Plants are being engineered for enhanced ethanol production; however, challenges remain in meeting the demand for bioenergy that is expected to double by 2030. Therefore, targeting carbon accumulation in the form of energy-dense oils in nonseed biomass is considered a superior alternative for bioenergy production. Although oils in the form of triacylglycerols (TAGs) are typically stored in seed tissues, various nonseed tissues such as mesocarp, tubers, stems and leaves also serve as storage tissues for TAG accumulation in plants. Moreover, the biomass of these tissues is generally far greater than seed biomass. In order to increase oil content in nonseed biomass for bioenergy and nutritional purposes, it is important to understand how such plants naturally accumulate TAG in nonseed tissues. Several molecular approaches, including transcriptomics, have been undertaken to elucidate the metabolic and regulatory mechanisms of carbon partitioning in oil-rich nonseed tissues. Such studies are expected to generate important transgenic tools that can be used to alter fatty acid metabolism and engineer plants to produce oil-rich biomass successfully. This review focuses on the potential of different oil-rich nonseed tissues and the strategies developed for enhancing oil biomass.

This project examined the association between dietary patterns, erythrocyte membrane fatty acids concentration, and Alzheimer’s-related genes in systemic inflammation, as indicated by C-reactive protein (CRP) levels, in order to achieve more comprehensive knowledge of how nutrition and genetics influence systemic inflammation among the elderly residents of Cache County, Utah. First, the associations between dietary patterns defined by Dietary Approaches to Stop Hypertension (DASH) and Mediterranean dietary patterns (MED) and the risk of having a high level of CRP were examined. This study showed that a healthy dietary pattern score was associated with CRP levels; a higher score reflecting the ideal DASH diet and MED diet was associated with a 26% and 27% reduction in the risk of having high CRP levels respectively. This association appeared stronger among overweight and obese individuals. Second, the association between erythrocyte membrane fatty acids (EMFAs) and elevated serum C-reactive protein (CRP) levels was examined. Those that had high EMFAs composition of palmitoleic acid and nervonic acids, both monounsaturated fatty acids (MUFAs), and dihomo-y-linolenic acid (DGLA), docosapentaenoic acid (DPA-6), docosahexaenoic acid (DHA), all polyunsaturated fatty acids (PUFAs), had an increased risk of having CRP elevation. In contrast, risk of CRP elevation was reduced in those that have highest levels of saturated fatty acids (SFAs) of margaric acid, stearic acid, and arachidic acid. These associations were generally observed to be stronger among women compared to men. Lastly, the study examined whether AD-related genes identified in previous genome-wide association studies are associated with elevated levels of inflammatory CRP. Results revealed a strong association between APOE-epsilon genotypes and CRP levels. Results also showed an association between major alleles of APOE rs439401, TOMM40 rs157580, and minor alleles MMP8 rs1892886, CR1 rs6656401, CR1 rs3818361, and CR1 rs4844609 that were associated with a risk of elevation of CRP. These associations were stronger among men compared to women. Reduction in the prevalence of AD could have tremendous importance; the results of this dissertation may help identify factors important to AD etiology and, in turn, provide valuable targets for prevention.

Fatty acid-binding proteins are small intracellular proteins with poorly defined functions in intracellular fatty acid transport. The adipocyte fatty acid-binding protein aP2 regulates systemic glucose and lipid metabolism. Using Affymetrix microarrays, we found that aP2, in addition to being abundantly expressed by adipocytes, is also expressed by airway epithelial cells. aP2 expression was markedly increased following stimulation of epithelial cells with the Th2 cytokines IL-4 and IL-13, and downregulated by the Th1 cytokine IFN-gamma. Regulation of aP2 mRNA expression by Th2 cytokines was dependent on STAT6, a transcription factor with a major regulatory role in allergic inflammation. We examined aP2 deficient mice in a model of allergic airway inflammation, and found that infiltration of leukocytes, especially eosinophils, into the airways was highly aP2 dependent. T cell priming and peritoneal allergy was unaffected by aP2 deficiency suggesting that aP2 was acting locally within the lung, and analysis of bone marrow chimeras implicated non-haematopoietic cells, most likely airway epithelial cells, as the site of aP2 action in allergic airway inflammation. Expression of the pro-inflammatory cytokines MCP-1 and IL-6 was impaired in cytokine activated aP2 deficient airway epithelial cells, while levels of the anti-inflammatory arachidonic acid metabolite 15-HETE was increased, providing a mechanism for the reduced airway inflammation in aP2 deficient mice. In addition to the immune functions of aP2, we found that the related fatty acid-binding protein mal1 was also upregulated by IL-4/IL-13 in airway epithelial cells, and mal1 deficient mice were protected against airway eosinophilia. Significantly, in comparison to single aP2 deficiency, mice with combined aP2-mal1 deficiency had augmented protection against airway inflammation, and bone marrow chimera experiments demonstrated that aP2-mal1 deficiency affected both non-haematopoeitic and haematopoeitic cells. In T cell priming experiments, aP2-mal1 deficiency resulted in defective cytokine profiles in antigen recall responses, suggesting compromised sensitisation to antigen as one mechanism for aP2-mal1 action in airway inflammation. Together, our data therefore demonstrates the crucial roles of fatty acid-binding proteins in airway epithelium, T cell priming and airway inflammation, and provides a new link between fatty acid signalling and allergy.

