RESUMEN
INTRODUCTION: Patients with hepatocyte nuclear factor-1 beta (HNF1B) mutations present a variable phenotype with two main symptoms: maturity onset diabetes of the young (MODY) and polycystic kidney disease (PKD). OBJECTIVES: Identification of serum metabolites specific for HNF1Bmut and evaluation of their role in disease pathogenesis. METHODS: We recruited patients with HNF1Bmut (N = 10), HNF1Amut (N = 10), PKD: non-dialyzed and dialyzed (N = 8 and N = 13); and healthy controls (N = 12). Serum fingerprinting was performed by LC-QTOF-MS. Selected metabolite was validated by ELISA (enzyme-linked immunosorbent assay) measurements and then biologically connected with HNF1B by in silico analysis. HepG2 were stimulated with lysophosphatidic acid (LPA) and HNF1B gene was knocked down (kd) by small interfering RNA. Transcriptomic analysis with microarrays and western blot measurements were performed. RESULTS: Serum levels of six metabolites including: arachidonic acid, hydroxyeicosatetraenoic acid, linoleamide and three LPA (18:1, 18:2 and 20:4), had AUC (the area under the curve) > 0.9 (HNF1Bmut vs comparative groups). The increased level of LPA was confirmed by ELISA measurements. In HepG2HNF1Bkd cells LPA stimulation lead to downregulation of many pathways associated with cell cycle, lipid metabolism, and upregulation of steroid hormone metabolism and Wnt signaling. Also, increased intracellular protein level of autotaxin was detected in the cells. GSK-3alpha/beta protein level and its phosphorylated ratio were differentially affected by LPA stimulation in HNF1Bkd and control cells. CONCLUSIONS: LPA is elevated in sera of patients with HNF1Bmut. LPA contributes to the pathogenesis of HNF1B-MODY by affecting Wnt/GSK-3 signaling.
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Glucógeno Sintasa Quinasa 3 , Enfermedades Renales Quísticas , Glucógeno Sintasa Quinasa 3/genética , Factor Nuclear 1-beta del Hepatocito/genética , Humanos , Lisofosfolípidos , Metabolómica , Mutación/genéticaRESUMEN
Cells sustain constant oxidative stress from both exogenous and endogenous sources. When unmitigated by antioxidant defenses, reactive oxygen species damage cellular macromolecules, including DNA. Oxidative lesions in both nuclear and mitochondrial DNA are repaired via the base excision repair (BER) pathway, initiated by DNA glycosylases. We have previously demonstrated that the BER glycosylase 8-oxoguanine DNA glycosylase (OGG1) plays a novel role in body weight maintenance and regulation of adiposity. Specifically, mice lacking OGG1 (Ogg1-/-) are prone to increased fat accumulation with age and consumption of hypercaloric diets. Conversely, transgenic animals with mitochondrially-targeted overexpression of OGG1 (Ogg1Tg) are resistant to age- and diet-induced obesity. Given these phenotypes of altered adiposity in the context of OGG1 genotype, we sought to determine if OGG1 plays a cell-intrinsic role in adipocyte maturation and lipid accumulation. Here, we report that preadipocytes from Ogg1-/- mice differentiate more efficiently and accumulate more lipids than those from wild-type animals. Conversely, OGG1 overexpression significantly blunts adipogenic differentiation and lipid accretion in both pre-adipocytes from Ogg1Tg mice, as well as in 3T3-L1 cells with adenovirus-mediated OGG1 overexpression. Mechanistically, changes in adipogenesis are accompanied by significant alterations in cellular PARylation, corresponding with OGG1 genotype. Specifically, deletion of OGG1 reduces protein PARylation, concomitant with increased adipogenic differentiation, while OGG1 overexpression significantly increases PARylation and blunts adipogenesis. Collectively, these data indicate a novel role for OGG1 in modulating adipocyte differentiation and lipid accretion. These findings have important implications to our knowledge of the fundamental process of adipocyte differentiation, as well as to our understanding of lipid-related diseases such as obesity.
