ABCD2 (D2) is a peroxisomal ATP binding cassette (ABC) transporter that is expressed in brain, adrenal and liver. D2 is transcriptionally regulated by key transcriptional factors that control lipid and glucose metabolism. Therefore, we examined its role in adipose tissue. These studies revealed that D2 is highly abundant in adipose tissue and upregulated during adipogenesis. However, D2 deficiency does not affect either adipogenesis or lipid accumulation. An examination of the lipid profile of adipose tissue revealed the accumulation of C20 and C22 fatty acids in D2 deficient (D2‐/‐) mice. When challenged with a diet enriched in erucic acid (C22:1, 10% kcal), this lipid accumulated in both liver and adipose tissue. Following 8 weeks of diet, D2‐/‐ mice showed increased adiposity, glucose intolerance, dyslipidemia and steatosis. Analysis of the hepatic lipid profile showed significant changes away from poly unsaturated fatty acids (PUFAs) and toward C18‐22 mono‐unsaturated fatty acids (MUFA). RT‐PCR of the mRNA from the adipose tissue and liver revealed significant changes in lipogenic (ACC, SCD1 & 2) and PUFA synthesis (Δ5 & 6‐desaturase) genes in D2‐/‐ mice. The molecular mechanisms by which D2 regulates lipid metabolism in adipose tissue remains unclear. To explore potential mechanisms, the subcellular localization of D2 in adipose tissue was determined. Our results demonstrated that D2 resides in a distinct subclass of peroxisomes that does not containing classical peroxisomal markers such as pex19 or PMP70, but are positive for pex14. In conclusion, our studies reveal a novel role of D2 and peroxisomes in the protection from disruptions of lipid metabolism induced by dietary erucic acid and that D2 resides in a unique compartment within adipocytes that plays a yet to be elucidated role in the regulation of lipid metabolism.

This thesis research started with analysis of oat fatty acids by using three different transmethylation methods. Basic sodium methoxide was compared with traditional acidic methanol for the total fatty acid analysis, while diazomethane was used to analyze free fatty acids. Epoxy FAs were readily hydrolyzed to dihydroxy fatty acids under the acidic condition, which suggest an overestimation of hydroxyl fatty acids and underestimation of epoxy fatty acids in previous analyses. The sodium methoxide method proved more reliable to quantify the oat seed fatty acid composition. CDC Dancer oat seed analyzed here was comprised mostly of palmitic acid (PA), oleic acid (OA) and the polyunsaturated fatty acid (PUFA) linoleic acid (LA) in quantities of 23%, 32%, and 37% of total seed FA, respectively. As well, the seed contained small quantities of another PUFA, α-linolenic (ALA) and several unusual oxygenated fatty acids (UFAs), Δ15-hydroxy fatty acid (15HFA) and epoxy fatty acids in quantities of 0.85%, 0.68%, and 2.3%, respectively. This thesis further aimed to identify and assemble all FAD2-like genes from an oat Expressed-Sequence Tag (EST) database using FAD2 and FAD2-like proteins from other organisms as query sequences in order to clone all putative FAD2-like genes-of-interest (GOIs) from oat. From the contig assemblies of retrieved oat ESTs, four distinct, putative genes were identified. From the Δ12-desaturase (FAD2) queries, a putative FAD2-like (AsFAD2) gene was identified; the Δ15-desaturase (FAD3) queries revealed two putative oat FAD3-like (AsFAD3-1 and AsFAD3-2) genes, while an ω-3 desaturase (FAD7) query identified a fourth putative full-length FAD6-like coding sequence of two possible lengths, AsFADX and AsFADX+. The GOIs were then subcloned into a yeast expression vector and functionally characterized. AsFAD2a and AsFAD2b both demonstrated Δ12 desaturation on 18:1-9c substrate. AsFAD3-1 had no activity on any substrates present, while AsFAD3-2 exhibited weak Δ15-desaturation activity specifically on 18:2-9c,12c. Finally, AsFADX converted 18:1-9c to 18:2-9c,12c, while AsFADX+ had no activity. Then, a comparative analysis of transcript levels of these GOIs via quantitative real-time PCR (qRT-PCR) was performed across oat germinating seed, root, leaf, and developing seed. AsFAD2 transcript abundance was generally much higher than AsFAD3-1 and AsFAD3-2 in all tissues. AsFAD3-1 mRNA level was highest in developing seed tissue, slightly lower in leaf tissue, and lowest in root. AsFAD3-2 mRNA was highest in germinating seed, and lowest in leaf tissue. In summary, the data produced from this thesis could be used to enhance breeding efforts for establishing oat cultivars with healthier oil content.

The omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA), in the free fatty acid (FFA) form, has been demonstrated to reduce adenoma number and size in patients with familial adenomatous polyposis. However, the mechanistic basis of the antineoplastic activity of EPA in the colorectum remains unclear. We tested the hypothesis that EPA-FFA negatively modulates synthesis of and signaling by prostaglandin (PG) E(2) in human colorectal cancer (CRC) cells. EPA-FFA induced apoptosis of cyclooxygenase (COX)-2-positive human HCA-7 CRC cells in vitro. EPA-FFA in cell culture medium was incorporated rapidly into phospholipid membranes of HCA-7 human CRC cells and acted as a substrate for COX-2, leading to reduced synthesis of PGE(2) and generation of PGE(3). Alone, PGE(3) bound and activated the PGE(2) EP4 receptor but with reduced affinity and efficacy compared with its "natural" ligand PGE(2). However, in the presence of PGE(2), PGE(3) acted as an antagonist of EP4 receptor-dependent 3',5' cyclic adenosine monophosphate induction in naturally EP4 receptor-positive LoVo human CRC cells and of resistance to apoptosis in HT-29-EP4 human CRC cells overexpressing the EP4 receptor. We conclude that EPA-FFA drives a COX-2-dependent "PGE(2)-to-PGE(3) switch" in human CRC cells and that PGE(3) acts as a partial agonist at the PGE(2) EP4 receptor.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23957号 / 農博第2506号 / 新制||農||1092(附属図書館) / 学位論文||R4||N5392(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 阪井 康能, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The role of dietary fatty acids and their subsequent effects on metabolism has received considerable attention in mammalian species. It is becoming increasingly clear that fatty acids have metabolic roles over and above their influence on energy density of the diet. Recent studies have linked changes in the fatty acyl composition of the plasma membrane, induced by the dietary fat profile, to alterations in both lipid and glucose metabolism. These dietary induced changes have profound effects on insulin action, glucose transport and enzyme activity that regulate triglyceride and fatty acid synthesis, factors that ultimately influence protein and lipid deposition of animals. Because of their high growth rate, broiler chickens have a high requirement for energy and the use of triglycerides as a major energy source has resulted in a fat carcass. A change in the glucose-insulin balance has been suggested as being the main reason for differences in adiposity between broilers selected for fatness or leanness. The hypotheses of this thesis is based on the finding that dietary polyunsaturated fatty acids (PUFA�s) increase the sensitivity of muscle tissue to insulin and this would presumably augment insulin-stimulated glucose uptake into muscle cells. Therefore, increasing the capacity of broiler muscle tissue to utilise glucose as its principal energy substrate would reduce the bird�s reliance on triglycerides and this inturn would result in a leaner carcass. The aims of this study are firstly to explore the role that dietary PUFA�s from the n-3 and n-6 series have on the growth and body composition of broiler chickens and secondly to determine the relationships between dietary fatty acid profile, tissue insulin sensitivity and lipid deposition. Because dietary fatty acids have been implicated in the modulation of hormones important for the growth and development of animals, a third aim of this thesis is to determine the effects of dietary n-3 and n-6 polyunsaturated fatty acids on pituitary and adrenal sensitivity. Since the modulation of metabolism by dietary fatty acids has been attributed to changes in the fatty acyl composition of the plasma membrane, the final aim of this study will be to investigate and characterise the molecular species of the breast muscle choline and ethanolamine phospholipids. Feeding either fish oil a source of n-3 PUFA�s or sunflower oil a source of n-6 PUFA�s fatty acids to broiler chickens resulted in a significant (P<0.01) reduction in the abdominal fat pad mass and a modest increase in breast muscle mass when compared to broilers fed edible tallow. Associated with the changes in carcass composition was an alteration in energy substrate utilisation. This was reflected by lower respiratory quotients and reduced triglyceride and insulin concentrations for the chickens fed the two PUFA diets. Coupled to the shift in energy metabolism was a significant (P<0.05) increase in the proportion of PUFA�s incorporated into the abdominal fat pad and breast muscle. The dietary fat supplements resulted in the incorporation of specific fatty acid subtypes. Feeding fish oil significantly increased the proportion of long-chain n-3 PUFA�s whereas feeding sunflower oil significantly increased the proportion of long-chain n-6 PUFA�s compared to tallow feeding whose tissues were dominated by a higher proportion of saturated fatty acids. It was further shown that dietary n-3 and n-6 PUFA�s enhanced glucose /insulin action. Feeding either fish oil or sunflower oil to broiler chickens increased insulin action when examined by an intravenous glucose tolerance test. The maximal insulin release in response to the glucose infusion was higher in the tallow fed group compared to either the sunflower oil or fish oil groups. To estimate the disappearance rate of glucose from the plasma and its incorporation into tissues, 2-deoxy-D-3H glucose was infused into each chicken. There were no significant differences in the clearance rate of 2-deoxy-D-3H glucose from the plasma. However, when measured under steady state conditions, the labelled glucose incorporation into the breast muscle was greater in birds fed fish oil compared to either tallow or sunflower oil feeding. The dietary fatty acid induced increase in insulin action suggests that the sensitivity of muscle cells to insulin was enhanced. This modulation of tissue sensitivity by dietary fatty acids was also shown to occur at the level of the pituitary. To provide an estimate of pituitary sensitivity, bolus GnRH and GHRH infusions were given on different days to chickens fed the three dietary treatments. Feeding sunflower oil (n-6 PUFA�s) increased the level of GH that was released in response to the GHRH infusion when compared to birds fed either tallow or fish oil (n-3 PUFA�s). This dietary fatty acid modulation appears to be specific to certain pituitary cell types as there was no effect on LH secretion following the GnRH infusion. Dietary fatty acid modulation of endocrine gland sensitivity is particular to the gland type. Although the dietary treatments mediated a distinct pattern in pituitary sensitivity to GHRH infusion, these same three diets did not influence adrenal sensitivity, as there was no difference in the corticosterone profile following either ACTH or CRF infusion. The previously observed physiological changes for the three dietary groups was expected to be positively correlated to an alteration of the plasma membrane phospholipids induced by the dietary fatty acids. Supplementation with fish oil (n-3 PUFA�s) significantly increased levels of both eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) into the choline (PC) and ethanolamine (PE) breast muscle phospholipids compared to either sunflower oil (n-6 PUFA�s) or tallow supplementation. The increase in n-3 PUFA incorporation was associated with a corresponding decrease in the proportion of arachidonic acid (AA; 20:4n-6) an event that would presumably alter substrate availability for the 1- and 2-series eicosanoids. However, feeding sunflower oil or tallow gave a molecular species profile that was remarkably similar in both fatty acid subtype and proportion. This suggests that the plasma membrane dynamics would be similar for these two dietary groups. Therefore, it is appears that factors other than a change in the fatty acyl- composition of the plasma membrane may be responsible for modifying the physiology of the broiler.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2002. / Härtill 4 uppsatser.

