Desmosterol Research Articles

SAT040 Administration Of A DHCR24 Inhibitor Reduces The Excessive Adrenal Steroidogenesis In db/db Mice Through Effects The Alteration Of Intracellular Cholesterol Metabolism

Abstract Disclosure: R. Kamigaki: None. H. Kameda: None. Y. Shibayama: None. H. Nomoto: None. K. Cho: None. A. Nakamura: None. S. Jin: None. K. Matoba: None. H. Miyoshi: None. T. Atsumi: None. Background and purpose: The circulating cortisol concentrations of patients with type 2 diabetes are high, suggesting that cortisol may be involved in diabetic complications. It has also been reported that the db/db mouse, a model of obesity and type 2 diabetes, has high circulating concentrations of steroid hormones. However, the mechanisms involved are unclear. In our previous study, we found that the expression of DHCR24, an enzyme that converts desmosterol (DES) to cholesterol (Chol) as part of de novo cholesterol synthesis, is upregulated in the adrenal glands of db/db mice. In the present study, we aimed to determine the effects of a DHCR24 inhibitor on adrenal steroidogenesis in db/db mice. Methods: Eight-week-old male db/db and db/+ mice were allocated to two groups, which were administered a weekly intraperitoneal injection of a DHCR24 inhibitor (U18666A; 10 mg/kg; (U) or PBS (P). Blood samples and their adrenal glands were collected 4 weeks later. We then compared the blood glucose, body mass, adrenal DES content, adrenal Chol content, and serum corticosterone (B) between the two groups. Sterols were extracted from adrenal tissue using the Folch method, and their concentrations were measured following picolinyl derivatization using liquid chromatography tandem-mass spectrometry. Results: There were no significant differences in the blood glucose concentrations or body masses of the P and U groups of both db/+ and db/db mice. The adrenal DES/adrenal mass ratio was higher in the db/db U group than in the db/db P group (db/db P vs. db/db U vs. db/+ P vs. db/+ U: 16.0±4.3 vs. 98.4±73.2 vs. 42.9±8.6 vs. 124.9±64.6 ng/mg, respectively; p<0.05). The adrenal Chol/adrenal mass and serum B concentration were lower in the db/db U and db/+ P groups than in the db/db P group (db/db P vs. db/db U vs. db/+ P vs. db/+ U: 2.1±0.2 vs. 1.3±0.2 vs. 1.1±0.6 vs. 1.3±0.2 μg/mg; and 232.8±20.1 vs. 189.6±13.1 vs. 126.3±20.6 vs. 111.2±25.8 ng/mL; respectively; all p<0.05). Conclusion: DHCR24 inhibition reduces cholesterol production and steroid hormone synthesis in the adrenal glands of db/db mice. This implies that the abnormal adrenal cholesterol metabolism in db/db mice plays a role in the greater adrenal steroidogenesis in type 2 diabetes, and that adrenal cholesterol metabolism may represent a novel therapeutic target for the affected patients. Presentation: Saturday, June 17, 2023

Characterization of Non-Cholesterol Sterols in Microglia Cell Membranes Using Targeted Mass Spectrometry.

Non-cholesterol sterols, as well as plant sterols, cross the blood-brain barrier and, thus, can be incorporated into cell membranes, affecting the cell's inflammatory response. The aim of our work was to develop an analytical protocol for a quantitative assessment of the sterol composition within the membrane microdomains of microglia. A protocol for cell membrane isolation using OptiPrepTM gradient ultracentrifugation, in combination with a targeted mass spectrometry (LC-MS/MS)-based assay, was developed and validated for the quantitative analysis of free sterols in microglia cell membranes. Utilizing an established LC-MS/MS assay, cholesterol and seven non-cholesterol sterols were analyzed with a limit of detection from 0.001 to 0.05 mg/L. Applying the detergent-free isolation of SIM-A9 microglia cell membranes, cholesterol (CH), desmosterol (DE), lanosterol (LA) stigmasterol (ST), beta-sitosterol (SI) and campesterol (CA) were quantified with coefficients of variations between 6 and 29% (fractions 4-6, n = 5). The highest concentrations of non-CH sterols within the microglia plasma membranes were found in the microdomain region (DE>LA>SI>ST>CA), with ratios to CH ranging from 2.3 to 435 lower abundancies. By applying our newly developed and validated analytical protocol, we show that the non-CH sterol concentration is about 38% of the total sterol content in microglia membrane microdomains. Further investigations must clarify how changes in the non-sterol composition influence membrane fluidity and cell signaling.

