Tesi sul tema "Metabolism of cholesterol derivatives"

Cholesterol is metabolized to a variety of important biological products in the body including bile acids and vitamin D. The present investigation is focused on enzymes that catalyze 7α-hydroxylation or 27-hydroxylation in the metabolism of cholesterol, oxysterols (side chain-hydroxylated derivatives of cholesterol) and vitamin D₃. The enzymes studied belong to the cytochrome P450 enzyme families CYP7 and CYP27.

The study describes purification of a cytochrome P450 enzyme fraction active in 7α-hydroxylation of 25-hydroxycholesterol, 27-hydroxycholesterol, dehydroepiandrosterone and pregnenolone from pig liver microsomes. Peptide sequence analysis indicated that this enzyme fraction contains an enzyme belonging to the CYP7B subfamily. The purified enzyme was not active towards cholesterol or testosterone. Purification and inhibition experiments suggested that hepatic microsomal 7α -hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone involves at least two enzymes, probably closely related.

The study shows that recombinantly expressed human and rat cholesterol 7α -hydroxylase (CYP7A) and partially purified pig liver cholesterol 7α -hydroxylase are active towards 20(S)-, 24-, 25- and 27-hydroxycholesterol. CYP7A was previously considered specific for cholesterol and cholestanol. The 7α -hydroxylation of 20(S)-, 25-, and 27-hydroxycholesterol in rat liver was significantly increased by treatment with cholestyramine, an inducer of CYP7A. Cytochrome P450 of renal origin showed 7α -hydroxylase activity towards 25- and 27-hydroxycholesterol, dehydroepiaundrosterone and pregnenolone but not towards 20(S)-, 24-hydroxycholesterol or cholesterol. The results indicate a physiological role for CYP7A as an oxysterol 7α -hydroxylase, in addition to the previously known human oxysterol 7α -hydroxylase CYP7B.

The role of renal sterol 27-hydroxylase (CYP27A) in the bioactivation of vitamin D₃ was studied with cytochrome P450 fractions purified from pig kidney mitochondria. Purification and inhibition experiments and experiments with a monoclonal antibody against CYP27A indicated that CYP27A plays a role in renal 25-hydroxyvitamin D₃ l α -hydroxylation.

The expression of CYP7A, CYP7B and CYP27A during development was studied. The levels of CYP27A in livers of newborn and six months old pigs were similar whereas the levels of CYP7A increased. The expression of CYP7B varied depending on the tissue. The expression of CYP7B increased with age in the liver whereas the CYP7B levels in kidney showed a marked age-dependent decrease.

The liver has a major role in the metabolism of cholesterol, being the main site of lipoprotein assembly and degradation and the only tissue where the metabolism of cholesterol to bile acids occurs. This provides the major pathway for the removal of cholesterol from the body. The results described in this thesis concern the use of specific enzyme inhibitors (58-035, Azacholesterol, Mevinolin) to determine the intracellular use of different sources of cholesterol in monolayers of rat hepatocytes. In particular, the fates of newly synthesized cholesterol from mevalonic acid and cholesterol derived from HDL2 were investigated. Incubation of hepatocyte monolayers with 58-035 resulted in the inhibition of esterification. In the presence of mevalonic acid as a cholesterol source, 58-035 stimulated bile acid synthesis. Azacholesterol inhibited bile acid synthesis, had no effect on cholesterol synthesis, and in the presence of mevalonic acid, stimulated secretion of cholesterol by the hepatocytes; it had no effect on cholesterol esterification. Mevinolin inhibited cholesterol synthesis and as a result inhibited esterification. HDL2, in the presence of mevinolin, was used as a cholesterol source. It stimulated bile acid synthesis and cholesterol esterification. Addition of 58-035 to the system resulted in the inhibition of both esterification and bile acid synthesis. Overall, the results indicated that different intracllular pools of free cholesterol exist and that the inter-relationships of these pools give a complex pattern of flux of intracellular cholesterol between various pathways in the rat hepatocyte.

