Number Of Lipid Droplets Research Articles

Oleaginous yeast Rhodotorula toruloides biomass effect on the metabolism of Arctic char (Salvelinus alpinus).

Sustainability issues arise when using fish oil and vegetable oils in fish feed production for aquaculture purposes. Microbial production of single cell oil is a potential alternative as a lipid ingredient in the production of fish feed. In this study, we replaced the vegetable oils with the oleaginous yeast R. toruloides biomass in the diet of Arctic char (S. alpinus) and investigated the effects on health and composition. Measurement of fish growth parameters showed a higher liver weight and hepatosomatic index in the experimental group of fish fed partly with yeast biomass compared to a control group fed a diet with vegetable oils. No significant differences in the lipid content of muscle and liver tissues were found. The fatty acid profiles in the muscle of both fish groups were similar while the experimental fish group had a higher amount of monounsaturated fatty acids in the liver. Histology of livers showed no significant difference in the number of lipid droplets. The size of hepatic lipid droplets seemed to be related to liver fat content. Quantification of metabolites in the liver revealed no differences between the fish groups while plasma metabolites involved in energy pathways such as alanine, 3-hydroxybutyrate, creatinine, serine, betaine, and choline were significantly higher in the experimental fish group.

Dysregulated lipolysis and lipophagy in lipid droplets of macrophages from high fat diet-fed obese mice.

Obesity is associated with lipid droplet (LD) accumulation, dysregulated lipolysis and chronic inflammation. Previously, the caspase recruitment domain‐containing protein 9 (CARD9) has been identified as a potential contributor to obesity‐associated abnormalities including cardiac dysfunction. In the current study, we explored a positive feedback signalling cycle of dysregulated lipolysis, CARD9‐associated inflammation, impaired lipophagy and excessive LD accumulation in sustaining the chronic inflammation associated with obesity. C57BL/6 WT and CARD9−/− mice were fed with normal diet (ND, 12% fat) or a high fat diet (HFD, 45% fat) for 5 months. Staining of LDs from peritoneal macrophages (PMs) revealed a significant increase in the number of cells with LD and the number of LD per cell in the HFD‐fed WT but not CARD9−/− obese mice. Rather, CARD9 KO significantly increased the mean LD size. WT obese mice showed down regulation of lipolytic proteins with increased diacylglycerol (DAG) content, and CARD9 KO normalized DAG with restored lipolytic protein expression. The build‐up of DAG in the WT obese mice is further associated with activation of PKCδ, NF‐κB and p38 MAPK inflammatory signalling in a CARDD9‐dependent manner. Inhibition of adipose triglyceride lipase (ATGL) by Atglistatin (Atg) resulted in similar effects as in CARD9−/− mice. Interestingly, CARD9 KO and Atg treatment enhanced lipophagy. In conclusion, HFD feeding likely initiated a positive feedback signalling loop from dysregulated lipolysis, CARD9‐dependent inflammation, impaired lipophagy, to excessive LD accumulation and sustained inflammation. CARD9 KO and Atg treatment protected against the chronic inflammation by interrupting this feedforward cycle.

ARP2/3 Regulates Fatty Acid Synthesis by Modulating Lipid Droplets' Motility.

The breakdown of lipid droplets (LDs) provides energy and contributes to the proliferation and migration of cancer cells. Recent studies have suggested that motility plays a key role in LD breakdown. However, the molecular mechanisms underlying LD motility were poorly characterized. In this study, we examined the function of microfilament-associated proteins 2 and 3 (ARP2 and ARP3) in regulating LDs’ motility in Hela cells. ARP2/3 mediated the LDs’ physical contact with F-actin and promoted the recruitment of Myosin Heavy Chain 9 (MYH9). MYH9 regulated the LD content by binding with LDs and ARP2/3. The number of LDs and TG content was increased after MYH9 interfered. The genes related to FA-related genes and neutral lipid synthesis-related genes were significantly increased (p < 0.05) when ARP2 and ARP3 were overexpressed. Bioinformatic analysis indicated that the high expression of ARP2/3 was associated with a poorer prognosis in cervical squamous cell carcinoma (CSCC). This study showed the effect of cytoskeletal filaments on LD metabolism in cancer cells.

