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Vol. 61, No. 3 Printed in U.S.A. 0021-972x/96/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1996 by The Endocrine Society Acipimox-Mediated Plasma Free Fatty Acid Depression per se Stimulates Growth Hormone (GH) Secretion in Normal Subjects and Potentiates the Response to Other GH-Releasing Stimuli* ROBERTO PEINO, FERNANDO CLARsA V. ALVAREZ, CARLOS CORDIDO, DIEGUEZ, ANGELA PERALVA, FELIPE F. CASANUEVA AND Hospital J. Canalejo (F.C.) and the Departments of Physiology (C.V.A., C.D.) and Medicine (A.P., R.P., F.F.C.), School of Medicine, and Complejo Hospitalario de Santiago, Santiago de Compostela University, Santiago de Compostela, Spain ABSTRACT Increases in plasma free fatty acids (FFA) inhibit the GH response to a variety of stimuli; however, the role of FFA depression in GH control is far from understood. In the present work, FFA reduction was obtained by the administration to normal subjects of acipimox, a lipid-lowering drug devoid of side-effects. Each subject tested underwent two paired tests. In one, acipimox was administered orally at a dose of 250 mg at -270 min and at a dose of 250 mg at -60 min; in the matched test, placebo was given at similar intervals. To induce GH release, four stimuli acting through different mechanisms were used: pyridostigmine (120 mg, orally) at -60 min, GHRH (1 cLg/kg, iv) at 0 min, GH-releasing peptide (GHRP-6; His-D-Trp-Ala-Trp-D-PheLys-NH,; 1 pg/kg, iv) at 0 min, and finally, GHRH plus GHRP-6 at the same doses at 0 min. GH secretion was analyzed as the area under the secretory curve (AUC; mean 2 SE, micrograms per L/120 min). Acipimox pretreatment aione (n = 6) induced a reduction in FFA levels compared with placebo treatment. The FFA reduction led to a sustained GH secretion that increased from 2.4 ? 1.8 pg/L at -120 min to 14.2 i 4.0 at 120 min. The GH AUC for placebo was 266 5 100, and that for acipimox was 1781 5 408 (P i 0.05). In the pyridostigmine-treated group (n = 61, the acipimox-pyridostigmine AUC (2046 -C 323) was higher (P < 0.05) than the placebo-pyridostigmine AUC (764 ? lOl), but was not different from the AUC of acipimox alone. Previous FFA reduction nearly doubled the GHRH-mediated GH secretion (n = 6; placebo-GHRH AUC, 1817 ? 365; acipimox-GHRH test, 3228 + 876; P < 0.05). A similar enhancement was observed when the stimulus employed was GHRP-6 (n = 6; placebo-GHRP-6 AUC, 2034 ? 295; acipimox-GHRP-6, 4827 2 703; P < 0.05). Furthermore, even the most potent GH stimulus known to date, i.e. GHRH plus GHRP-6, was enhanced by the FFA suppression (placeboGHRH-GHRP-6 AUC, 2034 + 277; acipimox-GHRH-GHRP-6,5809 ? 758; P < 0.05). The enhancing effect of lowering FFA levels was additive regardless of the stimulus employed. These results indicate that 1) FFA reduction per se stimulates GH secretion with a delayed time of action; 2) FFA reduction enhanced in an additive manner the GH secretion elicited by such different stimuli as pyridostigmine, GHRH, and GHRP-6; and 3) the observation that FFA reduction enhanced the response to the most potent GH stimulus, GHRH plus GHRP-6, suggests that FFA suppression acts by a separate mechanism. FFA reduction may have value in the clinical setting for assessing GH reserve. (J Clin Endocrinol Metub 81: 909913, 1996) G L-DOPA, deep sleep, physical exercise, or administration of GHRH or GHRP-6 (5-11). There is now compelling evidence indicating that the FFA blockade of GH secretion is exerted at the pituitary level, probably by direct inhibition of somatotroph function (11-13). More controversial is the role of FFA depression in GH control. The original study showing that FFA reduction by nicotinic acid administration stimulated GH release (14) was thought to be confounded by the intense side-effects of the drug. Other evidence that FFA depression was responsible for the secretion of GH came from use of the experimental drug BM 11.189,an adenosine derivative (15). The subsequent unavailability of the drug precluded further studies. Recent studies with acipimox, a nicotinic acid analog that blocks lipolysis and is devoid of side-effects (16, 17), have provided the best tool for further understanding the role of FFA depression in GH regulation. In the last few years considerable attention has been devoted to the series of hexapeptides devised by