빛 조절에 따른 금붕어 Carassius auratus의 송과체와 내장 멜라토닌의 일주기 리듬의 차이. 또한, 강력한 항산화제로 알려진 멜라토닌이 분비기관(송과선과 장)에 따라 그 작용에 차이가 있는지도 알아보고자 하였다. 금붕어의 원추체와 장에서 분비되는 멜라토닌의 일주기리듬에 차이가 있는지를 알아보기 위해 <멜라토닌 합성에 관여하는> 아랄킬아민과 멜라토닌 수용체 유전자, 혈중 N-아세틸트랜스퍼라제(AANAT)의 발현량을 측정하였다.
멜라토닌 농도의 변화가 관찰되었습니다. 멜라토닌 수용체는 한동안 분리되었습니다. 또한, 항산화제인 멜라토닌은 송과선과 장에서 분비됩니다.
H2O2) 농도의 변화도 AANAT 및 멜라토닌 수용체 유전자 발현 경향과 유사하였지만 송과선과 장조직 간에는 유의한 차이가 없었다.
Differential Daily Rhythms of Melatonin in the Pineal Gland and Gut of Goldfish,
Introduction
Light is one of the most important environmental factors affecting almost all organisms (Migaud et al., 2006). From the SCN, signals are then transmitted to photosensitive organs (Yoshikawa and Oishi, 1998; Yamazaki et al., 1999). Among the MT-Rs, MT-R1 is implicated in seasonal changes in physiology and behavior in vertebrates, including those in fish, and directly affects circadian rhythms (Reppert et al., 1996).
AANAT is known as a rate-limiting enzyme of the melatonin biosynthetic pathway (Klein et al., 2002). Furthermore, melatonin secretion and AANAT expression patterns in the intestines appear to vary depending on the species (Velarde et al., 2010).
Materials and methods
- Experimental fish, ophthalmectomy, and conditions
- Quantitative PCR (QPCR)
- Western blot analysis
- Melatonin concentration level
- H 2 O 2 concentration measurement
- Statistical analysis
The fish were killed by transection of the vertebral column (first sampling at 11 a.m.; ZT4) at 4-h sampling intervals (ZT4, ZT8, ZT12, ZT16, ZT20, and ZT24) to collect the pineal gland, whole intestine, and blood under dimmed conditions. light using an attenuated white fluorescent lamp. The pineal gland and intestines were removed from the fish, immediately frozen in liquid nitrogen and stored at 80 °C until analysis. The isolated pineal gland and whole intestinal tissues were then transferred to 6 ml of fresh dispersion buffer containing 0.25% trypsin (Type II-S from porcine pancreas; Sigma, USA).
The mixture of pineal gland and whole intestinal tissue was filtered and culture medium (neurobasal medium, without L-glutamine, containing 100 U/mL penicillin, 100 μg/mL streptomycin, 2.5 μg/mL fungizone, and 1 % fetal bovine serum, Gibco-BRL; to adjust mean osmolarity to goldfish plasma osmolarity, 353 mOs). The cell suspension was centrifuged at 800 × g for 10 min, and the cells were then resuspended in fresh culture medium. Cells from the pineal gland and intestine of 60 goldfish were counted and adjusted to a final concentration of approximately viable pineal gland cells/mL and viable intestinal cells/mL (as determined by trypan blue exclusion) in supplemental media .
The pineal gland and intestinal cells at a concentration of approximately 1.2 × 106 cells/ml were added in 24-well culture plates (SPL Life Sciences, Seoul, South Korea) and incubated at 22°C, 100% humidity and 5% atmospheric CO2 in a culture chamber (HB-302L; Hanbaek Scientipic Co., Gyeonggi, Korea). Then, cell suspension was centrifuged at 800 × g for 10 min, and the supernatant was removed and stored at 80 °C until total RNA extraction and analysis. Total RNA was extracted from the pineal gland and whole intestine by a TRIzol kit (Gibco/BRL, USA) according to manufacturer's protocol.
QPCR was performed to determine the relative expression levels of MT-R1 (GenBank accession no. AB481372 ), AANAT2 (GenBank accession no. GU205782 ), β-actin (GenBank accession no. AB039726 ), and GAPDH (GenBank accession no. AY641444 ) mRNA in the pineal gland and whole the gut. Total protein isolated from the pineal gland and whole intestine of goldfish was extracted using a T-PER® Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Inc., USA) following the manufacturer's protocol. Pineal gland and whole intestine samples were homogenized and centrifuged (4°C, 1,500 x g , 15 min), and plasma samples were separated by centrifugation.
Results
- Change of pineal gland AANAT2 expression levels
- Change of whole gut AANAT2 expression levels
- Change of pineal gland MT-R1 protein and mRNA expression levels
- MT-R1 protein and mRNA expression levels
- Melatonin concentration
- AANAT2 and MT-R1 expression in response to thermal stress AANAT2 and MT-R1 mRNA expression in both the pineal gland and gut
- H 2 O 2 concentration under thermal stress conditions
Changes in the expression levels of AANAT2 mRNA (A) in the pineal gland, for 2 days using different photoperiods [12L:12L (continuous), LL and DD] and ophthalmectomy (OP). Changes in expression levels of AANAT2 mRNA in the cultured pineal gland cells (B) using different photoperiods [12L:12L (continued), LL and DD]. Changes in the expression levels of AANAT2 mRNA (A) in the whole intestine, for 2 days using different photoperiods [12L:12L (continuous), LL and DD] and ophthalmectomy (OP).
