Stephanoside B Modulates PPARγ-Dependent Metabolic Genes and Lengthens the Circadian Bmal1 Oscillation Period in Differentiated Myotubes Revealed by Real-Time Bioluminescence

Stephanoside B Modulates PPARγ-Dependent Metabolic Genes and Lengthens the Circadian Bmal1 Oscillation Period in Differentiated Myotubes Revealed by Real-Time Bioluminescence | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Stephanoside B Modulates PPARγ-Dependent Metabolic Genes and Lengthens the Circadian Bmal1 Oscillation Period in Differentiated Myotubes Revealed by Real-Time Bioluminescence Papawee Saiki, Tatsunosuke Tomita, Saori Saori Yamamoto This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8951484/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Gymnema (G.) inodorum is a medicinal plant with anti-diabetic, anti-obesity, and anti-inflammatory properties traditionally consumed as tea in Southeast Asia. While bioactive stephanosides and gymnemic acids suppress adipocyte differentiation, their effects on skeletal muscle metabolism and circadian regulation remain unclear. Here, we investigated the actions of gymnemic acid (GiA-7) and stephanosides B and C in differentiated C2C12 myotubes. Stephanoside B selectively and concentration-dependently upregulated Pparg and Ppargc1a expression under basal conditions and after dexamethasone-induced circadian synchronization. It also enhanced expression of the core clock genes Nr1d1 , Per2 , and Cry1 , while repressing Bmal1 , consistent with known PPARγ–circadian interactions. To directly monitor circadian rhythmicity, we established a real-time Bmal1 -luciferase reporter gene assay in differentiated myotubes. Continuous bio-luminescence tracking over several days revealed that stephanoside B specifically lengthened the circadian period, demonstrating modulation of molecular clock function. Collectively, these results indicate that stephanoside B coordinately regulates metabolic gene expression and circadian rhythms in the skeletal muscle. Given the central role of muscle in systemic energy homeostasis and circadian regulation, these findings highlight the chrono-nutritional potential of G. inodorum extracts. Moreover, this study provides one of the first demonstrations of real-time circadian reporter analysis in differentiated myotubes, offering a novel platform to explore dietary compounds that influence muscle-specific metabolic and circadian processes. PPARγ signaling Metabolic gene regulation Circadian regulation Skeletal muscle metabolism Bmal1-luciferase reporter Stephanoside B Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Gymnema (G.) inodorum is a traditional edible leafy plant consumed as a vegetable and later developed into a tea in Southeast Asia. It has anti-diabetic, -obesity, and -inflammatory effects [ 1 – 3 ]. We previously found that bioactive gymnemic acid (GiA-7), as well as stephanosides B, and C in G. inodorum suppress adipocyte differentiation in 3T3-L1 cells by inhibiting expression of the peroxisome proliferator–activated receptor γ (PPARγ)-dependent gene and modulating downstream peroxisome proliferator–activated receptor γ co-activator 1 α ( Ppargc1a ) [ 4 ]. These compounds circulate systemically when orally ingested and reach peripheral tissues, including skeletal muscle. Although skeletal muscle expresses less PPARγ than adipose tissue, it is a key organ in systemic energy metabolism [ 5 ] and it functions as a heterodimer with the retinoid X receptor (RXR) [ 6 ]. Together with cofactors such as PGC-1α, PPARγ regulates transcription of metabolic genes involved in lipid oxidation and mitochondrial function [ 7 , 8 ]. Skeletal muscle also harbors an intrinsic circadian clock, consisting of transcriptional–translational feedback loops involving Brain and muscle ARNT-Like 1 ( Bmal1 ), Clock Circadian Regulator ( Clock ), Periods ( Per s), Cryptochromes ( Cry s), nuclear receptor subfamily 1, group d, member 1 ( Nr1d1 ) and RAR-related orphan receptor α ( Rorα ) [ 9 ]. This clock orchestrates daily rhythms of gene expression and metabolism. Notably, NR1D1 contributes to mitochondrial biogenesis and oxidative capacity, and its loss impairs mitochondrial content and exercise performance [ 10 ]. Emerging evidence has suggested reciprocal regulatory interactions between PPARγ signalling and circadian clock components. For example, PPARγ directly regulates Nr1d1 and aligns metabolic gene expression with circadian cues [ 11 ]. In contrast, PGC-1α induces Bmal1 expression through ROR/REV-ERB-response element (RORE) activation and integrates metabolic and circadian regulatory networks [ 12 ]. These findings together suggest that G. inodorum extracts could influence skeletal muscle metabolism and circadian regulation by modulating PPARγ, PGC-1α, and related pathways. However, the effects of G. inodorum derived compounds on skeletal muscle circadian mechanisms remain largely unexplored, and no study has yet examined their real-time influence on muscle clock gene oscillations. Therefore, we first assessed the effects of three compounds derived from G. inodorum on the expression of Pparg and Ppargc1a in differentiated C2C12 myotubes. We further analyzed a compound that upregulated both genes. Since circadian rhythms influence Pparg and Ppargc1a expression, we investigated the effects of these compounds on Pparg and Ppargc1a in C2C12 cells after circadian phase resetting. Thereafter, we evaluated the concentration-dependent effects of the compounds on the expression of the clock genes Bmal1 , Per2 , Nr1d1 and Cry1 under the same conditions. Finally, we established a Bmal1 -luciferase reporter gene assay in differentiated C2C12 myotubes to directly monitor real-time circadian oscillations. This experimental system enabled continuous measurement of Bmal1 promoter activity over several days, providing a novel analytical approach to investigate compounds that modulate molecular circadian regulation in skeletal muscle. This integrative approach provided new insights into the potential of G. inodorum derived triterpenoids, particularly stephanoside B, to modulate metabolic and circadian processes in muscle cells. Materials and methods Compounds and Reagents Harmine hydrochloride was obtained from Tokyo Kasei (TCI), Tokyo, Japan) [ 13 ], whereas GiA-7, stephanoside B, and stephanoside C were extracted from G. inodorum tea and purified to > 98% purity by high-performance liquid chromatography (HPLC). Their chemical structure were confirmed by nuclear magnetic resonance (NMR) spectroscopy as previously described [ 4 ]. Cell culture We cultured C2C12 mouse myoblasts (RIKEN BioResource Research Center, Tsukuba, Japan) in Dulbecco’s Modified Eagle’s Medium High Glucose with L-glutamine, phenol red and sodium pyruvate (DMEM; Fujifilm Wako Pure Chemical Corp., Osaka, Japan) containing 10% fetal bovine serum (FBS; Sigma-Aldrich Corp.) and 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Nacalai Tesque, Kyoto, Japan). The C2C12 cells were differentiated into myotubes using 2% horse serum (HS; Sigma-Aldrich Corp.) in DMEM [ 14 , 15 ] which was confirmed by the elongated, cylindrical, multinucleated morphological features of myotubes [ 16 ]. Cell proliferation assay We evaluated cell proliferation using a colorimetric assay of mitochondrial enzyme activity, which reflects the number of viable, metabolically active cells. We seeded 2 × 10 3 C2C12 myoblasts/well into 96-well flat-bottom microplates (Thermo Fisher Scientific Inc., Waltham, MA, USA) in DMEM and incubated them at 37°C under a 5% CO 2 atmosphere (standard conditions) until they reached confluence. The cells were incubated with or without 5, 10, 25, 50, or 100 µM extracts for 24 h. Subsequently, CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (MTS) (Promega