H2 O2 induces PP2A demethylation to downregulate mTORC1 signaling in HEK293 cells.

Mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase that functions as an ATP and amino acid sensor to govern cell growth and proliferation by mediating mitogen- and nutrient-dependent signal transduction. Protein phosphatase 2A (PP2A), a ubiquitously expressed serine/threonine phosphatase, negatively regulates mTOR signaling. Methylation of PP2A is catalyzed by leucine carboxyl methyltransferase-1 (LCMT1) and reversed by protein phosphatase methylesterase 1 (PME-1), which regulates PP2A activity and substrate specificity. However, whether PP2A methylation is related to mTOR signaling is still unknown. In this study, we examined the effect of PP2A methylation on mTOR signaling in HEK293 cells under oxidative stress. Our results show that oxidative stress induces PP2A demethylation and inhibits the mTORC1 signaling pathway. Next, we examined two strategies to block PP2A demethylation under oxidative stress. One strategy was to prevent PP2A demethylation using a PME-1 inhibitor; the other strategy was to activate PP2A methylation via overexpression of LCMT1. The results show that both the PME-1 inhibitor and LCMT1 overexpression prevent the mTORC1 signaling suppression induced by oxidative stress. Additionally, LCMT1 overexpression rescued cell viability and the mitochondrial membrane potential decrease in response to oxidative stress. These results demonstrate that H2 O2 induces PP2A demethylation to downregulate mTORC1 signaling. These findings provide a novel mechanism for the regulation of PP2A demethylation and mTORC1 signaling under oxidative stress.

Machine learning classifiers for screening nonalcoholic fatty liver disease in general adults.

Nonalcoholic fatty liver disease (NAFLD) is one of major causes of end-stage liver disease in the coming decades, but it shows few symptoms until it develops into cirrhosis. We aim to develop classification models with machine learning to screen NAFLD patients among general adults. This study included 14,439 adults who took health examination. We developed classification models to classify subjects with or without NAFLD using decision tree, random forest (RF), extreme gradient boosting (XGBoost) and support vector machine (SVM). The classifier with SVM was showed the best performance with the highest accuracy (0.801), positive predictive value (PPV) (0.795), F1 score (0.795), Kappa score (0.508) and area under the precision-recall curve (AUPRC) (0.712), and the second top of area under receiver operating characteristic curve (AUROC) (0.850). The second-best classifier was RF model, which was showed the highest AUROC (0.852) and the second top of accuracy (0.789), PPV (0.782), F1 score (0.782), Kappa score (0.478) and AUPRC (0.708). In conclusion, the classifier with SVM is the best one to screen NAFLD in general population based on the results from physical examination and blood testing, followed by the classifier with RF. Those classifiers have a potential to screen NAFLD in general population for physician and primary care doctors, which could benefit to NAFLD patients from early diagnosis.

Hepatic leucine carboxyl methyltransferase 1 (LCMT1) contributes to high fat diet-induced glucose intolerance through regulation of glycogen metabolism.

Impaired glucose regulation is one of the most important risk factors for type 2 diabetes mellitus (T2DM) and cardiovascular diseases, which have become a major public health issue worldwide. Dysregulation of carbohydrate metabolism in liver has been shown to play a critical role in the development of glucose intolerance but the molecular mechanism has not yet been fully understood. In this study, we investigated the role of hepatic LCMT1 in the regulation of glucose homeostasis using a liver-specific LCMT1 knockout mouse model. The hepatocyte-specific deletion of LCMT1 significantly upregulated the hepatic glycogen synthesis and glycogen accumulation in liver. We found that the liver-specific knockout of LCMT1 improved high fat diet-induced glucose intolerance and insulin resistance. Consistently, the high fat diet-induced downregulation of glucokinase (GCK) and other important glycogen synthesis genes were reversed in LCMT1 knockout liver. In addition, the expression of GCK was significantly upregulated in MIHA cells treated with siRNA targeting LCMT1 and improved glycogen synthesis. In this study, we provided evidences to support the role of hepatic LCMT1 in the development of glucose intolerance induced by high fat diet and demonstrated that inhibiting LCMT1 could be a novel therapeutic strategy for the treatment of glucose metabolism disorders.

An analysis of neurovascular disease markers in the hippocampus of Tupaia chinensis at different growth stages.

