Epigenetic Upregulation of H19 and AMPK Inhibition Concurrently Contribute to S-Adenosylhomocysteine Hydrolase Deficiency-Promoted Atherosclerotic Calcification.

BACKGROUND: S-adenosylhomocysteine (SAH) is a risk factor of cardiovascular disease; inhibition of SAH hydrolase (SAHH) results in SAH accumulation and induces endothelial dysfunction and atherosclerosis. However, the effect and mechanism of SAHH in atherosclerotic calcification is still unclear. We aimed to explore the role and mechanism of SAHH in atherosclerotic calcification. METHODS: The relationship between SAHH and atherosclerotic calcification was investigated in patients with coronary atherosclerotic calcification. Different in vivo genetic models were used to examine the effect of SAHH deficiency on atherosclerotic calcification. Human aortic and murine vascular smooth muscle cells (VSMCs) were cultured to explore the underlying mechanism of SAHH on osteoblastic differentiation of VSMCs. RESULTS: The expression and activity of SAHH were decreased in calcified human coronary arteries and inversely associated with coronary atherosclerotic calcification severity, whereas plasma SAH and total homocysteine levels were positively associated with coronary atherosclerotic calcification severity. Heterozygote knockout of SAHH promoted atherosclerotic calcification. Specifically, VSMC-deficient but not endothelial cell-deficient or macrophage-deficient SAHH promoted atherosclerotic calcification. Mechanistically, SAHH deficiency accumulated SAH levels and induced H19-mediated Runx2 (runt-related transcription factor 2)-dependent osteoblastic differentiation of VSMCs by inhibiting DNMT3b (DNA methyltransferase 3b) and leading to hypomethylation of the H19 promoter. On the contrary, SAHH deficiency resulted in lower intracellular levels of adenosine and reduced AMPK (AMP-activated protein kinase) activation. Adenosine supplementation activated AMPK and abolished SAHH deficiency-induced expression of H19 and Runx2 and osteoblastic differentiation of VSMCs. Finally, AMPK activation by adenosine inhibited H19 expression by inducing Sirt1 (sirtuin-1)-mediated histone H3 hypoacetylation and DNMT3b-mediated hypermethylation of the H19 promoter in SAHH deficiency VSMCs. CONCLUSIONS: We have confirmed a novel correlation between SAHH deficiency and atherosclerotic calcification and clarified a new mechanism that epigenetic upregulation of H19 and AMPK inhibition concurrently contribute to SAHH deficiency-promoted Runx2-dependent atherosclerotic calcification.

Gut Microbially Produced Indole-3-Propionic Acid Inhibits Atherosclerosis by Promoting Reverse Cholesterol Transport and Its Deficiency Is Causally Related to Atherosclerotic Cardiovascular Disease.

BACKGROUND: Accumulating evidence has shown that disorders in the gut microbiota and derived metabolites affect the development of atherosclerotic cardiovascular disease (ASCVD). However, which and how specific gut microbial metabolites contribute to the progression of atherosclerosis and the clinical relevance of their alterations remain unclear. METHODS: We performed integrated microbiome-metabolome analysis of 30 patients with coronary artery disease (CAD) and 30 age- and sex-matched healthy controls to identify CAD-associated microbial metabolites, which were then assessed in an independent population of patients with ASCVD and controls (n=256). We further investigate the effect of CAD-associated microbial metabolites on atherosclerosis and the mechanisms of the action. RESULTS: Indole-3-propionic acid (IPA), a solely microbially derived tryptophan metabolite, was the most downregulated metabolite in patients with CAD. Circulating IPA was then shown in an independent population to be associated with risk of prevalent ASCVD and correlated with the ASCVD severity. Dietary IPA supplementation alleviates atherosclerotic plaque development in ApoE-/- mice. In murine- and human-derived macrophages, administration of IPA promoted cholesterol efflux from macrophages to ApoA-I through an undescribed miR-142-5p/ABCA1 (ATP-binding cassette transporter A1) signaling pathway. Further in vivo studies demonstrated that IPA facilitates macrophage reverse cholesterol transport, correlating with the regulation of miR-142-5p/ABCA1 pathway, whereas reduced IPA production contributed to the aberrant overexpression of miR-142-5p in macrophages and accelerated the progression of atherosclerosis. Moreover, the miR-142-5p/ABCA1/reverse cholesterol transport axis in macrophages were dysregulated in patients with CAD, and correlated with the changes in circulating IPA levels. CONCLUSIONS: Our study identify a previously unknown link between specific gut microbiota-derived tryptophan metabolite and ASCVD. The microbial metabolite IPA/miR-142-5p/ABCA1 pathway may represent a promising therapeutic target for ASCVD.

