Cholesterol and fatty acyl-coenzymeA thioesters are signalling molecules with role in regulation of genes involved in lipid and glucose transport and metabolism. The studies described herein focused on three proteins that bind lipids and have different cellular functions: steroidogenic acute regulatory protein (StAR), hepatocyte nuclear factor-4a (HNF-4a) and acyl-CoA binding protein (ACBP). First, StAR mediates delivery of cholesterol to inner mitochondrial membrane in steroidogenesis by a poorly understood mechanism. In our studies, fluorescent NBD-cholesterol binding assays demonstrate that StAR binds cholesterol at two binding sites with 32 nM Kds and circular dichroism spectra show that cholesterol binding results in changes of StAR secondary structure. Fluorescent sterol exchange assays between donor and acceptor mitochondrial membranes indicate that StAR significantly increased the formation of rapidly transferable cholesterol domains. Second, HNF-4a, a nuclear receptor, had been shown to bind fatty acyl-CoAs as natural ligands with apparent low affinities obtained with radiolabeled ligand binding assays. Our fluorescence spectroscopy studies demonstrate that HNF-4a ligand binding domain (HNF-4aLBD) binds acyl-CoAs at a single binding site with Kds of 1.6-4 nM. Fluorescence resonance energy transfer (FRET) between HNF-4aLBD tryptophan residues and cis-parinaroyl-CoA yielded an intermolecular distance of 42 Â thus pointing to direct molecular interaction. Third, although ACBP has been detected in the nucleus, it is not known whether ACBP may directly and/or functionally interact with a nuclear acyl-CoA binding protein such as HNF-4a to regulate transcription. Our present studies in vitro and in intact cultured cells, including circular dichroism of HNF-4a in the presence of ACBP, coimmunoprecipitation of HNF-4a/ACBP complexes, ACBP and HNF-4a colocalization in nuclei of cells by confocal microscopy demonstrate a physical association of ACBP and HNF-4a. FRET microscopy data indicated an intermolecular distance of 53 Â between ACBP and HNF-4a in rat hepatoma cells. Functional assays (transactivation of an HNF4a-dependent reporter gene) showed significant increase in the presence of ACBP in two different cell lines. Expression of ACBP anti-sense RNA decreased HNF-4a-mediated transactivation, pointing to a role of ACBP in co-regulating HNF-4a-dependent transcription.

This study was conducted to examine the antioxidative effect of conjugated linoleic acid (CLA) in irradiated, cooked ground beef patties. The hypothesis was that CLA would be retained during irradiation and would reduce lipid oxidation that is caused by irradiation. The objective was to evaluate the effects of CLA alone and in combination with irradiation on lipid oxidation, fatty acid composition, cooking loss, moisture and fat content, and trained panel sensory evaluations of beef patties. CLA was added at 0, 1, 2, or 4% level during the grinding process. Addition of CLA during the grinding process increased CLA cis-9,trans-11 and CLA trans-10,cis-12 isomers in both irradiated and non-irradiated cooked ground beef patties (irradiated at 1.6 kGy) (P = 0.0001). Weight loss during cooking was greater in irradiated beef patties than in non-irradiated patties (P = 0.004). Irradiation reduced the serumy/bloody aromatic attribute and increased browned aromatic attribute, browned aftertaste, and wet dog/hairy aromatic attribute (P < 0.05). There was no significant main effect of irradiation on the basic tastes. The linoleic acid, CLA cis-9,trans-11, and CLA trans-10,cis-12 were decreased by irradiation (P < 0.05). Although irradiation decreased the CLA isomers, higher percentages of CLA isomers were retained in irradiated patties containing a 4% free fatty acid preparation of CLA (FFA-CLA), reflecting the ability of the FFA preparation to reduce lipid oxidation that is caused by irradiation. The thiobarbituric acid reactive substances (TBARS) values were significantly higher in irradiated, cooked ground beef patties than in non-irradiated ground beef patties (P = 0.004). Although the FFA-CLA was effective in reducing lipid oxidation that is caused by irradiation, it increased painty aromatic attribute, bitter taste, and astringent aftertaste due to the soapy flavor of the free fatty acid (all P < 0.05). The FFA-CLA decreased cooked beef/brothy and serumy/bloody aromatic attribute and browned aftertaste (all P < 0.05). The 1% triacylglycerol (TAG) preparation of CLA reduced TBARS in irradiated, cooked patties to levels seen in control, non-irradiated patties. The 1% TAG concentration also provided good retention of CLA in the cooked ground beef.

