0013-7227/90/1276-3087$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 6 Printed in U.S.A.
Mechanisms of Impaired Growth Hormone Secretion in Genetically Obese Zucker Rats: Roles of Growth Hormone-Releasing Factor and Somatostatin* GLORIA SHAFFER TANNENBAUMt, MARTINE LAPOINTE, WENDY GURD, AND JUDITH A. FINKELSTEIN Neuropeptide Physiology Laboratory, McGill University-Montreal Children's Hospital Research Institute, and the Departments of Pediatrics, Neurology and Neurosurgery, McGill University (G.S.T., M.L., W.G.), Montreal, Quebec, H3H 1P3 Canada; and the Department of Anatomy, Northeastern Ohio Universities College of Medicine (J.A.F.), Rootstown, Ohio 44272
ABSTRACT. GH secretion is markedly blunted in obesity; however, the mechanism(s) mediating this response remains to be elucidated. In the present study we examined the involvement of the two hypothalamic GH-regulatory hormones, GH-releasing factor (GRF) and somatostatin (SRIF), using the genetically obese male Zucker rat. Spontaneous GH, insulin, and glucose secretory profiles obtained from free moving, chronically cannulated rats revealed a marked suppression in amplitude and duration of GH pulses in obese Zucker rats compared to their lean littermates (mean 6-h plasma GH level, 3.9 ± 0.4 us. 21.5 ± 3.8 ng/ml; P < 0.001). Obese rats also exhibited significant hyperinsulinemia in the presence of normoglycemia. The plasma GH response to an iv bolus of 1 ng rat GRF-(1-29) NH2, administered during peak and trough periods of the GH rhythm, was significantly attenuated in obese rats at peak (137.4 ± 26.1 vs. 266.9 ± 40.7 ng/ml; P < 0.02), although not at trough, times. Passive immunization of obese rats with a specific antiserum to SRIF failed to restore the amplitude of GH pulses to normal values; the mean 6-h plasma GH level of obese rats given SRIF antiserum was not significantly different from that of obese rats administered normal sheep serum. Both pituitary wet weight
P
ITUITARY GH secretion has been shown to be blunted in both human and experimental obesity (1). In the genetically obese Zucker rat, a model of spontaneous obesity due to an autosomal recessive gene {fa/fa) (2), plasma GH levels are depressed compared to those in their lean (nonobese) littermates {Fa/—) (3, 4); however, the mechanism mediating this response has not been elucidated. Received July 30, 1990. Address all correspondence and requests for reprints to: Dr. Gloria S. Tannenbaum, Neuropeptide Physiology Laboratory, McGill University-Montreal Children's Hospital Research Institute, 2300 Tupper Street, Montreal, Quebec, H3H 1P3 Canada. * Presented in part at the 17th Annual Meeting of the Society for Neuroscience, New Orleans, LA, 1987. This work was supported by Grant MT-6837 (to G.S.T.) from the Medical Research Council of Canada and grants from the United Way and the Ohio Board of Regents (to J.A.F.). t Senior Scholar of the Fonds de la recherche en sante du Quebec.
and pituitary GH content and concentration were reduced in the obese group. Measurement of hypothalamic GRF immunoreactivity revealed a significant (P < 0.05) reduction in the mediobasal hypothalamic GRF content in obese rats (503.2 ± 60.1 pg/fragment) compared to that in lean controls (678.1 ± 50.2 pg/fragment), although no significant difference was observed in hypothalamic SRIF concentration. Peripheral SRIF immunoreactive levels were significantly (P < 0.01) elevated in both the pancreas and stomach of obese rats. These results demonstrate that the genetically obese Zucker rat exhibits 1) marked impairment in both spontaneous and GRF-induced GH release, which cannot be reversed by SRIF immunoneutralization, 2) significant reduction in pituitary GH concentration, 3) depressed hypothalamic GRF content, and 4) elevated gastric and pancreatic, but not hypothalamic, SRIF levels. The findings suggest that the defect in pituitary GH secretion observed in the genetically obese Zucker rat is due, at least partially, to insufficient stimulation by hypothalamic GRF, and that SRIF does not play a significant role. (Endocrinology 127: 3087-3095, 1990)
The defect in GH secretion associated with obesity could originate at the pituitary or hypothalamic level. In normal animals, the secretion of GH from the anterior pituitary is governed by an intricate interplay between two hypothalamic neuropeptides, the stimulatory GHreleasing factor (GRF) and the inhibitory hormone somatostatin (SRIF). In the rat these two neurohormones are released in reciprocal cycles into the hypophyseal portal circulation to generate an ultradian rhythm of GH secretion (5, 6). In addition, the neuroendocrine mechanism^) governing the rhythmic secretion of GH is subject to modulatory influences from metabolic factors (79). The possibility that one or both of these hypothalamic hormones might be involved in mediating the GH suppression observed in the obese Zucker rat has been considered. Several groups have measured steady state
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GRF AND SRIF IN GENETICALLY OBESE ZUCKER RATS
levels of SRIF in the hypothalamus of lean and obese Zucker rats, but the data are not consistent. A decrease (10, 11), increase (12), and no change (13-15) in hypothalamic SRIF levels of obese rats have been reported. While peripheral organs, such as the pancreas, of obese rats have been shown to contain more SRIF (10, 11), it is not known whether circulating SRIF levels are elevated. Fewer studies have used GRF. In in vitro studies using dispersed pituitary cells from obese and lean Zucker rats, GH responsiveness to GRF was attenuated in cells from obese rats compared to that of lean littermates (16, 17). In vivo, GRF-induced, but not basal, GH release was found to be decreased in obese rats (12, 17). However, these latter experiments were carried out under sodium pentobarbital anesthesia, which itself is known to have effects on both hypothalamic GRF and SRIF release (6, 18). Moreover, we have shown that the GH response to GRF in conscious rats can vary significantly, depending upon whether the stimulation is at a time of a spontaneously occurring GH peak or a trough period (5, 19). The aim of the present study, therefore, was to assess the physiological roles of both SRIF and GRF in mediating the abnormal GH secretion observed in obesity. To this end, we studied spontaneous and GRF-induced GH release in conscious, free moving, obese and lean Zucker rats and assessed the effects of immunoneutralization with anti-SRIF serum. In addition, we have measured plasma insulin and glucose levels, pituitary GH concentration, and hypothalamic and peripheral tissue levels of GRF and SRIF. Materials and Methods Animals and experimental procedure Adult male obese and nonobese littermate rats of the Zucker strain were obtained from a breeding colony at Northeastern Ohio Universities College of Medicine (Rootstown, OH). The initial animals in the colony were purchased from the Harriet G. Bird Memorial Laboratories (Stowe, MA) (2). Upon arrival, the rats were individually housed on a 12-h light, 12-h dark cycle (lights on between 0600-1800 h) in a temperature (22 ± 1 C)- and humidity-controlled room. At the time of surgery, the mean body weight was 332 ± 4 g for the nonobese group and 533 ± 14 g for the obese animals. Chronic intracardiac venous cannulae were implanted under sodium pentobarbital (50 mg/kg, ip) anesthesia, using a previously described technique (20). After surgery, the animals were placed directly in isolation test chambers, with Purina rat chow (Ralston-Purina, St. Louis, MO) and tap water available ad libitum. Body weight was monitored daily, and the animals were sampled when body weight returned to preoperative levels (usually within 7 days). On the test day, food was removed 1.5-2 h before the start of sampling and returned at the end. In the first experiment we obtained spontaneous GH, insulin, and glucose secretory profiles in obese and lean littermates.
Endo • 1990 Vol 127 • No 6
Blood samples (0.45 ml) were withdrawn every 15 min for periods of 6 h from 1000-1600 h. All blood samples were immediately centrifuged, and the plasma was separated and stored at —20 C for subsequent assay of GH, insulin, and glucose. To avoid hemodynamic disturbance, the red blood cells were resuspended in normal saline and returned to the animal after removal of the next blood sample. In the second experiment we examined pituitary GH responsiveness to GRF in obese and lean littermates. Free moving, chronically cannulated rats were administered rat (r) GRF-(129)NH2 (1 (ig/0.3 ml) iv at two different time points during the 6-h sampling period. The times of 1100 and 1300 h were chosen, since these times reflect typical peak and trough periods of GH secretion, respectively, as previously documented in our laboratory (5, 19, 20). The GRF peptide (lot CH23-25-31-10-16, kindly provided by Dr. P. Brazeau, Notre Dame Hospital, Montreal, Quebec, Canada) was diluted in normal saline just before use. To document the rapidity of the GH response to GRF, an additional blood sample was obtained 5 min after each injection of the peptide. In the third experiment, designed to assess the role of endogenous SRIF, one group of obese rats was administered 2 ml of a specific SRIF antiserum, iv, after removal of the first blood sample, while another group of obese animals received 2 ml normal sheep serum at the same time point. The SRIF antiserum was the same as that used in our previous passive immunization studies (5, 8, 9). At the termination of the experiments, the animals were killed by rapid decapitation. The brain was immediately removed, and the hypothalamus was dissected according to the following landmarks: anterior, posterior, and lateral borders and depth of dissection were at the optic chiasm, mammillary bodies, hypothalamic sulci, and 1-1.5 mm from the base of the brain, respectively. The hypothalamic fragments were immediately frozen on dry ice, weighed, boiled in 1 ml 2 N acetic acid for 5 min, and homogenized at 600-800 rpm using a PotterElvehjem homogenizer. The homogenate was then centrifuged at 2000 X g for 30 min, and the supernatant was removed and frozen at —20 C until subsequent assay of SRIF and GRF. Portions of the pancreas (body and tail) and stomach (pylorus) were simultaneously rapidly removed, frozen on dry ice, and weighed. Individual tissue fragments were added to 2 ml 2 N acetic acid, boiled for 5 min to inactivate degradative enzymes, and then homogenized using a Polytron (Brinkmann Instruments, Westbury, NY), centrifuged, and stored, as described above, for subsequent assay of SRIF. The pituitary gland was also removed, weighed, homogenized in 2 ml 0.05 M NaHCO3Na2CO3 buffer, pH 9.96, and centrifuged at 2000 X g for 30 min. The supernatant was frozen at —20 C until subsequent assay for GH. Hormone assays Plasma and pituitary GH concentrations were determined in duplicate by double antibody RIA, using materials supplied by the NIDDK (Bethesda, MD). The averaged plasma GH values are reported in terms of the rat GH reference preparation (rGH RP-2). The standard curve was linear between 0.62-320 ng/ml. All samples with values above 320 ng/ml were reassayed
