A 2x folic acid treatment affects epigenetics and dendritic spine densities in SHSY5Y cells

Many diseases are now associated with aberrant epigenetics and gene expression changes. Epigenetics can be modified by factors like diet. One dietary factor, folic acid, is consumed in various forms including supplements, energy drinks, and fortified grains. It was hypothesized high levels of folic acid would affect gene expression and enzyme activity of chromatin modifying enzymes as well as dendritic spine densities in a commonly utilized neuron model, the SHSY5Y cell. Decreased MBD2 and MECP2 were discovered upon treatment of SHSY5Y cells with a 2x folic acid dose. Corresponding decreases in dendritic spines were apparent in the 2x folic acid treated cells as well. Activity of DNMTs and H3K4 HMTs was altered. Further, H3K4me1, H3K4me3, H3K9Ac, and global DNA methylation were decreased in the 2x folic acid treated cells. Further studies are warranted to determine if the effects of excess folic acid are detrimental to organismal physiology.     

DNA methylation and histone deacetylation regulating insulin sensitivity due to chronic cold exposure

In this study, we investigated the causal relationship between chronic cold exposure and insulin resistance and the mechanisms of how DNA methylation and histone deacetylation regulate cold-reduced insulin resistance. 46 adult male mice from postnatal day 90–180 were randomly assigned to control group and cold-exposure group. Mice in cold-exposure group were placed at temperature from -1 to 4 °C for 30 days to mimic chronic cold environment. Then, fasting blood glucose, blood insulin level and insulin resistance index were measured with enzymatic methods. Immunofluorescent labeling was carried out to visualize the insulin receptor substrate 2 (IRS2), Obese receptor (Ob-R, a leptin receptor), voltage-dependent anion channel protein 1 (VDAC1), cytochrome C (cytC), 5-methylcytosine (5-mC) positive cells in hippocampal CA1 area. Furthermore, the expressions of some proteins mentioned above were detected with Western blot. The results showed: ① Chronic cold exposure could reduce the insulin resistance index (P < 0.01) and increase the number of IRS2 positive cells and Ob-R positive cells in hippocampus (P < 0.01). ② The expressions of mitochondrial energy-relative proteins, VDAC1 and cytC, were higher in cold-exposure group than in control group with both immunohistochemical staining and Western blot (P < 0.01). ③ Chronic cold exposure increased DNA methylation and histone deacetylation in the pyramidal cells of CA1 area and led to an increase in the expression of histone deacetylase 1 (HDAC1) and DNA methylation relative enzymes (P < 0.01). In conclusion, chronic cold exposure can improve insulin sensitivity, with the involvement of DNA methylation, histone deacetylation and the regulation of mitochondrial energy metabolism. These epigenetic modifications probably form the basic mechanism of cold-reduced insulin resistance.     

Let-7g induces granulosa cell apoptosis by targeting MAP3K1 in the porcine ovary

Follicular atresia mainly results from apoptosis of granulosa cells (GCs). Our previous microRNA array data indicated that the miRNA let-7g level increases significantly during porcine ovary follicular atresia. It is uncertain if GCs apoptosis is mediated by microRNA let-7g. In this study, the expression levels of the apoptosis-associated genes CASP3, BAX and BIM were significantly upregulated when let-7g mimic was transfected into porcine GCs, and the anti-apoptotic genes BCL-2 and MCL-1 were significantly downregulated. The apoptosis rate was measured by flow cytometry, and our results indicated that let-7g significantly enhanced GCs apoptosis. In further studies, we found that overexpression of let-7g induced the expression of FoxO1 in GCs and led to nuclear accumulation of dephosphorylated FoxO1. In addition, the effect of let-7g on FoxO1 expression and dephosphorylation resulted from repression of the expression of the MAP3K1 gene in porcine GCs. The site on MAP3K1 mRNA targeted by let-7g was confirmed by luciferase reporter assay. The anti-apoptotic effect of MAP3K1 was validated by silencing MAP3K1 using small interfering RNA technology. In conclusion, our data indicate that let-7g induces porcine GCs apoptosis by inhibiting the MAP3K1 gene, which promotes FoxO1 expression and dephosphorylation with nuclear accumulation.     

