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Here we use a combination of two-photon Fluorescence Lifetime Imaging Microscopy (FLIM) of NAD(P)H free/bound ratio in living HIs with post-fixation, immunofluorescence-based, cell-type identification. FLIM allowed to measure variations in the NAD(P)H free/bound ratio induced by glucose; immunofluorescence data allowed to identify single α and β cells; finally, matching of the two datasets allowed to assign metabolic shifts to cell identity. 312 α and 654 β cells from a cohort of 4 healthy donors, 15 total islets, were measured. Both α and β cells display a wide spectrum of responses, towards either an increase or a decrease in NAD(P)H free/bound ratio. Yet, if single-cell data are averaged according to the respective donor and correlated to donor insulin secretion power, a non-random distribution of metabolic shifts emerges: robust average responses of both α and β cells towards an increase of enzyme-bound NAD(P)H belong to the donor with the lowest insulin-secretion power; by contrast, discordant responses, with α cells shifting towards an increase of free NAD(P)H and β cells towards an increase of enzyme-bound NAD(P)H, correspond to the donor with the highest insulin-secretion power. Overall, data reveal neat anti-correlation of tissue metabolic responses with respect to tissue insulin secretion power., (© 2022. The Author(s).)

Metformin, a drug widely used in type 2 diabetes (T2D), has been shown to protect human β-cells exposed to gluco- and/or lipotoxic conditions and those in islets from T2D donors. We assessed whether metformin could relieve the human β-cell stress induced by pro-inflammatory cytokines (which mediate β-cells damage in type 1 diabetes, T1D) and investigated the underlying mechanisms using shotgun proteomics. Human islets were exposed to 50 U/mL interleukin-1β plus 1000 U/mL interferon-γ for 48 h, with or without 2.4 µg/mL metformin. Glucose-stimulated insulin secretion (GSIS) and caspase 3/7 activity were studied, and a shotgun label free proteomics analysis was performed. Metformin prevented the reduction of GSIS and the activation of caspase 3/7 induced by cytokines. Proteomics analysis identified more than 3000 proteins in human islets. Cytokines alone altered the expression of 244 proteins (145 up- and 99 down-regulated), while, in the presence of metformin, cytokine-exposure modified the expression of 231 proteins (128 up- and 103 downregulated). Among the proteins inversely regulated in the two conditions, we found proteins involved in vesicle motility, defense against oxidative stress (including peroxiredoxins), metabolism, protein synthesis, glycolysis and its regulation, and cytoskeletal proteins. Metformin inhibited pathways linked to inflammation, immune reactions, mammalian target of rapamycin (mTOR) signaling, and cell senescence. Some of the changes were confirmed by Western blot. Therefore, metformin prevented part of the deleterious actions of pro-inflammatory cytokines in human β-cells, which was accompanied by islet proteome modifications. This suggests that metformin, besides use in T2D, might be considered for β-cell protection in other types of diabetes, possibly including early T1D.

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., (© Georg Thieme Verlag KG Stuttgart · New York.)

In addition to genetic predisposition, environmental and lifestyle factors contribute to the pathogenesis of type 2 diabetes (T2D). Epigenetic changes may provide the link for translating environmental exposures into pathological mechanisms. In this study, we performed the first comprehensive DNA methylation profiling in pancreatic islets from T2D and non-diabetic donors. We uncovered 276 CpG loci affiliated to promoters of 254 genes displaying significant differential DNA methylation in diabetic islets. These methylation changes were not present in blood cells from T2D individuals nor were they experimentally induced in non-diabetic islets by exposure to high glucose. For a subgroup of the differentially methylated genes, concordant transcriptional changes were present. Functional annotation of the aberrantly methylated genes and RNAi experiments highlighted pathways implicated in β-cell survival and function; some are implicated in cellular dysfunction while others facilitate adaptation to stressors. Together, our findings offer new insights into the intricate mechanisms of T2D pathogenesis, underscore the important involvement of epigenetic dysregulation in diabetic islets and may advance our understanding of T2D aetiology.

Aims: Studies suggest that insulin-signaling molecules are present in the pancreatic islets. For this reason, the effects of insulin glulisine, insulin aspart and regular human insulin (RHI) on the function and molecular features of isolated human pancreatic islets were investigated., Methods: Human pancreatic islets were prepared by collagenase digestion and density-gradient purification of pancreata from multiple organ donors. Islets were then cultured for 48 h in the presence of 5.5 (normal) or 22.2 (high) mmol/L of glucose with and without glulisine, aspart and RHI (10 or 100 nmol/L). Functional (glucose-stimulated insulin secretion) and molecular (quantitative RT-PCR and immunoblot) studies were performed at the end of the different incubation conditions., Results: Glucose-stimulated insulin secretion was blunted in islets cultured in 22.2 mmol/L of glucose, with no significant effects from the exogenous added insulins. In islets maintained at 5.5 mmol/L of glucose, insulin receptor (IR) expression was reduced by low RHI, while phosphatidylinositol-3 kinase p110-alpha (PI3K) was enhanced by both concentrations of glulisine and aspart, and by high RHI. In islets preexposed to high glucose, IR expression was increased by both concentrations of aspart and RHI, but not by glulisine. Glulisine at high concentration significantly (P<0.05) increased PI3K expression. Glulisine and RHI significantly increased IRS-2 phosphorylation compared with control and aspart (P<0.05)., Conclusion: Insulin analogues have differential effects on the expression of insulin-signaling molecules in human pancreatic islets that are also dependent on the degree of glucose exposure., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

