Recent Advances in Understanding Proteins Involved in Lipid Droplet Formation,Growth and Fusion

Lipid droplets （LDs） were once viewed as simple, inert lipid micelles. However, they are now known to be organelles with a rich proteome involved in a myriad of cellular processes. LDs are heterogeneous in nature with different sizes and compositions of phospholipids, neutral lipids and proteins. This review takes a focused look at the roles of proteins involved in the regulation of LD formation, expansion, and morphology. The related proteins are summarized such as the fat-specific protein （Fsp27）, fat storage-inducing trans- membrane （FIT） proteins, seipin and ADP-ribosylation factor 1-coat protein complex I （Arf-COPI）. Finally, we present important challenges in LD biology for a deeper understanding of this dynamic organelle to be achieved.     

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.     

Evidence for the Involvement of Carnitine-Dependent Long-Chain Acyltransferases in Neuronal Triglyceride and Phospholipid Fatty Acid Turnover

Abstract: This study focuses on the potential involvement of carnitine palmitoyltransferase (CRT) on the phospholipid and triglyceride fatty acid turnover in neurons. This category of enzymes, which has been identified in several rat brain tissues, is well known for its role in modulating cellular fatty acid oxidation. Neuronal cell cultures from rat brain cortex incorporated radioactive palmitate or oleate into phospholipids and triglycerides. The largest fraction of radioactive fatty acids was recovered in phosphatidyl- choline followed by triglycerides and, to a lesser extent, phosphatidylethanolamine. CPT activity measured in neuronal lysates obtained from neurons treated with 40 μ M 2-tetradecylglycidic acid (TDGA) was almost completely abolished. Furthermore, between 2 and 10 μ M TDGA CPT activity dropped more rapidly than between 10 and 40 μ M. When the cells were pretreated with TDGA, the incorporation process of either radioactive fatty acid into triglycerides was dose-dependently suppressed. Radioactive fatty acid incorporation into phosphatidylcholine was significantly decreased in cells treated with TDGA. In contrast, phosphatidylethanolamine reacylation was essentially not affected by the CpT inhibitor. Similar results on the fatty acid incorporation into triglycerides and phospholipids were observed with neurons treated with palmitoyl- dl - aminocarnitine (PAC), a reversible CPT inhibitor, which does not consume free CoA. These effects do not seem to be the result of an inhibitory activity toward one of the steps involved in the acylation-deacylation process of triglycerides or phospholipids, as cellular lysates from TDGA-treated cells or lysates containing PAC incorporated radioactive fatty acids at rates comparable to controls. Our results suggest that CRT may be an important partner in the pathway of phospholipid and triglyceride fatty acid turnover in neurons.     

The TGL2 Gene of Saccharomyces cerevisiae Encodes an Active Acylglycerol Lipase Located in the Mitochondria

The Saccharomyces cerevisiae Tgl2 protein shows sequence homology to Pseudomonas triacylglycerol (TAG) lipases, but its role in the yeast lipid metabolism is not known. Using hemagglutinin-tagged Tgl2p purified from yeast, we report that this protein carries a significant lipolytic activity toward long-chain TAG. Importantly, mutant hemagglutinin-Tgl2p^S144A, which contains alanine 144 in place of serine 144 in the lipase consensus sequence (G/A)XSXG exhibits no such activity. Although cellular TAG hydrolysis is reduced in the tgl2 deletion mutant, overproduction of Tgl2p in this mutant leads to an increase in TAG degradation in the presence of fatty acid synthesis inhibitor cerulenin, but that of Tgl2p^S144A does not. This result demonstrates the lipolytic function of Tgl2p in yeast. Although other yeast TAG lipases are localized to lipid particles, Tgl2p is enriched in the mitochondria. The mitochondrial fraction purified from the TGL2-overexpressing yeast shows a strong lipolytic activity, which was absent in the tgl2 deletion mutant. Therefore, we conclude that Tgl2p is a functional lipase of the yeast mitochondria. By analyzing phenotypic effects of TGL2-deficient yeast, we also find that lipolysis-competent Tgl2p is required for the viability of cells treated with antimitotic drug. The addition of oleic acid, the product of Tgl2p-catalyzed lipolysis, fully complements the antimitotic drug sensitivity of the tgl2 null mutation. Thus, we propose that the mitochondrial Tgl2p-dependent lipolysis is crucial for the survival of cells under antimitotic drug treatment.     

