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Abstract Mitochondrial function plays an important role in the maintenance of male fertility. However, the mechanisms underlying mitochondrial defect-related infertility remain mostly unclear. Here we show that a deficiency of PARL (Parl −/−), a mitochondrial protease, causes complete arrest of spermatogenesis during meiosis I. PARL deficiency led to severe downregulation of proteins of respiratory chain complex IV in testes that did not occur in other tested organs, causing a deficit in complex IV activity and ATP production. Furthermore, Parl −/− testes showed an almost complete loss of HSD17B3, a protein of the sER responsible for the last step in testosterone synthesis. While testosterone production appeared to be restored by overexpression of HSD17B12, loss of the canonical testosterone synthesis led to an upregulation of luteinizing hormone (LH) and of LH-regulated responses. These results suggest an important impact of the downstream regulation of mitochondrial defects that manifest in a cell-type-specific manner and extend beyond mitochondria.

Macrophage cholesterol homeostasis is crucial for health and disease and has been linked to the lipid-peroxidizing enzyme arachidonate 15-lipoxygenase type B (ALOX15B), albeit molecular mechanisms remain obscure. We performed global transcriptome and immunofluorescence analysis in ALOX15B-silenced primary human macrophages and observed a reduction of nuclear sterol regulatory element-binding protein (SREBP) 2, the master transcription factor of cellular cholesterol biosynthesis. Consequently, SREBP2-target gene expression was reduced as were the sterol biosynthetic intermediates desmosterol and lathosterol as well as 25- and 27-hydroxycholesterol. Mechanistically, suppression of ALOX15B reduced lipid peroxidation in primary human macrophages and thereby attenuated activation of mitogen-activated protein kinase ERK1/2, which lowered SREBP2 abundance and activity. Low nuclear SREBP2 rendered both, ALOX15B-silenced and ERK1/2-inhibited macrophages refractory to SREBP2 activation upon blocking the NPC intracellular cholesterol transporter 1. These studies suggest a regulatory mechanism controlling macrophage cholesterol homeostasis based on ALOX15B-mediated lipid peroxidation and concomitant ERK1/2 activation.

Background Aortic stenosis has pathophysiological similarities with atherosclerosis, including the deposition of cholesterol‐containing lipoproteins. The resulting cholesterol crystals activate the NLRP3 (NOD‐like receptor protein 3) inflammasome, leading to inflammation and cardiovascular diseases. We aimed to investigate the cholesterol crystal dissolution rate (CCDR) of serum in patients with aortic stenosis and to assess the prognostic value of this biomarker. Methods and Results The study included 348 patients with aortic stenosis undergoing transcatheter aortic valve replacement. The CCDR was measured using flow cytometry to enumerate cholesterol crystals that were added to a serum solution, at baseline and after 2 hours of incubation. Based on the median CCDR, the cohort was stratified into high and low cholesterol crystal dissolvers. The incidence of the primary end point, a composite of 1‐year all‐cause mortality and major vascular complication, was significantly lower in the high CCDR group (7.3 per 100 person‐years) compared with the low CCDR group (17.0 per 100 person‐years, P=0.01). This was mainly driven by a lower 1‐year mortality rate in patients with a high CCDR (7.3 versus 15.1 per 100 person‐years, P=0.04). Unplanned endovascular interventions were significantly less frequent in high cholesterol crystal dissolvers (12.8 versus 22.6 per 100 person‐years, P=0.04). Although low‐density lipoprotein cholesterol levels were comparable in both groups (101.8±37.3 mg/dL versus 97.9±37.6 mg/dL, P=0.35), only patients with a low CCDR showed a benefit from statin treatment. In multivariate analysis, low CCDR (hazard ratio, 2.21 [95% CI, 0.99–4.92], P=0.04) was significantly associated with 1‐year mortality. Conclusions The CCDR is a novel biomarker associated with outcome in patients with aortic stenosis undergoing transcatheter aortic valve replacement. It may provide new insights into patients' anti‐inflammatory capacity and additional prognostic information beyond classic risk assessment.

Summary: Neurodegenerative, vascular, and dementia diseases are linked to dysregulations in cholesterol metabolism. Dietary plant sterols, or phytosterols, may interfere to neurodegeneration and cognitive decline, and have cholesterol-lowering, anti-inflammatory, and antioxidant qualities. Here, we investigated the potential associations between circulating cholesterol precursors and metabolites, triglycerides, and phytosterols with cognitive decline in older people by performing multivariate analysis on 246 participants engaged in a population-based prospective study. In our analysis we considered the potential effect of sex and APOEe4. We reveal particular dysregulations of diet-derived phytosterols and endogenous cholesterol synthesis and metabolism, and their variations over time linked to cognitive decline in the general population. These results are significant to the development of interventions to avoid cognitive decline in older adults and suggest that levels of circulating sterols should be taken into account when evaluating risk.

Oncosterone (6-oxo-cholestane-3β,5α-diol; OCDO) is an oncometabolite and a tumor promoter on estrogen receptor alpha-positive breast cancer (ER(+) BC) and triple-negative breast cancers (TN BC). OCDO is an oxysterol formed in three steps from cholesterol: 1) oxygen addition at the double bond to give α- or β- isomers of 5,6-epoxycholestanols (5,6-EC), 2) hydrolyses of the epoxide ring of 5,6-ECs to give cholestane-3β,5α,6β-triol (CT), and 3) oxidation of the C6 hydroxyl of CT to give OCDO. On the other hand, cholesterol can be hydroxylated by CYP27A1 at the ultimate methyl carbon of its side chain to give 27-hydroxycholesterol ((25R)-Cholest-5-ene-3beta,26-diol, 27HC), which is a tumor promoter for ER(+) BC. It is currently unknown whether OCDO and its precursors can be hydroxylated at position C27 by CYP27A1, as is the impact of such modification on the proliferation of ER(+) and TN BC cells. We investigated, herein, whether 27H-5,6-ECs ((25R)-5,6-epoxycholestan-3β,26-diol), 27H-CT ((25R)-cholestane-3β,5α,6β,26-tetrol) and 27H-OCDO ((25R)-cholestane-6-oxo-3β,5α,26-triol) exist as metabolites and can be produced by cells expressing CYP27A1. We report, for the first time, that these compounds exist as metabolites in humans. We give pharmacological and genetic evidence that CYP27A1 is responsible for their production. Importantly, we found that 27-hydroxy-OCDO (27H-OCDO) inhibits BC cell proliferation and blocks OCDO and 27-HC-induced proliferation in BC cells, showing that this metabolic conversion commutes the proliferative properties of OCDO into antiproliferative ones. These data suggest an unprecedented role of CYP27A1 in the control of breast carcinogenesis by inhibiting the tumor promoter activities of oncosterone and 27-HC.

Hypoxia contributes to numerous pathophysiological conditions including inflammation-associated diseases. We characterized the impact of hypoxia on the immunometabolic cross-talk between cholesterol and interferon (IFN) responses. Specifically, hypoxia reduced cholesterol biosynthesis flux and provoked a compensatory activation of sterol regulatory element-binding protein 2 (SREBP2) in monocytes. Concomitantly, a broad range of interferon-stimulated genes (ISGs) increased under hypoxia in the absence of an inflammatory stimulus. While changes in cholesterol biosynthesis intermediates and SREBP2 activity did not contribute to hypoxic ISG induction, intracellular cholesterol distribution appeared critical to enhance hypoxic expression of chemokine ISGs. Importantly, hypoxia further boosted chemokine ISG expression in monocytes upon infection with severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Mechanistically, hypoxia sensitized toll-like receptor 4 (TLR4) signaling to activation by SARS-CoV-2 spike protein, which emerged as a major signaling hub to enhance chemokine ISG induction following SARS-CoV-2 infection of hypoxic monocytes. These data depict a hypoxia-regulated immunometabolic mechanism with implications for the development of systemic inflammatory responses in severe cases of coronavirus disease-2019 (COVID-19).

Summary: Dysregulations in cholesterol metabolism are associated with neurodegenerative and vascular pathologies, and dementia. Diet-derived plant sterols (phytosterols) have cholesterol-lowering, anti-inflammatory, and antioxidant properties and may interfere with neurodegeneration and cognitive decline. Here we performed multivariate analysis in 720 individuals enrolled in a population-based prospective study to determine whether circulating cholesterol precursors and metabolites, triglycerides, and phytosterols, are associated with cognitive impairment and decline in the older population. We report specific dysregulations of endogenous cholesterol synthesis and metabolism, and diet-derived phytosterols, and their changes over time associated with cognitive impairment, and decline in the general population. These findings suggest circulating sterols levels could be considered in risk evaluation and are relevant for the development of strategies to prevent cognitive decline in older people.

