Magainin-AM2 improves glucose homeostasis and beta cell function in high-fat fed mice.

BACKGROUND: Magainin-AM2, a previously described amphibian host-defense peptide, stimulates insulin- and glucagon-like peptide-1-release in vitro. This study investigated anti-diabetic effects of the peptide in mice with diet-induced obesity and glucose intolerance. METHODS: Male National Institute of Health Swiss mice were maintained on a high-fat diet for 12-weeks prior to the daily treatment with magainin-AM2. Various indices of glucose tolerance were monitored together with insulin secretory responsiveness of islets at conclusion of study. RESULTS: Following twice daily treatment with magainin-AM2 for 15 days, no significant difference in body weight and food intake was observed compared with saline-treated high fat control animals. However, non-fasting blood glucose was significantly (P<0.05) decreased while plasma insulin concentrations were significantly (P<0.05) increased. Oral and intraperitoneal glucose tolerance and insulin secretion following glucose administration via both routes were significantly (P<0.05) enhanced. The peptide significantly (P<0.001) improved insulin sensitivity as well as the beta cell responses of islets isolated from treated mice to a range of insulin secretagogues. Oxygen consumption, CO2production, respiratory exchange ratio and energy expenditure were not significantly altered by sub-chronic administration of magainin-AM2 but a significant (P<0.05) reduction in fat deposition was observed. CONCLUSION: These results indicate that magainin-AM2 improves glucose tolerance, insulin sensitivity and islet beta cells secretory responsiveness in mice with obesity-diabetes. GENERAL SIGNIFICANCE: The activity of magainin-AM2 suggests the possibility of exploiting this peptide for treatment of type 2 diabetes.

Evaluation of the insulin-releasing and glucose-lowering effects of GPR120 activation in pancreatic ß-cells.

AIMS: To assess the potency and selectivity of various GPR120 agonists and to determine the cellular localization of GPR120 in clonal ß-cells and pancreatic islets. METHODS: Insulin secretion and alterations in intracellular Ca(2+) and cAMP response to glucose and GPR120 agonists, including endogenous agonists α-linolenic acid (ALA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and a synthetic analogue (GW-9508), were examined using clonal pancreatic BRIN-BD11 cells, mouse pancreatic islets and in vivo studies using NIH Swiss mice. Cytotoxicity was assessed by lactate dehydrogenase release. Cellular localization of GPR120 was explored by double-staining immunohistochemistry. RESULTS: The most potent and selective GPR120 agonist tested was ALA (half maximum effective concentration 1.2 × 10(-8) mol/l) with a maximum stimulation of insulin secretion of 53% at 10(-4) mol/l (p < 0.001) in BRIN-BD11 cells. Stimulation of insulin secretion was also observed with GW-9508 (6.4 × 10(-8) mol/l; 47%), EPA (7.9 × 10(-8) mol/l; 36%) and DHA (1.0 × 10(-7) mol/l; 50%). Results were corroborated by islet studies, with no evidence of cytotoxic effects. Dose-dependent insulin secretion by GPR120 agonists was glucose-sensitive and accompanied by significant elevations of intracellular Ca(2+) and cAMP. Immunocytochemistry showed GPR120 expression on BRIN-BD11 cells and was confined to islet ß-cells with no distribution on α-cells. Administration of GPR120 agonists (0.1 µmol/kg body weight) in glucose tolerance studies significantly reduced plasma glucose and augmented insulin release in mice. CONCLUSIONS: These results indicate that GPR120 is expressed on pancreatic ß-cells and that agonists at this receptor are potent insulin secretagogues with therapeutic potential for type 2 diabetes.

Evidence for inhibitory autocrine effects of proinsulin C-peptide on pancreatic ß-cell function and insulin secretion.

