SIRT1 regulates hepatic vldlr levels.

BACKGROUND: Endoplasmic reticulum (ER) stress-mediated increases in the hepatic levels of the very low-density lipoprotein (VLDL) receptor (VLDLR) promote hepatic steatosis by increasing the delivery of triglyceride-rich lipoproteins to the liver. Here, we examined whether the NAD(+)-dependent deacetylase sirtuin 1 (SIRT1) regulates hepatic lipid accumulation by modulating VLDLR levels and the subsequent uptake of triglyceride-rich lipoproteins. METHODS: Rats fed with fructose in drinking water, Sirt1-/- mice, mice treated with the ER stressor tunicamycin with or without a SIRT1 activator, and human Huh-7 hepatoma cells transfected with siRNA or exposed to tunicamycin or different inhibitors were used. RESULTS: Hepatic SIRT1 protein levels were reduced, while those of VLDLR were upregulated in the rat model of metabolic dysfunction-associated steatotic liver disease (MASLD) induced by fructose-drinking water. Moreover, Sirt1-/- mice displayed increased hepatic VLDLR levels that were not associated with ER stress, but were accompanied by an increased expression of hypoxia-inducible factor 1α (HIF-1α)-target genes. The pharmacological inhibition or gene knockdown of SIRT1 upregulated VLDLR protein levels in the human Huh-7 hepatoma cell line, with this increase abolished by the pharmacological inhibition of HIF-1α. Finally, SIRT1 activation prevented the increase in hepatic VLDLR protein levels in mice treated with the ER stressor tunicamycin. CONCLUSIONS: Overall, these findings suggest that SIRT1 attenuates fatty liver development by modulating hepatic VLDLR levels.

Thapsigargin and Tunicamycin Block SARS-CoV-2 Entry into Host Cells via Differential Modulation of Unfolded Protein Response (UPR), AKT Signaling, and Apoptosis.

BACKGROUND: SARS-Co-V2 infection can induce ER stress-associated activation of unfolded protein response (UPR) in host cells, which may contribute to the pathogenesis of COVID-19. To understand the complex interplay between SARS-Co-V2 infection and UPR signaling, we examined the effects of acute pre-existing ER stress on SARS-Co-V2 infectivity. METHODS: Huh-7 cells were treated with Tunicamycin (TUN) and Thapsigargin (THA) prior to SARS-CoV-2pp transduction (48 h p.i.) to induce ER stress. Pseudo-typed particles (SARS-CoV-2pp) entry into host cells was measured by Bright GloTM luciferase assay. Cell viability was assessed by cell titer Glo® luminescent assay. The mRNA and protein expression was evaluated by RT-qPCR and Western Blot. RESULTS: TUN (5 µg/mL) and THA (1 µM) efficiently inhibited the entry of SARS-CoV-2pp into host cells without any cytotoxic effect. TUN and THA's attenuation of virus entry was associated with differential modulation of ACE2 expression. Both TUN and THA significantly reduced the expression of stress-inducible ER chaperone GRP78/BiP in transduced cells. In contrast, the IRE1-XBP1s and PERK-eIF2α-ATF4-CHOP signaling pathways were downregulated with THA treatment, but not TUN in transduced cells. Insulin-mediated glucose uptake and phosphorylation of Ser307 IRS-1 and downstream p-AKT were enhanced with THA in transduced cells. Furthermore, TUN and THA differentially affected lipid metabolism and apoptotic signaling pathways. CONCLUSIONS: These findings suggest that short-term pre-existing ER stress prior to virus infection induces a specific UPR response in host cells capable of counteracting stress-inducible elements signaling, thereby depriving SARS-Co-V2 of essential components for entry and replication. Pharmacological manipulation of ER stress in host cells might provide new therapeutic strategies to alleviate SARS-CoV-2 infection.

Ghrelin regulates the endoplasmic reticulum stress signalling pathway in gestational diabetes mellitus.

