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Obesity poses significant challenges, necessitating comprehensive strategies for effective intervention. Bariatric Surgery (BS) has emerged as a crucial therapeutic approach, demonstrating success in weight loss and comorbidity improvement. This study aimed to evaluate the outcomes of BS in a cohort of 48 Uruguayan patients and investigate the interplay between BS and clinical and metabolic features, with a specific focus on FSTL1, an emerging biomarker associated with obesity and inflammation. We quantitatively analyzed BS outcomes and constructed linear models to identify variables impacting BS success. The study revealed the effectiveness of BS in improving metabolic and clinical parameters. Importantly, variables correlating with BS success were identified, with higher pre-surgical FSTL1 levels associated with an increased effect of BS on BMI reduction. FSTL1 levels were measured from patient plasma using an ELISA kit pre-surgery and six months after. This research, despite limitations of a small sample size and limited follow-up time, contributes valuable insights into understanding and predicting the success of BS, highlighting the potential role of FSTL1 as a useful biomarker in obesity.
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Cirugía Bariátrica , Biomarcadores , Proteínas Relacionadas con la Folistatina , Obesidad , Humanos , Proteínas Relacionadas con la Folistatina/sangre , Proteínas Relacionadas con la Folistatina/metabolismo , Femenino , Masculino , Cirugía Bariátrica/métodos , Adulto , Persona de Mediana Edad , Biomarcadores/sangre , Obesidad/cirugía , Obesidad/metabolismo , Uruguay/epidemiología , Estudios de Cohortes , Pérdida de Peso , Resultado del Tratamiento , Índice de Masa CorporalRESUMEN
Emerging evidence suggests that immune receptors may participate in many aging-related processes such as energy metabolism, inflammation, and cognitive decline. CD300f, a TREM2-like lipid-sensing immune receptor, is an exceptional receptor as it integrates activating and inhibitory cell-signaling pathways that modulate inflammation, efferocytosis, and microglial metabolic fitness. We hypothesize that CD300f can regulate systemic aging-related processes and ultimately healthy lifespan. We closely followed several cohorts of two strains of CD300f-/- and WT mice of both sexes for 30 months and observed an important reduction in lifespan and healthspan in knockout mice. This was associated with systemic inflammaging, increased cognitive decline, reduced brain glucose uptake observed by 18FDG PET scans, enrichment in microglial aging/neurodegeneration phenotypes, proteostasis alterations, senescence, increased frailty, and sex-dependent systemic metabolic changes. Moreover, the absence of CD300f altered macrophage immunometabolic phenotype. Taken together, we provide strong evidence suggesting that myeloid cell CD300f immune receptor contributes to healthy aging.
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Disfunción Cognitiva , Envejecimiento Saludable , Masculino , Femenino , Ratones , Animales , Macrófagos/metabolismo , Inflamación/metabolismo , Microglía/metabolismo , Ratones Noqueados , Disfunción Cognitiva/metabolismoRESUMEN
Significance: Sirtuins are NAD+-dependent histone deacetylases regulating important processes in cellular biology such as inflammation, metabolism, oxidative stress, and apoptosis. Recent Advances: Despite initially being discovered to regulate transcription and life span via histone deacetylase activities, emerging data continually uncover new targets and propose additional roles. Due to the outstanding importance of the sirtuins in the control of the inflammatory response, their roles in the pathogenesis of several inflammatory-based diseases have become an area of intense research. Although sirtuins have been traditionally regarded as anti-inflammatory players, several recent findings suggest that their role in inflammation is complex and that in some cases sirtuins can indeed promote inflammation. Critical Issues: In this article, we provide an update on the latest findings concerning the new mechanisms of action and concepts about the role of sirtuins during inflammation. We focus on the impact that inflammatory-based processes exert on the liver, adipose tissue, and nervous system as well as on macrophage function and activation. Also, we discuss available data pointing to the dual role that, in particular contexts, sirtuins may have on inflammation control. Future Directions: Since the knowledge of sirtuin impact on metabolism is continually expanding, new venues of research arise. Besides become being regarded as candidates of therapeutic targets, posttranscriptional control of sirtuin expression by means of microRNAs challenges our traditional concepts of sirtuin regulation; importantly, the emerging role of NAD+ metabolism in aging and longevity has added a new dimension to the interest in sirtuin function. Antioxid. Redox Signal. 39, 1185-1208.
