Confounding Effects of Tamoxifen: Cautionary and Practical Considerations for the Use of Tamoxifen-Inducible Mouse Models in Atherosclerosis Research-Brief Report.

BACKGROUND: In recent years, fate-mapping lineage studies in mouse models have led to major advances in vascular biology by allowing investigators to track specific cell populations in vivo. One of the most frequently used lineage tracing approaches involves tamoxifen-inducible CreERT-LoxP systems. However, tamoxifen treatment can also promote effects independent of Cre recombinase activation, many of which have not been fully explored. METHODS: To elucidate off-target effects of tamoxifen, male and female mice were either unmanipulated or injected with tamoxifen or corn oil. All mice received PCSK9 (proprotein convertase subtilisin/kexin type 9)-AAV (adeno-associated virus) injections and a modified Western diet to induce hypercholesterolemia. After 2 weeks, serum cholesterol and liver morphology were assessed. To determine the duration of any tamoxifen effects in long-term atherosclerosis experiments, mice received either 12 days of tamoxifen at baseline or 12 days plus 2 sets of 5-day tamoxifen boosters; all mice received PCSK9-AAV injections and a modified Western diet to induce hypercholesterolemia. After 24 weeks, serum cholesterol and aortic sinus plaque burden were measured. RESULTS: After 2 weeks of atherogenic treatment, mice injected with tamoxifen demonstrated significantly reduced serum cholesterol levels compared with uninjected- or corn oil-treated mice. However, there were no differences in PCSK9-mediated knockdown of LDL (low-density lipoprotein) receptors between the groups. Additionally, tamoxifen-treated mice exhibited significantly increased hepatic lipid accumulation compared with the other groups. Finally, the effects of tamoxifen remained for at least 8 weeks after completion of injections, with mice demonstrating persistent decreased serum cholesterol and impaired atherosclerotic plaque formation. CONCLUSIONS: In this study, we establish that tamoxifen administration results in decreased serum cholesterol, decreased plaque formation, and increased hepatic lipid accumulation. These alterations represent significant confounding variables in atherosclerosis research, and we urge future investigators to take these findings into consideration when planning and executing their own atherosclerosis experiments.

Tilianin improves lipid profile and alleviates atherosclerosis in ApoE-/- mice through up-regulation of SREBP2-mediated LDLR expression.

BACKGROUND: The huge global burden of atherosclerotic cardiovascular diseases (CVDs) represents an urgent unmet need for the development of novel therapeutics. Dracocephalum moldavica L. has been used as a traditional Uygur medicine to treat various CVDs for centuries. Tilianin is a major flavonoid component of D. moldavica L. and has potential for preventing atherosclerosis. However, the molecular mechanisms that tilianin attenuate atherosclerosis are far from fully understood. PURPOSES: The purpose of this study is to investigate the efficiency and underlying mechanisms of tilianin in controlling lipid profile and preventing atherogenesis. METHODS: The lipid-lowering effect of tilianin was evaluated in C57BL/6 and ApoE-/- mice by systematically determining serum biochemical parameters. The effects of tilianin on the atherosclerotic lesion were observed in aortic roots and whole aortas of ApoE-/- mice with oil red O staining. Caecal content from ApoE-/- mice were collected for 16S rRNA gene sequence analysis to assess the structure of the gut microbiota. The inhibition of hepatosteatosis was verified by histological examination, and a liver transcriptome analysis was performed to elucidate the tilianin-induced hepatic transcriptional alterations. Effects of tilianin on the expression and function of LDLR were examined in HepG2 cells and ApoE-/- mice. Further mechanisms underlying the efficacy of tilianin were investigated in HepG2 cells. RESULTS: Tilianin treatment improved lipid profiles in C57BL/6 and dyslipidemic ApoE-/- mice, especially reducing the serum LDL-cholesterol (LDL-C) level. Significant reductions of atherosclerotic lesion area and hepatosteatosis were observed in tilianin-treated ApoE-/- mice. The altered gut microbial composition in tilianin groups was associated with lipid metabolism and atherosclerosis. The liver transcriptome revealed that tilianin regulated the transcription of lipid metabolism-related genes. Then both in vitro and in vivo analyses revealed the potent effect of tilianin to enhance hepatic LDLR expression and its mediated LDL-C uptake. Further studies confirmed a critical role of SREBP2 in hepatic LDLR up-regulation by tilianin via increasing precursor and thus mature nuclear SREBP2 level. CONCLUSION: This study demonstrated the lipid-lowering effect of tilianin through SREBP2-mediated transcriptional activation of LDLR. Our findings reveal a novel anti-atherosclerotic mechanism of tilianin and underlie its potential clinical use in modulating CVDs with good availability and affordability.

Differential Effect of Dietary Supplementation with a Soybean Oil Enriched in Oleic Acid versus Linoleic Acid on Plasma Lipids and Atherosclerosis in LDLR-Deficient Mice.

