Short‐Term Effects of Dietary Fatty Acids on Muscle Lipid Composition and Serum Acylcarnitine Profile in Human Subjects

In cultured cells, palmitic acid (PA) and oleic acid (OA) confer distinct metabolic effects, yet, unclear, is whether changes in dietary fat intake impact cellular fatty acid (FA) composition. We hypothesized that short‐term increases in dietary PA or OA would result in corresponding changes in the FA composition of skeletal muscle diacylglycerol (DAG) and triacylglycerol (TAG) and/or the specific FA selected for β‐oxidation. Healthy males (N = 12) and females (N = 12) ingested a low‐PA diet for 7 days. After fasting measurements of the serum acylcarnitine (AC) profile, subjects were randomized to either high‐PA (HI PA) or low‐PA/high‐OA (HI OA) diets. After 7 days, the fasting AC measurement was repeated and a muscle/fat biopsy obtained. FA composition of intramyocellular DAG and TAG and serum AC was measured. HI PA increased, whereas HI OA decreased, serum concentration of 16:0 AC (P < 0.001). HI OA increased 18:1 AC (P = 0.005). HI PA was associated with a higher PA/OA ratio in muscle DAG and TAG (DAG: 1.03 ± 0.24 vs. 0.46 ± 0.08, P = 0.04; TAG: 0.63 ± 0.07 vs. 0.41 ± 0.03, P = 0.01). The PA concentration in the adipose tissue DAG (µg/mg adipose tissue) was 0.17 ± 0.02 in those receiving the HI PA diet (n = 6), compared to 0.11 ± 0.02 in the HI oa group (n = 4) (P = 0.067). The relative PA concentration in muscle DAG and TAG and the serum palmitoylcarnitine concentration was higher in those fed the high‐PA diet.     

Improvement in blood lipid levels by dietary sn-1,3-diacylglycerol in young women with variants of lipid transporters 54T-FABP2 and -493g-MTP

In a double-blind parallel-group study, serum lipids and visceral fat/total fat ratio in young women (n=49) with variants of lipid transporters, i.e., fatty acid binding protein 2 (FABP2) and microsomal triglyceride transfer protein (MTP), were analyzed by substituting dietary triacylglycerol (TAG) with sn-1,3-diacylglycerol (DAG). All subjects, including some with the hyperlipidemia-prone genotypes Ala54Thr of FABP2 and c-493g of MTP, received DAG or TAG (20 g/day) for 8 weeks. Reductions of serum lipids from weeks 4 to 8 in FABP2-Ala54Thr heterozygotes and MTP -493g homozygotes were significantly different between the DAG and TAG groups (p<0.05, p<0.01). Visceral fat/total fat (%), as determined by computed tomography (CT), was lower in FABP2-Ala54Thr heterozygotes (p<0.05) of the DAG group. The apoCII/CIII ratio was higher in the DAG group than in the TAG group (p<0.01). Other variants of lipid metabolism, including peroxisome proliferator activated receptors (PPARs) alpha and gamma and SREBP cleavage-activating protein (SCAP), were only slightly affected by dietary DAG. CONCLUSION: improvement of serum lipid profiles and visceral fat/total fat ratio (CT) was potentiated by DAG intake in subjects with hyperlipidemia-prone genotypes (Ala54Thr heterozygotes of FABP2 and -493g homozygotes of MTP).     

The Short‐Term Effect of Diacylglycerol Oil Consumption on Total and Dietary Fat Utilization in Overweight Women

