Identification of Novel Pathways That Control Farnesoid X Receptor-mediated Hypocholesterolemia

Farnesoid X receptor (FXR) plays important regulatory roles in bile acid, lipoprotein, and glucose homeostasis. Here, we have utilized Fxr^−/− mice and mice deficient in scavenger receptor class B type I (SR-BI), together with an FXR-specific agonist and adenovirus expressing hepatocyte nuclear factor 4α or constitutively active FXR, to identify the mechanisms by which activation of FXR results in hypocholesterolemia. We identify a novel pathway linking FXR to changes in hepatic p-JNK, hepatocyte nuclear factor 4α, and finally SR-BI. Importantly, we demonstrate that the FXR-dependent increase in SR-BI results in both hypocholesterolemia and an increase in reverse cholesterol transport, a process involving the transport of cholesterol from peripheral macrophages to the liver for excretion into the feces. In addition, we demonstrate that FXR activation also induces an SR-BI-independent increase in reverse cholesterol transport and reduces intestinal cholesterol absorption. Together, these data indicate that FXR is a promising therapeutic target for treatment of hypercholesterolemia and coronary heart disease.     

Increased Mitochondrial Fatty Acid Oxidation Is Sufficient to Protect Skeletal Muscle Cells from Palmitate-induced Apoptosis

The mechanisms underlying the protective effect of monounsaturated fatty acids (e.g. oleate) against the lipotoxic action of saturated fatty acids (e.g. palmitate) in skeletal muscle cells remain poorly understood. This study aimed to examine the role of mitochondrial long-chain fatty acid (LCFA) oxidation in mediating oleate''s protective effect against palmitate-induced lipotoxicity. CPT1 (carnitine palmitoyltransferase 1), which is the key regulatory enzyme of mitochondrial LCFA oxidation, is inhibited by malonyl-CoA, an intermediate of lipogenesis. We showed that expression of a mutant form of CPT1 (CPT1mt), which is active but insensitive to malonyl-CoA inhibition, in C2C12 myotubes led to increased LCFA oxidation flux even in the presence of high concentrations of glucose and insulin. Furthermore, similar to preincubation with oleate, CPT1mt expression protected muscle cells from palmitate-induced apoptosis and insulin resistance by decreasing the content of deleterious palmitate derivates (i.e. diacylglycerols and ceramides). Oleate preincubation exerted its protective effect by two mechanisms: (i) in contrast to CPT1mt expression, oleate preincubation increased the channeling of palmitate toward triglycerides, as a result of enhanced diacylglycerol acyltransferase 2 expression, and (ii) oleate preincubation promoted palmitate oxidation through increasing CPT1 expression and modulating the activities of acetyl-CoA carboxylase and AMP-activated protein kinase. In conclusion, we demonstrated that targeting mitochondrial LCFA oxidation via CPT1mt expression leads to the same protective effect as oleate preincubation, providing strong evidence that redirecting palmitate metabolism toward oxidation is sufficient to protect against palmitate-induced lipotoxicity.     

A Sequence Variation (I148M) in PNPLA3 Associated with Nonalcoholic Fatty Liver Disease Disrupts Triglyceride Hydrolysis

Obesity and insulin resistance are associated with deposition of triglycerides in tissues other than adipose tissue. Previously, we showed that a missense mutation (I148M) in PNPLA3 (patatin-like phospholipase domain-containing 3 protein) is associated with increased hepatic triglyceride content in humans. Here we examined the effect of the I148M substitution on the enzymatic activity and cellular location of PNPLA3. Structural modeling predicted that the substitution of methionine for isoleucine at residue 148 would restrict access of substrate to the catalytic serine at residue 47. In vitro assays using recombinant PNPLA3 partially purified from Sf9 cells confirmed that the wild type enzyme hydrolyzes emulsified triglyceride and that the I148M substitution abolishes this activity. Expression of PNPLA3-I148M, but not wild type PNPLA3, in cultured hepatocytes or in the livers of mice increased cellular triglyceride content. Cell fractionation studies revealed that ∼90% of wild type PNPLA3 partitioned between membranes and lipid droplets; substitution of isoleucine for methionine at position 148 did not alter the subcellular distribution of the protein. These data are consistent with PNPLA3-I148M promoting triglyceride accumulation by limiting triglyceride hydrolysis.     

