Accumulation Of Free Cholesterol Research Articles

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

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

Depletion of Cholesteryl Esters Causes Meibomian Gland Dysfunction-Like Symptoms in a Soat1-Null Mouse Model.

Previous studies on ablation of several key genes of meibogenesis related to fatty acid elongation, omega oxidation, and esterification into wax esters have demonstrated that inactivation of any of them led to predicted changes in the meibum lipid profiles and caused severe abnormalities in the ocular surface and Meibomian gland (MG) physiology and morphology. In this study, we evaluated the effects of Soat1 ablation that were expected to cause depletion of the second largest class of Meibomian lipids (ML)—cholesteryl esters (CE)—in a mouse model. ML of the Soat1-null mice were examined using liquid chromatography high-resolution mass spectrometry and compared with those of Soat1+/− and wild-type mice. Complete suppression of CE biosynthesis and simultaneous accumulation of free cholesterol (Chl) were observed in Soat1-null mice, while Soat1+/− mutants had normal Chl and CE profiles. The total arrest of the CE biosynthesis in response to Soat1 ablation transformed Chl into the dominant lipid in meibum accounting for at least 30% of all ML. The Soat1-null mice had clear manifestations of dry eye and MG dysfunction. Enrichment of meibum with Chl and depletion of CE caused plugging of MG orifices, increased meibum rigidity and melting temperature, and led to a massive accumulation of lipid deposits around the eyes of Soat1-null mice. These findings illustrate the role of Soat1/SOAT1 in the lipid homeostasis and pathophysiology of MG.

Aggregated LDL turn human macrophages into foam cells and induce mitochondrial dysfunction without triggering oxidative or endoplasmic reticulum stress.

Uptake of modified lipoproteins by macrophages turns them into foam cells, the hallmark of the atherosclerotic plaque. The initiation and progression of atherosclerosis have been associated with mitochondrial dysfunction. It is known that aggregated low-density lipoproteins (agLDL) induce massive cholesterol accumulation in macrophages in contrast with native LDL (nLDL) and oxidized LDL (oxLDL). In the present study we aimed to assess the effect of agLDL on the mitochondria and ER function in macrophage-derived foam cells, in an attempt to estimate the potential of these cells, known constituents of early fatty streaks, to generate atheroma in the absence of oxidative stress. Results show that agLDL induce excessive accumulation of free (FC) and esterified cholesterol in THP-1 macrophages and determine mitochondrial dysfunction expressed as decreased mitochondrial membrane potential and diminished intracellular ATP levels, without generating mitochondrial reactive oxygen species (ROS) production. AgLDL did not stimulate intracellular ROS (superoxide anion or hydrogen peroxide) production, and did not trigger endoplasmic reticulum stress (ERS) or apoptosis. In contrast to agLDL, oxLDL did not modify FC levels, but stimulated the accumulation of 7-ketocholesterol in the cells, generating oxidative stress which is associated with an increased mitochondrial dysfunction, ERS and apoptosis. Taken together, our results reveal that agLDL induce foam cells formation and mild mitochondrial dysfunction in human macrophages without triggering oxidative or ERS. These data could partially explain the early formation of fatty streaks in the intima of human arteries by interaction of monocyte-derived macrophages with non-oxidatively aggregated LDL generating foam cells, which cannot evolve into atherosclerotic plaques in the absence of the oxidative stress.

Probucol treatment is associated with an ABCA1-independent mechanism of cholesterol efflux to lipid poor apolipoproteins from foam cell macrophages