OBJECTIVE: We aimed to investigate the effects and mechanisms of the ghrelin-regulated endoplasmic reticulum stress (ERS) signalling pathway in gestational diabetes mellitus (GDM). METHODS: Pregnant female C57BL/6 mice were randomly divided into a normal group, GDM group (high-fat diet + STZ), GDM + ghrelin group (acyl ghrelin), and GDM + ghrelin + ghrelin inhibitor group ([D-lys3]-GHRP-6). We measured body weight, the intake of water and food, glucose, cholesterol, triglyceride and fasting insulin levels in each group. HE staining was used to observe the morphological changes in the pancreas. The TUNEL method was used to detect the apoptosis rate of islet cells. qPCR and Western boltting were performed to detect the relative expression levels of PERK, ATF6, IREIα, GRP78, CHOP and caspase-12, which are related to the ERS signalling pathway in the pancreas. Then, NIT-1 cells were cultured to verify whether ghrelin regulates ERS under high-glucose or tunicamycin conditions. RESULTS: Compared with the GDM group, the GDM + ghrelin group showed improved physical conditions and significantly decreased the fasting blood glucose, glucose tolerance, cholesterol, triglyceride and fasting insulin levels. Damaged islet areas were inhibited by ghrelin in the GDM group. The GDM + ghrelin group showed reduced ß-cell apoptosis compared to the GDM and GDM + ghrelin + ghrelin inhibitor groups. ERS-associated factors (PERK, ATF6, IREIα, GRP78, CHOP and caspase-12) mRNA and protein levels were obviously lower in the GDM + ghrelin group than in the GDM group, while expression levels were restored in the inhibitor group. Ghrelin treatment improved the high-glucose or tunicamycin-induced apoptosis, increased insulin levels and upregulation of GRP78, CHOP and caspase-12 in NIT-1 cells. CONCLUSION: Ghrelin suppressed ERS signalling and apoptosis in GDM mice and in NIT-1 cells. This study established a link between ghrelin and GDM, and the targeting of ERS with ghrelin represents a promising therapeutic strategy for GDM.

Identification of a novel RNA transcript TISPL upregulated by stressors that stimulate ATF4.

Cells sense, respond, and adapt to environmental conditions that cause stress. In a previous study using HeLa cells, we isolated reporter cells responding to the endoplasmic reticulum (ER) stress inducers, thapsigargin and tunicamycin, using a highly sensitive promoter trap vector system. Splinkerette PCR and 5' rapid amplification of cDNA ends (5' RACE) identified a novel transcript that is upregulated by ER stress. Its endogenous expression increased approximately 10-fold in response to thapsigargin and tunicamycin within 1 h, but was down-regulated after 4 h. Because the transcript starts from an intron of a long noncoding RNA known as LINC-PINT, we designated the newly identified transcript TISPL (transcript induced by stressors from LINC-PINTlocus). TISPL was also expressed under several other stress conditions. It was particularly increased > 10-fold upon glucose starvation and 7-fold by arsenite exposure. Furthermore, in silico analyses, including a ChIP-atlas search, revealed that there is an ATF4-binding region with a c/ebp-Atf response element (CARE) downstream of the transcription start site of TISPL. Based on these results, we hypothesized that TISPL may be induced by the phospho-eIF2α and ATF4- axis of the integrated stress response pathway, which is known to be activated by the stress conditions listed above. As expected, knockout of ATF4 abolished the stress-induced upregulation of TISPL. Our results indicate that TISPL may be a useful biomarker for detecting stress conditions that activate ATF4. Our highly sensitive trap vector system proved beneficial in discovering new biomarkers.

Endoplasmic Reticulum Stress Induces Vasodilation in Liver Vessels That Is Not Mediated by Unfolded Protein Response.

