Cholesterol crystallization within hepatocyte lipid droplets and its role in murine NASH

We recently reported that cholesterol crystals form in hepatocyte lipid droplets (LDs) in human and experimental nonalcoholic steatohepatitis. Herein, we assigned WT C57BL/6J mice to a high-fat (15%) diet for 6 months, supplemented with 0%, 0.25%, 0.5%, 0.75%, or 1% dietary cholesterol. Increasing dietary cholesterol led to cholesterol loading of the liver, but not of adipose tissue, resulting in fibrosing steatohepatitis at a dietary cholesterol concentration of ≥0.5%, whereas mice on lower-cholesterol diets developed only simple steatosis. Hepatic cholesterol crystals and crown-like structures also developed at a dietary cholesterol concentration ≥0.5%. Crown-like structures consisted of activated Kupffer cells (KCs) staining positive for NLRP3 and activated caspase 1, which surrounded and processed cholesterol crystal-containing remnant LDs of dead hepatocytes. The KCs processed LDs at the center of crown-like structures in the extracellular space by lysosomal enzymes, ultimately transforming into lipid-laden foam cells. When HepG2 cells were exposed to LDL cholesterol, they developed cholesterol crystals in LD membranes, which caused activation of THP1 cells (macrophages) grown in coculture; upregulation of TNF-alpha, NLRP3, and interleukin 1beta (IL1β) mRNA; and secretion of IL-1beta. In conclusion, cholesterol crystals form on the LD membrane of hepatocytes and cause activation and cholesterol loading of KCs that surround and process these LDs by lysosomal enzymes.     

Dietary modification dampens liver inflammation and fibrosis in obesity‐related fatty liver disease

Background: Alms1 mutant (foz/foz) mice develop hyperphagic obesity, diabetes, metabolic syndrome, and fatty liver (steatosis). High‐fat (HF) feeding converts pathology from bland steatosis to nonalcoholic steatohepatitis (NASH) with fibrosis, which leads to cirrhosis in humans. Objective: We sought to establish how dietary composition contributes to NASH pathogenesis. Design and Methods: foz/foz mice were fed HF diet or chow 24 weeks, or switched HF to chow after 12 weeks. Serum ALT, NAFLD activity score (NAS), fibrosis severity, neutrophil, macrophage and apoptosis immunohistochemistry, uncoupling protein (UCP)2, ATP, NF‐κB activation/expression of chemokines/adhesion molecules/fibrogenic pathways were determined. Result: HF intake upregulated liver fatty acid and cholesterol transporter, CD36. Dietary switch expanded adipose tissue and decreased hepatomegaly by lowering triglyceride, cholesterol ester, free cholesterol and diacylglyceride content of liver. There was no change in lipogenesis or fatty acid oxidation pathways; instead, CD36 was suppressed. These diet‐induced changes in hepatic lipids improved NAS, reduced neutrophil infiltration, normalized UCP2 and increased ATP; this facilitated apoptosis with a change in macrophage phenotype favoring M2 cells. Dietary switch also abrogated NF‐κB activation and chemokine/adhesion molecule expression, and arrested fibrosis by dampening stellate cell activation. Conclusion: Reversion to a physiological dietary composition after HF feeding in foz/foz mice alters body weight distribution but not obesity. This attenuates NASH severity and fibrotic progression by suppressing NF‐κB activation and reducing neutrophil and macrophage activation. However, adipose inflammation persists and is associated with continuing apoptosis in the residual fatty liver disease. Taken together, these findings indicate that other measures, such as weight reduction, may be required to fully reverse obesity‐related NASH.     

Macrophage Specific Caspase-1/11 Deficiency Protects against Cholesterol Crystallization and Hepatic Inflammation in Hyperlipidemic Mice

Background & Aims

While non-alcoholic steatohepatitis (NASH) is characterized by hepatic steatosis combined with inflammation, the mechanisms triggering hepatic inflammation are unknown. In Ldlr^-/- mice, we have previously shown that lysosomal cholesterol accumulation in Kupffer cells (KCs) correlates with hepatic inflammation and cholesterol crystallization. Previously, cholesterol crystals have been shown to induce the activation of inflammasomes. Inflammasomes are protein complexes that induce the processing and release of pro-inflammatory cytokines IL-1b and IL-18 via caspase-1 activation. Whereas caspase-1 activation is independent of caspase-11 in the canonical pathway of inflammasome activation, caspase-11 was found to trigger caspase-1-dependent IL-1b and IL-18 in response to non-canonical inflammasome activators. So far, it has not been investigated whether inflammasome activation stimulates the formation of cholesterol crystals. We hypothesized that inflammasome activation in KCs stimulates cholesterol crystallization, thereby leading to hepatic inflammation.

