Phospholipid transfer protein (PLTP) deficiency attenuates high fat diet induced obesity and insulin resistance.

Increased phospholipid transfer protein (PLTP) activity has been found to be associated with obesity, and metabolic syndrome in humans. However, whether or not PLTP has a direct effect on insulin sensitivity and obesity is largely unknown. Here we analyzed the effect by using PLTP knockout (PLTP-/-) mouse model. Although, PLTP-/- mice have normal body-weight-gain under chow diet, these mice were protected from high-fat-diet-induced obesity and insulin resistance, compared with wild type mice. In order to understand the mechanism, we evaluated insulin receptor and Akt activation and found that PLTP deficiency significantly enhanced phosphorylated insulin receptor and Akt levels in high-fat-diet fed mouse livers, adipose tissues, and muscles after insulin stimulation, while total Akt and insulin receptor levels were unchanged. Moreover, we found that the PLTP deficiency induced significantly more GLUT4 protein in the plasma membranes of adipocytes and muscle cells after insulin stimulation. Finally, we found that PLTP-deficient hepatocytes had less sphingomyelins and free cholesterols in the lipid rafts and plasma membranes than that of controls and this may provide a molecular basis for PLTP deficiency-mediated increase in insulin sensitivity. We have concluded that PLTP deficiency leads to an improvement in tissue and whole-body insulin sensitivity through modulating lipid levels in the plasma membrane, especially in the lipid rafts.

Synthesis and cardiovascular protective effects of quercetin 7-O-sialic acid.

Oxidative stress and inflammation play important roles in the pathogenesis of cardiovascular disease (CVD). Oxidative stress-induced desialylation is considered to be a primary step in atherogenic modification, and therefore, the attenuation of oxidative stress and/or inflammatory reactions may ameliorate CVD. In this study, quercetin 7-O-sialic acid (QA) was synthesized aiming to put together the cardiovascular protective effect of quercetin and the recently reported anti-oxidant and anti-atherosclerosis functions of N-acetylneuraminic acid. The biological efficacy of QA was evaluated in vitro in various cellular models. The results demonstrated that 50 µM QA could effectively protect human umbilical vein endothelial cells (HUVEC, EA.hy926) against hydrogen peroxide- or oxidized low-density lipoprotein-induced oxidative damage by reducing the production of reactive oxygen species. QA attenuated hydrogen peroxide-induced desialylation of HUVEC and lipoproteins. QA decreased lipopolysaccharide-induced secretion of tumour necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1), and it significantly reduced the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, TNF-α and MCP-1. Furthermore, QA effectively promoted cholesterol efflux from Raw 264.7 macrophages to apolipoprotein A-1 and high-density lipoprotein by up-regulating ATP-binding cassette transporter A1 and G1, respectively. Results indicated that the novel compound QA exhibited a better capacity than quercetin for anti-oxidation, anti-inflammation, cholesterol efflux promotion and biomolecule protection against desialylation and therefore could be a candidate compound for the prevention or treatment of CVD.

Cigarette smoke exposure impairs reverse cholesterol transport which can be minimized by treatment of hydrogen-saturated saline.

