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An adverse endogenous environment during early life predisposes the organism to develop metabolic disorders. We evaluated the impact of intake of an iso-caloric fructose rich diet (FRD) by lactating mothers (LM) on several metabolic functions of their male offspring. On postnatal d 1, ad libitum eating, lactating Sprague-Dawley rats received either 10% F (wt/vol; FRD-LM) or tap water (controls, CTR-LM) to drink throughout lactation. Weaned male offspring were fed ad libitum a normal diet, and body weight (BW) and food intake were registered until experimentation (60 d of age). Basal circulating levels of metabolic markers were evaluated. Both iv glucose tolerance and hypothalamic leptin sensitivity tests were performed. The hypothalamus was dissected for isolation of total RNA and Western blot analysis. Retroperitoneal (RP) adipose tissue was dissected and either kept frozen for gene analysis or digested to isolate adipocytes or for histological studies. FRD rats showed increased BW and decreased hypothalamic sensitivity to exogenous leptin, enhanced food intake (between 49-60 d), and decreased hypothalamic expression of several anorexigenic signals. FRD rats developed increased insulin and leptin peripheral levels and decreased adiponectinemia; although FRD rats normally tolerated glucose excess, it was associated with enhanced insulin secretion. FRD RP adipocytes were enlarged and spontaneously released high leptin, although they were less sensitive to insulin-induced leptin release. Accordingly, RP fat leptin gene expression was high in FRD rats. Excessive fructose consumption by lactating mothers resulted in deep neuroendocrine-metabolic disorders of their male offspring, probably enhancing the susceptibility to develop overweight/obesity during adult life., Facultad de Ciencias Exactas, Facultad de Ciencias Médicas

Neonatal treatment with monosodium glutamate (MSG) induces severe neuronal damage in selected brain areas, which in turn results in a number of neuroendocrine abnormalities during adult life. The present study was designed to determine what effects this partial and selective denervation of the hypothalamic-pituitary axis may have on the sensitivity of two key components of that axis, the median eminence (ME) and the anterior pituitary (AP). In order to test any changes in response that may occur after MSG treatment, the release of several peptide hormones from either the ME or the AP was evaluated in vitro, employing specific or general secretagogues. Male newborn pups of the Holtzman strain were injected with MSG every other day for 5 days, starting on day 2 of life; littermate controls received an injection of 10% NaCl. All animals were used when adult, at about 5-7 months of age. After decapitation, ME and AP tissues from control and MSG-treated rats were dissected out and incubated in vitro. Release of LHRH, SRIF, arginine vasopressin, and oxytocin from the ME was measured by direct RIA, under basal conditions and after stimulation with high K+ medium (28 mM). The results clearly indicate a marked hyperresponse of the release of each of the four neuropeptides by ME fragments from MSG-lesioned animals. The altered release was not attributable to changes in ME peptide content. In the case of the AP, the release of ACTH, LH, PRL, and GH in response to high K+ or, in some cases, to specific releasing factors, was evaluated in a dispersed cell preparation. The release of all four protein hormones was increased by high K+, and again the MSG-lesioned rats showed a pronounced hyperresponse. Corticotropin-releasing factor, at concentrations of 10(-9) and 10(-8) M enhanced ACTH release from control and MSG-lesioned rats, but the latter presented a marked hyperresponse. A similar observation on LH release was seen after stimulation with LHRH (10(-9) M). The results indicate that the neuronal damage induced by neonatal MSG treatment results in a generalized hyperresponsiveness in vitro to either specific or general secretagogues by ME or AP tissues, which may suggest the development of a denervation-type supersensitivity in the hypothalamic-pituitary axis. Since the release of these peptide hormones is sluggish in MSG-lesioned rats under in vivo conditions, it seems plausible to conclude that the major defect after the neurotoxin damage may reside in the loss of neurotransmitter systems normally innervating and regulating the activity of the peptidergic neurons.

