BBA - Molecular and Cell Biology of Lipids (v.1761, #10)

Cholesterol gallstones are solid calculi that form in the gallbladder from bile constituents and chiefly comprise cholesterol. Cholesterol gallstones are prevalent and costly for healthcare systems. In addition to various environmental factors, genetic risk contributes substantially to gallstone susceptibility. Candidate gene approaches to identify contributory genes are based on prior knowledge of gene–protein function. Whether selected from the entire genome or from limited genomic regions detected by experimental linkage analyses, thus far, candidate genes predominantly were related to lipid homeostasis. Alternatively, comprehensive review of available data suggests that a fundamental driving force underlying cholesterol gallstone formation is inflammation. Therefore, we predict that Lith genes in mice and LITH genes in humans also encode inflammatory molecules, their receptors and other mediators. Indeed, many Lith loci, defined experimentally using inbred mouse models, co-localise with genes that encode inflammation-related proteins. Systematic review of the literature reveals evidence consistent with inflammatory responses that may dictate each of the three cornerstones of cholesterol gallstone formation: biliary cholesterol supersaturation; cholesterol nucleation; gallbladder hypomotility. Genetically targeted inbred mice represent a powerful tool to interrogate the relationship between immune-related genes and gallstone susceptibility. We urge researchers to consider inflammation-related genes when designing population case-control genetic association studies pertaining to the genetic basis of gallstones. Immune and inflammatory events underlie each criterion necessary for cholesterol gallstone formation, which suggests that variation within the respective genes is fundamental for gallstone formation. In turn, inflammatory mediators may exert a spectrum of effects in response to genetic variation within lipid homeostatic genes.
Keywords: Cholelithiasis; Inflammation; Immunity; Quantitative trait locus; QTL; Lithogenic loci; Lith genes; Inbred mouse; Genetics;

Diabetes is a significant risk factor for cardiovascular diseases with the majority of these complications being attributed to coronary vascular pathology. However, both in humans and animal models of diabetes, an additional heart muscle specific disease in the absence of any vascular pathology has also been described. Even though diverse mechanisms have been suggested to explain the etiology of this diabetic heart disease, important roles of oxidative stress and cell death have been implicated behind this disorder. Apart from hyperglycemia, cardiac lipid overload is currently believed to be responsible for oxidative stress and cell death in the diabetic heart. Although lipotoxicity is considered a major player in precipitating cardiac cell death, most of the existing work revolves around saturated and monounsaturated fatty acids. Looking at the current western diet with its preponderance of ω-6 polyunsaturated fatty acids (PUFA), more emphasis should be placed on its role in the diabetic heart. In this review, we shall highlight the most intriguing and updated findings of the differential fatty acid classes including ω-6 PUFA and their established/probable roles on diabetic myocardial cell death.
Keywords: Heart; Diabetes; Omega-6 PUFA; Glutathione; Oxidative stress; Apoptosis;

AAV1-LPLS447X gene therapy reduces hypertriglyceridemia in apoE2 knock in mice by Jaap Rip; Ko Willems van Dijk; Jeroen A. Sierts; John J.P. Kastelein; Jaap Twisk; Jan Albert Kuivenhoven (1163-1168).
Intramuscular (IM) application of adeno-associated virus serotype 1 (AAV1) for the delivery of human lipoprotein lipase (hLPL) was previously shown efficacious in mice with chylomicronemia. The current study addresses whether AAV1-LPLS447X can reduce elevated triglyceride (TG) levels in mice with attenuated clearance of TG-rich remnant particles. Methods. Female mice, expressing human apoE2 but deficient for endogenous apoE (apoE2KI) received IM injections of AAV1-LPLS447X (n  = 6; 8 × 1012 gc/kg; 4-sites) or PBS (n  = 5). Following lipid monitoring, the mice were challenged with intravenous Intralipid injections, and sacrificed 3 months after treatment. Results. In the mice that received LPL gene therapy, a marked increase of post-heparin hLPL protein levels (averaging 517 ± 277 ng/mL vs. 4 ± 3 ng/mL in apoE2KI-untreated) induced 20% reductions of fasting plasma TG levels (p  < 0.05). This was accompanied by two-fold increased TG clearance rates after Intralipid administration at 6 weeks after treatment (p  < 0.05). Post-mortem analyses revealed increased levels of TG (2-fold, p  < 0.005) and cholesterol (1.7-fold, p  < 0.001) in the treated muscles. Conclusions. IM application of AAV1-LPLS447X is effective in reducing TG levels in a mouse model for type III dyslipidemia. Thus, hypertriglyceridemia caused by attenuated uptake of TG-rich lipoproteins can be alleviated by increasing lipolytic function of the skeletal muscle tissue.
Keywords: Lipoprotein lipase; Gene transfer; Adeno-associated virus; Dyslipidemia;

