Aging-dependent changes in the effect of daily melatonin supplementation on rat metabolic and behavioral responses.

Pineal melatonin secretion has been reported to commonly decrease with aging, whereas intra-abdominal adiposity, plasma insulin and plasma leptin levels tend to increase. We recently demonstrated that daily melatonin administration starting at middle age suppressed male rat intra-abdominal fat, plasma leptin and plasma insulin to youthful levels, suggesting that aging-related changes in pineal melatonin secretion and in energy regulation may be functionally related. Accordingly, we have now investigated the effects of daily melatonin treatment on energy regulation in young versus middle-aged male Sprague Dawley rats. Addition of melatonin to the drinking water (0.2 microg/mL) produced nocturnal and diurnal plasma melatonin concentrations in middle-aged rats (12 months) equivalent to those of young adult (5 months) rats. Administration of this melatonin dosage every day for 10 wk starting at 10 months of age suppressed (P < 0.01) relative intra-abdominal fat, non-fasted plasma insulin and plasma leptin by 27, 39, and 51%, respectively (vs. vehicle-treated controls). In contrast, administration of melatonin for 10 wk starting at 3 months of age did not significantly alter (P> 0.10) any of these parameters. The melatonin administration stimulated (102%, P < 0.001) behavioral responsiveness of the middle-aged rats in a test of response to novelty, restoring youthful levels, but did not significantly alter behavioral responsiveness of the young rats. These results suggest that suppression of intra-abdominal adiposity and plasma leptin and insulin levels and stimulation of behavioral responsiveness in response to daily exogenous melatonin begins at middle age, coincident with and likely dependent upon the aging-associated decline in endogenous pineal melatonin secretion. These results further suggest that appropriate melatonin supplementation may potentially provide therapy or prophylaxis not only for the insulin resistance, increased intra-abdominal fat and resulting pathologies that occur with aging, but also for some aging-associated behavioral changes.

Daily melatonin administration at middle age suppresses male rat visceral fat, plasma leptin, and plasma insulin to youthful levels.

Human and rat pineal melatonin secretion decline with aging, whereas visceral fat and plasma insulin levels increase. Melatonin modulates fat metabolism in some mammalian species, so these aging-associated melatonin, fat and insulin changes could be functionally related. Accordingly, we investigated the effects of daily melatonin supplementation to male Sprague-Dawley rats, starting at middle age (10 months) and continuing into old age (22 months). Melatonin was added to the drinking water (92% of which was consumed at night) at a dosage (4 microg/ml) previously reported to attenuate the aging-associated decrease in survival rate in male rats, as well as at a 10-fold lower dosage. The higher dosage produced nocturnal plasma melatonin levels in middle-aged rats which were 15-fold higher than in young (4 months) rats; nocturnal plasma melatonin levels in middle-aged rats receiving the lower dosage were not significantly different from young or middle-aged controls. Relative (% of body wt) retroperitoneal and epididymal fat, as well as plasma insulin and leptin levels, were all significantly increased at middle age when compared to young rats. All were restored within 10 weeks to youthful (4 month) levels in response to both dosages of melatonin. Continued treatment until old age maintained suppression of visceral (retroperitoneal + epididymal) fat levels. Plasma corticosterone and total thyroxine (T4) levels were not significantly altered by aging or melatonin treatment. Plasma testosterone, insulin-like growth factor I (IGF-I) and total triiodothyronine (T3) decreased by middle age; these aging-associated decreases were not significantly altered by melatonin treatment. Thus, visceral fat, insulin and leptin responses to melatonin administration may be independent of marked changes in gonadal, thyroid, adrenal or somatotropin regulation. Since increased visceral fat is associated with increased insulin resistance, diabetes, and cardiovascular disease, these results suggest that appropriate melatonin supplementation may potentially provide prophylaxis or therapy for some prominent pathologies associated with aging.

Effects of chronic nicotine treatment and withdrawal on hypothalamic proopiomelanocortin gene expression and neuroendocrine regulation.

