RESUMO
Potassium channels are widespread over all kingdoms and play an important role in the maintenance of cellular ionic homeostasis. Kv1.3 is a voltage-gated potassium channel of the Shaker family with a wide tissue expression and a well-defined pharmacology. In recent decades, experiments mainly based on pharmacological modulation of Kv1.3 have highlighted its crucial contribution to different fundamental processes such as regulation of proliferation, apoptosis, and metabolism. These findings link channel function to various pathologies ranging from autoimmune diseases to obesity and cancer. In the present review, we briefly summarize studies employing Kv1.3 knockout animal models to confirm such roles and discuss the findings in comparison to the results obtained by pharmacological modulation of Kv1.3 in various pathophysiological settings. We also underline how these studies contributed to our understanding of channel function in vivo and propose possible future directions.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana , Animais , Canais de PotássioRESUMO
The 66 kDa isoform of the mammalian Shc gene promotes adipogenesis, and p66Shc-/- mice accumulate less body weight than wild-type (WT) mice. As the metabolic consequences of the leaner phenotype of p66Shc-/- mice is debated, we hypothesized that gut microbiota may be involved. We confirmed that p66Shc-/- mice gained less weight than WT mice when on a high-fat diet (HFD), but they were not protected from insulin resistance and glucose intolerance. p66Shc deletion significantly modified the composition of gut microbiota and their modification after an HFD. This was associated with changes in gene expression of Il-1b and regenerating islet-derived protein 3 γ ( Reg3g) in the gut and in systemic trimethylamine N-oxide and branched chain amino acid levels, despite there being no difference in intestinal structure and permeability. Depleting gut microbiota at the end of HFD rendered both strains more glucose tolerant but improved insulin sensitivity only in p66Shc-/- mice. Microbiota-depleted WT mice cohoused with microbiota-competent p66Shc-/- mice became significantly more insulin resistant than WT mice cohoused with WT mice, despite no difference in weight gain. These findings reconcile previous inconsistent observations on the metabolic phenotype of p66Shc-/- mice and illustrate the complex microbiome-host-genotype interplay under metabolic stress.-Ciciliot, S., Albiero, M., Campanaro, S., Poncina, N., Tedesco, S., Scattolini, V., Dalla Costa, F., Cignarella, A., Vettore, M., Di Gangi, I. M., Bogialli, S., Avogaro, A., Fadini, G. P. Interplay between gut microbiota and p66Shc affects obesity-associated insulin resistance.
Assuntos
Microbioma Gastrointestinal , Resistência à Insulina , Obesidade/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/genética , Animais , Dieta Hiperlipídica/efeitos adversos , Feminino , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/genética , Obesidade/microbiologia , Proteínas Associadas a Pancreatite/genética , Proteínas Associadas a Pancreatite/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismoRESUMO
Macrophages are highly plastic and dynamic cells that exert much of their function through phagocytosis. Phagocytosis depends on a coordinated, finely tuned, and compartmentalized regulation of calcium concentrations. We examined the role of mitochondrial calcium uptake and mitochondrial calcium uniporter (MCU) in macrophage polarization and function. In primary cultures of human monocyte-derived macrophages, calcium uptake in mitochondria was instrumental for alternative (M2) macrophage polarization. Mitochondrial calcium uniporter inhibition with KB-R7943 or MCU knockdown, which prevented mitochondrial calcium uptake, reduced M2 polarization, while not affecting classical (M1) polarization. Challenging macrophages with E. coli fragments induced spikes of mitochondrial calcium concentrations, which were prevented by MCU inhibition or silencing. In addition, mitochondria remodelled in M2 macrophages during phagocytosis, especially close to sites of E. coli internalization. Remarkably, inhibition or knockdown of MCU significantly reduced the phagocytic capacity of M2 macrophages. KB-R7943, which also inhibits the membrane sodium/calcium exchanger and Complex I, reduced mitochondria energization and cellular ATP levels, but such effects were not observed with MCU silencing. Therefore, phagocytosis inhibition by MCU knockdown depended on the impaired mitochondrial calcium buffering rather than changes in mitochondrial and cellular energy status. These data uncover a new role for MCU in alternative macrophage polarization and phagocytic activity.
