The key defects characteristic of hyperglycemia in T2D include increased hepatic glucose production, a diminution of insulin secretion, and an absolute impairment in peripheral insulin action. The objective of my thesis was to investigate the molecular mechanisms leading to fatty acid induced β-cell dysfunction and determine the role of a novel transcriptional coregulator in the regulation of hepatic glucose homeostasis. The first part of my work focused on the chronic effects of fatty acids on human pancreatic β-cell function. Using microarray technology I established an important role for fatty acids in the pathogenesis of β-cell dysfunction. Accordingly chronic exposure of islets to oleate resulted in a significant reduction in glucose-stimulated insulin secretion and to an increase in the rate of reactive oxygen species generation. Additionally, pre-treatment of human islets with oleate led to a significant increase in the rate of oxidation of this fatty acid and to a significant decrease in glucose oxidation. My data indicate that chronic exposure of human islets to fatty acids activates inflammatory and metabolic pathways that lead to oxidative stress. In addition, the first part of my work demonstrated that fatty acids induce oxidative stress in vitro an effect that is preventable to a large extent by the use of antioxidants. In this setting and recapitulating the human islet data, fatty acids are causally linked to impaired insulin secretion, and the induction of oxidative stress. Our report demonstrated that oxidative stress plays a key role in the decrease in β-cell function induced by chronic lipotoxicity. My work also demonstrated that fatty acids are causally linked to the induction of endoplasmic reticulum stress in human islets. Finally, in the second part of my work I provide novel evidence for the role of PHIP in the regulation of hepatic gluconeogenesis. My work is the first to demonstrate that PHIP suppresses hepatic gluconeogenesis in vitro and in vivo. PHIP is amongst the few proteins that have ever been reported to suppress gluconeogenesis to date. PHIP thus represents a novel target for pharmaceutical intervention of diabetes and the suppression of hepatic glucose production.

Diet plays an important role in the development of chronic metabolic diseases (diabetes, obesity, cardiovascular disease) and as dietary fat consumption has increased, so has the incidence of these disorders. Metabolic syndrome, a clustering of risk factors that includes central obesity, increased plasma triacylglycerol (TG), elevated fasting glucose and glucose intolerance is perhaps the most notorious and aggressive. Animal and human studies indicate that bioactive fatty acids can influence cellular energy metabolism. Using susceptible rodent models (apoE-/- and LDLr-/- mice and Syrian Golden hamsters) this project investigated whether supplementation of a western type diet (WD) with bioactive fatty acids could improve hepatic lipid metabolism, plasma lipoprotein profiles or liver markers of lipogenesis. In mice, dietary supplementation with t-10, c-12 conjugated linoleic acid (CLA) decreased the weight gain induced by high fat diet compared with WD (p<0.01) and was accompanied by hyperinsulinemia (p<0.05) in the ApoE-/- and hypoadiponectinemia (p<0.01) in both mice strains. Although t-10, c-12 CLA supplementation increased plasma lipids and was associated with profound liver steatosis there was a reduction in atherosclerotic lesions in both mouse models (p<0.05). Analysis of mRNA and protein levels in the liver suggested that the differences in liver and plasma lipids may reflect inappropriate lipogenic response to t-10,c-12 CLA. In the high fat and fructose-fed hamster, the modulating role of fish fatty acids was investigated. The addition of DHA increased weight gain and adiposity compared to EPA and c-9, t-11 CLA supplementation. However, glucose tolerance was improved after 6 weeks of DHA supplementation (p? 0.01). Using [35S]methionine radiolabelling, DHA supplementation decreased apolipoprotein B100 synthesis and secretion. Newly synthesized cellular and secreted TG, as measured by [3H]glycerol incorporation, were also decreased with DHA supplementation. Although the effects of EPA were similar to those with DHA, the magnitude was generally lower. These results suggest that supplementation with fish fatty acids can improve several of the risk factors of the metabolic syndrome. Taken together, these observations indicate that some, but not all, bioactive fatty acids may be useful supplements for mediating cardiovascular risk factors.

