Cholesterol Supplementation Research Articles

Investigating the Role of Cholesterol and Lipid Trafficking in Mitotane Resistance in Adrenocortical Carcinoma

Introduction: Adrenocortical Carcinoma (ACC) is a rare aggressive cancer which carries a poor prognosis. Adjuvant mitotane improves survival but is limited by poor response rates and resistance following tumour recurrence. Mitotane’s efficacy has been attributed to intracellular accumulation of toxic free cholesterol (FC) predominantly through inhibition of cholesterol storage through SOAT1. Yet SOAT1 specific inhibitors demonstrate inferior efficacy to mitotane in inducing ACC cell death. We hypothesize that mitotane’s efficacy to induce toxic FC accumulation in ACC cells is also mediated through enhanced breakdown of stored cholesterol within intracellular lipid droplets (LDs). Methodology: ATCC-H295R (mitotane sensitive) and MUC-1 (mitotane resistant) ACC cells were evaluated for neutral lipid content using BODIPY493/503 under baseline and cholesterol loaded conditions using Amnis ImageStream, additionally cells were treated with mitotane (H295R - 20, 40, 50µM; MUC1 - 50, 100, 200µM) for 6hr. Analysis of LDs using CE-BODIPY and FA-BODIPY identified cholesterol ester (CE) and triacylglycerol (TAG)-containing LDs, respectively. Lipid droplet-associated proteins (LDAPs) Perilipin (PLIN) 1–4 and hormone sensitive lipase (HSL) were evaluated using western blotting and PCR. Lipid uptake receptors; SRB1, LDLR, LRP1 and CD36 were measured by flow cytometry. Results: Mitotane treatment, within therapeutic range, decreased staining for LDs significantly in H295R. This was also reflected by decreased expression of LDAPs, PLIN1 and PLIN3. The decrease in H295R LDs was associated with increased activation of HSL (pHSL and LIPE). However, this effect was only evident in MUC-1 at supratherapeutic mitotane (200µM). H295R and MUC-1 demonstrated similar overall LD numbers at baseline and under cholesterol supplementation. Expression of PLIN3 was high in both cell lines, while PLIN1, PLIN2 and PLIN4 demonstrated distinct LD profiles in each. Investigation of LD content showed that H295R preferentially store CEs while MUC-1 store only TAG, irrespective of cholesterol-loading. Mitotane treatment significantly reduces both CE and TAG LDs in H295R and MUC-1. Expression of lipid uptake receptors also demonstrated significant variability between cell lines including SRB1 and LRP1. Conclusion: We highlight that lipolysis through LD breakdown and activation of HSL represents a putative additional mechanism for mitotane induced FC cytotoxicity in ACC. We also demonstrate significant differences in cholesterol handling and LDAPs between mitotane sensitive and mitotane resistant models, in particular, the absence of CE LDs in MUC-1. We therefore propose a mechanism of resistance to mitotane through absent CE storage. Further understanding of cholesterol and lipid handling in ACC offers novel therapeutic exploitation, especially in the setting of mitotane resistant disease.

Dietary Cholesterol Supplements Disturb Copper Homeostasis in Multiple Organs in Rabbits: Aorta Copper Concentrations Negatively Correlate with the Severity of Atherosclerotic Lesions.

Dietary cholesterol causes atherosclerosis along with a reduction of copper concentrations in the atherosclerosis wall. This study was to determine the relationship between aorta copper concentrations and the severity of atherosclerotic lesions as well as copper homeostasis in multiple organs in cholesterol-fed rabbits. Male New Zealand white rabbits, 10-week-old and averaged 2.0 kg, were fed a diet containing 1% (w/w) cholesterol or the same diet without cholesterol as controls. Twelve weeks after the feeding, aortic atherosclerotic lesions, serum cholesterol, and multiple organ copper concentrations were measured. Compared to controls, rabbits fed cholesterol-supplemented diet displayed higher serum cholesterol levels and developed atherosclerosis. Copper concentrations in the cholesterol-fed rabbits were increased in the serum and kidney but decreased in the atherosclerosis wall and multiple organs, including heart, liver, spleen, and lung. Furthermore, aorta copper concentrations negatively correlated, respectively, with the severity of the atherosclerotic lesion (r = - 0.64, p = 0.01), the microscope atherosclerotic lesion area (r = - 0.60, p = 0.02), and the stenosis of the lumen (r = - 0.54, p = 0.04). Dietary cholesterol not only causes atherosclerosis but also disturbs copper homeostasis in multiple organ systems. The negative correlation between aorta copper concentrations and the severity of atherosclerotic lesions suggests a vicious cycle between copper reduction and the pathogenesis of atherosclerosis. These changes in copper homeostasis would be additive to atherosclerosis as a risk factor for cardiovascular disease in humans.

