Immunoreactive Insulin Secretion Research Articles

The Effect of Two Isocaloric and Energy-Matched Plant-Based and Processed-Meat Meals on Glucose Metabolism, Gastrointestinal Hormones, and Satiety in Subjects with T2D, Obese Subjects, and Healthy Controls—A Randomized Crossover Study

Objective: Gastrointestinal hormones play a key role in glucose metabolism and energy homeostasis. Methods: A randomized cross-over study was used to test the effects of two energy- (600 kcal) and macronutrient-matched plant-based and processed-meat meals (41% carbohydrates, 19% protein, and 40% lipids) on glucose metabolism, plasma concentrations of gastrointestinal hormones, and satiety in subjects with T2D (n=20), obese subjects (n=20) and healthy controls (n=20). Plasma concentrations of glucose, immunoreactive insulin, C-peptide, GLP-1, GIP, amylin and PYY, along with satiety, were determined at 0, 30, 60, 120 and 180 min. Repeated-measures ANOVA was used for statistical analysis. Results: An increase in stimulated secretion of immunoreactive insulin was observed in T2D and obese subjects (p=0.005, and p=0.045, respectively) after the plant-based meal. We observed an increase in stimulated secretion of C-peptide in all groups after the plant-based meal (p<0.001 for T2D, p=0.014 for obese subjects, and p=0.001 for healthy controls). An increase in postprandial concentrations of amylin was observed in T2D and healthy controls after the plant-based meal (p<0.001 for both groups). An increase in stimulated secretion of GLP-1 was observed in T2D obese subjects and healthy controls (p<0.001, and p=0.01, respectively) after the plant-based meal. A decrease in peak concentrations of GIP (at 60 min.), and an increase in peak concentrations of PYY, were observed in all groups after the plant-based meal. The participants in all groups reported greater satiety after the plant-based meal (p=0.004 for T2D, p<0.001 for obese subjects, and p<0.001 for healthy controls). Conclusions: Our study suggests that plant-based meals may be effective in increasing stimulated secretion of GLP-1, insulin, amylin and PYY, as well as in promoting satiety. Disclosure M. Klementova: None. L. Belinova: None. M. Haluzik: None. R. Pavlovicova: None. M. Hill: None. T. Pelikanova: None. H. Kahleova: None.

Incretin secretion and glucose metabolism in morbidly obese patients in the early and late periods after biliopancreatic diversion

To estimate the parameters of glucose metabolism and to assess the secretion of incretins in patients after biliopancreatic diversion (BPD) for morbid obesity (MO) in the early and late postoperative periods. The prospective part of the investigation included 22 patients with a body mass index of 35.8 to 68.4 kg/m2 and type 2 diabetes mellitus (T2DM). All the patients were examined before, 3 weeks and 3 months after BPD. The retrospective part covered 23 patients who were examined after BPD for MO; the postoperative period was 4.7 [2.3; 7.2] years. A control group consisted of 22 healthy, normal weight volunteers. A 75-g oral glucose tolerance test was carried out in all the groups to study the levels of glucose, immunoreactive insulin (IRI), glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon at 0, 30, 60, and 120 min. T2DM patients showed improvement in glucose metabolism just 3 weeks after BPD; following 3 months, they had normalized fasting blood glucose levels (5.6 [5.0; 6.0] mmol/l). During 3 months, glycated hemoglobin decreased from 7.5 [6.6; 8.5] to 5.7 [5.3; 5.9]%. In the early period following BPD, there was an increase in basal and postprandial GLP-1 levels associated with the peak IRI concentration. In the late period after BPD, the enhanced secretion of IRI and GLP-1 persisted, which was followed by a reduction in postprandial glucose levels in 4 of the 23 patients. T2DM remission does not depend on weight loss in the early period after BPD. In this period, the significant improvement of glucose metabolic parameters in patients with obesity and T2DM is associated with elevated GLP-1 levels. The altered incretin response is a stable effect of BPD and remains in its late period.