Introduction High-intensity focused ultrasound (HIFU) is an innovative noninvasive procedure for local ablation of different benign and malignant tumors. Preliminary data of animal studies suggest an ablation-associated immune response after HIFU that is induced by cell necrosis and release of intracellular components. The aim of this study is to evaluate if a HIFU-induced early sterile inflammatory reaction is initiated after ablation of uterine fibroids (UF) and pancreatic carcinoma (PaC) which might contribute to the therapeutic effect. Material and methods A hundred patients with PaC and 30 patients with UF underwent US-guided HIFU treatment. Serum markers of inflammation (leukocytes, CRP, IL-6) and LDH in both collectives as well as tumor markers CA 19-9, CEA and CYFRA in PaC patients were determined in sub-cohorts before and directly after HIFU (0, 2, 5 and 20 h post-ablation) as well as at 3, 6, 9 and 12 months follow-up. Peri-/post interventional imaging included contrast-enhanced MRI of both cohorts and an additional CT scan of PaC patients. Results An early post-ablation inflammatory response was observed in both groups with a significant increase of leukocytes, CRP and LDH within the first 20 h after HIFU. Interestingly, IL-6 was increased at 20 h after HIFU in PaC patients. A significant reduction of tumor volumes was observed during one year follow-up (p

Exposure to high ambient temperature has been shown to impair growth performance and to cause oxidative stress in broilers. This study investigated the hypothesis that supplementation with methionine (Met) as DL-Met (DLM) more than the National Research Council recommendations improves growth performance and alleviates oxidative stress in broilers exposed to high ambient temperature. One-day-old male Cobb-500 broilers (n = 68) were allotted to 4 groups and phase-fed 3 basal diets during days 1 to 10, 11 to 21, and 22 to 35. One group was kept under thermoneutral temperature conditions and received the basal diets with Met + cysteine (Cys) concentrations according to recommendations of NRC. The other 3 groups were kept in a room with an increased ambient temperature from week 3 to 5 and were fed either the basal diet or the basal diets supplemented with 2 levels of DLM in which Met + Cys concentrations exceeded NRC recommendations by around 20% (group DLM1) and 40% (group DLM2), respectively. As expected, the broilers exposed to high ambient temperature showed a lower feed intake, lower body weight gains, a higher feed:gain ratio, and biochemical indications of oxidative stress in comparison to broilers kept under thermoneutral temperature conditions. Supplementation of DLM did not improve the growth performance in broilers exposed to high ambient temperature. However, the broilers supplemented with DLM had increased concentrations of glutathione in liver and breast muscle (groups DLM1 and DLM2), increased concentrations of tocopherols in the liver (group DLM2), and reduced concentrations of 7α-hydroxycholesterol and 7-ketocholesterol in heat-processed thigh muscle (groups DLM1 and DLM2) in comparison to the control group exposed to high ambient temperature. Concentrations of thiobarbituric acid-reactive substances and vitamin C in plasma, liver, and muscle were not different between the 3 groups exposed to heat stress. Nevertheless, the study shows that supplementation of DLM in slight excess of the Met concentration required for maximum growth performance improved the antioxidant status in tissues and reduced the susceptibility of muscle toward oxidation in heat-stressed broilers.

Niemann-Pick type C (NPC)1 disease is a rare genetic condition in which the function of the lysosomal cholesterol transporter NPC1 protein is impaired. Consequently, sphingolipids and cholesterol accumulate in lysosomes of all tissues, triggering a cascade of pathological events that culminate in severe systemic and neurological symptoms. Lysosomal cholesterol accumulation is also a key factor in the development of atherosclerosis and NASH. In these two metabolic diseases, the administration of plant stanol esters has been shown to ameliorate cellular cholesterol accumulation and inflammation. Given the overlap of pathological mechanisms among atherosclerosis, NASH, and NPC1 disease, we sought to investigate whether dietary supplementation with plant stanol esters improves the peripheral features of NPC1 disease. To this end, we used an NPC1 murine model featuring a Npc1-null allele (Npc1nih), creating a dysfunctional NPC1 protein. Npc1nih mice were fed a 2% or 6% plant stanol ester-enriched diet over the course of 5 weeks. During this period, hepatic and blood lipid and inflammatory profiles were assessed. Npc1nih mice fed the plant stanol-enriched diet exhibited lower hepatic cholesterol accumulation, damage, and inflammation than regular chow-fed Npc1nih mice. Moreover, plant stanol consumption shifted circulating T-cells and monocytes in particular toward an anti-inflammatory profile. Overall, these effects were stronger following dietary supplementation with 6% stanols, suggesting a dose-dependent effect. The findings of our study highlight the potential use of plant stanols as an affordable complementary means to ameliorate disorders in hepatic and blood lipid metabolism and reduce inflammation in NPC1 disease.

Loss of pancreatic β-cell mass and function as a result of sustained ER stress is a core step in the pathogenesis of diabetes mellitus type 2. The complex control of β-cells and insulin production involves hedgehog (Hh) signaling pathways as well as cholesterol-mediated effects. In fact, data from studies in humans and animal models suggest that HDL protects against the development of diabetes through inhibition of ER stress and β-cell apoptosis. We investigated the mechanism by which HDL inhibits ER stress and apoptosis induced by thapsigargin, a sarco/ER Ca2+-ATPase inhibitor, in β-cells of a rat insulinoma cell line, INS1e. We further explored effects on the Hh signaling receptor Smoothened (SMO) with pharmacologic agonists and inhibitors. Interference with sterol synthesis or efflux enhanced β-cell apoptosis and abrogated the anti-apoptotic activity of HDL. During ER stress, HDL facilitated the efflux of specific oxysterols, including 24-hydroxycholesterol (OHC). Supplementation of reconstituted HDL with 24-OHC enhanced and, in cells lacking ABCG1 or the 24-OHC synthesizing enzyme CYP46A1, restored the protective activity of HDL. Inhibition of SMO countered the beneficial effects of HDL and also LDL, and SMO agonists decreased β-cell apoptosis in the absence of ABCG1 or CYP46A1. The translocation of the SMO-activated transcription factor glioma-associated oncogene GLI-1 was inhibited by ER stress but restored by both HDL and 24-OHC. In conclusion, the protective effect of HDL to counter ER stress and β-cell death involves the transport, generation, and mobilization of oxysterols for activation of the Hh signaling receptor SMO

Edible marine algae, or seaweeds, are a rich source of several bioactive compounds including phytosterols, carotenoids, and polysaccharides. Over the last decades, seaweed-derived constituents turned out to not only reside in the systemic circulation, but are able to cross the blood-brain barrier to exert neuro-active functions both in homeostatic and pathological conditions. Therefore, seaweed-derived constituents have gained increasing interest for their neuro-immunomodulatory and neuroprotective properties, rendering them interesting candidates for the management of several neurodegenerative disorders. In particular seaweed-derived phytosterols gained interest for the treatment of neurodegenerative disorders as they potentiate neuroplasticity, enhance phagocytic clearance of neurotoxic peptides and have anti-inflammatory properties. Though, the anti-inflammatory and anti-oxidative properties of other constituents including carotenoids, phenols and polysaccharides have recently gained more interest. In this review, we provide an overview of a selection of the described neuro-active properties of seaweed-derived constituents with a focus on phytosterols.

Inflammation is associated with changes in plasma lipids, lipoproteins, and cholesterol efflux capacity (CEC). It is unknown if the changes in lipids and lipoproteins during inflammation are related to changes in cholesterol absorption, synthesis, and bile acid synthesis. We, therefore, examined the effects of acute lipopolysaccharide (LPS)-induced transient systemic inflammation on lipids, lipoproteins, CEC, and markers of cholesterol metabolism. We also evaluated whether markers for cholesterol metabolism at baseline predict the intensity of the inflammatory response. Eight healthy young subjects received LPS infusion, and blood was sampled for the following 24 h. In addition to lipids, lipoproteins, and CEC, we also measured markers for cholesterol absorption and synthesis, bile acid synthesis, and inflammation. Compared with baseline, plasma total cholesterol, low-density lipoprotein cholesterol, and CEC decreased, while triglycerides increased in the 24 h following LPS infusion. TC-standardized levels of cholesterol synthesis markers (lathosterol, lanosterol, and desmosterol) and a bile acid synthesis marker (7α-OH-cholesterol) also decreased, with no changes in cholesterol absorption markers (campesterol, sitosterol, and cholestanol). Baseline TC-standardized levels of desmosterol and 7α-OH-cholesterol were positively correlated with concentrations of various inflammatory markers. Changes in TC-standardized desmosterol and 7α-OH-cholesterol were negatively correlated with concentrations of inflammatory markers. LPS infusion reduced endogenous cholesterol synthesis and bile acid synthesis in healthy young men.

