Rat Pancreatic Islet Cells Research Articles

Sodium nitroprusside inhibits glucose-induced insulin release by activating ATP-sensitive K + channels

Sodium nitroprusside (SNP) has been reported to be a potent stimulator of cGMP formation in different tissues, including pancreatic islets. The present study aimed at comparing the effects of sodium nitroprusside and dibutyryl cGMP on 86Rb outflow, 45Ca outflow, short-term 45Ca uptake, cytosolic Ca 2+ concentration and insulin release from rat pancreatic islet cells. The data indicate that cGMP potentiates whilst SNP inhibits the glucose-induced insulin release. This inhibitory effect appears to be mediated by the activation of ATP-sensitive K + channels leading to a decrease in Ca 2+ influx and subsequent reduction in cytosolic free Ca 2+ concentration. Whathever the exact mechanism(s) underlying the capacity of sodium nitroprusside to enhance the K + permeability of the B-cell membrane, the drug appears to be an unadequate pharmacological tool to characterize the involvement of cGMP in the insulin secretory process. The experimental results also suggest that cGMP potentiates glucose-induced insulin release without affecting ionic movements.

Rat and human pancreatic islet cells contain a calcium ion independent phospholipase A2 activity selective for hydrolysis of arachidonate which is stimulated by adenosine triphosphate and is specifically localized to islet .beta.-cells

The recent demonstration that myocardial Ca(2+)-independent phospholipase A2 exists as a complex of catalytic and regulatory polypeptides that is modulated by ATP has suggested a novel mechanisms through which alterations in glycolytic flux can be coupled to the generation of eicosanoids which facilitate insulin secretion. To determine the potential relevance of this mechanism, we examined the kinetic characteristics, substrate specificities, and cellular locus of phospholipase A2 activity in pancreatic islets. Rat pancreatic islets contain a Ca(2+)-independent phospholipase A2 activity which is optimal at physiologic pH, preferentially hydrolyzes phospholipid substrates containing a vinyl ether linkage at the sn-1 position, and prefers arachidonic acid compared to oleic acid in the sn-2 position. Rat islet Ca(2+)-independent phospholipase A2 activity is inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one and is stimulated by ATP. Purification of beta-cells from dispersed pancreatic islet cells by fluorescence-activated cell sorting demonstrated that beta-cells (but not non-beta-cells) contain Ca(2+)-independent, ATP-stimulated phospholipase A2 activity. Remarkably, clonal RIN-m5f insulinoma cells, which possess a defect in glucose-induced insulin secretion, contain a Ca(2+)-independent phospholipase A2 which is not modulated by alterations in ATP concentration. Collectively, these results and those of an accompanying paper [Ramanadham et al. (1993) Biochemistry (following paper in this issue)] implicate Ca(2+)-independent phospholipase A2 as a putative glucose sensor which can couple alterations in glycolytic metabolism to the generation of biologically active eicosanoids and thereby facilitate glucose-induced insulin secretion.

Phosphoinositide metabolism in a polyoma-BK-virus-transformed pancreatic islet cell line: evidence for constitutively activated phospholipase C.

We have characterized the phosphoinositide metabolism in a polyoma-BK-virus-transformed rat pancreatic islet cell line which has highly malignant characteristics, expresses viral T-antigen and has lost insulin-secreting capacity. After incorporation with [3H]inositol to isotopic equilibrium, all inositol metabolites were analyzed. When compared with normal pancreatic islets, increased levels of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3), inositol 1,3,4-trisphosphates and inositol tetrakisphosphate (Ins-P4), and decreased levels of phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP2) were found. The Ins-1,4,5-P3/PIP2 ratio increased, whereas the PIP2/PIP ratio was not altered after the transformation. In the pancreatic islet cell line there was a stable accumulation of inositol phosphates at 3.3 mM glucose. Glucose, KCl, cholecystokinin (CCK) and carbachol with and without LiCl were all without effect on the accumulation of inositol phosphates. Somatostatin inhibited the accumulation of inositol phosphates but a Ca(2+)-free/EDTA solution did not. Preincubation with cholera toxin or pertussis toxin inhibited the accumulation of inositol phosphates at 3.3 mM glucose except for Ins-P4, whereas no effect was observed on the phosphoinositides. NaF stimulated the accumulation of inositol phosphates, with a concomitant decrease in the phosphoinositides, whereas neomycin was without effect on the inositol phosphates. In normal pancreatic islets, pertussis toxin inhibited the CCK-induced increase in Ins-1,4,5-P3, whereas no effect was seen at 3.3 mM glucose. Finally, pertussis toxin inhibited the CCK-induced increase in the Ins-1,4,5-P3/PIP2 ratio in normal pancreatic islets. The same inhibition was also found in the pancreatic islet cell line at 3.3 mM glucose. We conclude that in the transformed pancreatic islet cell line the phosphoinositide hydrolysis is constitutively activated at the level of phospholipase C, with a substantial loss of regulatory control.