Immunological response induced by cryoablation against murine H22 hepatoma cell line in vivo

ObjectivesTo describe immunological consequences induced by cryoablation against H22 cells in vivo.MethodsAdult BALB/c mice underwent subcutaneous implantation of H22 cells. All of them were assigned into three groups randomly: group A (false surgery), group B (cryoablation) and group C (cryoablation plus Freund's adjuvant). Animals were sacrificed 1, 2 and 3 weeks after treatment. Serum IFN-γ and IL-4, Th1/Th2 in spleens and cytotoxicity were detected.ResultsCompared with that of group A, (1) INF-γ of group B was higher, but IL-4 was lower; cryoablation plus Freund's adjuvant enhanced these effects. (2) Th1/Th2 rose significantly in both group B and group C. (3) Strong cytolytic activity against H22 cells of group B and group C was found on day 7, 14 and 21.ConclusionsOur study showed a marked shift toward Th1 and IFN-γ expression after cryoablation, with an immuno-stimulatory effect against murine H22 hepatoma Cell.     

Dehydroepiandrosterone ameliorates H2O2-induced Leydig cells oxidation damage and apoptosis through inhibition of ROS production and activation of PI3K/Akt pathways

Dehydroepiandrosterone (DHEA) is widely used as a nutritional supplement, and administration of DHEA produces a number of beneficial effects in the elderly. Many researchers have suggested that DHEA exerts it function after conversion into more biologically active hormones in peripheral target cells. The actions of DHEA in Leydig cells, a major target cell of DHEA biotransformation in males, are not clear. The present study found that DHEA increased cell viability and decreased reactive oxygen species (ROS) and malondialdehyde contents in H₂O₂-induced Leydig cells. DHEA significantly increased the activities of superoxide dismutase, catalase and peroxidase, and decreased the DNA damage in H₂O₂-induced Leydig cells. Apoptosis was significant decreased in H₂O₂-induced Leydig cells after DHEA treatment. DHEA inhibited the loss of mitochondrial membrane potential (ΔΨm) and the upregulation of the caspase-3 protein level induced by H₂O₂ in Leydig cells. DHEA also reversed the decrease in PI3K and p-Akt protein levels induced by H₂O₂. These data showed that DHEA could ameliorate H₂O₂-induced oxidative damage by increasing anti-oxidative enzyme activities, which resulted in reduced ROS content, and decreased apoptosis, mainly by preventing the loss of ΔΨm and inhibiting caspase-3 protein levels via activation of PI3K/Akt signaling pathways. These results increase our understanding of the molecular mechanism of the anti-ageing effect of DHEA.     

Steroids from Ganoderma sinense as new natural inhibitors of cancer-associated mutant IDH1

Isocitrate dehydrogenase (IDH) is one of the key enzymes in the tricarboxylic acid cycle, and IDH mutations have been associated with many cancers, including glioblastoma, sarcoma, acute myeloid leukemia, etc. Three natural steroids 1–3 from Ganoderma sinense, a unique and rare edible-medicinal fungi in China, were found as potential IDH1 inhibitors by virtual ligand screening method. Among the three compounds, 3 showed the highest binding affinity to IDH1 with significant calculated binding free energy. Enzymatic kinetics demonstrated that 3 inhibited mutant enzyme in a noncompetitive manner. The half effective concentration of 3 for reducing the concentration of D-2HG in HT1080 cells was 35.97 μM. The levels of histone H3K9me3 methylation in HT1080 cells were reduced by treating with 3. Furthermore, knockdown of mutant IDH1 in HT1080 cells decreased the anti-proliferative sensitivity to 3. In short, our findings highlight that compound 3 may have clinical potential in tumor therapies as an effective inhibitor of mutant IDH1.     

Epigenetic determinants of phenotypic plasticity in Candida albicans

Epigenetics literally means heritable changes in gene expression without any modification in the DNA sequence. The field of epigenetics is revolutionising our understanding of basic fundamental principles behind the normal development and the diseased state of an individual. However, chromatin modifications during infection, wherein the pathogen interacts with its host, received comparatively little attention. Nevertheless, the role of epigenetics in the establishment of infectious diseases by breaching the host defense system is an emerging area of research. Epigenetic regulation impacts differentiation and expression of virulence attributes of a pathogen. For example, antigenic variations in parasites such as Giardia lamblia and Plasmodium falciparum are epigenetically determined. Similarly, chromatin modifying elements have been implicated in fungal morphogenesis and virulence. In particular, chromatin modifying enzymes including histone methyl transferases (KMTs), histone acetyl transferases (KATs), and histone deacetylases (KDACs) have been shown to epigenetically modulate pathogenicity of the human opportunistic pathogen Candida albicans. The significance of chromatin modifications has the potential for explaining the mechanistic basis for distinct lifestyles of the fungus. In this review, we summarize the existing body of evidence that emphasizes the importance of various chromatin modulations involved in providing phenotypic plasticity of the medically important fungal pathogen C. albicans.     

