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The effects of chronic sympathetic hyperinnervation on pancreatic beta-cell insulin secretion were investigated utilizing the in vitro perfused pancreas from transgenic mice. These mice exhibit islet hyperinnervation of sympathetic neurons resulting from overexpression of nerve growth factor in their beta-cells (1). The goal was to determine whether sympathetic hyperinnervation increased classic alpha-adrenergic inhibition of beta-cell insulin secretion or, in contrast, down-regulated beta-cell sensitivity to adrenergic input resulting in enhanced insulin secretion. Both fasting and fed blood sugars and pancreatic insulin content were normal in the transgenics. Response of the transgenic perfused pancreas to low glucose (7 mM) was primarily first phase and normal whereas high glucose (22 mM) caused enhanced, rather than reduced, insulin secretion of both first and second phases. The alpha-antagonist, phentolamine, caused a six-fold increase in glucose-stimulated insulin secretion from the control pancreas, an effect that was blunted for the transgenic pancreas. A similarly blunted response to phentolamine occurred when this agent was superimposed on a combined glucose-forskolin stimulus. (The positive effect on insulin secretion by phentolamine in normal beta-cell preparations has arguably been ascribed to non-specific ionic effects.) Therefore, as a test of possible changes in the ATP regulated K+ channel or the linked Ca++ channels, glyburide was perfused during glucose stimulation. Insulin secretion in response to glyburide was increased two fold in the control pancreas. However, with the transgenic pancreas, in contrast to the enhanced response to glucose, the effect of glyburide was almost completely inhibited. It is concluded that: 1) chronic adrenergic hyperinnervation results in enhanced glucose-stimulated insulin secretion by desensitization of a major alpha-adrenergic inhibitory site(s); and 2) adrenergic hyperinnervation acts directly or indirectly on ion flux to partially inhibit insulin release, an effect which is not desensitized. Since down-regulation of a single alpha-adrenergic receptor would be expected to desensitize both phenomena the observed differential desensitization indicates that different post receptor events or more than one adrenergic receptor are involved.

To evaluate the role of protein aggregation and calcium in the sorting of insulin for regulated vs. constitutive release from the intact pancreas, we targeted the expression of a monomeric mutant form of human (pro)insulin (B9/B27) to the pancreatic beta-cells of transgenic mice. This mutant insulin does not form dimers or hexamers, but can aggregate at high concentration in the presence of calcium. A homozygous line (171) was produced that expressed 55% of the total (pro)insulin message in their beta-cells as the mutant form and had normal pancreatic total (pro)insulin content [measured as immunoreactive insulin (IRI)]. Fasting glucose levels in these transgenics and in homozygous control mice expressing native human (pro)insulin were normal, although levels were abnormally elevated during ip glucose tolerance testing. In the presence of extracellular calcium, regulated IRI release from the isolated perfused pancreas of the transgenic mice was undetectable in the absence of secretagogues and responded with normal phasic kinetics when stimulated with increasing steps of glucose, with glucose plus isobutylmethylxanthine, or with arginine. Without extracellular calcium (0 calcium plus EGTA), normal pancreas did not release IRI in either the presence or absence of secretagogues. In contrast, without calcium or secretagogues, transgenic pancreas spontaneously and constitutively released IRI at high levels equivalent to those elicited by glucose (22 mM) plus calcium from normal pancreas. This release was partially inhibited by glucose or arginine. Constitutive secretion was acutely sensitive to calcium; inhibition occurred within minutes after the addition of calcium and quickly returned to its characteristic level (with overshoot) when calcium was subsequently removed. Somatostatin, at a concentration that caused 50% inhibition of normal glucose-stimulated secretion, did not affect constitutive release. Control pancreas from the transgenic mice, expressing native human (pro)insulin, responded normally to secretagogues and did not constitutively release hormone in the absence of calcium. It is concluded that expression of monomeric human insulin in pancreatic beta-cells from transgenic mice did not interfere with normal phenotypic insulin secretion, indicating that the functional secretory apparatus was not impaired. Constitutive secretion of IRI from the intact pancreas requires both the expression of a monomeric form of insulin and the absence of extracellular calcium, two conditions that reduce aggregation. These results are consistent with the hypothesis that protein aggregation favors sorting to the regulated pathway, whereas suppressed aggregation causes sorting for constitutive release.

Although information regarding insulin secretion usually is considered equivalent when generated in the mouse or the rat, it is established that the kinetics of insulin secretion from mouse and rat pancreatic beta cells differ. The mechanisms underlining these differences are not understood. The in vitro perfused pancreas and isolated islets of the mouse or rat were employed in this study to investigate the role of cyclic adenosine monophosphate (cAMP), a major positive modulator of beta-cell function, as one differentiating signal for the uniquely different insulin release from the beta cells of these commonly used rodents. Glucose-stimulated first-phase insulin release from the perfused pancreas of the rat was higher than the mouse when calculated per gram of pancreas or as fractional secretion, but this phase was identical in the two species when results were adjusted for total body weight. Whether related to insulin content, pancreatic weight or body weight, the rat pancreas responded to glucose with a progressively increasing second-phase insulin release compared to the mouse pancreas, which secreted a flat second-phase of lesser magnitude. Isolated islets from rat and mouse were comparable in insulin content whereas the basal cAMP level of mouse islets was less than half that of the rat. At submaximal stimulation with glucose or glucose + IBMX or forskolin, mouse islets exhibited lower cAMP levels to a given stimulus than the rat. In rat islets cAMP levels increased to approximately 1000 fmol per islet, although insulin secretion maximized by 100-150 fmol.(ABSTRACT TRUNCATED AT 250 WORDS)