There is a growing body of evidence that ER stress and the unfolded protein response (UPR) play a key role in numerous diseases. Impaired liver perfusion and ER stress often accompany each other in liver diseases. However, the exact impact of ER stress and UPR on the hepatic perfusion is not fully understood. The aim of this study was to disclose the effect of ER stress and UPR on the size of liver vessels and on the levels of Ca2+ and nitric oxide (NO), critical regulators of vascular tonus. This study was carried out in precisely cut liver tissue slices. Confocal microscopy was used to create 3D images of vessels. NO levels were determined either using either laser scan microscopy (LSM) in cells or by NO-analyser in medium. Ca2+ levels were analysed by LSM. We show that tunicamycin, an inducer of ER stress, acts similarly with vasodilator acetylcholine. Both exert a similar effect on the NO and Ca2+ levels; both induce significant vasodilation. Notably, this vasodilative effect persisted despite individual inhibition of UPR pathways-ATF-6, PERK, and IRE1-despite confirming the activation of UPR. Experiments with HUVEC cells showed that elevated NO levels did not result from endothelial NO synthase (eNOS) activation. Our study suggests that tunicamycin-mediated ER stress induces liver vessel vasodilation in an NO-dependent manner, which is mediated by intracellular nitrodilator-activatable NO store (NANOS) in smooth muscle cells rather than by eNOS.

Resolvin D1 alleviates apoptosis triggered by endoplasmic reticulum stress in IPEC-J2 cells.

BACKGROUND: Resolvin D1 (RvD1), a specialized pro-resolving lipid mediator (SPM), is derived from docosahexaenoic acid (DHA). It plays a key role in actively resolving inflammatory responses, which further reduces small intestinal damage. However, its regulation of the apoptosis triggered by endoplasmic reticulum (ER) stress in intestinal epithelial cells is still poorly understood. The intestinal porcine epithelial cells (IPEC-J2) were stimulated with tunicamycin to screen an optimal stimulation time and concentration to establish an ER stress model. Meanwhile, RvD1 (0, 1, 10, 20, and 50 nM) cytotoxicity and its impact on cell viability and the effective concentration for reducing ER stress and apoptosis were determined. Finally, the effects of RvD1 on ER stress and associated apoptosis were furtherly explored by flow cytometry analysis, AO/EB staining, RT-qPCR, and western blotting. RESULTS: The ER stress model of IPEC-J2 cells was successfully built by stimulating the cells with 1 µg/mL tunicamycin for 9 h. Certainly, the increased apoptosis and cell viability inhibition also appeared under the ER stress condition. RvD1 had no cytotoxicity, and its concentration of 1 nM significantly decreased cell viability inhibition (p= 0.0154) and the total apoptosis rate of the cells from 14.13 to 10.00% (p= 0.0000). RvD1 at the concentration of 1 nM also significantly reduced the expression of glucose-regulated protein 78 (GRP-78, an ER stress marker gene) (p= 0.0000) and pro-apoptotic gene Caspase-3 (p= 0.0368) and promoted the expression of B cell lymphoma 2 (Bcl-2, an anti-apoptotic gene)(p= 0.0008). CONCLUSIONS: Collectively, the results shed light on the potential of RvD1 for alleviating apoptosis triggered by ER stress, which may indicate an essential role of RvD1 in maintaining intestinal health and homeostasis.

Benefits and Pitfalls of a Glycosylation Inhibitor Tunicamycin in the Therapeutic Implication of Cancers.

The aberrant glycosylation is a hallmark of cancer progression and chemoresistance. It is also an immune therapeutic target for various cancers. Tunicamycin (TM) is one of the potent nucleoside antibiotics and an inhibitor of aberrant glycosylation in various cancer cells, including breast cancer, gastric cancer, and pancreatic cancer, parallel with the inhibition of cancer cell growth and progression of tumors. Like chemotherapies such as doxorubicin (DOX), 5'fluorouracil, etoposide, and cisplatin, TM induces the unfolded protein response (UPR) by blocking aberrant glycosylation. Consequently, stress is induced in the endoplasmic reticulum (ER) that promotes apoptosis. TM can thus be considered a potent antitumor drug in various cancers and may promote chemosensitivity. However, its lack of cell-type-specific cytotoxicity impedes its anticancer efficacy. In this review, we focus on recent advances in our understanding of the benefits and pitfalls of TM therapies in various cancers, including breast, colon, and pancreatic cancers, and discuss the mechanisms identified by which TM functions. Finally, we discuss the potential use of nano-based drug delivery systems to overcome non-specific toxicity and enhance the therapeutic efficacy of TM as a targeted therapy.

