Peroxiredoxin 4 (PRDX4): Its Critical in Vivo Roles in Animal Models of Metabolic Syndrome Ranging from Atherosclerosis to Nonalcoholic Fatty Liver Disease

Pathology International 2018; 68: 91–101 doi:10.1111/pin.12634

Review Article Peroxiredoxin 4 (PRDX4): Its critical in vivo roles in animal models of metabolic syndrome ranging from atherosclerosis to nonalcoholic fatty liver disease

Sohsuke Yamada1 and Xin Guo1,2 1Department of Pathology and Laboratory Medicine, Kanazawa Medical University, Ishikawa, Japan and 2Laboratory of Pathology, Hebei Cancer Institute, The Fourth Hospital of Hebei Medical University, Hebei, China

The peroxiredoxin (PRDX) family, a new family of proteins molecules and reactive oxygen species (ROS), such as with a pivotal antioxidative function, is ubiquitously synthe- superoxide anion (O2 ), hydrogen peroxide (H2O2), fi sized and abundantly identi ed in various organisms. In hydroxyl radicals (OH), and the concomitant generation of contrast to the intracellular localization of other family nitric oxide (NO), strongly contribute to the initiation and members (PRDX1/2/3/5/6), PRDX4 is the only known secre- fl tory form and protects against oxidative damage by scaveng- progression of chronic in ammatory diseases, such as 1,2 ing reactive oxygen species in both the intracellular diabetes mellitus (DM) or atherosclerosis. Oxygen (O2), (especially the endoplasmic reticulum) compartments and necessary for the vital activities of living organisms, is the extracellular space. We generated unique human PRDX4 paradoxically a highly reactive molecule and can easily form (hPRDX4) transgenic (Tg) mice on a C57BL/6J background the one-electron reduction state of O2 , subsequently and investigated the critical and diverse protective roles of resulting in the formation of H2O2, as shown in Fig. 1. PRDX4 against diabetes mellitus, atherosclerosis, insulin Increased oxidative stressors within the extracellular space resistance, and nonalcoholic fatty liver disease (NAFLD) as are considered a risk factor of atherosclerosis due to the well as evaluated its role in the intestinal function in various animal models. Our published data have shown that PRDX4 accumulation of oxidized low-density lipoprotein (oxLDL) as 3,4 helps prevent the progression of metabolic syndrome by well as the formation of ROS, a feature of chronic reducing local and systemic oxidative stress and synergisti- inflammatory processes. The vascular vessels range from cally suppressing steatosis, inflammatory reactions, and/or small to large sizes, with atherosclerotic lesions histopatho- – apoptotic activity. These observations suggest that Tg mice logically termed “arteriolosclerosis” to “atheroma”.5 7 DM is may be a useful animal model for studying the relevance of closely related to the pathophysiological aspects of arterio- oxidative stress on inflammation and the dysregulation losclerosis, and metabolic syndrome, manifesting as obe- of lipid/bile acid/glucose metabolism upon the progression of human metabolic syndrome, and that specific accelerators sity, dyslipidemia, type 2 DM and/or atherosclerosis, also of PRDX4 may be useful as therapeutic agents for ameliorat- has a tight correlation with the development of nonalcoholic – ing various chronic inflammatory diseases. fatty liver disease (NAFLD),6 9 which is an umbrella term covering a broad spectrum of clinicopathological presenta- Key words: animal model, chronic inflammatory disease, tions, ranging from simple steatosis to nonalcoholic steato- – human PRDX4 transgenic mice (Tg), metabolic syndrome, hepatitis (NASH).6 12 It is well known that NASH is not only peroxiredoxin (PRDX) 4 a more severe form of NAFLD, occasionally leading to end- stage liver diseases, including cirrhosis and/or hepatocellu- Accumulating evidence has revealed that oxidative stress lar carcinoma (HCC),8–12 but also that oxidative stress and and endoplasmic reticulum (ER) stress, including oxidized ER stress appear to be critically involved in these diseases’ processes, even in animal models.10–14 In fact, oxidative Correspondence: Sohsuke Yamada, MD, PhD, Department of stress appears, at least in part, to mechanistically illustrate Pathology and Laboratory Medicine, Kanazawa Medical University, the endothelial dysfunction, and the vascular inflammation 1-1 Uchinada, Ishikawa 920-0293, Japan. and complications especially in the initiation of the meta- – Email: [email protected] bolic syndrome/metabolic syndrome-related diseases.1 5 Received 12 November 2017. Accepted for publication 13 Furthermore, in addition to the target organs of metabolic December 2017. © 2018 Japanese Society of Pathology and John Wiley & Sons syndrome (arteries and liver), the small intestine with its Australia, Ltd extensive surface area is the first organ to encounter 92 S. Yamada and X. Guo

