Lipid Storage Droplets Research Articles

FABP7 is a key metabolic regulator in HER2+ breast cancer brain metastasis.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer patients is associated with increased incidence of breast cancer brain metastases (BCBM), but the mechanisms underlying this phenomenon remain unclear. Here, to identify brain-predominant genes critical for the establishment of BCBM, we conducted an in silico screening analysis and identified that increased levels of fatty acid binding protein 7 (FABP7) correlate with lower survival and higher incidence of brain metastases in breast cancer patients. We validated these findings using HER2+ BCBM cells compared to parental breast cancer cells. Importantly, through knockdown and overexpression assays we characterized the role of FABP7 in the BCBM process in vitro and in vivo. Our results uncover a key role of FABP7 in metabolic reprogramming of HER2+ breast cancer cells, supporting a glycolytic phenotype and storage of lipid droplets that enable their adaptation and survival in the brain microenvironment. Additionally, FABP7 is shown to be required for up-regulation of key metastatic genes and pathways, such as Integrins-Src and VEGFA, and for the growth of HER2+ breast cancer cells in the brain microenvironment in vivo. Together our results support FABP7 as a potential target for the treatment of HER2+ BCBM.

Lipid storage droplet protein 5 reduces sodium palmitate‑induced lipotoxicity in human normal liver cells by regulating lipid metabolism‑related factors.

Lipid storage droplet protein 5 (LSDP5) is specifically expressed in tissues with high oxidative metabolism such as liver and heart. The present study aimed to explored the role of LSDP5 in sodium palmitate-induced lipotoxicity in LO2 normal human liver cells. LO2 cells were treated with various concentrations of sodium palmitate (25, 50, 75, 100, 125 and 150 µmol/l) for 12, 24 and 48 h, and cell viability was determined by Cell Counting Kit-8. Subsequently, LO2 cells were exposed to 100 µmol/l sodium palmitate for 48 h to induce lipotoxicity (Model). Lipotoxicity Model LO2 cells were also transfected with pCMV5-LSDP5 overexpression vector, and reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP) and apoptotic rates were measured. The contents of non-esterified fatty acid (NEFA), malondialdehyde (MDA) and superoxide dismutase (SOD) were also measured. The expression levels of LSDP5, and apoptosis-, mitochondrial-, lipid metabolism-related factors were detected using reverse transcription-quantitative polymerase chain reaction and western blot assays. The results indicated that sodium palmitate exposure inhibited cell viability and induced lipotoxicity in LO2 cells. LSDP5 overexpression decreased ROS and apoptotic rates, and reduced NEFA and MDA content. LSDP5 transfection rescued the loss of MMP and elevated SOD content in lipotoxicity Model LO2 cells. In addition, LSDP5 upregulated the expression levels of B-cell lymphoma-2, acetyl-CoA carboxylase1/2 and fatty acid synthase (Fas), whereas the expression levels of activated-caspase-3, Bcl-2-associated X protein, cytochrome c, cytochrome c oxidase subunits IV, carnitine palmitoyltransferase 1a and peroxisome proliferator-activated receptors α levels were downregulated. LSDP5 may produce a protective effect on sodium palmitate-induced lipotoxicity in LO2 cells by regulating lipid metabolism-related factors.

Proteomics Analysis of Lipid Droplets from the Oleaginous Alga Chromochloris zofingiensis Reveals Novel Proteins for Lipid Metabolism

Chromochloris zofingiensis represents an industrially relevant and unique green alga, given its capability of synthesizing triacylglycerol (TAG) and astaxanthin simultaneously for storage in lipid droplets (LDs). To further decipher lipid metabolism, the nitrogen deprivation (ND)-induced LDs from C. zofingiensis were isolated, purified, and subjected to proteomic analysis. Intriguingly, many C. zofingiensis LD proteins had no orthologs present in LD proteome of the model alga Chlamydomonas reinhardtii. Seven novel LD proteins (i.e., two functionally unknown proteins, two caleosins, two lipases, and one l-gulonolactone oxidase) and the major LD protein (MLDP), which were all transcriptionally up-regulated by ND, were selected for further investigation. Heterologous expression in yeast demonstrated that all tested LD proteins were localized to LDs and all except the two functionally unknown proteins enabled yeast to produce more TAG. MLDP could restore the phenotype of mldp mutant strain and enhance TAG synthesis in wild-type strain of C. reinhardtii. Although MLDP and caleosins had a comparable abundance in LDs, they responded distinctly to ND at the transcriptional level. The two lipases, instead of functioning as TAG lipases, likely recycled polar lipids to support TAG synthesis. For the first time, we reported that l-gulonolactone oxidase was abundant in LDs and facilitated TAG accumulation. Moreover, we also proposed a novel working model for C. zofingiensis LDs. Taken together, our work unravels the unique characteristics of C. zofingiensis LDs and provides insights into algal LD biogenesis and TAG synthesis, which would facilitate genetic engineering of this alga for TAG improvement.

