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Deficient vascularization is a major driver of early islet graft loss and one of the primary reasons for the failure of islet transplantation as a viable treatment for type 1 diabetes. This study identifies the protein tyrosine phosphatase 1B (PTP1B) as a potential modulator of islet graft revascularization. We demonstrate that grafts of pancreatic islets lacking PTP1B exhibit increased revascularization, which is accompanied by improved graft survival and function, and recovery of normoglycemia and glucose tolerance in diabetic mice transplanted with PTP1B-deficient islets. Mechanistically, we show that the absence of PTP1B leads to activation of hypoxia-inducible factor 1α-independent peroxisome proliferator-activated receptor γ coactivator 1α/estrogen-related receptor α signaling and enhanced expression and production of vascular endothelial growth factor A (VEGF-A) by β cells. These observations were reproduced in human islets. Together, these findings reveal that PTP1B regulates islet VEGF-A production and suggest that this phosphatase could be targeted to improve islet transplantation outcomes., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Aims/hypothesis: Comprehensive characterisation of the interrelation between the peripancreatic adipose tissue and the pancreatic islets promises novel insights into the mechanisms that regulate beta cell adaptation to obesity. Here, we sought to determine the main pathways and key molecules mediating the crosstalk between these two tissues during adaptation to obesity by the way of an integrated inter-tissue, multi-platform analysis., Methods: Wistar rats were fed a standard or cafeteria diet for 30 days. Transcriptomic variations by diet in islets and peripancreatic adipose tissue were examined through microarray analysis. The secretome from peripancreatic adipose tissue was subjected to a non-targeted metabolomic and proteomic analysis. Gene expression variations in islets were integrated with changes in peripancreatic adipose tissue gene expression and protein and metabolite secretion using an integrated inter-tissue pathway and network analysis., Results: The highest level of data integration, linking genes differentially expressed in both tissues with secretome variations, allowed the identification of significantly enriched canonical pathways, such as the activation of liver/retinoid X receptors, triacylglycerol degradation, and regulation of inflammatory and immune responses, and underscored interaction network hubs, such as cholesterol and the fatty acid binding protein 4, which were unpredicted through single-tissue analysis and have not been previously implicated in the peripancreatic adipose tissue crosstalk with beta cells., Conclusions/interpretation: The integrated analysis reported here allowed the identification of novel mechanisms and key molecules involved in peripancreatic adipose tissue interrelation with beta cells during the development of obesity; this might help the development of novel strategies to prevent type 2 diabetes.

Insulin resistance and hyperinsulinemia are generally associated with obesity. Obese nondiabetic individuals develop a compensatory β-cell response to adjust insulin levels to the increased demand, maintaining euglycemia. Although several studies indicate that this compensation relies on structural changes, the existence of β-cell functional adaptations is incompletely understood. Here, we fed female mice with a high-fat diet (HFD) for 12 weeks. These animals became obese, hyperinsulinemic, insulin-resistant, and mildly glucose-intolerant while fed, and fasting glycemia was comparable in HFD and control mice. Islets from HFD animals exhibited increased β-cell mass and hypertrophy. Additionally, they had enhanced insulin gene expression and content and augmented glucose-induced insulin secretion. Electrophysiological examination of β-cells from both groups showed no differences in KATP channel open probability and conductance. However, action potentials elicited by glucose had larger amplitude in obese mice. Glucose-induced Ca²⁺ signals in intact islets, in isolated β-cells, and individual β-cells within islets were also increased in HFD mice. Additionally, a higher proportion of glucose-responsive cells was present in obese mice. In contrast, whole-cell Ca²⁺ current densities were similar in both groups. Capacitance measurements showed that depolarization-evoked exocytosis was enhanced in HFD β-cells compared with controls. Although this augment was not significant when capacitance increases of the whole β-cell population were normalized to cell size, the exocytotic output varied significantly when β-cells were distributed by size ranges. All these findings indicate that β-cell functional adaptations are present in the islet compensatory response to obesity.

Background/aims: Stimulation of insulin release by D-glucose is accompanied by Cl(-) and HCO(3)(-) efflux from pancreatic islet cells. The efflux of these anions may involve volume-regulated anion channels, including possibly TMEM16A, and the Na(+)-HCO(3)(-)-cotransporter SLC4A4. The present study was designed to explore the expression of both TMEM16A and SLC4A4 in human pancreatic islets., Methods: Pancreases were obtained from human cadaveric donors. Immunodetection of TMEM16A and SLC4A4 was performed by immunohistochemistry on sections of fixed pancreas, while real-time PCR for the study of corresponding gene expression was performed on RNA extracted from both total pancreatic pieces and isolated pancreatic islets., Results: RT-PCR yielded lower levels of SLC4A4 in isolated islets than in the total pancreas, whilst a mirror image prevailed for TMEM16A mRNA. Immunohistochemistry of human pancreas, however, indicated comparable immunostaining of SLC4A4 in insulin-producing cells and exocrine pancreatic cells, whilst that of TMEM16A appeared less pronounced in insulin-producing cells than in exocrine cells., Conclusion: The present findings support the view that, in humans like in rodent, the regulation of anion fluxes in insulin-producing cells may involve both SLC4A4 and TMEM16A., (Copyright © 2012 S. Karger AG, Basel.)

Aims/hypothesis: Transmembrane protein 27 (TMEM27) is a membrane protein cleaved and shed by pancreatic beta cells that has been proposed as a beta cell mass biomarker. Despite reports of its possible role in insulin exocytosis and cell proliferation, its function in beta cells remains controversial. We aimed to characterise the function of TMEM27 in islets and its potential use as a beta cell mass biomarker., Methods: To determine TMEM27 function, we studied TMEM27 gene expression and localisation in human healthy and diabetic islets, the correlation of its expression with cell cycle and insulin secretion genes in human islets, its expression in tungstate-treated rats, and the effects of its overproduction on insulin secretion and proliferation in a beta cell line and islets. To elucidate its utility as a beta cell mass biomarker, we studied TMEM27 cleavage in a beta cell line, islets and primary proximal tubular cells., Results: TMEM27 mRNA levels in islets are lower in diabetic donors than in controls. Its gene expression correlates with that of insulin and SNAPIN in human islets. TMEM27 expression is downregulated in islets of tungstate-treated rats, which exhibit decreased insulin secretion and increased proliferation. TMEM27 overproduction in a beta cell line and islets significantly enhanced glucose-induced insulin secretion, with modest or no effects on proliferation. Finally, TMEM27 is cleaved and shed by renal proximal tubular cells and pancreatic islets., Conclusions/interpretation: Our data support a role for TMEM27 in glucose-induced insulin secretion but not in cell proliferation. The finding that its cleavage is not specific to beta cells challenges the current support for its use as a potential beta cell mass biomarker.

