0013-7227/90/1275-2111$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 5 Printed in U.S.A.
Influence of Food Deprivation in the Rat on Hypothalamic Expression of Growth Hormone-Releasing Factor and Somatostatin* JOHN F. BRUNO, DAVID OLCHOVSKY, JEFFREY D. WHITEf, JOHN W. LEIDY, JINFEN SONG, AND MICHAEL BERELOWITZ Division of Endocrinology and Metabolism, Departments of Medicine (J.F.B., D.O., J.D.W., J.S., M.B.), Neurobiology/Behaviour (J.D.W.), and Physiology/Biophysics (M.B.), State University of New York, Stony Brook, New York 11794; and the Veterans Administration Medical Center/Department of Medicine, Marshall University School of Medicine (J.W.L.), Huntington, West Virginia 25701
hypothalamic SRIF content. The time course of hypothalamic prepro-GRF mRNA reduction was determined in groups of rats food-deprived for 24, 48, or 72 h and revealed a significant (30%) reduction of prepro-GRF mRNA (P < 0.05 us. fed) by 24 h, with maximal reduction (80%) by 48 h. Refeeding groups of animals for up to 72 h after they had been food-deprived for 72 h resulted in restoration of prepro-GRF mRNA levels to 50% of control levels by 24 h (P < 0.05 vs. fed) and a return to control values by 48 h. These data suggest that decreased GRF gene expression and possibly GRF release play a major role in the loss of pulsatile GH secretion seen in this model of nutrient deprivation. (Endocrinology 127: 2111-2116, 1990)
ABSTRACT. Food deprivation in the rat is associated with a reduction in serum GH levels characterized by suppression of high amplitude GH bursts and a decrease in the duration of secretory episodes. The mechanism(s) mediating this response is unknown. The present studies were designed to evaluate the role of hypothalamic factors potentially responsible for abnormal GH dynamics in food-deprived rats by measuring hypothalamic prepro-GH-releasing factor (GRF) and preprosomatostatin (SRIF) mRNA and peptide levels in adult male SpragueDawley rats after 72 h of food deprivation or free access to food. Hypothalamic prepro-GRF mRNA was reduced 80% in fooddeprived rats compared to that in fed controls (P < 0.001), while GRF content was unchanged. Levels of prepro-SRIF mRNA in food-deprived rats were similar to those in controls, as was
E
PISODIC pituitary GH secretion is regulated by opposing influences of the hypothalamic peptides GRF and somatostatin (SRIF). In the rat, major GH secretory episodes are characterized by high amplitude bursts that may reach 300-1000 ng/ml occurring at 3- to 4-h intervals throughout a 24-h period (1, 2). Food deprivation in the rat results in an inhibition of GH secretory episodes, with a significant reduction in high amplitude bursts that occurs as early as 24 h and is followed by a progressive decline in both the amplitude and duration of secretory episodes by 48 and 72 h (3). The mechanism(s) mediating this response is unknown. Decreased GH secretion in food-deprived rats could result from increased inhibitory SRIF influence on the somatotrope Received May 23, 1990. Address all correspondence and requests for reprints to: John F. Bruno, Ph.D., Division of Endocrinology and Metabolism, Department of Medicine, HSC T15, Room 060, State University of New York, Stony Brook, New York 11794. * This work was supported by NIH Research Grant AM-36831 (to M.B.) and Alcohol Drug Abuse and Mental Health Administration Grants MH-42074 and MH-00801 (to J.D.W.). t Recipient of a fellowship from the Aaron Diamond Foundation.
or from diminished GRF stimulatory tone. Increased SRIF has been circumstantially implicated based on in vivo immunoneutralization studies in which GH secretion was restored after the administration of specific SRIF antiserum to food-deprived rats (4,5). Such studies do not, however, provide evidence for a direct causeeffect relationship between SRIF and altered GH secretion, since SRIF alterations could be a secondary occurence and do not identify the tissue source of putative increases in SRIF. The present studies were designed to evaluate the role of hypothalamic factors potentially responsible for abnormal GH dynamics in food-deprived rats by comparing prepro-GRF and prepro-SRIF mRNA and peptide levels in hypothalamic extracts from adult male Sprague-Dawley rats allowed free access to food or food-deprived for periods up to 72 h.
Materials and Methods Animals and experimental procedures Male Sprague-Dawley rats (Taconic Farms, Germantown, NY), weighing 175-225 g, were housed in groups of two
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HYPOTHALAMIC GRF AND FOOD DEPRIVATION
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under constant temperature (22 C) and a 12-h light, 12-h dark cycle (lights on at 0700 h) and were provided free access to rat chow (Ralston-Purina, St. Louis, MO) and tap water. Animals were adapted for 5-7 days to the facility before all experimental procedures. Three sets of experiments comprised the present study.
described (8). Cartridge preconditioning and washing between extracts were slightly modified by the addition of chloroform and methanol washes to remove hypothalamic lipids that interfere with GRF recovery from the cartridge and GRF RIA.
72-h food deprivation. The effects of food deprivation on hypothalamic prepro-GRF and prepro-SRIF mRNA and peptide levels were determined in three separate self-contained experiments. Rats were deprived of food for 72 h or allowed free access to food; both groups had access to drinking water. Animals were weighed immediately before food deprivation, then before death 72 h later. Food deprivation resulted in a significant reduction in hypothalamic prepro-GRF mRNA, with no change in prepro-SRIF mRNA levels (see Results); therefore, further experiments were performed to evaluate the effects of varying duration of food deprivation and refeeding on changes in hypothalamic prepro-GRF mRNA.
GRF and SRIF were measured, as previously described, by double antibody RIA, using [125I]rat GRF (8) or [^IJTyr'-SRIF (9) prepared by the chloramine-T method and purified by reverse phase HPLC.
