The global prevalence of obesity and its associated metabolic disorders, such as type 2 diabetes mellitus (T2DM), represents a significant public health challenge [1]. The intricate relationship between obesity and hormonal regulation is a critical area of investigation, with a complex and often bidirectional interplay between metabolic state and hormonal levels [2, 3]. While hormones like leptin and insulin are primary regulators of appetite and metabolism, sex hormones also play a crucial and multifaceted role [4].

The association between obesity and sex hormone levels is highly context-dependent. One investigation found that the average estradiol level in obese premenopausal women was 32.8 pg/mL, compared to 39.8 pg/mL in non-obese women [5]. Estrogen is a potent regulator of core energy metabolism, influencing processes such as insulin secretion, insulin sensitivity, and hepatic glucose production [6, 7]. A deficiency in estrogen can thus promote metabolic dysfunction, predisposing individuals to obesity and T2DM [8].

Given the pronounced metabolic effects of estrogens, preclinical models offer a valuable platform to investigate their therapeutic potential. The ob/ob mouse model is a foundational tool in this research, as it serves as a well-established genetic model for obesity and T2DM [9]. These mice are homozygous for a spontaneous mutation in the leptin gene, leading to a complete deficiency of this key satiety hormone and a severe phenotype characterized by morbid obesity, hyperphagia, glucose intolerance, and hyperinsulinemia [10]. This model is particularly useful for studying the downstream physiological and morphological changes that occur once a state of severe metabolic dysfunction is established [11].

The pathologies of obesity extend to a host of profound morpho-functional changes in the primary organs of glucose regulation. In diet-induced obese (DIO) female mice, the pancreatic beta-cells initially undergo a robust compensation, exhibiting insulin hypersecretion and hyperinsulinemia to maintain normoglycemia [12, 13]. Concurrently, the alpha-cells, which produce glucagon, show a reduced area and impaired suppression of glucagon secretion during hyperglycaemia [13, 14]. However, this compensatory phase is ultimately overwhelmed, progressing to beta-cell failure driven by suppressed AMP-kinase (AMPK) activity, mitochondrial dysfunction, and elevated cholesterol synthesis within the islets [15]. At the cellular level, pancreatic islets express estrogen receptor alpha (Erα), ERβ, and G-protein-coupled estrogen receptor (GPER), enabling both transcriptional and rapid extranuclear estrogen signalling [16]. Activation of these receptors increases insulin gene expression and content [17], enhances glucose-stimulated insulin secretion [18], and protects beta-cells from oxidative and ER stress, thereby reducing apoptosis [19, 20]. Under conditions of increased metabolic demand or beta-cell stress, such as T2DM or partial pancreatomy, estrogens also promote islet beta-cell proliferation [21, 22], with ERα frequently implicated as a key driver of cell cycle entry [23]. While the magnitude of adult beta-cell replication is modest, incremental gains coupled with anti-apoptotic actions can meaningfully influence beta-cell mass over time.

The intestinal tract also undergoes significant pathology in obese models, contributing directly to systemic metabolic dysfunction [13, 24]. Maternal obesity, for instance, leads to offspring with an impaired gut epithelial barrier and a reduced villi-to-crypt ratio in the ileum, due to a decrease in villus height and an increase in crypt depth [25]. These morphological changes are linked to systemic inflammation. Furthermore, studies in morbidly obese individuals and mouse models demonstrate an accelerated rate of proximal intestinal glucose absorption, which is directly associated with increased expression of the glucose transporter Sodium–glucose co-transporter 1 (SGLT-1) [26, 27]. This enhanced absorption creates a hormonal profile that promotes persistent hyperinsulinemia and hyperglycaemia, a key mechanism of the “foregut theory” of T2DM progression [28]. In addition, estrogens influence the gastrointestinal tract, including barrier integrity and the enteroendocrine system [29, 30]. Enteroendocrine cells (EECs) secrete glucagon-like peptide-1 (GLP-1), peptide YY (PYY), and glucose-dependent insulinotropic polypeptide (GIP) to regulate appetite, insulin secretion, and energy balance, and their abundance and secretory tone are altered in obesity [31, 32]. Estradiol has been shown to modulate gut permeability and reshape the microbiota; changes linked to shifts in incretin signalling and whole body metabolism [33]. Despite this, comprehensive analyses of estrogen-induced intestinal morphometry and EEC distributions in genetic obesity remain sparse.

