Thioredoxin Domain Containing 5 (TXNDC5): Friend or Foe?

This review focuses on the thioredoxin domain containing 5 (TXNDC5), also known as endoplasmic reticulum protein 46 (ERp46), a member of the protein disulfide isomerase (PDI) family with a dual role in multiple diseases. TXNDC5 is highly expressed in endothelial cells, fibroblasts, pancreatic ß-cells, liver cells, and hypoxic tissues, such as cancer endothelial cells and atherosclerotic plaques. TXNDC5 plays a crucial role in regulating cell proliferation, apoptosis, migration, and antioxidative stress. Its potential significance in cancer warrants further investigation, given the altered and highly adaptable metabolism of tumor cells. It has been reported that both high and low levels of TXNDC5 expression are associated with multiple diseases, such as arthritis, cancer, diabetes, brain diseases, and infections, as well as worse prognoses. TXNDC5 has been attributed to both oncogenic and tumor-suppressive features. It has been concluded that in cancer, TXNDC5 acts as a foe and responds to metabolic and cellular stress signals to promote the survival of tumor cells against apoptosis. Conversely, in normal cells, TXNDC5 acts as a friend to safeguard cells against oxidative and endoplasmic reticulum stress. Therefore, TXNDC5 could serve as a viable biomarker or even a potential pharmacological target.

Lipidomic signatures discriminate subtle hepatic changes in the progression of porcine nonalcoholic steatohepatitis.

Recently, the development of nonalcoholic steatohepatitis (NASH) in common strains of pigs has been achieved using a diet high in saturated fat, fructose, cholesterol, and cholate and deficient in choline and methionine. The aim of the present work was to characterize the hepatic and plasma lipidomic changes that accompany the progression of NASH and its reversal by switching pigs back to a chow diet. One month of this extreme steatotic diet was sufficient to induce porcine NASH. The lipidomic platform using liquid chromatography-mass spectrometry analyzed 467 lipid species. Seven hepatic phospholipids [PC(30:0), PC(32:0), PC(33:0), PC(33:1), PC(34:0), PC(34:3) and PC(36:2)] significantly discriminated the time of dietary exposure, and PC(30:0), PC(33:0), PC(33:1) and PC(34:0) showed rapid adaptation in the reversion period. Three transcripts (CS, MAT1A, and SPP1) showed significant changes associated with hepatic triglycerides and PC(33:0). Plasma lipidomics revealed that these species [FA 16:0, FA 18:0, LPC(17:1), PA(40:5), PC(37:1), TG(45:0), TG(47:2) and TG(51:0)] were able to discriminate the time of dietary exposure. Among them, FA 16:0, FA 18:0, LPC(17:1) and PA(40:5) changed the trend in the reversion phase. Plasma LDL-cholesterol and IL12P40 were good parameters to study the progression of NASH, but their capacity was surpassed by hepatic [PC(33:0), PC(33:1), and PC(34:0)] or plasma lipid [FA 16:0, FA 18:0, and LPC(17:1)] species. Taken together, these lipid species can be used as biomarkers of metabolic changes in the progression and regression of NASH in this model. The lipid changes suggest that the development of NASH also affects peripheral lipid metabolism.NEW & NOTEWORTHY A NASH stage was obtained in crossbred pigs. Hepatic [PC(33:0), PC(33:1) and PC(34:0)] or plasma [FA 16:0, FA 18:0 and LPC(17:1)] species were sensitive parameters to detect subtle changes in development and regression of nonalcoholic steatohepatitis (NASH). These findings may delineate the liquid biopsy to detect subtle changes in progression or in treatments. Furthermore, phospholipid changes according to the insult-inducing NASH may play an important role in accepting or rejecting fatty livers in transplantation.

Lipoprotein Lipidomics as a Frontier in Non-Alcoholic Fatty Liver Disease Biomarker Discovery.

