Metabolic Profiling Changes Induced by Fermented Blackberries in High-Fat-Diet-Fed Mice Utilizing Gas Chromatography-Mass Spectrometry Analysis.

The aim of this study was to investigate the metabolic changes associated with the anti-obesity effects of fermented blackberry extracts in the liver tissues of high-fat-diet-fed mice using mass spectrometry-based metabolomics analysis. C57BL/6J mice were divided into eight groups: normal-diet-fed mice, high-fat-diet-fed mice, high-fat diet treated with blackberry extract, high-fat-diet mice treated with blackberry fermented by L. plantarum, and high-fat diet with blackberry fermented by L. brevis. After 12 weeks, the high-fat-diet group exhibited a greater increase in liver weight compared to the control group, and among the groups, the group administered with blackberry fermented with L. plantarum showed the most pronounced reduction in liver weight. As the primary organ responsible for amino acid metabolism, the liver is crucial for maintaining amino acid homeostasis. In our study, we observed that the levels of several essential amino acids, including isoleucine and valine, were decreased by the high-fat diet, and were recovered by administration of blackberry extract fermented with L. plantarum. Our results demonstrated the potential of blackberry extract fermented with L. plantarum as a functional material for metabolic disorders by restoring some of the amino acid metabolism disturbances induced by a high-fat diet.

Anti-Obesity Properties of Blackberries Fermented with L. plantarum JBMI F5 via Suppression of Adipogenesis Signaling Mechanisms.

Blackberries (Rubus fruticosus), which are known to include a variety of bioactive substances, have been extensively studied for their antioxidant properties. Blackberries possess multiple health beneficial effects, including anti-inflammation, anti-atherosclerosis, anti-tumor and immunomodulatory activity. However, the potential biological effects and precise molecular mechanisms of the fermented extracts remain largely unexplored. In this research, we demonstrate the effect of blackberries fermented with Lactobacillus for addressing obesity. We investigated the effect of blackberries fermented by Lactobacillus on mice fed a high-fat (60% kcal) diet for 12 weeks. Fermented blackberry administration reduced the body weight and epididymal fat caused by a high-fat diet compared to the obese group. The triglyceride and total cholesterol, which are blood lipid indicators, and the levels of leptin, which is an insulin resistance indicator, were significantly increased in the obese group but were significantly decreased in the fermented blackberries-treated group. Additionally, the expression of adipogenesis marker proteins, such as CEBPα, PPAR-γ and SREBP-1, was significantly increased in the obese group, whereas it was decreased in the fermented blackberries-treated group. These results suggest that fermented blackberries have a protective effect against high-fat-diet-induced obesity by inhibiting adipogenesis and are a potential candidate for the treatment of obesity.

Fermented Aloe arborescens Miller Leaf Extract Suppresses Acute Alcoholic Liver Injury via Antioxidant and Anti-Inflammatory Effects in C57BL/6J Mice.

This study confirmed the change in functional composition and alcohol-induced acute liver injury in Aloe arborescens after fermentation. An acute liver injury was induced by administration of ethanol (3 g/kg/day) to C57BL/6J mice for 5 days. A fermented A. arborescens Miller leaf (FAAL) extract was orally administered 30 minutes before ethanol treatment. After fermentation, the emodin content was approximately 13 times higher than that of the raw material. FAAL extract significantly attenuated ethanol-induced aspartate aminotransferase, alanine aminotransferase, and triglyceride increases in serum and liver tissue. Histological analysis revealed that FAAL extract inhibits inflammatory cell infiltration and fat accumulation in liver tissues. The cytochrome P450 2E1, superoxide dismutase, and glutathione (GSH), which involved in alcohol-induced oxidative stress, were effectively regulated by FAAL extract in serum and liver tissues, except for GSH. FAAL also maintained the antioxidant defense system by upregulating heme oxygenase 1 and nuclear factor erythroid 2-related factor 2 protein expression. In addition, FAAL extract inhibited the decrease in alcohol dehydrogenase and aldehyde dehydrogenase activity, which promoted alcohol metabolism and prevented the activation of inflammatory response. Our results suggest that FAAL could be used as a potential therapeutic agent for ethanol-induced acute liver injury.

Protective Effects of Fermented Paprika (Capsicum annuum L.) on Sodium Iodate-Induced Retinal Damage.

