Browsing by Subject "Lipids"
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Item Acute alcoholic fatty liver syndromes(1965-09-16) UnknownItem Barriers to population-wide lipid management and new tools for success(2023-02-24) Navar, Ann MarieItem Coronary heart disease in women: natural history and significance of lipids as risk factors(1991-10-17) Denke, Margo A.Item Development of a Lipidomics Platform to Discover Enzymatic Activities of Mycobacterial Proteins of Unknown Function(2020-05-01T05:00:00.000Z) Pasko, Breanna Lauren; Alto, Neal; Shiloh, Michael; Sperandio, Vanessa; McDonald, JeffreyMycobacterium tuberculosis (Mtb), a facultative intracellular pathogen, is responsible for 10 million new cases of tuberculosis and 1.5 million deaths annually. Mtb infection can be acute but also lifelong. Indeed, despite the devastating global impact on health, how Mtb is able to adapt to the human body and survive for years is unknown. To better understand the physiology of Mtb infection, exploring the functions of individual proteins produced by Mtb is critical. Less than half of the proteins encoded in the M. tuberculosis genome have been fully characterized either directly through experimentation or indirectly through annotation. Many of these unknown genes likely encode proteins important for pathogenesis and adaptation to host responses. I created a novel method to facilitate the identification of novel substrates and products using direct infusion tandem mass spectrometry (DI-MS/MS) analysis of nonpolar metabolite fluctuations after transient and rapid protein overexpression. I studied 24 proteins using this lipidomic method. Of these, several demonstrate unique patterns of metabolites secondary to rapid induction of protein expression, and work towards identifying their specific enzymatic activities is ongoing. I selected one protein, Cor (Rv1829), for further characterization as it was recently shown to be fundamental to carbon monoxide (CO) resistance and pathogenesis of Mtb during host infection. To study Cor in vitro, I purified recombinant Cor from E. coli and used activity based metabolic profiling (ABMP) to measure changes in mycobacterial metabolites exposed to Cor. In the ABMP assay, exposure of mycobacterial metabolites to Cor led to consumption of acetyl phosphate and accumulation of phosphatidic acid (PA). I developed direct enzymatic assays for Cor activity, and confirmed the consumption of acetyl phosphate. To assess substrate binding, I used isothermal titration calorimetry (ITC) and demonstrated binding of Cor to acetyl phosphate. Additionally, Cor interacted directly with cardiolipin (CL), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), and sulfatide on membrane lipid strips. I overexpressed Cor in M. smegmatis and Mtb and lipids were extracted and analyzed through DI-MS/MS to assess changes in lipid composition. I combined in vivo lipidomics and in vitro ABMP to biochemically characterize Cor and found that Cor is interacting with bacterial lipids. In vivo lipidomic analysis revealed an accumulation of PA and PG by 3 hours. Our findings suggest that Mtb Cor is interacting with lipids involved in the phospholipid biosynthesis pathway and modifying bacterial lipid composition by accumulating PG. These experiments provide mechanistic insight into the enzymatic function of Cor. Furthermore, the metabolomics and lipidomics approaches developed in this study can be broadly applied to study proteins of unknown function from other organisms.Item Elucidating the Role of Yeast Lipin (PAH1) in Lipid Droplet Biogenesis(2012-07-17) Adeyo, Oludotun; Goodman, Joel M.Lipid droplets are unique organelles important for a host of cellular functions including the storage of neutral lipids, but factors that regulate the biogenesis and maintenance of these organelles remain relatively unknown. The primary focus of this dissertation will be to understand the role of the phosphatidic acid hydrolase (Pah1p) in the biogenesis of lipid droplets in Saccharomyces cerevisiae. Pah1p is an enzyme that converts phosphatidic acid to diacylglycerol, and its absence or elimination of its catalytic activity results in the accumulation of neutral lipids within membranes of the endoplasmic reticulum. Furthermore, lipid droplet formation is facilitated by diacylglycerol through a mechanism that appears to be independent of diacylglycrol’s role as a substrate for triglyceride biosynthesis. Finally, lipid droplets originated from regions of the endoplasmic reticulum where the Pah1p activators were located. The second part of this dissertation will focus on the lipodystrophy related protein Fld1p and its association with lipid droplets. Droplets always associate with Fld1p, and in the absence of lipid droplets, Fld1p is localized as patches distributed throughout the endoplasmic reticulum. In addition, induced lipid droplets originate from these Fld1 