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Glutamine/glutamate metabolism rewiring in reprogrammed human hepatocyte-like cells.

ABSTRACT: Human dermal fibroblasts can be reprogrammed into hepatocyte-like (HEP-L) cells by the expression of a set of transcription factors. Yet, the metabolic rewiring suffered by reprogrammed fibroblasts remains largely unknown. Here we report, using stable isotope-resolved metabolic analysis in combination with metabolomic-lipidomic approaches that HEP-L cells mirrors glutamine/glutamate metabolism in primary cultured human hepatocytes that is very different from parental human fibroblasts. HEP-L cells diverge glutamine from multiple metabolic pathways into deamidation and glutamate secretion, just like periportal hepatocytes do. Exceptionally, glutamine contribution to lipogenic acetyl-CoA through reductive carboxylation is increased in HEP-L cells, recapitulating that of primary cultured human hepatocytes. These changes can be explained by transcriptomic rearrangements of genes involved in glutamine/glutamate metabolism. Although metabolic changes in HEP-L cells are in line with reprogramming towards the hepatocyte lineage, our conclusions are limited by the fact that HEP-L cells generated do not display a complete mature phenotype. Nevertheless, our findings are the first to characterize metabolic adaptation in HEP-L cells that could ultimately be targeted to improve fibroblasts direct reprogramming to HEP-L cells.

SUBMITTER: Ballester M

PROVIDER: S-EPMC6884617 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Glutamine/glutamate metabolism rewiring in reprogrammed human hepatocyte-like cells.

Ballester Maria M Sentandreu Enrique E Luongo Giovanna G Santamaria Ramon R Bolonio Miguel M Alcoriza-Balaguer Maria Isabel MI Palomino-Schätzlein Martina M Pineda-Lucena Antonio A Castell Jose J Lahoz Agustin A Bort Roque R

Scientific reports 20191129 1

Human dermal fibroblasts can be reprogrammed into hepatocyte-like (HEP-L) cells by the expression of a set of transcription factors. Yet, the metabolic rewiring suffered by reprogrammed fibroblasts remains largely unknown. Here we report, using stable isotope-resolved metabolic analysis in combination with metabolomic-lipidomic approaches that HEP-L cells mirrors glutamine/glutamate metabolism in primary cultured human hepatocytes that is very different from parental human fibroblasts. HEP-L cel ...[more]

PMID: 31784643

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Project description:Energy metabolism in proliferating cultured rat thymocytes was compared with that of freshly prepared non-proliferating resting cells. Cultured rat thymocytes enter a proliferative cycle after stimulation by concanavalin A and Lymphocult T (interleukin-2), with maximal rates of DNA synthesis at 60 h. Compared with incubated resting thymocytes, glucose metabolism by incubated proliferating thymocytes was 53-fold increased; 90% of the amount of glucose utilized was converted into lactate, whereas resting cells metabolized only 56% to lactate. However, the latter oxidized 27% of glucose to CO2, as opposed to 1.1% by the proliferating cells. Activities of hexokinase, 6-phosphofructokinase, pyruvate kinase and aldolase in proliferating thymocytes were increased 12-, 17-, 30- and 24-fold respectively, whereas the rate of pyruvate oxidation was enhanced only 3-fold. The relatively low capacity of pyruvate degradation in proliferating thymocytes might be the reason for almost complete conversion of glucose into lactate by these cells. Glutamine utilization by rat thymocytes was 8-fold increased during proliferation. The major end products of glutamine metabolism are glutamate, aspartate, CO2 and ammonia. A complete recovery of glutamine carbon and nitrogen in the products was obtained. The amount of glutamate formed by phosphate-dependent glutaminase which entered the citric acid cycle was enhanced 5-fold in the proliferating cells: 76% was converted into 2-oxoglutarate by aspartate aminotransferase, present in high activity, and the remaining 24% by glutamate dehydrogenase. With resting cells the same percentages were obtained (75 and 25). Maximal activities of glutaminase, glutamate dehydrogenase and aspartate aminotransferase were increased 3-, 12- and 6-fold respectively in proliferating cells; 32% of the glutamate metabolized in the citric acid cycle was recovered in CO2 and 61% in aspartate. In resting cells this proportion was 41% and 59% and in mitogen-stimulated cells 39% and 65% respectively. Addition of glucose (4 mM) or malate (2 mM) strongly decreased the rates of glutamine utilization and glutamate conversion into 2-oxoglutarate by proliferating thymocytes and also affected the pathways of further glutamate metabolism. Addition of 2 mM-pyruvate did not alter the rate of glutamine utilization by proliferating thymocytes, but decreased the rate of metabolism beyond the stage of glutamate significantly. Formation of acetyl-CoA in the presence of pyruvate might explain the relatively enhanced oxidation of glutamate to CO2 (56%) by proliferating thymocytes.

Dataset Information

Glutamine/glutamate metabolism rewiring in reprogrammed human hepatocyte-like cells.

Publications

Glutamine/glutamate metabolism rewiring in reprogrammed human hepatocyte-like cells.

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