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Adipogénesis , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Reparación del ADN , Células 3T3-L1 , Adipocitos/citología , Adipocitos/metabolismo , Animales , Diferenciación Celular , Regulación de la Expresión Génica , Metabolismo de los Lípidos , Ratones , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Due to the limited number of organ donors, 3D printing of organs is a promising technique. Tissue engineering is increasingly using xenogeneic material for this purpose. This study was aimed at assessing the safety of decellularized porcine pancreas, together with the analysis of the risk of an undesirable immune response. We tested eight variants of the decellularization process. We determined the following impacts: rinsing agents (PBS/NH3·H2O), temperature conditions (4 °C/24 °C), and the grinding method of native material (ground/cut). To assess the quality of the extracellular matrix after the completed decellularization process, analyses of the following were performed: DNA concentration, fat content, microscopic evaluation, proteolysis, material cytotoxicity, and most importantly, the Triton X-100 content. Our analyses showed that we obtained a product with an extremely low detergent content with negligible residual DNA content. The obtained results confirmed the performed histological and immuno-fluorescence staining. Moreover, the TEM microscopic analysis proved that the correct collagen structure was preserved after the decellularization process. Based on the obtained results, we chose the most favorable variant in terms of quality and biology. The method we chose is an effective and safe method that gives a chance for the development of transplant and regenerative medicine.
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Matriz Extracelular/fisiología , Páncreas/ultraestructura , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Bioimpresión/métodos , Células Cultivadas , Detergentes/química , Detergentes/farmacología , Matriz Extracelular/química , Fibroblastos/citología , Fibroblastos/fisiología , Ensayo de Materiales , Ratones , Octoxinol/química , Octoxinol/farmacología , Páncreas/citología , Polvos/química , Impresión Tridimensional , Proteómica , Control de Calidad , Porcinos , Ingeniería de Tejidos/normas , Andamios del Tejido/química , Andamios del Tejido/normasRESUMEN
The progression of non-alcoholic fatty liver (NAFL) into non-alcoholic steatohepatitis implicates multiple mechanisms, chief of which is mitochondrial dysfunction. However, the sequence of events underlying mitochondrial failure are still poorly clarified. In this work, male C57BL/6J mice were fed with a high-fat plus high-sucrose diet for 16, 20, 22, and 24 weeks to induce NAFL. Up to the 20th week, an early mitochondrial remodeling with increased OXPHOS subunits levels and higher mitochondrial respiration occurred. Interestingly, a progressive loss of mitochondrial respiration along "Western diet" feeding was identified, accompanied by higher susceptibility to mitochondrial permeability transition pore opening. Importantly, our findings prove that mitochondrial alterations and subsequent impairment are independent of an excessive mitochondrial reactive oxygen species (ROS) generation, which was found to be progressively diminished along with disease progression. Instead, increased peroxisomal abundance and peroxisomal fatty acid oxidation-related pathway suggest that peroxisomes may contribute to hepatic ROS generation and oxidative damage, which may accelerate hepatic injury and disease progression. We show here for the first time the sequential events of mitochondrial alterations involved in non-alcoholic fatty liver disease (NAFLD) progression and demonstrate that mitochondrial ROS are not one of the first hits that cause NAFLD progression.
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Mitocondrias/metabolismo , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Antioxidantes/metabolismo , Autofagia , Ésteres del Colesterol/metabolismo , Biología Computacional/métodos , Susceptibilidad a Enfermedades , Fibrosis , Hepatocitos/metabolismo , Metabolismo de los Lípidos , Hígado/metabolismo , Masculino , Ratones , Mitocondrias/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Oxidación-Reducción , Estrés Oxidativo , Triglicéridos/metabolismoRESUMEN
Stearoyl-CoA desaturase (SCD) is a rate-limiting enzyme that catalyzes the synthesis of monounsaturated fatty acids. It plays an important role in regulating skeletal muscle metabolism. Lack of the SCD1 gene increases the rate of fatty acid ß-oxidation through activation of the AMP-activated protein kinase (AMPK) pathway and the upregulation of genes that are related to fatty acid oxidation. The mechanism of AMPK activation under conditions of SCD1 deficiency has been unclear. In the present study, we found that the ablation/inhibition of SCD1 led to AMPK activation in skeletal muscle through an increase in AMP levels whereas muscle-specific SCD1 overexpression decreased both AMPK phosphorylation and the adenosine monophosphate/adenosine triphosphate (AMP/ATP) ratio. Changes in AMPK phosphorylation that were caused by SCD1 down- and upregulation affected NAD+ levels following changes in NAD+ -dependent deacetylase sirtuin-1 (SIRT1) activity and histone 3 (H3K9) acetylation and methylation status. Moreover, mice with muscle-targeted overexpression of SCD1 were more susceptible to high-fat diet-induced lipid accumulation and the development of insulin resistance compared with wild-type mice. These data show that SCD1 is involved in nucleotide (ATP and NAD+ ) metabolism and suggest that the SCD1-dependent regulation of muscle steatosis and insulin sensitivity are mediated by cooperation between AMPK- and SIRT1-regulated pathways. Altogether, the present study reveals a novel mechanism that links SCD1 with the maintenance of metabolic homeostasis and insulin sensitivity in skeletal muscle.
