As a control group, six normal subjects with a normal BMI were also studied. .... GHRH or the endogenous ligand for the GHRP-6 receptor as causative factors.
0021-972X/98/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1998 by The Endocrine Society
Vol. 83, No. 12 Printed in U.S.A.
Effect of Acute Pharmacological Reduction of Plasma Free Fatty Acids on Growth Hormone (GH) Releasing Hormone-Induced GH Secretion in Obese Adults with and without Hypopituitarism* FERNANDO CORDIDO, TERESA FERNANDEZ, TERESA MARTINEZ, ˜ ALVA, ROBERTO PEINO ´ , FELIPE F. CASANUEVA, AND ANGELA PEN CARLOS DIEGUEZ Department of Endocrinology, Hospital Juan Canalejo (F.C., T.F., T.M.) and University of La Corun˜a (F.C.), and University of Santiago (A.P., R.P., F.F.C., C.D.), La Corun˜a 15006 and Santiago, Spain ABSTRACT In obesity, there is a markedly decreased GH secretion. The diagnosis of GH deficiency (GHD) in adults is based on peak GH responses to stimulation tests. In the severely obese, peak GH levels after pharmacological stimulation are often in the range that is observed in hypopituitary patients. To distinguish obese subjects from GHD patients, it will be necessary to demonstrate that reduced GH responsiveness to a given test is reversible in the former, but not in the latter, group. Recent studies have shown that reduction of plasma free fatty acids (FFA) with acipimox in obese patients restores their somatotrope responsiveness. There are no data evaluating GH responsiveness to acipimox plus GHRH in obese adults with hypopituitarism. The aim of the present study was to evaluate the effect of acute pharmacological reduction of plasma FFA on GHRH-mediated GH secretion in obese normal subjects and obese adults with hypopituitarism. Eight obese patients with a body mass index of 34.2 6 1.2; eight obese adults with hypopituitarism, with a body mass index of 35.5 6 1.9; and six control subjects were studied. All the patients showed an impaired response to an insulin-tolerance test (0.15 U/kg, iv), with a peak GH secretion of less than 3 mg/L. Two tests were carried out. On one day, they were given GHRH (100 mg, iv, 0 min), preceded by placebo; and blood samples were taken every 15 min for 60 min. On the second day, they were given GHRH (100 mg, iv, 0 min), preceded by acipimox (250 mg, orally, at 2270 min and 260 min); and blood samples were taken every 15 min for 60 min.
I
N OBESITY, there is a markedly decreased GH secretion. For both children and adults, the greater the body mass index (BMI), the lower the GH response to provocative stimuli, including the response to GHRH (1–3). After weight reduction, as well as after short-term fasting, GH responsiveness significantly increases (4, 5). Recent studies have shown that acute reduction of plasma free fatty acids (FFA) with acipimox in obese patients restores their somatotrope responsiveness (6 – 8). There are no data evaluating GH responsiveness to acipimox plus GHRH in obese adults with hypopituitarism.
Received April 16, 1998. Revision received August 21, 1998. Accepted August 27, 1998. Address all correspondence and requests for reprints to: Dr. Fernando Cordido, Servicio de Endocrinologı´a, Hospital Juan Canalejo, Xubias de Arriba 84, 15006 La Corun˜a, Spain. * This work was supported by a grant from Xunta de Galicia (XUGA 9000/B98).