Obesity is today a problem that has reached epidemic proportions. One of the causes of obesity is the over-consumption of energy. Fat is the most energy-dense nutrient, where the quality seems to be more important for the development of the metabolic diseases than the quantity. The fatty acid composition in serum lipid fractions can be used to mirror the dietary fat quality. Stearoyl-CoA-desaturase (SCD) is an enzyme that converts saturated to monounsaturated fatty acids. A surrogate measure of SCD activity can be estimated as a fatty acid ratio; 16:1/16:0 (palmitoleic acid/palmitic acid) and 18:1/18:0 (oleic acid/stearic acid). The aim of this project was to investigate the intra-day variation in the SCD-ratio in humans eating a standardized diet. The results showed that triacylglycerol and nonesterified fatty acid fractions in serum lipids had a significant variance in the 16:1/16:0 ratio during the day, whereas 18:1/18:0 ratio in the same fractions did not exhibit the same pattern. In this study 16:1/16:0 ratio also seems to be a better marker than 18:1/18:0 ratio for estimating SCD activity. For further evaluation of the intra-day variation there need to be a more long-term study of the SCD-activity for a larger group of subjects.

A series of experiments was conducted to test the overall hypothesis that reducing the omega-6 (n-6) to omega-3 (n-3) fatty acid (FA) ratio in sow diets would improve sow reproductive performance (characterized by increases in numbers and body weight of piglets born alive and weaned) and would lessen the inflammatory responses of their offspring post weaning. Diets were wheat/barley based and consisted of a control (tallow based, similar to a standard production diet), 3 diets with plant oil based n-6:n-3 ratios (9:1P, 5:1P, and 1:1P) and a 5:1 fish oil diet (5:1F). The control diet had a ratio of 8:1, but contained approximately half the polyunsaturated FA content of the other diets. Sows were randomly assigned to a treatment diet on d 80 of gestation, and remained on that treatment for three consecutive reproductive cycles (gestation/lactation 1 = P1, gestation/lactation 2 = P2, gestation/lactation 3 = P3). Experiment 1 was designed to test the hypothesis that reducing the n-6:n-3 FA ratio in sow diets would increase circulating concentrations of n-3 FA’s in sows and in their offspring, and the passive immune status of piglets would be improved. Performance data was collected throughout P1 and P2 on 150 sows (n = 30/diet). Sow and piglet serum, colostrum and milk were analyzed for FA profiles, and colostrum and piglet serum were analyzed for immunoglobulin (Ig) A and IgG. In P1, birth weights were unaffected by diet (P > 0.05). Average piglet weaning weights (P = 0.02) and ADG (P = 0.01) however, were highest for piglets born to sows consuming the 9:1P and 5:1P diets. During P2, 5:1F sows consumed 10% less feed (P = 0.04), their piglets had reduced birth weights (P = 0.05), and average weaning weight was reduced by 0.8 kg (P = 0.04) relative to control or 5:1P sows. Colostral and piglet plasma IgA and IgG were unaffected by diet (P > 0.05). Colostrum FA profile patterns were similar to that of the sow diets. Serum n-3 FA’s were greatest in sows (P < 0.01) and piglets (P < 0.01) consuming 1:1P or 5:1F diets. Serum α-linolenic acid (ALA) was highest in the 1:1P sows and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were highest in the 5:1F sows. In piglet serum obtained prior to suckling, ALA and DHA did not differ among treatments (P > 0.05) but EPA was 2.5 times greater in the 1:1P group and 4 times greater in the fish group (P < 0.01) compared to those from the control diet. In post-suckle samples, ALA was highest in serum from 1:1P diet piglets (P < 0.01), and EPA and DHA were highest in piglet serum from the 5:1F sows (P < 0.01). Omega-3 FA’s can perturb lipid metabolism, specifically increasing the lipolytic activity of adipose tissue and thus the second experiment tested the hypothesis that high producing sows, consuming reduced n-6:n-3 ratios would have increased body fat mobilization. Twenty sows per diet, farrowing ≥ 11 piglets and nursing ≥ 10 piglets during P3, were used. Performance data on sows and piglets (such as weights, numbers, backfat changes) was collected throughout lactation and milk samples obtained on d 4 and d 16 of lactation. Jugular catheters were inserted into 8 sows from each of the 9:1P and 1:1P groups on d 5 of lactation and sows were challenged with a single injection of epinephrine followed by serial blood collections. Feed intake was highest for sows consuming the control (8.4 kg/d) and 5:1P (8.2 kg/d) diets and lowest for the sows fed the 1:1P (7.4 kg/d) and 5:1F (7.7 kg/d) diets (P = 0.05). Altering the n-6:n-3 FA ratio did not affect sow BW, piglet ADG, milk DM and N content or the total output of milk (P > 0.2). Sows consuming the 1:1P diet had greater backfat thickness (P < 0.05) and numerically higher plasma NEFA at baseline compared with the 9:1P sows (240 vs 93 uM; P = 0.16). When given epinephrine, 9:1P fed sows tended to have lower net incremental area under the curve (niAUC) glucose (P = 0.08) and numerically higher niAUC NEFA (P = 0.17) and glycerol (P = 0.15). A third experiment was conducted to test the hypothesis that piglets raised by sows consuming reduced n-6:n-3 ratios would have reduced inflammatory responses post-weaning. Piglets (n = 20/diet) raised by sows consuming the treatment diets described above for 2 gestation/lactation cycles (P2) were selected at weaning. Within diet group, pigs were randomized to either a challenge control group (saline injected) or to a lipopolysaccharide (LPS) injected group (n=10/challenge•diet-1). Piglets were fed a common starter diet for 6 days followed by saline or LPS injections on d 7. Rectal temperatures were recorded for 24 hrs and blood samples were collected at 0, 2, 6 and 12 hrs post injection for pro-inflammatory cytokine and blood urea nitrogen (BUN) analysis. Injecting LPS caused decreased feed intake and reduced ADG (P < 0.01), and increased temperature and cytokine production (P < 0.05). Piglets raised by sows consuming the 1:1P diet had elevated temperatures (P = 0.01; diet x challenge P > 0.05). Overall, circulating plasma ALA and EPA were increased in sows and piglets when sows were fed a 1:1 plant based ratio compared to the control or high n-6:n-3 ratio groups. Sows fed a ratio of 1:1 mobilized more body fat relative to those consuming the 9:1 ratio; there were no treatment effects on piglet growth. Reducing maternal n-6:n-3 FA ratios below 5:1 increased piglet body temperature prior to and during an LPS induced inflammatory challenge,. Reducing the sow dietary n-6:n-3 FA ratio below 5:1 may have detrimental effects on piglets due to over-stimulation of inflammatory responses.

The desert ant Cataglyphis fortis is one of the hyperthermophilic species of Cataglyphis. It lives in the Sahara desert and forages during the hottest hours of the day when it can get up to 70˚C in the sand. The body temperature of the ant during the foraging runs can reach a maximum of 55˚C. Since C.fortis is one of few eukaryotic hyperthermophilic species, its proteins probably have a high thermostability. Investigating the thermostability can give valuable information about the principles of protein folding and stability in hyperthermophiles.Fatty acid binding proteins (FABPs) have an important role in the cell taking up and transporting fatty acids and regulating metabolic and inflammatory pathways. FABPs have been extensively studied and structures from several species have been determined. The determined structures of all FABPs are very similar why thermostability studies of FABP from C.fortis are highly relevant.Fluorescence spectroscopy is an easy and fast method to measure intrinsic protein fluorescence. Tryptophans were genetically introduced into three different positions in FABP to be used as environmental sensitive probes. Complementing the measurement results with a model of the 3D structure of FABP from C.fortis gave additional information about the ligand binding.The (local) thermostability of the mutants can be detected by shift in wavelength maximum during temperature ramping experiments. All mutants are stabilised in the presence of fatty acids. The mutant with tryptophan positioned closest to the supposed ligand binding residues (Y11W) is most affected. The mutant with tryptophan situated farthest from the supposed binding residues (Y52W) shows a stabilisation of Tm less evident than for Y11W. Thus, the structural changes following fatty acid binding are more obvious in the environment close to the binding site.However, the third mutant C87W shows no significant stabilisation although positioned closer to the fatty acid binding site than Y52. This is probably due to the size difference between the original and introduced amino acid in the mutation. Since the high value of the starting λmax for C87W implies that C87W is quite exposed to the aqueous solvent, the residue is likely to not have subsumed in the protein tertiary structure.Further, the myristic acid stabilise the melting temperature of all the mutants while octanoic acid only has a local effect of Y11W increasing the cooperativity. This implies different binding properties and that myristic acid stabilise the entire protein while octanoic acid only has a local stabilisation effect around the ligand binding site.

