Browsing by Subject "cholesterol"
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Item Effects of a High Oleic Acid Beef Diet on Cardiovascular Disease Risk Factors of Human Subjects(2012-10-19) Adams, Thaddeus HunterThe consumption of high-fat hamburger enriched with saturated fatty acids (SFA) and trans-fatty acids (TFA) may increase risk factors for cardiovascular disease, whereas hamburger enriched with monounsaturated fatty acids (MUFA) may have the opposite effect. Ten mildly hypercholesterolemic men consumed five, 114-g hamburger patties per week for two consecutive phases. Participants consumed low-MUFA (high SFA) hamburger (MUFA:SFA = 0.95; produced from pasture-fed cattle) for 5 wk, consumed their habitual diets for 3 wk, and then consumed high-MUFA hamburger (MUFA:SFA = 1.31; produced from grain-fed cattle) for 5 wk. These MUFA:SFA were typical of ranges observed for retail ground beef. Relative to habitual levels and levels during the high-MUFA phase, the low-MUFA hamburger: increased plasma palmitic acid, palmitoleic acid, and triacylglycerols (P < 0.01); decreased HDL cholesterol (HDL-C) and LDL particle diameter percentile distributions (P < 0.05); and had no effect on LDL-C or plasma glucose (P > 0.10). Plasma palmitoleic acid was positively correlated with triacylglycerols (r = 0.90), VLDL-C (r = 0.73), and the LDL:HDL (r = 0.45), and was negatively correlated with plasma HDL-C (r = -0.58), whereas plasma palmitic, stearic, and oleic acid were negatively correlated with LDL particle diameter (all P <= 0.05). Because plasma palmitoleic acid was derived from [delta]9 desaturation of palmitic acid in the liver, we conclude that alterations in hepatic stearoyl-CoA desaturase activity may have been responsible for the variation in HDL-C and triacylglycerols caused by the low-MUFA and high-MUFA hamburgers. Cattle with a genetic predisposition to deposit MUFA in their lean and fat tissues, such as Wagyu cattle can be used to produce beef products that are especially enriched with oleic acid and lower in SFA and TFA, and feeding practices can further enhance the composition of beef fat. This indicates that ground beef or hamburger products can be produced that are naturally enriched with oleic acid, and conversely that certain production practices can impair the nutritional quality of beef fat. Finally, we cannot discern from this study design whether the high-MUFA hamburger reversed the effects of the low-MUFA hamburger, or whether the subjects gradually adapted to the elevated intake of total fat. It is clear, however, that the high-MUFA hamburger did not exacerbate any of the effects of the low-MUFA hamburger and can be viewed as at least neutral in its effects on HDL-C and triacylglycerols.Item Effects of Sterol Structure on Insect Herbivore Physiology, Biochemistry and Molecular Biology(2012-02-14) Jing, XiangfengSterols serve two important biological functions in animals - they act as cellular membrane components, and as the precursor to steroid hormones. Insects require a dietary source of sterol because they cannot synthesize sterols de novo. Cholesterol is the most common sterol in plant-feeding insects, but because plants contain very little cholesterol, plant-feeding insects must convert plant sterols into cholesterol. In this dissertation I investigate the effect of common and novel plant sterols and steroids found in a transgenic tobacco line on several caterpillar species. I also explore the metabolism of these sterols and steroids, and use a microarray approach to identify genes involved in sterol use and metabolism in plant-feeding insects. I also study cholesterol homeostasis using a grasshopper species. Modified tobacco plants containing a novel sterol profile negatively affected performance three different caterpillar species, especially in the second generation. Insects reared on modified plants contained less total sterols and cholesterol than those on control plants having normal sterol profile. Similar results were found using artificial diets containing atypical steroids, e.g., cholestanol and cholestan-3-one, identified in the tobacco plants that were fed to my experimental caterpillars. More importantly, the sterol/steroid ratio, but not their