GLP-1 and GIP are incretins known to affect beta-cell function and turnover. However, information on the direct actions of these hormones on human islet cells is limited. We tested the effects of acute (45min) or prolonged (2days) exposure to GLP-1 or GIP, alone or in combination, on the function and some molecular features of human islets isolated from non-diabetic and type 2 diabetic multiorgan donors. Acutely, both GLP-1 and, more markedly so, GIP, significantly potentiated glucose-stimulated insulin release, with no apparent synergic action. Some of these effects were observed with type 2 diabetic islets as well. Following prolonged exposure to the incretins, improved insulin secretion was observed, and transcription of insulin, PDX-1 and Bcl-2 was increased in both non-diabetic and diabetic islets, with the combination of GLP-1 and GIP showing more significant effects. Although it is still unclear at what extent these beta-cell direct actions of individual or combined incretins occur in-vivo in humans, nevertheless the results of the present study suggest that enhancing the exposure of pancreatic islets to circulating levels of both incretins may be useful for therapeutical purposes., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background and Aims: Pancreatic islet transplantation has become one of the potential treatments for type 1 diabetes. We evaluated functional and viability parameters of isolated islets in relation to donors clinical characteristics and preparation variables., Methods: Islets were isolated from 70 nondiabetic multiorgan donors of overall age of 62.5 +/- 15.9 years. There were 41 men and 29 women. Their mean body mass index (BMI) was 25.62 +/- 3.09 kg/m(2). We evaluated the islet number (IEQ/g pancreatic tissue) insulin release (IR; microU/islet/min) in response to 3.3 (g) or 16.7 (G) mmol/L glucose; calcium flux concentration (CFC); and islet cell viability., Results: IEQ was 5249 +/- 1505, with 73.7 +/- 14.96% viable islet cells. IR was 0.03 +/- 0.01 at g and 0.11 +/- 0.06 at G (stimulation index [S] = 3.24 +/- 1.96). CFC was 1.95 +/- 1.03 DeltaRFU. We observed positive correlations between viable cells and IR at g (R(2) = 0.260; P = .013), IR at G (R(2) = 0.165; P = .013), and CFC (R(2) = 0.175; P = .047). A positive correlation was documented between BMI and g (R(2) = 0.245; P = .016) and negative correlations between age with SI (R(2) = 0.188; P = .052) and cold ischemia time with IEQ (R(2) = 0.865; P = .0061)., Conclusions: These results showed that quality control of isolated human pancreatic islets allowed assessment of beta-cell function and survival before transplantation, revealing several important variables., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background and Aims: Islet transplantation is an attractive approach to treat type 1 diabetic patients. However, suboptimal islet engraftment still represents an unsolved problem. It has been shown that human islets release monocyte chemoattractant protein-1 (MCP-1), one of the most powerful macrophage chemokines, which may impair the fate of the transplant. The aim of this study was to evaluate the presence and role of MCP-1 in isolated human islets, including genotyping for a common polymorphism., Methods: Pancreatic islets were isolated by enzymatic digestion and gradient purification from 41 nondiabetic multiorgan donors. We measured MCP-1 mRNA expression by quantitative real- time reverse-transcriptase polymerization chain reaction, analyzed the MCP-1 single nucleotide polymorphism, -2518 G/A (SNP, rs 1024611) and evaluated glucose-stimulated insulin release (IR; microU/islet/min)., Results: MCP-1 mRNA expression was found in all studied batches of islets. Overall, IR was significantly higher at 16.7 mmol/L than 3.3 mmol/L glucose. We observed a significant negative correlation between MCP-1 mRNA expression and stimulation index (SI). We found that MCP-1 mRNA expression was significantly higher in CC and CT compared with TT genotype groups. Finally, SI was significant lower in the CC with respect to the TT genotype group., Conclusions: These data show that MCP-1 gene expression regulated by the -2518 G/A polymorphism, is correlated with glucose-stimulated insulin release. The study of MCP-1 expression and genotype on isolated islets before transplantation may be useful to understand the inflammatory response after infusion of human islets into patients with type 1 diabetes mellitus., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background: The C-allele of rs13266634 located in SLC30A8 (ZNT8) has been strongly associated with decreased insulin release and with type 2 diabetes (T2D) susceptibility in some but not all studies. To shed further light on this issue, we performed a meta-analysis of the association between rs13266634 and T2D in different ethnic groups and assessed the relationships between SLC30A8 genotypes and some properties of isolated human islets., Methods: From 32 original articles, a total of 77,234 control individuals and 44,945 subjects with T2D were studied in meta-analysis. To assess the relationships between SLC30A8 genotype and islet cell phenotype, insulin secretion in response to glucose, glucose plus arginine and glucose plus glibenclamide was determined in pancreatic islets isolated from 82 multiorgan donors genotyped for the rs13266634 polymorphism. Quantitative expression of SLC30A8, Insulin and Glucagon mRNA was also measured., Results: Overall, each SLC30A8 risk allele was associated with a 14% increased risk for T2D (P=2.78 x 10(-34)). The population risk of T2D attributable to this polymorphism was estimated at 9.5% in Europeans and 8.1% in East Asians. Basal and stimulated insulin secretion from human islets as well as islet expressions of SLC30A8, Insulin and Glucagon were not affected by the presence of the polymorphism. However, SLC30A8 expression was positively correlated with Insulin (r=0.75, P=6.43 x 10(-6)) and Glucagon (r: 0.70, P=4.89 x 10(-5)) levels., Conclusions: The SLC30A8 rs13266634 polymorphism is among the most confirmed genetic markers of T2D in Europeans and East Asians. In isolated human islets, the risk C-allele does not affect ex-vivo insulin secretion and SLC30A8 expression, which is correlated with that of insulin and glucagon., ((c) 2010. Published by Elsevier Inc.)

