0013-7227/92/1302-0920$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine
Vol. 130, No. 2 Printed in U.S.A.
Society
The Effect of Insulin-Induced Hypoglycemia Expression in the Hypothalamic-Pituitary-Adrenal of the Rat* BRUCE JOSEPH
G. ROBINSONt, A. MAJZOUB
KENNETH
MEALY$,
DOUGLAS
W. WILMORE,
on Gene Axis AND
Division of Endocrinology, Department of Medicine, Children’s Hospital (B.G.R., J.A.M.), and Surgical Nutrition Laboratory, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School (K.M., D. W. W.), Boston, Massachusetts 02115
ABSTRACT. This study has examined the effects of insulininduced hypoglycemia on expression of the CRH, arginine vasopressin, and POMC genes and corresponding peptides in freely moving, unanesthetized, male Sprague-Dawley rats. Animals were infused with 150 mM NaCl for 3 days before the experimental day and were then administered insulin (4 U/kg) or saline iv. In one experiment animals were killed 0, 30, 60, or 90 min after insulin or saline, and RNA was isolated from anterior pituitary, cerebral cortex, and punches of the hypothalamic paraventricular and supraoptic nuclei. In a second experiment, animals were killed 90 min after insulin or saline treatment, and RNA was isolated from whole hypothalami. RNA was analyzed by Northern blot. Plasma glucose fell from 106 * 5 to 38 + 2 mg/dl after insulin administration and remained low for the duration of the experiment. Plasma levels of ACTH, corticoster-
C
RH AND arginine vasopressin (AVP) are considered to be the principal stimulators of ACTH release after stress. Insulin-induced hypoglycemia is a potent physiological stimulus for the release of ACTH. It is unclear, however, the extent to which either CRH or AVP plays a role in this response. There have been several previous studies of the effects of insulin-induced hypoglycemia on activation of the hypothalamic-pituitary-adrenal (HPA) axis of the rat. Plotsky et al. (1) measured hypophyseal portal concentrations of CRH and AVP in hypoglycemic rats and found that AVP increased 1.9-fold, while CRH remained unchanged. Insulin-induced hypoglycemia has also been associated with elevated peripheral levels of plasma AVP (2), and recently, Received August 2, 1991. Address all correspondence and requests for reprints to: Dr. Joseph A. Majzoub, Endocrine Division, Children’s Hospital, 300 Longwood Avenue, Boston, Massachusetts 02115. * This work was supported by the Postgraduate Medical Foundation, University of Sydney, and NIH Grant ROl-DK-40170. t Present address: Molecular Genetics Unit, Kolling Institute, Royal North Shore Hospital, St. Leonard 2065, Australia. $ Present address: St. Vincent’s Hospital, Dublin, Ireland.
one, and vasopressin were lo-, 6-, and 4-fold higher, respectively, in the insulin-treated us. control animals (by analysis of variance, P < 0.0001 in all cases), while plasma CRH was unchanged. During hypoglycemia POMC mRNA levels were l&fold higher in the insulin-treated group (by analysis of variance, P < 0.025). In contrast, paraventricular nucleus, whole hypothalamic, and parietal cortex CRH mRNA and vasopressin mRNA were unchanged. These data support previous studies which indicated that POMC gene expression is increased by hypoglycemia. However, we found no evidence for an increase in paraventricular nucleus or cerebral cortex CRH mRNA expression during hypoglycemia-associated stimulation of the hypothalamic-pituitary-adrenal axis, suggesting that another factor(s) may mediate the observed increase in POMC gene expression. (Endocrinology 130: 920-925,1992)
the levels of peripheral plasma CRH, CRH mRNA (3), and POMC mRNA (4) have been reported to be elevated in rats after this stimulus. In addition, other investigators have reported elevated CRH levels in hypophyseal portal blood in response to insulin-induced hypoglycemia (5). The relative roles of CRH and AVP in mediating the response to insulin-induced hypoglycemia remain uncertain. It has been suggested that the role of AVP becomes predominant with severe hypoglycemia (6). All of these studies have been complicated by the fact that anesthetized animals have been used. To overcome this problem and that of animal stress due to handling, a study was performed in which the effects of insulin-induced hypoglycemia on HPA axis gene expression and hormone levels were assessed in freely moving, unanesthetized rats. Materials
and Methods
Adult male Sprague-Dawley rats were acclimated in our animal facility for 7 daysbefore the insertion of external jugular venous catheters under sodium pentobarbital anesthesia(50 mg/kg, ip). They were then transferred to individual metabolic 920
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cagesin a controlled environment room, with lights on at 0700 h and off at 1900h, and lines were kept patent with an infusion of 150 mM NaCl at 1 ml/h for 3 days before the experimental day. Animals were fasted overnight and then .received insulin (4 U/kg) or saline iv in a volume of lessthan 0.3 ml via the central line. Between 0800-1000 h, animals were killed by decapitation 0,30,60, or 90 min after insulin or salineinjection, within 30 set of a sublethal doseof sodium pentobarbital (50 mg/kg) iv, also via the central line. Trunk blood was collected into sodium heparin-containing tubes for the measurementof CRH, AVP, corticosterone, and glucoseand into EDTA-containing tubes for the measurement of ACTH. In the first experiment, punches of the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei, anterior pituitaries, and a sampleof parietal cortex were removed and frozen at -70 C, as previously described (7, 8). With the punching method employed,the entire PVN, including the parvocellular portion, wasreproducibly obtained (8). In the secondexperiment, whole hypothalami were collected from animals killed 90 min after insulin or saline administration.
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Statistics Resultsare expressedasthe meanf SEM. One- and two-way analyses of variance (ANOVA) were used for comparison of means,as appropriate. The Dunnett t test was used for posthoc comparisonswhen necessary.Differences were considered significant when P < 0.05. Results
Exp 1 Hormone levek;. The results of hormone determinations during insulin-induced hypoglycemia are shown in Fig. 1. Plasma glucose fell from 106 f 5 mg/dl in the control animals (mean + SEM) to 38 f 2 mg/dl 30 min after insulin and remained low for the duration of the experiment. Plasma levels of ACTH, corticosterone, and AVP were lo-, 6-, and 4- higher, respectively, in insulin-
RIA Plasma ACTH and corticosterone were assayedby RIA, using kits from Nichols (San Juan Capistrano, CA) and Cambridge Diagnostics(Billerica, MA), respectively. AVP and CRH peptide were measuredby RIA, using methodswe have previously described(9). The sensitivity was 1 pg/ml for AVP and 10 pg/ml for CRH.