Time course. To determine the time course of changes in hypothalamic prepro-GRF mRNA occurring as a result of nutrient deprivation, animals were either allowed free access to food (fed) or were killed after 24, 48, or 72 h of food deprivation (n = 6/group). All animals had free access to water, and body weights were recorded before food deprivation and at death. Refeeding after 72 h of food deprivation. The effect of refeeding on decreased hypothalamic prepro-GRF mRNA content after 72 h food deprivation was determined in groups of rats allowed free access to food or food-deprived for 72 h then permitted to refeed ad libitum for 24, 48, or 72 h (n = 6/group). All groups had free access to water and were weighed before and after food deprivation and again after refeeding. Tissue handling Rats were killed by decapitation, and hypothalami were rapidly dissected using landmarks to yield blocks weighing 2025 mg, as previously described (6), then immediately frozen on dry ice for later RNA extraction or individually extracted for SRIF or GRF RIA. For SRIF assay, hypothalami were placed in 2 N acetic acid, boiled for 3 min, homogenized by Polytron (Brinkmann Instruments, Westbury, NY) at setting 2 for 10 sec, and then centrifuged at 2000 X g for 30 min, and the supernatant was lyophilized. For hypothalamic nuclear SRIF determination, brains were processed, and nuclear areas were identified and removed by the method of Palkovitz (7). Paired nuclei from individual rats were expelled into a glass-glass microhomogenizer containing 100 or 200 jtl acetic acid, then thoroughly homogenized. Ten percent of the final homogenate volume was sampled for protein determination using the fluorescamine method. The remainder of the homogenate was boiled for 10 min, then centrifuged at 2000 X g for 30 min. The supernatant was frozen, lyophilized, then stored at -70 C for RIA. For GRF assay, hypothalami were homogenized in an acidic medium and extracted with octadecasilyl-silica cartridges (Bond Elut Cis, Analytichem, Harbor City, CA), as previously
Hormone assay
RNA extraction Total hypothalamic RNA was isolated from dissected hypothalamic tissue blocks using methods developed previously (10). Briefly, tissue was homogenized using a Polytron tissue homogenizer in a buffer containing 4 M guanidine isothiocyanate, 50 mM Tris (pH 7.5), 10 mM EDTA (Fisher Scientific, Springfield, NJ), 1% iV-lauroylsarcosine (Sigma, St. Louis, MO), and 1% 2-mercaptoethanol (Boehringer Mannheim Biochemicals, Indianapolis, IN). The samples were then extracted twice with phenol-chloroform, ethanol precipitated, subjected to DNase digestion, reextracted with phenol-chloroform, then chloroform extracted and ethanol precipitated. An average of 25 /xg RNA were recovered per hypothalamus. RNA concentrations were estimated based on absorbance at 260 nm, and identical concentrations of RNA from each sample were used for nuclease protection. Preparation of probes GRF cDNA constructs in pGEM 3 and SRIF cDNA constructs in pGEM 4 vectors were used to generate 32P-labeled antisense RNA for nuclease protection assays. The GRF riboprobe was transcribed in the presence of SP6 polymerase from a 215-basepair (bp) EcoRl-Hindlll fragment of the rat cDNA provided by Kelly E. Mayo (Northwestern University) after linearization with EcoRl (11). The SRIF riboprobe was transcribed in the presence of SP6 polymerase from a 461-bp Xbal/ Sau3A fragment of the rat cDNA provided by Jack E. Dixon (Purdue University) after linearization with Hindlll (12). Levels of /3-actin mRNA were measured using a 650-bp EcoRlHircdlll fragment of a rat /3-actin cDNA, provided by B. Patterson (NIH). This cDNA fragment was cloned into EcoRland ifindlll-digested pGEM-2 and, after linearization with EcoRl, was used to generate an antisense RNA probe, as previously described (13). Nuclease protection assay Five micrograms of total RNA from each hypothalamus were coprecipitated with 80,000 cpm 32P-labeled GRF riboprobe, or 2 ng total RNA were coprecipitated with either 50,000 cpm 32Plabeled SRIF riboprobe or 25,000 cpm 32P-labeled /3-actin riboprobe, prepared as described above. Samples were resuspended in hybridization buffer containing 40 mM 1,4-piperazine diethane sulfonic acid (pH 6.8), 72% formamide, 1 mM EDTA, and 0.4 M NaCl, boiled for 3 min, and hybridized at 45 C for 14-16 h. After hybridization, samples were subjected to com-
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HYPOTHALAMIC GRF AND FOOD DEPRIVATION bined RNase-A and -Tx digestion of nonhybridized probe at 30 C for 1 h. Stable hybrids were ethanol precipitated, denatured, and separated on 8% polyacrylamide-8 M urea gels. The dried gel was apposed to Kodak X-Omat x-ray film (Eastman Kodak, Rochester, NY) to generate an autoradiograph; exposure times ranged from 24-72 h. Autoradiographic densities were quantitated using an LKB laser densitometer (Rockville, MD) in the two-dimensional scan mode to obtain a densitometric value for the entire autoradiographic band.
FED
2113 FOOD DEPRIVED
|rRNA|PROBE
GRF
Analysis of data Results are expressed as the mean ± SEM. Comparisons of data between experimental groups were performed using oneway analysis of variance, followed by Fisher's least significant difference test.
Results
SRIF
Actin
72-h food deprivation Animals exposed to 72 h of food deprivation lost approximately 22% of their initial body weight, while fed controls gained almost 9% (Table 1). SRIF contents were similar in extracts of medial basal hypothalamus from 72-h food-deprived and control rats (80.5 ± 12.0 vs. 80.7 ± 17.1 ng/hypothalamus; n = 5). In addition, SRIF content in microdissected punches of periventricular nucleus, preoptic area, or median eminence was unchanged in food-deprived rats compared to that in fed controls (data not shown). GRF content in acidic extracts of hypothalami from food-deprived rats did not differ from that in control rats (2.87 ± 0.55 vs. 3.37 ± 0.93 ng/ hypothalamus; n = 5). Total hypothalamic RNA was isolated from 72-h fooddeprived and control rats, and prepro-GRF and preproSRIF mRNA contents were estimated using a solution hybridization/RNase protection assay. As shown in Fig. TABLE 1. Effect of food deprivation on body weight Exp
Group
n
Final BW (g)
% Change
15 15
263 ± 5 183 ± 4
+8.7 -22.5
272 235 220 196
±6 ±5 ±6 ±3
+7.1 -11.7 -17.0 -21.3
6 24
252 ± 3 179 ± 3
+9.1 -23.0
6 6 6 6
271 ± 4 202 ± 7 219 ± 4 234 ± 2
+7.5 +18.1 +25.1 +31.5
1) 72-hFD
Fed FD
2) Time course:
Fed 24-h FD 48-h FD 72-h FD
6 6 6 6
Fed 72-h FD Fed 24-h RF 48-h RF 72-h RF
3) Refeeding a) 72-h FD
b) Refeeding
Values are the mean ± SD; n is the number of animals. FD, Food deprived; RF, refeed.
FIG. 1. Effect of 72-h food deprivation on expression of hypothalamic prepro-GRF, prepro-SRIF, and /3-actin mRNA. Autoradiogram of a representative nuclease protection analysis for prepro-GRF (upper panel), prepro-SRIF {middle panel), and 0-actin mRNA (lower panel) in hypothalamic extracts from individual rats allowed free access to food (fed) or subjected to 72 h of food deprivation (food-deprived). The lane marked rRNA represents a negative control, showing the absence of hybridization of cRNA probes with E. co/i-derived ribosomal RNA. The lane designated probe indicates the mobility of cRNA probes undigested by RNase.
1, hypothalamic prepro-GRF mRNA levels were dramatically decreased in food-deprived rats compared to those in fed controls. In contrast, food deprivation failed to influence hypothalamic prepro-SRIF mRNA or /3-actin mRNA content in the same animals. When data were pooled from three independent experiments (Fig. 2) levels of hypothalamic prepro-GRF mRNA were decreased by 80% in food-deprived animals compared to those in fed controls (P < 0.001). Time course The time course of hypothalamic prepro-GRF mRNA reduction was established in groups of rats food-deprived for 24, 48, or 72 h. Food deprivation resulted in a timedependent reduction in body weight, with animals losing 12%, 17%, and 21% of initial body weight by 24, 48, and 72 h, respectively (Table 1). Total hypothalamic mRNA was extracted and subjected to nuclease protection as described above. As shown in Fig. 3, hypothalamic prepro-GRF mRNA was decreased by 30% (P < 0.05) as early as 24 h of food deprivation, with a maximal decrease of 80% by 48 h. Hypothalamic prepro-SRIF and /3-actin mRNA contents in food-deprived rats were similar to those in fed controls at all time points (data not shown).
