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Laboratoire des Intéractions Fonctionnelles en Neuroendocrinologie, INSERM U-501, Institut Fédératif Jean Roche (N.B., V.G., N.S., V.V., F.D., C.O., A.D.), 13916 Marseille Cedex 20, France; Departamentes de Phisiologia Anatomia y Produccìon Animal, Facultad Veterinaria, Campus Universitario (M.R.-G.), 27002 Lugo, Spain; Service d’Endocrinologie, Maladies Métaboliques et de la Nutrition, Hôpital Nord (V.G., F.D., C.O., A.D.), 13915 Marseille Cedex 20, France; and Exploration Fonctionnelle Endocrinologique, Hôpital Trousseau (Y.L.B.), 75571 Paris Cedex 12, France
Address all correspondence and requests for reprints to: Dr. A. Dutour, Laboratoire des Intéractions Fonctionnelles en Neuroendocrinologie, INSERM U-501, Institut Fédératif Jean Roche, boulevard P. Dramard, 13916 Marseille Cedex 20, France.
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Abstract |
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Immediately after initiation of Intralipid infusion, we observed a marked increase in the FFA concentration (2160 ± 200 vs. 295 ± 28 nmol/ml; P < 0.01) as well as a significant decrease in basal GH secretion (1.8 ± 0.1 vs. 2.5 ± 0.3 ng/ml; P < 0.05) and a drastic reduction of the GH response to iv GH-releasing hormone injection (4.8 ± 0.7 ng/ml in FFA group vs. 35.8 ± 9.7 ng/ml in saline group; P < 0.01). No change in plasma insulin-like growth factor I levels was observed. During the first 2 h of infusion, the GH decrease observed was concomitant with a significant increase in portal SRIH levels (22.1 ± 3.2 vs. 13 ± 1.6 pg/ml; P < 0.01). In rams actively immunized against SRIH, the effect of FFA on basal GH secretion was biphasic. During the first 90 min of infusion, the decrease in GH induced by FFA was significantly blunted in rams actively immunized against SRIH (57 ± 9% for immunized rams vs. 23.5 ± 2.5% for control rams). This corresponds to the period of increased SRIH portal levels. After this first 90-min period, no difference was seen between control and immunized rams.
Our results show that FFA exert their inhibitory action on the GH axis at both pituitary and hypothalamic levels, the latter mainly during the first 90 min, through increased SRIH secretion.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A classical metabolic/endocrine feedback loop between FFA and GH secretion has been suggested (8). GH has a direct lipolytic effect on adipose tissue, leading to the release of glycerol, FFA, and ketone bodies (9). In turn, metabolic signals such as endogenous FFA play an important role in neuroendocrine control of GH secretion. FFA elevation markedly reduces basal GH secretion and blocks GH secretion induced by pharmacological and physiological stimuli in humans, rats, sheep, and other species (10, 11, 12, 13). Conversely, pharmacological reduction in circulating FFA levels causes GH release (14). However, FFA mechanisms of action are not clearly understood. FFA may inhibit GH secretion either directly at the pituitary level and/or indirectly at the hypothalamic level through modulations of one or both of the GH regulatory neurohormones, GHRH and somatostatin (SRIH), a stimulator and an inhibitor, respectively, of GH secretion. There is clear evidence that in vitro FFA directly inhibit GH secretion from somatotroph cells in a dose- dependent manner, mainly by perturbing the function of plasma membrane integral proteins involved in the signaling pathway (15, 16). The persistence of FFA inhibition of GH release in rats with medial hypothalamic ablation and in rats with heterotopically transplanted pituitary suggest that in vivo FFA act also at least partly at the pituitary level (17). A SRIH-mediated mechanism of action of FFA has also been reported but is still controversial. In rats pretreated with anti-SRIH serum, the inhibitory effect of FFA on GHRH induced GH secretion was either completely abolished in one study (11) or was unaffected according to an other study (17).
There are experimental limitations in rodent models and growing interest in a clinical relevant animal model, such as the sheep, for the study of GH regulation (18). In this animal, it is possible to collect hypophysial portal blood (HPB) and directly assess the secretion of neurohormones into HPB under physiological conditions, specially without the biases induced by anesthesia. The aim of this work was to determine the effect of FFA on SRIH secretion and its participation in FFA regulation of GH secretion. We first studied the effects of an iv Intralipid infusion on basal and GHRH-stimulated GH levels, portal plasma SRIH levels, and insulin-like growth factor I (IGF-I) levels in intact rams chronically implanted with perihypophysial cannulas. To further determine the involvement of SRIH, the same experimental procedure was repeated in rams actively immunized against SRIH.
