Hepatic damage caused by long-term high cholesterol intake induces a dysfunctional restorative macrophage population in experimental NASH.

Excessive dietary cholesterol is preferentially stored in the liver, favoring the development of nonalcoholic steatohepatitis (NASH), characterized by progressive hepatic inflammation and fibrosis. Emerging evidence indicates a critical contribution of hepatic macrophages to NASH severity. However, the impact of cholesterol on these cells in the setting of NASH remains elusive. Here, we demonstrate that the dietary cholesterol content directly affects hepatic macrophage global gene expression. Our findings suggest that the modifications triggered by prolonged high cholesterol intake induce long-lasting hepatic damage and support the expansion of a dysfunctional pro-fibrotic restorative macrophage population even after cholesterol reduction. The present work expands the understanding of the modulatory effects of cholesterol on innate immune cell transcriptome and may help identify novel therapeutic targets for NASH intervention.

Long-Term Dietary Taurine Lowers Plasma Levels of Cholesterol and Bile Acids.

Cholesterol is an essential lipid in vertebrates, but excess blood cholesterol promotes atherosclerosis. In the liver, cholesterol is metabolized to bile acids by cytochrome P450, family 7, subfamily a, polypeptide 1 (CYP7A1), the transcription of which is negatively regulated by the ERK pathway. Fibroblast growth factor 21 (FGF21), a hepatokine, induces ERK phosphorylation and suppresses Cyp7a1 transcription. Taurine, a sulfur-containing amino acid, reportedly promotes cholesterol metabolism and lowers blood and hepatic cholesterol levels. However, the influence of long-term feeding of taurine on cholesterol levels and metabolism remains unclear. Here, to evaluate the more chronic effects of taurine on cholesterol levels, we analyzed mice fed a taurine-rich diet for 14-16 weeks. Long-term feeding of taurine lowered plasma cholesterol and bile acids without significantly changing other metabolic parameters, but hardly affected these levels in the liver. Moreover, taurine upregulated Cyp7a1 levels, while downregulated phosphorylated ERK and Fgf21 levels in the liver. Likewise, taurine-treated Hepa1-6 cells, a mouse hepatocyte line, exhibited downregulated Fgf21 levels and upregulated promoter activity of Cyp7a1. These results indicate that taurine promotes cholesterol metabolism by suppressing the FGF21/ERK pathway followed by upregulating Cyp7a1 expression. Collectively, this study shows that long-term feeding of taurine lowers both plasma cholesterol and bile acids, reinforcing that taurine effectively prevents hypercholesterolemia.

Our evolving understanding of how 27-hydroxycholesterol influences cancer.

Cholesterol has been implicated in the pathophysiology and progression of several cancers now, although the mechanisms by which it influences cancer biology are just emerging. Two likely contributing mechanisms are the ability for cholesterol to directly regulate signaling molecules within the membrane, and certain metabolites acting as signaling molecules. One such metabolite is the oxysterol 27-hydroxycholesterol (27HC), which is a primary metabolite of cholesterol synthesized by the enzyme Cytochrome P450 27A1 (CYP27A1). Physiologically, 27HC is involved in the regulation of cholesterol homeostasis and contributes to cholesterol efflux through liver X receptor (LXR) and inhibition of de novo cholesterol synthesis through the insulin-induced proteins (INSIGs). 27HC is also a selective modulator of the estrogen receptors. An increasing number of studies have identified its importance in cancer progression of various origins, especially in breast cancer. In this review, we discuss the physiological roles of 27HC targeting these two nuclear receptors and the subsequent contribution to cancer progression. We describe how 27HC promotes tumor growth directly through cancer-intrinsic factors, and indirectly through its immunomodulatory roles which lead to decreased immune surveillance and increased tumor invasion. This review underscores the importance of the cholesterol metabolic pathway in cancer progression and the potential therapeutic utility of targeting this metabolic pathway.

Pulmonary and vascular effects of acute ozone exposure in diabetic rats fed an atherogenic diet.