Methods

Ldlr ^-/- mice were transplanted (tp) with wild-type (Wt) or caspase-1/11^-/- (dKO) bone marrow and fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 12 weeks. In vitro, bone marrow derived macrophages (BMDM) from wt or caspase-1/11^-/- mice were incubated with oxLDL for 24h and autophagy was assessed.

Results

In line with our hypothesis, caspase-1/11^-/--tp mice had less severe hepatic inflammation than Wt-tp animals, as evident from liver histology and gene expression analysis in isolated KCs. Mechanistically, KCs from caspase-1/11^-/--tp mice showed less cholesterol crystals, enhanced cholesterol efflux and increased autophagy. In wt BMDM, oxLDL incubation led to disturbed autophagy activity whereas BMDM from caspase-1/11^-/- mice had normal autophagy activity.

Conclusion

Altogether, these data suggest a vicious cycle whereby disturbed autophagy and decreased cholesterol efflux leads to newly formed cholesterol crystals and thereby maintain hepatic inflammation during NASH by further activating the inflammasome.     

Genetic deletion or pharmacologic inhibition of the Nlrp3 inflammasome did not ameliorate experimental NASH

It has been postulated that inflammasomes, in particular the NLRP3 (NLR family pyrin domain containing 3) inflammasome, mediate the necroinflammation and fibrosis that characterize nonalcoholic steatohepatitis (NASH) by engaging innate immune responses. We aimed to investigate the impact of genetic deletion or pharmacologic inhibition of the NLRP3 inflammasome on experimental steatohepatitis. Global Nlrp3 KO (expected to inhibit the NLRP3 inflammasome) or Casp1 KO (expected to inhibit all inflammasomes) mice were compared to wild type controls after 6 months on a high-fat, high-cholesterol (HFHC, 1% cholesterol) diet known to induce fibrosing steatohepatitis. Additionally, wildtype mice on a HFHC diet (0.75% or 0.5% cholesterol) for 6 months were either treated or not treated with an oral, pharmacologic inhibitor of Nlrp3 (MCC950) that was delivered in the drinking water (0.3 mg/ml). We found that genetic deletion or pharmacologic inhibition of the NLRP3 inflammasome did not ameliorate any of the histological components of fibrosing NASH in HFHC-fed mice. Collectively, these results do not support NLRP3 inhibition as a potential target for human NASH.     

Ezetimibe ameliorates steatohepatitis via AMP activated protein kinase-TFEB-mediated activation of autophagy and NLRP3 inflammasome inhibition

Impairment in macroautophagy/autophagy flux and inflammasome activation are common characteristics of nonalcoholic steatohepatitis (NASH). Considering the lack of approved agents for treating NASH, drugs that can enhance autophagy and modulate inflammasome pathways may be beneficial. Here, we investigated the novel mechanism of ezetimibe, a widely prescribed drug for hypercholesterolemia, as a therapeutic option for ameliorating NASH. Human liver samples with steatosis and NASH were analyzed. For in vitro studies of autophagy and inflammasomes, primary mouse hepatocytes, human hepatoma cells, mouse embryonic fibroblasts with Ampk or Tsc2 knockout, and human or primary mouse macrophages were treated with ezetimibe and palmitate. Steatohepatitis and fibrosis were induced by feeding Atg7 wild-type, haploinsufficient, and knockout mice a methionine- and choline-deficient diet with ezetimibe (10 mg/kg) for 4 wk. Human livers with steatosis or NASH presented impaired autophagy with decreased nuclear TFEB and increased SQSTM1, MAP1LC3-II, and NLRP3 expression. Ezetimibe increased autophagy flux and concomitantly ameliorated lipid accumulation and apoptosis in palmitate-exposed hepatocytes. Ezetimibe induced AMPK phosphorylation and subsequent TFEB nuclear translocation, related to MAPK/ERK. In macrophages, ezetimibe blocked the NLRP3 inflammasome-IL1B pathway in an autophagy-dependent manner and modulated hepatocyte-macrophage interaction via extracellular vesicles. Ezetimibe attenuated lipid accumulation, inflammation, and fibrosis in liver-specific Atg7 wild-type and haploinsufficient mice, but not in knockout mice. Ezetimibe ameliorates steatohepatitis by autophagy induction through AMPK activation and TFEB nuclear translocation, related to an independent MTOR ameliorative effect and the MAPK/ERK pathway. Ezetimibe dampens NLRP3 inflammasome activation in macrophages by modulating autophagy and a hepatocyte-driven exosome pathway.     