Figure 1 The pathway of the detoxification of reactive oxygen species (ROS) by peroxiredoxin (PRDX). O2 is a highly reactive molecule and can easily form O2 (superoxide), subsequently undergoing dismutation to form H2O2 (hydrogen peroxide). The functional peroxidase activity of PRDXs is dependent on reduced forms of thioredoxin (redTRX) and/or glutathione (GSH). PRDX, peroxiredoxin; SOD, superoxide dismutase; GSSG, oxidized GSH; GPX, GSH peroxidase; OH, hydroxyl radicals; CoQ, coenzyme Q; p450, cytochrome p450; FAD, flavin adenine dinucleotide. nutrients, which likely plays an important role in the could be highly susceptible to hyperoxidation/inactivation 9,15,16 development of metabolic disorders as well. due to H2O2-mediated sulfonic acid formation on its perox- Taken together, these previous findings and our in vivo idatic Cys,30–34 PRDX4 might be a weak antioxidative data indicate that metabolic syndrome is an extremely enzyme compared to other thiol-dependent antioxidants in complex disease orchestrated by multiple molecular and the context of metabolic syndrome-related diseases. On the histopathologic factors, including not only oxidative stress other hand, PRDX4 is the only known secretory form located but also elevated levels of inflammatory cytokines and in not only the intracellular (especially the ER) but also the apoptotic activity and the translocation of either intestinal extracellular space, in contrast to the intracellular localization bacteria or microbial cell components.6–9,17–29 All research- of other family members (PRDX1, 2 and 6 are located in the ers should be aware of the fact that lipid/bile acid (BA)/ cytoplasm, and PRDX3 and 5 in mitochondria. Moreover, glucose metabolism with both local (each tissue) and several PRDXs, including PRDX4, have also been shown to whole-body functions plays a central, key role in the etiology be nuclear and are among the most abundant pro- of metabolic syndrome. teins).30,31,35,36 PRDX4 has an additional N-terminal region, Peroxiredoxin (PRDX), a new family of antioxidant en- which is unique to this enzyme, following a signal peptide zymes, is ubiquitously synthesized and has been abundantly that allows translocation across the ER membrane and is identified in various organisms.30,31 At least six distinct thereafter cleaved.30,31 Several previous studies have PRDX genes expressed in mammals possess high similari- focused on the molecular regulatory roles of PRDX4 in the ties in their molecular structures and contain a reactive nuclear factor kB(NF-kB),37 epidermal growth factor, and cysteine (Cys) in a conserved region near the N-terminus p53 or thromboxane A2 receptor cascade.36,38 More that forms Cys-sulfenic acid as an intermediate reaction recently, PRDX4 has been reported to play a pivotal role in 31–33 during the reduction of H2O2. As shown in Fig. 1, the disulfide bond formation (i.e. oxidative folding) of lip-

PRDXs regulate the H2O2 levels using thioredoxin (TRX) as oproteins in coordination with protein disulﬁde isomerase an electron donor, and their functional peroxidase activity is (PDI) and ER oxidoreductin 1 (ERO1) in the ER.39,40 dependent on the reduced forms of TRX (redTRX) and/or However, the detailed in vivo pathological and physiological glutathione (GSH).30–33 Fig. 1 summarizes the pathway of relevance of PRDX4 has remained unclear. the detoxiﬁcation of ROS by PRDXs. Within the PRDX We hypothesized that PRDX4 locally (intracellularly) and family, PRDX4 is a member of the 2-Cys-type PRDX family systemically (extracellularly) plays a critical, diverse role in and homologous to typical 2-Cys PRDX1 and 2, thus serving vivo in the protection against the initiation and development as a TRX-dependent peroxidase via a similar catalytic of metabolic syndrome manifesting as visceral obesity, DM, mechanism to PRDX1/2.31,34 On one hand, since PRDX4 atherosclerosis and/or NAFLD (i.e. disordered lipid/BA/

© 2018 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd PRDX4 in metabolic syndrome 93 glucose metabolism). We generated human PRDX4 NotI fragment containing hPRDX4 cDNA is inserted into the (hPRDX4) transgenic (Tg) mice and evaluated the in vivo NotI site of pcDNA3 (5.4 kb; Invitrogen, Life Technologies functions of PRDX4 in serial studies of various animal Japan, Tokyo, Japan), and the CMV enhancer/promoter of models,5–9 as summarized in Table 1. In the current review, the entire nucleic acid sequence displays extensive cross- we focus on the crucial in vivo roles of PRDX4 in various talk with other promoters since it contains many transcrip- chronic inflammatory diseases, with particular focus on DM, tion factor binding sites, such as NF-kB.5,7–9 However, as it atherosclerosis, and NAFLD. Our findings here suggest that is not a tissue-specific promoter, the protein expression of Tg mice may be a useful animal model for studying the the hPRDX4 transgene in each tissue will be affected by its relevance of oxidative stress in ROS- and/or chronic site of integration into the mouse genome (i.e., by chance). inflammation-induced human diseases, such as metabolic C57BL/6J mice (Charles River Laboratories, Yokohama, syndrome, and that PRDX4 may be a key factor involved in Japan) were used as the control WT mice in our serial the pathogenesis of metabolic syndrome. in vivo studies. Our data show that, in RT-PCR, the expression of endogenous mouse PRDX4 (mPRDX4) is clearly recog- ANIMAL MODELS OF METABOLIC SYNDROME USING nized in every tissue of non-treated Tg and WT mice and is THE UNIQUE Tg MICE identified particularly strongly in the pancreas, testes, liver, and brain.7,9,10 In contrast, the hPRDX4 expression in the Construction of Human PRDX4 (hPRDX4) Transgenic Tg mice is exclusively enhanced, especially in the pan- (Tg) Mice creas, aorta, liver, testes, heart, small intestine and – brain.5,7 9 In addition, a Western blotting analysis and Tg mice in a background of C57BL/6J wild-type (WT) immunohistochemical examination have demonstrated that – mice were generated as described elsewhere.5 9 The hPRDX4 is particularly highly expressed in non-treated Tg

