Advances in Clinical and Experimental Medicine

Title abbreviation: Adv Clin Exp Med
JCR Impact Factor (IF) – 1.727
Index Copernicus  – 166.39
MEiN – 70 pts

ISSN 1899–5276 (print)
ISSN 2451-2680 (online)
Periodicity – monthly

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Advances in Clinical and Experimental Medicine

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doi: 10.17219/acem/146775

Publication type: original article

Language: English

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Tyczewska M, Szyszka M, Jopek K, Ruciński M. Effects of Galp and alarin peptides on HPA axis gene expression and adrenal function: In vivo experiments [published online as ahead of print on March 11, 2022]. Adv Clin Exp Med. 2022. doi:10.17219/acem/146775

Effects of Galp and alarin peptides on HPA axis gene expression and adrenal function: In vivo experiments

Marianna Tyczewska1,A,B,C,D,E,F, Marta Szyszka1,B,C,F, Karol Jopek1,B,C,F, Marcin Ruciński1,A,E,F

1 Department of Histology and Embryology, Poznan University of Medical Sciences, Poland

Abstract

Background. Many experimental data indicate interactions between peptides involved in the control of food intake, energy homeostasis and adrenocortical hormone release. Glucocorticoids stimulate or inhibit the secretion of orexigenic and anorexigenic peptides, which in turn are involved in the regulation of adrenal growth, structure and function. Galanin-like peptide (Galp) and alarin (Ala) are involved in the regulation of food intake. Galp and Ala mRNAs have already been shown to be present in the arcuate nucleus (ARC) of the hypothalamus in both rats and mice.
Objectives. To investigate the expression of Ala, Galp and their receptors in the hypothalamus and pituitary and adrenal glands of the rat hypothalamic–pituitary–adrenal (HPA) axis after intraperitoneal administration of peptides in vivo.
Material and Methods. Experimental in vivo models were used: acute and long-term exposure to peptides.
Results. The expression of Galp, Ala, their receptors, and steroidogenesis enzymes was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Statistically significant expression changes were found in the hypothalamus and pituitary after 1-hour exposure to the peptides, such as a decrease in corticotropin-releasing hormone (CRH) expression after Ala, Galp and adrenocorticotropic hormone (ACTH) administration, and a decrease in the expression of receptors for galanin (Gal) (Galr1 and Galr2). In the pituitary, there was a statistically significant increase in the expression of Ala, Galr1, Galr2, and Galr3 receptors 1 h after Galp administration. In the adrenal glands, only a statistically significant decrease in Galr2 expression was observed after 1 h of Ala 0.5 administration. The mRNA expression of steroidogenesis enzymes also changed: for example, the expression of cholesterol desmolase increased 24 h after Ala peptide administration.
Conclusion. The results indicate that the peptides tested under in vivo conditions can alter the expression of the peptides tested, as well as of Galp, Ala and Gal receptors and steroidogenesis enzymes – Cyp11a1 (cholesterol desmolase), Cyp11b1 (11β-hydroxylase) and Cyp11b2 (aldosterone synthase).

Key words

adrenal gland, HPA axis, Galp, alarin (Ala), in vivo experiments

Tables


Table 1. Conventional qRT-PCR analyses of CRH. Oligonucleotide sequences for sense (S) and antisense (A) primers are shown. Hypoxanthine phosphorybosyl transferase (Hprt) was the reference gene

cDNA

Genbank

accession number

Primer

Primer sequence (5’-3’)

Position

PCR product size (bp)

CRH

NM_031019.1

S

A

GTACCTCGCAGAACAACAGT

CTTCACCCATGCGGATCAGA

113–132

340–359

247

POMC

NM_139326.2

S

A

TCACCACGGAAAGCAACCTG

CATGACGTACTTCCGGGGAT

231–250

339–358

128

Ala

NM_022633.1

S

A

TGCTCACAGGGGACGAGGA

CCGGAACATTCTTGTCCAC

200–218

429–447

248

Alarin

NM_022633.1

S

A

ACAGGTCCTCCACCTTTCC

CATTGACCTTTTGGTCATCCTTGG

205–233

314–337

133

Galr1

NM_012958.3

S

A

TTCATCGGGACAGCAACCA

GCCAAATACCACAACGACCA

755–774

974–994

239

Galr2

NM_019172.5

S

A

CATCCTGTGCTGCGTGCC

CTAGCCCCCAGATGAGCCC

251–269

468–487

236

Galr3

NM_019173.1

S

A

AGGACTGAGGAAGATGGCTGA

ATTGCCCACCATGCCCAAC

13–34

112–131

118

Cyp11a1

NM_017286

S

A

GATGACCTATTCCGCTTTGC

GTTGGCCTGGATGTTCTTG

592–611

930–948

357

Cyp11b1

NM_012537

S

A

AGAGTATCCTCCCGCATCG

GCCAGTCTGCCCCATTTAG

311–329

394–412

102

Cyp11b2

NM_012538.2

S

A

TGGCAGCACTAATAACTCAGG

AAAAGCCACCAACAGGGTAG

875–895

1131–1150

276

Hprt

NM_012583

S

A

CAGTCAACGGGGGACATAAAAG

ATTTTGGGGCTGTACTGCTTGA

391–412

515–536

146

qRT-PCR – quantitative real-time polymerase chain reaction; CRH – corticotropin-releasing hormone; POMC – proopiomelanocortin; Ala – alarin; Galp – galanin-like peptide; Galr1 – galanin receptor type 1; Galr2 – galanin receptor type 2; Galr3 – galanin receptor type 3; Cyp11a1 – cholesterol desmolase; Cyp11b1 – 11β-hydroxylase; Cyp11b2 – aldosterone synthase.

