Oxytocin infusion increases plasma insulin and glucagon levels and glucose production and uptake in the normal dog.

Infusion of oxytocin (50--500 microU/kg/min) into normal conscious dogs produces a rise in plasma glucose, insulin, and glucagon levels. These changes are accompanied by a prompt increase in glucose production followed by an increase in overall glucose uptake, as determined using 6-3H-glucose infusion.

Tolbutamide and glyburide differ in effectiveness to displace alpha- and beta-adrenergic radioligands in pancreatic islet cells and membranes.

Previous in vivo findings indicated that alpha-adrenergic blocking agents enhanced tolbutamide-induced insulin secretion, whereas beta-blockade attenuated it. In the present study, the interaction of tolbutamide and glyburide with the rat islet adrenergic receptors is examined directly by determining the effectiveness of these drugs to displace the specific alpha- and beta-adrenergic radioligands, [3H]-clonidine and [3H]-dihydroalprenolol (DHA). It was found that both tolbutamide and glyburide had affinity constants for the adrenergic receptors that were similar to those for the natural receptor ligands and powerful antagonists. Tolbutamide displaced both alpha- and beta-radioligands but had a higher affinity at the beta-receptor. Glyburide also displaced radioligands from both types of receptors but had a higher affinity for the alpha-receptor. This study suggests that these two sulfonylurea hypoglycemic agents may affect insulin secretion by different mechanisms.

Interaction of somatostatin, glucagon, and insulin on hepatic glucose output in the normal dog.

The interaction of insulin and glucagon during infusion of somatostatin (SRIF), which suppresses secretion of these hormones, was investigated in normal, postabsorptive, concious dogs. Hepatic glucose output (production) and over-all glucose uptake by the tissues was measured with 3-3H-glucose, administered by a priming injection along with a constant infusion. Infusion of SRIF (1.5-5.0 mug/min) for 90 minutes resulted in a moderate hypoglycemia associated with a decrease in glucose production. In some animals glucose production and plasma glucose levels returned to normal before the end of SRIF infusion. Glucose uptake tended to follow plasma glucose levels. Upon termination of SRIF infusion, glucose production and uptake and plasma glucose increased sharply.

On the mechanism of diazoxide-induced hyperglycemia.

Infusion of diazoxide (16.5 mg./kg. in 10 minutes) into normal unanesthetized dogs resulted in a prompt hyperglycemia due to increased hepatic glucose production as measured with a 3-3H-glucose primer-infusion technique. Plasma insulin and glucagon were decreased. Glucose uptake failed to increase. Diazoxide administration during period of alpha adrenergic receptor blockade with phentolamine still caused hyperglycemia and increased glucose production. Glucose uptake was inhibited despite adequate plasma insulin. Infusion of somatostatin along with insulin prevented the effects of diazoxide on plasma glucose and glucose production. It is concluded that diazoxide hyperglycemia is not due solely to decreased insulin secretion or increased epinephrine secretion and that glucagon is not a contributory factor. Diazoxide may act directly to increase glucose production and inhibit glucose uptake. Somatostatin appears capable of blocking the effect of diazoxide on glucose production by an unknown mechanism.

Preponderance of beta-adrenergic binding sites in pancreatic islet cells of the rat.

Although the alpha- and beta-adrenergic receptors have opposing effects on insulin secretion, the inhibitory influence of alpha-receptors appears to predominate. To determine if this was due to differences in number and affinity of receptors, isolated rat pancreatic islet cells were incubated with [3H]-dihydroalprenolol and [3H]-dihydroergocryptine as ligands for beta- and alpha-adrenergic binding sites. It was found that the number of beta-adrenergic binding sites was 143 fmol/mg islet protein with a Kd = 0.57 nM. The number of alpha-adrenergic binding sites was 53 fmol/mg protein with a Kd = 0.26 nM. Thus, there are 2.7 times as many beta-adrenergic binding sites as alpha-binding sites, and neither binding site number nor affinity is responsible for the predominant influence of the alpha-adrenergic receptors.

Glucose transport across ocular barriers of the streptozotocin-diabetic rat.

