Federico Mallo

Exendin-4 Potently Decreases Ghrelin Levels in Fasting Rats

Ghrelin is a potent orexigenic and adipogenic hormone that strongly influences fat deposition and the generation of hunger in obesity. Indeed, hyperghrelinemia appears to promote an increase in food intake as seen in Prader-Willi Syndrome (PWS). Exendin (Ex)-4 is an agonist of the glucagon-like peptide (GLP)-1 receptor (GLP-1r) that has anorexigenic and fat-reducing properties. Here, we report that Ex-4 reduces the levels of ghrelin by up to 74% in fasted rats. These effects are dose dependent and long lasting (up to 8 h), and they can be detected after both central and peripheral administration of Ex-4. Suppression of ghrelin was neither mimicked by GLP-1(7–36)-NH2 nor blocked by the GLP-1r antagonist Ex-(9–39). Moreover, it was independent of the levels of leptin and insulin. The decrease in ghrelin levels induced by Ex-4 may explain the reduced food intake in fasted rats, justifying the more potent anorexigenic effects of Ex-4 when compared with GLP-1. As well as the potential benefits of Ex-4 in type 2 diabetes, the potent effects of Ex-4 on ghrelin make it tempting to speculate that Ex-4 could offer a therapeutic option for PWS and other syndromes characterized by substantial amounts of circulating ghrelin.

Evidence for a Direct Pituitary Inhibition by Free Fatty Acids of in vivo Growth Hormone Responses to Growth Hormone-Releasing Hormone in the Rat

The aim of this study was to determinate whether elevations in circulating free fatty acids (FFA) inhibit in vivo growth hormone (GH) responses to GH-releasing hormone (GHRH) by increasing hypothalamic somatostatin release or by acting directly on the pituitary. Thus, we have studied the effect of an Intralipid-heparin infusion on in vivo GH responses to GHRH in normal rats, normal rats passively immunized with antisomatostatin antiserum, rats with medial hypothalamic ablation, and hypophysectomized rats bearing two hypophyses under the renal capsule. Administration of 1 ml of Intralipid (500 microliters at -30 min and 500 microliters at -25 min) plus heparin (50 IU at -15 min) induced a marked decrease in GH responses to both 1 and 5 micrograms/kg of GHRH (p less than 0.01 at 5, 10 and 15 min for GHRH alone vs. GHRH plus Intralipid). A similar degree of inhibition was obtained after the administration of antisomatostatin antiserum (750 microliters i.v. at -60 min) previous to a challenge with 5 micrograms/kg of GHRH plus 1 ml of Intralipid (p less than 0.05 at 5 and 15 min, and p less than 0.01 at 10 min for GHRH plus normal rabbit serum vs. GHRH plus Intralipid plus antisomatostatin antiserum). Furthermore, administration of 1 ml of Intralipid also markedly reduced GH responses to GHRH in rats with medial hypothalamic ablation (p less than 0.01 at 5, 10, 15 and 30 min for GHRH alone vs. GHRH plus Intralipid) as well as in hypophysectomized rats bearing two hypophyses under the renal capsule (p less than 0.01 at 5, 10 and 15 min for GHRH alone vs. GHRH plus Intralipid).(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of His-dTrp-Ala-Trp-dPhe-Lys-NH2 (GHRP-6)-lnduced GH Secretion in the Rat

His-dTrp-Ala-Trp-dPhe,Lys-NH2(GHRP-6) is a synthetic compound that releases GH in a dose-response and specific manner in several species and that may well be related to an endogenous compound of similar structure. The aim of this study was to investigate the in vivo GH responses to GHRP-6 in pentobarbital anesthetized rats. Specifically and in order to avoid the influence of endogenous GHRH and somatostatin secretion we studied the GH responses to GHRP-6 in animals with surgical ablation of the hypothalamus, confirmed by histological assessment, as well as in hypophysectomyzed-transplanted rats bearing two hypophyses under the renal capsule. Since it has been previously reported that rats pretreated with GHRH (10 micrograms/kg i.p. every 12 h for 15 days) rather than saline-treated rats have greater GH responses to acutely administered GHRH, we compared the self-potentiating effect of chronic GH pretreatment with GHRP-6 (10 micrograms/kg i.p. every 12 h). Furthermore we also studied the influence of estrogens, glucocorticoids, free fatty acids (FFA) and bombesin on somatotroph responsiveness to GHRP-6 in intact rats. We found a greater GH response to GHRP-6 in rats that underwent a surgical ablation of the hypothalamus 36 h prior to the test than in sham-operated rats. A direct stimulatory effect of GHRP-6 on in vivo GH secretion was demonstrated by a clear GH response to GHRP-6 in hypophysectomyzed-transplanted rats. In addition, we found a similar response whether the animals were pretreated with GHRH or GHRP-6 over the previous 2 weeks. Finally, we found that both estrogen- and testosterone-treated rats have greater GH responses to GHRP-6 than untreated rats. On the other hand, chronic dexamethasone administration, acute elevation of circulating FFA levels and bombesin administration markedly inhibited GH responses to GHRP-6. In contrast to the effects exerted on GH responses to GHRP-6 estrogen administration led to a decrease in GH responses to GHRH while dexamethasone did not affect the GH responses to GHRH, highlighting a differential regulation of these hormones on somatotroph responsiveness to these peptides.

