Defective in Cuticular Ridges (DCR) of Arabidopsis thaliana, a Gene Associated with Surface Cutin Formation, Encodes a Soluble Diacylglycerol Acyltransferase

A key step in the triacylglycerol (TAG) biosynthetic pathway is the final acylation of diacylglycerol (DAG) by DAG acyltransferase. In silico analysis has revealed that the DCR (defective in cuticular ridges) (At5g23940) gene has a typical HX4D acyltransferase motif at the N-terminal end and a lipid binding motif VX(2)GF at the middle of the sequence. To understand the biochemical function, the gene was overexpressed in Escherichia coli, and the purified recombinant protein was found to acylate DAG specifically in an acyl-CoA-dependent manner. Overexpression of At5g23940 in a Saccharomyces cerevisiae quadruple mutant deficient in DAG acyltransferases resulted in TAG accumulation. At5g23940 rescued the growth of this quadruple mutant in the oleate-containing medium, whereas empty vector control did not. Lipid particles were localized in the cytosol of At5g23940-transformed quadruple mutant cells, as observed by oil red O staining. There was an incorporation of 16-hydroxyhexadecanoic acid into TAG in At5g23940-transformed cells of quadruple mutant. Here we report a soluble acyl-CoA-dependent DAG acyltransferase from Arabidopsis thaliana. Taken together, these data suggest that a broad specific DAG acyltransferase may be involved in the cutin as well as in the TAG biosynthesis by supplying hydroxy fatty acid.

A Bifunctional Enzyme That Has Both Monoacylglycerol Acyltransferase and Acyl Hydrolase Activities

Monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol, the precursor of triacylglycerol biosynthesis and an important signaling molecule. Here, we describe the isolation and characterization of the peanut (Arachis hypogaea) MGAT gene. The soluble enzyme utilizes invariant histidine-62 and aspartate-67 residues of the acyltransferase motif for its MGAT activity. A sequence analysis revealed the presence of a hydrolase (GXSXG) motif, and enzyme assays revealed the presence of monoacylglycerol (MAG) and lysophosphatidylcholine (LPC) hydrolytic activities, indicating the bifunctional nature of the enzyme. The overexpression of the MGAT gene in yeast (Saccharomyces cerevisiae) caused an increase in triacylglycerol accumulation. Similar to the peanut MGAT, the Arabidopsis (Arabidopsis thaliana) homolog (At1g52760) also exhibited both acyltransferase and hydrolase activities. Interestingly, the yeast homolog lacks the conserved HX4D motif, and it is deficient in the acyltransferase function but exhibits MAG and LPC hydrolase activities. This study demonstrates the presence of a soluble MGAT/hydrolase in plants. The predicted three-dimensional homology modeling and substrate docking suggested the presence of two separate substrate (MAG and LPC)-binding sites in a single polypeptide. Our study describes a soluble bifunctional enzyme that has both MGAT and hydrolase functions.

Resveratrol: Anti-Obesity Mechanisms of Action

Resveratrol is a non-flavonoid polyphenol which belongs to the stilbenes group and is produced naturally in several plants in response to injury or fungal attack. Resveratrol has been recently reported as preventing obesity. The present review aims to compile the evidence concerning the potential mechanisms of action which underlie the anti-obesity effects of resveratrol, obtained either in cultured cells lines and animal models. Published studies demonstrate that resveratrol has an anti-adipogenic effect. A good consensus concerning the involvement of a down-regulation of C/EBPa and PPAR. in this effect has been reached. Also, in vitro studies have demonstrated that resveratrol can increase apoptosis in mature adipocytes. Furthermore, different metabolic pathways involved in triacylglycerol metabolism in white adipose tissue have been shown to be targets for resveratrol. Both the inhibition of de novo lipogenesis and adipose tissue fatty acid uptake mediated by lipoprotein lipase play a role in explaining the reduction in body fat which resveratrol induces. As far as lipolysis is concerned, although this compound per se seems to be unable to induce lipolysis, it increases lipid mobilization stimulated by beta-adrenergic agents. The increase in brown adipose tissue thermogenesis, and consequently the associated energy dissipation, can contribute to explaining the body-fat lowering effect of resveratrol. In addition to its effects on adipose tissue, resveratrol can also acts on other organs and tissues. Thus, it increases mitochondriogenesis and consequently fatty acid oxidation in skeletal muscle and liver. This effect can also contribute to the body-fat lowering effect of this molecule.