AIMS: Autocrine and paracrine regulatory mechanisms ensure integrated secretion of islet hormones that respond efficiently to changes in metabolic need. As proinsulin C-peptide exerts various biological effects and binds to cell membranes including insulin-secreting ß cells, its physiological role in insulin release was examined. METHODS: Insulin releasing activity of human and rat C-peptides were studied in the clonal pancreatic cell line, BRIN-BD11, with findings substantiated using isolated islets and in vivo studies employing SWISS TO mice. RESULTS: Acute exposure of clonal ß cells to human C-peptide resulted in concentration-dependent inhibitory effects on insulin secretion at 5.6 mM (p < 0.05-p < 0.001) and 16.7 mM (p < 0.01-p < 0.001) glucose. At physiologically relevant intra-islet concentrations (10(-9) -10(-6) M), C-peptide suppressed the insulin-secretory responses to a range of secretagogues acting at different points in the ß cell stimulus-secretion coupling pathway including alanine (p < 0.05), Ca(2+) (p < 0.001), arginine (p < 0.05), tolbutamide (p < 0.001), glucagon-like peptide 1 (GLP-1) (p < 0.001), isobutylmethylxanthine (IBMX) (p < 0.01) and KCl (p < 0.05). Similar results were obtained using isolated mouse pancreatic islets. Human C-peptide (3 × 10(-7) M, p < 0.001), somatostatin-14 (3 × 10(-7) M, p < 0.01) and diazoxide (300 µM, p < 0.001) reduced both alanine and glucose-stimulated insulin release by 43, 25 and 48%, respectively. The effects of human C-peptide were reproduced using rat C-peptide I and II. C-peptide also inhibited in vivo glucose-stimulated insulin release and impaired glucose tolerance in mice. CONCLUSIONS: C-peptide is a biologically active endogenous peptide hormone that exerts inhibitory autocrine effects on pancreatic ß-cell function. Mechanisms involving the activation of K(+) channels and a distal effect downstream of increased cytoplasmic Ca(2+) appear to be implicated in the inhibition of insulin secretion.

Evaluation of the insulin releasing and antihyperglycaemic activities of GPR55 lipid agonists using clonal beta-cells, isolated pancreatic islets and mice.

BACKGROUND AND PURPOSE: G-protein coupled receptor (GPR)55 is a novel lipid sensing receptor activated by both cannabinoid endogenous ligands (endocannabinoids) and other non-cannabinoid lipid transmitters. This study assessed the effects of various GPR55 agonists on glucose homeostasis. EXPERIMENTAL APPROACH: Insulin secretion and changes in intracellular Ca(2) (+) and cAMP in response to glucose and a range of GPR55 agonists [endogenous ligands (OEA, PEA), chemically synthetic cannabidiol (CBD) analogues (Abn-CBD, 0-1602), an analogue of rimonabant (AM-251) and antagonist (CBD)] were investigated in clonal BRIN-BD11 cells and mouse pancreatic islets. Cytotoxicity was assessed by LDH release, cellular localization by double-staining immunohistochemistry and in vivo effects assessed in mice. KEY RESULTS: The most potent and selective GPR55 agonist was the synthetic CBD analogue, Abn-CBD (pEC50 10.33), maximum stimulation of 67% at 10(-4) mol·L(-1) (P < 0.001) in BRIN-BD11 cells. AM-251 (pEC50 7.0), OEA (pEC50 7.0), 0-1602 (pEC50 7.3) and PEA (pEC50 6.0) stimulated insulin secretion. Results were corroborated by islet studies, with no cytotoxic effects. Concentration-dependent insulin secretion by GPR55 agonists was glucose-sensitive and accompanied by elevations of [Ca(2) (+) ]i (P < 0.01-P < 0.001) and cAMP (P < 0.05-P < 0.01). GPR55 agonists exhibited insulinotropic and glucose lowering activity in vivo. GPR55 was expressed on BRIN-BD11 cells and confined to islet beta cells with no distribution on alpha cells. CONCLUSION AND IMPLICATIONS: These results demonstrate GPR55 is distributed in pancreatic beta cells and is a strong activator of insulin secretion, with glucose-lowering effects in vivo. Development of agents agonizing the GPR55 receptor may have therapeutic potential in the treatment of type 2 diabetes.