Desmosterol: A natural product derived from macroalgae modulates inflammatory response and oxidative stress pathways in intestinal epithelial cells.

The serum level of cholesterol and its biosynthetic intermediates are critical indicators to access metabolism-related disorders in humans and animals. However, the molecular actions of these intermediates on gene functions and regulation remained elusive. Here, we show that desmosterol (DES) is the most abundant intermediate involved in cholesterol biosynthesis and is highly enriched in red/brown algae. It exerts a pivotal role in modulating core genes involved in oxidative stress and inflammatory response processes in the ileum epithelial cells (IPI-2I). We observed that the DES extracted from red algae did not affect IPI-2I cell growth or survival. A transcriptomic measurement revealed that the genes enrolled in the oxidative process and cholesterol homeostasis pathway were significantly down-regulated by DES treatment. Consistent with this notion, cellular reactive oxygen species (ROS) levels were markedly decreased in response to DES treatment. In contrast, key inflammatory genes including IL-6, TNF-α, and IFN-γ were remarkably upregulated in the RNA-seq analysis, as further confirmed by qRT-PCR. Given that DES is a specific agonist of nuclear receptor RORγ, we also found that DES caused the elevated expression of RORγ at mRNA and protein levels, suggesting it is a potential mediator under DES administration. Together, these results underscore the vital physiological actions of DES in inflammatory and oxidative processes possibly via RORγ, and may be helpful in anti-oxidation treatment and immunotherapy in the future.

Effects of Psychotropic Medication on Somatic Sterol Biosynthesis of Adult Mice.

Polypharmacy is commonly used to treat psychiatric disorders. These combinations often include drugs with sterol biosynthesis inhibiting side effects, including the antipsychotic aripiprazole (ARI), and antidepressant trazodone (TRZ). As the effects of psychotropic medications are poorly understood across the various tissue types to date, we investigated the effects of ARI, TRZ, and ARI + TRZ polypharmacy on the post-lanosterol biosynthesis in three cell lines (Neuro2a, HepG2, and human dermal fibroblasts) and seven peripheral tissues of an adult mouse model. We found that both ARI and TRZ strongly interfere with the function of 7-dehydrocholesterol reductase enzyme (DHCR7) and lead to robust elevation in 7-dehydrocholesterol levels (7-DHC) and reduction in desmosterol (DES) across all cell lines and somatic tissues. ARI + TRZ co-administration resulted in summative or synergistic effects across the utilized in vitro and in vivo models. These findings suggest that at least some of the side effects of ARI and TRZ are not receptor mediated but arise from inhibiting DHCR7 enzyme activity. We propose that interference with sterol biosynthesis, particularly in the case of simultaneous utilization of medications with such side effects, can potentially interfere with functioning or development of multiple organ systems, warranting further investigation.

Individual and simultaneous treatment with antipsychotic aripiprazole and antidepressant trazodone inhibit sterol biosynthesis in the adult brain

Polypharmacy, or the simultaneous use of multiple drugs to treat a single patient, is a common practice in psychiatry. Unfortunately, data on the health effects of commonly used combinations of medications are very limited. In this study, we therefore investigated the effects and interactions between two commonly prescribed psychotropic medications with sterol inhibiting side effects, trazodone (TRZ), an antidepressant, and aripiprazole (ARI), an antipsychotic. In vitro cell culture experiments revealed that both medications alone disrupted neuronal and astroglial sterol biosynthesis in dose-dependent manners. Furthermore, when ARI and TRZ were combined, exposure resulted in an additive 7-dehydrocholesterol (7-DHC) increase, as well as desmosterol (DES) and cholesterol decreases in both cell types. In adult mice, at baseline, we found that the three investigated sterols showed significant differences in distribution across the eight assessed brain regions. Furthermore, experimental mice treated with ARI or TRZ, or a combination of both medications for 8 days, showed strong sterol disruption across all brain regions. We show ARI or TRZ alone elevated 7-DHC and decreased DES levels in all brain regions, but with regional differences. However, the combined utilization of these two medications for 8 days did not lead to additive changes in sterol disturbances. Based on the complex roles of 7-DHC derived oxysterols, we conclude that individual and potentially simultaneous use of medications with sterol biosynthesis-inhibiting properties might have undesired side effects on the adult brain, with as yet unknown long-term consequences on mental or physical health.

