Clock Regulation of Metabolites Reveals Coupling between Transcription and Metabolism

Abstract

Summary The intricate connection between the circadian clock and metabolism remains poorly understood. We used high temporal resolution metabolite profiling to explore clock regulation of mouse liver and cell autonomous metabolism. In liver, ~50% of metabolites were circadian, with enrichment of nucleotide, amino acid, and methylation pathways. In U2 OS cells, 28% were circadian, including amino acids and NAD biosynthesis metabolites. Eighteen metabolites oscillated in both systems and a subset of these in primary hepatocytes. These 18 metabolites were enriched in methylation and amino acid pathways. To assess clock-dependence of these rhythms, we used genetic perturbation. BMAL1 knockdown diminished metabolite rhythms, while CRY1 or CRY2 perturbation generally shortened/lengthened rhythms, respectively. Surprisingly, CRY1 knockdown induced 8 h rhythms in amino acid, methylation, and vitamin metabolites, decoupling metabolite from transcriptional rhythms, with potential impact on nutrient sensing in vivo. These results provide the first comprehensive views of circadian liver and cell autonomous metabolism.

Extraction and Analysis of Pan-metabolome Polar Metabolites by Ultra Performance Liquid Chromatography–Tandem Mass Spectrometry (UPLC-MS/MS)

“…E.g., 1 × 10 6 U2OS cells were utilized in Krishnaiah et al, 2017.If extracting tissue, 2 ml safe-lock microfuge tubes should be utilized.…”

“…Thus, the COLMeD approach aimed to address this challenge and enabled a more comprehensive metabolite analysis across platforms including on a triple quadrupole mass spectrometer (Rhoades and Weljie, 2016). The LC-MS workflow on a triple quadrupole is described here and has been used to successfully study metabolomics in a circadian context in which 179 metabolites were successfully profiled and analyzed (Krishnaiah et al ., 2017). …”

“…The protocol described herein describes this workflow using targeted polar metabolite multiple reaction monitoring (MRM) from tissues and biofluids via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). This workflow has been utilized extensively for chronometabolic analysis (Krishnaiah et al ., 2017), with applications generalized to other types of analyses as well (Sengupta et al ., 2017; Sivanand et al ., 2017). …”

“…The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The above-described protocol has been adapted from (Rhoades and Weljie, 2016) and (Krishnaiah et al ., 2017). …”

“… Notes The above described extraction protocol has been utilized in the analysis of cell pellets (Krishnaiah et al ., 2017), liver (Krishnaiah et al ., 2017), whole Drosophila (Maguire et al ., 2015), and brain tissues (McGrath et al ., 2008) as well as other sample types such as plasma (Banoei et al ., 2017), serum (Beaudry et al ., 2016 and Hao et al ., 2016), cereberal spinal fluid biofluids, yeast (Tambellini et al ., 2013 and 2017) etc . We also strongly recommend that any results be validated by inclusion of a stable-isotope labeled internal standard prior to extraction or unlabeled standard analyzed in a similar matrix to confirm retention times and fragmentation patterns.…”