Cholesterol Biosynthetic Pathway Research Articles

Multi-tissue, selective PPARγ modulation of insulin sensitivity and metabolic pathways in obese rats.

Peroxisome proliferator-activated receptor-γ (PPARγ) ligands, including the insulin-sensitizing thiazolidinedione drugs, transcriptionally regulate hundreds of genes. Little is known about the relationship between PPARγ ligand-specific modulation of cellular mechanisms and insulin sensitization. We characterized the insulin sensitivity and multitissue gene expression profiles of lean and insulin-resistant, obese Zucker rats untreated or treated with one of four PPARγ ligands (pioglitazone, rosiglitazone, troglitazone, and AG-035029). We analyzed the transcriptional profiles of adipose tissue, skeletal muscle, and liver from the rats and determined whether ligand treatment insulin-sensitizing potency was related to ligand treatment-induced alteration of functional pathways. Ligand treatments improved insulin sensitivity in obese rats to varying degrees. Adipose tissue profiles revealed ligand treatment-selective modulation of inflammatory and branched-chain amino acid (BCAA) metabolic pathways, which correlated with ligand treatment-specific insulin-sensitizing potency. Skeletal muscle profiles showed that obese rats exhibited elevated expression of adipocyte and slow-twitch fiber markers, which further increased after ligand treatment, but the magnitude of the treatment-induced changes was not correlated with insulin sensitization. Although PPARγ ligand treatments heterogeneously improved dysregulated expression of cholesterol and fatty acid biosynthetic pathways in obese rat liver, these alterations were not correlated with ligand insulin-sensitizing potency. PPARγ ligand treatment-specific insulin-sensitizing potency correlated with modulation of adipose tissue inflammatory and BCAA metabolic pathways, suggesting a functional relationship between these pathways and whole body insulin sensitivity. Other PPARγ ligand treatment-induced functional pathway changes were detected in adipose tissue, skeletal muscle, and liver profiles but were not related to degree of insulin sensitization.

Morelloflavone from Garcinia dulcis as a Novel Biflavonoid Inhibitor of HMG‐CoA Reductase

Morelloflavone, a biflavonoid from Garcinia dulcis previously shown to have hypocholesterolemic activity, was examined for its effect on HMG-CoA reductase, the rate-limiting enzyme of the cholesterol biosynthetic pathway. By using the catalytic domain of house mouse HMG-CoA reductase, morelloflavone was found to inhibit the enzyme activity by competing with HMG-CoA whereas it was non-competitive towards NADPH. The inhibition constants (K(i)) with respect to HMG-CoA and NADPH were 80.87 ± 0.06 µm and 103 ± 0.07 µm, respectively. Both flavonoid subunits of this compound, naringenin and luteolin, equally competed with HMG-CoA with K(i) of 83.58 ± 4.37 µm and 83.59 ± 0.94 µm, respectively, and were also non-competitive with NADPH (K(i) of 182 ± 0.67 µm and 188 ± 0.14 µm, respectively). Due to these findings, we suggest that each subunit of morelloflavone would occupy the active site of the enzyme, thereby blocking access of its substrate. The present study thus demonstrates the ability of morelloflavone from G. dulcis to inhibit HMG-CoA reductase in vitro. As a result, this biflavonoid might serve as a new candidate for the future development of hypocholesterolemic agents.

Lycopene induces cell growth inhibition by altering mevalonate pathway and Ras signaling in cancer cell lines