The over-expression of the islet amyloid polypeptide (IAPP) gene could be a causal factor for islet amyloidosis and beta-cell destruction in Type 2 diabetes (T2DM). An IAPP gene promoter polymorphism, IAPP-132G to A, has been associated with T2DM in Spain. To investigate this polymorphism in other cohorts and in relation to therapy, DNA from 425 T2DM and 279 unrelated, non-diabetic UK subjects (ND) and 102 T2DM and 80 ND Finnish subjects was examined. The relationship of amyloid severity (percent amyloid/islet) to prevalence (number of islets affected) and the association of IAPP-132G/A with amyloid was determined in post-mortem pancreas from 38 T2DM subjects. The -132G/A was not associated with T2DM in the UK cohorts (4.5% T2DM; 3.2% ND) or associated with requirement for insulin therapy by 6 years. The mutation was and undetected in the Finnish samples but a new variant, -166T/C, was identified in 2 Finnish T2DM subjects. -132G/A was found in 2/38 diabetic, amyloid-containing and 3/19 ND, amyloid-free subjects. The islet amyloid severity was linearly correlated with the prevalence in T2DM. The IAPP-132G/A promoter polymorphism is not associated with T2DM, a requirement for insulin therapy or with the degree of islet amyloidosis in cohorts from the UK or Finland.

Objective: To study clinical characteristics, beta-cell function, HLA typing and mutations in the hepatocyte nuclear factor (HNF)-1alpha and HNF-4alpha genes in Type 1 diabetes mellitus (T1D) patients without pancreatic autoantibodies., Design and Methods: Twenty patients without pancreatic autoantibodies (Ab neg) and 20 with autoantibodies (Ab pos), age/gender matched, were included (age 17-34 years). Islet cell, glutamic acid decarboxylase, tyrosine phosphatase and insulin autoantibodies, basal and stimulated C-peptide were measured. HLA-DRB1-DQA1-DQB1 typing and screening for mutations in the HNF-1alpha and HNF-4alpha genes were performed., Results: No differences were found in clinical presentation, metabolic control and beta-cell function in the two groups (onset or after 12 months). DRB1*0301-DQA1*0501-DQB1*0201 was the most frequent haplotype in both groups but we found a higher proportion of protective T1D haplotypes and Asp(beta57) in the Ab neg group, but in all the cases in combination with susceptible T1D haplotypes. We found two previously reported polymorphisms (HNF-1alpha, Ala98Val; HNF-4alpha, Thr130Ile) in Ab neg and a new variant (Ser165Gly) in the HNF-4alpha gene in an Ab pos subject. Conclusions In a non-paediatric population with newly diagnosed T1D, the absence of islet antibodies does not imply clinical or metabolic differences when compared with those cases with islet antibodies. Despite a similar HLA-DR/DQ typing, the presence of protective alleles and molecular properties in a higher proportion in the Ab neg group suggests that these factors could modulate the presence or absence of islet antibodies. Variants in HNF-1alpha and HNF-4alpha are unlikely to be major contributors to the pathogenesis of diabetes in antibody-negative T1D.

The lysosomal enzyme iduronate-2-sulfatase (IDS) is expressed in pancreatic islets and is responsible for degradation of proteoglycans, such as perlecan and dermatan sulfate. To determine the role of IDS in islets, expression and regulation of the gene and localization of the enzyme were investigated in mouse pancreatic islets and clonal cells. The Ids gene was expressed in mouse islets and beta- and alpha-clonal cells, in which it was localized intracellularly in lysosomes. The transcriptional expression of Ids in mouse islets increased with glucose in a dose-dependent manner (11.5, 40.2, 88, and 179% at 5.5, 11.1, 16.7, and 24.4 mM, respectively, P < 0.01 for 16.7 and 24.4 mM glucose vs. 3 mM glucose). This increase was not produced by glyceraldehyde (1 mM) or 6-deoxyglucose (21.4 mM) and was blocked by the addition of mannoheptulose (21.4 mM). Neither insulin content nor secretory response to glucose (16.7 mM) was altered in mouse islets infected with lentiviral constructs carrying the IDS gene in sense orientation. Furthermore, no decrease in islet cell viability was observed in mouse islets carrying lentiviral contracts compared with controls. However, insulin content was reduced (35% vs. controls, P < 0.001) in islets infected with IDS antisense construct, while the secretory response of those islets to glucose was maintained. Inhibition of IDS by antisense infection led to an increase in lysosomal size and a high rate of insulin granule degradation via the crinophagic route in pancreatic beta-cells. We conclude that IDS is localized in lysosomes in pancreatic islet cells and expression is regulated by glucose. IDS has a potential role in the normal pathway of lysosomal degradation of secretory peptides and is likely to be essential to maintain pancreatic beta-cell function.

Aims/hypothesis: Cyclin-dependent kinase 4 (Cdk4) is crucial for beta cell development. A mutation in the gene encoding for Cdk4, Cdk4R24C, causes this kinase to be insensitive to INK4 cell cycle inhibitors and induces beta cell hyperplasia in Cdk4R24C knockin mice. We aimed to determine whether this Cdk4R24C mutation also affects proper islet function, and whether it promotes proliferation in human islets lentivirally transduced with Cdk4R24C cDNA., Methods: Our study was conducted on wild-type and Cdk4R24C knockin mice. Pancreases were morphometrically analysed. Intraperitoneal glucose tolerance tests and intravenous insulin tolerance tests were performed on wild-type and Cdk4R24C mice. We also did in vitro islet perifusion studies and islet metabolic labelling analysis. Human islets were transduced with Cdk4R24C cDNA., Results: Pancreatic islets from Cdk4R24C knockin mice exhibit a larger insulin-producing beta cell area and a higher insulin content than islets from wild-type littermates. Insulin secretion in response to glucose is faster and reaches a higher peak in Cdk4R24C mice without leading to hypoglycaemia. Conversion of pro-insulin into insulin and its intermediates is similar in Cdk4R24C and wild-type mice. Glucose utilisation and oxidation measured per islet were similar in both experimental groups. Insulin secretion was faster and enhanced in Cdk4R24C islets perifused with 16.7 mmol/l glucose, with slower decay kinetics when glucose returned to 2.8 mmol/l. Moreover, human islets expressing Cdk4R24C cDNA exhibited higher beta cell proliferation., Conclusions/interpretation: Despite their hyperplastic growth, Cdk4R24C insulin-producing islet cells behave like differentiated beta cells with regard to insulin production, insulin secretion in response to glucose, and islet glucose metabolism. Therefore Cdk4 could possibly be used to engineer a source of beta cell mass for islet transplantation.

Aims/hypothesis: Sodium tungstate has recently emerged as an effective oral treatment for diabetes. We examined the effects of tungstate administration in the beta-cell mass of the pancreas as well as its therapeutic potential., Methods: Sodium tungstate was administered via drinking water to healthy and neonatal streptozotocin (nSTZ)-diabetic rats for one month. The pancreas from each rat was removed and morphometric and immunocytochemical studies were carried out. The molecular mechanism of tungstate's action was also studied., Results: In nSTZ rats administration of this compound normalised glycaemia, and increased insulinaemia and islet insulin content. Blood glucose concentrations were normalised as early as on day 4 of treatment, and tungstate treatment produced a partial recovery of beta-cell mass. The rats remained normoglycaemic after tungstate withdrawal. Morphometric studies showed that the increase in beta-cell mass was not due to beta-cell hypertrophy but to hyperplasia, with an increase in islet density in treated diabetic rats. Tungstate treatment increased extra-islet beta-cell replication without modifying intra-islet beta-cell replication rates. Moreover, the treatment induced increases in insulin-positive cells located close to ducts; and in PDX-1 positive cells scattered in the exocrine tissue, suggesting active neogenesis. In islets from treated diabetic rats, tungstate is able to increase the phosphorylation state of PDX-1 through the activation of p38., Conclusion/interpretation: These observations indicate that tungstate treatment is able to regenerate a stable, functional pancreatic beta-cell population which leads to and maintains normoglycaemia.

