{'fraction_non_alphanumeric': 0.02621078701155751, 'fraction_numerical': 0.016717116125481563, 'mean_word_length': 5.190063875088716, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Mutual interaction between oxidative stress and endoplasmic reticulum stress in the pathogenesis of diseases specifically focusing on non-alcoholic fatty liver disease

Reactive oxygen species (ROS) are produced during normal physiologic processes with the consumption of oxygen. While ROS play signaling roles, when they are produced in excess beyond normal antioxidative capacity this can cause pathogenic damage to cells. The majority of such oxidation occurs in polyunsaturated fatty acids and sulfhydryl group in proteins, resulting in lipid peroxidation and protein misfolding, respectively. The accumulation of misfolded proteins in the endoplasmic reticulum (ER) is enhanced under conditions of oxidative stress and results in ER stress, which, together, leads to the malfunction of cellular homeostasis. Multiple types of defensive machinery are activated in unfolded protein response under ER stress to resolve this unfavorable situation. ER stress triggers the malfunction of protein secretion and is associated with a variety of pathogenic conditions including defective insulin secretion from pancreatic β-cells and accelerated lipid droplet formation in hepatocytes. Herein we use nonalcoholic fatty liver disease (NAFLD) as an illustration of such pathological liver conditions that result from ER stress in association with oxidative stress. Protecting the ER by eliminating excessive ROS via the administration of antioxidants or by enhancing lipid-metabolizing capacity via the activation of peroxisome proliferator-activated receptors represent promising therapeutics for NAFLD.


INTRODUCTION
Most eukaryotic cells, excluding enucleated cells such as mammalian red blood cells, synthesize proteins based on genetic information from nuclear DNA during their lifespan. Translated secretory and membrane proteins become functional when they undergo appropriate oxida tive folding, posttranslational modification, and cellular localization. While the redox status in the cytosol is in a reduced state, the extracellular space is largely in an oxi dized state. Premature secretion and exposure to an ex tracellular environment can cause aberrant conformational changes in proteins due to inappropriate disulfide bond formation. Hence, the appropriate oxidative folding of polypeptides by means of the oxidation of sulfhydryl groups to produce disulfide bridges in proteins within cells is a prerequisite process for producing secretory proteins and the membrane proteins prior to their secretion and translocation to the plasma membrane, respectively ( Figure  1). The signaling process for these types of oxidative protein folding reactions was not understood well until the end of the last century [1,2] . The accumulation of misfolded proteins in the endo plasmic reticulum (ER) causes ER stress, which then leads to the malfunction of the ER [3] . Without proper resolution of ER stress, affected cells become dysfunctional and, if not resolved properly, they die. To avoid this unfavorable scenario, multiple defensive machineries, referred to as an unfolded protein response (UPR), are activated under such conditions and play roles in preventing this and permit the cells to recover from this fatal situation [4,5] . Multiple factors, either internally or externally, can cause the accumulation of misfolded proteins in the ER [6,7] . Reactive oxygen species (ROS) are produced during con ventional physiological processes accompanied by oxygen consumption and the levels are enhanced under a variety of pathological conditions such as inflammation, high temperature, and a deficit in the antioxidative system, and result in the development of oxidative stress [8] .
Both low molecular weight antioxidant compounds and antioxidative enzymes function to control the levels of ROS and reduce their levels to acceptable ranges. However, the antioxidant levels in the ER are relatively low compared to the cytoplasm or other organelles, despite the robust production of hydrogen peroxide via active reduction oxidation (redox) reactions [9] . Oxidative stress perturbs the usual oxidative protein folding, which results in the ER stress and organ failure. Thus, among a variety of stressful conditions, oxidative stress can occur in any cell and is also responsible for ER stress, and they together lead to the development of a pathogenic state.
Herein we overview recent progress in our under standing of the relationships between oxidative stress and ER stress and attempt to clarify the pathogenic pathways that are involved, by focusing on fatty liver diseases.

