Browsing by Subject "Liver"
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Item Biochemical Characterization of Niemann-Pick C: a Disease of Cholesterol Transport(2012-08-15) Infante, Rodney Elwood; Brown, Michael S., M.D.Despite intense scientific interest, the mechanism by which cholesterol is transported between membrane compartments in animal cells remains obscure. One transport pathway begins in lysosomes where cholesterol is liberated from plasma lipoproteins that have entered the cell through receptor-mediated endocytosis. This cholesterol is transported from the lysosome to other cellular membranes to perform structural and regulatory roles. A clue to the mechanism of this cholesterol movement comes from observations in cells from patients with Niemann-Pick Type C (NPC) disease. These individuals accumulate large amounts of cholesterol throughout the body caused by mutations in either one of two genes encoding the lysosomal proteins NPC1 and NPC2. Unlike the membrane protein NPC1, evidence suggests that the soluble protein NPC2 is a cholesterol binding protein. In the course of isolating a cholesterol-homeostasis membrane protein that binds sterols, we encountered NPC1. Using rabbit membranes, an integral membrane protein that bound sterols was isolated with a 14,000-fold purification while maintaining 8% final yield. Mass spectrometry identified the protein to be NPC1. Recombinant human NPC1 was expressed, purified, and confirmed to be a high affinity sterol receptor. NPC1Õs sterol binding domain was localized to itÕs N-terminal luminal soluble domain, which can be prepared as a soluble protein of 240 amino acids, NPC1(NTD), that is secreted by cells. The binding properties of NPC1(NTD) binds cholesterol similar to NPC2 with a Kd of ~130nM. Cross-competition studies between purified NPC1(NTD) and the soluble NPC2 protein revealed differences in sterol specificity depicting the different parts of the cholesterol moiety the NPC proteins bind. [Keywords: Niemann-Pick C Disease; cholesterol trafficking; cholesterol binding; oxysterols; lysosomes; Niemann-Pick C1 protein; Niemann-Pick C2 protein]Item Calpain and lipopolysaccharide mediated hepatitis(2009-06-02) Rose, Robert EdwardThis study tested the role of the calcium dependent cytosolic protein calpain in neutrophilic hepatitis. We hypothesized that inhibition of calpain would protect against LPS-induced neutrophilic liver damage. To test our hypothesis, a reliable LPS-mediated hepatitis model to investigate the mechanisms of hepatic neutrophil infiltration following LPS administration was developed by repeat intravenous injection of LPS at a dose of 10 mg/kg to rats. Blood was collected for hematologic and biochemical analysis and multiple organs including liver were collected for evaluation of histopathologic changes. Flow cytometry was employed to investigate L-selectin (CD 62L) and MAC-1 (CD11b/18) expression on neutrophils both in vivo and in vitro. Significant hematologic changes included neutrophilia, elevation in neutrophil to lymphocyte ratio with toxic changes and left shift. Biochemical changes were observed in several liver (AST, GGT) and kidney (BUN) parameters generally at the earliest time points. Histopathology revealed a time-dependent neutrophil and mononuclear infiltration around the periportal areas in the single dose study and mid-zonal inflammation with multifocal coagulative necrosis in the repeated dose study. CD 11b was up-regulated both in vitro and in vivo. After development of a suitable model, the first goal was to investigate the role of the intracellular enzyme calpain in the development of neutrophilic hepatitis and midzonal necrosis. A second goal was to compare the observed protective effects of calpain inhibition with a relatively selective inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine and an inhibitor of coagulation, heparin. When compared to rats administered LPS alone, administration of calpain 1 inhibitor prior to LPS significantly reduced hepatic iNOS expression, hepatic neutrophil infiltration and attenuated midzonal hepatic necrosis. Administration of heparin and aminoguanidine prior to LPS also decreased liver iNOS expression, hepatic neutrophil infiltration and liver pathology comparable to calpain inhibition. Blood neutrophil activation, as measured by the neutrophil adhesion molecule CD11b integrin, was upregulated in all the LPS treated groups regardless of inhibitor administration. We conclude that amelioration of liver pathology via calpain inhibition is likely dependent on the down-regulation of iNOS expression in the rat model of LPS mediated hepatitis.Item Development and validation of microcystin biomarkers for exposure studies(2006-05) Billam, Madhavi; Wang, Jia-Sheng; Anderson, Todd; Pence, Barbara; Shen, Leslie; Smith, ErnestMicrocystins (MCs) are hepatotoxic cyanotoxins produced mainly by the cyanobacteria