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Leptin modulates norepinephrine-mediated melatonin synthesis in cultured rat pineal gland.

Peliciari-Garcia RA, Andrade-Silva J, Cipolla-Neto J, Carvalho CR - Biomed Res Int (2013)

Bottom Line: According to our data, cultured rat pineal glands express leptin receptor isoform b (Ob-Rb).In addition, the expression of inducible cAMP early repressor (ICER) was further induced by leptin challenge when associated with NE.In conclusion, leptin inhibition of pineal melatonin synthesis appears to be mediated by a reduction in AANAT activity and expression as well as by increased expression of Icer mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil. rodrigousp@gmail.com

ABSTRACT
Pineal melatonin synthesis can be modulated by many peptides, including insulin. Because melatonin appears to alter leptin synthesis, in this work we aimed to investigate whether leptin would have a role on norepinephrine- (NE-)mediated melatonin synthesis in cultured rat pineal glands. According to our data, cultured rat pineal glands express leptin receptor isoform b (Ob-Rb). Pineal expression of Ob-Rb mRNA was also observed in vivo. Administration of leptin (1 nM) associated with NE ( 1 µM) reduced melatonin content as well as arylalkylamine-N-acetyl transferase (AANAT) activity and expression in cultured pineal glands. Leptin treatment per se induced the expression of STAT3 in cultured pineal glands, but STAT3 does not participate in the leptin modulation of NE-mediated pineal melatonin synthesis. In addition, the expression of inducible cAMP early repressor (ICER) was further induced by leptin challenge when associated with NE. In conclusion, leptin inhibition of pineal melatonin synthesis appears to be mediated by a reduction in AANAT activity and expression as well as by increased expression of Icer mRNA. Peptidergic signaling within the pineal gland appears to be one of the most important signals which modulates melatonin synthesis; leptin, as a member of this system, is not an exception.

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Expression of Ob-Rb mRNA in male rat pineal gland. (a) In vivo qualitative expression of Ob-Rb mRNA, pool of six (6) pineal glands harvested in the middle of the dark phase. Data presented as arbitrary units. (b) Real-Time RT PCR analysis of in vivo and in vitro Ob-Rb mRNA expression in the rat pineal gland. Data are reported as the number of transcripts per number of ribosomal protein L37a (Rpl37a) molecules. Student's t-test, unpaired, and two-tailed, *P < 0.05 versus in vivo. n = 6 glands/group and each experiment was repeated 3 times.
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fig1: Expression of Ob-Rb mRNA in male rat pineal gland. (a) In vivo qualitative expression of Ob-Rb mRNA, pool of six (6) pineal glands harvested in the middle of the dark phase. Data presented as arbitrary units. (b) Real-Time RT PCR analysis of in vivo and in vitro Ob-Rb mRNA expression in the rat pineal gland. Data are reported as the number of transcripts per number of ribosomal protein L37a (Rpl37a) molecules. Student's t-test, unpaired, and two-tailed, *P < 0.05 versus in vivo. n = 6 glands/group and each experiment was repeated 3 times.

Mentions: Total RNA was isolated from rat pineal glands using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. DNase treatment was performed using Turbo DNA-free kit according to the kit's directions (Ambion, Austin, TX, USA). cDNA synthesis was performed using Super Script III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) from 1 μg of total RNA. 5 ng of the obtained cDNA was used in all qPCR assays, which were performed on the 7500HT Fast Real-Time PCR System, using Power SYBR Green (Applied Biosystems, Foster City, California, USA). Primer sequences for rat Tpoh, Aanat, and Hiomt have been previously published by our group [18], while specific primers assays for leptin receptor isoform b (Ob-Rb), Icer, and Rpl37a were designed from rat sequences available in the GenBank and are presented in Table 1. Absolute qPCR quantification was performed using DNA standards preparation (number of molecules) for each investigated gene [30]. Real-Time PCR data are reported as the number of transcripts per number of ribosomal protein L37a (Rpl37a) molecules. Qualitative PCR results (showed only in Figure 1(a)), show the expression of Ob-Rb mRNA/ng of total RNA (arbitrary units) in a pool of 6 pineal glands. For all gene expression analysis, except Figure 1(a), it was used 6 glands/group, and all experiments were repeated 3 times.


