Soumen Kanti Manna (Presenter)
Saha Institute of Nuclear Physics
Bio: After receiving my bachelor’s and master’s degrees in chemistry, I worked on elucidation of structure-function relationship and engineering of metalloproteins to receive my Ph.D. from Tata Institute of Fundamental Research, Mumbai, India. During my postdoctoral research at the Laboratory of Metabolism, NCI, I combined metabolomic and transcriptomic analysis to identify biomarkers for pathogenesis and environmental exposure as well as to elucidate the underlying mechanism. In addition, I was involved in metagenomics and dietary supplements research. Since 2014, I have joined as an Associate Professor in the Biophysics and Structural Genomics division of Saha Institute of Nuclear Physics, Kolkata, India, where I continue to work on elucidation of metabolic reprogramming associated with cancer, metabolic disorders and hepatobiliary diseases.
Authorship: Soumen Kanti Manna (1), Srujana Golla (2), Ying Chen (3), Kristopher W. Krausz (2), Vasilis Vassilou (3) and Frank J. Gonzalez (2).
(1) Saha Institute of Nuclear Physics, Kolkata, India (2) National Cancer Institute, Bethesda, USA, (3) Yale University, New Haven, USA
| Short Abstract Alcoholic liver disease (ALD) is known to progress via steatosis. Although factors contributing to the outcome are not fully understood, oxidative stress is known to be involved. Our recent study showed that glutathione-deficient (Gclm-null) mice, surprisingly, protected from alcoholic steatosis compared to wild-type mice. In order to investigate the mechanism, the reorganization of the polar metabolome was examined using HILIC-ESI-MS. The analysis revealed that shift in redox balance due to glutathione depletion takes place in tandem with redistribution of acetyl CoA flux into alternative metabolic pathways. This siphons alcohol-derived acetyl CoA away from de novo lipid biosynthesis leading to the observed protection of Gclm-null mice from alcoholic steatosis. |
Long Abstract
Introduction: Alcoholic liver disease is one of the leading causes of non-accidental deaths. Although it is well-known that the disease is initiated by lipid accumulation in liver, the molecular determinants of the extent of lipid accumulation, liver damage and final outcome are not well understood. However, it has been shown that oxidative stress plays an important role in pathogenesis of the disease. Understanding the role molecules involved in oxidative stress response in pathogenesis of alcoholic liver disease may lead to improvement of preventive and therapeutic strategies. Glutamate cysteine ligase (GCL), comprising a catalytic (GCLC) and a modifier (GCLM) subunit, catalyzes the rate-limiting step in glutathione biosynthesis and is key player in oxidative stress response. Our recent study showed that glutathione-deficient (Gclm-null) mice, surprisingly, protected from alcoholic steatosis compared to wild-type mice. In this study, metabolomic analysis was carried out to characterize the reorganization of the metabolic machinery associated with the observed protection in order to elucidate the underlying mechanism.
Methods: Male (10-12 weeks old) C57BL6/J (WT) and Gclm-null (KO) mice were pair-fed either control (carbohydrate-supplemented) modified Liber-DeCarli diet or ethanolic diet (2-5% v/v) for six weeks. Plasma biochemistry, liver histology, liver triglyceride and cholesterol ester levels were examined by following standard protocols at the end of 6-week feeding period. For the metabolomics study, metabolites were extracted from liver samples using chloroform-methanol-water biphasic extraction protocol to separate polar and non-polar metabolites. The unbiased profiling of polar metabolites was performed using 1.7µM Acquity UPLC BEH amide hydrophilic interaction liquid chromatography column coupled with a Xevo G2 ESIQTOF mass spectrometer (Waters Corporation, Milford, MA) in a randomized fashion. Mass spectrometric analysis was performed in both positive and negative ionization modes. MassLynx software (Waters Corporation) was used to acquire mass chromatograms and mass spectral data in centroid format. Chromatograms were manually inspected for chromatogram quality and retention time reproducibility across the run. MarkerLynx software (Waters Corporation) was used to deconvolute chromatograms, bin the data according to mass- retention time pairs and integrate the area under the peak. The intensity of each ion was normalized with respect to the either total ion count (TIC) or weight of the liver tissue used for extraction. The multivariate data matrix was analyzed by SIMCA-P+12 software (Umetrics, Kinnelon, NJ). Data quality inspection as well as distribution and unsupervised segregation of wild-type and Gclm-null on global metabolomic space were checked by principal components analysis (PCA) of the Pareto-scaled data. The supervised orthogonal projection to latent structures (OPLS) model was used identify ions contributing to discrimination of metabolic traits. Candidate metabolites for these ions were identified on the basis of accurate mass measurement using metabolomic databases such as METLIN (http://metlin.scripps.edu/) or HMDB (http://www.hmdb.ca/) and MS/MS fragmentation pattern. Identities of metabolites were confirmed using authentic standards. Finally, metabolites were quantified in multiple reactions monitoring mode on a XEVO triple quadruple mass spectrometer (Water Corporation) coupled with a 1.7µM Acquity UPLC BEH amide column. Metabolite concentrations thus determined were normalized with respect to the weight of the liver sample used for metabolite extraction to examine statistical significance. Statistical significance was tested using one-way ANOVA with Bonferroni’s correction for multiple testing.
Results: Our study showed earlier that following the 6-week alcohol feeding period, (i) KO mice are less susceptible to alcohol-induced hepatocyte damage, as reflected by plasma ALT levels; (ii) the steatosis observed in alcohol-fed WT mice was absent in KO mice; and (iii) alcohol feeding caused significant increase in total hepatic triglyceride (TG) content in WT mice, but had no effect on the hepatic TG content in KO mice. These results showed that, in spite of the impairment of glutathione biosynthesis, KO mice on alcoholic diet were protected from steatosis and liver injury.
The unsupervised analysis of metabolomic signatures revealed that genetic factor (Gclm knockout) has a stronger influence on the liver metabolome compared to the alcoholic diet. Interestingly, it also indicated that the effect of alcoholic diet on WT metabolome is stronger than that on the Gclm-null, mirroring trends observed for lipid accumulation and liver injury. Identification and quantitation of differential metabolic signatures revealed widespread reorganization of metabolic networks involving amino acid metabolism, nucleic acid metabolism and amino sugar metabolism. Although widespread, on careful analysis these changes were found to be interconnected. As expected, KO mice showed a huge depletion of GSH concentration reflecting change in redox balance. It was found that impairment of glutathione biosynthesis in KO liver builds-up glutamate concentration, which, in turn, leads to elevation of some nucleic acids and nucleotides that are synthesized using glutamate. While in WT liver ethanol-derived acetyl CoA could contribute to de novo fatty acid synthesis, the unavailability of NADPH due impairment of glutathione biosynthesis compromises the process in KO. In KO mice, acetyl CoA actually reacts with the glutamate to produce N-acetylglutamate and is also diverted to amino sugar metabolism, thus, creating an alternative sink for alcohol-derived acetyl CoA.
Conclusion: Metabolomic analysis revealed that in conjunction with shift in redox balance, reorganization of amino acid, nucleic acid, and amino sugar metabolism shunts acetyl CoA flux away from de novo lipid biosynthesis in ethanol-treated Glcm-null mice, and, thus, protects it from steatosis, the first stage of alcoholic liver disease.
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