Adipose tissue is a key regulator of energy metabolism and glucose homeostasis by promoting triglyceride storage and breakdown in various physiological states. Obesity, however, alters adipose tissue metabolism, inducing chronic inflammation, followed by excessive lipolysis. This results in higher systemic free fatty acid (FFA) levels, leading to desensitization of insulin signaling and ultimately to insulin resistance. Although the link between obesity and progression of insulin resistance and type 2 diabetes mellitus (T2DM) remains unclear, tumor necrosis factor-alpha (TNF-alpha) has been proposed to be a key player in promoting obesity-related development of T2DM through chronic inflammation of adipose tissue. TNF-alpha has direct and indirect mechanisms by which it elicits insulin resistance in adipocytes. TNF-alpha attenuates insulin signaling by directly inhibiting insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1). Indirectly, TNF-alpha activates signaling pathways to increase lipolysis and FFA release into circulation, leading to insulin resistance. Lipid droplet-associated fat specific protein 27 (FSP27) protects adipocytes from lipolysis by regulating the lipolytic capacity as well as transcription of adipose triglyceride lipase (ATGL). It has been observed that TNF-alpha promotes lipolysis by reducing the expression of FSP27 in murine adipocytes. The effect of TNF-alpha on lipolysis human adipocytes has also been studied; yet its effect on promoting insulin resistance in human adipocytes still remains elusive. In the present study, we examined the effect of FSP27 on TNF-alpha induced lipolysis and insulin resistance in human adipocytes. TNF-alpha enhanced lipolysis in cultured human adipocytes. In addition, TNF-alpha reduced the expression of endogenous FSP7 and the phosphorylation of AKT, inhibiting the activation of insulin signaling pathway in cultured human adipocytes. FSP27 overexpression, however, attenuated TNF-alpha induced lipolysis and restored activation of insulin signaling through phosphorylation of AKT in cultured human adipocytes. Taken together, these data suggest that FSP27 has a protective effect against TNF-alpha induced lipolysis and insulin resistance through regulating lipolysis and insulin signaling in human adipocytes.

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Triacylglycerol (TAG) is the main storage lipid in plants, found both in seed and non-seed tissues (e.g. root, leaves, mesocarp etc.). Plants use TAGs as a carbon and energy source during seed germination while humans use plant lipids for biofuel production, industrial feedstocks and nutrition. It is expected that by 2030 the demand for biodiesel will be doubled. To meet this demand it is important to understand the regulation of rate limiting reactions involved in TAG accumulation in nonseed tissues because of their higher biomass relative to the seed tissues. In this study, avocado (Persea americana) is used as a model organism because it is a basal angiosperm and can store up to 70% oil content in the form of TAG in its mesocarp, a nonseed tissue. Typically, the last acylation of diacylglycerol (DAG) to form TAG in seed tissues is catalyzed by diacylglycerol acyltransferases (DGAT) and/or phospholipid:diacylglycerol acyltransferases (PDAT). Based on the transcriptome of avocado, it is hypothesized that both DGAT and PDAT are responsible for catalyzing the terminal step of TAG biosynthesis in mesocarp of avocado. Fulllength coding sequences for DGAT1 and PDAT1 were identified based on the avocado transcriptome data and expressed in TAG-deficient yeast strain (SCY-1998) for complementation. Total lipid extracts from complemented yeast lines will be analyzed for presence of TAG. Furthermore, the enzyme activity and substrate specificity for PaDGAT1 and PaPDAT1 will be determined from microsomal preparations of avocado and eukaryotic expression systems containing the avocado transgenes. This study is expected to identify the enzymes responsible for the terminal acylation step in TAG synthesis in avocado, thereby contributing to the basic understanding of TAG accumulation in nonseed tissues.

No description available.