RNA isolation and blot hybridization Total RNA was isolated (10) from parietal cortex, anterior pituitary, whole hypothalami, and punchesof PVN asdescribed previously (7, 8), electrophoresedin 1.4% agarosecontaining 2.2 M formaldehyde, and blotted onto Genescreen(DuPont, Wilmington, DE). Hybridizations were performed using [a-“‘PI UTP-labeled antisense cRNA probes (11) at 65 C for 24 h. Riboprobe templates included the 700-basepair(bp) RsaI fragment of a rat CRH cDNA (8) encoding bases451-1838 of the rat CRH gene (12), the 350-bp fragment encoding bases21582500of the rat AVP gene(13,14), the mouse@actin cDNA (a generous gift from Dr. B. Spiegelman, Harvard Medical School), and a 900-bp fragment of the mousePOMC cDNA (15), all of which were subclonedinto the plasmid Bluescribe (Stratagene, La Jolla, CA). Plasmids were linearized, and labeled RNA was transcribed using either TB (CRH, AVP, and actin) or T7 (POMC) RNA polymerase (Stratagene). Blots were washed, as previously described (16), and exposed to Kodak X-AR film (Eastman Kodak, Rochester,NY) with intensifying screens.Autoradiograms and photographs of ethidium bromide-stained 18s ribosomalRNA on gelswere scanned with an LKB scanning densitometer (Rockville, MD). The amounts of CRH, AVP, and POMC mRNA were corrected for differences in recovery based on the amounts of both actin mRNA and 18s ribosomalRNA detected on the blots. mRNA levels were similar uisng either actin mRNA or 18s ribosomal RNA to correct for recovery and gel loading.
FIG. 1. Plasma hormone levels in response to insulin-induced cemia.
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treated us. control animals (by two-way ANOVA, P < 0.0001 in all cases), while plasma CRH was unchanged. The majority of animals remained asleep during the injection of insulin or saline and did not appear stressed at the time of death. POMC mRNA levels. During the period of hypoglycemia (30-90 min), POMC mRNA levels in anterior pituitary were 1.6fold higher in the insulin-treated group than in the controls (Figs. 2A and 3; by ANOVA, P < 0.025). There was no change in the size of POMC mRNA in the insulin-treated animals (Fig. 2A). CRH and AVP mRNA levels in PVN, SON, and cortex. CRH mRNA isolated from punches of PVN using skeletal muscle as a carrier tissue migrated as a broad-based band, approximately 1600 nucleotides long, just below 18s ribosomal RNA, which is 1874 nucleotides in length (17) (Fig. 2A, middle panel). In Fig. 2A, the concave nature of the top of the CRH mRNA band is probably due to a compression artifact caused by the slightly slower migrating 18s ribosomal RNA band. The appearance of CRH mRNA as a broad doublet may be due to its coisolation with skeletal muscle tissue, which contains large amounts of actin mRNA that comigrates with CRH
TIME
(mlnutcs)
FIG. 3. Quantitation of POMC, CRH, and AVP mRNA displayed in Fig. 2. 0, Controls; 0, insulin-treated animals. The levels of POMC, CRH, and AVP mRNA were corrected for the amount of either neuralspecific actin mRNA or 18s ribosomal RNA. The significance of POMC mRNA in insulin-treated us. control animals is indicated.
FIG. 2. Time course of the effect of insulin-induced hypoglycemia on HPA axis gene expression. A, Northern blot of anterior pituitary and PVN RNA isolated from animals killed 0 (immmediately), 30, 60, or 90 min after the injection of saline (C) or insulin (I). Blots were probed for POMC mRNA (top panel), CRH mRNA (middle panel), and AVP mRNA (lower panel). The arrow denotes the position of migration of l&S ribosomal RNA (1874 nucleotides). B, Northern blot of SON and cerebral cortex total RNA isolated from animals killed 0 (immediately), 30, 60, or 90 min after the injection of saline (C) or insulin (I). Blots were probed for AVP mRNA (upper panel) and CRH mRNA (lower panel).
mRNA (8) and could, thus, cause distortion of CRH mRNA into an apparent doublet. This explanation is supported by the fact that hypothalamic RNA isolated without added skeletal muscle contains a single broad CRH mRNA band lacking the appearance of a doublet (see below; Fig. 4A). It has been previously shown that CRH mRNA is not present in skeletal muscle (8). Thus, the CRH mRNA signal present in the PVN/skeletal muscle RNA preparations was derived from the PVN punch alone. Quantitation of these blots corrected for the amount of actin mRNA in each lane is shown in Fig. 3. There was no significant change in the amount of CRH mRNA in the
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increased from 87.3 + 13.7 to 512.6 + 22.6 pg/ml (P < 0.0001). There was no change in plasma CRH (control, 106.9 f 5.8 pg/ml; insulin-treated, 96.6 + 8.8 pg/ml) or AVP (control, 2.54 & 0.44 pg/ml; insulin-treated, 2.98 rt 0.22 pg/ml). The changes in plasma AVP levels were similar to those obtained in Exp 1, where there were no significant difference between the 0 and 90 min points (Fig. 1). Anterior pituitary POMC mRNA levels (Fig. 4) were significantly elevated in the insulin-treated us. control animals. CRH mRNA levels (corrected for actin mRNA) remained unchanged in this group of animals (Fig. 4). The levels of AVP mRNA in whole hypothalamus were unchanged by insulin-induced hypoglycemia (Fig. 4). The results of reprobing the same blot for actin mRNA are also shown in Fig. 4A, and quantitation of these blots with correction for actin mRNA is presented in Fig. 4B.
CICICI
POW
pit actin’
HT AVP
HT acti
Discussion
POMC
mRNA
CRH
mRNA
AVP
mRNA
4. Effect of insulin-induced hypoglycemia on HPA axis gene expression at 90 min. A, Northern blot of anterior pituitary and whole hypothalamic RNA isolated from animals killed 90 min after the injection of either saline (C) or insulin (I). The same pituitary blots were probed for POMC and actin mRNA, and the same hypothalamic (HT) blots were probed for CRH, AVP, and actin mRNA. B, Quantitation of anterior pituitary POMC mRNA and hypothalamic CRH and AVP mRNA from animals killed 90 min after the injection of either insulin or saline and corrected for the amount of actin mRNA. 0, Controls; W, insulin-treated animals. Results are expressed in arbitrary densitometric units. FIG.