Salvianolic acid B ameliorates non-alcoholic fatty liver disease by inhibiting hepatic lipid accumulation and NLRP3 inflammasome in ob/ob mice

Non-alcoholic fatty liver disease (NAFLD) has high occurrence in the global world, which poses serious threats to human health. Salvianolic acid B (SalB), an extract of the root of Salvia miltiorrhiza, has the protective effect on metabolic homeostasis. However, the mechanism is still unknown. In this study, we used ob/ob mice, a model of NAFLD, to explore the hepatoprotective effects of SalB. The results showed that SalB significantly reduced the body weights and liver weights, and ameliorated plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), hepatic free fatty acid (FFA), total cholesterol (TC) levels, and hepatic TG and TC levels in ob/ob mice. SalB reduced the number of lipid droplets and inhibited hepatic lipogenesis by regulating peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FASN), stearoyl-Co A desaturase 1 (SCD1), and cluster of differentiation 36 (CD36). Compared to ob/ob mice, the lower expressions of the pro-inflammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and F4/80, were observed after SalB treatment. Importantly, SalB treatment inhibited the activation of NLRP3 inflammasome and reduced the severity of liver inflammation. Our findings suggested that SalB improved NAFLD pathology in ob/ob mice by reducing hepatic lipid accumulation and NLRP3 inflammasome activation, which might be the potential hepatoprotective mechanism of SalB.

Insights into the control of taxane metabolism: Molecular, cellular, and metabolic changes induced by elicitation in Taxus baccata cell suspensions.

More knowledge is needed about the molecular/cellular control of paclitaxel (PTX) production in Taxus spp. cell cultures. In this study, the yield of this anticancer agent in Taxus baccata cell suspensions was improved 11-fold after elicitation with coronatine (COR) compared to the untreated cells, and 18-fold when co-supplemented with methyl-β-cyclodextrins (β-CDs). In the dual treatment, the release of taxanes from the producer cells was greatly enhanced, with 81.6% of the total taxane content being found in the medium at the end of the experiment. The experimental conditions that caused the highest PTX production also induced its maximum excretion, and increased the expression of taxane biosynthetic genes, especially the flux-limiting BAPT and DBTNBT. The application of COR, which activates PTX biosynthesis, together with β - CDs, which form inclusion complexes with PTX and related taxanes, is evidently an efficient strategy for enhancing PTX production and release to the culture medium. Due to the recently described role of lipid droplets (LDs) in the trafficking and accumulation of hydrophobic taxanes in Taxus spp. cell cultures, the structure, number and taxane storage capacity of these organelles was also studied. In elicited cultures, the number of LDs increased and they mainly accumulated taxanes with a side chain, especially PTX. Thus, PTX constituted up to 50-70% of the total taxanes found in LDs throughout the experiment in the COR + β - CD-treated cultures. These results confirm that LDs can store taxanes and distribute them inside and outside cells.

Mitochondria-targeted anti-oxidant AntiOxCIN4 improved liver steatosis in Western diet-fed mice by preventing lipid accumulation due to upregulation of fatty acid oxidation, quality control mechanism and antioxidant defense systems

Non-alcoholic fatty liver disease (NAFLD) is a health concern affecting 24% of the population worldwide. Although the pathophysiologic mechanisms underlying disease are not fully clarified, mitochondrial dysfunction and oxidative stress are key players in disease progression. Consequently, efforts to develop more efficient pharmacologic strategies targeting mitochondria for NAFLD prevention/treatment are underway. The conjugation of caffeic acid anti-oxidant moiety with an alkyl linker and a triphenylphosphonium cation (TPP+), guided by structure-activity relationships, led to the development of a mitochondria-targeted anti-oxidant (AntiOxCIN4) with remarkable anti-oxidant properties. Recently, we described that AntiOxCIN4 improved mitochondrial function, upregulated anti-oxidant defense systems, and cellular quality control mechanisms (mitophagy/autophagy) via activation of the Nrf2/Keap1 pathway, preventing fatty acid-induced cell damage. Despite the data obtained, AntiOxCIN4 effects on cellular and mitochondrial energy metabolism in vivo were not studied.In the present work, we proposed that AntiOxCIN4 (2.5 mg/day/animal) may prevent non-alcoholic fatty liver (NAFL) phenotype development in a C57BL/6J mice fed with 30% high-fat, 30% high-sucrose diet for 16 weeks. HepG2 cells treated with AntiOxCIN4 (100 μM, 48 h) before the exposure to supraphysiologic free fatty acids (FFAs) (250 μM, 24 h) were used for complementary studies. AntiOxCIN4 decreased body (by 43%), liver weight (by 39%), and plasma hepatocyte damage markers in WD-fed mice. Hepatic-related parameters associated with a reduction of fat liver accumulation (by 600%) and the remodeling of fatty acyl chain composition compared with the WD-fed group were improved. Data from human HepG2 cells confirmed that a reduction of lipid droplets size and number can be a result from AntiOxCIN4-induced stimulation of fatty acid oxidation and mitochondrial OXPHOS remodeling. In WD-fed mice, AntiOxCIN4 also induced a hepatic metabolism remodeling by upregulating mitochondrial OXPHOS, anti-oxidant defense system and phospholipid membrane composition, which is mediated by the PGC-1α-SIRT3 axis. AntiOxCIN4 prevented lipid accumulation-driven autophagic flux impairment, by increasing lysosomal proteolytic capacity.AntiOxCIN4 improved NAFL phenotype of WD-fed mice, via three main mechanisms: a) increase mitochondrial function (fatty acid oxidation); b) stimulation anti-oxidant defense system (enzymatic and non-enzymatic) and; c) prevent the impairment in autophagy. Together, the findings support the potential use of AntiOxCIN4 in the prevention/treatment of NAFLD.