Improving crop yield by generating stress tolerant plants is the enduring objective of this research. A small class of bioactive fatty acid derivatives, N-acylethanolamines (NAEs), including anandamide (NAE 20:4), an endocannabinoid receptor ligand, affects a wide range of physiological and behavioral functions in animals. In plants, NAEs to the exclusion of anandamide are found to be ubiquitous and abundant in seed tissues and are shown to be involved in mediating abscisic acid (ABA) -dependent or -independent stress responses. Early land plants such as Physcomitrella patens (moss) have been shown to tolerate abiotic stresses. We hypothesized that NAEs are involved in mediating stress responses in moss. Gas chromatography-mass spectrometry was employed in NAE detection and quantification in moss. Selective lipidomic approach revealed novel NAE metabolites. The endocannabinoid receptor ligand anandamide and its precursor molecules were detected and quantified. Exogenous treatment of NAE 12:0, NAE 20:4 and ABA showed a growth inhibitory effect for all three metabolites. NAE 20:4 was more potent than NAE 12:0 to degrees similar to the plant hormone ABA. In silico analyses of NAE catabolizing enzyme fatty acid amide hydrolase from Arabidopsis showed eight putative FAAH candidates in this moss. Candidates showed high similarities with plants as well as animal FAAH proteins. Primers specific to NAE pathway genes have been designed for expression analysis. Our recent identification of the ligand NAE 20:4 in this moss, provides us with a unique opportunity to address if 1) early land plants, such as mosses, retained the endocannabinoid signaling mechanism that is akin to animals but not to plants and 2) if such distinctive NAE profile and mechanism by which it may function in moss plant is responsible, in part, for their natural ability to resist high temperatures, dehydration, osmotic and salt stresses. Insights into unique lipids composition and signaling pathways that mosses acquire naturally, during their successful transition from water to land, may lead to development of tools necessary to enhance abiotic stress tolerance in vegetative tissues of higher plants and thus contribute to improvement of crop productivity.

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Technology Consultants: PharmDr. Ondřej Holas, Ph.D. Student: Simona Žemličková Title of thesis: Hybrid polymeric-lipid nanoparticles as drug carriers The work is focused on hybrid polymer-lipid nanoparticles, their advantages obtained from polymer and lipid part, purpose of surface modification, basic properties of nanoparticles, methods of preparation, modification of preparation conditions and use of nanoparticles in medicine. The aim of the experimental part was to prepare nanoparticles composed of polyester and lipid by emulsion evaporation method and nanoprecipitation. Two types of linear polymer poly (lactic-co-glycolic acid) and phosphatidylcholine were used in various ratios. The surfactant used for stabilization was poloxamer Pluronic® F127 and the organic solvents were ethyl acetate and acetone. Curcumin served as a model active substance. The effect of lipid and surfactant on the size and zeta potential of nanoparticles was evaluated. Modification of preparation conditions, which included many process parameters, also influenced the monitored parameters. Encapsulation effectivity and drug loading were also tested. Dissolution tests were performed. It was found that size of nanoparticles...

Lipid droplets in plants (also known as oil bodies, lipid bodies, or oleosomes) are well characterized in seeds, and oleosins, the major proteins associated with their surface, were shown to be important for stabilizing lipid droplets during seed desiccation and rehydration. However, lipid droplets occur in essentially all plant cell types, many of which may not require oleosin-mediated stabilization. The proteins associated with the surface of nonseed lipid droplets, which are likely to influence the formation, stability, and turnover of this compartment, remain to be elucidated. Here, we have combined lipidomic, proteomic, and transcriptomic studies of avocado (Persea americana) mesocarp to identify two new lipid droplet-associated proteins, which we named LDAP1 and LDAP2. These proteins are highly similar to each other and also to the small rubber particle proteins that accumulate in rubber-producing plants. An Arabidopsis (Arabidopsis thaliana) homolog to LDAP1 and LDAP2, At3g05500, was localized to the surface of lipid droplets after transient expression in tobacco (Nicotiana tabacum) cells that were induced to accumulate triacylglycerols. We propose that small rubber particle protein-like proteins are involved in the general process of binding and perhaps the stabilization of lipid-rich particles in the cytosol of plant cells and that the avocado and Arabidopsis protein members reveal a new aspect of the cellular machinery that is involved in the packaging of triacylglycerols in plant tissues.