Several evidences suggest that cancer cells have abnormal cholesterol biosynthetic pathways and prenylation of small guanosine triphosphatase proteins. Tomato lycopene has been suggested to have beneficial effects against certain types of cancer, including that of prostate, although the exact molecular mechanism(s) is unknown. We tested the hypothesis that lycopene may exert its antitumor effects through changes in mevalonate pathway and in Ras activation. Incubation of the Ras-activated prostatic carcinoma LNCaP cells with a 24 h lycopene treatment (2.5-10 μM) dose dependently reduced intracellular total cholesterol by decreasing 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase expression and by inactivating Ras, as evidenced by its translocation from cell membranes to cytosol. Concomitantly, lycopene reduced the Ras-dependent activation of nuclear factor-kappaB (NF-κB). Such a reduction was parallel to an inhibition of reactive oxygen species production and to a decrease in the phosphorylation ofc-jun N-terminal kinase, extracellular signal-regulated kinase 1/2 and p38. These effects were also accompanied by an arrest of cell cycle progression and by apoptosis induction, as evidenced by a decrease in cyclin D1 and phospho-AKT levels and by an increase in p21, p27 and p53 levels and in Bax:Bcl-2 ratio. The addition of mevalonate prevented the growth-inhibitory effects of lycopene as well as its increase in Ras cytoplasmatic accumulation and the subsequent changes in NF-κB. The ability of lycopene in inhibiting HMG-CoA reductase expression and cell growth and in inactivating Ras was also found in prostate PC-3, colon HCT-116 and HT-29 and lung BEN cancer cells. These findings provide a novel mechanistic insight into the growth-inhibitory effects of lycopene in cancer.

Down-regulation of the global regulator SATB1 by statins in COLO205 colon cancer cells

Special AT-rich sequence binding protein 1 (SATB1) regulates the expression of more than 1,000 genes in tumor cells. SATB1 expression has been implicated in metastasis, and its silencing results in reduced cancer progression and the reversion of metastatic cells to normal appearance. Therefore, any compound causing down-regulation of SATB1 expression or activity may be exploited for its therapeutic potential in terms of cancer regression. Earlier studies showed that the 3-hydroxy-3-methylglutaryl coenzymeA (HMG-CoA) reductase inhibitors (statin drugs), which are widely used to treat hypercholesterolemia, possess other pleotropic activities. These are now increasingly gaining attention for their cancer prevention abilities. However, the downstream interplay of the molecular mechanisms of such anti-cancer activities is unclear. Here, we show that SATB1 is down-regulated by statins in a time- and dose-dependent manner in COLO205 cells. This effect was statin-specific as the down-regulation of SATB1 was brought about by hydrophobic statins, such as simvastatin and fluvastatin, but not by hydrophilic pravastatin. Notably, treatment with mevalonate, an intermediate in the cholesterol and isoprenoid biosynthetic pathways, led to the inhibition of SATB1 down-regulation and cytotoxicity mediated by statins. Treatment with the proteasome inhibitors lactacystine and MG-132 inhibited the statin-mediated down-regulation of SATB1, suggesting that regulation occurs at the post-translational level. Thus, our results demonstrate a novel molecular mechanism for the anti-cancer activity of statin drugs in colon cancer cells, without invoking significant cytotoxicity.

The roles played by Aspergillus nidulans apoptosis-inducing factor (AIF)-like mitochondrial oxidoreductase (AifA) and NADH-ubiquinone oxidoreductases (NdeA-B and NdiA) in farnesol resistance

Farnesol (FOH) is a nonsterol isoprenoid produced by dephosphorylation of farnesyl pyrophosphate, a catabolite of the cholesterol biosynthetic pathway. These isoprenoids inhibit proliferation and induce apoptosis. Here, we show that Aspergillus nidulans AifA encoding the apoptosis-inducing factor (AIF)-like mitochondrial oxidoreductase plays a role in the function of the mitochondrial Complex I. Additionally, we demonstrated that ndeA-B and ndiA encode external and internal alternative NADH dehydrogenases, respectively, that have a function in FOH resistance. When exposed to FOH, the ΔaifA and ΔndeA strains have increased ROS production while ΔndeB, ΔndeA ΔndeB, and ΔndiA mutant strains showed the same ROS accumulation than in the absence of FOH. We observed several compensatory mechanisms affecting the differential survival of these mutants to FOH.

Quantitative Proteomics Analysis of Inborn Errors of Cholesterol Synthesis: Identification of Altered Metabolic Pathways in DHCR7 and SC5D Deficiency

