cholesterol synthesis

The brain contains a quarter of all cholesterol in the body, but has no access to cholesterol supplied by synthesis in the liver or by food absorption in the intestine . Therefore, cells in the brain rely entirely on cholesterol synthesis, a resource-intense process that requires energy, molecular oxygen and 21 enzymes . Previous studies indicate that neurons have a particularly high demand for cholesterol to form and maintain axons ), dendrites (Fan et al. 2002;) and synaptic connections . It has been estimated that neurons renew 20% of their cholesterol content daily (Dietschy and Turley 2004). So far, it is unknown, how these highly specialized cells meet their need for this essential lipid (Pfrieger 2003a). This question is of clinical importance, as deficits in cholesterol synthesis or transport cause neurodegeneration and severe neurological symptoms. Here, we compared cholesterol synthesis in neurons and different types of glial cells. Currently, this topic cannot be addressed in vivo, therefore we used serum-free primary cultures of immunoisolated cells. Our results reveal distinct precursor profiles and less efficient cholesterol synthesis in neurons compared to astrocytes suggesting that neurons delegate this costly metabolic pathway to astrocytes. Seven-day-old Wistar rats (animal facility, l'IFR des Neurosciences, Strasbourg, France) were decapitated according to institutional guidelines. Retinal ganglion cells (RGCs) and cerebellar neurons were purified by sequential immunopanning as described (Barres

Neurons have a high demand for cholesterol to develop and maintain membrane‐rich structures like axons, dendrites and synapses, but it remains unclear, whether they can satisfy their need by costly de novo synthesis. To address this, we compared cholesterol synthesis in serum‐free cultures of highly purified CNS neurons and glial cells from postnatal rats. We observed marked cell‐specific differences: Compared with glial cells, neurons showed different profiles of biosynthetic enzymes, post‐squalene precursors and cholesterol metabolites, and they produced cholesterol less efficiently, possibly because of very low levels of lanosterol‐converting enzymes. Astrocytes responded to inhibition of cholesterol synthesis with a much stronger up‐regulation of biosynthetic enzymes than neurons. Our results support the idea that neurons cannot produce cholesterol efficiently and that they depend on an external source of this lipid.

A s bstract. Measurement of mevalonic acid (MVA) concentrations in plasma or 24-h urine samples is shown to be useful in studies of the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and cholesterol synthesis. Plasma MVA con- centrations, measured either at 7-9 a.m. after an over- night fast, or throughout the 24-h cycle, were compared with cholesterol synthesis rates that were measured by the sterol balance method: plasma MVA concentrations were directly related to the rate of whole body cholesterol synthesis (r = 0.972; p < 0.001; n = 18) over a tenfold range of cholesterol synthesis rates. Moreover, hourly examination of MVA concentrations throughout the day demonstrated that interventions such as fasting or cholesterol feeding cause suppression of the postmidnight diurnal rise in plasma MVA concentrations, with little change in the base-line of the rhythm. Thus, the daily rise and fall of plasma MVA appears to reflect changes in tissues and organs, such as the liver and intestine, that are known to be most sensitive to regulation by fasting or by dietary cholesterol. The hypothesis that short-term regulation of HMG- CoA reductase in tissues is quickly reflected by corre- sponding variations in plasma MVA was tested by using a specific inhibitor of HMG-CoA reductase, mevinolin, to block MVA synthesis. Mevinolin caused a dose- dependent lowering of plasma MVA after a single dose; and in patients who received the drug twice a day for 4 wk, it decreased 24-h urinary MVA output. Significant

The expansion of a polyglutamine tract in the ubiquitously expressed huntingtin protein causes Huntington&#39;s disease (HD), a dominantly inherited neurodegenerative disease. We show that the activity of the cholesterol biosynthetic pathway is altered in HD. In particular, the transcription of key genes of the cholesterol biosynthetic pathway is severely affectedin vivoin brain tissue from HD mice and in human postmortem striatal and cortical tissue; this molecular dysfunction is biologically relevant because cholesterol biosynthesis is reduced in cultured human HD cells, and total cholesterol mass is significantly decreased in the CNS of HD mice and in brain-derived ST14A cells in which the expression of mutant huntingtin has been turned on. The transcription of the genes of the cholesterol biosynthetic pathway is regulated via the activity of sterol regulatory element-binding proteins (SREBPs), and we found an ∼50% reduction in the amount of the active nuclear form of SREBP in HD...

