Lipid Droplet Formation Research Articles

Seipin overexpression attenuates cerebral ischemia-reperfusion injury via preventing apoptosis and autophagy.

Ischemic cerebrovascular disease (ICVD) is one of three fatal diseases in humans, along with heart disease and malignant tumors. Cerebral ischemia/reperfusion injury (CI/RI) is the primary cause of ICVD. Recently, seipin was reported to be crucial for lipid droplet formation, hepatic steatosis, and axonal atrophy. However, the function and mechanism of seipin in CI/RI has not been explicitly stated. Oxygen-glucose deprivation/reoxygenation (OGD/R) hippocampal neuron cell line (HT-22) and middle cerebral artery occlusion (MCAO) in rats were established. The levels of apoptosis- and autophagy-related proteins and seipin were confirmed by western blot. Cell proliferation was assessed with CCK-8, and ischemic infarction and pathological structure were monitored by TTC and H&E staining, and tissue apoptosis was assessed through TUNEL assay. The proliferative activity was decreased, and apoptosis and autophagy pathways could also be induced in the OGD/R HT-22 cells. Seipin overexpression accelerated viability and inhibited apoptosis and autophagy in the OGD/R HT-22 cells. Moreover, the data revealed that seipin overexpression could also attenuate cerebral infarction, apoptosis. Autophagy pathways could be repressed by seipin in the MCAO rats. Seipin has a protective role against CI/RI in rats, and its mechanism might be relevant to the suppression of apoptosis and autophagy, suggesting that seipin might be a latent target for CI/RI.

Advances in regulation of hypoxia on adipocyte development and lipid metabolism

The growth, differentiation and proliferation of adipose cells run through the whole life process. Dysregulation of lipid metabolism in adipose cells affects adipose tissue immunity and systemic energy metabolism. Increasingly available data suggest that lipid metabolism is involved in regulating the occurrence and development of various diseases, such as hyperlipidemia, nonalcoholic fatty liver disease, diabetes and cancer, which pose a major threat to human and animal health. Hypoxia inducible factor (HIF) is a major transcription factor mediating oxygen receptors in tissues and organs. HIF can induce disease by regulating lipid synthesis, fatty acid metabolism and lipid droplet formation. However, due to the difference of hypoxia degree, time and mode of action, there is no conclusive conclusion whether it has harmful or beneficial effects on the development of adipocytes and lipid metabolism. This article summarizes the regulation of hypoxia stress mediated transcription regulators and regulation of adipocyte development and lipid metabolism, aiming to reveal the potential mechanism of hypoxia induced changes in adipocyte metabolism pathways.

Cadmium facilitates the formation of large lipid droplets via PLCβ2-DAG-DGKε-PA signal pathway in Leydig cells

Cadmium (Cd) exposure damages the reproductive system. Lipid droplets (LDs) play an important role in steroid-producing cells to provide raw material for steroid hormone. We have found that the LDs of Leydig cells exposed to Cd are bigger than those of normal cells, but the effects on steroidogenesis and its underlying mechanism remains unclear. Using Isobaric tag for relative and absolute quantitation (iTARQ) proteomics, phosphodiesterase beta-2 (PLCβ2) was identified as the most significantly up-regulated protein in immature Leydig cells (ILCs) and adult Leydig cells (ALCs) derived from male rats exposed to maternal Cd. Consistent with high expression of PLCβ2, the size of LDs was increased in Leydig cells exposed to Cd, accompanied by reduction in cholesterol and progesterone (P4) levels. However, the high PLCβ2 did not result in high diacylglycerol (DAG) level, because Cd exposure up-regulated diacylglycerol kinases ε (DGKε) to promote the conversion from DAG to phosphatidic acid (PA). Exogenous PA, which was consistent with the intracellular PA concentration induced by Cd, facilitated the formation of large LDs in R2C cells, followed by reduced P4 level in the culture medium. When PLCβ2 expression was knocked down, the increased DGKε caused by Cd was reversed, and then the PA level was decreased to normal. As results, large LDs returned to normal size, and the level of total cholesterol was improved to restore steroidogenesis. The accumulation of PA regulated by PLCβ2-DAG-DGKε signal pathway is responsible for the formation of large LDs and insufficient steroid hormone synthesis in Leydig cells exposed to Cd. These data highlight that LD is an important target organelle for Cd-induced steroid hormone deficiency in males.

