Joo-Hee Choi

New role of irisin in hepatocytes: The protective effect of hepatic steatosis in vitro

Irisin is a newly identified myokine related to exercise and the browning of white fat. Recently, it was reported that irisin serum levels are associated with intrahepatic triglyceride content, suggesting that it might have an important role in the liver. The aim of this study was to determine the role of irisin in hepatocytes. Specifically, the effect of recombinant irisin on palmitic acid (PA)-induced lipogenesis and its related signal pathways were examined in AML12 cells and mouse primary hepatocytes. In the present study, we observed the presence of irisin inside the cells in response to the treatment of recombinant irisin by flow cytometry and cell imaging technique. Recombinant irisin significantly inhibited the PA-induced increase in lipogenic markers ACC and FAS at the mRNA and protein levels, and prevented the PA-induced lipid accumulation in hepatocytes. Additionally, irisin inhibited the PA-induced increase in the expression, nuclear localization, and transcriptional activities of the master regulators of lipogenesis (LXRα and SREBP-1c). Moreover, irisin attenuated PA-induced oxidative stress, which was confirmed by measuring the expression of inflammatory markers (NFκB, COX-2, p38 MAPK, TNF, IL-6) and superoxide indicator (dihydroethidium). The preventive effects of irisin against lipogenesis and oxidative stress were mediated by the inhibition of protein arginine methyltransferase-3 (PRMT3). These findings suggested that irisin might have a beneficial role in the prevention of hepatic steatosis by altering the expression of lipogenic genes and attenuating oxidative stress in a PRMT3 dependent manner.

High glucose-induced O-GlcNAcylated carbohydrate response element-binding protein (ChREBP) mediates mesangial cell lipogenesis and fibrosis: the possible role in the development of diabetic nephropathy.

Background: Abnormal lipid synthesis and fibrosis can lead to diabetic nephropathy. Results: High glucose-induced O-GlcNAcylated ChREBP increases the expression of lipogenic and fibrotic proteins and induces lipid accumulation and fibrosis. Conclusion: O-GlcNAcylated ChREBP mediates lipogenesis and fibrosis in mesangial cells. Significance: This is the first report that O-glycosylated ChREBP plays a pathophysiological role in lipogenesis and fibrosis in mesangial cells. Carbohydrate response element-binding protein (ChREBP) is a transcription factor responsible for carbohydrate metabolism in the liver. However, the role of ChREBP in diabetic nephropathy has not been elucidated. Thus, we investigated the role of ChREBP in mesangial cells in diabetic nephropathy. Treatment with 25 mm glucose (high glucose; HG) increased cellular O-GlcNAc and O-GlcNAcylated ChREBP in mesangial cells compared with normal 5.5 mm glucose. O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino N-phenylcarbamate (PUGNAc), a drug that increases O-GlcNAc, augmented the expression of ChREBP targets, whereas DON, a drug that decreases O-GlcNAc and O-GlcNAcase overexpression, mitigated the increase with HG. O-GlcNAc augmented the protein stability, transcriptional activity, and nuclear translocation of ChREBP. HG treatment also stimulated lipid accumulation and the contents of triglyceride and cholesterol in mesangial cells. In addition, HG triggered expression of hypoxia-inducible factor 1-α, vascular endothelial growth factor, and extracellular matrix components related to nephrosclerosis. The ChREBP mutant, W130A, did not exhibit HG-induced lipid accumulation and fibrotic proteins, suggesting that the Trp-130 residue in the MCR3 domain is important in the development of glomerulosclerosis. O-GlcNAcylated ChREBP was elevated in mesangium cells of streptozotocin-induced diabetic rats. In conclusion, HG increased the O-GlcNAcylated ChREBP level, which resulted in lipid accumulation and up-regulation of fibrotic proteins in mesangial cells. These effects may lead mesangial cells to an ultimately pathological state.

Thioredoxin-interacting protein mediates hepatic lipogenesis and inflammation via PRMT1 and PGC-1α regulation in vitro and in vivo.