Non-alcoholic fatty liver disease (NAFLD) is a progressive liver disease characterized by the build-up of fat in the liver of individuals in the absence of alcohol consumption. This condition has become a burden in modern societies aggravated by the lack of appropriate predictive biomarkers (other than liver biopsy). To better understand this disease and to find appropriate biomarkers, a new technology has emerged in the last two decades with the ability to explore the unmapped role of lipids in this disease: lipidomics. This technology, based on the combination of chromatography and mass spectrometry, has been extensively used to explore the lipid metabolism of NAFLD. In this review, we aim to summarize the knowledge gained through lipidomics assays exploring tissues, plasma, and lipoproteins from individuals with NAFLD. Our goal is to identify common features and active pathways that could facilitate the finding of a reliable biomarker from this field. The most frequent observation was a variable decrease (1-9%) in polyunsaturated fatty acids in phospholipids and non-esterified fatty acids in NAFLD patients, both in plasma and liver. Additionally, a reduction in phosphatidylcholines is a common feature in the liver. Due to the scarcity of studies, further research is needed to properly detect lipoprotein, plasma, and tissue lipid signatures of NAFLD etiologies, and NAFLD subtypes, and to define the relevance of this technology in disease management strategies in the push toward personalized medicine.

TXNDC5 Plays a Crucial Role in Regulating Endoplasmic Reticulum Activity through Different ER Stress Signaling Pathways in Hepatic Cells.

The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is influenced by a number of variables, including endoplasmic reticulum stress (ER). Thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family and acts as an endoplasmic reticulum (ER) chaperone. Nevertheless, the function of TXNDC5 in hepatocytes under ER stress remains largely uncharacterized. In order to identify the role of TXNDC5 in hepatic wild-type (WT) and TXNDC5-deficient (KO) AML12 cell lines, tunicamycin, palmitic acid, and thapsigargin were employed as stressors. Cell viability, mRNA, protein levels, and mRNA splicing were then assayed. The protein expression results of prominent ER stress markers indicated that the ERN1 and EIF2AK3 proteins were downregulated, while the HSPA5 protein was upregulated. Furthermore, the ATF6 protein demonstrated no significant alterations in the absence of TXNDC5 at the protein level. The knockout of TXNDC5 has been demonstrated to increase cellular ROS production and its activity is required to maintain normal mitochondrial function during tunicamycin-induced ER stress. Tunicamycin has been observed to disrupt the protein levels of HSPA5, ERN1, and EIF2AK3 in TXNDC5-deficient cells. However, palmitic acid has been observed to disrupt the protein levels of ATF6, HSPA5, and EIF2AK3. In conclusion, TXNDC5 can selectively activate distinct ER stress pathways via HSPA5, contingent on the origin of ER stress. Conversely, the absence of TXNDC5 can disrupt the EIF2AK3 cascade.

Endoplasmic Reticulum Protein TXNDC5 Interacts with PRDX6 and HSPA9 to Regulate Glutathione Metabolism and Lipid Peroxidation in the Hepatic AML12 Cell Line.

Non-alcoholic fatty liver disease or steatosis is an accumulation of fat in the liver. Increased amounts of non-esterified fatty acids, calcium deficiency, or insulin resistance may disturb endoplasmic reticulum (ER) homeostasis, which leads to the abnormal accumulation of misfolded proteins, activating the unfolded protein response. The ER is the primary location site for chaperones like thioredoxin domain-containing 5 (TXNDC5). Glutathione participates in cellular oxidative stress, and its interaction with TXNDC5 in the ER may decrease the disulfide bonds of this protein. In addition, glutathione is utilized by glutathione peroxidases to inactivate oxidized lipids. To characterize proteins interacting with TXNDC5, immunoprecipitation and liquid chromatography-mass spectrometry were used. Lipid peroxidation, reduced glutathione, inducible phospholipase A2 (iPLA2) and hepatic transcriptome were assessed in the AML12 and TXNDC5-deficient AML12 cell lines. The results showed that HSPA9 and PRDX6 interact with TXNDC5 in AML12 cells. In addition, TXNDC5 deficiency reduced the protein levels of PRDX6 and HSPA9 in AML12. Moreover, lipid peroxidation, glutathione and iPLA2 activities were significantly decreased in TXNDC5-deficient cells, and to find the cause of the PRDX6 protein reduction, proteasome suppression revealed no considerable effect on it. Finally, hepatic transcripts connected to PRDX6 and HSPA9 indicated an increase in the Dnaja3, Mfn2 and Prdx5 and a decrease in Npm1, Oplah, Gstp3, Gstm6, Gstt1, Serpina1a, Serpina1b, Serpina3m, Hsp90aa1 and Rps14 mRNA levels in AML12 KO cells. In conclusion, the lipid peroxidation system and glutathione mechanism in AML12 cells may be disrupted by the absence of TXNDC5, a novel protein-protein interacting partner of PRDX6 and HSPA9.

