Postmenopausal high-gonadotropin-releasing hormone environment mediates pancreatic β-cell compensation via the cAMP-PKA-PDX1 pathway

Abstract

BACKGROUND

Menopause increases the risk of type 2 diabetes, which is typically attributed to estrogen deficiency; however, the metabolic impact of the concurrent postmenopausal surge in gonadotropin-releasing hormone (GnRH) remains undefined.

AIM

To investigate the direct regulatory role of GnRH in β-cell function.

METHODS

We assessed β-cell-specific GnRH receptor (GnRHR) expression in murine islets. The GnRH agonist gonadorelin was used to evaluate insulin secretion and downstream signaling pathways. Pharmacological and genetic manipulation approaches were applied to verify involvement of the cAMP-PKA-CREB1-PDX1 signaling cascade. In vivo, ovariectomized mice were used to model a high-endogenous GnRH environment, and db/db mice were used to assess responses under chronic metabolic stress.

RESULTS

GnRHR was expressed in murine β-cells. Gonadorelin increased insulin secretion via activation of the cAMP-PKA-CREB1-PDX1 signaling cascade, as confirmed by pharmacological and genetic interventions. In ovariectomized mice, elevated endogenous GnRH promoted compensatory β-cell expansion and hyperinsulinemia, thereby mitigating metabolic impairment. In db/db mice, chronic metabolic stress led to concurrent downregulation of GnRHR and insulin expression, although the GnRHR signaling axis remained responsive to stimulation.

CONCLUSION

GnRHR signaling contributes to pancreatic β-cell compensation through cAMP-dependent signaling, while this response appears limited by advanced β-cell structural deterioration.

Key Words: Gonadotropin-releasing hormone; Gonadotropin-releasing hormone receptor; Pancreatic β-cells; Insulin secretion; Menopause

Core Tip: This study examines the metabolic significance of elevated gonadotropin-releasing hormone (GnRH) in the postmenopausal state. We found that GnRH receptor is preferentially expressed in pancreatic β-cells and that GnRH enhances insulin secretion through the cAMP-PKA-PDX1 signaling pathway. These findings suggest that postmenopausal hypothalamic-pituitary-gonadal axis remodeling may contribute to β-cell compensation and provide a mechanistic link between reproductive endocrine aging and glucose homeostasis.

