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Fibroblast growth factor 21 (FGF21) is a hormone involved in the regulation of lipid, glucose, and energy metabolism. Although it is released mainly from the liver, in recent years it has been shown that it is a "myokine", synthesized in skeletal muscles after exercise and stress conditions through an Akt-dependent pathway and secreted for mediating autocrine and endocrine roles. To date, the molecular mechanism for the pathophysiological regulation of FGF21 production in skeletal muscle is not totally understood. We have previously demonstrated that muscle membrane depolarization controls gene expression through extracellular ATP (eATP) signaling, by a mechanism defined as "Excitation-Transcription coupling". eATP signaling regulates the expression and secretion of interleukin 6, a well-defined myokine, and activates the Akt/mTOR signaling pathway. This work aimed to study the effect of electrical stimulation in the regulation of both production and secretion of skeletal muscle FGF21, through eATP signaling and PI3K/Akt pathway. Our results show that electrical stimulation increases both mRNA and protein (intracellular and secreted) levels of FGF21, dependent on an extracellular ATP signaling mechanism in skeletal muscle. Using pharmacological inhibitors, we demonstrated that FGF21 production and secretion from muscle requires the activation of the P2YR/PI3K/Akt/mTOR signaling pathway. These results confirm skeletal muscle as a source of FGF21 in physiological conditions and unveil a new molecular mechanism for regulating FGF21 production in this tissue. Our results will allow to identify new molecular targets to understand the regulation of FGF21 both in physiological and pathological conditions, such as exercise, aging, insulin resistance, and Duchenne muscular dystrophy, all characterized by an alteration in both FGF21 levels and ATP signaling components. These data reinforce that eATP signaling is a relevant mechanism for myokine expression in skeletal muscle.

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The human epidermal melanocyte (hEM) are melanin-producing cells that provide skin pigmentation and protection against ultraviolet radiation. Although purinergic signaling is involved in skin biology and pathology, the presence of NTPDase members, as well as the rate of nucleotides degradation by melanocytes were not described yet. Therefore, in this study, we analyzed the expression of ectonucleotidases in hEM derived from discarded foreskin of male patients. The expression of purinergic enzymes was confirmed by mRNA and flow cytometry. Among the ectonucleotidases, ectonucleoside triphosphate diphosphohydrolase1 (NTPDase1) and ecto-5´-nucleotidase were the ectoenzymes with higher expressions. The hydrolysis rate for ATP, ADP, and AMP was low in comparison to other primary cells already investigated. The amount of ATP in the culture medium was increased after a scratch wound and decreased to basal levels in 48 h, while the NTPDase1 and P2X7 expressions increased. Therefore, it is possible to suggest that after cell injury, the ATP released by hEM into the extracellular space will be hydrolyzed by ectonucleotidases as the NTPDase1 that will control the levels of nucleotides in the skin micro-environment.