The transport kinetics across the plasma-aqueous and plasma-vitreous barriers were studied in normal and long-term streptozotocin-diabetic rats, using trace amounts of [14C]-L-glucose and [3H]-3-O-methyl-D-glucose. The former is passively transported while the latter uses the same transport-facilitating system as D-glucose. Transport rates of L-glucose were significantly higher in the diabetic rats, with ocular entry rates from the plasma being increased by 69% across both barriers. Thus, the data indicate that in experimental diabetes the passive permeability of the blood-ocular barriers is significantly increased. By contrast, calculated transport rate constants for 3-O-methyl-D-glucose, when adjusted for the hyperglycemia and the increased passive glucose movement, are not altered in the diabetic animal. Nevertheless, there is actually more mass D-glucose movement due to the prevailing hyperglycemia. The present study suggests that although streptozotocin diabetes alters plasma-ocular glucose transport, there is no direct impairment of glucose carrier function. Alterations in transport occurred at both ocular barriers, suggesting that involvement is general and that both the retinal pigment epithelium and the ciliary epithelium may be affected by the diabetes. It is unknown whether the increase in passive movement is related to the prevailing hyperglycemia or to insulin deficiency or other unknown factors.

Failure of injected growth hormone to increase plasma glucose and insulin concentration in the puppy.

The resistance of puppies to the diabetogenic effect of administered growth hormone is well known. The possibility that this involves an adaptive increase in insulin secretion was explored by determining the effect of injected growth hormone on plasma insulin concentration at various age periods. Bovine growth hormone (1 mg/kg per day for 4 days) administered to 5-month-old puppies had no effect on plasma glucose or insulin levels nor on the effectiveness of injected insulin to lower plasma glucose levels. At 8 and 12 months of age the growth hormone regimen increased plasma glucose and insulin levels and produced a resistance to the hypoglycemic effect of injected insulin. It is evident that the difference between the puppy and adult in response to growth hormone is not due to excessive secretion of insulin by the puppy.

Grwoth hormone, prolactin, and cortisol in dogs developing mammary nodules and an acromegaly-like appearance during treatment with medroxyprogesterone acetate.

PIP: Concentrations of (GH) growth hormone, (PRI) prolactin, cortisol, progesterone, and (MPA) medroxyprogesterone acetate were determined by RIA in blood sera collected from beagle bitches 17 months after initiating treatment with MPA (75 mg/kg.3 months; n = 12) MPA vehicle (controls; n = 12), or progesterone implants which produced physiological levels of progesterone (13.8 + or - 2.1 ng/ml; n = 12). In the MPA-treated bitches, mean MPA levels were 104 + or - 6 ng/ml, mean GH levels were elevated (9.5 + or - 3.0 vs. 0.4 + or - 0.1 ng/ml; P 0.01); mean PRL levels were unchanged (13.7 + or - 2.8 vs. 12.6 + or - 1.2 ng/ml); and mean cortisol levels were suppressed (1.7 + or - 0.2 vs. 13.7 + or - 1.4 ng/ml; P 0.01) in comparison to those in control animals. None of these parameters was significantly affected by progesterone treatment. External signs of an acromegaly-like condition and large mammary gland nodules (diameters, 5 mm) were noted in, and limited to, 9 bitches with elevated ( 2.5 ng/ml) GH levels (12.8 + or - 3.0 ng/ml). These were 8 MPA-treated bitches which developed the acromegal-like condition during treatment and 1 progesterone-treated bitch which appeared acromegalic before treatment and in which the condition was considered to have developed spontaneously. The data suggest that the acromegaly-like changes and large mammary nodules in dogs administered the contraceptive progestin MPA occurred as a result of MPA-induced elevations in GH. The results do not preclude the possibility that the MPA-induced suppression of cortisol and/or the direct action of MPA on the mammary glands also contributed to mammary nodule formation. MPA-treated dogs may also provide a unique experimental model for studying chronic elevations in endogenous GH levels and for testing compounds for their ability to suppress GH levels.^ieng

Radioimmunoassay of canine growth hormone: enzymatic radioiodination.