Beta-cell failure is a key step in the progression from metabolic disorder to overt type 2 diabetes (T2D). This failure is characterised by both secretory defects and loss of beta-cell mass, the latter most likely through increases in the rate of apoptosis. Although the mechanisms underlying these beta-cell defects are unclear, evidence suggests that chronic exposure of beta-cells to elevated fatty acid (FA) plays a role in disease development in genetically susceptible individuals. Furthermore, it has been postulated that endoplasmic reticulum (ER) stress signalling pathways (the unfolded protein response; UPR) play a role in FA-induced beta-cell dysfunction. The broad aim of this thesis was to explore the nature of these relationships. Experiments detailed in this thesis demonstrate that MIN6 beta-cells mount a comprehensive ER stress response with exposure to elevated saturated fatty acid palmitate, but not the unsaturated fatty acid, oleate, within the low elevated physiological range. This response was time-dependent and involved both transcriptional and translational changes in UPR transducers and targets. The differential activation of ER stress in MIN6 beta-cells by saturated, but not unsaturated FA species may represent a mechanism of differential beta-cell death described in many studies with these FA. Furthermore, these experiments describe defects in ER to Golgi trafficking with chronic palmitate treatment, but not oleate or thapsigagin treatment, identifying this as a potential mechanism by which palmitate treatment induces ER stress. Moreover, these studies have shown the relevance to ER stress to a whole body model of T2D by demonstrating UPR activation in the islets of the db/db mouse. In conclusion, studies detailed in this thesis have demonstrated that ER stress occurs in in vitro and in vivo models of beta-cell lipotoxicity and apoptosis. In addition, these studies have identified defects in ER to Golgi trafficking as a mechanism by which palmitate treatment induces ER stress. These studies highlight the importance of ER stress in the development of T2D.