ObjectiveProbucol is a cholesterol-lowering agent whose ability to prevent atherosclerosis is currently under study. Herein, we investigate the putative mechanism of probucol by observation of changes in cellular cholesterol efflux and lipid droplet morphology in macrophages. ResultsThe inhibitory activity of probucol was assessed in non-foam or foam cell macrophages expressing ABCA1 generated by treatment with fetal calf serum (FCS) alone or in combination with acetylated LDL, respectively. Probucol inhibited cholesterol efflux to apolipoprotein A-I (apoA-I) by 31.5±0.1% in THP-1 non-foam cells and by 18.5±0.2% in foam cells. In probucol-treated non-foam THP-1 cells, nascent high density lipoprotein (nHDL) particles with a diameter < 7 nm were generated, while in probucol-treated THP-1 foam cells nHDL particles of > 7 nm in diameter containing cholesterol were produced. Foam cells also displayed a significant accumulation of free cholesterol at the plasma membrane, as measured by percent cholestenone formed. Intracellularly, there was a significant decrease in lipid droplet number and an increase in size in probucol-treated THP-1 foam cells when compared to non-treated cells. ConclusionsWe report for the first time that probucol is unable to completely inhibit cholesterol efflux in foam cells to the same extent as in non-foam cells. Indeed, functional nHDL is released from foam cells in the presence of probucol. This difference in inhibitory effect could potentially be explained by changes in the plasma membrane pool as well as intracellular cholesterol storage independently of ABCA1.

The Cholesterol-Binding Translocator Protein (18 kDa) Regulates Hepatic Steatosis and Bile Acid Synthesis in Non-Alcoholic Fatty Liver Disease

Translocator protein (TSPO, 18kDa) levels increase in parallel with the evolution of simple steatosis (SS) to nonalcoholic steatohepatitis (NASH) in non-alcoholic fatty liver disease (NAFLD). However, TSPO function in SS and NASH is unknown. Loss of TSPO in hepatocytes in vitro downregulated acetyl-CoA acetyltransferase 2 (ACAT2) and increased free cholesterol (FC). FC accumulation induced endoplasmic reticulum stress via IRE1A and PERK/ATF4/CHOP pathways and autophagy. TSPO deficiency activated cellular adaptive antioxidant protection; this adaptation was lost upon excessive FC accumulation. 19-Atriol, a TSPO ligand, blocked cholesterol binding and recapitulated many of the alterations seen in TSPO-deficient cells. These data suggest that TSPO deficiency accelerated the progression of SS. In NASH, however, loss of TSPO ameliorated liver fibrosis through downregulation of bile acid synthesis by reducing CYP7A1 and CYP27A1 levels and increasing farnesoid X receptor expression. These studies indicate a dynamic and complex role for TSPO in the evolution of NAFLD.

Cholesterol Homeostasis: An In Silico Investigation into How Aging Disrupts Its Key Hepatic Regulatory Mechanisms.

Simple SummaryCardiovascular disease is the leading cause of death worldwide. High blood cholesterol levels are associated with an increased risk of this condition. How high levels of blood cholesterol result in cardiovascular disease during aging is a difficult question to answer. Computational models have been used over the years to help understand this problem, as they are capable of representing complex systems such as this. The aim of this work was to use a computational model of cholesterol metabolism to better understand why, in certain studies involving the oldest old (persons ≥ 85 years), high blood cholesterol levels have been associated with a decreased risk of death. Using our computational model, we found that key age-associated changes to how cholesterol is processed in the liver could be responsible for this observation. The findings from our work contribute to our understanding of cholesterol metabolism in older people and how treatments could be developed in the future to promote healthy aging.The dysregulation of intracellular cholesterol homeostasis is associated with several age-related diseases, most notably cardiovascular disease (CVD). Research in this area has benefitted from using computational modelling to study the inherent complexity associated with the regulation of this system. In addition to facilitating hypothesis exploration, the utility of modelling lies in its ability to represent an array of rate limiting enzymatic reactions, together with multiple feedback loops, which collectively define the dynamics of cholesterol homeostasis. However, to date no model has specifically investigated the effects aging has on this system. This work addresses this shortcoming by explicitly focusing on the impact of aging on hepatic intracellular cholesterol homeostasis. The model was used to investigate the experimental findings that reactive oxygen species induce the total activation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR). Moreover, the model explored the impact of an age-related decrease in hepatic acetyl-CoA acetyltransferase 2 (ACAT2). The model suggested that an increase in the activity of HMGCR does not have as significant an impact on cholesterol homeostasis as a decrease in hepatic ACAT2 activity. According to the model, a decrease in the activity of hepatic ACAT2 raises free cholesterol (FC) and decreases low-density lipoprotein cholesterol (LDL-C) levels. Increased acetyl CoA synthesis resulted in a reduction in the number of hepatic low-density lipoprotein receptors, and increased LDL-C, FC, and cholesterol esters. The rise in LDL-C was restricted by elevated hepatic FC accumulation. Taken together these findings have important implications for healthspan. This is because emerging clinical data suggest hepatic FC accumulation is relevant to the pathogenesis of non-alcoholic fatty liver disease (NAFLD), which is associated with an increased risk of CVD. These pathophysiological changes could, in part, help to explain the phenomenon of increased mortality associated with low levels of LDL-C which have been observed in certain studies involving the oldest old (≥85 years).

