AbstractHuman 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.