Table 1 Summary of the in vivo roles of PRDX4 in mouse models of metabolic syndrome

Model hPRDX4 Tg Mice References Oxidative stress (ROS)# Hyperglycemia# Hypoinsulinemia# Glucose tolerance" Ding et al., 2010 [ref. 5] SHDS-induced type 1 DM Insultis" Yamada et al., 2012 [ref. 6] Pancreatic islet areas" b-cell apoptosis# b-cell proliferation" Inflammatory cells/cytokines# Oxidative stress (ROS)# Atheroma# (Anti-atherogenic) HcD-induced Atherosclerosis Mf, SMCs and ECs apoptosis# Guo et al., 2012 [ref. 7] SMC migration/replication" Inflammatory cells/cytokines# Plaque stability" (Mf#, SMC", lipid core#, collagen", fibrous cap") Oxidative stress (ROS)## Insulin resistance# HFrDþSTZ-induced type 2 DM and NAFLD Glucose tolerance" Nabeshima et al., 2013 [ref. 8] NAFLD## (steatosis#, inflammation#, fibrosis#) Hepatocyte apoptosis# Stellate cell activation# Oxidative stress (ROS)# NAFLD# Total intestinal length!" Villi height!" MCDþHF-induced NAFLD Intestinal lipid accumulation" Nawata et al., 2016 [ref. 9] Enterocytes apoptosis# Enterocytes proliferation" Intestinal inflammation# hPRDX4, human peroxiredoxin 4 (PRDX4); SHDS, single high dose of streptozotocin (STZ); DM, diabetes mellitus; HcD, high-cholesterol diet; HFrD þ STZ, high fructose diet plus streptozotocin; NAFLD, nonalcoholic fatty liver disease; MCD þ HF, methionine- and choline-deﬁcient high fat diet; ROS, reactive oxygen species; Mf, macrophage; SMC, smooth muscle cell; EC, endothelial cell

© 2018 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd 94 S. Yamada and X. Guo tissues but is not expressed at all in non-treated WT formation, we generated Tg and apolipoprotein E (apoE)- mice.5–9 Biochemical examinations show no remarkable knockout (apoE/)mice(hPRDX4+/+/apoE/)bycrossing difference between WT and Tg mice in whole-body glucose apoE-KO mice.7 These atherosclerotic model experiments homeostasis, insulin resistance, or lipid profiles under basal wereperformedon8-week-oldmalemiceweighing20to conditions.5,8 Furthermore, neither physiologically nor mor- 22 g. ApoE / and hPRDX4+/+/apoE / mice are fed an HcD phologically significant differences between the two groups containing 1.25% cholesterol, 15.0% lard, and 0.5% sodium – of mice are evident. cholic acid (CA). They are sacrificed 12 weeks later.7,17 20 The experimental procedure is summarized in Fig. 2 as a schematic representation. The representative histopathology Streptozotocin-induced Type 1 DM mouse model of mouse aortic atheroma is available in our previous studies,7,18,23,28 showing a variably thickened intima with the To develop this model, WT and Tg male mice 8 weeks of accumulation of foamy macrophages (Mfs), migrating vascu- age weighing 20 to 22 g are fasted for 18 h and intraperito- lar smooth muscle cells (SMCs), T-lymphocyte infiltration, neally injected with a single high dose of streptozotocin central necrotic lipid cores, collagen accumulation, and cover- (SHDS) (Sigma-Aldrich, St. Louis, MO, USA) at 150 mg/kg ing fibrous cap formation. body weight (BW), dissolved in sodium citrate buffer (pH 4.5). Mice with blood glucose levels higher than 250 mg/dL are considered to be DM. Sham mice are given the same Special-diet-induced NAFLD and/or type 2 DM mouse volume of sodium citrate buffer. Animals were killed 1 or models 2 weeks after SHDS injection. The experimental procedure is summarized in Fig. 2 as a schematic representation. The Experiments were performed using 4- to 6-week-old male representative histopathology of the pancreas in type 1 DM WT and Tg mice weighing approximately 18 g. These mice mice is available in our previous studies,5,6 showing that the are fed a high-fructose diet (HFrD) (67% carbohydrates— pancreatic islet areas are reduced along with CD3-positive 98% of which is fructose—13% fat, and 20% protein; KBT T-lymphocyte infiltration and activated b-cell apoptosis. Oriental Corporation, Saga, Japan) for 4 weeks to generate peripheral insulin resistance and NAFLD, followed by a Hypercholesterolemia-induced atherosclerosis mouse single intraperitoneal injection (likely SHDS) of relatively low- model dose (100 mg/kg BW) freshly prepared STZ (Sigma-Aldrich) dissolved in sodium citrate buffer (pH 4.5) after fasting for In contrast to lipid metabolism in humans, the lipoprotein 18 h. The mice are then fed the same HFrD for a further four profile in mice is high-density lipoprotein (HDL)-dominant; as weeks, as described elsewhere.8,42 The animals were killed such, WT mice are essentially resistant to high-cholesterol four weeks after STZ injection. diet (HcD)-induced atheromatous plaque formation.5,17–29,41 As additional NAFLD animal models, 8-week-old male Thus, to achieve hypercholesterolemia-induced atheromatous WT and Tg mice are fed a methionine- and choline-deficient