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Sirtuinas , Humanos , Sirtuinas/metabolismo , NAD/metabolismo , Estrés Oxidativo , Envejecimiento/fisiología , InflamaciónRESUMEN
The sirtuin 6 has emerged as a regulator of acute and chronic immune responses. Recent findings show that SIRT6 is necessary for mounting an active inflammatory response in macrophages. In vitro studies revealed that SIRT6 is stabilized in the cytoplasm to promote tumor necrosis factor (TNFα) secretion. Notably, SIRT6 also promotes TNFα secretion by resident peritoneal macrophages upon lipopolysaccharide (LPS) stimulation in vivo. Although many studies have investigated SIRT6 function in the immune response through different genetic and pharmacological approaches, direct measurements of in vivo SIRT6 expression in immune cells by flow cytometry have not yet been performed. Here, we describe a step-by-step protocol for peritoneal fluid extraction, isolation, and preparation of peritoneal cavity cells, intracellular SIRT6 staining, and flow cytometry analysis to measure SIRT6 levels in mice peritoneal macrophages. By providing a robust method to quantify SIRT6 levels in different populations of macrophages, this method will contribute to deepening our understanding of the role of SIRT6 in immunity, as well as in other cellular processes regulated by SIRT6. Graphical abstract.
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The protein DBC1 is the main SIRT1 regulator known so far, and by doing so, it is involved in the regulation of energy metabolism, especially in liver and fat adipose tissue. DBC1 also has an important function in cell cycle progression and regulation in cancer cells, affecting tumorigenesis. We recently showed that during quiescence, non-transformed cells need DBC1 in order to re-enter and progress through the cell cycle. Moreover, we showed that deletion of DBC1 affects cell cycle progression during liver regeneration. This novel concept prompted us to evaluate the role of DBC1 during adult neurogenesis, where transition from quiescence to proliferation in neuronal progenitors is key and tightly regulated. Herein, we analyzed several markers of cell cycle expressed in the dentate gyrus of the hippocampus of controls and DBC1 KO adult mice. Our results suggest a reduced number of neuroblasts therein present, probably due to a decline of neuroblast generation or an impairment in neural differentiation. In agreement with this, we also found that adult DBC1 KO mice had a reduction in the volume of the granule cell layer of the dentate gyrus. Interestingly, behavioral analysis of KO and control mice revealed that deletion of DBC1 parallels to specific cognitive impairments, concerning learning and possibly memory formation. Our results show, for the first time, that DBC1 plays an active role in the nervous system. In particular, specific anatomical and behavioral changes are observed when is absent.
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Células-Madre Neurales , Neurogénesis , Ratones , Animales , Ratones Noqueados , Neurogénesis/fisiología , Hipocampo/metabolismo , Células-Madre Neurales/metabolismo , Cognición/fisiología , Giro Dentado , Ratones Endogámicos C57BLRESUMEN
Acute respiratory distress syndrome is associated with skeletal muscle compromise, which decreases survival and impairs functional capacity. A comparative analysis of peripheral and respiratory muscles' atrophy and dysfunction in acute lung injury (ALI) has not been performed. We aimed to evaluate diaphragmatic and peripheral muscle mass and contractility in an ALI animal model. ALI was induced in C57BL/6 mice by intratracheal lipopolysaccharides instillation. Muscle mass and in vitro contractility were evaluated at different time points in hindlimb soleus (slow-twitch) and extensor digitorum longus (EDL, fast-twitch), as well as in the main respiratory muscle diaphragm. Myogenic precursor satellite cell-specific transcription factor Pax7 expression was determined by Western blot. Lung injury was associated with atrophy of the three studied muscles, although it was more pronounced and persistent in the diaphragm. Specific contractility was reduced during lung injury in EDL muscle but restored by the time lung injury has resolved. Specific force was not affected in soleus and diaphragm. A persistent increase in Pax7 expression was detected in diaphragm and EDL muscles after induction of ALI, but not in soleus muscle. Different peripheral and respiratory skeletal muscles are distinctly affected during the course of ALI. Each of the studied muscles presented a unique pattern in terms of atrophy development, contractile dysfunction and Pax7 expression.