A developmental transition in growth control during zebrafish caudal fin development

A long-standing question in developmental biology is how do growing and developing animals achieve form and then maintain it. We have revealed a critical transition in growth control during zebrafish caudal fin development, wherein a switch from allometric to isometric growth occurs. This morphological transition led us to hypothesize additional physiological changes in growth control pathways. To test this, we fasted juvenile and adult zebrafish. Juvenile fins continued allometric growth until development of the mature bi-lobed shape was completed. In contrast, the isometric growth of mature adult fins arrested within days of initiating a fast. We explored the biochemical basis of this difference in physiology between the two phases by assessing the sensitivity to rapamycin, a drug that blocks a nutrient-sensing pathway. We show that the nutrition-independent, allometric growth phase is resistant to rapamycin at 10-fold higher concentrations than are effective at arresting growth in the nutrition-dependent, isometric growth phase. We thus link a morphological transition in growth control between allometric and isometric growth mechanisms to different physiological responses to nutritional state of the animal and finally to different pharmacological responses to a drug (rapamycin) that affects the nutrition-sensing mechanism described from yeast to human.     

雷帕霉素对小鼠原代肝细胞脂滴形态和脂滴表面蛋白表达的影响

目的:探讨雷帕霉素(Rapamycin)对小鼠原代肝细胞脂滴形态和脂滴表面蛋白表达的影响。方法:采用胶原酶灌注方法分离和培养小鼠原代肝细胞,采用100μM油酸诱导肝细胞内脂肪的合成。采用0、10、20、50μM的雷帕霉素处理肝细胞12 hr后,利用中性脂肪染料Bodipy493/503对肝细胞内的脂滴进行染色,荧光显微镜下观察细胞脂滴形态和数量。定量试剂盒检测细胞内甘油三酯(TG)的含量利用Western blot检测不同浓度雷帕霉素处理的小鼠原代肝细胞脂滴表面蛋白ADRP的表达水平。结果:成功分离和培养了小鼠原代肝细胞,使用油酸处理能够明显增加原代肝细胞内脂滴的数量。随着体外雷帕霉素处理浓度的增加,荧光显微镜下观察发现原代肝细胞内脂滴的数量呈现明显的下降趋势,甘油三酯的含量也呈见明确的下降趋势,在20μM浓度下就表现出显著性差异。Western blot结果显示雷帕霉素能够在抑制肝细胞内脂肪储积的同时降低脂滴表面蛋白ADRP的表达水平,并且随着雷帕霉素处理浓度的增加,其对ADRP表达的抑制越明显。结论:雷帕霉素能够抑制肝细胞内中性脂肪的储积,同时降低脂滴表面蛋白ADRP的表达水平。也间接说明了mTOR信号通路能够影响肝细胞内脂肪的储积,也为脂肪肝的防治提供了一个新的实验基础。     

Mechanism of lactose-proton cotransport in Escherichia coli. Kinetic results in terms of the site exposure model

Rigorous kinetic derivations are presented for the Site Exposure mechanism of lactose-proton cotransport in E. coli [J. Theor. Biol. (1978) 75, 35-50]. Proton translocation inwards is solely associated with the external exposure of the galactoside binding site. A symmetric dimer configuration of the transporter is proposed, resulting in two forms corresponding to the cis and the trans orientation of the binding sites. The cis to trans orientation is inherently unfavorable, induced only by transmembrane substrate gradients. Recently reported extensive kinetic data are straightforwardly predicted by this mechanism, including the complicated effects on the apparent affinity and maximal velocity of uptake exhibited by changes in the magnitude of the proton electrochemical gradient.     

The size and phospholipid composition of lipid droplets can influence their proteome

The proteomic makeup of lipid droplets (LDs) is believed to regulate the function of LDs, which are now recognized as important cellular organelles that are associated with many human metabolic disorders. However, factors that help determine LD proteome remain to be identified and characterized. Here we analyzed the phospholipid and protein composition of LDs isolated from wild type (WT) yeast cells, and also from fld1Δ, cds1, and ino2Δ mutant cells which produce ‘supersized’ LDs. LDs of fld1Δ and WT cells exhibited similar phospholipid profiles, whereas LDs of cds1 and ino2Δ strains had a higher (cds1) or lower (ino2Δ) percentage of phosphatidylcholine than those of WT, respectively. Unexpectedly, the presence of most known LD resident proteins was greatly reduced in the LD fraction isolated from cds1 and ino2Δ, including neutral lipid hydrolases. Consistent with this result, mobilization of neutral lipids was seriously impaired in these two strains. Contrary to the reduction of LD resident proteins, the Hsp90 family molecular chaperones, Hsc82 and Hsp82, were greatly increased in the LD fractions of cds1 and ino2Δ strains without changes at the level of expression. These data demonstrate the impact of LD phopholipids and size on the makeup of LD proteome.     

