A local glucagon-like peptide 1 (GLP-1) system in human pancreatic islets.

AIMS/HYPOTHESIS: Glucagon-like peptide 1 (GLP-1) is a major incretin, mainly produced by the intestinal L cells, with beneficial actions on pancreatic beta cells. However, while in vivo only very small amounts of GLP-1 reach the pancreas in bioactive form, some observations indicate that GLP-1 may also be produced in the islets. We performed comprehensive morphological, functional and molecular studies to evaluate the presence and various features of a local GLP-1 system in human pancreatic islet cells, including those from type 2 diabetic patients. METHODS: The presence of insulin, glucagon, GLP-1, proconvertase (PC) 1/3 and PC2 was determined in human pancreas by immunohistochemistry with confocal microscopy. Islets were isolated from non-diabetic and type 2 diabetic donors. GLP-1 protein abundance was evaluated by immunoblotting and matrix-assisted laser desorption-ionisation-time of flight (MALDI-TOF) mass spectrometry. Single alpha and beta cell suspensions were obtained by enzymatic dissociation and FACS sorting. Glucagon and GLP-1 release were measured in response to nutrients. RESULTS: Confocal microscopy showed the presence of GLP-1-like and PC1/3 immunoreactivity in subsets of alpha cells, whereas GLP-1 was not observed in beta cells. The presence of GLP-1 in isolated islets was confirmed by immunoblotting, followed by mass spectrometry. Isolated islets and alpha (but not beta) cell fractions released GLP-1, which was regulated by glucose and arginine. PC1/3 (also known as PCSK1) gene expression was shown in alpha cells. GLP-1 release was significantly higher from type 2 diabetic than from non-diabetic isolated islets. CONCLUSIONS/INTERPRETATION: We have shown the presence of a functionally competent GLP-1 system in human pancreatic islets, which resides in alpha cells and might be modulated by type 2 diabetes.

Evidence for a role of frataxin in pancreatic islets isolated from multi-organ donors with and without type 2 diabetes mellitus.

Frataxin (FXN) is a mitochondrial protein involved in iron metabolism and in the modulation of reactive oxygen and/or nitrogen species production. No information is currently available as for the role of frataxin in isolated human pancreatic islets. We studied islets from pancreases of multi-organ donors with (T2DM) and without (Ctrl) Type 2 diabetes mellitus. In these islets, we determined FXN gene and protein expression by qualitative and quantitative Real-Time RT-PCR, nitrotyrosine concentration, and insulin release in response to glucose stimulation (SI). FXN gene and protein were expressed in human islets, though the level of expression was much lower in T2DM islets. The latter also had lower insulin release and higher concentration of nitrotyrosine. A positive correlation was apparent between SI and FXN gene expression, while a negative correlation was found between nitrotyrosine islet concentration and FXN expression. Transfection of Ctrl islets with siRNA FXN caused reduction of FXN expression, increase of nitrotyrosine concentration, and reduction of insulin release. In conclusion, in human pancreatic islets FXN contributes to regulation of oxidative stress and insulin release in response to glucose. In islets from T2DM patients FXN expression is reduced while oxidative stress is increased and insulin release in response to glucose impaired.

Glucagon-like peptide-1 secretion in women with gestational diabetes mellitus during and after pregnancy.