La synthese endogene du cholesterol (CH) a ete etudie chez 81 nouveau-nes, ages de 4 mois (BRAS 1) ou de 11 a 12 mois (BRAS2), afin d'evaluer les effets a long terme d'un supplement de CH sur l'homeostasie du CH. BRAS 1 etait compose de 32 nouveau-nes recevant soit du lait humain (HUM) (6M, 7F), une formule a base de lait de vache (VAC) (6M, 3F) contenant 3.5 mg CH/dl, ou une formule a base de lait de vache modifiee (VACM) (6M, 7F) contenant 13.5 mg CH/dl, afin d'evaluer l'effet du supplement sur les taux de synthese du CH. BRAS2 etait compose de 49 autres enfants recevant soit HUM (11M, 6F), VAC (7M, 12F) ou VACM (6M, 7F) jusqu'a l'age de 6 mois dans le but d'evaluer une hypothese d'impression genetique. Ceci a ete realise en utilisant un design "cross-over" et en montant un defi journalier de 250 g de CH a l'age de 11 mois. Le taux d'incorporation de deuterium, provenant des reserves d'eau corporelle, dans la structure du CH a servi comme indice du taux de synthese fractionnel (TSF) de ce dernier sur une periode de 48 heures. Les niveaux de CH total et LDL etaient considerablement eleves dans HUM en comparaison avec VAC et VACM a l'age de 4 mois. La concentration sanguine du CH etait semblable a 11 et 12 mois. Le TSF etait 4 fois plus eleve dans VAC et VACM relatif a HUM, mais il n'y avait pas de difference entre VAC et VACM a 11 et 12 mois. Cependant, les TSF de 4 a 12 mois ont augmente dans HUM et baisse dans VAC et VACM. Nos resultats indiquent qu'independamment du contenu des dietes, le defi journalier de CH n'as pas eu d'effet considerable ni sur les taux de synthese, ni sur les niveaux de CH sanguin. Ces resultats appuient l'idee que le CH alimentaire n'a que des effets minimes sur le metabolisme a long terme du CH.

Bilirubin is a haem catabolite that is excreted through the hepatobiliary pathway and is therefore, commonly used as a biomarker of hepatic dysfunction and haemolysis in the clinical setting [1]. Although, bilirubin has been considered toxic [2], recent evidence suggests that mildly elevated circulating bilirubin concentrations may be protective against obesity, cardiovascular diseases (CVDs) and all-cause mortality [3–5]. Generally, the protective effects of bilirubin are attributed to its antioxidant potential [6–8], however, recent studies demonstrate that bilirubin modulates lipid metabolism and reduces adiposity, which could partly contribute to CVD protection [5,9–12]. However, a shortage of studies have examined the precise mechanisms of cholesterol metabolism and adiposity that could be affected by bilirubin. The main aims of this thesis were to: 1) determine whether hyperbilirubinaemia affects cholesterol synthesis, transport, and excretion; 2) explore bilirubin’s impact on body composition and bioenergetics including mitochondrial function in liver/skeletal muscle and changes in mitochondrial density and quality; 3) determine the effectiveness of oral Legalon® ingestion on circulating bilirubin concentrations, to investigate whether inducing mild hyperbilirubinaemia could impact circulating lipid concentrations in human participants. The first study measured the effect of hyperbilirubinaemia in mutant Gunn rats on circulating lipid concentrations, cholesterol synthesis, lipid excretion, and expression of hepatic genes/proteins involved in cholesterol metabolism. Female hyperbilirubinaemic (Gunn) rats had reduced serum cholesterol concentrations (0.60 ± 0.12 vs 1.56 ± 0.34 mM, P<0.001), elevated cholesterol synthesis (33.8 ± 3.77 vs 28.4 ± 5.73 % [13C]-cholesterol, P<0.05), enhanced LDL receptor (LDLr; P<0.01) expression, and increased biliary cholesterol excretion (232 ± 32.7 vs 141 ± 42.1 nmol hr-1 100g-1 bodyweight, P<0.001) compared to female normobilirubinaemic littermate (control) rats. These results indicate that female hyperbilirubinaemic Gunn rats have reduced circulating cholesterol in association with elevated LDLr expression. Increased LDLr expression and cholesterol synthesis is typical when hepatic cholesterol concentrations are decreased [13,14]. Therefore, increased cholesterol synthesis and LDLr expression observed in female Gunn rats may represent a counter-regulatory mechanism to maintain hepatic cholesterol content in the presence of elevated biliary cholesterol excretion [13,14]. The underlying mechanism explaining increased biliary lipid excretion in female Gunn rats remains unknown. However, this observation could be partly explained by greater relative biliary lipid (cholesterol+phospholipid) to bile acid excretion (0.33 ± 0.06 vs 0.24 ± 0.03 mol:mol, lipid:bile