Acid sphingomyelinase mediates the noise-induced liver disorder in mice.

Noise-induced structural and functional disorder of the liver has been realized, but the underlying mechanism remains to be characterized, which has limited the introduction of precautious measures. Over-activation of acid sphingomyelinase (ASM)/ceramide (Cer) pathway takes centre stage in hepatocyte injury entailed by various stimulus. We aimed to investigate whether it mediated the noise elicited liver disorder on infrastructure, lipid metabolism, apoptosis, and oxidative stress. Mice were exposed to broad band noise (20-20k Hz, 90-110 dB) for 1, 3, 5 or 7days by 3hr/d. Doxepin hydrochloride (DOX), an ASM inhibitor was given by 5mg/kg/d gavage. We showed that 5 or 7days intense, broad band noise exposure caused significant infrastructure derangement and lipid droplets storage in hepatocytes. The content of cholesterol, free fatty acids or triglyceride was increased significantly in liver tissue upon noise stimulation. Moreover, the noise promoted apoptosis and superoxide generation in hepatocytes significantly, enhancing activity of aspartate aminotransferase (AST) or alanine amino transferase (ALT) in serum. Acid sphingomyelinase activity and Cer generation in liver tissue were elevated by noise exposure, which was normalized with DOX administrated. Accordingly, DOX alleviated steatosis, apoptosis, oxidative stress and enzymatic change in hepatocytes or serum of noise exposed mice substantially. In summary, our results suggest the ASM/Cer pathway contributes to the broad band noise elicited liver damage in mice.

Interaction of Perilipin 5 with Mitochondria

Problems with lipid metabolism can lead to array of diseases such as fatty liver disease, obesity, and diabetes. Nearly one‐third of the world's population is obese or overweight, and struggle with these diseases. Cellular lipids are found in lipid storage droplets. Perilipins are a family of proteins that associate with the surface of lipid storage droplets and regulate their contents. We used CHO cells expressing perilipin 5 protein and assayed them using immunofluorescent microscopy to determine if mitochondria interact with perilipin 5 under different phosphorylation states. We hypothesized that the interaction of mitochondria and perilipin 5 was phosphorylation dependent. We used antibodies that detect either FLAG tagged and native perilipin 5 and Mitotracker red to stain cells and forskolin and IBMX to activate the PKA signaling pathway. Following one hour of stimulation, perilipin 5 appeared in the lipid droplets and cytosol of the cell but interactions with mitochondria were not apparent. In this model 3 hours of stimulation is required for perilipin 5 to translocate to the nucleus. Collectively these ongoing studies show that the cellular partitioning of perilipin 5 is in part phosphorylation dependent.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of the Perilipin 5 C‐Terminus

Many countries around the globe have been plagued with a recent epidemic in lipid‐associated diseases. These diseases include obesity, diabetes, and non‐alcoholic fatty liver disease among many others. The United States itself has fallen heavily victim to these ailments, placing first in worldwide rankings for obese persons and within the top fiftieth in rankings for the percentage of persons afflicted with diabetes. These diseases pose clear threats to modern society and having a greater understanding of the mechanistic background of them may lead to better treatments options for those afflicted. A common thread through all these devastating illnesses is their connection to lipids and more specifically lipid storage. Cellular lipids are stored in lipid storage droplets, and these droplets are coated by the perilipins, five regulatory proteins that have an integral role in regulating the breakdown of fats. One such of these family members is perilipin 3, a protein that's structure has been partially determined. In Perilipin 5 this structure has not been determined and is the current focus of this study. Previously a crystal structure of the C‐terminal region of perilipin 3 was determined for residues 206 to 431. These regions were aligned with perilipin 5 using BLAST and their analogous location within the Perilipin 5 DNA sequence was determined. This region of perilipin 5 is being expressed as a 6‐his fusion protein for subsequent characterization and structure determination. Using the predicative programs RaptorX and Phyre2 theoretical protein structures were compiled. These were labeled for regions of interest including a putative hydrophobic cleft found in perilipin 3 that was previously found to recruit that protein to lipid droplets. Collectively these findings indicate that it is highly likely that perilipin 5 has a similar structure to perilipin 3 in the carboxy terminus.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mapping Perilipin 5 interactions via Protein Crosslinking Studies