Figure 2 A schematic presentation of the experimental procedures in various mouse models of metabolic syndrome using the unique Tg mice. SHDS, single high dose of streptozotocin (STZ); HcD, high-cholesterol diet; apoE, apolipoprotein E; HFrD, high fructose diet; MCD þ HF, methionine- and choline-deﬁcient high fat diet.

© 2018 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd PRDX4 in metabolic syndrome 95 high-fat (MCD þ HF) diet (60% fat; KBT Oriental Corpora- of selective b-cell death at the onset of type 1 DM, typically tion) for 2 weeks, as described previously.7,8,43 STZ is not resulting from an autoimmune assault against b-cells by the injected in this model. The animals were killed in a fed state infiltration of mononuclear T-lymphocytes and cytotoxicity- two weeks later, and tissues—including the liver or small and inflammatory factor-induced processes.5,6,45 Actually, intestine—were excised. we have shown that the expression of many inflammatory In these NAFLD and/or type 2 DM (or insulin resistance) factors, including interleukin (IL)-1b, tumor necrosis factor mouse models, food consumption was determined using (TNF)-a, TNF receptor (TNFR) 2, Toll-like receptor (TLR) metabolic cages obtained from SUGIYAMA-GEN CO., Ltd. 3/4, and NFk-B, is significantly accelerated in WT mice after (Tokyo, Japan). The experimental procedures are summa- SHDS induction, while conversely, such inflammatory sig- rized in Fig. 2 as a schematic representation. The represen- naling is significantly reduced in the Tg pancreas. We can tative histopathology of the liver, jejunum, or pancreas in also verify the suppression of apoptotic cell morphology, NAFLD mice is available in our previous studies.7,8 NAFLD terminal deoxynucleotidyl transferase end-labeling (TUNEL) in mice typically manifests as vesicular steatosis, chronic staining and cleaved Caspase-3 activation in Tg islets.7 inflammation, hepatocellular ballooning, and fibrosis, asso- Furthermore, the expression of 8-hydroxy-20-deoxyguano- – ciated with lipid deposition, increased expression of hepatic sine (8-OHdG), as an oxidative stress marker,5,7,9,46 48 is aminotransferase, stimulated hepatocytic apoptosis, and much higher in WT and Tg islets after SHDS injection than stellate cell activation, reminiscent of human NASH. before treatment; however, at two weeks, the expression in the Tg mice is significantly lower than that in the WT mice. Taken together, these findings strongly suggest that NOVEL, PROTECTIVE IN VIVO FUNCTIONS OF PRDX4 hPRDX4 plays a protective, key role against SHDS-induced IN METABOLIC SYNDROME injury by inhibiting endothelial dysfunction, proinflammatory cytokines and cytotoxic T-cell infiltration, as well as by PRDX4 protects against SHDS-induced type 1 DM by preventing b-cell-derived ROS generation or scavenging of suppressing oxidative damage, inflammatory the generated ROS, particularly the extracellular pool of cytokines, and apoptotic activities throughout the ROS. The insulin-secreting b-cells in the Tg islets, where course of the disease hPRDX4 is specifically expressed, are significantly more protective against SHDS-induced insulitis and apoptosis We reported for the first time that,5,6 after SHDS injection, than those of WT mice. Additionally, the Tg islets are more Tg mice show significantly less hyperglycemia and hypo- likely to activate the proliferation of b-cells, since the insulinemia and a much faster response on the glucose specific expression of hPRDX4 might be able to accelerate tolerance test than treated WT mice, despite no marked replication of pluripotent cells to repair and remodel islets.5,6 differences in the insulin tolerance test. A histopathological Two different mechanisms have been proposed to explain observation showed that the Tg mice were significantly this unique effect of PRDX4 in the repression of apoptosis. more resistant to serious SHDS-induced injury than WT One mechanism involves the direct function of PRDX4 by mice and displayed accelerated reconstruction of the islets scavenging the increased ROS/oxidative stress due to high at two weeks while maintaining a high level of not only glucose levels (DM). The other involves the indirect function hPRDX4 but also endogenous mPRDXs expression, includ- of PRDX4 by preventing inflammatory cells (particularly, T ing mPRDX4. These data suggest that the overexpression lymphocytes) from infiltrating and/or reducing various proin- of hPRDX4 in Tg mice protects against critical injury to the flammatory mediators/cytokines and receptors/ligands islets by SHDS while increasing the resistance (or reducing involved in the cell survival and growth. PRDX4 may be the vulnerability) of endogenous mPRDXs expression, pathophysiologically relevant to the protection against type reminiscent of a cascade of the PRDX family. Also, PRDX4 1 DM in humans. Fig. 3 briefly summarizes the important has also been shown to heterodimerize with PRDX1. roles of PRDX4 in this SHDS-induced type 1 DM mouse Knowing the tendency of peroxiredoxins to oligomerize, a model. direct protein-protein interaction is likely to happen. In addition, hPRDX4 overexpression can suppress infiltration, particularly that of CD3-immunoreactive T lymphocytes. PRDX4 protects against atherosclerotic progression in STZ can be selectively accumulated in pancreatic b-cells hyperlipidemia-induced atheromatous and vulnerable via the low-affinity GLUT2 glucose transporter in the plasma plaque formation by suppressing oxidative damage and membrane.44 The transfer of the methyl group from STZ apoptosis (i.e., SHDS) to the DNA molecule causes DNA damage, resulting in the fragmentation of the DNA and b-cell death In addition, our data have confirmed the specific expres- (necrosis/apoptosis).44 Indeed, apoptosis is the main cause sions of hPRDX4 in vitro and in vivo in aortic vascular

