Bis(monoacylglycero)phosphate regulates oxysterol binding protein-related protein 11 dependent sterol trafficking

Bis(Monoacylglycero) Phosphate (BMP) is a unique phospholipid localized in late endosomes, a critical cellular compartment in low density lipoprotein (LDL)-cholesterol metabolism. In previous work, we demonstrated the important role of BMP in the regulation of macrophage cholesterol homeostasis. BMP exerts a protective role against the pro-apoptotic effect of oxidized LDL (oxLDL) by reducing the production of deleterious oxysterols. As the intracellular sterol traffic in macrophages is in part regulated by oxysterol binding protein (OSBP) and OSBP-related proteins (ORPs), we investigated the role of ORP11, localized at the Golgi-late endosomes interface, in the BMP-mediated protection from oxLDL/oxysterol cytotoxicity. Stably silencing of ORP11 in mouse RAW264.7 macrophages via a shRNA lentiviruses system had no effect on BMP production. However, ORP11 knockdown abrogated the protective action of BMP against oxLDL induced apoptosis. In oxLDL treated control cells, BMP enrichment was associated with reduced generation of 7-oxysterols, while these oxysterol species were abundant in the ORP11 knock-down cells. Of note, BMP enrichment in ORP11 knock-down cells was associated with a drastic increase in free cholesterol and linked to a decrease of cholesterol efflux. The expression of ATP-binding cassette-transporter G1 (ABCG1) was also reduced in the ORP11 knock-down cells. These observations demonstrate a cooperative function of OPR11 and BMP, in intracellular cholesterol trafficking in cultured macrophages. We suggest that BMP favors the egress of cholesterol from late endosomes via an ORP11-dependent mechanism, resulting in a reduced production of cytotoxic 7-oxysterols.Peer reviewe

Addendum: An Optimised Dual Extraction Method for the Simultaneous and Accurate Analysis of Polar Metabolites and Lipids Carried out on Single Biological Samples. Metabolites 2020, 10, 338

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An Optimized Dual Extraction Method for the Simultaneous and Accurate Analysis of Polar Metabolites and Lipids Carried out on Single Biological Samples

The functional understanding of metabolic changes requires both a significant investigation into metabolic pathways, as enabled by global metabolomics and lipidomics approaches, and the comprehensive and accurate exploration of specific key pathways. To answer this pivotal challenge, we propose an optimized approach, which combines an efficient sample preparation, aiming to reduce the variability, with a biphasic extraction method, where both the aqueous and organic phases of the same sample are used for mass spectrometry analyses. We demonstrated that this double extraction protocol allows working with one single sample without decreasing the metabolome and lipidome coverage. It enables the targeted analysis of 40 polar metabolites and 82 lipids, together with the absolute quantification of 32 polar metabolites, providing comprehensive coverage and quantitative measurement of the metabolites involved in central carbon energy pathways. With this method, we evidenced modulations of several lipids, amino acids, and energy metabolites in HepaRG cells exposed to fenofibrate, a model hepatic toxicant, and metabolic modulator. This new protocol is particularly relevant for experiments involving limited amounts of biological material and for functional metabolic explorations and is thus of particular interest for studies aiming to decipher the effects and modes of action of metabolic disrupting compounds

Queuosine biosynthesis is required for sinorhizobium meliloti-induced cytoskeletal modifications on HeLa Cells and symbiosis with Medicago truncatula.

Rhizobia are symbiotic soil bacteria able to intracellularly colonize legume nodule cells and form nitrogen-fixing symbiosomes therein. How the plant cell cytoskeleton reorganizes in response to rhizobium colonization has remained poorly understood especially because of the lack of an in vitro infection assay. Here, we report on the use of the heterologous HeLa cell model to experimentally tackle this question. We observed that the model rhizobium Sinorhizobium meliloti, and other rhizobia as well, were able to trigger a major reorganization of actin cytoskeleton of cultured HeLa cells in vitro. Cell deformation was associated with an inhibition of the three major small RhoGTPases Cdc42, RhoA and Rac1. Bacterial entry, cytoskeleton rearrangements and modulation of RhoGTPase activity required an intact S. meliloti biosynthetic pathway for queuosine, a hypermodifed nucleoside regulating protein translation through tRNA, and possibly mRNA, modification. We showed that an intact bacterial queuosine biosynthetic pathway was also required for effective nitrogen-fixing symbiosis of S. meliloti with its host plant Medicago truncatula, thus indicating that one or several key symbiotic functions of S. meliloti are under queuosine control. We discuss whether the symbiotic defect of que mutants may originate, at least in part, from an altered capacity to modify plant cell actin cytoskeleton

Ghrelin uses the GHS-R1a/Gi/cAMP pathway and induces differentiation only in mature osteoblasts. This ghrelin pathway is impaired in AIS patients

