Lipolysis in diabetic adipocytes: differences in response to growth hormone and adenosine

The sensitivity to lipolytic agents is altered in diabetic vs. control animals. Because of its role as a diabetogenic hormone and its ability to elicit lipolysis, GH was studied in isolated fat cells (IFC) from control and streptozotocin-diabetic (STZ-DM) rats. IFCs from the epididymal fat of 150 to 200-g normal and STZ-DM Holtzman rats were prepared by collagenase digestion. Lipolysis was measured by glycerol release after either incubation or perifusion with the following concentrations: epinephrine (EPI), 0.01-0.1 microM; theophylline, 0.01-1.0 mg/ml; adenosine deaminase (ADA), and bovine GH (bGH), 0.01-1.0 microgram/ml. Rats, rendered diabetic by STZ (65 mg/kg), were used on day 3. In a dose-response study comparing glycerol release from control and STZ-DM IFC, IFC were preincubated with 1.0 microgram/ml bGH and then incubated with varying concentrations of EPI or bGH. In STZ-DM, we noted increased lipolytic sensitivity to low concentrations of EPI or bGH. Furthermore, in perifusion, STZ-DM IFC did not require obligatory preincubation with bGH for optimal glycerol release. The addition of ADA increased glycerol release from incubated IFC (STZ-DM and controls). In both systems an enhanced lipolytic response to theophylline was seen in the presence of bGH in control and STZ-DM. It was thus concluded that IFC from normal animals do not respond to GH without preincubation. IFC from STZ-DM rats show a lipolytic response to GH without preincubation. Preincubation with GH increases the lipolytic response of IFC from STZ-DM to all lipolytic agents compared to control responses. In addition, ADA greatly enhanced lipolysis in IFC from STZ-DM compared to that in controls. Together these data demonstrate enhanced sensitivity to both lipolytic stimuli and adenosine suppression of lipolysis in IFC from STZ-DM.     

Development of responsiveness of young normal rats to growth hormone

Growth hormone (GH) exerts a biphasic effect on the membrane transport of amino acids and sugars in diaphragms from hypophysectomized rats. A stimulatory (insulin-like) effect of GH is observed for approximately 3 hr after the administration of the hormone, and then the diaphragm becomes “refractory” to further administration of the hormone for 24–48 hr.In the present study the in vitro responsiveness of diaphragms from young normal rats of different ages to bovine growth hormone (bGH) was studied by measuring the accumulation of α-aminoisobutyric acid (AIB), cycloleucine, and 3-O-methylglucose (3-OMG) and by determining the incorporation of phenylalanine into diaphragm proteins. bGH increased the uptake of AIB in 6, 10, 14, 18, and 22-day old and of cycloleucine in 10, 14, and 18-day old rats, respectively. The maximal stimulatory effect was seen in 18-day old rats and no effects were seen in 26 and 30-day old animals. A similar pattern of age-related responsiveness was also seen when the uptake of 3-OMG and the incorporation of phenylalanine were studied. The rate of accumulation of the non-utilizable amino acids, as well as the rate of incorporation of phenylalanine, decreased markedly with age. Fasting the rats for 20 hr did not change the principal age-related responsiveness of AIB transport to GH, but the magnitude of the hormone effect increased due to a lower uptake of AIB in control diaphragms. bGH had a dose-dependent effect on the accumulation of AIB in fasted 18-day old rats, the threshold concentration of bGH being 0.1 μg/ml.When diaphragms from 18-day old rats were incubated with bGH for various periods, the uptake of AIB was stimulated between 0 and 60 and 120 and 180 min, but not between 60 and 120 min after the start of the incubation. This observation indicated that the muscles were “refractory” to GH between 60 and 120 min. Preincubation of diaphragms from 21-day old rats with bGH for 3 hr did not inhibit the stimulatory effect of bGH added subsequently indicating that the duration of the “refractory phase” did not exceed 270 min in this group of rats. When diaphragms from fasted rats of different ages were preincubated in Krebs bicarbonate buffer for 3 hr, a stimulatory effect of bGH on AIB uptake was also observed in 26 and 30-day old rats, possibly because the influence of endogenous GH subsided during the preincubation period. These results suggest that diaphragms from “old” normal rats are insensitive to exogenous GH because the “refractory phases” become considerably prolonged with age.     