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GRF AND SRIF IN GENETICALLY OBESE ZUCKER RATS at dilutions ranging from 1:2 to 1:10. The intra- and interassay coefficients of variation were 9.1% and 12.1%, respectively, for duplicate samples of pooled plasma containing a mean GH concentration of 75.4 ng/ml. Plasma immunoreactive insulin (IRI) was measured by a dextran-coated charcoal method, using guinea pig antiporcine insulin serum (8). Purified crystalline rat insulin (lot 615-JE6-9, courtesy of Dr. R. Chance, Eli Lilly Co., Indianapolis, IN) served as a reference standard. The sensitivity of the assay was 0.16 ng/ml, and the intra- and interassay coefficients of variation were 8.4% and 14.3%, respectively, for duplicate samples of pooled plasma containing a mean IRI concentration of 1.25 ng/ml. Plasma glucose was measured by an automated glucose oxidase method (Glucose Analyzer 2, Beckman Instruments, Palo Alto, CA). Somatostatin-like immunoreactivity (SLI) was assayed by a previously reported double antibody RIA (7). On the day of the assay, the hypothalamic samples were recentrifuged at 900 X g for 10 min and then diluted at concentrations ranging from 1:100 to 1:200 depending on the origin of the tissue, and neutralized with 5 N NaOH using phenol red as a pH indicator. The SLI values are reported in terms of the somatostatin-14 reference standard (lot 83-57-3G-60-70, kindly provided by Dr. N. Ling, Salk Institute, La Jolla, CA). The intra- and interassay coefficients of variation were 10.8% and 14.1%, respectively, for pooled hypothalamic extracts containing a mean SRIF concentration of 92.1 pg/assay tube. The SRIF binding of plasma from rats treated with SRIF antiserum and normal sheep serum was assessed by determining the ability of aliquots of plasma obtained 6 h after the injections to bind [^I-TyrJSRIF. Plasma samples from each rat were diluted 1:100 in the SRIF RIA assay buffer, and binding to labeled SRIF was determined under conditions routinely used for RIA of SRIF (7). GRF-like immunoreactivity was measured by a newly developed double antibody RIA. The GRF antiserum was generated in sheep using synthetic rat GRF-(1-29)NH2. rGRF-(l-29)NH2 (8.9 Mg) dissolved in 2 ml 0.1 N HC1 and 1.9 ml 0.1 N NaOH was combined with 2.5 mg methylated BSA dissolved in 4.1 ml normal saline. This mixture was emulsified in an equal volume of complete Freund's adjuvant, and two sheep were injected sc at multiple sites. The animals received booster injections of rGRF-(l-29)NH2 emulsified in incomplete Freund's adjuvant at monthly intervals and were bled 2 weeks after each injection. Maximal titers of antisera were achieved by 7 weeks. In an RIA for rGRF-(l-43)OH, this antiserum, at a final dilution of 1:78,000, binds 35% of 125I-labeled rGRF. The antiserum shows no cross-reactivity with SRIF-14, SRIF-28 (Dr. N. Ling, Salk Institute), rGH, LH, FSH, PRL (NIDDK), calcitonin (Sandoz, Basel, Switzerland), insulin (Eli Lilly Co.), glucagon (Sigma, St. Louis, MO), ovine CRF, secretin, vasoactive intestinal peptide, and cholecystokinin-8 (Peninsula, Belmont, CA). rGRF-(l-43)OH (lot 115-194-11G-74-86-12G-70-88, Dr. N. Ling, Salk Institute) was iodinated with chloramine-T. The iodination mixture consisted of 3 ^g rGRF dissolved in 10 /nl 0.05 N acetic acid, 15 n\ 0.5 M phosphate buffer (pH 7.5), 1 mCi Na125I (IMS-30, Amersham, Oakville, Ontario, Canada), and 10 Mg chloramine-T in distilled H2O at a concentration of 1 mg/ml at room temperature. The reaction was stopped after 30
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sec by the addition of 5% BSA in 0.05 M phosphate buffer, pH 7.5 (200 fi\). [i25I]rGRF was purified by gel filtration on a Sephadex G-50 (fine) column (1.5 x 60 cm) in 0.1 N acetic acid with 0.1% BSA. Samples were neutralized with 5 N NaOH, using phenol red as an indicator, and centrifuged at 900 x g for 10 min before assay. Assays were performed in polystyrene tubes at 4 C. The assay buffer consisted of 10 mM NaHPO4-NaH2PO4 (pH 7.5), 0.2 M Na acetate, 25 mM EDTA, 150 mM NaCl, 0.1% Na azide (g/vol), 0.1% human serum albumin (g/vol), and 0.1% Triton X-100 (vol/vol) (Sigma) in distilled water, pH 7.2. The total volume of each tube was 600 nl. Separation of bound from free ligand was accomplished by the second antibody technique. Maximal sensitivity was achieved by delayed addition of labeled rGRF; the initial incubation was 24 h, with a subsequent incubation of 48 h after the addition of labeled rGRF. GRFlike immunoreactive values are reported in terms of the rat GRF-(1-43)OH reference preparation (lot 115-194-11G-7486-12G-70-88; Dr. N. Ling, Salk Institute) that was used for the standard curve of the assay. The sensitivity of the rGRF assay was 10 pg/tube, with half-maximal displacement of 135 pg/tube. The intra- and interassay coefficients of variation were 10.3% and 10.9%, respectively, for duplicate samples from pooled hypothalamic extracts containing a mean rGRF concentration of 142 pg/tube. Statistical analyses Analysis of variance for repeated measures and Student's t test were used for statistical comparisons between experimental groups. Student's paired t test was used to evaluate treatment effects within groups. P < 0.05 was considered significant. Results Spontaneous GH, IRI, and glucose secretory patterns in lean and obese Zucker rats Figure 1 illustrates individual plasma GH and IRI profiles in an obese rat compared to those in a lean littermate. Lean rats showed the typical pulsatile pattern of GH secretion; two episodes of GH secretion were evident during the 6-h sampling period (at 1030-1200 and 1350-1500 h), with most individual peak GH values above 90 ng/ml and trough levels below 1.2 ng/ml. Plasma IRI levels remained low and fluctuated minimally. In contrast, obese rats exhibited a marked suppression in amplitude and duration of spontaneous GH secretory bursts compared to lean littermates; the mean GH peak amplitude was reduced approximately 6-fold (20.8 ± 3.2 vs. 116.7 ± 31.0 ng/ml; P < 0.01). Generally, two brief episodes of GH secretion, separated by undetectable trough levels, were observed in obese rats, and the mean intervals between GH secretory episodes were similar in the two groups (2.9 ± 0.2 vs. 3.1 ± 0.2 h). Plasma IRI levels in these animals were markedly elevated and showed wide fluctuations (Fig. 1). Plasma glucose re-
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GRF AND SRIF IN GENETICALLY OBESE ZUCKER RATS
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caused a 3- to 6-fold increase in plasma GH levels, whereas injection during a trough period (1300 h) had only a minimal effect (Fig. 2A). In obese rats, the magnitude of the GH response to rGRF was markedly attenuated at peak, although not at trough, times compared to that in lean littermates (Fig. 2B). Figure 3 summarizes the GH responsiveness to GRF in lean and obese littermates. In lean animals, the mean plasma GH response at 1100 h (266.9 ± 40.7 ng/ml) was significantly greater than that at 1300 h (55.2 ± 12.6 ng/ml; P < 0.001). Obese rats also showed a significant time-dependent difference in GH responsiveness to rGRF (137.4 ± 26.1 vs. 54.1 ± 12.9 ng/ml; P < 0.05). However, comparisons between the two groups indicated that GRF-induced GH release in obese rats was significantly (P < 0.02) lower than that in lean littermates at peak times, although there was no difference at trough periods. Effects of SRIF antiserum on GH secretory dynamics in obese rats
»»«•»•< 1000
1100
1200
*-«
1300
M M
1400
I
1500
M
_ 1600
Time (hours)
FIG. 1. Individual representative 6-h plasma GH and 3-h plasma IRI profiles in an obese Zucker rat compared to those in a lean littermate. Obese animals exhibited a marked suppression of both amplitude and duration of spontaneous GH surges and significant elevation of plasma IRI levels.
mained stable in both groups. The mean 6-h plasma GH level of obese rats was significantly (P < 0.001) lower than that of lean littermatss (Table 1). Whereas obese rats showed a marked hyperinsulinemia over a 3-h sampling period, there was no significant difference in basal plasma glucose levels between the two groups (Table 1).
The GH secretory profiles of obese rats administered normal sheep serum continued to show a marked suppression of GH pulse amplitude (Fig. 4A). Administration of SRIF antiserum to obese rats caused an initial surge of GH release, but failed to restore the amplitude of GH pulses to that observed in lean animals (Fig. 4B). The mean 6-h plasma GH level of obese rats given SRIF antiserum was not significantly different from that of obese normal sheep serum-treated controls (Table 2). Immunoneutralization of endogenous SRIF also did not significantly alter the hyperinsulinemia observed in obese rats (Table 2). SRIF binding of rat plasma after SRIF antiserum administration The mean SRIF binding level of a 1:100 dilution of plasma of obese rats injected with SRIF antiserum was 47.4 ± 0.5%, and it was 4.9 ± 0.3% after normal sheep serum injection.
GH responsiveness to GRF in lean and obese Zucker rats
Effect of obesity on pituitary wet weight, GH content, and GH concentration
In lean animals, the iv administration of 1 /xg rGRF(1-29)NH2 during a time of peak GH secretion (1100 h)
Obese rats showed a significant (P < 0.05) atrophy of the pituitary gland compared to their lean littermates
TABLE 1. Mean plasma GH, insulin, and glucose levels in lean and obese Zucker rats
Experimental group
n
Final BW (g)
Mean 6-h plasma GH (ng/ml)
Mean 3-h plasma IRI (ng/ml)
Mean basal plasma glucose (mg/dl)
Lean Obese
8 12
385.8 ± 11.4 603.6 ± 16.3°
21.5 ± 3.8 3.9 ± 0.4°
0.47 ± 0.04 13.11 ± 2.21"
125.1 ± 6.8 123.9 ± 0.8
Values are the mean ± SE. P < 0.001 compared to lean littermates.
0
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GRF AND SRIF IN GENETICALLY OBESE ZUCKER RATS 400
Lean
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350
Peak Trough
300
200
E 100
300 „ B
Obese
200
Lean (11)
100
1000
1100
1200
1300
1400
1500
1600
Time (hours)
FIG. 2. Plasma GH response to lMgrGRF-(l-29)NH2,iv, administered during peak (1100 h) and trough (1300 h) periods of the GH rhythm. In lean rats (A), administration of rGRF during a time of peak secretion resulted in a 3- to 6-fold increase in plasma GH levels, whereas injection during trough periods had only a minimal effect. In obese rats (B), the magnitude of the GH response to rGRF was markedly attentuated at peak times. Arrows indicate the times of injections.
(Fig. 5). Both pituitary GH content and GH concentration were markedly reduced in obese rats compared to those in lean controls (Fig. 5). Hypothalamic GRF immunoreactivity in lean and obese Zucker rats Immunoreactive GRF levels in the two groups are shown in Table 3. Obese rats exhibited a significant decrease in hypothalamic GRF content compared to lean littermates. Hypothalamic and peripheral tissue immunoreactive SRIF levels in lean and obese Zucker rats Immunoreactive SRIF levels in the two groups are shown in Table 4. While the hypothalamic SLI concen-
Obese (10)
FIG. 3. Summary of GH responsiveness to rGRF in lean and obese rats. Both groups exhibited a time-dependent difference in their ability to respond to rGRF, with GRF-induced GH release being significantly greater during peak compared to trough times. However, in obese rats, there was a significant reduction in GH responsiveness to GRF at times of peak GH secretion, although not at trough periods. Each bar
represents the mean ± SE, and the number of animals in each group is shown in parentheses, a, P < 0.05 or less compared to GH release during peak periods in lean and obese rats, b, P < 0.02 compared to rGRF-induced GH release in lean rats at peak times.
tration was not altered by obesity, both pancreas and stomach SLI concentrations were significantly augmented in obese rats compared to lean littermates.