A Role For Lte1p (a Low Temperature Essential Protein Involved in Mitosis) in Proprotein Processing in the Yeast Secretory Pathway

We previously identified six single gene disruptions in Saccharomyces cerevisiae that allow enhanced immunoreactive insulin secretion primarily because of defective Kex2p-mediated endoproteolytic processing. Five eis mutants disrupted established VPS (vacuolar protein sorting) genes, The sixth, LTE1, is a Low Temperature (<15 degrees C) Essential gene encoding a large protein with potential guanine nucleotide exchange (GEF) domains. Lte1p functions as a positive regulator of the mitotic GTPase Tem1p, and overexpression of Tem1p suppresses the low temperature mitotic defect of lte1. By sequence analysis, Tem1p has highest similarity to Vps21p (yeast homolog of mammalian Rab5). Unlike TEM1, LTE1 is not restricted to mitosis but is expressed throughout the cell cycle. Lte1p function in interphase cells is largely unknown. Here we confirm the eis phenotype of lte1 mutant cells and demonstrate a defect in proalpha factor processing that is rescued by expression of full-length Lte1p but not a C-terminally truncated Lte1p lacking its GEF homology domain. Neither overexpression of Tem1p nor 13 other structurally related GTPases can suppress the secretory proprotein processing defect. However, overexpression of Vps21p selectively restores proprotein processing in a manner dependent upon the active GTP-bound form of the GTPase. By contrast, a vps21 mutant produces a synthetic defect with lte1 in proprotein processing, as well as a synthetic growth defect. Together, the data underscore a link between the mitotic regulator, Lte1p, and protein processing and trafficking in the secretory/endosomal system.

Intracellular depletion of insulin: a comparative study with palmitate, oleate and elaidate in INS-1 cells.

Free fatty acids (FFAs) deplete the intracellular insulin stores of pancreatic beta-cells. It has been suggested that this results from a lipotoxic dysregulation of both insulin secretion and insulin synthesis. In the present study, this hypothesis was tested within a 12-h time-course by directly relating the FFA-induced loss of intracellular insulin to corresponding parameters of insulin secretion and de novo biosynthesis. Palmitate, cis-monoenic oleate and the trans-monoenic elaidate were employed as model FFAs to elucidate potentially different effects due to chain length and configuration. INS-1 cells were incubated for 1, 4 or 12 h with 11.2 mmol/l glucose with 200 micromol/l palmitate, oleate or elaidate and compared with non-FFA-exposed controls with respect to content and secretion of immunoreactive insulin (IRI). Biosynthesis of insulin was monitored by pulse-labeling experiments and by Northern blot analysis. IRI content dropped by 50-60% after a short-term exposure with all FFAs employed (P< or =0.001). It tended to recover after 12 h of treatment with oleate and elaidate but not with palmitate. FFA treatment increased insulin secretion by 25% (P< or =0.05) which could not account quantitatively for the intracellular loss. FFA-induced changes in insulin biosynthesis did not correlate clearly with the FFA-induced intracellular loss. The FFA-induced loss of IRI is an acute effect independent of the FFA employed. It cannot be sufficiently explained by FFA-induced perturbances of IRI secretion and biosynthesis. We therefore postulate an additional FFA-triggered mechanism, e.g. intracellular IRI degradation.

Intracellular retention of newly synthesized insulin in yeast is caused by endoproteolytic processing in the Golgi complex.

An insulin-containing fusion protein (ICFP, encoding the yeast prepro-alpha factor leader peptide fused via a lysine-arginine cleavage site to a single chain insulin) has been expressed in Saccharomyces cerevisiae where it is inefficiently secreted. Single gene disruptions have been identified that cause enhanced immunoreactive insulin secretion (eis). Five out of six eis mutants prove to be vacuolar protein sorting (vps)8, vps35, vps13, vps4, and vps36, which affect Golgi<-->endosome trafficking. Indeed, in wild-type yeast insulin is ultimately delivered to the vacuole, whereas vps mutants secrete primarily unprocessed ICFP. Disruption of KEX2, which blocks intracellular processing to insulin, quantitatively reroutes ICFP to the cell surface, whereas loss of the Vps10p sorting receptor is without effect. Secretion of unprocessed ICFP is not based on a dominant secretion signal in the alpha-leader peptide. Although insulin sorting mediated by Kex2p is saturable, Kex2p functions not as a sorting receptor but as a protease: replacement of Kex2p by truncated secretory Kex2p (which travels from Golgi to cell surface) still causes endoproteolytic processing and intracellular insulin retention. Endoproteolysis promotes a change in insulin's biophysical properties. B5His residues normally participate in multimeric insulin packing; a point mutation at this position permits ICFP processing but causes the majority of processed insulin to be secreted. The data argue that multimeric assembly consequent to endoproteolytic maturation regulates insulin sorting in the secretory pathway.

