BBA - Molecular and Cell Biology of Lipids (v.1633, #1)

CTP:phosphocholine cytidylyltransferase (CCT) is a key regulatory enzyme in phosphatidylcholine (PtdCho) biosynthesis, and in mammals, there are two distinct genes that encode enzymes that catalyze this reaction. This work defines the structures of both the murine CCT genes (Pcyt1a and Pcyt1b) and identifies a new CCT protein, CCTβ3, with a unique amino terminus that arises from an alternate initiation exon. CCTα is expressed in all tissues, and is most abundant in liver, kidney and heart. A second CCTα transcript is described that initiates from a separate untranslated exon that is most highly expressed in testis. The CCTβ isoforms are most highly expressed in brain and reproductive tissues. CCTβ3 is not expressed in embryonic brain tissues, but is a significant transcript in the adult. These data suggest unique roles for the CCT protein isoforms in the differential regulation of PtdCho biosynthesis in specific tissues.
Keywords: CTP:phosphocholine cytidylyltransferase; Gene structure; Phosphatidylcholine; Alternate splicing;

Intestinal cholesterol absorption: identification of different binding proteins for cholesterol and cholesterol absorption inhibitors in the enterocyte brush border membrane by Werner Kramer; Frank Girbig; Daniel Corsiero; Katja Burger; Falk Fahrenholz; Christian Jung; Günter Müller (13-26).
Absorption of cholesterol from the intestine is a central part of body cholesterol homeostasis. The molecular mechanisms of intestinal cholesterol absorption and the proteins mediating membrane transport are not known. We therefore aimed to identify the proteins involved in intestinal cholesterol absorption across the luminal brush border membrane of small intestinal enterocytes. By photoaffinity labeling using photoreactive derivatives of cholesterol and 2-azetidinone cholesterol absorption inhibitors, an 80-kDa and a 145-kDa integral membrane protein were identified as specific binding proteins for cholesterol and cholesterol absorption inhibitors, respectively, in the brush border membrane of small intestinal enterocytes. The 80-kDa cholesterol-binding protein did not interact with cholesterol absorption inhibitors and vice versa; cholesterol or plant sterols did not interfere with the 145-kDa molecular target for cholesterol absorption inhibitors. Both proteins showed an identical tissue distribution and were exclusively found at the anatomical sites of cholesterol absorption—duodenum, jejunum and ileum. Neither stomach, cecum, colon, rectum, kidney, liver nor fat tissue expressed the 80- or 145-kDa binding proteins for cholesterol and cholesterol absorption inhibitors. Both proteins are different from the hitherto described candidate proteins for the intestinal cholesterol transporter,-SR-BI, ABC G5/ABC G8 or ABC A1. Our data strongly suggest that intestinal cholesterol absorption is not facilitated by a single transporter protein but occurs by a complex machinery. Two specific binding proteins for cholesterol (80 kDa) and cholesterol absorption inhibitors (145 kDa) of the enterocyte brush border membrane are probable protein constituents of the mechanism responsible for the intestinal absorption of cholesterol.
Keywords: Intestinal cholesterol absorption; Photocholesterol; 2-Azetidinone; Ezetimibe; Photoaffinity labeling; Cholesterol absorption inhibitor; Small intestine;

Glycerolipid synthesis in Chlorella kessleri 11h by Norihiro Sato; Mikio Tsuzuki; Akihiko Kawaguchi (27-34).
The fatty acid distributions at the sn-1 and sn-2 positions in major chloroplast lipids of Chlorella kessleri 11h, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), were determined to show the coexistence of both C16 and C18 acids at the sn-2 position, i.e. of prokaryotic and eukaryotic types in these galactolipids. For investigation of the biosynthetic pathway for glycerolipids in C. kessleri 11h, cells were fed with [14C]acetate for 30 min, and then the distribution of the radioactivity among glycerolipids and their constituent fatty acids during the subsequent chase period was determined. MGDG and DGDG were labeled predominantly as the sn-1-C18-sn-2-C16 (C18/C16) species as early as by the start of the chase, which suggested the synthesis of these lipids within chloroplasts via a prokaryotic pathway. On the other hand, the sn-1-C18-sn-2-C18 (C18/C18) species of these galactolipids gradually gained radioactivity at later times, concomitant with a decrease in the radioactivity of the C18/C18 species of phosphatidylcholine (PC). The change at later times can be explained by the conversion of the C18/C18 species of PC into galactolipids through a eukaryotic pathway. The results showed that C. kessleri 11h, distinct from most of other green algal species that were postulated mainly to use a prokaryotic pathway for the synthesis of chloroplast lipids, is similar to a group of higher plants designated as 16:3 plants in terms of the cooperation of prokaryotic and eukaryotic pathways to synthesize chloroplast lipids. We propose that the physiological function of the eukaryotic pathway in C. kessleri 11h is to supply chloroplast membranes with 18:3/18:3-MGDG for their functioning, and that the acquisition of a eukaryotic pathway by green algae was favorable for evolution into land plants.
Keywords: Chlorella kessleri; Chloroplast lipid; Eukaryotic pathway; Fatty acid; Glycerolipid synthesis; Prokaryotic pathway;

