There is increasing evidence that exposure to environmental toxicants may impact fertility, especially during critical windows of reproductive axis development. Hypothalamic gonadotropin-releasing hormone (GnRH) neurons, essential for puberty onset and fertility, originate from the olfactory placode and migrate toward the hypothalamus during development, making them particularly vulnerable to environmental insults. Cadmium (Cd), a widespread heavy metal, is well known for its gonadotoxicity, but its impact on human hypothalamic neuron development remains unclear. Using human fetal GnRH neuroblasts (FNCB4) we investigated the effects of Cd exposure on their morpho-functional and developmental features. Cd induced oxidative stress and COX2 mRNA upregulation, indicative of inflammatory pathway activation, which was accompanied by reduced cell migration and downregulation of motility-related genes. These effects were associated with F-actin disassembly and altered expression of adhesion molecules. Electrophysiological analyses showed that Cd altered membrane potential, increased capacitance and permeability, and disrupted gap junctional communication, as also confirmed by connexin-43 delocalization. Moreover, Cd significantly reduced the expression of specific GnRH neuronal markers, suggesting impaired functional maturation. Overall, our findings provide the first evidence that Cd may interfere with mechanisms crucially involved in human GnRH neuron development, adding new mechanistic insights into the comprehension of how early-life exposure to Cd may contribute to fertility concerns.

Abstract Background Diabetes, metabolic disorders and feeding behaviours continue to pose significant public health challenges. Calcium/calmodulin‐dependent protein kinase ID (CAMK1D) has recently emerged as a pivotal molecule potentially bridging peripheral metabolic control with central appetite regulation. Therefore, a comprehensive review was performed to critically evaluate and synthesize current evidence regarding the role of CAMK1D in diabetes, metabolic processes and feeding behaviours. Main text The review assessed both published results (263 non‐duplicate studies; across Pubmed, WebOfScience and EMBASE) and the grey literature (including 14 patents, 3 clinical trials). Results from 43 unique studies, 2 patents and 5 genome‐wide association studies were finally summarized. CAMK1D modulates both metabolic processes and feeding behaviours, exhibiting tissue‐specific dynamics and diverging regulatory control either in the central nervous system (i.e., hypothalamic nuclei regulating appetite and satiety) or in the periphery (i.e., pancreatic beta cells). Genetic studies highlighted significant associations between CAMK1D polymorphisms and increased susceptibility to diabetes, obesity and altered feeding behaviours. Conclusions CAMK1D represents an emerging molecular target with promising implications for the treatment of a wide range of clinical conditions. However, further large‐scale, mechanistic and longitudinal studies are warranted to validate its role across physiological and pathophysiological conditions, as well as to explore its future therapeutic potential.

Satellite cells (SCs) are recognized as the resident stem cells of adult skeletal muscles, essential for post-natal skeletal muscle growth and regeneration following focal myotrauma. In healthy muscle, SCs are quiescent, but in response to damage or growth signals, they become activated to proliferate and differentiate to form new myofibers. A small population self-renews to replenish the basal pool for future demands. The ability of SCs to precisely balance quiescence, self-renewal, and myogenic commitment/differentiation is essential for ensuring long-term muscle homeostasis and tissue maintenance. Their state and functionality are strictly regulated by different intrinsic and extrinsic cues, the latter deriving from the microenvironment in which SCs reside, known as the niche. The niche is a dynamic compartment where extracellular matrix components, soluble factors, mechanical stimuli, and multiple interacting cell populations modulate the morphological, molecular, and electrophysiological properties of SCs. This review provides an updated overview of the morpho-functional features of SCs and of non-myogenic stromal interstitial cells, highlighting their reciprocal crosstalk within the regenerative niche. Such stromal cells play a dual role, acting as "good" or "bad" cells: while functioning as nursing cells for SCs during muscle repair/regeneration via juxtracrine and paracrine interactions, their excessive accumulation and adoption of a fibrotic/fat phenotype may lead to aberrant tissue repair, compromising muscle function. A deeper understanding of SC biology and of collaborative spatiotemporal cell interactions in the healthy, damaged, and regenerative niche is essential to identify potential novel targets and to better address interventions for maintaining, restoring, or enhancing muscle regeneration capacity and mitigating the deleterious effects of extended, severe, or pathological muscle damage.