Both monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) play important roles in lipid metabolism, and diets enriched with either of these two fatty acids are associated with decreased cardiovascular risk. Conventional soybean oil (CSO), a common food ingredient, predominantly contains linoleic acid (LA; C18:2), a n-6 PUFA. Recently, a modified soybean oil (MSO) enriched in oleic acid (C18:1), a n-9 MUFA, has been developed, because of its improved chemical stability to oxidation. However, the effect of the different dietary soybean oils on cardiovascular disease remains unknown. To test whether diets rich in CSO versus MSO would attenuate atherosclerosis development, LDL receptor knock-out (LDLR-KO) mice were fed a Western diet enriched in saturated fatty acids (control), or a Western diet supplemented with 5% (w/w) LA-rich CSO or high-oleic MSO for 12 weeks. Both soybean oils contained a similar amount of linolenic acid (C18:3 n-3). The CSO diet decreased plasma lipid levels and the cholesterol content of VLDL and LDL by approximately 18% (p < 0.05), likely from increased hepatic levels of PUFA, which favorably regulated genes involved in cholesterol metabolism. The MSO diet, but not the CSO diet, suppressed atherosclerotic plaque size compared to the Western control diet (Control Western diet: 6.5 ± 0.9%; CSO diet: 6.4 ± 0.7%; MSO diet: 4.0 ± 0.5%) (p < 0.05), independent of plasma lipid level changes. The MSO diet also decreased the ratio of n-6/n-3 PUFA in the liver (Control Western diet: 4.5 ± 0.2; CSO diet: 6.1 ± 0.2; MSO diet: 2.9 ± 0.2) (p < 0.05), which correlated with favorable hepatic gene expression changes in lipid metabolism and markers of systemic inflammation. In conclusion, supplementation of the Western diet with MSO, but not CSO, reduced atherosclerosis development in LDLR-KO mice independent of changes in plasma lipids.

Sexual dimorphism: increased sterol excretion leads to hypocholesterolaemia in female hyperbilirubinaemic Gunn rats.

Circulating bilirubin is associated with reduced serum cholesterol concentrations in humans and in hyperbilirubinaemic Gunn rats. However, mechanisms contributing to hypocholesterolaemia remain unknown. Therefore, this study aimed to investigate cholesterol synthesis, transport and excretion in mutant Gunn rats. Adult Gunn and control rats were assessed for daily faecal sterol excretion using metabolic cages, and water was supplemented with [1-13 C]-acetate to determine cholesterol synthesis. Bile was collected to measure biliary lipid secretion. Serum and liver were collected for biochemical analysis and for gene/protein expression using RT-qPCR and western blot, respectively. Additionally, serum was collected and analysed from juvenile rats. A significant interaction of sex, age and phenotype on circulating lipids was found with adult female Gunn rats reporting significantly lower cholesterol and phospholipids. Female Gunn rats also demonstrated elevated cholesterol synthesis, greater biliary lipid secretion and increased total faecal cholesterol and bile acid excretion. Furthermore, they possessed increased hepatic low-density lipoprotein (LDL) receptor and SREBP2 expression. In contrast, there were no changes to sterol metabolism in adult male Gunn rats. This is the first study to demonstrate elevated faecal sterol excretion in female hyperbilirubinaemic Gunn rats. Increased sterol excretion creates a negative intestinal sterol balance that is compensated for by increased cholesterol synthesis and LDL receptor expression. Therefore, reduced circulating cholesterol is potentially caused by increased hepatic uptake via the LDL receptor. Future studies are required to further evaluate the sexual dimorphism of this response and whether similar findings occur in females with benign unconjugated hyperbilirubinaemia (Gilbert's syndrome). KEY POINTS: Female adult hyperbilirubinaemic (Gunn) rats demonstrated lower circulating cholesterol, corroborating human studies that report a negative association between bilirubin and cholesterol concentrations. Furthermore, female Gunn rats had elevated sterol excretion creating a negative intestinal sterol balance that was compensated for by elevated cholesterol synthesis and increased hepatic low-density lipoprotein (LDL) receptor expression. Therefore, elevated LDL receptor expression potentially leads to reduced circulating cholesterol levels in female Gunn rats providing an explanation for the hypocholesterolaemia observed in humans with elevated bilirubin levels. This study also reports a novel interaction of sex with the hyperbilirubinaemic phenotype on sterol metabolism because changes were only reported in females and not in male Gunn rats. Future studies are required to further evaluate the sexual dimorphism of this response and whether similar findings occur in females with benign unconjugated hyperbilirubinaemia (Gilbert's syndrome).

[Molecular mechanism of astragaloside â £ against atherosclerosis by regulating miR-17-5p and PCSK9/VLDLR signal pathway].

