A-Cell and gut glucagon in normal and depancreatized dogs. Inhibition by somatostatin and insulin.

The effect of somatostatin (SRIF) and of insulin on the plasma levels of immunoreactive glucagon (IRG) and glucose was examined in normal (N) and depancreatized (PX) dogs. The infusion of SRIF (3 microgram/min for 15 min) caused a rapid decrease of the total IRG measured by means of an antiglucanon serum (AGS 10) which cross reacts with extracts of intestinal mucosa. This decrease was due primarily to a fall in the IRG fraction measured by an antiserum (AGS 18) specific for the carboxyl terminus of pancreatic or A-cell IRG. When the dose of SRIF was increased to 10 microgram/min for 90 min, the difference between total and A-cell IRG in the systemic blood also decreased, indicating that other IRG fractions, such as gut IRG, had also been suppressed. The introduction of 50 ml of a 5% glucose solution into a loop of ileum was followed by an increase of gut IRG measured in the regional mesenteric blood. This response was suppressed by the infusion of SRIF (3 microgram/min). Insulin suppressed the basal level of total IRG, but did not alter the gut IRG response to glucose. The SRIF- and insulin-induced reduction in plasma IRG was not associated with a reduction in plasma glucose, suggesting that the high levels of total and A-cell IRG observed in depancreatized dogs were not essential for the maintenance of hyperglycemia.     

Effectiveness of somatostatin in regulating pancreatic splenic lobe hormone secretion following 99% pancreatectomy in adult chickens

In vivo studies were carried out in adult chickens in an attempt to evaluate the effectiveness of somatostatin (SRIF) in regulating hormone secretion from the splenic pancreatic lobe after 99% of the pancreatic mass was surgically ablated. Sixteen days after either sham operation or 99% pancreatectomy, birds were infused iv with SRIF (420 ng/min) alone and then glucose (59 mg/Kg/min) was superimposed on the infusate, or birds were infused iv with glucose alone and then SRIF was superimposed on the infusate. Serial blood samples were taken during the 16-day postoperative period and also at regular intervals during the 75-min observation period. Plasma was analyzed for glucose, insulin (IRI), glucagon (IRG), pancreatic polypeptide (IRAPP), and somatostatin (IRSRIF). Careful standardization of the SRIF radioimmunoassay, as well as analysis of the molecular form of circulating SRIF, indicated that "true" SRIF levels were being estimated in plasma of both groups of chickens. Normal-fed chickens have plasma SRIF levels of 1.12 +/- 0.07 ng/ml which increases 16 days after 99% pancreatectomy to 2.39 +/- 0.15 ng/ml plasma. The latter decreases by 55% with an overnight fast. Glucose infusion, superimposed upon a preexisting SRIF infusion in adult chickens, did not evoke an IRI response in the 99% depancreatized birds equal to that observed in sham-op controls. Although a full SRIF dose-response curve was not generated, the glucose data strongly suggest a reduced sensitivity of insulin-secreting cells to SRIF in pancreoprivic birds. Both bird groups were equally--and markedly--sensitive to the IRG-depressant effects of SRIF; in contrast, the depancreatized chickens were significantly more resistant to the APP-inhibitory effects of SRIF when compared to the sham-op control birds. Thus, 16 days after partial pancreatectomy, the hormone-release mechanisms appeared altered for IRI and IRAPP in response to SRIF. Data obtained when glucose infusions preceded SRIF infusions indicated that A-cell release of glucagon was much more sensitive to glucose (as a depression) in the partially depancreatized birds than in control birds. These same birds were significantly less responsive to the glucose-depressant effect on plasma APP levels. Thus, it appears that 99% pancreatectomy increases the sensitivity of the SRIF, IRI, and IRG release mechanisms in response to glucose 16 days after surgery. The insulin-to-glucagon (I/G) molar ratios indicative of metabolic anabolism can still be achieved by nutrients 16 days after partial pancreatectomy.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of interaction between insulin and glucagon on glucose turnover and FFA concentration in normal and depancreatized dogs

