Gerold M. Grodsky

A threshold distribution hypothesis for packet storage of insulin and its mathematical modeling

Phases of insulin release were studied in the perfused pancreas during a variety of glucose stimulation patterns. Patterns included staircase stimulations, constant prolonged single steps, restimulations, and ramp functions. Except at low concentrations, prolonged single steps of glucose elicited early spikes of insulin and a slowly rising second phase. Total insulin in the initial spikes increased with higher glucose concentrations. However, the time-related pattern of these spikes was similar in all cases; ratios of initial secretion rate to total insulin released were constant. Total insulin released in this early phase approximated a sigmoidal function of glucose concentration; mathematical differentiation of this function gave a skewed bell-shaped distribution curve. Staircase stimulations caused insulin to be released as a series of transient spikes which did not correlate with the increment of glucose but rather to the available insulin for a given glucose concentration minus that released in previous steps. The sum of total insulin released as spikes in a staircase series leading to a given glucose concentration was the same as when that concentration was used as a single step. Interrupted prolonged glucose infusions indicated the second phase of insulin release could prime the pancreas and that the first and second phases were interrelated. When glucose was perfused as ramp functions of slow, increasing, concentration, phasic response disappeared.A previous two-compartmental model was expanded to include a threshold or sensitivity distribution hypothesis. This hypothesis proposes that labile insulin is not stored in a homogeneous form but as packets with a bell-shaped distribution of thresholds to glucose. These packets respond quickly when their threshold levels to glucose are reached or exceeded. Data from single step stimulations were utilized for constructing a mathematical model which simulated satisfactorily the various stimulation patterns.

Cation requirements for insulin secretion in the isolated perfused pancreas.

The effect of cations on insulin release was studied in the isolated pancreas of the rat perfused with a synthetic perfusate consisting of albumin and buffer. The omission of calcium and magnesium ion completely inhibited insulin release stimulated by glucose. The ionic requirement was specific for calcium ion since perfusates containing calcium but no magnesium permitted normal insulin release by glucose, whereas perfusates containing magnesium but no calcium did not. The presence of only 0.2 mEq. per liter calcium was adequate to permit much, though not all, of the insulin response after glucose to occur. Potassium ion, when raised from 4 to 8 mEq. per liter, directly stimulated insulin release in the complete absence of glucose.

Insulin within islets is a physiologic glucagon release inhibitor.

To determine if glucagon secretion is under physiological control of intra-islet insulin, pancreata from normal rats were perfused at a 100 mg/dl glucose concentration with either guinea pig antiinsulin serum or normal guinea pig serum in a nonrecirculating system. Perfusion of antiserum was followed within 3 min by a significant rise in glucagon that reached peak levels three times the base-line values and assumed a hectic pattern that returned rapidly to base-line levels upon termination of the antiserum perfusion. Nonimmune guinea pig serum had no effect. To gain insight into the probable site of insulin neutralization, 125I-labeled human gamma-globulin was added to antiserum or nonimmune serum and perfused for 3 min. More than 83% of the radioactivity was recovered in the effluent within 3 min after termination of the infusion, and only 0.05 +/- 0.015% of the radioactivity injected was present in the pancreas 10 min after the perfusion. The maximal amount of insulin that could be completely bound to insulin antibody at a dilution and under conditions simulating those of the perfusion experiments was 20 mU/min. It is concluded that insulin maintains an ongoing restraint upon alpha cell secretion and in its absence causes hectic hypersecretion of glucagon. This restraint probably occurs largely in the intravascular compartment. Loss of this release-inhibiting action of insulin may account for initiation of hyperglucagonemia in insulin-deficient states.

Characterization of the Effects of Arginine and Glucose on Glucagon and Insulin Release from the Perfused Rat Pancreas

