Gonadotrophin-releasing hormone receptor autoantibodies induce polycystic ovary syndrome-like features in a rat model.

NEW FINDINGS: What is the central question of this study? Is there a causal relationship between gonadotrophin-releasing hormone (GnRH) receptor-activating autoantibodies and polycystic ovary syndrome (PCOS)? What is the main finding and its importance? Induction of GnRH receptor-activating autoantibodies in rats resulted in increased luteinizing hormone pulsatility and testosterone concentrations, disrupted oestrous cycles, increased atretic follicles, and activation of insulin signalling in the pituitary and ovary. These changes replicate those seen in humans with PCOS, suggesting that GnRH receptor-activating autoantibodies might be involved in the pathogenesis of PCOS. ABSTRACT: Gonadotrophin-releasing hormone receptor-activating autoantibodies (GnRHR-AAb) are associated with polycystic ovary syndrome (PCOS). In the present study, we examined the impact of GnRHR-AAb on reproductive function in GnRHR-immunized female rats. All immunized rats produced high titres of GnRHR-AAb targeting a dominant epitope located in the central region of the second extracellular loop of the GnRHR. Increased pulsatile luteinizing hormone secretion and testosterone concentrations were found in immunized rats. These rats exhibited disrupted oestrous cycles, increased ovarian follicular atresia, and activation of insulin signalling in the pituitary and ovary, as indicated by increased mRNA expressions of insulin receptor substrate, phosphatidylinositol 3-kinase and glucose transporter 1. No significant changes in inflammatory cytokines were detected in the ovarian tissue. These features mimic those observed in humans with PCOS. Our findings support the concept that chronic stimulation of the GnRHR by GnRHR-AAb, with an associated increase in pituitary luteinizing hormone secretion and ovarian androgen overproduction, might represent a new aetiological mechanism for PCOS.

Transcutaneous vagus nerve stimulation attenuates autoantibody-mediated cardiovagal dysfunction and inflammation in a rabbit model of postural tachycardia syndrome.

PURPOSE: Previous studies demonstrated M2 muscarinic acetylcholine receptor-activating autoantibodies (M2R-AAb) were present in some patients with postural tachycardia syndrome (POTS). This study examines how these autoantibodies might contribute to the pathophysiology of POTS, and whether low-level tragus stimulation (LLTS) can ameliorate autoantibody-mediated autonomic dysregulation in the rabbit. METHODS: Five New Zealand white rabbits were immunized with a M2R second extracellular loop peptide to produce cholinomimetic M2R-AAb. Tilt test and infusion studies were performed on conscious rabbits before immunization, 6 weeks after immunization, and 8 weeks after immunization with 2-week daily LLTS treatment. Each rabbit served as its own control. RESULTS: Compared to preimmune state, an enhanced heart rate increase and decreased parasympathetic activity upon tilting were observed in immunized rabbits. Furthermore, these rabbits demonstrated an attenuated heart rate-slowing response to infusion of the M2R orthosteric agonist arecaidine propargyl ester (APE), suggesting an inhibitory allosteric effect of M2R-AAb. There was also a significant increase in serum inflammatory cytokines in immunized rabbits. LLTS treatment suppressed the postural tachycardia, improved the sympathovagal balance with increased acetylcholine secretion, reduced the levels of inflammatory cytokines, and reversed the attenuated heart rate response to APE in immunized rabbits. No suppression of M2R-AAb expression by LLTS was found during this short-term study period. Receptor-modulating activity of M2R-AAb produced in immunized rabbits was confirmed with in vitro bioassay. CONCLUSIONS: Autoantibody inhibition of cholinergic ligand activity may be involved in the development of cardiovagal dysfunction and inflammation associated with POTS, both of which can be improved by vagal stimulation.

Low-level tragus stimulation improves autoantibody-induced hyperadrenergic postural tachycardia syndrome in rabbits.

