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. 2025 Jan 10;48(3):100830. doi: 10.1016/j.bj.2025.100830

Chronic low-dose REV-ERBs agonist SR9009 mitigates constant light-induced weight gain and insulin resistance via adipogenesis modulation

Ming-Yu Yang a,b, Hugo Y-H Lin c,d, Yi-Ywan M Chen e,f, Ming-Luen Hu g, I-Ya Chen a, Chao-Hui Yang b,h,i,⁎
PMCID: PMC12277734  PMID: 39800061

Abstract

Background

Obesity and circadian rhythm disruption are significant global health concerns, contributing to an increased risk of metabolic disorders. Both adipose tissue and circadian rhythms play critical roles in maintaining energy homeostasis, and their dysfunction is closely linked to obesity. This study aimed to assess the effects of chronic low-dose SR9009, a REV-ERB ligand, on circadian disruption induced by constant light exposure in mice.

Material and methods

Mice were exposed to constant light for eight weeks (LL mice), resulting in increased body weight, insulin resistance, white fat mass, and altered circadian clock gene expression. Low-dose SR9009 (10 mg/kg daily) was administered chronically to assess its impact on these metabolic disruptions.

Results

LL mice treated with SR9009 for eight weeks showed reduced weight gain, insulin resistance, and white fat mass but no significant impact on overall energy homeostasis. SR9009 suppressed Bmal1 expression and restored Rev-erbα and Rev-erbβ expression in white and brown adipose tissue (WAT and BAT). In vitro studies using 3T3-L1 cells indicated that SR9009 inhibited adipogenesis, leading to further investigation in vivo. SR9009 restored ChREBP1a and Srebp-1c expression in BAT but did not affect inflammatory cytokine or adipokine gene expression, nor did it restore Fasn, Pparγ, and Prom1 expression in both WAT and BAT.

Conclusions

These findings suggest that SR9009 may be a potential therapeutic approach for preventing weight gain and insulin resistance caused by circadian disruptions, likely through adipogenesis inhibition, though its effects on other metabolic pathways remain limited at low doses.

Keywords: REV-ERB, SR9009, Constant light exposure, Circadian clock genes, Adipogenesis

Highlights

  • •

    Constant light (LL) increases weight, insulin resistance, white fat, and disrupts clock.

  • •

    Chronic low-dose SR9009 reduced LL-induced weight gain and insulin resistance.

  • •

    SR9009 restored Bmal1 upregulation and Rev-erbs downregulation caused by LL.

  • •

    SR9009 inhibited adipogenesis in vitro and in vivo.

  • •

    SR9009 may prevent weight gain and insulin resistance caused by circadian disruption.

1. Introduction

Imagine waking up at midnight, eating a full meal, and heading to work while the rest of the world is sleeping. It may seem like a bizarre lifestyle, but this is the reality for millions of shift workers around the world. Circadian rhythms are crucial endogenous 24-h cycles that regulate various physiological and behavioral processes in living organisms, including metabolism, energy homeostasis, and glucose metabolism [[1], [2], [3]]. Epidemiological studies show that engaging in excessive activities or experiencing sleep disruption during traditional “rest" periods increases the risk of obesity, diabetes, cardiovascular disease, inflammation, and even cancer [[4], [5], [6], [7]].

Metabolic disorders are a growing concern worldwide, with obesity and diabetes being two of the most prevalent conditions. Metabolism and circadian rhythm are closely related since many metabolic pathways are regulated by circadian rhythm. In particular, obesity and insulin resistance have been linked to circadian clock dysregulation [8,9]. Animal experiments have shown that disrupting circadian rhythm causes glucose metabolism abnormalities [10], obesity, and leptin resistance [11], while mice with a deficiency in the circadian rhythm genes Clock or Bmal1 also exhibit metabolic disorders [[12], [13], [14]]. Disruptions to the circadian clock in human, such as those caused by shift work and irregular sleep patterns have been associated with a range of metabolic disorders [[15], [16], [17]]. Human experiments have found that forcing participants to work night shifts or rotating shifts significantly decreases their glucose sensitivity and increases inflammation [18]. This is due to the disruption of the body's circadian rhythm, which leads to decreased leptin and increased glucose and insulin levels. Therefore, readjusting the circadian rhythm may potentially alleviate these metabolic disorders.

Understanding the mechanisms underlying the relationship between the circadian clock and metabolic disorders such as obesity is essential for developing effective prevention and treatment strategies. One potential avenue for investigation is the role of REV-ERBα/β, a molecular clock component that plays a critical role in regulating circadian rhythms and metabolism. SR9009, also known as Stenabolic, is a synthetic compound that has been identified as a potent agonist of REV-ERBα/β. It enhances the activity of the circadian rhythm gene Rev-erb and suppresses BMAL1 activity, thereby stabilizing the body's intrinsic circadian rhythm. Animal experiments have shown that SR9009 has various effects, including reducing body weight, lowering cholesterol, triglycerides, and low-density lipoprotein [19,20], improving sleep and wakefulness regulation [21], reducing anxiety [22], decreasing inflammation [23,24], and improving heart function [25]. SR9009 has also been showed to improve glucose metabolism and insulin sensitivity in mice on a high-fat diet [26]. Low doses of SR9009 (10–40 mg) have been added to drugs used for human fitness purposes [27], but there have been no human trials, and the potential adverse effects on humans remain unknown. Importantly, while SR9009's actions are primarily attributed to its modulation of REV-ERBα/β, it has also been shown to have potent REV-ERB-independent effects in regulating metabolism and cellular processes, as demonstrated in recent studies [28,29]. In mouse experiments, SR9009 was found to increase exercise capacity by increasing the number of mitochondria in muscle cells [30]. The molecular regulatory mechanism of its action is still not fully understood.

