Search

Membrane-associated ring-CH-type finger 6 (MARCH6), also designated as TEB4 or RNF176, is an E3 ligase that is embedded in membranes of the endoplasmic reticulum where it ubiquitinates many substrate proteins to consign them to proteasome-mediated degradation. In recent years, MARCH6 has been identified as a key regulator of several metabolic pathways, including cholesterol and lipid droplet homeostasis, protein quality control, ferroptosis, and tumorigenesis. Despite its importance, there are currently no specific antibodies to detect and monitor MARCH6 levels in cultured cells and animals. Here, we address this deficiency by generating a monoclonal antibody that specifically detects MARCH6 in cultured cells of insect, mouse, hamster, and human origin, as well as in mouse tissues, with minimal cross-reactivity against other proteins. We then used this antibody to assess two properties of MARCH6. First, analysis of mouse tissues with this antibody revealed that the liver contained the highest levels of March6. Second, analysis of five different cell lines with this antibody showed that endogenous levels of MARCH6 are unchanged as the cellular content of cholesterol is varied. This reagent promises to be a useful tool in interrogating additional signaling roles of MARCH6.

Background: Cholesterol is an essential component of cell membranes whose local de novo biosynthesis may occur in response to additional cellular requirements, especially cell proliferation. In this study, we investigated: (1) the differential expression of the genes coding for the main enzymes involved in cholesterol biosynthesis (ACAT2, HMGCS1, HMGCR, FDFT1, SQLE, LSS, and NSDHL), or the proteins that control their activity (SREBF2, SCAP, and INSIG1), in patient-matched samples of breast cancer and adjacent histologically normal (HN) tissue; (2) their association with the expression of MKI67 or the histologic tumor grade (in particular, G2); (3) their association with recurrence-free survival (RFS). Methods: Nonparametric rank-based models for longitudinal data were applied to assess the differential gene expression between the tumor and the adjacent HN tissue from the same patient or between the classes of tumor grade. Spearman's rank correlation and Cox proportional hazard models were used to assess the correlation between the genes and their association with RFS. Results: Compared to the adjacent HN tissue, HMGCS1, HMGCR, SQLE, and NSDHL genes were more expressed in the tumor. Their expression progressively increased according to tumor grade and correlated positively with MKI67. ACAT2, HMGCR, and NSDHL genes were associated with a high risk of recurrence even when adjusted for age, tumor grade, or immunohistochemical Ki67. Conclusion: The findings indicated that some genes involved in cholesterol biosynthesis were more expressed in cancerous tissue, correlated positively with tumor grade and MKI67 expression, and were associated with RFS, thus substantiating the relationship between de novo cholesterol biosynthesis and tumor aggressiveness. [ABSTRACT FROM AUTHOR]

Membrane-associated ring-CH-type finger 6 (MARCH6), also designated as TEB4 or RNF176, is an E3 ligase that is embedded in membranes of the endoplasmic reticulum where it ubiquitinates many substrate proteins to consign them to proteasome-mediated degradation. In recent years, MARCH6 has been identified as a key regulator of several metabolic pathways, including cholesterol and lipid droplet homeostasis, protein quality control, ferroptosis, and tumorigenesis. Despite its importance, there are currently no specific antibodies to detect and monitor MARCH6 levels in cultured cells and animals. Here, we address this deficiency by generating a monoclonal antibody that specifically detects MARCH6 in cultured cells of insect, mouse, hamster, and human origin, as well as in mouse tissues, with minimal cross-reactivity against other proteins. We then used this antibody to assess two properties of MARCH6. First, analysis of mouse tissues with this antibody revealed that the liver contained the highest levels of March6. Second, analysis of five different cell lines with this antibody showed that endogenous levels of MARCH6 are unchanged as the cellular content of cholesterol is varied. This reagent promises to be a useful tool in interrogating additional signaling roles of MARCH6., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

To elucidate the transcriptomic changes of long noncoding RNAs (lncRNAs) in high-fat diet (HFD)-fed mice, we defined their hepatic transcriptome by RNA sequencing. Aberrant expression of 37 representative lncRNAs and 254 protein-coding RNAs was observed in the livers of HFD-fed mice with insulin resistance compared with the livers from control mice. Of these, 24 lncRNAs and 179 protein-coding RNAs were upregulated, whereas 13 lncRNAs and 75 protein-coding RNAs were downregulated. Functional analyses showed that the aberrantly expressed protein-coding RNAs were enriched in various lipid metabolic processes and in the insulin signaling pathway. Genomic juxtaposition and coexpression patterns identified six pairs of aberrantly expressed lncRNAs and protein-coding genes, consisting of five lncRNAs and five protein-coding genes. Four of these protein-coding genes are targeted genes upregulated by PPARα. As expected, the corresponding lncRNAs were significantly elevated in AML12 cells treated with palmitic acid or the PPARα agonist, WY14643. In Hepa1-6 cells, knockdown of NONMMUG027912 increased the cellular cholesterol level, the expression of cholesterol biosynthesis genes and proteins, and the HMG-CoA reductase activity. This genome-wide profiling of lncRNAs in HFD-fed mice reveals one lncRNA, NONMMUG027912, which is potentially regulated by PPARα and is implicated in the process of cholesterol biosynthesis.