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Materials and Methods |
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Experimental procedures
Three sets of experiments were carried out successively. For
each experiment, lipid emulsion (20% Intralipid, Pharmacia & Upjohn
AB, Uppsala, Sweden; containing 200 g fractionated soybean oil,
12 g fractionated ovolecitin, and 22 g glycerin in 1000 ml
oil in water emulsion) or 0.9% saline was infused continuously at the
rate of 0.5 ml/min. Human GHRH-(1–29)-NH2 (50 µg; Geref
50, Serono Pharma S.p.A., Rome, Italy) was administered in 2 ml 0.9%
saline as an iv bolus.
Exp 1. The aim of this experiment was to confirm in intact rams the effect of lipid infusion on plasma GH levels previously observed in ovariectomized ewes and to determine the effect of FFA on IGF-I levels. The animals (n = 8) were housed in individual pens placed immediately adjacent to each other. They were free to sit or stand and were exposed to natural lighting conditions. Two indwelling catheters were inserted in each jugular vein: one catheter for injection of heparin and Intralipid, and the other for collection of blood samples. On the following day, a 10-h infusion of lipid (n = 4) or saline (n = 4) was given between 1200–2200 h. A pituitary challenge of GHRH was administered to all animals at 2000 h. Jugular blood was collected every 15 min from 1000–2200 h. Blood samples were immediately centrifuged at 4 C for 10 min, and plasma was stored at -20 C until assayed for GH, FFA, and IGF-I.
Exp 2. The aim of this experiment was to determine the effects of an infusion of FFA on jugular GH and portal SRIH plasma levels in intact rams chronically implanted with perihypophysial cannulas. Fifteen days before the experiment, eight rams were anesthetized and prepared for portal blood sampling under general anesthesia, as previously described (19, 20). A twin cannula was implanted through the transnasal route in front of the long portal vessels, above the anterior pituitary gland. After 14 days, two catheters were inserted in each jugular vein: one catheter for injection of heparin and Intralipid, and the other for collection of peripheral blood. Two animals were placed side by side in two small pens. One day later, heparin (an initial dose of 25,000 IU followed by 5,000 IU every 30 min) was injected, and at 0700 h, a needle was inserted into the upper cannula to create a lesion of the hypophysial portal vessels. The resulting portal blood was collected through the lower cannula. A 5-h infusion of lipid (n = 4) or saline (n = 4) was given between 1200–1700 h. A pituitary challenge of GHRH was administered to all animals at 1600 h. Portal and jugular blood were collected every 15 min from 1000–1900 h. Samples were handled as described in Exp 1 until assayed for GH, portal SRIH, and FFA.
Exp 3. To determine the involvement of SRIH, the same experimental procedure was repeated in rams actively immunized against SRIH.
Immunization procedure
At the beginning of the study, animals (n = 12) were 3
months old and weighed 22.5 ± 0.6 kg. During the immunization
procedure (July 1997 to February 1998), they were housed at the
ENSA facility. Synthetic SRIH (Sanofi, Toulouse, France) was
coupled to BSA with glutaraldehyde (21). Two groups of six animals
received, respectively, five intradermic injections of SRIH immunogen
or saline mixed with Freund’s complete adjuvant. They received the
first (2 mg SRIH immunogen/animal) and the second (0.2 mg/animal)
injection at 2-month intervals and the three following injections (0.2
mg/animal) at 6-week intervals. The SRIH binding capacity of the serum
was repetitively tested to decide the number of SRIH immunogen
injections.
Characterization of SRIH binding capacity in serum
SRIH binding capacities in the serum of actively immunized
animals were determined using different conditions as previously
described (22). The dissociation constant (Kd) and the
binding capacity (Bmax) of each antiserum were calculated
using Scatchard coordinates (23).
Ten to 50 pg [125I]Tyr0-SRIH, prepared as previously described (24), were mixed with unlabeled synthetic SRIH in amounts ranging from 2 ng to 40 µg. One hundred microliters of serum from immunized animals were added to the tubes and incubated for 1 min at 37 C according to the method of Mariuyama et al. (25). At the end of the incubation period, free and antibody-bound iodinated SRIH were separated using the charcoal-dextran method.