INTRODUCTION

The trajectory of female reproductive aging involves the critical transition from perimenopause, characterized by declining ovarian function and hormonal instability, to the permanent ovarian senescence of menopause[1-5]. Clinical evidence has revealed that this transition is a metabolic “window of vulnerability”, underscored by a significantly increased incidence of type 2 diabetes mellitus in postmenopausal women. The primary driver of this metabolic decline is the loss of estrogen-mediated protection[6,7]. Physiologically, estrogen regulates metabolic homeostasis by promoting subcutaneous lipid storage, increasing insulin sensitivity, and protecting β-cells from oxidative stress[8,9]. Conversely, the irreversible estrogen deficit characteristic of menopause disrupts these processes, precipitating central obesity and dyslipidemia, which ultimately exacerbate systemic insulin resistance[10-13]. Following the onset of systemic insulin resistance, the maintenance of glucose homeostasis is critically dependent on the compensatory capacity of pancreatic β-cells[14]. Physiologically, this manifests as a mechanism to overcome peripheral resistance through compensatory hyperinsulinemia and β-cell proliferation[15,16]. Most postmenopausal women successfully maintain this metabolic equilibrium for extended periods[17]. However, in susceptible individuals, when the metabolic load exceeds the compensatory capacity, the progression to impaired glucose tolerance and overt type 2 diabetes mellitus ensues[18,19]. Clinically, hormone replacement therapy presents a dilemma; although it improves metabolic parameters, concerns regarding its long-term safety limits its widespread use[20,21]. Therefore, elucidating the underlying endogenous mechanisms is crucial: How does the postmenopausal body initiate and sustain this protective compensation? Elucidating these mechanisms is key to developing safer preventive and therapeutic strategies. To fully understand this endogenous protective mechanism, it is necessary to look beyond the traditional singular focus on “estrogen deficiency” and re-evaluate the comprehensive landscape of postmenopausal endocrine remodeling. Menopause represents not merely a deficiency state but also a bidirectional endocrine shift: As ovarian estrogen decreases, the removal of negative feedback on the hypothalamic-pituitary-gonadal (HPG) axis results in a compensatory surge in gonadotropin-releasing hormone (GnRH) and luteinizing hormone, characterized by sustained, high-amplitude pulsatile secretion[22-24]. While this hypergonadotropism has historically been viewed primarily as a marker of reproductive aging or a contributor to bone loss[25,26], emerging evidence suggests a paradigm shift. Current research indicates that these “reproductive” hormones have metabolic regulatory functions. For instance, FSH directly promotes UCP1-mediated thermogenesis in adipose tissue[23]. Given the capacity of the HPG axis to regulate metabolism, it is plausible that GnRH exerts similar effects. In support of this hypothesis, GnRH receptor (GnRHR) is widely expressed in peripheral metabolic organs[27,28], with historical histological data specifically confirming its presence within the pancreas[29]. Compelling clinical evidence indicates that patients receiving GnRH agonist (GnRHa) therapy often exhibit metabolic dysregulation and an increased risk of diabetes[30]. These converging lines of evidence strongly suggest that the “high GnRH" state is not merely a byproduct of aging but also a potential compensatory mechanism that facilitates adaptation to metabolic stress. Although GnRH and its receptor have been reported in several peripheral tissues, their functional relevance in pancreatic β-cell adaptation during postmenopausal endocrine remodeling remains poorly defined. In particular, whether the high endogenous GnRH milieu that accompanies ovarian failure contributes to β-cell compensation under metabolic stress has not been systematically examined. We therefore hypothesized that postmenopausal elevation of GnRH may participate in β-cell functional adaptation by activating receptor-dependent intracellular signaling. To test this hypothesis, we combined in vitro β-cell models with ovariectomized, high-fat-diet, and db/db mouse models to examine the relationship among GnRHR signaling, insulin secretion, and islet structural integrity.

MATERIALS AND METHODS

Cell culture

Murine insulinoma MIN6 cells and the pancreatic α-cell line αTC1-6 were provided by Xiamen University. The MIN6 cells were cultured in high-glucose DMEM (25 mmol/L; Gibco, NY, United States) supplemented with 15% fetal bovine serum (FBS), 50 μM β-mercaptoethanol, and 1% penicillin-streptomycin at 37 °C in 5% CO₂. αTC1-6 cells were maintained in DMEM containing 5.56 mmol/L glucose, 10% FBS, and 5 mmol/L HEPES under identical conditions. For signaling assays, the cells were treated with the GnRHa gonadorelin (TargetMol) or the antagonist cetrorelix (TargetMol) as indicated.

Animals and mouse models

Female C57BL/6J and BKS-db/db mice (6-8 weeks) were obtained from GemPharmatech (Guangdong, China) and housed in specific pathogen-free facilities. High-fat diet (HFD) model: To induce obesity and insulin resistance, C57BL/6J mice were randomized to receive either an HFD (60% kcal from fat; Research Diets) or a standard chow diet for 12 weeks. Type 2 diabetes model: Leptin receptor-deficient BKS-db/db mice were used as a spontaneous model of type 2 diabetes, with age-matched C57BL/6J mice serving as wild-type controls. Ovariectomy (OVX) model: To establish a physiological state of high endogenous GnRH, C57BL/6J and db/db mice underwent bilateral OVX or sham surgery. The animals were allowed to recover for 4 weeks post-surgery prior to metabolic characterization. All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Xiamen University, No. 3502Z202373104.