OXIDATIVE FOLDING OF SECRETORY AND MEMBRANE PROTEINS IN THE ER
Protein conformation is supported by several types of chemical bonds, among which the disulfide bonds formed between cysteine sulfhydryl groups are the primary deter minants of overall protein structure for secreted proteins and membrane proteins that face the extracellular space. Endoplasmic reticulum oxidoreductin 1 (ERO1), which contains flavin adenine dinucleotide (FAD) as a redox cofactor, is an evolutionarily conserved protein [10] and, in conjunction with molecular oxygen consumption, catalyzes disulfide formation in nascent proteins via protein disulfide isomerase (PDI) in the ER [11,12] . PDI is a member of the family of chaperone molecules that are specifically re sponsible for disulfide bond formation in proteins in the ER lumen [13,14] . In addition to PDI, several chaperone mole cules are also present in the ER lumen and participate in maintaining ER homeostasis [15] . Mammals have two ERO1 genes, ERO1α and ERO1β, that are transcriptionally regulated by the CCAATenhancerbinding protein ho mologous protein (CHOP) [16] . ERO1 first introduces a disulfide bond in PDI and its family members using mole cular oxygen as the oxidant [13,14] . Hydrogen peroxide is produced as a byproduct in this oxidative protein folding process. Because not only secretory proteins but also many membrane proteins that face the extracellular space must undergo oxidative protein folding in the ER, hydro gen peroxide is inevitably produced in the ERO1mediated sulfoxidation reaction. Thus, the ER is exposed to an oxi dative insult to a greater or lesser extent as the result of this type of oxidative protein folding [17] . ERO1 is prerequisite for oxidative protein folding Fujii J et al . Oxidative stress induces ER stress in yeast and the genetic ablation of the gene results in yeast that are hypersensitive to reducing agents such as dithiothreitol [18,19] . However the genetic ablation of genes encoding ERO1α and ERO1β cause only moderate effects in mice, e.g., a decrease in ERO1 activity blunts the cardiomyocyte inotropic response to adrenergic stimu lation and sensitized mice to adrenergic blockade [20] . This rather mild phenotype compared to the case of yeast can be explained by the presence of other redundant sulfoxi dase enzymes in mammalian cells. Quiescin sulfhydryl oxidase (QSOX), a flavoprotein, catalyzes disulfide for mation in PDI family members using an oxygen mole cule and produces hydrogen peroxide [21] . The enzymatic properties are somewhat similar to ERO1. However, since QSOX is localized primarily in the Golgi apparatus and secreted fluids it appears to be unique as a multi domain enzyme [22] . Mammalian cells also possess other sulfoxidases that introduce disulfide bonds to PDI by means of hydrogen peroxide or other oxidizing power, such as peroxiredoxin (PRDX)4 and glutathione peroxidase (GPX)7/GPX8 [23] , as described below.

STRESS
The accumulation of inappropriately folded proteins is caused by unfavorable conditions, including the excessive synthesis of proteins, the inhibition of protein glycosylation, and Ca 2+ depletion [24] . ER homeostasis is maintained by a variety of factors that include ionic and metabolic conditions. For example, ER is the organelle that stores millimolar ranges of Ca 2+ which is maintained by Ca 2+ ATPase localized at the ER membrane. Cardiac and skeletal muscle possess specialized forms for this Ca 2+ storage, designated as the sarcoplasmic reticulum (SR), that re gulates excitationcontraction coupling in these muscular tissues [25] . ATPase and leads to pathological conditions via ER stress [26] . Some antibiotics, e.g., tunicamycin, inhibit the formation of Nlinked glycosylation, leading to the suppression of the translocation of nascent proteins to the Golgi body [27] . Heat stress impairs the proper folding of proteins by affecting the free energy needed for correct protein folding and thereby produces misfolded proteins with a high frequency [28] . As a compensatory mechanism, heat shock proteins (HSPs), a large protein family, are induced at high temperatures and ameliorate protein folding via their ability to function as a chaperone. Repetitive or sustained stimulation of hormonal secretion, such as the case of insulin secretion under hyperglycemic conditions, is a sus pected cause for defected insulin production and βcell dysfunction, which typically leads to type Ⅱ diabetes [24] . Because the accumulation of misfolded proteins cau ses dysfunction of the ER and, if not properly resolved,  ultimately results in cell death, several systems in the UPR are activated to resolve the stress. Genes involved in ameliorating ER stress are largely induced by several mechanisms via sensor proteins that are localized on the ER membrane. Because this process has been overviewed extensively by experts in this research field [5,29] , we briefly mention major machineries and related matters here.

Control of protein synthesis on the rough ER
Both secretory proteins and membrane proteins are syn thesized on the rough ER where ribosomemRNA com plexes are attached. Eukaryotic initiation factor 2α (eIF2α) plays a key role in the regulation of the translational process from mRNA on the cytoplasmic surface of the rough ER. Upon ER stress, doublestranded RNAacti vated eIF2α kinaselike ER kinase (PERK) is activated upon autophosphorylation and phosphorylates eIF2α, which results in the selective translation of activating transcription factor 4 (ATF4) [30] . ATF4 then transcribes acti vating transcription factor 3 (ATF3), CHOP, and growth arrest and DNA damageinducible 34 (GADD34). In the meantime phosphorylated eIF2α causes the synthesis of many other protein to be suppressed and restricts further protein influx to the ER lumen via attenuating the guanine nucleotide exchange factor eIF2B [31] .

