Larsucosterol: endogenous epigenetic regulator for treating chronic and acute liver diseases.

Larsucosterol, a potent endogenous epigenetic regulator, has been reported to play a significant role in lipid metabolism, inflammatory responses, and cell survival. The administration of larsucosterol has demonstrated a reduction in lipid accumulation within hepatocytes and the attenuation of inflammatory responses induced by lipopolysaccharide (LPS) and TNFα in macrophages, alleviating LPS- and acetaminophen (ATMP)-induced multiple organ injury, and decreasing mortalities in animal models. Results from phase 1 and 2 clinical trials have shown that larsucosterol has potential as a biomedicine for the treatment of acute and chronic liver diseases. Recent evidence suggests that larsucosterol is a promising candidate for treating alcohol-associated hepatitis with positive results from a phase 2a clinical trial, and for metabolic dysfunction-associated steatohepatitis (MASH) from a phase 1b clinical trial. In this review, we present a culmination of our recent research efforts spanning two decades. We summarize the discovery, physiological and pharmacological mechanisms, and clinical applications of larsucosterol. Furthermore, we elucidate the pathophysiological pathways of metabolic dysfunction-associated steatotic liver diseases (MASLD), metabolic dysfunction-associated steatohepatitis (MASH), and acute liver injuries. A central focus of the review is the exploration of the therapeutic potential of larsucosterol in treating life-threatening conditions, including acetaminophen overdose, endotoxin shock, MASLD, MASH, hepatectomy, and alcoholic hepatitis.

Cholestenoic acid as endogenous epigenetic regulator decreases hepatocyte lipid accumulation in vitro and in vivo.

Cholestenoic acid (CA) has been reported as an important biomarker of many severe diseases, but its physiological and pathological roles remain unclear. This study aimed to investigate the potential role of CA in hepatic lipid homeostasis. Enzyme kinetic studies revealed that CA specifically activates DNA methyltransferases 1 (DNMT1) at low concentration with EC50 = 1.99 × 10-6 M and inhibits the activity at higher concentration with IC50 = 9.13 × 10-6 M, and specifically inhibits DNMT3a, and DNMT3b activities with IC50= 8.41 × 10-6 M and IC50= 4.89 × 10-6 M, respectively. In a human hepatocyte in vitro model of high glucose (HG)-induced lipid accumulation, CA significantly increased demethylation of 5mCpG in the promoter regions of over 7,000 genes, particularly those involved in master signaling pathways such as calcium-AMPK and 0.0027 at 6 h. RNA sequencing analysis showed that the downregulated genes are affected by CA encoding key enzymes, such as PCSK9, MVK, and HMGCR, which are involved in cholesterol metabolism and steroid biosynthesis pathways. In addition, untargeted lipidomic analysis showed that CA significantly reduced neutral lipid levels by 60% in the cells cultured in high-glucose media. Administration of CA in mouse metabolic dysfunction-associated steatotic liver disease (MASLD) models significantly decreases lipid accumulation, suppresses the gene expression involved in lipid biosynthesis in liver tissues, and alleviates liver function. This study shows that CA as an endogenous epigenetic regulator decreases lipid accumulation via epigenetic regulation. The results indicate that CA can be considered a potential therapeutic target for the treatment of metabolic disorders.NEW & NOTEWORTHY To our knowledge, this study is the first to identify the mitochondrial monohydroxy bile acid cholestenoic acid (CA) as an endogenous epigenetic regulator that regulates lipid metabolism through epigenome modification in human hepatocytes. The methods used in this study are all big data analysis, and the results of each part show the global regulation of CA on human hepatocytes rather than narrow point effects.

25-Hydroxycholesterol 3-sulfate is an endogenous ligand of DNA methyltransferases in hepatocytes.