Very few studies have investigated the interrelations between proprotein convertase subtilisin/kexin type 9 (PCSK9) metabolism, cholesterol synthesis, and cholesterol absorption. We aimed to address this issue in a large clinical trial of 245 patients with hypercholesterolemia. Serum lipids, PCSK9, lathosterol (cholesterol synthesis marker), campesterol, and sitosterol (cholesterol absorption markers) were measured before and 4–8 weeks after the start of treatment with PCSK9-antibodies (alirocumab or evolocumab). The patients had mean (standard error) LDL-cholesterol and PCSK9 concentrations of 3.87 (0.10) mmol/l and 356 (17) ng/ml, respectively. Eighty-four patients received no lipid-lowering pretreatment, 26 ezetimibe, 38 statins, and 97 ezetimibe + statins. Circulating PCSK9 increased in parallel with the potency of lipid-lowering pretreatment with circulating PCSK9 being highest in the ezetimibe + statin group (P < 0.001). Treatment with PCSK9-antibodies strongly decreased LDL-cholesterol, lathosterol, campesterol, and sitosterol (all P < 0.001) but hardly affected noncholesterol sterol to cholesterol ratios. Lipid-lowering pretreatment was not associated with the effects of PCSK9-antibodies on noncholesterol sterols (all P > 0.05). Summing up, circulating PCSK9 is increased by cholesterol synthesis and absorption inhibitors. Increased PCSK9 expression may partly explain the strong reductions of LDL-cholesterol achieved with PCSK9-antibodies after such pretreatment. On the other hand, treatment with PCSK9-antibodies does not significantly change the balance between cholesterol synthesis and absorption.

Regulating blood cholesterol (Chol) levels by pharmacotherapy has successfully improved cardiovascular health. There is growing interest in the role of Chol precursors in the treatment of diseases. One sterol precursor, desmosterol (Des), is a potential pharmacological target for inflammatory and neurodegenerative disorders. However, elevating levels of the precursor 7-dehydrocholesterol (7-DHC) by inhibiting the enzyme 7-dehydrocholesterol reductase is linked to teratogenic outcomes. Thus, altering the sterol profile may either increase risk toward an adverse outcome or confer therapeutic benefit depending on the metabolite affected by the pharmacophore. In order to characterize any unknown activity of drugs on Chol biosynthesis, a chemical library of Food and Drug Administration-approved drugs was screened for the potential to modulate 7-DHC or Des levels in a neural cell line. Over 20% of the collection was shown to impact Chol biosynthesis, including 75 compounds that alter 7-DHC levels and 49 that modulate Des levels. Evidence is provided that three tyrosine kinase inhibitors, imatinib, ponatinib, and masitinib, elevate Des levels as well as other substrates of 24-dehydrocholesterol reductase, the enzyme responsible for converting Des to Chol. Additionally, the mechanism of action for ponatinib and masitinib was explored, demonstrating that protein levels are decreased as a result of treatment with these drugs.

We previously demonstrated that oligomeric amyloid β42 (oAβ42) inhibits the mevalonate pathway impairing cholesterol synthesis and protein prenylation. Enzymes of the mevalonate pathway are regulated by the transcription factor SREBP-2. Here, we show that in several neuronal types challenged with oAβ42, SREBP-2 activation is reduced. Moreover, SREBP-2 activation is also decreased in the brain cortex of the Alzheimer's disease (AD) mouse model, TgCRND8, suggesting that SREBP-2 may be affected in vivo early in the disease. We demonstrate that oAβ42 does not affect enzymatic cleavage of SREBP-2 per se, but may impair SREBP-2 transport from the endoplasmic reticulum (ER) to the Golgi. Trafficking of SREBP-2 from the ER to the Golgi requires protein kinase B (Akt) activation. oAβ42 significantly reduces Akt phosphorylation and this decrease is responsible for the decline in SREBP-2 activation. Overexpression of constitutively active Akt prevents the effect of oAβ42 on SREBP-2 and the downstream inhibition of cholesterol synthesis and protein prenylation. Our work provides a novel mechanistic link between Aβ and the mevalonate pathway, which will impact the views on issues related to cholesterol, isoprenoids, and statins in AD. We also identify SREBP-2 as an indirect target of Akt in neurons, which may play a role in the cross-talk between AD and diabetes.

Background Inverse associations have been reported between serum 25-hydroxyvitamin D [25(OH)D] and circulating cholesterol concentrations in observational studies. Postulated mechanisms include reduced bioavailability of intestinal cholesterol and alterations in endogenous cholesterol synthesis. Objective To explore the effect of daily supplementation with 4000 IU/d vitamin D3 for 24 wk on surrogate biomarkers of cholesterol absorption (campesterol and β-sitosterol) and endogenous synthesis (lathosterol and desmosterol). Methods Ancillary study of The Vitamin D for Established Type 2 Diabetes (DDM2) trial. Patients with established type 2 diabetes (N = 127, 25–75 y, BMI 23–42 kg/m2) were randomly assigned to receive either 4000 IU vitamin D3 or placebo daily for 24 wk. Of participants without changes in cholesterol-lowering medications (n  = 114), plasma surrogate cholesterol absorption and endogenous synthesis biomarker concentrations were measured and merged with available measures of serum LDL cholesterol and HDL cholesterol concentrations. Results At week 24, vitamin D3 supplementation significantly increased 25(OH)D concentrations (+21.5 ± 13.4 ng/mL) but not insulin secretion rates (primary outcome of the parent study) as reported previously. In this ancillary study there was no significant effect of vitamin D3 supplementation on serum cholesterol profile or surrogate biomarkers of cholesterol absorption and endogenous synthesis. Compared with participants not treated with cholesterol-lowering medications, those who were treated exhibited a greater reduction in plasma campesterol concentrations in the vitamin D3 but not placebo group (P- interaction = 0.011). Analyzing the data on the basis of cholesterol absorption status (hypo- versus hyperabsorbers) or cholesterol synthesis status (hypo- versus hypersynthesizers) did not alter these results. Conclusions Vitamin D3 supplementation for 24 wk had no significant effect on surrogate biomarkers of cholesterol absorption or endogenous synthesis, consistent with the lack of effect on serum cholesterol profile. Vitamin D3 supplementation resulted in greater reduction in campesterol concentrations in participants not using compared with those using cholesterol-lowering medications. Further studies are required. This trial was registered at clinicaltrials.gov as NCT01736865. [ABSTRACT FROM AUTHOR]