SR-12813 (tetra-ethyl 2-(3,5-di-tert-butyl-4-hydroxyphenyl)ethenyl-1,1-bisphosphonate) lowers plasma cholesterol in five species. In this paper we investigate the underlying mechanism using Hep G2 cells. SR-12813 inhibited incorporation of tritiated water into cholesterol with an IC 50 of 1.2 M but had no effect on fatty acid synthesis. Furthermore, SR-12813 reduced cellular 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity with an IC 50 of 0.85 M. The inhibition of HMG-CoA reductase activity was rapid with a T1 ⁄2 of 10 min. After a 16-h incubation with SR-12813, mRNA levels of HMG-CoA reductase and low density lipoprotein (LDL) receptor were increased. The increased expression of LDL receptor translated into a higher LDL uptake, which can explain the primary hypocholesterolemic effect of SR-12813 in vivo. Western blot analysis indicated that the amount of HMG-CoA reductase protein rapidly decreased in the presence of SR-12813. Pulse-chase experiments with [ 35 S]methionine showed that the T1 ⁄2 of HMG-CoA reductase degradation decreased in the presence of SR-12813 from 90 to 20 min. Pre-incubation with 50 M of lovastatin did not prevent the effects of SR-12813 on HMG-CoA reductase degradation, indicating that the compound does not need mevalonate-derived regulators for its action. It is concluded that SR-12813 inhibits cholesterol synthesis mainly by an enhanced degradation of HMG-CoA reductase.

Adduction of ethylene glycol moieties to the 3-hydroxy position of cholesterol produces polyoxyethylated cholesterol (POEC), a water-soluble compound that suppresses cholesterol synthesis and esterification in cultured human fibroblasts. Feeding Sprague-Dawley rats a diet containing 2% (wt/wt) POEC with 10 ethoxy groups resulted in a 3-fold increase in hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity compared to activity in rats pair-fed a diet of standard rat chow. POEC with an average of 20 ethoxy groups (POEC-20) caused comparable changes in hepatic [2-14C]acetate incorporation into nonsaponifiable lipids under ad libitum feeding conditions, significantly reduced cholesterol absorption (18% vs 57%), and increased fecal excretion of neutral steroids (5.1 vs 2.0 mg/g food intake). POEC-20 also reduced cholesterol absorption in rats fed a diet enriched with 2% cholesterol (11% vs 31%). Histologic studies of intestinal mucosa and hepatic tissues from rats fed POEC showed no pathologic changes. These experiments indicate that POEC reduces cholesterol absorption and causes compensatory increases in hepatic cholesterol synthesis.

We have suggested previously, measuring 14C-acetate incorporation into free cholesterol, that oral administration of policosanol inhibits hepatic cholesterol biosynthesis in rats. Nevertheless, since acetate has limitations to study cholesterol synthesis in vivo, we now investigate rates of incorporation of labeled water into hepatic sterol after policosanol treatment. Absolute rates of incorporation of 3H-water in sterols were depressed by policosanol by about 20%, giving a more accurate degree of cholesterol biosynthesis inhibition in this species. Since policosanol did not inhibit labeled mevalonate incorporation into cholesterol in rat liver, we also studied the effect of policosanol on hydroxy-methylglutaryl-coenzyme A (HMG-CoA) reductase. Reductase activity assayed in microsomes treated with policosanol remained unchanged, suggesting that cholesterol synthesis is not inhibited by a direct action of policosanol on this enzyme.

Significant ambiguity exists in the scientific community with regard to the nomenclature of 26-hydroxylated oxysterols. Oxysterols constitute an important class of compounds that have biological roles in the regulation of cholesterol synthesis and as endogenous selective estrogen receptor modulators (SERMs). The ambiguity is attributable to deviations from clearly stated IUPAC rules and is likely to increase as more biologically active oxysterols are identified. This review provides a uniform approach to the naming of 26-hydroxylated sterols for those of current interest and for those on the horizon such as oxysterols of lanosterol that retain the unsaturation at C-24 and C-25 such as (E)-26-hydroxylanosterol. Using this molecule as a starting point, this review hopes to establish a common language to keep all investigators on the same page.

Background: In castration-resistant prostate cancer (CRPC), recent evidence has demonstrated the persistence of the intratumoral androgens. The multi-step androgen synthesis pathway originates from cholesterol, which can be obtained by cells from several major sources including intracellular synthesis through an enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). The inhibition of this enzyme by the use of statins has been investigated in prostate cancer as a possible therapeutic target for blocking the de novo androgen synthesis resulting in decreased tumor growth. However, the effectiveness of statins in CRPC has not been investigated. Methods: Castration-resistant C4-2 and androgen-sensitive LNCaP cells were treated with Simvastatin for 48 hours. Dose-dependent responses to Simvastatin were analyzed using cell proliferation and cytotoxicity assays. Cellular growth curve was generated using haemocytometer. HMGCR activity was assessed using 14 C-acetic acid detected by thin layer chromatography, and the protein expression was quantified using western blot analysis. Intracellular cholesterol and prostate specific antigen (PSA) levels were quantified using enzyme-linked immunosorbent assays (ELISA). Results: Significant decrease in cell viability and growth curve observed at 75 μM of Simvastatin compared to no treatment group in the castration-resistant C4-2 cells. HMGCR activity was significantly decreased up to 50% and 70% at 50 μM and 75 μM of Simvastatin respectively compared to the vehicle control in C4-2 cells. Simvastatin did not affect the protein expression. 80% decrease in the amount of total intracellular cholesterol levels was observed in 75 μM Simvastatin treatment group compared to vehicle control. PSA secretion levels were significantly reduced in the C4-2 cell line at 50 μM and 75 μM of Simvastatin compared to vehicle control. Conclusion: The inhibition of HMGCR via Simvastatin lowered the viability of castration-resistant C4-2 cells. Simvastatin's ability to limit the endogenous supply of cholesterol contributes to the effects seen in cell viability.