UNLABELLED: Gestational diabetes mellitus (GDM) predisposes women to future development of Type 2 diabetes mellitus (DM2) and the two conditions share similar metabolic alterations. Recent observations suggest that a defective glucose stimulated insulin secretion by glucagon-like peptide-1 (GLP- 1) plays a role in the pathogenesis of DM2. Whether such a defect is impaired in GDM remains to be ascertained. AIM: We have determined GLP-1 secretion in response to oral glucose tolerance test (OGTT) in GDM and normal glucose tolerance (NGT) during and after pregnancy. MATERIALS AND METHODS: 100-g-3h OGTT was performed in 12 GDM and 16 NGT women at 27.3 ± 4.1 weeks of gestation, for determination of plasma GLP-1, glucose, insulin, and C-peptide. Insulin sensitivity (ISI) and insulin secretion (first and second phase); as well as ISI-secretion index (ISSI) were also derived. RESULTS: NGT and GDM women were comparable for age pre-pregnancy body mass index (BMI) and weight gain. GDM had higher glucose area under the curve (AUC): 27,575.5 ± 3448 vs 20,685.88 ± 2715 mg/dl min (p<0.01), but lower first-phase insulin secretion (993.12±367 vs 1376.61 ± 423, p<0.05) and ISSI compared to controls (3873.23 ± 1185 vs 6232.13 ± 1734, p<0.001). When we examined GLP-1 mean levels in relation to mean glycemic values, GLP-1 secretion was inappropriately low with respect to mean glycemic values in GDM compared to NGT. At follow-up, AUCGLP-1 was significantly lower in post-partum GDM compared to post-partum NGT women (2542 ± 273 vs 10,092 ± 7367 pmol·l-1·min-1, p<0.05, respectively). CONCLUSIONS: Our study suggests that GLP-1 secretion in GDM women is inadequate for the prevailing glycemic levels both in pregnancy and post partum. Moreover, we cannot exclude that other important aspects of the incretin effect may be involved in GDM development.

G-protein-coupled receptor 40 (GPR40) expression and its regulation in human pancreatic islets: the role of type 2 diabetes and fatty acids.

BACKGROUND AND AIMS: GPR40 is a membrane-bound receptor paired with medium and long-chain fatty acids (FFA) as endogenous ligands. Its acute activation potentiates insulin secretion from beta cells, whereas prolonged binding might contribute to the deleterious effects of chronic exposure to FFA. Little information is available on the expression of GPR40 and its regulation in human islets (HI). MATERIAL AND METHODS: HI were prepared by enzymatic digestion and gradient separation from the pancreas of 20 non-diabetic (Ctrl) and 13 type 2 diabetic (T2DM) multiorgan donors, and functional and molecular studies were then performed. RESULTS: By qualitative and quantitative PCR experiments, mRNA expression was shown in HI. Both in T2DM islets and in Ctrl islets pre-exposed for 24 h to 1.0 mmol/l FFA (palmitate:oleate, 2:1), GPR40 mRNA expression was significantly reduced (p<0.01) in the T2DM cells as compared to Ctrl cells. A significant positive correlation was found between glucose-stimulated insulin secretion and GPR40 expression. CONCLUSIONS: These results show the expression of GPR40 in human pancreatic islets which are regulated by FFA. The finding that T2DM islets have a lower GPR40 expression, and the correlation of these genes with insulin secretion, raises the possibility of an involvement of GPR40 in human diabetes beta-cell dysfunction.

Autophagy in human type 2 diabetes pancreatic beta cells.

AIMS/HYPOTHESIS: Beta cell loss contributes to type 2 diabetes, with increased apoptosis representing an underlying mechanism. Autophagy, i.e. the physiological degradation of damaged organelles and proteins, may, if altered, be associated with a distinct form of cell death. We studied several features of autophagy in beta cells from type 2 diabetic patients and assessed the role of metabolic perturbation and pharmacological intervention. METHODS: Pancreatic samples were obtained from organ donors and isolated islets prepared both by collagenase digestion and density gradient centrifugation. Beta cell morphology and morphometry were studied by electron microscopy. Gene expression studies were performed by quantitative RT-PCR. RESULTS: Using electron microscopy, we observed more dead beta cells in diabetic (2.24 +/- 0.53%) than control (0.66 +/- 0.52%) samples (p < 0.01). Massive vacuole overload (suggesting altered autophagy) was associated with 1.18 +/- 0.54% dead beta cells in type 2 diabetic samples and with 0.36 +/- 0.26% in control samples (p < 0.05). Density volume of autophagic vacuoles and autophagosomes was significantly higher in diabetic beta cells. Unchanged gene expression of beclin-1 and ATG1 (also known as ULK1), and reduced transcription of LAMP2 and cathepsin B and D was observed in type 2 diabetic islets. Exposure of non-diabetic islets to increased NEFA concentration led to a marked increase of vacuole accumulation, together with enhanced beta cell death, which was associated with decreased LAMP2 expression. Metformin ameliorated autophagy alterations in diabetic beta cells and beta cells exposed to NEFA, a process associated with normalisation of LAMP2 expression. CONCLUSIONS/INTERPRETATION: Beta cells in human type 2 diabetes have signs of altered autophagy, which may contribute to loss of beta cell mass. To preserve beta cell mass in diabetic patients, it may be necessary to target multiple cell-death pathways.