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-
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FED
72h FD
9
1.0 -
0.0 0.0
Fed
Food Deprived
Group FIG. 2. Densitometric quantification of hypothalamic prepro-GRF mRNA levels in fed and 72-h food-deprived rats. Mean relative densitometric values (±SEM) were obtained from autoradiograms of nuclease protection analysis of three independent experiments for prepro-GRF, prepro-SRIF, and /3-actin mRNAs from fed (n = 15) and 72-h fooddeprived (n = 15) animals. *, P < 0.001 vs. fed.
Refeeding
The effect of refeeding on hypothalamic prepro-GRF mRNA levels was determined in groups of rats fooddeprived for 72 h then permitted to refeed ad libitum for 24, 48, or 72 h. Animals deprived of food for 72 h lost an average of 23% of body weight. Upon refeeding, animals gained weight in a time-dependent manner, with body weights returning to prestarvation levels in animals allowed to refeed for 72 h (Table 1). Animals deprived of food for 72 h displayed the expected 80% reduction in prepro-GRF mRNA levels (Fig. 4). Upon refeeding, prepro-GRF mRNA levels were restored to approximately 50% of control levels after 24 h (P < 0.05 vs. fed control) and returned to control values after 48 h (Fig. 4). Discussion Spontaneous episodes of GH release in the rat are markedly suppressed in response to prolonged food deprivation (3). The secretion of pituitary GH is normally
Fed
24h FD
48h FD
72h FD
Group FIG. 3. Time course of hypothalamic prepro-GRF mRNA changes after food deprivation. Upper panel, Autoradiogram of representative nuclease protection analysis for prepro-GRF mRNA in hypothalamic extracts of individual animals allowed free access to food (fed) or deprived of food for 24, 48, or 72 h (FD, food-deprived). Lower panel, Histogram of relative mean densitometric values (±SEM) from nuclease protection analysis for hypothalamic prepro-GRF mRNA from food deprivation time course (n = 6 rats/group). *, P < 0.05; **, P < 0.001 (vs. fed).
regulated by opposing influences of the hypothalamic peptides GRF and SRIF, which are secreted rhythmically into the hypophyseal-portal circulation over reciprocal 3- to 4-h intervals (1, 2). Suppressed GH secretion in food-deprived rats could, thus, be explained by altered hypothalamic peptide influences, either increased SRIF tone and/or suppressed GRF release. In the present study we have demonstrated a dramatic reduction in hypothalamic prepro-GRF mRNA levels in food-deprived rats. This diminution in prepro-GRF mRNA was unaccompanied by any change in the hypothalamic GRF peptide content, a finding that may indicate a parallel reduction in GRF secretion in this model of GH deficiency, as has previously been suggested for the discordance between prepro-GRF mRNA and peptide content seen in two models of GH deficiency, hypophy-
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HYPOTHALAMIC GRF AND FOOD DEPRIVATION 72h RF
FED
Fed
72h FD 24h RF 48h RF 72h RF
Group FIG. 4. The effect of refeeding after 72 h of food deprivation on hypothalamic prepro-GRF mRNA. Upper panel, Representative autoradiograph of nuclease protection analysis for hypothalamic preproGRF mRNA of individual animals allowed free access to food (fed) or food-deprived for 72 h (72h FD) then permitted to refeed for 24, 48, or 72 h (RF, refeed). Lower panel, Histogram of relative mean densitometric values (±SEM) from nuclease protection analysis for preproGRF mRNA for refeeding experiment (n = 6 rats/group). *, P < 0.001 vs. fed.
sectomy and hypothyroidism (14,15). Hypothalamic prepro-GRF mRNA content has previously been reported to be increased in hypophysectomized and hypothyroid rats and restored to control levels by GH administration (14, 15). These findings have been interpreted as supportive of a role for GH in physiological negative feedback regulation of GRF gene expression. Our present findings of decreased prepro-GRF mRNA associated with low GH levels suggest that such classical negative feedback regulation of GRF gene expression by GH is not operative during nutrient deprivation. Rather, our data suggest that decreased hypothalamic GRF may be the cause of reduced plasma GH levels in this model, rather than the result. In support of a hypothalamic, rather than a pituitary, defect causing reduced plasma GH levels, Tannenbaum et al. (16) have recently shown that GRF-induced GH release was enhanced in 72-h food-deprived rats compared to that in controls and that
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GH hyperresponsiveness to GRF persisted in perifused dispersed anterior pituitary cells. The suggestion that decreased hypothalamic GRF tone results in the marked suppression of GH secretory pulses previously described in association with food deprivation (3) is also supported by parallel modulation of GH and hypothalamic prepro-GRF mRNA. Thus, hypothalamic prepro-GRF mRNA was decreased by 80% in 72-h fooddeprived rats that show marked suppression of GH secretory bursts. Second, a significant decrease in preproGRF mRNA levels occurred as early as 24 h, at which point significant reductions in the amplitude of GH secretory bursts have previously been observed. Third, maximal decreases in prepro-GRF mRNA were observed by 48-72 h of food deprivation, a time when the amplitude and duration of GH secretory pulses are maximally suppressed. Finally, prepro-GRF mRNA levels return to control values within 48-72 h of refeeding, contemporaneously with restoration of the plasma GH secretory profile. Increased SRIF influence on pituitary somatrotropes could provide an alternate or additional explanation for the suppressed GH secretion seen in food-deprived rats. In several studies passive immunization of food-deprived rats with specific antiserum to SRIF has been shown to restore GH secretory episodes (4, 5). Increased SRIF content in gastrointestinal and pancreatic tissues of food-deprived animals and elevated plasma SRIF levels suggest that enhanced SRIF secretion could originate from peripheral sources (3). However, a recent study of food deprivation in hypothalamo-hypophyseal lesioned rats indicated that the hypothalamus was the likely source of SRIF involved in GH regulation in this model (17). In the present study hypothalamic prepro-SRIF mRNA and peptide contents did not differ in fooddeprived and control rats. However, an association of normal hypothalamic prepro-SRIF mRNA and peptide content with low circulating GH levels suggests that hypothalamic SRIF might be inappropriately elevated, since GH deficiency has previously been associated with reduced hypothalamic SRIF synthesis, content, and secretion (18, 19). It is, therefore, conceivable that hypothalamic SRIF synthesis and release are increased relative to ambient GH levels in food-deprived rats. Thus, taken together with our finding of decreased hypothalamic prepro-GRF mRNA (and, presumably, release) the inhibitory effect of normal levels of SRIF may override any stimulatory influence of GRF on GH and, thus, result in suppression of GH release that can be restored by immunoneutralization of SRIF. The peripheral signals sensed by the hypothalamus that modulate GRF and SRIF gene expression and secretion and ultimately GH release are unknown. However, since food deprivation is associated with changes
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in several metabolic and endocrine parameters, the possibility exists that one or more of these variables may be acting to modulate the expression of hypothalamic peptides involved in GH release. Thus, plasma FFA levels are elevated in food-deprived rats (20), and the GH response to exogenous GRF in lipid heparin-treated rats showing high plasma FFA levels is significantly depressed (21). A role for SRIF in mediating this response was proposed, since FFA-induced suppression of GH release was abolished by SRIF antiserum. Glucose has also been postulated as a metabolic factor modulating GH secretion (22). Spontaneous episodes of GH secretion in the rat are severely inhibited in response to 2-deoxyD-glucose (2DG)-induced intracellular glucopenia (23). However, unlike the GH response to FFA, suppression of GH secretion by 2DG was only partially restored by
immunization with SRIF antiserum. Decreased GRF release associated with increased SRIF release may be involved in regulating GH secretion in this model. Insulin, a potent inhibitor of GH release and synthesis (24, 25), is unlikely to be involved in modulating GH secretion in food-deprived animals, since GH secretion is markedly suppressed in both food-deprived and diabetic rats that are insulinopenic (4, 26) and in 2DG-treated rats that show normal insulin levels (23). Clearly, further studies are required to determine the mechanisms that regulate GH secretion in the food-deprived rat. In summary, we report a decrease in hypothalamic prepro-GRF mRNA levels in food-deprived rats that is unaccompanied by changes in GRF peptide content, a finding that may indicate decreased GRF secretion in this model of GH deficiency. The finding of reduced prepro-GRF mRNA (and, possibly, secretion) suggests that the marked suppression of GH secretory pulses observed in food-deprived animals can be explained in part by decreased hypothalamic GRF tone.