This study explores the regulatory effect of astragaloside Ⅳ on miR-17-5 p and its downstream proprotein convertase subtillisin/kexin type 9(PCSK9)/very low density lipoprotein receptor(VLDLR) signal pathway, aiming at elucidating the mechanism of astragaloside Ⅳ against atherosclerosis(AS). In cell experiment, oxidized low-density lipoprotein(ox-LDL) was used for endothelial cell injury modeling with vascular smooth muscle cells(VSMCs). Then cells were classified into the model group, miR-17-5 p inhibitor group, blank serum group, and astragaloside Ⅳ-containing serum group based on the invention. Afterward, cell viability and the expression of miR-17-5 p, VLDLR, and PCSK9 mRNA and protein in cells in each group were detected. In animal experiment, 15 C57 BL/6 mice were used as the control group, and 45 ApoE~(-/-) mice were classified into the model group, miR-17-5 p inhibitor group, and astragaloside Ⅳ group, with 15 mice in each group. After 8 weeks of intervention, the peripheral serum levels of interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α), and the expression of miR-17-5 p, VLDLR, and PCSK9 mRNA in the aorta of mice were detected. The pathological changes of mice in each group were observed. According to the cell experiment, VSMC viability in the miR-17-5 p inhibitor group and the astragaloside Ⅳ-containing serum group was higher than that in the model group(P<0.05). The mRNA and protein expression of miR-17-5 p and VLDLR in VSMCs in the miR-17-5 p inhibitor group and the astragaloside Ⅳ-containing serum group was lower than that in the model group(P<0.05), but the mRNA and protein expression of PCSK9 was higher than that in the model group(P<0.05). As for the animal experiment, the levels of IL-6 and TNF-α in the peripheral serum of the miR-17-5 p inhibitor group and the astragaloside Ⅳ group were lower(P<0.05) and the serum level of IL-10 was higher(P<0.05) than that of the model group. The mRNA expression of miR-17-5 p and VLDLR in the aorta in the miR-17-5 p inhibitor group and the astragaloside Ⅳ group was lower(P<0.05), and PCSK9 mRNA expression was higher(P<0.05) than that in the model group. Pathological observation showed mild AS in the miR-17-5 p inhibitor group and the astragaloside Ⅳ group. In summary, astragaloside Ⅳ can prevent the occurrence and development of AS. The mechanism is that it performs targeted regulation of miR-17-5 p, further affecting the PCSK9/VLDLR signal pathway, inhibiting vascular inflammation, and thus alleviating endothelial cell injury.

Dietary Neu5Ac Intervention Protects Against Atherosclerosis Associated With Human-Like Neu5Gc Loss-Brief Report.

Objective: Species-specific pseudogenization of the CMAH gene during human evolution eliminated common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) biosynthesis from its precursor N-acetylneuraminic acid (Neu5Ac). With metabolic nonhuman Neu5Gc incorporation into endothelia from red meat, the major dietary source, anti-Neu5Gc antibodies appeared. Human-like Ldlr-/-Cmah-/- mice on a high-fat diet supplemented with a Neu5Gc-enriched mucin, to mimic human red meat consumption, suffered increased atherosclerosis if human-like anti-Neu5Gc antibodies were elicited. Approach and Results: We now ask whether interventional Neu5Ac feeding attenuates metabolically incorporated Neu5Gc-mediated inflammatory acceleration of atherogenesis in this Cmah-/-Ldlr-/- model system. Switching to a Neu5Gc-free high-fat diet or adding a 5-fold excess of Collocalia mucoid-derived Neu5Ac in high-fat diet protects against accelerated atherosclerosis. Switching completely from a Neu5Gc-rich to a Neu5Ac-rich diet further reduces severity. Remarkably, feeding Neu5Ac-enriched high-fat diet alone has a substantial intrinsic protective effect against atherosclerosis in Ldlr-/- mice even in the absence of dietary Neu5Gc but only in the human-like Cmah-null background. Conclusions: Interventional Neu5Ac feeding can mitigate or prevent the red meat/Neu5Gc-mediated increased risk for atherosclerosis, and has an intrinsic protective effect, even in the absence of Neu5Gc feeding. These findings suggest that similar interventions should be tried in humans and that Neu5Ac-enriched diets alone should also be investigated further.

Identification of neolignans with PCSK9 downregulatory and LDLR upregulatory activities from Penthorum chinense and the potential in cholesterol uptake by transcriptional regulation of LDLR via SREBP2.

ETHNOPHARMACOLOGICAL RELEVANCE: Penthorum chinense has been used in East Asia for the treatment of cholecystitis, infectious hepatitis, jaundice and to treat liver problems. Recent evidences provided the potential for the clinical use of P. chinense in the treatment of metabolic disease. AIM OF THE STUDY: Based on the traditional use and recent evidences, we investigated the effects of constituents from P. chinense with modulation on proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein receptor (LDLR) expression, and the effect of the most active substance on cholesterol uptake, and genes relevant to lipid metabolism. MATERIALS AND METHODS: The isolation of compounds from the BuOH-soluble extract of 80% methanol extract of P. chinense was conducted using chromatographic methods and the structures were established by interpreting spectroscopic data. Quantitative real time-PCR, and Western blot analysis were performed to monitor the regulatory activity on PCSK9 and LDLR expression. PCSK9-LDLR binding interaction was also tested. The cholesterol uptake in hepatocyte was measured using 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI)-labeled LDL cholesterol. Additionally, gene network analysis of LDLR and responses of its target proteins were carried out to discover genes germane to the effect of active compound on HepG2 cells. Moreover, we performed protein-protein interaction analysis via String and constructed the compound target network using Cytoscape. RESULTS: Two new neolignans and 37 known compounds were characterized from P. chinense. Of the isolated compounds, (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one (3), penthorin A (4) and methyl gallate (25) were found to suppress PCSK9 mRNA expression with IC50 values of 5.13, 15.56 and 11.66 µM, respectively. However, all the isolated compounds were found to be inactive in PCSK9-LDLR interaction assay. Additionally, a dibenzoxepine-type lignan analog, (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one (3) demonstrated to upregulate LDLR mRNA and protein expression via transcriptional factor sterol regulatory element-binding protein 2 (SREBP2). Furthermore, (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one (3) increase the LDL-cholesterol uptake in DiI-LDL assay. CONCLUSION: (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one (3) seemed to increase potentially cholesterol uptake via the downregulation of PCSK9 and the activation of LDLR in hepatocytes. Moreover, SREBP2 was found to play an important role in regulation of PCSK9 and LDLR by (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one.