Diacylglycerol (DAG) is a natural component of edible oils with metabolic characteristics distinct from those of triacylglycerol (TAG). Consumption of DAG oil (containing >80% DAG) induces greater fat oxidation than consumption of TAG oil. We compared the effects of 4 days of DAG oil consumption with those of TAG oil consumption on total and dietary fat oxidation over 24 h in overweight women using a whole‐room respiratory chamber. Overweight (BMI (kg/m²) ≥25) females participated in this double‐blind, crossover‐controlled trial. The subjects consumed test diets containing either TAG or DAG oil as 15% of their total caloric intake (mean test oil intake was 33.0 ± 3.1 g/day) during each 4‐day treatment. Fat oxidation and energy expenditure were measured in a respiratory chamber on the 4th day of each treatment. Compared with TAG oil, DAG oil consumption significantly increased total fat oxidation and dietary fat oxidation in overweight subjects. Total energy expenditure (TEE) and carbohydrate (CHO) oxidation did not significantly differ between DAG oil and TAG oil consumption in overweight subjects. Compared with TAG oil, DAG oil consumption enhanced total fat oxidation and dietary fat oxidation in overweight subjects. The enhanced fat metabolism in overweight subjects that consumed DAG oil partly explains the greater loss of body weight and body fat related to DAG oil consumption in weight‐loss studies.     

Identification and characterization of DGA2, an acyltransferase of the DGAT1 acyl-CoA:diacylglycerol acyltransferase family in the oleaginous yeast Yarrowia lipolytica. New insights into the storage lipid metabolism of oleaginous yeasts

Triacylglycerols (TAG) and steryl esters (SE) are the principal storage lipids in all eukaryotic cells. In yeasts, these storage lipids accumulate within special organelles known as lipid bodies (LB). In the lipid accumulation-oriented metabolism of the oleaginous yeast Yarrowia lipolytica, storage lipids are mostly found in the form of TAG, and only small amounts of SE accumulate. We report here the identification of a new DAG acyltransferase gene, DGA2, homologous to the ARE genes of Saccharomyces cerevisiae. This gene encodes a member of the type 1 acyl-CoA:diacylglycerol acyltransferase family (DGAT1), which has not previously been identified in yeasts, but is commonly found in mammals and plants. Unlike the Are proteins in S. cerevisiae, Dga2p makes a major contribution to TAG synthesis via an acyl-CoA-dependent mechanism and is not involved in SE synthesis. This enzyme appears to affect the size and morphology of LB, suggesting a direct role of storage lipid proteins in LB formation. We report that the Are1p of Y. lipolytica was essential for sterol esterification, as deletion of the encoding gene (ARE1) completely abolished SE synthesis. Unlike its homologs in yeasts, YlARE1 has no DAG acyltransferase activity. We also reconsider the role and function of all four acyltransferase enzymes involved in the final step of neutral lipid synthesis in this oleaginous yeast.     

Variation of lipid classes among organs of the Northern krill Meganyctiphanes norvegica, with respect to reproduction.

Lipid content and class in the Northern krill Meganyctiphanes norvegica (digestive gland, stomach, gonad, fat body, abdomen) was investigated and correlated with sex and reproductive stage. Ready to spawn females, have high lipid content in ovaries, while in males and spent females, the major site of lipid deposits was the digestive gland, followed by the fat body. These differences among spawning and spent females are indicative of strong interactions between the ovary and digestive gland and the ovary and fat body during vitellogenesis. Triacylglycerols (TAG) were the major neutral lipid class with high levels in the digestive gland. The major phospholipid was phosphatidylcholine (PC) particularly in the muscular tissue of the abdomen. Phosphatidyl-ethanolamine (PE) and -serine-inositol (PS-PI), were present at intermediate levels. Reproductive males were depleted in TAG and diacylglycerols (DAG) in the digestive gland, gonad and fat body, and had 4 times lower cholesterol in the gonad than ready to spawn females. Furthermore, ready to spawn females had in the ovary higher amounts of TAG, DAG and phospholipids (PC, PE, PS-PI) than spent females. Linear relationships between lipid content and main lipid class (TAG, PC, PE, PS-PI) in different fractions of males and ready to spawn females showed that: (1). TAG was stored for both sexes in all cephalothorax fractions with highest values in the digestive gland and ovary fluid; (2). PC was accumulated for both sexes in the fat body and the gonad with a higher slope for females, with the highest values in the ovary fluid and in the abdomen of males and that (3). PS-PI was stored only in the ovary and abdomen of mature females. These results are discussed in terms of the strategy developed by Meganyctiphanes norvegica to allocate lipids to the next generation for optimised embryogenesis.     