Bongkrekic acid facilitates glycolysis in cultured cells and induces cell death under low glucose conditions

Bongkrekic acid (BKA) inhibits adenine nucleotide translocator (ANT) and suppresses ADP/ATP exchange in the mitochondrial inner membrane. Previously, we demonstrated that BKA exhibited cytotoxic effects on 4T1 tumor cells, depending on the cell number in the culture, but not on NIH3T3 cells. However, the cause of this differential sensitivity was unelucidated. Here we demonstrate that BKA reduced the O₂ consumption in both cell lines and increased the mitochondrial membrane potential, thereby facilitating glucose consumption. BKA reduced cellular ATP in 4T1 cells in a dose-dependent manner but not in NIH3T3 cells. The cellular ATP of 4T1 cells was decreased with a reduced glucose concentration in the media, but that of NIH3T3 cells remained constant. We also demonstrated that BKA-induced cell death in both cell lines in low glucose media; however, the susceptibility to the reduced glucose concentration was slightly higher in 4T1 cells, which may be attributed to the difference in the dependency on glycolysis as their energy source. These results indicate that 4T1 tumor cells rely heavily on glucose for energy production. Our data demonstrate that BKA disturbs ATP production in mitochondria and increases the susceptibility to a low glucose condition.     

Short and prolonged exposure to hyperglycaemia in human fibroblasts and endothelial cells: Metabolic and osmotic effects

High blood glucose levels are the main feature of diabetes. However, the underlying mechanism linking high glucose concentration to diabetic complications is still not fully elucidated, particularly with regard to human physiology. Excess of glucose is likely to trigger a metabolic response depending on the cell features, activating deleterious pathways involved in the complications of diabetes. In this study, we aim to elucidate how acute and prolonged hyperglycaemia alters the biology and metabolism in human fibroblasts and endothelial cells.We found that hyperglycaemia triggers a metabolic switch from oxidative phosphorylation to glycolysis that is maintained over prolonged time. Moreover, osmotic pressure is a major factor in the early metabolic response, decreasing both mitochondrial transmembrane potential and cellular proliferation. After prolonged exposure to hyperglycaemia we observed decreased mitochondrial steady-state and uncoupled respiration, together with a reduced ATP/ADP ratio. At the same time, we could not detect major changes in mitochondrial transmembrane potential and reactive oxygen species.We suggest that the physiological and metabolic alterations observed in healthy human primary fibroblasts and endothelial cells are an adaptive response to hyperglycaemia. The severity of metabolic and bioenergetics impairment associated with diabetic complications may occur after longer glucose exposure or due to interactions with cell types more sensitive to hyperglycaemia.     

The steroid hormone 20-hydroxyecdysone promotes switching from autophagy to apoptosis by increasing intracellular calcium levels

Autophagy regulates cell survival (or cell death in several cases), whereas apoptosis regulates cell death. However, the relationship between autophagy and apoptosis and the regulative mechanism is unclear. We report that steroid hormone 20-hydroxyecdysone (20E) promotes switching from autophagy to apoptosis by increasing intracellular calcium levels in the midgut of the lepidopteran insect Helicoverpa armigera. Autophagy and apoptosis sequentially occurred during midgut programmed cell death under 20E regulation, in which lower concentrations of 20E induced microtubule-associated protein 1 light chain 3–phosphatidylethanolamine (LC3–II, also known as autophagy-related gene 8, ATG8) expression and autophagy. High concentrations of 20E induced cleavage of ATG5 to NtATG5 and pro-caspase-3 to active caspase-3, which led to a switch from autophagy to apoptosis. Blocking autophagy by knockdown of ATG5, ATG7, or ATG12, or with the autophagy inhibitor 3-methyladenine, inhibited 20E-induced autophagy and apoptosis. Blocking apoptosis by using the apoptosis inhibitor Ac-DEVD-CHO did not prevent 20E-induced autophagy, suggesting that apoptosis relies on autophagy. ATG5 knockdown resulted in abnormal pupation and delayed pupation time. High concentrations of 20E induced high levels of intracellular Ca²⁺, NtATG5, and active caspase-3, which mediated the switch from autophagy to apoptosis. Blocking 20E-mediated increase of cellular Ca²⁺ caused a decrease of NtATG5 and active caspase-3 and repressed the transformation from autophagy to apoptosis, thereby promoting cell survival. 20E induces an increase in the concentration of intracellular Ca²⁺, thereby switching autophagic cell survival to apoptotic cell death.     

