N-Acylethanolamines (NAEs) are a class of bioactive lipids, and FAAH is one of the enzymes responsible for degrading NAEs in both plants and animals. in plants, FAAH appears to be closely associated with ABA, a phytohormone which has long been associated with plant stress responses, since the overexpression of FAAH in Arabidopsis results in ABA hypersensitivity. Therefore, it is reasonable to speculate that alterations in FAAH transcript levels will result in altered stress responses in plants. to investigate this hypothesis experiments were carried out in which wild type (WT), FAAH-overexpressing (OE), and T-DNA insertional FAAH knockouts of Arabidopsis (faah) were grown in MS media under stress conditions. the stress conditions tested included chilling stress, heavy metal stress induced by cadmium or copper, nutrient limitations induced by low phosphorus or low nitrogen, salt stress induced with NaCl, and osmotic stress induced with mannitol. the OE plants were consistently hypersensitive to all stress conditions in relation to wild type plants. Inactive FAAH overexpressors did not have the hypersensitivity to the salt and osmotic stress of the active OE plants and were instead tolerant to these stresses. FAAH2 (faah2) knockouts and FAAH 1 and 2 double knockouts (faah 1+2) were based on some root development parameters somewhat more tolerant than WT plants, but more sensitive in terms of shoot growth. Collectively the data suggests that FAAH activity may interact with stress-responsive pathways in plants, perhaps including pathways involving ABA.

The following doctoral thesis focuses on genetic and environmental factors that influence skeletal muscle metabolism in women with overweight or obesity. The effects of Acyl-CoA Synthetase Long-Chain 5 (ACSL5) genotype on fatty acid metabolism was studied in vitro, ex vivo and in vivo. The effects of the environmental toxicant mono-(2ethylhexyl) phthalate (MEHP) was also studied in vitro and in vivo. Statistical analyses illustrated how ACSL5 rs2419621 [T] allele carriers, with overweight or obesity had a greater reduction in their fat mass and visceral fat and greater increases in their percentage lean mass post diet/exercise intervention vs. non-carriers. This was paralleled with increased in vitro and in vivo fatty acid oxidation and ex vivo mitochondrial respiration within [T] allele carriers vs. non-carriers. Interestingly, it was noted that carriers of the polymorphism had increased levels of the ACSL5 683aa isoform in skeletal muscle, which was found to be localized in the mitochondria to a greater extent, playing a greater role in fatty acid oxidation vs. 739aa isoform. This explains in part why carriers of the polymorphism are more responsive to lifestyle interventions vs. non-carriers. Studies conducted in women with obesity who participated in the National Health and Nutrition Examination Survey (NHANES) demonstrated an association between increased urinary MEHP and increased plasma fatty acid levels. In vitro work in C2C12 myotubes exposed to MEHP displayed a reduction in fatty acid oxidation and mitochondrial respiration. An increase in basal glycolysis was paralleled with increased levels of hexokinase II protein expression in C2C12 myotubes exposed to increasing levels of MEHP. Thus, these results suggest that increased exposure to MEHP as well as urinary MEHP contributes towards dysfunction in glucose and fatty acid utilization at both the muscle and whole-body level. Hence, women with obesity may be more susceptible to the metabolic effects of MEHP, increasing their chances of metabolic dysfunction. The following thesis, provides a more comprehensive view on the effects of both genetics and environmental factors on metabolic response within women with overweight and obesity. This provides insights into factors that should be considered for personalized medicine, to improve treatment options for combatting this disease.

No description available.

Intact chloroplasts were isolated from Euglena gracilis variety bacillaris, and aliquots were exposed to several different chemical cross-linking reagents. The reagents penetrated the triple membrane of Euglena chloroplasts. This was shown by gradient acrylamide gel electrophoresis under denaturing conditions. The activity of the nonaggregated fatty acid synthetase of Euglena was located within the chloroplast stroma, and the effects of dimethylsuberimidate cross-linking on the activity of the enzyme system were examined. The acyl-carrier protein concentration in the chloroplast was measured at about 0.24 mM.

