Browsing by Subject "Liver"
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Item Challenges of field inhomogeneities and a method for compensation(2011-11) Styczynski Snyder, Angela LynnMRI of the body at 7 T has become possible only very recently. High fields bring the advantages of increased signal to noise ratio, resolution gains, faster image acquisition through better parallel imaging, improved and novel types of contrast, and greater spectral dispersion. There are many obstacles to be faced in the advancement to 7 T including field inhomogeneities, transmit inhomogeneities, and increased RF-absorption risk. Inhomogeneous B1+ is possibly the most significant obstacle currently facing 7 T body imaging in consistently producing clinical-quality images. In this work, the first demonstrations of MR imaging and spectroscopy (MRS) of the liver at 7 T are presented and evaluated with special attention given to parallel imaging. Also presented here is the first demonstration of imaging of the uterus at 7 T with particular emphasis on contrast between uterine layers. A comparison was done between 3 T and 7 T, and relaxation rates were mapped including higher rotating frame relaxations.SWIRLY (spatiotemporal-encoding with incremental refocusing along a trajectory) is a novel pulse sequence that employs a frequency and amplitude modulated excitation pulse in the presence of sinusoidal gradients to move a region of resonance through space along a spiral trajectory. The signal can be sequentially refocused and acquired in an entirely spatiotemporal manner such that no Fourier Transform is needed for reconstruction. Because each resonance region can be treated entirely independently, this sequence has incredible potential for addressing problems that are inherently spatial in nature, such as B1+ and B0 inhomogeneities.Item Dietary and Cellular Mechanisms Regulating Hepatocyte Proliferation and Cancer(2017-08) Ploeger, JonathanObesity is well-documented to promote the development of nonalcoholic fatty liver disease (NAFLD) including its more advanced stages such as non-alcoholic steatohepatitis, cirrhosis and hepatocellular carcinoma (HCC). While metabolic perturbations describing obesogenic progression from NAFLD to HCC have largely been investigated, our knowledge of the role lipolysis plays in this process is scant. This research project is aimed at understanding the role fasting lipid metabolism plays in pathologic features of carcinogenesis as well as synergistically combining with lifestyle factors to prevent obesity driven progression of NAFLD to HCC. To elucidate these features, we employed two seminal studies. The first study characterized the role of adipose triglyceride lipase (ATGL) in limiting a major cell cycle regulator, cyclin D1, and hepatocellular proliferation both in vitro and in vivo. We show that lipid catabolism via ATGL antagonizes cell proliferation. Additionally, we recapitulate these findings using a partial hepatectomy model to drive hepatocellular proliferation in vivo. In the second study, we conduct a long-term carcinogenesis study that examines the role of dietary fat composition and lifestyle factors that promote fasting lipid metabolism. Animals were calorically restricted (CR) or exposed to regular endurance exercise. Using the hepatic carcinogen diethylnitrosamine (DEN), we show CR prevents hepatic tumor formation independent of dietary fat composition. RNA sequencing of non-transformed liver tissues revealed changes in metabolic pathways and reduced inflammation, cytokine production, stellate cell activation and migration, and genes associated with liver injury and oncogenesis. Taken together, fasting hepatic lipid metabolism plays a significant role in mitigating proliferative effects often associated with overconsumption of calories. Furthermore, lifestyle factors that promote lipolysis in the liver robustly protected mice from developing tumors. Further investigation is warranted to define the molecular mechanisms ATGL plays in limiting hepatic proliferation as well as characterizing the role of ATGL and fasting in hepatic tumorigenesis.Item The Effect of Perfluorooctane Sulfonate (PFOS) and Choline Supplementation on Hepatic Steatosis in Sprague Dawley Rats(2017-05) Bagley, BradfordPerfluorooctane sulfonate (PFOS) is bioaccumulative and prevalent in the human population. PFOS induces hepatic steatosis in male rats at dietary exposures of 100 ppm via an unknown mechanism. In vitro, PFOS creates a choline ion complex. Choline deficiency induces hepatic steatosis in rats by decreasing VLDL secretion. The primary hypothesis was that a hepatic PFOS:choline ion complex causes steatosis that could be prevented by dietary choline supplementation. PFOS activation of steatosis related nuclear receptors (i.e., LXR, PXR, CAR, and PPAR-gamma) was investigated as a secondary hypothesis. To identify a choline dietary concentration, Sprague Dawley rats (5-6/sex/group) were fed control diet or 5X, 10X, or 15X basal choline diets for four weeks. The 5X diet was selected based on decreased body weights and body weight gains in the 10X (females only) and 15X groups. Sprague Dawley rats (12/sex/group) were fed control, choline