Lipid sensing and lipid sensors

Lipid droplets have been considered for a long time as inert intracytoplasmic deposits formed within cells under various conditions. Recently, new tools and new approaches have been used to visualize and study these intracellular structures. This revealed new aspects of lipid droplets biology and pointed out their organized structure and dynamic composition. In adipocytes, the specialized cell type for the storage of energy as fat, lipid droplets are particularly well-developed organelles and exhibit unique properties. Also discussed in this paper is the view that lipid droplets, through specific candidate constituents, can play a role in sensing the level of their lipid stores by adipocytes.     

Lipid droplets of neuroepithelial cells are a major calcium storage site during neural tube formation in chick and mouse embryos

In situ precipitation of calcium (Ca²⁺) with fluoride and antimonate shows that Ca²⁺-specific precipitate is localized almost exclusively within lipid droplets of neuroepithelial cells during neural tube formation in chick and mouse embryos. The density of Ca²⁺ precipitate within lipid droplets is generally greater in the apical ends of cells situated in regions of the neuroepithelium that are actively engaged in bending. These findings suggest that lipid droplets, in addition to providing a source of metabolic fuel for developing neuroepithelial cells, also serve as Ca²⁺-storage and-releasing sites during neurulation.This study was supported by grants from the NIH (NS23200), the BRSG fund of UMDNJ, and the Busch Fund of Rutgers University. Dr Bush was supported by a New Jersey State Postdoctoral Fellowship.     

P-glycoprotein and ‘lipid rafts’: some ambiguous mutual relationships (floating on them, building them or meeting them by chance?)

P-glycoprotein (P-gp) is an active membrane transporter responsible for cell detoxification against numerous amphiphilic compounds, leading to multidrug resistance in tumor cells. It displays entangled connections with its membrane environment since it recognizes its substrates within the cytosolic leaflet and it also translocates some endogenous lipids to the exoplasmic leaflet. Regarding its relationships with membrane microdomains, ‘lipid rafts’, a literature analysis concludes that (i) P-gp also exists in rafts and non-raft membrane domains, depending on the cell considered, the experimental conditions and the method used to test it; (ii) cholesterol has a positive influence on P-gp function, and this may be a direct effect of the free cholesterol present in membrane or an indirect effect mediated by the cholesterol-enriched microdomains; (iii) when present in rafts, P-gp interacts with protein partners regulating its activity; (iv) P-gp is a lipid translocase that handles the raft-constituting lipids with particular efficiency, and it also influences membrane trafficking in the cell. Received 18 November 2005; received after revision 23 December 2005; accepted 12 January 2006     

Triacylglycerol hydrolase: role in intracellular lipid metabolism

Recent scientific advances have revealed the identity of several enzymes involved in the synthesis, storage and catabolism of intracellular neutral lipid storage droplets. An enzyme that hydrolyzes stored triacylglycerol (TG), triacylglycerol hydrolase (TGH), was purified from porcine, human and murine liver microsomes. In rodents, TGH is highly expressed in liver as well as heart, kidney, small intestine and adipose tissues, while in humans TGH is mainly expressed in the liver, adipose and small intestine. TGH localizes to the endoplasmic reticulum and lipid droplets. The TGH genes are located within a cluster of carboxylesterase genes on human and mouse chromosomes 16 and 8, respectively. TGH hydrolyzes stored TG, and in the liver, the lipolytic products are made available for VLDL-TG synthesis. Inhibition of TGH activity also inhibits TG and apolipoprotein B secretion by primary hepatocytes. A role for TGH in basal TG lipolysis in adipocytes has been proposed. TGH expression and activity is both developmentally and hormonally regulated. A model for the function of TGH is presented and discussed with respect to tissue specific functions.     