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AIMS/HYPOTHESIS: Skeletal muscle is a key target organ for insulin's actions and is the main regulator of blood glucose. In obese individuals and animal models, there is a chronic low-grade inflammatory state affecting highly metabolic organs, leading to insulin resistance. We have described that adult skeletal muscle fibres can release ATP to the extracellular medium through pannexin-1 (PANX1) channels. Besides, it is known that high extracellular ATP concentrations can act as an inflammatory signal. Here, we propose that skeletal muscle fibres from obese mice release high levels of ATP, through PANX1 channels, promoting inflammation and insulin resistance in muscle cells. METHODS: C57BL/6J mice were fed with normal control diet (NCD) or high-fat diet (HFD) for 8 weeks. Muscle fibres were isolated from flexor digitorum brevis (FDB) muscle. PANX1-knockdown FDB fibres were obtained by in vivo electroporation of a short hairpin RNA Panx1 plasmid. We analysed extracellular ATP levels in a luciferin/luciferase assay. Gene expression was studied with quantitative real-time PCR (qPCR). Protein levels were evaluated by immunoblots, ELISA and immunofluorescence. Insulin sensitivity was analysed in a 2-NBDG (fluorescent glucose analogue) uptake assay, immunoblots and IPGTT. RESULTS: HFD-fed mice showed significant weight gain and insulin resistance compared with NCD-fed mice. IL-6, IL-1ß and TNF-α protein levels were increased in FDB muscle from obese mice. We observed high levels of extracellular ATP in muscle fibres from obese mice (197 ± 55 pmol ATP/µg RNA) compared with controls (32 ± 10 pmol ATP/µg RNA). ATP release in obese mice fibres was reduced by application of 100 µmol/l oleamide (OLE) and 5 µmol/l carbenoxolone (CBX), both PANX1 blockers. mRNA levels of genes linked to inflammation were reduced using OLE, CBX or 2 U/ml ATPase apyrase in muscle fibres from HFD-fed mice. In fibres from mice with pannexin-1 knockdown, we observed diminished extracellular ATP levels (78 ± 10 pmol ATP/µg RNA vs 252 ± 37 pmol ATP/µg RNA in control mice) and a lower expression of inflammatory markers. Moreover, a single pulse of 300 µmol/l ATP to fibres from control mice reduced insulin-mediated 2-NBDG uptake and promoted an elevation in mRNA levels of inflammatory markers. PANX-1 protein levels were increased two- to threefold in skeletal muscle from obese mice compared with control mice. Incubation with CBX increased Akt activation and 2-NBDG uptake in HFD fibres after insulin stimulation, rescuing the insulin resistance condition. Finally, in vivo treatment of HFD-fed mice with CBX (i.p. injection of 10 mg/kg each day) for 14 days, compared with PBS, reduced extracellular ATP levels in skeletal muscle fibres (51 ± 10 pmol ATP/µg RNA vs 222 ± 28 pmol ATP/µg RNA in PBS-treated mice), diminished inflammation and improved glycaemic management. CONCLUSIONS/INTERPRETATION: In this work, we propose a novel mechanism for the development of inflammation and insulin resistance in the skeletal muscle of obese mice. We found that high extracellular ATP levels, released by overexpressed PANX1 channels, lead to an inflammatory state and insulin resistance in skeletal muscle fibres of obese mice.

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Vascular smooth muscle cells (VSMCs) exhibit a high degree of plasticity when they undergo the progression from a normal to a disease condition, which makes them a potential target for evaluating early markers and for the development of new therapies. Purinergic signalling plays a key role in vascular tonus control, ATP being an inductor of vasoconstriction, whereas adenosine mediates a vasodilation effect antagonising the ATP actions. The control of extracellular ATP and adenosine levels is done by ectonucleotidases, which represent a potential target to be evaluated in the progression of cardiovascular diseases. In this study, we analysed the basal activity and expression of the ectonucleotidases in aortic rat VSMCs, and we further performed in silico analysis to determine the expression of those enzymes in conditions that mimicked vascular diseases. Cultured in vitro VSMCs showed a prominent expression of Entpd1 followed by Entpd2 and Nt5e (CD73) and very low levels of Entpd3. Slightly faster AMP hydrolysis was observed when compared to ATP and ADP nucleotides. In silico analysis showed that the ectonucleotidases were modulated after induction of conditions that can lead to vascular diseases such as, hypertensive and hypotensive mice models (Nt5e); exposition to high-fat (Entpd1 and Entpd2) or high-phosphate (Nt5e) diet; mechanical stretch (Entpd1, Entpd2 and Nt5e); and myocardial infarction (Entpd1). Our data show that VSMCs are able to efficiently metabolise the extracellular nucleotides generating adenosine. The modulation of Entpd1, Entdp2 and Nt5e in vascular diseases suggests these ectoenzymes as potential targets or markers to be investigated in future studies.

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Damage in fish activates retina repair that restores sight. The purinergic signalling system serves multiple homeostatic functions and has been implicated in cell cycle control of progenitor cells in the developing retina. We examined whether changes in the expression of purinergic molecules were instrumental in the proliferative phase after injury of adult zebrafish retinas with ouabain. P2RY1 messenger RNA (mRNA) increased early after injury and showed maximal levels at the time of peak progenitor cell proliferation. Extracellular nucleotides, mainly ADP, regulate P2RY1 transcriptional and protein expression. The injury-induced upregulation of P2RY1 is mediated by an autoregulated mechanism. After injury, the transcriptional expression of ecto-nucleotidases and ecto-ATPases also increased and ecto-ATPase activity inhibitors decreased Müller glia-derived progenitor cell amplification. Inhibition of P2RY1 endogenous activation prevented progenitor cell proliferation at two intervals after injury: one in which progenitor Müller glia mitotically activates and the second one in which Müller glia-derived progenitor cells amplify. ADPßS induced the expression of lin28a and ascl1a genes in mature regions of uninjured retinas. The expression of these genes, which regulate multipotent Müller glia reprogramming, was significantly inhibited by blocking the endogenous activation of P2RY1 early after injury. We consistently observed that the number of glial fibrillary acidic protein-BrdU-positive Müller cells after injury was larger in the absence than in the presence of the P2RY1 antagonist. Ecto-ATPase activity inhibitors or P2RY1-specific antagonists did not modify apoptotic cell death at the time of peak progenitor cell proliferation. The results suggested that ouabain injury upregulates specific purinergic signals which stimulates multipotent progenitor cell response.

