The effect of high-density lipoprotein (HDL) in protecting against atherosclerosis is usually attributed to its role in 'reverse cholesterol transport'. In this process, HDL particles mediate the efflux and the transport of cholesterol from peripheral cells to the liver for further metabolism and bile excretion. Thus, cell-surface receptors for HDL on hepatocytes are chief partners in the regulation of cholesterol homeostasis. A high-affinity HDL receptor for apolipoprotein A-I (apoA-I) was previously identified on the surface of hepatocytes. Here we show that this receptor is identical to the beta-chain of ATP synthase, a principal protein complex of the mitochondrial inner membrane. Different experimental approaches confirm this ectopic localization of components of the ATP synthase complex and the presence of ATP hydrolase activity at the hepatocyte cell surface. Receptor stimulation by apoA-I triggers the endocytosis of holo-HDL particles (protein plus lipid) by a mechanism that depends strictly on the generation of ADP. We confirm this effect on endocytosis in perfused rat liver ex vivo by using a specific inhibitor of ATP synthase. Thus, membrane-bound ATP synthase has a previously unsuspected role in modulating the concentrations of extracellular ADP and is regulated by a principal plasma apolipoprotein.

Glucagon-like peptide-1 (GLP-1)-based therapies control glycemia in type 2 diabetic (T2D) patients. However, in some patients the treatment must be discontinued, defining a state of GLP-1 resistance. In animal models we identified a specific set of ileum bacteria impairing the GLP-1-activated gut-brain axis for the control of insulin secretion and gastric emptying. Using prediction algorithms, we identified bacterial pathways related to amino acid metabolism and transport system modules associated to GLP-1 resistance. The conventionalization of germ-free mice demonstrated their role in enteric neuron biology and the gut-brain-periphery axis. Altogether, insulin secretion and gastric emptying require functional GLP-1 receptor and neuronal nitric oxide synthase in the enteric nervous system within a eubiotic gut microbiota environment. Our data open a novel route to improve GLP-1-based therapies.

We have investigated the cell death of a Chinese hamster ovary mutant (MT-58) with a thermo-sensitive CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of the CDP-choline pathway for phosphatidylcholine biosynthesis (Esko, J. D., Wermuth, M. M., and Raetz, C. R. H. (1981) J. Biol. Chem. 256, 7388-7393). After MT-58 cells were shifted to the restrictive temperature of 40 degrees C, the cytidylyltransferase was inactivated immediately leading to a decrease in phosphatidylcholine biosynthesis and cell death. DNA content and number of cells in the S phase decreased significantly in the dying MT-58 cells according to flow cytometrical analyses. The fragmentation of genomic DNA was detected by DNA ladders in agarose gel and release of the prelabeled genomic DNA into cytosolic fractions 14 h after the temperature shift. The dying cells underwent a dramatic reduction of cellular volume while maintaining the membrane containment of cellular contents. These events indicated that the inactivation of cytidylyltransferase triggered apoptosis in Chinese hamster ovary cells. This is the first report that apoptosis was induced in cultured cells, not by an added agent, but by a mutation in phospholipid biosynthesis.

We have previously shown that, among various isoprenoids, farnesol and geranylgeraniol specifically induced actin fiber disorganization, growth inhibition, and apoptosis in human lung adenocarcinoma A549 cells (Miquel, K., Pradines, A., and Favre, G. (1996) Biochem. Biophys. Res. Commun. 225, 869-876). Here we demonstrate that isoprenoidinduced apoptosis was preceded by an arrest in G 0 /G 1 phase. The isoprenoid effects were independent of protein prenylation and of mitogen-activated protein kinase activity. Moreover, geranylgeraniol and farnesol induced a rapid inhibition of phosphatidylcholine biosynthesis at the last step of the CDP-choline pathway controlled by choline phosphotransferase and not at the level of CTP:phosphocholine cytidylyltransferase, the key enzyme of the pathway. Inhibition of choline phosphotransferase was confirmed by in vitro assays on microsomal fractions, which clearly showed that the isoprenoids acted by competitive inhibition with the diacylglycerol binding. Exogenous phosphatidylcholine addition prevented all the biological effects of the isoprenoids, including actin fiber disorganization and apoptosis, suggesting that inhibition of phosphatidylcholine biosynthesis might be the primary event of the isoprenoid action. These data demonstrate the molecular mechanism of geranylgeraniol and farnesol effects and suggest that the mevalonate pathway, leading notably to prenylated proteins, might be linked to the control of cell proliferation through the regulation of phosphatidylcholine biosynthesis.