Effects of ezetimibe on markers of synthesis and absorption of cholesterol in high-risk patients with elevated C-reactive protein

AimsHigh-risk subjects with elevated C-reactive protein (CRP) are at high risk for cardiovascular events and frequently require potent statins or combined lipid-lowering therapy to achieve lipid targets and decrease inflammation. Our study aimed at evaluating the effects of three lipid-modifying therapies on LDL-cholesterol, CRP levels and markers of cholesterol absorption and synthesis.Main methodsA prospective intervention study was performed in high cardiovascular risk individuals receiving atorvastatin 10 mg daily for four weeks. Those with CRP ≥ 2.0 mg/L were randomized to another four-week treatment period with atorvastatin 40 mg, ezetimibe 10 mg or the combination of atorvastatin 40 mg / ezetimibe 10 mg. Lipids, markers of cholesterol absorption (campesterol and β-sitosterol), and synthesis (desmosterol), as well as CRP were quantified at baseline and end of study.Key findingsOne hundred and twenty two individuals were included. Atorvastatin alone or combined with ezetimibe reduced both LDL-cholesterol and CRP (P < 0.002 vs. baseline; Wilcoxon); ezetimibe did not modify CRP. Ezetimibe-based therapies reduced absorption markers and their ratios to cholesterol (P < 0.0001 vs. baseline, for all; Wilcoxon), whereas atorvastatin alone increased campesterol/cholesterol and β-sitosterol/cholesterol ratios (P < 0.05 vs. baseline; Wilcoxon). In addition, ezetimibe also increased desmosterol and desmosterol/cholesterol ratio (P < 0.0001 vs. baseline; Wilcoxon).SignificanceThese results contribute to understanding the link between cellular cholesterol homeostasis, inflammation and lipid-modifying therapies. Our findings highlight the broader benefit of combined therapy with a potent statin and ezetimibe decreasing inflammation, and preventing increase in cholesterol biosynthesis, an effect not observed with ezetimibe alone.     

Internalization of modified lipids by CD36 and SR-A leads to hepatic inflammation and lysosomal cholesterol storage in Kupffer cells

Background & Aims

Non-alcoholic steatohepatitis (NASH) is characterized by steatosis and inflammation, which can further progress into fibrosis and cirrhosis. Recently, we demonstrated that combined deletion of the two main scavenger receptors, CD36 and macrophage scavenger receptor 1 (MSR1), which are important for modified cholesterol-rich lipoprotein uptake, reduced NASH. The individual contributions of these receptors to NASH and the intracellular mechanisms by which they contribute to inflammation have not been established. We hypothesize that CD36 and MSR1 contribute independently to the onset of inflammation in NASH, by affecting intracellular cholesterol distribution inside Kupffer cells (KCs).

Methods & Results

Ldlr^−/− mice were transplanted with wild-type (Wt), Cd36^−/− or Msr1^−/− bone marrow and fed a Western diet for 3months. Cd36^−/−- and Msr1^−/−- transplanted (tp) mice showed a similar reduction in hepatic inflammation compared to Wt-tp mice. While the total amount of cholesterol inside KCs was similar in all groups, KCs of Cd36^−/−- and Msr1^−/−-tp mice showed increased cytoplasmic cholesterol accumulation, while Wt-tp mice showed increased lysosomal cholesterol accumulation.

Conclusion

CD36 and MSR1 contribute similarly and independently to the progression of inflammation in NASH. One possible explanation for the inflammatory response related to expression of these receptors could be abnormal cholesterol trafficking in KCs. These data provide a new basis for prevention and treatment of NASH.     

Ezetimibe Attenuates Atherosclerosis Associated with Lipid Reduction and Inflammation Inhibition

Background

Ezetimibe, as a cholesterol absorption inhibitor, has been shown protecting against atherosclerosis when combined with statin. However, side by side comparison has not been made to evaluate the beneficial effects of ezetimibe alone versus statin. Herein, the study aimed to test whether ezetimibe alone would exhibit similar effects as statin and the combination therapy would be necessary in a moderate lesion size.