We showed previously that arginine increased glucose production (R_a) and utilization (R_d) synchronously in normal dogs and suggested that this was due to concurrent insulin and glucagon release. In order to investigate the metabolic effects of coincidently elevated insulin and glucagon levels on R_a and R_d, glucagon was infused (1.55 μg/kg/hr) into normal dogs and into depancreatized dogs coincident with graded amounts of insulin (250–3000 μU/kg/min) until the metabolic response of the normal dog was achieved in depancreatized dogs. Main observations: Concurrent insulin and glucagon elevations increased glucose turnover (100%) in normal and depancreatized dogs while maintaining normoglycemia. Glucagon had no appreciable effect on peripheral glucose clearance in depancreatized dogs maintained on basal insulin. The effect of glucagon on R_a was not inhibited by concurrent insulin infusion at rates up to 3000 μU/kg/min. The effect of glucagon on R_a waned with time, indicating that a given insulin/glucagon ratio did not have a sustained effect. Near normal metabolic effects with respect to glucose turnover and FFA concentration were achieved in depancreatized dogs when the normal IRI response to glucagon was reproduced, indicating that the spike pattern of insulin release reflects not only the inherent secretory characteristic of β cells, but also serves an important glucoregulatory function. Glucagon induced an increase in ¹⁴C-glucose recycling, suggesting that it enhanced gluconeogenesis.     

Pancreatic polypeptide and other pancreatic hormones in spontaneously diabetic BB/W rats

The BB/W strain of rats develop spontaneous insulin-dependent diabetes. Diabetic BB/W rats have a marked insulinopenia and greatly diminished levels of insulin in their pancreas. Using a radioimmunoassay for rat pancreatic polypeptide (PP), we have examined the content of PP in extracts of the total pancreas and also the regional PP concentration of the three pancreatic lobes. Radioimmunoassays for glucagon, somatostatin (SRIF) and insulin were also made on these extracts. Compared with nondiabetic BB/W rat pancreas, pancreatic extracts from severely diabetic BB/W rats contained 30% as much PP, 31% as much glucagon, 19% as much SRIF, and 0.5% as much insulin. The rat PP radioimmunoassay was used to determine the elution pattern of PP-like antigens in gel chromatography fractions and to measure in vitro secretion of PP from perifused pancreatic slices obtained from diabetic and nondiabetic animals. PP-like immunoreactivity was observed in two zones in the elution from the gel columns when extracts from normal or diabetic rats were chromatographed. The major zone of immunoreactivity eluting at the volume expected for intact monometric rat PP accounted for 67% of the PP-like immunoreactivity in the case of nondiabetic rats and greater than 80% of the PP-like immunoreactivity found in extracts from severely diabetic rats. The minor zone of PP-like immunoreactivity eluted at a volume similar to the position of tetradecapeptide SRIF contained the remainder of detected PP-like immunoreactivity. Tissue slices from diabetic rats secreted more PP and glucagon than slices from nondiabetic rats when slices were perifused with a medium containing leucine, carbachol, and cholecystokinin, even though diabetic pancreas has smaller amounts of PP, glucagon, SRIF, and insulin. Stimulated insulin secretion was virtually absent when tissue slices from diabetic rats were perifused. These results indicate that in the BB/W diabetic rat: (a) pancreatic glucagon, PP, and SRIF are moderately decreased and insulin levels are drastically reduced, (b) lower levels of degraded or low molecular weight form of immunoreactive PP occurs in the diabetic rat pancreas compared to the normal rat, (c) the diabetic pancreas secretes more PP and glucagon and much less insulin than pancreas from nondiabetic rats when perifused under stimulating conditions. The diabetes occurring in the BB/W appears to be a severe type I diabetes characterized by reduced content of insulin, glucagon, SRIF, and PP in the pancreas of these animals. However, secretion of glucagon and PP were not reduced in this in vitro system.     

Somatostatin secretion from monolayer cultures of neonatal rat pancreas.

Monolayer cultures of neonatal rat pancreas have been characterized as an in vitro system for studying SRIF secretion. Marked 12- and 6-fold potentiation of SRIF release occurred with N-2-O-dibutyryl cAMP monosodium salt and theophylline, respectively. High glucose (300 mg/dl) stimulated SRIF release, whereas galactose was without effect. Exogenous insulin did not alter SRIF release, and the SRIF responses to theophylline and glucose were unaffected by the addition of antiinsulin serum to neutralize the insulin released by these agents. Arginine evoked a significant 2-fold increase in SRIF release. Exogenous glucagon produced slight but not significant stimulation of SRIF release. However, after exposure of the cultures to antiglucagon serum to diminish the concentration of glucagon in contact with the SRIF cells, exogenous glucagon produced a marked enhancement of SRIF secretion. These data suggest that glucose, arginine, glucagon, N-2-O-dibutyryl cAMP monosodium salt, and theophylline stimulate SRIF secretion, probably by direct effects on D cells or through mechanisms other than increased insulin secretion. Monolayer cultures of rat pancreas should provide a powerful in vitro system for studying pancreatic SRIF physiology.     