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., (Copyright 2009 Elsevier B.V. All rights reserved.)

Background: Chronic exposure to high free fatty acids (FFA) can lead to irreversible damage of beta-cell accounting for impaired insulin secretion. Multiple mechanisms concur in generating the damage, but activation of oxidative stress may contribute to the final toxic effect. To better understand the phenomenon of lipotoxicity in human beta-cells, we evaluated the effects of 24-h pre-culture with 1.0 mmol/l FFA on the function, survival and mRNA expression of several enzymes involved in the generation and scavenging of reactive oxygen species (ROS)., Material and Methods: Human islets, prepared by collagenase digestion and density gradient purification from 9 pancreases of multiorgan donors, were incubated for 24-h in the presence 1.0 mmol/l long-chain mixture (oleate:palmitate, 2:1) FFA, with or without 100 micromol/l IAC, a non-peptidyl low molecular weight radical scavenger. At the end of incubation period, insulin secretion was measured by static incubation, and mRNA expression of insulin, Cu/Zn-SOD, Mn-SOD, Catalase, Glutathione peroxidase (GSH-px) and HO-1 by quantitative Real-Time RT-PCR. Nitrotyrosine levels were determined by an ELISA technique., Results: As compared to control incubation (Ctrl, no FFA), exposure to FFA was associated with impaired insulin release and reduced insulin mRNA expression. The presence of IAC in the incubation medium increased insulin release significantly and prevented changes in mRNA expression. Exposure to FFA was associated with oxidative stress as indicated by a significant accumulation of nitrotyrosine and IAC restrained such an increase. mRNA expression of Cu/Zn-SOD, Mn-SOD, Catalase, GSH-Px, and HO-1 were all modified after FFA exposure. These changes were partially prevented in the presence of IAC., Conclusions: In human islets 24-h exposure to high FFA causes oxidative stress associated with changes of several enzymes involved in ROS scavenging. These effects were prevented by the use of an antioxidant molecule.

Aims/hypothesis: Insulin secretion in pancreatic islets is dependent upon mitochondrial function and production of ATP. The transcriptional coactivator peroxisome proliferator activated receptor gamma coactivator-1 alpha (protein PGC-1alpha; gene PPARGC1A) is a master regulator of mitochondrial genes and its expression is decreased and related to impaired oxidative phosphorylation in muscle from patients with type 2 diabetes. Whether it plays a similar role in human pancreatic islets is not known. We therefore investigated if PPARGC1A expression is altered in islets from patients with type 2 diabetes and whether this expression is influenced by genetic (PPARGC1A Gly482Ser polymorphism) and epigenetic (DNA methylation) factors. We also tested if experimental downregulation of PPARGC1A expression in human islets influenced insulin secretion., Methods: The PPARGC1A Gly482Ser polymorphism was genotyped in human pancreatic islets from 48 non-diabetic and 12 type 2 diabetic multi-organ donors and related to PPARGC1A mRNA expression. DNA methylation of the PPARGC1A promoter was analysed in pancreatic islets from ten type 2 diabetic and nine control donors. Isolated human islets were transfected with PPARGC1A silencing RNA (siRNA)., Results: PPARGC1A mRNA expression was reduced by 90% (p<0.005) and correlated with the reduction in insulin secretion in islets from patients with type 2 diabetes. After downregulation of PPARGC1A expression in human islets by siRNA, insulin secretion was reduced by 41% (p

We examined a possible protective effect of the nonpeptidyl low molecular weight radical scavenger IAC [bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decanedioate di-hydrochloride] on isolated human islet cells against isolation and culture oxidative stress. Islets isolated from pancreases of nondiabetic multiorgan donors by collagenase digestion were purified by density gradient centrifugation. After the isolation, islets were either exposed or not exposed for 7 days to 10 micromol/L IAC. We found that IAC markedly reduced oxidative stress and ameliorated islets function. These results suggest that the use of IAC could be an interesting pharmacological approach for the treatment of the islets before transplantation.

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.

Immune protection of artificial tissue by means of pancreatic islet microencapsulation is a very ambitious new approach to avoid life-long immune suppression. But the success in the utilization of the alginate-beads with incorporated islets is unfortunately limited. Some of the problems cannot be solved by a two-component system, so polymer encapsulation of the microbeads was tested to improve the properties. In the present paper a pure nanoencapsulation multilayer approach was tested in order to reduce the size of the capsule and possibly apply in the future a multilayer capsule with individual properties in each layer or region of the capsule. Different polycations were attached in a self-assembly process. The advantage in using the surface charge of islets as binding site for the polyions is the guarantee of complete coverage after the second layer. Release of insulin was determined to characterize the function of the islets after encapsulation as well as the permeability of the capsule. Fluorescence microscopy was used to visualize the polyelectrolyte layers. Finally by means of an immune assay, the protection capability of the capsule was proved. In these first measurements the encapsulation with a multilayer nanocapsule was shown to be a possible alternative to the more space-consuming and random islet-trapping microencapsulation.