Complete loss of pancreatic insulin function in insulin-dependent diabetes is thought to be due to an autoimmune cytokine-mediated destruction of the beta-cell. The effects of several classes of agents on interleukin-1 beta (IL-1 beta)-induced suppression of insulin secretion, beta-cell NAD levels, and beta-cell viability were examined. After overnight incubation of isolated rat islets with 15 U/ml IL-1 beta and 11 mM glucose, sequential hourly insulin secretory responses to the same glucose concentration, 22 mM glucose, and 22 mM glucose plus forskolin were severely inhibited to 10-37% of the control value. Islet NAD levels were also sharply reduced to 43% of the control value after 24-h exposure to IL-1 beta, but not after 1 or 3 h, demonstrating the same time course as that for inhibition of insulin secretion. Exposure to IL-1 beta also decreased islet cell viability measured as trypan blue exclusion. Only 1 mM N-methyl arginine, an inhibitor of nitric oxide synthase, completely protected all three parameters of beta-cell function from damage by IL-1 beta. Nicotinamide and thymidine prevented the IL-1 beta-induced loss of cell viability and suppression of NAD, but had no effect on sustaining insulin secretion. Antioxidants, steroids, and several neuropeptides also did not prevent inhibition or restore the secretory response. Thus, the loss of the secretory response appears to be more narrowly restricted to nitric oxide radical damage induced by exposure to IL-1B.

To evaluate the effect of chronically elevated adenylyl cyclase, we targeted the expression of a constitutively active mutant alpha-subunit (alpha s+) of Gs to the insulin-producing pancreatic beta-cells of transgenic mice. As assessed by the polymerase chain reaction, expression of alpha s+ mRNA was restricted to the transgenic pancreas. Histological analysis by light microscopy and immunohistochemistry for insulin, glucagon, and somatostatin appeared normal in transgenic islets. Pancreatic insulin content was quantitatively the same for alpha s+ transgenic and control mice. Comparisons of glucose homeostasis, insulin secretion, and islet cAMP revealed the expected differences between alpha s+ transgenic and control mice; in every case, however, responses to glucose alone were normal, and the differences were observed only when measurements were performed in the presence of isobutylmethylxanthine (IBMX), an inhibitor of cAMP phosphodiesterase. 1) In vivo, ip glucose tolerance was normal in alpha s+ transgenics; when ip glucose was preceded by administration of IBMX, the rise in blood glucose was approximately 33% less in the transgenic than in the control mice. 2) Insulin secretion from the perfused pancreas stimulated sequentially with 11 and 22 mM glucose caused characteristic first and second phase insulin release that did not differ between transgenic and control pancreases. IBMX increased biphasic insulin release from all pancreases, but caused a 2-fold greater than normal release from the transgenics. 3) Similarly, batch-incubated alpha s+ and control islets secreted equivalent amounts of insulin in the presence of glucose (22 mM) alone, whereas the combination of glucose plus IBMX was twice as effective on alpha s+ islets. 4) Islet cAMP levels paralleled insulin secretion; in the presence of IBMX, but not glucose alone, cAMP was increased 2-fold more in alpha s+ vs. control islets. We conclude that expression of constitutively active alpha s mutant in pancreatic beta-cells of transgenic mice is functionally effective, causing the physiological phenotype of increased islet cAMP and insulin secretion. However, these changes are uncovered only in the presence of IBMX; without IBMX, glucose homeostasis and islet function appear normal. This normalization, or counterregulation, of cAMP synthesis presumably is accomplished by a compensatory increase in cAMP degradation, possibly via increased activity of cAMP phosphodiesterase.

Investigation of intracellular pathways of stimulus-secretion signaling in vivo is possible by transgenic expression of agents known to influence specific biochemical interactions in the cells. The objective of the present study was to establish an experimental model for analyzing signal transduction mechanisms in pancreatic beta-cells in vivo, by expressing the cholera toxin A1 subunit under control of the insulin promoter, intending a constant activation of the Gs-protein, and thereby constant generation of cAMP. Surprisingly, the transgenic mice demonstrated mild hyperglycemia and hypoinsulinemia in vivo, and diminished glucose-induced insulin release from the in vitro perfused pancreas, whereas the pancreatic insulin content was normal. These observations suggest a deficiency in either the insulin release mechanisms or glucose recognition. Although the translated cholera toxin A1 subunit was biologically active, there was no increase in the islet content of cAMP. We conclude that the observed phenotype in the cholera toxin transgenic mice may be caused by a deleterious effect of the transgene itself on beta-cell function, or that counter regulatory mechanisms may compensate for the transgene-induced changes in intracellular enzymatic pathways.

The spontaneous decline of insulin secretion which occurs under a variety of secretory conditions is well documented and suggests a general desensitization of the secretory process distal to signal recognition. Accordingly, we have investigated the effects of agents thought to mobilize intracellular Ca++ on insulin secretion over 24 h, which includes periods of rising secretory activity (second phase) and desensitized secretory activity (third phase). During the first 3 h of glucose stimulation of freshly isolated rat islets, insulin secretion was strongly inhibited by 30 microM 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB) or 300 microM tetracaine hydrochloride (TC). However, when either of these agents was added for the first time to islets at h 20 when insulin secretion was at a low steady rate (third phase), insulin secretion was greatly enhanced. Both these inhibitory and stimulatory effects declined with continued administration. Removal of TMB and rechallenge with high glucose plus forskolin uncovered a residual inhibition in both chronically and acutely treated islets. Coadministration of forskolin with either TMB or TC blunted both inhibitory and stimulatory effects. Pertussis toxin pretreatment, however, did not alter subsequent response of islets to either agent. Thus TMB or TC have opposite, phase-dependent effects on glucose-stimulated insulin secretion. We postulate that potentiators of glucose-stimulated insulin secretion, which are increased during second phase, are most sensitive to inhibitory effects of TMB or TC, and that the low steady rate of third phase permits their stimulatory component(s) to become apparent.

