Cholesterol Precursors Research Articles

Free cholesterol, cholesterol precursor and plant sterol levels in atherosclerotic plaques are independently associated with symptomatic advanced carotid artery stenosis

Background and aimsCirculating sterols result either from cholesterol (CH) synthesis or intestinal uptake. They are mainly esterified and can be oxygenated. Sterols accumulate in atherosclerotic plaques whereby their clinical impact is uncertain. Here, we determined associations between circulating and plaque sterol levels in patients with advanced carotid artery stenosis in respect to a prior ischemic event and statin treatment. MethodsFree and esterified CH, CH precursors and plant sterols as well as oxysterols were quantified by liquid chromatography-tandem mass spectrometry in 63 consecutive patients undergoing carotid endarterectomy. ResultsCH, CH precursors, plant sterols and oxysterols accumulated in carotid artery plaques. Absolute circulating sterol levels were not predictive for their corresponding plaque levels. After normalisation to CH, plant sterol but not oxysterol levels correlated between plasma and plaques. Among the circulating sterols, oxysterols occurred proportionally less in plaques. Furthermore, CH and plant sterols were less esterified in plaques than in plasma.Patients who experienced a prior ischemic event (n = 29) and asymptomatic patients had, except for lanosterol, comparable circulating sterol levels. In contrast, the absolute plaque levels of free CH, CH precursors and plant sterols as well as oxysterols were increased in symptomatic compared to asymptomatic patients. These differences remained significant for free CH, precursors and 3 out of 4 analyzed plant sterols after adjustment to the most influencing covariates - statin treatment, type 2 diabetes and age. ConclusionsIncreased absolute plaque levels of free CH, precursors and plant sterols predict an ischemic event in patients with advanced carotid artery stenosis.

Phospholipids and cholesterol: Inducers of cancer multidrug resistance and therapeutic targets.

Lipids, phospholipids and cholesterol in particular, are the predominant components of the plasma membrane, wherein multidrug efflux transporters of the ATP-binding cassette (ABC) superfamily reside as integral pump proteins. In the current review, we discuss how lipids potently modulate the expression and activity of these multidrug efflux pumps, contributing to the development of the multidrug resistance (MDR) phenotype in cancer. The molecular mechanisms underlying this modulation of the MDR phenotype are pleiotropic. First, notwithstanding the high intra-and inter-tumor variability, MDR cells display an altered composition of plasma membrane phospholipids and glycosphingolipids, and are enriched with very long saturated fatty acid chains. This feature, along with the increased levels of cholesterol, decrease membrane fluidity, alter the spatial organization of membrane nano- and micro-domains, interact with transmembrane helices of ABC transporters, hence favoring drug binding and release. Second, MDR cells exhibit a peculiar membrane lipid composition of intracellular organelles including mitochondria and endoplasmic reticulum (ER). In this respect, they contain a lower amount of oxidizable fatty acids, hence being more resistant to oxidative stress and chemotherapy-induced apoptosis. Third, drug resistant cancer cells have a higher ratio of monosatured/polyunsatured fatty acids: this lipid signature reduces the production of reactive aldehydes with cytotoxic and pro-inflammatory activity and, together with the increased activity of anti-oxidant enzymes, limits the cellular damage induced by lipid peroxidation. Finally, specific precursors of phospholipids and cholesterol including ceramides and isoprenoids, are highly produced in MDR cells; by acting as second messengers, they trigger multiple signaling cascades that induce the transcription of drug efflux transporter genes and/or promote a metabolic reprogramming which supports the MDR phenotype. High-throughput lipidomics and computational biology technologies are a great tool in analyzing the tumor lipid signature in a personalized manner and in identifying novel biomarkers of drug resistance. Moreover, beyond the induction of MDR, lipid metabolism offers a remarkable opportunity to reverse MDR by using lipid analogues and repurposing lipid-targeting drugs (e.g. statins and aminobisphosphonates) that reprogram the lipid composition of drug resistant cells, hence rendering them drug sensitive.

