The endocrine secretion of human insulin and growth hormone by exocrine glands of the gastrointestinal tract.

The exocrine pancreas, liver, and submandibular glands of the rat were used to express and secrete two exogenous, human protein hormones (growth hormone and insulin) into blood at physiological concentrations. Transfection, expression, and secretion were achieved by the in vivo retrograde injection of plasmid DNA into the secretory ducts of these glands. Pancreatic acinar cells secreted physiological concentrations of growth hormone into the circulation, and its secretion was enhanced by cholinergic stimulation. A human insulin gene was engineered to allow normal processing of insulin in non-beta cells. With this gene, the secretion of human insulin by the exocrine pancreas normalized elevated blood glucose levels in diabetic rats. These in vivo observations demonstrate the utility of retrograde ductal administration of naked DNA into exocrine organs as a novel method for the regulated systemic delivery of protein-based pharmaceuticals.

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.

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.

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)

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.

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.

Thyrotropin-releasing hormone (TRH): immunohistochemical distribution in tadpole and frog brain.

The anatomical localization of immunoreactive TRH (IR-TRH) was demonstrated by the peroxidase-antiperoxidase technique in the brain and pituitary gland of larval and adult Rana catesbeiana. In the adult frog main sites of IR-TRH are perikarya and neuronal fibers in the preoptic and infundibular nuclei of the hypothalamus and in the amygdala and diagonal band of Broca of the telencephalon. In addition, TRH-positive neuronal fibers and endings were found in the septum, pallium, and brain stem as well as in the preoptico-hypophyseal tract, the external zone of the median eminence (which matures during late larval stages), and the pars nervosa; fibers were less extensive in the pars intermedia, and were absent from the pars distalis. In early larval stages, the magnocellular nucleus of the posterior preoptic area is the main site of immunoreactive perikarya. During late stages the extensive adult pattern of distribution of IR-TRH becomes established. The study represents the first immunohistochemical demonstration of IR-TRH in larval anurans, and serves as a basis for clarification of the neuroendocrine regulation of metamorphosis.

Protection by thymidine, an inhibitor of polyadenosine diphosphate ribosylation, of streptozotocin inhibition of insulin secretion.

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.

Direct effects of glucose on proinsulin synthesis and processing during desensitization.

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.

Progressive damage of cultured pancreatic islets after single early exposure to streptozocin.

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.

The third phase of in vitro insulin secretion. Evidence for glucose insensitivity.

In this study, in vitro B-cell models are described, which may be applicable for studying the reported B-cell desensitization produced by hyperglycemia in IDDM and NIDDM. Using a programmable perifusion/perfusion system, insulin secretion from perifused islets was measured at 10-30-min intervals for 24-50 h. After 3-4 h continuous glucose (11 mM), a new phase of insulin release occurs in which secretion declines to, and remains at, 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.

Effect of mammalian luteinizing hormone-releasing hormone on plasma testosterone in male alligators, with observations on the nature of alligator hypothalamic gonadotropin-releasing hormone.

Mature and subadult male alligators (30-50 kg body wt) were injected with a single dose (500 micrograms) of synthetic LH-RH. A blood sample was taken immediately before the hormone was injected and additional samples taken either at 24-hr or at 2-hr intervals up to 8 hr followed by a 24-hr sample. Testosterone concentrations in the blood were then determined by radioimmunoassay. LH-RH caused an increase in plasma testosterone in all animals by 2 hr. Plasma testosterone was still significantly higher in the LH-RH-injected animals than in the saline-injected group at 24 hr. Alligators, therefore, differ from other reptiles in that they respond to the mammalian hormone. Hypothalamic tissue from immature alligators was extracted and tested for the presence of immunoreactive LH-RH-like material using two different antisera. One of the antisera (IJ-29) did not cross-react with the tissue extract, whereas a good parallel displacement curve was obtained when the extract was tested against the other antiserum (R-42). Based on the known specificities of the antisera we conclude that alligator hypothalamic tissue contains an LH-RH-like peptide that differs from the mammalian hormone in at least the 8 position, and that alligator LH-RH may be similar to chicken LH-RH.

TRH stimulation of in vivo GH release in the domestic fowl. Influence of TRH metabolites and effect of age on in vitro degradation of TRH.