Considerable evidence suggest that some responses to smoking and nicotine are mediated by forebrain beta-endorphinergic opioid mechanisms. It has also been demonstrated that nicotine stimulates rat tuberoinfundibular dopaminergic activity. Since we have proposed that interactions between mediobasohypothalamic (MBH) dopaminergic and beta-endorphinergic mechanisms have a key role in neuroendocrine integration, we investigated the effects of chronic nicotine treatment and withdrawal on: (1) MBH concentrations of proopiomelanocortin (POMC, precursor for beta-endorphin biosynthesis) mRNA; (2) MBH concentrations of tyrosine hydroxylase (TH, rate limiting enzyme in catecholamine biosynthesis) mRNA; (3) corresponding serum prolacin, corticosterone, luteinizing hormone (LH), and testosterone concentrations. POMC and TH mRNA levels were measured by RNase protection/solution hybridization assay; serum hormone levels were measured by radioimmunoassay. Adult male rats received subcutaneous injections of either nicotine or saline during the dark period of each day on an increasing frequency (1-3 injections/day) and dosage (0.4-0.5 mg nicotine/kg body weight) schedule over 4 weeks. The rats were sacrificed after 4 weeks treatment and at 1, 3, 7, 14 and 21 days withdrawal. Chronic daily nicotine administration induced significant changes in serum corticosterone, serum prolactin, MBH TH mRNA, and MBH POMC mRNA concentrations that tended to persist through day 3 of withdrawal; serum prolactin and MBH POMC mRNA concentrations were suppressed whereas serum corticosterone and MBH TH mRNA concentrations were stimulated. None of the parameters were significantly different from control levels following 7 or more days of withdrawal from nicotine, except for a significant decrease of MBH POMC mRNA concentrations on day 21. Chronic daily nicotine or withdrawal did not significantly alter serum LH or testosterone concentrations. These results suggest that chronic nicotine inhibited POMC gene expression and thus, probably, biosynthesis of beta-endorphin and other opiomelanocortins. We hypothesize that suppression of forebrain beta-endorphin synthesis in response to long-term nicotine exposure produces a chronically opioid deficient condition which may play an important role in maintaining nicotine self-administration and in mediating some changes during the nicotine withdrawal syndrome.

Diurnal modulation of rat hypothalamic gonadotropin-releasing hormone release by melatonin in vitro.

The mechanism(s) by which melatonin (MEL) can regulate gonadotropin secretion remains unresolved. Accordingly, we used acute in vitro incubations of male rat hypothalamic tissues to investigate effects of MEL on hypothalamic gonadotropin-releasing hormone (GnRH) secretion. At 10:00 h (3.5 after lights on), addition of 1 nM MEL to the medium inhibited (p = 0.028) GnRH release from the median eminence (ME) by 24%, 100 nM MEL had no significant effect on GnRH release, and 10 microM MEL tended to inhibit (p = 0.056, 21%) GnRH release. At 15:00 h, none of these MEL dosages significantly altered GnRH release from the ME. When the effect of a lower range of MEL dosages was evaluated, 1 nM MEL again inhibited (24%, p = 0.032) GnRH release from the ME at 10:00 h, and a linear dose-response relationship between initially increasing dosages (0, 0.01, 0.1, 1 nM) of MEL treatment and decreasing GnRH release was evident (r = 0.88), although a further 10 fold increase in MEL dosage (10 nM) resulted in a loss of suppression by MEL. Treatment with these dosages of MEL did not significantly alter ME GnRH release at 15:00 h. In contrast to the results with the ME alone, treatment with 0.1 microM MEL increased (p = 0.018) GnRH release from the arcuate-median eminence region (ARC-ME) by 60.2% at 10:00 h, whereas treatment with either 0.001 or 10 microM MEL did not significantly affect GnRH release. Treatment with these dosages of MEL did not significantly alter ARC-ME GnRH release at 15:00 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Positive correlation between proopiomelanocortin and tyrosine hydroxylase mRNA levels in the mediobasohypothalamus of ovariectomized rats: response to estradiol replacement and withdrawal.

We have investigated putative dopaminergic regulation of opiomelanotropinergic activity in the arcuate/periarcuate mediobasohypothalamus (MBH) by assessing the changes in MBH tyrosine hydroxylase (TH; rate-limiting enzyme in catecholamine synthesis) and proopiomelanocortin (POMC; opiomelanotropin precursor) mRNA levels under conditions in which endogenous tuberinfundibular dopaminergic activity exhibits marked changes. Adult Sprague-Dawley rats were sacrificed at 09.00 and 15.00 h, and individual MBH POMC and TH cytoplasmic mRNA levels were simultaneously quantified by multiplex solution hybridization-RNase protection assay with protected fragments separated by polyacrylamide gel electrophoresis. In ovariectomized (OVX) rats treated for 3 days with low-dose estradiol (E2) implants (resulting in 18 +/- 4 pg E2/ml serum), the MBH levels of POMC and TH mRNAs were approximately 17 and 31% lower than those measured in OVX controls, respectively. In OVX rats implanted for 20 days with larger E2 implants (99 +/- 9 pg E2/ml serum), POMC and TH mRNA levels were approximately 29 and 41% lower than in OVX controls, respectively. Additional groups were exposed to the higher E2 dose for 20 days and then killed 10 or 20 days after removal of the E2 implant. In these rats, POMC mRNA levels rebounded to the same level seen in OVX controls, while TH mRNA levels even exceeded control values by 22-27%. TH and POMC mRNA levels did not change significantly between 09.00 and 15.00 h, except 10 days after removal of the E2 implants, when 09.00 h POMC mRNA levels were higher than the 15.00 h levels. MBH POMC and TH mRNA levels were positively correlated with each other within individual animals. This correlation is maintained when both POMC and TH mRNA levels are suppressed in response to both 3-day low-dose and 20-day high-dose E2 treatment. However, although rat MBH opiomelanotropinergic and tuberoinfundibular dopaminergic mRNA biosynthesis thus appear to be positively correlated, the coregulation or functional interactions of these two neuronal systems remain to be determined.