Diseases of the outer retina, including age-related macular degeneration (AMD), are major cause of permanent visual damage. The pathogenesis of AMD involves oxidative stress and damage of the retinal pigment epithelium. Capsicum annuum L. (paprika) fruits have been known as a source of vitamins, carotenoids, phenolic compounds, and metabolites with a well-known antioxidant activity, which have positive effects on human health and protection against AMD and cataracts. In this study, we investigated whether paprika (fermented (FP), yellow, and orange colored) fermented with Lactobacillus (L.) plantarum could increase the protective effect of retinal degeneration using in vitro and in vivo models. FP significantly increased cell survival and reduced levels of lactate dehydrogenase as well as intracellular reactive oxygen species (ROS) increase in SI (sodium iodate, NaIO3)-treated human retinal pigment epithelial (ARPE-19) cells. We developed a model of retinal damage in C57BL/6 mice using SI (30 mg/kg) via intraperitoneal injection. Seven days after SI administration, deformation and a decrease in thickness were observed in the outer nuclear layer, but improved by FP treatment. FP administration protected the SI-mediated reduction of superoxide dismutase and glutathione levels in the serum and ocular tissues of mice. The overproduction of cleaved poly(ADP-Ribose) Polymerase (PARP)1, caspase-3 and -8 proteins were significantly protected by FP in SI-treated cells and ocular tissues. In addition, we evaluated the potentiating effects of FP on antioxidants and their underlying mechanisms in RAW 264.7 cells. Lipopolysaccharide (LPS)-induced nitrite increase was markedly blocked by FP treatment in RAW 264.7 cells. Furthermore, FP reduced LPS-induced inducible nitric oxide synthase and cyclooxygenase-2 activation. The FP also enhanced the inhibitory effects on mitogen activated kinase signaling protein activation in ARPE-19 and RAW 264.7 cells and ocular tissues. There was no significant difference in total phenol and flavonoid content in paprika by fermentation, but the vitamin C content was increased in orange colored paprika, and protective effect against oxidative stress-mediated retinal damage was enhanced after fermentation. These results suggest that FP may be a potential candidate to protect against retinal degenerative diseases through the regulation of oxidative stress.

Fermentation of Blackberry with L. plantarum JBMI F5 Enhance the Protection Effect on UVB-Mediated Photoaging in Human Foreskin Fibroblast and Hairless Mice through Regulation of MAPK/NF-κB Signaling.

Chronic and extensive exposure of ultraviolet (UV)-irradiation causes human skin sunburn, inflammation, or photoaging, which is associated with downregulated collagen synthesis. This study investigated the effects of fermented blackberry (Rubus fruticosus B., FBB) by Lactobacillus plantarum JBMI F5 (LP) on UVB-induced photoaging in human foreskin fibroblast (Hs68) as well as in SKH-1 hairless mice. FBB pretreatment inhibited UVB-mediated type-1 procollagen degradation, matrix metalloproteinase (MMP)-1 and MMP-2 protein expression, and suppressed nuclear factor-κB (NF-κB) activation as well as mitogen-activated protein kinase (MAPK) phosphorylation in Hs68. In addition, FBB administration diminished the wrinkle formation in dorsal skin and epidermal thickening in UVB-irradiated hairless mice. Moreover, UVB-induced Type-1 procollagen reduction and antioxidant enzyme inactivation were reversed by FBB administration. These results suggest that FBB may have antiphotoaging effects on UVB-induced wrinkle formation by maintaining the extracellular matrix density in the dermis, which occurs via regulation of reactive oxygen species and related MAPK and NF-κB signaling. Therefore, FBB can be a potential candidate for protecting skin aging against UV irradiation.

Suppressive role of OGT-mediated O-GlcNAcylation of BAP1 in retinoic acid signaling.

BRCA1-associated protein 1 (BAP1) has been implicated in diverse biological functions, including tumor suppression. However, its regulation via glycosylation and its role in embryonic stem (ES) cells are poorly defined. BAP1 was recently reported to interact with O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT). Here, we confirmed the physical interaction and investigated its functional significance. The O-GlcNAcylation of BAP1, which requires OGT, was examined in vivo and in vitro, and was proven using alloxan, an OGT inhibitor. OGT promoted the BAP1-induced repression of retinoic acid (RA)-induced RA receptor (RAR) activation. The repressive activity of BAP1 was relieved by alloxan but exacerbated by PUGNAc, an O-GlcNAcase (OGA) inhibitor. Finally, we addressed the role of O-GlcNAcylation in the RA-induced differentiation of murine ES cells. Alkaline phosphatase staining revealed the cooperation of RA and alloxan for impairing the pluripotency of ES cells. This cooperation was also observed by measuring the size of embryonic bodies and the expression of Sox2, a pluripotency marker. Overall, our data suggest that OGT-mediated O-GlcNAcylation of BAP1 prefers the maintenance of pluripotency, whereas its inhibition facilitates RA-induced differentiation in ES cells.