Smith-Lemli-Opitz syndrome (SLOS) and lathosterolosis are malformation syndromes with cognitive deficits caused by mutations of 7-dehydrocholesterol reductase (DHCR7) and lathosterol 5-desaturase (SC5D), respectively. DHCR7 encodes the last enzyme in the Kandutsch-Russel cholesterol biosynthetic pathway, and impaired DHCR7 activity leads to a deficiency of cholesterol and an accumulation of 7-dehydrocholesterol. SC5D catalyzes the synthesis of 7-dehydrocholesterol from lathosterol. Impaired SC5D activity leads to a similar deficiency of cholesterol but an accumulation of lathosterol. Although the genetic and biochemical causes underlying both syndromes are known, the pathophysiological processes leading to the developmental defects remain unclear. To study the pathophysiological mechanisms underlying SLOS and lathosterolosis neurological symptoms, we performed quantitative proteomics analysis of SLOS and lathosterolosis mouse brain tissue and identified multiple biological pathways affected in Dhcr7(Delta3-5/Delta3-5) and Sc5d(-/-) E18.5 embryos. These include alterations in mevalonate metabolism, apoptosis, glycolysis, oxidative stress, protein biosynthesis, intracellular trafficking, and cytoskeleton. Comparison of proteome alterations in both Dhcr7(Delta3-5/Delta3-5) and Sc5d(-/-) brain tissues helps elucidate whether perturbed protein expression was due to decreased cholesterol or a toxic effect of sterol precursors. Validation of the proteomics results confirmed increased expression of isoprenoid and cholesterol synthetic enzymes. This alteration of isoprenoid synthesis may underlie the altered posttranslational modification of Rab7, a small GTPase that is functionally dependent on prenylation with geranylgeranyl, that we identified and validated in this study. These data suggested that although cholesterol synthesis is impaired in both Dhcr7(Delta3-5/Delta3-5) and Sc5d(-/-) embryonic brain tissues the synthesis of nonsterol isoprenoids may be increased and thus contribute to SLOS and lathosterolosis pathology. This proteomics study has provided insight into the pathophysiological mechanisms of SLOS and lathosterolosis, and understanding these pathophysiological changes will help guide clinical therapy for SLOS and lathosterolosis.

Abstract 36: Human ecdysoneless protein: Regulator of cholesterol homeostasis in pancreatic cancer

Abstract The human Ecdysoneless protein (hEcd) is a human orthologue of the D.melanogaster ecdysoneless protein, which is highly conserved from insects to mammals and identified as one of the factors regulating the synthesis of the steroid hormone ecdysone. Ecd is required autonomously throughout the body and its deficiency can lead to reproductive or developmental defects. hEcd, also known as human suppressor of GCR2 gene (hSGT) since it can rescue growth defects in S.cerevisiae mutants with a deletion of the growth control regulatory genes 2 (GCR2), a transcriptional activator of the glycolytic gene expression. Recent investigations revealed that hEcd interacts with murine double minute-2 and stabilizes p53 by inhibiting mdm-2 mediated degradation of p53. Another study done on MEFs generated from Ecd conditional knockout mice showed evidence that Ecd interacts with retinoblastoma protein at the pocket domain and competes with E2F for association with hypophosphorylated Rb, thereby regulating E2F target gene expression and cell cycle progression. In this study, we have analyzed the expression of Ecd in PanIN and Pancreatic adenocarcinoma tissues as well as pancreatic cancer cell lines. The studies show that Ecd is overexpressed in pancreatic cancer samples as compared to normal pancreas. Therefore based on its role as a cell cycle regulator and its increased expression in pancreatic tumors, we hypothesized that Ecd might act as a potential modulator contributing to the pathogenesis of pancreatic cancer cells. Stable knockdown clones of Ecd were established in CD/HPAF pancreatic cancer cell line using two different shRNA constructs, which led to reduced proliferation rate in vitro and decreased tumor weight in vivo. Further, microarray analysis of genes in Ecd knocked-down vs scrambled cells followed by bioinformatics analysis revealed that the major pathway affected due to Ecd downregulation is the cholesterol biosynthetic and regulatory pathway. The genes which were affected by more than two-fold in Ecd knocked-down cells, encoded key cholesterol biosynthetic pathway enzymes such as FDFT1, NSDHL, DHCR7 and CYP51A1 along with INSIG1, a protein which acts as a cholesterol level sensor in cells. This was further confirmed by Quantitative Real-Time PCR analysis. Flow-cytometric analysis for cholesterol-specific filipin staining showed increased cholesterol content in Ecd-kd cells vs the scrambled cells. Additionally, insulin stimulated GLUT-4 membrane translocation, one of the cholesterol-dependent pathways was also found to be affected with Ecd knockdown cells showing higher glucose uptake than the scrambled cells. Cholesterol being a major component of cells and a mediator of endocytic and cell signaling pathways, we conclude that Ecd contributes to pancreatic cancer pathogenesis by disrupting multiple cholesterol-dependent pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 36.

