Dietary Cholest-4-en-3-one, a Cholesterol Metabolite of Gut Microbiota, Alleviates Hyperlipidemia, Hepatic Cholesterol Accumulation, and Hyperinsulinemia in Obese, Diabetic db/db Mice.

Previous studies have shown that dietary cholest-4-en-3-one (4-cholestenone, 4-STN) exerts anti-obesity and lipid-lowering effects in mice. However, its underlying mechanisms are not fully understood. In the present study, we evaluated whether 4-STN supplementation would protect obese diabetic db/db mice from obesity-related metabolic disorders. After four weeks of feeding of a 0.25% 4-STN-containing diet, dietary 4-STN was found to have significantly alleviated hyperlipidemia, hepatic cholesterol accumulation, and hyperinsulinemia; however, the effect was not sufficient to improve hepatic triglyceride accumulation or obesity. Further analysis demonstrated that dietary 4-STN significantly increased the content of free fatty acids and neutral steroids in the feces of db/db mice, indicating that the alleviation of hyperlipidemia by 4-STN was due to an increase in lipid excretion. In addition, dietary 4-STN significantly reduced the levels of desmosterol, a cholesterol precursor, in the plasma but not in the liver, suggesting that normalization of cholesterol metabolism by 4-STN is partly attributable to the suppression of cholesterol synthesis in extrahepatic tissues. In addition, dietary 4-STN increased the plasma and hepatic levels of 4-STN metabolites cholestanol (5α-cholestan-3ß-ol) and coprostanol (5ß-cholestan-3ß-ol). Our results show that dietary 4-STN alleviates obesity-related metabolic disorders, such as hyperlipidemia, hepatic cholesterol accumulation, and hyperinsulinemia, in db/db mice.

Accumulation of oxysterols in the erythrocytes of COVID-19 patients as a biomarker for case severity.

BACKGROUND: Due to the high risk of COVID-19 patients developing thrombosis in the circulating blood, atherosclerosis, and myocardial infarction, it is necessary to study the lipidome of erythrocytes. Specifically, we examined the pathogenic oxysterols and acylcarnitines in the erythrocyte homogenate of COVID-19 patients. These molecules can damage cells and contribute to the development of these diseases. METHODS: This study included 30 patients and 30 healthy volunteers. The erythrocyte homogenate extract was analyzed using linear ion trap mass spectrometry combined with high-performance liquid chromatography. The concentrations of oxysterols and acylcarnitines in erythrocyte homogenates of healthy individuals and COVID-19 patients were measured. Elevated levels of toxic biomarkers in red blood cells could initiate oxidative stress, leading to a process known as Eryptosis. RESULTS: In COVID-19 patients, the levels of five oxysterols and six acylcarnitines in erythrocyte homogenates were significantly higher than those in healthy individuals, with a p-value of less than 0.05. The mean total concentration of oxysterols in the red blood cells of COVID-19 patients was 23.36 ± 13.47 µg/mL, while in healthy volunteers, the mean total concentration was 4.92 ± 1.61 µg/mL. The 7-ketocholesterol and 4-cholestenone levels were five and ten times higher, respectively, in COVID-19 patients than in healthy individuals. The concentration of acylcarnitines in the red blood cell homogenate of COVID-19 patients was 2 to 4 times higher than that of healthy volunteers on average. This finding suggests that these toxic biomarkers may cause the red blood cell death seen in COVID-19 patients. CONCLUSIONS: The abnormally high levels of oxysterols and acylcarnitines found in the erythrocytes of COVID-19 patients were associated with the severity of the cases, complications, and the substantial risk of thrombosis. The concentration of oxysterols in the erythrocyte homogenate could serve as a diagnostic biomarker for COVID-19 case severity.

Corrigendum: Reductive Power Generated by Mycobacterium leprae Through Cholesterol Oxidation Contributes to Lipid and ATP Synthesis.

[This corrects the article .].

Reductive Power Generated by Mycobacterium leprae Through Cholesterol Oxidation Contributes to Lipid and ATP Synthesis.

