β-hydroxybutyrate serves as a regulator in ketone body metabolism through lysine β-hydroxybutyrylation.

β-hydroxybutyrate (β-HB) may serve as a signaling metabolite in many physiological processes beyond a fuel source for tissues. However, whether and how it is involved in ketone body metabolism is still unknown. The present study aims to investigate the role of lysine β-hydroxybutyrylation (Kbhb) modification mediated by β-HB in regulating ketone body metabolic homeostasis both in vivo and in vitro. The starvation ketosis and type 1 diabetes mouse models were introduced to evaluate the influence of β-HB on Kbhb modification in mice. The lysine β-hydroxybutyrylation modifications of OXCT1 and HMGCS2, two rate-limiting enzymes involved in ketogenesis and utilization, showed a positive correlation with the level of β-HB both in vitro and in vivo. The modification levels of the enzymes increased during fasting but decreased after refeeding. However, the Kbhb modification level in all detected tissues showed minor change since the blood ketone body increased non-significantly in the type 1 diabetes mouse model. The in vitro experiments further indicated that mutation at the Kbhb modification site significantly inhibited the enzymatic activity of OXCT1 but not HMGCS2. SIRT1 and CBP were identified both in vitro and in vivo as potential Kbhb dehydrogenase and transferase for OXCT1, respectively. Lysine β-hydroxybutyrylation modification at lysine 421 of OXCT1 increases its enzyme activity during β-HB accumulation, accelerating the utilization of the ketone body and finally maintaining metabolism homeostasis. Our present study proposes a new ketone body metabolic regulatory mode primarily mediated by lysine β-hydroxybutyrylation modifications of OXCT1 during β-HB accumulation.