The development of the metabolic syndrome (MetS) and cardiovascular diseases have been suggested to be influenced more by the quality than the amount of dietary fat. The FA composition of serum lipids may be used as biomarkers of dietary fat quality. FAs can, however, also be endogenously synthesized by lipogenic enzymes such as elongases and desaturases. Three desaturases are important in humans: Stearoyl-CoA-desaturase (SCD), ∆6-desaturase (D6D) and ∆5-desaturase (D5D) and surrogate measures of desaturase activities can be estimated as product-to-precursor FA ratios. In this thesis, we demonstrated that high SCD, D6D and low D5D estimated activities predicted MetS 20 years later, as well as cardiovascular and total mortality during a maximum of 33.7 years. The relation between D5D and MetS was independent of lifestyle and BMI, while the relation between SCD, D6D and MetS was confounded by BMI. Serum proportions of palmitic (16:0), palmitoleic (16:1) and dihomo-γ-linoleic acids were higher and the serum proportion of linoleic acid (LA) lower at baseline in those individuals who developed MetS. Further, LA was inversely related to mortality, while palmitic, palmitoleic and dihomo-γ-linoleic acids were directly associated with mortality. We also demonstrated that a diet rich in saturated fat “induced” a similar serum FA pattern (including estimated desaturase activities) that was associated with MetS, cardiovascular disease and mortality. We also propose that the SCD ratio [16:1/16:0] might be a novel and useful marker of dietary saturated fat, at least in Western high-fat diets. Finally, genetic variations in the human SCD1 gene were linked to obesity and insulin sensitivity, results that agree with data in SCD1 deficient mice. This thesis suggests that dietary fat quality and endogenous desaturation may play a role in the development of metabolic and cardiovascular diseases and the results support current dietary guidelines.

Feline lipid metabolism is a topic for greater exploration due to this specie?s unique characteristics. Cats express limited Delta 6-desaturase activity necessary for conversion of linoleic acid (LA, 18:2n-6) to arachidonic acid (AA, 20:4n-6). The possibility exists that Gamma-linolenic acid (GLA, 18:3n-6) may serve as a precursor of AA in reproductive tissues especially if coupled with chain elongation and a functionally active Delta 5-desaturase. In addition no research has been conducted regarding feline reproductive Delta 8-desaturase activity as an alternate to the production of AA. To investigate desaturation activities, a group of 26 adult female cats were randomly assigned into 1 of 3 groups based on the diet fed: High Linoleic Acid (HL, n=7), Low Linoleic Acid (LL, n=9), and High Gamma-Linolenic Acid (GLA, n=10).The diets were fed for 300 days prior to ovariohysterectomy at which time EDTA plasma and ovarian, uterine, and subcutaneous adipose tissues were collected. Homogenates of each tissue were prepared and frozen in aliquots at -80 degrees C. Total lipids were extracted from the plasma and tissue homogenates followed by phospholipid (PL) fractionation via thin layer chromatography and fatty acid (FA) analyses by gas chromatography. The Shapiro-Wilks test was used to determine normal distribution of FA data followed by One-Way ANOVA and Tukey multiple comparisons (p<0.05). Plasma PLs were significantly increased in both GLA and dihomo-Gamma-linolenic acid (DGLA, 20:3n-6Delta8,11,14) in the GLA group and statistically increased in 20:2n-6 and 20:3n-6(Delta5,11,14) in the HL group. Uterine tissue homogenates had significantly increased amounts of DGLA and AA, however ovarian tissue showed an increase of only DGLA. Adipose tissue FAs showed significantly high amounts of DGLA in the GLA group. It is concluded that a high GLA diet results in increased AA in uterine, but not ovarian, tissues and thus may supply eicosanoid precursors in support of reproduction. The presence of increased amounts of 20:3n-6(Delta5,11,14) and not AA in the plasma and uterine tissues in the HL group suggests that Delta6-desaturase cannot be induced and that Delta8-desaturase is not active when feeding high dietary LA. Furthermore, the increase in DGLA may provide an adipose storage reservoir for additional conversion under times of metabolic need. These data support the presence of a functionally active Delta5-desaturase in uterine, but not ovarian, tissues. The findings also suggest that increased dietary GLA may be used to meet the AA requirements for reproduction in cats in the absence of an animal based pre-formed source of AA.