BACKGROUND: Cigarette smoke (CS) exposure impaired plasma lipid profiles by modification of apolipoproteins. Hydrogen (H2) has been proved effective on reducing oxidative stress or improving HDL functionalities in animal models or metabolic syndrome volunteers. This study was undertaken to explore the effects of CS exposure on reverse cholesterol transport (RCT) and the antioxidative effects of H2 treatment against CS exposure in mice transgenic for human cholesteryl ester transfer protein (CETP). METHODS: [(3)H]-cholesterol-laden macrophages were injected intraperitoneally into mice, and the samples of blood, bile, liver, and feces were collected for radioactivity determination to evaluate RCT. [(3)H]-cholesterol-laden macrophages were incubated with HDL isolated from different groups of mice, and the samples of cell medium supernatants were collected for evaluating the HDL functionality to elicit cholesterol efflux. RESULTS: CS exposure significantly decreased plasma HDL cholesterol level (HDL-C) by 22% and increased LDL cholesterol level (LDL-C) by 21% compared with the control group (p < 0.05, p < 0.01), while H2 treatment significantly improved the CS-impaired levels of TC, LDL-C and HDL-C by 10, 27 and 31%, respectively, compared with the CS group (p < 0.05, p < 0.01 and p < 0.05). Besides, CS exposure significantly decreased [(3)H] tracer concentrations in liver, bile and feces by 17, 35 and 48%, respectively, compared with the control group (p < 0.05 for liver and feces), while H2 treatment significantly improved them by 21, 72% and 89%, respectively, compared with the CS group (all p < 0.05). Furthermore, CS exposure significantly decreased the HDL functionality to elicit cholesterol efflux by 26% (p < 0.05), while H2 treatment also improved it by 32% (p < 0.05). We did not find any significant alterations in protein expressions of RCT involved genes. CONCLUSIONS: These findings provided direct evidence supporting the notion that CS exposure in vivo impairs plasma lipid profiles, HDL functionalities and macrophage-to-feces RCT pathway in CETP transgenic mice, all of which can be minimized by treatment of H2-saturated saline.

Molecular hydrogen stabilizes atherosclerotic plaque in low-density lipoprotein receptor-knockout mice.

Hydrogen (H(2)) attenuates the development of atherosclerosis in mouse models. We aimed to examine the effects of H(2) on atherosclerotic plaque stability. Low-density lipoprotein receptor-knockout (LDLR(-/-)) mice fed an atherogenic diet were dosed daily with H(2) and/or simvastatin. In vitro studies were carried out in an oxidized-LDL (ox-LDL)-stimulated macrophage-derived foam cell model treated with or without H(2). H(2) or simvastatin significantly enhanced plaque stability by increasing levels of collagen, as well as reducing macrophage and lipid levels in plaques. The decreased numbers of dendritic cells and increased numbers of regulatory T cells in plaques further supported the stabilizing effect of H(2) or simvastatin. Moreover, H(2) treatment decreased serum ox-LDL level and apoptosis in plaques with concomitant inhibition of endoplasmic reticulum stress (ERS) and reduction of reactive oxygen species (ROS) accumulation in the aorta. In vitro, like the ERS inhibitor 4-phenylbutyric acid, H(2) inhibited ox-LDL- or tunicamycin (an ERS inducer)-induced ERS response and cell apoptosis. In addition, like the ROS scavenger N-acetylcysteine, H(2) inhibited ox-LDL- or Cu(2+) (an ROS inducer)-induced reduction in cell viability and increase in cellular ROS. Also, H(2) increased Nrf2 (NF-E2-related factor-2, an important factor in antioxidant signaling) activation and Nrf2 small interfering RNA abolished the protective effect of H(2) on ox-LDL-induced cellular ROS production. The inhibitory effects of H(2) on the apoptosis of macrophage-derived foam cells, which take effect by suppressing the activation of the ERS pathway and by activating the Nrf2 antioxidant pathway, might lead to an improvement in atherosclerotic plaque stability.

Endoplasmic reticulum stress promotes macrophage-derived foam cell formation by up-regulating cluster of differentiation 36 (CD36) expression.

