Distribution Of Lipid Droplets Research Articles

Regulating interfacial structure of fat globules based on milk fat globule membrane with milk phospholipids to improve physicochemical properties and fat digestion of infant formula emulsions

Modulation of a suitable proportion of milk fat globule membrane (MFGM) and milk phospholipid (MPL) at the lipid droplet interface could be an effective strategy to improve the physicochemical properties and digestibility of infant formula emulsion (FE) systems that contain milk protein. The results showed that the distribution of lipid droplets became more uniform, and the adsorption of MFGM and MPL with different thicknesses was observed at the interface by adding different ratios of MFGM and MPL. Meantime, the stability of the FE was significantly increased than FE prepared by only protein-coated oil core, especially FE4 prepared by adding MFGM and MPL (1:3, w/w) as membrane components, the emulsifying activity index (48.28 m2/g) and emulsion stability index (73.08 min) reached the maximum. The emulsion prepared by phospholipid (PL) encapsulation oil core (FE5) exhibited lipid droplet aggregation and poor emulsion dispersion. The results of lipid digestion indicated that FE obtained with MFGM and MPL encapsulated lipid droplets maintained more PL ring structure similar to human milk structure during gastric digestion, which in turn promotes intestinal digestion of lipids by preventing the formation of protein aggregation. At the end of intestinal digestion, FE4 exhibited the highest lipolysis rate (86.48%) and release of free fatty acids (92.05 μmol/mL). Overall, the addition of a suitable ratio of MFGM to MPL, especially MFGM/MPL (1:3, w/w), contributes to the creation of a thin PL interface structure similar to that of human milk. This could facilitate fat degradation and fatty acid release by regulating the lipid droplet interface.

P-235 Lipid profile changes during neurodevelopment (ND) of mouse embryos cultured in vitro

Abstract Study question Does in vitro culture (IVC) induce changes in lipid profile of the embryo? Summary answer IVC induces changes in embryonic lipid homeostasis that subsequently affect placenta-brain axis (PBA) function and are then maintained in the brain of adult mice. What is known already During ART, embryos are exposed to oxidative stress (OS)-prone environment. Cellular lipids are particularly sensitive to OS. Lipids are a crucial component for proper brain development and any aberrancies in lipid profile of the embryo can affect neurodevelopment (ND) through the PBA, leading to functional and behavioural deficits during adulthood. In particular, deficiencies of ether-linked glicerophospolipids (e.g. plasmalogens), which are highly abundant in the brain, and polyunsaturated fatty acids (PUFAs), have been associated with psychiatric and ND disorders. Study design, size, duration C57BL6 mouse embryos were cultured in vitro for 72hs, and analysed or transferred to 25 recipient females for further development. From 6 pregnant mice, conceptuses (placentae and brain) were collected at near-term pregnancy (19dpc, n > 8/group). Remaining 8 pregnant mice were allowed to deliver and raise litters. Subsequently, brain was collected from male offspring at 4 month of age for lipid homeostasis evaluation. Control group consisted of embryos and offspring obtained from naturally mated C57BL6 mice. Participants/materials, setting, methods C57BL6 blastocysts (n > 15/group) were fixed and subjected to lipid analysis by Fourier transform infra-red (FTIR) and Coherent antistokes Raman spectroscopies (CARS). To reveal lipid changes indicative for ND disorders, selected PUFAs were analyzed by liquid chromatography-mass spectrometry (LC-MS) (n > 8/group) in placenta and brains from prenatal and adult mice. Additionally, placental expression of genes regulating biosynthesis of plasmalogens and proteome of adult males brain, was performed. Values were considered different when p < 0.05, Mean±SEM; Mann-Whitney test. Main results and the role of chance Our results show that IVC induced lipid composition changes in blastocyst, characterised by increased proportion of phospholipids, fatty amides and cholesterol (59±8.2 vs.44.7±8.8; 149±4.5vs. 104±12; 44±2.9vs. 27±4.6 sum of absorbance, p < 0.05); and lipid droplets (LDs) distribution changes, such as, increase size (2.3±0.11 vs 1.8±0.07 µm2, p = 0.0021), and number of large LDs (5.5±0.7 vs. 3.3±0.2, p = 0.006). LC-MS revealed increased levels of linolenic (LA), arachidonic (AA) and docosahexaenoic (DHA) acids in placenta (LA:15.49±1.7 vs. 6.92±2; AA:263.6±29.1 vs. 62.7±14.7 DHA:0±2.5 vs. 2.7±0.8 pmol/mg; p < 0.03) and foetal brain (LA:13.6±5.5 vs. 1.5±0.9; AA:240.5±100 vs. 39.3±9.6; 13.9±6.9 vs. 2.11±0.6 pmol/mg; p < 0.03) of IVC conceptuses, which may condition brain development and function. Conversely, levels of LA and AA were low in adult brain from IVC offspring (LA:4.4±1.5 vs 14.5±3.8; AA: 581±95 vs 1842±454 pmol/mg, p < 0.04). Additionally, the expression of genes regulating biosynthesis of plasmalogens was reduced in IVC placentae (Gnpat:0.2±0.04 vs 1.2±03; Tmem189:0.3±0.1 vs. 1.3±0.3 fold change; p ≤ 0.01). Proteome analysis demonstrated that pathways regulating lipid homeostasis, such as fatty acid oxidation and phospholipid binding and those involved in IL-17a signaling and neurogenesis were upregulated in brain of adult IVC offspring. Limitations, reasons for caution Our results may not entirely reflect what occurs in human embryo, as mouse oocytes have a higher lipid content than human oocyte. Differences in lipid content may cause an increased or decreased penetrance and exhibition of developmental changes of the embryos. Wider implications of the findings Being of non-genetic background, lipid changes induced by IVC may similarly threaten embryos regardless of species causing ND disorders. Our study sheds new light on non-genetic origins of ND disorders and will likely ignite further studies on embryonic lipid changes and their consequences, relevant for human pregnancy and neuropsychiatry. Trial registration number not applicable