The administration of acipimox induced a FFA reduction during the entire test. Normal control subjects had a mean peak (mg/L) of 23.8 6 4.8 after GHRH-induced GH secretion; previous acipimox administration increased GHRH-induced GH secretion, with a mean peak of 54.7 6 14.5. In obese patients, GHRH-induced GH secretion was markedly reduced, with a mean peak (mg/L) of 3.9 6 1; previous administration of acipimox markedly increased GHRH-mediated GH secretion, with a mean peak of 16.0 6 3.2 (P , 0.05). In obese adults with hypopituitarism, GHRH-induced GH secretion was markedly reduced, with a mean peak (mg/L) of 2 6 0.7; previous acipimox administration did not significantly modify GHRH-mediated GH secretion, with a mean peak of 3.3 6 1.1 (P , 0.05). The GH response of obese patients and obese adults with hypopituitarism was similar after GHRH alone. In contrast, the GH response after GHRH plus acipimox, was markedly decreased in obese adults with hypopituitarism (mean peak, 3.3 6 1.1), compared with obese patients (mean peak, 16.0 6 3.2) (P , 0.05) and control subjects (mean peak, 54.7 6 14.5) (P , 0.01). In conclusion, GH secretion, after GHRH-plus-acipimox administration, is reduced in obese adults with hypopituitarism patients, when compared with obese normal patients. Testing with GHRH plus acipimox is safe and is free from side effects and could be used for the diagnosis of GHD in adults. (J Clin Endocrinol Metab 83: 4350 – 4354, 1998)
It has been gradually realized that GH may have important physiological functions in adult man. Several studies have shown that GH deficiency (GHD) in adults is associated with abnormalities in body composition, metabolic derangements, and suboptimal physical performances; these impairments improve with GH replacement therapy (9 –11). In contrast to children, in adults with GHD, there are no auxological data to aid in diagnosis. Assessment of 24-h spontaneous GH secretion have not proved particularly useful, and in the clinical setting, we must therefore rely on pharmacological tests to establish the diagnosis of GHD. In this sense, it was recently recommended that the insulintolerance test (ITT) should be the diagnostic test of choice. However, this test may be contraindicated in some patients; and furthermore, in severely obese subjects, the responses to the ITT frequently overlap with those found in nonobese adult patients with GHD. To distinguish obese subjects from GHD patients, it will be necessary to demonstrate that re-
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PLASMA FFA AND GHRH-INDUCED GH SECRETION
duced GH responsiveness is reversible in the former but not in the latter group. We studied the potential value of acipimox to distinguish non-GHD obese subjects from GHD obese patients. The aim of the present study was to evaluate the effect of acute pharmacological reduction of plasma FFA on GHRHmediated GH secretion in obese normal subjects and obese adults with hypopituitarism. Subjects and Methods Eight obese patients [4 female, 4 male; 35–52 yr old (39.2 6 6 yr); with a BMI of 34.2 6 1.2 kg/m2] and eight obese adults with hypopituitarism [4 female, 4 male; 29 – 61 yr old (48.8 6 3.9 yr); with a BMI of 35.2 6 1.7 kg/m2] were studied (Table 1). Obese normal subjects and obese adults with hypopituitarism had a BMI greater than 30 kg/m2 (normal range, 20 –25 kg/m2). As a control group, six normal subjects with a normal BMI were also studied. None of the obese or control subjects had diabetes mellitus or other medical problems, nor were they taking any drugs. The diagnoses in the hypopituitary patients were: four nonfunctioning pituitary adenomas, one surgery-treated pituitary macroprolactinoma, one Sheehans’ syndrome, one empty sella turcica, and one idiopathic hypopituitarism. All the subjects showed an impaired response to an ITT (0.15 U/kg, iv), with a peak GH secretion of less than 3 mg/L. Where indicated, patients were receiving physiologic replacement doses of l-T4 and/or glucocorticoids and/or gonadal steroids. All subjects provided informed consent, and approval for this study was obtained from the hospital committee. The subjects had been eating a weight-maintaining diet for several weeks before the study. Each subject was studied on two different days, separated by at least one week. The tests were started at 0900 h, after an overnight fast, with the subjects recumbent. The first blood sample was obtained at 2270 min, and additional blood samples were obtained at appropriate intervals over the following 360 min of testing, through an indwelling catheter placed in a forearm vein, and kept patent by a slow infusion of saline. On one day, they were given GHRH (100 mg, iv, 0 min), preceded by placebo, and blood samples were taken every 15 min for 60 min. On the second day, they were given GHRH (100 mg, iv, 0 min), preceded by acipimox (250 mg, orally, at 2270 min and 260 min), and blood samples were taken every 15 min for 60 min. Serum GH was measured by RIA (Nichols Institute Diagnostics, San Juan Capistrano, CA) with a sensitivity of 0.04 mg/L and with intraassay coefficients of variation of 4.2%, 2.9%, and 2.8% for low, medium, and high plasma GH levels, respectively. FFA levels were determined by a enzymatic colorimetric method (NEFA-HA, Wako, Zaragoza, Spain). All samples from a given subject were analyzed in the same assay run. Hormone levels are presented as absolute values or as the mean GH peak. The area under the curve (AUC) was calculated by a trapezoidal method. Statistical analysis were performed by Wilcoxon rank sum between related groups and by Mann-Whitney tests between different groups. Results are expressed as mean 6 sem. P , 0.05 was considered significant.