Sensing and signaling to nucleus
The inositol requiring enzyme 1 (IRE1) was originally reported as a yeast membrane protein that was pre dicted to be a kinase involved in inositol metabolism [32] .
The potential role of IRE1 in the transmission of the unfolded protein signal across the ER or the inner nu clear membrane was then rediscovered later [33] . IRE1 undergoes activation via oligomerization in response to unfolded proteins and autophoshorylation. Active IRE1 then completes the splicing of the Xboxbindingprotein 1 (XBP1) precursor mRNA by its RNase activity [34] . The XBP1 protein translated from the mature XBP1 mRNA moves to the nucleus and then transcriptionally activates genes for a protein chaperone, such as the glucoseregulated protein of 78 kDa (GRP78 also referred to as Bip) [35] and PDIs [36] . Activating transcription factor 6 (ATF6) was also originally found in yeast [37] , but its localization in the ER membrane was recognized at later period. The localization of ATF6 to the ER membrane in its inactive form and its subsequent transcriptional activation in response to UPR have been reported [38] . ER stress stimulates the translocation of the precursor form of ATF6 to the Golgi membrane where Site1 (S1P) and the Site2 proteases (S2P) are located [39,40] . These proteases release the transcriptional activator domain from the precursors, and the resulting active ATF6 moves to the nucleus where it transcriptionally activates the corresponding genes [41] . Astrocytes specifically express a unique ER stress transducer, the old astrocyte specifically induced sub stance (OASIS), that regulates the signaling of UPR and contributes to the resistance to ER stress [42] .

ER-associated degradation of misfolded proteins
Misfolded proteins that are formed within the ER lumen undergo either refolding to the correct conformation in a chaperonemediated manner or undergo proteolytic degradation, designated as ERassociated degradation (ERAD) [43] . PDI is the first enzyme that was reported to catalyze protein folding [44] . Nearly 20 PDI family proteins are present in the ER lumen and are involved in correcting disulfide bonds and also the oxidative folding of nascent proteins in the ER in association with ERO1 and other sulfoxidases [45] . Some unfolded proteins are exported out from the ER lumen or the ER membrane, polyubiquitinated and then degraded by proteasomes [46] . ERAD pathways, which are further classified into ERADL, M, and C, are responsible for misfolded proteins in the ER lumen, within the mem brane, or on the cytosolic side of the ER membrane, re spectively [43] . RINGfinger ligase Hrd1, in association with other components appear to play a major role in ERADL and ERADM [47] . A retrotranslocation channel is formed by Hrd1 in complex with the partner molecule Hrd3 and are involved in the polyubiquitination of target proteins [48] . Thus, among the several candidate channel molecules, the Hrd1Hrd3 complex appears to play a bona fide role in polyubiquitination/protein translocation in the ERAD. Derin 1 (Der1), which reportedly initiates the export of aberrant proteins from the ER [49] , appears to aid luminal substrates to be moved to the Hrd1 channel. The potential involvement of other ubiquitin ligases that span the ER membrane in this manner has also been proposed.
The machinery responsible for ERAD is also involved in cholesterol homeostasis by controlling the protein levels of 3hydroxy3methylglutaryl coenzyme A (HMG CoA) reductase in the ER membrane [50] . HMG CoA reductase, which is embedded in the ER membrane, limits cholesterol synthesis. The presence of a sufficient amount of cho lesterol results in an accelerated degradation of HMG CoA reductase via the activation of polyubiquitination followed by proteasomecatalyzed degradation [51] , a process that is independent from ER stress. Hence, the ERAD system appears to be involved in, not only the proteolytic removal of misfolded proteins, but also regulation of the cholesterol metabolic process by means of the same ERassociated machinery. Moreover, because some ERresident proteins such as Bip [35] , PDI [36] , and PRDX4 [52,53] are reportedly present in the cytosol, the ERAD machinery may be partly responsible for the retrotranslocation of these proteins from the ER to the cytosol.

Induction of cell death
Cell death is induced when these protective mechanisms against the ER stress functions insufficiently and do not rescue ER function [54] . CHOP, also referred to as GADD153, is a developmentally regulated nuclear protein that has a strong sequence similarity to transcription factors C/ EBP. CHOP functions as a dominantnegative inhibitor of the activity of C/EBPlike proteins [55] . CHOP induces the production of ERO1α, which then activates the inositol 1,4,5triphosphate (IP3) receptor and stimulates the release of Ca 2+ from the ER via the IP3 receptor channel.
The increased levels of cytosolic Ca 2+ then trigger apop tosis in cells that are under ER stress [56] , and induction Fujii J et al . Oxidative stress induces ER stress of cell death is a wellestablished function of CHOP [57] . ERO1α was found to be enriched in the mitochondrial as sociated membrane (MAM), the interface area of the ER with mitochondria [58] , and may also regulate mitochondrial Ca 2+ flux via controlling redox homeostasis in the ER [59] . The proapoptotic roles of CHOP are supported by the finding that the disruption of the gene that encodes CHOP causes delays in the development of ER stressmediated diabetes and decreases the sensitivity of pancreatic islet cells to nitric oxide toxicity [60,61] . PERK and the IRE1 pathway activate CHOP, which, after translocation to the nucleus, inhibits the expression of antiapoptotic genes, such a BCL2, and activates the proapoptotic genes, Bim and DR5 [62,63] . Nevertheless, CHOP appears to have an additional function, which needs clarification [64] .

