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Effect of circadian rhythm on brain NAD: an MRS study at 7 T1Center for Biomedical Imaging, EPFL, Ecublens, Switzerland, 2Translation Research, Nestlé Health Science, Lausanne, Switzerland, 3Clinical Research Unit, Nestlé Research and Development, Lausanne, Switzerland
Synopsis
Our understanding of circadian rhythm has expanded to provide molecular insights into physiology and disease since the discovery of circadian clock.Pre-clinical experiments have shown that circadian rhythm is linked with the Nicotinamid Adenine Dinucleotide (NAD) levels and the redox ratio. However, clinical data is still lacking. In this study, we aim to study the effect of circadian rhythm on NAD levels in the human brain. By measuring the NAD levels in the occipital lobe with 31P-MRS, preliminary interim results suggest a negative correlation trend between cortisol level and total NAD level.
Introduction
The circadian rhythm (CIR) plays a vital role in regulating cellular, physiological and behavioral processes in health and disease. It was found that mammalian CIR are coordinated with metabolic activity through controlled expression of Nicotinamid Phosphoribosyltransferase (NAMPT)1. Regulation of NAMPT could result in oscillating NAD+, an important metabolite involved in bioenergetics and co-substrates for enzymes modulating cellular signaling processes2. The rhythmic oscillation of NAD+ could serve as a feedback timer by modulating the activities of NAD+-dependent enzymes, helping to establish the periodicity of the cycles3-4. Pre-clinical experiments have shown that CIR is directly linked with the NAD levels and the NAD redox ratio5. For example, mice liver data showed that level of NAD+ displayed circadian oscillations3, human ex-vivo data also showed that NAD oscillates over time in red blood cell6. However, no clinical study has been reported in human on the effect of the CIR on brain NAD levels. With the development of 31P-MRS at high magnetic field, NAD+ and NADH in human brain can be measured7, enabling the monitor of NAD level and the redox ratio changes.To explore whether and how CIR could influence the brain NAD level in human, we conducted a MRS study at 7T, where brain energy related metabolites were assessed at two different states during the day. Besides, cortisol level is tested to confirm that the experiment is done in different circadian conditions; brain lactate level is measured since lactate is an important energetic metabolite and that a 24h rhythm of lactate was observed in the somatosensory cortex with animals8. Moreover, Balloon Analogue Risk Test (BART)9 is implemented to explore possible associations between brain energetic status and reward behavior.
Methods
From the power analysis based on a previous study10, 25 subjects are planned to detect a similar effect size as observed in the red blood cells and mice livers3, 6. Considering the effects of age and gender on circadian function11, 25 male volunteers aging between 18 and 40 years old were recruited and 14 of them have been tested. Before the experiment, the participants are required to record their sleeping diary for one week to ensure regular sleeping habits. The experiment is implemented in fasted morning condition (8 am) and later afternoon condition (3 pm) during a day. For each session, the saliva sample of the volunteer is collected and analyzed for the cortisol level. Then, MR experiments are performed on a 7T/68cm MR scanner (Siemens Medical Solutions, Erlangen, Germany) with a 1H surface coil and a single-loop 31P coil (7cm-diameter) for the occipital lobe. 31P-MRS is performed by a pulse-acquire sequence (TR=3s, 320 averages) to measure 31P metabolites including NADH and NAD+. 1H-MRS is acquired by a short-TE STEAM sequence (TE/TM/TR=4.5/25/5500ms, 64 averages, voxel size = 35x20x25mm3) for neurochemical profiling including lactate. BART (Inquisit Lab) is implemented as a measure of individual risk-taking propensity. After the AM session, the participants are provided with one nutritional drink as breakfast (360kCal) for immediate consumption and 2 other drinks (720kCal) as lunch consumed at 12 pm; the participants are not allowed to consume other food or beverage until the PM session.All MR spectra were analyzed by LCModel. 31P metabolite concentrations were calculated assuming [γ-ATP] of 3mM. Unsuppressed water spectra were used for the quantification of 1H metabolites. Statistical analysis was done using paired t-test and Spearman correlation.