Mycobacterium tuberculosis suppresses host DNA repair to boost its intracellular survival

Mycobacterium tuberculosis (Mtb) triggers distinct changes in macrophages, resulting in the formation of lipid droplets that serve as a nutrient source. We discover that Mtb promotes lipid droplets by inhibiting DNA repair responses, resulting in the activation of the type-I IFN pathway and scavenger receptor-A1 (SR-A1)-mediated lipid droplet formation. Bacterial urease C (UreC, Rv1850) inhibits host DNA repair by interacting with RuvB-like protein 2 (RUVBL2) and impeding the formation of the RUVBL1-RUVBL2-RAD51 DNA repair complex. The suppression of this repair pathway increases the abundance of micronuclei that trigger the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway and subsequent interferon-β (IFN-β) production. UreC-mediated activation of the IFN-β pathway upregulates the expression of SR-A1 to form lipid droplets that facilitate Mtb replication. UreC inhibition via a urease inhibitor impaired Mtb growth within macrophages and invivo. Thus, our findings identify mechanisms by which Mtb triggers a cascade of cellular events that establish a nutrient-rich replicative niche.

Met stimulates ARID1A degradation and activation of the PI3K-SREBP1 signaling to promote milk fat synthesis in bovine mammary epithelial cells

Methionine (Met) can promote milk fat synthesis in bovine mammary epithelial cells (BMECs), but the potential molecular mechanism is largely unknown. In this report, we aim to explore the role and molecular mechanism of AT-rich interaction domain 1A (ARID1A) in milk fat synthesis stimulated by Met. ARID1A knockdown and activation indicated that ARID1A negatively regulated the synthesis of triglycerides, cholesterol and free fatty acids and the formation of lipid droplets in BMECs. ARID1A also negatively regulated the phosphorylation of PI3K and AKT proteins, as well as the expression and maturation of SREBP1. Met stimulated the phosphorylation of PI3K and AKT proteins, as well as the expression and maturation of SREBP1, while ARID1A gene activation blocked the stimulatory effects of Met. We further found that ARID1A was located in the nucleus of BMECs, and Met reduced the nuclear localization and expression of ARID1A. ARID1A gene activation blocked the stimulation of PI3K and SREBP1 mRNA expression by Met. In summary, our data suggests that ARID1A negatively regulates milk fat synthesis stimulated by Met in BMECs through inhibiting the PI3K-SREBP1 signaling pathway, which may provide some new perspectives for improving milk fat synthesis.

Stearoyl-CoA desaturase 1 inhibition impairs triacylglycerol accumulation and lipid droplet formation in colorectal cancer cells.

Increases in fatty acid (FA) biosynthesis meet the higher lipid demand by intensely proliferating cancer cells and promoting their progression. Stearoyl-CoA desaturase 1 (SCD1) is the key enzyme in FA biosynthesis, converting saturated FA (SFA) into monounsaturated FA (MUFA). Increases in the MUFA/SFA ratio and SCD1 expression have been observed in cancers of various origins and correlate with their aggressiveness. However, much is still unknown about the SCD1-dependent molecular mechanisms that promote specific changes in metabolic pathways of cancer cells. The present study investigated the involvement of SCD1 in shaping glucose and lipid metabolism in colorectal cancer (CRC) cells. Excess FAs that derive from de novo lipogenesis are stored in organelles, called lipid droplets (LDs), mainly in the form of triacylglycerol (TAG) and cholesteryl esters. LD accumulation is associated with key features of cancer development and progression. Consistent with our findings, the pharmacological inhibition of SCD1 activity affects CRC cell viability and impairs TAG accumulation and LD formation in these cells through the activation of lipolytic and lipophagic pathways. We showed that SCD1 suppression affects crucial lipogenic processes that promote lipid accumulation in CRC cells but in a sterol regulatory element-binding protein 1-independent manner. We propose that adenosine monophosphate-activated protein kinase contributes to these changes through the activation of lipolysis and inhibition of TAG synthesis. We also provide evidence of the involvement of SCD1 in the regulation of glucose uptake and utilization in CRC cells. These findings underscore the importance of SCD1 in regulating cellular processes that promote cancer development and progression.