acids, P<0.01) in female Gunn rats. Previous studies have established that organic anions including bilirubin glucuronides disrupt the capacity of bile acid micelles to excrete lipids in the bile [15]. Biliary excretion of bilirubin conjugates was decreased in female (13.1 ± 2.92 vs 33.5 ± 5.09 nmol hr-1 100g-1 bodyweight, P<0.001) and male (11.0 ± 2.43 vs 43.2 ± 12.8 nmol hr-1 100g-1 bodyweight, P<0.001) Gunn rats compared to controls, due to UGT1A1 dysfunction and the inability to conjugate bilirubin. Therefore, decreased biliary excretion of bilirubin conjugates, as observed in Gunn rats, may potentially facilitate the greater coupled excretion of biliary lipids to bile acids as demonstrated in this study. It should be noted that this conclusion does not completely explain the results reported here because Gunn rats demonstrated significant sexual dimorphism in cholesterol metabolism. Male Gunn rats exhibited a non-significant reduction in circulating cholesterol concentrations (1.41 ± 0.15 vs 1.56 ± 0.23, P=0.14) and increased biliary lipid:bile acid excretion (0.31 ± 0.07 vs 0.25 ± 0.04 mol:mol, lipid:bile acid, P=0.08) compared to male normobilirubinaemic littermate (control) rats, indicating that additional mechanisms, beyond bilirubin excretion, are involved. For example, UGT1A1, which conjugates bilirubin also conjugates and facilitates the excretion of sex hormones including oestrogen. Therefore, oestrogen concentrations may be elevated in female hyperbilirubinaemic rats and synergistically impact lipid metabolism [16,17]. The second study examined the effect of hyperbilirubinaemia in vitro and in vivo on mitochondrial function and body composition. Dual X-ray absorptiometry (DEXA) analysis revealed that female Gunn rats had significantly reduced fat mass (9.94 ± 5.35 vs 16.1 ± 6.65 g, P<0.05) and lean mass (140 ± 12.1 vs 160 ± 16.0 g, P<0.05) compared to littermate controls. Female Gunn rats consumed fewer calories per day (54.1 ± 6.38 vs 63.3 ± 6.95 kcal day-1, P<0.01). However, weight gain relative to calories consumed was reduced (8.09 ± 5.75 vs 14.9 ± 5.10 mg kcal-1, P<0.05) in female Gunn rats indicating that they are less energetically efficient. This led to the analysis of mitochondrial function in liver and skeletal muscle using high-resolution respirometry to ascertain the cause of reduced energetic efficiency. This analysis revealed that female Gunn rats exhibited increased oxidative phosphorylation (OXPHOS) relative to maximal noncoupled mitochondrial respiration (ETS) in hepatic mitochondria (0.78 ± 0.16 vs 0.62 ± 0.09 OXPHOS:ETS, P<0.05). The above findings were consistent with the effect of exogenous addition of unconjugated bilirubin (UCB) to control hepatic mitochondria, with 31.3 and 62.5 μM UCB increasing the OXPHOS: ETS ratio. However, exogenous UCB addition produced this effect by inhibiting ETS without affecting OXPHOS, indicating that UCB induces mitochondrial dysfunction at high concentrations. Conversely, no change in ETS (1130 ± 217 vs 1290 ± 373 pmol s-1 ng-1 citrate synthase (CS), P=0.16) or OXPHOS (901 ± 222 vs 796 ± 259 pmol s-1 ng-1 CS, P=0.36) was observed between female Gunn rats and controls. These data indicate that the greater OXPHOS:ETS ratios are a combination of increased OXPHOS and decreased ETS in female Gunn rats. Analysis of mitochondrial respiratory complexes revealed greater hepatic mitochondrial complex IV (CIV; P<0.01) expression in female Gunn rats. These findings support a conclusion that hepatic mitochondria have increased quality in female Gunn rats [18,19]. At present it remains unknown how this change in mitochondrial quality relates to reduced fat mass and energetic efficiency, however, the changes observed in female Gunn rats could represent an adaptation to bilirubin mediated inhibition of CIV as reported in vitro [20,21]. Otherwise, alterations in reproductive hormone metabolism in Gunn rats could also partially explain altered energetic states, as speculated in study one. Considering that hyperbilirubinaemia induced perturbed lipid metabolism and body composition in chapters one and two, study three sought to determine whether increasing bilirubin could alter circulating lipid profile in humans. The effect of Legalon®, containing the active ingredient silymarin, supplementation on circulating bilirubin concentrations and lipid status was investigated in a placebo controlled, single blind crossover clinical trial in healthy individuals (ACTRN12619001296123). Legalon® capsules containing 140 mg of silymarin were supplemented thrice daily (total