Size Of Lipid Droplets Research Articles

Atractylenolide III from Atractylodes macrocephala Koidz promotes the activation of brown and white adipose tissue through SIRT1/PGC-1α signaling pathway

BackgroundHypothermia is a complex pathophysiological response that can be life-threatening in low-temperature environment because of impaired thermoregulation. However, there is currently no clinically effective drugs that can prevent or treat this disease. Brown adipose tissue (BAT) activation or browning of white adipose tissue (WAT) is a promising therapeutic strategy to prevent or treat hypothermia. Atractylodes macrocephala Koidz extract (AE) and its active compound Atractylenolide III (AIII) has been reported to regulate glycolipid metabolism, which might be relevant to BAT activation. However, the thermogenic effect and mechanism of AE and AIII on adipose tissues have not been explored yet. Therefore, this study firstly investigated the role of AE and AIII on hypothermia by promoting heat production of BAT and WAT. PurposeTo explore the anti-cold effect of AE and AIII in cold exposure model and explore their biological function and mechanism underlying thermogenesis. MethodsThe effect of thermogenesis and anti-hypothermia of AE and AIII on C57BL/6J mice were evaluated with several experiment in cold environment, such as toxicity test, cold exposure test, metabolism estimation, histology and immunohistochemistry, and protein expression. Additionally, BAT, inguinal WAT (iWAT) and brown adipocytes were utilized to explore the mechanism of AE and AIII on thermogenesis in vivo and in vitro. Finally, SIRT1 agonist and inhibitor in brown adipocytes to verify that AIII activated BAT through SIRT1/PGC-1α pathway. ResultsBoth AE and AⅢ could significantly maintain the core body temperature and body surface temperature of mice during cold exposure. Besides, AE and AⅢ could significantly improve the capacity of total antioxidant and glucose, lipid metabolism of mice. In addition, AE and AIII reduced mitochondrial membrane potential and ATP content both in BAT and brown adipocytes, and decreased the size of lipid droplets. Moreover, AE and AⅢ promoted the expression of proteins related to heat production in BAT and iWAT. And AIII might activate BAT via SIRT1/PGC-1α pathway. ConclusionAE and AⅢ were potential candidate drugs that treated hypothermia by improving the heat production capacity of the mice. Mechanistically, they may activate SIRT1/PGC-1α pathway, thus enhancing the function of BAT, and promoting the browning of iWAT, to act as anti-hypothermia candidate medicine.

Asymmetrical Illumination Enables Lipid Droplets Segmentation in Caenorhabditis elegans Using Epi-Illumination Dark Field Microscopy

Lipid droplets are the major organelles for fat storage in a cell and analyzing lipid droplets in Caenorhabditis elegans (C. elegans) can shed light on obesity-related diseases in humans. In this work, we propose to use a label free scattering-based method, namely dark field microscopy, to visualize the lipid droplets with high contrast, followed by deep learning to perform automatic segmentation. Our method works through combining epi-illumination dark field microscopy, which provides high spatial resolution, with asymmetric illumination, which computationally rejects multiple scattering. Due to the raw data’s high quality, only 25 images are required to train a Convolutional Neural Network (CNN) to successfully segment lipid droplets in dense regions of the worm. The performance is validated on both healthy worms as well as those in starvation conditions, which alter the size and abundance of lipid droplets. Asymmetric illumination substantially improves CNN accuracy compared with standard dark field imaging from 70% to be 85%, respectively. Meanwhile, standard segmentation methods such as watershed and DIC object tracking (DICOT) failed to segment droplets due to the images’ complex label-free background. By successfully analyzing lipid droplets in vivo and without staining, our method liberates researchers from dependence on genetically modified strains. Further, due to the “open top” of our epi-illumination microscope, our method can be naturally integrated with microfluidic chips to perform large scale and automatic analysis.

Effect of Aerobic Exercise on Lipid Metabolism in Rats With NAFLD.

