Taurine-conjugated Ursodeoxycholic Acid Research Articles

Tauroursodeoxycholic Acid Inhibits Clostridioides difficile Toxin-Induced Apoptosis.

ABSTRACTC. difficile infection (CDI) is a highly inflammatory disease mediated by the production of two large toxins that weaken the intestinal epithelium and cause extensive colonic tissue damage. Antibiotic alternative therapies for CDI are urgently needed as current antibiotic regimens prolong the perturbation of the microbiota and lead to high disease recurrence rates. Inflammation is more closely correlated with CDI severity than bacterial burden, thus therapies that target the host response represent a promising yet unexplored strategy for treating CDI. Intestinal bile acids are key regulators of gut physiology that exert cytoprotective roles in cellular stress, inflammation, and barrier integrity, yet the dynamics between bile acids and host cellular processes during CDI have not been investigated. Here we show that several bile acids are protective against apoptosis caused by C. difficile toxins in Caco-2 cells and that protection is dependent on conjugation of bile acids. Out of 20 tested bile acids, taurine conjugated ursodeoxycholic acid (TUDCA) was the most potent inhibitor, yet unconjugated UDCA did not alter toxin-induced apoptosis. TUDCA treatment decreased expression of genes in lysosome associated and cytokine signaling pathways. TUDCA did not affect C. difficile growth or toxin activity in vitro whereas UDCA significantly reduced toxin activity in a Vero cell cytotoxicity assay and decreased tcdA gene expression. These results demonstrate that bile acid conjugation can have profound effects on C. difficile as well as the host and that conjugated and unconjugated bile acids may exert different therapeutic mechanisms against CDI.

Angiotensin II Causes β-Cell Dysfunction Through an ER Stress-Induced Proinflammatory Response.

The metabolic syndrome is associated with an increase in the activation of the renin angiotensin system, whose inhibition reduces the incidence of new-onset diabetes. Importantly, angiotensin II (AngII), independently of its vasoconstrictor action, causes β-cell inflammation and dysfunction, which may be an early step in the development of type 2 diabetes. The aim of this study was to determine how AngII causes β-cell dysfunction. Islets of Langerhans were isolated from C57BL/6J mice that had been infused with AngII in the presence or absence of taurine-conjugated ursodeoxycholic acid (TUDCA) and effects on endoplasmic reticulum (ER) stress, inflammation, and β-cell function determined. The mechanism of action of AngII was further investigated using isolated murine islets and clonal β cells. We show that AngII triggers ER stress, an increase in the messenger RNA expression of proinflammatory cytokines, and promotes β-cell dysfunction in murine islets of Langerhans both in vivo and ex vivo. These effects were significantly attenuated by TUDCA, an inhibitor of ER stress. We also show that AngII-induced ER stress is required for the increased expression of proinflammatory cytokines and is caused by reactive oxygen species and IP3 receptor activation. These data reveal that the induction of ER stress is critical for AngII-induced β-cell dysfunction and indicates how therapies that promote ER homeostasis may be beneficial in the prevention of type 2 diabetes.

Inhibition of endoplasmic reticulum stress improves coronary artery function in type 2 diabetic mice

What is the central question of this study? Endoplasmic reticulum (ER) stress has been reported to be involved in type 2 diabetes; however, the role of exacerbated ER stress in vascular dysfunction in type 2 diabetes remains unknown. What is the main finding and its importance? The main findings of this study are that ER stress is increased in the coronary arteries in type 2 diabetes, and inhibition of ER stress using taurine-conjugated ursodeoxycholic acid improves vascular function, which is associated with normalization of the myogenic response and endothelium-dependent relaxation. Vascular dysfunction is a major complication in type 2 diabetes. Although endoplasmic reticulum (ER) stress has been suggested to be a contributory factor in cardiovascular diseases, the relationship between ER stress and vascular dysfunction in type 2 diabetes remains unclear. Thus, in the present study, we examined whether ER stress contributes to coronary artery dysfunction and whether inhibition of ER stress ameliorates vascular function in type 2 diabetes. Type 2 diabetic mice and their control counterparts were treated with an ER stress inhibitor (taurine-conjugated ursodeoxycholic acid, 150mgkg(-1) day(-1) , by i.p. injection) for 2weeks or not treated. The myogenic response and endothelium-dependent relaxation were measured in pressurized coronary arteries. In type 2 diabetic mice, blood glucose and body weight were elevated compared with control mice. The myogenic response was potentiated and endothelium-dependent relaxation impaired in coronary arteries from the type 2 diabetic mice. Interestingly, treatment with the ER stress inhibitor normalized the myogenic responses and endothelium-dependent relaxation. These data were associated with an increase in ER stress marker expression or phosphorylation (IRE1-XBP-1 and PERK-eIF2α) in type 2 diabetic mice, which were reduced by treatment with the ER stress inhibitor. Inhibition of ER stress normalizes the myogenic response and improves vascular function in type 2 diabetes. Therefore, ER stress could be a potential target for cardiovascular diseases in diabetes mellitus.