The oxysterol sulfate, 25-hydroxycholesterol 3-sulfate (25HC3S), has been shown to play an important role in lipid metabolism, inflammatory response, and cell survival. However, the mechanism(s) of its function in global regulation is unknown. The current study investigates the molecular mechanism by which 25HC3S functions as an endogenous epigenetic regulator. To study the effects of oxysterols/sterol sulfates on epigenetic modulators, 12 recombinant epigenetic enzymes were used to determine whether 25HC3S acts as their endogenous ligand. The enzyme kinetic study demonstrated that 25HC3S specifically inhibited DNA methyltransferases (DNMTs), DNMT1, DNMT3a, and DNMT3b with IC50 of 4.04, 3.03, and 9.05 × 10-6 M, respectively. In human hepatocytes, high glucose induces lipid accumulation by increasing promoter CpG methylation of key genes involved in development of nonalcoholic fatty liver diseases. Using this model, whole genome bisulfate sequencing analysis demonstrated that 25HC3S converts the 5mCpG to CpG in the promoter regions of 1,074 genes. In addition, we observed increased expression of the demethylated genes, which are involved in the master signaling pathways, including MAPK-ERK, calcium-AMP-activated protein kinase, and type II diabetes mellitus pathways. mRNA array analysis showed that the upregulated genes encoded for key elements of cell survival; conversely, downregulated genes encoded for key enzymes that decrease lipid biosynthesis. Taken together, our results indicate that the expression of these key elements and enzymes are regulated by the demethylated signaling pathways. We summarized that 25HC3S DNA demethylation of 5mCpG in promoter regions is a potent regulatory mechanism.

Suppression of IGF binding protein-3 by palmitate promotes hepatic inflammatory responses.

IGF-binding protein-3 (IGFBP-3) is a liver-derived, anti-inflammatory molecule that is decreased in obesity, a key risk factor for nonalcoholic fatty liver disease (NAFLD). It was not known whether IGFBP-3 levels were altered in NAFLD, whether such alterations could be the result of lipotoxicity, and whether altered IGFBP-3 could affect pathways that are involved in hepatic and systemic inflammation. Serum IGFBP-3 was decreased in patients with NAFLD, whereas liver and circulating IL-8 levels were increased. Palmitate inhibited IGFBP-3 secretion by THP-1 macrophages and enhanced IL-8 expression. Exposure of palmitate-treated THP-1 macrophages to IGFBP-3-deficient conditioned medium led to a 20-fold increase in palmitate-induced IL-8 expression by hepatocytes. Conversely, overexpression of IGFBP-3 suppressed JNK and NF-κB activation and blocked palmitate-induced IL-8 expression in hepatocytes. Silencing IGFBP-3 in Huh7 cells enhanced JNK and NF-κB activity and increased palmitate-induced IL-8 secretion. These data indicate that IGFBP-3 serves as an anti-inflammatory brake in hepatocytes against JNK and NF-κB and limits their activation and downstream production of proinflammatory cytokines. Under lipotoxic conditions, palmitate inhibits hepatic macrophage secretion of IGFBP-3, thereby releasing the brake and enhancing palmitate-induced IL-8 synthesis and secretion.-Min, H.-K., Maruyama, H., Jang, B. K., Shimada, M., Mirshahi, F., Ren, S., Oh, Y., Puri, P., Sanyal, A. J. Suppression of IGF binding protein-3 by palmitate promotes hepatic inflammatory responses.

Activation of the GP130-STAT3 axis and its potential implications in nonalcoholic fatty liver disease.

The status of the GP130-STAT3 signaling pathway in humans with nonalcoholic fatty liver disease (NAFLD) and its relevance to disease pathogenesis are unknown. The expression of the gp130-STAT3 axis and gp130 cytokine receptors were studied in subjects with varying phenotypes of NAFLD including nonalcoholic steatohepatitis (NASH) and compared with lean and weight-matched controls without NAFLD. Gp130 and its downstream signaling element (Tyk2 and STAT3) expression were inhibited in obese controls whereas they were increased in NAFLD. IL-6 levels were increased in NASH and correlated with gp130 expression (P < 0.01). Palmitate inhibited gp130-STAT3 expression and signaling. IL-6 and palmitate inhibited hepatic insulin signaling via STAT3-dependent and independent mechanisms, respectively. STAT3 overexpression reversed palmitate-induced lipotoxicity by increasing autophagy (ATG7) and decreasing endoplasmic reticulum stress. These data demonstrate that the STAT3 pathway is activated in NAFLD and can worsen insulin resistance while protecting against other lipotoxic mechanisms of disease pathogenesis.

Semi-interpenetrating network (sIPN) co-electrospun gelatin/insulin fiber formulation for transbuccal insulin delivery.