This work aimed to study the intervention effect of exercise on lipid metabolism in NAFLD rats, provide a more scientific experimental basis for exploring and improving the theoretical system of exercise intervention in NAFLD, and further provide a theoretical research basis for clinical treatment of NAFLD. Forty healthy male Sprague Dawley rats were randomly divided into a blank control group (BC,14) and a model group (MO, 26). After 6°weeks of modeling, the MO group was randomly divided into the model control group (MC, 12) and the aerobic exercise group (AE, 12). Platform running intervention in group E was conducted at a slope of 0°, a speed of 15 m/min, 1 h/time, once a day, six times a week, and a day of rest, for 8°weeks in total. After the intervention, the liver tissues of rats were taken for pathological sections, and serum was taken and analyzed for TC, TG, LDL-C, HDL-C, and FFA levels. Under the light microscope, the liver tissue structure of rats in the BC group was complete and clear, the structure of liver lobules was clear and normal, the volume of hepatocytes was uniform, the nucleus was in the middle, and the cytoplasm was red-stained, and no steatosis of hepatocytes was found. The liver of rats in the MC group showed diffuse fatty lesions, disordered structure of hepatic lobules, disordered arrangement of hepatic cords, different sizes of hepatocytes, loose cytoplasm, and diffuse lipid droplets of different sizes in the cytoplasm. The accumulation of liver lipid droplets in the AE group was improved compared with the MC group, the number of fat vacuoles in hepatocytes was significantly reduced, and the degree of liver lipid deposition was reduced. Compared with the BC group, the content of TC, TG, LDL-C, and FFA in the serum of the MC group increased significantly (p < 0.01), and the content of HDL-C decreased significantly (p < 0.01). Compared with the MC group, the content of TC, TG, LDL-C, and FFA in the serum of the AE group decreased significantly (p < 0.01/p < 0.05), and the content of HDL-C increased significantly (p < 0.01). Therefore, moderate-intensity aerobic exercise has an intervention effect on lipid metabolism in NAFLD rats, which can be used as one of the means to treat NAFLD.

Juvenile hormone acts on male accessory gland function via regulating l-asparaginase expression and triacylglycerol mobilization in Aedes aegypti.

Hormones control the reproductive development of Aedes aegypti mosquitoes. The adult male reproductive process and mating behavior require adequate nutrients and energy. Understanding the molecular mechanism linking hormones, energy metabolism, and reproduction in male mosquitoes is important. In this study, we found that the size of the male accessory gland, an essential part of the male reproductive system, gradually increased after eclosion. However, it was significantly reduced in male mosquitoes deficient in methoprene-tolerant (Met), the receptor of juvenile hormone. Likewise, egg hatchability of females that mated with Met-depleted males showed the same downward trend. The mRNA level of the gene encoding accessory gland protein, l-asparaginase (ASNase), was reduced in Met dsRNA-treated males. Electrophoretic mobility shift assay and quantitative reverse transcription-PCR results revealed that Met was capable of binding directly to the promoter of ASNase and activated its transcription. RNA interference of ASNase in males resulted in the reduction of egg hatchability of the females with which they mated. These results showed that Met influenced the fecundity of male mosquitoes by directly upregulating the expression of the ASNase gene. Moreover, the levels of triacylglycerol and the sizes of lipid droplets were decreased by 72-78h after eclosion in the fat body cells, whereas both of them increased in Met-depleted male mosquitoes, indicating that Met knockdown reduced lipid catabolism. These data demonstrate that Met might influence the egg hatchability of females by regulating lipid metabolism and the development of the male accessory gland in male mosquitoes.

Betaine Promotes Fat Accumulation and Reduces Injury in Landes Goose Hepatocytes by Regulating Multiple Lipid Metabolism Pathways.

