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.
References
[1]
Albrecht U (2006) Orchestration of gene expression and physiology by the circadian clock. J Physiol Paris 100: 243–251.
Reppert SM, Weaver DR (2000) Comparing clockworks: mouse versus fly. J Biol Rhythms 15: 357–364.
[4]
Stanewsky R (2003) Genetic analysis of the circadian system in Drosophila melanogaster and mammals. J Neurobiol 54: 111–147.
[5]
Hardin PE (2011) Molecular genetic analysis of circadian timekeeping in Drosophila. Adv Genet 74: 141–173.
[6]
Allada R, Chung BY (2010) Circadian organization of behavior and physiology in Drosophila. Annual Review of Physiology 72: 605–624.
[7]
Abruzzi KC, Rodriguez J, Menet JS, Desrochers J, Zadina A, et al. (2011) Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression. Genes Dev 25: 2374–2386.
[8]
McDonald MJ, Rosbash M (2001) Microarray analysis and organization of circadian gene expression in Drosophila. Cell 107: 567–578.
[9]
Claudel T, Cretenet G, Saumet A, Gachon F (2007) Crosstalk between xenobiotics metabolism and circadian clock. FEBS Lett 581: 3626–3633.
[10]
Green CB, Takahashi JS, Bass J (2008) The meter of metabolism. Cell 134: 728–742.
[11]
Kang TH, Reardon JT, Kemp M, Sancar A (2009) Circadian oscillation of nucleotide excision repair in mammalian brain. Proc Natl Acad Sci U S A 106: 2864–2867.
[12]
Beaver LM, Hooven LA, Butcher SM, Krishnan N, Sherman KA, et al. (2010) Circadian clock regulates response to pesticides in Drosophila via conserved Pdp1 pathway. Toxicol Sci 115: 513–520.
[13]
Hooven LA, Sherman KA, Butcher S, Giebultowicz JM (2009) Does the clock make the poison? Circadian variation in response to pesticides. PLoS One 4: e6469.
[14]
Xu K, Zheng X, Sehgal A (2008) Regulation of feeding and metabolism by neuronal and peripheral clocks in Drosophila. Cell Metab 8: 289–300.
[15]
Krishnan N, Davis AJ, Giebultowicz JM (2008) Circadian regulation of response to oxidative stress in Drosophila melanogaster. Biochem Biophys Res Commun 374: 299–303.
[16]
Krishnan N, Kretzschmar D, Rakshit K, Chow E, Giebultowicz J (2009) The circadian clock gene period extends healthspan in aging Drosophila melanogaster. Aging 1: 937–948.
[17]
Krishnan N, Rakshit K, Chow ES, Wentzell JS, Kretzschmar D, et al. (2012) Loss of circadian clock accelerates aging in neurodegeneration-prone mutants. Neurobiol Dis 45: 1129–1135.
[18]
Kondratov RV, Kondratova AA, Gorbacheva VY, Vykhovanets OV, Antoch MP (2006) Early aging and age-related pathologies in mice deficient in BMAL1, the core component of the circadian clock. Genes Dev 20: 1868–1873.
[19]
Lu SC (2009) Regulation of glutathione synthesis. Mol Aspects Med 30: 42–59.
[20]
Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, et al. (2007) Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39: 44–84.
[21]
Hardeland R, Coto-Montes A, Poeggeler B (2003) Circadian rhythms, oxidative stress, and antioxidative defense mechanisms. Chronobiol Int 20: 921–962.
[22]
Ceriani MF, Hogenesch JB, Yanovsky M, Panda S, Straume M, et al. (2002) Genome-wide expression analysis in Drosophila reveals genes controlling circadian behavior. J Neurosci 22: 9305–9319.
[23]
Lin Y, Han M, Shimada B, Wang L, Gibler TM, et al. (2002) Influence of the period-dependent circadian clock on diurnal, circadian, and aperiodic gene expression in Drosophila melanogaster. Proc Natl Acad Sci U S A 99: 9562–9567.
[24]
Claridge-Chang A, Wijnen H, Naef F, Boothroyd C, Rajewsky N, et al. (2001) Circadian regulation of gene expression systems in the Drosophila head. Neuron 32: 657–671.