Primary islet isolation

Primary pancreatic islets were isolated from 12-week-old C57BL/6J mice using the collagenase IV digestion method with minor modifications. Pancreata were perfused in situ with 2 mg/mL collagenase IV, digested at 37 °C for 28 minutes, and purified through Histopaque-1077 density gradient centrifugation. Islets were hand-picked and cultured in RPMI 1640 supplemented with 10% FBS for 12 hours before the assays to ensure functional integrity.

Glucose-stimulated insulin secretion

Cells were preincubated in glucose-free Krebs-Ringer bicarbonate buffer for 30 minutes, followed by incubation for 1 hour in Krebs-Ringer bicarbonate buffer containing low (2.8 mmol/L) or high (16.7 mmol/L) concentrations of glucose with or without gonadorelin. Insulin levels in the supernatant were measured using a mouse insulin ELISA kit (ImmunoDiagnostics, Hong Kong, China) and normalized to the total cellular protein content.

Biochemical and molecular analysis

Cell viability: Cell viability was assessed using the MTT. Cells were treated with graded concentrations of gonadorelin (0-1000 μM) for 1 hour or 24 hours.

cAMP assay: Intracellular cAMP levels were measured using a cAMP ELISA kit (R&D Systems, MN, United States) following cell lysis in RIPA buffer.

Western blotting: The proteins were resolved by sodium-dodecyl sulfate gel electrophoresis and transferred to polyvinylidene fluoride membranes. The blots were incubated overnight with primary antibodies against GnRHR (Proteintech, 1:1000, China), PKA (ABclonal, 1:1000, China), CREB1 (ABclonal, 1:1000, China), PDX1 (ABclonal, 1:1000), GAPDH (Proteintech, 1:10000, China), and β-actin (ABclonal, 1:10000, China), followed by incubation with HRP-conjugated secondary antibodies. Band intensity was quantified using ImageJ.

Quantitative real-time polymerase chain reaction: Total RNA was extracted using TRIzol, and quantitative polymerase chain reaction was performed using SYBR Green. The specific primers for GnRHR were forward, 5’-GTCAGATCCGCTAGCATGGCTAACAATGCATCTCTTGAG-3’, and reverse, 5’-TTTGTAGTCGGATCCCAAAGAGAAATACCCATATATGAGT-3’.

Histological analysis and immunofluorescence staining

The pancreata were fixed in 4% paraformaldehyde and embedded in paraffin. Serial sections (5 μm) were then prepared.

Morphometry: Hematoxylin and eosin staining was performed to assess islet morphology. Islet number and cross-sectional area were quantified using ImageJ by investigators who were blinded to the experimental groups, with sections separated by at least 100 μm to avoid repetitive counting.

Immunofluorescence: Tissue sections were subjected to antigen retrieval in citrate buffer and incubated with primary antibodies against GnRHR, insulin, or glucagon, followed by incubation with Alexa Fluor-conjugated secondary antibodies. Fluorescence images were captured using a Leica confocal microscope under identical acquisition settings across groups (DAPI, 405 nm; insulin, 488 nm; GnRHR and glucagon, 647 nm).

Genetic manipulation

RNA interference: MIN6 cells were transfected with GnRHR-targeting small interfering RNA or negative control (100 nM) using Lipofectamine RNAiMAX.

Overexpression: MIN6 cells were transfected with the plasmid cytomegalovirus-GnRHR plasmid or empty vector (2.5 μg) using Lipofectamine 2000. Assays were conducted 36 hours after transfection.

In vivo metabolic test

Intraperitoneal glucose tolerance tests (IPGTTs, 2 g/kg glucose) and insulin tolerance tests (ITTs, 0.7 U/kg insulin) were performed after 16 hours or 4 hours of fasting, respectively. Blood glucose levels were monitored at 0, 30, 60, 90, and 120 minutes. Serum insulin levels during IPGTTs were measured at 0 and 30 minutes through ELISA[30].