A sensitive radioimmunoassay is described for the measurement of plasma concentrations of canine growth hormone (cGH) as low as 0.25 ng/ml. The assay utilizes enzymatically iodinated cGH and the double antibody technique. The mean plasma concentration of growth hormone in the normal dog after overnight fast is 1.75 plus or minus .17 ng/ml. Exogenous cGH was cleared from the plasma of both the normal and hypophysectomized dog with a mean half-life of 25.6 plus or minus 1.0 min and was distributed in a volume equal to 8.9% of the body weight. Insulin hypoglycemia produced a 3- to 5-fold increase in plasma GH in 4 of 6 dogs and arginine infusion failed to produce a statistically significant rise.

Evidence that the [3H]estradiol-binding protein in pancreas is localized in exocrine cells.

Extracts of rat pancreas contain significant amounts of an [3H]estradiol-binding protein. The amount of steroid-binding activity that could be measured varied considerably depending on the tonicity of the homogenizing medium. High speed supernatants of homogenates initially prepared in isotonic buffer contained about 10% of the binding activity as homogenates prepared in hypotonic buffer. Extraction with hypotonic buffer of pellets obtained by the isotonic procedure yielded most of the remaining [3H]estradiol-binding activity. In an attempt to avoid errors resulting from incomplete homogenization and to detect possible changes in intracellular distribution of [3H]estradiol-binding activity, pancreata were initially homogenized in isotonic buffer and centrifuged at high speed (100,000 X g; 1 hr). The pellet was then extracted with hypotonic buffer and centrifuged again at high speed, and both supernatants were analyzed for [3H]estradiol-binding and amylase activities. Two or 14 days after treatment of male rats with streptozotocin, no apparent decline or redistribution of [3H]estradiol-binding activity to the cytosol was noted despite extensive alteration of beta-islet cells, as determined by electron microscopic examination of sections of these pancreata and significant loss of insulin, as measured by RIA. Amylase activity was unaffected 2 days after streptozotocin treatment, but was depressed to about 1% of control levels at 14 days. Administration of insulin to the latter group of animals resulted in return of amylase to normal levels and a modest increase (approximately 50%) in [3H]estradiol-binding activity. Since amylase levels remained unchanged 2 days after streptozotocin treatment, during which time beta-islet cells were irreversibly altered, and amylase activity was restored to normal levels by insulin treatment after its depletion in chronically treated animals, it follows that neither amylase nor the [3H]estradiol-binding protein could have been associated with beta-islet cells. This was consistent with the observation that M cells (a tumor line of beta-cells only) and 14B cells (a cloned variant of this insulinoma) had neither detectable amounts of amylase nor [3H]estradiol-binding activity. To determine whether estrogen-binding activity was associated with any other type of islet cell, islets of Langerhans were isolated by the sedimentation procedure of Lacy and Kostianovsky. In this procedure, several washing steps are employed to separate the suspended acinar cells from the denser islets that sediment rapidly. During this isolation procedure, the cells from each wash were analyzed for protein, [3H]estradiol-binding protein, and amylase a

Effects of vasopressin on insulin secretion and inositol phosphate production in a hamster beta cell line (HIT).

The recent isolation of vasopressin (VP) from the rat and human pancreas led us to investigate the effects of VP on insulin secretion. In the SV 40-transformed hamster beta cell line (HIT), 0.1-1.0 nM VP caused rapid stimulation of insulin secretion. Slight but significant inhibition of insulin secretion was observed in the presence of 10 pM VP. These effects of VP on insulin secretion were paralleled by dose-dependent changes in inositol phosphate (IP) production, indicating mediation by V1-type VP receptors. VP stimulated IP3 production at 30 sec and production of IP1 by 60 sec. VP (0.1 nM to 1 microM) failed to stimulate the release or cellular content of cAMP, whereas forskolin was an effective stimulus. Forskolin and VP together caused at least additive stimulation of insulin secretion. Taken together, these observations indicate that VP is not acting via V2-mediated pathways. However, VP-induced stimulation of insulin and IP production were only slightly inhibited by a V1a pressor antagonist in 100- or 1,000-fold excess, indicating that VP effects are not mediated by V1a receptors. The V1 receptor involved may represent a V1b or a novel type of VP receptor. These observations suggest a potential physiological role of VP in regulating insulin secretion.

