Albino mice with the point mutation at the tyrosinase locus show high cholesterol diet-induced NASH susceptibility.

Non-alcoholic fatty liver disease (NAFLD) constitutes a metabolic disorder with high worldwide prevalence and increasing incidence. The inflammatory progressive state, non-alcoholic steatohepatitis (NASH), leads to liver fibrosis and carcinogenesis. Here, we evaluated whether tyrosinase mutation underlies NASH pathophysiology. Tyrosinase point-mutated B6 (Cg)-Tyrc-2J/J mice (B6 albino) and C57BL/6J black mice (B6 black) were fed with high cholesterol diet (HCD) for 10 weeks. Normal diet-fed mice served as controls. HCD-fed B6 albino exhibited high NASH susceptibility compared to B6 black, a phenotype not previously reported. Liver injury occurred in approximately 50% of B6 albino from one post HCD feeding, with elevated serum alanine aminotransferase and aspartate aminotransferase levels. NASH was induced following 2 weeks in severe-phenotypic B6 albino (sB6), but B6 black exhibited no symptoms, even after 10 weeks. HCD-fed sB6 albino showed significantly higher mortality rate. Histological analysis of the liver revealed significant inflammatory cell and lipid infiltration and severe fibrosis. Serum lipoprotein analysis revealed significantly higher chylomicron and very low-density lipoprotein levels in sB6 albino. Moreover, significantly higher small intestinal lipid absorption and lower fecal lipid excretion occurred together with elevated intestinal NPC1L1 expression. As the tyrosinase point mutation represents the only genetic difference between B6 albino and B6 black, our work will facilitate the identification of susceptible genetic factors for NASH development and expand the understanding of NASH pathophysiology.

RIPK1 Expression Associates With Inflammation in Early Atherosclerosis in Humans and Can Be Therapeutically Silenced to Reduce NF-κB Activation and Atherogenesis in Mice.

BACKGROUND: Chronic activation of the innate immune system drives inflammation and contributes directly to atherosclerosis. We previously showed that macrophages in the atherogenic plaque undergo RIPK3 (receptor-interacting serine/threonine-protein kinase 3)-MLKL (mixed lineage kinase domain-like protein)-dependent programmed necroptosis in response to sterile ligands such as oxidized low-density lipoprotein and damage-associated molecular patterns and that necroptosis is active in advanced atherosclerotic plaques. Upstream of the RIPK3-MLKL necroptotic machinery lies RIPK1 (receptor-interacting serine/threonine-protein kinase 1), which acts as a master switch that controls whether the cell undergoes NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells)-dependent inflammation, caspase-dependent apoptosis, or necroptosis in response to extracellular stimuli. We therefore set out to investigate the role of RIPK1 in the development of atherosclerosis, which is driven largely by NF-κB-dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NF-κB-dependent inflammation in early atherogenic lesions, and knocking down RIPK1 will reduce inflammatory cell activation and protect against the progression of atherosclerosis. METHODS: We examined expression of RIPK1 protein and mRNA in both human and mouse atherosclerotic lesions, and used loss-of-function approaches in vitro in macrophages and endothelial cells to measure inflammatory responses. We administered weekly injections of RIPK1 antisense oligonucleotides to Apoe-/- mice fed a cholesterol-rich (Western) diet for 8 weeks. RESULTS: We find that RIPK1 expression is abundant in early-stage atherosclerotic lesions in both humans and mice. Treatment with RIPK1 antisense oligonucleotides led to a reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control, P<0.01) and plasma inflammatory cytokines (IL-1α [interleukin 1α], IL-17A [interleukin 17A], P<0.05) in comparison with controls. RIPK1 knockdown in macrophages decreased inflammatory genes (NF-κB, TNFα [tumor necrosis factor α], IL-1α) and in vivo lipopolysaccharide- and atherogenic diet-induced NF-κB activation. In endothelial cells, knockdown of RIPK1 prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of IL1B, E-selectin, and monocyte attachment. CONCLUSIONS: We identify RIPK1 as a central driver of inflammation in atherosclerosis by its ability to activate the NF-κB pathway and promote inflammatory cytokine release. Given the high levels of RIPK1 expression in human atherosclerotic lesions, our study suggests RIPK1 as a future therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.

Dietary Oxysterol, 7-Ketocholesterol Accelerates Hepatic Lipid Accumulation and Macrophage Infiltration in Obese Mice.