scavenging the generated extracellular pool of ROS. Indeed, we detected lower local levels of 8-OHdG and oxidized LDL (ox-LDL) as well as systemic levels of TBARS in HcD-fed hPRDX4þ/þ/apoE/ mice than in apoE/ mice. Finally, significantly fewer TUNEL-positive Mfs, SMCs and endothelial cells (ECs) are noted in fewer atherosclerotic þ þ intimal lesions in hPRDX4 / /apoE / mice than apoE / mice, along with lower expression of Bax and Caspase-3, which are key apoptotic factors. Supporting those in vivo data, our in vitro experiments can show that the number of apoptotic cells including Mf is significantly lower in hPRDX4þ/þ/apoE/ mice than in apoE/ mice after the stimulation with acetyl-LDL and Acyl-CoA:cholesterol acyl- transferase (ACAT) inhibitor 58035 (i.e., free cholesterol- loading).7 Mf apoptosis may be an important feature of vulnerable plaque formation with an increased size of the necrotic lipid Figure 3 Summary of the critical roles of PRDX4 in type 1 DM. core due to weakened phagocytic clearance.23,50,51 How- This diagram depicts the crucial, protective roles of PRDX4 in the ever, apoptosis of ECs is also critical, as the initial step in SHDS-induced type 1 DM mouse model. PRDX4, peroxiredoxin 4; ROS, reactive oxygen species; DM, diabetes mellitus; SHDS, ROS-induced atherosclerosis involves endothelial damage, single high dose of streptozotocin (STZ); Mf, macrophage. the increased expression of adhesion molecules, and inflammatory cell migration.2,4 SMC apoptosis can lead to a loss of interstitial collagen fibers, resulting in unstable plaques that þ þ – SMCs and Mf isolated from hPRDX4 / /apoE / mice but are prone to rupture.23,50,52 54 Taken together, these findings not from apoE / mice.7 Intriguingly, we were able to show suggest that, since oxidative stress is closely associated with that the expression of the hPRDX4 transgene overcomes that apoptosis, PRDX4 may play a key role in ameliorating of the endogenous mPRDX4 gene in the significantly sup- atherosclerotic progression in HcD-fed apoE/ mice by þ þ pressed atherosclerotic plaques of hPRDX4 / /apoE / mice. protecting the aorta from oxidative stress and reducing As an additive effect, the circulating serum levels of hPRDX4 apoptosis of various vascular cells. þ þ protein are significantly elevated in hPRDX4 / /apoE / mice In conclusion, accumulating data suggest that the over- as well. Significantly decreased levels of circulating oxidant expression of hPRDX4 plays a crucial role in (i) ameliorating markers, serum thiobarbituric acid reactive substances atherosclerotic progression by reducing local and systemic – (TBARS),7 9 can also result in the subsequent suppression of oxidative stressors, decreasing the size of the central endothelial dysfunction and repressed turbulence, supporting necrotic core via repressing Mf apoptosis, and suppressing the protective effects of PRDX4 on attenuated atherosclerotic inflammatory cell migration through the decrease of EC progression and strengthened stable plaque formation in apoptosis; and (ii) reducing possible markers of plaque þ þ hPRDX4 / /apoE/ mice. instability via thickening the fibrous caps and enhancing the A detailed morphological observation shows that hyper- collagen-rich matrix by repressing SMC apoptosis. Fig. 4 cholesterolemia-induced Mf-rich atherosclerosis is signifi- summarizes the pivotal roles of PRDX4 in the atheroscle- cantly reduced, although more SMC-rich plaques are rotic mouse model. þ þ evident in hPRDX4 / /apoE / mice than apoE / mice. Their atheromatous formation is characterized by a thicker fibrous cap, a greater amount of collagen, and fewer central PRDX4 protects against the development of NAFLD, necrotic lipid cores than apoE/ mice, reminiscent of the type 2 DM, and metabolic syndrome by ameliorating structure of human stable plaques. As plaque vulnerability oxidative stress-induced injury in a nongenetic mouse is known to be the most critical factor in cardiac attack and model by feeding mice an HFrD after injecting STZ subsequently potential death,7,49 the specific induction of PRDX4 might be a potential therapeutic strategy to prevent Our laboratory detected the localized expression of hPRDX4 atherosclerotic progression and to possibly improve plaque exclusively in the liver of Tg mice by PCR, Western blotting, stability. Furthermore, the overexpression of hPRDX4 in the and immunohistochemistry after establishing an HFrD þ atherosclerotic aorta protects against chronic inflammation STZ-induced nongenetic mouse model.8 The CMV en- and the upregulation of proinflammatory cytokines by hancer/promoter in the hPRDX4 transgene is readily stimu- preventing inflammatory cell-derived ROS generation and lated by ROS and chronic inflammation during the

