HIIT and MICT attenuate high-fat diet-induced hepatic lipid accumulation and ER stress via the PERK-ATF4-CHOP signaling pathway

Fatty liver can be induced by dietary habits and lifestyle and is directly related to obesity. Although the benefits of exercise interventions for reduction of liver fat have recently been acknowledged, the underlying mechanisms remain unclear. Thus, our present study investigated the effects of high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT) on high-fat diet-induced hepatic lipid accumulation, and explored the role of endoplasmic reticulum (ER) stress signaling pathways. To establish an obesity model, rats were fed with a normal standard diet or a high-fat diet (45% kcal as fat). Then, both lean and obese rats were divided into three subgroups: sedentary control (LC, OC) groups, high-intensity interval training (LHI, OHI) groups, and moderated-intensity continuous training (LMI, OMI) groups (n = 10). Rats in the exercise group underwent a swimming training protocol for 8 weeks. After the experimental period, serum and liver tissues from different groups were dissected for morphological and biochemical analyses. The results showed that with HIIT and MICT interventions, body weight and serum inflammatory markers (e.g., MCP-1, IL-1β, and TNF-α) were reduced in obese rats. Interestingly, HIIT was more effective in ameliorating liver triglyceride content and enhancing mitochondrial metabolic-enzymatic activity than was MICT in obese rats. Both HIIT and MICT conferred beneficial properties through upregulating Nrf2 expression, improving antioxidant enzyme activities and reduction of hepatic ER stress, which may have been regulated by the Bip-mediated PERK-ATF4-CHOP pathway. In conclusion, our findings confirmed the effectiveness of HIIT and MICT, particularly HIIT, in mitigating hepatic lipid accumulation. (AU)

Critical role of regulator of calcineurin 1 in spinal cord injury

Spinal cord injury (SCI) is a severe clinical problem worldwide. The pathogenesis of SCI is complicated and much is unknown. The current study was designed to investigate the possible role of regulator of calcineurin 1 (RCAN1) in SCI and to explore the possible molecular mechanisms. Rats were injected with LVshRNAi-RCAN1 and then contusion-induced SCI was established. We found that RCAN1 was significantly increased in spinal cord of rats with SCI. Knockdown of RCAN1 markedly facilitated the structural and functional recovery in the spinal cord, as illustrated by decrease of lesion volume and increase of Basso, Beattie, and Bresnahan (BBB) and combined behavioral score (CBS) scores. Downregulation of RCAN1 suppressed the increase of pro-inflammatory cytokines, including IL-1β and TNF-α, and inhibited the increase of TUNEL-positive cell numbers and caspases 3 and 9 activities. The decrease of oxygen consumption rate and increase of expression of glucose-regulated protein 78 (GRP78) and phosphorylation of protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) in rats with SCI were inhibited by LVshRNAi-RCAN1. Moreover, knockdown of RCAN1 ameliorated oxidative stress in rats with SCI, as evidenced by decrease of TBA reactive substances (TBARS) and GSSG content and increase of glutathione (GSH) level. These results suggested that RCAN1 played an important role in SCI through regulation of various pathological processes. Overall, the data provide novel insights into the role of RCAN1 in SCI and novel therapeutic targets of the treatment of injury in the spinal cord (AU)

No disponible

A historical overview of protein kinase PKR: from its discovery and mechanism of action to its clinical and therapeutic implications / Una visión histórica de la proteína quinasa PKR: desde su descubrimiento y mecanismo de acción hasta sus implicaciones clínicas y terapéuticas

The protein kinase R (PKR, also called EIF2AK2) is an interferon-inducible double-stranded RNA protein kinase with multiple effects on cells. PKR plays an active part in the cellular response to numerous types of stress, with a critical role in the host's interferon-induced antiviral defence mechanisms. PKR has been extensively studied and documented for its relevance as a cell growth regulator, and more recently analysed in connection with metabolism, inflammatory processes, cancer, and neurodegenerative diseases. The present review will summarise, in chronological order, the state of the knowledge about this kinase as well as the contributions we have done at the National Centre of Biotechnology regarding the regulation and mechanisms of action of PKR. Specific mention will be made of the studies that the author leads at the University Hospital Complex of Granada, showing the importance that PKR has as a target of both conventional chemotherapeutics and novel drugs, and its potential as a biomarker and therapeutic target in various pathologies