Effect of virgin olive oil as spreadable preparation on atherosclerosis compared to dairy butter in Apoe-deficient mice.

Olive oil is the main source of lipid energy in the Mediterranean diet and there is strong evidence of its health benefits. The effect of extra virgin olive oil (EVOO) in the form of a preparation of spreadable virgin olive oil (S-VO) on the progression of atheroma plaques was investigated in Apoe-deficient mice, a model of accelerated atherosclerosis. METHODS: Two isocaloric Western purified diets containing 20% fat, either as S-VO or as dairy butter, were used to feed 28 males and 16 females of two-month-old Apoe-deficient mice for 12 weeks. S-VO was prepared by blending more than 75% virgin olive oil with other vegetal natural fat to obtain a solid fat. Plasma total cholesterol, triglycerides and HDL cholesterol were measured. Hepatic lipid droplets were analyzed. Areas of atherosclerotic aortic lesions were quantified in cross-sectional images of the proximal aorta and en face analysis of the whole aorta. RESULTS: Total plasma cholesterol was increased in mice on the butter-supplemented diet in both female and male mice compared to S-VO, and the ratio of TC/HDL-cholesterol was significantly lower in S-VO than in the butter diet, although only in males, and no differences in plasma triglycerides were observed. No significant differences in hepatic lipid droplets were observed between diets in either sex. Aortic lesion areas were significantly higher in mice consuming the butter versus the S-VO diet in both sexes. CONCLUSION: Extra virgin olive oil prepared in spreadable form maintained the delay in atheroma plaque progression compared to butter.

Hepatic subcellular distribution of squalene changes according to the experimental setting

Squalene is the main unsaponifiable component of virgin olive oil, the main source of dietary fat in Mediterranean diet, traditionally associated with a less frequency of cardiovascular diseases. In this study, two experimental approaches were used. In the first, New Zealand rabbits fed for 4 weeks with a chow diet enriched in 1% sunflower oil for the control group, and in 1% of sunflower oil and 0.5% squalene for the squalene group. In the second, APOE KO mice received either Western diet or Western diet enriched in 0.5% squalene for 11 weeks. In both studies, liver samples were obtained and analyzed for their squalene content by gas chromatography-mass spectrometry. Hepatic distribution of squalene was also characterized in isolated subcellular organelles. Our results show that dietary squalene accumulates in the liver and a differential distribution according to studied model. In this regard, rabbits accumulated in cytoplasm within small size vesicles, whose size was not big enough to be considered lipid droplets, rough endoplasmic reticulum, and nuclear and plasma membranes. On the contrary, mice accumulated in large lipid droplets, and smooth reticulum fractions in addition to nuclear and plasma membranes. These results show that the squalene cellular localization may change according to experimental setting and be a starting point to characterize the mechanisms involved in the protective action of dietary squalene in several pathologies

El receptor nuclear NOR-1 regula la activación de las células vasculares y el remodelado vascular en respuesta a estrés hemodinámico / The nuclear receptor NOR-1 regulates the activation of vascular cells and vascular remodelling in response to hemodynamic stress