The role of lipid scramblases in regulating lipid distributions at cellular membranes.

Glycerophospholipids, sphingolipids and cholesterol assemble into lipid bilayers that form the scaffold of cellular membranes, in which proteins are embedded. Membrane composition and membrane protein profiles differ between plasma and intracellular membranes and between the two leaflets of a membrane. Lipid distributions between two leaflets are mediated by lipid translocases, including flippases and scramblases. Flippases use ATP to catalyze the inward movement of specific lipids between leaflets. In contrast, bidirectional flip-flop movements of lipids across the membrane are mediated by scramblases in an ATP-independent manner. Scramblases have been implicated in disrupting the lipid asymmetry of the plasma membrane, protein glycosylation, autophagosome biogenesis, lipoprotein secretion, lipid droplet formation and communications between organelles. Although scramblases in plasma membranes were identified over 10 years ago, most progress about scramblases localized in intracellular membranes has been made in the last few years. Herein, we review the role of scramblases in regulating lipid distributions in cellular membranes, focusing primarily on intracellular membrane-localized scramblases.

MicroRNA-19a regulates milk fat metabolism by targeting SYT1 in bovine mammary epithelial cells

MicroRNAs (miRNAs) are important post-transcriptional factors involved in the regulation of gene expression and play crucial roles in biological processes related to milk fat metabolism. Our previous study revealed that miR-19a expression was significantly higher in the mammary epithelial cells of high-milk fat cows than in those of low-milk fat cows. However, the precise molecular mechanisms underlying these differences remain unclear. In this study, we found a high expression of miR-19a in the mammary tissues of dairy cows. The regulatory effects of miR-19a on bovine mammary epithelial cells (BMECs) were analyzed using cell counting kit-8 and 5-ethynyl-2′-deoxyuridine assays, which demonstrated that miR-19a significantly inhibited BMEC proliferation. Transfection of the miR-19a mimic into BMECs significantly upregulated the expression of milk fat marker genes LPL, SCAP, and SREBP1, promoting triglyceride (TG) synthesis and lipid droplet formation, whereas the miR-19a inhibitor exhibited the opposite function. TargetScan and miRWalk predictions revealed that synaptotagmin 1 (SYT1) is a target gene of miR-19a. A dual luciferase reporter gene assay, RT-qPCR, and western blot analyses revealed that miR-19a directly targets the 3′-untranslated region (UTR) of SYT1 and negatively regulates SYT1 expression. Functional validation revealed that overexpression of SYT1 in BMECs significantly downregulated the expression of LPL, SCAP, and SREBP1, and inhibited TG synthesis and lipid droplet formation. Conversely, the knockdown of SYT1 had the opposite effect. Altogether, miR-19a plays a crucial role in regulating the proliferation and differentiation of BMECs and regulates biological processes related to TG synthesis and lipid droplet formation by suppressing SYT1 expression. These findings provide a strong foundation for further research on the functional mechanisms underlying milk fat metabolism in dairy cows.

Molecular characterization of oleosin genes in Cyperus esculentus, a Cyperaceae plant producing oil in underground tubers.