establishment of NAFLD. The extreme antioxidative state and suppressed NAFLD in the model Tg mice are closely associated with fewer intrahepatic T lymphocytes and Kupffer cells and lower alanine aminotransferase (ALT) and expression of TNF-a and its receptor (TNFR). Taken together, these findings indicate that PRDX4 suppresses the progression of simple steatosis to NASH by blocking not only the ‘first hit (disordered hepatic lipid accumulation)’ but also the ‘second hits (metabolic, cytokine, and oxidative stressors)’, according to the ‘two-hit’ hypothesis,8,9,55 and should subsequently protect against the initiation to progression of type 2 DM. Regarding the third hit—apoptosis—our in vivo evidence has shown significantly fewer apoptotic hepatocytes in model Tg livers than model WT livers, similarly accompanied by reduced expression of the pro-apoptotic markers Bax and cleaved Caspase-3. Our supportive in vitro data further show

that oxidative stress (H2O2)-induced apoptosis is significantly reduced in cultured hepatocytes obtained from Tg mice. In addition to the anti-apoptotic effects of PRDX4, the model Tg liver also demonstrates significantly reduced expression of a-smooth muscle actin (a-SMA), a marker of stellate cell activation, and of collagen type I, a marker of hepatic Figure 4 Summary of the critical roles of PRDX4 in atherosclero- fibrosis/fibrogenesis. Indeed, as physiologically aberrant sis. This diagram depicts the beneficial, protective roles of PRDX4 apoptosis of hepatocytes is known to trigger the uncontrolled in the HcD-induced atherosclerosis mouse model. PRDX4, peroxir- fi f activation and differentiation of stellate cells into myo bro- edoxin 4; ROS, reactive oxygen species; M , macrophage; EC, fi fi endothelial cell; SMC, smooth muscle cell. blasts and subsequent liver brogenesis/ brosis, apoptotic hepatocytes potentially provide a critical ‘third hit’ that might drive the progression from NASH to cirrhosis and HCC.56 progression of mouse NAFLD and insulin resistance via In conclusion, PRDX4 overexpression plays critical key cross-talk with other promoters for other transcription factor roles in (i) ameliorating the local (intrahepatic) and systemic binding sites, such as the two activator protein 1 s (AP1s), (circulating) expression of oxidative stressors, (ii) preventing four NFkBs, and five cAMP response element binding the initiation of NAFLD and insulin resistance by reducing – proteins (CREBs).7 9 The intrahepatic overexpression of hepatic TG and FFA accumulation, and (iii) protecting hPRDX4 is therefore feasible in the model Tg mice. We also against the development of obesity, NASH, and type 2 DM found that, similar to the above atherosclerosis model, the by suppressing chronic inflammation, apoptosis, and fibro- expression of this activated transgene can overcome that of genesis, with its unique intracellular and extracellular the endogenous PRDX4 gene in modest NAFLD livers of Tg effects, as summarized in Fig. 5. Our observations support mice. In addition, the circulating (i.e., systemic) serum levels the utility of activators of PRDX4 as therapeutic agents for of soluble hPRDX4 are significantly elevated in model Tg ameliorating visceral obesity, NASH, and/or type 2 DM (i.e., mice. In that sense, the model establishment accelerates metabolic syndrome) by suppressing oxidative damage and oxidative stressors by increasing hepatic and blood ROS inflammatory signaling and by improving liver insulin sensi- levels in HFrD þ STZ-induced type 2 DM, NAFLD, and tivity throughout the course of these diseases. possibly metabolic syndrome, as shown by the intrahepatic expression of 8-OHdG, 4-hydroxy-2-nonenal (4-HNE), and systemic serum levels of TBARS, respectively. PRDX4 exerts beneficial effects on not only the hepatic The WT but not Tg mice showed particularly dramatic but also the intestinal function by protecting against increases in blood glucose and insulin levels, as well as oxidative stress-induced injury in an MCD þ HF-induced delayed glucose clearance, after STZ injection. This overt NAFLD mouse model dysfunction of the glucose/insulin metabolism may be due to the significant reduction in the insulin sensitivity, espe- In our second MCD þ HF diet-induced NAFLD mouse cially in the liver, due to significantly accumulated triglycer- model,9 an accumulating body of our data have confirmed ide (TG) and free fatty acid (FFA) levels, manifesting as the that the overexpression of intracellular (local) and secreted