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Plasmodium falciparum is the causative agent of the most dangerous form of malaria in humans. It has been reported that the P. falciparum genome encodes for a single ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), an enzyme that hydrolyzes extracellular tri- and di-phosphate nucleotides. The E-NTPDases are known for participating in invasion and as a virulence factor in many pathogenic protozoa. Despite its presence in the parasite genome, currently, no information exists about the activity of this predicted protein. Here, we show for the first time that P. falciparum E-NTPDase is relevant for parasite lifecycle as inhibition of this enzyme impairs the development of P. falciparum within red blood cells (RBCs). ATPase activity could be detected in rings, trophozoites, and schizonts, as well as qRT-PCR, confirming that E-NTPDase is expressed throughout the intraerythrocytic cycle. In addition, transfection of a construct which expresses approximately the first 500 bp of an E-NTPDase-GFP chimera shows that E-NTPDase co-localizes with the endoplasmic reticulum (ER) in the early stages and with the digestive vacuole (DV) in the late stages of P. falciparum intraerythrocytic cycle.

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Ecto-nucleotidase activity is involved in the infection process of Leishmania and various other parasites that enables modulation of host immune responses to promote disease progression. One of the enzymes responsible for this activity is the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase). The enzyme hydrolyzes nucleotides tri- and/or di-phosphate into monophosphate products, which are subsequently hydrolyzed into adenosine. These nucleotides can serve as purinergic signaling molecules involved in diverse cellular processes that govern immune responses. Given the importance of the extracellular metabolism of these nucleotides during intracellular pathogen infections, this study evaluates the role of ecto-nucleotidase activity during Leishmania infantum (L. infantum) infection in human macrophages. E-NTPDase protein expression and activity was evaluated in L. infantum during purine starvation, adenosine-enriched medium, or in the presence of an inhibitor of ecto-nucleotidases. Results show that E-NTPDase is expressed in L. infantum parasites, including on the cell membrane. Furthermore, functional activity of the enzyme was modulated according to the availability of adenosine in the medium. Purine starvation increased the hydrolytic capacity of nucleotides leading to higher infectivity, while growth in adenosine-enriched medium led to lower infectivity. Moreover, inhibiting E-NTPDase function decreased L. infantum infection in macrophages, suggesting the enzyme may serve as a ligand. Taken together, the ability of L. infantum to hydrolyze nucleotides is directly associated with increased infectivity in macrophages.

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The protozoan parasite Leishmania amazonensis is the etiological agent of cutaneous leishmaniasis. During its life cycle, the flagellated metacyclic promastigote forms are transmitted to vertebrate hosts by sandfly bites, and they develop into amastigotes inside macrophages, where they multiply. L. amazonensis possesses a bifunctional enzyme, called 3'-nucleotidase/nuclease (3'NT/NU), which is able to hydrolyze extracellular 3'-monophosphorylated nucleosides and nucleic acids. 3'NT/NU plays an important role in the generation of extracellular adenosine and has been described as a key enzyme in the acquisition of purines by trypanosomatids. Furthermore, it has been observed that 3'NT/NU also plays a valuable role in the establishment of parasitic infection. In this context, this study aimed to investigate the modulation of the 3'-nucleotidase (3'NT) activity of L. amazonensis by several nucleotides. It was observed that 3'NT activity is inhibited by micromolar concentrations of guanosine and guanine nucleotides. The inhibition promoted by 5'-GMP on the 3'NT activity of L. amazonensis is reversible and uncompetitive because the addition of the inhibitor decreased the kinetic parameters Km and Vmax. Finally, we found that the addition of 5'-GMP is able to reverse the stimulation promoted by 3'-AMP in a macrophage-parasite interaction assay. The determination of compounds that can inhibit the 3'NT activity of Leishmania is very important because this enzyme does not occur in mammals, making it a potential therapeutic target.

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