Bowers and co-workers (18, 191,of which GH-releasing peptide (His-DTrp-Ala-Trp-n-Phe-Lys-NH,, known asGHRP-6) is the most representative. GHRP-6 releasesGH through specific recep- H SECRETION is tightly linked to metabolic alterations as well as to variations in the intake or availability of lipids, amino acids, and carbohydrates (l-3). A classicfeedback relationship has been postulated between GH and the metabolically active component of lipids, i.e. the nonesterified fatty acids, also called free fatty acids (FFA). GH has a direct lipolytic effect on adipose tissue, leading to the release of glycerol, FFA, and ketone bodies (4). Pharmacological reduction of EEA is associatedwith GH release,whereas an increase in FFA reduces both basal and stimulated GH secretion (3). FFA elevations reduce or block GH secretion stimulated by a variety of stimuli or conditions, such as FFA depression, hypoglycemia, arginine infusion, protein meal, glucagon, Received May 18, 1995. Revision received July 18, August 1, 1995. Address all correspondence and requests for reprints nueva, M.D., Ph.D., P.O. Box 563, E-15780, Santiago Spain. *This work was supported by grants from the Fondo Sanitaria, Spanish Ministry of Health, and a Research Fundacion Ramon Areces. 1995. Accepted to: F. F. Casade Compostela, de Investigation Grant from the 909 910 PEINO tors (20-22) and by combined pituitary and hypothalamic stimulation (23, 24). This novel mechanism of action made GHRP-6 a convenient drug to study GH regulation in man (25-27). This study examined the effect of acipimox-induced FFA reduction on basal and stimulated GH secretion in normal subjects, using GH stimuli that operate at the hypothalamic (pyridostigmine), pituitary (GHRH), or combined (GHRP-6) levels. The aim of this study was to further define the mechanism of action by which FFA reduction operates to enhance GH secretion. JCE & M . 1996 Vol81 . No 3 ET AL. Placebo Placebo l Aciprmox 5-s-e 0 l Subjects 1-J I-v 1,10’1- o Placebo Aaplmox and Methods Eighteen normal male volunteers, aged 19.6 i- 0.3 yr (range, 18-23 yr) participated in this study after providing informed consent. All of them had normal life styles, were taking no medication, and were within 10% of their ideal body weight. The study was approved by the faculty bioethical committee. Tests were started at 0800 h after an overnight fast, with the subjects recumbent. An indwelling catheter was placed in a forearm vein and kept permeable with a slow infusion of 150 mmol/L NaCl. The first blood sample was obtained at -120 min, and additional blood samples were obtained at appropriate intervals over the following 4 h of testing. The paired tests were performed in random order 1 week apart, with each subject serving as his own control. Six of the volunteers were studied four times on different days. On day 1, placebo was administered at -270 and -60 min. On the second day, they received acipimox (Olbemox, Farmitalia, Barceloma, Spain) at a dose of 250 mg, orally, at -270 min and acipimox at a dose of 250 mg, orally, at -60 min. On the third day, they received pyridostigmine (Mestinon Roche, Madrid, Spain) at a dose of 120 mg, orally, at -60 min. Finally, on another day, they received 250 mg acipimox, orally, at -270 min and the same dose at -60 min plus 120 mg pyridostigmine, orally, at -60 min. A second group of six volunteers was tested for 2-day periods twice. On the first 2 days they were given GHRH [GHRH-(l-29)NHz; Geref, Serono, Madrid, Spain) at a dose of 1 pg/kg, iv, at 0 min, preceded by either placebo or acipimox pretreatment (250 mg, orally, at -270 min and 250 mg, orally, at -60 min). One month later, on each day, they were given GHRP-6 (His-o-Trp-Ala-Trp-o-Fhe-Lys-NH,; Peninsula Laboratories, Madrid, Spain), prepared as previously described (25), at a dose of 1 pg/kg, iv, at 0 min, preceded by either placebo or acipimox pretreatment (250 mg, orally, at -270 min and 250 mg, orally, at -60 min). The third group of six volunteers underwent two tests. On both occasions they received a combined administration of GHRH (1 pg/kg, iv) plus GHRP-6 (1 pg/kg, iv) at 0 min preceded by either placebo or, on the second day, acipimox pretreatment (250 mg, orally, at -270 min and 250 mg, orally, at -60 min). Serum GH concentrations were determined using a time-resolved fluoroimmunoassay (Delfia, Pharmacia, Barcelona, Spain) with a GH sensitivity of 0.02 pg/L and coefficients of variation of 6.3% (0.4 wg/L), 5.3% (10.2 pg/L), and 4.2% (43.4 pg/L). FFA levels were determined by a enzymatic calorimetric method (NEFA-HA, Wako, Zaragoza, Spain). All samples from a given subject were analyzed in the same assay run. Hormone levels are presented and analyzed as absolute values or as the mean GH peak. The areas under the secretory curve (AUC) were calculated by a trapezoidal method and compared between groups by the Wilcoxon rank test. The statistical level of significance was set at P < 0.05. Results The administration of acipimox induced a FFA reduction during the entire test (Fig. 1). The area under the curve (AUC) after placebo pretreatment (107.3 + 15.2 mmol/L +120 min) was significantly higher (P < 0.05) than that after acipimox pretreatment (25.6 + 7.8). The acipimox-induced FFA reduction elicited a sustained increase in GH levels from 2.4 2 1.8 pg/L at -120 min to 14.2 2 4.0 pg/L at the end of the test -120 -60 0 30 Minutes 60 90 120 FIG. 1. Mean + SE serum GH levels in six normal subjects after the administration of placebo in two different tests at 0 min. In one test, subjects were pretreated with acipimox at a dose of 250 mg, orally, at -270 min and at a dose of 250 mg, orally, at -60 min; in the matched test, placebo was administered at the same intervals. The FFA reduction was associated with higher (P < 0.05) GH values than those in placebo-pretreated subjects from -60 min and thereafter. 0 -120 Placebo -60 0 30 Minutes 60 90 120 FIG. 2. Mean ? SE serum GH levels in six normal subjects after the administration of pyridostigmine (120 mg, orally) at -60 min on 2 different days. In one test, subjects were pretreated with acipimox at a dose of 250 mg, orally, at -270 min and at a dose of 250 mg, orally, at -60 min; in the matched test, placebo was administered at the same intervals. (120 min), with a mean GH peak of 15.6 -C3.6 pg/L. The GH AUC was 266 -C 100 pg/L*120 min for placebo and 1781 t 408 pg/L.120 min for acipimox-treated subjects(P < 0.05;see Fig. 6). Administration of pyridostigmine (Fig. 2), a cholinergic drug that is thought to affect GH release by suppressing hypothalamic somatostatin, induced the expected moderate increase in GH levels, with a mean GH peak of 9.9 + 1.2 pg/L. When FFA were reduced by previous acipimox administration, an enhancement of pyridostigmine-mediated GH releasewas observed (mean GH peak, 18.3 -C1.7 pg/L). FFA DEPRESSION The effect of acipimox was simply additive, because the AUC of acipimox-pyridostigmine (2046 ? 323) was higher (P < 0.05) than that of placebo-pyridostigmine (764 -C 101), but was not statistically different from that of acipimox alone (1781 k 408; see Fig. 6). The pharmacological reduction in FFA also increased the GH releaseelicited by GHRH administration (Fig. 3). GHRHstimulated GH secretion in placebo-pretreated subjects (mean peak, 23.8 ? 4.8 pg/L) nearly doubled in acipimoxpretreated GHRH-stimulated subjects (mean peak, 54.5 + 14.3 pg/L). The action of FFA reduction was most evident when comparing the AUCs (see Fig. 6; 1817 2 365 for placebo-GHRH and 3228 2 876 for acipimox-GHRH; P < 0.05). A similar enhancement was observed when the stimulus employed was GHRP-6. As shown in Fig. 4, GHRP-6 induced a larger and more synchronized GH discharge than GHRH, with a mean peak of 33.3 2 5.8 pg/L, and the previous FFA drop due to acipimox pretreatment notably increased its effectiveness (mean peak, 64.9 2 9.0 pg/L). The placeboGHRP-6 AUC (2034 -C295) was significantly lower (P < 0.05) than that of acipimox-GHRP-6 (4827 2 703; see Fig. 6). The combined administration of GHRH and GHRP-6 in placebo-pretreated subjects induced the expected large GH discharge (mean peak, 58.3 -+-5.5 pg/L; AUC, 3791 + 277; Fig. 5). Even this potent stimulus was enhanced by FFA reduction causedby acipimox pretreatment (mean peak, 82.6 ?I 12.2 pg/L; AUC, 5809 2 758; P < 0.05). When the AUCs for all conditions in this study were compared (Fig. 6), the FFA reduction was seento further increase in an additive manner the responsesto all GH stimuli tested. The FFA reduction peusewas a more potent GH releaser than pyridostigmine and was as potent as the classicalGH secretagogues GHRH and GHRP-6. One subject in one test experienced a mild facial flushing 