CD14, a GPI-linked membrane protein, is a component of the lipopolysaccharide (LPS) receptor complex, one of the pattern-recognizing receptors (PRR) expressed by myeloid lineage cells. Here we report that CD14, the functionally linked toll-like receptor molecules, TLR2 and TLR4, and the associated molecule MD-2 are expressed in endocrine cells of the human pancreatic islets. CD14 expression in human pancreatic islets was determined by immunofluorescence staining of tissue sections and primary cultures, and confirmed by flow cytometry of dispersed normal islets and SV40-transformed islet cells (HP62). The latter cells synthesized and secreted CD14 in response to lipopolysaccharide (LPS) in a time- and dose-dependent manner. Reverse transcription polymerase chain reaction (RT-PCR)-Southern was positive for CD14, TLR2, TLR4 and MD-2 in human pancreas, purified islets and HP62 cells. In vitro experiments using rat islets (also positive for CD14 by RT-PCR) and HP62 cells showed that LPS regulates glucose-dependent insulin secretion and induces inflammatory cytokines [interleukin (IL)-1alpha, IL-6 and tumour necrosis factor (TNF)-alpha]. The functional expression of CD14 and associated molecules in islet beta cells adds a new pathway that islet cells may follow to adjust their function to endotoxaemia situations and become vulnerable to the inflammatory events that occur during diabetogenic insulitis.

Plasma insulin in healthy subjects shows regular oscillations, which are important for the hypoglycemic action of the hormone. In individuals with type 2 diabetes, these regular variations are altered, which has been implicated in the development of insulin resistance and hyperglycemia. The origin of the change is unknown, but derangement of the islet secretory pattern has been suggested as a contributing cause. In the present study, we show the dynamics of insulin release from individually perifused islets isolated from three subjects with type 2 diabetes. Insulin release at 3 mmol/l glucose was 10.5 +/- 4.5 pmol.g(-1).s(-1) and pulsatile (0.26 +/- 0.05 min(-1)). In islets from one subject, 11 mmol/l glucose transiently increased insulin release by augmentation of the insulin pulses without affecting the frequency. Addition of 1 mmol/l tolbutamide did not increase insulin release. In islets from the remaining subjects, insulin release was not affected by 11 mmol/l glucose. Tolbutamide transiently increased insulin release in islets from one subject. Insulin release from four normal subjects at 3 mmol/l glucose was 4.3 +/- 0.8 pmol.g(-1).s(-1) and pulsatile (0.23 +/- 0.03 min(-1)). At 11 mmol/l glucose, insulin release increased in islets from all subjects. Tolbutamide further increased insulin release in islets from two subjects. It is concluded that islets from the three individuals with type 2 diabetes release insulin in pulses. The impaired secretory response to glucose may be related to impaired metabolism before mitochondrial degradation of the sugar.

Mutations in the gene encoding hepatic nuclear factor 1-alpha (HNF1-alpha) cause a subtype of human diabetes resulting from selective pancreatic beta-cell dysfunction. We have analyzed mice lacking HNF1-alpha to study how this protein controls beta-cell-specific transcription in vivo. We show that HNF1-alpha is essential for the expression of glut2 glucose transporter and L-type pyruvate kinase (pklr) genes in pancreatic insulin-producing cells, whereas in liver, kidney, or duodenum tissue, glut2 and pklr expression is maintained in the absence of HNF1-alpha. HNF1-alpha nevertheless occupies the endogenous glut2 and pklr promoters in both pancreatic islet and liver cells. However, it is indispensable for hyperacetylation of histones in glut2 and pklr promoter nucleosomes in pancreatic islets but not in liver cells, where glut2 and pklr chromatin remains hyperacetylated in the absence of HNF1-alpha. In contrast, the phenylalanine hydroxylase promoter requires HNF1-alpha for transcriptional activity and localized histone hyperacetylation only in liver tissue. Thus, different HNF1-alpha target genes have distinct requirements for HNF1-alpha in either pancreatic beta-cells or liver cells. The results indicate that HNF1-alpha occupies target gene promoters in diverse tissues but plays an obligate role in transcriptional activation only in cellular- and promoter-specific contexts in which it is required to recruit histone acetylase activity. These findings provide genetic evidence based on a live mammalian system to establish that a single activator can be essential to direct nucleosomal hyperacetylation to transcriptional targets.

In a new experimental type 2 diabetic syndrome, a 40% reduction of pancreatic beta cells was observed by morphometric analysis. In diabetic islets, as compared to control islets, insulin release was decreased in response to high glucose but not to other stimuli, and total glucose oxidation and utilization were unchanged or slightly reduced. The extent of metabolic and functional impairment appeared proportional to the beta-cell loss. However, a substantial decrease was found in protein level and activity (by 77 and 60%, respectively, versus controls) of mitochondrial FAD-glycerophosphate dehydrogenase (mGDH), the key enzyme of the glycerophosphate shuttle. Interestingly, in diabetic islets, as recently reported for mGDH-deficient transgenic mice, definite functional alterations (mainly in response to D-glyceraldehyde) were only obtained upon pharmacological blockade of the second shuttle (i.e. malate-aspartate) responsible for mitochondrial transfer of reducing equivalents. In conclusion, in this diabetes model with reduction of beta-cell mass, the islets, despite decreased mGDH amount and activity, appear metabolically and functionally active in vitro, likely through the intervention of adaptive mechanisms, yet prone to failure in challenging situations.

We studied the contribution of the constitutive and the regulated pathways to the total secretion of islet amyloid polypeptide (IAPP) in human pancreatic islets after prolonged culture at either 5.5 or 24.4 mM glucose. In islets cultured in low concentrations of glucose, the secretion of IAPP in response to glucose was unaffected by brefeldin A (BFA) and completely blocked by ethyleneglycoltetraacetic acid. In islets cultured in high glucose concentrations, it was strongly inhibited by both agents. BFA had no effect on the glucose-induced insulin secretion. The determination of the islet peptide contents and the mRNA levels revealed a several-fold increase in the IAPP/insulin molar ratio of islets cultured in high glucose concentrations. Thus, prolonged exposure of human islets to high concentrations of glucose results in an increase in the synthesis of IAPP with respect to insulin. As a result, the release of IAPP through a mechanism sensitive to BFA is favored. These data support the hypothesis that IAPP and insulin are regulated in a noncoordinated way in human pancreatic islets.

Although P2 receptors for adenosine 5'-triphosphate (ATP) and/or adenosine 5'-diphosphate (ADP) have been characterized in mammalian pancreatic beta cells, no evidence for an insulin-secreting effect of P2 receptor agonists has been reported as yet in humans. The present study aimed at investigating whether P2 receptor agonists could stimulate insulin release in human pancreatic islets obtained from brain-dead organ donors. Experiments were performed using different glucose concentrations and insulin was measured by radioimmunoassay. When the glucose concentration (8.3 mmol/L) was slightly stimulating for insulin release, alpha,beta-methylene ATP (200 micromol/L) and ADPbetaS (50 micromol/L) similarly amplified insulin secretion: both compounds induced a threefold increase in insulin response. In the presence of a nonstimulating glucose concentration (3.0 mmol/L), only alpha,beta-methylene ATP could induce a significant 1.4-fold increase in insulin release, ADPbetaS being completely ineffective. These results give evidence that P2 receptor agonists are effective in stimulating insulin release in humans, the effect of the P2Y agonist being essentially glucose dependent.

