Estrogen receptor-related receptor (Esrra) induces ribosomal protein Rplp1-mediated adaptive hepatic translation during prolonged starvation.

Protein translation is an energy-intensive ribosome-driven process that is reduced during nutrient scarcity to conserve cellular resources. During prolonged starvation, cells selectively translate specific proteins to enhance their survival (adaptive translation); however, this process is poorly understood. Accordingly, we analyzed protein translation and mRNA transcription by multiple methods in vitro and in vivo to investigate adaptive hepatic translation during starvation. While acute starvation suppressed protein translation in general, proteomic analysis showed that prolonged starvation selectively induced translation of lysosome and autolysosome proteins. Significantly, the expression of the orphan nuclear receptor, estrogen-related receptor alpha (Esrra) increased during prolonged starvation and served as a master regulator of this adaptive translation by transcriptionally stimulating 60S acidic ribosomal protein P1 (Rplp1) gene expression. Overexpression or siRNA knockdown of Esrra expression in vitro or in vivo led to parallel changes in Rplp1 gene expression, lysosome/autophagy protein translation, and autophagy. Remarkably, we have found that Esrra had dual functions by not only regulating transcription but also controling adaptive translation via the Esrra/Rplp1/lysosome/autophagy pathway during prolonged starvation.

Metabolic switching of estrogen-related receptor alpha in breast cancer aggression.

Orphan nuclear receptor estrogen-related receptor alpha (ERRα) is an important regulator of energy metabolism, whereas its hyperactivation in breast cancer has been shown to regulate cell migration, proliferation, and tumour development. These findings suggest a fine balance in the status of ERRα in regulating metabolic homeostasis or promoting cancer progression. In this issue, Brindisi et al. have shown that ERRα is endogenously activated by cholesterol and caused breast cancer aggressiveness. This study also supports the anti-tumour mechanisms of cholesterol-lowering drugs such as statins.

Pharmacological inhibition of CFTR attenuates nonalcoholic steatohepatitis (NASH) progression in mice.

Nonalcoholic steatohepatitis (NASH) is considered a pivotal stage in nonalcoholic fatty liver disease (NAFLD) progression and increases the risk of end-stage liver diseases such as fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The etiology of NASH is multifactorial and identifying reliable molecular players has proven difficult. Presently, there are no approved drugs for NASH treatment, which has become a leading cause of liver transplants worldwide. Here, using public human transcriptomic NAFLD dataset, we uncover Cystic fibrosis transmembrane conductance receptor (CFTR) as a differentially expressed gene in the livers of human NASH patients. Similarly, murine Cftr expression was also found to be upregulated in two mouse models of diet-induced NASH. Furthermore, the pharmacological inhibition of CFTR significantly reduced NASH progression in mice and its overexpression aggravated lipotoxicity in human hepatic cells. These results, thus, underscore the involvement of murine Cftr in the pathogenesis of NASH and raise the intriguing possibility of its pharmacological inhibition in human NASH.

Spermidine-mediated hypusination of translation factor EIF5A improves mitochondrial fatty acid oxidation and prevents non-alcoholic steatohepatitis progression.

Spermidine is a natural polyamine that has health benefits and extends life span in several species. Deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH) are key enzymes that utilize spermidine to catalyze the post-translational hypusination of the translation factor EIF5A (EIF5AH). Here, we have found that hepatic DOHH mRNA expression is decreased in patients and mice with non-alcoholic steatohepatitis (NASH), and hepatic cells treated with fatty acids. The mouse and cell culture models of NASH have concomitant decreases in Eif5aH and mitochondrial protein synthesis which leads to lower mitochondrial activity and fatty acid ß-oxidation. Spermidine treatment restores EIF5AH, partially restores protein synthesis and mitochondrial function in NASH, and prevents NASH progression in vivo. Thus, the disrupted DHPS-DOHH-EIF5AH pathway during NASH represents a therapeutic target to increase hepatic protein synthesis and mitochondrial fatty acid oxidation (FAO) and prevent NASH progression.

Vitamin B12 and folate decrease inflammation and fibrosis in NASH by preventing syntaxin 17 homocysteinylation.