In human diabetes, inherent impaired insulin secretion can be exacerbated by desensitization of the beta cell by chronic hyperglycemia. Interest in this phenomenon has generated extensive studies in genetic or experimentally induced diabetes in animals and in fully in vitro systems, with often conflicting results. In general, although chronic glucose causes decreased beta-cell response to this carbohydrate, basal response and response to alternate stimulating agents are enhanced. Glucose-stimulated insulin synthesis can be increased or decreased depending on the system studied. Using a two-compartment beta-cell model of phasic insulin secretion, a unifying hypothesis is described which can explain some of the apparent conflicting data. This hypothesis suggests that glucose-desensitization is caused by an impairment in stimulation of a hypothetical potentiator singularly responsible for: 1) some of the characteristic phases of insulin secretion; 2) basal release; 3) potentiation of non-glucose stimulators; and 4) apparent "recovery" from desensitization. Review of some of the pathways that regulate insulin secretion suggest that phosphoinositol metabolism and protein kinase-C production are regulated similarly to the theoretical potentiator and their impairment is a major contributor to glucose desensitization in the beta cell.

We examined the effects of rat islet amyloid polypeptide (IAPP) on insulin biosynthesis and secretion by isolated rat islets of Langerhans. Culture of islets for 24 h in the presence of 10(-6) M IAPP and 5.5 mM glucose had no effect on insulin mRNA levels. Similarly, the rates of proinsulin biosynthesis were not altered in islets incubated in 10(-4)-10(-9) M IAPP and 5.5 mM glucose, nor was the rate of conversion of proinsulin to insulin changed at 10(-6) M IAPP. Addition of 10(-5) M IAPP to islets incubated in 11 mM glucose decreased the fractional insulin secretion rate; however, the secretion of newly synthesized proinsulin and insulin was not affected. These data indicate that it is unlikely that IAPP is a physiologically relevant modulator of insulin biosynthesis or secretion.

Glucose elevates both cyclic adenosine 3:5-monophosphate (cyclic AMP) and insulin secretion rapidly and in a parallel dose-dependent fashion in perifused rat islets. Theophylline stimulates cyclic AMP much more than glucose, yet secretion is much less. When the two agents are combined, cyclic AMP is similar to theophylline alone yet secretion is augmented synergistically. Glucose-induced cyclic AMP generation and insulin secretion are dependent on extracellular calcium. Theophylline-induced insulin secretion is also extracellular calcium-dependent; however, theophylline-induced cyclic AMP elevation is independent of extracellular calcium. Thus, extracellular calcium has multiple effects on insulin secretion, some of which appear unrelated to a terminal secretory process. When glucose is combined with theophylline at physiologic levels of extracellular calcium, both the first and second phases of secretion are prominent. At extracellular calcium levels of 0.05 mM, only the second phase is prominent whereas at 10 nM extracellular calcium (ethylene glycol bis(beta-aminoethyl ether)-N,-tetraacetic acid) only the first phase is prominent. A divalent cation ionophore (a23187, Eli Lilly), which transports calcium and magnesium ions across biological membranes, was used to elucidate further the role of calcium and magnesium. If the ionophore (10 muM) is perifused for 5 min at low extracellular calcium and magnesium, and physiologic calcium is then added, a sudden spike of insulin release occurs in the absence of cyclic AMP generation. Similar results were obtained with magnesium. When the ionophore is perifused for 30 min at low calcium and magnesium, insulin secretion again occurs in the absence of cyclic AMP generation. Electron microscopic examination of the B cells following perifusion with the ionophore shows no specific alterations. These observations suggest that: (a) glucose elevates cyclic AMP, but the latter acts primarily as a positive feed-forward modulator of glucose-induced insulin release; and (b) extracellular calcium has multiple effects on insulin secretion both upon, and independent of, the cyclic AMP system.

Kinetics of (45)Ca efflux and insulin release were studied in collagenase-isolated rat islets during 2-h perifusions with calcium-depleted (0.05 mM) bicarbonate-phosphate buffer containing 2.2 mM glucose. Addition of glucose (16.7 mM) suppressed (45)Ca efflux by 30%. Removal of glucose caused an "off response" of insulin release. The perifusion of a normal concentration of Ca (2.3 mM) greatly stimulated (45)Ca efflux, indicating Ca <--> (45)Ca exchange. When Ca and glucose were superimposed, the effects on (45)Ca efflux and insulin release depended upon the order of presentation of the stimuli: when Ca was added to an ongoing 16.7-mM glucose perifusion, biphasic patterns of (45)Ca and insulin release were seen; when glucose was superimposed on a Ca perifusion, an inhibition of the Ca-stimulated (45)Ca efflux occurred, and a reduced but clearly biphasic insulin response was seen. The subsequent insulin off response after with-drawal of the glucose was also reduced. Mathematical "peeling" of (45)Ca efflux curves from unstimulated islets suggests that there are at least two, and probably three, different intracellular Ca compartments (not including the extracellular sucrose space). At the beginning of perifusion, these three compartments (I, II, III) contain 25, 56, and 19% of the intracellular (45)Ca, and their rates of efflux are 6.7, 1.2, and 0.1%/min, respectively. Glucose appears to suppress efflux from the largest compartment (II); Ca appears to exchange with (45)Ca from a more inert compartment (III). The relationship between insulin and (45)Ca release is not stoichiometric.

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.

Freshly isolated rat islets and cultured hamster insulinoma cells (HIT T15) were incubated with a membrane-permeable octanoyl tripeptide (N-octanoyl-ASN-TYR-THR-NH2), which contains an acceptor sequence for ASN-linked glycosylation. Labeled octanoyltripeptide (125[I]TYR) was glycosylated by both islets and HIT cells. The carbohydrate moiety of this glycotripeptide was removed by N-glycanase indicating that glycotripeptide was formed in the lumen of endoplasmic reticulum and, subsequently was secreted via the route for secretory protein. Secretion of glycotripeptide began more rapidly than that of insulin newly synthesized from 3[H]leucine. At 30 min glycotripeptide secretion was already significant but, over a 3-h period, it never represented more than 21% of glycotripeptide produced. Glycotripeptide secretion was not affected by compounds shown to regulate insulin secretion (glucose, forskolin, EGTA and streptozotocin). Thus in beta cells, it appears that glycotripeptide secretion is unregulated and that its cellular secretory pathway is different from that for insulin.

