Distribution Of Lipid Droplets Research Articles

Alterations in the rainbow trout (Oncorhynchus mykiss) eggs exposed to ionizing radiation during induced androgenesis.

Ionizing radiation (IR) is applied to inactivate nuclear genome in the salmonid eggs to induce androgenetic development. However, it has been considered that doses of IR used to damage maternal chromosomes may also affect morphology of the eggs and decrease their developmental potential. Thus, the main goal of the present research was to assess alterations in the rainbow trout (Oncorhynchus mykiss) eggs caused by the high dose of IR administered during androgenesis. In the present research, rainbow trout eggs were irradiated with 350Gy of X-rays, inseminated and exposed to the high hydrostatic pressure (HHP) shock to develop as androgenetic doubled haploids (DHs). The distribution of lipid droplets in the irradiated and non-irradiated rainbow trout eggs, survival rates and morphology of larvae from androgenetic and control groups were compared. It has been observed that non-irradiated and irradiated eggs exhibited altered distribution of lipid droplets. Most of the eggs before IR treatment displayed rather equal distribution of the oil droplets. In turn, majority of eggs studied after irradiation had coalesced lipid droplets, a pattern found in eggs with reduced quality. Incidences of abnormally developed larvae were more frequently observed among fish that hatched from the irradiated eggs. Observed changes suggest X-rays applied for the genetic inactivation of rainbow trout eggs may lead to decrease of their developmental competence.

Effects of sera of rats fed with Huganqingzhi tablets on endoplasmic reticulum stress in a HepG2 cell model of nonalcoholic fatty liver disease

To investigate the effects of sera from rats fed with Huganqingzhi tablets (HGT) on endoplasmic reticulum (ER) stress in a steatotic hepatocyte model of free fatty acids (FFAs)-induced nonalcoholic fatty liver disease (NAFLD) and explore the possible mechanism. FFAs prepared by mixing oleic acid and palmitic acid at the ratio of 2:1. HepG2 cells were treated with the sera from rats fed with low-, moderate-or high-dose HGT (HGT sera) or sera of rats fed with fenofibrate (fenofibrate sera), followed by treatment with 1 mmol/L FFAs for 24 h to induce hepatic steatosis. Oil red O staining was used to observe the distribution of lipid droplets in the cells. The biochemical parameters including triglyceride (TG), lactated hydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using a commercial kit. The morphological changes of the ER in the cells were observed using transmission electron microscopy. The protein/mRNA expressions of ER stress-related signal molecules including GRP78, PERK, p-PERK, ATF6, ATF4, CASPASE-12, CHOP, XBP-1, PKC, and p-PKC-δ were detected using Western blotting and/or quantitative real-time PCR (qRT-PCR). The changes in the protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP were also detected in cells with transient transfection of PKC-δ siRNA for PKC-δ knockdown. Compared with the control cells, the cells treated with FFAs showed significantly increased levels of TG, AST, and ALT (P < 0.05). Compared with FFAs-treated cells, the cells pretreated with HGT sera or fenofibrate sera all showed significantly decreased TG, AST and ALT levels (P < 0.05), reduced accumulation of the lipid droplets (P < 0.05), and lowered protein or mRNA expression levels of GRP78, p-PERK, ATF6, ATF4, CHOP, CASPASE-12, XBP-1 and p-PKC-δ (P < 0.05). PKC-δ knockdown caused significantly reduced protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP in the cells with FFA-induced hepatic steatosis (P < 0.001); treatment with high-dose HGT serum more significantly reduced the expressions of GRP78 (P < 0.001) and P-PERK (P < 0.01) in FFAs-induced cells with PKC-δ knockdown. HGT serum can effectively prevent FFAs-induced steatosis in HepG2 cells by alleviating ER stress, in which PKC-δ may act as an important target.