Polarity-Sensitive Probe: Dual-Channel Visualization of the "Chameleon" Migration with the Assistance of Reactive Oxygen Species.

The real-time and differentiated visualization of the organelles is favorable for exploring the distribution and interaction. However, most visual probes emit monochromatic fluorescence and target a single organelle, which impedes the in-depth study of their interplay. To overcome this obstacle, we tactfully conceived a polarity-sensitive fluorescent DPDO-C that could accurately discriminate polarity changes in the cellular environment, exhibiting distinct fluorescence in lipid droplets (LDs) and mitochondria. Remarkably, the probe DPDO-C could migrate from mitochondria to LDs with the assistance of reactive oxygen species, which was conducive to further monitoring of the number and size of LDs as well as the interactions between LDs and other organelles. Moreover, the nuanced difference between normal and fatty liver tissues was also distinguished by two-color fluorescence imaging, which could act as a promising candidate for the early diagnosis of fatty liver.

Mitotane Targets Lipid Droplets to Induce Lipolysis in Adrenocortical Carcinoma.

IntroductionAdrenocortical carcinoma (ACC) is a rare aggressive cancer with low overall survival. Adjuvant mitotane improves survival but is limited by poor response rates and resistance. Mitotane’s efficacy is attributed to the accumulation of toxic free cholesterol, predominantly through cholesterol storage inhibition. However, targeting this pathway has proven unsuccessful. We hypothesize that mitotane-induced free-cholesterol accumulation is also mediated through enhanced breakdown of lipid droplets.MethodologyATCC-H295R (mitotane-sensitive) and MUC-1 (mitotane-resistant) ACC cells were evaluated for lipid content using specific BODIPY dyes. Protein expression was evaluated by immunoblotting and flow cytometry. Cell viability was measured by quantifying propidium iodide-positive cells following mitotane treatment and pharmacological inhibitors of lipolysis.ResultsH295R and MUC-1 cells demonstrated similar neutral lipid droplet numbers at baseline. However, evaluation of lipid machinery demonstrated distinct profiles in each model. Analysis of intracellular lipid droplet content showed H295R cells preferentially store cholesteryl esters, whereas MUC-1 cells store triacylglycerol. Decreased lipid droplets were associated with increased lipolysis in H295R and in MUC-1 at toxic mitotane concentrations. Pharmacological inhibition of lipolysis attenuated mitotane-induced toxicity in both models.ConclusionWe highlight that lipid droplet breakdown and activation of lipolysis represent a putative additional mechanism for mitotane-induced cytotoxicity in ACC. Further understanding of cholesterol and lipids in ACC offers potential novel therapeutic exploitation, especially in mitotane-resistant disease.

Olanzapine causes non-alcoholic fatty liver disease via inhibiting the secretion of apolipoprotein A5.