OBJECTIVE:GDF15 induces anorexia and visceral discomfort, regulating appetite, food intake and potentially metabolic responses. However, its role in eating disorders remains unexplored. METHOD:A total of 145 participants were recruited (60 patients with anorexia nervosa, 20 with bulimia nervosa, 13 with binge eating disorder, 52 participants from the general population). Ghrelin and GDF15 serum levels were measured with immunosorbent assay kits. RESULTS:Ghrelin was elevated in patients with an eating disorder compared to healthy controls (age and BMI-adjusted ANCOVA, F-value 4.084, p-value 0.008). GDF15 was significantly correlated with ghrelin (Spearman rho 0.430, p-value < 0.001) and BMI (rho = -0.176, p-value = 0.025). GDF15 predicted the BMI of patients with anorexia nervosa and individuals from the general population, again being elevated at lower BMI (linear regression beta -0.254, p-value 0.005). The effect of GDF15 on BMI was observed as under the mediation of ghrelin (direct effect -0.056, p-value 0.577; indirect effect -0.199, p-value < 0.001). CONCLUSIONS:Present results provide novel insights into the role of GDF15 in eating disorders, describing its serum level in this clinical population for the first time. In addition, a positive correlation between GDF15 and ghrelin serum levels is also reported for the first time.

Background: Growth Differentiation Factor 15 (GDF15) has been described as influencing skeletal physiology. Nevertheless, no systematic appraisal of the effect of GDF15 on skeletal muscle tissues has been developed to the present day. Objective: The aim of the present work was to review the evidence on the topic. Methods: In this preregistered systematic review (https://osf.io/wa8xr), articles were retrieved from MEDLINE/PubMed, EMBASE, and WebOfScience. Inclusion criteria comprised studies on humans or animal models, assessment of peripheral or local tissue GDF15 concentrations, as well as the direct expression of GDF15 in skeletal muscle, and direct or indirect correlates of GDF15 with physical activity/ sarcopenia/trophism/ function. Results: A total of 646 studies were retrieved, and 144 finally included. Molecular inducers or inhibitors of GDF15 in skeletal muscle tissues were described. GDF15 was reported to promote skeletal muscle health, metabolic homeostasis, and overall physical conditioning. In pathology, GDF15 seems to be correlated to the degree of muscle impairment and mitochondrial stress. GDF15 has also been described as having the potential to stratify patients based on clinical prognosis and functional outcome. Conclusion: A hormetic hypothesis for GDF15 on skeletal muscle was proposed. In fact, GDF15 exhibited beneficial effects when expressed at high levels facing acute stressors (i.e., “myoprotection”). Conversely, GDF15 exhibited maladaptive effects, such as chronic low-grade inflammation, when chronically expressed in pathological processes (e.g., obesity, aging). GDF15 may be a potential molecular target for disease-modifying interventions. The current review underscores the need for further research on GDF15 to elucidate its therapeutic potential across different pathological states. The study protocol, registered before data collection and analysis, can be retrieved at https://osf.io/wa8xr. It should be noted that the study deviated from the protocol after peer review, including other electronic databases beyond MEDLINE/PubMed alone.

Growth differentiation factor 15 (GDF15) is a TGF-β superfamily member involved in diverse physiological and pathological processes. It is expressed in various tissues and its circulating levels rise during exercise, aging, pregnancy, and conditions such as cancer, cardiovascular disease, and infections. The biological activities of GDF15, including anorexia and cachexia, are primarily mediated through the GFRAL receptor, localized in the brainstem and functioning via RET co-receptor recruitment. This signaling is crucial for energy homeostasis and nausea induction. Recent studies suggest a broader GFRAL distribution, potentially explaining GDF15's distinct roles. These findings sparked interest in leveraging GDF15-GFRAL pathways for therapeutic development. Two primary strategies include GDF15 analogues as GFRAL agonists for obesity treatment and GDF15-derived peptides as antagonists to counteract cancer-induced cachexia and related disorders. This review highlights advancements in understanding GDF15-GFRAL signaling and its implications, summarizing bioactive GDF15-derived molecules, their pharmacological applications, and offering insights into novel treatment avenues for GDF15-associated conditions.

