1. Introduction

Obesity is a global health concern closely associated with an increased risk of various chronic diseases, such as cardiovascular disorders, diabetes, and cancers [1]. As such, combating or reversing obesity has become a global challenge [2]. GPR30 (GPER), recognized as an estrogen membrane receptor, has its agonists confirmed to inhibit weight gain in obese mice and ameliorate metabolism [3, 4]. When activated in the pancreas, adipose tissue, liver, and skeletal muscles, GPR30 functions by inhibiting lipid synthesis and promoting lipid oxidation, reducing lipid accumulation [5, 6]. Moreover, GPR30 plays a role in blood glucose regulation by enhancing insulin secretion, improving glucose uptake, and reducing glucose production in nonadipose tissues such as the pancreas, skeletal muscle, and liver [7]. Additionally, the absence of GPR30 results in obesity, increased insulin resistance, and metabolic dysfunction in mice [8]. Therefore, activating GPR30 emerges as a promising new target for preventing weight gain and lipid accumulation.

The discovery of GPR30 agonists and antagonists has significantly deepened our understanding of GPR30’s function in physiology and disease. Activating GPR30 has been shown to inhibit weight gain and improve metabolism. Administration of G1 (G-1), a GPR30 receptor agonist with the chemical formula C₂₁H₁₈BrNO₃, to ovariectomized (OVX) estrogen-deficient mice results in weight reduction, decreased fat content, enhanced energy expenditure, and increased utilization of brown adipose tissue [9]. Preclinical studies suggest that G1 may prevent obesity through various mechanisms, such as promoting lipolysis and oxidation, inhibiting adipocyte proliferation and differentiation, and regulating the insulin signaling pathway [3]. Furthermore, G1 activation of GPR30 has improved glucose tolerance in OVX mice, maintaining glucose homeostasis by lowering fasting blood glucose and insulin levels [5]. Thus, G1 shows potential as a novel therapeutic for obesity and diabetes.

Research indicates that postmenopausal women experience decreased energy expenditure, increased fat deposition, insulin resistance, and impaired glucose and lipid metabolism [10, 11]. This leads to a higher incidence of obesity and insulin resistance (metabolic syndrome) in women compared to age-matched men [12]. One reason could be that premenopausal women’s estrogen levels offer protective effects against metabolic disorders. However, postmenopausal women lose the protective effects of estradiol (E2), leading to reduced insulin sensitivity in pancreatic β-cells, resulting in abnormal lipid metabolism, pathological obesity, and manifestations of metabolic syndrome. Studies have found that treating postmenopausal obese women with G1 activating the GPR30 receptor yields significant results [13–15].

However, dietary habits also play a vital role in the onset and progression of obesity. An imbalance between energy intake and expenditure is a primary reason for fat storage and weight gain [16]. High-fat diet (HFD)-induced obesity in mice more aptly simulates the obesity observed in modern patients consuming excessive fat and sugar [16, 17]. Given the noticeable weight loss effects post GPR30 receptor activation, we introduced a HFD as an intervention. In this study, we report the therapeutic effects of the GPR30 agonist G1 on menopausal mice on a HFD. Moreover, in our experiments, we unexpectedly found that administering G1 treatment under a HFD led to reduced glucose tolerance and increased insulin resistance in OVX mice, despite weight reduction. No studies have reported similar findings. Hence, we believe that the weight-reducing effects of GPR30 receptor activation might manifest side effects under HFD high-fat dietary intervention.

2. Materials and Methods

3. Results

3.1. G1 Treatment Accelerates Insulin Resistance in OVX Mice on a HFD

In this study, we investigated the effects of G1 treatment on glucose and lipid metabolism in OVX mice maintained on a HFD. Building on previous findings that G1 positively influences adipose tissue mobilization and weight management in obese OVX mice on a standard diet, we divided OVX mice into four groups: control chow, control chow + G1, HFD, and HFD + G1. Our observations revealed that the body weight of mice receiving G1 treatment alongside an HFD did not increase significantly over time, contrasting with the significant weight gain observed in the HFD group by the 6th week (p < 0.01, Figure 1(a)). These findings suggest that G1 possesses intrinsic antiobesity properties.

However, a notable trend emerged when examining fasting blood glucose levels. The HFD + G1 group exhibited a significant increase in fasting blood glucose starting from the second week, differing markedly from the gradual rise observed in the HFD group (Figure 1(b)). OGTTs further indicated the potential onset of glucose intolerance or insulin resistance (Figure S5C, D) mellitus in the HFD + G1 group, evidenced by elevated initial blood glucose levels and delayed glucose clearance (Figure 1(c)). Correspondingly, insulin levels were significantly higher in this group (Figure 1(d)). Calculations of the insulin resistance index confirmed increased insulin resistance in mice fed an HFD, with the effect being particularly pronounced in the G1-treated group (Figure 1(f)). Additionally, glucagon levels were elevated across all HFD groups, although they were slightly decreased in the G1-treated group (Figure 1(e)).

