{"paper_id":"0499acc9-783a-4112-84ea-bbd36a0eb327","body_text":"Quercetin and silibinin ameliorate insulin resistance by stimulating fat browning via the β3-AR/AMPK pathway in palmitate-exposed 3T3-F442A adipocytes | 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 Article Quercetin and silibinin ameliorate insulin resistance by stimulating fat browning via the β3-AR/AMPK pathway in palmitate-exposed 3T3-F442A adipocytes Yakun Ge, Aiping Liu, Fanwei Meng, Renwen Zhang, Junting Wang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7124697/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Quercetin (QC) and silibinin (SLB), natural bioactive compounds from medicinal plants, show potential in improving IR. However, the molecular mechanisms underlying their IR-attenuating effects through adipocyte browning remain not fully elucidated. In this study, molecular docking simulations were performed to characterize ligand-target interactions, binding conformations, and affinity energies between QC/SLB and key molecular targets. qRT-PCR and immunoblotting analyses were employed to quantify expression levels of β3-AR/AMPK pathway-associated genes and proteins. Notably, QC and SLB upregulated brown/beige adipocyte markers at transcriptional and translational levels: PGC-1α, PRDM16, and UCP1, and genes specific to beige adipose tissue like Tmem26, Tbx1, CD137, and Cited1. Mechanistically, these compounds enhanced lipolytic activity and β-oxidation throughβ3-AR-mediated activation of PKA, ATGL, CPT, ACO, and PPARα/γ signaling cascades. Overall, QC and SLB promote PA-induced adipocyte browning in IR-3T3-F442A cells by activating both the β3-AR/AMPK signaling pathways, highlighting their potential as therapeutic agents for metabolic disorders. Our findings collectively demonstrate the potential effectiveness of QC and SLB in ameliorating IR. Biological sciences/Biochemistry Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Drug discovery Biological sciences/Molecular biology QC SLB IR browning β3-AR/AMPK signaling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction IR, characterized by diminished biological responsiveness to insulin, serves as a central driver in the pathogenesis of metabolic disorders including type diabetes (T2D), obesity, polycystic ovarian syndrome, and cardiovascular diseases 1 , 2 . As a dynamic endocrine organ, adipose tissue exhibits dual functionality in energy homeostasis and IR development through three distinct subtypes: white adipose tissues (WAT), brown adipose tissues (BAT), and beige adipose tissues 3 . WAT primarily stores energy as triacylglycerol (TAG), whereas BAT specializes in thermogenic energy expenditure via uncoupling protein 1 (UCP1)-mediated mechanisms 4 . Beige adipocytes, emerging through the browning of WAT, demonstrate intermediate thermogenic capacity between white and brown fat, with their induction showing therapeutic potential for improving insulin sensitivity 5 , 6 . This phenotypic conversion enhances mitochondrial biogenesis and metabolic flexibility through coordinated activation of PPARα/γ, PGC1α, and AMPK signaling pathways 7 , 8 . Previous studies have highlighted that the following environmental and pharmacological interventions synergistically promoted adipose tissue remodeling: ⅰ) Chronic cold exposure and β-adrenergic agonists stimulate sympathetic activation of UCP1-dependent thermogenesis 9 , 10 ; ⅱ) medicinal plant-derived bioactive compounds offer safe alternatives for inducing beige adipogenesis 11 , 12 . Therefore, adipose tissue browning has emerged as a promising therapeutic axis for mitigating IR-associated comorbidities through metabolic reprogramming and inflammatory modulation 13 . Plant flavonoids have emerged as key bioactive compounds in metabolic regulation, with particular focus on their promising effects on IR and lipid metabolism 14 . These polyphenolic molecules exhibit multi-target functionality for mitigating IR including: ⅰ) Scavenging reactive oxygen species (ROS) to alleviate hyperglycemia-induced oxidative stress and improve insulin sensitivity 15 ; 2) modulating lipid profiles to alleviate IR by decreasing triglycerides/ low density lipoprotein (LDL) while elevating high density lipoprotein (HDL) cholesterol 16 ; Ⅲ) suppressing pro-inflammatory cytokines are implicated in IR pathogenesis 17 . Among natural flavonoids, QC and SLB exhibit unique therapeutic potential for alleviating IR. QC shows pleiotropic effects spanning blood pressure regulation to glucose homeostasis 18 , while SLB exhibits notable antioxidant, antiapoptotic, anti-inflammatory, and lipid-modulating capacities 19 , 20 . Moreover, previous research has shown that SLB effectively mitigated PA-induced IR, while QC counteracts tumor necrosis factor alpha (TNFα)-induced IR in mouse C2C12 myoblasts 21 , 22 . However, despite these promising in vitro findings, the mechanisms by which QC and SLB influence lipid accumulation and their potential effects on adipose tissue browning remain to be fully elucidated. In the present study, the beneficial effects of QC and SLB on metabolic functions were systematically investigated, with a particular focus on their IR-attenuating effects through adipocyte browning, providing new insights into natural product strategies for IR management. Results Effect of QC and SLB on metabolic activity and glucose uptake in PA-exposed adipocytes. To assess the putative cytotoxic properties of QC or SLB in 3T3-F442A preadipocytes, cellular models were subjected to combinatorial exposure with PA (0.75 mM) co-administered with pharmacologically relevant concentrations (5-160 µM) of test compounds for 24 h incubation. Subsequently, the impact of these flavonoids on PA-induced IR-3T3-F442A adipocytes was evaluated using the MTT assay. As shown in Fig. 1 A and B, QC and SLB intervention restored mitochondrial activity in a dose-dependent manner in PA-induced IR-3T3-F442A cells, with optimal therapeutic efficacy manifesting at a safe concentration (40 µM QC, 40% restoration; 80 µM SLB,35% restoration) relative to PA monotherapy 23 , 24 . Complementary assessment of intracellular ATP biosynthesis through luminometric quantification confirmed concordant metabolic rescue effects, demonstrating 22% elevation in ATP generation following QC/SLB treatment relative to PA-exposed controls (P < 0.001; Fig. 1 C), thereby corroborating MTT-derived metabolic parameters. Additionally, the glucose uptake capacity of IR-3T3-F442A adipocytes challenged with PA was systematically evaluated. As illustrated in Fig. 1 D, administration of PA at a concentration of 0.75 mM elicited a marked 15% suppression of glucose uptake relative to baseline levels. By contrast, pharmacological intervention with either QC (40 µM) or SLB (80 µM) exhibited significant enhancement of glucose transport efficiency, achieving 32 and 25% elevation respectively (P < 0.001) in IR-3T3-F442A cells. Effect of QC and SLB on lipid accumulation and mitochondrial bioenergetics in PA-exposed adipocytes. Pathological adipogenesis in WAT constitutes a pivotal etiological factor in the development of IR, as evidenced by seminal studies 25 – 27 . To elucidate the therapeutic potential of bioactive compounds, ORO histochemical staining with morphometric analysis was used to quantitatively assess the regulatory effects of QC and SLB on intracellular lipid deposition in an established IR-3T3-F442A adipocyte model. As shown in Fig. 2 A and B, PA challenge induced a 13.2% elevation in cytoplasmic lipid droplet formation (P < 0.001), consistent with characteristic metabolic perturbations observed in T2D. Notably, pharmacological administration of 40 µM QC or 80 µM SLB demonstrated substantial lipid-lowering efficacy, achieving 32.1 and 30.8% reductions in lipid accumulation respectively (P < 0.001). These findings collectively demonstrate that both phytochemicals effectively ameliorate PA-induced lipotoxic insults through mechanisms involving enhanced lipid catabolism. Mitochondrial bioenergetics exert a critical regulatory influence on the pathogenesis of IR 28 . To elucidate the therapeutic potential of QC or SLB in modulating mitochondrial dynamics within IR-3T3-F442A adipocytes, mitochondrial activity was quantitatively evaluated through MitoTracker Red CMXRos fluorescence staining. As shown in Fig. 2 C, confocal microscopy analysis revealed thatPA-induced IR adipocytes exhibited a marked augmentation in mitochondrial membrane potential, indicative of progressive restoration of mitochondrial bioenergetic capacity. Notably, pharmacological intervention with QC (40 µM) or SLB (80 µM) elicited a pronounced amplification of mitochondrial redox potential compared with PA-challenged controls. Complementary transcriptional profiling (Fig. 2 D) further demonstrated that both QC and SLB administration induced substantial transcriptional upregulation of nuclear-encoded mitochondrial biogenesis regulators, particularly Nrf1 and Tfam. These data collectively suggest that QC and SLB enhance mitochondrial oxidative phosphorylation efficiency through coordinated activation of the Nrf1/Tfam signaling axis, thereby facilitating the browning of insulin-resistant adipocytes. Effect of QC and SLB on fat browning in PA-exposed adipocytes. The pharmacological induction of WAT browning represents a promising therapeutic intervention for metabolic disorders 29 . To elucidate the molecular mechanisms underlying this process, comprehensive analyses of browning-specific genes and regulatory proteins were conducted in IR-3T3-F442A adipocytes following treatment with QC (40 µM) or SLB (80 µM). As shown in Fig. 3 A, IR adipocytes exposed to either compound exhibited marked upregulation of beige adipocyte-specific genes, including transmembrane protein 26 (Tmem26), T-box transcription factor 1 (Tbx1), tumor necrosis factor receptor superfamily member 9 (CD137), and Cbp/p300-interacting transactivator 1 (Cited1). Moreover, QC (40 µM) or SLB (80 µM) upregulated the expression levels of mRNA and protein for critical browning markers including PGC-1α, PRDM16, and UCP1 (Fig. 3 B and C). These data provide compelling evidence that both QC and SLB effectively promote the browning phenotype in 3T3-F442A adipocytes through coordinated activation of thermogenic transcriptional networks. Effect of QC and SLB on lipid metabolism in PA-exposed adipocytes. The present study investigated the effects of QC (40 µM) or SLB (80 µM) on lipid metabolism-related genes and proteins in IR-3T3-F442A adipocytes. The results demonstrated that QC/SLB treatment significantly upregulated the expression of carnitine palmitoyltransferas (CPT), 1-aminocyclopropane-1-carboxylate oxidase (ACO), and PPARα genes associated with fatty acid β-oxidation (Fig. 4 A). Additionally, genes encoding β3-AR, protein kinase A (PKA), and adipose triglyceride lipase (ATGL), which are involved in lipolytic pathways, showed marked elevation (Fig. 4 B). A pronounced increase was observed in the expression of β3-AR, PKA, and pAMPKα, which have been identified as key regulators in lipid metabolism and fat browning processes. Notably, the protein expression levels of β3-AR and PKA, along with the pAMPKα/AMPKα ratio, were significantly elevated (Fig. 4 C). QC and SLB activate adrenergic receptors. Computational docking protocols