Synergistic effect of Betulinic acid, Apigenin and Skimmianine (BASk) in high cholesterol diet rabbit: Involvement of CD36-TLR2 signaling pathway

BackgroundProgression of chronic inflammatory disease, atherosclerosis is a multifactorial process. Cluster of differentiation 36 (CD36) mediated downstream activation of Toll like receptor 2 (TLR2) and NLRP3 (Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3) inflammasome signaling pathway actively participates during chronic inflammation. Nowadays, synergistic combinations of bioactive compounds attained priority in the field of drug discovery and development as therapeutic agents. An investigation regarding the anti-inflammatory potential of a novel drug formulation, BASk which is a combination of three bioactive compounds Betulinic acid (B):Apigenin (A):Skimmianine (Sk) remains the focus area of this research study. We also elucidate the molecular mechanism behind the therapeutic potential of BASk through CD36 mediated activation TLR2-NLRP3 signaling pathway. MethodsOxLDL induced hPBMCs used to screen out a suitable combination of BASk via MTT, COX, LOX, NOS and MPO assays. Hypercholesterolemia is induced in rabbits by supplementing with 1% cholesterol + 0.5% cholic acid and treated with BASk (2:2:1) (5 mg/Kg) and atorvastatin (10 mg/Kg) for 60 days. CD36, TLR2, NLRP3, NFκB, cytokines, endothelial damage were quantified by reverse transcription, real time PCR, ELISA, flow cytometry and histopathology. ResultshPBMCs pretreated with BASk at 2:2:1 ratio significantly decreased the activities of COX, 15-LOX, NOS and MPO on OxLDL induction than quercetin. Down regulation of CD36, TLR2, MyD88, TRAF6 by BASk further buttressed NLRP3 inflammasome activation mediated by the transcription factor NFκB. This is in correlation with the effect of BASk by balancing pro (IL-1β, IL-18) and anti-inflammatory (TGF-β) mediators in the aortic endothelial cells. ConclusionBASk exerted its anti-inflammatory potential by reducing pro-inflammatory mediators during cholesterol supplementation via down regulating CD36 mediated TLR2 – NLRP3 inflammasome cascade. This deciphers a synergistic combination named BASk (2:2:1) as a novel drug formulation against chronic inflammatory disease, atherosclerosis.

Cholesterol Activates Cyclic AMP Signaling in Metaplastic Acinar Cells.

Cholesterol is a non-essential metabolite that exerts both structural and signaling functions. However, cholesterol biosynthesis is elevated, and actively supports, pancreatic carcinogenesis. Our previous work showed that statins block the reprogramming of mutant KRAS-expressing acinar cells, that spontaneously undergo a metaplastic event termed acinar-to-ductal metaplasia (ADM) to initiate carcinogenesis. Here we tested the impact of cholesterol supplementation on isolated primary wild-type acinar cells and observed enhanced ductal transdifferentiation, associated with generation of the second messenger cyclic adenosine monophosphate (cAMP) and the induction of downstream protein kinase A (PKA). Inhibition of PKA suppresses cholesterol-induced ADM ex vivo. Live imaging using fluorescent biosensors dissected the temporal and spatial dynamics of PKA activation upon cholesterol addition and showed uneven activation both in the cytosol and on the outer mitochondrial membrane of primary pancreatic acinar cells. The ability of cholesterol to activate cAMP signaling is lost in tumor cells. Qualitative examination of multiple normal and transformed cell lines supports the notion that the cAMP/PKA axis plays different roles during multi-step pancreatic carcinogenesis. Collectively, our findings describe the impact of cholesterol availability on the cyclic AMP/PKA axis and plasticity of pancreatic acinar cells.

Impaired cholesterol metabolism in the mouse model of cystic fibrosis. A preliminary study.