We demonstrated previously that the conversion rate of proinsulin to insulin in pancreatic islets progressively increased after prolonged prior exposure to glucose (11 mM) and that this effect could be blocked by cycloheximide. This study was designed to characterize further the time course and regulation of the proinsulin conversion process. The effects of prior exposure to glucose on proinsulin conversion were dose dependent (Km, approximately 7 mM glucose) and time dependent, taking approximately 3 h to reach the maximum rate. Glucose added at or after the subsequent [3H]leucine pulse was ineffective. Mannoheptulose, added during a 3-h exposure with glucose (11 mM), prevented glucose-induced activation of the proinsulin conversion process. L-Leucine (20 mM) was as effective as 11 mM glucose in activating conversion, whereas 2-alpha-ketoisocaproic acid (20 mM) or phorbol ester (50 nM) had little effect. Activation of proinsulin conversion by a 24-h exposure to glucose (11 mM) was reversed by a subsequent 3-h prior exposure to cycloheximide. alpha-Amanitin, an inhibitor of mRNA synthesis, did not influence the glucose-induced activation of proinsulin conversion when present during a 3-h exposure to glucose; however, it completely inhibited glucose-stimulated conversion when present during 24 h exposure. Results suggest that activation of the proinsulin conversion process is regulated by glucose metabolism rather than the glucose molecule per se and that other, but not all, secretagogues are effective. Conversion may require prior synthesis of a pool of converting enzyme(s) or other regulatory proteins whose turnover is relatively rapid (approximately 33 h) and whose mRNA is more stable (to 24 h).

A series of 3H-leucine pulse-labeling experiments was carried out with dispersed cells freshly isolated from transplanted rat insulinomas. After secreted fractions were separated, insulin was purified and specific activities were determined for both secreted and average cellular insulins. Labeling patterns in this line of tumor cells were compared with those previously established for isolated rat islets. With both tumors and islets, conversion of labeled proinsulin to insulin occurred to the same extent by 2.5 h, suggesting similar onset and half-time of proteolysis in these cells. However, total cellular insulin in tumors attained a threefold higher specific activity than in islets. Because total B-cell mass in these tumors was unknown, either a more rapid proinsulin biosynthesis or diminished cellular storage (or both) could lead to this faster fractional replacement of total stored insulin. Insulin secretion in these tumor cells was insensitive to high glucose but responded, albeit poorly, to leucine plus 3-isobutyl-1-methylxanthine (IBMX). Under all secretory conditions tested, tumor cells continuously secreted insulin at elevated fractional rates, which were slightly higher than fractional insulin secretory rates in maximally glucose-stimulated islets. In contrast with normal islets, newly synthesized insulin was stored homogeneously in tumor cells, and compartmental storage characteristics were not generated by incubation with either 20 mM glucose or leucine plus IBMX in the marking period. Thus, preferential secretion of insulin was never observed in tumor cells.(ABSTRACT TRUNCATED AT 250 WORDS)

With the use of isolated rat islet perfusion, levels of the islet cyclic adenosine 3' ,5' -monophosphate (cAMP) were compared with dynamic insulin secretion induced by tolbutamide and arginine. Tolbutamide elevated islet cAMP rapidly and augmented both glucose-induced islet cAMP levels and insulin secretion; arginine, however, did not elevate islet cAMP but did enhance glucose-induced insulin secretion. Since the latter result could have been modulated by cyclic guanosine 3' ,5' -monophosphate, this cyclic nucleotide was also measured and found to remain unchanged during stimulation of insulin secretion by arginine and a combination or arginine and glucose. Thus, the action of tolbutamide appears to be modulated in part by cAMP, whereas arginine appears to augment insulin secretion independently of cyclic nucleotides.

Tolbutamide (1 g/kg body wt) was administered to male rats for 3 days to determine the effects of this pretreatment on subsequent insulin biosynthesis and compartmental storage characteristics of freshly isolated islets. Islets were isolated 16 h after the last tolbutamide administration, at a time when fed plasma glucose concentrations were normal. Islet glucagon was unchanged but insulin content was significantly reduced (38 +/- 1.2 ng IRI/islet from seven untreated rats versus 7.9 +/- 1.2 ng IRI/islet from eight treated rats). After tolbutamide pretreatment, the rate of incorporation of 3H-leucine into islet proinsulin was unchanged, but the t1/2 of labeled proinsulin-to-insulin conversion was significantly (P less than 0.001) decreased from 36 to 20 min. After treatment, actual rates of glucose-stimulated insulin secretion were 50% lower, however, because due to the proportionately greater depletion of islet insulin content, the fractional rate of secretion was increased two-fold. After treatment, there was evidence of compartmental, heterogeneous insulin storage, and glucose still marked newly synthesized insulin for preferential release; however, the differential release of new and old insulin converged rapidly with time. Mathematical integration of the data suggested dilution of the newly synthesized insulin compartment with unlabeled insulin during the chase period, but additionally indicated more rapid mixing of newly synthesized with previously stored, unlabeled insulin. Thus, tolbutamide-treated rats partially compensated for acute insulin depletion by increasing the rate of proinsulin-to-insulin conversion, but not increasing the rate of proinsulin biosynthesis; doubling the glucose-stimulated fractional secretory rate of the depleted cellular insulin storage compartment; and retaining compartmental storage characteristics but mixing newly synthesized insulin more rapidly with the compartment of previously stored, unlabeled insulin.

To investigate compartmental properties of insulin storage and secretion, isolated rat islets were used for pulse-labeling experiments, after which proinsulin and insulin were purified rigorously. Processing of proinsulin to insulin neared completion by 3 h without additional loss of either radioactive peptide by cellular or extracellular proteolysis. The amount of labeled hormone rapidly diminished in islets; it was secreted at a higher fractional rate than immunoreactive insulin, resulting in secreted insulin's having a higher specific activity than the average cellular insulin. Newly synthesized insulin, therefore, was secreted preferentially. Changes in the specific activity of secreted and cellular insulin with time were consistent with changes predicted for islets containing 33% of their total insulin in a glucose-labile compartment. Predictions were based on steady-state analysis of a simple storage-limited representation of B cell function. Islets from either the dorsal or ventral part of the pancreas also contained 33% of their total insulin in a glucose-labile compartment. The same compartment was mobilized by 20 mM glucose, 50 mM potassium + 2 mM glucose, or 20 MM glucose + 1 mM 3-isobutylmethylxanthine as indicated by the specific activity ratio of secreted vs. cellular insulin, even though average secretion rates with these stimuli differed by more than threefold. In the absence of calcium, the effectiveness of 20 mM glucose as a secretagogue declined markedly, and the older stored insulin was preferentially mobilized because secreted insulin had a lower rather than a higher specific activity than cellular insulin. Results provide insight into the mechanisms of nonrandom mobilization and secretion of insulin form the B cell.