Age-Related Hypercholesterolemia and HMG-CoA Reductase Dysregulation: Sex Does Matter (A Gender Perspective)

Although cardiovascular diseases are less prevalent in premenopausal women than in men, their occurrence in women increases at the onset of menopause, and the loss of female sex hormones contributes to the striking increase in cardiovascular morbidity and mortality in postmenopausal women. We present here a description of age-related disruption of lipid homeostasis, which particularly affects 3-hydroxy 3-methylglutaryl Coenzyme A reductase, the key rate-limiting enzyme in the cholesterol biosynthetic pathway. We further discuss the age- and gender-related dysregulation of this enzyme, providing new evidence for the different mechanisms driving dyslipidemia in elderly men and women. In addition, we introduce pharmacological methods of regulating HMGR and maintaining cholesterol homeostasis.

Simvastatin enhances hippocampal long-term potentiation in C57BL/6 mice

Statins inhibit 3-hydroxy-3-methylglutaryl CoA reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway, and they are widely used to control plasma cholesterol levels and prevent cardiovascular disease. However, emerging evidence indicates that the beneficial effects of statins extend to the CNS. Statins have been shown to improve the outcome of stroke and traumatic brain injury, and statin use has been associated with a reduced prevalence of Alzheimer's disease (AD) and dementia. However, prospective studies with statins in AD have produced mixed results. Recently, we reported that simvastatin, a widely used statin in humans, enhances learning and memory in non-transgenic mice as well as in transgenic mice with AD-like pathology on a mixed genetic background. However, the cellular and molecular mechanisms underlying the beneficial effects of simvastatin on learning and memory remain elusive. The present study was undertaken to investigate the effect of acute simvastatin treatment on hippocampal long-term potentiation (LTP), a cellular model of learning and memory, in brain slices from C57BL/6 mice. Our results demonstrate that a prolonged in vitro simvastatin treatment for 2–4 h, but not a short-term 20-min exposure, significantly increases the magnitude of LTP at CA3–CA1 synapses without altering basal synaptic transmission or the paired-pulse facilitation ratio in hippocampal slices. Furthermore, we show that phosphorylation of Akt (protein kinase B) is increased significantly in the CA1 region following 2-hour treatment with simvastatin, and that inhibition of Akt phosphorylation suppresses the simvastatin-induced enhancement of LTP. These findings suggest activation of Akt as a molecular pathway for augmented hippocampal LTP by simvastatin treatment, and implicate enhancement of hippocampal LTP as a potential cellular mechanism underlying the beneficial effects of simvastatin on cognitive function.

Changes in cholesterol biosynthetic and transport pathways after excitotoxicity

The present study was carried out to elucidate changes in the gene expression and activity of cholesterol biosynthetic enzymes and transporters in the rat hippocampus after kainate excitotoxicity. Significantly increased cholesterol level was detected in the degenerating hippocampus, reaching double normal levels at 1 week after kainate injury. RT-PCR analyses of hippocampal homogenates showed significantly decreased mRNA expression of the transcription factor controlling cholesterol biosynthesis SREBP-2, and the rate-controlling enzyme HMG-CoA (3-hydroxy-3-methyl-glutaryl-CoA) reductase at all time points after kainate injection; and decreased lanosterol synthase and CYP51 at 1 and 2 weeks post-kainate injection respectively. GC-MS analyses showed a significant increase in cholesterol biosynthetic precursors lanosterol, desmosterol and 7-dehydrocholesterol at 1 day after kainate injection presumably reflecting biosysnthesis in injured neurons, and significant decreases in precursors at 1 and 2 weeks post-kainate injection, at time of gliosis in the degenerating hippocampus. Levels of cholesterol autooxidation including 7 ketocholesterol and cholesterol epoxides were elevated in the kainate lesioned hippocampus. Furthermore, loss of expression of the cholesterol transporter, ABCA1 was detected in neurons, but increased expression in astrocytes was detected after kainate lesions. The results suggest that increased cholesterol biosynthesis and loss of ABCA1 expression in injured neurons might result in increase in cholesterol in the degenerating hippocampus. The increased cholesterol might predispose to increased formation of cholesterol oxidation products which have been shown to be toxic to neurons.