Energy allocation strategy of skipjack tuna Katsuwonus pelamis during their reproductive cycle

The lipid composition of somatic and reproductive tissues was determined for female skipjack tuna Katsuwonus pelamis caught in the western Indian Ocean between latitude 10° N and 20° S and longitude 40° and 70° E. The highest total lipid (TL) contents were in the liver and gonads, with white muscle levels approximately three‐fold lower. Three lipid classes dominated: triacylglycerols (TAG), sterol esters and wax esters (SE‐WE) and phospholipids (PL). Collectively, these accounted for between 70 and 80% of TLs. Changes in lipid concentrations were evaluated over the maturation cycle. Immature fish had the lowest gonad and liver TL levels; concentrations of TL, TAG, SE‐WE and PL accumulated from immature to mature (spawning‐capable) phase, reflecting sustained vitellogenic activity of the liver and a transfer of lipids to developing oocytes from the onset of vitellogenesis. Gonado‐somatic and hepato‐somatic indices were positively correlated with each other and positively related to TL in the gonads and liver. Fulton's condition index and lipid concentrations in muscle did not vary significantly over the maturation cycle; fat content in the main storage tissues was undepleted as the ovary developed. Hence, K. pelamis apparently supports reproduction directly from food intake over the breeding season. In the gonads, reserve lipids (SE‐WE and TAG) and sterols were related to batch fecundity but this was not the case for somatic and hepatic tissues. These results suggest that K. pelamis utilizes an income breeding strategy.     

Challenging of AS160/TBC1D4 Alters Intracellular Lipid milieu in L6 Myotubes Incubated With Palmitate

The Akt substrate of 160 kDa (AS160) is a key regulator of GLUT4 translocation from intracellular depots to the plasma membrane in myocytes. Likely, AS160 also controls LCFAs transport, which requires relocation of fatty acid transporters. The aim of the present study was to determine the impact of AS160 knockdown on lipid milieu in L6 myotubes incubated with palmitate (PA). Therefore, we compared two different settings, namely: 1) AS160 knockdown prior to palmitate incubation (pre‐PA‐silencing, AS160^?/PA); 2) palmitate incubation with subsequent AS160 knockdown (post‐PA‐silencing, PA/AS160^?). The efficiency of AS160 silencing was checked at mRNA and protein levels. The expression and localization of FA transporters were determined using Western Blot and immunofluorescence analyses. Intracellular lipid content (FFA, DAG, TAG, and PL) and FA composition were estimated by GLC, whereas basal palmitate uptake was analyzed by means of scintigraphy. Both groups with silenced AS160 were characterized by a greater expression of FA transporters (FAT/CD36, FATP‐1, 4) which had contributed to an increased FA cellular influx. Accordingly, we observed that post‐PA‐silencing of AS160 resulted in a marked decrement in DAG, TAG, and PL contents, but increased FFA content (PA/AS160^? vs. PA). The opposite effect was observed in the group with pre‐PA‐silencing of AS160 in which AS160 knockdown did not affect the lipid pools (AS160^?/PA vs. PA). Our results indicate that post‐PA‐silencing of AS160 has a capacity to decrease the lipotoxic effect(s) of PA by decreasing the content of lipids (DAG and PL) that promote insulin resistance in myotubes. J. Cell. Physiol. 232: 2373–2386, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.

     

Mitochondrial lipids in Bufo arenarum full-grown oocytes

Both the content and composition of polar and neutral lipids from the mitochondrial fraction of ovarian full-grown Bufo arenarum oocytes were analysed in the present study. Triacylglycerols (TAG) represent 33% of the total lipids, followed by phosphatidylcholine (PC), free fatty acids (FFA) and phosphatidylethanolamine (PE). Diphosphatidylglycerol (DPG) or cardiolipin, a specific component of the inner mitochondrial membrane, represents about 4% of the total lipid content. Palmitic (16:0) and arachidonic (20:4n6) acids are the most abundant fatty acids in PC and PE, respectively. DPG is enriched in fatty acids with carbon chain lengths of 18, the principal component being linoleic acid. In phosphatidylinositol (PI), 20:4n6 and stearic acid (18:0) represent about 72 mol% of the total acyl group level. The main fatty acids in TAG are linoleic (18:2), oleic (18:1), and palmitic acids. The fatty acid composition of FFA and diacylglycerols (DAG) is similar, 16:0 being the most abundant acyl group. PE is the most unsaturated lipid and sphingomyelin (SM) has the lowest unsaturation index.     