patches. I conclude from this work that diacylglycerol facilitates lipid droplet formation and that Fld1p is somehow involved in the biogenesis or maintenance of these organelles.Item Essential hyperlipemia(1964-10-01) UnknownItem Fiber Orientation Modeling: a Method to Improve Quantitation of Intramyocellular Lipids in Human Subjects at 7 Tesla(2011-10-03) Khuu, Anthony N.; Malloy, Craig R.BACKGROUND: Increased intramyocellular lipid (IMCL) content in skeletal muscle has been suggested to be a biomarker for insulin resistance. As a noninvasive method of estimating IMCL, 1H MR spectroscopy of muscle fat has been a popular method for measuring the concentration of IMCLs, a goal highly desirable for research in the pathogenesis of type 2 diabetes. Extramyocellular lipids (EMCL) are often considered to be deposited along strands that are parallel to Bo (the applied field) whereas IMCL are assumed to be spherical droplets in the muscle cells’ cytoplasm. Resolution between IMCL and EMCL signals mainly results from the angle-dependant bulk susceptibility of the 2 geometric structures. However, IMCL signal is usually contaminated by a broad and asymmetrical EMCL . Conventional fitting methods usually assume that both the IMCL and EMCL signals to be symmetrical, represented by a single Lorentzian, Gaussian or Voigt (hybrid lineshape between Gaussian and Lorentzian) lineshapes. However, significant asymmetry in the resonance assigned to the methylene protons (-CH2-)n in extramyocellular lipids (EMCL) interfered with fitting the spectra. In this work, we explore another approach, named Fiber Orientation Modeling (FOM) by using the bulk susceptibility effect theory to accurately assess the lineshape of EMCL. METHODS: The distribution of EMCL strand orientation at any angle from 0 degrees to 90 degrees relative to Bo was described by a Gaussian function, centered at a specific angle and a width representing a dispersion of EMCL strands. The chemical shift OMEGA from each strand was translated by the well-known orientation dependence interaction OMEGA = 3cos2THETA-1, where THETA is the angle between EMCL and the applied field. As the result, the location and amplitude of individual curves representing each strand could be derived. Depend on the aforementioned Gaussian distribution, the combination of these individual curves generated a unique EMCL shape. In this work, spectral simulations were generated using muscle fiber orientation reported previously. The phantom experiment with a fat cylinder (representing EMCL) submerged in Intralipid(TM) solution (representing IMCL) was also performed to determine the maximal shift at 0 degrees and 90 degrees. Under IRB approval, single voxel and chemical shift images were acquired from soleus and gastrocnemius muscle of healthy human subjects at 7T (Phillips Medical system, Cleveland, Ohio). All the spectra were fitted with the hybrid Voigt lineshape and the experimental lineshape. RESULTS: In simulated spectra with dominant angle of 00 to the applied field and little dispersion, fitting with the Voigt lineshape accurately determined IMCL/EMCL ratio over a range of different linewidths. Increasing dispersion and central angle caused overestimation of IMCL/EMCL ratios, up to three-fold when fitted with the Voigt lineshape. The error was substantially reduced using our method. The improvement is also observed in phantom spectra and human spectra. Estimates of [IMCL]/[EMCL] were significantly improved by including variations in fiber orientation in the lineshape analysis (fiber orientation modeling, FOM). Calculated soleus [IMCL] using FOM, 4.43 ± 2.32 mmol/kg wet weight, was lower compared to most previous reports in soleus. The average orientation of EMCL was calculated to be 35 degrees relative to Bo with a dispersion width of 24 degrees CONCLUSION: Since prominent asymmetrical EMCL signal tends to contaminate into IMCL region, this interaction results in the amplitude-dependence of IMCL signal on the average orientation and dispersion of EMCL. As the result, the use of symmetrical lineshape tends to overestimate the IMCL signal if all strands of EMCL are not parallel to Bo and one another. By accounting for the angular dispersion& orientation, the fit would improve both the residual and the IMCL estimate.Item The Impact of Lipid Nanoparticle Chemistry on RNA Delivery and Therapeutic Outcomes(2022-12-01T06:00:00.000Z) Johnson, Lindsay Taylor; de Gracia Lux, Caroline; Siegwart, Daniel J.; Zhu, Hao; Hoshida, Yujin; Singal, Amit G.This dissertation aims to understand how two individual components of the traditional four-component lipid nanoparticle system, the PEG lipid component and the ionizable cationic lipid component, impact RNA delivery. To systematically investigate how PEG lipid chemistry impacted LNP formulation and RNA delivery, a series of linear-dendritic poly(ethylene glycol) (PEG) lipids were synthesized