PURPOSE: This work was aimed at developing a semi-interpenetrating network (sIPN) co-electrospun gelatin/insulin fiber scaffold (GIF) formulation for transbuccal insulin delivery. METHODS: Gelatin was electrospun into fibers and converted into an sIPN following eosin Y-initiated polymerization of polyethylene glycol diacrylate (PEG-DA). The cytocompatibility, degradation rate and mechanical properties were examined in the resulting sIPNs with various ratios of PEG-DA to eosin Y to find a suitable formulation for transbuccal drug delivery. Insulin was co-electrospun with gelatin into fibers and converted into an sIPN-GIF using this suitable formulation. The in vitro release kinetics of insulin was evaluated using ELISA. The bioactivity of released insulin was analyzed in 3T3-L1 preadipocytes using Western blotting and Oil Red O staining. The transbuccal permeability of released insulin was determined using an in vitro porcine oral mucosa model. RESULTS: The sIPN-GF formulation of GF cross-linked by PEG-DA (1% w/v) with eosin Y (5% v/v) possessed no cytotoxic effect, a moderate degradation rate with degradation half-life of 49 min, and a significant enhancement in mechanical properties. This formulation was used to fabricate sIPN-GIF. Insulin release was extended up to 4 h by sIPN-GIF. The released insulin successfully triggered intracellular AKT phosphorylation and induced adipocyte differentiation in 3T3-L1 preadipocytes. The transbuccal permeability of released insulin was determined on the order of 10(-7) cm/s. CONCLUSIONS: Insulin can be fabricated into an sIPN-GIF formulation following co-electrospinning and cross-linking without losing bioactivity. It proved the potential of this new formulation for transbuccal insulin delivery.

Sulfation of 25-hydroxycholesterol regulates lipid metabolism, inflammatory responses, and cell proliferation.

Intracellular lipid accumulation, inflammatory responses, and subsequent apoptosis are the major pathogenic events of metabolic disorders, including atherosclerosis and nonalcoholic fatty liver diseases. Recently, a novel regulatory oxysterol, 5-cholesten-3b, 25-diol 3-sulfate (25HC3S), has been identified, and hydroxysterol sulfotransferase 2B1b (SULT2B1b) has been elucidated as the key enzyme for its biosynthesis from 25-hydroxycholesterol (25HC) via oxysterol sulfation. The product 25HC3S and the substrate 25HC have been shown to coordinately regulate lipid metabolism, inflammatory responses, and cell proliferation in vitro and in vivo. 25HC3S decreases levels of the nuclear liver oxysterol receptor (LXR) and sterol regulatory element-binding proteins (SREBPs), inhibits SREBP processing, subsequently downregulates key enzymes in lipid biosynthesis, decreases intracellular lipid levels in hepatocytes and THP-1-derived macrophages, prevents apoptosis, and promotes cell proliferation in liver tissues. Furthermore, 25HC3S increases nuclear PPARγ and cytosolic IκBα and decreases nuclear NF-κB levels and proinflammatory cytokine expression and secretion when cells are challenged with LPS and TNFα. In contrast to 25HC3S, 25HC, a known LXR ligand, increases nuclear LXR and decreases nuclear PPARs and cytosol IκBα levels. In this review, we summarize our recent findings, including the discovery of the regulatory oxysterol sulfate, its biosynthetic pathway, and its functional mechanism. We also propose that oxysterol sulfation functions as a regulatory signaling pathway.

5-cholesten-3ß,25-diol 3-sulfate decreases lipid accumulation in diet-induced nonalcoholic fatty liver disease mouse model.

Sterol regulatory element-binding protein-1c (SREBP-1c) increases lipogenesis at the transcriptional level, and its expression is upregulated by liver X receptor α (LXRα). The LXRα/SREBP-1c signaling may play a crucial role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). We previously reported that a cholesterol metabolite, 5-cholesten-3ß,25-diol 3-sulfate (25HC3S), inhibits the LXRα signaling and reduces lipogenesis by decreasing SREBP-1c expression in primary hepatocytes. The present study aims to investigate the effects of 25HC3S on lipid homeostasis in diet-induced NAFLD mouse models. NAFLD was induced by feeding a high-fat diet (HFD) in C57BL/6J mice. The effects of 25HC3S on lipid homeostasis, inflammatory responses, and insulin sensitivity were evaluated after acute treatments or long-term treatments. Acute treatments with 25HC3S decreased serum lipid levels, and long-term treatments decreased hepatic lipid accumulation in the NAFLD mice. Gene expression analysis showed that 25HC3S significantly suppressed the SREBP-1c signaling pathway that was associated with the suppression of the key enzymes involved in lipogenesis: fatty acid synthase, acetyl-CoA carboxylase 1, and glycerol-3-phosphate acyltransferase. In addition, 25HC3S significantly reduced HFD-induced hepatic inflammation as evidenced by decreasing tumor necrosis factor and interleukin 1 α/ß mRNA levels. A glucose tolerance test and insulin tolerance test showed that 25HC3S administration improved HFD-induced insulin resistance. The present results indicate that 25HC3S as a potent endogenous regulator decreases lipogenesis, and oxysterol sulfation can be a key protective regulatory pathway against lipid accumulation and lipid-induced inflammation in vivo.

