Homocysteine (HCys), a sulfur-containing amino acid, is formed during the metabolism of methionine. An imbalance between the rate of production and the use of HCys during methionine metabolism can result in an increase in the plasma and urinary levels of HCys. HCys has been shown to be toxic to vascular endothelial cells through several pathways. Many earlier clinical studies have revealed an association between plasma HCys and cardiovascular and other diseases. In contrast, estrogens are suggested to lower the risk of cardiovascular disease. Several studies indicate that estrogen metabolites could be responsible for cardiovascular protection. It has been demonstrated that electrophilic estrogen quinones, E1(E2)-2,3-Q and E1(E2)-3,4-Q, can alkylate DNA as well as form conjugates with glutathione. I hypothesize that estrogen quinones generated in situ by oxidative enzymes, metal ions, or molecular oxygen can interact with HCys to form conjugates. This in turn could lower the levels of toxic HCys as well as quenching the reactive estrogen quinones, resulting in cardiovascular protective effects. To test the feasibility of a protective estrogen-HCys pathway, estrogen quinones were treated with HCys. Tandem mass spectrometry analysis of the assay mixture shows the formation of estrogen-HCys conjugates. Furthermore, incubation of catechol estrogens with myeloperoxidase (MPO) in the presence of HCys resulted in the formation of respective estrogen-HCys conjugates. The identities of estrogen-HCys conjugates in MPO assay extracts were confirmed by comparing them to pure synthesized estrogen-HCys standards. I propose that through conjugation estrogens could chemically regulate HCys levels; moreover these conjugates could be used as potential biomarkers in determining health.

Elevated homocysteine is an important risk factor that increases cerebrovascular and neurodegenerative disease morbidity. In mammals, B vitamin supplementation can reduce homocysteine levels. Whether, and how, hibernating mammals, that essentially stop ingesting B vitamins, maintain homocysteine metabolism and avoid cerebrovascular impacts and neurodegeneration remain unclear. Here, we compare homocysteine levels in the brains of torpid bats, active bats and rats to identify the molecules involved in homocysteine homeostasis. We found that homocysteine does not elevate in torpid brains, despite declining vitamin B levels. At low levels of vitamin B6 and B12, we found no change in total expression level of the two main enzymes involved in homocysteine metabolism (methionine synthase and cystathionine β-synthase), but a 1.85-fold increase in the expression of the coenzyme-independent betaine-homocysteine S-methyltransferase (BHMT). BHMT expression was observed in the amygdala of basal ganglia and the cerebral cortex where BHMT levels were clearly elevated during torpor. This is the first report of BHMT protein expression in the brain and suggests that BHMT modulates homocysteine in the brains of hibernating bats. BHMT may have a neuroprotective role in the brains of hibernating mammals and further research on this system could expand our biomedical understanding of certain cerebrovascular and neurodegenerative disease processes.

Betaine is the substrate of the liver- and kidney-specific betaine-homocysteine (Hcy) methyltransferase (BHMT), an alternate pathway for Hcy remethylation. We hypothesized that BHMT is a major pathway for homocysteine removal in cases of hyperhomocysteinaemia (HHcy). Therefore, we measured betaine in plasma and tissues from patients and animal models of HHcy of genetic and acquired cause. Plasma was collected from patients presenting HHcy without any Hcy interfering treatment. Plasma and tissues were collected from rat models of HHcy induced by diet and from a mouse model of cystathionine β-synthase (CBS) deficiency. S-adenosyl-methionine (AdoMet), S-adenosyl-homocysteine (AdoHcy), methionine, betaine and dimethylglycine (DMG) were quantified by ESI-LC-MS/MS. mRNA expression was quantified using quantitative real-time (QRT)-PCR. For all patients with diverse causes of HHcy, plasma betaine concentrations were below the normal values of our laboratory. In the diet-induced HHcy rat model, betaine was decreased in all tissues analysed (liver, brain, heart). In the mouse CBS deficiency model, betaine was decreased in plasma, liver, heart and brain, but was conserved in kidney. Surprisingly, BHMT expression and activity was decreased in liver. However, in kidney, BHMT and SLC6A12 expression was increased in CBS-deficient mice. Chronic HHcy, irrespective of its cause, induces betaine depletion in plasma and tissues (liver, brain and heart), indicating a global decrease in the body betaine pool. In kidney, betaine concentrations were not affected, possibly due to overexpression of the betaine transporter SLC6A12 where betaine may be conserved because of its crucial role as an osmolyte.