were executed to systematically assess the ligand-receptor binding thermodynamics, conformational orientations, and molecular recognition patterns between the small-molecule ligands QC or SLB and theβ3-AR target (Figs. 5 A and 5 B). Rigorous analysis of the molecular dynamics docking demonstrated that the QC ligand achieved a binding free energy of -8.4 kcal/mol with β3-AR, featuring multiple interacting residues. Similarly, the SLB ligand manifested superior binding thermodynamics (ΔG = -8.6 kcal/mol), engaging critical catalytic residues within the orthosteric pocket of the receptors. Both ligands demonstrated sustained stabilization (ΔG_< -5 kcal/mol) with minimal conformational fluctuations (root-mean-square deviation < 2.00 Å). These computational insights substantiate the formation of thermodynamically favorable binding complexes between the ligands and β3-AR, characterized by optimal pharmacophore complementarity. The binding conformations and detailed interactions of QC and SLB with β3-AR are depicted in Figs. 5 C and D. Specifically, QC formed non-covalent interactions with key amino acid residues, including Asn329, Asp114, Gly91, Trp330, Ala95, and Tyr333. In addition, SLB established six hydrogen bonds with Arg191, Trp330, and Asn329 residues on the receptor, along with three additional hydrogen bonds, further stabilizing its interaction. These results collectively indicate that both QC and SLB exhibit strong binding affinity for β3-AR, mediated by distinct yet effective non-covalent interactions. Effect of QC and SLB on adipose browning in PA-exposed adipocytes via the β3-AR/AMPKα signaling pathway. The regulatory roles ofβ3-AR and AMPK signaling pathways in IR pathogenesis have been extensively characterized 30 . To delineate the mechanistic basis of QC or SLB-induced adipose browning in PA-treated IR-3T3-F442A adipocytes, the β3-AR/AMPKα axis was investigated using the following pharmacological inhibitors: SR59230A (10 µM; β3-AR antagonist) and dorsomorphin (10 µM; AMPK antagonist). As illustrated in Figs. 6 A and B, dual pathway blockade substantially attenuated the expression of thermogenic markers (UCP-1, PGC-1αand PRDM16). Notably, both phytocompounds reversed this suppression, restoring brown adipocyte-specific gene signatures in a β3-AR/AMPKα-dependent manner. This restorative effect was abolished upon antagonist administration, indicating that QC or SLB promotes PA-induced fat browning in IR-3T3-F442A adipocytes by activating the β3-AR and AMPK signaling pathways (Fig. 7 ). Materials and methods Chemicals. SLB, QC, isobutylmethylxanthine (IBMX), dexamethasone (DEX), insulin, palmitic acid (PA), dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2-deoxy-2-[(7-nitro-1,2,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG, and Oil Red O (ORO) were obtained from Sigma-Aldrich (Merck KGaA). The β3-adrenergic receptor AR (β3-AR, ADRB3) antagonist SR59230A and the AMP activated protein kinase (AMPK) antagonist Dorsomorphin were purchased from MedChem Express. Isopropanol and chloroform were obtained from Shanghai Aladin Biochemical Technology Co., Ltd. Cell culture and differentiation. The 3T3-F442A murine embryonic fibroblast cell line (cat no. FY-22FN0241; Shanghai Fuyu Biotechnology Co., Ltd) was propagated in growth medium comprising Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Inc), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc), under standard culture conditions (37 ˚C, 5% CO₂, humidified atmosphere). Routine subculture was conducted at 2- or 3-day intervals upon attaining 80–90% monolayer confluence. For differentiation induction, cells were plated in six-well culture dishes at 2 x 10⁵ cells/well and left to proliferate to full confluence over 72 h. At confluence (designated Day 0), the growth medium was replaced with a differentiation induction cocktail containing DMEM with 10% FBS, 0.5 mM IBMX, 1 µM DEX, and 2 µg/ml insulin. After 48 h of exposure to differentiation stimuli, the induction medium was substituted with adipogenic maintenance medium (DMEM + 2 µg/ml insulin) for an additional 48 h. After differentiation culture, cells were reverted to standard growth medium (refreshed every two days) until terminal differentiation at Day 8, at which point experimental analyses were initiated. Preparation and processing conditions of a 3T3-F442A adipocyte model of IR. Differentiated and mature 3T3-F442A adipocytes were divided into the following four experimental cohorts: ⅰ) Control group; ⅱ) PA-induced IR model group; ⅲ) QC-therapeutic group; and ⅳ) SLB-therapeutic group. The control group was cultured in basal DMEM growth medium throughout the experimental timeline. The PA-induced IR model group underwent metabolic challenge through 24-h exposure to DMEM supplemented with 0.75 mM PA. In the QC-therapeutic group, cells were preconditioned with QC-enriched DMEM for 24 h prior to PA challenge, followed by cotreatment with QC during the 24-h PA induction phase, and subsequently maintained in QC-supplemented medium post-induction. Analogously, the SLB-therapeutic group received prophylactic treatment with SLB-fortified DMEM for 24 h, followed by concurrent administration with 0.75 mM PA in SLB-containing medium during the induction phase, culminating in a 24-h recovery period in SLB-supplemented conditions. Following completion of these temporally regulated pharmacological regimens, all cellular specimens were processed for downstream analytical procedures. MTT and ATP assays. Firstly, 3T3-F442A preadipocytes were plated in 96-well culture plates at an initial seeding density of 5 x10³ cells/well. The differentiation protocol was initiated upon achieving 70–80% cellular confluence. After differentiation and maturation, cells were randomly allocated into the following five experimental groups: ⅰ) untreated control group; ⅱ) PA-induced model group (0.75 mM PA); ⅲ) QC-therapeutic group (0.75 mM PA co-administered with 5-160 µM QC in geometric progression); ⅳ) SLB-therapeutic group (0.75 mM PA combined with 5-160 µM SLB); and ⅴ) reagent background control (cell-free system). Following experimental treatments, 20 µl MTT solution (5 mg/ml in PBS) was introduced to each well, with subsequent incubation at 37 ˚C for 4 h under standard culture conditions. The formazan crystals were solubilized by adding 150 µl DMSO following careful aspiration of culture supernatants. Optical density measurements were conducted at 570 nm using a multimode microplate reader (BioTek; Agilent Technologies, Inc.), with data normalized to untreated controls. To quantify ATP biosynthesis, ATP production analysis was conducted employing an ATP Generation Assay Kit (Sigma-Aldrich; Merk KGaA). The experimental protocol involved aliquoting 100 µl ATP detection reagent into test wells followed by a 5-min incubation period to stabilize baseline signal. Subsequently, 20 µl cellular lysate was introduced into each reaction chamber and homogenized through gentle agitation. Chemiluminescent signals were quantified using a multimode microplate detection system with 2-sec integration intervals between sequential measurements. Simultaneous protein quantification was performed using a BCA assay (Beyotime Institute of Biotechnology) to enable normalized ATP production values expressed as nmol/mg protein. Glucose uptake assay. The treated adipocytes were subjected to a 1-h incubation period at 37 ˚C in serum-free low-glucose DMEM medium containing diminished phenol red concentration, with supplementation with 100 µM 2-NBDG fluorescent glucose analog, under light-protected environmental conditions. Following the glucose uptake phase, cellular monolayers underwent three successive washes with ice-cold phosphate-buffered saline (PBS) to arrest the transport process. Quantitative fluorescence intensity analysis was performed using a multimode microplate reader configured with 485 nm excitation and 535 nm emission parameters. For experimental normalization, total protein content per well was determined through BCA protein assay (Beyotime Institute of Biotechnology), establishing specific fluorescence values/ unit protein mass. ORO staining and lipid content determination. The experimental adipocyte samples underwent ORO histochemical staining following an established protocol. Firstly, the ORO stock solution was prepared by dissolving 0.5 g crystalline ORO powder in 100 ml anhydrous isopropanol, followed by overnight storage at 4 ˚C. Prior to application, the stock solution was diluted with ultrapure water at a 3:2 (v/v) ratio, equilibrated to an ambient temperature for 20 min, filtered through a 0.45 µm pore-size membrane filter, and maintained under light-protected conditions. The cellular specimens were subjected to three sequential 5-min PBS washing cycles using gentle agitation. Subsequent fixation was achieved through incubation with 10% neutral-buffered formalin for 60 min at room temperature. Following triple ultrapure water rinses, the cells were immersed in freshly prepared ORO working solution for 30 min under standardized laboratory conditions. Post-staining cells were rinsed three times prior to microscopic examination and imaging. For quantitative lipid content, the intracellular ORO complexes were eluted in absolute isopropanol through 10-min orbital shaking. The resultant chromogenic solution was subjected to spectrophotometric analysis at λ = 570 nm employing a 96-well microplate reader system, with absorbance measurements normalized against reagent blanks. MitoTracker Red assay. Following cellular treatment, adipocytes underwent standardized immunofluorescence staining protocols. The existing culture medium was carefully aspirated prior to triple PBS washing. Cellular specimens were subsequently incubated with 200 nM MitoTracker Red CMXRos (Thermo Fisher Scientific) in serum-free DMEM medium (1 ml/well) under controlled conditions (37 ˚ C, 5% CO₂) for 30 min. Post-incubation, an additional 15-min maintenance phase was implemented under identical environmental parameters. Cellular fixation was achieved through 15-min exposure to 4% paraformaldehyde at ambient temperature, followed by thorough PBS rinsing. Membrane permeabilization was conducted using 0.2% Triton X-100 (10 min), succeeded by triple PBS washing cycles. Nuclear counterstaining was performed with 10 µg/ml Hoechst 33342 (Beyotime Institute of Biotechnology) in serum-free DMEM (5 min, room temperature). Following final PBS washes, specimens were mounted with 500 µl anti-fade reagent and visualized using inverted epifluorescence microscopy. Reverse transcription-quantitative PCR. The cellular sample (5 x 10 6 treated cells) was subjected to RNA extraction through optimized guanidinium thiocyanate-phenol-chloroform methodology. Following complete cell lysis using 1 ml Trizol® reagent (Beyotime Institute of Biotechnology; cat. no. R0016), the homogenate was centrifuged (12,000 x g, 5 min, 4 ˚C) to remove insoluble debris. Subsequent phase separation was achieved by chloroform supplementation (200 µl) with vigorous vortexing (30 sec) and incubation for 15 min at room temperature. Centrifugation under identical parameters yielded distinct phase stratification, from which 500 µl aqueous phase was aspirated for isopropanol precipitation (1:1 v/v ratio). The RNA pellet obtained after centrifugation (12,000 x g, 10 min, 4 ˚C) underwent sequential ethanol washes (75% v/v, 1 mL x 2) with intermediate centrifugation cycles (5 min each). Residual ethanol was removed by vacuum desiccation prior to resuspension in DEPC-treated ultrapure water. RNA purity and concentration were spectrophotometrically verified (NanoDrop™ 2000) through A260/A280 absorbance ratio quantification. Reverse transcription was performed using 1 µg total RNA with SweScript RT II First Strand cDNA Synthesis System (Wuhan Servicebio Technology Co., Ltd.) under manufacturer-specified conditions. Quantitative amplification was conducted on the ABI 7500 Real-Time PCR System with SYBR Green chemistry, employing the following thermal profile: initial denaturation (94 ˚C for 30 sec); 40 cycles of denaturation (94 ˚C for 5 sec), annealing (60 ˚C for 15 sec), and extension (72 ˚C for 30 sec). Cycle threshold (CT) values were normalized against the 18 S rRNA endogenous control, with relative expression levels calculated via the comparative CT method (2 −ΔΔCT ). Primer sequences are catalogued in Supplementary Table 1. In-silico analysis. The immunoblotting protocol commenced with generation of cellular lysates using RIPA buffer, followed by clarification through centrifugation at 12,000 x g for 20 min at 4 ˚C. Protein extracts were diluted in 5 x SDS loading buffer, denatured at 100 ˚C for 5 min, and resolved through discontinuous SDS-polyacrylamide gel electrophoresis employing 10 or 12% resolving gels. Electrophoretically separated proteins were subsequently transferred onto PVDF membranes under semi-dry conditions. Membranes underwent blocking in TBST supplemented with 5% non-fat dried milk for 60 min at an ambient temperature. Following three sequential TBST washes (5 min each), membranes were probed overnight at 4 ˚C with primary antibodies diluted in TBST/5% milk solution. The antibody (1:500 dilution in TBST/milk) panel comprised: Anti-GAPDH (cat. No. sc-137179; Santa Cruz Biotechnology, Inc.), anti-UCP1 (cat. no. 83870-1-RR; Wuhan Sanying Biotechnology), anti-PGC-1α (cat. no. 66369-1; Wuhan Sanying Biotechnology), anti-PRDM16 (cat. no. 83872-1-RR; Wuhan Sanying Biotechnology), anti-β3-AR (cat. no. ab76249; Abcam), anti-PKA (cat. no. 12232-1-AP; Wuhan Sanying Biotechnology), anti-AMPKα (cat. no. 66536-1-Ig; Wuhan Sanying Biotechnology), and anti-pAMPKα (cat. no. 83924-1-RR; Wuhan Sanying Biotechnology). Post-primary incubation, membranes received three TBST washes and were incubated for 60 min with species-matched HRP-conjugated secondary antibodies (anti-mouse/rabbit IgG, 1:1,000 dilution in TBST/milk). Chemiluminescent detection was performed using the MultiChemi Elite imaging platform. Quantitative densitometric analysis of immunoreactive bands was conducted via ImageJ software (1.54g, National Institute Health), with protein expression levels normalized against GAPDH as endogenous control. All experiments included triplicate biological replicates to ensure statistical robustness. Statistical analysis. Statistical analyses were performed utilizing one-wayANOVA, supplemented by Tukey’s honestly significant difference multiple comparison procedure, GraphPad Prism (v8.0.2; Dotmatics). Quantitative outcomes are expressed as the means ± standard error of measurement, derived from triplicate experimental iterations, P < 0.05 considered to indicate a statistically significant difference. Discussion Accumulating evidence indicates that PA concentrations spanning 0.2–0.75 mM reproducibly induce IR across diverse cellular models during 16–24 h exposure windows 31 , 32 . Notably, the PA-induced IR paradigm in adipocyte cultures has become a cornerstone experimental system for probing obesity-associated metabolic dysregulation 33 – 35 . In a recent investigation of 3T3-F442A adipocytes, PA administration triggered characteristic IR pathophysiology manifested as: ⅰ) compromised glucose transporter activity; ⅱ) aberrant lipid droplet deposition; ⅲ) suppressed mitochondrial respiration; and ⅳ) transcriptional downregulation of energy homeostasis regulators including β3-AR and AMPK. These mechanistic insights validate the translational relevance of this in vitro model for investigating systemic IR progression and obesity comorbidities in vivo 36 – 38 . The pharmacological induction of adipocyte browning has gained recognition as a novel therapeutic paradigm for addressing obesity-related metabolic disorders, particularly IR 39 , 40 . Capitalizing on the capacity to pharmacologically recapitulate a thermogenically active, BAT-like phenotype in vitro , the present study systematically evaluated the therapeutic efficacy of QC and SLB, a flavonoid glycoside and a sesquiterpene lactone respectively, as phytonutrient-derived bioactive compounds. The present experimental design specifically sought to elucidate the mechanistic basis underlying their potential application in the prevention and amelioration of obesity-associated metabolic dysregulation 41 . Flavonoids exhibit pleiotropic bioactivities with demonstrated therapeutic implications for metabolic syndrome management, as substantiated by recent pharmacological research 42 . The flavonoid QC has exhibited dose-dependent cytoprotective effects through Nrf2-mediated antioxidant response element activation, effectively attenuating ectopic lipid deposition while enhancing insulin sensitivity in oleic acid (OA)-challenged HepG2 hepatocytes, as evidenced by normalized glucose transporter 4 (GLUT4) trafficking and suppressed NF-κB-driven proinflammatory cytokine secretion 43 . Notably, QC administration in diet-induced obese murine models restores euglycemia through PGC-1α-mediated mitochondrial biogenesis, concurrent with gut microbiota remodeling characterized by an increased Bacteroidetes/Firmicutes ratio 44 . SLB exerts multi-target anti-inflammatory action via canonical signaling pathway modulation. In DSS-induced colitis models, SLB treatment maintains intestinal epithelial homeostasis through Wnt/β-catenin pathway inhibition, effectively curbing intestinal stem cell (ISC) hyperproliferation while preserving tight junction protein expression 45 . Mechanistically, the redox-modulating capacity reduces colonic ROS burden via NAD(P)H quinone oxidoreductase 1 (NQO1) induction, correlating with extended longevity in Drosophila through JNK pathway suppression 45 . Transcriptomic analyses reveal SLB-mediated downregulation of IL-6/STAT3 signaling cascades through competitive IKKβ inhibition, thereby attenuating MAPK/AP-1-driven inflammatory responses 46 . Hepatoprotective effects of SLB derivatives include modulation of hepatic sterol biosynthesis, as evidenced by reduced biliary cholesterol saturation index post-silymarin administration in both clinical trials and rodent models 47 . Recent findings implicate gut-liver axis modulation in the anti-steatotic action of silymarin, mediated through microbiota-dependent vitamin B12 biosynthesis and subsequent AMPK-driven lipolysis potentiation 44 . Flavonoids have been extensively documented to exert multifaceted regulatory effects on adipocyte physiology. Emerging evidence supports the therapeutic potential of flavonoid consumption in T2D management, particularly through glycemic regulation mechanisms 48 . Experimental studies utilizing C57BL/6 murine models have further demonstrated that dietary flavonoid supplementation significantly enhances insulin sensitivity under high-fat and high-fructose dietary conditions 49 – 51 . Wu et al. revealed that intracellular flavonoid accumulation in adipocytes upregulates insulin signaling pathways throughβ3-AR activation and AMPK phosphorylation, aligned with the present experimental data 52 . Paradoxically, while multiple studies have reported flavonoid-mediated inhibition of adipocyte differentiation and mitotic activity 53 – 55 , the findings of the present study concur with previous research by Zhang et al 56 , demonstrating that specific flavonoid derivatives (QC and SLB) exhibit selective attenuation of lipogenic pathways in WAT. The present study evaluates the stimulating effects of QC and SLB on the expression of beige-specific genes and proteins, thereby promoting fat browning in 3T3-F442A cells. Previous studies have emphasized the role of plant-based dietary components in promoting fat browning in BAT in rodent models 57 , as well as the direct impact of flavonoids on the browning of WAT in rats 58 . In line with these findings, prior research has demonstrated that the induction of UCP-2 expression in visceral WAT by QC is mediated through PPARα activity 59 . The findings of the present study further indicate that this process involves the activation of the AMPK signaling pathway, which is regulated by the β3-AR-PKA axis. Prior investigations have established thatβ3-AR activation in white adipocytes serves as a critical mediator of WAT browning 60 – 62 . The computational molecular docking analysis in the present study identified high-affinity interactions between QC/SLB and β3-AR, while functional studies corroborated that β3-AR signaling activation constitutes a central mechanism driving adipose browning. Notably, this work identifies AMPK as a central regulatory node in this process. Clinical evidence indicates that AMPK activity is markedly attenuated in adipose depots of patients with morbid obesity and comorbid IR 63 . Functioning as a master metabolic sensor, AMPK orchestrates BAT activation by stimulating adipogenic differentiation, preserving mitochondrial ultrastructure, and inducing transcriptional reprogramming toward a beige adipocyte phenotype 64 , 65 . The present experimental data revealed that QC/SLB treatment robustly upregulated AMPK expression, providing a mechanistic basis for the concomitant elevation of UCP-1 levels and driving browning in white adipocytes. Notably, pharmacological inhibition of AMPK and β3-AR abrogated these effects, confirming the indispensable function of the β3-AR/AMPK axis in modulating adipose browning. Collectively, these findings highlight the therapeutic potential of QC/SLB in enhancing adipose plasticity through WAT browning, thereby augmenting thermogenic capacity. Nonetheless, both PPARγ and SIRT1 play major roles in regulating cellular energy homeostasis and in response to IR 66 , 67 , and the involvement of the PPARγ and SIRT1 response to QC/SLB and systematic preclinical validation in vivo is required to comprehensively delineate the pharmacodynamic profile of QC/SLB in adipose browning regulation. In conclusion, the present experimental data elucidated that QC and SLB orchestrated lipid metabolic remodeling and triggered the phenotypic transition of white adipocytes toward a thermogenic lineage via activation of the β3-AR-AMPK signaling axis. As pleiotropic phytochemicals exhibiting multi-target bioactivity, QC and SLB emerge as promising translational candidates for developing pathophysiology-driven interventions against obesity and its metabolic sequelae. Declarations Ethics approval and consent to participate Not applicable. Conflicts of Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yuanxin Zhang reports financial support was provided by Jilin Provincial Science and Technology Department. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Patient consent for publication Not applicable. Funding This work was supported by Jilin province science and technology development plan project (grant number: 20210204026YY). Author Contribution Yakun Ge: Writing-original draft, Review & editing, Methodology, Formal analysis, Conceptualization. Aiping Liu: Investigation, Methodology, Formal analysis, Resources. Fanwei Meng: Investigation, Methodology. Renwen Zhang: Data curation, Methodology, Project administration. Junting Wang, Qiao Wang: Conceptualization, Methodology. Guanqiao Wu: Validation, Software. Yuanxin Zhang: Review & editing, Methodology, Formal analysis, Conceptualization, Supervision, Project administration, Funding acquisition, Visualization. Acknowledgements Not applicable. Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Li, M. et al. Trends in insulin resistance: insights into mechanisms and therapeutic strategy. Signal. Transduct. Target. Ther. 7 , 216 (2022). Lee, S. H., Park, S. Y. & Choi, C. S. Insulin resistance: from mechanisms to therapeutic strategies. 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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-7124697\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":495253009,\"identity\":\"4181aa3c-a00b-4488-9352-96a6bf599762\",\"order_by\":0,\"name\":\"Yakun Ge\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin University of Chemical Technology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yakun\",\"middleName\":\"\",\"lastName\":\"Ge\",\"suffix\":\"\"},{\"id\":495253010,\"identity\":\"092c485a-8238-4df2-81c1-34a6b9bf4e3d\",\"order_by\":1,\"name\":\"Aiping Liu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin University of Chemical Technology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Aiping\",\"middleName\":\"\",\"lastName\":\"Liu\",\"suffix\":\"\"},{\"id\":495253011,\"identity\":\"e6cc3596-d3ac-4dfa-99cf-9fe000633dd7\",\"order_by\":2,\"name\":\"Fanwei Meng\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin University of Chemical Technology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Fanwei\",\"middleName\":\"\",\"lastName\":\"Meng\",\"suffix\":\"\"},{\"id\":495253012,\"identity\":\"9b907026-fee6-44ae-bb07-711456ee1a84\",\"order_by\":3,\"name\":\"Renwen Zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin University of Chemical Technology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Renwen\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":495253013,\"identity\":\"17253373-3bca-4ba8-87bc-454edf64bd24\",\"order_by\":4,\"name\":\"Junting Wang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin University of Chemical Technology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Junting\",\"middleName\":\"\",\"lastName\":\"Wang\",\"suffix\":\"\"},{\"id\":495253015,\"identity\":\"936b935e-63c7-4358-9d96-6dbd49c2c802\",\"order_by\":5,\"name\":\"Qiao Dang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin University of Chemical Technology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Qiao\",\"middleName\":\"\",\"lastName\":\"Dang\",\"suffix\":\"\"},{\"id\":495253017,\"identity\":\"6cd1aff1-01b4-44a6-9f23-95556414cd58\",\"order_by\":6,\"name\":\"Guanqiao Wu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin University of Chemical Technology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Guanqiao\",\"middleName\":\"\",\"lastName\":\"Wu\",\"suffix\":\"\"},{\"id\":495253019,\"identity\":\"1654d96f-b1ba-4cea-a861-8a5e471eab7c\",\"order_by\":7,\"name\":\"Yuanxin Zhang\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYHACNhAhx9jO2ABiQEhitBgzNgMVHyBFS2IDM5AkSovBjfRnD37uqE1vbmZu/PyBwUZ2wwHmZw/wajlzIN2w98zx3MZmxmaJAwxpxhsOsJkb4NVyvOGYBG/bMZCWNqDDDiduOMDDJoFXy2HGNsm/bcfSGSFa/hOh5XgzmzRvW00CVMsBwlokzxxjk5ZtO2AI9ssZg2TjmYfZzPBq4QOGmOTbtjp5w/b2hx8qKuxk+443P8OrReEAmDrMYNgAdicQM+NTDwTyYJUMdQzyBBSOglEwCkbBCAYAmVFOJyFKI8wAAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Jilin University of Chemical Technology\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Yuanxin\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-07-14 22:38:14\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-7124697/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-7124697/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":88341748,\"identity\":\"6614bc7b-9c51-42b8-a2b4-e2762bd90def\",\"added_by\":\"auto\",\"created_at\":\"2025-08-05 12:51:19\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":559977,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eQC (a) and SLB (b) restored mitochondrial activity, as demonstrated by MTT and ATP production (c) assays, and enhanced glucose uptake (d) in PA-induced insulin resistance (IR)-3T3-F442A cells. Results are expressed as the mean ± SD of three independent experiments. \\u003csup\\u003e***\\u003c/sup\\u003eP \\u0026lt; 0.001 compared with the control group, and \\u003csup\\u003e#\\u003c/sup\\u003eP \\u0026lt; 0 .05, \\u003csup\\u003e##\\u003c/sup\\u003eP \\u0026lt; 0.01, \\u003csup\\u003e###\\u003c/sup\\u003eP \\u0026lt; 0.001 compared with the PA treatment group by the Student’s \\u003cem\\u003et\\u003c/em\\u003e test.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7124697/v1/569d9f4c3d6284425dab270e.png\"},{\"id\":88342799,\"identity\":\"657ac46d-315a-4cca-9e8a-f96a2cc49c0d\",\"added_by\":\"auto\",\"created_at\":\"2025-08-05 13:07:19\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":6981010,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eQC and SLB increase lipid droplet content, as indicated by ORO staining (a and b), and mitochondrial bioenergetics, as shown by MitoTracker Red CMXRo staining (c and d), in PA-exposed IR-3T3-F442A cells (images were taken at ×40 magnification, scale bars = 50 μm).\\u003cstrong\\u003e \\u003c/strong\\u003eValues are presented as the mean ± SD (n = 3). \\u003csup\\u003e*\\u003c/sup\\u003eP \\u0026lt; 0.05, \\u003csup\\u003e**\\u003c/sup\\u003eP \\u0026lt; 0.01, \\u003csup\\u003e***\\u003c/sup\\u003eP \\u0026lt; 0.001 compared with control by the Student’s \\u003cem\\u003et\\u003c/em\\u003e test. \\u003csup\\u003e#\\u003c/sup\\u003eP \\u0026lt; 0.05, \\u003csup\\u003e##\\u003c/sup\\u003eP \\u0026lt; 0.01, \\u003csup\\u003e###\\u003c/sup\\u003eP \\u0026lt; 0.001 compared with PA treatment group by the Student’s \\u003cem\\u003et\\u003c/em\\u003e test.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7124697/v1/c24ba58aa9f358674091f9d3.png\"},{\"id\":88342686,\"identity\":\"f6b88c59-be6c-45e8-a0a0-917b5eaa6e91\",\"added_by\":\"auto\",\"created_at\":\"2025-08-05 12:59:19\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":846807,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eQC and SLB increase the expression of beige-specific genes (a), the mRNA (b), and protein (c) levels of browning-specific markers in PA-exposed IR-3T3-F442A cells. Values are presented as the mean ± SD (n = 3). \\u003csup\\u003e*\\u003c/sup\\u003eP \\u0026lt; 0.05, \\u003csup\\u003e**\\u003c/sup\\u003eP \\u0026lt; 0.01, \\u003csup\\u003e***\\u003c/sup\\u003eP \\u0026lt; 0.001 compared with control by the Student’s \\u003cem\\u003et\\u003c/em\\u003e test. \\u003csup\\u003e#\\u003c/sup\\u003eP \\u0026lt; 0.05, \\u003csup\\u003e##\\u003c/sup\\u003eP \\u0026lt; 0.01, \\u003csup\\u003e###\\u003c/sup\\u003eP \\u0026lt; 0.001 compared with PA treatment group by the Student’s \\u003cem\\u003et\\u003c/em\\u003e test.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7124697/v1/c469275e95de0abdb8422ae8.png\"},{\"id\":88342693,\"identity\":\"61c052d7-f13b-4638-901f-b57106d4c716\",\"added_by\":\"auto\",\"created_at\":\"2025-08-05 12:59:19\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":796302,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eQC and SLB increase the expression of fatty acid β-oxidation (a)/lipolysis (b) genes and protein (c) levels of lipid metabolism and fat browning in PA-exposed IR in 3T3-F442A cells. Values are presented as the mean ± SD (n = 3). \\u003csup\\u003e*\\u003c/sup\\u003eP \\u0026lt; 0.05, \\u003csup\\u003e**\\u003c/sup\\u003eP \\u0026lt; 0.01, \\u003csup\\u003e***\\u003c/sup\\u003eP \\u0026lt; 0.001 compared with control by the Student’s \\u003cem\\u003et\\u003c/em\\u003e test. \\u003csup\\u003e#\\u003c/sup\\u003eP \\u0026lt; 0.05, \\u003csup\\u003e##\\u003c/sup\\u003eP \\u0026lt; 0.01, \\u003csup\\u003e###\\u003c/sup\\u003eP \\u0026lt; 0.001 compared with PA treatment group by the Student’s \\u003cem\\u003et\\u003c/em\\u003e test.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7124697/v1/08644edfa935366a862c9892.png\"},{\"id\":88342800,\"identity\":\"99731c61-bda5-412e-90b3-2dd714ad74be\",\"added_by\":\"auto\",\"created_at\":\"2025-08-05 13:07:19\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":2523060,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMolecular docking simulations of active compounds and targets. QC (a) and its predicted core targets (b). SLB (c) and its predicted core targets (d).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7124697/v1/dd32ab2c13363c7e326e86fe.png\"},{\"id\":88341772,\"identity\":\"d211af7d-2acb-40e7-a0c3-60e37738b9d3\",\"added_by\":\"auto\",\"created_at\":\"2025-08-05 12:51:20\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1320011,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eQC and SLB stimulate white adipose browning via the β3-AR/AMPKα signaling pathway in PA-exposed adipocytes. (a) The protein expression levels of brown adipocyte markers after treatment with dorsomorphin antagonist. (b) The protein expression levels of brown adipocyte markers after treatment with AMPK antagonist. Values are presented as the mean ±SD (n = 3). \\u003csup\\u003e*\\u003c/sup\\u003eP \\u0026lt; 0.05, \\u003csup\\u003e**\\u003c/sup\\u003eP \\u0026lt; 0.01, \\u003csup\\u003e***\\u003c/sup\\u003eP \\u0026lt; 0.001 compared with PA treatment group by the Student’s \\u003cem\\u003et\\u003c/em\\u003e test. \\u003csup\\u003e#\\u003c/sup\\u003eP \\u0026lt; 0.05, \\u003csup\\u003e##\\u003c/sup\\u003eP \\u0026lt; 0.01, \\u003csup\\u003e###\\u003c/sup\\u003eP \\u0026lt; 0.001 compared with antagonist treatment group by the Student’s \\u003cem\\u003et\\u003c/em\\u003e test.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7124697/v1/943db5d401f93f32286f7e52.png\"},{\"id\":88342688,\"identity\":\"1475bde9-222d-4b89-a0c4-3ab9fb9e6a58\",\"added_by\":\"auto\",\"created_at\":\"2025-08-05 12:59:19\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1277059,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSchematic diagram summarizes the molecular mechanism underlying the QC- and SLB-induced fat browning in 3T3-F442A adipocytes. Straight arrow refers to promotion.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7124697/v1/7df2214006ba42a5962b1765.png\"},{\"id\":95653876,\"identity\":\"0cfe21a2-ddf0-4742-afe1-3623c2010e77\",\"added_by\":\"auto\",\"created_at\":\"2025-11-11 16:03:47\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":14322584,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7124697/v1/edf29b2b-f006-4ab7-ae01-2a3d8e1e1304.pdf\"},{\"id\":88342691,\"identity\":\"f6309977-cefa-4720-93d8-4471b0458538\",\"added_by\":\"auto\",\"created_at\":\"2025-08-05 12:59:19\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":4999326,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryMaterial1.