Assuntos
Cálcio/metabolismo , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Mitocôndrias/metabolismo , Fagocitose/imunologia , Adolescente , Adulto , Sinalização do Cálcio , Inativação Gênica , Humanos , Imunidade Inata , Masculino , Adulto JovemRESUMO
AIMS/HYPOTHESIS: Oxygen radicals generated by p66Shc drive adipogenesis, but contradictory data exist on the role of p66Shc in the development of obesity and the metabolic syndrome. We herein explored the relationships among p66Shc, adipose tissue remodelling and glucose metabolism using mouse models and human adipose tissue samples. METHODS: In wild-type (WT), leptin-deficient (ob/ob), p66Shc(-/-) and p66Shc(-/-) ob/ob mice up to 30 weeks of age, we analysed body weight, subcutaneous and visceral adipose tissue histopathology, glucose tolerance and insulin sensitivity, and liver and muscle fat accumulation. A group of mice on a high fat diet (HFD) was also analysed. A parallel study was conducted on adipose tissue collected from patients undergoing elective surgery. RESULTS: We found that p66Shc(-/-) mice were slightly leaner than WT mice, and p66Shc(-/-) ob/ob mice became less obese than ob/ob mice. Despite their lower body weight, p66Shc(-/-) mice accumulated ectopic fat in the liver and muscles, and were glucose intolerant and insulin resistant. Features of adverse adipose tissue remodelling induced by obesity, including adipocyte enlargement, apoptosis, inflammation and perfusion were modestly and transiently improved by p66Shc (also known as Shc1) deletion. After 12 weeks of the HFD, p66Shc(-/-) mice were leaner than but equally glucose intolerant and insulin resistant compared with WT mice. In 77 patients, we found a direct correlation between BMI and p66Shc protein levels. Patients with low p66Shc levels were less obese, but were not protected from other metabolic syndrome features (diabetes, dyslipidaemia and hypertension). CONCLUSIONS/INTERPRETATION: In mice and humans, reduced p66Shc levels protect from obesity, but not from ectopic fat accumulation, glucose intolerance and insulin resistance.
Assuntos
Resistência à Insulina/genética , Obesidade/genética , Proteínas Adaptadoras da Sinalização Shc/genética , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Adiposidade/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/genética , Glicemia/metabolismo , Dieta Hiperlipídica , Feminino , Humanos , Insulina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Obesidade/metabolismo , Estresse Oxidativo/genética , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de SrcRESUMO
Three-dimensional hydrogel-based organ-like cultures can be applied to study development, regeneration, and disease in vitro. However, the control of engineered hydrogel composition, mechanical properties and geometrical constraints tends to be restricted to the initial time of fabrication. Modulation of hydrogel characteristics over time and according to culture evolution is often not possible. Here, we overcome these limitations by developing a hydrogel-in-hydrogel live bioprinting approach that enables the dynamic fabrication of instructive hydrogel elements within pre-existing hydrogel-based organ-like cultures. This can be achieved by crosslinking photosensitive hydrogels via two-photon absorption at any time during culture. We show that instructive hydrogels guide neural axon directionality in growing organotypic spinal cords, and that hydrogel geometry and mechanical properties control differential cell migration in developing cancer organoids. Finally, we show that hydrogel constraints promote cell polarity in liver organoids, guide small intestinal organoid morphogenesis and control lung tip bifurcation according to the hydrogel composition and shape.