Conservation of a Glycine-rich Region in the Prion Protein Is Required for Uptake of Prion Infectivity

Prion diseases are associated with the misfolding of the endogenously expressed prion protein (designated PrP^C) into an abnormal isoform (PrP^Sc) that has infectious properties. The hydrophobic domain of PrP^C is highly conserved and contains a series of glycine residues that show perfect conservation among all species, strongly suggesting it has functional and evolutionary significance. These glycine residues appear to form repeats of the GXXXG protein-protein interaction motif (two glycines separated by any three residues); the retention of these residues is significant and presumably relates to the functionality of PrP^C. Mutagenesis studies demonstrate that minor alterations to this highly conserved region of PrP^C drastically affect the ability of cells to uptake and replicate prion infection in both cell and animal bioassay. The localization and processing of mutant PrP^C are not affected, although in vitro and in vivo studies demonstrate that this region is not essential for interaction with PrP^Sc, suggesting these residues provide conformational flexibility. These data suggest that this region of PrP^C is critical in the misfolding process and could serve as a novel, species-independent target for prion disease therapeutics.     

O-Mycoloylated Proteins from Corynebacterium: AN UNPRECEDENTED POST-TRANSLATIONAL MODIFICATION IN BACTERIA*

O-Acylation of proteins was known only in a few eukaryotic proteins but never in bacteria. We demonstrate, using a combination of protein chemistry and mass spectrometry, the occurrence of three O-acylated polypeptides in Corynebacterium glutamicum, PorA, PorH, and an unknown small protein. The three polypeptides are O-substituted by mycolic acids, long chain α-alkyl and β-hydroxy fatty acids specifically produced by members of the Corynebacterineae suborder. To date these acids were described only as esterifying trehalose and arabinogalactan, and less frequently glycerol, important components of the highly impermeable outer barrier of Corynebacterineae. We show that the post-translational mycoloylation of PorA occurs at Ser-15 and is necessary for the pore-forming activity of C. glutamicum.     

Membrane microdomains and proteomics: lessons from tetraspanin microdomains and comparison with lipid rafts

Biological membranes are compartmentalized into microdomains that exhibit particular lipid and protein compositions. Membrane microdomains, such as tetraspanin-enriched microdomains and lipid rafts, have been suggested to play a role in a variety of physiological and pathological processes. Therefore, the characterization of the protein compositions of these microdomains, which is the focus of this review, appears to be a crucial step to better understanding their function. Proteomics has recently allowed the characterization of tetraspanin-enriched microdomains in colon cancer cells. This demonstrated the presence of different categories of membrane proteins and suggested a variation in the composition of tetraspanin-enriched microdomains during tumor progression. On the other hand, proteomics has permitted the identification of hundreds of proteins in lipid rafts of different origins. However, the diversity of methodologies in sample preparation and of strategies in protein identification led to a broad variability in the data obtained. These methodological issues are discussed. Moreover, proteomics has revealed that different sets of proteins were present in tetraspanin-enriched microdomains as compared with lipid rafts, strengthening the idea that these microdomains are distinct structures.     

Effect of Compositional Factors against the Thermal Oxidative Deterioration of Novel Food Emulsions

This work attempts to determine any relationship between certain endogenous parameters and the oxidative deterioration of protein-stabilized oil-in-water emulsions. The contribution of compositional factors (e.g., type and amount of emulsifier, fat phase, etc.) is further elucidated. Among 10% cottonseed o/w emulsions prepared by 1% emulsifier (Tween, sodium caseinate, or whey protein), lipid autoxidation (at 40°C) was much faster in the Tween emulsion than in the protein ones, with whey protein presenting a clear antioxidant effect. Increase in protein concentration (0.5–2% w/w) led to a decrease in droplet size but an increase in oxidative stability, in terms of conjugated diene hydroperoxides formation at 232 nm. The type of lipid phase significantly affected the rate of thermal oxidation at 60°C. In the most oxidatively vulnerable sunflower-oil-based emulsions, an increase in fat content (10–40%) resulted in a reduction of oxidative deterioration. By selecting a more concentrated emulsion (20% o/w, 2% emulsifier), in order to structurally approach real novel food products, any influence of the composition of the emulsifier (combination of Tween and sodium caseinate preparation) was subsequently tested. An increase in protein proportion in the emulsifier was found to inhibit proportionally the oxidative instability of the emulsions, as evaluated by the determination of both primary (conjugated diene and lipid hydroperoxides) and secondary [thiobarbituric acid-reactive substances (TBARS)] oxidation products.     

