Moderate aerobic training counterbalances the deleterious effect of undernutrition on oxidative balance and mitochondrial markers.

The state of Maternal Protein Malnutrition (MPM) is associated with several deleterious effects, including inflammatory processes and dysregulation in oxidative balance, which can promote neurodegeneration. On the other hand, it is known that aerobic exercise can promote systemic health benefits, combating numerous chronic diseases. Therefore, we evaluate the effect of aerobic exercise training (AET) on indicators of mitochondrial bioenergetics, oxidative balance, endoplasmic reticulum stress, and neurotrophic factor in the prefrontal cortex of malnourished juvenile Wistar rats. Pregnant Wistar rats were fed with a diet containing 17% or 8% casein during pregnancy and lactation. At 30 days of life, male offspring were divided into 4 groups: Low-Protein Control (LS), Low-Protein Trained (LT), Normoprotein Control (NS), and Normoprotein Trained (NT). The trained groups performed an AET for 4 weeks, 5 days a week, 1 h a day per session. At 60 days of life, the animals were sacrificed and the skeletal muscle, and prefrontal cortex (PFC) were removed to evaluate the oxidative metabolism markers and gene expression of ATF-6, GRP78, PERK and BDNF. Our results showed that MPM impairs oxidative metabolism associated with higher oxidative and reticulum stress. However, AET restored the levels of indicators of mitochondrial bioenergetics, in addition to promoting resilience to cellular stress. AET at moderate intensity for 4 weeks in young Wistar rats can act as a non-pharmacological intervention in fighting against the deleterious effects of a protein-restricted maternal diet.

Trueperella pyogenes pyolysin inhibits lipopolysaccharide-induced inflammatory response in endometrium stromal cells via autophagy- and ATF6-dependent mechanism.

Trueperella pyogenes (T. pyogenes) is a common opportunistic pathogen of many livestock and play an important regulation role during multibacterial infection and interaction with the host by its primary virulence factor pyolysin (PLO). The purpose of this study was to investigate the regulation role of PLO which serve as a combinational pathogen with lipopolysaccharide (LPS) during endometritis. In this study, the expression of bioactive recombinant PLO (rPLO) in a prokaryotic expression system and its purification are described. Moreover, we observed that rPLO inhibited the innate immune response triggered by LPS and that methyl-ß-cyclodextrin (MBCD) abrogated this inhibitory effect in goat endometrium stromal cells (gESCs). Additionally, we show from pharmacological and genetic studies that rPLO-induced autophagy represses gene expression by inhibiting NLRP3 inflammasome activation. Importantly, this study reported that ATF6 serves as a primary regulator of the cellular inflammatory reaction to rPLO. Overall, these observations suggest that T. pyogenes PLO could create an immunosuppressive environment for other pathogens invasion by regulating cellular signaling pathways.

VIP conditions human endometrial receptivity by privileging endoplasmic reticulum stress through ATF6α pathway.

Endometrial stromal cells undergo endoplasmic reticulum (ER) stress and unfolded protein response (UPR) during the decidualization linked with the inflammation and angiogenesis processes. Considering VIP (vasoactive intestinal peptide) induces the decidualization program, we studied whether modulates the ER/UPR pathways to condition both processes for embryo implantation. When Human Endometrial Stromal Cell line (HESC) were decidualized by VIP we observed an increased expression of ATF6α, an ER stress-sensor, and UPR markers, associated with an increase in IL-1ß production. Moreover, AEBSF (ATF6α -inhibitor pathway) prevented this effect and decreased the expansion index in the in vitro model of implantation. VIP-decidualized cells also favor angiogenesis accompanied by a strong downregulation in thrombospondin-1. Finally, ATF6α, VIP and VPAC2-receptor expression were reduced in endometrial biopsies from women with recurrent implantation failures in comparison with fertile. In conclusion, VIP privileged ATF6α-pathway associated with a sterile inflammatory response and angiogenesis that might condition endometrial receptivity.

Exhaustive acute exercise-induced ER stress is attenuated in IL-6-knockout mice.