Leptin modulates norepinephrine-mediated melatonin synthesis in cultured rat pineal gland.

Peliciari-Garcia RA, Andrade-Silva J, Cipolla-Neto J, Carvalho CR - Biomed Res Int (2013)

Expression of Ob-Rb mRNA in male rat pineal gland. (a) In vivo qualitative expression of Ob-Rb mRNA, pool of six (6) pineal glands harvested in the middle of the dark phase. Data presented as arbitrary units. (b) Real-Time RT PCR analysis of in vivo and in vitro Ob-Rb mRNA expression in the rat pineal gland. Data are reported as the number of transcripts per number of ribosomal protein L37a (Rpl37a) molecules. Student's t-test, unpaired, and two-tailed, *P < 0.05 versus in vivo. n = 6 glands/group and each experiment was repeated 3 times.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3713337&req=5

fig1: Expression of Ob-Rb mRNA in male rat pineal gland. (a) In vivo qualitative expression of Ob-Rb mRNA, pool of six (6) pineal glands harvested in the middle of the dark phase. Data presented as arbitrary units. (b) Real-Time RT PCR analysis of in vivo and in vitro Ob-Rb mRNA expression in the rat pineal gland. Data are reported as the number of transcripts per number of ribosomal protein L37a (Rpl37a) molecules. Student's t-test, unpaired, and two-tailed, *P < 0.05 versus in vivo. n = 6 glands/group and each experiment was repeated 3 times.
Mentions: Total RNA was isolated from rat pineal glands using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. DNase treatment was performed using Turbo DNA-free kit according to the kit's directions (Ambion, Austin, TX, USA). cDNA synthesis was performed using Super Script III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) from 1 μg of total RNA. 5 ng of the obtained cDNA was used in all qPCR assays, which were performed on the 7500HT Fast Real-Time PCR System, using Power SYBR Green (Applied Biosystems, Foster City, California, USA). Primer sequences for rat Tpoh, Aanat, and Hiomt have been previously published by our group [18], while specific primers assays for leptin receptor isoform b (Ob-Rb), Icer, and Rpl37a were designed from rat sequences available in the GenBank and are presented in Table 1. Absolute qPCR quantification was performed using DNA standards preparation (number of molecules) for each investigated gene [30]. Real-Time PCR data are reported as the number of transcripts per number of ribosomal protein L37a (Rpl37a) molecules. Qualitative PCR results (showed only in Figure 1(a)), show the expression of Ob-Rb mRNA/ng of total RNA (arbitrary units) in a pool of 6 pineal glands. For all gene expression analysis, except Figure 1(a), it was used 6 glands/group, and all experiments were repeated 3 times.

Bottom Line: According to our data, cultured rat pineal glands express leptin receptor isoform b (Ob-Rb).In addition, the expression of inducible cAMP early repressor (ICER) was further induced by leptin challenge when associated with NE.In conclusion, leptin inhibition of pineal melatonin synthesis appears to be mediated by a reduction in AANAT activity and expression as well as by increased expression of Icer mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil. rodrigousp@gmail.com

ABSTRACT
Pineal melatonin synthesis can be modulated by many peptides, including insulin. Because melatonin appears to alter leptin synthesis, in this work we aimed to investigate whether leptin would have a role on norepinephrine- (NE-)mediated melatonin synthesis in cultured rat pineal glands. According to our data, cultured rat pineal glands express leptin receptor isoform b (Ob-Rb). Pineal expression of Ob-Rb mRNA was also observed in vivo. Administration of leptin (1 nM) associated with NE ( 1 µM) reduced melatonin content as well as arylalkylamine-N-acetyl transferase (AANAT) activity and expression in cultured pineal glands. Leptin treatment per se induced the expression of STAT3 in cultured pineal glands, but STAT3 does not participate in the leptin modulation of NE-mediated pineal melatonin synthesis. In addition, the expression of inducible cAMP early repressor (ICER) was further induced by leptin challenge when associated with NE. In conclusion, leptin inhibition of pineal melatonin synthesis appears to be mediated by a reduction in AANAT activity and expression as well as by increased expression of Icer mRNA. Peptidergic signaling within the pineal gland appears to be one of the most important signals which modulates melatonin synthesis; leptin, as a member of this system, is not an exception.

Show MeSH