Introduction
Polycystic ovary syndrome (PCOS) is a prevalent endocrine and metabolic disorder among women of reproductive age, with a global occurrence estimated at approximately 15–20%, contingent upon the diagnostic criteria employed (1). Clinically, PCOS is characterized by androgen excess, ovulatory dysfunction, and polycystic ovarian morphology. A significant proportion of affected individuals also display metabolic abnormalities, with roughly 70% exhibiting insulin resistance (IR), which may elevate the risk of dyslipidemia, obesity, and type 2 diabetes (2). Moreover, PCOS is associated with a range of conditions including infertility, metabolic syndrome, impaired glucose tolerance, type 2 diabetes, cardiovascular disease, depression, obstructive sleep apnea (OSA), endometrial cancer, and metabolic dysfunction-associated steatotic liver disease (MASLD) (3).
Obesity represents a significant factor that exacerbates all hormonal and metabolic characteristics of PCOS, and its prevalence is rising globally (4). Obesity-induced insulin resistance, characterized by impaired cellular response to insulin signaling, is considered a primary mechanism underpinning metabolic complications associated with PCOS (5). Hyperinsulinemia, initially arising as a compensatory response to insulin resistance, has been reported to promote hyperandrogenemia and visceral adipose tissue (VAT) dysfunction. Prolonged exposure to hyperinsulinemia contributes to β-cell stress and eventual dysfunction (6). This phenomenon is frequently accompanied by a reduction in hepatic SHBG levels in insulin-resistant PCOS (7), which is attributed to insulin directly stimulating steroidogenesis in ovarian Theca cells via PI3K-dependent activation of 17α-hydroxylase (8). Furthermore, oxidative stress and inflammation may diminish insulin signaling and augment androgen excess, thereby potentially leading to metabolic and reproductive disturbance in PCOS (9).
Given that the precise etiologies of PCOS remain elusive, there is a need for experimental models that replicate all reproductive and metabolic alterations associated with PCOS (10). The in vivo model induced by letrozole (LET) is widely used as a control for assessing relevant physiological responses. LET, an aromatase inhibitor, inhibits the conversion of androgens to estrogens, resulting in persistent hyperandrogenemia and anovulation, which mirror the primary endocrine symptoms of PCOS (11). Nevertheless, while the LET-induced PCOS model predominantly mimics reproductive disturbances, metabolic phenotypes such as insulin resistance and hepatic steatosis may be less severe than those observed with obesity-associated PCOS (12). Recent evidence indicates that the addition of a high-fat diet (HFD) to LET-induced PCOS yields an in vivo phenotype more akin to obesity-associated PCOS, characterized by in increased body mass, elevated testosterone levels, more profound impairment of glucose and lipid metabolism, and exacerbated insulin resistance compared to LET administration alone (13). Moreover, under these combined conditions, decreased phosphorylation of proteins involved in insulin signaling pathways, such as insulin receptor (INSR)/insulin receptor substrate (IRS)/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) was observed not only in typical insulin-sensitive tissues but also in the ovaries, suggesting a potential mechanistic link between systemic metabolic stress and ovarian dysfunction (13).
Various studies employing HFD+LET rats have demonstrated that this combined model exhibits more severe metabolic disturbances and a more detrimental inflammatory and oxidative stress environment compared with the LV group. Administration of HFD in LET-induced PCOS mice resulted in elevated fasting insulin and blood glucose levels, increased HOMA-IR and triglycerides, heightened testosterone and malondialdehyde (MDA) levels, and a reduction in high-density lipoprotein (HDL) cholesterol. These changes exacerbate insulin resistance, dyslipidemia, and oxidative stress (14). Furthermore, the combination of HFD and LET provoked significant oxidative stress and inflammatory responses, culminating in the most profound impairment in metabolic and hormonal parameters, along with heightened expression of inflammatory genes and oxidative stress markers (15). Based on prior research, the HFD+LET model more accurately stimulates obesity-related PCOS and is valuable for investigating tissue-level molecular pathways linking metabolic dysfunction to ovarian pathology. Nevertheless, despite advancements in model development, there remains a paucity of evidence concerning rat models that concurrently encompass obesity-related metabolic disturbances and ovarian pathology. Consequently, the objective of this study is to develop a rat model that more faithfully mirrors the clinical manifestation of obesity-related PCOS by incorporating metabolic stress factor such as an HFD, and to perform multi-organ assessments of the liver, VAT, pancreas, and ovaries to explore the evaluate the potential utility of targeting the metabolic-reproductive axis in preclinical PCOS research.