Hypercholesterolemia and 3-hydroxy-3-methylglutaryl coenzyme A reductase regulation in aged female rats

Coronary heart disease is less prevalent in pre-menopausal women than in men, but increases at the onset of menopause. This delay is due to estrogen protective effects. The rise of cholesterolemia is one of the main risk factors for coronary disease. Since 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the rate-limiting enzyme of the cholesterol biosynthetic pathway, it plays a pivotal role in cholesterol homeostasis maintenance. Aim of this study is to investigate whether HMGR is involved in the cholesterolemia increase that occurs during aging, and to consider its potential role as a target for estrogen protective effects. “In vivo” studies have been performed using the livers of 12-month-old female rats (whose estrogen level decrease is comparable to the one detected at the occurrence of human menopause), 12-month-old female rats treated with 17-β-estradiol, and 3-month-old untreated male and female rats. The results indicated hypercholesterolemic status and a significant increase of HMGR activity according to a reduced activation of AMPK detected in treated rats compared to controls. Furthermore, 17-β estradiol treatment reduced HMGR activity restoring AMPK activation. These findings highlight the correlation between estrogen and HMGR short-term regulation, and suggest the presence of another mechanism underlying the protective role of estrogen in age-related diseases.

PCSK9 and heart disease: quieting an outdated metabolic moderator

vesicles [4]. Within early endosomes, acidification causes dissociation of the receptor–ligand complex, after which the LDLR recycles to the cell surface. The internalized LDL particles are degraded in lysosomes and liberated cholesterol is then used for the synthesis of membranes, steroid hormones, lipoproteins and bile acids. This process is subject to a negative-feedback mechanism that maintains tight control of cellular cholesterol levels [5]. Cholesterol influx results in transcriptional suppression of genes encoding cholesterol biosynthetic enzymes and LDLR, thus preventing potentially toxic cholesterol overaccumulation. Conversely, when cholesterol levels are depleted the expression of these same genes is stimulated. Cholesterol-lowering statin drugs modulate this regulatory circuit by directly inhibiting HMG-CoA reductase, the rate-controlling enzyme in the cholesterol biosynthetic pathway. I-LDL turnover studies in humans demonstrated an increased clearance rate of LDL from circulation following statin therapy, indicating increased liver LDLR function as a secondary beneficial effect of statins [6,7].

Neuroprotective Effects of Statins in an In Vitro Model of Alzheimer's Disease

Statins, used as cholesterol-lowering drugs, were reported to reduce the progression of Alzheimer's disease (AD). However, the molecular mechanisms underlying these findings remain to be clarified and it is not well understood whether this beneficial effect is due to simply lowering cholesterol levels. This study was aimed to investigate the neuroprotective effect of simvastatin and lovastatin, lipophilic statins that can transverse the blood brain barrier, against the toxicity triggered by the AD-associated amyloid-beta (Abeta) peptides and to analyze if such protection is cholesterol-independent. Using primary cultures of cortical neurons treated with Abeta1-40 peptide, we have demonstrated that pre-incubation with statins prevents the rise in cytosolic Ca2+ concentration and the accumulation of reactive oxygen species induced by Abeta through mechanisms independent of cholesterol reduction. The neuroprotective actions of statins were rather attributable to their ability to reduce isoprenyl intermediates levels in the cholesterol biosynthetic pathway since their effect was reversed by geranyl pyrophosphate while cholesterol addition was ineffective. Consequently, statins were shown to rescue cortical neurons from Abeta-40-induced caspase-3-dependent apoptosis. Moreover, our results revealed that simvastatin, at neuroprotective concentrations against Abeta-induced toxicity, is not able to activate Akt or ERK2, two signaling kinases with neuroprotective roles against apoptosis.