Silymarin decreases liver stiffness associated with gut microbiota in patients with metabolic dysfunction-associated steatotic liver disease: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Despite centuries of traditional use of silymarin for hepatoprotection, current randomized controlled trial (RCT) studies on the effectiveness of silymarin in managing metabolic dysfunction-associated steatotic liver disease (MASLD) are limited and inconclusive, particularly when it is administered alone. The low bioavailability of silymarin highlights the possible influence of gut microbiota on the effectiveness of silymarin; however, no human studies have investigated this aspect. OBJECTIVE: To determine the potential efficacy of silymarin in improving MASLD indicators and to investigate the underlying mechanisms related to gut microbiota. METHOD: In this 24-week randomized, double-blind, placebo-controlled trial, 83 patients with MASLD were randomized to either placebo (n = 41) or silymarin (103.2 mg/d, n = 42). At 0, 12, and 24 weeks, liver stiffness and hepatic steatosis were assessed using FibroScan, and blood samples were gathered for biochemical detection, while faecal samples were collected at 0 and 24 weeks for 16S rRNA sequencing. RESULTS: Silymarin supplementation significantly reduced liver stiffness (LSM, -0.21 ± 0.17 vs. 0.41 ± 0.17, P = 0.015) and serum levels of γ-glutamyl transpeptidase (GGT, -8.21 ± 3.01 vs. 1.23 ± 3.16, P = 0.042) and ApoB (-0.02 ± 0.03 vs. 0.07 ± 0.03, P = 0.023) but had no significant effect on the controlled attenuation parameter (CAP), other biochemical indicators (aminotransferases, total bilirubin, glucose and lipid parameters, hsCRP, SOD, and UA), physical measurements (DBP, SBP, BMI, WHR, BF%, and BMR), or APRI and FIB-4 indices. Gut microbiota analysis revealed increased species diversity and enrichment of Oscillospiraceae in the silymarin group. CONCLUSION: These findings suggest that silymarin supplementation could improve liver stiffness in MASLD patients, possibly by modulating the gut microbiota. TRIAL REGISTRATION: The trial was registered at the Chinese Clinical Trial Registry (ChiCTR2200059043).

Hepatic steatosis aggravates atherosclerosis via small extracellular vesicle-mediated inhibition of cellular cholesterol efflux.

BACKGROUND & AIMS: While it is recognized that non-alcoholic fatty liver disease (NAFLD) is associated with cardiovascular disease (CVD), how NAFLD affects the development and progression of CVD remains unclear and debatable. Hence, we aimed to determine the role of steatotic hepatocyte-derived small extracellular vesicles (sEVs) in foam cell formation and atherosclerosis progression. METHODS: sEVs from steatotic hepatocytes were isolated and characterized. MicroRNA (miRNA) deep sequencing was utilized to identify functional miRNA in sEVs. Lastly, we conducted a cross-sectional study on patients with NAFLD to validate these findings. RESULTS: Treatment of sEVs from steatotic hepatocytes promoted macrophage-derived foam cell formation and atherosclerosis progression via inhibition of ABCA1-mediated cholesterol efflux. Macrophage-specific deletion of Abca1 in ApoE-/- mice abolished the role of steatotic hepatocyte-derived sEVs in atherosclerosis progression. In addition, hepatocyte-specific deletion of Rab27a, which is the key GTPase regulating sEV release, significantly ameliorated high-fat, high-cholesterol diet-induced atherosclerosis progression in ApoE-/- mice. The miRNA deep sequencing results showed that miR-30a-3p was enriched in sEVs from steatotic hepatocytes. miR-30a-3p directly targeted the 3' untranslated region of ABCA1 to inhibit ABCA1 expression and cholesterol efflux. Treatment with antagomiR-30a-3p significantly attenuated atherosclerosis progression in high-fat, high-cholesterol diet-fed ApoE-/- mice. Moreover, serum sEVs from patients with NAFLD and sEV-miR-30a-3p expression were associated with decreased cholesterol efflux levels in foam cells. CONCLUSION: Steatotic hepatocyte-derived sEVs promote foam cell formation and facilitate atherogenesis via the miR-30a-3p/ABCA1 axis. Reducing sEV secretion by steatotic hepatocytes or targeting miR-30a-3p may be potential therapeutic approaches to slow the progression of NAFLD-driven atherosclerosis. IMPACT AND IMPLICATIONS: The presence of hepatic steatosis is strongly correlated with the risk of cardiovascular disease and cardiovascular events, yet the molecular mechanisms linking steatosis to progression of atherosclerosis are unclear. Herein, we identified small extracellular vesicles from steatotic hepatocytes as a trigger that accelerated the progression of atherosclerosis. Steatotic hepatocyte-derived small extracellular vesicles promoted foam cell formation via the miR-30a-3p/ABCA1 axis. Our findings not only provide mechanistic insight into non-alcoholic fatty liver disease-driven atherosclerosis but also provide potential therapeutic targets for patients with atherosclerosis.

Cohort Profile: Guangzhou Nutrition and Health Study (GNHS): A Population-Based Multi-Omics Study.

