Biphasic Insulin Release Research Articles

A new technique for hepatic portal vein catheterization in freely moving rats.

To perform kinetic studies of pancreatic hormones in freely moving rats, we have designed a new technique for hepatic portal vein catheterization through the left branch of the portal vein. Rats were followed up for 14-38 days. Catheter patency allowed blood sampling in 86% of rats on postoperative day 8 and in 55% on day 12. Rat growth, liver functions, and regional blood flows (measured with radiolabeled microspheres) were normal. In freely moving rats fasted for 16 h, portal venous concentrations in the basal state were: glucose, 112 +/- 4 mg/dl; insulin, 67 +/- 10 microU/ml; and glucagon, 480 +/- 74 pg/ml. These values remained stable for a 1-h period. Intravenous glucose infusion induced a biphasic insulin release, significant from the 1st min of infusion on, and a significant decline in glucagon level. Intravenous arginine stimulated both A and B cells. The portoperipheral hormone concentration gradient, which was detected in the basal state, increased during arginine infusion, particularly in regard to insulin. Ingestion of a mixed meal induced an early glucagon release and a sustained rise in insulin and glucose concentrations.

A role for endogenous prostaglandin E in biphasic pattern of insulin release in humans.

These studies were undertaken to evaluate in humans the possible physiological role of prostaglandins of the E series (PGE) in modulating insulin release and to assess whether endogenous PGE synthesis may account for the biphasic pattern of insulin secretion. We used a square-wave glucose stimulation previously determined to give maximal biphasic insulin release. Infusion of lysine acetylsalicylate to block the synthesis of endogenous PGE increased by twofold total insulin response to glucose and also converted insulin release to a multiphasic pattern. The infusion of exogenous PGE1 (0.2 microgram X kg-1 X min-1) or PGE2 (10 micrograms/min) in addition to lysine acetylsalicylate restored the typical biphasic pattern of insulin release and also decreased total insulin release to values similar to those of control studies. Infusion of either PGE1 or PGE2 in the absence of lysine acetylsalicylate reset insulin secretion to a lower level without altering the kinetics of release. On the basis of these results, it is hypothesized that endogenous PGE released in response to glucose stimulation exert an inhibiting effect on insulin release that becomes biphasic in appearance.

Cellular mechanisms of insulin release: the effects of vitamin D deficiency and repletion on rat insulin secretion.

To determine whether impaired insulin release from perifused rat islets of vitamin D-deficient (D-def) rats is a result of vitamin D-deficiency specifically or an associated decrease in food intake, we: 1) compared insulin release from islets of vitamin D-def rats with insulin release from islets of pair fed (pf) normal rats, and 2) measured the effects of 1,25(OH)2D3 treatment on food intake and insulin secretion from islets of D-def rats. Both vitamin D-def and pf normal rat islets showed significantly diminished insulin release in comparison with normal controls but were not different from each other. When D-def rats were repleted with 1,25(OH)2D3, food intake increased and insulin secretion improved during perifusion of rat islets. When D-def rats treated with 1,25(OH)2D3 were prevented from increasing their food intake in response to 1,25(OH)2D3 by pair feeding to a group of untreated D-def rats, insulin release from islets of treated rats was not significantly different from untreated D-def rats. To separate the effects of vitamin D deficiency from hypocalcemia, a group of vitamin D-def hypocalcemic rats was compared with a group of D-def normocalcemic rats. Normocalcemia did not reverse the defect in insulin release. In studies of cellular calcium uptake, both pf and D-def rat islets took up less calcium than normal islets but calcium uptake was not different between pf and D-def rat islets. Our studies suggest that vitamin D deficiency is associated with marked impairment of biphasic insulin release and that the decrease in food intake may account for this impairment at least in part.

Cellular mechanisms of insulin release. Effects of retinoids on rat islet cell-to-cell adhesion, reaggregation, and insulin release.