The constitutive secretory pathway is a major route for islet amyloid polypeptide secretion in neonatal but not adult rat islet cells.

Islet amyloid polypeptide (IAPP or amylin) is a normal secretory product of the pancreatic beta-cell that is cosecreted with insulin and is the major constituent of islet amyloid deposits in individuals with type 2 diabetes or insulinomas. We have previously reported that glucose stimulates IAPP, but not insulin secretion, from neonatal rat beta-cells when regulated secretion is prevented by use of calcium-free media, suggesting that IAPP secretion occurs via a constitutive secretory pathway. To directly test this hypothesis, we examined the effects of 2 substances-brefeldin A (BFA) and cycloheximide (CHX)-that are predicted to selectively block constitutive secretion on the release of IAPP-like immunoreactivity (IAPP-LI) and immunoreactive insulin (IRI) from neonatal rat islet cell monolayer cultures. When regulated release was prevented by use of calcium-free media, glucose-stimulated IAPP-LI release was nearly abolished by blocking constitutive release with 10 microg/ml BFA (mean +/- SD: 8.7 +/- 7.7 vs. 29.3 +/- 14.3 pmol/l; n = 5; P < 0.05), an inhibitor of constitutive vesicle formation. Similarly, calcium-independent, glucose-stimulated IAPP-LI secretion was markedly suppressed when new protein synthesis was blocked by administration of 20 microg/ml CHX (4.6 +/- 2.1 vs. 29.5 +/- 14.0 pmol/l; n = 5; P < 0.005). Secretion of IRI was low in the absence of calcium, and neither BFA nor CHX had any further effect. When calcium was added to the incubation media to allow regulated secretion of both IRI and IAPP-LI, both BFA (47.7 micro 8.7 vs. 80.7 micro 10.3 pmol/l; P < 0.001) and CHX (37.3 +/- 5.8 vs. 73.3 +/- 6.2 pmol/l; n = 5; P < 0.0001) inhibited glucose-stimulated IAPP-LI secretion by approximately 40%, but again had no inhibitory effect on IRI secretion. These data indicate that approximately 40% of glucose-stimulated IAPP-LI release occurs via a constitutive secretory pathway in neonatal rat islet cells. By contrast, in adult rat islets, glucose-stimulated IAPP-LI release was almost abolished in the absence of calcium (86 +/- 3% inhibition; P < 0.05) and unaffected by addition of BFA (275 +/- 28 vs. 205 +/- 89 pmol/l; NS) or CHX (160 +/- 20 vs. 205 +/- 89 pmol/l; NS), suggesting that constitutive secretion of IAPP does not occur in mature beta-cells. Collectively, these data suggest that a significant proportion of glucose-stimulated IAPP secretion from neonatal, but not adult, rat islet cells occurs via a constitutive secretory pathway.

Two novel immortal pancreatic beta-cell lines expressing and secreting human islet amyloid polypeptide do not spontaneously develop islet amyloid.