Key aspects of the basic agronomy Camelina sativa were evaluated under controlled environment conditions and at multiple field locations in 2011 and 2012. Camelina is a highly adaptable crop. It germinates well even under low water availability and has a great potential for yield compensation. The line CDI007 was the most promising genotype with the highest yield potential, the lowest glucosinolate content, and the highest tolerance to downy mildew. The optimum N rate for seed yield varied by year and location: 100 kg N/ha at Truro and Canning in 2011, 120-150 kg N/ha at Canning, Truro and New Glasgow, 160-200 kg N/ha at Fredericton in 2012. N was positively correlated with protein content, but negatively correlated with oil content. Application of sulphur increased protein content at all of the sites and yield at some of the sites. In general, camelina response to S was maximized when N was sufficient.

Co-immunoprecipitation (co-IP) followed by proteomic analysis was employed to examine the phosphoenolpyruvate carboxylase (PEPC) interactome of developing castor oil seed (COS) endosperm. Earlier studies suggested that immunologically unrelated 107-kDa plant-type and 118-kDa bacterial-type PEPCs (p107/PTPC and p118/BTPC, respectively) are subunits of an unusual ~910-kDa hetero-octameric Class-2 PEPC complex of developing COS. The current results confirm that a tight physical interaction occurs between p118 and p107 since p118 quantitatively co-IP’d with p107 following elution of COS extracts through an anti-p107-IgG immunoaffinity column. No PEPC activity or immunoreactive PTPC or BTPC polypeptides were detected in the corresponding flow-through fractions. Although BTPCs lack the N-terminal phosphorylation site characteristic of PTPCs, Pro-Q Diamond Phosphoprotein staining, immunoblotting with phospho-(Ser/Thr) Akt substrate IgG, and phosphate-affinity PAGE demonstrated that the co-IP’d p118 was significantly phosphorylated at unique Ser and/or Thr residue(s). The co-IP of p118 and p107 was not influenced by their phosphorylation status. As p118 phosphorylation appeared unchanged 48 h following elimination of photosynthate supply due to COS depodding, the signaling mechanisms responsible for photosynthate-dependent p107 phosphorylation differ from those controlling p118’s in vivo phosphorylation. A third PEPC polypeptide of ~110-kDa (p110; RcPPC1) co-IP’d with p118 and p107 when depodded COS was used. Analysis of RcPpc1’s full-length cDNA sequence revealed p110’s identity with PTPCs, but that a pair of unique amino-acid substitutions occurs in its N-terminal sequence that may render p110 non-phosphorylatable in vivo. The plastidial pyruvate dehydrogenase complex (PDCpl) was identified as a novel PEPC interactor. Subcellular fractionation indicated that p118 and p107 are strictly cytosolic, but that PDCpl is targeted to both the cytosol and leucoplast of developing COS. Thus, a putative cytosolic metabolon involving PEPC and PDCpl could function to channel carbon from phosphoenolpyruvate to acetyl-CoA and/or to recycle CO2 from PDCpl to PEPC. / Thesis (Master, Biology) -- Queen's University, 2007-09-26 15:57:52.216

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Dietary fatty acid (FA) composition and energy restriction (ER) independently affect serum leptin levels; however it is not known whether this correlates with changes in obese (ob) gene expression. Herein, we assessed whether dietary FA composition and ER influence white adipose tissue (WAT) ob mRNA by Northern analysis. Animals consumed diets containing tallow (BT), safflower oil (SO) or fish oil (FO) ad libitum or at 60% ad libitum intakes. Serum leptin values were not different between levels of energy intake. ER decreased weight gain and WAT weights, which positively correlated with serum leptin values. WAT ob mRNA levels were in the rank order: FO > SO > BT in depots of all groups with ER showing a lower level of ob mRNA. Data show similarity in ob mRNA levels between depots with discordance in circulating leptin levels. These data suggest that energy restriction exerts greater control over leptin production than dietary fat source.