Frog skin peptides (tigerinin-1R, magainin-AM1, -AM2, CPF-AM1, and PGla-AM1) stimulate secretion of glucagon-like peptide 1 (GLP-1) by GLUTag cells.

Skin secretions of several frog species contain host-defense peptides with multiple biological activities including in vitro and in vivo insulin-releasing actions. This study investigates the effects of tigerinin-1R from Hoplobatrachus rugulosus (Dicroglossidae) and magainin-AM1, magainin-AM2, caerulein precursor fragment (CPF-AM1) and peptide glycine leucine amide (PGLa-AM1) from Xenopus amieti (Pipidae) on GLP-1 secretion from GLUTag cells. Tigerinin-1R showed the highest potency producing a significant (P<0.05) increase in GLP-1 release at a concentration of 0.1nM for the cyclic peptide and 0.3nM for the reduced form. All peptides from X. amieti significantly (P<0.05) stimulated GLP-1 release at concentrations ⩾300nM with magainin-AM2 exhibiting the greatest potency (minimum concentration producing a significant stimulation=1nM). The maximum stimulatory response (3.2-fold of basal rate, P<0.001) was produced by CPF-AM1 at a concentration of 3µM. No peptide stimulated release of the cytosolic enzyme, lactate dehydrogenase from GLUTag cells at concentrations up to 3µM indicating that the integrity of the plasma membrane had been preserved. The data indicate that frog skin peptides, by stimulating GLP-1 release as well as direct effects on insulin secretion, show therapeutic potential as agents for the treatment of type 2 diabetes.

Tigerinin-1R: a potent, non-toxic insulin-releasing peptide isolated from the skin of the Asian frog, Hoplobatrachus rugulosus.

AIM: Characterization of peptides in the skin of the Vietnamese common lowland frog Hoplobatrachus rugulosus with the ability to stimulate insulin release in vitro and improve glucose tolerance in vivo. METHODS: Peptides in an extract of skin were purified by reversed-phase HPLC, and their abilities to stimulate the release of insulin and the cytosolic enzyme lactate dehydrogenase were determined using BRIN-BD11 clonal ß cells. Insulin-releasing potencies of synthetic peptides and their effects on membrane potential and intracellular Ca²âº concentration were also measured using BRIN-BD11 cells. Effects on glucose tolerance and insulin release in vivo were determined in mice fed a high-fat diet to induce obesity and insulin resistance. RESULTS: A cyclic dodecapeptide (RVCSAIPLPICH.NH2), termed tigerinin-1R, was isolated from the skin extract that lacked short-term cytotoxic and haemolytic activity but significantly (p < 0.01) stimulated the rate of release of insulin from BRIN-BD11 cells at concentrations ≥ 0.1 nM. The maximum response was 405% of the basal rate at 5.6 mM ambient glucose concentration and 290% of basal rate at 16.7 mM glucose. C-terminal α-amidation was necessary for high potency and a possible mechanism of action of the peptide-involved membrane depolarization and an increase in intracellular Ca²âº concentration. Administration of tigerinin-1R (75 nmol/kg body weight) to high fat-fed mice significantly (p < 0.05) enhanced insulin release and improved glucose tolerance during the 60-min period following an intraperitoneal glucose load. CONCLUSION: Tigerinin-1R is a potent, non-toxic insulin-releasing peptide that shows potential for development into an agent for the treatment of type 2 diabetes.

Brevinin-2-related peptide and its [D4K] analogue stimulate insulin release in vitro and improve glucose tolerance in mice fed a high fat diet.