BACKGROUND: The Guangzhou Nutrition and Health Study (GNHS) aims to assess the determinants of metabolic disease in nutritional aspects, as well as other environmental and genetic factors, and explore possible biomarkers and mechanisms with multi-omics integration. METHODS: The population-based sample of adults in Guangzhou, China (baseline: 40-83 years old; n = 5118) was followed up about every 3 years. All will be tracked via on-site follow-up and health information systems. We assessed detailed information on lifestyle factors, physical activities, dietary assessments, psychological health, cognitive function, body measurements, and muscle function. Instrument tests included dual-energy X-ray absorptiometry scanning, carotid artery and liver ultrasonography evaluations, vascular endothelial function evaluation, upper-abdomen and brain magnetic resonance imaging, and 14-d real-time continuous glucose monitoring tests. We also measured multi-omics, including host genome-wide genotyping, serum metabolome and proteome, gut microbiome (16S rRNA sequencing, metagenome, and internal transcribed spacer 2 sequencing), and fecal metabolome and proteome. RESULTS: The baseline surveys were conducted from 2008 to 2015. Now, we have completed 3 waves. The 3rd and 4th follow-ups have started but have yet to end. A total of 5118 participants aged 40-83 took part in the study. The median age at baseline was approximately 59.0 years and the proportion of female participants was about 69.4%. Among all the participants, 3628 (71%) completed at least one on-site follow-up with a median duration of 9.48 years. CONCLUSION: The cohort will provide data that have been influential in establishing the role of nutrition in metabolic diseases with multi-omics.

Hepatic Fibroblast Growth Factor 21 Is Involved in Mediating Functions of Liraglutide in Mice With Dietary Challenge.

BACKGROUND AND AIMS: Several studies have shown that expression of hepatic fibroblast growth factor 21 (FGF21) can be stimulated by glucagon-like peptide 1 (GLP-1)-based diabetes drugs. As GLP-1 receptor (GLP-1R) is unlikely to be expressed in hepatocytes, we aimed to compare such stimulation in mice and in mouse hepatocytes, determine the involvement of GLP-1R, and clarify whether FGF21 mediates certain functions of the GLP-1R agonist liraglutide. APPROACH AND RESULTS: Liver FGF21 expression was assessed in mice receiving a daily liraglutide injection for 3 days or in mouse primary hepatocytes (MPHs) undergoing direct liraglutide treatment. The effects of liraglutide on metabolic improvement and FGF21 expression were then assessed in high-fat diet (HFD)-fed mice and compared with the effects of the dipeptidyl-peptidase 4 inhibitor sitagliptin. Animal studies were also performed in Glp1r-/- mice and liver-specific FGF21-knockout (lFgf21-KO) mice. In wild-type mouse liver that underwent RNA sequencing and quantitative reverse-transcription PCR, we observed liraglutide-stimulated hepatic Fgf21 expression and a lack of Glp1r expression. In MPHs, liraglutide did not stimulate Fgf21. In mice with HFD-induced obesity, liraglutide or sitagliptin treatment reduced plasma triglyceride levels, whereas their effect on reducing body-weight gain was different. Importantly, increased hepatic FGF21 expression was observed in liraglutide-treated mice but was not observed in sitagliptin-treated mice. In HFD-fed Glp1r-/- mice, liraglutide showed no beneficial effects and could not stimulate Fgf21 expression. In lFgf21-KO mice undergoing dietary challenge, the body-weight-gain attenuation and lipid homeostatic effects of liraglutide were lost or significantly reduced. CONCLUSIONS: We suggest that liraglutide-stimulated hepatic Fgf21 expression may require GLP-1R to be expressed in extrahepatic organs. Importantly, we revealed that hepatic FGF21 is required for liraglutide to lower body weight and improve hepatic lipid homeostasis. These observations advanced our mechanistic understanding of the function of GLP-1-based drugs in NAFLD.

Inverse Association of Serum Adipsin with the Remission of Nonalcoholic Fatty-Liver Disease: A 3-Year Community-Based Cohort Study.

PURPOSES: Adipokine alterations contribute to the development and remission of nonalcoholic fatty-liver disease (NAFLD). Adipsin is one of the most abundant adipokines and is almost exclusively produced by adipocytes. However, data on adipsin in human NAFLD are limited and controversial. We performed this study to investigate the association between adipsin and the remission of NAFLD in middle-aged and elderly Chinese adults. METHODS: Whether adipsin is associated with the remission of NAFLD in a 3-year community-based prospective cohort study was investigated. Baseline levels of adipsin were measured in serum samples collected from 908 NAFLD participants. NAFLD was diagnosed using abdominal ultrasonography. Logistic regression analysis and a multiple stepwise logistic regression model including different variables were conducted to evaluate the association between serum adipsin levels and the remission of NAFLD. RESULTS: During a mean follow-up of 3.14 ± 0.36 years, 247 (27.20%) participants with NAFLD at baseline were in remission. At baseline, serum adipsin concentration was positively correlated with body mass index (r: 0.39, p < 0.001), insulin (r: 0.31, p < 0.001), and homeostasis model assessment of insulin resistance (r: 0.31, p < 0.001) and was inversely associated with NAFLD remission with a fully adjusted odds ratio (OR) of 0.28 (0.16-0.48) (p trend < 0.001). In a multiple stepwise logistic regression model, circulating adipsin independently predicted NAFLD remission (OR: 0.284, 95% confidence interval [CI]: 0.172-0.471, p for trend <0.001). The area under the receiver operating characteristic curve was 0.751 (95% CI: 0.717-0.785) (p < 0.001) for the prediction model of NAFLD remission. CONCLUSIONS: We provide evidence for an association between serum adipsin levels and the remission of NAFLD in a community-based prospective cohort study. Serum adipsin can be a potential biomarker for predicting NAFLD remission.