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Proteínas Quinasas Activadas por AMP/metabolismo , Nucleótidos de Adenina/metabolismo , Histonas/metabolismo , Músculo Esquelético/metabolismo , Sirtuina 1/metabolismo , Estearoil-CoA Desaturasa/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Acetilación , Animales , Línea Celular , Dieta Alta en Grasa , Regulación hacia Abajo , Regulación de la Expresión Génica , Histonas/genética , Humanos , Resistencia a la Insulina , Masculino , Ratones , Ratones Noqueados , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Sirtuina 1/genética , Estearoil-CoA Desaturasa/genéticaRESUMEN
Metabolic stress, such as lipotoxicity, affects the DNA methylation profile in pancreatic ß-cells and thus contributes to ß-cell failure and the progression of type 2 diabetes (T2D). Stearoyl-CoA desaturase 1 (SCD1) is a rate-limiting enzyme that is involved in monounsaturated fatty acid synthesis, which protects pancreatic ß-cells against lipotoxicity. The present study found that SCD1 is also required for the establishment and maintenance of DNA methylation patterns in ß-cells. We showed that SCD1 inhibition/deficiency caused DNA hypomethylation and changed the methyl group distribution within chromosomes in ß-cells. Lower levels of DNA methylation in SCD1-deficient ß-cells were followed by lower levels of DNA methyltransferase 1 (DNMT1). We also found that the downregulation of SCD1 in pancreatic ß-cells led to the activation of adenosine monophosphate-activated protein kinase (AMPK) and an increase in the activity of the NAD-dependent deacetylase sirtuin-1 (SIRT1). Furthermore, the physical association between DNMT1 and SIRT1 stimulated the deacetylation of DNMT1 under conditions of SCD1 inhibition/downregulation, suggesting a mechanism by which SCD1 exerts control over DNMT1. We also found that SCD1-deficient ß-cells that were treated with compound c, an inhibitor of AMPK, were characterized by higher levels of both global DNA methylation and DNMT1 protein expression compared with untreated cells. Therefore, we found that activation of the AMPK/SIRT1 signaling pathway mediates the effect of SCD1 inhibition/deficiency on DNA methylation status in pancreatic ß-cells. Altogether, these findings suggest that SCD1 is a gatekeeper that protects ß-cells against the lipid-derived loss of DNA methylation and provide mechanistic insights into the mechanism by which SCD1 regulates DNA methylation patterns in ß-cells and T2D-relevant tissues.
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ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN , Células Secretoras de Insulina/metabolismo , Estearoil-CoA Desaturasa/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/metabolismo , Acetilación , Animales , Línea Celular , Metilación de ADN/efectos de los fármacos , Regulación hacia Abajo , Silenciador del Gen , Histonas/metabolismo , Células Secretoras de Insulina/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Interferente Pequeño , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sirtuina 1/metabolismo , Espectrometría Raman , Estearoil-CoA Desaturasa/antagonistas & inhibidores , Estearoil-CoA Desaturasa/genética , Regulación hacia ArribaRESUMEN
Endocannabinoids are implicated in the control of glucose utilization and energy homeostasis by orchestrating pancreatic hormone release. Moreover, in some cell niches, endocannabinoids regulate cell proliferation, fate determination, and migration. Nevertheless, endocannabinoid contributions to the development of the endocrine pancreas remain unknown. Here, we show that α cells produce the endocannabinoid 2-arachidonoylglycerol (2-AG) in mouse fetuses and human pancreatic islets, which primes the recruitment of ß cells by CB1 cannabinoid receptor (CB1R) engagement. Using subtractive pharmacology, we extend these findings to anandamide, a promiscuous endocannabinoid/endovanilloid ligand, which impacts both the determination of islet size by cell proliferation and α/ß cell sorting by differential activation of transient receptor potential cation channel subfamily V member 1 (TRPV1) and CB1Rs. Accordingly, genetic disruption of TRPV1 channels increases islet size whereas CB1R knockout augments cellular heterogeneity and favors insulin over glucagon release. Dietary enrichment in ω-3 fatty acids during pregnancy and lactation in mice, which permanently reduces endocannabinoid levels in the offspring, phenocopies CB1R(-/-) islet microstructure and improves coordinated hormone secretion. Overall, our data mechanistically link endocannabinoids to cell proliferation and sorting during pancreatic islet formation, as well as to life-long programming of hormonal determinants of glucose homeostasis.