The present investigation describes the influence of partial, pharmacologic interruption of bile acid enterohepatic circulation on cholesterol and bile acid synthesis in germfree rats. Seven rats received a basal, semisynthetic diet and five rats received the basal diet supplemented with 5% cholestyramine. After 6 weeks, feces were collected in one 3-and one 4-day pool for analysis of bile acids and cholesterol. When the sampling period was concluded, the rats were killed and the liver microsomal fractions were isolated. The activities of HMG CoA reductase and 7a-hydroxylase were determined. The main fecal bile acids in the untreated rats were cholic acid and P-muricholic acid. During cholestyramine treatment cholic acid increased from 4.4 * 0.7 to 39.5k 5.6 mg/kg body weighdday and Pmuricholic acid from 4.5 2 0.6 to 7.7 2 0.9 mg/kg body

The present investigation describes the influence of partial, pharmacologic interruption of bile acid enterohepatic circulation on cholesterol and bile acid synthesis in germfree rats. Seven rats received a basal, semisynthetic diet and five rats received the basal diet supplemented with 5% cholestyramine. After 6 weeks, feces were collected in one 3-and one 4-day pool for analysis of bile acids and cholesterol. When the sampling period was concluded, the rats were killed and the liver microsomal fractions were isolated. The activities of HMG CoA reductase and 7a-hydroxylase were determined. The main fecal bile acids in the untreated rats were cholic acid and P-muricholic acid. During cholestyramine treatment cholic acid increased from 4.4 * 0.7 to 39.5k 5.6 mg/kg body weighdday and Pmuricholic acid from 4.5 2 0.6 to 7.7 2 0.9 mg/kg body

Hydroxy-Methyl-Glutaryl-CoA reductase (HMGCR) - the main enzyme of the cholesterol biosynthesis pathway - is mostly inhibited by statins in hepatocytes. In spite of the other tissues, liver utilizes cholesterol in different ways such as the synthesis of bile acids, excretion in to the intestine and synthesis of lipoproteins. Therefore, statins theoretically alter these pathways; although, there have not been such effects. In this review, we aim to show the roles of extra-hepatic tissues, in particular intestine, adipose and cutaneous tissues in providing the cholesterol after reduction of the whole body cholesterol content by statins.

Formula-fed infants present higher cholesterol synthesis rates and lower circulating cholesterol during the postnatal feeding period compared to breast-fed infants, though the mechanisms underlying this phenotype are not fully understood. Typical infant formulas contain vegetable-based fats, inherently including phytosterols (PS), which are structurally similar to cholesterol and may interfere with their absorption. A seven-day old piglets model was used to test the inhibitory effects of PS on cholesterol absorption during postnatal feeding. Following feeding for 21 days with milk-based formulas containing PS and cholesterol levels resembling those in formulas or human-milk, apparent cholesterol digestibility was analyzed in ileal digesta, and cholesterol, PS, and cholesterol synthesis markers were analyzed in plasma and liver samples. Ileal cholesterol digestibility content was increased in the piglets fed low PS formulas and the rate of the hepatic cholesterol synthesis, as determ...

Glycogen storage disease type 1a (GSD-1a) is a metabolic disorder characterized by fasting-induced hypoglycemia, hepatic steatosis, and hyperlipidemia. The mechanisms underlying the lipid abnormalities are largely unknown. To investigate these mechanisms seven GSD-1a patients and four healthy control subjects received an infusion of [1-13 C]acetate to quantify cholesterogenesis and lipogenesis. In a subset of patients, [1-13 C]valine was given to assess lipoprotein metabolism and [2-13 C]glycerol to determine whole body lipolysis. Cholesterogenesis was 274 Ϯ 112 mg/d in controls and 641 Ϯ 201 mg/d in GSD-1a patients (p Ͻ 0.01). Plasma triglyceride-palmitate derived from de novo lipogenesis was 7.1 Ϯ 9.4 and 86.3 Ϯ 42.5 mol/h in controls and patients, respectively (p Ͻ 0.01). Production of VLDL did not show a consistent difference between the groups, but conversion of VLDL into intermediate density lipoproteins was relatively retarded in all patients (0.6 Ϯ 0.5 pools/d) compared with controls (4.3 Ϯ 1.8 pools/d). Fractional catabolic rate of intermediate density lipoproteins was lower in patients (0.8 Ϯ 0.6 pools/d) compared with controls (3.1 Ϯ 1.5 pools/d). Whole body lipolysis was similar, i.e., 4.5 Ϯ 1.9 mol/kg/ min in patients and 3.8 Ϯ 1.9 mol/kg/min in controls. Hyperlipidemia in GSD-1a is associated with strongly increased lipid production and a slower relative conversion of VLDL to LDL.