Antibacterial Components and Modes of the Methanol-Phase Extract from Commelina communis Linn.

Infectious diseases caused by pathogenic bacteria severely threaten human health. Traditional Chinese herbs are potential sources of new or alternative medicine. In this study, we analyzed for the first time antibacterial substances in the methanol-phase extract from a traditional Chinese herb-Commelina communis Linn-which showed an inhibition rate of 58.33% against 24 species of common pathogenic bacteria. The extract was further purified using preparative high-performance liquid chromatography (Prep-HPLC), which generated four single fragments (Fragments 1 to 4). The results revealed that Fragment 1 significantly increased bacterial cell surface hydrophobicity and membrane permeability and decreased membrane fluidity, showing disruptive effects on cell integrity of Gram-positive and Gram-negative bacteria, such as Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus, and Salmonella enterica subsp., compared to the control groups (p < 0.05). In sum, 65 compounds with known functions in Fragment 1 were identified using liquid chromatography and mass spectrometry (LC-MS), of which quercetin-3-o-glucuronide was predominant (19.35%). Comparative transcriptomic analysis revealed multiple altered metabolic pathways mediated by Fragment 1, such as inhibited ABC transporters, ribosome, citrate cycle and oxidative phosphorylation, and upregulated nitrogen metabolism and purine metabolism, thereby resulting in the repressed bacterial growth and even death (p < 0.05). Overall, the results of this study demonstrate that Fragment 1 from C. communis Linn is a promising candidate against common pathogenic bacteria.

ER stress increases StarD5 expression by stabilizing its mRNA and leads to relocalization of its protein from the nucleus to the membranes.

StarD5 belongs to the StarD4 subfamily of steroidogenic acute regulatory lipid transfer (START) domain proteins. In macrophages, StarD5 is found in the cytosol and maintains a loose association with the Golgi. Like StarD1 and StarD4, StarD5 is known to bind cholesterol. However, its function and regulation remain poorly defined. Recently, it has been shown that its mRNA expression is induced in response to different inducers of endoplasmic reticulum (ER) stress. However, the molecular mechanism(s) involved in the induction of StarD5 expression during ER stress is not known. Here we show that in 3T3-L1 cells, the ER stressor thapsigargin increases intracellular free cholesterol due to an increase in HMG-CoA reductase expression. Activation of StarD5 expression is mediated by the transcriptional ER stress factor XBP-1. Additionally, the induction of ER stress stabilizes the StarD5 mRNA. Furthermore, StarD5 protein is mainly localized in the nucleus, and upon ER stress, it redistributes away from the nucleus, localizing prominently to the cytosol and membranes. These results reveal the increase in StarD5 expression and protein redistribution during the cell protective phase of the ER stress, suggesting a role for StarD5 in cholesterol metabolism during the ER stress response.

Subcellular localization and regulation of StarD4 protein in macrophages and fibroblasts.

StarD4 is a member of the StarD4 subfamily of START domain proteins with a characteristic lipid binding pocket specific for cholesterol. The objective of this study was to define StarD4 subcellular localization, regulation, and function. Immunobloting showed that StarD4 is highly expressed in the mouse fibroblast cell line 3T3-L1, in human THP-1 macrophages, Kupffer cells (liver macrophages), and hepatocytes. In 3T3-L1 cells and THP-1 macrophages, StarD4 protein appeared localized to the cytoplasm and the endoplasmic reticulum (ER). More specifically, in THP-1 macrophages StarD4 co-localized to areas of the ER enriched in Acyl-CoA:cholesterol acyltransferase-1 (ACAT-1), and was closely associated with budding lipid droplets. The addition of purified StarD4 recombinant protein to an in vitro assay increased ACAT activity 2-fold, indicating that StarD4 serves as a rate-limiting step in cholesteryl ester formation by delivering cholesterol to ACAT-1-enriched ER. In addition, StarD4 protein was found to be highly regulated and to redistribute in response to sterol levels. In summary, these observations, together with our previous findings demonstrating the ability of increased StarD4 expression to increase bile acid synthesis and cholesteryl ester formation, provide strong evidence for StarD4 as a highly regulated, non-vesicular, directional, intracellular transporter of cholesterol which plays a key role in the maintenance of intracellular cholesterol homeostasis.