Introducción: Estudios previos sugieren que la pérdida de función de NOR-1 modula la activación de las células musculares lisas vasculares (CMLV). En este estudio utilizamos un ratón que sobreexpresa NOR-1 en CMLV para analizar su efecto en la activación celular y en la hiperplasia de la íntima inducida por estrés hemodinámico. Métodos Para generar el modelo animal el ADNc de NOR-1 humano se situó bajo el control del promotor de SM22α. La expresión de NOR-1 se analizó mediante PCR a tiempo real, Western-blot, inmunohistoquímica e inmunocitoquímica, y su funcionalidad se determinó mediante ensayos de actividad luciferasa. Como índice de proliferación celular se determinó la incorporación de timidina tritiada. La carótida izquierda se sometió a ligadura y en secciones de la misma se realizaron análisis morfométricos e inmunohistoquímicos. Resultados El transgénico desarrollado exhibía niveles significativos de NOR-1 humano en la aorta y las arterias carótidas. En las CMLV de los animales transgénicos se detectó un aumento de la actividad transcripcional de la ciclina D2, una mayor actividad proliferativa y niveles incrementados de Myh10. En estos animales la ligadura de la carótida indujo mayor formación de neoíntima y de estenosis que en los animales control, en consonancia con el marcaje de Myh10 e histona H3 fosforilada. Conclusiones Estos resultados refuerzan el papel de NOR-1 en la proliferación de las CMLV y en el remodelado vascular, y permiten proponer este modelo como una herramienta útil para estudiar la implicación de este receptor en la función vascular y en enfermedades como la arteriosclerosis y la reestenosis

Introduction: Previous studies have shown that the loss of NOR-1 function modulates the activation of vascular smooth muscle cells (VSMC). In this study we use a mouse that over-expresses human NOR-1 in VSMC to analyze the effect of a gain of NOR-1 function on the activation ofVSMC and in the hyperplasia of the intima induced by hemodynamic stress. Methods: To generate the transgenic animal the human NOR-1 cDNA was placed under the control of the SM22promoter. The expression of NOR-1 was analyzed by real time PCR, Westernblot, immunohistochemistry and immunocitochemistry, and NOR-1 functionality was evaluated by luciferase activity assays. The incorporation of tritiated thymidine was determined as a cellproliferation index. The left carotid artery was ligated, and cross-sections were subjected tomorphometric and immunostaining analysis. Results: The transgenic mouse exhibited significant levels of human NOR-1 in aorta and carotidarteries. In aortic VSMC from transgenic mice an increase in the transcriptional activity of ciclinD2 was detected, as well as higher proliferative rates and increased levels of the marker Myh10.In these animals, carotid artery ligation induced a greater neointimal formation and a higherstenotic grade than in wild-type animals, in accordance with the labelling detected for Myh10and phosphorylated Histone H3.Conclusions: These results reinforce the role of NOR-1 in VSMC proliferation and in vascular remodelling, and allow us to propose this model as a useful tool to study the involvement ofNOR-1 in vascular function and in vascular diseases such as atherosclerosis and restenosis

Hiperhomocisteinemia. Panorama actual y contribución del ratón a su estudio / Hyperhomocysteinemia. Current overview and contribution of mice to research on the topic

La homocisteína (HCY) es un aminoácido cuya elevación sanguínea se asocia con el desarrollo de enfermedades de tipo vascular, neurológico y reproductivo. Sus niveles plasmáticos varían en función de la raza, el sexo, la edad y otros factores ambientales. En la presente revisión se aborda su metabolismo, su fisiopatología y las consecuencias clínicas de su elevación. Un énfasis especial se presta al empleo del ratón como modelo experimental en este campo, ya que su uso ha puesto de manifiesto la importancia de la dieta en la regulación de la HCY. Esto, unido al desarrollo de animales modificados genéticamente con hiperhomocisteinemia, está permitiendo una rápida caracterización de los mecanismos moleculares implicados en la acción in vivo de la HCY. Además, la combinación de estos modelos con otros modificados genéticamente permite definir la influencia de la combinación de factores de riesgo en el desarrollo de diversas patologías. A su vez, la exploración en estos nuevos modelos de factores ambientales y/o farmacológicos contribuye de este modo a explicar muchas de las evidencias epidemiológicas en humanos así como el tratamiento más adecuado para cada condición (AU)