Purpose: To investigate the influence of intensity versus total energy expenditure on neutrophilia and blood oxidative stress to acute exercise. Methods: Nine males (18-30 yr) completed one maximal (Max) and three submaximal exercise sessions: 1) 45 min at 10% above (LT+) lactate threshold (LT), 2) 45 min at 10% below (LT-) LT, and 3) 10% below LT until caloric expenditure equaled the 10%+ trial (LT-kcal). Blood was sampled before (PRE), immediately (POST), 1 h, and 2 h after exercise to measure neutrophils, myeloperoxidase, superoxide (O2-), neutrophil activation (O2-/neutrophils), ascorbic acid, uric acid, malondialdehyde, and lipid hydroperoxides. Results: Intensity-dependent neutrophilia occurred POST exercise with significant increases (P ≤ 0.05) after Max and LT+. A second neutrophilia wave occurred 2 h postexercise. Superoxide was elevated POST (Max) and 2 h post (Max and LT+). In contrast, O2-/neutrophils was increased at 2 h only (Max and LT +). These data indicate that immediately postexercise, total neutrophil number rather than activation best represents neutrophil-generated reactive species within blood. POST Max, ascorbic acid and uric acid were decreased indicating a blood oxidative stress occurred. Alternately, total energy expenditure was not related to any marker of neutrophilia or oxidative stress. Conclusion: Exercise intensity plays a major role in postexercise blood oxidative stress, whereas total exercise energy expenditure does not. Further, neutrophils recruited into circulation during exercise may impose a threshold dependent oxidative stress in blood plasma after exercise.

Advances in molecular medicine have produced a large amount of information about genes that translate to therapeutic molecules when expressed in living cells. There is an increasing interest in nonviral methods for gene delivery, to address all concerns on non-toxic, easy, and possibly efficient delivery systems. In this paper we introduced a new attractive approach for non-viral transferring of genetic materials on demand. By using lipofectin reagent (1:1 molar ratio of DOPE:DOTMA. DOPE: L-α-doleoyl posphatidylethanolamine; DOTMA: N-[1-(2,3-dideyloxy) propyl]-n,n,n-trimethylammonium chloride), the lipid/DNA complexes (lipoplexes) can be electrostatically adsorbed on the gold microelectrode surface. The resulting lipoplexes molecules can be subsequently removed from the surface by applying -1.0 V (vs. Ag/AgCl) in physiological phosphate buffer medium (pH 7.4). This electrochemically controlled-release process has been extensively examined by gel electrophoresis (GE), electrochemical quartz crystal microbalance (EQCM), infrared spectroscopy (IR), and square wave voltammetry (SWV) techniques. The lipoplex composition has been addressed for efficient gene delivery protocol, based on their different charge ratios. The results from different techniques coincided, as also verified by the repetitive control experiments. This in-vitro electrically - triggered release protocol for genetic material offers the current gene delivery arsenal a new, simple, and non-viral alternative.

Micro- and nanofabricated sample environments are useful tools for characterizing diffusion in confined aqueous environments. The steady-state diffusion and adsorption of unilamellar lipid vesicles in arrays of hydrophilic micropillars is investigated. Gradients in the coverage of fluorescently labeled, pillar-supported lipid films, formed from vesicle fusion, are determined from 3D z-stack images using confocal microscopy. The gradients are the result of preferential adsorption of vesicles near the tops of the pillars, which progressively deplete them from solution as they diffuse toward the base of the array. However, the increased propensity for vesicle adsorption near the pillar tops compared to the confined spaces between pillars results in the formation of confluent supported lipid bilayers at the pillar tops that resist the adsorption of additional vesicles while leaving the pillar surfaces below available for binding. This results in a reduction in the numbers of depleted vesicles compared to what one would anticipate based on diffusive fluxes. The resulting inhomogeneous spatial profiles of lipid structures on the pillars are the result of the system being maintained in a dissipative, nonequilibrium steady state during incubation of the pillar arrays in the vesicle solution, which is ultimately quenched by rinsing away the unbound, freely diffusing vesicles.