Type 2 diabetes is characterized by islet amyloid deposits, which are primarily composed of the amyloidogenic human form of islet amyloid polypeptide (IAPP, amylin). The mechanism of islet amyloido-genesis is not known, but other products (e.g., apolipoprotein E and perlecan) contained within islet amyloid may be necessary. Because rodent IAPP does not form islet amyloid, the currently available beta-cell lines are not useful for studying processes involved in amyloid formation. To develop a suitable in vitro cell system for the study of islet amyloid formation, we generated two new beta-cell lines that express the amyloidogenic human IAPP. We did this by crossbreeding human IAPP transgenic mice with RIP-Tag mice that develop islet tumors and then culturing one of these islet tumors from two separate offspring of this cross. The resultant 2350-2C0 and 2511 cell lines produce human as well as mouse IAPP-like immunoreactivity (IAPP-LI) and immunoreactive insulin (IRI). Incubation of both these cell lines with 16.7 mmol/l glucose resulted in a two- to fourfold increase in human IAPP-LI, mouse IAPP-LI, and IRI secretion compared with 1.67 mmol/l glucose and the combination of 16.7 mmol/l glucose and 10 mmol/l arginine, 0.1 mmol/l 3-isobutyl-1-methylxanthine (IBMX), and 5 micromol/l carbachol induced a >50-fold increase in the release of these peptides. The omission of calcium from the above secretagogue cocktail reduced secretion of all three peptides to only two- to sixfold higher than the 16.7 mmol/l glucose condition. Perifusion with 16.7 mmol/l glucose plus 0.1 mmol/l IBMX caused a biphasic secretion of human IAPP-LI and mouse IAPP-LI, as well as IRI, in both cell lines, with the peak of the first phase being five- to sixfold higher than the prestimulated 1.67 mmol/l glucose condition. Immunoelectron microscopic inspection of both 2350-2C0 and 2511 cells after 7 days of culture did not reveal the presence of amyloid fibrils, suggesting the need for other critical components. We conclude that we have established two novel beta-cell lines that produce and secrete human IAPP in a regulated manner. These cell lines will be a useful tool to investigate the secretion of human IAPP as well as the necessity of other components for islet amyloid formation.

Regulatable production of insulin from primary-cultured hepatocytes: insulin production is up-regulated by glucagon and cAMP and down-regulated by insulin

To utilize hepatocytes for insulin-producing surrogate cells, we devised a regulatory secretion system by placing proinsulin DNA under the regulatable promoter for phosphoenolpyruvate carboxykinase (PEPCK). The expression of PEPCK is down-regulated by insulin, and up-regulated by cAMP and glucagon. To express insulin in hepatocytes, we constructed an adenoviral insulin expression system. After infection, the hepatocytes secreted immunoreactive insulin (IRI) at an increasing rate. IRI secretion increased over four-fold upon stimulation with 300 microM cAMP and 500 microM of the cAMP-dependent phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). This increase was also observed with glucagon and IBMX. Production was augmented two-fold by the addition of wortmannin, phosphatidylinositol (PI)-3-kinase inhibitor, suggesting that inhibitory insulin signaling to the PEPCK promoter may be mediated through PI-3-kinase. Addition of exogenous insulin to the culture decreased insulin mRNA expression remarkably on Northern blot. Thus, by using a PEPCK promoter for insulin expression, we were able to up-regulate insulin production from hepatocytes with cAMP and glucagon, and down-regulate with insulin itself.

The IRI Release from Insulin Gene-Transduced Hepatic Cells Responds to Ambient Glucose Concentration

Insulin gene was induced into differentiated hepatic cell line. These cells released immunoreactive insulin (IRI) of which molecular weight was greater than mature insulin. The secretion of IRI was responsive to glucose concentration and inhibited by glucose metabolism antagonist, 2-deoxy glucose (2-DOG). The mRNA of GLUT2 and glucokinase (GK) were not detected in these cells by Northern blotting. The glucose metabolism process is supposed to play an important role in the glucose responsive IRI release. It is assumed that hepatic cells have metabolic “glucose sensor”, and insulin gene transduction to hepatic cells can be a physiological way of gene therapy for IDDM.

Regulatable production of mature insulin from a hepatocyte cell line: insulin production is up-regulated by cAMP and glucocorticoids, and down-regulated by insulin

We engineered a hepatoma cell line that produces an up-regulation of insulin in response to cAMP, dexamethasone, and retinoic acid, and a down-regulation in response to insulin. We devised a regulatory secretion system by placing proinsulin DNA under the regulatable promoter for phosphoenolpyruvate carboxykinase (PEPCK). To assess the ability to regulate insulin secretion, we used the rat hepatoma cell line, H4IIE. The H4IIE cells secreted immunoreactive insulin (IRI) constantly at a level of 1–3 fmol/10 6 cells/h. IRI increased approximately two-fold upon stimulation with 0.5 mM cAMP and five-fold with the addition of the cAMP-dependent phosphodiesterase inhibitor IBMX, as compared to baseline IRI secretion. IRI increased 18-fold by 1–500 nM dexamethasone together with cAMP and IBMX. Addition of exogenous insulin to the culture medium significantly decreased insulin mRNA expression on Northern blot.