Differential diurnal variations of anandamide and 2-arachidonoyl-glycerol levels in rat brain

The endogenous ligands of cannabinoid receptors, also known as endocannabinoids, have been implicated in many physiological and pathological processes of the central nervous system. Here we show that the levels of the two major endocannabinoids, anandamide and 2-arachidonoyl-glycerol (2-AG), in four areas of the rat brain, change dramatically between the light and dark phases of the day. While anandamide levels in the nucleus accumbens, pre-frontal cortex, striatum and hippocampus were significantly higher in the dark phase, the opposite was observed with 2-AG, whose levels were significantly higher during the light phase in all four regions. We found that the activity of the fatty acid amide hydrolase, which catalyzes the metabolism of anandamide, was significantly lower during the dark phase, thus providing a possible explaination for the increase in anandamide levels. However, the activities of monoacylglycerol lipase and diacylglycerol lipase, two of the possible enzymes catalyzing the degradation and biosynthesis of 2-AG, respectively, changed significantly only in the striatum. These data suggest that the levels of the two major endocannabinoids might be under the control of endogenous factors known to undergo diurnal variations, and underscore the different roles, suggested by previous studies, of anandamide and 2-AG in neurophysiological processes.Received 9 December 2003; received after revision 15 January 2004; accepted 20 January 2004     

The life cycle of neutral lipids: synthesis, storage and degradation

Triacylglycerols (TAGs), steryl esters (SEs) and wax esters (WEs) form the group of neutral lipids. Whereas TAGs are present in all types of cell, the occurrence of SEs in prokaryotes is questionable, and the presence of WEs as storage molecules is restricted to plants and a few bacteria. Here, we summarize recent knowledge on the formation, storage and degradation of TAGs and SEs in various cell types. We describe the biochemical pathways involved in TAG and SE synthesis and discuss the subcellular compartmentation of these processes. Recently, several novel enzymes governing the metabolism of storage lipids have been identified and characterized. Regulatory aspects of neutral lipid storage are just beginning to be understood. Finally, we describe consequences of defects in neutral lipid metabolism. Since severe diseases like atherosclerosis, obesity and type 2 diabetes are caused by lipid accumulation, mechanisms underlying neutral lipid synthesis, depot formation and mobilization are of major interest for curing such diseases that are increasingly associated with modern civilization. Received 18 January 2006; received after revision 7 March 2006; accepted 16 March 2006     

Anandamide induces cell death independently of cannabinoid receptors or vanilloid receptor 1: possible involvement of lipid rafts

Anandamide triggers various cellular activities by binding to cannabinoid (CB1/CB2) receptors or vanilloid receptor 1 (VR1). However, the role of these receptors in anandamide-induced apoptosis remains largely unknown. Here, we show that SR141716A, a specific inhibitor of cannabinoid receptor (CB1-R), did not block anandamide-induced cell death in endogenously CB1-R expressing cells. In addition, CB1-R-lacking Chinese hamster ovary (CHO) cells underwent cell death after anandamide treatment. SR144528, a specific inhibitor of CB2-R also failed to block anandamide-induced cell death in HL-60 cells. Capsazepine, a specific antagonist of VR1 could not prevent anandamide-induced cell death in constitutively and endogenously VR1 expressing PC12 cells. Moreover, anandamide noticeably triggered cell death in VR1-lacking human embryonic kidney (HEK) cells. In contrast, methyl-beta cyclodextrin (MCD), a membrane cholesterol depletor, completely blocked anandamide-induced cell death in a variety of cells, including PC12, C6, Neuro-2a, CHO, HEK, SMC, Jurkat and HL-60 cells. MCD also blocked anandamide-induced superoxide generation, phosphatidyl serine exposure and p38 MAPK/JNK activation. Thus, our data imply a novel role for of membrane lipid rafts in anandamide-induced cell death.     