Based on the idea of seeking health solutions for individuals who work long-term rotating shifts or night shifts, this study utilized mice exposed to constant light as a model for circadian rhythm disruption. We administered chronic low-dose SR9009 to investigate whether adjusting the body's circadian clock genes, specifically Bmal1 and Rev-erbα/β, could potentially prevent the occurrence of metabolic disorders. Unlike many studies that use a higher dose of 100 mg/kg, we selected a lower dose (10 mg/kg) to align with our goal of exploring SR9009 as a long-term preventative and health-maintenance strategy rather than as a short-term therapeutic intervention. Additionally, the cost of SR9009 remains a significant factor, and the use of lower doses could make such preventive applications more practical and accessible. The results of this study also demonstrated, as anticipated, that SR9009 can serve as a proactive strategy to prevent the occurrence of metabolic disorders in individuals with circadian rhythm disruption due to shift work.

2. Materials and methods

2.1. Animal studies and bioethical statement

Male C57BL/6 J mice weighing between 18 and 22 g and aged four weeks were obtained from the National Laboratory Animal Center (Taipei, Taiwan). The mice were housed in a temperature-controlled room with a 12-h light/dark cycle and provided with ad libitum access to normal chow diet and drinking water. After a two-week acclimatization period, the mice were randomly assigned to experimental groups and maintained under either a 12-h light/dark cycle or constant light conditions. The light intensity during the light on period was kept at approximately 270–320 lux (lm/m2). SR9009 (S684255, Toronto Research Chemicals, Toronto, Canada) was formulated in 15% Cremophor (C5135, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and administered intraperitoneally (i.p.) at a single dose of 10 mg/kg body weight or vehicle (15% Cremophor) at zeitgeber time (ZT) 4 daily for eight weeks. All procedures were approved and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Chang Gung Memorial Hospital (IACUC No. 2019062505).

2.2. Intraperitoneal glucose tolerance test (IPGTT)

After a 16-h fast (from 5 p.m. to 9 a.m.), an IPGTT was performed. Baseline glucose measurements were taken prior to the start of the test. Glucose tolerance was assessed by administering glucose (2 g/kg body weight) via i. p. injection, and blood glucose levels were monitored at 15, 30, 60, and 120 min using the Optium Xceed glucometer (Abbott, Abbott Park, Illinois, USA).

2.3. Serum biochemical measurement

Following a 16-h fast (from 5 p.m. to 9 a.m.), blood samples were collected via cardiac puncture. Serum levels of total cholesterol, high-density lipoprotein (HDL), corticosterone, and triglycerides were determined using ARKRAY SPOTCHEM chips (ARKRAY Inc., Kyoto, Japan) on a clinical chemistry analyzer (SPOTCHEM EZ SP-4430, ARKRAY Inc.).

2.4. Metabolic analysis

Metabolic analysis was performed using a metabolic cage (Phenomaster, TSE Systems, Bad Homburg, Germany) to measure food and water consumption, energy expenditure (Kcal/hr/Kg), volume of oxygen consumption (VO2, mL/Kg/hr), volume of carbon dioxide production (VCO2, mL/Kg/hr), and respiratory exchange ratio (RER, VCO2/VO2 ratio). Mice were individually housed and acclimatized to the metabolic cage one day prior to the measurements. The mice were acclimated for 24 h before being monitored at 23 °C for 72 h under a 12-h light/12-h dark cycle, followed by another 72 h at 6 °C under the same 12-h light/12-h dark cycle.

2.5. Locomotor activity/open field test

The locomotor activity/open-field test, conducted using the Multi Conditioning System (TSE Systems GmbH, Bad-Homburg, Germany), was employed to evaluate spontaneous locomotor activity and arousal in LL mice. Short-term locomotor activity was assessed during both the light phase (daytime, 9:00–13:00) and the dark phase (nighttime, 18:00–20:00). On the day of the experiment, mice were acclimated to the experimental room for 5 min before testing. Prior to each trial, the open-field arena was thoroughly cleaned with isopropyl alcohol to remove olfactory cues left by previous subjects. Each mouse was then placed in a black open-field box (30 × 30 × 30 cm) and allowed to explore freely without any external motivational constraints. The movements of the mice were recorded for 15 min using a video camera positioned above the open-field box. Behavioral parameters were analyzed, including moving and resting time, total distance traveled, rearing time and frequency in Z1, as well as time spent and distance traveled in the center versus the periphery of the arena.

