Differential Patterns in the Periodicity and Dynamics of Clock Gene Expression in Mouse Liver and Stomach

Abstract

The rhythmic recurrence of biological processes is driven by the functioning of cellular circadian clocks, operated by a set of genes and proteins that generate self-sustaining transcriptional-translational feedback loops with a free-running period of about 24 h. In the gastrointestinal apparatus, the functioning of the biological clocks shows distinct patterns in the different organs. The aim of this study was to evaluate the time-related variation of clock gene expression in mouse liver and stomach, two components of the digestive system sharing vascular and autonomic supply, but performing completely different functions. The authors analyzed the periodicity by cosinor analysis and the dynamics of variation by computing the fractional variation to assess the rate of change in gene expression. Five-week-old male Balb/c mice were exposed to 2 wks of 12-h light/12-h dark cycles, then kept in complete darkness for 3 d as a continuation of the dark span of the last light-dark cycle. The authors evaluated the expression of Bmal1, Clock, Cry1, Cry2, Per1, Per2, Per3, Rev-erbα, Rev-erbβ, Npas2, Timeless, Dbp, Csnk1d, and Csnk1e by using real-time quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) in mouse liver and stomach. A significant 24-h rhythmic component was found for 10 genes in the liver (Bmal1, Clock, Cry1, Per1, Per2, Per3, Rev-erbα, Rev-erbβ, Npas2, and Dbp), and for 9 genes in the stomach (Bmal1, Cry1, Per1, Per2, Per3, Rev-erbα, Rev-erbβ, Npas2, and Dbp). In particular, Clock showed marked rhythm differences between liver and stomach, putatively due to some compensation by Npas2. The acrophase of the original values of Bmal1, Per2, Per3, Rev-erbα, Rev-erbβ, Npas2, and Dbp expression was delayed in the stomach, and the average delay expressed as mean ± SD was 14.30 ± 7.94 degrees (57.20 ± 31.78 minutes). A statistically significant difference was found in the acrophases of Bmal1 (p = .015) and Npas2 (p = .011). Fractional variations provided significant circadian rhythms for nine genes in the liver (Bmal1, Per1, Per2, Per3, Rev-erbα, Rev-erbβ, Npas2, Timeless, and Dbp), and for seven genes in the stomach (Bmal1, Clock, Per2, Rev-erbα, Npas2, Dbp, and Csnk1e). The acrophase of the fractional variations of Bmal1, Per2, Per3, Rev-erbα, Rev-erbβ, and Dbp expression was delayed in the stomach, and the average delay expressed as mean ± SD was 19.10 ± 9.39 degrees (76.40 ± 37.59 minutes). A significantly greater fractional variation was found in the liver for Clock at 06:00 h (p = .034), Per1 at 02:00 h (p = .037), and Per3 at 02:00 h (p = .029), whereas the fractional variation was greater in the stomach for Clock at 10:00 h (p = .016), and for Npas2 at 02:00 h (p = .029) and at 06:00 h (p = .044). In conclusion, liver and stomach show different phasing and dynamics of clock gene expression, which are probably related to prevailing control by different driving cues, and allow them to keep going the various metabolic pathways and diverse functional processes that they manage. (Author correspondence: [email protected])

Keywords:

• Circadian rhythm
• Clock gene
• Liver
• Stomach

ACKNOWLEDGMENTS

We would like to thank Prof. Dr. Robert B. Sothern for his invaluable guidance.

Declaration of Interest: This work was supported by “Italian Ministry of Health” grant RC1203ME46 through the Department of Medical Sciences, Division of Internal Medicine and Chronobiology Unit, IRCCS Scientific Institute and Regional General Hospital “Casa Sollievo della Sofferenza,” Opera di Padre Pio da Pietrelcina, San Giovanni Rotondo (FG), Italy.

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.