Nedostatek vitaminu D a jeho zdravotní dopady

Vitamin D3 cholecalciferol is produced from its cholesterol precursors in the skin under the influence of ultraviolet calc. Its subsequent hydroxylation in the liver and kidneys leads to the formation of its most active metabolite calcitriol, which plays one of the key roles in the management of calcium phosphate metabolism. However, it also has the ability to regulate the function of a number of cells and tissues that express the vitamin D receptor. The most widespread method to evaluate the status of vitamin D in the body is to measure serum levels of its meta-bolite 25 hydroxyvitamin D - 25 (OH) D. Optimal values range between 75-125 nmoll / l. Its deficit is widespread in the human population worldwide and has a significant impact on the prevalence of metabolic bone diseases. Its deficiency may support the dysfunction of many other body systems. Ensuring optimal levels of vitamin D in the popula-tion is a challenge not only for health care and especially for government administration.

Estimating fetal cholesterol synthesis rates by cord blood analysis in intrauterine growth restriction and normally grown fetuses

BackgroundCholesterol 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.MethodsVenous 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.ResultsMean cholesterol levels were 25% lower in IUGR compared with CTRL (p < 0.0001). Lanosterol and lathosterol to cholesterol ratios were similar in IUGR and CTRL. In relation to cholesterol mean, desmosterol, 24S-hydroxycholesterol, and 27-hydroxycholesterol levels were higher by 30.0, 39.1 and 60.7%, respectively, in IUGR compared to CTRL (p < 0.0001). Equally, 5α-cholestanol, campesterol, and β-sitosterol to cholesterol ratios were higher in IUGR than in CTRL (17.2%, p < 0.004; 33.5%, p < 0.002; 29.3%, p < 0.021).ConclusionsCholesterol deficiency in IUGR is the result of diminished fetal de novo synthesis rates rather than diminished maternal supply. However, increased oxysterol- and phytosterol to cholesterol ratios suggest a lower sterol elimination rate. This is likely caused by a restricted hepatobiliary function. Understanding the fetal cholesterol metabolism is important, not only for neonatal nutrition, but also for the development of strategies to reduce the known risk of future cardiovascular diseases in the IUGR fetus.

Is 24(S)-hydroxycholesterol a potent modulator of cholesterol metabolism in Müller cells? An in vitro study about neuron to glia communication in the retina

Communication between neurons and glia plays a major role in nervous tissue homeostasis. It is thought to participate in tuning cholesterol metabolism to cellular demand, which is a critical issue for neuronal health. Cholesterol is a membrane lipid crucial for nervous tissue functioning, and perturbed regulation of its metabolism has been linked to several neurodegenerative disorders. In the brain, 24(S)-hydroxycholesterol (24S-OHC) is an oxysterol synthesized by neurons to eliminate cholesterol, and 24S-OHC has been shown to regulate cholesterol metabolism in astrocytes, glial cells which provide cholesterol to neurons. In the retina, 24S-OHC is also an elimination product of cholesterol produced by neurons, especially the retinal ganglion cells. However, it is not known whether Müller cells, the major macroglial cells of the retina, play the role of cholesterol provider for retinal neurons and whether they respond to 24S-OHC signaling, similarly to brain glial cells. In the present study, primary cultures of rat Müller cells were treated with 0, 0.5 or 1.5 μM 24S-OHC for 48 hours. The levels of cholesterol, precursors and oxysterols were quantified using gas chromatography coupled to flame-ionization detection or mass spectrometry. In addition, the expression of key genes related to cholesterol metabolism was analyzed using RTq-PCR. Müller cells were shown to express many genes linked to cholesterol metabolism, including genes coding for proteins implicated in cholesterol biosynthesis (HMGCR), cholesterol uptake and export via lipoproteins (LDL-R, SR-BI, ApoE and ABACA1) and regulation of cholesterol metabolism (SREBP2 and LXRβ). Cholesterol and several of its precursors and oxidative products were present. CYP27A1, the main retinal enzyme implicated in cholesterol elimination via oxysterol production, was quantified at low transcript levels but neither of its two typical products were detected in Müller cells. Furthermore, our results demonstrate that 24S-OHC has a strong hypocholesterolemic effect in Müller cells, leading to cholesterol depletion (-37 % at 1.5 μM). This was mediated by a decrease in cholesterol synthesis, as illustrated by reduced levels of cholesterol precursors: desmosterol (-38 % at 1.5 μM) and lathosterol (-84 % at 1.5 μM), and strong downregulation of HMGCR gene expression (2.4 fold decrease at 1.5μM). In addition, LDL-R and SR-BI gene expression were reduced in response to 24S-OHC treatment (2 fold and 1.6 fold at 1.5 μM, respectively), suggesting diminished lipoprotein uptake by the cells. On the contrary, there was a dramatic overexpression of ABCA1 transporter (10 fold increase at 1.5 μM), probably mediating an increase in cholesterol efflux. Finally, 24S-OHC induced a small but significant upregulation of the CYP27A1 gene. These data indicate that Müller cells possess the necessary cholesterol metabolism machinery and that they are able to sharply adjust their cholesterol metabolism in response to 24S-OHC, a signal molecule of neuronal cholesterol status. This suggests that Müller cells could be major players of cholesterol homeostasis in the retina via neuron-glia crosstalk.