Long-term treatment of olanzapine, the most widely-prescribed second-generation antipsychotic, remarkably increases the risk of non-alcoholic fatty liver disease (NAFLD), whereas the mechanism for olanzapine-induced NAFLD remains unknown. Excessive hepatic fat accumulation is the basis for the pathogenesis of NAFLD, which results from the disturbance of TG metabolism in the liver. Apolipoprotein A5 (ApoA5) is a key regulator for TG metabolism in vivo that promotes TG accumulation in hepatocytes, thereby resulting in the development of NAFLD. However, there are no data indicating the role of apoA5 in olanzapine-induced NAFLD. Therefore, this study aims to investigate the role of apoA5 in olanzapine-induced NAFLD. This study was carried out via animal studies, cell experiment, and ApoA5 gene knockdown experiment. Six-week-old male C57BL/6J mice were randomized into a control group, a low-dose group, and a high-dose group, which were treated by 10% DMSO, 3 mg/(kg·d) olanzapine, and 6 mg/(kg·d) olanzapine, respectively for 8 weeks. The lipid levels in plasma, liver function indexes, and expression levels of ApoA5 were detected. HepG2 cells were treated with 0.1% DMSO (control group), 25 μmol/L olanzapine (low-dose group), 50 μmol/L olanzapine (medium-dose group), and 100 μmol/L olanzapine (high-dose group) for 24 h. HepG2 cells pretreated with 100 μmol/L olanzapine were transfected with siRNA and scrambled siRNA (negative control), respectively. We observed the changes in lipid droplets within liver tissues and cells using oil red O staining and fat deposition in liver tissues using HE staining. The mRNA and protein levels of ApoA5 were determined by real-time PCR and Western blotting, respectively. After intervention with 3 and 6 mg/(kg·d) olanzapine for 8 weeks, there was no significant difference in body weight among the 3 groups (P>0.05). Olanzapine dose-dependently increased the plasma TG, ALT and AST levels, and reduced plasma ApoA5 levels (all P<0.05), whereas there was no significant difference in plasma cholesterol (HDL-C, LDL-C, and TC) levels among the 3 groups (all P>0.05). Olanzapine dose-dependently up-regulated ApoA5 protein levels in liver tissues (all P<0.05), but there was no significant change in ApoA5 mRNA expression among groups (P>0.05). In the control group, the structure of liver tissues was intact, the morphology of liver cells was regular, and only a few scattered lipid droplets were found in the cells. In the olanzapine-treated group, there was a large amount of lipid deposition in hepatocytes, and cells were balloon-like and filled with lipid droplet vacuoles. The nucleus located at the edge of cell, and the number of lipid droplets was increased significantly, especially in the high-dose group. Likewise, when HepG2 cells were treated with olanzapine for 24 h, the number and size of lipid droplets were significantly elevated in a dose-dependent manner. Moreover, olanzapine dose-dependently up-regulated ApoA5 protein levels in HepG2 cells (all P<0.05), but there was no significant difference in ApoA5 mRNA expression among groups (P>0.05). Compared with the HepG2 cells transfected with scrambled siRNA, the number and size of lipid droplets in HepG2 cells transfected with ApoA5 siRNA were significantly reduced. The short-term intervention of olanzapine does not significantly increase body weight of mice, but it can directly induce hypertriglyceridemia and NAFLD in mice. Olanzapine inhibits hepatic apoA5 secretion but does not affect hepatic apoA5 synthesis, resulting in the pathogenesis of NAFLD. Inhibition of apoA5 secretion plays a key role in the development of olanzapine-related NAFLD, which may serve as an intervention target for this disease.

Osteocalcin reduces fat accumulation and inflammatory reaction by inhibiting ROS-JNK signal pathway in chicken embryonic hepatocytes

Osteocalcin (OCN) has a function in preventing fatty liver hemorrhagic syndrome (FLHS) in poultry. The aim of this study was to investigate the effects of OCN on fat emulsion stimulated chicken embryonic hepatocytes and related signaling pathways. The primary chicken embryonic hepatocytes were isolated from the incubated 15-day (E15) pathogen free eggs and cultured with dulbecco's modified eagle medium (DMEM). After the hepatocyte density reached 80%, the cells were divided into 5 groups: control group (CONT), fat emulsion group (FE, 10% FE, v/v), FE with ucOCN at 1 ng/mL (FE-LOCN), 3 ng/mL (FE-MOCN), and 9 ng/mL (FE-HOCN). In addition, 2 mM N-Acetyl-L-cysteine (NAC) a reactive oxygen species (ROS) scavenger, and 5 μM SP600125, a Jun N-terminal kinase (JNK) inhibitor, were added separately in to the DMEM with 10% FE to test effects of FE on the function of ROS-JNK signal pathway. The number of hepatocytes, cell ultra-microstructure, viability, and apoptosis were detected after 48 h treatment, and the protein expressions and enzyme concentrations were detected after 72 h treatment. The results showed that, compared to the control group, FE increased the triglyceride (TG) concentration and lipid droplets (LDs) in chicken embryonic hepatocytes (P < 0.05), and induced hepatocytic edema with obviously mitochondrial swelling, membrane damage, and cristae rupture. FE also decreased ATP concentration, increased ROS concentrations and mitochondrial DNA (mtDNA) copy number, promoted inflammatory interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-α) concentrations and hepatocytic apoptosis rate, and raised phospho-c-Jun N-terminal kinase (p-JNK) protein expressions. Compared to the FE group, ucOCN significantly increased hepatocyte viability, reduced hepatocytic TG concentrations and LDs numbers, and alleviated hepatocytic edema and mitochondrial swelling. Furthermore, ucOCN significantly decreased ROS concentrations, increased ATP concentrations, reduced IL-1, IL-6, TNF-α concentrations and hepatocytic apoptosis rate, and inhibited p-JNK protein expressions (P < 0.05). NAC had the similar functions of ucOCN reduced the ROS concentration and inhibited the TNF-α protein expression and p-JNK/JNK ration. Similarly, SP600125 reduced p-JNK/JNK protein expression, IL-1, IL-6, TNF-α, and TG concentrations without effects on ROS concentration and hepatocytic apoptosis. These results suggest that ucOCN alleviates FE-induced mitochondrial damage, cellular edema, and apoptosis of hepatocytes. These results reveal that the functions of ucOCN in reducing fat accumulation and inflammatory reaction in chicken embryonic hepatocytes are mostly via inhibiting the ROS-JNK signal pathway.

