Type 2 diabetes mellitus (T2DM) is a disease of multi-complications affecting more than 20 million US adults. Hyperglycemia is the classic clinical feature of diabetes, and uncontrolled hyperglycemia leads to deadly health complications. Thus, control of blood glucose represents a major goal for diabetics. Human and rodent studies revealed another clinical feature; diabetics have low tissue and plasma levels of polyunsaturated fatty acids (PUFAs), an effect often attributed by impaired endogenous PUFA synthesis. In this context, rodent studies have revealed a possible link between PUFA synthesis and high fat diet induced obesity and diabetes. These studies have shown that obese and diabetic mice have low hepatic expression and activity of fatty acid elongase-5 (Elovl5), a key enzyme involved in the PUFA synthesis pathway. Over-expression of Elovl5 in livers of chow fed C57BL/6J mice decreased fasting blood glucose and increased hepatic glycogen contents. Therefore, my hypothesis for the current work is that elevated hepatic Elovl5 activity or improved hepatic PUFA synthesis will improve systemic and hepatic carbohydrate metabolism in a mouse model of diet induced obesity and diabetes. Using a recombinant adenovirus approach, we over-expressed Elovl5 in livers of high fat diets (60% calories derived from fat as lard, Research Diets) induced obese-diabetic mice. Elevated hepatic Elovl5 activity increased hepatic and plasma C��������������� PUFA contents, reduced homeostatic model assessment for insulin resistance (HOMA-IR), improved glucose tolerance and lowered fasting blood glucose to euglycemic levels in obese-diabetic mice. The mechanism for insulin mimetic effect of Elovl5 on hepatic glucose metabolism was correlated with increased phosphorylation of Akt-S��������, FoxO1-S�������� and PP2Acat-Y��������, decreased nuclear content of FoxO1, and decreased expression of Pck1 and G6Pase; important enzymes involved in gluconeogenesis (GNG) and glucose production. Phospho-FoxO1 is excluded from nuclei, ubiquitinated and degraded by the proteasome. Loss of nuclear FoxO1, due to its increased phosphorylation, leads to the reduction in the expression of key genes involved in gluconeogenesis, i.e., Pck1 and G6Pase. Using obese-diabetic mice liver extracts and HepG2 cells, I established that Elovl5 uses two mechanisms to control hepatic GNG. The first mechanism involves Elovl5 mediated increased Akt2-S�������� and FoxO1-S�������� phosphorylation via mTORC2-rictor pathway. The second mechanism involves Elovl5 mediated attenuation of de-phosphorylation of FoxO1 via PP2A inhibition. Together, these mechanisms increase FoxO1 phosphorylation status in livers of fasted obese-diabetic mice, lower hepatic FoxO1 nuclear abundance and FoxO1 capacity to sustain transcription of GNG genes and inhibit GNG and restore blood glucose levels in fasted obese-diabetic mice. Results of these studies showed Elovl5 corrected high fat diet induced hyperglycemia in C57BL/6J mice, identified the molecular mechanism of Elovl5 control of GNG and explained how Elovl5 or PUFA synthesis controls GNG. Therefore, these findings will be eventually helpful in developing a therapeutic target to combat hyperglycemia. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from July 27, 2012 - July 27, 2013

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Several factors including an unidentified endogenous substance were found to stimulate microsomal diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) from a microspore-derived cell suspension culture of oilseed rape (Brassica napus L. cv Jet Neuf). Mg2+ salts were found to stimulate microsomal DGAT 14 to 23-fold. ATP and CoA were also found to stimulate the enzyme 2.4 and 12 fold respectively, although the effects were decreased in the presence of high Mg2+ concentrations. While microsomal DGAT activity was only slightly increased by the concentration of exogenous diacylglycerol in the reaction mixture it was increased substantially by the addition of exogenous phosphatidate. Other phospholipids tested were not found to have this stimulatory effect. During attempts to investigate possible covalent modification of the enzyme, the soluble fraction obtained from cell suspension homogenate was found to contain a small metastable organic molecule(s) which stimulated DGAT activity. Stimulation of microsomal DGAT by this factor was concentration dependent but not dependent on preincubation time. / xiii, 95 leaves : ill. ; 28 cm.