CeOLE genes exhibit a tuber-predominant expression pattern and their mRNA/protein abundances are positively correlated with oil accumulation during tuber development. Overexpression could significantly increase the oil content of tobacco leaves. Oleosins (OLEs) are abundant structural proteins of lipid droplets (LDs) that function in LD formation and stabilization in seeds of oil crops. However, little information is available on their roles in vegetative tissues. In this study, we present the first genome-wide characterization of the oleosin family in tigernut (Cyperus esculentus L., Cyperaceae), a rare example accumulating high amounts of oil in underground tubers. Six members identified represent three previously defined clades (i.e. U, SL and SH) or six out of seven orthogroups (i.e. U, SL1, SL2, and SH1-3)proposed in this study. Comparative genomics analysis reveals that lineage-specific expansion of Clades SL and SH was contributed by whole-genome duplication and dispersed duplication, respectively. Moreover, presence of SL2 and SH3 in Juncus effuses implies their appearance sometime before Cyperaceae-Juncaceae divergence, whereas SH2 appears to be Cyperaceae specific. Expression analysis showed that CeOLE genes exhibit a tuber-predominant expression pattern and transcript levels are considerably more abundant than homologs in the close relative Cyperus rotundus. Moreover, CeOLE mRNA and protein abundances were shown to positively correlate with oil accumulation during tuber development. Additionally, two dominant isoforms (i.e. CeOLE2 and -5) were shown to locate in LDs as well as the endoplasmic reticulum of tobacco (Nicotiana benthamiana)leaves, and are more likely to function in homo and heteromultimers. Furthermore, overexpression of CeOLE2 and -5 in tobacco leaves could significantly increase the oil content, supporting their roles in oil accumulation. These findings provide insights into lineage-specific family evolution and putative roles of CeOLE genes in oil accumulation of vegetative tissues, which facilitate further genetic improvement for tigernut.

LncHLEF promotes hepatic lipid synthesis through miR-2188-3p/GATA6 axis and encoding peptides and enhances intramuscular fat deposition via exosome

Long noncoding RNAs (lncRNAs) have emergingly been implicated in mammalian lipid metabolism. However, their biological functions and regulatory mechanisms underlying adipogenesis remain largely elusive in chicken. Here, we systematically characterized the genome-wide full-length lncRNAs in the livers of pre- and peak-laying hens, and identified a novel intergenic lncRNA, lncHLEF, an RNA macromolecule with a calculated molecular weight of 433 kDa. lncHLEF was primarily distributed in cytoplasm of chicken hepatocyte and significantly up-regulated in livers of peak-laying hens. Functionally, lncHLEF could promote hepatocyte lipid droplet formation, triglycerides and total cholesterol contents. Mechanistically, lncHLEF could not only serve as a competitive endogenous RNA to modulate miR-2188-3p/GATA6 axis, but also encode three small functional polypeptides that directly interact with ACLY protein to enable its stabilization. Importantly, adeno-associated virus-mediated liver-specific lncHLEF overexpression resulted in increased hepatic lipid synthesis and intramuscular fat (IMF) deposition, but did not alter abdominal fat (AbF) deposition. Furthermore, hepatocyte lncHLEF could be delivered into intramuscular and abdominal preadipocytes via hepatocyte-secreted exosome to enhance intramuscular preadipocytes differentiation without altering abdominal preadipocytes differentiation. In conclusion, this study revealed that the lncHLEF could promote hepatic lipid synthesis through two independent regulatory mechanisms, and could enhance IMF deposition via hepatocyte-adipocyte communications mediated by exosome.

Fluorescence Imaging of Diabetic Cataract-Associated Lipid Droplets in Living Cells and Patient-Derived Tissues.

Diabetic cataract (DC) surgery carries risks such as slow wound healing, macular edema, and progression of retinopathy and is faced with a deficiency of effective drugs. In this context, we proposed a protocol to evaluate the drug's efficacy using lipid droplets (LDs) as the marker. For this purpose, a fluorescent probe PTZ-LD for LDs detection is developed based on the phenothiazine unit. The probe displays polarity-dependent emission variations, i.e., lower polarity leading to stronger intensity. Especially, the probe exhibits photostability superior to that of Nile Red, a commercial LDs staining dye. Using the probe, the formation of LDs in DC-modeled human lens epithelial (HLE) cells is validated, and the interplay of LDs-LDs and LDs-others are investigated. Unexpectedly, lipid transfer between LDs is visualized. Moreover, the therapeutic efficacy of various drugs in DC-modeled HLE cells is assessed. Ultimately, more LDs were found in lens epithelial tissues from DC patients than in cataract tissues for the first time. We anticipate that this work can attract more attention to the important roles of LDs during DC progression.