Objective: Data from prospective studies suggest a significant reduction in the risk of new diabetes from drug therapies containing angiotensin-converting enzyme (ACE) inhibitors. Since the renin-angiotensin system (RAS) has been found locally in several tissues and cells, including pancreatic islets, we hypothesized that the positive metabolic effects of ACE inhibitors may be due to a beneficial action of these compounds on insulin-secreting beta-cells., Design and Methods: Isolated human pancreatic islets were studied after 24 h of incubation with 22.2 mmol/l glucose, with or without the presence in the incubation medium of 0.5-6.0 mmol/l zofenoprilat or enalaprilat, ACE inhibitor drugs which differ by the presence of a sulphydryl or a carboxyl group in their structural formula. Functional and molecular studies were then performed to assess insulin secretion, redox balance, mRNA and protein expression., Results: Angiotensinogen, ACE and angiotensin type 1 receptor mRNA expression increased in islets cultured in high glucose; this was similarly prevented by the presence of either ACE inhibitor. As expected, preculture of human islets in high glucose determined a marked reduction in insulin secretion which was associated with enhanced oxidative stress, as shown by increased nitrotyrosine concentrations, and enhanced expression of protein kinase C beta and NADPH oxidase. The presence of either of the ACE inhibitors counteracted several of the deleterious effects of high glucose exposure, including reduction of insulin secretion and increased oxidative stress; zofenoprilat showed significantly more marked effects., Conclusions: These results showed that: (a) RAS molecules are present in human islets and their expression is sensitive to glucose concentration, (b) ACE inhibitors, and in particular zofenoprilat, protect human islets from glucotoxicity and (c) the effects of ACE inhibition are associated with decreased oxidative stress. Together, these findings provide evidence that the possible beneficial effects of ACE inhibitors in human diabetes are due, at least in part, to a protective action on pancreatic beta-cells.

To shed further light on the primary alterations of insulin secretion in type 2 diabetes and the possible mechanisms involved, we studied several functional and molecular properties of islets isolated from the pancreata of 13 type 2 diabetic and 13 matched nondiabetic cadaveric organ donors. Glucose-stimulated insulin secretion from type 2 diabetic islets was significantly lower than from control islets, whereas arginine- and glibenclamide-stimulated insulin release was less markedly affected. The defects were accompanied by reduced mRNA expression of GLUT1 and -2 and glucokinase and by diminished glucose oxidation. In addition, AMP-activated protein kinase activation was reduced. Furthermore, the expression of insulin was decreased, and that of pancreatic duodenal homeobox-1 (PDX-1) and forkhead box O1 (Foxo-1) was increased. Nitrotyrosine and 8-hydroxy-2'-deoxyguanosine concentrations, markers of oxidative stress, were significantly higher in type 2 diabetic than control islets, and they were correlated with the degree of glucose-stimulated insulin release impairment. Accordingly, 24-h exposure to glutathione significantly improved glucose-stimulated insulin release and decreased nitrotyrosine concentration, with partial recovery of insulin mRNA expression. These results provide direct evidence that the defects of insulin secretion in type 2 diabetic islets are associated with multiple islet cell alterations. Most importantly, the current study shows that the functional impairment of type 2 diabetic islets can be, at least in part, reversible. In this regard, it is suggested that reducing islet cell oxidative stress is a potential target of human type 2 diabetes therapy.

The direct effects of prolonged exposure to sulphonylureas on the function and survival of human islets are unknown. This study assessed the insulin content, glucose-stimulated insulin release, islet cell apoptosis, and mRNA expression of insulin and GLUT-1 in isolated human islets cultured in the presence of therapeutical concentrations of glimepiride (10 microM), glibenclamide (10 microM), or chlorpropamide (600 microM). Islets were prepared by collagenase digestion and density gradient purification from 18 multiorgan donors and were then exposed for 24 h to the different sulphonylureas. Insulin content decreased significantly following culture with any sulphonylurea compound. In response to an acute challenge with 3.3 and 16.7 mM glucose, insulin release from the control islets accounted for 1.9 +/- 0.5% and 4.9 +/- 1.7% of total insulin content (P<.01), respectively. Glucose responsiveness was preserved in islets precultured in the presence of glimepiride, whereas high glucose level did not elicit any significant increase of insulin secretion from islets preincubated with glibenclamide or chlorpropamide. These alterations were reverted by an additional 48-h incubation in drug-free conditions. The amount of apoptotic cells did not differ significantly among the experimental groups. Quantitative RT-PCR studies showed that, compared with the control islets, cells preincubated with glibenclamide or chlorpropamide had an increased expression of insulin mRNA, with no change in the expression of GLUT-1. In conclusion, prolonged exposure of human islets to different sulphonylureas causes different disturbances of islet cell function, with glimepiride showing milder effects, as compared with chlorpropamide and glibenclamide.

Several properties of pancreatic beta-cells in type 2 diabetes (T2D) were studied by using islets isolated from T2D subjects. Moreover, because metformin has protective effects on nondiabetic beta-cells exposed to high glucose or free fatty acid levels, we investigated its direct action on T2D islet cells. Diabetic islets were characterized by reduced insulin content, decreased amount of mature insulin granules, impaired glucose-induced insulin secretion, reduced insulin mRNA expression, and increased apoptosis with enhanced caspase-3 and -8 activity. These alterations were associated with increased oxidative stress, as shown by higher nitrotyrosine concentrations, increased expression of protein kinase C-beta2 and nicotinamide adenine dinucleotide phosphate reduced-oxidase, and changes in mRNA expression of manganese- superoxide dismutase, Cu/Zn-superoxide dismutase, catalase, and glutathione peroxidase. Twenty-four-hour incubation of T2D islets with metformin was associated with increased insulin content, increased number and density of mature insulin granules, improved glucose-induced insulin release, and increased insulin mRNA expression. Moreover, apoptosis was reduced, with concomitant decrease of caspase-3 and -8 activity. These changes were accompanied by reduction or normalization of several markers of oxidative stress. Thus, T2D islets have several functional and survival defects, which can be ameliorated by metformin; the beneficial effects of the drug are mediated, at least in part, by a reduction of oxidative stress.