Non-alcoholic fatty liver disease is strongly associated with obese and type 2 diabetes. It has been reported that an oxidized cholesterol, 7-ketocholesterol (7KC), might cause inflammatory response in macrophages and plasma 7KC concentration were higher in patients with cardiovascular diseases or diabetes. Therefore, we have decided to test whether small amount of 7KC in diet might induce hepatic steatosis and inflammation in two types of obese models. We found that addition of 0.01% 7KC either in chow diet (CD, regular chow diet with 1% cholesterol) or western type diet (WD, high fat diet with 1% cholesterol) accelerated hepatic neutral lipid accumulation by Oil Red O staining. Importantly, by lipid extraction analysis, it has been recognized that triglyceride rather than cholesterol species was significantly accumulated in CD+7KC compared to CD as well as in WD+7KC compared to WD. Immunostaining revealed that macrophages infiltration was increased in CD+7KC compared to CD, and also in WD+7KC compared to WD. These phenotypes were accompanied by inducing inflammatory response and downregulating fatty acid oxidation. Furthermore, RNA sequence analysis demonstrated that 7KC reduced expression of genes which related to autophagy process. Levels of LC3-II protein were decreased in WD+7KC compared to WD. Similarly, we have confirmed the effect of 7KC on acceleration of steatohepatitis in db/db mice model. Collectively, our study has demonstrated that small amount of dietary 7KC contributed to accelerate hepatic steatosis and inflammation in obese mice models.

Myeloid cells in liver and bone marrow acquire a functionally distinct inflammatory phenotype during obesity-related steatohepatitis.

OBJECTIVE: Bone marrow-derived myeloid cells accumulate in the liver as monocytes and macrophages during the progression of obesity-related non-alcoholic fatty liver disease (NAFLD) to steatohepatitis (NASH). Myeloid cells comprise heterogeneous subsets, and dietary overnutrition may affect macrophages in the liver and bone marrow. We therefore aimed at characterising in depth the functional adaptations of myeloid cells in fatty liver. DESIGN: We employed single-cell RNA sequencing to comprehensively assess the heterogeneity of myeloid cells in the liver and bone marrow during NAFLD, by analysing C57BL/6 mice fed with a high-fat, high-sugar, high-cholesterol 'Western diet' for 16 weeks. We also characterised NAFLD-driven functional adaptations of macrophages in vitro and their functional relevance during steatohepatitis in vivo. RESULTS: Single-cell RNA sequencing identified distinct myeloid cell clusters in the liver and bone marrow. In both compartments, monocyte-derived populations were largely expanded in NASH-affected mice. Importantly, the liver myeloid compartment adapted a unique inflammatory phenotype during NAFLD progression, exemplarily characterised by downregulated inflammatory calprotectin (S100A8/A9) in macrophage and dendritic cell subsets. This distinctive gene signature was also found in their bone marrow precursors. The NASH myeloid phenotype was principally recapitulated by in vitro exposure of bone marrow-derived macrophages with fatty acids, depended on toll-like receptor 4 signalling and defined a characteristic response pattern to lipopolysaccharide stimulation. This imprinted and stable NASH myeloid immune phenotype functionally determined inflammatory responses following acute liver injury (acetaminophen poisoning) in vivo. CONCLUSION: Liver myeloid leucocytes and their bone marrow precursors adapt a common and functionally relevant inflammatory signature during NAFLD progression.

Pioglitazone prevents cholesterol gallstone formation through the regulation of cholesterol homeostasis in guinea pigs with a lithogenic diet.

BACKGROUND: The cholesterol gallstones diseases (CGD) is highly correlated with metabolic syndrome and type 2 diabetes. The present study aimed to investigate preventive effects of pioglitazone (PIO), an antidiabetic drug, on the CGD in guinea pigs fed with a lithogenic diet (LD). METHODS: The guinea pigs were fed with the LD for 8 weeks. All guinea pigs were grouped as follows: low fat diet; LD; LD plus PIO (4 mg/kg); LD plus PIO (8 mg/kg); LD plus ezetimibe (EZE) (2 mg/kg). Gallbladder stones were observed using microscopy. The profile of biliary composition, and blood glucose, insulin and lipid were analyzed. The liver or ileum was harvested for determinations of hydroxyl-methyl-glutaryl-CoA reductase (HMGCR), sterol regulatory element-binding proteins 2 (SREBP2), 7α-hydroxylase (CYP7A1), adenosine triphosphate-binding cassette (ABC) sterol transporters G5 and G8 (ABCG5, ABCG8), bile salt export pump (BSEP), Niemann-Pick C1-Like 1 (NPC1L1) and acetyl-coenzyme A cholesterol acyltransferase (ACAT2) by Western blot. The gallbladders were used for histological examination. RESULTS: The LD successfully induced gallstone. Both pioglitazone and ezetimibe prevented gallstone formation, as well as hepatic and cholecystic damages. Pioglitazone significantly decreased HMGCR and SREBP2, but increased CYP7A1, ABCG5, ABCG8, and BSEP in the liver. Pioglitazone also remarkably decreased NPC1L1 and ACAT2, while increased ABCG5/8 in the intestine. The beneficial alterations of cholesterol and bile acids in the bile, as well as profile of glucose, insulin and lipid in the blood were found in the guinea pigs treated with pioglitazone. CONCLUSION: Pioglitazone has a noticeable benefit towards the CGD, which is involved in changes of synthesis, transformation, absorption, and transportation of cholesterol.