The cationic, alpha-helical frog skin antimicrobial peptide B2RP (brevinin-2-related peptide) shows sequence similarity to antimicrobial peptides belonging to the brevinin-2 family, but lacks the C-terminal cyclic heptapeptide domain (Cys-Lys-Xaa (4)-Cys). Synthetic B2RP produced a significant (p<0.05) stimulation of insulin release (148% of basal rate at a concentration of 1 muM with a maximum response of 222% of basal rate at a concentration of 3 muM) from BRIN-BD11 clonal beta-cells without increasing the release of the cytosolic enzyme, lactate dehydrogenase. Increasing cationicity of B2RP while maintaining amphipathicity by the substitution Asp (4) --> Lys enhanced the insulin-releasing potency (137% of basal rate at a concentration of 0.3 muM; p<0.05) with no stimulation of lactate dehydrogenase release. In contrast, the L18K, and D4K, L18K analogues were toxic to the cells, and the K16A analogue, with increased amphipathicity and hydrophobicity, showed reduced potency. Administration of [D4K]B2RP (100 nmol/kg body weight) to mice fed a high fat diet to induce obesity and insulin-resistance significantly (p<0.05) enhanced insulin release and improved glucose tolerance during the 60-minute period following an intraperitoneal glucose load (18 mmol/kg body weight). B2RP shows potential for development into an agent for the treatment of type 2 diabetes.

Soluble dietary fibre fraction of Trigonella foenum-graecum (fenugreek) seed improves glucose homeostasis in animal models of type 1 and type 2 diabetes by delaying carbohydrate digestion and absorption, and enhancing insulin action.

Trigonella foenum-graecum (fenugreek) seeds have been documented as a traditional plant treatment for diabetes. In the present study, the antidiabetic properties of a soluble dietary fibre (SDF) fraction of T. foenum-graecum were evaluated. Administration of SDF fraction (0 x 5 g/kg body weight) to normal, type 1 or type 2 diabetic rats significantly improved oral glucose tolerance. Total remaining unabsorbed sucrose in the gastrointestinal tract of non-diabetic and type 2 diabetic rats, following oral sucrose loading (2 x 5 g/kg body weight) was significantly increased by T. foenum-graecum (0 x 5 g/kg body weight). The SDF fraction suppressed the elevation of blood glucose after oral sucrose ingestion in both non-diabetic and type 2 diabetic rats. Intestinal disaccharidase activity and glucose absorption were decreased and gastrointestinal motility increased by the SDF fraction. Daily oral administration of SDF to type 2 diabetic rats for 28 d decreased serum glucose, increased liver glycogen content and enhanced total antioxidant status. Serum insulin and insulin secretion were not affected by the SDF fraction. Glucose transport in 3T3-L1 adipocytes and insulin action were increased by T. foenum-graecum. The present findings indicate that the SDF fraction of T. foenum-graecum seeds exerts antidiabetic effects mediated through inhibition of carbohydrate digestion and absorption, and enhancement of peripheral insulin action.

Aqueous extracts of husks of Plantago ovata reduce hyperglycaemia in type 1 and type 2 diabetes by inhibition of intestinal glucose absorption.

Plantago ovata has been reported to reduce postprandial glucose concentrations in diabetic patients. In the present study, the efficacy and possible modes of action of hot-water extracts of husk of P. ovata were evaluated. The administration of P. ovata (0.5 g/kg body weight) significantly improved glucose tolerance in normal, type 1 and type 2 diabetic rat models. When the extract was administered orally with sucrose solution, it suppressed postprandial blood glucose and retarded small intestinal absorption without inducing the influx of sucrose into the large intestine. The extract significantly reduced glucose absorption in the gut during in situ perfusion of small intestine in non-diabetic rats. In 28 d chronic feeding studies in type 2 diabetic rat models, the extract reduced serum atherogenic lipids and NEFA but had no effect on plasma insulin and total antioxidant status. No effect of the extract was evident on intestinal disaccharidase activity. Furthermore, the extract did not stimulate insulin secretion in perfused rat pancreas, isolated rat islets or clonal beta cells. Neither did the extract affect glucose transport in 3T3 adipocytes. In conclusion, aqueous extracts of P. ovata reduce hyperglycaemia in diabetes via inhibition of intestinal glucose absorption and enhancement of motility. These attributes indicate that P. ovata may be a useful source of active components to provide new opportunities for diabetes therapy.

Ocimum sanctum leaf extracts stimulate insulin secretion from perfused pancreas, isolated islets and clonal pancreatic beta-cells.

