Hexosamine biosynthetic pathway is required for glucose-induced <i>de novo</i> lipogenesis pathway activation in hepatocytes independent of protein O-GlcNAcylation

Objectives: De novo lipogenesis (DNL), the process of converting excess carbohydrates like glucose into fatty acids, is a key metabolic pathway in hepatocytes. The hexosamine biosynthetic pathway (HBP), a branch of glycolysis, produces UDP-N-acetylglucosamine (UDP-GlcNAc), a critical substrate for protein glycosylation. Although both pathways are individually well-characterized, their interaction in regulating hepatic lipid synthesis remains unclear. This study aims to elucidate the role of HBP in glucose-induced DNL activation and assess its dependence on protein O-GlcNAcylation. Methods: AML12 hepatocytes were cultured in low- or high-glucose media, with HBP inhibited pharmacologically (azaserine) or genetically (GFPT1 siRNA). Effects of protein O-GlcNAcylation were tested using OGT (OSMI) and OGA (TMG) inhibitors, while supplementation with glucosamine and N-acetylglucosamine evaluated the role of HBP intermediates. Gene and protein expression of DNL markers (SREBP-1c, Fasn, Acly) and GFPT1 were analyzed by qRT-PCR and Western blotting, and triglyceride accumulation was enzymatically measured. In vivo, male C57BL/6J mice were subjected to a fasting-refeeding protocol, with or without azaserine treatment, to evaluate hepatic DNL and HBP activity. Results: High glucose increased DNL-related gene expression and intracellular triglyceride levels, along with upregulation of HBP enzymes, particularly GFPT1. Inhibiting GFPT1 suppressed DNL activation and triglyceride accumulation in both cells and mice. Protein O-GlcNAcylation was elevated under high glucose, driven by increased OGT expression, but its inhibition paradoxically enhanced DNL, indicating a protein O-GlcNAcylation-independent mechanism. Supplementation with HBP intermediates restored DNL activation in GFPT1-inhibited cells, underscoring their role in promoting lipid synthesis. Mechanistically, ATF4 was identified as a critical upstream regulator of GFPT1, with its knockdown abolishing glucose-induced GFPT1 expression. ATF4 knockout mice similarly exhibited reduced GFPT1 expression and DNL activation, confirming its regulatory role in vivo. Conclusions: This study establishes that the HBP is essential for glucose-induced hepatic DNL activation through a protein O-GlcNAcylation-independent mechanism. HBP intermediates directly promote DNL, and ATF4-mediated GFPT1 upregulation links glucose metabolism to lipid synthesis. These findings offer new insights into hepatic metabolic regulation and potential therapeutic strategies for fatty liver diseases. This work was funded by US NIH Grant NIAAA R01AA026603 (to ZS) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.