Endoplasmic reticulum stress as a novel target to ameliorate epithelial-to-mesenchymal transition and apoptosis of human peritoneal mesothelial cells

Epithelial-to-mesenchymal transition (EMT) and apoptosis of peritoneal mesothelial cells are known to be the earliest mechanisms of peritoneal fibrosis in peritoneal dialysis (PD). Endoplasmic reticulum (ER) stress with an unfolded protein response is regarded to have a role in the development of organ fibrosis. To investigate the potential role of ER stress as a target to prevent and/or delay the development of peritoneal fibrosis, we examined the effect of ER stress on EMT or apoptosis of human peritoneal mesothelial cells (HPMCs) and elucidated the mechanisms underlying the protective effect of ER stress preconditioning on TGF-β1-induced EMT. ER stress inducers, tunicamycin (TM) and thapsigargin (TG), induced EMT with Smad2/3 phosphorylation, an increased nuclear translocation of β-catenin and Snail expression. Low concentrations of TM and TG did not induce apoptosis within 48 h; however, high concentrations of TM- (>1 ng/ml) and TG- (>1 nM) induced apoptosis at 12 h with a persistent increase in C/EBP homologous protein. TGF-β1 induced EMT and apoptosis in HPMCs, which was ameliorated by taurine-conjugated ursodeoxycholic acid, an ER stress blocker. Interestingly, pre-treatment with TM or TG for 4 h also protected the cells from TGF-β1-induced EMT and apoptosis, demonstrating the role of ER stress as an adaptive response to protect HPMCs from EMT and apoptosis. Peritoneal mesothelial cells isolated from PD patients displayed an increase in GRP78/94, which was correlated with the degree of EMT. These findings suggest that the modulation of ER stress in HPMCs could serve as a novel approach to ameliorate peritoneal damage in PD patients.

Inhibition of Endoplasmic Reticulum Stress Normalizes Augmented Myogenic Response in Coronary Arteris of the Spontaneously Hypertensive Rats

Endoplasmic reticulum (ER) stress has been shown to play a critical role in the pathogenesis of cardiovascular complications. However, the role and mechanisms of ER stress in hypertension remain unclear. Thus, we hypothesized that enhanced ER stress contributes to the maintenance of hypertension in spontaneously hypertensive rats (SHRs). We investigated this hypothesis by suppressing ER stress in SHRs and their normotensive controls, Wistar‐Kyoto rats (WKYs) with ER stress inhibitor (taurine‐conjugated ursodeoxycholic acid, TUDCA). Sixteen‐week old male SHRs and WKYs were treated with TUDCA (100 mg/kg/day, IP injection) or PBS (control, 300 μl/day, IP injection) for two weeks. There was a decrease in systolic blood pressure in SHR treated with TUDCA but not in SHR treated with PBS. The myogenic response is potentiated in coronary arteries of SHRs compared to WKYs. Interestingly, treatment of ER stress inhibitor normalized myogenic responses in SHRs. These data were associated with an increase in ER stress marker expression (Bip, CHOP, ATF6, XBP‐1, and phosphorylated‐eIF2α) in coronary arteries of SHRs, which were reduced by TUDCA treatment. In conclusion, ER stress inhibition decreases systolic blood pressure and normalizes myogenic response in SHRs. Moreover, ER stress inhibition reduces expression of ER stress markers. Therefore, we suggest that ER stress could be a potential target for hypertension.

Chaperones ameliorate beta cell dysfunction associated with human islet amyloid polypeptide overexpression.