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.

Increased secretion of 32,33 split proinsulin after intravenous glucose in glucose‐tolerant first‐degree relatives of patients with non‐insulin dependent diabetes of European, but not Asian, origin

The aetiology of non-insulin dependent diabetes is unknown, but defective insulin secretion is a feature. The disease also has a strong genetic basis and first-degree relatives of patients have an increased risk of future diabetes. To investigate whether beta-cell dysfunction is an early feature of the disease, we studied insulin secretion in healthy first-degree relatives of patients with non-insulin dependent diabetes. Each subject underwent a 1-hour intravenous glucose tolerance test (0.3 g/kg). Seventeen first-degree relatives of patients (10 of European and 7 of Asian (Indian subcontinent) origin) with normal glucose tolerance were compared with 17 matched control subjects with no family history of diabetes. Plasma immunoreactive insulin (IRI) was measured by radioimmunoassay, and specific insulin, intact and 32,33 split proinsulin were measured by specific immunoradiometric assays (IRMA) for the 1st phase (0-10 minutes) and 2nd phase (10-60 minutes) responses. Glucose and intermediary metabolites were measured enzymatically. Fasting concentrations of IRI, IRMA insulin, intact and 32,33 split proinsulin were similar in relatives and controls in each group. Fasting glucose levels were similar in European relatives and controls but lower in Asian relatives compared to their controls (mean +/- SE 4.9 +/- 0.2 vs 5.5 +/- 0.2 mmol/l, P < 0.05). Following intravenous glucose, European relatives had similar IRI and glucose levels to their controls. Secretion of 32,33 split proinsulin was increased in European relatives compared to their controls, significantly so for 2nd phase secretion (1st phase median (range): 71 (7-352) vs 55 (17-118) pmol/l min, NS; 2nd phase: 433 (115-1459) vs 234 (55-745) pmol/l min, P < 0.05). Secretion of IRMA insulin and intact proinsulin were similar in European relatives and controls (IRMA insulin: 1st phase 2757 (700-10,969) vs 2830 (632-4682) pmol/l min; 2nd phase 6387 (3006-15,865) vs 5284 (2060-18,605) pmol/l min; intact proinsulin: 1st phase 31 (13-113) vs 32 (16-72) pmol/l min; 2nd phase: 174 (87-737) vs 159 (97-298) pmol/l min). European relatives had a greater percentage of proinsulin-like molecules (intact + 32,33 split proinsulin) to total insulin (sum of IRMA insulin + intact + 32,33 split proinsulin) during the 2nd phase of secretion (9.1 (5.0-11.8) vs 5.9 (4.3-12.6)%, P < 0.05). In contrast, Asian relatives had similar secretion of IRI, IRMA insulin, intact and 32,33 split proinsulin to their controls. Glucose disappearance (KG) was similar in relatives and controls within each ethnic group (Europeans: relatives 725 +/- 101 vs controls 668 +/- 47/min; Asian: relatives 610 +/- 97 vs controls 783 +/- 936/min). Asian relatives had higher fasting circulating glycerol (65 +/- 7 vs 44 +/- 4 mumol/l, P < 0.05), non-esterified fatty acid (569 +/- 59 vs 375 +/- 64 mumol/l, P < 0.05) and 3-hydroxybutyrate levels (147 (44-187) vs 35 (21-57) mumol/l, P < 0.01) than their controls and this persisted following intravenous glucose. This difference was not observed in the European group. First-degree relatives of European patients with NIDDM possess early signs of beta-cell dysfunction, with increased and disproportionate secretion of 32,33 split proinsulin after intravenous glucose, whilst glucose tolerance is still normal.