Effect of syngeneic islet antigen administration on complement-dependent antibody-mediated cytotoxicity to islet cells and diabetes onset in diabetes-prone BB/OK rats.

Diabetes-prone BB/OK rats aged 30 to 35 days were subjected to three sequential intrasplenic injections of unfractionated homogenate prepared from Langerhans' islets of newborn syngeneic BB/OK rats. Syngeneic islet antigen administration resulted in increased complement-dependent antibody-mediated cytotoxicity (C'AMC) to rat pancreatic islet cells in serum, compared to buffer-treated control animals as detected by 51Cr-release assay. However, the increase of anti-islet-cell cytotoxicity neither impaired glucose tolerance nor affected the incidence of diabetes and the age at manifestation. In contrast to BB/OK rats developing diabetes, animals remaining long-term normoglycaemic did not show an enhancement of cytotoxicity to islet cells within twelve days after the first islet antigen injection as revealed retrospectively. In conclusion, humoral mediated anti-islet-cell cytotoxicity is not decisively involved in pancreatic beta-cell destruction and promotion of diabetes development in BB/OK rats, but animals becoming diabetic seem to be characterized by a stronger immunological reactivity upon syngeneic islet antigen challenge as indicated by an increase of anti-islet C'AMC compared to long-term normoglycaemic rats.

Oxygen radicals generated by the enzyme xanthine oxidase lyse rat pancreatic islet cells in vitro.

The endothelium-associated enzyme xanthine oxidase is known to generate reactive oxygen intermediates which may damage the surrounding tissue. We investigated whether reactive oxygen intermediates released by xanthine oxidase exert a toxic effect on isolated rat islet cells. The xanthine oxidase (25 mU/ml)/hypoxanthine (0.5 mmol/l) system released reactive oxygen intermediates in vitro as detected by luminol in a chemiluminescence analysing system. The addition of nicotinamide inhibited the release of reactive oxygen intermediates in a dose-dependent manner (50% inhibition at 20 mmol/l). Exposure of islet cells to enzyme generated reactive oxygen intermediates caused lysis of 39% of the cells within 15 h. Monitoring the mitochondrial function of islet cells by the conversion of tetrazolium bromide to its formazan product revealed a significant reduction of the respiratory activity down to 51% of that of the controls by 30 min after the initiation of the xanthine oxidase reaction. Mitochondrial dysfunction preceded plasma membrane damage. The addition of nicotinamide, a radical scavenger and inhibitor of the DNA repair enzyme poly(ADP-ribose) synthetase protected the islet cells from lysis and partially preserved their mitochondrial activity in the presence of reactive oxygen intermediates. We conclude that activation of the endothelial enzyme xanthine oxidase, known to be induced by mediators of immune cells or by episodes of ischaemia and reperfusion causes islet cell damage with subsequent cell death in early phases of pancreatic islet cell destruction.

Regulation of Na+/Ca2+ exchange in the rat pancreatic B cell.

