Tolbutamide-stimulated Insulin Release Research Articles

High heritability and genetic correlation of intravenous glucose- and tolbutamide-induced insulin secretion among non-diabetic family members of type 2 diabetic patients

The aim of this study was to estimate the heritability of quantitative measures of glucose regulation obtained from a tolbutamide-modified frequently sampled IVGTT (t-FSIGT) and to correlate the heritability of the glucose-stimulated beta cell response to the tolbutamide-induced beta cell response. In addition, single nucleotide polymorphisms (SNPs) having an exclusive effect on either glucose- or tolbutamide-stimulated insulin release were identified. Two hundred and eighty-four non-diabetic family members of patients with type 2 diabetes underwent a t-FSIGT with intravenous injection of glucose at t = 0 min and tolbutamide at t = 20 min. Measurements of plasma glucose, serum insulin and serum C-peptide were taken at 33 time points from fasting to 180 min. Insulin secretion rate, acute insulin response (AIR), disposition index (DI) after glucose and disposition index after tolbutamide (DIT), insulin sensitivity (SI), glucose effectiveness (SG) and beta cell responsiveness to glucose were calculated. A polygenic variance component model was used to estimate heritability, genetic correlations and associations. We found high heritabilities for acute insulin secretion subsequent to glucose stimulation (AIRglucose h (2) ± SE: 0.88 ± 0.14), but these were slightly lower after tolbutamide (AIRtolbutamide h (2) ± SE: 0.69 ± 0.14). We also estimated the heritabilities for SI (h (2) ± SE: 0.26 ± 0.12), SG (h (2) ± SE: 0.47 ± 0.13), DI (h (2) ± SE: 0.56 ± 0.14), DIT (h (2) ± SE: 0.49 ± 0.14) and beta cell responsiveness to glucose (h (2) ± SE: 0.66 ± 0.12). Additionally, strong genetic correlations were found between measures of beta cell response after glucose and tolbutamide stimulation, with correlation coefficients ranging from 0.77 to 0.88. Furthermore, we identified five SNPs with an exclusive effect on either glucose-stimulated (rs5215, rs1111875, rs11920090) or tolbutamide-stimulated (rs10946398, rs864745) insulin secretion. Our data demonstrate that both glucose- and tolbutamide-induced insulin secretions are highly heritable traits, which are largely under the control of the same genes.

Free fatty acid receptor 1 (FFA1R/GPR40) and its involvement in fatty-acid-stimulated insulin secretion

Free fatty acids (FFA) have generally been proposed to regulate pancreatic insulin release by an intracellular mechanism involving inhibition of CPT-1. The recently de-orphanized G-protein coupled receptor, FFA(1)R/GPR40, has been shown to be essential for fatty-acid-stimulated insulin release in MIN6 mouse insulinoma cells. The CPT-1 inhibitor, 2-bromo palmitate (2BrP), was investigated for its ability to interact with mouse FFA(1)R/GPR40. It was found to inhibit phosphatidyl inositol hydrolysis induced by linoleic acid (LA) (100 muM in all experiments) in HEK293 cells transfected with FFA(1)R/GPR40 and in the MIN6 subclone, MIN6c4. 2BrP also inhibited LA-stimulated insulin release from mouse pancreatic islets. Mouse islets were subjected to antisense intervention by treatment with a FFA(1)R/GPR40-specific morpholino oligonucleotide for 48 h. Antisense treatment of islets suppressed LA-stimulated insulin release by 50% and by almost 100% when islets were pretreated with LA for 30 min before applying the antisense. Antisense treatment had no effect on tolbutamide-stimulated insulin release. Confocal microscopy using an FFA(1)R/GPR40-specific antibody revealed receptor expression largely localized to the plasma membrane of insulin-producing cells. Pretreating the islets with LA for 30 min followed by antisense oligonucleotide treatment for 48 h reduced the FFA(1)R/GPR40 immunoreactivity to background levels. The results demonstrate that FFA(1)R/GPR40 is inhibited by the CPT-1 inhibitor, 2BrP, and confirm that FFA(1)R/GPR40 is indeed necessary, at least in part, for fatty-acid-stimulated insulin release.