dose of 420 mg silymarin) in a cohort of healthy males for two weeks. Two weeks of Legalon® supplementation did not change UCB concentrations compared to baseline (Legalon®: 12.5 ± 7.63 vs Baseline: 11.4 ± 4.14 μM, P=0.79). Secondary outcomes including lipid concentrations, inflammation, and total antioxidant status were also reported. Two weeks of Legalon® supplementation did not change serum cholesterol (4.80 ± 1.00 vs 4.88 ± 1.00 mM, P=0.19), triglyceride (1.07 ± 0.63 vs 1.04 ± 0.54 mM, P=0.79), C-reactive protein concentrations (1.74 ± 1.88 vs 0.92 ± 0.87 mg L-1, P=0.23) or serum antioxidant capacity (1194 ± 182 vs 1183 ± 201 mmol Fe2+ L-1, P=0.19) compared to baseline. Several clinical trials evaluating the impact of silymarin have reported changes to bilirubin concentrations following treatment [22–24]. However, these studies were conducted in patients with hepatic disease, which confounded bilirubin results, and with greater doses or different formulations of silymarin to that reported in this thesis. Although the results of this study demonstrated a negative finding, they are important because they represent the first attempt to use an orally administered, commercially approved, nutraceutical compound to increase bilirubin. These results provide important guidance to future studies that could utilise different doses or commercial preparations to induce a transient unconjugated hyperbilirubinaemia and test the impact on circulating cholesterol concentrations. In conclusion, this thesis contains three novel investigations that aimed to determine the impact of unconjugated hyperbilirubinaemia on cholesterol metabolism, synthesis and hepatic excretion; in addition to its effect on mitochondrial metabolism and body composition in Gunn rats. To determine whether these effects could be induced in humans, a nutraceutical with documented effects on circulating bilirubin was administered in a clinical trial, utilising a randomised, single-blind, crossover design. The results of this thesis suggest that bilirubin has the potential to modulate lipid and whole-body metabolism, particularly in female animals and provides the groundwork for additional studies that seek to reveal the mechanisms responsible for bilirubin’s effects. In addition, this thesis will support the discovery of more effective orally administered compounds that can modulate circulating bilirubin and lipid profile for protection against CVD.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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La maladie de Huntington (MH) est une maladie génétique autosomique dominante, causée par une augmentation du nombre de CAG sur le gène de la huntingtin. L’homéostasie du cholestérol cérébral est altérée dans la MH. L’expression de CYP46A1, enzyme neuronale catabolisant le cholestérol dans le cerveau, est diminuée dans le putamen des patients et dans le striatum de souris modèles de la MH. Après restauration de l’expression de CYP46A1 dans le striatum des souris Knock-In zQ175, les capacités locomotrices sont améliorées, l’agrégation de la huntingtine mutée est diminuée, l’atrophie neuronale est limitée et le métabolisme du cholestérol est stimulé. Une nouvelle signature transcriptionnelle est induite par CYP46A1, avec une restauration des voies impliquées dans l'autophagie, le protéasome, la communication synaptique et le transport axonal, connues pour leur dysfonctionnement dans la MH. CYP46A1 améliore la transmission synaptique et augmente la densité en épines synaptiques. L'élimination des agrégats par autophagie et par le protéasome est augmentée avec CYP46A1. Enfin, le transport de BDNF et de TrkB est amélioré par CYP46A1 dans un modèle in vitro de la MH. Ces résultats révèlent l'effet pléiotrope et bénéfique de la régulation du métabolisme du cholestérol dans la MH. Pour approfondir cette étude, une technique de tri cellulaire a été mise au point, afin de séparer les neurones et les astrocytes à partir de striatum de souris, pour étudier les régulations transcriptomique et lipidomique de ces deux populations. Cette étude permettra d’identifier de nouvelles cibles impliquées dans la neuroprotection par CYP46A1 et présentant un intérêt thérapeutique dans la MH