No abstract

During the orchestrated process leading to mature erythrocytes, reticulocytes must synthesize large amounts of hemoglobin, while eliminating numerous cellular components. Exosomes are small secreted vesicles that play an important role in this process of specific elimination. To understand the mechanisms of proteolipidic sorting leading to their biogenesis, we have explored changes in the composition of exosomes released by reticulocytes during their differentiation, in parallel to their physical properties. By combining proteomic and lipidomic approaches, we found dramatic alterations in the composition of the exosomes retrieved over the course of a 7-day in vitro differentiation protocol. Our data support a previously proposed model, whereby in reticulocytes the biogenesis of exosomes involves several distinct mechanisms for the preferential recruitment of particular proteins and lipids and suggest that the respective prominence of those pathways changes over the course of the differentiation process.

Hepatitis C virus (HCV) particles are known to be in complex with lipoproteins. As a result of this interaction, the low-density lipoprotein (LDL) receptor (LDLR) has been proposed as a potential entry factor for HCV; however, its implication in virus entry remains unclear. Here, we reinvestigated the role of the LDLR in the HCV life cycle by comparing virus entry to the mechanism of lipoprotein uptake. A small interfering RNA targeting the LDLR in Huh-7 cells reduced HCV infectivity, confirming that this receptor plays a role in the life cycle of HCV generated in cell culture. However, kinetics of internalization were much faster for lipoproteins than for infectious HCV particles. Furthermore, a decrease in HCV RNA replication was observed by blocking the LDLR with a specific antibody, and this was associated with an increase in the ratio of phosphatidylethanolamine to phosphatidylcholine in host cells. Nevertheless, a soluble form of the LDLR inhibited both HCV entry into the hepatocytes and its binding to the LDLR expressed on Chinese hamster ovary cells, suggesting a direct interaction between the HCV particle and the LDLR. Finally, we showed that modification of HCV particles by lipoprotein lipase (LPL) reduces HCV infectivity and increases HCV binding to LDLR. Importantly, LPL treatment also induced an increase in RNA internalization, suggesting that LDLR, at least in some conditions, leads to nonproductive internalization of HCV. Conclusion: The LDLR is not essential for infectious HCV particle entry, whereas the physiological function of this receptor is important for optimal replication of the HCV genome. (HEPATOLOGY 2012;55:998-1007) W ith 130 million chronically infected individuals worldwide, hepatitis C virus (HCV) infection accounts for a major cause of severe liver disease. 1 This virus primarily infects human hepatocytes, which, over time, leads to chronic inflammation, progressive fibrosis, and the development of hepatocellular carcinoma.HCV is a positive-stranded RNA virus belonging to the Flaviviridae family. 2 Increasing evidence indicates that HCV enters hepatocytes in a complex, tightly regulated process. 3 Heparan sulfate proteoglycans (HSPGs) could serve as an initial docking site for HCV attachment, and several cell-surface proteins have been described as specific entry factors for HCV; these include tetraspanin cluster of differentiation (CD)81, scavenger receptor BI (SRBI), and tight junction proteins claudin-1 and occludin. However, the precise role of these specific factors in HCV entry remains to be determined.The HCV particle is known to be associated with very-low-density lipoproteins (VLDLs). 4 This association with VLDL allows the virus to bind to target cells through lipoprotein receptors. 5 The low-density

Cell surface receptors for high-density lipoprotein (HDL) on hepatocytes are major partners in the regulation of cholesterol homeostasis. We recently identified a cell surface ATP synthase as a high-affinity receptor for HDL apolipoprotein A-I (apoA-I) on human hepatocytes. Stimulation of this ectopic ATP synthase by apoA-I triggered a low-affinity-receptor-dependent HDL endocytosis by a mechanism strictly related to the generation of ADP. This suggests that nucleotide G-protein-coupled receptors of the P2Y family are molecular components in this pathway. Only P2Y1 and P2Y13 are present on the membrane of hepatocytes. Using both a pharmacological approach and small interference RNA, we identified P2Y13 as the main partner in hepatic HDL endocytosis, in cultured cells as well as in situ in perfused mouse livers. We also found a new important action of the antithrombotic agent AR-C69931MX as a strong activator of P2Y13-mediated HDL endocytosis.