Cholesterol Accumulation as a Driver of Hepatic Inflammation Under Translational Dietary Conditions Can Be Attenuated by a Multicomponent Medicine.

Background: Non-alcoholic fatty liver disease (NAFLD) is a complex multifactorial disorder that is characterised by dysfunctional lipid metabolism and cholesterol homeostasis, and a related chronic inflammatory response. NAFLD has become the most common cause of chronic liver disease in many countries, and its prevalence continues to rise in parallel with increasing rates of obesity. Here, we evaluated the putative NAFLD-attenuating effects of a multicomponent medicine consisting of 24 natural ingredients: Hepar compositum (HC-24). Methods: Ldlr-/-.Leiden mice were fed a high-fat diet (HFD) with a macronutrient composition and cholesterol content comparable to human diets for 24 weeks to induce obesity-associated metabolic dysfunction, including hepatic steatosis and inflammation. HC-24 or vehicle control was administered intraperitoneally 3 times/week (1.5 ml/kg) for the last 18 weeks of the study. Histological analyses of liver and adipose tissue were combined with extensive hepatic transcriptomics analysis. Transcriptomics results were further substantiated with ELISA, immunohistochemical and liver lipid analyses. Results: HFD feeding induced obesity and metabolic dysfunction including adipose tissue inflammation and increased gut permeability. In the liver, HFD-feeding resulted in a disturbance of cholesterol homeostasis and an associated inflammatory response. HC-24 did not affect body weight, metabolic risk factors, adipose tissue inflammation or gut permeability. While HC-24 did not alter total liver steatosis, there was a pronounced reduction in lobular inflammation in HC-24-treated animals, which was associated with modulation of genes and proteins involved in inflammation (e.g., neutrophil chemokine Cxcl1) and cholesterol homeostasis (i.e., predicted effect on 'cholesterol' as an upstream regulator, based on gene expression changes associated with cholesterol handling). These effects were confirmed by CXCL1 ELISA, immunohistochemical staining of neutrophils and biochemical analysis of hepatic free cholesterol content. Intrahepatic free cholesterol levels were found to correlate significantly with the number of inflammatory aggregates in the liver, thereby providing a potential rationale for the observed anti-inflammatory effects of HC-24. Conclusions: Free cholesterol accumulates in the liver of Ldlr-/-.Leiden mice under physiologically translational dietary conditions, and this is associated with the development of hepatic inflammation. The multicomponent medicine HC-24 reduces accumulation of free cholesterol and has molecular and cellular anti-inflammatory effects in the liver.

Dietary phosphatidylcholine supplementation reduces atherosclerosis in Ldlr-/- male mice2.

Choline is an essential nutrient required for various biological processes. Eggs, dairy, and meat are rich in phosphatidylcholine (PC), whereas cereal and legumes are rich in free choline. Excess dietary choline leads to increase plasma trimethylamine N-oxide (TMAO). Epidemiological studies suggest that plasma TMAO is a biomarker for atherosclerosis and it has been suggested that a lower intake of eggs and meat would reduce choline consumption and thus reduce atherosclerosis development. To investigate whether the form of dietary choline influences atherosclerosis development in Ldlr-/-, we randomly fed Ldlr-/-male mice (aged 8 - 10 wk) one of the three 40% (calories) high fat diets (with 0.5% w/w of cholesterol): Control (0.1% w/w free-choline, CON), choline-supplemented (0.4% free-choline, CS), or PC-supplemented (0.1% free-choline and 0.3% choline from PC, PCS). After 12-wk of dietary intervention, the animals were euthanized and tissues and blood collected. Aortic atherosclerotic plaque area, plasma choline, lipid metabolites, and spleen and peripheral blood cell phenotypes were quantified. Surprisingly, the PCS group had significantly lower atherosclerotic lesions while having 2-fold higher plasma TMAO levels compared with both CON and CS groups (P<0.05). In the fasting state, we found that PCS decreased plasma very low-density lipoprotein-cholesterol (VLDL-C) and apolipoprotein B48 (APOB48), and increased plasma high-density lipoprotein-cholesterol (HDL-C). However, very low-density lipoprotein (VLDL) secretion was not affected by dietary treatment. We observed lower levels of circulating pro-atherogenic chemokines in the PCS group. Our study suggests that increased dietary PC intake does not induce a pro-atherogenic phenotype.

Dietary Fruit and Vegetable Supplementation Suppresses Diet-Induced Atherosclerosis in LDL Receptor Knockout Mice.