Air pollutants may increase risk for cardiopulmonary disease, particularly in susceptible populations with metabolic stressors such as diabetes and unhealthy diet. We investigated effects of inhaled ozone exposure and high-cholesterol diet (HCD) in healthy Wistar and Wistar-derived Goto-Kakizaki (GK) rats, a non-obese model of type 2 diabetes. Male rats (4-week old) were fed normal diet (ND) or HCD for 12 weeks and then exposed to filtered air or 1.0 ppm ozone (6 h/day) for 1 or 2 days. We examined pulmonary, vascular, hematology, and inflammatory responses after each exposure plus an 18-h recovery period. In both strains, ozone induced acute bronchiolar epithelial necrosis and inflammation on histopathology and pulmonary protein leakage and neutrophilia; the protein leakage was more rapid and persistent in GK compared to Wistar rats. Ozone also decreased lymphocytes after day 1 in both strains consuming ND (~50%), while HCD increased circulating leukocytes. Ozone increased plasma thrombin/antithrombin complexes and platelet disaggregation in Wistar rats on HCD and exacerbated diet effects on serum IFN-γ, IL-6, KC-GRO, IL-13, and TNF-α, which were higher with HCD (Wistar>GK). Ex vivo aortic contractility to phenylephrine was lower in GK versus Wistar rats at baseline(~30%); ozone enhanced this effect in Wistar rats on ND. GK rats on HCD had higher aortic e-NOS and tPA expression compared to Wistar rats. Ozone increased e-NOS in GK rats on ND (~3-fold) and Wistar rats on HCD (~2-fold). These findings demonstrate ways in which underlying diabetes and HCD may exacerbate pulmonary, systemic, and vascular effects of inhaled pollutants.

Hepatocyte ATF3 protects against atherosclerosis by regulating HDL and bile acid metabolism.

Activating transcription factor (ATF)3 is known to have an anti-inflammatory function, yet the role of hepatic ATF3 in lipoprotein metabolism or atherosclerosis remains unknown. Here we show that overexpression of human ATF3 in hepatocytes reduces the development of atherosclerosis in Western-diet-fed Ldlr-/- or Apoe-/- mice, whereas hepatocyte-specific ablation of Atf3 has the opposite effect. We further show that hepatic ATF3 expression is inhibited by hydrocortisone. Mechanistically, hepatocyte ATF3 enhances high-density lipoprotein (HDL) uptake, inhibits intestinal fat and cholesterol absorption and promotes macrophage reverse cholesterol transport by inducing scavenger receptor group B type 1 (SR-BI) and repressing cholesterol 12α-hydroxylase (CYP8B1) in the liver through its interaction with p53 and hepatocyte nuclear factor 4α, respectively. Our data demonstrate that hepatocyte ATF3 is a key regulator of HDL and bile acid metabolism and atherosclerosis.

Dietary Lipids Modulate Notch Signaling and Influence Adult Intestinal Development and Metabolism in Drosophila.

Tissue homeostasis involves a complex balance of developmental signals and environmental cues that dictate stem cell function. We found that dietary lipids control enteroendocrine cell production from Drosophila posterior midgut stem cells. Dietary cholesterol influences new intestinal cell differentiation in an Hr96-dependent manner by altering the level and duration of Notch signaling. Exogenous lipids modulate Delta ligand and Notch extracellular domain stability and alter their trafficking in endosomal vesicles. Lipid-modulated Notch signaling occurs in other nutrient-dependent tissues, suggesting that Delta trafficking in many cells is sensitive to cellular sterol levels. These diet-mediated alterations in young animals contribute to a metabolic program that persists after the diet changes. A low-sterol diet also slows the proliferation of enteroendocrine tumors initiated by Notch pathway disruption. Thus, a specific dietary nutrient can modify a key intercellular signaling pathway to shift stem cell differentiation and cause lasting changes in tissue structure and physiology.

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.

Derangement of intestinal epithelial cell monolayer by dietary cholesterol oxidation products.