The endoplasmic reticulum (ER) stress and inflammation relationship occurs at different levels and is essential for the adequate homeostatic function of cellular systems, becoming harmful when chronically engaged. Intense physical exercise enhances serum levels of interleukin 6 (IL-6). In response to a chronic exhaustive physical exercise protocol, our research group verified an increase of the IL-6 concentration and ER stress proteins in extensor digitorium longus (EDL) and soleus. Based on these results, we hypothesized that IL-6-knockout mice would demonstrate a lower modulation in the ER stress proteins compared to the wild-type mice. To clarify the relationship between exercise-induced IL-6 increased and ER stress, we studied the effects of an acute exhaustive physical exercise protocol on the levels of ER stress proteins in the skeletal muscles of IL-6-knockout (KO) mice. The WT group displayed a higher exhaustion time compared to the IL-6 KO group. After 1 h of the acute exercise protocol, the serum levels of IL-6 and IL-10 were enhanced in the WT group. Independent of the experimental group, the CHOP and cleaved caspase 12/total caspase 12 ratio in EDL as well as ATF6 and CHOP in soleus were sensitive to the acute exercise protocol. Compared to the WT group, the oscillation patterns over time of BiP in EDL and soleus as well as of peIF2-alpha/eIF2-alpha ratio in soleus were attenuated for the IL-6 KO group. In conclusion, IL-6 seems to be related with the ER stress homeostasis, once knockout mice presented attenuation of BiP in EDL and soleus as well as of pEiF2-alpha/EiF2-alpha ratio in soleus after the acute exhaustive physical exercise protocol.

Atrial natriuretic peptide attenuates endoplasmic reticulum stress in experimental acute pancreatitis.

Increasing evidence shows that the endoplasmic reticulum (ER) stress is an early event that injures pancreatic acinar cells and contributes to the pathogenesis of acute pancreatitis. In the present work we sought to establish whether atrial natriuretic peptide (ANP) alleviated ER stress in rats with cerulein-induced pancreatitis. The major components of the unfolded protein response (UPR) and their downstream effectors were assessed by immunoblotting or fluorimetry and the ultrastructure of ER evaluated by electron transmission microscopy. Cross-talk with autophagy was evaluated by beclin-1 expression. ANP reduced binding immunoglobulin protein (Bip) expression (UPR major controller) which under non-stress conditions keeps inactive the stress sensor proteins: protein kinase-like ER kinase (PERK), inositol-requiring enzyme-1 (IRE1) and activating transcription factor 6 (ATF6). Although ANP did not change PERK expression it decreased p-eIF2α and enhanced downstream effector CHOP, suggesting that ANP stimulates ER-dependent apoptosis. In accordance, ANP also decreased Bcl2 expression and enhanced proapoptotic proteins Bax and Bak. The atrial peptide enhanced ATF6 expression and although it did not affect IRE1/sXBP1 signaling, it increased caspase-2 activity, also involved in ER-dependent apoptosis. Furthermore, ANP decreased beclin-1 expression. The ultrastructure of the RE revealed decreased swelling and conserved ribosomes in the presence of ANP. Present findings support that ANP alleviates ER stress in acute pancreatitis by modulating the three branches of the UPR and stimulates ER-dependent apoptosis. Gaining insights into the modulation of ER stress may help to develop specific therapeutic strategies for acute pancreatitis and/or medical interventions at risk of its developing like endoscopic retrograde cholangiopancreatography.

Pre-pregnancy maternal obesity associates with endoplasmic reticulum stress in human umbilical vein endothelium.

Obesity associates with the endoplasmic reticulum (ER) stress-induced endothelial dysfunction. Pregnant women with pre-pregnancy maternal obesity (PGMO) may transfer this potential risk to their offspring; however, whether ER stress occurs and associates with foetoplacental endothelial dysfunction in PGMO is unknown. We studied the l-arginine transport and nitric oxide (NO) synthesis in human umbilical vein endothelial cells (HUVECs) from women with PGMO or with a normal pre-pregnancy weight. We analysed the expression and activation of the ER stress sensors protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6). PGMO associated with lower endothelial NO synthase activity due to increased Thr495-inhibitor and decreased Ser1177-stimulator phosphorylation. However, higher expression and activity of the human cationic amino acid transporter 1 was found. PGMO caused activation of PERK and its downstream targets eukaryotic initiation factor 2 (eIF2α), C/EBP homologous protein 10 (CHOP), and tribbles-like protein 3 (TRB3). Increased IRE1α protein abundance (but not its phosphorylation or X-box binding protein 1-mRNA splicing) and increased c-Jun N-terminal kinase 1 phosphorylation was seen in PGMO. A preferential nuclear location of the activating transcription factor 6 (ATF6) was found in HUVECs from PGMO. All the changes seen in PGMO were blocked by TUDCA but unaltered by tunicamycin. Thus, PGMO may determine a state of ER stress via upregulation of the PERK-eIF2α-CHOP-TRB3 axis signalling in HUVECs. This phenomenon results in foetoplacental vascular endothelial dysfunction at birth.