Induction of prolactin receptors in the liver is more closely related to the growth-promoting than to the lactogenic potency of peptides

The sex differentiated binding 125I-human prolactin (PRL) to rat liver membranes was studied and the present results extend our previous studies on induction of hepatic PRL receptors by growth hormone (GH). In prepubertal female rats, PRL receptor levels are low compared with those in mature female rat livers. Infusion of hGH during one week to 17-day-old female rats resulted in a receptor level typical of adult female rats. The time course of receptor disappearance in male rats treated with hGH was also studied. When the receptor-inducing hormone was removed, receptor levels in hGH-treated male rats returned to the normal level characteristic of male rats after approximately 96 h. The specificity of various GH-like and PRL-like hormones in PRL receptor induction was studied in hypophysectomized rats. The PRL-like hormones were identified by measuring their potency to displace 125I-hPRL from a receptor preparation obtained from female rat livers, and the GH-like hormones were identified by their potency to increase body weight in hypophysectomized rats. Using similar doses of hormones it was found that in vivo administration of growth-promoting peptides (rGH, hGH, bGH) induced PRL receptors, whereas lactogenic hormones (rPRL, hPL) had a very small or no effect on PRL receptor induction. This suggests that binding to a type of GH receptor is the first step in PRL receptor induction.     

Specific uptake of human growth hormone by the liver of severely hypothyroid rats

Severe thyroid hormone deficiency results in marked impairment of body growth. This is due, at least in part, to impaired growth hormone (GH) synthesis. We hae studied the possible effects of severe thyroid hormone deficiency on liver receptors for GH and for prolactin (PRL) by an in vivo technique. Female thyroidectomized (T) rats and age-paired controls (C) were injected iv with tracer amounts of biologically active monoiodinated hGH, alone or together with 200 micrograms/100 g bw of native hGH, bGH or oPRL. The liver uptake of labelled compounds, and the liver to serum radioactivity ratio was measured 20 min later. The liver to serum radioactivity ratio of C rats was decreased both by native bGH (purely somatogenic) and native oPRL (purely lactogenic). That of the T rats could only decrease with bGH. Such results confirm data obtained in vitro indicating that in the severely hypothyroid rat liver there is a marked decrease in lactogenic binding and strongly suggest that specific binding of growth hormone by the liver is not similarly affected.     

Specific binding of iodinated growth hormone to rat liver in vivo.

The specific uptake by rat liver of human (hGH) and bovine (bGH) GHs labeled with 125I was studied by an in vivo procedure. A significant reduction of the uptake was observed when labeled hormones were injected together with different amounts of the corresponding native GH. This reduction was dose dependent, and the concentration of native hormone that prevents 50% of the liver uptake of the labeled hormone was close to 12 microgram/100 g BW. In normal rats, only native hGH or bGH significantly decreased the liver uptake of [125I]iodo-bGH, while bovine PRL (oPRL) or heat-denatured bGH were inactive. The highest inhibition of the uptake of [125I]iodo-hGH by rat liver was obtained when this labeled hormone was injected either together with hGH or with bGH plus oPRL while partial displacement was observed with bGH or oPRL. These data suggest that hGH binds to both somatotropic and lactogenic sites in the liver of normal rats. In hypophysectomized animals, only the somatogenic binding sites could be detected.     

Effect of streptozotocin diabetes and insulin replacement on growth hormone in rats

The effects of insulin deprivation on the growth rate, plasma and pituitary growth hormone (GH) and GH synthesis were investigated in male Wistar rats. Diabetes was induced by administration of streptozotocin (STZ), 7 mg/100 g bw, and plasma and pituitary GH levels were measured by means of a specific radioimmunoassay. GH synthesis was determined in pituitaries by the in vitro incorporation of [3H] leucine into specific immunoprecipitates. The body weight and the pituitary GH content of normally developing rats showed an almost linear increase throughout the observation period, whereas diabetic rats stopped growing immediately after receiving STZ, and remained smaller than age-paired controls. Pituitary GH content remained within the control range through the 5 days following STZ administration and thereafter decreased reaching 10% of control values by the 30th day. Furthermore, pituitaries from diabetic rats incorporated [3H] leucine into r-GH at a greatly reduced rate, which could explain the diminished r-GH storage in pituitaries of diabetic rats. Plasma GH concentrations remained within the normal range for 10 days after STZ, thereafter plasma GH were markedly reduced. Insulin treatment prevented the metabolic changes, and restored normal levels of plasma and pituitary GH in diabetic rats. These findings indicate that diabetes, in rat, is characterized by an inhibitory effect on GH secretion, probably via a diminished GH synthesis by the pituitary gland.     