Synthesis and processing of radiolabelled rat insulin I and II were studied by pulse-labelling freshly isolated rat islets with [3H]leucine and chasing in 2 mM glucose for up to 270 min (which minimized insulin secretion, less than 1%/h). Islet samples were taken during the chase period and analyzed for their rat insulin I and II content by high-performance liquid chromatography. Prior to 60 min chase rat insulin I accounted for greater than 85% of the radiolabelled insulin present. With longer periods of chase, the relative percentage of rat insulin II progressively increased so that by completion of proinsulin to insulin processing the two labelled rat insulins were present in the same proportion as the relative immunoreactive content, approx. 60:40% insulin I/insulin II. Thus, although islets synthesize the two insulins in proportion to their relative immunoreactive content, rat insulin I and II are processed with different kinetics.

The effects of a vitamin D deficiency on insulin and glucagon release was determined in the isolated perfused rat pancreas by radioimmunoassay of the secreted proteins. During a 30-minute period of perfusion with glucose and arginine, pancreases from vitamin D-deficient rats exhibited a 48 percent reduction in insulin secretion compared to that for pancreases from vitamin D-deficient rats that had been replenished with vitamin D. Vitamin D status had no effect on pancreatic glucagon secretion. This result, along with the previously demonstrated presence in the pancreas of a vitamin D-dependent calcium-binding protein and cytosol receptor for the hormonal form of vitamin D, 1,25-dihydroxyvitamin D3, indicates an important role for vitamin D in the endocrine functioning of the pancreas.

To examine effects of streptozocin (STZ) on pancreatic islets in the absence of a functioning immune system, we examined isolated rat islets cultured for 96 h after a single 1-h exposure to STZ in vitro. In addition to an immediate and sustained suppression of insulin secretion, STZ also induced a progressive decline in insulin content per islet as well as in total islet tissue mass, characterized by a decrease in both islet number and volume. Viability studies show that STZ-induced cell death was also progressive and was not commensurate with loss of secretory function. Furthermore, media-transfer experiments demonstrate that decline of tissue mass is not due to accumulation of metabolite or degradation products in the media. After 96 h in culture, untreated islets showed a marked insulinogenic capacity that was inhibited more than fourfold by the initial STZ treatment. Progressive loss of glucagon content per islet suggests that STZ causes disruption of islet morphological integrity. These progressive sequelae observed in vitro indicate that several aspects of the time-delayed attack on the beta-cell by STZ are independent of a functioning immune system.

In short-term batch-incubation or perfusion experiments, we studied insulin release and associated 65Zn efflux from rat pancreatic islets loaded with 65Zn by 24-h tissue culture in low-glucose medium. The fractional basal insulin release and 65Zn efflux were 0.4% and 3% of total content/h/islet, respectively. Thus, basal 65Zn efflux was much greater than that to be accounted for if zinc was released proportionally with insulin release only; extragranular zinc flux was suggested. Two millimolar glucose, with or without 1 mM 3-isobutyl-1-methylxanthine (IBMX), affected neither insulin release nor associated 65Zn efflux. Twenty-five millimolar glucose produced a significant threefold increase in insulin release above baseline, but somewhat decreased 65Zn efflux at marginal significance. Glucose (25 mM) plus 1 mM IBMX provoked a high increase in insulin release and an associated 30% increase in fractional 65Zn efflux over basal. Calculations based on previous estimations of 65Zn distribution and equilibrium with islet zinc indicated that molar zinc efflux was more than sufficient to account for a 2-zinc-insulin hexamer. L-leucine (2 or 20 mM) plus 1 mM IBMX caused far greater 65Zn efflux for the amount of insulin released, indicating additional 65Zn mobilization not directly related to insulin secretion. To evaluate 65Zn efflux during inhibited insulin secretion, batch incubations were performed in 100% D2O or at 27 degrees C, conditions that inhibited insulin release stimulated by high glucose plus IBMX. These agents decreased the 65Zn efflux far below the basal value (35% and 50%, respectively) and greater than could be accounted for by the attendent inhibition of insulin secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of isolated islets labeled with radioactive leucine show that glucose at a critical time "marks" islets in such a way as to cause preferential release of newly synthesized insulin. The preferential release of insulin from marked islets is relatively independent of subsequent secretagogues or rates of insulin secretion. Previous kinetic studies have indicated that the critical time at which marking occurs is after proinsulin biosynthesis but before the secretory event. Thus, secretory cells may regulate the diversion of newly synthesized material for immediate release as it is approaching or transiting the Golgi apparatus.

The dicarboxylic amino acids, aspartic and glutamic acid, at their isoelectric pH, reduced aggregation of insulin solutions in vitro for 16 days during continuous agitation at 37 degrees C. Unprotected insulin solutions, when infused via a 14-day implantable infusion device in diabetic Chinese hamsters, controlled plasma glucose levels for only 2 days, followed by escape coincident with insulin aggregation. However, when insulin solutions were protected with glutamic acid, euglycemia was maintained for the 14-day life of the device.

