Increased expression of GAD65 and GABA in pancreatic beta-cells impairs first-phase insulin secretion.

The functional role of glutamate decarboxylase (GAD) and its product GABA in pancreatic islets has remained elusive. Mouse beta-cells express the larger isoform GAD67, whereas human islets express only the smaller isoform GAD65. We have generated two lines of transgenic mice expressing human GAD65 in pancreatic beta-cells (RIP7-hGAD65, Lines 1 and 2) to study the effect that GABA generated by this isoform has on islet cell function. The ascending order of hGAD65 expression and/or activity in beta-cells was Line 1 heterozygotes < Line 2 heterozygotes < Line 1 homozygotes. Line 1 heterozygotes have normal glucose tolerance, whereas Line 1 homozygotes and Line 2 heterozygotes exhibit impaired glucose tolerance and inhibition of insulin secretion in vivo in response to glucose. In addition, fasting levels of blood glucose are elevated and insulin is decreased in Line 1 homozygotes. Pancreas perfusion experiments suggest that GABA generated by GAD65 may function as a negative regulator of first-phase insulin secretion in response to glucose by affecting a step proximal to or at the K(ATP)(+) channel.

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.

Chronic sympathetic innervation of islets in transgenic mice results in differential desensitization of alpha-adrenergic inhibition of insulin secretion.

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.

Constitutive (pro)insulin release from pancreas of transgenic mice expressing monomeric insulin.

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.

Differences in insulin secretion between the rat and mouse: role of cAMP.

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)

Interrelationship of changes in islet nicotine adeninedinucleotide, insulin secretion, and cell viability induced by interleukin-1 beta.

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.

Constitutively active stimulatory G-protein alpha s in beta-cells of transgenic mice causes counterregulation of the increased adenosine 3',5'-monophosphate and insulin secretion.

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.

Functional effects of transgenic expression of cholera toxin in pancreatic beta-cells.

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.

Opposite, phase-dependent effects of 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester or tetracaine on islet function during three phases of glucose-stimulated insulin secretion.

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.

Effect on insulin production sorting and secretion by major histocompatibility complex class II gene expression in the pancreatic beta-cell of transgenic mice.

Expression of major histocompatibility complex (MHC) class II protein in islet beta-cells of transgenic mice causes severe diabetes without an attendant autoimmune component. Little is known of the aberrant beta-cell function and site of biological lesions responsible for the diabetic state. Therefore, changes in (pro)insulin production, processing, sorting, storage, and secretion were evaluated using the in vitro perfused pancreas from male hyperglycemic BALB/cBYJ Tg (O pinsproA alpha d pinsproA beta d) mice and a RIA capable of detecting mouse insulin or proinsulin with quantitative equivalency. Results were compared to control pancreases from normal BALB/cBYJ mice. Extractable pancreatic insulin plus proinsulin content in the transgenics was 4% of normal. Normal pancreases responded characteristically with a diphasic insulin release during 30-min stimulation by glucose, a response that was enhanced by subsequent forskolin. In contrast, hormone release from transgenic pancreases was undetectable; based on the sensitivity of the immunoassay, fractional secretion of the residual pancreatic hormone content from the transgenic pancreases was less than 25% of normal. Proinsulin or insulin constitutive release was also not detected in the absence or presence of glucose-containing stimuli even when experiments were extended to 3 h. In contrast, fractional secretion in response to nonglucose stimuli (carbachol-leucine and arginine-leucine) was greater than normal from the transgenic diabetic pancreases. Responses to glucose stimuli did not normalize even after 90 min in the absence of glucose. In other experiments, pancreases were stimulated with carbachol/leucine/forskolin for 90 min, and the proportion of proinsulin to insulin released by the regulated pathway was determined after Sep-Pak and HPLC separation of combined eluates. Proinsulin was undetectable (and, therefore, accounted for less than 10% of the total hormone secretion). It is concluded from the observations of hyperglycemia, low pancreatic insulin content, and impaired release that insulin production in the pancreas of the MHC diabetic transgenic is severely depressed. The limited insulin production and chronic hyperglycemia do not (as speculated) cause missorting to a constitutive pathway or impaired conversion of proinsulin to insulin, since a proportionately increased proinsulin release does not occur. Although the response of the secretory process to glucose-containing stimuli is almost completely destroyed, fractional secretion in response to nonglucose stimuli is enhanced. The possible contribution of hyperglycemia-induced beta-cell desensitization or specific lesions in the glucose recognition signals induced by MHC expression are discussed. Results suggest that expression of MHC class II protein causes highly specific beta-cell lesions which, in themselves, could be a contributing factor in human insulin-dependent diabetes.

Desensitization of the insulin-secreting beta cell.

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.

The role of Ca(2+)-related events in glucose-stimulated desensitization of insulin secretion.