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Endocannabinoides/metabolismo , Islotes Pancreáticos/embriología , Morfogénesis/fisiología , Receptor Cannabinoide CB1/metabolismo , Canales Catiónicos TRPV/metabolismo , Análisis de Varianza , Animales , Ácidos Grasos Omega-3/administración & dosificación , Femenino , Feto/metabolismo , Prueba de Tolerancia a la Glucosa , Procesamiento de Imagen Asistido por Computador , Islotes Pancreáticos/anatomía & histología , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , EmbarazoRESUMEN
Type 2 diabetes (T2D) is a complex disorder that is caused by a combination of genetic, epigenetic, and environmental factors. ß-cell failure and insulin resistance in peripheral tissues that are induced by lipid overload are main hallmarks of T2D. The mechanisms that link obesity-driven alterations of lipid metabolism and T2D are still elusive, thereby impeding the development of effective prevention and treatment strategies. Although genetic variants that have been identified in high-throughput studies comprise an appreciable proportion of the genetic component of T2D, they explain < 20% of the estimated heritability of T2D. A growing body of evidence suggests an intrinsic role for epigenetic modifications in the pathogenesis of T2D. The epigenetic regulation of gene expression in tissues that play a key role in the obesity-related development of T2D has been demonstrated, including PDX1 in pancreatic islets, PPARGC1A in skeletal muscles, ADIPOQ in adipose tissue, and TXNIP in the liver. The present review summarizes our current knowledge of crosstalk between the epigenetic control of gene expression, particularly via DNA methylation, toxic lipid mediators, and the pathogenesis of obesity-related T2D.
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Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/genética , Epigénesis Genética , Obesidad/complicaciones , Obesidad/genética , Adiponectina/metabolismo , Proteínas Portadoras/metabolismo , Metilación de ADN , Proteínas de Homeodominio/metabolismo , Humanos , Resistencia a la Insulina , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Transactivadores/metabolismoRESUMEN
During long-standing obesity and insulin resistance, pancreatic ß-cells adapt in order to meet the growing demand of the periphery for insulin. The development of type 2 diabetes requires parallel pathological processes, during which ß-cells are continuously exposed to an overabundant supply of specific lipid derivatives. The metabolic events that lead to inevitable ß-cell damage are not completely uncovered, and for the time being, our understanding of the dynamic endothelium-adipose tissue-ß-cell interactions is limited. Here, we explore various links between continuous obesity, adipose tissue spillover, a dysfunctional endothelium, and defects in islet angioarchitecture to elucidate the crosstalk between signaling systems, cellular mediators, and cell types that contribute to ß-cell failure through diverse actions of fatty acids. These molecular and biochemical mechanisms initiate critical rearrangements of the pancreatic vasculature, intraorgan lipid storage capacity, and inflammatory status that subsequently have severe repercussions on ß-cell function and promote diabetes.
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Tejido Adiposo/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Endotelio/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Obesidad/complicaciones , Diabetes Mellitus Tipo 2/patología , Endotelio/patología , Humanos , Resistencia a la Insulina , Obesidad/metabolismoRESUMEN
Studying organelles in isolation has been proven to be indispensable for deciphering the underlying mechanisms of molecular cell biology. However, observing organelles in intact cells with the use of microscopic techniques reveals a new set of different junctions and contact sites between them that contribute to the control and regulation of various cellular processes, such as calcium and lipid exchange or structural reorganization of the mitochondrial network. In recent years, many studies focused their attention on the structure and function of contacts between mitochondria and other organelles. From these studies, findings emerged showing that these contacts are involved in various processes, such as lipid synthesis and trafficking, modulation of mitochondrial morphology, endoplasmic reticulum (ER) stress, apoptosis, autophagy, inflammation and Ca 2 + handling. In this review, we focused on the physical interactions of mitochondria with the endoplasmic reticulum and plasma membrane and summarized present knowledge regarding the role of mitochondria-associated membranes in calcium homeostasis and lipid metabolism.