Chain flexibility of medicinal lipids determines their selective partitioning into lipid droplets

In guiding lipid droplets (LDs) to serve as storage vessels that insulate high-value lipophilic compounds in cells, we demonstrate that chain flexibility of lipids determines their selective migration in intracellular LDs. Focusing on commercially important medicinal lipids with biogenetic similarity but structural dissimilarity, we computationally and experimentally validate that LD remodeling should be differentiated between overproduction of structurally flexible squalene and that of rigid zeaxanthin and β-carotene. In molecular dynamics simulations, worm-like flexible squalene is readily deformed to move through intertwined chains of triacylglycerols in the LD core, whereas rod-like rigid zeaxanthin is trapped on the LD surface due to a high free energy barrier in diffusion. By designing yeast cells with either much larger LDs or with a greater number of LDs, we observe that intracellular storage of squalene significantly increases with LD volume expansion, but that of zeaxanthin and β-carotene is enhanced through LD surface broadening; as visually evidenced, the outcomes represent internal penetration of squalene and surface localization of zeaxanthin and β-carotene. Our study shows the computational and experimental validation of selective lipid migration into a phase-separated organelle and reveals LD dynamics and functionalization.

Abstract 3017: The poly-aneuploid cancer cell state utilizes lipid droplets as a mechanism of survival

Abstract Cancer is the second leading cause of all death in the world, killing over 10 million people in 2020. Metastatic cancer is responsible for 90% of all cancer related deaths. Metastatic disease remains incurable as a population of cancer cells within a tumor are or become resistant to all known natural and synthetic therapies. Despite decades of research, the source of this therapeutic resistance still remains largely undefined. We have identified that the polyaneuploid cancer cell (PACC) transition state is associated with therapeutic resistance. This state of cells are large in size, have high genomic content, and are the only cell remaining in tissue culture conditions after high dose chemotherapy. The PACC transition state has been documented in many different cancer types but their survival mechanisms have remained largely unexplored. We have discovered that the PACC transition state contains a higher number of lipid droplets (LDs) than the parental cells they are derived from. LDs are central anti-lipotoxic organelles that control fatty acid, diacylglycerol, cholesterol, and ceramide lipotoxicity by sequestering toxic lipids into inert triglycerides, cholesterol esters, and acylceramides. LDs are important in cancer as higher levels of LDs are associated with higher tumor aggressiveness and chemotherapy resistance. Utilizing TCGA data we discovered that PLIN1, an exclusive LD surface protein, is higher in prostate cancer patients with lower disease-free survival, higher Gleason score, and higher tumor stage. This indicates that more aggressive prostate cancer, which has been shown to have a higher number of PACCs in patient samples, is associated with higher levels of LDs. We hypothesize that the PACC state is surviving chemotherapeutic stress by sequestering toxic lipids into LDs to enhance cell survival. To determine when and how LDs form in the PACC transition state we used live cell imaging, western blotting, flow cytometry, and staining of key lipid droplet proteins. We utilized four different cancer cell lines (PC3, DU145, LNCaP, MDA-MB-231) with three different classes of chemotherapies (cisplatin, docetaxel, etoposide). We established that LDs form in the PACC transition state as early as three hours post chemotherapy treatment. This suggests that the PACC state utilizes a key mechanism of sequestering toxic lipids into LDs as soon as therapy is applied to avoid lipotoxicity and cell death. We also discovered that hormone-sensitive lipase (HSL), an enzyme that mobilizes fats stored in LDs, was down in the PACC state compared to parental cells, indicating that these LDs are not readily being broken down after formation. Our future work will focus on inhibiting LD formation to increase cellular lipotoxicity in the PACC transition state leading to PACC death and an increase in overall cancer cells killed. Citation Format: Laurie Gayle Kostecka, Sarah Amend, Kenneth Pienta. The poly-aneuploid cancer cell state utilizes lipid droplets as a mechanism of survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3017.