Assuntos
Bioimpressão , Organoides , Hidrogéis/química , Engenharia Tecidual/métodos , Polaridade Celular , PulmãoRESUMO
Mobilization of hematopoietic stem/progenitor cells (HSPC) from the bone marrow (BM) is impaired in diabetes. Excess oncostatin M (OSM) produced by M1 macrophages in the diabetic BM signals through p66Shc to induce Cxcl12 in stromal cells and retain HSPC. BM adipocytes are another source of CXCL12 that blunts mobilization. We tested a strategy of pharmacologic macrophage reprogramming to rescue HSPC mobilization. In vitro, PPAR-γ activation with pioglitazone switched macrophages from M1 to M2, reduced Osm expression, and prevented transcellular induction of Cxcl12 In diabetic mice, pioglitazone treatment downregulated Osm, p66Shc, and Cxcl12 in the hematopoietic BM, restored the effects of granulocyte-colony stimulation factor (G-CSF), and partially rescued HSPC mobilization, but it increased BM adipocytes. Osm deletion recapitulated the effects of pioglitazone on adipogenesis, which was p66Shc independent, and double knockout of Osm and p66Shc completely rescued HSPC mobilization. In the absence of OSM, BM adipocytes produced less CXCL12, being arguably devoid of HSPC-retaining activity, whereas pioglitazone failed to downregulate Cxcl12 in BM adipocytes. In patients with diabetes on pioglitazone therapy, HSPC mobilization after G-CSF was partially rescued. In summary, pioglitazone reprogrammed BM macrophages and suppressed OSM signaling, but sustained Cxcl12 expression by BM adipocytes could limit full recovery of HSPC mobilization.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Mobilização de Células-Tronco Hematopoéticas , Macrófagos/efeitos dos fármacos , PPAR gama/agonistas , Pioglitazona/farmacologia , Adipogenia , Animais , Células da Medula Óssea/fisiologia , Reprogramação Celular , Quimiocina CXCL12/biossíntese , Feminino , Humanos , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oncostatina M/antagonistas & inibidores , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/fisiologiaRESUMO
Skeletal muscle decellularization allows the generation of natural scaffolds that retain the extracellular matrix (ECM) mechanical integrity, biological activity, and three-dimensional (3D) architecture of the native tissue. Recent reports showed that in vivo implantation of decellularized muscles supports muscle regeneration in volumetric muscle loss models, including nervous system and neuromuscular junctional homing. Since the nervous system plays pivotal roles during skeletal muscle regeneration and in tissue homeostasis, support of reinnervation is a crucial aspect to be considered. However, the effect of decellularized muscles on reinnervation and on neuronal axon growth has been poorly investigated. Here, we characterized residual protein composition of decellularized muscles by mass spectrometry and we show that scaffolds preserve structural proteins of the ECM of both skeletal muscle and peripheral nervous system. To investigate whether decellularized scaffolds could per se attract neural axons, organotypic sections of spinal cord were cultured three dimensionally in vitro, in presence or in absence of decellularized muscles. We found that neural axons extended from the spinal cord are attracted by the decellularized muscles and penetrate inside the scaffolds upon 3D coculture. These results demonstrate that decellularized scaffolds possess intrinsic neurotrophic properties, supporting their potential use for the treatment of clinical cases where extensive functional regeneration of the muscle is required.
Assuntos
Matriz Extracelular/metabolismo , Imageamento Tridimensional/métodos , Músculo Esquelético/metabolismo , Proteômica/métodos , Engenharia Tecidual/métodos , Animais , Feminino , Humanos , Masculino , RatosRESUMO
Fabrication of three-dimensional (3D) structures and functional tissues directly in live animals would enable minimally invasive surgical techniques for organ repair or reconstruction. Here, we show that 3D cell-laden photosensitive polymer hydrogels can be bioprinted across and within tissues of live mice, using bio-orthogonal two-photon cycloaddition and crosslinking of the polymers at wavelengths longer than 850 nm. Such intravital 3D bioprinting-which does not create by-products and takes advantage of commonly available multiphoton microscopes for the accurate positioning and orientation of the bioprinted structures into specific anatomical sites-enables the fabrication of complex structures inside tissues of live mice, including the dermis, skeletal muscle and brain. We also show that intravital 3D bioprinting of donor-muscle-derived stem cells under the epimysium of hindlimb muscle in mice leads to the de novo formation of myofibres in the mice. Intravital 3D bioprinting could serve as an in vivo alternative to conventional bioprinting.