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Lesión Pulmonar Aguda , Enfermedades Musculares , Lesión Pulmonar Aguda/metabolismo , Animales , Atrofia , Ratones , Ratones Endogámicos C57BL , Contracción Muscular , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Músculo Esquelético/fisiología , Enfermedades Musculares/metabolismo , Músculos RespiratoriosRESUMEN
CCDC28B (coiled-coil domain-containing protein 28B) was identified as a modifier in the ciliopathy Bardet-Biedl syndrome (BBS). Our previous work in cells and zebrafish showed that CCDC28B plays a role regulating cilia length in a mechanism that is not completely understood. Here we report the generation of a Ccdc28b mutant mouse using CRISPR/Cas9 (Ccdc28b mut). Depletion of CCDC28B resulted in a mild phenotype. Ccdc28b mut animals i) do not present clear structural cilia affectation, although we did observe mild defects in cilia density and cilia length in some tissues, ii) reproduce normally, and iii) do not develop retinal degeneration or obesity, two hallmark features of reported BBS murine models. In contrast, Ccdc28b mut mice did show clear social interaction defects as well as stereotypical behaviors. This finding is indeed relevant regarding CCDC28B as a modifier of BBS since behavioral phenotypes have been documented in BBS. Overall, this work reports a novel mouse model that will be key to continue evaluating genetic interactions in BBS, deciphering the contribution of CCDC28B to modulate the presentation of BBS phenotypes. In addition, our data underscores a novel link between CCDC28B and behavioral defects, providing a novel opportunity to further our understanding of the genetic, cellular, and molecular basis of these complex phenotypes.
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Síndrome de Bardet-Biedl , Degeneración Retiniana , Animales , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/metabolismo , Cilios/metabolismo , Ratones , Fenotipo , Degeneración Retiniana/genética , Pez Cebra/genéticaRESUMEN
Acute and chronic inflammations are key homeostatic events in health and disease. Sirtuins (SIRTs), a family of NAD-dependent protein deacylases, play a pivotal role in the regulation of these inflammatory responses. Indeed, SIRTs have anti-inflammatory effects through a myriad of signaling cascades, including histone deacetylation and gene silencing, p65/RelA deacetylation and inactivation, and nucleotidebinding oligomerization domain, leucine rich repeat, and pyrin domaincontaining protein 3 inflammasome inhibition. Nevertheless, recent findings show that SIRTs, specifically SIRT6, are also necessary for mounting an active inflammatory response in macrophages. SIRT6 has been shown to positively regulate tumor necrosis factor alpha (TNFα) secretion by demyristoylating pro-TNFα in the cytoplasm. However, how SIRT6, a nuclear chromatin-binding protein, fulfills this function in the cytoplasm is currently unknown. Herein, we show by Western blot and immunofluorescence that in macrophages and fibroblasts there is a subpopulation of SIRT6 that is highly unstable and quickly degraded via the proteasome. Upon lipopolysaccharide stimulation in Raw 264.7, bone marrow, and peritoneal macrophages, this population of SIRT6 is rapidly stabilized and localizes in the cytoplasm, specifically in the vicinity of the endoplasmic reticulum, promoting TNFα secretion. Furthermore, we also found that acute SIRT6 inhibition dampens TNFα secretion both in vitro and in vivo, decreasing lipopolysaccharide-induced septic shock. Finally, we tested SIRT6 relevance in systemic inflammation using an obesity-induced chronic inflammatory in vivo model, where TNFα plays a key role, and we show that short-term genetic deletion of SIRT6 in macrophages of obese mice ameliorated systemic inflammation and hyperglycemia, suggesting that SIRT6 plays an active role in inflammation-mediated glucose intolerance during obesity.