Rosuvastatina, doença hepática gordurosa não alcoólica e ativação das células estreladas hepáticas em camundongos

Introdução: A atual epidemia de obesidade tem chamado a atenção para a doença hepática gordurosa não alcoólica (DHGNA). Atualmente não existe um medicamento para o tratamento da esteatose hepática, embora as estatinas sejam muito prescritas para pacientes obesos, este medicamento destina-se ao tratamento da hipercolesterolemia. Este trabalho teve como objetivo investigar os efeitos da rosuvastatina em um modelo de obesidade induzida por dieta, como foco principal a DHGNA e os marcadores hepáticos da lipogênese e beta-oxidação e ativação de células estreladas hepáticas (CEHs) em camundongos. Métodos: Camundongos machos C57BL/6 receberam dieta padrão (SC, 10% de energia como lipídios) ou dieta rica em gorduras (HF, 50% de energia como lipídios) durante 12 semanas. Em seguida, 7 semanas de tratamento, foram feitas, formando os grupos: SC, SCR (SC + rosuvastatina), HF e HFR (HF + rosuvastatina). As análises bioquímicas e técnicas moleculares foram aplicadas para abordar os resultados plasmáticos e moleculares. Resultados: O grupo HF apresentou maiores valores de insulina, colesterol total, triglicerídeos e leptina que o grupo SC, todos os quais foram reduzidos significativamente após o tratamento com Rosuvastatina no grupo HFR. O grupo HF apresentou maior percentual de esteatose, assim como maior ativação das CEHs, enquanto que a rosuvastatina provocou uma redução de 21% na esteatose hepática e atenuou a ativação das CEHs no grupo HFR. Em concordância com os achados histológicos, as expressões de SREBP-1 e PPAR-gama foram aumentados nos animais HF e reduzido após o tratamento no grupo HFR. Por outro lado, a expressão reduzida de PPAR-alfa e CPT-1 foram encontrados nos animais HF, sendo tais parâmetros restaurados após o tratamento no grupo HFR. Conclusão: A rosuvastatina atenua significativamente a ativação das células estreladas na obesidade induzida por dieta, afetando o equilíbrio dos PPARs na lipotoxicidade. Diante desses achados a Rosuvastatina pode ser indicada como alternativa para auxiliar o tratamento da esteatose hepática.

二脂酰甘油酰基转移酶基因(DGAT1)的沉默对烟草油脂合成的影响

二脂酰甘油酰基转移酶 (DGAT; EC 2.3.1.20) 是催化三脂酰甘油（TAG）合成的最后也是最关键步骤的酶。TAG是真核细胞中最重要的能量存储形式。在植物中，TAG主要在种子、花粉和许多物种的果实中积累。然而，DGAT1基因的转录本也存在于植物的其它器官中，这些器官包括根、茎、叶、花瓣、花粉囊、未成熟的角果、幼苗以及正在发芽的种子等。迄今为止，许多针对DGAT1基因的研究都集中于DGAT1基因的表达在对种子油脂的积累以及对种子中TAG的脂肪酸组成所起的作用上。在本研究中，我们通过构建烟草DGAT1基因带有内含子的发卡RNA（hpRNA）结构，使之在转基因烟草植株中表达双链RNA（dsRNA），利用RNAi原理达到使烟草内源DGAT1基因沉默的目的。转基因沉默烟草植株的获得将会为更好地研究DGAT1基因的功能奠定基础。本实验不仅研究分析了DGAT1基因的抑制对烟草种子油脂积累的影响，还对表现出沉默性状的转基因植株Sil7的不同器官中TAG的含量以及DGAT1的转录水平等进行了研究分析。此外，通过对转基因烟草不同株系种子中的主要贮藏物质——油脂、蛋白质和糖的含量测定，初步揭示出在烟草种子中三者生物合成代谢之间存在的相关性。主要研究结果如下： 采用烟草DGAT1基因的第615～1293碱基之间679bp的片段构建了能表达发卡RNA（hpRNA）结构的表达载体，并转化烟草（Nicotiana tabacum）Wisconsin 38。Northern杂交分析发现,与野生型对照烟草（WT）相比，在沉默植株的花和发育状态种子中DGAT1基因的转录水平有很大降低，这表明该发卡结构能够高效率地引起烟草DGAT1基因的沉默。此外，在对Sil1至Sil12共12株转基因烟草进行油脂含量分析的结果表明，其中有8株表现出油脂降低的性状，转基因沉默效率达到67％。这表明：利用RNAi的方法可对目标基因进行特异降解来研究基因的功能，因此是一个在研究基因的表达功能上十分有效的方法，而且已成为植物基因工程的有力工具。 为了研究DGAT1基因的沉默对转基因植株不同器官的影响，本实验分析了转基因植株Sil7的不同器官中TAG的含量和脂肪酸组成，并采用RT-PCR方法对野生型对照和转基因植株中DGAT1基因的转录水平进行了比较分析。研究发现，转基因植株不同器官中DGAT1基因转录水平的降低与各器官中TAG含量的减少呈正相关。由此看来，植物中DGAT1的表达水平与植物的各个器官内TAG的含量之间存在着一定的对应关系。此外，在转基因植株Sil7不同器官中依然能够产生TAG，这说明或者DGAT1酶活性丧失而由其它的酶（如DGAT2和PDAT）参与TAG的合成，或者DGAT1酶活性只是部分地受到影响。本实验还对Sil7的根、茎、叶、花瓣和种子中TAG的脂肪酸组成进行了分析，结果发现，与烟草野生型对照相比，在Sil7的这些器官中，除种子中TAG的脂肪酸组成无明显变化外，其余器官中TAG的18:3/18:2脂肪酸比例均有明显升高。 对其中8株转基因烟草种子进行油脂含量分析发现，在转基因烟草中由于DGAT1基因的沉默引起种子中TAG含量的减少，从而引起了种子平均千粒重的下降。而在TAG含量和种子平均千粒重下降的同时，种子中其它贮藏物质－蛋白质和糖类的含量却增加了。该实验结果表明：在烟草种子中TAG的生物合成与蛋白质和糖类物质的合成之间存在着负的相关性。