To characterize the mechanisms by which arginine and glucose affect pancreatic alpha and beta cell function, the effects of these agents over their full dose response, both alone and in various combinations, were studied using the perfused rat pancreas. Arginine (0-38 mM), in the absence of glucose, stimulated biphasic glucagon (IRG) secretion (Km approximately 3-4 mM) at concentrations less than 1 mM and caused nonphasic insulin (IRI) release (Km approximately 12-13 mM) but only at concentrations greater than 6 mM. Glucose (0-27.5 mM) alone stimulated biphasic IRI release (Km approximately 9-10 mM) at concentrations in excess of 5.5 mM and caused nonphasic inhibition of IRG secretion (Kt approximately 5-6 mM) at concentrations as low as 4.1 mM. These results demonstrate fundamental differences in pancreatic alpha and beta cell secretory patterns in response to glucose and arginine and suggest that glucagon secretion is more sensitive to the effect of both glucose and arginine. Various concentrations of arginine in the presence of 5.5 mM glucose stimulated biphasic IRG and IRI release: IRG responses were diminished and IRI responses were enhanced compared with those seen with arginine in the absence of glucose. Glucose (0-27.5 mM) in the presence of 3.2 or 19.2 mM arginine caused similar inhibition of IRG secretion (Km approximately 5-6 mM) and stimulation of IRI release (Km approximately 9-10 mM) as that seen with glucose alone, although greater IRG and IRI release occurred. This augmentation of IRI secretion was greater than that expected from mere additive effects of glucose and arginine. Classical Lineweaver-Burk analysis of these results indicates that glucose is a non-competitive inhibitor arginine-stimulated glucagon secretion and suggests that glucose and arginine affect pancreatic alpha and beta cell function via different mechanisms. In addition, comparison of simultaneous insulin and glucagon secretion patterns under various conditions suggests that endogenous insulin per se has little or no direct effect on IRG secretion and that endogenous glucagon does not appreciably affect pancreatic beta cell function.

Early Phase of Insulin Release

The early phase of insulin release in the first five minutes after intravenous administration of glucose, glucagon, and glucose-plus-glucagon was investigated systematically in various clinical conditions. In normal subjects there is an immediate release of insulin after glucose, glucagon, and glucose-plus-glucagon infusions. The latter combination produced the highest insulin levels. Of a group of nonobese subjects with diabetic heritage, some had impaired early release of insulin, but0 their mean response did not differ significantly from the normal group. Investigation of nonobese potential diabetics (offspring of two diabetic parents) revealed that as a group average they had decreased insulin levels during the early phase of insulin release, even though intravenous glucose tolerance was normal. Four of ten subjects had a normal response. Nonobese, noninsulin-dependent diabetics had no insulin response to infused glucose, but when glucagon was added to glucose a significant and rapid insulin discharge was observed. However, the magnitude of this response was about half that seen in normal subjects after glucose-plusglucagon. Finally, the early phase of insulin release was studied in obese nondiabetic subjects who demonstrated an exaggerated insulin release to each stimulus. Again, glucose-plusglucagon was the most potent stimulator of insulin release. It is postulated that impairment in the early phase of insulin release may be the first detectable abnormality of insulin secretion in diabetes mellitus and that glucagon has the capability of restoring this toward normal.

Effect of pulse administration of glucose or glucagon on insulin secretion in vitro

Abstract The isolated perfused pancreas of the rat was modified as follows from that previously used in this laboratory: Albumin buffer was substituted for rat plasma as perfusate, permitting controlled addition of agents with no interference from unknown plasma components; pulse experiments were performed in which agents were rapidly added and complete pancreatic eluates were collected at 30-sec. intervals, allowing measurement of the time relationship between stimulation and insulin response during a single passage of an agent through the pancreas. After addition of glucose, insulin secretion was prompt, occurring within 30 sec. Levels followed the rise and fall of glucose and notably there was no pancreatic memory, insulin secretion terminating 30 sec. after completion of the glucose pulse. Glucagon, containing glucagon-I 131 as an isotopic marker, directly stimulated insulin secretion in the absence of any added glucose. Insulin secretion occurred within 30 sec. after the glucagon entry in the pancreas and paralled glucagon concentration. As with the glucose-stimulated pancreas, there was no pancreatic memory, insulin secretion decreasing to basal levels within 30 sec. after the glucagon pulse was cleared. Glucagon did not affect the chromatographic integrity of simultaneously infused insulin-I 131 . Casein, an inhibitor of “insulinase”, did not duplicate the effect of glucagon. Theophylline, an inhibitor of cyclic AMP degradation, stimulated secretion in the absence of glucose. It was concluded that insulin secretion promptly responds to changes in glucose concentration and there is no significant pancreatic memory. Glucagon can also stimulate insulin secretion by a mechanism not requiring glucose or a sparing effect on insulin degradation, but possibly requiring the production of cyclic AMP.

Constitutional Nonhemolytic Hyperbilirubinemia in the Rat Defect of Bilirubin Conjugation

Summary 1. Broken-cell preparations of liver and of kidney from rats with constitutional nonhemolytic hyperbilirubinemia failed to synthesize bilirubin or o-aminophenol glucuronides. 2. Inhibition of β-glucuronidase with potassium saccharate failed to influence conjugation of bilirubin in these animals. 3. The evidence presented suggests that in constitutional nonhemolytic hyperbilirubinemia in rats, glucuronyl transferase activity is absent or inhibited.

A New Phase Of Insulin Secretion: How Will It Contribute to Our Understanding of β-Cell Function?