Effect of Drying Conditions and Jojoba Oil Incorporation on the Selected Physical Properties of Hydrogel Whey Protein-Based Edible Films.

Edible hydrogel coatings or films in comparison to conventional food packaging materials are characterized as thin layers obtained from biopolymers that can be applied or enveloped onto the surface of food products. The use of lipid-containing hydrogel packaging materials, primarily as edible protective coatings for food applications, is recognized for their excellent barrier capacity against water vapor during storage. With the high brittleness of waxes and the oxidation of different fats or oils, highly stable agents are desirable. Jojoba oil obtained from the jojoba shrub is an ester of long-chain fatty acids and monovalent, long-chain alcohols, which contains natural oxidants α, β, and δ tocopherols; therefore, it is resistant to oxidation and shows high thermal stability. The production of hydrogel films and coatings involves solvent evaporation, which may occur in ambient or controlled drying conditions. The study aimed to determine the effect of drying conditions (temperature from 20 to 70 °C and relative humidity from 30 to 70%) and jojoba oil addition at the concentrations of 0, 0.5, 1.0, 1.5, and 2.0% on the selected physical properties of hydrogel edible films based on whey protein isolate. Homogenization resulted in stable, film-forming emulsions with bimodal lipid droplet distribution and a particle size close to 3 and 45 µm. When higher drying temperatures were used, the drying time was much shorter (minimum 2 h for temperature of 70 °C and relative humidity of 30%) and a more compact structure, lower water content (12.00-13.68%), and better mechanical resistance (3.48-3.93 MPa) of hydrogel whey protein films were observed. The optimal conditions for drying hydrogel whey protein films are a temperature of 50 °C and an air humidity of 30% over 3 h. Increasing the content of jojoba oil caused noticeable color changes (total color difference increased from 2.00 to 2.43 at 20 °C and from 2.58 to 3.04 at 70 °C), improved mechanical elasticity (the highest at 60 °C from 48.4 to 101.1%), and reduced water vapor permeability (the highest at 70 °C from 9.00·10-10 to 6.35·10-10 g/m·s·Pa) of the analyzed films. The observations of scanning electron micrographs showed the heterogeneity of the film surface and irregular distribution of lipid droplets in the film matrix.

Effect of transglutaminase on gelation and functional proteins of mung bean protein isolate

This study aimed to improve mung bean protein’s gelation qualities via microbial transglutaminase (mTGase) cross-linking. The mTGase treatment significantly improved gel hardness and storage modulus (G′) at higher enzyme levels (2 IU/g), peaking hardness at 3 h. The scanning electron microscopy imaging demonstrated more cross-linked structures at 2 IU/g, evolving into a dense network by 3 h. The water-holding capacity for mTGase-treated samples (2 IU/g, 3 h, 55 °C) tripled to 3.77 ± 0.06 g/g versus control (1.24 ± 0.02 g/g), alongside a 15 % decrease in zeta potential (−30.84 ± 0.901 mV versus control’s −26.63 ± 0.497 mV) and an increase in emulsifying activity index to 4.519 ± 0.004 m2/g from 3.79 ± 0.01 m2/g (control). The confocal images showed a more uniform lipid droplet distribution in mTGase-treated samples, suggesting enhanced emulsifying activity. Thus, mTGase treatment significantly improved gel strength and emulsifying properties, making it ideal for plant-based seafood products.