3.2. G1 Therapy Promotes Lipolysis and Fat Utilization

We evaluated the impact of G1 therapy on fat mobilization by measuring triglycerides (TGs) and free fatty acids (FFA) in adipose tissue, serum, and liver. G1 treatment significantly reduced TG content in adipose tissue and promoted lipolysis, as evidenced by increased FFA levels in G1-treated mice on an HFD (Figures 2(f) and 2(i)). Serum analyses supported these findings, showing decreased TG and increased FFA levels in G1-treated mice (Figures 2(g) and 2(j)). Liver tissue analysis revealed reduced TG content and lipid accumulation in mice treated with G1 and fed an HFD (Figures 2(h) and 2(k)). Additionally, adipocyte diameter was significantly smaller in G1-treated mice on an HFD (Figures 2(a) and 2(b)), reinforcing the notion of enhanced lipid mobilization. Western blot analysis of adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and perilipin further supported increased lipolysis in G1-treated groups (Figures 2(c), 2(d), and 2(e), Figure S5A–C). Furthermore, Oil Red O staining of liver sections demonstrated a marked reduction in lipid accumulation in G1-treated groups (Figures 2(l) and 2(m)).

3.3. Identification of Metabolically Relevant Key Genes in Obese Postmenopausal Women

To understand the modifications in HFD-induced insulin resistance potentially mediated by G1 treatment, we analyzed adipose tissue samples from obese and normal-weight postmenopausal women. Differential gene expression analysis identified 444 DEGs, of which 81 were directly related to metabolism (Figure S1B–D). Protein–protein interaction network analysis revealed 10 hub genes; intriguingly, 8 of these were downregulated in obese postmenopausal women, with the exceptions of BHMT and BHMT2 (Figure S1E, F). KEGG pathway analysis suggested the downregulation of glycolipid metabolism pathways (Figure S1H). Moreover, weighted gene coexpression network analysis (WGCNA) identified BHMT2 and PC (pyruvate carboxylase) as the most metabolically relevant hub genes in this demographic (Figures 3(a), 3(d), and 3(e)).

3.4. Validation of Key Genes Using the GSE26637 Dataset

To validate our findings, we cross-referenced them with the GSE26637 dataset. Comparative analysis revealed that PC and BHMT2 were consistently associated with obesity and insulin resistance (Figures 3(b), 3(c), and 3(e)). The altered expression levels of PC and BHMT2 suggest a potential link to the development of insulin resistance in obese individuals. Gene set enrichment analysis (GSEA) further confirmed a significant correlation between the PC gene and metabolic pathways in insulin-resistant obese women (Figure 3(g)). In our insulin resistance model induced by an HFD, we observed a notable increase in PC expression. G1 treatment led to a modest upregulation of PC expression, and importantly, G1 treatment markedly enhanced PC expression levels in mice on an HFD. These findings were confirmed through Western blotting and quantitative PCR analyses (Figures 4(a), 4(b), and 4(c)).

3.5. Effect of G1 Treatment on Key Proteins of the Pyruvate Metabolic Pathway

We examined the impact of G1 treatment on key proteins within the pyruvate metabolic pathway. Levels of acetyl-CoA and oxaloacetate (OAA), crucial components of the tricarboxylic acid (TCA) cycle, were elevated in G1-treated mice, suggesting enhanced metabolic activity within these pathways. Additionally, the expression of citrate synthase (CS), an important enzyme in the TCA cycle, was downregulated under HFD conditions. This downregulation was attenuated in the presence of G1 treatment, and an upregulation was observed in the group treated with G1 alone (Figures 5(a) and 5(b)).

4. Discussion

This study presents the novel finding that the therapeutic effects of G-1 vary significantly between HFD and a normal diet (ND) in OVX mice, indicating a complex interplay among diet, metabolic regulation, and pharmacological intervention. Our results demonstrated that G-1 significantly reduced body weight in HFD mice, which is consistent with its known role as a specific agonist of the G protein–coupled estrogen receptor (GPER) [5, 18]. These findings align with previous studies indicating the involvement of GPER in regulating body weight and adiposity. However, the impact of G-1 on ND mice was minimal, suggesting that the presence of an obesity-inducing diet is a crucial factor in G-1’s therapeutic efficacy.