Statistical analysis

The data are presented as the mean ± SEM. Statistical comparisons were performed using Student’s t test (two groups) or one-way ANOVA with Tukey’s post hoc test (multiple groups). Two-way repeated-measures ANOVA was used for time-course data. P < 0.05 was considered to indicate statistical significance.

RESULTS

GnRHR is predominantly expressed and specifically localized in pancreatic β-cells

To elucidate the physiological role of GnRHR in islet function, we first characterized its expression profile. Western blot analysis revealed specific immunoreactive bands (60-70 kDa) in pancreatic tissues, which were consistent with the mRNA levels (Figure 1A-C)[31]. To determine the intraislet distribution, we compared GnRHR expression between murine β-cell (MIN6) and α-cell (αTC1-6) lines. Notably, GnRHR protein was highly enriched in MIN6 cells but nearly undetectable in the α-cell line (Figure 1D and E). This cellular specificity was confirmed by immunofluorescence, which revealed robust colocalization of GnRHR and insulin within pancreatic islets, with no significant overlap in insulin-negative regions (Figure 1F-H). These findings indicate that GnRHR is predominantly expressed in β-cells, providing a structural basis for direct regulation by GnRH.

Figure 1 Tissue distribution and β-cell-specific localization of the gonadotropin-releasing hormone receptor. A-C: Representative immunoblots and quantitative analysis of gonadotropin-releasing hormone receptor (GnRHR) protein expression in the murine pancreas compared with reproductive tissues (ovary and uterus). GAPDH was utilized as the loading control. ^bP < 0.01 vs ovary or adrenal, ^cP < 0.001 vs thyroid or uterus, and ^dP < 0.0001 vs thyroid, adrenal, ovary or uterus; D-G: Comparative analysis of GnRHR expression in murine β-cell (MIN6) vs α-cell (αTC1-6) lines. ^bP < 0.01 vs αTC1-6, ^dP < 0.0001 vs αTC1-6. H: Representative dual-immunofluorescence images displaying the colocalization of GnRHR (red) and insulin (green) within pancreatic islets. Nuclei were visualized through DAPI counterstaining (blue). Scale bar = 200 μm. Statistical analysis: Data are expressed as the mean ± SEM from three independent experiments. GnRHR: Gonadotropin-releasing hormone receptor.

GnRH promotes insulin secretion via the cAMP-PKA-CREB1-PDX1 axis under low-glucose conditions

In MIN6 cells incubated under basal glucose conditions (2.8 mmol/L), gonadorelin treatment elicited dose-dependent potentiation of insulin secretion, with a maximal effect observed at 10 μM (Figure 2A). This stimulatory effect was specifically mediated by GnRHR, as it was effectively abrogated by the antagonist cetrorelix[32]. Cytotoxicity was ruled out based on the results of MTT assays (Figure 2B and C). Mechanistically, gonadorelin triggered a rapid increase in intracellular cAMP, which was reduced by receptor blockade (Figure 2D). Western blot analysis showed that gonadorelin upregulated PKA and CREB1 expression, increasing the expression of PDX1, a key transcription factor for insulin synthesis (Figure 2E-H). These activating effects were reversed by cetrorelix, suggesting that the GnRHR-cAMP-PKA-PDX1 axis orchestrates basal insulin secretion[33].

Figure 2 Gonadorelin promotes basal insulin secretion by activating the gonadotropin-releasing hormone receptor-cAMP-PKA-PDX1 signaling axis. A: Dose-dependent effects of gonadorelin on insulin secretion in MIN6 cells under basal glucose conditions (2.8 mmol/L); B and C: Antagonism of the insulinotropic effect of gonadorelin by the specific gonadotropin-releasing hormone receptor antagonist cetrorelix; D: Intracellular cAMP levels in MIN6 cells stimulated with gonadorelin in the presence or absence of cetrorelix; E-H: Representative immunoblots and densitometric analysis of the expression and phosphorylation levels of PKA, CREB, and PDX1. β-actin was utilized as the loading control. Statistical analysis: Data are presented as the mean ± SEM (n = 3 independent experiments). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001. GnRH: Gonadotropin-releasing hormone.