Methods and Results

ApoE-/- male mice that were fed a saturated-fat supplemented diet were randomly assigned to different therapeutic regimens: vehicle, ezetimibe alone (10 mg/kg/day), atorvastatin (20 mg/kg/day) or combination of ezetimibe and atorvastatin through the drinking water. On 28 days, mice were sacrificed and aorta and sera were collected to analyze the atherosclerotic lesion and blood lipid and cholesterol levels. As a result, ezetimibe alone exerted similar protective effects on atherosclerotic lesion sizes as atorvastatin, which was mediated by lowering serum cholesterol concentrations, inhibiting macrophage accumulation in the lesions and reducing circulatory inflammatory cytokines, such as monocyte chemoattractant protein (MCP-1) and tumor necrosis factor (TNF-α). In contrast to ezetimibe administration, atorvastatin alone attenuated atherosclerotic lesion which is dependent on its anti-inflammation effects. There were no significance differences in lesion areas and serum concentrations of cholesterol, oxidized LDL and inflammatory cytokines between combination therapy and monotherapy (either ezetimibe or atorvastatin). There were significant correlations between the lesion areas and serum concentrations of cholesterol, MCP-1 and TNF-α, respectively. However, there were no significant correlations between the lesion areas and serum concentrations of TGF-β1 and oxLDL.

Conclusions

Ezetimibe alone played the same protection against a moderate atherosclerotic lesion as atorvastatin, which was associated with lowering serum cholesterol, decreasing circulating inflammatory cytokines, and inhibiting macrophage accumulation in the lesions.     

Pyruvate dehydrogenase kinase 4 mediates lipogenesis and contributes to the pathogenesis of nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a progressive disease and poses a high risk of severe liver damage. However, the pathogenesis of NASH is still unclear. Accumulation of lipid droplets and insulin resistance is the hallmark of NASH. Pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) plays key role in glucose metabolism via regulating the activity of pyruvate dehydrogenase complex (PDC). Here, we demonstrated a novel of PDK4 in NASH by regulating hepatic steatosis and insulin signaling pathway in methionine and choline deficient (MCD) diet induced NASH model. Hepatic PDK4 levels were highly induced in human patients with NASH and MCD diet fed mice, as well as in hepatocytes treated with oleic acid. The glucose and lipid metabolism were impaired in Pdk4^?/? mice. Pdk4 deficiency ameliorated the hepatic steatosis significantly in NASH mice. Pdk4^?/?-MCD mice had reduced liver weights and triglyceride (TG) levels. And Pdk4 deficiency dramatically reduced the expression of genes related to fatty acid uptake, synthesis and gluconeogenesis. In addition, elevated phosphorylated AMPK (p-AMPK), p-SAPK/JNK and diminished p-ERK, p-P38, p-Akt and p-mTOR/p-4EBP1 proteins were observed. In conclusion, our data indicated that PDK4 potentially contributes to the hepatic steatosis in NASH via regulating several signaling pathway and PDK4 may be a new therapeutic strategy against NAFLD.     

A truncating mutation of Alms1 reduces the number of hypothalamic neuronal cilia in obese mice

Primary cilia are ubiquitous cellular antennae whose dysfunction collectively causes various disorders, including vision and hearing impairment, as well as renal, skeletal, and central nervous system anomalies. One ciliopathy, Alström syndrome, is closely related to Bardet–Biedl syndrome (BBS), sharing amongst other phenotypic features morbid obesity. As the cellular and molecular links between weight regulation and cilia are poorly understood, we used the obese mouse strain foz/foz, bearing a truncating mutation in the Alström syndrome protein (Alms1), to help elucidate why it develops hyperphagia, leading to early onset obesity and metabolic anomalies. Our in vivo studies reveal that Alms1 localizes at the base of cilia in hypothalamic neurons, which are implicated in the control of satiety. Alms1 is lost from this location in foz/foz mice, coinciding with a strong postnatal reduction (～70%) in neurons displaying cilia marked with adenylyl cyclase 3 (AC3), a signaling protein implicated in obesity. Notably, the reduction in AC3‐bearing cilia parallels the decrease in cilia containing two appetite‐regulating proteins, Mchr1 and Sstr3, as well as another established Arl13b ciliary marker, consistent with progressive loss of cilia during development. Together, our results suggest that Alms1 maintains the function of neuronal cilia implicated in weight regulation by influencing the maintenance and/or stability of the organelle. Given that Mchr1 and Sstr3 localization to remaining cilia is maintained in foz/foz animals but known to be lost from BBS knockout mice, our findings suggest different molecular etiologies for the satiety defects associated with the Alström syndrome and BBS ciliopathies. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Economic evaluation of ezetimibe combined with simvastatin for the treatment of primary hypercholesterolaemia