Results
The preliminary interim data analysis with 14 subjects showed that the cortisol level was significantly higher in the morning, confirming that the experiment was done in different circadian conditions; the BART performance was significantly higher in the afternoon in which is coherent with previous research12 (table 1). Besides, it was found that total NAD was negatively correlated with the cortisol level (Figure 1).Discussion and conclusion
This is the first study to explore the relationship between CIR and brain metabolites such as NAD and lactate in human. The results of the project could contribute to a greater molecular understanding of the CIR in human brain and open therapeutic perspectives for diseases. Preliminary interim analysis showed a negative correlation trend between cortisol level and brain total NAD level. This preliminary result motivates further investigation upon the completion of the full study.Acknowledgements
This work was supported by Nestle Health Science. We acknowledge access to the facilities and expertise of the CIBM Center for Biomedical Imaging, a Swiss research center of excellence founded and supported by Lausanne University Hospital (CHUV), University of Lausanne (UNIL), Ecole polytechnique fédérale de Lausanne (EPFL), University of Geneva (UNIGE) and Geneva University Hospitals (HUG).References
1. Cambronne XA, Kraus WL. Location, Location, Location: Compartmentalization of NAD+ Synthesis and Functions in Mammalian Cells. Trends Biochem Sci. 2020 Oct;45(10):858-873. doi: 10.1016/j.tibs.2020.05.010. Epub 2020 Jun 25. PMID: 32595066; PMCID: PMC7502477.
2. Yang, Yue, and Anthony A. Sauve. "NAD+ metabolism: Bioenergetics, signaling and manipulation for therapy." Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 1864.12 (2016): 1787-1800. 3. Ramsey KM, Yoshino J, Brace CS, Abrassart D, Kobayashi Y, Marcheva B, Hong HK, Chong JL, Buhr ED, Lee C, Takahashi JS, Imai S, Bass J. Circadian clock feedback cycle through NAMPT-mediated NAD+ biosynthesis. Science. 2009 May 1;324(5927):651-4. doi: 10.1126/science.1171641. Epub 2009 Mar 19. PMID: 19299583; PMCID: PMC2738420.
4. Nakahata Y, Sahar S, Astarita G, Kaluzova M, Sassone-Corsi P. Circadian control of the NAD+ salvage pathway by CLOCK-SIRT1. Science. 2009 May 1;324(5927):654-7. doi: 10.1126/science.1170803. Epub 2009 Mar 12. PMID: 19286518; PMCID: PMC6501775.
5. Xie N, Zhang L, Gao W, Huang C, Huber PE, Zhou X, Li C, Shen G, Zou B. NAD+ metabolism: pathophysiologic mechanisms and therapeutic potential. Signal Transduct Target Ther. 2020 Oct 7;5(1):227. doi: 10.1038/s41392-020-00311-7. PMID: 33028824; PMCID: PMC7539288.
6. O'Neill JS, Reddy AB. Circadian clocks in human red blood cells. Nature. 2011 Jan 27;469(7331):498-503. doi: 10.1038/nature09702. PMID: 21270888; PMCID: PMC3040566.
7. Zhu XH, Lu M, Lee BY, Ugurbil K, Chen W. In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences. Proc Natl Acad Sci U S A. 2015 Mar 3;112(9):2876-81. doi: 10.1073/pnas.1417921112. Epub 2015 Feb 17. PMID: 25730862; PMCID: PMC4352772.
8. Shram N, Netchiporouk L, Cespuglio R. Lactate in the brain of the freely moving rat: voltammetric monitoring of the changes related to the sleep-wake states. Eur J Neurosci. 2002 Aug;16(3):461-6. doi: 10.1046/j.1460-9568.2002.02081.x. PMID: 12193189.
9. Lejuez, Carl W., et al. "Evaluation of a behavioral measure of risk taking: the Balloon Analogue Risk Task (BART)." Journal of Experimental Psychology: Applied 8.2 (2002): 75.
10. Xin L, Ipek Ö, Beaumont M, Shevlyakova M, Christinat N, Masoodi M, Greenberg N, Gruetter R, Cuenoud B. Nutritional Ketosis Increases NAD+/NADH Ratio in Healthy Human Brain: An in Vivo Study by 31P-MRS. Front Nutr. 2018 Jul 12;5:62. doi: 10.3389/fnut.2018.00062. PMID: 30050907; PMCID: PMC6052097.
11. Van Cauter E, Leproult R, Kupfer DJ. Effects of gender and age on the levels and circadian rhythmicity of plasma cortisol. J Clin Endocrinol Metab. 1996 Jul;81(7):2468-73. doi: 10.1210/jcem.81.7.8675562. PMID: 8675562.
12. Li M, Mai Z, Yang J, Zhang B, Ma N. Ideal Time of Day for Risky Decision Making: Evidence from the Balloon Analogue Risk Task. Nat Sci Sleep. 2020 Jul 16;12:477-486. doi: 10.2147/NSS.S260321. PMID: 32765144; PMCID: PMC7381795.
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