Lifestyle- and genetically induced disorders related to disturbances in cholesterol metabolism have shown the detrimental impact of excessive cholesterol levels on a plethora of pathological processes such as inflammation. In this context, two-hydroxypropyl-β-cyclodextrin (CD) is increasingly considered as a novel pharmacological compound to decrease cellular cholesterol levels due to its ability to increase cholesterol solubility. However, recent findings have reported contra-indicating events after the use of CD questioning the clinical applicability of this compound. Given its potential as a therapeutic compound in metabolic inflammatory diseases, in this study, we evaluated the inflammatory effects of CD administration in the context of cholesterol-induced metabolic inflammation in vivo and in vitro. The inflammatory and cholesterol-depleting effects of CD were first investigated in low-density lipoprotein receptor knockout (Ldlr-/) mice that were transplanted with Npc1nih or Npc1wt bone marrow and were fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 12 weeks, thereby creating an extreme model of lysosomal cholesterol-induced metabolic inflammation. In the final three weeks, these mice received daily injections of either control (saline) or CD subcutaneously. Subsequently, the inflammatory properties of CD were investigated in vitro in two macrophage cell lines and in murine bone marrow-derived macrophages (BMDMs). While CD administration improved cholesterol mobilization outside lysosomes in BMDMs, an overall pro-inflammatory profile was observed after CD treatment, evidenced by increased hepatic inflammation in vivo and a strong increase in cytokine release and inflammatory gene expression in vitro in murine BMDMs and macrophages cell lines. Nevertheless, this CD-induced pro-inflammatory profile was time-dependent, as short term exposure to CD did not result in a pro-inflammatory response in BMDM. While CD exerts desired cholesterol-depleting effects, its inflammatory effect is dependent on the exposure time. As such, using CD in the clinic, especially in a metabolic inflammatory context, should be closely monitored as it may lead to undesired, pro-inflammatory side effects.

Abstract In dialysis patients, cholesterol‐lowering therapy with statins is less effective than in other high‐risk patients. This may be explained by a shift from cholesterol synthesis toward cholesterol absorption. In line, markers of cholesterol absorption—such as campesterol—better predict atherosclerotic cardiovascular events than markers of cholesterol synthesis—such as lathosterol—in dialysis patients. To test the association between markers of cholesterol absorption such as campesterol—and markers of cholesterol synthesis—such as lathosterol—against cardiovascular events in non‐dialysis CKD patients. Altogether 251 patients not on lipid‐lowering agents were followed annually for the composite endpoint atherosclerotic cardiovascular disease (ASCVD) and all‐cause death. During follow‐up of 5.2 ± 2.1 years, 61 participants reached the primary endpoint atherosclerotic cardiovascular disease/all‐cause death [ASCVD/D], 47 participants suffered from ASCVD, and 46 participants died. In univariate Cox regression analysis, campesterol/lathosterol ratio did not significantly predict ASCVD/D (HR 0.643; 0.358–1.155; 3rd vs. 1st tertile), all‐cause death (HR 1.309; 0.604–2.838; 3rd vs. 1st tertile) nor ASCVD (HR 0.589; 0.311–1.118; 3rd vs. 1st tertile). We did not observe a shift from cholesterol synthesis to cholesterol absorption across the spectrum of non‐dialysis CKD. Campesterol/lathosterol ratio did not predict future ASCVD or all‐cause death in non‐dialysis CKD.

Background & AimsThe lysosomal enzyme, cathepsin D (CTSD) has been implicated in the pathogenesis of non-alcoholic steatohepatitis (NASH), a disease characterised by hepatic steatosis and inflammation. We have previously demonstrated that specific inhibition of the extracellular CTSD leads to improved metabolic features in Sprague-Dawley rats with steatosis. However, the individual roles of extracellular and intracellular CTSD in NASH are not yet known. In the current study, we evaluated the underlying mechanisms of extracellular and intracellular CTSD fractions in NASH-related metabolic inflammation using specific small-molecule inhibitors.MethodsLow-density lipoprotein receptor knock out (Ldlr-/-) mice were fed a high-fat, high cholesterol (HFC) diet for ten weeks to induce NASH. Further, to investigate the effects of CTSD inhibition, mice were injected either with an intracellular (GA-12) or extracellular (CTD-002) CTSD inhibitor or vehicle control at doses of 50 mg/kg body weight subcutaneously once in two days for ten weeks.ResultsLdlr-/- mice treated with extracellular CTSD inhibitor showed reduced hepatic lipid accumulation and an associated increase in faecal bile acid levels as compared to intracellular CTSD inhibitor-treated mice. Furthermore, in contrast to intracellular CTSD inhibition, extracellular CTSD inhibition switched the systemic immune status of the mice to an anti-inflammatory profile. In line, label-free mass spectrometry-based proteomics revealed that extra- and intracellular CTSD fractions modulate proteins belonging to distinct metabolic pathways.ConclusionWe have provided clinically translatable evidence that extracellular CTSD inhibition shows some beneficial metabolic and systemic inflammatory effects which are distinct from intracellular CTSD inhibition. Considering that intracellular CTSD inhibition is involved in essential physiological processes, specific inhibitors capable of blocking extracellular CTSD activity, can be promising and safe NASH drugs.

Abstract Background Cholesterol is an essential component in human development. In fetuses affected by intrauterine growth restriction (IUGR), fetal blood cholesterol levels are low. Whether this is the result of a reduced materno-fetal cholesterol transport, or due to low fetal de novo synthesis rates, remains a matter of debate. By analyzing cholesterol interbolites and plant sterols we aimed at deeper insights into transplacental cholesterol transport and fetal cholesterol handling in IUGR with potential targets for future therapy. We hypothesized that placental insufficiency results in a diminished cholesterol supply to the fetus. Methods Venous umbilical cord sera were sampled post-partum from fetuses delivered between 24 weeks of gestation and at full term. IUGR fetuses were matched to 49 adequate-for-age delivered preterm and term neonates (CTRL) according to gestational age at delivery. Cholesterol was measured by gas chromatography-flame ionization detection using 5a-cholestane as internal standard. Cholesterol precursors and synthesis markers, such as lanosterol, lathosterol, and desmosterol, the absorption markers, 5α-cholestanol and plant sterols, such as campesterol and sitosterol, as well as enzymatically oxidized cholesterol metabolites (oxysterols), such as 24S- or 27-hydroxycholesterol, were analyzed by gas chromatography-mass spectrometry, using epicoprostanol as internal standard for the non-cholesterol sterols and deuterium labeled oxysterols for 24S- and 27-hydroxycholesterol. Results Mean cholesterol levels were 25% lower in IUGR compared with CTRL (p

Large polyglutamine expansions in Ataxin-2 (ATXN2) cause multi-system nervous atrophy in Spinocerebellar Ataxia type 2 (SCA2). Intermediate size expansions carry a risk for selective motor neuron degeneration, known as Amyotrophic Lateral Sclerosis (ALS). Conversely, the depletion of ATXN2 prevents disease progression in ALS. Although ATXN2 interacts directly with RNA, and in ALS pathogenesis there is a crucial role of RNA toxicity, the affected functional pathways remain ill defined. Here, we examined an authentic SCA2 mouse model with Atxn2-CAG100-KnockIn for a first definition of molecular mechanisms in spinal cord pathology. Neurophysiology of lower limbs detected sensory neuropathy rather than motor denervation. Triple immunofluorescence demonstrated cytosolic ATXN2 aggregates sequestrating TDP43 and TIA1 from the nucleus. In immunoblots, this was accompanied by elevated CASP3, RIPK1 and PQBP1 abundance. RT-qPCR showed increase of Grn, Tlr7 and Rnaset2 mRNA versus Eif5a2, Dcp2, Uhmk1 and Kif5a decrease. These SCA2 findings overlap well with known ALS features. Similar to other ataxias and dystonias, decreased mRNA levels for Unc80, Tacr1, Gnal, Ano3, Kcna2, Elovl5 and Cdr1 contrasted with Gpnmb increase. Preterminal stage tissue showed strongly activated microglia containing ATXN2 aggregates, with parallel astrogliosis. Global transcriptome profiles from stages of incipient motor deficit versus preterminal age identified molecules with progressive downregulation, where a cluster of cholesterol biosynthesis enzymes including Dhcr24, Msmo1, Idi1 and Hmgcs1 was prominent. Gas chromatography demonstrated a massive loss of crucial cholesterol precursor metabolites. Overall, the ATXN2 protein aggregation process affects diverse subcellular compartments, in particular stress granules, endoplasmic reticulum and receptor tyrosine kinase signaling. These findings identify new targets and potential biomarkers for neuroprotective therapies.