Preliminary Investigations on the Health Benefits of Citrullus colocynthis (L.) Schrad Seeds

Aims: To evaluate the composition of Citrullus colocynthis (L.) Schrad seed as a means of assessing its health and possible therapeutic benefits.&#x0D; Study Design: Test-tube Lab Research.&#x0D; Place and Duration of Study: Federal Institute of Industrial Research, Oshodi, Nigeria, between June 2018 and March 2019.&#x0D; Methodology: Intact dehulled Citrullus colocynthis (L.) Schrad seeds were analyzed for their proximate and elemental content using standard methods and atomic absorption spectroscopy, respectively. The seed oil was extracted with n-hexane via cold maceration and the extracted oil was analyzed for its physiochemical properties. The fatty acid profile was determined using gas chromatography-mass spectrometry.&#x0D; Results: The proximate analysis values were determined to be 6.51%, 51.46%, 21.62%, 13.26%, 3.76% and 3.39% for the moisture, crude fat, crude protein, crude fiber, ash and total carbohydrate content respectively. Results of the elemental analysis show the seed contains; 3653.0322 mg/kg Na, 6639.7818 mg/kg K, 2329.0612 mg/kg Ca, 235.6057 mg/kg Fe, 5252.5884 mg/kg Mg, 27.9056 mg/kg Zn and 7.0068 mg/kg Pb. The predominant fatty acid detected with an area percentage of 20.31 was cis-11-octadecenoic acid. Other fatty acids detected include cis-9, cis-12-octadecadienoic acid, cis-9-octadecenoic, hexadecanoic acid, octadecanoic acid and icosanoic acid. Squalene, a biosynthetic precursor of cholesterol, was detected with an area percentage of 8.54.&#x0D; Conclusion: The evaluation of the compositional data provided evidential support for its beneficial health impact particularly in regards to nutritional and cardio-vascular health.

Lanosterol biosynthesis pathway (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Lanosterol is a precursor for cholesterol, which is synthesized primarily in the liver in a pathway often described as the mevalonate or HMG-CoA reductase pathway. The first two steps (formation of acetoacetyl CoA and the mitochondrial generation of (S)-3-hydroxy-3-methylglutaryl-CoA) are also associated with oxidation of fatty acids.

Octadecaneuropeptide (ODN) Induces N2a Cells Differentiation through a PKA/PLC/PKC/MEK/ERK-Dependent Pathway: Incidence on Peroxisome, Mitochondria, and Lipid Profiles.

Neurodegenerative diseases are characterized by oxidative stress, mitochondrial damage, and death of neuronal cells. To counteract such damage and to favor neurogenesis, neurotrophic factors could be used as therapeutic agents. Octadecaneuropeptide (ODN), produced by astrocytes, is a potent neuroprotective agent. In N2a cells, we studied the ability of ODN to promote neuronal differentiation. This parameter was evaluated by phase contrast microscopy, staining with crystal violet, cresyl blue, and Sulforhodamine 101. The effect of ODN on cell viability and mitochondrial activity was determined with fluorescein diacetate and DiOC6(3), respectively. The impact of ODN on the topography of mitochondria and peroxisomes, two tightly connected organelles involved in nerve cell functions and lipid metabolism, was evaluated by transmission electron microscopy and fluorescence microscopy: detection of mitochondria with MitoTracker Red, and peroxisome with an antibody directed against the ABCD3 peroxisomal transporter. The profiles in fatty acids, cholesterol, and cholesterol precursors were determined by gas chromatography, in some cases coupled with mass spectrometry. Treatment of N2a cells with ODN (10−14 M, 48 h) induces neurite outgrowth. ODN-induced neuronal differentiation was associated with modification of topographical distribution of mitochondria and peroxisomes throughout the neurites and did not affect cell viability and mitochondrial activity. The inhibition of ODN-induced N2a differentiation with H89, U73122, chelerythrine and U0126 supports the activation of a PKA/PLC/PKC/MEK/ERK-dependent signaling pathway. Although there is no difference in fatty acid profile between control and ODN-treated cells, the level of cholesterol and some of its precursors (lanosterol, desmosterol, lathosterol) was increased in ODN-treated cells. The ability of ODN to induce neuronal differentiation without cytotoxicity reinforces the interest for this neuropeptide with neurotrophic properties to overcome nerve cell damage in major neurodegenerative diseases.