Molecular probes for sensing the cholesterol composition of subcellular organelle membranes by Rong Wang; Masahiro Hosaka; Lu Han; Hiromi Yokota-Hashimoto; Masayuki Suda; Dai Mitsushima; Seiji Torii; Toshiyuki Takeuchi (1169-1181).
Neuroendocrine cells contain two types of secretagogue-regulated acidic compartments: secretory granules (SGs) and synaptic-like microvesicles (SLMVs), which can be identified by acidotropic probes such as acridine orange (AO) and DAMP. We investigated the accumulation of these probes in SGs and SLMVs as a function of glucose levels in the culture media using a pancreatic β-cell line MIN6. AO was accumulated in the low-glucose condition, but not in the high-glucose condition. The AO accumulation correlated well with the SLMV dynamics by glucose and DAMP was localized in the SGs. Because SG membranes are reportedly high in cholesterol, we prepared liposomes with increasing cholesterol levels. AO is well incorporated into liposomes having a 20 to 40 mol% cholesterol composition, whereas DAMP was so in those having over 40 mol% cholesterol levels. Indeed, when cholesterol was depleted from MIN6 SG membranes, DAMP incorporation decreased, instead AO was incorporated. In PC12 cells, AO incorporation into SGs was significant but DAMP incorporation was limited. Consistently, the cholesterol composition was found 37 to 39 mol% in the SG membrane of PC12 cells. We suggest that cholesterol-sensing probes, AO and DAMP, are useful tools for investigating cholesterol compositions in acidic organelle membranes.
Keywords: Acridine orange; DAMP; Secretory granule; Synaptic-like microvesicle; Membrane cholesterol; Liposome;

Caveolae and its structural protein caveolin-1 (Cav-1) are abundant in vascular endothelial cells (ECs) and have been suggested to contribute to cell signaling and cholesterol trafficking. This study investigated the effect of cholesterol on the movement of caveolae-related proteins in human umbilical vein ECs with use of caveolae functional proteomics. After cholesterol exposure to ECs for 2 to 4 h, caveolae were isolated and separated on 2-D protein gels. Among 40 protein spots revealed in caveolae fractions, the ATP synthase β subunit (ATPS-β), one of the 3 proteins enriched by cholesterol in caveolae, was confirmed by western blotting and confocal microscopy. Further, cholesterol exposure increased the level of ATPS-β, along with Cav-1 and cholesterol in caveolae. These effects could be blocked by cytochalasin B, an actin cytoskeleton disruptor. ATPS-β was physically associated with Cav-1, as demonstrated by co-immunoprecipitation and GST-Cav-1 fusion protein pull-down assay. Cholesterol increased the extracellular ATP release mediated by ATPS-β, since this action could be blocked by piceatannol or oligomycin, ATPS inhibitors. Thus, the ectopic localization of ATPS-β may participate in the energy balance of cells in response to the change in intracellular cholesterol levels.
Keywords: ATPS-β; Caveolae; Cholesterol; Proteomics; Endothelial cells;