Liver fatty acid binding protein (L-FABP) binds and translocates many lipophilic substrates within the cytoplasm including long chain fatty acids. Moreover it was reported that L-FABP possesses antioxidative properties within hepatocytes. However, the mechanism of L-FABP’s antioxidative activity remains to be determined. Peroxisome proliferator activated receptor (PPAR) agonists and antagonists can regulate L-FABP levels. However, it needs to be investigated how PPAR agonists and antagonists regulate L-FABP expression. And whether the altered expression of L-FABP by these agents will affect its antioxidative properties within hepatocytes remains unclear. In this thesis we employed clofibrate (PPARα agonist), MK886 (PPARα antagonist), and GW9662 (PPARγ antagonist) to elucidate the mechanism whereby PPAR regulate L-FABP expression and what effect such expression has on the antioxidant activity of L-FABP in CRL-1548 hepatoma cells. Clofibrate served to upregulate L-FABP expression while MK886 and GW9662 were employed to inhibit L-FABP expression. The principal findings revealed that clofibrate treatment enhanced L-FABP mRNA stability and transcription, which resulted in increased L-FABP levels, while MK866 and GW9662 reduced these levels. We also demonstrated that increases in L-FABP levels were associated with reduced cytosolic reactive oxygen species (ROS), while L-FABP siRNA knockdown resulted in a decrease in L-FABP expression and an associated increase in ROS levels. The antioxidant mechanism of recombinant rat L-FABP in the presence of a hydrophilic (AAPH) and lipophilic (AMVN) free radical generators was also evaluated. Recombinant rat L-FABP was produced in E. coli and its amino acid sequence was confirmed by MALDI QqTOF MS. Antioxidant activity was assayed using the thiobarbituric acid method. Ascorbic acid served as a positive control for the AAPH reaction while α-tocopherol was used as a positive control for the AMVN reaction. The antioxidant activity of recombinant L-FABP was greater when free radicals were generated with AAPH than AMVN. Oxidative modification of L-FABP included up to five methionine oxidative peptides with a total of 80 Da mass shift compared to native L-FABP. These findings suggest that the mechanism of L-FABP’s antioxidant activity involved the reaction of methionine with free radicals. In conclusion, L-FABP expression is regulated by PPAR agonists and antagonists through transcription and mRNA stability. Moreover, methionine residues appear to play an important role in the antioxidative activity of L-FABP.

This study was carried out to investigate the fatty acid composition of tumoral, marginal and normal breast tissue in female breast cancer patients. Patients were recruited from St. Boniface General Hospital. A pre-operative blood sample was drawn. After surgery, sections were obtained from tumoral, marginal and normal breast tissues for histology and biochemical analysis. Extracted lipids from marginal tissue were significantly higher than those in normal or tumoral tissue. The lipid profile in tumoral tissue was significantly different in terms of fatty acid composition compared to normal and marginal tissue with less linoleic and alpha linolenic acid and more long chain polyunsaturated fatty acid of omega-3 and omega-6 series. Marginal tissue showed significantly less alpha linolenic acid compared to normal tissue. An inverse correlation existed between plasma level of 22:6 n-3 and breast cancer stage. We found different lipid profile in tumoral tissue compared to normal and marginal tissue.

The key defects characteristic of hyperglycemia in T2D include increased hepatic glucose production, a diminution of insulin secretion, and an absolute impairment in peripheral insulin action. The objective of my thesis was to investigate the molecular mechanisms leading to fatty acid induced β-cell dysfunction and determine the role of a novel transcriptional coregulator in the regulation of hepatic glucose homeostasis. The first part of my work focused on the chronic effects of fatty acids on human pancreatic β-cell function. Using microarray technology I established an important role for fatty acids in the pathogenesis of β-cell dysfunction. Accordingly chronic exposure of islets to oleate resulted in a significant reduction in glucose-stimulated insulin secretion and to an increase in the rate of reactive oxygen species generation. Additionally, pre-treatment of human islets with oleate led to a significant increase in the rate of oxidation of this fatty acid and to a significant decrease in glucose oxidation. My data indicate that chronic exposure of human islets to fatty acids activates inflammatory and metabolic pathways that lead to oxidative stress. In addition, the first part of my work demonstrated that fatty acids induce oxidative stress in vitro an effect that is preventable to a large extent by the use of antioxidants. In this setting and recapitulating the human islet data, fatty acids are causally linked to impaired insulin secretion, and the induction of oxidative stress. Our report demonstrated that oxidative stress plays a key role in the decrease in β-cell function induced by chronic lipotoxicity. My work also demonstrated that fatty acids are causally linked to the induction of endoplasmic reticulum stress in human islets. Finally, in the second part of my work I provide novel evidence for the role of PHIP in the regulation of hepatic gluconeogenesis. My work is the first to demonstrate that PHIP suppresses hepatic gluconeogenesis in vitro and in vivo. PHIP is amongst the few proteins that have ever been reported to suppress gluconeogenesis to date. PHIP thus represents a novel target for pharmaceutical intervention of diabetes and the suppression of hepatic glucose production.