OXIDATIVE STRESS AND ER STRESS
Superoxide radicals are produced primarily via a one electron donation to molecular oxygen, which is mediated either enzymatic reactions such as oxygenases or non enzymatic reactions such as glycoxidation [65,66] ( Figure   2). While ROS, notably hydrogen peroxide, produced by stimuli function as signal regulators [67] , excessively pro duced ROS cause oxidative stress that can exert a variety of effects over cellular functions. Transferring an unpaired electron together with oxygen to an unsaturated lipid causes lipid peroxidation, which then triggers a radical chain reaction [68] . Superoxide is not reactive as a radical, but it may trigger the production of more reactive free radicals by a reaction referred to as Fenton chemistry, a process that is initiated by the donation of an electron to a transition metal ion, such as Fe 3+ [69] . The resultant Fe 2+ reacts with hydrogen peroxide, leading to the production of hydroxyl radicals, one of the most harmful molecules, which oxidizes lipids that contain unsaturated fatty acids, bases in nucleic acids, and proteins.

Sources for oxidative stress in the ER
Based on the amount of consumed oxygen molecules, mitochondria are generally regarded as the main source of ROS production. MAM appears to be the place where these organelles communicate and provide ROS to the ER from mitochondria [58] . NADPHoxidases (NOX) are another source of ROS and frequently cause oxidative stress, especially under inflammatory conditions [70] . After mitochondria, the ER is the organelle that consumes the most molecular oxygen because there are several oxy genases that are associated with this organelle. While most NOX members are located in the plasma membrane and produce superoxide as primary ROS, NOX4, a ERresident enzyme, faces the ER lumen and releases hydrogen per oxide by the twoelectron reduction of an oxygen mole cule [71] . Cytochrome P450 is largely associated with the ER membrane and releases ROS to the cytoplasmic side [72,73] . However, these genes are selectively expressed in tissues, such as the liver and steroidogenic organs.
In the meantime, oxygen molecule is also consumed inside the ER by ERO1 [74,75] , which is involved in oxidative protein folding in most cells, as mentioned above. In fact, the ER is the organelle that produces the highest levels of hydrogen peroxide [76] . The levels of hydrogen peroxide increase under conditions where the secretion of proteins is enhanced due to ERO1mediated oxidative protein Fujii J et al . Oxidative stress induces ER stress folding [77] . Insulin production is maintained at high levels under prolonged hyperglycemic conditions and hydrogen peroxide levels are also kept high, which may cause oxidative stress together with ER stress in pancreatic βcells and ultimately aberrant insulin secretion [78] . Hydrogen peroxide may also come from other cellular compartments or reactions other than ERO1catalysed PDI oxidation [79] .

Machineries maintaining ER homeostasis against oxidative stress
To avoid oxidative stress, ROS are either eliminated via interactions with low molecular weight antioxidants, such as glutathione (GSH) and vitamin C, or converted to lessreactive compunds by antioxidative enzymes. Superoxide is exclusively converted to hydrogen peroxide by superoxide dismutase (SOD). In the case of the eli mination of hydrogen peroxide, GSH peroxidase (GPX) and peroxiredoxin (PRDX), which are encoded by re spective gene families [80,81] , largely contribute to the reductive detoxification of hydrogen peroxide. While con ventional GPX utilizes GSH as the electron donor, PRDX largely utilizes thioredoxin for this purpose. Extracellular superoxide dismutase (SOD3) and the plasma form of GPX (GPX3) are produced in the ER in the same manner as other secretory proteins. However, while some of them may be retained in the ER lumen, most are excreted. Thus, only few antioxidative systems are present in the ER lumen compared to cytoplasm and mitochondria.