protein. Indeed, the Tg mice have shown a reduction in oxidation by decreasing the hepatic, intestinal, and circula- tory ROS levels, even after the establishment of MCD þ HF feeding-induced NAFLD and intestinal dysfunction, as demonstrated by a reduced level of DHE binding in hepatocytes/enterocytes and of TBARS in serum. Our laboratory noted the unique finding that intestinal lipid deposits in surface enterocytes, derived from TG, FFA, and cholesteryl ester (CE) accumulation, are significantly increased in model Tg mice, accompanied by the presence of fewer apoptotic and more proliferating epithelial cells, particularly in the crypts, than model WT mice. It has been suggested that, inversely, activated apoptosis of the intestinal multipotent stem cells can induce uncontrolled cell-renewal processes, including migration or differentiation, and the subsequent shortening of the jejunal villi height and the total length9,57; however, this has not actually been confirmed in model Tg mice. This dysregulated renewal process may also significantly reduce the intestinal fat absorption capacity at the mucosal surface, where nutrient absorption mainly takes Figure 5 Summary of the critical, protective roles of PRDX4 in place.28 Taken together, these findings support the above NAFLD and type 2 DM. This diagram depicts the critical, protective characteristic histopathological features of intestinal lipid roles of PRDX4 in the nongenetic (HFrD þ STZ-induced) mouse model of NAFLD and type 2 DM, and the MCD þ HF-induced accumulation in Tg mice. Furthermore, we observed fewer NAFLD mouse model. PRDX4, peroxiredoxin 4; ROS, reactive infiltrating jejunal macrophages, associated with a lower oxygen species; Mf, macrophage; NAFLD, nonalcoholic fatty liver expression of various inflammatory cytokines, in the intestines disease; DM, diabetes mellitus. ofmodelTgmicethaninthoseofmodelWTmice.The stimulation of inflammation and apoptosis in the intestinal (systemic) PRDX4 significantly reduces or even wholly epithelium plays an important role in disrupting the mucosal prevents damage due to NAFLD. We also provide the first integrity and barrier function protecting against enterobacterial evidence that the small intestine as well as the liver are invasion.58,59 A study of human NASH patients cited a higher spared by the antioxidant properties of PRDX4 and that the prevalence of small intestine bacterial overgrowth (SIBO) and overexpression of PRDX4 beneficially affects the intestinal a lower proportion of the phyla Bacteroidetes than healthy function while preventing the progression of NAFLD. In adult volunteers, with increased gut permeability.59 We are addition to the target organ (the liver), the small intestine is going to perform further experiments to address these also involved in a key role in the etiology of NASH, which is complicated issues, including assessing the relationship be- one aspect of human metabolic syndrome, manifesting as tween the progression of metabolic syndrome and the intesti- visceral obesity, dyslipidemia, type 2 DM, and atherosclero- nal bacterial counts and composition of the phyla/genera. – sis.6 9,12 Since the intestine is the first organ to encounter In conclusion, our serial in vivo study of the initiation and nutrients and serves as gatekeeper at the pathophysiologi- progression of NAFLD provides new evidence supporting cal interface between the body and the diet, it can play a several potential mechanisms by which PRDX4 overexpres- varied but crucial role in the metabolic processing of sion plays critical, key roles in (i) reducing local (intrahepatic nutrients along with efficient lipid and bile acid (BA) and intraintestinal) and systemic (circulating) oxidative absorption, partly via enterohepatic circulation.15,16 stressors; (ii) affecting beneficial intestinal function by Indeed, detailed morphological, biochemical, and molecu- suppressing crypt enterocyte apoptosis and inflammation lar observations have shown that, very similarly to findings and by protecting against the shortening of the total length in other mice models of metabolic syndrome, the specific and villi height; and (iii) synergistically reducing the severity expression of hPRDX4 in the small intestine as well as the of NAFLD. Both the small intestine and liver can be spared liver of Tg mice is evident exclusively after establishing this to some degree by the characteristic antioxidant properties MCD þ HF dietary mouse NAFLD model. We have further of PRDX4. Specific activators of PRDX4 may therefore offer shown that model Tg mice display a phenotype of signifi- therapeutic potential for ameliorating the progression of cant resistance to local and systemic inflammation/oxidative NAFLD and intestinal dysfunction by attenuating oxidative injury along with markedly higher levels of tissue (i.e., liver damage. Fig. 5 briefly summarizes the crucial in vivo roles and intestine) PRDX4 and circulating serum soluble PRDX4 of PRDX4 in these mouse NAFLD models.