Introduction: It is considered that Tupaia chinensis can replace laboratory primates in the study of nervous system diseases. To date, however, protein expression in the brain of Tupaia chinensis has not been fully understood. Method: Three age groups of T. chinensis-15 days, 3 months and 1.5 years-were selected to study their hippocampal protein expression profiles. Results: A significant difference was observed between the 15-day group and the other two age groups, where as there were no significant differences between the 3-month and 1.5-year age groups. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that differentially expressed proteins could be enriched in several pathways related to neurovascular diseases, such as metabolic pathways for Alzheimer's disease (AD), Huntington's disease, Parkinson's disease, and other diseases. The KEGG enrichment also showed that relevant protein involved in oxidative phosphorylation in the hippocampus of T. chinensis for 15days were downregulated, and ribosomal proteins (RPs) were upregulated, compared to those in the hippocampus of the other two age groups. Discussion: It was suggested that when the hippocampus of T. chinensis developed from day 15 to 3 months, the expression of oxidatively phosphorylated proteins and RPs would vary over time. Meanwhile, the hippocamppal protein expression profile of T. chinensis after 3 months had become stable. Moreover, the study underlines that, during the early development of the hippocampus of T. chinensis, energy demand increases while protein synthesis decreases. The mitochondria of T. chinensis changes with age, and the oxidative phosphorylation metabolic pathway of mitochondria is closely related to neurovascular diseases, such as stroke and cerebral ischemia.

Regulation PP2Ac methylation ameliorating autophagy dysfunction caused by Mn is associated with mTORC1/ULK1 pathway.

Manganese (Mn) exposure leads to autophagy dysfunction and causes neurodegenerative diseases such as Parkinson's syndrome and Alzheimer's disease. However, the mechanism of neurotoxicity of Mn has been less clear. The methylation of the protein phosphatase 2A catalytic subunit determines the dephosphorylation activity of protein phosphatase and plays an important role in autophagy regulation. In this investigation, we established a model of Mn (0-2000 µmol/L) exposure to N2a cells for 12 h, used the PPME-1 inhibitor ABL-127, and constructed an LCMT1-overexpressing N2a cell line. We also regulated the PP2Ac methylation level and explored the effect of PP2Ac methylation on Mn-induced (0-1000 µmol/L) N2a cellular autophagy. Our results showed that Mn > 500 µmol/L induced N2a cell damage and increased oxidative stress. Moreover, Mn modulated autophagy in N2a cells by downregulating PP2Ac methylation, which regulated mTORC1 signaling pathway activation. Both ABL-127 and LCMT1 overexpression can upregulate PP2Ac methylation in parallel with ameliorating N2a cell abnormal autophagy induced by Mn, Briefly, the upregulation of PP2Ac methylation can ameliorate the autophagy disorder of N2a by Mn and effectively alleviate Mn-induced cytotoxicity and oxidative stress, indicating that regulation of autophagy is a protective strategy against Mn-induced neurotoxicity.

[Expression, purification and functional assessment of asprosin inclusion body].

OBJECTIVE: The obtain purified recombinant asprosin and test its functions. METHODS: The recombinant plasmid of pET-22b-asprosin was constructed and transformed into competent E.coli BL (DE3) strain. After IPTG-induced expression, asprosin inclusion body was renatured by gradient urea and purified by Ni-NTA affinity chromatography column followed by removal of endotoxin to obtain recombinant asprosin for use in cells and animals experiments. C57 mice were injected intraperitoneally with the recombinant asprosin and blood glucose was detected using a blood glucose meter. Alamar Blue assay was used to evaluate of the effect of the recombinant asprosin on the viability of MIHA cells, and cellular glycogen content was detected using the anthrone method. RESULTS: At the absorbance at 600 nm of 0.8, induction of the recombinant host bacteria with 1 mmol/L IPTG at 37 ℃ for 4 h optimally induced the expression of asprosin inclusion body. After purification and endotoxin removal, the purity of the recombinant asprosin exceeded 95% with the content of endotoxin below 1 EU/mg. In C57 mice, intraperitoneal injection with recombinant asprosin significantly increased blood glucose level, which reached the peak level at 60 min following the injection (P=0.021) and recovered the normal level at 120 min (P=0.03). Treatment with the recombinant asprosin for 24 h did not cause obvious adverse effect on the viability of MIHA cells but significantly lowered glycogen content in the cells (P < 0.05). CONCLUSIONS: We successfully obtained recombinant asprosin using a prokaryotic expression system. The recombinant asprosin can decrease glycogen content in MIHA cells and increase blood glucose level in mice.

Systemic toxicity evaluation of novel tobacco products in Caenorhabditis elegans.