Knockdown of GnRHR abrogates the stimulatory effect and signaling activation of GnRH

To confirm receptor specificity, we knocked down GnRHR in MIN6 cells using small interfering RNA (Figure 3A-C). Functional analysis revealed that GnRHR silencing abolished the insulinotropic effect of gonadorelin (Figure 3D). Mechanistically, this blockade prevented the downstream induction of PKA, CREB1, and PDX1 (Figure 3E-I), establishing GnRHR as the essential mediator of gonadorelin-induced signaling and secretion.

Figure 3 Requirement of gonadotropin-releasing hormone receptor for gonadorelin-mediated signaling and insulin secretion. A-C: Validation of the knockdown efficiency of gonadotropin-releasing hormone receptor (GnRHR)-targeting small interfering RNA via western blotting in MIN6 cells; D: Effect of GnRHR silencing on the potentiation of insulin secretion by gonadorelin; E: Effect of GnRHR silencing on gonadorelin-induced intracellular cAMP production; F-I: Representative immunoblots and corresponding densitometric quantification demonstrating that GnRHR deficiency abolishes the gonadorelin-induced phosphorylation of PKA and CREB, as well as the upregulation of PDX1. Data are presented as the mean ± SEM (n = 3 independent biological replicates). ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001. GnRHR: Gonadotropin-releasing hormone receptor.

GnRHR overexpression sensitizes pancreatic β-cells to gonadorelin-induced signaling

To complement the loss-of-function data, we conducted gain-of-function studies. The overexpression of GnRHR (Figure 4A-C) significantly sensitized MIN6 cells to gonadorelin. Compared with vector control cells, GnRHR-overexpressing cells exhibited an increased signaling response, characterized by increased expression of PKA, p-CREB1, and PDX1 (Figure 4D-I). These findings indicate that receptor density acts as a rate-limiting determinant of the magnitude of the cAMP-PKA-PDX1 axis.

Figure 4 Overexpression of gonadotropin-releasing hormone receptor sensitizes β-cells to gonadorelin-induced signaling. A-C: Verification of gonadotropin-releasing hormone receptor (GnRHR) overexpression at the protein level following transfection with the plasmid cytomegalovirus-GnRHR plasmid in MIN6 cells; D-I: Representative immunoblots and corresponding densitometric analysis revealing the augmented phosphorylation of PKA and CREB, as well as the increased expression of PDX1, in GnRHR-overexpressing cells compared to empty vector controls upon gonadorelin stimulation. Data are expressed as the mean ± SEM (n = 3 independent biological replicates). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001. pCMV: Plasmid cytomegalovirus; GnRHR: Gonadotropin-releasing hormone receptor.

Dynamic and synchronous alterations in GnRHR and insulin expression in pancreatic β-cells under chronic metabolic stress

We investigated GnRHR dynamics under distinct metabolic states. In the HFD-induced obesity model (compensatory phase), mice exhibited significant weight gain and islet hypertrophy (Figure 5A-C). Quantitative analysis revealed a significant upregulation of GnRHR fluorescence intensity in the islets of the HFD group, which paralleled the increase in insulin expression. Analysis of the fluorescence ratio confirmed a strong positive correlation (synchronous enhancement) between GnRHR and insulin during this compensatory phase (Figure 5D-F). By contrast, in db/db mice (decompensation phase), GnRHR protein expression markedly decreased as β-cell function deteriorated (Figure 5G-J). This loss occurred synchronously with insulin depletion (Figure 5K-M). Thus, GnRHR expression is dynamically coupled to β-cell functional status: Upregulated during compensation and downregulated during exhaustion (“bidirectional synchronization”)[34].

