Abstract 670: A Metabolic Approach to Examining the Hexosamine Biosynthetic Pathway and Its Role in the Development of Diabetes-Associated Atherosclerosis

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Christina Petlura ◽  
Lisa Walter ◽  
Geoff Werstuck
Keyword(s):  
Mass Spectrometry ◽  
Mouse Model ◽  
Er Stress ◽  
Hepg2 Cells ◽  
Biosynthetic Pathway ◽  
Molecular Mechanisms ◽  
Imaging Mass Spectrometry ◽  
Current Evidence ◽  
Uridine Diphosphate ◽  
Hexosamine Biosynthetic Pathway

Introduction: Diabetes is a disease affecting millions of people worldwide, and is a major independent risk factor for cardiovascular disease (CVD). Despite a vast amount of research, the molecular mechanisms that link diabetes to CVD are not well understood. Current evidence suggests that increased flux through the hexosamine biosynthetic pathway (HBP) contributes to the development of hyperglycemia-associated diabetic complications. Our data suggest that increased HBP flux can induce vascular ER stress and accelerate atherogenesis in a mouse model. We hypothesized that this process can be attenuated by inhibiting the first and rate-limiting enzyme in the HBP - glutamine fructose-6-phosphate amidotransferase (GFAT) - using small molecules. Methods and Results: Using high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) we have developed a methodology to monitor and quantify the levels of the end product of the HBP, uridine diphosphate N -acetylglucosamine (UDP-GlcNAc). Treatment of HepG2 cells with glucosamine (0.2-5 mM), or adenovirus-directed overexpression of GFAT caused a 3-7 fold increase in UDP-GlcNAc ( P< 0.001 & P< 0.05, respectively). Inhibition of GFAT with three novel compounds - amrinone, lapachol or alloxan - decreased levels of UDP-GlcNAc by 1.5 ( P< 0.05), 3 ( P< 0.05) and 3.5-fold ( P< 0.001), respectively. Furthermore, we show that by modulating HBP flux, we can regulate ER stress levels in cultured HepG2 cells. The physiological relevance of this mechanism is supported by evidence of HBP augmentation in a hyperglycemic mouse model. Conclusions: These results support a role for the HBP in the development of atherosclerosis. Currently, MALDI imaging mass spectrometry is being performed on tissue sections to compare the levels of UDP-GlcNAc directly in hyperglycemic vs. normoglycemic mice. These studies may lead to the identification and validation of novel targets for the development of new pharmaceuticals to prevent diabetic atherosclerosis.

scholarly journals The Hexosamine Biosynthetic Pathway as a Therapeutic Target after Cartilage Trauma: Modification of Chondrocyte Survival and Metabolism by Glucosamine Derivatives and PUGNAc in an Ex Vivo Model

2021 ◽  
Vol 22 (14) ◽  
pp. 7247
Author(s):  
Jana Riegger ◽  
Julia Baumert ◽  
Frank Zaucke ◽  
Rolf E. Brenner
Keyword(s):  
Biosynthetic Pathway ◽  
Ex Vivo ◽  
Type Ii Collagen ◽  
Cartilage Explants ◽  
Uridine Diphosphate ◽  
Cell Protection ◽  
Ex Vivo Model ◽  
Human Cartilage ◽  
Hexosamine Biosynthetic Pathway ◽  
Cellular Processes

The hexosamine biosynthetic pathway (HBP) is essential for the production of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the building block of glycosaminoglycans, thus playing a crucial role in cartilage anabolism. Although O-GlcNAcylation represents a protective regulatory mechanism in cellular processes, it has been associated with degenerative diseases, including osteoarthritis (OA). The present study focuses on HBP-related processes as potential therapeutic targets after cartilage trauma. Human cartilage explants were traumatized and treated with GlcNAc or glucosamine sulfate (GS); PUGNAc, an inhibitor of O-GlcNAcase; or azaserine (AZA), an inhibitor of GFAT-1. After 7 days, cell viability and gene expression analysis of anabolic and catabolic markers, as well as HBP-related enzymes, were performed. Moreover, expression of catabolic enzymes and type II collagen (COL2) biosynthesis were determined. Proteoglycan content was assessed after 14 days. Cartilage trauma led to a dysbalanced expression of different HBP-related enzymes, comparable to the situation in highly degenerated tissue. While GlcNAc and PUGNAc resulted in significant cell protection after trauma, only PUGNAc increased COL2 biosynthesis. Moreover, PUGNAc and both glucosamine derivatives had anti-catabolic effects. In contrast, AZA increased catabolic processes. Overall, “fueling” the HBP by means of glucosamine derivatives or inhibition of deglycosylation turned out as cells and chondroprotectives after cartilage trauma.

