0021-972X/90/7005-1371$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright© 1990 by The Endocrine Society

Vol. 70, No. 5 Printed in U.S.A.

Growth Hormone Does not Inhibit Its Own Secretion during Prolonged Hypoglycemia in Man FABRIZIO BARBETTI, CASSANDRA CRESCENTI, MARCELLO NEGRI, FRIDA LEONETTI, ARMANDO GROSSI, AND GUIDO TAMBURRANO Cattedra di Endocrinologia I and Istituto di Clinica Medica II (M.N.), Universita la Sapienza, Rome, Italy

ABSTRACT. In man, continuous infusion of GH-releasing hormone (GHRH) does not sustain GH secretion, unlike prolonged hypoglycemia. To further evaluate this difference in the stimulation of GH release we measured GH concentrations for 3 h during prolonged insulin-induced hypoglycemia and GHRH(1-29)NH2 (100 fig/h) infusion in normal individuals. We also assessed the GH response to combined and separate administration of insulin and GHRH. Plasma GH levels increased during prolonged hypoglycemia and remained elevated for the third hour (22-24 iig/L). GH concentrations increased during GHRH infusion, peaked at 60 min (23.5 jtg/L), and rapidly declined. Thus, our findings confirmed that prolonged hypoglycemia, unlike GHRH infusion, sustained elevated GH levels and that these high levels did not appear to influence GH secretion from the pituitary. Changes in FFA did not account for the sustained GH secretion. FFA levels

G

H SECRETION is regulated by an integrated network involving hypothalamic releasing [GH-releasing hormone (GHRH)] and inhibiting (somatostatin) hormones, neurotransmitters, insulin-like growth factors, and a variety of metabolic substrates, such as glucose and FFA. GH is also believed to regulate its own secretion by an autofeedback mechanism. Studies in man demonstrated that a short term infusion of exogenous GH inhibits GHRH-stimulated GH release without increasing insulin-like growth factor-I levels (1, 2). Continuous infusion or repeated administration of GHRH (3, 4) increase GH concentrations, but with time decrease GH secretion, possibly due to an increase in somatostatin induced by high levels of circulating GH. In contrast, mild prolonged hypoglycemia induced by sc insulin administration elicits a long-lasting GH response (5). These findings indicate that moderately high levels of endogenous GH are ineffective in inhibiting GH secretion during a hypoglycemic period. Furthermore, insulin-induced hypoglycemia can stimulate GH secretion after a bolus Received October 31,1989. Address requests for reprints to: Dr. F. Barbetti, Diabetes Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Building 10, Room 8D50, Bethesda, Maryland 20892.

initially declined during insulin infusion, but after 3 h of hypoglycemia they returned to near-basal values (basal, 0.1 ± 0.02 g/ L; 180 min, 0.09 ± 0.02). The maximal GH concentration attained during the combined insulin and GHRH test was significantly higher than that with the insulin tolerance test or GHRH test (insulin plus GHRH, 71.9 ± 13.5; insulin tolerance test, 34.2 ± 2.9; P < 0.025; GHRH test, 27.9 ± 3.2; P < 0.02), indicating an additive effect on GH secretion. These data suggest that insulin-induced hypoglycemia stimulates GH secretion through a mechanism partly independent of GHRH. The release from somatostatin inhibition and stimulation through other neuropeptides (e.g. galanin) is suggested as possible causes of hypoglycemia-induced GH secretion. (J Clin Endocrinol Metab 70: 1371-1374, 1990)

(6) or an infusion of GHRH (7), suggesting that hypoglycemia stimulates GH secretion not only by inducing GHRH release but also by other mechanism(s). In this study we report the effects of prolonged hypoglycemia and GHRH infusion on GH secretion in normal volunteers. We also report the GH responses to a single stimulus of insulin and GHRH and a combination of both hormones. Materials and Methods Subjects and experimental design Fourteen normal volunteers (eight males and six females, aged 25-34 yr) participated in this study after informed consent was obtained. They were all within 10% of ideal body weight and were taking no medications. Study 1. Seven subjects were placed on an artificial pancreas (Biostator, Miles, Elkhart, IN) and were administered an iv infusion of insulin (Actrapid HM insulin, Novo, Copenhagen, Denmark; 0.83 mU/kg- min for 3 h). Hypoglycemia was induced and maintained at 2.5 mmol/L by glucose infusion. On a separate day, five of these subjects were given a GHRH infusion [GHRH-(1-29)NH2; Kabi-Vitrum Pierre, Stockholm, Sweden; 100 ng/h. for 3 h]. Blood samples were taken every 30 min for GH and FFA and every 5 min for blood glucose (BG) and immunoreactive insulin (IRI) determinations.