[25]
Hughes ME, Grant GR, Paquin C, Qian J, Nitabach MN (2012) Deep sequencing the circadian and diurnal transcriptome of Drosophila brain. Genome Res 22: 1266–1281.
[26]
Rutila JE, Suri V, Le M, So V, Rosbash M, et al. (1998) CYCLE is a second bHLH-PAS Clock protein essential for circadian rhythmicity and transcription of Drosophila period and timeless. Cell 93: 805–814.
[27]
Konopka RJ, Benzer S (1971) Clock mutants of Drosophila melanogaster. Proc Natl Acad Sci USA 68: 2112–2116.
[28]
Ling D, Salvaterra PM (2011) Robust RT-qPCR data normalization: validation and selection of internal reference genes during post-experimental data analysis. PLoS One 6: e17762.
[29]
Orr WC, Radyuk SN, Prabhudesai L, Toroser D, Benes JJ, et al. (2005) Overexpression of glutamate-cysteine ligase extends life span in Drosophila melanogaster. J Biol Chem 280: 37331–37338.
[30]
Rebrin I, Bayne AC, Mockett RJ, Orr WC, Sohal RS (2004) Free aminothiols, glutathione redox state and protein mixed disulphides in aging Drosophila melanogaster. Biochem J 382: 131–136.
[31]
Toroser D, Sohal RS (2005) Kinetic characteristics of native gamma-glutamylcysteine ligase in the aging housefly, Musca domestica L. Biochem Biophys Res Commun. 326: 586–593.
[32]
Chintapalli VR, Wang J, Dow JA (2007) Using FlyAtlas to identify better Drosophila melanogaster models of human disease. Nat Genet 39: 715–720.
[33]
Daines B, Wang H, Wang L, Li Y, Han Y, et al. (2011) The Drosophila melanogaster transcriptome by paired-end RNA sequencing. Genome Res 21: 315–324.
[34]
Luchak JM, Prabhudesai L, Sohal RS, Radyuk SN, Orr WC (2007) Modulating longevity in Drosophila by over- and underexpression of glutamate-cysteine ligase. Ann N Y Acad Sci 1119: 260–273.
[35]
Fraser JA, Kansagra P, Kotecki C, Saunders RD, McLellan LI (2003) The modifier subunit of Drosophila glutamate-cysteine ligase regulates catalytic activity by covalent and noncovalent interactions and influences glutathione homeostasis in vivo. J Biol Chem 278: 46369–46377.
[36]
Chen Y, Shertzer HG, Schneider SN, Nebert DW, Dalton TP (2005) Glutamate cysteine ligase catalysis: dependence on ATP and modifier subunit for regulation of tissue glutathione levels. J Biol Chem 280: 33766–33774.
[37]
Lee JI, Kang J, Stipanuk MH (2006) Differential regulation of glutamate-cysteine ligase subunit expression and increased holoenzyme formation in response to cysteine deprivation. Biochem J 393: 181–190.
[38]
Sawicki R, Singh SP, Mondal AK, Benes H, Zimniak P (2003) Cloning, expression and biochemical characterization of one Epsilon-class (GST-3) and ten Delta-class (GST-1) glutathione S-transferases from Drosophila melanogaster, and identification of additional nine members of the Epsilon class. Biochem J 370: 661–669.
[39]
Sykiotis GP, Bohmann D (2008) Keap1/Nrf2 signaling regulates oxidative stress tolerance and lifespan in Drosophila. Dev Cell 14: 76–85.
[40]
Wijnen H, Young MW (2006) Interplay of circadian clocks and metabolic rhythms. Annu Rev Genet 40: 409–448.
[41]
Sahar S, Sassone-Corsi P (2012) Regulation of metabolism: the circadian clock dictates the time. Trends Endocrinol Metab 23: 1–8.
[42]
Eckel-Mahan KL, Patel VR, Mohney RP, Vignola KS, Baldi P, et al. (2012) Coordination of the transcriptome and metabolome by the circadian clock. Proc Natl Acad Sci U S A 109: 5541–5546.
[43]
Dickinson DA, Levonen AL, Moellering DR, Arnold EK, Zhang H, et al. (2004) Human glutamate cysteine ligase gene regulation through the electrophile response element. Free Radic Biol Med 37: 1152–1159.