The spontaneous decline of insulin secretion (third phase) that occurs under a variety of secretory conditions is well documented and suggests a general impairment or desensitization of the secretory process. We have examined several aspects of Ca2+ flux as well as regulators of Ca-linked second messenger events in freshly isolated rat islets chronically stimulated with glucose over 24 h, a period that encompasses initial (hour 1), peak (hour 3), and subsequent impaired or desensitized (hour 20-22) secretion. In islets incubated for these periods in HB104 medium with 22 mM glucose, 45Ca2+ uptake did not vary (12.6 +/- 1.6 vs. 10.2 +/- 1.7 vs. 13.2 +/- 3.4 pmol Ca2+/islet.10 min at 1, 3, and 22 h, respectively). Chronic incubation in 2 mM glucose reduced total Ca2+ uptake at each of the time periods, but, again, uptake did not change with desensitization (9.8 +/- 1.4 vs. 6.6 +/- 2.1 vs. 7.8 +/- 2.3 pmol Ca2+/islet.10 min). In 11 mM glucose, the Ca channel antagonist verapamil (1-10 microM) reduced insulin secretion by 55-80% in a dose-dependent manner over 1-3 h; islets continuously exposed to verapamil escaped inhibition by 20 h even at the highest concentration. However, in islets first exposed to 10 microM verapamil only during 20-22 h, hourly insulin secretion was suppressed 25%, 45%, and 33% at 20, 21, and 22 h, respectively, indicating that glucose-desensitized islets were still sensitive to further inhibition of Ca channels. Staurosporine (1 microM), an inhibitor of protein kinase-C activity, progressively inhibited glucose-stimulated insulin secretion from 48% at 1 h to more than 80% by 3 h; again, this inhibitory effect was lost by 20 h of chronic staurosporine. When staurosporine was first administered at 20 h, insulin secretion was modestly suppressed and returned to control values in the next hour. With continuous glucose, the islet response to positive stimulation of endogenous C-kinase activity by carbachol was maintained. The Ca/calmodulin inhibitor trifluoroperazine also inhibited insulin secretion by 75-80% during 1-3 h and continued to exert inhibitory effects through 23 h of continuous administration. We conclude that even though insulin secretion has desensitized to glucose, 1) Ca2+ entry is unchanged and is still regulated by glucose, 2) voltage-dependent Ca channels are still sensitive to blockade by acute verapamil, but can desensitize to chronic verapamil; 3) stimulus-enhanced C-kinase activity may be especially labile during glucose-induced desensitization, while 4) possible Ca/calmodulin potentiation of secretion persists through the three secretory phases.(ABSTRACT TRUNCATED AT 400 WORDS)

Lack of islet amyloid polypeptide regulation of insulin biosynthesis or secretion in normal rat islets.

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.

Effect of glucagon or somatostatin on desensitized insulin secretion.

In this study we have examined the role of glucagon and somatostatin in regulating glucose-induced desensitization of insulin secretion from rat islets. Measured in batch incubations with medium routinely used to induce three phases of insulin secretion, secreted glucagon levels fell off over 24 h to 20% of peak secretion levels. Although more responsive to various secretagogues, somatostatin secretion also declined to the same degree. Thus, the A- and D-cells desensitize to chronic stimulation as does the B cell. In other experiments, added glucagon (10(-6) M) enhanced glucose (11 X 10(-3) M)-stimulated insulin secretion 34% in the first 3 h; however, islets became insensitive to continuous glucagon by 4 h. The exogenous glucagon did not prevent or delay glucose-induced desensitization of insulin secretion. When glucagon was administered as acute 1-h tests over continuous glucose administration, the degree of B-cell response did not differ in the 1st, 3rd, or 6th hours and appeared to increase in the 21st hour. When islets were perifused continuously with glucose (22 X 10(-3) M) plus 3 X 10(-7) M somatostatin, glucose-induced insulin secretion was suppressed 50% in the first 3 h, but this inhibitory effect disappeared after 6 h. Desensitization was slightly delayed, but not prevented. When somatostatin was administered as acute 1-h tests over continuous glucose perifusion, the B-cell response was relatively constant in the 3rd, 6th, and 21st hours. Results show that 1) islet release of glucagon and somatostatin desensitizes during constant stimulation; and 2) islet release of insulin desensitizes to chronic potentiation or inhibition, respectively, by these hormones. Furthermore, 3) changing B-cell sensitivity to either glucagon or somatostatin cannot account for observed desensitization of insulin secretion with chronic glucose exposure.

A new phase of insulin secretion. How will it contribute to our understanding of beta-cell function?

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)

Biosynthetic regulation of endogenous hamster insulin and exogenous rat insulin II in transfected HIT cells.

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.

Unregulated secretion of an exogenous glycotripeptide by rat islets and HIT cells.

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.

Glucose-regulated proinsulin processing in isolated islets from rat pancreas.

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).

Characteristics of desensitization of insulin secretion in fully in vitro systems.

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.