BACKGROUND & AIMS Non-alcoholic fatty liver disease (NAFLD) is strongly associated with obesity and type 2 diabetes. Thioredoxin-interacting protein (TXNIP) regulates the cellular redox state and metabolism and has been linked to many diseases, including diabetes. Therefore, we examined the role of TXNIP in hepatic steatosis in vitro and in vivo. METHODS Lipogenic and inflammatory proteins produced by hepatocytes treated with palmitic acid (PA) or transfected with TXNIP or Txnip siRNA were measured by Western blotting. Lipid accumulation was assessed using Oil Red O staining. Protein interactions were assessed by immunoprecipitation and proximity ligation assay. Hepatic protein levels were measured by Western blotting from wild type or Txnip(-/-) mice fed a high-fat diet (HFD) or chow diet. Livers from NAFLD patients were compared with normal liver by immunohistochemistry. RESULTS PA increased TXNIP, and inflammatory and lipogenic proteins in both AML12 and H4IIE cells. It also increased the peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α), which mediated the expression of lipogenic markers and lipid accumulation. In addition, PA increased protein arginine methyltransferase-1 (PRMT1) and PRMT1 siRNA abolished the increase in lipogenic markers with PGC-1α. Furthermore, TXNIP interacted with PRMT1 in PA-treated hepatocytes. In vivo, levels of lipogenic proteins, inflammatory molecules, PGC-1α, and PRMT1 were increased in the livers of HFD mice compared with those fed a chow diet, and were ameliorated in HFD Txnip(-/-) mice. Moreover, TXNIP, PRMT1, and PGC-1α were elevated in the livers of human NAFLD patients. CONCLUSIONS TXNIP mediates hepatic lipogenesis via PRMT1 and PGC-1α regulation and inflammation in vitro and in vivo, implying that targeting TXNIP and PRMT1 is a potential therapeutic approach for treatment of NAFLD.

Ubiquitination-dependent CARM1 degradation facilitates Notch1-mediated podocyte apoptosis in diabetic nephropathy.

Podocyte apoptosis induced by hyperglycemia is considered a critical factor in the development of diabetic nephropathy. Recent studies have implicated Notch signaling in podocyte apoptosis; however, its regulatory mechanisms are not fully understood. In this study, we found that high-glucose treatment increased Notch1 and Jagged-1 expression, the transcriptional activity of Hes, and podocyte apoptosis, and decreased the expression of coactivator-associated arginine methyltransferase 1 (CARM1) in rat podocytes. Transient transfection of CARM1 reversed high-glucose-induced Notch1 expression, the transcriptional activity of Hes, and podocyte apoptosis. Moreover, the silencing of CARM1 using siRNA increased Notch1 expression, the transcriptional activity of Hes, and podocyte apoptosis. However, the Glu(266)-mediated enzymatic activity of CARM1 was not necessary for Notch signaling activation and podocyte apoptosis. Here, we demonstrate that AMP-activated protein kinase alpha (AMPKα) and cannabinoid receptor 1 (CB1R) are regulated by CARM1 and that high-glucose-induced podocyte apoptosis is mediated by a CARM1-AMPKα-Notch1-CB1R signaling axis. We also show that high-glucose-induced CARM1 downregulation is due to ubiquitination-dependent CARM1 degradation. Finally, we demonstrate that CARM1 expression in podocytes was diminished in rats with streptozotocin-induced diabetes compared to vehicle-treated rats. Together, our data provide evidence that ubiquitination-dependent CARM1 degradation in podocytes in diabetes promotes podocyte apoptosis via Notch1 activation. Strategies to preserve CARM1 expression or reduce the enzymatic activity of a ubiquitin ligase specific for CARM1 could be used to prevent podocyte loss in diabetic nephropathy.