INTRODUCTION:Calcium/calmodulin-dependent protein kinase kinase 1 (CAMKK1) regulates energy homeostasis through AMP-activated protein kinase (AMPK). CAMKK1 has been implicated in appetite and satiety regulation; however, its role in obesity or type 2 diabetes mellitus (T2DM) remains unexplored. In this cross-sectional study, the primary aim was to confirm whether CAMKK1 is elevated in individuals with diabetes. The secondary aim was to investigate CAMKK1's molecular correlates. METHODS:CAMKK1 serum levels in individuals with obesity (n = 3,061), patients with T2DM (n = 4,910) and controls (n = 44,257) were retrieved and compared (age, body mass index-BMI and sex-adjusted ANCOVA). Pearson correlation coefficients and linear regression coefficients (age and BMI-adjusted) were computed. The moderation effect of diagnostic groups was also assessed. The interaction between factors was explored by mixed graphical models. RESULTS:CAMKK1 was elevated in patients with T2DM, in comparison to both individuals with obesity and controls (post hoc comparison, Tukey-adjusted p = 0.010 and p = 0.044, respectively). Across diagnostic groups, positive associations were observed between CAMKK1 and AMPK (min β > 0.400, max p < 0.001) or TNFα (min > β 0.070, max p < 0.001). A positive association with leptin (β = 0.010, p = 0.002) and ghrelin (β = 0.005, p = 0.048) was observed only within controls. Multivariate multivariable models confirmed that specific interactions between factors were disrupted in patients with T2DM (p < 0.001). CONCLUSION:These findings provide new insights into the role of CAMKK1 in obesity and T2DM. Future research may further explore CAMKK1's interplay with inflammatory pathways.

Growth Differentiation Factor 15 (GDF15) is a peptide from the transforming growth factor (TGF)-β superfamily, typically found at low levels in mammalian tissues, but significantly upregulated during cellular stress or injury. Initially recognised for its role in inducing anorexia and vomiting, GDF15 is now seen as a broader regulator of homeostasis, although its effects on gastrointestinal function remain unclear. This study examined GDF15's impact on the gastric fundus, a key region for appetite regulation. In ex vivo rodent gastric smooth muscle, GDF15 receptors were detected, and exposure to GDF15 caused smooth muscle depolarization, leading to increased mechanical activation. Morphological analyses revealed changes in the contractile apparatus, resembling cholinergic excitatory pathways. These effects were blocked by atropine, indicating muscarinic receptor involvement. Taken together, these findings suggest that GDF15 enhances gastric contractility by influencing cholinergic tone. Further studies will shed light on its mechanism of action and on the potential translational perspective of current results, elucidating whether elevated plasma levels of GDF15 observed in several physiological and pathological conditions can also have repercussions in gastric physiology, appetite regulation and weight loss.

In this letter, we put forward the light sheet fluorescence microscopy (LSFM) as a cutting-edge tool for 3D imaging of whole skeletal muscle, focusing on satellite cells (SCs). SCs represent the resident adult muscle stem cells, normally lying quiescent between the sarcolemma of the myofiber and the surrounding basal lamina. They typically express Pax-7 and, when activated following damage, they sequentially express specific myogenic regulatory factors including the myogenic determination factor, MyoD, thus starting differentiation towards multinucleated myofibers to repair injured tissue. The present analysis was performed on an ex vivo model of murine skeletal muscle injured by a forced eccentric contraction in isometric condition. The entire muscles were subjected to a tissue clearing and whole-mount staining process, enabling optical access and specific labeling across the entire intact sample. We performed labeling either with a fluorescent analog of standard hematoxylin and eosin, or with specific immunostaining against Pax-7 and MyoD. This proof of concept study demonstrates the feasibility of whole-muscle imaging with LSFM for the evaluation of the spatial arrangement of resting and activated SCs, overcoming the methodological limits of conventional 2D histology. This innovative experimental pipeline can be useful to test novel therapeutic approaches aimed at enhancing tissue regeneration and other biomedical/clinical applications.

Although photobiomodulation (PBM) therapy (i.e., the application of light with a 600-1100 nm wavelength using laser or light-emitting diode devices, a power density of less than 100 mW/cm2, and an energy density of less than 10 J/cm2 at the target) is emerging as a significant noninvasive strategy of promoting regeneration of damaged skeletal muscle tissue, its actual benefits remain debated. In particular, operating parameters exhibiting positive effects on regenerative muscle satellite stem cells need to be clearly identified. Hence, we investigated the effects of red PBM carried out by a laser diode (635 ± 10 nm; 0.4, 4, and 8 J/cm2; 4 mW/cm2; non-contact mode; continuous wave; single exposure) on murine myoblasts undergoing differentiation and on mature myotubes by combining morphological, biochemical, and functional analyses. Red PBM, especially with a 4 J/cm2 energy density, did not alter cell viability but successfully promoted the expression of myogenic transcription factors as myoblast determination protein 1 (MyoD) and myogenin, as well as myotube formation, mitochondrial metabolism, and biogenesis. Consistently, electrophysiological analyses of cell membrane passive properties and inward ion currents indicated the acquisition of a more differentiated phenotype in PBM-treated cells. Moreover, we found that PBM was able to enhance the release of extracellular vesicles (EVs) during cell differentiation according to a promyogenic phenotype. Red PBM treatment did not alter mature myotube viability and dimension while increasing their secretion of promyogenic EVs. Overall, this study provides experimental evidence supporting promyogenic effects of red PBM and the essential groundwork for further preclinical and clinical studies in the field of skeletal muscle regenerative medicine.

