Homocysteine Treatment Research Articles

Effect of Fetal Bovine Serum or Basic Fibroblast Growth Factor on Cell Survival and the Proliferation of Neural Stem Cells: The Influence of Homocysteine Treatment.

In vitro cell culture is a routinely used method which is also applied for in vitro modeling of various neurological diseases. On the other hand, media used for cell culture are often not strictly standardized between laboratories, which hinders the comparison of the obtained results. Here, we compared the effects of homocysteine (Hcy), a molecule involved in neurodegeneration, on immature cells of the nervous system cultivated in basal medium or media supplemented by either fetal bovine serum or basic fibroblast growth factor. The number of cells in basal media supplemented with basic fibroblast growth factor (bFGF) was 2.5 times higher in comparison to the number of cells in basal media supplemented with fetal bovine serum (FBS). We also found that the neuron-specific β-3-tubulin protein expression dose dependently decreased with increasing Hcy exposure. Interestingly, bFGF exerts a protective effect on β-3-tubulin protein expression at a concentration of 1000 µM Hcy compared to FBS-treated neural stem cells on Day 7. Supplementation with bFGF increased SOX2 protein expression two-fold compared to FBS supplementation. GFAP protein expression increased five-fold on Day 3 in FBS-treated neural stem cells, whereas on Day 7, bFGF increased GFAP expression two-fold compared to FBS-treated neural stem cells. Here, we have clearly shown that the selection of culturing media significantly influences various cellular parameters, which, in turn, can lead to different conclusions in experiments based on in vitro models of pathological conditions.

Homocysteine causes neuronal leptin resistance and endoplasmic reticulum stress.

Abnormally high serum homocysteine levels have been associated with several disorders, including obesity, cardiovascular diseases or neurological diseases. Leptin is an anti-obesity protein and its action is mainly mediated by the activation of its Ob-R receptor in neuronal cells. The inability of leptin to induce activation of its specific signaling pathways, especially under endoplasmic reticulum stress, leads to the leptin resistance observed in obesity. The present study examined the effect of homocysteine on leptin signaling in SH-SY5Y neuroblastoma cells expressing the leptin receptor Ob-Rb. Phosphorylation of the signal transducer and activator of transcription (STAT3) and leptin-induced STAT3 transcriptional activity were significantly inhibited by homocysteine treatment. These effects may be specific to homocysteine and to the leptin pathway, as other homocysteine-related compounds, namely methionine and cysteine, have weak effect on leptin-induced inhibition of STAT3 phosphorylation, and homocysteine has no impact on IL-6-induced activation of STAT3. The direct effect of homocysteine on leptin-induced Ob-R activation, analyzed by Ob-R BRET biosensor to monitor Ob-R oligomerization and conformational change, suggested that homocysteine treatment does not affect early events of leptin-induced Ob-R activation. Instead, we found that, unlike methionine or cysteine, homocysteine increases the expression of the endoplasmic reticulum (ER) stress response gene, a homocysteine-sensitive ER resident protein. These results suggest that homocysteine may induce neuronal resistance to leptin by suppressing STAT3 phosphorylation downstream of the leptin receptor via ER stress.

Primary human monocytes are stimulated in a pro-atherosclerotic manner in hyperhomocysteine conditions by attenuated PTEN phosphatase function