High-glucose-induced CARM1 expression regulates apoptosis of human retinal pigment epithelial cells via histone 3 arginine 17 dimethylation: Role in diabetic retinopathy

Hyperglycemia-induced apoptosis of retinal pigment epithelial (RPE) cells is considered to be involved in the progression of diabetic retinopathy. Histone arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) has emerged as an important histone modification involved in gene regulation. However, the role of PRMTs in diabetic retinopathy has not been elucidated. Here, we found that expression of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) was increased in the high-glucose treated human RPE cell line ARPE-19 and in the RPE layer of streptozotocin-treated rats. In addition, high-glucose induced apoptosis in ARPE-19 cells. To determine the function of CARM1 on RPE cell apoptosis, we performed gain- and loss-of-function studies. CARM1 overexpression increased apoptosis of RPE cells. In contrast, silencing of CARM1 expression by siRNA and pharmacological inhibition of CARM1 activity abolished high-glucose-induced RPE cell apoptosis. Furthermore, we found that inhibition of histone 3 arginine 17 (H3R17) asymmetric dimethylation attenuates both CARM1- and high-glucose-induced apoptosis in RPE cells. Together, these results show that high-glucose-induced CARM1 expression increases RPE cell apoptosis via H3R17 asymmetric dimethylation. Strategies to reduce CARM1 expression or enzymatic activity could be used to prevent apoptosis of RPE cells in the progression of diabetic retinopathy.

Hyperglycemia-induced GLP-1R downregulation causes RPE cell apoptosis

Glucagon-like peptide-1 receptor (GLP-1R) is closely associated with the onset of diabetes and its complications. However, its roles in diabetic retinopathy are unknown. Retinal pigment epithelial (RPE) cells are a crucial component of the outer blood-retina barrier and their death is related to the progression of diabetic retinopathy. Thus, we examined the pathophysiological role of GLP-1R in RPE cell apoptosis. We found that GLP-1R expression was lower in the isolated neuroretina and RPE cells of streptozotocin-treated rats than in vehicle-treated rats. High-glucose treatment also decreased GLP-1R expression in a human RPE cell line (ARPE-19 cells). GLP-1R was silenced in ARPE-19 cells, in order to elucidate the pathophysiological roles of GLP-1R. This increased intracellular reactive oxygen species (ROS) generation and activated p53-mediated Bax promoter and endoplasmic reticulum (ER) stress signaling. We also found that GLP-1R knockdown-mediated p53 expression was regulated by ER stress. Interestingly, antioxidant treatment and peroxiredoxin 1 (Prx1) overexpression attenuated GLP-1R knockdown-induced ER stress signaling and p53 expression. Finally, to confirm that GLP-1R activation has protective effects, ARPE-19 cells were treated with exendin-4, a synthetic GLP-1R agonist. This attenuated high-glucose-induced ROS generation, ER stress signaling, and p53 expression. Collectively, these results indicated that hyperglycemia decreases GLP-1R expression in RPE cells. Such a decrease generates intracellular ROS, which increases ER stress-mediated p53 expression, and subsequently causes apoptosis by increasing Bax promoter activity. Our data suggested that regulation of GLP-1R expression is a promising approach for the treatment of diabetic retinopathy.

Lactobacillus curvatus WiKim38 isolated from kimchi induces IL-10 production in dendritic cells and alleviates DSS-induced colitis in mice

Probiotics such as lactobacilli and bifidobacteria have healthpromoting effects by immune modulation. In the present study, we examined the immunomodulatory properties of Lactobacillus curvatus WiKim38, which was newly isolated from baechu (Chinese cabbage) kimchi. The ability of L. curvatus WiKim38 to induce cytokine production in bone marrow-derived dendritic cells (BMDCs) was determined by enzyme-linked immunosorbent assay. To evaluate the molecular mechanisms underlying L. curvatus Wikim38-mediated IL-10 production, Western blot analyses and inhibitor assays were performed. Moreover, the in vivo anti-inflammatory effects of L. curvatus WiKim38 were examined in a dextran sodium sulfate (DSS)-induced colitis mouse model. L. curvatus WiKim38 induced significantly higher levels of IL-10 in BMDCs compared with that induced by LPS. NF-κB and ERK were activated by L. curvatus WiKim38, and an inhibitor assay revealed that these pathways were required for L. curvatus WiKim38-induced production of IL-10 in BMDCs. An in vivo experiment showed that oral administration of L. curvatus WiKim38 increased the survival rate of mice with DSS-induced colitis and improved clinical signs and histopathological severity in colon tissues. Taken together, these results indicate that L. curvatus Wikim38 may have health-promoting effects via immune modulation, and may thus be applicable for therapy of various inflammatory diseases.