Microcystis spp. They are distributed in waterbodies worldwide, and the toxicity on exposure to MCs was reported worldwide in fish, animals and in humans for over a century. There are about 70 known variants of MCs to date and of them the most toxic and widely distributed MC is Microcystin-LR (MCLR). MCLR is hepatotoxic and a potent tumor promoter. Epidemiological studies in China have linked exposure to MCs with high incidence of liver cancer. Although analytical tools have been reported to detect MCs in water and in food samples, and biomarkers for biochemical alterations like inhibition of protein phosphatases (PP) have been proposed, to date, validation of these analytical methods for simultaneous measurement of MCs in environmental samples and in body fluids of exposed individuals has not yet been done. In this study, we have developed new methods and validated existing methods to detect MCLR and its biomarkers in body fluids of animals and human hepatic cells treated with different concentrations of MCLR. The methods thus validated were used to monitor the seasonal fluctuations in MCLR concentrations in two lakes of western Texas. Studies were also conducted to explore molecular level targets of MCLR in normal (THLE-2) and cancerous (HepG2) human hepatic cell lines. Effect of MCLR on cell proliferation was explored, and validated methods were used to detect alteration in PP activity in these cell lines on exposure to MCLR. Alteration in expression of apoptosis regulatory proteins like Bax, Bcl2, Bad and PP2A on exposure to MCLR was also studied by immunoblotting in these cell lines. Studies were also conducted to explore molecular level targets of MCLR on acute exposure to a single dose, and on subacute exposure to repeated doses of MCLR in F-344 rats. We observed a dose dependent alteration in expression of PP2A, Bax, Bcl2 and Bad in both acute and subchronic exposures, as quantified by western blotting and immunohistochemistry. The study also focused on alteration in levels of sphingolipids in serum on exposure to MCLR. In another experiment, the combinative toxic effect of MCLR along with the tumor initiator aflatoxin-B1 was studied in normal and hepatocarcinoma cell lines, and the mechanism involved was explored.Item Development and validation of microcystin biomarkers for exposure studies(Texas Tech University, 2006-05) Billam, Madhavi; Wang, Jia-Sheng; Pence, Barbara; Smith, Ernest; Shen, Leslie; Anderson, ToddMicrocystins (MCs) are hepatotoxic cyanotoxins produced mainly by the cyanobacteria Microcystis spp. They are distributed in waterbodies worldwide, and the toxicity on exposure to MCs was reported worldwide in fish, animals and in humans for over a century. There are about 70 known variants of MCs to date and of them the most toxic and widely distributed MC is Microcystin-LR (MCLR). MCLR is hepatotoxic and a potent tumor promoter. Epidemiological studies in China have linked exposure to MCs with high incidence of liver cancer. Although analytical tools have been reported to detect MCs in water and in food samples, and biomarkers for biochemical alterations like inhibition of protein phosphatases (PP) have been proposed, to date, validation of these analytical methods for simultaneous measurement of MCs in environmental samples and in body fluids of exposed individuals has not yet been done. In this study, we have developed new methods and validated existing methods to detect MCLR and its biomarkers in body fluids of animals and human hepatic cells treated with different concentrations of MCLR. The methods thus validated were used to monitor the seasonal fluctuations in MCLR concentrations in two lakes of western Texas. Studies were also conducted to explore molecular level targets of MCLR in normal (THLE-2) and cancerous (HepG2) human hepatic cell lines. Effect of MCLR on cell proliferation was explored, and validated methods were used to detect alteration in PP activity in these cell lines on exposure to MCLR. Alteration in expression of apoptosis regulatory proteins like Bax, Bcl2, Bad and PP2A on exposure to MCLR was also studied by immunoblotting in these cell lines. Studies were also conducted to explore molecular level targets of MCLR on acute exposure to a single dose, and on subacute exposure to repeated doses of MCLR in F-344 rats. We observed a dose dependent alteration in expression of PP2A, Bax, Bcl2 and Bad in both acute and subchronic exposures, as quantified by western blotting and immunohistochemistry. The study also focused on alteration in levels of sphingolipids in serum on exposure to MCLR. In another experiment, the combinative toxic effect of MCLR along with the tumor initiator aflatoxin-B1 was studied in normal and hepatocarcinoma cell lines, and the mechanism involved was explored.Item Dietary fat and antioxidant status relating to colon carcinogenesis(Texas Tech University, 