Effect of dietary carbohydrate-to-lipid ratios on growth and feed utilization in Chinese longsnout catfish (Leiocassis longirostris Gunther)

An 8-week growth trial was carried out in a semi-recirculation system at 26 +/- 0.5 degrees C to investigate the optimal dietary carbohydrate-to-lipid (CHO:L) ratio for carnivorous Chinese longsnout catfish (Leiocassis longirostris Gunther). Triplicate tanks of fish were assigned to each of five isocaloric and isonitrogenous diets with different carbohydrate-to-lipid ratios (0.75, 1.48, 1.98, 2.99 and 5.07). The results showed that a higher specific growth rate (SGR) and feed rate (FR) were observed in the fish fed diet ratios of 1.98 CHO:L (P < 0.05). Overloading dietary carbohydrate (5.07 CHO:L ratio) caused skeletal malformations. Apparent digestibility of dry matter (ADC(d)) significantly increased with dietary CHO:L ratio (P < 0.05), while significantly higher apparent digestibility of protein (ADC(p)) and apparent digestibility of energy (ACD(e)) was observed only in the 1.98 CHO:L group (P < 0.05). Whole body contents of dry matter, lipid and energy significantly increased as the CHO:L ratio decreased (P < 0.05). The hepatosomatic index (HSI) was highest at 1.98 CHO:L ratio (P < 0.05). Highest dietary CHO:L ratio resulted in lower liver glycogen, liver lipid, plasma glucose and plasma triacylglycerol (P < 0.05), whereas there was no significant difference in plasma total cholesterol (P > 0.05). High dietary CHO:L ratio caused pathological changes in fish morphology and liver histology. Based on maximum growth, the optimal carbohydrate-to-lipid ratio was 1.98 for Chinese longsnout catfish.

The impact of whey protein isolate on energy balance regulation

Using C57BL/6J mice fed whey protein isolate (WPI) enriched high fat (HFD) or low-fat diets (LFD), this study tested the hypothesis that WPI directly impacts on adiposity by influencing lipid metabolism. WPI suppressed HFD-induced body fat and increased lean mass at 8 weeks of dietary challenge despite elevated plasma triacylglycerol (TAG) levels, suggesting reduced TAG storage. WPI reduced HFD-associated hypothalamic leptin and insulin receptor (IR) mRNA expression, and prevented HFD-associated reductions in adipose tissue IR and glucose transporter 4 expression. These effects were largely absent at 21 weeks of HFD feeding, however WPI increased lean mass and cause a trend towards decreased fat mass, with notable increased Lactobacillus and decreased Clostridium gut bacterial species. Increasing the protein to carbohydrate ratio enhanced the above effects, and shifted the gut microbiota composition away from the HFD group. Seven weeks of WPI intake with a LFD decreased insulin signalling gene expression in the adipose tissue in association with an increased fat accumulation. WPI reduced intestinal weight and length, suggesting a potential functional relationship between WPI, gastro-intestinal morphology and insulin related signalling in the adipose. Extending the study to 15 weeks, did not affect adipose fat weight, but decreased energy intake, weight gain and intestinal length. The functionality of protein sensing lysophosphatidic acid receptor 5 (LPA5) in 3T3-L1 pre-adipocytes was assessed. Over-expression of the receptor in 3T3-L1 pre-adipocytes provided a growth advantage to the cells and suppressed cellular differentiation into mature fat cells. In conclusion, the data demonstrates WPI impacts on adiposity by influencing lipid metabolism in a temporal manner, resulting possibly due to changes in lean mass, hypothalamic and adipose gene expression, gut microbiota and gastrointestinal morphology. The data also showed LPA5 is a novel candidate in regulating of preadipocyte growth and differentiation, and may mediate dietary protein effects on adipose tissue.