Simple SummaryBetaine is a safe, effective, naturally occurring trimethylglycine, which is widely used as a nutritional supplement in animal husbandry. Early research showed betaine redistributes body fat by regulating systemic fatty acid metabolism. We previously found that betaine reduces sebum thickness and abdominal fat, and increases liver weight and triglyceride content, in Landes geese fed with high-energy carbohydrates. However, the mechanism underlying these effects remained unclear. Here, we present an in vitro Landes goose fatty liver model, which was developed using primary hepatocytes and a high-glucose medium simulating the conditions of high-energy carbohydrate feeding in vivo. We used this model to investigate the effects of betaine supplementation. Our results suggest that betaine increases lipid deposition, reduces lipid droplet size, and restores mitochondrial membrane potential (β-oxidation) and the expression of genes involved in lipid hydrolysis transfer genes. By contrast, the expression of fatty acid synthesis genes was down-regulated. The comprehensive effect led to an increase in total triglyceride in the liver, but a healthier steatosis phenotype. Overall, these results show that betaine could improve the quality of foie-gras while decreasing the injury to geese that results from overfeeding.Betaine is a well-established supplement used in livestock feeding. In our previous study, betaine was shown to result in the redistribution of body fat, a healthier steatosis phenotype, and an increased liver weight and triglyceride storage of the Landes goose liver, which is used for foie-gras production. However, these effects are not found in other species and strains, and the underlying mechanism is unclear. Here, we studied the underpinning molecular mechanisms by developing an in vitro fatty liver cell model using primary Landes goose hepatocytes and a high-glucose culture medium. Oil red-O staining, a mitochondrial membrane potential assay, and a qRT-PCR were used to quantify lipid droplet characteristics, mitochondrial β-oxidation, and fatty acid metabolism-related gene expression, respectively. Our in vitro model successfully simulated steatosis caused by overfeeding. Betaine supplementation resulted in small, well-distributed lipid droplets, consistent with previous experiments in vivo. In addition, mitochondrial membrane potential was restored, and gene expression of fatty acid synthesis genes (e.g., sterol regulatory-element binding protein, diacylglycerol acyltransferase 1 and 2) was lower after betaine supplementation. By contrast, the expression of lipid hydrolysis transfer genes (mitochondrial transfer protein and lipoprotein lipase) was higher. Overall, the results provide a scientific basis and theoretical support for the use of betaine in animal production.

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.

Edible film fabrication modified by freeze drying from whey protein isolate and sunflower oil: Functional property evaluation

The objective of the study was to simplify the composite film fabrication process. Different sunflower oil (SO) concentrations, 0.05, 0.10 and 0.15 (g/100 mL) were incorporated into whey protein isolate (WPI) matrix. The obtained WPI-SO solutions were dried using a freeze dryer to get a new composite powder. The powder was just mixed with water and glycerol; then it was cast to form WPI-SO based edible films. The effects of freeze drying and SO concentration on the optical, physical, thermal, barrier, mechanical and morphological properties of the novel WPI films were investigated. It was observed that films formed from reconstituted powder had enhanced barrier properties. Oil incorporation into the film matrix resulted in decreased lipid droplet size and increased opacity. In conclusion, edible films were produced successfully with improved functional properties. This situation proved that film production method, modified with freeze drying, has promising potential for biodegradable packaging industry.

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.

446-P: Lipid Droplet Protein PLIN1 Regulates Inflammatory Polarity in Human Macrophages and Is Involved in Atherosclerotic Plaque Development by Promoting Stable Lipid Storage

Background: Lipid droplet (LD) proteins play important roles in lipid accumulation and maturation in adipocytes. The relationship among PLIN family proteins and macrophage polarization in atherosclerosis has not been elucidated. Methods: The experiments used human artery tissues from 65 patients who had undergone a carotid endarterectomy. Cultured macrophages generated from healthy human peripheral blood mononuclear cells are utilized as well. Results: Plaque immunohistochemistry demonstrated co-expression of PLIN1 and PLIN2 in both symptomatic (n=31) and asymptomatic patients (n=34) . PLIN2 mRNA expression increased 3.38-fold in the symptomatic group compared with those from asymptomatic. PLIN1 was not expressed on small LDs at a shorter incubation (24 h) but was on large LDs at longer incubation (7 days) with oxidized LDL and VLDL, while PLIN2 was observed after 24 h and increased with a longer incubation in cultured M1 macrophage. In M2 macrophages, PLIN1 was seen as early as 24 h following incubation with VLDL, and LD size increased with longer incubation. PLIN1 overexpression increased the size of LDs in M1 macrophages, even after a short incubation, and reduced the RNA expression of TNFA, MMP2, ABCA1, and ABCG1 versus the M1 control. Conversely, silencing of PLIN1 in M2 macrophages had the contrary effects on LD size and RNA expression. Conclusion: There was a relationship among macrophage polarity, cytosolic LD size, and PLIN1/PLIN2 expression levels. PLIN2 was mainly expressed in arterial plaques in symptomatic stroke patients, and associated with the inflammatory phenotype of human macrophages, while PLIN1 expression is closely associated with plaque stability and the anti-inflammatory phenotype. Disclosure K.Cho: None. H.Miyoshi: Research Support; Abbott Japan Co., Ltd., Boehringer Ingelheim International GmbH, Daiichi Sankyo, Kowa Company, Ltd., LifeScan, Mitsubishi Tanabe Pharma Corporation, Novo Nordisk, Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Holdings Co., Ltd., Speaker's Bureau; Astellas Pharma Inc., Boehringer Ingelheim International GmbH, Eli Lilly and Company, Kowa Company, Ltd., Merck &amp; Co., Inc., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk, Ono Pharmaceutical Co., Ltd., Sanofi K.K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Holdings Co., Ltd. A.Nakamura: Research Support; Kissei Pharmaceutical Co., Ltd., MSD, Nippon Boehringer Ingelheim, Taisho Pharmaceutical Holdings Co., Ltd. T.Atsumi: Consultant; AbbVie Inc., AstraZeneca, MEDICAL &amp; BIOLOGICAL LABORATORIES CO., Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Ono Pharmaceutical Co., Ltd., Pfizer Inc., Research Support; AbbVie Inc., Alexion Pharmaceuticals, Inc., Astellas Pharma Inc., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo, Mitsubishi Tanabe Pharma Corporation, Nippon Boehringer Ingelheim Co. Ltd., Otsuka Pharmaceutical Co., Ltd., Pfizer Inc., Taiho Pharmaceutical Co. Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited, Speaker's Bureau; AbbVie Inc., Astellas Pharma Inc., Bristol-Myers Squibb Company, Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo, Eisai Co., Ltd., Eli Lilly and Company, Kyowa Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Novartis Pharma K.K., Pfizer Inc., Taiho Pharmaceutical Co. Ltd., Takeda Pharmaceutical Company Limited, UCB, Inc.