Induction of hepatic receptors for growth hormone (GH) and prolactin by GH infusion is sex independent

To determine whether induction of rat liver GH and PRL receptors by GH infusion is dependent upon the sex of the animal or whether or not the pituitary is intact, rat GH (rGH) or rat PRL (rPRL) was infused at approximately 200 micrograms/day for 7 days into male and female, intact and hypophysectomized rats, and the binding of radioiodinated bovine GH (bGH) and ovine PRL (oPRL) to liver microsomal membranes was measured. In females, bGH binding was reduced by hypophysectomy whether or not membranes were MgCl2 treated to remove endogenous ligand. However, in males, hypophysectomy caused an apparent 3-fold induction of bGH binding sites, which was absent in MgCl2-treated membranes, suggesting that the effect was due to receptor occupancy by endogenous rGH in the intact males. Hypophysectomy also lowered oPRL binding in females but had no effect in males. Infusion of rGH significantly induced binding sites for bGH and oPRL in all treatment groups, independently of sex or the presence of the pituitary, whereas rPRL infusion had no effect on either receptor type except for mild induction of bGH binding in hypophysectomized females. Serum somatomedin-C (SM-C), reduced 95% by hypophysectomy, was restored by rGH, but not rPRL, infusion. However, in intact animals of both sexes, rGH infusion significantly lowered SM-C levels by 30-40%; thus both bGH and oPRL binding in individual pituitary-intact rats were negatively correlated with serum SM-C. In contrast, in rGH-treated hypophysectomized rats, induced GH (but not PRL) binding sites showed significant positive correlation with SM-C levels. These results indicate that the induction of GH and PRL receptors by rGH occurs independently of SM-C generation, but suggest that newly induced GH receptors in GH-treated hypophysectomized rats may be involved in SM-C generation.     

Hypothalamic regulation of impaired growth hormone secretion in diabetic rats. 1. Studies in streptozotocin-induced diabetic rats.

Growth hormone (GH) secretion is blunted in diabetic rats. In the present experiment we observed that pituitary GH concentrations and the plasma GH response to an exogenous dose of growth hormone-releasing hormone (GHRH) is decreased in streptozotocin-induced diabetic rats (p less than 0.02) with respect to normal rats. In an attempt to determine if increased somatostatin (SRIF) secretion is responsible for the decreased GH secretion, we studied the effect of modulating SRIF tone on the GH response to GHRH in normal and streptozotocin-induced diabetic rats. Rats were pretreated with either normal sheep serum and saline (NSS+SAL), somatostatin antibodies (SRIF-Ab), or pyridostigmine (PD), an acetylcholinesterase inhibitor hypothesized to reduce hypothalamic SRIF secretion. Pretreatment of normal rats with SRIF-Ab or PD resulted in an increased GH response to exogenous GHRH in comparison to NSS+SAL-pretreated normal rats at 5 min postinjection. In contrast, pretreatment of diabetic rats with SRIF-Ab or PD did not alter the GH response to exogenous GHRH when compared to NSS+SAL-pretreated diabetic animals. These results suggest that the blunted GH response to exogenous GHRH observed in streptozotocin-induced diabetic rats may not be due to an increase of endogenous SRIF tone.     

Pituitary secretions related to adrenocorticotropic hormone induce sensitivity of adipose tissue to the insulin-like actions of growth hormone

In its initial encounter with growth hormone (GH) in vitro, epididymal fat excised from GH-deficient rats responds with an insulin-like increase in glucose metabolism. Tissues freshly excised from normal rats are refractory to the insulin-like effects of GH, but become sensitive immediately after surgical stress. Reversal of refractoriness is prevented by administration of the opioid antagonist, naloxone, just prior to stress, suggesting a possible role of beta-endorphin or related peptides. These experiments were undertaken to determine the source of these peptides which might equally well be released from the pituitary, adrenal medullae, or nerve endings in response to stress. Since adrenalectomy, like stress, also results in increased secretion of adrenocorticotropic hormone (ACTH) and related peptides, we studied the effects of GH on glucose oxidation in adipose tissue obtained from adrenalectomized rats and found a significant insulin-like response to GH in tissues studied 4 days after adrenalectomy. This effect was not due to GH deficiency, since plasma concentrations were only slightly reduced by adrenalectomy. Administration of naloxone (250 micrograms/rat), 30 or 60 min before sacrifice, or dexamethasone (100 micrograms/injection), 60 and 120 min before sacrifice, prevented a response to GH without affecting circulating levels of GH. The effects of adrenalectomy could not be reproduced by preincubation of adipose tissue from normal nonstressed rats with ACTH and beta-endorphin, but were duplicated by preincubation of adipose tissue for 15 min in medium in which pituitary glands had previously incubated in the presence of corticotropin-releasing hormone (0.1 microM) and arginine vasopressin (0.2 microM). Addition of naloxone (250 micrograms/ml) blocked this effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparing adiposity profiles in three mouse models with altered GH signaling.