Kinetic patterns of glucose-stimulated insulin secretion from the in vitro perfused pancreas were used to test different types of secretion models of similar complexity. A storage-limited, two-compartment model, modified slightly from that previously, was compared with signal-limited models incorporating delta or feedback characteristics. Mathematical relationships for all models were fixed by single-step, dose-response experiments and models were compared in a series of glucose test patterns including steps, step restimulations, staircases, pulses, ramps, and ramp restimulations. The work quantifies previously unreported characteristics of hypersensitivity and low-glucose rest/restimulation behavior in the pancreas. All models simulated staircase and ramp experiments. The two-compartment model contains an inherent hypersensitivity factor required for repeated pulse-type experiments. However, the kinetics of hypersensitivity were too rapid to be simulated in all types of pulse and ramp/pulse experiments by the restricted refilling characteristics as written into this model. The signal-limited, delta-feedback model did not inherently produce potentiation, but required add-on modification that then more closely simulated some pulse and ramp/pulse experiments. This model simulated experimental negative spikes, whereas the storage-limited model would require additional complexity to do so. These and other results suggest that both storage- and signal-limited models, although currently insufficient, could be elaborated to simulate available data. Therefore, a choice between the two to describe the underlying physiological mechanism of multiphasic insulin secretion is premature. The alternate possibility that the secretion mechanism may be reflected by a combination of the two models is presented.

Prolonged exposure of isolated islets to glucose (11 mM) results in desensitization of insulin secretion. In this study, both glucose-regulated proinsulin biosynthesis and its processing during glucose-induced desensitization were examined at critical periods during 24 h. Although insulin secretion declined at 24 h to one third the 3 h maximum rate, the total rate of proinsulin biosynthesis, assessed by [3H]leucine incorporation, was unchanged at 0, 3, and 24 h of glucose (11 mM) exposure. Total insulin recovery measured by immunoreactive insulin in islets and media after 24 h was approximately 172% of the initial islet content. After correction for insulin degradation, and since proinsulin biosynthesis was unchanged, the synthesis rate was calculated to be a constant 3.5 +/- 1.1%/h of the initial islet content. Results suggest that desensitization may occur at the release mechanism rather than at a step in glucose metabolism common for the stimulation of synthesis and secretion. In contrast, the conversion rate of proinsulin to insulin progressively increased with prolonged prior glucose exposure, the major increase occurring by 3 h of glucose preincubation. Low glucose (2 mM), when used during the 3-h prelabeling period, did not affect the conversion rate. Furthermore, cycloheximide added during the 3-h preexposure to glucose (11 mM) completely prevented glucose-induced activation of the conversion process. These results indicate that the conversion rate of proinsulin to insulin is a glucose-regulated process requiring synthesis of a pool of either converting enzyme(s) or other regulating protein before initiation of proinsulin synthesis.

Glucose, forskolin, 3-isobutyl-1-methylxanthine (IBMX), and dexamethasone were tested as regulators of proinsulin biosynthesis in HIT T-15 cells, which are glucose-responsive simian virus 40-transformed hamster beta-cells. Rate of [3H]leucine incorporation into proinsulin was increased as glucose concentrations were raised from 0 to 20 mM. Biosynthetic rate increases were significant after 48 but not at 4 or 24 h of glucose and were greater for proinsulin than for total extractable proteins. After 48 h, glucose-stimulated proinsulin biosynthesis was unaffected by 10(-6) M forskolin and/or 3 x 10(-5) M IBMX but was specifically and significantly inhibited by 10(-6) M dexamethasone. Four hours of exposure to dexamethasone had no effect. When cells were incubated for 24 h and then continuously labeled for an additional 24 h, cellular conversion of labeled proinsulin to insulin was increased by glucose, and this increase was reversed or inhibited by 10(-6) M dexamethasone. Therefore, proinsulin biosynthesis in transformed HIT T-15 cells is regulated in several ways by metabolites and hormones in a manner that compares with biosynthetic regulation in normal beta-cells.

When isolated rat islets were incubated with 10(-10) - 10(-6) M monensin, a sodium and proton ionophore, glucose-stimulated insulin release was inhibited in a concentration- and time-dependent manner. After removal of monensin, inhibition of insulin secretion persisted during stimulation with a variety of secretagogues, including 5 mM glucose plus 15 mM arginine, 20 mM glucose, and 20 mM glucose plus 1 mM 3-isobutyl-1-methylxanthine. Within the same low range of monensin concentrations, proteolytic conversion of newly synthesized proinsulin to insulin was also blocked. At each concentration, prohormone-to-hormone conversion was inhibited to almost the same extent as inhibition of insulin secretion. Therefore, both processes may have equal or common dependency on a subcellular ionic gradient. Although monensin decreased total insulin secretion, the glucose-regulated marking process was unaffected. Regardless of the monensin concentration or the overall rate of insulin secretion, the percentage of secreted newly synthesized versus older insulin remained the same, and the threefold differences in the fractional secretory rates of newly synthesized versus total insulin also remained the same. Thus, rather than specifically blocking protein traffic through the Golgi apparatus of the beta cell, monensin probably first inhibited insulin secretion by disrupting proton gradients in secretory vesicles and, thereby, also inhibited other processes occurring within this organelle.

This report has investigated desensitization of pancreatic B cell secretion, or diminution of the insulin response to chronic stimulation. Freshly isolated rat islets were continuously challenged with various secretagogues over 24 h either in batch incubation or in a computer-controlled, flow-through perifusion system. At various glucose concentrations, secretion rose to a peak level in the third hour, then dropped to a new desensitized secretory level which was 25% or less than that of the maximum rate. The amount of insulin secreted was glucose dependent although secretory kinetics were independent of the amount of hormone secreted. At all glucose concentrations the reduction in islet insulin content was not great enough to account for the observed degree of desensitization. Furthermore at hour 20, islets responded vigorously to an alternate stimulus, indicating insulin stores and islet secretory machinery were still capable of being stimulated. Addition of 3-isobutyl-1-methylxanthine or forskolin did not prevent glucose-induced desensitization. Insulin secretion desensitized similarly to nonglucose (alpha-ketoisocaproic acid) and nonfuel (phorbol ester) stimuli. Glucose potentiation of a terminal KIC response, although demonstrable after 20 h of chronic glucose, was diminished somewhat compared to that after 3 h of chronic glucose. Delaying glucose stimulation by 6 h reduced insulin secretion, yet desensitization persisted. Although insulin secretion entrained to a glucose signal which oscillated from 1.3-12.7 mM in sine wave pulses of 90-min frequency, desensitization was not prevented. Thus, desensitization occurred in response to glucose, nonglucose, and nonfuel stimuli and despite delayed or oscillating signals. We conclude that exhaustion of a finite insulin compartment is not the underlying defect in desensitized secretion and suggest that metabolic feedback or recruitment of multiple heterogeneous compartments may explain this phenomenon.