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Calcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Homeostasis , Metabolismo de los Lípidos , Mitocondrias/metabolismo , Animales , Apoptosis , Transporte Biológico , Membrana Celular/ultraestructura , Susceptibilidad a Enfermedades , Retículo Endoplásmico/ultraestructura , Humanos , Mitocondrias/ultraestructura , Dinámicas Mitocondriales , Transporte de ProteínasRESUMEN
AIMS/HYPOTHESIS: We aimed to identify microRNAs (miRNAs) under transcriptional control of the HNF1ß transcription factor, and investigate whether its effect manifests in serum. METHODS: The Polish cohort (N = 60) consisted of 11 patients with HNF1B-MODY, 17 with HNF1A-MODY, 13 with GCK-MODY, an HbA1c-matched type 1 diabetic group (n = 9) and ten healthy controls. Replication was performed in 61 clinically-matched British patients mirroring the groups in the Polish cohort. The Polish cohort underwent miRNA serum level profiling with quantitative real-time PCR (qPCR) arrays to identify differentially expressed miRNAs. Validation was performed using qPCR. To determine whether serum content reflects alterations at a cellular level, we quantified miRNA levels in a human hepatocyte cell line (HepG2) with small interfering RNA knockdowns of HNF1α or HNF1ß. RESULTS: Significant differences (adjusted p < 0.05) were noted for 11 miRNAs. Five of them differed between HNF1A-MODY and HNF1B-MODY, and, amongst those, four (miR-24, miR-27b, miR-223 and miR-199a) showed HNF1B-MODY-specific expression levels in the replication group. In all four cases the miRNA expression level was lower in HNF1B-MODY than in all other tested groups. Areas under the receiver operating characteristic curves ranged from 0.79 to 0.86, with sensitivity and specificity reaching 91.7% (miR-24) and 82.1% (miR-199a), respectively. The cellular expression pattern of miRNA was consistent with serum levels, as all were significantly higher in HNF1α- than in HNF1ß-deficient HepG2 cells. CONCLUSIONS/INTERPRETATION: We have shown that expression of specific miRNAs depends on HNF1ß function. The impact of HNF1ß deficiency was evidenced at serum level, making HNF1ß-dependent miRNAs potentially applicable in the diagnosis of HNF1B-MODY.
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Factor Nuclear 1-alfa del Hepatocito/metabolismo , Factor Nuclear 1-beta del Hepatocito/metabolismo , MicroARNs/sangre , MicroARNs/genética , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Hemoglobina Glucada/genética , Hemoglobina Glucada/metabolismo , Células Hep G2 , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 1-beta del Hepatocito/genética , Humanos , ARN Interferente Pequeño/genética , Curva ROC , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Ventricular arrhythmias are an important cause of mortality in the acute myocardial infarction (MI). To elucidate the effect of the omega-3 polyunsaturated fatty acids (PUFAs) on ventricular arrhythmias in acute nonreperfused MI, rats were fed with normal or eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA)-enriched diet for 3 weeks. Subsequently the rats were subjected to either MI induction or sham operation. ECG was recorded for 6 h after the operation and episodes of ventricular tachycardia/fibrillation (VT/VF) were identified. Six hours after MI epicardial monophasic action potentials (MAPs) were recorded, cardiomyocyte Ca(2+) handling was assessed and expression of proteins involved in Ca(2+) turnover was studied separately in non-infarcted left ventricle wall and infarct borderzone. EPA and DHA had no effect on occurrence of post-MI ventricular arrhythmias or mortality. Nevertheless, DHA but not EPA prevented Ca(2+) overload in LV cardiomiocytes and improved rate of Ca(2+) transient decay, protecting PMCA and SERCA function. Moreover, both EPA and DHA prevented MI-induced hyperphosphorylation of ryanodine receptors (RyRs) as well as dispersion of action potential duration (APD) in the left ventricular wall. In conclusion, EPA and DHA have no antiarrhythmic effect in the non-reperfused myocardial infarction in the rat, although these omega-3 PUFAs and DHA in particular exhibit several potential antiarrhythmic effects at the subcellular and tissue level, that is, prevent MI-induced abnormalities in Ca(2+) handling and APD dispersion. In this context further studies are needed to see if these potential antiarrhythmic effects could be utilized in the clinical setting. J. Cell. Biochem. 117: 2570-2582, 2016. © 2016 Wiley Periodicals, Inc.