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Study 2. Eight subjects underwent the following tests: 1) GHRH test [GHRH-(1-29)NH2; 100 ng, iv bolus); this GHRH dose was demonstrated to elicit maximal GH response (8); 2) insulin tolerance test (ITT; Actrapid HM; 0.1 IU/kg, iv bolus); and 3) a combination of the two tests (COMBIN; time zero = ITT; 30 min = GHRH test). Blood samples were collected for BG and GH determinations. All tests were performed in random order at intervals of at least 1 week. Assays

BG was assayed by the glucose oxidase method. FFA were determined by spectrophotometric analysis with reagents provided by Wako (Neuss, West Germany). GH was measured by double antibody RIA with reagents provided by Biodata (Milan, Italy); the lower detection limit was 0.25 Mg/L> with intra- and interassay coefficients of variation of 3.9% and 8.5%, respectively. IRI was assayed by a RIA method previously described (9). Results were expressed as the mean ± SEM. Statistical analysis was performed applying Student's t test for unpaired (study 1) and paired (studies 1 and 2) data when appropriate.

Results Study 1 The patterns of GH secretion during prolonged insulin and GHRH infusions are shown in Fig. 1. GH levels rapidly increased during GHRH administration, peaked at 60 min (23.5 ± 5.9), and declined during the last 2 h of infusion. In contrast, plasma GH levels increased

slowly over the initial 120 min of insulin infusion and remained elevated (22-24 jug/L) for the third hour of the study. Plasma GH values were significantly higher at the end of insulin infusion (22.6 ± 4.0) than at the end of GHRH infusion (6.9 ± 1.5; P < 0.025). FFA and BG values during prolonged hypoglycemia are listed in Table 1. FFA decreased during the first 120 min of the study, then returned to near-basal levels. IRI levels were 237 ± 35 pmol/L at 30 min of the insulin infusion and showed little variation throughout the period of hypoglycemia (data not shown). Study 2 GH levels after GHRH, insulin, and COMBIN (insulin/GHRH) bolus injections are shown in Fig. 2. The COMBIN test elicited a significantly greater GH response than both the ITT at 60 min (P < 0.05), 90 min (P < 0.02), and 120 min (P < 0.02) and the GHRH test at 60 min (P < 0.02) and 90 min (P < 0.05). The maximal GH level obtained during the COMBIN test was higher than that observed in the ITT or GHRH test [COMBIN, 71.9 ± 13.5; ITT, 34.2 ± 2.9 (P < 0.025); GHRH test, 27.9 ± 3.2 ( P < 0.02)]. Discussion Consistent with previous reports, we found plasma GH concentrations to increase during the first hour of continuous GHRH infusion and decline during the second GHRH ID—D) or

INSULIN

INFUSION

X 3 hs

120

150

3CH

25-

20FIG. 1. Mean (±SE) plasma GH responses to prolonged hypoglycemia (•) or GHRH infusion (100 /xg/h; • ) in normal subjects. *, P < 0.025, insulin infusion us. GHRH infusion at 180 min.

GH (M9/L)

15

ID-

-15 0

30

60

90 MIN

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GH SECRETION DURING PROLONGED HYPOGLYCEMIA TABLE 1. BG and plasma FFA during prolonged hypoglycemia in seven normal subjects Time (min) 0 30 60 90 120 150 180

BG (mmol/L) 4.5 ± 3.4 ± 2.4 ± 2.6 ± 2.3 ± 2.4 ± 2.6 ±

FPAs (g/L)