D-mannoheptulose was recently proposed to be transported into cells at the intervention of GLUT2. Since GLUT1, rather than GLUT2, represents the major carrier system for the transport of monosaccharides across the islet B-cell plasma membrane in human subjects, the uptake of D-mannoheptulose and its metabolic and secretory effects were investigated in human islets. The uptake of D-glucose reached much more rapidly a close-to-equilibrium value in isolated islets than in pieces of pancreas obtained from the same donor. The distribution space of D-[3H]mannoheptulose in the human islets largely exceeded that of [U-14C]sucrose, considered as an extracellular marker, and did not differ significantly from that of 3HOH. In the human islets, the heptose (10.0 mM) inhibited both D-[5-3H]glucose utilization and D-[U-14C] glucose oxidation, and decreased glucose-stimulated insulin release to the same extent as D-mannoheptulose hexaacetate. These findings indicate that a suitable radioactive analog of D-mannoheptulose could be used, in human like in rat islets, for preferential labelling of the endocrine moiety of the pancreatic gland.

Previous reports concerning the regulation of glucokinase expression in beta cells have been done using cell models from rodent origin. Evidence is lacking so far to implicate the same regulatory mechanisms in human cells. In this study, we investigate the effects of glucose on the expression of glucokinase using isolated human pancreatic islets. High glucose (16.7 mM), in a time-dependent manner, increases the amount of immunoreactive glucokinase (+150% after 7 days culture, P < 0.01) without apparent changes in glucokinase gene expression, suggesting that glucose exerts its effect at a posttranscriptional level. Mannose, but not the nonmetabolized hexoses, 3-O-methylglucose or 2-deoxyglucose, increases glucokinase protein content. Even though these findings are compatible with an involvement of signals derived from glucose metabolism, additional data argue against this hypothesis: (i) a glucokinase inhibitor (mannoheptulose) does not block glucose-induced increase in glucokinase content and (ii) other metabolic fuels (amino acids) are ineffective. We suggest that the glucose molecule, by mechanisms yet to be defined, but probably not involving its metabolism, regulates human glucokinase expression., (Copyright 2000 Academic Press.)

Human pancreatic islets were cultured for 63 hr at 2.8 or 16.7 mM D-glucose in the absence or presence of dexamethasone. In the 1.0 to 10 microM range, dexamethasone caused a concentration-related decrease in the FAD (flavin adenine dinucleotide)-linked mitochondrial glycerophosphate dehydrogenase (mGDH) mRNA content of the islets, and decreased both the mGDH content of the islets and the catalytic activity of the enzyme in islet homogenates, these effects being often more marked in islets cultured at 16.7 mM, rather than 2.8 mM, D-glucose. Even after culture in the presence of no more than 10 nM dexamethasone, namely under conditions in which the mGDH mRNA content and activity were both virtually unaffected, the corticosteroid restored the capacity of the beta-cells to display an increase in insulin output in response to a rise in D-glucose concentration in islets first cultured at 2.8 mM D-glucose but suppressed the insulinotropic action of the hexose in islets first cultured at 16.7 mM D-glucose. Whilst revealing an untoward effect of high concentrations of dexamethasone upon mGDH mRNA, content and activity in human islets, these findings also document a dual effect of a low concentration of the corticosteroid (10 nM) upon the secretory responsiveness of human islets to D-glucose, independently of any significant change in mGDH gene expression. It is proposed that such a dual action may account, in part at least, for both the well known increase in insulin output found in hypercorticism and the more recently discovered unfavourable direct effect of corticosteroid hormones on the secretory activity of islet beta-cells.

Effective cryopreservation of pancreatic islets would be valuable in several contexts: for the assessment of islet cell viability, the measurement of beta-cell function, and the maintenance of viability and sterility prior to islet transplantation. In this study, isolated rat islets were cryopreserved or not following overnight culture and the most suitable preservation solution for transportation between centers was sought. Unfrozen and frozen-and-thawed islets were allocated to each of four different groups: untreated controls; cultured overnight in RPMI at 37 degrees C; cold stored at 4 degrees C in RPMI for 18-24 h; and stored at 4 degrees C in University of Wisconsin (UW) solution for 18-24 h. The greatest cell viability, as assessed by ethidium bromide/acridine orange staining and image analysis, was observed when postthawed islets were cultured in RPMI, whereas the least viable samples were those that were stored in UW solution. Measurement of insulin content and secretion in static incubation assays using 2.8 and 16.7 mM glucose showed that all treated groups exhibited a significant insulin secretory response to glucose stimulation whereas the untreated frozen-thawed islets failed to show any response. The cryopreserved islets in each group were equally successful in reversing hyperglycemia in streptozotocin-treated allogeneic rats when grafted intraportally in sufficient numbers (2000-2500). The groups also showed a similar mean graft survival time of 6-7 days before rejection. However, the best experimental group (the postthaw cultured islets) failed to cure diabetic rats when grafted in a smaller numbers (<2000). These data demonstrate prompt and sustained function in cryopreserved islets when they were maintained by any of the methods studied if they were grafted in sufficient numbers. We conclude that cold storage of thawed cryopreserved islets using either RPMI or UW solution is an effective method for their transportation and/or storage, but does not reduce their immunogenicity before transplantation., (Copyright 1998 Academic Press.)

Reduction of amylin content and secretion in rat islets was attempted by transduction with an adenovirus bearing a 0.2-kb fragment of rat amylin cDNA inserted in the antisense orientation (AdCMV-alpha amylin). Exposure of islets to AdCMV-alpha amylin at a multiplicity of infection (moi) of 200 (1.2 x 10(7) pfu/ml) reduced amylin mRNA levels by 37 +/- 5% (p < 0.005), whereas infection with an adenovirus expressing the reporter gene of beta-galactosidase (AdCMV-lacz) did not modify amylin expression. Transduction with the antisense construct was specifically associated with the decrease (30 +/- 6%; p < 0.001) in the amylin content. Insulin content was unaltered in AdCMV-alpha amylin islets compared to AdCMV-lacz-transduced or untransduced cells. Basal amylin secretion (2.8 mM glucose) was 36 +/- 3% (p < 0.005) lower in AdCMV-alpha amylin islets than in untransduced or AdCMV-lacz-transduced islets. In contrast, no difference in amylin secretion in response to high glucose concentrations (16.7 mM) was detected in AdCMV-alpha amylin-transduced islets. Thus, a reduction of amylin content and basal secretion in islet cells can be achieved by the adenovirus-mediated expression of antisense RNA.

In the present study we investigate whether glucose concentration could have an effect on proinsulin biosynthesis and processing. We cultured control human islets under chronic high and low glucose concentrations. After the culture period, islets were pulse-labeled and chased for different periods of time. Proteins from islets were collected, insulin immunoprecipitated, and analyzed by alkaline-urea gel electrophoresis. We have found an accelerated rate of proinsulin conversion by those islets exposed to high glucose concentration (at 24.4 mM of glucose), but not by those islets cultured at low glucose concentration (at 5.5 mM of glucose). However, we do not observe any decrease or increase on newly proinsulin synthesis in any of these conditions.