Cerebrotendinous xanthomatosis (CTX) is caused by autosomal recessive loss-of-function mutations in CYP27A1, a gene encoding cytochrome p450 oxidase essential for bile acid synthesis, resulting in altered bile acid and lipid metabolism. Here, we aimed to identify metabolic aberrations that drive ongoing neurodegeneration in some patients with CTX despite chenodeoxycholic acid (CDCA) supplementation, the standard treatment in CTX. Using chromatographic separation techniques coupled to mass spectrometry, we analyzed 26 sterol metabolites in serum and cerebrospinal fluid (CSF) of patients with CTX and in one CTX brain. Comparing samples of drug naive patients to patients treated with CDCA and healthy controls, we identified 7α,12α-dihydroxycholest-4-en-3-one as the most prominently elevated metabolite in serum and CSF of drug naive patients. CDCA treatment substantially reduced or even normalized levels of all metabolites increased in untreated patients with CTX. Independent of CDCA treatment, metabolites of the 27-hydroxylation pathway were nearly absent in all patients with CTX. 27-hydroxylated metabolites accounted for ∼45% of total free sterol content in CSF of healthy controls but

Autosomal Dominant Hypercholesterolemia (ADH) is a genetic disorder caused by pathogenic variants in LDLR, APOB, PCSK9 and APOE genes. We sought to identify new candidate genes responsible for the ADH phenotype in patients without pathogenic variants in the known ADH-causing genes by focusing on a French family with affected and non-affected members who presented a high ADH polygenic risk score (wPRS). Linkage analysis, whole exome and whole genome sequencing resulted in the identification of variants p.(Pro398Ala) in CYP7A1, p.(Val1382Phe) in LRP6 and p.(Ser202His) in LDLRAP1. A total of 6 other variants were identified in 6 of 160 unrelated ADH probands: p.(Ala13Val) and p.(Aps347Asn) in CYP7A1; p.(Tyr972Cys), p.(Thr1479Ile) and p.(Ser1612Phe) in LRP6; and p.(Ser202LeufsTer19) in LDLRAP1. All six probands presented a moderate wPRS. Serum analyses of carriers of the p.(Pro398Ala) variant in CYP7A1 showed no differences in the synthesis of bile acids compared to the serums of non-carriers. Functional studies of the four LRP6 mutants in HEK293T cells resulted in contradictory results excluding a major effect of each variant alone. Within the family, none of the heterozygous for only the LDLRAP1 p.(Ser202His) variant presented ADH. Altogether, each variant individually does not result in elevated LDL-C; however, the oligogenic combination of two or three variants reveals the ADH phenotype.

Abstract Due to the obesity epidemic, non-alcoholic steatohepatitis (NASH) is a prevalent liver disease, characterized by fat accumulation and inflammation of the liver. However, due to a lack of mechanistic insight, diagnostic and therapeutic options for NASH are poor. Recent evidence has indicated cathepsin D (CTSD), a lysosomal enzyme, as a marker for NASH. Here, we investigated the function of CTSD in NASH by using an in vivo and in vitro model. In addition to diminished hepatic inflammation, inhibition of CTSD activity dramatically improved lipid metabolism, as demonstrated by decreased plasma and liver levels of both cholesterol and triglycerides. Mechanistically, CTSD inhibition resulted in an increased conversion of cholesterol into bile acids and an elevated excretion of bile acids via the feces, indicating that CTSD influences lipid metabolism. Consistent with these findings, treating Wt BMDMs with PepA in vitro showed a similar decrease in inflammation and an analogous effect on cholesterol metabolism. Conclusion: CTSD is a key player in the development of hepatic inflammation and dyslipidemia. Therefore, aiming at the inhibition of the activity of CTSD may lead to novel treatments to combat NASH.

The prevalence of metabolic disorders characterized by chronic inflammation has been on a sharp rise for decades. As such, tools that address metabolic and inflammatory dysregulation are of great importance. Plant stanols are well-known for reducing intestinal cholesterol absorption and may also have direct anti-inflammatory effects. In this study, our aim was to investigate to what extent the benefits of dietary plant stanol supplementation depend on dietary cholesterol intake in an experimental mouse model for cholesterol-induced metabolic inflammation. Here, we used Ldlr−/− mice transplanted with Npc1nih-derived bone marrow, featuring feature bone marrow-derived immune cells characterized by chronic inflammation induced by lysosomal lipid accumulation. Npc1nih- and Npc1wt-transplanted mice were placed on either a high fat, high cholesterol (HFC) or on a chow diet low in cholesterol, with or without 2% plant stanols supplementation. At the end of the study, the metabolic and inflammatory status of the mice was analyzed. Plant stanol supplementation to the HFC diet reduced liver cholesterol levels and improved lipid metabolism and liver inflammation, particularly in Npc1nih-tp mice. In contrast, plant stanol supplementation to the chow diet did not significantly improve the aforementioned parameters, though similar reductive trends to those in the HFC diet setting were observed regarding liver cholesterol accumulation and liver inflammatory markers. The effects of dietary plant stanol supplementation on dietary cholesterol-induced inflammation are largely dependent on dietary cholesterol intake. Future research should verify whether other models of metabolic inflammation exhibit similar stanol-related effects on inflammation.

We recently found that dietary supplementation with the seaweed Sargassum fusiforme, containing the preferential LXRβ-agonist 24(S)-saringosterol, prevented memory decline and reduced amyloid-β (Aβ) deposition in an Alzheimer’s disease (AD) mouse model without inducing hepatic steatosis. Here, we examined the effects of 24(S)-saringosterol as a food additive on cognition and neuropathology in AD mice. Six-month-old male APPswePS1ΔE9 mice and wildtype C57BL/6J littermates received 24(S)-saringosterol (0.5 mg/25 g body weight/day) (APPswePS1ΔE9 n = 20; C57BL/6J n = 19) or vehicle (APPswePS1ΔE9 n = 17; C57BL/6J n = 19) for 10 weeks. Cognition was assessed using object recognition and object location tasks. Sterols were analyzed by gas chromatography/mass spectrometry, Aβ and inflammatory markers by immunohistochemistry, and gene expression by quantitative real-time PCR. Hepatic lipids were quantified after Oil-Red-O staining. Administration of 24(S)-saringosterol prevented cognitive decline in APPswePS1ΔE9 mice without affecting the Aβ plaque load. Moreover, 24(S)-saringosterol prevented the increase in the inflammatory marker Iba1 in the cortex of APPswePS1ΔE9 mice (p < 0.001). Furthermore, 24(S)-saringosterol did not affect the expression of lipid metabolism-related LXR-response genes in the hippocampus nor the hepatic neutral lipid content. Thus, administration of 24(S)-saringosterol prevented cognitive decline in APPswePS1ΔE9 mice independent of effects on Aβ load and without adverse effects on liver fat content. The anti-inflammatory effects of 24(S)-saringosterol may contribute to the prevention of cognitive decline.

In this study, the hypothesis that supplementation with methionine (Met) as DL-Met (DLM) in excess of the National Research Council (NRC) recommendations improves the antioxidant system in broilers was investigated. Day-old male Cobb-500 broilers (n = 72) were divided into three groups which were fed a control diet or diets supplemented with two levels of DLM in which the concentrations of Met + Cys exceeded the recommendations of NRC by 15–20% (group DLM 1) or 30–40% (group DLM 2), respectively. The three groups of broilers did not show differences in body weight gains, feed intake, and feed conversion ratio. However, broilers of groups DLM 1 and DLM 2 had higher concentrations of glutathione (GSH) in liver and thigh muscle and lower concentrations of cholesterol oxidation products (COPs) in heat-processed thigh muscle than broilers of the control group. Concentrations of several oxidation products of phytosterols in heat-processed thigh muscle were also reduced in groups DLM 1 and DLM 2; however, the concentration of total oxidation products of phytosterols was not different between the three groups. The study shows that DLM supplementation improved the antioxidant status due to an increased formation of GSH and reduced the formation of COPs during heat-processing in thigh muscle.