References 1. 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 15:1952-7 2. Plotsky PM, Vale WW 1986 Patterns of growth hormone-releasing factor and somatostatin secretion into the hypophyseal-portal circulation of the rat. Science 230:461-3 3. Tannenbaum GS, Rorstad 0, Brazeau P 1979 Effects of prolonged food deprivation on the ultradian growth hormone rhythm and immunoreactive somatostatin tissue levels in the rat. Endocrinolology 104:1733-8 4. 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 102:1909-14 5. Hugues JN, Enjalbert A, Moyse E, Shu C, Voirol MJ, Sebaoun J, Epelbaum J 1986 Differential effects of passive immunization with somatostatin antiserum on adenohypophysial hormone secretions in starved rats. J Endocrinol 109:169-74 6. Berelowitz M, Firestone SL, Frohman LA 1981 Effects of growth
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hormone excess and deficiency on hypothalamic somatostatin content and release and on tissue somatostatin distribution. Endocrinology 109:714-9 Palkovitz M, Isolated removal of hypothalamic nuclei for neuroendocrinological and neurochemical studies. International Conference of Neurobiology of CNS-Hormone Interactions, Chapel Hill NC, 1979, pp 72-80 Leidy JW, Robbins RJ 1988 Growth hormone-releasing factor immunoreactivity in the hypothalamus and cortex of the rat: in vivo and in vitro studies. Endocrinology 122:1652-7 Berelowitz M, Maeda K, Harris S, Frohman LA 1980 The effect of alterations in the pituitary-thyroid axis on hypothalamic content and in vitro release of somatostatin-like immunoreactivity. Endocrinology 107:24-9 White JD, Stewart KD, McKelvy JF 1986 Measurement of neuroendocrine peptide mRNA in descrete brain regions. In: Conn PM (ed) Methods in Enzymology. Academic Press, Orlando, vol 124:548-60 Mayo KE, Cerelli GM, Rosenfeld MG, Evans RM 1985 Characterization of cDNA and genomic clones encoding the precursor of rat hypothalamic growth hormone-releasing factor. Nature 314:464-7
12. Funckes CL, Minth CD, Deschenes R, Magazin M, Tavianini MA, Sheets M, Collier K, Weith HL, Aron DC, Roos BA, Dixon JE 1983 Cloning and characterization of a mRNA-encoding rat preprosomatostatin. J Biol Chem 258:8781-7 13. Werner H, Woloschak M, Adamo M, Shen-Orr Z, Roberts CT, LeRoith D 1989 Developmental regulation of the rat like growth factor 1 receptor gene. Proc Natl Acad Sci USA 86:7451-5 14. Chomczynski P, Downs TR, Frohman LA 1988 Feedback regulation of growth hormone (GH)-releasing hormone gene expression by GH in rat hypothalamus. Mol Endocrinol 2:236-41 15. Downs TR, Chomczynski P, Frohman LA 1990 Effects of thyroid hormone deficiency and replacement on rat hypothalamic growth hormone (GH)-releasing hormone gene expression in vivo are mediated by GH. Mol Endocrinol 4:402-8 16. Tannenbaum GS, Painson JC, 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 124:1380-8 17. Mounier F, Bluet-Pajot MT, Durand D, Kordon C, Rasolonjanahary R, Epelbaum J 1989 Involvement of central somatostatin in the alteration of GH secretion in starved rats. Horm Res 31:26670 18. Berelowitz M, Shapiro B, Pimstone B, Kronheim S 1979 Growth hormone release inhibitory hormone-like immunoreactivity in pancreas and gut in streptozotocin diabetes in the rat and response to insulin administration. Clin Endocrinol (Oxf) 10:195-8 19. Patel YC, Wheatley T, Zingg HH 1980 Increased blood somatostatin concentration in streptozotocin diabetic rats. Life Sci 27:1563-70 20. Mlekusch W, Truppe W, Beyer W, Paletta B 1975 The effect of hunger on free fatty acid and corticosterone plasma levels in rats. Experimentia 31:1135-7 21. Imaki T, Shibasaki T, Masuda A, Hotta M, Yamauchi N, Demura H, Shizume K, Wakabayashi I, Ling N 1986 The effect of glucose and free fatty acids on growth hormone (GH)-releasing factormediated GH secretion in rats. Endocrinology 118:2390-4 22. Daughaday WH 1985 Regulation of growth hormone. In: Williams RH (ed) Textbook of Endocrinology. Saunders, Philadelphia, p 568 23. Painson JC, Tannenbaum GS 1985 Effects of intracellular glucopenia on pulsatile growth hormone secretion: mediation in part by somatostatin. Endocrinology 117:1132-8 24. Yamashita S, Melmed S 1986 Effects of insulin on rat anterior pituitary cells. Inhibition of growth hormone secretion and mRNA levels. Diabetes 35:440-7 25. Isaacs RE, Gardner DC, Baxter JD 1987 Insulin regulation of rat growth hormone gene expression. Endocrinology 120:2022-8 26. Tannenbaum GS 1981 Growth hormone secretory dynamics in streptozotocin diabetes: evidence of a role for endogenous circulating somatostatin. Endocrinology 108:76-82
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Vol. 127, No. 5 Printed in U.S.A.