PRMT1 and PRMT4 Regulate Oxidative Stress-Induced Retinal Pigment Epithelial Cell Damage in SIRT1-Dependent and SIRT1-Independent Manners

Oxidative stress-induced retinal pigment epithelial (RPE) cell damage is involved in the progression of diabetic retinopathy. Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) has emerged as an important histone modification involved in diverse diseases. Sirtuin (SIRT1) is a protein deacetylase implicated in the onset of metabolic diseases. Therefore, we examined the roles of type I PRMTs and their relationship with SIRT1 in human RPE cells under H2O2-induced oxidative stress. H2O2 treatment increased PRMT1 and PRMT4 expression but decreased SIRT1 expression. Similar to H2O2 treatment, PRMT1 or PRMT4 overexpression increased RPE cell damage. Moreover, the H2O2-induced RPE cell damage was attenuated by PRMT1 or PRMT4 knockdown and SIRT1 overexpression. In this study, we revealed that SIRT1 expression was regulated by PRMT1 but not by PRMT4. Finally, we found that PRMT1 and PRMT4 expression is increased in the RPE layer of streptozotocin-treated rats. Taken together, we demonstrated that oxidative stress induces apoptosis both via PRMT1 in a SIRT1-dependent manner and via PRMT4 in a SIRT1-independent manner. The inhibition of the expression of type I PRMTs, especially PRMT1 and PRMT4, and increased SIRT1 could be therapeutic approaches for diabetic retinopathy.

The incidence of epiglottic cysts in a cohort of adults with acute epiglottitis

Clin. Otolaryngol. 2010, 35, 18–24.

Anti-Inflammatory and Antioxidative Effects of Camellia japonica on Human Corneal Epithelial Cells and Experimental Dry Eye: In Vivo and In Vitro Study

Purpose To analyze the anti-inflammatory and antioxidative effects of Camellia japonica (CJ) on human corneal epithelial (HCE) cells and its therapeutic effects in a mouse model of experimental dry eye (EDE). Methods Camellia japonica extracts of varying concentrations (0.001%, 0.01%, and 0.1%) were used to treat HCE cells. Dichlorofluorescein diacetate (DCF-DA) and dihydroethidium (DHE) assays were performed. The production of peroxiredoxin (PRX) 1-6 and manganese-dependent superoxide dismutase (MnSOD) in HCE cells was assessed using Western blot analysis. Furthermore, eye drops containing 0.001%, 0.01%, or 0.1% CJ extract or a balanced salt solution (BSS) were applied to the EDE. Clinical parameters were measured 7 days after treatment. The levels of inflammatory markers and intracellular reactive oxygen species (ROS) were measured. Results Treatment with 0.01% and 0.1% CJ extracts decreased apoptosis in HCE cells. In addition, band intensities of PRX 1, 4, and 5, as well as MnSOD, after hydrogen peroxide (H2O2) treatment showed a significant improvement after pretreatment with 0.01% and 0.1% CJ extracts. Mice treated with 0.1% CJ extract showed significantly improved clinical parameters when compared to those of the EDE control and BSS groups. A significant decrease in the levels of inflammatory markers and intracellular ROS was observed in the 0.01% and 0.1% CJ extract groups. Conclusions Camellia japonica extracts promoted antioxidative protein expression and suppressed apoptosis in HCE cells. Furthermore, CJ extracts improved clinical signs of dry eye and reduced oxidative stress and the expression of inflammatory markers, suggesting that eye drops containing CJ extract could be used as an adjunctive treatment for dry eye.