Discussion The finding of a suppression of GH pulse amplitude and lower mean plasma GH levels in genetically obese Zucker rats compared to their lean littermates is consistent with previous observations using both single point determinations (3) and pulsatile release patterns over a 6-h sampling period (4). The lack of a difference in basal GH levels, reported in other studies (12, 17), is probably due to the confounding influence of anesthesia used in those experiments, thus emphasizing the importance of studying GH physiology in the conscious state. The lean male Zucker rats of the present study show an ultradian GH secretory pattern similar to that of Sprague-Dawley rats, as seen in numerous other studies since the first
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GRF AND SRIF IN GENETICALLY OBESE ZUCKER RATS
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Obese Zucker Rat + Normal Sheep Serum 90 r-
60 C
FIG. 4. Effects of passive immunization with SRIF antiserum on plasma GH profiles in obese Zucker rats (B) compared to those in obese rats given normal sheep serum (A). Administration of SRIF antiserum caused an initial surge of GH release, but failed to restore the amplitude of GH pulses to that of pulses in lean animals. Arrows indicate the times of injection.
30
0 C
H H I
•m*
IMIMI
o o Obese Zucker Rat + SRIF Antiserum
X
o
B
90
o CO
E
60
CO
30
1000
1200
1400
1600
1000
1400
1200
1600
Time (hours) TABLE 2. Effects of SRIF antiserum on mean plasma GH and IRI levels in obese Zucker rats Experimental group Obese + normal sheep serum Obese + SRIF antiserum
4 4
TABLE 3. Hypothalamic GRF immunoreactive levels in lean and obese Zucker rats
Mean 6-h plasma GH (ng/ml)
Mean 3-h plasma IRI (ng/ml)
Experimental group
MBH wet wt (mg)
GRF-like immunoreactivity (pg/fragment)
7.02 ± 2.30 9.16 ± 1.02
13.57 ± 3.0 17.53 ± 8.79
Lean Obese
16.0 ± 1.4 17.6 ± 1.1
678.1 ± 50.2 503.2 ± 60.1°
Values are the mean ± SE. MBH, Mediobasal hypothalamus. P < 0.05 compared to lean littermates.
Values are the mean ± SE.
0
TABLE 4. Hypothalamic and peripheral tissue SLI in lean and obese Zucker rats Pituitary Wet Weight (mg)
Pituitary GH Content (ug/gland)
Pituitary GH Concentration (ug/mg wet wt) I Lean (6)
Experimental group
Hypothalamic SLI (ng/mg wet wt)
Lean Obese
10.2 ± 0.9 8.7 ± 0.9
I Obese (8)
Pancreatic Stomach SLI SLI (ng/mg wet wt) (ng/mg wet wt) 0.58 ± 0.06 0.94 ± 0.13°
1.05 ± 0.06 1.38 ± 0.09"
Values are the mean ± SE. " P < 0.01 compared to lean littermates.
FIG. 5. Effects of obesity on pituitary weight, GH content, and GH concentration. Obese rats exhibited a significant reduction in both pituitary wet weight and pituitary GH content and concentration. Each bar represents the mean ± SE; the number of animals in each group is in parentheses. &,P< 0.05 or less compared to lean littermates.
description in 1976 (20), although the amplitude of the GH peaks in the Zucker strain appears to be slightly attenuated. The observation of hyperinsulinemia with normal blood glucose levels is a consistent feature of the genetically obese Zucker rat (21), although other studies of insulin levels in "fatty" rats have used only single point
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GRF AND SRIF IN GENETICALLY OBESE ZUCKER RATS determinations (22). The present results demonstrate that plasma insulin levels of obese rats are high and fluctuate widely compared to those in their lean littermates. While the origin of the fluctuations in insulin levels is not known, there did not appear to be any association with the GH secretory patterns. We previously reported in normal Sprague-Dawley rats that, in the absence of food during the testing period, plasma GH levels do not correlate with either glucose or insulin levels (23). Even though food was not available in the testing situation of the present study, the obese rats would be more likely to anticipate a meal during that time period because they are known to feed more during the light hours than lean animals (24), and this might account in part for the wide variations observed. The depressed plasma GH levels are reflective of the situation in the pituitary gland, where both the content and concentration of GH were significantly lower in obese us. lean rats. It is not known if the entire gland is affected or if the weight difference we observed is limited to a specific subdivision. No direct measurement of the number of somatotrophs in rats of the Zucker strain has been made. The finding that the amount of DNA per pituitary gland is the same in obese and lean Zucker rats (25) can be taken as indirect evidence that the total cell numbers are comparable, although relative distribution of cell types may vary. In vitro studies have shown that basal levels of GH release from pituitary cells in culture taken from lean and obese rats do not differ, but that GH responsiveness to GRF stimulation is attenuated in pituitary cells taken from obese animals (12, 16, 17). Our in vivo data in free moving, conscious rats also show attenuated sensitivity to GRF in obese rats, consistent with that previously reported in anesthetized animals (12, 17). Such an impairment in GH responsiveness to GRF has also been reported in obese humans (26). Both the lean and obese Zucker rats of the present study exhibited a time-dependent difference in GH responsiveness to GRF, with GRF-induced GH release being greater when GRF was administered during a peak compared to a trough period. These results are similar to those we previously reported in male Sprague-Dawley rats (5, 19). Since the variability in GH responsiveness to GRF has been shown to be due to interference by the cyclic increased release of endogenous SRIF (5), our findings suggest that SRIF secretion is also episodic in both lean and obese rats of the Zucker strain. However, a differential responsiveness between obese and lean rats was seen when GRF was administered at peak times of GH secretion, with obese rats exhibiting significantly lower GRF-induced GH release compared to their lean littermates. Since the high GH release observed in response to GRF administered at peak times is probably
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due to the additive effect of exogenous GRF to that of endogenous hypothalamic GRF, our data suggest that a reduction in endogenous hypothalamic GRF secretion may be a determining factor in the diminished GH secretion of the obese rats. Indeed, direct measurement of hypothalamic GRF levels in lean and obese rats of the present study showed a significant diminution of GRF immunoreactivity in the hypothalami of obese rats. It is also possible that the impaired responses to GRF are due to alterations in the GRF somatotroph receptor secondary to insufficient stimulation by hypothalamic GRF. Lack of exposure of pituitary somatotrophs to normal GRF signaling could conceivably alter GRF receptor sensitivity and perhaps account for the reduction in GH responsiveness to GRF. Such a mechanism still remains to be elucidated, although preliminary evidence suggests that this may not be the case; no differences in affinities or capacities of GRF-binding sites were found between lean and obese Zucker rat pituitaries (27). The possibility that the decreased pulsatile GH release in obese animals is a result of excessive S R I F (inhibitory) tone was addressed by two types of experiments: direct measurement of hypothalamic and peripheral tissue immunoreactive SRIF levels, and immunoneutralization of endogenous circulating SRIF by antibody injection. Neither experiment gave clear support to the concept of increased SRIF secretion in obese rats. While earlier studies had reported that genetically obese Zucker rats have decreased levels of hypothalamic SRIF (10, 11), more recent reports (13-15) are in agreement with our present data, which have failed to find a significant difference in hypothalamic SRIF concentration between obese and lean animals. However, we did observe elevated SRIF levels in the pancreas and stomach of obese Zucker rats, consistent with previous reports (10, 11, 14). Whether this increase in peripheral tissue SRIF represents changes in SRIF synthesis, storage, or release cannot be distinguished on the basis of the present studies. If the depressed GH levels in obese rats were produced by elevated circulating SRIF levels, treatment of obese rats by passive immunization with SRIF antibody would be expected to reverse the GH suppression, as shown previously in other situations of GH suppression in the rat, including starvation (28) and diabetes (8). However, administration of SRIF antiserum to obese rats failed to restore the amplitude of GH pulses to normal values. Mean 6-h plasma GH levels in obese rats given SRIF antiserum were no different from those in normal sheep serum-treated obese animals. As SRIF binding of a 1:100 dilution of plasma samples of animals treated with SRIF antiserum was approximately 50% throughout the experimental procedure, ineffective immunoneutralization is not likely to be the cause of the failure of SRIF
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GRF AND SRIF IN GENETICALLY OBESE ZUCKER RATS
antiserum to reverse the GH inhibition. These findings, therefore, suggest that increased SRIF release is not the mechanism mediating the attenuated GH secretion in the genetically obese Zucker rat. Additional support for this view comes from recent experiments in our laboratory in which we directly measured plasma levels of SLI in obese and lean Zucker rats and found no difference (Patel, Y. C., and G. S. Tannenbaum, unpublished observations). However, the possibility that an alteration of the SRIF somatotroph receptor could be involved still remains to be explored. Abnormal GH release patterns have been demonstrated in other experimental models of disordered metabolism in the rat; the genetically obese Zucker rat shares some characteristics with these types of rats, but does not duplicate any of them. For example, food deprivation (7) and both streptozotocin-induced (8) and spontaneous (29) diabetes result in a suppression of GH pulse amplitude. In contrast to the obese rats, starved and diabetic rats have elevated plasma SRIF levels (19, 30, 31) and exhibit enhanced responsiveness to GRF (19, 32, 33). Rats made obese by lesioning the ventromedial hypothalamic nuclei also have markedly depressed GH pulses (34), an effect probably due to destruction of GRFcontaining neurons in the mediobasal hypothalamus. Although the obese Zucker rats do exhibit lower hypothalamic GRF levels, this peptide is present and measurable. Whether the attenuated GH release in genetically obese Zucker rats follows or precedes the lower hypothalamic GRF levels cannot be distinguished on the basis of the present experiments, since our studies have been performed in adult rats only. GH itself appears to exert feedback regulation on hypothalamic GRF, as suggested by the finding that hypophysectomy results in depressed hypothalamic GRF content and release (35). Developmental studies currently underway should elucidate this relationship. Recent investigations have focused on the underlying molecular basis of the decreased plasma GH levels in obese Zucker rats. Using steady state levels of GH mRNA as a measure of gene expression, GH gene expression has been shown to be significantly lower in the obese rats (25) as early as 5 weeks of age (36). Results obtained from an in situ hybridization analysis indicate that each somatotroph contains fewer GH transcripts in the obese animals, rather than there being fewer somatotrophs present (37). Since GRF has been shown to stimulate transcription of the GH gene (38) and to increase GH mRNA levels in rat pituitary (39), it is possible that the decrease in hypothalamic GRF observed here in obese rats results in lowered GH gene expression and, consequently, could account for the lower steady state levels of GH mRNA reported. A recent preliminary study of hypothalamic gene expression in the genetically obese
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Zucker rat indicates that hypothalamic GRF mRNA is reduced, but hypothalamic SRIF mRNA is normal, in obese rats compared to that in lean controls (40). Thus, the gene expression and the peptide content data of the present report are in agreement. Several other factors that have been shown to regulate GH mRNA levels are also known to be abnormal in obese Zucker rats; these include insulin (22, 41), thyroid hormones (42-44), glucocorticoids (43-45), and insulin-like growth factor-1 (12, 46, 47). SRIF, however, appears to act at the level of release, rather than at the level of gene expression (47). Specific roles for any of these signals in modulating the hyposomatotropism of obesity have not yet been defined, but could be important. In summary, the results of the present study demonstrate that the genetically obese Zucker rat exhibits marked impairment in both spontaneous and GRF-induced GH release, which cannot be reversed by antiSRIF treatment. This hyposomatotropism is associated with a reduction in pituitary GH concentration, depressed hypothalamic GRF content, and elevated gastric and pancreatic, but not hypothalamic, SRIF levels. Taken together, these findings suggest that the defect in pituitary GH secretion in genetically obese Zucker rats is due, at least partially, to insufficient stimulation by hypothalamic GRF, and that SRIF does not play a significant role.