GLP-1(7-36)-amide and Exendin-4 Stimulate the HPA Axis in Rodents and Humans

Glucagon-like peptide-1 (GLP-1) is a potent insulinotropic peptide expressed in the gut and brain, which is secreted in response to food intake. The levels of GLP-1 within the brain have been related to the activity of the hypothalamic-pituitary-adrenal (HPA) axis, and hence, this peptide might mediate some responses to stress. Nevertheless, there is little information regarding the effects of circulating GLP-1 on the neuroendocrine control of HPA activity. Here, we have studied the response of corticoadrenal steroids to the peripheral administration of GLP-1 (7-36)-amide and related peptides [exendin (Ex)-3, Ex-4, and Ex-4(3-39)] in rats, mice, and humans. GLP-1 increases circulating corticosterone levels in a time-dependent manner, both in conscious and anaesthetized rats, and it has also increased aldosterone levels. Moreover, GLP-1 augmented cortisol levels in healthy subjects and diabetes mellitus (DM)-1 patients. The effects of GLP-1/Ex-4 on the HPA axis are very consistent after distinct means of administration (intracerebroventricular, iv, and ip), irrespective of the metabolic state of the animals (fasting or fed ad libitum), and they were reproduced by different peptides in this family, independent of glycaemic changes and their insulinotropic properties. Indeed, these effects were also observed in diabetic subjects (DM-1 patients) and in the DM-1 streptozotocin-rat or DM-2 muscle IGF-I receptor-lysine-arginine transgenic mouse animal models. The mechanisms whereby circulating GLP-1 activates the HPA axis remain to be elucidated, although an increase in ACTH after Ex-4 and GLP-1 administration implicates the central nervous system or a direct effect on the pituitary. Together, these findings suggest that GLP-1 may play an important role in regulating the HPA axis.

Exendin-4 increases blood glucose levels acutely in rats by activation of the sympathetic nervous system.

Exendin-4 (Ex-4), an agonist of the glucagon-like peptide-1 receptor (GLP-1R), shares many of the actions of GLP-1 on pancreatic islets, the central nervous system (CNS), and the gastrointestinal tract that mediates glucose homeostasis and food intake. Because Ex-4 has a much longer plasma half-life than GLP-1, it is an effective drug for reducing blood glucose levels in patients with type 2 diabetes mellitus (T2DM). Here, we report that acute administration of Ex-4, in relatively high doses, into either the peripheral circulation or the CNS, paradoxically increased blood glucose levels in rats. This effect was independent of the insulinotropic and hypothalamic-pituitary-adrenal activating actions of Ex-4 and could be blocked by a GLP-1R antagonist. Comparable doses of GLP-1 did not induce hyperglycemia, even when protected from rapid metabolism by a dipeptidyl peptidase IV inhibitor. Acute hyperglycemia induced by Ex-4 was blocked by hexamethonium, guanethidine, and adrenal medullectomy, indicating that this effect was mediated by sympathetic nervous system (SNS) activation. The potency of Ex-4 to elevate blood glucose waned with chronic administration such that after 6 days the familiar actions of Ex-4 to improve glucose tolerance were evident. These findings indicate that, in rats, high doses of Ex-4 activate a SNS response that can overcome the expected benefits of this peptide on glucose metabolism and actually raise blood glucose. These results have important implications for the design and interpretation of studies using Ex-4 in rats. Moreover, since there are many similarities in the response of the GLP-1R system across mammalian species, it is important to consider whether there is acute activation of the SNS by Ex-4 in humans.

