S. J. Bhathena

Reversal of Somatostatin Inhibition of Insulin and Glucagon Secretion

These studies were designed to elucidate the mechanism of inhibitory action of somatostatin (SRIF) on glucagon (IRG) and insulin (IRI) secretion. Studies were carried out in the unrecirculated isolated rat pancreas perfusion with arginine 19.2 mM and glucose 5.5 mM as stimulus primarily for IRG but also IRI secretion. The effects of excess Ca++ (15.2 mEq./L.) and excess K+ (12.8 mEq./L.) on IRG, IRI, and the SRIF-inhibited pancreas were studied. Ca++ excess in five perfusions strikingly stimulated IRG secretion (+92 per cent) but only stabilized IRI secretion compared with control perfusions. K+ excess (in seven perfusions) markedly inhibited IRG secretion (–39 per cent) while stimulating IRI secretion (+16 per cent). Restoration of normal concentrations of K+ resulted in a rebound of IRG to levels 120 per cent that of controls. SRIF, at concentrations from 0.1–20 ng./ml., produced inhibition of both IRG and IRI. In 11 perfusions, with SRIF at 10 ng./ml., IRG decreased more than IRI (–75.2 per cent IRG and –46.9 per cent IRI). In five perfusions, addition of Ca++ (15.2 mEq./L.) 10 minutes after SRIF was started resulted in a reversal of IRG inhibition to 69.4 per cent and IRI to 73.2 per cent of the arginine controls. The reversal by Ca++ of SRIF effect on IRG was greater at higher concentrations of Ca++, suggesting some form of competition. In four perfusions, excess K+ reversed SRIF-induced IRI inhibition to 79.6 per cent that of controls but had no effect on IRG inhibition. Studies in vitro with isolated islets revealed that SRIF (2 μg./ml.) inhibited 45Ca uptake of islets as did epinephrine (10−5 M). It was concluded that SRIF-induced inhibition of hormone release appears related to an action on Ca++ uptake.

Studies on submaxillary gland immunoreactive glucagon.

Abstract Biologically active immunoreactive glucagon is present in submaxillary gland of rat, mouse, guinea pig and human and can be extracted by saline adjusted to pH 2.8 with HCl. Chromatography on Sephadex G-150 indicates its molecular weight to be 29,000. It has similar immunologic characteristics as pancreatic glucagon. It is biologically active and elevates plasma glucose and insulin when injected intraperitoneally into rats. Compared to pancreatic glucagon, the hyperglycemic effect persists much longer. It competes with pancreatic glucagon for binding to specific glucagon receptors of rat liver plasma membranes. It is stable to pH changes, however, urea dissociates it into several smaller molecular weight fragments including that of 3500. It appears to be an aggregate of smaller glucagon molecules and is not responsible for immunoreactive glucagon in totally eviscerated rats. In vitro , the submaxillary gland does not release immunoreactive glucagon in response to arginine or glucose.

Reversible Impairment of Glucose-Induced Insulin Secretion in SHR/N-cp Rats: Genetic Model of Type II Diabetes

The SHR/N-cp rat is a new genetically obese model for non-insulin-dependent diabetes mellitus. Expression of the diabetes is enhanced by a high-sucrose (54%) diet. After 4 wk on the diet, the cp/cp rats weigh significantly more than their +/? controls, have postprandial hyperglycemia (> 400 mg/dl), and are hyperinsulinemic, with immunoreactive insulin (IRI) levels 10- to 20-fold > controls. Total pancreatic IRI tends to be increased 1.6-fold in the cp/cp rats (although not significantly). There is no increase in pancreatic proinsulin content as a percent of total IRI. Studies of in vitro pancreatic function were carried out with the isolated nonrecirculating perfused pancreas method. The cp/cp rats (n = 10) showed impaired or absent IRI responses to 16.5 mM glucose, whereas +/? rats (n = 9) responded with classic biphasic curves. Comparison of insulin secreted in 20 min revealed a > 53% decrease in IRI secretion in cp/cp rats (P < .05). A paradoxical hypersecretion of IRI at glucose concentrations of 0–2.7 mM was noted in cp/cp but not lean rats, i.e., 1.8 ± 0.2 mU/min IRI in cp/cp rats vs. 0.04 ± 0.007 mU/min in +/? rats. Perfusion of pancreases for 45 min with buffers containing no glucose resulted in restoration of a normal biphasic IRI response to 16.5 mM glucose in the cp/cp rats, whereas response in the lean rats was markedly reduced. Brisk IRI responses to 10 mM arginine in buffers with no glucose also occurred in cp/cp but not +/? rats. Glucagon secretion was relatively suppressed in the cp/cp rats. These findings are similar to those reported in glucose-infused normal rats and suggest that hyperglycemia per se may be responsible for the impaired β-cell responses to glucose in cp/cp rats.