The emerging role of the diet in the incidence of intestinal inflammatory diseases has stimulated research on the influence of eating habits with pro-inflammatory properties in inducing epithelial barrier disturbance. Cholesterol oxidation products, namely oxysterols, have been shown to promote and sustain oxidative/inflammatory reactions in human digestive tract. This work investigated in an in vitro model the potential ability of a combination of dietary oxysterols representative of a hyper-cholesterol diet to induce the loss of intestinal epithelial layer integrity. The components of the experimental mixture were the main oxysterols stemming from heat-induced cholesterol auto-oxidation, namely 7-ketocholesterol, 5α,6α-and 5ß,6ß-epoxycholesterol, 7α- and 7ß-hydroxycholesterol. These compounds added to monolayers of differentiated CaCo-2 cells in combination or singularly, caused a time-dependent induction of matrix metalloproteinases (MMP)-2 and -9, also known as gelatinases. The hyperactivation of MMP-2 and -9 was found to be associated with decreased levels of the tight junctions zonula occludens-1 (ZO-1), occludin and Junction Adhesion Molecule-A (JAM-A). Together with such a protein loss, particularly evident for ZO-1, a net perturbation of spatial localization of the three tight junctions was observed. Cell monolayer pre-treatment with the selective inhibitor of MMPs ARP100 or polyphenol (-)-epicathechin, previously shown to inhibit NADPH oxidase in the same model system, demonstrated that the decrease of the three tight junction proteins was mainly a consequence of MMPs induction, which was in turn dependent on the pro-oxidant property of the oxysterols investigated. Although further investigation on oxysterols intestinal layer damage mechanism is to be carried on, the consequent - but incomplete - prevention of oxysterols-dependent TJs alteration due to MMPs inhibition, avoided the loss of scaffold protein ZO-1, with possible significant recovery of intestinal monolayer integrity.

The cholesterol metabolite 27 hydroxycholesterol facilitates breast cancer metastasis through its actions on immune cells.

Obesity and elevated circulating cholesterol are risk factors for breast cancer recurrence, while the use of statins, cholesterol biosynthesis inhibitors widely used for treating hypercholesterolemia, is associated with improved disease-free survival. Here, we show that cholesterol mediates the metastatic effects of a high-fat diet via its oxysterol metabolite, 27-hydroxycholesterol. Ablation or inhibition of CYP27A1, the enzyme responsible for the rate-limiting step in 27-hydroxycholesterol biosynthesis, significantly reduces metastasis in relevant animal models of cancer. The robust effects of 27-hydroxycholesterol on metastasis requires myeloid immune cell function, and it was found that this oxysterol increases the number of polymorphonuclear-neutrophils and γδ-T cells at distal metastatic sites. The pro-metastatic actions of 27-hydroxycholesterol requires both polymorphonuclear-neutrophils and γδ-T cells, and 27-hydroxycholesterol treatment results in a decreased number of cytotoxic CD8+T lymphocytes. Therefore, through its actions on γδ-T cells and polymorphonuclear-neutrophils, 27-hydroxycholesterol functions as a biochemical mediator of the metastatic effects of hypercholesterolemia.High cholesterol is a risk factor for breast cancer recurrence. Here the authors show that cholesterol promotes breast cancer metastasis via its metabolite 27-hydroxycholesterol (27HC) that acts on immune myeloid cells residing at the distal metastatic sites, thus promoting an immune suppressive environment.

Silicon Alleviates Nonalcoholic Steatohepatitis by Reducing Apoptosis in Aged Wistar Rats Fed a High-Saturated Fat, High-Cholesterol Diet.