Many human health issues in the United States are related to lipid metabolism, heart disease, obesity, type II diabetes, and non‐alcoholic fatty liver disease among them. Nearly all cells store neutral lipids in lipid storage droplets. Perilipins are a family of proteins known to associate with these lipid droplets. In particular, perilipin 5 is more strongly expressed in highly oxidative tissues such as cardiac tissues and fasting liver. Previously, we have identified a cytosolic pool of perilipin 5 which exists on small, lipoprotein‐like lipid droplets. Based on that work we hypothesized that quantification of perilipin 5 on these lipid droplets could identify interaction partners and help provide clues to perilipin function. To test this, cells expressing perilipin 5 are disrupted and lipid droplets are isolated. Bis[sulfosuccinimidyl] suberate (BS3) is used to crosslink proteins on these microdroplets. These cross‐linked proteins can then be separated by SDS‐PAGE and proteins crosslinked to perilipin 5 identified by western blotting. Overall, this data will allow for identification of perilipin 5 interaction partners on these structures and help identify if perilipin 5 acts as a monomer or multimer on lipid droplets.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of a Shortened Form of the Perilipin 5 Protein

Many modern health issues arise from aberrant lipid metabolism, among these are type II diabetes and non‐alcoholic fatty liver disease. One commonality these diseases share is neutral lipid metabolism and storage. Regulation of neutral lipid metabolism is of vital importance in the body, and among the proteins responsible for such regulation are the perilipins. Perilipins are a family of conserved proteins that are found on the surface of lipid storage droplets and play a central role in the regulation of cellular neutral lipid metabolism. Perilipin 5, specifically, is expressed in tissues with a high capacity for fatty acid oxidation such as cardiac muscle, oxidative muscle, and fasting liver. Using western blotting and reverse transcriptase PCR, we have identified a splice variant of perilipin 5 which has been termed perilipin 5b. This shortened form of the protein retains the amino terminal 35 kDa of the protein but lacks the 4‐helix bundle found in the C‐terminus. Based on the reports in the literature, this protein would lack the carboxy terminal domains reported to be necessary for interactions with lipases and other proteins but would retain serine 155 that is requisite for PKA phosphorylation and translocation to the nucleus. We have constructed a carboxy terminal truncation of perilipin 5 and are expressing it in CHO cells to further define the function of this protein. When compared with CHO cellular expression of perilipin 5, 5b appears to have a higher number of smaller lipid droplets per cell. Collectively these data indicate that perilipin 5b plays a role in lipid storage, but its specific function of this shortened form is still unknown.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Roux-en-Y gastric bypass surgery reprograms enterocyte triglyceride metabolism and postprandial secretion in rats

ObjectiveRoux-en-Y gastric bypass (RYGB) surgery produces rapid and persistent reductions in plasma triglyceride (TG) levels associated with fewer cardiovascular events. The mechanisms of the reduction in systemic TG levels remain unclear. We hypothesized that RYGB reduces intestinal TG secretion via altered enterocyte lipid handling. MethodsRYGB or Sham surgery was performed in diet-induced obese, insulin-resistant male Sprague–Dawley rats. First, we tested whether RYGB reduced test meal-induced TG levels in the intestinal lymph, a direct readout of enterocyte lipid secretion. Second, we examined whether RYGB modified TG enterocyte secretion at the single lipid level and in comparison to other lipid subclasses, applying mass spectrometry lipidomics to the intestinal lymph of RYGB and Sham rats (0–21 days after surgery). Third, we explored whether RYGB modulated the metabolic characteristics of primary enterocytes using transcriptional and functional assays relevant to TG absorption, reesterification, storage in lipid droplets, and oxidation. ResultsRYGB reduced overall postprandial TG concentrations compared to Sham surgery in plasma and intestinal lymph similarly. RYGB reduced lymphatic TG concentrations more than other lipid subclasses, and shifted the remaining TG pool towards long-chain, unsaturated species. In enterocytes of fasted RYGB rats, lipid uptake was transcriptionally (Fatp4, Fabp2, Cd36) and functionally reduced compared to Sham, whereas TG reesterification genes were upregulated. ConclusionOur results show that RYGB substantially reduces intestinal TG secretion and modifies enterocyte lipid absorption and handling in rats. These changes likely contribute to the improvements in the plasma TG profile observed after RYGB in humans.