Glycerolipid synthesis in Chlorella kessleri 11 h by Norihiro Sato; Mikio Tsuzuki; Akihiko Kawaguchi (35-42).
In the accompanying paper, we demonstrated that Chlorella kessleri uses prokaryotic and eukaryotic pathways to synthesize sn-1-C18sn-2-C16 (C18/C16, prokaryotic lipids) and sn-1-C18sn-2-C18 (C18/C18, eukaryotic lipids) species, respectively, in chloroplast lipids such as monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG). In this study, to examine the effect of CO2 on lipid metabolism, we compared the fatty acid distributions at the sn-1 and sn-2 positions of each major lipid, i.e. MGDG, DGDG, phosphatidylcholine (PC), and phosphatidylethanolamine (PE), and the patterns of incorporation of [14C]acetate into fatty acids and lipids in vivo between cells of C. kessleri grown under ordinary air (low-CO2 cells) and ones grown under CO2-enriched air (high-CO2 cells). Low-CO2 cells, as compared with high-CO2 cells, showed elevated contents of 18:3(9,12,15), especially at both the sn-1 and sn-2 positions of MGDG and DGDG, and also at the sn-2 position of PC and PE. When the cells were labeled with [14C]acetate, slower rates of 18:3 synthesis in the respective major lipids with lower incorporation of 14C into total membrane lipids were observed in low-CO2 cells than in high-CO2 cells. These results thus indicate that the higher unsaturation levels in low-CO2 cells are at least partially due to repressed fatty acid synthesis, which promotes the desaturation of pre-existing fatty acids, rather than to up-regulation of desaturation activity. It was also noted that, in both MGDG and DGDG, the contents of eukaryotic lipids were higher at the expense of prokaryotic lipids in low-CO2 cells than in high-CO2 cells, suggesting relatively greater metabolic flow in the eukaryotic pathway compared to the prokaryotic pathway for galactolipid synthesis in low-CO2 cells. We propose that, together with the repression of fatty acid synthesis, the increased synthesis of C18/C18 species of galactolipids, which are suitable substrates for chloroplast desaturation, through the eukaryotic pathway, contributes to the higher contents of 18:3 in low-CO2 cells than in high-CO2 cells.
Keywords: Chlorella kessleri; CO2 condition; Eukaryotic lipid; Fatty acid desaturation; Prokaryotic lipid;

Nutritional regulation and role of peroxisome proliferator-activated receptor δ in fatty acid catabolism in skeletal muscle by Dorte Holst; Serge Luquet; Véronique Nogueira; Karsten Kristiansen; Xavier Leverve; Paul A. Grimaldi (43-50).
Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors primarily involved in lipid homeostasis. PPARδ displays strong expression in tissues with high lipid metabolism, such as adipose, intestine and muscle. Its role in skeletal muscle remains largely unknown. After a 24-h starvation period, PPARδ mRNA levels are dramatically up-regulated in gastrocnemius muscle of mice and restored to control level upon refeeding. The rise of PPARδ is accompanied by parallel up-regulations of fatty acid translocase/CD36 (FAT/CD36) and heart fatty acid binding protein (H-FABP), while refeeding promotes down-regulation of both genes. To directly access the role of PPARδ in muscle cells, we forced its expression and that of a dominant-negative PPARδ mutant in C2C12 myogenic cells. Differentiated C2C12 cells responds to 2-bromopalmitate or synthetic PPARδ agonist by induction of genes involved in lipid metabolism and increment of fatty acid oxidation. Overexpression of PPARδ enhanced these cellular responses, whereas expression of the dominant-negative mutant exerts opposite effects. These data strongly support a role for PPARδ in the regulation of fatty acid oxidation in skeletal muscle and in adaptive response of this tissue to lipid catabolism.
Keywords: Fatty acid; Gene expression; Fatty acid oxidation; Myotube;