Three mouse lines with altered growth hormone (GH) signaling were used to study GH's role in adiposity. Dwarf GH receptor knockout mice (GHR -/-) and bovine GH antagonist expressing mice (GHA) had an increased percent body fat with most of the excess fat mass accumulating in the subcutaneous region. Giant bovine GH expressing mice (bGH) had a reduced percent body fat. Only GHA mice consumed significantly more food per body weight. Serum leptin levels were significantly increased in GHA mice and decreased in bGH mice but unchanged in the GHR -/- mice. Interestingly, serum adiponectin levels were significantly increased in the GHR -/- and GHA lines but decreased in bGH mice. These data suggest that suppression or absence of GH action and enhanced GH action indeed have opposite metabolic effects in terms of adiposity. Interestingly, adiponectin levels were positively correlated with previously reported insulin sensitivity of these mice, but also positively correlated with adiposity, which is contrary to findings in other mouse models. Thus, adiponectin levels were negatively correlated with GH function suggesting a role for adiponectin in GH-induced insulin resistance.     

A single arginine residue determines species specificity of the human growth hormone receptor.

Although growth hormone (GH) receptors (GHRs) in many species bind human (h) GH as well as their own GH, the hGHR only binds primate GH. Arg43 in hGHR interacts with Asp171 of hGH. Nonprimates have a His in the position equivalent to residue 171 of primate GH and a Leu in position 43 of primate GHR. To determine whether Arg43 accounts for the species specificity of the hGHR, point mutations that changed Leu43 to Arg were introduced into the cDNAs encoding the bovine (b) GHR or the rat GH binding protein (GHBP) and these mutants or their wild-type (WT) counterparts were expressed in mouse L cells. Binding of hGH or bGH to transfected cells or to GHBP secreted into the incubation medium was assessed by displacement of 125I-labeled hGH. WT and mutant bGHR bound hGH with similar affinity, but the affinity of the mutant receptors for bGH was reduced 200-fold. Likewise, WT and mutant GHBP bound hGH with equal affinity, but only WT GHBP bound bGH. Cross-linking of 125I-labeled hGH to WT or mutant GHR produced a 141-kDa labeled complex whose appearance was blocked by unlabeled hGH, but bGH blocked cross-linking only to WT receptors. Both hGH and bGH stimulated tyrosine phosphorylation of a 95-kDa protein in cells transfected with WT GHR, but bGH was less effective in cells expressing mutant GHR. We conclude that incompatibility of Arg43 in the hGHR with His171 in nonprimate GH is the major determinant of species specificity.     

Prolactin (PRL) receptor induction in cultured rat hepatocytes: dual regulation by PRL and growth hormone

Although early work implicated PRL as the pituitary factor inducing rat hepatic PRL receptors, recent studies indicated that GH, not PRL, was responsible. The roles for these two hormones were evaluated on rat hepatocytes cultured in serum-free medium supplemented with insulin (1 microgram/ml), epidermal growth factor EGF (25 ng/ml), glucagon (500 ng/ml), cholera toxin (2 ng/ml), hydrocortisone (10(-8) M), and transferrin (1 microgram/ml) and changed daily. Ovine (o) PRL, bovine (b) GH, or human (h) GH were introduced after 2-4 days of culture, and PRL receptors were measured by determining [125I]hGH binding in the presence and absence of excess oPRL in a total particulate fraction pretreated with 3 M MgCl2. The specific binding of hGH (% per 100 micrograms protein) decreased by 8- to 10-fold (female, 17.9 +/- 0.2% to 1.5%; male, 7.0 +/- 0.1% to 0.7%) after 3 days in culture. When added after 3 days, hGH induced PRL receptors in both female and male cells with the effect being more gradual in the latter. Induction occurred with 10 ng/ml hGH and was maximal [11- to 13-fold control] at 250-1000 ng/ml. bGH and oPRL also induced PRL receptors with maximal levels attained at 250-500 ng/ml oPRL (3- to 4-fold control). The combined addition of oPRL (300 ng/ml) and bGH (300 ng/ml) yielded levels of induction comparable to that seen with hGH. Although hormone treatment restored PRL receptor levels to those seen in male rats, the much higher levels of female rats were not attained. Treatment of hepatocytes with hGH, bGH, or oPRL affected neither cell number (through 10 days of culture) nor PRL receptor affinity. At supramaximal doses hGH, PRL, and bGH down-regulated PRL receptors, but this was particularly noticeable for oPRL and hGH. 17 beta-Estradiol and testosterone added to male and female hepatocytes simultaneously with hGH had little or no effect on receptor induction. We conclude that hepatic PRL receptors are induced by both PRL and GH, each acting through its own receptor. The failure to restore receptor levels to those seen in female rats attests to the importance of other modulators. This dual regulation of the PRL receptor explains the unusual potency of hGH which binds to both PRL and GH receptors.     