Conversion of superoxide to hydrogen peroxide
Superoxide is converted to hydrogen peroxide by dismu tation, either by a spontaneous reaction between two superoxide molecules or by accelerated production via the SODcatalyzed reaction [82] . The latter reaction is about 3000 times faster than the spontaneous dismutation and causes superoxide levels to be decreased to less than 0.5% of that of the SOD1free erythrocytes [83] . However, the physiological relevance of SOD is sometimes a topic of debate because hydrogen peroxide, which is not a radical but is still a reactive compound, is the product of the catalytic conversion of superoxide. While hydrogen peroxide does not itself cause a radical chain reaction, on accepting one electron, hydrogen peroxide is converted to a highly toxic molecule, hydroxyl radical, via the Fenton reaction [69] . The Fenton reaction involves a oneelectron reduction of a transition metal ion in the preceding step and can be prevented by either the removal of the free transition metal ion or the elimination of the radical species that serves as the electron donor. Thus, the physiological significance of SOD can be rationally explained by the rapid elimination of superoxide before donating its un paired electron. The resulting hydrogen peroxide can be reduced immediately to water by peroxidases that abun dant in the body.
Among the three SOD isozymes present in mam malian cells, SOD1, which is localized in the cytoplasm and the intermembrane space in mitochondria as well, is expressed ubiquitously in the body and the best cha racterized from the biochemical and pathological view points [82] . A missense mutation in SOD1 is reportedly a cause of familial amyotrophic lateral sclerosis (ALS) [84] . More than 150 mutations have been identified in the SOD1 gene in familial ALS, and several model mice have been established by the transgenic overexpression of the mutant gene and have been employed in attempts to elucidate the etiology of ALS and to develop possible drugs [85] . On the contrary, the genetic ablation of SOD1 does not induce ALS in mice [86] , indicating that ALS de velops as a consequence of the gainoffunction of mu tant SOD1. Among several proposed mechanisms, an attractive hypothesis has been proposed from viewpoint of ER stressmediated motor neuron death as follows. ALSlinked mutations of SOD1 cause chronic ER stress through interactions with Der1, a component proposed for ERAD [87] , as described above. In addition it is noteworthy that, under zincdepleted conditions, the wildtype SOD1 interacts with Der1 [88,89] , suggesting that SOD1 plays a role in the interaction with Der1. Therefore SOD1 may be post translationally converted into a pathogenic molecule under zinc insufficient conditions and could also cause sporadic ALS. In the meantime, mutant SOD1 interacts with sub units of the coatomer coat protein Ⅱ (COPⅡ) complex, which is essential for ERGolgi transport [90] . Thus, mutant SOD1 may aggravate ER stress via the suppression of membrane transport from the ER to the Golgi body.

Reductive detoxification of hydrogen peroxide and oxidative folding by PRDX4
Because ERO1 and other oxidoreductases that are in volved in oxidative protein folding produce hydrogen per oxide, which would be expected to lead to ER stress via oxidative modification, especially sulfhydryls in nascent proteins. While peroxiredoxins exert peroxidase activity using the reducing power derived from reducing agents, such as thioredoxin, PRDX4 possesses a hydrophobic signal peptide [91] and is the only form present in the ER lumen [92,93] . PRDX4 eliminates peroxides in a thioredoxin dependent manner in vitro analogous to other family mem bers [94] , but the thioredoxinredox system is much less active in the ER compared to the cytosol. Hence, it is not likely that thioredoxin supports the elimination of hydrogen peroxide by PRDX4 in the ER.
PRDX4catalyzed disulfide formation in PDI proteins by means of hydrogen peroxide was first demonstrated in vitro [95,96] . PRDX4 utilizes hydrogen peroxide to oxi dize reactive sulfhydryls in the PDI, rescues an ERO1 deficiency in yeast [95] , and catalyzes hydrogen peroxide mediated oxidative protein folding in the ER [97] . Among the PDI family members, two proteins, P5 and ERp46, are preferential targets of PRDX4 [98] . While ERO1 utilizes molecular oxygen as an electron acceptor and produces hydrogen peroxide [95] , PRDX4 utilizes electrons from sulf hydryl groups in PDI family proteins and reduces hydrogen peroxide. Thus PRDX4 is beneficial from the standpoint of antioxidation as well [99] . PRDX4 regulates disulfide formation in glycerophosphodiester phosphodiesterase Fujii J et al . Oxidative stress induces ER stress (GDE)2, a membrane protein, and determines its cell sur face expression and consequently neurogenesis in mouse embryos as well [100] . The genetic ablation of PRDX4 causes a defect in the development of testis but otherwise shows a mode rate phenotypical abnormality in mice [101] . This can be attributed to a functional redundancy of other molecules in the ER. The induction of PRDX4 is observed during the terminal differentiation of B cells to plasma cells after a lipopolysaccharide treatment [102] . In PRDX4deficient plasma cells, large aggregates of IgM are actually formed in the ER. The ablation of both ERO1 and PRDX4 causes the consumption of ascorbic acid, which donates electrons for the hydroxylation of prolines in collagen, and results in atypical scurvy, which is characterized by an insufficient production of collagen [103] . The pancreas express PRDX4 abundantly [104] probably because protein secretion both as exocrine and endocrine mechanisms is critical in this organ. In fact mice that overexpress PRDX4 show protection against highdose streptozotocininduced dia betes in transgenic mice [105,106] . The biosynthesis and secretion of insulin are improved by the overexpression of PRDX4 in INS1E cells [107] . Moreover, atherosclerosis in apolipoprotein Eknockout mice is attenuated by the excessive expression of PRDX4 [108] . Issues regarding the physiological functions of PRDX4 other than ER sulfoxidase remain ambiguous and should be resolved. A portion of the PRDX4 is present in blood plasma [104] and is elevated under some pathological con ditions [109] . However, its release from cultivated cells is suppressed by oxidative stress [110] , so that the organ source and the mechanism responsible for the release of the PRDX4 are unknown. Moreover, sexually matured testes express a variant form of PRDX4 that is transcribed from the alternative exon 1 [111] . The translated variant protein does not possess a secretory signal peptide, which results in this protein being localized in the cytosol. The va riant form of PRDX4 exhibits an antioxidative function [112] , but its physiological significance in testes is unknown.