Drug-Induced Desensitization of Insulinotropic Actions of Sulfonylureas

KATP-channel-dependent and KATP-channel-independent insulin-releasing actions of the sulfonylurea, tolbutamide, were examined in the clonal BRIN-BD11 cell line. Tolbutamide stimulated insulin release at both nonstimulatory (1.1 mM) and stimulatory (16.7 mM) glucose. Under depolarizing conditions (16.7 mM glucose plus 30 mM KCl) tolbutamide evoked a stepwise KATP channel-independent insulinotropic response. Culture (18 h) with tolbutamide or the guanidine derivative BTS 67 582 (100 μM) markedly reduced (P < 0.001) subsequent responsiveness to acute challenge with tolbutamide, glibenclamide, and BTS 67 582 but not the imidazoline drug, efaroxan. Conversely, 18 h culture with efaroxan reduced (P < 0.001) subsequent insulinotropic effects of efaroxan but not that of tolbutamide, glibenclamide, or BTS 67 582. Culture (18 h) with tolbutamide reduced the KATP channel-independent actions of both tolbutamide and glibenclamide. Whereas culture with efaroxan exerted no effect on the KATP channel-independent actions of sulfonylureas, BTS 67 582 abolished the response of tolbutamide and inhibited that of glibenclamide. These data demonstrate that prolonged exposure to tolbutamide desensitizes both KATP-channel-dependent and -independent insulin-secretory actions of sulfonylureas, indicating synergistic pathways mediated by common sulfonylurea binding site(s).

Increased cytosolic calcium. A signal for sulfonylurea-stimulated insulin release from beta cells.

The mechanisms by which glyburide and tolbutamide signal insulin secretion were examined using a beta cell line (Hamster insulin-secreting tumor (HIT) cells). Insulin secretion was measured in static incubations, free cytosolic Ca2+ concentration ([Ca2+]i) was monitored in quin 2-loaded cells, and cAMP quantitated by radioimmunoassay. Insulin secretory dose-response curves utilizing static incubations fit a single binding site model and established that glyburide (ED50 = 112 +/- 18 nM) is a more potent secretagogue than tolbutamide (ED50 = 15 +/- 3 microM). Basal HIT cell [Ca2+]i was 76 +/- 7 nM (mean +/- S.E., n = 141) and increased in a dose-dependent manner with both glyburide and tolbutamide with ED50 values of 525 +/- 75 nM and 67 +/- 9 microM, respectively. The less active tolbutamide metabolite, carboxytolbutamide, had no effect on [Ca2+]i or insulin secretion. Chelation of extracellular Ca2+ with 4 mM EGTA completely inhibited the sulfonylurea-induced changes in [Ca2+]i and insulin release and established that the rise in [Ca2+]i came from an extracellular Ca2+ pool. The Ca2+ channel blocker, verapamil, inhibited glyburide- or tolbutamide-stimulated insulin release and the rise in [Ca2+]i at similar concentrations with IC50 values of 3 and 2.5 microM, respectively. At all concentrations tested, the sulfonylureas did not alter HIT cell cAMP content. These findings provide direct experimental evidence that glyburide and tolbutamide allow extracellular Ca2+ to enter the beta cell through verapamil-sensitive, voltage-dependent Ca2+ channels, causing a rise in [Ca2+]i which is the second messenger that stimulates insulin release.

Effects of fetal insulin secretory deficiency on metabolism in fetal lamb.

Fetal insulin secretion may be of importance in determining both fetal metabolic rate and glucose homeostasis in the resting state. To investigate this question, streptozocin (STZ) was injected into 10 late-gestation fetal lambs, and the effects of STZ on fetal pancreatic insulin storage and secretion, fetal metabolic rate, and umbilical glucose uptake were then studied. Fetal STZ injection caused a relative fetal hyperglycemia by 24 h after injection. Fetal hyperglycemia reached a maximum by 72 h and persisted for at least 10 days after injection. Neonates delivered after fetal injection were frankly diabetic. Fetal STZ injection was associated with complete suppression of both glucose- and tolbutamide-stimulated insulin release, although no changes in peripheral insulin concentration were observed when compared with controls. Fetal pancreatic insulin content was only 13% of that expected on the basis of gestational age. In a subgroup of 7 STZ-treated fetal lambs, fetal hyperglycemia was related to decrements in umbilical venoarterial difference of glucose, umbilical glucose uptake, and glucose-O2 quotient. No changes in maternal glucose homeostasis or in fetal O2 consumption were noted. The data suggest that deficient fetal insulin storage and secretion are associated with a decrement in exogenous fetal glucose entry but not in fetal metabolic rate. Whether the observed fetal changes relate to enhanced endogenous fetal glucose production with a passive decrease in maternofetal glucose transfer or are simply due to a decrease in overall fetal glucose utilization is not known. It is speculated that a quantitative decrease in pancreatic insulin secretion is responsible for the observed changes.