Effects of long-term glibenclamide administration on gastrointestinal and pancreatic hormones in normal fasting rats

We previously reported that sulfonylurea treatment reduces insulin (IRI), glucagon (IRG) and somatostatin (SRIF) release following metabolic stimuli from the isolated perfused pancreas of normal rats and that a reduction in IRI, IRG and SRIF pancreatic content was also observed. The present work was undertaken to investigate the effects of long-term glibenclamide treatment on the gastrointestinal content of gut hormones in normal rats. Moreover, the effects of sulfonylurea treatment on IRI, IRG, and SRIF pancreatic content were also analyzed and compared to the peripheral hormone plasma levels. Two groups of male Sprague-Dawley rats received glibenclamide (1 mg/kg/day per os; n = 14) or placebo (distilled water; n = 10) for 5 months, respectively. Tissue contents of IRI, IRG and SRIF in acid-ethanol extracts of pancreas and of gastric inhibitory peptide (GIP), vasoactive intestinal polypeptide (VIP), entero-glucagon (gut-GLI) and SRIF in acid-ethanol extracts of intestine were determined. Blood glucose and plasma pancreatic hormone levels were also measured. Glibenclamide treatment lowered the levels of IRI, IRG and SRIF in the pancreatic tissue; in the same way gut-GLI, SRIF and VIP intestinal concentrations were significantly reduced, whereas no significant inhibition was detected in intestinal GIP content. Blood glucose levels and IRI and SRIF plasma concentrations were similar in the two groups. IRG plasma levels were reduced in the sulfonylurea group. These findings might suggest that sulfonylurea suppresses hormone biosynthesis in a non-specific manner.     

The alpha cell response to glucose change during perfusion of anti-insulin serum in pancreas isolated from normal rats

Summary To determine the effect of neutralization of endogenous insulin upon the glucagon response to a rise and fall of glucose concentration, pancreata isolated from normal rats were perfused with either a potent anti-pork insulin guinea pig serum or a nonimmune guinea pig serum for 30 min. During this period glucose concentration was changed from 100 mg/dl to either 130, 180 or 80 mg/dl for 10 min. Antiserum perfusion at 100 mg/dl caused an approximately two-fold increase in glucagon which was not suppressed by an increase in glucose concentration to either 130 or 180 mg/dl, although glucagon secretion was significantly suppressed in the control experiments in which nonimmune serum was perfused. However, the 0.38±0.21 ng/min rise in glucagon secretion in response to a reduction in glucose concentration to 80 mg/dl in the control experiments was not abolished by antiserum perfusion but, instead, was enhanced (2.66±0.60 ng/min). These findings suggest that insulin may be required for glucose-mediated suppression of glucagon in the isolated pancreas of normal rats but not for stimulation of glucagon secretion by mild glucopenia. Alternatively, neutralization of insulin-mediated release-inhibition of glucagon secretion may simply have altered alpha cell responsiveness in a direction that desensitized it nonspecifically to suppression and sensitized it to stimulation.Senior Medical Investigator, Dallas Veterans Administration     

Effect of a hypoglycaemic agent M&B 39890A on glucagon secretion in isolated rat islets of Langerhans

Summary Administration of the compound M&B 39890A lowered serum glucose levels significantly (p<0.001) in genetically obese mice, while no effect on serum insulin levels was observed. In in vitro experiments with isolated rat islets of Langerhans M&B 39890A inhibited arginine-stimulated glucagon release at all concentrations tested (0.5, 5.0 and 50 mol/l). Insulin secretion was not inhibited by M&B 39890A (0.5 and 5.0 mol/l), but was slightly decreased at 50 mol/l. M&B 39890A (5 mol/l) also inhibited glucagon secretion in vitro in the presence of 2 mmol/l, 6 mmol/l and 20 mmol/l glucose, while exerting no effect on insulin secretion. These results suggest that the hypoglycaemic action of M&B 39890A may be due to its direct and selective effect on glucagon secretion; this appears to operate by a mechanism different to that of glucose.     