80 1 1 1 GHRH 0 Placebo Acipimox AND GH RELEASE 911 I 80 70 I GHRP-6 0 . -120 Placebo Aclpimox -60 T 0 30 60 SO 120 FIG. 4. Mean t- SE serum GH levels in six volunteers after the administration of GHRP-6 (1 fig/kg, iv) at 0 min on 2 different days. On one occasion, subjects were pretreated with acipimox at a dose of 250 mg, orally, at -270 min and at a dose of 250 mg, orally, at -60 min; in the matched test, placebo was administered at the same intervals. I 1 1 GHRH + GHRP- 6 100 90 -I 0 Placebo l Aciplmox 80 1 4 70- : g 60. b I 50- l -120 -60 0 30 Minutes 60 SO 120 FIG. 5. Mean t SE serum GH levels in six volunteers after the combined administration of GHRH (1 pg/kg, iv) plus GHRP-6 (1 pg/kg, iv) at 0 min on 2 different days. In one test, FFA levels were lowered by pretreatment with acipimox at a dose of 250 mg, orally, at -270 min and at a dose of 250 mg, orally, at -60 min; in the matched test, placebo was administered at the same intervals. -120 -60 0 30 Minutes FIG. 3. Mean t SE serum GH levels in administration of GHRH (1 pg/kg, iv) at On one occasion, subjects were pretreated of 250 mg, orally, at -270 min and at a -60 min; in the matched test, placebo same intervals. 60 SO 120 six volunteers after the 0 min on 2 different days. with acipimox at a dose dose of 250 mg, orally, at was administered at the 1.5 h after the first dose of acipimox. No side-effects were reported in the other tests Discussion FFA are common metabolites carried by the blood stream, and their levels oscillate widely during the day depending on PEINO ET AL. GHRH GHRP - 6 GHRH GHR+P - 6 FIG. 6. Mean t SE AUCs in five groups of six normal subjects stimulated with placebo, pyridostigmine, GHRH, GHRP-6, or GHRH plus GHRP-6 on 2 different days. In one test, the subjects were pretreated with acipimox (250 mg, orally, at -270 min and 250 mg, orally, at -60 min); in the matched test, placebo was administered at the same intervals. *, P < 0.05 us. the same stimulus without FFA reduction (placebo pretreated). +, P < 0.05, either higher or lower us. subjects treated with acipimox alone. the fed/fast state of the subject (28). These circulating compounds notably increase in some pathological states, and it is not known at present whether the abnormally high levels of FFA presented acutely in hypoxia and ketoacidosis or chronically in obesity and pregnancy could account for some of the morbid complications associated with such states. After their increasein plasma and despite the buffering effect of serum albumin, the amphiphilic FFA molecules rapidly partition into the cell plasma membrane, influencing the physicochemical state of lipid domains (29). After incorporation into the membrane, FFA perturb the bilayer structure of the membrane in a manner similar to some anesthetics, leading to alterations in membrane-cytoskeleton interactions and altering the functioning of the integral proteins (30-32). Considering that these integral membrane proteins are receptors, channels, or enzymatic systems implicated in transduction signals, it is easy to envision the degree of cell perturbation induced by an immoderate increasein plasmaFFA. Consistent with this view, it has been reported that FFA are able to alter such different cell functions as platelet aggregation, lymphocyte mitogenesis, or cell to cell substrate adhesion (33). We have also shown that FFA are capable of reversibly blocking the early intracellular signals elicited by epidermal growth factor in fibroblasts (34-36) and altering the gene expression of some hypothalamic hormones (37). Although the precise point of action of FFA is at present unknown, their effects are relevant and widespread. It hasbeen shown in vim that an increasein FFA blocks GH secretion elicited by all known stimuli (31, and this nonselective blockade is exerted directly on somatotroph cells (ll13). In vitro, FFA block, in minutes and in a dose-related manner, somatotroph function (11). Unfortunately, the effects on somatotroph function of acute FFA reduction have been not studied due to the unsuitability or unavailability of adequate lipid-lowering drugs. The use by Pontiroli and co-workers (16, 17) of acipimox, a new inhibitor of lipolysis JCE & M l 1996 Vol81 . No 3 devoid of significant side-effects in humans, opened a new way to perform such studies. In the present work, we took advantage