Background: Lipoapoptosis has been identified as a key event in the progression of nonalcoholic fatty liver disease (NAFLD), and hence, antiapoptotic agents have been recommended as a possible effective treatment for nonalcoholic steatohepatitis (NASH). Silicon, included in meat as a functional ingredient, improves lipoprotein profiles and liver antioxidant defenses in aged rats fed a high-saturated fat, high-cholesterol diet (HSHCD). However, to our knowledge, the antiapoptotic effect of this potential functional meat on the liver has never been tested.Objective: This study was designed to evaluate the effect of silicon on NASH development and the potential antiapoptotic properties of silicon in aged rats.Methods: One-year-old male Wistar rats weighing ∼500 g were fed 3 experimental diets containing restructured pork (RP) for 8 wk: 1) a high-saturated fat diet, as an NAFLD control, with 16.9% total fat, 0.14 g cholesterol/kg diet, and 46.8 mg SiO2/kg (control); 2) the HSHCD as a model of NASH, with 16.6% total fat, 16.3 g cholesterol/kg diet, and 46.8 mg SiO2/kg [high-cholesterol diet (Chol-C)]; and 3) the HSHCD with silicon-supplemented RP with amounts of fat and cholesterol identical to those in the Chol-C diet, but with 750 mg SiO2/kg (Chol-Si). Detailed histopathological assessments were performed, and the NAFLD activity score (NAS) was calculated. Liver apoptosis and damage markers were evaluated by Western blotting and immunohistochemical staining.Results: Chol-C rats had a higher mean NAS (7.4) than did control rats (1.9; P < 0.001). The score in Chol-Si rats (5.4) was intermediate and different from that in both other groups (P < 0.05). Several liver apoptosis markers-including hepatocyte terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate (dUTP) nick end labeling, cytosolic cytochrome c, apoptosis-inducing factor, caspases 9 and 3, and the mitochondrial Bcl-2-associated X protein (BAX)-to-B-cell lymphoma 2 (BCL2) ratio-were 9-45% lower in Chol-Si than in Chol-C rats (P < 0.05) and did not differ from values in the control group.Conclusions: Supplemental silicon substantially affects NASH development in aged male Wistar rats fed an HSHCD by partially blocking apoptosis. These results suggest that silicon-enriched RP could be used as an effective nutritional strategy in preventing NASH.

Chia Oil-Enriched Restructured Pork Effects on Oxidative and Inflammatory Status of Aged Rats Fed High Cholesterol/High Fat Diets.

Chia oil has the highest recognized α-linolenic acid (ALA) content. ALA is associated with beneficial changes in plasma lipids and the prevention of cardiovascular diseases. Present article aims to analyze the effect of Chia oil-enriched restructured pork (RP) on aged rats in a nonalcoholic steatohepatitis (NASH) model. Groups of six male Wistar rats (1-year old) were fed the experimental diets: control RP diet (C) noncholesterol high saturated; cholesterol-enriched high-saturated fat/high-cholesterol control RP diet (HC) with added cholesterol and cholic acid; and Chia oil- or Hydroxytyrosol RP cholesterol-enriched high-saturated fat/high cholesterol (CHIA and HxT). Total cholesterol, hepatosomatic index, Nrf2, antioxidant, and inflammation markers were determined. CHIA reduced the hypercholesterolemic effect by lowering levels similar to C; also, ameliorated redox index. CHIA, despite high polyunsaturated fatty acids (PUFA) content, reduced thiobarbituric acid reactive substances (TBARS) and induced the lowest SOD protein synthesis but not a reduction on its activity. Chia oil activated the Nrf2 to arrest the pro-oxidative response to cholesterol and aging. Endothelial nitric oxide synthase (eNOS) system was lower in HxT than in CHIA, suggesting its antiatherogenic activity and related protective effect against high PUFA. Increase in tumor necrosis factor alpha (TNFα) was partially blocked by CHIA. Chia oil has the ability to prevent oxidative damage and modify the inflammatory response, suggesting adequate regulation of the antioxidant system. Results stress the importance of incorporating ALA into the diet.

Activation of the Hypoxia Inducible Factor 1α Subunit Pathway in Steatotic Liver Contributes to Formation of Cholesterol Gallstones.