BACKGROUND: Epidemiologic studies suggest that fruit and vegetable (F&V) consumption is inversely associated with incidence of cardiovascular disease (CVD). However, evidence for causality is lacking, and the underlying mechanisms are not well understood. OBJECTIVES: We aimed to determine whether there is a causal relation between consuming high levels of F&V and prevention of atherosclerosis, the hallmark of CVD pathogenesis. Furthermore, the underlying mechanisms were determined. METHODS: Six-week-old male LDL receptor-knockout mice were randomly assigned to 3 diet groups (12 mice/group) for 20 wk: control (CON, 10% kcal fat, 0.20 g/kg cholesterol), atherogenic (Ath, 27% kcal fat, 0.55 g/kg cholesterol), and Ath supplemented with 15% F&V (Ath + FV) (equivalent to 8-9 servings/d in humans). F&V was added as a freeze-dried powder that was prepared from the 24 most commonly consumed F&Vs in the United States. Body weight, aortic atherosclerotic lesion area, hepatic steatosis area, serum lipid profile and proinflammatory cytokine TNF-α concentrations, gut microbiota, and liver TNF-α and fatty acid synthase (Fasn) mRNA concentrations were assessed. RESULTS: F&V supplementation did not affect weight gain. Mice fed the Ath + FV diet had a smaller aortic atherosclerotic lesion area (71.7% less) and hepatic steatosis area (80.7% less) than those fed the Ath diet (both P < 0.001) independent of impact on weight, whereas no difference was found between Ath + FV and CON groups in these 2 pathologic markers. Furthermore, F&V supplementation prevented Ath diet-induced dyslipidemia (high concentrations of serum TG and VLDL cholesterol and lower concentrations of HDL cholesterol), reduced serum TNF-α concentration (by 21.5%), suppressed mRNA expression of liver TNF-α and Fasn, and ameliorated Ath-induced gut microbiota dysbiosis. CONCLUSIONS: Our results indicate that consuming a large quantity and variety of F&Vs causally attenuates diet-induced atherosclerosis and hepatic steatosis in mice. These effects of F&Vs are associated with, and may be mediated through, improved atherogenic dyslipidemia, alleviated gut dysbiosis, and suppressed inflammation.

Ascorbic acid enhances low-density lipoprotein receptor expression by suppressing proprotein convertase subtilisin/kexin 9 expression.

Ascorbic acid, a water-soluble antioxidant, regulates various biological processes and is thought to influence cholesterol. However, little is known about the mechanisms underpinning ascorbic acid-mediated cholesterol metabolism. Here, we determined if ascorbic acid can regulate expression of proprotein convertase subtilisin/kexin 9 (PCSK9), which binds low-density lipoprotein receptor (LDLR) leading to its intracellular degradation, to influence low-density lipoprotein (LDL) metabolism. At cellular levels, ascorbic acid inhibited PCSK9 expression in HepG2 and Huh7 cell lines. Consequently, LDLR expression and cellular LDL uptake were enhanced. Similar effects of ascorbic acid on PCSK9 and LDLR expression were observed in mouse primary hepatocytes. Mechanistically, ascorbic acid suppressed PCSK9 expression in a forkhead box O3-dependent manner. In addition, ascorbic acid increased LDLR transcription by regulating sterol regulatory element-binding protein 2. In vivo, administration of ascorbic acid reduced serum PCSK9 levels and enhanced liver LDLR expression in C57BL/6J mice. Reciprocally, lack of ascorbic acid supplementation in L-gulono-γ-lactone oxidase deficient (Gulo-/-) mice increased circulating PCSK9 and LDL levels, and decreased liver LDLR expression, whereas ascorbic acid supplementation decreased PCSK9 and increased LDLR expression, ameliorating LDL levels in Gulo-/- mice fed a high fat diet. Moreover, ascorbic acid levels were negatively correlated to PCSK9, total and LDL levels in human serum samples. Taken together, these findings suggest that ascorbic acid reduces PCSK9 expression, leading to increased LDLR expression and cellular LDL uptake. Thus, supplementation of ascorbic acid may ameliorate lipid profiles in ascorbic acid-deficient species.

Butein Synergizes with Statin to Upregulate Low-Density Lipoprotein Receptor Through HNF1α-Mediated PCSK9 Inhibition in HepG2 Cells.

Downregulation of the low-density lipoprotein (LDL) receptor (LDLR) can lead to hypercholesterolemia and related conditions, including cardiovascular diseases. Statins are a class of LDL cholesterol-lowering agents and are best-selling medications for patients at high risk of developing cardiovascular diseases. Indeed, statins upregulate LDLR and proprotein convertase subtilisin/kexin type 9a (PCSK9), leading to LDLR lysosomal degradation, which interferes with the attenuation of hypercholesterolemia. In the present study, butein was found to decrease extracellular PCSK9 levels by reducing its mRNA expression, which was attributable to butein-mediated downregulation of HNF1α in HepG2 cells. Butein-mediated PCSK9 inhibition further reversed LDLR protein synthesis inhibition, which possibly occurred through butein-mediated inhibition of LDLR degradation. When treated as a combination of butein and a statin, butein reduced statin-mediated enhancement of PCSK9 protein expression. This resulted in a synergistic enhancement of LDLR protein expression, whereas butein alone marginally increased LDLR protein expression. These findings suggest that butein, a novel PCSK9 inhibitor, may be a potential alternative or adjunct to statin treatment.

Cigarette smoke exposure impairs lipid metabolism by decreasing low-density lipoprotein receptor expression in hepatocytes.