Objective

This study aims to assess the cost-effectiveness of ezetimibe plus simvastatin (E/S) versus atorvastatin or simvastatin monotherapy as second-line treatment of primary hypercholesterolaemia from the Dutch healthcare perspective.

Methods

The evaluation used a Markov model and patient data from the Dutch EASEGO study in which patients failing to reach goal low-density lipoprotein cholesterol levels on atorvastatin 10 mg or simvastatin 20 mg had their dose doubled or switched to ezetimibe 10 mg plus generic simvastatin 20 mg (E10/S20). The second scenario, based on Dutch guidelines, switched patients from simvastatin 40 mg to atorvastatin 40 mg, or ezetimibe 10 mg was added to simvastatin 40 mg (E10/S40). The key effectiveness input measure was change in total cholesterol/high-density lipoprotein ratio obtained from the EASEGO study. In conformity with published studies linking reduced lipid levels to reduced risk of cardiovascular events, the present model assumed that a lipid decrease with ezetimibe may be a signal for reduced risk of cardiovascular events. Model parameters were derived from published literature. Sensitivity analyses were performed for the key parameters.

Results

In the EASEGO scenario, incremental cost-effectiveness ratio for E10/S20 was €3497/quality-adjusted life-years (QALY) vs atorvastatin 20 mg and €26,417/QALY vs simvastatin 40 mg. In the Dutch guidelines scenario, E10/S40 was dominant (more effective and cost-saving) vs atorvastatin 40 mg. Varying model inputs had limited impact on the cost-effectiveness of E/S.

Conclusions

The analysis showed the cost-effectiveness of E/S versus atorvastatin 20 mg or simvastatin 40 mg (EASEGO scenario) at a threshold of €30,000/QALY and vs atorvastatin 40 mg was dominant (Dutch guidelines). Thus, E/S seems a valuable cost-effective second-line treatment option for patients not attaining lipid treatment goals.     

Cathepsin B inhibition ameliorates the non-alcoholic steatohepatitis through suppressing caspase-1 activation

Non-alcoholic fatty liver disease (NAFLD) has emerged as the most common chronic liver disease. NLRP3 inflammasome activation has been widely studied in the pathogenesis of NAFLD. Cathepsin B (CTSB) is a ubiquitous cysteine cathepsin, and the role of CTSB in the progression and development of NAFLD has received extensive concern. However, the exact roles of CTSB in the NAFLD development and NLRP3 inflammasome activation are yet to be evaluated. In the present study, we used methionine choline-deficient (MCD) diet to establish mice NASH model. CTSB inhibitor (CA-074) was used to suppress the expression of CSTB. Expressions of CTSB and caspase-1 were evaluated by immunohistochemical staining. Serum IL-1β and IL-18 levels were also determined. Palmitic acid was used to stimulate Kupffer cells (KCs), and protein expressions of CTSB, NLRP3, ASC (apoptosis-associated speck-like protein containing CARD), and caspase-1 in KCs were detected. The levels of IL-1β and IL-18 in the supernatant of KCs were evaluated by enzyme-linked immunosorbent assay (ELISA). Our results showed that CTSB inhibition improved the liver function and reduced hepatic inflammation and ballooning, and the levels of pro-inflammatory cytokines IL-1β and IL-18 were decreased. The expressions of CTSB and caspase-1 in liver tissues were increased in the NASH group. In in vitro experiments, PA stimulation could increase the expressions of CTSB and NLRP3 inflammasome in KCs, and CTSB inhibition downregulated the expression of NLRP3 inflammasome in KCs, when challenged by PA. Moreover, CTSB inhibition effectively suppressed the expression and activity of caspase-1 and subsequently secretions of IL-1β and IL-18. Collectively, these results suggest that CTSB inhibition limits NLRP3 inflammasome-dependent NASH formation through regulating the expression and activity of caspase-1, thus providing a novel anti-inflammatory signal pathway for the therapy of NAFLD.     