Homocysteine (Hcy) is an intermediate non-diet amino acid connecting methionine and folate cycles. Elevated total Hcy level in blood, denoted as hyperhomocysteinemia, has emerged as a prevalent and strong risk factor for multiple diseases including atherosclerotic vascular disease in coronary, cerebral, and peripheral vessels. Its detrimental effect on vascular system implies the potential application as an inhibitor of angiogenesis. However, the detailed mechanism is unveiled. Inhibitory effect of Hcy was assessed on vascular endothelial growth factor (VEGF) induced cell proliferation and migration with endothelial cell (EC) culture system. Its effect on angiogenesis was further examined in vitro and in vivo After Hcy treatment, key angiogenic factors were measured by RT-qPCR. Cellular skeletal structure was also evaluated by actin stress fiber staining. VEGF-induced human umbilical vein EC (HUVEC) proliferation and migration were dramatically down-regulated by Hcy in a dose-responsive manner. Hcy treatment significantly inhibited the VEGF-induced angiogenesis in vitro by tube formation assay and chick chorioallantoic membrane (CAM) vessel formation in vivo Key angiogenic factors like VEGFR1/2 and angiopoietin (Ang)1/2 were substantially reduced by Hcy in HUVEC- and VEGF-induced actin stress fiber cytoskeletal structure was abolished. We demonstrated that Hcy could inhibit angiogenesis by targetting key angiogenic factor and disruption of actin cytoskeleton which is crucial for cell migration.

The senescence of vascular endothelial cells (VECs) drives the occurrence and development of cardiovascular disease (CVD). Homocysteine (HCY) is a general risk factor for age-associated CVDs. Autophagy, an evolutionarily conserved lysosomal protein degradation pathway, serves a part in VEC senescence. The purpose of this study was to investigate the role of autophagy in HCY-induced endothelial cell senescence and explore novel mechanisms and therapeutic approaches for related CVDs. Human umbilical vein endothelial cells (HUVECs) were isolated from fresh umbilical cords of healthy pregnancies. Cell Counting Kit-8, flow cytometry and senescence-associated (SA) β-galactosidase (Gal) staining demonstrated that HCY induced HUVEC senescence by decreasing cell proliferation, arresting cell cycle and increasing the number of SA-β-Gal-positive cells. Stub-RFP-Sens-GFP-LC3 autophagy-related double fluorescence lentivirus revealed that HCY increased autophagic flux. Further, inhibition of autophagy using 3-methyladenine increased HCY-induced HUVEC senescence. By contrast, the induction of autophagy via rapamycin alleviated HCY-induced HUVEC senescence. Finally, the detection of reactive oxygen species (ROS) with ROS kit showed that HCY increased intracellular ROS, whereas induction of autophagy reduced intracellular ROS. In conclusion, HCY increased HUVEC senescence and upregulated autophagy; moderate autophagy could reverse HCY-induced cell senescence. Autophagy may alleviate HCY-induced cell senescence by decreasing intracellular ROS. This provides insight into the underlying mechanism of HCY-induced VEC senescence and potential treatments for age-associated CVDs.

Although hyperglycemia is the main instigator in the development of diabetic retinopathy, elevated circulating levels of a non-protein amino acid, homocysteine, are also associated with an increased risk of retinopathy. Homocysteine is recycled back to methionine by methylenetetrahydrofolate reductase (MTHFR) and/or transsulfurated by cystathionine β-synthase (CBS) to form cysteine. CBS and other transsulfuration enzyme cystathionine-γ-lyase (CSE), through desulfuration, generates H2S. Methionine cycle also regulates DNA methylation, an epigenetic modification associated with the gene suppression. The aim of this study was to investigate homocysteine and its metabolism in diabetic retinopathy.

Age related macular degeneration (ARMD) is a leading cause of blindness, particularly in persons above 60 yr of age. Homocysteine is implicated in many ocular diseases including ARMD. This study was undertaken to assess the status and relationship between plasma homocysteine, homocysteine - thiolactone, homocysteinylated protein and copper levels in patients with ARMD.

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.

Aims/Introduction:  To estimate nutritional risk factors for osteoporosis in patients with type 2 diabetes, bone mineral density, homocysteine level, and intakes and levels of Hcy-related vitamins including folate, vitamin B6 and vitamin B12 were analyzed in a cross-sectional study.