Oxidized low-density lipoprotein (ox-LDL) up-regulates CD36, a scavenger receptor responsible for macrophage uptake of ox-LDL without limitation. However, the precise underlying mechanism is not completely understood. Our previous study has demonstrated that ox-LDL induces endoplasmic reticulum (ER) stress in macrophages. The goal of this study was to explore the exact relationship between ER stress and macrophage-derived foam cell formation and whether ER stress would be involved in ox-LDL-induced CD36 up-regulation. Our results showed that ox-LDL-induced lipid accumulation in macrophages was promoted synergistically by ER stress inducer tunicamycin (TM), while attenuated by ER stress inhibitor 4-phenylbutyric acid (PBA). Ox-LDL caused CD36 up-regulation with concomitant activation of ER stress as assessed by phosphorylation of inositol-requiring kinase/endonuclease-1 (IRE-1) and protein kinase-like ER kinase (PERK), up-regulation of X-box-binding protein 1 (XBP1) and glucose-regulated protein 78 (GRP 78), and nuclear translocation of activating transcription factor 6 (ATF6). TM not only up-regulated CD36 alone but also synergized with ox-LDL to increase CD36 expression. Alleviation of ER stress with PBA and siRNA against ATF6, IRE1, and GRP78 mitigated ox-LDL-induced CD36 protein up-regulation. Moreover, administration of apoE(-/-) mice with PBA suppressed the up-regulation of CD36, phospho-IRE1, and GRP78 in macrophage-dense atherosclerotic lesions and in peritoneal macrophages. Additionally, CD36 silencing attenuated ox-LDL-induced nuclear translocation of ATF6, phosphorylation of IRE1 and up-regulation of XBP1 and GRP78. These data indicate that CD36-mediated ox-LDL uptake in macrophages triggers ER stress response, which, in turn, plays a critical role in CD36 up-regulation, enhancing the foam cell formation by uptaking more ox-LDL.

Hydrogen-rich water decreases serum LDL-cholesterol levels and improves HDL function in patients with potential metabolic syndrome.

We have found that hydrogen (dihydrogen; H2) has beneficial lipid-lowering effects in high-fat diet-fed Syrian golden hamsters. The objective of this study was to characterize the effects of H2-rich water (0.9-1.0 l/day) on the content, composition, and biological activities of serum lipoproteins on 20 patients with potential metabolic syndrome. Serum analysis showed that consumption of H2-rich water for 10 weeks resulted in decreased serum total-cholesterol (TC) and LDL-cholesterol (LDL-C) levels. Western blot analysis revealed a marked decrease of apolipoprotein (apo)B100 and apoE in serum. In addition, we found H2 significantly improved HDL functionality assessed in four independent ways, namely, i) protection against LDL oxidation, ii) inhibition of tumor necrosis factor (TNF)-α-induced monocyte adhesion to endothelial cells, iii) stimulation of cholesterol efflux from macrophage foam cells, and iv) protection of endothelial cells from TNF-α-induced apoptosis. Further, we found consumption of H2-rich water resulted in an increase in antioxidant enzyme superoxide dismutase and a decrease in thiobarbituric acid-reactive substances in whole serum and LDL. In conclusion, supplementation with H2-rich water seems to decrease serum LDL-C and apoB levels, improve dyslipidemia-injured HDL functions, and reduce oxidative stress, and it may have a beneficial role in prevention of potential metabolic syndrome.

Activating transcription factor 6 mediates oxidized LDL-induced cholesterol accumulation and apoptosis in macrophages by up-regulating CHOP expression.

AIM: This study was to explore whether activating transcription factor 6 (ATF6), an important sensor to endoplasmic reticulum (ER) stress, would mediate oxidized low-density lipoprotein (ox-LDL)- induced cholesterol accumulation and apoptosis in cultured macrophages and the underlying molecular mechanisms. METHODS: Intracellular lipid droplets and total cholesterol levels were assayed by oil red O staining and enzymatic colorimetry, respectively. Cell viability and apoptosis were determined using MTT assay and AnnexinV-FITC apoptosis detection kit, respectively. The nuclear translocation of ATF6 in cells was detected by immunofluorescence analysis. Protein and mRNA levels were examined by Western blot analysis and real time-PCR, respectively. ATF6 siRNA was transfected to RAW264.7 cells by lipofectamin. RESULTS: Exposure of cells to ox-LDL induced glucose-regulated protein 78 (GRP78). C/EBP homologous protein (CHOP), a key-signaling component of ER stress-induced apoptosis, was up-regulated in ox-LDL-treated cells. ATF6, a factor that positively regulates CHOP expression, was activated by ox-LDL in a concentration- and time- dependent manner. The role of the ATF6-mediated ER stress pathway was further confirmed through the siRNA-mediated knockdown of ATF6, which attenuated ox-LDL-induced upregulation of CHOP, cholesterol accumulation and apoptosis in macrophages. In addition, the phosphorylation of double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK), another factor that positively regulates CHOP expression, was induced in the presence of ox-LDL, and PERK-specific siRNA also inhibited the ox-LDL-induced upregulation of CHOP and apoptosis in RAW264.7 cells. CONCLUSION: These results demonstrate that ER stress-related proteins, particularly ATF6 and its downstream molecule CHOP, are involved in ox-LDL-induced cholesterol accumulation and apoptosis in macrophages.