Effect of nano extracts of olea europaea leaves, ficus carica and liraglutide in lipidemic liver of type 2 diabetic rat model

The study aimed to evaluate the impact of Ficus carica mixture and Olea europaea leaf nano extracts, and liraglutide, on liver tissue and serum lipids in type 2 diabetic male albino rat model. Forty rats were divided equally into 4 groups were used. Group 1 was the non-diabetic control group. The animals in Groups 2–4 was injected intraperitoneally with a single dose of 60 mg/kg b.w. Streptozotocin to induce a diabetic rat model. Group 2 served as a positive control for diabetes. 0.02 mg/kg b.w./day of Liraglutide gave to groups 3 and 4 and 4.8 ng/ml × 105 b.w./day of a mixture of the nano extracts, respectively. Eight weeks after treatment, the animals were sacrificed. Blood was collected for glucose analysis and serum low-density lipoprotein, high-density lipoprotein, total cholesterol, and triglycerides analysis, and the livers processed for histopathological examination. The elevated lipid profiles and blood glucose levels in diabetic group (Group 2) were significantly reduced (p < 0.001) following the administration of liraglutide and nano extracts in Groups 3 and 4. Progressive fatty acid changes were found in the liver sections, indicated by the deposition of various sizes of lipid droplets in most liver lobules, along with patchy hepatocyte necrosis. These pathological changes were ameliorated in the liraglutide- and nano-extract-treated rats. Treatment with the nano extracts resulted in significant power assays associated with recovery of hepatic histology and functional alterations, compared to liraglutide treatment.