PVN in response to hypoglycemia. In addition, CRH mRNA was isolated from parietal cortex, and its level was unchanged by hypoglycemia (Figs. 2B and 3). The level of AVP mRNA in punches of PVN and SON was also unchanged by hypoglycemia (Figs. 2, A and B, and 3). It is of interest that although total hypothalamic AVP mRNA content and size remained unchanged, peripheral plasma AVP levels were increased. Exp 2 Because of the possibility of inadequate sampling with the PVN-punching technique, 18 additional animals (9 controls and 9 insulin treated) were killed 90 min after the injection of insulin or saline and RNA prepared from whole hypothalami. Changes in plasma glucose and hormone levels in the control us. insulin-treated animals were similar to those in the initial study. Plasma glucose fell from 91.4 f 5.7 to 42.9 f 1.2 mg/dl with insulin treatment (P < O.OOOl), while plasma corticosterone increased from 10.6 f 2.6 to 57.2 + 2.9 pg/dl (P < 0.0001). Plasma ACTH levels
Previous studies investigating the effects of insulininduced hypoglycemia on the HPA axis have been complicated by the use of anesthetized animals. We have developed a model to study the effects of insulin-induced hypoglycemia on the activation of gene expression in the HPA axis in freely moving, unanesthetized rats. In this model, animals were given all treatments iv and remained undisturbed by handling. The experiments were all performed between 0800-1000 h, when rats are normally inactive. The majority of animals remained asleep during the injection of insulin or saline and did not appear stressed at the time of death. Our results demonstrate that the peripheral plasma levels of corticosterone, ACTH, and AVP all rose markedly after the induction of hypoglycemia. The rise in peripheral plasma AVP is in agreement with the study of Baylis and Robertson (2) and is consistent with the results of Plotsky et al. (l), who showed elevated levels of AVP in hypophyseal portal blood in response to hypoglycemia. It is thought that the peripheral plasma AVP is derived predominantly from the posterior pituitary gland; the magnitude of the component derived from the median eminence is unknown, although magnocellular neurons have been found to terminate in this area (18). The elevation in plasma ACTH levels is in agreement with previous reports (4). POMC mRNA levels were also increased by hypoglycemia to a level similar to that found by previous workers (4) and were associated with increased release of ACTH peptide into the circulation. POMC mRNA levels appeared to peak at 90 min, at which time all of these animals remained hypoglycemic. In contrast to the changes in POMC mRNA, the levels of both CRH and AVP mRNA did not change in either
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the PVN punches (at all time points) or the whole hypothalamus at the 90 min point. The consistent results obtained with these two methods suggests, as we have previously shown (8), that the entire PVN, including the parvocellular division, was present in the PVN punches, and that our inability to detect hypoglycemiacassociated changes in PVN CRH mRNA was not likely to be due to the incomplet: pollection of parvocellular PVN neurons. We have also previously shown, using in situ histohybridization (19), that the area included in the PVN punches does not include any CRH neurons other than those within the PVN. Thus, the PVN was the source of the CRH mRNA signal in these samples. PVN CRH peptide and mRNA have been shown to increase in response to adrenalectomy in previous immunocytochemical (20), in situ hybridization (21), and punch (8) studies. CRH in the parvocellular PVN thus clearly has a role in modulation of the HPA axis after adrenalectomy. The finding of no change in the level of CRH mRNA in response to hypoglycemia is in contrast to a previous report (3) which showed that CRH mRNA isolated from whole hypothalamus did increase with hypoglycemia in anesthetized Wistar rats. Comparable corticosterone and ACTH levels were achieved in both studies. However, in our study animals remained hypoglycemic for the duration of the experiment, providing a more prolonged stimulus than in the study of Suda et al. (3). Our experimental design also differed from that of Suda et al., in that our animals were unanesthetized Sprague-Dawley rats who were freely moving during the experiment and killed between 0800-1000 h, whereas those of Suda et al. were killed between 1200-1300 h. A further difference between the present study and that of Suda et al. (3) is that we assessedneuropeptide mRNA levels in samples of total RNA, as opposed to poly(A)selected mRNA (3). A potential error in quantitation using total RNA could be introduced if a change in the amount of rRNA relative to specific mRNAs was caused by the insulin-induced hypoglycemia. To avoid this potential problem, we measured changes in CRH, AVP, and POMC mRNAs relative to those of both actin mRNA and rRNA, and similar results were obtained with both methods. Since AVP has been shown to augment the ACTH response to CRH (22, 23), to increase in hypophyseal portal blood in response to hypoglycemia (l), and possibly to mediate the ACTH response to short term stress (18), the level of AVP mRNA in the PVN and whole hypothalamus was measured in an attempt to determine whether AVP synthesized in those sites contributed to the increase in POMC mRNA and activation of the HPA axis. PVN punches allowed separation of the AVP-containing neurons in the PVN from those previously identified in the SON and suprachiasmatic nucleus. However, the PVN contains both vasopressinergic magnocellular
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neurons, which project to the posterior pituitary, and parvocellular neurons, which project to the median eminence and release AVP into the hypophyseal portal circulation. Using a hyponatremic rat model, Dohanics et al. (24) suggested that magnocellular AVP does not play a role in mediating the ACTH response to insulin-induced hypoglycemia. Support for the role of parvocellular PVN AVP in mediating stress-induced ACTH release comes from the studies of Whitnall (18), which describe selective release of parvocellular AVP after insulin-induced hypoglycemia. Analysis of currently available data suggests that the parvocellular neurons express less than 0.5% of the total AVP in the PVN (14). Thus, only a very marked increase in the levels of parvocellular AVP mRNA would have been detected using the punching technique. Our finding of no change in PVN AVP mRNA should, therefore, be viewed in this light. We have previously shown that activation of vasopressin gene expression by osmotic stimulation is associated with an increase in both the content and poly(A) tail length of hypothalamic AVP mRNA (14). In the present study the lack of any such change in vasopressin mRNA size after insulin-induced hypoglycemia is consistent with a lack of activation of this gene in this setting. Given that the vast majority of AVP mRNA in these PVN punches is derived from magnocellular neurons, further studies will be required to determine if parvocellular PVN AVP gene expression is activated during stimulation of the HPA axis. This study, thus, found no evidence for PVN or cerebral cortex CRH gene activation during hypoglycemiaassociated stimulation of the HPA axis in unanesthetized animals. This suggests that other factors, such as catecholamines (25), may mediate the observed increase in POMC gene expression and the increase in ACTH and cortisol levels. We cannot exclude the possibility that CRH may play a permissive role in activation of the HPA axis in response to this stimulus, as has been previously suggested (26, 27), or that insulin-induced hypoglycemia stimulates the release of CRH peptide (5, 28, 29) without causing alterations in the level of CRH mRNA. While no change in whole PVN vasopressin mRNA levels was observed, further studies will be required to determine if parvocellular PVN vasopressin gene expression is activated during stimulation of the HPA axis in the rat. Acknowledgment We thank Ms. Ortencia Cortez for performing T. Suda for the CRH antiserum.