Onset of changes in glucose transport across ocular barriers in streptozotocin-induced diabetes.

In a previous study, measurements were made of facilitated and passive transport of glucose, using [3H]-3-O-methyl-D-glucose and [14C]-L-glucose, respectively, across blood-aqueous and blood-vitreous barriers in long-term streptozotocin-diabetic rats. It was found that passive transport was increased, while facilitated transport was decreased, possibly due to saturation of the transport system. The present study examines the appearance of these changes in glucose transport at various times following streptozotocin (STZ) injection. Passive transport, as indicated by the L-glucose rate constant, began to increase at about 10 days following induction of diabetes, stabilized at the elevated rates at 50-60 days and persisted during the 170-day period of observation. Rate constants for [3H]-3-O-methyl-D-glucose transport decreased within 1 day following induction of diabetes. Prevention of hyperglycemia by insulin treatment upon onset of diabetes prevented the latter changes ruling out a direct effect of STZ. Glucose infusion into normal rats produced a similar decrease in 3-O-methylglucose transport constants suggesting that hyperglycemia was responsible for the early decrease in facilitated transport found in the diabetic rats. It is speculated that increased passive transport of glucose may reflect an early loss in ocular barrier integrity. The later decrease in carrier facilitated transport cannot be explained by hyperglycemia alone and, thus, a loss in carrier function is suggested. Despite a decrease in facilitated transport, absolute glucose entry rates are increased in the diabetic due to elevated plasma glucose, which serves as an inward driving force, due to the significantly increased entry of glucose by the passive route.

The metabolic effects of oxytocin are mediated by a uterine type of receptor and are inhibited by oxytocin antagonist and by arginine vasopressin in the dog.

Infusion of oxytocin (OT) into normal dogs, in doses which produced plasma levels of OT in the physiological range, has been shown to increase plasma levels of glucose, insulin and glucagon and increase rates of glucose production and uptake. This study sought to determine whether there was a correlation between these metabolic effects and the oxytocic potency of four less potent oxytocic analogues when infused into normal dogs. The rank order of oxytocic potency of all 4 correlated well with the rise in plasma glucose levels, and in 3 of the 4 with the rise in plasma insulin levels. An antagonist of the oxytocic effect of OT suppressed the usual OT-induced rise in plasma glucose, insulin and glucagon as well as the increased glucose production and uptake. Arginine vasopressin (AVP) infusion, which by itself did not produce any metabolic effects, blocked completely the effects of OT infusion to raise plasma glucose and insulin levels and increase glucose production and uptake. The data suggest that the metabolic effects of OT in the dog are mediated by OT receptors that are similar to those producing the oxytocic effects. Whether the inhibition by AVP of the metabolic and hormonal effects of OT occurs at the receptor or post receptor level or via other mechanisms remains to be determined.

Increased release of cyclic adenosine monophosphate into jugular vein in response to isoproterenol administration.

Catecholamine administration elevates plasma cyclic AMP (cAMP) levels but the source of the cAMP is unknown. To determine possible sources, plasma cAMP levels were determined in blood vessels across the head, liver, kidney and lung in anesthetized dogs infused with the beta-adrenergic agonist, isoproterenol. Only the head showed an increased release of cAMP into the blood. The kidneys removed cAMP from the blood while liver and lung showed no change. This in vivo demonstration of release of cAMP from the head represents contributions from brain and facial muscles and may be a useful approach to study brain involvement in the action of various hormones and drugs.

Decreased insulin sensitivity and glucose tolerance in spontaneous canine hyperadrenocorticism.

Alterations in carbohydrate metabolism were evaluated in 60 dogs with untreated hyperadrenocorticism by measuring basal concentrations of plasma glucose and insulin and performing glucose and insulin tolerance tests. The 60 dogs could be divided into four groups based on paired glucose and insulin concentrations. Eight dogs had normal concentrations of both glucose and insulin. Twenty-four dogs (40 per cent) were euglycaemic with mild to moderate hyperinsulinaemia whereas 23 (38 per cent) had moderate hyperglycaemia with moderate to severe hyperinsulinaemia. The five dogs (8 per cent) with overt ketoacidotic diabetes mellitus had relative insulin deficiency. Of eight dogs tested, six had intravenous glucose intolerance; all of the dogs had increased total insulin secretion during glucose tolerance testing. Resistance to the hypoglycaemic effect of exogenous insulin was demonstrated in six of the seven dogs tested. In 20 dogs that had basal glucose and insulin again determined after correction of hyperadrenocorticism, mean concentrations of both glucose and insulin decreased into the normal range.