2.6. Real-time quantitative polymerase chain reaction (qPCR)

RNA was extracted from BAT, WAT and cultured cells using the RNeasy Mini Kit (Qiagen, GmbH, Germany), and cDNA synthesis was performed using the High Capacity cDNA Reverse Transcription Kit (Sigma-Aldrich, St. Louis, MO, USA). Real-time qPCR was carried out to measure gene expression of circadian clock genes, inflammatory cytokines and adipokines using TaqMan Gene Expression Assays ([Supplementary Table S1]; Thermo Fisher Scientific, Waltham, MA, USA). The expression of metabolism-related genes was carried out using SYBR® Green. [Supplementary Table S2] lists the gene names, GenBank accession numbers, amplicon sizes, sequences of forward and reverse primers, and assay location of the metabolism-related genes. The endogenous control was the expression of the mouse Actb (β-actin) gene. All reactions were run in the 7500 Fast Real-Time System (Applied Biosystems).

2.7. Cell culture and adipocyte differentiation

The 3T3-L1 cells were obtained from Bioresource Collection and Research Center (Hsinchu, Taiwan), and were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 4.5 g/L glucose and 10% calf bovine serum at 37 °C and 5% CO2. The induction of adipocyte differentiation was performed as previously described [31]. Briefly, 8 × 104 3T3-L1 cells were seeded in a 6-cm dish and incubated with DMEM supplemented with 10% fetal bovine serum (FBS), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μM dexamethasone (Sigma-Aldrich), 1 μg/mL insulin (Sigma-Aldrich), and 2 μM rosiglitazone (Sigma-Aldrich) for 3 days. SR9009 (1 or 10 μM, Toronto Research Chemicals) or dimethyl sulfoxide (DMSO) was added at day 1 of differentiation. After 3 days of differentiation, the medium was changed to DMEM supplemented with 10% FBS, 1 μg/mL insulin (Sigma-Aldrich), and SR9009. The medium was refreshed every two days until day 21. The differentiated cells were harvested at days 14 and 21 for Oil Red O staining.

2.8. Oil Red O staining

The harvested differentiated adipocytes were fixed in 10% formalin for 1 h, and then stained with Oil Red O solution (Sigma-Aldrich) for 8 min. Hematoxylin was used to counter stain the cells for 2 min. Adipocyte morphology was observed using an optical microscope, and ImageJ (v.1.50i, National Institute of Health, USA) was used to analyze the size and number of adipocytes.

2.9. Statistical analysis

Statistical analyses were conducted using IBM SPSS Statistics version 22.0 (IBM, Armonk, NY, USA) and GraphPad Prism version 7.04 (GraphPad, San Diego, CA, USA). Differences between two groups were evaluated using Student's t-test or Mann-Whitney test as appropriate. Results are presented as mean ± standard error of the mean (SEM). -ΔCt values were used for statistical analysis of qPCR results. Statistical significance was defined as p < 0.05 for all tests.

3. Results

3.1. Constant light exposure resulted in weight gain, insulin resistance, increased white fat mass, and decreased RER

To explore the impact of circadian rhythm disruption on metabolic regulation, we exposed C57BL/6 J mice to constant light (LL) for eight weeks, while controls remained under a 12-h light/dark cycle (LD). LL mice exhibited significant body weight gain starting from the first week of exposure, which persisted through the eighth week [Fig. 1A]. Furthermore, LL mice developed insulin resistance, as evidenced by impaired glucose tolerance in IPGTT compared to LD mice [Fig. 1B]. Notably, LL mice showed a significant increase in white fat mass [Fig. 1C], while brown fat mass remained unaffected [Supplementary Fig. S1A]. These results highlight that constant light exposure induces key characteristics of obesity and insulin resistance. Interestingly, the serum levels of total cholesterol, triglycerides, high-density lipoprotein, and corticosterone were not significantly different between LD and LL mice [Supplementary Figs. S1B–E], suggesting that these metabolic changes are not related to stress hormone levels.

Fig. 1.

Fig. 1

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The physiological changes in the mice after eight weeks of constant light exposure. (A) The body weight gain of LD and LL mice. The LL mice had a significantly higher body weight gain compared to the LD mice. (B) The results of IPGTT performed on LD and LL mice. The LL mice had higher insulin resistance compared to the LD mice. (C) The white fat mass of LD and LL mice. The LL mice had a significantly higher white fat mass compared to the LD mice. (D, E) The daily average food and water intakes of LD and LL mice at 23 °C. (F–I) The RER, oxygen volume, carbon dioxide volume, and energy expenditure of LD and LL mice during light and dark phase at 23 °C. ∗p < 0.05 and ∗∗p < 0.001 indicated in panels A and B are the comparison made between LD and LL mice of the same time point. In panels C and F, ∗ indicates statistical significance at p < 0.05. Abbreviations: LD: mice living under 12-h light/dark condition; LL: mice living under constant light condition; IPGTT: intraperitoneal glucose tolerance test; RER: respiratory exchange rate.