LSD-2 dysfunction induces dFoxO-dependent cell death in the wing of Drosophila melanogaster

Lipid storage droplet-2 (LSD-2) of Drosophila melanogaster is a member of the lipid storage droplet membrane surface-binding protein family. LSD-2 is detected in many specific tissues: germline precursor cells, fat body, and is associated with lipid metabolism, lipid storage, and regulation of lipid droplet transport. However, the roles of this gene in development remain unclear. To investigate these functions, we performed tissue-specific knockdown of Lsd-2 in Drosophila using the combination of GAL4/UAS system and RNAi. Here we report that the knockdown of Lsd-2 in the wing led to abnormal wing phenotype and cell death in the wing pouch of 3rd-instar larvae, suggesting an essential role of Lsd-2 in development of the Drosophila wing. This function of Lsd-2 is dependent on the transcription factor dFoxO, as dFoxO depletion suppresses cell death and the abnormal wing pattern formation induced by Lsd-2-knockdown. Furthermore, Lsd-2-knockdown up-regulated the expression of the dFoxO transcription target reaper, which constitutes a pro-apoptosis gene. This study provides the first evidence that Lsd-2-knockdown causes cell death mediated by dfoxO.

Triacylglycerol Metabolism in Drosophila melanogaster.

Triacylglycerol (TAG) is the most important caloric source with respect to energy homeostasis in animals. In addition to its evolutionarily conserved importance as an energy source, TAG turnover is crucial to the metabolism of structural and signaling lipids. These neutral lipids are also key players in development and disease. Here, we review the metabolism of TAG in the Drosophila model system. Recently, the fruit fly has attracted renewed attention in research due to the unique experimental approaches it affords in studying the tissue-autonomous and interorgan regulation of lipid metabolism in vivo Following an overview of the systemic control of fly body fat stores, we will cover lipid anabolic, enzymatic, and regulatory processes, which begin with the dietary lipid breakdown and de novo lipogenesis that results in lipid droplet storage. Next, we focus on lipolytic processes, which mobilize storage TAG to make it metabolically accessible as either an energy source or as a building block for biosynthesis of other lipid classes. Since the buildup and breakdown of fat involves various organs, we highlight avenues of lipid transport, which are at the heart of functional integration of organismic lipid metabolism. Finally, we draw attention to some "missing links" in basic neutral lipid metabolism and conclude with a perspective on how fly research can be exploited to study functional metabolic roles of diverse lipids.

20(S)-Protopanaxatriol Promotes Fatty Acid-Induced ER Stress and Apoptosis in Multiple Myeloma By Down-Regulating SCD1 Expression