Microorganism lipid droplets and biofuel development

Lipid droplet (LD) is a cellular organelle that stores neutral lipids as a source of energy and carbon. However, recent research has emerged that the organelle is involved in lipid synthesis, transportation, and metabolism, as well as mediating cellular protein storage and degradation. With the exception of multi-cellular organisms, some unicellular microorganisms have been observed to contain LDs. The organelle has been isolated and characterized from numerous organisms. Triacylglycerol (TAG) accumulation in LDs can be in excess of 50% of the dry weight in some microorganisms, and a maximum of 87% in some instances. These microorganisms include eukaryotes such as yeast and green algae as well as prokaryotes such as bacteria. Some organisms obtain carbon from CO2 via photosynthesis, while the majority utilizes carbon from various types of biomass. Therefore, high TAG content generated by utilizing waste or cheap biomass, coupled with an efficient conversion rate, present these organisms as bio-tech ‘factories’ to produce biodiesel. This review summarizes LD research in these organisms and provides useful information for further LD biological research and microorganism biodiesel development. [BMB Reports 2013; 46(12): 575-581]     

Human frame shift mutations affecting the carboxyl terminus of perilipin increase lipolysis by failing to sequester the adipose triglyceride lipase (ATGL) coactivator AB-hydrolase-containing 5 (ABHD5)

Perilipin (PLIN1) is a constitutive adipocyte lipid droplet coat protein. N-terminal amphipathic helices and central hydrophobic stretches are thought to anchor it on the lipid droplet, where it appears to function as a scaffold protein regulating lipase activity. We recently identified two different C-terminal PLIN1 frame shift mutations (Leu-404fs and Val-398fs) in patients with a novel subtype of partial lipodystrophy, hypertriglyceridemia, severe insulin resistance, and type 2 diabetes (Gandotra, S., Le Dour, C., Bottomley, W., Cervera, P., Giral, P., Reznik, Y., Charpentier, G., Auclair, M., Delépine, M., Barroso, I., Semple, R. K., Lathrop, M., Lascols, O., Capeau, J., O'Rahilly, S., Magré, J., Savage, D. B., and Vigouroux, C. (2011) N. Engl. J. Med. 364, 740-748.) When overexpressed in preadipocytes, both mutants fail to inhibit basal lipolysis. Here we used bimolecular fluorescence complementation assays to show that the mutants fail to bind ABHD5, permitting its constitutive coactivation of ATGL, resulting in increased basal lipolysis. siRNA-mediated knockdown of either ABHD5 or ATGL expression in the stably transfected cells expressing mutant PLIN1 reduced basal lipolysis. These insights from naturally occurring human variants suggest that the C terminus sequesters ABHD5 and thus inhibits basal ATGL activity. The data also suggest that pharmacological inhibition of ATGL could have therapeutic potential in patients with this rare but metabolically serious disorder.     

What can we learn about the lipid vesicle structure from the small-angle neutron scattering experiment?