To investigate mechanisms underlying biosynthetic regulation of an insulin gene, the rat insulin II gene was introduced into hamster beta-cells (HIT) by cotransfection with the neomycin phosphotransferase-selectable marker. The insulin gene fragment was 2.2 kilobases (kb) in length and contained all exons, introns, and approximately 700 base pairs (bp) of 5'-flanking DNA and 300 bp of 3'-flanking DNA. The HIT cell was known to have endogenous hamster insulin production under regulation by glucose and dexamethasone. In a pool of stably transfected cells (HIT M62pR2), rat insulin II and hamster insulin were produced at comparable rates. Glucose (20 mM) stimulated cellular [3H]leucine labeling of both hamster insulin and rat insulin II by approximately twofold. Addition of 10(-6) M dexamethasone to media containing 11.1 mM glucose inhibited biosynthesis of both hamster insulin and rat insulin II by greater than 90%. Thus, with both positive and negative biosynthetic regulation, changes in the cellular labeling of exogenous rat insulin II were qualitatively and quantitatively similar to those of the endogenous hamster insulin. These data suggest that the 2.2-kb rat insulin II gene fragment contained sufficient information for both expression and apparently "normal" biosynthetic regulation of exogenous rat insulin II (when compared with endogenous hamster insulin) in response to glucose and dexamethasone.

Diabetes mellitus in the adult Chinese hamster is characterized by subnormal pancreatic insulin release in vitro, decreased insulin content, and lack of obesity. The cause of the islet B-cell failure is not clear. We measured insulin, glucagon, and somatostatin release from in vitro perfused pancreases of young (mean age 10 and 20 weeks), genetically diabetic animals (subline AC, mean plasma glucose 8.0 and 16.6 mmol/l, respectively). Compared to age- and sex-matched normal hamsters (subline M, mean plasma glucose 5.3 mmol/l), the younger diabetic animals had a significantly elevated mean plasma glucose level, but net in vitro pancreatic release of insulin, glucagon, and somatostatin was normal. Pancreatic content of insulin and glucagon was also not significantly different from normal. At age 20 weeks, when the plasma glucose of the diabetic animals was even more elevated, pancreatic content and release of insulin were significantly subnormal, whereas glucagon and somatostatin release were normal, and pancreatic content of glucagon was normal. In a similar group of young (mean age 10 weeks) diabetic animals, non-fasting plasma insulin levels were within the normal range, but the corresponding glucose levels were excessive in most of the animals (13 out of 19). In conclusion, 10-week-old diabetic hamsters show mild hyperglycaemia which cannot be accounted for directly by decreased pancreatic release in response to a glucose plus arginine stimulus in vitro. Decreased ability of the B cell to respond in vivo to hyperglycaemia or peripheral resistance to insulin may contribute to later B-cell failure in the older diabetic hamster.

Glucose-induced insulin release from perfused rat pancreas was compared with glucose-induced changes in membrane potential of beta cells from mouse islets. Extracellular concentrations of Ca and Mg were varied as steps, simultaneously or separately, from 10% to 200% of normal in the presence of 11.1 mM glucose. A change in Ca induced a transient change in electrical activity paralleled by a transient change in insulin release. If the Ca/Mg ratio was maintained, steady-state insulin release remained constant between 10% and 200% Ca, while electrical activity showed alterations. Analysis of burst parameters indicated that increased or decreased Ca entry was balanced by decreased or increased excitability. It is postulated that the beta cell contains a compensator mechanism for the regulation of Ca influx.

Although initially described two decades ago, biphasic insulin secretion has gradually been understood to reflect beta-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 beta-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.(ABSTRACT TRUNCATED AT 250 WORDS)

It has been suggested that a result of streptozotocin (Sz)-initiated damage to beta-cell DNA is activation of the repair enzyme nuclear poly-ADP-ribose (p-ADPR) synthetase and subsequent depletion of cellular NAD. This reduction of NAD is presumed responsible for the impaired islet function. Using freshly isolated rat pancreatic islets, we have examined the ability of p-ADPR synthetase inhibitors, in particular the previously not studied thymidine, to block Sz-induced damage to the B cell. Islets were incubated in 2 mM glucose and 2 mM Sz with or without p-ADPR synthetase inhibitor and then challenged for 1 h with 25 mM glucose plus 25 microM forskolin or 100 nM phorbol ester without glucose. Sz treatment inhibited insulin secretion about 90% even when stimulated by the non-glucose-containing challenge, indicating that Sz caused a rapid and generalized lesion past glucose-specific signals. Thymidine maintained the insulin secretory response to glucose plus forskolin from Sz-treated islets as, or more, effectively than nicotinamide. In a dose-dependent manner, thymidine protected Sz-treated islets to 87% of normal islet secretion at the highest thymidine dose (20 mM). However, even protected islets could not sustain normal secretion; insulin secretion was significantly diminished in thymidine-protected islets challenged with a second consecutive 1-h glucose plus forskolin stimulus. As expected, the acute depletion of islet NAD content (approximately 50%, 1 h after Sz) was reversed to 90% of normal by thymidine. However, even in unprotected Sz-treated islets, NAD content gradually recovered over 48 h of culture in standard RPMI, although insulin secretion remained suppressed. Thus, thymidine may be a useful protective agent against chemically induced islet cell damage. Furthermore, a sustained suppression of NAD content does not explain Sz's permanent inhibition of islet secretory response. Other aspects of inhibited nuclear p-ADP-ribosylation are considered to explain protection against Sz damage to the beta-cell.