25-Hydroxycholesterol-3-sulfate attenuates inflammatory response via PPARγ signaling in human THP-1 macrophages.

The nuclear receptor peroxisome proliferator-activated receptors (PPARs) are important in regulating lipid metabolism and inflammatory responses in macrophages. Activation of PPARγ represses key inflammatory response gene expressions. Recently, we identified a new cholesterol metabolite, 25-hydroxycholesterol-3-sulfate (25HC3S), as a potent regulatory molecule of lipid metabolism. In this paper, we report the effect of 25HC3S and its precursor 25-hydroxycholesterol (25HC) on PPARγ activity and on inflammatory responses. Addition of 25HC3S to human macrophages markedly increased nuclear PPARγ and cytosol IκB and decreased nuclear NF-κB protein levels. PPARγ response element reporter gene assays showed that 25HC3S significantly increased luciferase activities. PPARγ competitor assay showed that the K(i) for 25HC3S was ∼1 µM, similar to those of other known natural ligands. NF-κB-dependent promoter reporter gene assays showed that 25HC3S suppressed TNFα-induced luciferase activities only when cotransfected with pcDNAI-PPARγ plasmid. In addition, 25HC3S decreased LPS-induced expression and release of IL-1ß. In the PPARγ-specific siRNA transfected macrophages or in the presence of PPARγ-specific antagonist, 25HC3S failed to increase IκB and to suppress TNFα and IL-1ß expression. In contrast to 25HC3S, its precursor 25HC, a known liver X receptor ligand, decreased nuclear PPARγ and cytosol IκB and increased nuclear NF-κB protein levels. We conclude that 25HC3S acts in macrophages as a PPARγ ligand and suppresses inflammatory responses via the PPARγ/IκB/NF-κB signaling pathway.

Cytosolic sulfotransferase 2B1b promotes hepatocyte proliferation gene expression in vivo and in vitro.

Cytosolic sulfotransferase 2B1b (SULT2B1b) catalyzes the sulfation of 3ß-hydroxysteroids and functions as a selective cholesterol and oxysterol sulfotransferase. Activation of liver X receptors (LXRs) by oxysterols has been known to be an antiproliferative factor. Overexpression of SULT2B1b impairs LXR's response to oxysterols, by which it regulates lipid metabolism. The aim of this study was to investigate in vivo and in vitro effects of SULT2B1b on liver proliferation and the underlying mechanisms. Primary rat hepatocytes and C57BL/6 mice were infected with adenovirus encoding SULT2B1b. Liver proliferation was determined by measuring the proliferating cell nuclear antigen (PCNA) immunostaining labeling index. The correlation between SULT2B1b and PCNA expression in mouse liver tissues was determined by double immunofluorescence. Gene expressions were evaluated by quantitative real-time PCR and Western blot analysis. SULT2B1b overexpression in mouse liver tissues increased PCNA-positive cells in a dose- and time-dependent manner. The increased expression of PCNA in mouse liver tissues was only observed in the SULT2B1b transgenic cells. Small interference RNA SULT2B1b significantly inhibited cell cycle regulatory gene expressions in primary rat hepatocytes. LXR activation by T0901317 effectively suppressed SULT2B1b-induced gene expression in vivo and in vitro. SULT2B1b may promote hepatocyte proliferation by inactivating oxysterol/LXR signaling.

Rapid and Visualized Detection of Virulence-Related Genes of Vibrio cholerae in Water and Aquatic Products by Loop-Mediated Isothermal Amplification.