SR-BI associates with ABCG1 and inhibits ABCG1-mediated cholesterol efflux from cells to high-density lipoprotein 3.

BACKGROUND: The single and combined effects of scavenger receptor-BI (SR-BI), ATP-binding cassette transporter (ABC) A1 and G1 on cholesterol efflux from Chinese Hamster Ovary (CHO) cells were investigated. RESULTS: When apolipoproteinA-I (apoA-I) was used as an acceptor, ABCA1 overexpression led to an increase in total cholesterol (TC) in medium which is attributable to a 2-fold increase in free cholesterol (FC) content. When high-density lipoprotein 3 (HDL3) was used as an acceptor, SR-BI overexpression not only promoted FC efflux, but also promoted the uptake of cholesteryl ester (CE) into cells, resulting in no TC varieties in medium. Overexpression of ABCG1 increased both the FC and CE levels in medium. However, when apoA-I and HDL3 were both used as acceptors, coexpression of SR-BI has no effect on ABCA1-mediated increased FC and TC accumulation in medium. Interestingly, coexpression of SR-BI with ABCG1 blocked the ABCG1-mediated cholesterol efflux to HDL3, mostly by promoting the reuptake of CE from the medium. Furthermore, co-immunoprecipitation experiments revealed that SR-BI interacted with ABCG1 in BHK cells overexpressing ABCG1 and SR-BI. CONCLUSIONS: We found SR-BI associates with ABCG1 and inhibits ABCG1-mediated cholesterol efflux from cells to HDL3.

Quercetin protects macrophages from oxidized low-density lipoprotein-induced apoptosis by inhibiting the endoplasmic reticulum stress-C/EBP homologous protein pathway.

Quercetin (QUE), a member of the bioflavonoid family, has been proposed to have antioxidative, anti-inflammatory and antihypertensive properties. This study was designed to investigate the protective effect of QUE on oxidized low-density lipoprotein (ox-LDL)-induced cytotoxicity in RAW264.7 macrophages and specifically the endoplasmic reticulum (ER) stress-C/EBP homologous protein (CHOP) pathway-mediated apoptosis. Our results showed that treatment with QUE (20, 40 and 80 µmol/L) significantly attenuated ox-LDL-induced cholesterol accumulation in macrophages and foam cell formation in a dose-dependent manner. Similar to tunicamycin (TM), a classical ER stress inducer, ox-LDL reduced cell viability and induced apoptosis in RAW264.7 macrophages. The cytotoxic effects of ox-LDL and TM were significantly inhibited by QUE treatment. Interestingly, we found that QUE also significantly suppressed the ox-LDL- and TM-induced activation of ER stress signaling events, including the phosphorylation of inositol-requiring enzyme 1 (IRE1), translocation of activating transcription factor 6 (ATF6) from the cytoplasm to the nucleus and upregulation of X-box-binding protein 1. In addition, exposure of RAW264.7 macrophages to ox-LDL or TM resulted in a significant increase in the expression of CHOP, a transcription factor regulated by IRE1 and ATF6 under conditions of ER stress, as well as a decrease in Bcl-2 transcript and protein concentrations. QUE blocked these effects in a dose-dependent manner. These data indicate that QUE can protect RAW264.7 cells from ox-LDL-induced apoptosis and that the mechanism at least partially involves its ability to inhibit the ER stress-CHOP signaling pathway.

