Glutaminolysis is Essential for Energy Production and Ion Transport in Human Corneal Endothelium

Zhang, Wenlin; Li, Hongde; Ogando, Diego G.; Li, Shimin; Feng, Matthew T.; Price, Francis W.; Tennessen, Jason M.; Bonanno, Joseph A.

doi:10.1016/j.ebiom.2017.01.004

Cited by 49 publications

(58 citation statements)

References 47 publications

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“…Our previous studies demonstrated that glutamine supplies approximately 50% of TCA cycle carbon chains in human corneal endothelium. 21 Consistent with this earlier observation, we find that 50% of citrate and approximately 35% of α-ketoglutarate (α-KG), fumarate, and malate carbon chains were labeled with U- 13 C-glucose-sourced carbon in Slc4a11 +/+ MCECs in the presence of unlabeled glutamine (Fig. 5C).…”

Section: Resultssupporting

confidence: 90%

Conditionally Immortal Slc4a11^−/− Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in Slc4a11^−/− Mouse Model

Zhang

Ogando

Kim

et al. 2017

Invest. Ophthalmol. Vis. Sci.

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PurposeTo establish conditionally immortal mouse corneal endothelial cell lines with genetically matched Slc4a11+/+ and Slc4a11−/− mice as a model for investigating pathology and therapies for SLC4A11 associated congenital hereditary endothelial dystrophy (CHED) and Fuchs' endothelial corneal dystrophy.MethodsWe intercrossed H-2Kb-tsA58 mice (Immortomouse) expressing an IFN-γ dependent and temperature-sensitive mutant of the SV40 large T antigen (tsTAg) with Slc4a11+/+ and Slc4a11−/− C57BL/6 mice. The growth characteristics of the cell lines was assessed by doubling time. Ion transport activities (Na+/H+ exchange, bicarbonate, lactate, and Slc4a11 ammonia transport) were analyzed by intracellular pH measurement. The metabolic status of the cell lines was assessed by analyzing TCA cycle intermediates via gas chromatography mass spectrometry (GC-MS).ResultsThe immortalized Slc4a11+/+ and Slc4a11−/− mouse corneal endothelial cells (MCECs) remained proliferative through passage 49 and maintained similar active ion transport activity. As expected, proliferation was temperature sensitive and IFN-γ dependent. Slc4a11−/− MCECs exhibited decreased proliferative capacity, reduced NH3:H+ transport, altered expression of glutaminolysis enzymes similar to the Slc4a11−/− mouse, and reduced proportion of TCA cycle intermediates derived from glutamine with compensatory increases in glucose flux compared with Slc4a11+/+ MCECs.ConclusionsThis is the first report of the immortalization of MCECs. Ion transport of the immortalized endothelial cells remains active, except for NH3:H+ transporter activity in Slc4a11−/− MCECs. Furthermore, Slc4a11−/− MCECs recapitulate the glutaminolysis defects observed in Slc4a11−/− mouse corneal endothelium, providing an excellent tool to study the pathogenesis of SLC4A11 mutations associated with corneal endothelial dystrophies and to screen potential therapeutic agents.

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Section: Resultssupporting

confidence: 90%

Conditionally Immortal Slc4a11^−/− Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in Slc4a11^−/− Mouse Model

Zhang

Ogando

Kim

et al. 2017

Invest. Ophthalmol. Vis. Sci.

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“…Decreased ratio of p-mTOR/mTOR as well as increased levels of LC3II/I in Slc4a11 KO corneal endothelia indicate that the autophagy initiation steps are present in these cells. This is consistent with previous studies that show defects in glutamine catabolism and decreased ATP production in KO 5,29 , which would favor increasing autophagy via reducing p-mTOR/mTOR ratio. The accumulation of P62, however indicates a downstream impairment of autophagy flux, and the lack of significant change in P62 levels in the presence of Bafilomycin A1 are consistent with impaired autophagy flux.…”

Section: Discussionsupporting

confidence: 93%

MitoROS due to loss ofSlc4a11in corneal endothelial cells induces ER stress, lysosomal dysfunction and impairs autophagy