The role of the splenic pancreatic lobe in regulating metabolic normalcy following 99% pancreatectomy in chickens

The functional competency of the splenic pancreatic lobe of adult chickens was evaluated by studying plasma and tissue levels of insulin (IRI), glucagon (IRG), somatostatin (IRSRIF), and avian pancreatic polypeptide (IRAPP) for 16 days following surgical removal of 99% of the pancreas. The rationale underlying this approach was to assess whether the splenic remnant tissue after 99% pancreatectomy was capable of maintaining apparent metabolic normalcy as has been suggested in the literature. Partial (99%) pancreatectomy resulted in almost immediate enlargement of the splenic remnant, immediate decreases in both plasma IRI and IRAPP, both of which subsequently reattained normal levels (IRI in 4 days and IRAPP in 16 days). Hyperinsulinemia persisted from Days 4-7 and then returned to normal the remaining 9 days. Plasma IRG levels increased markedly and progressively for the entire 16-day observation period. Plasma I/G molar ratios indicated a strong catabolic trend in the 99% depancreatized group approaching values of 0.4 by 4 hr postop, returning to normal by Day 5, only to decrease progressively again into another sustained catabolic mode of 0.8 or less for the additional 11 days. Splenic lobe hormone (total) content of IRI, IRG, and IRAPP increased significantly throughout the 16-day observation period; however, parenchymal enlargement of nonendocrine lobe tissue was so great that actual concentrations of IRI and IRG decreased significantly while IRAPP concentration appeared to remain constant at control levels. IRSRIF levels were very sensitive both to pancreatectomy (increasing 140% 16 days later) and to fasting (decreasing 52% after 48 hr). IRG levels increase with fasting and with the exception of IRAPP, pancreatectomy appeared to increase the "sensitivity" of the splenic remnant in responding to the fasting stimulus. The results suggest that very rapid enlargement of the remnant splenic lobe following 99% pancreatectomy is probably adequate to maintain an apparent metabolic normalcy in adult chickens. The depancreatized animal enters a strongly catabolic mode, due mainly to hyperglucagonemia, even though food intake is normal and body weight is constant (though at subcontrol levels) after 16 days. There is no evidence to support the suggestion that SRIF stimulates glucagon release.     

Somatostatin does not alter insulin-mediated glucose disposal

We examined the effect of somatostatin (SRIH) infusion on insulin-mediated glucose disposal (Rd) in normal young subjects (n = 8) to determine the influence of SRIH on insulin action. Paired 3-h euglycemic insulin clamp studies were performed in random order employing insulin alone (25 mU/m2 X min) or insulin with SRIH (250 micrograms/h) and replacement of basal glucagon (0.4 ng/kg X min). Basal plasma glucose, insulin, glucagon (IRG), and GH concentrations, hepatic glucose production, and Rd were similar on each occasion. Steady state (10-180 min) plasma insulin insulin alone, 283 +/- 10 (+/- SEM); insulin, IRG, and SRIH, 284 +/- 10 pmol/L) and glucagon levels (insulin alone, 84 +/- 7; insulin, IRG, and SRIH, 82 +/- 7 ng/L) were similar. Hepatic glucose production (insulin alone, 0.66 +/- 0.12; insulin, IRG, and SRIH, 0.78 +/- 0.48 mg/kg X min) and Rd (insulin alone, 8.16 +/- 0.62; insulin, IRG, and SRIH, 8.17 +/- 0.61 mg/kg X min) were not different at steady state. We conclude that SRIH infusion with glucagon replacement does not augment insulin-mediated glucose disposal in normal young subjects at physiological insulin levels.     

