Glucose metabolism through the hexosamine biosynthetic pathway drives hepatic de novo lipogenesis via promoting <i>N</i>-linked protein glycosylation

De novo lipogenesis (DNL) converts excess glucose into lipids, whereas the hexosamine biosynthetic pathway (HBP), a glycolytic branch, generates UDP-<i>N</i>-acetylglucosamine for protein glycosylation, including <i>O</i>-GlcNAcylation and <i>N</i>-linked glycosylation. Both pathways are active in hepatocytes and integral to glucose metabolism; however, their functional interplay remains unclear. Here, we investigated the role of HBP in hepatic DNL activation using both in vitro and in vivo models. AML12 hepatocytes were cultured in low- and high-glucose media with or without HBP blockade, both pharmacologically and genetically. For in vivo studies, male C57BL/6J mice were subjected to a fasting-refeeding regimen with or without intraperitoneal administration of azaserine, a competitive inhibitor of glutamine-fructose-6-phosphate transaminase 1 (GFPT1), the rate-limiting enzyme of the HBP. Our results demonstrated that, in AML12 cells, glucose exposure activated both DNL and HBP, leading to triacylglycerol (TAG) accumulation, whereas HBP inhibition ameliorated DNL and TAG accumulation. In mice, refeeding after a 24-h fasting induced hepatic DNL, which was abolished by HBP inhibition, indicating its mechanistic involvement in glucose-driven lipogenesis. Mechanistically, we identified ATF4 as a key regulator of GFPT1 upregulation under high-glucose conditions. As expected, both glucose-treated hepatocytes and livers from fasting-refed mice exhibited increased protein glycosylation. Notably, blocking <i>N</i>-linked glycosylation, but not <i>O</i>-GlcNAcylation, abolished glucose-induced DNL activation, indicating that HBP is essential for glucose-induced DNL pathway activation via promoting <i>N</i>-linked glycosylation, independent of <i>O</i>-GlcNAcylation. In conclusion, our findings establish that an intact HBP is required for glucose-induced hepatic DNL activation, primarily through promoting protein <i>N</i>-linked glycosylation.<b>NEW & NOTEWORTHY</b> High-glucose exposure activates both hepatic HBP and DNL pathways. The glucose metabolism into HBP is essential for the activation of the DNL pathway. ATF4 activation plays a mechanistic role in high glucose-induced HBP activation. HBP drives high glucose-induced hepatic DNL activation via promoting <i>N</i>-linked protein glycosylation.