Abstract 261: Robust Cardioprotection Afforded by a Previously Unrecognized Link between the Unfolded Protein Response and the Hexosamine Biosynthetic Pathway

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Zhao V Wang ◽  
Yingfeng Deng ◽  
Ningguo Gao ◽  
Zully Pedrozo ◽  
Dan Li ◽  
...  
Keyword(s):  
Unfolded Protein Response ◽  
Biosynthetic Pathway ◽  
Ischemia Reperfusion ◽  
Uridine Diphosphate ◽  
Hexosamine Biosynthetic Pathway ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Rate Limiting

Background: The hexosamine biosynthetic pathway (HBP) generates UDP-GlcNAc (uridine diphosphate N-acetylglucosamine) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in glucose metabolism and multiple diseases, regulation of the HBP remains largely undefined. Methods & Results: Here, we show that spliced Xbp1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers activation of the HBP by means of Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions known to promote O-GlcNAc modification. We go on to demonstrate that Xbp1s, acutely stimulated by ischemia/reperfusion (I/R) in heart, confers robust cardioprotection against I/R injury. We also show that HBP induction is required for this cardioprotective response. Mechanistically, HBP may mediate the adaptive branch of the UPR by activating autophagy and ER-associated degradation. Conclusion: These studies reveal that Xbp1s couples the UPR to the HBP, promoting robust cardioprotection during I/R.

scholarly journals Defining Changes in the Spatial Distribution and Composition of Brain Lipids in the Shiverer and Cuprizone Mouse Models of Myelin Disease

2018 ◽  
Vol 67 (3) ◽  
pp. 203-219 ◽  
Author(s):  
Rajanikanth J. Maganti ◽  
Xiaoping L. Hronowski ◽  
Robert W. Dunstan ◽  
Brian T. Wipke ◽  
Xueli Zhang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Spatial Distribution ◽  
Mouse Model ◽  
Morphological Changes ◽  
Imaging Mass Spectrometry ◽  
Ionization Mass ◽  
Brain Lipids ◽  
Esi Ms ◽  
Maldi Ims ◽  
Lipid Species

Myelin is composed primarily of lipids and diseases affecting myelin are associated with alterations in its lipid composition. However, correlation of the spatial (in situ) distribution of lipids with the disease-associated compositional and morphological changes is not well defined. Herein we applied high resolution matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS), immunohistochemistry (IHC), and liquid chromatography–electrospray ionization–mass spectrometry (LC-ESI-MS) to evaluate brain lipid alterations in the dysmyelinating shiverer (Shi) mouse and cuprizone (Cz) mouse model of reversible demyelination. MALDI-IMS revealed a decrease in the spatial distribution of sulfatide (SHexCer) species, SHexCer (d42:2), and a phosphatidylcholine (PC) species, PC (36:1), in white matter regions like corpus callosum (CC) both in the Shi mouse and Cz mouse model. Changes in these lipid species were restored albeit not entirely upon spontaneous remyelination after demyelination in the Cz mouse model. Lipid distribution changes correlated with the local morphological changes as confirmed by IHC. LC-ESI-MS analyses of CC extracts confirmed the MALDI-IMS derived reductions in SHexCer and PC species. These findings highlight the role of SHexCer and PC in preserving the normal myelin architecture and our experimental approaches provide a morphological basis to define lipid abnormalities relevant to myelin diseases.

scholarly journals Molecular portrait of prostate tissue probed by MALDI Imaging Mass Spectrometry

2021 ◽  
Author(s):  
Surya Kant Choubey ◽  
Amrita Mitra ◽  
RAJDEEP DAS ◽  
Pritilata Rout ◽  
Amit Kumar Mandal
Keyword(s):  
Mass Spectrometry ◽  
Citrate Synthase ◽  
Expression Profiles ◽  
Imaging Mass Spectrometry ◽  
Prostate Tissue ◽  
Human Prostate ◽  
Tissue Imaging ◽  
Current Evidence ◽  
Gold Standard Method ◽  
Aging Men

