Stewart A. Metz

Induction of Defective Insulin Secretion and Impaired Glucose Tolerance by Clonidine Selective Stimulation of Metabolic Alpha-adrenergic Pathways

Clonidine is an antihypertensive drug that markedly suppresses plasma catecholamine levels. In an attempt to investigate the effects of suppression of endogenous catecholamine secretion on glucose homeostasis and glucoregulatory hormones, we administered oral clonidine to 20 normal male subjects, two patients with transsections of the cervical spinal cord, and one patient with an insulinoma. Unexpectedly, after a single dose of 0.5 ing. given to normal subjects, insulin concentrations fell and plasma glucose rose 12 ± 1 mg./dl. (p < 0.001; n = 6) without an increase in glucagon. After four days of clonidine treatment, plasma glucose again was elevated (Δ = +11 ± 3 mg./dl.; p < 0.01), while basal plasma insulin and glucagon levels remained unchanged. The acute insulin response to an intravenous glucose injection of 5 gm. decreased from 33 ± 7 to 11 ± 2 μU./ml. (n = 8; p < 0.01), and KG declined from 0.80 ± 0.13 to 0.53 ± 0.06 per cent per minute (p < 0.01). Similar responses were seen in the paraplegic patients. In contrast, expected responses to clonidine were not observed in the insulinoma patient preoperatively; these normal responses were restored after resection of the tumor. All metabolic alterations induced by clonidine were reversed by infusion of phentolamine, an alpha-adrenergic blocking agent. We conclude that clonidine causes hyperglycemia and inhibits insulin secretion through an alpha-adrenergic mechanism. Since plasma catecholamine concentrations and vascular tone were markedly reduced by clonidine, this drug appears to selectively stimulate metabolic adrenergic pathways different from those operative in vasomotor regulation.

Inhibition of Prostaglandin E Synthesis Augments Glucose-induced Insulin Secretion in Cultured Pancreas

Monolayer cultures of neonatal rat pancreatic cells were examined to ascertain whether they synthesize prostaglandin E (PGE) and to determine the effects on insulin secretion caused by PGE and drugs that inhibit its synthesis. PGE release into the medium was observed. Sodium salicylate and ibuprofen (at drug concentrations similar to those achieved therapeutically in humans in vivo) inhibited PGE synthesis in a doseresponsive fashion to a maximum of 70–80% inhibition. Inhibition of PGE synthesis was accompanied by augmented insulin secretion. Both PGE synthesis inhibitors shifted the glucose dose-insulin response curves to the left at low glucose concentrations and augmented maximal insulin release at high glucose concentrations. Increments in glucose-induced insulin secretion induced by sodium salicylate correlated well (r = 0.89) with inhibition of PGE synthesis and addition of exogenous PGE1 to the cultures reversed the augmenting effects of the drug on insulin secretion. It is concluded that cultures of pancreatic cells synthesize PGE and that a function of PGE in these cultures appears to be a tonic negative modulation of glucose-induced insulin secretion.

A role for the lipoxygenase pathway of arachidonic acid metabolism in glucose- and glucagon-induced insulin secretion

Although the cyclo-oxygenase pathway of arachidonic acid (AA) metabolism inhibits glucose-stimulated insulin release through synthesis of prostaglandins, very little attention has been given to the effects of lipoxygenase pathway products on beta cell function. We have examined the effects of two structurally-dissimilar lipoxygenase inhibitors on insulin release from monolayer-cultured rat islet cells. Both nordihydroguaiaretic acid (NDGA, 20-50 microM) and BW755c (100-250 microM) caused a dose-responsive inhibition of glucose-induced insulin release. This inhibitory effect occurred despite concomitant inhibition of prostaglandin E synthesis. Lipoxygenase inhibitors also impeded cyclic AMP accumulation. Insulin and cyclic AMP release induced by glucagon were also blunted. These studies suggest the hypothesis that AA released in or near the beta cell is metabolized to lipoxygenase product(s) which have feed-forward properties important to glucose- and glucagon-stimulated cyclic nucleotide accumulation and insulin release.

Selective deficiency of 1,25-dihydroxycholecalciferol. A cause of isolated skeletal resistance to parathyroid hormone.

To investigate the role of vitamin D metabolites in the pathogenesis of pseudohypoparathyroidism, we studied an elderly man with a unique variant of the disease, which was characterized by hypocalcemia, elevated serum parathyroid hormone (513 +/- 13 pg per milliliter, mean +/- S.E.M., normal, less than 450) but normal renal responses (phosphate and cyclic AMP) to exogenous parathyroid extract. Treatment with parathyroid extract did not produce a calcemic effect, suggesting an isolated skeletal hyporesponsiveness to parathyroid hormone. Although 25-hydroxyvitamin D levels were not reduced, levels of 1,25-dihydroxycholecalciferol were extremely low (0.52 ng per deciliter; normal 3.3 +/- 0.06, S.D.). Treatment with 1,25-dihydroxycholecalciferol (1 microgram by mouth per day for four days) increased circulating levels to normal (4.60 ng per deciliter) and restored to normal the calcemic response to parathyroid (change in calcium 3.0 mg per deciliter). These data suggest that 1,25-dihydroxycholecalciferol deficiency may explain the skeletal resistance, but not the renal resistance, often present in classic pseudohypoparathyroidism.

