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Thesis--Wisconsin. / Vita. Includes bibliographical references (leaves 72-98).

Fatty acid transport across the plasma membrane is an important site of regulation in skeletal muscle lipid metabolism, and is governed by a number of fatty acid transport proteins including, CD36, FABPpm, and FATP1 and 4. While each transporter is capable of independently stimulating fatty acid transport, less is known about their specific functions under various metabolic conditions, although CD36 appears to be key. The purpose of this thesis was to examine skeletal muscle fatty acid metabolism in several rodent models where CD36 has been altered, particularly via whole body deletion, by muscle specific overexpression, or in the face of permanent redistribution of CD36 to the plasma membrane. Using these models, this thesis sought to answer the following questions: 1) Is caffeine-stimulated fatty acid oxidation CD36-dependent? 2) Does CD36 function in tandem with FABPpm, and does this enhance fatty acid uptake at the plasma membrane and/or influence the metabolic fate of incoming fatty acids? 3) Is intramuscular lipid distribution altered in a rodent model of obesity, in which CD36-mediated fatty acid uptake is increased? Specific novel findings include the following: 1) Caffeine-stimulated calcium release can elicit the translocation of a number of fatty acid transporters in skeletal muscle, but CD36 is essential for caffeine-induced increases in fatty acid uptake and oxidation. 2) In spite of difficulties associated with protein co-overexpression, it appears that simultaneous overexpression of CD36 and FABPpm enhances fatty acid transport across the plasma membrane, and that these transporters may collaborate to increase insulin-induced fatty acid esterification and AICAR-induced oxidation. 3) Finally, in the obese Zucker rat model, augmented CD36-dependent fatty acid transport into muscle in combination with elevated lipid supply, results in lipid accretion within the IMF region of muscle, an effect that could not be explained by compartment-specific changes in selected glycerolipid synthesizing enzymes. Taken together, these studies emphasize the importance of CD36 in the regulation of plasmalemmal fatty acid transport, and further elucidate the metabolic implication of CD36 alteration on overall skeletal muscle metabolism.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1962. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1963. / Typescript. Abstracted in Dissertation abstracts, v. 23 (1963) no. 9, p. 3111-2. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1968. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Little research has focused on the relationship between lecithin:cholesterol acyltransferase (LCAT) activity and cholesterol metabolism in dogs and cats. To study weight loss and cholesterol metabolism in dogs, four experimental weight-loss diets were fed to 12 obese female beagles for 8 wk in a partial crossover design (n = 6). High- (HGI) or low-glycemic index (LGI) starch and diacylglycerol or triacylglycerol oil were combined to compose diets with similar fatty acid (FA) profiles. Body weight was measured weekly. Fasted blood samples were drawn at wk1, wk4, and wk8 to measure plasma total (TC), unesterified (UC), and esterified cholesterol (EC) concentrations, LCAT activity, and FA composition of the phospholipid (PL) and EC fractions. All groups lost weight. UC increased from wk1 to wk4 (p < 0.05). LCAT activity increased from wk1 to wk4 and remained elevated at wk8 (p < 0.05). Plasma PL FA profiles reflected the diets fed with few diet or time effects. Plasma EC FA profiles reflected the specificity of LCAT for linoleic acid (LA) with minimal diet or time effects. We conclude that weight reduction in dogs occurs in conjunction with increased LCAT activity and altered plasma cholesterol fractions but not changes in plasma PL or EC FA profiles. To measure the activity and demonstrate the FA specificity of LCAT in felines fed varying types of fat, 29 female cats were fed diets enriched with high-oleic sunflower (n = 9), menhaden fish (n = 10), or safflower (n = 10) oil (8g oil/100g kibble) for 4 wk. Fasted blood samples were drawn at d0, d14, and d28 for determination of the blood parameters mentioned previously. LCAT and TC showed no time or diet effects. UC decreased at d28 compared to d0 and d14, while EC increased at d28 compared to d0 and d14 (all p < 0.05). Plasma EC FA profiles reflected the specificity of LCAT for LA with many diet and time effects but contained no docosahexanoic acid (DHA). We conclude that feline LCAT has no measurable affinity for DHA, but both feline and canine LCAT demonstrated specificity for LA regardless of diet fed.

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.