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Arritmias Cardíacas/tratamiento farmacológico , Ácidos Docosahexaenoicos/farmacología , Ácido Eicosapentaenoico/farmacología , Corazón/efectos de los fármacos , Infarto del Miocardio/complicaciones , Enfermedad Aguda , Animales , Arritmias Cardíacas/etiología , Arritmias Cardíacas/patología , Células Cultivadas , Masculino , Sustancias Protectoras/farmacología , Ratas , Ratas Endogámicas WKYRESUMEN
Autophagy is indispensable for the proper architecture and flawless functioning of pancreatic ß-cells. A growing body of evidence indicates reciprocal communication between autophagic pathways, apoptosis, and intracellular lipids. The way in which elevated levels of free saturated or unsaturated FAs contribute to progressive ß-cell failure remains incompletely understood. Stearoyl-CoA desaturase (SCD)1, a key regulatory enzyme in biosynthesis of MUFAs, was shown to play an important role in regulation of ß-cell function. Here, we investigated whether SCD1 activity is engaged in palmitate-induced pancreatic ß-cell autophagy. We found augmented apoptosis and diminished autophagy upon cotreatment of INS-1E cells with palmitate and an SCD1 inhibitor. Furthermore, we found that additional treatment of the cells with monensin, an inhibitor of autophagy at the step of fusion, exacerbates palmitate-induced apoptosis. Accordingly, diminished SCD1 activity affected the accumulation, composition, and saturation status of cellular membrane phospholipids and neutral lipids. Such an effect was accompanied by aberrant endoplasmic reticulum stress, mitochondrial injury, and decreases in insulin secretion and cell proliferation. Our data reveal a novel mechanism by which the inhibition of SCD1 activity affects autophagosome-lysosome fusion because of perturbations in cellular membrane integrity, thus leading to an aberrant stress response and ß-cell failure.
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Autofagia/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Palmitatos/farmacología , Estearoil-CoA Desaturasa/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Insulina/farmacología , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/enzimología , Células Secretoras de Insulina/metabolismo , Insulinoma , Lisosomas/metabolismo , Fusión de Membrana/efectos de los fármacos , Ácido Palmítico/farmacología , Fosfolípidos/metabolismo , Ratas , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismoRESUMEN
Obesity-related type 2 diabetes develops in individuals with the onset of ß-cell dysfunction. Pancreatic islet lipotoxicity is now recognized as a primary reason for the onset and progression of the disease. Such dysfunction is reflected by the aberrant secretory capacity and detrimental loss of ß-cell mass and survival. Elevated circulating serum fatty acid levels and disordered lipid metabolism management are particularly interesting in the search for biologically relevant triggers of ß-cell demise. Herein, we review various types of toxic lipid metabolites that may play a significant role in pancreatic islet failure. The lipotoxic effect on ß-cells depends on the type of lipid mediator (e.g., long-chain fatty acids, diacylglycerols, ceramides, phospholipids), cellular location of its action (e.g., endoplasmic reticulum, mitochondria), and associated-organelle conditions (e.g., membranes, vesicles). We also discuss various aspects of lipid action in ß-cells, including effects on metabolic pathways, stress responses (e.g., oxidative stress, endoplasmic reticulum stress, and autophagy), and gene expression.
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Diabetes Mellitus Tipo 2/fisiopatología , Islotes Pancreáticos/fisiopatología , Autofagia , Ceramidas/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/fisiología , Humanos , Mitocondrias/fisiologíaRESUMEN
Endocannabinoid signaling has been implicated in modulating insulin release from ß cells of the endocrine pancreas. ß Cells express CB1 cannabinoid receptors (CB1Rs), and the enzymatic machinery regulating anandamide and 2-arachidonoylglycerol bioavailability. However, the molecular cascade coupling agonist-induced cannabinoid receptor activation to insulin release remains unknown. By combining molecular pharmacology and genetic tools in INS-1E cells and in vivo, we show that CB1R activation by endocannabinoids (anandamide and 2-arachidonoylglycerol) or synthetic agonists acutely or after prolonged exposure induces insulin hypersecretion. In doing so, CB1Rs recruit Akt/PKB and extracellular signal-regulated kinases 1/2 to phosphorylate focal adhesion kinase (FAK). FAK activation induces the formation of focal adhesion plaques, multimolecular platforms for second-phase insulin release. Inhibition of endocannabinoid synthesis or FAK activity precluded insulin release. We conclude that FAK downstream from CB1Rs mediates endocannabinoid-induced insulin release by allowing cytoskeletal reorganization that is required for the exocytosis of secretory vesicles. These findings suggest a mechanistic link between increased circulating and tissue endocannabinoid levels and hyperinsulinemia in type 2 diabetes.