Perfluorononanoic acid (PFNA) alters lipid accumulation in bovine blastocysts after oocyte exposure during in vitro maturation

Perfluorononanoic acid (PFNA) is one of the perfluoroalkyl acids present in human tissues. In this study, effects on early embryo development after PFNA exposure were investigated using the bovine in vitro production system. Oocytes were exposed to PFNA during maturation in vitro (10 μg mL−1 and 0.1 μg mL−1), and then fertilized and cultured in parallel with control groups. Developmental parameters (cleavage, blastocyst formation) were followed and embryo quality evaluated (stage, grade). Embryos developed after exposure to 0.1 μg mL−1 were stained to distinguish nuclei, active mitochondria and neutral lipids. 10 μg mL−1 of PFNA had a severe negative effect on blastocyst formation (OR: 0.27 p < 0.05), an effect not observed at 0.1 μg mL−1. However, lipid droplet distribution was significantly altered in embryos exposed to 0.1 μg mL−1, suggesting a disturbance of lipid metabolism after exposure to sublethal levels of PFNA during oocyte maturation in vitro.

Lipid acquisition and tissue storage in hagfish: new insights from an ancient vertebrate.

Hagfishes are ancient vertebrates, which have the ability to tolerate nearly a year of food deprivation with energy during fasting maintained using lipid stores. While lipid transporters are evolutionarily conserved, there are relatively few studies examining mechanisms of lipid acquisition in teleosts and no reports in the primitive and evolutionarily important agnathans. We examined tissue lipid droplet distribution and used gut sac preparations to characterize uptake mechanisms of the monounsaturated fatty acid, oleic acid (OA; 18:1 cis-9), in the Pacific hagfish. OA absorption was carrier-mediated and demonstrated saturable concentration-dependent uptake with an affinity constant of 55 ± 7µM, and a maximal rate of uptake of 1311 ± 97pmol cm- 2h- 1. Additionally, regulation of intestinal transport was demonstrated as feeding significantly increased uptake. To further examine post-prandial effects on fatty acid transport, hagfish were dosed with bovine insulin, which had no effect on OA intestinal acquisition, but did lower plasma glucose. Overall, this is the first evidence for intestinal fatty acid acquisition in an agnathan, which are ideal models for understanding the evolution of nutrient transport processes in vertebrates, and are particularly suited to lipid research owing to their dependence upon this energy source during prolonged periods of fasting.

Effect of carbohydrates on lipid metabolism during porcine oocyte IVM.

Porcine oocytes contain a large amount of endogenous lipid, which is thought to function as an intracellular source of energy. The aim of this study was to determine the effects of stimulating or inhibiting lipid metabolism using l-carnitine or etomoxir respectively on the IVM of porcine oocytes cultured in media of varying carbohydrate composition. In the presence of pyruvate and lactate, exclusion of glucose inhibited oocyte nuclear and cytoplasmic maturation compared with oocytes matured in media containing low (1.5mM) and high (4.0mM) concentrations of glucose. In the absence of pyruvate and lactate in low-glucose medium only, a greater proportion of l-carnitine-treated oocytes progressed to the MII stage compared with untreated oocytes. The inclusion of pyruvate and lactate significantly altered the distribution of cytoplasmic lipid droplets and elevated the ATP content of oocytes, whereas the l-carnitine treatment did not. Further, the inhibitory effect of etomoxir on nuclear maturation was decreased in high- compared with low-glucose medium. The results indicate that carbohydrate substrates are absolutely necessary for effective porcine oocyte maturation, and that l-carnitine supplementation can only partially compensate for deficiencies in carbohydrate provision.

A microglial cell model for acyl-CoA oxidase 1 deficiency

Acyl-CoA oxidase 1 (ACOX1) deficiency is a rare and severe peroxisomal leukodystrophy associated with a very long-chain fatty acid (VLCFA) β–oxidation defect. This neurodegenerative disease lacks relevant cell models to further decipher the pathomechanisms in order to identify novel therapeutic targets. Since peroxisomal defects in microglia appear to be a key component of peroxisomal leukodystrophies, we targeted the Acox1 gene in the murine microglial BV-2 cell line. Using CRISPR/Cas9 gene editing, we generated an Acox1-deficient cell line and validated the allelic mutations, which lead to the absence of ACOX1 protein and enzymatic activity. The activity of catalase, the enzyme degrading H2O2, was increased, likely in response to the alteration of redox homeostasis. The mutant cell line grew more slowly than control cells without obvious morphological changes. However, ultrastructural analysis revealed an increased number of peroxisomes and mitochondria associated with size reduction of mitochondria. Changes in the distribution of lipid droplets containing neutral lipids have been observed in mutant cells; lipid analysis revealed the accumulation of saturated and monounsaturated VLCFA. Besides, expression levels of genes encoding interleukin-1 beta and 6 (IL-1β and IL-6), as well as triggering receptor expressed on myeloid cells 2 (Trem2) were found modified in the mutant cells suggesting modification of microglial polarization and phagocytosis ability. In summary, this Acox1-deficient cell line presents the main biochemical characteristics of the human disease and will serve as a promising model to further investigate the consequences of a specific microglial peroxisomal β–oxidation defect on oxidative stress, inflammation and cellular functions.