Lipid droplet detection in histopathological images using reinforcement learning

The identification and analysis of lipid droplets in pathological images are pivotal owing to the variations in their size, shading and shape. The development of an artificial intelligence-based automatic detection method can facilitate the analysis of complex pathological images and provide essential support to pathologists. In this paper, we introduce a novel approach for the automated detection of lipid droplets, employing a limited set of images and a modest application of reinforcement learning (RL). Several filters tailored to lipid droplet size and contrast are used in combination. Through the combination of multiple filters using RL, potential lipid droplet regions are identified within pathological images. Subsequently, a random forest classifier is employed to distinguish between normal and lipid droplet images. Evaluation guided by the expertise of two pathologists with over 10 years of clinical experience indicated the hierarchical extraction of lipid droplets with consistent size and shading in pathological tissue images utilizing RL. The proposed method successfully detected lipid droplets in pathological images and facilitated the determination of both the quantity and distribution of lipid droplets within cells. The results highlight the efficacy of the approach in lipid droplet detection. This method is also useful for small to medium-sized fat droplets, which are relatively difficult for humans to detect from their morphology.

Effect of LDL Extracted from Human Plasma on Membrane Stiffness in Living Endothelial Cells and Macrophages via Scanning Ion Conductance Microscopy.

Mechanical properties of living cells play a crucial role in a wide range of biological functions and pathologies, including atherosclerosis. We used low-stress Scanning Ion-Conductance Microscopy (SICM) correlated with confocal imaging and demonstrated the topographical changes and mechanical properties alterations in EA.hy926 and THP-1 exposed to LDL extracted from CVD patients' blood samples. We show that the cells stiffened in the presence of LDL, which also triggered caveolae formation. Endothelial cells accumulated less cholesterol in the form of lipid droplets in comparison to THP-1 cells based on fluorescence intensity data and biochemical analysis; however, the effect on Young's modulus is higher. The cell stiffness is closely connected to the distribution of lipid droplets along the z-axis. In conclusion, we show that the sensitivity of endothelial cells to LDL is higher compared to that of THP-1, triggering changes in the cytoskeleton and membrane stiffness which may result in the increased permeability of the intima layer due to loss of intercellular connections and adhesion.

Extracellular lipidosomes containing lipid droplets and mitochondria are released during melanoma cell division

BackgroundThe incidence of melanoma is increasing worldwide. Since metastatic melanoma is highly aggressive, it is important to decipher all the biological aspects of melanoma cells. In this context, we have previously shown that metastatic FEMX-I melanoma cells release small (< 150 nm) extracellular vesicles (EVs) known as exosomes and ectosomes containing the stem (and cancer stem) cell antigenic marker CD133. EVs play an important role in intercellular communication, which could have a micro-environmental impact on surrounding tissues.ResultsWe report here a new type of large CD133+ EVs released by FEMX-I cells. Their sizes range from 2 to 6 µm and they contain lipid droplets and mitochondria. Real-time video microscopy revealed that these EVs originate from the lipid droplet-enriched cell extremities that did not completely retract during the cell division process. Once released, they can be taken up by other cells. Silencing CD133 significantly affected the cellular distribution of lipid droplets, with a re-localization around the nuclear compartment. As a result, the formation of large EVs containing lipid droplets was severely compromised.ConclusionGiven the biochemical effect of lipid droplets and mitochondria and/or their complexes on cell metabolism, the release and uptake of these new large CD133+ EVs from dividing aggressive melanoma cells can influence both donor and recipient cells, and therefore impact melanoma growth and dissemination.

STK11 (LKB1) mutation suppresses ferroptosis in lung adenocarcinoma by facilitating monounsaturated fatty acid synthesis.