Objective: Peripheral benzodiazepine receptors (PBRs) are part of the mitochondrial permeability transition pore, and their activation may induce cell death. PBRs are expressed in human pancreatic islets, and cytokine-induced damage is accompanied by changes in their properties. We hypothesized that PBRs can have a role in human islet physiopathology, and evaluated the effects of prolonged exposure to two specific PBR ligands, PK11195 and Ro5-4864 on the function and survival of isolated human islets., Design: Isolated human islets were prepared from the pancreas of 25 multiorgan cadaveric donors and incubated for 12 h in the presence of PK11195 or Ro5-4864. Insulin secretion studies and apoptosis experiments were then performed, together with assessment of intracellular pathways involved in islet cell function and survival., Methods: Islets were prepared by enzymatic digestion and density gradient purification. Insulin secretion was assessed by the batch incubation method, and glucose oxidation was evaluated by the use of D-[U-(14)C]glucose. Apoptosis was studied using the TUNEL technique, ELISA methods, and electron microscopy evaluation. PCR experiments were performed by the use of specific primers., Results: Glucose-stimulated insulin release was significantly lower after exposure to PK11195 than after exposure to Ro5-4864. This was accompanied by reduced glucose oxidation and no major change of insulin or GLUT-1 mRNA expression. Apoptosis was higher in PK11195-exposed islets, and electron microscopy demonstrated the involvement of beta-cells. The apoptotic effects were prevented by bongkrekic acid and low-dose cyclosporin A, which stabilize the mitochondrial membrane, and were associated with no evident change of inducible nitric oxide synthase (iNOS), B-cell leukemia/lymphoma-2 (Bcl-2) or Bcl-2-associated X protein (Bax) expression. Caspase inhibition markedly reduced the amount of apoptosis, and the role of these proteases was confirmed by the increased activity of caspase-3 and caspase-9., Conclusions: Prolonged binding to PBRs may cause human beta-cells functional damage and apoptosis, a phenomenon which is prevented by stabilizing the mitochondrial membrane; occurs without changes of iNOS, Bax and Bcl-2 mRNA expression; and involves caspase activation. These results suggest an involvement of PBRs in human pancreatic beta-cell function and survival.

Because adverse effects of glucose were attributed to its increased routing through the hexosamine pathway (HBP), we inquired whether HBP activation affects pancreatic beta-cell survival. Exposure of human islets to high glucose resulted in increased apoptosis of beta-cells upon serum deprivation that was reversed by azaserine. Also, glucosamine, a direct precursor of the downstream product of the HBP, increased human beta-cells apoptosis upon serum deprivation, which was reversed by benzyl-2-acetamido-2-deoxy-alpha-d-galactopyranoside (BADGP), an inhibitor of protein O-glycosylation. These results were reproduced in RIN rat beta-cells. Glucosamine treatment resulted in inhibition of tyrosine-phosphorylation of the insulin receptor (IR), IRS-1, and IRS-2, which was associated with increased O-glycosylation. These changes caused impaired activation of the PI 3-kinase/Akt survival signaling that resulted in reduced GSK-3 and FOXO1a inactivation. BADGP reversed the glucosamine-induced reduction in insulin-stimulated phosphorylation of IR, IRS-1, IRS-2, Akt, GSK-3, and FOXO1a. Impaired FOXO1a inactivation sustained expression of the pro-apoptotic protein Bim, without affecting Bad, Bcl-XL, or Bcl-2 expression. These results indicate that hyperglycemia may increase susceptibility to apoptosis of human and rat beta-cell through activation of the HBP. Increased routing of glucose through this metabolic pathway results in impaired activation of the IR/IRSs/PI3-kinase/Akt survival pathway by induction of O-glycosylation of signaling molecules.

Many adverse effects of glucose were attributed to its increased routing through the hexosamine pathway (HBP). There is evidence for an autocrine role of the insulin signaling in beta-cell function. We tested the hypothesis that activation of the HBP induces defects in insulin biosynthesis by affecting the insulin-mediated protein translation signaling. Exposure of human pancreatic islets and RIN beta-cells to glucosamine resulted in reduction in glucose- and insulin-stimulated insulin biosynthesis, which in RIN beta-cells was associated with impairment in insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation at Tyr(608) and Tyr(628), which are essential for engaging phosphatidylinositol 3-kinase (PI 3-kinase). These changes were accompanied by impaired activation of PI 3-kinase, and activation of Akt/mammalian target of rapamycin/phosphorylated heat- and acid-stable protein-1/p70S6 kinase pathway. RIN beta-cells exposed to high glucose exhibited increased c-Jun N-terminal kinase (JNK) and ERK1/2 activity, which was associated with increased IRS-1 phosphorylation at serine (Ser)(307) and Ser(612), respectively, that inhibits coupling of IRS-1 to the insulin receptor and is upstream of the inhibition of IRS-1 tyrosine phosphorylation. Azaserine reverted the stimulatory effects of high glucose on JNK and ERK1/2 activity and IRS-1 phosphorylation at Ser(307) and Ser(612). Glucosamine mimicked the stimulatory effects of high glucose on JNK and ERK1/2 activity and IRS-1 phosphorylation at Ser(307) and Ser(612). Inhibition of JNK and MAPK kinase-1 activity reverted the negative effects of glucosamine on insulin-mediated protein synthesis. These results suggest that activation of the HBP accounts, in part, for glucose-induced phosphorylation at Ser(307) and Ser(612) of IRS-1 mediated by JNK and ERK1/2, respectively. These changes result in impaired coupling of IRS-1 and PI 3-kinase, and activation of the Akt/mammalian target of rapamycin/phosphorylated heat- and acid-stable protein-1/p70S6 kinase pathway.