Acidic extracellular pH promotes accumulation of free cholesterol in human monocyte-derived macrophages via inhibition of ACAT1 activity

Background and aimsIn focal areas of advanced human atherosclerotic lesions, the intimal fluid is acidic. An acidic medium impairs the ABCA1-mediated cholesterol efflux from macrophages, so tending to increase their content of free cholesterol, which is then available for esterification by the macrophage enzyme ACAT1. Here we investigated whether low extracellular pH would affect the activity of ACAT1. Methods– Human monocyte-derived macrophages were first incubated with acetyl-LDL at neutral and acidic conditions (pH 7.5, 6.5, and 5.5) to generate foam cells, and then the foam cells were incubated with [3H]oleate-BSA complexes, and the formation of [3H]oleate-labeled cholesteryl esters was measured. ACAT1 activity was also measured in cell-free macrophage extracts. Results– In acidic media, ACAT1-dependent cholesteryl [3H]oleate generation became compromised in the developing foam cells and their content of free cholesterol increased. In line with this finding, ACAT1 activity in the soluble cell-free fraction derived from macrophage foam cells peaked at pH 7, and gradually decreased under acidic pH with a rapid drop below pH 6.5. Incubation of macrophages under progressively more acidic conditions (until pH 5.5) lowered the cytosolic pH of macrophages (down to pH 6.0). Such intracellular acidification did not affect macrophage gene expression of ACAT1 or the neutral CEH. ConclusionsExposure of human macrophage foam cells to acidic conditions lowers their intracellular pH with simultaneous decrease in ACAT1 activity. This reduces cholesterol esterification and thus leads to accumulation of potentially toxic levels of free cholesterol, a contributing factor to macrophage foam cell death.

Disturbances in Cholesterol Homeostasis and Non-alcoholic Fatty Liver Diseases.

Non-alcoholic fatty liver disease (NAFLD) is a major health problem associated with obesity and other features of the metabolic syndrome including insulin resistance and dyslipidemia. The accumulation of lipids in hepatocytes causes liver damage and triggers inflammation, fibrosis, and cirrhosis. Beside fatty acids and triglycerides, evidence showed an increased accumulation of free cholesterol in the liver with subsequent toxic effects contributing to liver damage. The maintenance of cholesterol homeostasis in the body requires a balance between several pathways responsible for cholesterol synthesis, transport and conversion into bile acids. Intestinal absorption is also one of the major determinants of cholesterol homeostasis. The nature of changes in cholesterol homeostasis associated with NAFLD has been a subject of extensive investigations. In this article, we will attempt to provide a brief overview of the current knowledge about the disturbances in cholesterol metabolism associated with NAFLD and discuss how certain molecular targets of these pathways could be exploited for the treatment of this multifactorial disease.

Rationale and design of a randomised, double-blind, placebo-controlled, parallel-group, investigator-initiated phase 2a study to investigate the efficacy and safety of elobixibat in combination with cholestyramine for non-alcoholic fatty liver disease.