To further investigate how constant light exposure affects metabolic efficiency, we used metabolic cages to assess food/water intake and energy expenditure. LL mice displayed no significant difference in food or water consumption compared to LD controls [Fig. 1D and E], suggesting that the observed weight gain was not driven by increased caloric intake. However, we observed a significant reduction in respiratory exchange ratio (RER) during the dark phase in LL mice [Fig. 1F], indicating a shift toward lipid utilization over carbohydrates. Despite these metabolic shifts, O₂ consumption, CO₂ production, and energy expenditure were similar between LL and LD mice [Fig. 1G–I], indicating that altered energy homeostasis may not be the primary driver of weight gain in LL mice.

3.2. Constant light exposure alters locomotor activity

To evaluate the impact of constant light exposure on exploratory behavior, we subjected mice to the open-field test to assess their locomotor activity. LL mice exhibited significantly higher locomotor activity compared to the LD group [Fig. 2A]. This increase was evidenced by a marked rise in moving time [Fig. 2B], total distance traveled [Fig. 2C], number of rearings in Z1 [Fig. 2D], and the percentage of time spent in the center of the field [Fig. 2E]. The open-field test reveals that constant light exposure significantly alters behavior of LL mice. These findings suggest that constant light exposure substantially alters LL mice's behavior, likely due to disruptions in circadian rhythms or increased arousal.

Fig. 2.

Fig. 2

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Behavioral analysis of LD and LL mice in the open-field test. (A) Representative travel traces from the last 5 min of LD and LL mice in the arena. The moving time (B), total distance traveled (C), rearing frequencies in Z1 (D), and time spent in the center (E) were all significantly higher in LL mice compared to LD mice. ∗ indicates statistical significance at p < 0.05, evaluated by the Mann-Whitney test. Abbreviations: LD: mice living under 12-h light/dark cycle; LL: mice living under constant light condition.

3.3. Circadian clock gene dysregulation in constant light exposure

We next examined whether constant light-induced metabolic changes were associated with disruptions in circadian clock gene expression. Analysis of 12 core circadian genes revealed dysregulated gene expression patterns of Bmal1, Per1, Per2, Per3, Rev-erbα and Rev-erbβ in both WAT [Fig. 3] and BAT [Supplementary Fig. S2]. Notably, Bmal1 was significantly upregulated [Fig. 3A], while Rev-erbα and Rev-erbβ were downregulated at ZT12 in LL mice [Fig. 3I and J]. These alterations in circadian gene expression suggest that circadian rhythm disruption may play a critical role in the metabolic dysfunction observed in LL mice.

Fig. 3.

Fig. 3

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Expression pattern of 12 core circadian clock genes in white adipose tissue of mice after eight weeks of constant light exposure. The expression of Bmal1(A), Ck1ε(B), Clock(C), Cry 1(D), Cry2(E), Per1(F), Per2(G), Per3(H), Rev-erbα(I), Rev-erbβ(J), Rorα(K) and Tim(L) genes were examined at four Zeitgeber Times (ZTs) in the LD mice and at four Circadian Times (CTs) in LL mice. At ZT12, Bmal1(A) and Clock(C) were significantly upregulated, while Cry2(E), Per1(F), Per2(G), Per3(H), Rev-erbα(I), Rev-erbβ(J) and Rorα(K) were significantly downregulated in LL mice compared to LD mice. Each value represents the percentage change relative to the lowest expression level in the LD group, normalized to 100%. Data are mean ± SEM (n = 4 for each time point in each condition). ∗ and ∗∗ indicate statistical significance at p < 0.05 and p < 0.01, respectively, for differences between LD and LL mice at the same time point, evaluated by the Mann-Whitney test using values of ΔCt. Abbreviations: LD: mice living under 12-h light/dark condition; LL: mice living under constant light condition; SEM: standard error of the mean.

3.4. SR9009 mitigates weight gain, insulin resistance, and white fat mass accumulation induced by constant light exposure

Given the circadian gene disruptions observed, we hypothesized that modulating circadian rhythms could counteract the adverse metabolic effects of constant light exposure. Many studies have utilized a higher dose of SR9009 (100 mg/kg) to investigate its effects [19]. However, in this study, LL mice were treated daily with a lower dose of SR9009 (10 mg/kg, i. p.) once daily at ZT4 for 8 weeks. This lower dose was chosen to explore its potential as a long-term preventative and health-maintenance strategy rather than as a short-term therapeutic intervention. SR9009 significantly attenuated weight gain [Fig. 4A], improved glucose tolerance [Fig. 4B], and reduced white fat mass [Fig. 4C] compared to vehicle-treated LL mice. However, brown fat mass remained unaffected, suggesting that the protective effects of SR9009 were predominantly associated with white adipose tissue.

Fig. 4.

Fig. 4

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The physiological changes in the mice receiving SR9009 treatment and constant light exposure for eight weeks. (A) Mice that received SR9009 treatment while being exposed to constant light (LL + SR9009) showed a significant reduction in body weight gain compared to those that received the vehicle control (LL + Vehicle) for the same duration. (B) The constant light-induced insulin resistance was improved by SR9009. (C) The white fat mass of mice that received SR9009 treatment was significantly lower compared to those that did not receive the treatment (n = 16 for each group), indicating a potential weight-reducing effect. (D, E) The daily average food and water intakes of LD, LL + Vehicle and LL + SR9009 mice at 23 °C. (F–I) The RER, oxygen volume, carbon dioxide volume, and energy expenditure of LD, LL + Vehicle and LL + SR9009 mice during light and dark phase at 23 °C. In panels A and B, ∗ indicates the comparison made between LL + Vehicle and LL + SR9009 mice of the same time point. In panels C–I, ∗ and ∗∗ indicate statistical significance at p < 0.05 and p < 0.01, respectively. Abbreviations: LD: mice living under 12-h light/dark condition; LL + Vehicle: mice living under constant light condition and receiving 15% Cremophor treatment; LL + SR9009: mice living under constant light condition and receiving 10 mg/kg SR9009.