Investigating the effects of tauroursodeoxycholic acid (TUDCA) in mitigating endoplasmic reticulum stress and cellular responses in Pak choi.

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) within plant cells due to unfavourable conditions leads to ER stress. This activates interconnected pathways involving reactive oxygen species (ROS) and unfolded protein response (UPR), which play vital roles in regulating ER stress. The aim of this study is to investigate the underlying mechanisms of tunicamycin (TM) induced ER stress and explore the potential therapeutic applications of tauroursodeoxycholic acid (TUDCA) in mitigating cellular responses to ER stress in Pak choi (Brassica campestris subsp. chinensis). The study revealed that ER stress in Pak choi leads to detrimental effects on plant morphology, ROS levels, cellular membrane integrity, and the antioxidant defence system. However, treatment with TUDCA in TM-induced ER stressed Pak choi improved morphological indices, pigment contents, ROS accumulation, cellular membrane integrity, and antioxidant defence system restoration. Additionally, TUDCA also modulates the transcription levels of ER stress sensors genes, ER chaperone genes, and ER-associated degradation (ERAD) genes during ER stress in Pak choi. Furthermore, TUDCA has demonstrated its ability to alleviate ER stress, stabilize the UPR, reduce oxidative stress, prevent apoptosis, and positively influence plant growth and development. These results collectively comprehend TUDCA as a promising agent for mitigating ER stress-induced damage in Pak choi plants and provide valuable insights for further research and potential applications in crop protection and stress management.

Schisandrin B enhances embryo competence and potentially mitigates endoplasmic reticulum stress during porcine preimplantation development.

Endoplasmic reticulum (ER) stress induced by agents such as tunicamycin (TM) substantially impedes the developmental progression of porcine embryos. Lignan compounds such as Schisandrin B (Sch-B), may have the potential to mitigate this stress. However, there are few studies on the effects of Sch-B on embryo development. To address this research gap, this study evaluates the protective efficacy of Sch-B against TM-induced ER stress during pivotal stages of porcine embryogenesis. Notably, embryos treated with Sch-B exhibited pronounced resistance to TM-induced developmental arrest, particularly at the 4-cell stage, facilitating progression to the 8-cell stage and subsequent blastocyst formation. It was also observed that Sch-B effectively reduced reactive oxygen species (ROS) levels and improved mitochondrial membrane potential (MMP). Furthermore, Sch-B positively influenced the expression of several stress-related genes. These findings highlight the promising role of Sch-B in improving porcine embryo development and mitigating ER stress.

The Effects of N-Glycosylation on the Expression and Transport Activity of OATP1A2 and OATP2B1.

Organic anion transporting polypeptide (OATP)1A2 and OATP2B1 have potential N-glycosylation sites, but their influence remains unclear. This study aimed to identify the N-glycosylation sites of OATP1A2/2B1 and investigate their impact on the expression and function of OATP1A2/2B1. Human embryonic kidney cells expressing OATP1A2 or OATP2B1 (HEK293-OATP1A2/2B1) were exposed to tunicamycin, an N-glycosylation inhibitor, and a plasma membrane fraction (PMF) Western blot assay and an estrone 3-sulfate (E3S) uptake study were conducted. HEK293-OATP1A2/OATP2B1 cell lines with mutation(s) at potential N-glycosylation sites were established, and the Western blotting and uptake study were repeated. Tunicamycin reduced the PMF levels and E3S uptake of OATP1A2/OATP2B1. The Asn124Gln, Asn135Gln, and Asn492Gln mutations in OATP1A2 and Asn176Gln and Asn538Gln mutations in OATP2B1 reduced the molecular weights of the OATP molecules and their PMF levels. The PMF levels of OATP1A2 Asn124/135Gln, OATP1A2 Asn124/135/492Gln, and OATP2B1 Asn176/538Gln were further reduced. The maximum transport velocities of OATP1A2 Asn124Gln, OATP1A2 Asn135Gln, and OATP2B1 Asn176/538Gln were markedly reduced to 10 %, 4 %, and 10 % of the wild-type level, respectively. In conclusion, the N-glycans at Asn124 and Asn135 of OATP1A2 and those at Asn176 and Asn538 of OATP2B1 are essential for the plasma membrane expression of these molecules and also affect their transport function.