Vascular benefits of vitamin C supplementation against fine particulate air pollution in healthy adults: A double-blind randomised crossover trial.

Evidence on the health benefits of vitamin C supplementation in highly polluted areas has not been evaluated. We aimed to evaluate whether dietary vitamin C supplementation can improve vascular health linked to particulate matter (PM) exposure. A randomised double-blind crossover trial involving 58 health young adults was performed in Shijiazhuang, China in 2018. All subjects were randomly assigned to the vitamin C supplementation group (2000 mg/d) or placebo group for a week alternating with a 2 week washout period. Fifteen circulating biomarkers were measured. Linear mixed-effect model was applied to evaluate the effect of vitamin C supplementation on health outcomes. The average concentrations of PM2.5 and PM10 were 164.91 and 327.05 µg/m3, respectively. Vitamin C supplementation was significantly associated with a 19.47% decrease in interleukin-6 (IL-6), 17.30% decrease in tumour necrosis factor-a (TNF-α), 34.01% decrease in C-reactive protein (CRP), 3.37% decrease in systolic blood pressure (SBP) and 6.03% decrease in pulse pressure (PP). Furthermore, glutathione peroxidase (GSH-Px) was significantly increased by 7.15%. Sex-subgroup analysis showed that vitamin C significantly reduced TNF-α by 27.85% in male participants and significantly increased APOB by 6.28% and GSH-Px by 14.47% only in female participants. This study indicated that vitamin C supplementation may protect vascular vessels against PM exposure among healthy young adults in China.

Serum Bioavailable, Rather Than Total, 25-hydroxyvitamin D Levels Are Associated With Hepatocellular Carcinoma Survival.

BACKGROUND AND AIMS: Free and bioavailable 25-hydroxyvitamin D (25OHD) are emerging measurements of vitamin D status. It remains unclear whether circulating free or bioavailable 25OHD are relevant to hepatocellular carcinoma (HCC) prognosis. Our aim was to test the hypothesis that bioavailable 25OHD may be a better serum biomarker of vitamin D status than total 25OHD on the association with HCC survival. APPROACH AND RESULTS: We included 1,031 newly diagnosed, previously untreated patients with HCC from the Guangdong Liver Cancer Cohort enrolled between September 2013 and April 2017. Serum total 25OHD levels were measured using an electrochemiluminescence immunoassay. Serum-free 25OHD levels were measured using a two-step enzyme-linked immunosorbent assay. Bioavailable 25OHD levels were calculated from measured free 25OHD and albumin using a previously validated equation. Primary outcomes were liver cancer-specific (LCSS) and overall survival (OS). Cox proportional hazards models were performed to calculate the multivariable hazard ratios (HRs) and 95% confidence intervals (CIs). During a median follow-up of 726 days, 430 patients had deceased, including 393 deaths from HCC. In multivariable analyses, higher bioavailable 25OHD levels were significantly associated with better survival, independent of nonclinical and clinical prognostic factors including serum C-reactive protein, Barcelona Clinic Liver Cancer stage, and cancer treatment. The multivariable-adjusted HRs in the highest versus lowest quartile of bioavailable 25OHD levels were 0.69 (95% CI: 0.51, 0.93; P for trend = 0.014) for LCSS and 0.71 (95% CI: 0.53, 0.94; P for trend = 0.013) for OS. In contrast, neither total nor free 25OHD levels were associated with LCSS or OS. CONCLUSIONS: Higher bioavailable, rather than total, 25OHD levels were independently associated with improved survival in a population-based HCC cohort, suggesting a potential utility of bioavailable 25OHD in HCC prognosis.

Adropin regulates hepatic glucose production via PP2A/AMPK pathway in insulin-resistant hepatocytes.

Adropin as a secretory peptide has shown a protective role on the disorders of glucose and lipid metabolism. However, the role and mechanism of this peptide on the hepatic glucose production has remained unclear. Adropin knockout (KO) mice were generated to explore its effects on the enhanced hepatic glucose production in obesity. We found that compared to wild-type (WT) mice, adropin-KO mice developed the unbalanced enhanced hepatic glucose production in advance of the whole-body insulin resistance (IR) by high-fat diet (HFD). Mechanistically, adropin dissociated CREB-CRTC2 and FoxO1-PGC1α complex and reduced their binding to the promoters of G6Pase and PEPCK to decrease glucose production in IR. However, these effects were not observed in insulin-sensitive hepatocytes. Furthermore, in IR hepatocytes, dampened AMPK signaling was re-activated by adropin treatment via inhibition of PP2A. To further authenticate AMPK role in vivo, we administrated HFD-fed mice with AAV8-CA AMPKα and found that AMPK activation functionally restored the aberrant glucose production and IR induced by adropin-deficiency. This study provides evidence that adropin activates the AMPK pathway via inhibition of PP2A and decreases the liver glucose production in IR context. Therefore, adropin may represent a novel target for the prevention and treatment of diabetes.