A Tissue- and Temporal-Specific Autophagic Switch Controls Drosophila Pre-metamorphic Nutritional Checkpoints.

Properly timed production of steroid hormones by endocrine tissues regulates juvenile-to-adult transitions in both mammals (puberty) and holometabolous insects (metamorphosis). Nutritional conditions influence the temporal control of the transition, but the mechanisms responsible are ill defined. Here we demonstrate that autophagy acts as an endocrine organ-specific, nutritionally regulated gating mechanism to help ensure productive metamorphosis in Drosophila. Autophagy in the endocrine organ is specifically stimulated by nutrient restriction at the early, but not the late, third-instar larva stage. The timing of autophagy induction correlates with the nutritional checkpoints, which inhibit precocious metamorphosis during nutrient restriction in undersized larvae. Suppression of autophagy causes dysregulated pupariation of starved larvae, which leads to pupal lethality, whereas forced autophagy induction results in developmental delay/arrest in well-fed animals. Induction of autophagy disrupts production of the steroid hormone ecdysone at the time of pupariation not by destruction of hormone biosynthetic capacity but rather by limiting the availability of the steroid hormone precursor cholesterol in the endocrine cells via a lipophagy mechanism. Interestingly, autophagy in the endocrine organ functions by interacting with the endolysosome system, yet shows multiple features not fully consistent with a canonical autophagy process. Taken together, our findings demonstrate an autophagy mechanism in endocrine cells that helps shape the nutritional checkpoints and guarantee a successful juvenile-to-adult transition in animals confronting nutritional stress.

Altered Bile Transporter Expression and Cholesterol Metabolism in Children With Cholesterol and Pigment Gallstones.

We elucidated pathophysiology of pediatric gallstone disease by assessing liver expression of bile transporters in relation to bile acids and surrogates of cholesterol absorption and synthesis in serum and gallstones. RNA expression of canalicular bile transporters in liver biopsies from 32 pediatric gallstone patients and from 6 liver donors (controls) was measured by qRT-PCR (quantitative real-time reverse transcription polymerase chain reaction). Concentrations of cholesterol and precursors, plant sterols and bile acids in gallstones, and in serum of the patients and 82 healthy children were measured. Primary outcomes were the difference in RNA expressions and serum sterol profiles between patients and controls. Cholesterol stones (CS; n = 15) contained cholesterol >42% and pigment stones (PS; n = 17) <9% of weight. CS patients had markedly lower serum plant sterols (absorption) and higher cholesterol precursors (synthesis) than PS patients or healthy controls. CS contained several times more cholesterol precursors and less plant sterols relative to cholesterol than PS, which were enriched by primary bile acids (12-5.2-fold, P < 0.001). Liver RNA expression of ABCG5/G8 was similarly increased 2.5- to 1.8-fold (P < 0.002) in CS and PS patients, whereas PS patients had higher ABCB11 expression (P < 0.05). In PS bile acid concentration correlated with gallstone plant sterols (R = 0.83, P < 0.0001), and ABCG5 expression with ABCB11 expression (R = 0.27, P = 0.03). In CS, upregulation of ABCG5/G8 expression associates with low absorption and high gallstone content of cholesterol. In PS, activation of bile acid transport by ACBC11 interconnects with hepatic upregulation of ABCG5/G8 enriching PS with bile acids and plant sterols.