The presence of lactogen but not growth hormone binding sites in the isolated rat hepatocyte.

The binding of 125I-labelled human growth hormone (hGH) and bovine growth hormone (bGH) has been studied in hepatocytes isolated from female rats by perfusion with collagenase in situ. The cells appeared to retain normal membrane function, in that amino acid ([14C]alpha-aminoisobutyric acid) transport was both saturable and temperature-dependent. Amino acid ([14C]leucine) incorporation into protein was also linear over 3 h and was inhibited by cycloheximide. Binding of 125I-labelled hGH was dependent on time, temperature, hepatocyte concentration and hGH concentration. At 22 degrees C, binding reached a steady-state after 2-5 h and had a half-life of dissociation of 2-3 h. Hormone specificity studies indicated that binding was specific for hormones with prolactin-like activity (hGH, prolactins) and not for growth hormones themselves (bGH). Scatchard analysis revealed a single class of binding site with a binding capacity of 26-74+/-3-73 fmol/10(6) cells and a binding affinity of 1-24 X 10(9)+/-0-17 X 10(9) (S.E.M.) 1/mol (n=10). There was a significant sex difference in binding (female greater than male) and binding was subject to marked regulation by oestrogens (stimulation of binding) and by androgens (inhibition). The lactogen-binding sites, therefore, were comparable in many respects to those previously reported in rat liver membranes. No distinct GH binding sites were demonstrable as shown by the lack of specific binding by 125I-labelled bGH, purified either by Sephadex chromatography or by binding to and elution from GH receptors in rabbit liver membranes. The value of receptor purification of tracer for use in hormone binding studies was indicated by a substantial lowering of non-specific binding.     

Effect of zinc deficiency on serum somatomedin levels and skeletal growth in young rats

We have studied potential mechanisms by which zinc deficiency (ZD) may result in growth impairment in young animals. Dietary-induced ZD in young rats resulted in diminished skeletal growth as measured by tibial epiphyseal width. Treatment with bovine GH (bGH) did not increase skeletal growth suggesting GH resistance rather than GH deficiency in zinc-deficient rats. Serum levels of basic somatomedin (SM) were lower in zinc-deficient rats than in control rats receiving a zinc adequate diet, either ad libitum or in pair matched amounts, and were restored to normal by zinc repletion but not by bGH treatment, suggesting that SM production is impaired by ZD. There was a high correlation between tibial epiphyseal widths and serum or femur zinc concentrations. These findings, along with observations that despite similar levels of serum basic SM the bGH-treated zinc-deficient rats had smaller tibial epiphyseal widths than pair fed control rats, additionally suggest that the action of SM on skeletal growth is impaired by ZD.     

Induction of lactogenic receptors in liver of hypophysectomized rats treated with bovine growth hormone-monoclonal antibody complexes

Male hypophysectomized rats treated with bovine (b)GH-monoclonal antibody complexes showed enhanced weight gain compared with animals treated with bGH alone over a 12-day treatment period. Liver microsomes prepared from animals showing enhanced weight gain exhibited increased specific binding of human (h)GH. Studies on the specificity of these binding sites showed that they were lactogenic, 125I-labelled hGH being displaced by ovine prolactin, but not by non-mammalian growth hormones. In this respect they were similar to lactogenic binding sites in the liver of pregnant rats. Monoclonal antibodies to hGH blocked binding to lactogenic receptors to different extents. The pattern of such inhibition was similar, but not identical, for the receptors induced in hypophysectomized rats and those from pregnant rat liver. The evidence available suggests that the lactogenic receptors induced by bGH-monoclonal antibody complexes are not directly involved in the enhancement of growth.     