the RIAs, and
References 1. Plotsky PM, Bruhn TO, Vale W 1985 Hypophysiotropic regulation of adrenocorticotropin secretion in response to insulin-induced hypoglycemia. Endocrinology 117:323-329
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2. Baylis PH, Robertson GL 1980 Rat vasopressin response to insulininduced hypoglycemia. Endocrinology 107:1975-1979 3. Suda T, Tozawa F, Yamada M, Ushiyama T, Tomori N, Sumitomo T, Nakagami Y, Demura H, Shizume K 1988 Insulin-induced hypoglycemia increases corticotropin-releasing factor messenger ribonucleic acid levels in rat hypothalamus. Endocrinology 123:1371-1375 4. Tozawa F, Suda T, Yamada M, Ushiyama T, Tomori N, Sumitomo T. Nakaeami Y. Demura H. Shizume K 1988 Insulin-induced hypoglycemia increases proopiomelanocortin messenger ribonucleic acid levels in rat anterior pituitary gland. Endocrinology 122:1231-1235 5. Guillaume V, Grino M, Conte-Devolx B, Boudouresque F, Oliver C 1989 Corticotropin-releasing factor secretion increases in rat hypophysical portal blood during insulin-induced hypoglycemia. Neuroendocrinology 49:676-679 6. Caraty A, Grino M, Locatelli A, Guillaume V, Boudouresque F, Conte-Devolx B, Oliver C 1990 Insulin-induced hypoglycemia stimulates corticotropin-releasing factor and arginine vasopressin secretion into hypophysical portal blood of conscious, unrestrained rams. J Clin Invest 85:1716-1721 7. Robinson BG, Frim DM, Schwartz WJ, Majzoub JA 1988 Vasopressin mRNA in the suprachiasmatic nuclei: daily regulation of polyadenylate tail length. Science 241:342-344 8. Frim DM, Robinson BG, Pasieka KB, Majzoub JA 1990 Differential regulation of corticotropin releasing hormone mRNA in the hypothalamic paraventricular nucleus and cerebral cortex of the rat. Am J Physiol 258:E686-E692 9. Michie HR, Majzoub JA, O’Dwyer ST, Revhaug A, Wilmore DW 1990 Both cyclooxygenase-dependent and cyclooxygenase-independent pathways mediate the neuroendocrine response in humans. Surgery 108:254-261 10. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 185294-5299 11. Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR 1984 Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 12:7035-7056 12. Thompson RC, Seasholtz AF, Douglass JO, Herbert E 1987 The rat corticotropin-releasing hormone gene. Ann NY Acad Sci 512:111 13. Ivell R, Richter D 1984 Structure and comparison of the oxytocin and vasopressin genes from rat. Proc Nat1 Acad Sci USA 81:20062010 14. Carrazana DJ, Pasieka KB, Majzoub JA 1988 The vasopressin mRNA poly(A) tract is unusually long and increases during stimulation of vasopressin gene expression in uiuo. Mol Cell Biol 8~2267-2274 15. Roberts JL, Budarf ML, Baxter JD, Herbert E 1979 Selective reduction of proadrenocorticotropin/endorphin proteins and mes-
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18. 19.
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25.
26. 27. 28.
29.
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senger ribonucleic acid activity in mouse pituitary tumor cells by glucocorticoids. Biochemistry 184907-4915 Adler GK, Smas CM, Majzoub JA 1988 Expression and dexamethasone regulation of the human corticotropin-releasing hormone gene in a mouse anterior pituitary cell line. J Biol Chem 263:58465852 Chan YL, Gutell R, Roller HF, Wool IG 1984 The nucleotide sequence of a rat 18s ribosomal ribonucleic acid gene and a proposal for the secondary structure of 18s ribosomal ribonucleic acid. J Biol Chem 259:224-230 Whitnall MH 1989 Stress selectively activates the vasonressincontaining subset of corticotropin-releasing hormone neurons. Neuroendocrinolorn 50:702-707 Frim D, Majzoub TA, Localization of corticotropin-releasing hormone mRNA in the brain of the rat by in situ hybridization. 16th Annual Meeting of the Society for Neuroscience, Washington, DC, 1986, p 1388 Sawchenko PE 1987 Adrenalectomy-induced enhancement of CRF and vasopressin immunoreactivity in parvocellular neurosecretory neurons: anatomic, peptide, and steroid specificity. J Neurosci 7:1093-1106 Young WS 3d, Mezey E, Siegel RE 1986 Quantitative in situ hybridization histochemistry reveals increased levels of corticotropin-releasing factor mRNA after adrenalectomy in rats. Neurosci Lett 70:198-203 Gillies G, Lowry P 1979 Corticotrophin releasing factor may be modulated vasopressin. Nature 278:463-464 Rivier C, Vale W 1983 Interaction of corticotropin-releasing factor and arginine vasopressin on adrenocorticotropin secretion in uiuo. Endocrinology 113:939-942 Dohanics J, Hoffman GE, Verbalis JG 1991 Hyponatremia-induced inhibition of magnocellular neurons causes stressor-selective impairment of stimulated adrenocorticotropin secretion in rats. Endocrinology 128:331-340 Tomori N, Suda T, Nakagami Y, Tozawa F, Sumitomo T, Ushiyama T, Demura H, Shizume K 1989 Adrenergic modulation of adrenocorticotropin responses to insulin-induced hypoglycemia and corticotropin-releasing hormone. J Clin Endocrinol Metab 68:87-93 Dallman MF, Akana SF, Cascio CS, Darlington DN, Jacobson L, Levin N 1987 Regulation of ACTH secretion: variations on a theme of B. Recent Prog Horm Res 43:113-173 Plotsky PM 1987 Regulation of hypophysiotropic factors mediating ACTH secretion. Ann NY Acad Sci 512:205-217 Berkenbosch F, De Goeij DC, Tilders FJ 1989 Hypoglycemia enhances turnover of corticotropin-releasing factor and of vasopressin in the zona externa of the rat median eminence. Endocrinology 125:28-34 Widmaier EP, Plotsky PM, Sutton SW, Vale WW 1988 Regulation of corticotropin-releasing factor secretion in vitro by glucose. Am J Physiol 255:287-292
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Vol. 130, No. 2 Printed in U.S.A.