Thermal treatment reduces gliadin recognition by IgE, but a subsequent digestion and epithelial crossing permits recovery.

Wheat is one of the most important crops in the world in terms of human nutrition. With regards to health, some individuals exhibit wheat-related disorders such as food allergy to wheat (FAW). In this disorder, gluten is involved, particularly the gliadins which are among the main proteins responsible for FAW. Food processing, as well as digestibility and intestinal transport are key factors to consider since they may affect the allergenic potential of food allergens. Wheat is always consumed after heat processing and this step may impact epitope accessibility by inducing aggregation and may irreversibly destroy conformational epitopes. Our aim was to investigate the effects of heating and digestion on the structure of well-known allergens (total gliadins and α-gliadins) and their capacity to maintain their allergenic potential after crossing an intestinal barrier. The sizes of the processed (heated and heated/digested) proteins were characterized by laser light scattering and chromatographic reverse phase. The IgE-binding capacities of native and processed proteins were checked using a dot blot with sera from wheat allergenic patients. Furthermore, the abilities of these samples to cross the intestinal barrier and to induce mast cell degranulation were investigated by combining two in vitro cellular models, Caco-2 and RBL-SX38. The heat treatment of total gliadins and α-gliadins induced the production of large aggregates that were hardly recognized by IgE of patients in dot-blot. However, after limited pepsin hydrolysis, the epitopes were unmasked, and they were able to bind IgE again. Native proteins (gliadins and α-type) and processed forms were able to cross the Caco-2 cells in small amount. Permeability studies revealed the capacity of α-gliadins to increase paracellular permeability. In the RBL assay, the total native gliadins were able to trigger cell degranulation, but none of their processed forms. However after crossing the CaCo-2 monolayer, processed gliadins recovered their degranulation capacity to a certain extent. Total native gliadins remained the best allergenic form compared to α-type.

Effects of exendin-4 on islets from type 2 diabetes patients.

Exendin-4 is a dipeptidyl peptidase IV (DPP-IV)-resistant glucagon-like peptide 1 (GLP-1) mimetic and its synthetic counterpart, exenatide, is being used in the therapy of type 2 diabetes (T2DM). No information, however, is currently available as for the direct action of exendin-4 on human T2DM islets. In the present study, we exposed pancreatic islets prepared from non-diabetic and T2DM subjects to exendin-4 for 48 h and found that the compound had several, direct beneficial actions on insulin secretion and the expression of genes involved in beta-cell function and differentiation.

Beta-cell apoptosis in type 2 diabetes: quantitative and functional consequences.

Type 2 diabetes, the most common form of diabetes in humans, is characterized by impaired insulin secretion paralleled by a progressive decline in beta-cell function and chronic insulin resistance. Several authors have showed that in type 2 diabetes there is a reduction of islet and/or insulin-containing cell mass or volume. Regulation of the beta-cell mass appears to involve a balance of beta-cell replication and apoptosis but, at the molecular level, pancreatic beta-cell loss by apoptosis appears to play an important role in the development of insulin deficiency and the onset and/or progression of the disease. The mechanisms favoring apoptosis in type 2 diabetic pancreatic islets and new potential therapeutic approaches to prevent beta-cell death and maintain beta-cell mass are discussed.

Insulin secretion defects of human type 2 diabetic islets are corrected in vitro by a new reactive oxygen species scavenger.

Oxidative stress is a putative mechanism leading to beta-cell damage in type 2 diabetes. We studied isolated human pancreatic islets from type 2 diabetic and non-diabetic subjects, matched for age and body mass index. Evidence of increased oxidative stress in diabetic islets was demonstrated by measuring nitrotyrosine concentration and by electron paramagnetic resonance. This was accompanied by reduced glucose-stimulated insulin secretion, as compared to non-diabetic islets (Stimulation Index, SI: 0.9 +/- 0.2 vs. 2.0 +/- 0.4, P<0.01), and by altered expression of insulin (approximately -60%), catalase (approximately +90%) and glutathione peroxidase (approximately +140%). When type 2 diabetic islets were pre-exposed for 24 h to the new antioxidant bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate di-hydrochloride, nitrotyrosine levels, glucose-stimulated insulin secretion (SI: 1.6+/-0.5) and gene expressions improved/normalized. These results support the concept that oxidative stress may play a role in type 2 diabetes beta-cell dysfunction; furthermore, it is proposed that therapy with antioxidants could be an interesting adjunctive pharmacological approach to the treatment of type 2 diabetes.