Aerobic exercise training rescues protein quality control disruption on white skeletal muscle induced by chronic kidney disease in rats.

We tested whether aerobic exercise training (AET) would modulate the skeletal muscle protein quality control (PQC) in a model of chronic kidney disease (CKD) in rats. Adult Wistar rats were evaluated in four groups: control (CS) or trained (CE), and 5/6 nephrectomy sedentary (5/6NxS) or trained (5/6NxE). Exercised rats were submitted to treadmill exercise (60 min., five times/wk for 2 months). We evaluated motor performance (tolerance to exercise on the treadmill and rotarod), cross-sectional area (CSA), gene and protein levels related to the unfolded protein response (UPR), protein synthesis/survive and apoptosis signalling, accumulated misfolded proteins, chymotrypsin-like proteasome activity (UPS activity), redox balance and heat-shock protein (HSP) levels in the tibialis anterior. 5/6NxS presented a trend towards to atrophy, with a reduction in motor performance, down-regulation of protein synthesis and up-regulation of apoptosis signalling; increases in UPS activity, misfolded proteins, GRP78, derlin, HSP27 and HSP70 protein levels, ATF4 and GRP78 genes; and increase in oxidative damage compared to CS group. In 5/6NxE, we observed a restoration in exercise tolerance, accumulated misfolded proteins, UPS activity, protein synthesis/apoptosis signalling, derlin, HSPs protein levels as well as increase in ATF4, GRP78 genes and ATF6α protein levels accompanied by a decrease in oxidative damage and increased catalase and glutathione peroxidase activities. The results suggest a disruption of PQC in white muscle fibres of CKD rats previous to the atrophy. AET can rescue this disruption for the UPR, prevent accumulated misfolded proteins and reduce oxidative damage, HSPs protein levels and exercise tolerance.

Endoplasmic Reticulum Stress Markers and Their Possible Implications in Leprosy's Pathogenesis.

Mycobacterium leprae causes leprosy, a dermatoneurological disease which affects the skin and peripheral nerves. One of several cellular structures affected during M. leprae infection is the endoplasmic reticulum (ER). Infection by microorganisms can result in ER stress and lead to the accumulation of unfolded or poorly folded proteins. To restore homeostasis in the cell, the cell induces a series of signaling cascades known as the unfolded protein response called UPR (unfolded protein response). The present work is aimed at investigating the in situ expression of these markers in cutaneous lesions of clinical forms of leprosy and establish possible correlation expression patterns and types of lesion. A total of 43 samples from leprosy patients were analyzed by immunohistochemistry with monoclonal antibodies against GRP78/BiP, PERK, IRE1α, and ATF6. A statistically significant difference between the indeterminate, tuberculoid, and lepromatous clinical forms was detected, with high expression of GRP78/BiP, PERK, IRE1α, and ATF6 in tuberculoid forms (TT) when compared to lepromatous leprosy (LL) and indeterminate (I) leprosy. These results represent the first evidence of ER stress in samples of skin lesions from leprosy patients. We believe that they will provide better understanding of the complex pathogenesis of the disease and facilitate further characterization of the cascade of molecular events elicited during infection.

Protective effect of glutamine on the main and adjacent organs damaged by ischemia-reperfusion in rats.

Intestinal ischemia and reperfusion (I/R) causes cellular and tissue damage to the intestine and remote organs such as the liver. Increased production of ROS and nitric oxide and dysregulation of cytoprotective enzymes may be involved in intestinal I/R. The aim was to evaluate the protective effects of glutamine on the intestine and liver of rats with intestinal I/R injury. Twenty male Wistar rats (300 g) were divided into four groups: sham-operated (SO), glutamine + SO (G + SO), I/R, and glutamine + I/R (G + I/R). Occlusion of the SMA for 30 min was followed by 15-min reperfusion. Glutamine (25 mg/kg/day) was administered once daily 24 and 48 h before I/R induction. Blood and tissue of were collected for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, histopathological analysis, immunohistochemistry of IL-1ß and TNF-α, thiobarbituric acid reactive substance (TBARS) and nitric oxide, Nrf2/keap1, superoxide dismutase (SOD), NADPH quinone oxidoreductase1 (NQO1), inducible nitric oxide synthase (iNOS), heat shock protein (HSP70), glucose-regulated protein 78 (GRP78), and activating transcription factor 6 (ATF-6) by western blot. Statistic analysis by ANOVA-Student-Newman-Keuls test (mean ± SE) significantly was p < 0.05. Tissue damage, AST, ALT, IL-1ß, TNF-α, TBARS, NO, Keap1, iNOS, GRP78, and ATF-6 expression were significantly lower in the G + I/R group as compared to the I/R group. Expression of Nrf2, SOD, NQO1, and HSP70, was significantly higher in the G + I/R group as compared to I/R group. Pre-treatment with glutamine provided protection against oxidative damage in the intestine and liver in an experimental model of intestinal I/R.