Hypocholesterolemic Effects of Probiotic Mixture on Diet-Induced Hypercholesterolemic Rats.

Growing evidence has indicated that supplementation with probiotics improves lipid metabolism. We aimed to investigate the beneficial effects of a probiotics mixture (PM) of three strains belonging to the species Bifidobacterium (B. longum, B. lactis, and B. breve) and two strains belonging to the species Lactobacillus (L. reuteri and L. plantarum) on cholesterol-lowering efficacy in hypercholesterolemic rats. A hypercholesterolemic rat model was established by feeding a high-cholesterol diet for eight weeks. To test the effects of PM on hypercholesterolemia, hypercholesterolemic rats were assigned to four groups, which were treated daily with low (1.65 × 108 cfu/kg), medium (5.5 × 108 cfu/kg), or high (1.65 × 1010 cfu/kg) doses of probiotic mixture or simvastatin for eight weeks. Significant reductions of serum total cholesterol (TC), triacylglycerol (TG), and low-density lipoprotein (LDL)-cholesterol levels, but increases of high-density lipoprotein (HDL)-cholesterol were observed after supplementation of PM in hypercholesterolemic rats. In PM-supplemented hypercholesterolemic rats, hepatic tissue contents of TC and TG also significantly decreased. Notably, the histological evaluation of liver tissues demonstrated that PM dramatically decreased lipid accumulation. For their underlying mechanisms, we demonstrated that PM reduced expressions of cholesterol synthesis-related proteins such as sterol regulatory element-binding protein 1 (SREBP1), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) in the liver. Taken together, these findings suggest that PM has beneficial effects against hypercholesterolemia. Accordingly, our PM might be utilized as a novel therapeutic agent for the management of hypercholesterolemia.

Retracted: Histone H2B ubquitination regulates retinoic acid signaling through the cooperation of ASXL1 and BAP1.

Despite the importance of retinoic acid (RA) signaling and histone monoubiquitination in determining cell fate, the underlying mechanism linking the two processes is poorly explored. We describe that additional sex comb-like 1 (ASXL1) represses RA receptor activity by cooperating with BRCA1-associated protein 1 (BAP1), which contains the ubiquitin C-terminal hydrolase (UCH) domain. Both the UCH- and ASXL1-binding domains of BAP1 were required for cooperation. In contrast to Drosophila BAP1, mammalian BAP1 cleaved ubiquitin from histone H2B. As supported by BAP1 mutants, ASXL1 was critical for BAP1 recruitment to chromatin and its activation therein. ASXL1 requirement was supported using Asxl1 null mice embryonic fibroblasts. Both ASXL1 and BAP1 were downregulated during RA-induced P19 cell differentiation with concomitant increase of ubiquitinated H2B, leading to activation of Hox genes. Our data demonstrate the critical role of ASXL1 cooperation with BAP1 in cell differentiation through the regulation of RA signaling associated with H2B ubiquitination.

Positive regulation of additional sex comb-like 1 gene expression by the pluripotency factor SOX2.

Additional sex comb-like 1 (ASXL1) has been suggested to be an enhancer of trithorax and polycomb proteins, and functions as a dual co-regulator of retinoid acid (RA) signaling. However, the mechanism by which ASXL1 gene expression is regulated remains unresolved. Concomitant downregulation of both SOX2 and ASXL1 during the RA-induced differentiation of P19 cells prompted us to investigate the role of SOX2 in the regulation of ASXL1. Knockdown of SOX2 in SOX2-rich NT2 cells resulted in the reduction of ASXL1 expression, whereas SOX2 overexpression in SOX2-deficient H1299 cells increased ASXL1 expression. Using a cloned ASXL1-luciferase reporter, we demonstrated that SOX2 directly transactivates the ASXL1 promoter. Serial deletion and mutation studies mapped the SOX2 response element region in the ASXL1 promoter to -1600 to -1400 bp. We showed by chromatin immunoprecipitation assay that SOX2 directly binds to the ASXL1 promoter region. Finally, formation of embryonic bodies by ASXL1-depleted murine E14TG2a embryonic stem cells was significantly impaired, similar to SOX2-knockdown cells. From these results, we suggest that ASXL1 may be a direct target of SOX2 and may play a role in maintaining the pluripotency of stem cells.