Abstract Purpose Monocytes are essential for atherosclerosis and hereby crucial for the detrimental consequences of cardiovascular diseases like coronary heart disease. Interestingly, homocysteine (HCY) was identified as an idependent risk factor for atherosclerosis development. However, the specific implication of HCY on monocytes has not yet been clarified and was therefore investigated in the present study. Methods Primary human monocytes, isolated from peripheral blood through the immuno-magnetic method, were treated with a clinically relevant dose of 400 μM HCY for 24 hours. Monocyte migration was investigated with transwell migration assays. Adhesion to inflamed endothelium (HUVECs) was studied under static and physiological flow conditions. Expression of relevant molecules was quantified with Western Blot, RT-qPCR and FACS. 5-azacytidine (AZA) was used to inhibit DNA methyltransferase 1 (DNMT1) and SF1670 to inhibit PTEN. Results First, we studied monocyte migration phenotype. Under hyperhomocysteine conditons monocytes revealed elevated chemokinesis and enhance chemotaxis towards MCP-1. In addition, monocytes show a pro-adhesive phenotype under HCY treatment which was indicated by heightened adhesion to inflamed endothelial cells, both in static and physiological flow conditions. These data could be sustained by observation of HCY-induced augmented expression of adhesion molecule CD11a on primary human monocytes. Interestingly, we observed decreased PTEN phosphatase function and activity in HCY-treated monocytes, which was reflected downstream in increased activation of AKT at serine 473. Based on these data, it was also possible to induce the pro-migratory phenotype in monocytes by pharmacological inhibition of PTEN alone. Since HCY can modify the methylation status of PTEN and hereby regulates its expression, we inhibited DNMT1 with AZA which could prevent downregulation of PTEN expression by HCY treatment. In addition, through inhibition of DNMT1 HCY-induced aberrant migration and adhesion was rescued. In line with that, HCY-induced pro-migratory and pro-adhesive phenotype was also rescued by co-treatment with the cofactors (30 μM Vitamin B12 and 3 μM folic acid) responsible for homocysteine to methionine catabolism due to normalized PTEN expression. Conclusions The present work deciphers a previously unknown mechanism how an increased concentration of HCY induces a pro-atherosclerotic activation of monocytes. Accumulation of HCY leads to a methylation-dependent inactivation of PTEN phosphatase. This subsequently causes increased phosphorylation of AKT at serine 473, which results in an augmented migration behaviour of monocytes. At the same time, we were able to reduce HCY-induced aberrant monocyte activation by inhibiting DNTM1 or by interfering with HCY metabolism by adding vitamin B12 or folic acid. In summary, these findings provide a new approach to reset pro-atherosclerotic monocytes in hyperhomocysteinemic conditions. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): IZKF SEED Project 14/20

MiR-483-3p improves learning and memory abilities via XPO1 in Alzheimer's disease.

IntroductionAlzheimer's disease (AD), a common form of dementia, has been reported to influence 27 million individuals globally. Several risk factors including oxidative stress, gut microbiota imbalance, and cognitive activity are reported to be closely associated with the initiation or progression of AD. Although miR‐483‐3p was identified to be downregulated in AD patient serum. However, the biological role and mechanism of miR‐483‐3p remained unknown in AD. Here, we explored the role of miR‐483‐3p in AD.MethodsSprague–Dawley rats were injected with homocysteine (Hcy) to establish an AD animal model. The Morris water maze tests and contextual fear tests were conducted to assess the cognitive and memory abilities of rats. TUNEL staining was utilized to determine cell apoptosis. Luciferase reporter assay was used to evaluate the binding relation between miR‐483‐3p and exportin 1 (XPO1).ResultsHomocysteine treatment (400 μg/kg) induced the learning, cognitive and memory defects of rats. miR‐483‐3p was downregulated in Hcy‐treated rat hippocampus. Functionally, miR‐483‐3p alleviated cell apoptosis and impairments of learning and memory abilities in Hcy‐treated rats. In addition, miR‐483‐3p inhibited cell apoptosis and protein level of AD‐associated factors (APP, BACE1, and Aβ1‐42) in PC12 cells. In mechanism, miR‐483‐3p was confirmed to target XPO1 in PC12 cells. XPO1 displayed high level in rat hippocampus and was negatively correlated with miR‐483‐3p levels. Finally, XPO1 overexpression rescued the suppressive effect of miR‐483‐3p on cell apoptosis and protein levels of AD‐associated factors.ConclusionsmiR‐483‐3p alleviates neural cell apoptosis and impairments of learning and memory abilities by targeting XPO1 in AD.