BACKGROUND & AIMS: Several recent clinical studies have shown that serum homocysteine (Hcy) levels are positively correlated, while vitamin B12 (B12) and folate levels are negative correlated, with non-alcoholic steatohepatitis (NASH) severity. However, it is not known whether hyperhomocysteinemia (HHcy) plays a pathogenic role in NASH. METHODS: We examined the effects of HHcy on NASH progression, metabolism, and autophagy in dietary and genetic mouse models, patients, and primates. We employed vitamin B12 (B12) and folate (Fol) to reverse NASH features in mice and cell culture. RESULTS: Serum Hcy correlated with hepatic inflammation and fibrosis in NASH. Elevated hepatic Hcy induced and exacerbated NASH. Gene expression of hepatic Hcy-metabolizing enzymes was downregulated in NASH. Surprisingly, we found increased homocysteinylation (Hcy-lation) and ubiquitination of multiple hepatic proteins in NASH including the key autophagosome/lysosome fusion protein, Syntaxin 17 (Stx17). This protein was Hcy-lated and ubiquitinated, and its degradation led to a block in autophagy. Genetic manipulation of Stx17 revealed its critical role in regulating autophagy, inflammation and fibrosis during HHcy. Remarkably, dietary B12/Fol, which promotes enzymatic conversion of Hcy to methionine, decreased HHcy and hepatic Hcy-lated protein levels, restored Stx17 expression and autophagy, stimulated ß -oxidation of fatty acids, and improved hepatic histology in mice with pre-established NASH. CONCLUSIONS: HHcy plays a key role in the pathogenesis of NASH via Stx17 homocysteinylation. B12/folate also may represent a novel first-line therapy for NASH. LAY SUMMARY: The incidence of non-alcoholic steatohepatitis, for which there are no approved pharmacological therapies, is increasing, posing a significant healthcare challenge. Herein, based on studies in mice, primates and humans, we found that dietary supplementation with vitamin B12 and folate could have therapeutic potential for the prevention or treatment of non-alcoholic steatohepatitis.

Thyroid Hormone Decreases Hepatic Steatosis, Inflammation, and Fibrosis in a Dietary Mouse Model of Nonalcoholic Steatohepatitis.

Background: Nonalcoholic steatohepatitis (NASH) is characterized by hepatic steatosis, lobular inflammation, and fibrosis. Thyroid hormone (TH) reduces steatosis; however, the therapeutic effect of TH on NASH-associated inflammation and fibrosis is not known. This study examined the therapeutic effect of TH on hepatic inflammation and fibrosis during NASH and investigated THs molecular actions on autophagy and mitochondrial biogenesis. Methods: HepG2-TRß cells were treated with bovine serum albumin-conjugated palmitic acid (PA) to mimic lipotoxic conditions in vitro. Mice with NASH were established by feeding C57BL/6J mice Western diet with 15% fructose in drinking water for 16 weeks. These mice were administered triiodothyronine (T3)/thyroxine (T4) supplemented in drinking water for the next eight weeks. Results: In cultured HepG2-TRß cells, TH treatment increased mitochondrial respiration and fatty acid oxidation under basal and PA-treated conditions, as well as decreased lipopolysaccharides and PA-stimulated inflammatory and fibrotic responses. In a dietary mouse model of NASH, TH administration decreased hepatic triglyceride content (3.19 ± 0.68 vs. 8.04 ± 0.42 mM/g liver) and hydroxyproline (1.44 ± 0.07 vs. 2.58 ± 0.30 mg/g liver) when compared with mice with untreated NASH. Metabolomics profiling of lipid metabolites showed that mice with NASH had increased triacylglycerol, diacylglycerol, monoacylglycerol, and hepatic cholesterol esters species, and these lipid species were decreased by TH treatment. Mice with NASH also showed decreased autophagic degradation as evidenced by decreased transcription Factor EB and lysosomal protease expression, and accumulation of LC3B-II and p62. TH treatment restored the level of lysosomal proteins and resolved the accumulation of LC3B-II and p62. Impaired mitochondrial biogenesis was also restored by TH. The simultaneous restoration of autophagy and mitochondrial biogenesis by TH increased ß-oxidation of fatty acids. Additionally, the elevated oxidative stress and inflammasome activation in NASH liver were also decreased by TH. Conclusions: In a mouse model of NASH, TH restored autophagy and mitochondrial biogenesis to increase ß-oxidation of fatty acids and to reduce lipotoxicity, oxidative stress, hepatic inflammation, and fibrosis. Activating thyroid hormone receptor in the liver may represent an effective strategy for NASH treatment.