Exercise training reduces fatty acid availability and improves the insulin sensitivity of glucose metabolism

Aims/hypothesis - It is not known whether the beneficial effects of exercise training on insulin sensitivity are due to changes in hepatic and peripheral insulin sensitivity or whether the changes in insulin sensitivity can be explained by adaptive changes in fatty acid metabolism, changes in visceral fat or changes in liver and muscle triacylglycerol content. We investigated the effects of 6 weeks of supervised exercise in sedentary men on these variables. Subjects and methods - We randomised 17 sedentary overweight male subjects (age 50 ± 2.6 years, BMI 27.6 ± 0.5 kg/m2) to a 6-week exercise programme (n = 10) or control group (n = 7). The insulin sensitivity of palmitic acid production rate (Ra), glycerol Ra, endogenous glucose Ra (EGP), glucose uptake and glucose metabolic clearance rate were measured at 0 and 6 weeks with a two-step hyperinsulinaemic–euglycaemic clamp [step 1, 0.3 (low dose); step 2, 1.5 (high dose) mU kg−1 min−1]. In the exercise group subjects were studied >72 h after the last training session. Liver and skeletal muscle triacylglycerol content was measured by magnetic resonance spectroscopy and visceral adipose tissue by cross-sectional computer tomography scanning. Results - After 6 weeks, fasting glycerol, palmitic acid Ra (p = 0.003, p = 0.042) and NEFA concentration (p = 0.005) were decreased in the exercise group with no change in the control group. The effects of low-dose insulin on EGP and of high-dose insulin on glucose uptake and metabolic clearance rate were enhanced in the exercise group but not in the control group (p = 0.026; p = 0.007 and p = 0.04). There was no change in muscle triacylglycerol and liver fat in either group. Conclusions/interpretation - Decreased availability of circulating NEFA may contribute to the observed improvement in the insulin sensitivity of EGP and glucose uptake following 6 weeks of moderate exercise.

Risk prediction of prevalent diabetes in a Swiss population using a weighted genetic score--the CoLaus Study.

AIMS/HYPOTHESIS: Several susceptibility genes for type 2 diabetes have been discovered recently. Individually, these genes increase the disease risk only minimally. The goals of the present study were to determine, at the population level, the risk of diabetes in individuals who carry risk alleles within several susceptibility genes for the disease and the added value of this genetic information over the clinical predictors. METHODS: We constructed an additive genetic score using the most replicated single-nucleotide polymorphisms (SNPs) within 15 type 2 diabetes-susceptibility genes, weighting each SNP with its reported effect. We tested this score in the extensively phenotyped population-based cross-sectional CoLaus Study in Lausanne, Switzerland (n = 5,360), involving 356 diabetic individuals. RESULTS: The clinical predictors of prevalent diabetes were age, BMI, family history of diabetes, WHR, and triacylglycerol/HDL-cholesterol ratio. After adjustment for these variables, the risk of diabetes was 2.7 (95% CI 1.8-4.0, p = 0.000006) for individuals with a genetic score within the top quintile, compared with the bottom quintile. Adding the genetic score to the clinical covariates improved the area under the receiver operating characteristic curve slightly (from 0.86 to 0.87), yet significantly (p = 0.002). BMI was similar in these two extreme quintiles. CONCLUSIONS/INTERPRETATION: In this population, a simple weighted 15 SNP-based genetic score provides additional information over clinical predictors of prevalent diabetes. At this stage, however, the clinical benefit of this genetic information is limited.

L’impact d’une diète néonatale déficiente en nutriments essentiels à la défense antioxydante sur le métabolisme énergétique à long terme

Les prématurés subissent un stress oxydant qui résulte d’une défense antioxydante faible et/ou d’une charge oxydante. Des données suggèrent qu’un stress oxydant peut affecter le métabolisme énergétique et mener au syndrome métabolique. Hypothèse: Une faible défense antioxydante tôt dans la vie est suffisante pour affecter le métabolisme énergétique à long terme. Méthodes: Quatre groupes de cobayes (n=21) ont reçu entre leurs 3e et 7e jours de vie une diète standard (C-1sem, C-14sem) ou une diète déficiente (DC-1sem, DC-14sem). À 7 jours, les groupes C-1sem et DC-1sem ont été sacrifiés, le plasma et le foie collectés. Les groupes C-14sem et DC-14sem ont reçu la diète standard jusqu’à 14sem de vie. La glycémie et les triglycérides plasmatiques ont été mesurés à 1, 3, 11, et 13-14sem. La tolérance au glucose a été évaluée à 13sem. Les antioxydants hépatiques et les protéines régulant le métabolisme énergétique ont été analysés à 1 et 14sem. Résultats: Un statut redox oxydé du glutathion était associé avec la diète déficiente et était maintenu oxydé au moins jusqu’à 14sem (p<0.01). Les faibles niveaux de triglycérides plasmatiques et de glycémies, ainsi qu’une meilleure tolérance au glucose à 14sem (p<0.05) étaient associés avec un statut redox plus oxydé. Conclusion: Le faible taux de glutathion observé chez les prématurés a été reproduit dans notre modèle. Puisque nos données suggèrent un rôle protecteur d’un redox plus oxydé et que l’environnement redox est un important régulateur métabolique influençant le développement, il faudrait faire attention avant d’initier des traitements antioxydants agressifs chez les prématurés.