Association Between Soluble Epoxide Hydrolase and TRPC3 Channels in Pathological Cardiac Hypertrophy

Objectives Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Although previous studies have shown a positive correlation between homocysteine levels and the incidence of cardiac hypertrophy and remodeling, the molecular mechanisms by which homocysteine promotes the development of cardiac hypertrophy remain inadequately understood. In this study, we investigated the role of soluble epoxide hydrolase (sEH) and transient receptor potential isoform 3 (TRPC3) channels in homocysteine-induced cardiac hypertrophy with further elucidating the association between sEH and TRPC3. Methods H9c2 cardiomyocytes were exposed to different concentrations of homocysteine ranging from 25 to 200 μmol/L for different duration. The cell surface area was measured by phase contrast microscope and image analysis software. The mRNA and protein expressions of sEH, TRPC3 channels, and hypertrophic markers beta-smooth muscle myosin heavy chain (β-MHC) and atrial natriuretic peptide (ANP) were detected by quantitative polymerase chain reaction and western blot. Results Homocysteine treatment dose-dependently and time-dependently increased the expression of β-MHC and ANP in H9c2 cardiomyocytes. Both mRNA and protein levels of sEH and TRPC3 channels were unregulated by homocysteine. Inhibition of sEH with 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) significantly suppressed homocysteine-induced upregulation of β-MHC and ANP and attenuated the increase of cell size. Pyr3, a selective TRPC3 channel blocker, also exerted a strong anti-hypertrophic effect against homocysteine. Treatment with TPPU significantly suppressed the expression of TRPC3 channels in homocysteine-exposed H9c2 cells. Conclusions: Both sEH and TRPC3 channels are involved in homocysteine-induced hypertrophy of cardiomyocytes. TRPC3 channels may act as a downstream mediator for sEH activation induced by homocysteine to promote cardiac hypertrophy.

Homocysteine-induced neural tube defects in chick embryos via oxidative stress and DNA methylation associated transcriptional down-regulation of miR-124.

Although moderate homocysteine (HCY) elevation is associated with neural tube defects (NTDs), the underlying mechanisms have not been elucidated. In this study, we aimed to investigate that whether HCY-induced NTDs were associated with oxidative stress and methyl metabolism in chick embryos. The potential role of miR-124 in neurogenesis was also investigated. In this study, increased intracellular oxidative species and alterations in DNA methylation were observed following HCY treatment. This alteration coincided with decreases of Mn superoxide dismutase and glutathione peroxidase activities, as well as the expression of anti-rabbit DNA methyltransferase (DNMT) 1 and 3a. In addition, HCY induced significant decreases of S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) (P<0.05). N-acetyl-L-cysteine and choline ameliorated global DNA hypomethylation induced by HCY. MiR-124 levels were significantly suppressed by HCY (P<0.05), while elevated by 5-aza-2'-deoxycytidine (5-aza-dC). MiR-124 knockdown resulted in spina bifida occulta. Our research suggests that HCY-induced NTDs were associated with oxidative stress and methyl metabolism in chick embryos. MiR-124 down-regulation may occur via epigenetic mechanisms and contribute to HCY-induced NTDs in chick embryo models.

A low-cost mass spectrometry-based approach for quantifying purines in placental explants

Purine nucleotides serve as immediate building blocks for the synthesis of RNAs and DNA, energy carriers and co-substrates in the activation of many metabolites, regulators in cellular metabolism, and signaling molecules. Knowledge of the actual tissue-specific concentration of these metabolites and their synthesis rate is essential to studies focused on a tissue-specific metabolic rewiring in the pathogenesis of different diseases, including carcinogenesis. The existing data mainly characterize the purine concentration in body fluids and malignant cells. The protocols for estimating de novo purine biosynthesis usually required the usage of radioactive precursors and costly facilities like HPLC with tandem mass spectrometry or nuclear magnetic resonance. We developed a new simplified method to measure the level of purines by HPLC/ESI-MS, using a stable isotope glycine to label and measure newly synthesized purines. The developed method proved to be highly sensitive, interday repeatable, and intraday reproducible. We applied the method to measure purine concentration and the rate of purine synthesis in placental explants from the first and third trimesters of gestation and MCF7 cells cultivated with 20 μM and 40 μM homocysteine. Corresponding homocysteine concentration is typical for breast cancer and pregnancy-associated complications affecting the human placenta. The content of total purines in placental explants from the first trimester of gestation was around 9.0 μmol/g wet tissues at specified cultivation conditions. The newly formed purines comprised approximately 1% of total purines and decreased steadily in explants cultivated with 20 mM and 40 μM homocysteine. They reached the values characteristic for explants from the third trimester grown without homocysteine, 4.0 μmol/g wet tissues. In MCF7 cells, the newly formed purines comprised around 50% of total purines and decreased under homocysteine treatment. A low-cost mass spectrometry-based approach may be used to estimate the level and the rate of purine synthesis in eukaryotes’ tissues in a wide range of purines concentrations.