Acknowledgments We are grateful to Julie Temko for expert secretarial assistance, and to the NIDDK for the generous supply of rGH RIA materials.
References 1. Sims EAH, Danforth Jr E, Horton ES, Bray GA, Glennon JA, Salans LB 1973 Endocrine and metabolic effects of experimental obesity in man. Recent Prog Horm Res 29:457-496 2. Zucker LM, Zucker TF 1961 Fatty, a new mutation in the rat. J Hered 52:275-278 3. Martin RJ, Gahagan J 1977 Serum hormone levels and tissue metabolism in pair-fed lean and obese Zucker rats. Horm Metab Res 9:181-186 4. Finkelstein JA, Jervois P, Menadue M, Willoughby JO 1986 Growth hormone and prolactin secretion in genetically obese Zucker rats. Endocrinology 118:1233-1236 5. Tannenbaum GS, Ling N 1984 The interrelationship of growth hormone (GH)-releasing factor and somatostatin in generation of the ultradian rhythm of GH secretion. Endocrinology 115:19521957 6. Plotsky PM, Vale W 1985 Patterns of growth hormone-releasing factor and somatostatin secretion into hypophysial portal circulation of the rat. Science 230:461-463 7. Tannenbaum GS, Rorstad O, Brazeau P 1979 Effects of prolonged food deprivation on the ultradian growth hormone rhythm and immunoreactive somatostatin tissue levels in the rat. Endocrinology 104:1733-1738 8. Tannenbaum GS 1981 Growth hormone secretory dynamics in streptozotocin diabetes: evidence of a role for endogenous circulating somatostatin. Endocrinology 108:76-82 9. Painson JC, Tannenbaum GS 1985 Effects of intracellular glucopenia on pulsatile growth hormone secretion: mediation in part by
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GRF AND SRIF IN GENETICALLY OBESE ZUCKER RATS somatostatin. Endocrinology 117:1132-1138 10. Sheppard MC, Shapiro B, Hudson A, Pimstone BL 1980 Tissue and serum somatostatin-like immunoreactivity in lean and obese Zucker rats. Horm Metab Res 12:236-239 11. Voyles NR, Awoke S, Wade A, Bhathena SJ, Smith SS, Recant L 1982 Starvation increases gastrointestinal somatostatin in normal and obese Zucker rats: a possible regulatory mechanism. Horm Metab Res 14:392-395 12. Renier G, Gaudreau P, Deslauriers N, Petitclerc D, Brazeau P 1990 Dynamic of the GRF-induced GH response in genetically obese Zucker rats: influence of central and peripheral factors. Regul Peptides 28:95-106 13. Fletcher JM, Haggarty P, Wahle KWJ, Reeds PJ 1986 Hormonal studies of young lean and obese Zucker rats. Horm Metab Res 18:290-295 14. Voyles NR, Bhathena SJ, Kennedy B, Wilkins SD, Michaelis IV OE, Zalenski CM, Timmers KI, Recant L 1987 Tissue somatostatin levels in three models of genetic obesity in rats (42515). Proc Soc Exp Biol Med 185:49-54 15. DonCarlos LL, Ho RH, Finkelstein JA 1988 Immunocytochemical analysis of somatostatin in the hypothalamus of obese and nonobese Zucker rats. Brain Res 458:372-376 16. Heiman ML, Porter JR, Nekola MV, Murphy WA, Hartman AD, Lance VA, Coy DH 1985 Adenohypophyseal response to hypophysiotropic hormones in male obese Zucker rats. Am J Physiol 249:E380-E384 17. Renier G, Gaudreau P, Deslauriers N, Brazeau P 1989 In vitro and in vivo growth hormone responsiveness to growth hormone-releasing factor in male and female Zucker rats. Neuroendocrinology 50:454-459 18. Chihara K, Arimura A, Schally AV 1979 Immunoreactive somatostatin in rat hypophyseal portal blood: effects of anesthetics. Endocrinology 104:1434-1441 19. Tannenbaum GS, Painson J-C, Lengyel AMJ, Brazeau P 1989 Paradoxical enhancement of pituitary growth hormone (GH) responsiveness to GH-releasing factor in the face of high somatostatin tone. Endocrinology 124:1380-1388 20. Tannenbaum GS, Martin JB 1976 Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat. Endocrinology 98:562-570 21. Zucker LM, Antoniades HN 1972 Insulin and obesity in the Zucker genetically obese rat "fatty." Endocrinology 90:1320-1330 22. Martin RJ, Wangsness PJ, Gahagan JH 1978 Diurnal changes in serum metabolites and hormones in lean and obese Zucker rats. Horm Metab Res 10:187-192 23. Tannenbaum GS, Martin JB, Colle E 1976 Ultradian growth hormone rhythm in the rat: effects of feeding, hyperglycemia and insulin-induced hypoglycemia. Endocrinology 99:720-727 24. Becker EE, Grinker JA 1977 Meal patterns in the genetically obese Zucker rat. Physiol Behav 18:685-692 25. Ahmad I, Steggles AW, Carrillo AJ, Finkelstein JA 1989 Obesityand sex-related alterations in growth hormone messenger RNA levels. Mol Cell Endocrinol 65:103-109 26. Williams T, Berelowitz M, Joffe SN, Thorner MO, Rivier J, Vale W, Frohman LA 1984 Impaired growth hormone responses to growth hormone-releasing factor in obesity. A pituitary defect reversed with weight reduction. N Engl J Med 311:1403-1407 27. Abribat T, Finkelstein JA, Gaudreau P 1990 High and low affinity binding sites for [12SI-Tyr10]human growth hormone-releasing factor in Sprague-Dawley and Zucker rat pituitaries. Neuroendocrinology [Suppl 1] 52:119 (Abstract) 28. Tannenbaum GS, Epelbaum J, Colle E, Brazeau P, Martin JB 1978 Antiserum to somatostatin reverses starvation-induced inhibition of growth hormone but not insulin secretion. Endocrinology 102:1909-1914
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29. Tannenbaum GS, Colle E, Gurd W, Wanamaker L 1981 Dynamic time course studies of the spontaneously diabetic "BB" Wistar rat. I. Longitudinal profiles of plasma growth hormone, insulin and glucose. Endocrinology 109:1872-1879 30. Patel YC, Wheatley T, Zingg HH 1980 Increased blood somatostatin concentration in streptozotocin diabetic rats. Life Sci 27:1563-1570 31. Patel YC, Wheatley T, Malaisse-Lagae F, Orci L 1980 Elevated portal and peripheral blood concentration of immunoreactive somatostatin in spontaneously diabetic (BBL) Wistar rats. Diabetes 29:757-761 32. Locatelli V, Rovati S, Miyoshi H, Miiller EE 1984 Growth hormone hyperresponsiveness to human pancreatic growth hormone releasing hormone in streptozotocin-diabetic rats. Horm Metab Res 16:507 33. Serri O, Brazeau P 1987 Growth hormone responsiveness in vivo and in vitro to growth hormone releasing factor in the spontaneously diabetic BB Wistar rat. Neuroendocrinology 46:162-166 34. Eikelboom R, Tannenbaum GS 1983 Effects of obesity-inducing ventromedial hypothalamic lesions on pulsatile growth hormone and insulin secretion: evidence for the existence of a growth hormone-releasing factor. Endocrinology 112:212-219 35. Katakami H, Downs TR, Frohman LA 1987 Effect of hypophysectomy on hypothalamic growth hormone-releasing factor content and release in the rat. Endocrinology 120:1079-1082 36. Ahmad I, Steggles AW, Carrillo AJ, Finkelstein JA 1990 Developmental changes in levels of GH mRNA in Zucker rats. J Cell Biochem 43:59-66 37. Ahmad I, Steggles AW, Finkelstein JA 1990 In situ hybridization study of GH gene expression in lean and genetically obese Zucker rats. Anat Rec 226:5A (Abstract) 38. Barinaga M, Yamomoto G, Rivier C, Vale W, Evans R, Rosenfeld MG 1983 Transcriptional regulation of growth hormone gene expression by growth hormone-releasing factor. Nature 306:84-85 39. Gick GG, Zeytin FN, Brazeau P, Ling NC, Esch FS, Bancroft C 1984 Growth hormone-releasing factor regulates growth hormone mRNA in primary cultures of rat pituitary cells. Proc Natl Acad Sci USA 81:1553-1555 40. Finkelstein JA, Ahmad I, Steggles AW, Chomczynski P, Downs TR, Frohman LA 1990 Levels of growth hormone-releasing hormone mRNA and somatostatin mRNA in the hypothalamus of the genetically obese Zucker rat. J Cell Biochem 14F:40 (Abstract) 41. Yamashita S, Melmed S 1986 Effect of insulin on rat pituitary cells; inhibition of GH secretion and GH mRNA levels. Diabetes 35:440-447 42. Goldberg JR, Ehrmann B, Katzeff HL 1988 Altered triiodothyronine metabolism in Zucker fatty rats. Endocrinology 122:689-693 43. Evans RM, Birnberg NC, Rosenberg MG 1982 Glucocorticoid and thyroid hormones transcriptionally regulate growth hormone gene expression. Proc Natl Acad Sci USA 79:7659-7663 44. Martinoli MG, Pelletier G 1989 Thyroid and glucocorticoid hormone regulation of rat pituitary growth hormone messenger ribonucleic acid as revealed by in situ hybridization. Endocrinology 125:1246-1252 45. Guillaume-Gentil C, Rohner-Jeanrenaud F, Abramo F, Bestetti GE, Rossi GL, Jeanrenaud B 1990 Abnormal regulation of the hypothalamo-pituitary-adrenal axis in the genetically obese fa/fa rat. Endocrinology 126:1873-1879 46. Grzywa M 1984 Serum somatomedin activity and growth hormone secretion by cultured pituitary cells from genetically obese Zucker rats. Endokrynol Pol 35:105-111 47. Namba H, Morita S, Melmed S 1989 Insulin-like growth factor-I action on growth hormone secretion and messenger ribonucleic acid levels: interaction with somatostatin. Endocrinology 124:1794-1799
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Vol. 127, No. 6 Printed in U.S.A.