Corp., Madison, WI, USA) was added directly to the culture medium as described by the manufacturer, then the cells were incubated for 30 minutes under standard conditions. Absorbance at 495 nm was determined in four biological replicates using a Model 550 Microplate Reader (Bio-Rad Laboratories Inc., Hercules, CA, USA) to determine mitochondrial enzyme activity [ 17 ]. Cell proliferation (%) was calculated relative to that of the vehicle. Gene expression evaluation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) We seeded C2C12 myoblasts (1 × 10 5 /well) into 12-well plates (Iwaki Co., Ltd., Tokyo, Japan) in DMEM containing 10% FBS and incubated them at 37°C under a 5% CO 2 humidified atmosphere until they reached confluence. The medium was replaced with DMEM containing 2% HS to induce myogenic differentiation and refreshed every two days for a total of four days. After confirming myotube formation, the cells were incubated with the G. inodorum extracts in DMEM containing 10% FBS. We synchronized circadian gene expression by stimulating C2C12 myotubes with 100 nM water-soluble DEX in serum-free DMEM for 2 h before being exposed to the G. inodorum extracts for 24 h. The cells were washed twice with phosphate-buffered saline (PBS; Takara Bio Inc., Kusatsu, Japan) to remove residual medium. Total RNA was extracted using RNAiso Plus reagent (Takara Bio Inc.) as described by the manufacturer then reverse transcribed using PrimeScript™ RT Master Mix (Takara Bio Inc). Complementary (c) DNA was amplified by qRT-PCR using SYBR® Premix Ex Taq™ II (Takara Bio Inc.) on a StepOne™ Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) and the primers (Thermo Fisher Scientific Inc.) listed in Table S1 [ 18 , 19 ]. The cycling conditions for target sequences comprised initial denaturation at 95°C for 10 s, followed by 45 cycles of 95°C for 5 s, and 60°C for 10 s. All experiments included three biological replicates and qRT-PCR included two technical duplicates per RNA sample to ensure the reproducibility of gene expression. Relative gene expression was analyzed using the ΔΔCt method and normalized to that of Actin β ( Actb ) [ 20 ] and is expressed relative to the vehicle (control). Statistical analyses were conducted using EZR software [ 21 ]. One-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test was applied for pairwise comparisons. Results are expressed as mean ± SD, with significance indicated by p-values. Novel real-time bioluminescence monitoring of Bmal1 transcriptional activity in C2C12 cells We seeded 2 × 10 5 C2C12 myoblasts per well into 35 mm culture dishes (Iwaki Co., Ltd.) and maintained under standard culture conditions. Confluent cells were transfected with a Bmal1 -Luc reporter plasmid (containing the − 197 to + 27 bp region upstream of the transcription start site [ 20 ] using Lipofectamine™ 3000 (Thermo Fisher Scientific Inc.) as described by the manufacturer. On the following day the cells were incubated in differentiation medium, for 3 days to form myotubes. We synchronized circadian gene expression by incubating the transfected myotubes with either 100 nM water-soluble DEX or 50% HS in serum-free DMEM for 2 h [ 22 ]. The myotubes were incubated with or without G. inodorum extracts in DMEM containing 2% HS, 0.1 mM D-luciferin potassium salt (Fujifilm Wako Pure Chemical Industries), and 25 mM HEPES (Thermo Fisher Scientific Inc.). Real-time bioluminescence was measured at 37°C for 1 min [ 23 ] every 10 minutes for 7 consecutive days using a Kronos AB-2500 luminometer (ATTO Corporation, Tokyo, Japan) and circadian period length was analyzed as described [ 24 , 25 ]. Real-time bioluminescence monitoring stable Bmal1 -Luc NIH3T3 reporter cells A stable NIH3T3 cell line expressing a luciferase reporter under the control of the Bmal1 promoter was established to monitor circadian transcriptional activity [ 13 ]. We incubated 5 × 10 5 NIH3T3 cells seeded in 35 mm dishes (Iwaki Co., Ltd.) containing DMEM supplemented with 10% FBS, and at 37°C under a 5% CO 2 atmosphere for one day. Rhythmic Bmal1 expression was initiated and synchronized by incubating the cells with 100 nM DEX in serum-free DMEM for 2 h. The medium was replaced with DMEM containing 0.1 mM D-luciferin potassium salt and 25 mM HEPES, with or without G. inodorum extracts. Real-time bioluminescence intensity was evaluated using the Kronos AB-2500 luminometer and circadian period length was estimated as described [ 24 , 25 ]. Results Stephanoside B changed Pparg and Ppargc1a expression in differentiated C2C12 cells We previously found that GiA-7, and the stephanosides B and C extracted from G. inodorum suppress adipocyte differentiation without significant toxicity to 3T3-L1 cells. We used 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) assays to determine the cytotoxicity of the extracts in myotubes after 24 h. Figure S1 shows that GiA-7 at 50 and 100 µM and stephanoside C at 100 µM reduced cell viability to ~ 60%, whereas stephanoside B maintained viability at ~ 80%. Therefore, 25 µM was the maximal concentration of these extracts in subsequent experiments to ensure minimal cytotoxicity while assessing cellular responses. We analyzed the effects of 25 µM extracts for 24 h on the Pparg and Ppargc1a genes that express proteins with metabolic functions in differentiated C2C12 cells. Total RNA was isolated, reverse-transcribed, then Pparg and Ppargc1a expression was assessed using qRT-PCR. Figure 1 shows that stephanoside B significantly upregulated the expression of both genes. Stephanoside C significantly upregulated Ppargc1a gene expression but did not affect Pparg expression. Gymnemic acid-7 did not significantly change the expression of either gene. These results imply that GiA-7, and the stephanosides B and C affect skeletal muscle cells and adipocytes in different ways. Stephanoside B also upregulated Pparg and Ppargc1a expression in differentiated C2C12 cells stimulated with DEX. Because Pparg and Ppargc1a expression might fluctuate according to circadian phases, we considered literature indicating that these genes are expressed rhythmically throughout the day. Such rhythmic expression might have obscured changes induced by the extracts. Therefore, we synchronised the circadian clock in differentiated C2C12 cells using DEX, incubated them with 10 or 25 µM stephanoside B for 24 h, then evaluated the expression of Pparg and Ppargc1a . The expression of both Pparg and Ppargc1a in cells incubated with stephanoside B concentration-dependently increased compared with the vehicle (ethanol). Specifically, 10 and 25 µM stephanoside B respectively increased Pparg expression 1.5- and 1.9-fold relative to the vehicle (Fig. 2 ). These results indicated that stephanoside B consistently and concentration-dependently upregulated Pparg and Ppargc1a after resetting cellular circadian rhythms. 