endocrinology and Metabolism