This study aims to investigate cholesterol metabolism in a mouse model with cystic fibrosis (CF) by the comparison of affected homozygous versus wild type (WT) mice. In particular, we evaluated the effects of a diet enriched with cholesterol in both mice groups in comparison with the normal diet. To this purpose, beyond serum and liver cholesterol, we analyzed serum phytosterols as indirect markers of intestinal absorption of cholesterol, liver lathosterol as indirect marker of de novo cholesterol synthesis, liver cholestanol (a catabolite of bile salts synthesis) and the liver mRNA levels of LDL receptor (LDLR), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAR), acyl CoA:cholesterol acyl transferase 2 (ACAT2), cytochrome P450 7A1 (CYP7A1) and tumor necrosis factor alpha (TNFα). CF mice showed lower intestinal absorption and higher liver synthesis of cholesterol than WT mice. In WT mice, the cholesterol supplementation inhibits the synthesis of liver cholesterol and enhances its catabolism, while in CF mice we did not observe a reduction of LDLR and HMG-CoAR expression (probably due to an altered feed-back), causing an increase of intracellular cholesterol. In addition, we observed a further increase (5-fold) in TNFα mRNA levels. This preliminary study suggests that in CF mice there is a vicious circle in which the altered synthesis/secretion of bile salts may reduce the digestion/absorption of cholesterol. As a result, the liver increases the biosynthesis of cholesterol that accumulates in the cells, triggering inflammation and further compromising the metabolism of bile salts.

Mutation in the distal NPxY motif of LRP1 alleviates dietary cholesterol-induced dyslipidemia and tissue inflammation

The impairment of LDL receptor-related protein-1 (LRP1) in numerous cell types is associated with obesity, diabetes, and fatty liver disease. Here, we compared the metabolic phenotype of C57BL/6J wild-type and LRP1 knock-in mice carrying an inactivating mutation in the distal NPxY motif after feeding a low-fat diet or high-fat (HF) diet with cholesterol supplementation (HFHC) or HF diet without cholesterol supplementation. In response to HF feeding, both groups developed hyperglycemia, hyperinsulinemia, hyperlipidemia, increased adiposity, and adipose tissue inflammation and liver steatosis. However, LRP1 NPxY mutation prevents HFHC diet-induced hypercholesterolemia, reduces adipose tissue and brain inflammation, and limits liver progression to steatohepatitis. Nevertheless, this mutation does not protect against HFHC diet-induced insulin resistance. The selective metabolic improvement observed in HFHC diet-fed LRP1 NPxY mutant mice is due to an apparent increase of hepatic LDL receptor levels, leading to an elevated rate of plasma lipoprotein clearance and lower hepatic cholesterol levels. The unique metabolic phenotypes displayed by LRP1 NPxY mutant mice indicate an LRP1-cholesterol axis in modulating tissue inflammation. The LRP1 NPxY mutant mouse phenotype differs from phenotypes observed in mice with tissue-specific LRP1 inactivation, thus highlighting the importance of an integrative approach to evaluate how global LRP1 dysfunction contributes to metabolic disease development.

Membrane cholesterol regulates TRPV4 function, cytoskeletal expression, and the cellular response to tension

Despite the association of cholesterol with debilitating pressure-related diseases such as glaucoma, heart disease, and diabetes, its role in mechanotransduction is not well understood. We investigated the relationship between mechanical strain, free membrane cholesterol, actin cytoskeleton, and the stretch-activated transient receptor potential vanilloid isoform 4 (TRPV4) channel in human trabecular meshwork (TM) cells. Physiological levels of cyclic stretch resulted in time-dependent decreases in membrane cholesterol/phosphatidylcholine ratio and upregulation of stress fibers. Depleting free membrane cholesterol with m-β-cyclodextrin (MβCD) augmented TRPV4 activation by the agonist GSK1016790A, swelling and strain, with the effects reversed by cholesterol supplementation. MβCD increased membrane expression of TRPV4, caveolin-1, and flotillin. TRPV4 did not colocalize or interact with caveolae or lipid rafts, apart from a truncated ∼75 kDa variant partially precipitated by a caveolin-1 antibody. MβCD induced currents in TRPV4-expressing Xenopus laevis oocytes. Thus, membrane cholesterol regulates trabecular transduction of mechanical information, with TRPV4 channels mainly located outside the cholesterol-enriched membrane domains. Moreover, the biomechanical milieu itself shapes the lipid content of TM membranes. Diet, cholesterol metabolism, and mechanical stress might modulate the conventional outflow pathway and intraocular pressure in glaucoma and diabetes in part by modulating TM mechanosensing.

