Characterization of a lipid droplet protein from Yarrowia lipolytica that is required for its oleaginous phenotype

Oleaginous microorganisms are characterized by their ability to store high amounts of triacylglycerol (TAG) in intracellular lipid droplets (LDs). In this work, we characterized a protein of the oleaginous yeast Yarrowia lipolytica that is associated with LD and plays a role in the regulation of TAG storage. This protein is required for the oleaginous phenotype of Y. lipolytica because deletion of the coding gene results in a strongly reduced TAG content of the mutant. Therefore, we named it Oleaginicity Inducing LD protein, Oil1. Furthermore, a mutant overexpressing OIL1 accumulates more TAG than the wild type and is delayed in TAG lipolysis when this process is stimulated. We found that Oil1p plays a role in protecting the TAG content of the LD from degradation through lipases under conditions where the cell aims at building up its TAG reserves. Heterologous expression studies showed that Oil1p rescued the phenotype of a Saccharomyces cerevisiae mutant deleted for the perilipin-like protein Pln1p and that its expression in COS-7 cells resulted in increased TAG accumulation, similar to the phenotype of a perilipin 1 expressing control strain. Despite this phenotypical parallels to mammalian perilipins, Oil1p is not a member of this protein family and its activity does not depend on phosphorylation. Rather, our results suggest that ubiquitination might contribute to the function of Oil1p in Y. lipolytica and that a different mechanism evolved in this species to regulate TAG homeostasis.     

PAT蛋白抑制饥饿诱导的脂滴快速融合

目的:脂滴快速融合是增大脂滴直径的方式之一,但其研究相对少。本研究旨在建立脂滴快速融合的细胞模型,以便对其进行深入的生物学研究。方法:本研究使用大鼠肾成纤维细胞系NRK和小鼠前脂肪细胞系3T3-L1两种细胞系,先用油酸诱导细胞内产生大量脂滴,再使用饥饿缓冲液培养细胞,利用显微镜实时观测技术跟踪脂滴动态变化,建立脂滴快速融合的模型。而后在此模型中,加入自噬抑制剂或者以过表达CCT为阳性对照,过表达PAT蛋白(PLIN1、ADRP和TIP47),来探究它们在调控脂滴快速融合方面的功能。结果:饥饿缓冲液处理约3小时可诱导细胞发生脂滴快速融合,其融合速率很快,从脂滴接触到融合完成可发生在20秒内,显然不同于CIDE蛋白调控的缓慢脂滴融合过程。自噬抑制剂可以抑制自噬,但是并没有显著影响脂滴快速融合,说明饥饿诱导的脂滴快速融合不依赖于自噬。另发现,与过表达GFP相比,过表达定位于脂滴的GFP-CCT、GFP-PLIN1、GFP-ADRP或GFP-TIP47均能显著性抑制快速融合导致的脂滴变大的现象。结论:本研究建立了饥饿缓冲液诱导脂滴发生快速融合的细胞模型,并证明PAT蛋白(PLIN1、ADRP、TIP47)能抑制脂滴快速融合。     

Liver X receptor (LXR) regulates human adipocyte lipolysis

The Liver X receptor (LXR) is an important regulator of carbohydrate and lipid metabolism in humans and mice. We have recently shown that activation of LXR regulates cellular fuel utilization in adipocytes. In contrast, the role of LXR in human adipocyte lipolysis, the major function of human white fat cells, is not clear. In the present study, we stimulated in vitro differentiated human and murine adipocytes with the LXR agonist GW3965 and observed an increase in basal lipolysis. Microarray analysis of human adipocyte mRNA following LXR activation revealed an altered gene expression of several lipolysis-regulating proteins, which was also confirmed by quantitative real-time PCR. We show that expression and intracellular localization of perilipin1 (PLIN1) and hormone-sensitive lipase (HSL) are affected by GW3965. Although LXR activation does not influence phosphorylation status of HSL, HSL activity is required for the lipolytic effect of GW3965. This effect is abolished by PLIN1 knockdown. In addition, we demonstrate that upon activation, LXR binds to the proximal regions of the PLIN1 and HSL promoters. By selective knock-down of either LXR isoform, we show that LXRα is the major isoform mediating the lipolysis-related effects of LXR. In conclusion, the present study demonstrates that activation of LXRα up-regulates basal human adipocyte lipolysis. This is at least partially mediated through LXR binding to the PLIN1 promoter and down-regulation of PLIN1 expression.     

