RESUMEN
Mitochondria, similar to living cells and organelles, have a negative membrane potential, which ranges between (-108) and (150) mV as compared to (-70) and (-90) mV of the plasma membrane. Therefore, permeable lipophilic cations tend to accumulate in the mitochondria. Those cations which exhibit fluorescence activity after accumulation into energized systems are widely used to decipher changes in membrane potential by imaging techniques. Here we describe the use of two different dyes for labeling mitochondrial membrane potential (Δψm) in live cells. One is the lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocyanine iodide (JC-1), which alters reversibly its color from green (J-monomer, at its low concentration in the cytosol) to red (J-aggregates, at its high concentration in active mitochondria) with increasing mitochondrial membrane potential (Δψm). The other is MitoTracker® Orange, a mitochondrion-selective probe which passively diffuses across the plasma membrane and accumulates in active mitochondria depending on their Δψm. We show that in addition to changes in Δψm, these specific dyes can be used to follow alterations in mitochondrial distribution and mitochondrial network connectivity. We suggest that JC-1 is a preferable probe to compare between different cell types and cell state, as a red to green ratio of fluorescence intensities is used for analysis. This ratio depends only on the mitochondrial membrane potential and not on other cellular and/or mitochondrial-dependent or independent factors that may alter, for example, due to treatment or disease state. However, in cells labeled either with green or red fluorescence protein, JC-1 cannot be used. Therefore, other dyes are preferable. We demonstrate various applications of JC-1 and MitoTracker Orange staining to study mitochondrial abnormalities in different cell types derived from schizophrenia patients and healthy subjects.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Esquizofrenia/metabolismo , Bencimidazoles/química , Carbocianinas/química , Técnicas de Cultivo de Célula , Fibroblastos/metabolismo , Fibroblastos/patología , Colorantes Fluorescentes/química , Humanos , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Mitocondrias/patología , Prueba de Estudio Conceptual , Esquizofrenia/patología , Xantenos/químicaRESUMEN
Exocrine pancreas insufficiency is common in diabetic mellitus (DM) patients. Cellular stress is a prerequisite in the development of pancreatic pathologies such as acute pancreatitis (AP). The molecular mechanisms underlying exocrine pancreatic ER-stress in DM are largely unknown. We studied the effects of insulin and glucose (related to DM) alone and in combination with cerulein (CER)-induced stress (mimicking AP) on ER-stress unfolded protein response (UPR) in pancreatic acinar cells. Exocrine pancreas cells (AR42J) were exposed to high glucose (Glu, 25â¯mM) and insulin (Ins, 100â¯nM) levels with or without CER (10â¯nM). ER-stress UPR activation was analyzed at the transcript, protein, immunocytochemistry, western blotting, quantitative RT-PCR and XBP1 splicing, including; XBP1, sXBP1, ATF6, cleaved ATF6, IRE1-p, CHOP, Caspase-12 and Bax. Exocrine acinar cells exposed to high Ins or Ins+Glu concentrations (but not Glu alone) exhibited ER-stress UPR, demonstrated by significant increase of transcript and protein levels of downstream markers in the ATF6 and IRE1 transduction arms, including: sXBP1, cleaved ATF6, XBP1, CHOP, IRE1-p and caspase-12. UPR activation resulted in IRE1-p aggregation and nuclear trans-localization of cleaved activated ATF6 and sXBP1. Ins further aggravated UPR when cells were co-challenged with CER-induced stress, exacerbating the effects of CER alone. High Ins levels, typical to type-2-DM, activate the ER-stress UPR in pancreatic acinar cells, through the ATF6 and IRE1 pathways. This effect of Ins in naïve acinar cells further augments CER-induced UPR. Our data highlight molecular pathways through which DM enhances exocrine pancreas pathologies.