SREBP-2 activates transcription of all genes needed for cholesterol biosynthesis. To study SREBP-2 function in the intestine, we generated a mouse model (Vil-BP2−/−) in which Cre recombinase ablates SREBP-2 in intestinal epithelia. Intestines of Vil-BP2−/− mice had reduced expression of genes required for sterol synthesis, in vivo sterol synthesis rates, and epithelial cholesterol contents. On a cholesterol-free diet, the mice displayed chronic enteropathy with histological abnormalities of both villi and crypts, growth restriction, and reduced survival that was prevented by supplementation of cholesterol in the diet. Likewise, SREBP-2-deficient enteroids required exogenous cholesterol for growth. Blockade of luminal cholesterol uptake into enterocytes with ezetimibe precipitated acutely lethal intestinal damage in Vil-BP2−/− mice, highlighting the critical interplay in the small intestine of sterol absorption via NPC1L1 and sterol synthesis via SREBP-2 in sustaining the intestinal mucosa. These data show that the small intestine requires SREBP-2 to drive cholesterol synthesis that sustains the intestinal epithelia when uptake of cholesterol from the gut lumen is not available, and provide a unique example of cholesterol auxotrophy expressed in an intact, adult mammal.

Ion mobility-mass spectrometry (IM-MS) has proven to be a highly informative technique for the characterization of lipids from cells and tissues. We report the combination of hydrophilic-interaction liquid chromatography (HILIC) with traveling-wave IM-MS (TWIM-MS) for comprehensive lipidomics analysis. Main lipid categories such as glycerolipids, sphingolipids, and glycerophospholipids are separated on the basis of their lipid backbones in the IM dimension, whereas subclasses of each category are mostly separated on the basis of their headgroups in the HILIC dimension, demonstrating the orthogonality of HILIC and IM separations. Using our previously established lipid calibrants for collision cross-section (CCS) measurements in TWIM, we measured over 250 CCS values covering 12 lipid classes in positive and negative modes. The coverage of the HILIC-IM-MS method is demonstrated in the analysis of Neuro2a neuroblastoma cells exposed to benzalkonium chlorides (BACs) with C10 or C16 alkyl chains, which we have previously shown to affect gene expression related to cholesterol and lipid homeostasis. We found that BAC exposure resulted in significant changes to several lipid classes, including glycerides, sphingomyelins, phosphatidylcholines, and phosphatidylethanolamines. Our results indicate that BAC exposure modifies lipid homeostasis in a manner that is dependent upon the length of the BAC alkyl chain.

The effects of cholesterol-lowering drugs, including those that reduce cholesterol synthesis (statins) and those that reduce cholesterol absorption (ezetimibe), on cholesterol absorption and synthesis are well understood. Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors are a novel class of cholesterol-lowering drugs that robustly reduce LDL-cholesterol (LDL-C), but little is known about their effects on cholesterol absorption and synthesis. We evaluated how treatment with evolocumab, a fully human monoclonal IgG2 antibody to PCSK9, affects markers of cholesterol synthesis and absorption by measuring these markers in patients from an evolocumab clinical trial. At 2 weeks, changes in β-sitosterol/total cholesterol (TC) from baseline were 4% for placebo, 10% for evolocumab 140 mg (nonsignificant vs. placebo), and 26% for evolocumab 420 mg (P < 0.001 vs. placebo). Changes in campesterol/TC at week 2, relative to baseline between placebo and evolocumab, were all nonsignificant. Evolocumab had a modest effect on markers of cholesterol synthesis. At 2 weeks, changes in desmosterol/TC were 1% for placebo, 7% for evolocumab 140 mg (nonsignificant vs. placebo), and 15% for evolocumab 420 mg (P < 0.01 vs. placebo). Changes from baseline in lathosterol/TC at week 2 between placebo and evolocumab were nonsignificant. These results suggest that evolocumab has a modest effect on cholesterol synthesis and absorption despite significant LDL-C lowering.

Statins are competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. Statins reduce plasma cholesterol levels, but whether this is actually caused by inhibition of de novo cholesterol synthesis has not been clearly established. Using three different statins, we investigated the effects on cholesterol metabolism in mice in detail. Surprisingly, direct measurement of whole body cholesterol synthesis revealed that cholesterol synthesis was robustly increased in statin-treated mice. Measurement of organ-specific cholesterol synthesis demonstrated that the liver is predominantly responsible for the increase in cholesterol synthesis. Excess synthesized cholesterol did not accumulate in the plasma, as plasma cholesterol decreased. However, statin treatment led to an increase in cholesterol removal via the feces. Interestingly, enhanced cholesterol excretion in response to rosuvastatin and lovastatin treatment was mainly mediated via biliary cholesterol secretion, whereas atorvastatin mainly stimulated cholesterol removal via the transintestinal cholesterol excretion pathway. Moreover, we show that plasma cholesterol precursor levels do not reflect cholesterol synthesis rates during statin treatment in mice. In conclusion, cholesterol synthesis is paradoxically increased upon statin treatment in mice. However, statins potently stimulate the excretion of cholesterol from the body, which sheds new light on possible mechanisms underlying the cholesterol-lowering effects of statins.