Patterns of genomic instability in gastric cancer: clinical implications and perspectives.

In gastric cancer (GC) the loss of genomic stability represents a key molecular step that occurs early in the carcinogenesis process and creates a permissive environment for the accumulation of genetic and epigenetic alterations in tumor suppressor genes and oncogenes. It is widely accepted that GC can follow at least two major genomic instability pathways, microsatellite instability (MSI) and chromosome instability (CIN). MSI is responsible for a well-defined subset of GCs. CIN represents a more common pathway comprising heterogeneous subsets of GC. In addition to MSI and CIN, the CpG islands methylator phenotype (CIMP) plays an important role in gastric carcinogenesis. CIMP may lead to the transcriptional silencing of various genes in gastric carcinogenesis. Intriguingly, more recently in addition to CpG island hypermethylation, a global DNA demethylation, that precedes genomic damage, has been observed in GC. Thus, epigenetic alterations may play a relevant role in gastric carcinogenesis as alternative mechanisms. Evidence suggests that although MSI, CIN and CIMP phenotypes can be distinguished from one another, there might be some degree of overlap. This review describes our current knowledge of the instability pathways in gastric carcinogenesis and the potential clinical applications for different forms of genomic instability in GC.

The thermal aggregation of ovalbumin as large particles decreases its allergenicity for egg allergic patients and in a murine model.

Most egg-allergic children can tolerate extensively cooked eggs. Ovalbumin, a major allergen in egg whites, is prone to aggregate upon heating. This study compares ovalbumin's allergenicity when it is aggregated as large particles to ovalbumin in its native form. Immunoglobulins (Ig)-binding and the degranulation capacities of native and aggregated ovalbumin were measured with sera from egg-allergic children and from mice sensitized to native or aggregated ovalbumin. The influence of ovalbumin structure on Ig production upon sensitization and elicitation potency by challenge was also studied. We showed that heat aggregation of ovalbumin as large particles enhances IgG production and promotes IgG2a production (a shift toward the T helper 1 profile). Aggregated ovalbumin displayed lower Ig-binding and basophil-activation capacities for sera from both allergic patients and mice. This work illustrates the links between ovalbumin structure after heating and allergenicity potential using parameters from both the sensitization and elicitation phases of the allergic reaction.

Autoantibodies to CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) in Caucasian patients with diabetes: effects on insulin release from human islets.

The type II transmembrane glycoprotein CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) has been proposed as a mediator of insulin secretion from pancreatic beta-cells and as a candidate for autoimmune reactions in type 2 diabetes. We evaluated the presence of anti-CD38 autoantibodies in Caucasian patients with diabetes and investigated the effect of these antibodies on insulin secretion from isolated human pancreatic islets. The presence of anti-CD38 autoantibodies was evaluated by using Western blot analysis in 236 patients with type 2 diabetes (mean age 63 years), in 160 patients with type 1 diabetes (mean age 38 years), and in 159 nondiabetic subjects. Anti-CD38 autoantibody titers at least 3 SD above the mean value of the control group were found in 9.7% of type 2 diabetic patients and in 13.1% of type 1 diabetic patients (chi2 = 15.9, P = 0.0003 vs. 1.3% of control subjects). No significant differences were observed in sex distribution, current age, age at diabetes onset, BMI, fasting serum glucose, or glycemic control between anti-CD38+ and anti-CD38-diabetic patients in either the type 2 or type 1 diabetic groups. The effect of 23 anti-CD38- and 13 anti-CD38+ sera on insulin secretion at low (3.3 mmol/l) or high (16.7 mmol/l) medium glucose concentrations was evaluated in isolated human pancreatic islets. Data are medians (interquartile range). The anti-CD38+ sera potentiated insulin release both at low [95 (64) vs. 23 (12) microU/ml of control incubations, respectively, P < 0.0001] and high [271 (336) vs. a control of 55 (37) microU/ml, respectively, P = 0.001] medium glucose concentrations, whereas the anti-CD38- sera did not. Furthermore, in the pooled data from all 36 tested sera, insulin levels in the islet incubation medium were directly related to the anti-CD38 antibody titer. We conclude that autoantibodies to CD38 are associated with both type 1 and type 2 diabetes in Caucasian subjects. These autoantibodies exert a stimulatory effect on insulin secretion by cultured human islets. The role of this autoimmune reaction in the pathogenesis of diabetes remains to be elucidated.