We studied the effects of retinoids on islet cell-to-cell adhesiveness and glucose-induced insulin release from rat islets. For adhesion studies, islets were dispersed using low concentrations of trypsin. Thirteen cis-retinoic acid (13 cis-RA) was added to a suspension of 15 X 10(5) islet cells and adhesion of cells was quantitated using a hemocytometer. For functional studies, we measured biphasic insulin release from collagenase-isolated perifused islets, dispersed cells, and single large aggregates (clumps) of islet cells. Thirteen cis-RA (10(-4) M) stimulated insulin secretion at 9.7, 12.5, 16.7, and 27.7 mM glucose. Maximal effects of 13 cis-RA (174% of control) were evident during second phase release at 9.7 mM glucose. Thirteen cis-RA (10(-7) and 10(-6) M) caused cells to adhere to each other, and at higher concentrations, 13 cis-RA caused dispersed cells to reaggregate into a single clump. These retinoid-induced clumps were perifused in a Bio-Gel P-2 gel column. Secretion from the clump was twofold greater than from an equal number of perifused dispersed cells. Electron microscopic and freeze-fracture examination of the clump showed reaggregated cells to be intact and the presence of gap junctions between cells. In conclusion, 13 cis-RA has marked effects on islet cell-to-cell adhesiveness. Trypsin-dispersed cells reaggregated by 13 cis-RA have anatomical contacts and secrete more insulin as an aggregate than as dispersed cells. Thirteen cis-RA increases insulin release possibly by increasing adhesion or interactions between beta-cells.

A new in vitro model for studies of pancreatic polypeptide secretion and biochemistry.

A new model tissue (pseudoislet) is described for studies of pancreatic polypeptide (PP) secretion and biochemistry. It consists of islet-like aggregates of canine pancreatic endocrine cells which are formed and maintained on tissue culture. Immunocytochemical staining revealed that pseudoislets prepared from the duodenal end of the pancreas contained a predominance (40-60%) of F cells (the PP secreting cell). Also present were 10-25% exocrine cells and an equal proportion of A, B and D cells. Several studies were conducted to characterize the pseudoislets' capacity to secrete PP. Basal rates of PP release and the concentration of PP per pseudoislet remained constant during four weeks of culture. Stimulation at weekly intervals by carbachol (0.1 mM) resulted in a stable secretory rate for 2 weeks, that declined progressively at weeks 3 and 4. When studied in a perfusion system, carbachol-stimulated PP release occurred in a biphasic pattern, similar to the well-recognized biphasic release of insulin from perifused rat islets. Dose-response curves of four cholinergic agonists revealed clear differences in secretagogue activity. Acetylcholine and methacholine were found to be equipotent, followed in order of potency by carbachol and bethanechol. These histologic and secretory data show that canine pseudoislets are healthy tissues composed of a high proportion of F cells which secrete PP in response to cholinergic stimulation. The data suggest that the cultured canine pseudoislet model provides an excellent system useful in studies of PP secretion and biosynthesis.

The relative importance of first- and second-phase insulin secretion in countering the action of glucagon on glucose turnover in the conscious dog.

While it is known that glucagon induces a biphasic release of insulin when infused into a normal animal, it is not known whether the resultant pattern of insulin secretion has important metabolic consequences. To ascertain whether such is the case, glucagon was elevated fourfold in the presence of first-, second-, or combined first- and second-phase insulin release to determine ability of the latter hormone to antagonize the effect of glucagon on glucose turnover in the conscious dog. To separate the effects of the different phases of insulin release the “pancreatic clamp” technique, in which somatostatin is given to inhibit the endocrine pancreas and replacement amounts of insulin and glucagon are given intraportally, was used. In this way, a rise in glucagon (∼220 pg/ml) was brought about in the presence of simulated first-phase (peak IRI 25 μU/ml at 5 min; basal by 30 min), secondphase (peak IRI 19 μU/ml at 30 min and sustained elevation thereafter), or first- plus second-phase (peak IRI 33 μU/ml at 5 min; 17 μU/ml at 30 min and sustained elevation thereafter) insulin release. Optimal glycemic control required both first- and second-phase insulin release. A selective deficiency of first-phase release resulted in a transient (2 h) worsening of the glucagon-induced hyperglycemia (twofold the normal increment). This defect was attributable to a larger initial rise in glucose production (3.6 ± 0.6 mg/kg · min) than that observed when both phases of insulin release were present (0.9 ± 0.4 mg/kg · min). First-phase insulin release had no significant effect on glucose clearance. A selective deficiency of second-phase release resulted in marked (sixfold) and prolonged worsening of the glucagon-induced hyperglycemia. In this case, however, the hyperglycemia was primarily the result of a defect in glucose clearance. Glucose clearance fell by 29 ± 7% instead of rising by 30 ± 4% as it did when both firstand second-phase release were present. Glucose production was mildly elevated between 15 and 75 min when second-phase insulin release was deficient relative to that apparent when both the first- and secondphases of release were present; this also contributed to the abnormal hyperglycemia. We conclude that both phases of insulin release are vital to full counterregulation of the action of glucagon on glucose metabolism. First-phase insulin release is important to counter the quick effect of glucagon on glucose production, while second-phase insulin release is important to sustain that inhibition and to augment glucose utilization. Absence of first-phase release results in a transient (2 h) and moderate (20–30 mg/dl) glycemic defect while an absence of second-phase release results in a prolonged and dramatic (70–100 mg/dl) defect.