In type 2 diabetes, beta-cell dysfunction is thought to be due to several causes, one being the formation of toxic protein aggregates called islet amyloid, formed by accumulations of misfolded human islet amyloid polypeptide (hIAPP). The process of hIAPP misfolding and aggregation is one of the factors that may activate the unfolded protein response (UPR), perturbing endoplasmic reticulum (ER) homeostasis. Molecular chaperones have been described to be important in regulating ER response to ER stress. In the present work, we evaluate the role of chaperones in a stressed cellular model of hIAPP overexpression. A rat pancreatic beta-cell line expressing hIAPP exposed to thapsigargin or treated with high glucose and palmitic acid, both of which are known ER stress inducers, showed an increase in ER stress genes when compared to INS1E cells expressing rat IAPP or INS1E control cells. Treatment with molecular chaperone glucose-regulated protein 78 kDa (GRP78, also known as BiP) or protein disulfite isomerase (PDI), and chemical chaperones taurine-conjugated ursodeoxycholic acid (TUDCA) or 4-phenylbutyrate (PBA), alleviated ER stress and increased insulin secretion in hIAPP-expressing cells. Our results suggest that the overexpression of hIAPP induces a stronger response of ER stress markers. Moreover, endogenous and chemical chaperones are able to ameliorate induced ER stress and increase insulin secretion, suggesting that improving chaperone capacity can play an important role in improving beta-cell function in type 2 diabetes.

Endoplasmic reticulum stress contributes coronary artery dysfunction in type 2 diabetic mice (1086.7)

Background: Vascular dysfunction is a major complication in type 2 diabetes. Although endoplasmic reticulum (ER) stress has been suggested to be a contributing factor in cardiovascular diseases, the relationship between ER stress and type 2 diabetic vascular dysfunction remains unclear. Thus, in the present study, we examined whether ER stress contributes coronary artery dysfunction and ER stress inhibition ameliorates vascular function in type 2 diabetes. Methods and Results: Type 2 diabetic and their control mice were treated with or without ER stress inhibitors (taurine‐conjugated ursodeoxycholic acid, TUDCA, and 4‐phenylbutyric acid, 4‐PBA) for 2 weeks. In type 2 diabetic mice, blood glucose, body weight, and insulin level are elevated compared to control mice. The myogenic response is potentiated and endothelium‐dependent relaxation is impaired in type 2 diabetic mice. Interestingly, treatment of ER stress inhibitors normalized myogenic responses and endothelium dependent relaxation.These data were associated with an increase in ER stress marker expression (CHOP, ATF6, XBP‐1, and phosphorylated‐eIF2α) in heart and mesenteric resistance arteries in type 2 diabetic mice, which were reduced by treatment with ER stress inhibitors. Conclusion: ER stress inhibition normalizes myogenic response and improves vascular function in type 2 diabetes. Therefore, ER stress could be a potential target for cardiovascular diseases in diabetes mellitus.Grant Funding Source: supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF)

Neuronal cell death and regeneration in diseases associated with advanced glycation end-products accumulation.

Recent studies have shown that the glycation-associated damage is not limited to patients with diabetes. Because of their association with oxidative stress, advanced glycation end-products (AGEs) have also been implicated in many neurodegenerative diseases, such as Huntington disease, amyotrophic lateral sclerosis, and Alzheimer disease (Yan et al., 1996; Ma and Nicholson, 2004). As in other age-dependent neurodegenerative diseases of the brain, oxidative stress-associated age-dependent pathogenic processes are not unexpected in glaucoma because this disease is also more common in the elderly (Tezel et al. 2007). Growing evidence indicates that neuronal abnormalities including neuronal cell death are associated with the pathogenesis of early diabetic retinopathy. Our previous studies of human retinas indicate that mitochondrial- and caspase-dependent cell death pathways are associated with retinal neuronal cell degeneration in patients with diabetes (Oshitari et al., 2008). Brain-derived neurotrophic factor, neurotrophin-4 (NT-4), vascular endothelial growth factor (VEGF)120, VEGF164, taurine-conjugated ursodeoxycholic acid and citicoline had been studied and showed a survival effect on damaged retinal neurons induced by diabetic stress (Oshitari et al. 2003, 2010). It was also found that NT-4 had the best neuroprotective and regenerative effect under high glucose conditions (Oshitari et al, 2010). Earlier study indicated that the maximum rescue ratio of caspase-1, -3, -8, and -9 inhibitors in cultured retinas was 60% in damaged retinal ganglion cells (RGCs) (Oshitari and Adachi-Usami, 2003). Thus, at least 40% of neuronal cell death in damaged RGCs in cultured retinas should be related to caspase-independent cell death mechanisms. However, no reports have focused on caspase-independent cell death pathways under diabetic stress including AGEs exposure. Apoptosis-inducing factor (AIF) was the first mitochondrial protein shown to mediate cell death independent of caspase (Susin et al., 1996, 1999). It was initially characterized as a mitochondrial protein confined in the intermembrane space of healthy cells. In healthy cells, AIF is a mitochondrial flavin adenine dinucleotide (FAD)-dependent oxidoreductase that plays roles in redox control and oxidative phosphorylation (Modjtahedi et al., 2006).

