Cell-autonomous circadian clock of hepatocytes drives rhythms in transcription and polyamine synthesis

Abstract

The circadian clock generates daily rhythms in mammalian liver processes, such as glucose and lipid homeostasis, xenobiotic metabolism, and regeneration. The mechanisms governing these rhythms are not well understood, particularly the distinct contributions of the cell-autonomous clock and central pacemaker to rhythmic liver physiology. Through microarray expression profiling in Met murine hepatocytes (MMH)-D3, we identified over 1,000 transcripts that exhibit circadian oscillations, demonstrating that the cell-autonomous clock can drive many rhythms, and that MMH-D3 is a valid circadian model system. The genes represented by these circadian transcripts displayed both cophasic and antiphasic organization within a protein-protein interaction network, suggesting the existence of competition for binding sites or partners by genes of disparate transcriptional phases. Multiple pathways displayed enrichment in MMH-D3 circadian transcripts, including the polyamine synthesis module of the glutathione metabolic pathway. The polyamine synthesis module, which is highly associated with cell proliferation and whose products are required for initiation of liver regeneration, includes enzymes whose transcripts exhibit circadian oscillations, such as ornithine decarboxylase and spermidine synthase. Metabolic profiling revealed that the enzymatic product of spermidine synthase, spermidine, cycles as well. Thus, the cell-autonomous hepatocyte clock can drive a significant amount of transcriptional rhythms and orchestrate physiologically relevant modules such as polyamine synthesis.

Fig. 1.

Bioinformatics pipeline combines two analytic methods to increase circadian transcript yield. (A) Diagram depicting the pipeline component analytic methods: signal decomposition and model-matching algorithms. (B) Circadian transcripts identified by each analytic method (model-matching: blue, signal decomposition: red) and by the pipeline (purple) at decreasing resolution.

Fig. 2.

MMH-D3 displays circadian rhythms of transcription. (A) MMH-D3 circadian transcript calls for each arm of pipeline. (B–F) qPCR (blue) and microarray transcript (red and pink) for indicated clock genes. Error bars on qPCR traces represent SD of replicates (n = 3).

Fig. 3.

Circadian genes display co- and antiphasic organization within the mouse PPI network. (A) Discreet distributions of shortest path lengths of circadian and noncircadian nodes. (B) Mean resistance distances for all pairs of circadian genes within the largest connected component of the mouse PPI network, binned by phase difference. Error bars represent the 95% confidence interval around the estimates of the mean resistance distance for each bin. (C) Modules identified by the MATISSE algorithm (inside circle), which finds modules enriched for cophasic pairs, generally exhibit a biphasic pattern, as does the general distribution of phases across all circadian genes in the network (outer star chart). Colors represent the 0–23 (h) circadian phases. Circled annotation groups reflect enriched DAVID functional annotation clusters identified for the 1,130 MMH-D3 circadian transcripts. A larger version of these modules can be found in Fig. S5. (D) Specific cophasic module exhibiting the general biphasic pattern. The starred gene (*) Per2 was also found to be cycling in Kornmann et al. (5), where only systemic circadian signals were present.

Fig. 4.

Circadian rhythms of polyamine synthesis in both transcription and enzymatic activity. (A) Polyamine biosynthesis pathway. Ornithine is converted to putrescine (the first polyamine) by ornithine decarboxylase (ODC1). Putrescine is then converted to spermidine with the addition of decarboxylated S-adenosyl methionine (dcAdoMet) by spermidine synthase (SRM). Spermidine is then converted to spermine by spermine synthase (SMS). (B and C) Odc1 and Srm display circadian rhythms in MMH-D3 hepatocytes. (D) Spermidine displays circadian rhythmicity in MMH-D3 hepatocytes. Data points represent mean values for biological replicates (n = 3, except at hour 46, where n = 2) and error bars their SD.