Identification and characterization of adenovirus early region 1B-associated protein 5 as a surface marker on undifferentiated human embryonic stem cells.

Pluripotent human embryonic stem cells (hESCs) provide appropriate systems for developmental studies and prospective donor cell sources for regenerative medicine. Identification of surface markers specific to hESCs is a prerequisite for studying hESC biology and can be used to generate clinical-level donor cell preparations that are free from tumorigenic undifferentiated hESCs. We previously reported the generation of monoclonal antibodies that specifically recognize hESC surface antigens using a decoy immunization strategy. In this study, we show that monoclonal antibody 57-C11 recognizes a phosphorylated form of adenovirus early region 1B-associated protein 5 (E1B-AP5). E1B-AP5 is a nuclear RNA-binding protein, but we report that 57-C11-reactive E1B-AP5 is expressed on the surface of undifferentiated hESCs. In undifferentiated hESCs, 57-C11-reactive E1B-AP5 is localized to SSEA3-, SSEA4-, TRA-1-60-, TRA-1-81-, OCT4-, SOX2-, and NANOG-positive hESCs. In mixtures of undifferentiated hESCs and hESC-derived neurons, 57-C11 exclusively recognizes undifferentiated hESCs but not hESC-derived neuronal cells. Further, the expression of 57-C11-reactive E1B-AP5 decreases upon differentiation. Our results demonstrate that 57-C11-reactive E1B-AP5 is a novel surface molecule that is involved in the undifferentiated state of hESCs. As far as we know, this is the first report demonstrating that heterogeneous nuclear RNA-binding protein is expressed on the surface of undifferentiated hESCs.

ASXL1 represses retinoic acid receptor-mediated transcription through associating with HP1 and LSD1.

We previously suggested that ASXL1 (additional sex comb-like 1) functions as either a coactivator or corepressor for the retinoid receptors retinoic acid receptor (RAR) and retinoid X receptor in a cell type-specific manner. Here, we provide clues toward the mechanism underlying ASXL1-mediated repression. Transfection assays in HEK293 or H1299 cells indicated that ASXL1 alone possessing autonomous transcriptional repression activity significantly represses RAR- or retinoid X receptor-dependent transcriptional activation, and the N-terminal portion of ASXL1 is responsible for the repression. Amino acid sequence analysis identified a consensus HP1 (heterochromatin protein 1)-binding site (HP1 box, PXVXL) in that region. Systematic in vitro and in vivo assays revealed that the HP1 box in ASXL1 is critical for the interaction with the chromoshadow domain of HP1. Transcription assays with HP1 box deletion or HP1alpha knockdown indicated that HP1alpha is required for ASXL1-mediated repression. Furthermore, we found a direct interaction of ASXL1 with histone H3 demethylase LSD1 through the N-terminal region nearby the HP1-binding site. ASXL1 binding to LSD1 was greatly increased by HP1alpha, resulting in the formation of a ternary complex. LSD1 cooperates with ASXL1 in transcriptional repression, presumably by removing H3K4 methylation, an active histone mark, but not H3K9 methylation, a repressive histone mark recognized by HP1. This possibility was supported by chromatin immunoprecipitation assays followed by ASXL1 overexpression or knockdown. Overall, this study provides the first evidence that ASXL1 cooperates with HP1 to modulate LSD1 activity, leading to a change in histone H3 methylation and thereby RAR repression.

Reciprocal roles of SIRT1 and SKIP in the regulation of RAR activity: implication in the retinoic acid-induced neuronal differentiation of P19 cells.