Serine/threonine kinase 11 (STK11), a tumor suppressor gene, exhibits frequent mutations in lung adenocarcinoma (LUAD). However, the specific molecular mechanisms by which STK11 mutations exert an influence on the biosynthesis of monounsaturated fatty acids (MUFAs) and subsequently affect ferroptosis in LUAD remain indistinct. In this study, bioinformatic analysis was employed to probe into the linkage between STK11 and key inhibitory genes of ferroptosis, namely SLC7A11 and SCD1, in LUAD tissues. Quantitative reverse transcription polymerase chain reaction was employed to assess the expression of STK11 in both wild-type and mutant STK11 LUAD cells, cell counting kit-8 to assess cell viability, and flow cytometry to detect apoptosis. A transmission electron microscope was utilized to observe mitochondrial morphology, and Western blot to ascertain the protein expression of STK11, ferroptosis-related proteins, and the enzyme SCD1 involved in MUFA synthesis. Oil red O staining was employed to test the distribution of lipid droplets in cancer cells, and a lipid quantification method to measure the content of MUFAs. Commercial kits were employed to assess the levels of lipid reactive oxygen species, malondialdehyde, glutathione, and Fe2+ in cells. The result revealed a negative correlation between STK11 and SLC7A11 as well as SCD1, with STK11 expression downregulated in mutant STK11 LUAD cells. Furthermore, STK11 mutations were found to suppress ferroptosis in LUAD cells by affecting MUFA synthesis. Subsequent rescue assays demonstrated that STK11 mutations hindered ferroptosis by impacting the synthesis of MUFAs in LUAD cells. This study provided evidence that STK11 mutations suppressed ferroptosis in LUAD cells by promoting MUFA synthesis, thus offering a novel research direction in the management of LUAD.

Investigating ionizing radiation-induced changes in breast cancer cells using stimulated Raman scattering microscopy.

Altered lipid metabolism of cancer cells has been implicated in increased radiation resistance. A better understanding of this phenomenon may lead to improved radiation treatment planning. Stimulated Raman scattering (SRS) microscopy enables label-free and quantitative imaging of cellular lipids but has never been applied in this domain. We sought to investigate the radiobiological response in human breast cancer MCF7 cells using SRS microscopy, focusing on how radiation affects lipid droplet (LD) distribution and cellular morphology. MCF7 breast cancer cells were exposed to either 0 or 30Gy (X-ray) ionizing radiation and imaged using a spectrally focused SRS microscope every 24hrs over a 72-hr time period. Images were analyzed to quantify changes in LD area per cell, lipid and protein content per cell, and cellular morphology. Cell viability and confluency were measured using a live cell imaging system while radiation-induced lipid peroxidation was assessed using BODIPY C11 staining and flow cytometry. The LD area per cell and total lipid and protein intensities per cell were found to increase significantly for irradiated cells compared to control cells from 48 to 72hrs post irradiation. Increased cell size, vacuole formation, and multinucleation were observed as well. No significant cell death was observed due to irradiation, but lipid peroxidation was found to be greater in the irradiated cells than control cells at 72hrs. This pilot study demonstrates the potential of SRS imaging for investigating ionizing radiation-induced changes in cancer cells without the use of fluorescent labels.

Equal distribution of lipid droplets in daughter cells is regulated by microtubules

ABSTRACT During eukaryotic cell division, organelles are distributed between daughter cells through a dynamic process to ensure that cells can differentiate and perform their functions correctly. Uncovering the mode of lipid droplet (LD) distribution may help reveal the mechanism of membrane remodeling during cell division and lipid droplet function. Our results showed that LDs were equally distributed in both daughter cells during cytokinesis. Further experiments demonstrated that the key factor regulating the movement of LDs is the microtubule (MT)-resident protein KIF5B. Because the KIF5B structure lacks a hydrophilic region, we believe that there are proteins that mediate the interaction between LDs and KIF5B. Mass spectrometric detection of KIF5B-interacting proteins on the surface of LDs demonstrated that LDs were first wrapped by intermediate filaments forming a meshwork and then contacted with MTs to mediate lipid droplet movement during cytokinesis. Disruption of the homogeneous distribution of LDs may hinder cell proliferation and even lead to apoptosis.