Influence of Food Deprivation in the Rat on Hypothalamic Expression of Growth Hormone-Releasing Factor and Somatostatin* JOHN F. BRUNO, DAVID OLCHOVSKY, JEFFREY D. WHITEf, JOHN W. LEIDY, JINFEN SONG, AND MICHAEL BERELOWITZ Division of Endocrinology and Metabolism, Departments of Medicine (J.F.B., D.O., J.D.W., J.S., M.B.), Neurobiology/Behaviour (J.D.W.), and Physiology/Biophysics (M.B.), State University of New York, Stony Brook, New York 11794; and the Veterans Administration Medical Center/Department of Medicine, Marshall University School of Medicine (J.W.L.), Huntington, West Virginia 25701
hypothalamic SRIF content. The time course of hypothalamic prepro-GRF mRNA reduction was determined in groups of rats food-deprived for 24, 48, or 72 h and revealed a significant (30%) reduction of prepro-GRF mRNA (P < 0.05 us. fed) by 24 h, with maximal reduction (80%) by 48 h. Refeeding groups of animals for up to 72 h after they had been food-deprived for 72 h resulted in restoration of prepro-GRF mRNA levels to 50% of control levels by 24 h (P < 0.05 vs. fed) and a return to control values by 48 h. These data suggest that decreased GRF gene expression and possibly GRF release play a major role in the loss of pulsatile GH secretion seen in this model of nutrient deprivation. (Endocrinology 127: 2111-2116, 1990)
ABSTRACT. Food deprivation in the rat is associated with a reduction in serum GH levels characterized by suppression of high amplitude GH bursts and a decrease in the duration of secretory episodes. The mechanism(s) mediating this response is unknown. The present studies were designed to evaluate the role of hypothalamic factors potentially responsible for abnormal GH dynamics in food-deprived rats by measuring hypothalamic prepro-GH-releasing factor (GRF) and preprosomatostatin (SRIF) mRNA and peptide levels in adult male SpragueDawley rats after 72 h of food deprivation or free access to food. Hypothalamic prepro-GRF mRNA was reduced 80% in fooddeprived rats compared to that in fed controls (P < 0.001), while GRF content was unchanged. Levels of prepro-SRIF mRNA in food-deprived rats were similar to those in controls, as was
E
PISODIC pituitary GH secretion is regulated by opposing influences of the hypothalamic peptides GRF and somatostatin (SRIF). In the rat, major GH secretory episodes are characterized by high amplitude bursts that may reach 300-1000 ng/ml occurring at 3- to 4-h intervals throughout a 24-h period (1, 2). Food deprivation in the rat results in an inhibition of GH secretory episodes, with a significant reduction in high amplitude bursts that occurs as early as 24 h and is followed by a progressive decline in both the amplitude and duration of secretory episodes by 48 and 72 h (3). The mechanism(s) mediating this response is unknown. Decreased GH secretion in food-deprived rats could result from increased inhibitory SRIF influence on the somatotrope Received May 23, 1990. Address all correspondence and requests for reprints to: John F. Bruno, Ph.D., Division of Endocrinology and Metabolism, Department of Medicine, HSC T15, Room 060, State University of New York, Stony Brook, New York 11794. * This work was supported by NIH Research Grant AM-36831 (to M.B.) and Alcohol Drug Abuse and Mental Health Administration Grants MH-42074 and MH-00801 (to J.D.W.). t Recipient of a fellowship from the Aaron Diamond Foundation.
or from diminished GRF stimulatory tone. Increased SRIF has been circumstantially implicated based on in vivo immunoneutralization studies in which GH secretion was restored after the administration of specific SRIF antiserum to food-deprived rats (4,5). Such studies do not, however, provide evidence for a direct causeeffect relationship between SRIF and altered GH secretion, since SRIF alterations could be a secondary occurence and do not identify the tissue source of putative increases in SRIF. The present studies were designed to evaluate the role of hypothalamic factors potentially responsible for abnormal GH dynamics in food-deprived rats by comparing prepro-GRF and prepro-SRIF mRNA and peptide levels in hypothalamic extracts from adult male Sprague-Dawley rats allowed free access to food or food-deprived for periods up to 72 h.
Materials and Methods Animals and experimental procedures Male Sprague-Dawley rats (Taconic Farms, Germantown, NY), weighing 175-225 g, were housed in groups of two
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under constant temperature (22 C) and a 12-h light, 12-h dark cycle (lights on at 0700 h) and were provided free access to rat chow (Ralston-Purina, St. Louis, MO) and tap water. Animals were adapted for 5-7 days to the facility before all experimental procedures. Three sets of experiments comprised the present study.
described (8). Cartridge preconditioning and washing between extracts were slightly modified by the addition of chloroform and methanol washes to remove hypothalamic lipids that interfere with GRF recovery from the cartridge and GRF RIA.
72-h food deprivation. The effects of food deprivation on hypothalamic prepro-GRF and prepro-SRIF mRNA and peptide levels were determined in three separate self-contained experiments. Rats were deprived of food for 72 h or allowed free access to food; both groups had access to drinking water. Animals were weighed immediately before food deprivation, then before death 72 h later. Food deprivation resulted in a significant reduction in hypothalamic prepro-GRF mRNA, with no change in prepro-SRIF mRNA levels (see Results); therefore, further experiments were performed to evaluate the effects of varying duration of food deprivation and refeeding on changes in hypothalamic prepro-GRF mRNA.
GRF and SRIF were measured, as previously described, by double antibody RIA, using [125I]rat GRF (8) or [^IJTyr'-SRIF (9) prepared by the chloramine-T method and purified by reverse phase HPLC.