0.4 0.2 0.1 0.2 0.1 0.1 0.2

0.1 ± 0.08 ± 0.06 ± 0.06 ± 0.06 ± 0.07 ± 0.09 ±

0.02 0.02 0.01 0.01 0.01 0.02 0.02

COMBIN ITT

COMPIN GHRH-test

15 30

60

90

MIN FIG. 2. Mean (±SE) plasma GH responses to COMBIN (•) compared to ITT (O; upper graph) and GHRH test (•; lower graph) in seven normal subjects. *, P < 0.05; **, P < 0.02 (by Student's paired t test).

and third hours (3, 4). In contrast, during prolonged hypoglycemia we observed increased and sustained GH secretion until the end of the test. De Feo et al. (5) obtained similar results in a study examining the effects of mild, slowly induced hypoglycemia on glucose counterregulation. Thus, we suggest that persistent elevation of GH levels, which is a probable mechanism for the inhibition of GH release during GHRH infusion, do not appear to influence hypoglycemia-induced GH secretion. Rosenthal et al. found that the inhibitory effect of iv GH administration on GHRH-stimulated GH secretion is a time-dependent phenomenon that occurred at 4 h of infusion (10), but not at 2 h. Rosenbaum et al. (2) in a similar study found that GHRH-induced GH release is blunted by a 3-h continuous infusion of GH. Whether there is a correlation between the levels of circulating GH and the time of occurence of an inhibitory effect

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remains unknown. The plasma GH values attained with the GH infusion in these two studies (2, 10) were approximately 10-12 Mg/L, which is lower than the concentrations actually reached during GHRH infusion in this study. We and others have demonstrated a reduction of GH secretion within 1 h of GHRH infusion. Thus, it is most likely that higher levels of peripheral GH blunt the response to GHRH in a shorter period of time. Our data also indicate that prolonged hypoglycemia does not deplete releasable GH stores, which is one of the possible explanations for the lack of sustained GH secretion during GHRH infusion in man (4) or in perfused pituitary cells (4, 11). In a recent report, 14 days of continuous GHRH administration did not modify spontaneous or GHRH-induced GH secretion in normal man (12), providing evidence for the absence of hormone depletion or somatotroph desensitization with longstanding administration of GHRH. Our results indicate that sustained GH secretion by hypoglycemia is not solely mediated through the stimulation of GHRH, because we have observed differences with GHRH infusion. This conclusion is supported by the results of study 2, which shows an additive effect of GHRH (injected in a maximal dose) and insulin hypoglycemia on GH secretion. Page et al. (13) found a similar additive effect of GHRH and insulin-induced hypoglycemia on GH release. Many metabolic substrates, such as FFA, are influenced by insulin, and FFA play an important role in regulating GH secretion. Infusion of FFA blunts GHRHmediated GH secretion in man (14), and acute suppression of FFA by an antilipolytic agent (Acipimox) enhances the GH response to GHRH (15). It has been reported that the effect of FFA on GH secretion occurs in approximately 30 min (14). Therefore, it is unlikely that FFA, which returned to near-basal levels during the third hour of prolonged hypoglycemia, was responsible for the sustained GH secretion. It has been suggested that hypoglycemia stimulates GH secretion via the inhibition of somatostatinergic tone by increased catecholamine outflow (6). However, in vitro perfusion of rat hypothalamus with 3-O-methyl-D-glucose, 2-deoxy-D-glucose, or low concentrations of glucose stimulates somatostatin release (16). It has been recently demonstrated that galanin, a peptide with GH-releasing properties present in rat hypothalamus (17), when administered together with GHRH in man produces an additive effect on GH secretion of the same order of magnitude observed for hypoglycemia and GHRH stimulation (18). The possibility that hypoglycemia stimulates GH secretion through the indirect inhibition of somatostatin secretion or the stimulation of galanin or other neuropeptides (e.g. vasoactive

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intestinal peptide and motilin) should be taken into consideration.

9.

Acknowledgments

10.

We thank Dr. Richard F. Arakaki for revising the manuscript. The GHRH-(1-29)NH2 was a generous gift of Pierrel Kabi-Vitrum, Italy. 11.