Dependence of SARS-CoV-2 infection on cholesterol-rich lipid raft and endosomal acidification

Coronavirus disease 2019 is a kind of viral pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the mechanism whereby SARS-CoV-2 invades host cells remains poorly understood. Here we used SARS-CoV-2 pseudoviruses to infect human angiotensin-converting enzyme 2 (ACE2) expressing HEK293T cells and evaluated virus infection. We confirmed that SARS-CoV-2 entry was dependent on ACE2 and sensitive to pH of endosome/lysosome in HEK293T cells. The infection of SARS-CoV-2 pseudoviruses is independent of dynamin, clathrin, caveolin and endophilin A2, as well as macropinocytosis. Instead, we found that the infection of SARS-CoV-2 pseudoviruses was cholesterol-rich lipid raft dependent. Cholesterol depletion of cell membranes with methyl-β-cyclodextrin resulted in reduction of pseudovirus infection. The infection of SARS-CoV-2 pseudoviruses resumed with cholesterol supplementation. Together, cholesterol-rich lipid rafts, and endosomal acidification, are key steps of SARS-CoV-2 required for infection of host cells. Therefore, our finding expands the understanding of SARS-CoV-2 entry mechanism and provides a new anti-SARS-CoV-2 strategy.

Mevalonate Pathway-mediated ER Homeostasis Is Required for Haploid Stability in Human Somatic Cells.

The somatic haploidy is unstable in diplontic animals, but cellular processes determining haploid stability remain elusive. Here, we found that inhibition of mevalonate pathway by pitavastatin, a widely used cholesterol-lowering drug, drastically destabilized the haploid state in HAP1 cells. Interestingly, cholesterol supplementation did not restore haploid stability in pitavastatin-treated cells, and cholesterol inhibitor U18666A did not phenocopy haploid destabilization. These results ruled out the involvement of cholesterol in haploid stability. Besides cholesterol perturbation, pitavastatin induced endoplasmic reticulum (ER) stress, the suppression of which by a chemical chaperon significantly restored haploid stability in pitavastatin-treated cells. Our data demonstrate the involvement of the mevalonate pathway in the stability of the haploid state in human somatic cells through managing ER stress, highlighting a novel link between ploidy and ER homeostatic control.Key words: haploid, ER stress, Mevalonate pathway.

A Placebo-Controlled Pilot Exploration of Cholesterol Supplementation for Autistic Symptoms in Children with Low Cholesterol

This pilot placebo-controlled trial (NCT00965068, 07/28/2009) explored effects of cholesterol supplementation on autism spectrum disorder (ASD) symptoms in children with abnormally low cholesterol. Fifteen children age 4–12 with ASD and cholesterol <5th percentile for age/sex were randomized to 225 mg cholesterol vs. matched placebo twice daily for 12 weeks, followed by 12-week open extension. The cholesterol daily dose approximated the amount in two eggs. Measures included the Ohio Autism Clinical Impression Scale (OACIS, primary outcome), and Peabody Picture Vocabulary Test (PPVT-4). Effect sizes were emphasized over statistical tests. On the OACIS-Improvement score for overall ASD symptoms, both groups improved, with cholesterol showing nominally (nonsignificantly) more improvement. The cholesterol group improved more than placebo by medium to large effects in OACIS-I nonverbal communication (d = −0.643); and OACIS-I problems with anxiety and fear (d = −0.671). Moreover, cholesterol showed significantly more improvement than placebo in PPVT-4 receptive language (p = 0.040). During the 12-week open extension, children originally assigned placebo improved significantly more in OACIS-I social interaction (p = 0.026, d = 1.170) from end of randomized phase than those receiving cholesterol throughout and improved more by large effects in OACIS-I aberrant behavior (d = 0.924), and anxiety/fear (d = 0.925). Graphs suggest continued open-trial improvement for the cholesterol group. Adverse events were similar between treatments. Cholesterol supplementation of children with ASD and cholesterol below the 5th percentile appears safe. The effect on communication, social interaction, and anxiety/fear looks encouraging; deserving further study, possibly over a longer period or at higher doses.

