Browsing by Subject "Lipid Metabolism"
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Item Adipose tissue in health and disease: why should we care?(2019-01-25) Scherer, Philipp E.Item CDNA Cloning and Characterization of Enzymes That Synthesize Bile Acids, Vitamin D and Waxes(2006-05-15) Cheng, Jeffrey Binyan; Russell, David W.Countless enzymes are required for the synthesis of the diverse array of lipids found in nature. The identification and characterization of five different lipid metabolizing enzymes are reported here. The 3beta -hydroxy-delta 5-C27-steroid oxidoreductase (C27 3beta-HSD) enzyme catalyzes a step in bile acid synthesis. Subjects with mutations in the encoding gene fail to synthesize bile acids and develop liver disease. Fifteen patients were screened and twelve different mutations were identified in the C27 3beta -HSD gene. Vitamin D is required for normal bone metabolism and maintenance of serum calcium levels. The conversion of vitamin D into an active ligand requires 25-hydroxylation. I report here the identification by expression cloning of a cytochrome P450 (CYP2R1) with vitamin D 25-hydroxylase activity. A patient with low circulating levels of 25-hydroxyvitamin D and classic symptoms of vitamin D deficiency was identified. Molecular analysis of this individual revealed homozygosity for a transition mutation in the CYP2R1 gene causing the substitution of a proline for a leucine in the protein and eliminating vitamin D 25-hydroxylase enzyme activity. These data identify CYP2R1 as a biologically relevant vitamin D 25-hydroxylase and reveal the molecular basis of a human genetic disease, selective 25-hydroxyvitamin D deficiency. The reduction of fatty acids to fatty alcohols, by a fatty acyl-CoA reductase enzyme, is required for the synthesis of wax monoesters and ether lipids. Using a bioinformatics approach, the first two mammalian fatty acyl-CoA reductase genes (FAR1 and FAR2) were identified. The two mouse FAR enzymes, which share 57% sequence identity at the amino acid level, have differing substrate specificities and tissue distributions implying unique physiological roles for each. Wax monoesters are synthesized by the esterification of fatty alcohols and fatty acids. A mammalian enzyme that catalyzes this reaction has not been isolated. Here, I report the identification by expression cloning of a wax synthase gene. Co-expression of cDNAs specifying FAR1 and wax synthase led to the synthesis of wax monoesters. The data suggests that wax monoester synthesis in mammals involves a two step biosynthetic pathway catalyzed by fatty acyl-CoA reductase and wax synthase enzymes.Item Dissecting the Role of the Lipodystrophy Protein Seipin in the Biogenesis of the Lipid Droplet Organelle(2014-07-14) Cartwright, Bethany Rose; Sternweis, Paul C.; Chook, Yuh Min; Bickel, PerryLong thought to be little more than inert storage depots, lipid droplets have recently become recognized as unique, dynamic, regulated organelles that play an essential role in fat storage. Despite this increased interest, much remains unknown. Lipid droplets have been observed to emerge from the endoplasmic reticulum, but the available models for lipid droplet biogenesis are largely conceptual, with little to no evidence for specific mechanisms of droplet formation. Debate even continues within the field as to whether lipid droplet formation is a spontaneous process, driven by physicochemical and hydrophobic forces, or a regulated process driven by protein factors. The Goodman laboratory previously found evidence to suggest that seipin, mutated in the most severe cases of congenital generalized lipodystrophy, may be a key factor in the early stages of lipid droplet formation. Seipin resides at the junction between lipid droplets and the endoplasmic reticulum, and deletion of seipin results in both a drastic impediment to de novo droplet formation and a striking disorganization of droplet morphology. For my thesis work, I have explored several aspects of seipin’s role at the lipid droplet. I have studied the effects of seipin deletion on protein targeting to abnormal lipid droplets, through which I identified a unique effect of seipin on the regulation of lipase targeting. I have also analyzed the topology of the seipin complex itself through a series of deletion mutants, identifying regions that contribute to the localization, membrane association, and stability of the seipin complex. Furthermore, these studies have led to novel insights on the function of seipin, through the characterization of a remarkable N-terminal seipin mutation that presents with defects in droplet initiation but homogenous droplet morphology. I have therefore concluded that seipin plays two dissectible roles in lipid droplet formation: 1) promoting lipid droplet initiation and 2) regulating subsequent droplet morphology. Finally, I suggest hypotheses on the mechanisms by which seipin exerts these effects, proposing that the N-terminus of seipin may regulate lipin, a mouse lipodystrophy protein, to effect droplet initiation, while the bulk of the protein may