PKR, también llamada EIF2AK, es una proteína quinasa de respuesta a ARN de doble cadena inducida por interferón que participa en múltiples efectos en las células. PKR contribuye de forma activa en la respuesta celular a numerosos tipos de estrés, teniendo una importante función en el mecanismo de defensa antiviral del hospedador inducido por los interferones. PKR ha sido estudiada intensamente a lo largo del tiempo documentando su relevancia también como modulador del crecimiento celular, y más recientemente implicándola en metabolismo, procesos inflamatorios, cáncer y enfermedades neurodegenerativas. En esta revisión se resume de forma cronológica, el conocimiento adquirido sobre esta quinasa y nuestras contribuciones en el mecanismo de acción y regulación de PKR llevadas a cabo en el Centro Nacional de Biotecnología. Además, con especial interés, se describen los estudios que la autora lidera en el Complejo Hospitalario Universitario de Granada mostrando la importancia que PKR tiene como diana molecular de quimioterapéuticos convencionales y nuevos fármacos así como su potencial como biomarcador y diana terapéutica en varias enfermedades

Antitumoral activity of oncolytic vaccinia virus expressing the interferon-induced ds-RNA dependent protein kinase PKR / Actividad oncolítica del virus vaccinia que expresa la proteína quinasa PKR inducida por los interferones y dependiente de RNA bicatenario

Tumour cells generally become more susceptible to virus infection than normal cells due, in part, to a deficient interferon (IFN)-induced antiviral pathway. One of the key IFN-induced enzymes with potent antiviral action is the ds-RNA dependent protein kinase PKR, that once activated blocks protein synthesis, triggers apoptosis and prevents cell growth. Among viruses, vaccinia virus (VACV) lacking selected viral genes or armed with cytokines or tumour specific antigens has been used in preclinical and clinical studies as a therapeutic agent against different tumours. Here we showed in a mouse model of aggressive cancer by subcutaneous inoculation with prostateTRAMP-C1 cells, that a VACV recombinant expressing low levels of human PKR (VV-PKR) and lacking thymidine kinase (TK), is capable of reducing tumour burden when administered by a systemic route in immunocompetent C57/BL6 mice. In addition, expression of PKR was found to attenuate the virus, thus ensuring safety. A catalytically inactive enzyme PKR with a point mutation (K296R) induced similar oncolytic activity as the control virus lacking TK. These findings suggest that VACV recombinants expressing PKR are candidate vectors against cancer cells (AU)

Las células tumorales son generalmente mas susceptibles a la infección viral que las células normales debido, en parte, a tener deficiencias en el sistema de señalización por los interferones (IFN). Uno de los enzimas clave inducidos por IFN es la proteína quinasa PKR dependiente de RNA bicatenario, que una vez activada fosforila el factor de iniciación eIF-2 alfa lo que conlleva a una inhibición generalizada en la síntesis de proteínas, inducción de la apoptosis y prevención del crecimiento celular. Como agente terapéutico frente a distintos tumores se está utilizando en preclínica y clínica el virus vaccinia que carece de ciertos genes virales, expresa ciertas citoquinas o antígenos tumorales. En esta investigación demostramos en un modelo agresivo de cáncer en ratón por inoculación ex vivo de células tumorales de próstata TRAMP-C1, que se reduce el crecimiento tumoral tras la infección a ratones inmunocompetentes C57/BL6 por vía sistémica de un virus recombinante de vaccinia que expresa bajos niveles de PKR. Además, la expresión de PKR conduce a la atenuación del virus en los tejidos. Un mutante catalíticamente inactivo de PKR produce niveles similares de actividad oncolítica, pero con replicación viral en tejidos. Estos resultados indican que vectores recombinants de vaccinia que expresan PKR son candidatos como vectores para luchar contra tumores (AU)

Heme responsiveness in vitro is a common feature shared by the eukaryotic initiation factor 2á kinase family