IntroductionNon-alcoholic fatty liver disease (NAFLD) pathogenesis involves abnormal metabolism of cholesterol and hepatic accumulation of toxic free-cholesterol. Elobixibat (EXB) inhibits the ileal bile acid (BA) transporter. EXB and cholestyramine (CTM) facilitate the removal of free cholesterol from the liver by decreasing BA recirculation to the liver, thereby stimulating novel BA synthesis from cholesterol. In this randomised, double-blind, placebo-controlled, parallel-group, phase IIa study, we aim to provide a proof-of-concept assessment by evaluating the efficacy and safety of EXB in combination with CTM in patients with NAFLD.Methods and analysisA total of 100 adult patients with NAFLD, diagnosed based on low-density lipoprotein cholesterol (LDL-C) level of >120 mg/dL and liver fat content of ≥8% by MRI-based proton density fat fraction (MRI-PDFF), who meet the inclusion/exclusion criteria will be enrolled. The patients will be randomly assigned to receive the combination therapy of 10 mg EXB and 9 g CTM powder (4 g CTM), 10 mg EXB monotherapy, 9 g CTM powder monotherapy or a placebo treatment (n=25 per group). Blood tests and MRIs will be performed 16 weeks following treatment initiation. The primary study endpoint will be the absolute LDL-C level change at week 16 after treatment initiation. The exploratory endpoint will include absolute changes in the liver fat fraction as measured by MRI-PDFF. This proof-of-concept study will determine whether the combination therapy of EXB and CTM is effective and safe for patients with NAFLD.Ethics and disseminationEthics approval was obtained from the Ethics Committee of Yokohama City University Hospital before participant enrolment. The results of this study will be submitted for publication in international peer-reviewed journals and the key findings will be presented at international scientific conferences.Trial registration numberNCT04235205

Sterol-O-acyltransferase-1 has a role in kidney disease associated with diabetes and Alport syndrome

Defective cholesterol metabolism primarily linked to reduced ATP-binding cassette transporter A1 (ABCA1) expression is closely associated with the pathogenesis and progression of kidney diseases, including diabetic kidney disease and Alport Syndrome. However, whether the accumulation of free or esterified cholesterol contributes to progression in kidney disease remains unclear. Here, we demonstrate that inhibition of sterol-O-acyltransferase-1 (SOAT1), the enzyme at the endoplasmic reticulum that converts free cholesterol to cholesterol esters, which are then stored in lipid droplets, effectively reduced cholesterol ester and lipid droplet formation in human podocytes. Furthermore, we found that inhibition of SOAT1 in podocytes reduced lipotoxicity-mediated podocyte injury in diabetic kidney disease and Alport Syndrome in association with increased ABCA1 expression and ABCA1-mediated cholesterol efflux. Invivo, Soat1 deficient mice did not develop albuminuria or mesangial expansion at 10-12 months of age. However, Soat1 deficiency/inhibition in experimental models of diabetic kidney disease and Alport Syndrome reduced cholesterol ester content in kidney cortices and protected from disease progression. Thus, targeting SOAT1-mediated cholesterol metabolism may represent a new therapeutic strategy to treat kidney disease in patients with diabetic kidney disease and Alport Syndrome, like that suggested for Alzheimer's disease and cancer treatments.

Lipoprotein X in Cholestasis and Questionable Atherogenic Risk

Lipoprotein X is still an enigma for its lack of evidence as a risk association with atherosclerosis. Primary biliary cirrhosis is a chronic, autoimmune cholestatic liver disease characterized by the presence of T cells that destroy the biliary epithelium. In patients with primary biliary cirrhosis, total cholesterol levels may reach values between 500 and 1000 mg/dL, with cutaneous xanthomas similar to those observed in genetic hypercholesterolemias. The rise in cholesterol is largely due to increased serum levels of lipoprotein-X. It has a small amount of albumin, apoprotein AI, apoprotein Cs and apoprotein E and has no apoprotein B. The formation of lipoprotein-X results from regurgitation of bile lipids in plasma, as well as the accumulation of plasma phospholipids and free cholesterol, resulting from the lower activity of lecithin: acyltransferase cholesterol. Multicenter randomized trials are needed to assess the impact of lipid-lowering patients on lipid reduction and cardiovascular risk, as well as the effect on hepatic cholestasis. Treatment with statins is not indicated for lowering cholesterol in patients with primary biliary cirrhosis and dyslipidemia, as there is no evidence that these patients have high cardiovascular risk.