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease caused by abnormal CAG expansion on huntingtin’s gene. Recently, altered brain cholesterol homeostasis has been implicated in HD. Particularly, the expression of CYP46A1, the rate-limiting enzyme for cholesterol degradation in the brain, is decreased in patients’ putamen and in the striatum of HD mouse models. We restored CYP46A1 expression into the striatum of the zQ175 mice. Behavioral, neuropathological and molecular tests were performed and showed an improvement of locomotor activity and histological landmarks. Cholesterol homeostasis was restored with an increase of cholesterol degradation and synthesis. CYP46A1 induced a new transcriptional signature, with restoration of pathways involved in autophagy, proteasome, synaptic communication and axonal transport, which are known to be dysfunctional in HD. CYP46A1 improved synaptic transmission and spine density in the striatum of the zQ175 mice. Aggregate clearance mediated by autophagy and proteasome was increased after CYP46A1 expression. Finally, BDNF and TrkB transport were enhanced by CY46A1 in HD in vitro models. Overall, CYP46A1 restoration alleviates the zQ175 pathological phenotype through a global compensation. To gain further insights into CYP46A1 neuroprotection, a cell sorting strategy was set up to study the transcriptomic and lipidomic signature in purified neurons and astrocytes. This method will lead to a greater understanding of cell-type-specific regulations and cell communication. Altogether, this project gave new insights into the potential application of CYP46A1 restoration as a therapeutic strategy in HD

Ce travail de thèse s'articule autour de deux axes :1/ Les plastifiants, y compris les phtalates, ont été identifiés comme cancérigènes, mutagènes, reprotoxiques (CMR) de catégorie 1b et comme perturbateurs endocriniens. Le di-2-éthylhexyle phtalate (DEHP) est l'un des plastifiants les plus courants et est généralement associé au polychlorure de vinyle (PVC) dans les dispositifs médicaux. Le DEHP n'étant pas lié de manière covalente au PVC, il peut facilement migrer dans des matrices lipophiles et atteindre ensuite la circulation sanguine. Il est métabolisé par le foie en mono-2-éthylhexyle phtalate (MEHP), tout aussi toxique. Ces dernières années, des plastifiants alternatifs au DEHP ont été développés, notamment le di-2-éthylhexyl téréphtalate (DEHT), qui est métabolisé in vivo en mono-2-éthylhexyl téréphtalate (MEHT).La première partie de ce travail de thèse a consisté à développer des méthodes de dosage des plastifiants et de leurs métabolites dans diverses matrices biologiques, comme par exemple le plasma. Deux méthodes LC-MS/MS ont été développées pour la détermination du DEHP et du MEHP ainsi que des métabolites du DEHT. L'ionisation par spectrométrie de masse du DEHT étant très faible, une méthode LC-UV a été développée pour quantifier ce téréphtalate. Ces méthodes ont permis d'estimer le relargage du DEHP et du DEHT à partir de poches de sang et le dosage de leurs métabolites primaires dans les produits sanguins.2/ Les acides biliaires (AB) constituent une large famille de stéroïdes composée de nombreuses espèces. Ils sont synthétisés dans le foie et l'intestin et représentent la voie principale de catabolisme du cholestérol. Le 7a-hydroxy-4-cholesten-3-one (C4) est le précurseur des AB. Les AB jouent un rôle essentiel dans l'absorption des lipides mais également dans la signalisation cellulaire, étant ligands du récepteur nucléaire « Farnesoid X receptor » (FXR) et/ou du récepteur membranaire couplé aux protéines G, TGR5. Ces récepteurs, et donc leurs ligands, sont impliqués dans la régulation de l'homéostasie du glucose, des lipides et de la dépense énergétique. Toute modulation du profil des AB peut donc conduire à une modification de l'homéostasie métabolique.La deuxième partie de ce travail de thèse a consisté à développer deux méthodes de dosage par LC-MS/MS de 31 espèces d'AB et du C4 dans différentes matrices biologiques, dont le plasma. Une méthode spécifique permettant le dosage d'AB dérivés du LCA, récemment décrits, dans du contenu caecal est en cours d'optimisation. Ces méthodes ont permis d'analyser les variations du profil des AB dans divers contextes de la maladie cardiométabolique (obésité, insulino-résistance, diabète de type 2, NAFLD).Pour conclure, les méthodes d'analyse développées pour la quantification des plastifiants et des AB ont été validées et appliquées dans des études précliniques et cliniques. De manière intéressante, des données de la littérature ainsi que des essais préliminaires de transfection transitoire ont montré que des phtalates et leurs métabolites modulent l'activité du récepteur alpha activé par les proliférateurs de peroxysomes (PPARa) régulateur clé de l'homéostasie métabolique et de l'expression de l'enzyme CYP7A1 (enzyme majeure de la synthèse hépatique des AB). Les outils analytiques développés dans cette thèse permettent d'ouvrir des perspectives originales d'étude des effets des phtalates sur l'homéostasie métabolique via la régulation du métabolisme des AB.L'ensemble de ces travaux a permis de lier développements analytiques et applications dans le domaine de la biologie et de la santé