Upon infection, Mycobacterium leprae, an obligate intracellular bacillus, induces accumulation of cholesterol-enriched lipid droplets (LDs) in Schwann cells (SCs). LDs are promptly recruited to M. leprae-containing phagosomes, and inhibition of this process decreases bacterial survival, suggesting that LD recruitment constitutes a mechanism by which host-derived lipids are delivered to intracellular M. leprae. We previously demonstrated that M. leprae has preserved only the capacity to oxidize cholesterol to cholestenone, the first step of the normal cholesterol catabolic pathway. In this study we investigated the biochemical relevance of cholesterol oxidation on bacterial pathogenesis in SCs. Firstly, we showed that M. leprae increases the uptake of LDL-cholesterol by infected SCs. Moreover, fluorescence microscopy analysis revealed a close association between M. leprae and the internalized LDL-cholesterol within the host cell. By using Mycobacterium smegmatis mutant strains complemented with M. leprae genes, we demonstrated that ml1942 coding for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), but not ml0389 originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in M. leprae. The 3ß-HSD activity generates the electron donors NADH and NADPH that, respectively, fuel the M. leprae respiratory chain and provide reductive power for the biosynthesis of the dominant bacterial cell wall lipids phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL)-I. Inhibition of M. leprae 3ß-HSD activity with the 17ß-[N-(2,5-di-t-butylphenyl)carbamoyl]-6-azaandrost-4-en-3one (compound 1), decreased bacterial intracellular survival in SCs. In conclusion, our findings confirm the accumulation of cholesterol in infected SCs and its potential delivery to the intracellular bacterium. Furthermore, we provide strong evidence that cholesterol oxidation is an essential catabolic pathway for M. leprae pathogenicity and point to 3ß-HSD as a prime drug target that may be used in combination with current multidrug regimens to shorten leprosy treatment and ameliorate nerve damage.

The Cholesterol Metabolite Cholest-5-en-3-One Alleviates Hyperglycemia and Hyperinsulinemia in Obese (db/db) Mice.

Dietary sterols are catabolized into various substances in the intestinal tract. Dietary 3-oxo derivatives of cholesterol and plant sterols (e.g., cholest-4-en-3-one and campest-5-en-3-one) have been shown to have anti-obesity effects. In this study, we tested whether feeding cholest-5-en-3-one (5-cholestenone), a cholesterol metabolite, to db/db mice protects them from obesity-associated metabolic disorders. In db/db mice, dietary 5-cholestenone significantly alleviated hepatomegaly and elevated serum triglyceride levels; however, the effect was not sufficient to improve hepatic steatosis and obesity. On the other hand, hyperglycemia and severe hyperinsulinemia in control db/db mice were markedly attenuated in 5-cholestenone-fed db/db mice. The production of inflammatory cytokines, such as monocyte chemoattractant protein-1, interleukin-6, and tumor necrosis factor-alpha (TNFα), was decreased, suggesting that the suppressive actions of 5-cholestenone were attributable to the alleviation of chronic inflammation in db/db mice. Additionally, 5-cholestenone showed an inhibitory effect on TNFα-induced nuclear factor kappa B (NFκB) activation in the NFκB luciferase gene reporter assay. These results suggest that obesity-induced abnormal glucose metabolism could be alleviated in 5-cholestenone-fed db/db mice by reducing the production of inflammatory cytokines through suppression of the NFκB signaling pathway.

Reductive power generated by Mycobacterium leprae through cholesterol oxidation contributes to lipid and ATP synthesis

Upon infection, Mycobacterium leprae, an obligate intracellular bacillus, induces accumulation of cholesterol-enriched lipid droplets (LDs) in Schwann cells (SCs). LDs are promptly recruited to M. leprae-containing phagosomes, and inhibition of this process decreases bacterial survival, suggesting that LD recruitment constitutes a mechanism by which host-derived lipids are delivered to intracellular M. leprae. We previously demonstrated that M. leprae has preserved only the capacity to oxidize cholesterol to cholestenone, the first step of the normal cholesterol catabolic pathway. In this study we investigated the biochemical relevance of cholesterol oxidation on bacterial pathogenesis in SCs. Firstly, we showed that M. leprae increases the uptake of LDL-cholesterol by infected SCs. Moreover, fluorescence microscopy analysis revealed a close association between M. leprae and the internalized LDL-cholesterol within the host cell. By using Mycobacterium smegmatis mutant strains complemented with M. leprae genes, we demonstrated that ml1942 coding for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), but not ml0389 originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in M. leprae. The 3ß-HSD activity generates the electron donors NADH and NADPH that, respectively, fuel the M. leprae respiratory chain and provide reductive power for the biosynthesis of the dominant bacterial cell wall lipids phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL)-I. Inhibition of M. leprae 3ß-HSD activity with the 17ß-[N-(2,5-di-t-butylphenyl)carbamoyl]-6-azaandrost-4-en-3one (compound 1), decreased bacterial intracellular survival in SCs. In conclusion, our findings confirm the accumulation of cholesterol in infected SCs and its potential delivery to the intracellular bacterium. Furthermore, we provide strong evidence that cholesterol oxidation is an essential catabolic pathway for M. leprae pathogenicity and point to 3ß-HSD as a prime drug target that may be used in combination with current multidrug regimens to shorten leprosy treatment and ameliorate nerve damage.

Faecal Calprotectin and 7-α Cholestenone Levels in Microscopic Colitis: Experience from Edinburgh.