Increased expression of phosphorylated c-Jun and phosphorylated c-Jun N-terminal kinase associated with neuronal cell death in diabetic and high glucose exposed rat retinas

The aim of this study is to examine whether the increased expression of phosphorylated c-Jun (p-c-Jun) and phosphorylated c-Jun N-terminal kinase (p-JNK) are significantly associated with neuronal cell death in diabetic rat retinas and retinas exposed to high glucose. Retinas isolated from six adult male Sprague-Dawley rats and six streptozotocin-induced diabetic rats (DM) were cultured in serum-free medium. The explants from non-diabetic controls were cultured in normal-glucose (N) or high-glucose (HG) medium. Furthermore, neurotrophin-4 (NT-4) and Taurine-conjugated ursodeoxycholic acid (TUDCA) were incubated in HG medium. After 7 days, the numbers of regenerating neurites were counted per explant. After counting, the explants were fixed, cryosectioned, and stained by TUNEL, and also immunostained for p-c-Jun and p-JNK. The numbers of TUNEL-positive, p-c-Jun- and p-JNK-immunopositive cells in the GCL were significantly higher and the numbers of regenerating neurites were significantly lower in the HG and the DM groups than in the N groups. In the HG groups supplemented with NT-4 and TUDCA, the numbers of TUNEL-positive, p-c-Jun- and p-JNK-immunopositive cells were significantly lower and the numbers of neurites were significantly higher than in the HG group without NT-4 and TUDCA. Increased expression of p-c-Jun and p-JNK is associated with neuronal cell death in diabetic rat retinas and retinas exposed to high glucose. Neuroprotective effect of TUDCA and NT-4 is correlated with the suppression of p-c-Jun and p-JNK expression. These results provide a better understanding of the neurodegenerative process underlying DR.

Involvement of Endoplasmic Reticulum Stress in Albuminuria Induced Inflammasome Activation in Renal Proximal Tubular Cells

Albuminuria contributes to the progression of tubulointerstitial fibrosis. Although it has been demonstrated that ongoing albuminuria leads to tubular injury manifested by the overexpression of numerous proinflammatory cytokines, the mechanism remains largely unknown. In this study, we found that the inflammasome activation which has been recognized as one of the cornerstones of intracellular surveillance system was associated with the severity of albuminuria in the renal biopsies specimens. In vitro, bovine serum albumin (BSA) could also induce the activation of NLRP3 inflammasome in the cultured kidney epithelial cells (NRK-52E). Since there was a significant overlap of NLRP3 with the ER marker calreticulin, the ER stress provoked by BSA seemed to play a crucial role in the activation of inflammasome. Here, we demonstrated that the chemical chaperone taurine-conjugated ursodeoxycholic acid (TUDCA) which was proved to be an enhancer for the adaptive capacity of ER could attenuate the inflammasome activation induced by albuminuria not only in vitro but also in diabetic nephropathy. Taken together, these data suggested that ER stress seemed to play an important role in albuminuria-induced inflammasome activation, elimination of ER stress via TUDCA might hold promise as a novel avenue for preventing inflammasome activation ameliorating kidney epithelial cells injury induced by albuminuria.