Mechanisms of Impaired Growth Hormone Secretion in Genetically Obese Zucker Rats: Roles of Growth Hormone-Releasing Factor and Somatostatin* GLORIA SHAFFER TANNENBAUMt, MARTINE LAPOINTE, WENDY GURD, AND JUDITH A. FINKELSTEIN Neuropeptide Physiology Laboratory, McGill University-Montreal Children's Hospital Research Institute, and the Departments of Pediatrics, Neurology and Neurosurgery, McGill University (G.S.T., M.L., W.G.), Montreal, Quebec, H3H 1P3 Canada; and the Department of Anatomy, Northeastern Ohio Universities College of Medicine (J.A.F.), Rootstown, Ohio 44272
ABSTRACT. GH secretion is markedly blunted in obesity; however, the mechanism(s) mediating this response remains to be elucidated. In the present study we examined the involvement of the two hypothalamic GH-regulatory hormones, GH-releasing factor (GRF) and somatostatin (SRIF), using the genetically obese male Zucker rat. Spontaneous GH, insulin, and glucose secretory profiles obtained from free moving, chronically cannulated rats revealed a marked suppression in amplitude and duration of GH pulses in obese Zucker rats compared to their lean littermates (mean 6-h plasma GH level, 3.9 ± 0.4 us. 21.5 ± 3.8 ng/ml; P < 0.001). Obese rats also exhibited significant hyperinsulinemia in the presence of normoglycemia. The plasma GH response to an iv bolus of 1 ng rat GRF-(1-29) NH2, administered during peak and trough periods of the GH rhythm, was significantly attenuated in obese rats at peak (137.4 ± 26.1 vs. 266.9 ± 40.7 ng/ml; P < 0.02), although not at trough, times. Passive immunization of obese rats with a specific antiserum to SRIF failed to restore the amplitude of GH pulses to normal values; the mean 6-h plasma GH level of obese rats given SRIF antiserum was not significantly different from that of obese rats administered normal sheep serum. Both pituitary wet weight
P
ITUITARY GH secretion has been shown to be blunted in both human and experimental obesity (1). In the genetically obese Zucker rat, a model of spontaneous obesity due to an autosomal recessive gene {fa/fa) (2), plasma GH levels are depressed compared to those in their lean (nonobese) littermates {Fa/—) (3, 4); however, the mechanism mediating this response has not been elucidated. Received July 30, 1990. Address all correspondence and requests for reprints to: Dr. Gloria S. Tannenbaum, Neuropeptide Physiology Laboratory, McGill University-Montreal Children's Hospital Research Institute, 2300 Tupper Street, Montreal, Quebec, H3H 1P3 Canada. * Presented in part at the 17th Annual Meeting of the Society for Neuroscience, New Orleans, LA, 1987. This work was supported by Grant MT-6837 (to G.S.T.) from the Medical Research Council of Canada and grants from the United Way and the Ohio Board of Regents (to J.A.F.). t Senior Scholar of the Fonds de la recherche en sante du Quebec.
and pituitary GH content and concentration were reduced in the obese group. Measurement of hypothalamic GRF immunoreactivity revealed a significant (P < 0.05) reduction in the mediobasal hypothalamic GRF content in obese rats (503.2 ± 60.1 pg/fragment) compared to that in lean controls (678.1 ± 50.2 pg/fragment), although no significant difference was observed in hypothalamic SRIF concentration. Peripheral SRIF immunoreactive levels were significantly (P < 0.01) elevated in both the pancreas and stomach of obese rats. These results demonstrate that the genetically obese Zucker rat exhibits 1) marked impairment in both spontaneous and GRF-induced GH release, which cannot be reversed by SRIF immunoneutralization, 2) significant reduction in pituitary GH concentration, 3) depressed hypothalamic GRF content, and 4) elevated gastric and pancreatic, but not hypothalamic, SRIF levels. The findings suggest that the defect in pituitary GH secretion observed in the genetically obese Zucker rat is due, at least partially, to insufficient stimulation by hypothalamic GRF, and that SRIF does not play a significant role. (Endocrinology 127: 3087-3095, 1990)
The defect in GH secretion associated with obesity could originate at the pituitary or hypothalamic level. In normal animals, the secretion of GH from the anterior pituitary is governed by an intricate interplay between two hypothalamic neuropeptides, the stimulatory GHreleasing factor (GRF) and the inhibitory hormone somatostatin (SRIF). In the rat these two neurohormones are released in reciprocal cycles into the hypophyseal portal circulation to generate an ultradian rhythm of GH secretion (5, 6). In addition, the neuroendocrine mechanism^) governing the rhythmic secretion of GH is subject to modulatory influences from metabolic factors (79). The possibility that one or both of these hypothalamic hormones might be involved in mediating the GH suppression observed in the obese Zucker rat has been considered. Several groups have measured steady state
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levels of SRIF in the hypothalamus of lean and obese Zucker rats, but the data are not consistent. A decrease (10, 11), increase (12), and no change (13-15) in hypothalamic SRIF levels of obese rats have been reported. While peripheral organs, such as the pancreas, of obese rats have been shown to contain more SRIF (10, 11), it is not known whether circulating SRIF levels are elevated. Fewer studies have used GRF. In in vitro studies using dispersed pituitary cells from obese and lean Zucker rats, GH responsiveness to GRF was attenuated in cells from obese rats compared to that of lean littermates (16, 17). In vivo, GRF-induced, but not basal, GH release was found to be decreased in obese rats (12, 17). However, these latter experiments were carried out under sodium pentobarbital anesthesia, which itself is known to have effects on both hypothalamic GRF and SRIF release (6, 18). Moreover, we have shown that the GH response to GRF in conscious rats can vary significantly, depending upon whether the stimulation is at a time of a spontaneously occurring GH peak or a trough period (5, 19). The aim of the present study, therefore, was to assess the physiological roles of both SRIF and GRF in mediating the abnormal GH secretion observed in obesity. To this end, we studied spontaneous and GRF-induced GH release in conscious, free moving, obese and lean Zucker rats and assessed the effects of immunoneutralization with anti-SRIF serum. In addition, we have measured plasma insulin and glucose levels, pituitary GH concentration, and hypothalamic and peripheral tissue levels of GRF and SRIF. Materials and Methods Animals and experimental procedure Adult male obese and nonobese littermate rats of the Zucker strain were obtained from a breeding colony at Northeastern Ohio Universities College of Medicine (Rootstown, OH). The initial animals in the colony were purchased from the Harriet G. Bird Memorial Laboratories (Stowe, MA) (2). Upon arrival, the rats were individually housed on a 12-h light, 12-h dark cycle (lights on between 0600-1800 h) in a temperature (22 ± 1 C)- and humidity-controlled room. At the time of surgery, the mean body weight was 332 ± 4 g for the nonobese group and 533 ± 14 g for the obese animals. Chronic intracardiac venous cannulae were implanted under sodium pentobarbital (50 mg/kg, ip) anesthesia, using a previously described technique (20). After surgery, the animals were placed directly in isolation test chambers, with Purina rat chow (Ralston-Purina, St. Louis, MO) and tap water available ad libitum. Body weight was monitored daily, and the animals were sampled when body weight returned to preoperative levels (usually within 7 days). On the test day, food was removed 1.5-2 h before the start of sampling and returned at the end. In the first experiment we obtained spontaneous GH, insulin, and glucose secretory profiles in obese and lean littermates.
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Blood samples (0.45 ml) were withdrawn every 15 min for periods of 6 h from 1000-1600 h. All blood samples were immediately centrifuged, and the plasma was separated and stored at —20 C for subsequent assay of GH, insulin, and glucose. To avoid hemodynamic disturbance, the red blood cells were resuspended in normal saline and returned to the animal after removal of the next blood sample. In the second experiment we examined pituitary GH responsiveness to GRF in obese and lean littermates. Free moving, chronically cannulated rats were administered rat (r) GRF-(129)NH2 (1 (ig/0.3 ml) iv at two different time points during the 6-h sampling period. The times of 1100 and 1300 h were chosen, since these times reflect typical peak and trough periods of GH secretion, respectively, as previously documented in our laboratory (5, 19, 20). The GRF peptide (lot CH23-25-31-10-16, kindly provided by Dr. P. Brazeau, Notre Dame Hospital, Montreal, Quebec, Canada) was diluted in normal saline just before use. To document the rapidity of the GH response to GRF, an additional blood sample was obtained 5 min after each injection of the peptide. In the third experiment, designed to assess the role of endogenous SRIF, one group of obese rats was administered 2 ml of a specific SRIF antiserum, iv, after removal of the first blood sample, while another group of obese animals received 2 ml normal sheep serum at the same time point. The SRIF antiserum was the same as that used in our previous passive immunization studies (5, 8, 9). At the termination of the experiments, the animals were killed by rapid decapitation. The brain was immediately removed, and the hypothalamus was dissected according to the following landmarks: anterior, posterior, and lateral borders and depth of dissection were at the optic chiasm, mammillary bodies, hypothalamic sulci, and 1-1.5 mm from the base of the brain, respectively. The hypothalamic fragments were immediately frozen on dry ice, weighed, boiled in 1 ml 2 N acetic acid for 5 min, and homogenized at 600-800 rpm using a PotterElvehjem homogenizer. The homogenate was then centrifuged at 2000 X g for 30 min, and the supernatant was removed and frozen at —20 C until subsequent assay of SRIF and GRF. Portions of the pancreas (body and tail) and stomach (pylorus) were simultaneously rapidly removed, frozen on dry ice, and weighed. Individual tissue fragments were added to 2 ml 2 N acetic acid, boiled for 5 min to inactivate degradative enzymes, and then homogenized using a Polytron (Brinkmann Instruments, Westbury, NY), centrifuged, and stored, as described above, for subsequent assay of SRIF. The pituitary gland was also removed, weighed, homogenized in 2 ml 0.05 M NaHCO3Na2CO3 buffer, pH 9.96, and centrifuged at 2000 X g for 30 min. The supernatant was frozen at —20 C until subsequent assay for GH. Hormone assays Plasma and pituitary GH concentrations were determined in duplicate by double antibody RIA, using materials supplied by the NIDDK (Bethesda, MD). The averaged plasma GH values are reported in terms of the rat GH reference preparation (rGH RP-2). The standard curve was linear between 0.62-320 ng/ml. All samples with values above 320 ng/ml were reassayed