Society
The Effect of Insulin-Induced Hypoglycemia Expression in the Hypothalamic-Pituitary-Adrenal of the Rat* BRUCE JOSEPH
G. ROBINSONt, A. MAJZOUB
KENNETH
MEALY$,
DOUGLAS
W. WILMORE,
on Gene Axis AND
Division of Endocrinology, Department of Medicine, Children’s Hospital (B.G.R., J.A.M.), and Surgical Nutrition Laboratory, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School (K.M., D. W. W.), Boston, Massachusetts 02115
ABSTRACT. This study has examined the effects of insulininduced hypoglycemia on expression of the CRH, arginine vasopressin, and POMC genes and corresponding peptides in freely moving, unanesthetized, male Sprague-Dawley rats. Animals were infused with 150 mM NaCl for 3 days before the experimental day and were then administered insulin (4 U/kg) or saline iv. In one experiment animals were killed 0, 30, 60, or 90 min after insulin or saline, and RNA was isolated from anterior pituitary, cerebral cortex, and punches of the hypothalamic paraventricular and supraoptic nuclei. In a second experiment, animals were killed 90 min after insulin or saline treatment, and RNA was isolated from whole hypothalami. RNA was analyzed by Northern blot. Plasma glucose fell from 106 * 5 to 38 + 2 mg/dl after insulin administration and remained low for the duration of the experiment. Plasma levels of ACTH, corticoster-
C
RH AND arginine vasopressin (AVP) are considered to be the principal stimulators of ACTH release after stress. Insulin-induced hypoglycemia is a potent physiological stimulus for the release of ACTH. It is unclear, however, the extent to which either CRH or AVP plays a role in this response. There have been several previous studies of the effects of insulin-induced hypoglycemia on activation of the hypothalamic-pituitary-adrenal (HPA) axis of the rat. Plotsky et al. (1) measured hypophyseal portal concentrations of CRH and AVP in hypoglycemic rats and found that AVP increased 1.9-fold, while CRH remained unchanged. Insulin-induced hypoglycemia has also been associated with elevated peripheral levels of plasma AVP (2), and recently, Received August 2, 1991. Address all correspondence and requests for reprints to: Dr. Joseph A. Majzoub, Endocrine Division, Children’s Hospital, 300 Longwood Avenue, Boston, Massachusetts 02115. * This work was supported by the Postgraduate Medical Foundation, University of Sydney, and NIH Grant ROl-DK-40170. t Present address: Molecular Genetics Unit, Kolling Institute, Royal North Shore Hospital, St. Leonard 2065, Australia. $ Present address: St. Vincent’s Hospital, Dublin, Ireland.
one, and vasopressin were lo-, 6-, and 4-fold higher, respectively, in the insulin-treated us. control animals (by analysis of variance, P < 0.0001 in all cases), while plasma CRH was unchanged. During hypoglycemia POMC mRNA levels were l&fold higher in the insulin-treated group (by analysis of variance, P < 0.025). In contrast, paraventricular nucleus, whole hypothalamic, and parietal cortex CRH mRNA and vasopressin mRNA were unchanged. These data support previous studies which indicated that POMC gene expression is increased by hypoglycemia. However, we found no evidence for an increase in paraventricular nucleus or cerebral cortex CRH mRNA expression during hypoglycemia-associated stimulation of the hypothalamic-pituitary-adrenal axis, suggesting that another factor(s) may mediate the observed increase in POMC gene expression. (Endocrinology 130: 920-925,1992)
the levels of peripheral plasma CRH, CRH mRNA (3), and POMC mRNA (4) have been reported to be elevated in rats after this stimulus. In addition, other investigators have reported elevated CRH levels in hypophyseal portal blood in response to insulin-induced hypoglycemia (5). The relative roles of CRH and AVP in mediating the response to insulin-induced hypoglycemia remain uncertain. It has been suggested that the role of AVP becomes predominant with severe hypoglycemia (6). All of these studies have been complicated by the fact that anesthetized animals have been used. To overcome this problem and that of animal stress due to handling, a study was performed in which the effects of insulin-induced hypoglycemia on HPA axis gene expression and hormone levels were assessed in freely moving, unanesthetized rats. Materials
and Methods
Adult male Sprague-Dawley rats were acclimated in our animal facility for 7 daysbefore the insertion of external jugular venous catheters under sodium pentobarbital anesthesia(50 mg/kg, ip). They were then transferred to individual metabolic 920
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cagesin a controlled environment room, with lights on at 0700 h and off at 1900h, and lines were kept patent with an infusion of 150 mM NaCl at 1 ml/h for 3 days before the experimental day. Animals were fasted overnight and then .received insulin (4 U/kg) or saline iv in a volume of lessthan 0.3 ml via the central line. Between 0800-1000 h, animals were killed by decapitation 0,30,60, or 90 min after insulin or salineinjection, within 30 set of a sublethal doseof sodium pentobarbital (50 mg/kg) iv, also via the central line. Trunk blood was collected into sodium heparin-containing tubes for the measurementof CRH, AVP, corticosterone, and glucoseand into EDTA-containing tubes for the measurement of ACTH. In the first experiment, punches of the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei, anterior pituitaries, and a sampleof parietal cortex were removed and frozen at -70 C, as previously described (7, 8). With the punching method employed,the entire PVN, including the parvocellular portion, wasreproducibly obtained (8). In the secondexperiment, whole hypothalami were collected from animals killed 90 min after insulin or saline administration.
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Statistics Resultsare expressedasthe meanf SEM. One- and two-way analyses of variance (ANOVA) were used for comparison of means,as appropriate. The Dunnett t test was used for posthoc comparisonswhen necessary.Differences were considered significant when P < 0.05. Results
Exp 1 Hormone levek;. The results of hormone determinations during insulin-induced hypoglycemia are shown in Fig. 1. Plasma glucose fell from 106 f 5 mg/dl in the control animals (mean + SEM) to 38 f 2 mg/dl 30 min after insulin and remained low for the duration of the experiment. Plasma levels of ACTH, corticosterone, and AVP were lo-, 6-, and 4- higher, respectively, in insulin-
RIA Plasma ACTH and corticosterone were assayedby RIA, using kits from Nichols (San Juan Capistrano, CA) and Cambridge Diagnostics(Billerica, MA), respectively. AVP and CRH peptide were measuredby RIA, using methodswe have previously described(9). The sensitivity was 1 pg/ml for AVP and 10 pg/ml for CRH.