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Exocitosis , Quinasa 1 de Adhesión Focal/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Receptor Cannabinoide CB1/metabolismo , Vesículas Secretoras/metabolismo , Animales , Ácidos Araquidónicos/farmacología , Agonistas de Receptores de Cannabinoides/farmacología , Línea Celular , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Endocannabinoides/genética , Endocannabinoides/metabolismo , Endocannabinoides/farmacología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Quinasa 1 de Adhesión Focal/genética , Glicéridos/farmacología , Humanos , Hiperinsulinismo/genética , Hiperinsulinismo/metabolismo , Hiperinsulinismo/patología , Insulina/genética , Secreción de Insulina , Ratones , Ratones Noqueados , Alcamidas Poliinsaturadas/farmacología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/genética , Vesículas Secretoras/genéticaRESUMEN
Investigating the potential of human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) in in vitro heart models is essential to develop cardiac regenerative medicine. iPSC-CMs are immature with a fetal-like phenotype relative to cardiomyocytes in vivo. Literature indicates methods for enhancing the structural maturity of iPSC-CMs. Among these strategies, nanofibrous scaffolds offer more accurate mimicry of the functioning of cardiac tissue structures in the human body. However, further research is needed on the use of nanofibrous mats to understand their effects on iPSC-CMs. Our research aimed to evaluate the suitability of poly(ε-caprolactone) (PCL) and polyurethane (PU) nanofibrous mats with different elasticities as materials for the maturation of iPSC-CMs. Analysis of cell morphology and orientation and the expression levels of selected genes and proteins were performed to determine the effect of the type of nanofibrous mats on the maturation of iPSC-CMs after long-term (10-day) culture. Understanding the impact of 3D structural properties in in vitro cardiac models on induced pluripotent stem cell-derived cardiomyocyte maturation is crucial for advancing cardiac tissue engineering and regenerative medicine because it can help optimize conditions for obtaining more mature and functional human cardiomyocytes.
Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Nanofibras , Poliésteres , Poliuretanos , Andamios del Tejido , Humanos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Poliuretanos/química , Poliésteres/química , Nanofibras/química , Diferenciación Celular/efectos de los fármacos , Andamios del Tejido/química , Ingeniería de Tejidos/métodos , Células CultivadasRESUMEN
Cardiac hypertrophy is accompanied by molecular remodeling that affects different cellular pathways, including fatty acid (FA) utilization. In the present study, we show that cardiac lipid metabolism is differentially regulated in response to physiological (endurance training) and pathological [abdominal aortic banding (AAB)] hypertrophic stimuli. Physiological hypertrophy was accompanied by an increased expression of lipogenic genes and the activation of sterol regulatory element-binding protein-1c and Akt signaling. Additionally, FA oxidation pathways regulated by AMP-activated protein kinase (AMPK) and peroxisome proliferator activated receptor-α (PPARα) were induced in trained hearts. Cardiac lipid content was not changed by physiological stimulation, underlining balanced lipid utilization in the trained heart. Moreover, pathological hypertrophy induced the AMPK-regulated oxidative pathway, whereas PPARα and expression of its downstream targets, i.e., acyl-CoA oxidase and carnitine palmitoyltransferase I, were not affected by AAB. In contrast, pathological hypertrophy leads to cardiac triglyceride (TG) and diacylglycerol (DAG) accumulation, although the expression of lipogenic genes and the levels of FA transport proteins (CD36 and FATP) were not changed or reduced compared with the sham group. A possible explanation for this phenomenon is a decrease in lipolysis, as evidenced by the increased content of adipose triglyceride lipase inhibitor G0S2, the increased phosphorylation of hormone-sensitive lipase at Ser(565), and the decreased protein levels of DAG lipase that attenuate TG and DAG contents. The increased TG and DAG accumulation observed in AAB-induced hypertrophy might have lipotoxic effects, thereby predisposing to cardiomyopathy and heart failure in the future.
Asunto(s)
Corazón/fisiología , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/fisiopatología , Lipogénesis/genética , Condicionamiento Físico Animal/fisiología , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica/fisiología , Hipertrofia Ventricular Izquierda/diagnóstico por imagen , Lipasa/genética , Lipasa/metabolismo , Lipogénesis/fisiología , Masculino , PPAR alfa/genética , PPAR alfa/metabolismo , Resistencia Física/fisiología , Distribución Aleatoria , Ratas , Ratas Wistar , Ultrasonografía , Regulación hacia Arriba/fisiologíaRESUMEN
We previously showed that knockdown of the anaplerotic enzyme pyruvate carboxylase in the INS-1 832/13 insulinoma cell line inhibited glucose-stimulated insulin release and glucose carbon incorporation into lipids. We now show that knockdown of fatty acid synthase (FAS) mRNA and protein also inhibits glucose-stimulated insulin release in this cell line. Levels of numerous phospholipids, cholesterol esters, diacylglycerol, triglycerides and individual fatty acids with C14-C24 side chains were acutely lowered about 20% in glucose-stimulated pyruvate carboxylase knockdown cells over a time course that coincides with insulin secretion. In FAS knockdown cells glucose carbon incorporation into lipids and the levels of the subclasses of phospholipids and cholesterol ester species were lower by 20-30% without inhibition of glucose oxidation. These studies suggest that rapid lipid modification is essential for normal glucose-stimulated insulin secretion.