Asunto(s)
Células Acinares/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Respuesta de Proteína Desplegada , Células Acinares/efectos de los fármacos , Animales , Línea Celular Tumoral , Ceruletida/farmacología , Estrés del Retículo Endoplásmico , Glucosa/metabolismo , Glucosa/farmacología , Insulina/farmacología , Islotes Pancreáticos/efectos de los fármacos , RatasRESUMEN
Irisin is a novel secreted myokine, encoded by the fibronectin type III domain-containing protein 5 (FNDC5) precursor gene. Irisin plays a role in the female reproductive system in pregnancy and in embryonic development, and is associated with fetal size. It is expressed in the ovary, placenta and neonatal cord serum. We studied whether maternal and neonatal FNDC5 genetic polymorphisms are associated with preterm birth (PTB). Blood for DNA analysis was collected from Israeli mothers (nâ¯=â¯315) and from umbilical veins of their respected idiopathic preterm (24-36â¯weeks) and control term (>37â¯weeks) newborns (nâ¯=â¯161). Genotypes of maternal and neonatal FNDC5 polymorphisms (rs726344 and rs1746661) were determined by restriction fragment length polymorphism analysis. Genotype-phenotype associations were analyzed using SPSS program. The Frequency of FNCD5 rs726344 G alleles in the Israeli cohort is 82%. We found significant FNCD5 rs726344 genotype frequencies control and PTB groups. Women bearing the FNDC5 rs726344 GG genotype had 2.18 fold ([CI] 1.193-4.008, pâ¯=â¯0.01) higher chance to deliver at term compared to both AG and AA genotypes (adjusting to age, gravidity, parity, weight percentile per gestational age and gender of newborn). Neonates carrying the FNDC5 rs726344 GG genotype had 2.24 fold ([CI] 0.979-5.134, pâ¯=â¯0.05) higher chance to be born at term compared to either AG or AA genotypes (adjusting to parity, previous abortions and weight percentile per gestational age). There was no significant association of the rs1746661 polymorphism with PTB. Thus, we determined FNDC5 polymorphisms frequencies in the Israeli population and demonstrated that maternal and neonatal FNDC5 rs726344 polymorphism is significantly associated with increased risk for PTB.
Asunto(s)
Fibronectinas/genética , Nacimiento Prematuro/genética , Adulto , Alelos , Estudios de Cohortes , Femenino , Fibronectinas/sangre , Fibronectinas/metabolismo , Frecuencia de los Genes/genética , Estudios de Asociación Genética/métodos , Predisposición Genética a la Enfermedad/genética , Humanos , Recién Nacido , Israel , Masculino , Neuropéptidos/genética , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
INTRODUCTION: Pancreatic pathologies are characterized by a progressive fibrosis process. Pancreatic stellate cells (PSC) play a crucial role in pancreatic fibrogenesis. Endoplasmic reticulum (ER) stress emerges as an important determinant of fibrotic remodeling. Overload of fatty acids (FA), typical to obesity, may lead to lipotoxic state and cellular stress. AIM: To study the effect of different lipolytic challenges on pancreatic ER stress and PSC activation. METHODS: Primary PSCs were exposed to different FAs, palmitate (pal) and oleate (ole), at pathophysiological concentrations typical to obese state, and in acute caerulein-induced stress (cer). PSC activation and differentiation were analyzed by measuring fat accumulation (oil-red staining and quantitation), proliferation (cells count) and migration (wound- healing assay). PSC differentiation markers (α-sma, fibronectin, tgf-ß and collagen secretion), ER stress unfolded protein response and immune indicators (Xbp1, CHOP, TNF-α, IL-6) were analyzed at the transcript and protein expression levels (quantitative RT-PCR and western blotting). RESULTS: PSC exposure to pal and ole FAs (500µM) increased significantly fat accumulation. Proliferation and migration analysis demonstrated that ole FA retained PSC activation, while exposure to pal FA significantly halted proliferation rate and delayed migration. Cer significantly augmented PSC differentiation markers α- sma, fibronectin and collagen, and ER stress and inflammation markers including Xbp1, CHOP, TNF-α and IL-6. The ole FA treatment significantly elevated PSC differentiation markers α-sma, fibronectin and collagen secretion. PSC ER stress was demonstrated following pal treatment with significant elevation of Xbp1 splicing and CHOP levels. CONCLUSION: Exposure to pal FA halted PSC activation and differentiation and elevated ER stress markers, while cer and ole exposure significantly induced activation, differentiation and fibrosis. Thus, dietary FA composition should be considered and optimized to regulate PSC activation and differentiation in pancreatic pathologies.