Regulation of folate and methionine metabolism by multisite phosphorylation of human methylenetetrahydrofolate reductase

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the irreversible conversion of 5,10-methylene-tetrahydrofolate (THF) to 5-methyl-THF, thereby committing one-carbon units to the methionine cycle. While MTHFR has long been known to be allosterically inhibited by S-adenosylmethionine (SAM), only relatively recently has N-terminal multisite phosphorylation been shown to provide an additional layer of regulation. In vitro, the multiply phosphorylated form of MTHFR is more sensitive to allosteric inhibition by SAM. Here we sought to investigate the kinases responsible for MTHFR multisite phosphorylation and the physiological function of MTHFR phosphorylation in cells. We identified DYRK1A/2 and GSK3A/B among the kinases that phosphorylate MTHFR. In addition, we found that MTHFR phosphorylation is maintained by adequate cellular SAM levels, which are sensed through the C-terminal SAM binding domain of MTHFR. To understand the function of MTHFR phosphorylation in cells, we generated MTHFR CRISPR knockin mutant lines that effectively abolished MTHFR phosphorylation and compared them with the parental cell lines. Whereas the parental cell lines showed increased 5-methyl-THF production in response to homocysteine treatment, the knockin cell lines had high basal levels of 5-methyl-THF and did not respond to homocysteine treatment. Overall, our results suggest that MTHFR multisite phosphorylation coordinates with SAM binding to inhibit MTHFR activity in cells.

Cytokine Treatment for Myocarditis May Directly Impact Cardiomyocytes Negatively

The purpose of our study was to determine if direct administration of CXCL1/KC to cardiomyocytes causes negative changes to cell density or proliferation. This molecule has been shown to reduce inflammation in certain instances. Homocysteine models the direct effect of an inflammatory agent on cardiomyocytes. Our question was whether these molecules directly impact cell density through an interaction with the cell proliferation process. We hypothesized that cells treated with CXCL1/KC would maintain the same cell density as untreated cells. In contrast, cells treated with Homocysteine or both Homocysteine and CXCL1/KC, were expected to have a higher cell density that than that of untreated cells. To test these hypotheses, HL-1 cardiomyocytes were cultured and treated in chambers on a glass slide. DAPI staining, which allows individual nuclei to be counted, was viewed under fluorescence and pictures were analyzed using Image J. Our hypotheses were rejected because there was a significant difference between the Homocysteine group and both the Homocysteine+CXCL1/KC and CXCL1/KC groups. This was extended to signify that the cell density of the CXCL1/KC and Homocysteine+CXCL1/KC groups was decreased cell proliferation had been reduced. The difference between the CXCL1/KC and Homocysteine groups likely indicates effects possessed by each treatment. Comparing results from the Homocysteine+CXCL1/KC treatment to the Homocysteine treatment, Homocysteine treatment increased cell density enough so that the decreased proliferation which may have been caused by the CXCL1/KC was significant compared to the Homocysteine sample. Future studies could examine additional applicable models such as mouse heart tissue or a living mouse.

Nuclear factor erythroid 2-related factor 2 activation mediates hyperhomocysteinemia-associated lipolysis suppression in adipocytes.