Background Diabetes mellitus (DM) has been shown to increase the risk of Alzheimer's disease (AD). Downregulation of selective Alzheimer's disease indicator-1 (seladin-1) occurs in the cerebral regions affected by AD. However, inconsistent results have been reported for the relationship between seladin-1 levels and AD. The effect of DM on serum seladin-1 levels in AD is unknown. The present study is aimed to investigate serum seladin-1 levels in diabetic and non-diabetic patients with AD. Methods Forty-six patients with AD and 25 healthy volunteers over 65 years of age were included in this study. The patients were divided into three groups-those with AD only, those with DM and AD, and control groups. Demographic characteristics and serum seladin-1 levels were compared among the groups. Results There was no statistically significant difference in seladin-1 levels in the AD only group compared to the control group (p = 0.376). However, seladin-1 levels were significantly lower in the DM and AD group compared to the AD only and control groups (p = 0.002, p = 0.001; respectively). Negative correlations were present between seladin-1 and fasting glucose, postprandial glucose, HbA1c, and insulin (p < 0.05; all). Conclusion Decreased serum seladin-1 values in the presence of DM and inverse correlations with diabetic parameters in patients with AD, together with a non-significant difference from the control group, suggests that seladin-1 may be altered only in the presence of DM in patients with AD. Lower serum seladin-1 levels may also play a role in the pathogenesis of AD in patients with DM.

Urinary excretion of mevalonic acid was investigated as an indicator of cholesterol synthesis. In normolipemic volunteers, excretion of mevalonic acid averaged 3.51 f 0.59 (SD) pg,/kg x day'; (n = 24) and was not different from patients with hypercholesterolemia (3.30 f 0.92 pdkg x day'; n = 24). In patients with cerebrotendineous xanthomatosis, the excretion was significantly higher (8.55 f 1.92 pg/kg x day'; n = 6, P < 0.001) but comparable to volunteers treated with cholestyramine (6.69 f 2.6 pglkg x day'; n = 5). A significant correlation was found between 24-h excretion of mevalonic acid and cholesterol synthesis (r = 0.835; n = 35; P < 0.001). The coefficient of variation of excretion of mevalonic acid during 3 consecutive days was small (9.8%; n = 7). However, urinary output of mevalonic acid was significantly higher during the night (164 f 14 pg/12-h) than during the day (129 f 9 ~g A 2-h ; n = 11; P < 0.05). In patients treated with simvastatin (40 mg/day) for 6 weeks, the ratio of mevalonic acid to creatinine in a morning urine sample decreased significantly compared to pretreatment values (1 10 f 25 pg/g vs. 66 f 25 pug; P < 0.001). Furthermore, the ratio of mevalonic acid to creatinine in a morning urine sample correlated with the ratio from the 24h collection period (r = 0.714; n = 34; P < 0.001). The results indicate that the analysis of urinary mevalonic acid, either in 24h collection or in a single morning sample, is an attractive method for evaluation of long and very short term changes of the rates of cholesterol synthesis.-lindent, B., A. Simatupang, M. T. Dotti, A.

The underlying cause of cellular degeneration in the substantia nigra of patients with Parkinson disease has not been clearly established. With the objective of investigating whether metabolic abnormalities would be detected in peripheral non-neuronal cells, we began assessing key metabolic parameters in skin fibroblasts of these patients. The present report focuses on the finding of a remarkably reduced cholesterol biosynthetic capability of fibroblasts from patients with Parkinson disease. 14C-Acetate incorporation into cholesterol of these fibroblasts was 27.8 +/- 9.4% that observed in normal fibroblasts, and the reduced cholesterol synthesis was confirmed by measuring the activity of the rate-limiting enzyme HMGCoA reductase which averaged 6.64 +/- 2.50 nmol/h/mg protein in the patient&#39;s fibroblasts compared to 14.70 +/- 0.69 nmol/h/mg protein in the control fibroblasts. Cholesterol esterifying activity, as cholesteryl oleate formed from 14C-oleate, of the fibroblasts from P...