Background: Recent studies have demonstrated that antiadrenergic autoantibodies are involved in the pathophysiology of postural orthostatic tachycardia syndrome (POTS). Objective: The purpose of this study was to test the hypothesis that transcutaneous low-level tragus stimulation (LLTS) ameliorates autoantibody-induced autonomic dysfunction and inflammation in a rabbit model of autoimmune POTS. Methods: Six New Zealand white rabbits were co-immunized with peptides from the α1-adrenergic and ß1-adrenergic receptors to produce sympathomimetic antibodies. The tilt test was performed on conscious rabbits before immunization, 6 weeks after immunization, and 10 weeks after immunization with 4-week daily LLTS treatment. Each rabbit served as its own control. Results: An enhanced postural heart rate increase in the absence of significant change in blood pressure was observed in immunized rabbits, confirming our previous report. Power spectral analysis of heart rate variability during the tilt test showed a predominance of sympathetic over parasympathetic activity in immunized rabbits as reflected by markedly increased low-frequency power, decreased high-frequency power, and increased low-to-high-frequency ratio. Serum inflammatory cytokines were also significantly increased in immunized rabbits. LLTS suppressed the postural tachycardia, improved the sympathovagal balance with increased acetylcholine secretion, and attenuated the inflammatory cytokine expression. Antibody production and activity were confirmed with in vitro assays, and no antibody suppression by LLTS was found in this short-term study. Conclusion: LLTS improves cardiac autonomic imbalance and inflammation in a rabbit model of autoantibody-induced hyperadrenergic POTS, suggesting that LLTS may be used as a novel neuromodulation therapy for POTS.

Autoimmune activation of the GnRH receptor induces insulin resistance independent of obesity in a female rat model.

Polycystic ovary syndrome (PCOS), a metabolic and reproductive disease, is frequently associated with type 2 diabetes. We have demonstrated activating autoantibodies (AAb) directed toward the second extracellular loop (ECL2) of the gonadotropin-releasing hormone receptor (GnRHR) are present in a significant subgroup of PCOS patients. It is unclear whether GnRHR-AAb can induce peripheral tissue insulin resistance (IR) in animal models. Sixteen rats were divided equally into a GnRHR ECL2 peptide-immunized group (IMM group) and a control group (CON group). Sera GnRHR-AAb titer, luteinizing hormone (LH), and testosterone (T) were higher in IMM rats compared with CON rats. No significant difference in fasting blood glucose was observed between the two groups. However, the plasma glucose level at other time points of the IMM group was higher than that of the CON group during an intraperitoneal glucose tolerance test (IPGTT) and an insulin tolerance test (ITT) (p < 0.01). These data support the likelihood of the GnRHR-AAb induction of glucose intolerance and IR. Compared with the CON group, the IMM group showed a significant increase in insulin-stimulated phosphorylation of IRS-1 (p-IRS-1 S636/639) and a decrease in insulin-stimulated phosphorylation of Akt (p-AKT S473). Expression of the glucose transport genes including GLUT-2 in liver and GLUT-4 in white adipose tissue and skeletal muscle was significantly decreased in IMM rats compared with the CON rats. Serum levels of proinflammatory cytokines (TNF-α, IL-1α, and IL-18) were increased, while anti-inflammatory cytokines (IL-4 and IL-10) were decreased in the IMM group. Taken together, elevated GnRHR-AAb enhanced LH, hyperandrogenism, and inflammation. These changes are likely related to the observed peripheral tissue IR through the downregulation of the insulin-stimulated IRS/PI3K/Akt/Glut signaling pathway.

Increased testosterone and proinflammatory cytokines in patients with polycystic ovary syndrome correlate with elevated GnRH receptor autoantibody activity assessed by a fluorescence resonance energy transfer-based bioassay.

PURPOSE: The recently identified agonistic autoantibodies (AAb) to the gonadotropin-releasing hormone receptor (GnRHR) are a novel investigative and therapeutic target for polycystic ovary syndrome (PCOS). In this study, we used a new cell-based fluorescence resonance energy transfer (FRET) bioassay to analyze serum GnRHR-AAb activity and examine its relationship with testosterone and proinflammatory cytokines in patients with PCOS. METHODS: Serum samples from 33 PCOS patients, 39 non-PCOS ovulatory infertile controls and 30 normal controls were tested for GnRHR-AAb activity and proinflammatory cytokines in a FRET-based bioassay and multiplex bead-based immunoassay, respectively. Correlation was analyzed using the Spearman's correlation test. RESULTS: Serum GnRHR-AAb activity was significantly higher in the PCOS patients than for the ovulatory infertile (p < 0.05) and normal (p < 0.01) controls. GnRHR-AAb were positive in 39% of PCOS patients, 10% of ovulatory infertile controls, and 0% of normal controls. PCOS IgG-induced GnRHR activation was specifically blocked by the GnRHR antagonist cetrorelix. Serum levels of proinflammatory cytokines interleukin-2, interleukin-6, interferon-γ, and tumor necrosis factor-α were significantly increased in PCOS patients compared with ovulatory infertile and normal controls (p < 0.01). Correlation analysis demonstrated positive correlations of GnRHR-AAb activity with testosterone and proinflammatory cytokine levels in the PCOS group. CONCLUSIONS: Elevated GnRHR-AAb activity, as assessed by a new FRET assay, is associated with increased testosterone and proinflammatory cytokines in PCOS, suggesting autoimmune activation of GnRHR may contribute to the pathogenesis of this common disorder.