RNA isolation and blot hybridization Total RNA was isolated (10) from parietal cortex, anterior pituitary, whole hypothalami, and punchesof PVN asdescribed previously (7, 8), electrophoresedin 1.4% agarosecontaining 2.2 M formaldehyde, and blotted onto Genescreen(DuPont, Wilmington, DE). Hybridizations were performed using [a-“‘PI UTP-labeled antisense cRNA probes (11) at 65 C for 24 h. Riboprobe templates included the 700-basepair(bp) RsaI fragment of a rat CRH cDNA (8) encoding bases451-1838 of the rat CRH gene (12), the 350-bp fragment encoding bases21582500of the rat AVP gene(13,14), the mouse@actin cDNA (a generous gift from Dr. B. Spiegelman, Harvard Medical School), and a 900-bp fragment of the mousePOMC cDNA (15), all of which were subclonedinto the plasmid Bluescribe (Stratagene, La Jolla, CA). Plasmids were linearized, and labeled RNA was transcribed using either TB (CRH, AVP, and actin) or T7 (POMC) RNA polymerase (Stratagene). Blots were washed, as previously described (16), and exposed to Kodak X-AR film (Eastman Kodak, Rochester,NY) with intensifying screens.Autoradiograms and photographs of ethidium bromide-stained 18s ribosomalRNA on gelswere scanned with an LKB scanning densitometer (Rockville, MD). The amounts of CRH, AVP, and POMC mRNA were corrected for differences in recovery based on the amounts of both actin mRNA and 18s ribosomalRNA detected on the blots. mRNA levels were similar uisng either actin mRNA or 18s ribosomal RNA to correct for recovery and gel loading.
FIG. 1. Plasma hormone levels in response to insulin-induced cemia.
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treated us. control animals (by two-way ANOVA, P < 0.0001 in all cases), while plasma CRH was unchanged. The majority of animals remained asleep during the injection of insulin or saline and did not appear stressed at the time of death. POMC mRNA levels. During the period of hypoglycemia (30-90 min), POMC mRNA levels in anterior pituitary were 1.6fold higher in the insulin-treated group than in the controls (Figs. 2A and 3; by ANOVA, P < 0.025). There was no change in the size of POMC mRNA in the insulin-treated animals (Fig. 2A). CRH and AVP mRNA levels in PVN, SON, and cortex. CRH mRNA isolated from punches of PVN using skeletal muscle as a carrier tissue migrated as a broad-based band, approximately 1600 nucleotides long, just below 18s ribosomal RNA, which is 1874 nucleotides in length (17) (Fig. 2A, middle panel). In Fig. 2A, the concave nature of the top of the CRH mRNA band is probably due to a compression artifact caused by the slightly slower migrating 18s ribosomal RNA band. The appearance of CRH mRNA as a broad doublet may be due to its coisolation with skeletal muscle tissue, which contains large amounts of actin mRNA that comigrates with CRH
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FIG. 3. Quantitation of POMC, CRH, and AVP mRNA displayed in Fig. 2. 0, Controls; 0, insulin-treated animals. The levels of POMC, CRH, and AVP mRNA were corrected for the amount of either neuralspecific actin mRNA or 18s ribosomal RNA. The significance of POMC mRNA in insulin-treated us. control animals is indicated.
FIG. 2. Time course of the effect of insulin-induced hypoglycemia on HPA axis gene expression. A, Northern blot of anterior pituitary and PVN RNA isolated from animals killed 0 (immmediately), 30, 60, or 90 min after the injection of saline (C) or insulin (I). Blots were probed for POMC mRNA (top panel), CRH mRNA (middle panel), and AVP mRNA (lower panel). The arrow denotes the position of migration of l&S ribosomal RNA (1874 nucleotides). B, Northern blot of SON and cerebral cortex total RNA isolated from animals killed 0 (immediately), 30, 60, or 90 min after the injection of saline (C) or insulin (I). Blots were probed for AVP mRNA (upper panel) and CRH mRNA (lower panel).
mRNA (8) and could, thus, cause distortion of CRH mRNA into an apparent doublet. This explanation is supported by the fact that hypothalamic RNA isolated without added skeletal muscle contains a single broad CRH mRNA band lacking the appearance of a doublet (see below; Fig. 4A). It has been previously shown that CRH mRNA is not present in skeletal muscle (8). Thus, the CRH mRNA signal present in the PVN/skeletal muscle RNA preparations was derived from the PVN punch alone. Quantitation of these blots corrected for the amount of actin mRNA in each lane is shown in Fig. 3. There was no significant change in the amount of CRH mRNA in the
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increased from 87.3 + 13.7 to 512.6 + 22.6 pg/ml (P < 0.0001). There was no change in plasma CRH (control, 106.9 f 5.8 pg/ml; insulin-treated, 96.6 + 8.8 pg/ml) or AVP (control, 2.54 & 0.44 pg/ml; insulin-treated, 2.98 rt 0.22 pg/ml). The changes in plasma AVP levels were similar to those obtained in Exp 1, where there were no significant difference between the 0 and 90 min points (Fig. 1). Anterior pituitary POMC mRNA levels (Fig. 4) were significantly elevated in the insulin-treated us. control animals. CRH mRNA levels (corrected for actin mRNA) remained unchanged in this group of animals (Fig. 4). The levels of AVP mRNA in whole hypothalamus were unchanged by insulin-induced hypoglycemia (Fig. 4). The results of reprobing the same blot for actin mRNA are also shown in Fig. 4A, and quantitation of these blots with correction for actin mRNA is presented in Fig. 4B.
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4. Effect of insulin-induced hypoglycemia on HPA axis gene expression at 90 min. A, Northern blot of anterior pituitary and whole hypothalamic RNA isolated from animals killed 90 min after the injection of either saline (C) or insulin (I). The same pituitary blots were probed for POMC and actin mRNA, and the same hypothalamic (HT) blots were probed for CRH, AVP, and actin mRNA. B, Quantitation of anterior pituitary POMC mRNA and hypothalamic CRH and AVP mRNA from animals killed 90 min after the injection of either insulin or saline and corrected for the amount of actin mRNA. 0, Controls; W, insulin-treated animals. Results are expressed in arbitrary densitometric units. FIG.