SUN-216 Investigating the Role of the Liver X Receptor in Potentiating Mitotane Therapy in Adrenocortical Carcinoma

Introduction: Adrenocortical Carcinoma is a rare aggressive cancer which carries a poor prognosis. Adjuvant mitotane improves survival but is limited by a narrow therapeutic window and severe adverse effects. Liver X receptors (LXRs), part of the nuclear receptor superfamily are highly expressed in adrenal tissue and mediate transcellular and intracellular cholesterol homeostasis. We hypothesise that LXRα inhibition increases toxic lipid accumulation in adrenocortical cancer cells and potentiates the adrenolytic effect of mitotane. Methodology: ATCC-H295R and MUC1 ACC cells and were pre-treated with the LXRα inverse agonist SR9243 5µM and antagonist GSK2033 5µM followed by mitotane treatment (20, 40, 50μΜ) for 6 hours. Cholesterol-methyl-β-cyclodextrin treatment was carried out 1hr prior to mitotane. H295R cells were transfected with a LXR⍺ dominant negative construct using lipofectamine. Cell death was assessed using annexin/PI staining and proliferation using MTT assay. Free cholesterol (FC) levels were assayed using filipin staining and lipid droplets via BODIPY® and analysed on the Amnis ImageStream® imaging cytometer. Downstream targets ABCA1 and ABCG1 were evaluated by qRT-PCR. Lipid droplet associated proteins PLIN 1-4 and hormone sensitive lipase (HSL) expression were evaluated using western blotting. Results: Downstream reduction of ABCA1 and ABCG1 expression confirmed LXRα blockade. Mitotane effectively induced dose-dependent H295R apoptotic cell death which was potentiated pharmacologically and genetically by LXR⍺ inhibition. In line with these findings, cholesterol-methyl-β-cyclodextrin treatment increased cell death in H295R and MUC1 cells. In addition to inducing cell death, LXR⍺ inhibition decreased proliferation of both cell lines. An increase in FC and a decrease in cholesterol esters was observed following mitotane treatment in H295R cells. This was accompanied by decreased lipid droplet numbers confirmed by lower expression of lipid droplet associated proteins, PLIN1-3. These effects were potentiated when mitotane was combined with LXRα inhibition. We demonstrate increased HSL activity, which was associated with higher SOAT-1 expression and increasing toxic FC accumulation. Investigation of lipid droplet content BODIPY® of both cell lines showed H295R cells preferentially store cholesterol esters and MUC1 cells store triacylglycerides.Conclusion: We propose a mechanism for enhancing mitotane’s efficacy as an adrenolytic through increased free cholesterol via LXR⍺inhibition. Targeting the LXRα, its putative ligands, or associated lipid mediators may present a novel therapeutic approach in the setting of primary and metastatic ACC.

Abnormal LAMP1 glycosylation may play a role in Niemann-Pick disease, type C pathology.

A hallmark of Niemann-Pick disease, type C (NPC) is the progressive degeneration of Purkinje neurons in the cerebellum caused by the accumulation of free cholesterol and glycosphingolipids in the lysosome. Recent studies suggest that the state of glycosylation of lysosomal membrane proteins may play a role in disease progression. Our study has identified the presence of a highly glycosylated form of Lysosome Associated Membrane Protein 1 (LAMP1) that correlated spatiotemporally with Purkinje neuron loss. This form of LAMP1 was predominantly localized to activated microglia; showing a ~5-fold increase in surface labeling by FACS analysis. This suggests a potential role for LAMP1 in the neuro-inflammatory process in these mice during disease progression. Analysis of other mouse models of neurodegeneration that exhibit neuro-inflammation showed little or no presence of this glycosylated form of LAMP1, suggesting this observation for LAMP1 is specific to NPC disease. Furthermore, early treatment of Npc1-/- mice with 2-hydroxypropyl-β-cyclodextrin (HPβCD), significantly prevented the appearance of the glycosylated LAMP1 in the cerebellum of Npc1-/- mice at 7 weeks, consistent with the prevention of neuro-inflammation in mice treated with this drug. Treatment of Npc1-/- mice with HPβCD at 7 weeks, after disease onset, did not reverse or prevent further appearance of the hyperglycosylated LAMP1, demonstrating that once this aspect of neuro-inflammation began, it continued despite the HPβCD treatment. Analysis of LAMP1 in cerebellar tissue of NPC1 patients showed a small level of hyperglycosylated LAMP1 in the tissue, however, this was not seen in the CSF of patients.