Small-angle neutron scattering (SANS) on the unilamellar vesicle (ULV) populations (diameter 500 and 1,000 Å) in D₂O was used to characterize lipid vesicles from dimyristoylphosphatidylcholine (DMPC) at three phases: gel L_β′, ripple P_β′ and liquid L_α. Parameters of vesicle populations and internal structure of the DMPC bilayer were characterized on the basis of the separated form factor (SFF) model. Vesicle shape changes from nearly spherical in the L_α phase to elliptical in the P_β′ and L_β′ phases. This is true for vesicles prepared via extrusion through pores with the diameter 500 Å. Parameters of the internal bilayer structure (thickness of the membrane and the hydrophobic core, hydration and the surface area of the lipid molecule) were determined on the basis of the hydrophobic–hydrophilic (HH) approximation of neutron scattering length density across the bilayer ρ(x) and of the step function (SF) approximation of ρ(x). DMPC membrane thickness in the L_α phase (T=30°C) demonstrates a dependence on the membrane curvature for extruded vesicles. Prepared via extrusion through 500 Å diameter pores, vesicle population in the L_α phase has the following characteristics: average value of minor semi-axis 266±2 Å, ellipse eccentricity 1.11±0.02, polydispersity 26%, thickness of the membrane 48.9±0.2 Å and of the hydrophobic core 19.9±0.4 Å, surface area 60.7±0.5 Ų and number of water molecules 12.8±0.3 per DMPC molecule. Vesicles prepared via extrusion through pores with the diameter 1,000 Å have polydispersity of 48% and membrane thickness of 45.5±0.6 Å in the L_α phase. SF approximation was used to describe the DMPC membrane structure in L_β′ (T=10°C) and P_β′ (T=20°C) phases. Extruded DMPC vesicles in D₂O have membrane thickness of 49.6±0.5 Å in the L_β′ phase and 48.3±0.6 Å in the P_β′ phase. The dependence of the DMPC membrane thickness on temperature was restored from the SANS experiment.     

Nitrogen-starvation triggers cellular accumulation of triacylglycerol in Metarhizium robertsii

Nitrogen starvation can induce cellular triacylglycerol (TAG) accumulation in different organisms with an unclear mechanism. In this study, we performed nutrient starvation and lipid droplet (LD) proteomics analyses of the filamentous fungus Metarhizium robertsii. Our results indicated that nitrogen starvation activated cell autophagic activity but inhibited the internalization of LDs into vacuoles for degradation. LD proteomic analyses identified an array of differentially accumulated proteins including autophagy-related (ATG) proteins, heat shock proteins, TAG metabolic and phospholipid biosynthetic enzymes when the fungus was grown in different nutrient conditions. In contrast to the highly activated MrATG8, the ATG proteins involved in vacuolar LD internalization were down-regulated after nitrogen starvation. Cellular TAG contents were increased in different ATG-gene null mutants of M. robertsii. In addition, TAG increase could be due to the up-regulation of TAG biogenesis along with the down-regulation of TAG catabolic enzymes in fungal cells after nitrogen deprivation. The data of this study benefit our understanding of the mechanism of nitrogen starvation induced TAG increase in different cells.     

Lipolytic activity in submerged cultures of Issatchenkia orientalis

The ability of the yeast Issatchenkia orientalis CECT 10688 to secrete lipolytic activity in submerged culture was investigated. The yeast was grown in a complex medium supplemented with a fixed concentration of several lipidic compounds (triglycerides, fatty acids). Maximum enzyme activity around 70–80 U cm⁻³ was produced in cultures supplemented with tributyrin. An optimum tributyrin concentration of 10 g dm⁻³ was selected. Several surfactants were added to the cultures, but no significant increase in activity was detected. Finally, the effect of the type of carbon source on lipolytic enzyme production was studied. The best results were obtained with glucose and fructose (60–80 U cm⁻³), while rather low enzyme activity was found in cultures grown on lactose and maltose (about 20 U cm⁻³).     

Characterization of Large Unilamellar Vesicles as Models for Studies of Lipid Peroxidation Initiated by Azocompounds

The aim of this work was to characterize large unilamellar vesicles (LUVETs) prepared by a hand-driven extrusion device in order to use them for studies of lipid peroxidation and antioxidant activity. Vesicle structure and size were examined by electron microscopy. Lipid and antioxidant content was determined before and after the extrusion procedure. Then LUVETs were subjected to autoxidation initiated by both the lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile) and the water-soluble 2,2'-azobis(2-amidinopropane hydrochloride) azocompounds. The results demonstrated that: i) LUVETs prepared with lipid concentrations ranging between 25 and 150 mM were essentially unilamellar and reasonably homogeneous, with an average diameter of 90 nm; ii) the phospholipid, cholesterol and antioxidant amounts retained by filters were about 10-15%; iii) LUVETs were suitable for autoxidation studies initiated by the water-soluble azocompound both in the absence and presence of antioxidants. The lipid-soluble azocompound could be used only at low concentrations and its vesicle content had to be determined since part of the initiator was not incorporated into the lipid bilayer. These data suggest that LUVETs seem to be recommended for studies of lipid peroxidation and antioxidant activity.