Isoliquiritigenin Ameliorates High-Fat Diet-Induced Obesity in Mice by Activating Brown Adipose Tissue

Excess energy in the body is primarily stored in white adipocytes [1]. The overaccumulation of white adipose tissue (WAT) is not only a hallmark of obesity but is also closely linked to various chronic metabolic diseases, such as type 2 diabetes (T2D) [2], hypertension [3], and cardiovascular diseases [4]. Moreover, obesity-induced adipose tissue inflammation, characterized by elevated macrophage infiltration and pro-inflammatory cytokine production (e.g., TNF-α, IL-6), plays a crucial role in the pathogenesis of obesity-associated metabolic disorders. This inflammatory response exacerbates insulin resistance, adipocyte dysfunction, and systemic metabolic derangements, further contributing to the development of diseases such as T2D and cardiovascular diseases [5]. In contrast, brown adipocytes and beige adipocytes can enhance energy metabolism through the production of heat mediated by uncoupling protein 1 (UCP1). These adipocytes contribute to non-shivering thermogenesis, which consumes stored energy and helps regulate energy balance [6]. Therefore, a comprehensive investigation of the regulatory molecules and mechanisms of thermogenesis in brown adipose tissue (BAT) is crucial for developing strategies to combat obesity and its associated metabolic disorders, including T2D, hypertension, and cardiovascular diseases.

Natural products and their phytochemicals have garnered significant interest, particularly for their potential roles in regulating fat metabolism [7]. Researchers have high expectations for the efficacy, safety, and stability of these natural products in this context. Among them, natural flavonoids are a primary focus of research due to their widespread occurrence in plants and their status as one of the most abundant polyphenolic compounds in the human diet [8]. Several in vitro and in vivo studies have now demonstrated significant effects of flavonoids on fat metabolism [9]. Licorice (Glycyrrhiza glabra), a pivotal herb in traditional Chinese medicine with over 2000 years of history, contains isoliquiritigenin (ISL), a bioactive chalcone renowned for its potent antioxidant [10], anti-inflammatory [11], antibacterial [12], and anticancer properties [13]. ISL has been demonstrated to effectively inhibit the activation of the NLRP3 inflammasome and reduce inflammation in adipose tissue induced by a high-fat diet (HFD), owing to its potent anti-inflammatory effects [14]. Further studies have found that ISL interrupts the inflammatory signaling between adipocytes and macrophages independently of the inflammasome and reduces fibrosis in adipose tissue by modulating several innate immune responses [15]. Additionally, ISL has been found to affect metabolic disorders related to obesity, such as hepatic steatosis and insulin resistance [16]. ISL can diminish inflammation in adipose tissue and reduce the risk of metabolic syndrome by modifying the gut microbiota composition [17]. Notably, the combination of berberine and ISL synergistically improves adipose inflammation and obesity-induced insulin resistance [18]. ISL also enhances the differentiation potential of adipose-derived stem cells into beige adipocytes by inhibiting the JNK signaling pathway [19]. These findings suggest that ISL holds significant promise as a therapeutic agent against obesity, showing positive results in modulating diseases associated with fat metabolism, although further investigation is needed to fully understand its effects and underlying mechanisms.

Thus, we hypothesized that ISL ameliorates HFD-induced obesity in mice by activating BAT. To test this hypothesis, we aimed to investigate the effects of ISL on thermogenesis in BAT and its potential role in preventing the whitening of BAT induced by HFD. Furthermore, we explored whether ISL regulates adipose deposition through mechanisms involving the expression of NNAT and SGK1. This study seeks to provide insights into the therapeutic potential of ISL for obesity.