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7124697/v1/5eb42910359bd5401b272fde.pdf\"},{\"id\":88341752,\"identity\":\"29e328bf-9b39-43aa-9e9c-52cca7b604bb\",\"added_by\":\"auto\",\"created_at\":\"2025-08-05 12:51:19\",\"extension\":\"xlsx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":30661,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryMaterial2.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7124697/v1/290d228e85ee79c6aa85c383.xlsx\"},{\"id\":88342689,\"identity\":\"677fbb50-c68d-41a2-9374-64ac4f4b68d6\",\"added_by\":\"auto\",\"created_at\":\"2025-08-05 12:59:19\",\"extension\":\"docx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":14558,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryMaterial3.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7124697/v1/6fb444e2769c3e5226a56a42.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Quercetin and silibinin ameliorate insulin resistance by stimulating fat browning via the β3-AR/AMPK pathway in palmitate-exposed 3T3-F442A adipocytes\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eIR, characterized by diminished biological responsiveness to insulin, serves as a central driver in the pathogenesis of metabolic disorders including type diabetes (T2D), obesity, polycystic ovarian syndrome, and cardiovascular diseases\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e. As a dynamic endocrine organ, adipose tissue exhibits dual functionality in energy homeostasis and IR development through three distinct subtypes: white adipose tissues (WAT), brown adipose tissues (BAT), and beige adipose tissues\\u003csup\\u003e\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003e. WAT primarily stores energy as triacylglycerol (TAG), whereas BAT specializes in thermogenic energy expenditure via uncoupling protein 1 (UCP1)-mediated mechanisms\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e. Beige adipocytes, emerging through the browning of WAT, demonstrate intermediate thermogenic capacity between white and brown fat, with their induction showing therapeutic potential for improving insulin sensitivity\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e. This phenotypic conversion enhances mitochondrial biogenesis and metabolic flexibility through coordinated activation of PPARα/γ, PGC1α, and AMPK signaling pathways\\u003csup\\u003e\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e. Previous studies have highlighted that the following environmental and pharmacological interventions synergistically promoted adipose tissue remodeling: ⅰ) Chronic cold exposure and β-adrenergic agonists stimulate sympathetic activation of UCP1-dependent thermogenesis\\u003csup\\u003e\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u003c/sup\\u003e; ⅱ) medicinal plant-derived bioactive compounds offer safe alternatives for inducing beige adipogenesis\\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u003c/sup\\u003e. Therefore, adipose tissue browning has emerged as a promising therapeutic axis for mitigating IR-associated comorbidities through metabolic reprogramming and inflammatory modulation\\u003csup\\u003e\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003cp\\u003ePlant flavonoids have emerged as key bioactive compounds in metabolic regulation, with particular focus on their promising effects on IR and lipid metabolism\\u003csup\\u003e\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u003c/sup\\u003e. These polyphenolic molecules exhibit multi-target functionality for mitigating IR including: ⅰ) Scavenging reactive oxygen species (ROS) to alleviate hyperglycemia-induced oxidative stress and improve insulin sensitivity\\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003e; 2) modulating lipid profiles to alleviate IR by decreasing triglycerides/ low density lipoprotein (LDL) while elevating high density lipoprotein (HDL) cholesterol\\u003csup\\u003e\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u003c/sup\\u003e; Ⅲ) suppressing pro-inflammatory cytokines are implicated in IR pathogenesis\\u003csup\\u003e\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u003c/sup\\u003e. Among natural flavonoids, QC and SLB exhibit unique therapeutic potential for alleviating IR. QC shows pleiotropic effects spanning blood pressure regulation to glucose homeostasis\\u003csup\\u003e\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u003c/sup\\u003e, while SLB exhibits notable antioxidant, antiapoptotic, anti-inflammatory, and lipid-modulating capacities\\u003csup\\u003e\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u003c/sup\\u003e. Moreover, previous research has shown that SLB effectively mitigated PA-induced IR, while QC counteracts tumor necrosis factor alpha (TNFα)-induced IR in mouse C2C12 myoblasts\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e\\u003c/sup\\u003e. However, despite these promising \\u003cem\\u003ein vitro\\u003c/em\\u003e findings, the mechanisms by which QC and SLB influence lipid accumulation and their potential effects on adipose tissue browning remain to be fully elucidated. In the present study, the beneficial effects of QC and SLB on metabolic functions were systematically investigated, with a particular focus on their IR-attenuating effects through adipocyte browning, providing new insights into natural product strategies for IR management.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e\\u003cb\\u003eEffect of QC and SLB on metabolic activity and glucose uptake in PA-exposed adipocytes.\\u003c/b\\u003e To assess the putative cytotoxic properties of QC or SLB in 3T3-F442A preadipocytes, cellular models were subjected to combinatorial exposure with PA (0.75 mM) co-administered with pharmacologically relevant concentrations (5-160 \\u0026micro;M) of test compounds for 24 h incubation. Subsequently, the impact of these flavonoids on PA-induced IR-3T3-F442A adipocytes was evaluated using the MTT assay. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA and B, QC and SLB intervention restored mitochondrial activity in a dose-dependent manner in PA-induced IR-3T3-F442A cells, with optimal therapeutic efficacy manifesting at a safe concentration (40 \\u0026micro;M QC, 40% restoration; 80 \\u0026micro;M SLB,35% restoration) relative to PA monotherapy\\u003csup\\u003e\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e. Complementary assessment of intracellular ATP biosynthesis through luminometric quantification confirmed concordant metabolic rescue effects, demonstrating 22% elevation in ATP generation following QC/SLB treatment relative to PA-exposed controls (P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC), thereby corroborating MTT-derived metabolic parameters.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eAdditionally, the glucose uptake capacity of IR-3T3-F442A adipocytes challenged with PA was systematically evaluated. As illustrated in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD, administration of PA at a concentration of 0.75 mM elicited a marked 15% suppression of glucose uptake relative to baseline levels. By contrast, pharmacological intervention with either QC (40 \\u0026micro;M) or SLB (80 \\u0026micro;M) exhibited significant enhancement of glucose transport efficiency, achieving 32 and 25% elevation respectively (P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) in IR-3T3-F442A cells.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eEffect of QC and SLB on lipid accumulation and mitochondrial bioenergetics in PA-exposed adipocytes.\\u003c/b\\u003e Pathological adipogenesis in WAT constitutes a pivotal etiological factor in the development of IR, as evidenced by seminal studies\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR26\\\" citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u003c/sup\\u003e. To elucidate the therapeutic potential of bioactive compounds, ORO histochemical staining with morphometric analysis was used to quantitatively assess the regulatory effects of QC and SLB on intracellular lipid deposition in an established IR-3T3-F442A adipocyte model. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA and B, PA challenge induced a 13.2% elevation in cytoplasmic lipid droplet formation (P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001), consistent with characteristic metabolic perturbations observed in T2D. Notably, pharmacological administration of 40 \\u0026micro;M QC or 80 \\u0026micro;M SLB demonstrated substantial lipid-lowering efficacy, achieving 32.1 and 30.8% reductions in lipid accumulation respectively (P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001). These findings collectively demonstrate that both phytochemicals effectively ameliorate PA-induced lipotoxic insults through mechanisms involving enhanced lipid catabolism.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eMitochondrial bioenergetics exert a critical regulatory influence on the pathogenesis of IR\\u003csup\\u003e\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u003c/sup\\u003e. To elucidate the therapeutic potential of QC or SLB in modulating mitochondrial dynamics within IR-3T3-F442A adipocytes, mitochondrial activity was quantitatively evaluated through MitoTracker Red CMXRos fluorescence staining. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC, confocal microscopy analysis revealed thatPA-induced IR adipocytes exhibited a marked augmentation in mitochondrial membrane potential, indicative of progressive restoration of mitochondrial bioenergetic capacity. Notably, pharmacological intervention with QC (40 \\u0026micro;M) or SLB (80 \\u0026micro;M) elicited a pronounced amplification of mitochondrial redox potential compared with PA-challenged controls. Complementary transcriptional profiling (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD) further demonstrated that both QC and SLB administration induced substantial transcriptional upregulation of nuclear-encoded mitochondrial biogenesis regulators, particularly Nrf1 and Tfam. These data collectively suggest that QC and SLB enhance mitochondrial oxidative phosphorylation efficiency through coordinated activation of the Nrf1/Tfam signaling axis, thereby facilitating the browning of insulin-resistant adipocytes.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eEffect of QC and SLB on fat browning in PA-exposed adipocytes.\\u003c/b\\u003e The pharmacological induction of WAT browning represents a promising therapeutic intervention for metabolic disorders\\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e. To elucidate the molecular mechanisms underlying this process, comprehensive analyses of browning-specific genes and regulatory proteins were conducted in IR-3T3-F442A adipocytes following treatment with QC (40 \\u0026micro;M) or SLB (80 \\u0026micro;M). As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA, IR adipocytes exposed to either compound exhibited marked upregulation of beige adipocyte-specific genes, including transmembrane protein 26 (Tmem26), T-box transcription factor 1 (Tbx1), tumor necrosis factor receptor superfamily member 9 (CD137), and Cbp/p300-interacting transactivator 1 (Cited1). Moreover, QC (40 \\u0026micro;M) or SLB (80 \\u0026micro;M) upregulated the expression levels of mRNA and protein for critical browning markers including PGC-1α, PRDM16, and UCP1 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB and C). These data provide compelling evidence that both QC and SLB effectively promote the browning phenotype in 3T3-F442A adipocytes through coordinated activation of thermogenic transcriptional networks.