Regenerating gene 4 promotes chemoresistance of colorectal cancer by affecting lipid droplet synthesis and assembly.

Regenerating gene 4 (REG4) has been proved to be carcinogenic in some cancers, but its manifestation and possible carcinogenic mechanisms in colorectal cancer (CRC) have not yet been elucidated. Our previous study found that the drug resistance of CRC cells may be closely linked to their fat metabolism. To explore the role of REG4 in CRC and its association with lipid droplet formation and chemoresistance. We conducted a meta-analysis and bioinformatics and pathological analyses of REG4 expression in CRC. The effects of REG4 on the phenotypes and related protein expression were also investigated in CRC cells. We detected the impacts of REG4 on the chemoresistance and lipid droplet formation in CRC cells. Finally, we analyzed how REG4 regulated the transcription and proteasomal degradation of lipogenic enzymes in CRC cells. Compared to normal mucosa, REG4 mRNA expression was high in CRC (P < 0.05) but protein expression was low. An inverse correlation existed between lymph node and distant metastases, tumor-node-metastasis staging or short overall survival and REG4 mRNA overexpression (P < 0.05), but vice versa for REG4 protein expression. REG4-related genes included: Chemokine activity; taste receptors; protein-DNA and DNA packing complexes; nucleosomes and chromatin; generation of second messenger molecules; programmed cell death signals; epigenetic regulation and DNA methylation; transcription repression and activation by DNA binding; insulin signaling pathway; sugar metabolism and transfer; and neurotransmitter receptors (P < 0.05). REG4 exposure or overexpression promoted proliferation, antiapoptosis, migration, and invasion of DLD-1 cells in an autocrine or paracrine manner by activating the epidermal growth factor receptor-phosphoinositide 3-kinase-Akt-nuclear factor-κB pathway. REG4 was involved in chemoresistance not through de novo lipogenesis, but lipid droplet assembly. REG4 inhibited the transcription of acetyl-CoA carboxylase 1 (ACC1) and ATP-citrate lyase (ACLY) by disassociating the complex formation of anti-acetyl (AC)-acetyl-histone 3-AC-histone 4-inhibitor of growth protein-5-si histone deacetylase;-sterol-regulatory element binding protein 1 in their promoters and induced proteasomal degradation of ACC1 or ACLY. REG4 may be involved in chemoresistance through lipid droplet assembly. REG4 reduces expression of de novo lipid synthesis key enzymes by inhibiting transcription and promoting ubiquitination-mediated proteasomal degradation.

Concept of lipid droplet biogenesis

Lipid droplets (LD) are functionally conserved fat storage organelles found in all cell types. LDs have a unique structure comprising of a hydrophobic core of neutral lipids (fat), triacylglycerol (TAG) and cholesterol esters (CE) surrounded by a phospholipid monolayer. LD surface is decorated by a multitude of proteins and enzymes rendering this compartment functional. Accumulating evidence suggests that LDs originate from discrete ER-subdomains, demarcated by the lipodystrophy protein seipin, however, the mechanisms of which are not well understood. LD biogenesis factors together with biophysical properties of the ER membrane orchestrate spatiotemporal regulation of LD nucleation and growth at specific ER subdomains in response to metabolic cues. Defects in LD formation manifests in several human pathologies, including obesity, lipodystrophy, ectopic fat accumulation, and insulin resistance. Here, we review recent advances in understanding the molecular events during initial stages of eukaryotic LD assembly and discuss the critical role of factors that ensure fidelity of this process.