Multiple myeloma (MM), is a hematological malignancy characterized by the accumulation of clonal malignant plasma cells. Nowadays more and more studies concern that alert metabolism including glycolysis, glutaminolysis and lipid metabolism has potent in vivo anticancer activity in multiple myeloma. While glycolysis and glutaminolysis was well established, lipid metabolism of MM is poorly understood and there is a need for a new low-toxic therapy that selectively target MM.Lipid metabolism studies such as on the use of inhibitors of fatty acid synthesis and their effects on MM cell survival have been reported. Our study showed the increase of the expression of stearoyl CoA desaturase 1 (SCD1) and the elevated fatty acid biosynthesis in MM cells (Fig.1A, p<0.001). We found that SCD1 is overexpression in MM patients' samples and associated with clinical stage of myeloma (Fig 1B, p<0.05). Then we examined the level of lipid droplets(LDs) in MM cells, and the high level of LDs detected in MM cells demonstrated the lipid accumulation in MM (Fig.1C). Stable depletion of SCD1 inhibited fatty acid biosynthesis and decreased LDs levels and this reduction of LDs remained at low levels in MM cells (Fig.1D, E). These results suggest that MM cell growth party relies on SCD1-mediated fatty acid metabolism.The finding that 20(S)-protopanaxatriol(PPT) has significant effect on inhibiting the transcription of lipogenic genes have reported. Western blotting analysis shows that PPT decreased SCD1 protein levels in RPMI-8226, ARH-77 cell lines. In addition, PPT treatment decreased fatty acid biosynthesis and blocked lipid storage in lipid droplets(LDs) (Fig.1F). The proportion of saturated and monounsaturated was also decreased after treatment (Fig. 1G). Given that 20(S)-protopanaxatriol(PPT) has lipid-lowering effect in MM, we hypothesized that PPT exerts anti-myeloma effects by disrupting lipogenesis. In vitro experiments demonstrate the significant effect of PPT on decreasing proliferation and inducing apoptosis in multiple myeloma (Fig.1H). Supplementation with the SCD1 enzymatic product, oleic acid, rescued MM cells from PPT cell killing and SCD1 silencing, decreasing levels of SCD1 inhibition induced apoptosis and proliferation inhibition (Fig.1I). The results of Western Blot Analysis show a positive correlation between SCD1 inhibition and endoplasmic reticulum stress (ER stress) (Fig.1J). In addition, PPT can obviously induce ER stress after inhibiting SCD1, while ER-stress inhibitor TUDCA can significantly reverse the induced apoptosis of PPT treatment in MM cells (Fig.1K, p<0.05). These results suggest that excessive endoplasmic reticulum stress is the main cause of PPT induced apoptosis.In summary, our studies reveal that regulation of fatty acid metabolism in MM cells is an essential target. We show that the redeployed drug PPT killed MM cells by decreasing SCD1 protein levels and promoting fatty acid-induced ER stress. This study is relevant to the wider context of multiple myeloma therapeutics that developing therapeutics which can disrupt fatty acid biosynthesis. To our knowledge, this is the first study to describe the aglycone of ginsenosides 20(S)-protopanaxatriol with demonstrable anti-myeloma activity that target fatty acid biosynthesis [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Plin4-Dependent Lipid Droplets Hamper Neuronal Mitophagy in the MPTP/p-Induced Mouse Model of Parkinson's Disease.

Epidemiological studies have shown that both lipid metabolism disorder and mitochondrial dysfunction are correlated with the pathogenesis of neurodegenerative diseases (NDDs), including Parkinson’s disease (PD). Emerging evidence suggests that deposition of intracellular lipid droplets (LDs) participates in lipotoxicity and precedes neurodegeneration. Perilipin family members were recognized to facilitate LD movement and cellular signaling interactions. However, the direct interaction between Perilipin-regulated LD deposition and mitochondrial dysfunction in dopaminergic (DA) neurons remains obscure. Here, we demonstrate a novel type of lipid dysregulation involved in PD progression as evidenced by upregulated expression of Plin4 (a coating protein and regulator of LDs), and increased intracellular LD deposition that correlated with the loss of TH-ir (Tyrosine hydroxylase-immunoreactive) neurons in the MPTP/p-induced PD model mouse mesencephalon. Further, in vitro experiments showed that inhibition of LD storage by downregulating Plin4 promoted survival of SH-SY5Y cells. Mechanistically, reduced LD storage restored autophagy, leading to alleviation of mitochondrial damage, which in turn promoted cell survival. Moreover, the parkin-poly-Ub-p62 pathway was involved in this Plin4/LD-induced inhibition of mitophagy. These findings were further confirmed in primary cultures of DA-nergic neurons, in which autophagy inhibitor treatment significantly countermanded the ameliorations conferred by Plin4 silencing. Collectively, these experiments demonstrate that a dysfunctional Plin4/LD/mitophagy axis is involved in PD pathology and suggest Plin4-LDs as a potential biomarker as well as therapeutic strategy for PD.

Evolutionary and genomic analysis of the caleosin/peroxygenase (CLO/PXG) gene/protein families in the Viridiplantae.