Chitosan oligosaccharides promote reverse cholesterol transport and expression of scavenger receptor BI and CYP7A1 in mice.

Chitosan oligosaccharides (COS) are beneficial in improving plasma lipids and diminishing atherosclerotic risks. In this study, we examined the effects of COS on reverse cholesterol transport (RCT) in C57BL/6 mice. (3)H-cholesterol-laden macrophages were injected intraperitoneally into mice fed with various dosage of COS (250, 500, 1000 mg/kg mouse weight, respectively) or vehicle by gastric gavages. Plasma lipid level was determined and (3)H-cholesterol was traced in plasma, liver, bile and feces. The effects of COS on hepatic cholesterol 7 alpha-hydroxylase (CYP7A1) and scavenger receptor BI (SR-BI) expression were also investigated. COS administration led to a significant decrease in plasma total cholesterol and low-density lipoprotein (LDL) cholesterol and a significant increase in peritoneal macrophage-derived (3)H-cholesterol in liver and bile as well as in feces. Liver protein expressions of CYP7A1, SR-BI and LDL receptor (LDL-R) were improved in a dosage-dependent manner in COS-administered mice. Our findings provide the first in vivo demonstration of a positive role for COS in RCT pathway and hepatic CYP7A1 and SR-BI expression in mice. Additionally, the LDL cholesterol lowering effect might be relative to hepatic LDL-R expression stimulated by COS in mice.

Hydrogen decreases athero-susceptibility in apolipoprotein B-containing lipoproteins and aorta of apolipoprotein E knockout mice.

OBJECTIVE: It is to characterize the underlying molecular mechanisms of the anti-atherosclerotic effects of hydrogen (dihydrogen; H(2)), a novel antioxidant. In particular, to examine the effects of hydrogen on athero-susceptibility in lipoproteins and aorta of apolipoprotein E knockout (apoE-/-) mice. METHODS AND RESULTS: Plasma analysis by enzymatic method and spectrophotometric measurement showed that eight weeks intraperitoneally injection of hydrogen-saturated saline remarkably decreased plasma total and non-high-density lipoprotein (non-HDL) cholesterol, and malondialdehyde in apoE-/- mice fed either chow or high fat diet. Western blot analysis showed hydrogen treatment reduced the contents of apolipoprotein B (apoB), a major protein constituent of non-HDL in either plasma or hepatic tissues. Moreover, ELISA assay revealed that the production of tumor necrosis factor-α and interleukin-6 were significantly suppressed by hydrogen in RAW264.7 macrophages, after stimulation with the isolated non-HDL from treated or untreated mice. Immunohistochemistry of aortic valve sections revealed that hydrogen suppressed the expression of several proinflammatory factors and decreased vessel wall infiltration of macrophages. Besides, real-time PCR and Western blot analysis disclosed that hepatic scavenger receptor class B type I (SR-BI), ATP-binding cassette (ABC) transporters ABCG8, ABCB4, ABCB11, and macrophage SR-BI, were all induced by hydrogen treatment. Finally arterial wall lipid disposition displayed by oil red O staining was reduced significantly in aortic root and whole aorta en face in hydrogen administrated mice. In addition, hydrogen significantly improved HDL functionality in C57BL/6J mice assessed in two independent ways, namely (i) stimulation of cholesterol efflux from macrophage foam cells by measuring HDL-induced [(3)H]cholesterol efflux, and (ii) protection against LDL oxidation as a measure of Cu(2+)-induced TBARS formation. CONCLUSION: These results reveal that administration of hydrogen-saturated saline decreases athero-susceptibility in apoB-containing lipoprotein and aortic atherosclerosis in apoE-/- mice and improves HDL functionality in C57BL/6J mice.