Na+/Ca2+ exchange in the B cell was recently characterized by measuring intracellular-Na(+)-dependent 45Ca2+ uptake in isolated rat pancreatic islet cells. The aim of the present study was to investigate the regulation of this process. Extracellular pH (pHo) and intracellular pH (pHi) markedly affected Na+/Ca2+ exchange. A fall of 0.04 unit in pHi decreased the exchange by 45%, whereas a rise of 0.13 unit increased the uptake by 70%. Mitochondrial poisons (oligomycin, antimycin A and 2,4-dinitrophenol) inhibited reverse Na+/Ca2+ exchange by about 25-50%. The exchanger displayed a low Q10 (temperature coefficient), indicating that it is only indirectly dependent on metabolic energy. The phorbol ester phorbol 12-myristate 13-acetate did not affect Na+/Ca2+ exchange. Likewise, lowering the extracellular K+ concentration did not inhibit 45Ca2+ uptake. In conclusion, the pHi and the metabolic state of the cell may represent important modulatory signals by which insulin secretagogues such as glucose could regulate reverse Na+/Ca2+ exchange in the B cell. The process does not appear to co-transport K+ nor to be influenced by protein kinase C.

Transformed rat pancreatic islet-cell lines established by BK virus infection in vitro.

Single-cell suspensions of primary rat pancreatic islet cells were infected with BK virus (BKV) prototype (Gardner) and the naturally occurring BKV strain (TU), isolated from human urine. These viral strains have different sequences in their non-coding control regions which contain promoter-enhancer elements and origin of DNA replication. Uninfected cell cultures disintegrated within 3 weeks, while a varying number of virus-infected cultures were immortalized and a majority exhibited focal areas of 2-dimensional growth. A significantly higher proportion of the BKV (TU)- than of the BKV (Gardner)-infected cultures were immortalized. Large tumor (T) antigen expression was evident in virus-infected cultures from day 4 post infection (p.i.), while insulin secretion decreased steadily during the first weeks of culture. Two cell lines were established from BKV (TU)-infected cultures. Line 5A4 was contact-inhibited, growing as a dense monolayer, while 6A3 demonstrated foci of 2-dimensional growth. Both cell lines have retained their morphology and T-antigen expression for approximately 130 passages. At high passages a low level of intracellular insulin, but no secretion into media, was detected. Based on standard biological tests (reduced serum requirements, growth at low cell density, anchorage-independent growth and tumor induction in nude mice) both cell lines have a fully transformed phenotype, but 6A3 appears to be more malignantly transformed. Since both cell lines were established simultaneously from the same primary cells with the same virus batch, they provide an opportunity to study the transforming mechanisms of BKV in a relative context.

Effects of CCK-8 on the cytoplasmic free calcium concentration in isolated rat islet cells

The C-terminal octapeptide of cholecystokinin (CCK-8) is known to stimulate insulin secretion. We examined its effects on the cytoplasmic free calcium concentration ([Ca 2+] IC) in isolated rat pancreatic islet cells. At 8.3 mM glucose and 1.28 mM Ca 2+, CCK-8 (100 nM) rapidly increased [Ca 2+] IC to a short-lived peak, whereafter the [Ca 2+] IC, within 1.5 minutes, fell to values below baseline. CCK-8 also rapidly increased the [Ca 2+] IC at 3.3 mM glucose and in a calcium deficient medium. However, either at low glucose or in the absence of extracellular Ca 2+, the post-peak [Ca 2+] IC did not fall below baseline levels. The CCK A receptor antagonist, L-364,718 (20 nM), inhibited the effects of CCK-8 on [Ca 2+] IC. The results suggest that CCK-8 in islet cells liberates calcium from intracellular stores by activating CCK A receptors.