Time course. To determine the time course of changes in hypothalamic prepro-GRF mRNA occurring as a result of nutrient deprivation, animals were either allowed free access to food (fed) or were killed after 24, 48, or 72 h of food deprivation (n = 6/group). All animals had free access to water, and body weights were recorded before food deprivation and at death. Refeeding after 72 h of food deprivation. The effect of refeeding on decreased hypothalamic prepro-GRF mRNA content after 72 h food deprivation was determined in groups of rats allowed free access to food or food-deprived for 72 h then permitted to refeed ad libitum for 24, 48, or 72 h (n = 6/group). All groups had free access to water and were weighed before and after food deprivation and again after refeeding. Tissue handling Rats were killed by decapitation, and hypothalami were rapidly dissected using landmarks to yield blocks weighing 2025 mg, as previously described (6), then immediately frozen on dry ice for later RNA extraction or individually extracted for SRIF or GRF RIA. For SRIF assay, hypothalami were placed in 2 N acetic acid, boiled for 3 min, homogenized by Polytron (Brinkmann Instruments, Westbury, NY) at setting 2 for 10 sec, and then centrifuged at 2000 X g for 30 min, and the supernatant was lyophilized. For hypothalamic nuclear SRIF determination, brains were processed, and nuclear areas were identified and removed by the method of Palkovitz (7). Paired nuclei from individual rats were expelled into a glass-glass microhomogenizer containing 100 or 200 jtl acetic acid, then thoroughly homogenized. Ten percent of the final homogenate volume was sampled for protein determination using the fluorescamine method. The remainder of the homogenate was boiled for 10 min, then centrifuged at 2000 X g for 30 min. The supernatant was frozen, lyophilized, then stored at -70 C for RIA. For GRF assay, hypothalami were homogenized in an acidic medium and extracted with octadecasilyl-silica cartridges (Bond Elut Cis, Analytichem, Harbor City, CA), as previously
Hormone assay
RNA extraction Total hypothalamic RNA was isolated from dissected hypothalamic tissue blocks using methods developed previously (10). Briefly, tissue was homogenized using a Polytron tissue homogenizer in a buffer containing 4 M guanidine isothiocyanate, 50 mM Tris (pH 7.5), 10 mM EDTA (Fisher Scientific, Springfield, NJ), 1% iV-lauroylsarcosine (Sigma, St. Louis, MO), and 1% 2-mercaptoethanol (Boehringer Mannheim Biochemicals, Indianapolis, IN). The samples were then extracted twice with phenol-chloroform, ethanol precipitated, subjected to DNase digestion, reextracted with phenol-chloroform, then chloroform extracted and ethanol precipitated. An average of 25 /xg RNA were recovered per hypothalamus. RNA concentrations were estimated based on absorbance at 260 nm, and identical concentrations of RNA from each sample were used for nuclease protection. Preparation of probes GRF cDNA constructs in pGEM 3 and SRIF cDNA constructs in pGEM 4 vectors were used to generate 32P-labeled antisense RNA for nuclease protection assays. The GRF riboprobe was transcribed in the presence of SP6 polymerase from a 215-basepair (bp) EcoRl-Hindlll fragment of the rat cDNA provided by Kelly E. Mayo (Northwestern University) after linearization with EcoRl (11). The SRIF riboprobe was transcribed in the presence of SP6 polymerase from a 461-bp Xbal/ Sau3A fragment of the rat cDNA provided by Jack E. Dixon (Purdue University) after linearization with Hindlll (12). Levels of /3-actin mRNA were measured using a 650-bp EcoRlHircdlll fragment of a rat /3-actin cDNA, provided by B. Patterson (NIH). This cDNA fragment was cloned into EcoRland ifindlll-digested pGEM-2 and, after linearization with EcoRl, was used to generate an antisense RNA probe, as previously described (13). Nuclease protection assay Five micrograms of total RNA from each hypothalamus were coprecipitated with 80,000 cpm 32P-labeled GRF riboprobe, or 2 ng total RNA were coprecipitated with either 50,000 cpm 32Plabeled SRIF riboprobe or 25,000 cpm 32P-labeled /3-actin riboprobe, prepared as described above. Samples were resuspended in hybridization buffer containing 40 mM 1,4-piperazine diethane sulfonic acid (pH 6.8), 72% formamide, 1 mM EDTA, and 0.4 M NaCl, boiled for 3 min, and hybridized at 45 C for 14-16 h. After hybridization, samples were subjected to com-
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HYPOTHALAMIC GRF AND FOOD DEPRIVATION bined RNase-A and -Tx digestion of nonhybridized probe at 30 C for 1 h. Stable hybrids were ethanol precipitated, denatured, and separated on 8% polyacrylamide-8 M urea gels. The dried gel was apposed to Kodak X-Omat x-ray film (Eastman Kodak, Rochester, NY) to generate an autoradiograph; exposure times ranged from 24-72 h. Autoradiographic densities were quantitated using an LKB laser densitometer (Rockville, MD) in the two-dimensional scan mode to obtain a densitometric value for the entire autoradiographic band.
FED
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|rRNA|PROBE
GRF
Analysis of data Results are expressed as the mean ± SEM. Comparisons of data between experimental groups were performed using oneway analysis of variance, followed by Fisher's least significant difference test.
Results
SRIF
Actin
72-h food deprivation Animals exposed to 72 h of food deprivation lost approximately 22% of their initial body weight, while fed controls gained almost 9% (Table 1). SRIF contents were similar in extracts of medial basal hypothalamus from 72-h food-deprived and control rats (80.5 ± 12.0 vs. 80.7 ± 17.1 ng/hypothalamus; n = 5). In addition, SRIF content in microdissected punches of periventricular nucleus, preoptic area, or median eminence was unchanged in food-deprived rats compared to that in fed controls (data not shown). GRF content in acidic extracts of hypothalami from food-deprived rats did not differ from that in control rats (2.87 ± 0.55 vs. 3.37 ± 0.93 ng/ hypothalamus; n = 5). Total hypothalamic RNA was isolated from 72-h fooddeprived and control rats, and prepro-GRF and preproSRIF mRNA contents were estimated using a solution hybridization/RNase protection assay. As shown in Fig. TABLE 1. Effect of food deprivation on body weight Exp
Group
n
Final BW (g)
% Change
15 15
263 ± 5 183 ± 4
+8.7 -22.5
272 235 220 196
±6 ±5 ±6 ±3
+7.1 -11.7 -17.0 -21.3
6 24
252 ± 3 179 ± 3
+9.1 -23.0
6 6 6 6
271 ± 4 202 ± 7 219 ± 4 234 ± 2
+7.5 +18.1 +25.1 +31.5
1) 72-hFD
Fed FD
2) Time course:
Fed 24-h FD 48-h FD 72-h FD
6 6 6 6
Fed 72-h FD Fed 24-h RF 48-h RF 72-h RF
3) Refeeding a) 72-h FD
b) Refeeding
Values are the mean ± SD; n is the number of animals. FD, Food deprived; RF, refeed.
FIG. 1. Effect of 72-h food deprivation on expression of hypothalamic prepro-GRF, prepro-SRIF, and /3-actin mRNA. Autoradiogram of a representative nuclease protection analysis for prepro-GRF (upper panel), prepro-SRIF {middle panel), and 0-actin mRNA (lower panel) in hypothalamic extracts from individual rats allowed free access to food (fed) or subjected to 72 h of food deprivation (food-deprived). The lane marked rRNA represents a negative control, showing the absence of hybridization of cRNA probes with E. co/i-derived ribosomal RNA. The lane designated probe indicates the mobility of cRNA probes undigested by RNase.
1, hypothalamic prepro-GRF mRNA levels were dramatically decreased in food-deprived rats compared to those in fed controls. In contrast, food deprivation failed to influence hypothalamic prepro-SRIF mRNA or /3-actin mRNA content in the same animals. When data were pooled from three independent experiments (Fig. 2) levels of hypothalamic prepro-GRF mRNA were decreased by 80% in food-deprived animals compared to those in fed controls (P < 0.001). Time course The time course of hypothalamic prepro-GRF mRNA reduction was established in groups of rats food-deprived for 24, 48, or 72 h. Food deprivation resulted in a timedependent reduction in body weight, with animals losing 12%, 17%, and 21% of initial body weight by 24, 48, and 72 h, respectively (Table 1). Total hypothalamic mRNA was extracted and subjected to nuclease protection as described above. As shown in Fig. 3, hypothalamic prepro-GRF mRNA was decreased by 30% (P < 0.05) as early as 24 h of food deprivation, with a maximal decrease of 80% by 48 h. Hypothalamic prepro-SRIF and /3-actin mRNA contents in food-deprived rats were similar to those in fed controls at all time points (data not shown).