As global obesity rates continue to rise, this condition has emerged as a significant public health challenge, contributing to a range of comorbidities, including diabetes, cardiovascular diseases, and certain types of cancer [20,21,22]. Therefore, identifying safe and effective weight loss interventions has become increasingly important. Recent research underscores the vital role of BAT in energy metabolism and weight management. Unlike white adipose tissue, which primarily stores excess energy, BAT has the unique ability to generate heat through thermogenesis, increasing the basal metabolic rate and enhancing insulin sensitivity, potentially mitigating obesity-related health risks [23,24]. In this context, licorice, widely utilized in traditional Chinese medicine, has garnered attention for its therapeutic benefits [25,26]. Licorice contains ISL, a bioactive chalcone recognized for its antioxidant, anti-inflammatory, and anticancer properties [11]. The integrative approach of utilizing compounds such as ISL from licorice may offer promising avenues for enhancing BAT activity and supporting weight management efforts. To date, the potential of ISL to alleviate HFD-induced obesity in mice by activating BAT remains unexplored.

This study employed gavage administration of ISL to HFD mice to investigate its effects on the activity and function of BAT. The results indicated that, in comparison to the control group, ISL treatment significantly reduced the body weight of HFD mice. This finding is consistent with previous research demonstrating that ISL treatment can attenuate weight gain induced by HFD [14,17,27]. Furthermore, existing studies have shown that extracts of licorice active components can decrease the weight of various adipose depots and the size of adipocytes in the body [28]. Specifically, ISL treatment has been found to significantly lower the weight of mesenteric and perirenal adipose tissues in HFD mice [29]. In line with these observations, our study also noted a significant decrease in the adipose weight-to-body weight ratio in HFD mice following ISL treatment, suggesting that the reduction in body weight may be attributed to decreased lipid deposition in adipose, likely due to the effects of ISL treatment. Moreover, related research indicates that numerous active components from natural medicines can significantly suppress the expression of key adipogenic genes in adipocytes [28,30,31,32], thereby potentially preventing obesity. In this study, ISL treatment led to a significant downregulation of adipogenic genes, accompanied by a reduction in the volume of BAT adipocytes and an increase in the number of small, multifocal lipid droplets. These findings further substantiate the substantial role of ISL, as an extract of licorice active components, in inhibiting lipid deposition.

Numerous studies have indicated that natural flavonoid compounds facilitate the browning of white adipose tissue (WAT) and the thermogenic activity of brown adipose tissue (BAT), thereby modulating lipid metabolism and exerting anti-obesity effects [33,34]. Mitochondria serve as the sole site for UCP1-mediated uncoupling, and any perturbation in mitochondrial function exerts a profound influence on BAT thermogenesis [35]. Notably, ISL has been observed to play a pivotal role in promoting mitochondrial biogenesis and enhancing BAT thermogenesis [34,36]. Consistent with these findings, our study also observed that ISL treatment significantly enhanced the expression of UCP1, UCP2, and other thermogenic genes in the BAT of HFD mice. Additionally, in vitro experiments using C3H10T1/2 cells further validated the promoting effect of ISL on thermogenesis in brown adipocytes. Moreover, ISL has been shown to influence mitochondrial function and biogenesis, with PGC1α appearing to be a key regulatory factor in these processes [37,38,39]. In our study, ISL treatment significantly increased the expression of PGC1α in BAT, thereby confirming its role in promoting mitochondrial biogenesis in BAT. Additionally, pathway enrichment analysis of differentially expressed genes revealed the significant activation of pathways such as the TCA cycle, oxidative phosphorylation, and fatty acid degradation, highlighting the potential impact of ISL on thermogenic and metabolic pathways. In summary, this study provides evidence that ISL treatment can effectively reduce body weight and adipose tissue mass in HFD-induced obese mice, likely through promoting thermogenesis and mitochondrial biogenesis in BAT. These effects are associated with the upregulation of thermogenic genes and PGC1α expression in BAT. Furthermore, our findings are consistent with observations that ISL increases cell viability by reducing apoptosis and oxidative stress through the activation of the Nrf2/HO-1 pathway. These insights contribute to a deeper understanding of the multifaceted benefits of ISL in various biological contexts, including obesity and metabolic disorders [40]. Therefore, ISL may serve as a potential therapeutic agent in the prevention and treatment of obesity and related metabolic disorders.