Figures


Fig. 1. Relative mRNA expression of the galanin-like peptide (Galp), alarin (Ala) and mRNA receptors in the hypothalamus after the administration of the peptides (Galp/Ala, 1 h, 24 h and 2 days). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the mRNA expression levels. Data points were presented as medians, 1st and 3rd quartiles (boxes) and ranges (whiskers – minimal and maximal values without outliers). Statistical analysis of the data was performed using the Kruskal–Wallis test and Dunn’s post hoc test. Different letters in the graphs indicate significant differences between specific groups. Outliers were also presented on the graphs. Statistical differences detected with post hoc test were indicated with letters, where different letters correspond to statistical significance (p < 0.05) between groups.
ACTH – adrenocorticotropic hormone; DMSO – dimethyl sulfoxide; Galr – galanin receptor.
Fig. 2. Relative mRNA expression of the galanin-like peptide (Galp), alarin (Ala) and mRNA receptors in the pituitary gland after the administration of the peptides (Galp/Ala, 1 h, 24 h and 2 days). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the mRNA expression levels. Data points were presented as medians, 1st and 3rd quartiles (boxes) and ranges (whiskers – minimal and maximal values without outliers). Statistical analysis of the data was performed using the Kruskal–Wallis test and Dunn’s post hoc test. Different letters in the graphs indicate significant differences between specific groups. Outliers were also presented on the graphs. Statistical differences detected with post hoc test were indicated with letters, where different letters correspond to statistical significance (p < 0.05) between groups.
ACTH – adrenocorticotropic hormone; DMSO – dimethyl sulfoxide; Galr – galanin receptor.
Fig. 3. Relative mRNA expression of the corticotropin-releasing hormone (CRH) and proopiomelanocortin (POMC) mRNAs after the administration of the peptides (galanin-like peptide (Galp)/alarin (Ala), 1 h, 24 h and 2 days). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the mRNA expression levels. Data points were presented as medians, 1st and 3rd quartiles (boxes) and ranges (whiskers – minimal and maximal values without outliers). Statistical analysis of the data was performed using the Kruskal–Wallis test and Dunn’s post hoc test. Different letters in the graphs indicate significant differences between specific groups. Outliers were also presented on the graphs. Statistical differences detected with post hoc test were indicated with letters, where different letters correspond to statistical significance (p < 0.05) between groups.
ACTH – adrenocorticotropic hormone; DMSO – dimethyl sulfoxide.
Fig. 4. Relative mRNA expression of the galanin-like peptide (Galp), alarin (Ala) and receptors mRNAs in the adrenal gland after the administration of the peptides (Galp/Ala, 1 h, 24 h and 2 days). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the mRNA expression levels. Data points were presented as medians, 1st and 3rd quartiles (boxes) and ranges (whiskers – minimal and maximal values without outliers). Statistical analysis of the data was performed using the Kruskal–Wallis test and Dunn’s post hoc test. Different letters in the graphs indicate significant differences between specific groups. Outliers were also presented on the graphs. Statistical differences detected with post hoc test were indicated with letters, where different letters correspond to statistical significance (p < 0.05) between groups.
ACTH – adrenocorticotropic hormone; DMSO – dimethyl sulfoxide; Galr – galanin receptor.
Fig. 5. Relative mRNA expression of the Cyp11a1, Cyp11b1 and Cyp11b2 mRNAs in the adrenal gland after the administration of the peptides (galanin-like peptide (Galp)/alarin (Ala), 1 h, 24 h and 2 days). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the mRNA expression levels. Data points were presented as medians, 1st and 3rd quartiles (boxes) and ranges (whiskers – minimal and maximal values without outliers). Statistical analysis of the data was performed using the Kruskal–Wallis test and Dunn’s post hoc test. Different letters in the graphs indicate significant differences between specific groups. Outliers were also presented on the graphs. Statistical differences detected with post hoc test were indicated with letters, where different letters correspond to statistical significance (p < 0.05) between groups.
ACTH – adrenocorticotropic hormone; DMSO – dimethyl sulfoxide; Cyp11a1 – cholesterol desmolase; Cyp11b1 – 11β-hydroxylase; Cyp11b2 – aldosterone synthase.
Fig. 6. Level of corticosterone and ACTH in blood after peptides administration (galanin-like peptide (Galp)/alarin (Ala), 1 h, 24 h and 2 days). The analysis was performed with enzyme-linked immunosorbent assay (ELISA). Data points were presented as medians, 1st and 3rd quartiles (boxes) and ranges (whiskers – minimal and maximal values without outliers). Statistical analysis of the data was performed using the Kruskal–Wallis test and Dunn’s post hoc test. Different letters in the graphs indicate significant differences between specific groups
ACTH – adrenocorticotropic hormone; DMSO – dimethyl sulfoxide.

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