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Results
The administration of acipimox induced an FFA reduction during the entire test. In normal subjects, the AUC (mmol/ Lz60 min) after placebo treatment (107 6 15.2 mmol/Lz60 min) was higher than after acipimox pretreatment (25.6 6 7.8 mmol/Lz60 min) (P , 0.05). In obese subjects, the AUC (mmol/Lz60 min) after placebo pretreatment (104.4 6 8.5 mmol/Lz60 min) was significantly higher (P , 0.05) than after acipimox pretreatment (28.3 6 5.2 mmol/Lz60 min) (P , 0.05). In obese adults with hypopituitarism, the AUC after placebo pretreatment (71.1 6 9.8 mmol/Lz60 min) was significantly higher (P , 0.05) than after acipimox pretreatment (25.2 6 4.1 mmol/Lz60 min) (P , 0.05). Normal control subjects had a mean peak of 23.8 6 4.8 mg/L after GHRH-induced GH secretion (range, 9.5–38.4 mg/L). When FFA were reduced by previous acipimox administration, there was an increased GHRH-induced GH secretion, with a mean peak of 54.7 6 14.5 mg/L (range, 13.7–113 mg/L). In obese subjects, GHRH-induced GH secretion was markedly reduced, with a mean peak of 3.9 6 1 mg/L (range, 2.8 – 6.2 mg/L). When FFA were reduced by previous acipimox administration, a markedly increased GHRH-mediated GH secretion was observed, with a mean peak of 16.0 6 3.2 mg/L (range, 7.4 –36 mg/L; P , 0.05) (Fig. 1). In obese adults with hypopituitarism, GHRH-induced GH secretion was markedly reduced, with a mean peak of 2 6 0.7 mg/L (range, 0.2– 4.0 mg/L). In contrast with obese normal subjects, the previous acipimox administration in obese adults with hypopituitarism did not significantly modify GHRH-mediated GH release, with a mean peak of 3.3 6 1.1 mg/L (range, 0.1–7.1 mg/L; P, not significant) (Fig. 2). When we compare the response of obese normal and obese adults with hypopituitarism, after GHRH alone, it was similar in both groups (P, not significant), with a mean GH peak of 3.9 6 1.0 mg/L and 2.0 6 0.7 mg/L for obese normal and obese adults with hypopituitarism, respectively. In contrast, when we compare the response of obese normal and obese adults with hypopituitarism after GHRH-plus-acipimox pretreatment, it was markedly decreased in obese adults with hypopituitarism (P , 0.05) and even further decreased when compared with normal control subjects (P , 0.01), with a mean peak of 3.3 6 1.1 mg/L for obese hypopituitary, a mean
TABLE 1. Clinical characteristics of obese hypopituitary patients Patient
Age (yr)
Sex
Height (cm)
Weight (kg)
BMI (kg/m2)
1
53
F
151.0
73
32.0
54 62 47 29 61 43 41 48.8 6 3.9
F M F M M M F
153.4 169.0 160.0 163.5 156.5 156.0 167 159.6 6 2.3
76.5 101.5 116 85 93 81 98 90.5 6 5.1
30.2 35.6 45.3 31.8 38 33.8 35 35.2 6 1.7
2 3 4 5 6 7 8 Mean 6
SEM
Diagnosis
Sheehans’ syndrome NFA Idiopathic NFA MacroPRL ES NFA NFA
Hormonal treatment
T, G T, C, T, G T, C, C, G C, T, T, C, T, C,
G G G G G
Previous treatment
Peak GH (ug/L)
None
0.9
S, R None S S None S S
0.5 2.5 0.7 2 1 1.9 1.8
NFA, Nonfunctioning pituitary adenoma; ES, empty sella; MacroPRL, macroprolactinoma; C, corticosteroids; T, thyroxine; G, gonadal steroids; S, surgery; R, radiation therapy.