Dynamics of pancreatic insulin release in young Zucker rats: a heterozygote effect.

Total pancreatic insulin, dynamic insulin response to glucose (325 mg/dl), or tolbutamide (40 mg/dl) using the isolated perfused pancreas preparation and body composition were determined for 2- and 4-wk-old homozygous lean Fa/Fa, heterozygous lean Fa/fa, unknown lean Fa/??, and homozygous obese fa/fa female Zucker rats. At 2-wk, obese rats (body fat greater than 16%) released significantly more insulin than homozygous lean rats during first (min 10-16) and second phases (min 17-70) (61 vs. 30 ng and 637 vs. 255 ng, respectively). Plasma insulinemia was 177 microU/ml in obese, compared with 51 microU/ml in homozygous lean rats. The dynamic response of unknown lean followed the pattern of either the homozygous lean or the obese rats. There was no significant difference in total pancreatic insulin among any of the groups at 2 wk of age (Fa/Fa, 1.72; Fa/??, 2.01; and Fa/Fa, 1.80 micrograms). At 4 wk, the dynamic response by obese (2.98 micrograms) was similar to that of heterozygous (3.31 micrograms), both being significantly greater than the homozygous lean (1.14 micrograms) or unknown lean rats (1.77 microgram). Total pancreatic insulin in 4-wk obese (19.8 micrograms) was greater than homozygous lean rats (14.5 micrograms). Tolbutamide-stimulated insulin release was significantly greater in 4-wk obese than homozygous lean rats. Exposure to tolbutamide reduced by 60% the second-phase insulin release in both obese and homozygous lean rats during a subsequent 40-min glucose (325 mg/dl) perfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of somatostatin on insulin and glucagon in patients with insulinoma

Effects of somatostatin on fasting and arginine-or tolbutamide-stimulated insulin release were studied in four patients with insulinoma. Somatostatin (bolus or bolus + infusion) reduced fasting insulin values in all patients; insulin response to tolbutamide was partially reduced in two patients; somatostatin bolus impaired the insulin response to arginine. Fasting glucagon levels and glucagon response to arginine were also reduced by somatostatin. These results indicate the potential usefulness of somatostatin in the diagnosis of insulinoma even if its effect on insulin is only partial.

Hyperinsulinemic Hypoglycemia: Effect of Epinephrine Suppression

The inhibitory effect of epinephrine on basal and tolbutamide-stimulated insulin release was studied in 5 patients with hyperinsulinemic hypoglycemia. Epinephrine inhibited both basal and tolbutamide-induced insulin release in patients with beta-cell adenoma and hyperplasia, but failed to inhibit insulin release in a patient with beta-cell carcinoma. The inhibition of basal insulin with epinephrine was maximum in patients with beta-cell hyperplasia. This differential inhibitory effect of epinephrine on insulin release may prove to be a useful screening test in the preoperative diagnosis of the nature of the lesion producing hyperinsulinemia.

The inhibitoty effect of DDT on insulin secretion in mice

A single oral dose of 50 mg/kg of DDT administered to mice 18 hr prior to experimentation was found to reduce glucose tolerance without affecting the basal blood concentration of glucose. Also, the treatment inhibited both d-glucose and tolbutamide-stimulated insulin release by isolated pancreatic islets. Hyperglycemia was not noted during the 18-hr period after DDT. The effect of DDT on glucose tolerance persisted for at least 1 week. Reduced pancreatic secretory activity after DDT administration is consistent with the increased activity of gluconeogenic enzymes as well as decreased glycogen synthetase activity reported by earlier workers.

The relative roles of calcium, phosphorus, and parathyroid hormone in glucose- and tolbutamide-mediated insulin release.