Metabolic cage analysis showed a slight increase in food and water intake in SR9009-treated LL mice compared to LD controls, but these differences were not statistically significant [Fig. 4D and E]. Despite the slightly higher intake, SR9009 treatment still effectively reduced weight gain. Notably, SR9009 treatment did not restore the lowered RER in LL mice [Fig. 4F], and there were no significant differences in O₂ consumption, CO₂ production, or energy expenditure between SR9009-and vehicle-treated groups [Fig. 4G–I]. These findings suggest that SR9009 primarily mitigates weight gain through mechanisms other than energy expenditure modulation.

3.5. SR9009 did not reverse behavioral changes induced by constant light exposure

To evaluate the potential of SR9009 to mitigate behavioral alterations caused by constant light exposure, we performed an open-field test on LD, LL + Vehicle, and LL + SR9009 groups. SR9009 treatment failed to reduce the heightened locomotor activity observed in the LL + Vehicle group [Fig. 5A]. Specifically, moving time [Fig. 5B], total distance traveled [Fig. 5C], rearings in Z1 [Fig. 5D], and time spent in the center of the field [Fig. 5E] remained elevated and unaffected. These results indicate that SR9009 is ineffective in counteracting the effects of constant light exposure on locomotor activity and exploratory behavior.

Fig. 5.

Fig. 5

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Behavioral analysis of mice receiving SR9009 treatment under constant light exposure for eight weeks in the open-field test. (A) Representative travel traces from the last 5 min of LD, LL + Vehicle, and LL + SR9009 mice in the arena. The moving time (B), total distance traveled (C), rearing frequencies in Z1 (D), and time spent in the center of the arena (E) were not improved by SR9009 treatment. ∗ and ∗∗ denote statistical significance at p < 0.05 and p < 0.01, respectively, as evaluated by the Mann-Whitney test. Abbreviations: LD: mice living under 12-h light/dark cycle; LL + Vehicle: mice living under constant light condition and receiving 15% Cremophor treatment; LL + SR9009: mice living under constant light condition and receiving 10 mg/kg SR9009 treatment.

3.6. SR9009 restores circadian clock gene expression in adipose tissues

To investigate whether SR9009 exerts its effects by modulating circadian gene expression, we analyzed the expression patterns of six clock genes that were altered in WAT and BAT under LL conditions. Treatment with SR9009 significantly reduced Bmal1 expression in WAT at ZT or CT 12 [Fig. 6A] and in BAT [Supplementary Fig. S3A]. Additionally, SR9009 partially restored the expression of Per3, Rev-erbα, and Rev-erbβ in WAT [Fig. 6D–F] and fully restored these genes in BAT [Supplementary Figs. S3E–F]. These findings demonstrate that SR9009 modulates circadian clock gene expression, particularly by suppressing Bmal1 and enhancing Rev-erb expression, which may contribute to the observed reductions in weight gain and insulin resistance.

Fig. 6.

Fig. 6

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The expression pattern of six core circadian clock genes in the white adipose tissue of mice treated with SR9009 and exposed to constant light for eight weeks. The expression of Bmal1(A), Per1(B), Per2(C), Per3(D), Rev-erbα(E) and Rev-erbβ(F) genes were examined at four Zeitgeber Times (ZTs) in the LD mice and at four Circadian Times (CTs) in LL + Vehicle and LL + SR9009 mice. At ZT or CT 12, the upregulated expression of Bmal1(A) in LL + Vehicle mice was decreased by SR9009 treatment, while the downregulated Per1(B), Per2(C), Per3(D), Rev-erbα(E) and Rev-erbβ(F) were restored by SR9009 treatment. Each value represents the percentage change relative to the lowest expression level in the LD group, normalized to 100%. Data are mean ± SEM (n = 4 for each time point in each condition). ∗ and ∗∗ indicate statistical significance at p < 0.05 and p < 0.01, respectively, for differences between LL + Vehicle and LL + SR9009 mice at the same time point, evaluated by the Mann-Whitney test using values of ΔCt. Abbreviations: LD: mice living under 12-h light/dark condition; LL + Vehicle: mice living under constant light condition and receiving 15% Cremophor treatment; LL + SR9009: mice living under constant light condition and receiving 10 mg/kg SR9009; SEM: standard error of the mean.

