published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy

Background and objectives: Growing bodies of evidence demonstrate that adipocyte fatty acid binding protein (A-FABP) mediates the pathogenesis of atherosclerosis through its direct impacts on macrophages. Loss-of-function study was conducted by utilizing peritoneal macrophages derived from A-FABP knockout (KO) mice, to investigate the role of A-FABP in autophagy and macrophage foam cell formation. Key findings: 1. No morphological changes between the peritoneal macrophages derived from A-FABP knockout (KO) or their wild-type (WT) littermates. 2. Foam cell formation was successfully induced by the treatment of acetylated low-density lipoproteins (LDL) in peritoneal macrophages derived from A-FABP WT and KO mice. 3. LDL treatment induces autophagy in peritoneal macrophages from both A-FABP WT and KO mice. 4. The extent of LDL-induced autophagy is reduced in peritoneal macrophages of WT mice and is accompanied by increased lipid droplet accumulation when compared with A-FABP KO mice. Conclusions: A-FABP is a suppressor of autophagy and contributes to the attenuation of cholesterol efflux, subsequently resulting in enhancement of lipid droplets accumulation in peritoneal macrophages. A-FABP mediates the formation of macrophage foam cell via the suppression of autophagy. The results suggest that A-FABP is a potential therapeutic target to suspend the progression of atherosclerosis and remit the atherosclerotic lesion. / published_or_final_version / Medicine / Master / Master of Medical Sciences

Flax (Linum usitatissimum L.) is one of the richest plant sources of omega-3 fatty acids praised for their health benefits. In this study, the extent of the genetic variability for genes encoding stearoyl-ACP desaturase (SAD), fatty acid desaturase 2 (FAD2) and 3 (FAD3) was determined by sequencing the six paralogous genes from 120 flax accessions representing a broad range of germplasm including some EMS mutant lines. A total of 6 alleles for sad1 and sad2, 21 for fad2a, 5 for fad2b, 15 for fad3a and 18 for fad3b were identified. Deduced amino acid sequences of the alleles predicted 4, 2, 3, 4, 6, and 7 isoforms, respectively. Allele frequencies varied greatly across genes. Fad3a, with 110 SNPs and 19 indels, and fad3b, with 50 SNPs and 5 indels, showed the highest levels of genetic variation. While most of the SNPs and all the indels were silent mutations, both genes carried non-sense SNP mutations resulting in premature stop codons, a feature not observed in sad and fad2 genes. Some alleles and isoforms discovered in induced mutant lines were absent in the natural germplasm. Correlation of these genotypic data with fatty acid composition data of 120 flax accessions phenotyped in six field experiments revealed statistically significant correlations of some of the SAD and FAD isoforms on fatty acid composition, oil content and iodine value. The novel allelic variants and isoforms identified for the six desaturases will be a resource for the development of oilseed flax with unique and useful fatty acid profiles. / October 2015

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

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.

Replacement of saturated fatty acids (SFA) with monounsaturated fatty acids (MUFA) or polyunsaturated fatty acids (PUFA) impacts risk of atherosclerosis and cardiovascular disease (CVD). However, although dietary fatty acids (DFA) have been established as an important factor related to CVD, their exact mechanisms of action have not been clearly established. One of the possible mechanisms is that DFA convert to fatty acid ethanolamides (FAEs), such as oleoylethanolamide (OEA), palmitoylethanolamide (PEA) and arachidonoylethanolamide (AEA), which are thought to associate with lipid signalling, fat oxidation and appetite control. Hence, the objectives of this thesis were to identify the impact of diets containing corn oil, canola oil, DHA + canola oil and fish oil on plasma and organ levels of FAEs as well as energy metabolism and lipid profiles in Syrian Golden hamsters. Forty-eight hamsters were provided diets containing 6% treatment oil for 30 d before sacrifice. Across all diets, in proximal small intestine and liver, animals fed canola oil showed higher (p<0.05) levels of OEA than corn oil and fish oil fed groups, but no difference compared to those fed DHA +canola oil. In plasma, fish oil fed animals showed higher (p<0.05) OEA and PEA levels and lower (p<0.05) AEA levels compared to all other groups. Feed intakes (g/d), oxygen consumption (ml/g) and body composition of total fat (%) and mass (g) did not differ across groups. However, energy expenditure associated with fat oxidation (%) was higher (p<0.01) in canola oil and DHA + canola oil fed hamsters compared to those consuming corn oil and fish oil. Also, body composition of fish oil fed animals showed a lower (p<0.01) total lean mass (g) compared to other three groups and a lower (p<0.01) total mass (g) compared to DHA + canola oil diets, but no difference compared to animals fed the canola oil diet. None of the treatments had any effect on triglyceride (TG) or C-reactive protein (CRP) levels. The fish oil group showed a higher (p<0.01) plasma total cholesterol (TC) levels than all other three groups. No differences existed between DHA + canola oil and fish oil groups in HDL or Non-HDL levels, but these levels were different (p<0.01) compared to corn oil group and canola oil groups. To conclude, different DFA affect whole body energetics and plasma lipid profiles. Also DFA produced marked shifts in plasma and organ levels of OEA, PEA and AEA. These dietary induced shifts in FAEs may translate into discernable changes in energy expenditure and lipid levels which in turn influence CVD risk.