Inflammatory bowel disease (IBD) is a clinical condition of the gastrointestinal tract that has significant incidence in childhood. Major symptoms include abdominal pain, dyspepsia, delayed gastric emptying, anorexia, diarrhea and weight loss. IBD etiopathogenesis is multifactorial, with a proven involvement of cytokines. In this regard, cytokines like resistin and adiponectin produced by adipose tissue play a crucial role in inflammation. Particularly, resistin seems related to IBD severity and is considered a promising marker of disease occurrence and progression. Unraveling its mechanism of action and downstream effectors is mandatory when designing novel therapies. This preclinical study aims to further elucidate the action of resistin in causing functional gastrointestinal alterations, comparing it with the well-defined effect of adiponectin. To this end, we carried out electrophysiological analysis on murine gastric fundus. We found that resistin, similarly to adiponectin, increases smooth muscle cell (SMC) capacitance, indicative of cell surface remodeling, which is consistent with relaxation. However, contrary to adiponectin, resistin unalters membrane potential and inward Ca2+ entry and scarcely affects outward current, suggesting its inefficacy in markedly modifying electrical phenomena on the SMC membrane. This outcome, supporting the role of resistin in gastrointestinal distention, as observed in IBD, rules out a strikingly direct effect on SMCs.

Holotomography (HT) is a cutting-edge fast live-cell quantitative label-free imaging technique. Based on the principle of quantitative phase imaging, it combines holography and tomography to record a three-dimensional map of the refractive index, used as intrinsic optical and quantitative imaging contrast parameter of biological samples, at a sub-micrometer spatial resolution. In this study HT has been employed for the first time to analyze the changes of fibroblasts differentiating towards myofibroblasts - recognized as the main cell player of fibrosis - when cultured in vitro with the pro-fibrotic factor, namely transforming growth factor-beta 1. In parallel, F-actin, vinculin, alpha-smooth muscle actin, phospho-myosin light chain 2, type-1 collagen, peroxisome proliferator-activated receptor-gamma coactivator-1 alpha expression and mitochondria were evaluated by confocal laser scanning microscopy. Plasmamembrane passive properties and transient receptor potential canonical channels' currents were also recorded by whole-cell patch-clamp. The fluorescence images and electrophysiological results have been compared to the data obtained by HT and their congruence has been discussed. HT turned out to be a valid approach to morphologically distinguish fibroblasts from well differentiated myofibroblasts while obtaining objective measures concerning volume, surface area, projection area, surface index and dry mass (i.e., the mass of the non-aqueous content inside the cell including proteins and subcellular organelles) of the entire cell, nuclei and nucleoli with the major advantage to monitor outer and inner features in living cells in a non-invasive, rapid and label-free approach. HT might open up new research opportunities in the field of fibrotic diseases.Research Highlights Holotomography (HT) is a label-free laser interferometric imaging technology exploiting the intrinsic optical property of cells namely refractive index (RI) to enable a direct imaging and analysis of whole cells or intracellular organelles. HT turned out a valid approach to distinguish morphological features of living unlabeled fibroblasts from differentiated myofibroblasts. HT provided quantitative information concerning volume, surface area, projection area, surface index and dry mass of the entire fibroblasts/myofibroblasts, nuclei and nucleoli. Holotomographic microscopy was employed for the first time to detect morphological features of living unlabeled fibroblasts and myofibroblasts turning out as a valid approach. It provided quantitative measures concerning volume, surface area, projection area, surface index and dry mass of the entire cells, nuclei and nucleoli. image