Although initially described two decades ago, biphasic insulin secretion has gradually been understood to reflect (β-cell rate sensitivity, be important in minimizing overinsulinization in normal individuals, be defective in non-insulin-dependent diabetes mellitus (NIDDM), and be useful as an early predictor in prediabetic individuals. Recently, a third phase of insulin secretion has been observed in fully in vitro islets or pancreatic preparations. This phase is characterized as a spontaneous decline of secretion (desensitization) during 24 h of sustained exposure to glucose or other secretagogues and does not appear to be simply an artifact of in vitro preparations. The impaired secretion is localized to the final release process in that neither glucose-stimulated proinsulin synthesis nor its conversion to insulin is affected. The mechanisms responsible for the third phase of reduced secretion are unknown. Kinetic evidence suggests it is not caused by emptying of a single finite insulin storage compartment but does not exclude the possibility that the decreased release reflects depletion of threshold-sensitive β-cells recruited at a given secretagogue level. Alternatively, the third phase may reflect inhibition of a priming or terminal insulin-release process by metabolic feedback. Because several secretagogues cause similar third-phase impaired release, even in the absence of glucose, desensitization probably occurs at a common fundamental site in the secretory site (e.g., calcium metabolism). Preliminary studies indicate the third phase is not the result of a paracrine effect by other islet hormones or of a change in muscarinic regulation. Whether other neurologic effectors are involved requires further investigation. Experiments examining the effect of glucose-induced third-phase desensitization on other secretagogues emphasize that priming (potentiation) and desensitization of insulin secretion are occurring simultaneously and that interpretations of results can be highly dependent on the design of the experiment. The possibility that the third phase of insulin secretion may involve mechanisms (waning of priming; desensitization) that relate to glucose-induced β-cell desensitization in NIDDM is discussed. Ultimate appreciation of the significance of the third phase of insulin secretion may develop, as was true for biphasic secretion, with our increasing understanding of the underlying processes in human diabetes.

The Third Phase of In Vitro Insulin Secretion Evidence For Glucose Insensitivity

In this study, in vitro B-cell models are described, which may be applicable for studying the reported B-cell desensitization produced by hyperglycemia in IDDM and NIDDM. Using a programmable perifusion/ perfusion system, insulin secretion from perifused islets was measured at 10–30-min intervals for 24–50 h. After 3–4 h continuous glucose (11 mM), a new phase of insulin release occurs in which secretion declines to, and remains at, ∼25% maximal release. Results were similar when using: (1) perifused islets embedded in Cytodex 3, or Bio-Gel P-2,100–200 mesh; (2) batchincubated islets with hourly changes of medium; and (3) the isolated pancreas perfused for 8 h. Three different media, Hana HB104 (fortified, fully defined medium), RPMI-1640 + 10% FBS, and perfusion bufferalbumin, were used. Despite reduced secretion to continuous glucose, each system responded vigorously to an acute stimulation with glucose-forskolin. Decreased secretion was primarily caused by decreased secretagogue efficiency (reduced fractional secretion). Prolonged stimulation with glucose or glucose-IBMX produced a similar waning of secretion regardless of the amount of insulin released. It is concluded that the third phase of insulin secretion may represent a secretagogue-induced, signal desensitization of the B-cell, rather than exhaustion of a B-cell compartment of Stored insulin.

Iontophoresis of monomeric insulin analogues in vitro: effects of insulin charge and skin pretreatment.

The aim of this study was to investigate the influence of association state and net charge of human insulin analogues on the rate of iontophoretic transport across hairless mouse skin, and the effect of different skin pretreatments on said transport. No insulin flux was observed with anodal delivery probably because of degradation at the Ag/AgCl anode. The flux during cathodal iontophoresis through intact skin was insignificant for human hexameric insulin, and only low and variable fluxes were observed for monomeric insulins. Using stripped skin on the other hand, the fluxes of monomeric insulins with two extra negative charges were 50-100 times higher than that of hexameric human insulin. Introducing three additional charges led to a further 2-3-fold increase in flux. Wiping the skin gently with absolute alcohol prior to iontophoresis resulted in a 1000-fold increase in transdermal transport of insulin relative to that across untreated skin, i.e. to almost the same level as stripping the skin. The alcohol pretreatment reduced the electrical resistance of the skin, presumably by lipid extraction. In conclusion, monomeric insulin analogues with at least two extra negative charges can be iontophoretically delivered across hairless mouse skin, whereas insignificant flux is observed with human, hexameric insulin. Wiping the skin with absolute alcohol prior to iontophoresis gave substantially improved transdermal transport of monomeric insulins resulting in clinically relevant delivery rates for basal treatment.