Salicylic acid- and ethylene-dependent effects of the ER stress-inducer tunicamycin on the photosynthetic light reactions in tomato plants.

Plant hormones such as ethylene (ET) and salicylic acid (SA) have an elementary role in the regulation of ER stress and unfolded protein response (UPR) in plants via modulating defence responses or inducing oxidative stress. Chloroplasts can be sources and targets of reactive oxygen species (ROS) that affect photosynthetic efficiency, which has not been investigated under tunicamycin (Tm)-induced ER stress. In this study, the direct and indirect effects of Tm on chloroplastic ROS production were first investigated in leaves of wild-type tomato (Solanum lycopersicum L.) plants. Secondly changes in activities of photosystem II and I were analysed under Tm exposure and after application of the chemical chaperone 4-phenylbutyrate (PBA) in different genotypes, focusing on the regulatory role of SA and ET Tm treatments significantly but indirectly induced ROS production in tomato leaves and in parallel it decreased the effective quantum yield of PSII [Y(II)] and PSI [Y(I)], as well as the photochemical quenching coefficient (qP) and the quantum yield of non-photochemical energy dissipation in PSI due to acceptor-side limitation [Y(NA)]. At the same time, Tm increased non-photochemical quenching (NPQ) and cyclic electron flow (CEF) in tomato leaves after 24 h. However, the photosynthetic activity of the SA hydroxylase-overexpressing NahG tomato plants was more severely affected by Tm as compared to wild-type and ET-insensitive Never ripe (Nr) plants. These results suggest the protective role of SA in the regulation of photosynthetic activity contributing to UPR and the survival of plants under ER stress. Interestingly, the activation of photoprotective mechanisms by NPQ was independent of SA but dependent on active ET signalling under ER stress, whereas CEF was reduced by ET due to its higher ratio in Nr plants.

Kimchi attenuates endoplasmic reticulum stress-induced hepatic steatosis in HepG2 cells and C57BL/6N mice.

Kimchi is a traditional fermented food that contains abundant nutrients and functional ingredients with various health benefits. We previously reported that kimchi active components suppress hepatic steatosis caused by endoplasmic reticulum (ER) stress in vitro and in vivo. Therefore, we assessed the effect of kimchi on the inhibition of hepatic steatosis caused by ER stress in HepG2 cells and C57BL/6N mice to verify the hypothesis that kimchi may potentially inhibit nonalcoholic fatty liver disease. We investigated the effect of kimchi on cell viability and triglyceride concentrations in cells and on lipid profile, lipid accumulation, and expression of related genes in cells and mice with hepatic steatosis. A mechanistic study was also performed using the liver X receptor α agonist T0901317 and the AMP-activated protein kinase agonist AICAR. Kimchi was noncytotoxic and effectively reduced triglyceride concentrations and suppressed hepatic steatosis-related gene expression in cells and mice. Additionally, kimchi recovered weight loss, lowered the serum and liver tissue lipid profiles, suppressed lipid accumulation, and reduced the effects of T0901317 and AICAR on lipogenic gene expression in tunicamycin-treated mice. Our results highlight that kimchi could prevent hepatic steatosis caused by ER stress in cells and mice.

DEL-1: a promising treatment for AMD-associated ER stress in retinal pigment epithelial cells.