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FED
72h FD
9
1.0 -
0.0 0.0
Fed
Food Deprived
Group FIG. 2. Densitometric quantification of hypothalamic prepro-GRF mRNA levels in fed and 72-h food-deprived rats. Mean relative densitometric values (±SEM) were obtained from autoradiograms of nuclease protection analysis of three independent experiments for prepro-GRF, prepro-SRIF, and /3-actin mRNAs from fed (n = 15) and 72-h fooddeprived (n = 15) animals. *, P < 0.001 vs. fed.
Refeeding
The effect of refeeding on hypothalamic prepro-GRF mRNA levels was determined in groups of rats fooddeprived for 72 h then permitted to refeed ad libitum for 24, 48, or 72 h. Animals deprived of food for 72 h lost an average of 23% of body weight. Upon refeeding, animals gained weight in a time-dependent manner, with body weights returning to prestarvation levels in animals allowed to refeed for 72 h (Table 1). Animals deprived of food for 72 h displayed the expected 80% reduction in prepro-GRF mRNA levels (Fig. 4). Upon refeeding, prepro-GRF mRNA levels were restored to approximately 50% of control levels after 24 h (P < 0.05 vs. fed control) and returned to control values after 48 h (Fig. 4). Discussion Spontaneous episodes of GH release in the rat are markedly suppressed in response to prolonged food deprivation (3). The secretion of pituitary GH is normally
Fed
24h FD
48h FD
72h FD
Group FIG. 3. Time course of hypothalamic prepro-GRF mRNA changes after food deprivation. Upper panel, Autoradiogram of representative nuclease protection analysis for prepro-GRF mRNA in hypothalamic extracts of individual animals allowed free access to food (fed) or deprived of food for 24, 48, or 72 h (FD, food-deprived). Lower panel, Histogram of relative mean densitometric values (±SEM) from nuclease protection analysis for hypothalamic prepro-GRF mRNA from food deprivation time course (n = 6 rats/group). *, P < 0.05; **, P < 0.001 (vs. fed).
regulated by opposing influences of the hypothalamic peptides GRF and SRIF, which are secreted rhythmically into the hypophyseal-portal circulation over reciprocal 3- to 4-h intervals (1, 2). Suppressed GH secretion in food-deprived rats could, thus, be explained by altered hypothalamic peptide influences, either increased SRIF tone and/or suppressed GRF release. In the present study we have demonstrated a dramatic reduction in hypothalamic prepro-GRF mRNA levels in food-deprived rats. This diminution in prepro-GRF mRNA was unaccompanied by any change in the hypothalamic GRF peptide content, a finding that may indicate a parallel reduction in GRF secretion in this model of GH deficiency, as has previously been suggested for the discordance between prepro-GRF mRNA and peptide content seen in two models of GH deficiency, hypophy-
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HYPOTHALAMIC GRF AND FOOD DEPRIVATION 72h RF
FED
Fed
72h FD 24h RF 48h RF 72h RF
Group FIG. 4. The effect of refeeding after 72 h of food deprivation on hypothalamic prepro-GRF mRNA. Upper panel, Representative autoradiograph of nuclease protection analysis for hypothalamic preproGRF mRNA of individual animals allowed free access to food (fed) or food-deprived for 72 h (72h FD) then permitted to refeed for 24, 48, or 72 h (RF, refeed). Lower panel, Histogram of relative mean densitometric values (±SEM) from nuclease protection analysis for preproGRF mRNA for refeeding experiment (n = 6 rats/group). *, P < 0.001 vs. fed.
sectomy and hypothyroidism (14,15). Hypothalamic prepro-GRF mRNA content has previously been reported to be increased in hypophysectomized and hypothyroid rats and restored to control levels by GH administration (14, 15). These findings have been interpreted as supportive of a role for GH in physiological negative feedback regulation of GRF gene expression. Our present findings of decreased prepro-GRF mRNA associated with low GH levels suggest that such classical negative feedback regulation of GRF gene expression by GH is not operative during nutrient deprivation. Rather, our data suggest that decreased hypothalamic GRF may be the cause of reduced plasma GH levels in this model, rather than the result. In support of a hypothalamic, rather than a pituitary, defect causing reduced plasma GH levels, Tannenbaum et al. (16) have recently shown that GRF-induced GH release was enhanced in 72-h food-deprived rats compared to that in controls and that
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GH hyperresponsiveness to GRF persisted in perifused dispersed anterior pituitary cells. The suggestion that decreased hypothalamic GRF tone results in the marked suppression of GH secretory pulses previously described in association with food deprivation (3) is also supported by parallel modulation of GH and hypothalamic prepro-GRF mRNA. Thus, hypothalamic prepro-GRF mRNA was decreased by 80% in 72-h fooddeprived rats that show marked suppression of GH secretory bursts. Second, a significant decrease in preproGRF mRNA levels occurred as early as 24 h, at which point significant reductions in the amplitude of GH secretory bursts have previously been observed. Third, maximal decreases in prepro-GRF mRNA were observed by 48-72 h of food deprivation, a time when the amplitude and duration of GH secretory pulses are maximally suppressed. Finally, prepro-GRF mRNA levels return to control values within 48-72 h of refeeding, contemporaneously with restoration of the plasma GH secretory profile. Increased SRIF influence on pituitary somatrotropes could provide an alternate or additional explanation for the suppressed GH secretion seen in food-deprived rats. In several studies passive immunization of food-deprived rats with specific antiserum to SRIF has been shown to restore GH secretory episodes (4, 5). Increased SRIF content in gastrointestinal and pancreatic tissues of food-deprived animals and elevated plasma SRIF levels suggest that enhanced SRIF secretion could originate from peripheral sources (3). However, a recent study of food deprivation in hypothalamo-hypophyseal lesioned rats indicated that the hypothalamus was the likely source of SRIF involved in GH regulation in this model (17). In the present study hypothalamic prepro-SRIF mRNA and peptide contents did not differ in fooddeprived and control rats. However, an association of normal hypothalamic prepro-SRIF mRNA and peptide content with low circulating GH levels suggests that hypothalamic SRIF might be inappropriately elevated, since GH deficiency has previously been associated with reduced hypothalamic SRIF synthesis, content, and secretion (18, 19). It is, therefore, conceivable that hypothalamic SRIF synthesis and release are increased relative to ambient GH levels in food-deprived rats. Thus, taken together with our finding of decreased hypothalamic prepro-GRF mRNA (and, presumably, release) the inhibitory effect of normal levels of SRIF may override any stimulatory influence of GRF on GH and, thus, result in suppression of GH release that can be restored by immunoneutralization of SRIF. The peripheral signals sensed by the hypothalamus that modulate GRF and SRIF gene expression and secretion and ultimately GH release are unknown. However, since food deprivation is associated with changes
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in several metabolic and endocrine parameters, the possibility exists that one or more of these variables may be acting to modulate the expression of hypothalamic peptides involved in GH release. Thus, plasma FFA levels are elevated in food-deprived rats (20), and the GH response to exogenous GRF in lipid heparin-treated rats showing high plasma FFA levels is significantly depressed (21). A role for SRIF in mediating this response was proposed, since FFA-induced suppression of GH release was abolished by SRIF antiserum. Glucose has also been postulated as a metabolic factor modulating GH secretion (22). Spontaneous episodes of GH secretion in the rat are severely inhibited in response to 2-deoxyD-glucose (2DG)-induced intracellular glucopenia (23). However, unlike the GH response to FFA, suppression of GH secretion by 2DG was only partially restored by
immunization with SRIF antiserum. Decreased GRF release associated with increased SRIF release may be involved in regulating GH secretion in this model. Insulin, a potent inhibitor of GH release and synthesis (24, 25), is unlikely to be involved in modulating GH secretion in food-deprived animals, since GH secretion is markedly suppressed in both food-deprived and diabetic rats that are insulinopenic (4, 26) and in 2DG-treated rats that show normal insulin levels (23). Clearly, further studies are required to determine the mechanisms that regulate GH secretion in the food-deprived rat. In summary, we report a decrease in hypothalamic prepro-GRF mRNA levels in food-deprived rats that is unaccompanied by changes in GRF peptide content, a finding that may indicate decreased GRF secretion in this model of GH deficiency. The finding of reduced prepro-GRF mRNA (and, possibly, secretion) suggests that the marked suppression of GH secretory pulses observed in food-deprived animals can be explained in part by decreased hypothalamic GRF tone.