Pro-inflammatory cytokines enhance ERAD and ATF6α pathway activity in salivary glands of Sjögren's syndrome patients.

Salivary gland (SG) acinar-cells are susceptible to endoplasmic reticulum (ER) stress related to their secretory activity and the complexity of synthesized secretory products. SGs of Sjögren's syndrome patients (SS)-patients show signs of inflammation and altered proteostasis, associated with low IRE1α/XBP-1 pathway activity without avert increases in apoptosis. Acinar-cells may avoid apoptosis by activation of the ATF6α pathway and ER-associated protein degradation (ERAD). The aim of this study was to evaluate the role of pro-inflammatory cytokines in ATF6α pathway/ERAD activation and cell viability in labial salivary glands (LSG) of SS-patients. In biopsies from SS-patients increased ATF6α signaling pathway activity, as evidenced by generation of the ATF6f cleavage fragment, and increased expression of ERAD machinery components, such as EDEM1, p97, SEL1L, gp78, UBE2J1, UBE2G2, HERP and DERLIN1, were observed compared to controls. Alternatively, for pro- (active-caspase-3) and anti-apoptotic (cIAP2) markers no significant difference between the two experimental groups was detected. Increased presence of ATF6f and ERAD molecules correlated significantly with increased expression of pro-inflammatory cytokines. These observations were corroborated in vitro in 3D-acini treated with TNF-α and/or IFN-γ, where an increase in the expression and activation of the ATF6α sensor and ERAD machinery components was detected under ER stress conditions, while changes in cell viability and caspase-3 activation were not observed. Cytokine stimulation protected cells from death when co-incubated with an ERAD machinery inhibitor. Alternatively, when cytokines were eliminated from the medium prior to ERAD inhibition, cell death increased, suggesting that the presence of pro-inflammatory cytokines in the medium is essential to maintain cell viability. In conclusion, the ATF6α pathway and the ERAD machinery are active in LSG of SS-patients. Both were also activated by TNF-α and IFN-γ in vitro in 3D-acini and aided in preventing apoptosis. IFN-γ levels were elevated in SS-patients and UPR responses triggered in vitro by this cytokine closely matched those observed in LSG from SS-patients, suggesting that cytokines may induce ER stress.

HIV infection and antiretroviral therapy lead to unfolded protein response activation.

BACKGROUND: The unfolded protein response (UPR) is one of the pathways triggered to ensure quality control of the proteins assembled in the endoplasmic reticulum (ER) when cell homeostasis is compromised. This mechanism is primarily composed of three transmembrane proteins serving as stress sensors: PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1). These three proteins' synergic action elicits translation and transcriptional downstream pathways, leading to less protein production and activating genes that encode important proteins in folding processes, including chaperones. Previous reports showed that viruses have evolved mechanisms to curtail or customize this UPR signaling for their own benefit. However, HIV infection's effect on the UPR has scarcely been investigated. METHODS: This work investigated UPR modulation by HIV infection by assessing UPR-related protein expression under in vitro and in vivo conditions via Western blotting. Antiretroviral (ARV) drugs' influence on this stress response was also considered. RESULTS: In in vitro and in vivo analyses, our results confirm that HIV infection activates stress-response components and that ARV therapy contributes to changes in the UPR's activation profile. CONCLUSIONS: This is the first report showing UPR-related protein expression in HIV target cells derived directly from HIV-infected patients receiving different ARV therapies. Thus, two mechanisms may occur simultaneously: interference by HIV itself and the ARV drugs' pharmacological effects as UPR activators. New evidence of how HIV modulates the UPR to enhance its own replication and secure infection success is also presented.

A novel ER stress-independent function of the UPR in angiogenesis.

Tumors rely on the unfolded protein response (UPR) and angiogenesis to survive the metabolic stress of hypoxia. Karali et al. (2014) revealed that VEGF signaling engages UPR sensors in an unconventional manner that is independent of endoplasmic reticulum (ER) stress, mediated by mTOR signaling to promote endothelial cell survival and angiogenesis.

Implication of eIF2α kinase GCN2 in induction of apoptosis and endoplasmic reticulum stress-responsive genes by sodium salicylate.

