The metabolism of DβHB in mitochondria is stereospecific to the D isoform. Under normal physiological conditions, the level of DβHB is low but becomes dramatically elevated during starvation, increased fatty acid metabolism or pathological conditions such as diabetes. From its site of production in the liver, DβHB is released into the blood and circulated for utilization by other tissues. In general, the rate of ketone body usage in the brain is proportional to the concentration in the circulation [59]. Circulating DβHB readily crosses the blood-brain barrier and enters brain mitochondria where it is metabolized by mitochondrial β-hydroxybutyrate dehydrogenase to acetoacetate, which is subsequently converted to acetyl-coenzyme A to feed into the tricarboxylic acid cycle (TCA) cycle. The intermediate products generated from this cycle, NADH and succinate, in turn feed into the electron transport chain to subsequently generate ATP at complex V. Through this metabolic pathway, DβHB is an excellent alternative source of energy in the brain when glycolysis is not operative or when glucose supply is depleted such as during starvation [59].

Male C57Bl/6 mice (∼21 weeks-old) was infused subcutaneously (1 µl/h) with either DβHB (1.6 mmol/kg in saline) or vehicle (saline) using implanted osmotic minipumps. One day after surgery, each animal group was further subdivided into two groups to receive nine i.p. injections of either saline or 3-NP (50 mg/kg) at 12 h intervals. Five hours after the last 3-NP injection, animals were intracardially perfused with 4% paraformaldehyde. As compared to control (A), 3-NP induced significant cell loss (pale region) as seen here with Nissl stain (B). This lesion was not detectable in the group that received DβHB infusion (C). DβHB by itself did not affect the baseline level (not shown) of Nissl staining. Immunoreactivity for MAC-1/CD11b was observed after 3-NP treatment (E, H) in the lesioned area (outlined) as compared to the saline control group (D, G). Although 3-NP also induced discrete regions of increased microglia reaction in some animals that received DβHB (F, I), the immunoreactivity was not as dramatic as compared to the 3-NP treated group (E, H). Panel J summarizes the death rate and numbers of animals with striatal lesions induced by 3-NP in the groups that received either saline or DβHB infusion. Panel K demonstrates comparable magnitude of complex II inhibition in the two 3-NP treated groups, indicating that DβHB did not interfere with the function of complex II per se.

Male C57Bl/6 mice (∼21 weeks-old) were treated with 3-NP and DβHB as described in Figure 1. Five hours after the last 3-NP injection, mice were assessed for locomotor activities using infrared photobeam chambers. In the group that received saline infusion (n = 12), 3-NP injection induced dramatically impaired locomotor movements. In contrast, these abnormalities were attenuated in the 3-NP group that received DβHB infusion (n = 7). Units expressed as % of the saline injected control group (not shown) ± SEM. *p<0.05 as compared to the 3-NP treated mice that receive saline infusion.

Six week old male transgenic (Tg)-R6/2 mice and non-transgenic (Ntg) littermates were infused with either saline vehicle or DβHB as described in Figure 2. Osmotic minipumps were replaced every two weeks for the entire study period. Locomotor activities were assessed using infrared photobeams chambers. Tg-R6/2 mice displayed significant impairment in locomotor movements. In contrast, these abnormalities were delayed and less severe in the Tg group treated with DβHB (n = 7). DβHB did not affect locomotor function of Ntg mice (not shown). Units expressed as % control of Ntg-Saline group at seven weeks old (n = 12). *p<0.05 as compared to the respective Tg-saline groups (n = 7) at the same time points.

Animals from Figure 4 were monitored twice daily for their survival rate (A). These mice were considered to reach their end stage if they were unable to right themselves after being placed on their back or died overnight. DβHB significantly extended the survival time in Tg-R6/2 mice. n = 12 (Ntg-saline), n = 7 (Tg-saline), n = 7 (Tg-DβHB). p<0.001 Kaplan-Meier analysis between Tg-sal and Tg- DβHB groups. These animals were also assessed weekly for glucose levels after six hours of fasting (B). One animal from the Tg-saline group survived up to 14 weeks and exhibited an elevated glucose level.

Animals recovered from the survival study were perfused and striatal sections were obtained and immunostained for histone H4 acetylation. Immunoreactivity was visualized by incubation in 3,3′-diaminobenzidine . There was less immunoreactivity of histone H4 acetylation in the transgenic animals as compared to the Ntg mice. DβHB treatment restored H4 acetylation in the Tg animals. n = 2 representative animals per group.

(Panel A): Dose-response study of DβHB and sodium butyrate (0.25 mM-4 mM) in PC12 cells with inducible expression of Htt^Q103 upon addition of tebufenozide (1 µM) using immunoblotting. Two microgram of histones extracted from Hela cells treated with 5 mM sodium butyrate (obtained from Upstate Biotechnology Inc.) was used as a positive control (“+C”). Levels of histone acetylation were quantified in cells with mutant (Panel B, Htt^Q103) or normal (Panel C, Htt^Q25) huntingtin 48 h after induction in the absence (“0”) or presence of equimolar concentrations (4 mM) of DβHB (“D”), LβHB (“L”) or sodium butyrate (“Na”) and compared with the non-tebufenozide treated control group (“C”). Data represent n = 6–10 per group (pooled from three separate experiments). *p<0.01 as compared to the control group (“C”) without tebufenozide treatment.

Hela nuclear extract was used as a source of class I and II HDAC activity (Panel A) where as recombinant human Sir1 was used as a source of class III HDAC activity (Panel B). HDAC activities were assessed in the absence (control, “C”) or presence of 4 mM DβHB (“D”), LβHB (“L”) or sodium butyrate (“Na”, an HDAC I and II inhibitor), 1 µM trichostatin A (“TA”, an HDAC I and II inhibitor) or 1 mM nicotinamide (“Nic”, an HDAC III inhibitor). Activities were quantified based on fluorescence intensity and expressed as arbitrary fluorescence unit (AFU). n = 6–16 per group from 5 independent experiments.