Lipid droplet dynamics in healthy and pyometra-affected canine endometrium

BackgroundAccumulation of lipid droplets (LDs) was recently observed in pyometra-affected uteri. As data about their nature and function are missing we intended to compare the localization, quality and quantity of LDs in canine healthy and pyometra-affected tissues and in an in vitro model.Methods and resultsWe characterized LDs in healthy and pyometra uterine tissue samples as well as in canine endometrial epithelial cells (CEECs) in vitro by means of histochemistry, immunohistochemistry, transmission electron microscopy, western blot, and RT-qPCR. Oil Red O (ORO) staining and quantification as well as p-phenylenediamine staining showed a higher number of LDs in epithelial cells of pyometra samples. Immunohistochemistry revealed that the amount of LDs coated by perilipin2 (PLIN2) protein was also higher in pyometra samples. Transmission electron microscopy showed an increase of LD size in surface and glandular epithelial cells of pyometra samples. In cell culture experiments with CEECs, supplementation with oleic acid alone or in combination with cholesterol lead to an increased LD accumulation. The expression of PLIN2 at protein and mRNA level was also higher upon oleic acid supplementation. Most LDs were double positive for ORO and PLIN2. However, ORO positive LDs lacking PLIN2 coating or LDs positive for PLIN2 but containing a lipid class not detectable by ORO staining were identified.ConclusionsWe found differences in the healthy and pyometra-affected endometrium with respect to LDs size. Moreover, several kinds of LDs seem to be present in the canine endometrium. In vitro studies with CEECs could show their responsiveness to external lipids. Since epithelial cells reacted only to oleic acid stimulation, we assume that the cyclic lipid accumulation in the canine endometrium is based mainly on triglycerides and might serve as energy provision for the developing early embryo. Further studies are necessary to verify the complex role of lipids in the healthy and pyometra-affected canine endometrium.

Prostaglandin 2α Promotes Autophagy and Mitochondrial Energy Production in Fish Hepatocytes.

Fatty liver, characterized by excessive lipid droplet (LD) accumulation in hepatocytes, is a common physiological condition in humans and aquaculture species. Lipid mobilization is an important strategy for modulating the number and size of cellular LDs. Cyclooxygenase (COX)-mediated arachidonic acid derivatives are known to improve lipid catabolism in fish; however, the specific derivatives remain unknown. In the present study, we showed that serum starvation induced LD degradation via autophagy, lipolysis, and mitochondrial energy production in zebrafish hepatocytes, accompanied by activation of the COX pathway. The cellular concentration of PGF2α, but not other prostaglandins, was significantly increased. Administration of a COX inhibitor or interference with PGF2α synthase abolished serum deprivation-induced LD suppression, LD–lysosome colocalization, and expression of autophagic genes. Additionally, exogenous PGF2α suppressed the accumulation of LDs, promoted the accumulation of lysosomes with LD and the autophagy marker protein LC3A/B, and augmented the expression of autophagic genes. Moreover, PGF2α enhanced mitochondrial accumulation and ATP production, and increased the transcript levels of β-oxidation- and mitochondrial respiratory chain-related genes. Collectively, these findings demonstrate that the COX pathway is implicated in lipid degradation induced by energy deprivation, and that PGF2α is a key molecule triggering autophagy, lipolysis, and mitochondrial development in zebrafish hepatocytes.

1458-P: Lipophagy Is Constitutively Active in Pancreatic Beta Cells and Maintains Insulin Secretion

Lipid droplets (LDs) , organelles important for intracellular lipid metabolism, are actively formed in rat and human adult beta cells and affect islet function and health. Acute mobilization of LDs by adipose triglyceride lipase in beta cells is known to support insulin secretion. As LD degradation by lysosomal acid lipase (LIPA) through lipophagy is an alternative pathway for LD catabolism, we addressed whether LIPA regulates beta cell function. LDs in human beta cells are associated with autophagy (LC3) and lysosomal (LAMP1) markers. LIPA suppression via siRNA and LIPA inhibitor (lalistat2) in INS1 cells increased LD size (DMSO 0.5 vs. Lali2 1.5 µm2/cell, p&amp;lt;0.05, p value by Student’s t test unless specified otherwise) and number (Scr 2/cell vs. siLIPA 14/cell, p&amp;lt;0.05; DMSO 4.5/cell vs. Lali2 16/cell, p&amp;lt;0.05) . Rat and human beta cells treated with Lali2 also showed increased in LD area and/or number of LDs per cell (p&amp;lt;0.05) . In all three models, LIPA suppression increased TG contents confirming constitutively active lipophagy. While acute inhibition of LIPA by Lali2 had little effect on glucose-stimulated insulin secretion (GSIS) , chronic (over 48 h) LIPA suppression by Lali2 or siRNA reduced GSIS from INS1 cells, rat islets, and human islets. Lentiviral shRNA transduction in human pseudoislets decreased insulin secretion in response to glucose (41 ± 10.5% of Scr, p&amp;lt;0.05) and to KCl (61.3 ±16.1% of Scr, p&amp;lt;0.05) in vitro. When Scr and shLIPA human pseudoislets were transplanted under kidney capsules of NSG mice on Western diet, human insulin secretion in response to intraperitoneal glucose load was blunted in mice carrying shLIPA islets compared with shScr control (2-Way ANOVA, p&amp;lt;0.05) . In summary, lipophagy is constitutively active in human and rat beta cells and serves as a pathway for LD catabolism that is acutely insensitive to glucose. However, LIPA mediated LD catabolism is needed for beta cell function in a long-term. Disclosure S.Liu: None. B.R.Brennecke: None. U.Yang: None. J.A.Ankrum: None. Y.Imai: None. Funding National Institute of Diabetes andDigestive and Kidney Diseases (R01=DK090490) U.S. Department of Veterans Affairs (IBX005107)

Functional yogurt fermented by two-probiotics regulates blood lipid and weight in a high-fat diet mouse model.