Brassica napus is known to contain an endogenous and soluble stearoyl-acyl carrier protein (^918:0-ACP) desaturase, but does not express a palmitic (16:0)-ACP desaturase. Levels of 16:0 are low in canola oil and are associated with enhanced cholesterol biosynthesis in humans. In an attempt to further reduce the saturated fatty acid (SFA) content of canola oil, B. Napus L. cv Westar was transformed with a cDNA encoding a ^916:0-ACP desaturase from cat's claw (Doxantha unguis-cati L.). Arabidopsis thaliana was also transformed with this cDNA. Transformation of both oilseeds resulted in increased production of palmitoleic acid (^16:1) and many other effects of fatty acid composition. Overall, the SFA content did not decrease in either oilseed and investigation to why this effect occurred was examined using transgenic B. napus. Molecular genetic testing on second generation B. napus also determined the plants contained the cDNA of interest and were transcribing the cDNA. / xxi, 155 leaves : ill. ; 29 cm.

Cyclopropanation of the unsaturated fatty acid moieties of membrane phospholipids is a commonly observed phenomenon in a number of bacterial systems. The cyclopropane fatty acids are usually synthesized during and after the transition from exponential growth to stationary phase, or under such environmental conditions as acidic culture pH, low oxygen tension or high salt concentrations. S-Adenosylmethionine, the ubiquitous methyl group donor, provides the methylene bridge carbon in the reaction catalyzed by cyclopropane fatty acid synthase. Also formed in the reaction is S-adenosylhomocysteine, a potent inhibitor of cyclopropane fatty acid synthase, which is degraded by S-adenosylhomocysteine nucleosidase. This work provides evidence for at least two modes of regulation of lactobacillic acid synthesis, the cyclopropane fatty acid formed from cis-vaccenic acid (cis-11,12-octadecenoic acid), in Lactobacillus piantarum.