In Vivo Evaluation of the Cidec R46S Polymorphism for an Obesity QTL, tabw2a

ObjectivesA quantitative trait locus (QTL), tabw2a, was linked to obesity in TALLYHO/Jng (TH) mice, a polygenic model of obesity and type 2 diabetes. In combination with fine mapping information, we used whole genome sequencing data of TH mice to search positional candidate genes for tabw2a. From this search, the cell death-inducing DFFA-like effector c (Cidec) gene, containing a SNP identified as deleterious (likely to affect protein function) by both SIFT and PROVEAN, was raised as a potential candidate for tabw2a. CIDEC is a lipid droplet protein involved in the regulation of cellular lipid droplet size and lipid storage in adipocytes. There was one nucleotide substitution in the coding sequence of Cidec in TH mice, 136 C > A, compared to C57BL/6J (B6) reference sequence, that results in an amino acid substitution R46S (Arginine to Serine). The aim of this study was to evaluate the Cidec R46S polymorphism in vivo for the tabw2a QTL.MethodsWe generated knock-in mice where the TH allele (S46) is exchanged for the B6 allele (R46) at the Cidec gene on a B6 background. Targeting, ES clone selection, blastocyst injection, and breeding of ES cell chimeras were performed by Transgenics Core facility at Pennington Biomedical Research Center (Baton Rouge, LA). We bred the chimeras to B6 mice for 10 generation and collected heterozygous mice with germline transmission of the knock-in allele of Cidec S46. We then interbred the heterozygous mice and obtained homozygous mice for the knock-in allele of Cidec S46. Male and female Cidec R46S knock-in and B6 mice were weaned onto chow and high fat (HF) diets (D12266B, Research Diets) at 4 weeks of age and maintained on the same diets. At 20 weeks of age, body composition including fat and lean mass was measured using EchoMRI-100 whole body composition analyzer (Houston, TX).ResultsOn chow, no genotype effects were shown on adiposity between knock-in and B6 mice for both sexes. However, on HF diets, for both males and females, knock-in homozygous mice had significantly larger body fat mass than B6, although their body weights were comparable.ConclusionsWe conclude that the Cidec gene is a likely candidate for the obesity effect of tabw2a, and that Cidec S46 variant contributes to the obesity susceptibility.Funding SourcesAmerican Heart Association and National Institutes of Health.

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)

Unraveling the Roles of Mitochondria and Peroxisomes in Lipid Droplet Utilization in Tetrahymena thermophila

There is growing recognition of the lipid droplet as a dynamic hub for intracellular metabolic pathways. However, the cellular mechanisms that facilitate lipid droplet utilization remain unclear. In an effort to unravel these mechanisms, our experiments examine lipid droplets in the single‐celled ciliate Tetrahymena thermophila, an evolutionarily divergent organism with a rich history of experimentation. Like other organisms, Tetrahymenaaccumulate lipid droplets in response to starvation. Within 3 hours of starving Tetrahymena in 10mM Tris buffer (pH 7.4), lipid droplet size and number increase, remain elevated for an additional 72 hours, and then decline. We hypothesize that starved Tetrahymena mobilize lipids in lipid droplets for oxidation in the mitochondria or peroxisomes. Pulse chase experiments using a fluorescent fatty acid analogue (red C12) demonstrate lipid trafficking to lipid droplets in starved Tetrahymena. Further experiments show modest co‐localization of the red C12 fatty acid analogue with fluorescent markers of mitochondria, and at later time points, the peroxisomes. Chlorpromazine, an inhibitor of peroxisomal fatty acid metabolism, significantly decreased the viability and metabolic activity of starved Tetrahymena,as measured by cell density, trypan blue staining, and MTT reduction. On the other hand, inhibition of fatty acid import into mitochondria with the drug etomoxir elicited smaller decreases in viability and metabolic activity. Together, our experiments suggest that lipid mobilization in lipid droplets and fatty acid oxidation in peroxisomes and mitochondria contribute to cell survival in nutrient‐scarce environments.

Formation of Nascent Lipid Droplets at Specialized ER subdomains

Lipid droplets (LDs) are neutral lipid containing organelles enclosed in a single monolayer of phospholipids. LD formation begins with the accumulation of neutral lipids within the bilayer of the endoplasmic reticulum (ER) membrane. It is not known how the sites of formation of nascent LDs in the ER membrane are determined. Here we show that multiple C2 domain–containing transmembrane proteins, MCTP1 and MCTP2, are at sites of LD formation in specialized ER subdomains. We show that the transmembrane domain of these proteins is similar to a reticulon homology domain. Like reticulons, MCTPs tubulate the ER membrane and favor highly curved regions of the ER. Our data indicate that the transmembrane domains (TMD) of MCTP promote LD biogenesis by increasing LD number. MCTPs colocalize with seipin, a protein involved in LD biogenesis, but form more stable microdomains in the ER. The MCTP C2 domains bind charged lipids and regulate LD size, likely by mediating ER–LD contact sites. Together, our data indicate that MCTPs form specialized subdomains within ER tubules that regulate LD biogenesis, size, and ER–LD contacts. Interestingly, MCTP punctae are associated with other organelles as well, suggesting that these proteins may play a more general role in linking tubular ER to organelle contact sites.

Adapalene induces adipose browning through the RARβ-p38 MAPK-ATF2 pathway.