Background: Type 1 diabetes results from the destruction of pancreatic beta-cell as a consequence of an autoimmune process. To date, information on the properties of islets isolated from type 1 diabetic patients is very scant., Methods: Some immunological and functional properties of islets prepared from the pancreas of type 1 diabetic patients were studied shortly after the isolation and after a period of culture in euglycemic condition., Results: Compared to control islets, freshly prepared type 1 diabetic islets released a significantly higher amount of cytokines (pg/mL) into the culture medium (TNF-alpha: 112.9 +/- 5.6 vs 75.6 +/- 24.4; INF-gamma: 286.9 +/- 26.9 vs 58.6 +/- 6.2; IL-10: 41.8 +/- 4.3 vs 10.1 +/- 3.2; TGF-1 beta: 294.0 +/- 20.6 vs 45.1 +/- 3.5); had a significantly higher chemotactic index (1.9 +/- 0.2 vs 1.2 +/- 0.1); showed reduced insulin release (% of insulin content) in response to glucose (2.8 +/- 0.7 vs 5.3 +/- 1.9), arginine (3.0 +/- 0.6 vs 5.6 +/- 1.0), and glibenclamide (2.9 +/- 0.7 vs 5.4 +/- 0.9); and exhibited decreased glucose oxidation capability and diminished mRNA expression of glucokinase, aldolase, pyruvate kinase, and mitochondrial glycerolphosphate dehydrogenase. Ten days after isolation, a normalization of cytokine release (TNF-alpha: 55.7 +/- 2.3; INF-gamma: 53.9 +/- 4.3; IL-10: 8.6 +/- 0.7; TGF-1 beta: 60.7 +/- 12.4) and chemotactic index (1.3 +/- 0.2) were observed. Moreover, there was an improvement of insulin secretion (3.8 +/- 0.3, 4.7 +/- 0.6 and 3.5 +/- 0.2 respectively in response to glucose, arginine, and glibenclamide) and glucose oxidation, and a partial recovery of the measured mRNA expressions., Conclusions: These novel results, obtained with islets prepared from patients with type 1 diabetes, demonstrate that even after months after diabetes diagnosis, a period of culture of the islets in a more favorable environment has beneficial effects on the islet function., (Copyright 2004 John Wiley & Sons, Ltd.)

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.

Peroxisome proliferator-activated receptors (PPARs) are a subgroup of the superfamily of nuclear receptors, with three distinct main types: alpha, beta and gamma (subdivided into gamma(1) and gamma(2)). Recently, the presence of PPARgamma has been reported in human islets. Whether other PPAR types can be found in human islets, how islet PPARgamma mRNA expression is regulated by the metabolic milieu, their role in insulin secretion, and the effects of a PPARgamma agonist are not known. In this study, human pancreatic islets were prepared by collagenase digestion and density gradient purification from nonobese adult donors. The presence of PPAR mRNAs was assessed by RT-PCR, and the effect was evaluated of exposure for up to 24 h to either 22.2 mmol/l glucose and/or 0.25, 0.5, or 1.0 mmol/l long-chain fatty acid mixture (oleate to palmitate, 2:1). PPARbeta and, to a greater extent, total PPARgamma and PPARgamma(2) mRNAs were expressed in human islets, whereas PPARalpha mRNA was not detected. Compared with human adipose tissue, PPARgamma mRNA was expressed at lower levels in the islets, and PPARbeta at similar levels. The expression of PPARgamma(2) mRNA was not affected by exposure to 22.2 mmol/l glucose, whereas it decreased markedly and time-dependently after exposure to progressively higher free fatty acids (FFA). This latter effect was not affected by the concomitant presence of high glucose. Exposure to FFA caused inhibition of insulin mRNA expression, glucose-stimulated insulin release, and reduction of islet insulin content. The PPARgamma agonists rosiglitazone and 15-deoxy-Delta-(12,14)prostaglandin J(2) prevented the cytostatic effect of FFA as well as the FFA-induced changes of PPAR and insulin mRNA expression. In conclusion, this study shows that PPARgamma mRNA is expressed in human pancreatic islets, with predominance of PPARgamma(2); exposure to FFA downregulates PPARgamma(2) and insulin mRNA expression and inhibits glucose-stimulated insulin secretion; exposure to PPARgamma agonists can prevent these effects.

