Metabolic Reprogramming by Hexosamine Biosynthetic and Golgi N-Glycan Branching Pathways

Most mammalian growth factor receptors and solute transporters are co-translationally N-glycosylated. N-glycans are branched and elongated in the Golgi, and their interaction with lectins regulates cell surface residency and activity of transmembrane glycoproteins. The Golgi branching N-acetylglucosaminyltransferases, Mgat1, 2, 4, 5 and 6, require a common donor substrate uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) generated de novo by the hexosamine biosynthetic pathway (HBP) from glucose, glutamine and acetyl-CoA, as well as from GlcNAc salvage pathway. In this thesis I describe evidence for cell-autonomous regulation of cellular metabolism by the Golgi N-glycan branching pathway. Induced expression of Mgat1, Mgat5 or Mgat6, and GlcNAc supplementation to the HBP, increased central metabolites in an additive manner. I show that UDP-GlcNAc levels are also sensitive to dietary GlcNAc supplementation in vivo, increasing nutrient uptake and promoting anabolic metabolism via the Golgi N-glycan branching pathway. Chronic oral GlcNAc supplementation in C57BL/6 mice increased hepatic UDP-GlcNAc and N-glycan branching on liver glycoproteins. Furthermore, GlcNAc supplementation altered levels of numerous hepatic metabolites, insulin and glucagon balance, and promoted excess lipid storage, as well as body-weight increase, without affecting food intake or energy expenditure. In cultured cells, GlcNAc enhanced uptake of glucose, glutamine and fatty acids, and elevated fatty acid synthesis and storage in an N-glycan-dependent manner. Mgat5-/- mice exhibit a lean phenotype, and oral GlcNAc rescued fat accumulation, consistent with functional redundancy of N-glycan branches. However, fat accumulation in this context was rescued at the expense of lean mass, suggesting that Mgat5 plays a role in lean to fat body composition. These results suggest that GlcNAc reprograms cellular metabolism by enhancing nutrient uptake and lipid storage through the HBP and UDP-GlcNAc supply to the Golgi N-glycan branching pathway.Ph.D

Electric ion dispersion as a new type of mass spectrometer

At the 2014 Fields-MPrime Industrial Problem Solving Workshop, PerkinElmer presented a design problem for mass spectrometry. Traditionally mass spectrometry is done via three methods: using magnetic fields to deflect charged particles whereby different masses bend differently; using a time-of-flight procedure where particles of different mass arrive at different times at a target; and using an electric quadrupole that filters out all masses except for one very narrow band. The challenge posed in the problem was to come up with a new design for mass spectrometry that did not involve magnetic fields and where mass-fractions could be measured in an entire sample on a continuous basis. We found that by sending the sample particles down a channel of line charges that oscillations would be induced with a spatial wave-length being mass-dependent thereby allowing different masses to be separated spatially and potentially detected on a continuous basis, without the use of magnetic fields. In this paper, we present the analysis of our design and illustrate how this principle could be used for mass spectrometry

BMP signaling induces visceral endoderm differentiation of XEN cells and parietal endoderm

The extraembryonic endoderm of mammals is essential for nutritive support of the fetus and patterning of the early embryo. Visceral and parietal endoderm are major subtypes of this lineage with the former exhibiting most, if not all, of the embryonic patterning properties. Extraembryonic endoderm (XEN) cell lines derived from the primitive endoderm of mouse blastocysts represent a cell culture model of this lineage, but are biased towards parietal endoderm in culture and in chimeras. In an effort to promote XEN cells to adopt visceral endoderm character we have mimicked different aspects of the in vivo environment. We found that BMP signaling promoted a mesenchymal-to-epithelial transition of XEN cells with up-regulation of E-cadherin and down-regulation of vimentin. Gene expression analysis showed the differentiated XEN cells most resembled extraembryonic visceral endoderm (exVE), a subtype of VE covering the extraembryonic ectoderm in the early embryo, and during gastrulation it combines with extraembryonic mesoderm to form the definitive yolk sac. We found that laminin, a major component of the extracellular matrix in the early embryo, synergised with BMP to promote highly efficient conversion of XEN cells to exVE. Inhibition of BMP signaling with the chemical inhibitor, Dorsomorphin, prevented this conversion suggesting that Smad1/5/8 activity is critical for exVE induction of XEN cells. Finally, we show that applying our new culture conditions to freshly isolated parietal endoderm (PE) from Reichert\u27s membrane promoted VE differentiation showing that the PE is developmentally plastic and can be reprogrammed to a VE state in response to BMP. Generation of visceral endoderm from XEN cells uncovers the true potential of these blastocyst-derived cells and is a significant step towards modelling early developmental events ex vivo. © 2011 Elsevier Inc

BMP signaling induces visceral endoderm differentiation of XEN cells and parietal endoderm

