The effects of aging on biosynthetic processes in the rat hypothalamic osmoregulatory neuroendocrine system

Abstract

Elderly people exhibit a diminished capacity to cope with osmotic challenges such as dehydration. We have undertaken a detailed molecular analysis of arginine vasopressin (AVP) biosynthetic processes in the supraoptic nucleus (SON) of the hypothalamus and secretory activity in the posterior pituitary of adult (3 months) and aged (18 months) rats, to provide a comprehensive analysis of age-associated changes to the AVP system. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, we identified differences in pituitary peptides, including AVP, in adult and aged rats under both basal and dehydrated states. In the SON, increased Avp gene transcription, coincided with reduced Avp promoter methylation in aged rats. Based on transcriptome data, we have previously characterized a number of novel dehydration-induced regulatory factors involved in the response of the SON to osmotic cues. We found that some of these increase in expression with age, while dehydration-induced expression of these genes in the SON was attenuated in aged rats. In summary, we show that aging alters the rat AVP system at the genome, transcriptome, and peptidome levels. These alterations however did not affect circulating levels of AVP in basal or dehydrated states.

Keywords: Aging; Gene expression; Methylation; Peptidomics; Supraoptic nucleus; Vasopressin.

Fig. 1

Comparison of metabolic parameters in adult and aged rats. Physiological parameters—(A) body weight, (B) food intake, (C) water intake, (D) urine output, (E) extrarenal water secretion, and (F) urine osmolality—were recorded in adult (3-month old) and aged (18-month old) male rats (n = 6) housed in metabolic cages. **, p < 0.01; ***, p < 0.001 by the 2-way analysis of variance with Bonferroni post hoc test.

Fig. 2

Effect of aging on the rat AVP system. Adult and aged male rats were subjected to dehydration for 3 days and AVP measures were performed. (A) Body weights were recorded before and after dehydration (n = 11–14). (B) Plasma osmolality was measured (n = 19–23) by freezing point depression. The effect of aging on Avp expression was examined at the transcriptional level in control and dehydrated adult and aged rats. (C, D) The RNA expression level of Avp (both heteronuclear [hnAvp] and mature form) was examined by qRT-PCR (n = 6). AVP measures were performed on NILs and plasma extracts. (E) AVP content in NILs was measured by ELISA (n = 9–10). (F) Plasma AVP level was determined by radioimmunoassay (n = 9). *, p < 0.05; ***, p < 0.001 by the 2-way analysis of variance with Bonferroni post hoc test. ^#, p < 0.05 by unpaired t-test. Abbreviations: AVP, arginine vasopressin; DH, dehydrated; NIL, neurointermediate lobe.

Fig. 3

Peptide analysis in aged rats. Adult and aged male rats were subjected to dehydration for 3 days, and NIL peptide measures were performed by MALDI-TOF MS. (A–E) Signal intensity of peptides in NILs was measured by MALDI-TOF MS in individual tissue extracts from adult and aged rats in basal and dehydrated states (n = 11–16). (A) Control and dehydrated peptide profiles are easily classified by PCA according to PC1 (∼60% of variance) in adult rats; PCA plot is shown for the first 3 PCs. (B) Loading plot indicates that a small subset of peptides contributes to differences between control and dehydrated adult rats, among which are masses matching AVP, acetylated alpha-MSH, and di-acetylated alpha-MSH. (C) The AVP and OT signal decreases significantly, while the alpha-MSH signals and proSAAS (221-237) increase significantly with dehydration in adult rats. (D, E) The effect of age on peptide signals in the NIL. (D) The OT signals decreased, while the signals of POMC-derived peptides increased in aged compared to adult rats. (E) Peptide signals in the NIL of adult and aged rats subjected to 3 days of dehydration. The AVP-copeptin signal increased, whereas the proSAAS signal decreased with age. (F) Peptides uniquely altered as a function or aging or by just dehydration. (G) Relative RNA expression of proSAAS and OT in the SON of adult and aged rats. *p < 0.05 by unpaired t-test or peptide names beginning with * by Kruskal-Wallis tests. Abbreviations: AVP, arginine vasopressin; DH, dehydrated; NIL, neurointermediate lobe; PCA, principal component analysis; POMC, proopiomelanocortin.

Fig. 4

Epigenetic changes in Avp gene promoter in aging. Methylation status of the Avp promoter in the SON of control and dehydrated adult and aged male rats. (A) Relative mRNA expression of Dnmt1, Dnmt3a, Tet1, Tet2, and Tet3 was determined by qRT-PCR. (B) Diagram showing 7 CpG sites on the Avp promoter that were examined by colony-based PCR. (C) Representative tile diagrams showing the methylation status of 7 CpG sites for individual clones of the Avp promoter extracted from the SON. (D) Percentage of global methylation on this region of the Avp promoter in control and dehydrated adult and aged rats. (E) Percentage methylation of individual CpG sites on the Avp promoter in control and dehydrated adult and aged rats. *, p < 0.05; **, p < 0.01; ***, p < 0.001 by the 2-way analysis of variance with Tukey's post hoc test. ^#, p < 0.05 by unpaired t-test. Abbreviation: SON, supraoptic nucleus.

Fig. 5

Effect of aging on the expression of osmotically induced genes. Relative mRNA expression of genes involved in hyperosmotic stress in the SON of the hypothalamus in control and 3-day dehydrated adult and aged rats. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 by the 2-way analysis of variance with Bonferroni post hoc test. ^#, p < 0.05 by unpaired t-test. Abbreviations: DH, dehydrated; SON, supraoptic nucleus.

Fig. 6

Effect of aging on transcriptional and post-transcriptional Avp gene expression. The effect of aging on Avp poly(A) tail length in the SON in control and dehydrated adult and aged rats. (A) Diagram of poly(A) tail assay design for the rat Avp gene. (B) poly(A) tail length of the Avp mRNA was examined using a PCR-based poly(A) tail assay. Abbreviations: Ad, adult; Ag, aged; DH, dehydrated; RT, reverse transcription; SON, supraoptic nucleus.

Fig. 7

Modeling of molecular events in the SON in aging and dehydrated rats. (A) How aging may alter Avp expression in the SON compared to younger counterparts. (B) Proposed differential regulation of Avp expression in the SON as a function of both aging and dehydration. Abbreviation: SON, supraoptic nucleus.