Differential Effects of 2-Hydroxypropyl-Cyclodextrins on Lipid Accumulation in Npc1-Null Cells.

Niemann–Pick disease type C (NPC) is an autosomal recessive disorder characterized by abnormal accumulation of free cholesterol and sphingolipids in lysosomes. The iminosugar miglustat, which inhibits hexosylceramide synthesis, is used for NPC treatment, and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), a cyclic oligosaccharide derivative, is being developed to treat NPC. Moreover, therapeutic potential of 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD) was shown in NPC models, although its mechanism of action remains unclear. Here, we investigated the effects of HP-β-CD, HP-γ-CD, and their homolog 2-hydroxypropyl-α-cyclodextrin (HP-α-CD) on lipid accumulation in Npc1-null Chinese hamster ovary (CHO) cells compared with those of miglustat. HP-β-CD and HP-γ-CD, unlike HP-α-CD, reduced intracellular free cholesterol levels and normalized the lysosome changes in Npc1-null cells but not in wild-type CHO cells. In contrast, miglustat did not normalize intracellular free cholesterol accumulation or lysosome changes in Npc1-null cells. However, miglustat decreased the levels of hexosylceramide and tended to increase those of sphingomyelins in line with its action as a glucosylceramide synthase inhibitor in both Npc1-null and wild-type CHO cells. Interestingly, HP-β-CD and HP-γ-CD, unlike HP-α-CD, reduced sphingomyelins in Npc1-null, but not wild-type, cells. In conclusion, HP-β-CD and HP-γ-CD reduce the accumulation of sphingolipids, mainly sphingomyelins, and free cholesterol as well as lysosome changes in Npc1-null, but not in wild-type, CHO cells.

SIRT6 inhibits cholesterol crystal-induced vascular endothelial dysfunction via Nrf2 activation

Sirtuin 6 (SIRT6), a nicotinamide adenine dinucleotide-dependent deacetylase, participates in various age-related disorders, such as dyslipidemia and cardiovascular diseases. Recent studies have revealed that minute cholesterol crystals (CCs), which are generated after excess free cholesterol accumulation, form not only in mature atherosclerotic plaques but also extremely early in atherosclerosis. Since endothelial dysfunction is an early feature of atherogenesis, this study was designed to investigate the role of SIRT6 in minute CC-induced endothelial dysfunction and the related mechanism. We found that minute CCs could be endocytosed by endothelial cells (ECs), which then decreased nitric oxide (NO) levels and endothelial nitric oxide synthase (eNOS) activity and expression, upregulated the expression of adhesion molecules and enhanced monocyte adhesion to ECs. In addition, minute CCs significantly suppressed SIRT6 expression in ECs. Moreover, the overexpression of SIRT6 could mitigate minute CC-induced endothelial dysfunction. In addition, the expression of Nuclear factor erythroid2-related factor2 (Nrf2) was suppressed after minute CC treatment, whereas SIRT6 overexpression reversed this decrease in Nrf2 expression. More importantly, Nrf2 activation also notably attenuated minute CC-induced endothelial dysfunction. In vivo experiments further indicated that endothelium-specific SIRT6 depletion impaired vascular endothelial function and suppressed Nrf2 expression in hyperlipidemic mice. Taken together, these results indicate that SIRT6 rescues minute CC-induced endothelial dysfunction partly via Nrf2 activation.

Liver X Receptors Regulate Cholesterol Metabolism and Immunity in Hepatic Nonparenchymal Cells.