Clonidine or xylazine as provocative tests for growth hormone secretion in the dog.

Base-line values of plasma growth hormone (GH) are low in most species, requiring provocative tests to assess GH deficiency. Clonidine, an antihypertensive drug, and its analogue, xylazine, a sedative hypnotic, were found to stimulate GH secretion. Administration (IV) of clonidine to conscious healthy, dogs at doses of 30, 16.5, and 3 microgram/kg produced significant increases in plasma GH by 15 minutes and the effects subsided by 120 minutes. Plasma glucose concentration increased slowly with all doses, but less so and for shorter duration in dogs given the 3 microgram/kg dose. Xylazine increased plasma GH when injected at doses of 300 and 100 microgram/kg, but not at 30 microgram/kg. Plasma glucose increased only with the 300 microgram/kg dose. The alpha-adrenergic blocker, phentolamine, markedly attenuated these responses. Thus, both clonidine and xylazine, when used at appropriate doses, can stimulate GH secretion, with minimal effect on plasma glucose and without causing significant sedation.

Suppression of growth hormone secretion in spontaneous canine hyperadrenocorticism and its reversal after treatment.

The plasma growth hormone response to the provocative agent, xylazine, was assessed in 4 dogs with spontaneous hyperadrenocorticism, before and after therapy. Before treatment of the hyperadrenocorticism, no significant increase in growth hormone concentration occurred in any of the dogs following the administration of xylazine. A significant increase in growth hormone concentrations following xylazine administration occurred in 2 of the 3 dogs with hypophysis (pituitary)-dependent hyperadrenocorticism after treatment with mitotane (o,p'-DDD) and in 1 dog after surgical removal of a hyperfunctional adrenal adenoma. Although the impaired growth hormone response persisted in 1 dog, the administration of xylazine was repeated in this animal after only 3 weeks of mitotane therapy; it is likely that growth hormone unresponsiveness would reverse if the hyperadrenocorticism were controlled for a longer period. These findings demonstrate that in dogs, as in persons, the excessive production of endogenous corticosteroids associated with either hypophysis-dependent hyperadrenocorticism or hyperfunctional adrenal tumor can induce suppression of growth hormone release which is reversible following treatment.

Thymic abnormalities and growth hormone deficiency in dogs.

A high frequency of occurrence of a wasting disease, unthriftiness, and retarded growth was observed in a group of inbred Weimaraner dogs. Affected pups had a small thymus gland, with a marked absence of thymic cortex. A litter of eight pups from a sire and dam that were known to have produced affected offspring was chosen for further study. The pups had normal concentrations of WBC and gamma-globulins and were able to produce antibody in response to Brucella abortus. Two pups in the litter developed a wasting syndrome and responded well to therapy with thymosin fraction 5. One pup that survived the wasting syndrome had a significant (P < 0.05) depression of its lymphocyte blastogenic response to phytohemagglutinin compared with its surviving littermates. Pups from this litter also lacked a normal increase in plasma growth hormone concentration after the injection of clonidine HCl. These pups had concurrent abnormalities of the thymus-dependent immune function and in growth hormone metabolism. The syndrome in these pups has some features in common with the syndrome in the Ames or Snell-Bagg strains of immunodeficient dwarf mice.

Acromegaly associated with transient overproduction of growth hormone in a dog.

A 6-year-old female crossbred Belgian Shepherd Dog with features of acromegaly was monitored for almost 4 years. The history of frequent and excessive administration of a progestational agent suggested that the progestational drug induced the acromegaly. During the monitoring period. soft tissue changes diminished and there was normalization of several factors: plasma growth hormone concentration, response of plasma insulin and glucose to an oral glucose load, and response of plasma glucose hormone to the injection of insulin.