Effects of physical exercise on glycemic metabolism, aerobic capacity and structure of brown adipose tissue in diabetic animals

The ability of brown adipose tissue (BAT) and aerobic physical exercise (AET) to improve energy metabolism is widely known. Since AET has been used as an insulin- sensitizing therapy for diabetes, here we tested whether the effects of AET on the glycemic metabolism and aerobic capacity of diabetic animals are accompanied by structural changes in BAT. Adult male C57BL6/J mice were assigned into groups: non-diabetic sedentary (NDS, n=6), non-diabetic trained (NDT, n=6), diabetic sedentary (DS, n=4) and diabetic trained (DT, n=7). Diabetes was induced by high-fat and -fructose diet and intraperitoneal injection of streptozotocin (120 mg/g). AET consisted of 8-wk running session of 60 min at 60% of maximal speed, 5 days/wk. Experimental procedures were approved by Ethics Committees from Faculty of Medicine and from School of Arts, Science and Humanities of University of São Paulo (#1473/2020 and #001/2020, respectively). Initial body weight (BW) did not differ among groups, however the DS and DT groups had lower final BW compared with NDS. The BW gain of DS group was lower compared with NDS group. Food consumption (Kcal/animal/day) was higher in DT group compared with NDS and NDT. The DS and DT groups had higher fasting blood glucose, and DS group had greater area under the curve on the glucose tolerance test than the NDS and NDT groups. Indirect calorimetry measurement at rest revealed no difference in the variables oxygen uptake (VO2), carbon dioxide expiration (VCO2), respiratory exchange ratio (RER), energy expenditure (EE), carbohydrate and fat oxidation among groups. During maximal exercise test, NDT and DT increased the time to exhaustion, maximal speed, intensity corresponding to the VO2máx (iVO2) compared with NDS and DS. An increase in VO2máx and VCO2máx was observed in NDT group compared with DS and DT group. No differences were observed in the weight of white adipose tissue depots (retroperitoneal, periepididymal and subcutaneous), interscapular brown adipose tissue (iBAT), skeletal muscle (gastrocnemius and soleus), and other organs (heart, lung, and kidney). Morphometric analysis of the iBAT revealed that the area, diameter, volume, mass, frequency of lipid droplet distribution by size (small, medium, large, and very large) and amount of lipid droplets did not differ among groups, however, collagen deposition in the iBAT increased in the DS and DT groups compared with NDS group. In conclusion, the AET-induced improvement in glucose tolerance and aerobic capacity in diabetic animals is not accompanied by changes in morphometry and collagen deposition in the iBAT. This study was supported by grants from the São Paulo Research Foundation (FAPESP) #2020/15748-4 and #2020/12005-0. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

NS5A domain I antagonises PKR to facilitate the assembly of infectious hepatitis C virus particles.

Hepatitis C virus NS5A is a multifunctional phosphoprotein comprised of three domains (DI, DII and DIII). DI and DII have been shown to function in genome replication, whereas DIII has a role in virus assembly. We previously demonstrated that DI in genotype 2a (JFH1) also plays a role in virus assembly, exemplified by the P145A mutant which blocked infectious virus production. Here we extend this analysis to identify two other conserved and surface exposed residues proximal to P145 (C142 and E191) that exhibited no defect in genome replication but impaired virus production. Further analysis revealed changes in the abundance of dsRNA, the size and distribution of lipid droplets (LD) and the co-localisation between NS5A and LDs in cells infected with these mutants, compared to wildtype. In parallel, to investigate the mechanism(s) underpinning this role of DI, we assessed the involvement of the interferon-induced double-stranded RNA-dependent protein kinase (PKR). In PKR-silenced cells, C142A and E191A exhibited levels of infectious virus production, LD size and co-localisation between NS5A and LD that were indistinguishable from wildtype. Co-immunoprecipitation and in vitro pulldown experiments confirmed that wildtype NS5A domain I (but not C142A or E191A) interacted with PKR. We further showed that the assembly phenotype of C142A and E191A was restored by ablation of interferon regulatory factor-1 (IRF1), a downstream effector of PKR. These data suggest a novel interaction between NS5A DI and PKR that functions to evade an antiviral pathway that blocks virus assembly through IRF1.

Effects of Nutritionally Induced Obesity on Metabolic Pathways of Zebrafish.

Human obesity has become a global epidemic that can lead to many metabolic diseases, including insulin resistance, type 2 diabetes, dyslipidemia, hypertension and nonalcoholic fatty liver. The development of obesity is closely associated with excess food intake and energy imbalance, family history, lifestyle, psychology and other factors, but molecular mechanisms underlying the induction and development of obesity remain to be intensively studied under a variety of internal and external pathogenesis conditions. In this study, we generated two obesity models of zebrafish that were treated with a high-fat diet (HFD) or an overfeeding diet (DIO). Both HFD and DIO zebrafish exhibited higher levels of lipid accumulation, fat distribution, microvascular steatosis and ectopic accumulation of lipid droplets in liver and muscle than normal diet (NOD) fish. The comparison of transcriptome sequencing data for the livers of HFD, DIO and NOD groups identified common and specific genes and signaling pathways that are potentially associated with zebrafish obesity induced by HFD and/or DIO. These findings provide clues for further understanding the mechanisms of obesity development and preventing nutritionally induced obesity through targeting the common signaling pathways and biological processes.