Isolated perfused Brockmann body as a model for studying pancreatic endocrine secretion

The release of insulin, glucagon, and somatostatin was investigated in a newly developed in vitro perfusion preparation of the splenic Brockmann body (principal pancreatic islet) of the channel catfish (Ictalurus punctatus). The preparation was viable for up to 7 h by functional and morphological criteria. Glucose evoked a biphasic release of insulin and somatostatin, but had no effect on the release of glucagon. Arginine at basal glucose concentration caused a marked release of insulin and glucagon, but only a minor release of somatostatin.

Phenytoin inhibition of insulin release. Studies on the involvement of Ca2+ fluxes in rat pancreatic islets.

The mechanism by which phenytoin inhibits insulin release was studied. Insulin release and 45Ca2+ efflux were measured during perifusion of collagenase isolated rat islets after 2-day maintenance in tissue culture in the presence of a trace amount of 45Ca2+. Islets maintained in the absence of the isotope were used to measure 45Ca2+ uptake over 5 min. Glucose (16.7 mM) induced a biphasic release of insulin, which was accompanied by a biphasic increase in the rate of 45Ca2+ efflux above basal. Phenytoin (80 microM) added during second phase rapidly inhibited insulin release. In contrast, phenytoin added together with glucose failed to affect first phase, but reduced second phase release by 64%. Phenytoin failed to affect basal 45Ca2+ efflux in the presence or absence of Ca2+, nor did it interfere with the inhibitory effect of high glucose on Ca2+ efflux. The drug did not affect basal Ca2+ uptake, but significantly reduced glucose-induced Ca2+ uptake. Glucose utilization was not inhibited by phenytoin. It is suggested that phenytoin inhibits glucose-stimulated insulin release by interfering with Ca2+ uptake via voltage-dependent Ca2+ channels. The pattern of inhibition of insulin release appears to favor this conclusion as the second phase is more dependent on the stimulation of Ca2+ uptake than is the first phase.

Insulin Release from the Isolated Perfused Rat Pancreas Containing Insulinomata Induced by Streptozotocin and Nicotinamide: Effects of Glucose and Responses to Tumor Removal

The kinetics of insulin secretion in response to glucose were studied in the in vitro perfused rat pancreas before and after removal of islet cell tumors induced by streptozotocin and nicotinamide. In addition, insulin secretion before and after tumor removal was compared with that from normal pancreata before and after sham operations, respectively. Thus, the two pancreas preparations were subjected to repeated perfusions with glucose. Perfusion of pancreata containing tumors with 8.4 mM glucose resulted in biphasic release in a pattern similar to that of normal pancreas. However, both basal and stimulated insulin secretion of tumor-bearing pancreata were greater than either those of pancreata from which tumors had been excluded by ligature or those of normal pancreata before sham operation. A second increase in the concentration of glucose from 2.8 to 8.4 mM also produced a biphasic release of insulin from extratumoral pancreata as well as from sham-operated normal pancreata. However, the insulin secretory response to glucose of extratumoral pancreatic tissue was less than that of control pancreatic tissue. Our findings indicate that pancreatic islet cell tumors induced by streptozotocin and nicotinamide respond to glucose with typical biphasic insulin release. Thus, chemically induced rat insulinomata may provide a readily available and valuable model of insulin-secreting tissue, analogous to normal islets. Furthermore, our study suggests that the B cell function of pancreata containing tumors is inhibited by the preexisting tumor-induced hyperinsulinism or by its metabolic consequences.