Caffeine prevents restenosis and inhibits vascular smooth muscle cell proliferation through the induction of autophagy.

Caffeine is among the most highly consumed substances worldwide, and it has been associated with decreased cardiovascular risk. Although caffeine has been shown to inhibit the proliferation of vascular smooth muscle cells (VSMCs), the mechanism underlying this effect is unknown. Here, we demonstrated that caffeine decreased VSMC proliferation and induced macroautophagy/autophagy in an in vivo vascular injury model of restenosis. Furthermore, we studied the effects of caffeine in primary human and mouse aortic VSMCs and immortalized mouse aortic VSMCs. Caffeine decreased cell proliferation, and induced autophagy flux via inhibition of MTOR signaling in these cells. Genetic deletion of the key autophagy gene Atg5, and the Sqstm1/p62 gene encoding a receptor protein, showed that the anti-proliferative effect by caffeine was dependent upon autophagy. Interestingly, caffeine also decreased WNT-signaling and the expression of two WNT target genes, Axin2 and Ccnd1 (cyclin D1). This effect was mediated by autophagic degradation of a key member of the WNT signaling cascade, DVL2, by caffeine to decrease WNT signaling and cell proliferation. SQSTM1/p62, MAP1LC3B-II and DVL2 were also shown to interact with each other, and the overexpression of DVL2 counteracted the inhibition of cell proliferation by caffeine. Taken together, our in vivo and in vitro findings demonstrated that caffeine reduced VSMC proliferation by inhibiting WNT signaling via stimulation of autophagy, thus reducing the vascular restenosis. Our findings suggest that caffeine and other autophagy-inducing drugs may represent novel cardiovascular therapeutic tools to protect against restenosis after angioplasty and/or stent placement.

Role of AKR1B10 and AKR1B8 in the pathogenesis of non-alcoholic steatohepatitis (NASH) in mouse.

Non-alcoholic steatohepatitis (NASH) is a clinically important spectrum of non-alcoholic fatty liver disease (NAFLD) in humans. NASH is a stage of NAFLD progression wherein liver steatosis accompanies inflammation and pro-fibrotic events. Presently, there are no approved drugs for NASH, which has become a leading cause of liver transplant worldwide. To discover novel drug targets for NASH, we analyzed a human transcriptomic NASH dataset and found Aldo-keto reductase family 1 member B10 (AKR1B10) as a significantly upregulated gene in livers of human NASH patients. Similarly murine Akr1b10 and Aldo-keto reductase family 1 member B8 (Akr1b8) gene, which is a murine ortholog of human AKR1B10, were also found to be upregulated in a mouse model of diet-induced NASH. Furthermore, pharmacological inhibitors of AKR1B10 significantly reduced the pathological features of NASH such as steatosis, inflammation and fibrosis in mouse. In addition, genetic silencing of both mouse Akr1b10 and Akr1b8 significantly reduced the expression of proinflammatory cytokines from hepatocytes. These results, thus, underscore the involvement of murine AKR1B10 and AKR1B8 in the pathogenesis of murine NASH and raise an intriguing possibility of a similar role of AKR1B10 in human NASH.

Early induction of hepatic deiodinase type 1 inhibits hepatosteatosis during NAFLD progression.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD) comprises a spectrum ranging from hepatosteatosis to progressive nonalcoholic steatohepatitis that can lead to cirrhosis. Humans with low levels of prohormone thyroxine (T4) have a higher incidence of NAFLD, and thyroid hormone treatment is very promising in all patients with NAFLD. Deiodinase type 1 (Dio1) is a hepatic enzyme that converts T4 to the bioactive T3 and therefore regulates thyroid hormone availability within hepatocytes. We investigated the role of this intrahepatic regulation during the progression of NAFLD. METHODS: We investigated hepatic thyroid hormone metabolism in two NAFLD models: wild-type mice fed a Western diet with fructose and Leprdb mice fed a methionine- and choline-deficient diet. AAV8-mediated liver-specific Dio1 knockdown was employed to investigate the role of Dio1 during the progression of NAFLD. Intrahepatic thyroid hormone levels, deiodinase activity, and metabolic parameters were measured. RESULTS: Dio1 expression and activity were increased in the early stages of NAFLD and were associated with an increased T3/T4 ratio. Prevention of this increase by AAV8-mediated liver-specific Dio1 knockdown increased hepatic triglycerides and cholesterol and decreased the pACC/ACC ratio and acylcarnitine levels, suggesting there was lower ß-oxidation. Dio1 siRNA KD in hepatic cells treated with fatty acids showed increased lipid accumulation and decreased oxidative phosphorylation. CONCLUSION: Hepatic Dio1 gene expression was modulated by dietary conditions, was increased during hepatosteatosis and early NASH, and regulated hepatic triglyceride content. These early adaptations likely represent compensatory mechanisms that reduce hepatosteatosis and prevent NASH progression.