with modulated hydrophobic domains. The chemical structure of the hydrophobic domain did not impact the formulation of 5A2-SC8 LNPs, including nanoparticle size, RNA encapsulation, and stability. However, the chemical structure did affect RNA delivery efficacy both in vitro and in vivo. The chemical structure of the hydrophobic domain of the PEG lipids impacted the escape of 5A2-SC8 LNPs from endosomes at early cell incubation time points. Overall, the results indicated that PEG lipid anchoring and chemical structure modulated RNA delivery. Although most LNPs accumulate in the liver after intravenous administration (suggesting that liver delivery is straightforward), it was observed that two similar LNP formulations (5A2-SC8 and 3A5-SC14 LNPs) resulted in distinct RNA delivery within the liver organ. Despite both LNPs possessing similar physical properties, the ability to silence RNA in vitro, strong accumulation within the liver, and sharing a pKa of 6.5, only 5A2-SC8 LNPs were able to functionally deliver RNA to hepatocytes. Protein corona analysis indicated that 5A2-SC8 LNPs bind Apolipoprotein E (ApoE), which can drive LDL-R receptor mediated endocytosis in hepatocytes. In contrast, the surface of 3A5-SC14 LNPs was enriched in Albumin but depleted in ApoE, which likely led to Kupffer cell delivery and detargeting of hepatocytes. In an aggressive MYC-driven liver cancer model, 5A2-SC8 LNPs carrying let-7g miRNA were able to significantly extend survival compared the non-treatment group. Since disease targets exist in an organ- and cell-type specific manner, the clinical development of RNA LNP therapeutics will require an improved understanding of LNP cellular tropism within organs. Overall, the results from this work illustrates the importance of understanding the cellular localization of RNA delivery and incorporating further checkpoints when choosing nanoparticles beyond biochemical and physical characterization, as small changes in the chemical composition of LNPs can have an impact on both the biofate of LNPs and therapeutic outcomes.Item Insig-Mediated Regulation of Hepatic Lipid Synthesis(2007-05-22) Engelking, Luke James; Brown, Michael S.Cholesterol synthesis in mammals is tightly regulated by end-product feedback inhibition. 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes a rate determining reaction that is highly regulated by transcriptional and post-transcriptional mechanisms. As cellular cholesterol accumulates, the transcription of HMGR mRNA is suppressed and the proteosomal degradation of HMGR protein is accelerated. The sterol-regulated transcription of HMGR and other lipogenic genes is controlled by sterol regulatory element binding proteins (SREBPs). These membrane-bound transcription factors are escorted by SREBP cleavage activating protein (SCAP) from the endoplasmic reticulum (ER) to the Golgi apparatus where SREBPs are proteolytically processed to their active forms. In cultured cells, feedback inhibition of SREBP processing is mediated by Insigs. When sterols accumulate, Insigs block SREBP activation by retaining SCAP in the ER. Insigs also mediate rapid, sterol-dependent turnover of HMGR protein. When sterols accumulate, Insigs bind to HMGR and stimulate its ubiquitination and degradation. Although Insigs are key regulators of cholesterol homeostasis in cultured cells, their role in the intact mammal was undefined. To explore this question, gain-of-function and loss-of-function analyses were performed by studying the livers of genetically engineered mice. First, transgenic mice that overexpress Insig-1 in liver (TgInsig-1) were generated. In the livers of TgInsig-1 mice, nuclear SREBPs (nSREBPs) were reduced and SREBP processing was supersensitive to inhibition by feeding high-cholesterol diets. The block in SREBP processing reduced the mRNA levels of SREBP target genes. Levels of HMGR protein were reduced and declined further with cholesterol feeding. Next, knockout mice that lack Insig-1, Insig-2, or both Insigs were generated. In the livers of Insig double knockout mice, cholesterol and triglycerides accumulated to high levels, and despite their accumulation, nSREBPs and mRNAs of SREBP target genes were not suppressed. SREBP processing was insensitive to inhibition by feeding high-cholesterol diets. HMGR protein levels were increased and failed to decline with cholesterol feeding. As a consequence of Insig overexpression or deficiency and the respective effect on SREBPs and HMGR, hepatic cholesterol and fatty acid synthesis in living animals was decreased in TgInsig-1 mice and increased in Insig double knockout mice. These studies indicate that Insigs are essential regulators of hepatic lipid synthesis.Item Metabolic Regulation at Sub-Organelle Length Scales: Inter-Organelle Contacts and Lipid Droplets(2021-09-29) Rogers, Sean W.; Radhakrishnan, Arun; Henne, W. Mike; Liou, Jen; Rosen, Michael K.For cells