This thesis has two main focuses:1/ Plasticizers, including phthalates, have been identified as category 1b carcinogenic, mutagenic and reprotoxic (CMR) and as endocrine disruptors. Di-2-ethylhexyl phthalate (DEHP) is one of the most common plasticizers and is generally associated with polyvinyl chloride (PVC) in medical devices. As DEHP is not covalently bound to PVC, it can easily migrate into lipophilic matrices and then reach the bloodstream. It is metabolized by the liver into mono-2-ethylhexyl phthalate (MEHP), which is just as toxic. In recent years, alternative plasticizers to DEHP have been developed, notably di-2-ethylhexyl terephthalate (DEHT), which is metabolized in vivo to mono-2-ethylhexyl terephthalate (MEHT).The first part of this thesis involved developing methods for measuring plasticizers and their metabolites in various biological matrices, such as plasma. Two LC-MS/MS methods were developed for the determination of DEHP and MEHP as well as DEHT metabolites. As the ionization in mass spectrometry of DEHT is very low, a LC-UV method was developed to quantify this terephthalate. These methods have made it possible to estimate the release of DEHP and DEHT from blood bags and to measure their primary metabolites in blood products.2/ Bile acids (BA) are a large family of steroids made up of numerous species. They are synthesized in the liver and intestine and represent the main route of cholesterol catabolism. 7a-hydroxy-4-cholesten-3-one (C4) is the precursor of BA. BA play an essential role in lipid absorption but also in cell signaling, as they are ligands for the nuclear receptor 'Farnesoid X receptor' (FXR) and/or the G protein-coupled membrane receptor, TGR5. These receptors, and hence their ligands, are involved in glucose homeostasis, lipid homeostasis and energy expenditure. Any modulation of the BA profile can therefore lead to changes in metabolic homeostasis. The second part of this thesis involved developing two LC-MS/MS assay methods for 31 BA species and C4 in different biological matrices, including plasma. A specific method for the determination of recently described BA derived from LCA in caecal contents is currently being optimized. These methods have made it possible to analyze variations in the BA profile in various cardiometabolic disease contexts (obesity, insulin resistance, type 2 diabetes, NAFLD).In conclusion, the analytical methods developed for quantifying plasticizers and BA have been validated and applied in preclinical and clinical studies. Interestingly, data from the literature and preliminary transient transfection assays have shown that phthalates and their metabolites modulate the activity of the peroxisome proliferator-activated receptor alpha (PPARa), a key regulator of metabolic homeostasis and expression of CYP7A1 (a major enzyme in hepatic BA synthesis). The analytical tools developed in this thesis open up original perspectives for studying the effects of phthalates on metabolic homeostasis via the regulation of BA metabolism. All of this work has made it possible to link analytical developments and applications in the field of biology and health

To examine the role that lipoprotein surface charge plays in cholesterol metabolism in vivo, we characterized the effects of an intravenous injection of an uncharged phospholipid (phosphatidylcholine, PC (∼8 mg/kg)) or an anionic phospholipid (phosphatidylinositol, PI (∼9 mg/kg)) into fasted rabbits. The PI-injection significantly (P < 0.05) increased the negative surface charge of the lipoproteins. The clearance of tritiated-cholesterol from the PI-injected rabbit-plasma was ∼50% greater when compared to controls (P < 0.05). The PI-injection also prevented the formation of cholesteryl ester. To determine how increased lipoprotein PI content may regulate cholesterol clearance by the liver, cell culture studies using a human hepatoma cell-line were undertaken. In vitro enrichment of human plasma or high density lipoproteins with PI caused a ∼2 fold stimulation in cholesterol cellular uptake, relative to controls. PI-enrichment did not affect cellular cholesterol-uptake from low density lipoproteins. These results suggest that lipoprotein PI levels may affect intravascular cholesterol transport.