[44]
Suh JH, Shenvi SV, Dixon BM, Liu H, Jaiswal AK, et al. (2004) Decline in transcriptional activity of Nrf2 causes age-related loss of glutathione synthesis, which is reversible with lipoic acid. Proc Natl Acad Sci U S A 101: 3381–3386.
[45]
Fraser JA, Saunders RD, McLellan LI (2002) Drosophila melanogaster glutamate-cysteine ligase activity is regulated by a modifier subunit with a mechanism of action similar to that of the mammalian form. J Biol Chem 277: 1158–1165.
[46]
Limón-Pacheco JH, Gonsebatt ME (2010) The Glutathione System and its Regulation by Neurohormone Melatonin in the Central Nervous System. Cent Nerv Syst Agents Med Chem 10(4): 287–297.
[47]
Dringen R, Hirrlinger J (2003) Glutathione pathways in the brain. Biol Chem 384: 505–516.
[48]
Dickinson DA, Forman HJ (2002) Glutathione in defense and signaling: lessons from a small thiol. Ann N Y Acad Sci 973: 488–504.
[49]
Maher P (2006) Redox control of neural function: background, mechanisms, and significance. Antioxid Redox Signal 8: 1941–1970.
[50]
Forman HJ, Zhang H, Rinna A (2009) Glutathione: overview of its protective roles, measurement, and biosynthesis. Mol Aspects Med 30: 1–12.
[51]
Edgar RS, Green EW, Zhao Y, van Ooijen G, Olmedo M, et al. (2012) Peroxiredoxins are conserved markers of circadian rhythms. Nature 485: 459–464.
[52]
Low WY, Ng HL, Morton CJ, Parker MW, Batterham P, et al. (2007) Molecular evolution of glutathione S-transferases in the genus Drosophila. Genetics 177: 1363–1375.
[53]
Matzkin LM (2008) The molecular basis of host adaptation in cactophilic Drosophila: molecular evolution of a glutathione S-transferase gene (GstD1) in Drosophila mojavensis. Genetics 178: 1073–1083.
[54]
Hochmuth CE, Biteau B, Bohmann D, Jasper H (2011) Redox regulation by Keap1 and Nrf2 controls intestinal stem cell proliferation in Drosophila. Cell Stem Cell 8: 188–199.
[55]
Wijnen H, Naef F, Boothroyd C, Claridge-Chang A, Young MW (2006) Control of daily transcript oscillations in Drosophila by light and the circadian clock. PLoS Genet 2: e39.
[56]
Keegan KP, Pradhan S, Wang JP, Allada R (2007) Meta-analysis of Drosophila circadian microarray studies identifies a novel set of rhythmically expressed genes. PLoS Computational Biology 3: e208.
[57]
Taghert PH, Shafer OT (2006) Mechanisms of clock output in the Drosophila circadian pacemaker system. J Biol Rhythms 21: 445–457.
[58]
Jackson FR (2011) Glial cell modulation of circadian rhythms. Glia 59: 1341–1350.
[59]
Kula-Eversole E, Nagoshi E, Shang Y, Rodriguez J, Allada R, et al. (2009) Surprising gene expression patterns within and between PDF-containing circadian neurons in Drosophila. Proc Natl Acad Sci U S A 107: 13497–13502.
[60]
Radyuk SN, Rebrin I, Luchak JM, Michalak K, Klichko VI, et al. (2009) The catalytic subunit of Drosophila glutamate-cysteine ligase is a nucleocytoplasmic shuttling protein. J Biol Chem 284: 2266–2274.
[61]
Radyuk SN, Gambini J, Borras C, Serna E, Klichko VI, et al. (2012) Age-dependent changes in the transcription profile of long-lived Drosophila over-expressing glutamate cysteine ligase. Mech Ageing Dev 133: 401–413.
[62]
Gachon F, Firsov D (2011) The role of circadian timing system on drug metabolism and detoxification. Expert Opin Drug Metab Toxicol 7: 147–158.
[63]
Xu K, DiAngelo JR, Hughes ME, Hogenesch JB, Sehgal A (2011) The circadian clock interacts with metabolic physiology to influence reproductive fitness. Cell Metab 13: 639–654.