Ho, Tik Shun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 88-102). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.vii / Table of Contents --- p.viii / List of Table --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiii / Chapter CHAPTER 1 --- General Introduction --- p.1 / Chapter 1.1 --- Fatty Acid Synthase (FAS) - 7-domain multifunctional enzyme --- p.1 / Chapter 1.1.1 --- Functions --- p.1 / Chapter 1.1.2 --- Structure --- p.2 / Chapter 1.2 --- Fatty Acid biosynthesis reactions --- p.4 / Chapter 1.3 --- Malonyl Coenzyme A - An important mediator in lipogenesis --- p.7 / Chapter 1.4 --- FAS expression in different histotypes --- p.8 / Chapter 1.4.1 --- FAS in normal cells --- p.8 / Chapter 1.4.2 --- FAS in pathological cells --- p.8 / Chapter 1.4.3 --- Tumor-associated FAS (Oncogenic antigen-519) in cancer cells --- p.9 / Chapter 1.5 --- FAS signaling models in breast and prostate cancers --- p.12 / Chapter 1.5.1 --- Association between FAS and PI3K／Akt pathway --- p.12 / Chapter 1.5.2 --- Hypothetical model of FAS hyperactivity in breast and prostate cancer cells --- p.13 / Chapter 1.6 --- FAS inhibition to tackle cancer cell growth --- p.15 / Chapter 1.6.1 --- FAS inhibitors --- p.15 / Chapter 1.6.1.1 --- Cerulenin --- p.16 / Chapter 1.6.1.2 --- C75 --- p.17 / Chapter 1.6.2 --- Small interfering RNA --- p.17 / Chapter 1.7 --- FAS inhibition to enhance chemoresistant cancer cells sensitivity to drugs --- p.19 / Chapter 1.8 --- Hypothesis --- p.20 / Chapter CHAPTER 2 --- Methods and Materials --- p.21 / Chapter 2.1 --- Chemicals and antibodies --- p.21 / Chapter 2.2 --- Cell cultures --- p.21 / Chapter 2.3 --- MTT assay --- p.22 / Chapter 2.4 --- 5-Bromo-2'-deoxyuridine (BrdU)-labeling cell proliferation assay --- p.22 / Chapter 2.5 --- Cytotoxicity detection assay of LDH release --- p.23 / Chapter 2.6 --- DNA flow cytometry --- p.23 / Chapter 2.7 --- Confocal micocropy --- p.24 / Chapter 2.8 --- Immunoblot analysis --- p.24 / Chapter 2.8.1 --- Preparation of protein lysates --- p.24 / Chapter 2.8.2 --- Immunoblotting --- p.25 / Chapter 2.9 --- Caspase inhibitor studies --- p.26 / Chapter 2.10 --- Analysis of mitochondrial membrane potential --- p.26 / Chapter 2.11 --- Determination of caspase activities --- p.27 / Chapter 2.12 --- siRNA transfection --- p.27 / Chapter 2.13 --- Statistical analysis --- p.28 / Chapter CHAPTER 3 --- Results --- p.29 / Chapter 3.1 --- Cytostatic & cytotoxic studies of FAS inhibitors on human cancer cells --- p.29 / Chapter 3.1.1 --- Cerulenin and C75 suppress cell growth of different cancer histotypes --- p.29 / Chapter 3.1.2 --- Cerulenin and C75 suppress cell growth of A-375 dose- and time-dependently --- p.32 / Chapter 3.1.3 --- Cerulenin and C75 exert cytotoxic effect on A-375 but not normal skin HS68 cells --- p.36 / Chapter 3.1.4 --- Cerulenin and C75 arrest cell cycle progression and induce apoptosis with DNA Fragmentation --- p.39 / Chapter 3.2 --- Mechanistic studies of FAS inhibitors in A-375 cells --- p.46 / Chapter 3.2.1 --- Cerulenin and C75 induce caspase-dependent apoptosis --- p.46 / Chapter 3.2.2 --- Cerulenin- and C75-induced apoptosis involve extrinsic death receptor pathway --- p.52 / Chapter 3.2.3 --- Cerulenin- and C75-induced apoptosis involve intrinsic mitochondrial pathway --- p.57 / Chapter 3.2.4 --- Extrinsic death receptor pathway serves as a pioneer and links with intrinsic mitochondrial pathway in cerulenin- and C75-induced apoptosis --- p.65 / Chapter 3.3 --- Small interfering RNA on Fatty Acid Synthase (FAS siRNA) --- p.68 / Chapter 3.3.1 --- FAS siRNA induces PARP cleavage --- p.68 / Chapter 3.3.2 --- FAS siRNA triggers caspase-dependent apoptosis as FAS inhibitors --- p.70 / Chapter CHAPTER 4 --- Discussion --- p.72 / Chapter CHAPTER 5 --- Future Prospect --- p.85 / Chapter CHAPTER 6 --- References --- p.88

Bibliography: leaves 267-345. / xi, 345, [61] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines the regulation of interleukin-1[Beta] and tumor necrosis factor[alpha] synthesis by fatty acids and eicosanoids by examining the mediator synthesis following dietary fatty acid manipulation / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1998?

