Enhanced Insulin Release Research Articles

(Pro-) insulin biosynthesis and release of newly synthesized (pro-) insulin from isolated islets of rat pancreas in the presence of amino acids and sulphonylureas.

The influence of arginine, lysine, tolbutamide and glibenclamide on (pro-) insulin biosynthesis and release of newly synthesized (pro-) insulin was studied in isolated islets of rat pancreas. Islets were incubated with 3H-leucine and glucose in the presence and absence of the test agents. Proinsulin and insulin of islets and incubation media were separated by gel filtration on Sephadex G 50. Estimations were carried out for radioactivity and immunoreactivity for insulin. All four substances were able to enhance insulin release whereas no stimulation of leucine incorporation into (pro-) insulin was found. Arginine and tolbutamide even markedly reduced (pro-) insulin aynthesis. Conversion of proinsulin to insulin was not affected by any of the test agents. For studying the influence of the 4 substances on secretion of newly synthesized (pro-) insulin two experimental models were used: 1) Labelling of the islets in the presence of the test agents, followed by uniform stimulation with glucose alone in the presence or absence of Ca++. 2) Addition of the 4 test substances after uniform prelabelling of the islets. 1) Presence of arginine and sulfonylureas during labelling resulted in a significantly enhanced relative fractional release of newly synthesized (pro-) insulin, although the bulk of secreted hormone appeared to stem from the storage pool also under these conditions. The enhanced fractional release was persistent also during the postlabelling period when the islets had been labelled in the presence of arginine or glibenclamide. On the other hand, a decreased release of newly synthesized (pro-) insulin was obsevered during the postlabelling period in islets labelled in the presence of tolbutamide. Lysine was without significant effects in both periods. Omission of calcium ions during the postlabelling period inhibited the release of both immunoreactive and radioactive hormone. 2) When amino acids or sulphonylureas were added after prelabelling no significant changes were found in the specific radioactivity of released (pro-) insulin or in the fractional release of newly synthesized hormone. Enhanced release of fresh granules from the beta cell might explain the increased fractional release of newly synthesized (pro-)insulin when lebelling is carried out in the presence of arginine and sulphonylureas, especially glibenclamide.

Effects of alpha-ketomonocarboxylic acids upon insulin secretion and metabolism of isolated pancreatic islets.

In perifused isolated pancreatic islets alpha-ketoisocaproic acid (KIC) or alpha-ketocaproic acid (KC) induced a high insulin secretion rate and a steep increase of the fluorescence of reduced pyridine pyridine nucleotides [NAD(P)H] which fell again to almost prestimulatory levels 6 min after medium change. Insulin release in response to alpha-ketooctanoic (KO) acid started slowly and was accompanied by a decrease of the NAD(P)H-fluorescence trace. Beta-phenylpyruvate which is known to initiate insulin release also caused a fluorescence decrease. Alpha-keto-isovaleric (KIV) acid or pyruvate had no significant effects upon insulin secretion or NAD(P)H-fluorescence. In contrast to l-leucine, l-norleucine or l-valine did not enhance insulin release or fluorescence of NAD(P)H. KIV, alpha-keto-beta-methylvaleric acid (KMV), KIC and KC raised the production their corresponding amino acids by islet cells. From these results it is concluded that alpha-ketomonocarboxylic acids as such trigger insulin release by acting upon receptor sites which differ from those occupied by amino acids.

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.

Dynamics of Insulin Release and Microtubular‐Microfilamentous System

Abstract. Colchicine failed to inhibit and actually enhanced insulin release evoked by gliclazide at a low glucose concentration (0.3 mg/ml). The facilitating action of colchicine was invariably present, whatever the concentration of (2.10−5 to 10−3 M) and length of exposure to (25 to 120 min.) the mitotic spindle‐inhibitor. Colchicine, and similarly cytochalasin B, also minimized the reduction in insulin output otherwise observed during the second of two successive stimulations with sulphonylurea. These data suggest that the short‐lived secretory response evoked by sulphonylurea corresponds to a modality of insulin release which does not require the oriented intracellular transport of secretory granules.