Single-Cell Analysis of Morphological and Metabolic Heterogeneity in Euglena gracilis by Fluorescence-Imaging Flow Cytometry.

Microalgal biofuels and biomass have ecofriendly advantages as feedstocks. Improved understanding and utilization of microalgae require large-scale analysis of the morphological and metabolic heterogeneity within populations. Here, with Euglena gracilis as a model microalgal species, we evaluate how fluorescence- and brightfield-derived-image-based descriptors vary during environmental stress at the single-cell level. This is achieved with a new multiparameter fluorescence-imaging cytometric technique that allows the assaying of thousands of cells per experiment. We track morphological changes, including the intensity and distribution of intracellular lipid droplets, and pigment autofluorescence. The combined fluorescence-morphological analysis identifies new metrics not accessible with traditional flow cytometry, including the lipid-to-cell-area ratio (LCAR), which shows promise as an indicator of oil productivity per biomass. Single-cell metrics of lipid productivity were highly correlated ( R2 > 0.90, p < 0.005) with bulk oil extraction. Such chemomorphological atlases of algal species can help optimize growth conditions and selection approaches for large-scale biomass production.

Distinct lipid droplet characteristics and distribution unmask the apparent contradiction of the athlete's paradox

ObjectiveIntramyocellular lipid (IMCL) storage negatively associates with insulin resistance, albeit not in endurance-trained athletes. We investigated the putative contribution of lipid droplet (LD) morphology and subcellular localization to the so-called athlete's paradox. MethodsWe performed quantitative immunofluorescent confocal imaging of muscle biopsy sections from endurance Trained, Lean sedentary, Obese, and Type 2 diabetes (T2DM) participants (n = 8/group). T2DM patients and Trained individuals were matched for IMCL content. Furthermore we performed this analysis in biopsies of T2DM patients before and after a 12-week exercise program (n = 8). ResultsWe found marked differences in lipid storage morphology between trained subjects and T2DM: the latter group mainly store lipid in larger LDs in the subsarcolemmal (SS) region of type II fibers, whereas Trained store lipid in a higher number of LDs in the intramyofibrillar (IMF) region of type I fibers. In addition, a twelve-week combined endurance and strength exercise program resulted in a LD phenotype shift in T2DM patients partly towards an ‘athlete-like’ phenotype, accompanied by improved insulin sensitivity. Proteins involved in LD turnover were also more abundant in Trained than in T2DM and partly changed in an ‘athlete-like’ fashion in T2DM patients upon exercise training. ConclusionsOur findings provide a physiological explanation for the athlete's paradox and reveal LD morphology and distribution as a major determinant of skeletal muscle insulin sensitivity.

Small rubber particle proteins from Taraxacum brevicorniculatum promote stress tolerance and influence the size and distribution of lipid droplets and artificial poly(cis-1,4-isoprene) bodies.

Natural rubber biosynthesis occurs on rubber particles, i.e. organelles resembling small lipid droplets localized in the laticifers of latex-containing plant species, such as Hevea brasiliensis and Taraxacum brevicorniculatum. The latter expresses five small rubber particle protein (SRPP) isoforms named TbSRPP1-5, the most abundant proteins in rubber particles. These proteins maintain particle stability and are therefore necessary for rubber biosynthesis. TbSRPP1-5 were transiently expressed in Nicotiana benthamiana protoplasts and the proteins were found to be localized on lipid droplets and in the endoplasmic reticulum, with TbSRPP1 and TbSRPP3 also present in the cytosol. Bimolecular fluorescence complementation confirmed pairwise interactions between all proteins except TbSRPP2. The corresponding genes showed diverse expression profiles in young T. brevicorniculatum plants exposed to abiotic stress, and all except TbSRPP4 and TbSRPP5 were upregulated. Young Arabidopsis thaliana plants that overexpressed TbSRPP2 and TbSRPP3 tolerated drought stress better than wild-type plants. Furthermore, we used rubber particle extracts and standards to investigate the affinity of the TbSRPPs for different phospholipids, revealing a preference for negatively charged head groups and 18:2/16:0 fatty acid chains. This finding may explain the effect of TbSRPP3-5 on the dispersity of artificial poly(cis-1,4-isoprene) bodies and on the lipid droplet distribution we observed in N. benthamiana leaves. Our data provide insight into the assembly of TbSRPPs on rubber particles, their role in rubber particle structure, and the link between rubber biosynthesis and lipid droplet-associated stress responses, suggesting that SRPPs form the basis of evolutionarily conserved intracellular complexes in plants.