Interestingly, although G-1 exhibited antiobesity effects, it simultaneously increased fasting blood glucose levels, impaired glucose tolerance, and elevated the insulin resistance index in HFD mice. These adverse effects were accompanied by increased mobilization of fatty acids, suggesting enhanced lipid metabolism under HFD conditions. The underlying mechanism appears to involve accelerated β-oxidation of fatty acids and increased gluconeogenesis, possibly due to G-1’s modulation of metabolic pathways in the liver [19, 20]. These observations highlight a potential deleterious effect of G-1 under HFD conditions, which could undermine its therapeutic benefits.

Conversely, other studies have reported beneficial effects of G-1 on glucose homeostasis, suggesting that specific experimental conditions—including diet type and timing of intervention—significantly influence G-1’s metabolic outcomes [18]. For instance, Sharma et al. found that G-1 treatment in OVX mice reduced fasting blood glucose and insulin levels and improved glucose tolerance, thereby reducing HOMA-IR and enhancing peripheral insulin action to maintain glucose homeostasis. However, in their study, G-1 treatment started after inducing obesity with an HFD, and the mice were fed an ND during the treatment period [21]. In contrast, our study administered G-1 immediately after ovariectomy and continued HFD feeding for 6 weeks during treatment. We observed that G-1 treatment did not significantly affect blood glucose levels in ND-fed mice, unlike in HFD-fed mice. This suggests that dietary context and timing of G-1 administration are critical factors influencing its metabolic effects.

The molecular mechanisms underpinning the observed metabolic effects of G-1 are multifaceted. G-1’s activation of GPER is known to influence various signaling pathways, including those involved in insulin secretion and lipid metabolism [22]. Under normal dietary conditions, G-1 can activate the ERK and PI3K pathways to regulate blood glucose levels, maintaining glucose and lipid homeostasis [6]. Interestingly, the activation of the ERK and PI3K pathways is mutually exclusive, which may help balance glucose levels and insulin secretion. However, under an HFD, this balance appears disrupted, leading to increased fatty acid mobilization and altered glucose metabolism. Specifically, our study suggests that G-1 enhances fatty acid β-oxidation and activates PC, driving the gluconeogenic pathway and increasing blood glucose levels.

Targeting the interaction between ATGL, HSL, and perilipin, ATGL is the primary enzyme that initiates lipolysis by hydrolyzing triglycerides into diglycerides and free fatty acids, playing a crucial role in both short-term and long-term energy regulation [22]. Following ATGL’s action, HSL further catalyzes the breakdown of diglycerides into monoglycerides and free fatty acids and also has some capacity to hydrolyze monoglycerides, releasing additional free fatty acids. Perilipin is a regulatory protein on the surface of lipid droplets that helps protect them from lipase attack [23]. When stored fat needs to be mobilized, the phosphorylation state of perilipin changes, allowing ATGL and HSL to access and break down the lipids within the droplets. Regarding hormonal regulation, epinephrine regulates HSL activity and ATGL by phosphorylating perilipin and activating protein kinase A (PKA), thereby promoting the release of fatty acids [24]. In our study, we found that ATGL protein expression was upregulated in the G1-treated group, suggesting that G1 promotes the breakdown of triglycerides, especially in the context of a HFD. This upregulation of ATGL anp may be the main reason for the accumulation of lipid droplets in adipose tissue. The results showed that G1 treatment increased the phosphorylation level of perilipin, while HSL expression did not show significant changes.

Through literature review, we learned that when GPR30 is activated, it may regulate various intracellular functions through multiple pathways such as the cAMP signaling pathway, PI3K/Akt signaling pathway, or MAPK signaling pathway [25]. Literature indicates that GPR30 activation can increase cAMP levels, which is a key molecule for activating PKA, and PKA can phosphorylate perilipin [26]. Therefore, the activation of GPR30 promotes the phosphorylation of perilipin through this mechanism, thereby affecting fatty acid mobilization. Studies have also shown that the phosphorylation of perilipin is closely related to fatty acid release; it facilitates enzymes such as ATGL and HSL to access fatty acids for lipolysis by regulating the surface structure of lipid droplets [27].

Moreover, our research delved into the changes in the expression and activity of key metabolic enzymes, including ATGL and PEPCK. Despite the reduction in circulating free fatty acids and hepatic lipid deposition, G1 treatment led to increased acetyl-CoA levels and PC activity, contributing to elevated blood glucose levels and insulin resistance in HFD mice. These findings suggest that while G1 promotes lipolysis through upregulating ATGL and promoting perilipin phosphorylation, it also influences gluconeogenic pathways via increased PEPCK expression and PC activity.