Bioinformatics analyses of caleosin/peroxygenases (CLO/PXG) demonstrated that these genes are present in the vast majority of Viridiplantae taxa for which sequence data are available. Functionally active CLO/PXG proteins with roles in abiotic stress tolerance and lipid droplet storage are present in some Trebouxiophycean and Chlorophycean green algae but are absent from the small number of sequenced Prasinophyceaen genomes. CLO/PXG-like genes are expressed during dehydration stress in Charophyte algae, a sister clade of the land plants (Embryophyta). CLO/PXG-like sequences are also present in all of the >300 sequenced Embryophyte genomes, where some species contain as many as 10–12 genes that have arisen via selective gene duplication. Angiosperm genomes harbour at least one copy each of two distinct CLO/PX isoforms, termed H (high) and L (low), where H-forms contain an additional C-terminal motif of about 30–50 residues that is absent from L-forms. In contrast, species in other Viridiplantae taxa, including green algae, non-vascular plants, ferns and gymnosperms, contain only one (or occasionally both) of these isoforms per genome. Transcriptome and biochemical data show that CLO/PXG-like genes have complex patterns of developmental and tissue-specific expression. CLO/PXG proteins can associate with cytosolic lipid droplets and/or bilayer membranes. Many of the analysed isoforms also have peroxygenase activity and are involved in oxylipin metabolism. The distribution of CLO/PXG-like genes is consistent with an origin >1 billion years ago in at least two of the earliest diverging groups of the Viridiplantae, namely the Chlorophyta and the Streptophyta, after the Viridiplantae had already diverged from other Archaeplastidal groups such as the Rhodophyta and Glaucophyta. While algal CLO/PXGs have roles in lipid packaging and stress responses, the Embryophyte proteins have a much wider spectrum of roles and may have been instrumental in the colonisation of terrestrial habitats and the subsequent diversification as the major land flora.

Whole-genome RNAi screen identifies methylation-related genes influencing lipid metabolism in Caenorhabditis elegans

Lipid droplets (LDs) are highly conserved multifunctional cellular organelles and aberrant lipid storage in LDs can lead to many metabolic diseases. However, the molecular mechanisms governing lipid dynamic changes remain elusive, and the high-throughput screen of genes influencing LD morphology was limited by lacking specific LD marker proteins in the powerful genetic tool Caenorhabditis elegans. In this study, we established a new method to conduct whole-genome RNAi screen using LD resident protein DHS-3 as a LD marker, and identified 78 genes involved in significant LD morphologic changes. Among them, mthf-1, as well as a series of methylation-related genes, was found dramatically influencing lipid metabolism. SREBP-1 and SCD1 homologs in C. elegans were involved in the lipid metabolic change of mthf-1(RNAi) worms, and the regulation of ATGL-1 also contributed to it by decreasing triacylglycerol (TAG) hydrolysis. Overall, this study not only identified important genes involved in LD dynamics, but also provided a new tool for LD study using C. elegans, with implications for the study of lipid metabolic diseases.

Characterization of a Short Form of Perilipin 5

Many modern health issues arise from aberrant lipid metabolism. Among these are type II diabetes and non‐alcoholic fatty liver disease. One commonality these diseases share is neutral lipid metabolism and storage. Perilipins are a family of five conserved proteins that are found on the surface of lipid storage droplets and play a central role in the regulation of cellular neutral lipid metabolism. Perilipin 5 is expressed in tissues with a high capacity for fatty acid oxidation such as heart, oxidative muscle, and fasting liver. Using western blotting and reverse transcriptase PCR we have identified a shortened form of perilipin 5 we have termed perilipin 5B. This short form of the protein retains the amino terminal 35 kDa of the protein but lacks the 4 helix bundle found in the C‐terminus. Based on reports in the literature, this protein would lack the carboxy terminal domains reported to be necessary for interactions with lipases and other proteins but would retain serine 155 that is requisite for PKA phosphorylation and translocation to the nucleus. We have constructed a carboxy terminal truncation of perilipin 5 and are expressing it in CHO cells to further define the function of this protein. Like the full length protein, this truncation targets to lipid storage droplets. Collectively these studies indicate that perilipin 5B plays a role in lipid storage, but its function is still unknown.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of phosphorylation state on perilipin 5 localization and trafficking.