Dietary cholesterol supplementation and inhibitory factor 1 serum levels in two dizygotic Smith-Lemli-Opitz syndrome twins: a case report

BackgroundSmith-Lemli-Opitz syndrome (SLOS) is a rare genetic neurodevelopmental disorder caused by the defect in the 7-dehydrocholesterol reductase. This defect leads to the deficiency of cholesterol biosynthesis with accumulation of 7-dehydrocholesterol. Inhibitory factor 1 (IF1) is a well-known mitochondrial protein. Recently, it has been discovered in the human serum where it is reported to be involved in the HDL-cholesterol intake. Here we report the IF1 presence in the serum of two paediatric SLOS dizygotic twins treated with dietary cholesterol supplementation.Case presentationThe patients showed a typical phenotype. They started dietary supplementation with cholesterol when 2 months old. The cholesterol intake was periodically titrated on the basis of weight increase and the twin 1 required a larger supplementation than the twin 2 during the follow-up. When 6.4-year-old, they underwent IF1 assay that was 7-fold increased in twin 2 compared to twin 1 (93.0 pg/ml vs 13.0 pg/ml, respectively).ConclusionsWe report, for the first time, the presence of circulating IF1 in the serum of SLOS patients, showing different levels among them. Our findings confirm that IF1 could be a novel research target in cholesterol-related disorders and also in SLOS, and could contribute to the general debate on IF1 as a new modulator of cholesterol levels.

Subcellular diversion of cholesterol by gain- and loss-of-function mutations in PMP22.

Abnormalities of the peripheral myelin protein 22 (PMP22) gene, including duplication, deletion and point mutations are a major culprit in Type 1 Charcot-Marie-Tooth (CMT) diseases. The complete absence of PMP22 alters cholesterol metabolism in Schwann cells, which likely contributes to myelination deficits. Here, we examined the subcellular trafficking of cholesterol in distinct models of PMP22-linked neuropathies. In Schwann cells from homozygous Trembler J (TrJ) mice carrying a Leu16Pro mutation, cholesterol was retained with TrJ-PMP22 in the Golgi, alongside a corresponding reduction in its plasma membrane level. PMP22 overexpression, which models CMT1A caused by gene duplication, triggered cholesterol sequestration to lysosomes, and reduced ATP-binding cassette transporter-dependent cholesterol efflux. Conversely, lysosomal targeting of cholesterol by U18666A treatment increased wild type (WT)-PMP22 levels in lysosomes. Mutagenesis of a cholesterol recognition motif, or CRAC domain, in human PMP22 lead to increased levels of PMP22 in the ER and Golgi compartments, along with higher cytosolic, and lower membrane-associated cholesterol. Importantly, cholesterol trafficking defects observed in PMP22-deficient Schwann cells were rescued by WT but not CRAC-mutant-PMP22. We also observed that myelination deficits in dorsal root ganglia explants from heterozygous PMP22-deficient mice were improved by cholesterol supplementation. Collectively, these findings indicate that PMP22 is critical in cholesterol metabolism, and this mechanism is likely a contributing factor in PMP22-linked hereditary neuropathies. Our results provide a basis for understanding how altered expression of PMP22 impacts cholesterol metabolism.

Altered Lipid Domains Facilitate Enhanced Pulmonary Vasoconstriction after Chronic Hypoxia.

