Effect of Hypoglycemia on the Content of Pituitary Growth Hormone (GH) and Hypothalamic Growth Hormone-Releasing Factor (GHRF) in the Rat S. H. KATZ, A. P. S. DHARIWAL, AND S. M. M C C A N N 1 Departments of Physiology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania, and University of Texas Southwestern Medical School at Dallas, Texas 75235

ABSTRACT. The effect of insulin-induced hypoglycemia on the content of growth hormone (GH) in the pituitary and growth hormone-releasing factor (GHRF) in the hypothalamus of adult male rats was studied. GH was estimated by the tibial epiphyseal cartilage test and GHRF activity was determined by the depletion of pituitary GH evoked by intravenous (iv) administration of hypothalamic extracts into normal male rats. Initial studies revealed that a doseresponse relationship existed between the logdose of ovine hypothalamic extract administered and the degree of depletion of pituitary GH in recipient rats. Cerebral cortical extract was in-

active. A dose of crystalline insulin (1 U/kg) was administered intraperitoneally, and blood sugar, pituitary GH and hypothalamic GHRF were estimated 1 hr later. Control rats received an equal volume of saline. The treatment with insulin induced a profound hypoglycemia which was accompanied by a significant depletion in pituitary GH and an even greater depletion in hypothalamic GHRF. It was concluded that the hypoglycemic stimulus to GH secretion acts via release of GHRF, which then triggers GH release from the pituitary gland. (Endocrinology 81: 333, 1967)

NUMBER of experimental investigations have produced evidence for a hypothalamic mechanism which controls the secretion of pituitary growth hormone (GH) (1). This control appears to be mediated by a specific GH-releasing factor (GHRF) since crude hypothalamic extracts appear to stimulate GH secretion by the pituitary both in vivo and in vitro (2-6). The purification of GHRF and its separation from other releasing factors affords further evidence for this view (7-9). Another series of experimental investigations have dealt with a number of stimuli which apparently cause an increase in the secretion of GH (10-15). Insulin-induced hypoglycemia has been found to be one of

A

Received January 30, 1967. Supported by Grants AM 10073-2 and TD 109-04 from the USPHS. 1 Please send reprint requests to: Dr. S. M. McCann, Department of Physiology, Univ. of Texas Southwestern Medical School, Dallas, Texas 75235.

the most rapid, potent and consistent stimuli associated with increases in the plasma GH levels in the primate (10, 14, 15) and decreases in the content of anterior pituitary GH activity in the rat (16, 17). The possible relationship between these physiological stimuli that are associated with depletion of pituitary GH content and hypothalamic GHRF activity has been investigated in two different studies. First, Meites and Fiel reported that during starvation in the rat there is a depletion of pituitary GH which is accompanied by a depletion of GHRF activity in the median eminence region of the hypothalamus (18). Second, Krulich and McCann utilized the more acute stimulus provided by insulin hypoglycemia in the hypophysectomized rat to demonstrate a circulating GHRF activity that could be eliminated by posterior median eminence lesions (19). These same lesions had already been found to block the depletion of pituitary GH activity which follows hypoglycemia (20). The present