The Effects of Long‐ or Medium‐Chain Fat Diets on Glucose Tolerance and Myocellular Content of Lipid Intermediates in Rats

Accumulation of triacylglycerols (TAGs) and acylcarnitines in skeletal muscle upon high‐fat (HF) feeding is the resultant of fatty acid uptake and oxidation and is associated with insulin resistance. As medium‐chain fatty acids (MCFAs) are preferentially β‐oxidized over long‐chain fatty acids, we examined the effects of medium‐chain TAGs (MCTs) and long‐chain TAGs (LCTs) on muscle lipid storage and whole‐body glucose tolerance. Rats fed a low‐fat (LF), HFLCT, or an isocaloric HFMCT diet displayed a similar body weight gain over 8 weeks of treatment. Only HFLCT increased myocellular TAG (42.3 ± 4.9, 71.9 ± 6.7, and 48.5 ± 6.5 µmol/g for LF, HFLCT, and HFMCT, respectively, P < 0.05) and long‐chain acylcarnitine content (P < 0.05). Neither HF diet increased myocellular diacylglycerol (DAG) content. Intraperitoneal (IP) glucose tolerance tests (1.5 g/kg) revealed a significantly decreased glucose tolerance in the HFMCT compared to the HFLCT‐fed rats (802 ± 40, 772 ± 18, and 886 ± 18 area under the curve for LF, HFLCT, and HFMCT, respectively, P < 0.05). Finally, no differences in myocellular insulin signaling after bolus insulin injection (10 U/kg) were observed between LF, HFLCT, or HFMCT‐fed rats. These results show that accumulation of TAGs and acylcarnitines in skeletal muscle in the absence of body weight gain do not impede myocellular insulin signaling or whole‐body glucose intolerance.     

Metabolic rate in diapause and nondiapause brown locust eggs correlated with embryonic development

Insects use dormancy to survive adverse conditions. Brown locust Locustana pardalina (Walk.) eggs offer a convenient model to study dormancy (diapause and quiescence), which contributes to their survival under arid conditions. The metabolic rates of developing nondiapause, diapause and quiescent eggs are compared in the present study using closed‐system respirometry. The embryo becomes committed to continue development and hatch or to enter diapause 6 days after the eggs are placed on moist soil. The metabolic rate of nondiapause eggs increases exponentially until hatching, whereas that of diapause eggs is low and stable. The metabolic rate of diapause laboratory eggs (1.9 ± 0.6 µL CO₂ mg^?1 h^?1) is significantly higher than that of field eggs (0.5 ± 0.3 µL CO₂ mg^?1 h^?1), although the ranges of metabolic rate overlap and the embryos are all in late anatrepsis. The metabolic rate of quiescent eggs is similar to that of diapause eggs but decreases with time. Low metabolic rates during arrested development allow eggs to persist over long periods before hatching.     

Lipid uptake by insect oocytes

Approximately 30-40% of the dry weight of an insect egg consists of lipid, mostly triacylglycerol (TAG). Although this lipid is essential for the energy needed by the developing embryo, little is known about the mechanism that leads to the accumulation of TAG in the insect egg. Insect oocytes can readily synthesize TAG from free fatty acids (FFAs) and glycerol, however, de novo synthesis of FAs by the oocyte is marginal. Hence, FAs have to be imported from the fat body or the diet. Insect hemolymph contains two lipoproteins that transport lipids, lipophorin and vitellogenin. Both are taken up via endocytosis by the oocyte, however, this provides only about 10% of the egg's lipid reserves. The rest is unloaded from circulating lipoprotein particles at the oocyte surface in the form of diacylglycerol (DAG), the major lipid transport form in insects, or as FFA. The mechanism of lipoprotein unloading at the oocyte surface is currently unclear. Possible roles of the lipid transfer particle (LTP), FA transporters, and lipoprotein lipase activity are discussed.     