Abstract 38: Endoplasmic Reticulum Stress Pathway-mediated Apoptosis is Involved in Ang II Induced Abdominal Aortic Aneurysm

Backgroud— Abdominal aortic aneurysms (AAA) represent a unique and dramatic example of vessel wall remodeling characterized by degeneration of the elastic media. Apoptosis of vascular smooth muscle cells plays an important role in the pathogenesis of AAA. However, the potential mechanism remains poorly understood. Endoplasmic reticulum stress (ER stress)-induced apoptosis has been proved to be one of the important pathogenesis of cardiovascular disease. However, whether ER stress is involved in AAA is still not known. We assessed the hypothesis that ER-associated apoptosis is involved in Angiotensin II (Ang II)-induced AAA in apolipoprotein E-deficient mice. Methods and Results— Mice were infused with Ang II (1000 ng/kg per minute) with or without ER stress inhibitor (taurine-conjugated ursodeoxycholic acid) for 4 weeks. Mice infused with Ang II displayed an increase in aortic diameter. Detection of apoptosis was performed with the TUNEL assay. We performed Western blot and Real-time PCR to analyze indicators of ER molecule chaperone and ER-associated apoptosis. Glucose Regulated Proteins 78 and 94 (GRP78/BiP and GRP94), the ER chaperone, were up-regulated significantly in AAA compared to control. Furthermore, the hallmarks of ER-associated apoptosis, C/EBP homologous protein (CHOP), caspase-12 and PERK-eIF2-ATF4 signaling pathway were found to have activated in the AAA. The inhibition of ER stress significantly decreased maximal aortic diameter by 31% and abdominal aortic weight by 35% ( P <0.05, respectively). ER stress inhibitor also reduced GRP 78, CHOP and caspase-12 expression ( P <0.05, respectively). Taken together, these results suggested that apoptosis induced by ER stress may contribute to the development of AAA. Conclusions— ER stress response is involved in the pathogenesis of Ang II induced AAA in apolipoprotein E-deficient mice. ER stress inhibition attenuates AAA formation during Ang II infusion in apolipoprotein E-deficient mice. Therefore, ER stress could be a potential target for AAA.

Abstract 518: Inhibition of Endoplasmic Reticulum Stress Reduces Aneurysm Induction in Different Aneuryms Models

Abdominal aorta aneurysm (AAA) is a major cause of mortality in men. The role of endoplasmic reticulum (ER) stress in the pathogenesis of AAA is unknown. Human AAA specimens from diabetic patients were found to have increased expression of ER stress markers. The inhibition of ER stress signaling with (taurine-conjugated ursodeoxycholic acid, Tudca) administration reduces the development of aneurysm in Ang-II infused and calcium chloride (CaCl2)-induced mouse models. Type 2 diabetic (db-/db-) and control (db-/db+) mice were infused with angiotensin II (Ang-II, 1000ng/kg/min) and treated with or without the ER stress inhibitor Tudca for 4 weeks. Mice infused with Ang-II displayed an increase in blood pressure, plasma cholesterol and CRP levels, which were reduced after ER stress inhibition. Human AAA and Ang-II-induced AAA are characterized by an increase in lumen, cross sectional area and were associated with increased macrophage infiltration, expression of the ER stress marker CHOP and elastic fiber disruption, and these indicators of aneurysm disease were reduced by Tudca treatment. To determine the effect of ER stress inhibition on aneurysm formation in a model that did not exhibit blood pressure and metabolic changes, we exposed the adventitial surface of carotid arteries of db-/db- and db-/db+ mice to CaCl2 and then treated the mice with or without Tudca. Our results indicate that in the CaCl2 induced carotid artery aneurysm model that treatment with Tudca significantly reduced carotid artery aneurysm formation. These results provide evidence in support of a role for that ER stress activation in the induction of aneurysm in type 2 diabetes.

AGEs Induce Cell Death via Oxidative and Endoplasmic Reticulum Stresses in Both Human SH-SY5Y Neuroblastoma Cells and Rat Cortical Neurons

Advanced glycation endproducts (AGEs) are elevated in aging and neurodegenerative diseases such as Alzheimer's disease (AD), and they can stimulate the generation of reactive oxygen species (ROSs) via NADPH oxidase, induce oxidative stress that lead to cell death. In the current study, we investigated the molecular events underlying the process that AGEs induce cell death in SH-SY5Y cells and rat cortical neurons. We found: (1) AGEs increase intracellular ROSs; (2) AGEs cause cell death after ROSs increase; (3) oxidative stress-induced cell death is inhibited via the blockage of AGEs receptor (RAGE), the down-regulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and the increase of scavenging by anti-oxidant alpha-lipoic acid (ALA); (4) endoplasmic reticulum (ER) stress was triggered by AGE-induced oxidative stress, resulting in the activation of C/EBP homologous protein (CHOP) and caspase-12 that consequently initiates cell death, taurine-conjugated ursodeoxycholic acid (TUDCA) inhibited AGE-induced ER stress and cell death. Blocking RAGE-NADPH oxidase, and RAGE-NADPH oxidase-ROSs and ER stress scavenging pathways could efficiently prevent the oxidative and ER stresses, and consequently inhibited cell death. Our results suggest a new prevention and or therapeutic approach in AGE-induced cell death.

