A series of experiments was conducted to test the overall hypothesis that reducing the omega-6 (n-6) to omega-3 (n-3) fatty acid (FA) ratio in sow diets would improve sow reproductive performance (characterized by increases in numbers and body weight of piglets born alive and weaned) and would lessen the inflammatory responses of their offspring post weaning. Diets were wheat/barley based and consisted of a control (tallow based, similar to a standard production diet), 3 diets with plant oil based n-6:n-3 ratios (9:1P, 5:1P, and 1:1P) and a 5:1 fish oil diet (5:1F). The control diet had a ratio of 8:1, but contained approximately half the polyunsaturated FA content of the other diets. Sows were randomly assigned to a treatment diet on d 80 of gestation, and remained on that treatment for three consecutive reproductive cycles (gestation/lactation 1 = P1, gestation/lactation 2 = P2, gestation/lactation 3 = P3). Experiment 1 was designed to test the hypothesis that reducing the n-6:n-3 FA ratio in sow diets would increase circulating concentrations of n-3 FA’s in sows and in their offspring, and the passive immune status of piglets would be improved. Performance data was collected throughout P1 and P2 on 150 sows (n = 30/diet). Sow and piglet serum, colostrum and milk were analyzed for FA profiles, and colostrum and piglet serum were analyzed for immunoglobulin (Ig) A and IgG. In P1, birth weights were unaffected by diet (P > 0.05). Average piglet weaning weights (P = 0.02) and ADG (P = 0.01) however, were highest for piglets born to sows consuming the 9:1P and 5:1P diets. During P2, 5:1F sows consumed 10% less feed (P = 0.04), their piglets had reduced birth weights (P = 0.05), and average weaning weight was reduced by 0.8 kg (P = 0.04) relative to control or 5:1P sows. Colostral and piglet plasma IgA and IgG were unaffected by diet (P > 0.05). Colostrum FA profile patterns were similar to that of the sow diets. Serum n-3 FA’s were greatest in sows (P < 0.01) and piglets (P < 0.01) consuming 1:1P or 5:1F diets. Serum α-linolenic acid (ALA) was highest in the 1:1P sows and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were highest in the 5:1F sows. In piglet serum obtained prior to suckling, ALA and DHA did not differ among treatments (P > 0.05) but EPA was 2.5 times greater in the 1:1P group and 4 times greater in the fish group (P < 0.01) compared to those from the control diet. In post-suckle samples, ALA was highest in serum from 1:1P diet piglets (P < 0.01), and EPA and DHA were highest in piglet serum from the 5:1F sows (P < 0.01). Omega-3 FA’s can perturb lipid metabolism, specifically increasing the lipolytic activity of adipose tissue and thus the second experiment tested the hypothesis that high producing sows, consuming reduced n-6:n-3 ratios would have increased body fat mobilization. Twenty sows per diet, farrowing ≥ 11 piglets and nursing ≥ 10 piglets during P3, were used. Performance data on sows and piglets (such as weights, numbers, backfat changes) was collected throughout lactation and milk samples obtained on d 4 and d 16 of lactation. Jugular catheters were inserted into 8 sows from each of the 9:1P and 1:1P groups on d 5 of lactation and sows were challenged with a single injection of epinephrine followed by serial blood collections. Feed intake was highest for sows consuming the control (8.4 kg/d) and 5:1P (8.2 kg/d) diets and lowest for the sows fed the 1:1P (7.4 kg/d) and 5:1F (7.7 kg/d) diets (P = 0.05). Altering the n-6:n-3 FA ratio did not affect sow BW, piglet ADG, milk DM and N content or the total output of milk (P > 0.2). Sows consuming the 1:1P diet had greater backfat thickness (P < 0.05) and numerically higher plasma NEFA at baseline compared with the 9:1P sows (240 vs 93 uM; P = 0.16). When given epinephrine, 9:1P fed sows tended to have lower net incremental area under the curve (niAUC) glucose (P = 0.08) and numerically higher niAUC NEFA (P = 0.17) and glycerol (P = 0.15). A third experiment was conducted to test the hypothesis that piglets raised by sows consuming reduced n-6:n-3 ratios would have reduced inflammatory responses post-weaning. Piglets (n = 20/diet) raised by sows consuming the treatment diets described above for 2 gestation/lactation cycles (P2) were selected at weaning. Within diet group, pigs were randomized to either a challenge control group (saline injected) or to a lipopolysaccharide (LPS) injected group (n=10/challenge•diet-1). Piglets were fed a common starter diet for 6 days followed by saline or LPS injections on d 7. Rectal temperatures were recorded for 24 hrs and blood samples were collected at 0, 2, 6 and 12 hrs post injection for pro-inflammatory cytokine and blood urea nitrogen (BUN) analysis. Injecting LPS caused decreased feed intake and reduced ADG (P < 0.01), and increased temperature and cytokine production (P < 0.05). Piglets raised by sows consuming the 1:1P diet had elevated temperatures (P = 0.01; diet x challenge P > 0.05). Overall, circulating plasma ALA and EPA were increased in sows and piglets when sows were fed a 1:1 plant based ratio compared to the control or high n-6:n-3 ratio groups. Sows fed a ratio of 1:1 mobilized more body fat relative to those consuming the 9:1 ratio; there were no treatment effects on piglet growth. Reducing maternal n-6:n-3 FA ratios below 5:1 increased piglet body temperature prior to and during an LPS induced inflammatory challenge,. Reducing the sow dietary n-6:n-3 FA ratio below 5:1 may have detrimental effects on piglets due to over-stimulation of inflammatory responses.