Anopheles gambiae lipophorin: characterization and role in lipid transport to developing oocyte

Lipid transport in arthropods is achieved by highly specialized lipoproteins, which resemble those described in vertebrate blood. Here, we describe purification and characterization of the lipid-apolipoprotein complex, lipophorin (Lp), in the malaria vector mosquito Anopheles gambiae. We also describe the Lp-mediated lipid transfer to developing eggs and the distribution of the imported lipid in developing embryos. The density of the Lp complex was 1.135 g/ml with an apparent molecular weight of 630 kDa. It is composed of two major polypeptides, apoLp I (260 kDa) and apoLp II (74 kDa) and composed of 50% protein, 48% lipid and 2% carbohydrate (w/w). Hydrocarbon, cholesterol, phosphatidyl choline, phosphatidyl ethanolamine, cholesteryl ester and diacylglyceride were the major Lp-associated lipids. Using fluorescently tagged lipids, we observed patterns that suggest that in live developing oocytes, the Lp was taken up by a receptor-mediated endocytic process. Such process was blocked at low temperature and in the presence of excess unlabeled Lp, but not by bovine serum albumin. Imported Lp was segregated in the spherical yolk bodies (mean size 1.8 microm) and distributed evenly in the cortex of the oocyte. In embryonic larvae, before hatching, a portion of the fatty acid in vesicles was found evenly distributed along the body, whereas portion of phospholipids was accumulated in the intestine.     

Dynamics of cortisol and the development of the glucocorticoid function in the early ontogenesis of Atlantic salmon Salmo salar

The dynamics of cortisol were studied during the embryonic and early postembryonic development of Atlantic salmon Salmo salar (in brood obtained from the same pair of spawners, but with differing hatching dates). The initial level of maternal cortisol in the eggs after fertilization comprised of 25.6 ± 2.2 ng/g, and decreased continuously in the development process to 1.1 ± 0.2 ng/g by the 95th day after fertilization (t1) or the 18th day after hatching (t2). The first increase in the concentration of endogenous cortisol to 6.1 ± 1.2 ng/g was recorded at t2 = 24 days. Then the level of cortisol increased constantly and reached 32.8 ng/g by t1 = 149 (t2 = 72). A comparison of the hormone dynamics in two offspring groups from the same pare of spawners, differing in the longevity of embryogenesis, demonstrated that the level of cortisol increased after hatching more intensely in fishes with shorter embryogenesis. An injection of DL-aminoglutethimide, a compound preventing the synthesis of cortisol, significantly decreased the concentration of cortisol for the first time at the day of t1 = 101 (t2 = 24), i.e. at the first moment of the increase of endogenous cortisol. The effect from the adrenaline injection as a stress hormone was observed only at the day of t1 = 149 (t2 = 72), i.e., during the transition to the fry stages of development. Problems concerning when steroidogenesis begins, of the formation of the hypothalamic-hypophyseal-interrenal axis as a single hormone-competent ensemble, and of the development the stressed response in salmon juveniles are discussed.     

Receptor-mediated endocytosis of lipid and lipophorin by the larval fat body, adult ovary and testis in the wax moth Galleria mellonella