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GRF AND SRIF IN GENETICALLY OBESE ZUCKER RATS at dilutions ranging from 1:2 to 1:10. The intra- and interassay coefficients of variation were 9.1% and 12.1%, respectively, for duplicate samples of pooled plasma containing a mean GH concentration of 75.4 ng/ml. Plasma immunoreactive insulin (IRI) was measured by a dextran-coated charcoal method, using guinea pig antiporcine insulin serum (8). Purified crystalline rat insulin (lot 615-JE6-9, courtesy of Dr. R. Chance, Eli Lilly Co., Indianapolis, IN) served as a reference standard. The sensitivity of the assay was 0.16 ng/ml, and the intra- and interassay coefficients of variation were 8.4% and 14.3%, respectively, for duplicate samples of pooled plasma containing a mean IRI concentration of 1.25 ng/ml. Plasma glucose was measured by an automated glucose oxidase method (Glucose Analyzer 2, Beckman Instruments, Palo Alto, CA). Somatostatin-like immunoreactivity (SLI) was assayed by a previously reported double antibody RIA (7). On the day of the assay, the hypothalamic samples were recentrifuged at 900 X g for 10 min and then diluted at concentrations ranging from 1:100 to 1:200 depending on the origin of the tissue, and neutralized with 5 N NaOH using phenol red as a pH indicator. The SLI values are reported in terms of the somatostatin-14 reference standard (lot 83-57-3G-60-70, kindly provided by Dr. N. Ling, Salk Institute, La Jolla, CA). The intra- and interassay coefficients of variation were 10.8% and 14.1%, respectively, for pooled hypothalamic extracts containing a mean SRIF concentration of 92.1 pg/assay tube. The SRIF binding of plasma from rats treated with SRIF antiserum and normal sheep serum was assessed by determining the ability of aliquots of plasma obtained 6 h after the injections to bind [^I-TyrJSRIF. Plasma samples from each rat were diluted 1:100 in the SRIF RIA assay buffer, and binding to labeled SRIF was determined under conditions routinely used for RIA of SRIF (7). GRF-like immunoreactivity was measured by a newly developed double antibody RIA. The GRF antiserum was generated in sheep using synthetic rat GRF-(1-29)NH2. rGRF-(l-29)NH2 (8.9 Mg) dissolved in 2 ml 0.1 N HC1 and 1.9 ml 0.1 N NaOH was combined with 2.5 mg methylated BSA dissolved in 4.1 ml normal saline. This mixture was emulsified in an equal volume of complete Freund's adjuvant, and two sheep were injected sc at multiple sites. The animals received booster injections of rGRF-(l-29)NH2 emulsified in incomplete Freund's adjuvant at monthly intervals and were bled 2 weeks after each injection. Maximal titers of antisera were achieved by 7 weeks. In an RIA for rGRF-(l-43)OH, this antiserum, at a final dilution of 1:78,000, binds 35% of 125I-labeled rGRF. The antiserum shows no cross-reactivity with SRIF-14, SRIF-28 (Dr. N. Ling, Salk Institute), rGH, LH, FSH, PRL (NIDDK), calcitonin (Sandoz, Basel, Switzerland), insulin (Eli Lilly Co.), glucagon (Sigma, St. Louis, MO), ovine CRF, secretin, vasoactive intestinal peptide, and cholecystokinin-8 (Peninsula, Belmont, CA). rGRF-(l-43)OH (lot 115-194-11G-74-86-12G-70-88, Dr. N. Ling, Salk Institute) was iodinated with chloramine-T. The iodination mixture consisted of 3 ^g rGRF dissolved in 10 /nl 0.05 N acetic acid, 15 n\ 0.5 M phosphate buffer (pH 7.5), 1 mCi Na125I (IMS-30, Amersham, Oakville, Ontario, Canada), and 10 Mg chloramine-T in distilled H2O at a concentration of 1 mg/ml at room temperature. The reaction was stopped after 30
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sec by the addition of 5% BSA in 0.05 M phosphate buffer, pH 7.5 (200 fi\). [i25I]rGRF was purified by gel filtration on a Sephadex G-50 (fine) column (1.5 x 60 cm) in 0.1 N acetic acid with 0.1% BSA. Samples were neutralized with 5 N NaOH, using phenol red as an indicator, and centrifuged at 900 x g for 10 min before assay. Assays were performed in polystyrene tubes at 4 C. The assay buffer consisted of 10 mM NaHPO4-NaH2PO4 (pH 7.5), 0.2 M Na acetate, 25 mM EDTA, 150 mM NaCl, 0.1% Na azide (g/vol), 0.1% human serum albumin (g/vol), and 0.1% Triton X-100 (vol/vol) (Sigma) in distilled water, pH 7.2. The total volume of each tube was 600 nl. Separation of bound from free ligand was accomplished by the second antibody technique. Maximal sensitivity was achieved by delayed addition of labeled rGRF; the initial incubation was 24 h, with a subsequent incubation of 48 h after the addition of labeled rGRF. GRFlike immunoreactive values are reported in terms of the rat GRF-(1-43)OH reference preparation (lot 115-194-11G-7486-12G-70-88; Dr. N. Ling, Salk Institute) that was used for the standard curve of the assay. The sensitivity of the rGRF assay was 10 pg/tube, with half-maximal displacement of 135 pg/tube. The intra- and interassay coefficients of variation were 10.3% and 10.9%, respectively, for duplicate samples from pooled hypothalamic extracts containing a mean rGRF concentration of 142 pg/tube. Statistical analyses Analysis of variance for repeated measures and Student's t test were used for statistical comparisons between experimental groups. Student's paired t test was used to evaluate treatment effects within groups. P < 0.05 was considered significant. Results Spontaneous GH, IRI, and glucose secretory patterns in lean and obese Zucker rats Figure 1 illustrates individual plasma GH and IRI profiles in an obese rat compared to those in a lean littermate. Lean rats showed the typical pulsatile pattern of GH secretion; two episodes of GH secretion were evident during the 6-h sampling period (at 1030-1200 and 1350-1500 h), with most individual peak GH values above 90 ng/ml and trough levels below 1.2 ng/ml. Plasma IRI levels remained low and fluctuated minimally. In contrast, obese rats exhibited a marked suppression in amplitude and duration of spontaneous GH secretory bursts compared to lean littermates; the mean GH peak amplitude was reduced approximately 6-fold (20.8 ± 3.2 vs. 116.7 ± 31.0 ng/ml; P < 0.01). Generally, two brief episodes of GH secretion, separated by undetectable trough levels, were observed in obese rats, and the mean intervals between GH secretory episodes were similar in the two groups (2.9 ± 0.2 vs. 3.1 ± 0.2 h). Plasma IRI levels in these animals were markedly elevated and showed wide fluctuations (Fig. 1). Plasma glucose re-
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caused a 3- to 6-fold increase in plasma GH levels, whereas injection during a trough period (1300 h) had only a minimal effect (Fig. 2A). In obese rats, the magnitude of the GH response to rGRF was markedly attenuated at peak, although not at trough, times compared to that in lean littermates (Fig. 2B). Figure 3 summarizes the GH responsiveness to GRF in lean and obese littermates. In lean animals, the mean plasma GH response at 1100 h (266.9 ± 40.7 ng/ml) was significantly greater than that at 1300 h (55.2 ± 12.6 ng/ml; P < 0.001). Obese rats also showed a significant time-dependent difference in GH responsiveness to rGRF (137.4 ± 26.1 vs. 54.1 ± 12.9 ng/ml; P < 0.05). However, comparisons between the two groups indicated that GRF-induced GH release in obese rats was significantly (P < 0.02) lower than that in lean littermates at peak times, although there was no difference at trough periods. Effects of SRIF antiserum on GH secretory dynamics in obese rats
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Time (hours)
FIG. 1. Individual representative 6-h plasma GH and 3-h plasma IRI profiles in an obese Zucker rat compared to those in a lean littermate. Obese animals exhibited a marked suppression of both amplitude and duration of spontaneous GH surges and significant elevation of plasma IRI levels.
mained stable in both groups. The mean 6-h plasma GH level of obese rats was significantly (P < 0.001) lower than that of lean littermatss (Table 1). Whereas obese rats showed a marked hyperinsulinemia over a 3-h sampling period, there was no significant difference in basal plasma glucose levels between the two groups (Table 1).
The GH secretory profiles of obese rats administered normal sheep serum continued to show a marked suppression of GH pulse amplitude (Fig. 4A). Administration of SRIF antiserum to obese rats caused an initial surge of GH release, but failed to restore the amplitude of GH pulses to that observed in lean animals (Fig. 4B). The mean 6-h plasma GH level of obese rats given SRIF antiserum was not significantly different from that of obese normal sheep serum-treated controls (Table 2). Immunoneutralization of endogenous SRIF also did not significantly alter the hyperinsulinemia observed in obese rats (Table 2). SRIF binding of rat plasma after SRIF antiserum administration The mean SRIF binding level of a 1:100 dilution of plasma of obese rats injected with SRIF antiserum was 47.4 ± 0.5%, and it was 4.9 ± 0.3% after normal sheep serum injection.
GH responsiveness to GRF in lean and obese Zucker rats
Effect of obesity on pituitary wet weight, GH content, and GH concentration
In lean animals, the iv administration of 1 /xg rGRF(1-29)NH2 during a time of peak GH secretion (1100 h)
Obese rats showed a significant (P < 0.05) atrophy of the pituitary gland compared to their lean littermates
TABLE 1. Mean plasma GH, insulin, and glucose levels in lean and obese Zucker rats
Experimental group
n
Final BW (g)
Mean 6-h plasma GH (ng/ml)
Mean 3-h plasma IRI (ng/ml)
Mean basal plasma glucose (mg/dl)
Lean Obese
8 12
385.8 ± 11.4 603.6 ± 16.3°
21.5 ± 3.8 3.9 ± 0.4°
0.47 ± 0.04 13.11 ± 2.21"
125.1 ± 6.8 123.9 ± 0.8
Values are the mean ± SE. P < 0.001 compared to lean littermates.
0
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GRF AND SRIF IN GENETICALLY OBESE ZUCKER RATS 400
Lean
3091
350
Peak Trough
300
200
E 100
300 „ B
Obese
200
Lean (11)
100
1000
1100
1200
1300
1400
1500
1600
Time (hours)
FIG. 2. Plasma GH response to lMgrGRF-(l-29)NH2,iv, administered during peak (1100 h) and trough (1300 h) periods of the GH rhythm. In lean rats (A), administration of rGRF during a time of peak secretion resulted in a 3- to 6-fold increase in plasma GH levels, whereas injection during trough periods had only a minimal effect. In obese rats (B), the magnitude of the GH response to rGRF was markedly attentuated at peak times. Arrows indicate the times of injections.
(Fig. 5). Both pituitary GH content and GH concentration were markedly reduced in obese rats compared to those in lean controls (Fig. 5). Hypothalamic GRF immunoreactivity in lean and obese Zucker rats Immunoreactive GRF levels in the two groups are shown in Table 3. Obese rats exhibited a significant decrease in hypothalamic GRF content compared to lean littermates. Hypothalamic and peripheral tissue immunoreactive SRIF levels in lean and obese Zucker rats Immunoreactive SRIF levels in the two groups are shown in Table 4. While the hypothalamic SLI concen-
Obese (10)
FIG. 3. Summary of GH responsiveness to rGRF in lean and obese rats. Both groups exhibited a time-dependent difference in their ability to respond to rGRF, with GRF-induced GH release being significantly greater during peak compared to trough times. However, in obese rats, there was a significant reduction in GH responsiveness to GRF at times of peak GH secretion, although not at trough periods. Each bar
represents the mean ± SE, and the number of animals in each group is shown in parentheses, a, P < 0.05 or less compared to GH release during peak periods in lean and obese rats, b, P < 0.02 compared to rGRF-induced GH release in lean rats at peak times.
tration was not altered by obesity, both pancreas and stomach SLI concentrations were significantly augmented in obese rats compared to lean littermates.