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Inflamación , Macrófagos , Sirtuinas , Animales , Citoplasma/metabolismo , Inflamación/genética , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Ratones , Obesidad/metabolismo , Sirtuinas/genética , Sirtuinas/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
CD38 enzymatic activity regulates NAD+ and cADPR levels in mammalian tissues, and therefore has a prominent role in cellular metabolism and calcium homeostasis. Consequently, it is reasonable to hypothesize about its involvement in cardiovascular physiology as well as in heart related pathological conditions. AIM: To investigate the role of CD38 in cardiovascular performance, and its involvement in cardiac electrophysiology and calcium-handling. METHODS AND RESULTS: When submitted to a treadmill exhaustion test, a way of evaluating cardiovascular performance, adult male CD38KO mice showed better exercise capacity. This benefit was also obtained in genetically modified mice with catalytically inactive (CI) CD38 and in WT mice treated with antibody 68 (Ab68) which blocks CD38 activity. Hearts from these 3 groups (CD38KO, CD38CI and Ab68) showed increased NAD+ levels. When CD38KO mice were treated with FK866 which inhibits NAD+ biosynthesis, exercise capacity as well as NAD+ in heart tissue decreased to WT levels. Electrocardiograms of conscious unrestrained CD38KO and CD38CI mice showed lower basal heart rates and higher heart rate variability than WT mice. Although inactivation of CD38 in mice resulted in increased SERCA2a expression in the heart, the frequency of spontaneous calcium release from the sarcoplasmic reticulum under stressful conditions (high extracellular calcium concentration) was lower in CD38KO ventricular myocytes. When mice were challenged with caffeine-epinephrine, CD38KO mice had a lower incidence of bidirectional ventricular tachycardia when compared to WT ones. CONCLUSION: CD38 inhibition improves exercise performance by regulating NAD+ homeostasis. CD38 is involved in cardiovascular function since its genetic ablation decreases basal heart rate, increases heart rate variability and alters calcium handling in a way that protects mice from developing catecholamine induced ventricular arrhythmias.
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ADP-Ribosil Ciclasa 1/metabolismo , Calcio , Glicoproteínas de Membrana/metabolismo , NAD , ADP-Ribosil Ciclasa 1/genética , Animales , Arritmias Cardíacas/etiología , Arritmias Cardíacas/metabolismo , Calcio/metabolismo , Catecolaminas/metabolismo , Tolerancia al Ejercicio , Frecuencia Cardíaca , Masculino , Mamíferos/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , NAD/metabolismoRESUMEN
Chronic metabolic diseases, like obesity, type II diabetes and atherosclerosis often involve a low-grade and sterile systemic inflammatory state, in which activation of the pro-inflammatory transcription factor NF-kB and the NLRP3 inflammasome play a major role. It is well established that genetic inhibition of the NLRP3 inflammasome ameliorates acute and chronic inflammation. Indeed, accumulating experimental evidences in murine models and also in humans suggest that inhibition of the NLRP3 inflammasome might be a suitable approach to tackle the deleterious effects of chronic metabolic diseases. In this work, we explored our previously synthesized nitroalkene-Trolox™ derivative named NATx0, as a non-conventional anti-inflammatory strategy to treat chronic inflammatory diseases, such as obesity-induced glucose intolerance. We found that NATx0 inhibited NF-kB nuclear translocation and pro-inflammatory gene expression in macrophages in vitro. In addition, treatment with NATx0 prevented NLRP3 inflammasome activation after LPS/ATP stimulation in macrophages in vitro. When tested acutely in vivo, NATx0 inhibited neutrophil recruitment in zebrafish larvae, and also diminished IL-1ß production after LPS challenge in mice. Finally, when NATx0 was administered chronically to diet-induced obese mice, it decreased muscle tissue inflammation and glucose intolerance, leading to improved glucose homeostasis. In conclusion, we propose that this novel nitroalkene-Trolox derivative is a suitable tool to tackle acute and chronic inflammation in vitro and in vivo mainly due to inhibition of NF-kB/NLRP3 activation.