Perilipin and adipophilin expression in lipid loaded macrophages

Lipid-filled macrophages (foam cells) are a defining feature of atherosclerotic plaques. Foam cells contain lipid droplet-associated proteins that in other cell types regulate lipid turnover. In foam cell such proteins may directly affect lipid droplet formation and lipid efflux. Differentiated primary human monocytes or THP-1 cells were lipid loaded by incubation with aggregated low density lipoproteins (AgLDL) or VLDL resulting in macrophage foam cells with predominantly cholesterol ester or triglyceride-rich lipid droplets, respectively. Lipid droplets were isolated and major proteins identified by mass spectrometry, among them the apolipoprotein B-48 receptor that has not previously been recognized in this context. Expression of two proteins, perilipin and adipophilin, was quantified by Western blots of cell lysates. Perilipin content decreased and adipophilin increased with lipoprotein lipid loading regardless of intracellular neutral lipid composition. This protein expression pattern may hinder lipid turnover in macrophage foam cells, thereby increasing lipid content of atherosclerotic plaques.     

Sulfhydration of perilipin 1 is involved in the inhibitory effects of cystathionine gamma lyase/hydrogen sulfide on adipocyte lipolysis

Hydrogen sulfide (H₂S) is a novel adipokine mediating glucose uptake, lipid storage and mobilization, thus contributing to the genesis of obesity and associated diseases. Our previous work demonstrated that H₂S inhibited isoproterenol-stimulated lipolysis by reducing the phosphorylation of perilipin 1 (plin-1), a lipid-droplet protein blocking lipase access. How H₂S modulates plin-1 phosphorylation is still unclear. Our present study found that an H₂S donor slightly increased adipose tissue weight and reduced lipolysis in mice; by contrast, deleting the key H₂S generation enzyme cystathionine gamma lyase (CSE) in adipocytes lowered adipose accumulation and enhanced lipolysis. Intriguingly, an H₂S donor induced sulfhydration of plin-1 but not hormone-sensitive lipase, and CSE deletion abolished the post-translational modification of plin-1. During isoproterenol-stimulated lipolysis, plin-1 sulfhydration was associated with reduced phosphorylation, and removing sulfhydration by dithiothreitol recovered the phosphorylation. Finally, plin-1 knockout abolished the effect of H₂S on lipolysis, which indicates that plin-1 sulfhydration is a major direct target of H₂S in lipolysis. We have identified a new post-translation modification, sulfhydration (direct action by H₂S) of plin-1, causing reduced phosphorylation then decreased lipolysis. This finding also highlights a novel molecular regulatory mechanism of lipolysis.     

A proposed model of fat packaging by exchangeable lipid droplet proteins

Humans have evolved mechanisms of efficient fat storage to survive famine, but these mechanisms contribute to obesity in our current environment of plentiful food and reduced activity. Little is known about how animals package fat within cells. Five related structural proteins serve roles in packaging fat into lipid droplets. The proteins TIP47, S3-12, and OXPAT/MLDP/PAT-1 move from the cytosol to coat nascent lipid droplets during rapid fat storage. In contrast, perilipin and adipophilin constitutively associate with lipid droplets and play roles in sustained fat storage and regulation of lipolysis. Different tissues express different complements of these lipid droplet proteins. Thus, the tissue-specific complement of these proteins determines how tissues manage lipid stores.     

Adipocyte responses to adrenaline and insulin in active and former sportsmen

The rates of lipolysis and lipogenesis in adipocytes, isolated from biopsy samples of subcutaneous fat, was assessed by estimation of glycerol release during a 30-min incubation, and of the incorporation of 14C-glucose into lipids during a 1-h incubation at 37 degrees C, respectively. The subjects were six highly-qualified, active endurance sportsmen, eight former endurance sportsmen of international class, and six untrained young men. In the active sportsmen the basal rate of lipolysis was about half of that in the previously-active sportsmen and the untrained subjects, but after the addition of adrenaline (10(-4) or 5 x 10(-4) mol.l-1) the lipolysis rate was the highest. No differences were observed in the lipolytic rates in the former sportsmen compared to the untrained subjects. Gases of a comparatively high level of lipogenesis were found in the trained subjects. The addition of insulin (9 microU.ml-1) to isolated adipocytes caused a significant augmentation of individual rates of lipogenesis in the active sportsmen and the untrained persons but not in the previously-active sportsmen. In comparison with the active sportsmen, the previously active sportsmen revealed an increased basal rate of lipolysis and a reduced sensitivity to the lipogenic action of insulin. These findings suggest that these changes may have had significance in avoiding an increase of adipose tissue after a decrease in energy expenditure due to a change in physical activity.     

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.     