Figure 5 Synchronous downregulation of gonadotropin-releasing hormone receptor and insulin expression in pancreatic β-cells under chronic metabolic stress. A-F: Alterations in gonadotropin-releasing hormone receptor (GnRHR) expression in high-fat diet-induced obese mice. Body weight trajectories of C57BL/6J mice fed a standard chow (control) or high-fat diet (A). Representative double immunofluorescence staining of pancreatic islets for GnRHR (red) and insulin (green). Nuclei were counterstained with DAPI (blue). Scale bar = 200 μm (B). Quantitative analysis of islet morphology and protein expression: Mean islet area (C); mean fluorescence intensity (MFI) of GnRHR (D); MFI of insulin (E); and the ratio of GnRHR to insulin fluorescence (F); G-M: Alterations in GnRHR expression in db/db diabetic mice. Metabolic characterization of db/m (control) and db/db mice: Body weight (G) and fasting blood glucose levels (H). Representative immunofluorescence images of islets stained for GnRHR (red) and insulin (green) (I). Quantitative analysis of diabetic islets: Mean islet area (J); MFI of GnRHR (K); MFI of insulin (L); and the ratio of GnRHR to insulin fluorescence (M). Data are presented as the mean ± SEM. Statistical significance was determined by Student’s t test. ^aP < 0.05, ^bP < 0.01, and ^dP < 0.0001. HFD: High-fat diet; GnRHR: Gonadotropin-releasing hormone receptor.

Gonadorelin potentiates insulin secretion via activation of the GnRHR-cAMP-PKA-PDX1 signaling axis under high-glucose conditions

To simulate postprandial hyperglycemia, we evaluated the effects of gonadorelin under high-glucose conditions (16.7 mmol/L). Gonadorelin exerted a synergistic effect on glucose-stimulated insulin secretion (GSIS), further enhancing release beyond the high-glucose baseline (Figure 6A and B). This potentiation remained strictly receptor-dependent, as it was abolished by cetrorelix (Figure 6C). Mechanistically, the GnRHR signaling pathway remained functional; however, gonadorelin further upregulated PKA activity, CREB1 phosphorylation, and PDX1 expression despite the high metabolic background (Figure 6D-H). Short-term high-glucose exposure did not alter GnRHR protein levels, excluding rapid desensitization. These results indicate that gonadorelin augments secretory capacity via the canonical axis even under hyperglycemic stress.

Figure 6 Gonadorelin potentiates glucose-stimulated insulin secretion in MIN6 cells. A: Dose-response analysis of gonadorelin-mediated insulin secretion under hyperglycemic conditions (16.7 mmol/L glucose); B: Comparison of absolute insulin release levels, demonstrating the synergistic effect of gonadorelin on basal glucose-stimulated insulin secretion; C: Pharmacological inhibition of the gonadorelin-mediated insulinotropic effect by the specific gonadotropin-releasing hormone receptor antagonist cetrorelix; D-H: Representative immunoblots and corresponding densitometric quantification of the phosphorylation and expression of signaling effectors (PKA, CREB1, and PDX1) under high-glucose conditions. Statistical analysis: Data are expressed as the mean ± SEM (n = 3 independent biological replicates). Statistical significance was determined using one-way ANOVA, followed by Tukey’s post hoc test. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001.

High-endogenous-GnRH milieu is associated with β-cell compensation in vivo

To investigate the role of the GnRH axis in vivo, we established sham, OVX, db/db, and OVX + db/db mouse models. OVX and OVX + db/db mice exhibited elevated luteinizing hormone levels (a surrogate for high GnRH levels) and reduced estradiol levels, confirming the high-GnRH/hypoestrogenic profile (Figure 7A-D). Metabolic profiling revealed that while estrogen deficiency exacerbated glucose intolerance and insulin resistance in OVX mice, the insulin secretory profile revealed a divergent trend. Compared with the sham control mice, the OVX mice exhibited significantly increased GSIS, which is indicative of compensatory hyperinsulinemia (Figure 7E-J). Because OVX induces broad HPG axis remodeling, this result should be interpreted as an endocrine-associated compensatory response rather than as evidence of an isolated GnRH-dominant effect.