Prolonged effect of single carnitine administration on fasted carnitine-deficient JVS mice regarding their locomotor activity and energy expenditure by Meng Xian Li; Goichiro Yoshida; Masahisa Horiuchi; Keiko Kobayashi; Takeyori Saheki (1191-1199).
Carnitine is an essential cofactor for the oxidation of fatty acid in the mitochondria and an efficient therapeutics for primary carnitine deficiency. We herein analyzed the prolonged effects of carnitine on the reduced locomotor activity and energy metabolism of fasted carnitine-deficient juvenile visceral steatosis (jvs /) mice. We found that a single carnitine administration to 24-h fasted jvs / mice in the morning increased both the locomotor activity and oxygen consumption at night not only on the same day, but also on the next day, when the carnitine levels in the blood and tissues were already as low as at the original carnitine-deficient state. We also found that fat utilization for energy production significantly increased under fasting even in jvs / mice and was stimulated in the carnitine-administrated fasted jvs / mice at night, in comparison to that observed in the saline-administered jvs / mice, at least for 2 days even under the low plasma and tissue carnitine levels. These results suggest that the low tissue carnitine levels are therefore not the sole rate-limiting factor of general fatty acid oxidation in carnitine-deficient jvs / mice.
Keywords: Carnitine; Fasting; Fatty acid oxidation; Energy expenditure; Juvenile visceral steatosis (jvs); Locomotor activity;

Homo- and hetero-dimerization of LPA/S1P receptors, OGR1 and GPR4 by Alexander Zaslavsky; Lisam Shanjukumar Singh; Haiyan Tan; Huawen Ding; Zicai Liang; Yan Xu (1200-1212).
G protein coupled receptors (GPCRs) form homo- and hetero-dimers or -oligomers, which are functionally important. Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are bioactive lysophopholipids involved in diverse biological processes. We have examined homo- and hetero-dimerization among three major LPA receptors (LPA1–3), three major S1P receptors (S1P1–3), as well as OGR1 and GPR4. Using LacZ complementation assays, we have shown that LPA receptors form homo- and hetero-dimers within the LPA receptor subgroup and hetero-dimers with other receptors (S1P1–3 and GPR4). In addition, we have found that although GPR4 and OGR1 share more than 50% homology, GPR4 forms strong homo- and hetero-dimers with LPA and S1P receptors, but OGR1 forms very weak homo-dimer and relatively weak hetero-dimers with other receptors. Using chimeric receptors between GPR4 and OGR1, we have shown that different domains of GPR4 receptor are involved in its dimerization with different GPCRs and more than one domain may be involved in some of the complex formation. Our results suggest that when studying a signal transduction induced by a stimulus, not only is the expression and activation of its own receptor(s), but also the status of the interacting receptors should be taken into consideration.
Keywords: G protein couple receptors (GPCRs); β-galactosidase (LacZ); Lysophosphatidic acid (LPA); LPA receptors (LPA1, LPA2 and LPA3); Sphingosine-1-phosphate (S1P); S1P receptors (S1P1, S1P2, and S1P3); OGR1 and GPR4;

Squalene epoxidase (SE) is one of the most highly regulated enzymes of the cholesterol biosynthesis pathway. Here we identify the molecular basis for SREBP-2 synergy with NF-Y as the prime regulator of SE gene transcription. As expected cholesterol markedly suppressed transcriptional activity, while SREBP-1a, -1c and -2 activated it. Knock down of SREBP-2 mRNA resulted in an 85% reduction in SE expression. Interspecies comparison of SE promoter sequences identified two conserved putative NF-Y sites that were found to be important for maximal SREBP dependent gene activation and one novel conserved sterol response element (SRE). Altogether three novel SREs were identified within a 205 bp region of the SE promoter. Each of the SREs was capable of binding SREBP-2 but mutation of all three, singly or in combination, did not completely eliminate the SREBP response. Our results demonstrate the critical dependence of this 205 bp region for sterol dependent regulation of SE and uncover a possible framework for SREBP–promoter interaction, including a potent synergy with NF-Y that may be of principal importance.
Keywords: Squalene epoxidase; Cholesterol; SREBP; NF-Y;