3.7. SR9009 suppresses adipogenesis in 3T3-L1 cells

To investigate whether the reduction in white fat mass observed with SR9009 treatment involved direct effects on adipogenesis, we treated 3T3-L1 cells with SR9009. SR9009 (10 μM) significantly inhibited adipocyte differentiation, as shown by reduced Oil Red O staining [Fig. 7A]. Quantification confirmed that SR9009-treated cells had a significantly lower lipid accumulation compared to controls [Fig. 7B], suggesting that SR9009 may inhibit adipogenesis, contributing to the reduction in white adipose tissue observed in vivo.

Fig. 7.

Fig. 7

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SR9009 suppressed adipocyte adipogenesis in 3T3-L1 cells. (A) Representative images of Oil Red O staining in differentiated adipocytes from 3T3-L1 cells treated with and without SR9009 for 14 and 21 days (magnification × 200). (B) The area positive for Oil Red O staining was quantified using ImageJ software. Data are presented as mean ± SEM. ∗ indicates statistical significance at p < 0.05. Abbreviation: SEM: standard error of the mean.

3.8. SR9009 selectively modulates metabolism-related genes

SR9009 exerted selective effects on adipogenesis and metabolism-related genes in WAT and BAT [Fig. 8]. In BAT, SR9009 restored the expression of key adipogenesis regulators ChREBP1a and Srebp-1c [Fig. 8A and B], while genes such as Fasn, Pparγ, and Prom1 were unaffected [Fig. 8C–E]. In WAT, SR9009 downregulated genes [Fig. 8F–I] related to mitochondrial function (Sirt3), endoplasmic reticulum (ER) stress (Atf6, Xbp1), and fatty acid oxidation (Pparα), indicating that SR9009 may reduce metabolic stress and improve efficiency. However, oxidative stress, nutrient sensing, and thermogenesis-related genes in BAT, such as Nrf 2, mTOR, and Ucp1, remained unchanged [Fig. 8J–L], indicating tissue-specific gene modulation.

Fig. 8.

Fig. 8

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The expression of metabolism-related genes in the adipose tissue of mice treated with SR9009 and exposed to constant light for eight weeks. The expression of six adipogenesis-related genes (A–E), one mitochondria-related gene (F), two ER stress response-related genes (G, H), two fatty acid oxidation-related genes (I, J), and two thermogenesis-related genes (K, L) was altered by constant light exposure and was differentially expressed, either restored or further downregulated, after SR9009 treatment. Each value represents the percentage change relative to the lowest expression level in the LD group, normalized to 100%. Data are mean ± SEM (n = 16 for each condition).∗ and ∗∗ indicate statistical significance at p < 0.05 and p < 0.01, respectively, evaluated by the Mann-Whitney test using values of ΔCt. Abbraviations: BAT: brown adipose tissue; WAT: white adipose tissue; LD: mice living under 12-h light/dark condition; LL + Vehicle: mice living under constant light condition and receiving 15% Cremophor treatment; LL + SR9009: mice living under constant light condition and receiving 10 mg/kg SR9009. ER: Endoplasmic reticulum.

4. Discussion

In developed countries, overweight, obesity, and diabetes are prevalent public health concerns. Global estimates suggest that around 35% of the population is overweight, 12% is obese, and 425 million adults have diabetes [32,33]. Although overeating, poor diet, and lack of exercise have been traditionally linked to these conditions, these factors do not fully explain the high prevalence (25%–40%) of metabolic diseases. Recent research has indicated that sleep and circadian rhythm disruption also play a significant role. Circadian rhythms are vital in regulating metabolic processes, and disruptions in these rhythms increase the risk for metabolic disorders. While lifestyle interventions, such as regular exercise and a balanced diet, can help mitigate these risks, pharmacological interventions targeting circadian rhythms could offer additional benefits.

This study utilized mice exposed to constant light for eight weeks to simulate the circadian rhythm disturbances commonly observed in long-term shift or night shift workers. Consistent with these conditions, we observed typical weight gain and heightened insulin resistance in LL mice. Additionally, the LL mice displayed increased white fat mass accumulation and dysregulation of core circadian clock genes, specifically the upregulation of Bmal1 and the downregulation of Rev-erbα and Rev-erbβ in both WAT [Fig. 3] and BAT [Supplementary Fig. S3]. Treatment with SR9009 modulated these disruptions by significantly reducing Bmal1 expression and partially or fully restoring Rev-erbα and Rev-erbβ expression, particularly in WAT [Fig. 6] and BAT [Supplementary Fig. S3]. These findings provide direct evidence that SR9009 alleviates circadian clock gene dysregulation caused by LL conditions, thus supporting its role in restoring circadian rhythm. These results align with previous studies linking circadian disruption to metabolic disorders [11,34,35] and underscore the critical role of circadian regulation in maintaining metabolic health. For instance, the Clock protein PER2 interacts with PPARα, inhibiting Bmal1 and suppressing adipocyte adipogenesis [36,37]. Mice with deficiencies in Clock, Bmal1, or Rev-erb genes exhibit various metabolic disorders [[12], [13], [14],38,39]. Human studies have also shown that circadian disruption reduces leptin levels and increases glucose and insulin levels [40]. Interestingly, PER2 and REV-ERBα expression in subcutaneous adipose tissue significantly increase following weight loss in obese individuals [39], [41]. Treatments like melatonin and metformin that improve circadian rhythm activity have been shown to enhance glucose tolerance and reduce obesity [42]. Similarly, REV-ERB agonists, such as SR9009, significantly reduce fat mass and improve hyperlipidemia and hyperglycemia in diet-induced obese mice [19]. In light of our findings, we propose that readjusting the circadian rhythm, as demonstrated by the modulation of clock gene expression with SR9009, may serve as a therapeutic strategy to alleviate metabolic symptoms caused by circadian imbalance.