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Diabetes Mellitus Tipo 2 , Intolerancia a la Glucosa , Animales , Intolerancia a la Glucosa/tratamiento farmacológico , Inflamasomas , Inflamación/tratamiento farmacológico , Interleucina-1beta , Lipopolisacáridos , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR , Obesidad/tratamiento farmacológico , Vitamina E , Pez CebraRESUMEN
Motor neuron degeneration and neuroinflammation are the most striking pathological features of amyotrophic lateral sclerosis (ALS). ALS currently has no cure and approved drugs have only a modest clinically therapeutic effect in patients. Drugs targeting different deleterious inflammatory pathways in ALS appear as promising therapeutic alternatives. Here, we have assessed the potential therapeutic effect of an electrophilic nitroalkene benzoic acid derivative, (E)-4-(2-nitrovinyl) benzoic acid (BANA), to slow down paralysis progression when administered after overt disease onset in SOD1G93A rats. BANA exerted a significant inhibition of NF-κB activation in NF-κB reporter transgenic mice and microglial cell cultures. Systemic daily oral administration of BANA to SOD1G93A rats after paralysis onset significantly decreased microgliosis and astrocytosis, and significantly reduced the number of NF-κB-p65-positive microglial nuclei surrounding spinal motor neurons. Numerous microglia bearing nuclear NF-κB-p65 were observed in the surrounding of motor neurons in autopsy spinal cords from ALS patients but not in controls, suggesting ALS-associated microglia could be targeted by BANA. In addition, BANA-treated SOD1G93A rats after paralysis onset showed significantly ameliorated spinal motor neuron pathology as well as conserved neuromuscular junction innervation in the skeletal muscle, as compared to controls. Notably, BANA prolonged post-paralysis survival by ~30%, compared to vehicle-treated littermates. These data provide a rationale to therapeutically slow paralysis progression in ALS using small electrophilic compounds such as BANA, through a mechanism involving microglial NF-κB inhibition.
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Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Nitrobenzoatos/uso terapéutico , Esclerosis Amiotrófica Lateral/mortalidad , Esclerosis Amiotrófica Lateral/patología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Células HT29/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/efectos de los fármacos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Ratas , Médula Espinal/efectos de los fármacos , Médula Espinal/patologíaRESUMEN
Sirtuin 6, SIRT6, is critical for both glucose and lipid homeostasis and is involved in maintaining genomic stability under conditions of oxidative DNA damage such as those observed in age-related diseases. There is an intense search for modulators of SIRT6 activity, however, not many specific activators have been reported. Long acyl-chain fatty acids have been shown to increase the weak in vitro deacetylase activity of SIRT6 but this effect is modest at best. Herein we report that electrophilic nitro-fatty acids (nitro-oleic acid and nitro-conjugated linoleic acid) potently activate SIRT6. Binding of the nitro-fatty acid to the hydrophobic crevice of the SIRT6 active site exerted a moderate activation (2-fold at 20 µm), similar to that previously reported for non-nitrated fatty acids. However, covalent Michael adduct formation with Cys-18, a residue present at the N terminus of SIRT6 but absent from other isoforms, induced a conformational change that resulted in a much stronger activation (40-fold at 20 µm). Molecular modeling of the resulting Michael adduct suggested stabilization of the co-substrate and acyl-binding loops as a possible additional mechanism of SIRT6 activation by the nitro-fatty acid. Importantly, treatment of cells with nitro-oleic acid promoted H3K9 deacetylation, whereas oleic acid had no effect. Altogether, our results show that nitrated fatty acids can be considered a valuable tool for specific SIRT6 activation, and that SIRT6 should be considered as a molecular target for in vivo actions of these anti-inflammatory nitro-lipids.
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Ácidos Grasos/farmacología , Nitrocompuestos/farmacología , Sirtuinas/metabolismo , Acetilación , Humanos , Estrés Oxidativo , Conformación Proteica , Sirtuinas/química , Sirtuinas/genéticaRESUMEN
Cardiovascular diseases are among the main causes of morbimortality in the adult population. Among them, hypertension is a leading cause for stroke, heart disease and kidney failure. Also, as a result of arterial wall weakness, hypertension can lead to the development of dissecting aortic aneurysms, a rare but often fatal condition if not readily treated. In this work, we investigated the role of DBC1 in the regulation of vascular function in an ANGII-induced hypertension mouse model. We found that WT and DBC1 KO mice developed hypertension in response to ANGII infusion. However, DBC1 KO mice showed increased susceptibility to develop aortic dissections. The effect was accompanied by upregulation of vascular remodeling factors, including MMP9 and also VEGF. Consistent with this, we found decreased collagen deposition and elastic fiber fragmentation, suggesting that increased expression of MMPs in DBC1 KO mice weakens the arterial wall, promoting the formation of aortic dissections during treatment with ANGII. Finally, DBC1 KO mice had reduced cell proliferation in the intima-media layer in response to ANGII, paralleled with an impairment to increase wall thickness in response to hypertension. Furthermore, VSMC purified from DBC1 KO mice showed impaired capacity to leave quiescence, confirming the in vivo results. Altogether, our results show for the first time that DBC1 regulates vascular response and function during hypertension and protects against vascular injury. This work also brings novel insights into the molecular mechanisms of the development of aortic dissections.