Rapidly multiplying cancer cells synthesize greater amounts of cholesterol to build their membranes. Cholesterol-lowering drugs (statins) are currently in clinical trials for anticancer chemotherapy. However, given at higher doses, statins cause serious side effects by inhibiting the formation of other biologically important molecules derived from mevalonate. Sterol 14α-demethylase (CYP51), which acts 10 steps downstream, is potentially a more specific drug target because this portion of the pathway is fully committed to cholesterol production. However, screening a variety of commercial and experimental inhibitors of microbial CYP51 orthologs revealed that most of them (including all clinical antifungals) weakly inhibit human CYP51 activity, even if they display high apparent spectral binding affinity. Only one relatively potent compound, (R)-N-(1-(3,4′-difluorobiphenyl-4-yl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VFV), was identified. VFV has been further tested in cellular experiments and found to decrease proliferation of different cancer cell types. The crystal structures of human CYP51-VFV complexes (2.0 and 2.5 Å) both display a 2:1 inhibitor/enzyme stoichiometry, provide molecular insights regarding a broader substrate profile, faster catalysis, and weaker susceptibility of human CYP51 to inhibition, and outline directions for the development of more potent inhibitors.

The gut microbiota influences many aspects of host metabolism. We have previously shown that the presence of a gut microbiota remodels lipid composition. Here we investigated how interaction between gut microbiota and dietary lipids regulates lipid composition in the liver and plasma, and gene expression in the liver. Germ-free and conventionally raised mice were fed a lard or fish oil diet for 11 weeks. We performed lipidomics analysis of the liver and serum and microarray analysis of the liver. As expected, most of the variation in the lipidomics dataset was induced by the diet, and abundance of most lipid classes differed between mice fed lard and fish oil. However, the gut microbiota also affected lipid composition. The gut microbiota increased hepatic levels of cholesterol and cholesteryl esters in mice fed lard, but not in mice fed fish oil. Serum levels of cholesterol and cholesteryl esters were not affected by the gut microbiota. Genes encoding enzymes involved in cholesterol biosynthesis were downregulated by the gut microbiota in mice fed lard and were expressed at a low level in mice fed fish oil independent of microbial status. In summary, we show that gut microbiota-induced regulation of hepatic cholesterol metabolism is dependent on dietary lipid composition.

Cholesterol and its biosynthetic pathway intermediates and derivatives are required for many developmental processes including membrane biogenesis, transmembrane receptor signaling, steroid biogenesis, nuclear receptor activation, and posttranslational modification of hedgehog (Hh) proteins. To perform such multifaceted tasks depends on stringent regulation of expression of cholesterol biosynthetic enzymes (CBEs). We established for a whole organism, for the first time, the 3D expression pattern of all genes required for cholesterol biosynthesis (CBS), starting from acetyl-CoA and ending with cholesterol. This data was produced by high-throughput in situ hybridization on serial sections through the mouse fetus. The textually annotated image data were seamlessly integrated into the METscout and GenePaint public databases. This novel information helps in the understanding of why CBEs are expressed at particular locations within the fetus. For example, strong CBE expression is detected at sites of cell proliferation and also where cell growth increases membrane surface, such as in neurons sprouting axons and forming synapses. The CBE data also sheds light on the spatial relationship of cells and tissue that express sonic Hh (Shh) and produce cholesterol, respectively. We discovered that not all cells expressing Shh are capable of CBS. This finding suggests novel ways by which cholesterylation of Shh is regulated.

SREBP cleavage-activating protein (Scap) is an endoplasmic reticulum membrane protein required for cleavage and activation of sterol regulatory element-binding proteins (SREBPs), which activate the transcription of genes in sterol and fatty acid biosynthesis. Liver-specific loss of Scap is well tolerated; hepatic synthesis of sterols and fatty acids is reduced, but mice are otherwise healthy. To determine whether Scap loss is tolerated in the intestine, we generated a mouse model (Vil-Scap−) in which tamoxifen-inducible Cre-ERT2, a fusion protein of Cre recombinase with a mutated ligand binding domain of the human estrogen receptor, ablates Scap in intestinal mucosa. After 4 days of tamoxifen, Vil-Scap− mice succumb with a severe enteropathy and near-complete collapse of intestinal mucosa. Organoids grown ex vivo from intestinal crypts of Vil-Scap− mice are readily killed when Scap is deleted by 4-hydroxytamoxifen. Death is prevented when culture medium is supplemented with cholesterol and oleate. These data show that, unlike the liver, the intestine requires Scap to sustain tissue integrity by maintaining the high levels of lipid synthesis necessary for proliferation of intestinal crypts.

Squalene synthase (SS) catalyzes the biosynthesis of squalene, the first specific intermediate in the cholesterol biosynthetic pathway. To test the feasibility of lowering plasma cholesterol by inhibiting hepatic SS, we generated mice in which SS is specifically knocked out in the liver (L-SSKO) using Cre-loxP technology. Hepatic SS activity of L-SSKO mice was reduced by >90%. In addition, cholesterol biosynthesis in the liver slices was almost eliminated. Although the hepatic squalene contents were markedly reduced in L-SSKO mice, the hepatic contents of cholesterol and its precursors distal to squalene were indistinguishable from those of control mice, indicating the presence of sufficient centripetal flow of cholesterol and/or its precursors from the extrahepatic tissues. L-SSKO mice showed a transient liver dysfunction with moderate hepatomegaly presumably secondary to increased farnesol production. In a fed state, the plasma total cholesterol and triglyceride were significantly reduced in L-SSKO mice, primarily owing to reduced hepatic VLDL secretion. In a fasted state, the hypolipidemic effect was lost. mRNA expression of liver X receptor α target genes was reduced, while that of sterol-regulatory element binding protein 2 target genes was increased. In conclusion, liver-specific ablation of SS inhibits hepatic cholesterol biosynthesis and induces hypolipidemia without increasing significant mortality.