Hyperhomocysteinemia may be a risk factor for endothelial dysfunction. Folate and vitamin B12 regulate the homocysteine metabolic process. This study aimed to evaluate the associations between subsequent events of adverse pregnancy outcome and early variables of homocysteine, folate, and vitamin B12 in pregnant women.

BACKGROUND: There is accumulated evidence that plasma concentration of the sulfur-containing amino-acid homocysteine (Hcy) is a prognostic marker for cardiovascular morbidity and mortality. Both fasting levels of Hcy and post methionine loading levels are used as prognostic markers. The aim of the present study was to investigate the existence of a daily rhythm in plasma Hcy under strictly controlled nutritional and sleep-wake conditions. We also investigated if the time during which methionine loading is performed, i.e., morning or evening, had a different effect on the resultant plasma Hcy concentration. METHODS: Six healthy men aged 23-26 years participated in 4 experiments. In the first and second experiments, the daily rhythm in Hcy as well as in other amino acids was investigated under a normal or an inverse sleep-wake cycle. In the third and fourth, Hcy concentrations were investigated after a morning and evening methionine loading. To standardize food consumption in the first two experiments, subjects received every 3 hours 150 ml of specially designed low-protein liquid food (Ensure(R) formula). RESULTS: In both the first and second experiments there was a significant daily rhythm in Hcy concentrations with a mid-day nadir and a nocturnal peak. Strikingly different 24-h patterns were observed in methionine, leucine, isoleucine and tyrosine. In all, the 24-h curves revealed a strong influence of both the sleep-wake cycle and the feeding schedule. Methionine loading resulted in increased plasma Hcy levels during both morning and evening experiments, which were not significantly different from each other. CONCLUSIONS: There is a daily rhythm in plasma concentration of the amino acid Hcy, and this rhythm is independent of sleep-wake and food consumption. In view of the fact that increased Hcy concentrations may be associated with increased cardiovascular risks, these findings may have clinical implications for the health of rotating shift workers.

Diabetic retinopathy (DR) is the most common cause of blindness in people under the age of 65. Unfortunately, the current screening process for DR restricts the population that can be evaluated and the disease goes undetected until irreversible damage occurs. Herein, we aimed to evaluate homocysteine (Hcy) as a biomarker for DR screening. Hcy levels were measured by enzyme-linked immuno sorbent assay (ELISA) and immunolocalization methods in the serum, vitreous and retina of diabetic patients as well as in serum and retina of different animal models of DM representing type 1 diabetes (streptozotocin (STZ) mice, Akita mice and STZ rats) and db/db mice which exhibit features of human type 2 diabetes. Our results revealed increased Hcy levels in the serum, vitreous and retina of diabetic patients and experimental animal models of diabetes. Moreover, optical coherence tomography (OCT) and fluorescein angiography (FA) were used to evaluate the retinal changes in mice eyes after Hcy-intravitreal injection into normal wild-type (WT) and diabetic (STZ) mice. Hcy induced changes in mice retina which were aggravated under diabetic conditions. In conclusion, our data reported Hcy as a strong candidate for use as a biomarker in DR screening. Targeting the clearance of Hcy could also be a future therapeutic target for DR.

Homocysteine (Hcy) is an amino acid that requires vitamins B12 and folic acid for its metabolism. Vitamins B12 and folic acid deficiencies lead to hyperhomocysteinemia (HHcy, elevated Hcy), which is linked to the development of diabetic retinopathy (DR), age-related macular degeneration (AMD), and Alzheimer's disease (AD). The goal of the current study was to explore inflammation as an underlying mechanism of HHcy-induced pathology in age related diseases such as AMD, DR, and AD. Mice with HHcy due to a lack of the enzyme cystathionine-β-synthase (CBS) and wild-type mice were evaluated for microglia activation and inflammatory markers using immuno-fluorescence (IF). Tissue lysates isolated from the brain hippocampal area from mice with HHcy were evaluated for inflammatory cytokines using the multiplex assay. Human retinal endothelial cells, retinal pigment epithelial cells, and monocyte cell lines treated with/without Hcy were evaluated for inflammatory cytokines and NFκB activation using the multiplex assay, western blot analysis, and IF. HHcy induced inflammatory responses in mouse brain, retina, cultured retinal, and microglial cells. NFκB was activated and cytokine array analysis showed marked increase in pro-inflammatory cytokines and downregulation of anti-inflammatory cytokines. Therefore, elimination of excess Hcy or reduction of inflammation is a promising intervention for mitigating damage associated with HHcy in aging diseases such as DR, AMD, and AD.