Chronic hypoxia (CH) augments depolarization-induced pulmonary vasoconstriction through superoxide-dependent, Rho kinase-mediated Ca2+ sensitization. Nicotinamide adenine dinucleotide phosphate oxidase and EGFR (epidermal growth factor receptor) signaling contributes to this response. Caveolin-1 regulates the activity of a variety of proteins, including EGFR and nicotinamide adenine dinucleotide phosphate oxidase, and membrane cholesterol is an important regulator of caveolin-1 protein interactions. We hypothesized that derangement of these membrane lipid domain components augments depolarization-induced Ca2+ sensitization and resultant vasoconstriction after CH. Although exposure of rats to CH (4 wk, ∼380 mm Hg) did not alter caveolin-1 expression in intrapulmonary arteries or the incidence of caveolae in arterial smooth muscle, CH markedly reduced smooth muscle membrane cholesterol content as assessed by filipin fluorescence. Effects of CH on vasoreactivity and superoxide generation were examined using pressurized, Ca2+-permeabilized, endothelium-disrupted pulmonary arteries (∼150 μm inner diameter) from CH and control rats. Depolarizing concentrations of KCl evoked greater constriction in arteries from CH rats than in those obtained from control rats, and increased superoxide production as assessed by dihydroethidium fluorescence only in arteries from CH rats. Both cholesterol supplementation and the caveolin-1 scaffolding domain peptide antennapedia-Cav prevented these effects of CH, with each treatment restoring membrane cholesterol in CH arteries to control levels. Enhanced EGF-dependent vasoconstriction after CH similarly required reduced membrane cholesterol. However, these responses to CH were not associated with changes in EGFR expression or activity, suggesting that cholesterol regulates this signaling pathway downstream of EGFR. We conclude that alterations in membrane lipid domain signaling resulting from reduced cholesterol content facilitate enhanced depolarization- and EGF-induced pulmonary vasoconstriction after CH.

Choline and phosphatidylcholine, but not methionine, cysteine, taurine and taurocholate, eliminate excessive gut mucosal lipid accumulation in Atlantic salmon (Salmo salar L)

Excessive enterocyte lipid accumulation, with the suggested term lipid malabsorption syndrome (LMS), is frequently observed in Atlantic salmon (Salmo salar L), in small fish in fresh water as well as in large fish in seawater. The symptoms indicate insufficient supply of components involved in lipid assimilation. The questions addressed in the present work were whether dietary supply of components involved in phospholipid and sterol metabolism might prevent LMS. Atlantic salmon (35 fish, 330 g per 600 L tank) were fed a low fish meal diet (LF) as such or supplemented with taurocholate at two levels (3.5 and 6.9 g/kg), cholesterol (2.0 g/kg), taurine (0.8 g/kg), phosphatidylcholine (15.1 g/kg), choline (3.7 g/kg), cysteine (0.8 g/kg) or methionine (1.0 g/kg). A high fish meal diet (HF) was also included. The overall growth rate of the fish was high (TGC>4.2) with no significant effects of diet. Fish fed the LF diet showed increased relative weight of the pyloric and mid intestine and excessive lipid accumulation in the enterocytes, characteristics which were nearly absent in fish fed the HF diet and the LF diet supplemented with choline and phosphatidylcholine. The phosphatidylcholine supplemented diet showed significantly higher lipid digestibility than the LF diet. None of the other supplements eliminated the signs of excessive enterocyte lipid accumulation. Phosphatidylcholine down-regulated pcyt1a, involved in the phosphatidylcholine synthesis, and both choline and phosphatidylcholine induced apoaIV, important in lipoprotein assembly, and markedly suppressed the lipid droplet marker plin2. Methionine supplementation did not stimulate endogenous synthesis of choline. Cholesterol supplementation suppressed sterol uptake and de novo cholesterol synthesis, and induced sterol efflux from the intestinal mucosa. Taurocholate and taurine induced their respective metabolic pathways. All feed supplements, in particular cholesterol and cysteine, down-regulated genes related to antiviral, chemokine, antigen presentation, immunoglobulinfunctions, as well as of extracellular proteases. The results of this study confirm the results from our previous study showing that choline or phosphatidylcholine is a necessary ingredient in low fish meal diets.

Cholesterol Induces Nrf-2- and HIF-1α-Dependent Hepatocyte Proliferation and Liver Regeneration to Ameliorate Bile Acid Toxicity in Mouse Models of NASH and Fibrosis.