INTRIGUING TOPICS FOR FUTURE STUDIES AND Society of Pathology, and the Pathology Research Award CONCLUDING REMARKS ON THE UNIQUE PRDX4 Lecture at 63th annual meeting of The Japanese Society of Tg MICE Pathology, by Sohsuke Yamada in 2017. We would like to thank Dr. and Prof. Junichi Fujii, Metabolic syndrome is a complex, multifactorial disease, Department of Biomolecular Function, Graduate School of orchestrated by diet type affecting glucose/lipid/BA metabo- Medical Science, Yamagata University, Yamagata 990- lism, oxidative stress, insulin resistance, inflammatory cell 9585; Dr. and Prof. Yasuyuki Sasaguri, Department of infiltration, cytokine levels and/or apoptotic activities in Pathology and Cell Biology, School of Medicine, University various organs, translocation of either intestinal bacteria or of Occupational and Environmental Health, Kitakyushu 807- microbial cell components, and other factors. There is a 8555, Japan; Dr. and Prof. Akihide Tanimoto, Department growing body of evidence supporting the innate link between of Pathology, Field of Oncology, Graduate School of metabolic syndrome and the only known secretory member Medical and Dental Sciences, Kagoshima University, Ka- of the PRDX antioxidant family (PRDX4), which has not only goshima 890-8544, Japan; Dr. and Prof. Kimitoshi Kohno, local but also whole-body functions. Therefore, clinically, Asahi-Matsumoto Hospital, Kitakyushu, 800-0242, Japan; specific accelerators of PRDX4 may prove useful as thera- and Dr. Ke-Yong Wang, Shared-Use Research Center, peutic agents for protecting against the initiation and pro- School of Medicine, University of Occupational and Environ- gression of DM, atherosclerosis, NAFLD, and subsequently mental Health, Kitakyushu 807-8555, Japan, for their metabolic syndrome in humans by suppressing oxidative constructive and helpful suggestions/comments and excel- damage and pro-apoptotic or inflammatory factors through- lent technical assistance. out the course of these diseases. By contrast, one paper This work was supported in part by a grant from the MSD more recently published has reported that the plasma Life Science Foundation, Public Interest Incorporated Foun- PRDX4 levels are significantly higher in patients with dation, Japan (to S.Y.); by Grants-in-Aid for Scientific prediabetes.60 In line with that, we have also found that Research (24790394 and 16K08750) from the Ministry of patients with type 2 DM show significantly higher serum Education, Culture, Sports, Science and Technology, To- hPRDX4 levels than healthy adult volunteers,8 potentially kyo, Japan (to S.Y.); and by grants from National Natural due to the greater demand for antioxidant defense in type 2 Science Foundation of China (Number 81402490), Natural DM. Nevertheless, PRDX4 would have pathophysiological Science Foundation of Hebei Province (Number relevance for protecting against metabolic syndrome in H2016206170), and high-level talent support project of humans. The unique Tg mouse model will be useful for Hebei Province (Number CG2015003011) (to X.G.). We studying the associations of the locally and systemically would like to thank Hiroko Isagai, Hana Nishimura, and antioxidant properties of PRDX4 overexpression with lipid/ Naoko Tasaki for their expert technical assistance. BA/glucose metabolism and may also be a promising novel animal model of human metabolic syndrome. A recently published paper from another Japanese labora- DISCLOSURE STATEMENT tory using PRDX4 knockout mice (PRDX4/y), whose spermatogenic cells are more susceptible to apoptosis via None declared. oxidative damage than those of WT PRDX4þ/y male mice, þ/y reported that the levels of PRDX4 protein in PRDX4 male REFERENCES miceweresignificantly higher in nearly all tissues, especially /y those of the testes and pancreas, than those in PRDX4 1 Iuliano L. 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