P3.25: INTESTINAL CHOLESTEROL ABSORPTION IS PRESERVED AFTER ISOLATED INTESTINAL TRANSPLANTATION DESPITE INCREASED CHOLESTEROL SYNTHESIS

Introduction. We aimed to investigate cholesterol metabolism in relation to nutritional status and serum lipids after isolated intestinal transplantation (ITX). Methods. Seven patients who had undergone isolated ITX including ascending colon at median age of 17.3 (range, 5.9–52) years due to intestinal failure caused by chronic intestinal pseudo obstruction (n=5) and total intestinal aganglionosis (n=2) were included median 3.5 (IQR, 2.9–7.4) years after ITX. Blood samples were analyzed for fat-soluble vitamins (A, E, D-25-OH), prealbumin, and cholesterol (total, LDL, HDL) and triglyceride levels. In six patients and 14 heathy age- and gender-matched controls, cholesterol metabolism was assessed with serum non-cholesterol sterols. The ratios to cholesterol (102 x µmol/mmol of cholesterol) of the serum cholesterol precursors (cholestenol, desmosterol and lathosterol) reflect cholesterol synthesis, while those of plant sterols (campesterol, sitosterol, and avenasterol) and cholestanol reflect cholesterol absorption efficiency. Lathosterol/cholestanol ratio reflects the balance between cholesterol absorption and synthesis and campesterol/cholestanol ratio reflects the plant sterol intake. Results. Patients were on tacrolimus-based maintenance immunosuppression without current signs of acute or chronic transplant rejection. All patients had weaned off PN median 39 days (IQR 21–215) after ITX and had a median body mass-index of 18.2 (IQR, 16.6–19.1) kg/m2. The levels of fat-soluble vitamins and prealbumin were mostly in normal range while plasma HDL cholesterol was low and triglyceride levels were increased in most patients (Table 1). Cholesterol precursor levels were significantly increased while plant sterols and cholestanol were not significantly different between patients and controls (Table 2). Increased lathosterol/cholestanol-ratio suggest that cholesterol synthesis markedly predominated absorption in patients’ whole body cholesterol homeostasis while the campesterol/cholestanol ratio suggest that the plant sterol intake was decreased in patients compared to controls (Table 2). Conclusion. Following isolated ITX, intestinal cholesterol absorption efficiency is preserved, while cholesterol synthesis increases likely to compensate malabsorption of bile acids. Satisfactory nutritional status, normal fat-soluble vitamin levels and preserved cholesterol absorption suggest that the compensatory increase was sufficient to maintain adequate lipid absorption.

Upregulation of Scavenger Receptor B1 Is Required for Steroidogenic and Nonsteroidogenic Cholesterol Metabolism in Prostate Cancer.

Aberrant cholesterol metabolism is increasingly appreciated to be essential for prostate cancer initiation and progression. Transcript expression of the high-density lipoprotein-cholesterol receptor scavenger receptor B1 (SR-B1) is elevated in primary prostate cancer. Hypothesizing that SR-B1 expression may help facilitate malignant transformation, we document increased SR-B1 protein and transcript expression in prostate cancer relative to normal prostate epithelium that persists in lethal castration-resistant prostate cancer (CRPC) metastasis. As intratumoral steroid synthesis from the precursor cholesterol can drive androgen receptor (AR) pathway activity in CRPC, we screened androgenic benign and cancer cell lines for sensitivity to SR-B1 antagonism. Benign cells were insensitive to SR-B1 antagonism, and cancer line sensitivity inversely correlated with expression levels of full-length and splice variant AR. In androgen-responsive CRPC cell model C4-2, SR-B1 antagonism suppressed cholesterol uptake, de novo steroidogenesis, and AR activity. SR-B1 antagonism also suppressed growth and viability and induced endoplasmic reticulum stress and autophagy. The inability of exogenous steroids to reverse these effects indicates that AR pathway activation is insufficient to overcome cytotoxic stress caused by a decrease in the availability of cholesterol. Furthermore, SR-B1 antagonism decreased cholesterol uptake, growth, and viability of the AR-null CRPC cell model PC-3, and the small-molecule SR-B1 antagonist block lipid transport-1 decreased xenograft growth rate despite poor pharmacologic properties. Overall, our findings show that SR-B1 is upregulated in primary and castration-resistant disease and is essential for cholesterol uptake needed to drive both steroidogenic and nonsteroidogenic biogenic pathways, thus implicating SR-B1 as a novel and potentially actionable target in CRPC. SIGNIFICANCE: These findings highlight SR-B1 as a potential target in primary and castration-resistant prostate cancer that is essential for cholesterol uptake needed to drive steroidogenic and nonsteroidogenic biogenic pathways.