15(S)-Lipoxygenase-1 associates with neutral lipid droplets in macrophage foam cells: evidence of lipid droplet metabolism

15(S)-lipoxygenase-1 (15-LO-1) was present in the whole-cell homogenate of an acute human monocytic leukemia cell line (THP-1). Additionally, 15-LO-1 was detected on neutral lipid droplets isolated from THP-1 foam cells. To investigate if 15-LO-1 is active on lipid droplets, we used the mouse leukemic monocytic macrophage cell line (RAW 264.7), which are stably transfected with human 15-LO-1. The RAW 15-LO-1 cells were incubated with acetylated low density lipoprotein to generate foam cells. 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], the major 15-LO-1 metabolite of arachidonic acid, was produced in the 15-LO-1 RAW but not in the mock transfected cells when incubated with arachidonic acid. Lipid droplets were isolated from the cells and incubated with arachidonic acid, and production of 15(S)-HETE was measured over 2 h. 15(S)-HETE was produced in the incubations with the lipid droplets, and this production was attenuated when the lipid droplet fraction was subjected to enzyme inactivation through heating. Efflux of 15(S)-HETE from cholesteryl ester-enriched 15-LO RAW cells, when lipid droplets are present, was significantly reduced compared with that from cells enriched with free cholesterol (lipid droplets are absent). We propose that 15-LO-1 is present and functional on cytoplasmic neutral lipid droplets in macrophage foam cells, and these droplets may act to accumulate the anti-inflammatory lipid mediator 15(S)-HETE.     

Effects of Ezetimibe on Endothelial Progenitor Cells and Microparticles in High-Risk Patients

Imbalance on endothelial turnover can predict cardiovascular outcomes. We aimed at evaluating the effects of lipid-modifying therapies on circulating endothelial progenitor cells (EPCs), endothelial microparticles (EMPs), and platelet microparticles (PMPs) in high cardiovascular risk subjects with elevated C-reactive protein (CRP). Sixty-three individuals with coronary heart disease (CHD) or CHD risk equivalent on stable statin therapy, with LDL-cholesterol <100 mg/dL and CRP ≥2.0 mg/L were selected. After a 4-week run-in period with atorvastatin 10 mg, those with persistent CRP ≥2.0 mg/L were randomized to another 4-week treatment period with atorvastatin 40 mg, ezetimibe 10 mg or atorvastatin 40 mg/ezetimibe 10 mg. EPC (CD34⁺/CD133⁺/KDR⁺), EMP (CD51⁺), and PMP (CD42⁺/CD31⁺) were quantified by flow cytometry. Atorvastatin 40 mg and atorvastatin 40 mg/ezetimibe 10 mg reduced LDL-cholesterol (P < 0.001, paired T test, vs. baseline). Combined therapy, but not ezetimibe reduced CRP. CD34⁺/KDR⁺ EPC were reduced after ezetimibe alone (P = 0.011 vs. baseline, Wilcoxon test) or combined with atorvastatin (P = 0.016 vs. baseline, Wilcoxon test). In addition, ezetimibe increased CD51⁺ EMP (P = 0.017 vs. baseline, Wilcoxon test). No correlations between these markers and LDL-cholesterol or CRP were observed. These results contribute to understand the link between inflammation and vascular homeostasis and highlight the broader benefit of statins decreasing inflammation and preventing microparticles release, an effect not observed with ezetimibe alone.     

Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux

Peroxisome proliferator-activated receptor delta (PPARδ) is involved in regulation of energy homeostasis. Activation of PPARδ markedly increases fecal neutral sterol secretion, the last step in reverse cholesterol transport. This phenomenon can neither be explained by increased hepatobiliary cholesterol secretion, nor by reduced cholesterol absorption. To test the hypothesis that PPARδ activation leads to stimulation of transintestinal cholesterol efflux (TICE), we quantified it by intestine perfusions in FVB mice treated with PPARδ agonist GW610742. To exclude the effects on cholesterol absorption, mice were also treated with cholesterol absorption inhibitor ezetimibe or ezetimibe/GW610742. {"type":"entrez-nucleotide","attrs":{"text":"GW601742","term_id":"299511820","term_text":"GW601742"}}GW601742 treatment had little effect on plasma lipid levels but stimulated both fecal neutral sterol excretion (∼200%) and TICE (∼100%). GW610742 decreased intestinal Npc1l1 expression but had no effect on Abcg5/Abcg8. Interestingly, expression of Rab9 and LIMPII, encoding proteins involved in intracellular cholesterol trafficking, was increased upon PPARδ activation. Although treatment with ezetimibe alone had no effect on TICE, it reduced the effect of GW610742 on TICE. These data show that activation of PPARδ stimulates fecal cholesterol excretion in mice, primarily by the two-fold increase in TICE, indicating that this pathway provides an interesting target for the development of drugs aiming at the prevention of atherosclerosis.     