Changes of expression levels of AANAT2 mRNA in the cultured whole intestinal cells (B) using various photoperiods [12L:12L (Continued), LL and DD]. In the DD and OP groups, MT-R1 mRNA expression also decreased significantly in the second-day cycle compared to the first-day cycle. In contrast to the pineal gland, MT-R1 mRNA expression in the DD and OP groups did not differ significantly between the first day cycle and the second day cycle.
Changes in the expression levels of MT-R1 protein (A) and MT-R1 mRNA (B) in the pineal gland over 2 days using different photoperiods [12L:12L (continued), LL and DD] and ophthalmectomy (OP ) . Changes in the expression levels of MT-R1 mRNA in the cultured pineal cells (C) using different photoperiods [12L:12L (continued), LL and DD]. Changes in the expression levels of MT-R1 protein (A) and MT-R1 mRNA (B) in the whole intestine over 2 days using different photoperiods [12L:12L (continued), LL and DD] and ophthalmectomy ( ON) .
Changes in the expression levels of MT-R1 mRNA in the cultured whole intestinal cells (C) using different photoperiods [12L:12L (continued), LL and DD]. Changes in melatonin levels in the pineal gland (A), plasma (B) and whole intestine (C) of goldfish under different photoperiods [12L:12L (continued), LL and DD] and ophthalmectomy (OP), as measured by plate reader. The H2O2 concentrations in the pineal gland and intestines of the thermal stress group (30°C) were significantly higher than those of the control group (22°C) (Fig. 8).
Changes in the levels of AANAT2 mRNA in goldfish pineal gland (A) and in whole intestine (B) under different temperature conditions, 22°C (control) and 30°C (thermal stress) during the natural photoperiod (SNP). Changes in the levels of MT-R1 mRNA in goldfish pineal gland (A) and in whole intestine (B) under different temperature conditions, 22°C (control) and 30°C (thermal stress) during the natural photoperiod (12L: 12D).
![Fig. 5. Changes in the levels of melatonin in the pineal gland (A), plasma (B) and whole gut (C) of goldfish under various photoperiods [12L:12L (Cont.), LL and DD] and ophthalmectomy (OP) as measured by plate reader](https://thumb-ap.123doks.com/thumbv2/123dokinfo/10569023.0/40.892.320.574.507.752/changes-levels-melatonin-goldfish-various-photoperiods-ophthalmectomy-measured.webp)
Discussion
In other words, this study suggests that the gut can regulate its own circadian rhythm, without using light signals, through AANAT2 and MT-1R mRNA expression. In contrast, pineal gland and plasma AANAT2 and MT-R1 mRNA were significantly higher during the second day cycle compared to the first day cycle (ZT4 ZT24). A similar decrease in the amplitude of the oscillation of AANAT2 mRNA expression and activity was found in chickens exposed to continuous light or dark conditions (Klein et al., 1997).
This study also determined that the diurnal secretion pattern of melatonin in the pineal gland, plasma, and gut corresponded to the observed changes in AANAT2 and MT-R1 mRNA expression across all experimental groups. Notably, these results found no difference in melatonin concentrations between the DD and OP groups, as fish in the OP condition were effectively deprived of light signals, similar to the DD condition. In addition, melatonin diurnal rhythms in the pineal gland and plasma are similar, but gut melatonin rhythms are consistently observed across all experimental groups (LL, DD or OP).
Again, these results indicate that the gut has its own biorhythms for melatonin secretion and that the synthesis of melatonin and AANAT2 in the gut is regulated by the circadian clock and includes non-photic cues. In both organs, AANAT2 and MT-R1 mRNA expression increased in response to heat stress, indicating that oxidative stress induced by high water temperatures induced an antioxidant response in fish. To examine the effects of oxidative stress on goldfish under heat stress conditions, H2O2 levels in the pineal gland and gut were measured in this experiment.
Significantly higher H2O2 levels were noted in the tissues in the thermal stress groups (30°C) than in tissues in the control groups (22°C). Our results suggested that the pineal gland and intestine exhibit antioxidant functions under thermal stress conditions, and that the two tissues have the same antioxidant mechanisms. To summarize, these studies found that: 1) AANAT2 expression, MT-R1 mRNA and protein expression, and melatonin concentrations in the pineal gland and plasma are controlled by light, 2) dermal melatonin differs from pineal .. glandular melatonin, possibly because it possesses an autonomous circadian rhythm, which is not affected by light, 3) the function of melatonin as an antioxidant does not differ between photosensitive (pineal gland) and non-photosensitive tissue (gut).
Acknowledgements
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Effect of artificial lighting conditions on diurnal melatonin profiles in the gut of surface-dwelling carp (Catla catla) Biol. Effects of LED spectral sensitivity on circadian rhythm-related genes in the yellowtail clownfish, Amphiprion clarkii.