An unbalanced diet is a major environmental factor affecting the onset and exacerbation of PCOS. To emulate this condition in preclinical research, rodents are frequently administered a HFD to induce obesity and insulin resistance, thereby replicating associated metabolic characteristics (5). These findings suggest that the combined effect of elevated fat intake and hyperandrogenemia may compound adverse metabolic and reproductive outcomes, indicating that disruption in insulin signaling and inflammation in metabolic tissues could underpin the pathophysiology, as suggested by (16). Nevertheless, the molecular mechanism by which metabolic stress precipitates ovarian dysfunction under these combined conditions remains insufficiently understood. The liver, adipose tissue, and ovaries engage in complex interactions, with insulin signaling pathways such as IRS-1/AKT/glucose transporter 4 (GLUT4) recognized as critical components of this relationship (8). Additionally, the pancreas may serve as a pivotal nexus within this axis, since insulin secretion in response to chronic metabolic stress can result in pancreatic β-cell dysfunction, thereby intensifying systemic insulin resistance and ovarian dysfunction, as indicated by (17).
Therefore, in this study, we hypothesized that the combination of a HFD and LET administration would exacerbate impaired insulin signaling in key metabolic tissues, resulting in a concomitant aggravation of metabolic disorders and ovarian lesions. Accordingly, we seek to evaluate how HFD exacerbates metabolic dysfunction and ovarian lesions, elucidate the metabolic-reproductive axis, and confirm the role of insulin resistance in PCOS within the context of obesity. Furthermore, our objectives include not only establishing a rat model of PCOS but also to clarify how tissue-level impairment of insulin signaling is associated with reproductive abnormalities.
Discussion
In this study, a rat model of obese PCOS was established through the combination of LET with an HFD. This methodology is intended to better emulate the clinical spectrum of obese PCOS, characterized by mutually reinforcing metabolic and reproductive dysfunctions. While LET alone can induce hyperandrogenism and ovarian dysfunction, the inclusion of an HFD in this model resulted in more severe metabolic disturbances, such as insulin resistance, β-cell dysfunction, hepatic steatosis, and adipocyte hypertrophy (13,20). Conversely, body weight and fasting blood glucose levels did not differ significantly between the HLV and LV groups, suggesting that these systemic parameters were primarily influenced by LET-induced hyperandrogenism and were not further aggravated by HFD. Histological assessment demonstrated notable liver fat accumulation, cellular damage, and adipocyte hypertrophy. Immunostaining of pancreatic tissue confirmed a reduction in insulin-positive β-cell areas. Additionally, key components of the insulin signaling pathway, including IRS-1, AKT, and GLUT4, were showed significant reductions in liver and VAT at the molecular level relative to CON group, indicating the presence of insulin signaling disorders that were already evident in the LV group and not significantly further intensified by HFD at the transcript level, as no significant LV-HLV difference was detected for INSR, IRS-1, IRS-2, AKT, PI3K, or GLUT4.
Concomitantly, the expression of genes involved in adipogenesis SREBP-1c and ACC (significantly higher in HLV than LV) and FAS (elevated vs. CON but not significantly different between LV and HLV) and its synthesis (PEPCK, G6Pase) was increased, indicating a more extensive regulatory defect in glucose and lipid metabolism than observed in the LV group (21). In reproductive terms, this model exhibited typical ovarian features of PCOS, including decreased corpora lutea, increased follicular cysts, and abnormal ovarian morphology, suggesting follicular inactivity. These phenomena indicate an interaction between insulin resistance and ovarian dysfunction, affirming this as a phenotypic hallmark of metabolic PCOS (22). Consistent with prior studies, HFD alone causes weight gain and impaired glucose tolerance (23), while the combination of HFD with LET significantly exacerbated insulin resistance, as evidenced by elevated fasting glucose and insulin levels (23). Notably, previous research has demonstrated that the concurrent presence of HFD and hyperandrogenism leads to a more severe impairment of insulin sensitivity than either condition alone. Although earlier studies indicated LET alone typically causes only mild insulin resistance (13,24), our findings suggest that the simultaneous administration of excessive dietary fat markedly amplifies metabolic disturbances.