The effect of naturally produced volatile fatty acids (VFAs) on the removal of 2, 4-D from a wastewater during the denitrification process was studied in this thesis. The VFAs were generated from an anaerobic digester using soya flour solution as a synthetic feed. The digester was operated at an SRT and HRT of 10 days. The pH (4.8 ± 0.2) and temperature (32 ± 3 ℃) of the digester were not controlled. A mean VFA concentration of 3153 ± 801 mg/L was achieved with acid speciation results of acetic (51.4 %), propionic (27.5 %), n-butyric (19.6 %) and iso-valeric (1.4 %). The specific VFA production rate was 0.014 mg VFA/mg VSS/day. The extent of the digestion process converting the substrate from a particulate to soluble form was evaluated as the specific TOC solubilization rate (0.007 mg TOC/mg VSS/day), soluble COD production rate (0.022 mg SCOD/mg VSS/day) and percent VSS reduction (14 %). The low solubilization rate is possibly due to high feed solids (3.4%) which led to a heavily overloaded bioreactor. It also suggests that the particulate substrate was not entirely amenable to solubilization. The acclimation of 2, 4-D degrading bacteria was developed successfully in an SBR fed with sewage and 2, 4-D (30-100 mg/L) as carbon and energy sources. A mean MLSS of 3653 ± 547 mg/L and an SRT of 20 ± 9 days were observed during the research period. The settleability of the SBR sludge was excellent evidenced by a low sludge volume index (SVI) of 101 ± 50 mL/g and less than 5 mg/L of effluent suspended solids. The specific 2, 4-D degradation rate was 0.046 ± 0.018 mg/mg MLSS/day. However, the removal of 2, 4-D during 60 minutes of non-aerated phase was negligible while more than 90 % of the 2, 4-D was removed within 240 minutes of the aerated phase. The successful degradation of 2, 4-D is related to the length of the acclimation period, as the acclimation period increased, the specific biodegradation rate increased. A biosorption study using ultrasound pre-treatment of the SBR acclimated biomass suggested that less than 10 % of the removal of 2, 4-D was due to biosorption, while more than 90 % removal of the 2, 4-D was likely due to biodegradation. Denitrification batch tests (using SBR-acclimated biomass) demonstrated that the addition of a digester effluent rich in naturally-produced VFAs increased both the 2 specific denitrification rate and the 2, 4-D degradation efficiency, as compared to that using 2, 4-D as a sole carbon source. In particular, the specific denitrification rates increased from 0.0119 ± 0.0039 to 0.0192 ± 0.0079 to 0.024 ± 0.003 g NO₃-N/g VSS per day, when using 2, 4-D alone, 2, 4-D plus natural VFAs and natural VFAs alone as carbon sources. The percent 2, 4-D removal increased from 28.33 ± 11.88 using 2, 4-D alone as a carbon source to 54.17 ± 21.89 using 2, 4-D plus natural VFAs as carbon sources. The specific 2, 4-D degradation rate and 2, 4-D removal efficiency of unacclimated biomass were 2.0 to 2.5 times less than those of the acclimated biomass. Natural VFAs and synthetic VFAs were found to be identical in denitrification batch tests in terms of their use as a carbon source. The mean specific denitrification and VFA-C consumption rates as well as the mean specific 2, 4-D degradation rate derived from experiments using natural VFAs and 2, 4-D as carbon sources were close to the valuess from experiments using synthetic VFAs and 2, 4-D as carbon sources. Further exploration of 2, 4-D degradation behaviour with pulsed additions of NO₃-N did not find further significant 2, 4-D removal, although almost all of NO₃-N was used by the end of the experimental run due to endogenous carbon sources used for cell maintenance and growth. However, the higher the concentration of biomass used in the denitrification batch system, the larger the amount of 2, 4-D degraded and the faster the VFA-C and NO₃-N were consumed. Further research with respect to optimisation of the acid-phase anaerobic digestion process (e.g. to adjust SRT and HRT or to lower the solid content of synthetic feed) would improve the specific VFA production rate and the solubilization rate. More research on the SBR could be carried out to investigate its maximum 2, 4-D removal capability as well as the removal of other structurally related herbicides. Attempts could be made to stimulate the growth of denitrifiers in the SBR (e.g. to add certain amounts of NO₃-N according to proper C: N ratios or to increase the length of non-aerated time). More microbiological studies of 2, 4-D degrading bacteria may also be helpful to understand the combined SBR/denitrification and 2, 4-D degradation process. More theoretical aspects of modelling kinetics could be developed to apply the combined process in-situ at 2, 4-D contaminated sites.