Alterations of plasma opioid activity in human diabetics

Plasma opioid levels were determined in 9 obese non-diabetic subjects, their 8 age matched controls, and in 29 diabetic patients; 10 maintained on diet alone, 6 on an oral hypoglycemic agent (chlorpropamide) and 13 treated with insulin. Five age matched controls for the diabetic groups were also studied for comparison. beta-endorphin and met-enkephalin levels were measured by radioimmunoassay. Enkephalin-like activity was measured by a receptor assay. Among the study groups, diabetic patients receiving insulin showed a 64% elevation of plasma beta-endorphins and diabetic patients on chlorpropamide showed a 121% increase in enkephalin-like activity. There were no statistically significant differences in the plasma met-enkephalin values in the treatment groups though levels were decreased (p less than 0.05) in diabetics vs non-diabetics. The pathophysiological importance of these alterations remains to be elucidated.

Characteristics of the Interaction of the Glucagon Receptor, cAMP, and Insulin Secretion in Parent Cells and Clone 5F of a Cultured Rat Insulinoma

Rat insulinoma cells, which grow in culture and secrete insulin, were used to study the mechanism of stimulation of insulin release by glucagon. The parent cell line (RIN-m) and a clone that secretes high levels of insulin (5F) had been shown to possess specific receptors for glucagon. Glucagon (1 μM) stimulated a rapid increase in cyclic adenosine 35-monophosphate (cAMP) that was followed by an increase in insulin secretion in both cell lines. The concentration of glucagon necessary for half-maximal stimulation of cAMP was 50 μM in parent and approximately 0.5 μM in 5F, whereas the concentration required to inhibit binding by 50% was 0.5 nM and 30 nM, respectively. In 5F, the dose-response relationships for cAMP and insulin secretion were superimposable. The glucagon effects on insulin secretion and cAMP did not require either glucose or amino acids in the incubation media. No refractoriness to glucagon stimulation of cAMP or insulin was noted. It may be concluded that (1) there are significant differences between glucagon binding and glucagon responses in parent cells and clone 5F, (2) there are glucagon receptors that are not coupled to adenylate cyclase, and (3) cAMP mediates glucagon-stimulated insulin release.

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

SummaryEviscerated 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 μU/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 μU/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.

Potassium Ion Concentration Alters Glucagon Secretion Independently of Insulin Secretion in the Isolated Rat Pancreas

SummaryIn the arginine-stimulated perfused rat pancreas, elevated concentrations of potassium ion inhibited glucagon secretion while stimulating the secretion of insulin. Decreased potassium ion produced the reverse effect. The observed inverse correlation between changes in insulin and glucagon secretion (r = -0.64; p<0.001) was suggestive of local interactions between islet hormones, and prompted us to determine whether potassium-induced changes in glucagon secretion were dependent upon concurrent changes in insulin release. We found that when insulin secretion was greatly suppressed, either through acute induction of diabetes with streptozotocin or by utilization of a glucose-free perfusate, no qualitative differences in glucagon responsiveness to altered potassium ion were evident, although the amplitude of these glucagon changes was enhanced. Similarly, when exogenous insulin (20,000 mU/l) was added to the perfusate in order to render potassium-induced changes in endogenous insulin secretion insignificant, glucagon responsiveness to altered potassium ion was maintained. Exogenous insulin alone had no effect on arginine-stimulated glucagon secretion. We conclude that any indirect effects of potassium ion on arginine-stimulated glucagon secretion are not mediated by insulin, but could be related to changes in somatostatin secretion.

IMPAIRMENT OF INSULIN SECRETION DURING EXPERIMENTAL POTASSIUM DEPLETION IS NOT CORRECTED BY THE PROSTAGLANDIN SYNTHESIS INHIBITOR, INDOMETHACIN

The effect of potassium depletion on glucose tolerance, plasma insulin and plasma glucagon was studied in six normal young female subjects. Negative potassium balance was induced by a diet low in potassium, together with frusemide (40 mg/day), for 3 days. Studies were performed during a period of potassium depletion and were repeated during potassium depletion in five subjects taking indomethacin (150 mg/day), an inhibitor of prostaglandin biosynthesis. Mean plasma potassium concentration was reduced from 4·2 ± 0·1 mmol/l to 3·3 ± 0·1 mmol/l, and was 3·2 ± 0·1 mmol/l during administration of indomethacin. Potassium depletion had no significant effect on the levels of plasma glucose, either fasting or following a 100 g oral glucose load, although the peak rise in plasma glucose after oral glucose was delayed (from 30 to 60 min). There was a decrease in the fasting plasma insulin concentration from 10·0 ± 1·3 mu/l to 6·7 ± 0·6 mu/l and a significant suppression of the early (30 min) insulin response to oral glucose from 126·0 ± 23·5 mu/l to 74·0 ± 19·2 mu/l. The insulin: glucose ratio during the first 60 min following oral glucose was significantly decreased from 43·7 ± 7·3 mu insulin/mmol glucose to 30·6 ± 7·3 mu insulin/mmol glucose. Furthermore, the suppression of plasma glucagon secretion that normally follows oral glucose was not observed. Administration of indomethacin during potassium depletion had no significant effect on plasma glucose, insulin or glucagon concentrations. These data indicate that short‐term potassium depletion in normal young females impairs the early insulin response to oral glucose but does not significantly alter overall glucose tolerance. Failure of an indomethacin effect suggests that the defect in insulin secretion may not be mediated by an increased synthesis of prostaglandins.