Increased plasma homocysteinemia is considered a risk factor of dementia, including Alzheimer's disease (AD) and vascular dementia. However, the reason elevated plasma homocysteinemia increases the risk of dementia remains unknown. A pathological hallmark of AD is neurofibrillary tangles (NFTs) that consist of pathologically phosphorylated tau proteins. The effect of homocysteine (Hcy) on tau aggregation was explored using human neuroblastoma M1C cells that constitutively express human wild-type tau (4R0N) under the control of a tetracycline off system, primary mouse cultured neurons, and by inducing hyperhomocysteinemia in a mouse model of tauopathy (HHCy mice). A wide range of Hcy concentrations (10-1000 µM) increased total tau and phosphorylated tau protein levels. Hcy activated glycogen synthase kinase 3, and cyclin dependent kinase 5, major tau phosphokinases, and inactivated protein phosphatase 2A, a main tau phosphatase. Hcy exhibited cytotoxic effects associated with enhanced activation of caspase. Truncation of tau in the C-terminus, the cleavage site of caspase 3 (i.e., D421, detected by the TauC3 antibody) was also increased. Total tau, phosphorylated tau, as well as C-terminal cleaved tau were increased in the sarkosyl insoluble tau fraction. Hcy also increased the level of tau oligomers, as indicated by the tau oligomer complex 1 (TOC1) antibody that specifically identifies oligomeric tau species, in the tris insoluble, sarkosyl soluble fraction. The levels of TOC1-positive oligomeric tau were increased in brain lysates from HHCy mice, and treating HHCy mice with S-adenosylmethionine, an intermediate of Hcy, reduced the levels of oligomeric tau to control levels. These observations suggest that Hcy increases the levels of phosphorylated tau as well as truncated tau species via caspase 3 activation, and enhanced tau oligomerization and aggregation.

Homocysteine concentrations are a read-out of methionine metabolism and have been related to changes in lifespan in animal models. In humans, high homocysteine concentrations are an important predictor of age related disease. We aimed to explore the association of homocysteine with familial longevity by testing whether homocysteine is lower in individuals that are genetically enriched for longevity. We measured concentrations of total homocysteine in 1907 subjects from the Leiden Longevity Study consisting of 1309 offspring of nonagenarian siblings, who are enriched with familial factors promoting longevity, and 598 partners thereof as population controls. We found that homocysteine was related to age, creatinine, folate, vitamin B levels and medical history of hypertension and stroke in both groups (all p<0.001). However, levels of homocysteine did not differ between offspring enriched for longevity and their partners, and no differences in the age-related rise in homocysteine levels were found between groups (p for interaction 0.63). The results suggest that homocysteine metabolism is not likely to predict familial longevity.

Homocysteine is an independent risk factor for coronary, cerebral, and peripheral vascular diseases. Recent studies have shown that levels of homocysteine are elevated in patients with impaired hepatic function, but the precise role of homocysteine in the development of hepatic dysfunction is unclear. In this study, we examined the effect of homocysteine on hepatocyte proliferation in vitro. Our results demonstrated that homocysteine inhibited hepatocyte proliferation by up-regulating protein levels of p53 as well as mRNA and protein levels of p21(Cip1) in primary cultured hepatocytes. Homocysteine induced cell growth arrest in p53-positive hepatocarcinoma cell line HepG2, but not in p53-null hepatocarcinoma cell line Hep3B. A p53 inhibitor pifithrin-α inhibited the expression of p21(Cip1) and attenuated homocysteine-induced cell growth arrest. Homocysteine induced TRB3 expression via endoplasmic reticulum stress pathway, resulting in Akt dephosphorylation. Knock-down of endogenous TRB3 significantly suppressed the inhibitory effect of homocysteine on cell proliferation and the phosphorylation of Akt. LiCl reversed homocysteine-mediated cell growth arrest by inhibiting TRB3-mediated Akt dephosphorylation. These results demonstrate that both TRB3 and p21(Cip1) are critical molecules in the homocysteine signaling cascade and provide a mechanistic explanation for impairment of liver regeneration in hyperhomocysteinemia.

