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Obesity, a disease characterized by excess adipose tissue (AT), is a growing worldwide epidemic. The Centers for Disease Control and Prevention (CDC), in 2017-2018, reported the prevalence of obesity in adults in the United States was 42.4% . Obesity increases the risk for many other serious health conditions such as type 2 diabetes, cardiovascular diseases, stroke, and some cancers. In individuals with obesity, the hypertrophic expansion of adipocytes, the main cell type within AT, is not matched by new vessel formation, leading to AT hypoxia. As a result, hypoxia inducible factor-1⍺ (HIF-1⍺) accumulates in adipocytes inducing a transcriptional program that upregulates profibrotic genes and biosynthetic enzymes such as lysyl oxidase (LOX) synthesis. This excess synthesis and crosslinking of extracellular matrix (ECM) components cause AT fibrosis. Although fibrosis is a hallmark of obese AT, the role of fibroblasts, cells known to regulate fibrosis in other fibrosis-prone tissues, is not well studied. Adipocytes are mechanoresponsive and affected by different microenvironmental cues, including hypoxia and mechanical (un)loading. Yet, no study has focused on the role of the aforementioned factors on the adipocyte mechanical response, including actin cytoskeletal remodeling. This dissertation aims to develop an in vitro model of healthy/diseased AT to explore the effect of microenvironmental cues on adipocyte function and actin cytoskeletal remodeling. The first aim is to study (1) the crosstalk between fibroblasts and adipocytes in a co-culture model and (2) the effect of hypoxia on the ras homolog gene family member A (RhoA)/Rho-associated coiled-coil kinases (ROCK) mechanical pathway and actin cytoskeletal remodeling in adipocytes. We confirmed that hypoxia creates a diseased phenotype by inhibiting adipocyte maturation and inducing actin stress fiber formation facilitated by myocardin-related transcription factor A (MRTF-A/MKL1) nuclear translocation. The second aim explores the effects of mechanical unloading (simulated microgravity) on key adipocyte functions and actin cytoskeletal remodeling. This study demonstrated that mechanical unloading enhances adipocyte maturation via increased lipogenesis and lipolysis and cortical actin remodeling, which together further enhanced glucose uptake. However, disrupting cortical actin remodeling by using inhibitors or exposure to a high concentration of free fatty acids (FFAs) diminished enhanced adipocyte functions observed in simulated microgravity. Overall, the results of these studies support the importance of microenvironmental cues on adipocyte actin cytoskeletal remodeling. Therefore, targeting mechanical pathways that regulate actin cytoskeletal remodeling can be used to improve adipocyte function and AT metabolism and possibly treat related diseases such as type 2 diabetes and obesity. / Bioengineering

The role of membrane-intrinsic enzymes of lipid metabolism in complex biological processes is being realized through comprehensive structure function studies. Detailed analysis of substrate-enzyme interactions occurring within the restrictive membrane environment has proved to be exceedingly challenging. Using detergent micelles, we describe a detailed model for substrate recognition and binding by the outer-membrane intrinsic enzyme PagP from Escherichia coli. PagP is an 8-stranded antiparallel β-barrel that transfers a palmitoyl group from a phospholipid molecule to lipid A, the endotoxin component of lipopolysaccharide. This simple modification provides bacterial resistance to host antimicrobial peptides and attenuates the inflammatory response signalled through the host toll-like receptor 4 pathway. We describe a molecular embrasure and a crenel, which display weakened transmembrane β-strand hydrogen bonding, to provide site-specific routes for lateral entry of substrates into the PagP active site. A Tyr147 localized to the L4 loop gates the entry of the phospholipid substrate through the crenel, while lipid A enters via the embrasure. The side chains of the catalytic residues that are located in the extracellular loops point towards the central axis of the enzyme, directly above the active site. An acyl-chain binding pocket known as the hydrocarbon ruler is buried within the transmembrane β-barrel structure, and is optimized to accommodate a 16-carbon saturated palmitate chain. The hydrocarbon ruler, therefore, accounts for PagP's stringent selectivity for a palmitate chain. Substituting Gly88 lining the floor of the hydrocarbon ruler with residues possessing linear, unbranched, aliphatic side chains changes the selectivity of PagP to utilize shorter acyl chains. The serendipitous discovery of an exciton interaction between Trp66 and Tyr26 at the floor of the hydrocarbon ruler provides an intrinsic spectroscopic probe to monitor the methylene unit acyl-chain resolution of PagP. A compromised acyl chain resolution of the Gly88Cys mutant is attributed to an unexpected decrease of the Cys sulfhydryl group pKa within the β-barrel interior, resulting in a burying of a charged thiolate within the PagP core. The structural perturbation associated with the Cys thiolate extinguishes the exciton and expands the acyl-chain selectivity. These molecular details of lateral lipid diffusion and acyl-chain selection provide the first such example for any membrane-intrinsic enzyme of lipid metabolism. / Thesis / Doctor of Philosophy (PhD)

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Marine oils are used as ration additives to provide omega-3 fatty acids to dairy cows. Supplementing dairy cows with omega-3 fatty acid-rich feeds does not easily increase quantities in milk fat of dairy cows because polyunsaturated fatty acids are biohydrogenated in the rumen. Lipid encapsulation of omega-3 fatty acids provides protection from biohydrogenation in the rumen and allows them to be available for absorption and utilization in the small intestine. Lactating cows were supplemented with rumen protected algae biomass or algal oil in a 4 × 4 Latin Square. Feeding lipid encapsulated algae supplements increased docosahexaenoic acid content in milk fat while not adversely impacting milk fat yield; however, docosahexaenoic acid was preferentially esterified into plasma phospholipid, limiting its incorporation into milk fat. In the second study, triglyceride emulsions of oils enriched in either oleic, linoleic, linolenic, or docosahexaenoic acids were intravenously infused to avoid confounding effects of triglyceride esterification patterns in the small intestine and to compare mammary uptake. Milk transfer of fatty acids delivered as intravenous triglyceride emulsions was reduced with increased chain length and unsaturation. Increased target fatty acids were evident in plasma phospholipid, suggesting re-esterification in the liver. Transfer efficiencies were 37.8, 27.6, and 10.9±5.4% for linoleic, linolenic, and docosahexaenoic acid. Both liver and mammary mechanisms may regulate transfer of long-chain polyunsaturates. Intracellular fatty acid binding proteins (FABP) are cytoplasmic proteins that are hypothesized to be essential for fatty acid transport and metabolism by accelerating longchain fatty acid uptake and targeting to intracellular organelles, such as the endoplasmic reticulum for triglyceride esterification. FABP3 mRNA is highly expressed in bovine mammary and heart tissue, but is not present in MAC-T cells, a bovine mammary epithelial cell line. When overexpressed in MAC-T cells, FABP3 does not appear to be rate-limiting for fatty acid uptake in vitro and did not alter lipid metabolism. The function of FABP3 in the mammary gland remains unclear. / Ph. D.