Th2 cytokines have a partial, direct protective effect on the function and survival of isolated human islets exposed to combined proinflammatory and Th1 cytokines.

Studies in rodents have suggested that Th2 and Th3 cytokines can be effective in reducing proinflammatory and Th1 cytokine-induced islet damage. Whether this is the case with human islets and might be due to a direct action of Th2 and Th3 cytokines is not known. In the present study, we evaluated whether Th2 (500 U/ml IL-4 plus 100 U/ml IL-10) or Th3 (5 ng/ml TGF-1beta) cytokines may prevent the derangements induced on isolated human islets by prolonged (12 or 72 h) exposure to combined proinflammatory (50 U/ml IL-1beta, 1000 U/ml TNF alpha) and Th1 (1000 U/ml interferon gamma) cytokines. Compared with control islets, cells preincubated for 12 or 72 h with proinflammatory and Th1 cytokines showed a significant decrease of glucose-stimulated insulin secretion and a significant increase of nitrites production. The addition of IL-4 plus IL-10 or TGF-1beta in the medium prevented these cytostatic effects in the 12-h incubation experiments, but not after the 72-h exposure period. IL-1beta, interferon gamma, and TNF alpha caused no major change in either islet cell survival or Bcl-2 and Bax mRNA expression after a 12-h incubation; however, a marked increase in the amount of dead cells, with a major decrease of Bcl-2 mRNA expression, was observed after 72 h. The presence of Th2, but not of Th3, cytokines significantly reduced beta-cell death, without any major effect on Bcl-2 and Bax mRNA expression. These results suggest that Th2 and (at lower extent) Th3 cytokines may have a partial, direct protective effect on isolated human islets exposed to the cytostatic and cytotoxic action of proinflammatory and Th1 cytokines.

Bovine islets are less susceptible than human islets to damage by human cytokines.

BACKGROUND: The potential benefits of islet xenografting in type 1 diabetes include the intriguing, but still unanswered, possibility that the grafted xenoislets may be less subjected to human autoimmune attack. Cytokines may play a major role in the pathogenesis of autoimmune diabetes by causing impairment of insulin release and pancreatic islet cell toxicity. METHODS: We compared insulin secretion, islet cell death and survival, inducible nitric oxide synthase (iNOS) mRNA expression, nitrite production, and Bcl-2 and Bax mRNA expression in isolated human and large mammal (bovine) islets exposed to 50 U/ml recombinant human interleukin-1, 1,000 U/ml recombinant human tumor necrosis factor-alpha and 1,000 U/ml recombinant human interferon-gamma. RESULTS: After 24-hr exposure, a marked decrease of glucose-stimulated insulin secretion was observed with human, but not with bovine islets. After 48-hr exposure, human, but not bovine, pancreatic islets showed a significantly higher percentage of apoptotic cells compared to controls. Treatment of human islets with human cytokines induced up-regulation of iNOS mRNA, increased levels of nitrites, and down-regulation of Bcl-2 mRNA, with unchanged levels of Bax mRNA. These parameters were not affected by cytokines in bovine islets. CONCLUSIONS: Bovine islets are less susceptible than human islets to the effects of human cytokines, which may be a potential advantage of xenotransplantation.

Effects of low-dose VOSO(4) on age-related changes in glucose homeostasis in rats.