Endoplasmic Reticulum Stress Is Involved in Cardiac Damage and Vascular Endothelial Dysfunction in Hypertensive Mice

Cardiac damage and vascular dysfunction are major causes of morbidity and mortality in hypertension. In the present study, we explored the beneficial therapeutic effect of endoplasmic reticulum (ER) stress inhibition on cardiac damage and vascular dysfunction in hypertension. Mice were infused with angiotensin II (400 ng/kg per minute) with or without ER stress inhibitors (taurine-conjugated ursodeoxycholic acid and 4-phenylbutyric acid) for 2 weeks. Mice infused with angiotensin II displayed an increase in blood pressure, cardiac hypertrophy and fibrosis associated with enhanced collagen I content, transforming growth factor-β1 (TGF-β1) activity, and ER stress markers, which were blunted after ER stress inhibition. Hypertension induced ER stress in aorta and mesenteric resistance arteries (MRA), enhanced TGF-β1 activity in aorta but not in MRA, and reduced endothelial NO synthase phosphorylation and endothelium-dependent relaxation (EDR) in aorta and MRA. The inhibition of ER stress significantly reduced TGF-β1 activity, enhanced endothelial NO synthase phosphorylation, and improved EDR. The inhibition of TGF-β1 pathway improved EDR in aorta but not in MRA, whereas the reduction in reactive oxygen species levels ameliorated EDR in MRA only. Infusion of tunicamycin in control mice induced ER stress in aorta and MRA, and reduced EDR by a TGF-β1-dependent mechanism in aorta and reactive oxygen species-dependent mechanism in MRA. ER stress inhibition reduces cardiac damage and improves vascular function in hypertension. Therefore, ER stress could be a potential target for cardiovascular diseases.

Oxidized lipid derived aldehyde, 4‐hydroxy‐trans‐2‐nonenal causes vascular inflammation by inducing endoplasmic reticulum stress

Oxidized lipoproteins‐derived aldehydes, such as 4‐hydroxy‐trans‐2‐nonenal (HNE) increase endothelial activation, however, mechanisms that mediate the effects of lipid oxidation products on the endothelium remain unclear. We observed HNE modified ER proteins in atherosclerotic lesions of apoE‐null mice. In vitro, treatment of endothelial cells (EC) and macrophages with HNE led to the modification of several proteins that colocalize with the endoplasmic reticulum (ER). HNE also caused the splicing of XBP‐1, an increase in the phosphorylation of PERK and eIF2á, and induction of ATF3 and ATF4. Phosphorylation of eIF2á by HNE was prevented by transfection with PERK siRNA. Treatment with HNE was also associated with a significant increase in the expression of ER chaperones, GRP78 and HERP. Pre‐treatment with a chemical chaperone, phenylbutyric acid (PBA) or adenoviral transfection with ATF6 attenuated HNE‐induced monocyte adhesion and IL‐8 induction. IL‐8 induction was also prevented by IRE1 siRNA. Moreover, PBA and taurine‐conjugated ursodeoxycholic acid (TUDCA) attenuated HNE‐induced leukocyte rolling and their firm adhesion to the endothelium in rat cremaster muscle. These data suggest that endothelial activation by HNE is mediated in part by ER stress. ER stress, induced by oxidized lipids may be a significant cause of vascular inflammation during atherogenesis.

