Chronic treatment with dexamethasone alters clock gene expression and melatonin synthesis in rat pineal gland at night

Figures & data

Table 1 Primers sequences for real-time (quantitative) RT-PCR assays of rat mRNA samples

Gene name	GeneBank N.	Amp Len	Primer	Sequences
Tpoh	NM_001100634	90	Forward	5′-CCTGGCTTCTCTTGGAGCTTC-3′
			Reverse	5′-ATCTTGCTTGCACAGTCCAAAC-3′
Aanat	NM_012818.2	89	Forward	5′-CCGGAGGATGCCACCA-3′
			Reverse	5′-CCGGATCTCATCCAAGTGGA-3′
Hiomt	NM_144759.2	100	Forward	5′-CCGCACCCACTTCCTGTC-3′
			Reverse	5′-CAAGAATGAAGAGGTCAGCGC-3′
Bmal1	NM_024362.2	77	Forward	5′-CCGATGACGAACTGAAACACC-3′
			Reverse	5′-TCTTCCCTCGGTCACATCCT-3′
Rev-erbα	NM_145775.1	81	Forward	5′-AGGTGACCCTGCTTAAGGCTG-3′
			Reverse	5′-ACTGTCTGGTCCTTCACGTTGA-3′
Per1	NM_001034125.1	71	Forward	5′-CTGCCTCAGGCCCTCGA-3′
			Reverse	5′-GTCCGAGTGGCCAGGATCTT-3′
Per2	NM_031678.1	75	Forward	5′-GCAGCCTTTCGATTATTCTCCC-3′
			Reverse	5′-GGACCAGCTAGTGTCCAGTGTG-3′
Cry1	NM_198750.2	74	Forward	5′-TTCGCCGGCTCTTCCAA-3′
			Reverse	5′-ATTGGCATCAAGGTCCTCAAGA-3′
Cry2	NM_133405.1	76	Forward	5′-TCAGCGTGAATGCAGGCA-3′
			Reverse	5′-AGGGCAGTAGCAGTGGAAGAAC-3′
InsR	NM_017071	90	Forward	5′-TCTGATTGTGCTATATGAAGTGAGCTATC-3′
			Reverse	5′-CCGCTCCAGGGCAAAAT-3′
Adcy1	NM_001107239.1	77	Forward	5′-TGCGGCTGGAAGATGAGAA-3′
			Reverse	5′-TCATCTCCATGGCGACATTC-3′
Glut1	NM_138827.1	111	Forward	5′-ACCCTGCATCTCATTGGTCT-3′
			Reverse	5′-GGCCACGATACTCAGATAGGAC-3′
Actb	NM_031144	97	Forward	5′-CTAGGAGCCAGGGCAGTAATCT-3′
			Reverse	5′-AAGACCTCTATGCCAACACAGTG-3′
Rpl37a	NM_001108801	93	Forward	5′-CGCTAAGTACACTTGCTCCTTCTG-3
			Reverse	5′-GCCACTGTTTTCATGCAGGAAC-3′
Actg1	NM_001127449	80	Forward	5′-TACCCTATTGAGCACGGCAT-3′
			Reverse	5′-CGCAGCTCGTTGTAGAAGGT-3′

Table 2 Effects of dexamethasone treatment on final body weight, the percentage of periepididymal fat pad, glycemia, insulin and HOMA. Eight rats were examined for each group

Groups	Control		DEX
	Mean	SE	Mean	SE
Initial body weight (g)	211.9	3.45	210.4	4.10
Final body weight (g)	229.4	4.46	174.9^*	3.98
% periepididymal fat pad (g/g body weight×100)	0.63	0.05	0.85^*	0.03
Glycemia (mg/dL)	86.33	2.49	91.67	1.72
Insulin (ng/mL)	0.65	0.14	5.28^*	0.23
HOMA	3.47	0.02	29.88^*	0.02

Notes: Mean values were significantly different from those of the control group (^*P<0.05; Student’s t-test).

Abbreviations: DEX, dexamethasone; HOMA, homeostasis model assessment; SE, standard error.

Figure 1 L/D glucose cycle profile of control rats (lines with circles) and treated with dexamethasone (lines with squares).

Notes: Animals were installed in 12 h L/D cycle, fed commercial Labina chow and water ad libitum. Blood drops were collected from the tail of the animal by capillarity in both experimental groups at 1 h intervals over a 24 h period. The ordinate values are expressed in mg/dL, and the white and black bars of the abscissa indicate the light and dark phases of the L/D cycle respectively. The results are represented as mean ± SEM. Two-way ANOVA, Bonferroni’s post-tests, ^*P<0.05, ^**P<0.01 (n=8 for both groups).
Abbreviations: ANOVA, analysis of variance; DEX, dexamethasone; L/D, light/dark; ZT, Zeitgeber time.