BACKGROUND: Age-related macular degeneration (AMD) is an irreversible eye disease that can cause blurred vision. Regular exercise has been suggested as a therapeutic strategy for treating AMD, but how exercise improves AMD is not yet understood. This study investigated the protective effects of developmental endothelial locus-1 (DEL-1), a myokine upregulated during exercise, on endoplasmic reticulum (ER) stress-induced injury in retinal pigment epithelial cells. METHODS: We evaluated the levels of AMPK phosphorylation, autophagy markers, and ER stress markers in DEL-1-treated human retinal pigment epithelial cells (hRPE) using Western blotting. We also performed cell viability, caspase 3 activity assays, and autophagosome staining. RESULTS: Our findings showed that treatment with recombinant DEL-1 dose-dependently reduced the impairment of cell viability and caspase 3 activity in tunicamycin-treated hRPE cells. DEL-1 treatment also alleviated tunicamycin-induced ER stress markers and VEGF expression. Moreover, AMPK phosphorylation and autophagy markers were increased in hRPE cells in the presence of DEL-1. However, the effects of DEL-1 on ER stress, VEGF expression, and apoptosis in tunicamycin-treated hRPE cells were reduced by AMPK siRNA or 3-methyladenine (3-MA), an autophagy inhibitor. CONCLUSIONS: Our study suggests that DEL-1, a myokine, may have potential as a treatment strategy for AMD by attenuating ER stress-induced injury in retinal pigment epithelial cells.

Triplex glycan quantification by metabolic labeling with isotopically labeled glucose in yeast.

Mass spectrometry-based approaches encompass a powerful collection of tools for the analysis biological molecules, including glycans and glycoconjugates. Unlike most traditional bioanalytical methods focusing on these molecules, mass spectrometry is especially suited for multiplexing, by utilizing stable-isotope labeling. Indeed, stable isotope-based multiplexing can be regarded as the gold-standard approach in reducing noise and uncertainty in quantitative mass spectrometry and quantitative analyses generally. The increasing sophistication and depth of biological questions being asked continue to challenge the practitioners of mass spectrometry method development. To understand the biological relevance of glycans, many stable isotope labeling-based mass spectrometry methods have been developed. Based on the duplex MILPIG (metabolic isotope labeling of polysaccharides with isotopic glucose), we establish here a novel triplex isotope labeling method using baker's yeast as the model system. Two differentially isotope-labeled glucoses (medium: 1-13C1 and heavy: 1,2-13C2), in addition to natural abundance glucose (light), were successfully used to label each monosaccharide ring in N-linked glycans in three different cell culture conditions, that, after sample mixing, resulted in a predictable triplet spectrum amenable for relative quantitation. We demonstrate excellent accuracy and precision of relative quantitation for a 1:1:1 mixture of glycans labeled in such a fashion. In addition, we applied triplex MILPIG to interrogate differential N-glycan profiles in tunicamycin-treated and control yeast cells and show that different N-glycans respond differently to tunicamycin.

The tunicamycin derivative TunR2 exhibits potent antibiotic properties with low toxicity in an in vivo Mycobacterium marinum-zebrafish TB infection model.

Tunicamycins (TUN) are well-defined, Streptomyces-derived natural products that inhibit protein N-glycosylation in eukaryotes, and by a conserved mechanism also block bacterial cell wall biosynthesis. TUN inhibits the polyprenylphosphate-N-acetyl-hexosamine-1-phospho-transferases (PNPT), an essential family of enzymes found in both bacteria and eukaryotes. We have previously published the development of chemically modified TUN, called TunR1 and TunR2, that have considerably reduced activity on eukaryotes but that retain the potent antibacterial properties. A mechanism for this reduced toxicity has also been reported. TunR1 and TunR2 have been tested against mammalian cell lines in culture and against live insect cells but, until now, no in vivo evaluation has been undertaken for vertebrates. In the current work, TUN, TunR1, and TunR2 are investigated for their relative toxicity and antimycobacterial activity in zebrafish using a well-established Mycobacterium marinum (M. marinum) infection system, a model for studying human Mycobacterium tuberculosis infections. We also report the relative ability to activate the unfolded protein response (UPR), the known mechanism for the eukaryotic toxicity observed with TUN treatment. Importantly, TunR1 and TunR2 retained their antimicrobial properties, as evidenced by a reduction in M. marinum bacterial burden, compared to DMSO-treated zebrafish. In summary, findings from this study highlight the characteristics of recently developed TUN derivatives, mainly TunR2, and its potential for use as a novel anti-bacterial agent for veterinary and potential medical purposes.