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Enfermedades Cardiovasculares/genética , Proteínas de Ciclo Celular/genética , Hipertensión/genética , Proteínas del Tejido Nervioso/genética , Lesiones del Sistema Vascular/genética , Angiotensina II/efectos adversos , Animales , Enfermedades Cardiovasculares/patología , Proliferación Celular/genética , Modelos Animales de Enfermedad , Humanos , Hipertensión/inducido químicamente , Hipertensión/patología , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Noqueados , Factor A de Crecimiento Endotelial Vascular/genética , Lesiones del Sistema Vascular/patologíaRESUMEN
The protein Deleted in Breast Cancer-1 is a regulator of several transcription factors and epigenetic regulators, including HDAC3, Rev-erb-alpha, PARP1 and SIRT1. It is well known that DBC1 regulates its targets, including SIRT1, by protein-protein interaction. However, little is known about how DBC1 biological activity is regulated. In this work, we show that in quiescent cells DBC1 is proteolytically cleaved, producing a protein (DN-DBC1) that misses the S1-like domain and no longer binds to SIRT1. DN-DBC1 is also found in vivo in mouse and human tissues. Interestingly, DN-DBC1 is cleared once quiescent cells re-enter to the cell cycle. Using a model of liver regeneration after partial hepatectomy, we found that DN-DBC1 is down-regulated in vivo during regeneration. In fact, WT mice show a decrease in SIRT1 activity during liver regeneration, coincidentally with DN-DBC1 downregulation and the appearance of full length DBC1. This effect on SIRT1 activity was not observed in DBC1 KO mice. Finally, we found that DBC1 KO mice have altered cell cycle progression and liver regeneration after partial hepatectomy, suggesting that DBC1/DN-DBC1 transitions play a role in normal cell cycle progression in vivo after cells leave quiescence. We propose that quiescent cells express DN-DBC1, which either replaces or coexist with the full-length protein, and that restoring of DBC1 is required for normal cell cycle progression in vitro and in vivo. Our results describe for the first time in vivo a naturally occurring form of DBC1, which does not bind SIRT1 and is dynamically regulated, thus contributing to redefine the knowledge about its function.
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Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Técnicas de Inactivación de Genes , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Ciclo Celular/genética , Humanos , Regeneración Hepática/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Peso Molecular , Unión Proteica/genética , Dominios Proteicos , Proteolisis , Sirtuina 1/metabolismoRESUMEN
Cellular senescence is an endpoint of chemotherapy, and targeted therapies in melanoma and the senescence-associated secretory phenotype (SASP) can affect tumor growth and microenvironment, influencing treatment outcomes. Metabolic interventions can modulate the SASP, and an enhanced mitochondrial energy metabolism supports resistance to therapy in melanoma cells. Herein, we assessed the mitochondrial function of therapy-induced senescent melanoma cells obtained after exposing the cells to temozolomide (TMZ), a methylating chemotherapeutic agent. Senescence induction in melanoma was accompanied by a substantial increase in mitochondrial basal, ATP-linked, and maximum respiration rates and in coupling efficiency, spare respiratory capacity, and respiratory control ratio. Further examinations revealed an increase in mitochondrial mass and length. Alterations in mitochondrial function and morphology were confirmed in isolated senescent cells, obtained by cell-size sorting. An increase in mitofusin 1 and 2 (MFN1 and 2) expression and levels was observed in senescent cells, pointing to alterations in mitochondrial fusion. Silencing mitofusin expression with short hairpin RNA (shRNA) prevented the increase in mitochondrial length, oxygen consumption rate and secretion of interleukin 6 (IL-6), a component of the SASP, in melanoma senescent cells. Our results represent the first in-depth study of mitochondrial function in therapy-induced senescence in melanoma. They indicate that senescence increases mitochondrial mass, length and energy metabolism; and highlight mitochondria as potential pharmacological targets to modulate senescence and the SASP.