International audienceWe have examined the Acylated Ghrelin (AG)/Gi pathway in different human osteoblastic cell lines. We have found that: 1) AG induces differentiation/mineralization only in mature osteoblasts; 2) the expression of GHS-R1a increases up to the mature cell stage, 3) the action is mediated via the GHS-R/Gi/cAMP pathway only in mature osteoblasts, and 4) osteoblastic cells from adolescent idiopathic scoliosis (AIS) are resistant to the AG/Gi/cAMP pathway. Altogether, these results suggested that AG uses the GHS-R1a/Gi/cAMP pathway to induce differentiation in mature osteoblasts only. This pathway is impaired in AIS osteoblasts. Understanding AG-specific pathways involved in normal and pathological osteoblasts may be useful for developing new treatments for pathologies such as AIS or osteoporosi

Chronic apelin treatment improves hepatic lipid metabolism in obese and insulin-resistant mice by an indirect mechanism

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Stable Isotope Labeling Highlights Enhanced Fatty Acid and Lipid Metabolism in Human Acute Myeloid Leukemia

International audienceBackground: In Acute Myeloid Leukemia (AML), a complete response to chemotherapy is usually obtained after conventional chemotherapy but overall patient survival is poor due to highly frequent relapses. As opposed to chronic myeloid leukemia, B lymphoma or multiple myeloma, AML is one of the rare malignant hemopathies the therapy of which has not significantly improved during the past 30 years despite intense research efforts. One promising approach is to determine metabolic dependencies in AML cells. Moreover, two key metabolic enzymes, isocitrate dehydrogenases (IDH1/2), are mutated in more than 15% of AML patient, reinforcing the interest in studying metabolic reprogramming, in particular in this subgroup of patients. Methods: Using a multi-omics approach combining proteomics, lipidomics, and isotopic profiling of [U-13C] glucose and [U-13C] glutamine cultures with more classical biochemical analyses, we studied the impact of the IDH1 R132H mutation in AML cells on lipid biosynthesis. Results: Global proteomic and lipidomic approaches showed a dysregulation of lipid metabolism, especially an increase of phosphatidylinositol, sphingolipids (especially few species of ceramide, sphingosine, and sphinganine), free cholesterol and monounsaturated fatty acids in IDH1 mutant cells. Isotopic profiling of fatty acids revealed that higher lipid anabolism in IDH1 mutant cells corroborated with an increase in lipogenesis fluxes. Conclusions: This integrative approach was efficient to gain insight into metabolism and dynamics of lipid species in leukemic cells. Therefore, we have determined that lipid anabolism is strongly reprogrammed in IDH1 mutant AML cells with a crucial dysregulation of fatty acid metabolism and fluxes, both being mediated by 2-HG (2-Hydroxyglutarate) production

Effect of EPA on muscle lipid metabolism.

<p>(A) Intramuscular triglycerides content in red gastrocnemius of ND (n = 9), HFD (n = 10) and HFD+EPA (n = 10) mice. (B) ¹⁴C-palmitate complete β-oxidation and mRNA expression of (C) CPT1b and (D) UCP3 in soleus muscle of ND (n = 9), HFD (n = 6–10) and HFD+EPA mice (n = 6–10). Results are mean ±SEM and were normalized to the ND group (100%) for B, C and D, *p<0.05, **p<0.01, ***p<0.001</p

Plasma lipid composition.

<p>SFA (Saturated Fatty Acids), MUFAs (Mono-Unsaturated Fatty Acids) and PUFAs (Poly-Unsaturated Fatty Acids) were measured by gas-liquid chromatography in plasma of fasted mice after 10 weeks feeding with ND (black column, n = 6), HFD (white column, n = 12) or HFD+EPA (grey column, n = 9). Data are expressed as mean ±SEM. *p<0.05, **p<0.01, ***p<0.001 vs ND; # p<0.05, ## p<0.01, ### p<0.001 vs HFD. ns: not significant</p

Protective effect of EPA supplementation on the HFD-induced obesity.

<p>(A) Body weight in mice after 10-week feeding with a ND (n = 12), HFD (n = 14) or HFD+EPA(n = 14). Results represent mean ±SEM. *p<0.05, **p<0.01, ***p<0.001 vs ND; and ### p<0.001 vs HFD, ns: not significant. (B) fat and lean mass and (C) fat pads weights of subcutaneous (Subcut), perigonadal (Perigon) and mesenteric (Mes) adipose tissue in mice fed for 10 weeks with a ND or N (n = 12), HFD or H (n = 14) or HFD+EPA or E (n = 14) Results represent mean ±SEM. ***p<0.001, (D) Representative photographs of H&E staining of liver section of mice fed the different diets for 10 weeks (bar = 200 µm).</p