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Oxysterols, as metabolites of cholesterol, play a key role in cholesterol homeostasis, autophagosome formation, and regulation of immune responses. Disorders in oxysterol metabolism are closely related to the pathogenesis of neurodegenerative diseases. To systematically investigate the profound molecular regulatory mechanisms of neurodegenerative diseases, it is necessary to quantify oxysterols and their metabolites in central and peripheral biospecimens simultaneously and accurately. However, there are a lot of unsolved problems with the existing methods, such as the hindrance of applying a single method to different biological specimens or the challenge of simultaneous quantification due to differential groups on the ends of the oxysterol side chains. Herein, according to the physicochemical properties and structure of oxysterols, an optimized liquid chromatography-tandem mass spectrometry method for the quantification of oxysterols was established by optimizing the sample preparation process, chromatographic conditions, mobile phase pH, and solvent selection. Seven oxysterols were detected by this method, including 27-hydroxycholesterol, 7α-hydroxycholesterol, 7α,27-dihydroxycholesterol, 7-dehydrocholesterol, 7α-hydroxy-3-oxo-4-cholestenoic acid, 3-hydroxy-5-cholestenoic acid, and 24(S)-hydroxycholesterol. Non-derivatization extraction with methyl tert-butyl ether was used for different biospecimens, followed by simultaneous chromatographic separation of oxysterols on a phenyl hexyl column. By repeated validation, this method exhibited satisfactory linearity, precision, recovery, sensitivity, repeatability, and stability, and it was successfully applied to the detection of oxysterols in the plasma, cerebral cortex, and liver of mouse. In summary, our optimized method enables concurrent analysis and quantification of oxysterols and their metabolites in various biospecimens, presenting a broad range of applicability.

Lysosomal dysfunction has been increasingly linked to disease and normal ageing 1 , 2 . Lysosomal membrane permeabilization (LMP), a hallmark of lysosome-related diseases, can be triggered by diverse cellular stressors 3 . Given the damaging contents of lysosomes, LMP must be rapidly resolved, although the underlying mechanisms are poorly understood. Here, using an unbiased proteomic approach, we show that LMP stimulates a phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway for rapid lysosomal repair. Upon LMP, phosphatidylinositol-4 kinase type 2α (PI4K2A) accumulates rapidly on damaged lysosomes, generating high levels of the lipid messenger phosphatidylinositol-4-phosphate. Lysosomal phosphatidylinositol-4-phosphate in turn recruits multiple oxysterol-binding protein (OSBP)-related protein (ORP) family members, including ORP9, ORP10, ORP11 and OSBP, to orchestrate extensive new membrane contact sites between damaged lysosomes and the endoplasmic reticulum. The ORPs subsequently catalyse robust endoplasmic reticulum-to-lysosome transfer of phosphatidylserine and cholesterol to support rapid lysosomal repair. Finally, the lipid transfer protein ATG2 is also recruited to damaged lysosomes where its activity is potently stimulated by phosphatidylserine. Independent of macroautophagy, ATG2 mediates rapid membrane repair through direct lysosomal lipid transfer. Together, our findings identify that the PITT pathway maintains lysosomal membrane integrity, with important implications for numerous age-related diseases characterized by impaired lysosomal function. Lysosomal membrane damage triggers a lipid signalling pathway that repairs lysosomes via lipid transport at newly established endoplasmic reticulum–lysosomal membrane contact sites.

Autophagy is an intracellular degradation system for cytoplasmic constituents which is mediated by the formation of a double-membrane organelle termed the autophagosome and its subsequent fusion with the lysosome/vacuole. The formation of the autophagosome requires membrane from the endoplasmic reticulum (ER) and is tightly regulated by a series of autophagy-related (ATG) proteins and lipids. However, how the ER contacts autophagosomes and regulates autophagy remain elusive in plants. In this study, we identified and demonstrated the roles of Arabidopsis oxysterol-binding protein—related protein 2A (ORP2A) in mediating ER–autophagosomal membrane contacts and autophagosome biogenesis. We showed that ORP2A localizes to both ER—plasma membrane contact sites (EPCSs) and autophagosomes, and that ORP2A interacts with both the ER-localized VAMP-associated protein (VAP) 27-1 and ATG8e on the autophagosomes to mediate the membrane contact sites (MCSs). In ORP2A artificial microRNA knockdown (KD) plants, seedlings display retarded growth and impaired autophagy levels. Both ATG1a and ATG8e accumulated and associated with the ER membrane in ORP2A KD lines. Moreover, ORP2A binds multiple phospholipids and shows colocalization with phosphatidylinositol 3-phosphate (PI3P) in vivo. Taken together, ORP2A mediates ER–autophagosomal MCSs and regulates autophagy through PI3P redistribution.