Ocimum sanctum leaves have previously been reported to reduce blood glucose when administered to rats and humans with diabetes. In the present study, the effects of ethanol extract and five partition fractions of O. sanctum leaves were studied on insulin secretion together with an evaluation of their mechanisms of action. The ethanol extract and each of the aqueous, butanol and ethylacetate fractions stimulated insulin secretion from perfused rat pancreas, isolated rat islets and a clonal rat beta-cell line in a concentration-dependent manner. The stimulatory effects of ethanol extract and each of these partition fractions were potentiated by glucose, isobutylmethylxanthine, tolbutamide and a depolarizing concentration of KCl. Inhibition of the secretory effect was observed with diazoxide, verapamil and Ca2+ removal. In contrast, the stimulatory effects of the chloroform and hexane partition fractions were associated with decreased cell viability and were unaltered by diazoxide and verapamil. The ethanol extract and the five fractions increased intracellular Ca2+ in clonal BRIN-BD11 cells, being partly attenuated by the addition of verapamil. These findings indicated that constituents of O. sanctum leaf extracts have stimulatory effects on physiological pathways of insulin secretion which may underlie its reported antidiabetic action.

Skin secretions of Rana saharica frogs reveal antimicrobial peptides esculentins-1 and -1B and brevinins-1E and -2EC with novel insulin releasing activity.

Skin secretions of Rana saharica were evaluated for the isolation and characterisation of novel insulinotropic peptides. Crude secretions obtained from young adult frogs by mild electrical stimulation of the dorsal skin surface were purified by reverse phase HPLC yielding 80 fractions. In acute 20-min incubations with glucose responsive BRIN-BD11 cells, fractions 36-43, 46-54 and 57-63 significantly stimulated insulin release by 2- to 8-fold compared with 5.6 mM glucose alone. Pooled fractions in the latter two bands were rechromatographed to reveal 9 homogenous peaks, which elicited significant 1.3- to 3.5-fold increases in insulin release (P < 0.05). Structural analysis of the most potent non-toxic peptides was performed by mass spectrometry and automated Edman degradation. This revealed four major insulin-releasing peaks with molecular masses of 2,676.9 Da, 3,519.3 Da, 4,920.4 Da and 4,801.2 Da respectively. These peptides were found to be identical to brevinin-1E, brevinin-2EC, esculentin-1 and esculentin-1B, which belong to the group of antimicrobial peptides isolated from skin secretions of various Rana frog species. Preliminary studies on the mechanism underlying the insulinotropic actions of esculentins-1 and -1B suggested possible involvement of both cyclic AMP-protein kinase A and -C-dependent G-protein sensitive pathways. These data indicate that the skin secretions of Rana saharica frogs contain bioactive molecules with significant insulin-releasing activity. Relatives of the brevinin/esculentin peptide family merit further investigation as novel insulin secretagogues.

Characterization of naturally occurring peptides in the skin secretion of Rana pipiens frog reveal pipinin-1 as the novel insulin-releasing agent.

Naturally occurring insulinotropic peptides were isolated from the skin secretions of Rana pipiens frogs. Crude secretions (50 mg; 5-10 frogs) obtained by mild electrical stimulation of the dorsal skin surface were purified by reversed-phase high-performance liquid chromatography (HPLC) yielding 80 fractions. In acute incubations with glucose-responsive BRIN-BD11 cells, fractions 40-47 (band 1) and fractions 60-65 (band 2) showed significant 1.7-6.7-fold increases in insulin-releasing activity (P < 0.001) compared with 5.6 mm glucose alone. Pooled fractions in bands 1 and 2 were rechromatographed yielding a total of seven peaks capable of subsequent 1.2-1.8-fold stimulation of insulin release. Final purification by HPLC to single homogenous peaks revealed one prominent peptide (peak 4.1) with insulin-releasing activity which lacked effects on cell viability. Electrospray mass spectrometric analysis of this peptide indicated molecular mass of 2562.6 Da. Determination of the primary amino acid sequence of this peptide revealed a 24-amino acid sequence: FLPIIAGVAAKVFPKIFCAISKKC. Database search showed a 100% homology to histamine-releasing pipinin-1. In conclusion, this study revealed the skin secretions of Rana pipiens to be a rich source of insulin-releasing peptides. The discovery of insulinotropic activity for pipinin-1, initially characterized as an antimicrobial is interesting and merits further investigation.