3.3. Stephanoside B changed clock gene expression in differentiated C2C12 cells with reset circadian clocks. We examined expression of the core clock genes, Bmal1 , Per2 , Cry1 and Nr1d1 in differentiated C2C12 myotubes with DEX-induced reset circadian rhythms. Figure 3 shows the qRT-PCR results of cDNA reverse transcribed from the RNA samples described above. A peroxisome proliferator response element (PPRE) in the promoter region of Nr1d1 is transcriptionally regulated by PPARγ combined with RXR. Consistent with this, stephanoside B concentration-dependently increased Nr1d1 expression in DEX-stimulated cells compared with controls 24 h after DEX stimulation. The expression of Per2 and Cry1 was also elevated under these conditions. In contrast, the expression of Bmal1 , which is repressed by NR1D1 via retinoic acid-related orphan receptor response elements (ROREs), concentration-dependently decreased. Stephanoside B elongated Bmal1 period length in differentiated C2C12 cells and NIH3T3 fibroblasts. Analyses of clock gene expression at one time point (24 h) suggested that stephanoside B should influence the rhythmic expression of the circadian clock. We considered that the transcriptional changes induced by stephanoside B might be associated with changes in the oscillatory rhythms of clock gene expression. Therefore, we established a real-time reporter assay system in myotubes that were differentiated from C2C12 cells. We optimized the previous transfection strategy because Bmal1 expression was more stable when the Bmal1 reporter plasmid was introduced before, than after differentiation (data not shown). Harmine can elongate the period in other cells [ 13 , 25 ] or tissues [ 26 ] and we found similar effects on C2C12 cells. We then compared synchronization using DEX or 50% HS [ 27 ] and found that Bmal1 rhythms were more stable using DEX (Supplementary Figure S2). We differentiated C2C12 cells by transfection with Bmal1 –luciferase ( Bmal1 –Luc) reporter plasmids. The differentiated cells were then synchronised by incubation with DEX for 2 h, after which the culture fluid was replaced with medium containing luciferin supplemented with extracts. Bioluminescence was then monitored in real time (Fig. 4 ). Using this transient expression system, circadian oscillations of clock gene reporter activity were robust for several days. Furthermore, 25 µM stephanoside B elongated the circadian period to 25.7 h in both C2C12 and NIH3T3 cells. These results indicate that this phenomenon is not restricted to C2C12 myotubes (Supplementary Figure S3). Together, these results demonstrate that our optimized real-time Bmal1 monitoring system provides a reliable platform to evaluate nutrient-derived modulators of muscle circadian rhythms. Discussion Here, we investigated the effects of GiA-7, and stephanosides B and C extracted from G. inodorum on skeletal muscle cells, with focus particularly on energy metabolism and the molecular circadian clock. Among the compounds tested, stephanoside B upregulated Pparg expression in differentiated C2C12 myotubes, whereas GiA-7 and stephanoside C had no significant effects. Notably, this response contrasts with our previous observations in adipocytes, highlighting a clear cell type–specific difference in the biological actions of G. inodorum –derived compounds. The mechanistic basis for these cell type–specific responses awaits elucidation.The expression of PPARγ is abundant in adipose tissue and the liver [ 28 ], but lower in skeletal muscle [ 29 , 30 ]. Nevertheless, muscle PPARγ might function in the regulation of energy metabolism [ 31 ]. The transcription of Nr1d1 can be directly upregulated via the PPRE element by PPARγ [ 32 ]. NR1D1 not only functions in the regulation of cellular redox states but also acts as a transcription factor that directly suppresses Bmal1 expression via the RORE [ 33 , 34 ]. Our results are consistent with these findings, because stephanoside B induced concentration-dependently increases in Pparg and Ppargc1a expression together with upregulated Nr1d1 and a corresponding decrease in Bmal1 expression. Together, PPARγ and PGC-1α regulate the expression of many genes and their upregulation should result in upregulated downstream genes. We speculate that the mechanisms through which stephanoside B regulates Pparg differ from those in adipocytes, which warrants further mechanistic investigation. Circadian genes expressed in skeletal muscle regulate the expression of GLUT4 and other proteins involved in glucose metabolism, which contributes to the maintenance of physiological homeostasis [ 35 ]. These considerations highlight the value of experimental systems that can monitor clock gene expression in muscle. In this study, we successfully established the real-time Bmal1 -luciferase monitoring system in differentiated C2C12 myotubes. To our knowledge, this represents one of the first demonstrations of real-time circadian promoter activity tracking in fully differentiated skeletal muscle cells. The system allowed for the continuous observation of circadian oscillations and period changes induced by compounds in a differentiated muscle model relevant to physiology. Taken together with our previous findings that G. inodorum extracts suppress adipocyte differentiation, the present study revealed that stephanoside B can modulate the circadian period in muscle via PPARγ-associated regulation of clock gene expression. G. inodorum has long been consumed as a food plant and tea, and is widely associated with metabolic health benefits. From a nutritional biochemistry perspective, modulation of skeletal muscle circadian timing by plant-derived triterpenoids may represent an additional mechanism through which dietary components influence metabolic homeostasis. Our findings suggest a novel potential influence of G. inodorum on skeletal muscle physiology by modulating the molecular circadian clock. Conclusions We investigated the effects of three G. inodorum extracts on skeletal muscle metabolism and circadian regulation. Among them, stephanoside B selectively upregulated Pparg and Ppargc1a expression in differentiated C2C12 myotubes under basal conditions and after circadian resetting. Consistent with these transcriptional changes, stephanoside B modulated core clock gene expression by inducing Nr1d1 , Per2 , and Cry1 while repressing Bmal1 . Using the newly established real-time Bmal1 –luciferase reporter system in differentiated C2C12 myotubes, we directly demonstrated that stephanoside B lengthened the circadian period, providing dynamic evidence that a dietary plant-derived compound has potential for modulation of the muscle-intrinsic molecular clock properties. These findings indicated that stephanoside B exerts regulatory effects on skeletal muscle by enhancing PPARγ/PGC-1α signaling and influencing molecular circadian rhythms. Given the central role of skeletal muscle in systemic energy metabolism and its contribution to whole-body circadian organization, the ability of stephanoside B to influence metabolic gene networks and circadian rhythms suggests a potential mechanism that may contribute to previously reported metabolic effects of G. inodorum . Collectively, our study identifies a novel chrono-nutritional action of G. inodorum derived compounds and establishes a real-time analytical platform for exploring nutritionally driven regulation of molecular circadian rhythms specifically in muscle. Declarations Contribution statement Papawee Saiki: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Data curation, Formal analysis, Conceptualization. Tatsunosuke Tomita: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. Saori Yamamoto: Methodology, Investigation Declaration of Competing Interest There are no conflicts to declare. Acknowledgement We thank the National Institute of Advanced Industrial Science and Technology (AIST) for support. We also thank Katsutaka Oishi and Tomoki Abe for their helpful discussions. 