supplemented (CS), 100 ppm PFOS, or 100 ppm PFOS + CS diets for three weeks. The male PFOS (±CS) rats developed hepatic steatosis, decreased mean serum cholesterol, and increased liver choline concentrations; the supplemented diet did not prevent hepatic steatosis. Female rats did not have these findings, even though serum and liver PFOS concentrations were similar to the males. In vitro, 400 µM PFOS did not inhibit choline kinase activity, which does not support the primary hypothesis. Regarding the secondary hypothesis, there was no activation (LXR, PXR, and CAR) or very weak activation (PPAR-gamma) by PFOS in a luciferase-linked assay. Also, liver mRNA activated by these nuclear receptors were not upregulated in rats fed PFOS. There are no clear data from this project that support the primary or secondary hypothesis. However, increased hepatic choline concentrations in the male PFOS rats correlates with the primary hypothesis. This finding and the sex-related difference in PFOS-induced hepatic steatosis warrant further investigation.Item Engineering a three-dimensional culture system for the directed differentiation of pluripotent stem cells toward a hepatocyte-like cell fate(2012-11) Owens, Derek JasonBecause stem cells have the ability to self-renew and differentiate into more specialized cell types, they hold enormous potential in the fields of regenerative and personalized medicine as well as providing a model system for studying development in vitro. Stem cells with the capacity to differentiate to hepatocytes, the functional cells of the liver, have potential applications in the pharmaceutical industry in high-throughput drug toxicity screening and in clinical settings in bioartificial liver devices or as candidates for transplantation to treat end-stage liver disease. However, these applications rely on the ability to generate and differentiate the stem cells to functionally mature hepatocytes in a robust and reproducible manner. We have recently optimized a multistage directed differentiation protocol for guiding human embryonic stem (hES) cells and rat multipotent adult progenitor cells (rMAPCs), among other stem cell types, toward a hepatic fate. We also recently showed that rMAPCs, which are isolated from the bone marrow of post-natal rats and exhibit the ability to self-renew and differentiate to all three lineages, can be cultured as three-dimensional aggregates without losing their potency or self-renewal capacity. In this study, we report three-dimensional aggregate-based culture systems that enhance the differentiation of rMAPCs and hES cells to hepatocyte-like cells. rMAPCs were allowed to self-assemble into undifferentiated aggregates before being differentiated via the four-step directed differentiation protocol. Compared to adherent monolayer cultures, differentiation as aggregates resulted in significantly higher expression of liver-specific transcripts, including albumin, and increased secretion of albumin and urea. The differentiated cell aggregates also demonstrated functional activities of primary hepatocytes, as demonstrated by pentoxyresorufin O-dealkylation (PROD) and ethoxyresorufin O-dealkylation (EROD), and ultrastructural features of hepatocytes by electron microscopy. A similar three-dimensional culture system likewise enhanced the differentiation of hES cells. HSF6 cells differentiated as a monolayer culture were dissociated and allowed to self-assemble into three-dimensional spheroids in an extended differentiation culture. Compared cells maintained in the monolayer culture, cells within the spheroids exhibited significantly higher expression levels of liver-enriched transcripts and proteins, including Albuming, PEPCK, and ASGPR-1. Cells in the spheroids demonstrated hepatic functions EROD, PROD, and biliary accumulation of fluorescein diacetate metabolite and ultrastructural characteristics of hepatocytes by electron microscopy. Finally, whole-genome transcriptome analysis was performed to investigate the expression profile of liver-specific sets of genes, including the hepatocyte nuclear factors (HNFs), cytochrome P450s (CYP450s), and UDP-glucuronosyltransferases (UGTs), during differentiation. Cells in the spheroid were shown to have overall increased expression levels of most of the genes in these families, although the expression levels were still lower than in adult liver. The transcriptome analysis was also used to identify genes that change during establishment of the spheroid culture that may play a role in the enhanced differentiation status of the cells; multiple members of the aldo/keto reductase (AKR) and metallothionein (MT) families were found to have much higher expression in spheroids than in monolayer culture. These studies demonstrate the ability of three-dimensional, scalable culture systems to enhance the differentiation of pluripotent stem cells toward a hepatic fate and to maintain the differentiated phenotype for extended culture. With modifications to further enhance the maturity of stem cell-derived hepatocyte-like cells, these systems may facilitate the translation of stem cell generated tissues to technology.Item Hepatotoxic and Immunomodulatory Transcriptome Responses to Aflatoxin