Cancer cells increase lipogenesis for their proliferation and the activation of sterol regulatory element-binding proteins (SREBPs) has a central role in this process. SREBPs are inhibited by a complex composed of INSIG proteins, SREBP cleavage-activating protein (SCAP) and sterols in the endoplasmic reticulum. Regulation of the interaction between INSIG proteins and SCAP by sterol levels is critical for the dissociation of the SCAP–SREBP complex from the endoplasmic reticulum and the activation of SREBPs 1 , 2 . However, whether this protein interaction is regulated by a mechanism other than the abundance of sterol—and in particular, whether oncogenic signalling has a role—is unclear. Here we show that activated AKT in human hepatocellular carcinoma (HCC) cells phosphorylates cytosolic phosphoenolpyruvate carboxykinase 1 (PCK1), the rate-limiting enzyme in gluconeogenesis, at Ser90. Phosphorylated PCK1 translocates to the endoplasmic reticulum, where it uses GTP as a phosphate donor to phosphorylate INSIG1 at Ser207 and INSIG2 at Ser151. This phosphorylation reduces the binding of sterols to INSIG1 and INSIG2 and disrupts the interaction between INSIG proteins and SCAP, leading to the translocation of the SCAP–SREBP complex to the Golgi apparatus, the activation of SREBP proteins (SREBP1 or SREBP2) and the transcription of downstream lipogenesis-related genes, proliferation of tumour cells, and tumorigenesis in mice. In addition, phosphorylation of PCK1 at Ser90, INSIG1 at Ser207 and INSIG2 at Ser151 is not only positively correlated with the nuclear accumulation of SREBP1 in samples from patients with HCC, but also associated with poor HCC prognosis. Our findings highlight the importance of the protein kinase activity of PCK1 in the activation of SREBPs, lipogenesis and the development of HCC. Phosphorylation of INSIG1 and INSIG2 by PCK1 leads to a reduction in the binding of sterols, the activation of SREBP1 and SREBP2 and the downstream transcription of lipogenesis-associated genes that promote tumour growth.

Osteoarthritis—the most common form of age-related degenerative whole-joint disease 1 —is primarily characterized by cartilage destruction, as well as by synovial inflammation, osteophyte formation and subchondral bone remodelling 2 , 3 . However, the molecular mechanisms that underlie the pathogenesis of osteoarthritis are largely unknown. Although osteoarthritis is currently considered to be associated with metabolic disorders, direct evidence for this is lacking, and the role of cholesterol metabolism in the pathogenesis of osteoarthritis has not been fully investigated 4 – 6 . Various types of cholesterol hydroxylases contribute to cholesterol metabolism in extrahepatic tissues by converting cellular cholesterol to circulating oxysterols, which regulate diverse biological processes 7 , 8 . Here we show that the CH25H–CYP7B1–RORα axis of cholesterol metabolism in chondrocytes is a crucial catabolic regulator of the pathogenesis of osteoarthritis. Osteoarthritic chondrocytes had increased levels of cholesterol because of enhanced uptake, upregulation of cholesterol hydroxylases (CH25H and CYP7B1) and increased production of oxysterol metabolites. Adenoviral overexpression of CH25H or CYP7B1 in mouse joint tissues caused experimental osteoarthritis, whereas knockout or knockdown of these hydroxylases abrogated the pathogenesis of osteoarthritis. Moreover, retinoic acid-related orphan receptor alpha (RORα) was found to mediate the induction of osteoarthritis by alterations in cholesterol metabolism. These results indicate that osteoarthritis is a disease associated with metabolic disorders and suggest that targeting the CH25H–CYP7B1–RORα axis of cholesterol metabolism may provide a therapeutic avenue for treating osteoarthritis. In experimentally induced osteoarthritis, chondrocytes show increased uptake and metabolism of cholesterol, implicating the CH25H–CYP7B1–RORα axis in the pathogenesis of osteoarthritis.

Cholesterol homeostasis has a pivotal function in regulating immune cells. Here we show that apolipoprotein E (apoE) deficiency leads to the accumulation of cholesterol in the cell membrane of dendritic cells (DC), resulting in enhanced MHC-II-dependent antigen presentation and CD4 + T-cell activation. Results from WT and apoE KO bone marrow chimera suggest that apoE from cells of hematopoietic origin has immunomodulatory functions, regardless of the onset of hypercholesterolemia. Humans expressing apoE4 isoform (ε4/3–ε4/4) have increased circulating levels of activated T cells compared to those expressing WT apoE3 (ε3/3) or apoE2 isoform (ε2/3–ε2/2). This increase is caused by enhanced antigen-presentation by apoE4-expressing DCs, and is reversed when these DCs are incubated with serum containing WT apoE3. In summary, our study identifies myeloid-produced apoE as a key physiological modulator of DC antigen presentation function, paving the way for further explorations of apoE as a tool to improve the management of immune diseases. Cholesterol homeostasis can modulate immunity via multiple pathways. Here the authors show that apolipoprotein E, an important regulator of cholesterol, produced by myeloid cells can regulate T cell activation by controlling the antigen presentation activity of dendritic cells in both humans and mice.