In an effort to better understand the phenomenon of lipotoxicity in human beta-cells, we evaluated the effects of 48-h preculture with 1.0 or 2.0 mmol/l free fatty acid (FFA) (2:1 oleate to palmitate) on the function and survival of isolated human islets and investigated some of the possible mechanisms. Compared with control islets, triglyceride content was significantly increased and insulin content and glucose-stimulated insulin release were significantly reduced in islets precultured with increased FFA concentrations. These changes were accompanied by a significant reduction of glucose utilization and oxidation. By cell death detection techniques, it was observed that exposure to FFAs induced a significant increase of the amount of dead cells. Electron microscopy showed the involvement of beta-cells, with morphological appearance compatible with the presence of apoptotic phenomena. FFA-induced islet cell death was blocked by inhibition of upstream caspases and partially prevented by inhibiton of ceramide synthesis or serine protease activity, whereas inhibition of nitric oxide synthesis had no effect. RT-PCR studies revealed no major change of iNOS and Bax mRNA expression and a marked decrease of Bcl-2 mRNA expression in the islets cultured with FFA. Thus, prolonged exposure to FFAs has cytostatic and pro-apoptotic effects on human pancreatic beta-cells. The cytostatic action is likely to be due to the FFA-induced reduction of intraislet glucose metabolism, and the proapoptotic effects are mostly caspase mediated, partially dependent on ceramide pathway, and possibly Bcl-2 regulated.

Type 2 (non-insulin-dependent) diabetes results from decreased insulin action in peripheral target tissues (insulin resistance) and impaired pancreatic beta-cell function. These defects reflect both genetic components and environmental risk factors. Recently, the common Gly(972)-->Arg amino acid polymorphism of insulin receptor substrate 1 (Arg(972) IRS-1) has been associated with human type 2 diabetes. In this study, we report on some functional and morphological properties of isolated human islets carrying the Arg(972) IRS-1 polymorphism. Insulin content was lower in variant than control islets (94 +/- 47 vs. 133 +/- 56 microU/islet; P < 0.05). Stepwise glucose increase (1.7 to 16.7 mmol/l) significantly potentiated insulin secretion from control islets, but not Arg(972) IRS-1 islets, with the latter also showing a relatively lower response to glyburide and a significantly higher response to arginine. Proinsulin release mirrored insulin secretion, and the insulin-to-proinsulin ratio in response to arginine was significantly lower from Arg(972) IRS-1 islets than from control islets. Glucose utilization and oxidation did not differ in variant and wild-type islets at both low and high glucose levels. Electron microscopy showed that Arg(972) IRS-1 beta-cells had a severalfold greater number of immature secretory granules and a lower number of mature granules than control beta-cells. In conclusion, Arg(972) IRS-1 islets have reduced insulin content, impaired insulin secretion, and a lower amount of mature secretory granules. These alterations may account for the increased predisposition to type 2 diabetes in individuals carrying the Gly(972)-->Arg amino acid polymorphism of IRS-1.

Human pancreatic islets from eight donors were incubated for 48 h in the presence of 2.0 mmol/l free fatty acid (FFA) (oleate to palmitate, 2 to 1). Insulin secretion was then assessed in response to glucose (16.7 mmol/l), arginine (20 mmol/l), and glyburide (200 micromol/l) during static incubation or by perifusion. Glucose oxidation and utilization and intra-islet triglyceride content were measured. The effect of metformin (2.4 microg/ml) was studied because it protects rat islets from lipotoxicity. Glucose-stimulated but not arginine- or glyburide-stimulated insulin release was significantly lower from FFA-exposed islets. Impairment of insulin secretion after exposure to FFAs was mainly accounted for by defective early-phase release. In control islets, increasing glucose concentration was associated with an increase in glucose utilization and oxidation. FFA incubation reduced both glucose utilization and oxidation at maximal glucose concentration. Islet triglyceride content increased significantly after FFA exposure. Addition of metformin to high-FFA media prevented impairment in glucose-mediated insulin release, decline of first-phase insulin secretion, and reduction of glucose utilization and oxidation without significantly affecting islet triglyceride accumulation. These results show that lipotoxicity in human islets is characterized by selective loss of glucose responsiveness and impaired glucose metabolism, with a clear defect in early-phase insulin release. Metformin prevents these deleterious effects, supporting a direct protective action on human beta-cells.

Immunoprotection of pancreatic islets for successful allo- or xenotransplantation without chronic immunosuppression is an attractive, but still elusive, approach for curing type 1 diabetes. It was recently shown that, even in the absence of fibrotic overgrowth, other factors, mainly insufficient nutrition to the core of the islets, represent a major barrier for long-term survival of intraperitoneal microencapsulated islet grafts. The use of dispersed cells might contribute to solve this problem due to the conceivably easier nutritional support to the cells. In the present study, purified bovine islets, prepared by collagenase digestion and density gradient purification, and dispersed bovine islet cells, obtained by trypsin and DNAsi (viability > 90%), were entrapped into either 2% (w/v) sodium alginate (commonly used for encapsulation purposes) or (dispersed islet cells only) macroporous gelatin microcarriers (CulthiSpher-S, commonly used for the production of biologicals by animal cells). Insulin release studies in response to glucose were performed within 1 week and after 1 month from preparation of the varying systems and showed no capability of dispersed bovine islet cells within sodium alginate microcapsules to sense glucose concentration changes. On the contrary, bovine islet cells entrapped in CulthiSpher-S microcarriers showed maintained capacity of increasing insulin secretion upon enhanced glucose concentration challenge. In this case, insulin release was approximately 60% of that from intact bovine islets within sodium alginate microcapsules. MTT and hematoxylineosin staining of islet cell-containing microcarriers showed the presence of viable and metabolically active cells throughout the study period. This encouraging functional data prompted us to test whether the microcarriers could be immunoisolated for potential use in transplantation. The microcarriers were embedded within 3% sodium alginate, which was then covered with a poly-L-lysine layer and a final outer alginate layer. Maintained insulin secretion function of this system was observed, which raises the possibility of using microencapsulated CulthiSpher-S microcarriers, containing dispersed pancreatic islet cells, in experimental transplantation studies., (Copyright 2001 John Wiley & Sons, Inc.)