BACKGROUND & AIMS: Hypoxia-inducible factor 1α subunit (HIF1A) is a transcription factor that controls the cellular response to hypoxia and is activated in hepatocytes of patients with nonalcoholic fatty liver disease (NAFLD). NAFLD increases the risk for cholesterol gallstone disease by unclear mechanisms. We studied the relationship between HIF1A and gallstone formation associated with liver steatosis. METHODS: We performed studies with mice with inducible disruption of Hif1a in hepatocytes via a Cre adenoviral vector (inducible hepatocyte-selective HIF1A knockout [iH-HIFKO] mice), and mice without disruption of Hif1a (control mice). Mice were fed a diet rich in cholesterol and cholate for 1 or 2 weeks; gallbladders were collected and the number of gallstones was determined. Livers and biliary tissues were analyzed by histology, quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, and immunoblots. We measured concentrations of bile acid, cholesterol, and phospholipid in bile and rates of bile flow. Primary hepatocytes and cholangiocytes were isolated and analyzed. HIF1A was knocked down in Hepa1-6 cells with small interfering RNAs. Liver biopsy samples from patients with NAFLD, with or without gallstones, were analyzed by quantitative reverse-transcription polymerase chain reaction. RESULTS: Control mice fed a diet rich in cholesterol and cholate developed liver steatosis with hypoxia; levels of HIF1A protein were increased in hepatocytes around central veins and 90% of mice developed cholesterol gallstones. Only 20% of the iH-HIFKO mice developed cholesterol gallstones. In iH-HIFKO mice, the biliary lipid concentration was reduced by 36%, compared with control mice, and bile flow was increased by 35%. We observed increased water secretion from hepatocytes into bile canaliculi to mediate these effects, resulting in suppression of cholelithogenesis. Hepatic expression of aquaporin 8 (AQP8) protein was 1.5-fold higher in iH-HIFKO mice than in control mice. Under hypoxic conditions, cultured hepatocytes increased expression of Hif1a, Hmox1, and Vegfa messenger RNAs (mRNAs), and down-regulated expression of AQP8 mRNA and protein; AQP8 down-regulation was not observed in cells with knockdown of HIF1A. iH-HIFKO mice had reduced inflammation and mucin deposition in the gallbladder compared with control mice. Liver tissues from patients with NAFLD with gallstones had increased levels of HIF1A, HMOX1, and VEGFA mRNAs, compared with livers from patients with NAFLD without gallstones. CONCLUSIONS: In steatotic livers of mice, hypoxia up-regulates expression of HIF1A, which reduces expression of AQP8 and concentrates biliary lipids via suppression of water secretion from hepatocytes. This promotes cholesterol gallstone formation. Livers from patients with NAFLD and gallstones express higher levels of HIF1A than livers from patients with NAFLD without gallstones.

Silkworm pupae oil exerts hypercholesterolemic and antioxidant effects in high-cholesterol diet-fed rats.

BACKGROUND: Silkworm pupae is a good resource of edible oil that is especially rich in unsaturated fatty acids and is considered to be an excellent dietary supplement for hyperlipidemia. RESULTS: Groups fed a high-cholesterol diet (HCD) with silkworm pupae oil (SPO) supplementation (1, 2, or 4 mL kg-1 day-1 ) orally had significantly lower levels of serum total cholesterol (P < 0.05) and low-density lipoprotein cholesterol (P < 0.05) compared to the HCD group. With regard to antioxidant parameters, except for levels of glutathione peroxidase (GSH-Px) in the liver, 2 and 4 mL kg-1 day-1 of SPO supplementation leaded to higher total antioxidant capacity (P < 0.05), superoxide dismutase (P < 0.05) and GSH-Px levels (P < 0.05), as well as lower malondialdehyde levels (P < 0.05), both in serum and liver compared to the HCD group. CONCLUSION: The results of the present study indicate that supplementation with SPO can improve lipid profiles and alleviate oxidative stress in high-cholesterol diet-fed rats. © 2016 Society of Chemical Industry.

Dietary fat and gut microbiota interactions determine diet-induced obesity in mice.