These results reveal a paradoxical effect of G1, wherein it both ameliorates lipid accumulation and aggravates glucose metabolism disorders depending on the dietary environment. The increased acetyl-CoA levels may enhance gluconeogenesis, leading to elevated blood glucose and insulin resistance. Therefore, our findings highlight the complex metabolic effects of G1 treatment, indicating that it accelerates lipolysis by upregulating ATGL and promoting perilipin phosphorylation but also affects glucose metabolism in a way that may require careful consideration in therapeutic contexts.

Research has shown that a HFD can negatively impact the expression and activity of the PI3K/Akt signaling pathway [28]. Chronic consumption of an HFD is associated with insulin resistance and altered glucose metabolism, partly due to impaired PI3K/Akt signaling in insulin-sensitive tissues such as the liver, muscle, and adipose tissue [29, 30]. Specifically, HFD can lead to decreased PI3K activity and reduced Akt phosphorylation, which contributes to diminished glucose uptake and increased gluconeogenesis [31]. This is consistent with our previous study [32].

In this context, GPR30 may play a beneficial role by modulating the PI3K/Akt pathway to alleviate the metabolic disturbances induced by an HFD [33]. Activation of GPR30 has been shown to enhance PI3K activity and Akt phosphorylation, thereby improving insulin signaling and glucose uptake. For instance, the GPR30 agonist G-1 can improve glucose metabolism through activation of the PI3K/Akt pathway in insulin-resistant models [34].

In our study, G1 treatment may counteract the adverse effects of an HFD by activating GPR30 and subsequently stimulating the PI3K/Akt signaling pathway. This activation could restore PI3K activity that is suppressed by an HFD, leading to improved insulin sensitivity and metabolic function. By enhancing PI3K/Akt signaling, GPR30 activation might promote glucose uptake in peripheral tissues and inhibit excessive gluconeogenesis, thereby mitigating hyperglycemia and insulin resistance associated with an HFD.

However, our findings also reveal a complex role of G1 treatment. While G1 promotes lipolysis by upregulating ATGL and promoting perilipin phosphorylation, it also leads to increased levels of acetyl-CoA and enhanced activity of gluconeogenic enzymes like PEPCK and PC. This paradoxical effect suggests that GPR30 activation via G1 influences multiple metabolic pathways, some of which may exacerbate glucose metabolism disorders despite improvements in lipid metabolism.

Therefore, while GPR30 activation through the PI3K/Akt pathway may alleviate some of the negative impacts of an HFD on insulin signaling and glucose uptake, it also highlights the complexity of metabolic regulation. The interplay between enhanced lipolysis and altered glucose metabolism underscores the need for a nuanced understanding of GPR30’s role in metabolic processes. Further research is necessary to elucidate the precise mechanisms by which GPR30 and the PI3K/Akt pathway interact to modulate the effects of an HFD and to determine how these pathways can be targeted for therapeutic interventions in metabolic disorders.

In conclusion, our study provides compelling evidence that the metabolic effects of G-1 are highly dependent on dietary conditions. While G-1 shows promise in reducing adiposity, its potential adverse effects on glucose metabolism under an HFD highlight the need for caution and further investigation. These findings emphasize the importance of understanding the intricate relationship between diet, metabolic pathways, and pharmacological interventions in developing effective and safe therapeutic strategies. Future research should focus on delineating the specific mechanisms by which G-1 interacts with dietary factors to modulate metabolism and exploring the potential of diet-specific treatment regimens to optimize therapeutic outcomes.

Consent

The authors have nothing to report.

Disclosure

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Author Contributions

All authors reviewed the manuscript. Da Liu, Yi-Da Tang, Mingqi Zheng, and Congcong Lu: contributions to the conception; design of the work; the acquisition, analysis, and interpretation of data. Wenyao Wang, Mengdan Miao, Xiangbin Meng, and Fangfang Ma: the creation of new software used in the work; Wei Wang, Gang Liu, and Jing Li have drafted the work or substantively revised it. Yinge Zhan, Yajuan Yin, Mei Wei, and Yaohua Zhang: Preparation, creation and presentation of the published work, specifically visualization/ data presentation. Da Liu and Yi-Da Tang: Acquisition of the financial support for the project leading to this publication. All authors read and approved the manuscript. Da Liu and Mingqi Zheng contributed equally to this work and should be considered co-first authors.

Funding

This work was supported by the National Natural Science Foundation of China (81825003 and 82270376); the Natural Science Foundation of Hebei Province (H2020206420, H2022206295, and H2021206399); the Key Research and Development Project of Hebei Province (22377719D); the Hebei Province Finance Department Project (LS202201); the Key Science and Technology Research Program of Hebei Provincial Health Commission (20210065); and the National Key Research and Development Program (2020YFC2004700);