Lipid Peroxidation Product 4-Hydroxy-trans-2-nonenal Causes Endothelial Activation by Inducing Endoplasmic Reticulum Stress

Lipid peroxidation products, such as 4-hydroxy-trans-2-nonenal (HNE), cause endothelial activation, and they increase the adhesion of the endothelium to circulating leukocytes. Nevertheless, the mechanisms underlying these effects remain unclear. We observed that in HNE-treated human umbilical vein endothelial cells, some of the protein-HNE adducts colocalize with the endoplasmic reticulum (ER) and that HNE forms covalent adducts with several ER chaperones that assist in protein folding. We also found that at concentrations that did not induce apoptosis or necrosis, HNE activated the unfolded protein response, leading to an increase in XBP-1 splicing, phosphorylation of protein kinase-like ER kinase and eukaryotic translation initiation factor 2α, and the induction of ATF3 and ATF4. This increase in eukaryotic translation initiation factor 2α phosphorylation was prevented by transfection with protein kinase-like ER kinase siRNA. Treatment with HNE increased the expression of the ER chaperones, GRP78 and HERP. Exposure to HNE led to a depletion of reduced glutathione and an increase in the production of reactive oxygen species (ROS); however, glutathione depletion and ROS production by tert-butyl-hydroperoxide did not trigger the unfolded protein response. Pretreatment with a chemical chaperone, phenylbutyric acid, or adenoviral transfection with ATF6 attenuated HNE-induced monocyte adhesion and IL-8 induction. Moreover, phenylbutyric acid and taurine-conjugated ursodeoxycholic acid attenuated HNE-induced leukocyte rolling and their firm adhesion to the endothelium in rat cremaster muscle. These data suggest that endothelial activation by HNE is mediated in part by ER stress, induced by mechanisms independent of ROS production or glutathione depletion. The induction of ER stress may be a significant cause of vascular inflammation induced by products of oxidized lipids.

Endoplasmic reticulum stress is a mediator of posttransplant injury in severely steatotic liver allografts

Hepatic steatosis continues to present a major challenge in liver transplantation. These organs have been shown to have increased susceptibility to cold ischemia/reperfusion (CIR) injury in comparison with otherwise comparable lean livers; the mechanisms governing this increased susceptibility to CIR injury are not fully understood. Endoplasmic reticulum (ER) stress is an important link between hepatic steatosis, insulin resistance, and metabolic syndrome. In this study, we investigated ER stress signaling and blockade in the mediation of CIR injury in severely steatotic rodent allografts. Steatotic allografts from genetically leptin-resistant rodents had increased ER stress responses and increased markers of hepatocellular injury after liver transplantation into strain-matched lean recipients. ER stress response components were reduced by the chemical chaperone taurine-conjugated ursodeoxycholic acid (TUDCA), and this resulted in an improvement in the allograft injury. TUDCA treatment decreased nuclear factor kappa B activation and the proinflammatory cytokines interleukin-6 and interleukin-1β. However, the predominant response was decreased expression of the ER stress cell death mediator [CCAAT/enhancer-binding protein homologous protein (CHOP)]. Furthermore, activation of inflammation-associated caspase-11 was decreased, and this linked ER stress/CHOP to proinflammatory cytokine production after steatotic liver transplantation. These data confirm ER stress in steatotic allografts and implicate this as a mediating mechanism of inflammation and hepatocyte death in the steatotic liver allograft.

Changes in Hepatic Gene Expression upon Oral Administration of Taurine-Conjugated Ursodeoxycholic Acid in ob/ob Mice

Nonalcoholic fatty liver disease (NAFLD) is highly prevalent and associated with considerable morbidities. Unfortunately, there is no currently available drug established to treat NAFLD. It was recently reported that intraperitoneal administration of taurine-conjugated ursodeoxycholic acid (TUDCA) improved hepatic steatosis in ob/ob mice. We hereby examined the effect of oral TUDCA treatment on hepatic steatosis and associated changes in hepatic gene expression in ob/ob mice. We administered TUDCA to ob/ob mice at a dose of 500 mg/kg twice a day by gastric gavage for 3 weeks. Body weight, glucose homeostasis, endoplasmic reticulum (ER) stress, and hepatic gene expression were examined in comparison with control ob/ob mice and normal littermate C57BL/6J mice. Compared to the control ob/ob mice, TUDCA treated ob/ob mice revealed markedly reduced liver fat stained by oil red O (44.2±5.8% vs. 21.1±10.4%, P<0.05), whereas there was no difference in body weight, oral glucose tolerance, insulin sensitivity, and ER stress. Microarray analysis of hepatic gene expression demonstrated that oral TUDCA treatment mainly decreased the expression of genes involved in de novo lipogenesis among the components of lipid homeostasis. At pathway levels, oral TUDCA altered the genes regulating amino acid, carbohydrate, and drug metabolism in addition to lipid metabolism. In summary, oral TUDCA treatment decreased hepatic steatosis in ob/ob mice by cooperative regulation of multiple metabolic pathways, particularly by reducing the expression of genes known to regulate de novo lipogenesis.