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Supplementary Files supplementdata.docx abstractfig.jpg Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 23 Mar, 2026 Reviews received at journal 22 Mar, 2026 Reviews received at journal 11 Mar, 2026 Reviewers agreed at journal 01 Mar, 2026 Reviewers agreed at journal 27 Feb, 2026 Reviewers invited by journal 27 Feb, 2026 Editor assigned by journal 25 Feb, 2026 Submission checks completed at journal 25 Feb, 2026 First submitted to journal 23 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8951484","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":598771843,"identity":"8009d074-c4be-4058-aa17-ede1a3e41246","order_by":0,"name":"Papawee Saiki","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYHADxmaGhAoGHiiPjVgtZ4Ba2IjXwsDMwNhGhGJz9rMHP/NU2DDItx9uNng4z05Gfn4D84sPDHx5uLRY9uQlS/OcSWMwOJPYnJC4LZnH4BgDm+UMBrZiXFoMDuQYSOe2Ha7fwJDYfCBxGzOPARsDmzHQR4kNuLScf2P8O/fffwb5/odALXPqeeTbCGm5kWMmndtwgIHhBshhDYd5GI4xMD/Gp8Vyxrs06z/HkoF6HzYbJBw7DvRLYhvjDAPcfjHnzz18c0aNHdBh6Y8lf9RU28s3Hz784UPFMZwhZgCPbgRgbJNgMDiWQIoWBuYPDAw1OLWMglEwCkbBiAMAvGFUF4QkILgAAAAASUVORK5CYII=","orcid":"","institution":"National Institute of Advanced Industrial Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Papawee","middleName":"","lastName":"Saiki","suffix":""},{"id":598771844,"identity":"c708a136-ba53-4d5f-acea-48c7d2f1ffa0","order_by":1,"name":"Tatsunosuke Tomita","email":"","orcid":"","institution":"National Institute of Advanced Industrial Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Tatsunosuke","middleName":"","lastName":"Tomita","suffix":""},{"id":598771845,"identity":"48593e51-91d3-47d7-a1c3-ba5e83c31b8f","order_by":2,"name":"Saori Saori Yamamoto","email":"","orcid":"","institution":"National Institute of Advanced Industrial Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Saori","middleName":"Saori","lastName":"Yamamoto","suffix":""}],"badges":[],"createdAt":"2026-02-24 01:23:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8951484/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8951484/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103933257,"identity":"2461b12c-c910-4024-8a87-7644c899ccb7","added_by":"auto","created_at":"2026-03-04 17:04:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":141984,"visible":true,"origin":"","legend":"\u003cp\u003eStephanoside B upregulated \u003cem\u003ePparg\u003c/em\u003e and \u003cem\u003ePpargc1a \u003c/em\u003eexpression. We incubated C2C12 cells with vehicle (solvent) or extracts (25 μM) for 24 h, then total RNA was obtained from cell lysates. Expression of (A) \u003cem\u003ePparg\u003c/em\u003eand (B)\u003cem\u003e Ppargc1a\u003c/em\u003e analyzed by qRT-PCR. Panels (A) and (B) are derived from same set of samples. Horizontal axes show GiA-7 (a) stephanosides B (b), and C (c), respectively. Vertical axes indicate expression normalized to \u003cem\u003eActb\u003c/em\u003eand relative to vehicle (set as 1). Data are shown as means ± SD of four biological replicates per condition. Effects of each compound were compared with vehicle. Values with *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001 were considered statistically significant (Dunnett’s post hoc test).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8951484/v1/2f4d4fb5ec8ba84222bd895c.png"},{"id":104401588,"identity":"6db9be88-773b-494b-a4e1-809d4dbdb496","added_by":"auto","created_at":"2026-03-11 12:13:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":149176,"visible":true,"origin":"","legend":"\u003cp\u003eStephanoside B concentration-dependently upregulated \u003cem\u003ePparg\u003c/em\u003e and \u003cem\u003ePpargc1a \u003c/em\u003eexpression in C2C12 cells stimulated with DEX and influenced clock gene expression. We incubated C2C12 cells with 100 nM DEX for 2 h followed by 24 h without (vehicle) or with 10 or 25 µM stephanoside B. Total RNA was extracted from cell lysates then gene expression was analyzed using qRT-PCR. (A), \u003cem\u003ePparg\u003c/em\u003e; (B), \u003cem\u003ePpargc1a\u003c/em\u003e. All panels are derived from same set of samples. X axes, stephanoside B concentrations. Y axis, relative expression normalized to Actb and expressed relative to vehicle (set at 1). Data are shown as means ± SD of four biological replicates per condition. Effects of each compound were compared with vehicle. Values with *P \u0026lt; 0.05; **P \u0026lt; 0.001 were considered statistically significant (Dunnett’s post hoc test).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8951484/v1/ee9bb0db8df51c4b1a1d9fc0.png"},{"id":103933259,"identity":"31ac4c22-95c5-438a-8615-eea204e31f52","added_by":"auto","created_at":"2026-03-04 17:04:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":286090,"visible":true,"origin":"","legend":"\u003cp\u003eStephanoside B altered clock gene expression in differentiated C2C12 cells. We stimulated C2C12 cells with 100 nM DEX for 2 h followed by incubation for 24 h with vehicle (0), 10 or 25 μM stephanoside B. Total RNA was extracted from cell lysates, then we analyzed gene expression using qPCR. (A), \u003cem\u003eBmal1\u003c/em\u003e; (B), \u003cem\u003eNr1d1\u003c/em\u003e; (C) \u003cem\u003eCry1\u003c/em\u003e; (D) \u003cem\u003ePer2\u003c/em\u003e: All panels are derived from the same set of samples. X axis, concentrations of stephanoside B. Y axis, relative gene expression normalized to \u003cem\u003eActb\u003c/em\u003e and expressed relative to vehicle (set to 1). Data are shown as means ± SD of four biological replicates per condition. Effects of each compound were compared with vehicle. Values with *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001 were considered statistically significant (Dunnett’s post hoc test).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8951484/v1/d46da70b5eaddd00bb69c36f.png"},{"id":103933261,"identity":"4692b3e1-b4ef-4053-b35a-1af9f2f025f4","added_by":"auto","created_at":"2026-03-04 17:04:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":93490,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of stephanoside B at 10 and 25 µM on \u003cem\u003eBmal1\u003c/em\u003e-luciferase activity in transfected C2C12 cells. (A) Period length estimated from curve fitting analysis. (B) Real-time bioluminescence profiles.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8951484/v1/a16f54ca120dfb6fd8f87047.png"},{"id":104408486,"identity":"48eeac19-37fe-4995-89f1-13fb1354353e","added_by":"auto","created_at":"2026-03-11 12:42:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1118665,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8951484/v1/246aef96-a748-4c33-b290-021f301fdfdf.pdf"},{"id":103933260,"identity":"3f4a2a4c-6f1b-4398-9605-36fb8e122053","added_by":"auto","created_at":"2026-03-04 17:04:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":387576,"visible":true,"origin":"","legend":"","description":"","filename":"supplementdata.docx","url":"https://assets-eu.researchsquare.com/files/rs-8951484/v1/2b8b33af55ef7fff01930b7d.docx"},{"id":103933262,"identity":"a1cb2c45-f346-4c3f-9f94-669b0d78f10a","added_by":"auto","created_at":"2026-03-04 17:04:27","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":471294,"visible":true,"origin":"","legend":"","description":"","filename":"abstractfig.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8951484/v1/08e9f799b68356c9295f4af0.