Shyam

Ogando

Choi

et al. 2020

Preprint

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Recent studies from Slc4a11 KO mice have identified mitochondrial dysfunction as a major contributor toward oxidative stress and cell death in Congenital Hereditary Endothelial Dystrophy. Here we asked if this stress activated autophagy in the Slc4a11 KO cell line and in KO mouse endothelial tissue. Early indicators of autophagy, phospho-mTOR and LC3-II indicated activation, however P62 was elevated suggesting an impairment of autophagy flux. The activity and the number of lysosomes, the organelle responsible for the final degradation of autophagy substrates, were found to be reduced in the KO. In addition, the expression of the master regulator of lysosomal function and biogenesis, TFEB, was significantly reduced in the KO corneal endothelia. Also, we observed increased Unfolded Protein Response, as well as elevated expression of ER stress markers, BIP and CHOP. To test if lysosomal and ER stress stems from elevated mitochondrial ROS, we treated Slc4a11 KO corneal endothelial cells with the mitochondrial ROS quencher, MitoQ. MitoQ restored lysosomal enzymes as well as TFEB, reduced ER stress, and increased autophagy flux. MitoQ injections of Slc4a11 KO mice decreased corneal edema, the major phenotype associated with CHED. We conclude that mitochondrial ROS causes ER stress and lysosomal dysfunction with impairment of autophagy in Slc4a11 KO corneal endothelium. Our study is the first to identify the presence as well as cause of lysosomal dysfunction and ER stress in an animal model of CHED, and to characterize inter-organelle relationship in a corneal cell type.

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“…9 Corneal endothelium is one of the most metabolically active tissues in the human body. 10 Dissecting the biologic functions of metabolites and mitochondria in cHCEC cell fate decision will provide essential cues to further improve cell-based therapy. Mitochondria are poised to play an essential role.…”

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confidence: 99%

Metabolites Interrogation in Cell Fate Decision of Cultured Human Corneal Endothelial Cells

Hamuro

Numa

Fujita

et al. 2020

Invest. Ophthalmol. Vis. Sci.

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Aiming to clarify the metabolic interrogation in cell fate decision of cultured human corneal endothelial cells (cHCECs). METHODS. To analyze the metabolites in the culture supernatants (CS), 34 metabolome measurements were carried out for mature differentiated and a variety of cHCECs with cell state transition through a facility service. Integrated proteomics research for cell lysates by liquid chromatography−tandem mass spectrometry (LC-MS/MS) was performed for 3 aliquots of each high-quality or low-quality cHCEC subpopulations (SP). The investigations for the focused genes involved in cHCEC metabolism were performed by using DAVID and its options "KEGG_PATHWAY." RESULTS. The clusters of metabolites coincided well with the distinct content of CD44−/+ SPs. Both secreted pyruvic acid and lactic acid in the CS were negatively correlated with the content of high-quality SPs. Lactic acid and pyruvic acid in the CS exhibited the positive correlation with that of Ile, Leu, and Ser, whereas the negative correlation was with glutamine. Platelet-derived growth factor-ββ in the CS negatively correlated with lactic acid in CS, indicating indirectly the positive correlation with the content of CD44−/+ SPs. Upregulated glycolytic enzymes and influx of glutamine to the tricarboxylic acid cycle may be linked with a metabolic rewiring converting oxidative metabolism in mature differentiated CD44−/+SPs into a glycolytic flux-dependent state in immature SPs with cell state transition. CONCLUSIONS. The findings suggest that the cell fate decision of cHCECs may be dictated at least partly through metabolic rewiring.

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Glutaminolysis is Essential for Energy Production and Ion Transport in Human Corneal Endothelium

Cited by 49 publications

References 47 publications

Conditionally Immortal Slc4a11^−/− Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in Slc4a11^−/− Mouse Model

Conditionally Immortal Slc4a11^−/− Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in Slc4a11^−/− Mouse Model

MitoROS due to loss ofSlc4a11in corneal endothelial cells induces ER stress, lysosomal dysfunction and impairs autophagy

Metabolites Interrogation in Cell Fate Decision of Cultured Human Corneal Endothelial Cells

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Glutaminolysis is Essential for Energy Production and Ion Transport in Human Corneal Endothelium

Cited by 49 publications

References 47 publications

Conditionally Immortal Slc4a11−/− Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in Slc4a11−/− Mouse Model

Conditionally Immortal Slc4a11−/− Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in Slc4a11−/− Mouse Model

MitoROS due to loss ofSlc4a11in corneal endothelial cells induces ER stress, lysosomal dysfunction and impairs autophagy

Metabolites Interrogation in Cell Fate Decision of Cultured Human Corneal Endothelial Cells

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Conditionally Immortal Slc4a11^−/− Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in Slc4a11^−/− Mouse Model

Conditionally Immortal Slc4a11^−/− Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in Slc4a11^−/− Mouse Model