Nonalcoholic steatohepatitis (NASH) is currently one of the most common liver diseases worldwide. The toxic effects of lipids and bile acids contribute to NASH. The regenerative pathway in response to damage to the liver includes activation of the inflammatory process and priming of hepatocytes to proliferate to restore tissue homeostasis. However, the effects of cholesterol on bile acid toxicity, inflammation, and fibrosis remain unknown. We have used two mouse models of bile acid toxicity to induce liver inflammation and fibrosis. A three-week study was conducted using wild-type mice receiving an atherogenic diet (1% (w/w) cholesterol and 0.5% (w/w) cholic acid) and its separate constituents. Mdr2-/- mice were fed a high-cholesterol-enriched diet or standard AIN-93 diet for 6 weeks. We measured serum transaminase levels to assess liver tissue necrosis and fibrosis; iNOS, SAA1, SAA2, and F4/80 levels to determine liver inflammation; PCNA and HGF levels to evaluate proliferative response; and Nrf-2, HIF-1α, and downstream gene expression to establish protective responses. In both studies, high bile acid levels increased serum transaminases and liver fibrosis, whereas cholesterol supplementation attenuated these effects. Cholesterol supplementation activated survival and the robustness of HIF-1α and Nrf-2 gene expression in hepatocytes, induced liver inflammation and hepatocyte proliferation, and inhibited stellate cell hyperplasia and fibrosis. In conclusion, our data show for the first time that cholesterol intake protects against bile acid liver toxicity. The balance between hepatic cholesterol and bile acid levels may be of prognostic value in liver disease progression and trajectory.

Tembusu virus enters BHK-21 cells through a cholesterol-dependent and clathrin-mediated endocytosis pathway

Tembusu virus (TMUV) is a newly emerging flavivirus and has caused significant economic loss to the poultry industry in China. To date, the entry of TMUV into host cells remains poorly understood. Here, the mechanism of TMUV entry into BHK-21 cells was investigated. The depletion of cellular cholesterol by methyl-β-cyclodextrin led to a significant decline in the titers and RNA levels of the infectious TMUV. This reduction was restored by supplementation of exogenous cholesterol. Membrane cholesterol depletion mainly blocked viral internalization but not attachment. However, viral infection was unaffected by genistein treatment or caveolin-1 silencing by small interfering RNA. In addition, clathrin-mediated endocytosis might be utilized in TMUV entry given that the viral infection was inhibited by knockdown of clathrin heavy chain and treatment of chlorpromazine (CPZ). Moreover, the number of internalized virus particles decreased under CPZ treatment. Dynasore inhibited TMUV entry suggesting a role for dynamin. Our results reveal that TMUV entry into BHK-21 cells is dependent on cholesterol, clathrin and dynamin but not caveolae.

Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome

Primary cilia are antenna‐like organelles on the surface of most mammalian cells that receive sonic hedgehog (Shh) signaling in embryogenesis and carcinogenesis. Cellular cholesterol functions as a direct activator of a seven‐transmembrane oncoprotein called Smoothened (Smo) and thereby induces Smo accumulation on the ciliary membrane where it transduces the Shh signal. However, how cholesterol is supplied to the ciliary membrane remains unclear. Here, we report that peroxisomes are essential for the transport of cholesterol into the ciliary membrane. Zellweger syndrome (ZS) is a peroxisome‐deficient hereditary disorder with several ciliopathy‐related features and cells from these patients showed a reduced cholesterol level in the ciliary membrane. Reverse genetics approaches revealed that the GTP exchange factor Rabin8, the Rab GTPase Rab10, and the microtubule minus‐end‐directed kinesin KIFC3 form a peroxisome‐associated complex to control the movement of peroxisomes along microtubules, enabling communication between peroxisomes and ciliary pocket membranes. Our findings suggest that insufficient ciliary cholesterol levels may underlie ciliopathies.

Evaluation of various parameters of consumption indices of the silkworm hybrids administered with cholesterol content during 4th instar silkworms

In the present investigation an attempt has been made to study the various parameters of consumption indices in relation to the different concentration of cholesterol supplementation during 4th instar silkworm of FC1XFC2 and FC4XFC3 hybrids. The food consumption comprises the ingesta and digesta during 4th instar showed the level of ingesta was significantly higher compare to digesta in all the cholesterol treated batches in all 4 days, but on 4th day the rate of food consumption represents the highest rate of ingesta in the selected hybrids. Similarly the approximate digestibility was maintained ranging from 50-85% in cholesterol treated batches, whereas the efficiency of conversion of food ingested and efficiency of conversion of food digested were low ranging from 5-38% in FC1XFC2 and FC4XFC3 silkworm hybrids. The consumption index and coefficient of metabolism are the two nutritional parameters indicates the maximum attainment ranging from 0.8-0.9 ratio in all the days in all the cholesterol treatment concentration, whereas the growth rate was constantly maintained during 4th instar ranging from 0-0.05 throughout the instar in FC1XFC2 and FC4XFC3 hybrids. The pattern of changes in the ingesta and digesta of 5th instar silkworm, but cholesterol supplemented during 4th instar extended the above parameters ranging from 9-14g from first day to day seventh, whereas the digesta falls within a range of 9g during 5th instar in both the hybrids. The efficiency of conversion of food ingested, efficiency of conversion of food digested and approximate digestibility of 5th instar silkworm hybrids treated with three doses of cholesterol during 4th instar a showed the magnitude of changes above the level of 50% in FC4XFC3 hybrid, but the FC1XFC2 represents less than level of 50% except 5th day and 7th day during 5th instar. The consumption index, growth rate and co-efficient of metabolism were calculated during 5th instar the ratio was ranging from 0-1 in all the three parameters except consumption index and co-efficient of metabolism on 7th day ranging from 6-7 and 2-3 on respectively on the last day of 5th instar in FC1XFC2 and FC4XFC3 hybrids.