Ultrastructure Study of the Stored Lipid Reserves in Gyrodactylus gasterostei (Monogenea) Using Confocal and Transmission Electron Microscopy.

This study examines the distribution and depletion of stored lipids in Gyrodactylus gasterostei Gläser, 1974, migrating off its three-spine stickleback host Gasterosteus aculeatus L., with the prospect that it might prove informative for interpreting the biology of other gyrodactylids species more generally. Nonfeeding life cycle stages, such as the dispersal stages of parasites, are dependent for survival upon finite energy reserves gathered during feeding phases. Thus, those individuals with more limited reserves will die sooner and consequently have less time available to find a new host once detached. At this stage, the principal energy reserves in gyrodactylids are stored as large lipid droplets. Confocal laser scanning microscopy has been used to investigate the distribution of lipid droplets in Gyrodactylus, which have migrated off their fish host, testing the hypothesis that these droplets function as a proxy for the nutritional state. This study demonstrated that the lipid droplets were particularly associated with the gut and that there is a significant variability in the volume of stored lipid carried out by each individual. Transmission electron microscopy showed that gyrodactylids carry lipid droplets at all stages of their life cycle, including at release from the birth pore. It is likely that transferring worms requires stored energy reserves to survive in the event of failure to establish contact with a new host. These reserves could allow the parasite to survive without a host for several days.

Association between white opaque substance under magnifying colonoscopy and lipid droplets in colorectal epithelial neoplasms.

AIMTo examine the association between white opaque substance (WOS) and histologically verified lipid droplets in colorectal epithelial neoplasms.METHODSWe reviewed colonoscopy records at our institution from 2014 to 2016 and identified cases of endoscopically or surgically resected colorectal epithelial neoplasms observed by magnifying narrow-band imaging (M-NBI) colonoscopy. Immunohistochemistry was used to stain tumors with a monoclonal antibody specific to adipophilin as a marker of lipids. The expression and distribution of adipophilin were compared between WOS-positive and WOS-negative lesions and among tumors classified by histologic type and depth of invasion.RESULTSUnder M-NBI colonoscopy, 81 lesions were positive for WOS and 48 lesions were negative for WOS. The rate of adipophilin expression was significantly higher in WOS-positive lesions (95.1%) than in WOS-negative lesions (68.7%) (P = 0.0001). The incidence of deep adipophilin expression was higher in WOS-positive lesions (24.7%) than in WOS-negative lesions (4.2%) (P = 0.001). The incidence of deep expression was predominant among cancers with massive submucosal invasion (62.5%) compared to adenoma (7.2%) and high-grade dysplasia or cancers with slight submucosal invasion (12.7%) (P = 0.0001).CONCLUSIONThe distribution of lipid droplets may be closely associated with the visibility of WOS under M-NBI colonoscopy, and with histologic grade and depth of tumor invasion.