Ghrelin secretion is inhibited by either somatostatin or cortistatin in humans

Ghrelin possesses endocrine and non-endocrine actions mediated by the GH Secretagogue (GHS)-Receptors (GHS-R). The regulation of ghrelin secretion is still largely unknown. Somatostatin (SRIF) modulates central and gastroenteropancreatic hormonal secretions and functions. SRIF actions are partially shared by cortistatin (CST), a natural SRIF analogue, that binds all SRIF receptors and also GHS-R. Herein, we studied the effects of SRIF-14 or CST-14 (2.0 micro g/kg/h i.v. over 120 min) and of placebo on ghrelin, GH, insulin, glucagon and glucose levels in 6 normal young men. Placebo unaffected GH, insulin, glucagon, glucose and ghrelin levels. SRIF and CST similarly inhibited (p < 0.05) spontaneous GH secretion of about 90%. After SRIF or CST withdrawal, GH levels recovered to baseline levels. Both SRIF and CST similarly inhibited (p<0.01) insulin secretion of about 45%. In both sessions, after SRIF or CST withdrawal, insulin overrode baseline levels. Both SRIF and CST similarly inhibited (p < 0.01) glucagon levels of about 40%. After SRIF or CST withdrawal, glucagon persisted lower (p < 0.05) than at baseline. Neither SRIF nor CST modified glucose levels. Both SRIF and CST similarly inhibited (p < 0.01) circulating ghrelin levels of about 55%. Ghrelin levels progressively decreased from time +15 min, reaching the nadir at 120 and 105 min for SRIF and CST, respectively. Even 30 min after SRIF or CST withdrawal, ghrelin levels persisted lower (p < 0.05) than those at baseline. In conclusion, this study first shows that SRIF and CST strongly inhibits ghrelin secretion that, differently from GH and insulin secretion, persists inhibited even after stopping the infusion of SRIF or CST.     

Changes in glucagon do not play an essential role in the glucoregulatory responses to mild hyperinsulinemia in dogs

To examine whether an increase in the glucagon concentration is essential for restoring hepatic glucose output following moderate decrements in blood glucose, we used isotope dilution techniques in trained conscious dogs (n = 5) to measure glucose production (Ra) and glucose utilization (Rd) during mild hyperinsulinemia (19 +/- 1 mU/l). In Study A, when insulin was infused to raise plasma insulin (IRI) from 13 +/- 2 to 19 +/- 1 mU/l, basal glucose (93 +/- 3 mg/dl) fell at a rate of 0.37 +/- 0.06 mg/dl/min over 30 min. Ra fell from 2.8 +/- 0.4 mg/kg/min by 0.5 +/- 0.1 mg/kg/min at 20 min (P less than 0.05), but recovered to baseline by 30 min; glucagon (IRG) fell transiently but returned to baseline by 45 min. In Study B, endogenous secretion of IRI and IRG was suppressed by infusion of somatostatin (0.2 microgram/kg/min), while peripheral concentrations were maintained constant by replacing glucagon (0.65 ng/kg/min) and insulin (0.225 mU/kg/min). Steady-state baseline plasma IRI, IRG, glucose and glucose turnover rates were similar to Study A; hyperinsulinemia was then induced as in Study A. Glucose fell by 0.78 +/- 0.19 mg/dl/min over 30 min and, as in Study A, Ra decreased transiently, but recovered to baseline by 30 min. The restoration of Ra occurred in study B despite constant IRG, and preceded later increments in cortisol and catecholamines at 60-90 min. Thus, in both studies A and B, Ra recovered to baseline without an increase in IRG and before the onset of significant hypoglycemia (glucose 83 +/- 1 and 70 +/- 1 mg/dl).(ABSTRACT TRUNCATED AT 250 WORDS)     

Sulfonylurea-induced inhibition of glucagon secretion from the perfused rat pancreas: evidence for a direct,non-paracrine effect

Summary The effects of sulfonylurea on glucagon secretion were characterized in the perfused rat pancreas using glibenclamide (1 g/ml) or tolazamide (10 g/ml) in the presence of 3.3 mmol/1 glucose. Glucagon release, which was unaffected by glibenclamide at 2.75 mmol/1 calcium, was suppressed at 1.19 and 0.64 mmol/l but transiently stimulated at 0.25 mmol/l extracellular calcium. The insulinogenic effect of glibenclamide at 0.64 and 0.25 mmol/1 calcium was enhanced by 35% and 89%, respectively, compared to the response at 2.75 mmol/1 calcium. The stimulatory effect of the compound on somatostatin secretion, however, was lost at the lower calcium levels. The effects of tolazamide at 2.75 and 0.64 mmol/1 calcium mimicked those of glibenclamide, thus indicating that our results with the latter compound may be representative for all sulfonylureas. In pancreata from insulin-deficient alloxan diabetic rats, glibenclamide completely lost its inhibitory effect on glucagon release at 0.64 mmol/1 calcium. Inhibition was not restored by adding insulin (25 U/1) to the perfusate. However, when diabetic rats had been treated with insulin for 6–7 days, glibenclamide suppressed glucagon release at low calcium levels in the absence of stimulated insulin and somatostatin release. It is concluded that, at low calcium concentrations, sulfonylureas suppress glucagon secretion by a direct action on the A cell and not through paracrine interactions by insulin and somatostatin. Prolonged insulin deficiency impairs the sulfonylurea action on glucagon secretion.     