B1 in the Turkey (Meleagris gallopavo)(2015-05) Monson, MelissaHepatoxicity and immunotoxicity from dietary exposure to aflatoxin B1 (AFB1) adversely affect poultry health and production. Domestic turkeys (Meleagris gallopavo) are especially sensitive to AFB1 since they have a deficiency in glutathione-mediated detoxification of the reactive AFB1 intermediate. Changes in gene expression can be used to characterize the molecular mechanisms of toxicity; transcriptome analysis allows investigation of differential expression at the genome-wide level. In this research, Illumina RNA-sequencing (RNA-seq) was used to examine transcriptome responses to AFB1 exposure in the turkey. As the liver is the primary site of AFB1 activation and toxicity, the effects of dietary AFB1 on the domestic turkey liver transcriptome was first investigated by sequencing 4 pooled libraries representing 3 individuals for each of 4 treatment groups. As detailed in Chapter 2, predicted transcripts were de novo assembled and differential expression analysis identified significant effects on transcripts from genes involved in apoptosis, cell cycle regulation and lipid metabolism (like E3 ubiquitin-protein ligase Mdm2 and lipoprotein lipase). In Chapter 3, RNA-seq and de novo transcriptome assembly were performed on 3 individual spleen samples per treatment group (n = 12) collected from the same AFB1 challenge trial. Significant down-regulation of antimicrobial genes (like beta-defensin 1) and up-regulation of cytotoxic and antigen presentation genes (such as granzyme A) were observed after AFB1 treatment. Another aspect of these studies was to evaluate the ability of a Lactobacillus-based dietary probiotic to reduce AFB1-effects in the liver and spleen. Addition of probiotics during AFB1 exposure modulated expression in both tissues. Many AFB1-induced expression changes were not mitigated in liver, and although probiotics had some amelioratory effects in the spleen, they were also broadly suppressive of immune genes. Multiple genes impacted in the spleen transcriptome belonged to the Major Histocompatibility Complex (MHC), a region of the genome with genes essential to immune functions. The functions and expression patterns of many of the genes located in the turkey MHC have not been characterized. A single-gene investigation (Chapter 4) characterized expression patterns of 29 MHC genes in the domestic turkey and provided first evidence for expression of B-butyrophilin 2 in muscle tissue. Understanding these expression profiles will help determine MHC gene functions and provide background for expression changes from immunological challenges like AFB1. Unlike the domestic turkey, Eastern wild turkeys (M. g. silvestris) are more resistant to aflatoxicosis due at least in part to their ability to detoxify AFB1. In Chapter 5, an in ovo exposure model was utilized to directly compare the effects of AFB1 exposure in domestic and wild turkey embryos. Embryonic exposure has applicability to poultry production since AFB1 can be maternally transferred into eggs. RNA-seq datasets from embryonic liver tissue in domestic (n = 24) and wild (n =15) turkeys were mapped to a MAKER turkey gene set. Differential expression and pathway analysis identified conserved effects on cell cycle regulators (like E3 ubiquitin-protein ligase Mdm2) and variable effects in genes encoding detoxifying and anti-oxidant enzymes (like glutathione S-transferases) in domestic and wild turkeys. Overall, transcriptome analysis identified hepatic and splenic responses to AFB1, evaluated the use of probiotics, directly compared domestic and wild turkeys, and provided gene targets for future investigation of the molecular mechanisms of aflatoxicosis.Item The Role Of Acyl-Coa Thioesterase 1 In Hepatic Lipid Metabolism(2017-05) Franklin, MalloryThe liver is an essential organ for maintaining homeostasis and is vital for storage, synthesis, oxidation, and recirculation of lipid in fed and fasted states. In response to fasting, fatty acids (FAs) flux from adipose tissue to the liver and are converted to acyl-CoAs for incorporation into complex lipids or transportation into the mitochondria for oxidation. The latter process is orchestrated by a group of proteins that are transcriptional targets of peroxisome proliferator activated receptor α (PPARα). However, little is known about hepatic acyl-CoA thioesterase 1 (ACOT1), a member of the broader acyl-CoA thioesterase family that catalyzes the conversion of acyl-CoAs back to FAs and coenzyme A. Thus, this research is aimed to understand the role of ACOT1 in fasting lipid metabolism. To investigate its physiological importance, we employed adenovirus-mediated knockdown, overexpression in tissue culture, as well as generation of a whole-body Acot1 knockout mouse line. Our results show that ACOT1 preferentially hydrolyzes acyl-CoA molecules that are destine for mitochondrial β-oxidation. As such, acute Acot1 knockdown results in reduced liver triglyceride (TG) and enhanced FA oxidation in vivo and in vitro. Increased FA oxidation correlated to greater hepatic glucose production and storage. Additionally, we determined that ACOT1 regulates PPARα by providing FA ligands. As