INTRODUCTION: Microscopic colitis (MC) is an important cause of chronic, watery diarrhoea. Currently, there is no specific biomarker available to guide diagnosis. The use of faecal calprotectin (FCP) as a potential marker has been addressed in only a few studies. Further, bile acid malabsorption (BAM) often accompanies MC. Current practice recommends the selenium-labelled homocholic acid-taurine (SeHCAT) test, but at our centre, 7 alpha-hydroxy-4-cholesten-3-one (7αC) is used as a simpler and less expensive alternative to SeHCAT, with values over 22ng/mL indicating BAM. This study aims to evaluate the use of FCP as a biomarker in the diagnosis of MC and the role of 7αC in detecting concomitant BAM with MC. METHODS: Pathology records were retrospectively reviewed for patients diagnosed with collagenous colitis (CC) between 2000 and 2018 and lymphocytic colitis (LC) between 1995 and 2011. FCP and 7αc results, if measured within 6 months of pathological diagnosis, were extracted for analysis. RESULTS: Between 2000 and 2018, 646 CC cases were confirmed on histology. Of 646 patients, 147 had FCP measured; in 111 (75.5%) FCP was elevated with mean levels 238.1µg/g (SD±273.0); 140/646 had 7αC measured; 16 (11.4%) indicated BAM. Mean levels were 10.2ng/mL (SD±9.4). During a 21-year period (1995-2011), 204 LC diagnoses were made: 14/204 had FCP measured; 8 (57.1%) were elevated. Mean levels were 128.4µg/g (SD±107.7). Of 204 LC patients, 20 had 7αC measured, 5 (25%) indicating BAM. Mean levels were 13.95ng/mL (SD±9.4). DISCUSSION: Both CC and LC were associated with raised FCP during the diagnostic phase, supporting the potential role of its use in clinical practice. Furthermore, we present results of using 7αC in identifying BAM amongst patients with MC. In our cohort, low levels of 7αC suggest relatively low concordance of BAM with MC.

Cholesterol-oxidase-magnetic nanobioconjugates for the production of 4-cholesten-3-one and 4-cholesten-3, 7-dione.

Cholesterol oxidase(ChOx) enzyme isolated from Pseudomonas aeruginosa PseA(ChOxP) and Rhodococcus erythropolis MTCC 3951(ChOxR) strains as well as a commercial variant produced by Streptomyces sp.(ChOxS) were immobilized on silane modified iron(II, III)oxide magnetic nanoparticles(MNP) by covalent coupling methods. The nanobiocatalysts in case of ChOxP, ChOxR and ChOxS, retained 71, 91 and 86% of cholesterol oxidase activity respectively, as compared to their soluble counterparts. The catalytic efficiency of the immobilized enzymes on nanoparticles was more than 2.0 times higher than the free enzyme. They also showed enhanced pH and thermal stability. After 10 cycles of operation, the MNP-bioconjugates retained 50, 52 and 51% of residual activity in case of ChOxP, ChOxR and ChOxS respectively. The presence of enzyme on nanoparticles was confirmed by FTIR, SEM and TEM. The nanobiocatalysts were used for the biotransformation of cholesterol and 7-ketocholesterol to 4-cholesten-3-one and 4-cholesten-3, 7-dione respectively, which are industrially and medically important steroid precursors.

Research on the determination of cholesterol efflux from foam cells mediated by apoA-1 using HPLC analysis / 重庆医学

Objective To establish a high performance liquid chromatography (HPLC) method for determining the choles‐terol efflux from macrophage‐derived foam cells mediated by apolipoprotein A‐1(apoA‐1) .Methods Human THP‐1 monocytic cells ,pre‐treated with 160 nmol/L phorbol‐12‐myristate acetate (PMA) for 24 h to differentiate into adherence macrophages ,then incubated with 50 μg/mL acetylated low density lipoprotein (ac‐LDL) for 48 h to induce foam cells formation ,then added apoA‐1 for 24 h .THP‐1‐derived macrophage foam cells were identified by oil red O‐staining ,and the cellular cholesterol content by meas‐ured by HPLC method .Cholesterol efflux from macrophage foam cells was determined by HPLC analysis and liquid scintillation counting ,respectively .Results Oil red O‐stainable lipid droplet accumulation were observed in entire cytoplasm of THP‐1‐derived macrophage foam cells .Measuring cellular cholesterol content showed that free cholesterol ,total cholesterol and cholesterol ester content in macrophage foam cells were increased remarkably than PMA group macrophages (P<0 .01) .After treated with ac‐LDL for 48 h ,the macrophage foam cells accumulated 80 .25 μg/mg cell protein and 47 .65 μg/mg cell protein respectively ,and the cho‐lesterol ester accounted for 59 .38% of the cellular total cholesterol (P<0 .01) .The ratio of cholesterol efflux reached 5 .63% and 7 .08% respectively by HPLC analysis and liquid scintillation counting using apoA‐1 mediation (P<0 .01) .Conclusion Combina‐tion of an enzymatic catalysis and HPLC method for determining cholesterol efflux from foam cells is successfully established in this study , thus providing a technical foundation for the further study of cellular lipid homeostasis .