La contamination de la nutrition parentérale par l’ascorbylperoxyde perturbe le métabolisme énergétique chez le cochon d'inde nouveau-né

L'exposition à la lumière des solutions de nutrition parentérale (NP) génère des peroxydes tels que l'H2O2 et l'ascorbylperoxyde (AscOOH). Cette absence de photo-protection provoque une augmentation des triglycérides (TG) plasmatique chez les enfants prématurés et chez un modèle animal, ayant un stress oxydatif et une stéatose hépatique indépendante de l’exposition au H2O2. Nous pensons que l'AscOOH est l'agent actif conduisant à l'élévation des TG. Le but est d'investiguer le rôle de l'AscOOH sur les métabolismes du glucose et des lipides à l'aide d'un modèle animal néonatal de NP.

Modalities of exercise training on liver fat accretion and inflammatory markers in ovariectomized rats

Les facteurs de risque des maladies cardiovasculaires, telle, que la détérioration du profil lipidique, deviennent plus prononcés après la ménopause, ce qui fait de la maladie coronarienne, l’une des principales causes de décès chez les femmes ménopausées. Une proportion importante de femmes prennent du poids après la ménopause en particulier dans la région abdominale entraînant par conséquent des perturbations métaboliques. Des données récentes suggèrent également que l’absence des œstrogènes observée à la ménopause favorise le développement de la stéatose hépatique. Cette dernière a été incriminée pour incriminée dans le développement de la résistance à l'insuline, et est de ce fait considérée comme une composante hépatique du syndrome métabolique. Il est impératif d'établir des stratégies visant à contrecarrer l'accumulation de graisse dans le foie et l’accroissement du tissu adipeux chez les femmes ménopausées, en tenant compte que l'utilisation de l'hormonothérapie substitutive est de nos jours moins soutenue. Les quatre études de la présente thèse ont été conduites pour tenter de fournir des informations sur le traitement et la prévention de l’augmentation de la masse graisseuse et de la stéatose hépatique qu’entraîne la suppression des œstrogènes, à travers les modifications du mode de vie (diète et exercice physique) chez la rate ovariectomizée (Ovx); un modèle animal de la ménopause. Dans les deux premières études nous nous sommes concentrés sur l’augmentation de la masse graisseuse et sa reprise suite à une perte de poids. Dans la première étude, nous avons montré que les rates Ovx qui ont suivi un programme de restriction alimentaire (FR) ont diminué significativement (P < 0.01) leur poids corporel, leur contenu en graisses intra-abdominales ainsi que leurs triacylglycérols (TAG) hépatiques, comparativement aux rates Ovx nourries à la diète normale. De plus, l’entraînement en résistance (RT) a prévenu la reprise de poids corporel ainsi que l’accroissement du tissu adipeux et l’accumulation de lipides dans le foie des rates Ovx, après l’arrêt du régime amaigrissant. Les résultats de la deuxième étude ont confirmé l'efficacité de la restriction alimentaire associée à l’entraînement en résistance (FR + RT) dans la réduction du poids corporel, des lipides dans le foie et le tissu adipeux chez les rates Ovx. Tenant compte des résultats de notre première étude, l’entraînement en résistance seulement a constitué un atout pour atténuer le poids corporel et la masse grasse reprise par les rates Ovx suite à un programme de perte de poids (FR + RT); bien que l'impact ait été moindre comparé au maintien seul de la restriction alimentaire. De la même manière que la supplémentation en œstrogènes, les résultats de la troisième étude indiquent que l'entraînement en endurance mené concurremment avec l’ovariectomie a significativement atténué l'accumulation de lipides dans le foie ainsi que dans le tissu adipeux. Toutefois, l’entraînement en endurance effectué avant l'ovariectomie n'a pas protégé contre l'accumulation des graisses qu’entraîne l'ovariectomie, si celui-ci est interrompu après l'ovariectomie. Enfin, pour compléter les résultats antérieurs, nous avons montré dans la quatrième étude que l’expression des gènes impliqués dans la synthèse de lipide; SREBP-1c, SCD-1, ChREBP, et ACC dans le foie a augmenté après le retrait des œstrogènes, tandis qu’une diminution (P < 0.01) des niveaux d'ARNm de PPAR-α a été observée. De plus, l'expression hépatique des gènes des cytokines pro-inflammatoires incluant IKKβ, IL-6 ainsi que le contenu protéinique de NF-кB étaient augmentés (P < 0.01) chez les rates Ovx par rapport aux rates ayant subi une Ovx simulée (Sham). Toutes ces perturbations ont été améliorées avec la supplémentation en œstrogènes seulement, ainsi qu'avec l'entraînement en endurance seulement. Dans l'ensemble, nos résultats indiquent que l'exercice physique (en résistance ou en endurance) a un impact significatif sur la réduction de l'accumulation des lipides dans le foie et dans le tissu adipeux des rates Ovx. De plus, chez les rates Ovx, l’entraînement en endurance mimerait les effets des œstrogènes sur l'expression des gènes impliqués dans l'accumulation de lipides et l’inflammation préclinique dans le foie.

