Circadian regulation of glutathione levels and biosynthesis in Drosophila melanogaster

Abstract

Circadian clocks generate daily rhythms in neuronal, physiological, and metabolic functions. Previous studies in mammals reported daily fluctuations in levels of the major endogenous antioxidant, glutathione (GSH), but the molecular mechanisms that govern such fluctuations remained unknown. To address this question, we used the model species Drosophila, which has a rich arsenal of genetic tools. Previously, we showed that loss of the circadian clock increased oxidative damage and caused neurodegenerative changes in the brain, while enhanced GSH production in neuronal tissue conferred beneficial effects on fly survivorship under normal and stress conditions. In the current study we report that the GSH concentrations in fly heads fluctuate in a circadian clock-dependent manner. We further demonstrate a rhythm in activity of glutamate cysteine ligase (GCL), the rate-limiting enzyme in glutathione biosynthesis. Significant rhythms were also observed for mRNA levels of genes encoding the catalytic (Gclc) and modulatory (Gclm) subunits comprising the GCL holoenzyme. Furthermore, we found that the expression of a glutathione S-transferase, GstD1, which utilizes GSH in cellular detoxification, significantly fluctuated during the circadian day. To directly address the role of the clock in regulating GSH-related rhythms, the expression levels of the GCL subunits and GstD1, as well as GCL activity and GSH production were evaluated in flies with a null mutation in the clock genes cycle and period. The rhythms observed in control flies were not evident in the clock mutants, thus linking glutathione production and utilization to the circadian system. Together, these data suggest that the circadian system modulates pathways involved in production and utilization of glutathione.

Figure 1. Circadian regulation of GSH levels in Drosophila heads.

(A) Daily changes in GSH levels in wild type CS males. Data represents average values ± SEM obtained from 4 independent bio-replicates (total N = 8). Data were analyzed by a 1-way ANOVA and Bonferroni's post-tests where an asterisk marks significantly lower values relative to ZT 0 (p<0.05). White horizontal bar marks the time when light is on; black bar denotes darkness. (B) GSH levels were altered in per⁰¹ and cyc⁰¹ mutants such that no statistical difference was detected between time points where control CS flies showed a peak (ZT 0) and a trough (ZT 8). Bars represent average values ± SEM obtained from 3–4 independent bio-replicates (± SEM). Data in (B) were analyzed by a 2-way ANOVA and Bonferroni's post-tests. Different subscript letters indicate significant difference between treatment groups. ZT = Zeitgeber Time.

Figure 2. Circadian regulation of Gclc and Gclm mRNA expression levels in fly heads.

There is a significant rhythm in Gclc (A) and Gclm (B) mRNA but not in GS mRNA profile (C). Data for (A–C) were analyzed by a 1-way ANOVA and Bonferroni's post-tests, and an asterisk marks significantly higher values relative to the lowest value (p<0.05). In cyc⁰¹ mutants, the peak in Gclc (D) and Gclm (E) is abolished while GS mRNA is not affected (F). In per⁰¹ mutants, the trough-to-peak differences in Gclc (G) and Gclm (H) are abolished while GS is not changed (I). Different subscript letters in (D–I) indicate a significant difference between treatment groups. All graphs are average values obtained from 3–5 independent bio-replicates (± SEM) and normalized to ZT 0 or ZT 4 as appropriate.

Figure 3. Circadian expression of GCLc isoforms.

Daily oscillations in (A) total Gclc mRNA levels were also significant when (B) Gclc-RA and (C) Gclc-RB isoforms were measured separately using isoform-specific primers. The two isoforms share the same coding regions, but have distinct 5′ UTR regions. All graphs are average values obtained from 3 independent bio-repeats each normalized to the time point with the lowest expression. An asterisk indicates significant difference from the trough based on a 1-way ANOVA and Bonferroni's post-tests (p<0.05).

Figure 4. Circadian rhythm in Gclm expression persists in constant darkness.

(A) tim and (B) Gclm mRNA expression show a circadian rhythm in heads of CS flies on the second day of constant darkness. An asterisk indicates a significant difference in the expression level between the trough of each gene and the peak (p<0.05). (C) No significant rhythm was detected in Gclc mRNA levels in wild type flies. Data represents average values obtained from 3 independent bio-replicates (± SEM) and normalized to ZT 0. Significance was calculated by a 1-way ANOVA and Bonferroni's multiple comparison post-tests. CT = Circadian Time. Shaded horizontal bars indicate subjective day.

Figure 5. Profiles of GCL proteins and their ratio over the circadian day in the heads of wild type CS males.

(A) GCLm and (B) GCLc protein levels based on average densitometry of signals obtained on Western blots with anti-GCLc or anti-GCLm antibodies normalized to signals obtained with anti-actin antibodies. Each replicate was normalized to the time point with the lowest expression. (C) Ratio of GCLc to GCLm protein over the circadian day in wild type CS males. (A–C) Data represent average values ± SEM obtained from 8 immunoblots performed with 4 independent bio-replicates. Statistical significance was determined by a 1-way ANOVA and Dunnett's post-test as denoted by asterisks (p<0.05).

Figure 6. Circadian regulation of GCL enzymatic activity.

(A) Daily profile of GCL activity in heads of CS flies as measured by the formation of the GCL product, γ-GC. Data represents average values ± SEM obtained from 4 independent bio-replicates (total N = 16). An asterisk indicates a significant difference between the peak and trough time points calculated by 1-way ANOVA and Bonferroni post-tests. (B) GCL activity was altered in per⁰¹ and cyc⁰¹ mutants such that no statistical difference was detected between time points where control CS flies showed peak at (ZT 0) and trough (ZT 8). Bars show average values ± SEM obtained from 4–5 independent bio-replicates (total N = 16). Data in (B) are analyzed by 2-way ANOVA and Bonferroni's post-tests. Different subscript letters indicate significant differences between treatment groups (p<0.05).

Figure 7. Circadian regulation of GstD1 expression.

(A) A circadian rhythm in GstD1 mRNA levels was detected in wild type (CS) flies with a peak at ZT 8 significantly different from the trough at ZT 20 (p<0.01). (B) No significant difference was observed between ZT 8 and ZT 20 in per⁰¹ and cyc⁰¹ flies while the difference was observed in CS heads (p<0.01). Data represent average values (± SEM) obtained from 3 independent bio-replicates and normalized to ZT 0. Data were analyzed by a 2-way ANOVA and Bonferroni's post-tests. Different subscript letters indicate significant difference between treatment groups.