Sitosterolemia (STSL) is a sterol storage disorder characterized by very high plasma plant sterol (PS) and 5α-stanol levels, and leads to premature atherosclerosis, xanthomas, macrothrombocytopenia and endocrine disruption. Ezetimibe (EZE), a sterol absorption inhibitor, reduces plasma PS levels in STSL but its effect on tissue pool of sterols has not been investigated yet. The research objectives were to assess if EZE reduces whole body sitosterol and cholesterol pool sizes, improves cholesterol homeostasis, enhance hematologic profile and reduce endocrine disruption in STSL. EZE effects on circulating levels of cholestanol and its precursors (cholesterol and bile acid derivative 7α-hydroxy-4-cholesten-3-one, 7α-H-C4) relative to exogenous stanols (sitostanol) were also studied. Eight STSL patients were taken off EZE for 14 wks. After 4 wks off EZE they received intravenous doses of D7-sitosterol and 18O-cholesterol for sterol pool sizes assessments, and oral doses of 13C-cholesterol and deuterium oxide to measure fractional cholesterol absorption and synthesis rates. EZE (10 mg/d) was resumed and stable isotopes testing repeated. Measurement parameters included isotopic sterol enrichments, blood cell count, plasma and red blood cell (RBC) PS, cholesterol and its precursor (lathosterol), 5α-stanols and plasma 7α-H-C4, and thyroid hormones levels. EZE reduced plasma levels of sitosterol and total cholesterol, whole body sitosterol and cholesterol pool sizes and fractional cholesterol absorption rate while increasing cholesterol synthesis, production and clearance rates. EZE increased platelet count and decreased platelet size without affecting RBC indices of size or mass. A substantial decrease in circulating sitostanol but moderate decrease of cholestanol was noted with EZE. EZE increased lathosterol but not 7α-H-C4, suggesting increases in cholesterol biosynthesis and thus precursor availability for synthesis of cholestanol. In summary, EZE reduces body stores of PS and cholesterol, and increases cholesterol turnover by reducing cholesterol absorption and enhancing its synthesis and clearance. EZE reduces circulating PS and 5α-stanol levels, and improves macrothrombocytopenia and thyroid disruption. Endogenous cholestanol in STSL is mainly derived from cholesterol but not bile acid synthesis pathway. These data suggest that EZE may reduce the risks of developing premature atherosclerosis, bleeding and hormone disruption, thereby reinforcing the rationale for the use of EZE in treatment of STSL.
February 2015

In vitro studies have shown that the major membrane phospholipid phosphatidylcholine (PC) can positively influence the incorporation of cholesterol in lipid membranes. The influence of PC on the cellular trafficking of LDL-derived free cholesterol was investigated. Sterol regulatory-defective (SRD)-4 cells are Chinese hamster ovary (CHO)-derived fibroblasts that display vastly elevated rates for the synthesis and catabolism of PC. SRD-4 cells harbor two known gene mutations: a mutation in the functional allele for SCAP, resulting in defective feedback suppression of cholesterol biosynthesis; and a loss-of-function mutation in the functional allele for acyl-CoA:cholesterol acyl transferase (ACAT), an endoplasmic reticulum (ER)-localized enzyme that esterifies free cholesterol. Incubation of SRD-4 cells with 50 µg/ml low density lipoprotein (LDL) for 18 h resulted in lysosomal accumulation of free cholesterol as revealed by filipin staining. This accumulation was not evident following LDL treatment of parental CHO7 cells, and was blunted in SRD-2 cells that express a constitutively-active form of SREBP-2 and overproduce cholesterol but have functional ACAT activity. Treatment of SRD-2 cells with LDL in the presence of an ACAT inhibitor 58-035 resulted in robust lysosomal cholesterol accumulation that was reversible upon drug washout, supporting that cholesterol trafficking in cholesterol-loaded cells is dependent on ACAT activity and, more specifically, ER free cholesterol levels. Lysosomal accumulation of LDL-derived cholesterol was prevented in SRD-4 cells supplemented with lyso-PC (50 µM), a substrate for PC synthesis through the reacylation pathway, and also in cells treated with bromoenol lactone (BEL), an inhibitor of phospholipase A2 implicated in bulk PC turnover. In a counter study, lysosomal LDL-derived cholesterol accumulation was induced in parental CHO-7 cells using R-propranolol, which inhibits the conversion of phosphatidic acid to diacylglycerol (DAG), a substrate in the CDP-choline pathway. This blockage was also relieved through co-treatment with lyso-PC. These studies support that PC to free cholesterol ratios in downstream organellar membranes can influence cholesterol trafficking out of lysosomal compartments in cholesterol-loaded cells.