S.06.01 - Synaptic basis of cocaine addiction

Organotypic culture of testicular fragments from 7-day-old male pigs (Polish White Large) was used. Tissues were treated with an antagonist of G-protein coupled estrogen receptor (GPER) (G-15; 10 nM), and bisphenol A (BPA), and its analogs (TBBPA, TCBPA; 10 nM) alone or in combination and analyzed using electron and light (stainings for collagen fibers, lipid droplet and autophagy markers) microscopes. In addition, mRNA and protein abundances and localization of molecules required for miRNA biogenesis and function (Drosha, Exportin 5; EXPO5, Dicer, and Argonaute 2; AGO2) were assessed together with calcium ion (Ca2+) and estradiol concentrations. Regardless of GPER blockade and/or treatment with BPA, TBBPA and TCBPA, there were no changes in Leydig cell morphology. Also, there were no changes in lipid droplet content and distribution but there were changes in lipid and autophagy protein abundance. In the interstitial tissue, there was an increase of collagen content, especially after treatment with BPA analogs and G-15 + BPA. Independent of the treatment, there was downregulation of EXPO5 and Dicer genes but the Drosha and AGO2 genes were markedly upregulated as a result of treatment with G-15 + BPA and TCBPA, respectively. There was always a lesser abundance of EXPO5 and AGO2 proteins regardless of treatment. There was markedly greater abundances of Drosha after G-15 + BPA treatment, and this also occurred for Dicer after treatment with G-15 + TCBPA. Immunolocalization of miRNA proteins indicated there was a cytoplasmic-nuclear pattern in control and treated cells. There was an increase of Ca2+ concentrations after treatment with G-15 and BPA analogs. Estradiol secretion decreased after antagonist and chemical treatments when these were administered alone, however, there was an increase in estradiol secretion after treatment with combinations of these compounds.

SOCS3 expression in SF1 cells regulates adrenal differentiation and exercise performance.

Many hormones/cytokines are secreted in response to exercise and cytokine signaling may play a pivotal role in the training adaptations. To investigate the importance of cytokine signaling during vertical ladder climbing, a resistance exercise model, we produced mice lacking SOCS3 protein exclusively in steroidogenic factor-1 (SF1) cells (SF1 Socs3 KO mice). SF1 expression is found in steroidogenic cells of the adrenal cortex and gonads, as well as in neurons of the ventromedial nucleus of the hypothalamus. Histological markers of the fetal adrenal zone (or X-zone in rodents) were still present in adult males and postpartum SF1 Socs3 KO females, suggesting a previously unrecognized effect of SOCS3 on the terminal differentiation of the adrenal gland. This change led to a distinct distribution of lipid droplets along the adrenal cortex. Under basal conditions, adult SF1 Socs3 KO mice exhibited similar adrenal weight, and plasma ACTH and corticosterone concentrations. Nonetheless, SF1 Socs3 KO mice exhibited a blunted ACTH-induced corticosterone secretion. The overall metabolic responses induced by resistance training remained unaffected in SF1 Socs3 KO mice, including changes in body adiposity, glucose tolerance and energy expenditure. However, training performance and glucose control during intense resistance exercise were impaired in SF1 Socs3 KO mice. Furthermore, a reduced counter-regulatory response to 2-deoxy-d-glucose was observed in mutant mice. These findings revealed a novel participation of SOCS3 regulating several endocrine and metabolic aspects. Therefore, cytokine signaling in SF1 cells exerts an important role to sustain training performance possibly by promoting the necessary metabolic adjustments during exercise.

Effect of sodium chloride on growth and lipid accumulation of Chlorella protothecoides CS-41

With basal medium, we studied the growth status, lipid droplet distribution, total lipid content of Chlorella protothecoides CS-41 treated with different concentrations of sodium chloride (0, 150, 300 and 600 mmol/L) by optical microscopy, electron microscopy, confocal laser focusing and Nile red staining. Results show that the addition of NaCl affected the growth of Chlorella protothecoides CS-41. With the increase of NaCl concentration, the growth rate of Chlorella was inhibited. Chlorella cell wall became thicker, and lipid droplets increased. At the early stage, the amount of lipid droplets in the 600 mmol/L NaCl culture was the highest, but at the late-log stage, the amount of lipid droplets increased with the increase of the biomass of culture in 150 and 300 mmol/L NaCl culture. At the stable stage, biomass (dry weight) in 300 mmol/L NaCl culture was 73.55% of that in the control, but the total lipid content was 2.22 times higher than that in the control. A certain concentration of sodium chloride treatment can significantly increase the lipid content of Chlorella protothecoides CS-41.

Pronounced limb and fibre type differences in subcellular lipid droplet content and distribution in elite skiers before and after exhaustive exercise.