Dietary Supplementation with Hazelnut Oil Reduces Serum Hyperlipidemia and Ameliorates the Progression of Nonalcoholic Fatty Liver Disease in Hamsters Fed a High-Cholesterol Diet.

Objective: Hazelnut oil (HO) is rich in monounsaturated fatty acids and polyunsaturated fatty acids. This study intended to analyze the effects of hazelnut oil supplementation on the serum lipid profile and nonalcoholic fatty liver disease in hamsters fed a high-cholesterol (HC) diet. Methods: Hamsters were fed a basic diet (control group) and an HC diet (HC group) for 16 weeks (n = 10 in each group). Hamsters were fed an HC diet for four weeks to induce hyperlipidemia and were then fed an HC diet enriched with 5% (low-dose HC + HO group; n = 10) and 10% HO (high-dose HC + HO group; n = 10) for 12 weeks. Serum lipid levels, hepatic changes (including steatosis, inflammation, and fibrosis), and hepatic prooxidant-antioxidant status (malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione S-transferase (GST)) were evaluated after the treatment period. Results: Hamsters in the control group showed normal serum lipid profiles, normal liver function, and moderate glycogen storage without hepatic steatosis. Hamsters in the HC group showed severe hyperlipidemia, severe hepatic steatosis, and moderate steatohepatitis (mononuclear cell and neutrophil infiltration, oval cell hyperplasia, and fibrosis). Compared to the HC group, both the low-dose and the high-dose HC + HO groups showed a significant reduction of hyperlipidemia (serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and very-low-density lipoprotein cholesterol (VLDL-C levels)) and improved liver function (serum glutamic-oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT)). Additionally, compared to the HC group, intrahepatic triglyceride accumulation (IHTC) was significantly higher in the HC + HO group, while the incidence of steatohepatitis was significantly lower. The intake of the HC diet was associated with a higher level of lipid peroxidation (malondialdehyde, MDA) and a lower concentration of hepatic antioxidant enzymes (SOD, GPx, and GST), and all these factors were partially improved in the low-dose and high-dose HC + HO groups. Conclusions: Our findings indicate that the intake of HO reduced serum hyperlipidemia and oxidative stress and ameliorated the progression of nonalcoholic fatty liver disease in hamsters fed a high-cholesterol diet.

Antioxidant and antihyperlipidemic effects of Ajwa date (Phoenix dactylifera L.) extracts in rats fed a cholesterol-rich diet.

This study investigated the chemical composition, in vivo antioxidant, and antihyperlipidemic potential of Ajwa date polyphenol extract (DPE). Chemical analysis revealed that the Ajwa dates contain substantial amounts of carbohydrates, energy, potassium, iron, polyphenols, and flavonoids. In vivo studies showed that feeding rats with cholesterol-rich diets significantly (p ≤ 0.05) increased their body and liver weights, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), very-low-density lipoprotein cholesterol (VLDL-C), and triglycerides (TG) in plasma and liver, and reduced the high-density lipoprotein cholesterol (HDL-C) content and antioxidant enzyme activities. However, oral administration of 25, 50, and 100 mg DPE/kg body weight to hypercholestrolemic rats, significantly (p ≤ 0.05) reduced their body and liver weights, total hepatic cholesterol, LDL-C, VLDL-C, and triglycerides. Furthermore, treatment with DPE improved (p ≤ 0.05) the HDL-C concentration and antioxidant enzyme activity in a concentration-dependent fashion, thereby regulating lipid profiles, enhancing the antioxidant defense system. Overall, DPE showed significant (p ≤ 0.05) nutritional, antioxidants, and antihyperlipidemic benefits. PRACTICAL APPLICATIONS: Coronary heart disease is the leading cause of death in the world. Consumption of fruits and vegetables rich in polyphenol have shown to exert cardio-protective effect. This work revealed that phenolic extracts of Ajwa dates have positive impacts on the health as it reduced TC, LDL-C, and lipids VLDL-C and improved HDL-C and the antioxidant defense system in rats. The findings of this study could aid in the profound understanding of the nutritional and health potentials of Ajwa dates and thus could help in utilization of these valuable fruits for the prevention and curing of cardiovascular diseases.