Feeds that maximize reproductive potential are needed to ensure success of captive broodstock. Nutritional requirements for somatic growth of juvenile fishes differ from nutritional requirements of mature fishes and are largely species-specific. Broodstock nutritional research has focused primarily on lipid and fatty acid requirements and their effects on reproductive conditioning due to the importance of these nutrients in providing metabolic energy and structural elements, i.e. for phospholipids for embryonic development. Development of suitable broodstock feeds are limited by not knowing fatty acid requirements for many species. Once requirements are identified, plant, grain, marine, algal, and fungal lipid sources can be blended to develop least-cost diet formulations. The objectives of this dissertation are to (1) evaluate white bass Morone chrysops ovum fatty acid composition and reproductive performance after feeding maternal broodstock graded levels of squid to fish oil; (2) evaluate flax, canola, and corn oils as alternatives to marine oil(s) in white bass maternal broodstock diets; (3) determine extent to which grain oils can replace marine oils in female white bass broodstock diets in order to maintain reproductive performance and quantify fatty acid utilization of larvae with different initial fatty acid profiles; and (4) assess dietary supplementation of 20:4n-6, 20:5n-3, and 22:6n-3 concentrates to boost reproductive performance of female white bass fed primarily plant oil-based lipid sources. Of the marine and plant oils tested, menhaden fish oil provided female white bass broodstock the fatty acids (~3.9% n-3 long-chain polyunsaturated fatty acids; LC-PUFA; dry matter basis) necessary to maximize embryonic survival; however, flax oil, due to its low 18:2n-6 and high 18:3n-3 content, showed promise as a suitable plant oil candidate for partial if not complete marine oil substitution in female white bass broodstock feeds. Differential responses in embryonic and larval survival resulted in comparable total larval yields at 5 days post hatch (DPH) after feeding female broodstock graded levels (0, 33, 67, or 100%) flax to fish oils for 30 weeks prior to spawning. At the end of the endogenous feeding period, fatty acid compositions of flax and fish oil-fed broodstock progeny deviated from initial ova composition. Although n-3 LC-PUFA from menhaden fish oil are essential for embryonic survival, sunshine bass appear to have lower n-3 LC-PUFA requirements after hatch. Larval survival was highly dependent on the presence of C18 PUFA present due to flax oil inclusion in maternal diets. Embryonic survival of progeny produced from broodstock fed dietary saturated fatty acid-rich plant lipids supplemented with intact LC-PUFA concentrates (~3.4% n-3 LC-PUFA; dry matter basis) was similar to that of the broodstock fed the menhaden fish oil control diet containing 4.8% n-3 LC-PUFA. Although the dietary requirement for n-3 LC-PUFA was reduced by feeding these LC-PUFA concentrates in combination with plant lipids, menhaden fish oil is still the most viable option for least cost broodstock diet formulations intended for white bass.

Seafood represents the most important source of long-chain polyunsaturated fatty acids (LC-PUFAs) in the human diet. However, consuming fish can present risks from persistent organic pollutants (POPs) that bioaccumulate in edible tissues following dietary exposure. In farmed fish, POPs accumulate as a result of feeding diets based on fish oil (FO). Fish oil substitution can reduce POP accumulation, but also results in loss of beneficial LC-PUFAs. Fish oil-based finishing diets at the end of production can restore LC-PUFAs, but this strategy also increases POPs. The present study assessed the use of saturated fatty acid (SFA)-rich lipids to replace fish oil in grow-out feeds in conjunction with a fish oil-rich finishing diet to determine if this strategy could produce hybrid striped bass with equal production performance, equivalent LC-PUFA levels, and reduced POP concentrations. Triplicate tanks of hybrid striped bass were raised on diets containing fish oil (100% FO), fish oil spiked with additional POPs (100% FO Spike), or blends (50/50 or 25/75) of FO and coconut (CO) or palm (PO) oils (50% CO, 50% PO, 75% CO, 75% PO) with and without an eight week finishing period with the 100% FO diet prior to harvest. Production performance, fillet LC-PUFA, and POP content were assessed. Production performance was not adversely affected by any of the feeding regimens. However, fatty acid profile was altered, with fillets of fish consuming less fish oil having lower LC-PUFA and POP levels. Finishing yielded a modest increase in fillet LC-PUFAs and POPs, but POPs accumulated more readily than LC-PUFAs during finishing. However, harvest fillet POP and LC-PUFA levels in the experimental groups were lower relative to levels in the 100% FO group. Replacing fish oil in aquafeeds can produce fish with reduced LC-PUFAs, and also reduced POPs. Feeding fish oil results in more rapid accumulation of POPs than LC-PUFA. Overall, the 75% fish oil replacement feeds yielded fish with the highest ratio of LC-PUFAs to POPs. Despite lower LC-PUFA content, fillets of fish fed the 75% fish oil replacement feeds could be incorporated into a weekly meal plan with other dietary sources of LC-PUFAs to meet dietary recommendations for these essential nutrients.