Excess dietary cholesterol intake and the dysregulation of cholesterol metabolism are associated with the pathogenesis and progression of nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and fibrosis. Hepatic accumulation of free cholesterol induces activation of nonparenchymal cells, including Kupffer cells, macrophages, and hepatic stellate cells, which leads to persistent inflammation and fibrosis. The nuclear receptors liver X receptor α (LXRα) and LXRβ act as negative regulators of cholesterol metabolism through the induction of hepatocyte cholesterol catabolism, excretion, and the reverse cholesterol transport pathway. Additionally, LXRs exert an anti-inflammatory effect in immune cell types, such as macrophages. LXR activation suppresses acute hepatic inflammation that is mediated by Kupffer cells/macrophages. Acute liver injury, diet-induced steatohepatitis, and fibrosis are exacerbated by significant hepatic cholesterol accumulation and inflammation in LXR-deficient mice. Therefore, LXRs regulate hepatic lipid metabolism and immunity and they are potential therapeutic targets in the treatment of hepatic inflammation that is associated with cholesterol accumulation.

AdipoRon Attenuates Wnt Signaling by Reducing Cholesterol-Dependent Plasma Membrane Rigidity

The increasing prevalence of adult and adolescent obesity and its associated risk of colorectal cancer reinforces the urgent need to elucidate the underlying mechanisms contributing to the promotion of colon cancer in obese individuals. Adiponectin is an adipose tissue-derived adipokine, whose levels are reduced during obesity. Both epidemiological and preclinical data indicate that adiponectin suppresses colon tumorigenesis. We have previously demonstrated that both adiponectin and AdipoRon, a small-molecule adiponectin receptor agonist, suppress colon cancer risk in part by reducing the number of Lgr5+ stem cells in mouse colonic organoids. However, the mechanism by which the adiponectin signaling pathway attenuates colon cancer risk remains to be addressed. Here, we have hypothesized that adiponectin signaling supports colonic stem cell maintenance through modulation of the biophysical properties of the plasma membrane (PM). Specifically, we investigated the effects of adiponectin receptor activation by AdipoRon on the biophysical perturbations linked to the attenuation of Wnt-driven signaling and cell proliferation as determined by LEF luciferase reporter assay and colonic organoid proliferation, respectively. Using physicochemical sensitive dyes, Di-4-ANEPPDHQ and C-laurdan, we demonstrated that AdipoRon decreased the rigidity of the colonic cell PM. The decrease in membrane rigidity was associated with a reduction in PM free cholesterol levels and the intracellular accumulation of free cholesterol in lysosomes. These results suggest that adiponectin signaling plays a role in modulating cellular cholesterol homeostasis, PM biophysical properties, and Wnt-driven signaling. These findings are noteworthy because they may in part explain how obesity drives colon cancer progression.

ATP-binding cassette A1 deficiency causes cardiolipin-driven mitochondrial dysfunction in podocytes.

Fibroblasts from patients with Tangier disease carrying ATP-binding cassette A1 (ABCA1) loss-of-function mutations are characterized by cardiolipin accumulation, a mitochondrial-specific phospholipid. Suppression of ABCA1 expression occurs in glomeruli from patients with diabetic kidney disease (DKD) and in human podocytes exposed to DKD sera collected prior to the development of DKD. We demonstrated that siRNA ABCA1 knockdown in podocytes led to reduced oxygen consumption capabilities associated with alterations in the oxidative phosphorylation (OXPHOS) complexes and with cardiolipin accumulation. Podocyte-specific deletion of Abca1 (Abca1fl/fl) rendered mice susceptible to DKD, and pharmacological induction of ABCA1 improved established DKD. This was not mediated by free cholesterol, as genetic deletion of sterol-o-acyltransferase-1 (SOAT1) in Abca1fl/fl mice was sufficient to cause free cholesterol accumulation but did not cause glomerular injury. Instead, cardiolipin mediates ABCA1-dependent susceptibility to podocyte injury, as inhibition of cardiolipin peroxidation with elamipretide improved DKD in vivo and prevented ABCA1-dependent podocyte injury in vitro and in vivo. Collectively, we describe a pathway definitively linking ABCA1 deficiency to cardiolipin-driven mitochondrial dysfunction. We demonstrated that this pathway is relevant to DKD and that ABCA1 inducers or inhibitors of cardiolipin peroxidation may each represent therapeutic strategies for the treatment of established DKD.