Assuntos
Bioimpressão , Impressão Tridimensional , Engenharia Tecidual/métodos , Animais , Hidrogéis/administração & dosagem , Hidrogéis/química , Interações Hidrofóbicas e Hidrofílicas , Raios Infravermelhos , Injeções , Camundongos , Microscopia de Fluorescência por Excitação MultifotônicaRESUMO
Diabetes impairs the mobilization of hematopoietic stem/progenitor cells (HSPCs) from the bone marrow (BM), which can worsen the outcomes of HSPC transplantation and of diabetic complications. In this study, we examined the oncostatin M (OSM)-p66Shc pathway as a mechanistic link between HSPC mobilopathy and excessive myelopoiesis. We found that streptozotocin-induced diabetes in mice skewed hematopoiesis toward the myeloid lineage via hematopoietic-intrinsic p66Shc. The overexpression of Osm resulting from myelopoiesis prevented HSPC mobilization after granulocyte colony-stimulating factor (G-CSF) stimulation. The intimate link between myelopoiesis and impaired HSPC mobilization after G-CSF stimulation was confirmed in human diabetes. Using cross-transplantation experiments, we found that deletion of p66Shc in the hematopoietic or nonhematopoietic system partially rescued defective HSPC mobilization in diabetes. Additionally, p66Shc mediated the diabetes-induced BM microvasculature remodeling. Ubiquitous or hematopoietic restricted Osm deletion phenocopied p66Shc deletion in preventing diabetes-associated myelopoiesis and mobilopathy. Mechanistically, we discovered that OSM couples myelopoiesis to mobilopathy by inducing Cxcl12 in BM stromal cells via nonmitochondrial p66Shc. Altogether, these data indicate that cell-autonomous activation of the OSM-p66Shc pathway leads to diabetes-associated myelopoiesis, whereas its transcellular hematostromal activation links myelopoiesis to mobilopathy. Targeting the OSM-p66Shc pathway is a novel strategy to disconnect mobilopathy from myelopoiesis and restore normal HSPC mobilization.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Mielopoese/genética , Oncostatina M/genética , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/genética , Adulto , Idoso , Animais , Transplante de Medula Óssea , Quimiocina CXCL12/genética , Diabetes Mellitus/metabolismo , Feminino , Fator Estimulador de Colônias de Granulócitos , Mobilização de Células-Tronco Hematopoéticas , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Oncostatina M/metabolismo , Transdução de Sinais , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo , Células-TroncoRESUMO
AIMS: Diabetes is associated with an excess release of neutrophil extracellular traps (NETs) and an enhanced NETosis, a neutrophil cell death programme instrumental to anti-microbial defences, but also involved in tissue damage. We herein investigated whether the antidiabetic drug metformin protects against NETosis. METHODS: We measured NET components in the plasma of patients with pre-diabetes who were randomized to receive metformin or placebo for 2 months. To control for the effect on glucose, we also measured NET components in the plasma of patients with type 2 diabetes before and after treatment with insulin or dapagliflozin. In vitro, we used static and dynamic imaging with advanced live confocal two-photon microscopy to evaluate the effects of metformin on cellular events during NETosis. We examined putative molecular mechanisms by monitoring chromatin decondensation and DNA release in vitro. RESULTS: Metformin, as compared to placebo, significantly reduced the concentrations of NET components elastase, proteinase-3, histones and double strand DNA, whereas glucose control with insulin or dapagliflozin exerted no significant effect. In vitro, metformin prevented pathologic changes in nuclear dynamics and DNA release, resulting in a blunted NETosis in response to phorbol myristate acetate and calcium influx. Metformin prevented membrane translocation of PKC-ßII and activation of NADPH oxidase in neutrophils, both of which diminished the NETosis response. CONCLUSIONS: Metformin treatment reduced the concentrations of NET components independently from glucose control. This effect was reproducible in vitro and was related to the inhibitory effect exerted by metformin on the PKC-NADPH oxidase pathway.