Figure 2 Plasma insulin profile in control rats (white bar) and rats treated with dexamethasone (black bars).

Notes: Animals were installed in 12 h light/12 h dark cycle, fed commercial Labina chow and water ad libitum. The animals were euthanized at the indicated times ZT (ZT17, ZT18, ZT19, ZT20, ZT21, and ZT22), and blood samples were collected and measured by radioimmunoassay (see Materials and methods section). The results are represented as mean ± SEM. Two-way ANOVA (with Bonferroni posttests), ^*P<0.05, ^***P<0.001 (n=6 for both groups).
Abbreviations: ANOVA, analysis of variance; DEX, dexamethasone; ZT, Zeitgeber time.

Figure 3 (A) Evaluation of plasma glucose during the glucose tolerance test. Blood samples were collected from control and dexamethasone-treated rats at ZT2 at baseline (0 min) and at 1, 5, 20, and 50 min after glucose injection. The results are represented as mean ± SEM. Two-way ANOVA, Bonferroni’s post-tests ^*P<0.05, ^**P<0.01, ^***P<0.001 (n=8 for both groups). (B) Evaluation of plasma glucose during the insulin tolerance test (K_ITT). Glucose was measured in control rats and rats treated with dexamethasone at ZT2 (0 min) and at 4, 8, 12, and 16 min after insulin injection. Student’s t-test ^#P<0.05.

Notes: The plasma glucose values were calculated by the rate of decay of plasma glucose over time and K_ITT was calculated (n=8 for both groups).
Abbreviations: ANOVA, analysis of variance; DEX, dexamethasone; ZT, Zeitgeber time.

Figure 4 AANAT activity, melatonin content, and plasma level of corticosterone were evaluated in Control and dexamethasone-treated rats (lines graph with circles and squares for Control and dexamethasone-treated rats, respectively).

Notes: The results are represented as mean ± SEM. Quantification of AANAT activity, the values of AANAT activity over time were organized in the form of linear graph (A) at the indicated ZT (ZT12, ZT14, ZT16, ZT18, ZT20, ZT22, and ZT24), the results are represented as mean ± SEM (Student’s t-test ^*P<0.05), and bar graph (Control rats [white bars] and rats treated with dexamethasone [black bars]) by calculating the mean total of AANAT activity (the mean total of AANAT activity ± SEM) (Student’s t-test ^*P<0.05) (B) The values of melatonin quantification are expressed in nanograms/gland and represented as mean total of melatonin content (Student’s t-test ^*P<0.05) (C) at the indicated ZT (ZT21, and ZT22). Plasma levels of corticosterone are expressed in nanograms/mL (D) by calculating the mean total and represented as mean ± SEM (Student’s t-test ^*P<0.05) (n=6 for both groups).
Abbreviations: AANAT, Arylalkylamine N-acetyltransferase; DEX, dexamethasone; ZT, Zeitgeber time.

Figure 5 Expression of Tph, Hiomt, Aanat, AC1, and B-adrenergic receptor in pineal glands isolated from control (line graph with circles) and treated with dexamethasone rats (line graph with squares).

Notes: Animals were installed in 12 h light/dark cycle, fed with commercial Labina chow and water ad libitum. The animals were sacrificed at ZT17, ZT18, ZT19, ZT20, ZT21, and ZT22 and the pineal gland was collected and processed for extraction of total RNA and mRNA detection by real-time polymerase chain reaction of Tph (A), Hiomt (B), Aanat (C), AC1 (D), and β-adrenergic receptor (E). Gene expression values are represented as mean ± SEM. Two-way ANOVA (with Bonferroni posttests), ^*P<0.05, ^**P<0.01 (n=6 for both groups).
Abbreviations: AANAT, Arylalkylamine N-acetyltransferase; AC1, Adenylate cyclase; ANOVA, analysis of variance; ZT, Zeitgeber time.

Figure 6 Expression of Bmal1, Rev-erbA, Per1, Per2, Cry1, Cry2, Inrs, and Glut1 in pineal glands isolated from control (white bar) and treated with dexamethasone rats (filled bars).

Notes: Animals were installed in 12 h light/dark cycle, fed with commercial Labina chow, and water ad libitum. The animals were killed at the times indicated and the pineal gland was removed and processed for extraction of total RNA and mRNA detection by real-time polymerase chain reaction of Bmal1 (A), Rev-erbA (B), Per1 (C), Per2 (D), Cry1 (E), Cry2 (F), Inrs (G), and Glut1 (H). Gene expression values are represented as mean ± SEM. Two-way ANOVA (with Bonferroni posttests), ^*P<0.05, ^**P<0.01, ^***P<0.001 (n=6 for both groups).
Abbreviations: ANOVA, analysis of variance; ZT, Zeitgeber time.