Hyperhomocysteinemia (HHcy) is associated with suppressed lipolytic response in adipocytes/adipose tissue, however, the underlying mechanism remains to be extensively studied. Nuclear factor erythroid 2-related factor 2 (Nrf2), a master transcriptional factor regulating antioxidant generation, has been recently reported to mediate lipid metabolism. Employing both fully differentiated 3T3-L1 adipocytes and male C57BL/6 mice, in the present study, we investigated the potential involvement of Nrf2 activation in HHcy-mediated lipolytic suppression. Our results showed that homocysteine (Hcy) treatment resulted in suppressed lipolysis, evidenced by increased intracellular triglyceride (TG) accumulation, decreased glycerol and free fatty acid (FFA) in fully differentiated 3T3-L1 adipocytes. Interestingly, Hcy exposure was associated with Nrf2 activation in adipocytes. Further studies showed that Nrf2 knockdown via siRNA transfection ameliorated Hcy-induced glycerol release in adipocytes. On the contrary, Nrf2 activators, epigallocatechin gallate (EGCG) and tert-butylhydroquinone (t-BHQ), increased intracellular TG content and decreased glycerol release in adipocytes. Importantly, our in vitro observations were corroborated by our in vivo findings, in which Hcy feeding (0.1% wt/vol) for four weeks induced Nrf2 expression in adipose tissue and lowered circulating FFA and glycerol levels in mice. Furthermore, EGCG injection (5 mg/kg/d) decreased circulating glycerol levels in comparison to the control group in mice. In conclusion, these results indicated that Nrf2 activation in response to HHcy plays an important role in mediating Hcy-suppressed lipolysis in adipocytes.

Effect of standard percutaneous nephroscope combined with bladder soft lens lithotomy in the treatment of serum Hcy, β2MG in kidney stone

Objective To discuss the effect of standard percutaneous nephroscope combined with bladder soft lens lithotomy in the treatment of serum homocysteine (Hcy), beta 2-microglobulin (β2MG) in kidney stone. Methods A total of 100 patients with kidney stone were selected from December 2013 to December 2016 in our hospital, and all patients were randomly divided into double lens and renal lens group, 50 cases in each group, renal lens group was given standard percutaneous nephrolithotomy (PCNL) treatment, the double lens group was given standard percutaneous nephroscope combined with bladder soft lens lithotomy treatment. Intraoperative blood loss, operation, bowel function recovery, length of hospital stay, the issue of stone removal, complications and pain before and after treatment, serum Hcy, β2MG levels were statistical analyzed. Results The intraoperative blood loss, bowel function recovery, length of hospital stay, the incidence of complication of double lens group were significantly lower than thosed of renal lens group, the first phase stone clearance rate, operation time of the former were significantly higher than those of the latter, the differences were statistically significant (P<0.05). The 1, 4 d after treatment, VAS scores, serum Hcy levels of double lens and renal lens group compared with before treatment were increased, the serum β2MG level was increased, but the change of former was significantly more than that of the latter, the difference was statistically significant (P<0.05). Conclusions Standard percutaneous nephroscope combined with bl- adder soft lens lithotomy can improve the treatment of patients with kidney stones and reduce complications and surgical trauma, and it can improve the level of serum Hcy, β2MG, it is beneficial to patients' pain relief, recovery, but it still need to improve the relevant operation technology. Key words: Kidney Calculi; Nephrostomy, Percutaneous; Cystoscopes; beta 2-Microglobulin

Quantification of S-adenosylmethionine and S-adenosylhomocysteine in human placenta and placental explants under homocysteine treatment

S-Adenosylmethionine (SAM) and S-adenosylhomocysteine(SAH) are essential compounds in methionine cycle. SAM is a universal donor in majority of methyltransferase reactions while SAH is a universal product and a potent inhibitor of methyltransferase reactions. The reactions of methionine cycle and connected with them folate-related reactions and transsulfuration pathway are implicated in various complications of pregnancy. Their role in placental metabolism and responsiveness to hyperhomocysteinemia is scarcely investigated though hyperhomocysteinemia is a risk factor in several obstetric pathologies. In this paper, a simple and fast method based on LC/MS technology is applied for simultaneous SAM and SAH quantification in human placenta and placental explants. The developed method proved to be highly sensitive, inter-day repeatable and intra-day reproducible. The content of SAM and SAH in term human placenta is in the range 8.33–18.73 and 5.20–8.74nmol/g wet tissue, respectively. SAM concentration doubles while SAH decreases under treatment of cultivated placental explants with clinically relevant 20μM homocysteine in comparison with placental tissue with ultimate impact on velocity of numerous methyltransferase reactions.