Human sirtuin 1 (SIRT1) is a NAD(+)-dependent deacetylase that participates in cell death/survival, senescence and metabolism. Although its substrates are well characterized, no direct regulators have been defined. Here, we show that SIRT1 associates with SKI-interacting protein (SKIP) and modulates its activity as a coactivator of retinoic acid receptor (RAR). Binding assays indicated that SKIP interacts with RAR in a RA-dependent manner, through a region that overlaps the binding site for SIRT1. SKIP augmented the transcriptional activation activity of RAR by cooperating with SRC-1, and SIRT1 suppressed SKIP/SRC-1-enhanced RAR transactivation activity. The suppression was dependent on the deacetylase activity of SIRT1 and was enhanced by a SIRT1 activator, resveratrol. In contrast, the suppression was relieved by SIRT1 knockdown, overexpression of SKIP and treatment with a SIRT1 inhibitor, splitomicin. Upon SKIP overexpression, the recruitment of SIRT1 to the endogenous RARbeta2 promoter was severely impaired, and SKIP was recruited to the promoter instead. Finally, resveratrol treatment inhibited RA-induced neuronal differentiation of P19 cells, accompanied by reductions in the neuronal marker nestin and a RAR target gene, RARbeta2. This inhibition was relieved by either knockdown of SIRT1 or overexpression of SKIP. These data suggest that SIRT1 and SKIP play reciprocal roles in the regulation of RAR activity, which is implicated in the regulation of RA-induced neuronal differentiation of P19 cells.

Utilization of the recombinant human beta-carotene-15,15'-monooxygenase gene in Escherichia coli and mammalian cells.

In animals, beta-carotene 15,15'-monooxygenase (BCMO) is the key enzyme involved in the metabolism of plant beta-carotene to retinal. In the present study, we utilized beta-carotene-producing Escherichia coli to screen for mutants with higher BCMO activity which was monitored by color changes derived from beta-carotene cleavage. Recombinant wild-type and T381L mutant BCMO proteins were purified to near homogeneity in E. coli, and their enzymatic activities were determined by HPLC analysis. The catalytic efficiency for beta-carotene and retinal production of the mutant were 1.5-fold and 1.7-fold higher than those of wild-type, respectively. Further BCMO function in mammalian cells was analyzed by a retinoic acid receptor reporter assay, which responds to the metabolic conversion of beta-carotene to retinoic acid in vivo. Overall, these tools can be used to screen more active BCMO for the industrial and pharmacological purpose of retinal production from beta-carotene.

Transcriptional regulation of the p73 gene, a member of the p53 family, by early growth response-1 (Egr-1).

To elucidate the regulatory mechanism of p73 gene expression, we analyzed the human p73 promoter and found three putative Egr-1-binding sites located upstream of exon 1 (-1728, -321, and -38). The Egr-1 responsiveness of these sites was analyzed by transient transfection assays using 5'- and 3'-serial truncations of the p73 promoter, subcloned in a CAT reporter vector. The functional significance of the region was further confirmed by an electrophoretic mobility shift assay using the Egr-1 protein synthesized in vitro and a [32P]-labeled middle site sequence, followed by competition with unlabeled wild-type or mutant oligonucleotides and supershift assays using an anti-Egr-1 antibody. When induced by either the nitric oxide donor NOC-18 or the PPARgamma agonist troglitazone, Egr-1 bound to the p73 promoter, as assessed by chromatin immunoprecipitation assays, accompanied by increased expression of p73. MTT assays revealed that cell growth was significantly inhibited on treating the cells with troglitazone. Overall, our results provide direct evidence that Egr-1 positively regulated p73 expression by binding to its promoter in vivo, consistent with Egr-1 and p73 being involved in p53-independent tumor suppression.

FHL2 mediates p53-induced transcriptional activation through a direct association with HIPK2.

To understand the molecular mechanism underlying HIPK2 regulation of the transcriptional activation by p53, we sought to identify the protein that interacts with HIPK2. From our yeast two-hybrid screen, we found that four and a half LIM domains 2 (FHL2) could bind to the C-terminal half of HIPK2. Further assays in yeast mapped the minimal interaction domain to amino acids 812-907 in HIPK2. The interaction was confirmed using a GST pull-down assay in vitro, and an immunoprecipitation (IP) assay and fluorescence microscopy in vivo. FHL2 alone spread throughout both the cytoplasm and nucleus but was redistributed to dot-like structures in the nucleus when HIPK2 was coexpressed in HEK293 cells. When tethered to the Gal4-responsive promoter through the Gal4 DBD fusion, FHL2 showed autonomous transcriptional activity that was enhanced by wild-type HIPK2, but not by the kinase-defective mutant. In addition, FHL2 increased the p53-dependent transcriptional activation and had an additive effect on the activation when coexpressed with HIPK2, which was again not observed with the kinase-defective mutant of HIPK2. Finally, we found a ternary complex of p53, HIPK2, and FHL2 using IP, and their recruitment to the p53-responsive p21Waf1 promoter in chromatin IP assays. Overall, our findings indicate that FHL2 can also regulate p53 via a direct association with HIPK2.