Inhibition of Na/Ca exchange in pancreatic islet cells by 3′,4′-dichlorobenzamil

Na/Ca exchange may play a role in Ca 2+ extrusion from the pancreatic B cell. The role played by the exchanger was examined by characterizing the effects of 3′-4′-dichlorobenzamil on ionic fluxes and insulin release in normal rat pancreatic islet cells. 3′,4′-Dichlorobenzamil potently inhibited 45Ca uptake mediated by reverse Na/Ca exchange ( ic 50: 18 μm) in islet cells. The drug failed to decrease intracellular pH but reduced 86Rb outflow from perifused islets. The effects of glucose and 3′,4′-dichlorobenzamil on 86Rb outflow were not additive. The drug potently blocked 45Ca uptake through voltage-sensitive Ca 2+ channels (IC 50: 7.5μM). In the presence of extracellular Ca 2+ and 3′,4′-dichlorobenzamil, glucose lost part of its ability to reduce 45Ca outflow. The drug failed to affect the secondary rise in 45Ca outflow induced by the sugar. In the absence of extracellular Ca 2+,3′,4′-dichlorobenzamil induced a delayed inhibition of 45Ca outflow, the effect of the sugar and the drug being not additive. This effect of 3′,4′-dichlorobenzamil and its ability to impair the inhibitory effect of glucose were reproduced by the removal of extracellular Na + and disappeared under the latter experimental condition. 3′,4′-Dichlorobenzamil did not affect insulin release in the absence of glucose but significantly increased glucose-induced insulin release when used at a high concentration. It is concluded that 3′,4′-dichlorobenzamil is a potent inhibitor of the process of Na/Ca exchange in the pancreatic B cell. Unfortunately, the drug is of poor specificity and blocks, in the same range of concentrations, both K + channels and voltage-sensitive Ca 2+ channels. The data also indicate that glucose inhibits 45Ca outflow from pancreatic islets to a great extent (at least 75%) by inhibiting Na/Ca exchange. The type of Na/Ca exchange that is inhibited by glucose, remains to be elucidated

Dual effect of 1.4-dihydropyridines on Ca2+ inflow into rat pancreatic islet cells

The present study aimed at comparing the effects of nifedipine, a dihydropyridine "Ca2+ antagonist", and of BAY K 8644, a dihydropyridine "Ca2+ agonist", on the short term (5 min) 45Ca uptake and the cytosolic Ca2+ concentration of rat pancreatic islet cells incubated in the presence of physiological concentrations of glucose. Nanomolar concentrations of nifedipine increased the short term 45Ca uptake while micromolar concentrations decreased it. Cd2+, an inorganic Ca2+ channel blocker, reduced the stimulatory effect of nifedipine. Low concentrations of BAY K 8644 stimulated 45Ca uptake whereas high concentrations decreased it. In contrast, verapamil, a phenylalkylamine type calcium antagonist, only provoked a dose-dependent reduction in 45Ca uptake. Lastly, low concentrations of both nifedipine and BAY K 8644 raised the fluorescence intensity of fura 2 loaded islet cells. These findings indicate that nifedipine and BAY K 8644 may exhibit agonistic and antagonistic actions on B-cell voltage-dependent Ca2+ channels. This dualistic behaviour is markedly concentration-dependent and appears to be inherent to the 1,4-dihydropyridine compounds.

Activation of mechanical responses in leukocytes

Different kinds of leukocytes undergo cytoskeleton-dependent mechanical responses associated with their specific physiological functions. We have investigated cellular stiffening of several types of leukocytes using a method which measures the force resisting cellular indentation. We have found that lymphocytes stiffen in response to crosslinking cell surface antigens in a process associated with the much studied capping and patching processes. Further studies of myosin-deficient mutants of the ameba Dictyostelium discoideum suggest that this stiffening process results from a myosin dependent contractile process. Rat basophilic leukemia cells and pancreatic islet cells stiffen when triggered to secrete. The function of these cytoskeleton dependent processes is now unknown, but, at least in the islet cells, may be related to a regulation of the rate of secretion. Primary neutrophils stiffen in response to the chemotactic agent, fMet-Leu-Phe. This stiffening may be responsible for retention of these cells in the pulmonary microcirculation during response to inflammation. These observations pose the challenge of determining the structural basis, mechanism, and physiological function of each of these cellular responses.