CMyBP-C was decreased via KLHL3-mediated proteasomal degradation in congenital heart diseases

Cardiac myosin binding protein C (cMyBP-C) is a cardiac structural and regulatory protein; mutations of cMyBP-C are frequently associated with hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Cardiac special transcription factors may regulate the expression of cMyBP-C. However, the role of cMyBP-C in congenital heart diseases (CHD) remains poorly understood. In the current study, western blotting and the MRM approach showed that cMyBP-C expression was significantly reduced in fetuses with CHD compared to those without. Furthermore, we found that cMyBP-C interacted with KLHL3 by immunoprecipitation and immunofluorescence, and the degradation of cMyBP-C was caused by KLHL3-mediated ubiquitination. In addition, homocysteine (Hcy, a risk factor of CHD) treatment caused a decrease in cMyBP-C and an increase in KLHL3 expression, and the proteasome inhibitor MG132 reversed the Hcy-induced reduction of cMyBP-C expression. Finally, we verified that reduced cMyBP-C by Hcy promoted apoptosis in cardiomyocytes. These results demonstrate that Hcy decreases the expression of cMyBP-C through a KLHL3-mediated ubiquitin–proteasome pathway, and thereby influences heart development.

MicroRNA-217 suppresses homocysteine-induced proliferation and migration of vascular smooth muscle cells via N-methyl-D-aspartic acid receptor inhibition.

Hyperhomocysteine has become a critical risk for atherosclerosis and can stimulate proliferation and migration of vascular smooth muscle cells (VSMCs). N-methyl-D-aspartic acid receptor (NMDAR) is a receptor of homocysteine and mediates the effects of homocysteine on VSMCs. Bioinformatics analysis has shown NMDAR is a potential target of microRNA-217 (miR-217), which exerts multiple functions in cancer tumorigenesis and carotid plaque progression. In this study, we sought to investigate the role of miR-217 in VSMCs phenotype transition under homocysteine exposure and elucidate its effect on atherosclerotic plaque formation. After treating with several doses of homocysteine (0-8×10(-4) mol/L) for 24hours, the expression of miR-217 in HA-VSMCs and rat aortic VSMCs was not altered. Intriguingly, the expression of NMDAR mRNA and protein was reduced by homocysteine in a dose-dependent manner. Transfection of miR-217 mimic significantly inhibited the proliferation and migration of VSMCs with homocysteine treatment, while transfection of miR-217 inhibitor promoted VSMCs migration. Moreover, miR-217 mimic down-regulated while miR-217 inhibitor up-regulated NMDAR protein expression but not NMDAR mRNA expression. Through luciferase reporter assay, we showed that miR-217 could directly bind to the 3'-UTR of NMDAR. MiR-217 mimic transfection also released the inhibition of cAMP-response element-binding protein (CREB)-PGC-1α signalling induced by homocysteine. Additionally, restoration of PGC-1α expression via AdPGC-1α infection markedly suppressed VSMCs proliferation through the degradation of NADPH oxidase (NOX1) and reduction of reactive oxygen species (ROS). Collectively, our study identified the role of miR-217 in regulating VSMCs proliferation and migration, which might serve as a target for atherosclerosis therapy.

Alleviation of hepatic fat accumulation by betaine involves reduction of homocysteine via up-regulation of betaine-homocysteine methyltransferase (BHMT)

We investigated the anti-lipogenic effect of betaine in rats fed methionine and choline-deficient diet (MCD). Intake of MCD for 3 wk resulted in a significant accumulation of hepatic lipids, which was prevented by betaine supplementation in drinking water (1%). Phosphorylation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), sterol regulatory element-binding protein-1c (SREBP-1c), and liver kinase B1 (LKB1) was inhibited by MCD intake, and these changes were all inhibited by betaine feeding. Meanwhile, betaine supplementation reversed the reduction of methionine and S-adenosylmethionine (SAM), and the elevation of homocysteine levels in the liver, which could be attributable to the induction of betaine-homocysteine methyltransferase (BHMT) and methionine adenosyltransferase (MAT). Different cell lines were used to clarify the role of homocysteine on activation of the AMPK pathway. Homocysteine treatment decreased pAMPK, pACC, pSREBP-1c and pLKB1 in HepG2 cells. Metformin-induced activation of AMPK was also inhibited by homocysteine. Treatment with hydroxylamine, a cystathionine β-synthase inhibitor, resulted in a reduction of pAMPK, pACC and pSREBP-1c, accompanied by an elevation of intracellular homocysteine. Betaine treatment prevented the homocysteine-induced reduction of pAMPK, pACC, pSREBP-1c and pLKB1 in H4IIE cells, but not in HepG2 cells. Also the elevation of cellular homocysteine and inhibition of protein expression of BHMT were prevented by betaine only in H4IIE cells which express BHMT. The results suggest that the beneficial effect of betaine against hepatic lipid accumulation may be attributed, at least in part, to the depletion of homocysteine via up-regulation of BHMT in hepatocytes.

