Anticonvulsant Effect of Time-Restricted Feeding in a Pilocarpine-Induced Seizure Model: Metabolic and Epigenetic Implications

Abstract

A new generation of antiepileptic drugs has emerged; however, one-third of epilepsy patients do not properly respond to pharmacological treatments. The purpose of the present study was to investigate whether time-restricted feeding (TRF) has an anticonvulsant effect and whether this restrictive diet promotes changes in energy metabolism and epigenetic modifications in a pilocarpine-induced seizure model. To resolve our hypothesis, one group of rats had free access to food and water ad libitum (AL) and a second group underwent a TRF schedule. We used the lithium-pilocarpine model to induce status epilepticus (SE), and behavioral seizure monitoring was analyzed. Additionally, an electroencephalography (EEG) recording was performed to verify the effect of TRF on cortical electrical activity after a pilocarpine injection. For biochemical analysis, animals were sacrificed 24 h after SE and hippocampal homogenates were used to evaluate the proteins related to metabolism and chromatin structure. Our results showed that TRF had an anticonvulsant effect as measured by the prolonged latency of forelimb clonus seizure, a decrease in the seizure severity score and fewer animals reaching SE. Additionally, the power of the late phase EEG recordings in the AL group was significantly higher than the TRF group. Moreover, we found that TRF is capable of inducing alterations in signaling pathways that regulate energy metabolism, including an increase in the phosphorylation of AMP dependent kinase (AMPK) and a decrease in the phosphorylation of Akt kinase. Furthermore, we found that TRF was able to significantly increase the beta hydroxybutyrate (β-HB) concentration, an endogenous inhibitor of histone deacetylases (HDACs). Finally, we found a significant decrease in HDAC activity as well as an increase in acetylation on histone 3 (H3) in hippocampal homogenates from the TRF group. These findings suggest that alterations in energy metabolism and the increase in β-HB mediated by TRF may inhibit HDAC activity, thus increasing histone acetylation and producing changes in the chromatin structure, which likely facilitates the transcription of a subset of genes that confer anticonvulsant activity.

Keywords: AMP kinase; Akt kinase; HDACs inhibition; anticonvulsant; beta-hydroxybutyrate; histone 3 acetylation; pilocarpine.

Figure 1

Influence of the time-restricted feeding (TRF) model on body weight, food intake, glucose, and β-hydroxybutyrate in the fasted/refed state and the ratio among kilocalorie consumption and basal metabolic rate (BMR) in rats. (A) Schematic representation of the experimental procedure of the dietary schedule and status epilepticus (SE) induction. Body weight of TRF rats showed a significant decrease at all time points measured (B); moreover, there is a reduction in food intake and the ratio of caloric intake/metabolic rate compared with that of ad libitum (AL)-fed animals (D,F). Regarding biochemical parameters, the blood glucose concentration showed no significant difference in the fasted state; however, AL-fed rats showed an inability to metabolize glucose at 15 and 20 days, which does not occur in TRF animals (C). Interestingly, the blood β-hydroxybutyrate concentration was high during the TRF schedule and it was maintained even in the refed state, even though it is lower than during the fasted state (E). Data are expressed as the mean ± SD from each determination (n = 30, **p < 0.01; ***p < 0.001).

Figure 2

TRF model decreases liver weight, but not brain weight. At the end of the dietary schedule, the mean liver weight of TRF rats showed a significant decrease compared with that of AL-fed animals (B,D); on the other hand, brain weight was not influenced by the time restricted feeding model (A,C). Data are expressed as the mean ± SD from each determination (n = 10, ***p < 0.001).

Figure 3

Effects on metabolic related signaling pathways during TRF schedule in brain and liver tissues. Immunoblots from hippocampal rat brain homogenates that followed dietary restriction revealed a significant increase in AMP dependent kinase (AMPK) phosphorylation at threonine Thr172 content compared with that of their respective AL-fed control animals. Conversely, homogenates from TRF rats showed a significant decrease in Akt phosphorylation at serine 473 compared with that of their AL-fed controls (A,B). Additionally, similar results were obtained in liver homogenates, where there was an increase in AMPK phosphorylation and a decrease in Akt phosphorylation (C,D). Samples were normalized with total AMPK or Akt. Bar charts are the semiquantitative optical densities of immunostained bands (B). Data are expressed as the mean ± SD from each determination (n = 8, ***p < 0.001).

Figure 4

TRF inhibits seizure susceptibility measured by behavioral analysis. TRF animals showed an increased latency (A), a significant decrease in the mean seizure score (B) and a smaller number of animals reaching status epilepticus (SE) (C) compared with that of AL-fed rats. Moreover, there was a positive statistically significant correlation among high β-HB levels and decreased latency to the first seizure and a negative correlation between high β-HB levels and a reduction in seizure score in rats subjected to TRF (D). Data are expressed as the mean ± SD from each determination (n = 30 for behavioral analysis, *p < 0.05; ***p < 0.001; n = 10 for Pearson’s or Spearman’s correlation test, p < 0.05 and p < 0.01, respectively).

Figure 5

Representative electroencephalography (EEG) signal and FTT analysis from AL and TRF pilocarpine-injected rats. The upper tracings show typical EEG recordings before administration of pilocarpine in AL rats (A) and TRF rats (B) (n = 5 each). The numbers at the left of each record indicate the time after the application of pilocarpine. Plots of the mean power spectra as a function of time for the EEG recording taken from rats injected with pilocarpine. Power values were calculated as the percent of the 45 min baseline power recorded before pilocarpine injection, assigned as a value of 100% (data not shown). (C) Fast fourier transformations (FFT) of the 1–50 Hz frequency spectrum. (D) FFT of the 50–100 Hz frequency spectrum. The red symbol represents the mean data from AL rats and the blue symbol represents the mean values from TRF rats. AL: (1) Baseline period, (2) 32, (3) 65, and (4) 75 min, time after the application of pilocarpine and TRF: (1) Baseline period, (2) 15, (3) 40, and (4) 75 min, time after the application of pilocarpine. Data are expressed as the mean ± SEM (*p < 0.05).

Figure 6

TRF promotes epigenetic changes in the hippocampus through inhibition of HDAC activity and posttranslational modifications on histone 3 (H3). Total HDAC activity was measured in four groups (AL, TRF, AL-SE and TRF-SE). There was a statistically significant decrease in HDAC activity in TRF-subjected animals compared with that of AL-fed animals (A); additionally, the same result was observed in TRF-SE animals compared with that of AL-SE rats (D). Representative immunoblots show that there was a significant increase in the acetylation of H3K9ac and H3K14ac in the nuclear extract from TRF rats compared with that of AL-fed rats (B,C). Interestingly, the same results were observed in pilocarpine injected animals subjected to TRF compared with that of pilocarpine-injected animals fed AL (E,F). Nuclear fraction samples were normalized with total H3 protein. Bar charts are the semiquantitative optical densities of immunostained bands (B). Data are expressed as the mean ± SD from each determination (*p < 0.05; ***p < 0.001). The total activity assay is from four independent animals and representative blots are from eight independent animals.