The effects of low doses of vanadyl sulfate (0.2 mg/ml in the drinking water) on the age-related impairment of glucose homeostasis in Sprague-Dawley rats were investigated. VOSO(4) administration was initiated in 5-month-old animals and lasted 3 months. Thus, in 8-month-old rats, we investigated glucose metabolism in vivo and insulin secretory function in vitro. Results showed that VOSO(4) allowed the disposal of an oral glucose load at lower insulin levels than in age-matched controls. No significant changes were found in muscle glucose transporter (GLUT-4) levels or in glycogen content upon VOSO(4) treatment. Islets isolated from VOSO(4)-treated rats released less insulin than control islets, but showed a better preserved sensitivity to secretagogues, in terms of incremental release over basal release, secretory efficiency, and maintenance of the priming effect of glucose. In conclusion, chronic low-dose VOSO(4) treatment facilitates insulin action by a mechanism independent of muscle GLUT-4 levels and helps preserve the appropriate sensitivity of beta cells to stimuli, thereby preventing age-dependent functional alterations.

The biguanide compound metformin prevents desensitization of human pancreatic islets induced by high glucose.

Pancreatic islet desensitization by high glucose concentrations is a temporary and reversible state of beta-cell refractoriness to glucose (and possibly other secretagogues), due to repeated or prolonged pre-exposure to increased glucose concentrations. We evaluated whether the oral antidiabetic agent metformin affects this phenomenon in isolated, human pancreatic islets, and whether the possible effects of the biguanide are influenced by the presence of a sulphonylurea, glyburide. Islets prepared from five human pancreases were incubated for 24 h in M199 culture medium containing either 5.5 or 22.2 mmol/l glucose, with or without a therapeutic concentration (2.4 microg/ml) of metformin. Then, the islets were challenged with either 3.3 mmol/l glucose, 16.7 mmol/l glucose, or 3.3 mmol/l glucose + 10 mmol/l arginine, and insulin release was measured. After incubation in the absence of metformin, the human islets exposed to 22.2 mmol/l glucose showed no significant increase in insulin release when challenged with 16.7 mmol/l glucose (confirming that hyperglycemia desensitizes pancreatic beta-cells). In the presence of metformin, the islets fully maintained the ability to significantly increase their insulin release in response to glucose, even when previously exposed to 22.2 mmol/l glucose. No major effect on arginine-induced insulin release was observed, whatever the culture conditions. The protective action of metformin was observed also when glyburide was present in the incubation medium, whereas the sulphonylurea alone did not affect insulin release from the islets previously exposed to high glucose concentrations. These in vitro results suggest that metformin can prevent the desensitization of human pancreatic islets induced by prolonged exposure to increased glucose concentrations.

Effects of glibenclamide and metformin (alone or in combination) on insulin release from isolated human pancreatic islets.

Isolated human pancreatic islets were prepared by collagenase digestion and density gradient purification, and the effects of glibenclamide (0.5 and 5.0 mumol/l) and metformin (20 and 200 mumol/l), alone or in combination, on insulin release were evaluated at varying glucose concentrations. At 3.3 mmol/l glucose level, the addition of 5.0 mumol/l glibenclamide or 5.0 mumol/l glibenclamide plus 200 mumol/l metformin caused a significant increase of insulin release, compared with glucose alone. At 16.7 mmol/l glucose concentration, a significant increase of insulin secretion, compared with glucose alone, was produced by the addition of either 5.0 mumol/l glibenclamide, 200 mumol/l metformin, or both 5.0 mumol/l glibenclamide and 200 mumol/l metformin. The effect of the combination of the two drugs was significantly higher than that with either drug used alone. Thus, glibenclamide was shown to have an insulinotropic effect on human islets at both low and high glucose concentrations, and metformin at high glucose concentrations. A possible synergistic effect of glibenclamide and metformin at high glucose concentrations is also suggested.

An alternative and simple method to consistently prepare viable isolated human islets for clinical transplantation.

We describe a method to consistently prepare human islets for transplantation. By combining a simple collagenase digestion method and a density gradient purification system, we were able to obtain successful isolations (>/=200,000 islet equivalents, >/=50% purity) in 69% of processed glands. No reagent of animal source was used. Isolated islets were morphologically well maintained and functionally competent, with sterility confirmed in 97% of cases. Two patients were transplanted with islets prepared by this method; graft function was demonstrated for a few months. Improved simplicity and consistency, together with adequate quality of the preparations, are the main features of this isolation method.