The Hepatitis C Virus-induced NLRP3 Inflammasome Activates the Sterol Regulatory Element-binding Protein (SREBP) and Regulates Lipid Metabolism

Hepatitis C virus (HCV) relies on host lipids and lipid droplets for replication and morphogenesis. The accumulation of lipid droplets in infected hepatocytes manifests as hepatosteatosis, a common pathology observed in chronic hepatitis C patients. One way by which HCV promotes the accumulation of intracellular lipids is through enhancing de novo lipogenesis by activating the sterol regulatory element-binding proteins (SREBPs). In general, activation of SREBPs occurs during cholesterol depletion. Interestingly, during HCV infection, the activation of SREBPs occurs under normal cholesterol levels, but the underlying mechanisms are still elusive. Our previous study has demonstrated the activation of the inflammasome complex in HCV-infected human hepatoma cells. In this study, we elucidate the potential link between chronic hepatitis C-associated inflammation and alteration of lipid homeostasis in infected cells. Our results reveal that the HCV-activated NLRP3 inflammasome is required for the up-regulation of lipogenic genes such as 3-hydroxy-3-methylglutaryl-coenzyme A synthase, fatty acid synthase, and stearoyl-CoA desaturase. Using pharmacological inhibitors and siRNA against the inflammasome components (NLRP3, apoptosis-associated speck-like protein containing a CARD, and caspase-1), we further show that the activation of the NLRP3 inflammasome plays a critical role in lipid droplet formation. NLRP3 inflammasome activation in HCV-infected cells enables caspase-1-mediated degradation of insulin-induced gene proteins. This subsequently leads to the transport of the SREBP cleavage-activating protein·SREBP complex from the endoplasmic reticulum to the Golgi, followed by proteolytic activation of SREBPs by S1P and S2P in the Golgi. Typically, inflammasome activation leads to viral clearance. Paradoxically, here we demonstrate how HCV exploits the NLRP3 inflammasome to activate SREBPs and host lipid metabolism, leading to liver disease pathogenesis associated with chronic HCV.     

Changes in cholesterol absorption and cholesterol synthesis caused by ezetimibe and/or simvastatin in men

This study evaluates changes in cholesterol balance in hypercholesterolemic subjects following treatment with an inhibitor of cholesterol absorption or cholesterol synthesis or coadministration of both agents. This was a randomized, double blind, placebo-controlled, four-period crossover study to evaluate the effects of coadministering 10 mg ezetimibe with 20 mg simvastatin (ezetimibe/simvastatin) on cholesterol absorption and synthesis relative to either drug alone or placebo in 41 subjects. Each treatment period lasted 7 weeks. Ezetimibe and ezetimibe/simvastatin decreased fractional cholesterol absorption by 65% and 59%, respectively (P < 0.001 for both relative to placebo). Simvastatin did not significantly affect cholesterol absorption. Ezetimibe and ezetimibe/simvastatin increased fecal sterol excretion (corrected for dietary cholesterol), which also represents net steady state cholesterol synthesis, by 109% and 79%, respectively (P < 0.001). Ezetimibe, simvastatin, and ezetimibe/simvastatin decreased plasma LDL-cholesterol by 20, 38, and 55%, respectively. The coadministered therapy was well tolerated. The decreases in net cholesterol synthesis and increased fecal sterol excretion yielded nearly additive reductions in LDL-cholesterol for the coadministration of ezetimibe and simvastatin.     