Cholesterol to cholestenone oxidation by ChoG, the main extracellular cholesterol oxidase of Rhodococcus ruber strain Chol-4.

The choG ORF of Rhodococcus ruber strain Chol-4 (referred from now as Chol-4) encodes a putative extracellular cholesterol oxidase. In the Chol-4 genome this ORF is located in a gene cluster that includes kstD3 and hsd4B, showing the same genomic context as that found in other Rhodococcus species. The putative ChoG protein is grouped into the class II of cholesterol oxidases, close to the Rhodococcus sp. CECT3014 ChoG homolog. The Chol-4 choG was cloned and expressed in a CECT3014 ΔchoG host strain in order to assess its ability to convert cholesterol into cholestenone. The RT-PCR analysis showed that choG gene was constitutively expressed in all the conditions assayed, but a higher induction could be inferred when cells were growing in the presence of cholesterol. A Chol-4 ΔchoG mutant strain was still able to grow in minimal medium supplemented with cholesterol, although at a slower rate. A comparative study of the removal of both cholesterol and cholestenone from the culture medium of either the wild type Chol-4 or its choG deletion mutant revealed a major role of ChoG in the extracellular production of cholestenone from cholesterol and, therefore, this enzyme may be related with the maintenance of a convenient supply of cholestenone for the succeeding steps of the catabolic pathway.

A DSC and FTIR spectroscopic study of the effects of the epimeric 4-cholesten-3-ols and 4-cholesten-3-one on the thermotropic phase behaviour and organization of dipalmitoylphosphatidylcholine bilayer membranes: comparison with their 5-cholesten analogues.

We present the results of a comparative differential calorimetric and Fourier transform infrared spectroscopic study of the effect of cholesterol and five of its analogues on the thermotropic phase behaviour and organization of dipalmitoylphosphatidylcholine bilayer membranes. These sterols/steroids differ in both the nature and stereochemistry of the polar head group at C3 (ßOH, αOH or C=O) and in the position of the double bond (C4-C5 in ring A or C5-C6 in ring B). In the three Δ(5) sterols/steroid series, the concentration of these compounds required to abolish the DPPC pretransition, inversely related to their relative ability to disorder gel state DPPC bilayers, decreases in the order ßOH>αOH>C=O and these differences in concentration are significant. However, in the Δ(4) series, these concentrations are more similar, regardless of polar head group nature or stereochemistry. Similarly, the residual enthalpy of the main phase transition of DPPC at 50 mol.% sterol/steroid, which is inversely related to the miscibility of these compounds in the DPPC bilayer, also increases in the order ßOH>αOH>C=O, but this effect is attenuated in the Δ(4) as opposed to the Δ(5) series. Both of these results indicate that the presence of a double bond at C4-C5 in ring A, as compared to a C5-C6 double bond in ring B, reduces the effect of variations in the structure of the polar group at C3 on the properties of the host DPPC bilayer. The movement of the double bond from C5 to C4 in the two sterol pairs results in a greater decrease in the temperature and enthalpy of both the pretransition and the main phase transition, whereas the opposite result is observed in the ketosteroid pair. Similarly, the ability of these compounds to order the DPPC hydrocarbon chains decreases in the order ßOH>αOH>C=O in both series of compounds, but in the two sterol pairs, hydrocarbon chain ordering is greater for the Δ(5) than the Δ(4) sterols, whereas the opposite is the case for the steroid pair. All of these results indicate that the typical effects of sterols/steroids in increasing the packing density and thermal stability of fluid lipid bilayers are optimal when an OH group rather than C=O group is present at C3, and that this OH group is more effective in the equatorial rather than the axial orientation. We can explain all of our sterol results by noting that the shift of the double bond from Δ(5) to Δ(4) introduces of a bend in ring A, which in turn destroys the coplanarity of the steroid fused ring system and reduces the goodness of sterol packing in the host DPPC bilayer. However, this conformational change should also occur in the ketosteroid pair, yet our experimental results indicate that the presence of the Δ(4) double bond is less disruptive than a double bond at Δ(5). We suggest that the presence of keto-enol tautomerism in the conjugated Δ(4) ketosteroid, but not in the nonconjugated Δ(5) compound, may provide additional H-bonding opportunities to adjacent DPPC molecules in the bilayer, which can overcome the unfavourable conformational change in ring A induced by the Δ(4) double bond.