References 1. 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 15:1952-7 2. Plotsky PM, Vale WW 1986 Patterns of growth hormone-releasing factor and somatostatin secretion into the hypophyseal-portal circulation of the rat. Science 230:461-3 3. Tannenbaum GS, Rorstad 0, Brazeau P 1979 Effects of prolonged food deprivation on the ultradian growth hormone rhythm and immunoreactive somatostatin tissue levels in the rat. Endocrinolology 104:1733-8 4. 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 102:1909-14 5. Hugues JN, Enjalbert A, Moyse E, Shu C, Voirol MJ, Sebaoun J, Epelbaum J 1986 Differential effects of passive immunization with somatostatin antiserum on adenohypophysial hormone secretions in starved rats. J Endocrinol 109:169-74 6. Berelowitz M, Firestone SL, Frohman LA 1981 Effects of growth
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hormone excess and deficiency on hypothalamic somatostatin content and release and on tissue somatostatin distribution. Endocrinology 109:714-9 Palkovitz M, Isolated removal of hypothalamic nuclei for neuroendocrinological and neurochemical studies. International Conference of Neurobiology of CNS-Hormone Interactions, Chapel Hill NC, 1979, pp 72-80 Leidy JW, Robbins RJ 1988 Growth hormone-releasing factor immunoreactivity in the hypothalamus and cortex of the rat: in vivo and in vitro studies. Endocrinology 122:1652-7 Berelowitz M, Maeda K, Harris S, Frohman LA 1980 The effect of alterations in the pituitary-thyroid axis on hypothalamic content and in vitro release of somatostatin-like immunoreactivity. Endocrinology 107:24-9 White JD, Stewart KD, McKelvy JF 1986 Measurement of neuroendocrine peptide mRNA in descrete brain regions. In: Conn PM (ed) Methods in Enzymology. Academic Press, Orlando, vol 124:548-60 Mayo KE, Cerelli GM, Rosenfeld MG, Evans RM 1985 Characterization of cDNA and genomic clones encoding the precursor of rat hypothalamic growth hormone-releasing factor. Nature 314:464-7
12. Funckes CL, Minth CD, Deschenes R, Magazin M, Tavianini MA, Sheets M, Collier K, Weith HL, Aron DC, Roos BA, Dixon JE 1983 Cloning and characterization of a mRNA-encoding rat preprosomatostatin. J Biol Chem 258:8781-7 13. Werner H, Woloschak M, Adamo M, Shen-Orr Z, Roberts CT, LeRoith D 1989 Developmental regulation of the rat like growth factor 1 receptor gene. Proc Natl Acad Sci USA 86:7451-5 14. Chomczynski P, Downs TR, Frohman LA 1988 Feedback regulation of growth hormone (GH)-releasing hormone gene expression by GH in rat hypothalamus. Mol Endocrinol 2:236-41 15. Downs TR, Chomczynski P, Frohman LA 1990 Effects of thyroid hormone deficiency and replacement on rat hypothalamic growth hormone (GH)-releasing hormone gene expression in vivo are mediated by GH. Mol Endocrinol 4:402-8 16. Tannenbaum GS, Painson JC, 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 124:1380-8 17. Mounier F, Bluet-Pajot MT, Durand D, Kordon C, Rasolonjanahary R, Epelbaum J 1989 Involvement of central somatostatin in the alteration of GH secretion in starved rats. Horm Res 31:26670 18. Berelowitz M, Shapiro B, Pimstone B, Kronheim S 1979 Growth hormone release inhibitory hormone-like immunoreactivity in pancreas and gut in streptozotocin diabetes in the rat and response to insulin administration. Clin Endocrinol (Oxf) 10:195-8 19. Patel YC, Wheatley T, Zingg HH 1980 Increased blood somatostatin concentration in streptozotocin diabetic rats. Life Sci 27:1563-70 20. Mlekusch W, Truppe W, Beyer W, Paletta B 1975 The effect of hunger on free fatty acid and corticosterone plasma levels in rats. Experimentia 31:1135-7 21. Imaki T, Shibasaki T, Masuda A, Hotta M, Yamauchi N, Demura H, Shizume K, Wakabayashi I, Ling N 1986 The effect of glucose and free fatty acids on growth hormone (GH)-releasing factormediated GH secretion in rats. Endocrinology 118:2390-4 22. Daughaday WH 1985 Regulation of growth hormone. In: Williams RH (ed) Textbook of Endocrinology. Saunders, Philadelphia, p 568 23. Painson JC, Tannenbaum GS 1985 Effects of intracellular glucopenia on pulsatile growth hormone secretion: mediation in part by somatostatin. Endocrinology 117:1132-8 24. Yamashita S, Melmed S 1986 Effects of insulin on rat anterior pituitary cells. Inhibition of growth hormone secretion and mRNA levels. Diabetes 35:440-7 25. Isaacs RE, Gardner DC, Baxter JD 1987 Insulin regulation of rat growth hormone gene expression. Endocrinology 120:2022-8 26. Tannenbaum GS 1981 Growth hormone secretory dynamics in streptozotocin diabetes: evidence of a role for endogenous circulating somatostatin. Endocrinology 108:76-82
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