Chlorogenic acid enhances PPARγ-mediated lipogenesis through preventing Lipin 1 nuclear translocation in Staphylococcus aureus-exposed bovine mammary epithelial cells

Chlorogenic acid (CGA) as one of the most ubiquitously dietary polyphenolic compounds, has been reported to have various antimicrobial effects and exhibit strong anti-inflammatory ability. Staphylococcus aureus is a gram-positive bacterium that can induce mastitis. However, the mechanism through which S. aureus infection affects lipid synthesis and whether CGA have protective effect on S. aureus reduced lipid synthesis is not fully understood. In this study, the internalization of S. aureus reduced intracellular lipid droplet formation, decreased the levels of intracellular triacylglycerol, total cholesterol and 7 types of fatty acid and downregulated the expression of lipogenic genes FAS, ACC, and DGAT1 in bovine mammary epithelial cells (BMECs). In addition, we found that S. aureus intracellular infection attenuated mTORC1 activation resulting in Lipin 1 nuclear localization. Remarkablely, S. aureus infection-mediated repression of lipid synthesis related to the mTORC1 signaling and Lipin 1 nuclear localization can be alleviated by CGA. Thus, our findings provide a novel mechanism by which lipid synthesis is regulated under S. aureus infection and the protective effects of CGA on lipid synthesis in BMECs.

INVESTIGATING THE ROLE OF LIPID DROPLETS IN MEDULLOBLASTOMA TUMOURS

Abstract AIMS Medulloblastomas (MBs) are the most common paediatric brain tumour which originates in the cerebellum. It is classified into four subgroups named: sonic hedgehog, wingless, Group 3 and Group 4. Current treatment for MBs includes surgery, followed by high levels of chemotherapy and radiotherapy. This treatment regime is highly aggressive for the developing brain and produces several significant side effects. Therefore, research into new treatments or improving those already available is crucial to improving patients of life. METHOD It is well-documented that cancer cells have the ability to reprogram their metabolism to support rapid proliferation. These metabolic adaptations will provide the cells with the ability to use alternative sources of energy such as glutamine or lipids. Lipid Droplets (LDs) are highly dynamic cytoplasmic organelles that, under homeostatic conditions, are key in the storage of excess lipids within the cell to provide a buffer against lipotoxicity. Recent research has also begun to describe LDs as more than simple storage molecules but instead as function- ally active organelles. Increases in LD formation have been reported in several cancers such as Glioblastoma RESULTS In this study, we observed that aggressive MB cells have significantly more LDs when compared to non- aggressive MB cells. Further, oleic acid stimulation induced LD formation in MB cells. Finally, in silico gene expression analysis was performed to evaluate the expression of multiple components of the de novo lipid synthesis pathway in medulloblastoma. CONCLUSIONS Our work demonstrates that aggressive MB cells overexpress LDs and therefore identifies them as a possible novel target for MB treatment.

Rottlerin impairs early and late steps of Toxoplasma gondii infection in human trophoblast cells and villous explants

Congenital toxoplasmosis, caused by the opportunistic protozoan parasite T. gondii, can cause stillbirths, miscarriages and fetal abnormalities, as well as encephalitis and chorioretinitis in newborns. Available treatment options rely on antiparasitic drugs that have been linked to serious side effects, high toxicity and the development of drug-resistant parasites. The search for alternative therapeutics to treat this disease without acute toxicity for the mother and child is essential for the advancement of current therapeutic procedures. The present study aimed to unravel the mode of the anti-T. gondii action of Rottlerin, a natural polyphenol with multiple pharmacological properties described. Herein, we further assessed the antiparasitic activity of Rottlerin against T. gondii infection on the human trophoblastic cells (BeWo cells) and, for the first time, on human villous explants. We found that non-cytotoxic doses of Rottlerin impaired early and late steps of parasite infection with an irreversible manner in BeWo cells. Rottlerin caused parasite cell cycle arrest in G1 phase and compromised the ability of tachyzoites to infect new cells, thus highlighting the possible direct action on parasites. An additional and non-exclusive mechanism of action of Rottlerin involves the modulation of host cell components, by affecting lipid droplet formation, mitochondrial function and upregulation of the IL-6 and MIF levels in BeWo cells. Supporting our findings, Rottlerin also controlled T. gondii proliferation in villous explants with low toxicity and reduced the IL-10 levels, a cytokine associated with parasite susceptibility. Collectively, our results highlighted the potential use of Rottlerin as a promising tool to prevent and/or treat congenital toxoplasmosis.