Fiber and phenolic compounds contribution to the hepatoprotective effects of mango diets in rats fed high cholesterol/sodium cholate.

The present study evaluated the contribution of mango fiber (MF) and mango phenolic compounds (MP) to the hepatoprotective effect of freeze-dried mango pulp (FDM) cultivar (cv.) "Ataulfo" diets in high cholesterol/sodium cholate (HCC)-fed rats. Male Wistar rats were fed with a HCC diet for 12 weeks, either untreated, or supplemented with MF, MP, FDM, or a control diet (no HCC; n = 6/group). All mango treatments significantly decreased hepatic cholesterol deposition and altered its fatty acid profile, whereas MF and MP mitigated adipose tissue hypertrophy. MF caused a lower level of proinflammatory cytokines (IL-1α/ß, IFN-γ, TNF-α) whereas FDM increased the anti-inflammatory ones (IL-4, 6, 10). Mango treatments increased catalase (CAT) activity and its mRNA expression; superoxide dismutase (SOD) activity was normalized by MF and FDM, but its activity was unrelated to its hepatic mRNA expression. Changes in CAT and SOD mRNA expression were unrelated to altered Nrf2 mRNA expression. Higher hepatic PPARα and LXRα mRNA levels were found in MP and MF. We concluded that MF and MP are highly bioactive, according to the documented hepatoprotection in HCC-fed rats; their mechanism of action appears to be related to modulating cholesterol and fatty acid metabolism as well as to stimulating the endogenous antioxidant system.

Dietary Intake of Fatty Acids, Total Cholesterol, and Stomach Cancer in a Chinese Population.

To investigate the associations between dietary fatty acids and cholesterol consumption and stomach cancer (SC), we analyzed data from a population-based case-control study with a total of 1900 SC cases and 6532 controls. Dietary data and other risk or protective factors were collected by face-to-face interviews in Jiangsu Province, China, from 2003 to 2010. Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using multiple unconditional logistic regression models and an energy-adjusted method. The joint associations between dietary factors and known risk factors on SC were examined. We observed positive associations between dietary saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and total cholesterol and the development of SC, comparing the highest versus lowest quarters. Increased intakes of dietary SFAs (p-trend = 0.005; aOR, 1.11; 95% CI, 1.01-1.22 with a 7 g/day increase as a continuous variable) and total cholesterol (p-trend < 0.001; aOR, 1.13; 95% CI, 1.06-1.22 with a 250 mg/day increase as a continuous variable) were monotonically associated with elevated odds of developing SC. Our results indicate that dietary SFAs, MUFAs, and total cholesterol are associated with stomach cancer, which might provide a potential dietary intervention for stomach cancer prevention.

Vertical Sleeve Gastrectomy Attenuates the Progression of Non-Alcoholic Steatohepatitis in Mice on a High-Fat High-Cholesterol Diet.

OBJECTIVE: To determine whether vertical sleeve gastrectomy (VSG) attenuates fibrosis in mice on a high-fat high-cholesterol (HFHC) diet. BACKGROUND: Bariatric surgery mitigates non-alcoholic steatohepatitis in 85-90% of obese patients. While animal models demonstrate similar results on a high-fat diet, none have observed the effects of bariatric surgery on a combined HFHC diet. METHODS: Mice on a HFHC diet were used to confirm the development of hepatic fibrosis at 8 (n = 15) and 24 (n = 15) weeks. A separate cohort of mice on a HFHC diet for 12 weeks was subjected to either VSG (n = 18) or sham (n = 12) operations and remained on a HFHC diet for an additional 20 weeks. Changes in weight, dyslipidemia, and the development of steatosis and fibrosis were documented. Serum was obtained for bile acid analysis by liquid chromatography and mass spectrometry, while hepatic gene expression by RT-PCR was performed to evaluate intrahepatic lipid metabolism. RESULTS: Hepatic steatosis and fibrosis developed after 8 weeks on the HFHC diet. After VSG, mice demonstrated a sustained decrease in weight with a significant decrease in fibrosis compared to sham mice. Serum total cholesterol, HDL, and LDL were significantly reduced following surgery, while serum bile acids were significantly elevated. Intra-hepatic cholesterol excretion was not upregulated based on hepatic gene expression of CYP7A1 and ABCG5/8. CONCLUSIONS: VSG attenuates the development of hepatic fibrosis in diet-induced obese mice, presumably through enhancement of cholesterol elimination at the intestinal level.