Thyrotropin-releasing hormone (TRH) stimulated in vivo growth hormone (GH) release in conscious and anesthetized young domestic fowl. The administration of the presumed metabolites of TRH, deamido-TRH (TRH-OH) and histidyl-proline diketopiperazine (HPD), was followed by small but significant (p less than 0.05) increases in the plasma concentrations of GH in both conscious and anesthetized chicks. However, the ability of TRH-OH or HPD to stimulate GH secretion was less than that observed with a 100-fold lower dose of TRH. The administration of either TRH-OH or HPD with TRH increased the GH response over that observed with TRH alone. The ability of chicken plasma to degrade exogenous TRH in vitro was determined by measuring immunoreactive TRH (IR-TRH) content and by assessing the ability of the incubated samples to increase the plasma concentration of GH when administered to young fowl. The in vitro half-life of TRH was estimated to be 9.8 (by immunoassay) and 9.6 (by a biological index) min for plasma from adult male chickens and 23.9 (by immunoassay) and 20.2 (by biological index) min for plasma from 6-week-old chicks. This difference in degradation may account, at least in part, for the observed age-related decrease in the plasma concentration of GH in birds and for the diminished GH responsiveness of adult birds to exogenous TRH.

Characterisation of somatostatin and TRH release by rat hypothalamic and cerebral cortical neurons maintained on a capillary membrane perfusion system.

We have investigated the nature of the immunoreactive somatostatin (SS) and thyrotropin-releasing hormone (TRH) produced by long-term capillary perfusion cultures of rat fetal cortical and hypothalamic cells. Dispersed cortical and hypothalamic cells from 17-day-old fetal rats were injected on to the outer surfaces of separate capillary membrane perfusion systems. Recirculating nutrient medium (Minimum Essential Medium with added glucose, antibiotics and 10% fetal calf serum) was then perfused via the capillary lumen at a rate of 1.5 ml/min and was changed three times weekly. Medium reservoirs, gaseous exchange coils and capillary columns were maintained in a 95% air/5% CO2 environment with 100% humidity. After 6 and 12 days in continuous perfusion, both cortical and hypothalamic cells demonstrated immuno-reactive SS release following 60 mMK+ depolarization (5- to 7-fold increase from basal secretion levels of 15-20 pg/3 X 10(7) cells/10 min). This response was clearly calcium dependent since it was abolished during washes with Ca2+-free Krebs-Ringer bicarbonate solution. Affinity purified material from pooled neuronal perfusates showed three distinct peaks of somatotropin release-inhibiting factor (SRIF) immunoreactivity following polyacrylamide gel chromatography on Biogel-P10. The dominant form coeluted with synthetic tetradecapeptide-somatostatin (SS-14) and a smaller amount (c 30%) coeluted with synthetic SS-28. (The SS-14 antibody used showed equimolar cross reactivity with SS-28). A larger form of immunoreactive material was also detected with an apparent molecular weight of about 11,500 daltons. Hypothalamic and cortical perfusates produced similar electrophoretic patterns of immunoreactive SS (ISS).(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in thyrotropin-releasing hormone levels in alligator stomach during fasting.

Stomach tissue of the American alligator, Alligator mississippiensis, contains substantial levels of thyrotropin-releasing hormone (TRH) which behaves identically to the synthetic hormone on radioimmunoassay (RIA) and high performance liquid chromatography (HPLC). Fasting induces a marked increase in gastric tissue levels of this hypophysiotropic hormone, but is without effect on hypothalamic content, suggesting a physiological role for TRH in gastric function of this vertebrate.

Regulation of thyrotropin-releasing hormone secretion from frog skin.

The regulation of TRH secretion from skin fragments of the leopard frog, Rana pipiens, was examined under various conditions. TRH secretion is under dual neurotransmitter control with stimulatory noradrenergic and inhibitory dopaminergic components; other neurotransmitters were generally without effect. Membrane-depolarizing conditions required the presence of calcium to cause TRH release consistent with a stimulus-secretion coupling mechanism. However, the norepinephrine-stimulated response appeared to be independent of external Ca++. TRH secretion was unresponsive to somatostatin, T3, and dibutyryl cAMP, substances which may affect TRH secretion in the mammalian hypothalamus. Anuran skin is a complex secretory tissue, but the presence of large quantities of TRH and other biologically active neuropeptides makes this tissue an attractive model to study the regulation of neuropeptide secretion.