Conjugation is essential for the anticholestatic effect of NorUrsodeoxycholic acid in taurolithocholic acid-induced cholestasis in rat liver

NorUDCA (24-norursodeoxycholic acid), the C₂₃-homolog of ursodeoxycholic acid (UDCA), showed remarkable therapeutic effects in cholestatic Mdr2 (Abcb4) (multidrug resistance protein 2/ATP-binding cassette b4) knockout mice with sclerosing/fibrosing cholangitis. In contrast to UDCA, norUDCA is inefficiently conjugated in human and rodent liver, and conjugation has been discussed as a key step for the anticholestatic action of UDCA in cholestasis. We compared the choleretic, anticholestatic, and antiapoptotic properties of unconjugated and taurine-conjugated UDCA (C₂₄) and norUDCA (C₂₃) in isolated perfused rat liver (IPRL) and in natrium/taurocholate cotransporting polypeptide (Ntcp)-transfected human hepatoma (HepG2) cells. Taurolithocholic acid (TLCA) was used to induce a predominantly hepatocellular cholestasis in IPRL. Bile flow was determined gravimetrically; bile acids determined by gas chromatography and liquid chromatography/tandem mass spectrometry; the Mrp2 model substrate, 2,4-dinitrophenyl-S-glutathione (GS-DNP) was determined spectrophotometrically; and apoptosis was determined immunocytochemically. The choleretic effect of C₂₃-bile acids was comparable to their C₂₄-homologs in IPRL. In contrast, TnorUDCA, but not norUDCA antagonized the cholestatic effect of TLCA. Bile flow (percent of controls) was 8% with TLCA-induced cholestasis, and unchanged by coinfusion of norUDCA (14%). However, it was increased by TnorUDCA (83%), UDCA (73%) and TUDCA (136%). Secretion of GS-DNP was markedly reduced by TLCA (5%), unimproved by norUDCA (4%) or UDCA (17%), but was improved modestly by TnorUDCA (26%) or TUDCA (58%). No apoptosis was observed in IPRL exposed to low micromolar TLCA, but equivalent antiapoptotic effects of TUDCA and TnorUDCA were observed in Ntcp-HepG2 cells exposed to TLCA. Conjugation is essential for the anticholestatic effect of norUDCA in a model of hepatocellular cholestasis. Combined therapy with UDCA and norUDCA may be superior to UDCA or norUDCA monotherapy in biliary disorders in which hepatocyte as well as cholangiocyte dysfunction contribute to disease progression.

Linking endoplasmic reticulum stress to cell death in hepatocytes: roles of C/EBP homologous protein and chemical chaperones in palmitate-mediated cell death.

Prolonged endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) have been linked to apoptosis via several mechanisms, including increased expression of C/EBP homologous protein (Chop). Increased long-chain fatty acids, in particular saturated fatty acids, induce ER stress, Chop expression, and apoptosis in liver cells. The first aim of the present study was to determine the role of Chop in lipid-induced hepatocyte cell death and liver injury induced by a methionine-choline-deficient diet. Albumin-bound palmitate increased Chop gene and protein expression in a dose-dependent fashion in H4IIE liver cells. siRNA-mediated silencing of Chop in H4IIE liver cells reduced thapsigargin-mediated cell death by approximately 40% and delayed palmitate-mediated cell death, but only at high concentrations of palmitate (400-500 microM). Similar results were observed in primary hepatocytes isolated from Chop-knockout mice. Indices of liver injury were also not reduced in Chop-knockout mice provided a methionine-choline-deficient diet. To ascertain whether ER stress was linked to palmitate-induced cell death, primary hepatocytes were incubated in the absence or presence of the chemical chaperones taurine-conjugated ursodeoxycholic acid or 4-phenylbutyric acid. The presence of either of these chemical chaperones protected liver cells from palmitate-mediated ER stress and cell death, in part, via inhibition of JNK activation. These data suggest that ER stress is linked to palmitate-mediated cell death via mechanisms that include JNK activation.