The current dietary recommendations for optimal health are designed to increase our intake of two bioactive omega-3 (n-3) fatty acids, eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), abundant naturally in fatty fish such as salmon. Health authorities recommend that the general population consume two to three fatty fish meals per week (1) for optimal health and for the prevention of cardiovascular disease. However, some modern Western societies consume only modest amounts of fish and seafood (2;3). Land based vegetable oils may provide an alternative to meet these needs. Linseed and canola oils are rich in alpha-linolenic acid (ALA, 18:3n-3) (4). ALA can be converted endogenously to EPA and DHA and suggests that increasing the dietary intake of ALA may increase the conversion and accumulation of DHA in tissues and plasma. However, elevated dietary intakes of ALA in animals and humans results in an increased level of EPA in tissues yet there is little or no change in the level of DHA (5-7). The current consensus is that the synthesis of DHA from ALA in humans is limited yet the mechanisms involved in regulating the accumulation of DHA in tissues are poorly understood. The reputed rate-limiting enzyme in the conversion of fatty acids is delta 6 desaturase (D6D). ALA is a substrate for D6D and undergoes a series of desaturation and elongation reactions to yield n-3 long chain polyunsaturated fatty acids (LCPUFA). The final step in the synthesis of DHA from ALA involves translocation of its immediate fatty acid precursor, 24:6n-3 from the endoplasmic reticulum to the peroxisome to be partially beta-oxidised to yield DHA. The involvement of multiple enzymes in the desaturation-elongation pathway, and the integration of other pathways, such as phospholipid biosynthesis, suggests there are various steps that may regulate the accumulation of DHA in cell membranes. This thesis aimed to examine the possible regulatory steps in the conversion of fatty acids to LCPUFA, particularly in the synthesis of DHA from n-3 fatty acid precursors. The human hepatoma cell line, HepG2, was used as an in vitro cell system to examine the accumulation of individual fatty acids and their metabolites in isolation from other competing fatty acid substrates. The accumulation of linoleic acid (LA, 18:2n-6) and ALA in HepG2 cell phospholipids following supplementation with increasing concentrations of each respective fatty acid correlated with that described in vivo, as was the accumulation of their conversion products. The accumulation of DHA in cells supplemented with ALA reached a plateau at concentrations above 5 micro g/ml and paralleled the accumulation of 24:6n-3 in cell phospholipids, suggesting that the delta 6 desaturation of 24:6n-3 was prevented by increasing concentrations of ALA, thereby limiting the accumulation of DHA. The accumulation of DHA in cells supplemented with eicosapentaenoic acid (EPA, 20:5n-3) or docosapentaenoic acid (DPA, 22:5n-3) was significantly greater than the level of DHA that accumulated in cells supplemented with ALA. However, regardless of substrate, the level of DHA in cell membranes reached a plateau at substrate concentrations above 5 micro g/ml. This thesis further aimed to examine the effect of fatty acid supplementation on the mRNA expression of D6D in HepG2 cells. The expression and activity of D6D mRNA is subject to nutritional and hormonal regulation. The mRNA expression of D6D in HepG2 cells following supplementation with oleic acid (OA, 18:1n-9), LA, ALA, arachidonic acid (AA, 20:4n-6) or EPA was examined by real time RT PCR. The expression of D6D mRNA was reduced by up to 50% in cells supplemented with OA, LA, ALA , AA or EPA compared with control cells and suggests that fatty acids modulate the expression of the key enzyme involved in the conversion of fatty acids. The effect of fatty acid co-supplementation on the fatty acid composition of HepG2 cell phospholipids was also examined in an attempt to gain insights into the role of D6D and the enzymes involved in peroxisomal beta-oxidation on the accumulation of DHA from n-3 fatty acid precursors. The reduction in the accumulation of DHA in cells co-supplemented with DPA and docosatetraenoic acid (DTA, 22:4n-6) was greater than in cells co-supplemented with DPA and LA, suggesting that peroxisomal beta-oxidation may have a greater role in determining the accumulation of DHA from DPA than the activity of D6D. Further investigation should be directed towards understanding the role that peroxisomal beta-oxidation may play in the synthesis of DHA from precursor fatty acids. The fatty acid composition of cell membranes in vivo is a result of several physiological processes including dietary intake, phospholipids biosynthesis and fatty acid conversion as well as catabolic processes. This thesis demonstrates that a greater understanding of the regulation of the conversion of fatty acids will help to define dietary approaches that enhance the synthesis of n-3 LCPUFA from n-3 fatty acid precursors to lead to improved outcomes for health.

Mitochondrial fatty acid �-oxidation disorders are a group of clinically and biochemically heterogeneous defects mainly associated with intolerance to catabolic stress. The diseases are potentially fatal, but treatable and the prognosis for most diagnosed disorders is generally favourable. Early diagnosis is thus important to prevent morbidity and mortality. This project describes an improved and validated quantitative fibroblast acylcarnitine profile assay for the investigation of suspected fatty acid �-oxidation disorders. Intact cells were incubated with deuterium-labelled hexadecanoate and L-carnitine, and the accumulated acylcarnitines in the medium analysed using electrospray tandem mass spectrometry. This modified procedure is less demanding technically, requires fewer cells and better reflects the in vivo acylcarnitine status than previously published methods. Mitochondrial fatty acid �-oxidation is coupled to the respiratory chain. Functional defects of one pathway may lead to secondary alterations in flux through the other. The diagnostic specificity and the prognostic potential of the in vitro acylcarnitine profile assay were investigated in fibroblasts from 14 normal controls, 38 patients with eight enzyme deficiencies of fatty acid �-oxidation presenting with various phenotypes, and 16 patients with primary respiratory chain defects including both isolated and multiple enzyme complex defects. All fatty acid �-oxidation deficient cell lines revealed disease-specific acylcarnitine profiles related to the sites of defects irrespective of the severity of symptoms or of different mutation. Preliminary studies suggested a correlation between severity of symptoms and higher concentrations of long-chain acylcarnitine species. However, the fibroblast acylcarnitine profiles from some patients with respiratory chain defects were similar to those of controls, whereas others had abnormal profiles resembling those found in fatty acid �-oxidation disorders. In vitro acylcarnitine profiling is useful for the detection of fatty acid �-oxidation deficiencies, and perhaps the prediction of disease severity and prognostic evaluation facilitating decisions of therapeutic intervention and genetic counselling. However, abnormal profiles do not exclusively indicate these disorders, and primary defects of the respiratory chain remain a possibility. Awareness of this diagnostic pitfall will aid in the selection of subsequent confirmatory tests and therapeutic options.