PVN in response to hypoglycemia. In addition, CRH mRNA was isolated from parietal cortex, and its level was unchanged by hypoglycemia (Figs. 2B and 3). The level of AVP mRNA in punches of PVN and SON was also unchanged by hypoglycemia (Figs. 2, A and B, and 3). It is of interest that although total hypothalamic AVP mRNA content and size remained unchanged, peripheral plasma AVP levels were increased. Exp 2 Because of the possibility of inadequate sampling with the PVN-punching technique, 18 additional animals (9 controls and 9 insulin treated) were killed 90 min after the injection of insulin or saline and RNA prepared from whole hypothalami. Changes in plasma glucose and hormone levels in the control us. insulin-treated animals were similar to those in the initial study. Plasma glucose fell from 91.4 f 5.7 to 42.9 f 1.2 mg/dl with insulin treatment (P < O.OOOl), while plasma corticosterone increased from 10.6 f 2.6 to 57.2 + 2.9 pg/dl (P < 0.0001). Plasma ACTH levels
Previous studies investigating the effects of insulininduced hypoglycemia on the HPA axis have been complicated by the use of anesthetized animals. We have developed a model to study the effects of insulin-induced hypoglycemia on the activation of gene expression in the HPA axis in freely moving, unanesthetized rats. In this model, animals were given all treatments iv and remained undisturbed by handling. The experiments were all performed between 0800-1000 h, when rats are normally inactive. The majority of animals remained asleep during the injection of insulin or saline and did not appear stressed at the time of death. Our results demonstrate that the peripheral plasma levels of corticosterone, ACTH, and AVP all rose markedly after the induction of hypoglycemia. The rise in peripheral plasma AVP is in agreement with the study of Baylis and Robertson (2) and is consistent with the results of Plotsky et al. (l), who showed elevated levels of AVP in hypophyseal portal blood in response to hypoglycemia. It is thought that the peripheral plasma AVP is derived predominantly from the posterior pituitary gland; the magnitude of the component derived from the median eminence is unknown, although magnocellular neurons have been found to terminate in this area (18). The elevation in plasma ACTH levels is in agreement with previous reports (4). POMC mRNA levels were also increased by hypoglycemia to a level similar to that found by previous workers (4) and were associated with increased release of ACTH peptide into the circulation. POMC mRNA levels appeared to peak at 90 min, at which time all of these animals remained hypoglycemic. In contrast to the changes in POMC mRNA, the levels of both CRH and AVP mRNA did not change in either
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the PVN punches (at all time points) or the whole hypothalamus at the 90 min point. The consistent results obtained with these two methods suggests, as we have previously shown (8), that the entire PVN, including the parvocellular division, was present in the PVN punches, and that our inability to detect hypoglycemiacassociated changes in PVN CRH mRNA was not likely to be due to the incomplet: pollection of parvocellular PVN neurons. We have also previously shown, using in situ histohybridization (19), that the area included in the PVN punches does not include any CRH neurons other than those within the PVN. Thus, the PVN was the source of the CRH mRNA signal in these samples. PVN CRH peptide and mRNA have been shown to increase in response to adrenalectomy in previous immunocytochemical (20), in situ hybridization (21), and punch (8) studies. CRH in the parvocellular PVN thus clearly has a role in modulation of the HPA axis after adrenalectomy. The finding of no change in the level of CRH mRNA in response to hypoglycemia is in contrast to a previous report (3) which showed that CRH mRNA isolated from whole hypothalamus did increase with hypoglycemia in anesthetized Wistar rats. Comparable corticosterone and ACTH levels were achieved in both studies. However, in our study animals remained hypoglycemic for the duration of the experiment, providing a more prolonged stimulus than in the study of Suda et al. (3). Our experimental design also differed from that of Suda et al., in that our animals were unanesthetized Sprague-Dawley rats who were freely moving during the experiment and killed between 0800-1000 h, whereas those of Suda et al. were killed between 1200-1300 h. A further difference between the present study and that of Suda et al. (3) is that we assessedneuropeptide mRNA levels in samples of total RNA, as opposed to poly(A)selected mRNA (3). A potential error in quantitation using total RNA could be introduced if a change in the amount of rRNA relative to specific mRNAs was caused by the insulin-induced hypoglycemia. To avoid this potential problem, we measured changes in CRH, AVP, and POMC mRNAs relative to those of both actin mRNA and rRNA, and similar results were obtained with both methods. Since AVP has been shown to augment the ACTH response to CRH (22, 23), to increase in hypophyseal portal blood in response to hypoglycemia (l), and possibly to mediate the ACTH response to short term stress (18), the level of AVP mRNA in the PVN and whole hypothalamus was measured in an attempt to determine whether AVP synthesized in those sites contributed to the increase in POMC mRNA and activation of the HPA axis. PVN punches allowed separation of the AVP-containing neurons in the PVN from those previously identified in the SON and suprachiasmatic nucleus. However, the PVN contains both vasopressinergic magnocellular
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neurons, which project to the posterior pituitary, and parvocellular neurons, which project to the median eminence and release AVP into the hypophyseal portal circulation. Using a hyponatremic rat model, Dohanics et al. (24) suggested that magnocellular AVP does not play a role in mediating the ACTH response to insulin-induced hypoglycemia. Support for the role of parvocellular PVN AVP in mediating stress-induced ACTH release comes from the studies of Whitnall (18), which describe selective release of parvocellular AVP after insulin-induced hypoglycemia. Analysis of currently available data suggests that the parvocellular neurons express less than 0.5% of the total AVP in the PVN (14). Thus, only a very marked increase in the levels of parvocellular AVP mRNA would have been detected using the punching technique. Our finding of no change in PVN AVP mRNA should, therefore, be viewed in this light. We have previously shown that activation of vasopressin gene expression by osmotic stimulation is associated with an increase in both the content and poly(A) tail length of hypothalamic AVP mRNA (14). In the present study the lack of any such change in vasopressin mRNA size after insulin-induced hypoglycemia is consistent with a lack of activation of this gene in this setting. Given that the vast majority of AVP mRNA in these PVN punches is derived from magnocellular neurons, further studies will be required to determine if parvocellular PVN AVP gene expression is activated during stimulation of the HPA axis. This study, thus, found no evidence for PVN or cerebral cortex CRH gene activation during hypoglycemiaassociated stimulation of the HPA axis in unanesthetized animals. This suggests that other factors, such as catecholamines (25), may mediate the observed increase in POMC gene expression and the increase in ACTH and cortisol levels. We cannot exclude the possibility that CRH may play a permissive role in activation of the HPA axis in response to this stimulus, as has been previously suggested (26, 27), or that insulin-induced hypoglycemia stimulates the release of CRH peptide (5, 28, 29) without causing alterations in the level of CRH mRNA. While no change in whole PVN vasopressin mRNA levels was observed, further studies will be required to determine if parvocellular PVN vasopressin gene expression is activated during stimulation of the HPA axis in the rat. Acknowledgment We thank Ms. Ortencia Cortez for performing T. Suda for the CRH antiserum.