Lipophorin (Lp) in the hemolymph of insects is known to selectively deliver lipids from sites of absorption or synthesis to sites of storage and utilization, such as the fat body, ovary and testis; however, no study regarding this has been reported in Galleria mellonella. In the present study, we examined the process by which Lp is taken up into the larval fat body, adult ovary and adult testis, and the transfer of lipid by Lp to these tissues in Galleria mellonella. To investigate the involvement of a receptor in Lp endocytosis, the larval fat body, adult ovary and adult testis were incubated for 1 h at room temperature with fluorescein isothiocyanate (FITC)‐Lp, FITC‐Lp plus unlabeled Lp, and FITC‐Lp plus suramin, a receptor endocytic inhibitor. The amounts of FITC‐Lp in the three tissues were significantly decreased in the presence of unlabeled Lp and suramin, indicating that endocytosis of Lp by the tissues is mediated by a receptor. To examine the transfer of lipid by Lp, the tissues were incubated for 1 h at room temperature with 1,1′‐dilinoleyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI)‐Lp, DiI‐Lp plus unlabeled Lp, and DiI‐Lp plus suramin. The transfer of lipid by Lp was inhibited in the presence of unlabeled Lp and suramin, which is consistent with a receptor‐mediated process. Our results show that the transfer process of lipid by Lp and uptake of Lp itself is by receptor‐mediated endocytosis.     

Respiration rates of subitaneous eggs from a marine calanoid copepod: monitored by nanorespirometry

The oxygen consumption rate during embryogenesis of Acartia tonsa subitaneous eggs were measured at different temperatures (10, 15, 17, 21, 24 and 28°C) with nanorespirometry. The oxygen consumption was constant during the embryogenesis but increased rapidly at hatching time. The mean ± SD oxygen consumption rate increased exponentially with temperature and ranged from 0.09 ± 0.04 (10°C) to 0.54 ± 0.09 nmol O₂ egg⁻¹ h⁻¹ (28°C). The mean ± SD Q₁₀-value was 2.51 ± 0.15. Calculations of energy consumption during embryogenesis ranged from 1.86 to 18.28 mJ depending on temperature and development time. We conclude that the effect of temperature on oxygen consumption rate was far less important than the prolonged development time when calculating the energy consumed during embryogenesis.     

Overexpression of a phosphatidic acid phosphatase type 2 leads to an increase in triacylglycerol production in oleaginous <Emphasis Type="Italic">Rhodococcus</Emphasis> strains

Oleaginous Rhodococcus strains are able to accumulate large amounts of triacylglycerol (TAG). Phosphatidic acid phosphatase (PAP) enzyme catalyzes the dephosphorylation of phosphatidic acid (PA) to yield diacylglycerol (DAG), a key precursor for TAG biosynthesis. Studies to establish its role in lipid metabolism have been mainly focused in eukaryotes but not in bacteria. In this work, we identified and characterized a putative PAP type 2 (PAP2) encoded by the ro00075 gene in Rhodococcus jostii RHA1. Heterologous expression of ro00075 in Escherichia coli resulted in a fourfold increase in PAP activity and twofold in DAG content. The conditional deletion of ro00075 in RHA1 led to a decrease in the content of DAG and TAG, whereas its overexpression in both RHA1 and Rhodococcus opacus PD630 promoted an increase up to 10 to 15 % by cellular dry weight in TAG content. On the other hand, expression of ro00075 in the non-oleaginous strain Rhodococcus fascians F7 promoted an increase in total fatty acid content up to 7 % at the expense of free fatty acid (FFA), DAG, and TAG fractions. Moreover, co-expression of ro00075/atf2 genes resulted in a fourfold increase in total fatty acid content by a further increase of the FFA and TAG fractions. The results of this study suggest that ro00075 encodes for a PAP2 enzyme actively involved in TAG biosynthesis. Overexpression of this gene, as single one or with an atf gene, provides an alternative approach to increase the biosynthesis and accumulation of bacterial oils as a potential source of raw material for biofuel production.     