Effect of piceatannol-rich passion fruit seed extract on human glyoxalase I–mediated cancer cell growth

Passion fruit seed extract (PFSE), a product rich in stilbenes such as piceatannol and scirpusin B, has various physiological effects. It is unclear whether PFSE and its stilbene derivatives inhibit cancer cell proliferation via human glyoxalase I (GLO I), the rate-limiting enzyme for detoxification of methylglyoxal. We examined the anticancer effects of PFSE in two types of human cancer cell lines with different GLO I expression levels, NCI–H522 cells (highly-expressed GLO I) and HCT116 cells (lowly-expressed GLO I). PFSE and its stilbenes inhibited GLO I activity. In addition, PFSE and its stilbenes supressed the cancer cell proliferation of NCI–H522 cells more than HCT116 cells. These observations suggest that PFSE can provide a novel anticancer strategy for prevention and treatment.     

Metabolic reorganization in winter: Regulation of pyruvate dehydrogenase (PDH) during long-term freezing and anoxia

Wood frogs, Rana sylvatica, can undergo prolonged periods of whole body freezing during winter, locking as much as 65–70% of total body water into extracellular ice and imposing both anoxia and dehydration on their cells. Metabolic rate depression (MRD) is an adaptation used by R. sylvatica to survive these environmental stresses, where a finite amount of ATP generated through anaerobic metabolism is directed towards maintaining pro-survival functions, while most ATP-expensive cellular processes are temporarily reduced in function. Pyruvate dehydrogenase (PDH) is a vital metabolic enzyme that links anaerobic glycolysis to the aerobic TCA cycle and is an important regulatory site in MRD. PDH enzymatic activity is regulated via reversible protein phosphorylation in response to energetic demands of cells. This study explored the posttranslational regulation of PDH at three serine sites (S232, S293, S300) on the catalytic E1α subunit along with protein expression of four pyruvate dehydrogenase kinases (PDHK1-4) in response to 24 h Freezing, 8 h Thaw, 24 h Anoxia, and 4 h Recovery in the liver and skeletal muscle of R. sylvatica using Luminex multiplex technology and western immunoblotting. Overall, inhibitory regulation of PDH was evident during 24 h Freezing and 24 h Anoxia, which could indicate a notable reduction in glycoytic flux and carbon entry into the tricarboxylic acid cycle as part of MRD. Furthermore, the expression of PDHK1-4 and phosphorylation of PDH at S232, S293, and S300 were highly tissue and stress-specific, indicative of how different tissues respond differently to stress within the same organism.     

Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer metastasis and chemo/radioresistance via the PI3K/Akt/mTOR signaling pathway

Prostate cancer (CaP) is the second leading malignancy in men. The role of epithelial cell adhesion molecule (EpCAM), also known as CD326, in CaP progression and therapeutic resistance is still uncertain. Here, we aimed to investigate the roles of EpCAM in CaP metastasis and chemo/radioresistance. Expression of EpCAM in CaP cell lines and human CaP tissues was assessed using immunofluorescence and immunohistochemistry, respectively. EpCAM was knocked down (KD) in PC-3, DU145 and LNCaP-C4-2B cells using small interfering RNA (siRNA), and KD results were confirmed by confocal microscope, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by proliferation and colony formation assays. The invasive potential was assessed using a matrigel chamber assay. Tumorigenesis potential was measured by a sphere formation assay. Chemo-/radiosensitivity were measured using a colony formation assay. Over-expression of EpCAM was found in primary CaP tissues and lymph node metastases including cancer cells and surrounding stromal cells. KD of EpCAM suppressed CaP proliferation and invasive ability, reduced sphere formation, enhanced chemo-/radiosensitivity, and down-regulated E-cadherin, p-Akt, p-mTOR, p-4EBP1 and p-S6K expression in CaP cells. Our findings suggest that EpCAM plays an important role in CaP proliferation, invasion, metastasis and chemo-/radioresistance associated with the activation of the PI3K/Akt/mTOR signaling pathway and is a novel therapeutic target to sensitize CaP cells to chemo-/radiotherapy.     

