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Introduction: Galectin-3 is a pro-fibrotic β-galactoside binding lectin highly expressed in fibrotic liver and implicated in hepatic fibrosis. Selvigaltin (previously known as GB1211) is a novel orally active galectin-3 small molecule inhibitor that has high affinity for galectin-3 (human K D = 25 nM; rabbit K D = 12 nM) and high oral bioavailability in rabbits and man. In this study the efficacy of selvigaltin was investigated in a high fat diet (HFD) rabbit model of metabolic-associated steatohepatitis (MASH)., Methods: Male New Zealand White rabbits were individually caged under standard conditions in a temperature and humidity-controlled room on a 12 h light/darkness cycle. After 1 week of regular diet (RD), rabbits were randomly assigned for 8 or 12 weeks to different groups: RD/vehicle, RD/selvigaltin, HFD (8 weeks), HFD/vehicle and HFD/selvigaltin (0.3, 1.0, 5.0 or 30 mg/kg selvigaltin with vehicle/selvigaltin p.o. dosed therapeutically q.d. 5 days per week from week 9 or 12). Liver inflammation, steatosis, ballooning, and fibrosis was measured via blood metabolic markers, histomorphological evaluation [Oil Red O, Giemsa, Masson's trichome, picrosirius red (PSR) and second harmonic generation (SHG)], and mRNA and protein expression., Results: Steatosis, inflammation, ballooning, and fibrosis were all increased from RD to HFD/vehicle groups. Selvigaltin demonstrated target engagement by significantly decreasing galectin-3 levels in the liver as measured via immunohistochemistry and mRNA analysis. Selvigaltin dose-dependently reduced biomarkers of liver function (AST, ALT, bilirubin), inflammation (cells foci), and fibrosis (PSR, SHG), as well as decreasing the mRNA and protein expression of several key inflammation and fibrosis biomarkers (e.g., IL6, TGFβ3, SNAI2, collagen). Doses of 1.0 or 5.0 mg/kg demonstrated consistent efficacy across most biological endpoints supporting the current clinical doses of selvigaltin being investigated in liver disease., Discussion: Selvigaltin significantly reduced hepatic inflammation and fibrosis in an HFD rabbit model of MASH following therapeutic dosing for 4 weeks in a dose-dependent manner. These data support the human selvigaltin dose of 100 mg b.i.d. that has been shown to reduce key liver biomarkers during a clinical study in liver cirrhosis., Competing Interests: Author(s) IH, FZ, HL, and RS were employed by Galecto Biotech AB. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from Galecto Biotech AB. The funder was involved in the study design, interpretation of data, the writing of this article, and the decision to submit it for publication., (Copyright © 2024 Comeglio, Guarnieri, Filippi, Cellai, Acciai, Holyer, Zetterberg, Leffler, Kahl-Knutson, Sarchielli, Morelli, Maggi, Slack and Vignozzi.)

Metabolic syndrome (MetS) is known to be associated to inflammation and alteration in the hypothalamus, a brain region implicated in the control of several physiological functions, including energy homeostasis and reproduction. Previous studies demonstrated the beneficial effects of testosterone treatment (TTh) in counteracting some MetS symptoms in both animal models and clinical studies. This study investigated the effect of TTh (30 mg/kg/week for 12 weeks) on the hypothalamus in a high-fat diet (HFD)-induced animal model of MetS, utilizing quantitative RT-PCR and immunohistochemical analyses. The animal model recapitulates the human MetS features, including low testosterone/gonadotropin plasma levels. TTh significantly improved MetS-induced hypertension, visceral adipose tissue accumulation, and glucose homeostasis derangements. Within hypothalamus, TTh significantly counteracted HFD-induced inflammation, as detected in terms of expression of inflammatory markers and microglial activation. Moreover, TTh remarkably reverted the HFD-associated alterations in the expression of important regulators of energy status and reproduction, such as the melanocortin and the GnRH-controlling network. Our results suggest that TTh may exert neuroprotective effects on the HFD-related hypothalamic alterations, with positive outcomes on the circuits implicated in the control of energy metabolism and reproductive tasks, thus supporting a possible role of TTh in the clinical management of MetS.