Discussion The finding of a suppression of GH pulse amplitude and lower mean plasma GH levels in genetically obese Zucker rats compared to their lean littermates is consistent with previous observations using both single point determinations (3) and pulsatile release patterns over a 6-h sampling period (4). The lack of a difference in basal GH levels, reported in other studies (12, 17), is probably due to the confounding influence of anesthesia used in those experiments, thus emphasizing the importance of studying GH physiology in the conscious state. The lean male Zucker rats of the present study show an ultradian GH secretory pattern similar to that of Sprague-Dawley rats, as seen in numerous other studies since the first
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GRF AND SRIF IN GENETICALLY OBESE ZUCKER RATS
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Endo • 1990 Vol 127 • No 6
Obese Zucker Rat + Normal Sheep Serum 90 r-
60 C
FIG. 4. Effects of passive immunization with SRIF antiserum on plasma GH profiles in obese Zucker rats (B) compared to those in obese rats given normal sheep serum (A). Administration of SRIF antiserum caused an initial surge of GH release, but failed to restore the amplitude of GH pulses to that of pulses in lean animals. Arrows indicate the times of injection.
30
0 C
H H I
•m*
IMIMI
o o Obese Zucker Rat + SRIF Antiserum
X
o
B
90
o CO
E
60
CO
30
1000
1200
1400
1600
1000
1400
1200
1600
Time (hours) TABLE 2. Effects of SRIF antiserum on mean plasma GH and IRI levels in obese Zucker rats Experimental group Obese + normal sheep serum Obese + SRIF antiserum
4 4
TABLE 3. Hypothalamic GRF immunoreactive levels in lean and obese Zucker rats
Mean 6-h plasma GH (ng/ml)
Mean 3-h plasma IRI (ng/ml)
Experimental group
MBH wet wt (mg)
GRF-like immunoreactivity (pg/fragment)
7.02 ± 2.30 9.16 ± 1.02
13.57 ± 3.0 17.53 ± 8.79
Lean Obese
16.0 ± 1.4 17.6 ± 1.1
678.1 ± 50.2 503.2 ± 60.1°
Values are the mean ± SE. MBH, Mediobasal hypothalamus. P < 0.05 compared to lean littermates.
Values are the mean ± SE.
0
TABLE 4. Hypothalamic and peripheral tissue SLI in lean and obese Zucker rats Pituitary Wet Weight (mg)
Pituitary GH Content (ug/gland)
Pituitary GH Concentration (ug/mg wet wt) I Lean (6)
Experimental group
Hypothalamic SLI (ng/mg wet wt)
Lean Obese
10.2 ± 0.9 8.7 ± 0.9
I Obese (8)
Pancreatic Stomach SLI SLI (ng/mg wet wt) (ng/mg wet wt) 0.58 ± 0.06 0.94 ± 0.13°
1.05 ± 0.06 1.38 ± 0.09"
Values are the mean ± SE. " P < 0.01 compared to lean littermates.
FIG. 5. Effects of obesity on pituitary weight, GH content, and GH concentration. Obese rats exhibited a significant reduction in both pituitary wet weight and pituitary GH content and concentration. Each bar represents the mean ± SE; the number of animals in each group is in parentheses. &,P< 0.05 or less compared to lean littermates.
description in 1976 (20), although the amplitude of the GH peaks in the Zucker strain appears to be slightly attenuated. The observation of hyperinsulinemia with normal blood glucose levels is a consistent feature of the genetically obese Zucker rat (21), although other studies of insulin levels in "fatty" rats have used only single point
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GRF AND SRIF IN GENETICALLY OBESE ZUCKER RATS determinations (22). The present results demonstrate that plasma insulin levels of obese rats are high and fluctuate widely compared to those in their lean littermates. While the origin of the fluctuations in insulin levels is not known, there did not appear to be any association with the GH secretory patterns. We previously reported in normal Sprague-Dawley rats that, in the absence of food during the testing period, plasma GH levels do not correlate with either glucose or insulin levels (23). Even though food was not available in the testing situation of the present study, the obese rats would be more likely to anticipate a meal during that time period because they are known to feed more during the light hours than lean animals (24), and this might account in part for the wide variations observed. The depressed plasma GH levels are reflective of the situation in the pituitary gland, where both the content and concentration of GH were significantly lower in obese us. lean rats. It is not known if the entire gland is affected or if the weight difference we observed is limited to a specific subdivision. No direct measurement of the number of somatotrophs in rats of the Zucker strain has been made. The finding that the amount of DNA per pituitary gland is the same in obese and lean Zucker rats (25) can be taken as indirect evidence that the total cell numbers are comparable, although relative distribution of cell types may vary. In vitro studies have shown that basal levels of GH release from pituitary cells in culture taken from lean and obese rats do not differ, but that GH responsiveness to GRF stimulation is attenuated in pituitary cells taken from obese animals (12, 16, 17). Our in vivo data in free moving, conscious rats also show attenuated sensitivity to GRF in obese rats, consistent with that previously reported in anesthetized animals (12, 17). Such an impairment in GH responsiveness to GRF has also been reported in obese humans (26). Both the lean and obese Zucker rats of the present study exhibited a time-dependent difference in GH responsiveness to GRF, with GRF-induced GH release being greater when GRF was administered during a peak compared to a trough period. These results are similar to those we previously reported in male Sprague-Dawley rats (5, 19). Since the variability in GH responsiveness to GRF has been shown to be due to interference by the cyclic increased release of endogenous SRIF (5), our findings suggest that SRIF secretion is also episodic in both lean and obese rats of the Zucker strain. However, a differential responsiveness between obese and lean rats was seen when GRF was administered at peak times of GH secretion, with obese rats exhibiting significantly lower GRF-induced GH release compared to their lean littermates. Since the high GH release observed in response to GRF administered at peak times is probably
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due to the additive effect of exogenous GRF to that of endogenous hypothalamic GRF, our data suggest that a reduction in endogenous hypothalamic GRF secretion may be a determining factor in the diminished GH secretion of the obese rats. Indeed, direct measurement of hypothalamic GRF levels in lean and obese rats of the present study showed a significant diminution of GRF immunoreactivity in the hypothalami of obese rats. It is also possible that the impaired responses to GRF are due to alterations in the GRF somatotroph receptor secondary to insufficient stimulation by hypothalamic GRF. Lack of exposure of pituitary somatotrophs to normal GRF signaling could conceivably alter GRF receptor sensitivity and perhaps account for the reduction in GH responsiveness to GRF. Such a mechanism still remains to be elucidated, although preliminary evidence suggests that this may not be the case; no differences in affinities or capacities of GRF-binding sites were found between lean and obese Zucker rat pituitaries (27). The possibility that the decreased pulsatile GH release in obese animals is a result of excessive S R I F (inhibitory) tone was addressed by two types of experiments: direct measurement of hypothalamic and peripheral tissue immunoreactive SRIF levels, and immunoneutralization of endogenous circulating SRIF by antibody injection. Neither experiment gave clear support to the concept of increased SRIF secretion in obese rats. While earlier studies had reported that genetically obese Zucker rats have decreased levels of hypothalamic SRIF (10, 11), more recent reports (13-15) are in agreement with our present data, which have failed to find a significant difference in hypothalamic SRIF concentration between obese and lean animals. However, we did observe elevated SRIF levels in the pancreas and stomach of obese Zucker rats, consistent with previous reports (10, 11, 14). Whether this increase in peripheral tissue SRIF represents changes in SRIF synthesis, storage, or release cannot be distinguished on the basis of the present studies. If the depressed GH levels in obese rats were produced by elevated circulating SRIF levels, treatment of obese rats by passive immunization with SRIF antibody would be expected to reverse the GH suppression, as shown previously in other situations of GH suppression in the rat, including starvation (28) and diabetes (8). However, administration of SRIF antiserum to obese rats failed to restore the amplitude of GH pulses to normal values. Mean 6-h plasma GH levels in obese rats given SRIF antiserum were no different from those in normal sheep serum-treated obese animals. As SRIF binding of a 1:100 dilution of plasma samples of animals treated with SRIF antiserum was approximately 50% throughout the experimental procedure, ineffective immunoneutralization is not likely to be the cause of the failure of SRIF
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GRF AND SRIF IN GENETICALLY OBESE ZUCKER RATS
antiserum to reverse the GH inhibition. These findings, therefore, suggest that increased SRIF release is not the mechanism mediating the attenuated GH secretion in the genetically obese Zucker rat. Additional support for this view comes from recent experiments in our laboratory in which we directly measured plasma levels of SLI in obese and lean Zucker rats and found no difference (Patel, Y. C., and G. S. Tannenbaum, unpublished observations). However, the possibility that an alteration of the SRIF somatotroph receptor could be involved still remains to be explored. Abnormal GH release patterns have been demonstrated in other experimental models of disordered metabolism in the rat; the genetically obese Zucker rat shares some characteristics with these types of rats, but does not duplicate any of them. For example, food deprivation (7) and both streptozotocin-induced (8) and spontaneous (29) diabetes result in a suppression of GH pulse amplitude. In contrast to the obese rats, starved and diabetic rats have elevated plasma SRIF levels (19, 30, 31) and exhibit enhanced responsiveness to GRF (19, 32, 33). Rats made obese by lesioning the ventromedial hypothalamic nuclei also have markedly depressed GH pulses (34), an effect probably due to destruction of GRFcontaining neurons in the mediobasal hypothalamus. Although the obese Zucker rats do exhibit lower hypothalamic GRF levels, this peptide is present and measurable. Whether the attenuated GH release in genetically obese Zucker rats follows or precedes the lower hypothalamic GRF levels cannot be distinguished on the basis of the present experiments, since our studies have been performed in adult rats only. GH itself appears to exert feedback regulation on hypothalamic GRF, as suggested by the finding that hypophysectomy results in depressed hypothalamic GRF content and release (35). Developmental studies currently underway should elucidate this relationship. Recent investigations have focused on the underlying molecular basis of the decreased plasma GH levels in obese Zucker rats. Using steady state levels of GH mRNA as a measure of gene expression, GH gene expression has been shown to be significantly lower in the obese rats (25) as early as 5 weeks of age (36). Results obtained from an in situ hybridization analysis indicate that each somatotroph contains fewer GH transcripts in the obese animals, rather than there being fewer somatotrophs present (37). Since GRF has been shown to stimulate transcription of the GH gene (38) and to increase GH mRNA levels in rat pituitary (39), it is possible that the decrease in hypothalamic GRF observed here in obese rats results in lowered GH gene expression and, consequently, could account for the lower steady state levels of GH mRNA reported. A recent preliminary study of hypothalamic gene expression in the genetically obese
Endo • 1990 Voll27«No6
Zucker rat indicates that hypothalamic GRF mRNA is reduced, but hypothalamic SRIF mRNA is normal, in obese rats compared to that in lean controls (40). Thus, the gene expression and the peptide content data of the present report are in agreement. Several other factors that have been shown to regulate GH mRNA levels are also known to be abnormal in obese Zucker rats; these include insulin (22, 41), thyroid hormones (42-44), glucocorticoids (43-45), and insulin-like growth factor-1 (12, 46, 47). SRIF, however, appears to act at the level of release, rather than at the level of gene expression (47). Specific roles for any of these signals in modulating the hyposomatotropism of obesity have not yet been defined, but could be important. In summary, the results of the present study demonstrate that the genetically obese Zucker rat exhibits marked impairment in both spontaneous and GRF-induced GH release, which cannot be reversed by antiSRIF treatment. This hyposomatotropism is associated with a reduction in pituitary GH concentration, depressed hypothalamic GRF content, and elevated gastric and pancreatic, but not hypothalamic, SRIF levels. Taken together, these findings suggest that the defect in pituitary GH secretion in genetically obese Zucker rats is due, at least partially, to insufficient stimulation by hypothalamic GRF, and that SRIF does not play a significant role.