Cyclooxygenase expression is elevated in retinoic acid-differentiated U937 cells by Yan J. Jiang; Tian-Rui Xu; Biao Lu; David Mymin; Edwin A. Kroeger; Tom Dembinski; Xi Yang; Grant M. Hatch; Patrick C. Choy (51-60).
Cyclooxygenase (COX) is the rate-limiting enzyme for the biosynthesis of prostaglandins in monocytes/macrophages. The COX-1 is constitutively expressed in most tissues and may be involved in cellular homeostasis, whereas the COX-2 is an inducible enzyme that may play an important role in inflammation and mitogenesis. When U937 monocytic cells were incubated with retinoic acid (RA) for 48 h, cell differentiation took place with concomitant increases in prostaglandin E2 (PGE2) production and COX activity. In this study, the mechanism of RA (all-trans- or 9-cis-RA)-induced enhancement of PGE2 biosynthesis in U937 cells was examined. Treatment of cells with all-trans- or 9-cis-RA up to 48 h caused an increase in PGE2 production in a time- and dose-dependent manner. Both RA isomers caused the enhancement of PGE2 production and the up-regulation of COX-1 expression at the protein and mRNA levels. The increase in COX-1 mRNA was found to precede the increase in COX-1 protein expression. Interestingly, the COX-2 protein and COX-2 mRNA were not detected in U937 cells, and their levels remained undetectable during the entire course of RA treatment. We conclude that treatment of U937 cells by RA for 48 h caused the initiation of cell differentiation, which was found to be concomitant with a significant increase in PGE2 production mediated via the up-regulation of COX-1 mRNA and protein expression.
Keywords: U937 cell; Cyclooxygenase-1; Cyclooxygenase-2; Retinoic acid; Prostaglandin E2 production; Cell differentiation;

Endocannabinoids and related fatty acid amides, and their regulation, in the salivary glands of the lone star tick by Filomena Fezza; Jack W. Dillwith; Tiziana Bisogno; James S. Tucker; Vincenzo Di Marzo; John R. Sauer (61-67).
The salivary glands and saliva from the lone star tick Amblyomma americanum (L.) were analyzed for the presence of the two endogenous agonists of cannabinoid receptors, N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), as well as of the anandamide congener, N-palmitoylethanolamine (PEA), an anti-inflammatory and analgesic mediator that is inactive at cannabinoid receptors. Two very sensitive mass-spectrometric techniques were used for this purpose. Both 2-AG and PEA, as well as other N-acylethanolamines (NAEs), were identified in salivary glands, but anandamide was below detection. The levels of 2-AG were considerably higher in the salivary glands of partially fed than replete females. Ex vivo gland stimulation with arachidonic acid increased the levels of 2-AG, but not of PEA or other NAEs, and caused the formation of anandamide and of the potent analgesic compound N-arachidonoylglycine. Instead, the amounts of anandamide, 2-AG and PEA were not influenced by treatment of salivary glands with dopamine, which stimulates saliva secretion. The possible biosynthetic precursors of anandamide, PEA and other NAEs were also detected in salivary glands, whereas only PEA was detected in tick saliva. These data demonstrate for the first time that the salivary glands of an obligate ectoparasite species can make endocannabinoids and/or related congeners with analgesic and anti-inflammatory activity, which possibly participate in the inhibition of the host defense reactions.
Keywords: Endocannabinoid; Cannabinoid; Parasite; Eicosanoid; Anandamide; N-acylethanolamine; 2-Arachidonoylglycerol;

In yeast sterol biosynthesis the 3-keto reductase protein (Erg27p) is required for oxidosqualene cyclase (Erg7p) activity by C. Mo; P. Milla; K. Athenstaedt; R. Ott; G. Balliano; G. Daum; M. Bard (68-74).
In Saccharomyces cerevisiae, the 3-keto reductase (Erg27p) encoded by ERG27 gene is one of the key enzymes involved in the C-4 demethylation of the sterol intermediate, 4,4-dimethylzymosterol. The oxidosqualene cyclase (Erg7p) encoded by the ERG7 gene converts oxidosqualene to lanosterol, the first cyclic component of sterol biosynthesis. In a previous study, we found that erg27 strains grown on cholesterol- or ergosterol-supplemented media did not accumulate lanosterol or 3-ketosterols but rather squalene, oxidosqualene, and dioxidosqualene intermediates normally observed in ERG7 (oxidosqualene cyclase) mutants. These results suggested a possible interaction between these two enzymes. In this study, we present evidence that Erg27p interacts with Erg7p, facilitating the association of Erg7p with lipid particles (LPs) and preventing digestion of Erg7p both in the endoplasmic reticulum (ER) and LPs. We demonstrate that Erg27p is required for oxidosqualene cyclase (Erg7p) activity in LPs, and that Erg27p co-immunoprecipitates with Erg7p in LPs but not in microsomal fractions. While Erg27p is essentially a component of the ER, it can also be detected in LPs. In erg27 strains, a truncated Erg7p mislocalizes to microsomes. Restoration of Erg7p enzyme activity and LPs localization was achieved in an erg27 strain transformed with a plasmid containing a wild-type ERG27 allele. We suggest that the physical interaction of Erg27p with Erg7p is an essential regulatory tool in yeast sterol biosynthesis.
Keywords: Saccharomyces cerevisiae; Ergosterol; Oxidosqualene; Erg7p; Erg27p;