The revascularization of islets of Langerhans transplanted in heterotopic sites like the liver by portal vein embolization or the renal subcapsular space is a major process necessary for the viability of grafted cells. This process has been extensively studied by different techniques and the results have shown that islet revascularization is an early phenomenon that takes place soon after transplantation. In this report we have analyzed by a double indirect immunofluorescence technique, the revascularization process of purified endocrine islet beta-cells transplanted in the renal subcapsular space of syngeneic rats. Lewis rats were grafted with islets cultured for 24 h, with a suspension of purified beta-cells cultured for 24 h, and with a suspension of purified beta plus nonbeta-cells cultured for 24 h. Rats were killed at different days after implantation and the kidney bearing the grafts were snap frozen and immunohistochemically stained with a rabbit anti factor VIII antiserum (which labels endothelial cells). Immunocytochemical analysis revealed that cultured islets completed revascularization by days 3-5 after transplantation, as shown by the detection of capillary endothelial cells within and surrounding the islets. Within purified endocrine beta-cell grafts, the presence of numerous endothelial cells was not observed until days 10-14, indicating that revascularization of beta-cells with host vessels is not such an early phenomenon as it takes place in whole isolated islets. Conversely, the addition of a population of endocrine nonbeta-cells to the purified islet cell grafts, partially accelerated the revascularization of pure beta-cell grafts, which showed the presence of abundant capillary endothelial cells already at day 7 after transplantation, indicating that some other unidentified factors besides the absence of endothelial cells may explain the retardation of beta-cell grafts revascularization.

The cDNA fragments coding for the FAD-, glycerophosphate- and calcium-binding domains of mitochondrial glycerophosphate dehydrogenase (mGDH) were synthetized using RNA extracted from freshly isolated pancreatic islets of a normal subject and two-non-insulin-dependent diabetic patients. Single strand conformation polymorphism analysis of the PCR products yielded the same mobility as control cDNA probes. Likewise, the nucleotide sequence and corresponding amino acid sequence were identical to the normal gene bank sequence. These findings argue against the presence, in pancreatic islets, of an mGDH isoform distinct from that previously characterized in extrapancreatic organs.

Considerable interest has recently focused on the possible role of alterations in mitochondrial activity and mutations in the mitochondrial genome for the development of non-insulin-dependent diabetes. Our study aimed at investigating the normal mitochondrial respiratory chain activity of nonpurified and purified islet cells to further explore whether some diabetic states are associated with alterations of mitochondrial oxidative processes. For this purpose, pancreatic islets were isolated from Wistar rats. Unpurified islet cells were obtained in the presence of trypsin and DNAse, and purified beta and non-beta cells were prepared by autofluorescence-activated sorting using a flowcytometer. Intact cell respiration and substrate oxidation in digitonin-permeabilized cells were measured polarographically with a Clark oxygen electrode in a micro-water-jacketed cell. Specific activity of the individual complexes of the respiratory chain was determined spectrophotometrically in unpurified islet cells. The relative amount of mitochondrial (mtDNA) and nuclear (nDNA) DNA in all three cell populations and in rat brain and skeletal muscle was estimated by dot blotting. The intact cell respiration of unpurified islet cells corresponds to the mean of values obtained for beta and non-beta islet cells. Oxidation rates of different substrates by permeabilized beta cells were lower than those for unpurified and non-beta cells. The amount of mtDNA relative to nDNA was similar in all three groups of cells, and was also similar to that obtained from brain and skeletal muscle. In summary, we have described mitochondrial respiratory chain activity in unpurified, beta, and non-beta islet cells. Our results represent an initial step in investigating the potential pathogenic role that alterations in oxidative phosphorylation could play in some diabetic states.

Cryopreservation is an effective method of islet storage and may facilitate clinical trials of islet transplantation. It was the aim of the present study to evaluate the in vitro viability of cryopreserved rat islets, including the response to nonglucose secretagogues and glucose oxidation. After pancreatic digestion via intraductal injection of collagenase, 75- to 200-micron Wistar rat islets were handpicked and cultured in RPMI 1640 (glucose 11.1 mmol/L) and randomized into two groups: control (cultured 20 to 24 hours at 37 degrees C) and cryopreserved (after 20 to 24 hours of culture at 37 degrees C, islets were cryopreserved according to Rajotte's protocol: freezing velocity, -0.25 degree C/min; thawing velocity, 200 degrees C/min). In the two groups, we evaluated recovery, insulin content per islet, staining viability (ethidium bromide/orange acridine; semiquantitative scoring, measuring the viable area of the islet from 0 = less viable to 3 = more viable), insulin secretion after glucose and nonglucose secretagogues, and oxidation of D-[U-14C]glucose. The results for the control group were always higher for the following: recovery (95.4% +/- 1.2% v 83.0% +/- 2.1%, P = .00), insulin content (2,203.9 +/- 335.2 v 1,443.3 +/- 171.8 microU/islet, P = .03), insulin secretion after 5.5 mmol/L glucose (61.3 +/- 8.0 v 28.3 +/- 3.4 microU/islet/90 min, p = .00), 16.7 mmol/L glucose (151.4 +/- 16.1 v 98.7 +/- 14.1 microU/islet/90 min, p = .03), 10 mmol/L L-leucine +10 mmol/L L-glutamine (125.6 +/- 27.9 v 56.8 +/- 6.4 microU/islet/90 min, P = .05), and 10 mmol/L L-arginine (202.5 +/- 27.5 v 128.8 +/- 14.2 microU/islet/90 min, P = .01), and glucose oxidation at 5.5 mmol/L (12.5 +/- 1.1 v 7.9 +/- 0.6 pmol/islet/120 min, P = .00) and at 16.7 mmol/L (26.1 +/- 2.6 v 14.3 +/- 1.6 pmol/islet/120 min, P = .00). No significant differences in staining viability were found between groups (2.35 and 2.48, respectively, P = .55). However, cryopreserved and control islets showed a significant increase in insulin secretion and glucose oxidation after increasing the glucose concentration from 5.5 to 16.7 mmol/L. We conclude that when glucose is increased, cryopreserved islets keep the capacity to increase insulin secretion, but cryopreservation produces a significant decrease in several islet viability characteristics. This decrease may be due to a decline of beta-cell number per islet and/or a decrease in the content of insulin per beta cell.

Intracellular pathways by which glucose regulates islet amyloid polypeptide (IAPP) gene expression in pancreatic islets were studied. IAPP mRNA levels were threefold higher in islets cultured with 16.7 mM glucose compared with control (5.5 mM glucose). Mannose and amino acids but not 2-deoxyglucose or 6-deoxyglucose mimicked the effect of glucose. Mannoheptulose (a glycolysis inhibitor) and verapamil and diazoxide (which affect calcium signaling pathway) abolished the difference in islet IAPP mRNA content between high and low glucose. At low glucose, IAPP mRNA levels were increased 1.9-fold in islets treated with forskolin or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) but not with 12-O-tetradecanoylphorbol 13-acetate. Insulin mRNA levels were 1.6-fold higher in islets cultured at high glucose than controls; glucose metabolism was required, whereas no effects of cAMP or diazoxide were observed. IAPP and insulin were cosecreted into the media. We conclude that glucose regulation of IAPP mRNA abundance requires intracellular metabolism of the hexose and that calcium may serve as a mediator of this effect; cAMP but not protein kinase C possibly participates in this regulation.