Evidence for the involvement of Na/Ca exchange in glucose-induced insulin release from rat pancreatic islets.

Glucose-induced inhibition of Ca(++) extrusion from the beta-cell may contribute to the rise in cytosol Ca(++) that leads to insulin release. To study whether interference with Na/Ca exchange is involved in this inhibition the effects of glucose were compared to those of ouabain. This substance inhibits Na/K ATPase, decreases the transmembrane Na(+) gradient in islets, and thus interferes with Na/Ca exchange. Collagenase isolated rat islets were maintained for 2 d in tissue culture with a trace amount of (45)Ca(++). Insulin release and (45)Ca(++) efflux were then measured during perifusion. In Ca(++)-deprived medium (to avoid changes in tissue specific radioactivity) 16.7 mM glucose inhibited (45)Ca(++) efflux. Initially 1 mM ouabain inhibited (45)Ca(++) efflux in a similar fashion, the onset being even faster than that of glucose. The effects of 16.7 mM glucose and ouabain were not additive, indicating that both substances may interfere with Na/Ca exchange. In the presence of Ca(++), 16.7 mM glucose induced biphasic insulin release. Ouabain alone caused a gradual increase of insulin release. Again, the effects of ouabain and 16.7 mM glucose were not additive. In contrast, at a submaximal glucose concentration (7 mM) ouabain enhanced both phases of release. An important role for Na/Ca exchange is suggested from experiments in which Ca(++) was removed at the time of glucose-stimulation (16.7 mM). The resulting marked inhibition of insulin release was completely overcome during first phase by ouabain added at the time of Ca(++) removal; second phase was restored to 60%. This could be due to the rapid inhibitory action of ouabain on Ca(++) efflux thereby preventing loss of cellular calcium critical for glucose to induce insulin release. It appears, therefore, that interference with Na/Ca exchange is an important event in the stimulation of insulin release by glucose.

Development of insulin release by fetal rat pancreas in vitro: effects of glucose, amino acids, and theophylline.

The dynamics of insulin release by pieces of fetal rat pancreas from 17.5 to 21.5 days of gestation was measured in an in vitro perifusion system. Interactions between glucose, theophylline, and a mixture of 12 amino acids at physiologic concentration (mix. A.A.) were studied. On day 17.5, 13.9 mM glucose induced only a small (10 min) early phase of insulin release. The late phase of insulin secretion appeared on day 18.5 and gradually increased as the gestation proceeded. The mix. A.A. (9 mM) or theophylline (5 mM) potentiated the two phases of insulin release induced by 13.9 mM glucose from days 18.5 to 21.5 of gestation. On day 21.5, the combination of theophylline and mix. A.A. at 2.2 mM glucose stimulated insulin release by fetal pancreas. The dose-dependent curves of the early phase of insulin release, due to glucose or glucose and mix. A.A. (9 mM), showed half maximal responses in the term fetal pancreas at glucose concentrations of 7.2 and 4 mM, respectively. The values were 7.9 and 5.9 mM for the late phase. The results indicate that the mechanisms controlling biphasic insulin release develop during the late fetal life in the rat. Transition from the fetal to adult type of insulin secretion more likely parallels quantitative, rather than qualitative, changes within the B cells.

Temporal changes in islet polymerized and depolymerized tubulin during biphasic insulin release

In the present study, a colchicine binding assay was used to measure changes in islet polymerized and depolymerized tubulin at intervals characterizing the biphasic pattern of glucose-induced insulin release i.e., 2.5, 5.5, 10.5, and 30.5 min. At 2.5 min during the rapid onset of insulin release, a significant increase from 24% to 33% in polymerized tubulin content was observed. This increase in polymerized tubulin was followed by a reduction that temporally correlated with the disappearance of first phase release. Second phase release was also associated with a shift in equilibrium favoring tubulin polymerization at longer incubation periods. The effect of cytochalasin B on the equilibrium between polymerized and depolymerized tubulin was evaluated at a submaximal glucose concentration of 16.5 m M. Under these conditions, cytochalasin B increased polymerized tubulin content in a manner that parallels its enhancing effect on insulin release. These results indicate that the rapid events associated with the biphasic pattern of insulin release are temporally correlated with changes in polymerized tubulin.