The hypoglycaemic and insulinotropic activity of Tinospora crispa: studies with human and rat islets and HIT-T15 B cells

Summary In Malaysia, Tinospora crispa extract is taken orally by Type 2 (non-insulin-dependent) diabetic patients to treat hyperglycaemia. We have evaluated the claimed hypoglycaemic property by adding aqueous extract to the drinking water of normal and alloxan-diabetic rats. After one week, fasting blood glucose levels were significantly (p<0.01) lower and serum insulin levels were significantly (p<0.01) higher in treated diabetic animals (10.4±1.0 mmol/l and 12.8±1.1 U/ml respectively) compared to untreated diabetic controls (17.4±1.7 mmol/l and 8.0±0.7 U/ml respectively). The insulinotropic action of T. crispa was further investigated in vitro using isolated human or rat islets of Langerhans and HIT-T15 cells. In static incubations with rat islets and HIT-T15 B cells, the extract induced a dosage dependent stimulation and potentiation of basal and glucose-stimulated insulin secretion respectively. This insulinotropic effect was also evident in perifused human and rat islets and HIT-T5 B-cells. The observations that (i) in all three models insulin secretory rates rapidly returned to basal levels on removal of the extract and (ii) in rat islets, a second challenge with T. crispa induced an additional, stimulated response, are all consistent with physiological release of insulin by B cells. Moreover, the rate of HIT-T15 glucose utilisation was not affected by incubation with T. crispa, suggesting that the cells were viable throughout. These are the first studies to provide biochemical evidence which substantiates the traditional claims for an oral hypoglycaemic effect of Tinospora crispa, and which also show that the hypoglycaemic effect is associated with increased insulin secretion.     

Somatostatin, insulin and glucagon after arginine stimulation in active and treated acromegaly

Ten acromegalic patients, 28-71 years old, were compared with 10 normal controls, 21-39 years old. In another study, 7 patients with active acromegaly, 19-70 years old, were investigated before and 4-9 months following transsphenoidal adenectomy and radiation. They were all investigated following an arginine infusion (0.5 g/kg/20 min). Although the mean plasma somatostatin (somatotrophin release inhibiting factor (SRIF] was somewhat higher in acromegalic patients compared to normal controls (mean basal values 21 +/- 3.8 and 16.6 +/- 2.1 pmol/l, respectively), the difference was not significant. The patients had higher serum insulin (peak values 118 +/- 23.9 and 63 +/- 11.8 mU/l, respectively) and lower plasma glucagon (peak values 171 +/- 29.0 and 310 +/- 52.7 pmol/l, respectively). Plasma SRIF increased during arginine infusion, but the concentrations were similar before and following the operation (mean basal values 18.2 +/- 2.6 and 15.2 +/- 2.3 pmol/l, respectively). Serum insulin was significantly higher before the operation (peak values 154 +/- 38.8 and 91 +/- 24.9 mU/l, respectively). Plasma glucagon was similar before and after the operation (peak values 143 +/- 23.4 and 127 +/- 22.7 pmol/l, respectively). Plasma SRIF is similar in active acromegaly and normal controls, and in acromegaly before and following treatment, despite differences in serum growth hormone (GH), serum insulin and plasma glucagon. This points towards a modulating role for GH on plasma SRIF, possibly by affecting the other islet cell hormones.     