EFFECT OF STARVATION ON LIPOPHORIN DENSITY IN FIFTH INSTAR LARVAL Manduca sexta

Lipophorin (Lp) is a major insect lipoprotein and is responsible for lipid transport between organs. In this study, the effect of starvation on Lp properties was analyzed in larval Manduca sexta during the fifth instar. Lp hemolymph concentrations in larvae at days 1 and 2 were around 2–3 mg/ml and at day 3 it increased to 8 mg/ml. When larvae were starved for 24 h, they did not grow, but their body mass and hemolymph volume did not decrease significantly. Differences in Lp densities were observed. In fed larvae, from days 1 to 4, two major Lp populations were found with densities of 1.124 ± 0.002 (high density Lp‐larval₁, HDLp‐L₁) and 1.141 ± 0.002 g/ml (HDLp‐L₂). When larvae were starved for 24 h, only one Lp population was present, with density 1.114 ± 0.001 g/ml (HDLp‐L_s). When larvae were abdominally ligated at day 1 or 2 of fifth instar, only HDLp‐L_s was found after 24 h, indicating that the formation of this HDLp population was not dependent on any factor released by head. On the other hand, larvae that were ligated at day 3 showed the same Lp populations as the fed ones. In 24‐h starved larvae, lipid load in Lp was higher as compared to the fed controls. In 24‐h ligated larvae Lp lipid content increased when ligation was performed on day 1 or 2, but not on day 3. So, different responses to starvation can be observed depending on the developmental phase of the same larval instar.     

The storage of nutritional resources during vitellogenesis of Panstrongylus megistus (Hemiptera: Reduviidae): the pathways of lipophorin in lipid delivery to developing oocytes

In this work, we have analyzed the pathways by which lipophorin (Lp) delivers its lipid cargo to developing oocytes of Panstrongylus megistus, a hematophagous vector of Chagas’ disease. Lp, vitellin, total lipids and proteins were measured in ovarian tissues at different stages of the reproductive cycle. Localization of Lp in developing oocytes, mainly at their cortical area, was demonstrated by immunofluorescence assays using an anti-Lp antibody labeled with FITC. In vivo approaches injecting fluorescently labeled Lp to follow the course of the entire particle (Lp-DiI or Lp-Oregon Green) or its lipid cargo (Lp-Bodipy-FA) were monitored by laser scanning confocal microscopy. Significant increases in the amounts of lipids, proteins and vitellin were observed in ovarian tissue with the progress of vitellogenesis. Unexpectedly, an increase in the amount of Lp was also observed. The experiments in vivo demonstrated that the uptake of fluorescent Lp labeled on its protein or lipid moiety by developing oocytes occurred very fast, being impaired at low temperatures. The co-injection of fluorescent Lp and vitellogenin (Vg) showed that both particles co-localized inside yolk bodies, confirming the endocytic pathway for Lp. When the fate of lipids transferred to oocytes was evaluated in vitellogenic females by co-injecting Lp-Bodipy-FA and Lp-DiI, the signal for Bodipy-FA was found in both lipid droplets and yolk bodies. In contrast, in injected females kept at 4 °C the fluorescence was reduced, being observed exclusively in lipid droplets, implying that lipid transfer to the oocyte was diminished but not abolished. Taken together, the results demonstrate that in the hematophagous P. megistus, the storage of lipid resources by developing oocytes occurs by the convergence of different pathways by which Lp maximizes the delivery of its lipid cargo. In addition, it was also shown that, to some extent, lipids stored in the oocyte lipid droplets can also originate from endocytosed Vg. The relevance of these events in the context of the physiology of reproduction in P. megistus is discussed.     

高效薄层色谱测定湛江等鞭金藻培养过程脂质变化

【目的】研究湛江等鞭金藻(Isochrysis zhangjiangensis)由氮元素丰富至限制培养过程中脂质成分的变化。【方法】利用高效薄层色谱(HPTLC)分离分析微藻中脂质。【结果】随着培养基中营养物质的消耗,细胞逐渐处于胁迫状态,在这种状态下,细胞大量积累贮存脂质—甘油三脂(TAG),组成生物膜系统的单半乳糖甘油二脂(MGDG)、硫代异鼠李糖甘油二脂(SQDG)、磷脂酰甘油(PG)和磷脂酰胆碱(PC)含量降低,而游离脂肪酸(FFA)、甘油二脂(DAG)、双半乳糖甘油二脂(DGDG)、磷脂酰肌醇(PI)和磷脂酰乙醇胺(PE)则相对稳定。【结论】HPTLC可作为一种简便、可靠的微藻中脂质分离分析方法,为研究微藻油脂代谢途径以及甘油三脂(TAG)的调控积累提供有效手段。