Characterization of Lipid Droplets and Functional Analysis of Lipid Droplet-Associated Proteins in Dictyostelium discoideum

Lipid droplets (LDs) are the universal storage form of fat as a reservoir of metabolic energy in animals, plants, bacteria and single celled eukaryotes. Dictyostelium LD formation was investigated in response to the addition of different nutrients to the growth medium. LDs were induced by adding exogenous cholesterol, palmitic acid (PA) as well as growth in bacterial suspension, while glucose addition fails to form LDs. Among these nutrients, PA addition is most effective to stimulate LD formation, and depletion of PA from the medium caused LD degradation. The neutral lipids incorporated into the LD-core are composed of triacylglycerol (TAG), steryl esters, and an unknown neutral lipid (UKL) species when the cells were loaded simultaneously with cholesterol and PA. In order to avoid the contamination with other cellular organelles, the LD-purification method was modified. The isolated LD fraction was analysed by mass spectrometry and 100 proteins were identified. Nineteen of these appear to be directly involved in lipid metabolism or function in regulating LD morphology. Together with a previous study, a total of 13 proteins from the LD-proteome were confirmed to localize to LDs after the induction with PA. Among the identified LD-proteins, the localization of Ldp (lipid droplet membrane protein), GPAT3 (glycerol-3-phosphate acyltransferase 3) and AGPAT3 (1-acylglycerol-3-phosphate-acyltransferase 3) were further verified by GFP-tagging at the N-termini or C-termini of the respective proteins. Fluorescence microscopy demonstrated that PA-treatment stimulated the translocation of the three proteins from the ER to LDs. In order to clarify DGAT (diacylglycerol acyltransferase) function in Dictyostelium, the localization of DGAT1, that is not present in LD-proteome, was also investigated. GFP-tagged DGAT1 localized to the ER both, in the presence and absence of PA, which is different from the previously observed localization of GFP-tagged DGAT2, which almost exclusively binds to LDs. The investigation of the cellular neutral lipid level helps to elucidate the mechanism responsible for LD-formation in Dictyostelium cells. Ldp and two short-chain dehydrogenases, ADH (alcohol dehydrogenase) and Ali (ADH-like protein), are not involved in neutral lipid biosynthesis. GPAT, AGPAT and DGAT are three transferases responsible for the three acylation steps of de novo TAG synthesis. Knock-out (KO) of AGPAT3 and DGAT2 did not affect storage-fat formation significantly, whereas cells lacking GPAT3 or DGAT1 decreased TAG and LD accumulation dramatically. Furthermore, DGAT1 is responsible for the accumulation of the unknown lipid UKL. Overexpression of DGAT2 can rescue the reduced TAG content of the DGAT1-KO mutant, but fails to restore UKL content in these cells, indicating that of DGAT1 and DGAT2 have overlapping functions in TAG synthesis, but the role in UKL formation is unique to DGAT1. Both GPAT3 and DGAT1 affect phagocytic activity. Mutation of GPAT3 increases it but a DGAT1-KO decreases phagocytosis. The double knockout of DGAT1 and 2 also impairs the ability to grow on a bacterial lawn, which again can be rescued by overexpression of DGAT2. These and other results are incorporated into a new model, which proposes that up-regulation of phagocytosis serves to replenish precursor molecules of membrane lipid synthesis, whereas phagocytosis is down-regulated when excess fatty acids are used for storage-fat formation.  

