Negative regulation of neuromedin U mRNA expression in the rat pars tuberalis by melatonin

Abstract

The pars tuberalis (PT) is part of the anterior pituitary gland surrounding the median eminence as a thin cell layer. The characteristics of PT differ from those of the pars distalis (PD), such as cell composition and gene expression, suggesting that the PT has a unique physiological function compared to the PD. Because the PT highly expresses melatonin receptor type 1, it is considered a mediator of seasonal and/or circadian signals of melatonin. Expression of neuromedin U (NMU) that is known to regulate energy balance has been previously reported in the rat PT; however, the regulatory mechanism of NMU mRNA expression and secretion in the PT are still obscure. In this study, we examined both the diurnal change of NMU mRNA expression in the rat PT and the effects of melatonin on NMU in vivo. In situ hybridization and quantitative PCR analysis of laser microdissected PT samples revealed that NMU mRNA expression in the PT has diurnal variation that is high during the light phase and low during the dark phase. Furthermore, melatonin administration significantly suppressed NMU mRNA expression in the PT in vivo. On the other hand, 48 h fasting did not have an effect on PT-NMU mRNA expression, and the diurnal change of NMU mRNA expression was maintained. We also found the highest expression of neuromedin U receptor type 2 (NMUR2) mRNA in the third ventricle ependymal cell layer, followed by the arcuate nucleus and the spinal cord. These results suggest that NMU mRNA expression in the PT is downregulated by melatonin during the dark phase and shows diurnal change. Considering that NMU mRNA in the PT showed the highest expression level in the brain, PT-NMU may act on NMUR2 in the brain, especially in the third ventricle ependymal cell layer, with a circadian rhythm.

Figure 1. NMU mRNA expression regions in the brain of male Wistar rats.

(A) Microphotograph of NMU mRNA expression regions detected by ISH staining on free-floating brain sections with DIG-labeled cRNA probes for rat whole brain. A strong signal was detected in the PT. No signal was detected in the other areas, including the VMH and ARC. (B) Magnified image of the boxed area in A. NMU mRNA-expressing cells were widely distributed throughout the PT. (C) No signal for NMU mRNA expression was detected in the SCN. (D) Sense RNA probes to NMU generated no specific signal. Scale bar: 200 µm. PT, pars tuberalis; ARC, arcuate nucleus; VMH, ventromedial hypothalamic nucleus; 3V, third ventricle; SCN, suprachiasmatic nucleus. (E) qPCR analysis of NMU in the PT, PD, SCN, ARC, and VMH using LMD samples. High expression was detected in the PT, low expression was detected in the PD and SCN, and no expression was detected in the ARC and VMH. All values are means ± S.E.M. (n = 4).

Figure 2. NMU mRNA-expressing cell types in the PT.

Microphotograph of NMU mRNA expression detected by double staining by HNPP fluorescent ISH for NMU mRNA and by fluorescent IHC for TSH in fixed, frozen 5-µm sections. (A) Microphotograph of NMU mRNA expression detected by HNPP fluorescent ISH (red). NMU mRNA-expressing cells were widely distributed in the PT. (B) Microphotograph of TSH detected by fluorescent IHC staining. Many TSH-immunostained cells were distributed throughout the PT (green). (C) Microphotograph of merged pictures of double staining for NMU mRNA expression, which was detected by HNPP fluorescent ISH and TSH detected by fluorescent IHC. Yellow color represents the merged signal for NMU mRNA and TSH production. Although almost all NMU-expressing cells showed TSH immunoreactivity (arrows, yellow), cells exhibiting only TSH immunostaining were also found (arrowheads, green). (D) Magnified image of the boxed area in C. NMU mRNA-expressing and TSH producing cells (arrows, yellow) and cells producing only TSH (arrowheads, green) exist. Scale bars: 50 µm. PT, pars tuberalis; ME, median eminence.

Figure 3. Diurnal change of NMU mRNA expression in the rat PT.

(A, B) Microphotograph of NMU mRNA expression detected by ISH staining on fresh frozen sections. The staining signal in the PT (arrows) was high at ZT6 (light phase) and low at ZT18 (dark phase). (C) Sense RNA probes for NMU generated no specific signal. Scale bar: 200 µm. PT, pars tuberalis; ARC, arcuate nucleus; 3V, third ventricle. (D) qPCR analysis of NMU using LMD samples of the PT at ZT0, 6, 12, 18, and 24. The expression level fluctuated in a time-dependent manner. NMU mRNA expression was low at ZT0 and gradually increased, and then reached a peak at ZT12. The level was decreased at ZT18 and remained low at ZT24. All values are means ± S.E.M. (n = 4). Significant differences were detected by one-way analysis of variance (P = 0.0004) and Tukey’s multiple comparison test (** P<0.01, *** P<0.001).

Figure 4. Effects of melatonin on NMU mRNA expression in the PT, PD, and SCN.

(A–D) Microphotograph of NMU mRNA expression detected by ISH in the PT of control and melatonin-treated groups at ZT6 and ZT18. (A, B) Staining signal in the PT was decreased in the melatonin-treated group compared to the sham group at ZT6. (C, D) In the ZT18 samples, a similar staining intensity was observed in the sham and melatonin-treated groups. Scale bar: 200 µm. PT, pars tuberalis; ARC, arcuate nucleus; 3V, third ventricle. (E) qPCR analysis of NMU in the PT was performed for the sham and melatonin groups at ZT6 and ZT18 using LMD samples. Two-way ANOVA revealed a significant interaction between melatonin treatment and time point (P<0.05) and that the NMU mRNA expression level of the ZT6 melatonin group was significantly higher than that of the ZT6 melatonin, ZT18 control, and ZT18 melatonin groups (one way ANOVA, P<0.01). (F, G) qPCR analysis for NMU in the SCN and PD performed for the sham and melatonin-treated rats at ZT6 and ZT18. NMU mRNA expression in the SCN and PD were considerably lower and not influenced by time or melatonin treatment.

Figure 5. Effects of fasting on NMU mRNA expression in PT.

qPCR analysis of NMU in the PT performed for the sham and fasting groups at ZT6 and ZT18. The NMU mRNA expression level was not changed when the sham group was compared with the fasting group at both sampling times, ZT6 (fasting from ZT6 to ZT6 for 48 h) and ZT18 (fasting from ZT18 to ZT18 for 48 h), and the diurnal change was maintained even under fasting conditions (high at ZT6, low at ZT18, P<0.01; ZT6 control vs. ZT18 control, ZT6 fasting vs. ZT18 fasting).

Figure 6. NMU receptor type 2 mRNA expression in the rat brain.

(A) Microphotograph of NMU mRNA expression detected by ISH in the fixed frozen sections of the rat brain. Staining signal was detected in the EC of the 3V (arrows). Upper part of the EC of 3V did not show NMUR2 expression (arrowheads). (B) Enlarged view of A. The staining signal was found in the EC of the 3V (arrows), but not in the bottom of the 3V, adjacent to the median eminence (arrowheads). (C) Sense RNA probes to NMUR2 generated no specific signal. Scale bar: 200 µm. PT, pars tuberalis; ARC, arcuate nucleus; 3V, third ventricle; EC, ependymal cell layer of the third cerebroventricle. (D) qPCR analysis of NMUR2 in the PT, PD, SCN, ARC, EC, and SC at ZT6 and ZT18. NMUR2 expression was detected in the EC at the highest level, followed by the ARC and SC, and was detected in the SCN at a considerably lower level. NMUR2 expression was not detected in the PT and PD. NMUR2 expression did not change at either time point in the SCN, ARC, EC, and SC.