Homocysteine is an amino acid whose plasma levels are associated with the development of vascular, neurologic and reproductive diseases. Plasma levels show a wide range of values according to age, sex, race and other environmental factors. The present review discusses homocysteine metabolism and physiopathology and the clinical consequences of increased levels of this substance. Special emphasis has been placed on the use of mice as an experimental animal model in this field, since their use has unveiled the contribution of dietary modifications on plasma homocysteine levels. These findings, together with the generation of genetically-modified mice as models of hyperhomocysteinemia, are allowing rapid progress to be made in the characterization of the in vivo molecular mechanisms of homocysteine action. Crosses among these genetically-modified mice and others with different deleted genes will increase knowledge of the influence of the combination of several risk factors on pathological development. In these models, research into new environmental or pharmacological factors may yield results that could explain epidemiological findings in humans and help in the design of new treatments for specific clinical settings (AU)

Conocimiento de la acción biológica del aceite de oliva virgenextra mediante el uso del ratón carente de la apolipoproteína E / Knowledge of the Biological Actions of Extra Virgin Olive Oil Gained From Mice Lacking Apolipoprotein E

La baja incidencia de enfermedades cardiovascularesen los países de la Cuenca Mediterránea, donde elaceite de oliva es la principal fuente de grasa en la alimentación,ha motivado un mejor conocimiento de sucomposición química y el desarrollo de aceites enriquecidosen sus componentes minoritarios. En esta revisiónse recopilan los efectos de diferentes preparaciones delaceite de oliva sobre el desarrollo de la aterosclerosis yel valor pronóstico para la enfermedad de los parámetrosplasmáticos mediante el empleo de un ratón modificadogenéticamente en el que ésta se desarrolla espontáneamente.Las limitaciones del modelo por sus diferenciasmorfológicas y fisiológicas con el hombre se minimizanante la similitud de ambos genomas y el avance de conocimientoque posibilita, ya que efectuar en humanoslas intervenciones recopiladas habría requerido 400 años.Confirmando la tradición de los pueblos mediterráneos,se ha verificado la eficacia del aceite de oliva virgen consumidoprudentemente y en dietas con bajo contenido encolesterol por la relativa escasez de productos de origenanimal. Además, la exploración con herramientas de genómicaha identificado nuevos marcadores de respuestaal aceite. En conclusión, la investigación multidisciplinariadel aceite de oliva virgen extra permite ampliar el conocimientode sus propiedades biológicas (AU)

The low incidence of cardiovascular disease in countriesbordering the Mediterranean basin, where olive oil is themain source of dietary fat, has stimulated interest in thechemical composition of olive oil and in the productionof other oils enriched with its minor components.This review summarizes what has been learned aboutthe effects of different olive oil preparations on thedevelopment of atherosclerosis and about the prognosticvalue of associated plasma variables in the diseasefrom experiments on genetically modified mice thatspontaneously develop atherosclerosis. The limitations ofthis animal model associated with its morphological andphysiological differences with humans are minimized bythe similarity of the two genomes and by the potential forincreased understanding attainable, given that the dietaryinterventions reported here would have taken 400 years toachieve in humans. As observed in traditional Mediterraneanpopulations, it has been confirmed that extra virgin olive oilis beneficial when consumed judiciously and in a diet thatis low in cholesterol due to the relative scarcity of animalproducts. Furthermore, the use of genomic techniqueshas led to the identification of new markers of responseto olive oil. In conclusion, multidisciplinary research intoextra virgin olive oil is expanding our knowledge of thesubstance’s biological properties (AU)