Dynamics of insulin release and microtubular-microfilamentous system. IV. Effect of colchicine upon sulphonylurea-induced insulin secretion.

Abstract. Colchicine failed to inhibit and actually enhanced insulin release evoked by gliclazide at a low glucose concentration (0.3 mg/ml). The facilitating action of colchicine was invariably present, whatever the concentration of (2.10‐5 to 10‐3 M) and length of exposure to (25 to 120 min.) the mitotic spindle‐inhibitor. Colchicine, and similarly cyto‐chalasin B, also minimized the reduction in insulin output otherwise observed during the second of two successive stimulations with sulphonylurea. These data suggest that the short‐lived secretory response evoked by sulphonylurea corresponds to a modality of insulin release which does not require the oriented intracellular transport of secretory granules.

Insulin response to glucose infusion during pregnancy. A prospective study of high and low insulin responders with normal carbohydrate tolerance.

ABSTRACT A decreased insulin response to glucose administration has been suggested to be a prerequisite for the development of diabetes mellitus. Factors that increase the demand for insulin in the organism may precipitate diabetes in subjects with a low insulin response to a glucose infusion test (GIT). Since it is well-known that pregnancy is a diabetogenic factor, its effect on the carbohydrate metabolism of subjects with a low insulin response was studied. During pregnancy, the insulin response of the low responders was enhanced as in the controls, but at all stages the insulin response was significantly less than in the controls. None of the subjects developed glucose intolerance during pregnancy. The fasting blood glucose and plasma insulin levels and the k-value in intravenous glucose tolerance tests (IVGTT) were modified according to similar patterns in both groups. The sensitivity to endogenous insulin was significantly greater in the low insulin responders but was reduced to a greater extent than in the controls towards the end of pregnancy. In four of the 11 low insulin responders the initial insulin response to glucose in the last trimester was lower than in mid-pregnancy. This occurred only in one out of 14 high insulin responders. It is suggested that gestational diabetes occurs in those low insulin responders who demonstrate either a dramatic decrease in insulin sensitivity, or limitations in the enhancement of insulin release, or, more likely both conditions.

Gastrointestinal Hormones and Insulin Secretion

When the subject was reviewed in this journal three years ago1 the conclusion was reached that the enhanced insulin release after ingestion of glucose, as compared with an intravenous application, is caused by hormones released from the intestinal tract. However, the available data did not indicate whether this insulin release after oral food intake was due to one of the known hormones (gastrin, secretin, cholecystokinin, serotonin or enteroglucagon) or to the combined action of more than one of these hormones or to a still unidentified insulin-releasing factor, hypothetically called incretin. More work has been done in this field, and the . . .

The effect of secretin on pancreatic glucoregulatory hormones in man.

Secretin was given intravenously to four normal men as a 150 U pharmacologic pulse. To an additional four men, during hyperglycemia, secretin was administered as a 5 U pulse with an infusion of 1.2 U per minute for 30 minutes, a rate shown to produce plasma secretin levels similar to those found after oral glucose load. Although insulin increased an average of 7 and 12 fold during the respective infusions, secretin did not significantly alter glucagon levels. These data suggest that, in humans, secretin influences glucose utilization by enhancing insulin release without producing a concomitant change in glucagon.

Insulin release from cultured fetal human pancreas.