Use of the fab fragment for immimoneutralization of somatostatin in the isolated perfused human pancreas

The role of the somatostatin-secreting D cell in the islet remains controversial. The present study was undertaken to determine whether infusion of the Fab fragment of a highly sensitive somatostatin monoclonal antibody into the isolated, perfused human pancreas would influence insulin secretion. Single-pass perfusion was performed in pancreata obtained from cadaveric organ donors using a modified Krebs-media with 3.9 mM glucose. Sequential test periods separated by basal periods were performed with either somatostatin monoclonal antibody Fab fragment (SFab), somatostatin-14 (SS-14), or a combined infusion. Immunoneutralization of intraislet somatostatin with SFab resulted in a significant increase in both immunoreactive insulin (IRI) (1,122 ± 497 pM) (p < 0.05) and immunoreactive C-peptide (IRC-P) secretion (146 ± 53 pM) (p < 0.05). Infusion of SS-14 resulted in inhibition of both IRI secretion (−3,372 ± 1,360 pM) (p < 0.05) and IRC-P secretion (−708 ±220 pM) (p < 0.05). Combined infusion of SFab and SS-14 reversed the inhibitory effect of exogenous SS-14 on IRI and IRC-P secretion. The data suggest that intraislet somatostatin has an inhibitory role in the regulation of B-cell secretion in the human islet and demonstrates that the Fab fragment of the somatostatin monoclonal antibody is an effective tool for immunoneutralization studies in the human pancreas. In addition, immunostaining of the donor pancreata demonstrated the presence of somatostatin-immunoreactive endocrine cells interspersed throughout the islet core and mantle. The demonstrated proximity of somatostatin-immunoreactive endocrine cells to B cells lends anatomic support to the concept that intraislet somatostatin influences insulin secretion in the human islet.

Glucose-dependent insulinotropic polypeptide stimulated insulin release from a tumor-derived β-cell line (βTC3)

The beta TC3 tumor cell line was examined for the presence of functional glucose-dependent insulinotropic polypeptide (GIP) receptors. Increasing amounts of natural porcine GIP decreased the binding of HPLC-purified [125I]GIP to beta TC3 cells in a concentration-dependent manner. Displacement of GIP was significant at concentrations as low as 500 pM, and the radioligand was fully displaced at 100 nM. GIP(1-30) produced a displacement of [125I]GIP comparable with that produced by GIP(1-42), and glucagon yielded 20% displacement at a concentration of 1 microM but was without effect at 100 mM. Incubation of beta TC3 cells in the presence of glucose concentrations of 2-20 mM yielded a concentration-dependent stimulation of immunoreactive insulin (IRI) release. GIP and glucagon-like peptide-I(7-36) amide (tGLP-I) at concentrations of 1 nM or greater significantly stimulated IRI release in the presence of 2 mM glucose. The threshold glucose concentration for GIP-stimulated IRI release from beta TC3 cells was 0.5 mM, and maximal potentiation of IRI release by GIP occurred at 5 mM glucose. Somatostatin significantly inhibited GIP-stimulated IRI release in the presence of 5 mM glucose. It is concluded that beta TC3 cells have functional GIP receptors and may provide a useful model for the study of IRI secretion at the cellular level.

Inhibitory actions of a somatostatin analog SMS 201-995 on endocrine secretion from the isolated perfused stomach and pancreas of the rat.