Assuntos
Biomarcadores/sangue , Diabetes Mellitus Tipo 2/metabolismo , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Hipoglicemiantes/farmacologia , Inflamação/prevenção & controle , Metformina/farmacologia , Adulto , Compostos Benzidrílicos/administração & dosagem , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Quimioterapia Combinada , Feminino , Glucosídeos/administração & dosagem , Humanos , Hipoglicemiantes/uso terapêutico , Inflamação/etiologia , Inflamação/metabolismo , Masculino , Metformina/uso terapêutico , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Neutrófilos/patologiaRESUMO
The molecular bases of sperm thermotaxis, the temperature-oriented cell motility, are currently under investigation. Thermal perception relies on a subclass of the transient receptor potential [TRP] channels, whose member TRPV1 is acknowledged as the heat sensing receptor. Here we investigated the involvement of TRPV1 in human sperm thermotaxis. We obtained semen samples from 16 normozoospermic subjects attending an infertility survey programme, testis biopsies from 6 patients with testicular germ cell cancer and testis fine needle aspirates from 6 patients with obstructive azoospermia undergoing assisted reproductive technologies. Expression of TRPV1 mRNA was assessed by RT-PCR. Protein expression of TRPV1 was determined by western blot, flow cytometry and immunofluorescence. Sperm motility was assessed by Sperm Class Analyser. Acrosome reaction, apoptosis and intracellular-Ca2+ content were assessed by flow cytometry. We found that TRPV1 mRNA and protein were highly expressed in the testis, in both Sertoli cells and germ-line cells. Moreover, compared to no-gradient controls at 31°C or 37°C (Ctrl 31°C and Ctrl 37°C respectively), sperm migration towards a temperature gradient of 31-37°C (T gradient) in non-capacitated conditions selected a higher number of cells (14,9 ± 4,2×106 cells T gradient vs 5,1± 0,3×106 cells Ctrl 31°C and 5,71±0,74×106 cells Ctrl 37°C; P = 0,039). Capacitation amplified the migrating capability towards the T gradient. Sperms migrated towards the T gradient showed enriched levels of both TRPV1 protein and mRNA. In addition, sperm cells were able to migrate toward a gradient of capsaicin, a specific agonist of TRPV1, whilst capsazepine, a specific agonist of TRPV1, blocked this effect. Finally, capsazepine severely blunted migration towards T gradient without abolishing. These results suggest that TRPV1 may represent a facilitating mediator of sperm thermotaxis.
Assuntos
Infertilidade Masculina/genética , Neoplasias Embrionárias de Células Germinativas/genética , Espermatozoides/fisiologia , Canais de Cátion TRPV/genética , Resposta Táctica , Neoplasias Testiculares/genética , Adulto , Azoospermia/genética , Azoospermia/metabolismo , Azoospermia/patologia , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Humanos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Neoplasias Embrionárias de Células Germinativas/metabolismo , Neoplasias Embrionárias de Células Germinativas/patologia , Motilidade dos Espermatozoides , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Canais de Cátion TRPV/metabolismo , Resposta Táctica/efeitos dos fármacos , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologia , Adulto JovemRESUMO
INTRODUCTION: Epidemiological studies suggest that statins may promote the development or exacerbation of diabetes, but whether this occurs through inhibition of insulin secretion is unclear. This lack of understanding is partly due to the cellular models used to explore this phenomenon (cell lines or pooled islets), which are non-physiologic and have limited clinical transferability. METHODS: Here, we study the effect of simvastatin on insulin secretion using single-islet cultures, an optimal compromise between biological observability and physiologic fidelity. We develop and validate a microfluidic device to study single-islet function ex vivo, which allows for switching between media of different compositions with a resolution of seconds. In parallel, fluorescence imaging provides real-time analysis of the membrane voltage potential, cytosolic Ca2+ dynamics, and insulin release during perfusion under 3 or 11 mM glucose. RESULTS: We found that simvastatin reversibly inhibits insulin secretion, even in high-glucose. This phenomenon is very rapid (<60 s), occurs without affecting Ca2+ concentrations, and is likely unrelated to cholesterol biosynthesis and protein isoprenylation, which occur on a time span of hours. CONCLUSIONS: Our data provide the first real-time live demonstration that a statin inhibits insulin secretion in intact islets and that single islets respond differently from cell lines on a short time scale. FUNDING: University of Padova, EASD Foundation.