The relative contributions of Ca++, phosphorus, and parathyroid hormone (PTH) on insulin secretion were evaluated in three groups of dogs. Dogs were studied with glucose infusions (group I) or standard intravenous glucose tolerance tests (IVGTT) (group II) before and after the development of diet-induced hypophosphatemia. Mean serum phosphorus levels for both groups fell from 4.1 to 1.1 mg/100 ml. Animals in group I demonstrated a fall in glucose disappearance rates (Kg) from 5.3+/-0.6% min to 3.5+/-0.5% after induction of hypophosphatemia (P less than 0.001). Mean insulin response was significantly greater in the hypophosphatemic animals than in controls in this group. In group II animals, mean insulin areas obtained during the IVGTT increased from 1,426+/-223 to 2,561+/-141 muU/ml/60 min after induction of hypophosphatemia, and were unaffected by Ca++ or PTH administration. Ca++ administration, but not hypophosphatemia or PTH infusion, increased significantly the mean insulin response to tolbutamide. Secondary hyperparathyroidism was induced by dietary manipulation in four dogs (group III). Mean PTH values increased from 71.4+/-2.1 to 3,012+/-372 pg/ml (P less than 0.001). Mean insulin response to an IVGTT was similar to group III animals, but increased from 1,352+/-128 to 1,894+/-360 muU/ml/60 min after the excessive dietary phosphorus was reduced for 3 mo, and plasma phosphorus fell from 3.2+/-0.1 to 2.8+/-0.3 mg/100 ml. PTH values decreased to 647+/-53 pg/ml. The insulin response to tolbutamide was comparable to that in group II animals, but increased significantly after calcium administration. Immunoreactive insulin disappearance rates were unaffected by hypophosphatemia or diet-induced secondary hyperparathyroidism. These data demonstrate that hypophosphatemia is associated with an augmented glucose-stimulated insulin release, without any effect on tolbutamide-stimulated insulin release. Hypercalcemia produces an augmented tolbutamide-stimulated insulin release with no apparent effect on glucose-stimulated insulin release. Finally, PTH does not appear to be an insulin antagonist and has no apparent effect on either glucose- or tolbutamide-stimulated insulin release in animals with dietary-induced secondary hyperparathyroidism.

Effects of drugs on glucose and tolbutamide-stimulated insulin release from isolated rat islets of Langerhans

The effects of acetylsalicylic acid, sulphadimidine, phenylbutazone and chlorpromazine on glucose- and tolbutamide-stimulated insulin release by isolated rat islets of Langerhans have been investigated. Acetylsalicylic acid did not influence glucose-stimulated insulin secretion; although at 2.5 mM it potentiated the effect of tolbutamide. Sulphadimidine potentiated the effects of glucose and tolbutamide on insulin secretion, whereas phenylbutazone was found to augment glucose-stimulated insulin release and to antagonize the tolbutamide effect. The effects of glucose and tolbutamide on insulin secretion were inhibited by low concentrations of chlorpromazine (0.005 mM–0.01 mM) and were stimulated by high concentrations (0.5 mM–1 mM). These results are discussed in relation to the possibilities that certain drugs may affect tolbutamide-stimulated insulin release either by altering the binding of tolbutamide to the B-cell or to other proteins, or by affecting the cyclic AMP system of the B-cell.

Inhibition of insulin and glucagon release from the perfused rat pancreas by cyproheptadine (Periactinol, Nuran).

The tricyclic compound cyproheptadine (Periactinol, Nuran) inhibited glucose-induced insulin release from the perfused rat pancreas. Tolbutamide-stimulated insulin release was significantly reduced in the presence and completely suppressed in the absence of a substimulatory glucose concentration (5 mM). Arginine produced a slow rise of insulin release, which was completely abolished by cyproheptadine. Furthermore the biphasic glucagon release due to the stimulus was inhibited. Oxidation of 14C-glucose in isolated islets was unaltered in the presence of cyproheptadine, and pyruvate added to the perfusion medium failed to reverse the inhibitory effect on glucose induced insulin release, indicating that impaired glucose metabolism is not responsible for the inhibition. In addition, the inhibition remained unchanged when phentolamine was present, suggesting that the effect is not mediated by inhibitory adrenergic alpha receptors. Theophylline, in contrast, partly overcame the inhibition. When the calcium concentration of the medium was enhanced, the inhibitory effect of cyproheptadine was still visible, although the relative inhibition had become smaller. The results suggest that cyproheptadine blocks insulin release by affecting a fundamental step of the stimulus-secretion coupling common to peptide hormones. A participation of a calcium-antagonizing effect in the inhibition is discussed.