Inhibition of homocysteine-induced endoplasmic reticulum stress and endothelial cell damage by l-serine and glycine

Hyperhomocysteinemia is an independent risk factor for several cardiovascular diseases. The use of vitamins to modulate homocysteine metabolism substantially lowers the risk by reducing plasma homocysteine levels. In this study, we evaluated the effects of l-serine and related amino acids on homocysteine-induced endoplasmic reticulum (ER) stress and endothelial cell damage using EA.hy926 human endothelial cells. Homocysteine treatment decreased cell viability and increased apoptosis, which were reversed by cotreatment with l-serine. l-Serine inhibited homocysteine-induced ER stress as verified by decreased glucose-regulated protein 78kDa (GRP78) and C/EBP homologous protein (CHOP) expression as well as X-box binding protein 1 (xbp1) mRNA splicing. The effects of l-serine on homocysteine-induced ER stress are not attributed to intracellular homocysteine metabolism, but instead to decreased homocysteine uptake. Glycine exerted effects on homocysteine-induced ER stress, apoptosis, and cell viability that were comparable to those of l-serine. Although glycine did not affect homocysteine uptake or export, coincubation of homocysteine with glycine for 24h reduced the intracellular concentration of homocysteine. Taken together, l-serine and glycine cause homocysteine-induced endothelial cell damage by reducing the level of intracellular homocysteine. l-Serine acts by competitively inhibiting homocysteine uptake in the cells. However, the mechanism(s) by which glycine lowers homocysteine levels are unclear.

Hyperhomocysteinemia activates the aryl hydrocarbon receptor/CD36 pathway to promote hepatic steatosis in mice.

Hyperhomocysteinemia (HHcy) is associated with liver diseases such as fatty liver and hepatic fibrosis; however, the underlying mechanism is still largely unknown. The current study aimed to explore the signaling pathway involved in HHcy-induced hepatic steatosis (HS). C57BL/6 mice were fed a high-methionine diet (HMD) for 4 and 8 weeks to establish the HHcy mouse model. Compared to a chow diet, the HMD induced hepatic steatosis and elevated hepatic expression of CD36, a fatty acid transport protein. The increased CD36 expression was associated with activation of the aryl hydrocarbon receptor (AHR). In primary cultured hepatocytes, high levels of homocysteine (Hcy) treatment up-regulated CD36 and increased subsequent lipid uptake; both were significantly attenuated by small interfering RNA (siRNA) knockdown of CD36 and AHR. Chromatin immunoprecipitation assay revealed that Hcy promoted binding of AHR to the CD36 promoter, and transient transfection assay demonstrated markedly increased activity of the AHR response element by Hcy, which was ligand dependent. Mass spectrometry revealed significantly increased hepatic content of lipoxin A4 (LXA4 ), a metabolite of arachidonic acid, in HMD-fed mice. Furthermore, overexpression of 15-oxoprostaglandin 13-reductase 1, a LXA4 inactivation enzyme, inhibited Hcy-induced AHR activation, lipid uptake, and lipid accumulation. Moreover, LXA4 -induced up-regulation of CD36 and lipid uptake was inhibited by AHR siRNA in vitro in hepatocytes. Finally, treatment with an AHR antagonist reversed HHcy-induced lipid accumulation by inhibiting the AHR-CD36 pathway in mice. HHcy activates the AHR-CD36 pathway by increasing hepatic LXA4 content, which results in hepatic steatosis. (Hepatology 2016;64:92-105).