We investigated the blood lipid regulation effects and mechanism of a functional Natto yogurt in a high-fat diet-induced hyperlipidemia mouse model. Natto yogurt was characteristically fermented by Bacillus natto and Lactobacillus plantarum with milk-soy dual protein as substrates. After 5 weeks of Natto yogurt consumption, the body weight, fat, and liver weight of mice were significantly improved, while serum levels of TG, TC, LDL, ALT, TBIL, and TBA were reduced. Natto yogurt significantly decreased the area of liver fat infiltration and the number of lipid droplets. In mechanism, we found that Natto yogurt can inhibit fatty acid synthesis and enhance fatty acid catabolism by regulating the expression of PPARα, PPARγ, CD36 and FAS in the liver. Moreover, Natto yogurt increased the ratio of Bacteroidetes to Firmicutes in the intestine. These results provide a possibility for Natto yogurt as a dual protein functional food to prevent and treat hyperlipidemia and obesity. PRACTICAL APPLICATIONS: Traditional-fermented yogurt promotes nutritional absorption and reduces blood pressure and fat, while Bacillus natto and its fermented food have been proved to play a significant role in improving cardiovascular and cerebrovascular diseases and obesity. Therefore, we developed a new dual protein functional yogurt (Natto yogurt) fermented by B. natto and Lactobacillus plantarum with milk and soy as substrates. We found that Natto yogurt could notably regulate blood lipid by inhibiting the synthesis of fatty acids, accelerating the catabolism of fatty acids, reducing liver damage, and increasing the abundance of beneficial intestinal microorganisms. This study suggested that Natto yogurt could improve hyperlipidemia and obesity as a safe, effective, and healthy functional food.

Pyroptosis-related genes regulate proliferation and invasion of pancreatic cancer and serve as the prognostic signature for modeling patient survival

ObjectivePancreatic ductal adenocarcinoma (PDAC) has high mortality and poor prognosis. Pyroptosis can influence the prognosis of patients by regulating the proliferation, invasion, and metastasis of cancer cells. However, the role of pyroptosis-related genes (PRGs) in PDAC remains unclear.MethodsIn this study, based on the Cancer Genome Atlas (TCGA) cohort of PDAC samples, univariate Cox analysis and LASSO regression analysis were used to screen the prognostic PRGs and establish the gene signature. To further evaluate the functional significance of CASP4 and NLRP1 in PDAC, we also conducted an in vitro study to explore the mechanism of CASP4 and NLRP1 regulating the occurrence and development of PDAC. Finally, we investigated the relationship between CASP4 and NLRP1 expression levels and drug sensitivity in pancreatic cancer cells.ResultsA risk prediction model based on CASP4 and NLRP1 was established, which can distinguish high-risk patients from low-risk patients (P < 0.001). Both internal validation and external GEO data sets validation demonstrate good predictive capability of the model (AUC = 0.732, AUC = 0.802, AUC = 0.632, P < 0.05). In vitro, CCK8 and Transwell assay suggested that CASP4 may accelerate the progression of PDAC by promoting proliferation and migration of pancreatic cancer cells, while NLRP1 has been found to have tumor suppressive effect. It should be noted that knockdown of CASP4 reduced the level of coke death, the expression levels of acetyl-CoA carboxylase, FASN, SREBP-1 and SREBP-2 were decreased, and the number of lipid droplets was also significantly reduced. Moreover, the enrichment of signaling pathways showed that NLRP1 was significantly correlated with MAPK and RAS/ERK signaling pathways, and knocking down NLRP1 could indeed up-regulate p-ERK expression. Finally, high expression of CASP4 and low expression of NLRP1 increased the sensitivity of pancreatic cancer cells to ERK inhibitors.ConclusionsIn especial, CASP4 can promote tumor progression by promoting the synthesis and accumulation of fatty acids, while NLRP1 acts on RAS/ERK signaling pathway. Both of genes play an important role in the diagnosis and treatment of PDAC, which may also affect the inhibitors of MAPK/ERK efficiency.

MicroRNA-206 inhibits HCV proliferation through depressing ACC1 lipid synthesis signalling pathway.