Four distinct eukaryotic initiation factor 2á (eIF2á) kinases phosphorylate eIF2á at Ser-51 and regulate protein synthesis in response to cellular stress conditions. This kinase family includes the hemin-regulated inhibitor (HRI); the doublestranded RNA-dependent kinase (PKR); the GCN2 protein kinase; and the endoplasmic reticulum-resident kinase (PERK). HRI mediates protein synthesis inhibition in heme-deficient reticulocyte lysates. Although HRI contains two putative heme regulatory motifs (HRMs) that are not present in other eIF2á kinases, the significance of these motifs in heme regulation is not clear. In fact, it had been characterized two novel eIF2á kinases from Schizosaccharomyces pombe that lacked any of the HRMs, but were sensitive to heme regulation in vitro. To investigate the importance of different regions in the regulation of HRI by heme, specific HRI mutants were generated, and kinase activities and heme responsiveness were analyzed in vitro. Mutational analysis indicated that the heme regulatory motifs were spread around some regions in the HRI catalytic domain, outside of the HRMs. In accordance with these results, both the autokinase and the eIF2á kinase activities of three distinct eIF2á kinases, including the human PKR, the mouse GCN2 and the Drosophila PERK were inhibited in vitro by hemin. Although the known regulatory mechanisms of these eIF2á kinases are very different, the data reported here indicate that all known eIF2á kinases are regulated in vitro by hemin. This finding provides evidence that hemin represents a regulatory mechanism unique to eIF2á kinases and underscores the role of hemin in the translational regulation of eukaryotic cells

Las cuatro eIF2á quinasas eucarióticas fosforilan el residuo Ser-51 de la subunidad alfa del factor de iniciación 2 y regulan la síntesis de proteínas en respuesta a situaciones de estrés celular. Esta familia de proteínas quinasas está formada por el inhibidor regulado por hemina (HRI); la quinasa dependiente de RNA de doble cadena (PKR); la proteína quinasa GCN2 y la quinasa residente en el retículo endoplásmico (PERK). El HRI inhibe la síntesis de proteínas en lisados de reticulocitos de conejo deficientes de hemina. Aunque el HRI contiene dos supuestos motivos reguladores de hemina (HRMs), que no están presentes en las otras eIF2á quinasas, no está claro aún el papel de estos motivos en la regulación por hemina. De hecho, se han caracterizado dos nuevas eIF2á quinasas de Schizosaccharomyces pombe que carecen de dichos HRMs, pero son sensibles a la regulación por hemina in vitro. Un análisis mutacional indicó que los motivos reguladores de hemina estaban dispersos a lo largo del dominio catalítico, fuera de los HRMs. De acuerdo con estos resultados, las actividades autoquinasa y eIF2á quinasa de tres eIF2á quinasas distintas, la PKR humana, la GCN2 de ratón y la PERK de Drosophila, se inhibían por hemina in vitro. Aunque los mecanismos de regulación de todas estas eIF2á quinasas son muy diferentes, nuestros resultados indican que todas las eIF2á quinasas se regulan por hemina in vitro. Este descubrimiento soporta la evidencia de que la hemina representa un mecanismo de regulación específico de las eIF2á quinasas, y subraya su papel en la regulación de la traducción de células eucarióticas

Deficiencias en piruvato quinasa y anemias hemolíticas / Pyruvate kinase deficiencies and hemolytic anemias

La piruvato quinasa cataliza la transformación de fosfoenolpiruvato y ADP en piruvato y ATP. La enzima, que aparece en todas las células vivas, es clave en la ruta central del metabolismo de carbohidratos. La deficiencia en piruvato quinasa, debida a una mutación en el gen PK-LR, origina alteraciones únicamente en los eritrocitos, porque estas células no son capaces de compensar el defecto enzimático. Por ello, la deficiencia de esta enzima es causa principal de la anemia hemolítica no esferocítica que puede provocar incluso la muerte de los pacientes. Las dificultades en la caracterización bioquímica de la PK nos ha llevado a estudiar la enzimopatía mediante técnicas de Biología Molecular. El trabajo se realizó sobre 10 pacientes con deficiencia en piruvato quinasa eritrocitaria. Mediante análisis molecular se han encontrado 11 mutaciones diferentes en los 17 alelos mutados: tres de estas mutaciones, G694A, A1150G y G1154A, no han sido previamente descritas. A las mutaciones que originan fuertes modificaciones en la estructura local de la molécula, observadas mediante estudios de modelización molecular, como consecuencia de un desajuste en el balance de las cargas eléctricas o por impedimento estérico, corresponden valores disminuidos de la actividad de la enzima en aquellos pacientes portadores de dichas mutaciones (AU)