The most notable finding of our study is that chronic low-dose administration of SR9009 (a REV-ERB ligand) effectively mitigated the metabolic dysfunction induced by constant light exposure. SR9009 reduced weight gain, improved insulin sensitivity, and decreased white fat mass, likely through its effects on circadian clock gene expression and adipogenesis inhibition. Previous animal experiments have suggested that SR9009 can regulate the expression of circadian rhythm genes Bmal1, Per, and Rev-erbα, leading to weight loss and decreased insulin resistance [19,43,44]. However, these studies utilized high doses and frequencies of administration (100 mg/kg body weight twice daily) and employed mouse models induced with obesity through high-fat diets. In contrast, our study focused solely on disrupting circadian rhythm through light exposure and employed a chronic (eight weeks), low-dose (10 mg/kg body weight once daily) treatment approach. Considering the health implications for shift or night workers, the safety of SR9009 is a crucial consideration. Recent research has indicated that administering high doses of SR9009 in animal studies can lead to liver damage and adverse effects [45]. Additionally, taking into account the affordability of the medication, we chose to use a low-dose regimen of SR9009. Importantly, our study demonstrates that low-dose SR9009 can achieve these effects without the toxicity associated with higher doses. This is particularly relevant for long-term use in managing metabolic disorders, as lower doses may offer a safer therapeutic window. It is also important to note that while the beneficial effects of SR9009 have been primarily attributed to its REV-ERB-dependent mechanisms, recent studies have highlighted its REV-ERB-independent effects on metabolism and other cellular processes, indicating that its full therapeutic potential may extend beyond modulation of the circadian clock [28,29].

Our study showed that constant light exposure significantly altered locomotor activity and exploratory behavior in mice, as evidenced by increased moving time, total distance traveled, rearings in Z1, and time spent in the center of the field [Fig. 2]. These findings align with previous studies showing that continuous light exposure disrupts circadian rhythms, leading to heightened locomotor behavior without significantly affecting other parameters, such as memory or baseline anxiety levels [46,47]. The observed behavioral changes may be attributed to circadian dysregulation and associated neurochemical imbalances. Notably, heightened locomotor activity could also impact metabolism and energy balance, potentially influencing body weight and metabolic homeostasis. SR9009 treatment, however, failed to mitigate these alterations, as LL + SR9009 mice exhibited similarly elevated activity levels as the LL + Vehicle group [Fig. 5]. While SR9009 has been reported to suppress circadian-driven locomotor rhythms under normal conditions [22], its efficacy in reversing behavioral changes induced by constant light exposure remains unclear. Some studies suggest that SR9009's mechanisms may operate independently of the circadian clock [48], which could explain its lack of effect in this context. These results highlight the complexity of constant light-induced behavioral changes and suggest that interventions targeting both circadian and non-circadian pathways may be necessary to counteract these effects.

Interestingly, SR9009 did not significantly alter energy expenditure or inflammatory markers [Supplementary Fig. S4], which contrasts with previous studies using higher doses. This suggests that the primary mechanism of SR9009 in our study is through modulating adipogenesis rather than altering energy balance or inflammation. Previous studies have demonstrated the anti-inflammatory effects of SR9009, but they used a dosage of 100 mg/kg body weight [24,49]. Therefore, one possible explanation for our observed results is that the lower dosage of SR9009 we used may not have yielded significant improvements. From a long-term perspective of health maintenance, using a low dosage is sufficient to stabilize the expression of circadian clock genes disrupted by circadian rhythm dysregulation, leading to reduced weight gain and insulin resistance while minimizing drug toxicity. Should this be considered a successful outcome? However, from a therapeutic perspective, more severe cases of obesity characterized by pronounced energy imbalance, inflammation, and diminished adipokines may necessitate higher doses of SR9009 for intervention. Striking a balance between health maintenance, treatment efficacy, and drug toxicity or side effects may pose a significant challenge when translating these findings to human applications in the future.