OBJECTIVE: Gut microbiota may promote positive energy balance; however, germfree mice can be either resistant or susceptible to diet-induced obesity (DIO) depending on the type of dietary intervention. We here sought to identify the dietary constituents that determine the susceptibility to body fat accretion in germfree (GF) mice. METHODS: GF and specific pathogen free (SPF) male C57BL/6N mice were fed high-fat diets either based on lard or palm oil for 4 wks. Mice were metabolically characterized at the end of the feeding trial. FT-ICR-MS and UPLC-TOF-MS were used for cecal as well as hepatic metabolite profiling and cecal bile acids quantification, respectively. Hepatic gene expression was examined by qRT-PCR and cecal gut microbiota of SPF mice was analyzed by high-throughput 16S rRNA gene sequencing. RESULTS: GF mice, but not SPF mice, were completely DIO resistant when fed a cholesterol-rich lard-based high-fat diet, whereas on a cholesterol-free palm oil-based high-fat diet, DIO was independent of gut microbiota. In GF lard-fed mice, DIO resistance was conveyed by increased energy expenditure, preferential carbohydrate oxidation, and increased fecal fat and energy excretion. Cecal metabolite profiling revealed a shift in bile acid and steroid metabolites in these lean mice, with a significant rise in 17ß-estradiol, which is known to stimulate energy expenditure and interfere with bile acid metabolism. Decreased cecal bile acid levels were associated with decreased hepatic expression of genes involved in bile acid synthesis. These metabolic adaptations were largely attenuated in GF mice fed the palm-oil based high-fat diet. We propose that an interaction of gut microbiota and cholesterol metabolism is essential for fat accretion in normal SPF mice fed cholesterol-rich lard as the main dietary fat source. This is supported by a positive correlation between bile acid levels and specific bacteria of the order Clostridiales (phylum Firmicutes) as a characteristic feature of normal SPF mice fed lard. CONCLUSIONS: In conclusion, our study identified dietary cholesterol as a candidate ingredient affecting the crosstalk between gut microbiota and host metabolism.

Intestinal CREBH overexpression prevents high-cholesterol diet-induced hypercholesterolemia by reducing Npc1l1 expression.

OBJECTIVE: The transcription factor cyclic AMP-responsive element-binding protein H (CREBH, encoded by Creb3l3) is highly expressed in the liver and small intestine. Hepatic CREBH contributes to glucose and triglyceride metabolism by regulating fibroblast growth factor 21 (Fgf21) expression. However, the intestinal CREBH function remains unknown. METHODS: To investigate the influence of intestinal CREBH on cholesterol metabolism, we compared plasma, bile, fecal, and tissue cholesterol levels between wild-type (WT) mice and mice overexpressing active human CREBH mainly in the small intestine (CREBH Tg mice) under different dietary conditions. RESULTS: Plasma cholesterol, hepatic lipid, and cholesterol crystal formation in the gallbladder were lower in CREBH Tg mice fed a lithogenic diet (LD) than in LD-fed WTs, while fecal cholesterol output was higher in the former. These results suggest that intestinal CREBH overexpression suppresses cholesterol absorption, leading to reduced plasma cholesterol, limited hepatic supply, and greater excretion. The expression of Niemann-Pick C1-like 1 (Npc1l1), a rate-limiting transporter mediating intestinal cholesterol absorption, was reduced in the small intestine of CREBH Tg mice. Adenosine triphosphate-binding cassette transporter A1 (Abca1), Abcg5/8, and scavenger receptor class B, member 1 (Srb1) expression levels were also reduced in CREBH Tg mice. Promoter assays revealed that CREBH directly regulates Npc1l1 expression. Conversely, CREBH null mice exhibited higher intestinal Npc1l1 expression, elevated plasma and hepatic cholesterol, and lower fecal output. CONCLUSION: Intestinal CREBH regulates dietary cholesterol flow from the small intestine by controlling the expression of multiple intestinal transporters. We propose that intestinal CREBH could be a therapeutic target for hypercholesterolemia.

High cholesterol diet leads to oxidant load and peroxidation in the rabbit kidney tissue.