In the ovarian tissue of HLV rats, suppression of follicle formation, increased follicular atresia, and decreased corpora lutea were observed, consistent with a pattern similar to that reported for LET-induced PCOS (25). Our results also confirmed the potential for an HFD to exacerbate these reproductive abnormalities, consistent with the notion that insulin resistance impairs granulosa cell function and suppresses FSH receptor signaling, thereby worsening ovarian function under metabolic stress (26). To support these results, ovarian immunofluorescence analysis revealed increased AMH and CYP19A1 signals in both the LV and HLV groups, with the strongest changes observed in the HLV group. Although these markers do not independently contribute to ovarian steroid production, the changes in their distribution and intensity suggest that the combined effects of endocrine and metabolic stress may have altered the follicular microenvironment.
The histological examination of liver tissues in the HLV group revealed both microcellular and macrocellular fat deposits that are characteristic of MASLD. This finding aligns with previous research indicating that the PCOS-HFD model exacerbates hepatic steatosis under combined metabolic and endocrine stress conditions (27,28). Molecular analysis corroborated these observations by demonstrating increased expression levels of SREBP-1c and its downstream targets, FASN and ACC (29). These molecules serve as key regulators of hepatic de novo lipogenesis and are closely linked to lipid dysregulation observed in insulin resistance (30). It is noteworthy that administration of LET alone induced only mild hepatic fat accumulation. However, when combined with an HFD, it resulted in a more pronounced fatty liver phenotype.
Pancreatic β-cell damage in HLV rats was evidenced by a significant decrease in insulin-positive areas in both the LV and HLV groups compared with CON, although neither parameter differed significantly between the LV and HLV groups. These findings indicate pancreatic islet alterations in the PCOS groups but do not establish the functional state of insulin secretion or its temporal progression. Previous studies have proposed that compensatory hyperinsulinemia associated with insulin resistance may contribute to hyperandrogenism and may eventually be followed by β-cell dysfunction (31). However, it is unclear whether this sequence occurred in this model in practice, because circulating insulin and androgen levels were not measured in the present study.
In VAT, adipocyte hypertrophy was observed in both LV and HLV groups, with a more pronounced increase in the HLV group, suggesting combined effects of excessive diet and a hyperandrogenic environment. This observation was accompanied by increased expression of adipogenic genes, including SREBP-1c, and ACC (significantly higher in HLV than LV) and FAS (elevated vs. CON but not significantly different between LV and HLV), indicating accelerated fat accumulation and a tissue state associated with local inflammation, hypoxia, and worsened systemic insulin resistance (32). It is noteworthy that our 7-week HFD protocol captures early VAT remodeling, primarily hypertrophy and lipogenic gene activation, rather than established inflammatory infiltration, which typically requires prolonged HFD feeding (16+ weeks) to become histologically evident (33). Collectively, these findings suggest that early VAT remodeling contributes to systemic insulin resistance within this model and may indirectly facilitate the progression of ovarian failure. Genetic and protein data imply that alterations in PI3K/AKT signaling in the liver and VAT of HLV rats impaired insulin signaling. In VAT, the significant reduction in GLUT4 expression indicates compromised insulin-stimulated glucose uptake. In the liver, hepatocytes predominantly utilize GLUT2 for bidirectional glucose transport (34). The decrease in GLUT4 mRNA may reflect low-level hepatocyte expression or contributions from hepatic non-parenchymal cell populations (35). This pattern supports broader impairment of insulin signaling across metabolic tissues rather than a specific hepatocyte glucose uptake.