Studies on persistent circulating immunoreactive glucagon (IRG) and immunoreactive insulin (IRI) found in eviscerated rats with a functional liver

Summary Eviscerated rats (animals without gastrointestinal tract or pancreas, but with intact liver and kidneys) are diabetic with blood glucose levels of 287 ± 10 mg% (n = 35) 24 h after surgery. Immunoreactive insulin (IRI) and immunoreactive glucagon (IRG) persisted in these animals at plasma levels of 36 ± 4 U/ml and 0.29 ± 0.02 ng/ml, respectively. Twenty-four h fasted sham-operated controls, on the other hand, had blood glucose levels of 101 ± 3mg%, plasma IRI levels of 62 ± 8 U/ml and plasma IRG levels of 0.38 ± 0.05 ng/ml (n = 21). IRG levels were not increased in eviscerated animals by surgical stress, fasting, arginine infusion, or insulin-induced hypoglycaemia, nor did they decrease following somatostatin infusion. IRI levels were similarly unresponsive. An unexplained decrease in IRG followed arginine infusion. Gel filtration studies showed plasma IRI and IRG to consist mainly of the larger molecular weight components with little of the smaller native hormone species. The disappearance rates of injected ¹²⁵I-insulin and ¹²⁵I-glucagon did not differ from sham-operated controls. Removal of the submaxillary glands from eviscerated animals had no effect on the circulating levels of IRG. Bilateral nephrectomy doubled plasma IRG. It is suggested that persistent IRG and IRI in eviscerated rats represents retained immunoreactive materials with slow rates of degradation, although an unresponsive extravisceral source of IRG can not be ruled out.     

Oxytocin stimulates glucagon and insulin secretion in fetal and neonatal sheep

In adults of several species arginine vasopressin (AVP) and oxytocin (OT) stimulate pancreatic secretion of immunoreactive plasma glucagon (IRG). In fetal sheep AVP is an important stress hormone and may be simultaneously secreted with OT; however, their effects on IRG secretion are not known. We sought to determine if AVP and/or OT affected pancreatic IRG secretion in fetal and neonatal sheep. Either AVP or OT was infused for 30 min in chronically catheterized fetal and neonatal sheep, obtaining peripheral arterial and/or portal venous blood samples before; 10, 15, and 30 min during; and 15, 30, and 60 min after infusion for measurements of blood gases, hematocrit, IRG, immunoreactive plasma insulin (IRI) and plasma glucose. AVP did not affect IRG or IRI in fetal sheep (mean +/- SE, 133 +/- 1 days gestation), but small increases occurred in portal venous blood of lambs (2-49 days old). In contrast, OT (4.6 +/- 0.3 mU/min.kg; n = 12) increased fetal plasma IRG from 72 +/- 5 to 86 +/- 6 and 97 +/- 7 pg/ml (P less than 0.001) and IRI from 16 +/- 2 to 20 +/- 3 and 20 +/- 2 microU/ml (P less than 0.02) at 15 and 30 min, respectively; 157 +/- 11 microU OT/min.kg had no effect. In lambs (2-49 days old), 3.0 mU OT/min.kg increased arterial (n = 15) IRG from 139 +/- 19 to 367 +/- 43 and 483 +/- 76 pg/ml (P less than 0.01) and portal IRG (n = 8) from 167 +/- 39 to 341 +/- 72 and 502 +/- 148 pg/ml (P less than 0.01), respectively. Arterial and portal IRI also rose (P less than 0.01) from 36 +/- 4 to 82 +/- 12 and 105 +/- 32 microU/ml and from 29 +/- 5 to 65 +/- 13 and 51 +/- 7 microU/ml, respectively. Glucose was unchanged in all experiments. In fetal and neonatal sheep, AVP has minimal effects on IRG and IRI release. In contrast, OT increases both substantially; furthermore, there is a difference in fetal and neonatal responsiveness. OT may be important in modulating glucagon and insulin secretion during and after parturition.     

Novel long-acting somatostatin analog with endocrine selectivity: potent suppression of growth hormone but not of insulin