Characterization of the process of sodium-calcium exchange in pancreatic islet cells

Na(+)-Ca2+ exchange may play a role in Ca2+ extrusion from the pancreatic B-cell. The characteristics of the process working in its reverse mode were examined in normal rat pancreatic islet cells. Isosmotical replacement of extracellular Na+ by sucrose induced a concentration-dependent increase in 45Ca uptake, displaying a pharmacological sensitivity compatible with an uptake mediated by Na(+)-Ca2+ exchange. Glucose, up to 2.8 mM, stimulated reverse Na(+)-Ca2+ exchange. Likewise, membrane depolarization activated the process but only under raised intracellular Na+ activity. In conclusion, the B-cell Na(+)-Ca2+ exchange displays properties similar to those observed in other cells: reversibility and sensitivity to membrane potential. When working in its reverse mode the exchanger displays a quite large capacity. The role played by the exchanger in the process of insulin release warrants further investigation.

The occurrence and receptor specificity of endogenous opioid peptides within the pancreas and liver of the rat. Comparison with brain

Our observations that opioid peptides have direct effects on islet insulin secretion and liver glucose production prompted a search for endogenous opiates and their receptors in these peripheral tissues. Mu-, delta- and kappa-receptor-active opiates were demonstrated in brain, pancreas and liver extracts by displacement studies using selective ligands for the three opiate receptor subtypes [( 3H][D-Ala2,MePhe4,Gly5-ol]enkephalin, [3H][D-Ala2,D-Leu5]enkephalin and [3H]dynorphin respectively). Receptor-active opiates in brain extracts exhibited a stronger preference for delta-opiate-receptor sites than for mu and kappa sites. Pancreatic extract opiates demonstrated a similar activity at mu and delta sites, but substantially less at kappa sites. Liver extracts displayed similar selectivity for all three sites. The affinities of the receptor-active opiates for mu-, delta- and kappa-receptor subtypes displayed a rank order of potency: brain much greater than pancreas greater than liver. Total immunoreactive beta-endorphin and [Met5]enkephalin levels in liver and hepatocytes were greater than those in brain. Immunoreactive [Met5]enkephalin levels in pancreas were similar to, but beta-endorphin levels were substantially higher than, those in brain. Delta and kappa opiate-binding sites of high affinity were identified in crude membrane preparations of islets of Langerhans, but no specific opiate-binding sites could be demonstrated in liver membrane preparations. Immunoreactive dynorphin and beta-endorphin were demonstrated by immunogold labelling in rat pancreatic islet cells. No positive staining of liver sections for opioids was observed. These results suggest that the tissue content of opiate-receptor-active compounds in the pancreas and the liver is very significant and could contribute to the regulation of normal blood glucose levels.

Interferon-gamma sensitizes rat pancreatic islet cells to lysis by cytokines and cytotoxic cells

Immunoregulatory polypeptides (cytokines) produced by mononuclear cells of the immune system can inhibit insulin secretion and may be cytotoxic to pancreatic islet beta cells. We used a modified 51Cr release cytotoxicity assay to define the interactive cytotoxic effects of interleukin-1 (IL-1), tumor necrosis factor (TNF), and interferon gamma (IFN-gamma) on rat islet cells in monolayer culture. We here demonstrate that (a) IFN-gamma, but not IL-1 or TNF, can sensitize islet cells to subsequent cytolysis by other cytokines (IL-1 or TNF); (b) the cytolytic effect of sequential addition of IFN-gamma, then IL-1 or TNF (approximately 35-40% 51Cr release) is similar to that produced by the concurrent addition of IFN-gamma plus IL-1 or TNF; (c) the priming effect of IFN-gamma persists for 3 to 6 or more days after its removal; and (d) islet cells preincubated with IFN-gamma are also more sensitive to cytolysis by splenic mononuclear cells from diabetes-prone BB/Wor rats. These findings suggest that IFN-gamma produced by activated T lymphocytes and monocytic cells infiltrating islets in Type 1 diabetes may play a direct and important role in sensitizing beta cells to damage by other cytokines (IL-1, TNF) and cytotoxic cells in the immune/inflammatory infiltrate.