Lipid particle composition of the yeast Yarrowia lipolytica depends on the carbon source

Lipid particles (LP) of all types of cells are a depot of neutral lipids. The present investigation deals with the isolation of LP from the yeast Yarrowia lipolytica and the characterization of their lipid and protein composition. Properties of LP varied depending on the carbon source. LP from glucose-grown cells revealed a mean diameter of 650 nm with a hydrophobic core mainly formed of triacylglycerols (TAG) and a minor amount of steryl esters (SE). Oleic acid was the major fatty acid species esterified in LP. When cells were grown on oleic acid, LP size increased 3.8-fold, the particles exhibited a significantly lower ratio of TAG to SE, and the relative amount of oleic acid in LP lipids increased compared to cells grown on glucose. Analysis of LP proteins revealed an increasing number of polypeptides when cells were shifted from glucose- to oleic acid-containing medium. Twenty-one major LP proteins were identified under both growth conditions, and additional nine polypeptides were specific for growth on oleic acid. Identification of these proteins by MS and comparison of the deduced ORFs to those from Saccharomyces cerevisiae revealed that most proteins of Y. lipolytica LP are involved in lipid metabolism. LP proteins specific for growth on oleic acid are also enzymes involved in lipid metabolism, but some of them are also components of the intracellular traffic machinery. Thus, proteom analysis of LP proteins suggests involvement of this compartment in different cell biological processes.     

Cellular isoform of the prion protein PrPc in human intestinal cell lines: genetic polymorphism at codon 129, mRNA quantification and protein detection in lipid rafts

The cellular isoform of the normal prion protein PrP(c), encoded by the PRNP gene, is expressed in human intestinal epithelial cells where it may represent a potential target for infectious prions. We have sequenced the PRNP gene in Caco-2 and HT-29 parental and clonal cell lines, and found that these cells have a distinct polymorphism at codon 129. HT-29 cells are homozygous Met/Met, whereas Caco-2 cells are heterozygous Met/Val. The 129Val variant was also detected in Caco-2 mRNAs. Real-time PCR quantifications revealed that PrP(c) mRNAs were more expressed in HT-29 cells than in Caco-2 cells. These data were confirmed by studying the expression of PrP(c) in plasma membranes and lipid rafts prepared from these cells. Overall, these results may be important in view of using human intestinal cell lines Caco-2 and HT-29 as cellular in vitro models to study the initial steps of prion propagation after oral inoculation.     

Involvement of apoptosis and autophagy in the death of RPMI 8226 multiple myeloma cells by two enantiomeric sigma receptor ligands

Over-expression of σ receptors by many tumor cell lines makes ligands for these receptors attractive as potential chemotherapeutic drugs. Enantiomeric piperazines (S)-4 and (R)-4 were prepared as potential σ-receptor ligands in a chiral pool synthesis starting from (S)- and (R)-aspartate. Both compounds showed high affinities for the σ₁ and σ₂ receptors. In the human multiple myeloma cell line RPMI 8226, a line expressing high levels of σ receptors, both compounds inhibited cell proliferation with IC₅₀ values in the low μM range. No chiral differentiation between either the σ receptor binding affinity or the cytotoxicity of the two enantiomers was observed. Both compounds induced apoptosis, which was evidenced by nuclear condensation, binding of annexin-V to phosphatidylserine in the outer leaf of the cell membrane, cleavage products of poly(ADP-ribose) polymerase-1 (PARP-1) and caspase-8 as well as the expression of bcl₂ family members bax, bad and bid. However, apoptosis appeared to be caspase independent. Increased levels of the phosphorylated form of the microtubule associated protein light chain 3-II (LC3-II), an autophagosome marker, gave evidence that both compounds induced autophagy. However, further data (e.g., treatment with wortmannin) indicate that autophagy is incomplete and not cytoprotective. Lipid peroxidation (LPO) was observed in RPMI 8226 cells treated with the two compounds, and the lipid antioxidant α-tocopherol attenuated LPO. Interestingly, α-tocopherol reduced significantly both apoptosis and autophagy induced by the compounds. These results provide evidence that, by initiating LPO and changes in mitochondrial membrane potential, both compounds induce apoptosis and autophagy in RPMI 8226 cells.