An analog of somatostatin, SMS 201-995 (SMS; Sandostatin), has been shown to have increased potency in the inhibition of hormone secretion in vivo. In this study, the effect of SMS on the secretion of immunoreactive insulin, gastrin and gastric somatostatin was examined in the rat using in situ vascularly perfused organ preparations. The analog was found to inhibit both basal somatostatin and gastrin secretion from the perfused stomach in a concentration-dependent manner. SMS (9.4 x 10(-8) mol/l) also inhibited somatostatin release stimulated by gastric inhibitory polypeptide (GIP), but did not suppress gastrin release under the same conditions; an opposite effect was obtained when l-isoproterenol was used to stimulate somatostatin release. In the perfused pancreas both SS-14 (6.1 x 10(-9) mol/l) and SMS (9.4 x 10(-9) mol/l) inhibited the GIP (2 x 10(-9) mol/l) or acetylcholine (1 x 10(-6) mol/l), but not 17.8 x 10(-9) mol/l glucose-stimulated insulin secretion. Insulin secretion stimulated by 17.8 x 10(-3) mol/l glucose was unaffected by either peptide at this concentration. These results show that SMS is a potent inhibitor of gastric and pancreatic endocrine secretion. However, the inhibitory effect of SMS on gastric endocrine secretion was uncoupled in the presence of different secretagogues, suggesting that the action of SMS was dependent on the activation of different mechanisms or pathways in the stomach. In the pancreas, SMS appears to be acting on the beta-cell via a similar mechanism as somatostatin.

Mumps and Coxsackie B3 virus infection of human fetal pancreatic islet-like cell clusters.

Islet-like cell clusters (ICCs) prepared from human fetal pancreases were infected with mumps or coxsackie B3 virus. Double-labeled antibody technique showed that the viruses infected both insulin-secreting and other pancreatic cells and that secretion of immunoreactive insulin into the culture medium of the mumps virus-infected cells had already ceased on day 7. The mumps virus-infected ICC clusters produced virus for 14 days, and the mumps virus antigen was detected in the ICCs through the whole 22-day observation period. The coxsackie B3 virus-infected ICCs contained cells highly positive for viral antigen during the first 2 days after infection, and the infectious virus was detected in the culture medium for 22 days. This in vitro model indicates that mumps and coxsackie B3 viruses infect human fetal pancreatic endocrine cells and are able to alter beta-cell function. Coxsackie B3 virus infection in ICCs is lytical and seems to lead to rapid cell destruction, but long-lasting, restricted mumps virus infection in human fetal pancreatic ICCs offers an interesting model to study the effects of viral infection in the endocrine pancreas and beta cell.

The Effects of Body Fat Distribution on Glucose Tolerance in Overweight Subjects

To evaluate the pathophysiological significance of intra-abdominal fat accumulation in Japanese subjects with mild to moderate overweight, 107 subjects (56 men and 51 women, aged 16-68 years) with body mass index (BMI, kg/m2) of 17-39 (mean +/- SD, 25 +/- 4.3) were evaluated. Subjects with disorders which affect glucose metabolism, such as thyroid, adrenal, liver, and kidney diseases, were excluded. A 75 g oral glucose tolerance test (OGTT) was performed in all subjects, and venous samples were obtained before 15, 30, 60, 90 and 120 min after the glucose load for plasma glucose (PG), immunoreactive insulin (IRI) and C-peptide immunoreactivity (CPR) measurements. In 72 of the subjects, plasma free fatty acid (FFA) level at fasting was also determined. The degree of visceral fat accumulation was evaluated using a CT-scan by the method reported by Fujioka et al. (Metabolism, 36: 54-59, 1987), and intra-abdominal fat area/subcutaneous fat area (V/S ratio) was obtained. V/S ratio and BMI correlated positively in subjects with BMI less than 25 (17 men and 16 women, aged 28-62 years) but they did not correlate at all with each other in those with BMI greater than or equal to 25 (39 men and 35 women, aged 16-68 years). Based on this finding, the possible adverse effects of increased intra-abdominal fat on glucose metabolism were investigated in the subjects with BMI greater than or equal to 25. For this purpose, the correlation of V/S ratio with fasting PG (FPG), fasting IRI (FIRI), fasting CPR (FCPR), FPG/FIRI, FFA, or PG area (sigma PG) and sigma PG/sigma IRI at OGTT was analyzed. V/S ratio positively correlated with FPG, sigma PG, FPG/FIRI and sigma PG/sigma IRI but not with FFA. The correlation between V/S ratio and FIRI or FCPR was significant in the subjects with V/S ratio greater than or equal to 0.8 in men and greater than or equal to 0.4 in women. In sharp contrast to V/S ratio, BMI did not correlate at all with any of these metabolic indices. We conclude that in Japanese subjects with mild overweight to moderate obesity, intra-abdominal fat accumulation, but not the increase in the degree of obesity, accompanies worsening of glucose tolerance. Because PG elevation relative to IRI secretion is progressively greater with increasing V/S ratio, it is suggested that the deleterious effects of intra-abdominal fat accumulation can be attributed to increased insulin resistance.