Cholesterol 24-Hydroxylase: An Enzyme of Cholesterol Turnover in the Brain

Cholesterol 24-hydroxylase is a highly conserved cytochrome P450 that is responsible for the majority of cholesterol turnover in the vertebrate central nervous system. The enzyme is expressed in neurons, including hippocampal and cortical neurons that are important for learning and memory formation. Disruption of the cholesterol 24-hydroxylase gene in the mouse reduces both cholesterol turnover and synthesis in the brain but does not alter steady-state levels of cholesterol in the tissue. The decline in synthesis reduces the flow of metabolites through the cholesterol biosynthetic pathway, of which one, geranylgeraniol diphosphate, is required for learning in the whole animal and for synaptic plasticity in vitro. This review focuses on how the link between cholesterol metabolism and higher-order brain function was experimentally established.

The Antifungal Agent Itraconazole Induces the Accumulation of High Mannose Glycoproteins in Macrophages

Bacterial lipopolysaccharide (LPS) is a key mediator in the development of Gram-negative septic shock, which is a major health problem. The effect of LPS on myeloid cells is mediated by a multicomplex receptor system in which CD14, a glycosylphosphatidylinositol-anchored glycoprotein, and Toll-like receptor 4 are the major players. We have found that incubation of macrophages with itraconazole (ICZ), an azole antifungal commonly used in humans, altered both the expression and glycosylation of CD14. This glycoprotein, which is endo H-resistant in untreated cells, becomes endo H-sensitive following ICZ treatment. The effect of ICZ on glycan processing was observed in all newly synthesized glycoproteins as indicated by incorporation of [2-(3)H]mannose. In addition, cells treated with ICZ increased surface concanavalin A (ConA) binding, corroborating an increase in high mannose surface glycoproteins. Although the glycosylation pattern of CD14 was altered, this glycoprotein was delivered to the cell surface or was secreted. Moreover, it appeared functional as demonstrated by the release of LPS-induced tumor necrosis factor-alpha under conditions specific for a CD14-mediated activation process. The effect of ICZ on glycosylation was not dependent on inhibition of the cholesterol biosynthetic pathway and was specific for this drug because other azole antifungals, such as ketoconazole and econazole, did not alter glycan processing. These results suggest a possible secondary effect of ICZ that impacts the processing of glyconjugates and may alter cellular function and homeostasis.

Abstract: P718 CLINICAL DOSE OF PITAVASTATIN PREVENTS FOAM CELL FORMATION IN RAW264.7 MACROPHAGES BY OXIDIZED LOW DENSITY LIPOPROTEIN

Biosynthesis of the isoprenols, geraniol, farnesol, nerolidol, and geranylgeraniol, from [2-14C]mevalonic acid is described in the fly, Sarcophaga bullata. Each was identified by thin-layer and gas chromatographic, infra-red spectroscopic, and mass spectrometric analyses.These isoprenols underwent structural rearrangement when acid hydrolysis was used to isolate them from their pyrophosphates. Most of the farnesyl pyrophosphate was converted to nerolidol, while only a fraction of the geranyl pyrophosphate was changed to its isomers. Since farnesol is the major isoprenol product of the in vivo incubation, we conclude that this insect is capable of isoprenoid biosynthesis at least to this point, and that the block in the cholesterol biosynthetic pathway lies beyond.

Effects of lanosterol on in vitro maturation of porcine oocytes

FF-MAS and T-MAS sterols, intermediaries in the cholesterol biosynthetic pathway present in all cells, may represent the physiological signal that instructs the oocyte to reinitiate meiosis. The purpose of this study was to examine the hypothesis that exogenous lanosterol could be included in the sterol biosynthetic pathway from acetate to cholesterol and induce resumption of meiosis in oocytes cultured in vitro. Porcine oocytes were in vitro matured in medium supplemented with different concentrations of lanosterol. First, after 22 h of in vitro maturation, cumulus cells were recovery and Δ7-Reductase gene expression was quantified using real-time PCR. Second, after 44 h of in vitro maturation, chromatin configuration was evaluated using Hoechst 33342. Lipid content was also evaluated at 22 and 44 h of in vitro maturation using Nile red staining. The results showed that the addition of lanosterol increased the Δ7-Reductase gene expression and influenced resumption of meiosis using 50 and 100 μM, as well as enhancing higher cytoplasmic lipid accumulation after in vitro maturation. The results demonstrate that exogenous lanosterol acts in the sterol biosynthetic pathway from acetate to cholesterol and it was responsible for a higher resumption of meiosis in porcine oocytes cultured in vitro.