Asunto(s)
Ácido Graso Sintasas/genética , Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Metabolismo de los Lípidos , Piruvato Carboxilasa/genética , Animales , Línea Celular Tumoral , Ácido Graso Sintasas/metabolismo , Técnicas de Silenciamiento del Gen , Insulinoma/metabolismo , Piruvato Carboxilasa/metabolismo , RatasRESUMEN
Protein kinase C (PKC) activation induced by diacylglycerols (DAGs) is one of the sequels of the dysregulation of intramuscular lipid metabolism and is thought to play an important role in the development of insulin resistance (IR). We tested the hypothesis that DAGs with different acyl chains have different biological effects and that DAG species enriched in monounsaturated fatty acids (MUFA) act as better activators of PKC. The experiments were performed in vitro on C2C12 myotubes treated with palmitate (16:0), stearate (18:0) or oleate (18:1) and in vivo on the skeletal muscles of rats fed high-fat (HF), high-tristearin (TS) or high-triolein (TO) diets. To define the importance of endogenously synthesized MUFA on DAG-induced PKCθ activation, we performed experiments on stearoyl-CoA desaturase 1 knockout mice (SCD1-/-) as well. The results show that the content of total DAGs and the levels of saturated DAG species are significantly increased in both insulin-resistant (16:0, HF and TO) and highly insulin-sensitive (18:0 and TS) groups. An increase in MUFA-containing DAGs levels was most constantly related to increase in PKCθ membrane translocation and IR. In the muscles of MUFA-deficient SCD1-/- mice, the DAG content and the induction of PKCθ translocation by the HF diet were significantly reduced. Collectively, our data from both the cell and animal experiments show that DAGs composed of 16:1 and/or 18:1, rather than the levels of total or saturated DAGs, are related to PKCθ membrane translocation. Moreover, our results show that the availability of dietary MUFA and/or the activity of endogenous desaturases play an important role in muscle DAG accumulation.
Asunto(s)
Diglicéridos/metabolismo , Ácidos Grasos Monoinsaturados/metabolismo , Isoenzimas/metabolismo , Trastornos del Metabolismo de los Lípidos/metabolismo , Músculo Esquelético/metabolismo , Proteína Quinasa C/metabolismo , Animales , Línea Celular , Grasas de la Dieta/efectos adversos , Grasas de la Dieta/farmacología , Diglicéridos/genética , Ácidos Grasos Monoinsaturados/farmacología , Isoenzimas/genética , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Trastornos del Metabolismo de los Lípidos/genética , Trastornos del Metabolismo de los Lípidos/patología , Masculino , Ratones , Ratones Noqueados , Músculo Esquelético/patología , Proteína Quinasa C/genética , Proteína Quinasa C-theta , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Ratas , Ratas Wistar , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismoRESUMEN
The Wnt signaling pathway plays an important role in morphogenesis, differentiation, cell survival and proliferation. Wnt activators are secreted proteins that work in an auto-, para- and endocrine manner and their synthesis, secretion and transport are tightly regulated. Frizzled/LRP is the main receptor complex in the canonical Wnt pathway. Its activation triggers ß-catenin translocation to the nucleus and increases activity of TCF transcription factor. Disruption in Wnt signaling has been found in many pathophysiological states such as different types of cancer, neurodegenerative diseases and metabolic disorders. Recent studies revealed the important role of Wnt signaling in maintaining carbohydrate and lipid homeostasis. Activation of the Frizzled/LRP receptor complex leads to increase in the activity of transcription factors and nuclear receptors that regulate expression of genes involved in lipid utilization (PPARδ, RAR, LXR) and inhibits adipogenesis. The Wnt signaling pathway is also involved in the regulation of gluconeogenesis and glycolysis. This review summarizes the current state of knowledge about mechanisms that regulate canonical Wnt signaling and its role in cell metabolism regulation.