Experimental procedure
The experimental protocol was conducted in February 1998 under
the same conditions as those in Exp 1. Twelve days after the last
injection of immunogen or vehicle, two indwelling catheters were
inserted in each jugular vein; one catheter for injection of heparin
and Intralipid, and the other for collection of peripheral blood. On
the following day, a 5-h infusion of lipid (immunized, n = 3;
controls, n = 3) or saline (immunized, n = 2; controls,
n = 3) was given between 1200–1700 h. A pituitary challenge of
GHRH was administered to all animals at 1600 h. Jugular blood was
collected every 15 min from 1000–1900 h. Seven days later, the same
protocol was repeated (saline for the animals that had received FFA and
reciprocally). Samples were handled as described in Exp 1 until assayed
for peripheral GH and IGF-I. Blood sampling during saline infusion was
used as a control for lipid infusion as well as for measurement of
basal IGF-I to characterize the immunization effects.
Hormone assays
The GH RIA was performed in duplicate using reagents provided by
the NIDDK, Hormone Distribution Program (Bethesda MD). Ovine GH 1–4
was used as the standard, and the least detectable concentration of GH
was 0.5 ng/ml plasma. The intra- and interassay coefficients of
variation were 7% and 11%, respectively.
Before SRIH RIA, peptides were extracted from plasma with 2 vol acetone/20 mM HCl as previously described (24). The SRIH RIA was performed in duplicate in portal and jugular plasma extracts using [125I]Tyr0-SRIH as radioligand. The antiserum (no. 2044) was a gift from Dr. C. Rougeot (INSERM U-207, Paris, France). The intra- and interassay coefficients of variation were 8% and 10%, respectively, and the least detectable concentration was 5 pg/ml plasma.
Plasma IGF-I was measured with a previously reported assay system (26) previously tested on sheep plasma (22). Briefly, samples were gel filtered in acetic acid on columns of Ultrogel AcA54 (Sepracor/IBF s.a., Villeneuve la Garenne, France) to separate IGFs from their binding proteins. Recombinant human IGF-I was provided by Ciba-Geigy Ltd. (Basel, Switzerland) and was used as standard and tracer after iodination by the chloramine-T method. IGF-I was assayed by RIA using anti-IGF-I antiserum prepared by Dr. Closset (Liege, Belgium). Unknown samples were studied at three concentrations, each in duplicate plus one nonspecific binding tube. Intra- and interassay coefficients of variation were 4.8% and 10%, respectively.
Plasma FFA was measured with an enzymatic colorimetric kit (Wako Chemicals, Neuss, Germany).
Statistical analysis
All data are reported as the mean ± SEM. Data
were analyzed by periods. In Exp 1, mean plasma GH values were
calculated during the 2-h period before infusion (base), the 8-h period
during saline or FFA infusion (infusion), and the 2-h period after GHRH
iv injection (GHRH). For Exp 2 and 3, the infusion period was divided
in two periods. In Exp 2, mean plasma GH and SRIH values were
calculated during the 2-h period before infusion (base), the first
2 h of infusion (period 1), and the 2 consecutive h of infusion
(period 2), 1 h after iv GHRH injection (GHRH), and 2 h after
stopping the infusion (Rebound). In Exp 3, as SRIH immunization induced
a slight increase in basal GH levels (4.8 ± 0.3 vs.
3.6 ± 0.2; P < 0.01), mean plasma GH values
during lipid infusion were expressed as a percentage of saline values;
for each ram, each GH value of the lipid infusion experiment was
divided by the mean plasma GH values of the saline experiment during
the corresponding period.
The mean percentage ± SEM were calculated during the 1-h period before infusion (base), the first 1.30 h of infusion (period 1), and the 2.30 consecutive h of infusion (period 2).
Statistical analysis between infusion (period 1 or 2), GHRH, or rebound periods vs. the basal period was performed for each group of animals using paired Student’s t test (with computer software StatView 512, Brain Power, Inc., Calabasas, CA). Statistical analysis between Intralipid infusion vs. saline infusion was performed for each period using unpaired Student’s t test. P < 0.05 was considered significant.