Trans-Golgi network sorting

The trans-Golgi network (TGN) is a major secretory pathway sorting station that directs newly synthesized proteins to different subcellular destinations. The TGN also receives extracellular materials and recycled molecules from endocytic compartments. In this review, we summarize recent progress on understanding TGN structure and the dynamics of trafficking to and from this compartment. Protein sorting into different transport vesicles requires specific interactions between sorting motifs on the cargo molecules and vesicle coat components that recognize these motifs. Current understanding of the various targeting signals and vesicle coat components that are involved in TGN sorting are discussed, as well as the molecules that participate in retrieval to this compartment in both yeast and mammalian cells. Besides proteins, lipids and lipid-modifying enzymes also participate actively in the formation of secretory vesicles. The possible mechanisms of action of these lipid hydrolases and lipid kinases are discussed. Finally, we summarize the fundamentally different apical and basolateral cell surface delivery mechanisms and the current facts and hypotheses on protein sorting from the TGN into the regulated secretory pathway in neuroendocrine cells. Received 2 November 2000; received after revision 19 February 2001; accepted 19 February 2001     

Ultrastructural evidence of a secretory process in the rat pineal gland.

Pericapillar spaces of the rat pineal gland belong to the most active sites of this organ. Neighborhood of sympathetic nerve endings, capillaries and pinealocyte processes facilitates possibly the synthesis and the secretion of methoxyindoles. Lipid droplets migrate through the pinealocyte cellular processes towards the terminal enlargement or poles, where they are secreted into the pericapillar space, and the possibility that indoleamines are included in the lipid droplets has been discussed.     

Transmembrane movements of lipids

Summary Membranes allow the rapid passage of uncharged lipids. Phospholipids on the other hand diffuse very slowly from one monolayer to another with a half-time of several hours. This slow spontaneous movement in a pure lipid bilayer can be selectively modulated in biological membranes by intrinsic proteins. In microsomes, and probably in bacterial membranes, non-specific phospholipid flippases allow the rapid redistribution of newly synthesized phospholipids. In eukaryotic plasma membranes, aminophospholipid translocase selectively pumps phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the outer to the inner leaflet and establishes a permanent lipid asymmetry. The discovery of an aminophospholipid translocase in chromaffin granules proves that eukaryotic organelles may also contain lipid translocators.     

Opposite actions of testosterone and progesterone on UCP1 mRNA expression in cultured brown adipocytes

The brown adipose tissue (BAT) thermogenic response to diet-induced obesity and cold has been found to be gender dependent. In the present work, we aimed to investigate the effects of the main physiological male and female sex hormones, i.e. testosterone, progesterone and 17-β-estradiol, on the expression of uncoupling protein 1 (UCP1) – the main mediator of BAT thermogenesis – and on UCP2 and lipid accumulation in rodent brown adipocytes differentiated in culture. Testosterone-treated cells showed fewer and smaller lipid droplets than control cells and a dose-dependent inhibition of UCP1 mRNA expression, under adrenergic stimulation by norepinephrine (NE). These effects were reverted by the androgen receptor antagonist flutamide, suggesting they are dependent, at least in part, on the androgen receptor. Progesterone- and 17-β-estradiol-treated cells showed more and larger lipid droplets and progesterone stimulated NE-induced UCP1 mRNA expression at the lower concentration tested, but not at higher concentrations, suggesting that for brown adipocytes, this hormone is dose dependent. 17-β-Estradiol did not have any remarkable effect either on UCP1 or UCP2 mRNA expression. Interestingly, the specific progesterone receptor antagonist RU486 induced UCP1 and UCP2 mRNAs, including UCP1 mRNA expression in non-NE-treated brown adipocytes, suggesting a profound effect of this anti-progestagen on brown adipocyte thermogenic capacity. Thus, are conclude that testosterone, 17-β-estradiol, progesterone and RU486 have distinct actions on brown adipocytes, thus modulating UCP1 and UCP2 mRNA expression and/or lipid accumulation, and that sex hormones are factors that may explain in part the gender-dependent BAT thermogenic response. Received 24 June 2002; received after revision 20 August 2002; accepted 26 August 2002 RID="*" ID="*"Corresponding author.     