absolute amount in the diets, determined the negative effects. Caterpillar species could convert stigmasterol, a common plant sterol, into cholesterol. They could also convert cholestan-3-one into cholestanol and epicholestanol, although this ability varied among different species. A microarray study, that focused on gene expression in midgut tissue, indicated that stigmasterol, cholestanol and cholestan-3-one could induce different gene expression level, and that cholestan-3-one caused a the largest pool of genes to be regulated. The genes possibly involved in the metabolism of stigmasterol and cholestan-3-one were reported. These findings are important in directing further research on the potential application of plant sterol modification to control pests in agricultural systems. Insect herbivores could behaviorally regulate the intake of several nutrients, but they could not regulate their sterol intake. They did, however, practice cholesterol homeostasis, by postingestively regulating tissue sterol levels, even when feeding on diets with high cholesterol content. Collectively, the results from this dissertation provide unique insight into cholesterol regulation, which is difficult to achieve in mammals that are capable of synthesizing their own sterols.Item Inhibition of cholesterol biosynthesis under hypoxia(Texas A&M University, 2006-04-12) Tan, QiulinOxygen balance is very important and tightly regulated in mammals. Under hypoxia, hypoxia inducible factor 1(HIF-1) dimerizes with hypoxia inducible factor 1? (HIF-) and activates expression of several genes. Using a mammalian two hybrid assay, we found that HIF-1 interacted with sterol response element binding protein 1a (SREBP1a). SREBP1a regulates transcription of HMG-CoA reductase via binding to the sterol response element (SRE) in the promoter region. HMG-CoA reductase is the rate-limiting enzyme in cholesterol biosynthesis. The interaction between SREBP1a and HIF-1suggests that HIF-1 may play an important role in regulation of cholesterol biosynthesis. We tested the effects of hypoxia on the HMG-CoA reductase. We found that hypoxia caused suppression of SRE-driven luciferase reporter gene expression. HMG-CoA reductase mRNA levels decreased under hypoxia in both hepatoma cells and mouse primary hepatocytes. Electrophoretic mobility shift assay showed that HIF-1 blocked binding of SREBP1a to the SRE sequence in vitro. Ectopic expression of HIF-1 suppressed the SRE- driven luciferase reporter gene expression in BPR cells (HIF-1). Our results suggest that hypoxia inhibits cholesterol biosynthesis by suppressing SREBP1a-regulated gene expression and this suppression is caused by the blockage of SREBP1a binding to SRE sequence by HIF-1.Item Ligand binding proteins: roles in ligand transfer and activation of nuclear receptors(Texas A&M University, 2004-09-30) Petrescu, Anca DanielaCholesterol 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 ? 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 ? 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.Item PPARs: Potential Mechanisms Regulating Blood Lipid and Lipoprotein Concentrations at Rest and Following Exercise in the Obese(2011-10-21) Greene, Nicholas PerryObesity is associated with greater rates of cardiovascular disease, dyslipidemia and dysfunctional lipid metabolism. Exercise may provide an effective therapeutic tool to ameliorate dyslipidemia. However, how exercise attenuates dyslipidemia with obesity is not fully understood. Additionally, whether acute exercise or exercise training is the primary driver of such changes in this population is unknown. Furthermore, mechanisms mediating these exercise responses are not elucidated. The peroxisome proliferator-activated receptors (PPARs) provide a likely mechanism through enhanced expression of oxidative metabolism and cholesterol transport proteins augmenting fatty acid oxidation and cholesterol transport. Study one describes blood lipid and lipoprotein responses to acute aerobic exercise and exercise training in obese men and women. The primary measured effects include: increased HDL-C in men following 12 wks exercise training, and a shift from HDL3-C to HDL2-C, with concomitantly reduced HDL-C mean density and LDL3-C