|Glucose is metabolized through several pathways; glycolysis, pentose phosphate pathway, and hexosamine biosynthesis
pathway (HBP). Orexin system plays a central role of integration of sleep/wake in broad spectrum of neural-metabolic
physiology. Orexin-A and -B, are produced from prepro-orexin encoded by HCRT/Hcrt gene. Despite enormous efforts,
authentic methods successfully used to induce other hypothalamic peptide neurons could not generate the orexin neurons
from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We succeeded in generating functional orexin
neurons from mouse ESCs and human iPSCs by adding ManNAc, an intermediate of HBP. The induced orexin neurons were
sensitive to glucose, and possessed the ability to respond to neurotransmitter and peptides such as GABA, TRH, ghrelin,
and leptin. CpGs of the Hcrt genome locus was hypomethylated, which was associated with H3/H4 higher acetylation.
Concomitantly, histone acetyltransferase, p300, CBP and Mgea5 (O-GlcNAcase) were colocalized at the Hcrt gene locus in
the orexin neurons. In the orexin non-expressing cells, hypoacetylation of H3/H4 and hyper O-GlcNAc modification were
observed at the Hcrt gene locus, which were occupied with Ogt and Sirt1. Therefore, stream of HBP is sensed in the epigenetic
mechanism to switch the Hcr gene from inactive state to active state. These results indicated that the multi-layer epigenetic
regulation in the generation of orexin neurons. The established method will be useful for development of orexin neurons for
regenerative medicine, and the induced orexin neurons will provide a strong tool for the development of medical applications
for diseases related to the orexin system.