of acipimox to obtain a well tolerated reduction in FFA levels, which lasted for hours, to further understand the mechanism of action of those compounds in both basal and stimulated GH secretion. The first observation of the present work is that FFA reduction per sewas able to significantly stimulate GH secretion. In fact, FFA reduction-mediated GH releasewas greater than 7 pg/L at 0 min and thereafter. FFA reduction was by no means a weak GH stimulus, considering that both the mean GH peak and the AUC were higher than those after pyridostigmine treatment and as large as those induced by either GHRH or GHRP-6, both administered at a saturating dose. Perhaps the most interesting observation of the present work was the long period needed for FFA reduction before releasing GH and the long lasting effect, with GH levels still rising at 120 min when the test was ended. This delayed effect is peculiar to FFA reduction and different from those of other stimuli such asGHRH and hypoglycemia, perhaps reflecting an intrinsic characteristic of the action of FFA. Conversely, to observe a blockade of GH secretion, FFA levels must be elevated for a long period before there is an effect on the GH response to stimuli (11). Acipimox-mediated FFA reduction was able to enhance the GH releaseelicited by four GH stimuli, each thought to act by a different mechanism. For example, pyridostigmine, an indirect cholinergic agonist, is widely accepted for releasing GH, operating at the hypothalamic level and through the inhibition of somatostatin release (38). GHRH acts directly at the pituitary, stimulating somatotroph cells. The main action of GHRP-6 is exerted at the hypothalamic level through undetermined mediators (24), with an ancillary stimulatory action at the somatotroph cell (20). Despite these different mechanismsof action, FFA reduction enhanced the GH secretory response in a consistent and additive manner. This additive action, i.e. the GH released by acipimox plus stimulus was the arithmetical sum of the GH released by acipimox alone and the stimulus alone, suggeststhat FFA reduction operates to release GH by mechanisms different from reduction of somatostatin release, release of endogenous GHRH, or release of the endogenous ligand of the GHRP-6 receptor. The observation that FFA reduction was able to further increase the effectiveness of GHRH plus GHRP-6, again in an additive manner, particularly emphasizes that the reduction in plasma FFA acts by a separate mechanism. Although that mechanism is not known, based on our previous work we postulated that a sustained plasma FFA reduction leads to a parallel reduction in the amount of FFA molecules residing in the plasma membrane, altering the physicochemical state of receptors, channels, or any other integral protein and making the somatotroph cell more sensitive to any stimulus. Data from cell biology studies support this working hypothesis. Plasma-borne FFA molecules already residing in the cell plasma membrane are not covalently linked; on the contrary, they are included in the bilayer as wedges (39). If the gradient of FFA from plasma toward membrane is not maintained, FFA molecules quickly disappear from the plasma membrane, altering its state (34). FFA DEPRESSION Considerable experimental work is needed to test this hypothesis. In conclusion, acipimox-induced FFA depression peu se resulted in delayed GH release.Furthermore, FFA reduction enhanced, in an additive manner, GH secretion elicited by diverse stimuli, including pyridostigmine, GHRH, GHRP-6, and GHRH plus GHRP-6. These results suggest that FFA reduction actsthrough a mechanism separatefrom that of the other stimuli. Considerable work is needed before it can be ascertained whether this GH stimulus can be used to assess the GH secretory reserve in the clinical setting. Acknowledgment The expert knowledged. technical assistance of Ms. Mary Lage is gratefully ac- References 1. Dieguez C, Page MD, Scanlon MF. 1988 Growth hormone neuroregulation and its alterations in disease states. Clin Endocrinol (0x0. 28:109-143. 2. Casanueva FF. 1992 Physiology of growth hormone secretion and action. Endocrinol Metab Clin North Am. 21:483-517. 3. Dieguez C, Casanueva FF. 1995 Influence of metabolic substrates and obesity on growth hormone secretion. 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