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eStephanoside B Modulates PPARγ-Dependent Metabolic Genes and Lengthens the Circadian Bmal1 Oscillation Period in Differentiated Myotubes Revealed by Real-Time Bioluminescence\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eGymnema (G.) inodorum\u003c/em\u003e is a traditional edible leafy plant consumed as a vegetable and later developed into a tea in Southeast Asia. It has anti-diabetic, -obesity, and -inflammatory effects [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. We previously found that bioactive gymnemic acid (GiA-7), as well as stephanosides B, and C in \u003cem\u003eG. inodorum\u003c/em\u003e suppress adipocyte differentiation in 3T3-L1 cells by inhibiting expression of the peroxisome proliferator\u0026ndash;activated receptor γ (PPARγ)-dependent gene and modulating downstream peroxisome proliferator\u0026ndash;activated receptor γ co-activator 1 α (\u003cem\u003ePpargc1a\u003c/em\u003e) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese compounds circulate systemically when orally ingested and reach peripheral tissues, including skeletal muscle. Although skeletal muscle expresses less PPARγ than adipose tissue, it is a key organ in systemic energy metabolism [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and it functions as a heterodimer with the retinoid X receptor (RXR) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Together with cofactors such as PGC-1α, PPARγ regulates transcription of metabolic genes involved in lipid oxidation and mitochondrial function [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSkeletal muscle also harbors an intrinsic circadian clock, consisting of transcriptional\u0026ndash;translational feedback loops involving Brain and muscle ARNT-Like 1 (\u003cem\u003eBmal1\u003c/em\u003e), Clock Circadian Regulator (\u003cem\u003eClock\u003c/em\u003e), Periods (\u003cem\u003ePer\u003c/em\u003es), Cryptochromes (\u003cem\u003eCry\u003c/em\u003es), nuclear receptor subfamily 1, group d, member 1 (\u003cem\u003eNr1d1\u003c/em\u003e) and RAR-related orphan receptor α (\u003cem\u003eRorα\u003c/em\u003e) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This clock orchestrates daily rhythms of gene expression and metabolism. Notably, NR1D1 contributes to mitochondrial biogenesis and oxidative capacity, and its loss impairs mitochondrial content and exercise performance [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEmerging evidence has suggested reciprocal regulatory interactions between PPARγ signalling and circadian clock components. For example, PPARγ directly regulates \u003cem\u003eNr1d1\u003c/em\u003e and aligns metabolic gene expression with circadian cues [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In contrast, PGC-1α induces \u003cem\u003eBmal1\u003c/em\u003e expression through ROR/REV-ERB-response element (RORE) activation and integrates metabolic and circadian regulatory networks [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These findings together suggest that \u003cem\u003eG. inodorum\u003c/em\u003e extracts could influence skeletal muscle metabolism and circadian regulation by modulating PPARγ, PGC-1α, and related pathways.\u003c/p\u003e \u003cp\u003eHowever, the effects of \u003cem\u003eG. inodorum\u003c/em\u003e derived compounds on skeletal muscle circadian mechanisms remain largely unexplored, and no study has yet examined their real-time influence on muscle clock gene oscillations. Therefore, we first assessed the effects of three compounds derived from \u003cem\u003eG. inodorum\u003c/em\u003e on the expression of \u003cem\u003ePparg\u003c/em\u003e and \u003cem\u003ePpargc1a\u003c/em\u003e in differentiated C2C12 myotubes. We further analyzed a compound that upregulated both genes. Since circadian rhythms influence \u003cem\u003ePparg\u003c/em\u003e and \u003cem\u003ePpargc1a\u003c/em\u003e expression, we investigated the effects of these compounds on \u003cem\u003ePparg\u003c/em\u003e and \u003cem\u003ePpargc1a\u003c/em\u003e in C2C12 cells after circadian phase resetting. Thereafter, we evaluated the concentration-dependent effects of the compounds on the expression of the clock genes \u003cem\u003eBmal1\u003c/em\u003e, \u003cem\u003ePer2\u003c/em\u003e, \u003cem\u003eNr1d1\u003c/em\u003e and \u003cem\u003eCry1\u003c/em\u003e under the same conditions.\u003c/p\u003e \u003cp\u003eFinally, we established a \u003cem\u003eBmal1\u003c/em\u003e-luciferase reporter gene assay in differentiated C2C12 myotubes to directly monitor real-time circadian oscillations. This experimental system enabled continuous measurement of \u003cem\u003eBmal1\u003c/em\u003e promoter activity over several days, providing a novel analytical approach to investigate compounds that modulate molecular circadian regulation in skeletal muscle. This integrative approach provided new insights into the potential of \u003cem\u003eG. inodorum\u003c/em\u003e derived triterpenoids, particularly stephanoside B, to modulate metabolic and circadian processes in muscle cells.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eCompounds and Reagents\u003c/p\u003e \u003cp\u003eHarmine hydrochloride was obtained from Tokyo Kasei (TCI), Tokyo, Japan) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], whereas GiA-7, stephanoside B, and stephanoside C were extracted from \u003cem\u003eG. inodorum\u003c/em\u003e tea and purified to \u0026gt;\u0026thinsp;98% purity by high-performance liquid chromatography (HPLC). Their chemical structure were confirmed by nuclear magnetic resonance (NMR) spectroscopy as previously described [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCell culture\u003c/p\u003e \u003cp\u003eWe cultured C2C12 mouse myoblasts (RIKEN BioResource Research Center, Tsukuba, Japan) in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium High Glucose with L-glutamine, phenol red and sodium pyruvate (DMEM; Fujifilm Wako Pure Chemical Corp., Osaka, Japan) containing 10% fetal bovine serum (FBS; Sigma-Aldrich Corp.) and 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Nacalai Tesque, Kyoto, Japan). The C2C12 cells were differentiated into myotubes using 2% horse serum (HS; Sigma-Aldrich Corp.) in DMEM [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] which was confirmed by the elongated, cylindrical, multinucleated morphological features of myotubes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCell proliferation assay\u003c/p\u003e \u003cp\u003eWe evaluated cell proliferation using a colorimetric assay of mitochondrial enzyme activity, which reflects the number of viable, metabolically active cells. We seeded 2 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e C2C12 myoblasts/well into 96-well flat-bottom microplates (Thermo Fisher Scientific Inc., Waltham, MA, USA) in DMEM and incubated them at 37\u0026deg;C under a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere (standard conditions) until they reached confluence. The cells were incubated with or without 5, 10, 25, 50, or 100 \u0026micro;M extracts for 24 h. Subsequently, CellTiter 96\u003csup\u003e\u0026reg;\u003c/sup\u003e AQueous One Solution Cell Proliferation Assay (MTS) (Promega Corp., Madison, WI, USA) was added directly to the culture medium as described by the manufacturer, then the cells were incubated for 30 minutes under standard conditions. Absorbance at 495 nm was determined in four biological replicates using a Model 550 Microplate Reader (Bio-Rad Laboratories Inc., Hercules, CA, USA) to determine mitochondrial enzyme activity [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Cell proliferation (%) was calculated relative to that of the vehicle.\u003c/p\u003e \u003cp\u003eGene expression evaluation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR)\u003c/p\u003e \u003cp\u003eWe seeded C2C12 myoblasts (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e/well) into 12-well plates (Iwaki Co., Ltd., Tokyo, Japan) in DMEM containing 10% FBS and incubated them at 37\u0026deg;C under a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified atmosphere until they reached confluence. The medium was replaced with DMEM containing 2% HS to induce myogenic differentiation and refreshed every two days for a total of four days. After confirming myotube formation, the cells were incubated with the \u003cem\u003eG. inodorum\u003c/em\u003e extracts in DMEM containing 10% FBS. We synchronized circadian gene expression by stimulating C2C12 myotubes with 100 nM water-soluble DEX in serum-free DMEM for 2 h before being exposed to the \u003cem\u003eG. inodorum\u003c/em\u003e extracts for 24 h. The cells were washed twice with phosphate-buffered saline (PBS; Takara Bio Inc., Kusatsu, Japan) to remove residual medium.