Cholesterol and desmosterol dancing to the beat of a different drug.

Cholesterol and desmosterol dancing to the beat of a different drug.

A calorimetric and spectroscopic comparison of the effects of cholesterol and its immediate biosynthetic precursors 7-dehydrocholesterol and desmosterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes

We performed differential scanning calorimetric (DSC) and Fourier transform infrared (FTIR) spectroscopic studies of the effects of cholesterol (CHOL), 7-dehydrocholeterol (7DHC) and desmosterol (DES) on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine (DPPC) bilayer membranes. 7DHC and DES are the immediate biosynthetic precursors of CHOL in the Kandutch–Russell and Bloch pathways and 7DHC and DES differ in structure from CHOL only by the presence of an additional double bond at C7 of ring B or C24 of the alkyl side chain, respectively. Our DSC results indicate that the incorporation of all three sterols produces comparable decreases in the temperature of the pretransition of DPPC, but CHOL decreases its cooperativity and enthalpy more strongly than 7DHC and especially DES. These findings indicate that all three sterols decrease the thermal stability of gel phase DPPC bilayers but that 7DHC and especially DES are less miscible in them. However, the incorporation of CHOL and DES produce comparable increases in the temperature of the broad component of the main phase transition of DPPC while 7DHC decreases it, but again CHOL produces greater decreases in its cooperativity and enthalpy then 7DHC and especially DES. These results indicate that CHOL and DES stabilize the sterol-rich domains of fluid DPPC bilayers, but that 7DHC and especially DES are less miscible in them. Our FTIR spectroscopic results indicate that CHOL increases the rotational conformational order of fluid DPPC bilayers to a somewhat and markedly greater degree than DES and 7DHC, respectively, consistent with our DSC findings. Our spectroscopic results also indicate that although all three sterols produce comparable degrees of H-bonding (hydration) of the DPPC ester carbonyl groups in fluid bilayers, CHOL is again found to be fully soluble in gel state DPPC bilayers at low temperatures, whereas 7DHC and especially DES are not. In general, we find that 7DHC and DES incorporation produce considerably different effects on DPPC bilayer membranes. In particular, the presence of an additional double bond at C7 or C24 produces a marked reduction in the ability of 7DHC to order fluid DPPC bilayers and in the miscibility of DES in such bilayers, respectively. These different effects may be the biophysical basis for the reduction of these double bonds in the last steps of CHOL biosynthesis, and for the deleterious biological effects of the accumulation of these sterols in vivo.

4,4'-Diethylaminoethoxyhexestrol dihydrochloride

The bis-diethylaminoethylether of hexestrol has been shown to be hypocholesteremic by inhibiting the reduction of desmosterol to cholesterol.[...]

A comparison of the behavior of cholesterol and selected derivatives in mixed sterol–phospholipid Langmuir monolayers: a fluorescence microscopy study

Eukaryotic cells require sterols to achieve normal structure and function of their plasma membranes, and deviations from normal sterol composition can perturb these features and compromise cellular and organism viability. The Smith–Lemli–Opitz syndrome (SLOS) is a hereditary metabolic disease involving cholesterol (CHOL) deficiency and abnormal accumulation of the CHOL precursor, 7-dehydrocholesterol (7DHC). In this study, the interactions of CHOL and the related sterols desmosterol (DES) and 7DHC with l-α-dipalmitoylphosphatidylcholine (DPPC) monolayers were compared. Pressure–area isotherms and fluorescence microscopy were used to study DPPC monolayers containing 0, 10, 20, or 30 mol% sterol. Similar behavior was noted for CHOL- and DES-containing DPPC monolayers with both techniques. However, while 7DHC gave isotherms similar to those obtained with the other sterols, microscopy indicated limited domain formation with DPPC, indicating that 7DHC packs somewhat differently in DPPC membranes compared to CHOL and DES. These results are discussed in relation to SLOS pathobiology.