Hepatocyte-specific IL11 cis-signaling drives lipotoxicity and underlies the transition from NAFLD to NASH.

IL11 is important for fibrosis in non-alcoholic steatohepatitis (NASH) but its role beyond the stroma in liver disease is unclear. Here, we investigate the role of IL11 in hepatocyte lipotoxicity. Hepatocytes highly express IL11RA and secrete IL11 in response to lipid loading. Autocrine IL11 activity causes hepatocyte death through NOX4-derived ROS, activation of ERK, JNK and caspase-3, impaired mitochondrial function and reduced fatty acid oxidation. Paracrine IL11 activity stimulates hepatic stellate cells and causes fibrosis. In mouse models of NASH, hepatocyte-specific deletion of Il11ra1 protects against liver steatosis, fibrosis and inflammation while reducing serum glucose, cholesterol and triglyceride levels and limiting obesity. In mice deleted for Il11ra1, restoration of IL11 cis-signaling in hepatocytes reconstitutes steatosis and inflammation but not fibrosis. We found no evidence for the existence of IL6 or IL11 trans-signaling in hepatocytes or NASH. These data show that IL11 modulates hepatocyte metabolism and suggests a mechanism for NAFLD to NASH transition.

Gut microbiota and their metabolites in the progression of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver disorder worldwide. It comprises a spectrum of conditions that range from steatosis to non-alcoholic steatohepatitis, with progression to cirrhosis and hepatocellular carcinoma. Currently, there is no FDA-approved pharmacological treatment for NAFLD. The pathogenesis of NAFLD involves genetic and environmental/host factors, including those that cause changes in intestinal microbiota and their metabolites. In this review, we discuss recent findings on the relationship(s) of microbiota signature with severity of NAFLD and the role(s) microbial metabolites in NAFLD progression. We discuss how metabolites may affect NAFLD progression and their potential to serve as biomarkers for NAFLD diagnosis or therapeutic targets for disease management.

Protocol to Generate Senescent Cells from the Mouse Hepatic Cell Line AML12 to Study Hepatic Aging.

Previously developed senescent primary fibroblast models have limited relevance to study age-related changes in metabolically active tissues such as the liver. Here, we describe a protocol to generate senescent cells from the mouse hepatic cell line, AML12. These senescent cells exhibit molecular and metabolic signatures that are similar to those observed in livers from aged mice. These senescent AML12 cells should be a useful in vitro model to study the metabolic effects of aging in the liver. For complete details on the use and execution of this protocol, please refer to Singh et al. (2020).

Decreased autophagy and fuel switching occur in a senescent hepatic cell model system.

Although aging in the liver contributes to the development of chronic liver diseases such as NAFLD and insulin resistance, little is known about the molecular and metabolic details of aging in hepatic cells. To examine these issues, we used sequential oxidative stress with hydrogen peroxide to induce premature senescence in AML12 hepatic cells. The senescent cells exhibited molecular and metabolic signatures, increased SA-ßGal and γH2A.X staining, and elevated senescence and pro-inflammatory gene expression that resembled livers from aged mice. Metabolic phenotyping showed fuel switching towards glycolysis and mitochondrial glutamine oxidation as well as impaired energy production. The senescent AML12 cells also had increased mTOR signaling and decreased autophagy which likely contributed to the fuel switching from ß-oxidation that occurred in normal AML12 cells. Additionally, senescence-associated secretory phenotype (SASP) proteins from conditioned media of senescent cells sensitized normal AML12 cells to palmitate-induced toxicity, a known pathological effect of hepatic aging. In summary, we have generated senescent AML12 cells which displayed the molecular hallmarks of aging and also exhibited the aberrant metabolic phenotype, mitochondrial function, and cell signaling that occur in the aged liver.