In this study, we investigated steroidogenic gene mRNA expression in human vaginas and verified the ability of human vagina smooth muscle cells (hvSMCs) to synthesize androgens from upstream precursor dehydroepiandrosterone (DHEA). As a readout for androgen receptor (AR) activation, we evaluated the mRNA expression of various androgen-dependent markers. hvSMCs were isolated from vagina tissues of women undergoing surgery for benign gynecological diseases. In these cells, we evaluated mRNA expression of several steroidogenic enzymes and sex steroid receptors using real time reverse transcription-polymerase chain reaction. Androgen production was quantified with liquid chromatography tandem-mass spectrometry (LC-MS/MS). In vaginal tissues, AR mRNA was significantly less expressed than estrogen receptor α, whereas in hvSMCs, its mRNA expression was higher than progestin and both estrogen receptors. In hvSMCs and in vaginal tissue, when compared to ovaries, the mRNA expression of proandrogenic steroidogenic enzymes (HSD3β1/β2, HSD17β3/β5), along with 5α-reductase isoforms and sulfotransferase, resulted as being more abundant. In addition, enzymes involved in androgen inactivation were less expressed than in the ovaries. The LC-MS/MS analysis revealed that, in hvSMCs, short-term DHEA supplementation increased Δ4-androstenedione levels in spent medium, while increasing testosterone and DHT secretion after longer incubation. Finally, androgenic signaling activation was evaluated through AR-dependent marker mRNA expression, after DHEA and T stimulation. This study confirmed that the human vagina is an androgen-target organ with the ability to synthesize androgens, thus providing support for the use of androgens for local symptoms of genitourinary syndrome in menopause., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Chronic inflammation is involved in the genitourinary syndrome of menopause (GSM) and beneficial effects of androgens in the vagina have been described. We investigated the potential involvement of human vagina smooth muscle cells (hvSMCs) in the inflammatory response and the immunomodulatory effect of androgen receptor (AR) agonist dihydrotestosterone (DHT). HvSMCs isolated from menopausal women were evaluated for sex steroids receptors and toll-like receptors mRNA expression, and left untreated or treated in vitro with lipopolysaccharide (LPS) or IFNγ, in the presence or absence of DHT. We evaluated mRNA expression (by RT-PCR) and secretion in cell culture supernatants (by a bead-based immunoassay) of pro-inflammatory markers. Nuclear translocation of NF-κB (by immunofluorescence) and cell surface HLA-DR expression (by flow cytometry) were also evaluated. Similar experiments were repeated in rat vSMCs (rvSMCs). In hvSMCs and rvSMCs, AR was highly expressed. DHT pre-treatment inhibited LPS-induced mRNA expression of several pro-inflammatory mediators (i.e. COX2, IL-6, IL-12A and IFNγ), effect significantly blunted by AR antagonist bicalutamide. DHT significantly counteracted the secretion of IL-1RA, IL-2, IL-5, IL-15, FGF, VEGF and TNFα. LPS-induced NF-κB nuclear translocation was significantly inhibited by DHT, an effect counteracted by bicalutamide. DHT pre-treatment significantly decreased IFNγ-induced expression of HLA-DR, mRNA expression of iNOS, COX2 and MCP1, and secretion of IL-1, IL-2, IL-5, IL-6, MCP1 and GCSF. Similar effects were observed in rvSMCs. The activation of AR suppresses the inflammatory response in hvSMCs, reducing their potential to be involved in the initiation and maintaining of inflammation, thus representing a therapeutic strategy in conditions, such as the GSM.

Metabolic syndrome (MetS) clusters cardiovascular and metabolic risk factors along with hypogonadism and erectile dysfunction. Lifestyle modifications including physical exercise (PhyEx) are well-known treatments for this condition. In this study, we analyzed the effect of PhyEx on hypothalamic-pituitary-testis axis and erectile function by use of an animal MetS model, previously established in rabbits fed a high-fat diet (HFD). Rabbits fed a regular diet (RD) were used as controls. A subset of both groups was trained on a treadmill. HFD rabbits showed typical MetS features, including HG (reduced T and LH) and impairment of erectile function. PhyEx in HFD rabbits completely restored plasma T and LH and the penile alterations. At testicular and hypothalamic levels, an HFD-induced inflammatory status was accompanied by reduced T synthesis and gonadotropin-releasing hormone (GnRH) immunopositivity, respectively. In the testis, PhyEx normalized HFD-related macrophage infiltration and increased the expression of steroidogenic enzymes and T synthesis. In the hypothalamus, PhyEx normalized HFD-induced gene expression changes related to inflammation and glucose metabolism, restored GnRH expression, particularly doubling mRNA levels, and regulated expression of molecules related to GnRH release (kisspeptin, dynorphin). Concerning MetS components, PhyEx significantly reduced circulating cholesterol and visceral fat. In multivariate analyses, cholesterol levels resulted as the main factor associated with MetS-related alterations in penile, testicular, and hypothalamic districts. In conclusion, our results show that PhyEx may rescue erectile function, exert anti-inflammatory effects on hypothalamus and testis, and increase LH levels and T production, thus supporting a primary role for lifestyle modification to combat MetS-associated hypogonadism and erectile dysfunction.

Exendin-4 is a molecule currently used, in its synthetic form exenatide, for the treatment of type 2 diabetes mellitus. Exendin-4 binds and activates the Glucagon-Like Peptide-1 Receptor (GLP-1R), thus inducing insulin release. More recently, additional biological properties have been associated to molecules that belong to the GLP-1 family. For instance, Peptide YY and Vasoactive Intestinal Peptide have been found to affect cell adhesion and migration and our previous data have shown a considerable actin cytoskeleton rearrangement after exendin-4 treatment. However, no data are currently available on the effects of exendin-4 on tumor cell motility. The aim of this study was to investigate the effects of this molecule on cell adhesion, differentiation and migration in two neuroblastoma cell lines, SH-SY5Y and SK-N-AS. We first demonstrated, by Extra Cellular Matrix cell adhesion arrays, that exendin-4 increased cell adhesion, in particular on a vitronectin substrate. Subsequently, we found that this molecule induced a more differentiated phenotype, as assessed by i) the evaluation of neurite-like protrusions in 3D cell cultures, ii) the analysis of the expression of neuronal markers and iii) electrophysiological studies. Furthermore, we demonstrated that exendin-4 reduced cell migration and counteracted anchorage-independent growth in neuroblastoma cells. Overall, these data indicate for the first time that exendin-4 may have anti-tumoral properties.