The present studies were designed to test if estrogens influence basal and angiotensin II (AII)-stimulated ACTH release and the binding characteristics of AII receptors in isolated anterior pituitary (AP) cells from estrogen-deplete and estrogen-replete rats incubated in vitro. AP cells were obtained from various adult donors: randomly cycling (rc), 10-day ovariectomized (Ovx), Ovx with estradiol restored at a circulating physiological level (Ovx + lEB), Ovx with estradiol at a supraphysiological circulating level (Ovx + hEB); and male (m) rats. The amount of ACTH released under basal conditions was similar in the rc, Ovx + lEB, and m groups, although this amount was significantly greater (P less than 0.02) than that in the Ovx and Ovx + hEB groups. The ACTH-releasing activity (CRA) of AII was concentration dependent (10(-9)-10(-6) M) in all cells. The rank order of the CRA of AII in the groups varied as follows: rc = Ovx + lEB greater than Ovx = Ovx + hEB = m. The slopes of the AII responses were similar. Estradiol addition in vitro did not modify either basal or AII-stimulated ACTH release in any group of cells. AII binding studies indicated that AP cells from donors with different circulating estradiol levels had similar apparent equilibrium dissociation constants (Kd, 16-30 nM). However, the maximum binding capacities in AP cells from the m, Ovx, and Ovx + hEB groups (40, 51, and 49 fmol/10(6) cells, respectively) were significantly lower (P less than 0.05) than those in the rc and Ovx + lEB groups (77 and 81 fmol/10(6) cells, respectively). In summary, these studies indicate that 1) circulating levels of estradiol are able to modulate spontaneous ACTH release by dispersed AP cells; and 2) circulating estradiol, at a lower or higher level than that during the estrous cycle, decreases the in vitro ACTH-releasing activity of AII, possibly through a reduction in the number of AP AII receptors. These results further suggest that estrogen is likely to have a physiological role in corticotropic function.

The present studies were designed to evaluate the binding characteristics of arginine vasopressin (AVP), using rat anterior pituitary dispersed cells, in correlation with the biological activity of the peptide. Synthetic AVP released ACTH from dispersed anterior pituitary cells in a concentration-dependent fashion, with a minimal effective dose of 10(-10) M. [3H] AVP, the ligand for binding studies, showed full biological activity at different concentrations. Saturable and high affinity binding sites for [3H]AVP were obtained using dispersed anterior pituitary cells. Specific binding reached equilibrium by 180 min at 22 C, and rapid and complete dissociation was obtained after the addition of excess AVP. Scatchard analysis of the data indicated a single class of binding sites, with an apparent Kd of 1.63 nM and a binding capacity (Bmax) of 10.7 fmol/10(6) cells, at 37 C. When cells were incubated at 22 C, Kd (7.63 nM) and Bmax (39 fmol/10(6) cells) values were within the same order of magnitude. AVP effectively inhibited [3H]AVP binding with an IC50 of 1.5 X 10(-7) M, while oxytocin showed a somewhat higher IC50 (0.9 X 10(-6) M) and did not achieve complete inhibition of binding. An AVP analog with a ring substitution ( Asu1 ,6 AVP) showed decreased displacement capacity. Both oxytocin and the AVP analog showed weak ACTH-releasing activity compared to synthetic AVP. This indicates that modifications in the tail and/or ring portion of the AVP molecule reduce both the binding affinity and ACTH-releasing activities of these peptides. Other structurally unrelated peptides with intrinsic ACTH-releasing activity, such as rat corticotropin-releasing factor and angiotensin II, had no affinity for AVP-binding sites. The results indicate that specific AVP-binding sites in anterior pituitary cells are closely correlated with the intrinsic ability of the peptide to elicit ACTH release.

Many reports indicate that serotonin (5-HT) has a role in the regulation of the secretion of ACTH and related peptides. The present study was designed to evaluate in vitro a possible direct effect of 5-HT upon ACTH release from the anterior pituitary and also to determine if any interactions between the amine and different peptides with corticotropin-releasing activity (CRA) take place at the pituitary level. Dispersed anterior pituitary cells from donor adult male rats were incubated in the presence of 5-HT, quipazine (a 5-HT agonist), and metergoline (METER; a 5-HT receptor blocker) at different concentrations and also with synthetic arginine vasopressin (AVP) and corticotropin-releasing factor at several concentrations, alone or combined with some of the drugs mentioned above. In addition, several concentrations of acidic median eminence (ME) extracts were run alone or in combination with METER. The results indicate that 5-HT consistently elicited a small, but significant, release of ACTH in a dose-dep...