Formulation and Characterization of Quercetin-loaded Oil in Water Nanoemulsion and Evaluation of Hypocholesterolemic Activity in Rats.

Due to poor water solubility and high susceptibility to chemical degradation, the applications of quercetin have been limited. This study investigated the effects of pH on the formation of quercetin-loaded nanoemulsion (NQ) and compared the hypocholesterolemic activity between quercetin and NQ to utilize the quercetin as functional food ingredient. NQ particle size exhibited a range of 207⁻289 nm with polydispersity index range (<0.47). The encapsulation efficiency increased stepwise from 56 to 92% as the pH increased from 4.0 to 9.0. Good stability of NQ was achieved in the pH range of 6.5⁻9.0 during 3-month storage at 21 and 37 °C. NQ displayed higher efficacy in reducing serum and hepatic cholesterol levels and increasing the release of bile acid into feces in rats fed high-cholesterol diet, compared to quercetin alone. NQ upregulated hepatic gene expression involved in bile acid synthesis and cholesterol efflux, such as cholesterol 7 alpha-hydroxylase (CYP7A1), liver X receptor alpha (LXRα), ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette sub-family G member 1 (ABCG1). These results suggest at least partial involvement of hepatic bile acid synthesis and fecal cholesterol excretion in nanoemulsion quercetin-mediated beneficial effect on lipid abnormalities.

Egg Consumption in Relation to Glioma: A Case-Control Study.

BACKGROUND AND AIMS: Data on the link between egg consumption and brain tumors are limited. The objective of this study was to investigate the association between egg consumption and odds of glioma in Iranian adults. METHODS: In this hospital-based case-control study, 128 newly-diagnosed cases of glioma and 256 age- and sex-matched controls were enrolled from hospitals. Patients with pathologically confirmed glioma (without any other cancers) were considered eligible. We collected data on dietary intakes, including egg consumption, using a 126-item validated FFQ. Egg consumption was computed from all foods containing this food as their ingredients. Participants were categorized into tertiles of egg consumption. Conditional logistic regression was used to examine the association between egg consumption and glioma. RESULTS: After adjustment for age, sex and energy intake, individuals in the middle tertile of egg consumption were 58% (OR 0.42; 95% CI 0.24, 0.73) less likely to have glioma compared with those in the lowest tertile. Further adjustment for other potential confounders strengthened the association; such that participants with the greatest consumption of egg intake were 62% (0.38; 0.18, 0.76) less likely to have glioma compared with those with the lowest consumption. Additional controlling for dietary intakes did not change the association significantly (0.39; 0.18, 0.85). CONCLUSION: We found an inverse association between egg consumption at the amount of almost 2 eggs/week and odds of glioma. Further studies are required to examine this association.

Lipoprotein Profile in Aged Rats Fed Chia Oil- or Hydroxytyrosol-Enriched Pork in High Cholesterol/High Saturated Fat Diets.

Restructuring pork (RP) by adding new functional ingredients, like Chia oil (one of the richest natural source of α-linolenic acid) or hydroxytyrosol (HxT) (potent antioxidant), both with hypolipidemic activities, is one of the strategies that may help to reduce the potential negative effects of high meat products consumption. The aim of this study was to evaluate the Chia oil- or HxT-enriched-RP effect on the lipoprotein profile of aged rats fed high-fat, high-energy, and cholesterol-enriched diets. RP samples were prepared by mixing lean pork and lard with or without Chia oil (152.2 g/kg fresh matter) or HxT (3.6 g/kg fresh matter). Diets were prepared by mixing a semisynthetic diet with freeze-dried RP. Groups of 1-year male Wistar rats were fed the following experimental diets for 8 weeks: C, control-RP diet; HC, cholesterol-enriched-RP diet; and Chia oil-RP (CHIA) and HxT, Chia oil- or hydroxytyrosol-RP, cholesterol-enriched diet. Plasma lipid, lipoprotein profile, SREBP-1c protein, and low-density lipoproteins (LDL) receptor gene (Ldlr) expressions were evaluated. Compared to C diet, the HC diet increased plasma cholesterol, triglycerides, free fatty acids, total lipids, and SREBP-1c expression, but reduced Ldlr expression and significantly modified the lipoprotein profile, giving rise to the presence of high levels of atherogenic cholesterol-enriched very low-density lipoproteins (VLDL) particles. Compared to the HC diet, the HxT diet did not produce significant changes in feed intake but it reduced the body weight. Chia oil and HxT partially arrested the negative effects of the high-fat, high-energy, and cholesterol-enriched meat-based diets on lipemia and lipoproteinemia, mostly by reducing the amount of cholesterol content in VLDL (60% and 74% less in CHIA and HxT vs. HC, respectively) and the VLDL total mass (59% and 63% less in CHIA and HxT vs. HC, respectively). Free fatty acids (FFA) significantly correlated with adipose tissue weight and VLDL total mass (both p < 0.05), and plasma triglycerides, phospholipids, total lipids, and SREBP-1c (all p < 0.001), suggesting the important role of FFA in lipoprotein metabolism. Results support the recommendation to include these ingredients in pork products addressed to reduce the presence of increased atherogenic particles in aged people at CVD risk consuming large amounts of pork.