With crude oil demand rising and supplies being depleted, alternative energy, specifically biofuels, are of intense scientific interest. Current plant crop based biofuels suffer from several problems, most importantly the use of land needed for food. Cyanobacteria offer a solution to this problem as they do not compete with land for food and produce hydrocarbons that can be used as biofuels. Upon examination of metabolic pathways competing with hydrocarbon synthesis, it appeared that cyanobacteria lacked the major fatty acid degradative metabolic pathway β-oxidation, generally thought to be a universally occurring pathway. Lack of this pathway in cyanobacteria was confirmed by employing a range of analytical techniques. Bioinformatic analysis suggested that potential enzymes with β-oxidation activity were involved in other metabolic pathways. A sensitive assay was set up to detect acyl- CoAs, the substrates of β-oxidation, using liquid chromatography triple quadrupole mass spectrometry. None could be detected in cyanobacteria. No enzymatic activity from the rate-limiting acyl-CoA dehydrogenase/oxidase could be detected in cyanobacterial extracts. It was found that radiolabeled fatty acids fed to cyanobacteria were utilised for lipid membranes as opposed to being converted to CO2 by respiration or into other compounds by the TCA cycle. An element of the β-oxidation pathway, E. coli acyl-CoA synthetase was ectopically expressed in a strain of cyanobacteria and implications of the introduction of acyl-CoA synthesis were assessed. Finally, the regulation of the fatty acid biosynthetic pathway was investigated. It was determined that under conditions of excess fatty acid, the transcription of acetyl-CoA carboxylase and enoyl-ACP reductase was repressed and acyl-ACP synthetase involved in fatty acid recycling was induced. These results were discussed in relation to fatty acid oxidation and hydrocarbon biosynthesis in other organisms.

Recent investigations suggest that reducing stearoyl CoA desaturase (SCD) 1 expression confers protection against obesity and insulin resistance, whereas others show that increasing SCD1 expression protects cells from lipotoxicity. The overall aim of this thesis was to establish the role of SCD1 expression in fatty acid metabolism and insulin stimulated glucose disposal in skeletal muscle. In vitro and in vivo studies were conducted to investigate the relationship between fatty acid subtype, SCD1 expression and fuel metabolism. The role of fatty acid subtype on fatty acid metabolite accumulation and insulin resistance was initially examined in rats. Rats were provided with a low fat diet or a high fat diet consisting of predominantly saturated (SAT) or polyunsaturated fatty acids (PUFA). Rats fed a SAT diet were insulin resistant and had increased skeletal muscle diacylglycerol content whereas rats fed a PUFA diet retained insulin sensitivity and accumulated triacylglycerol rather than diacylglycerol. Interestingly, SCD1 mRNA and protein content were elevated in SAT rats compared with PUFA fed and control fed rats, indicating a possible involvement of SCD1 in the aetiology of insulin resistance. Subsequently, SCD1 expression was examined in the skeletal muscle of various rodent models of genetic and diet-induced obesity. SCD1 content was consistently upregulated in the skeletal muscle of obese rodents. To determine whether SCD1 contributes to or protects from fatty-acid induced insulin resistance, SCD1 levels were transiently altered in L6 skeletal muscle myotubes. Short interfering (si) RNA was used to decrease SCD1 content and a pcDNA3.1/HygromSCD1 vector was introduced to increase SCD1 content. Reducing SCD1 protein resulted in marked esterification of exogenous fatty acids into diacylglycerol and ceramide. Insulin-stimulated Akt (acute transforming retrovirus thymoma) phosphorylation and 2-deoxyglucose uptake were reduced with SCD1 siRNA. Exposure of L6 myotubes to palmitate abolished insulin-stimulated glucose uptake in both control and SCD1 siRNA myotubes. Transient overexpression of SCD1 resulted in triacylglycerol esterification but attenuated ceramide and diacylglycerol accumulation and protected myotubes from fatty acid-induced insulin resistance. Further, these changes were associated with reduced phosphorylation of c-Jun Amino-Terminal Kinase (JNK) and the inhibitor of IêB kinase (IKK), both of which impair insulin signalling. These studies indicated that SCD1 protects from cellular toxicity in L6 myotubes by preventing excessive accumulation of bioactive lipid metabolites. Collectively, these experiments indicate that increasing SCD1 expression may be a protective mechanism designed to prevent insulin resistance in obese phenotypes.