Chronic food restriction (FR), which retards many aging processes, enhances the endogenous diurnal peak of plasma total corticosterone (B) in young rats. Although the FR-dependent enhancement of total B disappears in aged rats, increased levels of the bioavailable fraction, free B, appear to be maintained. In young rats, we previously found that the FR-induced increase in the diurnal peak of total B is associated with increased adrenal response to corticotropin, also known as adrenocorticotropic hormone (ACTH). Here we show that the FR-enhanced adrenal response of total B to ACTH disappears with age but that the enhanced response of free B is maintained. We measured the endogenous diurnal peak and the response to ACTH of total and free B in 10-, 16-, and 22-month-old ad-libitum fed and FR male Fischer 344 rats in the afternoon, when plasma B peaks. At 10 and 16 months, FR rats showed enhanced total plasma B responses to ACTH relative to ad-libitum fed rats, but not at 22 months. By contrast, the response of free B to ACTH was enhanced by FR at all ages. The effect of FR on patterns of endogenous total and free diurnal B in these three age groups paralleled the ACTH-response data. The enhanced adrenocortical response of FR rats to ACTH does not reflect an increased expression of ACTH-receptor (ACTH-R) mRNA, because ACTH-R mRNA/microg adrenal RNA and ACTH-R mRNA/mg adrenal weight did not differ between ad-libitum fed and FR rats at any age.

Chronic food restriction (FR) of rats and mice results in moderate hyperadrenocorticism, which may play a role in activating cellular mechanisms that retard aging. Previously, we reported that the FR-induced hyperadrenocorticism is not due to an activated hypothalamo-pituitary unit. Therefore, we investigated in a series of experiments whether adrenal responsiveness to adrenocorticotropic hormone (ACTH), in vitro and in vivo, is enhanced by FR. Three mo-old male Fischer 344 rats were either given free access to food (AL rats) or restricted to 60% of food consumed by AL rats (FR rats) from 6 wk of age. They were killed by decapitation in the morning (AM) and afternoon (PM) when endogenously circulating corticosterone levels are at their nadir and peak, respectively. In vitro, adrenal glands from FR rats (1.5 mo FR) produced more corticosterone per mg at all doses of ACTH than those from AL rats in both the AM and PM (diet main effect, P < 0.001). FR (1.5 to 2.5 mo) also enhanced adrenal responsiveness to physiologic (diet main effect, P < 0.05) and superphysiologic (diet main effect, P < 0.001) levels of ACTH administered in vivo to dexamethasone-treated rats. ACTH-receptor (ACTH-R) mRNA, normalized to adrenal mass or to total RNA, was not influenced by FR (1.5 mo). However, adrenal ACTH-R mRNA, as well as adrenal mass, per unit body weight was greater in FR than in AL rats (diet main effect, P < 0.001). These results indicate that enhanced adrenocortical responsiveness to ACTH plays a major role in the hyperadrenocortical state of chronically FR rats.

Because neuroendocrine mechanisms may contribute to the antiaging effects of food restriction (FR), we measured the effect of FR on mRNAs encoding anterior pituitary (AP) tropic hormones. Slot blots or RNase protection assays were done on AP RNA from 3-, 6-, 12-, 18- and 24-mo-old male F344 rats consuming food ad libitum (AL) or food restricted (FR; to 60% of AL food intake) from 6 wk. Both AL and FR rats gained body weight during the study (P < 0.05), but FR rats weighed approximately 40% less (P < 0.0001). Messenger RNA levels were expressed in two ways, i.e., per total AP and per microgram total AP RNA. Proopiomelanocortin (POMC) mRNA/microg RNA was higher (P < 0.0005) in FR than in AL rats at all ages. Thyroid-stimulating hormone (TSH) beta mRNA declined with age (P < 0.05) in AL but not FR rats and was reduced by FR up to 12 mo (P < 0.01). Growth hormone (GH) mRNA/microg RNA declined with age (P < 0.05) in AL but not FR rats, and total GH mRNA in the AP was reduced by FR at early ages (P < 0.05). FR reduced prolactin (PRL) mRNA and its age-related increase (P < 0.0005). Levels of luteinizing hormone (LH) beta and follicle-stimulating hormone (FSH) beta mRNAs did not differ between AL and FR rats until 12 mo, but thereafter rose in FR (LH beta mRNA; P < 0.01, FSH beta mRNA; P < 0.05). Many of these changes in gene expression corroborate previously reported hormonal changes in FR rodents and mutant mice with extended life spans, and thus provide further support for the hypothesis that an altered hormonal milieu contributes to the antiaging effects of food restriction.

The aim of this study was to determine if long-term treatment with melatonin (MEL), a purported anti-aging agent, was as effective as calorie restriction (CR) in modulating immune parameters in aging Fischer 344 male rats. Splenic lymphocytes were isolated from 17-month-old rats that, beginning at 6 weeks of age, were treated with MEL (4 or 16 μg/ml in drinking water) and from 17-month-old rats fed ad libitum (AL) or rats fed a CR diet (55% of AL intake). The number of splenic T cell populations and T cell subsets was measured by flow cytometry, the proliferative response of splenocytes to Concanavalin A (Con A) and lipopolysaccharide (LPS) was measured by [3H]thymidine incorporation, and the induction of cytokine production (IL-2 and IFN-γ) was measured by ELISA assay. In addition, the level of the natural killer (NK) cell activity was assessed by fluorimetric assay. CR rats had a higher number of lymphocytes expressing the naïve T cell marker (CD3 OX22) than AL rats (P < 0.05). CR rats also showed greater induction of proliferative response, IL-2 and IFN-γ levels following Con A simulation, and NK cell activity than AL rats (P < 0.05). MEL-treated rats did not differ from AL rats in any of these parameters or in any other measurement. These results indicate that MEL treatment is unable to modulate immune function in a manner comparable with that of CR.