Cholesterol and lipoprotein metabolic regulation by dietary fat and cholesterol was studied in guinea pigs fed 15% fat (w/w) diets differing in fat saturation (saturated lard, monounsaturated olive oil, or polyunsaturated corn oil) and cholesterol amount (Basal (0.01%), Low (0.08%), Medium (0.17%), or High (0.33%)). Absorbed cholesterol intakes provided 6, 50, 100, and 200% respectively the mass of endogenous cholesterol synthesis. Dietary fat and cholesterol interactions affected plasma lipoprotein cholesterol concentrations, hepatic LDL binding, LDL composition, and hepatic free and esterified cholesterol levels. Hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity was independently suppressed by olive-oil based diets and cholesterol intakes above Basal levels, reflecting the regulation of cholesterol biosynthesis by hepatic cholesterol levels. Cholesterol-mediated down-regulation of hepatic LDL receptors correlated with plasma LDL levels, consistent with the primary role of the receptor in LDL uptake. In general, regulatory effects of fat type was more prominent with moderate cholesterol intakes, and diets containing lard or olive oil were hypercholesterolemic as compared to corn oil. Homeostatic regulatory mechanisms maintained plasma total and LDL cholesterol concentrations within modest ranges with moderate cholesterol intakes; however, High cholesterol intake resulted in large increments in plasma total and LDL cholesterol levels. Plasma HDL cholesterol concentrations were interactively increased by dietary cholesterol and fat type, although the changes were modest. HDL apo A-I fractional catabolic rates were not altered by dietary treatment and specific hepatic HDL binding did not constitute a significant catabolic route. Dietary cholesterol affected apo A-I mRNA levels in a tissue-specific manner such that intestinal apo A-I mRNA levels did not change but hepatic levels were increased; however, apo A-I mRNA abundance did not correlate with plasma HDL cholesterol concentrations, suggesting that post-transcriptional events may be more important determinants of apo A-I production than transcript abundance. Significant interactions between dietary fat saturation and cholesterol amount were demonstrated to alter regulatory mechanisms which maintain plasma cholesterol homeostasis. Where independent dietary fat and cholesterol effects occurred, cholesterol amount was generally a more significant dietary regulator than fat type.

Studies of aortic plaque reveal the presence of tissue macrophages filled with cholesteryl esters. To study lipoprotein metabolism of in vivo, maturated human macrophages, I isolated cells from human peritoneal effluent. Population analysis using cytochemistry showed substantial numbers of acid-esterase positive monocytic cells, lymphocytes, leukocytes and erythrocytes. Substantial variation in cell populations existed among patients. Human peritoneal cells degraded low density lipoproteins (LDL) and acetylated LDL (AcLDL) by high affinity, receptor-mediated processes. AcLDL degradation saturated at 15 ug protein/ml and LDL degradation saturated at 11 ug protein/ml. Positive correlation of the percentages of monocytic cells with the degradation values (LDL, r =.710; AcLDL, r =.725) and a degradation assay using cells isolated by Lymphoprep showed that the monocytic cells substantially contributed to the degradation of LDL. AcLDL degradation was calcium independent and inhibited by fucoidin. LDL degradation was calcium dependent and very low density lipoprotein and apoE-containing high density lipoprotein (HDL) competed with LDL for receptor uptake; apoE-free HDL, AcLDL and fucoidin did not reduce LDL degradation. Both receptors were pronase-sensitive and degradation was dependent upon lysosomal activity. ACAT activity analysis showed that pre-incubation of cells with LDL or AcLDL stimulated ACAT activity. ACAT activity was greatest for cells preincubated using AcLDL and fresh medium was necessary to maintain the ACAT activity values beyond 24 hrs. LDL-stimulated ACAT activity declined as time was increased above 24 hrs. Flow cytometry analysis of a total cell population and the Lymphoprep-isolated cells revealed a heterogenous T cell population, the presence of monocyte/macrophages, suggested that some of the cells present were activated and confirmed cytochemistry analysis demonstrating that Lymphoprep concentrated the mononuclear cells. Human peritoneal macrophages formed foam cells when incubated in the presence of AcLDL or LDL for 72 hrs. The formation of foam cells in the presence of LDL was dependent upon cell exposure time to the medium. Foam cell formation in the presence of LDL was accompanied by dense vacuolization and in the demonstrated absence of the oxidation of LDL the oil red O stainable material collected outside the vacuoles.