This thesis investigates the contributions of fatty acids (FA) to adipokine dysregulation and inflammation. Differentiated 3T3-L1 adipocytes were treated with palmitic, stearic, palmitoleic, and oleic acids and changes in adipokine gene expression were measured. Here it was determined that saturated FA (SFA) increased the expression of RANTES and monounsaturated FA (MUFA) decreased the expression of RANTES and IL-6; demonstrating that FA differentially regulate adipokine expression. Relationships between plasma levels of SFA, MUFA and C-reactive protein (CRP) were also identified in a human observational study, further demonstrating the link between FA and inflammation Moreover, an association was also found between stearoyl-CoA desaturase 1 (SCD1) activity and CRP, demonstrating that SCD1 activity contributes to the inflammatory state. Genetic variation in SCD1 was also found to alter plasma FA and CRP levels, thus contributing to systemic inflammation. Taken together, these results demonstrate that SFA and MUFA influence adipokine dysregulation and systemic inflammation.

Based on their highly bioactive properties in membrane phospholipids, there is growing recognition that dietary n-3 polyunsaturated fatty acids (PUFA) may be of significant benefit in the prevention and treatment of many lifestyle related pathologies, however direct evidence is lacking. The fat-1 transgenic mouse, a genetic model of n-3 PUFA enrichment, is a useful tool in nutritional research which has provided enhanced insight into the health effects of lifelong n-3 PUFA exposure. However, the influence of timing of n-3 PUFA exposure on health related outcomes remains unclear. This thesis describes the functional characterization of the novel Cre recombinase dependent inducible fat-1 (iFat-1) transgene. In the presence of Cre, the iFat-1 transgene was found to reduce phospholipid n-6/n-3 PUFA ratios both in vitro (100%) and in vivo (upwards of 70%), suggesting that the iFat-1 transgene has potential application to address temporal effects of n-3 PUFA in health and disease. / Canadian Institutes of Health Research - Frederick Banting and Charles Best Canada Graduate Scholarship, Sun Life Financial

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22472号 / 工博第4733号 / 新制||工||1739(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 梅田 眞郷, 教授 浜地 格, 教授 跡見 晴幸 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

No description available.

The gastrointestinal tract (GIT) regulates nutrient uptake, secretes hormones and has a crucial gut flora and enteric nervous system. Of relevance for these functions are the G protein-coupled receptors (GPCRs) and the solute carriers (SLCs). The Adhesion GPCR subfamily is known to mediate neural development and immune system functioning, whereas SLCs transport e.g. amino acids, fatty acids (FAs) and drugs over membranes. We aimed to comprehensively characterize Adhesion GPCR and SLC gene expression along the rat GIT. Using qPCR we measured expression of 78 SLCs as well as all 30 Adhesion GPCRs in a twelve-segment GIT model. 21 of the Adhesion GPCRs had a widespread (≥5 segments) or ubiquitous (≥11 segments) expression. Restricted expression patterns were characteristic for most group VII members. Of the SLCs, we found the majority (56 %) of these transcripts to be expressed in all GIT segments. SLCs were predominantly found in the absorption-responsible gut regions. Both Adhesion GPCRs and SLCs were widely expressed in the rat GIT, suggesting important roles. The distribution of Adhesion GPCRs defines them as a potential pharmacological target. FAs constitute an important energy source and have been implicated in the worldwide obesity increase. FAs and their ratios – indices for activities of e.g. the desaturase enzymes SCD-1 (SCD-16, 16:1n-7/16:0), D6D (18:3n-6/18:2n-6) and D5D (20:4n-6/20:3n-6) – have been associated with e.g. overall mortality and BMI. We examined whether differences in FAs and their indices in five lipid fractions contributed to obesity susceptibility in rats fed a high fat diet (HFD), and the associations of desaturase indices between lipid fractions in animals on different diets. We found that on a HFD, obesity-prone (OP) rats had a higher SCD-16 index and a lower linoleic acid (LA) proportions in subcutaneous adipose tissue (SAT) than obesity-resistant rats. Desaturase indices were significantly correlated between many of the lipid fractions. The higher SCD-16 may indicate higher SCD-1 activity in SAT in OP rats, and combined with lower LA proportions may provide novel insights into HFD-induced obesity. The associations between desaturase indices show that plasma measurements can serve as proxies for some lipid fractions, but the correlations seem to be affected by diet and weight gain.

No description available.