Figure 7 High-endogenous-gonadotropin-releasing hormone milieu is associated with β-cell compensation in vivo. A: Experimental workflow illustrating the grouping and surgical interventions (sham and ovariectomy) in wild-type and db/db mice; B: Progressive body weight trajectories across the four experimental cohorts; C and D: Serum concentrations of luteinizing hormone (C) and estradiol (D), confirming the successful establishment of the high-gonadotropin-releasing hormone and hypoestrogenic model; E-H: Systemic glucose and insulin homeostasis assessment: Representative curves and corresponding areas under the curve for intraperitoneal glucose tolerance tests (E and F) and insulin tolerance tests (G and H); I and J: Functional evaluation of β-cell compensation: Serum insulin concentrations during the intraperitoneal glucose tolerance tests (I); fold change in insulin secretion (30 minutes vs 0 minute post-glucose challenge), reflecting augmented secretory responsiveness in ovariectomy mice relative to sham controls (J). Data are expressed as the mean ± SEM (n = 8-10 animals per group). ^aP < 0.05, ^bP < 0.01, and ^dP < 0.0001. OVX: Ovariectomy; WT: Wild-type; AUC: Area under the curve.

GnRH-associated compensatory signaling is limited by islet structural deterioration in advanced diabetes

In the OVX + db/db dual model, western blot analysis showed that PKA, CREB1, and PDX1 expression remained higher than that in db/db mice alone, suggesting that components of the GnRH-associated signaling axis were still detectable under severe metabolic stress (Figure 8A-D). However, this molecular response was not accompanied by a clear improvement in systemic glucose homeostasis. Histological examination revealed marked islet disorganization, reduced islet area, and a decreased β-to-α cell area ratio in db/db cohorts (Figure 8E-I). These findings suggest that the functional consequence of GnRH-associated signaling may depend on preserved islet architecture and β-cell integrity. Because β-cell proliferation and apoptosis were not directly assessed in this study, these structural observations should be interpreted as associative rather than as direct evidence that GnRH regulates β-cell mass or turnover.

Figure 8 Gonadotropin-releasing hormone-associated compensatory signaling is limited by islet structural deterioration in advanced diabetes. A-D: Representative immunoblots and densitometric quantification of PKA, CREB1, and PDX1 in isolated islets; E: Representative hematoxylin and eosin staining of pancreatic sections; arrowheads denote islet atrophy and structural disarray in diabetic cohorts (scale bars: 100 μm and 50 μm). F and G: Quantitative analysis of mean islet area (F) and total islet number (G); H: Β-to-α cell area ratio; I: Dual-immunofluorescence staining for insulin (red) and glucagon (green). Nuclei were counterstained with DAPI (blue). Scale bar: 200 μm. Data are expressed as the mean ± SEM (n = 8-10 animals per group). ^aP < 0.05, ^bP < 0.01, and ^dP < 0.0001. OVX: Ovariectomy.