to properly respond to environmental changes, cellular interiors must be exquisitely organized both spatially and temporally. In particular, metabolism must be spatially coordinated so metabolites are appropriately shunted into either storage or growth. Despite our understanding of how membrane-bound organelles organize metabolic processes, little is known about how metabolic regulation occurs at sub-organelle length scales. At these length scales, physical interactions between the endoplasmic reticulum (ER) and other organelles at ER-membrane-contact-sites (ER-MCSs) are now recognized as sub-organelle hubs for the regulation of metabolic processes. Our work uses the nucleus-vacuole-junction (NVJ) in S. cerevisiae (yeast) as a model ER-MCS to further an understanding about potential general functions of ER-MCSs. We have noted that the NVJ, a physical connection between the nuclear-ER and the vacuole, is a hub for lipid metabolic enzymes and regulators. When yeast are exposed to low glucose conditions, the NVJ recruits several metabolic proteins, including the enzyme Hmg1. Hmg1 catalyzes the conversion of HMG-CoA to mevalonate and is the rate-limiting enzyme in sterol biogenesis. We noted that Hmg1 is less catalytically active when Nvj1, the protein that recruits Hmg1 to the NVJ, is genetically ablated, or when Nvj1 lacks a minimal motif required to recruit Hmg1. Hmg1 NVJ partitioning is accompanied by its assembly into high molecular weight species, which may underlie its increase in enzymatic efficiency. Indeed, artificial tetramerization of Hmg1 overcomes the deficiencies of an Nvj1 knock-out. During Hmg1 partitioning, mevalonate is preferentially shunted into synthesis of sterol-esters (SEs), which are storage lipids found in large cytoplasmic organelles, lipid droplets (LDs). Coordinately, glucose starvation promotes the degradation of triglycerides (TAGs), the other major lipid species contained in LDs. We found that the SE/TAG imbalance in LDs during glucose starvation leads to a phase separation of SEs from a liquid to liquidcrystalline state. Upon SE phase separation, the proteome of LDs is considerably changed. Collectively, our studies of the NVJ have identified a novel function for an ER-MCS and connected it to a lipid metabolic circuit that controls the proteome of LDs.Item [News](1982-08-23) Williams, AnnItem Plasma lipid abnormalities associated with diabetes mellitus(1977-12-22) Bilheimer, David W.Item Role of lipids and lipotoxicity in diabetic renal disease(2001-05-03) Levi, MosheItem SCAP, Insig, and Cholesterol Interactions in Mammalian Cells(2007-05-22) Feramisco, Jamison Derek; Brown, Michael S.; Goldstein, Joseph L.; Roth, Michael G.; Ranganathan, Rama; Albanesi, Joseph P.Cholesterol synthesis in mammalian cells is highly regulated by an end-product feedback mechanism. The transcription of genes necessary for both fatty acid and cholesterol production are controlled by sterol regulatory element binding proteins (SREBPs). The critical regulatory step is the proteolytic release of SREBPs from their inactive membrane bound form. Soon after translation, SREBPs bind SREBP cleavage activating protein (SCAP), a polytopic membrane protein of the endoplasmic reticulum (ER). In sterol depleted situations, SCAP escorts SREBPs to the Golgi, where SREBPs are cleaved and can move freely to the nucleus and activate the numerous enzymes of cellular lipid homeostasis. When cellular sterol levels rise, the SCAP/SREBP complex binds to an ER resident protein named Insig. Upon binding to Insig, the movement of the SCAP/SREBP complex to the Golgi is inhibited, thus halting cholesterol and fatty acid synthesis. The mechanism by which the cell senses sterol levels has been long unknown. Radhakrishnan et al. and Adams et al. demonstrated that SCAP itself binds cholesterol and thus may act directly to sense cellular sterol levels and mediate the end-product feedback control of SREBPs. The goal of this thesis is to elucidate the molecular details of the interactions between SCAP, Insig and cholesterol. My thesis experimentally details the membrane topology of human Insig-1 and shows that it is a polytopic integral membrane protein of the ER with six transmembrane spanning segments. In addition, the amino and carboxy-termini of Insig are both facing the cytosol. Furthermore, crucial residues of Insig that are important for SCAP interaction are identified. My thesis has also defined distinct amino acids of SCAP that are essential for its role as a protein that binds Insig and as a protein that has the ability to bind cholesterol. An aspartic acid in the middle of transmembrane six is necessary for sterol regulated binding to Inisg, while residues in transmembrane segments one and three of SCAP are crucial for cholesterol binding both in vivo and in vitro.Item [Southwestern News](2003-08-08) Shields, AmyItem [UT News](1985-08-08) Bosler, Tommy Joy