Other proteins involved in disulfide formation in the ER
Several other proteins that are present in the ER lumen also appear to participate in disulfide formation [113] . GPX7 and GPX8 exhibit sulfoxidase activity in the ER [81,114] , and GPX8 is reportedly more selective for the reduction of hydrogen peroxide produced by ERO1. GPX8 appears to form a complex with Ero1α and exerts peroxidase activity that prevents the diffusion of Ero1αderived hydrogen peroxide into and out of the rough ER [115] . Similar to PRDX4, GPX7 and GPX8 are not expressed in β cells but improves insulin secretion when overexpressed in INS1E βcells [116] . Vitamin K epoxide reductase (VKOR) catalyzes the reduction of vitamin K 2,3epoxide and vita min K to vitamin K hydroquinone, which is required for the γglutamyl carboxylation of coagulation factors [117] . VKOR is an ER protein consisting of four transmembrane helices, employs an electron transfer pathway similar to the bacterial homologue and preferably couples with membranebound thioredoxinlike redox partners [118] . The combined depletion of ERO1, PRDX4, and VKOR actually causes cell death, and VKOR alone is capable of supporting cell viability and protein secretion in the absence of ERO1 and PRDX4 activities [119] . Thus, VKOR appears to function sufficiently in oxidative protein folding and compensates for the lack of other sulfoxidases.

Low molecular redox compounds
Some small molecules other than proteins are also repor tedly involved in redox homeostasis in the ER. GSH is the most abundant nonprotein thiol in cells and plays pleiotropic roles, such as in antioxidation and in the detoxi fication of toxicants [120] . The antioxidative functions of GSH are effectively expressed by a donation of electrons to peroxides via GPX [81] . Hence, a GSH insufficiency triggers a redox imbalance and makes cells more vulnerable to an oxidative insult, leading to cell death in severe cases. ER contains 15 mM GSH [121] which reportedly supports the secretion of proteins by its redox capacity. However, GSH depletion, which is caused by expressing an exogenous GSHmetabolizing enzyme in the ER lumen, shows no measurable effect on the induction of UPR, suggesting the existence of an alternative molecule capable of supporting redox reactions in the ER [122] . Ascorbic acid is also present abundantly in the ER and appears to support oxidative protein folding. As mentioned above, a double deficiency of ERO1 and PRDX4 causes atypical scurvy in mice due to ascorbic acid consumption and defects in collagen synthesis [103] . However, the genetic ablation of gulonolactone oxidase (GULO), which is the enzyme that catalyzes the last step of ascorbic synthesis but which is lacking in primates, shows normal collagen status in mouse skin [123] . In mice that lack aldehyde re ductase encoded by AKR1A, ascorbic acid synthesis is decreased to approximately 10% of wildtype mice [124] but normal collagen synthesis in the fibrotic kidney after unilateral ureteral obstruction was observed [125] . Thus, these reports suggest the presence of other electron donors, other than ascorbic acid, to support collagen syn thesis.

Nonalcoholic fatty liver disease (NAFLD) is a condition
where the fat content in the liver exceeds 5%10% by weight without a history of an excessive ingestion of alcohol [126] . A variety of metabolic dysfunctions including diabetes, dyslipidemia, and the metabolic syndrome are associated with NAFLD, which then degenerates to non alcoholic steatohepatitis (NASH), a condition associated with inflammation and fibrotic liver damage [127] .