Polyunsaturated fatty acids are major structural components of plant chloroplast and endoplasmic reticulum membranes. Two fatty acid desaturases (designated FAD2 and FAD3) desaturate 75% of the fatty acids in the endoplasmic reticulum. The w -6 fatty acid desaturase (FAD2) may be responsible for cold acclimation response, since polyunsaturated phospholipids are important in helping maintain plant viability at lowered temperatures. To study regulation of FAD2 gene expression in cotton, a FAD2 gene was isolated from two genomic libraries using an Arabidopsis FAD2 hybridization probe and a cotton FAD2 5¢ -flanking region gene-specific probe, respectively. A cotton FAD2 gene was found to be in two overlapping genomic clones by physical mapping and DNA sequencing. The cloned DNA fragments are identical in size to cotton FAD2 genomic DNA fragments shown by genomic blot hybridization. The cotton FAD2 coding region has 1,155 bp with no introns and would encode a putative polypeptide of 384 amino acids. The cotton FAD2 enzyme has a high identity of 75% with other plant FAD2 enzymes. The enzyme has three histidine-rich motifs that are conserved in all plant membrane desaturases. These histidine boxes may be the iron-binding domains for reduction of oxygen during desaturation. To confirm that this FAD2 enzyme is functional, a plasmid construct containing the cotton FAD2 coding region was transformed into Saccharomyces cerevisiae. The transformed yeast cells were able to catalyze the conversion of oleic acid (C18:1) into linoleic acid (C18:2). The FAD2 gene contains an intron of 2,967 bp in its 5¢ -flanking region, 11 bp upstream from the initiation codon. The intron could be essential for transcriptional regulation of FAD2 gene expression. Several putative promoter elements occur in the 5¢ -flanking region of this gene. A potential TATA basal promoter element occurs at 41 bp upstream from the cap site. Two presumptive helix-loop-helix (bHLH) motifs that may be seed-specific promoter elements are located at 109 bp and 135 bp upstream from the potential cap site.

La capacité des microalgues à accumuler des quantités importantes de lipides de réserve font de ces organismes de bons candidats pour envisager une production durable de biocarburants (biodiesel). Cependant, des verrous d’ordre technologique et biologique persistent avant d’atteindre une production économiquement viable. Dans le but de mieux comprendre les mécanismes et biosynthèse et d’accumulation des lipides chez les microalgues et de proposer des voies d’amélioration biotechnologiques, nous avons développé deux approches expérimentales complémentaires en utilisant la microalgue Chlamydomonas reinhardtii comme modèle. La première a été de caractériser par des techniques de protéomique et de lipidomique la composition des gouttelettes lipidiques s’accumulant en réponse à une carence en azote. Les données de protéomique nous ont permis de montrer que les gouttelettes lipidiques étaient des structures cellulaires dynamiques impliquées non seulement dans le stockage, mais aussi dans la biosynthèse, la remobilisation et le « trafficking » des lipides. Les protéines identifiées au cours de cette étude nous fournissent des gènes cibles d’intérêt pour mieux comprendre les voies de biosynthèse des triacylglycérols et accroître l’accumulation d’huile. La seconde approche, de génétique formelle, a consisté à rechercher puis à caractériser des mutants isolés à partir d’une banque de mutants d’insertion de C. reinhardtii. Deux mutants d’intérêt, l’un affecté dans la composition en acides gras (crfad7) et l’autre capable d’accumuler des lipides en l’absence de stress (coa1, pour constitutive oil accumulator 1), ont été isolés. / The ability of microalgae to accumulate high amounts of reserve lipids makes these organisms good candidates for the production of sustainable biofuel (biodiesel). However, both technological and biological bottlenecks remain to be overcome before profitable production is reached. With the aim to better understand lipid metabolic pathways in microalgae and further propose new strategies for biotechnological improvement, we have developed two complementary experimental approaches in the model microalga Chlamydomonas reinhardtii. As a first approach, we performed a proteomic and lipidomic characterization of oil bodies isolated from nitrogen-deprived cells. Based on proteomic data, we have concluded that oil bodies are dynamic structures involved not only in the storage, but also in oil biosynthesis, degradation and lipid homeostasis. The proteins identified in this study should provide useful targets for genetic studies aiming at increasing our understanding of triacylglycerol synthesis and further improve intracellular oil accumulation. The second approach, based on the development of a forward genetic screen, aimed at searching and further characterizing mutants isolated from a C. reinhardtii insertion library. Two mutants of interest, one affected in the fatty acid composition (crfad7), the other (coa1, for constitutive oil accumulator 1) able to accumulate reserve lipids in the absence of stress, have been isolated. The crfad7 mutant, affected in the expression of the unique ω3 fatty acid desaturase present in the C. reinhardtii genome, has been complemented and subjected to extensive phenotypical characterization.