Although lipid droplets in skeletal muscle are an important energy source during endurance exercise, our understanding of lipid metabolism in this context remains incomplete. Using transmission electron microscopy, two distinct subcellular pools of lipid droplets can be observed in skeletal muscle - one beneath the sarcolemma and the other between myofibrils. At rest, well-trained leg muscles of cross-country skiers contain 4- to 6-fold more lipid droplets than equally well-trained arm muscles, with a 3-fold higher content in type 1 than in type 2 fibres. During exhaustive exercise, lipid droplets between the myofibrils but not those beneath the sarcolemma are utilised by both type 1 and 2 fibres. These findings provide insight into compartmentalisation of lipid metabolism within skeletal muscle fibres. Although the intramyocellular lipid pool is an important energy store during prolonged exercise, our knowledge concerning its metabolism is still incomplete. Here, quantitative electron microscopy was used to examine subcellular distribution of lipid droplets in type 1 and 2 fibres of the arm and leg muscles before and after 1h of exhaustive exercise. Intermyofibrillar lipid droplets accounted for 85-97% of the total volume fraction, while the subsarcolemmal pool made up 3-15%. Before exercise, the volume fractions of intermyofibrillar and subsarcolemmal lipid droplets were 4- to 6-fold higher in leg than in arm muscles (P<0.001). Furthermore, the volume fraction of intermyofibrillar lipid droplets was 3-fold higher in type 1 than in type 2 fibres (P<0.001), with no fibre type difference in the subsarcolemmal pool. Following exercise, intermyofibrillar lipid droplet volume fraction was 53% lower (P=0.0082) in both fibre types in arm, but not leg muscles. This reduction was positively associated with the corresponding volume fraction prior to exercise (R2 =0.84, P<0.0001). No exercise-induced change in the subsarcolemmal pool could be detected. These findings indicate clear differences in the subcellular distribution of lipid droplets in the type 1 and 2 fibres of well-trained arm and leg muscles, as well as preferential utilisation of the intermyofibrillar pool during prolonged exhaustive exercise. Apparently, the metabolism of lipid droplets within a muscle fibre is compartmentalised.

Myosin-1 inhibition by PClP affects membrane shape, cortical actin distribution and lipid droplet dynamics in early Zebrafish embryos.

Myosin-1 (Myo1) represents a mechanical link between the membrane and actin-cytoskeleton in animal cells. We have studied the effect of Myo1 inhibitor PClP in 1–8 cell Zebrafish embryos. Our results indicate a unique involvement of Myo1 in early development of Zebrafish embryos. Inhibition of Myo1 (by PClP) and Myo2 (by Blebbistatin) lead to arrest in cell division. While Myo1 isoforms appears to be important for both the formation and the maintenance of cleavage furrows, Myo2 is required only for the formation of furrows. We found that the blastodisc of the embryo, which contains a thick actin cortex (~13 μm), is loaded with cortical Myo1. Myo1 appears to be crucial for maintaining the blastodisc morphology and the actin cortex thickness. In addition to cell division and furrow formation, inhibition of Myo1 has a drastic effect on the dynamics and distribution of lipid droplets (LDs) in the blastodisc near the cleavage furrow. All these results above are effects of Myo1 inhibition exclusively; Myo2 inhibition by blebbistatin does not show such phenotypes. Therefore, our results demonstrate a potential role for Myo1 in the maintenance and formation of furrow, blastodisc morphology, cell-division and LD organization within the blastodisc during early embryogenesis.

Downsizing cumulus cell layers to improve cryotolerance of germinal vesicle-stage bovine oocytes

This study was undertaken to investigate whether complete removal or downsizing of the cumulus cell layers in germinal vesicle (GV)-stage bovine cumulus-oocyte complexes (COCs) can improve blastocyst development rate following Cryotop vitrification. Downsized COCs (196 μm in mean diameter) and denuded oocytes (141 μm in mean diameter) were prepared by vortex-mixing of full-sized COCs (330 μm in mean diameter) retrieved from abattoir-derived ovaries. Nuclear maturation rates, assessed by the first polar body extrusion, after vitrification and the subsequent 22-h IVM were comparable (61.9–62.9%). Approximately one-third (30.5–31.2%) of the matured oocytes derived from the downsized COCs could develop into high quality blastocysts after 6-h IVF and 8-d IVC, while 13.4 and 23.7% of the matured oocytes derived from denuded oocytes and full-size COCs reached to the blastocysts, respectively. Cytoplasmic lipid droplets of matured oocytes in vitrification group were more clustered with decreased number and increased size of the droplets, when compared to those in fresh control group. However, individual oocyte culture in well-of-the well system suggested that change of lipid droplet distribution in the matured oocytes had no adverse effect on their subsequent developmental competence up to the blastocyst stage. In conclusion, Cryotop vitrification of downsized GV-stage bovine COCs allowed blastocyst yields as high as >30%.