Inflammatory responses by the innate and adaptive immune systems protect against infections and are essential to health and survival. Many diseases including atherosclerosis, osteoarthritis, rheumatoid arthritis, psoriasis, and obesity involve persistent chronic inflammation. Currently available anti-inflammatory agents, including non-steroidal anti-inflammatory drugs, steroids, and biologics, are often unsafe for chronic use due to adverse effects. The development of effective non-toxic anti-inflammatory agents for chronic use remains an important research arena. We previously reported that oral administration of Oxy210, a semi-synthetic oxysterol, ameliorates non-alcoholic steatohepatitis (NASH) induced by a high-fat diet in APOE*3-Leiden.CETP humanized mouse model of NASH and inhibits expression of hepatic and circulating levels of inflammatory cytokines. Here, we show that Oxy210 also inhibits diet-induced white adipose tissue inflammation in APOE*3-Leiden.CETP mice, evidenced by the inhibition of adipose tissue expression of IL-6, MCP-1, and CD68 macrophage marker. Oxy210 and related analogs exhibit anti-inflammatory effects in macrophages treated with lipopolysaccharide in vitro, mediated through inhibition of toll-like receptor 4 (TLR4), TLR2, and AP-1 signaling, independent of cyclooxygenase enzymes or steroid receptors. The anti-inflammatory effects of Oxy210 are correlated with the inhibition of macrophage polarization. We propose that Oxy210 and its structural analogs may be attractive candidates for future therapeutic development for targeting inflammatory diseases.

Breast cancer is the most frequent cancer among women. In 2018, it is estimated that 627,000 women died from breast cancer. This is approximately 15% of all cancer deaths among women (WHO 2018). Breast cancer is a multifactorial chronic disease. While important progress has been made to treat patients, many questions regarding aspects of this disease relating to carcinogenesis are still open. During carcinogenesis, cells exhibit cholesterol homeostasis deregulation. This results in an accumulation of intracellular cholesterol, which is required to sustain their high growth rate. Cholesterol efflux and influx are two metabolic pathways that are necessary to prevent cholesterol accumulation in the cells. Liver X receptors (LXRs) are nuclear receptors that, upon activation, induce the expression of ABC transporters, responsible for promoting cholesterol efflux, and the expression of IDOL (inducible degrader of low-density lipoprotein receptor), in charge of reducing cholesterol influx. Oxysterols, oxygenated derivatives of cholesterol formed through different pathways, have been discovered as LXR-specific ligands. Some oxysterols are involved in tumor formation while others are considered anti-tumor agents. In the present review, we discuss the involvement of cholesterol, oxysterols and LXRs in breast cancer pathophysiology, with an emphasis on the biological effects of LXR ligands.

HIF-1α exerts both detrimental and beneficial actions in atherosclerosis. While there is evidence that HIF-1α could be pro-atherogenic within the atheromatous plaque, experimental models of atherosclerosis suggest a more complex role that depends on the cell type expressing HIF-1α. In atheroma plaques, HIF-1α is stabilized by local hypoxic conditions and by the lipid microenvironment. Macrophage exposure to oxidized LDLs (oxLDLs) or to necrotic plaque debris enriched with oxysterols induces HIF-1α -dependent pathways. Moreover, HIF-1α is involved in many oxLDL-induced effects in macrophages including inflammatory response, angiogenesis and metabolic reprogramming. OxLDLs activate toll-like receptor signaling pathways to promote HIF-1α stabilization. OxLDLs and oxysterols also induce NADPH oxidases and reactive oxygen species production, which subsequently leads to HIF-1α stabilization. Finally, recent investigations revealed that the activation of liver X receptor, an oxysterol nuclear receptor, results in an increase in HIF-1α transcriptional activity. Reciprocally, HIF-1α signaling promotes triglycerides and cholesterol accumulation in macrophages. Hypoxia and HIF-1α increase the uptake of oxLDLs, promote cholesterol and triglyceride synthesis and decrease cholesterol efflux. In conclusion, the impact of HIF-1α on cholesterol homeostasis within macrophages and the feedback activation of the inflammatory response by oxysterols via HIF-1α could play a deleterious role in atherosclerosis. In this context, studies aimed at understanding the specific mechanisms leading to HIF-1α activation within the plaque represents a promising field for research investigations and a path toward development of novel therapies.

Diabetic kidney disease (DKD) is a leading cause of end stage renal disease with unmet clinical demands for treatment. Lipids are essential for cell survival; however, renal cells have limited capability to metabolize overloaded lipids. Dyslipidaemia is common in DKD patients and renal ectopic lipid accumulation is associated with disease progression. Unveiling the molecular mechanism involved in renal lipid regulation is crucial for exploring potential therapeutic targets. In this review, we focused on the mechanism underlying cholesterol, oxysterol and fatty acid metabolism disorder in the context of DKD. Specific regulators of lipid accumulation in different kidney compartment and TREM2 macrophages, a lipid‐related macrophages in DKD, were discussed. The role of sodium‐glucose transporter 2 inhibitors in improving renal lipid accumulation was summarized.