Alterations of the adrenocorticotropin-cortisol axis in normal weight bulimic women: evidence for a central mechanism.

We studied pituitary-adrenal function in eight women with normal weight bulimia and seven normal women by measuring plasma ACTH and serum cortisol levels at 20-min intervals for 24 h and the responses to human CRH (hCRH) and to a noon meal. The bulimic women had increased 24-h transverse mean plasma ACTH [1.09 +/- 0.06 (+/- SE) vs. 0.75 +/- 0.14 pmol/L; P less than 0.05] and serum cortisol (235 +/- 21 vs. 152 +/- 9 nmol/L; P less than 0.005) concentrations. While the 24-h ACTH and cortisol pulse frequencies were unaltered, the bulimic women had higher (P less than 0.05) mean peak ACTH (1.46 +/- 0.09 vs. 1.03 +/- 0.15 pmol/L) and cortisol values (331 +/- 33 vs. 239 +/- 17 nmol/L). Despite having higher mean and peak plasma ACTH and serum cortisol values, the bulimic women had a blunted response of both ACTH (P less than 0.001) and cortisol (P less than 0.005) to hCRH, which included a lower mean maximal plasma ACTH response, decreased (P less than 0.05) integrated area under the ACTH response curve (161 +/- 12 vs. 231 +/- 23 pmol/min.L), a lower (P less than 0.05) maximum cortisol response (284 +/- 35 vs. 413 +/- 19 nmol/L), and decreased (P less than 0.05) area under the cortisol curve (11.1 +/- 1.9 vs. 15.9 +/- 1.3 X 10(3) nmol/min.L). The circadian variations of both ACTH and cortisol were maintained in the bulimic women; the nadir and acrophase times were similar to those of the normal women. However, the rise in serum cortisol that occurred within 1 h after the lunch meal in the normal women (104 +/- 35 nmol) did not occur in the bulimic women (P less than 0.05). These data demonstrate that marked changes in hypothalamic-pituitary-adrenal function occur in bulimia in the absence of weight disturbance and suggest central activation of CRH and/or synergistic factors as well as alterations in signals from gut to brain in this syndrome.

Human fetal hypothalamic GnRH neurosecretion: dopaminergic regulation in vitro.

An in-vitro perifusion system was used to investigate GnRH release from fetal (21-23 weeks gestation) human hypothalami in response to dopamine (DA) and the DA receptor antagonist haloperidol. Administration of 1 mumol/l DA during five perifusions in which 1 mumol/l haloperidol was added to the medium failed to alter basal GnRH release. In contrast DA evoked a rapid and sustained 95.8 +/- 20.3% increase (P less than 0.01) in GnRH release during five matching perifusions with medium containing the alpha-adrenergic antagonist phentolamine. While exposure to 0.01 mumol/l DA failed to alter basal GnRH release during three perifusions, 0.1 mumol/l DA elicited a 145.7 +/- 65.2% increase (P less than 0.05) in GnRH release in three matching perifusions, indicating a dose-dependent effect. These studies demonstrate that DA can stimulate in-vitro release of GnRH from the mid-gestation fetal human hypothalamus by a DA receptor mediated mechanism.

Gonadotropin-releasing hormone neurosecretion in the human hypothalamus: in vitro regulation by dopamine.

An in vitro perifusion system was used to investigate GnRH release from adult human hypothalami in response to dopamine (DA) and the DA receptor antagonist haloperidol (HAL). Administration of a 1-microM pulse of DA consistently elicited a mean +/- SE 218 +/- 59% increase (P less than 0.05; n = 5) in GnRH release, whereas 1 microM HAL had no effect. Administration of 1 microM DA during three perifusions in which 1 microM HAL was added to the medium failed to alter basal GnRH release. In contrast, DA did evoke an acute 98 +/- 39% increase (P less than 0.06) in GnRH release during three matching perifusions with medium containing the alpha-adrenergic antagonist phentolamine. These studies demonstrate that DA can stimulate in vitro release of GnRH from the adult human hypothalamus by a DA receptor-mediated mechanism.

Progesterone and 20 alpha-hydroxyprogesterone stimulate the in vitro release of GnRH by the isolated mediobasal hypothalamus.

An in vitro perifusion system was used to investigate the effect of progesterone (P4) and 20 alpha-hydroxyprogesterone (20 alpha-OHP) on the release of GnRH from isolated hypothalamic tissue of the adult ovariectomized estradiol-17 beta primed rat. Pulse delivery of P4 stimulated GnRH release not only from the isolated mediobasal hypothalamus-anterior hypothalamus-preoptic area (MBH-AHPOA) complex but also from the MBH alone. Release of GnRH from the MBH was also increased by 20 alpha-OHP. These results suggest that the in vivo increase of circulating P4 or 20 alpha-OHP associated with the initiation of the preovulatory LH surge may play a role in regulating the timing or amplitude of this surge by directly stimulating the release of GnRH from the MBH.