1993-05) Tsai, Shwu-yarEpidemiological evidence has linked dietary fat with colorectal cancer in humans but with mixed results. Studies using animal models have shown that high fat diets containing predominantly corn oil, beef tallow or lard induce colon tumorigenesis to a greater extent than corresponding low fat diets; however, these findings are still inconclusive. Recently, some research indicated differences according to the types of fat used and support the concept that diets high in unsaturated fatty acids have a greater tumor-promoting capability than diets high in saturated fatty acids. The mechanism was proposed that free radicals were involved in colon carcinogenesis. Therefore, a polyunsaturated fatty acid (PUFA) diet would increase antioxidant activity to prevent free radical damage. Several studies have indicated that dietary lipids influence the liver microsomal mixed function oxidase system. It has been reported that the elevation of dietary polyunsaturated fatty acid intake increases the activity of the liver microsomal enzymes responsible for carcinogen metabolism. Some studies directed the diet-related effect on the susceptibility of colonic cells to nuclear-damaging agents. However, there have been very few studies on the effects of dietary fat and nuclear aberrations due to xenobiotics challenge. The purpose of this study was to examine the effect of amount and type of dietary fat on the (1) colon mucosal antioxidant status; (2) liver microsomal demethylase activity; (3) liver microsomal and cytosolic mutagenic activation; and (4) colonic epithelial nuclear aberrations during colon carcinogenesis.Item Feeding tylosin in attempting to control liver abscesses(Texas Tech University, 1969-05) Simpson, Kenneth EdwardNOT AVAILABLEItem 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 Liver abscess effect on certain production and carcass traits in fattening beef cattle(Texas Tech University, 1966-08) Powell, Murphy DallasThe primary objective of this analysis was to determine the effects, if any, of liver abscesses on midpoint gain, total gain, dressing percentage and martling score of beef cattle.Item The Liver-Derived Endocrine Hormone FGF21 Alters Metabolism and Diurnal Behavior via the Nervous System(2012-07-09) Bookout, Angie Lynn; Mangelsdorf, David J.Fuel acquisition is essential to survival. During privation, the body protects glucose concentrations acutely by glycogenolysis, and later by gluconeogenesis and ketogenesis. Additionally, animals alter daily behavioral patterns to seek food, but eventually reduce energetically costly activities (growth, reproduction, locomotion). Little is known about the mechanisms that orchestrate and coordinate these physiological and behavioral responses to starvation. The liver-derived endocrine hormone fibroblast growth factor 21 (FGF21) is induced in chronic fasting and acts as a global starvation signal. Previous studies focusing on FGF21 as an anti-diabetic drug indicate that FGF21 coordinates whole-body fat utilization and energy expenditure. However, its basic physiological role is underexplored. // Acute injection of recombinant FGF21 quickly elicits a coordinated program between tissues resulting in reduced plasma insulin and gluconeogenic and thermogenic gene expression programs in liver and brown adipose, effects that require an intact animal. Mice with chronic FGF21 overexpression (FGF21tg) are smaller in size, females are infertile, and if fasted, they undergo torpor, an energy-conserving process. Taken together, these data suggest that FGF21 may exert some effects through the nervous system. // To explore this idea, I utilized anatomically-guided laser capture microdissection followed by quantitative, real-time PCR to profile expression of the FGF receptor/co-receptor family in specific hypothalamic nuclei of mice. Surprisingly, the FGFR1-IIIc/βKlotho complex is found in the suprachiasmatic nucleus (SCN), area postrema (AP), nucleus tractus solitarii (NTS), and nodose ganglion (cell body of vagus nerve), implicating roles in circadian and metabolic regulation. // Results of surgical, pharmacological, and genetic strategies indicate the vagus senses circulating FGF21, resulting in adrenergic efferent responses that reduce insulin secretion, while a different adrenergic site modulates liver and brown adipose gene expression. //Analyses of the effects of FGF21 on the SCN, the body’s master clock, using running wheels show FGF21tg mice have dramatically altered circadian activity, likely as a consequence of inhibiting SCN output functions. Deletion of βKlotho specifically from the SCN rescues this behavior in addition to growth defects of FGF21tg mice. To date, this is the first description of a liver-derived endocrine hormone that affects such diverse aspects of the starvation response by acting on the nervous system. [Keywords: FGF21. Circadian, metabolism, starvation, hypothalamus, PPAR, diabetes, fasting]Item