ABSTRACT Monocytes from HIV-infected patients produce increased levels of inflammatory cytokines, which are associated with chronic immune activation and AIDS progression. Chronic immune activation is often not restored even in patients showing viral suppression under ART. Therefore, new therapeutic strategies to control inflammation and modulate immune activation are required. Hydroxypropyl-beta-cyclodextrin (HP-BCD) is a cholesterol-sequestering agent that has been reported to be safe for human use in numerous pharmaceutical applications and that has been shown to inactivate HIV in vitro and to control SIV infection in vivo. Since cellular cholesterol content or metabolism has been related to altered cellular activation, we evaluated whether HP-BCD treatment could modulate monocyte response to inflammatory stimuli. Treatment of monocytes isolated from HIV-positive and HIV-negative donors with HP-BCD inhibited the expression of CD36 and TNF-α after LPS stimulation, independent of raft disruption. Accordingly, HP-BCD-treated cells showed significant reduction of TNF-α and IL-10 secretion, which was associated with lower mRNA expression. LPS-induced p38MAPK phosphorylation was dampened by HP-BCD treatment, indicating this pathway as a target for HP-BCD-mediated anti-inflammatory response. The expression of HLA-DR was also reduced in monocytes and dendritic cells treated with HP-BCD, which could hinder T cell activation by these cells. Our data suggest that, besides its well-known antiviral activity, HP-BCD could have an immunomodulatory effect, leading to decreased inflammatory responses mediated by antigen-presenting cells, which may impact HIV pathogenesis and AIDS progression. IMPORTANCE Chronic immune activation is a hallmark of HIV infection and is often not controlled even in patients under antiretroviral therapy. Indeed, chronic diseases with inflammatory pathogenesis are being reported as major causes of death for HIV-infected persons. Hydroxypropyl-beta cyclodextrin (HP-BCD) is a cholesterol-sequestering drug that inhibits HIV replication and infectivity in vitro and in vivo. Recent studies have demonstrated the importance of cholesterol metabolism and content in different inflammatory conditions; therefore, we investigated the potential of HP-BCD as an immunomodulatory drug, regulating the activation of cells from HIV-infected patients. Treatment of monocytes with HP-BCD inhibited the expression and secretion of receptors and mediators that are usually enhanced in HIV patients. Furthermore, we investigated the molecular mechanisms associated with the immunomodulatory effect of HP-BCD. Our results indicate that, besides reducing viral replication, HP-BCD treatment may contribute to modulation of chronic immune activation associated with AIDS.

Arachidonate 15-lipoxygenase (ALOX15) and arachidonate 15-lipoxygenase, type B (ALOX15B) catalyze the dioxygenation of polyunsaturated fatty acids and are upregulated in human alternatively activated macrophages (AAMs) induced by Th2 cytokine interleukin-4 (IL-4) and/or interleukin-13. Known primarily for roles in bioactive lipid mediator synthesis, 15-lipoxygenases (15-LOXs) have been implicated in various macrophage functions including efferocytosis and ferroptosis. Using a combination of inhibitors and siRNAs to suppress 15-LOX isoforms, we studied the role of 15-LOXs in cellular cholesterol homeostasis and immune function in naïve and AAMs. Silencing or inhibiting the 15-LOX isoforms impaired sterol regulatory element binding protein (SREBP)-2 signaling by inhibiting SREBP-2 processing into mature transcription factor and reduced SREBP-2 binding to sterol regulatory elements and subsequent target gene expression. Silencing ALOX15B reduced cellular cholesterol and the cholesterol intermediates desmosterol, lanosterol, 24,25-dihydrolanosterol, and lathosterol as well as oxysterols in IL-4-stimulated macrophages. In addition, attenuating both 15-LOX isoforms did not generally affect IL-4 gene expression but rather uniquely impacted IL-4-induced CCL17 production in an SREBP-2-dependent manner resulting in reduced T cell migration to macrophage conditioned media. In conclusion, we identified a novel role for ALOX15B, and to a lesser extent ALOX15, in cholesterol homeostasis and CCL17 production in human macrophages.

BACKGROUND:The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) is a known modulator of inflammation and ligand to both, pro-inflammatory cannabinoid receptor 1 (CB1) and anti-inflammatory CB2. While the role of both receptors in atherogenesis has been studied extensively, the significance of 2-AG for atherogenesis is less well characterized. METHODS:The impact of 2-AG on atherogenesis was studied in two treatment groups of ApoE-/- mice. One group received the monoacylglycerol lipase (MAGL)-inhibitor JZL184 [5 mg/kg i.p.], which impairs 2-AG degradation and thus causes elevated 2-AG levels, the other group received vehicle for four weeks. Simultaneously, both groups were fed a high-cholesterol diet. The atherosclerotic plaque burden was assessed in frozen sections through the aortic sinus following oil red O staining and infiltrating macrophages were detected by immunofluorescence targeting CD68. In vitro, the effect of 2-AG on B6MCL macrophage migration was assessed by Boyden chamber experiments. Transcription of adhesion molecules and chemokine receptors in macrophages was assessed by qPCR. RESULTS:As expected, application of the MAGL-inhibitor JZL184 resulted in a significant increase in 2-AG levels in vascular tissue (98.2 ± 16.1 nmol/g vs. 27.3 ± 4.5 nmol/g; n = 14-16; p < 0.001). ApoE-/- mice with elevated 2-AG levels displayed a significantly increased plaque burden compared to vehicle treated controls (0.44 ± 0.03 vs. 0.31 ± 0.04; n = 14; p = 0.0117). This was accompanied by a significant increase in infiltrating macrophages within the atherosclerotic vessel wall (0.33 ± 0.02 vs. 0.27 ± 0.01; n = 13-14; p = 0.0076). While there was no alteration to the white blood counts of JZL184-treated animals, 2-AG enhanced macrophage migration in vitro by 1.8 ± 0.2 -fold (n = 4-6; p = 0.0393) compared to vehicle, which was completely abolished by co-administration of either CB1- or CB2-receptor-antagonists. qPCR analyses of 2-AG-stimulated macrophages showed an enhanced transcription of the chemokine CCL5 (1.59 ± 0.23 -fold; n = 5-6; p = 0.0589) and its corresponding receptors CCR1 (2.04 ± 0.46 -fold; n = 10-11; p = 0.0472) and CCR5 (2.45 ± 0.62 -fold; n = 5-6; p = 0.0554). CONCLUSION:Taken together, elevated 2-AG levels appear to promote atherogenesis in vivo. Our data suggest that 2-AG promotes macrophage migration, possibly by the CCL5-CCR5/CCR1 axis, and thereby contributes to vascular inflammation. Thus, decreasing vascular 2-AG levels might represent a promising therapeutic strategy in patients suffering from atherosclerosis and coronary heart disease.

ABCG5 (G5) and ABCG8 (G8) form a sterol transporter that acts in liver and intestine to prevent accumulation of dietary sterols. Mutations in either G5 or G8 cause sitosterolemia, a recessive disorder characterized by sterol accumulation and premature coronary atherosclerosis. Hepatic G5G8 mediates cholesterol excretion into bile, but the function and relative importance of intestinal G5G8 has not been defined. To determine the role of intestinal G5G8, we developed liver-specific (L-G5G8−/−), intestine-specific (I-G5G8−/−), and total (G5G8−/−) KO mice. Tissue levels of sitosterol, the most abundant plant sterol, were >90-fold higher in G5G8−/− mice than in WT animals. Expression of G5G8 only in intestine or only in liver decreased tissue sterol levels by 90% when compared with G5G8−/− animals. Biliary sterol secretion was reduced in L-G5G8−/− and G5G8−/− mice, but not in I-G5G8−/− mice. Conversely, absorption of plant sterols was increased in I-G5G8−/− and G5G8−/− mice, but not in L-G5G8−/− mice. Reverse cholesterol transport, as assessed from the fraction of intravenously administered 3H-cholesterol that appeared in feces, was reduced in G5G8−/−, I-G5G8−/−, and L-G5G8−/− mice. Thus, G5G8 expression in both the liver and intestine protects animals from sterol accumulation, and intestinal G5G8 contributes to extrahepatic cholesterol efflux in mice.

Reduced plasma LDL-cholesterol is a hallmark of hyperthyroidism and is caused by transcriptional stimulation of LDL receptors in the liver. Here, we investigated whether thyroid hormone (TH) actions involve other mechanisms that may also account for the reduction in LDL-cholesterol, including effects on proprotein convertase subtilisin/kexin type 9 (PCSK9) and bile acid synthesis. Twenty hyperthyroid patients were studied before and after clinical normalization, and the responses to hyperthyroidism were compared with those in 14 healthy individuals after 14 days of treatment with the liver-selective TH analog eprotirome. Both hyperthyroidism and eprotirome treatment reduced circulating PCSK9, lipoprotein cholesterol, apoB and AI, and lipoprotein(a), while cholesterol synthesis was stable. Hyperthyroidism, but not eprotirome treatment, markedly increased bile acid synthesis and reduced fibroblast growth factor (FGF) 19 and dietary cholesterol absorption. Eprotirome treatment, but not hyperthyroidism, reduced plasma triglycerides. Neither hyperthyroidism nor eprotirome treatment altered insulin, glucose, or FGF21 levels. TH reduces circulating PSCK9, thereby likely contributing to lower plasma LDL-cholesterol in hyperthyroidism. TH also stimulates bile acid synthesis, although this response is not critical for its LDL-lowering effect.