The mechanism of lipopolysaccharide administration-induced cognitive function impairment caused by glucose metabolism disorder in adult rats

ObjectiveThis essay aims to make investigation on the mechanism of glucose metabolism disorder and Lipopolysaccharide administration-induced cognitive function impairment in adult rats with surgery. Methods: Divide the objects, 40 male Sprague-Dawley rats at the age of 9 months, into 4 groups. Provide unilateral nephrectomy surgery and/or lipopolysaccharide intraperitoneal injection. Postoperative cognitive function evaluation would be tested by the Morris water maze. Rats with Postoperative Cognitive Dysfunction (POCD) were scanned to analyze the brain glucose metabolism by means of 18F-FDG PET/CT. Phosphatidylinositol 3-Kinase (PI3K), Protein Kinase β (AKT), Insulin Substrates Receptor-2 (IRS-2) and Glucose Transporter 4 (GLUT4) were detected as well. Data will be captured through gene expression in POCD rats via Quantitative Real-Time PCR (QRT-PCR). On the other side, Western Blot was used to measure the expression levels of IRS-2, p-IRS-2, p-PI3K, PI3K, p-AKT, AKT, GLUT4, and p-GLUT4. Results: During the Morris water maze test, the staging time (latency) of rats in each group was becoming short gradually as the training progressed. The incubation time of Day 5 of each group was shorter than that of Day 1 (P < 0.05). On the Day 3 after the surgery, the average target quadrant residence time of Group S+L (100 μg/Kg) was shorter, compared with Group C, L and S. Of which, the average number of perforation was reduced greater than that of Group C (P < 0.05). The average swimming speed of the groups is of no distinct difference (P > 0.05). After the operation, there was no great difference shown among the subjects (P > 0.05) in the average residence time of the target quadrant, the mean number of passages, and the mean swimming speed. On Day 3, the average latency of Group S+L (100 μg/Kg) was longer than Group C (P < 0.05) in the working memory test after the operation. The average latency of rats in Group L and S was showed longer than that in Group C, with tiny difference (P > 0.05). In the 7-Day working memory test, the average latency of the rats in Group L, S and S+L (100 μg/Kg) was obviously longer than that in Group C. Comparing to preoperative rats, POCD rats of Group S+L (100 μg/Kg) were scanned by 18F-FDG PET/CT three days later after the operation. Its SUVmax of the frontal and temporal lobe areas were decreased significantly (P < 0.05). However, difference degree was not significantly shown in the SUVmax between Group C and the preoperative rats (P > 0.05). In comparison with the gene expression of of Group C, the PI3K, IRS-2, AKT and GLUT4 mRNA genes are the key genes in the insulin signaling pathways of the hippocampus of the POCD rats. The expression level was reduced. The expression level of all protein of PI3K, IRS-2, GLUT4 and AKT in the POCD rats was of no great contrast with that in Group C. But for IRS-2 protein, the phosphorylation level has increased, and meanwhile decreased for AKT, PI3K and GLUT4 proteins (P < 0.05). Conclusions: Adult SD rats cognitive dysfunction model treated with unilateral nephrectomy combined and 100 μg/kg LPS intraperitoneal injection were led to abnormal both brain glucose metabolism and insulin expression. The proved phenomenal results signal pathway-related proteins PI3K, IRS-2, AKT and GLUT4. It reached the conclusion that surgical trauma, rather than anesthesia, leads to impaired cognitive function. PI3K, IRS-2, AKT, and GLUT4pathway of brain can be partial explanations of the pathogenesis of POCD.     

Ins and Outs of Interpreting Lipidomic Results

Membrane lipids are essential for life; however, research on how cells regulate cell lipid composition has been falling behind for quite some time. One reason was the difficulty in establishing analytical methods able to cope with the cell lipid repertoire. Development of a diversity of mass spectrometry-based technologies, including imaging mass spectrometry, has helped to demonstrate beyond doubt that the cell lipidome is not only greatly cell type dependent but also highly sensitive to any pathophysiological alteration such as differentiation or tumorigenesis. Interestingly, the current popularization of metabolomic studies among numerous disciplines has led many researchers to rediscover lipids. Hence, it is important to underscore the peculiarities of these metabolites and their metabolism, which are both radically different from protein and nucleic acid metabolism. Once differences in lipid composition have been established, researchers face a rather complex scenario, to investigate the signaling pathways and molecular mechanisms accounting for their results. Thus, a detail often overlooked, but of crucial relevance, is the complex networks of enzymes involved in controlling the level of each one of the lipid species present in the cell. In most cases, these enzymes are redundant and promiscuous, complicating any study on lipid metabolism, since the modification of one particular lipid enzyme impacts simultaneously on many species. Altogether, this review aims to describe the difficulties in delving into the regulatory mechanisms tailoring the lipidome at the activity, genetic, and epigenetic level, while conveying the numerous, stimulating, and sometimes unexpected research opportunities afforded by this type of studies.