Seafood is the number one source of essential fatty acids, particularly, long-chain polyunsaturated fatty acids (LC-PUFA) in the human diet. As global population growth eventually surpasses what the world's wild commercial stocks can provide, reliance on the aquaculture industry to expand production will continue to increase in order to meet the demands of consumers worldwide. Currently, fluctuations in supply and cost coupled with environmental sustainability and contaminant concerns have motivated the aquaculture industry to research alternative lipid sources and feeding strategies in order to reduce the reliance on marine-derived resources. For most cultured species, replacing fish oil with terrestrial plant-based lipid sources is a minor dietary modification that has little consequence on production performance. However, fish raised on these plant-based lipid alternatives contain considerably higher medium chain polyunsaturated fatty acids (MC-PUFA) and n-6 fatty acids and less beneficial LC-PUFA and n-3 fatty acids within the fillets, thus negatively impacting the nutritional value of cultured seafood to the consumer. In order to alleviate this problem, producers can employ finishing strategies to restore fillet LC-PUFA content prior to harvest. As a complement to this approach, provision of dietary saturated fatty acids (SFA) and/or monounsaturated fatty acids (MUFA) in lieu of MC-PUFA appears to maximize the retention of LC-PUFA deposition during the grow-out period and may increase deposition during finishing. Accordingly, my objectives were to 1) assess whether the SFA, MUFA, and MC-PUFA content of the alternative lipid affected LC-PUFA levels in Nile Tilapia fed reduced fish oil feeds; and 2) using the optimal alternative lipid identified in the first objective, assess increasing fish oil replacement rates in conjunction with finishing to maximize product nutritional value and minimize fish oil usage in Nile Tilapia culture. To address the first objective, I assessed production performance and tissue composition of Nile Tilapia fed diets containing fish oil or blends of fish oil and various soybean-derived alternative lipids. Quadruplicate tanks of juvenile Nile Tilapia were fed diets containing fish oil (FISH, high in LC-PUFA) or a 50:50 blend of fish oil and standard (STD-SO, high in MC-PUFA), saturated fatty acid-enriched (SFA-SO, high in SFA), low α-linolenic (LO-ALA-SO, high in MC-PUFA), or hydrogenated (HYD-SO, high in MUFA) soybean oil for 16 weeks. Partial replacement of fish oil with soybean oils did not significantly affect production performance with the exception of the HYD-SO diet which yielded significantly reduced growth efficiency in comparison with some of the experimental diets, though not the FISH control. Despite distinctly different dietary fatty acid profiles, fillet fatty acid composition was similar among fish fed the FISH, SFA-SO, and HYD-SO diets. However, feeding the STD-SO and LO-ALA-SO diets resulted in significant enrichment of less desirable MC-PUFA and n-6 fatty acids within the fillet. Fillet LC-PUFA levels were equivalent among all groups despite the 50% reduction in dietary LC-PUFA intake among fish fed the soybean oil-based feeds. Based on these results, incorporation of STD-SO, SFA-SO, or LO-ALA-SO could be used as partial replacements for fish oil in Nile Tilapia feeds without impairing production performance, though SFA-rich soybean oils appeared to be the best alternative for maintaining a more "fish oil"-associated fillet fatty acid profile. Accordingly, the SFA-enriched soybean oil was selected for further study in the second objective trial that evaluated the effects of graded levels of fish oil replacement without or without implementation of finishing periods on production performance and fillet fatty acid composition. Nile Tilapia were fed feeds containing 100% fish oil (100-FO), the previously assessed SFA-enriched soybean oil (100-SFA-SO), or blends of fish oil and SFA-enriched soybean oil (50-SFA-SO, 75-SFA-SO). Triplicate groups of fish were fed the aforementioned diets exclusively throughout the feeding trial (100-SFA-SO unfinished, 75-SFA-SO unfinished, 50-SFA-SO unfinished) or in conjunction with 4 or 8 weeks of finishing with the 100-FO feed (100-SFA-SO + 4 wks, 100-SFA-SO + 8 wks, 75-SFA-SO + 4 wks, 75-SFA-SO + 8 wks, 50-SFA-SO + 4 wks, 