Roles of oxidative/ER stress in fatty liver disease
Oxidative stress is commonly regarded as "the second hit" for NASH development [128] . Signaling pathways for the ER stressinduced inflammatory process to NAFLD de Fujii J et al . Oxidative stress induces ER stress velopment has been extensively overviewed [129] . Oxidative stress triggers ER stress by stimulating the formation of misfolded proteins, and they together coordinately trigger NAFLD [130,131] . Below we briefly summarize the processes for lipid droplet accumulation triggered by these types of stress.
Sterol regulatory elementbinding proteins (SREBPs) are the main transcriptional regulatory factors that con trol the synthesis of fatty acids and cholesterol under the control of the sterol status in the ER membrane [51] . Two SREBP genes, SREBP1 and SREBP2, are present in mammals. SREBP1 is alternatively transcribed into two forms SREBP1a and SREBP1c and is mainly involved in fat ty acid synthesis. SREBP2 regulates a series of genes that are responsible for cholesterol synthesis. SREBPs are mem brane proteins predominantly exposed to the cytoplasmic side and bind to another ER protein, the SREBP cleavage activating protein (SCAP), which is located in the ER membrane [132] . Intermembrane domains on the SCAP protein bind cholesterol, which enable the formation of the SCAP/SREBP complex. Insig1, another ER membrane protein, block the lateral movement of SCAP/SREBP into COPⅡcoated vesicles on ER membranes and prevent them from reaching the Golgi body [51,133] . Upon the disso ciation of cholesterol due to an insufficient presence within the ER membrane, the SCAP/SREBP complex is released from Insig1, translocated to the Golgi apparatus and then cleaved by S1P and S2P there [134] . This proteolytic activation recruits the cytoplasmic domain of the proteins to the nucleus, which then results in the induction of the expression of a series of lipogenic genes [135] . As described above, S1P and S2P are processing enzymes for the ER membranebound transcriptional regulatory protein ATF6 and activate it under conditions of ER stress [41] . Because the proteolytic machinery is shared, ER stress actually upregulates SREBP1c, leading to the accumulation of lipid in hepatic cells [136] . Glycogen synthase kinase (GSK)3 appears to be involved in signaling downstream of ER stress [137] . In the meantime HMG CoA reductase limits cholesterol synthesis, irrespective of ER stress. The protein levels of HMG CoA reductase are indirectly regulated by cholesterol via an Insigmediated reaction; i.e., the presence of sufficient amounts of cholesterol consequently drives the polyubiquitination of HMG CoA reductase and proteolytic degradation by proteasomes [50] . The fact that ER stress induces the activation of SREBPs, in turn, suggests that SREBPmediated lipo genesis also activated under conditions of oxidative stress. In fact, oxidative stress induces SREBP1c activation and lipid accumulation [138] . Thus, oxidative stress and ER stress interdependently stimulates the de novo synthesis and accumulation of triglycerides and cholesterol but, on another front, inhibits the secretion of lipoproteins [139] . Because cell cultures are typically performed under atmos pheric oxygen, the spontaneous activation of SREBP1 and the associated expression of genes fatty acid syn thase (FAS), acetylCoA carboxylase (ACC), and stearoyl CoA desaturase (SCD)1 are observed in primary hepa tocytes and this is greatly enhanced in SOD1knockout hepatocytes [140] . For the excretion of triglyceriderich lipoproteins, appropriate oxidative folding of the apoB protein is essential [141,142] . In fact, oxidative stress appears to suppress lipoprotein secretion, which would be most likely caused via the misfolding of apoB and an impaired microsomal transfer protein function [143] . Stimulation of lipogenesis and the inhibition of lipoprotein secretion would cooperatively elevate lipid droplet accumulation, which would consequently result in the development of liver steatosis (Figure 3). This interdependent work of dealing with oxidative stress and ER stress in liver steatosis is further supported by recent observations showing the double knockout of SOD1 and PRDX4 result in aggravated liver damage compared to singly knockout mice [144] .
An examination of fatty acid metabolism in NAFLD patients indicates that the inability of the liver to regulate changes in lipogenesis during the transition from the fasted to fed state is the underlying mechanism responsible for this [145] . Fasting induces the formation of lipid droplets, not only in the liver but also other tissues that are dominantly involved in active βoxidation. While feeding a high fat diet leads to lipid droplet accumulation in the liver [146] and intestinal epithelia [143] more intensely in SOD1knockout mice than the wildtype mice, fasting induces severe and irreversible damage not only to the liver but to other aerobic organs as well [147,148] . Thus fasting may aggravate oxidative damage in these organs and become a serious pathogenic factor for NAFLD.
These observations raise the next query, i.e., why is lipogenesis elevated as a consequence of UPR in cases of ER stress if lipid accumulation is unfavorable for the liver? We have a clue to this, in that feeding a lard containing highcalorie diet increases the accumulation of lipid droplets but improves the longevity of the SOD1 deficient mice compared to mice fed a normal diet [149] . This is unexpected because lipid accumulation is generally recognized to be an exacerbating factor for liver function. Based on these observations we hypothesized that lipid droplets that accumulate in response to oxidative stress may have a protective role against the hepatotoxic effects of ROS. Experimental data actually suggest that lipids that transiently accumulate in the liver have a protective function against oxidative injury caused by a liver toxicant thioacetamide in mice and by hydrogen peroxide in culti vated cells [150] . Thus, the accumulation of lipids under ER stress conditions may also be regarded as an adoptive response of hepatocytes to oxidative stress conditions [151] , although liver steatosis at a more advanced stage is hazardous and should be treated appropriately.