Indiana University-Purdue University Indianapolis (IUPUI) / This dissertation studies the effects of fatty acids on prostate cancer. Prostate cancer is one of the most common malignant diseases in males in the U.S. Because of the slow progression of this disease, early intervention methods, especially, dietary fatty acid interventions are considered very important to control the disease in early stages. This study describes how the depletion of the enzyme for endogenous fatty acid synthesis, fatty acid synthase, influences the expression of enzymes that metabolize dietary fatty acids and show how dietary fatty acids affect prostate cancer protein expression and function. Fatty acid synthase is an oncoprotein overexpressed in prostate cancer and its expression is suppressed with omega-3 fatty acid treatment. This study finds that the depletion of fatty acid synthase by siRNA knockdown induces suppression of cyclooxygenase-2 and fatty acid desaturase-1. Our results also show that fish oil (omega-3 fatty acid), but not oleic acid (omega-9 fatty acid), suppresses prostate cancer cell viability. Assessment of fatty acid synthesis activity indicates that oleic acid is a more potent inhibitor than fish oil of de novo fatty acid biosynthesis. In addition, the inhibition of its activity occurs over several days while its effects on cell viability occur within 24 hours. To better understand this relationship, label free LC-MS/MS based mass spectrometry was carried out to determine global proteomic and phosphoproteomic profiles of the prostate cell line PC3, with longitudinal treatment with fish oil or oleic acid. With short-term fish oil treatment, sequestosome-1was elevated. Prolonged treatment induced downregulation of microseminoprotein, a proinflammation factor, as well as proteins in the glycolysis pathway. In the phosphoproteomics study, we confidently identified 828 phosphopeptides from 361 phosphoproteins. Quantitative comparison between fish oil or oleic acid treated groups and the untreated group suggests that the fish oil induces changes in phosphorylation of proteins involved in the pathways associated with cell viability and metabolic processes, with fish oil inducing significant decreases in the levels of phospho-PDHA1Ser232 and phospho-PDHA1Ser300 and they were accompanied by an increase in PDH activity, suggesting a role for n-3 polyunsaturated fatty acids in controlling the balance between lipid and glucose oxidation.

Indiana University-Purdue University Indianapolis (IUPUI) / Polyacetylenic natural products, compounds containing multiple carbon-carbon triple bonds, have been found in a large collection of organisms. Radiochemical tracer studies have indicated that these bioactive metabolites are synthesized from fatty acid precursors through a series of uncharacterized desaturation and acetylenation steps. To date, there are three main pathways believed to be involved in acetylenic natural product biosynthesis. However, it is apparent that the crepenynic acid pathway is the origin of a vast majority of the known plant and fungal acetylenic products. This investigation provides concrete evidence that the polyacetylenic natural products found in the fungus Fistulina hepatica and the medicinal plant species Echinacea purpurea are biosynthesized from crepenynic acid. Through heterologous expression in Yarrowia lipolytica, two acetylenases capable of producing crepenynic acid were identified from E. purpurea. Furthermore, heterologous expression of two diverged desaturases isolated from F. hepatica, uncovered a ∆12-acetylenase and the first multifunctional enzyme capable of ∆14-/∆16- desaturation and ∆14-acetylenation.

Indiana University-Purdue University Indianapolis (IUPUI) / Fatty acid elongation is the extension of de novo synthesized fatty acids through a series of four reactions analogous to those of fatty acid synthase. ELOs catalyze the first reaction in the elongation pathway through the condensation of an acyl group with a two carbon unit derived from malonyl-CoA. This study uses the condensing enzyme, EloA, from the cellular slime mold, Dictyostelium discoideum as a model for the family of ELOs. EloA has substrate specificity for monounsaturated and saturated C16 fatty acids and catalyzes the elongation of 16:1Δ9 to 18:1Δ11. Site-directed mutagenesis was used to change residues highly conserved among the ELO family to examine their potential role in the condensation reaction. Mutant EloAs were expressed in yeast and fatty acid methyl esters prepared from total cellular lipids were analyzed by gas chromatography/mass spectrometry. Sixteen out of twenty mutants had a decrease in 18:1Δ11 production when compared to the wild-type EloA with little to no activity observed in ten mutants, four mutants had within 20% of wild-type activity, and six mutants had 10-60% of wild-type activity. Immunoblot studies using anti-EloA serum were used to determine if the differences in elongation activity were related to changes in protein expression for each mutant. Analysis of immunoblots indicated that those mutants with little to no activity, with the exception of T130A and Q203A, had x comparable protein expression to the wild-type. Further research included the solubilization of the His6-ELoA fusion protein and preliminary work toward the isolation of the tagged protein and the use of a radiolabeled condensation assay to determine the activity of the eluted protein. Preliminary results indicated that the protein was solubilized but the eluted protein showed no activity when examined by a condensation assay. The work presented here contributes to a better understanding of the role of certain amino acid residues in the activity of EloA and serves as a stepping-stone for future EloA isolation work.