The ketogenic diet suppresses the cathepsin E expression induced by kainic acid in the rat brain

Abstract

Purpose: The ketogenic diet has long been used to treat epilepsy, but its mechanism is not yet clearly understood. To explore the potential mechanism, we analyzed the changes in gene expression induced by the ketogenic diet in the rat kainic acid (KA) epilepsy model.

Materials and methods: KA-administered rats were fed the ketogenic diet or a normal diet for 4 weeks, and microarray analysis was performed with their brain tissues. The effects of the ketogenic diet on cathepsin E messenger ribonucleic acid (mRNA) expression were analyzed in KA-administered and normal saline-administered groups with semi-quantitative and real-time reverse transcription polymerase chain reaction (RT-PCR). Brain tissues were dissected into 8 regions to compare differential effects of the ketogenic diet on cathepsin E mRNA expression. Immunohistochemistry with an anti-cathepsin E antibody was performed on slides of hippocampus obtained from whole brain paraffin blocks.

Results: The microarray data and subsequent RT-PCR experiments showed that KA increased the mRNA expression of cathepsin E, known to be related to neuronal cell death, in most brain areas except the brain stem, and these increases of cathepsin E mRNA expression were suppressed by the ketogenic diet. The expression of cathepsin E mRNA in the control group, however, was not significantly affected by the ketogenic diet. The change in cathepsin E mRNA expression was greatest in the hippocampus. The protein level of cathepsin E in the hippocampus of KA-administered rat was elevated in immunohistochemistry and the ketogenic diet suppressed this increase.

Conclusion: Our results showed that KA administration increased cathepsin E expression in the rat brain and its increase was suppressed by the ketogenic diet.

Fig. 1

Changes of body weight and serum β-hydroxybutyrate. (A) Groups fed the ketogenic diet (NS-KD and KA-KD) gained less body weight than the control groups fed a normal diet (NS-ND and KA-ND). (B) The serum level of β-hydroxybutyrate increased due to the ketogenic diet in NS-KD and KA-KD rats. NS-ND, normal saline, normal diet; NS-KD, normal saline, ketogenic diet; KA-ND, kainic acid, normal diet; KA-KD, kainic acid, ketogenic diet.

Fig. 2

Semiquantitative RT-PCR analysis of cathepsin E mRNA expression in whole brain tissue. Cathepsin E expression was lower in the KA-KD group than in the KA-ND group. RT-PCR, reverse transcription polymerase chain reaction; mRNA, messenger ribonucleic acid; KA-KD, kainic acid, ketogenic diet; KA-ND, kainic acid, normal diet.

Fig. 3

Cathepsin E mRNA expression in different brain areas. Rat brains were dissected into 8 compartments. (A) In the semiquantitative RT-PCR analysis, the ketogenic diet down-regulated cathepsin E expression in the rats given KA, but not in those given normal saline. (B) In the real-time RT-PCR, KA increased cathepsin E expression in all brain areas examined except the brain stem, and the ketogenic diet suppressed the KA-induced cathepsin E expression. The hippocampus showed the most prominent change in cathepsin E expression. Data represent the relative amount of cathepsin E mRNA of each group compared to NS-ND in the same brain area. ^*indicates statistical significance (^*p < 0.05, ^**p < 0.01). HASP, hypothalamus, amygdala, septum, and preoptic area. mRNA, messenger ribonucleic acid; RT-PCR, reverse transcription polymerase chain reaction; NS-ND, normal saline, normal diet; NS-KD, normal saline, ketogenic diet; KA-ND, kainic acid, normal diet; KA-KD, kainic acid, ketogenic diet.

Fig. 4

Immunohistochemistry of cathepsin E in rat hippocampus. After i.p. administration of normal saline (A and B) or KA (12 mg/kg in C and D, 25 mg/kg in D, E, G and H), rats were fed a normal diet (A, C, E and G) or the ketogenic diet (B, D, F and H). The CA3 region of the rat hippocampus of the NS-ND (A) and the NS-KD (B) equally showed very few cathepsin-E positive neurons. In the lower-dose KA-ND rat (C), a small number of neurons expressed cathepsin E, while those of the lower-dose KA-KD rat (D) distributed more sparsely. In the CA3 region of the higher-dose KA-ND rat (E), widespread expression of cathepsin E by neurons were observed, in contrast to a marked decrease in the cathepsin E immunoreactivity in the same area of the higher-dose KA-KD rat (F). Details of higher-dose KA-ND neurons exhibit pyknotic changes (G, arrowheads), whereas neurons of the higher-dose KA-KD rat had round, euchromatic nuclei (H, arrowheads). A, B, G, and H were counterstained (scale bar = 500 µm in A, 100 µm in G; A through F are in a same degree of magnification, so are G and H). KA, kainic acid; NS-ND, NS-ND, normal saline, normal diet; NS-KD, normal saline, ketogenic diet; KA-KD, kainic acid, ketogenic diet; CA3, cornu ammonis.