Estrogen-Related Receptor Alpha: An Under-Appreciated Potential Target for the Treatment of Metabolic Diseases.

The estrogen-related receptor alpha (ESRRA) is an orphan nuclear receptor (NR) that significantly influences cellular metabolism. ESRRA is predominantly expressed in metabolically-active tissues and regulates the transcription of metabolic genes, including those involved in mitochondrial turnover and autophagy. Although ESRRA activity is well-characterized in several types of cancer, recent reports suggest that it also has an important role in metabolic diseases. This minireview focuses on the regulation of cellular metabolism and function by ESRRA and its potential as a target for the treatment of metabolic disorders.

Inhibiting Interleukin 11 Signaling Reduces Hepatocyte Death and Liver Fibrosis, Inflammation, and Steatosis in Mouse Models of Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: We studied the role of interleukin 11 (IL11) signaling in the pathogenesis of nonalcoholic steatohepatitis (NASH) using hepatic stellate cells (HSCs), hepatocytes, and mouse models of NASH. METHODS: We stimulated mouse and human fibroblasts, HSCs, or hepatocytes with IL11 and other cytokines and analyzed them by imaging, immunoblot, and functional assays and enzyme-linked immunosorbent assays. Mice were given injections of IL11. Mice with disruption of the interleukin 11 receptor subunit alpha1 gene (Il11ra1-/-) mice and Il11ra1+/+ mice were fed a high-fat methionine- and choline-deficient diet (HFMCD) or a Western diet with liquid fructose (WDF) to induce steatohepatitis; control mice were fed normal chow. db/db mice were fed with methionine- and choline-deficient diet for 12 weeks and C57BL/6 NTac were fed with HFMCD for 10 weeks or WDF for 16 weeks. Some mice were given intraperitoneal injections of anti-IL11 (X203), anti-IL11RA (X209), or a control antibody at different timepoints on the diets. Livers and blood were collected; blood samples were analyzed by biochemistry and liver tissues were analyzed by histology, RNA sequencing, immunoblots, immunohistochemistry, hydroxyproline, and mass cytometry time of flight assays. RESULTS: HSCs incubated with cytokines produced IL11, resulting in activation (phosphorylation) of ERK and expression of markers of fibrosis. Livers of mice given injections of IL11 became damaged, with increased markers of fibrosis, hepatocyte cell death and inflammation. Following the HFMCD or WDF, livers from Il11ra1-/- mice had reduced steatosis, fibrosis, expression of markers of inflammation and steatohepatitis, compared to and Il11ra1+/+ mice on the same diets. Depending on the time of administration of anti-IL11 or anti-IL11RA antibodies to wild-type mice on the HFMCD or WDF, or to db/db mice on the methionine and choline-deficient diet, the antibodies prevented, stopped, or reversed development of fibrosis and steatosis. Blood samples from Il11ra1+/+ mice fed the WDF and given injections of anti-IL11 or anti-IL11RA, as well as from Il11ra1-/- mice fed WDF, had lower serum levels of lipids and glucose than mice not injected with antibody or with disruption of Il11ra1. CONCLUSIONS: Neutralizing antibodies that block IL11 signaling reduce fibrosis, steatosis, hepatocyte death, inflammation and hyperglycemia in mice with diet-induced steatohepatitis. These antibodies also improve the cardiometabolic profile of mice and might be developed for the treatment of NASH.

Thyroid hormone receptor and ERRα coordinately regulate mitochondrial fission, mitophagy, biogenesis, and function.