The neuroendocrine control of reproduction is strictly coordinated at the central level by the pulsatile release of gonadotropin-releasing hormone (GnRH) by the hypothalamic GnRH neurons. Alterations of the GnRH-network, especially during development, lead to long-term reproductive and systemic consequences, also causing infertility. Recent evidence shows that benzo[a]pyrene (BaP), a diffuse pollutant that can play a role as an endocrine disruptor, affects gonadal function and gamete maturation, whereas data demonstrating its impact at hypothalamic level are very scarce. This study investigated the effects of BaP (10 μM) in a primary cell culture isolated from the human fetal hypothalamus (hfHypo) and exhibiting a clear GnRH neuron phenotype. BaP significantly decreased gene and protein expression of both GnRH and kisspeptin receptor (KISS1R), the master regulator of GnRH neuron function. Moreover, BaP exposure increased phospho-ERK1/2 signaling, a well-known mechanism associated with KISS1R activation. Interestingly, BaP altered the electrophysiological membrane properties leading to a significant depolarizing effect and it also significantly increased GnRH release, with both effects being not affected by kisspeptin addition. In conclusion, our findings demonstrate that BaP may alter GnRH neuron phenotype and function, mainly interfering with KISS1R signaling and GnRH secretion and therefore with crucial mechanisms implicated in the central neuroendocrine control of reproduction.

Mechanomimetic materials are particularly attractive for modeling in vitro fibroblast to myofibroblast (Myof) transition, a key process in the physiological repair of damaged tissue, and recognized as the core cellular mechanism of pathological fibrosis in different organs. In vivo, mechanical stimuli from the extracellular matrix (ECM) are crucial, together with cell-cell contacts and the pro-fibrotic transforming growth factor (TGF)-β1, in promoting fibroblast differentiation. Here, we explore the impact of hydrogels made by polyacrylamide with different composition on fibroblast behavior. By appropriate modulation of the hydrogel composition (e.g. adjusting the crosslinker content), we produce and fully characterize three kinds of scaffolds with different Young modulus (E). We observe that soft hydrogels (E < 1 kPa) induced fibroblast differentiation better than stiffer ones, also in the absence of TGF-β1. This study provides a readily accessible biomaterial platform to promote Myof generation. The easy approach used and the commercial availability of the monomers make these hydrogels suitable to a wide range of biomedical applications combined with high reproducibility and simple preparation protocols.

Adipokines are peptide hormones produced by the adipose tissue involved in several biological functions. Among adipokines, adiponectin (ADPN) has antidiabetic and anti-inflammatory properties. It can also modulate food intake at central and peripheral levels, acting on hypothalamus and facilitating gastric relaxation. ADPN exerts its action interacting with two distinct membrane receptors and triggering some well-defined signaling cascades. The ceramidase activity of ADPN receptor has been reported in many tissues: it converts ceramide into sphingosine. In turn, sphingosine kinase (SK) phosphorylates it into sphingosine-1 phosphate (S1P), a crucial mediator of many cellular processes including contractility. Using a multidisciplinary approach that combined biochemical, electrophysiological and morphological investigations, we explored for the first time the possible role of S1P metabolism in mediating ADPN effects on the murine gastric fundus muscle layer. By using a specific pharmacological inhibitor of SK2, we showed that ADPN affects smooth muscle cell membrane properties and contractile machinery via SK2 activation in gastric fundus, adding a piece of knowledge to the action mechanisms of this hormone. These findings help to identify ADPN and its receptors as new therapeutic targets or as possible prognostic markers for diseases with altered energy balance and for pathologies with fat mass content alterations.

Pheochromocytomas/paragangliomas (PPGLs) are neuroendocrine tumours, mostly resulting from mutations in predisposing genes. Mutations of succinate dehydrogenase (SDH) subunit B (SDHB) are associated with high probability of metastatic disease. Since bioelectrical properties and signalling in cancer are an emerging field, we investigated the metabolic, functional and electrophysiological characteristics in human succinate dehydrogenase subunit B (SDHB)-deficient pheochromocytoma cells. These cells exhibited reduced SDH function with elevated succinate-to-fumarate ratio and reduced intracellular ATP levels. The analysis of membrane passive properties revealed a more hyperpolarized membrane potential and a lower cell capacitance of SDHB-deficient cells compared to the parental ones. These bioelectrical changes were associated with reduced proliferation and adhesion capacity of SDHB-deficient cells. Only in SDHB-deficient cells, we also observed an increased amplitude of potassium currents suggesting an activation of ATP-sensitive potassium channels (KATP). Indeed, exposure of the SDHB-deficient cells to glibenclamide, a specific KATP inhibitor, or to ATP caused normalization of potassium current features and altered proliferation and adhesion. In this work, we show for the first time that reduced intracellular ATP levels in SDHB-deficient chromaffin cells impaired cell bioelectrical properties, which, in turn, are associated with an increased cell aggressiveness. Moreover, we first ever demonstrated that glibenclamide not only reduced the outward potassium currents in SDHB-deficient cells but increased their growth capacity, reduced their ability to migrate and shifted their phenotype towards one more similar to that of parental one.