serve to regulate the access of phospholipids to the lipid droplet surface.Item Examination of Abnormal Dolichol Metabolism in Infantile Batten Disease Caused by Palmitoyl Protein Thioesterase-1 (PPT1) Deficiency(2004-08-19) Cho, Steve Kyungrae; Lehrman, Mark A.The neuronal ceroid lipofuscinosis (NCLs, also known collectively as Batten disease) are a group of lysosomal storage disorders characterized by the accumulation of autofluorescent storage material in the brain. Although a number of genes underlying different forms of NCL have been cloned, the underlying mechanism for the neurodegeneration is still unknown. The most severe form of NCL (infantile NCL) is caused by mutations in the CLN1/PPT1 gene, which encodes a soluble lysosomal hydrolase (palmitoyl protein thioesterase-1) that removes fatty acids from lipid modified proteins in the lysosome. It has been postulated that abnormal dolichol metabolism might be involved in NCL pathogenesis because high levels of dolichol phosphate (Dol-P) and lipid linked oligosaccharides (LLOs) accumulate in NCL patients including infantile NCL. Here, a possible relationship between fatty acid and dolichol metabolism in the neuropathogenesis of NCL has been explored by analyzing LLOs from mouse models of Batten disease and other non-NCL lysosomal storage diseases (LSDs), and by characterizing a unique fusion protein consisting of PPT1 and a dolichol metabolizing enzyme (DOLPP1) in Schizosaccharomyces pombe, which we named pdf1 (for ppt1-dolpp1 fusion1). To do this, first, I characterized S. pombe pdf1 by ablating the pdf1 gene and studying the function of each of the proteins (PPT1 and DOLPP1) independently. These results revealed that PPT1 and DOLPP1 may be co-regulated in lower organisms but the functional relationship in higher eukaryotes remains unclear. To further explore the relationship between PPT1 and DOLPP1 in mammalian cells, I cloned the mammalian ortholog of DOLPP1 and subsequently characterized the mouse Dolpp1p. Finally, I characterized and analyzed LLOs in various mouse models of NCL by FACE (fluorophore assisted carbohydrate electrophoresis). It was shown that LLOs accumulated in PPT1-deficient mouse brain and the level of LLO accumulation was 14.5-fold higher as compared to wild type brain. Despite the striking accumulation of LLOs in PPT1-deficient brain compared to age-matched controls, I also found that the LLOs encompassed only 0.3% of the autofluorescent storage material by mass. Therefore, the abnormal dolichol catabolism is most likely a secondary phenotype to PPT1 deficiency during the pathogenesis of infantile Batten disease.Item Genetic Reduction of Cholesterol Synthesis in the Mouse Brain Does Not Affect Amyloid Formation in an Alzheimer’s Disease Model, but Does Extend Lifespan(2011-08-10) Warren, Rebekkah Lynn; Russell, David W.In vitro alterations in cellular cholesterol content or synthesis affect the cleavage of amyloid precursor protein (APP) to amyloidogenic peptides characteristic of Alzheimer’s disease (AD). To determine whether a decrease in cholesterol synthesis would affect APP processing in vivo, we crossed cholesterol 24-hydroxylase knockout (KO) mice, which exhibit a 50 percent reduction in sterol synthesis, with transgenic mice (B6.Cg-Tg(APPswe, PSEN1E9)85Dbo/J) that develop AD and followed progression of the disease and lipid metabolism in the offspring. APP expression and amyloid plaque deposition in the cortex and hippocampus of 3- to 15-month-old male and female AD mice were similar in the presence and absence of cholesterol 24-hydroxylase. At 15 months of age, a modest but statistically significant decline in insoluble A-beta 40 and A-beta 42 peptide levels was detected in the hippocampus but not cortex of KO/AD mice versus WT/AD mice. Amyloid plaque accumulation did not affect brain sterol or fatty acid synthesis rates in 24-hydroxylase WT or KO mice. Unexpectedly, loss of one or two 24-hydroxylase alleles increased longevity in AD mice. These studies suggest that reducing de novo cholesterol synthesis in the brain will not substantially alter the course of AD, but may confer a survival advantage.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 Metabolic Regulation of Protein Phosphorylation and Acetylation(2018-07-12) Zhang, Menglu; Kohler, Jennifer J.; Tu, Benjamin; Nijhawan, Deepak; Yu, HongtaoCelluar metabolism can influence phosphorylation and acetylation modifications on proteins as part of an intricate network of cellular and organismal regulation. We have investigated one molecular mechanism through which protein phosphorylation and acetylation can be regulated based on metabolic status and how metabolic enzymes are regulated by nutrient availability. In the first part of this study, we report that a simple enzyme involved in acetate utilization, Acetyl-CoA synthetase 2 (ACSS2), promotes systemic fat storage and utilization through selective regulation of genes involved in lipid metabolism. We reveal that mice lacking ACSS2 exhibit a significant reduction in body weight and hepatic steatosis in a diet-induced obesity model. ACSS2 