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.

Replacement of saturated fatty acids (SFA) with monounsaturated fatty acids (MUFA) or polyunsaturated fatty acids (PUFA) impacts risk of atherosclerosis and cardiovascular disease (CVD). However, although dietary fatty acids (DFA) have been established as an important factor related to CVD, their exact mechanisms of action have not been clearly established. One of the possible mechanisms is that DFA convert to fatty acid ethanolamides (FAEs), such as oleoylethanolamide (OEA), palmitoylethanolamide (PEA) and arachidonoylethanolamide (AEA), which are thought to associate with lipid signalling, fat oxidation and appetite control. Hence, the objectives of this thesis were to identify the impact of diets containing corn oil, canola oil, DHA + canola oil and fish oil on plasma and organ levels of FAEs as well as energy metabolism and lipid profiles in Syrian Golden hamsters. Forty-eight hamsters were provided diets containing 6% treatment oil for 30 d before sacrifice. Across all diets, in proximal small intestine and liver, animals fed canola oil showed higher (p<0.05) levels of OEA than corn oil and fish oil fed groups, but no difference compared to those fed DHA +canola oil. In plasma, fish oil fed animals showed higher (p<0.05) OEA and PEA levels and lower (p<0.05) AEA levels compared to all other groups. Feed intakes (g/d), oxygen consumption (ml/g) and body composition of total fat (%) and mass (g) did not differ across groups. However, energy expenditure associated with fat oxidation (%) was higher (p<0.01) in canola oil and DHA + canola oil fed hamsters compared to those consuming corn oil and fish oil. Also, body composition of fish oil fed animals showed a lower (p<0.01) total lean mass (g) compared to other three groups and a lower (p<0.01) total mass (g) compared to DHA + canola oil diets, but no difference compared to animals fed the canola oil diet. None of the treatments had any effect on triglyceride (TG) or C-reactive protein (CRP) levels. The fish oil group showed a higher (p<0.01) plasma total cholesterol (TC) levels than all other three groups. No differences existed between DHA + canola oil and fish oil groups in HDL or Non-HDL levels, but these levels were different (p<0.01) compared to corn oil group and canola oil groups. To conclude, different DFA affect whole body energetics and plasma lipid profiles. Also DFA produced marked shifts in plasma and organ levels of OEA, PEA and AEA. These dietary induced shifts in FAEs may translate into discernable changes in energy expenditure and lipid levels which in turn influence CVD risk.

The triglyceride-free fatty acid (TG-FFA) cycle was studied in white adipose tissue. The major aims of the study were 1) to see if the rate of TG-FFA cycling (i.e. FFA reesterification) and the sensitivity properties (see Newsholme and Crabtree, 1976, Biochem. Soc. Symp. 41, 61-109) were affected by various treatments, and 2) to measure the rate of cycling in vivo and assess its contribution to the metabolic rate of an animal. There are two ways of estimating the rate of TG-FFA cycling; the first is based on the release of glycerol and FFA from the tissue, and the second on the synthesis of the glycerol and FFA moieties of triglyceride. Experimental agreement between the two methods is very good. It is shown that the rate of TG glycerol synthesis can be estimated by measuring the incorporation of tritium from tritiated water into the TG-glycerol moiety; this method is used to study the TG-FFA cycle in vivo. Experimental results indicated that the rate of TG-FFA cycling in white adipose tissue in vitro and in vivo is affected by various short- and long-term treatments. However, the reesterification of FFA in adipose tissue can only account for perhaps ~1% of the basal metabolic rate of a mouse, and perhaps 4% of the increase in osygen consumption observed in fenoterol-treated mice. The equations of Newsholme and Crabtree (1976) describing the sensitivity properties of substrate cycles are extended and used to show that the TG-FFA cycle increases the sensitivity of control of FFA release from adipose tissue. The degree of sensitivity attainable is variable depending on the treatment used. The use of tritiated water for estimating TG-FFA cycling is tentatively extended to brown adipose tissue. It is suggested that the rate of cycling could be used as an indicator of sympathetic activity in brown and white adipose tissue.