Cholesterol Biosynthesis and Uptake in Developing Neurons.

Brain cholesterol biosynthesis, a separate and distinct process from whole-body cholesterol homeostasis, starts during embryonic development. To gain a better understanding of the neuronal and glial contributions to the brain cholesterol pool, we studied this process in control, Dhcr7-/-, and Dhcr24-/- cell cultures. Our LC-MS/MS method allowed us to measure several different sterol intermediates and cholesterol during neuronal differentiation. We found that developing cortical neurons rely on endogenous cholesterol synthesis and utilize ApoE-complexed cholesterol and sterol precursors from their surroundings. Both developing neurons and astrocytes release cholesterol into their local environment. Our studies also uncovered that developing neurons produced significantly higher amounts of cholesterol per cell than the astrocytes. Finally, we established that both neurons and astroglia preferentially use the Bloch sterol biosynthesis pathway, where desmosterol is the immediate precursor to cholesterol. Overall, our studies suggest that endogenous sterol synthesis in developing neurons is a critical and complexly regulated homeostatic process during brain development.

Alterations in Cholesterol Metabolism and Genetics as Key Players in Mild Cognitive Impairment (P15-023-19)

ObjectivesAlteration of circulating cholesterol precursors and oxidation products, in the form of sterols and oxysterols, has been demonstrated in Alzheimer’s disease (AD) progression and its preclinical stage, mild cognitive impairment (MCI). However, limited species of metabolites were detected with the methods that do not discriminate between free and esterified forms, the former of which is the biologically active form. This study was aimed to identify MCI-associated differences in cholesterol metabolism by detecting a broad range of cholesterol synthetic and metabolic profiles in free form and their interplay with genetic variations for cholesterol homeostasis. MethodsWe conducted a pilot case–control study in a well-characterized Chinese cohort entitled the Effects and Mechanism Investigation of Cholesterol and Oxysterol on Alzheimer’s disease (EMCOA) study including 100 MCI cases, each matched to one control participants. A broad range of non-esterified circulating cholesterol synthetic precursors and oxidation products were measured by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). A total of 21 tag single nucleotide polymorphisms (TagSNPs) in ApoE, CYP27A1, CYP46A1, CH25H, CYP7A1 and CYP7B1 was selected and genotyped by the Sequenom MassArray system. ResultsA total of 31 non-esterified cholesterol metabolites including 9 precursors and 22 oxysterols were detected in serum. We found significantly higher levels of 1 precursor and 11 oxysterols whereas lower levels of 3 oxysterols in MCI cases as compared to the controls. The random forest analysis showed that a biomarker panel of 20 lipids classified MCI patients with area under the curve (AUC) of 0.794 (95% CI = 0.694–0.868). With respect to SNPs, a QTL analysis identified 8 SNPs that were significantly associated with levels of 5 precursors and 14 oxysterols. ConclusionsThe findings indicate that cholesterol metabolism is disrupted in MCI patients and correlating their profiles with genotype may provide productive insight into cognitive dysfunction and lead to new treatment strategies. Funding SourcesThis work was supported by the State Key Program of the National Natural Science Foundation of China (Grant No. 81330065). Supporting Tables, Images and/or Graphs▪▪▪▪▪

Novel Approaches for Detection Fluorescent-Labeled by Cellvizio Lab System on Hippocampal CA1 Region

Neurosteroids have been identified in the 1981. Dehydroepiandrosterone sulphate (DHEAS) is one of the vital neurosteroids that de novo synthesized in the nervous system from cholesterol precursor (Baulieu &amp; Robel, 1998). The aim of the study is to develop a method for fluorescence labelling. Alexa Fluor 488 dye with DHEAS antibody can binds the DHEAS antibody in the rat brain monitored by Cellvizio Lab System. DHEAS antibody (IgG isotype antibodies) was fluorescently conjugated by an amine-reactive compound, Alexa Fluor 5-SDP ester 488 dye. The resultant Alexa Fluor 488-conjugated antibodies were collected and analyzed by UV-Vis spectrophotometer instrument. The absorbance of the protein-dye conjugate at 280 nm and 494 nm were measured. Then, the degree of labeling (DOL) was calculated to achieve the desired results. Fluorescence labelling were carried out into the CA1 region of hippocampus Sprague-Dawley rat. We reported that the conjugation was successful. Optimal labeling depending on degree of labeling (DOL) needs some necessity to achieve and effective binding to the target neurosteroid, DHEAS. Cellvizio Lab system connected with Fiber Fluorescence Microscopy (FFM) probe is presented as a new approach in real-time imaging of DHEAS. In conclusion, we have developed a new method of DHEAS-Alexa Fluor fluorescence labelling to visualize and evaluate the changes of DHEAS fluorescence level in the rat hippocampus. This novel approach as a diagnostic tool and can be used to better understand the mechanisms and functions of DHEAS and other neurosteroids in future research.