ATP-binding cassette transporter A1 (ABCA1) mediates cellular cholesterol efflux in the brain and influences whole brain cholesterol homeostasis. Activation of liver X receptors (LXRs), transcription factors that increase the expression of cholesterol transport genes including ABCA1, reduces neuroinflammation and pathology in neurodegenerative animal models suggesting that in addition to its involvement in cholesterol transport, ABCA1 may play a role in modulating the inflammatory response in the brain. We investigated the cell-type specific role of ABCA1 in neuroinflammation in vivo using mice specifically lacking brain ABCA1 (ABCA1−B/−B) as well as mice lacking neuronal (ABCA1−N/−N) and astrocytic (ABCA1−Ast/−Ast) ABCA1. ABCA1−B/−B mice exhibit cortical astrogliosis, increased inflammatory gene expression as well as activation of mitogen-activated protein kinases (MAPKs) following acute lipopolysaccharide (LPS) administration. Microglia cultured from ABCA1−B/−B mice exhibit augmented LPS-induced secretion of tumor necrosis factor α (TNFα) and decreased phagocytic activity, indicating an increase in a pro-inflammatory response. ABCA1−N/−N mice develop astrogliosis but show no change in inflammatory gene expression. Intriguingly, ABCA1−Ast/−Ast mice show neither astrogliosis nor elevated expression of inflammatory markers. Cortical apolipoprotein E (apoE) levels are reduced in ABCA1−Ast/−Ast but not in ABCA1−N/−N mice, providing in vivo evidence for the specific role of astrocyte ABCA1 in regulating brain apoE levels. Interestingly, cortical neuronal death is increased in 17 month-old ABCA1−B/−B mice but not in ABCA1−N/−N or ABCA1−Ast/−Ast mice. Our findings suggest that coordinated ABCA1 activity across neurons and glial cells influences neuroinflammation and neurodegeneration.

APOE ε4 (apoE4) polymorphism is the main genetic determinant of sporadic Alzheimer’s disease (AD). A dietary approach (Fortasyn) including docosahexaenoic acid, eicosapentaenoic acid, uridine, choline, phospholipids, folic acid, vitamins B12, B6, C, and E, and selenium has been proposed for dietary management of AD. We hypothesize that the diet could inhibit AD-like pathologies in apoE4 mice, specifically cerebrovascular and connectivity impairment. Moreover, we evaluated the diet effect on cerebral blood flow (CBF), functional connectivity (FC), gray/white matter integrity, and postsynaptic density in aging apoE4 mice. At 10–12 months, apoE4 mice did not display prominent pathological differences compared to wild-type (WT) mice. However, 16–18-month-old apoE4 mice revealed reduced CBF and accelerated synaptic loss. The diet increased cortical CBF and amount of synapses and improved white matter integrity and FC in both aging apoE4 and WT mice. We demonstrated that protective mechanisms on vascular and synapse health are enhanced by Fortasyn, independent of apoE genotype. We further showed the efficacy of a multimodal translational approach, including advanced MR neuroimaging, to study dietary intervention on brain structure and function in aging.

BACKGROUND:Toll-like receptors (TLR) of the innate immune system have been closely linked with the development of atherosclerotic lesions. TLR9 is activated by unmethylated CpG motifs within ssDNA, but also by CpG motifs in nucleic acids released during vascular apoptosis and necrosis. The role of TLR9 in vascular disease remains controversial and we sought to investigate the effects of a proinflammatory TLR9 stimulation in mice. METHODS AND FINDINGS:TLR9-stimulation with high dose CpG ODN at concentrations between 6.25 nM to 30 nM induced a significant proinflammatory cytokine response in mice. This was associated with impaired reendothelialization upon acute denudation of the carotid and increased numbers of circulating endothelial microparticles, as a marker for amplified endothelial damage. Chronic TLR9 agonism in apolipoprotein E-deficient (ApoE-/-) mice fed a cholesterol-rich diet increased aortic production of reactive oxygen species, the number of circulating endothelial microparticles, circulating sca-1/flk-1 positive cells, and most importantly augmented atherosclerotic plaque formation when compared to vehicle treated animals. Importantly, high concentrations of CpG ODN are required for these proatherogenic effects. CONCLUSIONS:Systemic stimulation of TLR9 with high dose CpG ODN impaired reendothelialization upon acute vascular injury and increased atherosclerotic plaque development in ApoE-/- mice. Further studies are necessary to fully decipher the contradictory finding of TLR9 agonism in vascular biology.

BACKGROUND:The endocannabinoid 2-arachidonoylglycerol (2-AG) is a known modulator of inflammation. Despite its high concentration in vascular tissue, the role of 2-AG in atherogenesis has not yet been examined. METHODS:ApoE-deficient mice were sublethally irradiated and reconstituted with bone marrow from mice with a myeloid-specific knockout of the 2-AG synthesising enzyme diacylglycerol lipase α (Dagla) or control bone marrow with an intact 2-AG biosynthesis. After a cholesterol-rich diet for 8 weeks, plaque size and plaque morphology were examined in chimeric mice. Circulating inflammatory cells were assessed by flow cytometry. Aortic tissue and plasma levels of endocannabinoids were measured using liquid chromatography-multiple reaction monitoring. RESULTS:Mice with Dagla-deficient bone marrow and circulating myeloid cells showed a significantly reduced plaque burden compared to controls. The reduction in plaque size was accompanied by a significantly diminished accumulation of both neutrophil granulocytes and macrophages in atherosclerotic lesions of Dagla-deficient mice. Moreover, CB2 expression and the amount of oxidised LDL within atherosclerotic lesions was significantly reduced. FACS analyses revealed that levels of circulating inflammatory cells were unaltered in Dagla-deficient mice. CONCLUSIONS:Myeloid synthesis of the endocannabinoid 2-AG appears to promote vascular inflammation and atherogenesis. Thus, myeloid-specific disruption of 2-AG synthesis may represent a potential novel therapeutic strategy against atherosclerosis.

Plant sterols such as sitosterol and campesterol are frequently administered as cholesterol-lowering supplements in food. Recently, it has been shown in mice that, in contrast to the structurally related cholesterol, circulating plant sterols can enter the brain. We questioned whether the accumulation of plant sterols in murine brain is reversible. After being fed a plant sterol ester-enriched diet for 6 weeks, C57BL/6NCrl mice displayed significantly increased concentrations of plant sterols in serum, liver, and brain by 2- to 3-fold. Blocking intestinal sterol uptake for the next 6 months while feeding the mice with a plant stanol ester-enriched diet resulted in strongly decreased plant sterol levels in serum and liver, without affecting brain plant sterol levels. Relative to plasma concentrations, brain levels of campesterol were higher than sitosterol, suggesting that campesterol traverses the blood-brain barrier more efficiently. In vitro experiments with brain endothelial cell cultures showed that campesterol crossed the blood-brain barrier more efficiently than sitosterol. We conclude that, over a 6-month period, plant sterol accumulation in murine brain is virtually irreversible.

Moderately elevated levels of plasma plant sterols have been suspected to be causally involved in atherosclerosis. The aim of this study was to investigate whether plant sterols and other markers of sterol metabolism predicted all-cause and cardiovascular mortality in participants of the Ludwigshafen Risk and Cardiovascular health (LURIC) study. A total of 1,257 individuals who did not use statins and at baseline had a mean (± SD) age of 62.8 (± 11.0) years were included in the present analysis. Lathosterol, cholestanol, campesterol, and sitosterol were measured to estimate cholesterol synthesis and absorption. The mean (± SD) time of the follow-up for all-cause and cardiovascular mortality was 7.32 (± 2.3) years. All-cause (P = 0.001) and cardiovascular (P = 0.006) mortality were decreased in the highest versus the lowest lathosterol to cholesterol tertile. In contrast, subjects in the third cholestanol to cholesterol tertile had increased all-cause (P < 0.001) and cardiovascular mortality (P = 0.010) compared with individuals in the first tertile. The third campesterol to cholesterol tertile was associated with increased all-cause mortality (P = 0.025). Sitosterol to cholesterol tertiles were not significantly related to all-cause or cardiovascular mortality. The data suggest that high absorption and low synthesis of cholesterol predict increased all-cause and cardiovascular mortality in LURIC participants.