BACKGROUND: Cigarette smoke (CS) exposure impairs serum lipid profiles and the function of vascular endothelial cells, which accelerates the atherosclerosis. However, the precise mechanism and effect on the expression of low-density lipoprotein receptor (LDLR) in the liver by CS exposure is still unclear. METHODS: In this study, adult male C57BL/6 J mice were divided into three groups, with one group being exposed to CS for 6 weeks. HepG2 cells were treated with CS extract at concentrations of 1, 2.5, 5, and 10%. RESULTS: The serum levels of total cholesterol (TC), triglycerides (TGs), and low-density lipoprotein cholesterol (LDL-C) for the CS-exposure group were significantly higher than those in the control group (P < 0.05). Moreover, CS exposure decreased the LDLR expression in the hepatocytes and promoted inflammation in the blood vessel walls. Melatonin was intraperitoneally injected at 10 mg/kg/d for 6 weeks alongside CS exposure, and this significantly decreased the levels of TC, TGs, and LDL-C and decreased the expression of intercellular adhesion molecule-1 and the infiltration of cluster determinant 68-cells. In vitro, CS extract prepared by bubbling CS through phosphate-buffered saline decreased the LDLR expression in HepG2 cells in a time- and concentration-dependent manner, and this effect was prevented by pretreatment with 100 µM melatonin. CONCLUSIONS: In conclusion, CS exposure impaired lipid metabolism and decreased LDLR expression in hepatocytes, and these effects could be prevented by melatonin supplementation. These findings implied that melatonin has the potential therapeutic applicability in the prevention of lipid metabolic disorder in smokers.

White tea and its active polyphenols lower cholesterol through reduction of very-low-density lipoprotein production and induction of LDLR expression.

Emerging in vivo and vitro data suggest that white tea extract (WTE) is capable of favourably modulating metabolic syndrome, especially by ameliorating abnormal lipid metabolism. Microarray-based gene expression profiling was performed in HepG2 cells to analyze the effects of WTE from a systematic perspective. Gene Ontology and pathway analysis revealed that WTE significantly affected pathways related to lipid metabolism. WTE significantly downregulated apolipoprotein B (APOB) and microsomal triglyceride transfer protein (MTTP) expression and thereby reduced the production of very-low-density lipoprotein. In the meanwhile, WTE stimulated low-density lipoprotein-cholesterol (LDL-c) uptake through targeting low-density lipoprotein receptor (LDLR), as a consequence of the activation of sterol regulatory element-binding protein 2 (SREBP2) and peroxisome proliferator-activated receptor δ (PPARδ). Furthermore, WTE significantly downregulated triglycerides synthetic genes and reduced intracellular triglycerides accumulation. Besides, we demonstrated that the tea catechins epigallocatechin-3-gallate (EGCG) and epicatechin-3-gallate (ECG) are abundant in WTE and contribute to the regulation of cholesterol metabolism related genes, including LDLR, MTTP and APOB. Our findings suggest white tea plays important roles in ameliorating abnormal lipid metabolism in vitro.

Himalayan Nettle Girardinia diversifolia as a Candidate Ingredient for Pharmaceutical and Nutraceutical Applications-Phytochemical Analysis and In Vitro Bioassays.

Girardinia diversifolia, also known as Himalayan nettle, is a perennial herb used in Nepal to make fiber as well as in traditional medicine for the treatment of several diseases. To date, phytochemical studies and biological assays on this plant are scarce. Thus, in the present work, the G. diversifolia extracts have been evaluated for their potential pharmaceutical, cosmetic and nutraceutical uses. For this purpose, detailed phytochemical analyses were performed, evidencing the presence of phytosterols, fatty acids, carotenoids, polyphenols and saponins. The most abundant secondary metabolites were ß- and γ-sitosterol (11 and 9% dw, respectively), and trans syringin (0.5 mg/g) was the most abundant phenolic. Fatty acids with an abundant portion of unsaturated derivatives (linoleic and linolenic acid at 22.0 and 9.7 mg/g respectively), vitamin C (2.9 mg/g) and vitamin B2 (0.12 mg/g) were also present. The antioxidant activity was moderate while a significant ability to inhibit acetylcholinesterase (AChE), butyrilcholinesterase (BuChE), tyrosinase, α-amylase and α-glucosidase was observed. A cytotoxic effect was observed on human ovarian, pancreatic and hepatic cancer cell lines. The effect in hepatocarcinoma cells was associated to a downregulation of the low-density lipoprotein receptor (LDLR), a pivotal regulator of cellular cholesterol homeostasis. These data show the potential usefulness of this species for possible applications in pharmaceuticals, nutraceuticals and cosmetics.

Association of single nucleotide polymorphisms with dyslipidemia in antiretroviral exposed HIV patients in a Ghanaian population: A case-control study.