Metabolic Regulation by Fibroblast Growth Factor 21(2011-12-12) Dutchak, Paul Anthony; Kilewer, Steven A.Fibroblast growth factor 21 (FGF21) is a secreted hormone that can beneficially regulate glucose and lipid homeostasis. Through a reverse endocrinology approach, we uncovered that FGF21 expression is transcriptionally regulated by the peroxisome proliferator activated-receptor alpha (PPARa) in liver. PPARa is a member of the nuclear hormone receptor superfamily that is physiologically activated by increased fatty acid mobilization to liver during fasting, and regulates the genetic program whereby lipids are converted to ketone bodies through a process known as ketogenesis. Here, I show the effects of FGF21 as a fasting hormone that is expressed in liver and contributes to the regulation of adipose tissue and hepatic ketogenesis during the fasted state. Using in vitro and in vivo methods to investigate the effects of FGF21, a model whereby FGF21 stimulates lipolysis in adipose tissue was generated. Intriguingly, using our FGF21 transgenic mice, I observed the expression of many genes involved in lipogenesis was highly induced in adipose tissue in an FGF21-dependent manner. Moreover, many of these lipogenic genes were found to be down-regulated in adipose of the FGF21 knockout mouse. The inhibition of lipogenic genes in adipose tissue was associated with increased SUMOylation of PPARg protein in this tissue. Using a feeding-fasting paradigm, I found that FGF21 expression in the liver and adipose tissue was rhythmic, peaking in liver prior to feeding and peaking in the adipose after feeding. Furthermore, the induction of FGF21 by PPARg ligands suggested a unique function for this protein in adipose, independent from its role in the fasted state. To assess the contribution of FGF21 to the anti-diabetic properties of PPARg agonists (ie. thiazolidinediones), diet-induced obese wild type and Fgf21-/- mice were treated with the TZD rosiglitazone. Rosiglitazone produced a significant increase in adipose FGF21 expression, but decreased hepatic FGF21 mRNA and circulating FGF21 protein. These data suggest that FGF21 functions as an autocrine factor within adipose tissue. Moreover, the therapeutic effects of rosiglitazone as an insulin sensitizer were lost in the Fgf21-/- mouse, as assessed by glucose and insulin tolerance tests. Several other effects of rosiglitazone were lost in the Fgf21-/- mice, including increased adipose mass, edema, and PPARg target gene expression in the adipose. These data indicated that PPARg can control the expression of FGF21, which functions as a feed-forward mechanism to stimulate PPARg target genes and PPARg dependent physiology. Since PPARg can be modified by SUMO on two different sites on the protein, in vitro experiments were performed to show that PPARg is SUMOylated at Lysine-107, a previously identified negative regulator of its transcriptional activity. Importantly, I found that treatment of Fgf21-/- adipocytes with FGF21 reduced the amount of SUMOylated PPARg, thereby allowing it to be it an active state. Collectively, these data reveal that FGF21 has two independent roles in regulating metabolism in vivo: as a hepatic endocrine hormone that is induced during the fasting response through PPARa, and as an adipose autocrine/paracrine factor that is induced in a feed-forward loop to stimulate PPARg activity.Item Purification of Native and Recombinant NPC1(2008-12-23) Dale, Jarrod Donald; Goldstein, JosephThe Niemann-Pick, Type C1 protein (NPC1) is required for the transport of lipoproteinderived cholesterol from lysosomes to endoplasmic reticulum. The 1278-amino acid, polytopic membrane protein has not been purified, and its mechanism of action is unknown. We encountered NPC1 in a search for a membrane protein that binds 25-hydroxycholesterol (25-HC) and other oxysterols. Described here is the initial purification of rabbit NPC1 using a classical biochemical approach and an analysis of the sterol binding properties of native and recombinant NPC1. Our purification yielded a membrane-bound 25-HC-binding protein which was purified more than 14,000-fold from rabbit liver membranes. This protein was identified as NPC1 by mass spectroscopy. We prepared recombinant human NPC1 and confirmed its ability to bind oxysterols, including those with a hydroxyl group on the 24, 25, or 27 positions. Hydroxyl groups on the 7, 19, or 20 positions failed to confer binding. Initial characterization of the sterol binding properties showed specific binding for 25-HC; however, we were unable to demonstrate significant binding of NPC1 to cholesterol using our current experimental conditions. The availability of assays to measure NPC1 sterol binding in vitro may further the understanding of intracellular sterol transport.Item Timing Matters: The Role of Circadian Clock Genes In Development and