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KATZ, DHARIWAL AND McCANN

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above, except that they were fed oranges and moistened Wayne Lab Blox mash ad lib. DILUENT Fourteen days after hypophysectomy they CONTROL were used for assaying GH activity. \ Two series of experiments were performed. The first series was designed to determine if a 250 dose-response relationship existed between the dose of hypothalamic extract injected and the depletion of pituitary GH of recipient rats. The second series of experiments was performed to determine if there was a difference in the content of GH in the pituitary and GHRF in the hypothalami of rats rendered 200 hypoglycemic by insulin in comparison to the content in saline-injected controls. For the first series of experiments, crude ovine SME extract was prepared by the method of Dhariwal et al. (7) and was dissolved in 0.1M (pH 5.0) ammonium acetate buffer. Using the general method of GHRF assay developed by Pecile et al. (4) and modified by Krulich et al. (6), increasing doses of either 4.5, 13.5 and 0 4.5 (3.5 40.5 (log scale) 40.5 mg/kg, or a diluent control, all of equal DOSE SME EXTRACT (mg/kg) volume (0.5 ml), were infused iv into 4 groups of 7 rats each. Thirty min later these recipient FIG. 1. Relationship between dose of crude ovine rats were lightly etherized and sacrificed. Their SME extract and pituitary GH activity of recipient anterior pituitaries were weighed on a torsion rats as indicated by the width of the tibial epiphy- balance, pooled and frozen at — 40 C until the seal cartilage of hypophysectomized rats. Vertical time of assay for GH content. bars indicate the standard error of the mean. In the second series of experiments, 2 groups of 21 rats each served as donors of median experiments were designed to investigate eminence, anterior pituitary and cerebral corfurther the role of GHRF as a specific tex. Each experimental group of 21 rats received an intraperitoneal (ip) injection of inmediator for the effects of hypoglycemia sulin (Lilly-Iletin) at a dose of 1 U/kg in order upon the release of pituitary GH by deter- to produce hypoglycemia. Sixty min after the mining the effect of insulin-induced hypo- injection these rats were lightly etherized and glycemia on the content of GHRF stored in a blood sample was removed from every the stalk-median eminence (SME) region fourth rat in order to measure its blood sugar with the autoanalyzer. All rats were then of the rat. A preliminary report of this work sacrificed by decapitation. Each anterior pituhas appeared (21). itary and SME region was removed, weighed on a torsion balance, frozen at —40 C, and Materials and Methods saved for extraction and assay. A duplicate For all experiments, male rats weighing control group received a saline injection ip, 200-225 g, of the Sherman strain (West Jersey and the identical procedure was followed, exBiological Supply Co., Haddonfield, N. J.), cept that fragments of brain cortex were also were maintained in the animal quarters for 2 removed from these donors and saved for exdays prior to experimentation. During this traction. interim they were fed Wayne Lab Blox pellets The hypothalamic and cortical tissues from ad lib. and were subjected to a constant tem- the various groups of donors were homogenized perature of 24 C and controlled lighting for 12 in 0.1N HC1, centrifuged at 5000 X g for 20 hr/day (lights on 7 AM-7 PM). Hypophysec- min, decanted and brought to a final concentratomized female rats of the Sprague-Dawley tion of 3.0 median eminences/0.5 ml dose, and strain, weighing 80-85 g, were obtained 1 an equivalent dosage by weight of brain cortex/ week post hypophysectomy from the Hor- 0.5 ml dose. All operations in this procedure mone Assay Laboratories, Chicago, 111. These were carried out at 4 C. rats were subjected to the same conditions as Brain extracts from these donors were ad-

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August 1967

HYPOGLYCEMIA AND HYPOTHALAMIC GHRF

ministered to other normal intact male rats in groups of 7 in order to assay the GHRF activity of these extracts. They were treated exactly as described above in the assay for crude ovine SME extracts. In each experiment pituitary GH content from rats which had received hypothalamic extract from insulin-treated rats was compared with that of rats which had received hypothalamic extract from the saline-injected controls in order to determine the GHRF activity by the depletion of pituitary GH. In addition, the pituitary GH content of the rats which had received insulin or saline was also estimated. The "tibia test" method of Greenspan et al. (22) was used to assess the GH activity throughout all experiments. This consisted of the administration of saline extracts of anterior pituitary glands in doses of 2 mg ip once daily to the female hypophysectomized rats already described. The animals were sacrificed on the 4th day. Two doses of bovine reference standard GH (NIH B-6) with a 5-fold difference in dose were also administered in each experiment. Six or 7 hypophysectomized rats were used in each group. Ten measurements of tibial epiphyseal cartilage plate width were made for each rat, and the results were used to calculate the percentage changes of GH activity of the various groups of pituitaries. In addition, Student's i-test was used to calculate the significance of the differences in cartilage width between experimental and control groups of pituitary extracts. All statistical operations were performed on a CDC 160-A computer. Results Effect of varying doses of ovine hypothalamic extract on pituitary GH activity. In the first

experiment an attempt was made to determine if increasing doses of crude ovine SME extract would produce a graded depletion of pituitary GH. Over the range of doses utilized, 4.5, 13.5 and 40.5 mg/kg body weight, a linear log-dose response relationship was obtained between the dose of extract injected and the depletion of pituitary GH as indicated by narrowing of the epiphyseal cartilage plate (Fig. 1). The regression equation which describes this relationship is as follows:

PITUITARIES FROM RECIPIENT BRAIN EXTRACT TREATED RATS

335 DONOR PITUITARIES

25 50 fit

?s

ta.100

Saline

Median Eminence

Insulin Median Eminence

Saline Injected

Insulin Injected

FIG. 2. Effect of insulin-induced hypoglycemia on the content of pituitary GH and the GHRF activity of the hypothalamus. The right panel indicates the depletion of pituitary GH in the insulininjected rats. Saline-injected rats are considered to show 0% decrease in pituitary GH. The left panel indicates the % decrease in pituitary GH of recipient rats after injection of brain extracts from saline- or insulin-treated animals. Pituitary GH in animals injected with cerebral cortical extract is considered as 0% decrease. Note that increasing depletion of pituitary GH reflects increases in GHRF.

slope (55.64 + 15.21) is significantly different (p