deficiency reduces dietary lipid absorption by the intestine, and perturbs repartitioning and utilization of triglycerides from adipose tissue to the liver due to lowered expression of lipid transporters and fatty acid oxidation genes. In this manner, ACSS2 promotes the systemic storage or metabolism of fat according to the fed or fasted state. Targeting ACSS2 may therefore offer therapeutic benefit for the treatment of fatty liver disease. We also report that ACSS2 may play a critical role in the development of pancreatic cancer. We have demonstrated that ACSS2 expression in a KRas-driven mouse model of pancreatic ductal adenocarcinoma (PDAC) showed that ACSS2 was absent in normal pancreatic tissue but expressed at very high levels in precancerous lesions of PDAC. The absence of ACSS2 in mouse pancreatic cancer models reduced the tumor burdens, and ACSS2 expression is correlated with tumor size. These data indicate that ACSS2 has a potential function in the development of PDAC. The experiments reported in the first two chapters of this thesis were performed in close collaboration with a former postdoc in the lab, Dr. Zhiguang Huang. In the second part of the study, we report that methylation of Protein Phosphatase 2A (PP2A) may play a critical role in regulating cell growth and autophagy. We have reconstituted the methylation activity of leucine carboxyl methyltransferase 1 (LCMT-1) in vitro and determined the kinetic parameters of LCMT-1-catalyzed methylation of PP2A. We reveal that LCMT-1 might be a "SAM sensor" as it is very sensitive to the SAM/SAH ratio. Methionine deprivation study in cell lines revealed that methionine depletion boosts PP2A demethylation. We further conducted a high-throughput screen to identify potent and specific small molecule inhibitors of LCMT-1.Item Nonalcoholic fatty liver disease (NAFLD) turns 38: what have we learned?(2018-10-05) Horton, Jay D.Item Role for Lipids in the Cellular Transmission of α-Synuclein(2015-07-30) Peres, Yair; Michaely, Peter A.; Thomas, Philip J.; DeBose-Boyd, Russell A.; Albanesi, Joseph P.The presynaptic protein α-Synuclein (α-Syn) abnormally aggregates in the brains of Parkinson's Disease patients. Evidence suggest a transcellular transfer of an oligomeric form of the protein (seed) in a prion-like fashion. The mechanism underlying cell entry is unclear, but studies have implicated the cell surface glycan HSPG followed by macropinocytosis, as a possible uptake route. Secretion and uptake of aggregated α-Syn was reported to be associated with lipid vesicles in cells. Monomeric α-Syn was found to tubulate membranes in-vitro. This work aims to study the significance of membrane association and tubulation by α-Syn to its effect on uptake by cells. Uptake of lipid-associated α-Syn seeds was significantly more efficient than lipid-free uptake. Furthermore, seeds in the presence of monomers and lipids, which formed tubules in-vitro, were more readily internalized than seeds and lipids in the absence of monomers. Lipid associated α-Syn was internalized by cells in an HSPG dependent manner as evident from competitive inhibition and enzymatic digestion experiments. Tubule associated seeds may constitute an efficient mechanism of pathological propagation of synucleinopathies. This mechanism should be considered in any therapeutic approach targeting the inhibition of α-Syn intercellular transfer.Item Role of Ceramidase in Regulating Hepatic Lipid Metabolism and Whole Body Insulin Sensitivity(2017-03-16) Xia, Jonathan Y.; Bickel, Perry; Scherer, Philipp; Holland, William L.; Goodman, Joel M.; Horton, Jay D.Concomitant with the rise in obesity, the prevalence of non-alcoholic fatty liver disease (NAFLD), a newly emerging obesity-related disorder, has also been rising steadily. NAFLD is a chronic liver disease that ranges histologically from simple steatosis in its mildest form to non-alcoholic steatohepatitis (NASH) in the more severe form, which is characterized by hepatocyte inflammation and fibrosis. In recent years, sphingolipids have garnered increasing attention for their role in the development of diabetes and metabolic syndrome. Specifically, the accumulation of ceramides in liver was shown to be associated with both hepatic insulin resistance and NAFLD. We recently demonstrated that the adipose-derived secretory factor adiponectin promotes an increase in ceramide catabolism, which is dependent on adiponectin receptors 1 and 2. The associated ceramidase activity promotes ceramide degradation and generation of sphingosine 1-phosphate (S1P). To further investigate the tissue-specific effects of the receptor associated ceramidase activity, we have developed transgenic mice that inducibly express adiponectin receptors 1 and 2 under the control of a tetracycline response element. Hepatic overexpression of adiponectin receptors improved whole body glucose metabolism and prevented hepatic steatosis. Furthermore, we found that catabolism of hepatic ceramides improved insulin signaling in the adipocyte, which suggests the existence of a "cross-talk" between liver and adipose tissue.