ABSTRACT: Vibrio cholerae can cause pandemic cholera in humans. The bacterium resides in aquatic environments worldwide. Continuous testing of V. cholerae contamination in water and aquatic products is imperative for food safety control and human health. In this study, a rapid and visualized method was developed for the first time based on loop-mediated isothermal amplification (LAMP) for detection of the important virulence-related genes ace, zot, cri, and nanH for toxins and the infectious process of V. cholerae. Three pairs of molecular probes targeting each of these genes were designed and synthesized. The one-step LAMP reaction was conducted at 65°C for 40 min. Positive results were inspected by the production of a light green color under visible light or green fluorescence under UV light (302 nm). Limit of detection of the LAMP method ranged from 1.85 to 2.06 pg per reaction of genomic DNA or 2.50 × 100 to 4.00 × 102 CFU per reaction for target genes of cell culture of V. cholerae, which was more sensitive than standard PCR. Inclusivity and exclusivity of the LAMP method were 100% for all target genes. The method showed similar high efficiency to a certain extent in rapid testing of spiked or collected specimens of water and aquatic products. Target genes were detected by absence from all water samples from various sources. However, high occurrences of the nanH gene were observed in intestinal samples derived from four species of fish and one species of shellfish, indicating a risk of potentially toxic V. cholerae in commonly consumed aquatic products. The results in this study provide a potential tool for rapid and visualized detection of V. cholerae in water and aquatic products.

25-Hydroxycholesterol 3-Sulfate Recovers Acetaminophen Induced Acute Liver Injury via Stabilizing Mitochondria in Mouse Models.

Acetaminophen (APAP) overdose is one of the most frequent causes of acute liver failure (ALF). N-acetylcysteine (NAC) is currently being used as part of the standard care in the clinic but its usage has been limited in severe cases, in which liver transplantation becomes the only treatment option. Therefore, there still is a need for a specific and effective therapy for APAP induced ALF. In the current study, we have demonstrated that treatment with 25-Hydroxycholesterol 3-Sulfate (25HC3S) not only significantly reduced mortality but also decreased the plasma levels of liver injury markers, including LDH, AST, and ALT, in APAP overdosed mouse models. 25HC3S also decreased the expression of those genes involved in cell apoptosis, stabilized mitochondrial polarization, and significantly decreased the levels of oxidants, malondialdehyde (MDA), and reactive oxygen species (ROS). Whole genome bisulfite sequencing analysis showed that 25HC3S increased demethylation of 5mCpG in key promoter regions and thereby increased the expression of those genes involved in MAPK-ERK and PI3K-Akt signaling pathways. We concluded that 25HC3S may alleviate APAP induced liver injury via up-regulating the master signaling pathways and maintaining mitochondrial membrane polarization. The results suggest that 25HC3S treatment facilitates the recovery and significantly decreases the mortality of APAP induced acute liver injury and has a synergistic effect with NAC in propylene glycol (PG) for the injury.

Cholesterol Metabolites 25-Hydroxycholesterol and 25-Hydroxycholesterol 3-Sulfate Are Potent Paired Regulators: From Discovery to Clinical Usage.

Oxysterols have long been believed to be ligands of nuclear receptors such as liver × receptor (LXR), and they play an important role in lipid homeostasis and in the immune system, where they are involved in both transcriptional and posttranscriptional mechanisms. However, they are increasingly associated with a wide variety of other, sometimes surprising, cell functions. Oxysterols have also been implicated in several diseases such as metabolic syndrome. Oxysterols can be sulfated, and the sulfated oxysterols act in different directions: they decrease lipid biosynthesis, suppress inflammatory responses, and promote cell survival. Our recent reports have shown that oxysterol and oxysterol sulfates are paired epigenetic regulators, agonists, and antagonists of DNA methyltransferases, indicating that their function of global regulation is through epigenetic modification. In this review, we explore our latest research of 25-hydroxycholesterol and 25-hydroxycholesterol 3-sulfate in a novel regulatory mechanism and evaluate the current evidence for these roles.

High Glucose Induces Lipid Accumulation via 25-Hydroxycholesterol DNA-CpG Methylation.

This work investigates the relationship between high-glucose (HG) culture, CpG methylation of genes involved in cell signaling pathways, and the regulation of carbohydrate and lipid metabolism in hepatocytes. The results indicate that HG leads to an increase in nuclear 25-hydroxycholesterol (25HC), which specifically activates DNA methyltransferase-1 (DNMT1), and regulates gene expression involved in intracellular lipid metabolism. The results show significant increases in 5mCpG levels in at least 2,225 genes involved in 57 signaling pathways. The hypermethylated genes directly involved in carbohydrate and lipid metabolism are of PI3K, cAMP, insulin, insulin secretion, diabetic, and NAFLD signaling pathways. The studies indicate a close relationship between the increase in nuclear 25HC levels and activation of DNMT1, which may regulate lipid metabolism via DNA CpG methylation. Our results indicate an epigenetic regulation of hepatic cell metabolism that has relevance to some common diseases such as non-alcoholic fatty liver disease and metabolic syndrome.