Adiponectin (ADPN), a hormone produced by adipose tissue, facilitates gastric relaxation and can be a satiety signal in the network connecting peripheral organs and the central nervous system for feeding behavior control. Here, we performed preclinical research by morpho-functional analyses on murine gastric fundus smooth muscle to add insights into the molecular mechanisms underpinning ADPN action. Moreover, we conducted a clinical study to evaluate the potential use of ADPN as a biomarker for eating disorders (ED) based on the demonstrated gastric alterations and hormone level fluctuations that are often associated with ED. The clinical study recruited patients with ED and healthy controls who underwent blood draws for ADPN dosage and psychopathology evaluation tests. The findings of this basic research support the ADPN relaxant action, as indicated by the smooth muscle cell membrane pro-relaxant effects, with mild modifications of contractile apparatus and slight inhibitory effects on gap junctions. All of these actions engaged the ADPN/nitric oxide/guanylate cyclase pathway. The clinical data failed to unravel a correlation between ADPN levels and the considered ED, thus negating the potential use of ADPN as a valid biomarker for ED management for the moment. Nevertheless, this adipokine can modulate physiological eating behavior, and its effects deserve further investigation.

Hypoxia-inducible factor (HIF)-1α represents an oxygen-sensitive subunit of HIF transcriptional factor, which is usually degraded in normoxia and stabilized in hypoxia to regulate several target gene expressions. Nevertheless, in the skeletal muscle satellite stem cells (SCs), an oxygen level-independent regulation of HIF-1α has been observed. Although HIF-1α has been highlighted as a SC function regulator, its spatio-temporal expression and role during myogenic progression remain controversial. Herein, using biomolecular, biochemical, morphological and electrophysiological analyses, we analyzed HIF-1α expression, localization and role in differentiating murine C2C12 myoblasts and SCs under normoxia. In addition, we evaluated the role of matrix metalloproteinase (MMP)-9 as an HIF-1α effector, considering that MMP-9 is involved in myogenesis and is an HIF-1α target in different cell types. HIF-1α expression increased after 24/48 h of differentiating culture and tended to decline after 72 h/5 days. Committed and proliferating mononuclear myoblasts exhibited nuclear HIF-1α expression. Differently, the more differentiated elongated and parallel-aligned cells, which are likely ready to fuse with each other, show a mainly cytoplasmic localization of the factor. Multinucleated myotubes displayed both nuclear and cytoplasmic HIF-1α expression. The MMP-9 and MyoD (myogenic activation marker) expression synchronized with that of HIF-1α, increasing after 24 h of differentiation. By means of silencing HIF-1α and MMP-9 by short-interfering RNA and MMP-9 pharmacological inhibition, this study unraveled MMP-9's role as an HIF-1α downstream effector and the fact that the HIF-1α/MMP-9 axis is essential in morpho-functional cell myogenic commitment.

BACKGROUND:Adiponectin (Adn), released by adipocytes and other cell types such as skeletal muscle, has insulin-sensitizing and anti-inflammatory properties. Sphingosine 1-phosphate (S1P) is reported to act as effector of diverse biological actions of Adn in different tissues. S1P is a bioactive sphingolipid synthesized by the phosphorylation of sphingosine catalyzed by sphingosine kinase (SK) 1 and 2. Consolidated findings support the key role of S1P in the biology of skeletal muscle. METHODS AND RESULTS:Here we provide experimental evidence that S1P signalling is modulated by globular Adn treatment being able to increase the phosphorylation of SK1/2 as well as the mRNA expression levels of S1P4 in C2C12 myotubes. These findings were confirmed by LC-MS/MS that showed an increase of S1P levels after Adn treatment. Notably, the involvement of S1P axis in Adn action was highlighted since, when SK1 and 2 were inhibited by PF543 and ABC294640 inhibitors, respectively, not only the electrophysiological changes but also the increase of oxygen consumption and of aminoacid levels induced by the hormone, were significantly inhibited. CONCLUSION:Altogether, these findings show that S1P biosynthesis is necessary for the electrophysiological properties and oxidative metabolism of Adn in skeletal muscle cells.