Hepatic Expression of PEMT, but Not Dietary Choline Supplementation, Reverses the Protection against Atherosclerosis in Pemt-/-/Ldlr-/- Mice.

Background: Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine to phosphatidylcholine. Pemt-/-/low density lipoprotein receptor (Ldlr)-/- mice have significantly reduced plasma lipids and are protected against atherosclerosis. Recent studies have shown that choline can be metabolized by the gut flora into trimethylamine-N-oxide (TMAO), which is an emerging risk factor for atherosclerosis. Objective: The objective of this study was to determine whether ectopic hepatic PEMT expression or choline supplementation would promote atherosclerosis in Pemt-/-/Ldlr-/- mice. Methods: Male 8- to 10-wk-old Pemt+/+/Ldlr-/- (SKO) and Pemt-/-/Ldlr-/- (DKO) mice were injected with an adeno-associated virus (AAV) expressing green fluorescent protein (GFP) or human PEMT and fed a Western diet (40% of calories from fat, 0.5% cholesterol) for 8 wk. In a separate experiment, 8- to 10-wk-old SKO and half of the DKO male mice were fed a Western diet with normal (3 g/kg) choline for 12 wk. The remaining DKO mice [choline-supplemented (CS) DKO] were fed a CS Western diet (10 g choline/kg). Plasma lipid concentrations, choline metabolites, and aortic atherosclerosis were measured. Results: Plasma cholesterol, plasma TMAO, and aortic atherosclerosis were reduced by 60%, 40%, and 80%, respectively, in DKO mice compared with SKO mice. AAV-PEMT administration increased plasma cholesterol and TMAO by 30% and 40%, respectively, in DKO mice compared with AAV-GFP-treated DKO mice. Furthermore, AAV-PEMT-injected DKO mice developed atherosclerotic lesions similar to SKO mice. In the second study, there was no difference in atherosclerosis or plasma cholesterol between DKO and CS-DKO mice. However, plasma TMAO concentrations were increased 2.5-fold in CS-DKO mice compared with DKO mice. Conclusions: Reintroducing hepatic PEMT reversed the atheroprotective phenotype of DKO mice. Choline supplementation did not increase atherosclerosis or plasma cholesterol in DKO mice. Our data suggest that plasma TMAO does not induce atherosclerosis when plasma cholesterol is low. Furthermore, this is the first report to our knowledge that suggests that de novo choline synthesis alters TMAO status.

Effect of resveratrol on seric vascular endothelial growth factor concentrations during atherosclerosis. / Efecto del resveratrol sobre las concentraciones séricas del factor de crecimiento endotelial vascular durante la aterosclerosis.

INTRODUCTION: Although it is known that resveratrol has anti-inflammatory and anti-atherogenic actions, its effect on vascular endothelial growth factor (VEGF) in atherosclerosis is unknown. OBJECTIVE: To evaluate the effect of resveratrol on serum concentrations of VEGF during the progression and evolution of atherosclerosis, as well as and its evolution over time in rabbits fed with a cholesterol diet. MATERIALS AND METHODS: A total of 48 New Zealand white male rabbits were randomly divided into four groups of 12 rabbits: group1 (control): standard diet (commercial rabbit food); group2: cholesterol diet (0.5% cholesterol); group3 (control resveratrol): standard diet (commercial rabbit food) and resveratrol (2mg/Kg); group4: cholesterol diet (0.5% cholesterol) and resveratrol (2mg/Kg), for 12weeks. Blood samples of overnight-fasted rabbits were collected at baseline and the sixth and twelfth weeks, and the lipid profile, VEGF, and C-reactive protein (CRP) levels were determined. Half of the animals were sacrificed on the sixth or twelfth week, and the aorta was dissected for histological studies. RESULTS: VEGF and CRP levels were significantly higher in groups2 and 4 than in groups1 and 3, respectively, from the 6th week (p<0.001). VEGF and CRP were significantly lower in group4 than in group2 on 12th week (p<0.004). Supplementation of resveratrol reduced the formation of atherosclerotic lesions. CONCLUSIONS: Serum VEGF and CRP levels are early markers of atherosclerosis. Oral supplementation of resveratrol exerts anti-inflammatory and anti-atherosclerotic effects, decreasing serum concentrations of VEGF and CRP and the formation and evolution of atherosclerotic lesions.