This pathway is central to insulin-mediated metabolic regulation, and its downregulation may lead to decreased peripheral glucose uptake, increased hepatic glucose production, and lipotoxicity (36). Furthermore, increased expression of inflammatory cytokines, such as TNF-α, IL-1β, and IL-18, in the liver may lead to inflammation and fat accumulation in HLV rats, thereby further aggravating hepatic insulin resistance and promoting a shift toward lipogenic metabolism. Notably, TNF-α and IL-1β levels were significantly higher in the HLV group than in the LV group (P<0.01 for both; Fig. 6D), whereas IL-18 showed the opposite pattern, with significantly higher levels in the LV group (2.25±0.21) than in the HLV group (1.44±0.12; P<0.0001; Fig. 6D), although both PCOS groups remained elevated relative to CON (1.00±0.20) although this LV-HLV difference was statistically significant. Previous reports suggest that elevated hepatic G6Pase and PEPCK levels indicate increased endogenous glucose production (37). In this context, compensatory hyperinsulinemia associated with impaired glucose regulation has been proposed to promote ovarian androgen production and contribute to follicular dysfunction (38). However, as described above, whether a hyperinsulinemia-hyperandrogenism pathway occurred in the HLV group cannot be determined from the present study. Accordingly, the pancreatic β-cell alterations observed in the HLV group should be interpreted as structural evidence of islet injury rather than as evidence of a defined temporal sequence involving earlier hyperinsulinemia and subsequent β-cell decompensation. These mechanisms demonstrate how metabolic and reproductive dysfunction interlock in PCOS accompanied by obesity. Taken together, the HLV rat model is thought to more clearly reproduce the metabolic and reproductive abnormalities in obese PCOS, in contrast to the conventional model that only uses LET. Furthermore, this model can be a useful platform for preclinical evaluation of treatment strategies targeting multiple pathologies simultaneously, integrating insulin resistance, hepatic steatosis, dyslipidemia, and PCOS into a complex phenotype (39). What is remarkable about this model is that these changes are detected in multiple organs, including the liver, VAT, pancreas, and ovaries, allowing the structural and molecular aspects of the metabolism-reproduction axis to be assessed in a single experimental system. Collectively, these findings illustrate a coordinated multi-organ cascade underlying the HLV phenotype. LET-induced hyperandrogenism and HFD-induced metabolic stress jointly suppress hepatic and VAT insulin signaling, as reflected by the concurrent downregulation of INSR, IRS-1, and AKT in both tissues; this impairment is accompanied by increased hepatic gluconeogenic (PEPCK, G6Pase) and lipogenic (SREBP-1c, FASN, ACC) gene expression, promoting hepatic steatosis. In contrast, VAT GLUT4 downregulation and adipocyte hypertrophy further exacerbates peripheral insulin resistance (34–36). In parallel, pancreatic islet alterations were observed, including reduced insulin-positive area and increased apoptosis in both PCOS groups, while ovarian abnormalities were reflected by altered CYP19A1 and AMH expression, and disrupted follicular morphology. Together, these hepatic alterations with the VAT, pancreas and ovaries indicate a coordinated metabolic-reproductive phenotype in the LET and HFD model. A schematic summary of the coordinated metabolic and reproductive alterations observed in the HLV rat model is presented in Fig. 7.
Furthermore, beyond therapeutic screening, this model may also be instrumental in investigating adipokine imbalance, particularly decreased adiponectin and elevated leptin and resistin that characterize PCOS-associated VAT dysfunction. This dysfunction has been linked to endoplasmic reticulum stress and mitochondrial disturbance, which have been shown to contribute to the progression of metabolic and reproductive abnormalities in PCOS (40,41). For example, developing diverse therapeutic approaches, such as insulin-sensitizing agents like metformin, endocrine-regulating and antioxidant therapies like melatonin to alleviate oxidative stress, promoting ovarian follicle development, and modulating the gut-liver-ovarian axis through probiotics and natural product therapies-may enhance the potential to simultaneously improve metabolic balance and reproductive function (42–44). Despite these advantages, the present study has several limitations. Direct clinical application may be limited by physiological differences between rodents and humans, notably in estrous cycle dynamics, ovarian physiology, and fat distribution (45). Furthermore, although pathway-related alterations at the mRNA and protein levels have been identified, additional validation through immunohistochemistry and phosphorylation-specific markers, such as p-AKT and p-INSR, are necessary to substantiate our findings regarding impaired insulin signaling (46). Serum insulin and androgen concentrations and HOMA-IR were not directly measured in this study; therefore, the proposed pathway from compensatory hyperinsulinemia to hyperandrogenism remains hypothetical and requires future validation through direct hormonal and metabolic assessments. Finally, future studies should focus on the temporal development and reversibility of the PCOS phenotype. Longitudinal monitoring of hormonal profiles, fertility outcomes, ovarian reserve markers such as AMH, and gut microbial communities will facilitate the elucidation of underlying mechanisms and improve cross-transferability of the findings.
In conclusion, this study effectively reproduces the key metabolic features of PCOS with LET is administered in combination with an HFD. This model provides useful experimental evidence linking insulin signaling disorders to ovarian dysfunction and may serve as a suitable preclinical platform for studying the mechanism of obesity-related PCOS evaluating treatments. However, the hypothesized pathway from compensatory hyperinsulinemia to hyperandrogenism was not directly validated by serum insulin, androgen, or HOMA-IR measurements in the present study; future studies incorporating these direct hormonal and metabolic assessments are warranted to confirm this temporal sequence.