Analysis of Islet Cell Surface Antigen Reactions with Islet Cell Surface Antibodies (ICSA)

Islet cell surface antibodies (ICSA) have been detected in the sera of many patients with insulin-dependent diabetes mellitus (IDDM). They have also been demonstrated to affect the plasma membranes of beta-cells in vitro. To determine the pathogenetic role of ICSA in IDDM, we studied their prevalence and their relationship to lymphoblastogenesis (LBG) in diabetes as well as in other autoimmune diseases. Furthermore, islet cell antigens (IAg) were characterized from rat pancreatic islet cells, using an affinity column consisting of human IgGs including ICSA. ICSA titers were measured by indirect immunofluorescence. Sera were determined as ICSA-positive when they reacted to more than 10% of 50-100 cells. The LBG investigation was carried out after a 4-day incubation with phytohemagglutinin (PHA), pokeweed mitogen (PWM), or concanavalin A (Con A). The LBG induced by IAg was investigated after an 8-day incubation. Lymphocytes included 75% CD3-positive cells and 5% CD20-positive cells. IAg were purified from ICSA-positive IgG coupled to CNBr-activated sepharose 4B. The prevalence of ICSA was 39% in patients with IDDM (11/28), 15% in non-insulin dependent diabetes mellitus (NIDDM) (16/109), 14% in Graves' disease (3/22), 29% in Hashimoto's disease (5/17), 12% in rheumatoid arthritis (3/25), 20% in systemic lupus erythematosus (SLE) (7/35), and 33% in Sjögren's syndrome (2/6). No ICSA were detected in 27 normal subjects. Although sera from the patients with autoimmune diseases contained antinuclear antibodies, antithyroid antibodies and/or rheumatoid factor, there was no relationship between the prevalence of such antibodies in patients with ICSA and those without it. In 3 patients (60%) with ICSA-positive IDDM, the lymphoblastogenic responses to PHA and PWM were decreased. A similar decrease was observed when comparing ICSA-positive NIDDM to ICSA-negative NIDDM (PHA: p less than 0.05; PWM: p less than 0.01). However, there was no relationship between HbA1 and the LBG response or between HbA1 and the presence of ICSA. The relative molecular weights (Mr.) of the IAg reacting with ICSA-positive IgG were around 67, 64, 55, and 20K in all but three patients with diabetes mellitus. IAg with a Mr. around 30K or less than 14K were also demonstrated in some patients with diabetes mellitus. The Mr. of IAg was the same in three patients with autoimmune disease as in diabetes mellitus, but it differed in three other similar patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of Prolactin and Glycosylated Prolactin on (Pro)insulin Synthesis and Insulin Release from Cultured Rat Pancreatic Islets

In order to study the possible differential effects of the nonglycosylated and glycosylated forms of prolactin on insulin content and secretion in pancreatic islets, neonatal rat pancreatic islets were exposed for 6 days in vitro to 2 micrograms/ml of nonglycosylated ovine prolactin (oPRL), or to 2 micrograms/ml of glycosylated oPRL (G-oPRL). oPRL stimulated a significant increase (p less than 0.01) in the total amount of insulin released into the medium over the 6 day culture period; however, G-oPRL had no effect. Islets cultured for 6 days in the presence of oPRL showed no increase in the amount of DNA per islet. However, there was a significant (p less than 0.007) increase in the amount of total protein synthesized by the islets exposed to oPRL. These findings suggest that the effect of oPRL on neonatal rat pancreatic islet cells is a nonspecific effect. The nonglycosylated form of PRL may play a role in B-cell function by promoting protein synthesis, which results in augmented insulin synthesis.