50-SFA-SO + 8 wks) for a total of 20 weeks. Production performance was unaffected by dietary inclusion of SFA-enriched soybean oil when fed exclusively or in combination with fish oil, though growth performance was lower than observed in the previous trial and likely confounded by behavioral interactions and frequent spawning. After 12 weeks of consuming the SFA-enriched soybean oil grow-out diets, fillet levels of n-3 LC-PUFA were not statistically different from 100-FO control levels despite different levels of dietary inclusion. However, the high dietary levels of SFA in the experimental feeds did not translate into increased fillet SFA content, suggesting selective retention of LC-PUFA at the expense of fillet SFA. Finishing for 4 or 8 weeks increased fillet n-3 LC-PUFA content in all groups, though it appears that the 50- and 75-SFA-SO diets were more successful in maintaining acceptable health promoting n-3:n-6 ratios. Based on these results, SFA-enriched soybean oil-based feeds can be used as a cost-saving measure during grow-out, and the effects of these feeds on fillet fatty acid profile can be reversed to a considerable extent in as little as 4 weeks by implementing a finishing period prior to harvest. This approach is a promising strategy for minimizing fish oil usage while maximizing product value of cultured Nile Tilapia.

Factors affecting energy transfer to higher trophic levels can determine the survival and production of commercially important species and thus the success of fisheries management regimes. Juvenile salmon experience particularly high mortality during their early marine residence, but the root causes of this mortality remain uncertain. One potential contributing factor is the food quality encountered at this critical time. The nutritionally vital essential fatty acids (EFA) docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3) are essential to all marine heterotrophs, and their availability has the potential to affect energy transfer through a limitation-driven food quality effect. Assessing variability in DHA and EPA in an ecologically important prey species of juvenile salmon could give insight into the prevalence and severity of food quality effects. On the west coast of Vancouver Island (WCVI), one such species is the calanoid copepod Calanus marshallae. This omnivorous species possesses a high grazing capacity and the ability to store large amounts of lipids. As it is also an important prey item for a diverse array of predators, including juvenile Pacific salmon, C. marshallae plays a key role in energy transfer from phytoplankton to high-trophic iv consumers. This study quantified spatiotemporal variability in the quality of C. marshallae as prey for higher trophic levels using three polyunsaturated fatty acid indicators: DHA:EPA, %EFA and PUFA:SFA (polyunsaturated fatty acids to saturated fatty acids). Samples were collected on the WCVI in May and September of 2010 and May 2011. The environmental parameters included in the analysis were the phase of the Pacific Decadal Oscillation (PDO), sea surface temperature (SST), latitude, station depth, and season (spring versus late summer). Despite a phase shift in the PDO from positive to negative, overall means of the fatty acid indicators did not vary between May 2010 and May 2011. Same-station %EFA values rarely fluctuated more than 5%. DHA:EPA ratios were more variable but without a discernable pattern, while PUFA:SFA ratios decreased in shelf stations and increased offshore. Contrary to expectations, fatty acid indicators showed a weak positive correlation or no relationship with SST, nor was there a relationship with latitude. The narrow temperature range observed across all stations suggests that temperature may not play a significant role in PUFA availability off the WCVI. There were, however, significant relationships between the fatty acid indicators and bottom depth and season. Shelf and slope stations showed significantly higher %EFA and PUFA:SFA than did offshore stations (depth >800 m), with this gradient appearing stronger in May than September. While the food quality represented by C. marshallae was consistent across all shelf stations, the lower food quality observed offshore could potentially affect juvenile salmon growth along the WCVI where the shelf narrows to less than 5 km. / Graduate / dpbevan@uvic.ca