Activation of α7nAChR via vagus nerve prevents obesity-induced insulin resistance via suppressing endoplasmic reticulum stress-induced inflammation in Kupffer cells.

Obesity is a major risk factor for type 2 diabetes mellitus and insulin resistance (IR). In the state of obesity, excess fat accumulates in the liver, a key organ in systemic metabolism, altering the inflammatory and metabolic signals contributing substantially to the development of hepatic IR. Current therapies for these metabolic disorders have not been able to reverse their rapidly rising prevalence. One of the reasons is that the effects of existing drugs are predominantly non-lasting [1,2]. The vagus nerve (VN) is known to play an essential role in maintaining metabolic homeostasis while decreased VN activity has been suggested to contribute to obesity associated metabolic syndrome [3,4]. Several studies have reported that activation of α7 nicotinic acetylcholine receptor (α7nAChR) cholinergic signaling with or without VN intervention has protective effects against obesity-related inflammation and other metabolic complications [5]. However, the molecular mechanisms are still not elucidated. Exaggerated endoplasmic reticulum (ER) stress and consequent dysregulated inflammation has been implicated in the development of lipid accumulation and IR [6]. Whether targeting α7nAChR can regulate IR through these pathways is rarely reported. Accordingly, the present proposal posits that activation of the α7nAChR by VNS attenuates ER stress induced inflammation, thus ameliorating hepatic IR in Kupffer cell. We will focus on the specific interaction between vagal cholinergic activity and the modulation of ER stress induced inflammation via the α7nAChR associated pathway during IR development. Recently, the Endocrine Society has emphasized the absence of specific evidence from basic science, clinical, and epidemiological literature to assess current knowledge regarding underlying mechanisms of obesity [7]. In this proposal, we assign a significant role to α7nAChR in obesity-induced hepatic IR, and suggest a possible therapeutic strategy with VNS intervention.

The Role of GnRH Receptor Autoantibodies in Polycystic Ovary Syndrome.

OBJECTIVE: Is polycystic ovary syndrome (PCOS) associated with activating autoantibodies (AAb) to the second extracellular loop (ECL2) of gonadotropin-releasing hormone receptor (GnRHR)? DESIGN AND METHODS: We retrospectively screened sera from 40 patients with PCOS and 14 normal controls (NCs) with regular menses using enzyme-linked immunosorbent assay (ELISA) for the presence of GnRHR-ECL2-AAb. We obtained similar data from 40 non-PCOS ovulatory but infertile patients as a control group (OIC) of interest. We analyzed GnRHR-ECL2-AAb activity in purified immunoglobulin (Ig)G using a cell-based GnRHR bioassay. RESULTS: The mean ELISA value in the PCOS group was markedly higher than the NC (P = .000036) and the OIC (P = .0028) groups. IgG from a sample of 5 PCOS subjects, in contrast to a sample of 5 OIC subjects, demonstrated a dose-dependent increase in GnRHR-stimulating activity qualitatively similar to the acute action of the natural ligand GnRH and the synthetic agonist leuprolide. The GnRHR antagonist cetrorelix significantly suppressed (P < .01) the elevated GnRHR activity induced by IgG from 7 PCOS patients while the IgG activity level from 7 OIC subjects was unchanged. Five other OIC subjects had relatively high ELISA values at or above the 95% confidence limits. On further study, 3 had normal or low activity while 2 had elevated IgG-induced GnRHR activity. One suppressed with cetrorelix while the other did not. The copresence of PCOS IgG increased the responsiveness to GnRH and shifted the dosage response curve to the left (P < .01). CONCLUSIONS: GnRHR-ECL2-AAb are significantly elevated in patients with PCOS compared with NCs. Their presence raises important etiological, diagnostic, and therapeutic implications.