Untersuchungen zur Dynamik des Lipid-Metabolismus von Dictyostelium discoideum

Die soziale Waldamöbe Dictystelium discoideum ist ein etablierter Modellorganismus zur Erforschung grundlegender zellbiologischer Prozesse. Innerhalb der letzten Jahre konnte dabei insbesondere das Wissen zum Lipidmetabolismus umfassend erweitert werden. In diesem Zusammenhang spielt besonders eine Enzymgruppe eine wichtige Rolle: die LC/VLC-Acyl-CoA-Synthetasen. Diese übernehmen dabei die Aufgabe Fettsäuren zu aktivieren, um sie so dem Zellmetabolismus überhaupt erst zugänglich zu machen. D. discoideum verfügt über insgesamt vier dieser Enzyme: FcsA, FcsB, FcsC und das Bubblegum-Enzym Acsbg1. Während die FcsA und FcsB bereits in vorangegangenen Arbeiten untersucht wurden, werden die FcsC und die Acsbg1 in dieser Arbeit erstmals biologisch charakterisiert. Untersuchungen zur subzellulären Lokalisation der Proteine zeigen, dass die meisten LC/VLC-Acyl-CoA-Synthetase auf Endosomen und im Cytoplasma gefunden werden können (FcsA, FcsC und Acsbg1), während die FcsB als Transmembranprotein über das ER zu den Peroxisomen transportiert wird. Die Acsbg1 akkumuliert dabei zusätzlich an der Plasmamembran. Funktionell konnte gezeigt werden, dass neben der FcsA auch die Acsbg1 an der Bereitstellung von Acyl-CoA für Triacylglyceridsynthese beteiligt ist. Dabei besitzt die FcsA die Hauptenzymaktivität und kompensiert den Verlust der Acsbg1 in acsbg1- Zellen. In fcsA-/acsbg1- Zellen dagegen kommt der Verlust der Acsbg1 durch eine zusätzliche Verringerung des TAG-Gehaltes der Doppel-KOs im Vergleich zu fcsA- Zellen zum tragen. Alle vier Enzyme beeinflussen die Phagozytose. Dabei zeigen fcsA- und fcsC- Zellen eine gesteigerte Phagozytose in Gegenwart von der gesättigten Fettsäure Palmitinsäure im Kulturmedium. Auch der knockout der Acsbg1 wirkt sich positiv auf die Phagozytoserate aus, jedoch kommt auch nur dieser zum tragen, wenn neben der Acsbg1 auch die FcsA ausgeschaltet wird. Die FcsB dagegen zeigt eine dramatische Reduktion der Partikelaufnahme in nicht Fettsäure gefütterten Zellen. Durch die Zugabe einer exogenen Fettsäure kann dieser Effekt nicht kompensiert werden. Auch der zusätzliche Verlust der FcsA-Enzymaktivität verändert dieses Verhalten in Palmitinsäure inkubierten Zellen nicht. In fcsA-/fcsB- konnte zudem ein Defekt beim Abbau von Triacylglyceriden gefunden werden. Dieser Defekt liefert erste Hinweise für ein Modell, das den Abbau von LD gespeicherten Lipiden durch Autophagozytose in D. discoideum beschreibt. Peroxisomen sind wichtige Organellen für die Detoxifikation und die Oxidation von Fettsäuren. Durch das Ausschalten der Acaa1, der Thiolase, die den letzten Schritt der β-Oxidation in Peroxisomen katalysiert, zeigte sich ein verlangsamter Triacylglycerol-Abbau sowie eine verringerte Degradation des Etherlipids UKL und von Sterolestern, was auf eine Beteiligung der Peroxisomen beim Abbau von langkettigen Fettsäuren schließen lässt. Bei dem Versuch durch das Ausschalten des pex19-Gens eine Zelllinie zu generieren, die keine Peroxisomen besitzt, wurde die Organelle überraschender Weise, wenn auch mit einer vom Wildtyp abweichenden Morphologie, weiterhin vorgefunden. Dieser Befund korrelierte mit dem Resultat, dass trotzdem das pex19-Gen erfolgreich unterbrochen wurde, dennoch eine intakte Kopie des Gens nachgewiesen werden konnte. Dementsprechend sollte die erschaffene pex19- Zelllinie als knockdown und nicht als knockout gewertet werden. Der pex19 knockdown zeigte beim Abbau von Triacylglyceriden eine ähnliche Verlangsamung wie acaa1- Zellen. Zusätzlich wurde eine Verringerung der Synthese des Etherlipids UKL beobachtet, was darauf hindeutet, dass dieses Lipid im Peroxisom gebildet wird. Auch die Phagozytose und das Wachstum auf Bakterienrasen waren im pex19 knockdown dramatisch reduziert. Durch die Überexpression von Pex19-GFP im knockdown Hintergrund konnten die physiologischen Defekte in den meisten so generierten Zelllinien ausgeglichen werden. Lipid Droplets sind Organellen, die in Eukaryoten und Prokaryoten als Speicher für Neutralfette dienen und ebenfalls als Ort der Lipidsynthese fungieren. Um diese Aufgaben erfüllen zu können, besitzen sie auf ihrer Oberfläche Proteine, die für die Regulierung dieser Prozesse notwendig sind. Durch die weiterführende Analyse von Kandidatenproteinen, die durch eine proteomische Analyse von aufgereinigten LDs identifiziert wurden, konnte für vier weitere Proteine (Plsc1, Net4, Lip5 und Nsdhl) die LD-Assoziation durch GFP-Fusionsproteine bestätigt werden. Bei der Charakterisierung von plsc1 knockouts zeigte sich eine verminderte Fähigkeit beim Wachstum auf Bakterienrasen sowie eine erhöhte Phagozytoserate in Gegenwart einer exogenen Fettsäure, was auf eine Involvierung des Proteins in die Phospholipidsynthese hindeutet. Die bisher einzige identifizierte LD-assoziierte Lipase Lip5 nimmt nur eine untergeordnete Rolle bei der Hydrolyse von Triacylglycerolen und Sterolestern ein, da in KO-Mutanten nur ein milder Defekt beim Abbau beider Substanzen beobachtet werden konnte. Die LD-Lokalisation von Net4 ist evolutionär konserviert und kann nicht nur in D. discoideum beobachtet werden, sondern auch in humanen Zellen. Welche Funktion das Protein auf der LD-Oberfläche ausübt, konnte nicht geklärt werden. Allerdings kann ein direkter Einfluss auf den TAG- und Sterolaufbau ausgeschlossen werden. LDs stehen in engem Kontakt mit anderen Organellen, die in den Lipidmetabolismus involviert sind, wie mit den Mitochondrien oder dem ER. Durch Perilipin-Hybridproteine können künstliche, stabile Verbindungen zwischen LDs und diesen Organellen hergestellt werden. Dabei zeigte Perilipin ein sehr starkes Targeting-Potenzial, durch welches es notwendig war, als zweite Hybridhälfte ein Transmembranprotein zu wählen. Die Analyse eines Hybrids, das eine dauerhafte Verbindung von LDs und dem ER herstellt, wies dabeieine Reduktion der LD-Größe auf, wobei der Gesamt-TAG-Gehalt der Zellen unbeeinflusst blieb. Durch die starke Affinität von Perilipin für die Assoziation an LDs konnten durch die Generierung von Hybriden andere Proteine an die LD-Oberfläche dirigiert werden. Auf diese Weise konnte erfolgreich die LC-Acyl-CoA-Synthetase FcsA auf das LD transplantiert werden.