Previous studies suggest an interdependent relationship between liver and intestine for cholesterol elimination from the body. We hypothesized that a combination of ursodiol (Urso) and ezetimibe (EZ) could increase biliary secretion and reduce cholesterol reabsorption, respectively, to promote cholesterol excretion. Treatment with Urso increased hepatic ABCG5 ABCG8 (G5G8) protein and both biliary and fecal sterols in a dose-dependent manner. To determine whether the drug combination (Urso-EZ) further increased cholesterol excretion, mice were treated with Urso alone or in combination with two doses of EZ. EZ produced an additive and dose-dependent increase in fecal neutral sterol (FNS) elimination in the presence of Urso. Finally, we sequentially treated wide-type and G5G8-deficient mice with Urso and Urso-EZ to determine the extent to which these effects were G5G8 dependent. Although biliary and FNS were invariably lower in G5G8 KO mice, the relative increase in FNS following treatment with Urso alone or the Urso-EZ combination was not affected by genotype. In conclusion, Urso increases G5G8, biliary cholesterol secretion, and FNS and acts additively with EZ to promote fecal sterol excretion. However, the stimulatory effect of these agents was not G5G8 dependent.

Highlights • Lysophosphatidylcholine (LPC) is associated with oxidation of low density lipoproteins (LDL) and atherosclerosis. • We elucidated RNA profiling from LPC treated THP-1 derived macrophages using RNA sequencing method. • The LPC affected the expression of genes involved in cholesterol synthesis pathways. • LPCs with saturated acyl groups is involved in cholesterol biosynthesis. Abstract Lysophosphatidylcholine (LPC), a major component of oxidized low-density lipoprotein, is associated with atherosclerosis, obesity, stroke, and cancer. However, the direction and mechanism of this relationship remains unclear. In this study, we conducted RNA profiling in THP-1 derived macrophages treated with LPC and uncovered a relationship between LPC and the cholesterol biosynthesis pathway. Principal component analysis (PCA) of RNA profiling showed that untreated THP-1 cells and those treated with 10, 20, or 40 µM LPC were distinctly distributed. Functional annotation revealed that LPC affected the expression of genes involved in cytokine–cytokine receptor interaction, TNF signaling, and MAPK signaling. Interestingly, LPC also altered the expression of 11 genes involved in cholesterol synthesis such as those in terpenoid backbone biosynthesis and steroid biosynthesis pathways. This increased gene expression occurred in a dose-dependent manner in response to LPC treatment. Especially, LPC with saturated acyl groups enhanced the expression of these genes compared to LPC with unsaturated acyl groups, and similar results were shown in response to saturated and unsaturated free fatty acids. Our findings demonstrate that LPCs with saturated acyl groups induce the expression of genes involved in cholesterol biosynthesis and may have implications for cholesterol related diseases. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) cells exhibit an increase in the protein expression of β-arrestin-2 (βarr2) coincident with perinuclear accumulation of free cholesterol. Arrestins are proteins that both serve as broad signaling regulators and contribute to G-protein coupled receptor internalization after agonist stimulation. The hypothesis of this study is that βarr2 is an important component in the mechanisms leading to cholesterol accumulation characteristic of CF cells. To test this hypothesis, epithelial cells stably expressing GFP-tagged βarr2 (βarr2-GFP) and respective GFP-expressing control cells (cont-GFP) were analyzed by filipin staining. The βarr2-GFP cells show a late endosomal/lysosomal cholesterol accumulation that is identical to that seen in CF cells. This βarr2-mediated accumulation is sensitive to Rp-cAMPS treatment, and depleting βarr2 expression in CF-model cells by shRNA alleviates cholesterol accumulation compared with controls. Cftr/βarr2 double knockout mice also exhibit wild-type (WT) levels of cholesterol synthesis, and WT profiles of signaling protein expression have previously been shown to be altered in CF due to cholesterol-related pathways. These data indicate a significant regulatory role for βarr2 in the development of CF-like cholesterol accumulation and give further insight into cholesterol processing mechanisms. An impact of βarr2 expression on Niemann-Pick type C-1 (NPC1)-containing organelle movement is proposed as the mechanism of βarr2-mediated alterations on cholesterol processing. It is concluded that βarr2 expression contributes to altered cholesterol trafficking observed in CF cells.

Enterocyte cholesterol homeostasis reflects aggregated rates of sterol synthesis, efflux, and uptake from plasma and gut lumen. Cholesterol synthesis and LDL uptake are coordinately regulated by sterol regulatory element-binding proteins (SREBP), whereas sterol efflux is regulated by liver X receptors (LXR). How these processes are coordinately regulated in enterocytes, the site of cholesterol absorption, is not well understood. Here, we treat mice with ezetimibe to investigate the effect of blocking cholesterol absorption on intestinal SREBPs, LXRs, and their effectors. Ezetimibe increased nuclear SREBP-2 8-fold. HMG-CoA reductase (HMGR) and LDL receptor (LDLR) mRNA levels increased less than 3-fold, whereas their protein levels increased 30- and 10-fold, respectively. Expression of inducible degrader of LDLR (IDOL), an LXR-regulated gene that degrades LDLRs, was reduced 50% by ezetimibe. Coadministration of ezetimibe with the LXR agonist T0901317 abolished the reduction in IDOL and prevented the increase in LDLR protein. Ezetimibe-stimulated LDLR expression was independent of proprotein convertase subtilisin/kexin type 9 (PSCK9), a protein that degrades LDLRs. To maintain cholesterol homeostasis in the face of ezetimibe, enterocytes boost LDL uptake by increasing LDLR number, and they boost sterol synthesis by increasing HMGR and other cholesterologenic genes. These studies reveal a hitherto undescribed homeostatic network in enterocytes triggered by blockade of cholesterol absorption.