Cold stimulation has been shown to reduce NNAT expression in adipose tissue, and NNAT knockout mice exhibit significantly enhanced thermogenic capacity [41]. The overexpression of NNAT promotes the adipogenic differentiation of 3T3-L1 cells [42], and NNAT expression in obese mice is significantly negatively correlated with classical metabolic pathways such as oxidative phosphorylation [43]. These findings suggest that NNAT expression promotes lipid deposition and suppresses thermogenesis in adipose tissue. In the present study, ISL treatment significantly decreased NNAT expression in the BAT of HFD mice, promoting BAT thermogenesis. This finding is consistent with previous studies and suggests that ISL regulates lipid deposition and thermogenesis in BAT by modulating NNAT expression. SGK1 exhibits a strong correlation with obesity-associated inflammation [44], and its overexpression is associated with adipocyte dysfunction, potentially contributing to the development of obesity, diabetes, and metabolic syndrome [45]. As an epigenetic regulatory factor, SGK1 promotes the differentiation and lipid deposition of bovine preadipocytes by modulating the phosphorylation and the expression of FOXO1, thereby improving meat quality [46]. In this study, ISL treatment significantly reduced SGK1 expression in the BAT of HFD-fed mice and suppressed the expression of its differentiation-related genes, aligning with previous findings. However, the precise mechanisms by which ISL regulates genes such as NNAT and SGK1 to influence thermogenesis in BAT remain unclear. Further investigation is required to uncover the detailed molecular pathways and interactions involved in this process. Gaining a deeper understanding of these mechanisms could shed light on the regulation of energy expenditure and provide potential strategies for modulating metabolic health.

Our study demonstrates that ISL significantly attenuates HFD-induced obesity in mice by increasing the expression of thermogenesis-related genes and proteins in BAT. Key metabolic pathways, including the TCA cycle, oxidative phosphorylation, and fatty acid degradation, are modulated by ISL. Notably, gene expression analysis reveals that ISL downregulates the expression of NNAT and SGK1 in BAT, implicating these genes as potential molecular targets mediating ISL’s effects on BAT function. Collectively, these findings highlight the therapeutic potential of ISL in managing obesity and related metabolic disorders, offering a foundation for further investigation into its molecular mechanisms.

We wish to thank the Chongqing Key Laboratory of Herbivore Science, the Chongqing Key Laboratory of Forage and Herbivore, the Chongqing Engineering Research Center for Herbivores Resource Protection and Utilization, and the Chongqing Herbivore Engineering Research Center, Chongqing 400715, China, for their invaluable support and resources.

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms26041616/s1↗.

Writing—Review and Editing, L.Z., H.Y. and Y.Z.; Writing—Original Draft, L.Z. and M.L.; Validation, Resources, L.Z. and M.L.; Methodology, L.Z., M.L., H.Y. and Y.Z.; Formal Analysis, L.Z. and M.L.; Data Curation, L.Z., M.L., Q.Z. and X.F.; Conceptualization, L.Z., M.L., Q.Z., X.F., H.Y. and Y.Z.; Funding acquisition, Y.Z. All authors have read and agreed to the published version of the manuscript.

All animal experiments followed the Southwest University Institutional Animal Care and Use Committee (IACUC-20230227-01) regulations.

Not applicable.

The data analyzed during the current study are available from the corresponding author on reasonable request.

The authors declare no conflicts of interest.

This work was financially supported by the National Natural Science Foundation of China (No. 32372834), the National Key Research and Development Program of China (No. 2022YFD1300202), Chongqing Modern Agricultural Industry Technology System (CQMAITS202313), the Southwest University Graduate Student Research Innovation Project (No. SWUS23107).