Dyslipidemia is a potential complication of long-term usage of antiretroviral therapy (ART) and also known to be associated with genetic factors. The host genetic variants associated with dyslipidemia in HIV patients on ART in Ghana have not been fully explored. The study constituted a total of 289 HIV-infected patients on stable ART for at least a year. Fasting blood was collected into EDTA tube for lipids measurement. Lipid profiles were used to define dyslipidemia based on the NCEP-ATP III criteria. HIV-infected subjects were categorized into two groups; those with dyslipidemia (cases) (n = 90; 31.1%) and without dyslipidemia (controls)(n = 199; 68.9%). Four candidate single nucleotide polymorphism (SNP) genes (ABCA1-rs2066714, LDLR-rs6511720, APOA5-rs662799 and DSCAML1-rs10892151) were determined. Genotyping was performed on isolated genomic DNA of study participants using PCR followed by a multiplex ligation detection reaction (LDR). The percentage of the population who had the rare homozygote alleles for rs6511720 (T/T), rs2066714 (G/G), rs10892151 (T/T) and rs662799 (G/G) among case subjects were 5.5%, 14.4%, 6.6% and 10.0% whiles 2.0% 9.1%, 6.5% and 4.0% were observed among control subjects. There were statistically significant differences in the genotypic prevalence of APOA5 (p = 0.0357) and LDLR polymorphisms (p = 0.0387) between case and control subjects. Compared to the AA genotype of the APOA5 polymorphisms, individuals with the rare homozygote genotype [aOR = 2.38, 95%CI(1.06-6.54), p = 0.004] were significantly associated with an increased likelihood of developing dyslipidemia after controlling for age, gender, treatment duration, CD4 counts and BMI. Moreover, individuals with the rare homozygous genotype of ABCA1 (G/G) [aOR = 10.7(1.3-88.7), p = 0.0280] and LDLR (rs6511720) G>T [aOR = 61.2(7.6-493.4), p<0.0001) were more likely to have high levels of total cholesterol levels. Our data accentuate the presence of SNPs in four candidate genes and their association with dyslipidemia among HIV patients exposed to ART in the Ghanaian population, especially variants in APOA5-rs662799 and LDLR rs6511720 respectively. These findings provide baseline information that necessitates a pre-symptomatic strategy for monitoring dyslipidemia in ART-treated HIV patients. There is a need for longitudinal studies to validate a comprehensive number of SNPs and their associations with dyslipidemia.

Dietary Choline or Trimethylamine N-oxide Supplementation Does Not Influence Atherosclerosis Development in Ldlr-/- and Apoe-/- Male Mice.

BACKGROUND: Choline, an essential nutrient, is required for cell membranes, lipoprotein secretion, and methyl-group metabolism. Recently, it has been proposed that excess dietary choline consumption is metabolized to trimethylamine (TMA) by the gut microbiota; TMA is then oxidized to trimethylamine N-oxide (TMAO) in the liver. Epidemiological studies have clearly shown a positive correlation between plasma TMAO concentrations and cardiovascular events. Furthermore, some studies have shown an association between excess dietary choline, plasma TMAO concentrations, and atherosclerotic lesion size in apoE knockout (Apoe-/-) mice. OBJECTIVE: The aim of this study was to further investigate the relation between dietary choline and atherosclerosis in 2 atherogenic mouse models, the LDL receptor knockout (Ldlr-/-) and Apoe-/- mice. METHODS: Six feeding trials were performed in Ldlr-/- (40% high-fat diet) and Apoe-/- (unpurified diet) male mice, aged 8-10 wk. Mice randomly received control diet (0.1% choline), or choline- (1% choline), betaine- (0.1% choline and 0.9% betaine), or TMAO- (0.1% choline and 0.12% or 0.2% TMAO) supplemented diet for ≤28 wk. After the dietary intervention, the animals were killed and tissues and blood collected. Aortic atherosclerotic plaque area, plasma lipids, and choline metabolites were quantified. RESULTS: In Ldlr-/- mice, dietary supplementation for 8 wk with choline or TMAO increased plasma TMAO concentrations by 1.6- and 4-fold, respectively. After 16 wk, there was a 2-fold increase in plasma TMAO after dietary TMAO supplementation. In Apoe-/- mice, dietary supplementation with choline, betaine, or TMAO for 12 wk did not increase plasma TMAO concentrations. However, choline and TMAO supplementation for 28 wk significantly increased plasma TMAO concentrations by 1.8- and 1.5-fold, respectively. Contrary to predictions, atherosclerotic lesion size was not altered by any of the dietary interventions, irrespective of mouse model. CONCLUSIONS: In our study, high intakes of dietary choline or TMAO supplementation did not influence atherosclerosis development in Ldlr-/- or Apoe-/- male mice.

Effects of Chromium and Carnitine Co-supplementation on Body Weight and Metabolic Profiles in Overweight and Obese Women with Polycystic Ovary Syndrome: a Randomized, Double-Blind, Placebo-Controlled Trial.

The primary aim of our study was to determine the influence of taking chromium plus carnitine on insulin resistance, with a secondary objective of evaluating the influences on lipid profiles and weight loss in overweight subjects with polycystic ovary syndrome (PCOS). In a 12-week randomized, double-blind, placebo-controlled clinical trial, 54 overweight women were randomly assigned to receive either supplements (200 µg/day chromium picolinate plus 1000 mg/day carnitine) or placebo (27/each group). Chromium and carnitine co-supplementation decreased weight (- 3.6 ± 1.8 vs. - 1.0 ± 0.7 kg, P < 0.001), BMI (- 1.3 ± 0.7 vs. - 0.3 ± 0.3 kg/m2, P < 0.001), fasting plasma glucose (FPG) (- 5.1 ± 6.0 vs. - 1.1 ± 4.9 mg/dL, P = 0.01), insulin (- 2.0 ± 1.4 vs. - 0.2 ± 1.2 µIU/mL, P < 0.001), insulin resistance (- 0.5 ± 0.4 vs. - 0.04 ± 0.3, P < 0.001), triglycerides (- 18.0 ± 25.2 vs. + 5.5 ± 14.4 mg/dL, P < 0.001), total (- 17.0 ± 20.3 vs. + 3.6 ± 12.0 mg/dL, P < 0.001), and LDL cholesterol (- 13.3 ± 19.2 vs. + 1.4 ± 13.3 mg/dL, P = 0.002), and elevated insulin sensitivity (+ 0.007 ± 0.005 vs. + 0.002 ± 0.005, P < 0.001). In addition, co-supplementation upregulated peroxisome proliferator-activated receptor gamma (P = 0.02) and low-density lipoprotein receptor expression (P = 0.02). Overall, chromium and carnitine co-supplementation for 12 weeks to overweight women with PCOS had beneficial effects on body weight, glycemic control, lipid profiles except HDL cholesterol levels, and gene expression of PPAR-γ and LDLR. Clinical trial registration number: http://www.irct.ir: IRCT20170513033941N38.