Cholesterol 24S-hydroxylase (CYP46A1) is of key importance for cholesterol homeostasis in the brain. This enzyme seems to be resistant toward most regulatory factors and at present no drug effects on its activity have been described. The crystal structures of the substrate-free and substrate-bound CYP46A1 were recently determined (Mast et al., Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principal cholesterol hydroxylase in the brain. Proc. Natl. Acad. Sci. USA. 2008. 105: 9546–9551). These structural studies suggested that ligands other than sterols can bind to CYP46A1. We show here that the antifungal drug voriconazole binds to the enzyme in vitro and inhibits CYP46A1-mediated cholesterol 24-hydroxylation with a Ki of 11 nM. Mice treated with daily intraperitoneal injections of voriconazole for 5 days had high levels of voriconazole in the brain and significantly reduced brain levels of 24S-hydroxycholesterol. The levels of squalene, lathosterol, and HMG-CoA reductase mRNA were reduced in the brain of the voriconazole-treated animals as well, indicating a reduced cholesterol synthesis. Most of this effect may be due to a reduced utilization of cholesterol by CYP46A1. One of the side-effects of voriconazole is visual disturbances. Because CYP46A1 is also expressed in the neural retina, we discuss the possibility that the inhibition of CYP46A1 by voriconazole contributes to these visual disturbances.

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.

Cholesterol homeostasis is of emerging therapeutic importance for Alzheimer's disease (AD). Agonists of liver-X-receptors (LXRs) stimulate several genes that regulate cholesterol homeostasis, and synthetic LXR agonists decrease neuropathological and cognitive phenotypes in AD mouse models. The cholesterol transporter ABCG1 is LXR-responsive and highly expressed in brain. In vitro, conflicting reports exist as to whether ABCG1 promotes or impedes Aβ production. To clarify the in vivo roles of ABCG1 in Aβ metabolism and brain cholesterol homeostasis, we assessed neuropathological and cognitive outcome measures in PDAPP mice expressing excess transgenic ABCG1. A 6-fold increase in ABCG1 levels did not alter Aβ, amyloid, apolipoprotein E levels, cholesterol efflux, or cognitive performance in PDAPP mice. Furthermore, endogenous murine Aβ levels were unchanged in both ABCG1-overexpressing or ABCG1-deficient mice. These data argue against a direct role for ABCG1 in AD. However, excess ABCG1 is associated with decreased levels of sterol precursors and increased levels of SREBP-2 and HMG-CoA-reductase mRNA, whereas deficiency of ABCG1 leads to the opposite effects. Although functions for ABCG1 in cholesterol efflux and Aβ metabolism have been proposed based on results with cellular model systems, the in vivo role of this enigmatic transporter may be largely one of regulating the sterol biosynthetic pathway.

The H2 allele of APOC1, giving rise to increased gene expression of apolipoprotein C-I (apoC-I), is in genetic disequilibrium with the APOE4 allele and may provide a major risk factor for Alzheimer's disease (AD). We found that apoC-I protein is present in astrocytes and endothelial cells within hippocampal regions in both human control and AD brains. Interestingly, apoC-I colocalized with β-amyloid (Aβ) in plaques in AD brains, and in vitro experiments revealed that aggregation of Aβ was delayed in the presence of apoC-I. Moreover, apoC-I was found to exacerbate the soluble Aβ oligomer-induced neuronal death. To establish a potential role for apoC-I in cognitive functions, we used human (h) APOC1+/0 transgenic mice that express APOC1 mRNA throughout their brains and apoC-I protein in astrocytes and endothelial cells. The hAPOC1+/0 mice displayed impaired hippocampal-dependent learning and memory functions compared with their wild-type littermates, as judged from their performance in the object recognition task (P = 0.012) and in the Morris water maze task (P = 0.010). ApoC-I may affect learning as a result of its inhibitory properties toward apoE-dependent lipid metabolism. However, no differences in brain mRNA or protein levels of endogenous apoE were detected between transgenic and wild-type mice. In conclusion, human apoC-I expression impairs cognitive functions in mice independent of apoE expression, which supports the potential of a modulatory role for apoC-I during the development of AD.

Some studies have suggested that a modest increase of plant sterol levels is a risk factor for coronary artery disease (CAD). We studied the relationship between plant sterol levels and CAD risk in a prospective nested case-control study consisting of 373 cases and 758 controls. Sitosterol and campesterol concentrations did not differ between cases and controls [sitosterol, 0.21 vs. 0.21 mg/dl (P = 0.1); campesterol, 0.31 vs. 0.32 mg/dl (P = 0.5)]. The sitosterol-to-cholesterol ratio was significantly lower in cases than in controls (1.19 vs. 1.29 μg/mg; P = 0.008), whereas the campesterol-to-cholesterol ratio did not differ significantly (1.78 vs. 1.88 μg/mg; P = 0.1). Plant sterol concentrations correlated positively with cholesterol levels and inversely with body mass index and triglyceride and lathosterol concentrations. Among individuals in the highest tertile of the sitosterol concentration, the unadjusted odds ratio (OR) for future CAD was 0.75 [95% confidence interval (CI) = 0.56–1.01]. After adjustment for traditional risk factors, the OR was 0.79 (95% CI = 0.56–1.13). For the campesterol concentration, the unadjusted OR was 0.95 (95% CI = 0.71–1.29) and the adjusted OR was 0.97 (95% CI = 0.68–1.39). In this large prospective study, higher levels of plant sterols, at least in the physiological range, do not appear to be adversely related to CAD in apparently healthy individuals.

Results of previous studies have shown that ezetimibe (10 mg/day) reduces LDL cholesterol in patients with mild hypercholesterolemia on a normal-cholesterol diet (dietary intake of 200–500 mg/day) by 16–22%. However, the LDL cholesterol-lowering effect of ezetimibe in subjects with an extremely low dietary cholesterol intake (vegetarians) has not been studied. We conducted a randomized, double-blind, placebo-controlled, two-phase crossover study in 18 healthy pure vegetarians to assess the effect of ezetimibe (10 mg/day) on plasma lipids, cholesterol absorption, and its synthesis. Treatment periods lasted 2 weeks each, with an intervening 2 week washout period. Fractional cholesterol absorption was determined using the continuous dual stable isotope feeding method. Mean dietary cholesterol intake in the pure vegetarians was extremely low and averaged 29.4 ± 16.8 and 31.4 ± 14.4 mg/day during the placebo and ezetimibe administration phases, respectively. Fractional cholesterol absorption during the placebo phase was 48.2 ± 8.2% and was decreased by 58% during ezetimibe treatment to 20.2 ± 6.2% (P < 0.001). This change in intestinal cholesterol absorption was followed by a significant reduction in LDL cholesterol of 17.3%. In individuals with extremely low dietary cholesterol intake, treatment with ezetimibe (10 mg/day) leads to a significant reduction of cholesterol absorption and a clinically relevant decrease of plasma LDL cholesterol, comparable to that of subjects with a normal dietary cholesterol intake. Thus, the lipid-lowering effect of ezetimibe is mediated mainly through a reduction of the absorption of endogenous (biliary) cholesterol.

The ATP binding cassette transporters ABCG5 (G5) and ABCG8 (G8) limit the accumulation of neutral sterols by restricting sterol uptake from the intestine and promoting sterol excretion into bile. Humans and mice lacking G5 and G8 (G5G8−/−) accumulate plant sterols in the blood and tissues. However, despite impaired biliary cholesterol secretion, plasma and liver cholesterol levels are lower in G5G8−/− mice than in wild-type littermates. To determine whether the observed changes in hepatic sterol metabolism were a direct result of decreased biliary sterol secretion or a metabolic consequence of the accumulation of dietary noncholesterol sterols, we treated G5G8−/− mice with ezetimibe, a drug that reduces the absorption of both plant- and animal-derived sterols. Ezetimibe feeding for 1 month sharply decreased sterol absorption and plasma levels of sitosterol and campesterol but increased cholesterol in both the plasma (from 60.4 to 75.2 mg/dl) and the liver (from 1.1 to 1.87 mg/g) of the ezetimibe-treated G5G8−/− mice. Paradoxically, the increase in hepatic cholesterol was associated with an increase in mRNA levels of HMG-CoA reductase and synthase.Together, these results indicate that pharmacological blockade of sterol absorption can ameliorate the deleterious metabolic effects of plant sterols even in the absence of G5 and G8.

The major pathway for the removal of cholesterol from the body is via secretion into the bile. Three members of the ATP binding cassette (ABC) family, ABCG5 (G5), ABCG8 (G8), and ABCB4 (MDR2), are required for the efficient biliary export of sterols. Here, we examined the interdependence of these three ABC transporters for biliary sterol secretion. Biliary lipid levels in mice expressing no MDR2 (Mdr2−/− mice) were compared with those of Mdr2−/− mice expressing 14 copies of a human G5 (hG5) and hG8 transgene (Mdr2−/−;hG5G8Tg mice). Mdr2−/− mice had only trace amounts of biliary cholesterol and phospholipids. The Mdr2−/−;hG5G8Tg mice had biliary cholesterol levels as low as those of Mdr2−/− mice. Thus, MDR2 expression is required for G5G8-mediated biliary sterol secretion. To determine whether the reduction in fractional absorption of dietary sterols associated with G5G8 overexpression is secondary to the associated increase in biliary cholesterol, we compared the fractional absorption of sterols in Mdr2−/−;hG5G8Tg and hG5G8Tg animals. Inactivation of MDR2 markedly attenuated the reduction in fractional sterol absorption associated with G5G8 overexpression.These results are consistent with the notion that increased biliary cholesterol secretion contributes to the reduction in fractional sterol absorption associated with G5G8 overexpression.