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eEffect of QC and SLB on lipid metabolism in PA-exposed adipocytes.\\u003c/b\\u003e The present study investigated the effects of QC (40 \\u0026micro;M) or SLB (80 \\u0026micro;M) on lipid metabolism-related genes and proteins in IR-3T3-F442A adipocytes. The results demonstrated that QC/SLB treatment significantly upregulated the expression of carnitine palmitoyltransferas (CPT), 1-aminocyclopropane-1-carboxylate oxidase (ACO), and PPARα genes associated with fatty acid β-oxidation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). Additionally, genes encoding β3-AR, protein kinase A (PKA), and adipose triglyceride lipase (ATGL), which are involved in lipolytic pathways, showed marked elevation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB). A pronounced increase was observed in the expression of β3-AR, PKA, and pAMPKα, which have been identified as key regulators in lipid metabolism and fat browning processes. Notably, the protein expression levels of β3-AR and PKA, along with the pAMPKα/AMPKα ratio, were significantly elevated (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eQC and SLB activate adrenergic receptors.\\u003c/b\\u003e Computational docking protocols were executed to systematically assess the ligand-receptor binding thermodynamics, conformational orientations, and molecular recognition patterns between the small-molecule ligands QC or SLB and theβ3-AR target (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA and \\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB). Rigorous analysis of the molecular dynamics docking demonstrated that the QC ligand achieved a binding free energy of -8.4 kcal/mol with β3-AR, featuring multiple interacting residues. Similarly, the SLB ligand manifested superior binding thermodynamics (ΔG = -8.6 kcal/mol), engaging critical catalytic residues within the orthosteric pocket of the receptors. Both ligands demonstrated sustained stabilization (ΔG_\\u0026lt; -5 kcal/mol) with minimal conformational fluctuations (root-mean-square deviation\\u0026thinsp;\\u0026lt;\\u0026thinsp;2.00 \\u0026Aring;). These computational insights substantiate the formation of thermodynamically favorable binding complexes between the ligands and β3-AR, characterized by optimal pharmacophore complementarity.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eThe binding conformations and detailed interactions of QC and SLB with β3-AR are depicted in Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC and D. Specifically, QC formed non-covalent interactions with key amino acid residues, including Asn329, Asp114, Gly91, Trp330, Ala95, and Tyr333. In addition, SLB established six hydrogen bonds with Arg191, Trp330, and Asn329 residues on the receptor, along with three additional hydrogen bonds, further stabilizing its interaction. These results collectively indicate that both QC and SLB exhibit strong binding affinity for β3-AR, mediated by distinct yet effective non-covalent interactions.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eEffect of QC and SLB on adipose browning in PA-exposed adipocytes via the β3-AR/AMPKα signaling pathway.\\u003c/b\\u003e The regulatory roles ofβ3-AR and AMPK signaling pathways in IR pathogenesis have been extensively characterized\\u003csup\\u003e\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003e. To delineate the mechanistic basis of QC or SLB-induced adipose browning in PA-treated IR-3T3-F442A adipocytes, the β3-AR/AMPKα axis was investigated using the following pharmacological inhibitors: SR59230A (10 \\u0026micro;M; β3-AR antagonist) and dorsomorphin (10 \\u0026micro;M; AMPK antagonist). As illustrated in Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA and B, dual pathway blockade substantially attenuated the expression of thermogenic markers (UCP-1, PGC-1αand PRDM16). Notably, both phytocompounds reversed this suppression, restoring brown adipocyte-specific gene signatures in a β3-AR/AMPKα-dependent manner. This restorative effect was abolished upon antagonist administration, indicating that QC or SLB promotes PA-induced fat browning in IR-3T3-F442A adipocytes by activating the β3-AR and AMPK signaling pathways (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cp\\u003e\\u003cb\\u003eChemicals.\\u003c/b\\u003e SLB, QC, isobutylmethylxanthine (IBMX), dexamethasone (DEX), insulin, palmitic acid (PA), dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2-deoxy-2-[(7-nitro-1,2,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG, and Oil Red O (ORO) were obtained from Sigma-Aldrich (Merck KGaA). The β3-adrenergic receptor AR (β3-AR, ADRB3) antagonist SR59230A and the AMP activated protein kinase (AMPK) antagonist Dorsomorphin were purchased from MedChem Express. Isopropanol and chloroform were obtained from Shanghai Aladin Biochemical Technology Co., Ltd.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eCell culture and differentiation.\\u003c/b\\u003e The 3T3-F442A murine embryonic fibroblast cell line (cat no. FY-22FN0241; Shanghai Fuyu Biotechnology Co., Ltd) was propagated in growth medium comprising Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Inc), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc), under standard culture conditions (37 ˚C, 5% CO₂, humidified atmosphere). Routine subculture was conducted at 2- or 3-day intervals upon attaining 80\\u0026ndash;90% monolayer confluence. For differentiation induction, cells were plated in six-well culture dishes at 2 x 10⁵ cells/well and left to proliferate to full confluence over 72 h. At confluence (designated Day 0), the growth medium was replaced with a differentiation induction cocktail containing DMEM with 10% FBS, 0.5 mM IBMX, 1 \\u0026micro;M DEX, and 2 \\u0026micro;g/ml insulin. After 48 h of exposure to differentiation stimuli, the induction medium was substituted with adipogenic maintenance medium (DMEM\\u0026thinsp;+\\u0026thinsp;2 \\u0026micro;g/ml insulin) for an additional 48 h. After differentiation culture, cells were reverted to standard growth medium (refreshed every two days) until terminal differentiation at Day 8, at which point experimental analyses were initiated.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003ePreparation and processing conditions of a 3T3-F442A adipocyte model of IR.\\u003c/b\\u003e Differentiated and mature 3T3-F442A adipocytes were divided into the following four experimental cohorts: ⅰ) Control group; ⅱ) PA-induced IR model group; ⅲ) QC-therapeutic group; and ⅳ) SLB-therapeutic group. The control group was cultured in basal DMEM growth medium throughout the experimental timeline. The PA-induced IR model group underwent metabolic challenge through 24-h exposure to DMEM supplemented with 0.75 mM PA. In the QC-therapeutic group, cells were preconditioned with QC-enriched DMEM for 24 h prior to PA challenge, followed by cotreatment with QC during the 24-h PA induction phase, and subsequently maintained in QC-supplemented medium post-induction. Analogously, the SLB-therapeutic group received prophylactic treatment with SLB-fortified DMEM for 24 h, followed by concurrent administration with 0.75 mM PA in SLB-containing medium during the induction phase, culminating in a 24-h recovery period in SLB-supplemented conditions. Following completion of these temporally regulated pharmacological regimens, all cellular specimens were processed for downstream analytical procedures.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eMTT and ATP assays.\\u003c/b\\u003e Firstly, 3T3-F442A preadipocytes were plated in 96-well culture plates at an initial seeding density of 5 x10\\u0026sup3; cells/well. The differentiation protocol was initiated upon achieving 70\\u0026ndash;80% cellular confluence. After differentiation and maturation, cells were randomly allocated into the following five experimental groups: ⅰ) untreated control group; ⅱ) PA-induced model group (0.75 mM PA); ⅲ) QC-therapeutic group (0.75 mM PA co-administered with 5-160 \\u0026micro;M QC in geometric progression); ⅳ) SLB-therapeutic group (0.75 mM PA combined with 5-160 \\u0026micro;M SLB); and ⅴ) reagent background control (cell-free system). Following experimental treatments, 20 \\u0026micro;l MTT solution (5 mg/ml in PBS) was introduced to each well, with subsequent incubation at 37 ˚C for 4 h under standard culture conditions. The formazan crystals were solubilized by adding 150 \\u0026micro;l DMSO following careful aspiration of culture supernatants. Optical density measurements were conducted at 570 nm using a multimode microplate reader (BioTek; Agilent Technologies, Inc.), with data normalized to untreated controls.\\u003c/p\\u003e\\u003cp\\u003eTo quantify ATP biosynthesis, ATP production analysis was conducted employing an ATP Generation Assay Kit (Sigma-Aldrich; Merk KGaA). The experimental protocol involved aliquoting 100 \\u0026micro;l ATP detection reagent into test wells followed by a 5-min incubation period to stabilize baseline signal. Subsequently, 20 \\u0026micro;l cellular lysate was introduced into each reaction chamber and homogenized through gentle agitation. Chemiluminescent signals were quantified using a multimode microplate detection system with 2-sec integration intervals between sequential measurements. Simultaneous protein quantification was performed using a BCA assay (Beyotime Institute of Biotechnology) to enable normalized ATP production values expressed as nmol/mg protein.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eGlucose uptake assay.\\u003c/b\\u003e The treated adipocytes were subjected to a 1-h incubation period at 37 ˚C in serum-free low-glucose DMEM medium containing diminished phenol red concentration, with supplementation with 100 \\u0026micro;M 2-NBDG fluorescent glucose analog, under light-protected environmental conditions. Following the glucose uptake phase, cellular monolayers underwent three successive washes with ice-cold phosphate-buffered saline (PBS) to arrest the transport process. Quantitative fluorescence intensity analysis was performed using a multimode microplate reader configured with 485 nm excitation and 535 nm emission parameters. For experimental normalization, total protein content per well was determined through BCA protein assay (Beyotime Institute of Biotechnology), establishing specific fluorescence values/ unit protein mass.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eORO staining and lipid content determination.