OBJECTIVES: Sodium salicylate (NaSal) can disturb cell viability by affecting the activity of multiple cellular molecules. In this work, we investigated the involvement of stress-responsive kinase GCN2 in regulating cell death and expression of stress genes in mouse embryonic fibroblasts (MEFs) upon exposure to NaSal. METHODS: Cell viability was assayed using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) method, and apoptosis was evaluated by annexin V and propidium iodide staining. A polymerase chain reaction (PCR) array approach was used to analyse differential expression of a panel of 84 endoplasmic reticulum (ER) stress-associated genes. Gene reporter assays were carried out to determine activity of ER stress element (ERSE), and the protein levels of activating transcription factor 6 (ATF6) and C/EBP homologous protein (CHOP) were determined by western blot. KEY FINDINGS: NaSal treatment resulted in reduction of cellular viability and induction of apoptosis in wild-type but not Gcn2(-/-) cells. Many genes with important functions in protein synthesis/degradation, transcriptional regulation and apoptosis were induced by NaSal and most of these were dependent on GCN2. The activation of ERSE within Ddit3 and the production of CHOP and ATF6 induced by NaSal required GCN2. CONCLUSIONS: Our data provide evidence for the involvement of GCN2 in apoptosis and gene expression triggered by NaSal, and contributes to the understanding of molecular events occurring in NaSal-treated cells.

Rotavirus infection induces the unfolded protein response of the cell and controls it through the nonstructural protein NSP3.

The unfolded protein response (UPR) is a cellular mechanism that is triggered in order to cope with the stress caused by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). This response is initiated by the endoribonuclease inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), and PKR-like ER kinase, which increase the expression of the genes involved in the folding and degradation processes and decrease the protein input into the ER by inhibiting translation. It has been shown that viruses both induce and manipulate the UPR in order to protect the host cells from an ER stress-mediated death, thus permitting the translation of viral proteins and the efficient replication of the virus. To understand the cellular events that occur during the rotavirus replication cycle, we examined the activation of the three UPR arms following infection, using luciferase reporters driven by promoters of the ER stress-responsive genes and real-time reverse transcription-PCR to determine the levels of the stress-induced mRNAs. Our findings indicated that during rotavirus infection two of the three arms of the UPR (IRE1 and ATF6) become activated; however, these pathways are interrupted at the translational level by the general inhibition of protein synthesis caused by NSP3. This response seems to be triggered by more than one viral protein synthesized during the replication of the virus, but not by the viral double-stranded RNA (dsRNA), since cells transfected with psoralen-inactivated virions, or with naked viral dsRNA, did not induce UPR.

Fine-tuning of the unfolded protein response: Assembling the IRE1alpha interactome.

Endoplasmic reticulum (ER) stress is a hallmark feature of secretory cells and many diseases, including cancer, neurodegeneration, and diabetes. Adaptation to protein-folding stress is mediated by the activation of an integrated signal transduction pathway known as the unfolded protein response (UPR). The UPR signals through three distinct stress sensors located at the ER membrane-IRE1alpha, ATF6, and PERK. Although PERK and IRE1alpha share functionally similar ER-luminal sensing domains and both are simultaneously activated in cellular paradigms of ER stress in vitro, they are selectively engaged in vivo by the physiological stress of unfolded proteins. The differences in terms of tissue-specific regulation of the UPR may be explained by the formation of distinct regulatory protein complexes. This concept is supported by the recent identification of adaptor and modulator proteins that directly interact with IRE1alpha. In this Review, we discuss recent evidence supporting a model where IRE1alpha signaling emerges as a highly regulated process, controlled by the formation of a dynamic scaffold onto which many regulatory components assemble.

Applications of counter-current chromatography in organic synthesis purification of heterocyclic derivatives of lapachol.

This work describes the application of counter-current chromatography (CCC) as a useful, fast and economic alternative for the isolation and purification of heterocyclic derivatives from lapachol and beta-lapachone, two naturally occurring compounds from Tabebuia species, and nor-beta-lapachone, a synthetic congener of lapachol. The discussed data comprise four examples of purification of synthetic reactions with different solvent systems - the mixture of the oxazole and the imidazole from beta-lapachone; the quinoxaline from nor-beta-lapachone; and the purification of the N-oxides from the quinoxaline and the phenazine from nor-beta-lapachone from their respective not fully reacted substrates by means of aqueous reversed- and normal-phase elution modes and non-aqueous solvent systems. Traditional purification of these reaction products by silica gel column chromatography demanded a large amount of solvent and time and, in some cases, serious degradation of the products occurred, leading to low yield of the reaction. High-speed counter-current chromatography (HSCCC) was used as an alternative to optimize the process and raise the yield of the reactions.