The inflammatory response in mammals is characterized by the synthesis in the liver of several N-linked serum glycoproteins called acute-phase reactants. In C57BL/6J mice, turpentine-induced inflammation was accompanied by increases in 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity, dolichol synthesis, and dolichyl phosphoryl mannose synthesis. Cholesterol feeding, but not fasting, prevented these inflammation-induced increases in reductase activity and dolichol synthesis. However, the rate of incorporation of [3H]mannose into total serum glycoproteins was not affected by the high-cholesterol diet, and this rate increased during acute inflammation in control and cholesterol-fed mice.

Human studies have shown diurnal rhythms of cholesterol and bile acid synthesis, but a better understanding of the role of the circadian system in cholesterol homeostasis is needed for the development of targeted interventions to improve metabolic health. Therefore, we performed a systematic literature search on the diurnal rhythms of cholesterol synthesis and absorption markers and of bile acid synthesis markers. We also examined the diurnal rhythms of the cholesterol synthesis markers lathosterol and desmosterol, and of the cholesterol absorption markers cholestanol, campesterol, and sitosterol in serum samples from the Bispebjerg study. These samples were collected every three hours over a 24-h period in healthy males (n = 24) who consumed low-fat meals. The systematic search identified sixteen papers that had examined the diurnal rhythms of the cholesterol synthesis markers lathosterol (n = 3), mevalonate (n = 9), squalene (n = 2), or the bile acid synthesis marker 7α-hydroxy-4-cholesten-3-one (C4) (n = 4). Results showed that lathosterol, mevalonate, and squalene had a diurnal rhythm with nocturnal peaks, while C4 had a diurnal rhythm with daytime peaks. Furthermore, cosinor analyses of the serum samples showed a significant diurnal rhythm for lathosterol (cosinor p < 0.001), but not for desmosterol, campesterol, sitosterol, and cholestanol (cosinor p > 0.05). In conclusion, cholesterol synthesis and bile acid synthesis have a diurnal rhythm, though no evidence for a diurnal rhythm of cholesterol absorption was found under highly standardised conditions. More work is needed to further explore the influence of external factors on the diurnal rhythms regulating cholesterol homeostasis.

We have developed cultures of smooth muscle cells (SMC) isolated from arterial hypercholesterolemic chicks (cholesterol-SMC). These cultures are suitable for the study at the molecular level of the changes in arterial SMC induced by a cholesterol diet. By using a strong dose of cholesterol (5%) for 10 d, we obtained very proliferative SMC which became foam cells after 30 d in culture. On the other hand, SMC cultures isolated from control-fed chicks had a lower growth rate than the SMC ones under the same culture conditions. DNA synthesis was fourfold greater in cholesterol-SMC than in control-SMC cultures. Intracellular cholesterol concentrations were the same in both cholesterol and control SMC during the first 14 d of culture but afterward increased in differing ways: after 20 d of culture the cholesterol-SMC increased their cholesterol content to double the control. We give here the results obtained from transmission electron microscopy, lipid analysis, proliferation studies, DNA, RNA and protein synthesis, and then discuss their implications.

Hydroxy-Methyl-Glutaryl-CoA reductase (HMGCR) - the main enzyme of the cholesterol biosynthesis pathway - is mostly inhibited by statins in hepatocytes. In spite of the other tissues, liver utilizes cholesterol in different ways such as the synthesis of bile acids, excretion in to the intestine and synthesis of lipoproteins. Therefore, statins theoretically alter these pathways; although, there have not been such effects. In this review, we aim to show the roles of extra-hepatic tissues, in particular intestine, adipose and cutaneous tissues in providing the cholesterol after reduction of the whole body cholesterol content by statins.

The expansion of a polyglutamine tract in the ubiquitously expressed huntingtin protein causes Huntington&#39;s disease (HD), a dominantly inherited neurodegenerative disease. We show that the activity of the cholesterol biosynthetic pathway is altered in HD. In particular, the transcription of key genes of the cholesterol biosynthetic pathway is severely affectedin vivoin brain tissue from HD mice and in human postmortem striatal and cortical tissue; this molecular dysfunction is biologically relevant because cholesterol biosynthesis is reduced in cultured human HD cells, and total cholesterol mass is significantly decreased in the CNS of HD mice and in brain-derived ST14A cells in which the expression of mutant huntingtin has been turned on. The transcription of the genes of the cholesterol biosynthetic pathway is regulated via the activity of sterol regulatory element-binding proteins (SREBPs), and we found an ∼50% reduction in the amount of the active nuclear form of SREBP in HD...