The interaction network of the proteasome assembly chaperone PSMD9 regulates proteostasis.

Functional networks in cells are created by physical, genetic, and regulatory interactions. Mapping them and annotating their functions by available methods remains a challenge. We use affinity purification mass spectrometry (AP-MS) coupled with SLiMFinder to discern such a network involving 26S proteasome non-ATPase regulatory subunit 9 (PSMD9), a chaperone of proteasome assembly. Approximately 20% of proteins within the PSMD9 interactome carry a short linear motif (SLiM) of the type 'EXKK'. The binding of purified PSMD9 with the peptide sequence ERKK, proteins heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNPA2B1; containing ERKK), and peroxiredoxin-6 (PRDX6; containing EAKK) provided proof of principle for this motif-driven network. The EXKK motif in the peptide primarily interacts with the coiled-coil N domain of PSMD9, a unique interaction not reported for any coiled-coil domain. PSMD9 knockout (KO) HEK293 cells experience endoplasmic reticulum (ER) stress and respond by increasing the unfolded protein response (UPR) and reducing the formation of aggresomes and lipid droplets. Trans-expression of PSMD9 in the KO cells rescues lipid droplet formation. Overexpression of PSMD9 in HEK293 cells results in reduced UPR, and increased lipid droplet and aggresome formation. The outcome argues for the prominent role of PSMD9 in maintaining proteostasis. Probable mechanisms involve the binding of PSMD9 to binding immunoglobulin protein (BIP/GRP78; containing EDKK), an endoplasmic reticulum chaperone and key regulator of the UPR, and fatty acid synthase (FASN; containing ELKK), involved in fatty acid synthesis/lipid biogenesis. We propose that PSMD9 acts as a buffer in the cellular milieu by moderating the UPR and enhancing aggresome formation to reduce stress-induced proteotoxicity. Akin to waves created in ponds that perpetuate to a distance, perturbing the levels of PSMD9 would cause ripples down the networks, affecting final reactions in the pathway, one of which is altered proteostasis.

Centrins control chicken cone cell lipid droplet dynamics through lipid-droplet-localized SPDL1

As evolutionarily conserved organelles, lipid droplets (LDs) carry out numerous functions and have various subcellular localizations in different cell types and species. In avian cone cells, there is a single apically localized LD. We demonstrated that CIDEA (cell death inducing DFFA like effector a) and microtubules promote the formation of the single LD in chicken cone cells. Centrins, which are well-known centriole proteins, target to the cone cell LD via their C-terminal calcium-binding domains. Centrins localize on cone cell LDs with the help of SPDL1-L (spindle apparatus coiled-coil protein 1-L), a previously uncharacterized isoform of the kinetochore-associated dynein adaptor SPDL1. The loss of CETN3 or overexpression of a truncated CETN1 abrogates the apical localization of the cone cell LD. Simulation analysis showed that multiple LDs or a single mispositioned LD reduces the light sensitivity. Collectively, our findings identify a role of centrins in the regulation of cone cell LD localization, which is important for the light sensitivity of cone cells.

Effects of choline deficiency and supplementation on lipid droplet accumulation in bovine primary liver cells in vitro

