Long-Term Dietary Taurine Lowers Plasma Levels of Cholesterol and Bile Acids.

Cholesterol is an essential lipid in vertebrates, but excess blood cholesterol promotes atherosclerosis. In the liver, cholesterol is metabolized to bile acids by cytochrome P450, family 7, subfamily a, polypeptide 1 (CYP7A1), the transcription of which is negatively regulated by the ERK pathway. Fibroblast growth factor 21 (FGF21), a hepatokine, induces ERK phosphorylation and suppresses Cyp7a1 transcription. Taurine, a sulfur-containing amino acid, reportedly promotes cholesterol metabolism and lowers blood and hepatic cholesterol levels. However, the influence of long-term feeding of taurine on cholesterol levels and metabolism remains unclear. Here, to evaluate the more chronic effects of taurine on cholesterol levels, we analyzed mice fed a taurine-rich diet for 14-16 weeks. Long-term feeding of taurine lowered plasma cholesterol and bile acids without significantly changing other metabolic parameters, but hardly affected these levels in the liver. Moreover, taurine upregulated Cyp7a1 levels, while downregulated phosphorylated ERK and Fgf21 levels in the liver. Likewise, taurine-treated Hepa1-6 cells, a mouse hepatocyte line, exhibited downregulated Fgf21 levels and upregulated promoter activity of Cyp7a1. These results indicate that taurine promotes cholesterol metabolism by suppressing the FGF21/ERK pathway followed by upregulating Cyp7a1 expression. Collectively, this study shows that long-term feeding of taurine lowers both plasma cholesterol and bile acids, reinforcing that taurine effectively prevents hypercholesterolemia.

Adrenic acid induces oxidative stress in hepatocytes.

Adrenic acid (ADA), which is an endogenously synthesized polyunsaturated free fatty acid, was significantly increased in nonalcoholic fatty liver disease (NAFLD) patients and NAFLD-model mice compared with the corresponding controls in our previous study. To elucidate the involvement of ADA in NAFLD and nonalcoholic steatohepatitis (NASH), we examined ADA-induced lipotoxicity in human hepatocarcinoma HepG2 cells. The ROS production in HepG2 cells was increased by exposure to ADA. It was also shown that the treatment with ADA decreased cell viability in a dose-dependent manner. The N-Acetyl-L-Cysteine pretreatment counteracted this ADA-induced ROS production and cell death. Furthermore, ADA modulated the expressions of SOD2, HO-1 and Gpx1 as antioxidant enzymes. These findings suggest that ADA could induce oxidative stress accompanied by cell death, providing new insights into lipotoxicity that is involved in the pathogenesis of NAFLD and NASH.

Noninvasive and Safe Cell Viability Assay for Breast Cancer MCF-7 Cells Using Natural Food Pigment.

A dye exclusion test (DET) was performed to determine the viability of human breast cancer cells MCF-7, using natural food pigments as compared with trypan blue (TB), a typical synthetic dye for DET known to exhibit teratogenicity and cytotoxicity. We demonstrated that Monascus pigment (MP) is noninvasive to living cells and can effectively stain only dead cells. This study is the first verification of the applicability of MP to cancer cells. The appropriate MP concentration was 0.4% (0.02% as the concentration of pure MP) and all the dead cells were stained within 10 min. We found that the cell proliferation or the reduced nicotinamide adenine dinucleotide (NADH) activity of living cells was maintained over 48 h. Although 0.1% TB did not show an increase in dead cells, a marked decrease in NADH activity was confirmed. In addition, even when MP coexisted with cisplatin, staining of dead cells was maintained for 47 h, indicating stability to drugs (reagents). The cost of MP is estimated to be about 1/10 of TB. The fact that MP can be used as a cell viability determination reagent for Euglena and Paramecium, as shown in preceding papers, and also for MCF-7, as shown in this paper, indicates the possibility of application in more cells of different species.

Nutlin-3a suppresses poly (ADP-ribose) polymerase 1 by mechanisms different from conventional PARP1 suppressors in a human breast cancer cell line.

Poly (ADP-ribose) polymerase 1 (PARP1) plays important roles in single strand DNA repair. PARP1 inhibitors enhance the effects of DNA damaging drugs in homologous recombination-deficient tumors including tumors with breast cancer susceptibility gene (BRCA1) mutation. Nutlin-3a, an analog of cis-imidazoline, inhibits degradation of murine double minute 2 (MDM2) and stabilizes p53. We previously reported that nutlin-3a induces PARP1 degradation in p53-dependent manner in mouse fibroblasts, suggesting nutlin-3a may be a PARP1 suppressor. Here, we investigated the effects of nutlin-3a on PARP1 in MCF-7, a human breast cancer cell line. Consistent with our previous results, nutlin-3a reduced PARP1 levels in dose- and time-dependent manners in MCF-7 cells, but this reduction was suppressed in p53 knockdown cells. RITA, a p53 stabilizer that binds to p53 itself, failed to reduce PARP1 protein levels. Moreover, transient MDM2 knockdown repressed nutlin-3a-mediated PARP1 reduction. The MG132 proteasome inhibitor, and knockdown of checkpoint with forkhead and ring finger domains (CHFR) and ring finger protein 146 (RNF146), E3 ubiquitin ligases targeting PARP1, suppressed nutlin-3a-induced PARP1 reduction. Short-term nutlin-3a treatment elevated the levels of PARylated PARP1, suggesting nutlin-3a promoted PARylation of PARP1, thereby inducing its proteasomal degradation. Furthermore, nutlin-3a-induced PARP1 degradation enhanced DNA-damaging effects of cisplatin in BRCA1 knockdown cells. Our study revealed that nutlin-3a is a PARP1 suppressor that induces PARP1 proteasomal degradation by binding to MDM2 and promoting autoPARylation of PARP1. Further analysis of the mechanisms in nutlin-3a-induced PARP1 degradation may lead to the development of novel PARP1 suppressors applicable for cancers with BRCA1 mutation.