Empagliflozin reduces endoplasmic reticulum stress associated TXNIP/NLRP3 activation in tunicamycin-stimulated aortic endothelial cells.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have proven to be of therapeutic significance for cardiovascular diseases beyond the treatment of type 2 diabetes. Recent studies have demonstrated the beneficial effects of SGLT2i on endothelial cell (EC) dysfunction, but the underlying cellular mechanisms remain to be clarified. In this study, we sought to understand the effect of empagliflozin (EMPA; Jardiance®) on cell homeostasis and endoplasmic reticulum (ER) stress signaling. ER stress was induced by tunicamycin (Tm) in human abdominal aortic ECs treated with EMPA over 24 h. Tm-induced ER stress caused increases in the protein expression of thioredoxin interacting protein (TXNIP), NLR-family pyrin domain-containing protein 3 (NLRP3), C/EBP homologous protein (CHOP), and in the ratio of phospho-eIF2α/eIF2α. EMPA (50-100 µM) resulted in a dampened downstream activation of ER stress as seen by the reduced expression of CHOP and TXNIP/NLRP3 in a dose-dependent manner. Nuclear factor erythroid 2-related factor 2 (nrf2) translocation was also attenuated in EMPA-treated ECs. These results suggest that EMPA improves redox signaling under ER stress which in turn attenuates the activation of TXNIP/NLRP3.

Tangeretin enhances pancreatic beta-TC-6 function by ameliorating tunicamycin-induced cellular perturbations.

BACKGROUND: Pancreatic beta cell health and its insulin-secreting potential are severely compromised under the diabetic condition. One of the key mediators of beta cell dysfunction is endoplasmic reticulum (ER) stress. Pharmacological intervention of ER stress and associated complications in pancreatic beta cells may be an effective strategy for the management of diabetes. In the present study, we evaluated the efficacy of tangeretin, a citrus pentamethoxyflavone, in the alleviation of ER stress and associated perturbations in pancreatic Beta-TC-6 cell lines. METHODS AND RESULTS: Tunicamycin (pharmacological ER stress inducer) at subtoxic levels was observed to induce beta cell dysfunction by upregulation of intracellular ROS levels, lowering mitochondrial number/biogenesis and membrane potential, elevation of UPR markers, XBP-1, GADD153, and ER resident chaperones. Treatment with tangeretin was successful in improving the beta cell function by lowering the ROS levels and improving the mitochondrial biogenesis and mitochondrial membrane potential. Tangeretin also downregulated the expression levels of XBP-1, GADD153, and ER resident chaperones. GLUT2 expression, however, did not undergo any significant change under ER stress. We also observed altered expression of Pdx-1, TRB3, and p-Akt under the ER stress condition. Tangeretin augmented the expression levels of Pdx-1, and p-Akt while curtailing the expression of TRB3 in beta cells. Tunicamycin treatment suppressed the insulin levels, however, co-treatment with tangeretin could only marginally improve the levels. CONCLUSION: Targeting ER stress and associated pathways in pancreatic Beta-TC-6 cell lines by tangeretin can be an effective strategy for improving beta cell function.

Endoplasmic Reticulum Stress Disrupts Mitochondrial Bioenergetics, Dynamics and Causes Corneal Endothelial Cell Apoptosis.