We examined the mechanism by which prior glucose exposure accelerates conversion of proinsulin to insulin in pancreatic islets. Using actinomycin A, introduced at different times after a 3H-leucine pulse to block the intracellular transport of proinsulin, transport was found to be slightly accelerated after prolonged glucose. However, computer simulation of pulse-chase experiments showed that activation of conversion is mostly due to increased converting activity. Measurement of islet procathepsin B by immunoprecipitation indicated synthesis of this enzyme was stimulated about two fold after glucose. Pre-exposure with tunicamycin inhibited the acceleration of conversion, suggesting that the converting system requires glycosylation.

Uptake of 65Zn and distribution of 65Zn, total zinc, and insulin were measured in rat islets and islet granules under different conditions of islet culture. Specific activity of islet zinc (65Zn/zinc) was less than 15% that of extracellular zinc even after 48 h. In contrast, once in the islet, 65Zn approached 70% of equilibrium with granular zinc in 24 h and apparent equilibrium by 48 h. During a 24-h culture, at either high or low glucose, reduction of both islet zinc and insulin occurred. However, zinc depletion was greater than that predicted if zinc loss was proportional to insulin depletion and occurred only from the granular compartment, which represents only one third of the total islet zinc. Extension of culture to 48 h caused additional insulin depletion, but islet zinc was unchanged. Omission of calcium during the 48-h culture caused a predicted increase in insulin retention, presumably by inhibiting secretion; however, zinc retention was not increased proportionately. Pretreatment of rats with tolbutamide caused a massive depletion of insulin stored in isolated islets, with little change in total islet zinc; subsequent culture of these islets resulted in a greater loss of granular zinc than predicted from the small loss of granular insulin. None of the conditions tested affected the percentage of either 65Zn or total zinc that was distributed in the islet granules. Results show that zinc exists in a metabolically labile islet compartment(s) as well as in secretory granules; and extra-granular zinc, although not directly associated with insulin storage, may act as a reservoir for granular zinc and may regulate insulin synthesis, storage, and secretion in ways as yet unknown.

Insulin release from perfused rat pancreas was compared with membrane potentials of single beta-cells from perifused mouse islets during glucose stimulation (11.1 mM) in the presence of varying Ca and Mg concentrations. Depolarization was associated with insulin release and hyperpolarization with its suppression, irrespective of Ca concentration. After sudden reduction of Ca and Mg (to 0.05 and 0.01 mM, respectively), glucose-stimulated insulin release was maintained while the cell membrane depolarized, leading to a reversed pattern of burst activity. Readdition of Ca and Mg caused suppression of insulin release that paralleled hyperpolarization of the cell membrane. This suppression was transient, lasting < 5 min, and was due mainly to readdition of Ca. Patterns of insulin release during reduction of Ca and Mg in the presence of valinomycin (1 microM), diphenylhydantoin (25 microgram/ml), and ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (0.037 and 1.0 mM) were also studied. In conclusion, the relative concentrations of Ca and Mg and the membrane potential per se are important in the release of insulin. Also, under certain experimental conditions, Ca can block glucose-stimulated insulin release, possibly by increasing K+ permeability.

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, approximately 25% maximal release. Results were similar when using: perifused islets embedded in Cytodex 3, or Bio-Gel P-2, 100-200 mesh; batchincubated islets with hourly changes of medium; and the isolated pancreas perfused for 8 h. Three different media, Hana HB 104 (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 secret-agogue-induced, signal desensitization of the B-cell, rather than exhaustion of a B-cell compartment of stored insulin.

The kinetics of zinc release and insulin secretion were compared in perifused islets previously loaded for 24 h with 65Zn. In the absence of detectable insulin secretion, fractional basal 65Zn release was constant at 3-5%/h throughout the experimental periods, suggesting that basal efflux of zinc is primarily from a single extragranular compartment. Glucose (25 mM) alone caused a prompt release of both 65Zn and insulin. Previous studies using cell fractionation showed that approximately one third of islet 65Zn is in the granular fraction. Thus, if zinc efflux represents only that being cosecreted from the granule, a 65Zn to insulin fractional secretion ratio of approximately 0.33 would be expected. However, the ratio of 65Zn to insulin fractional secretion was only 0.16, indicating that glucose not only causes cosecretion of zinc associated with stored insulin, but also independently decreases 65Zn efflux from extragranular sources. At low glucose concentrations, 3-isobutyl-1-methylxanthine (IBMX; 1 mM) did not affect either insulin or 65Zn efflux; however, the combination of glucose plus IBMX caused a large and parallel biphasic release of both 65Zn and insulin at ratios approximating 0.33. Similar results were obtained with glucose plus forskolin. Leucine (20 mM) plus IBMX caused a characteristic rapid secretion of insulin which waned with time. Efflux of 65Zn was also prompt; however, the ratio of secreted 65Zn to insulin gradually increased during stimulation from a near-theoretical value for cosecretion at the initial peak to 0.70 by the end of the stimulation. 65Zn efflux was sustained even after termination of the leucine plus IBMX stimulus. alpha-Ketoisocaproic acid caused a similar stimulation and waning of insulin secretion, but with a consistent 65Zn to insulin fractional secretion ratio of about 0.33. With both leucine and alpha-ketoisocaproic acid, poststimulatory 65Zn efflux remained elevated. Perifusion of unlabeled zinc (1 mM; a concentration that inhibits insulin secretion) caused prompt and sustained release of 65Zn; efflux remained high at least 30 min after termination of zinc administration. Results expand previous batch islet experiments to show that zinc and insulin can be temporally cosecreted at ratios quantitatively consistent with their storage ratio in the granules without conservation or intracellular reutilization. In addition, there is 65Zn efflux from nongranular compartments which is stimulated or inhibited, depending on the secretagogue.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of varying exogenous zinc concentrations on insulin secretion by the perfused pancreas and short-term cultured islets was studied. No significant effect was observed at several supraphysiologic concentrations unless islets were cultured with 200 microM added zinc. These results suggest that exposure of the pancreas to non-physiologic zinc levels is necessary in order to influence the insulin secretory process.