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.

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.

Background: Certain clinical conditions are associated with inappropriately high levels of circulating glucagon. To date, little information is available about the direct effects of prolonged exposure of human islet cells to pancreatic glucagon. In the present study we evaluated the function, antigenicity and survival of human islets exposed for 24 h to human pancreatic glucagon., Methods: We prepared human islets of Langerhans by collagenase digestion and density-gradient purification, incubated them for 24 h with 44 or 430 pmol/l pancreatic glucagon at physiological (5.5 mmol/l) glucose level, and evaluated their insulin release function, which was then compared with that obtained from islets kept at high (11.1 mmol/l) glucose concentration. In addition, aliquots of the islets were evaluated to assess their chemotactic properties towards human monocyte-macrophage cells, and their potency to induce cytokine release from human lymphocytes. Finally, survival of the islet cells cultured under varying conditions was evaluated, and an assessment was performed of mRNA expression of Bcl-2 and Bax proteins., Results: The insulin secretion results demonstrated that, compared to the control islets, the islets previously exposed to either 44 or 430 pmol/l glucagon exhibited changes in insulin release in response to glucose, consisting of augmented secretion at low glucose challenge, and no further significant increase at high glucose stimulation, similar to the effects observed with islets pre-cultured with high glucose. These effects were reversible, as documented by the recovery of normal islet sensitivity to glucose after an additional 24-h culture in medium lacking glucagon. Compared to control islets, the culture medium from islets pre-cultured with high glucagon or high glucose showed an increased chemotactic potency towards human monocyte-macrophage cells. In addition, human lymphocytes released a greater amount of tumour necrosis factor alpha when co-cultured with the islets pre-exposed to high glucagon or high glucose, whereas no significant difference was observed (in comparison with control islets) as regards the release of gamma-interferon, interleukin-2 and interleukin-10. The TUNEL technique and RT-PCR showed, respectively, no major difference in cell survival and expression of mRNA encoding for Bcl-2 and Bax protein between control islets and islets kept for 24 h in the presence of high glucagon or high glucose., Conclusions: Our results show that in vitro exposure of human islets to pancreatic glucagon for 24 h causes changes in the function and antigenicity of isolated human islets that are similar to the changes observed after pre-culture with increased glucose levels. Under our experimental conditions, these changes were not accompanied by any evidence of cytotoxicity., (Copyright 2000 John Wiley & Sons, Ltd.)

This study evaluated the release of Th1 and Th2 cytokines from human lymphomononuclear cells (LMC) in response to purified human (HI) or bovine (BI) islets, and the role of long-term (3-4 weeks) islet culture and removal of monocyte-macrophage cells. The results showed that HI and BI caused a similar increase of the release of gamma interferon (IFN), IL-2 and IL-6 from LMC, whereas BI had a more marked effect than HI on IL-10 release. Culturing the islets had possible positive effects (reduction of IFN and IL-2), but also potentially negative effects (increase of TNF). Removal of monocyte-macrophage cells determined a significant reduction of IL-6, IL-10 and TNF production in response to xeno-islets.

The adipocyte-derived hormone leptin has been reported to inhibit, have no effect, or potentiate insulin secretion in-vitro; these effects mainly depend on the species considered, the concentrations used, and the length of exposure. We investigated the direct effects of recombinant human leptin (HL) on human pancreatic beta cell function by studying insulin secretion (IS), hexokinase and glucokinase activity and Km, and potassium channel permeability in purified human islets (HI). In acute experiments, no effect of 1, 5, 20, or 50 ng/ml HL on glucose or arginine stimulated insulin release was found, whereas 500 ng/ml HL caused a significant decrease of glucose induced IS. After 24h pre-culture with either 20 or 500 ng/ml HL, a significant reduction of glucose (but not arginine) stimulated IS was observed. Exposure to leptin caused a significant increase of potassium channel permeability, whereas hexokinase and glucokinase activity and Km remained unchanged. These results suggest that physiological human leptin concentration is able to importantly affect glucose (but not arginine) stimulated insulin release from human islets only after prolonged exposure. This effect is probably mediated by changes of potassium channel permeability, and is not accompanied by modifications of glucose phosphorylating enzymes properties., (Copyright 1999 Academic Press.)

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.

Current information on pancreatic islet sulfonylurea receptors has been obtained with laboratory animal pancreatic beta cells or stable beta-cell lines. In the present study, we evaluated the properties of sulfonylurea receptors of human islets of Langherans, prepared by collagenase digestion and density-gradient purification. The binding characteristics of labeled glibenclamide to pancreatic islet membrane preparations were analyzed, displacement studies with several oral hypoglycemic agents were performed, and these latter compounds were tested as for their insulinotropic action on intact human islets. [3H]glibenclamide saturable binding was shown to be linear at < or =0.25 mg/ml protein; it was both temperature and time dependent. Scatchard analysis of the equilibrium binding data at 25 degrees C indicated the presence of a single class of saturable, high-affinity binding sites with a Kd value of 1.0+/-0.07 nM and a Bmax value of 657+/-48 fmol/mg of proteins. The displacement experiments showed the following rank order of potency of the oral hypoglycemic agents we tested: glibenclamide = glimepiride > tolbutamide > chlorpropamide >> metformin. This binding potency order was parallel with the insulinotropic potency of the evaluated compounds.

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.