The modifications of cholesterol metabolism associated with aging are ill-defined. The objective of this study was to define age-associated alterations of the different metabolic pathways controlling cholesterol homeostasis by analyzing circulating sterols. Methods: We analyzed serum samples collected from 201 adult (75 male, 126 female) subjects within the epidemiological MICOL study (Multicentrica Italiana Colelitiasi). The age range was 38-79 years; 103 had evidence of gallstones. The concentrations of the different sterols, recognized as markers of the main pathways of cholesterol homeostasis, were analyzed by gas chromatography-mass spectrometry, including lathosterol (synthesis), campesterol and sitosterol (absorption), and 7α-hydroxy-4-cholesten-3-one (degradation to bile acids). Results: A significant direct correlation was detected between age and cholesterol levels (r=0.34, P,0.01). The lathosterol/cholesterol ratio was lower in older age quartiles (P,0.05 by analysis of variance), with an inverse correlation between the lathosterol/cholesterol ratio and age (r=-0.32, P,0.01). Such correlation was particularly evident in females. The campesterol/cholesterol and sitosterol/cholesterol ratios were inversely correlated with aging in control, but not in gallstone patients. The levels of 7α-hydroxy-4-cholesten-3-one were not correlated with age. Conclusion: These data show a reduction of cholesterol synthesis with aging which is associated with increased circulating cholesterol levels. The finding might be related to a reduced metabolic need for cholesterol in advancing age, leading to a downregulation of the main mechanisms of cholesterol intake in the liver. A different age-related behavior was observed in gallstone-free versus gallstone patients regarding cholesterol absorption. The possible implications in terms of the pharmacological management of hypercholesterolemia in the elderly remain to be defined.

We have developed cultures of smooth muscle cells (SMC) isolated from arterial hypercholesterolemic chicks (cholesterol-SMC). These cultures are suitable for the study at the molecular level of the changes in arterial SMC induced by a cholesterol diet. By using a strong dose of cholesterol (5%) for 10 d, we obtained very proliferative SMC which became foam cells after 30 d in culture. On the other hand, SMC cultures isolated from control-fed chicks had a lower growth rate than the SMC ones under the same culture conditions. DNA synthesis was fourfold greater in cholesterol-SMC than in control-SMC cultures. Intracellular cholesterol concentrations were the same in both cholesterol and control SMC during the first 14 d of culture but afterward increased in differing ways: after 20 d of culture the cholesterol-SMC increased their cholesterol content to double the control. We give here the results obtained from transmission electron microscopy, lipid analysis, proliferation studies, DNA, RNA and protein synthesis, and then discuss their implications.

Although two-dimensional electrophoresis (2D-GE) remains the basis for many ecotoxicoproteomic analyses, newer non-gel-based methods are beginning to be applied to overcome throughput and coverage limitations of 2D-GE. The overall objective of our research was to apply a comprehensive, liquid chromatography− tandem mass spectrometry (LC-MS/MS)-based proteomic approach to identify and quantify differentially expressed hepatic proteins from female fathead minnows exposed to fadrozole, a potent inhibitor of estrogen synthesis. Female fathead minnows were exposed to 0 (control), 0.04, and 1.0 μg of fadrozole/L of water for 4 days, and proteomic analysis was performed. Proteins were extracted and digested, and proteolytic peptides were separated via high-resolution one-or two-dimensional (1-D or 2-D) ultrapressure liquid chromatography (UPLC) and analyzed by tandem mass spectrometry. Mass spectra were searched against the National Center for Biotechnology Information (NCBI) ray-finned fish (Actinopterygii) database, resulting in identification of 782 unique proteins by single-dimension UPLC. When multidimensional LC analysis (2-D) was performed, an average increase of 1.9× in the number of identified proteins was observed. Differentially expressed proteins in fadrozole exposures were consistent with changes in liver function, including a decline in concentrations of vitellogenin as well as other proteins associated with endocrine function and cholesterol synthesis. Overall, these results demonstrate that a gel-free, label-free proteomic analysis method can successfully be utilized to determine differentially expressed proteins in small fish species after toxicant exposure.

Cancer cells have altered metabolism and, in some cases, an increased demand for cholesterol. It is important to identify novel, rational treatments based on biology, and cellular cholesterol metabolism as a potential target for cancer is an innovative approach. Toward this end, we focused

We have developed cultures of smooth muscle cells (SMC) isolated from arterial hypercholesterolemic chicks (cholesterol-SMC). These cultures are suitable for the study at the molecular level of the changes in arterial SMC induced by a cholesterol diet. By using a strong dose of cholesterol (5%) for 10 d, we obtained very proliferative SMC which became foam cells after 30 d in culture. On the other hand, SMC cultures isolated from control-fed chicks had a lower growth rate than the SMC ones under the same culture conditions. DNA synthesis was fourfold greater in cholesterol-SMC than in control-SMC cultures. Intracellular cholesterol concentrations were the same in both cholesterol and control SMC during the first 14 d of culture but afterward increased in differing ways: after 20 d of culture the cholesterol-SMC increased their cholesterol content to double the control. We give here the results obtained from transmission electron microscopy, lipid analysis, proliferation studies, DNA, RNA and protein synthesis, and then discuss their implications.