Administration of hydrogen-saturated saline decreases plasma low-density lipoprotein cholesterol levels and improves high-density lipoprotein function in high-fat diet-fed hamsters.

Hydrogen (dihydrogen; H(2)) has an antiatherosclerotic effect in apolipoprotein (apo) E knockout mice. The goals of this study were to further characterize the effects of H(2) on the content, composition, and biological activities of plasma lipoproteins in golden hamsters. Plasma analysis by enzymatic method and fast protein liquid chromatography showed that 4-week intraperitoneal injection of hydrogen-saturated saline remarkably decreased plasma total cholesterol and low-density lipoprotein (LDL) cholesterol levels in high-fat diet-fed hamsters. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of apolipoproteins from ultracentrifugally isolated plasma lipoproteins revealed a marked decrease of apo B100 and apo B48 in LDL. A profound decrease of apo E level in very low-density lipoprotein was also observed. Besides, we determined the functional quality of high-density lipoprotein (HDL) particles isolated from H(2)-treated and control mice. H(2) significantly improved HDL functionality assessed in 2 independent ways, namely, (1) stimulation of cholesterol efflux from macrophage foam cells by measuring HDL-induced [(3)H]cholesterol efflux and (2) protection against LDL oxidation as a measure of Cu(2+)-induced thiobarbituric acid reactive substances formation. Administration of hydrogen-saturated saline decreases plasma LDL cholesterol and apo B levels and improves hyperlipidemia-injured HDL functions, including the capacity of enhancing cellular cholesterol efflux and playing antioxidative properties, in high-fat diet-fed hamsters.

[Differential time attachment: optimization of the adherent time to obtain mouse bone marrow-derived endothelial progenitor cells].

The different biological functions were studied in mouse bone marrow-derived endothelial progenitor cells isolated by differential time attachment to obtain the optimal adherent time in this study. Density gradient centrifugation-isolated bone marrow mononuclear cells were seeded on the fibronectin-coated dish. The 1-day cultured unattached cells were seeded on the second dish for 2 more days. Then unattached cells in the second dish were seeded on the third dish. The cells on 3 dishes were defined as 1-day adherent cells, 3-day adherent cells and 3-day unattached cells, respectively. After 20-day culture, the biological functions, such as the percentage of biomarkers, the ability of adhesion, and the ability of forming tubes in vitro were analyzed. The results showed that the percentages of positive CD34, FLK-1, and CD34/FLK-1 expressions in 1-day attached cells were significantly increased compared to those in the 3-day adherent or unattached cells (P < 0.01), which showed the strongest adhesion ability. The expression of eNOS in 1- or 3-day adherent cells was significantly higher than that in 3-day unattached cells (P < 0.01). The expression of VEGF in 3-day adherent cells was significantly higher than that in 1-day adherent cells or 3-day unattached cells (P < 0.01). These results suggest the biological functions of 1-day adherent cells are significantly stronger than that of 3-day adherent or unattached cells. VEGF expression in 3-day adherent cells is higher than that in 1-day adherent cells or 3-day unattached cells. The expression of eNOS in 1-day adherent cells or 3-day adherent cells is higher than that in 3-day unattached cells. The optimal adherent time to obtain mouse bone marrow-derived endothelial progenitor cells is 1-3 d.

[A study of correlation between the change of chromosome centromeric dots and habitual abortions].

To study the correlation between chromosome centromeric dots and habitual abortions, Cd variation, Cd loss,maximum diameter of Cd and Cd-NOR of 38 habitual abortion patients and 42 healthy persons were measured, compared and analysed with Cd-banding technique. It was found that the frequencies of Cd variation and Cd loss were obviously higher and maximum diameter of Cd was smaller in habitual abortion patients than those in healthy persons. The increase of frequencies of Cd variation and Cd loss and the decrease of maximum diameter of Cd might be the causes affecting habitual abortions.