Considering the major role of insulin signaling on fatty acid synthesis via stimulation of lipogenic enzymes, differential effects of insulin signaling on individual carbon fluxes for fatty acid synthesis have been investigated by comparing the individual lipogenic fluxes in WT and IRS-1 knockout (IRS-1 KO) brown adipocytes. Results from experiments on WT and IRS-1 KO cells incubated with [5-¹³C] glutamine were consistent with the existence of reductive carboxylation pathway. Analysis of isotopomer distribution of nine metabolites related to the lipogenic routes from glucose and glutamine in IRS-1 KO cells using [U-¹³C] glutamine as compared to that in WT cells indicated that flux through reductive carboxylation pathway was diminished while flux through conventional TCA cycle was stimulated due to absence of insulin signaling in IRS-1 KO cells. This observation was confirmed by quantitative estimation of individual lipogenic fluxes in IRS-1 KO cells and their comparison with fluxes in WT cells. Thus, these results suggest that glutamine’s substantial contribution to fatty acid synthesis can be directly manipulated by controlling the flux through reductive carboxylation of alpha-ketoglutarate to citrate using hormone (insulin). / Singapore-MIT Alliance (SMA)

Thesis (Ph.D. (Environmental Science)--North-West University, Potchefstroom Campus, 2007.

As nontoxic biobased surfactants derived from plant oils and cellulose or starch, saccharide-fatty acid esters are widely used in cosmetics, food, and pharmaceutical industries due to their biocompatibility, biodegradability as well as antimicrobial activity. Generally, saccharide-fatty acid esters are synthesized chemically under high pressure, temperature and the presence of alkaline or acid catalysts leading to low-quality products (chemo-degradation of double bonds and oxygenated moieties) and large amounts of byproducts. In contrast, biocatalytic synthesis enhances sustainability: near-ambient pressure and temperature, the absence of toxic, acids and bases catalysts, and improved selectivity of products. For lipase-catalyzed synthesis under nearly anhydrous conditions, the major hurdle to be overcome is the poor miscibility of the acyl donor and acceptor substrates, resulting in slow reaction rates. Although several approaches such as, the employments of organic solvents, complexation agents, and ionic liquids, have been reported in the literature, a robust solution is desperately needed. This study focused on employing immobilized lipases under completely solvent-free conditions to synthesize saccharide-fatty acid esters using the ester products to enhance miscibility. Experimentally, metastable saccharide particles with a diameter of 10-100 micron-sized suspensions of saccharide were formed in oleic acid-rich ester mixtures initially for synthesis of saccharide-fatty acid esters in packed bed bioreactor containing immobilized lipases. Water, a by-product that limits ester yield by promoting hydrolysis, was removed via free evaporation. In this dissertation, a bioreactor system was developed for the eco-friendly solvent-free, immobilized lipase-catalyzed synthesis of biobasaed surfactants utilizing suspensions as reaction medium with 88 wt% in 6 days; the performance of the bioreactor systems developed for Objective 1 was optimized through water concentration control and interval time with 91 wt% in 4.8 days; and to improve design of bioreactor system developed in Objective 1 by in-line filter and derive a mathematical model to describe the production of esters by the bioreactor systems developed. Finally, 84 wt% ester content was achieved in 8.4 days.

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.