Anthocyanins regulate serum adipsin and visfatin in patients with prediabetes or newly diagnosed diabetes: a randomized controlled trial.

BACKGROUND: Epidemiological studies have suggested that adipsin and visfatin are associated with the development of type 2 diabetes. This is the first study to investigate the effects of supplementation with purified anthocyanins on serum adipsin and visfatin in patients with prediabetes or newly diagnosed diabetes. METHODS: A total of 160 participants with prediabetes or newly diagnosed diabetes (40-75 years old) were given 320 mg anthocyanins or placebo daily for 12 weeks in a randomized trial. Serum adipsin, serum visfatin, lipids and glycated hemoglobin A1c (HbA1c) were measured. The areas under the curve (AUCs) for glucose, insulin and C-peptide were determined before-and after-treatment by a standard 3-h 75 g oral glucose tolerance test (OGTT). RESULTS: Relatively significant increases in serum adipsin (net change 0.15 µg/mL [0.03, 0.27], p = 0.018) and decreases in visfatin (-3.5 ng/mL [-6.69, -0.31], p = 0.032) were observed between the anthocyanins and placebo groups. We also observed significant improvements in HbA1c (-0.11% [-0.22, -0.11], p = 0.033), apolipoprotein A-1 (apo A-1) (0.12 g/L [0.03, 0.21], p = 0.012) and apolipoprotein B (apo B) (-0.07 g/L [-0.14, -0.01], p = 0.033) in response to the anthocyanins intervention. CONCLUSION: Purified anthocyanins supplementation for 12 weeks increased serum adipsin and decreased serum visfatin in patients with prediabetes or newly diagnosed diabetes. Trial registration ClinicalTrials.gov, identifier: NCT02689765.

Egg consumption associated with all-cause mortality in rural China: a 14-year follow-up study.

BACKGROUND: Dietary recommendations regarding egg intake remain controversial topic for public health. We hypothesized that there was a positive association between egg consumption and all-cause mortality. METHODS: To test this hypothesis, we enrolled 9885 adults from a community-based cohort in Anhui Province, China during 2003-05. Egg consumption was assessed by food questionnaire. Stratified analyses were performed for age, sex, body mass index (BMI), blood pressure, smoking, drinking and laboratory tests. RESULTS: After an average follow-up of 14.1 years, 9444 participants were included for analysis. A total of 814 deaths were recorded. Participants' BMI and lipid profile had no significantly difference between three egg consumption groups. BMI was 21.6±2.7 of the whole population, especially BMI>24 was only 17.3%. A bivariate association of egg consumption >6/week with increased all-cause mortality was observed compared with ≤6/week (RR: 1.35, 95% CI: 1.05, 1.73, P = 0.018). A significant interaction was observed for BMI ≥ 21.2 kg/m2 vs. BMI<21.2 kg/m2 (P for interaction: 0.001). No other significant interactions were found. CONCLUSIONS: In this study, consuming >6 eggs/week increased risk of all-cause mortality, even among lean participants, especially who with BMI ≥ 21.2 kg/m2. Eggs are an easily accessible and constitute an affordable food source in underdeveloped regions. Consuming <6 eggs/week may be the most suitable intake mode.

The 6-month effect of whole soy and purified isoflavones daidzein on thyroid function-A double-blind, randomized, placebo controlled trial among Chinese equol-producing postmenopausal women.

The aim of this study was to examine the effect of whole soy and purified daidzein on markers of thyroid function in Chinese postmenopausal women who were equol-producers. Total 270 eligible women were randomized to either one of the three isocaloric supplements: 40 g soy flour (whole soy group), 40 g low-fat milk powder +63 mg daidzein (daidzein group) or 40 g low-fat milk powder (placebo) daily for 6 months. Serum thyroid markers were tested at baseline and 6 months for thyroid stimulating hormone, free triiodothyronine, reverse triiodothyronine and free thyroxine (FT4). There was no significant difference in the 6-month changes of thyroid markers among the three groups. Subgroup analysis among women with lowered thyroid function suggested a modest decrease of FT4. This randomized controlled trial among Chinese equol-producing postmenopausal women indicates the consumption of whole soy and purified daidzein at the provided dosages are safe and have no detrimental effect on thyroid function.

Inhibition of S-Adenosylhomocysteine Hydrolase Induces Endothelial Dysfunction via Epigenetic Regulation of p66shc-Mediated Oxidative Stress Pathway.