Acknowledgments We are grateful to Julie Temko for expert secretarial assistance, and to the NIDDK for the generous supply of rGH RIA materials.
References 1. Sims EAH, Danforth Jr E, Horton ES, Bray GA, Glennon JA, Salans LB 1973 Endocrine and metabolic effects of experimental obesity in man. Recent Prog Horm Res 29:457-496 2. Zucker LM, Zucker TF 1961 Fatty, a new mutation in the rat. J Hered 52:275-278 3. Martin RJ, Gahagan J 1977 Serum hormone levels and tissue metabolism in pair-fed lean and obese Zucker rats. Horm Metab Res 9:181-186 4. Finkelstein JA, Jervois P, Menadue M, Willoughby JO 1986 Growth hormone and prolactin secretion in genetically obese Zucker rats. Endocrinology 118:1233-1236 5. Tannenbaum GS, Ling N 1984 The interrelationship of growth hormone (GH)-releasing factor and somatostatin in generation of the ultradian rhythm of GH secretion. Endocrinology 115:19521957 6. Plotsky PM, Vale W 1985 Patterns of growth hormone-releasing factor and somatostatin secretion into hypophysial portal circulation of the rat. Science 230:461-463 7. Tannenbaum GS, Rorstad O, Brazeau P 1979 Effects of prolonged food deprivation on the ultradian growth hormone rhythm and immunoreactive somatostatin tissue levels in the rat. Endocrinology 104:1733-1738 8. Tannenbaum GS 1981 Growth hormone secretory dynamics in streptozotocin diabetes: evidence of a role for endogenous circulating somatostatin. Endocrinology 108:76-82 9. Painson JC, Tannenbaum GS 1985 Effects of intracellular glucopenia on pulsatile growth hormone secretion: mediation in part by
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GRF AND SRIF IN GENETICALLY OBESE ZUCKER RATS somatostatin. Endocrinology 117:1132-1138 10. Sheppard MC, Shapiro B, Hudson A, Pimstone BL 1980 Tissue and serum somatostatin-like immunoreactivity in lean and obese Zucker rats. Horm Metab Res 12:236-239 11. Voyles NR, Awoke S, Wade A, Bhathena SJ, Smith SS, Recant L 1982 Starvation increases gastrointestinal somatostatin in normal and obese Zucker rats: a possible regulatory mechanism. Horm Metab Res 14:392-395 12. Renier G, Gaudreau P, Deslauriers N, Petitclerc D, Brazeau P 1990 Dynamic of the GRF-induced GH response in genetically obese Zucker rats: influence of central and peripheral factors. Regul Peptides 28:95-106 13. Fletcher JM, Haggarty P, Wahle KWJ, Reeds PJ 1986 Hormonal studies of young lean and obese Zucker rats. Horm Metab Res 18:290-295 14. Voyles NR, Bhathena SJ, Kennedy B, Wilkins SD, Michaelis IV OE, Zalenski CM, Timmers KI, Recant L 1987 Tissue somatostatin levels in three models of genetic obesity in rats (42515). Proc Soc Exp Biol Med 185:49-54 15. DonCarlos LL, Ho RH, Finkelstein JA 1988 Immunocytochemical analysis of somatostatin in the hypothalamus of obese and nonobese Zucker rats. Brain Res 458:372-376 16. Heiman ML, Porter JR, Nekola MV, Murphy WA, Hartman AD, Lance VA, Coy DH 1985 Adenohypophyseal response to hypophysiotropic hormones in male obese Zucker rats. Am J Physiol 249:E380-E384 17. Renier G, Gaudreau P, Deslauriers N, Brazeau P 1989 In vitro and in vivo growth hormone responsiveness to growth hormone-releasing factor in male and female Zucker rats. Neuroendocrinology 50:454-459 18. Chihara K, Arimura A, Schally AV 1979 Immunoreactive somatostatin in rat hypophyseal portal blood: effects of anesthetics. Endocrinology 104:1434-1441 19. Tannenbaum GS, Painson J-C, Lengyel AMJ, Brazeau P 1989 Paradoxical enhancement of pituitary growth hormone (GH) responsiveness to GH-releasing factor in the face of high somatostatin tone. Endocrinology 124:1380-1388 20. Tannenbaum GS, Martin JB 1976 Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat. Endocrinology 98:562-570 21. Zucker LM, Antoniades HN 1972 Insulin and obesity in the Zucker genetically obese rat "fatty." Endocrinology 90:1320-1330 22. Martin RJ, Wangsness PJ, Gahagan JH 1978 Diurnal changes in serum metabolites and hormones in lean and obese Zucker rats. Horm Metab Res 10:187-192 23. Tannenbaum GS, Martin JB, Colle E 1976 Ultradian growth hormone rhythm in the rat: effects of feeding, hyperglycemia and insulin-induced hypoglycemia. Endocrinology 99:720-727 24. Becker EE, Grinker JA 1977 Meal patterns in the genetically obese Zucker rat. Physiol Behav 18:685-692 25. Ahmad I, Steggles AW, Carrillo AJ, Finkelstein JA 1989 Obesityand sex-related alterations in growth hormone messenger RNA levels. Mol Cell Endocrinol 65:103-109 26. Williams T, Berelowitz M, Joffe SN, Thorner MO, Rivier J, Vale W, Frohman LA 1984 Impaired growth hormone responses to growth hormone-releasing factor in obesity. A pituitary defect reversed with weight reduction. N Engl J Med 311:1403-1407 27. Abribat T, Finkelstein JA, Gaudreau P 1990 High and low affinity binding sites for [12SI-Tyr10]human growth hormone-releasing factor in Sprague-Dawley and Zucker rat pituitaries. Neuroendocrinology [Suppl 1] 52:119 (Abstract) 28. Tannenbaum GS, Epelbaum J, Colle E, Brazeau P, Martin JB 1978 Antiserum to somatostatin reverses starvation-induced inhibition of growth hormone but not insulin secretion. Endocrinology 102:1909-1914
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29. Tannenbaum GS, Colle E, Gurd W, Wanamaker L 1981 Dynamic time course studies of the spontaneously diabetic "BB" Wistar rat. I. Longitudinal profiles of plasma growth hormone, insulin and glucose. Endocrinology 109:1872-1879 30. Patel YC, Wheatley T, Zingg HH 1980 Increased blood somatostatin concentration in streptozotocin diabetic rats. Life Sci 27:1563-1570 31. Patel YC, Wheatley T, Malaisse-Lagae F, Orci L 1980 Elevated portal and peripheral blood concentration of immunoreactive somatostatin in spontaneously diabetic (BBL) Wistar rats. Diabetes 29:757-761 32. Locatelli V, Rovati S, Miyoshi H, Miiller EE 1984 Growth hormone hyperresponsiveness to human pancreatic growth hormone releasing hormone in streptozotocin-diabetic rats. Horm Metab Res 16:507 33. Serri O, Brazeau P 1987 Growth hormone responsiveness in vivo and in vitro to growth hormone releasing factor in the spontaneously diabetic BB Wistar rat. Neuroendocrinology 46:162-166 34. Eikelboom R, Tannenbaum GS 1983 Effects of obesity-inducing ventromedial hypothalamic lesions on pulsatile growth hormone and insulin secretion: evidence for the existence of a growth hormone-releasing factor. Endocrinology 112:212-219 35. Katakami H, Downs TR, Frohman LA 1987 Effect of hypophysectomy on hypothalamic growth hormone-releasing factor content and release in the rat. Endocrinology 120:1079-1082 36. Ahmad I, Steggles AW, Carrillo AJ, Finkelstein JA 1990 Developmental changes in levels of GH mRNA in Zucker rats. J Cell Biochem 43:59-66 37. Ahmad I, Steggles AW, Finkelstein JA 1990 In situ hybridization study of GH gene expression in lean and genetically obese Zucker rats. Anat Rec 226:5A (Abstract) 38. Barinaga M, Yamomoto G, Rivier C, Vale W, Evans R, Rosenfeld MG 1983 Transcriptional regulation of growth hormone gene expression by growth hormone-releasing factor. Nature 306:84-85 39. Gick GG, Zeytin FN, Brazeau P, Ling NC, Esch FS, Bancroft C 1984 Growth hormone-releasing factor regulates growth hormone mRNA in primary cultures of rat pituitary cells. Proc Natl Acad Sci USA 81:1553-1555 40. Finkelstein JA, Ahmad I, Steggles AW, Chomczynski P, Downs TR, Frohman LA 1990 Levels of growth hormone-releasing hormone mRNA and somatostatin mRNA in the hypothalamus of the genetically obese Zucker rat. J Cell Biochem 14F:40 (Abstract) 41. Yamashita S, Melmed S 1986 Effect of insulin on rat pituitary cells; inhibition of GH secretion and GH mRNA levels. Diabetes 35:440-447 42. Goldberg JR, Ehrmann B, Katzeff HL 1988 Altered triiodothyronine metabolism in Zucker fatty rats. Endocrinology 122:689-693 43. Evans RM, Birnberg NC, Rosenberg MG 1982 Glucocorticoid and thyroid hormones transcriptionally regulate growth hormone gene expression. Proc Natl Acad Sci USA 79:7659-7663 44. Martinoli MG, Pelletier G 1989 Thyroid and glucocorticoid hormone regulation of rat pituitary growth hormone messenger ribonucleic acid as revealed by in situ hybridization. Endocrinology 125:1246-1252 45. Guillaume-Gentil C, Rohner-Jeanrenaud F, Abramo F, Bestetti GE, Rossi GL, Jeanrenaud B 1990 Abnormal regulation of the hypothalamo-pituitary-adrenal axis in the genetically obese fa/fa rat. Endocrinology 126:1873-1879 46. Grzywa M 1984 Serum somatomedin activity and growth hormone secretion by cultured pituitary cells from genetically obese Zucker rats. Endokrynol Pol 35:105-111 47. Namba H, Morita S, Melmed S 1989 Insulin-like growth factor-I action on growth hormone secretion and messenger ribonucleic acid levels: interaction with somatostatin. Endocrinology 124:1794-1799
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