Bindings of PPARγ ligand-binding domain with 5-cholesten-3ß, 25-diol, 3-sulfate: accurate prediction by molecular simulation.

Peroxisome proliferator-activated receptor gamma (PPARγ) has recently been identified as an attractive target for atherosclerosis intervention. Given potential relevance of 5-cholesten-3ß, 25-diol, 3-sulphate (CHOS) and PPARγ, an integrated docking method was used to study their interaction mechanisms, with the full considerations to distinct CHOS conformations and dynamic ensembles of PPARγ ligand-binding domain (PPARγ-LBD). The results revealed that this novel platform is satisfactory to the accurate determination of binding profiles, and the binding pattern of CHOS is rather similar as those of current PPARγ full/partial agonists. CHOS contributes to the stabilization of the AF2 and ß-sheet surfaces of PPARγ-LBD and promotes the configuration adjustment of Ω loop, in order to inhibit the Cdk5-mediated PPARγ phosphorylation. Nonetheless, there are clear differences in term of occupation of full or partial agonist-like binding models. The energetic and geometric analyses further revealed that CHOS may be fond of partial agonist-like binding, and its sulfonic group and carbon skeleton are helpful for the binding process. We hope that the results will aid our understanding of recognitions involving CHOS with PPARγ-LBD and warrant the further aspects to pharmacological experiments.Communicated by Ramaswamy H. Sarma.

Prophage-Related Gene VpaChn25_0724 Contributes to Cell Membrane Integrity and Growth of Vibrio parahaemolyticus CHN25.

Vibrio parahaemolyticus is a leading seafood-borne pathogen that can cause acute gastroenteritis and even death in humans. In aquatic ecosystems, phages constantly transform bacterial communities by horizontal gene transfer. Nevertheless, biological functions of prophage-related genes in V. parahaemolyticus remain to be fully unveiled. Herein, for the first time, we studied one such gene VpaChn25_0724 encoding an unknown hypothetical protein in V. parahaemolyticus CHN25. This gene deletion mutant ΔVpaChn25_0724 was constructed by homologous recombination, and its complementary mutant ΔVpaChn25_0724-com was also obtained. The ΔVpaChn25_0724 mutant exhibited a sever defect in growth and swimming motility particularly at lower temperatures. Biofilm formation and cytotoxicity capacity of V. parahaemolyticus CHN25 was significantly lowered in the absence of VpaChn25_0724. Comparative secretomic analysis revealed an increase in extracellular proteins of ΔVpaChn25_0724, which likely resulted from its damaged cell membrane. Comparison of transcriptome data showed twelve significantly altered metabolic pathways in ΔVpaChn25_0724, suggesting inactive transport and utilization of carbon sources, repressed energy production and membrane biogenesis in ΔVpaChn25_0724. Comparative transcriptomic analysis also revealed several remarkably down-regulated key regulators in bacterial gene regulatory networks linked to the observed phenotypic variations. Overall, the results here facilitate better understanding of biological significance of prophage-related genes remaining in V. parahaemolyticus.

Bile acids as regulatory molecules.

In the past, bile acids were considered to be just detergent molecules derived from cholesterol in the liver. They were known to be important for the solubilization of cholesterol in the gallbladder and for stimulating the absorption of cholesterol, fat-soluble vitamins, and lipids from the intestines. However, during the last two decades, it has been discovered that bile acids are regulatory molecules. Bile acids have been discovered to activate specific nuclear receptors (farnesoid X receptor, preganane X receptor, and vitamin D receptor), G protein coupled receptor TGR5 (TGR5), and cell signaling pathways (c-jun N-terminal kinase 1/2, AKT, and ERK 1/2) in cells in the liver and gastrointestinal tract. Activation of nuclear receptors and cell signaling pathways alter the expression of numerous genes encoding enzyme/proteins involved in the regulation of bile acid, glucose, fatty acid, lipoprotein synthesis, metabolism, transport, and energy metabolism. They also play a role in the regulation of serum triglyceride levels in humans and rodents. Bile acids appear to function as nutrient signaling molecules primarily during the feed/fast cycle as there is a flux of these molecules returning from the intestines to the liver following a meal. In this review, we will summarize the current knowledge of how bile acids regulate hepatic lipid and glucose metabolism through the activation of specific nuclear receptors and cell signaling pathways.