Perilipins are a family of five proteins found on the surface of lipid storage droplets in nearly all tissues. These proteins act as cofactors for lipases and scaffolding for other proteins involved in lipid metabolism. In addition to the lipid droplet surface, members of the perilipin family have been found in the cytosol, endoplasmic reticulum, plasma membrane and mitochondria. The localization of these proteins is in part determined by their phosphorylation state. Recently perilipin 5 has been shown to localize to the nucleus when phosphorylated by PKA on serine 155 and interact with the peroxisome proliferator‐activated receptor gamma coactivator alpha (PGC‐1a). In silico analysis of a phosphomimetic mutant (Ser155Asp) indicate that this site may contain a nuclear localization sequence. Using a CHO model cell line we observed that translocation to the nucleus could be stimulated by treatment with forskolin and isobutylmethylxanthine (IBMX) and occurred independent of treating cells with oleic acid to promote triacylglycerol deposition. We are currently investigating the affects of stimulating other phosphorylation pathways to determine if they also phosphorylate perilipin 5 and affect subcellular localization. These data have the potential to gain insights to the regulation of perilipin 5 trafficking into the nucleus and therefore interacting with PGC‐1a regulating mitochondrial biogenesis.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The role of PLIN protein in healthy lipid storage and lipid droplet expansion.

In skeletal muscle, fatty acids are stored in vesicle-like organelles known as lipid droplets (LDs). The importance of how lipids are stored in skeletal muscle has emerged from studies demonstrating a strong association between muscle lipids and insulin sensitivity. This article is protected by copyright. All rights reserved

Dihydroceramide is a key metabolite that regulates autophagy and promotes fibrosis in hepatic steatosis model

Non-alcoholic fatty liver disease (NAFLD) is an increasingly common chronic liver disease worldwide. Sphingolipids are a family of lipids that play essential roles as critical regulators in metabolic disorders. Some sphingolipids are known key factors in metabolic dysfunction. However, the precise effect of dihydroceramide on NAFLD remains unknown. Here, we report how dihydroceramide in autophagosome accumulation activates fibrogenesis in human liver Chang cells treated with free fatty acids (FFA). According to LC/MS lipid profiling, FFA increased the levels of sphingolipids and triacylglycerol (TG). To demonstrate the potential role of dihydroceramide metabolism in autophagy, several sphingolipid synthesis inhibitors were used. Increased dihydroceramide led to impairment of autophagic flux, resulting in increased TG storage in lipid droplets (LD) and upregulated expression of fibrosis markers. Hepatic stellate cells (HSCs, LX-2 cells) were co-cultured with Chang cells to assess the potential fibrogenic response to dihydroceramide, Treatment with rapamycin recovered autophagic flux in Chang cells and fibrogenesis in the co-culture system. Our results identified a critical function of dihydroceramide metabolism in autophagy. It could play an important role in the progression of NAFLD associated with lipid over-accumulation. Therefore, preventing autophagic flux by regulating dihydroceramide could be a potential strategic approach for providing therapy for NAFLD.

Polystyrene nanoplastics inhibit reproduction and induce abnormal embryonic development in the freshwater crustacean Daphnia galeata

We assayed the toxicity of polystyrene nanoparticles (PS-NP, 52 nm) to Daphnia galeata. Survival and reproduction were significantly decreased in individuals exposed to 5 mg/L of PS-NP for 5 days, and embryos showed abnormal development, including a low hatching rate. Using fluorescence confocal microscopy, we recorded the transfer of PS-NP from the external surface of the body to the internal organs, including the thoracic appendices, ovaries, caudal appendices, and brood chamber, as well as PS-NP storage in lipid droplets. Although embryos were exposed to PS-NP in the brood chamber, they did not internalize PS-NP. Exposed D. galeata adults that were not pregnant stored significantly fewer lipid droplets than did the control group, and the lipid droplets that they did store were smaller; meanwhile, there were no significant changes in lipid storage in exposed pregnant individuals. Some embryos showed a high level of lipid storage, a response that occurs when embryos experience an abnormal state, and these embryos showed a very low hatching rate. However, the offspring of exposed adults showed normal survival and lipid storage. This study provides visual evidence that confirms the transfer and effects of PS-NP in Daphnia species, and suggests a relationship between toxicity and lipid storage.