We describe the development of a method for the extraction and analysis of 62 sterols, oxysterols, and secosteroids from human plasma using a combination of HPLC-MS and GC-MS. Deuterated standards are added to 200 μl of human plasma. Bulk lipids are extracted with methanol:dichloromethane, the sample is hydrolyzed using a novel procedure, and sterols and secosteroids are isolated using solid-phase extraction (SPE). Compounds are resolved on C18 core-shell HPLC columns and by GC. Sterols and oxysterols are measured using triple quadrupole mass spectrometers, and lathosterol is measured using GC-MS. Detection for each compound measured by HPLC-MS was ∪ 1 ng/ml of plasma. Extraction efficiency was between 85 and 110%; day-to-day variability showed a relative standard error of

Treatment of Sprague-Dawley rats with AY9944, an inhibitor of 3β-hydroxysterol-Δ7-reductase (Dhcr7), leads to elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in all biological tissues, mimicking the key biochemical hallmark of Smith-Lemli-Opitz syndrome (SLOS). Fourteen 7-DHC-derived oxysterols previously have been identified as products of free radical oxidation in vitro; one of these oxysterols, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), was recently identified in Dhcr7-deficient cells and in brain tissues of Dhcr7-null mouse. We report here the isolation and characterization of three novel 7-DHC-derived oxysterols (4α- and 4β-hydroxy-7-DHC and 24-hydroxy-7-DHC) in addition to DHCEO and 7-ketocholesterol (7-kChol) from the brain tissues of AY9944-treated rats. The identities of these five oxysterols were elucidated by HPLC-ultraviolet (UV), HPLC-MS, and 1D- and 2D-NMR. Quantification of 4α- and 4β-hydroxy-7-DHC, DHCEO, and 7-kChol in rat brain, liver, and serum were carried out by HPLC-MS using d7-DHCEO as an internal standard. With the exception of 7-kChol, these oxysterols were present only in tissues of AY9944-treated, but not control rats, and 7-kChol levels were markedly (>10-fold) higher in treated versus control rats. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the pathogenesis of SLOS.—.

The vertebrate retina has multiple demands for utilization of cholesterol and must meet those demands either by synthesizing its own supply of cholesterol or by importing cholesterol from extraretinal sources, or both. Unlike the blood-brain barrier, the blood-retina barrier allows uptake of cholesterol from the circulation via a lipoprotein-based/receptor-mediated mechanism. Under normal conditions, cholesterol homeostasis is tightly regulated; also, cholesterol exists in the neural retina overwhelmingly in unesterified form, and sterol intermediates are present in minimal to negligible quantities. However, under certain pathological conditions, either due to an inborn error in cholesterol biosynthesis or as a consequence of exposure to selective inhibitors of enzymes in the cholesterol pathway, the ratio of sterol intermediates to cholesterol in the retina can rise dramatically and persist, in some cases resulting in progressive degeneration that significantly compromises the structure and function of the retina. Although the relative contributions of de novo synthesis versus extraretinal uptake are not yet known, herein we review what is known about these processes and the dynamics of cholesterol in the vertebrate retina and indicate some future avenues of research in this area.

Summary: Background & aims: The bile acid pool influences intestinal cholesterol absorption because this process is strictly dependent on micellar solubilization, which is disrupted by plant sterols (PS). Plasma lipid variation relates to promoter variant −204A > C (rs3808607) of the CYP7A1 gene encoding for 7α-hydroxylase, an enzyme for bile acid synthesis. We hypothesized that this polymorphism would be associated with variability in lipid responses to PS. Methods: We investigated 67 subjects (31 AA and 36 AC + CC) with lipid responses to PS documented in two studies. To assess the functionality of the −204A > C variant, electrophoretic mobility gel shift assays were performed and luciferase reporter plasmids containing the promoter were transfected into HepG2 cells. Results: Compared to AA-subjects, C-carriers showed significantly higher adjusted mean reductions in total cholesterol (0.14 versus 0.43 mmol/L, P = 0.042) and increases in lathosterol-to-cholesterol ratios (0.10 versus 0.75, P = 0.013). The C-construct caused a 78% promoter activity increase and gel-shift assays showed lower affinity for nuclear transcription factors, while in silico experiments predicted a binding site for inhibitory nuclear factors RXR-CAR. Conclusions: Results suggest that promoter −204A > C variant is associated with enhanced CYP7A1 activity. Increased intestinal bile acids and ensuing more efficient cholesterol absorption might explain why C-allele carriers show enhanced cholesterol lowering and increased feedback cholesterol synthesis to PS intervention. [Copyright &y& Elsevier]

Background: Inverse associations have been reported between serum 25-hydroxyvitamin D [25(OH)D] and circulating cholesterol concentrations in observational studies. Postulated mechanisms include reduced bioavailability of intestinal cholesterol and alterations in endogenous cholesterol synthesis., Objective: To explore the effect of daily supplementation with 4000 IU/d vitamin D3 for 24 wk on surrogate biomarkers of cholesterol absorption (campesterol and β-sitosterol) and endogenous synthesis (lathosterol and desmosterol)., Methods: Ancillary study of The Vitamin D for Established Type 2 Diabetes (DDM2) trial. Patients with established type 2 diabetes (N = 127, 25-75 y, BMI 23-42 kg/m2) were randomly assigned to receive either 4000 IU vitamin D3 or placebo daily for 24 wk. Of participants without changes in cholesterol-lowering medications (n = 114), plasma surrogate cholesterol absorption and endogenous synthesis biomarker concentrations were measured and merged with available measures of serum LDL cholesterol and HDL cholesterol concentrations., Results: At week 24, vitamin D3 supplementation significantly increased 25(OH)D concentrations (+21.5 ± 13.4 ng/mL) but not insulin secretion rates (primary outcome of the parent study) as reported previously. In this ancillary study there was no significant effect of vitamin D3 supplementation on serum cholesterol profile or surrogate biomarkers of cholesterol absorption and endogenous synthesis. Compared with participants not treated with cholesterol-lowering medications, those who were treated exhibited a greater reduction in plasma campesterol concentrations in the vitamin D3 but not placebo group (P-interaction = 0.011). Analyzing the data on the basis of cholesterol absorption status (hypo- versus hyperabsorbers) or cholesterol synthesis status (hypo- versus hypersynthesizers) did not alter these results., Conclusions: Vitamin D3 supplementation for 24 wk had no significant effect on surrogate biomarkers of cholesterol absorption or endogenous synthesis, consistent with the lack of effect on serum cholesterol profile. Vitamin D3 supplementation resulted in greater reduction in campesterol concentrations in participants not using compared with those using cholesterol-lowering medications. Further studies are required.This trial was registered at clinicaltrials.gov as NCT01736865., (Copyright © The Author(s) on behalf of the American Society for Nutrition 2020.)