Dietary Supplementation with Galactooligosaccharides Attenuates High-Fat, High-Cholesterol Diet-Induced Glucose Intolerance and Disruption of Colonic Mucin Layer in C57BL/6 Mice and Reduces Atherosclerosis in Ldlr-/- Mice.

BACKGROUND: A Western-type diet (WD), rich in fat and cholesterol but deficient in fiber, induces development of diabetes and atherosclerosis. Colonic bacteria use the gut's mucous lining as an alternate energy source during periods of fiber deficiency, resulting in intestinal barrier erosion. OBJECTIVE: We hypothesized that supplementing a WD with galactooligosaccharide (GOS) fiber would attenuate WD-induced mucin layer disruption and attenuate development of metabolic diseases. METHODS: C57BL/6 mice (both sexes, 8-10 wk of age) were fed a standard rodent diet (TD7012, reference) or a high-fat, high-cholesterol-containing WD (TD88137, 21% fat, 0.15% cholesterol, 19.5% caesin) or a WD supplemented with 5% GOS fiber (TD170432, WD + GOS) for 16 wk. WD-fed mice that were gavaged daily with curcumin (100 mg/kg) served as positive controls. Glucose tolerance, colonic mucin layer, gene expression, and circulating macrophage/neutrophil levels were determined. Hyperlipidemic Ldlr-/- mice (both sexes, 8-10 wk of age) fed a WD with or without GOS supplementation (for 16 wk) were used to assess plasma LPS and atherosclerosis. Effects of dietary supplementation on different parameters were compared for each genotype. RESULTS: Compared with a WD, glucose tolerance was significantly improved in male C57BL/6 mice fed a WD + GOS (mean ± SEM: AUC = 53.6 ± 43.9 compared with 45.4 ± 33.3 g ⋅ min/dL; P = 0.015). Continuity of colonic mucin layer (MUC-2 expression) was improved in mice receiving GOS supplementation, indicating improved intestinal barrier. GOS supplementation also reduced circulating macrophages (30% decrease) and neutrophils (60% decrease), suggesting diminished systemic inflammation. In Ldlr-/- mice, GOS supplementation significantly reduced plasma LPS concentrations (mean ± SEM: 0.81 ±  0.43 EU/mL compared with 0.32 ± 0.26 EU/mL, P   < 0.0001, in females and 0.56 ± 0.24 EU/mL compared with 0.34 ± 0.12 EU/mL, P = 0.036, in males), improved glucose tolerance in male mice, and attenuated atherosclerotic lesion area (mean ± SEM: 54.2% ± 6.19% compared with 43.0% ± 35.12%, P   = 0.0006, in females and 54.6% ± 3.99% compared with 43.1% ± 8.11%, P = 0.003, in males). CONCLUSIONS: GOS fiber supplementation improves intestinal barrier in C57BL/6 and Ldlr-/- mice and significantly attenuates WD-induced metabolic diseases and, therefore, may represent a novel strategy for management of these diseases.

Niacin protects against abdominal aortic aneurysm formation via GPR109A independent mechanisms: role of NAD+/nicotinamide.

AIMS: Chronic adventitial and medial infiltration of immune cells play an important role in the pathogenesis of abdominal aortic aneurysms (AAAs). Nicotinic acid (niacin) was shown to inhibit atherosclerosis by activating the anti-inflammatory G protein-coupled receptor GPR109A [also known as hydroxycarboxylic acid receptor 2 (HCA2)] expressed on immune cells, blunting immune activation and adventitial inflammatory cell infiltration. Here, we investigated the role of niacin and GPR109A in regulating AAA formation. METHODS AND RESULTS: Mice were supplemented with niacin or nicotinamide, and AAA was induced by angiotensin II (AngII) infusion or calcium chloride (CaCl2) application. Niacin markedly reduced AAA formation in both AngII and CaCl2 models, diminishing adventitial immune cell infiltration, concomitant inflammatory responses, and matrix degradation. Unexpectedly, GPR109A gene deletion did not abrogate the protective effects of niacin against AAA formation, suggesting GPR109A-independent mechanisms. Interestingly, nicotinamide, which does not activate GPR109A, also inhibited AAA formation and phenocopied the effects of niacin. Mechanistically, both niacin and nicotinamide supplementation increased nicotinamide adenine dinucleotide (NAD+) levels and NAD+-dependent Sirt1 activity, which were reduced in AAA tissues. Furthermore, pharmacological inhibition of Sirt1 abrogated the protective effect of nicotinamide against AAA formation. CONCLUSION: Niacin protects against AAA formation independent of GPR109A, most likely by serving as an NAD+ precursor. Supplementation of NAD+ using nicotinamide-related biomolecules may represent an effective and well-tolerated approach to preventing or treating AAA.