Adrenergic regulation of clock gene expression in mouse liver - PubMed (original) (raw)

Adrenergic regulation of clock gene expression in mouse liver

Hideyuki Terazono et al. Proc Natl Acad Sci U S A. 2003.

Abstract

A main oscillator in the suprachiasmatic nucleus (SCN) conveys circadian information to the peripheral clock systems for the regulation of fundamental physiological functions. Although polysynaptic autonomic neural pathways between the SCN and the liver were observed in rats, whether activation of the sympathetic nervous system entrains clock gene expression in the liver has yet to be understood. To assess sympathetic innervation from the SCN to liver tissue, we investigated whether injection of adrenaline/noradrenaline (epinephrine/norepinephrine) or sympathetic nerve stimulation could induce mPer gene expression in mouse liver. Acute administration of adrenaline or noradrenaline increased mPer1 but not mPer2 expression in the liver of mice in vivo and in hepatic slices in vitro. Electrical stimulation of the sympathetic nerves or adrenaline injection caused an elevation of bioluminescence in the liver area of transgenic mice carrying mPer1 promoter-luciferase. Under a light-dark cycle, destruction of the SCN flattened the daily rhythms of not only mPer1, mPer2, and mBmal1 genes but also noradrenaline content in the liver. Daily injection of adrenaline, administered at a fixed time for 6 days, recovered oscillations of mPer2 and mBmal1 gene expression in the liver of mice with SCN lesion on day 7. Sympathetic nerve denervation by 6-hydroxydopamine flattened the daily rhythm of mPer1 and mPer2 gene expression. Thus, on the basis of the present results, activation of the sympathetic nerves through noradrenaline and/or adrenaline release was a factor controlling the peripheral clock.

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Figures

Fig. 2.

Effect of adrenaline on mPer1 gene expression in liver slices in vitro. mPer1 mRNA was expressed in a manner dependent on the concentration of adrenaline (A), phenylephrine (B), and isoproterenol (C). The vertical values reflect the relative mRNA levels of mPer1 or mPer2 as the ratio to β-actin mRNA levels. mPer1 or mPer2 levels in the vehicle (V)-treated group are set at 1.0. Each point represents three to six animals. *, P < 0.05; Dunnett's test. (D and E) Effect of mitogen-associated protein kinase (MAPK) kinase (D) and protein kinase A (PKA) (E) inhibitor on adrenaline-induced mPer1 gene expression. Each point represents three to six animals. *, P < 0.05; Student's t test. (F) In vitro study of the effect of adrenaline (0.1 μg/ml, for 60 min) on RLU and mPer1 mRNA expression in the liver of transgenic mice carrying the mPer1 promoter-luciferase. Both bioluminescence (RLU) and RT-PCR products of the intrinsic mPer1 gene transcript were measured in hepatic slices (12 slices from three animals for each point). Images of mPer1 and β-actin RT-PCR products after electrophoresis are shown above each figure. **, P < 0.01, Student's t test.

Fig. 1.

Effect of adrenaline on mPer expression in mouse liver. In the histograms, the vertical values reflect the relative mRNA levels of mPer1 or mPer2 as the ratio to β-actin mRNA levels. mPer1 or mPer2 levels in the vehicle-treated group are set at 1.0. Images of mPer1, mPer2, and β-actin RT-PCR products after electrophoresis are shown above each histogram. (A) Adrenaline treatment dose-dependently effects the expression of mPer1 but not mPer2 at ZT 3 (three to nine animals per point). (B) Time course of mPer1 and mPer2 gene expression after adrenaline injection (three to nine animals per point). (C) Adrenaline and noradrenaline comparison of the expression of mPer1 mRNA (three six animals per point). (D) Phase-dependent response of mPer1 gene expression with adrenaline injection (three to five animals per point). **, P < 0.01; *, P < 0.05; Student's t test.

Fig. 3.

(A) Daily rhythm of bioluminescence in the liver of _mPer1_-luciferase transgenic mice. Sampling times were ZT 3, 7, 15, and 20 (three animals per point). Luminescence (RLU) is significantly higher at ZT 15 than at ZT 3 (**, P < 0.01; Dunnett's test). (B) Summary of _mPer1_-luc luminescence after electrical stimulation (EST) as described in the legend of Fig. 4. Prestimulation luminescence was measured at ZT 3, and post-EST luminescence was measured 60 min later. **, P < 0.01; Student's t test. (C) Luminescence (RLU) in the mouse liver 60 min after adrenaline (2 mg/kg) or saline injection at ZT 3 (three animals per point). **, P < 0.01; Student's t test.

Fig. 4.

Visualization of elevated mPer1 transcription by electrical stimulation (EST) of the sympathetic nerve. The splanchnic nerve hepatic branch was electrically stimulated for 30 min (3 Hz, 5 V, 5 ms). (Upper Left) Luminescence before stimulation. (Upper Right) Luminescence after stimulation. Luminescence was recorded by a two-dimensional photon-counting camera. Luminescence in the liver was increased without affecting the front and rear paws. Images displayed with pseudocolor represent accumulated photo counts. The site of EST is shown in detail in Lower.

Fig. 5.

Effect of 6-OHDA injection on mPer1 (B) and mPer2 (C) gene expression in the liver. (A) Images of mPer1, mPer2, and β-actin RT-PCR products obtained from saline-injected or 6-OHDA-injected mice after electrophoresis. (B and C) Each animal was injected with either saline (○, three animals per point) or 6-OHDA (•, four or five animals per point) at ZT 12 on days 1 and 3. On day 4, animals were killed at each ZT. Relative mRNA level of mPer1 or mPer2 is shown as the ratio to β-actin mRNA level. Dotted lines represent duplicate data at ZT 5 in the respective groups.

Fig. 6.

Daily pattern of noradrenaline content in the liver of SCN-lesioned (▵) (three or four animals per point) or intact (○) (three animals per point) mice. Dotted lines represent duplicate data at ZT 5 and 11 in the respective groups.

Fig. 7.

Effect of daily adrenaline injection on mPer1 (B), mPer2 (C), and mBmal1 (D) gene expression in the liver of SCN-lesioned, arrhythmic mice. Shown are images after electrophoresis (A) and relative mRNA levels (B–D)of mPer1, mPer2, mBmal1, and β-actin RT-PCR products obtained from intact mice (○, three to six animals per point), daily saline-injected SCN-lesioned mice (▵, three to six animals per point), and daily adrenaline-injected SCN-lesioned mice (•, three to five animals per point). Relative mRNA level of mPer1, mPer2, or mBmal1 is shown as the ratio to β-actin mRNA level. Dotted lines represent duplicate data at ZT 3, 5, 7, and 23 in the respective groups.

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Adrenergic regulation of clock gene expression in mouse liver - PubMed (original) (raw)