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Results |
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Exp 1: effect of lipid infusion on jugular plasma levels of GH and
IGF-I
Lipid infusion significantly decreased plasma GH concentrations
(3.4 ± 0.3 vs. 9.5 ± 0.8 ng/ml;
P < 0.01; Fig. 1
). The
decrease in plasma GH concentration occurred immediately after the
beginning of lipid infusion and was already significant during the
initial 2-h period of lipid infusion (9.5 ± 0.8 ng/ml during the
basal period vs. 5.4 ± 0.7 ng/ml during the initial
2-h period of lipid infusion; P < 0.01). Compared with
the saline infusion group, the response of plasma GH to GHRH injection
was significantly reduced by lipid infusion (peak, 45.8 ± 13.7
vs. 97.8 ± 20.5 ng/ml, respectively; P
< 0.01). No change was observed in IGF-I concentration after lipid
infusion (121.2 ± 31.3 vs. 174.2 ± 14.9
ng/ml).
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). The maximal increase in SRIH levels
was observed during the first 2 h of infusion. Portal SRIH levels
started to increase 20 min after the beginning of lipid infusion and
peaked after 80 min. During period 2, a small increase in SRIH levels
was also observed in the animal perfused with saline; this increase did
not reach significance, but the difference between saline- and
lipid-infused animals during this period was not significant. After
GHRH injection, the usual increase in SRIH due to short feedback was
observed in both groups.
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and Fig. 3. Circulating antibodies were able to
immunoneutralize about 1–10 µg SRIH/ml plasma, i.e.
5,000–50,000 times more than reported endogenous peripheral and
hypophysial portal blood SRIH levels (24) (Fig. 3A). All of the
antisera bound 30–40% of
[125I]Tyr0-SRIH-14 at a final
dilution ranging from 1:25,000 to 1:150,000 under the RIA conditions
described above. The mean Kd of the antiserum was 2.3
± 0.9 x 10-6 mol/liter, and each milliliter was
able to bind 25 ± 7.9 pmol SRIH as calculated on the Scatchard
plot. None of the antisera showed any cross-reactivity (<0.001%) with
GHRH.
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, A and B). Moreover, lipid infusion
decreased the basal plasma GH concentration significantly in control
rams as well as in immunized rams (Fig. 4). However, in the control
group, the decrease in plasma GH concentrations occurred immediately
after the beginning of lipid infusion, whereas in the immunized group,
the effect of lipid on basal GH secretion was biphasic. During the
first 90 min after the beginning of infusion, the decrease in GH was
significantly blunted in rams actively immunized against SRIH (57
± 9% for immunized rams vs. 23.5 ± 2.5% for control
rams). The maximum of inhibition was reached only 105 min after the
beginning of infusion. During the following second period (105–240 min
of infusion), the effect of lipid on basal GH secretion was maximal and
identical in both groups (24.8 ± 3.2% for immunized rams
vs. 23.5 ± 2.5% for control rams). A marked rebound
in plasma GH concentration was observed after the end of lipid infusion
in the control group (peak, 17.8 ± 4.2 ng/ml; basal, 3.1 ±
0.3 ng/ml) and to a lesser extent in the immunized group (peak,
11.5 ± 4.4 ng/ml; basal, 5.3 ± 0.7 ng/ml). In the control
group, GH levels rose immediately and peaked 45 min after the end of
lipid infusion, whereas in the immunized group, the rise in GH was
delayed, starting at 30 min and peaking at 90 min after the end of
lipid infusion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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On the other hand, the reality of FFA effects on the somatotroph axis at the hypothalamic level was until now controversial, with only indirect data available. In the rhesus monkey, nicotinic acid-induced FFA suppression leads to increased GH; this effect of nicotinic acid in GH was significantly blunted by intracerebroventricular injection of a small amount of oleic acid (32), providing indirect evidence of a hypothalamic site of action for FFA in modulating GH secretion. Passive immunization against SRIH in rats produced conflicting results. Imaki et al. (11) have shown that pretreatment with anti-SRIH serum completely abolished the inhibitory effect of FFA on GHRH-induced GH secretion. Conversely, in another study the FFA inhibitory effect was unaffected by the administration of anti-SRIH antiserum (17). The reason for this discrepancy may be related to the difference in the timing of the two experiments. The inhibitory effect of FFA on GHRH-induced GH secretion was studied in the first case 2 min after the injection of FFA, whereas in the second case GHRH was injected 30 min later. As suggested by Alvarez et al. (17), these two experiments suggest that the involvement of SRIH is limited to a short early period. Our results obtained in sheep confirm this hypothesis.