The pancreas from human fetuses of 55-140 days gestation was grown on grids in organ culture dishes. Insulin was shown to be released at no earlier than 63 days gestation, but the amount increased thereafter with gestational age. The insulin content of the explants varied considerably. However, it appeared clear that pancreas from younger fetuses (55 and 63 days gestation) had much less insulin content than those from older fetuses (99-140 days gestation). Whereas 16.7 mM glucose alone caused no increase in insulin release, when used with 10 mM theophylline, a marked release occurred. Tolbutamide (1.85 mivr) plus glucose (16.7 mM), and tolbutamide (3.7 mM) plus glucose (1.67 mM) plus theophylline (10 mM) were also effective. No effect upon insulin release was produced by glucose (1.67 mM) plus theophylline (10 mM), by glucose (1.67 HIM) plus tolbutamide (3.7 mM), or by glucose (16.7 mM) plus arginine (11 mM). {Endocrinology 91: 1133, 1972) T N THE HUMAN fetus, pancreatic beta cells -*• may be distinguished by the presence of aldehyde-fuchsin positive granules during the third month of gestation (1). During the fourth-fifth months of gestation, accumulation of secretory granules in the capillary poles of the beta cells suggests active insulin synthesis and probably release (2). The ultrastructural features of alpha, beta, delta and exocrine acinar cells of the fetal human pancreas have been described in a recent report (3). Circulating insulin has been measured in the human fetus as early as 12 weeks of gestation (4,5). Glucagon, but not glucose or tolbutamide, enhanced insulin release from pancreatic islets isolated from human fetuses of 12-16 weeks of gestation (6). Recently, it has been reported that insulin release was significantly enhanced from human fetal pancreatic slices by a number of agents (arginine, leucine, glucagon, theophylline, dibutyryladenosine cyclic 3',5'-monophosphate, etc.) but not by glucose (7). These fetuses were of 1624 weeks gestation. Immunoassayable insulin and glucagon have been demonstrated in slices of fetal human pancreas by the 50th day of gestation; neither hormone was found in the pancreas of a 2 5-day-old fetus (L. D. Schaeffer, M. W. Wilder, and R. H. Williams, in preparaReceived January 4, 1972. 1 Supported in part by PHS grants AM-O5O2O and AM-02456. 2 Supported in part by PHS Fellowship 1F03 AM-47142-01A1 and Research Career Development Award 1K04 AM-47142-01 from the National Institute of Arthritis and Metabolic Diseases. tion). We now report studies on organ cultures of fetal human pancreas as an approach to the investigation of insulin release in the developing human pancreas. Materials and Methods Human fetuses were obtained at therapeutic abortion, except for one fetus obtained from an ectopic pregnancy. Gestational ages, estimated from crown to rump length, ranged from 55-140 days (8). The pancreas was identified with a dissecting microscope, removed and immediately transported in isotonic saline to the laboratory. Excess peripancreatic tissue was removed. The pancreas was cut into pieces about 2 mm thick and placed on a stainless steel grid in an organ culture dish. Medium 199 (9), supplemented with fetal calf serum (15% v/v) and neomycin (50 ngm/ml), was added. As measured by a Technicon Auto Analyzer, glucose concentration in this medium was about 115 mg/100 ml. Cultures were maintained at 37 C in an atmosphere of 5% CO2 and 95% air. Culture medium was usually changed every other day and saved for cellulose radioimmunoassay of immunoreactive insulin (IRI), using a Novo human insulin standard (10). Insulin was extracted from some explants by the method of Davoren (11). Some explants were also examined after hemotoxylin-eosin and aldehyde-fuchsin stains. During the second week of culture, studies of insulin release in the presence of glucose, tolbutamide, arginine, or theophylline as stimulatory agents were performed in Krebs-Ringer phosphate buffer (KRP), pH 7.4, with 2% (v/v) fetal calf serum. In each study, the culture was incubated first in a baseline medium of KRP containing 1.67 mM glucose for 1530-min periods. The medium was then changed to KRP containing the test substance and incubation was continued for another 60 min (in a few cases 30 min, for which IRI release was not significantly

The stimulus-secretion coupling of glucose-induced insulin release. 8. Combined effects of glucose and sulfonylureas.

