Temporal patterns of lipoperoxidation and antioxidant enzymes are modified in the hippocampus of vitamin A-deficient rats

Abstract

Animals can adapt their behavior to predictable temporal fluctuations in the environment through both, memory-and-learning processes and an endogenous time-keeping mechanism. Hippocampus plays a key role in memory and learning and is especially susceptible to oxidative stress. In compensation, antioxidant enzymes activity, such as Catalase (CAT) and Glutathione peroxidase (GPx), has been detected in this brain region. Daily rhythms of antioxidant enzymes activity, as well as of glutathione and lipid peroxides levels, have been described in brain. Here, we investigate day/night variations in lipoperoxidation, CAT, and GPx expression and activity, as well as the temporal fluctuations of two key components of the endogenous clock, BMAL1 and PER1, in the rat hippocampus and evaluate to which extent vitamin A deficiency may affect their amplitude or phase. Holtzman male rats from control, vitamin A-deficient, and vitamin A-refed groups were sacrificed throughout a 24-h period. Daily levels of clock proteins, lipoperoxidation, CAT and GPx mRNA, protein, and activity, were determined in the rat hippocampus obtained every 4 or 5 h. Gene expression of RARalpha and RXRbeta was also quantified in the hippocampus of the three groups of rats. Our results show significant daily variations of BMAL1 and PER1 protein expression. Rhythmic lipoperoxidation, CAT, and GPx, expression and activity, were also observed in the rat hippocampus. Vitamin A deficiency reduced RXRbeta mRNA level, as well as the amplitude of BMAL1 and PER1 daily oscillation, phase-shifted the daily peak of lipoperoxidation, and had a differential effect on the oscillating CAT and GPx mRNA, protein, and activity. Learning how vitamin A deficiency affects the circadian gene expression in the hippocampus may have an impact on the neurobiology, nutritional and chronobiology fields, emphasizing for the first time the importance of nutritional factors, such as dietary micronutrients, in the regulation of circadian parameters in this brain memory-and-learning-related region.

Figure 1. CAT and GPx mRNA levels in the hippocampus of control, vitamin A-deficient and vitamin A-refed rats

Bars represent mean ± standard error of four animals per group performed in duplicates. Statistical analysis was performed using one-way ANOVA followed by Tukey test. *P<0,02; **P<0,05.

Figure 2. Day-night cycles of lipoperoxidation in the hippocampus of control, vitamin A-deficient and vitamin A-refed rats

MDA was measured by the thiobarbituric acid method. Each value represents the mean ± SE of two pools of three left hippocampi each. Horizontal bars represent the distribution of light (open) and dark (closed) phases of the 24 h photoperiod. ZT is zeitgeber time, with ZT=0 when light is on. Statistical analysis was performed using one-way ANOVA followed by Tukey test with *P<0,05; **P<0,01 and ***P<0,001 when compared indicated means with the corresponding maximal value in each group.

Figure 3. Daily rhythms of CAT mRNA expression (A), protein levels (B) and enzymatic activity (C), in the hippocampus of control, vitamin A-deficient and vitamin A-refed rats

Each value on the curve represents the mean ± SE of three pools of two right hippocampi each (mRNA levels), or two pools of three left hippocampi each (protein levels and enzymatic activity), at a given ZT (with ZT=0 when light is on). Horizontal bars represent the distribution of light (open) and dark (closed) phases of the 24-h photoperiod. Statistical analysis was performed using one-way ANOVA followed by Tukey test with *P<0,05; **P<0,01 and ***P<0,001 when indicated means were compared to the corresponding maximal value in each group.

Figure 3. Daily rhythms of CAT mRNA expression (A), protein levels (B) and enzymatic activity (C), in the hippocampus of control, vitamin A-deficient and vitamin A-refed rats

Each value on the curve represents the mean ± SE of three pools of two right hippocampi each (mRNA levels), or two pools of three left hippocampi each (protein levels and enzymatic activity), at a given ZT (with ZT=0 when light is on). Horizontal bars represent the distribution of light (open) and dark (closed) phases of the 24-h photoperiod. Statistical analysis was performed using one-way ANOVA followed by Tukey test with *P<0,05; **P<0,01 and ***P<0,001 when indicated means were compared to the corresponding maximal value in each group.