Packaging of fat: an evolving model of lipid droplet assembly and expansion

Lipid droplets (LDs) are organelles found in most types of cells in the tissues of vertebrates, invertebrates, and plants, as well as in bacteria and yeast. They differ from other organelles in binding a unique complement of proteins and lacking an aqueous core but share aspects of protein trafficking with secretory membrane compartments. In this minireview, we focus on recent evidence supporting an endoplasmic reticulum origin for LD formation and discuss recent findings regarding LD maturation and fusion.     

Perilipin5基因功能结构域载体的构建及亚细胞定位分析

目的:依据perilipin5的功能结构域,构建含perilipin5截断体的真核表达载体,并研究它们的亚细胞定位。方法:以小鼠肝脏cDNA文库为模板,PCR扩增出perilipin5的全长及功能结构域,将之分别装载入真核表达载体PCMV5中,并引入HA标签。酶切和测序鉴定,脂质体法将构建的质粒转染293T细胞,Western blot验证表达,免疫荧光检测标记HA,于荧光显微镜下观察perilipin5各结构域的亚细胞定位。结果:构建的质粒序列正确,转染细胞后可检测到HA-perilipin5融合蛋白的表达,免疫荧光显示含有1-188aa结构域的perilipin5截断体可定位于脂滴表面,1-188aa一旦缺失perilipin5的截断体则弥散于胞内。结论:包含perilipin5功能结构域的真核表达载体构建成功,perilipin5的1-188aa与其脂滴定位密切相关。     

Lipolysis response to endoplasmic reticulum stress in adipose cells

In obesity and diabetes, adipocytes show significant endoplasmic reticulum (ER) stress, which triggers a series of responses. This study aimed to investigate the lipolysis response to ER stress in rat adipocytes. Thapsigargin, tunicamycin, and brefeldin A, which induce ER stress through different pathways, efficiently activated a time-dependent lipolytic reaction. The lipolytic effect of ER stress occurred with elevated cAMP production and protein kinase A (PKA) activity. Inhibition of PKA reduced PKA phosphosubstrates and attenuated the lipolysis. Although both ERK1/2 and JNK are activated during ER stress, lipolysis is partially suppressed by inhibiting ERK1/2 but not JNK and p38 MAPK and PKC. Thus, ER stress induces lipolysis by activating cAMP/PKA and ERK1/2. In the downstream lipolytic cascade, phosphorylation of lipid droplet-associated protein perilipin was significantly promoted during ER stress but attenuated on PKA inhibition. Furthermore, ER stress stimuli did not alter the levels of hormone-sensitive lipase and adipose triglyceride lipase but caused Ser-563 and Ser-660 phosphorylation of hormone-sensitive lipase and moderately elevated its translocation from the cytosol to lipid droplets. Accompanying these changes, total activity of cellular lipases was promoted to confer the lipolysis. These findings suggest a novel pathway of the lipolysis response to ER stress in adipocytes. This lipolytic activation may be an adaptive response that regulates energy homeostasis but with sustained ER stress challenge could contribute to lipotoxicity, dyslipidemia, and insulin resistance because of persistently accelerated free fatty acid efflux from adipocytes to the bloodstream and other tissues.     

Temporal and spatial assembly of lipid droplet-associated proteins in 3T3-L1 preadipocytes

This study aimed to investigate the relationship between newly formed lipid droplets and lipid droplet surface proteins, including perilipin, adipose differentiation related protein (ADRP), and p200 kDa protein (p200) in 3T3-L1 preadipocytes, during lipogenesis. Sterol ester was used to induce nascent lipid droplets in 3T3-L1 preadipocytes and the sequence of lipids and lipid droplet surface proteins was studied using a combination of immunohistochemistry and Nile red staining/Oil red O. We demonstrated that, although most growing lipid droplets appeared to have a lipid core surrounded by a fluorescent rim of ADRP, perilipin, and p200, tiny protein aggregates of ADRP, perilipin, or p200 could also be found to occur in the absence of lipid accumulation. In addition, ADRP associated with nascent lipid droplets prior to that of perilipin or p200. We provide evidence that lipid droplet surface proteins, especially ADRP and perilipin, are important in serving as a nucleation center for the assembly of lipid to form nascent lipid droplets.     

Lipid droplets: a classic organelle with new outfits

Lipid droplets are depots of neutral lipids that exist virtually in any kind of cell. Recent studies have revealed that the lipid droplet is not a mere lipid blob, but a major contributor not only to lipid homeostasis but also to diverse cellular functions. Because of the unique structure as well as the functional importance in relation to obesity, steatosis, and other prevailing diseases, the lipid droplet is now reborn as a brand new organelle, attracting interests from researchers of many disciplines.     