Somatostatin, also known as somatotropin release-inhibiting factor (SRIF), is a natural cyclic peptide inhibitor of pituitary, pancreatic, and gastrointestinal secretion. Its long-acting analogs are in clinical use for treatment of various endocrine syndromes and gastrointestinal anomalies. These analogs are more potent inhibitors of the endocrine release of GH, glucagon, and insulin than the native SRIF; hence, they do not display considerable physiological selectivity. Our goal was to design effective and physiologically selective SRIF analogs with potential therapeutic value. We employed an integrated approach consisting of screening of backbone cyclic peptide libraries constructed on the basis of molecular modeling of known SRIF agonists and of high throughput receptor binding assays with each of the five cloned human SRIF receptors (hsst1-5). By using this approach, we identified a novel, high affinity, enzymatically stable, and long-acting SRIF analog, PTR-3173, which binds with nanomolar affinity to human SRIF receptors hsst2, hsst4, and hsst5. The hsst5 and the rat sst5 (rsst5) forms have the same nanomolar affinity for this analog. In the human carcinoid-derived cell line BON-1, PTR-3173 inhibits forskolin-stimulated cAMP accumulation as efficiently as the drug octreotide, indicating its agonistic effect in this human cell system. In hormone secretion studies with rats, we found that PTR-3173 is 1000-fold and more than 10,000-fold more potent in inhibiting GH release than glucagon and insulin release, respectively. These results suggest that PTR-3173 is the first highly selective somatostatinergic analog for the in vivo inhibition of GH secretion, with minimal or no effect on glucagon and insulin release, respectively.     

Effect of glucagon antibodies on plasma glucose,insulin and somatostatin in the fasting and fed rat

Summary A potent high-titre glucagon antibody pool was used to induce a state of acute glucagon deficiency in order to investigate the importance of glucagon in maintaining euglycaemia in the fed and fasted anaesthetised rat. Binding characteristics of the antiserum and evidence of its neutralisation of the biological effects of exogenous glucagon are described. The amount of antibody administered was capable of neutralising up to 12 times the total content of glucagon (approximately 1nmol) in the rat pancreas. The hyperglycaemic response to 1.43 nmol exogenous glucagon was significantly inhibited in the rat by glucagon antibodies given intravenously or intraperitoneally (p < 0.001). However, no changes in plasma glucose occurred in rats fasted 16 h (4.35±0.1 mmol/l or 24 h (4.0±0.05 mmol/l) after antibody administration. The same dose of glucagon antibodies produced no change in plasma glucose (6.1±0.2 mmol/l), immunoreactive insulin (1.85±0.05 g/l) or immunoreactive somatostatin (110±30 ng/l) in rats after antibody administration. Antibody excess, equivalent to a binding capacity for glucagon of 40 nmol/l in the plasma of recipient animals, was demonstrable at all times after passive immunisation. The absence of any affect on glucose concentrations following immunoneutralisation of glucagon suggests that glucagon secretion may not be a major factor in the maintenance of euglycaemia in the rat.     

The mechanism of vagal nerve stimulation of glucagon and insulin secretion in the dog

The mechanism of vagal nerve stimulation of glucagon (IRG) and insulin (IRI) secretion was investigated in halothane-anesthetized dogs. Both ventral and dorsal branches of the thoracic vagi were stimulated electrically (10 Hz, 5 msec, 13.5 mA, 10 min) below the heart. Arterial and superior pancreaticoduodenal venous plasma were sampled, superior pancreaticoduodenal venous plasma flow was measured, and net pancreatic output of IRG and IRI were calculated. During vagal nerve stimulation (n = 15) net pancreatic output of IRG doubled (delta = +0.83 +/- 0.28 ng/min, P less than 0.01; baseline = 0.81 +/- 0.15 ng/ min) and IRI quadrupled (delta = +3.5 +/- 1.5 mU/min, P less than 0.025; baseline = 1.1 +/- 0.3 mU/min). Arterial glucose levels increased by 7 +/- 2 mg/dl from 108 +/- 3 mg/dl (P less than 0.005). After atropine pretreatment (n = 7), the pancreatic IRI response to vagal nerve stimulation was +0.71 +/- 0.28 mU/min (P less than 0.025), a reduction of 80%. In contrast, atropine pretreatment changed neither the IRG response (delta = +0.87 +/- 0.36 ng/min; P less than 0.05) nor the arterial glucose response (delta = +9 +/- 3 mg/dl; P less than 0.025) to vagal nerve stimulation. Hexamethonium pretreatment (n = 9) abolished the pancreatic IRG response (delta = +0.13 +/- 0.11 ng/min; NS), the arterial glucose response (delta = +0.5 +/- 1.9 mg/dl; NS) and the pancreatic IRI response (delta = +0.16 +/- 0.31 mU/min; NS) to vagal nerve stimulation. It is concluded that vagal nerve stimulation in the dog produces a moderate increase of IRG secretion, mediated by a nonmuscarinic (peptidergic?) mechanism, and a marked increase of IRI secretion, mediated by a muscarinic mechanism. Both responses are dependent on nicotinic transmission.