Gastric inhibitory polypeptide mechanisms of augmenting insulin secretion

The cellular mechanisms whereby gastric inhibitory polypeptide (GIP) augments glucose-dependent insulin secretion remains poorly defined. Since glucose-dependent insulin secretion is modulated by membrane associated phospholipase A2 (PLA 2) and intracellular lipoxygenase (LPX) and cyclooxygenase (CO) we hypothesize that GIP's augmentation of insulin secretion involves these enzyme systems. Neonatal rat pancreatic islet cell cultures were preincubated with 5.6mM glucose for 60 minutes. The cultures were then stimulated for 60 minutes with 16mM glucose alone or with GIP with or without the addition of PLA 2, LPX, and CO inhibitors. Insulin secretion significantly increased (P<0.05) when the glucose concentration was raised from 5.6 to 16mM glucose and this was further augmented by the addition of GIP (P<0.05). PLA 2 inhibitors significantly (P<0.025) decreased 16mM glucose insulin secretion but this was restored by the simultaneous addition of GIP. LPX inhibitors significantly (P<0.01) decreased glucose-dependent insulin secretion and this decrease parsisted despite the addition of GIP. Simultaneous treatment of islet cell cultures with GIP and CO inhibitors yielded insulin responses that were indistinguishable from CO inhibition alone. These studies suggest that GIP exerts its influence in part by modulating membrane associated PLA 2 activity. Furthermore, the formation of intracellular LPX products appears to be a pivotal step in the insulinotrophic action of GIP.

A cytotoxic monoclonal autoantibody from the BB rat which binds an islet cell surface protein

The BB rat provides an excellent animal model for type 1 (insulin-dependent) diabetes mellitus. Cytotoxic autoantibodies against pancreatic β cells have been found in the sera of both patients with type 1 (insulin-dependent) diabetes and BB rats. These antibodies have been implicated in the pathogenesis of the disease. In this study, a monoclonal autoantibody, designated KT1, has been developed by the fusion of spleen cells from a BB rat and a mouse myeloma cell line. KT1 was found to be of the immunoglobulin M isotype and reacted specifically with islet cells. In microcytotoxicity assays KT1 was shown to mediate complement-dependent lysis of approximately 30% of a rat insulinoma cell line and 25% of rat pancreatic islet cells in culture. It did not cause lysis of the other cell lines tested. KT1 has been demonstrated, by indirect immunofluorescence, to bind specifically to a cell surface antigen on live and acetone-fixed islet cell cultures from Wistar rat neonates and to rat insulinoma cells. Western blotting experiments revealed reaction to a 68-kDa protein from rat insulinoma cell extracts. This monoclonal antibody may have clinical relevance as it exhibits properties similar to the islet cell surface antibodies present in the sera of BB rats.

Effects of Glucagonlike Peptide l-(7–36) on Release of Insulin, Glucagon, and Somatostatin by Rat Pancreatic Islet Cell Monolayer Cultures

Glucagonlike peptide I (GLP-I-(7-36] is cleaved from proglucagon in ileal epithelial cells and increases in human plasma after nutrient ingestion. This peptide has been shown to stimulate insulin secretion in vitro and in vivo and thus potentially acts as an incretin. To characterize its action on islet cells, the release of insulin, glucagon, and somatostatin by rat pancreatic islet monolayer cultures at varying concentrations of GLP-I-(7-36) was measured. The interaction of GLP-I-(7-36) with nutrient substrates was assessed by adding amino acids and differing glucose concentrations to the cultures. Islet cell cultures (n = 5) were incubated for 1 h in medium containing 1.67 or 16.7 mM glucose or 1.67 mM glucose supplemented with amino acids and GLP-I-(7-36) at 10(-13)-10(-7) M. Hormone release was compared with control cultures containing no GLP-I-(7-36); 1.67-16.7 mM glucose with and without GLP-I-(7-36) at 10(-11) M; and 1.67, 3.3, 8.3, or 11.1 mM glucose alone or supplemented with amino acids, GLP-I-(7-36) 10(-11) M, or both amino acids and GLP-I-(7-36). In medium with 1.67 or 16.7 mM glucose or 1.67 mM glucose and amino acids, GLP-I-(7-36) increased insulin secretion two- to threefold over control at concentrations of 10(-9), 10(-11), and 10(-12) M, respectively. In medium with increasing concentrations of glucose, GLP-I-(7-36) at 10(-11) M significantly increased insulin secretion at glucose concentrations greater than or equal to 3.34 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of glucagonlike peptide I-(7-36) on release of insulin, glucagon, and somatostatin by rat pancreatic islet cell monolayer cultures

Effects of glucagonlike peptide I-(7-36) on release of insulin, glucagon, and somatostatin by rat pancreatic islet cell monolayer cultures