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Senescencia Celular , Metabolismo Energético , GTP Fosfohidrolasas/metabolismo , Melanoma Experimental/metabolismo , Mitocondrias/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , GTP Fosfohidrolasas/genética , Silenciador del Gen , Interleucina-6/genética , Interleucina-6/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , Mitocondrias/genética , Mitocondrias/patología , Dinámicas Mitocondriales/efectos de los fármacos , Dinámicas Mitocondriales/genética , Proteínas de Neoplasias/genética , Temozolomida/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: Atherosclerosis is characterized by chronic low-grade inflammation with concomitant lipid accumulation in the arterial wall. Anti-inflammatory and anti-atherogenic properties have been described for a novel class of endogenous nitroalkenes (nitrated-unsaturated fatty acids), formed during inflammation and digestion/absorption processes. The lipid-associated antioxidant α-tocopherol is transported systemically by LDL particles including to the atheroma lesions. To capitalize on the overlapping and complementary salutary properties of endogenous nitroalkenes and α-tocopherol, we designed and synthesized a novel nitroalkene-α-tocopherol analogue (NATOH) to address chronic inflammation and atherosclerosis, particularly at the lesion sites. EXPERIMENTAL APPROACH: We synthesized NATOH, determined its electrophilicity and antioxidant capacity and studied its effects over pro-inflammatory and cytoprotective pathways in macrophages in vitro. Moreover, we demonstrated its incorporation into lipoproteins and tissue both in vitro and in vivo, and determined its effect on atherosclerosis and inflammatory responses in vivo using the Apo E knockout mice model. KEY RESULTS: NATOH exhibited similar antioxidant capacity to α-tocopherol and, due to the presence of the nitroalkenyl group, like endogenous nitroalkenes, it exerted electrophilic reactivity. NATOH was incorporated in vivo into the VLDL/LDL lipoproteins particles to reach the atheroma lesions. Furthermore, oral administration of NATOH down-regulated NF-κB-dependent expression of pro-inflammatory markers (including IL-1ß and adhesion molecules) and ameliorated atherosclerosis in Apo E knockout mice. CONCLUSIONS AND IMPLICATIONS: In toto, the data demonstrate a novel pharmacological strategy for the prevention of atherosclerosis based on a creative, natural and safe drug delivery system of a non-conventional anti-inflammatory compound (NATOH) with significant potential for clinical application.
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Antiinflamatorios/farmacología , Antioxidantes/farmacología , Aterosclerosis/tratamiento farmacológico , Ciclopentanos/farmacología , Inflamación/tratamiento farmacológico , Nitrocompuestos/farmacología , alfa-Tocoferol/análogos & derivados , alfa-Tocoferol/farmacología , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/química , Antioxidantes/síntesis química , Antioxidantes/química , Aterosclerosis/metabolismo , Citocinas/antagonistas & inhibidores , Citocinas/metabolismo , Femenino , Inflamación/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Estructura Molecular , Células RAW 264.7RESUMEN
Inflammation plays a major role in the onset and development of chronic non-communicable diseases like obesity, cardiovascular diseases and cancer. Combined, these diseases represent the most common causes of death worldwide, thus development of novel pharmacological approaches is crucial. Electrophilic nitroalkenes derived from fatty acids are formed endogenously and exert anti-inflammatory actions by the modification of proteins involved in inflammation signaling cascades. We have developed novel nitroalkenes derived from α-tocopherol aiming to increase its salutary actions by adding anti-inflammatory properties to a well-known nutraceutical. We synthesized and characterized an α-tocopherol-nitroalkene (NATOH) and two hydrosoluble analogues derived from Trolox (NATxME and NATx0). We analyzed the kinetics of the Michael addition reaction of these compounds with thiols in micellar systems aiming to understand the effect of hydrophobic partition on the reactivity of nitroalkenes. We studied NATxME in vitro showing it exerts non-conventional anti-inflammatory responses by inducing Nrf2-Keap1-dependent gene expression and inhibiting the secretion of NF-κB dependent pro-inflammatory cytokines. NATxME was also effective in vivo, inhibiting neutrophil recruitment in a zebrafish model of inflammation. This work lays the foundation for the rational design of a new therapeutic strategy for the prevention and treatment of metabolic and inflammation-related diseases.