Genistein affects lipogenesis and lipolysis in isolated rat adipocytes

Genistein is a phytoestrogen found in several plants eaten by humans and food-producing animals and exerting a wide spectrum of biological activity. In this experiment, the impact of genistein on lipogenesis and lipolysis was studied in isolated rat adipocytes. Incubation of the cells (10⁶ cells/ml in plastic tubes at 37°C with Krebs-Ringer buffer, 90 min) with genistein (0.01, 0.3, 0.6 and 1 mM) clearly restricted (1 nM) [U-¹⁴C]glucose conversion to total lipids in the absence and presence of insulin. When [¹⁴C]acetate was used as the substrate for lipogenesis, genistein (0.01, 0.1 and 1 mM) exerted a similar effect. Thus, the anti-lipogenetic action of genistein may be an effect not only of alteration in glucose transport and metabolism, but this phytoestrogen can also restrict the fatty acids synthesis and/or their estrification. Incubation of adipocytes with estradiol at the same concentrations also resulted in restriction of lipogenesis, but the effect was less marked. Genistein (0.1 and 1 mM) augmented basal lipolysis in adipocytes. This process was strongly restricted by insulin (1 μM) and H-89 (an inhibitor of protein kinase A; 50 μM) and seems to be primarily due to the inhibitory action of the phytoestrogen on cAMP phosphodiesterase in adipocytes. Genistein at the smallest concentration (0.01 mM) augmented epinephrine-stimulated (1 μM) lipolysis but failed to potentiate lipolysis induced by forskolin (1 μM) or dibutyryl-cAMP (1 mM). These results suggest genistein action on the lipolytic pathways before activation of adenylate cyclase. The restriction of lipolysis stimulated by several lipolytic agents – epinephrine, forskolin and dibutyryl-cAMP were observed when adipocytes were incubated with genistein at highest concentrations (0.1 and 1 mM). These results prove the inhibitory action of this phyestrogen on the final steps of the lipolytic cascade, i.e. on protein kinase A or hormone sensitive lipase. Estradiol, added to the incubation medium, did not affect lipolysis. It can be concluded that genistein significantly affects lipogenesis and lipolysis in isolated rat adipocytes.     

Quantitative electron microscopy shows uniform incorporation of triglycerides into existing lipid droplets

The lipid droplet (LD) is an organelle with a lipid ester core and a surface phospholipid monolayer. The mechanism of LD biogenesis is not well understood. The present study aimed to elucidate the LD growth process, for which we developed a new electron microscopic method that quantifies the proportion of existing and newly synthesized triglycerides in individual LDs. Our method takes advantage of the reactivity of unsaturated fatty acids and osmium tetroxide, which imparts LDs an electron density that reflects fatty acid composition. With this method, existing triglyceride-rich LDs in 3Y1 fibroblasts were observed to incorporate newly synthesized triglycerides at a highly uniform rate. This uniformity and its persistence even after microtubules were depolymerized suggest that triglycerides in fibroblasts are synthesized in the local vicinity of individual LDs and then incorporated. In contrast, LDs in 3T3-L1 adipocytes showed heterogeneity in the rate at which lipid esters were incorporated, indicating different mechanisms of LD growth in fibroblasts and adipocytes.     

Targets for TNF-alpha-induced lipolysis in human adipocytes

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha)-induced lipolysis may be important for insulin resistance in both obesity and cachexia. In rodent cells TNF-alpha enhances lipolysis through down-regulation of the expression of the membrane proteins Galpha(i) and the lipid droplet-associated protein perilipin (PLIN). In human (but not murine) adipocytes TNF-alpha stimulates lipolysis through the mitogen activated protein kinases (MAPKs) p44/42 and JNK although it is unclear whether this is mediated via PLIN and/or Galpha(i). METHODS: Galpha(i) and PLIN as down-stream effectors of MAPKs were assessed in human adipocytes stimulated with TNF-alpha in the absence or presence of specific MAPK inhibitors. RESULTS: A 48-h incubation with TNF-alpha resulted in a pronounced increase in lipolysis, which was paralleled by a decrease in the mRNA and protein expression of PLIN. Both these effects were inhibited in a concentration-dependent manner in the presence of MAPK inhibitors specific for p44/42 (PD98059) and JNK (SP600125). However, TNF-alpha did not affect Galpha(i) mRNA or protein expression. Furthermore, experiments with pertussis toxin demonstrated that inhibition of Galpha(i) signaling did not affect TNF-alpha-mediated lipolysis. CONCLUSIONS: Our results suggest that TNF-alpha-mediated lipolysis is dependent on down-regulation of PLIN expression via p44/42 and JNK. This could be an important mechanism for the development of insulin resistance in both obesity and cachexia. However, in contrast to findings in rodent cells, Galpha(i) does not appear to be essential for TNF-alpha-induced lipolysis in human adipocytes.