Another indirect experimental approach also suggests that SRIH may not mediate the FFA effect in humans; FFA reduction induced by acipimox, a lipid-lowering drug that reduces FFA levels, was shown to enhance GH secretion induced by pyridostigmine in an additive manner. This additive effect prompted the researchers to conclude that FFA and pyridostigmine alter GH secretion through different mechanisms and, therefore, that FFA action does not involve SRIH release, as pyridostigmine has been suggested to act mainly through increased SRIH secretion (33). However, we have previously shown that in sheep, acute administration of neostigmine, a cholinesterase inhibitor closely related to pyridostigmine, alters GH secretion through increased GHRH release and does not trigger any change in hypothalamic SRIH release (24).
The FFA cellular mechanism of action on the hypothalamus is still unknown. Oomura et al. (34) have shown that glucose-sensitive neurons of the ventromedial nucleus (VMH) and lateral hypothalamic areas respond to direct application of FFA by intraneuronal iontophoresis. Therefore, FFA may activate FFA-sensitive neurons in lateral hypothalamic areas or may inhibit the activity of FFA-responsive neurons in the VMH and, in turn, stimulate somatostatinergic neurons in the anterior preoptic area. Furthermore, a connection between the VMH and the anterior preoptic area has been demonstrated anatomically (35). No study has investigated whether SRIH-containing neurons of periventricular nucleus directly respond to FFA.
At the pituitary level, the cellular mechanisms of FFA action have been extensively studied; an alteration of the binding affinity of pituitary receptors (GHRH or SRIH receptors) and a perturbation of the postreceptor mechanism have been demonstrated. Indeed, the amphiphilic FFA molecule rapidly partitions into the cell membrane and incorporates into the lipid bilayer (36). FFA incorporation alters the bilayer structure of the membrane in a manner similar to the effect of some anesthetics (37, 38, 39). Moreover, Renier et al. (40) have shown that caprylic acid inhibits GH secretion in rats by reducing the affinity of GHRH for its pituitary binding sites and by inducing changes in transmembrane signaling. It decreases basal and GHRH-induced cAMP release as well as the ability of the ionophore A23187 to stimulate GH secretion. As the action of A23187 does not involve GHRH receptors, it gives indirect evidence that FFA act also through GHRH receptor-independent mechanisms such as adenylate cyclase-cAMP and calcium channel systems (41, 42). This GHRH receptor-independent mechanism has been confirmed recently by Perez et al. (16), who demonstrated that in cultured GH3 cells, cis-unsaturated FFA, such as oleic acid, were able to block the calcium signal elicited by a saturating dose of TRH and to inhibit inositol 1,4,5-triphosphate generation, suggesting either a perturbation in phospholipase C activation or an interference in the interaction of phospholipase C with phosphatidylinositol 4,5-bisphosphate. The inhibitory action of oleic acid on THR-mediated early signals in GH3 cells was paralleled by the inhibition of GH secretion. The cis unsaturated FFA such as oleic acid possess an angular structure that perturbs their normal packing when inserted into lipid bilayers, altering the function of plasma membrane integral proteins.
We have previously shown that GHRH is the major hypothalamic neurohormone involved in the control of GH secretion in sheep. GHRH is involved in the regulation of GH induced by all pharmacological stimuli tested, such as neostigmine or tianeptine, and by various physiological stimuli, such as stress. SRIH appeared to play a role in preserving the depletion of pituitary stores of GH and responsiveness to GHRH (18). Indeed, until now we had never been able to characterize a direct role of SRIH in the control of GH secretion in sheep. These results demonstrating the involvement of SRIH in the regulation of GH by FFA are interesting because they suggest that SRIH may play an important role in the metabolic regulation of GH secretion in sheep, although in our study, the level of FFA is 4 times higher than that found in obese sheep (43). Furthermore, in sheep after long term food restriction, it has been shown that the increase in GH secretion is probably related to decreased SRIH release and that food restriction has no effect on GHRH secretion (44).
In conclusion, we have shown here that FFA have a more complex effect on the somatotroph axis than previously suggested, displaying an action at both hypothalamic and pituitary levels. These results could help to define new pharmacological approaches to restore somatotrope function in obesity.
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Acknowledgments |
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Footnotes |
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Received July 8, 1998.
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References |
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-melanocytes
stimulating hormone and adrenocorticotropic hormone. J Clin Invest 44:1672–1678
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; n = 4) or saline infusion (
; n = 4).
Values are the mean ± SEM for the 2-h period before
infusion (base), the 8-h period during saline or FFA infusion
(infusion), and the 2-h period after GHRH iv injection (GHRH).
Comparisons were made between infusion or GHRH periods and the basal
period for each group of animals (**, P < 0.01)
and between Intralipid infusion and saline infusion for each period
(++, P < 0.01).