As shown by recent reports, number of obese people in recent years has been on the increase, there are about 4 million people in Taiwan who are considered to be overweight. World Health Organization (WHO) and United States Center for Disease Control and Prevention (CDC) publicly announced that: Obesity will be the greatest health killer of this century, its damage to personal health is comparable to that of cigarettes. Obesity can cause heart problems, diabetes, artery diseases, high blood pressure, increased chances of cancer occurrence, condition increase and deteriora- tion of Alzheimer¡¦s disease, gall bladder diseases, and shortening of life span. The cause of obesity is due to a fault in adipocytes metabolism functions, and because of this, research into adipocytes molecular regulation is becoming more popular and valued. The process of adipogenesis, the formation of adipose tissue, has become better understood by the studies of several cell types that can be induced to undergo differentiation into adipocytes. The first, and the best characterized, model of adipogenesis in vitro is the 3T3-L1 cell line, a substrain of Swiss 3T3 mouse cell line. 3T3-L1 cells propagated under normal conditions have a fibroblastic phenotype. However, when treated with a combination of dexamethasone, isobutylmethylxanthine (IBMX or MIX) and insulin, 3T3-L1 cells adopt a rounded phenotype and within 5 days begin to accumulate lipids intracellularly in the form of lipid droplets. Treatment of cells with dexamethasone activates the transcription factor CCAAT/enhancer -binding protein £] (C/EBP£]). IBMX inhibits soluble cyclic nucleotide phosphodiesterases and results in increased intracellular cAMP levels. At the nuclear level, treatment with IBMX results in activation of the related transcription factor C/EBP£_. Immediately after exposure to exogenous inducers, the gene expression of C/EBP£] and C/EBP£_ significantly and transiently increases, C/EBP£] and C/EBP£_ may also regulate the expression of C/EBP£\ and PPAR£^. C/EBP£\ and PPAR£^ are considered to play a prominent role in regulating the gene expression of proteins necessary for the development fo the functional mature adipocyte. Within 3 days of exposure to inducers, the cells undergo two rounds of mitosis, termed mitotic clonal expansion, which are required for differentiation. Insulin or insulin-like growth factor-1 promote adipocyte differentiation by activating PI3-kinase and Akt activity. Modulation of the activity of the forkhead transcription factor Foxo1 appears to be necessary for insulin to promote adipocyte differentiation. C/EBP£\ and PPAR£^ direct the final phase of adipogenesis by activating expression of adipocyte-specific genes, such as fatty acid synthetase, fatty acid binding protein, leptin and adiponectin. The identification of regulators of adipogenesis raises the prospect of preventing or reversing obesity through pharmacological means. My research is aimed at investigating the adipocytes differentiation and regeneration adaptive mechanisms of mice 3T3L-1 preadipocytes and human processed lipoaspirate cells (PLA). By using adipocytes culture techniques in conjunction with adipocytes growth induction and gene delivery techniques to further study obesity related genes, POMC and PTEN, and downstream regulators , PPAR£^ and Adiponectin, in regards to their roles in the process of adipocytes differentiation.