The purpose of this study was to examine whether a synthetic estrogen, ethinyl estradiol (EE2), could therapeutically modulate the metabolic dysfunction in this genetic model. The investigation was designed not only to assess the functional outcome of the intervention via a glucose tolerance test (GTT) but, more importantly, to identify the precise morphological and cellular substrates of its action. Using immunofluorescence of pancreatic sections from EE2-treated ob/ob mice, we assessed islet areas, beta/alpha compartments, alpha-cell localization, and beta-cell proliferation. We also conducted intestinal analysis (crypt/villus morphometry and EEC distribution). By linking systemic metabolic outcomes with specific morphological changes in these key organs, this study aims to provide comprehensive insights into the potential of estrogenic compounds for treating obesity and related metabolic diseases.

Estrogens are well-established regulators of energy balance and glucose homeostasis, particularly in females [21, 40], yet their capacity to coordinate adaptive remodelling of both pancreatic islets and the enteroendocrine system in obesity remains poorly defined [41, 42]. In this study, we demonstrate that treatment of ob/ob mice with the synthetic estrogen EE2 produces coordinated improvements in body weight, glucose control, islet architecture, and gut endocrine cell composition. Collectively, these findings suggest that EE2 induces an integrated metabolic adaptation involving both central appetite regulation and peripheral endocrine remodelling, rather than isolated tissue-specific effects.

EE2 treatment produced a rapid and sustained reduction in body weight, accompanied by a marked decrease in energy intake. Because ob/ob mice lack functional leptin, these effects demonstrate that estrogen can suppress appetite through leptin-independent mechanisms, consistent with previous studies in this model [43, 44]. Although circulating leptin levels were not measured in the present study, the established leptin-deficient phenotype of ob/ob mice supports a model in which estrogen signalling can bypass leptin deficiency by engaging parallel hypothalamic circuits involved in energy balance regulation. However, direct assessment of leptin would provide additional mechanistic insight. Estrogens are known to act centrally through hypothalamic ERα-expressing neurons to regulate feeding behaviour [45], in part via modulation of neuropeptide Y (NPY) pathways [46]. The present data therefore support the concept that estrogenic signalling provides an alternative satiety pathway when leptin signalling is absent or impaired. Although 17beta-estradiol has been reported to reduce body weight by increasing energy expenditure in other models [47], the consistent reduction in food intake observed here indicates that appetite suppression is a major contributor to the weight-lowering effect of EE2 in ob/ob mice. This is particularly relevant given that estrogen and leptin pathways converge on kisspeptinergic neurons [48], suggesting that estrogen may compensate for defective leptin signalling to restore satiety.

Beyond its effects on body weight, EE2 exerted profound effects on glucose metabolism. EE2-treated mice displayed sustained reductions in fasting glycaemia and markedly improved glucose tolerance, consistent with previous studies showing that estrogens and estrogen derivatives enhance insulin sensitivity and preserve islet function [42, 49]. These findings reinforce the therapeutic potential of EE2 in metabolic disease [9, 42, 50]. Although appetite suppression clearly contributes to improved metabolic status [46], the present data indicate that EE2 also acts directly on glucose-regulating tissues.

Glucose tolerance was significantly improved in EE2-treated mice, as reflected by reduced glucose excursion and AUC during GTT. This improvement likely reflects both enhanced peripheral insulin action and improved pancreatic endocrine function. Mechanistically, this dual improvement suggests that EE2 may act both upstream at the level of systemic insulin sensitivity and downstream at the level of islet endocrine responsiveness, thereby amplifying glucose clearance through complementary pathways. Estrogen increases insulin sensitivity in skeletal muscle and adipose tissue [49] while simultaneously preserving beta-cell survival, proliferation, and glucose-stimulated insulin secretion [19, 40, 41, 51]. This is further supported by studies in ERα knockout mice, which demonstrate impaired insulin responsiveness in peripheral tissues when estrogen signalling is disrupted [52]. Together, these observations indicate that EE2 promotes glucose homeostasis through both improved insulin sensitivity and preservation of functional beta-cell mass.

At the islet level, EE2 induced marked structural remodelling. Although total islet and beta-cell area were unchanged, EE2 significantly increased beta-cell proliferation and islet density and shifted islet size distribution toward larger islet clusters. These changes are consistent with enhanced islet remodelling, in line with previous reports showing that estrogens stimulate beta-cell proliferation and protect against apoptosis [19, 21, 41, 53]. The reduction in medium-sized islets and increase in large islets in EE2-treated mice further support a transition toward a more mature and metabolically competent islet population. These structural adaptations are consistent with estrogen-driven activation of proliferative and survival signalling pathways within beta-cells, potentially involving ERα-mediated modulation of cell cycle and anti-apoptotic pathways [53].

EE2 also significantly reduced alpha-cell area. Obesity and insulin resistance in humans are associated with alpha-cell expansion and hyperglucagonemia, which contribute to excessive hepatic glucose output and worsening hyperglycaemia [54]. Although alpha-cell dynamics in rodent models can differ [55, 56], limiting alpha-cell expansion in ob/ob mice is likely to improve insulin–glucagon balance and support glycaemic control. This suggests that EE2 may contribute to improved glucose homeostasis not only through enhanced insulin output but also through suppression of inappropriate glucagon-driven hepatic glucose production [49]. The combination of reduced alpha-cell area and increased beta-cell proliferation, therefore, represents a favourable reorganisation of islet endocrine architecture.