The aim of this research was to isolate and characterize novel strains of marine protists with potential to commercially produce PUFAs. Twelve trips were made visiting 10 different locations in the Canadian Maritime Provinces. Sixty-nine strains were isolated and screened for biomass and fatty acid production. Those meeting specific criteria were selected for further investigation including characterization by 18S rDNA sequencing. Isolate ONC-KTB-56 produced the greatest amount of biomass (1 807 mg L-1) and fatty acids (24.6% dry weight). Of the total fatty acids, ARA, EPA and DHA comprised 0.89, 1.22 and 4.7 percent, respectively. Isolate ONC-KTB-14 produced 1 704 mg L-1 dry biomass with 5.4 percent fatty acids including 1.44, 1.35 and 37.5 percent, ARA, EPA and DHA, respectively. Through optimization of culture conditions biomass, fatty acid content and the proportions of specific fatty acids can be increased. With such optimization, there is potential for isolates ONC-KTB-14 and ONC-KTB-56 to be grown at a commercial scale for production of PUFAs.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18343号 / 農博第2068号 / 新制||農||1024(附属図書館) / 学位論文||H26||N4850(農学部図書室) / 31201 / 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 植田 充美, 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

Yes / We investigated red blood cell (RBC) PUFA profiles, and the predictive value of RBC EPA content for tumour EPA exposure and clinical outcomes, in the EMT study, a randomised trial of EPA in patients awaiting colorectal cancer (CRC) liver metastasis surgery (A.J. Cockbain et al., 2014). There was a significant increase in RBC EPA in the EPA group (n=43; median intervention 30 days; mean absolute 1.26 [±0.14]% increase; P<0.001), but not in the placebo arm (n=45). EPA incorporation varied widely in EPA users and was not explained by treatment duration or compliance. There was little evidence of ‘contamination’ in the placebo group. The EPA level predicted tumour EPA content (r=0.36; P=0.03). Participants with post-treatment EPA ≥1.22% (n=49) had improved OS compared with EPA <1.22% (n=29; HR 0.42[95%CI 0.16–0.95]). RBC EPA content should be evaluated as a biomarker of tumour exposure and clinical outcomes in future EPA trials in CRC patients.