Figure 3. Daily rhythms of CAT mRNA expression (A), protein levels (B) and enzymatic activity (C), in the hippocampus of control, vitamin A-deficient and vitamin A-refed rats

Each value on the curve represents the mean ± SE of three pools of two right hippocampi each (mRNA levels), or two pools of three left hippocampi each (protein levels and enzymatic activity), at a given ZT (with ZT=0 when light is on). Horizontal bars represent the distribution of light (open) and dark (closed) phases of the 24-h photoperiod. Statistical analysis was performed using one-way ANOVA followed by Tukey test with *P<0,05; **P<0,01 and ***P<0,001 when indicated means were compared to the corresponding maximal value in each group.

Figure 4. Daily rhythms of GPx mRNA expression (A), protein levels (B), and enzymatic activity (C), in the hippocampus of control, vitamin A-deficient and vitamin A-refed rats

Each value on the curve represents the mean ± SE of three pools of two right hippocampi each (mRNA levels), or two pools of three left hippocampi each (protein levels and enzymatic activity), at a given ZT (with ZT=0 when light is on). Horizontal bars represent the distribution of light (open) and dark (closed) phases of the 24-h photoperiod. Statistical analysis was performed using one-way ANOVA followed by Tukey test with *P<0,05; **P<0,01 and ***P<0,001 when indicated means were compared to the corresponding maximal value in each group.

Figure 4. Daily rhythms of GPx mRNA expression (A), protein levels (B), and enzymatic activity (C), in the hippocampus of control, vitamin A-deficient and vitamin A-refed rats

Each value on the curve represents the mean ± SE of three pools of two right hippocampi each (mRNA levels), or two pools of three left hippocampi each (protein levels and enzymatic activity), at a given ZT (with ZT=0 when light is on). Horizontal bars represent the distribution of light (open) and dark (closed) phases of the 24-h photoperiod. Statistical analysis was performed using one-way ANOVA followed by Tukey test with *P<0,05; **P<0,01 and ***P<0,001 when indicated means were compared to the corresponding maximal value in each group.

Figure 4. Daily rhythms of GPx mRNA expression (A), protein levels (B), and enzymatic activity (C), in the hippocampus of control, vitamin A-deficient and vitamin A-refed rats

Each value on the curve represents the mean ± SE of three pools of two right hippocampi each (mRNA levels), or two pools of three left hippocampi each (protein levels and enzymatic activity), at a given ZT (with ZT=0 when light is on). Horizontal bars represent the distribution of light (open) and dark (closed) phases of the 24-h photoperiod. Statistical analysis was performed using one-way ANOVA followed by Tukey test with *P<0,05; **P<0,01 and ***P<0,001 when indicated means were compared to the corresponding maximal value in each group.

Figure 5. Schematic representation of RARE, RXRE and E-box sites on the 5′ regulatory region of CAT and GPx genes

The accession # for the sequences taken from the PubMed database are: CAT (Acc#: AH004967) and GPx (Acc#: AB004231). Arrows indicate the first translation codon, gray boxes represent exons, white circles are RAREsites, dashed circles RXREs, black ovals perfect E-box (CACGTG) sites and white ovals are E-box-like (CATATG, CATGTG or CACTTG) sites. Grey oval on rGPx promoter represents a RORE site. Negative (−) numbers indicate regulatory sites positions relative to the start of translation (+1).

Figure 6. Transcript levels of RARα and RXRβ in the hippocampus of control, vitamin A-deficient and vitamin A-refed rats

mRNA levels were determined by Real Time PCR and normalized to β-actin. Each bar represents the mean ± SE of 4 samples in triplicates with *P<0,05 and ***P<0,001 in comparison to control.

Figure 7. Daily rhythms of BMAL1 and PER2 protein levels in the hippocampus of control, vitamin A-deficient and vitamin A-refed rats

A) Densitometric quantitation of the Immunoblots representative data. Each value on the curve represents the percentage (%) of maximal protein expression at a given ZT, with ZT=0 when light is on. Horizontal bars represent the distribution of light (open) and dark (closed) phases of the 24-h photoperiod. B) Immunoblot analysis of protein extracted from control, vitamin A-deficient, and vitamin A-refed rat hippocampi obtained at ZT2, ZT6, ZT10, ZT14, ZT18 and ZT22.

Figure 7. Daily rhythms of BMAL1 and PER2 protein levels in the hippocampus of control, vitamin A-deficient and vitamin A-refed rats

A) Densitometric quantitation of the Immunoblots representative data. Each value on the curve represents the percentage (%) of maximal protein expression at a given ZT, with ZT=0 when light is on. Horizontal bars represent the distribution of light (open) and dark (closed) phases of the 24-h photoperiod. B) Immunoblot analysis of protein extracted from control, vitamin A-deficient, and vitamin A-refed rat hippocampi obtained at ZT2, ZT6, ZT10, ZT14, ZT18 and ZT22.