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FIG. 1. A, Mean 6 SE serum GH levels in obese subjects after the administration of placebo (f) or acipimox (E); B, mean 6 SE serum FFA levels in obese subjects after the administration of placebo (f) or acipimox (E).
JCE & M • 1998 Vol 83 • No 12
FIG. 2. A, Mean 6 SE serum GH levels in obese hypopituitary patients after the administration of placebo (f) or acipimox (E); B, mean 6 SE serum FFA levels in obese hypopituitary patients after the administration of placebo (f) or acipimox (E).
PLASMA FFA AND GHRH-INDUCED GH SECRETION
4353
peak of 16.0 6 3.2 mg/L for obese subjects, and a mean peak of 54.7 6 14.5 mg/L for normal control subjects (Fig. 3). Individually considered (mean peak GH secretion after GHRH-plus-acipimox pretreatment), there was a clear difference between normal controls and hypopituitary patients, and a less clear difference between obese and hypopituitary patients (the lowest response for obese normal subjects was 7.4 mg/L, and the highest response for obese hypopituitary patients was 7.1 mg/L) (Fig. 4). One control subject, one obese patient, and one obese adult with hypopituitarism experienced a mild transient facial flushing after the first dose of acipimox. No side effects were reported in the other tests. Discussion
This study demonstrates that GHRH-plus-acipimox administration results in a lower GH secretion in a group of obese adults with hypopituitarism, compared with a group of obese normal subjects of similar age, without any other pathology. Obesity-related limitation of GH secretion, in response to all stimuli, has attracted considerable study. Although enhancement of GH clearance is a contributing factor (12), the main altered mechanism in obesity is the impaired GH secretion, either stimulated or spontaneous (13). The primary cause of this alteration could be an altered hypothalamus, abnormal pituitary function, or a perturbation of the peripheral signals acting at either the pituitary or hypothalamic level. In obese subjects, both exogenous GHRH- and GHRP (growth hormone releasing peptide)-6-induced GH secretion are blocked, ruling out a secretory deficit of endogenous GHRH or the endogenous ligand for the GHRP-6 receptor as
FIG. 3. Mean 6 SE serum GH levels after the administration of GHRH plus acipimox in normal control subjects (f), obese normal subjects (E), and obese hypopituitary patients (F).
FIG. 4. Individual mean peak serum GH levels after the administration of GHRH plus acipimox in normal control subjects (f), obese subjects (E), and obese hypopituitary patients (F).