Toxin Responses(2009-05-15) Qu, XiaoyuMost members of the PAS (PER-ARNT-SIM) protein family are transcription factors, mediating development and adaptive responses to the environment, such as circadian rhythms and toxin responses. Because the PAS domain mediates protein-protein interactions and functional cross-talk between distinct biological processes, we hypothesized that PAS genes in the circadian clockworks, namely Per1 and Per2, may be involved in development and toxin responses, which are modulated by other PAS members. To explore the possible role of clock genes in development, we examined mammary epithelial cells in vitro and the mouse mammary gland in vivo for evidences of changes in clock gene expression during different stages of development and differentiation. Our results showed that Per1 and Bmal1 expression were up-regulated in differentiated HC-11 cells, whereas Per2 mRNA levels were higher in undifferentiated cells. A similar differentiation-dependent profile of clock gene expression was observed in mouse mammary glands; Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, whereas Per2 expression was higher in proliferating virgin and early pregnant glands. These data suggest that circadian clock genes may play a role in mouse mammary gland development. To examine clock gene function in toxin responses, we evaluated whether disruption or inhibition of Per1 and/or Per2 alters toxin-induced activity of the AhR signaling pathway in the mouse mammary gland and liver. We assessed the activation of the AhR signaling pathway in response to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototypical AhR agonist, by analyzing the mRNA abundance of its two target genes, cytochrome P450, subfamily I, polypeptide 1 (Cyp1A1) and Cyp1B1. Our results showed that the targeted disruption of Per1, but not Per2, significantly increases the TCDD-induced p450 expression in the mammary gland and liver in vivo. Similar changes in TCDD-mediated p450 expression were observed in vitro using mammary primary cultures of mammary cells derived from from Per1ldc, Per2ldc and Per1ldc/Per2ldc mutant mice and Hepa1c1c7 cells subjected to siRNA-mediated inhibition of Per1 or Per2. These discoveries suggest that the clock gene Per1 may modulate toxin responses perhaps by functioning as a negative regulator for TCDD-mediated activation of the AhR signaling pathway.Item Tracing organic matter pathways in marine food webs using fatty acids and compound specific stable isotope analysis(2015-08) Smith, Stephanie Denise; McClelland, James W.; Dunton, Kenneth H; Walther, Benjamin DOrganic matter inputs to the marine environment vary over seasonal and spatial scales, altering the type and availability of food sources for marine consumers. It is important to identify diet in order to understand basic ecology, characterize trophic interactions, and predict consequences of biotic and abiotic change within a community. Methods of direct observation of diet and feeding can be difficult, so indirect methods have been developed such as analysis of gut contents and fecal pellets. However, these methods only represent a snapshot of the last meal, and provide information about what was ingested, but not what was actually incorporated into consumer tissues. Therefore, biogeochemical approaches such as fatty acid (FA) and stable isotope analyses have been developed, which provide a time-integrated measure of diet. Further, stable isotope measurements of specific FA markers can be used to identify carbon sources, and can be applied to a variety of food web studies (Iverson et al., 2004). The purpose of this research is to examine the linkages between organic carbon sources and trophic transfer by consumers. To achieve this, we use FA biomarkers and compound specific stable isotope analysis (CSIA) to trace carbon cycling. This study has two main components: environmental sampling and experimental research. Chapter 1 demonstrates the use of these tools for elucidating seasonal trophic linkages in invertebrates collected from the Alaskan Arctic coast. Overall, invertebrate diets were characterized by terrestrial, detrital, and carnivorous sources in winter and spring, with a shift toward autochthonous diatom-based diets in summer. Our results demonstrate the importance of terrestrial organic carbon as a subsistence food source in winter, whereas in situ production in summer was critical for accumulating FA stores rich in essential FAs. Chapter 2 is an experimental feeding study designed to quantify the incorporation rates of 18:2n-6 from diet to tissue in Atlantic croaker. Liver tissues accumulated FAs more quickly than muscle tissues, but both tissues reached equilibrium at 5 to 7 weeks. From these experiments, quantitative assessments of diet sources can be made with confidence when using FAs to understand trophic interactions of Atlantic croaker and other similar species.