Obesity is a disorder of energy imbalance and the most prevalent nutritional diseases in developed countries. Besides, obesity is also strongly associated with health problems such as type 2 diabetes (NIDDM), hypertension, hyperlipidaemia, cardiovascular diseases and cancers. However, the defects in lipid metabolism underlying obesity-related disorders are extremely complicated. Thus, extensive studies on the mechanism of endogenous fatty acids synthesis would be one of the keys to elucidate molecular pathogenesis of obesity. In liver or adipose, fatty acid synthase (FAS) utilizes acetyl-CoA, malonyl-CoA and NADPH to synthesize long-chain fatty acids (C16 or C18), which can be converted to triglycerides and stored as fat. During lactation, thioesterase II (TE II) expresses in mammary glands and interacts with FAS to produce medium-chain fatty acid (primarily C10) in milk, which provides immune protection and energy for the newborn. TE II causes premature termination of fatty acid synthesis catalyzed by FAS and releases medium-chain fatty acids. Unlike long-chain fatty acids, medium-chain fatty acids can enter mitochondria directly for beta-oxidation to generate ATP, thus provide energy more efficiently. Since TE II gene expression is under strict regulation, we utilized adenovirus gene transfer techniques to deliver and express TE II in hepatocytes. It was postulated that expression of TE II in hepatocytes might result in the increase of ATP and reduction of long-chain fatty acids, subsequently decrease the fat production. Recombinant adenovirus was used as gene delivery system for TE II because of its high titer, wide host range, and transduction efficiency. In the present study, we have generated and characterized the recombinant Ad-TE II by PCR, western blot analysis, and enzymatic assay, respectively. By using Ad-GFP, we have determined the optimal multiplicity of infection (MOI) for adenovirus to infect HepG2 cells is about 100-200. Adenovirus-mediated TE II expression in hepatocytes was demonstrated by western blot as well as TE II enzymatic assay. We have demonstrated that the adenovirus-mediated TE II expression was slightly cytotoxic to hepatocytes. Besides, an increase of free fatty acids, asparate transaminase, lactate dehydrogenase levels, as well as ATP synthesis was also noted in the TE II-expressed hepatocytes. The enhanced the release of asparate transaminase (AST/GOT) and lactate dehydrogenase (LDH) after TE II expression in the hepatocytes further supported its cytotoxcity to hepatocytes. In the future, we will carry out experiments to further characterize the effects of TE II expression on cellular lipid metabolism through adenovirus gene delivery. We hope that the present studies will not only provide further insights into mammalian lipid metabolism, but also enable us to evaluate the therapeutic potential of TE II on the treatment of obesity and its related disorders.

This study was conducted to examine the antioxidative effect of conjugated linoleic acid (CLA) in irradiated, cooked ground beef patties. The hypothesis was that CLA would be retained during irradiation and would reduce lipid oxidation that is caused by irradiation. The objective was to evaluate the effects of CLA alone and in combination with irradiation on lipid oxidation, fatty acid composition, cooking loss, moisture and fat content, and trained panel sensory evaluations of beef patties. CLA was added at 0, 1, 2, or 4% level during the grinding process. Addition of CLA during the grinding process increased CLA cis-9,trans-11 and CLA trans-10,cis-12 isomers in both irradiated and non-irradiated cooked ground beef patties (irradiated at 1.6 kGy) (P = 0.0001). Weight loss during cooking was greater in irradiated beef patties than in non-irradiated patties (P = 0.004). Irradiation reduced the serumy/bloody aromatic attribute and increased browned aromatic attribute, browned aftertaste, and wet dog/hairy aromatic attribute (P < 0.05). There was no significant main effect of irradiation on the basic tastes. The linoleic acid, CLA cis-9,trans-11, and CLA trans-10,cis-12 were decreased by irradiation (P < 0.05). Although irradiation decreased the CLA isomers, higher percentages of CLA isomers were retained in irradiated patties containing a 4% free fatty acid preparation of CLA (FFA-CLA), reflecting the ability of the FFA preparation to reduce lipid oxidation that is caused by irradiation. The thiobarbituric acid reactive substances (TBARS) values were significantly higher in irradiated, cooked ground beef patties than in non-irradiated ground beef patties (P = 0.004). Although the FFA-CLA was effective in reducing lipid oxidation that is caused by irradiation, it increased painty aromatic attribute, bitter taste, and astringent aftertaste due to the soapy flavor of the free fatty acid (all P < 0.05). The FFA-CLA decreased cooked beef/brothy and serumy/bloody aromatic attribute and browned aftertaste (all P < 0.05). The 1% triacylglycerol (TAG) preparation of CLA reduced TBARS in irradiated, cooked patties to levels seen in control, non-irradiated patties. The 1% TAG concentration also provided good retention of CLA in the cooked ground beef.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Greek alphabet delta in title. Includes bibliographical references (leaves 136-155). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Oregon, 2003. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 94-104). Also available for download via the World Wide Web; free to University of Oregon users.