The effects of vitamin A nutritional status on glutathione levels and microsomal lipid peroxidation in rat lung

Summary In vitamin A-deficient rats, the glutathione level in lung was diminished and microsomal lipid peroxidation much increased. In vitamin A-loaded animals, however, both were depressed below control. Thus vitamin A protection against lipid peroxidation is independent of glutathione.Acknowledgments. We thank Dr L. Y. Y. Fong and Mr David Y. H. Woo for preparing the animals used in this research, for retinol determinations and for valuable discussions, and also the China Medical Board of New York and the University of Hong Kong for the award of a Fellowship to V.P.     

Putting endotoxin to work for us: Monophosphoryl lipid A as a safe and effective vaccine adjuvant

The development of non-infectious subunit vaccines greatly increases the safety of prophylactic immunization, but also reinforces the need for a new generation of immunostimulatory adjuvants. Because adverse effects are a paramount concern in prophylactic immunization, few new adjuvants have received approval for use anywhere in the developed world. The vaccine adjuvant monophosphoryl lipid A is a detoxified form of the endotoxin lipopolysaccharide, and is among the first of a new generation of Toll-like receptor agonists likely to be used as vaccine adjuvants on a mass scale in human populations. Much remains to be learned about this compound’s mechanism of action, but recent developments have made clear that it is unlikely to be simply a weak version of lipopolysaccharide. Instead, monophosphoryl lipid A’s structure seems to have fortuitously retained several functions needed for stimulation of adaptive immune responses, while shedding those associated with pro-inflammatory side effects. Received 25 April 2008; received after revision 05 June 2008; accepted 10 June 2008     

Lipid droplet dynamics in budding yeast

Eukaryotic cells store excess fatty acids as neutral lipids, predominantly triacylglycerols and sterol esters, in organelles termed lipid droplets (LDs) that bulge out from the endoplasmic reticulum. LDs are highly dynamic and contribute to diverse cellular functions. The catabolism of the storage lipids within LDs is channeled to multiple metabolic pathways, providing molecules for energy production, membrane building blocks, and lipid signaling. LDs have been implicated in a number of protein degradation and pathogen infection processes. LDs may be linked to prevalent human metabolic diseases and have marked potential for biofuel production. The knowledge accumulated on LDs in recent years provides a foundation for diverse, and even unexpected, future research. This review focuses on recent advances in LD research, emphasizing the diverse physiological roles of LDs in the model system of budding yeast.     

Confocal microscopy and biochemical analysis reveal spatial and functional separation between anandamide uptake and hydrolysis in human keratinocytes

The signaling activity of anandamide (AEA) is terminated by its uptake across the cellular membrane and subsequent intracellular hydrolysis by the fatty acid amide hydrolase (FAAH). To date, the existence of an AEA membrane transporter (AMT) independent of FAAH activity remains questionable, although it has been recently corroborated by pharmacological and genetic data. We performed confocal microscopy and biochemical analysis in human HaCaT keratinocytes, in order to study the cellular distribution of AMT and FAAH. We found that FAAH is intracellularly localized as a punctate staining partially overlapping with the endoplasmic reticulum. Consistently, subcellular fractionation and reconstitution of vesicles from membranes of different compartments demonstrated that FAAH activity was localized mainly in microsomal fractions, whereas AMT activity was almost exclusively in plasma membranes. These results provide the first morphological and biochemical evidence to support the view that transport and hydrolysis are two spatially and functionally distinct processes in AEA degradation.Received 11 October 2004; received after revision 24 November 2004; accepted 7 December 2004     

核受体LXRa靶基因研究进展

核受体的研究近几年已有飞速的发展。LXRa(Liver X Receptor a)是一种与脂类代谢有关的核受体。研究发现LXRa的靶基因具有调节脂类的吸收、运输、转化和生物合成的功能,此外,LXRa在糖类的代谢等方面也有重要的调控作用。研究结果表明LXRa与动脉粥样硬化的发生有非常密切的关系。目前利用LXRa为靶标进行抗动脉粥样硬化药物筛选已经成为一个重要的研究方法。     