Striatal Cholesterol Precursors Are Altered with Age in Female Huntington's Disease Model Mice.

Cholesterol is necessary for proper neurodevelopment and neuronal health. The brain relies on neural and astrocytic de novo cholesterol synthesis. Huntington's disease presents with altered levels of cholesterol precursors however it is unknown when the disruption in this molecular pathway occurs and whether Manganese (Mn) may alter these metabolic alterations. To examine the effect of Mn exposure on cholesterol biosynthesis in pre-manifest and manifest Huntington's disease mice. 12-week (pre-manifest) male and female and 42-week old (manifest) female YAC128 and littermate control (WT) mice received 3 subcutaneous Mn or vehicle injections. Animals were sacrificed 24 hours after the final injection and striatum, cerebral cortex and cerebellum were collected to measure cholesterol and cholesterol precursors using LC/MS-MS. Striatal desmosterol and cholesterol are increased in pre-manifest HD females compared to age-matched WT female mice. Striatal lanosterol, 8-DHC and desmosterol and cholesterol are reduced in manifest HD females compared to age-and sex-matched WT mice with minimal effects in the cortex and cerebellum. Mn treatment had no effect in the pre-manifest or manifest female brain except reduced lanosterol levels in the cortex of pre-manifest female mice. Neither Mn or HD altered brain cholesterol precursor levels in the pre-manifest HD or WT male mouse. Cholesterol biosynthesis is impaired in early disease stage in female HD mice only and continues throughout disease. These alterations appear largely striatal-specific. Acute systemic exposure to Mn did not significantly alter cholesterol biosynthesis in the striatum at any disease stage.

Placental ESRRG-CYP19A1 Expressions and Circulating 17-Beta Estradiol in IUGR Pregnancies.

Introduction: Sex steroids are regulating factors for intrauterine growth. 17-β Estradiol (E2) is particularly critical to a physiological pregnancy, as increased maternal E2 was correlated to lower fetal weight at delivery. The placenta itself is a primary source of estrogens, synthetized from cholesterol precursors. Cytochrome P450 aromatase (encoded by CYP19A1 gene) is a rate-limiting enzyme for E2 biosynthesis. CYP19A1 transcription is supported by Estrogen Related-Receptor Gamma (ERRγ- ESRRG gene), which thus has an indirect role in placental steroidogenesis. Here we investigated maternal E2 levels and placental CYP19A1 and ESRRG expressions in pregnancies with IntraUterine Growth Restriction (IUGR).Methods: Singleton pregnancies were studied. E2 was measured in maternal plasma by electrochemiluminescence in 16 term controls and 11 IUGR (classified by umbilical artery doppler pulsatility index) at elective cesarean section, and also in 13 controls during pregnancy at a gestational age comparable to IUGR. CYP19A1 and ESRRG expressions were analyzed in placental tissue. Maternal/fetal characteristics, placental and molecular data were compared among study groups and tested for correlations.Results: Maternal E2 plasma concentrations were significantly decreased in IUGR compared to controls at delivery. When analyzing normal pregnancies at a gestational age similar to IUGR, E2 levels were not different to pathological cases. However, E2 levels at delivery positively correlated with placental efficiency. Placental CYP19A1 levels were significantly higher in IUGR placental tissue vs. controls, and specifically increased in female IUGR placentas. ESRRG expression was not different among groups.Discussion: We report a positive correlation between 17-β Estradiol levels and placental efficiency, that might indicate a disrupted steroidogenesis in IUGR pregnancies. Moreover, we show alterations of CYP19A1 expression in IUGR placentas, possibly indicating a compensatory effect to the adverse IUGR intrauterine environment, also depending on fetal sex. Further studies are needed to deeper investigate IUGR alterations in the complex interaction among molecules involved in placental steroidogenesis.