Novel insulin-releasing peptides in the skin of Phyllomedusa trinitatis frog include 28 amino acid peptide from dermaseptin BIV precursor.

OBJECTIVE: The granular glands of amphibians have long been known to produce many biologically active compounds. The aim of this study was to isolate and characterize insulinotropic peptides from the skin of Phyllomedusa trinitatis frog. METHODS AND RESULTS: Crude secretions obtained by mild electrical stimulation of the dorsal skin surface were purified by reverse phase HPLC yielding 80 fractions. In acute incubations with glucose-responsive BRIN-BD11 cells, fractions 39-40 (band 1) and fractions 43-46 (band 2) significantly stimulated insulin release by 1.5 to 2.5-fold. Pooled fractions in bands 1 and 2 were rechromatographed to 4 homogeneous peaks, each with insulin-releasing activity. Mass spectrometry analysis was successfully completed for 3 peptides, indicating 2996.4, 3379.9, and 8326.4 Da. The sequence of the 2996.4 Da peptide was determined as ALWKDILKNVGKAAGKAVLNTVTDMVNQ. This 28-amino-acid peptide has 100% homology with the C-terminal of the 75-amino-acid dermaseptin BIV precursor of a family of structurally related antimicrobial peptides in the skin of the Phyllomedusinae subfamily. CONCLUSION: These data demonstrate that the defensive skin secretions of P. trinitatis contain biologically active peptides, which may have mammalian counterparts and merit further investigation as insulin secretagogues.

Isolation and characterisation of an unexpected class of insulinotropic peptides in the skin of the frog Agalychnis litodryas.

Skin secretions of the frog Agalychnis litodryas were evaluated for the isolation and characterisation of novel insulinotropic peptides. Crude secretions obtained from young adult frogs by mild electrical stimulation of the dorsal skin surface were purified by reverse-phase high-performance liquid chromatography (HPLC) yielding 70 fractions. In acute 20-min incubations with glucose responsive BRIN-BD11 cells, fractions 39-42 (band 1) and fractions 44-46 (band 2) significantly stimulated insulin release by 2-3.5-fold compared with 5.6 mM glucose alone. Pooled fractions in band 1 and band 2 were rechromatographed to reveal 20 homogenous peptide peaks, which elicited significant 1.5-4-fold increases in insulin release. Mass spectrometry analyses indicated molecular masses of between 1649.2 and 4988.9 Da. The two peptides with the greatest insulin-releasing activity were directly subjected to N-terminal amino acid sequence analysis. The sequence of the 3020 Da peptide, called frog skin insulinotropic peptide or FSIP, was determined as AVWKDFLKNIGKAAGKAVLNSVTDMVNE, which has 79% homology with the C-terminal of the 75 amino acid dermaseptin BIV precursor. A partial N-terminal sequence was determined for the 2546.2 Da peptide as MLADVFEKIMGD... These data indicate that the skin secretions of A. litodryas frogs contain biologically active peptides which merit further evaluation as a new class of insulin secretagogues.

Brevinin-1 and multiple insulin-releasing peptides in the skin of the frog Rana palustris.