Comparative behavior of sterols in phosphatidylcholine-sterol monolayer films

The ability of sterols other than cholesterol (CHOL) to support membrane functions in membranes that normally contain CHOL as the primary, if not sole, sterol may be due, in part, to how well such sterols can mimic CHOL’s behavior and physical properties in membranes. We compared the mixing properties of CHOL, 7-dehydrocholesterol (7DHC), and desmosterol (DES) in egg phosphatidylcholine-sterol monolayer films containing 10, 20, and 30 mol percent sterol, measuring pressure-area isotherms on a Langmuir–Blodgett trough with the aqueous, buffered subphase maintained at 37°C. Under the conditions employed, the pressure-area isotherms for all three sterols were similar, with 7DHC exhibiting slightly larger molecular areas on the water surface at all compositions. These results are discussed in the context of the ability of sterols such as 7DHC and DES to substitute structurally and functionally for CHOL in biological membranes.

Drosophila melanogaster does not dealkylate [14C]sitosterol

Drosophila melanogaster was unable to dealkylate and convert [14C]sitosterol to cholesterol and no evidence was found for conversion of [14C]desmosterol to cholesterol. Therefore,D. melanogaster is incapable of dealkylating and converting C28 and C29 phytosterols to cholesterol.

Preparation of (26-13C)desmosterol.

Preparation of (26-13C)desmosterol.

The effect of 3-methylcholanthrene on postlanosterol cholesterol biosynthetic pathways in mouse skin.

Repeated topical application of 3-methylcholanthrene to the backs of BALB/c mice lowered the tissue levels of lathosterol and provitamin D3, intermediates in one of the cholesterol biosynthetic pathways (the delta 7 pathway), though the activities of lathosterol 5-desaturase and provitamin D3 7-reductase were similar to those of control animals. These results seemed to indicate that carcinogen treatment exerted a depressive effect at some earlier step(s) than lathosterol synthesis. However, the content of cholesterol in mouse skin was not lowered in these animals, suggesting that another biosynthetic pathway might be activated. When diazacholesterol, which is known to inhibit the conversion of desmosterol to cholesterol, was administered together with the carcinogen, a marked accumulation of desmosterol was observed compared to animals given only diazacholesterol. Since desmosterol is an intermediate in the pathway in which the delta 24 double bond is reduced at the final step (the delta 24 pathway), this seemed to suggest that the delta 24 pathway was activated by carcinogen treatment.

Desmosterol in CSF

Desmosterol in CSF

The occurrence of desmosterol in myelin of developing rats

The occurrence of desmosterol in myelin of developing rats

Induced myotonia in man and goat.

Abstract Treatment of 41 hypercholesterolemic patients with the cholesterol-lowering agent, diazacholesterol, an analogue of cholesterol, was associated with the development of muscular spasms in 30 individuals; electromyography (EMG) disclosed myotonia in 27 of 28 patients so tested. Associated with the clinical and EMG myotonia were alterations in the body sterol composition, as determined by gas-liquid chromatography (GLC). The plasma of all but one of the 36 patients so studied showed a fall in total sterols, while the plasma, erythrocyte membrane, and the one muscle sample examined were marked by the appearance of desmosterol, the immediate precursor of cholesterol. Discontinuation of diazacholesterol therapy was accompanied by the disappearance of clinical and EMG manifestations of myotonia, a marked reduction in plasma and erythrocyte desmosterol, and a concomitant rise in total sterol levels. Administration of diazacholesterol to normal, myotonic, and hybrid goats was associated with the appearance of myotonia or the worsening of pre-existing myotonia, a decline in plasma total sterols, and the appearance of desmosterol in plasma, erythrocyte membrane, and muscle. The findings suggest that diazacholesterol may cause clinical and electromyographic findings similar to those observed in the human myotonic disorders and in myotonic goats. The role of altered sterol synthesis in the pathogenesis of the findings is now being investigated.