causative factors. On the other hand, pyridostigmine, a drug thought to reduce somatostatinergic tone (14, 15), increases GHRH-stimulated GH secretion in obese subjects (16). This effect of pyridostigmine suggests that an enhanced somatostatinergic tone could partially explain the altered somatotroph function in obese patients, but because the GH responses after pyridostigmine or hypoglycemia are lower than those observed in normal-weight subjects, this higher somatostatinergic tone is unable to fully explain the obesityaltered secretory mechanisms. FFA reduction, with acipimox [a lipid-lowering drug with minimal side-effects (notably, increased pyridostigmine-, GHRH-, and GHRH plus GHRP6-mediated GH secretion)] restoring the level of this secretion to 50 –70% of normal (6). These and other results indicate that elevated FFA levels play an important role in causing GH insufficiency in obesity, and they suggest that treatment with inhibitors of lypolisis could be useful in restoring somatotroph function (7, 8, 17). The approach to the diagnosis of adult GHD must include a high index of suspicion. It should include adults who, in childhood, received GH for pituitary dwarfism and adults with known or suspected pituitary disease (18, 19). Because of its pronounced intraindividual fluctuation determinations of basal plasma concentrations of GH, it is of no value for the diagnosis of GHD, and considerable overlap with healthy control groups precludes determination of IGF-I and IGFBP-3 (20, 21), analysis of GH pulses (22), and/or GH determination in pooled serum samples (22) or in urine (23). Therefore, most investigators agree that stimulation tests are indispensable in defining GHD in adults (20 –22). The ITT is the diagnostic test of choice for adult GHD (24). The criteria for profound GHD is met if the patient is symptomatic of hypoglycemia, the blood glucose is below 2.2 mmol/L, and
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CORDIDO ET AL.
the peak GH response is less than 3 mg/L (18). It is in these patients (who, for the most part, had peak GH response to ITT of less than 3 mg/L) that beneficial responses with GH treatment have been observed (10, 11, 25). The ITT may not be the best tool for the diagnosis of GHD in adults; its reproducibility has been questioned in normal adults (26). A reference for the ITT will be difficult to establish, and it may be impossible to evaluate the possible influences of sex, age, body composition, and cortisol levels in ITT-stimulated GH levels, because patients find the ITT unpleasant. Furthermore, the ITT is potentially hazardous (27). In contrast to this, no severe side effects and only minor transitory complaints were recorded with the GHRH-plus-acipimox test. Other tests, including GHRH, could be used in the diagnosis of adult GHD. GHRH-stimulated secretion of GH has been found to be reduced in a group of patients with pituitary macroadenomas, and this confirmed partial pituitary insufficiency, compared with a control group. However, in an individual patient, this maneuver, even in the presence of partial pituitary insufficiency, may suggest (but does not prove) additional GHD (28). Obesity is a very important confounding factor, as shown by our study of a markedly decreased GHRH-induced GH secretion in both obese normal patients and obese adults with hypopituitarism. We have shown that, with the addition of acipimox, it is possible to distinguish GHRH-induced GH secretion of obese normal patients from obese adults with hypopituitarism. This study, to our knowledge, is the first report of GH responses to acipimox plus GHRH in obese adults with hypothalamicpituitary lesions. The results of this study suggest that patients with hypothalamic-pituitary disease have a markedly significant decreased GH response to GHRH plus acipimox, even when obesity is taken into account. Therefore, it can be concluded that our group of patients with pituitary disease is different from a comparable group of obese adults, in that they displayed a significantly lower mean peak GH level after GHRH-plus-acipimox administration. In conclusion, this study has demonstrated a significantly blunted peak GH response to GHRH in both obese normal and obese hypopituitary patients. The use of acipimox in conjunction with GHRH in obese hypopituitary patients is an effective, safe, and side-effect-free method for overcoming the inhibition of GH response associated with obesity. Further studies, assessing this test in a large group of patients and controls, are needed, to establish its reliability in the clinical setting. References 1. Kopelman PG, Noonan K. 1987 Growth hormone response to low dose intravenous injections of growth hormone releasing factor in obese and normal weight women. Clin Endocrinol (Oxf). 27:727–733. 2. Loche S, Pintor C, Cappa M, et al. 1989 Pyridostigmine counteracts the blunted growth hormone response to growth hormone-releasing hormone of obese children. Acta Endocrinol (Copenh). 120:624 – 628. 3. Cordido F, Casanueva FF, Dieguez C. 1989 Cholinergic receptor activation by
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