Our initial hypothesis was that SR9009 could ameliorate metabolic abnormalities caused by circadian rhythm disruption through its effects on energy balance, inflammation, and adipokines, as demonstrated in previous studies [24], [50], [51], [52]. However, it became evident that the effects of chronic low-dose SR9009 treatment differ from those observed with high-dose treatment. To further investigate the effectiveness of low-dose SR9009 in reducing weight gain, we conducted an examination of its impact on white adipocyte differentiation using 3T3-L1 cells. Remarkably, we observed a significant inhibition of adipocyte differentiation with a concentration of 10 μM SR9009. It has been suggested that SR9009 influences adipocyte differentiation involve the modulation of crucial molecular pathways in adipogenesis, such as PPARγ [37]. Our results also demonstrated the selective effects of SR9009 on adipogenesis- and metabolism-related genes in both BAT and WAT. In BAT, SR9009 treatment notably restored the expression of key adipogenesis regulators, ChREBP1a and Srebp-1c, which are important for lipid metabolism and energy balance. However, genes involved in fatty acid synthesis and adipocyte differentiation, remained unaffected. In WAT, SR9009 further downregulated genes related to mitochondrial function, ER stress, and fatty acid oxidation, suggesting a role for SR9009 in reducing metabolic stress and possibly improving metabolic efficiency. Interestingly, genes related to oxidative stress response, nutrient sensing, and thermogenesis in BAT remained unchanged, indicating that the effect of SR9009 may be more focused on lipid metabolism rather than energy expenditure or stress responses in BAT. These findings highlight the tissue-specific actions of SR9009, particularly its ability to modulate gene expression linked to lipid metabolism and stress responses, which may contribute to its anti-obesity effects. Further investigation into these pathways could provide deeper insight into the mechanisms through which SR9009 exerts its metabolic benefits.

The major limitation of this study is that, while our findings suggest SR9009 alleviates obesity by mitigating circadian clock disturbances, this study does not definitively establish whether these disturbances are a cause or consequence of obesity. Obesity itself is known to disrupt peripheral circadian clocks [53], complicating the interpretation of the observed effects. Moreover, our experiments primarily focused on 3T3-L1 cells and a constant light-induced obesity model, which, while effective, may not fully capture the complexity of circadian regulation in vivo. Although we observed weight reduction with SR9009 treatment in mice exposed to constant light for 8 weeks, we did not evaluate its effects in clock gene knockout models. Such models could provide clearer insights into the interplay between SR9009, circadian clocks, and obesity. Future studies are needed to validate whether SR9009's anti-obesity effects are mediated through direct circadian clock modulation or other mechanisms, further elucidating its role in metabolic regulation.

In addition, there are several other limitations to this study. First, SR9009 needs to be dissolved in 15% Cremophor, which is an oil-based vehicle. Although we included a control group treated with 15% Cremophor alone, it is unclear whether the effects of the oil vehicle might mask the benefits of SR9009. Second, the study focused solely on the effects of constant light exposure and did not investigate the effects of other circadian disruptions, such as jet lag or irregular sleep patterns. Third, we used only a low dose of SR9009, and the impact on energy and inflammation may not be sufficiently pronounced. Future experiments incorporating higher doses should be conducted to establish toxicity and assess potential side effects. Fourth, insulin levels were not measured in this study, limiting our ability to directly assess insulin sensitivity and determine whether the impaired glucose tolerance observed under constant light exposure reflects insulin resistance. Fifth, actogram data were not included in this study due to the lack of appropriate equipment at our institute, which limits our ability to directly assess circadian activity in LL-housed mice. Finally, it is worth noting that the study investigated the effects of one specific REV-ERB ligand, and it remains uncertain whether other compounds that modulate the circadian clock would yield similar effects.

In summary, our findings suggest chronic low-dose administration of the REV-ERB ligand SR9009 effectively alleviated weight gain and insulin resistance caused by constant light exposure. By targeting circadian clock pathways with REV-ERB ligands like SR9009 may offer a promising therapeutic approach for preventing or treating metabolic disorders associated with circadian disruption. Future studies should explore the long-term effects of SR9009 and evaluate its potential in clinical settings.

5. Conclusions

Our study demonstrates that constant light exposure disrupts circadian clock gene expression, leading to metabolic changes such as weight gain, insulin resistance, and increased white fat mass in mice. Chronic low-dose SR9009 treatment effectively mitigates these adverse effects, likely through its modulation of circadian clock genes and suppression of adipogenesis. These findings suggest that SR9009 may serve as a potential therapeutic agent for circadian rhythm-related metabolic disorders.

CRediT authorship contribution statement

Ming-Yu Yang: conceptualization, funding acquisition, supervision, confirm the authenticity of all the raw data, writing - original draft, writing - review & editing. Hugo Y.-H. Lin: investigation, validation. Yi-Ywan M. Chen: conceptualization, validation. Ming-Luen Hu: conceptualization, data curation, writing - review & editing. I-Ya Chen: formal analysis, methodology, project administration. Chao-Hui Yang: conceptualization, funding acquisition, supervision, confirm the authenticity of all the raw data, writing - review & editing. All authors have read and approved the final manuscript.

Availability of data and materials

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Funding

This work was supported by Chang Gung Memorial Hospital, Taiwan [grant numbers CMRPD8J0011, CMRPD8J0012, CMRPD8J0013, and CMRPD8P0011 to Ming-Yu Yang]; and the National Science and Technology Council (Ministry of Science and Technology), Taiwan [grant number NSTC 111-2635-B-182-001 to Ming-Yu Yang and MOST 107-2314-B-182A-083, MOST 109-2314-B-182A-016, and MOST 110-2314-B-182A-102 to Chao-Hui Yang].

Conflict of interest statement

The authors declare no competing interests.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bj.2025.100830.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Multimedia component 1
mmc1.docx (2.8MB, docx)

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Associated Data

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Supplementary Materials

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Data Availability Statement

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

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