Cinnamaldehyde exerts vasculoprotective effects in hypercholestrolemic rabbits.

The effects of cinnamaldehyde (CIN), a commonly consumed food flavor, against high-cholesterol diet (HCD)-induced vascular damage in rabbits were evaluated. Male New Zealand rabbits (n = 24) were allocated to four groups at random: control, fed with standard rabbit chow; CIN, fed with standard diet and administered CIN; HCD, fed with 1% cholesterol-enriched diet; and HCD-CIN, fed with HCD and treated with CIN. CIN was orally given at a dose of (10 mg/kg/day) concomitantly with each diet type from day 1 until the termination of the experimental protocol (4 weeks). HCD elicited significant elevations in serum levels of total cholesterol (TC), triglycerides (TGs), and high- and low-density lipoprotein cholesterol (HDL-C and LDL-C, respectively) compared with control rabbits. Moreover, aortic levels of nitric oxide metabolites (NOx) and antioxidant enzyme activities were significantly lower, while aortic levels of malondialdehyde (MDA) and myeloperoxidase (MPO) activity were significantly higher, in HCD-fed rabbits relative to control animals. CIN administration mitigated or completely reversed HCD-induced metabolic alterations, vascular oxidative stress, and inflammation. Moreover, CIN ameliorated HCD-induced vascular functional and structural irregularities. Aortic rings from HCD-CIN group showed improved relaxation to acetylcholine compared to aortas from HCD group. Moreover, CIN decreased atherosclerotic lipid deposition and intima/media (I/M) ratio of HCD aortas. CIN-mediated effects might be related to its ability to attenuate the elevated aortic mRNA expression of cholesteryl ester transfer protein (CETP) and MPO in HCD group. Interestingly, the vasculoprotective effects of CIN treatment in the current study do not seem to be mediated via Nrf2-dependent mechanisms. In conclusion, CIN may mitigate the development of atherosclerosis in hypercholestrolemic rabbits via cholesterol-lowering, antiinflammatory and antioxidant activities.

Lactobacillus plantarum LRCC 5273 isolated from Kimchi ameliorates diet-induced hypercholesterolemia in C57BL/6 mice.

This study was designed to select potent cholesterol-lowering probiotic strains on HepG2 cell and investigate the effect of selected strain, Lactobacillus plantarum LRCC 5273 and LRCC 5279 in hypercholesterolemic mice. In the results, LP5273 group showed significantly reduced total and LDL cholesterol compared to HCD group. In addition to significantly up-regulated hepatic mRNA expression of LXR-α and CYP7A1, intestinal LXR-α and ABCG5 were significantly up-regulated in LP5273 group. With activation of hepatic and intestinal LXR-α and its target genes, fecal cholesterol and bile acid excretion were increased in LP5273 fed mice. These results suggest that LP5273 ameliorates hypercholesterolemia in mice through the activation of hepatic and intestinal LXR-α, resulting in enhancement of fecal cholesterol and bile acids excretion in the small intestine. The results of present study suggest mechanistic evidences for hypocholesterolemic effects of L. plantarum spp., and may contribute to future researches for prevention of hypercholesterolemia and cardiovascular disease.

Low or excess levels of dietary cholesterol impaired immunity and aggravated inflammation response in young grass carp (Ctenopharyngodon idella).