OBJECTIVES: The aim of this study was to investigate possible effects of high cholesterol diet on oxidant/antioxidant status in rabbit kidney tissues. BACKGROUND: Although a number of experimental animal models have suggested that hyperlipidemia is associated with progressive kidney failure data remain sparse on the role of dietary cholesterol intake on kidney disease. METHODS: Twelve male New Zealand albino rabbits were randomly divided into two groups (control and cholesterol). Both groups were fed on a standard laboratory diet. Animals in the cholesterol group additionally received cholesterol (1 g/kg/day), orally. The study period was 12 weeks. Activities of catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), nitric oxide synthase (NOS), xanthine oxidase (XO), paraoxonase (PON), adenosine deaminase (ADA) enzymes and levels of malondialdehyde (MDA) and nitric oxide (NO) were measured in kidney tissue samples. Histological examination of the kidney tissue samples was also done. RESULTS: SOD, GSH-Px and XO enzyme activities were found to be decreased and NOS and PON activities increased significantly in cholesterol group compared to controls. As an indication of oxidation, MDA levels were found to be increased in cholesterol group. Histological examination revealed some derangements in the kidney tissue. CONCLUSION: High cholesterol diet creates oxidant load and causes peroxidation, which in turn, leads derangements in the rabbit kidney tissue (Tab. 2, Fig. 2, Ref. 69).

Cellular cholesterol accumulation modulates high fat high sucrose (HFHS) diet-induced ER stress and hepatic inflammasome activation in the development of non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH), is the form of non-alcoholic fatty liver disease posing risk to progress into serious long term complications. Human and pre-clinical models implicate cellular cholesterol dysregulation playing important role in its development. Mouse model studies suggest synergism between dietary cholesterol and fat in contributing to NASH but the mechanisms remain poorly understood. Our laboratory previously reported the primary importance of hepatic endoplasmic reticulum cholesterol (ER-Chol) in regulating hepatic ER stress by comparing the responses of wild type, Ldlr-/-xLcat+/+ and Ldlr-/-xLcat-/- mice, to a 2% high cholesterol diet (HCD). Here we further investigated the roles of ER-Chol and ER stress in HFHS diet-induced NASH using the same strains. With HFHS diet feeding, both WT and Ldlr-/-xLcat+/+ accumulate ER-Chol in association with ER stress and inflammasome activation but the Ldlr-/-xLcat-/- mice are protected. By contrast, all three strains accumulate cholesterol crystal, in correlation with ER-Chol, albeit less so in Ldlr-/-xLcat-/- mice. By comparison, HCD feeding per se (i) is sufficient to promote steatosis and activate inflammasomes, and (ii) results in dramatic accumulation of cholesterol crystal which is linked to inflammasome activation in Ldlr-/-xLcat-/- mice, independent of ER-Chol. Our data suggest that both dietary fat and cholesterol each independently promote steatosis, cholesterol crystal accumulation and inflammasome activation through distinct but complementary pathways. In vitro studies using palmitate-induced hepatic steatosis in HepG2 cells confirm the key roles by cellular cholesterol in the induction of steatosis and inflammasome activations. These novel findings provide opportunities for exploring a cellular cholesterol-focused strategy for treatment of NASH.

Murine Norovirus Infection Variably Alters Atherosclerosis in Mice Lacking Apolipoprotein E.

Macrophages play a key role in the development of atherosclerosis. Murine noroviruses (MNV) are highly prevalent in research mouse colonies and infect macrophages and dendritic cells. Our laboratory found that MNV4 infection in mice lacking the LDL receptor alters the development of atherosclerosis, potentially confounding research outcomes. Therefore, we investigated whether MNV4 likewise altered atherosclerosis in ApoE(-/-) mice. In the presence of oxidized LDL, MNV4 infection of ApoE(-/-) bone marrow-derived macrophages increased the gene expression of the inflammatory markers inducible nitric oxide synthase, monocyte chemoattractant protein 1, and IL6. In addition, proteins involved in cholesterol transport were altered in MNV4-infected ApoE -/- bone marrow-derived macrophages and consisted of increased CD36 and decreased ATP-binding cassette transporter A1. MNV4 infection of ApoE(-/-) mice at 12 wk of age (during the development of atherosclerosis) had a variable effect on atherosclerotic lesion size. In one study, MNV4 significantly increased atherosclerotic plaque area whereas in a second study, no effect was observed. Compared with controls, MNV4-infected mice had higher circulating Ly6C-positive monocytes, and viral RNA was detected in the aortas of some mice, suggesting potential mechanisms by which MNV4 alters disease progression. Plaque size did not differ when ApoE -/- mice were infected at 4 wk of age (early during disease development) or in ApoE -/- mice maintained on a high-fat, high-cholesterol diet. Therefore, these data show that MNV4 has the potential to exert a variable and unpredictable effect on atherosclerosis in ApoE(-/-) mice. We therefore propose that performing experiments in MNV-free mouse colonies is warranted.