such, supplementation with a PPARα synthetic ligand rescues the Acot1 knockdown phenotype. Furthermore, Acot1 overexpression increases PPARα activity only when ACOT1 is catalytically active. Together these data suggest that ACOT1 regulates PPARα through its hydrolysis product. We also discovered that ACOT1 translocates to the nucleus during prolonged fasting, potentially to provide a local pool of FAs to activate PPARα. Thus, acute Acot1 knockdown solicits enhanced FA oxidation, yet reduces PPARα target gene expression. Complications of this disconnect between metabolism and gene expression was evident by increased oxidative stress and inflammation, often seen in fibrotic and cirrhotic stages of non-alcoholic fatty liver disease (NAFLD), when Acot1 was knocked down. To further our investigation of ACOT1, we compared whole-body Acot1 knockout mice to their wild type littermates. We demonstrate that Acot1 knockout lead reduces adiposity, by decreasing adipocyte size and increasing adipocyte number. Acot1 knockout also reduced hepatic TG, providing protection from oxidative stress and inflammation that precedes TG accumulation. However, Acot1 knockout reduced glucose tolerance suggesting impaired glucose homeostasis. These results suggest Acot1 knockout impaired lipid storage potential, increasing lipid intermediates, and contributing to glucose intolerance. Taken together, hepatic ACOT1 regulates FA oxidation and protects from oxidative stress and inflammation, whereas whole-body ACOT1 contributes to lipid storage.Item Triacylglycerol hydrolysis and fatty acid partitioning in the liver(2013-05) Tholen, Jillian TheresaThe liver is a vital organ for the homeostatic control of both carbohydrate and lipid metabolism, functioning as a major regulatory site of energy storage, processing, and transformation. This project focused on liver lipid metabolism--it's production and trafficking of very low density density lipoproteins in particular. Specifically, we investigated whether VLDL trafficking is affected by 1) glucose and/or insulin, and 2) source of fatty acid (de novo or exogenous). As these were two distinct questions, each required its own sub-study, both of which are outlined separately below. Although it is well known that glucose and insulin influence the metabolism of fatty acids (FA) and the secretion of very low-density lipoprotein (VLDL), little is understood about how they regulate the partitioning of FA hydrolyzed from hepatic stored triacylglycerol (TAG) to different metabolic pathways. The aims of this study were to investigate the effects of differing concentrations of glucose and insulin on TAG hydrolysis and the subsequent partitioning of FA to pathways of secretion and oxidation in primary hepatocytes. We utilized primary mouse hepatocytes for pulse-chase experiments using [1-14C] oleate to examine the turnover and partitioning of the labeled FA. In addition to looking at the effects of differing glucose and insulin concentrations, we wanted to investigate whether any effects differed following acute (6 h) vs. chronic (24 h) treatment. Although we did not observe any acute effects of either glucose or insulin on TAG hydrolysis or partitioning of hydrolyzed FA, chronic insulin exposure decreased both FA oxidation and TAG secretion. These results suggest that insulin and glucose do not acutely influence TAG hydrolysis or channeling of hydrolyzed FA, but longer exposure to insulin reduces FA oxidation and secretion. There is a great deal of evidence suggesting that before being incorporated into VLDL for secretion, hepatic FA are first directed to storage in the cytosolic lipid droplet pool, and must subsequently be mobilized and channeled to assembly into VLDL. It remains unclear, however, whether the liver stores FA from different sources in distinct lipid droplet pools. Furthermore, should distinct storage pools exist, it is unknown whether they are preferentially channeled to different pathways, such as VLDL assembly. We performed experiments utilizing [1-14C] acetate and [1-14C] palmitate to represent FA derived from de novo lipogenesis and exogenous uptake, respectively. There was no difference between percentage of cellular acetate (via conversion to long chain FA) or palmitate secreted into the media at 4, 8, and 16 h in primary hepatocytes. However, we consistently saw a greater percentage of the label from acetate (de novo FA) secreted into the media after 2 h. Following the same 2 h labeling period in primary hepatocytes, we observed no difference between the partitioning of [1-14C] acetate and [1-14C] palmitate to the microsomal (ER) and lipid droplet fractions. Finally, to examine in vivo partitioning of the different FA, we injected mice with labeled FA and isolated their livers to quantify the contribution of the respective FA to cellular TAG and phospholipids. There was no difference in the incorporation of differentially sourced FA in liver TAG or phospholipid fractions. The results of these studies suggest that given the choice between FA taken up from exogenous sources and FA synthesized de novo, the liver may acutely preferentially secrete FA from de novo synthesis, however more work is needed to confirm this finding.