the RIAs, and
References 1. Plotsky PM, Bruhn TO, Vale W 1985 Hypophysiotropic regulation of adrenocorticotropin secretion in response to insulin-induced hypoglycemia. Endocrinology 117:323-329
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2. Baylis PH, Robertson GL 1980 Rat vasopressin response to insulininduced hypoglycemia. Endocrinology 107:1975-1979 3. Suda T, Tozawa F, Yamada M, Ushiyama T, Tomori N, Sumitomo T, Nakagami Y, Demura H, Shizume K 1988 Insulin-induced hypoglycemia increases corticotropin-releasing factor messenger ribonucleic acid levels in rat hypothalamus. Endocrinology 123:1371-1375 4. Tozawa F, Suda T, Yamada M, Ushiyama T, Tomori N, Sumitomo T. Nakaeami Y. Demura H. Shizume K 1988 Insulin-induced hypoglycemia increases proopiomelanocortin messenger ribonucleic acid levels in rat anterior pituitary gland. Endocrinology 122:1231-1235 5. Guillaume V, Grino M, Conte-Devolx B, Boudouresque F, Oliver C 1989 Corticotropin-releasing factor secretion increases in rat hypophysical portal blood during insulin-induced hypoglycemia. Neuroendocrinology 49:676-679 6. Caraty A, Grino M, Locatelli A, Guillaume V, Boudouresque F, Conte-Devolx B, Oliver C 1990 Insulin-induced hypoglycemia stimulates corticotropin-releasing factor and arginine vasopressin secretion into hypophysical portal blood of conscious, unrestrained rams. J Clin Invest 85:1716-1721 7. Robinson BG, Frim DM, Schwartz WJ, Majzoub JA 1988 Vasopressin mRNA in the suprachiasmatic nuclei: daily regulation of polyadenylate tail length. Science 241:342-344 8. Frim DM, Robinson BG, Pasieka KB, Majzoub JA 1990 Differential regulation of corticotropin releasing hormone mRNA in the hypothalamic paraventricular nucleus and cerebral cortex of the rat. Am J Physiol 258:E686-E692 9. Michie HR, Majzoub JA, O’Dwyer ST, Revhaug A, Wilmore DW 1990 Both cyclooxygenase-dependent and cyclooxygenase-independent pathways mediate the neuroendocrine response in humans. Surgery 108:254-261 10. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 185294-5299 11. Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR 1984 Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 12:7035-7056 12. Thompson RC, Seasholtz AF, Douglass JO, Herbert E 1987 The rat corticotropin-releasing hormone gene. Ann NY Acad Sci 512:111 13. Ivell R, Richter D 1984 Structure and comparison of the oxytocin and vasopressin genes from rat. Proc Nat1 Acad Sci USA 81:20062010 14. Carrazana DJ, Pasieka KB, Majzoub JA 1988 The vasopressin mRNA poly(A) tract is unusually long and increases during stimulation of vasopressin gene expression in uiuo. Mol Cell Biol 8~2267-2274 15. Roberts JL, Budarf ML, Baxter JD, Herbert E 1979 Selective reduction of proadrenocorticotropin/endorphin proteins and mes-
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senger ribonucleic acid activity in mouse pituitary tumor cells by glucocorticoids. Biochemistry 184907-4915 Adler GK, Smas CM, Majzoub JA 1988 Expression and dexamethasone regulation of the human corticotropin-releasing hormone gene in a mouse anterior pituitary cell line. J Biol Chem 263:58465852 Chan YL, Gutell R, Roller HF, Wool IG 1984 The nucleotide sequence of a rat 18s ribosomal ribonucleic acid gene and a proposal for the secondary structure of 18s ribosomal ribonucleic acid. J Biol Chem 259:224-230 Whitnall MH 1989 Stress selectively activates the vasonressincontaining subset of corticotropin-releasing hormone neurons. Neuroendocrinolorn 50:702-707 Frim D, Majzoub TA, Localization of corticotropin-releasing hormone mRNA in the brain of the rat by in situ hybridization. 16th Annual Meeting of the Society for Neuroscience, Washington, DC, 1986, p 1388 Sawchenko PE 1987 Adrenalectomy-induced enhancement of CRF and vasopressin immunoreactivity in parvocellular neurosecretory neurons: anatomic, peptide, and steroid specificity. J Neurosci 7:1093-1106 Young WS 3d, Mezey E, Siegel RE 1986 Quantitative in situ hybridization histochemistry reveals increased levels of corticotropin-releasing factor mRNA after adrenalectomy in rats. Neurosci Lett 70:198-203 Gillies G, Lowry P 1979 Corticotrophin releasing factor may be modulated vasopressin. Nature 278:463-464 Rivier C, Vale W 1983 Interaction of corticotropin-releasing factor and arginine vasopressin on adrenocorticotropin secretion in uiuo. Endocrinology 113:939-942 Dohanics J, Hoffman GE, Verbalis JG 1991 Hyponatremia-induced inhibition of magnocellular neurons causes stressor-selective impairment of stimulated adrenocorticotropin secretion in rats. Endocrinology 128:331-340 Tomori N, Suda T, Nakagami Y, Tozawa F, Sumitomo T, Ushiyama T, Demura H, Shizume K 1989 Adrenergic modulation of adrenocorticotropin responses to insulin-induced hypoglycemia and corticotropin-releasing hormone. J Clin Endocrinol Metab 68:87-93 Dallman MF, Akana SF, Cascio CS, Darlington DN, Jacobson L, Levin N 1987 Regulation of ACTH secretion: variations on a theme of B. Recent Prog Horm Res 43:113-173 Plotsky PM 1987 Regulation of hypophysiotropic factors mediating ACTH secretion. Ann NY Acad Sci 512:205-217 Berkenbosch F, De Goeij DC, Tilders FJ 1989 Hypoglycemia enhances turnover of corticotropin-releasing factor and of vasopressin in the zona externa of the rat median eminence. Endocrinology 125:28-34 Widmaier EP, Plotsky PM, Sutton SW, Vale WW 1988 Regulation of corticotropin-releasing factor secretion in vitro by glucose. Am J Physiol 255:287-292
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