Side chain oxidizedoxysterols have a unique ability to traverse lipophilic membranes. We tested thehypothesis that there is a net flux of 27-hydroxycholesterol from the circulationinto the brain using plasma samples collected from the internal jugular vein andan artery of healthy male volunteers. Two independent studies were performed, onein which total levels of 27-hydroxycholesterol were measured and one in which thefree fraction of 27-hydroxycholesterol was measured. In the majority of subjectsstudied, the level of 27-hydroxycholesterol was higher in the artery than in thevein, and uptake from the circulation was calculated to be about 5 mg/24h.The distribution of 27-hydroxycholesterol in human brain was found to beconsistent with an extracerebral origin, with a concentration gradient from thewhite to the gray matter

In a recent publication [Diestel, A., O. Aktas, D. Hackel, I. Häke, S. Meier, C. S. Raine, R. Nitsch, F. Zipp, and O. Ullrich. 2003. Activation of microglial poly (ADP-ribose)-polymerase-1 by cholesterol breakdown products during neuroinflammation: a link between demyelination and neuronal damage. J. Exp. Med. 198: 1729–1740], extremely high levels of 7-oxocholesterol were reported in cerebrospinal fluid (CSF) of 11 patients with multiple sclerosis (MS) [7.4 ± 0.3 mg/l (mean ± SEM)]. The corresponding level of 12 subjects with other kinds of neurological diseases was reported to be 0.5 ± 0.1 mg/l. Such high levels of 7-oxocholesterol were found to cause neuronal damage of living brain tissues. Using a highly accurate method for an assay of 7-oxocholesterol based on isotope dilution-mass spectrometry and anaerobic conditions during workup, we found that the level of 7-oxocholesterol in CSF from 29 Swedish patients with MS was only 1.2 μg/l (median, ranging from 0.4 to 4.6 μg/l), less than 1/1,000th of the previously reported level. The level of 7-oxocholesterol in CSF from 24 Swedish control patients was 0.9 μg/l (0.3–2.3 μg/l), slightly but significantly lower than the CSF level in MS patients (P = 0.002). In vitro-induced lipid peroxidation of the endogenous cholesterol in CSF increased the level of 7-oxygenated cholesterol metabolites, particularly 7-oxocholesterol, up to ∼0.3 mg/l.These results are discussed in relation to the fact that 7-oxygenated steroids are easily artificially formed by autoxidation of cholesterol during workup procedures and analysis of sterols and oxysterols from biological samples.

24S-hydroxycholesterol is a side-chain oxidized oxysterol formed in the brain that is continuously crossing the blood-brain barrier to reach the circulation. There may be an opposite flux of 27-hydroxycholesterol, which is formed to a lower extent in the brain than in most other organs. Here we measured cholesterol, lathosterol, 24S- and 27-hydroxycholesterol, and plant sterols in four different brain areas of deceased Alzheimer's disease (AD) patients and controls. 24S-hydroxycholesterol was decreased and 27-hydroxycholesterol increased in all the brain samples from the AD patients. The difference was statistically significant in four of the eight comparisons. The ratio of 27-hydroxycholesterol to 24S-hydroxycholesterol was significantly increased in all brain areas of the AD patients and also in the brains of aged mice expressing the Swedish Alzheimer mutation APP751. Cholesterol 24S-hydroxylase and 27-hydroxylase protein was not significantly different between AD patients and controls. A high correlation was observed between the levels of 24S-hydroxycholesterol and lathosterol in the frontal cortex of the AD patients but not in the controls. Most probably the high levels of 27-hydroxycholesterol are due to increased influx of this steroid over the blood-brain barrier and the lower levels of 24S-hydroxycholesterol to decreased production.The high correlation between lathosterol and 24-hydroxycholesterol is consistent with a close coupling between synthesis and metabolism of cholesterol in the frontal cortex of the AD brain.

The recent identification of the aberrant transport proteins ABCG5 and ABCG8 resulting in sitosterolemia suggests that intestinal uptake of cholesterol is an unselective process, and that discrimination between cholesterol and plant sterols takes place at the level of sterol efflux from the enterocyte. Although plant sterols are structurally very similar to cholesterol, differing only in their side chain length, they are absorbed from the intestine to a markedly lower extent. In order to further evaluate the process of discrimination, three different sterols (cholesterol, campesterol, sitosterol) and their corresponding 5α-stanols (cholestanol, campestanol, sitostanol) were compared concerning their concentration in the proximal small intestine, in serum, and in bile after a single oral dose of deuterated compounds.The data obtained support the hypothesis that i) the uptake of sterols and stanols is an extremely rapid process, ii) discrimination probably takes place on the level of reverse transport back into the gut lumen, iii) plant stanols are taken up, but not absorbed to a measurable extent, and iv) the process of discrimination probably also exists at the level of biliary excretion. The range of structural alterations that decrease intestinal absorption and increase biliary excretion is: 1) campesterol, 2) cholestanol-sitosterol, and 3) campestanol-sitostanol.

The present study investigated the role of apolipoprotein E (apoE) phenotype on intestinal cholesterol absorption and cholesterol synthesis. Studies were carried out in eight subjects homozygous for the apoE4 and 12 subjects homozygous for the E2 allele (six normocholesterolemic volunteers and six patients with type III hyperlipoproteinemia). Cholesterol absorption did not differ between the three groups of subjects and averaged 38 ± 2% (mean ± SEM) in normolipemic E2/2, 37 ± 4% in type III hyperlipemic E2/2, and 41 ± 3% in E4/4 subjects, respectively. Dietary intake of fat and cholesterol had no influence on cholesterol absorption efficiency. A positive correlation between efficiency of cholesterol absorption and the ratio of campesterol to cholesterol in plasma, an indirect marker for cholesterol absorption, was observed after combining the results of the three groups (r = 0.504; P < 0.02). Bile acid and total cholesterol synthesis were also not affected by the different apoE alleles, but the well-known relationship between body weight and cholesterol synthesis was noticed (r = 0.574; P < 0.01).Thus, the present study provides evidence that the efficiency of intestinal absorption and synthesis of cholesterol in humans are not related to the apoE phenotype.

Cholesterol is implicated to play a role in Alzheimer disease pathology. Therefore, the concentrations of cholesterol, its precursors, and its degradation products in brain homogenates of aging wild-type and β-amyloid precursor protein transgenic mice carrying the Swedish mutation (APP23) were analyzed. Among the sterols measured, lanosterol is the first common intermediate of two different pathways, which use either desmosterol or lathosterol as the predominant precursors for de novo synthesis of brain cholesterol. In young mice, cholesterol is mainly synthesized via the desmosterol pathway, while in aged mice, lathosterol is the major precursor. 24S-hydroxycholesterol (cerebrosterol), which plays a key role in the removal of cholesterol from the brain, modestly increased during aging.No differences in the levels of cholesterol, its precursors, or its metabolites were found between wild-type and APP23 transgenic mice. Moreover, the levels of the exogenous plant sterols campesterol and sitosterol were significantly elevated in the brains of APP23 animals at age 12 and 18 months. This time point coincides with abundant plaque formation.

The inflammatory component of non-alcoholic steatohepatitis (NASH) can lead to irreversible liver damage. Therefore there is an urgent need to identify novel interventions to combat hepatic inflammation. In mice, omitting cholesterol from the diet reduced hepatic inflammation. Considering the effects of plant sterol/stanol esters on cholesterol metabolism, we hypothesized that plant sterol/stanol esters reduces hepatic inflammation. Indeed, adding plant sterol/stanol esters to a high-fat-diet reduced hepatic inflammation as indicated by immunohistochemical stainings and gene expression for inflammatory markers. Finally, adding sterol/stanol esters lowered hepatic concentrations of cholesterol precursors lathosterol and desmosterol in mice, which were highly elevated in the HFD group similarly as observed in severely obese patients with NASH. In vitro, in isolated LPS stimulated bone marrow derived macrophages desmosterol activated cholesterol efflux whereas sitostanol reduced inflammation. This highly interesting observation that plant sterol/stanol ester consumption leads to complete inhibition of HFD-induced liver inflammation opens new venues in the treatment and prevention of hepatic inflammation.