Schnyder corneal dystrophy-associated UBIAD1 mutations cause corneal cholesterol accumulation by stabilizing HMG-CoA reductase.

Schnyder corneal dystrophy (SCD) is a rare genetic eye disease characterized by corneal opacification resulted from deposition of excess free cholesterol. UbiA prenyltransferase domain-containing protein-1 (UBIAD1) is an enzyme catalyzing biosynthesis of coenzyme Q10 and vitamin K2. More than 20 UBIAD1 mutations have been found to associate with human SCD. How these mutants contribute to SCD development is not fully understood. Here, we identified HMGCR as a binding partner of UBIAD1 using mass spectrometry. In contrast to the Golgi localization of wild-type UBIAD1, SCD-associated mutants mainly resided in the endoplasmic reticulum (ER) and competed with Insig-1 for HMGCR binding, thereby preventing HMGCR from degradation and increasing cholesterol biosynthesis. The heterozygous Ubiad1 G184R knock-in (Ubiad1G184R/+) mice expressed elevated levels of HMGCR protein in various tissues. The aged Ubiad1G184R/+ mice exhibited corneal opacification and free cholesterol accumulation, phenocopying clinical manifestations of SCD patients. In summary, these results demonstrate that SCD-associated mutations of UBIAD1 impair its ER-to-Golgi transportation and enhance its interaction with HMGCR. The stabilization of HMGCR by UBIAD1 increases cholesterol biosynthesis and eventually causes cholesterol accumulation in the cornea.

PNPLA3 I148M Variant Impairs Liver X Receptor Signaling and Cholesterol Homeostasis in Human Hepatic Stellate Cells.

The patatin‐like phospholipase domain‐containing protein 3 (PNPLA3) I148M variant predisposes to hepatic steatosis and progression to advanced liver injury with development of fibrosis, cirrhosis, and cancer. Hepatic stellate cells (HSCs) drive the wound healing response to chronic injury, and lack of liver X receptor (LXR) signaling exacerbates liver fibrogenesis by impairing HSC cholesterol homeostasis. However, the contribution of the I148M variant to this process is still unknown. We analyzed LXR expression and transcriptional activity in primary human HSCs and overexpressing LX‐2 cells according to PNPLA3 genotype (wild type [WT] versus I148M). Here we demonstrate that LXRα protein increased whereas LXR target gene expression decreased during in vitro activation of primary human HSCs. Notably, LXRα levels and signaling were reduced in primary I148M HSCs compared to WT, as displayed by decreased expression of LXR target genes. Moreover, reduced expression of cholesterol efflux and enzymes generating oxysterols was associated with higher total and free cholesterol accumulation whereas endogenous cholesterol synthesis and uptake were diminished in I148M HSCs. Luciferase assays on LXR response element confirmed decreased LXR transcriptional activity in I148M HSCs; in contrast the synthetic LXR agonist T0901317 replenished LXR functionality, supported by adenosine triphosphate‐binding cassette subfamily A member 1 (ABCA1) induction, and reduced collagen1α1 and chemokine (C‐C motif) ligand 5 expression. Conversely, the peroxisome proliferator‐activated receptor gamma (PPARγ) agonist rosiglitazone had only partial effects on the LXR target gene ABCA1, and neither diminished expression of proinflammatory cytokines nor increased de novo lipogenic genes in I148M HSCs. Conclusion: As a consequence of reduced PPARγ activity, HSCs carrying I148M PNPLA3 show impaired LXR signaling, leading to cholesterol accumulation. The use of a specific LXR agonist shows beneficial effects for diminishing sustained HSC activation and development of liver fibrogenesis.