in women. Acute exercise of 400 kcal duration performed before and after training, yielded a decreased TC: HDL-C ratio in men, which was unaffected by training. Thus, the primary exercise-based treatment for dyslipidemia with obesity appears to be exercise training. In study two, PPAR? and PGC-1? content were significantly enhanced after acute exercise, whereas PPAR? and AMPK? content were augmented only after training. These effects were seen with concomitantly increased content of target proteins involved in oxidative and lipoprotein metabolism including lipoprotein lipase, CPT-I, COX-IV, and FAT/CD36. PPAR? expression was correlated with total and LDL-cholesterol concentrations. AMPK? expression was correlated with the concentration of HDL-C and its subfractions, suggesting regulation of blood cholesterols by PPAR? and AMPK?. Study three demonstrates comparative responses to high volume resistance exercise (RE) in lean and obese Zucker rats. RE enhanced PPAR? expression regardless of phenotype, but PGC-1? in obese only. Mitochondrial biogenesis was enhanced in lean animals only, indicating PPAR? and PGC-1? content is disconnected from mitochondrial biogenesis with obesity. These studies enhance our understanding of exercise as a therapeutic tool in treating dyslipidemia and dysregulated lipid metabolism often associated with obesity. They further demonstrate the necessity for exercise training to attenuate dyslipidemia, while illustrating PPAR-mediated augmentations in oxidative and lipoprotein metabolism following exercise with obesity.Item Secondary Structural and Functional Studies of Rotavirus NSP4 and Caveolin-1 Peptide-Peptide Interactions(2011-02-22) Schroeder, Megan ElizabethThe rotavirus NSP4 protein is the first described viral enterotoxin. Abundant data from our laboratory reveals that NSP4 binds both the N- and C-termini of caveolin- 1 (aa2-31 and 161-178, respectively). Yeast two-hybrid and peptide binding analysis mapped the caveolin-1 binding site to three hydrophobic residues within the amphipathic a-helix, enterotoxic peptide domain (aa114-135). The research studies herein utilized peptides to investigate the interaction between NSP4 and caveolin-1. Peptides were synthesized corresponding to the amphipathic a-helix and caveolin-1 binding domain of NSP4 (aa112-140) and to the N- (aa2-20 and 19-40) and C- (161-178) termini of caveolin-1, and were utilized in structural and functional studies. Fluorescence binding assays revealed that NSP4 (aa112-140) binds to the N-terminus (aa19-40) of caveolin-1 with a stronger affinity than the C-terminus (aa161-178). In addition, this assay further delineated the NSP4 binding domain on caveolin-1 to aa19-40. Secondary structural changes following NSP4-caveolin-1 peptide-peptide interactions were investigated by circular dichroism analysis. Changes in a-helix formation were observed only upon interaction of the NSP4112-140 peptide with the C-terminal caveolin-1 peptide (C-Cav161- 178). The NSP4112-140 peptide contains a potential cholesterol recognition amino acid consensus (CRAC) sequence. Therefore this peptide was examined for cholesterol binding. Results of the binding assay revealed NSP4 binds cholesterol with a Kd of 7.67 +/- 1.49nM and this interaction occurs via aa112-140. Mutation of amino acid residues within the CRAC motif resulted in weaker binding affinities between each of the corresponding mutant peptides and cholesterol. NSP4 peptides containing mutations within the hydrophobic and charged faces of the amphipathic a-helix, enterotoxic peptide and caveolin-1 binding domain of NSP4 were examined for changes in secondary structure as well as diarrhea induction in mouse pups. Circular dichroism analysis revealed that mutation of hydrophobic residues resulted in a decrease in a-helix formation, whereas mutation of acidic and basic charged residues caused little to no change in a-helical content. When tested for diarrhea induction in mouse pups, the peptides containing mutations of either the hydrophobic or basic charged residues did not cause diarrhea. Taken together, the results of this research suggest a complex interplay between NSP4 secondary structure, caveolin-1 and cholesterol binding and diarrheagenic function.