Purpose: Endoplasmic reticulum (ER) and mitochondrial stress are independently associated with corneal endothelial cell (CEnC) loss in many corneal diseases, including Fuchs' endothelial corneal dystrophy (FECD). However, the role of ER stress in mitochondrial dysfunction contributing to CEnC apoptosis is unknown. The purpose of this study is to explore the crosstalk between ER and mitochondrial stress in CEnC. Methods: Human corneal endothelial cell line (HCEnC-21T) and human corneal endothelial tissues were treated with ER stressor tunicamycin. ER stress-reducing chemical 4-phenyl butyric acid (4-PBA) was used in HCEnC-21T after tunicamycin. Fuchs' corneal endothelial cell line (F35T) was used to determine differential activation of ER stress with respect to HCEnC-21T at the baseline. ER stress, mitochondrial-mediated intrinsic apoptotic, mitochondrial fission, and fusion proteins were determined using immunoblotting and immunohistochemistry. Mitochondrial bioenergetics were assessed by mitochondrial membrane potential (MMP) loss and ATP production at 48 hours after tunicamycin. Mitochondria dynamics (shape, area, perimeter) were also analyzed at 24 hours using transmission electron microscopy. Results: Treatment of HCEnC-21T cell line with tunicamycin activated three ER stress pathways (PERK-eIF2α-CHOP, IRE1α-XBP1, and ATF6), reduced cell viability, upregulated mitochondrial-mediated intrinsic apoptotic molecules (cleaved caspase 9, caspase 3, PARP, Bax, cytochrome C), downregulated anti-apoptotic Bcl-2 protein, initiated mitochondrial dysfunction by loss of MMP and lowering of ATP production, and caused mitochondrial swelling and fragmentation with increased expression of mitochondrial fission proteins (Fis1 and p-Drp1). Fuchs' CEnC (F35T) cell line also showed activation of the ER stress-related proteins (p-eIF2α, GRP78, CHOP, XBP1) compared to HCEnC-21T at the baseline. The 4-PBA ameliorated cell loss and reduced cleaved caspase 3 and 9, thereby rescuing tunicamycin-induced cell death but not mitochondrial bioenergetics in HCEnC-21T cell line. Conclusions: Tunicamycin-induced ER stress disrupts mitochondrial bioenegetics, dynamics and contributes to the loss of CEnC viability. This novel study highlights the importance of ER-mitochondria crosstalk and its contribution to CEnCs apoptosis, seen in many corneal diseases, including FECD.

CHOP upregulation and dysregulation of the mature form of the SNAT2 amino acid transporter in the placentas from small for gestational age newborns.

BACKGROUND: The placentas from newborns that are small for gestational age (SGA; birth weight < -2 SD for gestational age) may display multiple pathological characteristics. A key determinant of fetal growth and, therefore, birth weight is placental amino acid transport, which is under the control of the serine/threonine kinase mechanistic target of rapamycin (mTOR). The effects of endoplasmic reticulum (ER) stress on the mTOR pathway and the levels of amino acid transporters are not well established. METHODS: Placentas from SGA and appropriate for gestational age (AGA) newborns and the human placental BeWo cell line exposed to the ER stressor tunicamycin were used. RESULTS: We detected a significant increase in the levels of C/EBP homologous protein (CHOP) in the placentas from SGA newborns compared with those from AGA newborns, while the levels of other ER stress markers were barely affected. In addition, placental mTOR Complex 1 (mTORC1) activity and the levels of the mature form of the amino acid transporter sodium-coupled neutral amino acid transporter 2 (SNAT2) were also reduced in the SGA group. Interestingly, CHOP has been reported to upregulate growth arrest and DNA damage-inducible protein 34 (GADD34), which in turn suppresses mTORC1 activity. The GADD34 inhibitor guanabenz attenuated the increase in CHOP protein levels and the reduction in mTORC1 activity caused by the ER stressor tunicamycin in the human placental cell line BeWo, but it did not recover mature SNAT2 protein levels, which might be reduced as a result of defective glycosylation. CONCLUSIONS: Collectively, these data reveal that GADD34A activity and glycosylation are key factors controlling mTORC1 signaling and mature SNAT2 levels in trophoblasts, respectively, and might contribute to the SGA condition. Video Abstract.