BACKGROUND: Elevated levels of S-adenosylhomocysteine (SAH), the precursor of homocysteine, are positively associated with the risk of cardiovascular disease and with the development and progression of atherosclerosis. However, the role of SAH in endothelial dysfunction is unclear. METHODS: Apolipoprotein E-deficient ( apoE-/-) mice received dietary supplementation with the SAH hydrolase (SAHH) inhibitor adenosine dialdehyde or were intravenously injected with a retrovirus expressing SAHH shRNA. These 2 approaches, along with the heterozygous SAHH gene knockout ( SAHH+/-) mouse model, were used to elevate plasma SAH levels and to examine the role of SAH in aortic endothelial dysfunction. The relationship between plasma SAH levels and endothelial dysfunction was also investigated in human patients with coronary artery disease and healthy control subjects. RESULTS: Plasma SAH levels were increased in SAHH+/- mice and in apoE-/- mice after dietary administration of adenosine dialdehyde or intravenous injection with SAHH shRNA. SAHH+/- mice or apoE-/- mice with SAHH inhibition showed impaired endothelium-dependent vascular relaxation and decreased nitric oxide bioavailability after treatment with acetylcholine; this was completely abolished by the administration of the endothelial nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester. Furthermore, SAHH inhibition induced production of reactive oxygen species and p66shc expression in the mouse aorta and human aortic endothelial cells. Antioxidants and p66shc siRNA prevented SAHH inhibition-induced generation of reactive oxygen species and attenuated the impaired endothelial vasomotor responses in high-SAH mice. Moreover, inhibition of SAHH induced hypomethylation in the p66shc gene promoter and inhibited expression of DNA methyltransferase 1. Overexpression of DNA methyltransferase 1, induced by transduction of an adenovirus, was sufficient to abrogate SAHH inhibition-induced upregulation of p66shc expression. Finally, plasma SAH levels were inversely associated with flow-mediated dilation and hypomethylation of the p66shc gene promoter and positively associated with oxidative stress levels in patients with coronary artery disease and healthy control subjects. CONCLUSIONS: Our findings indicate that inhibition of SAHH results in elevated plasma SAH levels and induces endothelial dysfunction via epigenetic upregulation of the p66shc-mediated oxidative stress pathway. Our study provides novel molecular insight into mechanisms of SAH-associated endothelial injury that may contribute to the development of atherosclerosis. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT03345927.

Dietary fruit and vegetable intake, gut microbiota, and type 2 diabetes: results from two large human cohort studies.

BACKGROUND: Little is known about the inter-relationship among fruit and vegetable intake, gut microbiota and metabolites, and type 2 diabetes (T2D) in human prospective cohort study. The aim of the present study was to investigate the prospective association of fruit and vegetable intake with human gut microbiota and to examine the relationship between fruit and vegetable-related gut microbiota and their related metabolites with type 2 diabetes (T2D) risk. METHODS: This study included 1879 middle-age elderly Chinese adults from Guangzhou Nutrition and Health Study (GNHS). Baseline dietary information was collected using a validated food frequency questionnaire (2008-2013). Fecal samples were collected at follow-up (2015-2019) and analyzed for 16S rRNA sequencing and targeted fecal metabolomics. Blood samples were collected and analyzed for glucose, insulin, and glycated hemoglobin. We used multivariable linear regression and logistic regression models to investigate the prospective associations of fruit and vegetable intake with gut microbiota and the association of the identified gut microbiota (fruit/vegetable-microbiota index) and their related fecal metabolites with T2D risk, respectively. Replications were performed in an independent cohort involving 6626 participants. RESULTS: In the GNHS, dietary fruit intake, but not vegetable, was prospectively associated with gut microbiota diversity and composition. The fruit-microbiota index (FMI, created from 31 identified microbial features) was positively associated with fruit intake (p < 0.001) and inversely associated with T2D risk (odds ratio (OR) 0.83, 95%CI 0.71-0.97). The FMI-fruit association (p = 0.003) and the FMI-T2D association (OR 0.90, 95%CI 0.84-0.97) were both successfully replicated in the independent cohort. The FMI-positive associated metabolite sebacic acid was inversely associated with T2D risk (OR 0.67, 95%CI 0.51-0.86). The FMI-negative associated metabolites cholic acid (OR 1.35, 95%CI 1.13-1.62), 3-dehydrocholic acid (OR 1.30, 95%CI 1.09-1.54), oleylcarnitine (OR 1.77, 95%CI 1.45-2.20), linoleylcarnitine (OR 1.66, 95%CI 1.37-2.05), palmitoylcarnitine (OR 1.62, 95%CI 1.33-2.02), and 2-hydroglutaric acid (OR 1.47, 95%CI 1.25-1.72) were positively associated with T2D risk. CONCLUSIONS: Higher fruit intake-associated gut microbiota and metabolic alteration were associated with a lower risk of T2D, supporting the public dietary recommendation of adopting high fruit intake for the T2D prevention.

Protocatechuic Acid Inhibits Vulnerable Atherosclerotic Lesion Progression in Older Apoe-/- Mice.