Thyroid hormone receptor ß1 (THRB1) and estrogen-related receptor α (ESRRA; also known as ERRα) both play important roles in mitochondrial activity. To understand their potential interactions, we performed transcriptome and ChIP-seq analyses and found that many genes that were co-regulated by both THRB1 and ESRRA were involved in mitochondrial metabolic pathways. These included oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, and ß-oxidation of fatty acids. TH increased ESRRA expression and activity in a THRB1-dependent manner through the induction of the transcriptional coactivator PPARGC1A (also known as PGC1α). Moreover, TH induced mitochondrial biogenesis, fission, and mitophagy in an ESRRA-dependent manner. TH also induced the expression of the autophagy-regulating kinase ULK1 through ESRRA, which then promoted DRP1-mediated mitochondrial fission. In addition, ULK1 activated the docking receptor protein FUNDC1 and its interaction with the autophagosomal protein MAP1LC3B-II to induce mitophagy. siRNA knockdown of ESRRA, ULK1, DRP1, or FUNDC1 inhibited TH-induced autophagic clearance of mitochondria through mitophagy and decreased OXPHOS. These findings show that many of the mitochondrial actions of TH are mediated through stimulation of ESRRA expression and activity, and co-regulation of mitochondrial turnover through the PPARGC1A-ESRRA-ULK1 pathway is mediated by their regulation of mitochondrial fission and mitophagy. Hormonal or pharmacologic induction of ESRRA expression or activity could improve mitochondrial quality in metabolic disorders.

Loss of ULK1 increases RPS6KB1-NCOR1 repression of NR1H/LXR-mediated Scd1 transcription and augments lipotoxicity in hepatic cells.

Lipotoxicity caused by saturated fatty acids (SFAs) induces tissue damage and inflammation in metabolic disorders. SCD1 (stearoyl-coenzyme A desaturase 1) converts SFAs to mono-unsaturated fatty acids (MUFAs) that are incorporated into triglycerides and stored in lipid droplets. SCD1 thus helps protect hepatocytes from lipotoxicity and its reduced expression is associated with increased lipotoxic injury in cultured hepatic cells and mouse models. To further understand the role of SCD1 in lipotoxicity, we examined the regulation of Scd1 in hepatic cells treated with palmitate, and found that NR1H/LXR (nuclear receptor subfamily 1 group H) ligand, GW3965, induced Scd1 expression and lipid droplet formation to improve cell survival. Surprisingly, ULK1/ATG1 (unc-51 like kinase) played a critical role in protecting hepatic cells from SFA-induced lipotoxicity via a novel mechanism that did not involve macroautophagy/autophagy. Specific loss of Ulk1 blocked the induction of Scd1 gene transcription by GW3965, decreased lipid droplet formation, and increased apoptosis in hepatic cells exposed to palmitate. Knockdown of ULK1 increased RPS6KB1 (ribosomal protein S6 kinase, polypeptide 1) signaling that, in turn, induced NCOR1 (nuclear receptor co-repressor 1) nuclear uptake, interaction with NR1H/LXR, and recruitment to the Scd1 promoter. These events abrogated the stimulation of Scd1 gene expression by GW3965, and increased lipotoxicity in hepatic cells. In summary, we have identified a novel autophagy-independent role of ULK1 that regulates NR1H/LXR signaling, Scd1 expression, and intracellular lipid homeostasis in hepatic cells exposed to a lipotoxic environment.

Hyperhomocysteinemia causes ER stress and impaired autophagy that is reversed by Vitamin B supplementation.

Hyperhomocysteinemia (HHcy) is a well-known risk factor for stroke; however, its underlying molecular mechanism remains unclear. Using both mouse and cell culture models, we have provided evidence that impairment of autophagy has a central role in HHcy-induced cellular injury in the mouse brain. We observed accumulation of LC3B-II and p62 that was associated with increased MTOR signaling in human and mouse primary astrocyte cell cultures as well as a diet-induced mouse model of HHcy, HHcy decreased lysosomal membrane protein LAMP2, vacuolar ATPase (ATP6V0A2), and protease cathepsin D, suggesting that lysosomal dysfunction also contributed to the autophagic defect. Moreover, HHcy increased unfolded protein response. Interestingly, Vitamin B supplementation restored autophagic flux, alleviated ER stress, and reversed lysosomal dysfunction due to HHCy. Furthermore, the autophagy inducer, rapamycin was able to relieve ER stress and reverse lysosomal dysfunction caused by HHcy in vitro. Inhibition of autophagy by HHcy exacerbated cellular injury during oxygen and glucose deprivation and reperfusion (OGD/R), and oxidative stress. These effects were prevented by Vitamin B co-treatment, suggesting that it may be helpful in relieving detrimental effects of HHcy in ischemia/reperfusion or oxidative stress. Collectively, these findings show that Vitamin B therapy can reverse defects in cellular autophagy and ER stress due to HHcy; and thus may be a potential treatment to reduce ischemic damage caused by stroke in patients with HHcy.