Under strictly Framework Convention on Tobacco Control, novel tobacco products are going to be promising alterations to consumers and manufactures. Even though the novel tobacco products have been considered less harmful than traditional tobaccos, there is a few knowledges about the subsequent substances during consume and their impacts to the consumers due to short introduction into the market. Thus, the present study aims to investigate the adverse effects of novel tobacco products on Caenorhabditis elegans(C. elegans) and to provide relevant references for novel tobacco products toxicity research and assessment. C. elegans individuals at L4 stage were exposed to different kinds of novel tobacco products, including electronic cigarettes liquid (e-liquid), the extract of e-cig aerosol (e-aerosol), mint and black tea flavor snus. After specific exposure time, the multiple toxic endpoints of C. elegans were measured, including acute toxicity, locomotion behavior, body length, and life-span. The oxidative stress was tested too. According to acute toxicity assays, the half lethal dose of four novel tobacco products calculated from theoretical nicotine concentration, ranked as follows e-liquid (0.29 mg/ml) > the extract of e-cig aerosol (0.43 mg/ml) > mint flavor snus (1.20 mg/ml) > black tea flavor snus (1.50 mg/ml). The equivalent lethal rate 5%~20% of four novel tobacco products were applied to following experiments. These novel tobacco products damaged nematode's locomotion including head thrashing and body bending, the damage was most evident in two flavors of snus. The similar trends were found in reproductive performance investigation. At tested concentrations, the retardation development of C. elegans was found throughout all stages with peak blockage at adulthood. Life-span tests showed that novel tobacco products at 5% lethal rate seemed no significant effect on affected the life-span of nematodes, with snus shortened the lifespan of C. elegans at 20% lethal rate. Imaging stress response indicted four types of tobacco productions causing stress response in C. elegans. Exposed to either 5% or 20% lethal levels (5% and 20%), the percentages of worms with DAF-16 redistribution among all groups varied, with higher frequencies in both snus. Summary, novel tobacco products caused multiple adverse impacts to C. elegans, including acute toxicity, locomotion behavior disruption, brood size reduction, development retardation, and life-span reduction. The toxicity was associated with both the feature and concentration of tobacco products, and oxidative stress was the main mechanism.

Involvement of gap junctions in astrocyte impairment induced by manganese exposure.

Glutamate excitotoxicity, characterized as excessive glutamate stress, is considered to be involved in cerebral ischaemia, brain trauma, and neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Glutamate homeostasis disruption was highlighted in Mn neurotoxicity caused by high levels of Mn. Astrocytes, accounting for approximately 50% of the neuronal cells in the central nervous system and maintain glutamate homeostasis, are sensitive to neurotoxicity induced by Mn exposure. Astrocytes are tightly coupled with gap junctions (GJ), which are comprised of connexins, mainly connexin43 (Cx43). The gap junctional intercellular communication (GJIC) pathway allows small signal molecules, such as glutamate, ATP (adenosine triphosphate, ATP) and tropic factors, etc., to transfer between adjacent cells. Evidence has shown that astrocytes execute the bystander effect during cell death through the GJIC pathway. However, the pathogenic mechanism of the gap junction underlying glutamate neurotoxicity induced by manganese exposure has not been elucidated yet. In the present study, primary astrocytes were cultured and then exposed to different levels of Mn (ranging from 0 to 1000 µM) for 4/16 h to investigate the function of the GJIC in apoptosis induced by Mn. The cellular toxicity was confirmed by cell viability and apoptotic percentage through MTT assay and flow cytometry (FC). The levels of intracellular/extracellular glutamate were measured by high-performance liquid chromatography (HPLC). The fluorescent dye, Lucifer Yellow (LY), was used to assess the status of gap junctions among astrocytes after Mn exposure. The protein/gene expression of major gap junctional forming protein, Cx43, was also investigated. Cell viability was distinctly reduced when exposed to 500 and 1000 µM MnCl2 compared with control cells at both time points. The percentage of apoptosis was significantly increased among all detected Mn levels (125, 500 and 1000 µM MnCl2) of exposure (p < 0.05) with a concentration-dependent manner at either time point. Mn administration for 4/16 h also caused a remarkable intracellular/extracellular glutamate increase in a concentration-dependent manner for extracellular glutamate levels (p < 0.01). Gap junctions were prominently inhibited by Mn with Cx43 protein shown as shortening of the LY dye transfer distance at both time points. In-cell western blot indicated that Mn caused a decrease in Cx43 protein/gene expression in a dose-dependent manner. These results suggested that the gap junction intercellular communication and its forming protein, Cx43, are likely involved in glutamate excitotoxicity induced by Mn exposure.