A particularly notable finding was the increase in PYY-positive cells within the islets of EE2-treated mice. We previously demonstrated that PYY not only suppresses appetite but also directly protects pancreatic beta-cells by reducing apoptosis and preserving islet morphology [13]. PYY plays an important role in energy and glucose regulation and supports islet function. In vitro, PYY protects beta-cells from DNA damage [57], and in vivo, it preserves islet integrity [58]. The increase in islet PYY expression observed here raises the possibility that PYY acts as an intra-islet paracrine mediator linking estrogen signalling to beta-cell survival and functional adaptation, thereby providing a local mechanism of islet protection independent of systemic gut-derived effects [57]. In contrast, SST-positive delta cells were unchanged, suggesting that EE2 selectively remodels alpha- and beta-cell compartments rather than broadly altering all endocrine populations [59]. Collectively, these data indicate that EE2 promotes a more favourable islet phenotype by reducing alpha-cell mass, enhancing beta-cell renewal, and increasing PYY-mediated cytoprotection.

EE2 also induced substantial remodelling of the intestinal enteroendocrine system. Obesity impairs EEC function and reduces GLP-1 secretion [60, 61]. In EE2-treated mice, GLP-1-positive cells were significantly increased in both crypts and villi. Given the well-established actions of GLP-1 on insulin secretion, appetite suppression, and beta-cell survival [62], this likely contributes to the improved glucose tolerance and reduced food intake observed. Similarly, GIP-positive cells were increased, particularly within the crypt compartment. GIP plays an important role in amplifying insulin secretion and regulating glucose metabolism [63], and its upregulation may further support metabolic control [64]. Importantly, the concurrent upregulation of GLP-1 and GIP suggests that EE2 does not act on a single incretin pathway but rather induces a broader reprogramming of enteroendocrine differentiation toward an insulinotropic and metabolically favourable profile. Although GIP is predominantly produced by K cells in the proximal small intestine [65], the present analysis focused on the ileum to enable a coordinated assessment of multiple EEC populations within a single intestinal region, particularly GLP-1 and PYY-expressing L cells [66]. Taken together, these findings support a coordinated gut–pancreas endocrine axis response to EE2, in which structural and functional remodelling of both tissues act synergistically to improve metabolic homeostasis.

Crypts and villi form the structural and functional units of the small intestine, with crypts housing proliferative progenitors and villi containing differentiated absorptive and endocrine cells [67]. Deeper crypts reflect increased proliferative demand, a feature of obesity-associated gut remodelling [68, 69]. EE2 significantly reduced crypt depth while increasing GLP-1 and GIP-positive EECs, suggesting that estrogen relieves obesity-induced hyperproliferation and promotes efficient differentiation toward metabolically beneficial EEC lineages. Estrogen signalling via ERα, ERβ, and GPER has been shown to influence intestinal architecture [70–72], supporting a direct role for EE2 in coordinating gut endocrine remodelling. The concurrent modulation of gut hormone-producing cells and islet architecture following EE2 treatment supports a model of integrated gut–pancreas axis regulation, whereby both direct actions on pancreatic tissue and indirect enteroendocrine signalling may contribute to the observed metabolic improvements. Complementary quantitative approaches, such as Western blotting, would further strengthen the validation of these findings in future studies. A limitation of the present study is the absence of a lean, normal-weight control group. While comparisons between untreated and EE2-treated ob/ob mice reveal treatment effects within a disease context, they do not determine whether EE2 restores parameters to normal physiological levels. Inclusion of a healthy control group in future studies would allow clearer assessment of the extent of normalisation. In addition, circulating estradiol/EE2 concentrations were not measured, and administration via drinking water may introduce variability in dosing. Therefore, the physiological relevance of the selected EE2 dose cannot be fully established, and the observed effects likely reflect pharmacological estrogen exposure rather than precise restoration of endogenous hormone levels.

In conclusion, EE2 treatment in ob/ob mice produced significant improvements in body weight, energy intake, and glycaemic control, accompanied by coordinated remodelling of pancreatic islets and the enteroendocrine system. Increased beta-cell proliferation, reduced alpha-cell area, and enhanced expression of GLP-1, GIP, and PYY together define a favourable gut–islet endocrine environment that supports glucose homeostasis. These findings provide a mechanistic framework for estrogen-driven gut–islet remodelling and highlight the therapeutic potential of estrogenic compounds in obesity-associated diabetes and beta-cell dysfunction in females.