ß-hydroxybutyrate protects hepatocytes against endoplasmic reticulum stress in a sirtuin 1-independent manner.

ß-hydroxybutyrate (BHB), a major ketone body in mammals, is produced from fatty acids through mitochondrial fatty acid oxidation in hepatocytes. To elucidate the role of BHB in the hepatic endoplasmic reticulum (ER), we examined the effects of BHB on hepatic ER stress induced by tunicamycin. In mouse hepatoma Hepa1c1c7 cells, BHB treatment suppressed the protein expression of ER stress responsive genes and increased cell viability, while reducing the protein expression of apoptosis inducible genes, without causing any alterations in the protein expression of sirtuin 1 (SIRT1) or the phosphorylation of AMP-activated protein kinase. The intraperitoneal administration of BHB also reduced the protein expression of ER stress responsive genes in mouse livers. In human hepatoma HepG2 cells, the protein expression levels of ER stress responsive genes were increased by the partial inhibition of BHB production with siRNA targeting endogenous 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) lyase, whereas they were decreased by promoting BHB production with fenofibrate. These findings revealed that BHB helps to suppress hepatic ER stress via a SIRT1-independent pathway, and it might be possible to manipulate ER stress by regulating BHB production genetically or pharmacologically.

Identification of WWP1 as an obesity-associated E3 ubiquitin ligase with a protective role against oxidative stress in adipocytes.

White adipose tissue (WAT) is not only the main tissue for energy storage but also an endocrine organ that secretes adipokines. Obesity is the most common metabolic disorder and is related to alterations in WAT characteristics, such as chronic inflammation and increasing oxidative stress. WW domain containing E3 ubiquitin protein ligase 1 (WWP1) is a HECT-type ubiquitin E3 ligase that has been implicated in various pathologies. In the present study, we found that WWP1 was upregulated in obese WAT in a p53-dependent manner. To investigate the functions of WWP1 in adipocytes, a proteome analysis of WWP1 overexpression (OE) and knockdown (KD) 3T3-L1 cells was performed. This analysis showed a positive correlation between WWP1 expression and the abundance of several antioxidative proteins. Thus, we measured reactive oxygen species (ROS) in WWP1 OE and KD cells. Consistent with the proteome results, WWP1 OE reduced ROS levels, whereas KD increased them. These findings indicate that WWP1 is an obesity-inducible E3 ubiquitin ligase that can protect against obesity-associated oxidative stress in WAT.

Differential Metabolic Responses to Adipose Atrophy Associated with Cancer Cachexia and Caloric Restriction in Rats and the Effect of Rikkunshito in Cancer Cachexia.

Despite the similar phenotypes, including weight loss, reduction of food intake, and lower adiposity, associated with caloric restriction (CR) and cancer cachexia (CC), CC is a progressive wasting syndrome, while mild CR improves whole body metabolism. In the present study, we compared adipose metabolic changes in a novel rat model of CC, mild CR (70% of the food intake of control rats, which is similar to the food consumption of CC rats), and severe CR (30% of the food intake of controls). We show that CC and severe CR are associated with much smaller adipocytes with significantly lower mitochondrial DNA content; but, that mild CR is not. CC and both mild and severe CR similarly upregulated proteins involved in lipolysis. CC also downregulated proteins involved in fatty acid biosynthesis, but mild CR upregulated these. These findings suggest that CC might impair de novo fatty acid biosynthesis and reduce mitochondrial biogenesis, similar to severe CR. We also found that rikkunshito, a traditional Japanese herbal medicine, does not ameliorate the enhanced lipolysis and mitochondrial impairment, but rather, rescues de novo fatty acid biosynthesis, suggesting that rikkunshito administration might have partially similar effects to mild CR.

Alterations in metabolic pathways in gastric epithelial cells infected with Helicobacter pylori.

Helicobacter pylori (H. pylori), which is a spiral-shaped Gram-negative microaerobic bacterium, is a causative pathogen. The entry of H. pylori into gastric epithelial cells involves various host signal transduction events, and its virulence factors can also cause a variety of biological responses. In this study, AGS human gastric carcinoma cells were infected with CagA-positive H. pylori strain ATCC43504, and then the metabolites in the AGS cells after the 2-, 6- and 12-h infections were analyzed by GC/MS-based metabolomic analysis. Among 67 metabolites detected, 11 metabolites were significantly altered by the H. pylori infection. The metabolite profiles of H. pylori-infected AGS cells were evaluated on the basis of metabolite pathways, and it was found that glycolysis, tricarboxylic acid (TCA) cycle, and amino acid metabolism displayed characteristic changes in the H. pylori-infected AGS cells. At 2 h post-infection, the levels of many metabolites related to TCA cycle and amino acid metabolism were lower in H. pylori-infected AGS cells than in the corresponding uninfected AGS cells. On the contrary, after 6-h and 12-h infections the levels of most of these metabolites were higher in the H. pylori-infected AGS cells than in the corresponding uninfected AGS cells. In addition, it was shown that the H. pylori infection might regulate the pathways related to isocitrate dehydrogenase and asparagine synthetase. These metabolite alterations in gastric epithelial cells might be involved in H. pylori-induced biological responses; thus, our findings are important for understanding H. pylori-related gastric diseases.