\u003c/p\u003e \u003cp\u003eTotal RNA was extracted using RNAiso Plus reagent (Takara Bio Inc.) as described by the manufacturer then reverse transcribed using PrimeScript\u0026trade; RT Master Mix (Takara Bio Inc). Complementary (c) DNA was amplified by qRT-PCR using SYBR\u0026reg; Premix Ex Taq\u0026trade; II (Takara Bio Inc.) on a StepOne\u0026trade; Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) and the primers (Thermo Fisher Scientific Inc.) listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The cycling conditions for target sequences comprised initial denaturation at 95\u0026deg;C for 10 s, followed by 45 cycles of 95\u0026deg;C for 5 s, and 60\u0026deg;C for 10 s. All experiments included three biological replicates and qRT-PCR included two technical duplicates per RNA sample to ensure the reproducibility of gene expression. Relative gene expression was analyzed using the ΔΔCt method and normalized to that of \u003cem\u003eActin β\u003c/em\u003e (\u003cem\u003eActb\u003c/em\u003e) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and is expressed relative to the vehicle (control). Statistical analyses were conducted using EZR software [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. One-way analysis of variance (ANOVA) followed by Dunnett\u0026rsquo;s post hoc test was applied for pairwise comparisons. Results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, with significance indicated by p-values.\u003c/p\u003e \u003cp\u003eNovel real-time bioluminescence monitoring of Bmal1 transcriptional activity in C2C12 cells\u003c/p\u003e \u003cp\u003eWe seeded 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e C2C12 myoblasts per well into 35 mm culture dishes (Iwaki Co., Ltd.) and maintained under standard culture conditions. Confluent cells were transfected with a \u003cem\u003eBmal1\u003c/em\u003e-Luc reporter plasmid (containing the \u0026minus;\u0026thinsp;197 to +\u0026thinsp;27 bp region upstream of the transcription start site [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] using Lipofectamine\u0026trade; 3000 (Thermo Fisher Scientific Inc.) as described by the manufacturer. On the following day the cells were incubated in differentiation medium, for 3 days to form myotubes. We synchronized circadian gene expression by incubating the transfected myotubes with either 100 nM water-soluble DEX or 50% HS in serum-free DMEM for 2 h [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The myotubes were incubated with or without \u003cem\u003eG. inodorum\u003c/em\u003e extracts in DMEM containing 2% HS, 0.1 mM D-luciferin potassium salt (Fujifilm Wako Pure Chemical Industries), and 25 mM HEPES (Thermo Fisher Scientific Inc.). Real-time bioluminescence was measured at 37\u0026deg;C for 1 min [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] every 10 minutes for 7 consecutive days using a Kronos AB-2500 luminometer (ATTO Corporation, Tokyo, Japan) and circadian period length was analyzed as described [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eReal-time bioluminescence monitoring stable \u003cem\u003eBmal1\u003c/em\u003e-Luc NIH3T3 reporter cells\u003c/p\u003e \u003cp\u003eA stable NIH3T3 cell line expressing a luciferase reporter under the control of the \u003cem\u003eBmal1\u003c/em\u003e promoter was established to monitor circadian transcriptional activity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. We incubated 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e NIH3T3 cells seeded in 35 mm dishes (Iwaki Co., Ltd.) containing DMEM supplemented with 10% FBS, and at 37\u0026deg;C under a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere for one day. Rhythmic \u003cem\u003eBmal1\u003c/em\u003e expression was initiated and synchronized by incubating the cells with 100 nM DEX in serum-free DMEM for 2 h. The medium was replaced with DMEM containing 0.1 mM D-luciferin potassium salt and 25 mM HEPES, with or without \u003cem\u003eG. inodorum\u003c/em\u003e extracts. Real-time bioluminescence intensity was evaluated using the Kronos AB-2500 luminometer and circadian period length was estimated as described [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eStephanoside B changed \u003cem\u003ePparg\u003c/em\u003e and \u003cem\u003ePpargc1a\u003c/em\u003e expression in differentiated C2C12 cells\u003c/p\u003e \u003cp\u003eWe previously found that GiA-7, and the stephanosides B and C extracted from \u003cem\u003eG. inodorum\u003c/em\u003e suppress adipocyte differentiation without significant toxicity to 3T3-L1 cells. We used 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) assays to determine the cytotoxicity of the extracts in myotubes after 24 h. Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e shows that GiA-7 at 50 and 100 \u0026micro;M and stephanoside C at 100 \u0026micro;M reduced cell viability to ~\u0026thinsp;60%, whereas stephanoside B maintained viability at ~\u0026thinsp;80%. Therefore, 25 \u0026micro;M was the maximal concentration of these extracts in subsequent experiments to ensure minimal cytotoxicity while assessing cellular responses.\u003c/p\u003e \u003cp\u003eWe analyzed the effects of 25 \u0026micro;M extracts for 24 h on the \u003cem\u003ePparg\u003c/em\u003e and \u003cem\u003ePpargc1a\u003c/em\u003e genes that express proteins with metabolic functions in differentiated C2C12 cells. Total RNA was isolated, reverse-transcribed, then \u003cem\u003ePparg\u003c/em\u003e and \u003cem\u003ePpargc1a\u003c/em\u003e expression was assessed using qRT-PCR. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows that stephanoside B significantly upregulated the expression of both genes. Stephanoside C significantly upregulated \u003cem\u003ePpargc1a\u003c/em\u003e gene expression but did not affect \u003cem\u003ePparg\u003c/em\u003e expression. Gymnemic acid-7 did not significantly change the expression of either gene. These results imply that GiA-7, and the stephanosides B and C affect skeletal muscle cells and adipocytes in different ways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eStephanoside B also upregulated \u003cem\u003ePparg\u003c/em\u003e and \u003cem\u003ePpargc1a\u003c/em\u003e expression in differentiated C2C12 cells stimulated with DEX.\u003c/p\u003e \u003cp\u003eBecause \u003cem\u003ePparg\u003c/em\u003e and \u003cem\u003ePpargc1a\u003c/em\u003e expression might fluctuate according to circadian phases, we considered literature indicating that these genes are expressed rhythmically throughout the day. Such rhythmic expression might have obscured changes induced by the extracts. Therefore, we synchronised the circadian clock in differentiated C2C12 cells using DEX, incubated them with 10 or 25 \u0026micro;M stephanoside B for 24 h, then evaluated the expression of \u003cem\u003ePparg\u003c/em\u003e and \u003cem\u003ePpargc1a\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe expression of both \u003cem\u003ePparg\u003c/em\u003e and \u003cem\u003ePpargc1a\u003c/em\u003e in cells incubated with stephanoside B concentration-dependently increased compared with the vehicle (ethanol). Specifically, 10 and 25 \u0026micro;M stephanoside B respectively increased \u003cem\u003ePparg\u003c/em\u003e expression 1.5- and 1.9-fold relative to the vehicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results indicated that stephanoside B consistently and concentration-dependently upregulated \u003cem\u003ePparg\u003c/em\u003e and \u003cem\u003ePpargc1a\u003c/em\u003e after resetting cellular circadian rhythms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3.3. Stephanoside B changed clock gene expression in differentiated C2C12 cells with reset circadian clocks.\u003c/p\u003e \u003cp\u003eWe examined expression of the core clock genes, \u003cem\u003eBmal1\u003c/em\u003e, \u003cem\u003ePer2\u003c/em\u003e, \u003cem\u003eCry1\u003c/em\u003e and \u003cem\u003eNr1d1\u003c/em\u003e in differentiated C2C12 myotubes with DEX-induced reset circadian rhythms. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the qRT-PCR results of cDNA reverse transcribed from the RNA samples described above. A peroxisome proliferator response element (PPRE) in the promoter region of \u003cem\u003eNr1d1\u003c/em\u003e is transcriptionally regulated by PPARγ combined with RXR. Consistent with this, stephanoside B concentration-dependently increased \u003cem\u003eNr1d1\u003c/em\u003e expression in DEX-stimulated cells compared with controls 24 h after DEX stimulation. The expression of \u003cem\u003ePer2\u003c/em\u003e and \u003cem\u003eCry1\u003c/em\u003e was also elevated under these conditions. In contrast, the expression of \u003cem\u003eBmal1\u003c/em\u003e, which is repressed by NR1D1 via retinoic acid-related orphan receptor response elements (ROREs), concentration-dependently decreased.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eStephanoside B elongated \u003cem\u003eBmal1\u003c/em\u003e period length in differentiated C2C12 cells and NIH3T3 fibroblasts.\u003c/p\u003e \u003cp\u003eAnalyses of clock gene expression at one time point (24 h) suggested that stephanoside B should influence the rhythmic expression of the circadian clock. We considered that the transcriptional changes induced by stephanoside B might be associated with changes in the oscillatory rhythms of clock gene expression. Therefore, we established a real-time reporter assay system in myotubes that were differentiated from C2C12 cells. We optimized the previous transfection strategy because \u003cem\u003eBmal1\u003c/em\u003e expression was more stable when the \u003cem\u003eBmal1\u003c/em\u003e reporter plasmid was introduced before, than after differentiation (data not shown). Harmine can elongate the period in other cells [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] or tissues [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and we found similar effects on C2C12 cells. We then compared synchronization using DEX or 50% HS [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and found that \u003cem\u003eBmal1\u003c/em\u003e rhythms were more stable using DEX (Supplementary Figure S2).\u003c/p\u003e \u003cp\u003eWe differentiated C2C12 cells by transfection with \u003cem\u003eBmal1\u003c/em\u003e\u0026ndash;luciferase (\u003cem\u003eBmal1\u003c/em\u003e\u0026ndash;Luc) reporter plasmids. The differentiated cells were then synchronised by incubation with DEX for 2 h, after which the culture fluid was replaced with medium containing luciferin supplemented with extracts. Bioluminescence was then monitored in real time (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Using this transient expression system, circadian oscillations of clock gene reporter activity were robust for several days. Furthermore, 25 \u0026micro;M stephanoside B elongated the circadian period to 25.7 h in both C2C12 and NIH3T3 cells. These results indicate that this phenomenon is not restricted to C2C12 myotubes (Supplementary Figure S3). Together, these results demonstrate that our optimized real-time \u003cem\u003eBmal1\u003c/em\u003e monitoring system provides a reliable platform to evaluate nutrient-derived modulators of muscle circadian rhythms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we investigated the effects of GiA-7, and stephanosides B and C extracted from \u003cem\u003eG. inodorum\u003c/em\u003e on skeletal muscle cells, with focus particularly on energy metabolism and the molecular circadian clock. Among the compounds tested, stephanoside B upregulated \u003cem\u003ePparg\u003c/em\u003e expression in differentiated C2C12 myotubes, whereas GiA-7 and stephanoside C had no significant effects. Notably, this response contrasts with our previous observations in adipocytes, highlighting a clear cell type\u0026ndash;specific difference in the biological actions of \u003cem\u003eG. inodorum\u003c/em\u003e\u0026ndash;derived compounds. The mechanistic basis for these cell type\u0026ndash;specific responses awaits elucidation.The expression of PPARγ is abundant in adipose tissue and the liver [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], but lower in skeletal muscle [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Nevertheless, muscle PPARγ might function in the regulation of energy metabolism [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The transcription of \u003cem\u003eNr1d1\u003c/em\u003e can be directly upregulated via the PPRE element by PPARγ [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. NR1D1 not only functions in the regulation of cellular redox states but also acts as a transcription factor that directly suppresses \u003cem\u003eBmal1\u003c/em\u003e expression via the RORE [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Our results are consistent with these findings, because stephanoside B induced concentration-dependently increases in \u003cem\u003ePparg\u003c/em\u003e and \u003cem\u003ePpargc1a\u003c/em\u003e expression together with upregulated \u003cem\u003eNr1d1\u003c/em\u003e and a corresponding decrease in \u003cem\u003eBmal1\u003c/em\u003e expression. Together, PPARγ and PGC-1α regulate the expression of many genes and their upregulation should result in upregulated downstream genes. We speculate that the mechanisms through which stephanoside B regulates \u003cem\u003ePparg\u003c/em\u003e differ from those in adipocytes, which warrants further mechanistic investigation. Circadian genes expressed in skeletal muscle regulate the expression of GLUT4 and other proteins involved in glucose metabolism, which contributes to the maintenance of physiological homeostasis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These considerations highlight the value of experimental systems that can monitor clock gene expression in muscle.\u003c/p\u003e \u003cp\u003eIn this study, we successfully established the real-time \u003cem\u003eBmal1\u003c/em\u003e-luciferase monitoring system in differentiated C2C12 myotubes. To our knowledge, this represents one of the first demonstrations of real-time circadian promoter activity tracking in fully differentiated skeletal muscle cells. The system allowed for the continuous observation of circadian oscillations and period changes induced by compounds in a differentiated muscle model relevant to physiology.\u003c/p\u003e \u003cp\u003eTaken together with our previous findings that \u003cem\u003eG. inodorum\u003c/em\u003e extracts suppress adipocyte differentiation, the present study revealed that stephanoside B can modulate the circadian period in muscle via PPARγ-associated regulation of clock gene expression. \u003cem\u003eG. inodorum\u003c/em\u003e has long been consumed as a food plant and tea, and is widely associated with metabolic health benefits. From a nutritional biochemistry perspective, modulation of skeletal muscle circadian timing by plant-derived triterpenoids may represent an additional mechanism through which dietary components influence metabolic homeostasis. Our findings suggest a novel potential influence of \u003cem\u003eG. inodorum\u003c/em\u003e on skeletal muscle physiology by modulating the molecular circadian clock.