Few studies have comprehensively examined amphibian granular gland secretions for novel insulinotropic peptides. This study involved isolation and characterisation of biologically active peptides from the skin secretions of Rana palustris frogs. Crude secretions obtained by mild electrical stimulation from the dorsal skin surface were purified by reversed-phase HPLC on a semipreparative Vydac C18 column, yielding 80 fractions. These fractions were assayed for insulin-releasing activity using glucose-responsive BRIN-BD11 cells. Acute 20 min incubations were performed in Krebs Ringer bicarbonate buffer supplemented with 5.6 mmol/l glucose in the absence (control) and presence of various fractions. Fractions 29-54 and fractions 68-75 showed significant 2.0-6.5-fold increases in insulin-releasing activity (P<0.001). The fractions showing most prominent insulinotropic activity were further purified to single homogeneous peaks, which, on testing, evoked 1.5-2.8-fold increases in insulin release (P<0.001). The structures of the purified peptides were determined by mass spectrometry and N-terminal amino acid sequencing. Electrospray ionisation ion-trap mass spectrometry analysis revealed molecular masses of 2873.5-8560.4 Da. Sufficient material was isolated to determine the primary amino acid sequence of the 2873.5 Da peptide, revealing a 27 amino acid sequence, ALSILRGLEKLAKMGIALTNCKATKKC, repressing palustrin-1c. The database search for this peptide showed a 48% homology with brevinin-1, an antimicrobial peptide isolated from various Rana species, which itself stimulated insulin release from BRIN-BD11 cells in a concentration-dependent manner. In conclusion, the skin secretions of R. palustris frogs contain a novel class of peptides with insulin-releasing activity that merit further investigation.

Evidence for a sustained increase in clonal beta-cell basal intracellular Ca2+ levels after incubation in the presence of newly diagnosed Type-1 diabetic patient sera. Possible role in serum-induced inhibition of insulin secretion.

We have previously reported that newly diagnosed Type-1 diabetic patient sera potently suppressed insulin secretion from a clonal rat pancreatic beta-cell line (BRIN BD11) but did not alter cell viability. Here, we report that apoptosis in BRIN BD11 cells incubated in various sera types (fetal calf serum (FCS), normal human serum and Type-1 diabetic patient) was virtually undetectable. Although low levels of necrosis were detected, these were not significantly different between cells incubated in sera from different sources. ATP levels were reduced by approximately 30% while nitrite production increased twofold from BRIN BD11 cells incubated for 24 h in the presence of Type-1 diabetic patient sera compared with normal human sera. Additionally, ATP levels were reduced by approximately 40% and DNA fragmentation increased by more than 20-fold in BRIN BD11 cells incubated in FCS in the presence of a pro-inflammatory cytokine cocktail (interleukin-1beta, tumour necrosis factor-alpha and interferon-gamma), compared with cells incubated in the absence of cytokines. Nitric oxide production from BRIN BD11 cells was markedly increased (up to 10-fold) irrespective of sera type when the cytokine cocktail was included in the incubation medium. Type-1 diabetic patient sera significantly (P<0.001) raised basal levels of intracellular free Ca(2+ )concentration ([Ca(2+)](i)) in BRIN BD11 cells after a 24-h incubation. The alteration in [Ca(2+)](i) concentration was complement dependent, as removal of the early complement components C1q and C3 resulted in a significant reduction (P<0.01) of sera-induced [Ca(2+)](i )changes. We propose that the mechanism of Type-1 diabetic patient sera-induced inhibition of insulin secretion from clonal beta-cells may involve complement-stimulated elevation of [Ca(2+)](i) which attenuates the nutrient-induced insulin secretory process possibly by desensitizing the cell to further changes in Ca(2+).

Secretion of glycated insulin from pancreatic beta-cells in diabetes represents a novel aspect of beta-cell dysfunction and glucose toxicity.

Hyperglycaemia, a significant pathophysiological state in diabetes mellitus, may contribute to defective pancreatic beta-cell function, secretion and action of insulin through glycation of important regulatory proteins. This paper highlights recent data supporting the concept that pancreatic beta-cell dysfunction is associated with increased glycation of functional proteins. The pancreatic beta-cell provides a highly favourable environment for the intracellular glycation of insulin which is a relatively rapid, glucose-dependent process. Using a novel radioimmunoassay and immunocytochemical techniques, glycated insulin has been shown to be stored and secreted from pancreatic beta-cells in both human and animal models of diabetes. Glycated insulin represents a significant proportion of total circulating insulin in type 2 diabetes and may have impaired metabolic clearance compared with native insulin. Since glycation of insulin disturbs normal cellular function and results in decreased biological activity, it may play a significant contributory role in the insulin resistance and glucose intolerance of type 2 diabetes. Further studies are necessary to evaluate the possible significance of glycated insulin in both the pathophysiology of diabetes and future therapeutic approaches.