Insulin, glucagon, somatostatin, and thyrotropin-releasing hormone content and secretion by perifused fetal rat islets during culture

In the neonatal period of the rat, pancreatic thyrotropin-releasing hormone content decreases and the sensitivity of insulin secretion to glucose increases. In adult rat islets, TRH inhibits glucose-induced insulin release. The aim of this study was to investigate whether a high TRH content and release can be part of the explanation for the functional immaturity of neonatal islets. For that purpose, we have measured the tissue content and the secretion of immunoreactive insulin, glucagon, somatostatin and TRH in islets from 21.5-day-old rat fetuses cultured for up to one week. Insulin, glucagon and somatostatin content increased during one week of culture in the presence of 11.1 mmol/l glucose. The TRH content decreased during culture, but did not equal adult values. Insulin, glucagon and somatostatin responses to glucose were present after one week of culture. Glucose had no effect on TRH release in cultured fetal islets, but inhibited TRH release in adult islets. We conclude that glucose can stimulate insulin secretion without inhibiting TRH release, but that a decrease in islet TRH content and a sensitization of TRH secretion to glucose may be important in the full maturation of fetal pancreatic islets.

Endocrine Function of the Pancreas and Gastrin Secretion in Patients with a Kidney Transplant&lt;sup&gt;1&lt;/sup&gt;

In 24 patients with a kidney transplant and in 10 healthy subjects the test meal response of immunoreactive insulin (IRI), glucagon (IR-G), pancreatic polypeptide (IR-PP) and gastrin (IR-Ga) was studied. Patients with a kidney transplant showed significantly elevated basal plasma glucagon levels but normal levels of plasma IRI, IR-PP and IR-Ga. After administration of a test meal a significantly suppressed response of IRI, IR-PP and IR-Ga secretion was observed both in the patients treated with azathioprine as well as those treated with ciclosporin. In patients of the ciclosporin group the compromised response of IR-Ga and IRI secretion was significantly more marked than in subjects in the azathioprine group. From the results obtained in this paper it follows that the type of immunosuppressive drugs (azathioprine or ciclosporin) seems to be of minor importance in the pathogenesis of the described endocrine abnormalities.

Co-secretion of carboxypeptidase H and insulin from isolated rat islets of Langerhans

The release of carboxypeptidase H activity from isolated rat islets was determined and compared to the secretion of immunoreactive insulin. Analysis of pancreatic islet cells sorted into beta and non-beta types indicated that approx. 80% of islet carboxypeptidase H activity is present in the beta cell. The release of both insulin and carboxypeptidase H was stimulated markedly by increasing the glucose concentration in the medium from 2.8 to 28 mM. The fractional release was in accordance with the observed cellular distribution of both proteins. The secretory response was biphasic with time, with an initial rapid transient phase of release within 5 min, followed by a more sustained response. The concentration-dependencies of glucose stimulation of release of insulin and carboxypeptidase H were similar, with a threshold for stimulation around 5.6 mM-glucose and maximal stimulatory response at 16.7-28 mM-glucose. The release of both proteins was inhibited by 20 mM-mannoheptulose, removal of Ca2+ from the medium and addition of 1 microM-noradrenaline. The combination of 10 mM-4-methyl-2-oxopentanoate and 10 mM-glutamine stimulated the release of carboxypeptidase H and insulin, as did 3-isobutyl-1-methylxanthine and 350 microM-tolbutamide in the presence of glucose. It is evident that carboxypeptidase H is released from the pancreatic beta-cell by an exocytotic process from the same intracellular compartment as insulin. The release of carboxypeptidase H by a constitutive process was at best equivalent to 0.4%/h, or less than 2% of the maximal rate of release via the regulated pathway. It is concluded that carboxypeptidase H can be used as a sensitive index of beta-cell secretion and an alternative marker to the insulin-related peptides.