WITHDRAWN: Ultrastructure study of the stored lipid reserves in Gyrodactylus gasterostei Glüser, 1974 using confocal microscopy

This study examines the distribution and depletion of stored lipids in Gyrodactylus gasterostei Glaser, 1974 migrating off its three-spine stickleback host Gasterosteus aculeatus L., with the prospect that it might prove informative for interpreting the biology of other gyrodactylids species more generally. Non feeding life-cycle stages, such as the dispersal stages of parasites, are dependent for survival upon finite energy reserves gathered during feeding phases. Thus, those individuals with more limited reserves will die sooner and consequently have less time available to find a new host once detached. At this stage, the principal energy reserves in gyrodactylids are stored as large lipids droplets. Confocal laser scanning microscopy has been used to investigate the distribution of lipid droplets in Gyrodactylus, which have migrated off their fish host, testing the hypothesis that these droplets function as a proxy for the nutritional state. This study demonstrated that the lipid droplets were particularly associated with the gut and that there is a significant variability in the volume of stored lipid carried out by each individual. Transmission electron microscopy showed that gyrodactylids carry lipid droplets at all stages of their life cycle, including at release from the birth pore. It is likely that transferring worms require stored energy reserves to survive in the event of failure to establish contact with a new host. These reserves could allow the parasite to survive without a host for several days.

Fat Cell and Fatty Acid Turnover in Obesity.

The ratio of free fatty acid (FFA) turnover decreases significantly with the expansion of white adipose tissue. Adipose tissue and dietary saturated fatty acid levels significantly correlate with an increase in fat cell size and number. Inhibition of adipose triglyceride lipase leads to an accumulation of triglyceride, whereas inhibition of hormone-sensitive lipase leads to the accumulation of diacylglycerol. The G0/G1 switch gene 2 increases lipid content in adipocytes and promotes adipocyte hypertrophy through the restriction of triglyceride turnover. Excess triacylglycerols (TAGs), sterols and sterol esters are surrounded by the phospholipid monolayer surface and form lipid droplets. Following the release of lipid droplets from endoplasmic reticulum, cytoplasmic lipid droplets increase their volume either by local TAG synthesis or by homotypic fusion. The number and the size of lipid droplet distribution is correlated with obesity. Obesity-associated adipocyte death exhibits feature of necrosis-like programmed cell death. NOD-like receptors family pyrin domain containing 3 (NLRP3) inflammasome-dependent caspase-1 activation in hypertrophic adipocytes induces obese adipocyte death by pyroptosis. Actually adipocyte death may be a prerequisite for the transition from hypertrophic to hyperplastic obesity. Major transcriptional factors, CCAAT/enhancer-binding proteins beta and delta, play a central role in the subsequent induction of critical regulators, peroxisome-proliferator-activated receptor gamma, CCAAT/enhancer-binding protein alpha and sterol regulatory element-binding protein 1, in the transcriptional control of adipogenesis in obesity.Collectively, in this chapter the concept of adipose tissue remodeling in response to adipocyte death or adipogenesis, and the complexity of lipid droplet interactions with the other cellular organelles are reviewed. Furthermore, in addition to lipid droplet growth, the functional link between the adipocyte-specific lipid droplet-associated protein and fatty acid turn-over is also debated.

VMP1 Establishes ER-Microdomains that Regulate Membrane Contact Sites and Autophagy.

The endoplasmic reticulum (ER) regulates organelle dynamics through the formation of membrane contact sites (MCS). Here we describe that VMP1, a multispanning ER-resident protein involved in autophagy, is enriched in ER micro-domains that are in close proximity to diverse organelles in HeLa and Cos-7 cells. These VMP1 puncta are highly dynamic, moving in concert with lipid droplets, mitochondria and endosomes. Some of these micro-domains are associated with ER sliding events and also with fission events of mitochondria and endosomes. VMP1-depleted cells display increased ER-mitochondria MCS and altered mitochondria morphology demonstrating a role in the regulation of MCS. Additional defects in ER structure and lipid droplets size and distribution are consistent with a more general function of VMP1 in membrane remodeling and organelle function. We hypothesize that in autophagy VMP1 is required for the correct morphogenesis of the omegasome by regulating MCS at the site of autophagosome formation.