Betaine-homocysteine methyltransferase (BHMT) catalyzes the remethylation of homocysteine. BHMT is highly expressed in the human liver. In the liver, BHMT catalyzes up to 50% of homocysteine metabolism. Understanding the relationship between BHMT genetic polymorphisms and function might increase our understanding of the role of this reaction in homocysteine remethylation and in S-adenosylmethionine-dependent methylation. To help achieve those goals, we measured levels of BHMT enzyme activity and immunoreactive protein in 268 human hepatic surgical biopsy samples from adult subjects as well as 73 fetal hepatic tissue samples obtained at different gestational ages. BHMT protein levels were correlated significantly (p<0.001) with levels of enzyme activity in both fetal and adult tissues, but both were decreased in fetal tissue when compared with levels in the adult hepatic biopsies. To determine possible genotype-phenotype correlations, 12 tag SNPs for BHMT and the closely related BHMT2 gene were selected from SNPs observed during our own gene resequencing studies as well as from HapMap. These SNPs data were used to genotype DNA from the adult hepatic surgical biopsy samples, and genotype-phenotype association analysis was performed. Three SNPs (rs41272270, rs16876512, and rs6875201), located 28kb upstream, in the 5'-UTR and in intron 1 of BHMT, respectively, were significantly correlated with both BHMT activity (p=3.41E-8, 2.55E-9 and 2.46E-10, respectively) and protein levels (p=5.78E-5, 1.08E-5 and 6.92E-6, respectively). We also imputed 230 additional SNPs across the BHMT and BHMT2 genes, identifying an additional imputed SNP, rs7700790, that was also highly associated with hepatic BHMT enzyme activity and protein. However, none of the 3 genotyped or one imputed SNPs displayed a "shift" during electrophoretic mobility shift assays. These observations may help us to understand individual variation in the regulation of BHMT in the human liver and its possible relationship to variation in methylation.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) and homocysteine (Hcy) have been linked to inflammation and Alzheimer's disease (AD). Using a case-control design, we examined their independent effects and interactions with cardiovascular disease equivalent (CVDE), on AD risk.

Diabetic retinopathy is a progressive disease, and one of the key metabolic abnormalities in the pathogenesis of diabetic retinopathy, mitochondrial damage, is also influenced by the duration of hyperglycemia. Mitochondrial quality control involves a coordination of mitochondrial dynamics, biogenesis and removal of the damaged mitochondria. In diabetes, these processes are impaired, and the damaged mitochondria continue to produce free radicals. Diabetic patients also have high homocysteine and reduced levels of hydrogen sulfide, and hyperhomocysteinemia is shown to exacerbate diabetes-induced mitochondrial damage and worsen their dynamics. This study aims to investigate the temporal relationship between hyperhomocysteinemia and retinal mitochondrial quality control in diabetic retinopathy.

Although a high-fat diet (HFD) induces gut dysbiosis and cardiovascular system remodeling, the precise mechanism is unclear. We hypothesize that HFD instigates dysbiosis and cardiac muscle remodeling by inducing matrix metalloproteinases (MMPs), which leads to an increase in white adipose tissue, and treatment with lactobacillus (a ketone body donor from lactate; the substrate for the mitochondria) reverses dysbiosis-induced cardiac injury, in part, by increasing lipolysis (PGC-1α, and UCP1) and adipose tissue browning and decreasing lipogenesis. To test this hypothesis, we used wild type (WT) mice fed with HFD for 16 weeks with/without a probiotic (PB) in water. Cardiac injury was measured by CKMB activity which was found to be robust in HFD-fed mice. Interestingly, CKMB activity was normalized post PB treatment. Levels of free fatty acids (FFAs) and methylation were increased but butyrate was decreased in HFD mice, suggesting an epigenetically governed 1-carbon metabolism along with dysbiosis. Levels of PGC-1α and UCP1 were measured by Western blot analysis, and MMP activity was scored via zymography. Collagen histology was also performed. Contraction of the isolated myocytes was measured employing the ion-optic system, and functions of the heart were estimated by echocardiography. Our results suggest that mice on HFD gained weight and exhibited an increase in blood pressure. These effects were normalized by PB. Levels of fibrosis and MMP-2 activity were robust in HFD mice, and treatment with PB mitigated the fibrosis. Myocyte calcium-dependent contraction was disrupted by HFD, and treatment with PB could restore its function. We conclude that HFD induces dysbiosis, and treatment with PB creates eubiosis and browning of the adipose tissue.