Continuous administration of growth hormone does not prevent the decrease of IGF-I gene expression in zinc-deprived rats despite normalization of liver GH binding

To determine the role of reduced liver GH binding (GHR) in the decreased IGF-I observed in zinc-deficient (ZD) animals, we investigated the effects of GHR restoration on growth, insulin-like growth factor I (IGF-I) and its binding proteins (IGFBPs) in ZD rats. Rats were fed for 4 weeks a zinc-deficient diet (ZD Zn, 0 ppm) or a Zinc-normal diet (pair-fed or PF; Zn, 75 ppm). ZD rats received continuous s.c. infusion of bovine growth hormone (bGH) (100 microg/d) for the 4 weeks or for the last week of the study. Compared with pair-fed rats, zinc deficiency produced attenuated weight gain (-43%, P < 0.001), lower serum IGF-I and liver IGF-I mRNA (-52%, P < 0.001 and -44%, P < 0.05), lower serum IGFBPs (IGFBP-3 -66%, IGFBP-4 -48%, 34-29 kDa IGFBP cluster -53%, P < 0.05), lower liver GHR and its mRNA (-20 and -34%, P < 0.05) and lower serum growth hormone binding protein (GHBP) and its mRNA (-56 and -48%, P < 0.05; all comparisons vs PF rats). Exogenous bGH given continuously normalized the liver GHR, serum GHBP and their liver mRNAs, as well as circulating IGFBPs. Despite restoration of GHR and GHBP to normal, growth, serum IGF-I and its liver mRNA were not stimulated by GH infusion in ZD rats, indicating that IGF-I synthesis requires the presence of zinc in addition to GH, and that the lack of growth-promoting action of GH in zinc-deprived rats results from a defect beyond GH binding to its liver receptors.     

Growth hormone treatment increases oestrogen receptor concentration in the guinea-pig uterus.

Growth hormone does not act as a body growth-promoting hormone during the postnatal period in the guinea-pig. To determine whether it affects uterine and mammary growth, guinea-pigs of 8-9 weeks of age were treated with either recombinant bovine GH (bGH; 0.5 mg per animal) or vehicle for 10 days. Uterine and mammary weights were not changed by treating the animals with bGH. The amount of available cytosolic oestradiol receptor per unit of uterine weight or DNA content, or in whole uteri was increased in bGH-treated animals (7.3- to 14.0-fold) when compared with controls. The nuclear uterine oestradiol receptor concentration was 3.3- to 6.7-fold higher than in controls. The dissociation constant values did not differ between control and bGH-treated animals, suggesting that uterine oestradiol receptors are regulated by GH through changes in the number of binding sites rather than alteration of their binding affinity. Mammary growth and oestradiol receptor levels were unaffected by bGH treatment. The results of this investigation demonstrate that injected bGH selectively affects the oestradiol receptor level in guinea-pig uterine tissue.     

Hypothalamic regulation of impaired growth hormone secretion in diabetic rats. 2. Studies in spontaneously diabetic BB Worcester rats.

In the present study we investigated the effects of modulating endogenous somatostatin (SRIF) on the GH response to growth hormone-releasing hormone (GHRH) in spontaneously diabetic BB/Wor rats and nondiabetic littermates. Plasma growth hormone (GH) concentrations following injection of GHRH (500 ng/kg, i.v.) were measured in the rats after pretreatment with either normal sheep serum+saline (NSS+SAL), somatostatin antibody (SRIF-Ab), or pyridostigmine bromide (PD), an acetylcholine esterase inhibitor hypothesized to decrease hypothalamic SRIF tone. The GH response to GHRH in spontaneous diabetic rats pretreated with NSS+SAL was significantly lower (p less than 0.05) than the response observed in the nondiabetic group. SRIF-Ab pretreatment reversed the blunted GH response observed in the diabetic rats. However, PD pretreatment was not effective. These results indicate that the blunted GH response observed in BB/Wor diabetic rats is reversed by neutralization of endogenous SRIF with SRIF-Ab and leads to the conclusion that SRIF plays an active role in modulating GH secretion in spontaneously diabetic rats. The failure of PD to modulate the GH response suggests this acetylcholine agonist is ineffective in this animal paradigm.