FAILURE OF SOMATOSTATIN TO INHIBIT TOLBUTAMIDE-INDUCED INSULIN SECRETION IN PATIENTS WITH INSULINOMAS: A POSSIBLE DIAGNOSTIC TOOL

The effects of somatostatin on tolbutamide-stimulated insulin release were studied in 4 patients with insulin-producing tumors of the pancreas and in 6 normal subjects. In contrast to its effective inhibition of insulin release in normal subjects, somatostatin, without exception, failed to inhibit tolbutamide-induced insulin release in the patients with pancreatic beta-cell tumors. This differential effect of somatostatin may prove useful in the diagnosis of insulin-producing tumors of the pancreas.

Carbohydrate Content and Regulation following Injection of Different Glycogenolytic Enzymes

The effect of injection of glycogenolytic enzymes on tissue glycogen, blood glucose and plasma insulin was studied in mice. No effects were observed following phosphorylase, whereas the hydrolytic enzymes, alpha-amylase and acid amyloglucosidase depressed liver glycogen. In addition acid amyloglucosidase induced a decrease in blood glucose, a slight elevation of plasma insulin and a marked increase in tolbutamide-stimulated insulin release. At the doses given none of the enzymes affected muscle glycogen. Amyloglucosidase pretreatment markedly enhanced insulin release induced by glibenclamide, leucine, isoleucine, lysine and glucose whereas insulin release stimulated by IPNA, ACTH, glucagon and "CCK-PZ" was unaffected. Injection of acid amyloglucosidase has a profound influence on carbohydrate content and regulation in mice. It is suggested that the dependence or independence of amyloglucosidase activity among the insulin secretagogues tested might reflect different or partially different mechanisms in the process of insulin secretion.

Effect of somatostatin on plasma glucose and insulin responses to glucagon and tolbutamide in man.

ABSTRACT To characterize further the mechanism by which somatostatin lowers plasma glucose levels in man, we studied its effect on glucagon-induced glycogenolysis in 6 normal subjects. Additionally, we examined the effect of somatostatin on glucagonand tolbutamide-stimulated insulin release. During infusion of somatostatin (250 μg iv bolus followed by a sustained infusion of 500 μg/hr), plasma glucose responses to glucagon (1 mg iv) exceeded those seen after glucagon administration alone. The resultant hyperglycemia (190–200 mg/100 ml) was unaffected by administration of tolbutamide (1 g iv) and persisted for 15 min after stopping the somatostatin infusion. Plasma insulin remained at basal levels throughout the infusion, despite administration of glucagon and tolbutamide (with coexistent hyperglycemia). Within 15 min after stopping the somatostatin infusion, plasma insulin rose abruptly and plasma glucose declined toward basal levels. These studies indicate that, in man: 1) the hypoglycemic effect of soma...

Effect of chlorpromazine on glucose and tolbutamide-stimulated insulin release in the adrenalectomized rat

The influence of chlorpromazine pretreatment on the insulin response to glucose and tolbutamide was investigated in adrenalectomized rats. Chlorpromazine pretreatment (10 mg/kg, ip) reduced the capacity of the animals to secrete insulin in response to intravenously administered glucose and tolbutamide. Furthermore, it appeared that in the adrenalectomized rat, the insulin response to tolbutamide was not transient but multiphasic and prolonged. The data suggest that the reduction in glucose tolerance in rats following chlorpromazine pretreatment may be due, in part, to a reduced beta cell responsiveness.

Effects of amino acids on insulin release from excised rabbit pancreas.

The influence of amino acids, tolbutamide and glucose on the release of insulin from excised rabbit pancreas into Krebs buffer was studied. Glucose stimulated insulin release and a dose response relationship was demonstrated. Tolbutamide stimulated insulin release in the absence of glucose in the buffer. The amino acids arginine, lysine, leucine and histidine when tested in buffer containing either no glucose or glucose 0.5 mg/ml failed to stimulate insulin release. However, when the amino acids were tested in buffer containing glucose1.5 mg/ml or tolbutamide 125 μg/ml, they caused significant enhancement of insulin release. No essential difference of insulin releasing potency was demonstrated among the individual amino acids tested. Epinephrine inhibited insulin release stimulated by glucose and by glucoseamino acid. It is concluded that at least the major effect of amino acids on insulin release is dependent upon the presence of a primary stimulant (e.g., glucose or tolbutamide). (Endocrinology 84: 835,...