Applications and limitations of measurement of 15-keto,13,14-dihydro prostaglandin E2 in human blood by radioimmunoassay.

It has been anticipated that the inherent limitations of radioimmunoassays for prostaglandin E (PGE) would be obviated by assays for its major circulating metabolite, 15-keto, 13,14-dihydro PGE2) which has a longer half-life in blood. We examined the effects of PGE2 infusion and alterations in lipolysis in vivo, and of clotting, prolonged storage and hemolysis in vitro, on KH2-PGE2 immunoreactivity in unextracted human plasma and serum samples. Indeed KH2-PGE2 levels rose several hundred fold during infusions of PGE2 at doses which cause little or no increment in peripheral PGE levels. During stimulation of lipolysis by infusions of epinephrine, apparent KH2-PGE2 levels rose five-fold. However, the dilution curve of plasma obtained during stimulation of lipolysis was not parallel to the standard curve; furthermore, apparent KH2-PGE2 levels were correlated strongly with free fatty acid (FFA) levels, suggesting that FFAs cross-reacted in the RIA weakly but significantly due to their very high molar concentration in blood. Clotting and prolonged storage of samples, but not hemolysis, also caused marked apparent increments in KH2-PGE2 levels. Competition curves using dilutions of such samples were again not parallel to the standard curves in plasma or buffer, but resembled dilution curves of samples containing high levels of FFA. These results suggest that handling of human blood samples for KH2-PGE2 measurement must be carefully standardized to avoid significant artifacts which presumably are due in part to fatty acids released from triglyceride stores in vivo or from disrupted membrane phospholipids in vitro. Unextracted plasma appears to be unsatisfactory for use in this RIA.

Ectopic secretion of chorionic gonadotropin by a lung carcinoma: Pituitary gonadotropin and subunit secretion and prolonged chemotherapeutic remission☆

The ability of tumor markers to improve cancer therapy is not established. We studied a man with a human chorionic gonadotropin (HCG)-secreting large cell carcinoma of the lung and gynecomastia. Preoperatively, levels of HCG (109 ng/ml), its alpha and beta subunits (3.2 and 21 ng/ml, respectively) and plasma estradiol were elevated. Despite apparently complete tumor resection and total resolution of gynecomastia, HCG titers remained elevated (3.3 ng/ml), heralding tumor recurrence three weeks later. Because the pathophysiologic consequences of the ectopic secretion of HCG on pituitary function are not established, we administered 100 microgram of gonadotropin-releasing hormone (LHRH) and observed a markedly delayed increase in pituitary gonadotropins. Early chemotherapy, guided by persistence of HCG, reduced HCG to undetectable levels, restored to normal the response to LHRH and resulted in a distinctly unusual 30-month complete remission. Use of HCG as a tumor marker levels is more sensitive than the symptom of gynecomastia and may permit detection of small, potentially curable tumor foci.

Modulation of insulin secretion by cyclic AMP and prostaglandin E: The effects of theophylline, sodium salicylate and tolbutamide

Abstract Cyclic AMP (cAMP) and prostaglandin E (PGE) are acknowledged to be local modulators of insulin secretion in response to physiologic stimuli. We have attempted to define the mechanisms of action of distinct classes of pharmacologic insulin secretagogues (nonsteroidal anti-inflammatory drugs, methylxanthines and sulfonylureas) by examining their dependence on changes in cAMP and PGE accumulation in monolayer cultures of neonatal rat pancreatic cells. As we have previously observed, sodium salicylate (SS, 20 mg/dl) stimulated insulin secretion in proportion to the ambient glucose concentration. SS potently inhibited PGE synthesis and decreased cAMP accumulation; exogenous PGE 1 reversed the effect on insulin secretion. Similar results were found using ibuprofen, a structurally dissimilar inhibitor of prostaglandin synthesis. Theophylline (1.5 mM) also stimulated insulin secretion in a fashion dependent on glucose concentration. However, in contrast to SS, theophylline (THEO) augmented cAMP accumulation without reducing PGE synthesis. Furthermore, the effects of THEO and SS on insulin release were additive. Exogenous PGE 1 did not reverse the effects of THEO on insulin secretion. The effects of sulfonylureas were markedly different from those of both SS and THEO. Stimulation of insulin release by tolbutamide (TOLB, 2 mg/dl) was accompanied by only minor and inconsistent inhibition of PGE synthesis. The effect of TOLB was not reversed by exogenous PGE 1 and was largely independent of ambient glucose concentration. Additionally, the stimulatory effect of SS on insulin secretion was additive to that of a maximally effective concentration of TOLB. Accumulation of cAMP could also not be implicated in TOLB action. Tolbutamide failed to augment cAMP generation, and its stimulatory action on insulin secretion was additive to that of a maximally stimulatory concentration of THEO. Virtually identical results were observed with a second sulfonylurea, chlorpropamide. We conclude that stimulation of insulin secretion by certain distinct classes of pharmacologic agents may be defined by changes in cAMP synthesis in some cases and in PGE availability in others. In the case of the sulfonylureas, other mechanisms must be sought.