Glycogen storage disease type 1a (GSD-1a) is a metabolic disorder characterized by fasting-induced hypoglycemia, hepatic steatosis, and hyperlipidemia. The mechanisms underlying the lipid abnormalities are largely unknown. To investigate these mechanisms seven GSD-1a patients and four healthy control subjects received an infusion of [1-13 C]acetate to quantify cholesterogenesis and lipogenesis. In a subset of patients, [1-13 C]valine was given to assess lipoprotein metabolism and [2-13 C]glycerol to determine whole body lipolysis. Cholesterogenesis was 274 Ϯ 112 mg/d in controls and 641 Ϯ 201 mg/d in GSD-1a patients (p Ͻ 0.01). Plasma triglyceride-palmitate derived from de novo lipogenesis was 7.1 Ϯ 9.4 and 86.3 Ϯ 42.5 mol/h in controls and patients, respectively (p Ͻ 0.01). Production of VLDL did not show a consistent difference between the groups, but conversion of VLDL into intermediate density lipoproteins was relatively retarded in all patients (0.6 Ϯ 0.5 pools/d) compared with controls (4.3 Ϯ 1.8 pools/d). Fractional catabolic rate of intermediate density lipoproteins was lower in patients (0.8 Ϯ 0.6 pools/d) compared with controls (3.1 Ϯ 1.5 pools/d). Whole body lipolysis was similar, i.e., 4.5 Ϯ 1.9 mol/kg/ min in patients and 3.8 Ϯ 1.9 mol/kg/min in controls. Hyperlipidemia in GSD-1a is associated with strongly increased lipid production and a slower relative conversion of VLDL to LDL.

Hydroxy-Methyl-Glutaryl-CoA reductase (HMGCR) - the main enzyme of the cholesterol biosynthesis pathway - is mostly inhibited by statins in hepatocytes. In spite of the other tissues, liver utilizes cholesterol in different ways such as the synthesis of bile acids, excretion in to the intestine and synthesis of lipoproteins. Therefore, statins theoretically alter these pathways; although, there have not been such effects. In this review, we aim to show the roles of extra-hepatic tissues, in particular intestine, adipose and cutaneous tissues in providing the cholesterol after reduction of the whole body cholesterol content by statins.

Incorporation of 13C from a dietary precursor into cholesterol was studied in neonatal rats. Rats were given uniformly I3Cenriched polyunsaturated fatty acids intragastrically and total lipid extracts of liver and brain were analyzed by I3C-NMR 1,4, 8, and 15 days later. I3C-enrichment was detected in brain but not in liver cholesterol. Maximal I3C-labeling was observed 4 days after injection of the label. Spectra revealed that 70% of newly incorporated I3C had I3C as an adjacent neighbor, the other 30% had I2C as the neighbor. Double quantum NMR revealed the arrangement in the cholesterol skeleton of the I3C-l3C pairs transferred from precursors to cholesterol. Desmosterol, an intermediate of cholesterol synthesis, was identified in the spectra of brain lipids. Comparison of 13C-13C unit arrangements in both cholesterol and desmosterol allowed carbons 26 and 27 of desmosterol to be unambiguously assigned.

In female frogs (Rana Esculenta) during gametogenesis the cholesterol synthesized in the liver by 3-hydroxy-3-methylglutaryl coenzyme A reductase is mostly exported into the blood and taken up by the oocytes. In order to understand the fate of the neosynthesized cholesterol, female and male frogs and estrogenized male controls were injected with the labelled precursor14C mevalonate. In females and in estrogenized controls, mevalonate-derived radioactivity is found in a plasmatic lipoprotein that has been identified as vitellogenin by immunological detection. The increased 3-hydroxy-3-methylglutaryl coenzyme A reductase activity present in females in Fall is likely to be committed to provide cholesterol for the lipidation of this cholesterol-rich protein.

The expansion of a polyglutamine tract in the ubiquitously expressed huntingtin protein causes Huntington's disease (HD), a dominantly inherited neurodegenerative disease. We show that the activity of the cholesterol biosynthetic pathway is altered in HD. In particular, the transcription of key genes of the cholesterol biosynthetic pathway is severely affected in vivo in brain tissue from HD mice and in human postmortem striatal and cortical tissue; this molecular dysfunction is biologically relevant because cholesterol biosynthesis is reduced in cultured human HD cells, and total cholesterol mass is significantly decreased in the CNS of HD mice and in brain-derived ST14A cells in which the expression of mutant huntingtin has been turned on. The transcription of the genes of the cholesterol biosynthetic pathway is regulated via the activity of sterol regulatory element-binding proteins (SREBPs), and we found an ϳ50% reduction in the amount of the active nuclear form of SREBP in HD cells and mouse brain tissue. As a consequence, mutant huntingtin reduces the transactivation of an SRE-luciferase construct even under conditions of SREBP overexpression or in the presence of an exogenous N-terminal active form of SREBP. Finally, the addition of exogenous cholesterol to striatal neurons expressing mutant huntingtin prevents their death in a dosedependent manner. We conclude that the cholesterol biosynthetic pathway is impaired in HD cells, mice, and human subjects, and that the search for HD therapies should also consider cholesterol levels as both a potential target and disease biomarker.

Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5α-cholestanol are widely used as surrogate markers of cholesterol synthesis and absorption, respectively. Increasing numbers of laboratories utilize a broad spectrum of well-established and recently developed methods for the determination of cholesterol and non-cholesterol sterols (NCS). In order to evaluate the quality of these measurements and to identify possible sources of analytical errors our group initiated the first international survey for cholesterol and NCS. The cholesterol and NCS survey was structured as a two-part survey which took place in the years 2013 and 2014. The first survey part was designed as descriptive, providing information about the variation of reported results from different laboratories. A set of two lyophilized pooled sera (A and B) was sent to twenty laboratories specialized in chromatographic lipid analysis. The different sterols were quantified either by gas chromatography-flame ionization detection, gas chromatography-or liquid chromatography-mass selective detection. The participants were requested to determine cholesterol and NCS concentrations in the provided samples as part of their normal laboratory routine. The second part was designed as interventional survey. Twenty-two laboratories agreed to participate and received again two different lyophilized pooled sera (C and D). In contrast to the first international survey, each participant received standard stock solutions with defined concentrations of cholesterol and NCS. The participants were requested to use diluted calibration solutions from the provided standard stock solutions for quantification of cholesterol and NCS. In both surveys, each laboratory used its own internal standard (5α-cholestane, epicoprostanol or deuterium labelled sterols). Main outcome of the survey was, that unacceptably high interlaboratory variations for cholesterol and NCS concentrations are reported, even when the individual laboratories used the same calibration material. We discuss different sources of errors and recommend all laboratories analysing cholesterol and NCS to participate in regular quality control programs.

SLC13A5 is a Na +-coupled transporter for citrate that is expressed in the plasma membrane of specific cell types in the liver, testis, and brain. It is an electrogenic transporter with a Na + :citrate 3− stoichiometry of 4:1. In humans, the Michaelis constant for SLC13A5 to transport citrate is ~600 µM, which is physiologically relevant given that the normal concentration of citrate in plasma is in the range of 150–200 µM. Li + stimulates the transport function of human SLC13A5 at concentrations that are in the therapeutic range in patients on lithium therapy. Human SLC13A5 differs from rodent Slc13a5 in two important aspects: the affinity of the human transporter for citrate is ~30-fold less than that of the rodent transporter, thus making human SLC13A5 a low-affinity/high-capacity transporter and the rodent Slc13a5 a high-affinity/low-capacity transporter. In the liver, SLC13A5 is expressed exclusively in the sinusoidal membrane of the hepatocytes, where it plays a role in the uptake of circulating citrate from the sinusoidal blood for metabolic use. In the testis, the transporter is expressed only in spermatozoa, which is also only in the mid piece where mitochondria are located; the likely function of the transporter in spermatozoa is to mediate the uptake of citrate present at high levels in the seminal fluid for subsequent metabolism in the sperm mitochondria to generate biological energy, thereby supporting sperm motility. In the brain, the transporter is expressed mostly in neurons. As astrocytes secrete citrate into extracellular medium, the potential function of SLC13A5 in neurons is to mediate the uptake of circulating citrate and astrocyte-released citrate for subsequent metabolism. Slc13a5-knockout mice have been generated; these mice do not have any overt phenotype but are resistant to experimentally induced metabolic syndrome. Recently however, loss-of-function mutations in human SLC13A5 have been found to cause severe epilepsy and encephalopathy early in life. Interestingly, there is no evidence of epilepsy or encephalopathy in Slc13a5-knockout mice, underlining the significant differences in clinical consequences of the loss of function of this transporter between humans and mice. The markedly different biochemical features of human SLC13A5 and mouse Slc13a5 likely contribute to these differences between humans and mice with regard to the metabolic consequences of the transporter deficiency. The exact molecular mechanisms by which the functional deficiency of the citrate transporter causes epilepsy and impairs neuronal development and function remain to be elucidated, but available literature implicate both dysfunction of GABA (γ-aminobutyrate) signaling and hyperfunction of NMDA (N-methyl-D-aspartate) receptor signaling. Plausible synaptic mechanisms linking loss-of-function mutations in SLC13A5 to epilepsy are discussed.