Administration of nicotinamide mononucleotide improves oocyte quality of obese mice.

Obesity has become a common health concern around the world. Maternal obesity could cause poor reproductive outcomes due to chronic ovarian inflammation and decreased oocyte quality. However, the strategies to improve the poor reproductive outcomes of obese females have not been fully studied. In this study, we aimed to explore the effects and underlying mechanisms of nicotinamide mononucleotide (NMN) on oocyte quality and reproductive performance of obese mice. The obese mouse model was established by feeding high-fat diet which was confirmed by body weight record, fasting blood glucose test and oral glucose tolerance test. The expression of ovary development related genes and inflammation related genes, including Lhx8, Bmp4, Adgre1, Ccl2, TNF-α, Gal-3, Clec10a and IL-10 in ovaries and the expression of Bax and Sod1 in oocytes were detected using quantitative reverse transcription PCR (RT-qPCR). The adipose size of abdominal fat tissue was determined with haematoxylin and eosin (H&E) staining. Immunofluorescence staining was performed to measure the ROS level, spindle/chromosome structure, mitochondrial function, actin dynamics and DNA damage of oocytes. The administration of NMN restored ovarian weight and reduced the adipose size of abdominal fat tissue and ovarian inflammation in high fat diet (HFD) mice. Furthermore, NMN treatment improved the oocytes quality partially by restoring the mitochondrial function and actin dynamics, reducing meiotic defects, DNA damage and ROS level and lipid droplet distribution of oocytes in HFD mice. On the long-term effect, NMN restored offspring body weight of HFD mice. NMN could improve the oocyte quality of HFD-induced obese mice.

P-158 Beneficial effects of platelet rich plasma for human germinal vesicle oocyte in vitro maturation

Abstract Study question Can platelet rich plasma (PRP) supplementation to the medium improve outcomes of in vitro maturation of germinal vesicle (GV) oocytes? Summary answer PRP as a component of in vitro maturation medium benefits the GV oocyte maturation rate, the mitochondrial membrane potential and the distribution of lipid droplets. What is known already The European Society of Human Reproduction and Embryology presents several strategies for maintaining fertility in women, one of which is in vitro maturation (IVM) of oocytes. Despite more than 5,000 children have been born after using the IVM method, the mechanisms involved in meiotic arrest and resumption in oocytes are not fully understood. Hormones and growth factors are components of a complex regulation system which play a mediator role in ovarian function and are involved in meiotic resumption. PRP is a rich source of growth factors and can be effectively used to supplement the medium for in vitro oocyte maturation. Study design, size, duration Oocyte maturity was assessed after denudation, and immature gametes at the GV stage (n = 84) were randomly divided into 4 groups for IVM (24 - 48h): 1- without cumulus cells (CC) and PRP, 2 – with PRP without CC, 3 - with CC without PRP, 4 – with CC and PRP. The maturation rate of the oocytes was evaluated in 24 or 48h. The study was carried out between January 2020 to December 2021. Participants/materials, setting, methods This prospective study included 30 female patients 33±3.6 years old. Women received exogenous gonadotropins as part of a short protocol of superovulation induction. About 15% oocytes were at the germinal vesicle stage after denudation of retrieved gametes. PRP was prepared by two-step centrifugation method and was added (5%) to the maturation media. Primary endpoints were oocyte maturation rate, mitochondrial membrane potential and lipid droplets distribution evaluated by using JC-1 and Nile Red staining respectively. Main results and the role of chance The highest maturation rate was in group 4 (85%) compared with groups 1-3, where the rates were 9.5; 36.4 and 47.6, respectively (p &amp;lt; 0.05). The biggest number of cells with high mitochondrial potential was in oocytes matured with PRP and cumulus cells (4.5 ± 0.7) compared with other groups (1.3 ± 0.3; 3.0 ± 0.5 and 2.7 ± 0.4 for groups 1-3, respectively) (p &amp;lt; 0.05). The distribution of lipid droplets, typical of in vivo matured metaphase II oocytes, was the largest in group 1 (75%) (p &amp;lt; 0.05). There were no differences in this parameter between the other groups. Limitations, reasons for caution Limited number of GV oocytes in the study group. Further studies are recommended and should include group of patients with different age range. Wider implications of the findings PRP injection into ovarian tissue causes its rejuvenation. Our results confirm for the first time that PRP supplementation to the medium of oocyte in vitro maturation can increase the maturation rate, mitochondrial activity, and improve lipid droplets distribution in human oocytes. These finding are valuable for improvement of IVM medium. Trial registration number Not applicable