The present study explored the effect of cholesterol on the immunity and inflammation response in the immune organs (head kidney, spleen and skin) of young grass carp (Ctenopharyngodon idella) fed graded levels of dietary cholesterol (0.041-1.526%) for 60 days and then infected with Aeromonas hydrophila for 14 days. The results showed that low levels of cholesterol (1) depressed the innate immune components [lysozyme (LZ), acid phosphatase (ACP), complements and antimicrobial peptides] and adaptive immune component [immunoglobulin M (IgM)], (2) up-regulated the mRNA levels of pro-inflammatory cytokines [interleukin 1ß (IL-1ß), IL-6, IL-8, IL-12p35, IL-12p40, IL-15, IL-17D, tumor necrosis factor α (TNF-α) and interferon γ2 (IFN-γ2)], partly due to the activated nuclear factor kappa B (NF-κB) signalling, and (3) down-regulated the mRNA levels of anti-inflammatory cytokines [IL-4/13B, IL-10, IL-11, transforming growth factor (TGF)-ß1 and TGF-ß2], partly due to the suppression of target of rapamycin (TOR) signalling in the immune organs of young grass carp. Interestingly, dietary cholesterol had no influences on the IκB kinase α (IKKα) and IL-4/13A mRNA levels in the head kidney, spleen and skin, the IL-1ß and IL-12p40 mRNA levels in the spleen and skin, or the ß-defensin-1 mRNA level in the skin of young grass carp. Additionally, low levels of cholesterol increased the skin haemorrhage and lesion morbidity. In summary, low levels of cholesterol impaired immunity by depressing the innate and adaptive immune components, and low levels of cholesterol aggravated the inflammation response via up-regulating the expression of pro-inflammatory cytokines as well as down-regulating the expression of anti-inflammatory cytokines partly through the modulation of NF-κB and TOR signalling in the immune organs of fish. Similar to the low level of cholesterol, the excess level of dietary cholesterol impaired immunity and aggravated inflammation response in the immune organs of fish. Finally, based on the percent weight gain (PWG), the ability against skin haemorrhage and lesions as well as the LZ activity in the head kidney and the ACP activity in the spleen, the optimal dietary cholesterol levels for young grass carp were estimated as 0.721, 0.826, 0.802 and 0.772% diet, respectively.

Neuroprotective Effects of the Methanol Extract of Kimchi, a Korean Fermented Vegetable Food, Mediated Via Suppression of Endoplasmic Reticulum Stress and Caspase Cascade Pathways in High-Cholesterol Diet-Fed Mice.

Endoplasmic reticulum (ER) stress-related unfolded peptide accumulation is closely associated with the development of neurodegenerative diseases known as protein misfolding disorders. The antioxidative properties of kimchi, a traditional Korean fermented vegetable dish, have been well established. In this study, the neuroprotective effects of the kimchi methanol extract (KME) were examined in high-cholesterol diet (HCD)-fed mice. The animals were fed a HCD, with oral administration of either KME (KME group, 200 mg·kg bw-1·day-1, n = 10) or distilled water (Control group, n = 10) for 8 weeks. Compared with the levels in the control group, the reactive oxygen species, peroxynitrite, and lipid peroxidation levels in the brain were significantly decreased in the KME group (P < .05), whereas the glutathione level was increased (P < .05). In addition, the ER stress biomarkers, phospho-eukaryotic initiation factor 2 subunit α, glucose-regulated protein 78, X-box binding protein 1, inositol-requiring enzyme 1, and C/EBP homologous protein and the nuclear factor-kappaB-mediated inflammation were significantly reduced in the KME group (P < .05). In contrast, the expression levels of antioxidative enzymes regulated by nuclear factor erythroid 2-related factor-2 were elevated (P < .05). The amyloid-beta expression levels of the KME group were lower than that of the control group (P < .05). Moreover, the expression levels of Bcl-2-associated X, and caspases-3 and -9 were downregulated, with a concomitant upregulation of B cell lymphoma 2 (P < .05). Accordingly, KME provide neuronal cell protection via suppressing ER stress and caspase cascade signaling.

Bile acid detoxifying enzymes limit susceptibility to liver fibrosis in female SHRSP5/Dmcr rats fed with a high-fat-cholesterol diet.

During middle age, women are less susceptible to nonalcoholic steatohepatitis (NASH) than men. Thus, we investigated the underlying molecular mechanisms behind these sexual differences using an established rat model of NASH. Mature female and male stroke-prone spontaneously hypertensive 5/Dmcr rats were fed control or high-fat-cholesterol (HFC) diets for 2, 8, and 14 weeks. Although HFC-induced hepatic fibrosis was markedly less severe in females than in males, only minor gender differences were observed in expression levels of cytochrome P450 enzymes (CYP)7A1, CYP8B1 CYP27A1, and CYP7B1, and multidrug resistance-associated protein 3, and bile salt export pump, which are involved in fibrosis-related bile acid (BA) kinetics. However, the BA detoxification-related enzymes UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) 2A1, and the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR), were strongly suppressed in HFC-fed males, and were only slightly changed in HFC-diet fed females. Expression levels of the farnesoid X receptor and its small heterodimer partner were similarly regulated in a gender-dependent fashion following HFC feeding. Hence, the pronounced female resistance to HFC-induced liver damage likely reflects sustained expression of the nuclear receptors CAR and PXR and the BA detoxification enzymes UGT and SULT.