Adipose browning has recently been reported to be a novel therapeutic strategy for obesity. Because the retinoic acid receptor (RAR) is a potential target involved in browning, adapalene (AD), an anti-acne agent with RAR agonism, was examined in detail for its effects on adipose browning and the underlying mechanisms in vitro and in vivo. AD upregulated the expression of adipose browning-related markers in a concentration-dependent manner, promoted mitochondrial biogenesis, increased oxygen consumption rates, and lowered lipid droplet sizes in differentiated 3T3/L1 white adipocytes. Among the three retinoic acid receptors (RARα, RARβ, and RARγ), knockdown of the gene encoding RARβ mitigated AD-induced adipose browning. Similarly, LE135 (a selective RARβ antagonist) attenuated AD action, suggesting that AD promotes adipose browning through RARβ. Sequential phosphorylation of p38 mitogen-activated protein kinase (MAPK) and activating transcription factor 2 (ATF2) was critical for AD-induced adipose browning, based on the observations that either SB203580 (a p38 MAPK inhibitor) or ATF2 siRNA reduced the effects of AD. In vivo browning effects of AD were confirmed in C57BL/6J mice and high-fat diet-induced obese (DIO) mice after oral administration of AD either acutely or chronically. This study identifies new actions of AD as an adipose browning agent and demonstrates that RARβ activation followed by increased phosphorylation of p38 MAPK and ATF2 appears to be a key mechanism of AD action.

Impairment of transcription factor Gcr1p binding motif perturbs OPI3 transcription in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the transcription factor GCR1 plays a vital role in carbohydrate metabolism and in the current study we tried to elucidate its role in lipid metabolism. In silico analysis revealed the upstream activation sequence (UAS) in the promoter region of OPI3 possessed six conserved recognition sequences for Gcr1p and the ChIP assay confirmed the binding of Gcr1p on the OPI3 promoter region. The real-time quantitative polymerase chain reaction and promoter-reporter activity revealed a substantial reduction in OPI3 expression and was supported with decreased phosphatidylcholine (PC) level that is rescued with exogenous choline supplementation in gcr1∆ cells. Simultaneously, there was an increase in triacylglycerol level, accompanied with increased number and size of lipid droplets in gcr1∆ cells. The expression of pT1, pT2 truncations in opi3∆ cells revealed the -1 to -500 bp in the promoter region is essential for the activation of OPI3 transcription. The mutation specifically at UASCT box (-265) in the OPI3 promoter region displayed a reduction in the PC level and the additional mutation at UASINO (-165) further reduced the PC level. Collectively, our data suggest that the GCR1 transcription factor also regulates the OPI3 expression and has an impact on lipid homeostasis.

Choline Kinase Alpha2 Promotes Lipid Droplet Lipolysis in Non-Small-Cell Lung Carcinoma.

BackgroundRapid tumor growth inevitably results in energy stress, including deficiency of glutamine, a critical amino acid for tumor cell proliferation. However, whether glutamine deficiency allows tumor cells to use lipid droplets as an energy resource and the mechanism underlying this potential regulation remain unclear.MethodsWe purified lipid droplets from H322 and H358 human non-small-cell lung cancer (NSCLC) cells under glutamine deprivation conditions and performed immunoblotting to determine the binding of choline kinase (CHK) α2 to lipid droplets. Immunofluorescence was used to quantify lipid droplet numbers and sizes. Immunoprecipitation and immunoblotting were performed to examine AMPK activation and CHKα2 phosphorylation. Cellular fatty acid levels, mitochondrial acetyl coenzyme A and ATP production, and cell apoptosis and proliferation were measured. Immunohistochemical analyses were performed to determine the expression levels of ACC pS79 and CHKα2 pS279 in tumor specimens from NSCLC patients. The prognostic value of ACC pS79 and CHKα2 pS279 was assessed using the Kaplan-Meier method and Cox regression models.ResultsGlutamine deficiency induces AMPK-mediated CHKα2 S279 phosphorylation, which promotes the binding of CHKα2 to lipid droplets, resulting in recruitment of cytosolic lipase ATGL and autophagosomes and subsequent lipolysis of lipid droplets to sustain tumor cell survival and proliferation. In addition, the levels of ACC pS79 and CHKα S279 were much higher in human NSCLC specimens than in their adjacent normal tissues and positively correlated with each other. Notably, ACC pS79 and CHKα pS279 expression levels alone were associated with poor prognosis of NSCLC patients, and combined values of both phosphorylation levels were correlated with worse prognosis of the patients.ConclusionCHKα2 plays a critical role in lipolysis of lipid droplets in NSCLC. ACC pS79 and CHKα2 pS279 alone or in combination can be used as prognostic markers in NSCLC.