DGAT2 Inhibition Potentiates Lipid Droplet Formation To Reduce Cytotoxicity in APOL1 Kidney Risk Variants.

Two variants in the gene encoding apolipoprotein L1 (APOL1) that are highly associated with African ancestry are major contributors to the large racial disparity in rates of human kidney disease. We previously demonstrated that recruitment of APOL1 risk variants G1 and G2 from the endoplasmic reticulum to lipid droplets leads to reduced APOL1-mediated cytotoxicity in human podocytes. We used CRISPR-Cas9 gene editing of induced pluripotent stem cells to develop human-derived APOL1G0/G0 and APOL1G2/G2 kidney organoids on an isogenic background, and performed bulk RNA sequencing of organoids before and after treatment with IFN-γ. We examined the number and distribution of lipid droplets in response to treatment with inhibitors of diacylglycerol O-acyltransferases 1 and 2 (DGAT1 and DGAT2) in kidney cells and organoids. APOL1 was highly upregulated in response to IFN-γ in human kidney organoids, with greater increases in organoids of high-risk G1 and G2 genotypes compared with wild-type (G0) organoids. RNA sequencing of organoids revealed that high-risk APOL1G2/G2 organoids exhibited downregulation of a number of genes involved in lipogenesis and lipid droplet biogenesis, as well as upregulation of genes involved in fatty acid oxidation. There were fewer lipid droplets in unstimulated high-risk APOL1G2/G2 kidney organoids than in wild-type APOL1G0/G0 organoids. Whereas DGAT1 inhibition reduced kidney organoid lipid droplet number, DGAT2 inhibition unexpectedly increased organoid lipid droplet number. DGAT2 inhibition promoted the recruitment of APOL1 to lipid droplets, with associated reduction in cytotoxicity. Lipogenesis and lipid droplet formation are important modulators of APOL1-associated cytotoxicity. Inhibition of DGAT2 may offer a potential therapeutic strategy to attenuate cytotoxic effects of APOL1 risk variants.

Rubicon deficiency exacerbates fasting-induced hepatic steatosis

Abstract Objective: Rubicon is an inhibitory interacting protein of the autophagy-related protein Uvrag. We previously showed that Rubicon deficiency promotes autophagic flux in vivo and that autophagy can degrade lipid droplets. This study aimed to investigate the effects of Rubicon deficiency on fasting-induced hepatic steatosis. Methods: Two-month-old wild-type (WT) and Rubicon-deficient mice were subjected to feeding or fasting for 24 hours to induce hepatic steatosis. The distribution of liver lipid droplets was revealed by oil red O staining. Hepatic and plasma triglyceride, non-esterified fatty acid (NEFA), and cholesterol levels were detected using commercially available kits. Real-time reverse transcriptase-polymerase chain reaction was performed to analyze the mRNA expression of genes related to lipid metabolism in the liver. Western blot was conducted to assess autophagy-related protein levels in the liver. The animal experiments were approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University, China. Results: We showed that under fasting conditions, Rubicon-deficient mice had more lipid droplets in the liver than WT controls. Consistent with these results, the hepatic triglyceride, NEFA, and cholesterol levels in fasted Rubicon-deficient mice were significantly higher than those of fasted WT controls. The levels of SREBP-1, a key regulator of lipid synthesis, were significantly lower in livers from fasted WT mice than those of fed WT mice. However, the decrease in SREBP-1 in fasted mice was attenuated by Rubicon deficiency. Western blot analysis demonstrated that the fasting-induced increase in autophagic flux was amplified by Rubicon deficiency. Finally, we showed that Rubicon deficiency in mice led to elevated plasma triglyceride and NEFA acid levels under fasting conditions. Conclusion: Rubicon deficiency exacerbates fasting-induced hepatic steatosis in mice.