Receptors and ligands for autocrine growth pathways are up-regulated when pancreatic cancer cells are adapted to serum-free culture.

Overexpression of autocrine growth factors and their receptors has been reported in many human cancers. The study of autocrine-regulated pathways using in vitro culture systems can be hindered by the presence of fetal bovine serum in culture medium. A human pancreatic cancer cell line (HPAF) was slowly weaned from its dependence on fetal bovine serum and subsequently maintained in serum-free conditions. Growth factor secretion studies showed that production of autocrine growth factors such as transforming growth factor alpha, gastrin-releasing peptide, and insulin-like growth factor I from weaned cells increased three times compared with nonweaned cells (p < 0.01). The epidermal growth factor and gastrin-releasing peptide receptor densities were also increased in weaned cells (2 times and 2.5 times, respectively, p < 0.05). The proliferation of weaned cells cultured continuously in the same medium was significantly greater than of nonweaned cells (p < 0.05). Collectively, these data indicate that weaned pancreatic cancer cells can proliferate in the absence of serum by up-regulating autocrine pathways.

Functional enhancement of electrofusion-derived BRIN-BD11 insulin-secreting cells after implantation into diabetic mice.

Electrofusion-derived BRIN-BD11 cells are glucose-sensitive insulin-secreting cells which provide an archetypal bioengineered surrogate beta-cell for insulin replacement therapy in diabetes mellitus. 5x10(6) BRIN-BD11 cells were implanted intraperitoneally into severely hyperglycaemic (>24 mmol/l) streptozotocin-induced insulin-treated diabetic athymic nude (nu/nu) mice. The implants reduced hyperglycaemia such that insulin injections were discontinued by 5-16 days (<17 mmol/l) and normoglycaemia (<9 mmol/l) was achieved by 7-20 days. Implanted cells were removed after 28 days and re-established in culture. After re-culture for 20 days, glucose-stimulated (16.7 mmol/l) insulin release was enhanced by 121% (p<0.001) compared to non-implanted cells. Insulin responses to glucagon-like peptide-1 (10(-9) mol/l), cholecystokinin-8 (10(-8) mol/l) and L-alanine (10 mmol/l) were increased by 32%, 31% and 68% respectively (p<0.05-0.01). Insulin content of the cells was 148% greater at 20 days after re-culture than before implantation (p<0.001), but basal insulin release (at 5.6 mmol/l glucose) was not changed. After re-culture for 40 days, insulin content declined to 68% of the content before implantation (p<0.01), although basal insulin release was unchanged. However, the insulin secretory responses to glucose, glucagon-like peptide-1, cholecystokinin-8 and L-alanine were decreased after 40 days of re-culture to 65%, 72%, 73% and 42% respectively of the values before implantation (p<0.05-0.01). The functional enhancement of electrofusion-derived surrogate beta-cells that were re-cultured for 20 days after implantation and restoration of normoglycaemia indicates that the in vivo environment could greatly assist beta-cell engineering approaches to therapy for diabetes.

Detection of glycated gastric inhibitory polypeptide within the intestines of diabetic obese (ob/ob) mice.

Gastric inhibitory polypeptide (GIP) is produced within endocrine cells of the small intestine and released into the circulation upon nutrient ingestion. This study has quantified the levels of this insulinotropic peptide in the intestines of lean and diabetic obese ob/ob mice and estimated the proportion that is glycated. The total intestinal GIP concentration and content of the diabetic mice were significantly greater (p < 0.01) than that of control animals. Affinity chromatographic separation and side-viewing GIP radioimmunoassay demonstrated that approx 20% of the GIP extracted from intestines of ob/ob mice was present in glycated form. Less than 2% of intestinal GIP was glycated in lean mice. In conclusion substantial quantities of glycated GIP exist within the intestines of diabetic ob/ob mice, suggesting that this may be a contributing factor to the physiological disarray of this syndrome.