Highly Sensitive Two-Photon Lipid Droplet Tracker for In Vivo Screening of Drug Induced Liver Injury.

Lipid droplets (LDs) are lipid-abundant organelles found in most cell lines and primarily consist of neutral lipids. They serve as a repository of various lipids and are associated with many cellular metabolic processes, including energy storage, membrane synthesis, and protein homeostasis. LDs are prominent in a variety of diseases related to lipid regulation, including obesity, fatty liver disease, diabetes, and atherosclerosis. To monitor LD dynamics in live samples, we developed a highly selective two-photon fluorescent tracker for LDs (LD1). It exhibited outstanding sensitivity with a remarkable two-photon-action cross section (Φδmax > 600 GM), photostability, and low cytotoxicity. In human hepatocytes and in vivo mouse liver tissue imaging, LD1 showed very bright fluorescence with high LD selectivity and minimized background signal to evaluate the stages of nonalcoholic fatty liver disease. Interestingly, we demonstrated that the liver sinusoid morphology became narrower with increasing LD size and visualized the dynamics including fusion of the LDs in vivo. Moreover, real-time and dual-color TPM imaging with LD1 and a two-photon lysosome tracker could be a useful predictive screening tool in the drug development process to monitor impending drug-induced liver injury inducing drug candidates.

Palmitate induces DNA damage and senescence in human adipocytes in vitro that can be alleviated by oleic acid but not inorganic nitrate.

Hypertrophy in white adipose tissue (WAT) can result in sustained systemic inflammation, hyperlipidaemia, insulin resistance, and onset of senescence in adipocytes. Inflammation and hypertrophy can be induced in vitro using palmitic acid (PA). WAT adipocytes have innately low β-oxidation capacity, while inorganic nitrate can promote a beiging phenotype, with promotion of β-oxidation when cells are exposed to nitrate during differentiation.We hypothesized that treatment of human adipocytes with PA in vitro can induce senescence, which might be attenuated by nitrate treatment through stimulation of β-oxidation to remove accumulated lipids. Differentiated subcutaneous and omental adipocytes were treated with PA and nitrate and senescence markers were analyzed.PA induced DNA damage and increased p16INK4a levels in both human subcutaneous and omental adipocytes in vitro. However, lipid accumulation and lipid droplet size increased after PA treatment only in subcutaneous adipocytes. Thus, hypertrophy and senescence seem not to be causally associated. Contrary to our expectations, subsequent treatment of PA-induced adipocytes with nitrate did not attenuate PA-induced lipid accumulation or senescence. Instead, we found a significantly beneficial effect of oleic acid (OA) on human subcutaneous adipocytes when applied together with PA, which reduced the DNA damage caused by PA treatment.

Flazin as a Lipid Droplet Regulator against Lipid Disorders.

Lipid disorders are closely related to numerous metabolic diseases, and lipid droplets (LDs) have been considered as a new target for regulating lipid metabolism. Dietary intervention and nutraceuticals provide safe and long-term beneficial effects for treating metabolic diseases. Flazin is a diet-derived bioactive constituent mainly existing in fermented foods, of which the lipid metabolism improvement function has not been studied. In this study, the effect of flazin on lipid regulation at both cell level and organelle level was investigated. Lipidomic profiling showed that flazin significantly decreased cellular triglyceride (TG) by 12.0–22.4% compared with modeling groups and improved the TG and free fatty acid profile. LD staining revealed that flazin efficiently reduced both cellular neutral lipid content by 17.4–53.9% and LD size by 10.0–35.3%. Furthermore, nanoelectrospray ionization mass spectrometry analysis proved that flazin exhibited a preferential suppression of LD TG and regulated LD morphology, including a size decrease and surface property improvement. An evaluation of related gene expression suggested the mechanism to be lipolysis promotion and lipogenesis inhibition. These findings indicated that flazin might be an LD regulator for reversing lipid metabolism disturbance. Moreover, the strategy proposed in this study may contribute to developing other nutraceuticals for treating lipid disorder-related metabolic diseases.