ABCA2: a candidate regulator of neural transmembrane lipid transport

Studies in the past years have implicated multispan transmembrane transport molecules of the ATP binding cassette (ABC) transporter family in cellular lipid export processes. The prototypic ABC transporter ABCA1 has recently been demonstrated to act as a major facilitator of cellular cholesterol and phospholipid export. Moreover, the transporter ABCA4 (ABCR) plays a pivotal role in retinaldehyde processing, and ABCA3 has recently implicated in lung surfactant processing. These pioneering observations have directed considerable attention to the A subfamily of ABC proteins. ABCA2 is the codefining member of the ABC A-transporter subclass. Although known for some time, it was not until recently that its complete molecular structure was established. Unlike other ABC A-subfamily members, ABCA2 is predominantly expressed in the brain and neural tissues. The unique expression profile together with available structural data suggest roles for this largest known ABC protein in neural transmembrane lipid export. Received 31 January 2002; received after revision 11 March 2002; accepted 11 March 2002     

Enzymatic reduction of fatty acids and acyl-CoAs to long chain aldehydes and alcohols

Summary The properties of enzymatic systems involved in the synthesis of long chain aldehydes and alcohols have been reviewed. Fatty acid and acyl-CoA reductases are widely distributed and generate fatty alcohols for ether lipid and was ester synthesis as well as fatty aldehydes for bacterial bioluminescence. Fatty alcohol is generally the major product of fatty acid reduction in crude or membrane systems, although reductases which release fatty aldehydes as products have also been purified. The reduction of fatty acid proceeds through the ATP-dependent formation of acyl intermediates such as acyl-CoA and acyl protein, followed by reduction to aldehyde and alcohol with NAD(P)H. In most cases, both the rate of fatty acid conversion and acyl chain specificity of the reaction are determined at the level of reduction of the intermediate. The reduction of fatty acids represents the major pathway for the control of the synthesis of fatty aldehydes and alcohols. Several other enzymatic reactions involved in lipid degradation also release fatty aldehydes but do not apear to play an important role in long chain alcohol synthesis.     

Effect of hydrogen peroxide on the binding of Merocyanine 540 to human erythrocytes

The fluorescent dye Merocyanine 540 (MC540) is often used as a probe to monitor the molecular packing of phospholipids in the outer leaflet of biomembranes. In a previous study we showed that the increased staining of erythrocytes with a perturbed membrane structure was mainly due to an increase in the fluorescence yield of cell-bound MC540, rather than to an increase of the number of bound molecules. Erythrocytes and ghosts exposed to continuous fluxes of H₂O₂ exhibited pronounced lipid peroxidation. Further, red blood cells subjected to this form of oxidative stress also showed increased staining with MC540. It appeared that this was caused by a strong increase in binding of MC540, together with a slight red shift of the fluorescence emission maximum and a small increase in the fluorescence yield of bound MC540. The changed MC540 binding characteristics were not observed when lipid peroxidation was suppressed by the presence of the antioxidant BHT in the incubation medium. However, open ghosts exposed to H₂O₂ showed no increase of MC540 binding, excluding a direct involvement of lipid peroxidation. Measurement of fluorescence emission spectra and gel filtration studies showed that MC540 can bind to H₂O₂-exposed hemoglobin. Experiments with erythrocytes lysed in hypotonic medium after exposure to H₂O₂ revealed that peroxidation of lipids with H₂O₂ induced a non-specific permeabilization of the plasma membrane to MC540, thereby allowing MC540 to bind to the oxidatively denatured, more hydrophobic hemoglobin. These results indicate that conclusions about packing of phospholipids in the outer leaflet of the membrane based on increased MC540-staining should be drawn with care. Received 27 September 1996; received after revision 5 November 1996; accepted 27 November 1996