RESUMO
Upon activation, neutrophils undergo histone citrullination by protein arginine deiminase (PAD)4, exocytosis of chromatin and enzymes as neutrophil extracellular traps (NETs), and death. In diabetes, neutrophils are primed to release NETs and die by NETosis. Although this process is a defense against infection, NETosis can damage tissue. Therefore, we examined the effect of NETosis on the healing of diabetic foot ulcers (DFUs). Using proteomics, we found that NET components were enriched in nonhealing human DFUs. In an independent validation cohort, a high concentration of neutrophil elastase in the wound was associated with infection and a subsequent worsening of the ulcer. NET components (elastase, histones, neutrophil gelatinase-associated lipocalin, and proteinase-3) were elevated in the blood of patients with DFUs. Circulating elastase and proteinase-3 were associated with infection, and serum elastase predicted delayed healing. Neutrophils isolated from the blood of DFU patients showed an increased spontaneous NETosis but an impaired inducible NETosis. In mice, skin PAD4 activity was increased by diabetes, and FACS detection of histone citrullination, together with intravital microscopy, showed that NETosis occurred in the bed of excisional wounds. PAD4 inhibition by Cl-amidine reduced NETting neutrophils and rescued wound healing in diabetic mice. Cumulatively, these data suggest that NETosis delays DFU healing.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Armadilhas Extracelulares/fisiologia , Cicatrização/fisiologia , Idoso , Animais , Células Cultivadas , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/imunologia , Pé Diabético/imunologia , Pé Diabético/patologia , Pé Diabético/fisiopatologia , Feminino , Humanos , Elastase de Leucócito/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Fatores de Tempo , Cicatrização/imunologiaRESUMO
Recent studies disclosed a cross talk between testis and bone. By the action of LH, Leydig cells are able to modulate bone metabolism through testosterone and insulin-like factor 3. Moreover, LH modulates the Leydig expression of CYP2R1, the key enzyme involved in vitamin D (Vit D) 25-hydroxylation. However, pathways regulating CYP2R1 expression have been poorly investigated. The cross talk from the bone to the testis of the vitamin D 25-hydroxylase CYP2R1 involves osteocalcin (OC), which is produced by the osteoblasts and stimulates the production of testosterone by the Leydig cells through its putative receptor GPRC6A, a cation-sensing G-protein-coupled receptor. The aim of this study was to investigate the possible action of OC on CYP2R1 expression and 25-hydroxy Vit D (25-OH Vit D) production in a mouse Leydig cell line (MA-10). After confirmation of the expression of GPRC6A by MA-10, we found that stimulation with either human chorionic gonadotropin or uncarboxylated-OC (ucOC) increases CYP2R1 protein expression in a dose-dependent manner and, in turn, increases the release of 25-OH Vit D in culture medium. This effect was abolished by receptor blockade with, respectively, anti-LH receptor and anti-GPRC6A antibodies. Moreover, both agonists converged to phosphorylation of Erk1/2 by a likely differential action on second messengers. Human chorionic gonadotropin induced slow "tonic" increase of intercellular calcium and accumulation of cAMP, whereas ucOC mainly induced phasic increase of cell calcium. Supporting these findings, we found that serum ucOC positively correlated with 25-OH Vit D levels in 40 overweight male patients and 21 controls. Altogether, our results suggest that OC contributes with LH to 25-OH Vit D production by Leydig cells.