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe investigated the effects of three \u003cem\u003eG. inodorum\u003c/em\u003e extracts on skeletal muscle metabolism and circadian regulation. Among them, stephanoside B selectively upregulated \u003cem\u003ePparg\u003c/em\u003e and \u003cem\u003ePpargc1a\u003c/em\u003e expression in differentiated C2C12 myotubes under basal conditions and after circadian resetting. Consistent with these transcriptional changes, stephanoside B modulated core clock gene expression by inducing \u003cem\u003eNr1d1\u003c/em\u003e, \u003cem\u003ePer2\u003c/em\u003e, and \u003cem\u003eCry1\u003c/em\u003e while repressing \u003cem\u003eBmal1\u003c/em\u003e. Using the newly established real-time \u003cem\u003eBmal1\u003c/em\u003e\u0026ndash;luciferase reporter system in differentiated C2C12 myotubes, we directly demonstrated that stephanoside B lengthened the circadian period, providing dynamic evidence that a dietary plant-derived compound has potential for modulation of the muscle-intrinsic molecular clock properties. These findings indicated that stephanoside B exerts regulatory effects on skeletal muscle by enhancing PPARγ/PGC-1α signaling and influencing molecular circadian rhythms. Given the central role of skeletal muscle in systemic energy metabolism and its contribution to whole-body circadian organization, the ability of stephanoside B to influence metabolic gene networks and circadian rhythms suggests a potential mechanism that may contribute to previously reported metabolic effects of \u003cem\u003eG. inodorum\u003c/em\u003e. Collectively, our study identifies a novel chrono-nutritional action of \u003cem\u003eG. inodorum\u003c/em\u003e derived compounds and establishes a real-time analytical platform for exploring nutritionally driven regulation of molecular circadian rhythms specifically in muscle.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eContribution statement\u003c/p\u003e\n\u003cp\u003ePapawee Saiki: Writing – review \u0026amp; editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Data curation, Formal analysis, Conceptualization. Tatsunosuke Tomita: Writing – review \u0026amp; editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. Saori Yamamoto: Methodology, Investigation\u003c/p\u003e\n\u003cp\u003eDeclaration of Competing Interest\u003c/p\u003e\n\u003cp\u003eThere are no conflicts to declare.\u003c/p\u003e\n\u003cp\u003eAcknowledgement\u003c/p\u003e\n\u003cp\u003eWe thank the National Institute of Advanced Industrial Science and Technology (AIST) for support. We also thank Katsutaka Oishi and Tomoki Abe for their helpful discussions.\u003c/p\u003e\n\u003cp\u003eSupporting Information\u003c/p\u003e\n\u003cp\u003eThe Supporting Information is available free of charge at\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eNo primary research results, software or code have been included, and no new data were generated or analyzed as part of this review.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSrinuanchai W, Nooin R, Pitchakarn P, Karinchai J, Suttisansanee U, Chansriniyom C, Jarussophon S, Temviriyanukul P, Nuchuchua O (2021) Inhibitory effects of Gymnema inodorum (Lour.) Decne leaf extracts and its triterpene saponin on carbohydrate digestion and intestinal glucose absorption. 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Int J Mol Sci, 22\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFontaine C, Dubois G, Duguay Y, Helledie T, Vu-Dac N, Gervois P, Soncin F, Mandrup S, Fruchart JC, Fruchart-Najib J, Staels B (2003) The orphan nuclear receptor Rev-Erbalpha is a peroxisome proliferator-activated receptor (PPAR) gamma target gene and promotes PPARgamma-induced adipocyte differentiation. J Biol Chem 278:37672\u0026ndash;37680\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang S, Liu CH, Wang ZX, Fu ZJ, Britton WR, Blomfield AK, Kamenecka TM, Dunaief JL, Solt LA, Chen J (2022) REV-ERBa regulates age-related and oxidative stress-induced degeneration in retinal pigment epithelium via NRF2. Redox Biol, 51\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSengupta S, Yang G, O'Donnell JC, Hinson MD, McCormack SE, Falk MJ, La P, Robinson MB, Williams ML, Yohannes MT, Polyak E, Nakamaru-Ogiso E, Dennery PA (2016) The circadian gene Rev-erbα improves cellular bioenergetics and provides preconditioning for protection against oxidative stress. Free Radical Bio Med 93:177\u0026ndash;189\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGutierrez-Monreal MA, Harmsen JF, Schrauwen P, Esser KA (2020) Ticking for Metabolic Health: The Skeletal-Muscle Clocks, Obesity, 28 S46\u0026ndash;S54\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"PPARγ signaling, Metabolic gene regulation, Circadian regulation, Skeletal muscle metabolism, Bmal1-luciferase reporter, Stephanoside B","lastPublishedDoi":"10.21203/rs.3.rs-8951484/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8951484/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eGymnema (G.) inodorum\u003c/em\u003e is a medicinal plant with anti-diabetic, anti-obesity, and anti-inflammatory properties traditionally consumed as tea in Southeast Asia. While bioactive stephanosides and gymnemic acids suppress adipocyte differentiation, their effects on skeletal muscle metabolism and circadian regulation remain unclear. Here, we investigated the actions of gymnemic acid (GiA-7) and stephanosides B and C in differentiated C2C12 myotubes. Stephanoside B selectively and concentration-dependently upregulated \u003cem\u003ePparg\u003c/em\u003e and \u003cem\u003ePpargc1a\u003c/em\u003e expression under basal conditions and after dexamethasone-induced circadian synchronization. It also enhanced expression of the core clock genes \u003cem\u003eNr1d1\u003c/em\u003e, \u003cem\u003ePer2\u003c/em\u003e, and \u003cem\u003eCry1\u003c/em\u003e, while repressing \u003cem\u003eBmal1\u003c/em\u003e, consistent with known PPARγ\u0026ndash;circadian interactions. To directly monitor circadian rhythmicity, we established a real-time \u003cem\u003eBmal1\u003c/em\u003e-luciferase reporter gene assay in differentiated myotubes. Continuous bio-luminescence tracking over several days revealed that stephanoside B specifically lengthened the circadian period, demonstrating modulation of molecular clock function. Collectively, these results indicate that stephanoside B coordinately regulates metabolic gene expression and circadian rhythms in the skeletal muscle. Given the central role of muscle in systemic energy homeostasis and circadian regulation, these findings highlight the chrono-nutritional potential of \u003cem\u003eG. inodorum\u003c/em\u003e extracts. Moreover, this study provides one of the first demonstrations of real-time circadian reporter analysis in differentiated myotubes, offering a novel platform to explore dietary compounds that influence muscle-specific metabolic and circadian processes.\u003c/p\u003e","manuscriptTitle":"Stephanoside B Modulates PPARγ-Dependent Metabolic Genes and Lengthens the Circadian Bmal1 Oscillation Period in Differentiated Myotubes Revealed by Real-Time Bioluminescence","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-04 17:04:20","doi":"10.21203/rs.3.rs-8951484/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-23T09:02:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-22T15:39:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-11T16:42:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"291646677485479028846559008674971757774","date":"2026-03-01T10:59:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"263964153603458235081427223889072437919","date":"2026-02-27T10:39:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-27T09:55:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-25T06:43:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-25T06:38:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2026-02-24T01:06:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6d2e6415-502e-40e4-bca2-b709965d4fd5","owner":[],"postedDate":"March 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T13:25:31+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-04 17:04:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8951484","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8951484","identity":"rs-8951484","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}