The extraembryonic endoderm of mammals is essential for nutritive support of the fetus and patterning of the early embryo. Visceral and parietal endoderm are major subtypes of this lineage with the former exhibiting most, if not all, of the embryonic patterning properties. Extraembryonic endoderm (XEN) cell lines derived from the primitive endoderm of mouse blastocysts represent a cell culture model of this lineage, but are biased towards parietal endoderm in culture and in chimeras. In an effort to promote XEN cells to adopt visceral endoderm character we have mimicked different aspects of the in vivo environment. We found that BMP signaling promoted a mesenchymal-to-epithelial transition of XEN cells with up-regulation of E-cadherin and down-regulation of vimentin. Gene expression analysis showed the differentiated XEN cells most resembled extraembryonic visceral endoderm (exVE), a subtype of VE covering the extraembryonic ectoderm in the early embryo, and during gastrulation it combines with extraembryonic mesoderm to form the definitive yolk sac. We found that laminin, a major component of the extracellular matrix in the early embryo, synergised with BMP to promote highly efficient conversion of XEN cells to exVE. Inhibition of BMP signaling with the chemical inhibitor, Dorsomorphin, prevented this conversion suggesting that Smad1/5/8 activity is critical for exVE induction of XEN cells. Finally, we show that applying our new culture conditions to freshly isolated parietal endoderm (PE) from Reichert\u27s membrane promoted VE differentiation showing that the PE is developmentally plastic and can be reprogrammed to a VE state in response to BMP. Generation of visceral endoderm from XEN cells uncovers the true potential of these blastocyst-derived cells and is a significant step towards modelling early developmental events ex vivo. © 2011 Elsevier Inc

Probing the Hexosamine Biosynthetic Pathway in Human Tumor Cells by Multitargeted Tandem Mass Spectrometry

Cancer progression is accompanied by increases in glucose and glutamine metabolism, providing the carbon and nitrogen required in downstream anabolic pathways. Fructose-6P, glutamine, and acetyl-CoA are central metabolites and substrates of the hexosamine biosynthesis pathway (HBP) to UDP-<i>N</i>-acetylglucosamine (UDP-GlcNAc), an essential high-energy donor for protein glycosylation. Golgi and cytosolic glycosylation pathways are sensitive to UDP-GlcNAc levels, which in turn regulates metabolic homeostasis in a poorly understood manner. To study the hexosamine biosynthesis pathway in cancer cells, we developed a targeted approach for cellular metabolomics profiling by liquid chromatography–tandem mass spectrometry. Human cervical (HeLa) and prostate cancer (PC-3) cell lines were cultured in medium with increasing concentrations of glucose, glutamine, or GlcNAc to perturb the metabolic network. Principal component analysis indicated trends that were further analyzed as individual metabolites and pathways. HeLa cell metabolism was predominantly glycolytic, while PC-3 cells showed a greater dependency on extracellular glutamine. In both cell lines, UDP-GlcNAc levels declined with glucose but not glutamine starvation, whereas glutamine abundance increased UDP-GlcNAc levels 2–3-fold. GlcNAc supplementation increased UDP-GlcNAc 4–8-fold in both HeLa and PC-3 cells. GlcNAc supplementation in HeLa cells induced nonmonotonic changes in NADH/NAD+, NADPH/NADP+, reactive oxygen species, and reduced/oxidized glutathione. In PC-3 cells, GlcNAc supplementation also increased glucose and glutamine uptake and catabolism. Our results suggest that stimulation of the HBP in cancer cells regulates metabolism and redox potential, which might be exploited to target cancer cells

The hexosamine biosynthetic pathway couples growth factor-induced glutamine uptake to glucose metabolism

Glucose and glutamine serve as the two primary carbon sources in proliferating cells, and uptake of both nutrients is directed by growth factor signaling. Although either glucose or glutamine can potentially support mitochondrial tricarboxylic acid (TCA) cycle integrity and ATP production, we found that glucose deprivation led to a marked reduction in glutamine uptake and progressive cellular atrophy in multiple mammalian cell types. Despite the continuous presence of growth factor and an abundant supply of extracellular glutamine, interleukin-3 (IL-3)-dependent cells were unable to maintain TCA cycle metabolite pools or receptor-dependent signal transduction when deprived of glucose. This was due at least in part to down-regulation of IL-3 receptor α (IL-3Rα) surface expression in the absence of glucose. Treatment of glucose-starved cells with N-acetylglucosamine (GlcNAc) to maintain hexosamine biosynthesis restored mitochondrial metabolism and cell growth by promoting IL-3-dependent glutamine uptake and metabolism. Thus, glucose metabolism through the hexosamine biosynthetic pathway is required to sustain sufficient growth factor signaling and glutamine uptake to support cell growth and survival