Metabolic disorders are often associated with male hypogonadotropic hypogonadism, suggesting that hypothalamic defects involving GnRH neurons may impair the reproductive function. Among metabolic factors hyperglycemia has been implicated in the control of the reproductive axis at central level, both in humans and in animal models. To date, little is known about the direct effects of pathological high glucose concentrations on human GnRH neurons. In this study, we investigated the high glucose effects in the human GnRH-secreting FNC-B4 cells. Gene expression profiling by qRT-PCR, confirmed that FNC-B4 cells express GnRH and several genes relevant for GnRH neuron function (KISS1R, KISS1, sex steroid and leptin receptors, FGFR1, neuropilin 2, and semaphorins), along with glucose transporters (GLUT1, GLUT3, and GLUT4). High glucose exposure (22 mM; 40 mM) significantly reduced gene and protein expression of GnRH, KISS1R, KISS1, and leptin receptor, as compared to normal glucose (5 mM). Consistent with previous studies, leptin treatment significantly induced GnRH mRNA expression at 5 mM glucose, but not in the presence of high glucose concentrations. In conclusion, our findings demonstrate a deleterious direct contribution of high glucose on human GnRH neurons, thus providing new insights into pathogenic mechanisms linking metabolic disorders to reproductive dysfunctions.

Glucagon-like peptide-1 (GLP-1) is an insulinotropic peptide with neurotrophic properties, as assessed in animal cell models. Exendin-4, a GLP-1 analogue, has been recently approved for the treatment of type 2 diabetes mellitus. The aim of this study was to morphologically, structurally, and functionally characterize the differentiating actions of exendin-4 using a human neuronal cell model (i.e., SH-SY5Y cells). We found that exendin-4 increased the number of neurites paralleled by dramatic changes in intracellular actin and tubulin distribution. Electrophysiological analyses showed an increase in cell membrane surface and in stretch-activated-channels sensitivity, an increased conductance of Na(+) channels and amplitude of Ca(++) currents (T- and L-type), typical of a more mature neuronal phenotype. To our knowledge, this is the first demonstration that exendin-4 promotes neuronal differentiation in human cells. Noteworthy, our data support the claimed favorable role of exendin-4 against diabetic neuropathy as well as against different neurodegenerative diseases.

Estrogen exerts neuroprotective effects and reduces beta-amyloid accumulation in models of Alzheimer's disease (AD). A few years ago, a new neuroprotective gene, i.e. seladin-1 (for selective AD indicator-1), was identified and found to be down-regulated in AD vulnerable brain regions. Seladin-1 inhibits the activation of caspase-3, a key modulator of apoptosis. In addition, it has been demonstrated that the seladin-1 gene encodes 3beta-hydroxysterol Delta24-reductase, which catalyzes the synthesis of cholesterol from desmosterol. We have demonstrated previously that in fetal neuroepithelial cells, 17beta-estradiol (17betaE2), raloxifene, and tamoxifen exert neuroprotective effects and increase the expression of seladin-1. The aim of the present study was to elucidate whether seladin-1 is directly involved in estrogen-mediated neuroprotection. Using the small interfering RNA methodology, significantly reduced levels of seladin-1 mRNA and protein were obtained in fetal neuroepithelial cells. Seladin-1 silencing determined the loss of the protective effect of 17betaE2 against beta-amyloid and oxidative stress toxicity and caspase-3 activation. A computer-assisted analysis revealed the presence of half-palindromic estrogen responsive elements upstream from the coding region of the seladin-1 gene. A 1490-bp region was cloned in a luciferase reporter vector, which was transiently cotransfected with the estrogen receptor alpha in Chinese hamster ovarian cells. The exposure to 17betaE2, raloxifene, tamoxifen, and the soy isoflavones genistein and zearalenone increased luciferase activity, thus suggesting a functional role for the half-estrogen responsive elements of the seladin-1 gene. Our data provide for the first time a direct demonstration that seladin-1 may be considered a fundamental mediator of the neuroprotective effects of estrogen.

Neuroblastoma (NB) is the most common extracranial tumor in children and accounts for around 15% of all paediatric oncology deaths. The treatment of NB includes surgery, chemotherapy, and radiotherapy. Unfortunately, most children with NB present with advanced disease, and more than 60% of patients with high-risk features will have a poor prognosis despite intensive therapy. Agonists of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) have been shown to have pleiotropic effects, including antineoplastic effects. The studies that addressed the role and the possible mechanism(s) of action of PPARgamma in NB cells are reviewed.