MicroRNAs are considered to play important roles in cell biological and pathological progress. microRNA-206 (miR-206) was reported to participate in lipogenesis, and lipid droplets were necessary for the life cycle of HCV proliferation. Whether miR-206 was associated with HCV proliferation and the potential mechanism are not clear. In this study, we firstly identified that miR-206 could inhibit HCV proliferation at the RNA and protein level. Bioinformatical prediction of target genes binding to miR-206 was performed to investigate whether inhibiting function was due to a lipogenesis pathway. Then, the acetyl-CoA carboxylase 1 (ACC1) gene was selected as target gene of miR-206. The dual-luciferase reporter assay results showed that luciferase significantly decreased in cells transfected with 3'-UTR of the ACC1 gene and miR-206. The RNA and protein levels of the ACC1 gene and its pathway genes were significantly lower in cells transfected with miR-206 than in controls. Furthermore, the lipid droplet numbers also significantly decreased in cells transfected with miR-206. In conclusion, miR-206 could inhibit HCV proliferation through depressing ACC1 lipogenesis pathway and decreasing the lipid droplet numbers. miR-206 might be used as anti-HCV biochemical drug in the future.

Global effects of pharmacologic inhibition of OSBP in human umbilical vein endothelial cells

Oxysterol-binding protein (OSBP) is a cholesterol/PI4P exchanger at contacts of the endoplasmic reticulum (ER) with trans-Golgi network (TGN) and endosomes. Several central endothelial cell (EC) functions depend on adequate cholesterol distribution in cellular membranes. Here we elucidated the effects of pharmacologic OSBP inhibition on the lipidome and transcriptome of human umbilical vein endothelial cells (HUVECs). OSBP was inhibited for 24 h with 25 nM Schweinfurthin G (SWG) or Orsaponin (OSW-1), followed by analyses of cellular cholesterol, 27-hydroxy-cholesterol, and triacylglycerol concentration, phosphatidylserine synthesis rate, the lipidome, as well as lipid droplet staining and western analysis of OSBP protein. Next-generation RNA sequencing of the SWG-treated and control HUVECs and angiogenesis assays were performed. Protein-normalized lipidomes of the inhibitor-treated cells revealed decreases in glycerophospholipids, the most pronounced effect being on phosphatidylserines and the rate of their synthesis, as well as increases in cholesteryl esters, triacylglycerols and lipid droplet number. Transcriptome analysis of SWG-treated cells suggested ER stress responses apparently caused by disturbed cholesterol exit from the ER, as indicated by suppression of cholesterol biosynthetic genes. OSBP was associated with the TGN in the absence of inhibitors and disappeared therefrom in inhibitor-treated cells in a time-dependent manner, coinciding with OSBP reduction on western blots. Prolonged treatment with SWG or OSW-1 inhibited angiogenesis in vitro. To conclude, inhibition of OSBP in primary endothelial cells induced multiple effects on the lipidome, transcriptome changes suggesting ER stress, and disruption of in vitro angiogenic capacity. Thus, OSBP is a crucial regulator of EC lipid homeostasis and angiogenic capacity.

AgNPs Aggravated Hepatic Steatosis, Inflammation, Oxidative Stress, and Epigenetic Changes in Mice With NAFLD Induced by HFD.

The recent development of silver nanoparticles (AgNPs) has sparked increased interest in biomedical and pharmaceutical applications, leading to the possibility of human exposure. The liver is the primary target organ in the metabolism and transport of nanoparticles. Non-alcoholic fatty liver disease (NAFLD) is the most common and leading cause of hepatic metabolic syndrome with approximately 15% of patients will develop into non-alcoholic steatohepatitis, fibrosis, cirrhosis, and eventually hepatocellular carcinoma. Thus, the potential hepatotoxicity of AgNPs on NAFLD development and progression should be of great concern. Herein, we explored the potential hepatic effect of a single intravenously injected dose of 0.5, 2.5, and 12.5 mg/kg BW on the liver function of high-fat-diet (HFD)-fed mice for 7 days. AgNP treatment increased serum levels of alanine aminotransferase, aspartate transaminase, triglycerides and cholesterols, the number of lipid droplets, and the contents of triglycerides and cholesterols in NAFLD mice livers compared to HFD-fed mice. The mechanism of AgNP-induced worsen hepatotoxicity in mice is associated with hyperactivation of SREBP-1c-mediated de novo lipogenesis and liver inflammation. Additionally, HFD-fed mice treated with AgNPs had significantly higher oxidative damage and lower global DNA methylation and DNA hydroxymethylation than NAFLD mice. This study suggests that AgNP treatment exacerbated HFD-induced hepatic steatosis, liver inflammation, oxidative stress, and epigenetic changes in mice, which is relevant to the risk of AgNP exposure on NAFLD development and progression.