NSun2 Deficiency Protects Endothelium From Inflammation via mRNA Methylation of ICAM-1

Vascular endothelial inflammation, including the expression of intercellular adhesion molecule 1 (ICAM-1), is a key event in vascular diseases. However, the mechanisms underlying the regulation of ICAM-1 are largely unknown. To investigate the mechanisms on the regulation of ICAM-1 by NOP2/Sun domain family, member 2 (NSun2)-mediated mRNA methylation and the impact of NSun2-ICAM-1 regulatory process in vascular inflammation and allograft arteriosclerosis. By using in vitro, in cells, and in vivo methylation assays, we showed that the tRNA methyltransferase NSun2 methylated the ICAM-1 mRNA. Methylation by NSun2 promoted the translation of ICAM-1, thereby increasing the adhesion of leukocytes to endothelial cells. Tumor necrosis factor-α or homocysteine activated the methyltransferase activity of NSun2 by repressing the phosphorylation of NSun2 by Aurora-B. The levels of ICAM-1 induction and of leukocyte adhesion to vascular endothelium observed with homocysteine treatment in wild-type rats were markedly decreased in NSun2(-/-) rats. In a rat model of aortic allograft, the lack of donor NSun2 impaired the formation of allograft arteriosclerosis. NSun2 upregulates the expression of ICAM-1 by methylating ICAM-1 mRNA. This regulatory process impacts on vascular inflammation and allograft arteriosclerosis.

Homocysteine elicits an M1 phenotype in murine macrophages through an EMMPRIN-mediated pathway.

Hyperhomocysteinemia (HHcy) is associated with inflammatory diseases and is known to increase the production of reactive oxygen species (ROS), matrix metalloproteinase (MMP)-9, and inducible nitric oxide synthase, and to decrease endothelial nitric oxide production. However, the impact of HHcy on macrophage phenotype differentiation is not well-established. It has been documented that macrophages have 2 distinct phenotypes: the "classically activated/destructive" (M1), and the "alternatively activated/constructive" (M2) subtypes. We hypothesize that HHcy increases M1 macrophage differentiation through extracellular matrix metalloproteinase inducer (EMMPRIN), a known inducer of matrix metalloproteinases. murine J774A.1 and Raw 264.7 macrophages were treated with 100 and 500 μmol/L Hcy, respectively, for 24 h. Samples were analyzed using Western blotting and immunocytochemistry. Homocysteine treatment increased cluster of differentiation 40 (CD40; M1 marker) in J774A.1 and Raw 264.7 macrophages. MMP-9 was induced in both cell lines. EMMPRIN protein expression was also increased in both cell lines. Blocking EMMPRIN function by pre-treating cells with anti-EMMPRIN antibody, with or without Hcy, resulted in significantly lower expression of CD40 in both cell lines by comparison with the controls. A DCFDA assay demonstrated increased ROS production in both cell lines with Hcy treatment when compared with the controls. Our results suggest that HHcy results in an increase of the M1 macrophage phenotype. This effect seems to be at least partially mediated by EMMPRIN induction.

Neuroprotection and mechanisms of atractylenolide III in preventing learning and memory impairment induced by chronic high-dose homocysteine administration in rats

Studies demonstrated that chronic high-dose homocysteine administration induced learning and memory impairment in animals. Atractylenolide III (Aen-III), a neuroprotective constituent of Atractylodis macrocephalae Koidz, was isolated in our previous study. In this study, we investigated potential benefits of Aen-III in preventing learning and memory impairment following chronic high-dose homocysteine administration in rats. Results showed that administration of Aen-III significantly ameliorated learning and memory impairment induced by chronic high-dose homocysteine administration in rats, decreased homocysteine-induced reactive oxygen species (ROS) formation and restored homocysteine-induced decrease of phosphorylated protein kinase C expression level. Moreover, Aen-III protected primary cultured neurons from apoptotic death induced by homocysteine treatment. This study provides the first evidence for the neuroprotective effect of Aen-III in preventing learning and impairment induced by chronic administration of homocysteine. Aen-III may have therapeutic potential in treating homocysteine-mediated cognitive impairment and neuronal injury.