Therapeutics to combat against NAFLD
Multiple processes appear to underlie the pathogenesis of NAFLD, so that a variety of agents and treatment may be applicable for therapeutic purposes. Antioxidants directly eliminate ROS and result in the suppression of oxidative stress and consequently ER stress. Hydrophobic antioxidants, such as vitamin E [68] and coenzyme Q10 [152] , and their derivatives and hydrophilic antioxidants, such as vitamin C (ascorbic acid) and Nacetylcysteine, a precursor for cysteine and GSH [153] , may be promising agents for use in ameliorating the effects of NAFLD. In fact, vitamin E and polyphenol have been reported to be useful in the treatment of NAFLD patients, while the issue of whether vitamin C is beneficial is ambiguous at this moment [154,155] . While antioxidants decrease the levels of pathogenic ROS directly, it would be helpful if cellular capacity to resist malfunctions in lipid metabolism could be additionally en hanced. In this sense, peroxisome proliferatoractivated receptors (PPARs) and their binding partner retinoid Xreceptors (RXR), appear to have roles in maintaining lipid homeostasis and, hence, represent promising targets for ameliorating the pathogenesis of NAFLD [126] . PPAR family proteins of which PPARα, PPARβ/δ, PPARγ are members, can be either activated or inhibited by lipid metabolites and other lipophilic agents in vivo. Regarding the distribution of PPARs in tissues, the highest expression of PPARα is observed in brown adipose tissue, liver, kidney, and heart [156] . The expression of PPARγ is higher in adipose tissues than other tissues. Compared to these isoforms, the expression of the PPARβ/δ isoform is rather ubiquitous. Mice in which PPARα is knocked out are viable and fertile and show no detectable gross phenotypic de fects [157] . Contrary to PPARα knockout mice, a PPARγ deficiency showed impaired placental vascularization, leading to embryonic death by embryonic day E10.0 [158] . Similarly, the genetic ablation of PPARβ/δ also impairs the placenta and leads to embryonic death at E9.5 E10.5 [159] . The roles of PPARβ/δ are less established compared to those of PPARα and PPARγ. In 2003 it was reported that PPARδ contributes to fat metabolism [160,161] , which then attracted more interest in PPARδ than before. For example, PPARδ activation enhances fatty acid oxidation and rescues ER stress in pancreatic βcells [162] . The PPARα/δ agonist GFT505 prevents high fat dietinduced liver steatosis and protects the liver from inflammatory reactions in mice [163] . The activation of PPARβ/δ by GW501516 also prevents the inflammation associated with ER stress in skeletal muscle cells and ameliorates insulin resistance in mice through an adenosine mono phosphateactivated protein kinase (AMPK)dependent mechanism [164] . The PPARβ/δ agonist GW0742 also re portedly attenuates ER stress by improving hepatic energy metabolism in the livers of high fat dietadministered mice [165] . The effectiveness of agonists for PPARα/δ has been confirmed in NASH patients as well [166] . Thus, the application of PPAR agonists, notably those for PPARβ/δ isoforms, appears to be promising therapeutics for the treatment of NAFLD [167] . While the molecular mechanisms for how PPARβ/δ controls insulin signaling are largely Oxidative stress ER stress Figure 3 Coordinate action of oxidative stress and endoplasmic reticulum stress in liver steatosis. Excessively produced reactive oxygen species (ROS) cause the misfolding of proteins in the endoplasmic reticulum (ER), which leads to ER stress. The precursor forms of activating transcription factor 6 (ATF6) and sterol regulatory element-binding proteins (SREBPs) in the ER membrane are translocated to the Golgi body by an independent mechanism but are proteolytically activated there by site-1 protease (S1P) and S2P. The transcriptionally active ATF6 and SREBP then move to the nucleus. While ATF6 exerts a protective function by activating the genes involved in the unfolded protein response, SREBPs activates genes that are involved in lipogenesis and steroidogenesis, which may lead to the development of nonalcoholic fatty liver disease. ROS: Reactive oxygen species; ER: Endoplasmic reticulum; ATF6: Activating transcription factor 6; SREBP: Sterol regulatory element-binding protein; S1P: Site-1 protease; UPR: Unfolded protein response.
Fujii J et al . Oxidative stress induces ER stress unknown, a stable interaction of PPARβ/δ with nuclear Tcell protein tyrosine phosphatase 45 (TCPTP45) isoform has been demonstrated as the most upstream component that resolves the downregulation of insulin signaling [168] .
Forthcoming experiments directed at elucidating the path way in more detail would provide a clearer vision on this issue.
As described above, the activation of PPARβ/δ is a largely promising area of therapeutics for fatty live dis eases in animal experiments [169] . However, it should be remembered that the most potent and specific activator for PPARβ/δ, GW501516, also accelerates intestinal adenocarcinoma in Apc min mice that are predisposed to de veloping intestinal polyposis [170] . The activation of PPARβ/δ by GW501516 is also associated with the induction of cancer development, while the PPARγ agonist GW7845 causes a moderate delay in tumor formation [171] . At the present stage, the activation of PPARβ/δ does not always lead to consistent results; i.e., it is either procarcinogenic or anticarcinogenic, depending on carcinogenic model and the animals being used [172,173] . Therefore the development of activating agents that exert a beneficial action on PPARβ/δ, but do not have other side effects, is awaited for using synthetic activators for therapeutic purposes in the treatment of fatty liver diseases.

PERSPECTIVES
Oxidative stress and ER stress are frequently linked and become pathogenic in some diseases such as NAFLD [174] . Antioxidants suppress oxidative stress directly and also subsequent ER stress and, hence, can mitigate the causal factors for NAFLD. In the meantime, the activation of PPARs renders the liver more resistant against stress caused by lipotoxicity. Coordinated treatment with these agents may exert therapeutic effects more efficiently in NAFLD as well as in other diseases that are caused by aberrant lipid metabolism.