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Alquenos/síntesis química , Alquenos/farmacología , Antiinflamatorios/síntesis química , Antiinflamatorios/farmacología , Transducción de Señal , Tocoferoles/síntesis química , Tocoferoles/farmacología , Alquenos/química , Animales , Antiinflamatorios/química , Cromanos/síntesis química , Cromanos/química , Cromanos/farmacología , Cinética , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Micelas , Infiltración Neutrófila/efectos de los fármacos , Células RAW 264.7 , Tocoferoles/química , Pez CebraRESUMEN
Bardet-Biedl syndrome is a model ciliopathy. Although the characterization of BBS proteins has evidenced their involvement in cilia, extraciliary functions for some of these proteins are also being recognized. Importantly, understanding both cilia and cilia-independent functions of the BBS proteins is key to fully dissect the cellular basis of the syndrome. Here we characterize a functional interaction between BBS4 and the secreted protein FSTL1, a protein linked to adipogenesis and inflammation among other functions. We show that BBS4 and cilia regulate FSTL1 mRNA levels, but BBS4 also modulates FSTL1 secretion. Moreover, we show that FSTL1 is a novel regulator of ciliogenesis thus underscoring a regulatory loop between FSTL1 and cilia. Finally, our data indicate that BBS4, cilia and FSTL1 are coordinated during the differentiation of 3T3-L1 cells and that FSTL1 plays a role in this process, at least in part, by modulating ciliogenesis. Therefore, our findings are relevant to fully understand the development of BBS-associated phenotypes such as obesity.
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Diferenciación Celular/genética , Cilios/genética , Cilios/metabolismo , Proteínas Relacionadas con la Folistatina/biosíntesis , Proteínas Relacionadas con la Folistatina/genética , Regulación de la Expresión Génica , Proteínas/metabolismo , Células 3T3-L1 , Adipocitos/citología , Adipocitos/metabolismo , Adipogénesis/genética , Animales , Técnicas de Silenciamiento del Gen , Espacio Intracelular/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos , Proteínas/genéticaRESUMEN
Macrophages exert potent effector functions against invading microorganisms but constitute, paradoxically, a preferential niche for many bacterial strains to replicate. Using a model of infection by Salmonella Typhimurium, we have identified a molecular mechanism regulated by the nuclear receptor LXR that limits infection of host macrophages through transcriptional activation of the multifunctional enzyme CD38. LXR agonists reduced the intracellular levels of NAD+ in a CD38-dependent manner, counteracting pathogen-induced changes in macrophage morphology and the distribution of the F-actin cytoskeleton and reducing the capability of non-opsonized Salmonella to infect macrophages. Remarkably, pharmacological treatment with an LXR agonist ameliorated clinical signs associated with Salmonella infection in vivo, and these effects were dependent on CD38 expression in bone-marrow-derived cells. Altogether, this work reveals an unappreciated role for CD38 in bacterial-host cell interaction that can be pharmacologically exploited by activation of the LXR pathway.
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Receptores X del Hígado/metabolismo , Macrófagos/metabolismo , NAD/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Infecciones por Salmonella/metabolismo , Salmonella typhimurium/patogenicidad , ADP-Ribosil Ciclasa 1/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Femenino , Masculino , Ratones , Células RAW 264.7RESUMEN
Nicotinamide adenine dinucleotide (NAD) levels decrease during aging and are involved in age-related metabolic decline. To date, the mechanism responsible for the age-related reduction in NAD has not been elucidated. Here we demonstrate that expression and activity of the NADase CD38 increase with aging and that CD38 is required for the age-related NAD decline and mitochondrial dysfunction via a pathway mediated at least in part by regulation of SIRT3 activity. We also identified CD38 as the main enzyme involved in the degradation of the NAD precursor nicotinamide mononucleotide (NMN) in vivo, indicating that CD38 has a key role in the modulation of NAD-replacement therapy for aging and metabolic diseases.