The effect of angiotensin-converting enzyme inhibition on inflammatory and angiogenic factors in hypercholesterolemia.

BACKGROUND: Renin-angiotensin system (RAS) can be activated during hyperlipidemia. Angiotensin II increases the migration of monocytes, cytokine levels, and gene expressions of VEGF and VCAM-1. With this in mind, the present work attempted to investigate the effect of angiotensin-converting enzyme (ACE) inhibition on VEGF, VCAM-1, and nitric oxide (NO) serum levels in hypercholesterolemic rats. METHODS: Forty male Wistar rats were divided into 4 groups including normal diet+saline injection (control), hypercholesterol diet+saline injection, normal diet+captopril injection, and hypercholesterol diet+captopril injection. Before and after the beginning of the diet and after the treatment, the serum levels of cholesterol, triglycerides, LDL, HDL, and NO were measured. Finally, gene expressions of VCAM-1 and VEGF in the vascular cells from aorta were determined. RESULTS: Hypercholesterolemic diet increased the serum levels of cholesterol, LDL (p<0.001), triglycerides (p<0.01) and decreased HDL (p<0.001). Captopril caused a reduction in the serum levels of cholesterol, LDL (p<0.001), and triglycerides (p<0.05) as well as an increase in HDL levels (p<0.01). Although the serum levels of NO decreased after hypercholesterolemic diet (p<0.001), no significant change was observed after the treatment. Increased gene expressions of VEGF (p<0.05) and VCAM-1 (p<0.01) in hypercholesterolemia were regressed in captopril treated rats (p<0.01 and p<0.05, respectively). CONCLUSION: Captopril, an ACE inhibitor, improves hyperlipidemia and prevents from overexpression of genes for VEGF and VCAM-1, that are implicated in the inflammation and angiogenesis.

Impact of SCP-2/SCP-x gene ablation and dietary cholesterol on hepatic lipid accumulation.

While a high-cholesterol diet induces hepatic steatosis, the role of intracellular sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x) proteins is unknown. We hypothesized that ablating SCP-2/SCP-x [double knockout (DKO)] would impact hepatic lipids (cholesterol and cholesteryl ester), especially in high-cholesterol-fed mice. DKO did not alter food consumption, and body weight (BW) gain decreased especially in females, concomitant with hepatic steatosis in females and less so in males. DKO-induced steatosis in control-fed wild-type (WT) mice was associated with 1) loss of SCP-2; 2) upregulation of liver fatty acid binding protein (L-FABP); 3) increased mRNA and/or protein levels of sterol regulatory element binding proteins (SREBP1 and SREBP2) as well as increased expression of target genes of cholesterol synthesis (Hmgcs1 and Hmgcr) and fatty acid synthesis (Acc1 and Fas); and 4) cholesteryl ester accumulation was also associated with increased acyl-CoA cholesterol acyltransferase-2 (ACAT2) in males. DKO exacerbated the high-cholesterol diet-induced hepatic cholesterol and glyceride accumulation, without further increasing SREBP1, SREBP2, or target genes. This exacerbation was associated both with loss of SCP-2 and concomitant downregulation of Ceh/Hsl, apolipoprotein B (ApoB), MTP, and/or L-FABP protein expression. DKO diminished the ability to secrete excess cholesterol into bile and oxidize cholesterol to bile acid for biliary excretion, especially in females. This suggested that SCP-2/SCP-x affects cholesterol transport to particular intracellular compartments, with ablation resulting in less to the endoplasmic reticulum for SREBP regulation, making more available for cholesteryl ester synthesis, for cholesteryl-ester storage in lipid droplets, and for bile salt synthesis and/or secretion. These alterations are significant findings, since they affect key processes in regulation of sterol metabolism.