The composition, size, and peptide structure of a fatty acid synthetase aggregate from etiolated Euglena gracilis was studied. The fatty acid synthetase was a lipoprotein containing about 40% lipid. Low-angle laser light scattering of the native fatty acid synthetase yielded a molecular weight of 6 · 106 up to concentrations of about 30 μg fatty acid synthetase/ml; at higher concentrations, the molecular weight increased to 11 · 106. Viscometry of the synthetase solutions yielded results that suggested that the asymmetric fatty acid synthetase aggregate formed a 'dimer' at concentrations above 30 μg fatty acid synthetase/ml by side-to-side interaction. The peptide structure of the fatty acid synthetase prepared in the presence of a variety of proteinase inhibitors included at least six peptides of Mr 150000 or less. More than 68% of the protein was in peptides of less than Mr 150000. N-terminal amino acid analysis gave eight different residues all present in integral amounts, seven at about 11% and one at 24% of the total α-N-dansyl amino acids. The Euglena-aggregated fatty acid synthetase appears to be a very large true multienzyme complex.

No description available.

<p> Diabetes mellitus is a major independent risk factor for cardiovascular disease and stroke. However, the molecular and cellular mechanisms by which diabetes contributes to the development of vascular disease are not fully understood. We have shown that conditions of hyperglycemia are associated with accumulation of intracellular glucosamine, a downstream metabolite of glucose. Our findings indicate that elevated levels of intracellular glucosamine can promote inflammation and lipid accumulation - the hallmark features of atherosclerosis - in vascular cells and HepG2 cells.</p> <p> Here I demonstrate that exposure of HepG2 cells to the branched chain fatty acid, valproic acid, increases cellular resistance to glucosamine-induced lipid accumulation and nuclear factor-KB activation. In vivo I show that hyperglycemic apolipoprotein E-deficient (ApoE-/-) mice fed a diet supplemented with 625 mg/kg valproic acid have significantly reduced lesion volumes relative to non-supplemented controls. Valproate supplementation has no apparent effect on the plasma levels of glucose, or lipids, nor does it affect the expression of ER chaperones. Significant reductions were observed in total hepatic lipids(> 50.4%) and hepatic glycogen synthase kinases (GSK)-3β activity (> 55.8%) in mice fed the valproate supplemented diet.</p> <p> In vitro I demonstrate that valproic acid directly inhibits GSK-3α/β. Also pretreatment with novel GSK-3 inhibitors protects primary mouse hepatocytes from glucosamine-induced unesterified cholesterol accumulation. I further establish the role of GSK-3 by showing that GSK-3-deficient mouse embryonic fibroblasts do not accumulate unesterified cholesterol after glucosamine treatment. Dietary supplementation with 2-ethylbutyric acid, a novel and potent GSK-3 inhibitor in vitro, did not reduce lesion development in hyperglycemic ApoE-/- mice and significantly increased atherosclerosis in normoglycemic mice. This may be a side effect attributed to multiple cellular pathways controlled by GSK-3 .</p> <p> In conclusion, I have identified a pathway involving glucosamine-induced cellular dysfunction that leads to accelerated hyperglycemia-associated atherosclerosis. This pathway involves GSK-3, which regulates glucosamine-induced unesterified cholesterol accumulation.</p> / Thesis / Doctor of Philosophy (PhD)

No description available.