DISCUSSION

The present study provides evidence that gonadorelin enhances insulin secretion in β-cells through activation of the GnRHR-cAMP-PKA-CREB1-PDX1 signaling cascade. This pathway was supported by pharmacological inhibition, receptor knockdown, and receptor overexpression experiments. However, the current data should be interpreted as evidence for one functional signaling axis rather than a complete delineation of all GnRHR-dependent mechanisms in β-cells. The upstream G-protein coupling of GnRHR, including the potential involvement of Gαs-dependent signaling, was not directly examined. Moreover, whether GnRH interacts with canonical GSIS mechanisms, such as KATP channel closure, membrane depolarization, calcium influx, or insulin granule exocytosis, remains to be determined. Under high-glucose conditions, gonadorelin further enhanced insulin secretion, but the relative magnitude of this effect appeared lower than that observed under basal glucose conditions. We therefore interpret the proposed ceiling effect as a possible explanation rather than a directly demonstrated mechanism. This observation also refines the translational interpretation of the study: GnRH signaling should not be considered an acute glucose-lowering strategy for advanced hyperglycemia, but rather a candidate pathway that may help preserve or extend β-cell compensatory capacity before severe structural deterioration occurs[35-37]. The in vivo findings also require cautious interpretation. In the OVX model, increased insulin secretion occurred in the context of broad HPG axis remodeling. Ovarian failure alters multiple endocrine factors simultaneously, including GnRH, luteinizing hormone, follicle-stimulating hormone (FSH), and estradiol; therefore, the present in vivo data cannot fully isolate the independent contribution of GnRH. We did not measure serum FSH concentrations or analyze the correlation between FSH and insulin levels in OVX mice. Thus, the increased insulin secretion observed in OVX mice should be interpreted as a net endocrine-associated compensatory response, in which GnRH signaling may contribute to β-cell adaptation, rather than definitive evidence that GnRH predominates over FSH-mediated effects. Future studies using β-cell-specific GnRHR deletion, selective GnRH modulation, and parallel assessment of FSH and GnRH activity will be required to define their relative contributions[38,39]. The present findings further suggest that the functional impact of GnRH-associated signaling depends on the preservation of β-cell integrity. The current db/db data represent the examined disease stage and do not define the complete temporal trajectory of GnRHR expression from early compensation to late decompensation. In the HFD model, which reflects an earlier compensatory metabolic state, GnRHR expression increased in parallel with insulin expression. By contrast, in the db/db model examined here, GnRHR and insulin expression were both reduced, consistent with a more advanced decompensated state. Longitudinal studies in db/db mice at different ages will be needed to determine whether GnRHR initially increases during early hyperinsulinemia and subsequently decreases during β-cell failure. In advanced db/db mice, activation of downstream signaling components was not sufficient to restore systemic glucose regulation, likely because severe islet disorganization and reduced β-cell area limited the functional output of the pathway. Nevertheless, this interpretation is based primarily on histological and morphometric observations. We did not directly evaluate β-cell proliferation or apoptosis using Ki67 staining, TUNEL assay, or lineage-tracing approaches. Therefore, the current study cannot determine whether GnRH directly regulates β-cell mass, survival, or turnover. Future work should address whether GnRH signaling modulates β-cell renewal under postmenopausal metabolic stress[40]. These findings have potential translational implications but should be interpreted cautiously. The present data do not support the direct clinical use of systemic GnRHas for diabetes treatment. Systemic GnRHa administration may initially induce a pituitary flare effect with transient gonadotropin release, including FSH elevation, and therefore cannot spatially isolate pancreatic β-cell effects from pituitary-dependent endocrine responses[41]. The direct pancreatic action proposed here is supported mainly by in vitro β-cell and islet experiments, in which pituitary signaling is absent. Instead, our findings suggest that the GnRH-GnRHR axis may represent a regulatory pathway involved in β-cell adaptation. Further studies are needed to determine whether β-cell-selective modulation of GnRHR signaling or downstream cAMP-dependent pathways can preserve β-cell function without inducing systemic reproductive endocrine effects.

CONCLUSION

In conclusion, this study suggests that GnRHR signaling participates in pancreatic β-cell compensation during postmenopausal endocrine remodeling. Activation of this pathway enhances insulin secretion through cAMP-PKA-PDX1 signaling, while the relative effect under high-glucose conditions and the in vivo contribution of GnRH should be interpreted within the broader endocrine context of ovarian failure. These data provide a mechanistic link between HPG axis remodeling and β-cell adaptation, but further studies are required to define FSH-GnRH interactions, upstream G-protein coupling, temporal GnRHR changes during diabetes progression, and direct effects on β-cell mass and turnover.

ACKNOWLEDGEMENTS

We thank all members of the Wei Wang’s team for their support and technical assistance.