To elucidate the transcriptomic changes of long noncoding RNAs (lncRNAs) in high-fat diet (HFD)-fed mice, we defined their hepatic transcriptome by RNA sequencing. Aberrant expression of 37 representative lncRNAs and 254 protein-coding RNAs was observed in the livers of HFD-fed mice with insulin resistance compared with the livers from control mice. Of these, 24 lncRNAs and 179 protein-coding RNAs were upregulated, whereas 13 lncRNAs and 75 protein-coding RNAs were downregulated. Functional analyses showed that the aberrantly expressed protein-coding RNAs were enriched in various lipid metabolic processes and in the insulin signaling pathway. Genomic juxtaposition and coexpression patterns identified six pairs of aberrantly expressed lncRNAs and protein-coding genes, consisting of five lncRNAs and five protein-coding genes. Four of these protein-coding genes are targeted genes upregulated by PPARα. As expected, the corresponding lncRNAs were significantly elevated in AML12 cells treated with palmitic acid or the PPARα agonist, WY14643. In Hepa1-6 cells, knockdown of NONMMUG027912 increased the cellular cholesterol level, the expression of cholesterol biosynthesis genes and proteins, and the HMG-CoA reductase activity. This genome-wide profiling of lncRNAs in HFD-fed mice reveals one lncRNA, NONMMUG027912, which is potentially regulated by PPARα and is implicated in the process of cholesterol biosynthesis., (Copyright © 2019 Chen et al. Published by The American Society for Biochemistry and Molecular Biology, Inc.)

Very few studies have investigated the interrelations between proprotein convertase subtilisin/kexin type 9 (PCSK9) metabolism, cholesterol synthesis, and cholesterol absorption. We aimed to address this issue in a large clinical trial of 245 patients with hypercholesterolemia. Serum lipids, PCSK9, lathosterol (cholesterol synthesis marker), campesterol, and sitosterol (cholesterol absorption markers) were measured before and 4-8 weeks after the start of treatment with PCSK9-antibodies (alirocumab or evolocumab). The patients had mean (standard error) LDL-cholesterol and PCSK9 concentrations of 3.87 (0.10) mmol/l and 356 (17) ng/ml, respectively. Eighty-four patients received no lipid-lowering pretreatment, 26 ezetimibe, 38 statins, and 97 ezetimibe + statins. Circulating PCSK9 increased in parallel with the potency of lipid-lowering pretreatment with circulating PCSK9 being highest in the ezetimibe + statin group ( P < 0.001). Treatment with PCSK9-antibodies strongly decreased LDL-cholesterol, lathosterol, campesterol, and sitosterol (all P < 0.001) but hardly affected noncholesterol sterol to cholesterol ratios. Lipid-lowering pretreatment was not associated with the effects of PCSK9-antibodies on noncholesterol sterols (all P > 0.05). Summing up, circulating PCSK9 is increased by cholesterol synthesis and absorption inhibitors. Increased PCSK9 expression may partly explain the strong reductions of LDL-cholesterol achieved with PCSK9-antibodies after such pretreatment. On the other hand, treatment with PCSK9-antibodies does not significantly change the balance between cholesterol synthesis and absorption.

Regulating blood cholesterol (Chol) levels by pharmacotherapy has successfully improved cardiovascular health. There is growing interest in the role of Chol precursors in the treatment of diseases. One sterol precursor, desmosterol (Des), is a potential pharmacological target for inflammatory and neurodegenerative disorders. However, elevating levels of the precursor 7-dehydrocholesterol (7-DHC) by inhibiting the enzyme 7-dehydrocholesterol reductase is linked to teratogenic outcomes. Thus, altering the sterol profile may either increase risk toward an adverse outcome or confer therapeutic benefit depending on the metabolite affected by the pharmacophore. In order to characterize any unknown activity of drugs on Chol biosynthesis, a chemical library of Food and Drug Administration-approved drugs was screened for the potential to modulate 7-DHC or Des levels in a neural cell line. Over 20% of the collection was shown to impact Chol biosynthesis, including 75 compounds that alter 7-DHC levels and 49 that modulate Des levels. Evidence is provided that three tyrosine kinase inhibitors, imatinib, ponatinib, and masitinib, elevate Des levels as well as other substrates of 24-dehydrocholesterol reductase, the enzyme responsible for converting Des to Chol. Additionally, the mechanism of action for ponatinib and masitinib was explored, demonstrating that protein levels are decreased as a result of treatment with these drugs., (Copyright © 2018 Wages et al.)

We previously demonstrated that oligomeric amyloid β 42 (oAβ 42 ) inhibits the mevalonate pathway impairing cholesterol synthesis and protein prenylation. Enzymes of the mevalonate pathway are regulated by the transcription factor SREBP-2. Here, we show that in several neuronal types challenged with oAβ 42 , SREBP-2 activation is reduced. Moreover, SREBP-2 activation is also decreased in the brain cortex of the Alzheimer's disease (AD) mouse model, TgCRND8, suggesting that SREBP-2 may be affected in vivo early in the disease. We demonstrate that oAβ 42 does not affect enzymatic cleavage of SREBP-2 per se, but may impair SREBP-2 transport from the endoplasmic reticulum (ER) to the Golgi. Trafficking of SREBP-2 from the ER to the Golgi requires protein kinase B (Akt) activation. oAβ 42 significantly reduces Akt phosphorylation and this decrease is responsible for the decline in SREBP-2 activation. Overexpression of constitutively active Akt prevents the effect of oAβ 42 on SREBP-2 and the downstream inhibition of cholesterol synthesis and protein prenylation. Our work provides a novel mechanistic link between Aβ and the mevalonate pathway, which will impact the views on issues related to cholesterol, isoprenoids, and statins in AD. We also identify SREBP-2 as an indirect target of Akt in neurons, which may play a role in the cross-talk between AD and diabetes., (Copyright © 2018 by the American Society for Biochemistry and Molecular Biology, Inc.)