Cholesterol Metabolism: the Main Pathway Acting Downstream of Cytochrome P450 Oxidoreductase in Skeletal Development of the Limb

Cytochrome P450 oxidoreductase (POR) is the obligate electron donor for all microsomal cytochrome P450 enzymes, which catalyze the metabolism of a wide spectrum of xenobiotic and endobiotic compounds. Point mutations in POR have been found recently in patients with Antley-Bixler-like syndrome, which includes limb skeletal defects. In order to study P450 function during limb and skeletal development, we deleted POR specifically in mouse limb bud mesenchyme. Forelimbs and hind limbs in conditional knockout (CKO) mice were short with thin skeletal elements and fused joints. POR deletion occurred earlier in forelimbs than in hind limbs, leading additionally to soft tissue syndactyly and loss of wrist elements and phalanges due to changes in growth, cell death, and skeletal segmentation. Transcriptional analysis of E12.5 mouse forelimb buds demonstrated the expression of P450s involved in retinoic acid, cholesterol, and arachidonic acid metabolism. Biochemical analysis of CKO limbs confirmed retinoic acid excess. In CKO limbs, expression of genes throughout the whole cholesterol biosynthetic pathway was upregulated, and cholesterol deficiency can explain most aspects of the phenotype. Thus, cellular POR-dependent cholesterol synthesis is essential during limb and skeletal development. Modulation of P450 activity could contribute to susceptibility of the embryo and developing organs to teratogenesis.

M1723 Highly Sensitive Metabolome Analysis of Cholesterol and Bile Acid Biosynthetic Pathways By LC-ESI-MS/MS

Background: Alcoholic chronic liver disease (ACLD) is one of the most common forms of acquired immunodeficiency. Aim: To evaluate ex vivo toll-like receptor (TLR) 2 and TLR4 innate immune response in stable ACLD. Methods: Blood was collected from 26 males with ACLD in an outpatient hepatology clinic and from 10 controls. Serum was used for lipopolysaccharide (LPS), sCD14, LPS-binding protein(LBP), tumour necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) quantification. mRNA expression of TLR2, TLR4, MD2, CD14, TNF-α and IL-10 was evaluated in anti-CD11b positive selected monocytes. Monocyte primary cultures were stimulated with zymosan (TLR2 agonist) or LPS (TLR4 agonist), and TNF-α production analyzed. Results: ACLD patients presented increased circulating LPS (+22.5±4.1%), LBP (+60.6±12.2%) and sCD14 (+23.5±4.6%), but with no differences in TNF-α and IL-10. TNF-αmRNA expression was decreased in monocytes from ACLD patients (-50.1±8.0%), with no significant differences in the other studied genes. Zymosan, but not LPS, induced TNF-α production by monocytes was blunted in ACLD (-55.2±10.9%). Results were similar in Child-Pugh A and B/C patients. See figure *=p<0.05 vs control group; #= p<0.05 vs production at time 0h. Conclusions: Patients with ACLD present an attenuation of TLR2-mediated innate immune response. This was not related with decreased monocyte TLR2 expression or impaired TLR4 signaling pathways, being already present in the early stages of disease. Attenuation of TLR2 activation may constitute a mechanism for acquired immunodeficiency.

Sterol regulatory element binding proteins (SREBPs): controllers of lipid synthesis and cellular uptake.

Mammalian cells use an exquisitely sensitive mechanism to control the amount of cholesterol and fatty acids in their membranes. This process relies on a feedback system that adjusts the rates of transcription of genes encoding the low density lipoprotein receptor and multiple enzymes in the cholesterol and fatty acid biosynthetic pathways. When cellular cholesterol levels are depleted, these genes are all transcribed in abundant amounts, and their transcription is repressed when sterols build up within the cell. Until recently, the mechanism of this regulation was elusive. How do cells sense the level of a membrane-embedded lipid such as cholesterol and how is this information transmitted to the nucleus where gene transcription is regulated? Answers are now beginning to emerge from the study of a newly discovered family of transcription-regulating proteins called sterol regulatory element binding proteins.