Rumen-protected choline (RPC) supplementation in the periparturient period has in some instances prevented and alleviated fatty liver disease in dairy cows. Mechanistically, however, it is unclear how choline prevents the accumulation of lipid droplets (LD) in liver cells. In this study, primary liver cells isolated from liver tissue obtained via puncture biopsy from 3 nonpregnant mid-lactation multiparous Holstein cows (∼160 d postpartum) were used. Analyses of LD via oil red O staining, protein abundance via Western blotting, and phospholipid content and composition measured by thin-layer chromatography and HPLC/mass spectrometry were performed in liver cells cultured in choline-deficient medium containing 150 μmol/L linoleic acid for 24 h. In a subsequent experiment, lipophagy was assessed in liver cells cultured with 30, 60, or 90 µmol/L choline-chloride. All data were analyzed statistically using SPSS 20.0 via t-tests or one-way ANOVA. Compared with liver cells cultured in Dulbecco's Modified Eagle Medium alone, choline deficiency increased the average diameter of LD (1.59 vs. 2.10 µm), decreased the proportion of small LD (<2 µm) from 75.3% to 56.6%, and increased the proportion of large LD (>4 µm) from 5.6% to 15.0%. In addition, the speed of LD fusion was enhanced by the absence of choline. Among phospholipid species, the phosphatidylcholine (PC) content of liver cells decreased by 34.5%. Seventeen species of PC (PC [18:2_22:6], PC [15:0_16:1], PC [14:0_20:4], and so on) and 6 species of lysophosphatidylcholine (LPC; LPC [15:0/0:0]), PC (22:2/0:0), LPC (20:2/0:0), and so on] were decreased, while PC (14:1_16:1) and LPC (0:0/20:1) were increased. Choline deficiency increased the triglyceride (TAG) content (0.57 vs. 0.39 μmol/mg) in liver cells and increased the protein abundance of sterol regulatory element binding protein 1, sterol regulatory element binding protein cleavage activation protein, and fatty acid synthase by 23.5%, 17%, and 36.1%, respectively. Upon re-supplementation with choline, the phenotype of LD (TAG content, size, proportion, and phospholipid profile) was reversed, and the ratio of autophagy marker LC3II/LC3I protein was significantly upregulated in a dose-dependent manner. Overall, at least in vitro in mid-lactation cows, these data demonstrated that PC synthesis is necessary for normal LD formation, and both rely on choline availability. According to the limitation of the source of liver cells used, further work should be conducted to ascertain that these effects are applicable to liver cells from postpartum cows, the physiological stage where the use of RPC has been implemented for the prevention and treatment of fatty liver.

Unanticipated Enhancement of Intestinal TG Output by Apoc3 ASO Inhibition.

The objective of this study was to investigate whether apoC3 (apolipoprotein C3) inhibition with an antisense oligonucleotide (ASO) modulates intestinal triglyceride secretion. Sprague-Dawley rats were treated with subcutaneous injections of apoC3 ASO 25 mg/kg twice weekly or inactive ASO for 4 weeks before the assessment of lymph flow, triglyceride and apoB48 appearance in the lymph. Rats were surgically implanted with catheters in the mesenteric lymph duct and duodenum. Following an overnight fast, an intraduodenal lipid bolus (1.5-mL intralipid) was administered. Lymph fluid was collected for the following 4 hours to compare effects on lymph flow, lymph triglyceride and apoB48 concentration, and secretion. To assess suppression of apoC3 expression and protein abundance by apoC3 ASO compared with inactive ASO (placebo), intestinal and hepatic tissues were collected from a subset of animals before (fasting) and after an enteral lipid bolus (post-lipid). ApoC3 ASO significantly reduced apoC3 mRNA expression in the liver compared with inactive ASO (fasting: 42%, P=0.0048; post-lipid: 66%, P<0.001) and in the duodenum (fasting: 29%, P=0.0424; post-lipid: 53%, P=0.0120). As expected, plasma triglyceride also decreased significantly (fasting: 74%, P<0.001; post-lipid: 33%, P=0.0276). Lymph flow and cumulative lymph volume remained unchanged following apoC3 ASO therapy; however, lymph triglyceride, but not apoB48 output, increased by 38% (ANOVA, P<0.001). Last, no changes were observed in stool triglyceride, intestinal fat (quantified via oil red O staining), and expression of mRNAs involved in triglyceride synthesis, lipid droplet formation, and chylomicron transport and secretion. Despite the marked reduction in plasma triglyceride concentration that occurs with apoC3 ASO inhibition, intestinal triglyceride output surprisingly increased rather than decreased. These data demonstrate that the reduction of intestinal triglyceride output does not contribute to the potent plasma triglyceride-lowering observed with this novel therapy for hypertriglyceridemia. Further studies are required to explore the mechanism of this intestinal effect.