A Role for Endogenous Prostaglandins in Defective Glucose Potentiation of Nonglucose Insulin Secretagogues in Diabetics

Noninsulin dependent diabetics have insulin responses to nonglucose secretagogues that are subnormal for their plasma glucose levels. Since endogenous prostaglandins have been implicated in the abnormal insulin responses to glucose in diabetics, the present study was performed to explore whether prostaglandins might also play a role in the defective insulin responses to nonglucose stimuli. We examined the effects of infusions of either prostaglandin E2 (PGE2) or sodium salicylate (SS), a PG synthesis inhibitor, on the acute insulin responses (AIRs) to arginine and isoproterenol and on the glucose potentiation of the insulin response to arginine in both normal and diabetic subjects. The AIR to arginine was augmented by SS in diabetics (SS = 61 +/- 12 microunits/ml, control = 37 +/- 5 microunits/ml, n = 11, p less than .01). SS, however, had no effect on the AIR to arginine in normal subjects (SS = 39 +/- 4 microunits/ml. control = 34 +/- 4 microunits/ml, n = 6, p = ns). Similarly, SS augmented the AIR to an isoproterenol pulse in diabetics (SS = 38 +/- 9 microunits/ml, control = 18 +/- 3, n = 9, p less than .05) but not in normal subjects (SS = 19 +/- 4 microunits/ml, control = 21 +/- 4 microunits/ml, n = 8, p = ns), suggesting a SS-sensitive defect in the insulin response to these nonglucose stimuli in diabetics. Conversely, PGE2 inhibited the AIR to arginine in diabetics (PGE = 28 +/- 5 microunits/ml, control = 39 +/- 7 microunits/ml, n = 7, p less than .05), but not in normal subjects (PGE = 74 +/- 7 microunits/ml, control = 80 +/- 14 microunits/ml, n = 5, p = ns). The effect of SS on glucose potentiation of the AIR to arginine was studied by measuring the AIR to arginine at two different levels of plasma glucose, one before and one after an insulin infusion, with glucose potentiation defined as the ratio delta AIR/delta prestimulus glucose. Glucose potentiation was significantly less in diabetics than in normals and SS significantly improved glucose potentiation toward normal values in diabetics but did not change glucose potentiation in normals. These findings suggest that endogenous PGs may play a role in the defective glucose potentiation of the AIR to nonglucose secretagogues in diabetics resulting in impaired insulin responses to these stimuli. This defect is partially reversible by an inhibitor of PG synthesis.

Feedback moduiaticn of gilcose-induced insulin secretion by arachidonic acid meiabol: Possible molecular mechanisms and relevance to diabetes mellitus

Recent evidence suggests that glucose stimulation of insulin release may trigger a classic negative feedback loop involving local release of an inhibitor of beta cell function. One or more metabolite of arachidonic acid could comprise such putative system. Several metabolic events triggered by glucose-induced stimulus-secretion coupling (such as calcium influx, membrane turnover, augmented reduced pyridine nucleotide or glutathione levels, and alterations in toxic oxygen radical availability) would be expected to alter arachidonic acid release and subsequent metabolism via the lipoxygenase or cyclo-oxygenase pathways. At lease one arachidonate derivative (prostaglandin E) inhibits insulin secretion, and several inhibitors of prostaglandin synthesis augment glucose-induced insulin release in normal subjects and type II diabetics. Development of more selective inhibitors of arachidonate metabolism could represent a new approach to therapeutic manipulation of beta cell function.

Effects of clonidine on hormone and substrate responses to hypoglycemia.

We have reported that clonidine (CLON) reduces basal norepinephrine (NE) levels but has no effect on basal epinephrine (E) levels. To study the effects of CLON on stimulated NE and E secretion, six normal men received a single dose of CLON followed by induction of hypoglycemia with insulin. Despite the presence of a mean hypoglycemic nadir of 44 mg/100 ml, there was no rise in E and NE levels during CLON treatment. To determine the effects of such catecholamine inhibition on the counterregulation of hypoglycemia, additional experiments were performed since the mean glucose nadir during CLON in the initial studies was slightly (9 mg/100 ml) less deep than in controls. When subjects on CLON received higher doses of insulin, glucose responses were identical to control responses; CLON still reduced the 0 to 60‐min catecholamine response by 83% (p < 0.02). Glucagon secretion was not impaired, but CLON blunted the early rate of glucose recovery in the first 15 min after the glucose nadir. We conclude that CLON inhibits the catecholamine (but not the glucagon) rise during insulin‐induced hypoglycemia. Selective inhibition of catecholamine secretion does not inhibit the glucogan response but leads to definite, although transient, inhibition of glucose recovery after hypoglycemia.