SREBP-2 activates transcription of all genes needed for cholesterol biosynthesis. To study SREBP-2 function in the intestine, we generated a mouse model ( Vil-BP2 -/- ) in which Cre recombinase ablates SREBP-2 in intestinal epithelia. Intestines of Vil-BP2 -/- mice had reduced expression of genes required for sterol synthesis, in vivo sterol synthesis rates, and epithelial cholesterol contents. On a cholesterol-free diet, the mice displayed chronic enteropathy with histological abnormalities of both villi and crypts, growth restriction, and reduced survival that was prevented by supplementation of cholesterol in the diet. Likewise, SREBP-2-deficient enteroids required exogenous cholesterol for growth. Blockade of luminal cholesterol uptake into enterocytes with ezetimibe precipitated acutely lethal intestinal damage in Vil-BP2 -/- mice, highlighting the critical interplay in the small intestine of sterol absorption via NPC1L1 and sterol synthesis via SREBP-2 in sustaining the intestinal mucosa. These data show that the small intestine requires SREBP-2 to drive cholesterol synthesis that sustains the intestinal epithelia when uptake of cholesterol from the gut lumen is not available, and provide a unique example of cholesterol auxotrophy expressed in an intact, adult mammal., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Ion mobility-mass spectrometry (IM-MS) has proven to be a highly informative technique for the characterization of lipids from cells and tissues. We report the combination of hydrophilic-interaction liquid chromatography (HILIC) with traveling-wave IM-MS (TWIM-MS) for comprehensive lipidomics analysis. Main lipid categories such as glycerolipids, sphingolipids, and glycerophospholipids are separated on the basis of their lipid backbones in the IM dimension, whereas subclasses of each category are mostly separated on the basis of their headgroups in the HILIC dimension, demonstrating the orthogonality of HILIC and IM separations. Using our previously established lipid calibrants for collision cross-section (CCS) measurements in TWIM, we measured over 250 CCS values covering 12 lipid classes in positive and negative modes. The coverage of the HILIC-IM-MS method is demonstrated in the analysis of Neuro2a neuroblastoma cells exposed to benzalkonium chlorides (BACs) with C10 or C16 alkyl chains, which we have previously shown to affect gene expression related to cholesterol and lipid homeostasis. We found that BAC exposure resulted in significant changes to several lipid classes, including glycerides, sphingomyelins, phosphatidylcholines, and phosphatidylethanolamines. Our results indicate that BAC exposure modifies lipid homeostasis in a manner that is dependent upon the length of the BAC alkyl chain., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

The effects of cholesterol-lowering drugs, including those that reduce cholesterol synthesis (statins) and those that reduce cholesterol absorption (ezetimibe), on cholesterol absorption and synthesis are well understood. Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors are a novel class of cholesterol-lowering drugs that robustly reduce LDL-cholesterol (LDL-C), but little is known about their effects on cholesterol absorption and synthesis. We evaluated how treatment with evolocumab, a fully human monoclonal IgG2 antibody to PCSK9, affects markers of cholesterol synthesis and absorption by measuring these markers in patients from an evolocumab clinical trial. At 2 weeks, changes in β-sitosterol/total cholesterol (TC) from baseline were 4% for placebo, 10% for evolocumab 140 mg (nonsignificant vs. placebo), and 26% for evolocumab 420 mg (P < 0.001 vs. placebo). Changes in campesterol/TC at week 2, relative to baseline between placebo and evolocumab, were all nonsignificant. Evolocumab had a modest effect on markers of cholesterol synthesis. At 2 weeks, changes in desmosterol/TC were 1% for placebo, 7% for evolocumab 140 mg (nonsignificant vs. placebo), and 15% for evolocumab 420 mg (P < 0.01 vs. placebo). Changes from baseline in lathosterol/TC at week 2 between placebo and evolocumab were nonsignificant. These results suggest that evolocumab has a modest effect on cholesterol synthesis and absorption despite significant LDL-C lowering., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)

Statins are competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. Statins reduce plasma cholesterol levels, but whether this is actually caused by inhibition of de novo cholesterol synthesis has not been clearly established. Using three different statins, we investigated the effects on cholesterol metabolism in mice in detail. Surprisingly, direct measurement of whole body cholesterol synthesis revealed that cholesterol synthesis was robustly increased in statin-treated mice. Measurement of organ-specific cholesterol synthesis demonstrated that the liver is predominantly responsible for the increase in cholesterol synthesis. Excess synthesized cholesterol did not accumulate in the plasma, as plasma cholesterol decreased. However, statin treatment led to an increase in cholesterol removal via the feces. Interestingly, enhanced cholesterol excretion in response to rosuvastatin and lovastatin treatment was mainly mediated via biliary cholesterol secretion, whereas atorvastatin mainly stimulated cholesterol removal via the transintestinal cholesterol excretion pathway. Moreover, we show that plasma cholesterol precursor levels do not reflect cholesterol synthesis rates during statin treatment in mice. In conclusion, cholesterol synthesis is paradoxically increased upon statin treatment in mice. However, statins potently stimulate the excretion of cholesterol from the body, which sheds new light on possible mechanisms underlying the cholesterol-lowering effects of statins., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)