\\u003c/b\\u003e The experimental adipocyte samples underwent ORO histochemical staining following an established protocol. Firstly, the ORO stock solution was prepared by dissolving 0.5 g crystalline ORO powder in 100 ml anhydrous isopropanol, followed by overnight storage at 4 ˚C. Prior to application, the stock solution was diluted with ultrapure water at a 3:2 (v/v) ratio, equilibrated to an ambient temperature for 20 min, filtered through a 0.45 \\u0026micro;m pore-size membrane filter, and maintained under light-protected conditions. The cellular specimens were subjected to three sequential 5-min PBS washing cycles using gentle agitation. Subsequent fixation was achieved through incubation with 10% neutral-buffered formalin for 60 min at room temperature. Following triple ultrapure water rinses, the cells were immersed in freshly prepared ORO working solution for 30 min under standardized laboratory conditions. Post-staining cells were rinsed three times prior to microscopic examination and imaging. For quantitative lipid content, the intracellular ORO complexes were eluted in absolute isopropanol through 10-min orbital shaking. The resultant chromogenic solution was subjected to spectrophotometric analysis at λ\\u0026thinsp;=\\u0026thinsp;570 nm employing a 96-well microplate reader system, with absorbance measurements normalized against reagent blanks.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eMitoTracker Red assay.\\u003c/b\\u003e Following cellular treatment, adipocytes underwent standardized immunofluorescence staining protocols. The existing culture medium was carefully aspirated prior to triple PBS washing. Cellular specimens were subsequently incubated with 200 nM MitoTracker Red CMXRos (Thermo Fisher Scientific) in serum-free DMEM medium (1 ml/well) under controlled conditions (37 ˚ C, 5% CO₂) for 30 min. Post-incubation, an additional 15-min maintenance phase was implemented under identical environmental parameters. Cellular fixation was achieved through 15-min exposure to 4% paraformaldehyde at ambient temperature, followed by thorough PBS rinsing. Membrane permeabilization was conducted using 0.2% Triton X-100 (10 min), succeeded by triple PBS washing cycles. Nuclear counterstaining was performed with 10 \\u0026micro;g/ml Hoechst 33342 (Beyotime Institute of Biotechnology) in serum-free DMEM (5 min, room temperature). Following final PBS washes, specimens were mounted with 500 \\u0026micro;l anti-fade reagent and visualized using inverted epifluorescence microscopy.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eReverse transcription-quantitative PCR.\\u003c/b\\u003e The cellular sample (5 x 10\\u003csup\\u003e6\\u003c/sup\\u003e treated cells) was subjected to RNA extraction through optimized guanidinium thiocyanate-phenol-chloroform methodology. Following complete cell lysis using 1 ml Trizol\\u0026reg; reagent (Beyotime Institute of Biotechnology; cat. no. R0016), the homogenate was centrifuged (12,000 x g, 5 min, 4 ˚C) to remove insoluble debris. Subsequent phase separation was achieved by chloroform supplementation (200 \\u0026micro;l) with vigorous vortexing (30 sec) and incubation for 15 min at room temperature. Centrifugation under identical parameters yielded distinct phase stratification, from which 500 \\u0026micro;l aqueous phase was aspirated for isopropanol precipitation (1:1 v/v ratio).\\u003c/p\\u003e\\u003cp\\u003eThe RNA pellet obtained after centrifugation (12,000 x g, 10 min, 4 ˚C) underwent sequential ethanol washes (75% v/v, 1 mL x 2) with intermediate centrifugation cycles (5 min each). Residual ethanol was removed by vacuum desiccation prior to resuspension in DEPC-treated ultrapure water. RNA purity and concentration were spectrophotometrically verified (NanoDrop\\u0026trade; 2000) through A260/A280 absorbance ratio quantification. Reverse transcription was performed using 1 \\u0026micro;g total RNA with SweScript RT II First Strand cDNA Synthesis System (Wuhan Servicebio Technology Co., Ltd.) under manufacturer-specified conditions. Quantitative amplification was conducted on the ABI 7500 Real-Time PCR System with SYBR Green chemistry, employing the following thermal profile: initial denaturation (94 ˚C for 30 sec); 40 cycles of denaturation (94 ˚C for 5 sec), annealing (60 ˚C for 15 sec), and extension (72 ˚C for 30 sec). Cycle threshold (CT) values were normalized against the 18 S rRNA endogenous control, with relative expression levels calculated via the comparative CT method (2\\u003csup\\u003e\\u0026minus;ΔΔCT\\u003c/sup\\u003e). Primer sequences are catalogued in Supplementary Table\\u0026nbsp;1.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eIn-silico analysis.\\u003c/b\\u003e The immunoblotting protocol commenced with generation of cellular lysates using RIPA buffer, followed by clarification through centrifugation at 12,000 x g for 20 min at 4 ˚C. Protein extracts were diluted in 5 x SDS loading buffer, denatured at 100 ˚C for 5 min, and resolved through discontinuous SDS-polyacrylamide gel electrophoresis employing 10 or 12% resolving gels. Electrophoretically separated proteins were subsequently transferred onto PVDF membranes under semi-dry conditions. Membranes underwent blocking in TBST supplemented with 5% non-fat dried milk for 60 min at an ambient temperature. Following three sequential TBST washes (5 min each), membranes were probed overnight at 4 ˚C with primary antibodies diluted in TBST/5% milk solution. The antibody (1:500 dilution in TBST/milk) panel comprised: Anti-GAPDH (cat. No. sc-137179; Santa Cruz Biotechnology, Inc.), anti-UCP1 (cat. no. 83870-1-RR; Wuhan Sanying Biotechnology), anti-PGC-1α (cat. no. 66369-1; Wuhan Sanying Biotechnology), anti-PRDM16 (cat. no. 83872-1-RR; Wuhan Sanying Biotechnology), anti-β3-AR (cat. no. ab76249; Abcam), anti-PKA (cat. no. 12232-1-AP; Wuhan Sanying Biotechnology), anti-AMPKα (cat. no. 66536-1-Ig; Wuhan Sanying Biotechnology), and anti-pAMPKα (cat. no. 83924-1-RR; Wuhan Sanying Biotechnology). Post-primary incubation, membranes received three TBST washes and were incubated for 60 min with species-matched HRP-conjugated secondary antibodies (anti-mouse/rabbit IgG, 1:1,000 dilution in TBST/milk). Chemiluminescent detection was performed using the MultiChemi Elite imaging platform. Quantitative densitometric analysis of immunoreactive bands was conducted via ImageJ software (1.54g, National Institute Health), with protein expression levels normalized against GAPDH as endogenous control. All experiments included triplicate biological replicates to ensure statistical robustness.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eStatistical analysis.\\u003c/b\\u003e Statistical analyses were performed utilizing one-wayANOVA, supplemented by Tukey\\u0026rsquo;s honestly significant difference multiple comparison procedure, GraphPad Prism (v8.0.2; Dotmatics). Quantitative outcomes are expressed as the means\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard error of measurement, derived from triplicate experimental iterations, P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 considered to indicate a statistically significant difference.\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eAccumulating evidence indicates that PA concentrations spanning 0.2\\u0026ndash;0.75 mM reproducibly induce IR across diverse cellular models during 16\\u0026ndash;24 h exposure windows\\u003csup\\u003e\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e\\u003c/sup\\u003e. Notably, the PA-induced IR paradigm in adipocyte cultures has become a cornerstone experimental system for probing obesity-associated metabolic dysregulation\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR34\\\" citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u003c/sup\\u003e. In a recent investigation of 3T3-F442A adipocytes, PA administration triggered characteristic IR pathophysiology manifested as: ⅰ) compromised glucose transporter activity; ⅱ) aberrant lipid droplet deposition; ⅲ) suppressed mitochondrial respiration; and ⅳ) transcriptional downregulation of energy homeostasis regulators including β3-AR and AMPK. These mechanistic insights validate the translational relevance of this \\u003cem\\u003ein vitro\\u003c/em\\u003e model for investigating systemic IR progression and obesity comorbidities in vivo\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR37\\\" citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003cp\\u003eThe pharmacological induction of adipocyte browning has gained recognition as a novel therapeutic paradigm for addressing obesity-related metabolic disorders, particularly IR\\u003csup\\u003e\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e\\u003c/sup\\u003e. Capitalizing on the capacity to pharmacologically recapitulate a thermogenically active, BAT-like phenotype \\u003cem\\u003ein vitro\\u003c/em\\u003e, the present study systematically evaluated the therapeutic efficacy of QC and SLB, a flavonoid glycoside and a sesquiterpene lactone respectively, as phytonutrient-derived bioactive compounds. The present experimental design specifically sought to elucidate the mechanistic basis underlying their potential application in the prevention and amelioration of obesity-associated metabolic dysregulation\\u003csup\\u003e\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003cp\\u003eFlavonoids exhibit pleiotropic bioactivities with demonstrated therapeutic implications for metabolic syndrome management, as substantiated by recent pharmacological research\\u003csup\\u003e\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e\\u003c/sup\\u003e. The flavonoid QC has exhibited dose-dependent cytoprotective effects through Nrf2-mediated antioxidant response element activation, effectively attenuating ectopic lipid deposition while enhancing insulin sensitivity in oleic acid (OA)-challenged HepG2 hepatocytes, as evidenced by normalized glucose transporter 4 (GLUT4) trafficking and suppressed NF-κB-driven proinflammatory cytokine secretion\\u003csup\\u003e\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e\\u003c/sup\\u003e. Notably, QC administration in diet-induced obese murine models restores euglycemia through PGC-1α-mediated mitochondrial biogenesis, concurrent with gut microbiota remodeling characterized by an increased Bacteroidetes/Firmicutes ratio\\u003csup\\u003e\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e\\u003c/sup\\u003e. SLB exerts multi-target anti-inflammatory action via canonical signaling pathway modulation. In DSS-induced colitis models, SLB treatment maintains intestinal epithelial homeostasis through Wnt/β-catenin pathway inhibition, effectively curbing intestinal stem cell (ISC) hyperproliferation while preserving tight junction protein expression\\u003csup\\u003e\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e\\u003c/sup\\u003e. Mechanistically, the redox-modulating capacity reduces colonic ROS burden via NAD(P)H quinone oxidoreductase 1 (NQO1) induction, correlating with extended longevity in Drosophila through JNK pathway suppression\\u003csup\\u003e\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e\\u003c/sup\\u003e. Transcriptomic analyses reveal SLB-mediated downregulation of IL-6/STAT3 signaling cascades through competitive IKKβ inhibition, thereby attenuating MAPK/AP-1-driven inflammatory responses\\u003csup\\u003e\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e\\u003c/sup\\u003e. Hepatoprotective effects of SLB derivatives include modulation of hepatic sterol biosynthesis, as evidenced by reduced biliary cholesterol saturation index post-silymarin administration in both clinical trials and rodent models\\u003csup\\u003e\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e\\u003c/sup\\u003e. Recent findings implicate gut-liver axis modulation in the anti-steatotic action of silymarin, mediated through microbiota-dependent vitamin B12 biosynthesis and subsequent AMPK-driven lipolysis potentiation\\u003csup\\u003e\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003cp\\u003eFlavonoids have been extensively documented to exert multifaceted regulatory effects on adipocyte physiology. Emerging evidence supports the therapeutic potential of flavonoid consumption in T2D management, particularly through glycemic regulation mechanisms\\u003csup\\u003e\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e\\u003c/sup\\u003e. Experimental studies utilizing C57BL/6 murine models have further demonstrated that dietary flavonoid supplementation significantly enhances insulin sensitivity under high-fat and high-fructose dietary conditions\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR50\\\" citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e\\u003c/sup\\u003e. Wu et al. revealed that intracellular flavonoid accumulation in adipocytes upregulates insulin signaling pathways throughβ3-AR activation and AMPK phosphorylation, aligned with the present experimental data\\u003csup\\u003e\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e\\u003c/sup\\u003e. Paradoxically, while multiple studies have reported flavonoid-mediated inhibition of adipocyte differentiation and mitotic activity\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR54\\\" citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e\\u003c/sup\\u003e, the findings of the present study concur with previous research by Zhang et al\\u003csup\\u003e\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e\\u003c/sup\\u003e, demonstrating that specific flavonoid derivatives (QC and SLB) exhibit selective attenuation of lipogenic pathways in WAT.