Lipid metabolism in women

Differences in whole-body lipid metabolism between men and women are indicated by lower-body fat accumulation in women but more marked accumulation of fat in the intra-abdominal visceral fat depots of men. Circulating blood lipid concentrations also show gender-related differences. These differences are most marked in premenopausal women, in whom total cholesterol, LDL-cholesterol and triacylglycerol concentrations are lower and HDL-cholesterol concentration is higher than in men. Tendency to accumulate body fat in intra-abdominal fat stores is linked to increased risk of CVD, metabolic syndrome, diabetes and other insulin-resistant states. Differential regional regulation of adipose tissue lipolysis and lipogenesis must underlie gender-related differences in the tendency to accumulate fat in specific fat depots. However, empirical data to support current hypotheses remain limited at the present time because of the demanding and specialist nature of the methods used to study adipose tissue metabolism in human subjects. In vitro and in vivo data show greater lipolytic sensitivity of abdominal subcutaneous fat and lesser lipolytic sensitivity of femoral and gluteal subcutaneous fat in women than in men. These differences appear to be due to fewer inhibitory alpha adrenergic receptors in abdominal regions and greater a adrenergic receptors in gluteal and femoral regions in women than in men. There do not appear to be major gender-related differences in rates of fatty acid uptake (lipogenesis) in different subcutaneous adipose tissue regions. In visceral fat rates of both lipolysis and lipogenesis appear to be greater in men than in women; higher rates of lipolysis may be due to fewer alpha adrenergic receptors in this fat depot in men. Fatty acid uptake into this depot in the postprandial period is approximately 7-fold higher in men than in women. Triacylglycerol concentrations appear to be a stronger cardiovascular risk factor in women than in men, with particular implications for cardiovascular risk in diabetic women. The increased triacylglycerol concentrations observed in women taking hormone-replacement therapy (HRT) may explain the paradoxical findings of increased rates of CVD in women taking HRT that have been reported from recent primary and secondary prevention trials of HRT.