Adipose tissue is well-recognized as an endocrine organ which secretes a variety of bioactive molecules, including angiotensin II and its precursor angiotensinogen (Agt). There is mounting evidence linking the adipose renin-angiotensin system (RAS) and diet to obesity and obesity-related disorders. However, research addressing dietary regulation and function of adipose RAS is limited, and the specific mechanisms by which PUFAs modulate the endocrine function of adipose tissue remain largely unclear. There are several potential mechanisms that may mediate PUFA effects on Agt, including toll-like receptor signalling, prostaglandins or PPAR-gamma. Thus, we propose to investigate whether PUFAs differentially modulate Agt expression and secretion and to examine possible mechanisms by which PUFA alter Agt expression using the 3T3-L1 cell line. Differentiated 3T3-L1 adipocytes were treated with arachidonic acid (AA), eicosapentaenoic acid (EPA), AA + EPA, or vehicle (C) for 48 hours. Results showed a significant increase in intracellular Agt protein following treatment with PUFAs. Agt secretion, however, was only increased by AA. Interestingly, there is a dose-dependent decrease in Agt protein levels by EPA suggesting that a minimum concentration of n-3 PUFAs is required to elicit an Agt response. Agt mRNA levels were measured by RT-PCR and results showed a significant increase in Agt mRNA in response to treatment with AA but not EPA. These findings suggest that Agt regulation by PUFAs is complex and occurs both post-transcriptionally and post-translationally. Changes in mRNA stability may account for the observed effects of PUFAs. Adipocytes were treated with the transcriptional inhibitor actinomycin D (Act D) and Agt mRNA expression was measured over time. Total RNA was also measured at each time point to ensure that Act D treatment was effectively decreasing transcription. Agt mRNA expression was not significantly altered by treatment with EPA while treatment with AA increased Agt mRNA levels. These results suggest that Agt mRNA stability is differentially increased by n-6 but not n-3 PUFAs. Although there are clear effects of AA on Agt secretion and mRNA stability, the signaling pathways mediating this response remain to be determined, and additional studies are necessary to further dissect the underlying mechanisms of this regulation.

Adipose tissue is well-recognized as an endocrine organ which secretes a variety of bioactive molecules, including angiotensin II and its precursor angiotensinogen (Agt). There is mounting evidence linking the adipose renin-angiotensin system (RAS) and diet to obesity and obesity-related disorders. However, research addressing dietary regulation and function of adipose RAS is limited, and the specific mechanisms by which PUFAs modulate the endocrine function of adipose tissue remain largely unclear. There are several potential mechanisms that may mediate PUFA effects on Agt, including toll-like receptor signalling, prostaglandins or PPAR-gamma. Thus, we propose to investigate whether PUFAs differentially modulate Agt expression and secretion and to examine possible mechanisms by which PUFA alter Agt expression using the 3T3-L1 cell line.Differentiated 3T3-L1 adipocytes were treated with arachidonic acid (AA), eicosapentaenoic acid (EPA), AA + EPA, or vehicle (C) for 48 hours. Results showed a significant increase in intracellular Agt protein following treatment with PUFAs. Agt secretion, however, was only increased by AA. Interestingly, there is a dose-dependent decrease in Agt protein levels by EPA suggesting that a minimum concentration of n-3 PUFAs is required to elicit an Agt response. Agt mRNA levels were measured by RT-PCR and results showed a significant increase in Agt mRNA in response to treatment with AA but not EPA. These findings suggest that Agt regulation by PUFAs is complex and occurs both post-transcriptionally and post-translationally.Changes in mRNA stability may account for the observed effects of PUFAs. Adipocytes were treated with the transcriptional inhibitor actinomycin D (Act D) and Agt mRNA expression was measured over time. Total RNA was also measured at each time point to ensure that Act D treatment was effectively decreasing transcription. Agt mRNA expression was not significantly altered by treatment with EPA while treatment with AA increased Agt mRNA levels. These results suggest that Agt mRNA stability is differentially increased by n-6 but not n-3 PUFAs. Although there are clear effects of AA on Agt secretion and mRNA stability, the signaling pathways mediating this response remain to be determined, and additional studies are necessary to further dissect the underlying mechanisms of this regulation.

The biological activity of α-linolenic acid (ALA) is poorly understood and primarily associated with its conversion to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). This study used the Δ6 desaturase knockout (D6KO) mouse, which lacks Δ6 desaturase and therefore cannot convert ALA, to evaluate the independent effects of ALA on preventing non-alcoholic fatty liver disease (NAFLD). First, the capacity of very long chain fatty acids to rescue the D6KO lipid profile was established. Next, to evaluate the independent effects of ALA, D6KO or wild-type mice were fed diets containing lard, canola, flaxseed, or fish oil. Following treatment, liver phospholipid fatty acid composition was evaluated and livers were scored for steatosis and inflammation. Glucose tolerance was also evaluated. D6KO mice fed ALA-rich diets had lower liver lipid accumulation, lower hepatic inflammation (8 weeks) and improved glucose tolerance (20 weeks) relative to lard-fed D6KO mice. Overall, this thesis supports an independent biological role for ALA. / D.W.L. Ma is funded by the Canola Council of Canada, Natural Sciences and Engineering Research Council of Canada and the Canada Foundation for Innovation Leaders Opportunity Fund with matching from the Ontario Research Fund; Jessica Monteiro is funded Ontario Graduate Scholarship.

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

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.