BACKGROUND: Normalization of arterial inflammation inhibits atherosclerosis. The preventive role for protocatechuic acid (PCA) in early-stage atherosclerosis is well recognized; however, its therapeutic role in late-stage atherosclerosis remains unexplored. OBJECTIVE: We investigated whether PCA inhibits vulnerable atherosclerosis progression by normalizing arterial inflammation. METHODS: Thirty-wk-old male apolipoprotein E-deficient (Apoe-/-) mice with vulnerable atherosclerotic lesions in the brachiocephalic artery were fed the AIN-93G diet alone (control) or supplemented with 0.003% PCA (wt:wt) for 20 wk. Lesion size and composition, IL-1ß, and NF-κB in the brachiocephalic arteries, and serum lipid profiles, oxidative status, and proinflammatory cytokines (e.g., IL-1ß, monocyte chemoattractant protein-1, and serum amyloid A) were measured. Moreover, the effect of PCA on the inflammation response was evaluated in efferocytic macrophages from C57BL/6J mice. RESULTS: Compared with the control treatment, dietary PCA supplementation significantly reduced lesion size (27.5%; P < 0.05) and also improved lesion stability (P < 0.05) as evidenced by increased thin fibrous cap thickness (31.7%) and collagen accumulation (58.3%), reduced necrotic core size (37.6%) and cellular apoptosis (73.9%), reduced macrophage accumulation (45.1%), and increased vascular smooth muscle cell accumulation (51.5%). Moreover, PCA supplementation inhibited IL-1ß expression (53.7%) and NF-κB activation (64.4%) in lesions. However, PCA supplementation did not change serum lipid profiles, total antioxidant capacity, and inflammatory cytokines. In efferocytic macrophages, PCA at 0.5 and 1 µmol/L inhibited Il1b/IL-1ß mRNA (27.2-46.5%) and protein (29.2-49.6%) expression and NF-κB activation (67.0-80.3%) by upregulation of MER proto-oncogene tyrosine kinase (MERTK) and inhibition of mitogen-activated protein kinase 3/1 (MAPK3/1). Strikingly, the similar pattern of the MERTK and MAPK3/1 changes in lesional macrophages of mice after PCA intervention in vivo was recapitulated. CONCLUSION: PCA inhibits vulnerable lesion progression in mice, which might partially be caused by normalization of arterial inflammation by upregulation of MERTK and inhibition of MAPK3/1 in lesional macrophages.

Dietary Cyanidin-3-Glucoside Attenuates High-Fat-Diet-Induced Body-Weight Gain and Impairment of Glucose Tolerance in Mice via Effects on the Hepatic Hormone FGF21.

BACKGROUND: Dietary polyphenols including anthocyanins target multiple organs. OBJECTIVE: We aimed to assess the involvement of glucagon-like peptide 1 (GLP-1), leptin, insulin and fibroblast growth factor 21 (FGF21) in mediating metabolic beneficial effects of purified anthocyanin cyanidin-3-glucoside (Cy3G). METHODS: Intestinal proglucagon gene (Gcg; encoding GLP-1) and liver Fgf21 expression were assessed in 6-wk-old male C57BL-6J mice fed a low-fat-diet (LFD; 10% of energy from fat), alone or with 1.6 mg Cy3G/L in drinking water for 3 wk [experiment (Exp.) 1; n = 5/group]. Similar mice were fed the LFD or a high-fat diet (HFD; 60% energy from fat) with or without Cy3G for 20 wk. Half of the mice administered Cy3G also received 4 broad-spectrum antibiotics (ABs) in drinking water between weeks 11 and 14, for a total of 6 groups (n = 8/group). Metabolic tolerance tests were conducted between weeks 2 and 16. Relevant hormone gene expression and plasma hormone concentrations were assessed mainly at the end of 20 wk (Exp. 2). RESULTS: In Exp. 1, Cy3G administration increased ileal but not colonic Gcg level by 2-fold (P < 0.05). In Exp. 2, Cy3G attenuated HFD-induced body-weight gain (20.3% at week 16), and improved glucose tolerance (26.5% at week 15) but not insulin tolerance. Although Cy3G had no effect on glucose tolerance in LFD mice, LFD/Cy3G/AB mice showed better glucose tolerance than LFD/Cy3G mice (23%). In contrast, HFD/Cy3G/AB mice showed worse glucose tolerance compared with HFD/Cy3G mice (15%). Beneficial effects of Cy3G in HFD mice were not associated with changes in plasma leptin, insulin or GLP-1 concentrations. However, Cy3G increased hepatic Fgf21 expression in mice in Exp. 1 by 4-fold and attenuated Fgf21 overexpression in HFD mice (Exp. 2, 22%), associated with increased expression of genes that encode FGFR1 and ß-klotho (>3-fold, P < 0.05). CONCLUSIONS: Dietary Cy3G may reduce body weight and exert metabolic homeostatic effects in mice via changes in hepatic FGF21.

Serum Bioavailable and Free 25-Hydroxyvitamin D Levels, but Not Its Total Level, Are Associated With the Risk of Mortality in Patients With Coronary Artery Disease.