Benign prostatic hyperplasia (BPH) is the most common condition in aging men, associated with lower urinary tract symptoms. BPH has been suggested to be a risk factor for certain urologic cancers, but the current evidence is inconsistent. The gold-standard method for the diagnosis of BPH is histopathology. Histopathology displays the cellular morphologies of tissues wherein characteristic changes pertaining specifically to BPH can be identified. However, the onset of BPH might be associated with minimal phenotypic changes in cellular morphologies in tissues that histopathology might not be able to detect successfully. Therefore, to understand the onset of a disease and its pathogenesis it is important to investigate the detailed molecular profiles associated with the disease that might help in the diagnosis and to understand the insights of the disease pathogenesis. Over the last decade, imaging mass spectrometry has been used to explore the spatial distribution and expression profiles of several molecules with their two-dimensional heterogeneity retained across the tissues. In the present study, using MALDI mass spectrometry based tissue imaging platform, we observed the expression of several proteins across human prostate tissue sections diagnosed with BPH. The proteins were identified and characterized using tissue proteomics approach. We could successfully identify the on-tissue distribution of ribonuclease T2, vinculin, isoform 2 of tropomyosin alpha-3 chain and mitochondrial citrate synthase proteins using this approach. Therefore, imaging mass spectrometry might be a potential tool to complement the findings of histopathology in diagnosis of BPH in human prostate tissues.

scholarly journals Detecting early myocardial ischemia in rat heart by MALDI imaging mass spectrometry

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Aleksandra Aljakna Khan ◽  
Nasim Bararpour ◽  
Marie Gorka ◽  
Timothée Joye ◽  
Sandrine Morel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Morphological Changes ◽  
Imaging Mass Spectrometry ◽  
Post Mortem ◽  
Molecular Changes ◽  
Ischemic Region ◽  
Maldi Ims

AbstractDiagnostics of myocardial infarction in human post-mortem hearts can be achieved only if ischemia persisted for at least 6–12 h when certain morphological changes appear in myocardium. The initial 4 h of ischemia is difficult to diagnose due to lack of a standardized method. Developing a panel of molecular tissue markers is a promising approach and can be accelerated by characterization of molecular changes. This study is the first untargeted metabolomic profiling of ischemic myocardium during the initial 4 h directly from tissue section. Ischemic hearts from an ex-vivo Langendorff model were analysed using matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) at 15 min, 30 min, 1 h, 2 h, and 4 h. Region-specific molecular changes were identified even in absence of evident histological lesions and were segregated by unsupervised cluster analysis. Significantly differentially expressed features were detected by multivariate analysis starting at 15 min while their number increased with prolonged ischemia. The biggest significant increase at 15 min was observed for m/z 682.1294 (likely corresponding to S-NADHX—a damage product of nicotinamide adenine dinucleotide (NADH)). Based on the previously reported role of NAD+/NADH ratio in regulating localization of the sodium channel (Nav1.5) at the plasma membrane, Nav1.5 was evaluated by immunofluorescence. As expected, a fainter signal was observed at the plasma membrane in the predicted ischemic region starting 30 min of ischemia and the change became the most pronounced by 4 h. Metabolomic changes occur early during ischemia, can assist in identifying markers for post-mortem diagnostics and improve understanding of molecular mechanisms.

scholarly journals Imaging Mass Spectrometry: A New Tool to Assess Molecular Underpinnings of Neurodegeneration

Metabolites ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 135 ◽  
Author(s):  
Kevin Chen ◽  
Dodge Baluya ◽  
Mehmet Tosun ◽  
Feng Li ◽  
Mirjana Maletic-Savatic
Keyword(s):  
Mass Spectrometry ◽  
Neurodegenerative Diseases ◽  
Molecular Mechanisms ◽  
New Technologies ◽  
A Priori ◽  
Imaging Mass Spectrometry ◽  
The Past ◽  
Wide Range ◽  
Lateral Sclerosis

Neurodegenerative diseases are prevalent and devastating. While extensive research has been done over the past decades, we are still far from comprehensively understanding what causes neurodegeneration and how we can prevent it or reverse it. Recently, systems biology approaches have led to a holistic examination of the interactions between genome, metabolome, and the environment, in order to shed new light on neurodegenerative pathogenesis. One of the new technologies that has emerged to facilitate such studies is imaging mass spectrometry (IMS). With its ability to map a wide range of small molecules with high spatial resolution, coupled with the ability to quantify them at once, without the need for a priori labeling, IMS has taken center stage in current research efforts in elucidating the role of the metabolome in driving neurodegeneration. IMS has already proven to be effective in investigating the lipidome and the proteome of various neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, Huntington’s, multiple sclerosis, and amyotrophic lateral sclerosis. Here, we review the IMS platform for capturing biological snapshots of the metabolic state to shed more light on the molecular mechanisms of the diseased brain.

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