Amino acid stimulation of insulin secretion from the in situ perfused eel pancreas; Modification by somatostatin, adrenaline, and theophylline

Amino acid stimulation of insulin secretion, and its modification by somatostatin, adrenaline and theophylline, has been studied using the in situ perfused eel pancreas. Lysine, leucine, and phenylalanine at 10 m M caused biphasic insulin release, while histidine produced a weak, nonphasic response by comparison. Lysine was the most effective secretagogue in this series. Somatostatin (10 and 100 ng/ml) and adrenaline (10 and 50 ng/ml) significantly reduced total insulin secretion in response to 10 m M lysine. At the lower concentration the hormones were without effect on first-phase insulin secretion but significantly lowered second phase secretion. At the higher concentration, however, first phase secretion was significantly reduced, and second phase secretion virtually abolished. Theophylline (2 m M), increased basal insulin secretion in a biphasic manner and significantly potentiated 10 m M lysine-stimulated insulin secretion. These data indicate an important role of somatostatin and adrenaline in the control of B-cell function in teleosts and further, suggest that insulin-secretory responses, as in mammals, may be cyclic AMP dependent.

Stimulation of insulin release in the absence of extracellular calcium by isobutylmethylxanthine and its inhibition by somatostatin.

It has been suggested that somatostatin may inhibit insulin release by interfering with pancreatic islet calcium uptake. To further investigate this hypothesis, the effect of somatostatin on insulin release was examined under conditions where islet uptake of calcium would be unlikely to occur. The phosphodiesterase inhibitor, isobutylmethylxanthine (0.75 mM), was found to stimulate biphasic insulin release from rat pancreases perfused in vitro in the absence of added extracellular calcium on a background of 0.3 mM EGTA And 8 mM glucose; these results support previous suggestions that methylxanthine phosphodiesterase inhibitors may stimulate insulin release by increasing islet cytosol free calcium through translocation of bound (stored) intraislet calcium. Somatostatin (1.0 muM) completely inhibited both phases of isobutylmethylxanthine-stimulated insulin release. Since uptake of extracellular calcium by islets was unlikely under the present experimental conditions, these results suggest that somatostatin inhibition of insulin release is probably due to interference with a cAMP-dependent translocation of intraislet calcium, or to interference with some other effects of cAMP or an effect of calcium itself rather than to interference with islet calcium uptake.

Tolbutamide stimulation and inhibition of insulin release: studies of the underlying ionic mechanisms in isolated rat islets.

The effects of tolbutamide on insulin release, 45Ca2+ uptake and 86Rb+ efflux were studied in isolated rat islets. At a low glucose concentration (75 mg/dl), tolbutamide (20-500 microgram/ml) produced a rapid, dose-dependent increase in insulin release from perifused islets. After 30-40 min however, the rate of secretion as well as the potentiating effect of theophylline were inversely related to the concentration of sulphonylurea. The monophasic release of insulin triggered by tolbutamide (100 microgram/ml) at low glucose could be evoked again by removing and reintroducing the drug, or by temporarily withdrawing calcium or adding cobalt to the medium. Tolbutamide (20 microgram/ml) accelerated and potentiated the biphasic insulin release in response to a secondary stimulation by glucose (150 mg/dl). By contrast, 100 microgram/ml tolbutamide reduced the releasing effect of glucose to a slow increase in secretion rates. Theophylline normalized the second phase of release, but did not restore the rapid phase. Tolbutamide stimulated 45Ca2+ influx (2 min-uptake) in islet cells; this effect was maximum immediately after addition of the drug and decreased later on, exhibiting a monophasic pattern. Glucose stimulation of Ca2+ uptake (5 min) was reduced in the presence of 100 microgram/ml tolbutamide. At a low glucose concentration, tolbutamide reversibly reduced 86Rb+ efflux (tracer of K+) from islet cells, without altering the further inhibition of this efflux by a later glucose increase. It is suggested that tolbutamide depolarizes B cells partially by reducing their K+ permeability. This depolarization leads to opening of voltage-dependent calcium channels and the resulting Ca2+ influx triggers insulin release. The important and maintained depolarization by high concentrations of tolbutamide may secondarily inactivate these channels and cause a decrease in Ca2+ influx. This could explain the monophasic release of insulin and the refractoriness of B cells to subsequent glucose stimulation.