Distribution and behavior of lipid droplets in hepatic cells analyzed by variations of citochemical technique and scanning electron microscopy

Toxicity evaluations involve the analysis of multiple biomarkers. In this study, the liver, target organ analyzed by treatments with iron concentrations, indicated the accumulation of lipids as a response. Considering that the distribution of lipids in an organ is directly related to the induction of inflammatory processes by aquatic contaminants, this study proposes to carry out an integrative investigation of the behavior and the distribution of lipids in the liver tissue. Techniques of light and electron microscopy were performed in order to propose a new way of assessing and quantifying the distribution of lipid droplets, also presenting methodological alternatives that can be chosen by the reader according to the interests and resources available. Thus, it is assumed that the method begins with the fixation of the liver with Glutaraldehyde 2,5% in PBS 0,1 M and continues with post fixation with osmium tretoxide 1%, which marks lipids. For this proposition, two inclusion methodologies were performed to histological analyses in Historesin and ultrastructural analyses in EMBeed 812. For light microscopy (LM) analyses, cuts were obtained with 2,5 micrometers thickness, which were stained with (1) Mayers hematoxylin and (2) toluidine blue. The images obtained were processed in software Image J Fiji to evidence the lipid distribution in liver.•Cytological reactions with osmium tetroxide constitute low complexity methods that allow the optimization of the localization, identification and quantification of lipid droplets in the liver tissue when analyzed under the conventional light microscope.•Samples included in EMBeed 812 resin commonly used in Transmission Electron Microscopy can be analyzed by SEM-BEC, as complementary analyses for the detection of lipids.•Using SEM-BEC and conventional light microscopy, it is possible to quantify the area occupied by lipid droplets using Image J Fiji software, as these are contrasted due to the reaction with osmium tetroxide.

Three-dimensional structural analysis of mitochondria composing each subtype of fast-twitch muscle fibers in chicken.

In a previous study, the three-dimensional structures of mitochondria in type I and type IIb muscle fibers of chicken were analyzed. The study reported differences in the shape of the mitochondria and the distribution of lipid droplets. In this study, we three-dimensionally analyzed mitochondria and lipid droplets of type II muscle fiber subtypes IIa, IIb, and IIc of chicken lateral iliotibial muscle in the same field of view using correlative light electron microscopy (CLEM) and array tomography methods. The reconstructed images showed that the mitochondria of type IIa muscle fiber were thick and aligned along the myofibrils, and many lipid droplets were embedded in the mitochondria. The mitochondria of type IIb muscle fibers were intermittent, aligned along the myofibrils, and showed contact between adjacent horizontal mitochondria. No lipid droplets were observed in type IIb muscle fiber. In type IIc muscle fiber, we observed irregularly shaped mitochondria with small diameters aligned along the myofibrils. Lipid droplets not only were embedded in the mitochondria but also existed independently in some cases. The combination of array tomography and CLEM methods enabled three-dimensional electron microscopic observation of mitochondria in different subtypes of type II muscle fibers. The subtypes of type II muscle fibers differed in mitochondrial occupancy and morphology and in lipid droplet distribution, and characteristics that had been demonstrated biochemically were also demonstrated ultrastructurally.

An ultrasensitive lipid droplet-targeted NIR emission fluorescent probe for polarity detection and its application in liver disease diagnosis.

Compared to normal cells, cancer cells require more energy supply during proliferation and metabolism. In living cells, in addition to mitochondria, lipid droplets are also an important organelle for providing energy. Studies have shown that the number and distribution of lipid droplets change significantly during the production of lesions in cells. At this stage, the predisposing factors for the development of cellular lesions are not clear, thus leading to limitations in the early diagnosis and treatment of diseases such as liver injury, fatty liver, and hepatitis. To meet the urgent challenge, we used a near-infrared emission fluorescent probe SSR-LDs based on the intramolecular charge transfer effect (ICT) to detect polarity changes within intracellular lipid droplets. The probe SSR-LDs has ultra-sensitive polarity sensitivity, excellent chemical stability and photo-stability. In addition, by comparing normal and cancer cells through cell imaging experiments, we found that the robust probe has the ability to sensitively monitor the changes in lipid droplet polarity in the living cells. More importantly, using the constructed fluorescent probe, we have achieved an in vitro fluorescence detection of liver injury and fatty liver, and the detection of hepatitis at the in vivo level. The unique fluorescent probe SSR-LDs is expected to serve as a powerful tool for the medical diagnosis of diseases related to lipid droplet polarity.