Evaluation of economic traits of selected FC1XFC2 and FC4XFC3 double hybrids treated with different doses of cholesterol during IV and V instar silkworm Bombyx mori L

The effect of different concentration of cholesterol treatment during 4th instar and economic characters were better manifested in larval weight except larval duration and significant improvement in cocoon characters and egg characters were observed in FC1XFC2 hybrid. Similarly, FC4XFC3 hybrid has been subjected for the cholesterol treatment during 4th instar showed an unchanged larval duration in control and treated silkworms and enhancement in larval weight, cocoon characters and filament characters were highly significant except denier and renditta at 0.01 M concentration showed a negative relationship with reference to cholesterol supplementation. The egg characters of FC4XFC3 hybrid responded relatively better in the performance of egg hatching and fecundity. The impact of supplementation of cholesterol during 5th instar silkworm hybrid namely, FC1XFC2 contributed to the improvement of all the economic parameters comprising larval characters, cocoon characters, filament characters and egg characters except unchanged larval duration in control and untreated batches. On the other hand, FC4XFC3 hybrid were subjected for the application of different concentration of cholesterol treatment during 5th instar and expression and manifestation of the larval characters and cocoon characters were responded significantly except larval duration and cocoon weight at 0.1 M concentration showed relatively low in the cocoon weight compare to control batches. The filament length and weight of FC4XFC3 hybrid showed a consistent increase in the improvement of the traits, whereas the denier and renditta were inversely related to control and negatively correlated but at 0.1M concentration showed positive trend with respect to Renditta as observed in the investigation. The egg characters namely, fecundity and egg hatching in relation to the cholesterol administration and its impact leads to the enhancement in the rate of traits of selected bivoltine.

A calcified chronic total occlusion preclinical model.

To create an experimental chronic total occlusion (CTO) model with calcification by dietary modification (cholesterol, calcium carbonate, vitamin D) and local injection of pro-calcification factors (dipotassium phosphate, calcium chloride, and bone morphogenetic protein-2 [BMP-2]). Percutaneous revascularization of CTOs frequently fails in heavily calcified occlusions. Development of novel approaches requires a reproducible preclinical model of calcified CTO. CTOs were created in 18 femoral arteries of 9 New Zealand White rabbits using the thrombin injection model. Dietary interventions included a high cholesterol diet (0.5% or 0.25%), calcium carbonate (150 mg × 3-5 days/week), and vitamin D (50,000 U × 3-5 days/week). In selected animals, BMP-2 (1-4 μg), dipotassium phosphate, and calcium chloride were injected locally at the time of CTO creation. Animals were sacrificed at 2 weeks (n = 4 arteries), 6 weeks (n = 4 arteries), and 10-12 weeks (n = 14 arteries). CTOs showed evidence of chronic lipid feeding (foam cells) and chronic inflammation (intimal/medial fibrosis and microvessels, inflammatory cells, internal elastic lamina disruption). In calcium/vitamin D supplemented rabbits, mineralization (calcification and/or ossification) was evident as early as 2 weeks post CTO creation, and in 78% of the overall arteries. Mineralization changes were not present in the absence of calcium/vitamin D dietary supplements. Mineralization occurred in 85% of BMP-treated arteries and 60% of arteries without BMP. Complex mineralization occurs in preclinical CTO models with dietary supplementation of cholesterol with vitamin D and calcium.