Intracellular pathways involved in glucose stimulation of IAPP gene expression were studied in human pancreatic islets. Glucose (16.7 mM), but not mannose, caused a 2.3-fold increase in IAPP mRNA levels; this effect was inhibited by actinomycin D. In the presence of the non-metabolizable 6-deoxyglucose (16.7 mM) IAPP mRNA levels were markedly depleted. Both mannoheptulose and verapamil blocked glucose-induced stimulation of the IAPP gene. The magnitude of the insulin gene response to glucose was smaller (1.3-fold); none of the above-mentioned agents had significant effects on insulin mRNA content. Tunicamycin elicited a 2.4- and 2.7-fold increase in IAPP mRNA levels in the low and high glucose media, respectively; however, it did not change insulin mRNA. It had no effect on rat IAPP or insulin mRNAs, either. We conclude that IAPP gene expression is regulated by signals derived from glucose metabolism and that intracellular calcium may be involved in this response. IAPP and insulin genes are not co-regulated in cultured human pancreatic islets.

Human islets from an adult subject with nesidioblastosis were isolated and used to perform in vitro studies. Isolated nesidioblastotic islets showed an increased basal rate of insulin secretion (nesidioblastotic islets, 81.3 +/- 6.4 vs. control islets, from cadaveric organ normal donors, 10.2 +/- 0.9 microU/islet/90 min) without any further release with increasing glucose concentration. In addition, islets isolated from the pancreas with nesidioblastosis contained more insulin than control islets, 1,547.0 +/- 128.7 vs. 935.0 +/- 51.7 microU/islet, and the levels of insulin mRNA were also higher than those measured in controls. Interestingly, the serum of the subject with nesidioblastosis contained autoantibodies that stained brightly and selectively a population of islet cells whose distribution coincided with that expected of alpha cells. In summary, pancreatic islets in nesidioblastosis display beta-cell functional abnormalities. Moreover, the finding of antibodies against islet cells is a common feature in the serum of nesidioblastotic subjects; nevertheless, their pathological significance warrants further investigation.

Cryopreservation of islets of Langerhans is a necessary procedure since human pancreatic islet transplantation has become a reality for the clinical treatment of Type I, insulin-dependent diabetes mellitus. Although successful cryopreservation of rodent and human islets is a well-established technique for islet storage after isolation and purification, little is known about the influence of the freeze-thaw procedure on the islets' potential to induce angiogenesis and revascularization, a major process necessary for the viability of grafted cells. In this study, the revascularization process of cryopreserved islets transplanted in the liver and in the renal subcapsular space of diabetic and nondiabetic rats is analyzed by a double indirect immunofluorescence technique. Frozen-thawed pancreatic islets were cooled slowly to -40 degrees C, stored at -196 degrees C, and thawed rapidly. Lewis rat were grafted with either Lewis (isografts) or Wistar (allografts) overnight-cultured and frozen-thawed islets obtained by collagenase digestion. Rats were killed different days after implantation, and the livers and kidneys bearing the grafted islets were snap-frozen and immunohistochemically stained with a double immunofluorescence technique using a rabbit anti-factor VIII antiserum (which labels endothelial cells) and a guinea pig anti-insulin antibody. Overnight-cultured islet grafts completed revascularization by Days 4-7 after transplantation, as shown by the detection of endothelial cells within and surrounding the islets. The identical staining pattern of revascularization was observed in islets frozen-thawed before transplantation. It is concluded that islet cryopreservation is a suitable technique for long-term storage prior to transplantation since it does not interfere with the neovascularization process of islet grafts.

The aim of the study was to determine the appropriate dose of subcutaneous insulin to induce "beta-cell rest" without any hypoglycemic risk, as the first stop in the investigation of its potential effect in preventing or delaying clinical diabetes mellitus onset in high-risk subjects. Four subjects at high risk for type I diabetes mellitus (first-degree relatives, islet cell antibodies (ICA)-positive, and with diminished first-phase insulin secretion) were compared with four healthy individuals. After hospitalization, urinary C-peptide excretion (UCP) and 24-hour serum profiles for glucose were measured before and after administration of NPH insulin 0.1, 0.2, and 0.3 U x kg body weight per day subcutaneously in a single dose on 4 consecutive days. After insulin 0.1 U x kg body weight, a significant inhibition of endogenous insulin secretion was observed in high-risk subjects, but not in control subjects. There was no further inhibition when a higher insulin dose (0.2 and 0.3) was administered. A sustained beta-cell rest was obtained after 3, 6, and 12 months of treatment with 0.1 U x kg body weight per day as outpatient therapy in high-risk subjects. With this dose, no subject developed hypoglycemia (plasma glucose <50 mg/dL), whereas this adverse effect was detected after 0.2 and 0.3 U x kg body weight in both groups. In conclusion, our results indicate that administration of NPH insulin 0.1 U x kg bodyweight per day induces beta-cell rest without the undesirable effect of hypoglycemic episodes. This is a preliminary study to investigate the potential beneficial effect of insulin in preventing or delaying type I diabetes mellitus in subjects at high risk for the disease.

Western blotting of pancreatic islet extracts from either hereditarily diabetic Goto-Kakizaki rats (GK rats) or animals injected with streptozotocin during the neonatal period (STZ rats) demonstrated a pronounced decrease of immunoreactive mitochondrial glycerophosphate dehydrogenase (m-GDH), when compared to results obtained in islets from control rats. By contrast, the islet glucokinase protein content was either unaffected (GK rats) or much less severely decreased than that of m-GDH (STZ rats). These findings indicate that the impaired activity of m-GDH previously documented in islet homogenates from diabetic rats coincides with a decreased content of this enzyme in the endocrine pancreas.

GLUT2 glucose transporter mRNA has been shown to be underexpressed in pancreatic islets of numerous animal models of non-insulin-dependent diabetes mellitus (NIDDM). It has been proposed that this molecular defect contributes to the pathogenesis of diabetes, although information concerning the expression of GLUT2 in human pancreatic islet tissue is lacking. In contrast to the high abundance of GLUT2 in rat islets, human islets were found to express distinctly low levels of this glucose transporter mRNA and protein. Thus, a sensitive competitive reverse transcription-polymerase chain reaction assay was developed to quantify human GLUT2 mRNA. We obtained pancreases from 4 human organ donors with previously diagnosed NIDDM and 11 nondiabetic donors and found no significant differences in GLUT2 mRNA between the two groups. GLUT2 mRNA was 0.24 +/- 0.08 amol/micrograms RNA (mean +/- SE) in pancreases from humans with diabetes and 0.27 +/- 0.06 amol/microgram RNA in those without this diagnosis. Similarly, human pancreatic islet GLUT2 protein was measured by immunoblot and found to be present at similar levels in two individuals with diabetes relative to six control samples. These results thus demonstrate the existence of species differences in the abundance of islet GLUT2 mRNA and protein. Furthermore, the analysis of islet GLUT2 in a small sample of human organ donors with and without diabetes raises the possibility that decreased beta-cell GLUT2 may not represent a widespread feature of humans with NIDDM.