RATIONALE: Bioavailable and free 25-hydroxyvitamin D (25(OH)D) are emerging measurements of vitamin D. Whether serum bioavailable or free 25(OH)D level is associated with mortality in patients with coronary artery disease (CAD) is unknown. OBJECTIVE: Our aim is to determine the potential association between serum total, bioavailable, and free 25(OH)D levels and the risk of mortality among patients with CAD. METHODS AND RESULTS: We measured serum 25(OH) levels in 1387 patients with angiographically confirmed CAD from the Guangdong Coronary Artery Disease Cohort. Serum DBP (vitamin D-binding protein) levels were measured using a polyclonal immunoassay, and serum-free 25(OH)D levels were measured using a 2-step immunoassay. Bioavailable 25(OH)D levels were calculated using a previously validated formula. By the median follow-up time of 6.7 years, 205 patients had died, including 134 deaths from cardiovascular diseases. In multivariate analyses, low serum bioavailable 25(OH)D level was significantly associated with increased risks of mortality, independent of established cardiovascular risk factors, features and treatments of CAD, factors associated with vitamin D and mineral metabolism, and CRP (C-reactive protein). The multivariable-adjusted hazard ratios across quartiles of bioavailable 25(OH)D were 1.79, 1.35, 1.36, and 1.00 for all-cause mortality ( P for trend=0.01) and 2.58, 1.85, 1.73, and 1.00 for cardiovascular mortality ( P for trend=0.001), respectively. Serum-free 25(OH)D level was inversely associated with the risk of mortality, with the extreme-quartile hazard ratios of 1.64 for all-cause mortality ( P for trend=0.024) and 1.97 for cardiovascular mortality ( P for trend=0.013). In contrast, serum total 25(OH)D level was not significantly associated with all-cause mortality or cardiovascular mortality. CONCLUSIONS: Lower serum bioavailable and free 25(OH)D levels rather than total 25(OH)D level are independently associated with an increased risk of all-cause mortality and cardiovascular mortality in a population-based CAD cohort.

Urinary equol is associated with bioavailable testosterone but not total testosterone in women.

Little is known about the association between equol and bioavailable testosterone (BT) in adults. In this study, we examined the associations of urinary equol concentrations with serum concentrations of total, bioavailable and free testosterone (FT), dehydroepiandrosterone sulfide (DHEAS), free androgen index (FAI) and sex hormone-binding globulin (SHBG). This cross-sectional study included 1,904 women with a mean age of 59.7 years. Urinary equol concentrations were measured using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The serum androgenic indices and SHBG were also determined. Overall, urinary equol tended to be inversely associated with bioactive forms of androgenic indices (BT, FT or FAI) but not with total testosterone (TT) or DHEAS. Urinary equol was also positively associated with SHBG. In multi-covariate-adjusted analyses stratified by menopausal status, graded and inverse associations between urinary equol and bioactive forms of androgenic indices (BT, FT and FAI) were observed in postmenopausal women (all p-trends < 0.05), but not in premenopausal women. A significant positive association between urinary equol and SHBG was observed only in postmenopausal women. No significant associations were observed between urinary equol and TT or DHEAS in either group. A path analysis indicated that these associations of equol with androgens in postmenopausal women might be mediated by SHBG. In conclusion, urinary equol exhibited graded and inverse associations with BT or FT, but not TT in women. However, further longitudinal studies of human patients are needed to confirm these results and overcome the limitations of cross-sectional studies.

Plasma Magnesium Concentrations and Risk of Incident Cancer in Adults with Hypertension: A Nested Case-Control Study.

OBJECTIVE: The association between plasma magnesium and risk of incident cancer remains inconclusive in previous studies. We aimed to investigate the prospective relationship of baseline plasma magnesium concentrations with the risk of incident cancer and to examine possible effect modifiers. METHODS: A nested case-control study with 228 incident cancer cases and 228 matched controls was conducted using data from the China Stroke Primary Prevention Trial (CSPPT), a randomized, double-blind, controlled trial, conducted from May 2008 to August 2013. Study outcomes included incident cancer and its subtypes. RESULTS: When plasma magnesium concentrations were assessed as quartiles, a significantly higher incident risk of total cancer was found in participants in quartile 1 (<0.76 mmol/L; odds ratio [OR] = 2.70; 95% CI: 1.33-5.49) and quartile 4 (≥0.89 mmol/L; OR = 2.05; 95% CI: 1.12-3.76), compared with those in quartile 3 (0.83 to <0.89 mmol/L). In cancer site-specific analyses, similar trends were found for gastrointestinal cancer, esophageal cancer, gastric cancer, breast cancer, lung cancer, and other cancers. Furthermore, none of the variables, including age, sex, current smoking status, current alcohol intake, BMI, systolic blood pressure, and total cholesterol levels at baseline significantly modified the association between plasma magnesium and cancer risk. CONCLUSIONS: Both low and high plasma magnesium concentrations were significantly associated with an increased incident risk of cancer, compared with the reference concentrations of 0.83 to <0.89 mmol/L among hypertensive adults.