\\u003c/p\\u003e\\u003cp\\u003eThe present study evaluates the stimulating effects of QC and SLB on the expression of beige-specific genes and proteins, thereby promoting fat browning in 3T3-F442A cells. Previous studies have emphasized the role of plant-based dietary components in promoting fat browning in BAT in rodent models\\u003csup\\u003e\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e\\u003c/sup\\u003e, as well as the direct impact of flavonoids on the browning of WAT in rats\\u003csup\\u003e\\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e58\\u003c/span\\u003e\\u003c/sup\\u003e. In line with these findings, prior research has demonstrated that the induction of UCP-2 expression in visceral WAT by QC is mediated through PPARα activity\\u003csup\\u003e\\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e\\u003c/sup\\u003e. The findings of the present study further indicate that this process involves the activation of the AMPK signaling pathway, which is regulated by the β3-AR-PKA axis.\\u003c/p\\u003e\\u003cp\\u003ePrior investigations have established thatβ3-AR activation in white adipocytes serves as a critical mediator of WAT browning\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR61\\\" citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e60\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR62\\\" class=\\\"CitationRef\\\"\\u003e62\\u003c/span\\u003e\\u003c/sup\\u003e. The computational molecular docking analysis in the present study identified high-affinity interactions between QC/SLB and β3-AR, while functional studies corroborated that β3-AR signaling activation constitutes a central mechanism driving adipose browning. Notably, this work identifies AMPK as a central regulatory node in this process. Clinical evidence indicates that AMPK activity is markedly attenuated in adipose depots of patients with morbid obesity and comorbid IR\\u003csup\\u003e\\u003cspan citationid=\\\"CR63\\\" class=\\\"CitationRef\\\"\\u003e63\\u003c/span\\u003e\\u003c/sup\\u003e. Functioning as a master metabolic sensor, AMPK orchestrates BAT activation by stimulating adipogenic differentiation, preserving mitochondrial ultrastructure, and inducing transcriptional reprogramming toward a beige adipocyte phenotype\\u003csup\\u003e\\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e64\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR65\\\" class=\\\"CitationRef\\\"\\u003e65\\u003c/span\\u003e\\u003c/sup\\u003e. The present experimental data revealed that QC/SLB treatment robustly upregulated AMPK expression, providing a mechanistic basis for the concomitant elevation of UCP-1 levels and driving browning in white adipocytes. Notably, pharmacological inhibition of AMPK and β3-AR abrogated these effects, confirming the indispensable function of the β3-AR/AMPK axis in modulating adipose browning. Collectively, these findings highlight the therapeutic potential of QC/SLB in enhancing adipose plasticity through WAT browning, thereby augmenting thermogenic capacity. Nonetheless, both PPARγ and SIRT1 play major roles in regulating cellular energy homeostasis and in response to IR\\u003csup\\u003e\\u003cspan citationid=\\\"CR66\\\" class=\\\"CitationRef\\\"\\u003e66\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR67\\\" class=\\\"CitationRef\\\"\\u003e67\\u003c/span\\u003e\\u003c/sup\\u003e, and the involvement of the PPARγ and SIRT1 response to QC/SLB and systematic preclinical validation in vivo is required to comprehensively delineate the pharmacodynamic profile of QC/SLB in adipose browning regulation.\\u003c/p\\u003e\\u003cp\\u003eIn conclusion, the present experimental data elucidated that QC and SLB orchestrated lipid metabolic remodeling and triggered the phenotypic transition of white adipocytes toward a thermogenic lineage via activation of the β3-AR-AMPK signaling axis. As pleiotropic phytochemicals exhibiting multi-target bioactivity, QC and SLB emerge as promising translational candidates for developing pathophysiology-driven interventions against obesity and its metabolic sequelae.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003ch2\\u003eEthics approval and consent to participate\\u003c/h2\\u003e\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eConflicts of Interest\\u003c/strong\\u003e\\u003cp\\u003eThe authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yuanxin Zhang reports financial support was provided by Jilin Provincial Science and Technology Department. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003ch2\\u003ePatient consent for publication\\u003c/h2\\u003e\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\u003c/p\\u003e\\u003ch2\\u003eFunding\\u003c/h2\\u003e\\u003cp\\u003eThis work was supported by Jilin province science and technology development plan project (grant number: 20210204026YY).\\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eYakun Ge: Writing-original draft, Review \\u0026amp; editing, Methodology, Formal analysis, Conceptualization. Aiping Liu: Investigation, Methodology, Formal analysis, Resources. Fanwei Meng: Investigation, Methodology. Renwen Zhang: Data curation, Methodology, Project administration. Junting Wang, Qiao Wang: Conceptualization, Methodology. Guanqiao Wu: Validation, Software. Yuanxin Zhang: Review \\u0026amp; editing, Methodology, Formal analysis, Conceptualization, Supervision, Project administration, Funding acquisition, Visualization.\\u003c/p\\u003e\\u003ch2\\u003eAcknowledgements\\u003c/h2\\u003e\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\u003ch2\\u003eData Availability\\u003c/h2\\u003e\\u003cp\\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eLi, M. et al. Trends in insulin resistance: insights into mechanisms and therapeutic strategy. \\u003cem\\u003eSignal. Transduct. Target. Ther.\\u003c/em\\u003e \\u003cb\\u003e7\\u003c/b\\u003e, 216 (2022).\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eLee, S. H., Park, S. Y. \\u0026amp; Choi, C. S. Insulin resistance: from mechanisms to therapeutic strategies. \\u003cem\\u003eDiabetes Metab. J.\\u003c/em\\u003e \\u003cb\\u003e46\\u003c/b\\u003e, 15\\u0026ndash;37 (2022).\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eCoelho, M., Oliveira, T. \\u0026amp; Fernandes, R. Biochemistry of adipose tissue: an endocrine organ. \\u003cem\\u003eArch. Med. Sci.\\u003c/em\\u003e \\u003cb\\u003e9\\u003c/b\\u003e, 191\\u0026ndash;200 (2003).\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eFr\\u0026uuml;hbeck, G., Becerril, S., S\\u0026aacute;inz, N. \\u0026amp; Garrastachu, P. Garc\\u0026iacute;a-Velloso. M. J. BAT: a new target for human obesity? \\u003cem\\u003eTrends Pharmacol. Sci.\\u003c/em\\u003e \\u003cb\\u003e30\\u003c/b\\u003e, 387\\u0026ndash;396 (2009).\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eSidossis, L. \\u0026amp; Kajimura, S. 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Cycle\\u003c/em\\u003e. \\u003cb\\u003e7\\u003c/b\\u003e, 3669\\u0026ndash;3679 (2008).\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"QC, SLB, IR, browning, β3-AR/AMPK signaling pathway\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7124697/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7124697/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eQuercetin (QC) and silibinin (SLB), natural bioactive compounds from medicinal plants, show potential in improving IR. However, the molecular mechanisms underlying their IR-attenuating effects through adipocyte browning remain not fully elucidated. In this study, molecular docking simulations were performed to characterize ligand-target interactions, binding conformations, and affinity energies between QC/SLB and key molecular targets. qRT-PCR and immunoblotting analyses were employed to quantify expression levels of β3-AR/AMPK pathway-associated genes and proteins. Notably, QC and SLB upregulated brown/beige adipocyte markers at transcriptional and translational levels: PGC-1α, PRDM16, and UCP1, and genes specific to beige adipose tissue like Tmem26, Tbx1, CD137, and Cited1. Mechanistically, these compounds enhanced lipolytic activity and β-oxidation throughβ3-AR-mediated activation of PKA, ATGL, CPT, ACO, and PPARα/γ signaling cascades. Overall, QC and SLB promote PA-induced adipocyte browning in IR-3T3-F442A cells by activating both the β3-AR/AMPK signaling pathways, highlighting their potential as therapeutic agents for metabolic disorders. Our findings collectively demonstrate the potential effectiveness of QC and SLB in ameliorating IR.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Quercetin and silibinin ameliorate insulin resistance by stimulating fat browning via the β3-AR/AMPK pathway in palmitate-exposed 3T3-F442A adipocytes\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-08-05 12:51:14\",\"doi\":\"10.21203/rs.3.rs-7124697/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"a80f4ca8-425c-4534-b058-32f757a34db6\",\"owner\":[],\"postedDate\":\"August 5th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[{\"id\":52591601,\"name\":\"Biological sciences/Biochemistry\"},{\"id\":52591602,\"name\":\"Biological sciences/Cell biology\"},{\"id\":52591603,\"name\":\"Health sciences/Diseases\"},{\"id\":52591604,\"name\":\"Biological sciences/Drug discovery\"},{\"id\":52591605,\"name\":\"Biological sciences/Molecular biology\"}],\"tags\":[],\"updatedAt\":\"2025-11-06T13:38:30+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-08-05 12:51:14\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7124697\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7124697\",\"identity\":\"rs-7124697\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}