Glucogenic supply increases ovulation rate by modifying follicle recruitment and subsequent development of preovulatory follicles without effects on ghrelin secretion

This study determined the effects of short-term energy inputs on ghrelin secretion and possible links with changes in the follicle population or the ovulation rate. Oestrous cycle was synchronized in 16 Manchega sheep using progestagen sponges and cloprostenol. Half of the animals were treated from days 0 to 4 by the oral administration, twice daily, of 200 ml of a glucogenic mixture containing 70% of glycerol, 20% of 1,2-propanediol and 10% of water; the control group received 200 ml water. The mean (+/-S.E.M.) plasma glucose increased immediately after the first administration (3.9+/-0.3 vs 3.0+/-0.1 mmol/l in control group, P<0.05), remaining statistically different during the treatment. However, plasma ghrelin levels were similar in both groups. On the other hand, the results indicated that short-term energy inputs modify ovulation rate (1.9+/-0.1 vs 1.3+/-0.2 in control group, P<0.05) by increasing the number of follicles able to be selected to ovulate during the period of treatment (>or=4 mm in size; 5.9+/-0.6 vs 4.3+/-0.4 at day 2, P<0.05). After sponge withdrawal, the number of these follicles decreased throughout follicular phase (5.8+/-0.8 to 1.5+/-0.4, P<0.0005) while the number of large follicles increased (>or=6 mm in size; 0.8+/-0.4 to 2.0+/-0.3, P<0.05); this would indicate an active growth of preovulatory follicles that were not found in the control group. Thus, the increases of ovulation rate by high-energy inputs would be caused by an enhancement in the developmental competence of preovulatory follicles.

Gut hormones ghrelin, PYY, and GLP-1 in the regulation of energy, balance, and metabolism

The first hormone discovered in the gastrointestinal tract was secretin, isolated from duodenal mucosa. Some years later, two additional gastrointestinal hormones, gastrin and cholecystokinin (CCK), were discovered, but it was not until the 1970s that gastrointestinal endocrinology studies became more prevalent, resulting in the discovery of many more hormones. Here, we examine the role of gut hormones in energy balance regulation and their possible use as pharmaceutical targets for obesity.

Retinoic acid inhibits in vivo thyroid-stimulating hormone secretion.

Retinoids are needed for normal growth and development. Retinoic acid (RA), an active metabolite of vitamin A, acts through nuclear receptors that belongs to the superfamily which also includes the T3 receptors and 1-25-dihydroxyvitamin D receptor. In order to assess whether RA is a regulator of in vivo thyroid-stimulating hormone (TSH) secretion, we studied the effect of RA administration on spontaneous basal TSH secretion and TSH responses to TRH in either euthyroid or hypothyroid rats. We found that rats treated with RA showed a decrease in spontaneous basal TSH levels and TSH responses to TRH. Similarly, RA administration to hypothyroid rats led to a decrease on TSH responses to TRH. Our data suggests that RA plays an important inhibitory role on in vivo secretion and this effect is unrelated to the thyroid status of the animals.

Effect of Retinoic Acid Deficiency on in vivo and in vitro GH Responses to GHRH in Male Rats

Retinoic acid has recently been shown to increase growth hormone (GH)-gene transcription rate and GH synthesis in vitro. To investigate the role retinoic acid plays in the neuroregulation of GH secretion we have studied GH responses to growth hormone-releasing hormone (GHRH) in retinoic acid-deficient rats. Compared to normally fed male rats, retinoic acid-deficient rats showed a marked impairment in body weight, which was statistically significant after 3 weeks and maximal after 5-6 weeks (p less than 0.001). Yet, in vivo GH responses to different doses of GHRH (1, 5 and 25 micrograms/kg) in pentobarbital-anesthesized rats were similar in both groups. Also, in vitro GH responses to GHRH, forskolin, and KCl were similar in perfused pituitary cells taken from control and retinoic acid-deficient rats. However, further studies carried out in freely-moving rats showed the typical GH secretory pattern usually found in male rats of the control group, while retinoic acid-deficient rats displayed a highly variable GH secretory pattern with GH peaks of much lower amplitude. Finally, after gel electrophoresis of in vitro 35S-labelled proteins, no differences were observed in the molecular forms of GH. Considering these findings on normal pituitary responsiveness and alterations in GH pulsatility, our data suggest that retinoic acid deficiency leads to an alteration in the neuroregulation of GH secretion at the central level.

Metabolic clearance rate of biosynthetic growth hormone after endogenous growth hormone suppression with a somatostatin analogue in chronic renal failure patients and control subjects

OBJECTIVE Several disturbances in the regulation of growth hormone secretion have been reported in chronic renal failure. The general assumption is that an altered hormonal clearance is at the basis of such GH alterations. Nevertheless, details of GH elimination kinetics in uraemia are not available. To clarify the role played by the kidney in its catabolism, GH elimination kinetics were studied in uraemic and control subjects after suppression of endogenous secretion of GH.