Abstract. Sulfonylureas such as BS4231 and HB419 stimulate insulin secretion in pieces of rat pancreatic tissue. Their insulinotropic effect requires the presence of a sufficient amount of calcium in the incubation medium. Both sulfonylureas markedly stimulate calcium uptake by isolated islets of Langerhans, their stimulant action being more marked in the absence of glucose or at low glucose concentration than at higher glucose levels. Theophylline alone does not stimulate insulin secretion, but it significantly enhances insulin release in the presence of BS4231. This finding is interpreted as another indication that sulfonylureas, like glucose or leucine, favour calcium accumulation in the beta cell by modifying the balance between calcium influx and efflux across the plasma membrane. Heavy water, a microtubules‐stabilizer, inhibits the insulinotropic action of sulfonylureas, but does not affect BS4231‐induced calcium uptake. These data suggest that the insulinotropic action of sulfonylureas results from the accumulation of calcium in the beta cell, and the subsquent activation of the microtubular‐microfilamentous system involved in the emiocytosis of insulin secretory granules.

Metabolic state, pancreatic insulin content and B-cell morphology of normoglycemie spiny mice (Acomys cahirinus): indications for an impairment of insulin secretion.

Plasma glucose, plasma IRI and pancreatic IRI content were measured and B-cell morphology was studied in fasted, fed and glucose-injected normoglycemic spiny mice of varying ages. Both high and low plasma IRI concentrations were observed at all ages, irrespective of the degree of pancreatic stimulation. In old but not in young animals, plasma IRI concentrations correlated with body weight, suggesting that accelerated weight gain was secondary to hyperinsulinemia. Pancreatic IRI content was higher than in other strains of non-diabetic mice and increased with age but was unrelated to either plasma IRI concentrations, or to body weight individual age groups. Relative to pancreatic IRI content, plasma IRI concentrations of spiny mice were lower than those of normal or obese-hyperglycemic mice of other strains. The B-cells showed an unusual degree of granulation. Pale granules predominated and had a characteristic tendency to fusion and confluence. With the exception of 1 out of 200 animals (in the islets of which emiocytosis was seen), no signs of enhanced insulin release could be observed. Neo-formation ofβ-granules by the Golgi complex of already well granulated cells was the only feature consistently associated with pancreatic stimulation by glucose injection. The data are in agreement with the hypothesis of a B-cell defect resulting in an impairment of insulin secretion and also suggest that the B-cells of normoglycemic spiny mice may contain two compartments of insulin: one which is accessible to secretory stimulation and has a rapid turnover and one which is relatively inaccessible to stimulation and accumulates with age. Available data do not allow for the identification of the postulated defect or for the anatomical or physiological definition of the two compartments of insulin.

Insulin biosynthesis and secretion--a still unsettled topic.

The mechanism of insulin biosynthesis and secretion is only partially understood, and new evidence suggests that its complexity is greater than previously thought. Thus, ultrastructural evidence indicates the presence of a discontinuous, microvesicular transfer of secretory material between rough endoplasmic reticulum and Golgi complex, and further suggests that more than one morphologic mode of insulin release must be considered. Similarly, biochemical evidence suggests that stimulation of insulin release involves a multicomponent system including cyclic 3′, 5′ AMP, α and β autonomous nervous receptors, Ca++ and at least two types of substrate effects, one of them specific for certain substrates only. The complexity of the stimulatory mechanism involved is particularly well illustrated by studies with metabolic inhibitors — for example, mannoheptulose inhibits glucose-induced stimulation of insulin release, as expected, but enhances insulin release induced by tolbutamide. D-2-deoxyglucose inhibits glucose-induced but enhances pyruvate-induced or tolbutamide-induced release of the hormone.

Effect of arginine on insulin release in vitro

The effect of arginine on insulin release was studied by observing the effect of glucose and arginine, both singly and together, on the release of immuno insulin from pieces of rabbit pancreas incubated in bicarbonate buffer. Arginine alone (1.0–3.0 mg./ml.) failed to stimulate insulin release above control levels, whereas the same pancreas when subsequently exposed to a glucose concentration of 3.0 or 3.5 mg./ml. showed a convincing response. When ariginine was tested in buffer fortified with 1.5 mg./ml. glucose, it significantly enhanced insulin release. These data suggest that arginine influences insulin release by enhancing the stimulatory effect of the glucose metabolite(s) which triggers insulin release in the B-cell.