References 1. Rosenthal SM, Hulse JA, Kaplan SL, Grumbach MM. Exogenous growth hormone inhibits growth hormone-releasing factor-induced growth hormone secretion in normal men. J Clin Invest. 1976;77:176-80. 2. Rosenbaum M, Loche S, Balzano S, Gertner JM. Short-term MethGH infusion inhibits somatotroph response to growth hormone releasing hormone (1-44). Metabolism. 1988;37:131-5. 3. Losa M, Bock L, Schopohl J, Stalla GK, Muller OA, von Werder K. Growth hormone releasing factor infusion does not sustain elevated GH levels in normal subjects. Acta Endocrinol (Copenh). 1984;107:462-70. 4. Gelato MC, Rittmaster RS, Pescovitz OH, et al. Growth hormone responses to continuous infusions of growth hormone-releasing hormone. J Clin Endocrinol Metab. 1985;61:223-8. 5. De Feo P, Perriello G, De Cosmo S, et al. Comparison of glucose counterregulation during short-term and prolonged hypoglycemia in normal humans. Diabetes. 1986;35:563-9. 6. Shibasaki T, Hotta M, Masuda A, et al. Plasma GH responses to GHRH and insulin-induced hypoglycemia in man. J Clin Endocrinol Metab. 1985;60:1265-7. 7. Vance ML, Kaiser DL, Rivier J, Vale W, Thorner MO. Dual effects of growth hormone (GH)-releasing hormone infusion in normal men: somatotroph desensitization and increase in releasable GH. J Clin Endocrinol Metab. 1986;62:591-4. 8. Gelato MC, Pescovitz OH, Cassorla F, Loriaux DL, Merriam GR. Dose response relationships for the effects of growth hormone

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releasing factor-(1-44)-NH2 in young adult men and women. J Clin Endocrinol Metab. 1984;59:197-201. Andreani D, Fallucca F, Javicoli M, Tamburrano G, Menzinger G. Insulin, glucagon and growth hormone in primary adult mixedema. Diabetologia. 1974;10:7-ll. Rosenthal SM, Kaplan SL, Grumbach MM. Short term continuous intravenous infusion of growth hormone (GH) inhibits GH-releasing hormone-induced GH secretion: a time-dependent effect. J Clin Endocrinol Metab. 1989;68:1101-5. Badger TM, Millard WJ, McCormick GF, Bowers CY, Martin JB. The effects of growth hormone (GH)-releasing peptides on GH secretion in perifused pituitary cells of adult male rats. Endocrinology. 1984;115:1432-8. Vance ML, Kaiser DL, Martha Jr PM, et al. Lack of in vivo somatotroph desensitization or depletion after 14 days of continuous growth hormone (GH)-releasing hormone administration in normal men and a GH-deficient boy. J Clin Endocrinol Metab. 1989;68:22-8. Page MD, Koppeschaar HPF, Edwards CA, Dieguez C, Scanlon MF. Additive effects of growth hormone releasing factor and insulin hypoglycaemia on growth hormone release in man. Clin Endocrinol (Oxf). 1987;26:589-95. Imaki T, Shibasaki T, Shizume K, et al. The effect of free fatty acids on growth hormone (GH)-releasing hormone-mediated GH secretion in man. J Clin Endocrinol Metab. 1985;60:290-3. Lanzi R, Pontiroli AE, Pozza G. Additional effect of methionyl growth hormone (Met-GH) and free fatty acids in the inhibition of GH response to GHRH [Abstract]. Proc of the 71st Annual Meet of The Endocrine Soc. 1989;222. Berelowitz M, Dudlack D, Frohman LA. Release of somatostatinlike immunoreactivity from incubated rat hypothalamus and cerebral cortex. Effect of glucose and glucoregulatory hormones. J Clin Invest. 1982;69:1293-301. Ottlecz A, Snyder GD, McCann SM. Regulatory role of galanin in control of hypothalamic-anterior pituitary function. Proc Natl Acad Sci USA. 1988;85:9861-5. Davis TME, Burrin JM, Bloom SR. Growth hormone (GH) release in response to GH-releasing hormone in man is 3-fold enhanced by galanin. J Clin Endocrinol Metab. 1987;65:1248-52.

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Growth hormone does not inhibit its own secretion during prolonged hypoglycemia in man.

In man, continuous infusion of GH-releasing hormone (GHRH) does not sustain GH secretion, unlike prolonged hypoglycemia. To further evaluate this diff...
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