The hexosamine biosynthetic pathway (HBP) is a nutrient-sensing metabolic pathway that produces the activated amino sugar UDP-N-acetylglucosamine, a critical substrate for protein glycosylation. Despite its biological significance, little is known about the regulation of HBP flux during nutrient limitation. Here, we report that amino acid or glucose shortage increase GFAT1 production, the first and rate-limiting enzyme of the HBP. GFAT1 is a transcriptional target of the activating transcription factor 4 (ATF4) induced by the GCN2-eIF2α signalling pathway. The increased production of GFAT1 stimulates HBP flux and results in an increase in O-linked β-N-acetylglucosamine protein modifications. Taken together, these findings demonstrate that ATF4 provides a link between nutritional stress and the HBP for the regulation of the O-GlcNAcylation-dependent cellular signalling.The regulation of metabolic fluxes is a key process in the cellular response to changes in nutrient availability, and dysregulation of this process may contribute to the development of various diseases 1,2 . The hexosamine biosynthetic pathway (HBP) plays a central role in sensing the nutritional status of the cell, since it integrates molecules coming from carbohydrates, fatty acids, amino acids and nucleotides metabolism 3 . The HBP converts fructose-6-phosphate, a glucose derivative, into UDP-N-acetylglucosamine (UDP-GlcNAc), a central nucleotide sugar acting as a donor substrate for the glycosylation of proteins and lipids. In addition, UDP-GlcNAc is used for the O-GlcNAcylation of proteins, i.e. the addition of N-acetylglucosamine to their serine and threonine residues. Similarly to phosphorylation, O-GlcNAcylation regulates cellular activities, such as signalling and transcription 4 . It has been proposed that O-GlcNAcylated proteins modulate cellular responses to nutrient deprivation and stress, and that their dysregulation is implicated in a wide range of pathologies 5 . Despite these wide-ranging biological effects, the mechanisms underlying the adaptation of the HBP flux to nutrient availability remains poorly understood.The first and rate-limiting enzyme of the HBP, namely glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1), which drives the HBP flux, is involved in various physiopathological processes 6-9 . GFAT1 activity is inhibited by UDP-GlcNAc, the end product of the HBP 10 , and the AMP-activated protein kinase-mediated phosphorylation 11 . In contrary, GFAT1 is stimulated upon phosphorylation by protein kinase A and calcium/ calmodulin-dependent protein kinase II 12 . More recently, it was shown that GFAT1 production is upregulated by a transcription factor of the unfolded protein response (UPR), namely the spliced form of X-box binding protein 1 (XBP1s), resulting in the stimulation of the HBP flux and protein O-GlcNAcylation 13 . These authors unveiled in this way an intrinsic link between the HBP and disruption of the endoplasmic reticulum (ER) homeostasis, i.e. ER stress, which activates the UPR.UPR signalling is mediat...