Conflicting data were previously reported concerning the effect of starvation upon the glucokinase protein and glucokinase mRNA content of pancreatic islets. In the present study, conducted in fed rats and animals starved for 48 h, the catalytic activity of glucokinase in islet homogenates and the content of this enzyme in islets were both decreased by starvation to the same relative extent. These findings support the view that the altered metabolic and secretory response to D-glucose found in islets from starved rats may be attributable, in part at least, to an apparent repression of glucokinase.

Although CD5 + B lymphocytes are mostly committed to the production of polyreactive natural autoantibodies, CD5 + B lymphocytes committed to the production of somatically mutated and monoreactive high-affinity IgM autoantibodies have been also shown. Increased proportions of CD5 + B lymphocytes in some autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM), have been noticed. The present study was undertaken to analyse the differences between CD5 + and CD5- B lymphocyte subsets for production of IDDM-related autoantibodies, i.e. anti-human insulin antibodies (IA) and anti-human islet cell antibodies (ICA). For this purpose, Epstein-Barr Virus (EBV)-transformation of FACS cell-sorted CD5 + and CD5- B lymphocytes and unfractionated enriched B lymphocytes from nine IDDM patients treated exclusively with recombinant human insulin, and from four healthy control subjects was performed; a mean of 102-216 microcultures with a mean of 1,000-2,333 cells/microculture for each B-lymphocyte fraction and individual was established. Data show that both CD5 + and CD5- B-lymphocyte subsets from either normal subjects or from IDDM patients receiving recombinant human insulin, contain B lymphocytes committed to the production of IA-IgM as a common element of their repertoire. In contrast, cells committed to the production of IA-IgG were only detected among the CD5- B lymphocyte subset from some IDDM patients. Only one microculture, out of a total of 6,211 screened (from control subjects and patients), in the CD5- B-cell subset from a recently-diagnosed IDDM patient, was found to produce ICA-IgM lambda. This might suggest that the frequency of circulating B lymphocytes committed to the production of ICA is very low even in IDDM patients bearing serum ICA. EBV-transformed B cells producing the ICA-IgM lambda were stabilized and cloned by somatic hybridization technique. This ICA-IgM lambda human monoclonal antibody, designated HY1-MB91, is not polyreactive, but shows a restricted reactivity with human pancreatic islets, failing to react with other human tissues including cerebellar cortex, and lacking rheumatoid factor and anti-DNA antibody activities. It also lacks reactivity with pancreatic islets from other mammalian species (rat, mouse and monkey) as well as with other rat tissues, including cerebellar cortex. The antigen recognized by HY1-MB91 antibody in human islet cells is a cytoplasmic component mostly found in beta cells.

An insulin response to glucose is required to correct hyperglycemia. Two proteins, the glucose transporter GLUT-2 and the glucose-phosphorylating enzyme glucokinase, have been implicated in the control of glucose metabolism in beta cells. To study the role of glucose transporter GLUT-2 in the regulation of insulin secretion and in the development of diabetes mellitus, we have obtained transgenic mice expressing high levels of GLUT-2 antisense RNA in beta cells. Western blot analysis showed an 80% reduction in GLUT-2 protein in the beta cells of these animals. Islets from transgenic mice showed impaired glucose-stimulated insulin secretion. In addition, much higher levels of blood glucose were detected in transgenic mice than in controls when glucose tolerance tests were performed. These results suggest that the reduction of GLUT-2 in the pancreas could be a crucial step in the development of diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM), in which only the pancreatic beta cells are destroyed by the autoimmune response, is the paradigm of organ-specific autoimmunity. As a result of a combination of factors, the number of immunohistologic/cellular/molecular studies of pancreas in IDDM is very limited. We report here studies conducted in the pancreata of two IDDM patients: one newly diagnosed (case 1) and one long standing (case 2). In case 1, we demonstrated the presence of morphologically normal viable beta cells without evidence of viral infection. In both cases the expression of the autoantigens defined by islet cell Abs and by glutamic acid decarboxylase was markedly reduced in the islet cells whereas expression of hsp60, another putative autoantigen, was normal. Over-expression of HLA class I was detected in 58% of the islets in pancreatic sections and in cultured beta cells in case 1 and also in 30% of islets in case 2 but it was not restricted to any insular cell type. In case 1, there was "inappropriate" HLA class II expression in islets cells but it was a rare finding and not beta cell specific. The analysis of the correlation between class I overexpression, residual insulin, and insulitis suggests that the first event is the increase of HLA class I expression. Of adhesion molecules, ICAM-1, VLA, VCAM, and LFA-3 were normal and only ICAM-1 was moderately overexpressed in and around the islets of case 1 insulitis, as was detected by immunofluorescence which showed that 18% of the islets of case 1 had CD8+ lymphocytes as the predominant population. Reverse transcription-PCR demonstrated moderate V beta skewing and the profile of cytokines expected in CTLs: IL-2, IL-4, IL-10, and IFN-gamma negative, perforin positive. In addition, IFN-alpha, IFN-beta, and IL-6 transcripts were detected in the case 1 pancreas, consistent with the existence of a silent viral infection. Overall, the results indicated that, differently from spontaneous animal models of diabetes, in the pancreas of IDDM patients there are no elements of the inductive phase of the autoimmune response.

The mechanisms by which interleukin-1 (IL-1) exerts destructive action on the pancreatic islet beta-cells remain elusive. Fragmentation of DNA leading to the activation of poly(ADP-ribose) synthetase was investigated in the present study, by assessing the nuclear response to cytokines in rat pancreatic islets. Nuclear fractions display Mg(2+)-dependent poly(ADP-ribose) synthetase activity catalyzing the incorporation of [adenine-U-14C]NAD, with Ka and Km for Mg2+ and NAD amounting to 0.86 mM and 0.43 mM, respectively. Exposure of the nuclear fraction to rIL-1 beta (10 IU/ml) provoked DNA strand breaks and increased nuclear poly(ADP-ribose) synthetase activity (148.4%, P < 0.01). In intact islets, this nuclear response was observed after 18 h culture in medium containing rIL-1 beta, with a concomitant decrease in NAD (88.5%). Brief periods of pre-incubation (90 min) with rIL-1 beta were unable to induce any nuclear activity. Under these conditions, the presence of IFN-alpha (24 U/ml) and TNF (120 U/ml) was necessary to induce a response to rIL-1 beta. Under the latter experimental conditions, a decrease in NAD content was also observed. The nuclear effects of IL-1 beta were modified by nicotinamide (10 mM), an inhibitor of poly(ADP-ribose) synthetase. It is thus conceivable that an increase in poly(ADP-ribose) synthetase activity together with DNA break is implicated in the beta cytotoxic effect of interleukin-1 beta.

Increased circulating insulin and glucagon levels are a common observation in patients with cirrhosis, as well as in portal hypertensive models. Hyperinsulinemia and hyperglucagonemia may be caused either by increased beta- and alpha-cell secretion or by defective hepatic clearance of these hormones. To elucidate whether an abnormal endocrine pancreatic function might contribute to the hyperinsulinism or to the hyperglucagonism observed in chronic portal hypertension, insulin and glucagon secretion were measured in vitro in isolated pancreatic islets from rats with partial portal vein ligated and rats with cirrhosis caused by carbon tetrachloride poisoning. Both rats with partial portal vein ligation and rats with cirrhosis caused by carbon tetrachloride poisoning exhibited hyperinsulinism and hyperglucagonism as compared with control rats. Isolated pancreatic islets from both experimental portal hypertensive models showed an impaired insulin secretion after glucose stimulation. On the contrary, glucagon secretion was significantly increased, and there was a markedly enhanced response to arginine. This increased in vitro glucagon production could not be corrected, even in the presence of high glucose concentrations in the incubation medium. Therefore our data show that although hyperglucagonism in rats with partial portal vein ligation and in rats with cirrhosis caused by carbon tetrachloride poisoning is promoted by an enhanced alpha-cell secretion, hyperinsulinism is associated with impaired beta-cell secretion.