Phospholipid-hydroperoxide glutathione peroxidase (GPx-4) localization in resting platelets, and compartmental change during platelet activation by Caroline Januel; Fatima-Zahra El Hentati; Martine Carreras; John R. Arthur; Catherine Calzada; Michel Lagarde; Evelyne Véricel (1228-1234).
Seleno-glutathione peroxidases are an important family of antioxidant enzymes, that include the phospholipid hydroperoxide glutathione peroxidase (GPx-4), an enzyme that reduces lipid hydroperoxides in membranes. The essential characteristics of platelet GPx-4 were found to be the same as the GPx-4 from other tissues. To explore the subcellular expression of GPx-4 in human platelets, we first investigated both its activity and localization in subcellular fractions. About 47% of the total cell enzyme activity was found in the membrane fractions, 29% in the mitochondria and 23% in the cytosol fractions. The same subcellular distribution of GPx-4 protein was demonstrated in resting platelets. This distribution data was further established by confocal microscopy. Of major potential biological significance, this distribution changed when platelets were activated. Confocal immunofluorescence microscopy localized mainly GPx-4 to membranes in contrast to cytoplasm in the resting cells. Based on these results we propose that cytoplasmic GPx-4 could be moved to the membrane for protection during platelet activation. This enzyme would then be important to maintain the integrity of platelet function in vascular system stressed by oxidative reactions.
Keywords: Glutathione peroxidase; Platelet; Subcellular fraction; Confocal microscopy; Platelet activation;

LDL receptor gene transcription is selectively induced by t10c12-CLA but not by c9t11-CLA in the human hepatoma cell line HepG2 by Robert Ringseis; Bettina König; Beatrice Leuner; Sandra Schubert; Norbert Nass; Gabriele Stangl; Klaus Eder (1235-1243).
Conjugated linoleic acids (CLA) have attracted scientific interest due to their potential beneficial effects on atherosclerosis. Recently, a mixture of CLA isomers was demonstrated to upregulate LDL receptor expression in the human hepatoma cell line HepG2. However, the underlying mechanisms remain to be resolved. Thus, the aim of this study was to elucidate how CLA mediates upregulation of LDL receptor in HepG2 cells and whether this upregulation is isomer-specific. The results revealed that LDL receptor promoter activity and mRNA expression were strongly induced upon treatment with t10c12-CLA (P  < 0.05), whereas c9t11-CLA and linoleic acid (LA) had no effect. In addition, only treatment with t10c12-CLA markedly induced mRNA expression of SREBP-2 and HMG-CoA reductase and slightly induced that of SREBP-1 (P  < 0.05). Using SREBP-2 knockdown cells, we could demonstrate that the effect of t10c12-CLA on LDL receptor gene transcription was significantly reduced when compared to control cells (P  < 0.05). When using SREBP-1 knockdown cells the effect of t10c12-CLA on LDL receptor mRNA only slightly decreased compared to control cells. In addition, using different deletion constructs of the LDL receptor gene promoter we showed that the induction of the LDL receptor by t10c12-CLA is independent of the AP-1 motif in the LDL receptor promoter. In conclusion, the present study revealed that transcriptional activation of the LDL receptor gene by t10c12-CLA is dependent on the upregulation of SREBP-2 and is probably due to the activation of the SRE-1 in the LDL receptor gene promoter in HepG2 cells. Thus, the decreased plasma cholesterol levels in response to CLA as observed in a limited number of animal and human studies might be explained by an enhanced uptake of VLDL and LDL cholesterol via hepatic LDL receptors. However, it provides no explanation for the outcome of most human studies reporting unaltered or even increased plasma and LDL cholesterol concentrations in response to supplementation with CLA.
Keywords: Conjugated linoleic acid; LDL receptor; Atherosclerosis; SREBP-2; SREBP-1;