The Gram-negative bacterium 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 can live on long chain fatty acids as its sole carbon source. However, the rate of fatty acid uptake was reduced after starvation (Mangroo, 1992). In this study, it was found that this starvation effect of reducing oleate uptake rate could only be observed in the initial minute post starvation. The starvation effect was not due to the depletion of its substrates ATP or reduced coenzyme A. Neither was the effect caused by a lowered acyl-CoA synthetase level. This reduction in oleate uptake rate can be reversed by incubation in oleate. It has been reported that lactate activated oleate uptake in 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 (Mangroo and Gerber, 1993). In the present study, it was discovered that when [9, 10-³H]oleate was used as the radioactive tracer in uptake assay, the majority of the radioactivity effluxed from the cells. It was found that lactate did not have an effect on the overall oleate uptake. Its apparent activation on oleate uptake was due to the reduction in the efflux rate of radioactive probes, instead of increasing the uptake rate of oleate. Acyl-CoA synthetase in 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 has always been described as a protein of around 45 kDa (Kameda and Nunn, 1981; Kameda 𝘦𝘵 𝘢𝘭., 1986). However, the 𝘧𝘢𝘥𝘋 gene that encodes the 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 acyl-GoA synthetase predicts a 62 kDa protein (Black 𝘦𝘵 𝘢𝘭., 1992; Fulda 𝘦𝘵 𝘢𝘭., 1994). This new form of acyl-CoA synthetase has been partially purified. This form, which has a size of around 62 kDa, can be converted to its 45 kDa counterpart in a Triton X-1 00 and temperature dependent manner. This induced processing of the 62 kDa enzyme was found to be inhibited by oleate, a natural substrate of acyl-CoA synthetase. The two forms of acyl-CoA synthetase have also been shown to be immunologically related. A much simpler purification scheme was developed for the 45 kDa acyl-CoA synthetase using the membrane bound form of acyl-CoA synthetase. The selective extraction of the 62 kDa acyl-CoA synthetase from the membrane, together with the induced processing into the 45 kDa form has provided the basis for this comparatively simple process. The availability of the expression system made the low yield associated with this simple process acceptable. Using oleate as substrate, the Kₘ and Vₘₐₓ for the 45 kDa acyl-CoA synthetase were determined to be 85 ± 18μM and 1550 ± 165 nmole/min/mg protein respectively, whereas the Kₘ and Vₘₐₓ for the 62 kDa enzyme were determined to be 38 ± 12μM and 633 ± 79 nmole/min/mg protein respectively. This suggested that the 62 kDa enzyme has higher affinity towards oleate than its 45 kDa counterpart under standard assay conditions while the ratio of Vₘₐₓ /Kₘ remained relatively constant. Evidence suggesting Triton X-1 00 activates acyl-CoA synthetase by providing a surface for catalysis was presented. This interaction between acyl-CoA synthetase and the Triton X-1 00 I oleate mixed micelle could be affected by the surface charge of the mixed micelle. / Thesis / Master of Science (MS)

Adipositas und die damit verbundenen Folgeerkrankungen sind eine der zentralen Gesund-heitsherausforderungen unserer Zeit. Dauerhafte Adipositas führt zu einer Dysregulation fettgewebseigener Peptidhormone. Diese sogenannten Adipokine stellen ein Verbindungsglied zwischen Fettgewebsakkumulation und den vielfältigen Adipositaskomplikationen des gesamten Organismus dar. Adipocyte fatty acid binding protein (AFABP) wurde in den letzten Jahren als zirkulierendes Adipokin mit diabetogenen, proinflammatorischen und proateriosklerotischen Effekten etabliert. Zu Beginn der Dissertation lagen unzureichende Erkenntnisse über die Elimination von AFABP sowie die Regulation des Adipokins bei eingeschränkter Nierenfunktion vor. Aus diesem Grund untersucht die vorliegende Arbeit die AFABP-Regulation in Abhängigkeit von der Nierenfunktion in 532 Patienten mit chronischer Niereninsuffizienz (Studienpopulation 1) und 32 Patienten mit akuter Nierenfunktionsverminderung nach Nephrektomie (Studienpopulation 2). In beiden Kohorten stiegen die medianen AFABP-Serumkonzentrationen mit abfallender Nierenfunktion an. Zudem waren Marker der Nierenfunktion in beiden Studienpopulationen die stärksten unabhängigen Prädiktoren für zirkulierendes AFABP. Untersuchungen aus der Arbeitsgruppe zur AFABP-Regulation in einem Rattenmodell der akuten Niereninsuffizienz unterstützen die klinischen Studienergebnisse. Zusammenfassend zeigen diese Ergebnisse zum ersten Mal signifikant steigende AFABP-Serumspiegel bei chronischer und akuter Nierenfunktionsstörung, sowie bei akutem Abfall der Nierenfunktion. Diese Befunde stützen die Hypothese, dass AFABP renal eliminiert wird. Inwiefern AFABP darüber hinaus in die Pathogenese der chronischen Niereninsuffizienz eingreift, muss in weiterführenden Studien beleuchtet werden.