Different dynamics of insulin secretion in the perfused pancreas of mouse and rat

The dynamics of insulin release were studied in the perfused pancreas of rats and mice. Perfusion of the rat pancreas with 20 mM D-glucose resulted in the classical biphasic release of insulin with a rising second phase. However, in normal C57BL/KsJ-mice and non-inbred mice, whether fed or starved, the second phase was nearly constant. The secretory dynamics of KsJ-mice were essentially the same, whether the glucose concentration was 30 or 20 mM, whether the medium contained 2.56 or mM Ca2+, and whether or not the medium was supplemented with 5 mM pyruvate, 5 mM glutamate, and 5 mM fumarate. Insulin secretion in these mice was almost totally inhibited by omission of Ca2+, and was markedly enhanced by 3-isobutyl-1-methylxanthine. Insulin release during the constant phase was reversed by lowering the glucose concentration. A second rise of glucose from 3 to 20 mM produced a secretory pattern very similar to the first response. These studies indicate that the dynamics of insulin secretion are somewhat different in rats and mice. Since similar results were obtained with C57BL/KsJ-mice and non-inbred mice, the liability of KsJ-mice to develop beta-cell failure when stressed by the mutated db gene is not related to the constancy of the second insulin secretory phase.

Biphasic Insulin Release in Rat Islets of Langerhans and the Role of Intracellular Ca++Stores*

The role of extracellular Ca++ in the development of biphasic insulin release has been studied previously by using Verapamil to block the glucose-induced stimulation of Ca++ uptake. It was found that glucose-stimulated uptake of Ca++ from the extracellular fluid was important for the second phase of insulin release but had no role in the first phase. In the current study, the role of intracellular Ca++ stores in biphasic insulin release has been studied. Isolated rat islets were maintained in tissue culture for 46 h with normal (1 HIM) or high (5 RIM) phosphate concentrations. The intracellular calcium content of islets maintained in high phosphate was 50 times greater than the content of control islets. Insulin release was studied by perifusion, and the Ca++ concentration in the medium was decreased to 0.1 mM during stimulation with high glucose to minimize the effect of extracellular Ca++. The islets maintained in normal phosphate responded with a transient subnormal first phase insulin release. In cont...

Ultracytochemical calcium distribution in B cells in relation to biphasic glucose-stimulated insulin release by the perfused rat pancreas

Ultracytochemical calcium distribution in B cells in relation to biphasic glucose-stimulated insulin release by the perfused rat pancreas

Ultracytochemical calcium distribution in B cells in relation to biphasic glucose-stimulated insulin release by the perfused rat pancreas.

Calcium distribution in B cells of the isolated perfused rat pancreas was examined by the pyroantimonate precipitation technique in relation to the insulin secretory pattern of the perfused pancreas in response to 3 mM or 20 mM D-glucose or 20 mM D-glucose in calcium-depleted ethylene glycol tetra-acetic acid (EGTA) medium. Perfusion fixation after various time intervals from 3 to 30 min allowed appropriate relation to secretory phases. Qualitative and quantitative evaluation of the precipitation patterns revealed a significant increase in cell membrane associated percipitates after 3--5 min of perfusion with 20 mM glucose compared with the results after perfusion with 3 mM glucose. After 10--30 min of perfusion with 20 mM glucose there was an additional significant increase in precipitates located in the cytoplasm and the halos of the secretory granules. Perfusion with 20 mM glucose in calcium-deprived EGTA medium strongly reduced the number of precipitates within the B cells. The results suggest that cell membrane associated calcium may be involved in exocytosis, and by its sudden increase may trigger the first phase of insulin secretion. The calcium stores in the cytoplasm and the granules may be of importance for long-term regulation of insulin release.

Defective calcium handling and insulin release in islets from diabetic Chinese hamsters

In pancreatic islets from normal Chinese hamsters preloaded with 45Ca2+, glucose-induced biphasic insulin release was associated with increased 45Ca2+ efflux; islets from diabetic hamsters showed decreased insulin release and no increase in 45Ca2+ efflux. The lack of stimulated 45Ca2+ efflux persisted even when glucose-induced insulin release was potentiated by 3-isobutyl-1-methylxanthine. Since glucose-stimulated 45Ca2+ uptake by diabetic islets was not impaired, a defect in intracellular Ca2+ handling may be involved in the defective insulin release of the diabetic Chinese hamster.