Resveratrol protects against atherosclerosis,but does not add to the antiatherogenic effect of atorvastatin,in APOE*3-Leiden.CETP mice

Resveratrol is a major constituent of traditional Asian medicinal herbs and red wine and is suggested to be a potential antiatherosclerotic drug due to its proposed hypolipidemic, anti-inflammatory and antioxidative properties. The aim of this study was to evaluate whether resveratrol protects against atherosclerosis development in APOE*3-Leiden.CETP (E3L.CETP) mice and adds to the antiatherogenic effect of mild statin treatment, currently the most widely used antiatherogenic therapy. E3L.CETP mice were fed a cholesterol-rich diet without (control) or with resveratrol (0.01% w/w), atorvastatin (0.0027% w/w) or both for 14 weeks. During the study plasma lipid, inflammatory and oxidative stress parameters were determined. Resveratrol reduced atherosclerotic lesion area (?52%) in the aortic root, comparable to atorvastatin (?40%) and the combination of both drugs (?47%). The collagen/macrophage ratio in the atherosclerotic lesion, a marker of plaque stability, was increased by resveratrol (+108%), atorvastatin (+124%) and the combination (+154%). Resveratrol decreased plasma cholesterol levels (?19%) comparable to atorvastatin (?19%) and the combination (?22%), which was completely confined to (very)low-density lipoprotein cholesterol levels in all groups. Post hoc analyses showed that the antiatherogenic effect of atorvastatin could be explained by cholesterol lowering, while the antiatherosclerotic effect of resveratrol could be attributed to factors additional to cholesterol lowering. Markers of inflammation and oxidative stress were not different, but resveratrol improved macrophage function. We conclude that resveratrol potently reduces atherosclerosis development and induces a more stable lesion phenotype in E3L.CETP mice. However, under the experimental conditions tested, resveratrol does not add to the antiatherogenic effect of atorvastatin.     

RORα and 25-Hydroxycholesterol Crosstalk Regulates Lipid Droplet Homeostasis in Macrophages

Nuclear hormone receptors have important roles in the regulation of metabolic and inflammatory pathways. The retinoid-related orphan receptor alpha (Rorα)-deficient staggerer (sg/sg) mice display several phenotypes indicative of aberrant lipid metabolism, including dyslipidemia, and increased susceptibility to atherosclerosis. In this study we demonstrate that macrophages from sg/sg mice have increased ability to accumulate lipids and accordingly exhibit larger lipid droplets (LD). We have previously shown that BMMs from sg/sg mice have significantly decreased expression of cholesterol 25-hydroxylase (Ch25h) mRNA, the enzyme that produces the oxysterol, 25-hydroxycholesterol (25HC), and now confirm this at the protein level. 25HC functions as an inverse agonist for RORα. siRNA knockdown of Ch25h in macrophages up-regulates Vldlr mRNA expression and causes increased accumulation of LDs. Treatment with physiological concentrations of 25HC in sg/sg macrophages restored lipid accumulation back to normal levels. Thus, 25HC and RORα signify a new pathway involved in the regulation of lipid homeostasis in macrophages, potentially via increased uptake of lipid which is suggested by mRNA expression changes in Vldlr and other related genes.     

Ezetimibe markedly attenuates hepatic cholesterol accumulation and improves liver function in the lysosomal acid lipase-deficient mouse,a model for cholesteryl ester storage disease

Lysosomal acid lipase (LAL) plays a critical role in the intracellular handling of lipids by hydrolyzing cholesteryl esters (CE) and triacylglycerols (TAG) contained in newly internalized lipoproteins. In humans, mutations in the LAL gene result in cholesteryl ester storage disease (CESD), or in Wolman disease (WD) when the mutations cause complete loss of LAL activity. A rat model for WD and a mouse model for CESD have been described. In these studies we used LAL-deficient mice to investigate how modulating the amount of intestinally-derived cholesterol reaching the liver might impact its mass, cholesterol content, and function in this model. The main experiment tested if ezetimibe, a potent cholesterol absorption inhibitor, had any effect on CE accumulation in mice lacking LAL. In male Lal^−/− mice given ezetimibe in their diet (20 mg/day/kg bw) for 4 weeks starting at 21 days of age, both liver mass and hepatic cholesterol concentration (mg/g) were reduced to the extent that whole-liver cholesterol content (mg/organ) in the treated mice (74.3 ± 3.4) was only 56% of that in those not given ezetimibe (133.5 ± 6.7). There was also a marked improvement in plasma alanine aminotransferase (ALT) activity. Thus, minimizing cholesterol absorption has a favorable impact on the liver in CESD.