In order to assess the functional properties of human pancreatic islets we have evaluated insulin secretion, glucose metabolism and insulin mRNA synthesis. Particular attention was given to evaluate how culture and exposure to high glucose concentrations "in-vitro" influence the subsequent function of these cells. Islets were cultured for 1 and 7 days at 5.5 or 16.7 mM glucose concentrations. Insulin response to glucose, glucose+forskolin and leucine+glutamine was evaluated in freshly isolated and cultured islets. Catabolism of D-glucose was studied measuring D-(6-14C) glucose oxidation and D-(5-3H) glucose utilization. Northern blot analysis was performed to measure insulin mRNA levels. In freshly isolated islets there a low insulin response to all stimulus, but especially to nutrient secretagogues. Total glucose utilization was also lower than expected, with preferential disturbances in mitochondrial oxidative events. Independently of glucose concentration, culture improved insulin release, glucose catabolism and especially glucose oxidation, and insulin mRNA synthesis. After 7-days culture, high glucose concentration in the medium exerted beneficial effects on the overall functional activities of human pancreatic islets. Our study, 1) indicates that functional properties, initially impaired in freshly isolated human islets, improve after culture and become comparable to those previously proposed for rodent islets; and 2) argues against high glucose concentrations producing a deleterious effect on human beta-cell function. Rather they point to the concept that high glucose levels are beneficial for human islets function after long-term exposure in-vitro.

Dithizone (DTZ) is a selective stain for pancreatic islets which facilitates their identification, being of special interest in human islet isolation assessment. Nevertheless, there are few studies concerning its potential toxic effects on islet function. In our study, we have evaluated the effects of DTZ (dissolved in dimethyl sulfoxide [DMSO] 1% w/v) at three different concentrations (2, 10, and 100 micrograms/ml) on insulin response to glucose in human and rat islets. Likewise, we studied the effect of incubation time, in the presence of DTZ at the above-mentioned concentrations, on insulin release. Only when DTZ was employed at low concentrations and for a short period of incubation (10 min) was there no impairment of pancreatic islet function. Moreover, even at this low concentration, DTZ became deleterious for islet function when the incubation period with the dye was prolonged for 30 min. Culture (24 h) of previously stained islets produced a partial recovery of insulin response. In conclusion, our findings indicate (a) DTZ should not be employed to collect islets for functional studies because of its deleterious effect on beta-cell function, (b) DTZ's deleterious effects on beta-cell function should be considered if this dye is used to purify islets by fluorescence-activated cell sorting for transplantation.

Islets were isolated by automatic digestion from non-diabetic cadaveric organ donors and from Type 2 (non-insulin-dependent) diabetic subjects. The activity of FAD-glycerophosphate dehydrogenase, but not that of either glutamate dehydrogenase, glutamate-oxalacetate transaminase or glutamate-pyruvate transaminase, was lower in Type 2 diabetic patients than control subjects. Hexokinase, glucokinase and glutamate decarboxylase activities were also measured in islets from control subjects. The utilization of D-[5-3H]glucose, oxidation of D-[6-14C]glucose and release of insulin evoked by D-glucose were all lower in Type 2 diabetic patients than control subjects. The secretory response to the combination of L-leucine and L-glutamine appeared less severely affected. Islets from Type 2 diabetic patients may thus display enzymatic, metabolic and secretory anomalies similar to those often observed in animal models of Type 2 diabetes, including a deficiency of beta-cell FAD-linked glycerophosphate dehydrogenase, the key enzyme of the glycerol phosphate shuttle.

This study investigated the effect of glucose on islet amyloid polypeptide secretion, content, and mRNA synthesis of human pancreatic islets. The release of islet amyloid polypeptide from fresh isolated islets in response to glucose was parallel to that of insulin. The islet amyloid polypeptide-to-insulin molar ratios in response to 5.5 and 16.7 mM glucose were 1:16 and 1:15 respectively. Islets were cultured for 1 and 7 days at two different glucose concentrations (5.5 and 16.7 mM). The islet amyloid polypeptide response to the 1-day culture was similar to that of the fresh islets; however, after the 7-day culture the islet amyloid polypeptide and insulin secretory responses to glucose were dissociated. The insulin response of islets to a high-glucose stimulus was significantly (P < 0.001) increased, whereas the islet amyloid polypeptide response of islets to the same stimulus was blunted. The IAPP content was greater than insulin content in a molar ratio (1:50 to 1:30) after long exposure of islets to concentrations of high glucose even though the increase was significant for both peptides (P < 0.005). Northern blot analysis of each cultured condition showed an increase of both mRNA IAPP and insulin signals after exposure of islets at 16.7 mM glucose, the maximum mRNA expression being after long exposure to high-glucose concentrations. Quantification of both signals by densitometry showed a greater increase for islet amyloid polypeptide than for insulin. These findings suggest that IAPP can be accumulated in beta-cells after long exposure of human islets to high-glucose concentrations, because glucose increases IAPP synthesis but not secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Pancreatic islet GLUT2 mRNA is known to be regulated in vitro and in vivo by glucose. We have investigated several potential mechanisms mediating the response of islet GLUT2 to glucose. GLUT2 mRNA and protein were measured from isolated rat islets cultured for up to 24 h under selected conditions. Glucose at 11 mM stimulated GLUT2 mRNA 10-fold compared with 2 mM glucose, with no additional increase at 16.7 mM glucose, whereas maximal 4-fold induction of the protein was attained with 16 mM glucose. Time course studies showed a 2.5-fold induction of GLUT2 mRNA apparent after only 8 h of culture at 16.7 mM glucose. Glycolysis inhibitor mannoheptulose suppressed the stimulatory effect of 16.7 mM glucose on GLUT2 mRNA and protein. Metabolizable sugars mannose and glyceraldehyde enhanced transporter mRNA levels, in contrast with the lack of stimulation by nonmetabolizable 2-deoxy-D-glucose. Stimulation by different sugars and glycolysis inhibition led to analogous changes of proinsulin mRNA, suggesting that common signaling mechanisms are shared in glucose regulation of proinsulin and GLUT2 gene expression. Preexposure to mannoheptulose, however, failed to suppress glucose-stimulated insulin release. Tunicamycin, a glycoprotein synthesis inhibitor, did not block the effect of 16 mM glucose on GLUT2 mRNA levels. RNA and protein synthesis inhibitors actinomycin and cycloheximide abolished the enhancing effects of high glucose on GLUT2 mRNA. These findings indicate that glucose metabolism, but not glycoprotein synthesis or substrate interaction with the transporter protein, is instrumental in the stimulatory effects of glucose on beta-cell GLUT2 mRNA accumulation. In addition, ongoing RNA and protein synthesis are required for this effect.