The isoflavones genistein and daidzein increase hepatic concentration of thyroid hormones and affect cholesterol metabolism in middle-aged male rats

We examined whether isoflavones interfere with thyroid homeostasis, increase hepatic thyroid hormone concentrations and affect cholesterol metabolism in middle-aged (MA) male rats. Thirteen-month-old Wistar rats were injected subcutaneously with 35 mg/kg b.w./day of genistein, daidzein or vehicle (controls) for four weeks. Hepatic Dio1 gene expression was up-regulated by 70% (p < 0.001 for both) and Dio1 enzyme activity increased by 64% after genistein (p < 0.001) and 73% after daidzein treatment (p < 0.0001). Hepatic T3 was 75% higher (p < 0.05 for both), while T4 increased only after genistein treatment. Serum T4 concentrations were 31% lower in genistein- and 49% lower in dadzein-treated rats (p < 0.001 for both) compared with controls. Hepatic Cyp7a1 gene expression was up-regulated by 40% after genistein and 32% after daidzein treatment (p < 0.05 for both), in agreement with a 7α-hydroxycholesterol increase of 50% (p < 0.01) and 88% (p < 0.001), respectively. Serum 24- and 27-hydroxycholesterol were 30% lower (p < 0.05 for both), while only 24-hydroxycholesterol was decreased in the liver by 45% after genistein (p < 0.05) and 39% (p < 0.01) after dadzein treatment. Serum concentration of the cholesterol precursor desmosterol was 32% (p < 0.05) lower only after dadzein treatment alone, while both isoflavones elevated this parameter in the liver by 45% (p < 0.01). In conclusion, isoflavones increased T3 availability in the liver of MA males, despite decreasing serum T4. Hepatic increase of T3 possibly contributes to activation of the neutral pathway of cholesterol degradation into bile acids in the liver. While isoflavones obviously have the potential to trigger multiple mechanisms involved in cholesterol metabolism and oxysterol production, they failed to induce any hypocholesterolemic effect.

A Tissue-Specific and Temporally-Regulated Autophagic Switch Controls a Drosophila Nutritional Checkpoint

Properly timed production of steroid hormones by endocrine tissues regulates juvenile-to-adult transitions in both mammals (puberty) and holometabolous insects (metamorphosis). Nutritional conditions influence the temporal control of the transition, but the mechanisms responsible are ill-defined. Here we demonstrate that autophagy acts as an endocrine organ-specific, nutritionally-regulated, gating mechanism to help ensure productive metamorphosis in Drosophila. Autophagy in the endocrine organ is specifically stimulated by nutrient restriction (NR) at the early, but not the late L3 stage, which suppresses precocious metamorphosis in undersized larvae. Induction of autophagy disrupts production of the steroid hormone ecdysone not by destruction of biosynthetic capacity, but rather by limiting the availability of the steroid hormone precursor cholesterol. Interestingly, autophagy functions by interacting with endo/lysosome system, yet shows multiple features not fully consistent with a canonical autophagy process. Taken together, our findings demonstrate an autophagy mechanism in endocrine cells that helps guarantee a successful juvenile-to-adult transition in animals confronting nutritional stress.

Autophagy-Mediated Cholesterol Trafficking Controls Steroid Production

Steroid hormones are important signaling molecules that regulate growth and drive the development of many cancers. These factors act as long-range signals that systemically regulate the growth of the entire organism, whereas the Hippo/Warts tumor-suppressor pathway acts locally to limit organ growth. We show here that autophagy, a pathway that mediates the degradation of cellular components, also controls steroid production. This process is regulated by Warts (in mammals, LATS1/2) signaling, via its effector microRNA bantam, in response to nutrients. Specifically, autophagy-mediated mobilization and trafficking of the steroid precursor cholesterol from intracellular stores controls the production of the Drosophila steroid ecdysone. Furthermore, we also show that bantam regulates this process via the ecdysone receptor and Tor signaling, identifying pathways through which bantam regulates autophagy and growth. The Warts pathway thus promotes nutrient-dependent systemic growth during development by autophagy-dependent steroid hormone regulation (ASHR). These findings uncover an autophagic trafficking mechanism that regulates steroid production.